Production and degradation of polyhydroxyalkanoates in waste environment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, S.Y.; Choi, J.

1999-06-01

Polyhydroxyalkanoates (PHAs) are energy/carbon storage materials accumulated under unfavorable growth condition in the presence of excess carbon source. PHAs are attracting much attention as substitute for non-degradable petrochemically derived plastics because of their similar material properties to conventional plastics and complete biodegradability under natural environment upon disposal. In this paper, PHA production and degradation in waste environment as well as its role in biological phosphorus removal are reviewed. In biological phosphorus removal process, bacteria accumulating polyphosphate (poly P) uptake carbon substrates and accumulate these as PHA by utilizing energy from breaking down poly P under anaerobic conditions. In the followingmore » aerobic condition, accumulated PHA is utilized for energy generation and for the regeneration of poly P. PHA production from waste has been investigated in order to utilize abundant organic compounds in waste water. Since PHA content and PHA productivity that can be obtained are rather low, PHA production from waste product should be considered as a coupled process for reducing the amount of organic waste. PHAs can be rapidly degraded to completion in municipal anaerobic sludge by various microorganisms.« less

Impact of phosphate limitation on PHA production in a feast-famine process.

PubMed

Korkakaki, Emmanouela; van Loosdrecht, Mark C M; Kleerebezem, Robbert

2017-12-01

Double-limitation systems have shown to induce polyhydroxyalkanoates (PHA) production in chemostat studies limited in e.g. carbon and phosphate. In this work the impact of double substrate limitation on the enrichment of a PHA producing community was studied in a sequencing batch process. Enrichments at different C/P concentration ratios in the influent were established and the effect on the PHA production capacity and the enrichment community structure was investigated. Experimental results demonstrated that when a double substrate limitation is imposed at a C/P ratio in the influent in a range of 150 (C-mol/mol), the P-content of the biomass and the specific substrate uptake rates decreased. Nonetheless, the PHA storage capacity remained high (with a maximum of 84 wt%). At a C/P ratio of 300, competition in the microbial community is based on phosphate uptake, and the PHA production capacity is lost. Biomass specific substrate uptake rates are a linear function of the cellular P-content, offering advantages for scaling-up the PHA production process due to lower oxygen requirements. Copyright © 2017 Elsevier Ltd. All rights reserved.

Transcriptional and Posttranscriptional Control of Phaseolin and Phytohemagglutinin Gene Expression in Developing Cotyledons of Phaseolus vulgaris.

PubMed

Chappell, J; Chrispeels, M J

1986-05-01

The expression of phaseolin and phytohemagglutinin (PHA) in the developing cotyledons of a normal (Greensleeves) and a PHA-deficient (Pinto 111) cultivar of Phaseolus vulgaris was investigated. Phaseolin mRNA translational activity and abundance were present at similar levels in both cultivars. In contrast, PHA mRNA translational activity and abundance in Pinto 111 were less than 1% of the levels measured in Greensleeves. Using nuclear runoff assays, the transcription rate of phaseolin gene sequences was similar in both cultivars. The transcription rate of PHA gene sequences in Pinto 111 was only 20% of that measured in Greensleeves. Comparison of the transcription rates with the relative mRNA amounts measured in RNA blot hybridizations indicated that the normally expressed storage protein gene mRNAs were very stable with half-lives greater than several days. Because a low level of PHA gene transcription in Pinto 111 was measurable but no PHA mRNA accumulated, these results suggest that the PHA deficiency in Pinto 111 is due to a reduced transcription rate and possibly an instability of the mRNA.

Analysis of poly-β-hydroxyalkonates (PHA) during the enhanced biological phosphorus removal process using FTIR spectroscopy.

PubMed

Li, Wei-hua; Mao, Qin-yan; Liu, Yi-xin; Sheng, Guo-ping; Yu, Han-qing; Huang, Xian-huai; Liu, Shao-geng; Ling, Qi; Yan, Guo-bing

2014-06-01

Enhanced biological phosphorus removal (EBPR) is the main phosphorus removal technique for wastewater treatment. During the anaerobic-aerobic alternative process, the activated sludge experienced the anaerobic storage of polyhydroxy-β-alkonates (PHA) and aerobic degradation, corresponding the infrared peak intensity of sludge at 1 740 cm(-1) increased in the aerobic phase and declined in the anaerobic phase. Compared with PHA standard, this peak was indentified to attribute the carbonyl of PHA. The overlapping peaks of PHA, protein I and II bands were separated using Gaussian peak fitting method. The infrared peak area ratios of PHA versus protein I had a good relationship with the PHA contents measured by gas chromatography, and the correlation coefficient was 0.873. Thus, the ratio of the peak area of PHA versus protein I can be considered as the indicator of the PHA content in the sludge. The infrared spectra of 1 480-1 780 cm(-1) was selected, normalized and transferred to the absorption data. Combined with the chromatography analysis of PHA content in the sludge sample, a model between the Fourier-transform infrared spectroscopy (ETIR) spectra of the sludge and PHA content was established, which could be used for the prediction of the PHA content in the unknown sample. The PHA content in the sludge sample could be acquired by the infrared spectra of the sludge sample and the established model, and the values fitted well with the results obtained from chromatograph. The results would provide a novel analysis method for the rapid characterization and quantitative determination of the intracellular PHA content in the activated sludge.

The Effect of Acclimation to Sublethal Temperature on Subsequent Susceptibility of Sitophilus zeamais Mostchulsky (Coleoptera: Curculionidae) to High Temperatures

PubMed Central

Lü, Jianhua; Zhang, Huina

2016-01-01

Heat treatment is a popular alternative to synthetic pesticides in disinfesting food-processing facilities and empty grain storages. Sitophilus zeamais Mostchulsky is one of the most cosmopolitan and destructive insects found in empty grain storage facilities and processing facilities. The effect of acclimation in S. zeamais adults to sublethal high temperature on their subsequent susceptibility to high temperatures was investigated. S. zeamais adults were acclimated to 36°C for 0 (as a control), 1, 3, and 5 h, and then were exposed at 43, 47, 51, and 55°C for different time intervals respectively. Acclimation to sublethal high temperature significantly reduced subsequent susceptibility of S. zeamais adults to lethal high temperatures of 43, 47, 51, and 55°C, although the mortality of S. zeamais adults significantly increased with increasing exposure time at lethal high temperatures. The mortality of S. zeamais adults with 1, 3, and 5 h of acclimation to 36°C was significantly lower than that of S. zeamais adults without acclimation when exposed to the same lethal high temperatures. The present results suggest that the whole facility should be heated to target lethal high temperature as soon as possible, avoiding decreasing the control effectiveness of heat treatment due to the acclimation in stored product insects to sublethal temperature. PMID:27462906

Increased Flow of Fatty Acids toward β-Oxidation in Developing Seeds of Arabidopsis Deficient in Diacylglycerol Acyltransferase Activity or Synthesizing Medium-Chain-Length Fatty Acids1

PubMed Central

Poirier, Yves; Ventre, Giovanni; Caldelari, Daniela

1999-01-01

Synthesis of polyhydroxyalkanoates (PHAs) from intermediates of fatty acid β-oxidation was used as a tool to study fatty acid degradation in developing seeds of Arabidopsis. Transgenic plants expressing a peroxisomal PHA synthase under the control of a napin promoter accumulated PHA in developing seeds to a final level of 0.06 mg g−1 dry weight. In plants co-expressing a plastidial acyl-acyl carrier protein thioesterase from Cuphea lanceolata and a peroxisomal PHA synthase, approximately 18-fold more PHA accumulated in developing seeds. The proportion of 3-hydroxydecanoic acid monomer in the PHA was strongly increased, indicating a large flow of capric acid toward β-oxidation. Furthermore, expression of the peroxisomal PHA synthase in an Arabidopsis mutant deficient in the enzyme diacylglycerol acyltransferase resulted in a 10-fold increase in PHA accumulation in developing seeds. These data indicate that plants can respond to the inadequate incorporation of fatty acids into triacylglycerides by recycling the fatty acids via β-oxidation and that a considerable flow toward β-oxidation can occur even in a plant tissue primarily devoted to the accumulation of storage lipids. PMID:10594123

Increased flow of fatty acids toward beta-oxidation in developing seeds of Arabidopsis deficient in diacylglycerol acyltransferase activity or synthesizing medium-chain-length fatty acids.

PubMed

Poirier, Y; Ventre, G; Caldelari, D

1999-12-01

Synthesis of polyhydroxyalkanoates (PHAs) from intermediates of fatty acid beta-oxidation was used as a tool to study fatty acid degradation in developing seeds of Arabidopsis. Transgenic plants expressing a peroxisomal PHA synthase under the control of a napin promoter accumulated PHA in developing seeds to a final level of 0. 06 mg g(-1) dry weight. In plants co-expressing a plastidial acyl-acyl carrier protein thioesterase from Cuphea lanceolata and a peroxisomal PHA synthase, approximately 18-fold more PHA accumulated in developing seeds. The proportion of 3-hydroxydecanoic acid monomer in the PHA was strongly increased, indicating a large flow of capric acid toward beta-oxidation. Furthermore, expression of the peroxisomal PHA synthase in an Arabidopsis mutant deficient in the enzyme diacylglycerol acyltransferase resulted in a 10-fold increase in PHA accumulation in developing seeds. These data indicate that plants can respond to the inadequate incorporation of fatty acids into triacylglycerides by recycling the fatty acids via beta-oxidation and that a considerable flow toward beta-oxidation can occur even in a plant tissue primarily devoted to the accumulation of storage lipids.

Strategies for efficiently selecting PHA producing mixed microbial cultures using complex feedstocks: Feast and famine regime and uncoupled carbon and nitrogen availabilities.

PubMed

Oliveira, Catarina S S; Silva, Carlos E; Carvalho, Gilda; Reis, Maria A

2017-07-25

Production of polyhydroxyalkanoates (PHAs) by open mixed microbial cultures (MMCs) has been attracting increasing interest as an alternative technology to PHA production by pure cultures, due to the potential for lower costs associated with the use of open systems (eliminating the requirement for sterile conditions) and the utilisation of cheap feedstock (industrial and agricultural wastes). Such technology relies on the efficient selection of an MMC enriched in PHA-accumulating organisms. Fermented cheese whey, a protein-rich complex feedstock, has been used previously to produce PHA using the feast and famine regime for selection of PHA accumulating cultures. While this selection strategy was found efficient when operated at relatively low organic loading rate (OLR, 2g-CODL -1 d -1 ), great instability and low selection efficiency of PHA accumulating organisms were observed when higher OLR (ca. 6g-CODL -1 d -1 ) was applied. High organic loading is desirable as a means to enhance PHA productivity. In the present study, a new selection strategy was tested with the aim of improving selection for high OLR. It was based on uncoupling carbon and nitrogen supply and was implemented and compared with the conventional feast and famine strategy. For this, two selection reactors were fed with fermented cheese whey applying an OLR of ca. 8.5g-CODL -1 (with 3.8g-CODL -1 resulting from organic acids and ethanol), and operated in parallel under similar conditions, except for the timing of nitrogen supplementation. Whereas in the conventional strategy nitrogen and carbon substrates were added simultaneously at the beginning of the cycle, in the uncoupled substrates strategy, nitrogen addition was delayed to the end of the feast phase (i.e. after exogenous carbon was exhausted). The two different strategies selected different PHA-storing microbial communities, dominated by Corynebacterium and a Xantomonadaceae, respectively with the conventional and the new approaches. The new strategy originated a more efficient PHA-production process than the conventional one (global PHA productivity of 6.09g-PHAL -1 d -1 and storage yield of 0.96 versus 2.55g-PHAL -1 d -1 and 0.86, respectively). Dissociation between the feast to famine length ratio (F/F) and storage efficiency was shown to be possible with the new strategy, allowing selection of an efficient PHA-storing culture with complex feedstock under high organic loading rates. Copyright © 2016 Elsevier B.V. All rights reserved.

Ralstonia eutropha's Poly(3-hydroxybutyrate)(PHB) polymerase PhaC1 and PHB depolymerase PhaZa1 are phosphorylated in vivo.

PubMed

Jüngert, Janina R; Patterson, Cameron; Jendrossek, Dieter

2018-04-20

In this study, we screened PHB synthase PhaC1 and PHB depolymerase PhaZa1 of Ralstonia eutropha for the presence of phosphorylated residues during the PHB accumulation and PHB degradation phase. Thr373 of PHB synthase PhaC1 was phosphorylated in the stationary growth phase but was not modified in the exponential and PHB accumulation phases. Ser35 of PHB depolymerase PhaZa1 was identified in phosphorylated form both in the exponential and in the stationary growth phase. Additional phosphosites were identified for both proteins in sample-dependent forms. Site-directed mutagenesis of the codon for Thr373 and other phosphosites of PhaC1 revealed a strong negative impact on PHB synthase activity. Modification of Thr26 and Ser35 of PhaZa1 reduced the ability of R. eutropha to mobilize PHB in the stationary growth phase. Our results show that phosphorylation of PhaC1 and PhaZa1 can be important for modulation of the activities of PHB synthase and PHB depolymerase. Importance Polyhydroxybutyrate (PHB) and related polyhydroxyalkanoates (PHAs) are important intracellular carbon and energy storage compounds in many prokaryotes. The accumulation of PHB or PHAs increases the fitness of cells during periods of starvation and other stress conditions. The simultaneous presence of poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) and PHB depolymerase (PhaZa1) on synthesized PHB granules in Ralstonia eutropha (alternative designation Cupriavidus necator ) has been previously shown in several laboratories. These findings imply that the activities of PHB synthase and PHB depolymerase should be regulated to avoid a futile cycle of simultaneous synthesis and degradation of PHB. Here, we addressed this question by identifying phosphorylation sites on PhaC1 and PhaZa1 and by site-directed mutagenesis of identified residues. Furthermore, we conducted in vitro and in vivo analysis of PHB synthase activity and PHB contents. Copyright © 2018 American Society for Microbiology.

Impact of nitrogen feeding regulation on polyhydroxyalkanoates production by mixed microbial cultures.

PubMed

Silva, Fernando; Campanari, Sabrina; Matteo, Stefania; Valentino, Francesco; Majone, Mauro; Villano, Marianna

2017-07-25

A sequencing batch reactor (SBR) is typically used for selecting mixed microbial cultures (MMC) for polyhydroxyalkanoate (PHA) production. Since many waste streams suitable as process feedstock for PHA production are nitrogen-deficient, a nutrient supply in the SBR is typically required to allow for efficient microbial growth. The scope of this study was to devise a nitrogen feeding strategy which allows controlling the nitrogen levels during the feast and famine regime of a lab-scale SBR, thereby selecting for PHA-storing microorganisms. At the beginning of the cycle the reactor was fed with a synthetic mixture of acetic and propionic acids at an overall organic load rate of 8.5gCODL -1 d -1 (i.e. 260CmmolL -1 d -1 ), whereas nitrogen (in the form of ammonium sulphate) was added either simultaneously to the carbon feed (coupled feeding strategy) or after the end of the feast phase (uncoupled feeding strategy). As a main result, PHA production was more than doubled (up to about 1300±64mgCODL -1 ) when carbon and nitrogen were separately fed and the higher PHA production also corresponded to an 82% increase in the polymer HV content (up to 20±1%, wtwt -1 ). Three SBR runs were performed with the uncoupled carbon and nitrogen feeding at different carbon to nitrogen (C/N) ratios (of 14.3, 17.9, and 22.3CmolNmol -1 , respectively) which were varied by progressively reducing the concentration of the nitrogen feeding. In spite of a comparable PHA storage yield at 14.3 and 17.9CmolNmol -1 (0.41±0.05 gCOD PHA gCOD VFA -1 and 0.38±0.05 gCOD PHA gCOD VFA -1 , respectively), the storage response of the selected MMC significantly decreased when the C/N ratio was set at the highest investigated value. Notably, an increase in this parameter also resulted in a change in the HV content in the polymer regardless the composition of the organic acids solution. Copyright © 2016 Elsevier B.V. All rights reserved.

PHB granules are attached to the nucleoid via PhaM in Ralstonia eutropha.

PubMed

Wahl, Andreas; Schuth, Nora; Pfeiffer, Daniel; Nussberger, Stephan; Jendrossek, Dieter

2012-11-16

Poly(3-hydroxybutyrate) (PHB) granules are important storage compounds of carbon and energy in many prokaryotes which allow survival of the cells in the absence of suitable carbon sources. Formation and subcellular localization of PHB granules was previously assumed to occur randomly in the cytoplasm of PHB accumulating bacteria. However, contradictionary results on subcellular localization of PHB granules in Ralstonia eutropha were published, recently. Here, we provide evidence by transmission electron microscopy that PHB granules are localized in close contact to the nucleoid region in R. eutropha during growth on nutrient broth. Binding of PHB granules to the nucleoid is mediated by PhaM, a PHB granule associated protein with phasin-like properties that is also able to bind to DNA and to phasin PhaP5. Over-expression of PhaM resulted in formation of many small PHB granules that were always attached to the nucleoid region. In contrast, PHB granules of ∆phaM strains became very large and distribution of granules to daughter cells was impaired. Association of PHB granules to the nucleoid region was prevented by over-expression of PhaP5 and clusters of several PHB granules were mainly localized near the cell poles. Subcellular localization of PHB granules is controlled in R. eutropha and depends on the presence and concentrations of at least two PHB granule associated proteins, PhaM and PhaP5.

Quantitative measurement of the growth rate of the PHA-producing photosynthetic bacterium Rhodocyclus gelatinous CBS-2[PolyHydroxyAlkanoate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wolfrum, E.J.; Weaver, P.F.

Researchers at the National Renewable Energy Laboratory (NREL) have been investigating the use of model photosynthetic microorganisms that use sunlight and two-carbon organic substrates (e.g., ethanol, acetate) to produce biodegradable polyhydroxyalkanoate (PHA) copolymers as carbon storage compounds. Use of these biological PHAs in single-use plastics applications, followed by their post-consumer composting or anaerobic digestion, could impact petroleum consumption as well as the overloading of landfills. The large-scale production of PHA polymers by photosynthetic bacteria will require large-scale reactor systems utilizing either sunlight or artificial illumination. The first step in the scale-up process is to quantify the microbial growth rates andmore » the PHA production rates as a function of reaction conditions such as nutrient concentration, temperature, and light quality and intensity.« less

Optimization of Water/Oil/Surfactant System for Preparation of Medium-Chain-Length Poly-3-Hydroxyalkanoates (mcl-PHA)-Incorporated Nanoparticles via Nanoemulsion Templating Technique.

PubMed

Ishak, K A; Annuar, M Suffian M; Ahmad, N

2017-12-01

Polymeric nanoparticles gain a widespread interest in food and pharmaceutical industries as delivery systems that encapsulate, protect, and release lipophilic compounds such as omega-3 fatty acids, fat-soluble vitamins, carotenoids, carvedilol, cyclosporine, and ketoprofen. In this study, medium-chain-length poly-3-hydroxyalkanoate (mcl-PHA)-incorporated nanoparticle was developed via facile organic solvent-free nanoemulsion templating technique. The water content (W/surfactant-to-oil (S/O)), S/O, and Cremophor EL-to-Span 80 (Cremo/Sp80) ratios were first optimized using response surface methodology (RSM) to obtain nanoemulsion template prior to incorporation of mcl-PHA. Their effects on nanoemulsion formation were investigated. The mcl-PHA-incorporated nanoparticle system showed a good preservation capability of β-carotene and extended storage stability.

Bulking sludge for PHA production: energy saving and comparative storage capacity with well-settled sludge.

PubMed

Wen, Qinxue; Chen, Zhiqiang; Wang, Changyong; Ren, Nanqi

2012-01-01

Two acetate-fed sequencing batch reactors (SBR) were operated under an aerobic dynamic feeding (ADF) model (SBR#2) and with anaerobic phase before aerobic phase (SBR#1) to select mixed cultures with a high polyhydroxyalkanoates (PHA) storage response. Although kinetic selection based on storage response should bring about a predominance of floc-formers, a bulking sludge with storage response comparable to well-settled sludge was steadily established. An anaerobic phase was introduced before the aerobic phase in the ADF model to improve the sludge settleability (SBR #1), however, due to the consequent increased feast/famine ratio, the performance of SBR #1, in terms of both the maximum PHB (polyhydroxybutyrate) cell content and deltaPHB, was lower than that of SBR #2. SBR #2 gradually reached a steady state while SBR #1 failed suddenly after 50 days of operation. The maximum specific substrate uptake rate and storage rate for the selected bulking sludge were 0.4 Cmol Ac/(Cmol X x hr) and 0.18 Cmol Ac/(Cmol PHB x hr), respectively, resulting a yield of 0.45 Cmol PHB/(Cmol Ac) in SBR #2 in the culture enrichment phase. A maximum PHB content of 53% of total suspended solids and PHB storage rate of 1.36 Cmol Ac/(Cmol PHB x hr) was achieved at 10.2 hr in batch accumulation tests under nitrogen starvation. The results indicated that it was feasible to utilize filamentous bacteria to accumulate PHA with a rate comparable to well-settled sludge. Furthermore, the lower dissolved oxygen demand of filamentous bacteria would save energy required for aeration in the culture enrichment stage.

New Insights into PhaM-PhaC-Mediated Localization of Polyhydroxybutyrate Granules in Ralstonia eutropha H16

PubMed Central

Bresan, Stephanie

2017-01-01

ABSTRACT The formation and localization of polyhydroxybutyrate (PHB) granules in Ralstonia eutropha are controlled by PhaM, which interacts both with the PHB synthase (PhaC) and with the bacterial nucleoid. Here, we studied the importance of proline and lysine residues of two C-terminal PAKKA motifs in PhaM for their importance in attaching PHB granules to DNA by in vitro and in vivo methods. Substitution of the lysine residues but not of the proline residues resulted in detachment of formed PHB granules from the nucleoid. Instead, formation of PHB granule clusters at polar regions of the rod-shaped cells and an unequal distribution of PHB granules to daughter cells were observed. The formation of PHB granules was studied by the expression of chromosomally anchored gene fusions of fluorescent proteins with PhaM and PhaC in different backgrounds. PhaM and PhaC fusions showed a distinct colocalization at formed PHB granules in the nucleoid region of the wild type. In a ΔphaC background, PhaM and the catalytically inactive PhaCC319A protein were not able to form fluorescent foci, indicating that correct positioning requires the formation of PHB. Furthermore, time-lapse experiments revealed that PhaC and PhaM proteins detach from formed PHB granules at later stages, resulting in a nonhomogeneous population of PHB granules. This could explain why growth of individual PHB granules stops under PHB-permissive conditions at a certain size. IMPORTANCE PHB granules are storage compounds for carbon and energy in many prokaryotes. Equal distribution of accumulated PHB granules during cell division is therefore important for optimal fitness of the daughter cells. In R. eutropha, PhaM is responsible for maximal activity of PHB synthase, for initiation of PHB granule formation at discrete regions in the cells, and for association of formed PHB granules with the nucleoid. Here we found that four lysine residues of C-terminal PhaM sequence motifs are essential for association of PHB granules with the nucleoid. Furthermore, we followed PHB granule formation by time-lapse microscopy and provide evidence for aging of PHB granules that is manifested by detachment of previously PHB granule-associated PhaM and PHB synthase. PMID:28389545

PHB granules are attached to the nucleoid via PhaM in Ralstonia eutropha

PubMed Central

2012-01-01

Background Poly(3-hydroxybutyrate) (PHB) granules are important storage compounds of carbon and energy in many prokaryotes which allow survival of the cells in the absence of suitable carbon sources. Formation and subcellular localization of PHB granules was previously assumed to occur randomly in the cytoplasm of PHB accumulating bacteria. However, contradictionary results on subcellular localization of PHB granules in Ralstonia eutropha were published, recently. Results Here, we provide evidence by transmission electron microscopy that PHB granules are localized in close contact to the nucleoid region in R. eutropha during growth on nutrient broth. Binding of PHB granules to the nucleoid is mediated by PhaM, a PHB granule associated protein with phasin-like properties that is also able to bind to DNA and to phasin PhaP5. Over-expression of PhaM resulted in formation of many small PHB granules that were always attached to the nucleoid region. In contrast, PHB granules of ∆phaM strains became very large and distribution of granules to daughter cells was impaired. Association of PHB granules to the nucleoid region was prevented by over-expression of PhaP5 and clusters of several PHB granules were mainly localized near the cell poles. Conclusion Subcellular localization of PHB granules is controlled in R. eutropha and depends on the presence and concentrations of at least two PHB granule associated proteins, PhaM and PhaP5. PMID:23157596

Polyhydroxyalkanoate-associated phasins as phylogenetically heterogeneous, multipurpose proteins.

PubMed

Maestro, Beatriz; Sanz, Jesús M

2017-11-01

Polyhydroxyalkanoates (PHAs) are natural polyesters of increasing biotechnological importance that are synthesized by many prokaryotic organisms as carbon and energy storage compounds in limiting growth conditions. PHAs accumulate intracellularly in form of inclusion bodies that are covered with a proteinaceous surface layer (granule-associated proteins or GAPs) conforming a network-like surface of structural, metabolic and regulatory polypeptides, and configuring the PHA granules as complex and well-organized subcellular structures that have been designated as 'carbonosomes'. GAPs include several enzymes related to PHA metabolism (synthases, depolymerases and hydroxylases) together with the so-called phasins, an heterogeneous group of small-size proteins that cover most of the PHA granule and that are devoid of catalytic functions but nevertheless play an essential role in granule structure and PHA metabolism. Structurally, phasins are amphiphilic proteins that shield the hydrophobic polymer from the cytoplasm. Here, we summarize the characteristics of the different phasins identified so far from PHA producer organisms and highlight the diverse opportunities that they offer in the Biotechnology field. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Large sensitivity in land carbon storage due to geographical and temporal variation in the thermal response of photosynthetic capacity.

PubMed

Mercado, Lina M; Medlyn, Belinda E; Huntingford, Chris; Oliver, Rebecca J; Clark, Douglas B; Sitch, Stephen; Zelazowski, Przemyslaw; Kattge, Jens; Harper, Anna B; Cox, Peter M

2018-06-01

Plant temperature responses vary geographically, reflecting thermally contrasting habitats and long-term species adaptations to their climate of origin. Plants also can acclimate to fast temporal changes in temperature regime to mitigate stress. Although plant photosynthetic responses are known to acclimate to temperature, many global models used to predict future vegetation and climate-carbon interactions do not include this process. We quantify the global and regional impacts of biogeographical variability and thermal acclimation of temperature response of photosynthetic capacity on the terrestrial carbon (C) cycle between 1860 and 2100 within a coupled climate-carbon cycle model, that emulates 22 global climate models. Results indicate that inclusion of biogeographical variation in photosynthetic temperature response is most important for present-day and future C uptake, with increasing importance of thermal acclimation under future warming. Accounting for both effects narrows the range of predictions of the simulated global land C storage in 2100 across climate projections (29% and 43% globally and in the tropics, respectively). Contrary to earlier studies, our results suggest that thermal acclimation of photosynthetic capacity makes tropical and temperate C less vulnerable to warming, but reduces the warming-induced C uptake in the boreal region under elevated CO 2 . © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

Impact of Ralstonia eutropha's poly(3-Hydroxybutyrate) (PHB) Depolymerases and Phasins on PHB storage in recombinant Escherichia coli.

PubMed

Eggers, Jessica; Steinbüchel, Alexander

2014-12-01

The model organism for polyhydroxybutyrate (PHB) biosynthesis, Ralstonia eutropha H16, possesses multiple isoenzymes of granules coating phasins as well as of PHB depolymerases, which degrade accumulated PHB under conditions of carbon limitation. In this study, recombinant Escherichia coli BL21(DE3) strains were used to study the impact of selected PHB depolymerases of R. eutropha H16 on the growth behavior and on the amount of accumulated PHB in the absence or presence of phasins. For this purpose, 20 recombinant E. coli BL21(DE3) strains were constructed, which harbored a plasmid carrying the phaCAB operon from R. eutropha H16 to ensure PHB synthesis and a second plasmid carrying different combinations of the genes encoding a phasin and a PHB depolymerase from R. eutropha H16. It is shown in this study that the growth behavior of the respective recombinant E. coli strains was barely affected by the overexpression of the phasin and PHB depolymerase genes. However, the impact on the PHB contents was significantly greater. The strains expressing the genes of the PHB depolymerases PhaZ1, PhaZ2, PhaZ3, and PhaZ7 showed 35% to 94% lower PHB contents after 30 h of cultivation than the control strain. The strain harboring phaZ7 reached by far the lowest content of accumulated PHB (only 2.0% [wt/wt] PHB of cell dry weight). Furthermore, coexpression of phasins in addition to the PHB depolymerases influenced the amount of PHB stored in cells of the respective strains. It was shown that the phasins PhaP1, PhaP2, and PhaP4 are not substitutable without an impact on the amount of stored PHB. In particular, the phasins PhaP2 and PhaP4 seemed to limit the degradation of PHB by the PHB depolymerases PhaZ2, PhaZ3, and PhaZ7, whereas almost no influence of the different phasins was observed if phaZ1 was coexpressed. This study represents an extensive analysis of the impact of PHB depolymerases and phasins on PHB accumulation and provides a deeper insight into the complex interplay of these enzymes. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Impact of Ralstonia eutropha's Poly(3-Hydroxybutyrate) (PHB) Depolymerases and Phasins on PHB Storage in Recombinant Escherichia coli

PubMed Central

Eggers, Jessica

2014-01-01

The model organism for polyhydroxybutyrate (PHB) biosynthesis, Ralstonia eutropha H16, possesses multiple isoenzymes of granules coating phasins as well as of PHB depolymerases, which degrade accumulated PHB under conditions of carbon limitation. In this study, recombinant Escherichia coli BL21(DE3) strains were used to study the impact of selected PHB depolymerases of R. eutropha H16 on the growth behavior and on the amount of accumulated PHB in the absence or presence of phasins. For this purpose, 20 recombinant E. coli BL21(DE3) strains were constructed, which harbored a plasmid carrying the phaCAB operon from R. eutropha H16 to ensure PHB synthesis and a second plasmid carrying different combinations of the genes encoding a phasin and a PHB depolymerase from R. eutropha H16. It is shown in this study that the growth behavior of the respective recombinant E. coli strains was barely affected by the overexpression of the phasin and PHB depolymerase genes. However, the impact on the PHB contents was significantly greater. The strains expressing the genes of the PHB depolymerases PhaZ1, PhaZ2, PhaZ3, and PhaZ7 showed 35% to 94% lower PHB contents after 30 h of cultivation than the control strain. The strain harboring phaZ7 reached by far the lowest content of accumulated PHB (only 2.0% [wt/wt] PHB of cell dry weight). Furthermore, coexpression of phasins in addition to the PHB depolymerases influenced the amount of PHB stored in cells of the respective strains. It was shown that the phasins PhaP1, PhaP2, and PhaP4 are not substitutable without an impact on the amount of stored PHB. In particular, the phasins PhaP2 and PhaP4 seemed to limit the degradation of PHB by the PHB depolymerases PhaZ2, PhaZ3, and PhaZ7, whereas almost no influence of the different phasins was observed if phaZ1 was coexpressed. This study represents an extensive analysis of the impact of PHB depolymerases and phasins on PHB accumulation and provides a deeper insight into the complex interplay of these enzymes. PMID:25281380

Formation of Polyhydroxyalkanoate in Aerobic Anoxygenic Phototrophic Bacteria and Its Relationship to Carbon Source and Light Availability▿

PubMed Central

Xiao, Na; Jiao, Nianzhi

2011-01-01

Aerobic anoxygenic phototrophic bacteria (AAPB) are unique players in carbon cycling in the ocean. Cellular carbon storage is an important mechanism regulating the nutrition status of AAPB but is not yet well understood. In this paper, six AAPB species (Dinoroseobacter sp. JL1447, Roseobacter denitrificans OCh 114, Roseobacter litoralis OCh 149, Dinoroseobacter shibae DFL 12T, Labrenzia alexandrii DFL 11T, and Erythrobacter longus DSMZ 6997) were examined, and all of them demonstrated the ability to form the carbon polymer polyhydroxyalkanoate (PHA) in the cell. The PHA in Dinoroseobacter sp. JL1447 was identified as poly-beta-hydroxybutyrate (PHB) according to evidence from Fourier transform infrared spectroscopy, differential scanning calorimetry, and 1H nuclear magnetic resonance spectroscopy examinations. Carbon sources turned out to be critical for PHA production in AAPB. Among the eight media tested with Dinoroseobacter sp. JL1447, sodium acetate, giving a PHA production rate of 72%, was the most productive carbon source, followed by glucose, with a 68% PHA production rate. Such PHA production rates are among the highest recorded for all bacteria. The C/N ratio of substrates was verified by the experiments as another key factor in PHA production. In the case of R. denitrificans OCh 114, PHA was not detected when the organism was cultured at C/N ratios of <2 but became apparent at C/N ratios of >3. Light is also important for the formation of PHA in AAPB. In the case of Dinoroseobacter sp. JL1447, up to a one-quarter increase in PHB production was observed when the culture underwent growth in a light-dark cycle compared to growth completely in the dark. PMID:21908634

Microbial Cometabolism and Polyhydroxyalkanoate Co-polymers.

PubMed

Ray, Subhasree; Kalia, Vipin Chandra

2017-03-01

Polyhydroxyalkanoate (PHAs) are natural, biodegradable biopolymers, which can be produced from renewable materials. PHAs have potential to replace petroleum derived plastics. Quite a few bacteria can produce PHA under nutritional stress. They generally produce homopolymers of butyrate i.e., polyhydroxybutyrate (PHB), as a storage material. The biochemical characteristics of PHB such as brittleness, low strength, low elasticity, etc. make these unsuitable for commercial applications. Co-polymers of PHA, have high commercial value as they overcome the limitations of PHBs. Co-polymers can be produced by supplementing the feed with volatile fatty acids or through hydrolysates of different biowastes. In this review, we have listed the potential bacterial candidates and the substrates, which can be co-metabolized to produce PHA co-polymers.

Acclimation of mechanical and hydraulic functions in trees: impact of the thigmomorphogenetic process

PubMed Central

Badel, Eric; Ewers, Frank W.; Cochard, Hervé; Telewski, Frank W.

2015-01-01

The secondary xylem (wood) of trees mediates several functions including water transport and storage, mechanical support and storage of photosynthates. The optimal structures for each of these functions will most likely differ. The complex structure and function of xylem could lead to trade-offs between conductive efficiency, resistance to embolism, and mechanical strength needed to count for mechanical loading due to gravity and wind. This has been referred to as the trade-off triangle, with the different optimal solutions to the structure/function problems depending on the environmental constraints as well as taxonomic histories. Thus, the optimisation of each function will lead to drastically different anatomical structures. Trees are able to acclimate the internal structure of their trunk and branches according to the stress they experience. These acclimations lead to specific structures that favor the efficiency or the safety of one function but can be antagonistic with other functions. Currently, there are no means to predict the way a tree will acclimate or optimize its internal structure in support of its various functions under differing environmental conditions. In this review, we will focus on the acclimation of xylem anatomy and its resulting mechanical and hydraulic functions to recurrent mechanical strain that usually result from wind-induced thigmomorphogenesis with a special focus on the construction cost and the possible trade-off between wood functions. PMID:25954292

Physiological responses of horses to a treadmill simulated speed and endurance test in high heat and humidity before and after humid heat acclimation.

PubMed

Marlin, D J; Scott, C M; Schroter, R C; Harris, R C; Harris, P A; Roberts, C A; Mills, P C

1999-01-01

To investigate whether horses were able to acclimate to conditions of high temperature and humidity, 5 horses of different breeds were trained for 80 min on 15 consecutive days on a treadmill at 30 degrees C and 80%RH. Training consisted of a combination of long duration low-intensity exercise, medium duration medium intensity exercise and short duration high intensity exercise. Between training sessions the horses were maintained at 11+/-3 degrees C and 74+/-2%RH. Before (PRE-ACC) and after acclimation (POST-ACC) the horses undertook a simulated Competition Exercise Test (CET), designed to represent the Speed and Endurance Test of a 3-day event, at 30 degrees C/80%RH. Maximal oxygen uptake (VO2PEAK) was not changed following acclimation (PRE-ACC 141+/-8 ml/min/kg bwt vs. POST-ACC 145+/-9 ml/min/kg bwt [STPD], P>0.05). Following acclimation, 4 of the 5 horses were able to complete a significantly greater amount of Phase D in the CET (PRE-ACC 6.3+/-0.3 min vs. POST-ACC 7.3+/-0.3 min, P<0.05; target time = 8 min). Resting body temperatures (pulmonary artery [TPA], rectal [TREC] and tail-skin [TTSK] temperatures) were all significantly lower following acclimation. During exercise, metabolic heat production (M) and heat dissipation (HD), for the same exercise duration, were both significantly lower following acclimation (P<0.05), although heat storage (HS) was significantly higher (P<0.05). The higher heat storage following acclimation was associated with a lower TTSK for a given TPA and a decreased total fluid loss (% bodyweight, P<0.05). Plasma volume was not changed following acclimation. The relationship of sweating rate (SR) to TPA or TTSK on either the neck or the gluteal region was not significantly altered by acclimation, although the onset of sweating occurred at a lower TPA or TTSK following acclimation (P<0.05). The horses in the present study showed a number of physiological adaptations to a period of 15 days of exposure to high heat and humidity consistent with a humid heat acclimation response. These changes were mostly similar to those reported to occur in man and other species and were consistent with thermal acclimation and an increased thermotolerance, leading to an improved exercise tolerance. It is concluded that a 15 day period of acclimation is beneficial for horses from cooler and or drier climates, that have to compete in hot humid conditions and that this may redress, to some extent, the decrement in exercise tolerance seen in nonacclimated horses and reduce the risk of heat related disorders, such as heat exhaustion.

Enhanced rhamnolipid production in Burkholderia thailandensis transposon knockout strains deficient in polyhydroxyalkanoate (PHA) synthesis.

PubMed

Funston, Scott J; Tsaousi, Konstantina; Smyth, Thomas J; Twigg, Matthew S; Marchant, Roger; Banat, Ibrahim M

2017-12-01

Microbially produced rhamnolipids have significant commercial potential; however, the main bacterial producer, Pseudomonas aeruginosa, is an opportunistic human pathogen, which limits biotechnological exploitation. The non-pathogenic species Burkholderia thailandensis produces rhamnolipids; however, yield is relatively low. The aim of this study was to determine whether rhamnolipid production could be increased in Burkholderia thailandensis through mutation of genes responsible for the synthesis of the storage material polyhydroxyalkanoate (PHA), thereby increasing cellular resources for the production of rhamnolipids. Potential PHA target genes were identified in B. thailandensis through comparison with known function genes in Pseudomonas aeruginosa. Multiple knockout strains for the phbA, phbB and phbC genes were obtained and their growth characteristics and rhamnolipid and PHA production determined. The wild-type strain and an rhamnolipid (RL)-deficient strain were used as controls. Three knockout strains (ΔphbA1, ΔphbB1 and ΔphbC1) with the best enhancement of rhamnolipid production were selected for detailed study. ΔphbB1 produced the highest level of purified RL (3.78 g l -1 ) compared to the wild-type strain (1.28 g l -1 ). In ΔphbB1, the proportion of mono-rhamnolipid was also increased compared to the wild-type strain. The production of PHA was reduced by at least 80% in all three phb mutant strains, although never completely eliminated. These results suggest that, in contrast to Pseudomonas aeruginosa, knockout of the PHA synthesis pathway in Burkholderia thailandensis could be used to increase rhamnolipid production. The evidence of residual PHA production in the phb mutant strains suggests B. thailandensis possesses a secondary unelucidated PHA synthesis pathway.

Community proteomics provides functional insight into polyhydroxyalkanoate production by a mixed microbial culture cultivated on fermented dairy manure.

PubMed

Hanson, Andrea J; Guho, Nicholas M; Paszczynski, Andrzej J; Coats, Erik R

2016-09-01

Polyhydroxyalkanoates (PHAs) are bio-based, biodegradable polyesters that can be produced from organic-rich waste streams using mixed microbial cultures (MMCs). To maximize PHA production, MMCs are enriched for bacteria with a high polymer storage capacity through the application of aerobic dynamic feeding (ADF) in a sequencing batch reactor (SBR), which consequently induces a feast-famine metabolic response. Though the feast-famine response is generally understood empirically at a macro-level, the molecular level is less refined. The objective of this study was to investigate the microbial community composition and proteome profile of an enriched MMC cultivated on fermented dairy manure. The enriched MMC exhibited a feast-famine response and was capable of producing up to 40 % (wt. basis) PHA in a fed-batch reactor. High-throughput 16S rRNA gene sequencing revealed a microbial community dominated by Meganema, a known PHA-producing genus not often observed in high abundance in enrichment SBRs. The application of the proteomic methods two-dimensional electrophoresis and LC-MS/MS revealed PHA synthesis, energy generation, and protein synthesis prominently occurring during the feast phase, corroborating bulk solution variable observations and theoretical expectations. During the famine phase, nutrient transport, acyl-CoA metabolism, additional energy generation, and housekeeping functions were more pronounced, informing previously under-determined MMC functionality under famine conditions. During fed-batch PHA production, acetyl-CoA acetyltransferase and PHA granule-bound phasin proteins were in increased abundance relative to the SBR, supporting the higher PHA content observed. Collectively, the results provide unique microbial community structural and functional insight into feast-famine PHA production from waste feedstocks using MMCs.

Enrichment of a mixed microbial culture for polyhydroxyalkanoates production: Effect of pH and N and P concentrations.

PubMed

Montiel-Jarillo, Gabriela; Carrera, Julián; Suárez-Ojeda, María Eugenia

2017-04-01

Polyhydroxyalkanoates (PHA) are biopolymers that can be an alternative against conventional plastics. The study reported herein evaluated the enrichment of a mixed microbial culture (MMC) operated under feast/famine regime and different pHs in a sequencing batch reactor (SBR) using acetate as sole carbon source to produce polyhydroxyalkanoates (PHAs). The enrichment step was evaluated at controlled pH of 7.5 and also without pH control (averaged value of 9.0). The acetate uptake rate (-q S ) of both enrichments at the end of the experimental period exhibited similar behaviour being about 0.18CmolAcCmolX -1 h -1 and 0.19CmolAcCmolX -1 h -1 for SBR-A and SBR-B, respectively. However, the PHA-storing capacity of the biomass enriched without pH control was better, exhibiting a maximum PHA content of 36% (gPHAg -1 VSS) with a PHA production rate (q PHA ) of 0.16CmolPHACmolX -1 h -1 . Batch experiments were performed to evaluate PHA-storing capacity of the enriched culture at different pHs and nutrients concentrations. In the pH experiments (without nutrient limitation), it was found that in the absence of controlled pH, the enriched biomass exhibited a PHA content of 44% gPHAg -1 VSS with -q S and PHA to substrate yield (Y PHA/Ac ) of 0.57CmolAcCmolX -1 h -1 and 0.33CmolPHACmolAc -1 , respectively. Regarding the experiments at variable nutrients concentration (pH ranging 8.8 to 9.2), the results indicate that the PHA content in the enriched biomass is significantly higher being around 51% gPHAg -1 VSS under nitrogen limitation. This work demonstrated the feasibility of the enrichment of a MMC with PHA storage ability without pH control. Results also suggest that better PHAs contents and substrate uptake rates are obtained without controlling the pH in the accumulation step. Finally, this work also highlights the importance of understanding the role of nutrients concentration during the accumulation step. Copyright © 2017 Elsevier B.V. All rights reserved.

Link between microbial composition and carbon substrate-uptake preferences in a PHA-storing community

PubMed Central

Albuquerque, Maria G E; Carvalho, Gilda; Kragelund, Caroline; Silva, Ana F; Barreto Crespo, Maria T; Reis, Maria A M; Nielsen, Per H

2013-01-01

The microbial community of a fermented molasses-fed sequencing batch reactor (SBR) operated under feast and famine conditions for production of polyhydroxyalkanoates (PHAs) was identified and quantified through a 16 S rRNA gene clone library and fluorescence in situ hybridization (FISH). The microbial enrichment was found to be composed of PHA-storing populations (84% of the microbial community), comprising members of the genera Azoarcus, Thauera and Paracoccus. The dominant PHA-storing populations ensured the high functional stability of the system (characterized by high PHA-storage efficiency, up to 60% PHA content). The fermented molasses contained primarily acetate, propionate, butyrate and valerate. The substrate preferences were determined by microautoradiography-FISH and differences in the substrate-uptake capabilities for the various probe-defined populations were found. The results showed that in the presence of multiple substrates, microbial populations specialized in different substrates were selected, thereby co-existing in the SBR by adapting to different niches. Azoarcus and Thauera, primarily consumed acetate and butyrate, respectively. Paracoccus consumed a broader range of substrates and had a higher cell-specific substrate uptake. The relative species composition and their substrate specialization were reflected in the substrate removal rates of different volatile fatty acids in the SBR reactor. PMID:22810062

Genomic Analysis of Storage Protein Deficiency in Genetically Related Lines of Common Bean (Phaseolus vulgaris)

PubMed Central

Pandurangan, Sudhakar; Diapari, Marwan; Yin, Fuqiang; Munholland, Seth; Perry, Gregory E.; Chapman, B. Patrick; Huang, Shangzhi; Sparvoli, Francesca; Bollini, Roberto; Crosby, William L.; Pauls, Karl P.; Marsolais, Frédéric

2016-01-01

A series of genetically related lines of common bean (Phaseolus vulgaris L.) integrate a progressive deficiency in major storage proteins, the 7S globulin phaseolin and lectins. SARC1 integrates a lectin-like protein, arcelin-1 from a wild common bean accession. SMARC1N-PN1 is deficient in major lectins, including erythroagglutinating phytohemagglutinin (PHA-E) but not α-amylase inhibitor, and incorporates also a deficiency in phaseolin. SMARC1-PN1 is intermediate and shares the phaseolin deficiency. Sanilac is the parental background. To understand the genomic basis for variations in protein profiles previously determined by proteomics, the genotypes were submitted to short-fragment genome sequencing using an Illumina HiSeq 2000/2500 platform. Reads were aligned to reference sequences and subjected to de novo assembly. The results of the analyses identified polymorphisms responsible for the lack of specific storage proteins, as well as those associated with large differences in storage protein expression. SMARC1N-PN1 lacks the lectin genes pha-E and lec4-B17, and has the pseudogene pdlec1 in place of the functional pha-L gene. While the α-phaseolin gene appears absent, an approximately 20-fold decrease in β-phaseolin accumulation is associated with a single nucleotide polymorphism converting a G-box to an ACGT motif in the proximal promoter. Among residual lectins compensating for storage protein deficiency, mannose lectin FRIL and α-amylase inhibitor 1 genes are uniquely present in SMARC1N-PN1. An approximately 50-fold increase in α-amylase inhibitor like protein accumulation is associated with multiple polymorphisms introducing up to eight potential positive cis-regulatory elements in the proximal promoter specific to SMARC1N-PN1. An approximately 7-fold increase in accumulation of 11S globulin legumin is not associated with variation in proximal promoter sequence, suggesting that the identity of individual proteins involved in proteome rebalancing might also be determined at the translational level. PMID:27066039

Poly-β-hydroxybutyrate Metabolism Is Unrelated to the Sporulation and Parasporal Crystal Protein Formation in Bacillus thuringiensis.

PubMed

Wang, Xun; Li, Zhou; Li, Xin; Qian, Hongliang; Cai, Xia; Li, Xinfeng; He, Jin

2016-01-01

Poly-3-hydroxybutyrate (PHB) is a natural polymer synthesized by many bacteria as a carbon-energy storage material. It was accumulated maximally prior to the spore formation but was degraded during the process of sporulation in Bacillus thuringiensis. Intriguingly, B. thuringiensis also accumulates large amounts of insecticidal crystal proteins (ICPs) during sporulation, which requires considerable input of carbon and energy sources. How PHB accumulation affects sporulation and ICP formation remains unclear to date. Intuitively, one would imagine that accumulated PHB provides the energy required for ICP formation. Yet our current data indicate that this is not the case. First, growth curves of the deletion mutants of phaC (encoding the PHB synthase) and phaZ (encoding the PHB depolymerase) were found to be similar to the parent strain BMB171; no difference in growth rate could be observed. In addition we further constructed the cry1Ac10 ICP gene overexpression strains of BMB171 (BMB171-cry), as well as its phaC and phaZ deletion mutants ΔphaC-cry and ΔphaZ-cry to compare their spore and ICP production rates. Again, not much change of ICP production was observed among these strains either. In fact, PHB was still degraded in most ΔphaZ-cry cells as observed by transmission electron microscopy. Together these results indicated that there is no direct association between the PHB accumulation and the sporulation and ICP formation in B. thuringiensis. Some other enzymes for PHB degradation or other energy source may be responsible for the sporulation and/or ICP formation in B. thuringiensis.

Effect of carbon source type on intracellular stored polymers during endogenous denitritation (ED) treating landfill leachate.

PubMed

Miao, Lei; Wang, Shuying; Li, Baikun; Cao, Tianhao; Zhang, Fangzhai; Wang, Zhong; Peng, Yongzhen

2016-09-01

Glycogen accumulating organisms (GAOs) capable of storing organic compounds as polyhydroxyalkanoate (PHA) have been used for endogenous denitritation (ED), but the effect of carbon sources type on nitrogen removal performance of GAOs treating landfill leachate is unclear. In this study, a successful ED system treating landfill leachate (COD/NH4(+)-N (C/N): 4) without external carbon source addition was applied. The mature leachate with C/N of 1 was used as the feeding base solution, with acetate, propionate, and glucose examined as the carbon sources, and their effects on yields and compositions of PHA produced by GAOs were determined and associated with nitrogen removal performance. In the case of sole carbon source, acetate was much easier to be stored than propionate and glucose, which led to a higher nitrogen removal efficiency. Glucose had the lowest amount of PHA storage and led to the lowest performance. In the case of composite carbon sources (two scenarios: acetate + propionate; acetate + propionate + glucose), GAOs stored sufficient PHA and exhibited similar nitrogen removal efficiencies. Moreover, type of carbon source influenced the compositions of PHA. The polyhydroxybutyrate (PHB) fraction in PHA was far more than polyhydroxyvalerate (PHV) in all tests. PHV was synthesized only when acetate existed in carbon source. The microbial diversity analysis revealed that Proteobacteria was the most abundant phylum. Among the 108 genera detected in this ED system, the genera responsible for denitritation were Thauera, Paracoccus, Ottowia and Comamonadaceae_unclassified, accounting for 46.21% of total bacteria. Especially, Paracoccus and Comamonadaceae_unclassified transformed the carbon source into PHA for denitritation, and carried out endogenous denitritation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cyanobacterial PHA Production—Review of Recent Advances and a Summary of Three Years’ Working Experience Running a Pilot Plant

PubMed Central

Troschl, Clemens; Meixner, Katharina; Drosg, Bernhard

2017-01-01

Cyanobacteria, as photoautotrophic organisms, provide the opportunity to convert CO2 to biomass with light as the sole energy source. Like many other prokaryotes, especially under nutrient deprivation, most cyanobacteria are able to produce polyhydroxyalkanoates (PHAs) as intracellular energy and carbon storage compounds. In contrast to heterotrophic PHA producers, photoautotrophic cyanobacteria do not consume sugars and, therefore, do not depend on agricultural crops, which makes them a green alternative production system. This review summarizes the recent advances in cyanobacterial PHA production. Furthermore, this study reports the working experience with different strains and cultivating conditions in a 200 L pilot plant. The tubular photobioreactor was built at the coal power plant in Dürnrohr, Austria in 2013 for direct utilization of flue gases. The main challenges were the selection of robust production strains, process optimization, and automation, as well as the CO2 availability. PMID:28952505

T cell activation and proliferation following acute exercise in human subjects is altered by storage conditions and mitogen selection.

PubMed

Siedlik, Jacob A; Deckert, Jake A; Benedict, Stephen H; Bhatta, Anuja; Dunbar, Amanda J; Vardiman, John P; Gallagher, Philip M

2017-07-01

Recent work investigating exercise induced changes in immunocompetence suggests that some of the ambiguity in the literature is resultant from different cell isolation protocols and mitogen selection. To understand this effect, we compared post-exercise measures of T cell activation and proliferation using two different stimulation methods (costimulation through CD28 or stimulation with phytohaemagglutinin [PHA]). Further, we investigated whether exercise induced changes are maintained when T cell isolation from whole blood is delayed overnight in either a room temperature or chilled (4°C) environment. As expected, an increased proliferation response was observed post-exercise in T cells isolated from whole blood of previously trained individuals immediately after blood collection. Also, cells stimulated with PHA after resting overnight in whole blood were not adversely impacted by the storage conditions. In contrast, allowing cells to rest overnight in whole blood prior to stimulation through CD28, lessened the proliferation observed by cells following exercise rendering both the room temperature and chilled samples closer to the results seen in the control condition. Changes in early markers of activation (CD25), followed a similar pattern, with activation in PHA stimulated cells remaining fairly robust after overnight storage; whereas cell activation following stimulation through CD3+CD28 was disproportionately decreased by the influence of overnight storage. These findings indicate that decisions regarding cell stimulation methods need to be paired with the timeline for T cell isolation from whole blood. These considerations will be especially important for field based studies of immunocompetence where there is a delay in getting whole blood samples to a lab for processing as well as clinical applications where a failure to isolate T cells in a timely manner may result in loss of the response of interest. Copyright © 2017 Elsevier B.V. All rights reserved.

Enhanced Polyhydroxybutyrate Production for Long-Term Spaceflight Applications

NASA Technical Reports Server (NTRS)

Putman, Ryan J.; Rahman, Asif; Miller, Charles D.; Hadi, Masood Z.

2015-01-01

Synthetic biology holds the promise of advancing long term space fight by the production of medicine, food, materials, and energy. One such application of synthetic biology is the production of biomaterials, specifically polyhydroxyalkanoates (PHAs), using purposed organisms such as Escherichia coli. PHAs are a group of biodegradable bioplastics that are produced by a wide variety of naturally occurring microorganisms, mainly as an energy storage intermediate. PHAs have similar melting point to polypropylene and a Youngs modulus close to polystyrene. Due to limited resources and cost of transportation, large-scale extraction of biologically produced products in situ is extremely cumbersome during space flight. To that end, we are developing a secretion systems for exporting PHA from the cell in order to reduce unit operations. PHAs granules deposited inside bacteria are typically associated with proteins bound to the granule surface. Phasin, a granule bound protein, was targeted for type I secretion by fusion with HlyA signal peptide for indirect secretion of PHAs. In order to validate our secretion strategy, a green fluorescent protein (GFP) was tagged to the PHA polymerase enzyme (phaC), this three part gene cassette consists of phaA and phaB and are required for PHA production. Producing PHAs in situ during space flight or planet colonization will enable mission success by providing a valuable source of biomaterials that can have many potential applications thereby reducing resupply requirements. Biologically produced PHAs can be used in additive manufacturing such as three dimensional (3D) printing to create products that can be made on demand during space flight. After exceeding their lifetime, the PHAs could be melted and recycled back to 3D print other products. We will discuss some of our long term goals of this approach.

Thermal acclimation of leaf respiration of tropical trees and lianas: response to experimental canopy warming, and consequences for tropical forest carbon balance.

PubMed

Slot, Martijn; Rey-Sánchez, Camilo; Gerber, Stefan; Lichstein, Jeremy W; Winter, Klaus; Kitajima, Kaoru

2014-09-01

Climate warming is expected to increase respiration rates of tropical forest trees and lianas, which may negatively affect the carbon balance of tropical forests. Thermal acclimation could mitigate the expected respiration increase, but the thermal acclimation potential of tropical forests remains largely unknown. In a tropical forest in Panama, we experimentally increased nighttime temperatures of upper canopy leaves of three tree and two liana species by on average 3 °C for 1 week, and quantified temperature responses of leaf dark respiration. Respiration at 25 °C (R25 ) decreased with increasing leaf temperature, but acclimation did not result in perfect homeostasis of respiration across temperatures. In contrast, Q10 of treatment and control leaves exhibited similarly high values (range 2.5-3.0) without evidence of acclimation. The decrease in R25 was not caused by respiratory substrate depletion, as warming did not reduce leaf carbohydrate concentration. To evaluate the wider implications of our experimental results, we simulated the carbon cycle of tropical latitudes (24°S-24°N) from 2000 to 2100 using a dynamic global vegetation model (LM3VN) modified to account for acclimation. Acclimation reduced the degree to which respiration increases with climate warming in the model relative to a no-acclimation scenario, leading to 21% greater increase in net primary productivity and 18% greater increase in biomass carbon storage over the 21st century. We conclude that leaf respiration of tropical forest plants can acclimate to nighttime warming, thereby reducing the magnitude of the positive feedback between climate change and the carbon cycle. © 2014 John Wiley & Sons Ltd.

Influence of acclimation to sublethal temperature on heat tolerance of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) exposed to 50°C.

PubMed

Lü, Jianhua; Liu, Shuli

2017-01-01

Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) is a serious pest of stored agricultural products and one of the most common insects found in grain storage and food processing facilities. Heat treatment has been revisited to control stored-product insects as a potential alternative to methyl bromide for disinfesting mills and food-processing facilities. The influence of acclimation of T. castaneum adults, pupae, larvae, and eggs to sublethal temperatures of 36, and 42°C on their subsequent susceptibility to lethal temperature of 50°C was respectively investigated. The acclimation of T. castaneum eggs, larvae, pupae, and adults to 36, and 42°C significantly decreased their subsequent susceptibility to lethal high temperature of 50°C. The influence of acclimation to 42°C was significantly greater than that of acclimation to 36°C. The most influential acclimation times at 42°C for mortality of T. castaneum eggs, larvae, pupae, and adults were 15, 5, 5, and 5 h, respectively, and their corresponding mortality were 41.24, 5.59, 20.19, and 4.48%, compared to 100% mortality of T. castaneum eggs, larvae, pupae, and adults without acclimation when exposed to 50°C for 35 min, respectively. The present results have important implications for developing successful heat treatment protocols to control T. castaneum, improving disinfestation effectiveness of heat treatment and understanding insect response to high temperatures.

Smart polyhydroxyalkanoate nanobeads by protein based functionalization.

PubMed

Dinjaski, Nina; Prieto, M Auxiliadora

2015-05-01

The development of innovative medicines and personalized biomedical approaches calls for new generation easily tunable biomaterials that can be manufactured applying straightforward and low-priced technologies. Production of functionalized bacterial polyhydroxyalkanoate (PHA) nanobeads by harnessing their natural carbon-storage granule production system is a thrilling recent development. This branch of nanobiotechnology employs proteins intrinsically binding the PHA granules as tags to immobilize recombinant proteins of interest and design functional nanocarriers for wide range of applications. Additionally, the implementation of new methodological platforms regarding production of endotoxin free PHA nanobeads using Gram-positive bacteria opened new avenues for biomedical applications. This prompts serious considerations of possible exploitation of bacterial cell factories as alternatives to traditional chemical synthesis and sources of novel bioproducts that could dramatically expand possible applications of biopolymers. In the 21st century, we are coming into the age of personalized medicine. There is a growing use of biomaterials in the clinical setting. In this review article, the authors describe the use of natural polyhydroxyalkanoate (PHA) nanoparticulates, which are formed within bacterial cells and can be easily functionalized. The potential uses would include high-affinity bioseparation, enzyme immobilization, protein delivery, diagnostics etc. The challenges of this approach remain the possible toxicity from endotoxin and the high cost of production. Copyright © 2015 Elsevier Inc. All rights reserved.

Bio-plastic (poly-hydroxy-alkanoate) production from municipal sewage sludge in the Netherlands: a technology push or a demand driven process?

PubMed

Bluemink, E D; van Nieuwenhuijzen, A F; Wypkema, E; Uijterlinde, C A

Valorisation of components from municipal 'waste' water and sewage sludge gets more and more attention in order to come to a circular economy by developing an efficient 'waste' to value concept. On behalf of the transition team 'Grondstoffenfabriek' ('Resource factory') a preliminary research was performed for all the Dutch water boards to assess the technical and economical feasibility of poly-hydroxy-alkanoate (PHA)-production from sewage sludge, a valuable product to produce bio-plastics. This study reveals that the production of bio-plastics from sewage sludge is feasible based on technical aspects, but not yet economically interesting, even though the selling price is relatively close to the actual PHA market price. (Selling price is in this particular case the indicative cost effective selling price. The cost effective selling price covers only the total production costs of the product.) Future process optimization (maximizing the volatile fatty acids production, PHA storage capacity, etc.) and market developments are needed and will result in cost reductions of the various sub-processes. PHA-production from sewage sludge at this stage is just a technology; every further research is needed to incorporate the backward integration approach, taking into account the market demand including associated product quality aspects.

Quantitative determination of microbial activity and community nutritional status in estuarine sediments: evidence for a disturbance artifact

NASA Technical Reports Server (NTRS)

Findlay, R. H.; Pollard, P. C.; Moriarty, D. J.; White, D. C.

1985-01-01

In estuarine sediments with a high degree of vertical heterogeneity in reduced substrate and terminal electron acceptor concentrations, the method of exposure of the microbiota to labeled substrates can introduce a "disturbance artifact" into measures of metabolic activity. The detection of this artifact is based on quantitative measurement of the relative rates of incorporation of [14C]acetate into phospholipid fatty acids (PLFA) and endogenous storage lipid, poly-beta-hydroxyalkanoate (PHA). Previous studies have shown that PLFA synthesis measures cellular growth and that PHA synthesis measures carbon accumulation (unbalanced growth). The "disturbance artifact" of exposure to [14C]acetate was demonstrated by comparing injection of a core with the usual or pore-water replacement or slurry techniques. Only injection of labeled substrate allowed detection of preassay disturbance of the sediment with a garden rake. The raking increased PLFA synthesis with little effect to differences in concentration or distribution of [14C]acetate in the 10-min incubation. Bioturbation induced by sand dollar feeding in estuarine sediment could be detected in an increased PLFA/PHA ratio which was due to decreased PHA synthesis if the addition of labeled substrate was by the injection technique. Addition of labeled precursors to sediment by slurry or pore-water replacement induces greater disturbance artifacts than injection techniques.

The Multiple Functions of Common Microbial Carbon Polymers, Glycogen and PHB, during Stress Responses in the Non-Diazotrophic Cyanobacterium Synechocystis sp. PCC 6803.

PubMed

Damrow, Ramon; Maldener, Iris; Zilliges, Yvonne

2016-01-01

Classical microbial carbon polymers such as glycogen and polyhydroxybutyrate (PHB) have a crucial impact as both a sink and a reserve under macronutrient stress conditions. Most microbial species exclusively synthesize and degrade either glycogen or PHB. A few bacteria such as the phototrophic model organism Synechocystis sp. PCC 6803 surprisingly produce both physico-chemically different polymers under conditions of high C to N ratios. For the first time, the function and interrelation of both carbon polymers in non-diazotrophic cyanobacteria are analyzed in a comparative physiological study of single- and double-knockout mutants (ΔglgC; ΔphaC; ΔglgC/ΔphaC), respectively. Most of the observed phenotypes are explicitly related to the knockout of glycogen synthesis, highlighting the metabolic, energetic, and structural impact of this process whenever cells switch from an active, photosynthetic 'protein status' to a dormant 'glycogen status'. The carbon flux regulation into glycogen granules is apparently crucial for both phycobilisome degradation and thylakoid layer disassembly in the presence of light. In contrast, PHB synthesis is definitely not involved in this primary acclimation response. Moreover, the very weak interrelations between the two carbon-polymer syntheses indicate that the regulation and role of PHB synthesis in Synechocystis sp. PCC 6803 is different from glycogen synthesis.

Optimization of nitrogen and phosphorus limitation for better biodegradable plastic production and organic removal using single fed-batch mixed cultures and renewable resources.

PubMed

Din, M F M; Ujang, Z; van Loosdrecht, M C M; Ahmad, A; Sairan, M F

2006-01-01

The process for the production of biodegradable plastic material (polyhydroxyalkanoates, PHAs) from microbial cells by mixed-bacterial cultivation using readily available waste (renewable resources) is the main consideration nowadays. These observations have shown impressive results typically under high carbon fraction, COD/N and COD/P (usually described as nutrient-limiting conditions) and warmest temperature (moderate condition). Therefore, the aim of this work is predominantly to select mixed cultures under high storage responded by cultivation on a substrate - non limited in a single batch reactor with shortest period for feeding and to characterize their storage response by using specific and kinetics determination. In that case, the selected-fixed temperature is 30 degrees C to establish tropical conditions. During the accumulated steady-state period, the cell growth was inhibited by high PHA content within the cells because of the carbon reserve consumption. From the experiments, there is no doubt about the PHA accumulation even at high carbon fraction ratio. Apparently, the best accumulation occurred at carbon fraction, 160 +/- 7.97 g COD/g N (PHAmean, = 44.54% of dried cells). Unfortunately, the highest PHA productivity was achieved at the high carbon fraction, 560 +/- 1.62 g COD/g N (0.152 +/- 0.17 g/l. min). Overall results showed that with high carbon fraction induced to the cultivation, the PO4 and NO3 can remove up to 20% in single cultivation.

Insights into the role of heat shock protein 72 to whole-body heat acclimation in humans

PubMed Central

Amorim, Fabiano Trigueiro; Fonseca, Ivana T; Machado-Moreira, Christiano A; Magalhães, Flávio de Castro

2015-01-01

Abstract Heat acclimation results in systemic and cellular adaptions that reduce the negative effect of heat and, consequently, the risk of heat illness. Although the classical changes observed with heat acclimation lead to increased tolerance to exercise in the heat by reducing heat storage (reflected in reduced core and skin temperatures) and increasing whole-body capacity for heat dissipation (greater plasma volume, sweat output, and skin blood flow), it appears that heat acclimation also induces changes at the cellular level that might increase tolerance of the whole organism to a higher core temperature for the development of fatigue. Thermotolerance is a process that involves increased resilience to an otherwise lethal heat stress that follows a sublethal exposure to heat. Thermotolerance is believed to be the result of increased content of heat shock proteins (Hsp), specially a member of the 70 kDa family, Hsp72 kDa. In humans, we and others have reported that heat acclimation increases intracellular Hsp72 levels. This increase in intracellular Hsp72 could improve whole-body organism thermotolerance by maintaining intestinal epithelial tight junction barriers, by increasing resistance to gut-associated endotoxin translocation, or by reducing the inflammatory response. In this review, we will initially provide an overview of the physiological adaptations induced by heat acclimation and emphasize the main cellular changes that occur with heat acclimation associated with intracellular accumulation of Hsp72. Finally, we will present an argument for a role of whole-body heat acclimation in augmenting cellular thermotolerance, which may protect vital organs from deleterious effects of heat stress in humans. PMID:27227070

Identification of proteins associated with polyhydroxybutyrate granules from Herbaspirillum seropedicae SmR1--old partners, new players.

PubMed

Tirapelle, Evandro F; Müller-Santos, Marcelo; Tadra-Sfeir, Michelle Z; Kadowaki, Marco A S; Steffens, Maria B R; Monteiro, Rose A; Souza, Emanuel M; Pedrosa, Fabio O; Chubatsu, Leda S

2013-01-01

Herbaspirillum seropedicae is a diazotrophic ß-Proteobacterium found associated with important agricultural crops. This bacterium produces polyhydroxybutyrate (PHB), an aliphatic polyester, as a carbon storage and/or source of reducing equivalents. The PHB polymer is stored as intracellular insoluble granules coated mainly with proteins, some of which are directly involved in PHB synthesis, degradation and granule biogenesis. In this work, we have extracted the PHB granules from H. seropedicae and identified their associated-proteins by mass spectrometry. This analysis allowed us to identify the main phasin (PhaP1) coating the PHB granule as well as the PHB synthase (PhbC1) responsible for its synthesis. A phbC1 mutant is impaired in PHB synthesis, confirming its role in H. seropedicae. On the other hand, a phaP1 mutant produces PHB granules but coated mainly with the secondary phasin (PhaP2). Furthermore, some novel proteins not previously described to be involved with PHB metabolism were also identified, bringing new possibilities to PHB function in H. seropedicae.

Identification of Proteins Associated with Polyhydroxybutyrate Granules from Herbaspirillum seropedicae SmR1 - Old Partners, New Players

PubMed Central

Tirapelle, Evandro F.; Müller-Santos, Marcelo; Tadra-Sfeir, Michelle Z.; Kadowaki, Marco A. S.; Steffens, Maria B. R.; Monteiro, Rose A.; Souza, Emanuel M.; Pedrosa, Fabio O.; Chubatsu, Leda S.

2013-01-01

Herbaspirillum seropedicae is a diazotrophic ß-Proteobacterium found associated with important agricultural crops. This bacterium produces polyhydroxybutyrate (PHB), an aliphatic polyester, as a carbon storage and/or source of reducing equivalents. The PHB polymer is stored as intracellular insoluble granules coated mainly with proteins, some of which are directly involved in PHB synthesis, degradation and granule biogenesis. In this work, we have extracted the PHB granules from H. seropedicae and identified their associated-proteins by mass spectrometry. This analysis allowed us to identify the main phasin (PhaP1) coating the PHB granule as well as the PHB synthase (PhbC1) responsible for its synthesis. A phbC1 mutant is impaired in PHB synthesis, confirming its role in H. seropedicae. On the other hand, a phaP1 mutant produces PHB granules but coated mainly with the secondary phasin (PhaP2). Furthermore, some novel proteins not previously described to be involved with PHB metabolism were also identified, bringing new possibilities to PHB function in H. seropedicae. PMID:24086439

Micro-topographic hydrologic variability due to vegetation acclimation under climate change

NASA Astrophysics Data System (ADS)

Le, P. V.; Kumar, P.

2012-12-01

Land surface micro-topography and vegetation cover have fundamental effects on the land-atmosphere interactions. The altered temperature and precipitation variability associated with climate change will affect the water and energy processes both directly and that mediated through vegetation. Since climate change induces vegetation acclimation that leads to shifts in evapotranspiration and heat fluxes, it further modifies microclimate and near-surface hydrological processes. In this study, we investigate the impacts of vegetation acclimation to climate change on micro-topographic hydrologic variability. The ability to accurately predict these impacts requires the simultaneous considerations of biochemical, ecophysiological and hydrological processes. A multilayer canopy-root-soil system model coupled with a conjunctive surface-subsurface flow model is used to capture the acclimatory responses and analyze the changes in dynamics of structure and connectivity of micro-topographic storage and in magnitudes of runoff. The study is performed using Light Detection and Ranging (LiDAR) topographic data in the Birds Point-New Madrid floodway in Missouri, U.S.A. The result indicates that both climate change and its associated vegetation acclimation play critical roles in altering the micro-topographic hydrological responses.

Characterization of binding preference of polyhydroxyalkanoate biosynthesis-related multifunctional protein PhaM from Ralstonia eutropha.

PubMed

Ushimaru, Kazunori; Tsuge, Takeharu

2016-05-01

The binding preference of a polyhydroxyalkanoate (PHA) biosynthesis-related multifunctional protein from Ralstonia eutropha (PhaMRe) was characterized. In vitro activity assay showed that PHA synthase from R. eutropha (PhaCRe) was activated by the presence of PhaMRe but PHA synthase from Aeromonas caviae (PhaCAc) was not. Additionally, in vitro assays of protein-protein interactions demonstrated that PhaMRe interacted with PhaCRe directly, but did not interact with PhaCAc. These results suggest that the protein-protein interaction is important for the activation of PhaC by PhaMRe. Further analyses indicated that PhaMRe has little or no direct interaction with the PHA polymer chain. Subsequently, PHA biosynthesis genes (phaA Re, phaB Re, and phaC Re/phaC Ac) and the phaM Re gene were introduced into recombinant Escherichia coli and cultivated for PHA accumulation. Contrary to our expectations, the expression of PhaMRe decreased PHA accumulation and changed the morphology of PHA granules to be microscopically obscure shape in PhaCRe-expressing E. coli. No change in the amount of P(3HB) or the morphology of granules by PhaMRe expression was observed in PhaCAc-expressing E. coli. These observations suggest that PhaMRe affects cellular physiology through the PhaM-PhaC interaction.

Phasin Proteins Activate Aeromonas caviae Polyhydroxyalkanoate (PHA) Synthase but Not Ralstonia eutropha PHA Synthase

PubMed Central

Ushimaru, Kazunori; Motoda, Yoko; Numata, Keiji

2014-01-01

In this study, we performed in vitro and in vivo activity assays of polyhydroxyalkanoate (PHA) synthases (PhaCs) in the presence of phasin proteins (PhaPs), which revealed that PhaPs are activators of PhaC derived from Aeromonas caviae (PhaCAc). In in vitro assays, among the three PhaCs tested, PhaCAc was significantly activated when PhaPs were added at the beginning of polymerization (prepolymerization PhaCAc), whereas the prepolymerization PhaCRe (derived from Ralstonia eutropha) and PhaCDa (Delftia acidovorans) showed reduced activity with PhaPs. The PhaP-activated PhaCAc showed a slight shift of substrate preference toward 3-hydroxyhexanoyl-CoA (C6). PhaPAc also activated PhaCAc when it was added during polymerization (polymer-elongating PhaCAc), while this effect was not observed for PhaCRe. In an in vivo assay using Escherichia coli TOP10 as the host strain, the effect of PhaPAc expression on PHA synthesis by PhaCAc or PhaCRe was examined. As PhaPAc expression increased, PHA production was increased by up to 2.3-fold in the PhaCAc-expressing strain, whereas it was slightly increased in the PhaCRe-expressing strain. Taken together, this study provides evidence that PhaPs function as activators for PhaCAc both in vitro and in vivo but do not activate PhaCRe. This activating effect may be attributed to the new role of PhaPs in the polymerization reaction by PhaCAc. PMID:24584238

Analysis of Two Polyhydroxyalkanoate Synthases in Bradyrhizobium japonicum USDA 110

PubMed Central

Mongiardini, Elías J.; Pérez-Giménez, Julieta; Parisi, Gustavo; Lodeiro, Aníbal R.

2013-01-01

Bradyrhizobium japonicum USDA 110 has five polyhydroxyalkanoate (PHA) synthases (PhaC) annotated in its genome: bll4360 (phaC1), bll6073 (phaC2), blr3732 (phaC3), blr2885 (phaC4), and bll4548 (phaC5). All these proteins possess the catalytic triad and conserved amino acid residues of polyester synthases and are distributed into four different PhaC classes. We obtained mutants in each of these paralogs and analyzed phaC gene expression and PHA production in liquid cultures. Despite the genetic redundancy, only phaC1 and phaC2 were expressed at significant rates, while PHA accumulation in stationary-phase cultures was impaired only in the ΔphaC1 mutant. Meanwhile, the ΔphaC2 mutant produced more PHA than the wild type under this condition, and surprisingly, the phaC3 transcript increased in the ΔphaC2 background. A double mutant, the ΔphaC2 ΔphaC3 mutant, consistently accumulated less PHA than the ΔphaC2 mutant. PHA accumulation in nodule bacteroids followed a pattern similar to that seen in liquid cultures, being prevented in the ΔphaC1 mutant and increased in the ΔphaC2 mutant in relation to the level in the wild type. Therefore, we used these mutants, together with a ΔphaC1 ΔphaC2 double mutant, to study the B. japonicum PHA requirements for survival, competition for nodulation, and plant growth promotion. All mutants, as well as the wild type, survived for 60 days in a carbon-free medium, regardless of their initial PHA contents. When competing for nodulation against the wild type in a 1:1 proportion, the ΔphaC1 and ΔphaC1 ΔphaC2 mutants occupied only 13 to 15% of the nodules, while the ΔphaC2 mutant occupied 81%, suggesting that the PHA polymer is required for successful competitiveness. However, the bacteroid content of PHA did not affect the shoot dry weight accumulation. PMID:23667236

Bacillus cereus-type polyhydroxyalkanoate biosynthetic gene cluster contains R-specific enoyl-CoA hydratase gene.

PubMed

Kihara, Takahiro; Hiroe, Ayaka; Ishii-Hyakutake, Manami; Mizuno, Kouhei; Tsuge, Takeharu

2017-08-01

Bacillus cereus and Bacillus megaterium both accumulate polyhydroxyalkanoate (PHA) but their PHA biosynthetic gene (pha) clusters that code for proteins involved in PHA biosynthesis are different. Namely, a gene encoding MaoC-like protein exists in the B. cereus-type pha cluster but not in the B. megaterium-type pha cluster. MaoC-like protein has an R-specific enoyl-CoA hydratase (R-hydratase) activity and is referred to as PhaJ when involved in PHA metabolism. In this study, the pha cluster of B. cereus YB-4 was characterized in terms of PhaJ's function. In an in vitro assay, PhaJ from B. cereus YB-4 (PhaJ YB4 ) exhibited hydration activity toward crotonyl-CoA. In an in vivo assay using Escherichia coli as a host for PHA accumulation, the recombinant strain expressing PhaJ YB4 and PHA synthase led to increased PHA accumulation, suggesting that PhaJ YB4 functioned as a monomer supplier. The monomer composition of the accumulated PHA reflected the substrate specificity of PhaJ YB4 , which appeared to prefer short chain-length substrates. The pha cluster from B. cereus YB-4 functioned to accumulate PHA in E. coli; however, it did not function when the phaJ YB4 gene was deleted. The B. cereus-type pha cluster represents a new example of a pha cluster that contains the gene encoding PhaJ.

Two Polyhydroxyalkanoate Synthases from Distinct Classes from the Aromatic Degrader Cupriavidus pinatubonensis JMP134 Exhibit the Same Substrate Preference.

PubMed

Jiang, Xuan; Luo, Xi; Zhou, Ning-Yi

2015-01-01

Cupriavidus pinatubonensis JMP134 utilizes a variety of aromatic substrates as sole carbon sources, including meta-nitrophenol (MNP). Two polyhydroxyalkanoate (PHA) synthase genes, phaC1 and phaC2, were annotated and categorized as class I and class II PHA synthase genes, respectively. In this study, both His-tagged purified PhaC1 and PhaC2 were shown to exhibit typical class I PHA synthase substrate specificity to make short-chain-length (SCL) PHA from 3-hydroxybutyryl-CoA and failed to make medium-chain-length (MCL) PHA from 3-hydroxyoctanoyl-CoA. The phaC1 or phaC2 deletion strain could also produce SCL PHA when grown in fructose or octanoate, but the double mutant of phaC1 and phaC2 lost this ability. The PhaC2 also exhibited substrate preference towards SCL substrates when expressed in Pseudomonas aeruginosa PAO1 phaC mutant strain. On the other hand, the transcriptional level of phaC1 was 70-fold higher than that of phaC2 in MNP-grown cells, but 240-fold lower in octanoate-grown cells. Further study demonstrated that only phaC1 was involved in PHA synthesis in MNP-grown cells. These findings suggested that phaC1 and phaC2 genes were differentially regulated under different growth conditions in this strain. Within the phaC2-containing gene cluster, a single copy of PHA synthase gene was present clustering with genes encoding enzymes in the biosynthesis of PHA precursors. This is markedly different from the genetic organization of all other previously reported class II PHA synthase gene clusters and this cluster likely comes from a distinct evolutionary path.

Two Polyhydroxyalkanoate Synthases from Distinct Classes from the Aromatic Degrader Cupriavidus pinatubonensis JMP134 Exhibit the Same Substrate Preference

PubMed Central

Jiang, Xuan; Luo, Xi; Zhou, Ning-Yi

2015-01-01

Cupriavidus pinatubonensis JMP134 utilizes a variety of aromatic substrates as sole carbon sources, including meta-nitrophenol (MNP). Two polyhydroxyalkanoate (PHA) synthase genes, phaC1 and phaC2, were annotated and categorized as class I and class II PHA synthase genes, respectively. In this study, both His-tagged purified PhaC1 and PhaC2 were shown to exhibit typical class I PHA synthase substrate specificity to make short-chain-length (SCL) PHA from 3-hydroxybutyryl-CoA and failed to make medium-chain-length (MCL) PHA from 3-hydroxyoctanoyl-CoA. The phaC1 or phaC2 deletion strain could also produce SCL PHA when grown in fructose or octanoate, but the double mutant of phaC1 and phaC2 lost this ability. The PhaC2 also exhibited substrate preference towards SCL substrates when expressed in Pseudomonas aeruginosa PAO1 phaC mutant strain. On the other hand, the transcriptional level of phaC1 was 70-fold higher than that of phaC2 in MNP-grown cells, but 240-fold lower in octanoate-grown cells. Further study demonstrated that only phaC1 was involved in PHA synthesis in MNP-grown cells. These findings suggested that phaC1 and phaC2 genes were differentially regulated under different growth conditions in this strain. Within the phaC2-containing gene cluster, a single copy of PHA synthase gene was present clustering with genes encoding enzymes in the biosynthesis of PHA precursors. This is markedly different from the genetic organization of all other previously reported class II PHA synthase gene clusters and this cluster likely comes from a distinct evolutionary path. PMID:26544851

PhaR, a Negative Regulator of PhaP, Modulates the Colonization of a Burkholderia Gut Symbiont in the Midgut of the Host Insect, Riptortus pedestris.

PubMed

Jang, Seong Han; Jang, Ho Am; Lee, Junbeom; Kim, Jong Uk; Lee, Seung Ah; Park, Kyoung-Eun; Kim, Byung Hyun; Jo, Yong Hun; Lee, Bok Luel

2017-06-01

Five genes encoding PhaP family proteins and one phaR gene have been identified in the genome of Burkholderia symbiont strain RPE75. PhaP proteins function as the surface proteins of polyhydroxyalkanoate (PHA) granules, and the PhaR protein acts as a negative regulator of PhaP biosynthesis. Recently, we characterized one phaP gene to understand the molecular cross talk between Riptortus insects and Burkholderia gut symbionts. In this study, we constructed four other phaP gene-depleted mutants (Δ phaP1 , Δ phaP2 , Δ phaP3 , and Δ phaP4 mutants), one phaR gene-depleted mutant, and a phaR -complemented mutant (Δ phaR/phaR mutant). To address the biological roles of four phaP family genes and the phaR gene during insect-gut symbiont interaction, these Burkholderia mutants were fed to the second-instar nymphs, and colonization ability and fitness parameters were examined. In vitro , the Δ phaP3 and Δ phaR mutants cannot make a PHA granule normally in a stressful environment. Furthermore, the Δ phaR mutation decreased the colonization ability in the host midgut and negatively affected the host insect's fitness compared with wild-type Burkholderia -infected insects. However, other phaP family gene-depleted mutants colonized well in the midgut of the fifth-instar nymph insects. However, in the case of females, the colonization rate of the Δ phaP3 mutant was decreased and the host's fitness parameters were decreased compared with the wild-type-infected host, suggesting that the environment of the female midgut may be more hostile than that of the male midgut. These results demonstrate that PhaR plays an important role in the biosynthesis of PHA granules and that it is significantly related to the colonization of the Burkholderia gut symbiont in the host insects' midgut. IMPORTANCE Bacterial polyhydroxyalkanoate (PHA) biosynthesis is a complex process requiring several enzymes. The biological roles of PHA granule synthesis enzymes and the surface proteins of PHA granules during host-gut symbiont interactions are not fully understood. Here, we report the effects on colonization ability in the host midguts and the fitness of host insects after feeding Burkholderia mutant cells (four phaP -depleted mutants and one phaR -depleted mutant) to the host insects. Analyses of both synthesized PHA granule amounts and CFU numbers suggest that the phaR gene is closely related to synthesis of the PHA granule and the colonization of the Burkholderia gut symbiont in the host insect's midgut. Like our previous report, this study also supports the idea that the environment of the host midgut may not be favorable to symbiotic Burkholderia cells and that PHA granules may be required to adapt in the host midgut. Copyright © 2017 American Society for Microbiology.

PhaR, a Negative Regulator of PhaP, Modulates the Colonization of a Burkholderia Gut Symbiont in the Midgut of the Host Insect, Riptortus pedestris

PubMed Central

Jang, Seong Han; Jang, Ho Am; Lee, Junbeom; Kim, Jong Uk; Lee, Seung Ah; Park, Kyoung-Eun; Kim, Byung Hyun; Jo, Yong Hun

2017-01-01

ABSTRACT Five genes encoding PhaP family proteins and one phaR gene have been identified in the genome of Burkholderia symbiont strain RPE75. PhaP proteins function as the surface proteins of polyhydroxyalkanoate (PHA) granules, and the PhaR protein acts as a negative regulator of PhaP biosynthesis. Recently, we characterized one phaP gene to understand the molecular cross talk between Riptortus insects and Burkholderia gut symbionts. In this study, we constructed four other phaP gene-depleted mutants (ΔphaP1, ΔphaP2, ΔphaP3, and ΔphaP4 mutants), one phaR gene-depleted mutant, and a phaR-complemented mutant (ΔphaR/phaR mutant). To address the biological roles of four phaP family genes and the phaR gene during insect-gut symbiont interaction, these Burkholderia mutants were fed to the second-instar nymphs, and colonization ability and fitness parameters were examined. In vitro, the ΔphaP3 and ΔphaR mutants cannot make a PHA granule normally in a stressful environment. Furthermore, the ΔphaR mutation decreased the colonization ability in the host midgut and negatively affected the host insect's fitness compared with wild-type Burkholderia-infected insects. However, other phaP family gene-depleted mutants colonized well in the midgut of the fifth-instar nymph insects. However, in the case of females, the colonization rate of the ΔphaP3 mutant was decreased and the host's fitness parameters were decreased compared with the wild-type-infected host, suggesting that the environment of the female midgut may be more hostile than that of the male midgut. These results demonstrate that PhaR plays an important role in the biosynthesis of PHA granules and that it is significantly related to the colonization of the Burkholderia gut symbiont in the host insects' midgut. IMPORTANCE Bacterial polyhydroxyalkanoate (PHA) biosynthesis is a complex process requiring several enzymes. The biological roles of PHA granule synthesis enzymes and the surface proteins of PHA granules during host-gut symbiont interactions are not fully understood. Here, we report the effects on colonization ability in the host midguts and the fitness of host insects after feeding Burkholderia mutant cells (four phaP-depleted mutants and one phaR-depleted mutant) to the host insects. Analyses of both synthesized PHA granule amounts and CFU numbers suggest that the phaR gene is closely related to synthesis of the PHA granule and the colonization of the Burkholderia gut symbiont in the host insect's midgut. Like our previous report, this study also supports the idea that the environment of the host midgut may not be favorable to symbiotic Burkholderia cells and that PHA granules may be required to adapt in the host midgut. PMID:28341680

Effects of temperature acclimation on Pacific bluefin tuna (Thunnus orientalis) cardiac transcriptome.

PubMed

Jayasundara, Nishad; Gardner, Luke D; Block, Barbara A

2013-11-01

Little is known about the mechanisms underpinning thermal plasticity of vertebrate hearts. Bluefin tuna hearts offer a unique model to investigate processes underlying thermal acclimation. Their hearts, while supporting an endothermic physiology, operate at ambient temperature, and are presented with a thermal challenge when migrating to different thermal regimes. Here, we examined the molecular responses in atrial and ventricular tissues of Pacific bluefin tuna acclimated to 14°C, 20°C, and 25°C. Quantitative PCR studies showed an increase in sarcoplasmic reticulum Ca(2+) ATPase gene expression with cold acclimation and an induction of Na(+)/Ca(2+)-exchanger gene at both cold and warm temperatures. These data provide evidence for thermal plasticity of excitation-contraction coupling gene expression in bluefin tunas and indicate an increased capacity for internal Ca(2+) storage in cardiac myocytes at 14°C. Transcriptomic analysis showed profound changes in cardiac tissues with acclimation. A principal component analysis revealed that temperature effect was greatest on gene expression in warm-acclimated atrium. Overall data showed an increase in cardiac energy metabolism at 14°C, potentially compensating for cold temperature to optimize bluefin tuna performance in colder oceans. In contrast, metabolic enzyme activity and gene expression data suggest a decrease in ATP production at 25°C. Expression of genes involved in protein turnover and molecular chaperones was also decreased at 25°C. Expression of genes involved in oxidative stress response and programmed cell death suggest an increase in oxidative damage and apoptosis at 25°C, particularly in the atrium. These findings provide insights into molecular processes that may characterize cardiac phenotypes at upper thermal limits of teleosts.

To Be or Not To Be a Poly(3-Hydroxybutyrate) (PHB) Depolymerase: PhaZd1 (PhaZ6) and PhaZd2 (PhaZ7) of Ralstonia eutropha, Highly Active PHB Depolymerases with No Detectable Role in Mobilization of Accumulated PHB

PubMed Central

Sznajder, Anna

2014-01-01

The putative physiological functions of two related intracellular poly(3-hydroxybutyrate) (PHB) depolymerases, PhaZd1 and PhaZd2, of Ralstonia eutropha H16 were investigated. Purified PhaZd1 and PhaZd2 were active with native PHB granules in vitro. Partial removal of the proteinaceous surface layer of native PHB granules by trypsin treatment or the use of PHB granules isolated from ΔphaP1 or ΔphaP1-phaP5 mutant strains resulted in increased specific PHB depolymerase activity, especially for PhaZd2. Constitutive expression of PhaZd1 or PhaZd2 reduced or even prevented the accumulation of PHB under PHB-permissive conditions in vivo. Expression of translational fusions of enhanced yellow fluorescent protein (EYFP) with PhaZd1 and PhaZd2 in which the active-site serines (S190 and Ser193) were replaced with alanine resulted in the colocalization of only PhaZd1 fusions with PHB granules. C-terminal fusions of inactive PhaZd2(S193A) with EYFP revealed the presence of spindle-like structures, and no colocalization with PHB granules was observed. Chromosomal deletion of phaZd1, phaZd2, or both depolymerase genes had no significant effect on PHB accumulation and mobilization during growth in nutrient broth (NB) or NB-gluconate medium. Moreover, neither proteome analysis of purified native PHB granules nor lacZ fusion studies gave any indication that PhaZd1 or PhaZd2 was detectably present in the PHB granule fraction or expressed at all during growth on NB-gluconate medium. In conclusion, PhaZd1 and PhaZd2 are two PHB depolymerases with a high capacity to degrade PHB when artificially expressed but are apparently not involved in PHB mobilization in the wild type. The true in vivo functions of PhaZd1 and PhaZd2 remain obscure. PMID:24907326

To be or not to be a poly(3-hydroxybutyrate) (PHB) depolymerase: PhaZd1 (PhaZ6) and PhaZd2 (PhaZ7) of Ralstonia eutropha, highly active PHB depolymerases with no detectable role in mobilization of accumulated PHB.

PubMed

Sznajder, Anna; Jendrossek, Dieter

2014-08-01

The putative physiological functions of two related intracellular poly(3-hydroxybutyrate) (PHB) depolymerases, PhaZd1 and PhaZd2, of Ralstonia eutropha H16 were investigated. Purified PhaZd1 and PhaZd2 were active with native PHB granules in vitro. Partial removal of the proteinaceous surface layer of native PHB granules by trypsin treatment or the use of PHB granules isolated from ΔphaP1 or ΔphaP1-phaP5 mutant strains resulted in increased specific PHB depolymerase activity, especially for PhaZd2. Constitutive expression of PhaZd1 or PhaZd2 reduced or even prevented the accumulation of PHB under PHB-permissive conditions in vivo. Expression of translational fusions of enhanced yellow fluorescent protein (EYFP) with PhaZd1 and PhaZd2 in which the active-site serines (S190 and Ser193) were replaced with alanine resulted in the colocalization of only PhaZd1 fusions with PHB granules. C-terminal fusions of inactive PhaZd2(S193A) with EYFP revealed the presence of spindle-like structures, and no colocalization with PHB granules was observed. Chromosomal deletion of phaZd1, phaZd2, or both depolymerase genes had no significant effect on PHB accumulation and mobilization during growth in nutrient broth (NB) or NB-gluconate medium. Moreover, neither proteome analysis of purified native PHB granules nor lacZ fusion studies gave any indication that PhaZd1 or PhaZd2 was detectably present in the PHB granule fraction or expressed at all during growth on NB-gluconate medium. In conclusion, PhaZd1 and PhaZd2 are two PHB depolymerases with a high capacity to degrade PHB when artificially expressed but are apparently not involved in PHB mobilization in the wild type. The true in vivo functions of PhaZd1 and PhaZd2 remain obscure. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

A Patatin-Like Protein Associated with the Polyhydroxyalkanoate (PHA) Granules of Haloferax mediterranei Acts as an Efficient Depolymerase in the Degradation of Native PHA

PubMed Central

Liu, Guiming; Hou, Jing; Cai, Shuangfeng; Zhao, Dahe; Cai, Lei; Han, Jing; Zhou, Jian

2015-01-01

The key enzymes and pathways involved in polyhydroxyalkanoate (PHA) biosynthesis in haloarchaea have been identified in recent years, but the haloarchaeal enzymes for PHA degradation remain unknown. In this study, a patatin-like PHA depolymerase, PhaZh1, was determined to be located on the PHA granules in the haloarchaeon Haloferax mediterranei. PhaZh1 hydrolyzed the native PHA (nPHA) [including native polyhydroxybutyrate (nPHB) and native poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (nPHBV) in this study] granules in vitro with 3-hydroxybutyrate (3HB) monomer as the primary product. The site-directed mutagenesis of PhaZh1 indicated that Gly16, Ser47 (in a classical lipase box, G-X-S47-X-G), and Asp195 of this depolymerase were essential for its activity in nPHA granule hydrolysis. Notably, phaZh1 and bdhA (encoding putative 3HB dehydrogenase) form a gene cluster (HFX_6463 to _6464) in H. mediterranei. The 3HB monomer generated from nPHA degradation by PhaZh1 could be further converted into acetoacetate by BdhA, indicating that PhaZh1-BdhA may constitute the first part of a PHA degradation pathway in vivo. Interestingly, although PhaZh1 showed efficient activity and was most likely the key enzyme in nPHA granule hydrolysis in vitro, the knockout of phaZh1 had no significant effect on the intracellular PHA mobilization, implying the existence of an alternative PHA mobilization pathway(s) that functions effectively within the cells of H. mediterranei. Therefore, identification of this patatin-like depolymerase of haloarchaea may provide a new strategy for producing the high-value-added chiral compound (R)-3HB and may also shed light on the PHA mobilization in haloarchaea. PMID:25710370

Alcoholytic Cleavage of Polyhydroxyalkanoate Chains by Class IV Synthases Induced by Endogenous and Exogenous Ethanol

PubMed Central

Hyakutake, Manami; Tomizawa, Satoshi; Mizuno, Kouhei; Abe, Hideki

2014-01-01

Polyhydroxyalkanoate (PHA)-producing Bacillus strains express class IV PHA synthase, which is composed of the subunits PhaR and PhaC. Recombinant Escherichia coli expressing PHA synthase from Bacillus cereus strain YB-4 (PhaRCYB-4) showed an unusual reduction of the molecular weight of PHA produced during the stationary phase of growth. Nuclear magnetic resonance analysis of the low-molecular-weight PHA revealed that its carboxy end structure was capped by ethanol, suggesting that the molecular weight reduction was the result of alcoholytic cleavage of PHA chains by PhaRCYB-4 induced by endogenous ethanol. This scission reaction was also induced by exogenous ethanol in both in vivo and in vitro assays. In addition, PhaRCYB-4 was observed to have alcoholysis activity for PHA chains synthesized by other synthases. The PHA synthase from Bacillus megaterium (PhaRCBm) from another subgroup of class IV synthases was also assayed and was shown to have weak alcoholysis activity for PHA chains. These results suggest that class IV synthases may commonly share alcoholysis activity as an inherent feature. PMID:24334666

Purification, immobilization, and characterization of nattokinase on PHB nanoparticles.

PubMed

Deepak, Venkataraman; Pandian, Suresh babu Ram Kumar; Kalishwaralal, Kalimuthu; Gurunathan, Sangiliyandi

2009-12-01

In this study, nattokinase was purified from Bacillus subtilis using ion exchange chromatography and immobilized upon polyhydroxybutyrate (PHB) nanoparticles. A novel strain isolated from industrial dairy waste was found to synthesize polyhydroxyalkanoates (PHA) and the strain was identified as Brevibacterium casei SRKP2. PHA granules were extracted from 48 h culture and the FT-IR analysis characterized them as PHB, a natural biopolymer from B. casei. Nanoprecipitation by solvent displacement technique was used to synthesize PHB nanoparticles. PHB nanoparticles were characterized using transmission electron microscopy and particle size ranged from 100-125 nm. Immobilization of nattokinase upon PHB nanoparticles resulted in a 20% increase in the enzyme activity. Immobilization also contributed to the enhanced stability of the enzyme. Moreover, the activity was completely retained on storage at 4 degrees C for 25 days. The method has proven to be highly simple and can be implemented to other enzymes also.

Identification of the Haloarchaeal Phasin (PhaP) That Functions in Polyhydroxyalkanoate Accumulation and Granule Formation in Haloferax mediterranei

PubMed Central

Cai, Shuangfeng; Cai, Lei; Liu, Hailong; Liu, Xiaoqing; Han, Jing; Zhou, Jian

2012-01-01

The polyhydroxyalkanoate (PHA) granule-associated proteins (PGAPs) are important for PHA synthesis and granule formation, but currently little is known about the haloarchaeal PGAPs. This study focused on the identification and functional analysis of the PGAPs in the haloarchaeon Haloferax mediterranei. These PGAPs were visualized with two-dimensional gel electrophoresis (2-DE) and identified by matrix-assisted laser desorption ionization–tandem time of flight mass spectrometry (MALDI-TOF/TOF MS). The most abundant protein on the granules was identified as a hypothetical protein, designated PhaP. A genome-wide analysis revealed that the phaP gene is located upstream of the previously identified phaEC genes. Through an integrative approach of gene knockout/complementation and fermentation analyses, we demonstrated that this PhaP is involved in PHA accumulation. The ΔphaP mutant was defective in both PHA biosynthesis and cell growth compared to the wild-type strain. Additionally, transmission electron microscopy results indicated that the number of PHA granules in the ΔphaP mutant cells was significantly lower, and in most of the ΔphaP cells only a single large granule was observed. These results demonstrated that the H. mediterranei PhaP was the predominant structure protein (phasin) on the PHA granules involved in PHA accumulation and granule formation. In addition, BLASTp and phylogenetic results indicate that this type of PhaP is exclusively conserved in haloarchaea, implying that it is a representative of the haloarchaeal type PHA phasin. PMID:22247127

24 CFR 982.355 - Portability: Administration by receiving PHA.

Code of Federal Regulations, 2011 CFR

2011-04-01

... by the receiving PHA. (1) If funding is available under the consolidated ACC for the receiving PHA... consolidated ACC for the receiving PHA tenant-based program. (2) HUD may require that the receiving PHA absorb... families to the receiving PHA from funds available under the initial PHA ACC. (2) HUD may provide...

24 CFR 982.355 - Portability: Administration by receiving PHA.

Code of Federal Regulations, 2010 CFR

2010-04-01

... by the receiving PHA. (1) If funding is available under the consolidated ACC for the receiving PHA... consolidated ACC for the receiving PHA tenant-based program. (2) HUD may require that the receiving PHA absorb... families to the receiving PHA from funds available under the initial PHA ACC. (2) HUD may provide...

Mutations Derived from the Thermophilic Polyhydroxyalkanoate Synthase PhaC Enhance the Thermostability and Activity of PhaC from Cupriavidus necator H16

PubMed Central

Chen, Wen-Ming; Lai, Yung-Wei; Chang, Rey-Chang

2012-01-01

The thermophile Cupriavidus sp. strain S-6 accumulated polyhydroxybutyrate (PHB) from glucose at 50°C. A 9.0-kbp EcoRI fragment cloned from the genomic DNA of Cupriavidus sp. S-6 enabled Escherichia coli XL1-Blue to synthesize PHB at 45°C. Nucleotide sequence analysis showed a pha locus in the clone. The thermophilic polyhydroxyalkanoate (PHA) synthase (PhaCCsp) shared 81% identity with mesophilic PhaC of Cupriavidus necator H16. The diversity between these two strains was found dominantly on their N and C termini, while the middle regions were highly homologous (92% identity). We constructed four chimeras of mesophilic and thermophilic phaC genes to explore the mutations related to its thermostability. Among the chimeras, only PhaCH16β, which was PhaCH16 bearing 30 point mutations derived from the middle region of PhaCCsp, accumulated a high content of PHB (65% [dry weight]) at 45°C. The chimera phaCH16β and two parental PHA synthase genes were overexpressed in E. coli BLR(DE3) cells and purified. At 30°C, the specific activity of the chimera PhaCH16β (172 ± 17.8 U/mg) was 3.45-fold higher than that of the parental enzyme PhaCH16 (50 ± 5.2 U/mg). At 45°C, the half-life of the chimera PhaCH16β (11.2 h) was 127-fold longer than that of PhaCH16 (5.3 min). Furthermore, the chimera PhaCH16β accumulated 1.55-fold (59% [dry weight]) more PHA content than the parental enzyme PhaCH16 (38% [dry weight]) at 37°C. This study reveals a limited number of point mutations which enhance not only thermostability but also PhaCH16 activity. The highly thermostable and active PHA synthase will provide advantages for its promising applications to in vitro PHA synthesis and recombinant E. coli PHA fermentation. PMID:22408158

Modified pseudomonas oleovorans phaC1 nucleic acids encoding bispecific polyhydroxyalkanoate polymerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Srienc, Friedrich; Jackson, John K.; Somers, David A.

A genetically engineered Pseudomonas oleovorans phaC1 polyhydroxyalkanoate (PHA) polymerase having tailored substrate specificity is provided. The modified PHA polymerase is preferably a "bispecific" PHA polymerase capable of copolymerizing a short chain length monomer and a medium chain length monomer is provided. Methods for making the modified PHA polymerase and for making nucleic acids encoding the modified PHA polymerase are also disclosed, as are methods of producing PHA using the modified PHA polymerase. The invention further includes methods to assay for altered substrate specificity.

High Polyhydroxybutyrate Production in Pseudomonas extremaustralis Is Associated with Differential Expression of Horizontally Acquired and Core Genome Polyhydroxyalkanoate Synthase Genes

PubMed Central

Catone, Mariela V.; Ruiz, Jimena A.; Castellanos, Mildred; Segura, Daniel; Espin, Guadalupe; López, Nancy I.

2014-01-01

Pseudomonas extremaustralis produces mainly polyhydroxybutyrate (PHB), a short chain length polyhydroxyalkanoate (sclPHA) infrequently found in Pseudomonas species. Previous studies with this strain demonstrated that PHB genes are located in a genomic island. In this work, the analysis of the genome of P. extremaustralis revealed the presence of another PHB cluster phbFPX, with high similarity to genes belonging to Burkholderiales, and also a cluster, phaC1ZC2D, coding for medium chain length PHA production (mclPHA). All mclPHA genes showed high similarity to genes from Pseudomonas species and interestingly, this cluster also showed a natural insertion of seven ORFs not related to mclPHA metabolism. Besides PHB, P. extremaustralis is able to produce mclPHA although in minor amounts. Complementation analysis demonstrated that both mclPHA synthases, PhaC1 and PhaC2, were functional. RT-qPCR analysis showed different levels of expression for the PHB synthase, phbC, and the mclPHA synthases. The expression level of phbC, was significantly higher than the obtained for phaC1 and phaC2, in late exponential phase cultures. The analysis of the proteins bound to the PHA granules showed the presence of PhbC and PhaC1, whilst PhaC2 could not be detected. In addition, two phasin like proteins (PhbP and PhaI) associated with the production of scl and mcl PHAs, respectively, were detected. The results of this work show the high efficiency of a foreign gene (phbC) in comparison with the mclPHA core genome genes (phaC1 and phaC2) indicating that the ability of P. extremaustralis to produce high amounts of PHB could be explained by the different expression levels of the genes encoding the scl and mcl PHA synthases. PMID:24887088

Impact of Medium on the Development and Physiology of Pseudomonas fluorescens Biofilms on Polyurethane Paint

DTIC Science & Technology

2012-02-01

including P. fluorescens are known to make several types of intracellular storage granules, including polyhydroxyalkanoates (PHAs), polyphosphates, and... polyhydroxyalkanoates in Pseudomonas putida KT2442 and the fundamental role of PhaZ depolymerase for the metabolic balance. Environ Microbiol. 12(1... polyhydroxyalkanoates by bacteria. Biotechnol Lett. 11(7):471-476. Herigstad B, Hamilton M, Heersink J. 2001. How to optimize the drop plate method for

Novel polyhydroxyalkanoate copolymers produced in Pseudomonas putida by metagenomic polyhydroxyalkanoate synthases.

PubMed

Cheng, Jiujun; Charles, Trevor C

2016-09-01

Bacterially produced biodegradable polyhydroxyalkanoates (PHAs) with versatile properties can be achieved using different PHA synthases (PhaCs). This work aims to expand the diversity of known PhaCs via functional metagenomics and demonstrates the use of these novel enzymes in PHA production. Complementation of a PHA synthesis-deficient Pseudomonas putida strain with a soil metagenomic cosmid library retrieved 27 clones expressing either class I, class II, or unclassified PHA synthases, and many did not have close sequence matches to known PhaCs. The composition of PHA produced by these clones was dependent on both the supplied growth substrates and the nature of the PHA synthase, with various combinations of short-chain-length (SCL) and medium-chain-length (MCL) PHA. These data demonstrate the ability to isolate diverse genes for PHA synthesis by functional metagenomics and their use for the production of a variety of PHA polymer and copolymer mixtures.

Localization of Poly(3-Hydroxybutyrate) (PHB) Granule-Associated Proteins during PHB Granule Formation and Identification of Two New Phasins, PhaP6 and PhaP7, in Ralstonia eutropha H16

PubMed Central

Pfeiffer, Daniel

2012-01-01

Poly(3-hydroxybutyrate) (PHB) granules are covered by a surface layer consisting of mainly phasins and other PHB granule-associated proteins (PGAPs). Phasins are small amphiphilic proteins that determine the number and size of accumulated PHB granules. Five phasin proteins (PhaP1 to PhaP5) are known for Ralstonia eutropha. In this study, we identified three additional potential phasin genes (H16_B1988, H16_B2296, and H16_B2326) by inspection of the R. eutropha genome for sequences with “phasin 2 motifs.” To determine whether the corresponding proteins represent true PGAPs, fusions with eYFP (enhanced yellow fluorescent protein) were constructed. Similar fusions of eYFP with PhaP1 to PhaP5 as well as fusions with PHB synthase (PhaC1), an inactive PhaC1 variant (PhaC1-C319A), and PhaC2 were also made. All fusions were investigated in wild-type and PHB-negative backgrounds. Colocalization with PHB granules was found for all PhaC variants and for PhaP1 to PhaP5. Additionally, eYFP fusions with H16_B1988 and H16_B2326 colocalized with PHB. Fusions of H16_B2296 with eYFP, however, did not colocalize with PHB granules but did colocalize with the nucleoid region. Notably, all fusions (except H16_B2296) were soluble in a ΔphaC1 strain. These data confirm that H16_B1988 and H16_B2326 but not H16_B2296 encode true PGAPs, for which we propose the designation PhaP6 (H16_B1988) and PhaP7 (H16_B2326). When localization of phasins was investigated at different stages of PHB accumulation, fusions of PhaP6 and PhaP7 were soluble in the first 3 h under PHB-permissive conditions, although PHB granules appeared after 10 min. At later time points, the fusions colocalized with PHB. Remarkably, PHB granules of strains expressing eYFP fusions with PhaP5, PhaP6, or PhaP7 localized predominantly near the cell poles or in the area of future septum formation. This phenomenon was not observed for the other PGAPs (PhaP1 to PhaP4, PhaC1, PhaC1-C319A, and PhaC2) and indicated that some phasins can have additional functions. A chromosomal deletion of phaP6 or phaP7 had no visible effect on formation of PHB granules. PMID:22923598

Localization of poly(3-hydroxybutyrate) (PHB) granule-associated proteins during PHB granule formation and identification of two new phasins, PhaP6 and PhaP7, in Ralstonia eutropha H16.

PubMed

Pfeiffer, Daniel; Jendrossek, Dieter

2012-11-01

Poly(3-hydroxybutyrate) (PHB) granules are covered by a surface layer consisting of mainly phasins and other PHB granule-associated proteins (PGAPs). Phasins are small amphiphilic proteins that determine the number and size of accumulated PHB granules. Five phasin proteins (PhaP1 to PhaP5) are known for Ralstonia eutropha. In this study, we identified three additional potential phasin genes (H16_B1988, H16_B2296, and H16_B2326) by inspection of the R. eutropha genome for sequences with "phasin 2 motifs." To determine whether the corresponding proteins represent true PGAPs, fusions with eYFP (enhanced yellow fluorescent protein) were constructed. Similar fusions of eYFP with PhaP1 to PhaP5 as well as fusions with PHB synthase (PhaC1), an inactive PhaC1 variant (PhaC1-C319A), and PhaC2 were also made. All fusions were investigated in wild-type and PHB-negative backgrounds. Colocalization with PHB granules was found for all PhaC variants and for PhaP1 to PhaP5. Additionally, eYFP fusions with H16_B1988 and H16_B2326 colocalized with PHB. Fusions of H16_B2296 with eYFP, however, did not colocalize with PHB granules but did colocalize with the nucleoid region. Notably, all fusions (except H16_B2296) were soluble in a ΔphaC1 strain. These data confirm that H16_B1988 and H16_B2326 but not H16_B2296 encode true PGAPs, for which we propose the designation PhaP6 (H16_B1988) and PhaP7 (H16_B2326). When localization of phasins was investigated at different stages of PHB accumulation, fusions of PhaP6 and PhaP7 were soluble in the first 3 h under PHB-permissive conditions, although PHB granules appeared after 10 min. At later time points, the fusions colocalized with PHB. Remarkably, PHB granules of strains expressing eYFP fusions with PhaP5, PhaP6, or PhaP7 localized predominantly near the cell poles or in the area of future septum formation. This phenomenon was not observed for the other PGAPs (PhaP1 to PhaP4, PhaC1, PhaC1-C319A, and PhaC2) and indicated that some phasins can have additional functions. A chromosomal deletion of phaP6 or phaP7 had no visible effect on formation of PHB granules.

Homologous functional expression of cryptic phaG from Pseudomonas oleovorans establishes the transacylase-mediated polyhydroxyalkanoate biosynthetic pathway.

PubMed

Hoffmann, N; Steinbüchel, A; Rehm, B H

2000-11-01

Various pseudomonads are capable of the synthesis of polyhydroxyalkanoate (PHA), composed of medium chain length (MCL) 3-hydroxy fatty acids (C6-C14), when grown on simple carbon sources such as, for example, gluconate or acetate. In Pseudomonas putida, the fatty acid de novo synthesis and PHA synthesis are linked by the transacylase PhaG. Southern hybridization experiments with digoxigenin-labeled phaG(Pp) from P. putida and genomic DNA from various pseudomonads indicate that phaG homologues are present in various other pseudomonads. Although P. oleovorans does not accumulate PHA(MCL) from non-related carbon sources, its genomic DNA reveals a strong hybridization signal. We employed PCR to amplify this phaG homologue. The respective PCR product comprising the coding region of phaG(Po) was cloned into pBBR1MCS-2, resulting in plasmid pBHR84. DNA sequencing revealed that putative PhaG(Po) from P. oleovorans exhibited about 95% amino acid sequence identity to PhaG(Pp) from P. putida. Reverse transcriptase-PCR analysis demonstrated that phaG(Po) was not transcribed even tinder inducing conditions, i.e. in the presence of gluconate as carbon source, whereas induction of phaG(Pp) transcription was obtained in P. putida. When octanoate was used as sole carbon source, only low levels of phaG mRNA were detected in P. putida. Plasmid pBHR84 complemented the phaG-negative mutant PhaG(N)-21 from P. putida. Interestingly, reintroduction of phaG(Po) under lac promoter control into the natural host P. oleovorans established PHA(MCL) synthesis from non-related carbon sources in this bacterium. These data indicated that phaG(Po) in P. oleovorans is not functionally expressed and does not exert its original function.

Engineering microorganisms for improving polyhydroxyalkanoate biosynthesis.

PubMed

Chen, Guo-Qiang; Jiang, Xiao-Ran

2017-11-20

Biosynthesis of polyhydroxyalkanoates (PHA) has been studied since the 1920s. The biosynthesis pathways have been well understood and various attempts have been made to improve the PHA biosynthesis efficiency. Recent progresses have been focused on systematic improvements on PHA biosynthesis including changing growth pattern for rapid proliferation, engineering to enlarge cell sizes for more PHA accumulation space, reprogramming the PHA synthesis pathways using optimized RBS and promoter, redirecting metabolic flux to PHA synthesis using CRISPR/Cas9 tools, and very importantly, the employment of non-traditional host such as halophiles for reduced complexity on PHA production. All of the efforts should lead to ultrahigh PHA accumulation, controllable PHA compositions and molecular weights, open and continuous PHA production with gravity separation processes, resulting in competitive PHA production cost. Copyright © 2017 Elsevier Ltd. All rights reserved.

Class IV polyhydroxyalkanoate (PHA) synthases and PHA-producing Bacillus.

PubMed

Tsuge, Takeharu; Hyakutake, Manami; Mizuno, Kouhei

2015-08-01

This review highlights the recent investigations of class IV polyhydroxyalkanoate (PHA) synthases, the newest classification of PHA synthases. Class IV synthases are prevalent in organisms of the Bacillus genus and are composed of a catalytic subunit PhaC (approximately 40 kDa), which has a PhaC box sequence ([GS]-X-C-X-[GA]-G) at the active site, and a second subunit PhaR (approximately 20 kDa). The representative PHA-producing Bacillus strains are Bacillus megaterium and Bacillus cereus; the nucleotide sequence of phaC and the genetic organization of the PHA biosynthesis gene locus are somewhat different between these two strains. It is generally considered that class IV synthases favor short-chain-length monomers such as 3-hydroxybutyrate (C4) and 3-hydroxyvalerate (C5) for polymerization, but can polymerize some unusual monomers as minor components. In Escherichia coli expressing PhaRC from B. cereus YB-4, the biosynthesized PHA undergoes synthase-catalyzed alcoholytic cleavage using endogenous and exogenous alcohols. This alcoholysis is thought to be shared among class IV synthases, and this reaction is useful not only for the regulation of PHA molecular weight but also for the modification of the PHA carboxy terminus. The novel properties of class IV synthases will open up the possibility for the design of new PHA materials.

Modelling of microbial polyhydroxyalkanoate surface binding protein PhaP for rational mutagenesis.

PubMed

Zhao, Hongyu; Yao, Zhenyu; Chen, Xiangbin; Wang, Xinquan; Chen, Guo-Qiang

2017-11-01

Phasins are unusual amphiphilic proteins that bind to microbial polyhydroxyalkanoate (PHA) granules in nature and show great potential for various applications in biotechnology and medicine. Despite their remarkable diversity, only the crystal structure of PhaP A h from Aeromonas hydrophila has been solved to date. Based on the structure of PhaP A h , homology models of PhaP A z from Azotobacter sp. FA-8 and PhaP TD from Halomonas bluephagenesis TD were successfully established, allowing rational mutagenesis to be conducted to enhance the stability and surfactant properties of these proteins. PhaP A z mutants, including PhaP A z Q38L and PhaP A z Q78L, as well as PhaP TD mutants, including PhaP TD Q38M and PhaP TD Q72M, showed better emulsification properties and improved thermostability (6-10°C higher melting temperatures) compared with their wild-type homologues under the same conditions. Importantly, the established PhaP homology-modelling approach, based on the high-resolution structure of PhaP A h , can be generalized to facilitate the study of other PhaP members. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Production of microbial polyester by fermentation of recombinant microorganisms.

PubMed

Lee, S Y; Choi, J I

2001-01-01

Polyhydroxyalkanoates (PHAs) can be produced from renewable sources and are biodegradable with similar material properties and processibility to conventional plastic materials. With recent advances in our understanding of the biochemistry and genetics of PHA biosynthesis and cloning of the PHA biosynthesis genes from a number of different bacteria, many different recombinant bacteria have been developed to improve PHA production for commercial applications. For enhancing PHA synthetic capacity, homologous or heterologous expression of the PHA biosynthetic enzymes has been attempted. Several genes that allow utilization of various substrates were transformed into PHA producers, or non-PHA producers utilizing inexpensive carbon substrate were transformed with the PHA biosynthesis genes. Novel PHAs have been synthesized by introducing a new PHA biosynthesis pathway or a new PHA synthase gene. In this article, recent advances in the production of PHA by recombinant bacteria are described.

Modulation of the interface between polyester and spent coffee grounds in polysaccharide membranes: Preparation, cell proliferation, antioxidant activity and tyrosinase activity.

PubMed

Wu, Chin-San

2017-09-01

The structural, antioxidant and cytocompatibility properties of membranes prepared from polyhydroxyalkanoate (PHA) and spent coffee ground (SCG) blends (PHA/SCG) were studied. Acrylic acid-grafted PHA (PHA-g-AA) was used to enhance the desirable characteristics of these membranes, which had better tensile properties than the corresponding PHA/SCG membranes. The water resistance of the PHA-g-AA/SCG membranes was greater than that of the PHA/SCG membranes, and a cytocompatibility evaluation with mouse normal tail fibroblasts (FBs) indicated that both materials were nontoxic. Cell cycle assays of FBs on PHA/SCG and PHA-g-AA/SCG membrane samples were not affected by the DNA content related to damage. Moreover, SCG enhanced the saccharide and polyphenol contents, and antioxidant properties, of the PHA-g-AA/SCG and PHA/SCG membranes. Therefore, we analysed the effects of these compounds' membranes on melanogenesis in B16-F10 melanoma cells. The results demonstrated that PHA/SCG and PHA-g-AA/SCG membranes reduced cellular tyrosinase activities in vitro. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression of lectin genes during seed development in normal and phytohemagglutinin-deficient cultivars of Phaseolus vulgaris.

PubMed

Staswick, P; Chrispeels, M J

1984-01-01

Phytohemagglutinin (PHA), the major lectin of the common bean Phaseolus vulgaris, is synthesized during the development of the seeds. In most cultivars PHA makes up 5-10% of the total seed protein, but certain cultivars do not contain PHA. In vivo labeling of a normal cultivar (Greensleeves) and a PHA-minus cultivar (Pinto 111) showed that PHA was not synthesized in the PHA-minus cultivar. To find out whether the lack of synthesis was due to the absence of mRNA for PHA, recombinant cDNA clones for PHA were obtained. Total poly(A)+ RNA was isolated from cotyledons of developing seeds of Greensleeves and used to direct cDNA synthesis. The double stranded cDNA was cloned in pUC8 and transformants of Escherichia coli screened with pPVL134, a recombinant plasmid which contains the complete coding sequence for a PHA-like protein. Two weakly hybridizing clones (pSC1 and pSC2) were selected. Hybrid selection experiments showed that these two clones selected mRNAs which could be translated into polypeptides identical in size to PHA and recognized by antibodies to PHA. The recombinant pPVL134 selected mRNA which translated into polypeptides which were slightly smaller than those of PHA, and poorly recognized by antibodies to PHA. The recombinant clones were used to demonstrate that the genes for PHA and for the PHA-like protein are under temporal control during seed development. The cultivar Pinto 111, which has no detectable PHA, also has greatly reduced levels of mRNA for PHA. However, the gene for the PHA-like protein encoded by pPVL134 is expressed to the same degree in the cultivars Greensleeves and Pinto 111.

Characterization and Functional Analyses of R-Specific Enoyl Coenzyme A Hydratases in Polyhydroxyalkanoate-Producing Ralstonia eutropha

PubMed Central

Kawashima, Yui; Cheng, Wen; Mifune, Jun; Orita, Izumi; Nakamura, Satoshi

2012-01-01

A genome survey of polyhydroxyalkanoate (PHA)-producing Ralstonia eutropha H16 detected the presence of 16 orthologs of R-specific enoyl coenzyme A (enoyl-CoA) hydratase, among which three proteins shared high homologies with the enzyme specific to enoyl-CoAs of medium chain length encoded by phaJ4 from Pseudomonas aeruginosa (phaJ4Pa). The recombinant forms of the three proteins, termed PhaJ4aRe to PhaJ4cRe, actually showed enoyl-CoA hydratase activity with R specificity, and the catalytic efficiencies were elevated as the substrate chain length increased from C4 to C8. PhaJ4aRe and PhaJ4bRe showed >10-fold-higher catalytic efficiency than PhaJ4cRe. The functions of the new PhaJ4 proteins were investigated using previously engineered R. eutropha strains as host strains; these strains are capable of synthesizing poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from soybean oil. Deletion of phaJ4aRe from the chromosome resulted in significant decrease of 3HHx composition in the accumulated copolyester, whereas no change was observed with deletion of phaJ4bRe or phaJ4cRe, indicating that only PhaJ4aRe was one of the major enzymes supplying the (R)-3HHx-CoA monomer through β-oxidation. Introduction of phaJ4aRe or phaJ4bRe into the R. eutropha strains using a broad-host-range vector enhanced the 3HHx composition of the copolyesters, but the introduction of phaJ4cRe did not. The two genes were then inserted into the pha operon on chromosome 1 of the engineered R. eutropha by homologous recombination. These modifications enabled the biosynthesis of P(3HB-co-3HHx) composed of a larger 3HHx fraction without a negative impact on cell growth and PHA production on soybean oil, especially when phaJ4aRe or phaJ4bRe was tandemly introduced with phaJAc from Aeromonas caviae. PMID:22081565

An integrative study on biologically recovered polyhydroxyalkanoates (PHAs) and simultaneous assessment of gut microbiome in yellow mealworm.

PubMed

Ong, Su Yean; Kho, Hui-Pheng; Riedel, Sebastian L; Kim, Seok-Won; Gan, Chee-Yuen; Taylor, Todd D; Sudesh, Kumar

2018-01-10

Polyhydroxyalkanoates (PHAs) are produced in microbes as a source of carbon and energy storage. They are biodegradable and have properties similar to synthetic plastics, which make them an interesting alternative to petroleum-based plastics. In this study, a refined method of recovering PHA from Cupriavidus necator biomass was proposed by incorporating the use of the yellow mealworm (the larval phase of the mealworm beetle, Tenebrio molitor) as partial purification machinery, followed by washing of the fecal pellets with distilled water and sodium hydroxide. The PHA contents of the cells used in this study were 55wt% (produced from palm olein) and 60 wt% (produced from waste animal fats). The treatment of distilled water and NaOH further increased the purity of PHA to 94%. In parallel, analysis of the 16S rRNA metagenomic sequencing of the mealworm gut microbiome has revealed remarkable changes in the bacterial diversity, especially between the mealworms fed with cells produced from palm olein and waste animal fats. This biological recovery of PHA from cells is an attempt to move towards a green and sustainable process with the aim of reducing the use of harmful solvents and strong chemicals during polymer purification. The results obtained show that - purities of >90%, without a reduction in the molecular weight, can be obtained through this integrative biological recovery approach. In addition, this study has successfully shown that the cells, regardless of their origins, were readily consumed by the mealworms, and there is a correlation between the feed type and the mealworm gut microbiome. Copyright © 2017 Elsevier B.V. All rights reserved.

Optimization of enhanced biological phosphorus removal after periods of low loading.

PubMed

Miyake, Haruo; Morgenroth, Eberhard

2005-01-01

Enhanced biological phosphorus removal is a well-established technology for the treatment of municipal wastewater. However, increased effluent phosphorus concentrations have been reported after periods (days) of low organic loading. The purpose of this study was to evaluate different operating strategies to prevent discharge of effluent after such low-loading periods. Mechanisms leading to these operational problems have been related to the reduction of polyphosphate-accumulating organisms (PAOs) and their storage compounds (polyhydroxy alkanoates [PHA]). Increased effluent phosphorus concentrations can be the result of an imbalance between influent loading and PAOs in the system and an imbalance between phosphorus release and uptake rates. The following operating conditions were tested in their ability to prevent a reduction of PHA and of overall biomass during low organic loading conditions: (a) unchanged operation, (b) reduced aeration time, (c) reduced sludge wastage, and (d) combination of reduced aeration time and reduced sludge wastage. Experiments were performed in a laboratory-scale anaerobic-aerobic sequencing batch reactor, using acetate as the carbon source. Without operational adjustments, phosphorus-release rates decreased during low-loading periods but recovered rapidly. Phosphorus-uptake rates also decreased, and the recovery typically required several days to increase to normal levels. The combination of reduced aeration time and reduced sludge wastage allowed the maintenance of constant levels of both PHA and overall biomass. A mathematical model was used to explain the influence of the tested operating conditions on PAO and PHA concentrations. While experimental results were in general agreement with model predictions, the kinetic expression for phosphorus uptake deviated significantly for the first 24 hours after low-loading conditions. Mechanisms leading to these deviations need to be further investigated.

Backup Expression of the PhaP2 Phasin Compensates for phaP1 Deletion in Herbaspirillum seropedicae, Maintaining Fitness and PHB Accumulation

PubMed Central

Alves, Luis P. S.; Teixeira, Cícero S.; Tirapelle, Evandro F.; Donatti, Lucélia; Tadra-Sfeir, Michelle Z.; Steffens, Maria B. R.; de Souza, Emanuel M.; de Oliveira Pedrosa, Fabio; Chubatsu, Leda S.; Müller-Santos, Marcelo

2016-01-01

Phasins are important proteins controlling poly-3-hydroxybutyrate (PHB) granules formation, their number into the cell and stability. The genome sequencing of the endophytic and diazotrophic bacterium Herbaspirillum seropedicae SmR1 revealed two homologous phasin genes. To verify the role of the phasins on PHB accumulation in the parental strain H. seropedicae SmR1, isogenic strains defective in the expression of phaP1, phaP2 or both genes were obtained by gene deletion and characterized in this work. Despite of the high sequence similarity between PhaP1 and PhaP2, PhaP1 is the major phasin in H. seropedicae, since its deletion reduced PHB accumulation by ≈50% in comparison to the parental and ΔphaP2. Upon deletion of phaP1, the expression of phaP2 was sixfold enhanced in the ΔphaP1 strain. The responsive backup expression of phaP2 partially rescued the ΔphaP1 mutant, maintaining about 50% of the parental PHB level. The double mutant ΔphaP1.2 did not accumulate PHB in any growth stage and showed a severe reduction of growth when glucose was the carbon source, a clear demonstration of negative impact in the fitness. The co-occurrence of phaP1 and phaP2 homologous in bacteria relatives of H. seropedicae, including other endophytes, indicates that the mechanism of phasin compensation by phaP2 expression may be operating in other organisms, showing that PHB metabolism is a key factor to adaptation and efficiency of endophytic bacteria. PMID:27242754

Backup Expression of the PhaP2 Phasin Compensates for phaP1 Deletion in Herbaspirillum seropedicae, Maintaining Fitness and PHB Accumulation.

PubMed

Alves, Luis P S; Teixeira, Cícero S; Tirapelle, Evandro F; Donatti, Lucélia; Tadra-Sfeir, Michelle Z; Steffens, Maria B R; de Souza, Emanuel M; de Oliveira Pedrosa, Fabio; Chubatsu, Leda S; Müller-Santos, Marcelo

2016-01-01

Phasins are important proteins controlling poly-3-hydroxybutyrate (PHB) granules formation, their number into the cell and stability. The genome sequencing of the endophytic and diazotrophic bacterium Herbaspirillum seropedicae SmR1 revealed two homologous phasin genes. To verify the role of the phasins on PHB accumulation in the parental strain H. seropedicae SmR1, isogenic strains defective in the expression of phaP1, phaP2 or both genes were obtained by gene deletion and characterized in this work. Despite of the high sequence similarity between PhaP1 and PhaP2, PhaP1 is the major phasin in H. seropedicae, since its deletion reduced PHB accumulation by ≈50% in comparison to the parental and ΔphaP2. Upon deletion of phaP1, the expression of phaP2 was sixfold enhanced in the ΔphaP1 strain. The responsive backup expression of phaP2 partially rescued the ΔphaP1 mutant, maintaining about 50% of the parental PHB level. The double mutant ΔphaP1.2 did not accumulate PHB in any growth stage and showed a severe reduction of growth when glucose was the carbon source, a clear demonstration of negative impact in the fitness. The co-occurrence of phaP1 and phaP2 homologous in bacteria relatives of H. seropedicae, including other endophytes, indicates that the mechanism of phasin compensation by phaP2 expression may be operating in other organisms, showing that PHB metabolism is a key factor to adaptation and efficiency of endophytic bacteria.

Cloning of phaCAB genes from thermophilic Caldimonas manganoxidans in Escherichia coli for poly(3-hydroxybutyrate) (PHB) production.

PubMed

Lin, Ji-Hong; Lee, Ming-Chieh; Sue, You-Sheng; Liu, Yung-Chuan; Li, Si-Yu

2017-08-01

PHB biosynthesis pathway, consisting of three open reading frames (ORFs) that encode for β-ketothiolase (phaA Cma , 1179 bp), acetoacetyl-CoA reductase (phaB Cma , 738 bp), and PHA synthase (phaC Cma , 1694 bp), of Caldimonas manganoxidans was identified. The functions of PhaA, PhaB, and PhaC were demonstrated by successfully reconstructing PHB biosynthesis pathway of C. manganoxidans in Escherichia coli, where PHB production was confirmed by OD 600 , gas chromatography, Nile blue stain, and transmission electron microscope (TEM). The protein sequence alignment of PHB synthases revealed that phaC Cma shares at least 60% identity with those of class I PHB synthase. The effects of PhaA, PhaB, and PhaC expression levels on PHB production were investigated. While the overexpression of PhaB is found to be important in recombinant E. coli, performances of PHB production can be quantified as follows: PHB concentration of 16.8 ± 0.6 g/L, yield of 0.28 g/g glucose, content of 74%, productivity of 0.28 g/L/h, and Mw of 1.41 MDa.

Identification of a multifunctional protein, PhaM, that determines number, surface to volume ratio, subcellular localization and distribution to daughter cells of poly(3-hydroxybutyrate), PHB, granules in Ralstonia eutropha H16.

PubMed

Pfeiffer, Daniel; Wahl, Andreas; Jendrossek, Dieter

2011-11-01

A two-hybrid approach was applied to screen for proteins with the ability to interact with PHB synthase (PhaC1) of Ralstonia eutropha. The H16_A0141 gene (phaM) was identified in the majority of positive clones. PhaM (26.6 kDa) strongly interacted with PhaC1 and with phasin PhaP5 but not with PhaP1 or other PHB granule-associated proteins. A ΔphaM mutant accumulated only one or two large PHB granules instead of three to six medium-sized PHB granules of the wild type, and distribution of granules to daughter cells was disordered. All three phenotypes (number, size and distribution of PHB granules) were reversed by reintroduction of phaM. Purified PhaM revealed DNA-binding properties in gel mobility shift experiments. Expression of a fusion of the yellow fluorescent protein (eYfp) with PhaM resulted in formation of many small fluorescent granules that were bound to the nucleoid region. Remarkably, an eYfp-PhaP5 fusion localized at the cell poles in a PHB-negative background and overexpression of eYfp-PhaP5 in the wild type conferred binding of PHB granules to the cell poles. In conclusion, subcellular localization of PHB granules in R. eutropha depends on a concerted expression of at least three PHB granule-associated proteins, namely PhaM, PhaP5 and PHB synthase PhaC1. © 2011 Blackwell Publishing Ltd.

Production, process optimization and molecular characterization of polyhydroxyalkanoate (PHA) by CO2 sequestering B. cereus SS105.

PubMed

Maheshwari, Neha; Kumar, Madan; Thakur, Indu Shekhar; Srivastava, Shaili

2018-04-01

Carbon dioxide sequestering bacterial strains were previously isolated from free air CO 2 enriched (FACE) soil. In the present study, these strains were screened for PHA accumulation and Bacillus cereus SS105 was found to be the most prominent PHA accumulating strain on sodium bicarbonate and molasses as carbon source. This strain was further characterized by Spectrofluorometric method and Confocal microscopy after staining with Nile red. PHA granules in inclusion bodies were visualized by Transmission Electron Microscopy. The PHA and its monomer composition were characterized by GC-MS followed by FTIR and NMR. The genetic basis of PHA production was confirmed by the amplification, cloning and analysis of PHA biosynthesis genes phaR, phaB and phaC from B. cereus with the degenerate primers. The PHA production was further optimized by Response Surface Methodology and the percent increase observed after optimization was 55.16% (w/v). Copyright © 2018 Elsevier Ltd. All rights reserved.

Interface design and reinforced features of arrowroot (Maranta arundinacea) starch/polyester-based membranes: Preparation, antioxidant activity, and cytocompatibility.

PubMed

Wu, Chin-San; Liao, Hsin-Tzu

2017-01-01

The structural, mechanical, antioxidant, and cytocompatibility properties of membranes prepared from the polyhydroxyalkanoate (PHA) and arrowroot (Maranta arundinacea) starch powder (ASP) blend (PHA/ASP) were studied. The acrylic acid-grafted PHA (PHA-g-AA) and the coupling agent treated ASP (TASP) were used to enhance the desired characteristics of these membranes. The PHA-g-AA/TASP membranes had better mechanical properties than the PHA/ASP membrane. This effect was attributed to greater compatibility between the grafted PHA and TASP. The water resistance of the PHA-g-AA/TASP membranes was greater than that of the PHA/ASP membranes, and a cytocompatibility evaluation with human foreskin fibroblasts (FBs) indicated that both materials were nontoxic. Moreover, both ASP and TASP enhanced the polyphenol content and antioxidant properties of the membranes. PHA-g-AA/TASP and PHA/ASP membranes had better antioxidant activity than the control group. Copyright © 2016 Elsevier B.V. All rights reserved.

Whole genome amplification approach reveals novel polyhydroxyalkanoate synthases (PhaCs) from Japan Trench and Nankai Trough seawater.

PubMed

Foong, Choon Pin; Lau, Nyok-Sean; Deguchi, Shigeru; Toyofuku, Takashi; Taylor, Todd D; Sudesh, Kumar; Matsui, Minami

2014-12-24

Special features of the Japanese ocean include its ranges of latitude and depth. This study is the first to examine the diversity of Class I and II PHA synthases (PhaC) in DNA samples from pelagic seawater taken from the Japan Trench and Nankai Trough from a range of depths from 24 m to 5373 m. PhaC is the key enzyme in microorganisms that determines the types of monomer units that are polymerized into polyhydroxyalkanoate (PHA) and thus affects the physicochemical properties of this thermoplastic polymer. Complete putative PhaC sequences were determined via genome walking, and the activities of newly discovered PhaCs were evaluated in a heterologous host. A total of 76 putative phaC PCR fragments were amplified from the whole genome amplified seawater DNA. Of these 55 clones contained conserved PhaC domains and were classified into 20 genetic groups depending on their sequence similarity. Eleven genetic groups have undisclosed PhaC activity based on their distinct phylogenetic lineages from known PHA producers. Three complete DNA coding sequences were determined by IAN-PCR, and one PhaC was able to produce poly(3-hydroxybutyrate) in recombinant Cupriavidus necator PHB-4 (PHB-negative mutant). A new functional PhaC that has close identity to Marinobacter sp. was discovered in this study. Phylogenetic classification for all the phaC genes isolated from uncultured bacteria has revealed that seawater and other environmental resources harbor a great diversity of PhaCs with activities that have not yet been investigated. Functional evaluation of these in silico-based PhaCs via genome walking has provided new insights into the polymerizing ability of these enzymes.

Accumulation of PHA granules in Cupriavidus necator as seen by confocal fluorescence microscopy.

PubMed

Mravec, Filip; Obruca, Stanislav; Krzyzanek, Vladislav; Sedlacek, Petr; Hrubanova, Kamila; Samek, Ota; Kucera, Dan; Benesova, Pavla; Nebesarova, Jana

2016-05-01

Many bacteria are capable of accumulating intracellular granules of polyhydroxyalkanoates (PHA). In this work, we developed confocal microscopy analysis of bacterial cells to study changes in the diameters of cells as well as PHA granules during growth and PHA accumulation in the bacterium Cupriavidus necator H16 (formerly Ralstonia eutropha). The cell envelope was stained by DiD(®) fluorescent probe and PHA granules by Nile Red. Signals from both probes were separated based on their spectral and fluorescence life-time properties. During growth and PHA accumulation, bacterial cells increased their length but the width of the cells remained constant. The volume fraction of PHA granules in cells increased during PHA accumulation, nevertheless, its value did not exceed 40 vol. % regardless of the PHA weight content. It seems that bacterial cultures lengthen the cells in order to control the PHA volume portion. However, since similar changes in cell length were also observed in a PHA non-accumulating mutant, it seems that there is no direct control mechanism, which regulates the prolongation of the cells with respect to PHA granules volume. It is more likely that PHA biosynthesis and the length of cells are influenced by the same external stimuli such as nutrient limitation. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A microbial polyhydroxyalkanoates (PHA) based bio- and materials industry.

PubMed

Chen, Guo-Qiang

2009-08-01

Biopolyesters polyhydroxyalkanoates (PHA) produced by many bacteria have been investigated by microbiologists, molecular biologists, biochemists, chemical engineers, chemists, polymer experts and medical researchers. PHA applications as bioplastics, fine chemicals, implant biomaterials, medicines and biofuels have been developed and are covered in this critical review. Companies have been established or involved in PHA related R&D as well as large scale production. Recently, bacterial PHA synthesis has been found to be useful for improving robustness of industrial microorganisms and regulating bacterial metabolism, leading to yield improvement on some fermentation products. In addition, amphiphilic proteins related to PHA synthesis including PhaP, PhaZ or PhaC have been found to be useful for achieving protein purification and even specific drug targeting. It has become clear that PHA and its related technologies are forming an industrial value chain ranging from fermentation, materials, energy to medical fields (142 references).

Methodes de decomposition pour la planification a moyen terme de la production hydroelectrique sous incertitude

NASA Astrophysics Data System (ADS)

Carpentier, Pierre-Luc

In this thesis, we consider the midterm production planning problem (MTPP) of hydroelectricity generation under uncertainty. The aim of this problem is to manage a set of interconnected hydroelectric reservoirs over several months. We are particularly interested in high dimensional reservoir systems that are operated by large hydroelectricity producers such as Hydro-Quebec. The aim of this thesis is to develop and evaluate different decomposition methods for solving the MTPP under uncertainty. This thesis is divided in three articles. The first article demonstrates the applicability of the progressive hedging algorithm (PHA), a scenario decomposition method, for managing hydroelectric reservoirs with multiannual storage capacity under highly variable operating conditions in Canada. The PHA is a classical stochastic optimization method designed to solve general multistage stochastic programs defined on a scenario tree. This method works by applying an augmented Lagrangian relaxation on non-anticipativity constraints (NACs) of the stochastic program. At each iteration of the PHA, a sequence of subproblems must be solved. Each subproblem corresponds to a deterministic version of the original stochastic program for a particular scenario in the scenario tree. Linear and a quadratic terms must be included in subproblem's objective functions to penalize any violation of NACs. An important limitation of the PHA is due to the fact that the number of subproblems to be solved and the number of penalty terms increase exponentially with the branching level in the tree. This phenomenon can make the application of the PHA particularly difficult when the scenario tree covers several tens of time periods. Another important limitation of the PHA is caused by the fact that the difficulty level of NACs generally increases as the variability of scenarios increases. Consequently, applying the PHA becomes particularly challenging in hydroclimatic regions that are characterized by a high level of seasonal and interannual variability. These two types of limitations can slow down the algorithm's convergence rate and increase the running time per iteration. In this study, we apply the PHA on Hydro-Quebec's power system over a 92-week planning horizon. Hydrologic uncertainty is represented by a scenario tree containing 6 branching stages and 1,635 nodes. The PHA is especially well-suited for this particular application given that the company already possess a deterministic optimization model to solve the MTPP. The second article presents a new approach which enhances the performance of the PHA for solving general Mstochastic programs. The proposed method works by applying a multiscenario decomposition scheme on the stochastic program. Our heuristic method aims at constructing an optimal partition of the scenario set by minimizing the number of NACs on which an augmented Lagrangean relaxation must be applied. Each subproblem is a stochastic program defined on a group of scenarios. NACs linking scenarios sharing a common group are represented implicitly in subproblems by using a group-node system index instead of the traditional scenario-time index system. Only the NACs that link the different scenario groups are represented explicitly and relaxed. The proposed method is evaluated numerically on an hydroelectric reservoir management problem in Quebec. The results of this experiment show that our method has several advantages. Firstly, it allows to reduce the running time per iteration of the PHA by reducing the number of penalty terms that are included in the objective function and by reducing the amount of duplicated constraints and variables. In turn, this allows to reduce the running time per iteration of the algorithm. Secondly, it allows to increase the algorithm's convergence rate by reducing the variability of intermediary solutions at duplicated tree nodes. Thirdly, our approach reduces the amount of random-access memory (RAM) required for storing Lagrange multipliers associated with relaxed NACs. The third article presents an extension of the L-Shaped method designed specifically for managing hydroelectric reservoir systems with a high storage capacity. The method proposed in this paper enables to consider a higher branching level than conventional decomposition method enables. To achieve this, we assume that the stochastic process driving random parameters has a memory loss at time period t = tau. Because of this assumption, the scenario tree possess a special symmetrical structure at the second stage (t > tau). We exploit this feature using a two-stage Benders decomposition method. Each decomposition stage covers several consecutive time periods. The proposed method works by constructing a convex and piecewise linear recourse function that represents the expected cost at the second stage in the master problem. The subproblem and the master problem are stochastic program defined on scenario subtrees and can be solved using a conventional decomposition method or directly. We test the proposed method on an hydroelectric power system in Quebec over a 104-week planning horizon. (Abstract shortened by UMI.).

A proteome analysis of freezing tolerance in red clover (Trifolium pratense L.).

PubMed

Bertrand, Annick; Bipfubusa, Marie; Castonguay, Yves; Rocher, Solen; Szopinska-Morawska, Aleksandra; Papadopoulos, Yousef; Renaut, Jenny

2016-03-10

Improvement of freezing tolerance of red clover (Trifolium pratense L.) would increase its persistence under cold climate. In this study, we assessed the freezing tolerance and compared the proteome composition of non-acclimated and cold-acclimated plants of two initial cultivars of red clover: Endure (E-TF0) and Christie (C-TF0) and of populations issued from these cultivars after three (TF3) and four (TF4) cycles of phenotypic recurrent selection for superior freezing tolerance. Through this approach, we wanted to identify proteins that are associated with the improvement of freezing tolerance in red clover. Freezing tolerance expressed as the lethal temperature for 50 % of the plants (LT50) increased markedly from approximately -2 to -16 °C following cold acclimation. Recurrent selection allowed a significant 2 to 3 °C increase of the LT50 after four cycles of recurrent selection. Two-dimensional difference gel electrophoresis (2D-DIGE) was used to study variations in protein abundance. Principal component analysis based on 2D-DIGE revealed that the largest variability in the protein data set was attributable to the cold acclimation treatment and that the two genetic backgrounds had differential protein composition in the acclimated state only. Vegetative storage proteins (VSP), which are essential nitrogen reserves for plant regrowth, and dehydrins were among the most striking changes in proteome composition of cold acclimated crowns of red clovers. A subset of proteins varied in abundance in response to selection including a dehydrin that increased in abundance in TF3 and TF4 populations as compared to TF0 in the Endure background. Recurrent selection performed indoor is an effective approach to improve the freezing tolerance of red clover. Significant improvement of freezing tolerance by recurrent selection was associated with differential accumulation of a small number of cold-regulated proteins that may play an important role in the determination of the level of freezing tolerance.

A CAM- and starch-deficient mutant of the facultative CAM species Mesembryanthemum crystallinum reconciles sink demands by repartitioning carbon during acclimation to salinity

PubMed Central

Haider, Muhammad Sajjad; Barnes, Jeremy D.; Cushman, John C.; Borland, Anne M.

2012-01-01

In the halophytic species Mesembryanthemum crystallinum, the induction of crassulacean acid metabolism (CAM) by salinity requires a substantial investment of resources in storage carbohydrates to provide substrate for nocturnal CO2 uptake. Acclimation to salinity also requires the synthesis and accumulation of cyclitols as compatible solutes, maintenance of root respiration, and nitrate assimilation. This study assessed the hierarchy and coordination of sinks for carbohydrate in leaves and roots during acclimation to salinity in M. crystallinum. By comparing wild type and a CAM-/starch-deficient mutant of this species, it was sought to determine if other metabolic sinks could compensate for a curtailment in CAM and enable acclimation to salinity. Under salinity, CAM deficiency reduced 24 h photosynthetic carbon gain by >50%. Cyclitols were accumulated to comparable levels in leaves and roots of both the wild type and mutant, but represented only 5% of 24 h carbon balance. Dark respiration of leaves and roots was a stronger sink for carbohydrate in the mutant compared with the wild type and implied higher maintenance costs for the metabolic processes underpinning acclimation to salinity when CAM was curtailed. CAM required the nocturnal mobilization of >70% of primary carbohydrate in the wild type and >85% of carbohydrate in the mutant. The substantial allocation of carbohydrate to CAM limited the export of sugars to roots, and the root:shoot ratio declined under salinity. The data suggest a key role for the vacuole in regulating the supply and demand for carbohydrate over the day/night cycle in the starch-/CAM-deficient mutant. PMID:22219316

Screening of polyhydroxyalkanoate-producing bacteria and PhaC-encoding genes in two hypersaline microbial mats from Guerrero Negro, Baja California Sur, Mexico.

PubMed

Martínez-Gutiérrez, Carolina A; Latisnere-Barragán, Hever; García-Maldonado, José Q; López-Cortés, Alejandro

2018-01-01

Hypersaline microbial mats develop through seasonal and diel fluctuations, as well as under several physicochemical variables. Hence, resident microorganisms commonly employ strategies such as the synthesis of polyhydroxyalkanoates (PHAs) in order to resist changing and stressful conditions. However, the knowledge of bacterial PHA production in hypersaline microbial mats has been limited to date, particularly in regard to medium-chain length PHAs (mcl-PHAs), which have biotechnological applications due to their plastic properties. The aim of this study was to obtain evidence for PHA production in two hypersaline microbial mats of Guerrero Negro, Mexico by searching for PHA granules and PHA synthase genes in isolated bacterial strains and environmental samples. Six PHA-producing strains were identified by 16S rRNA gene sequencing; three of them corresponded to a Halomonas sp. In addition, Paracoccus sp., Planomicrobium sp. and Staphylococcus sp. were also identified as PHA producers. Presumptive PHA granules and PHA synthases genes were detected in both sampling sites. Moreover, phylogenetic analysis showed that most of the phylotypes were distantly related to putative PhaC synthases class I sequences belonging to members of the classes Alphaproteobacteria and Gammaproteobacteria distributed within eight families, with higher abundances corresponding mainly to Rhodobacteraceae and Rhodospirillaceae. This analysis also showed that PhaC synthases class II sequences were closely related to those of Pseudomonas putida , suggesting the presence of this group, which is probably involved in the production of mcl-PHA in the mats. According to our state of knowledge, this study reports for the first time the occurrence of phaC and phaC1 sequences in hypersaline microbial mats, suggesting that these ecosystems may be a novel source for the isolation of short- and medium-chain length PHA producers.

[Increasing reductant NADPH content via metabolic engineering of PHB synthesis pathway in Synechocystis sp. PCC 6803].

PubMed

Xie, Juan; Zhou, Jie; Zhang, Haifeng; Li, Yin

2011-07-01

Cyanobacteria have become attractive hosts for renewable chemicals production. The low productivity, however, prevents it from industrial application. Reductant NAD(P)H availability is a chief hurdle for the production of reductive metabolites in microbes. To increase NADPH content in Synechocystis sp. PCC 6803, PHB synthase encoding gene phaC and phaE in Synechocystis was inactivated by replacing phaC&E genes with chloromycetin resistance cassette via homologous recombination. PCR analysis showed that mutant S.delta phaC&E with complete genome segregation was generated. The comparison between growth curves of S.wt and S.delta phaC&E indicated the knockout of phaC & phaE genes did not affect obviously the cell growth. Gas chromatography analysis showed that the accumulation of PHB in wild type was about 2.3% of the dry cell weight, whereas no PHB was detected in the mutant S.delta phaC&E. The data indicated that inactivation of PHB synthase gene phaC and phaE interrupted the synthesis of PHB. Further comparative study of wild type and mutant demonstrated that NADPH content in S.delta phaC&E was obviously increased. On the third day, the NADPH content in S.delta phaC&E was up to 1.85 fold higher than that in wild type. These results indicated that deleting PHB synthase gene phaC and phaE not only can block the synthesis of PHB, but also can save NADPH to contribute reductant sink in cyanobacteria. Hence, the engineered cyanobacterial strain S.delta phaC&E, in which carbon flux was redirected and NADPH was increased, will be a potential host strain for chemicals production in cyanobacteria.

Identification and analysis of putative polyhydroxyalkanoate synthase (PhaC) in Pseudomonas fluorescens.

PubMed

Lim, Ju Hyoung; Rhie, Ho-Gun; Kim, Jeong Nam

2018-05-11

Pseudomonas fluorescens KLR101 was found to be capable of producing polyhydroxyalkanoate (PHA) using various sugars and fatty acids with carbon numbers ranging from 2 to 6. PHA granules mainly consisted of poly(3-hydroxybutyrate) homopolymer and/or poly(3-hydroxybutyrate- co -3-hydroxyvalerate) copolymer. Genomic DNA of P. fluorescens was fractionated and cloned into a lambda library, in which a 5.8-kb fragment hybridized to a heterologous phaC probe from Ralstonia eutropha was identified. In vivo expression in Klebsiella aerogenes KC2671 (pUMS), restriction mapping, Southern hybridization experiments, and sequencing data revealed that PHA biosynthesis by P. fluorescens relied upon a polypeptide encoded by a 1,683-bp non-operonal ORF, which was preceded by a possible -24/-12 promoter and highly similar to DNA sequences of a gene encoding PHA synthase in the genus Pseudomonas . In vivo expression of the putative PHA synthase gene ( phaC Pf ) in a recombinant Escherichia coli strain was investigated by using glucose and decanoate as substrates. E. coli ( phaC Pf + , pUMS) grown in medium containing glucose accumulated PHA granules mainly consisting of 3-hydroxybutyrate, whereas only a trace amount of 3-hydroxydecanoate was detected from E. coli fadR mutant ( phaC Pf + ) grown in medium containing decanoate. In vitro enzymatic assessment experiments showed that 3-hydroxybutyryl-CoA was efficiently used as a substrate of purified PhaC Pf , suggesting that the putative PHA synthase of P. fluorescens mainly utilizes short-chain-length PHA precursors as a substrate.

Discovery of a new polyhydroxyalkanoate synthase from limestone soil through metagenomic approach.

PubMed

Tai, Yen Teng; Foong, Choon Pin; Najimudin, Nazalan; Sudesh, Kumar

2016-04-01

PHA synthase (PhaC) is the key enzyme in the production of biodegradable plastics known as polyhydroxyalkanoate (PHA). Nevertheless, most of these enzymes are isolated from cultivable bacteria using traditional isolation method. Most of the microorganisms found in nature could not be successfully cultivated due to the lack of knowledge on their growth conditions. In this study, a culture-independent approach was applied. The presence of phaC genes in limestone soil was screened using primers targeting the class I and II PHA synthases. Based on the partial gene sequences, a total of 19 gene clusters have been identified and 7 clones were selected for full length amplification through genome walking. The complete phaC gene sequence of one of the clones (SC8) was obtained and it revealed 81% nucleotide identity to the PHA synthase gene of Chromobacterium violaceum ATCC 12472. This gene obtained from uncultured bacterium was successfully cloned and expressed in a Cupriavidus necator PHB(-)4 PHA-negative mutant resulting in the accumulation of significant amount of PHA. The PHA synthase activity of this transformant was 64 ± 12 U/g proteins. This paper presents a pioneering study on the discovery of phaC in a limestone area using metagenomic approach. Through this study, a new functional phaC was discovered from uncultured bacterium. Phylogenetic classification for all the phaCs isolated from this study has revealed that limestone hill harbors a great diversity of PhaCs with activities that have not yet been investigated. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

PhaM Is the Physiological Activator of Poly(3-Hydroxybutyrate) (PHB) Synthase (PhaC1) in Ralstonia eutropha

PubMed Central

Pfeiffer, Daniel

2014-01-01

Poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) is the key enzyme of PHB synthesis in Ralstonia eutropha and other PHB-accumulating bacteria and catalyzes the polymerization of 3-hydroxybutyryl-CoA to PHB. Activity assays of R. eutropha PHB synthase are characterized by the presence of lag phases and by low specific activity. It is assumed that the lag phase is caused by the time necessary to convert the inactive PhaC1 monomer into the active dimeric form by an unknown priming process. The lag phase can be reduced by addition of nonionic detergents such as hecameg [6-O-(N-heptyl-carbamoyl)-methyl-α-d-glucopyranoside], which apparently accelerates the formation of PhaC1 dimers. We identified the PHB granule-associated protein (PGAP) PhaM as the natural primer (activator) of PHB synthase activity. PhaM was recently discovered as a novel type of PGAP with multiple functions in PHB metabolism. Addition of PhaM to PHB synthase assays resulted in immediate polymerization of 3HB coenzyme A with high specific activity and without a significant lag phase. The effect of PhaM on (i) PhaC1 activity, (ii) oligomerization of PhaC1, (iii) complex formation with PhaC1, and (iv) PHB granule formation in vitro and in vivo was shown by cross-linking experiments of purified proteins (PhaM, PhaC1) with glutardialdehyde, by size exclusion chromatography, and by fluorescence microscopic detection of de novo-synthesized PHB granules. PMID:24212577

PhaM is the physiological activator of poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) in Ralstonia eutropha.

PubMed

Pfeiffer, Daniel; Jendrossek, Dieter

2014-01-01

Poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) is the key enzyme of PHB synthesis in Ralstonia eutropha and other PHB-accumulating bacteria and catalyzes the polymerization of 3-hydroxybutyryl-CoA to PHB. Activity assays of R. eutropha PHB synthase are characterized by the presence of lag phases and by low specific activity. It is assumed that the lag phase is caused by the time necessary to convert the inactive PhaC1 monomer into the active dimeric form by an unknown priming process. The lag phase can be reduced by addition of nonionic detergents such as hecameg [6-O-(N-heptyl-carbamoyl)-methyl-α-D-glucopyranoside], which apparently accelerates the formation of PhaC1 dimers. We identified the PHB granule-associated protein (PGAP) PhaM as the natural primer (activator) of PHB synthase activity. PhaM was recently discovered as a novel type of PGAP with multiple functions in PHB metabolism. Addition of PhaM to PHB synthase assays resulted in immediate polymerization of 3HB coenzyme A with high specific activity and without a significant lag phase. The effect of PhaM on (i) PhaC1 activity, (ii) oligomerization of PhaC1, (iii) complex formation with PhaC1, and (iv) PHB granule formation in vitro and in vivo was shown by cross-linking experiments of purified proteins (PhaM, PhaC1) with glutardialdehyde, by size exclusion chromatography, and by fluorescence microscopic detection of de novo-synthesized PHB granules.

[Effect of phytohemagglutinin (PHA) from Yunnan white kidney bean on development of mouse embryos].

PubMed

Zhang, Lifen; Wang, Changmei; Yang, Mingjie; Zhang, Tian; Wang, Minkang

2011-06-01

To study the effect of different concentration of phytohemagglutinin (PHA) on mouse embryo development. In experiment 1, crude and purified PHA extracted from Yunnan white kidney bean with different concentration were added into M16 culture medium, the final concentration of PHA were: 50, 100, 200, 500, 1 000, 2 000 and 5 000 mg x L(-1) respectively. 2-cell stage embryos were collected and cultured in PHA containing or control medium for 72-96 h and their development were recorded. In experiment 2, different stage of embryos from 1-cell to blastocyst were treated by different concentrations of PHA same as experiment 1 and 10 000 mg x L(-1) in culture medium for 24 h before washing and cultured in M16 + PVA without PHA to blastocyst or hatching blastocyst stage. Low concentrations PHA at 50-100 mg x L(-1) promoted embryo development and increased the number of blastocyst stage embryos. In contrast, high concentrations of PHA (> 1 000 mg x L(-1)) blocked the embryos development from 1-cell to blastocyst stage and showed apoptosis morphology or death. Depending on the concentrations, PHA from white kidney bean shown promotion or inhibition on mouse embryo development. 1-cell stage embryo shown more sensitive to PHA treatment than that of later stage embryos. Pretreatment 24 h in PHA containing medium can influence the further development of embryos. Low concentrations of PHA is benefit to embryo development, but high concentrations of PHA (> 1 000 mg x L(-1)) will block of the development of embryos.

Molecular analysis of a phytohemagglutinin-defective cultivar of Phaseolus vulgaris L.

PubMed

Vitale, A; Ceriotti, A; Bollini, R

1985-10-01

The seeds of Phaseolus vulgaris cv. Pinto III are known to lack detectable amounts of phytohemagglutinin (PHA) and to accumulate very reduced levels of PHA mRNA compared with normal cultivars. Using PHA complementary-DNA clones and monospecific antibodies we analyzed cv. Pinto III genomic DNA and cotyledonary proteins synthesized both in vitro and in vivo. We detected genomic DNA sequences that hybridize with complementary-DNA clones for the two different classes of PHA polypeptides (PHA-E and PHA-L), at levels comparable to a normal bean cultivar. This indicates that the cv. Pinto III phenotype is not the result of a large deletion of the PHA structural genes. Messenger RNA isolated from cv. Pinto III developing cotyledons synthesizes in vitro very small amounts of a protein which is recognized by antibodies specific for PHA, and gives, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a single band with molecular weight similar but not identical to that of PHA-L polypeptides. This protein is also synthesized in vivo at a very reduced level, less than 1% compared with PHA in normal cultivars, and has mitogenic activity comparable to that of the PHA-L subunit, while it shows very weak erythroagglutinating activity. The initial steps in the synthesis and processing of this protein are identical to those already identified for PHA polypeptides. The cv. Pinto III protein could be either a PHA-L polypeptide whose synthesis is not affected by the mutation or a PHA-like lectin present normally at low levels in P. vulgaris.

Food waste conversion to microbial polyhydroxyalkanoates.

PubMed

Nielsen, Chad; Rahman, Asif; Rehman, Asad Ur; Walsh, Marie K; Miller, Charles D

2017-11-01

Polyhydroxyalkanoates (PHAs) are biopolymers with desirable material properties similar to petrochemically derived plastics. PHAs are naturally produced by a wide range of microorganisms as a carbon storage mechanism and can accumulate to significantly high levels. PHAs are an environmentally friendly alternative to their petroleum counterparts because they can be easily degraded, potentially reducing the burden on municipal waste systems. Nevertheless, widespread use of PHAs is not currently realistic due to a variety of factors. One of the major constraints of large-scale PHA production is the cost of carbon substrate for PHA-producing microbes. The cost of production could potentially be reduced with the use of waste carbon from food-related processes. Food wastage is a global issue and therefore harbours immense potential to create valuable bioproducts. This article's main focus is to examine the state of the art of converting food-derived waste into carbon substrates for microbial metabolism and subsequent conversion into PHAs. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

PHB Biosynthesis Counteracts Redox Stress in Herbaspirillum seropedicae.

PubMed

Batista, Marcelo B; Teixeira, Cícero S; Sfeir, Michelle Z T; Alves, Luis P S; Valdameri, Glaucio; Pedrosa, Fabio de Oliveira; Sassaki, Guilherme L; Steffens, Maria B R; de Souza, Emanuel M; Dixon, Ray; Müller-Santos, Marcelo

2018-01-01

The ability of bacteria to produce polyhydroxyalkanoates such as poly(3-hydroxybutyrate) (PHB) enables provision of a carbon storage molecule that can be mobilized under demanding physiological conditions. However, the precise function of PHB in cellular metabolism has not been clearly defined. In order to determine the impact of PHB production on global physiology, we have characterized the properties of a Δ phaC1 mutant strain of the diazotrophic bacterium Herbaspirillum seropedicae . The absence of PHB in the mutant strain not only perturbs redox balance and increases oxidative stress, but also influences the activity of the redox-sensing Fnr transcription regulators, resulting in significant changes in expression of the cytochrome c -branch of the electron transport chain. The synthesis of PHB is itself dependent on the Fnr1 and Fnr3 proteins resulting in a cyclic dependency that couples synthesis of PHB with redox regulation. Transcriptional profiling of the Δ phaC1 mutant reveals that the loss of PHB synthesis affects the expression of many genes, including approximately 30% of the Fnr regulon.

24 CFR 983.254 - Vacancies.

Code of Federal Regulations, 2010 CFR

2010-04-01

... vacancy (and notwithstanding the reasonable good faith efforts of the PHA to fill such vacancies), the PHA... on the PHA waiting list referred by the PHA. (3) The PHA and the owner must make reasonable good faith efforts to minimize the likelihood and length of any vacancy. (b) Reducing number of contract...

24 CFR 982.103 - PHA application for funding.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 24 Housing and Urban Development 4 2012-04-01 2012-04-01 false PHA application for funding. 982... URBAN DEVELOPMENT SECTION 8 TENANT BASED ASSISTANCE: HOUSING CHOICE VOUCHER PROGRAM Funding and PHA Application for Funding § 982.103 PHA application for funding. (a) a PHA must submit an application for...

Comparative life cycle assessment and financial analysis of mixed culture polyhydroxyalkanoate production.

PubMed

Gurieff, Nicholas; Lant, Paul

2007-12-01

A life cycle assessment and financial analysis of mixed culture PHA (PHA(MC)) and biogas production was undertaken based on treating an industrial wastewater. Internal rate of return (IRR) and non-renewable CO(2)eq emissions were used to quantify financial viability and environmental impact. PHA(MC) was preferable to biogas production for treating the specified industrial effluent. PHA(MC) was also financially attractive in comparison to pure culture PHA production. Both PHA production processes had similar environmental impacts that were significantly lower than HDPE production. A large potential for optimisation exists for the PHA(MC) process as financial and environmental costs were primarily due to energy use for downstream processing. Under the conditions used in this work PHA(MC) was shown to be a viable biopolymer production process and an effective industrial wastewater treatment technology. This is the first study of its kind and provides valuable insight into the PHA(MC) process.

New PHA products using unrelated carbon sources

PubMed Central

Matias, Fernanda; de Andrade Rodrigues, Maria Filomena

2011-01-01

Polyhydroxyalkanoates (PHA) are natural polyesters stored by a wide range of bacteria as carbon source reserve. Due to its chemical characteristics and biodegradability PHA can be used in chemical, medical and pharmaceutical industry for many human purposes. Over the past years, few Burkholderia species have become known for production of PHA. Aside from that, these bacteria seem to be interesting for discovering new PHA compositions which is important to different industrial applications. In this paper, we introduce two new strains which belong either to Burkholderia cepacia complex (Bcc) or genomovar-type, Burkholderia cepacia SA3J and Burkholderia contaminans I29B, both PHA producers from unrelated carbon sources. The classification was based on 16S rDNA and recA partial sequence genes and cell wall fatty acids composition. These two strains were capable to produce different types of PHA monomers or precursors. Unrelated carbon sources were used for growth and PHA accumulation. The amount of carbon source evaluated, or mixtures of them, was increased with every new experiment until it reaches eighteen carbon sources. As first bioprospection experiments staining methods were used with colony fluorescent dye Nile Red and the cell fluorescent dye Nile Blue A. Gas chromatography analysis coupled to mass spectrometry was used to evaluate the PHA composition on each strain cultivated on different carbon sources. The synthesized polymers were composed by short chain length-PHA (scl-PHA), especially polyhydroxybutyrate, and medium chain length-PHA (mcl-PHA) depending on the carbon source used. PMID:24031764

A novel biological recovery approach for PHA employing selective digestion of bacterial biomass in animals.

PubMed

Ong, Su Yean; Zainab-L, Idris; Pyary, Somarajan; Sudesh, Kumar

2018-03-01

Polyhydroxyalkanoate (PHA) is a family of microbial polyesters that is completely biodegradable and possesses the mechanical and thermal properties of some commonly used petrochemical-based plastics. Therefore, PHA is attractive as a biodegradable thermoplastic. It has always been a challenge to commercialize PHA due to the high cost involved in the biosynthesis of PHA via bacterial fermentation and the subsequent purification of the synthesized PHA from bacterial cells. Innovative enterprise by researchers from various disciplines over several decades successfully reduced the cost of PHA production through the efficient use of cheap and renewable feedstock, precisely controlled fermentation process, and customized bacterial strains. Despite the fact that PHA yields have been improved tremendously, the recovery and purification processes of PHA from bacterial cells remain exhaustive and require large amounts of water and high energy input besides some chemicals. In addition, the residual cell biomass ends up as waste that needs to be treated. We have found that some animals can readily feed on the dried bacterial cells that contain PHA granules. The digestive system of the animals is able to assimilate the bacterial cells but not the PHA granules which are excreted in the form of fecal pellets, thus resulting in partial recovery and purification of PHA. In this mini-review, we will discuss this new concept of biological recovery, the selection of the animal model for biological recovery, and the properties and possible applications of the biologically recovered PHA.

Metabolic shift of polyphosphate-accumulating organisms with different levels of polyphosphate storage.

PubMed

Acevedo, B; Oehmen, A; Carvalho, G; Seco, A; Borrás, L; Barat, R

2012-04-15

Previous studies have shown that polyphosphate-accumulating organisms (PAOs) are able to behave as glycogen-accumulating organisms (GAOs) under different conditions. In this study we investigated the behavior of a culture enriched with Accumulibacter at different levels of polyphosphate (poly-P) storage. The results of stoichiometric ratios Gly(degraded)/HAc(uptake), PHB(synthesized)/HAc(uptake), PHV(synthesized)/HAc(uptake) and P(release)/HAc(uptake) confirmed a metabolic shift from PAO metabolism to GAO metabolism: PAOs with high poly-P content used the poly-P to obtain adenosine tri-phosphate (ATP), and glycogen (Gly) to obtain nicotinamide adenine dinucleotide (NADH) and some ATP. In a test where poly-P depletion was imposed on the culture, all the acetate (HAc) added in each cycle was transformed into polyhydroxyalkanoate (PHA) despite the decrease of poly-P inside the cells. This led to an increase of the Gly(degraded)/HAc(uptake) ratio that resulted from a shift towards the glycolytic pathway in order to compensate for the lack of ATP formed from poly-P hydrolysis. The shift from PAO to GAO metabolism was also reflected in the change in the PHA composition as the poly-P availability decreased, suggesting that polyhydroxyvalerate (PHV) is obtained due to the consumption of excess reducing equivalents to balance the internal NADH, similarly to GAO metabolism. Fluorescence in situ hybridization analysis showed a significant PAO population change from Type I to Type II Accumulibacter as the poly-P availability decreased in short term experiments. This work suggests that poly-P storage levels and GAO-like metabolism are important factors affecting the competition between different PAO Types in enhanced biological phosphorus removal systems. Copyright © 2012 Elsevier Ltd. All rights reserved.

The improvement of fibroblast growth on hydrophobic biopolyesters by coating with polyhydroxyalkanoate granule binding protein PhaP fused with cell adhesion motif RGD.

PubMed

Dong, Ying; Li, Ping; Chen, Chong-bo; Wang, Zhi-hui; Ma, Ping; Chen, Guo-Qiang

2010-12-01

Polyhydroxyalkanoates (PHA), a family of biopolyesters, have been studied as tissue engineering biomaterials due to their adjustable mechanical properties, biodegradability and tissue compatibility. Amphiphilic PHA granule binding protein PhaP has been shown to be able to bind to hydrophobic surfaces of polymers, especially PHA, via strong hydrophobic interaction. Genes of PhaP and RGD peptides, which are a cell adhesion motif recognized by many cell surface receptors, were successfully expressed and obtained as a pure fusion protein PhaP-RGD in Escherichia coli DH5α. When films of poly(3-hydroxybutyrate-co-3-hydroxy- hexanoate) (PHBHHx), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and polylactic acid (PLA) were coated with PhaP-RGD, their surface hydrophilicities were all increased compared with their corresponding naked (non-coated) films, respectively. Among the three biopolyesters, PHBHHx demonstrated the strongest affinity to PhaP. In vitro study showed that mouse fibroblasts L929 and mouse embryonic fibroblasts NIH/3T3 attached better and grew faster on all three PhaP-RGD coated films compared with their related behaviors on PhaP coated and non-coated films, respectively. Both fibroblasts attached and grew very well on PhaP-RGD coated PHBHHx, PHBV and PLA, even in their serum-free medium, while the non-coated and PhaP coated biopolyesters poorly supported the cell growth if the two fibroblasts were incubated in their serum free medium. These results indicated that PhaP-RGD could be used as a coating material to improve cell growth on hydrophobic biopolyesters for implant tissue engineering purposes. Copyright © 2010 Elsevier Ltd. All rights reserved.

Effects of carbon sources on the enrichment of halophilic polyhydroxyalkanoate-storing mixed microbial culture in an aerobic dynamic feeding process

PubMed Central

Cui, You-Wei; Zhang, Hong-Yu; Lu, Peng-Fei; Peng, Yong-Zhen

2016-01-01

Microbial polyhydroxyalkanoate (PHA) production serves as a substitute for petroleum-based plastics. Enriching mixed microbial cultures (MMCs) with the capacity to store PHA is a key precursor for low-cost PHA production. This study investigated the impact of carbon types on enrichment outcomes. Three MMCs were separately fed by acetate sodium, glucose, and starch as an enriching carbon source, and were exposed to long-term aerobic dynamic feeding (ADF) periods. The PHA production capacity, kinetics and stoichiometry of the enrichments, the PHA composition, and the microbial diversity and community composition were explored to determine carbon and enrichment correlations. After 350-cycle enriching periods under feast-famine (F-F) regimes, the MMCs enriched by acetate sodium and glucose contained a maximum PHA content of 64.7% and 60.5% cell dry weight (CDW). The starch-enriched MMC only had 27.3% CDW of PHA. High-throughput sequencing revealed that non-PHA bacteria survived alongside PHA storing bacteria, even under severe F-F selective pressure. Genus of Pseudomonas and Stappia were the possible PHA accumulating bacteria in acetate-enriched MMC. Genus of Oceanicella, Piscicoccus and Vibrio were found as PHA accumulating bacteria in glucose-enriched MMC. Vibrio genus was the only PHA accumulating bacteria in starch-enriched MMC. The community diversity and composition were regulated by the substrate types. PMID:27485896

Molecular Diagnostic for Prospecting Polyhydroxyalkanoate-Producing Bacteria.

PubMed

Montenegro, Eduarda Morgana da Silva; Delabary, Gabriela Scholante; Silva, Marcus Adonai Castro da; Andreote, Fernando Dini; Lima, André Oliveira de Souza

2017-05-25

The use of molecular diagnostic techniques for bioprospecting and microbial diversity study purposes has gained more attention thanks to their functionality, low cost and quick results. In this context, ten degenerate primers were designed for the amplification of polyhydroxyalkanoate synthase ( phaC ) gene, which is involved in the production of polyhydroxyalkanoate (PHA)-a biodegradable, renewable biopolymer. Primers were designed based on multiple alignments of phaC gene sequences from 218 species that have their genomes already analyzed and deposited at Biocyc databank. The combination of oligos phaCF3/phaCR1 allowed the amplification of the expected product (PHA synthases families types I and IV) from reference organisms used as positive control (PHA producer). The method was also tested in a multiplex system with two combinations of initiators, using 16 colonies of marine bacteria (pre-characterized for PHA production) as a DNA template. All amplicon positive organisms ( n = 9) were also PHA producers, thus no false positives were observed. Amplified DNA was sequenced ( n = 4), allowing for the confirmation of the pha C gene identity as well its diversity among marine bacteria. Primers were also tested for screening purposes using 37 colonies from six different environments. Almost 30% of the organisms presented the target amplicon. Thus, the proposed primers are an efficient tool for screening bacteria with potential for the production of PHA as well to study PHA genetic diversity.

Analysis of polyhydroxyalkanoate (PHA) synthase gene and PHA-producing bacteria in activated sludge that produces PHA containing 3-hydroxydodecanoate.

PubMed

Yang, Chao; Zhang, Wei; Liu, Ruihua; Zhang, Chi; Gong, Ting; Li, Qiang; Wang, Shufang; Song, Cunjiang

2013-09-01

Activated sludge is an alternative to pure cultures for polyhydroxyalkanoate (PHA) production due to the presence of many PHA-producing bacteria in activated sludge community. In this study, activated sludge was submitted to aerobic dynamic feeding in a sequencing batch reactor. During domestication, the changes of bacterial community structure were observed by terminal restriction fragment length polymorphism analysis. Furthermore, some potential PHA-producing bacteria, such as Thauera, Acinetobacter and Pseudomonas, were identified by denaturing gradient gel electrophoresis analysis. The constructed PHA synthase gene library was analyzed by DNA sequencing. Of the 80 phaC genes obtained, 76 belonged to the Class I PHA synthase, and four to the Class II PHA synthase. Gas chromatography-mass spectrometry analysis showed that PHA produced by activated sludge was composed of three types of monomers: 3-hydroxybutyrate, 3-hydroxyvalerate and 3-hydroxydodecanoate (3HDD). This is the first report of production of medium-chain-length PHAs (PHAMCL ) containing 3HDD by activated sludge. Further studies suggested that a Pseudomonas strain may play an important role in the production of PHAMCL containing 3HDD. Moreover, a Class II PHA synthase was found to have a correlation with the production of 3HDD-containing PHAMCL . © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Effects of carbon sources on the enrichment of halophilic polyhydroxyalkanoate-storing mixed microbial culture in an aerobic dynamic feeding process

NASA Astrophysics Data System (ADS)

Cui, You-Wei; Zhang, Hong-Yu; Lu, Peng-Fei; Peng, Yong-Zhen

2016-08-01

Microbial polyhydroxyalkanoate (PHA) production serves as a substitute for petroleum-based plastics. Enriching mixed microbial cultures (MMCs) with the capacity to store PHA is a key precursor for low-cost PHA production. This study investigated the impact of carbon types on enrichment outcomes. Three MMCs were separately fed by acetate sodium, glucose, and starch as an enriching carbon source, and were exposed to long-term aerobic dynamic feeding (ADF) periods. The PHA production capacity, kinetics and stoichiometry of the enrichments, the PHA composition, and the microbial diversity and community composition were explored to determine carbon and enrichment correlations. After 350-cycle enriching periods under feast-famine (F-F) regimes, the MMCs enriched by acetate sodium and glucose contained a maximum PHA content of 64.7% and 60.5% cell dry weight (CDW). The starch-enriched MMC only had 27.3% CDW of PHA. High-throughput sequencing revealed that non-PHA bacteria survived alongside PHA storing bacteria, even under severe F-F selective pressure. Genus of Pseudomonas and Stappia were the possible PHA accumulating bacteria in acetate-enriched MMC. Genus of Oceanicella, Piscicoccus and Vibrio were found as PHA accumulating bacteria in glucose-enriched MMC. Vibrio genus was the only PHA accumulating bacteria in starch-enriched MMC. The community diversity and composition were regulated by the substrate types.

Effects of carbon sources on the enrichment of halophilic polyhydroxyalkanoate-storing mixed microbial culture in an aerobic dynamic feeding process.

PubMed

Cui, You-Wei; Zhang, Hong-Yu; Lu, Peng-Fei; Peng, Yong-Zhen

2016-08-03

Microbial polyhydroxyalkanoate (PHA) production serves as a substitute for petroleum-based plastics. Enriching mixed microbial cultures (MMCs) with the capacity to store PHA is a key precursor for low-cost PHA production. This study investigated the impact of carbon types on enrichment outcomes. Three MMCs were separately fed by acetate sodium, glucose, and starch as an enriching carbon source, and were exposed to long-term aerobic dynamic feeding (ADF) periods. The PHA production capacity, kinetics and stoichiometry of the enrichments, the PHA composition, and the microbial diversity and community composition were explored to determine carbon and enrichment correlations. After 350-cycle enriching periods under feast-famine (F-F) regimes, the MMCs enriched by acetate sodium and glucose contained a maximum PHA content of 64.7% and 60.5% cell dry weight (CDW). The starch-enriched MMC only had 27.3% CDW of PHA. High-throughput sequencing revealed that non-PHA bacteria survived alongside PHA storing bacteria, even under severe F-F selective pressure. Genus of Pseudomonas and Stappia were the possible PHA accumulating bacteria in acetate-enriched MMC. Genus of Oceanicella, Piscicoccus and Vibrio were found as PHA accumulating bacteria in glucose-enriched MMC. Vibrio genus was the only PHA accumulating bacteria in starch-enriched MMC. The community diversity and composition were regulated by the substrate types.

Mixed culture polyhydroxyalkanoate (PHA) synthesis from nutrient rich wet oxidation liquors.

PubMed

Wijeyekoon, Suren; Carere, Carlo R; West, Mark; Nath, Shresta; Gapes, Daniel

2018-09-01

Organic waste residues can be hydrothermally treated to produce organic acid rich liquors. These hydrothermal liquors are a potential feedstock for polyhydroxyalkanoate (PHA) production. We investigated the effect of dissolved oxygen concentration and substrate feeding regimes on PHA accumulation and yield using two hydrothermal liquors derived from a mixture of primary and secondary municipal wastewater treatment sludge and food waste. The enriched culture accumulated a maximum of 41% PHA of cell dry weight within 7 h; which is among the highest reported for N and P rich hydrothermal liquors. Recovered PHA was 77% polyhydroxybutyrate and 23% polyhydroxyvalerate by mass. The families Rhodocyclaceae (84%) and Saprospiraceae (20.5%) were the dominant Proteobacteria (73%) in the enriched culture. The third most abundant bacterial genus, Bdellovibrio, includes species of known predators of PHA producers which may lead to suboptimal PHA accumulation. The PHA yield was directly proportional to DO concentration for ammonia stripped liquor (ASL) and inversely proportional to DO concentration for low strength liquor (LSL). The highest yield of 0.50 Cmol PHA/Cmol substrate was obtained for ASL at 25% DO saturation. A progressively increasing substrate feeding regime resulted in increased PHA yields. These findings demonstrate that substrate feeding regime and oxygen concentration can be used to control the PHA yield and accumulation rate thereby enhancing PHA production viability from nutrient rich biomass streams. Copyright © 2018 Elsevier Ltd. All rights reserved.

Molecular Diagnostic for Prospecting Polyhydroxyalkanoate-Producing Bacteria

PubMed Central

Montenegro, Eduarda Morgana da Silva; Delabary, Gabriela Scholante; da Silva, Marcus Adonai Castro; Andreote, Fernando Dini; Lima, André Oliveira de Souza

2017-01-01

The use of molecular diagnostic techniques for bioprospecting and microbial diversity study purposes has gained more attention thanks to their functionality, low cost and quick results. In this context, ten degenerate primers were designed for the amplification of polyhydroxyalkanoate synthase (phaC) gene, which is involved in the production of polyhydroxyalkanoate (PHA)—a biodegradable, renewable biopolymer. Primers were designed based on multiple alignments of phaC gene sequences from 218 species that have their genomes already analyzed and deposited at Biocyc databank. The combination of oligos phaCF3/phaCR1 allowed the amplification of the expected product (PHA synthases families types I and IV) from reference organisms used as positive control (PHA producer). The method was also tested in a multiplex system with two combinations of initiators, using 16 colonies of marine bacteria (pre-characterized for PHA production) as a DNA template. All amplicon positive organisms (n = 9) were also PHA producers, thus no false positives were observed. Amplified DNA was sequenced (n = 4), allowing for the confirmation of the phaC gene identity as well its diversity among marine bacteria. Primers were also tested for screening purposes using 37 colonies from six different environments. Almost 30% of the organisms presented the target amplicon. Thus, the proposed primers are an efficient tool for screening bacteria with potential for the production of PHA as well to study PHA genetic diversity. PMID:28952531

Structure of polyhydroxyalkanoate (PHA) synthase PhaC from Chromobacterium sp. USM2, producing biodegradable plastics.

PubMed

Chek, Min Fey; Kim, Sun-Yong; Mori, Tomoyuki; Arsad, Hasni; Samian, Mohammed Razip; Sudesh, Kumar; Hakoshima, Toshio

2017-07-13

Polyhydroxyalkanoate (PHA) is a promising candidate for use as an alternative bioplastic to replace petroleum-based plastics. Our understanding of PHA synthase PhaC is poor due to the paucity of available three-dimensional structural information. Here we present a high-resolution crystal structure of the catalytic domain of PhaC from Chromobacterium sp. USM2, PhaC Cs -CAT. The structure shows that PhaC Cs -CAT forms an α/β hydrolase fold comprising α/β core and CAP subdomains. The active site containing Cys291, Asp447 and His477 is located at the bottom of the cavity, which is filled with water molecules and is covered by the partly disordered CAP subdomain. We designated our structure as the closed form, which is distinct from the recently reported catalytic domain from Cupriavidus necator (PhaC Cn -CAT). Structural comparison showed PhaC Cn -CAT adopting a partially open form maintaining a narrow substrate access channel to the active site, but no product egress. PhaC Cs -CAT forms a face-to-face dimer mediated by the CAP subdomains. This arrangement of the dimer is also distinct from that of the PhaC Cn -CAT dimer. These findings suggest that the CAP subdomain should undergo a conformational change during catalytic activity that involves rearrangement of the dimer to facilitate substrate entry and product formation and egress from the active site.

24 CFR 968.330 - PHA performance and evaluation report.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false PHA performance and evaluation... 250 or More Public Housing Units) § 968.330 PHA performance and evaluation report. For any FFY in which a PHA has received assistance under this subpart, the PHA shall submit a Performance and...

Mechanical properties, biocompatibility, and biodegradation of cross-linked cellulose acetate-reinforced polyester composites.

PubMed

Wu, Chin-San

2014-05-25

Composites of treated (cross-linked) cellulose acetate (t-CA) and acrylic acid-grafted poly(hydroxyalkanoate) (PHA-g-AA/t-CA) exhibited noticeably superior mechanical properties compared with PHA/CA composites due to greater compatibility between the two components. The dispersion covering of t-CA in the PHA-g-AA matrix was highly homogeneous as a result of condensation reactions. Human lung fibroblasts (FBs) were seeded on these two series of composites to characterize the biocompatibility properties. In a time-dependent course, the FB proliferation results demonstrated higher performance from the PHA/CA series of composites than from the PHA-g-AA/t-CA composites. The water resistance of PHA-g-AA/t-CA was higher than that of PHA/CA, although the weight loss of both composites buried in Acetobacter pasteurianus (A. pasteurianus) indicated that they were both biodegradable, especially at higher levels of cellulose acetate substitution. The PHA/CA and PHA-g-AA/t-CA composites were more biodegradable than pure PHA, implying a strong connection between cellulose acetate content and biodegradability. Copyright © 2014 Elsevier Ltd. All rights reserved.

Metabolic engineering for microbial production and applications of copolyesters consisting of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates.

PubMed

Zou, Xiang Hui; Chen, Guo-Qiang

2007-02-12

Poly(hydroxyalkanoate)s (PHAs) are a class of microbially synthesized polyesters that combine biological properties, such as biocompatibility and biodegradability, and non-bioproperties such as thermoprocessability, piezoelectricity, and nonlinear optical activity. PHA monomer structures and their contents strongly affect the PHA properties. Using metabolic engineering approaches, PHA structures and contents can be manipulated to achieve controllable monomer and PHA cellular contents. This paper focuses on metabolic engineering methods to produce PHA consisting of 3-hydroxybutyrate (3HB) and medium-chain-length 3-hydroxyalkanoates (3HA) in recombinant microbial systems. This type of copolyester has mechanical and thermal properties similar to conventional plastics such as poly(propylene) and poly(ethylene terephthalate) (PET). In addition, pathways containing engineered PHA synthases have proven to be useful for enhanced PHA production with adjustable PHA monomers and contents. The applications of PHA as implant biomaterials are briefly discussed here. In the very near term, metabolic engineering will help solve many problems in promoting PHA as a new type of plastic material for many applications.

A new biological recovery approach for PHA using mealworm, Tenebrio molitor.

PubMed

Murugan, Paramasivam; Han, Lizhu; Gan, Chee-Yuen; Maurer, Frans H J; Sudesh, Kumar

2016-12-10

Bacterial polyhydroxyalkanoates (PHA) are expensive partly due to the recovery and purification processes. Thus, many studies have been carried out in order to minimize the cost. Here we report on the use of mealworm, which is the larva of mealworm beetle (Tenebrio molitor) to recover PHA granules from Cupriavidus necator. Mealworms were shown to readily consume the freeze-dried C. necator cells and excrete the PHA granules in the form of whitish feces. Further purification using water, detergent and heat resulted in almost 100% pure PHA granules. Comparison with chloroform extraction showed no signs of reduction in the molecular weight and dispersion of the PHA molecules. Scanning electron microscopy and dynamic light scattering measurements revealed that the biologically recovered PHA granules retained their native spherical morphology. The PHA granules were subjected to a battery of tests to determine their purity and properties in comparison to the chloroform extracted PHA. This study has demonstrated the possibility of using mealworms as a biological agent to partially purify the PHA granules. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular weight-dependent degradation of D-lactate-containing polyesters by polyhydroxyalkanoate depolymerases from Variovorax sp. C34 and Alcaligenes faecalis T1.

PubMed

Sun, Jian; Matsumoto, Ken'ichiro; Tabata, Yuta; Kadoya, Ryosuke; Ooi, Toshihiko; Abe, Hideki; Taguchi, Seiichi

2015-11-01

Polyhydroxyalkanoate depolymerase derived from Variovorax sp. C34 (PhaZVs) was identified as the first enzyme that is capable of degrading isotactic P[67 mol% (R)-lactate(LA)-co-(R)-3-hydroxybutyrate(3HB)] [P(D-LA-co-D-3HB)]. This study aimed at analyzing the monomer sequence specificity of PhaZVs for hydrolyzing P(LA-co-3HB) in comparison with a P(3HB) depolymerase from Alcaligenes faecalis T1 (PhaZAf) that did not degrade the same copolymer. Degradation of P(LA-co-3HB) by action of PhaZVs generated dimers, 3HB-3HB, 3HB-LA, LA-3HB, and LA-LA, and the monomers, suggesting that PhaZVs cleaved the linkages between LA and 3HB units and between LA units. To provide a direct evidence for the hydrolysis of these sequences, the synthetic methyl trimers, 3HB-3HB-3HB, LA-LA-3HB, LA-3HB-LA, and 3HB-LA-LA, were treated with the PhaZs. Unexpectedly, not only PhaZVs but also PhaZAf hydrolyzed all of these substrates, namely PhaZAf also cleaved LA-LA linkage. Considering the fact that both PhaZs did not degrade P[(R)-LA] (PDLA) homopolymer, the cleavage capability of LA-LA linkage by PhaZs was supposed to depend on the length of the LA-clustering region in the polymer chain. To test this hypothesis, PDLA oligomers (6 to 40 mer) were subjected to the PhaZ assay, revealing that there was an inverse relationship between molecular weight of the substrates and their hydrolysis efficiency. Moreover, PhaZVs exhibited the degrading activity toward significantly longer PDLA oligomers compared to PhaZAf. Therefore, the cleaving capability of PhaZs used here toward the D-LA-based polymers containing the LA-clustering region was strongly associated with the substrate length, rather than the monomer sequence specificity of the enzyme.

A common active site of polyhydroxyalkanoate synthase from Bacillus cereus YB-4 is involved in polymerization and alcoholysis reactions.

PubMed

Hyakutake, Manami; Tomizawa, Satoshi; Mizuno, Kouhei; Hisano, Tamao; Abe, Hideki; Tsuge, Takeharu

2015-06-01

Polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRCYB4) catalyzes not only PHA polymerization but also alcoholytic cleavage of PHA chains. The alcoholysis activity of PhaRCYB4 is expressed when a hydroxyacyl-CoA monomer is absent but an alcohol compound is present. In this study, we performed alanine mutagenesis of the putative catalytic triad (Cys(151), Asp(306), and His(335)) in the PhaCYB4 subunit to identify the active site residues for polymerization and alcoholysis activities. Individual substitution of each triad residue with alanine resulted in loss of both polymerization and alcoholysis activities, suggesting that these residues are commonly shared between polymerization and alcoholysis reactions. The loss of activity was also observed following mutagenesis of the triad to other amino acids, except for one PhaRCYB4 mutant with a C151S substitution, which lost polymerization activity but still possessed cleavage activity towards PHA chains. The low-molecular-weight PHA isolated from the PhaRCYB4(C151S)-expressing strain showed a lower ratio of alcohol capping at the P(3HB) carboxy terminus than did that from the wild-type-expressing strain. This observation implies that hydrolysis activity of PhaRCYB4 might be elicited by the C151S mutation.

24 CFR 941.205 - PHA contracts.

Code of Federal Regulations, 2011 CFR

2011-04-01

... PUBLIC HOUSING DEVELOPMENT PHA Eligibility and Program Requirements § 941.205 PHA contracts. (a) ACC... into by the PHA shall provide for compliance with the provisions of the ACC. (b) Contract forms. HUD...

24 CFR 941.205 - PHA contracts.

Code of Federal Regulations, 2010 CFR

2010-04-01

... PUBLIC HOUSING DEVELOPMENT PHA Eligibility and Program Requirements § 941.205 PHA contracts. (a) ACC... into by the PHA shall provide for compliance with the provisions of the ACC. (b) Contract forms. HUD...

Natural and engineered polyhydroxyalkanoate (PHA) synthase: key enzyme in biopolyester production.

PubMed

Zou, Huibin; Shi, Mengxun; Zhang, Tongtong; Li, Lei; Li, Liangzhi; Xian, Mo

2017-10-01

With the finite supply of petroleum and increasing concern with environmental issues associated with their harvest and processing, the development of more eco-friendly, sustainable alternative biopolymers that can effectively fill the role of petro-polymers has become a major focus. Polyhydroxyalkanoate (PHA) can be naturally produced by many species of bacteria and the PHA synthase is believed to be key enzyme in this natural pathway. Natural PHA synthases are diverse and can affect the properties of the produced PHAs, such as monomer composition, molecular weights, and material properties. Moreover, recent studies have led to major advances in the searching of PHA synthases that display specific properties, as well as engineering efforts that offer more efficient PHA synthases, increased PHA compound production, or even novel biopolyesters which cannot be naturally produced. In this article, we review the updated information of natural PHA synthases and their engineering strategies for improved performance in polyester production. We also speculate future trends on the development of robust PHA synthases and their application in biopolyester production.

Polyhydroxyalkanoate (PHA) accumulation potential and PHA-accumulating microbial communities in various activated sludge processes of municipal wastewater treatment plants.

PubMed

Sakai, K; Miyake, S; Iwama, K; Inoue, D; Soda, S; Ike, M

2015-01-01

To clarify the polyhydroxyalkanoate (PHA) accumulation potential and the PHA-accumulating microbial community structure in activated sludge in municipal wastewater treatment plants (WWTPs) and to identify their influential factors. Nine activated sludge samples were collected from municipal WWTPs employing various biological treatment processes. In acetate-fed 24-h batch experiments under aerobic and nitrogen- and phosphorus-limited conditions, polyhydroxybutyrate (PHB) content of activated sludge increased from 0-1·3 wt% to 7·9-24 wt%, with PHB yields of 0·22-0·50 C-mol 3-hydroxybutyrate (C-mol acetate)(-1). Microbial community analyses found that activated sludge samples that accumulated >20 wt% of PHB after 24-h PHA accumulation experiments had >5·0 × 10(8) copies g(-1)-mixed liquor-suspended solid of phaC genes. Results indicated that (i) activated sludge in municipal WWTPs can accumulate up to approx. 20 wt% of PHA without enrichment processes, (ii) PHA accumulation potential of activated sludge varied depending on the operational conditions (treatment processes) of WWTPs, and (iii) phaC gene number can provide a simple indication of PHA accumulation potential. This is the first study to compare the PHA accumulation potential and PHA-accumulating microbial communities in activated sludge of various treatment processes. Our findings may be useful for enhancing the resource recovery potential of wastewater treatment systems. © 2014 The Society for Applied Microbiology.

The plasma membrane H+-ATPase gene family in Solanum tuberosum L. Role of PHA1 in tuberization.

PubMed

Stritzler, Margarita; Muñiz García, María Noelia; Schlesinger, Mariana; Cortelezzi, Juan Ignacio; Capiati, Daniela Andrea

2017-10-13

This study presents the characterization of the plasma membrane (PM) H+-ATPases in potato, focusing on their role in stolon and tuber development. Seven PM H+-ATPase genes were identified in the Solanum tuberosum genome, designated PHA1-PHA7. PHA genes show distinct expression patterns in different plant tissues and under different stress treatments. Application of PM H+-ATPase inhibitors arrests stolon growth, promotes tuber induction, and reduces tuber size, indicating that PM H+-ATPases are involved in tuberization, acting at different stages of the process. Transgenic potato plants overexpressing PHA1 were generated (PHA1-OE). At early developmental stages, PHA1-OE stolons elongate faster and show longer epidermal cells than wild-type stolons; this accelerated growth is accompanied by higher cell wall invertase activity, lower starch content, and higher expression of the sucrose-H+ symporter gene StSUT1. PHA1-OE stolons display an increased branching phenotype and develop larger tubers. PHA1-OE plants are taller and also present a highly branched phenotype. These results reveal a prominent role for PHA1 in plant growth and development. Regarding tuberization, PHA1 promotes stolon elongation at early stages, and tuber growth later on. PHA1 is involved in the sucrose-starch metabolism in stolons, possibly providing the driving force for sugar transporters to maintain the apoplastic sucrose transport during elongation. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Regulation of Polyhydroxybutyrate Synthesis in the Soil Bacterium Bradyrhizobium diazoefficiens

PubMed Central

Quelas, J. I.; Mesa, S.; Mongiardini, E. J.; Jendrossek, D.

2016-01-01

ABSTRACT Polyhydroxybutyrate (PHB) is a carbon and energy reserve polymer in various prokaryotic species. We determined that, when grown with mannitol as the sole carbon source, Bradyrhizobium diazoefficiens produces a homopolymer composed only of 3-hydroxybutyrate units (PHB). Conditions of oxygen limitation (such as microoxia, oxic stationary phase, and bacteroids inside legume nodules) were permissive for the synthesis of PHB, which was observed as cytoplasmic granules. To study the regulation of PHB synthesis, we generated mutations in the regulator gene phaR and the phasin genes phaP1 and phaP4. Under permissive conditions, mutation of phaR impaired PHB accumulation, and a phaP1 phaP4 double mutant produced more PHB than the wild type, which was accumulated in a single, large cytoplasmic granule. Moreover, PhaR negatively regulated the expression of phaP1 and phaP4 as well as the expression of phaA1 and phaA2 (encoding a 3-ketoacyl coenzyme A [CoA] thiolases), phaC1 and phaC2 (encoding PHB synthases), and fixK2 (encoding a cyclic AMP receptor protein [CRP]/fumarate and nitrate reductase regulator [FNR]-type transcription factor of genes for microoxic lifestyle). In addition to the depressed PHB cycling, phaR mutants accumulated more extracellular polysaccharides and promoted higher plant shoot dry weight and competitiveness for nodulation than the wild type, in contrast to the phaC1 mutant strain, which is defective in PHB synthesis. These results suggest that phaR not only regulates PHB granule formation by controlling the expression of phasins and biosynthetic enzymes but also acts as a global regulator of excess carbon allocation and symbiosis by controlling fixK2. IMPORTANCE In this work, we investigated the regulation of polyhydroxybutyrate synthesis in the soybean-nodulating bacterium Bradyrhizobium diazoefficiens and its influence in bacterial free-living and symbiotic lifestyles. We uncovered a new interplay between the synthesis of this carbon reserve polymer and the network responsible for microoxic metabolism through the interaction between the gene regulators phaR and fixK2. These results contribute to the understanding of the physiological conditions required for polyhydroxybutyrate biosynthesis. The interaction between these two main metabolic pathways is also reflected in the symbiotic phenotypes of soybeans inoculated with phaR mutants, which were more competitive for nodulation and enhanced dry matter production by the plants. Therefore, this knowledge may be applied to the development of superior strains to be used as improved inoculants for soybean crops. PMID:27208130

24 CFR 982.629 - Homeownership option: Additional PHA requirements for family search and purchase.

Code of Federal Regulations, 2014 CFR

2014-04-01

... PHA requirements for family search and purchase. 982.629 Section 982.629 Housing and Urban Development...: Additional PHA requirements for family search and purchase. (a) The PHA may establish the maximum time for a family to locate a home, and to purchase the home. (b) The PHA may require periodic family reports on the...

24 CFR 982.629 - Homeownership option: Additional PHA requirements for family search and purchase.

Code of Federal Regulations, 2010 CFR

2010-04-01

... PHA requirements for family search and purchase. 982.629 Section 982.629 Housing and Urban Development...: Additional PHA requirements for family search and purchase. (a) The PHA may establish the maximum time for a family to locate a home, and to purchase the home. (b) The PHA may require periodic family reports on the...

24 CFR 982.629 - Homeownership option: Additional PHA requirements for family search and purchase.

Code of Federal Regulations, 2011 CFR

2011-04-01

... PHA requirements for family search and purchase. 982.629 Section 982.629 Housing and Urban Development...: Additional PHA requirements for family search and purchase. (a) The PHA may establish the maximum time for a family to locate a home, and to purchase the home. (b) The PHA may require periodic family reports on the...

24 CFR 982.629 - Homeownership option: Additional PHA requirements for family search and purchase.

Code of Federal Regulations, 2012 CFR

2012-04-01

... PHA requirements for family search and purchase. 982.629 Section 982.629 Housing and Urban Development...: Additional PHA requirements for family search and purchase. (a) The PHA may establish the maximum time for a family to locate a home, and to purchase the home. (b) The PHA may require periodic family reports on the...

24 CFR 982.629 - Homeownership option: Additional PHA requirements for family search and purchase.

Code of Federal Regulations, 2013 CFR

2013-04-01

... PHA requirements for family search and purchase. 982.629 Section 982.629 Housing and Urban Development...: Additional PHA requirements for family search and purchase. (a) The PHA may establish the maximum time for a family to locate a home, and to purchase the home. (b) The PHA may require periodic family reports on the...

Polyhydroxyalkanoate production potential of heterotrophic bacteria in activated sludge.

PubMed

Inoue, Daisuke; Suzuki, Yuta; Uchida, Takahiro; Morohoshi, Jota; Sei, Kazunari

2016-01-01

This study was conducted to evaluate the polyhydroxyalkanoate (PHA) production potential of cultivable heterotrophic bacteria in activated sludge by genotypic and phenotypic characterizations. A total of 114 bacterial strains were isolated from four activated sludge samples taken from a lab-scale sequencing batch reactor and three wastewater treatment processes of two municipal wastewater treatment plants. PCR detection of the phaC genes encoding class I and II PHA synthase revealed that 15% of the total isolates possessed phaC genes, all of which had the closest similarities to known phaC genes of α- and β-Proteobacteria and Actinobacteria. PHA production experiments under aerobic and nitrogen-limited conditions showed that 68% of the total isolates were capable of producing PHA from at least one of the six substrates used (acetate, propionate, lactate, butyrate, glucose and glycerol). Genotypic and phenotypic characterizations revealed that 75% of the activated sludge bacteria had PHA production potential. Our results also indicated that short-chain fatty acids would be the preferable substrates for PHA production by activated sludge bacteria, and that there might be a variety of unidentified phaC genes in activated sludge. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Polyhydroxyalkanoate production by a novel bacterium Massilia sp. UMI-21 isolated from seaweed, and molecular cloning of its polyhydroxyalkanoate synthase gene.

PubMed

Han, Xuerong; Satoh, Yasuharu; Kuriki, Yumi; Seino, Teruyuki; Fujita, Shinji; Suda, Takanori; Kobayashi, Takanori; Tajima, Kenji

2014-11-01

We successfully isolated one microorganism (UMI-21) from Ulva, a green algae that contains starch. The strain UMI-21 can produce polyhydroxyalkanoate (PHA) from starch, maltotriose, or maltose as a sole carbon source. Taxonomic studies and 16S rDNA sequence analysis revealed that strain UMI-21 was phylogenetically related to species of the genus Massilia. The PHA content under the cultivation condition using a 10-L jar fermentor was 45.5% (w/w). This value was higher than that obtained after cultivation in a flask, suggesting the possibility of large-scale PHA production by UMI-21 from starch. A major issue for the industrial production of microbial PHAs is the very high production cost. Starch is a relatively inexpensive substrate that is also found in abundant seaweeds such as Ulva. Therefore, the strain isolated in this study may be very useful for producing PHA from seaweeds containing polysaccharides such as starch. In addition, a 3.7-kbp DNA fragment containing the whole PHA synthase gene (phaC) was obtained from the strain UMI-21. The results of open reading frame (ORF) analysis suggested that the DNA fragment contained two ORFs, which were composed of 1740 (phaC) and 564 bp (phaR). The deduced amino acid sequence of PhaC from strain UMI-21 shared high similarity with PhaC from Ralstonia eutropha, which is a representative PHA-producing bacterium with a class I PHA synthase. This is the first report for the cloning of the PHA synthase gene from Massilia species. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Identification of a new polyhydroxyalkanoate (PHA) producer Aquitalea sp. USM4 (JCM 19919) and characterization of its PHA synthase.

PubMed

Ng, Lee-Mei; Sudesh, Kumar

2016-11-01

Aquitalea sp. USM4 (JCM 19919) was isolated from a freshwater sample at Lata Iskandar Waterfall in Perak, Malaysia. It is a rod-shaped, gram-negative bacterium with high sequence identity (99%) to Aquitalea magnusonii based on 16S rRNA gene analysis. Aquitalea sp. USM4 also possessed a PHA synthase gene (phaC), which had amino acid sequence identity of 77-78% to the PHA synthase of Chromobacterium violaceum ATCC12472 and Pseudogulbenkiania sp. NH8B. PHA biosynthesis results showed that wild-type Aquitalea sp. USM4 was able to accumulate up to 1.5 g/L of poly(3-hydroxybutyrate), [P(3HB)]. The heterologous expression of the PHA synthase gene of Aquitalea sp. USM4 (phaC Aq ) in Cupriavidus necator PHB - 4 had resulted in PHA accumulation up to 3.2 g/L of P(3HB). It was further confirmed by 1 H nuclear magnetic resonance (NMR) analysis that Aquitalea sp. USM4 and C. necator PHB - 4 transformant were able to produce PHA containing 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB) and 3-hydroxy-4-methylvalerate (3H4MV) monomers from suitable precursor substrates. Interestingly, relatively high PHA synthase activity of 863 U/g and 1402 U/g were determined in wild-type Aquitalea sp. USM4 and C. necator PHB - 4 transformant respectively. This is the first report on the member of genus Aquitalea as a new PHA producer as well as in vitro and in vivo characterization of a novel PHA synthase from Aquitalea sp. USM4. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Variations in temperature acclimation effects on glycogen storage, hypoxia tolerance and swimming performance with seasonal acclimatization in juvenile Chinese crucian carp.

PubMed

Yang, Yang; Cao, Zhen-Dong; Fu, Shi-Jian

2015-07-01

The aim of this study was to test whether temperature acclimation (10 vs 20 °C) effects on tissue glycogen content, hypoxia tolerance, and swimming performance of Chinese crucian carp (Carassius auratus) varied with seasonal acclimatization (winter vs spring) and potential combined interactions. Both the routine metabolic rate (MO(2rout)) and critical oxygen tension (P(crit)) of the MO(2rout) increased significantly with temperature, whereas the seasonal acclimatization showed no significant effect. Only the high temperature group that acclimatized in spring showed a significantly higher aquatic surface respiration (ASR(crit)) value compared with the other three groups. Fish in spring tended to show ASR behavior at higher oxygen tension compared with those in winter, which might have been caused by a more active lifestyle. Time to show LOE prolonged by 25-34% under low temperature. Spring fish showed 20% shorter LOE duration at 10 °C, whereas the difference tended to vanish at 20 °C. Glycogen contents in both liver and muscle were higher in winter than spring. The liver and muscle glycogen content decreased by 5-42% after exposure to anoxic conditions, whereas the magnitude was much smaller in spring. When fish swam in normoxic conditions, fish in higher temperatures showed higher critical swimming speed (Ucrit) than low temperature (5.49 vs 3.74 BL s(-1) in winter and 4.27 vs 3.21 BL s(-1) in spring), whereas fish in winter also showed higher U(crit) than fish in spring for each temperature. However, when fish swam in hypoxic waters, fish in higher temperatures showed a more profound decrease (52-61%) in U(crit) compared to those in lower temperature (25-27%). Fish in lower temperatures that had acclimatized in winter showed the highest U(crit), which might have been caused by higher glycogen storage. The present study suggested that both glycogen storage and alterations in lifestyle had profound effects on hypoxia tolerance and swimming performance, which resulted in a profound difference between seasons and acclimation temperatures. Copyright © 2015 Elsevier Inc. All rights reserved.

Dynamics of polyhydroxyalkanoate accumulation in aerobic granules during the growth-disintegration cycle.

PubMed

Gobi, K; Vadivelu, V M

2015-11-01

The polyhydroxyalkanoate (PHA) accumulation dynamics in aerobic granules that undergo the growth-disintegration cycle were investigated. Four sequencing batch reactors (SBR) were inoculated with aerobic granules at different stages of development (different sizes). Different sizes of aerobic granules showed varying PHA contents. Thus, further study was conducted to investigate the diffusion of substrate and oxygen on PHA accumulation using various organic loading rates (OLR) and aeration rates (AR). An increase in OLR from 0.91 to 3.64kg COD/m(3)day increased the PHA content from 0.66 to 0.87g PHA/g CDW. Meanwhile, an AR increase from 1 to 4L/min only accelerated the maximum PHA accumulation without affecting the PHA content. However, the PHA composition only changes with AR, while the hydroxyvalerate (HV) content increased at a higher AR. Copyright © 2015 Elsevier Ltd. All rights reserved.

Polyhydroxyalkanoate recovery and effect of in situ extracellular polymeric substances removal from aerobic granules.

PubMed

Gobi, K; Vadivelu, V M

2015-01-01

Polyhydroxyalkanoate (PHA) recovery from aerobic granules was investigated using four cell digestion agents, namely, sodium hypochlorite, sodium hydroxide, acetone and sodium chloride. Simultaneously, the removal of extracellular polymeric substances (EPS) and its effect on PHA yield were investigated. The highest PHA recovery yield was obtained using sodium hypochlorite, accounting for 89% cell dry weight (CDW). The highest PHA was recovered after the sodium hypochlorite completely removed the EPS from the aerobic granules. The average molecular weight (Mw) of the PHA recovered using sodium hypochlorite was 5.31 × 10(5)g/mol with only 1.8% molecular weight degradation. The energy and duration analysis for PHA recovery revealed that the sodium hypochlorite method required the least amount of energy and time at 0.0561 MJ/g PHA and 26 h, respectively. The PHA that was recovered was a P3(HB-co-HV) co-polymer. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification and Biochemical Evidence of a Medium-Chain-Length Polyhydroxyalkanoate Depolymerase in the Bdellovibrio bacteriovorus Predatory Hydrolytic Arsenal

PubMed Central

Martínez, Virginia; de la Peña, Fernando; García-Hidalgo, Javier; de la Mata, Isabel; García, José Luis

2012-01-01

The obligate predator Bdellovibrio bacteriovorus HD100 shows a large set of proteases and other hydrolases as part of its hydrolytic arsenal needed for its predatory life cycle. We present genetic and biochemical evidence that open reading frame (ORF) Bd3709 of B. bacteriovorus HD100 encodes a novel medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase (PhaZBd). The primary structure of PhaZBd suggests that this enzyme belongs to the α/β-hydrolase fold family and has a typical serine hydrolase catalytic triad (serine-histidine-aspartic acid) in agreement with other PHA depolymerases and lipases. PhaZBd has been extracellularly produced using different hypersecretor Tol-pal mutants of Escherichia coli and Pseudomonas putida as recombinant hosts. The recombinant PhaZBd has been characterized, and its biochemical properties have been compared to those of other PHA depolymerases. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. It is also affected by the reducing agent dithiothreitol and nonionic detergents like Tween 80. PhaZBd is an endoexohydrolase that cleaves both large and small PHA molecules, producing mainly dimers but also monomers and trimers. The enzyme specifically degrades mcl-PHA and is inactive toward short-chain-length polyhydroxyalkanoates (scl-PHA) like polyhydroxybutyrate (PHB). These studies shed light on the potentiality of these predators as sources of new biocatalysts, such as an mcl-PHA depolymerase, for the production of enantiopure hydroxyalkanoic acids and oligomers as building blocks for the synthesis of biobased polymers. PMID:22706067

Cloning of the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes and use of these genes for enhanced production of Poly(3-hydroxybutyrate) in Escherichia coli.

PubMed

Choi, J I; Lee, S Y; Han, K

1998-12-01

Polyhydroxyalkanoates (PHAs) are microbial polyesters that can be used as completely biodegradable polymers, but the high production cost prevents their use in a wide range of applications. Recombinant Escherichia coli strains harboring the Ralstonia eutropha PHA biosynthesis genes have been reported to have several advantages as PHA producers compared with wild-type PHA-producing bacteria. However, the PHA productivity (amount of PHA produced per unit volume per unit time) obtained with these recombinant E. coli strains has been lower than that obtained with the wild-type bacterium Alcaligenes latus. To endow the potentially superior PHA biosynthetic machinery to E. coli, we cloned the PHA biosynthesis genes from A. latus. The three PHA biosynthesis genes formed an operon with the order PHA synthase, beta-ketothiolase, and reductase genes and were constitutively expressed from the natural promoter in E. coli. Recombinant E. coli strains harboring the A. latus PHA biosynthesis genes accumulated poly(3-hydroxybutyrate) (PHB), a model PHA product, more efficiently than those harboring the R. eutropha genes. With a pH-stat fed-batch culture of recombinant E. coli harboring a stable plasmid containing the A. latus PHA biosynthesis genes, final cell and PHB concentrations of 194.1 and 141.6 g/liter, respectively, were obtained, resulting in a high productivity of 4.63 g of PHB/liter/h. This improvement should allow recombinant E. coli to be used for the production of PHB with a high level of economic competitiveness.

How Does Poly(hydroxyalkanoate) Affect Methane Production from the Anaerobic Digestion of Waste-Activated Sludge?

PubMed

Wang, Dongbo; Zhao, Jianwei; Zeng, Guangming; Chen, Yinguang; Bond, Philip L; Li, Xiaoming

2015-10-20

Recent studies demonstrate that, besides being used for production of biodegradable plastics, poly(hydroxyalkanoate) (PHA) that is accumulated in heterotrophic microorganisms during wastewater treatment has another novel application direction, i.e., being utilized for enhancing methane yield during the anaerobic digestion of waste-activated sludge (WAS). To date, however, the underlying mechanism of how PHA affects methane production remains largely unknown, and this limits optimization and application of the strategy. This study therefore aims to fill this knowledge gap. Experimental results showed that with the increase of sludge PHA levels from 21 to 184 mg/g of volatile suspended solids (VSS) the methane yield linearly increased from 168.0 to 246.1 mL/g of VSS (R(2) = 0.9834). Compared with protein and carbohydrate (the main components of a cell), PHA exhibited a higher biochemical methane potential on a unit VSS basis. It was also found that the increased PHA not only enhanced cell disruption of PHA cells but also benefited the soluble protein conversion of both PHA- and non-PHA cells. Moreover, the reactor fed with higher PHA sludge showed greater sludge hydrolysis and acidification than those fed with the lower PHA sludges. Further investigations using fluorescence in situ hybridization and enzyme analysis revealed that the increased PHA enhanced the abundance of methanogenic Archaea and increased the activities of protease, acetate kinase, and coenzyme F420, which were consistent with the observed methane yield. This work provides insights into PHA-involved WAS digestion systems and may have important implications for future operation of wastewater treatment plants.

Production of polyhydroxyalkanoates (PHA) by bacterial consortium from excess sludge fermentation liquid at laboratory and pilot scales.

PubMed

Jia, Qianqian; Xiong, Huilei; Wang, Hui; Shi, Hanchang; Sheng, Xinying; Sun, Run; Chen, Guoqiang

2014-11-01

The generation of polyhydroxyalkanoates (PHA) from excess sludge fermentation liquid (SFL) was studied at lab and pilot scale. A PHA-accumulated bacterial consortium (S-150) was isolated from activated sludge using simulated SFL (S-SFL) contained high concentration volatile fatty acids (VFA) and nitrogen. The maximal PHA content accounted for 59.18% in S-SFL and dropped to 23.47% in actual SFL (L-SFL) of the dry cell weight (DCW) at lab scale. The pilot-scale integrated system comprised an anaerobic fermentation reactor (AFR), a ceramic membrane system (CMS) and a PHA production bio-reactor (PHAR). The PHA content from pilot-scale SFL (P-SFL) finally reached to 59.47% DCW with the maximal PHA yield coefficient (YP/S) of 0.17 g PHA/g COD. The results indicated that VFA-containing SFL was suitable for PHA production. The adverse impact of excess nitrogen and non-VFAs in SFL might be eliminated by pilot-scale domestication, which might resulted in community structure optimization and substrate selective ability improvement of S-150. Copyright © 2014 Elsevier Ltd. All rights reserved.

Combined polyhydroxyalkanoates (PHA) and 1,3-propanediol production from crude glycerol: Selective conversion of volatile fatty acids into PHA by mixed microbial consortia.

PubMed

Burniol-Figols, Anna; Varrone, Cristiano; Le, Simone Balzer; Daugaard, Anders Egede; Skiadas, Ioannis V; Gavala, Hariklia N

2018-06-01

Crude glycerol is an important by-product of the biodiesel industry, which can be converted into volatile fatty acids (VFA) and/or 1,3-propanediol (1,3-PDO) by fermentation. In this study, a selective conversion of VFA to polyhydroxyalkanoates (PHA) was attained while leaving 1,3-PDO in the supernatant by means of mixed microbial consortia selection strategies. The process showed highly reproducible results in terms of PHA yield, 0.99 ± 0.07 C mol PHA/C mol S (0.84 g COD PHA/g COD S), PHA content (76 ± 3.1 g PHA/100 g TSS) and 1,3-PDO recovery (99 ± 2.1%). The combined process had an ultimate yield from crude glycerol of 0.19 g COD PHA and 0.42 g COD 1,3-PDO per g of input COD. The novel enrichment strategy applied for selectively transforming fermentation by-products into a high value product (PHA) demonstrates the significance of the enrichment process for targeting specific bio-transformations and could potentially prove valuable for other biotechnological applications as well. Copyright © 2018 Elsevier Ltd. All rights reserved.

Synthesis of High-Molecular-Weight Polyhydroxyalkanoates by Marine Photosynthetic Purple Bacteria

PubMed Central

Higuchi-Takeuchi, Mieko; Morisaki, Kumiko; Toyooka, Kiminori; Numata, Keiji

2016-01-01

Polyhydroxyalkanoate (PHA) is a biopolyester/bioplastic that is produced by a variety of microorganisms to store carbon and increase reducing redox potential. Photosynthetic bacteria convert carbon dioxide into organic compounds using light energy and are known to accumulate PHA. We analyzed PHAs synthesized by 3 purple sulfur bacteria and 9 purple non-sulfur bacteria strains. These 12 purple bacteria were cultured in nitrogen-limited medium containing acetate and/or sodium bicarbonate as carbon sources. PHA production in the purple sulfur bacteria was induced by nitrogen-limited conditions. Purple non-sulfur bacteria accumulated PHA even under normal growth conditions, and PHA production in 3 strains was enhanced by nitrogen-limited conditions. Gel permeation chromatography analysis revealed that 5 photosynthetic purple bacteria synthesized high-molecular-weight PHAs, which are useful for industrial applications. Quantitative reverse transcription polymerase chain reaction analysis revealed that mRNA levels of phaC and PhaZ genes were low under nitrogen-limited conditions, resulting in production of high-molecular-weight PHAs. We conclude that all 12 tested strains are able to synthesize PHA to some degree, and we identify 5 photosynthetic purple bacteria that accumulate high-molecular-weight PHA molecules. Furthermore, the photosynthetic purple bacteria synthesized PHA when they were cultured in seawater supplemented with acetate. The photosynthetic purple bacteria strains characterized in this study should be useful as host microorganisms for large-scale PHA production utilizing abundant marine resources and carbon dioxide. PMID:27513570

Neural correlates of an attentional bias to health-threatening stimuli in individuals with pathological health anxiety.

PubMed

Mier, Daniela; Bailer, Josef; Ofer, Julia; Kerstner, Tobias; Zamoscik, Vera; Rist, Fred; Witthöft, Michael; Diener, Carsten

2017-05-01

An attentional bias to health-threat stimuli is assumed to represent the primary pathogenetic factor for the development and maintenance of pathological health anxiety (PHA; formerly termed "hypochondriasis"). However, little is known about the neural basis of this attentional bias in individuals with PHA. A group of patients with PHA, a group of depressed patients and a healthy control group completed an emotional Stroop task with health-threat (body symptom and illness) words and neutral control words while undergoing functional MRI. We included 33 patients with PHA, 28 depressed patients and 31 controls in our analyses. As reflected in reaction times, patients with PHA showed a significantly stronger attentional bias to health-threat words than both control groups. In addition, patients with PHA showed increased amygdala and rostral anterior cingulate cortex activation for body symptom, but not for illness words. Moreover, only in patients with PHA amygdala activation in response to symptom words was positively associated with higher arousal and more negative valence ratings of the body symptom word material. A control group of patients with an anxiety disorder but without PHA would have helped to define the specificity of the results for PHA. The attentional bias observed in patients with PHA is associated with hyperactivation in response to body symptom words in brain regions that are crucial for an arousal-related fear response (e.g., the amygdala) and for resolving emotional interference (e.g., the rostral anterior cingulate cortex). The findings have important implications for the nosological classification of PHA and suggest the application of innovative exposure-based interventions for the treatment of PHA.

Neural correlates of an attentional bias to health-threatening stimuli in individuals with pathological health anxiety

PubMed Central

Mier, Daniela; Bailer, Josef; Ofer, Julia; Kerstner, Tobias; Zamoscik, Vera; Rist, Fred; Witthöft, Michael; Diener, Carsten

2017-01-01

Background An attentional bias to health-threat stimuli is assumed to represent the primary pathogenetic factor for the development and maintenance of pathological health anxiety (PHA; formerly termed “hypochondriasis”). However, little is known about the neural basis of this attentional bias in individuals with PHA. Methods A group of patients with PHA, a group of depressed patients and a healthy control group completed an emotional Stroop task with health-threat (body symptom and illness) words and neutral control words while undergoing functional MRI. Results We included 33 patients with PHA, 28 depressed patients and 31 controls in our analyses. As reflected in reaction times, patients with PHA showed a significantly stronger attentional bias to health-threat words than both control groups. In addition, patients with PHA showed increased amygdala and rostral anterior cingulate cortex activation for body symptom, but not for illness words. Moreover, only in patients with PHA amygdala activation in response to symptom words was positively associated with higher arousal and more negative valence ratings of the body symptom word material. Limitations A control group of patients with an anxiety disorder but without PHA would have helped to define the specificity of the results for PHA. Conclusion The attentional bias observed in patients with PHA is associated with hyperactivation in response to body symptom words in brain regions that are crucial for an arousal-related fear response (e.g., the amygdala) and for resolving emotional interference (e.g., the rostral anterior cingulate cortex). The findings have important implications for the nosological classification of PHA and suggest the application of innovative exposure-based interventions for the treatment of PHA. PMID:28234209

Insights into the surface topology of polyhydroxyalkanoate synthase: self-assembly of functionalized inclusions.

PubMed

Hooks, David O; Rehm, Bernd H A

2015-10-01

The polyhydroxyalkanoate (PHA) synthase catalyzes the synthesis of PHA and remains attached to the hydrophobic PHA inclusions it creates. Although this feature is actively exploited to generate functionalized biobeads via protein engineering, little is known about the structure of the PHA synthase. Here, the surface topology of Ralstonia eutropha PHA synthase was probed to inform rational protein engineering toward the production of functionalized PHA beads. Surface-exposed residues were detected by conjugating biotin to inclusion-bound PHA synthase and identifying the biotin-conjugated lysine and cysteine residues using peptide fingerprinting analysis. The identified sites (K77, K90, K139, C382, C459, and K518) were investigated as insertion sites for the generation of new protein fusions. Insertions of FLAG epitopes into exposed sites K77, K90, K139, and K518 were tolerated, retaining >65 % of in vivo activity. Sites K90, K139, and K518 were also tested by insertion of the immunoglobulin G (IgG)-binding domain (ZZ), successfully producing PHA inclusions able to bind human IgG in vitro. Although simultaneous insertions of the ZZ domain into two sites was permissive, insertion at all three lysine sites inactivated the synthase. The K90/K139 double ZZ insertion had the optimum IgG-binding capacity of 16 mg IgG/g wet PHA beads and could selectively purify the IgG fraction from human serum. Overall, this study identified surface-exposed flexible regions of the PHA synthase which either tolerate protein/peptide insertions or are critical for protein function. This further elucidates the structure and function of PHA synthase and provides new opportunities for generating functionalized PHA biobeads.

Force measurements in the medial meniscus posterior horn attachment: effects of anterior cruciate ligament removal.

PubMed

Markolf, Keith L; Jackson, Steven R; McAllister, David R

2012-02-01

Tears of the medial meniscus posterior horn attachment (PHA) occur clinically, and an anterior cruciate ligament (ACL)-deficient knee may be more vulnerable to this injury. The PHA forces from applied knee loadings will increase after removal of the ACL. Controlled laboratory study. A cap of bone containing the medial meniscus PHA was attached to a load cell that measured PHA tensile force. Posterior horn attachment forces were recorded before and after ACL removal during anteroposterior (AP) laxity testing at ±200 N and during passive knee extension tests with 5 N·m tibial torque and varus-valgus moment. Selected tests were also performed with 500 N joint load. For AP tests with no joint load, ACL removal increased laxity between 0° and 90° and increased PHA force generated by applied anterior tibial force between 30° and 90°. For AP tests with an intact ACL, application of joint load approximately doubled PHA forces. Anteroposterior testing of ACL-deficient knees was not possible with joint load because of bone cap failures from high PHA forces. Removal of the ACL during knee extension tests under joint load significantly increased PHA forces between 20° and 90° of flexion. For unloaded tests with applied tibial torque and varus-valgus moment, ACL removal had no significant effect on PHA forces. Applied anterior tibial force and external tibial torque were loading modes that produced relatively high PHA forces, presumably by impingement of the medial femoral condyle against the medial meniscus posterior horn rim. Under joint load, an ACL-deficient knee was particularly susceptible to PHA injury from applied anterior tibial force. Because tensile forces developed in the PHA are also borne by meniscus tissue near the attachment site, loading mechanisms that produce high PHA forces could also produce complete or partial radial tears near the posterior horn, a relatively common clinical observation.

Data on partial polyhydroxyalkanoate synthase genes (phaC) mined from Aaptos aaptos marine sponge-associated bacteria metagenome.

PubMed

Amelia, Tan Suet May; Amirul, Al-Ashraf Abdullah; Bhubalan, Kesaven

2018-02-01

We report data associated with the identification of three polyhydroxyalkanoate synthase genes (phaC) isolated from the marine bacteria metagenome of Aaptos aaptos marine sponge in the waters of Bidong Island, Terengganu, Malaysia. Our data describe the extraction of bacterial metagenome from sponge tissue, measurement of purity and concentration of extracted metagenome, polymerase chain reaction (PCR)-mediated amplification using degenerate primers targeting Class I and II phaC genes, sequencing at First BASE Laboratories Sdn Bhd, and phylogenetic analysis of identified and known phaC genes. The partial nucleotide sequences were aligned, refined, compared with the Basic Local Alignment Search Tool (BLAST) databases, and released online in GenBank. The data include the identified partial putative phaC and their GenBank accession numbers, which are Rhodocista sp. phaC (MF457754), Pseudomonas sp. phaC (MF437016), and an uncultured bacterium AR5-9d_16 phaC (MF457753).

Carbon dioxide sequestration by chemolithotrophic oleaginous bacteria for production and optimization of polyhydroxyalkanoate.

PubMed

Kumar, Manish; Gupta, Asmita; Thakur, Indu Shekhar

2016-08-01

The present work involved screening of a previously reported carbon concentrating oleaginous bacterial strain Serratia sp. ISTD04 for production of PHA and optimization of process parameters for enhanced PHA and biomass generation. The selected bacterial strain was screened for PHA production based on Nile red staining followed by visualization under fluorescence microscope. Spectrofluorometric measurement of Nile red fluorescence of the bacterial culture was also done. Confirmatory analysis of PHA accumulation by GC-MS revealed the presence of 3-hydroxyvalerate. Detection of characteristic peaks in the FT-IR spectrum further confirmed the production of PHA by the bacterium. Response Surface Methodology was used for optimization of pH and carbon sources' concentrations for higher PHA production. There was almost a 2 fold increase in the production of PHA following optimization as compared to un-optimized condition. The study thus establishes the production of PHA by Serratia sp. ISTD04. Copyright © 2016 Elsevier Ltd. All rights reserved.

Characterization of medium-chain-length polyhydroxyalkanoate biosynthesis by Pseudomonas mosselii TO7 using crude glycerol.

PubMed

Liu, Ming-Hsu; Chen, Yi-Jr; Lee, Chia-Yin

2018-03-01

Polyhydroxyalkanoates (PHAs) are biopolyesters produced by microorganisms that are environmentally friendly. PHAs can be used to replace traditional plastic to reduce environmental pollution in various fields. PHA production costs are high because PHA must be produced from a carbon substrate. The purpose of this study was to find the strain that can used the BDF by-product as the sole carbon source to produce high amounts of medium-chain-length PHA. Three isolates were evaluated for potential PHA production by using biodiesel-derived crude glycerol as the sole carbon source. Among them, Pseudomonas mosselii TO7 yielded high PHA content. The PHA produced from P. mosselii TO7 were medium-chain-length-PHAs. The PHA content of 48% cell dry weight in 48 h with a maximum PHA productivity of 13.16 mg PHAs L -1  h -1 . The narrow polydispersity index value of 1.3 reflected the homogeneity of the polymer chain, which was conducive to industrial applications.

The use of NaCl addition for the improvement of polyhydroxyalkanoate production by Cupriavidus necator.

PubMed

Passanha, Pearl; Kedia, Gopal; Dinsdale, Richard M; Guwy, Alan J; Esteves, Sandra R

2014-07-01

External stress factors in the form of ionic species or temperature increases have been shown to produce a stress response leading to enhanced PHA production. The effect of five different NaCl concentrations, namely 3.5, 6.5, 9, 12 and 15 g/l NaCl on PHA productivity using Cupriavidus necator has been investigated alongside a control (no added NaCl). A dielectric spectroscopy probe was used to measure PHA accumulation online in conjunction with the chemical offline analysis of PHA. The highest PHA production was obtained with the addition of 9 g/l NaCl, which yielded 30% higher PHA than the control. Increasing the addition of NaCl to 15 g/l was found to inhibit the production of PHA. NaCl addition can therefore be used as a simple, low cost, sustainable, non toxic and non reactive external stress strategy for increasing PHA productivity. Copyright © 2014 Elsevier Ltd. All rights reserved.

24 CFR 902.22 - Physical inspection of PHA projects.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 24 Housing and Urban Development 4 2013-04-01 2013-04-01 false Physical inspection of PHA projects... URBAN DEVELOPMENT PUBLIC HOUSING ASSESSMENT SYSTEM Physical Condition Indicator § 902.22 Physical inspection of PHA projects. (a) The inspection, generally. The PHA's score for the physical condition...

24 CFR 902.22 - Physical inspection of PHA projects.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 24 Housing and Urban Development 4 2014-04-01 2014-04-01 false Physical inspection of PHA projects... URBAN DEVELOPMENT PUBLIC HOUSING ASSESSMENT SYSTEM Physical Condition Indicator § 902.22 Physical inspection of PHA projects. (a) The inspection, generally. The PHA's score for the physical condition...

24 CFR 902.22 - Physical inspection of PHA projects.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 24 Housing and Urban Development 4 2012-04-01 2012-04-01 false Physical inspection of PHA projects... URBAN DEVELOPMENT PUBLIC HOUSING ASSESSMENT SYSTEM Physical Condition Indicator § 902.22 Physical inspection of PHA projects. (a) The inspection, generally. The PHA's score for the physical condition...

24 CFR 902.22 - Physical inspection of PHA projects.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 24 Housing and Urban Development 4 2011-04-01 2011-04-01 false Physical inspection of PHA projects... URBAN DEVELOPMENT PUBLIC HOUSING ASSESSMENT SYSTEM Physical Condition Indicator § 902.22 Physical inspection of PHA projects. (a) The inspection, generally. The PHA's score for the physical condition...

RNA-Seq Analysis Provides Insights for Understanding Photoautotrophic Polyhydroxyalkanoate Production in Recombinant Synechocystis Sp.

PubMed Central

Lau, Nyok-Sean; Foong, Choon Pin; Kurihara, Yukio; Sudesh, Kumar; Matsui, Minami

2014-01-01

The photosynthetic cyanobacterium, Synechocystis sp. strain 6803, is a potential platform for the production of various chemicals and biofuels. In this study, direct photosynthetic production of a biopolymer, polyhydroxyalkanoate (PHA), in genetically engineered Synechocystis sp. achieved as high as 14 wt%. This is the highest production reported in Synechocystis sp. under photoautotrophic cultivation conditions without the addition of a carbon source. The addition of acetate increased PHA accumulation to 41 wt%, and this value is comparable to the highest production obtained with cyanobacteria. Transcriptome analysis by RNA-seq coupled with real-time PCR was performed to understand the global changes in transcript levels of cells subjected to conditions suitable for photoautotrophic PHA biosynthesis. There was lower expression of most PHA synthesis-related genes in recombinant Synechocystis sp. with higher PHA accumulation suggesting that the concentration of these enzymes is not the limiting factor to achieving high PHA accumulation. In order to cope with the higher PHA production, cells may utilize enhanced photosynthesis to drive the product formation. Results from this study suggest that the total flux of carbon is the possible driving force for the biosynthesis of PHA and the polymerizing enzyme, PHA synthase, is not the only critical factor affecting PHA-synthesis. Knowledge of the regulation or control points of the biopolymer production pathways will facilitate the further use of cyanobacteria for biotechnological applications. PMID:24466058

Genetic Analysis of Comamonas acidovorans Polyhydroxyalkanoate Synthase and Factors Affecting the Incorporation of 4-Hydroxybutyrate Monomer

PubMed Central

Sudesh, Kumar; Fukui, Toshiaki; Doi, Yoshiharu

1998-01-01

The polyhydroxyalkanoate (PHA) synthase gene of Comamonas acidovorans DS-17 (phaCCa) was cloned by using the synthase gene of Alcaligenes eutrophus as a heterologous hybridization probe. Complete sequencing of a 4.0-kbp SmaI-HindIII (SH40) subfragment revealed the presence of a 1,893-bp PHA synthase coding region which was followed by a 1,182-bp β-ketothiolase gene (phaACa). Both the translated products of these genes showed significant identity, 51.1 and 74.2%, respectively, to the primary structures of the products of the corresponding genes in A. eutrophus. The arrangement of PHA biosynthesis genes in C. acidovorans was also similar to that in A. eutrophus except that the third gene, phaB, coding for acetoacetyl-coenzyme A reductase, was not found in the region downstream of phaACa. The cloned fragment complemented a PHA-negative mutant of A. eutrophus, PHB−4, resulting in poly-3-hydroxybutyrate accumulation of up to 73% of the dry cell weight when fructose was the carbon source. The heterologous expression enabled the incorporation of 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate monomers. The PHA synthase of C. acidovorans does not appear to show any preference for 4-hydroxybutyryl-coenzyme A as a substrate. This leads to the suggestion that in C. acidovorans, it is the metabolic pathway, and not the specificity of the organism’s PHA synthase, that drives the incorporation of 4HB monomers, resulting in the efficient accumulation of PHA with a high 4HB content. PMID:9726894

In vitro synthesis of polyhydroxyalkanoate (PHA) incorporating lactate (LA) with a block sequence by using a newly engineered thermostable PHA synthase from Pseudomonas sp. SG4502 with acquired LA-polymerizing activity.

PubMed

Tajima, Kenji; Han, Xuerong; Satoh, Yasuharu; Ishii, Ayako; Araki, Yuji; Munekata, Masanobu; Taguchi, Seiichi

2012-04-01

Recently, we succeeded in isolating a thermotolerant bacterium, Pseudomonas sp. SG4502, which is capable of accumulating polyhydroxyalkanoate (PHA) even at 55 °C, as a source of thermostable enzymes. In this study, we cloned a pha locus from the bacterium and identified two genes encoding PHA synthases (PhaC1(SG) and PhaC2(SG)). Two mutations, Ser324Thr and Gln480Lys, corresponding to those of a lactate (LA)-polymerizing enzyme (LPE) from mesophilic Pseudomonas sp. 61-3 were introduced into PhaC1(SG) to evaluate the potential of the resulting protein as a "thermostable LPE". The mutated PhaC1(SG) [PhaC1(SG)(STQK)] showed high thermal stability in synthesizing P(LA-co-3HB) in an in vitro reaction system under a range of high temperatures. Requirement of 3HBCoA as a priming unit for LA polymerization by the LPE has been suggested in both of the in vitro and in vivo experiments. Based on the finding, the PhaC1(SG)(STQK)-mediated synthesis of a LA-based copolymer with a block sequence was achieved in the in vitro system by sequential feeding of the corresponding two substrates. This in vitro reaction system using the thermostable LPE provides us with a versatile way to synthesize the various types of LA-based copolymers with desired sequence patterns, random or block, depending on the way of supplying hydroxyalkanoates (mixed or sequential feeding).

PHB Biosynthesis Counteracts Redox Stress in Herbaspirillum seropedicae

PubMed Central

Batista, Marcelo B.; Teixeira, Cícero S.; Sfeir, Michelle Z. T.; Alves, Luis P. S.; Valdameri, Glaucio; Pedrosa, Fabio de Oliveira; Sassaki, Guilherme L.; Steffens, Maria B. R.; de Souza, Emanuel M.; Dixon, Ray; Müller-Santos, Marcelo

2018-01-01

The ability of bacteria to produce polyhydroxyalkanoates such as poly(3-hydroxybutyrate) (PHB) enables provision of a carbon storage molecule that can be mobilized under demanding physiological conditions. However, the precise function of PHB in cellular metabolism has not been clearly defined. In order to determine the impact of PHB production on global physiology, we have characterized the properties of a ΔphaC1 mutant strain of the diazotrophic bacterium Herbaspirillum seropedicae. The absence of PHB in the mutant strain not only perturbs redox balance and increases oxidative stress, but also influences the activity of the redox-sensing Fnr transcription regulators, resulting in significant changes in expression of the cytochrome c-branch of the electron transport chain. The synthesis of PHB is itself dependent on the Fnr1 and Fnr3 proteins resulting in a cyclic dependency that couples synthesis of PHB with redox regulation. Transcriptional profiling of the ΔphaC1 mutant reveals that the loss of PHB synthesis affects the expression of many genes, including approximately 30% of the Fnr regulon. PMID:29599762

Recovery of polyhydroxyalkanoates from municipal secondary wastewater sludge.

PubMed

Kumar, Manish; Ghosh, Pooja; Khosla, Khushboo; Thakur, Indu Shekhar

2018-05-01

In the current study, the feasibility of utilizing municipal secondary wastewater sludge for Polyhydroxyalkanoate (PHA) extraction was improved by optimization of various parameters (temperature, duration and concentration of sludge solids). Optimized process parameters resulted in PHA recovery of 0.605 g, significantly higher than un-optimized conditions. The characterization of PHA was carried out by GC-MS, FT-IR and NMR ( 1 H and 13 C) spectroscopy. The PHA profile was found to be dominated by mcl PHA (58%) along with other diverse PHA. The results of the present study show rich diversity of PHA extracted from a raw material which is readily available at minimal cost. In conclusion, exploring the potential of wastes for production of bioplastics not only reduces the cost of bioplastic production, but also provides a sustainable means for waste management. Copyright © 2018 Elsevier Ltd. All rights reserved.

24 CFR 983.252 - PHA information for accepted family.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 24 Housing and Urban Development 4 2011-04-01 2011-04-01 false PHA information for accepted family... family. (a) Oral briefing. When a family accepts an offer of PBV assistance, the PHA must give the family... of how the program works; and (2) Family and owner responsibilities. (b) Information packet. The PHA...

24 CFR 982.555 - Informal hearing for participant.

Code of Federal Regulations, 2010 CFR

2010-04-01

... allowance schedule. (iii) A determination of the family unit size under the PHA subsidy standards. (iv) A... appropriate for the family unit size under the PHA subsidy standards, or the PHA determination to deny the... with HQS because of the family size. (8) A determination by the PHA to exercise or not to exercise any...

24 CFR 941.501 - HUD review of PHA performance; sanctions.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false HUD review of PHA performance... URBAN DEVELOPMENT PUBLIC HOUSING DEVELOPMENT Performance Review § 941.501 HUD review of PHA performance; sanctions. (a) HUD determination. HUD shall carry out such reviews of the performance of each PHA as may be...

24 CFR 983.252 - PHA information for accepted family.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false PHA information for accepted family... family. (a) Oral briefing. When a family accepts an offer of PBV assistance, the PHA must give the family... of how the program works; and (2) Family and owner responsibilities. (b) Information packet. The PHA...

24 CFR 982.403 - Terminating HAP contract when unit is too small.

Code of Federal Regulations, 2010 CFR

2010-04-01

... Dwelling Unit: Housing Quality Standards, Subsidy Standards, Inspection and Maintenance § 982.403... change in family composition, the PHA must issue the family a new voucher, and the family and PHA must...-based certificate program. (2) The PHA must issue the family a new voucher, and the family and PHA must...

Synthesis of polyhydroxyalkanoate from palm oil and some new applications.

PubMed

Sudesh, Kumar; Bhubalan, Kesaven; Chuah, Jo-Ann; Kek, Yik-Kang; Kamilah, Hanisah; Sridewi, Nanthini; Lee, Yan-Fen

2011-03-01

Polyhydroxyalkanoate (PHA) is a potential substitute for some petrochemical-based plastics. This biodegradable plastic is derived from microbial fermentation using various carbon substrates. Since carbon source has been identified as one of the major cost-absorbing factors in PHA production, cheap and renewable substrates are currently being investigated as substitutes for existing sugar-based feedstock. Plant oils have been found to result in high-yield PHA production. Malaysia, being the world's second largest producer of palm oil, is able to ensure continuous supply of palm oil products for sustainable PHA production. The biosynthesis and characterization of various types of PHA using palm oil products have been described in detail in this review. Besides, by-products and waste stream from palm oil industry have also demonstrated promising results as carbon sources for PHA biosynthesis. Some new applications in cosmetic and wastewater treatment show the diversity of PHA usage. With proper management practices and efficient milling processes, it may be possible to supply enough palm oil-based raw materials for human consumption and other biotechnological applications such as production of PHA in a sustainable manner.

Influence of aerobic and anoxic microenvironments on polyhydroxyalkanoates (PHA) production from food waste and acidogenic effluents using aerobic consortia.

PubMed

Reddy, M Venkateswar; Mohan, S Venkata

2012-01-01

The functional role of aerobic and anoxic microenvironments on polyhydroxyalkanoates (PHA) production using food waste (UFW) and effluents from acidogenic biohydrogen production process (FFW) were studied employing aerobic mixed culture as biocatalyst. Anoxic microenvironment documented higher PHA production, while aerobic microenvironment showed higher substrate degradation. FFW showed higher PHA accumulation (39.6%) than UFW (35.6%) due to ready availability of precursors (fatty acids). Higher fraction of poly-3-hydroxy butyrate (PHB) was observed compared to poly-3-hydroxy valerate (PHV) in the accumulated PHA in the form of co-polymer [P3(HB-co-HV)]. Dehydrogenase, phosphatase and protease enzymatic activities were monitored during process operation. Integration with fermentative biohydrogen production yielded additional substrate degradation under both aerobic (78%) and anoxic (72%) microenvironments apart from PHA production. Microbial community analysis documented the presence of aerobic and facultative organisms capable of producing PHA. Integration strategy showed feasibility of producing hydrogen along with PHA by consuming fatty acids generated during acidogenic process in association with increased treatment efficiency. Copyright © 2011 Elsevier Ltd. All rights reserved.

Potential for mcl-PHA production from nonanoic and azelaic acids.

PubMed

Gillis, James; Ko, Kenton; Ramsay, Juliana A; Ramsay, Bruce A

2018-01-01

Greater than 65% of canola and high-oleic soy oil fatty acids is oleic acid, which is readily converted to nonanoic (NA) and azelaic (AzA) acids by ozonolysis. NA is an excellent substrate for medium-chain-length polyhydroxyalkanoate (mcl-PHA) production but AzA has few uses. Pseudomonas citronellolis DSM 50332 and Pseudomonas fluorescens ATCC 17400, both able to produce mcl-PHA from fatty acids and to grow on AzA as the sole source of carbon and energy, were assessed for the accumulation of mcl-PHA from AzA and NA. In N-limited shake flasks using NA, P. citronellolis produced 32% of its dry biomass as mcl-PHA containing 78% 3-hydroxynonanoate with 22% 3-hydroxyheptanoate. Pseudomonas fluorescens produced only 2% PHA. N-limited P. citronellolis on AzA produced 20% dry weight PHA containing 75% 3-hydroxydecanoate and 25% 3-hydroxyoctanoate, indicative of de novo synthesis. Although selective pressure, including β-oxidation inhibition, under well-controlled (chemostat) conditions was applied to P. citronellolis, no side-chain carboxyl groups were detected. It was concluded that one or more of FabG and PhaJ or the PHA synthase cannot catalyze reactions involving ω-carboxy substrates. However, a process based on oleic acid could be established if Pseudomonas putida was engineered to grow on AzA.

Engineering Halomonas spp. as A Low-Cost Production Host for Production of Bio-surfactant Protein PhaP.

PubMed

Lan, Lu-Hong; Zhao, Han; Chen, Jin-Chun; Chen, Guo-Qiang

2016-12-01

Halomonas spp. have been studied as a low cost production host for producing bulk materials such as polyhydroxyalkanoates (PHA) bioplastics, since they are able to grow at high pH and high NaCl concentration under unsterile and continuous conditions without microbial contamination. In this paper, Halomonas strain TD is used as a host to produce a protein named PHA phasin or PhaP which has a potential to be developed into a bio-surfactant. Four Halomonas TD expression strains are constructed based on a strong T7-family expression system. Of these, the strain with phaC deletion and chromosomal expression system resulted in the highest production of PhaP in soluble form, reaching 19% of total cellular soluble proteins and with a yield of 1.86 g/L in an open fed-batch fermentation process. A simple "heat lysis and salt precipitation" method is applied to allow rapid PhaP purification from a mixture of cellular proteins with a PhaP recovery rate of 63%. It clearly demonstrated that Halomonas TD could be used for high yield expression of a bio-surfactant protein PhaP for industrial application in an economical way. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Photoautotrophic Polyhydroxybutyrate Granule Formation Is Regulated by Cyanobacterial Phasin PhaP in Synechocystis sp. Strain PCC 6803

PubMed Central

Hauf, Waldemar; Watzer, Björn; Roos, Nora; Klotz, Alexander

2015-01-01

Cyanobacteria are photoautotrophic microorganisms which fix atmospheric carbon dioxide via the Calvin-Benson cycle to produce carbon backbones for primary metabolism. Fixed carbon can also be stored as intracellular glycogen, and in some cyanobacterial species like Synechocystis sp. strain PCC 6803, polyhydroxybutyrate (PHB) accumulates when major nutrients like phosphorus or nitrogen are absent. So far only three enzymes which participate in PHB metabolism have been identified in this organism, namely, PhaA, PhaB, and the heterodimeric PHB synthase PhaEC. In this work, we describe the cyanobacterial PHA surface-coating protein (phasin), which we term PhaP, encoded by ssl2501. Translational fusion of Ssl2501 with enhanced green fluorescent protein (eGFP) showed a clear colocalization to PHB granules. A deletion of ssl2501 reduced the number of PHB granules per cell, whereas the mean PHB granule size increased as expected for a typical phasin. Although deletion of ssl2501 had almost no effect on the amount of PHB, the biosynthetic activity of PHB synthase was negatively affected. Secondary-structure prediction and circular dichroism (CD) spectroscopy of PhaP revealed that the protein consists of two α-helices, both of them associating with PHB granules. Purified PhaP forms oligomeric structures in solution, and both α-helices of PhaP contribute to oligomerization. Together, these results support the idea that Ssl2501 encodes a cyanobacterial phasin, PhaP, which regulates the surface-to-volume ratio of PHB granules. PMID:25911471

Controlling microbial PHB synthesis via CRISPRi.

PubMed

Li, Dan; Lv, Li; Chen, Jin-Chun; Chen, Guo-Qiang

2017-07-01

Microbial polyhydroxyalkanoates (PHA) are a family of biopolyesters with properties similar to petroleum plastics such as polyethylene (PE) or polypropylene (PP). Polyhydroxybutyrate (PHB) is the most common PHA known so far. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a technology recently developed to control gene expression levels in eukaryotic and prokaryotic genomes, was employed to regulate PHB synthase activity influencing PHB synthesis. Recombinant Escherichia coli harboring an operon of three PHB synthesis genes phaCAB cloned from Ralstonia eutropha, was transformed with various single guided RNA (sgRNA with its guide sequence of 20-23 bases) able to bind to various locations of the PHB synthase PhaC, respectively. Depending on the binding location and the number of sgRNA on phaC, CRISPRi was able to control the phaC transcription and thus PhaC activity. It was found that PHB content, molecular weight, and polydispersity were approximately in direct and reverse proportion to the PhaC activity, respectively. The higher the PhaC activity, the more the intracellular PHB accumulation, yet the less the PHB molecular weights and the wider the polydispersity. This study allowed the PHB contents to be controlled in the ranges of 1.47-75.21% cell dry weights, molecular weights from 2 to 6 millions Dalton and polydispersity of 1.2 to 1.43 in 48 h shake flask studies. This result will be very important for future development of ultrahigh molecular weight PHA useful to meet high strength application requirements.

Safety testing of GM-rice expressing PHA-E lectin using a new animal test design.

PubMed

Poulsen, Morten; Schrøder, Malene; Wilcks, Andrea; Kroghsbo, Stine; Lindecrona, Rikke Hvid; Miller, Andreas; Frenzel, Thomas; Danier, Jürgen; Rychlik, Michael; Shu, Qingyao; Emami, Kaveh; Taylor, Mark; Gatehouse, Angharad; Engel, Karl-Heinz; Knudsen, Ib

2007-03-01

The 90-day animal study is the core study for the safety assessment of genetically modified foods in the SAFOTEST project. The model compound tested in the 90-day study was a rice variety expressing the kidney bean Phaseolus vulgaris lectin agglutinin E-form (PHA-E lectin). Female Wistar rats were given a nutritionally balanced purified diet with 60% parental rice, 60% PHA-E rice or 60% PHA-E rice spiked with 0.1% recombinant PHA-E lectin for 90 days. This corresponded to a mean daily PHA-E lectin intake of approximately 0, 30 and 100mg/kg body weight for each group, respectively. The spiking was used to increase the specificity and to demonstrate the sensitivity of the study. A range of biological, biochemical, microbiological and pathological parameters were examined and significant differences in weight of small intestine, stomach and pancreas and plasma biochemistry were seen between groups. Included in this paper are also data from the molecular characterisation and chemical analysis of the PHA-E rice, from the construction and production of the PHA-E lectin, and from the preceding 28-day in vivo study where the toxicity of the pure PHA-E lectin was determined. In conclusion, the combined use of information from the compositional analysis, the 28-day study and the characterisation of the PHA-E rice and the PHA-E lectin has improved the design of the 90-day study. The spiking procedure has facilitated the interpretation of the results of the study and transferred it into a valuable tool for the future safety testing of genetically modified foods.

Quantification of polyhydroxyalkanoates in mixed and pure cultures biomass by Fourier transform infrared spectroscopy: comparison of different approaches.

PubMed

Isak, I; Patel, M; Riddell, M; West, M; Bowers, T; Wijeyekoon, S; Lloyd, J

2016-08-01

Fourier transform infrared (FTIR) spectroscopy was used in this study for the rapid quantification of polyhydroxyalkanoates (PHA) in mixed and pure culture bacterial biomass. Three different statistical analysis methods (regression, partial least squares (PLS) and nonlinear) were applied to the FTIR data and the results were plotted against the PHA values measured with the reference gas chromatography technique. All methods predicted PHA content in mixed culture biomass with comparable efficiency, indicated by similar residuals values. The PHA in these cultures ranged from low to medium concentration (0-44 wt% of dried biomass content). However, for the analysis of the combined mixed and pure culture biomass with PHA concentration ranging from low to high (0-93% of dried biomass content), the PLS method was most efficient. This paper reports, for the first time, the use of a single calibration model constructed with a combination of mixed and pure cultures covering a wide PHA range, for predicting PHA content in biomass. Currently no one universal method exists for processing FTIR data for polyhydroxyalkanoates (PHA) quantification. This study compares three different methods of analysing FTIR data for quantification of PHAs in biomass. A new data-processing approach was proposed and the results were compared against existing literature methods. Most publications report PHA quantification of medium range in pure culture. However, in our study we encompassed both mixed and pure culture biomass containing a broader range of PHA in the calibration curve. The resulting prediction model is useful for rapid quantification of a wider range of PHA content in biomass. © 2016 The Society for Applied Microbiology.

24 CFR 968.335 - HUD review of PHA performance.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false HUD review of PHA performance. 968... 250 or More Public Housing Units) § 968.335 HUD review of PHA performance. (a) HUD determination. At least annually, HUD shall carry out such reviews of the performance of each PHA as may be necessary or...

Enhancing the stability of colloidal silver nanoparticles using polyhydroxyalkanoates (PHA) from Bacillus circulans (MTCC 8167) isolated from crude oil contaminated soil.

PubMed

Phukon, Pinkee; Saikia, Jyoti Prasad; Konwar, Bolin Kumar

2011-09-01

Polyhydroxyalkanoate (PHA) was produced by growing Bacillus circulans (MTCC 8167) in the specific detection medium. The identification of the polymer as PHA was confirmed by fluorescence microscopy. The PHA was purified and characterized using FT-IR. The silver nanoparticles (SNP) were synthesized from AgNO3 in the dispersed colloids of PHA (0.085%) using NaBH4 (sodium borohydrate as reducing agent). The stability was tested using wave length scanning with a UV-Vis spectrophotometer and finally with transmission electron microscopy. The PHA stabilized solution was found to be stable for 30 days as against the low stability of silver nanoparticles (SNP) solution alone. Copyright © 2011 Elsevier B.V. All rights reserved.

Peoria Housing Authority(PHA) Weatherization Training Project

DOE Office of Scientific and Technical Information (OSTI.GOV)

Phillip Chrismon; Jason Dollarhide

2011-12-31

The DOE Weatherization Training Project's goal is to obtain a solid foundation of administrative and technical knowledge so the Peoria Housing Authority (PHA) can establish and implement a successful Weatherization Program by 2011. The DOE weatherization Training Project's two objectives are to (1) build PHA's capabilities by (2) developing its staff members capacities via the acquisition of weatherization skills and competencies. The impacts from this project include: (a) the improvement and expansion of PHA staff skills, (b) the overall enhancement of the quality of the PHA workforce, which will (c) foster employment, (d) the ability to properly weatherize PHA housingmore » stock, tribal buildings, and tribal members houses, which will (e) result in reduced energy use, and (f) improved tribal and household economies.« less

Detection of the enzymatically-active polyhydroxyalkanoate synthase subunit gene, phaC, in cyanobacteria via colony PCR.

PubMed

Lane, Courtney E; Benton, Michael G

2015-12-01

A colony PCR-based assay was developed to rapidly determine if a cyanobacterium of interest contains the requisite genetic material, the PHA synthase PhaC subunit, to produce polyhydroxyalkanoates (PHAs). The test is both high throughput and robust, owing to an extensive sequence analysis of cyanobacteria PHA synthases. The assay uses a single detection primer set and a single reaction condition across multiple cyanobacteria strains to produce an easily detectable positive result - amplification via PCR as evidenced by a band in electrophoresis. In order to demonstrate the potential of the presence of phaC as an indicator of a cyanobacteria's PHA accumulation capabilities, the ability to produce PHA was assessed for five cyanobacteria with a traditional in vivo PHA granule staining using an oxazine dye. The confirmed in vivo staining results were then compared to the PCR-based assay results and found to be in agreement. The colony PCR assay was capable of successfully detecting the phaC gene in all six of the diverse cyanobacteria tested which possessed the gene, while exhibiting no undesired product formation across the nine total cyanobacteria strains tested. The colony PCR quick prep provides sufficient usable DNA template such that this assay could be readily expanded to assess multiple genes of interest simultaneously. Copyright © 2015 Elsevier Ltd. All rights reserved.

Sequence analysis, structure prediction and functional validation of phaC1/phaC2 genes of Pseudomonas sp. LDC-25 and its importance in polyhydroxyalkanoate accumulation

USDA-ARS?s Scientific Manuscript database

Polyhydroxyalkanoates (PHAs) are attractive biomaterials in both conventional medical devices and tissue engineering. PHA synthase is responsible for catalyzing the formation of polyhydroxyalkanoates (PHA), but its structural information is limited. Hence, the focus of this study is to predict 3D mo...

24 CFR 880.507 - Default by PHA and/or owner (private-owner/PHA projects).

Code of Federal Regulations, 2011 CFR

2011-04-01

... projects). (a) Rights of Owner if PHA defaults under Agreement or Contract. The ACC, the Agreement and the... obligations to enter into the Contract. (b) Rights of HUD if PHA defaults under ACC. The ACC will provide that..., HUD will continue to pay annual contributions in accordance with the terms of the ACC and the Contract...

24 CFR 943.120 - What programs of a PHA are included in a consortium's functions?

Code of Federal Regulations, 2010 CFR

2010-04-01

... PHA under an ACC with HUD; and (5) Any grant programs of the PHA in connection with its Section 8 or... in paragraph (a) of this section, the consortium must cover the PHA's whole program under the ACC with HUD for that category, including all dwelling units and all funding for that program under the ACC...

24 CFR 943.120 - What programs of a PHA are included in a consortium's functions?

Code of Federal Regulations, 2011 CFR

2011-04-01

... PHA under an ACC with HUD; and (5) Any grant programs of the PHA in connection with its Section 8 or... in paragraph (a) of this section, the consortium must cover the PHA's whole program under the ACC with HUD for that category, including all dwelling units and all funding for that program under the ACC...

24 CFR 880.507 - Default by PHA and/or owner (private-owner/PHA projects).

Code of Federal Regulations, 2010 CFR

2010-04-01

... projects). (a) Rights of Owner if PHA defaults under Agreement or Contract. The ACC, the Agreement and the... obligations to enter into the Contract. (b) Rights of HUD if PHA defaults under ACC. The ACC will provide that..., HUD will continue to pay annual contributions in accordance with the terms of the ACC and the Contract...

24 CFR 901.220 - Resident participation in competitive proposals to manage the housing of a PHA.

Code of Federal Regulations, 2010 CFR

2010-04-01

... competitive proposals to manage the housing of a PHA. 901.220 Section 901.220 Housing and Urban Development... § 901.220 Resident participation in competitive proposals to manage the housing of a PHA. (a) When a competitive proposal to manage the housing of a PHA in substantial default is solicited in a Request for...

Production and optimization of polyhydroxyalkanoates from non-edible Calophyllum inophyllum oil using Cupriavidus necator.

PubMed

Arumugam, A; Senthamizhan, S G; Ponnusami, V; Sudalai, S

2018-06-01

Polyhydroxyalkanoates (PHA) are biodegradable polymers found in the cellular masses of a wide range of bacterial species and the demand for PHA is steadily growing. In this work we have produced PHA from a low-cost substrate, Calophyllum inophyllum oil, using Cupriavidus necator. Effects of various process parameters such as Oil concentration, Nitrogen source and inoculum size on the production of PHA were studied using Response Surface Methodology. A quadratic equation was used in the model to fit the experimental data. It was found that the model could satisfactorily predict the PHA yield (R 2 =99.17%). Linear, quadratic and interaction terms used in the model were found to be statistically significant. Maximum PHA yield of 10.6gL -1 was obtained under the optimized conditions of oil concentration - 17.5%, inoculum concentration - 50mL/L and nitrogen content - 1.125gL -1 , respectively. The product obtained was characterized using FTIR and NMR to confirm that it was PHA. The results demonstrate that C. inophyllum oil, a non-edible oil, can be potentially used as a low-cost substrate for the production of PHA. Copyright © 2018 Elsevier B.V. All rights reserved.

Polymodal Responses in C. elegans Phasmid Neurons Rely on Multiple Intracellular and Intercellular Signaling Pathways

PubMed Central

Zou, Wenjuan; Cheng, Hankui; Li, Shitian; Yue, Xiaomin; Xue, Yadan; Chen, Sixi; Kang, Lijun

2017-01-01

Animals utilize specialized sensory neurons enabling the detection of a wide range of environmental stimuli from the presence of toxic chemicals to that of touch. However, how these neurons discriminate between different kinds of stimuli remains poorly understood. By combining in vivo calcium imaging and molecular genetic manipulation, here we investigate the response patterns and the underlying mechanisms of the C. elegans phasmid neurons PHA/PHB to a variety of sensory stimuli. Our observations demonstrate that PHA/PHB neurons are polymodal sensory neurons which sense harmful chemicals, hyperosmotic solutions and mechanical stimulation. A repulsive concentration of IAA induces calcium elevations in PHA/PHB and both OSM-9 and TAX-4 are essential for IAA-sensing in PHA/PHB. Nevertheless, the PHA/PHB neurons are inhibited by copper and post-synaptically activated by copper removal. Neuropeptide is likely involved in copper removal-induced calcium elevations in PHA/PHB. Furthermore, mechanical stimulation activates PHA/PHB in an OSM-9-dependent manner. Our work demonstrates how PHA/PHB neurons respond to multiple environmental stimuli and lays a foundation for the further understanding of the mechanisms of polymodal signaling, such as nociception, in more complex organisms. PMID:28195191

Biopolymer - A beginning towards back to nature

NASA Astrophysics Data System (ADS)

Gautam, S.; Gautam, A.

2018-05-01

Biopolymer is regarded as a polymer which can be biodegradable. Polyhydroxyalkanoates (PHAs) is one of the biopolymer which can be recovered from biomass. PHAs are naturally conserved in the cytoplasm of the bacterial cell during the growth. Bacteria/microbes store their energy from carbon sources in the form of hydrocarbons. Intracellular stored compounds are tightly linked with entire cell resulting difficulty of separation. The work aims to extract PHAs from biomass effectively. Chemical and mechanical separation of PHA can be done from biomass. A pretreatment of cells before chemical and mechanical separation is also effective for separation of PHA and has been carried out. Chemical extraction of PHA includes digestion of cell wall in acidic or alkaline medium and releasing PHA in broth, later sedimentation recovers PHA. In recent work different chemical methods were carried out to extract PHA of medium chain length. In one of these, sodium hypochlorite was used to denature the protein and chloroform was used for extraction of purified PHA. A recovery upto 96.6%, PHA by dried weight of cell, was obtained which is quite high comparing to reported literature. Other chemical disruption by sodium chloride, sodium hydroxide and hydrogen peroxide with and without pretreatment have also been carried out.

Effect of gamma irradiation on cell lysis and polyhydroxyalkanoate produced by Bacillus flexus

NASA Astrophysics Data System (ADS)

Divyashree, M. S.; Shamala, T. R.

2009-02-01

Bacillus flexus cultivated on sucrose and sucrose with plant oil such as castor oil produced polyhydroxybutyrate (PHB), a homopolymer of polyhydroxyalkanoate (PHA) and PHA copolymer (containing hydroxybutyrate and hexanoate), respectively. Gamma irradiation of these cells (5-40 kGy) resulted in cell damage and aided in the isolation of 45% and 54% PHA on biomass weight, correspondingly. Molecular weight of PHB increased from 1.5×10 5 to 1.9×10 5 after irradiation (10 kGy), with marginal increase of tensile strength from 18 to 20 MPa. At the same irradiation dosage, PHA copolymer showed higher molecular weight increase from 1.7×10 5 to 2.3×10 5 and tensile strength from 20 to 35 MPa. GC, GC-MS, FTIR and 1H NMR were used for the characterization of PHA. Gamma irradiation seems to be a novel technique, to induce cross-linking and molecular weight increase of PHA copolymer and aid in easy extractability of intracellular PHA, simultaneously.

Effect of famine-phase reduced aeration on polyhydroxyalkanoate accumulation in aerobic granules.

PubMed

Vjayan, T; Vadivelu, V M

2017-12-01

The effects of variable aeration in the famine period on polyhydroxyalkanoate (PHA) accumulation in aerobic granules were investigated. Results showed that regardless of the aeration rates used during famine period, all aerobic granules achieved a similar chemical oxygen demand removal and PHA content. The decrease in famine-period aeration rates accelerated the maximum PHA accumulation together with increase in granular size and settling ability. The PHA-accumulating microorganisms were found to have shifted closer to the surface of the granules when the aeration rate was reduced. Moreover, PHA compositional changes occurred, where the hydroxyvalerate content had increased with the reduction in aeration rate. Ultimately, the results indicate that the requirement of aeration for PHA accumulation in aerobic granules is highly insignificant in the famine phase. PHA production in aerobic granules under zero aeration in the famine period may result in an energy input reduction of up to 74%. Copyright © 2017 Elsevier Ltd. All rights reserved.

Polyhydroxyalkanoate production as a side stream process on a municipal waste water treatment plant.

PubMed

Pittmann, T; Steinmetz, H

2014-09-01

This work describes the production of polyhydroxyalkanoates (PHAs) as a side stream process on a municipal waste water treatment plant (WWTP) at different operation conditions. Therefore various tests were conducted regarding a high PHA production and stable PHA composition. Influence of substrate concentration, temperature, pH and cycle time of an installed feast/famine-regime were investigated. The results demonstrated a strong influence of the operating conditions on the PHA production. Lower substrate concentration, 20°C, neutral pH-value and a 24h cycle time are preferable for high PHA production up to 28.4% of cell dry weight (CDW). PHA composition was influenced by cycle time only and a stable PHA composition was reached. Copyright © 2014 Elsevier Ltd. All rights reserved.

Biosynthesis of Polyhydroxyalkanoate from Steamed Soybean Wastewater by a Recombinant Strain of Pseudomonas sp. 61-3.

PubMed

Hokamura, Ayaka; Yunoue, Yuko; Goto, Saki; Matsusaki, Hiromi

2017-08-08

Pseudomonas sp. 61-3 accumulates a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer, poly(3-hydroxybutyrate- co -3-hydroxyalkanoate) [P(3HB- co -3HA)], consisting of 3HA units of 4-12 carbon atoms. Pseudomonas sp. 61-3 possesses two types of PHA synthases, PHB synthase (PhbC) and PHA synthases (PhaC1 and PhaC2), encoded by the phb and pha loci, respectively. The P(94 mol% 3HB- co -6 mol% 3HA) copolymer synthesized by the recombinant strain of Pseudomonas sp. 61-3 ( phbC :: tet ) harboring additional copies of phaC1 gene is known to have desirable physical properties and to be a flexible material with moderate toughness, similar to low-density polyethylene. In this study, we focused on the production of the P(3HB- co -3HA) copolymer using steamed soybean wastewater, a by-product in brewing miso , which is a traditional Japanese seasoning. The steamed soybean wastewater was spray-dried to produce a powder (SWP) and used as the sole nitrogen source for the synthesis of P(3HB- co -3HA) by the Pseudomonas sp. 61-3 recombinant strain. Hydrolyzed SWP (HSWP) was also used as a carbon and nitrogen source. P(3HB- co -3HA)s with relatively high 3HB fractions could be synthesized by a recombinant strain of Pseudomonas sp. 61-3 ( phbC :: tet ) harboring additional copies of the phaC1 gene in the presence of 2% glucose and 10-20 g/L SWP as the sole nitrogen source, producing a PHA concentration of 1.0-1.4 g/L. When HSWP was added to a nitrogen- and carbon-free medium, the recombinant strain could synthesize PHA without glucose as a carbon source. The recombinant strain accumulated 32 wt% P(3HB- co -3HA) containing 80 mol% 3HB and 20 mol% medium-chain-length 3HA with a PHA concentration of 1.0 g/L when 50 g/L of HSWP was used. The PHA production yield was estimated as 20 mg-PHA/g-HSWP, which equates to approximately 1.0 g-PHA per liter of soybean wastewater.

Polyhydroxyalkanoate synthesis by mixed microbial consortia cultured on fermented dairy manure: Effect of aeration on process rates/yields and the associated microbial ecology.

PubMed

Coats, Erik R; Watson, Benjamin S; Brinkman, Cynthia K

2016-12-01

Polyhydroxyalkanoates (PHAs) are biodegradable polymers that can substitute for petroleum-based plastics in a variety of applications. One avenue to commercial PHA production involves coupling waste-based synthesis with the use of mixed microbial consortia (MMC). In this regard, production requires maximizing the enrichment of a MMC capable of feast-famine PHA synthesis, with the metabolic response induced through imposition of aerobic-dynamic feeding (ADF) conditions. However, the concept of PHA production in complex matrices remains unrefined; process operational improvements are needed, along with an enhanced understanding of the MMC. Research presented herein investigated the effect of aeration on feast-famine PHA synthesis, with four independent aeration state systems studied; MMC were fed volatile fatty acid (VFA)-rich fermented dairy manure. Regardless of the aeration state, all MMC exhibited a feast-famine response based on observed carbon cycling. Moreover, there was no statistical difference in PHA synthesis rates, with q PHA ranging from 0.10 to 0.19 CmmolPHA gVSS -1 min -1 ; VFA uptake rates exhibited similar statistical indifferences. PHA production assessments on the enriched MMC resulted in maximum intracellular concentrations ranging from 22.5 to 90.7% (mgPHA mgVSS -1 ); at maximum concentration, the mean hydroxyvalerate mol content was 73 ± 0.6%. While a typical feast-famine dissolved oxygen (DO) pattern was observed at maximum aeration, less resolution was observed at decreasing aeration rates, suggesting that DO may not be an optimal process monitoring parameter. At lower aeration states, nitrogen cycling patterns, supported by molecular investigations targeting AOBs and NOBs, indicate that NO 2 and NO 3 sustained feast-famine PHA synthesis. Next-generation sequencing analysis of the respective MMC revealed numerous and diverse genera exhibiting the potential to achieve PHA synthesis, suggesting functional redundancy embedded in the diverse MMC. Ultimately, results demonstrate that aeration can be controlled in waste-based ADF systems to sustain PHA production potential, while enriching for a diverse MMC that exhibits potential functional redundancy. Reduced aeration could also enhance cost competitiveness of waste-based PHA production, with potential further benefits associated with nitrogen treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Biosynthesis of Polyhydroxyalkanoate from Steamed Soybean Wastewater by a Recombinant Strain of Pseudomonas sp. 61-3

PubMed Central

Hokamura, Ayaka; Yunoue, Yuko; Goto, Saki; Matsusaki, Hiromi

2017-01-01

Pseudomonas sp. 61-3 accumulates a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer, poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) [P(3HB-co-3HA)], consisting of 3HA units of 4–12 carbon atoms. Pseudomonas sp. 61-3 possesses two types of PHA synthases, PHB synthase (PhbC) and PHA synthases (PhaC1 and PhaC2), encoded by the phb and pha loci, respectively. The P(94 mol% 3HB-co-6 mol% 3HA) copolymer synthesized by the recombinant strain of Pseudomonas sp. 61-3 (phbC::tet) harboring additional copies of phaC1 gene is known to have desirable physical properties and to be a flexible material with moderate toughness, similar to low-density polyethylene. In this study, we focused on the production of the P(3HB-co-3HA) copolymer using steamed soybean wastewater, a by-product in brewing miso, which is a traditional Japanese seasoning. The steamed soybean wastewater was spray-dried to produce a powder (SWP) and used as the sole nitrogen source for the synthesis of P(3HB-co-3HA) by the Pseudomonas sp. 61-3 recombinant strain. Hydrolyzed SWP (HSWP) was also used as a carbon and nitrogen source. P(3HB-co-3HA)s with relatively high 3HB fractions could be synthesized by a recombinant strain of Pseudomonas sp. 61-3 (phbC::tet) harboring additional copies of the phaC1 gene in the presence of 2% glucose and 10–20 g/L SWP as the sole nitrogen source, producing a PHA concentration of 1.0–1.4 g/L. When HSWP was added to a nitrogen- and carbon-free medium, the recombinant strain could synthesize PHA without glucose as a carbon source. The recombinant strain accumulated 32 wt% P(3HB-co-3HA) containing 80 mol% 3HB and 20 mol% medium-chain-length 3HA with a PHA concentration of 1.0 g/L when 50 g/L of HSWP was used. The PHA production yield was estimated as 20 mg-PHA/g-HSWP, which equates to approximately 1.0 g-PHA per liter of soybean wastewater. PMID:28952548

24 CFR 982.302 - Issuance of voucher; Requesting PHA approval of assisted tenancy.

Code of Federal Regulations, 2013 CFR

2013-04-01

... family is selected, or when a participant family wants to move to another unit, the PHA issues a voucher to the family. The family may search for a unit. (b) If the family finds a unit, and the owner is willing to lease the unit under the program, the family may request PHA approval of the tenancy. The PHA...

24 CFR 982.302 - Issuance of voucher; Requesting PHA approval of assisted tenancy.

Code of Federal Regulations, 2012 CFR

2012-04-01

... family is selected, or when a participant family wants to move to another unit, the PHA issues a voucher to the family. The family may search for a unit. (b) If the family finds a unit, and the owner is willing to lease the unit under the program, the family may request PHA approval of the tenancy. The PHA...

24 CFR 982.302 - Issuance of voucher; Requesting PHA approval of assisted tenancy.

Code of Federal Regulations, 2010 CFR

2010-04-01

... family is selected, or when a participant family wants to move to another unit, the PHA issues a voucher to the family. The family may search for a unit. (b) If the family finds a unit, and the owner is willing to lease the unit under the program, the family may request PHA approval of the tenancy. The PHA...

24 CFR 982.302 - Issuance of voucher; Requesting PHA approval of assisted tenancy.

Code of Federal Regulations, 2014 CFR

2014-04-01

... family is selected, or when a participant family wants to move to another unit, the PHA issues a voucher to the family. The family may search for a unit. (b) If the family finds a unit, and the owner is willing to lease the unit under the program, the family may request PHA approval of the tenancy. The PHA...

24 CFR 982.302 - Issuance of voucher; Requesting PHA approval of assisted tenancy.

Code of Federal Regulations, 2011 CFR

2011-04-01

... family is selected, or when a participant family wants to move to another unit, the PHA issues a voucher to the family. The family may search for a unit. (b) If the family finds a unit, and the owner is willing to lease the unit under the program, the family may request PHA approval of the tenancy. The PHA...

Engineering Mycobacteria for the Production of Self-Assembling Biopolyesters Displaying Mycobacterial Antigens for Use as a Tuberculosis Vaccine.

PubMed

Lee, Jason W; Parlane, Natalie A; Rehm, Bernd H A; Buddle, Bryce M; Heiser, Axel

2017-03-01

Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis or Mycobacterium bovis and still remains one of the world's biggest global health burdens. Recently, engineered polyhydroxyalkanoate (PHA) biobeads that were produced in both Escherichia coli and Lactococcus lactis and displayed mycobacterial antigens were found to induce significant cell-mediated immune responses in mice. We observed that such PHA beads contained host cell proteins as impurities, which we hypothesized to have the potential to induce immunity. In this study, we aimed to develop PHA beads produced in mycobacteria (mycobacterial PHA biobeads [MBB]) and test their potential as a TB vaccine in a mouse model. As a model organism, nonpathogenic Mycobacterium smegmatis was engineered to produce MBB or MBB with immobilized mycobacterial antigens Ag85A and ESAT-6 on their surface (A:E-MBB). Three key enzymes involved in the poly(3-hydroxybutyric acid) pathway, namely, β-ketothiolase (PhaA), acetoacetyl-coenzyme A reductase (PhaB), and PHA synthase (PhaC), were engineered into E. coli - Mycobacterium shuttle plasmids and expressed in trans Immobilization of specific antigens to the surface of the MBB was achieved by creating a fusion with the PHA synthase which remains covalently attached to the polyester core, resulting in PHA biobeads displaying covalently immobilized antigens. E-MBB, and an M. smegmatis vector control (MVC) were used in a mouse immunology trial, with comparison to phosphate-buffered saline (PBS)-vaccinated and Mycobacterium bovis BCG-vaccinated groups. We successfully produced MBB and A:E-MBB and used them as vaccines to induce a cellular immune response to mycobacterial antigens. IMPORTANCE Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis or Mycobacterium bovis and still remains one of the world's biggest global health burdens. In this study, we produced polyhydroxyalkanoate (PHA) biobeads in mycobacteria and used them as vaccines to induce a cellular immune response to mycobacterial antigens. Copyright © 2017 Lee et al.

Engineering Mycobacteria for the Production of Self-Assembling Biopolyesters Displaying Mycobacterial Antigens for Use as a Tuberculosis Vaccine

PubMed Central

Lee, Jason W.; Parlane, Natalie A.; Rehm, Bernd H. A.; Buddle, Bryce M.

2017-01-01

ABSTRACT Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis or Mycobacterium bovis and still remains one of the world's biggest global health burdens. Recently, engineered polyhydroxyalkanoate (PHA) biobeads that were produced in both Escherichia coli and Lactococcus lactis and displayed mycobacterial antigens were found to induce significant cell-mediated immune responses in mice. We observed that such PHA beads contained host cell proteins as impurities, which we hypothesized to have the potential to induce immunity. In this study, we aimed to develop PHA beads produced in mycobacteria (mycobacterial PHA biobeads [MBB]) and test their potential as a TB vaccine in a mouse model. As a model organism, nonpathogenic Mycobacterium smegmatis was engineered to produce MBB or MBB with immobilized mycobacterial antigens Ag85A and ESAT-6 on their surface (A:E-MBB). Three key enzymes involved in the poly(3-hydroxybutyric acid) pathway, namely, β-ketothiolase (PhaA), acetoacetyl-coenzyme A reductase (PhaB), and PHA synthase (PhaC), were engineered into E. coli-Mycobacterium shuttle plasmids and expressed in trans. Immobilization of specific antigens to the surface of the MBB was achieved by creating a fusion with the PHA synthase which remains covalently attached to the polyester core, resulting in PHA biobeads displaying covalently immobilized antigens. MBB, A:E-MBB, and an M. smegmatis vector control (MVC) were used in a mouse immunology trial, with comparison to phosphate-buffered saline (PBS)-vaccinated and Mycobacterium bovis BCG-vaccinated groups. We successfully produced MBB and A:E-MBB and used them as vaccines to induce a cellular immune response to mycobacterial antigens. IMPORTANCE Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis or Mycobacterium bovis and still remains one of the world's biggest global health burdens. In this study, we produced polyhydroxyalkanoate (PHA) biobeads in mycobacteria and used them as vaccines to induce a cellular immune response to mycobacterial antigens. PMID:28087528

Carbon flux to growth or polyhydroxyalkanoate synthesis under microaerophilic conditions is affected by fatty acid chain-length in Pseudomonas putida LS46.

PubMed

Blunt, Warren; Dartiailh, Christopher; Sparling, Richard; Gapes, Daniel; Levin, David B; Cicek, Nazim

2018-05-24

Economical production of medium-chain length polyhydroxyalkanoates (mcl-PHA) is dependent on efficient cultivation processes. This work describes growth and mcl-PHA synthesis characteristics of Pseudomonas putida LS46 when grown on medium-chain length fatty acids (octanoic acid) and lower-cost long-chain fatty acids (LCFAs, derived from hydrolyzed canola oil) in microaerophilic environments. Growth on octanoic acid ceased when the oxygen uptake rate was limited by the oxygen transfer rate, and mcl-PHA accumulated to 61.9% of the cell dry mass. From LCFAs, production of non-PHA cell mass continued at a rate of 0.36 g L -1  h -1 under oxygen-limited conditions, while mcl-PHA accumulated simultaneously to 31% of the cell dry mass. The titer of non-PHA cell mass from LCFAs at 14 h post-inoculation was double that obtained from octanoic acid in bioreactors operated with identical feeding and aeration conditions. While the productivity for octanoic acid was higher by 14 h, prolonged cultivation on LCFAs achieved similar productivity but with twice the PHA titer. Simultaneous co-feeding of each substrate demonstrated the continued cell growth under microaerophilic conditions characteristic of LCFAs, and the resulting polymer was dominant in C8 monomers. Furthermore, co-feeding resulted in improved PHA titer and volumetric productivity compared to either substrate individually. These results suggest that LCFAs improve growth of P. putida in oxygen-limited environments and could reduce production costs since more non-PHA cell mass, the cellular factories required to produce mcl-PHA and the most oxygen-intensive cellular process, can be produced for a given oxygen transfer rate.

The role of the maturation stage of thymocytes on the phytohemagglutinin and concanavalin A responses in thymocyte and lymph node lymphocyte cocultures in guinea pig. Effects of pretreatment of thymocytes with thymosin or levamisole.

PubMed

Soppi, E

1981-02-01

Guinea pig thymocytes (TH) and lymph node lymphocytes (LNL) synergized optimally in both phytohemagglutinin (PHA) and concanavalin (Con A) responses in mixtures containing 0.3 x 10(6) TH and 0.2 x 10(6) LNL. Using discontinuous albumin gradient centrifugation thymocytes were separated into two subpopulations (F4 and F6) at different stages of maturation. Immature, PHA and Con A unresponsive F6 thymocytes synergized significantly only in the PHA response. More mature, PHA and Con A responsive F4 thymocytes cooperated well in the Con A response, but only a small synergy was observed in the PHA response. Pretreatment of the unfractionated thymocytes with the low concentration (0.05 microgram/ml) of thymosin decreased significantly their capacity to interact with LNL in both PHA and Con A responses. Preincubation of F4 thymocytes with the high concentration (200 microgram/ml) of thymosin increased the synergy in PHA response. All other combinations with thymosin or levamisole and thymocytes were ineffective on the mitogenic responses in the TH and LNL cocultures. Altogether, the results how that the thymocyte populations that induce synergy in PHA, or in (PHA and) Con A responses represent the two subpopulations of thymocytes with different maturation stages. The differential effects of the two concentrations of thymosin on the thymocytes support further the concept that the thymocytes synergizing in responses to both mitogens are more mature than those synergizing only PHA response. Thus, the ability of thymocytes to interact with LNL is dependent on the maturation stage of thymocytes, and can be utilized to study the differentiation of thymocytes.

Improved intracellular PHA determinations with novel spectrophotometric quantification methodologies based on Sudan black dye.

PubMed

Porras, Mauricio A; Villar, Marcelo A; Cubitto, María A

2018-05-01

The presence of intracellular polyhydroxyalkanoates (PHAs) is usually studied using Sudan black dye solution (SB). In a previous work it was shown that the PHA could be directly quantified using the absorbance of SB fixed by PHA granules in wet cell samples. In the present paper, the optimum SB amount and the optimum conditions to be used for SB assays were determined following an experimental design by hybrid response surface methodology and desirability-function. In addition, a new methodology was developed in which it is shown that the amount of SB fixed by PHA granules can also be determined indirectly through the absorbance of the supernatant obtained from the stained cell samples. This alternative methodology allows a faster determination of the PHA content (involving 23 and 42 min for indirect and direct determinations, respectively), and can be undertaken by means of basic laboratory equipment and reagents. The correlation between PHA content in wet cell samples and the spectra of the SB stained supernatant was determined by means of multivariate and linear regression analysis. The best calibration adjustment (R 2  = 0.91, RSE: 1.56%), and the good PHA prediction obtained (RSE = 1.81%), shows that the proposed methodology constitutes a reasonably precise way for PHA content determination. Thus, this methodology could anticipate the probable results of the above mentioned direct PHA determination. Compared with the most used techniques described in the scientific literature, the combined implementation of these two methodologies seems to be one of the most economical and environmentally friendly, suitable for rapid monitoring of the intracellular PHA content. Copyright © 2018 Elsevier B.V. All rights reserved.

Synthetic Biology of Polyhydroxyalkanoates (PHA).

PubMed

Meng, De-Chuan; Chen, Guo-Qiang

Microbial polyhydroxyalkanoates (PHA) are a family of biodegradable and biocompatible polyesters which have been extensively studied using synthetic biology and metabolic engineering methods for improving production and for widening its diversity. Synthetic biology has allowed PHA to become composition controllable random copolymers, homopolymers, and block copolymers. Recent developments showed that it is possible to establish a microbial platform for producing not only random copolymers with controllable monomers and their ratios but also structurally defined homopolymers and block copolymers. This was achieved by engineering the genome of Pseudomonas putida or Pseudomonas entomophiles to weaken the β-oxidation and in situ fatty acid synthesis pathways, so that a fatty acid fed to the bacteria maintains its original chain length and structures when incorporated into the PHA chains. The engineered bacterium allows functional groups in a fatty acid to be introduced into PHA, forming functional PHA, which, upon grafting, generates endless PHA variety. Recombinant Escherichia coli also succeeded in producing efficiently poly(3-hydroxypropionate) or P3HP, the strongest member of PHA. Synthesis pathways of P3HP and its copolymer P3HB3HP of 3-hydroxybutyrate and 3-hydroxypropionate were assembled respectively to allow their synthesis from glucose. CRISPRi was also successfully used to manipulate simultaneously multiple genes and control metabolic flux in E. coli to obtain a series of copolymer P3HB4HB of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB). The bacterial shapes were successfully engineered for enhanced PHA accumulation.

Effect of various refrigeration temperatures on quality of shell eggs.

PubMed

Shin, Daekeun; Narciso-Gaytán, Carlos; Regenstein, Joe M; Sánchez-Plata, Marcos X

2012-05-01

The objective of this study was to evaluate the effects of low storage temperatures on shell egg quality. Approximately 2100 shell eggs were collected and stored at - 1.1, 0.6, 2.2, 3.9, 5.6 and 7.2 °C for up to 4 weeks. Eighteen eggs at each storage temperature were evaluated after 0, 2, 7, 14, 21 and 28 days of storage. Haugh units (HU), yolk index (YI), albumen pH (pHA), yolk pH (pHY) and angel food cake density (CD) were measured. Shell egg quality tended to be preserved better at below 2.2 °C, as high HU and YI values relative to eggs stored at 7.2 °C were determined on day 28. However, storage at - 1.1 °C tended to cause the opposite effect, especially highly declined HU values over time. Significantly different HU values of shell eggs were measured after 14 days of storage, with eggs stored at 0.6 and 2.2 °C having the highest HU values, 80.42 and 77.97 respectively. A lower temperature limit for shell egg storage could be established between 0.6 and 2.2 °C, as both temperatures showed the highest HU values, 77.88 and 77.60 respectively, after 28 days of storage. Copyright © 2011 Society of Chemical Industry.

Cloning and heterologous expression of a novel subgroup of class IV polyhydroxyalkanoate synthase genes from the genus Bacillus.

PubMed

Mizuno, Kouhei; Kihara, Takahiro; Tsuge, Takeharu; Lundgren, Benjamin R; Sarwar, Zaara; Pinto, Atahualpa; Nomura, Christopher T

2017-01-01

Many microorganisms harbor genes necessary to synthesize biodegradable plastics known as polyhydroxyalkanoates (PHAs). We surveyed a genomic database and discovered a new cluster of class IV PHA synthase genes (phaRC). These genes are different in sequence and operon structure from any previously reported PHA synthase. The newly discovered PhaRC synthase was demonstrated to produce PHAs in recombinant Escherichia coli.

Producing microbial polyhydroxyalkanoate (PHA) biopolyesters in a sustainable manner.

PubMed

Koller, Martin; Maršálek, Lukáš; de Sousa Dias, Miguel Miranda; Braunegg, Gerhart

2017-07-25

Sustainable production of microbial polyhydroxyalkanoate (PHA) biopolyesters on a larger scale has to consider the "four magic e": economic, ethical, environmental, and engineering aspects. Moreover, sustainability of PHA production can be quantified by modern tools of Life Cycle Assessment. Economic issues are to a large extent affected by the applied production mode, downstream processing, and, most of all, by the selection of carbon-rich raw materials as feedstocks for PHA production by safe and naturally occurring wild type microorganisms. In order to comply with ethics, such raw materials should be used which do not interfere with human nutrition and animal feed supply chains, and shall be convertible towards accessible carbon feedstocks by simple methods of upstream processing. Examples were identified in carbon-rich waste materials from various industrial braches closely connected to food production. Therefore, the article shines a light on hetero-, mixo-, and autotrophic PHA production based on various industrial residues from different branches. Emphasis is devoted to the integration of PHA-production based on selected raw materials into the holistic patterns of sustainability; this encompasses the choice of new, powerful microbial production strains, non-hazardous, environmentally benign methods for PHA recovery, and reutilization of waste streams from the PHA production process itself. Copyright © 2016 Elsevier B.V. All rights reserved.

Synthesis and function of polyhydroxyalkanoates in anaerobic syntrophic bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

McInerney, M.J.; Amos, D.A.; Kealy, K.S.

1992-12-31

Anaerobic syntrophic bacteria degrade fatty acids and some aromatic compounds which are important intermediates in the degradation of organic matter to CO{sub 2} and CH{sub 4} in methanogenic environments. Several of the described syntrophic species produce poly-{beta}-hydroxyalkanoate (PHA) suggesting that the synthesis and use of PHA is important in their physiology. In the fatty acid-degrading, syntrophic bacterium, Syntrophomonas wolfei, PHA is made during exponential phase of growth and used after growth has stopped and substrate levels are low. Altering the carbon to nitrogen ratio of the medium does not affect the amount of PHA made or its monomeric composition. Itmore » is hypothesized that PHA serves as an endogenous energy source for syntrophic bacteria when the concentrations of hydrogen or acetate are too high for the degradation of the growth substrate to be thermodynamically favorable. In S. wolfei, PHA is synthesized by two routes, the direct incorporation of 3-ketoacyl-coenzyme A (CoA) generated in {beta}-oxidation without cleavage of a C-C bond, and by the condensation and subsequent reduction of two acetyl-CoA molecules. Genes that encode for the synthesis of PHA in S. wolfei have been cloned into Escherichia coli in order to understand the molecular mechanisms that regulate PHA synthesis. 61 refs., 1 fig., 4 tabs.« less

Potential and Prospects of Continuous Polyhydroxyalkanoate (PHA) Production

PubMed Central

Koller, Martin; Braunegg, Gerhart

2015-01-01

Together with other so-called “bio-plastics”, Polyhydroxyalkanoates (PHAs) are expected to soon replace established polymers on the plastic market. As a prerequisite, optimized process design is needed to make PHAs attractive in terms of costs and quality. Nowadays, large-scale PHA production relies on discontinuous fed-batch cultivation in huge bioreactors. Such processes presuppose numerous shortcomings such as nonproductive time for reactor revamping, irregular product quality, limited possibility for supply of certain carbon substrates, and, most of all, insufficient productivity. Therefore, single- and multistage continuous PHA biosynthesis is increasingly investigated for production of different types of microbial PHAs; this goes for rather crystalline, thermoplastic PHA homopolyesters as well as for highly flexible PHA copolyesters, and even blocky-structured PHAs consisting of alternating soft and hard segments. Apart from enhanced productivity and constant product quality, chemostat processes can be used to elucidate kinetics of cell growth and PHA formation under constant process conditions. Furthermore, continuous enrichment processes constitute a tool to isolate novel powerful PHA-producing microbial strains adapted to special environmental conditions. The article discusses challenges, potential and case studies for continuous PHA production, and shows up new strategies to further enhance such processes economically by developing unsterile open continuous processes combined with the application of inexpensive carbon feedstocks. PMID:28955015

Prolonged post-hyperventilation apnea in two young adults with hyperventilation syndrome

PubMed Central

2013-01-01

Background The prognosis of hyperventilation syndrome (HVS) is generally good. However, it is important to proceed with care when treating HVS because cases of death following hyperventilation have been reported. This paper was done to demonstrate the clinical risk of post-hyperventilation apnea (PHA) in patients with HVS. Case presentation We treated two patients with HVS who suffered from PHA. The first, a 21-year-old woman, had a maximum duration of PHA of about 3.5 minutes and an oxygen saturation (SpO2) level of 60%. The second patient, a 22-year-old woman, had a maximum duration of PHA of about 3 minutes and an SpO2 level of 66%. Both patients had loss of consciousness and cyanosis. Because there is no widely accepted regimen for treating patients with prolonged PHA related to HVS, we administered artificial ventilation to both patients using a bag mask and both recovered without any after effects. Conclusion These cases show that some patients with HVS develop prolonged PHA or severe hypoxia, which has been shown to lead to death in some cases. Proper treatment must be given to patients with HVS who develop PHA to protect against this possibility. If prolonged PHA or severe hypoxemia arises, respiratory assistance using a bag mask must be done immediately. PMID:23594702

Conversion of post consumer polyethylene to the biodegradable polymer polyhydroxyalkanoate.

PubMed

Guzik, Maciej W; Kenny, Shane T; Duane, Gearoid F; Casey, Eoin; Woods, Trevor; Babu, Ramesh P; Nikodinovic-Runic, Jasmina; Murray, Michael; O'Connor, Kevin E

2014-05-01

A process for the conversion of post consumer (agricultural) polyethylene (PE) waste to the biodegradable polymer medium chain length polyhydroxyalkanoate (mcl-PHA) is reported here. The thermal treatment of PE in the absence of air (pyrolysis) generated a complex mixture of low molecular weight paraffins with carbon chain lengths from C8 to C32 (PE pyrolysis wax). Several bacterial strains were able to grow and produce PHA from this PE pyrolysis wax. The addition of biosurfactant (rhamnolipids) allowed for greater bacterial growth and PHA accumulation of the tested strains. Some strains were only capable of growth and PHA accumulation in the presence of the biosurfactant. Pseudomonas aeruginosa PAO-1 accumulated the highest level of PHA with almost 25 % of the cell dry weight as PHA when supplied with the PE pyrolysis wax in the presence of rhamnolipids. The change of nitrogen source from ammonium chloride to ammonium nitrate resulted in faster bacterial growth and the earlier onset of PHA accumulation. To our knowledge, this is the first report where PE is used as a starting material for production of a biodegradable polymer.

Crystallization and preliminary X-ray analysis of a novel thermoalkalophilic poly(3-hydroxybutyrate) depolymerase (PhaZ7) from Paucimonas lemoignei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kapetaniou, Evangelia G.; Braaz, Reinhard; Jendrossek, Dieter

2005-05-01

A novel thermoalkalophilic depolymerase, PhaZ7, from P. lemoignei was crystallized by the microdialysis technique. Crystals belong to space group C2 and diffract to 2.75 Å resolution at a synchrotron source. Polyhydroxyalkanoates (PHA) are biodegradable polyesters that have attracted commercial and academic interest as environmentally friendly materials. A number of enzymes are able to degrade polyhydroxyalkanoates to water-soluble products. PhaZ7 poly(3-hydroxybutyrate) (PHB) depolymerase (EC 3.1.1.75), a 342-amino-acid hydrolase from the PHA-degrading bacterium Paucimonas lemoignei, has been found to possess substrate specificity for amorphous PHA. PhaZ7 was crystallized by the microdialysis method. Thin rod-like crystals were grown in low ionic strength solutionmore » and found to belong to the monoclinic space group C2, with unit-cell parameters a = 225.8, b = 46.5, c = 171.3, β = 128.9°. A complete data set was collected to 2.75 Å resolution at 100 K using synchrotron radiation.« less

Emerging bone tissue engineering via Polyhydroxyalkanoate (PHA)-based scaffolds.

PubMed

Lim, Janice; You, Mingliang; Li, Jian; Li, Zibiao

2017-10-01

Polyhydroxyalkanoates (PHAs) are a class of biodegradable polymers derived from microorganisms. On top of their biodegradability and biocompatibility, different PHA types can contribute to varying mechanical and chemical properties. This has led to increasing attention to the use of PHAs in numerous biomedical applications over the past few decades. Bone tissue engineering refers to the regeneration of new bone through providing mechanical support while inducing cell growth on the PHA scaffolds having a porous structure for tissue regeneration. This review first introduces the various properties PHA scaffold that make them suitable for bone tissue engineering such as biocompatibility, biodegradability, mechanical properties as well as vascularization. The typical fabrication techniques of PHA scaffolds including electrospinning, salt-leaching and solution casting are further discussed, followed by the relatively new technology of using 3D printing in PHA scaffold fabrication. Finally, the recent progress of using different types of PHAs scaffold in bone tissue engineering applications are summarized in intrinsic PHA/blends forms or as composites with other polymeric or inorganic hybrid materials. Copyright © 2017 Elsevier B.V. All rights reserved.

24 CFR 982.202 - How applicants are selected: General requirements.

Code of Federal Regulations, 2010 CFR

2010-04-01

... but not limited to 24 CFR part 5, subpart L, protection for victims of domestic violence, dating violence, and stalking, and with PHA policies stated in the PHA administrative plan and the PHA plan. The...

Phase angle as a nutritional evaluation tool in all stages of chronic liver disease.

PubMed

Peres, W A F; Lento, D F; Baluz, K; Ramalho, A

2012-01-01

Malnutrition is commonly and frequently under-diagnosed in clinical settings in patients with chronic liver disease (CLD) due to the limitations of nutritional evaluation methods in this population. We hypothesized that the bioelectrical impedance analysis derived phase angle (BIA-derived PhA) might be considered as a nutritional indicator in CLD since it represents either cell death or malnutrition characterized by changes in cellular membrane integrity. The aim of this study was to evaluate the BIA-derived PhA as a nutritional evaluation tool in all stages of CLD, including chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). Liver-related death and survival were evaluated. A total of 66 patients were enrolled in a cross-sectional study. For the nutritional diagnosis, mid-arm circumference (MAC), triceps skinfold thickness (TST), mid-arm muscle circumference (MAMC) and Subject Global Assessment (SGA) were evaluated. Biochemical and clinical evaluations were performed. Our results showed that PhA was higher in well-nourished patients, according to SGA and in the patients without hepatic encephalopathy. PhA correlated significantly with MAMC, MAC and albumin and was inversely correlated with age. No correlation was found between PhA values and the Child-Pugh score and ascites. PhA was strongly associated with survival and PhA ≤ 5.18º with relative risk increase of 2.5 for death. We conclude that the BIA-derived PhA is a relevant nutritional evaluation tool in chronic hepatitis, liver cirrhosis and HCC and the role of PhA in the prediction of survival in CLD should be examined further in a controlled study.

Rapid spectrofluorometric screening of poly-hydroxyalkanoate-producing bacteria from microbial mats.

PubMed

Berlanga, Mercedes; Montero, M T; Fernández-Borrell, Jordi; Guerrero, Ricardo

2006-06-01

Microbial mat ecosystems are characterized by both seasonal and diel fluctuations in several physicochemical variables, so that resident microorganisms must frequently adapt to the changing conditions of their environment. It has been pointed out that, under stress conditions, bacterial cells with higher contents of poly-hydroxyalkanoates (PHA) survive longer than those with lower PHA content. In the present study, PHA-producing strains from Ebro Delta microbial mats were selected using the Nile red dying technique and the relative accumulation of PHA was monitored during further laboratory cultivation. The number of heterotrophic isolates in trypticase soy agar (TSA) was ca. 107 colony-forming units/g microbial mat. Of these, 100 randomly chosen colonies were replicated on mineral salt agar limited in nitrogen, and Nile red was added to the medium to detect PHA. Orange fluorescence, produced upon binding of the dye to polymer granules in the cell, was detected in approximately 10% of the replicated heterotrophic isolates. The kinetics of PHA accumulation in Pseudomonas putida, and P. oleovorans were compared with those of several of the environmental isolates spectrofluorometry. PHA accumulation, measured as relative fluorescence intensity, resulted in a steady-state concentration after 48 h of incubation in all strains assayed. At 72 h, the maximum fluorescence intensity of each strain incubated with glucose and fructose was usually similar. MAT-28 strain accumulated more PHA than the other isolates. The results show that data obtained from environmental isolates can highly improve studies based on modeling-simulation programs, and that microbial mats constitute an excellent source for the isolation of PHA-producing strains with industrial applications.

Production and characterization of a biodegradable poly (hydroxybutyrate-co-hydroxyvalerate) (PHB-co-PHV) copolymer by moderately haloalkalitolerant Halomonas campisalis MCM B-1027 isolated from Lonar Lake, India.

PubMed

Kulkarni, S O; Kanekar, P P; Nilegaonkar, S S; Sarnaik, S S; Jog, J P

2010-12-01

Several microorganisms produce polyhydroxyalkanoates (PHA). They are accumulated intracellularly as energy storage compounds. The PHAs are of interest because of their potential in biomedical applications. Halophilic bacteria and archaea are known to produce polyhydroxybutyrate (PHB). This paper describes production of a biodegradable copolymer, PHB-co-PHV by a moderately haloalkalitolerant Halomonas campisalis, isolated from Lonar Lake, India. The production of PHA was in the range of 45-81% on dry cell weight basis when the organism was grown in a production medium containing 1% (w/v) maltose and 0.1% (w/v) yeast extract, at pH ranging from 6 to 9 with an inoculum density of 10(5)-10(7) cells/ml of medium, for incubation period of 15-30 h and at 37 degrees C. The polymer produced by the organism is a hydroxyester with molecular weight of 1.3014 x 10(6). Its melting temperature was 171 degrees C. The (1)H NMR analysis revealed that the polymer was a copolymer of PHB-co-PHV. This could be achieved by providing simple carbon source viz. maltose. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Aerobic dynamic feeding as a strategy for in situ accumulation of polyhydroxyalkanoate in aerobic granules.

PubMed

Gobi, K; Vadivelu, V M

2014-06-01

Aerobic dynamic feeding (ADF) strategy was applied in sequencing batch reactor (SBR) to accumulate polyhydroxyalkanoate (PHA) in aerobic granules. The aerobic granules were able to remove 90% of the COD from palm oil mill effluent (POME). The volatile fatty acids (VFAs) in the POME are the sole source of the PHA accumulation. In this work, 100% removal of propionic and butyric acids in the POME were observed. The highest amount of PHA produced in aerobic granules was 0.6833mgPHA/mgbiomass. The PHA formed was identified as a P (hydroxybutyrate-co-hydroxyvalerate) P (HB-co-HV). Copyright © 2014 Elsevier Ltd. All rights reserved.

Methods for the Isolation of Genes Encoding Novel PHA Metabolism Enzymes from Complex Microbial Communities.

PubMed

Cheng, Jiujun; Nordeste, Ricardo; Trainer, Maria A; Charles, Trevor C

2017-01-01

Development of different PHAs as alternatives to petrochemically derived plastics can be facilitated by mining metagenomic libraries for diverse PHA cycle genes that might be useful for synthesis of bio-plastics. The specific phenotypes associated with mutations of the PHA synthesis pathway genes in Sinorhizobium meliloti and Pseudomonas putida, allows the use of powerful selection and screening tools to identify complementing novel PHA synthesis genes. Identification of novel genes through their function rather than sequence facilitates the functional proteins that may otherwise have been excluded through sequence-only screening methodology. We present here methods that we have developed for the isolation of clones expressing novel PHA metabolism genes from metagenomic libraries.

Advances in Polyhydroxyalkanoate (PHA) Production.

PubMed

Koller, Martin

2017-11-02

This editorial paper provides a synopsis of the contributions to the Bioengineering special issue "Advances in Polyhydroxyalkanoate (PHA) Production". It illustrates the embedding of the issue's individual research articles in the current global research and development landscape related to polyhydroxyalkanoates (PHA). The article shows how these articles are interrelated to each other, reflecting the entire PHA process chain including strain selection, metabolic and genetic considerations, feedstock evaluation, fermentation regimes, process engineering, and polymer processing towards high-value marketable products.

PHA Productivity and Yield of Ralstonia eutropha When Intermittently or Continuously Fed a Mixture of Short Chain Fatty Acids

PubMed Central

Chakraborty, Panchali; Muthukumarappan, Kasiviswanathan; Gibbons, William R.

2012-01-01

The research described in this present study was part of a larger effort focused on developing a dual substrate, dual fermentation process to produce Polyhydroxyalkanoate (PHA). The focus of this study was developing and optimizing a strategy for feeding a mixture of SCFAs (simulated ARF) and maximizing PHA production in a cost-effective way. Three different feeding strategies were examined in this study. The substrate evaluated in this study for the growth phase of R. eutropha was condensed corn solubles, a low-value byproduct of the dry-mill, corn ethanol industry. The culture was grown to high cell densities in nitrogen-supplemented condensed corn solubles media in 5 L bioreactors. The overall growth rate of R. eutropha was 0.2 h−1. The 20 mL ARF feeding every 3 h from 48 to 109 h strategy gave the best results in terms of PHA production. PHA productivity (0.0697 g L−1 h−1), PHA concentration (8.37 g L−1), and PHA content (39.52%) were the highest when ARF was fed every 3 h for 61 h. This study proved that condensed corn solubles can be potentially used as a growth medium to boost PHA production by R. eutropha thus reducing the overall cost of biopolymer production. PMID:23118512

Polyhydroxyalkanoate accumulation ability and associated microbial community in activated sludge-derived acetate-fed microbial cultures enriched under different temperature and pH conditions.

PubMed

Inoue, Daisuke; Suzuki, Yuta; Sawada, Kazuko; Sei, Kazunari

2018-03-01

The influence of temperature and pH during enrichment on the polyhydroxyalkanoate (PHA) accumulation ability and composition of PHA-accumulating microorganisms (PHAAMOs) in enrichment cultures was investigated. Enrichment of PHAAMOs from activated sludge was conducted in acetate-fed sequencing batch reactors using a feast-famine regime under different temperature (20°C, 28°C, and 36°C) and pH (controlled at 7.2 or not) conditions. PHA accumulation ability, which was evaluated in nitrogen- and phosphorus-deficient 24-h single-batch cultures, was greatly enhanced by enrichment, irrespective of the temperature and pH. Enrichment at 20°C or 28°C and without pH control seemed most appropriate for strong PHA accumulation. Analyses of the PHAAMO composition by the clone library method targeting phaC genes, which encode the class I and II PHA synthases, revealed that Burkholderiales were the dominant PHAAMOs in the seed sludge, while Rhodocyclales, specifically Azoarcus spp. and Thauera spp., were dominant after enrichment without pH control, showing a strong ability to accumulate PHA. The results indicated that Azoarcus spp. and Thauera spp. are key PHAAMOs in an enrichment culture based on the feast-famine method, with high PHA accumulation ability. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Phasins, Multifaceted Polyhydroxyalkanoate Granule-Associated Proteins

PubMed Central

Mezzina, Mariela P.

2016-01-01

Phasins are the major polyhydroxyalkanoate (PHA) granule-associated proteins. They promote bacterial growth and PHA synthesis and affect the number, size, and distribution of the granules. These proteins can be classified in 4 families with distinctive characteristics. Low-resolution structural studies and in silico predictions were performed in order to elucidate the structure of different phasins. Most of these proteins share some common structural features, such as a preponderant α-helix composition, the presence of disordered regions that provide flexibility to the protein, and coiled-coil interacting regions that form oligomerization domains. Due to their amphiphilic nature, these proteins play an important structural function, forming an interphase between the hydrophobic content of PHA granules and the hydrophilic cytoplasm content. Phasins have been observed to affect both PHA accumulation and utilization. Apart from their role as granule structural proteins, phasins have a remarkable variety of additional functions. Different phasins have been determined to (i) activate PHA depolymerization, (ii) increase the expression and activity of PHA synthases, (iii) participate in PHA granule segregation, and (iv) have both in vivo and in vitro chaperone activities. These properties suggest that phasins might play an active role in PHA-related stress protection and fitness enhancement. Due to their granule binding capacity and structural flexibility, several biotechnological applications have been developed using different phasins, increasing the interest in the study of these remarkable proteins. PMID:27287326

Phasins, Multifaceted Polyhydroxyalkanoate Granule-Associated Proteins.

PubMed

Mezzina, Mariela P; Pettinari, M Julia

2016-09-01

Phasins are the major polyhydroxyalkanoate (PHA) granule-associated proteins. They promote bacterial growth and PHA synthesis and affect the number, size, and distribution of the granules. These proteins can be classified in 4 families with distinctive characteristics. Low-resolution structural studies and in silico predictions were performed in order to elucidate the structure of different phasins. Most of these proteins share some common structural features, such as a preponderant α-helix composition, the presence of disordered regions that provide flexibility to the protein, and coiled-coil interacting regions that form oligomerization domains. Due to their amphiphilic nature, these proteins play an important structural function, forming an interphase between the hydrophobic content of PHA granules and the hydrophilic cytoplasm content. Phasins have been observed to affect both PHA accumulation and utilization. Apart from their role as granule structural proteins, phasins have a remarkable variety of additional functions. Different phasins have been determined to (i) activate PHA depolymerization, (ii) increase the expression and activity of PHA synthases, (iii) participate in PHA granule segregation, and (iv) have both in vivo and in vitro chaperone activities. These properties suggest that phasins might play an active role in PHA-related stress protection and fitness enhancement. Due to their granule binding capacity and structural flexibility, several biotechnological applications have been developed using different phasins, increasing the interest in the study of these remarkable proteins. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Development of a Novel Process Integrating the Treatment of Sludge Reject Water and the Production of Polyhydroxyalkanoates (PHAs).

PubMed

Frison, Nicola; Katsou, Evina; Malamis, Simos; Oehmen, Adrian; Fatone, Francesco

2015-09-15

Polyhydroxyalkanoates (PHAs) from activated sludge and renewable organic material can become an alternative product to traditional plastics since they are biodegradable and are produced from renewable sources. In this work, the selection of PHA storing bacteria was integrated with the side stream treatment of nitrogen removal via nitrite from sewage sludge reject water. A novel process was developed and applied where the alternation of aerobic-feast and anoxic-famine conditions accomplished the selection of PHA storing biomass and nitrogen removal via nitrite. Two configurations were examined: in configuration 1 the ammonium conversion to nitrite occurred in the same reactor in which the PHA selection process occurred, while in configuration 2 two separate reactors were used. The results showed that the selection of PHA storing biomass was successful in both configurations, while the nitrogen removal efficiency was much higher (almost 90%) in configuration 2. The PHA selection degree was evaluated by the volatile fatty acid (VFA) uptake rate (-qVFAs) and the PHA production rate (qPHA), which were 239 ± 74 and 89 ± 7 mg of COD per gram of active biomass (Xa) per hour, respectively. The characterization of the biopolymer recovered after the accumulation step, showed that it was composed of 3-hydroxybutyrate (3HB) (60%) and 3-hydroxyvalerate (3HV) (40%). The properties associated with the produced PHA suggest that they are suitable for thermoplastic processing.

Divergence of water balance mechanisms and acclimation potential in body color morphs of Drosophila ananassae.

PubMed

Parkash, Ravi; Aggarwal, Dau Dayal; Lambhod, Chanderkala; Singh, Divya

2014-01-01

Drosophila ananassae is a desiccation sensitive species, but the physiological basis of its abundance in the drier subtropical areas is largely unknown. We tested the hypothesis whether body color morphs of D. ananassae differ in the mechanistic basis of water conservation as well as desiccation acclimation potential, consistent with their distribution under dry or wet habitats. We observed reduced rate of water loss consistent with the greater desiccation potential of dark morph as compared with light morph, despite lack of quantitative differences in cuticular lipid mass between them. Dark morph evidenced greater wet and dry mass (∼1.17-fold) as well as higher hemolymph content (∼1.70-fold) and (∼17%) dehydration tolerance to sustain longer survival under desiccation stress (LT50 17.5 hr) as compared with light morph (LT50 4.3 hr). We found significant differences in the storage of energy metabolites in the body color morphs of D. ananassae, that is, carbohydrate content was significantly higher (∼0.18 mg/mg dry mass) in the dark morph as compared to light morph, but greater (∼0.05 mg/mg dry mass) body lipid content was evident in the light morph. Under desiccation stress, dark and light morphs utilized mainly carbohydrates but also lipids to a lesser extent. However, the rate of utilization of energy metabolites did not vary between dark and light morphs. Further, the dark morph consumed higher energy content derived from carbohydrates under desiccation stress as compared with the light morph. Finally, we found contrasting patterns of acclimation to desiccation stress in the two body color morphs, that is, increase in desiccation survival (4.7 hr), as well as in dehydration tolerance (∼6%) due to acclimation of the dark morph but no such effects were observed in the light morph. Thus, divergence in water balance mechanisms as well as acclimation potential reflects evolved physiological adaptations of the dark morph under drier but of the light morph to wet climatic conditions. © 2013 Wiley Periodicals, Inc.

Enhanced polyhydroxyalkanoate (PHA) production from the organic fraction of municipal solid waste by using mixed microbial culture.

PubMed

Colombo, Bianca; Favini, Francesca; Scaglia, Barbara; Sciarria, Tommy Pepè; D'Imporzano, Giuliana; Pognani, Michele; Alekseeva, Anna; Eisele, Giorgio; Cosentino, Cesare; Adani, Fabrizio

2017-01-01

In Europe, almost 87.6 million tonnes of food waste are produced. Despite the high biological value of food waste, traditional management solutions do not consider it as a precious resource. Many studies have reported the use of food waste for the production of high added value molecules. Polyhydroxyalkanoates (PHAs) represent a class of interesting bio-polyesters accumulated by different bacterial cells, and has been proposed for production from the organic fraction of municipal solid waste (OFMSW). Nevertheless, until now, no attention has been paid to the entire biological process leading to the transformation of food waste to organic acids (OA) and then to PHA, getting high PHA yield per food waste unit. In particular, the acid-generating process needs to be optimized, maximizing OA production from OFMSW. To do so, a pilot-scale Anaerobic Percolation Biocell Reactor (100 L in volume) was used to produce an OA-rich percolate from OFMSW which was used subsequently to produce PHA. The optimized acidogenic process resulted in an OA production of 151 g kg -1 from fresh OFMSW. The subsequent optimization of PHA production from OA gave a PHA production, on average, of 223 ± 28 g kg -1 total OA fed. Total mass balance indicated, for the best case studied, a PHA production per OFMSW weight unit of 33.22 ± 4.2 g kg -1 from fresh OFMSW, corresponding to 114.4 ± 14.5 g kg -1 of total solids from OFMSW. PHA composition revealed a hydroxybutyrate/hydroxyvalerate (%) ratio of 53/47 and Mw of 8∙10 5 kDa with a low polydispersity index, i.e. 1.4. This work showed how by optimizing acidic fermentation it could be possible to get a large amount of OA from OFMSW to be then transformed into PHA. This step is important as it greatly affects the total final PHA yield. Data obtained in this work can be useful as the starting point for considering the economic feasibility of PHA production from OFMSW by using mixed culture.

Quantitative ‘Omics Analyses of Medium Chain Length Polyhydroxyalkanaote Metabolism in Pseudomonas putida LS46 Cultured with Waste Glycerol and Waste Fatty Acids

PubMed Central

Fu, Jilagamazhi; Sharma, Parveen; Spicer, Vic; Krokhin, Oleg V.; Zhang, Xiangli; Fristensky, Brian; Cicek, Nazim; Sparling, Richard; Levin, David. B.

2015-01-01

Transcriptomes and proteomes of Pseudomonas putida LS46 cultured with biodiesel-derived waste glycerol or waste free fatty acids, as sole carbon sources, were compared under conditions that were either permissive or non-permissive for synthesis of medium chain length polyhydroxyalkanoates (mcl-PHA). The objectives of this study were to elucidate mechanisms that influence activation of biopolymer synthesis, intra-cellular accumulation, and monomer composition, and determine if these were physiologically specific to the carbon sources used for growth of P. putida LS46. Active mcl-PHA synthesis by P. putida LS46 was associated with high expression levels of key mcl-PHA biosynthesis genes and/or gene products including monomer-supplying proteins, PHA synthases, and granule-associated proteins. ‘Omics data suggested that expression of these genes were regulated by different genetic mechanisms in P. putida LS46 cells in different physiological states, when cultured on the two waste carbon sources. Optimal polymer production by P. putida LS46 was primarily limited by less efficient glycerol metabolism during mcl-PHA synthesis on waste glycerol. Mapping the ‘Omics data to the mcl-PHA biosynthetic pathway revealed significant variations in gene expression, primarily involved in: 1) glycerol transportation; 2) enzymatic reactions that recycle reducing equivalents and produce key mcl-PHA biosynthesis pathway intermediates (e.g. NADH/NADPH, acetyl-CoA). Active synthesis of mcl-PHAs was observed during exponential phase in cultures with waste free fatty acids, and was associated with the fatty acid beta-oxidation pathway. A putative Thioesterase in the beta-oxidation pathway that may regulate the level of fatty acid beta-oxidation intermediates, and thus carbon flux to mcl-PHA biosynthesis, was highly up-regulated. Finally, the data suggested that differences in expression of selected fatty acid metabolism and mcl-PHA monomer-supplying enzymes may play a role in determining the monomer composition of mcl-PHA polymers. Understanding the relationships between genome content, gene and gene product expression, and how these factors influence polymer synthesis, will aid in optimization of mcl-PHA production by P. putida LS46 using biodiesel waste streams. PMID:26544181

The cell agglutination agent, phytohemagglutinin-L, improves the efficiency of somatic nuclear transfer cloning in cattle (Bos taurus).

PubMed

Du, Fuliang; Shen, Perng-Chih; Xu, Jie; Sung, Li-Ying; Jeong, B-Seon; Lucky Nedambale, Tshimangadzo; Riesen, John; Cindy Tian, X; Cheng, Winston T K; Lee, Shan-Nan; Yang, Xiangzhong

2006-02-01

One of the several factors that contribute to the low efficiency of mammalian somatic cloning is poor fusion between the small somatic donor cell and the large recipient oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity on fusion rate, and subsequent developmental potential of cloned bovine embryos. The toxicity of PHA was established by examining its effects on the development of parthenogenetic bovine oocytes treated with different doses (Experiment 1), and for different durations (Experiment 2). The effective dose and duration of PHA treatment (150 microg/mL, 20 min incubation) was selected and used to compare membrane fusion efficiency and embryo development following somatic cell nuclear transfer (Experiment 3). Cloning with somatic donor fibroblasts versus cumulus cells was also compared, both with and without PHA treatment (150 microg/mL, 20 min). Fusion rate of nuclear donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61% (P < 0.05), and from 59 to 88% (P < 0.05) with cumulus cell nuclear donors. The nuclear transfer (NT) efficiency per oocyte used was improved following PHA treatment, for both fibroblast (13% versus 22%) as well as cumulus cells (17% versus 34%; P < 0.05). The cloned embryos, both with and without PHA treatment, were subjected to vitrification and embryo transfer testing, and resulted in similar survival (approximately 90% hatching) and pregnancy rates (17-25%). Three calves were born following vitrification and embryo transfer of these embryos; two from the PHA-treated group, and one from non-PHA control group. We concluded that PHA treatment significantly improved the fusion efficiency of somatic NT in cattle, and therefore, increased the development of cloned blastocysts. Furthermore, within a determined range of dose and duration, PHA had no detrimental effect on embryo survival post-vitrification, nor on pregnancy or calving rates following embryo transfer.

Integrated analysis of gene expression and metabolic fluxes in PHA-producing Pseudomonas putida grown on glycerol.

PubMed

Beckers, Veronique; Poblete-Castro, Ignacio; Tomasch, Jürgen; Wittmann, Christoph

2016-05-03

Given its high surplus and low cost, glycerol has emerged as interesting carbon substrate for the synthesis of value-added chemicals. The soil bacterium Pseudomonas putida KT2440 can use glycerol to synthesize medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHA), a class of biopolymers of industrial interest. Here, glycerol metabolism in P. putida KT2440 was studied on the level of gene expression (transcriptome) and metabolic fluxes (fluxome), using precisely adjusted chemostat cultures, growth kinetics and stoichiometry, to gain a systematic understanding of the underlying metabolic and regulatory network. Glycerol-grown P. putida KT2440 has a maintenance energy requirement [0.039 (mmolglycerol (gCDW h)(-1))] that is about sixteen times lower than that of other bacteria, such as Escherichia coli, which provides a great advantage to use this substrate commercially. The shift from carbon (glycerol) to nitrogen (ammonium) limitation drives the modulation of specific genes involved in glycerol metabolism, transport electron chain, sensors to assess the energy level of the cell, and PHA synthesis, as well as changes in flux distribution to increase the precursor availability for PHA synthesis (Entner-Doudoroff pathway and pyruvate metabolism) and to reduce respiration (glyoxylate shunt). Under PHA-producing conditions (N-limitation), a higher PHA yield was achieved at low dilution rate (29.7 wt% of CDW) as compared to a high rate (12.8 wt% of CDW). By-product formation (succinate, malate) was specifically modulated under these regimes. On top of experimental data, elementary flux mode analysis revealed the metabolic potential of P. putida KT2440 to synthesize PHA and identified metabolic engineering targets towards improved production performance on glycerol. This study revealed the complex interplay of gene expression levels and metabolic fluxes under PHA- and non-PHA producing conditions using the attractive raw material glycerol as carbon substrate. This knowledge will form the basis for the development of future metabolically engineered hyper-PHA-producing strains derived from the versatile bacterium P. putida KT2440.

Effects of 12-o-tetradecanoyl-phorbol-13-acetate (TPA) on the colony growth of human T lymphocytes.

PubMed Central

Foa, R; Lusso, P; Fierro, M T; Giubellino, M C; Ferrando, M L; Pegoraro, L

1984-01-01

The T colony promoting activity of 12-o-tetradecanoyl-phorbol-13-acetate (TPA) was assessed in a double layer culture assay which is dependent on the simultaneous presence of phytohaemagglutinin (PHA) and a leucocyte rich underlayer. TPA (10(-8) M) incorporated in the overlayer in place of PHA was capable of promoting T cell growth in the form of clusters in all 37 experiments performed and in the form of colonies in more than 50% of the samples tested. However, the T colony promoting activity of TPA alone was markedly less evident and consistent than that of PHA (mean 13 +/- 19.9 s.d. colonies vs 168 +/- 78.6). TPA concentrations of 10(-6) M, 10(-9) M and 10(-10) M were practically ineffective. On the other hand, the number of colonies obtained when both TPA 10(8) M and PHA were incorporated in the overlayer was significantly higher (P less than 0.01) than that observed with PHA alone (mean 250 +/- 108.2 vs 178 +/- 84.5 colonies). When TPA concentrations of 10(-9) M and 10(-10) M were used in addition to PHA, the enhancing effect was less evident, while an inhibition of T colony growth was observed with TPA 10(-6) M + PHA. TPA 10(-8) M was also capable of enhancing T colony growth when incorporated in the leucocyte rich underlayer (222 +/- 98.6 vs 172 +/- 80.9 colonies). In all cultures with TPA the peak of growth was delayed compared with that of control experiments with PHA. These findings demonstrate that TPA, particularly when co-cultured with PHA, is an effective T colony promoting agent. The observation that the number of colonies formed in the presence of TPA plus PHA is higher than the sum of those observed with the two stimulators independently, suggests that their synergistic effect may be mediated via the production of colony stimulating soluble factors. PMID:6610514

Metabolic Engineering of Poly(3-Hydroxyalkanoates): From DNA to Plastic

PubMed Central

Madison, Lara L.; Huisman, Gjalt W.

1999-01-01

Poly(3-hydroxyalkanoates) (PHAs) are a class of microbially produced polyesters that have potential applications as conventional plastics, specifically thermoplastic elastomers. A wealth of biological diversity in PHA formation exists, with at least 100 different PHA constituents and at least five different dedicated PHA biosynthetic pathways. This diversity, in combination with classical microbial physiology and modern molecular biology, has now opened up this area for genetic and metabolic engineering to develop optimal PHA-producing organisms. Commercial processes for PHA production were initially developed by W. R. Grace in the 1960s and later developed by Imperial Chemical Industries, Ltd., in the United Kingdom in the 1970s and 1980s. Since the early 1990s, Metabolix Inc. and Monsanto have been the driving forces behind the commercial exploitation of PHA polymers in the United States. The gram-negative bacterium Ralstonia eutropha, formerly known as Alcaligenes eutrophus, has generally been used as the production organism of choice, and intracellular accumulation of PHA of over 90% of the cell dry weight have been reported. The advent of molecular biological techniques and a developing environmental awareness initiated a renewed scientific interest in PHAs, and the biosynthetic machinery for PHA metabolism has been studied in great detail over the last two decades. Because the structure and monomeric composition of PHAs determine the applications for each type of polymer, a variety of polymers have been synthesized by cofeeding of various substrates or by metabolic engineering of the production organism. Classical microbiology and modern molecular bacterial physiology have been brought together to decipher the intricacies of PHA metabolism both for production purposes and for the unraveling of the natural role of PHAs. This review provides an overview of the different PHA biosynthetic systems and their genetic background, followed by a detailed summation of how this natural diversity is being used to develop commercially attractive, recombinant processes for the large-scale production of PHAs. PMID:10066830

Site-directed saturation mutagenesis of polyhydroxylalkanoate synthase for efficient microbial production of poly[(R)-2-hydroxybutyrate].

PubMed

Hori, Chiaki; Oishi, Kenta; Matsumoto, Ken'ichiro; Taguchi, Seiichi; Ooi, Toshihiko

2018-06-01

In our previous study, artificial polyhydroxyalkanoate (PHA) poly[(R)-2-hydroxybutyrate] [P(2HB)] was successfully biosynthesized from racemic 2HB in recombinant Escherichia coli using an engineered PHA synthase, PhaC1 Ps (S325T/Q481K). Although P(2HB) has promising material properties, the low level of polymer production was a drawback. In this study, we performed directed evolution of PhaC1 Ps towards enhanced P(2HB) accumulation in E. coli by site-directed dual saturation mutagenesis at the positions 477 and 481, which was known for their potential in enhancing natural PHA accumulation. By using a screening on agar plates with Nile red, eight colonies were isolated which produced a greater amount of P(2HB) compared to a colony expressing the parent enzyme PhaC1 Ps (S325T/Q481K). Among them, the cells expressing PhaC1 Ps (S325T/S477R/Q481G) [ST/SR/QG] accumulated polymer at the highest level (up to 2.9-fold). As seen in PhaC1 Ps (ST/SR/QG), glycine and basic amino acid residues (K or R) were frequently found at the two positions of the select mutated enzymes. The enzymatic activity of PhaC1 Ps (ST/SR/QG) toward 2HB-CoA was approximately 3-fold higher than that of the parent enzyme. Additionally, expression levels of the select mutated enzymes were lower than the parent. These results indicated that PhaC1 Ps mutagenesis at the positions 477 and 481 increased specific activity toward 2HB-CoA and it could result in the enhanced production of P(2HB). Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic.

PubMed

Madison, L L; Huisman, G W

1999-03-01

Poly(3-hydroxyalkanoates) (PHAs) are a class of microbially produced polyesters that have potential applications as conventional plastics, specifically thermoplastic elastomers. A wealth of biological diversity in PHA formation exists, with at least 100 different PHA constituents and at least five different dedicated PHA biosynthetic pathways. This diversity, in combination with classical microbial physiology and modern molecular biology, has now opened up this area for genetic and metabolic engineering to develop optimal PHA-producing organisms. Commercial processes for PHA production were initially developed by W. R. Grace in the 1960s and later developed by Imperial Chemical Industries, Ltd., in the United Kingdom in the 1970s and 1980s. Since the early 1990s, Metabolix Inc. and Monsanto have been the driving forces behind the commercial exploitation of PHA polymers in the United States. The gram-negative bacterium Ralstonia eutropha, formerly known as Alcaligenes eutrophus, has generally been used as the production organism of choice, and intracellular accumulation of PHA of over 90% of the cell dry weight have been reported. The advent of molecular biological techniques and a developing environmental awareness initiated a renewed scientific interest in PHAs, and the biosynthetic machinery for PHA metabolism has been studied in great detail over the last two decades. Because the structure and monomeric composition of PHAs determine the applications for each type of polymer, a variety of polymers have been synthesized by cofeeding of various substrates or by metabolic engineering of the production organism. Classical microbiology and modern molecular bacterial physiology have been brought together to decipher the intricacies of PHA metabolism both for production purposes and for the unraveling of the natural role of PHAs. This review provides an overview of the different PHA biosynthetic systems and their genetic background, followed by a detailed summation of how this natural diversity is being used to develop commercially attractive, recombinant processes for the large-scale production of PHAs.

Selecting optimal feast-to-famine ratio for a new polyhydroxyalkanoate (PHA) production system fed by valerate-dominant sludge hydrolysate.

PubMed

Hao, Jiuxiao; Wang, Hui; Wang, Xiujin

2018-04-01

The feast-to-famine ratio (F/F) represents the extent of selective pressure during polyhydroxyalkanoate (PHA) culture selection. This study evaluated the effects of F/F on a new PHA production system by an enriched culture with valerate-dominant sludge hydrolysate and selected the optimal F/F. After the original F/F 1/3 was modified to 1/1, 1/2, 1/4, and 1/5, F/F did not affect their lengths of feast phase, but affected their biomass growth behaviors during the famine phase and PHA-producing abilities. The optimal F/F was 1/2, and compared with 1/3, it increased the maximal PHA content and the fraction of 3-hydroxyvalerate (3HV) and 3-hydroxy-2-methylvalerate (3H2MV) monomers, with higher productivity and better polymer properties. Although F/F 1/2 impaired the advantage of the dominant genus Delftia, it improved the PHA production rate while decreased biomass growth rate, meanwhile enhancing the utilization and conversion of valerate. These findings indicate that in contrast to previous studies using acetate-dominant substrate for PHA production, the new system fed by valerate-dominant substrate can adopt a higher F/F.

Cyclic adenosine 3′,5′-monophosphate in human lymphocytes. Alterations after phytohemagglutinin stimulation

PubMed Central

Smith, Jay W.; Steiner, Alton L.; Newberry, W. Marcus; Parker, Charles W.

1971-01-01

We have studied cyclic adenosine 3′,5′-monophosphate (cyclic AMP) concentrations in human peripheral blood lymphocytes after stimulation with phytohemagglutinin (PHA), isoproterenol, prostaglandins, and aminophylline. Purified lymphocytes were obtained by nylon fiber chromatography, and low speed centrifugation to remove platelets. Cyclic AMP levels were determined by a highly sensitive radioimmunoassay. At concentrations of 0.1-1.0 mmoles/liter isoproterenol and aminophylline produced moderate increases in cyclic AMP concentrations, whereas prostaglandins produced marked elevations. High concentrations of PHA produced 25-300% increases in cyclic AMP levels, alterations being demonstrated within 1-2 min. The early changes in cyclic AMP concentration appear to precede previously reported metabolic changes in PHA-stimulated cells. After 6 hr cyclic AMP levels in PHA-stimulated cells had usually fallen to the levels of control cells. After 24 hr the level in PHA-stimulated cells was characteristically below that of the control cells. Adenyl cyclase, the enzyme which converts ATP to cyclic AMP, was measured in lymphocyte homogenates. Adenyl cyclase activity was rapidly stimulated by fluoride, isoproterenol, prostaglandins, and PHA. Since adenyl cyclase is characteristically localized in external cell membranes, our results are consistent with an initial action of PHA at this level. PMID:4395563

24 CFR 960.605 - How PHA administers service requirements.

Code of Federal Regulations, 2010 CFR

2010-04-01

... Service Activities or Self-Sufficiency Work Activities § 960.605 How PHA administers service requirements... economic self-sufficiency requirements for public housing residents. (b) Administration of qualifying community service or self-sufficiency activities for residents. The PHA may administer qualifying community...

Advances in Polyhydroxyalkanoate (PHA) Production

PubMed Central

2017-01-01

This editorial paper provides a synopsis of the contributions to the Bioengineering special issue “Advances in Polyhydroxyalkanoate (PHA) Production”. It illustrates the embedding of the issue’s individual research articles in the current global research and development landscape related to polyhydroxyalkanoates (PHA). The article shows how these articles are interrelated to each other, reflecting the entire PHA process chain including strain selection, metabolic and genetic considerations, feedstock evaluation, fermentation regimes, process engineering, and polymer processing towards high-value marketable products. PMID:29099065

Inhibitory effects of p-cresol and p-hydroxy anisole dimers on expression of the cyclooxygenase-2 gene and lipopolysaccharide-stimulated activation of nuclear factor-κB in RAW264.7 cells.

PubMed

Murakami, Yukio; Kawata, Akifumi; Ito, Shigeru; Katayama, Tadashi; Fujisawa, Seiichiro

2014-01-01

Phenolic compounds, particularly dihydroxybiphenyl-related compounds, possess efficient anti-oxidative and anti-inflammatory activity. We investigated the anti-inflammatory activity of 2,2'-dihydroxy-5,5'-dimethylbiphenol (p-cresol dimer), 2,2'-dihydroxy-5,5'-dimethoxybiphenol (pHA dimer), p-cresol, p-hydroxyanisole (pHA) and 2-t-butyl-4-hydroxyanisole (BHA). The cytotoxicity of the investigated compounds against RAW264.7 cells was determined using a cell counting kit (CCK-8). Their inhibitory effects on cyclooxygenase-2 (Cox2) mRNA expression stimulated by lipopolysaccharide (LPS) were determined using northern blot analysis, and their inhibition of LPS-stimulated nuclear factor-kappa B (Nf-κb) activation was evaluated using enzyme-linked immunosorbent assay-like microwell colorimetric transcription factor activity assay. The molecular orbital energy was calculated on the basis of density function theory BLYP/6-31G*. The cytotoxicity of the compounds declined in the order pHA dimer > p-cresol dimer > BHA > p-cresol > pHA. The inhibitory effect on Cox2 expression and Nf-κb activation was enhanced by p-cresol dimer and pHA dimer, particularly the former, suggesting potent anti-inflammatory activity, whereas p-cresol and pHA showed weak activity, and BHA no activity. Both p-cresol dimer and pHA dimer were highly electronegative, as determined by quantum chemical calculations. Dimerization of p-cresol and pHA enhances their anti-inflammatory activity. p-Cresol dimer and pHA dimer, particularly the former, are potent anti-inflammatory agents. Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Molecular analysis of two phytohemagglutinin genes and their expression in Phaseolus vulgaris cv. Pinto, a lectin-deficient cultivar of the bean.

PubMed

Voelker, T A; Staswick, P; Chrispeels, M J

1986-12-01

Phytohemagglutinin (PHA), the seed lectin of the common bean, Phaseolus vulgaris, is encoded by two highly homologous, tandemly linked genes, dlec1 and dlec2, which are coordinately expressed at high levels in developing cotyledons. Their respective transcripts translate into closely related polypeptides, PHA-E and PHA-L, constituents of the tetrameric lectin which accumulates at high levels in developing seeds. In the bean cultivar Pinto UI111, PHA-E is not detectable, and PHA-L accumulates at very reduced levels. To investigate the cause of the Pinto phenotype, we cloned and sequenced the two PHA genes of Pinto, called Pdlec1 and Pdlec2, and determined the abundance of their respective mRNAs in developing cotyledons. Both genes are more than 90% homologous to the normal PHA genes found in other cultivars. Pdlec1 carries a 1-bp frameshift mutation close to the 5' end of its coding sequence. Only very truncated polypeptides could be made from its mRNA. The gene Pdlec2 encodes a polypeptide, which resembles PHA-L and its predicted amino acid sequence agrees with the available Pinto PHA amino acid sequence data. Analysis of the mRNA of developing cotyledons revealed that the Pdlec1 message is reduced 600-fold, and Pdlec2 mRNA is reduced 20-fold with respect to mRNA levels in normal cultivars. A comparison of the sequences which are upstream from the coding sequence shows that Pdlec2 has a 100-bp deletion compared to the other genes (dlec1, dlec2 and Pdlec1). This deletion which contains a large tandem repeat may be responsible for the low level of expression of Pdlec2. The very low expression of Pdlec1 is as yet unexplained.

76 FR 80377 - Notice of Submission of Proposed Information Collection to OMB Additional On-Site Data Collection...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-12-23

... with PHA staff and reviews of client files and administrative data collected by the PHA. The results of... reviews of client files and administrative data collected by the PHA. The results of the site visits will...

Structure and Activity Changes of Phytohemagglutinin from Red Kidney Bean (Phaseolus vulgaris) Affected by Ultrahigh-Pressure Treatments.

PubMed

Lu, Yunjun; Liu, Cencen; Zhao, Mouming; Cui, Chun; Ren, Jiaoyan

2015-11-04

Phytohemagglutin (PHA), purified from red kidney beans (Phaseolus vulgaris) by Affi-Gel blue affinity chromatography, was subjected to ultrahigh-pressure (UHP) treatment (150, 250, 350, and 450 MPa). The purified PHA lost its hemagglutination activity after 450 MPa treatment and showed less pressure tolerance than crude PHA. However, the saccharide specificity and α-glucosidase inhibition activity of the purified PHA did not change much after UHP treatment. Electrophoresis staining by periodic acid-Schiff (PAS) manifested that the glycone structure of purified PHA remained stable even after 450 MPa pressure treatment. However, electrophoresis staining by Coomassie Blue as well as circular dichroism (CD) and differential scanning calorimetry (DSC) assay proved that the protein unit structure of purified PHA unfolded when treated at 0-250 MPa but reaggregates at 250-450 MPa. Therefore, the hemagglutination activity tends to be affected by the protein unit structure, while the stability of the glycone structure contributed to the remaining α-glucosidase inhibition activity.

Advanced functionalization of polyhydroxyalkanoate via the UV-initiated thiol-ene click reaction.

PubMed

Tajima, Kenji; Iwamoto, Kosuke; Satoh, Yasuharu; Sakai, Ryosuke; Satoh, Toshifumi; Dairi, Tohru

2016-05-01

Polyhydroxyalkanoates (PHAs) incorporating vinyl-bearing 3-hydroxyalkanoates were prepared in 8.5-12.9 g L(-1) yield. The molar ratios (0-16 mol%) of the vinyl-bearing 3-hydroxyalkanoate derivatives were controlled by the continuous feeding of undecylenate at various concentrations. Subsequently, the PHAs were functionalized by UV-initiated thiol-ene click reaction and chemical modification. (1)H NMR spectra suggested that 3-mercaptopropionic acid and 2-aminoethanethiol were successfully introduced into the vinyl-bearing PHA. Subsequently, chemical modification using fluorescein or a fibronectin active fragment (GRGDS) was attempted. The former yielded a PHA derivative capable of emitting fluorescence under UV irradiation, which was useful for determining the miscibility of PHA in a composite film comprising poly-ʟ-lactic acid (PLLA) and PHA. In the latter case, PHA bearing GRGDS peptides exhibited cell adhesiveness, suggesting that its biocompatibility was improved upon peptide introduction. Taken together, the UV-initiated thiol-ene click reaction was demonstrated to be useful in PHA modification.

Bone Regeneration of Hydroxyapatite with Granular Form or Porous Scaffold in Canine Alveolar Sockets

PubMed Central

JANG, SEOK JIN; KIM, SE EUN; HAN, TAE SUNG; SON, JUN SIK; KANG, SEONG SOO; CHOI, SEOK HWA

2017-01-01

This study was undertaken to assess bone regeneration using hydroxyapatite (HA). The primary focus was comparison of bone regeneration between granular HA (gHA) forms and porous HA (pHA) scaffold. The extracted canine alveolar sockets were divided with three groups: control, gHA and pHA. Osteogenic effect in the gHA and pHA groups showed bone-specific surface and bone mineral density to be significantly higher than that of the control group (p<0.01). Bone volume fraction, bone mineral density, and amount of connective tissue related to disturbing osseointegration of the gHA group was higher than in the pHA group. Quantity of new bone formation of the pHA group was higher than that of the gHA group. This study demonstrated that gHA and pHA are potentially good bone substitutes for alveolar socket healing. For new bone formation during 8 weeks' post-implantation, HA with porous scaffold was superior to the granular form of HA. PMID:28438860

Increasing polyhydroxyalkanoate (PHA) yields from Cupriavidus necator by using filtered digestate liquors.

PubMed

Passanha, Pearl; Esteves, Sandra R; Kedia, Gopal; Dinsdale, Richard M; Guwy, Alan J

2013-11-01

The production of polyhydroxyalkanoates (PHAs) using digestate liquor as culture media is a novel application to extend the existing uses of digestates. In this study, two micro-filtered digestates (0.22 μm) were evaluated as a source of complex culture media for the production of PHA by Cupriavidus necator as compared to a conventional media. Culture media using a mixture of micro-filtered liquors from food waste and from wheat feed digesters showed a maximum PHA accumulation of 12.29 g/l PHA, with 90% cell dry weight and a yield of 0.48 g PHA/g VFA consumed, the highest reported to date for C. necator studies. From the analysis of the starting and residual media, it was concluded that ammonia, potassium, magnesium, sulfate and phosphate provided in the digestate liquors were vital for the initial growth of C. necator whereas copper, iron and nickel may have played a significant role in PHA accumulation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Scary symptoms? Functional magnetic resonance imaging evidence for symptom interpretation bias in pathological health anxiety.

PubMed

Yan, Zhimin; Witthöft, Michael; Bailer, Josef; Diener, Carsten; Mier, Daniela

2017-08-12

Patients with pathological health anxiety (PHA) tend to automatically interpret bodily sensations as sign of a severe illness. To elucidate the neural correlates of this cognitive bias, we applied an functional magnetic resonance imaging adaption of a body-symptom implicit association test with symptom words in patients with PHA (n = 32) in comparison to patients with depression (n = 29) and healthy participants (n = 35). On the behavioral level, patients with PHA did not significantly differ from the control groups. However, on the neural-level patients with PHA in comparison to the control groups showed hyperactivation independent of condition in bilateral amygdala, right parietal lobe, and left nucleus accumbens. Moreover, patients with PHA, again in comparison to the control groups, showed hyperactivation in bilateral posterior parietal cortex and left dorsolateral prefrontal cortex during incongruent (i.e., harmless) versus congruent (i.e., dangerous) categorizations of body symptoms. Thus, body-symptom cues seem to trigger hyperactivity in salience and emotion processing brain regions in PHA. In addition, hyperactivity in brain regions involved in cognitive control and conflict resolution during incongruent categorization emphasizes enhanced neural effort to cope with negative implicit associations to body-symptom-related information in PHA. These results suggest increased neural responding in key structures for the processing of both emotional and cognitive aspects of body-symptom information in PHA, reflecting potential neural correlates of a negative somatic symptom interpretation bias.

Transient pseudohypoaldosteronism in infancy secondary to urinary tract infection.

PubMed

Abraham, Mary B; Larkins, Nicholas; Choong, Catherine S; Shetty, Vinutha B

2017-05-01

Hyponatraemia with hyperkalaemia in infancy is a typical presentation of congenital adrenal hyperplasia. In the presence of pyelonephritis, the same biochemical picture can occur with transient type 1 pseudohypoaldosteronism (PHA-1) also termed type 4 renal tubular acidosis. Recognition of PHA-1 enables appropriate management thus avoiding unnecessary investigations and treatment. To improve awareness of this condition, we present a case series to highlight the clinical and biochemical features of PHA-1. A retrospective chart review of patients diagnosed with transient PHA-1 at a tertiary children's hospital in Western Australia was conducted. Five male infants (32 days to 6 months) with transient PHA-1 were identified. Failure to thrive was the most common symptom with hyponatraemia on presentation. Two infants had antenatally diagnosed bilateral hydronephrosis and urinary tract infection (UTI) on admission. Two infants were treated for congenital adrenal hyperplasia and received hydrocortisone. All infants had UTI and required parenteral antibiotics. The condition was transient and hyponatraemia corrected by day 4 in all infants. There was no correlation between plasma sodium and aldosterone levels. The severity of PHA-1 was independent of the underlying renal anomaly. Four infants had hydronephrosis and vesicoureteric reflux. Surgical intervention was required in two infants. PHA-1 may be precipitated by UTI or urinary tract anomalies in early infancy. Urine analysis should be performed in infants with hyponatraemia. Diagnosis of PHA-1 facilitates appropriate renal investigations to reduce long-term morbidity. © 2017 Paediatrics and Child Health Division (The Royal Australasian College of Physicians).

Mediterranean Diet and Phase Angle in a Sample of Adult Population: Results of a Pilot Study

PubMed Central

Barrea, Luigi; Muscogiuri, Giovanna; Macchia, Paolo Emidio; Di Somma, Carolina; Falco, Andrea; Savanelli, Maria Cristina; Colao, Annamaria; Savastano, Silvia

2017-01-01

The Mediterranean diet is a healthy dietary pattern known to actively modulate the cell membrane properties. Phase angle (PhA) is a direct measure by Bioelectrical Impedance Analysis (BIA) used as marker of cell membrane integrity. Both food behaviour and PhA are influenced by age, sex and body weight. The aim of this study was to cross-sectionally evaluate the association between the adherence to Mediterranean diet and PhA in 1013 healthy adult patients stratified according to sex, age, and body mass index (BMI). The adherence to the Mediterranean diet was evaluated using the PREvención con DIeta MEDiterránea (PREDIMED) questionnaire. PhA was calculated by BIA phase-sensitive system (50 kHz BIA 101 RJL, Akern Bioresearch, Florence, Italy Akern). In both sexes, at ROC analysis a PREDIMED score ≥ 6 predicted a PhA beyond the median value. At the multivariate analysis, among PREDIMED score, age, and BMI, the PREDIMED score was the major determinant of PhA, explaining 44.5% and 47.3% of PhA variability, in males and females respectively (p < 0.001). A novel association was reported between the adherence to the Mediterranean diet and PhA, independently of sex, age, and body weight. This association uncovered a new potential benefit of the Mediterranean diet on health outcomes, as in both sexes higher adherence to the Mediterranean diet was associated to larger PhAs, as expression of cell membrane integrity. PMID:28218645

Mediterranean Diet and Phase Angle in a Sample of Adult Population: Results of a Pilot Study.

PubMed

Barrea, Luigi; Muscogiuri, Giovanna; Macchia, Paolo Emidio; Di Somma, Carolina; Falco, Andrea; Savanelli, Maria Cristina; Colao, Annamaria; Savastano, Silvia

2017-02-17

The Mediterranean diet is a healthy dietary pattern known to actively modulate the cell membrane properties. Phase angle (PhA) is a direct measure by Bioelectrical Impedance Analysis (BIA) used as marker of cell membrane integrity. Both food behaviour and PhA are influenced by age, sex and body weight. The aim of this study was to cross-sectionally evaluate the association between the adherence to Mediterranean diet and PhA in 1013 healthy adult patients stratified according to sex, age, and body mass index (BMI). The adherence to the Mediterranean diet was evaluated using the PREvención con DIeta MEDiterránea (PREDIMED) questionnaire. PhA was calculated by BIA phase-sensitive system (50 kHz BIA 101 RJL, Akern Bioresearch, Florence, Italy Akern). In both sexes, at ROC analysis a PREDIMED score ≥ 6 predicted a PhA beyond the median value. At the multivariate analysis, among PREDIMED score, age, and BMI, the PREDIMED score was the major determinant of PhA, explaining 44.5% and 47.3% of PhA variability, in males and females respectively ( p < 0.001). A novel association was reported between the adherence to the Mediterranean diet and PhA, independently of sex, age, and body weight. This association uncovered a new potential benefit of the Mediterranean diet on health outcomes, as in both sexes higher adherence to the Mediterranean diet was associated to larger PhAs, as expression of cell membrane integrity.

Design of the polar neutron-imaging aperture for use at the National Ignition Facility.

PubMed

Fatherley, V E; Barker, D A; Fittinghoff, D N; Hibbard, R L; Martinez, J I; Merrill, F E; Oertel, J A; Schmidt, D W; Volegov, P L; Wilde, C H

2016-11-01

The installation of a neutron imaging diagnostic with a polar view at the National Ignition Facility (NIF) required design of a new aperture, an extended pinhole array (PHA). This PHA is different from the pinhole array for the existing equatorial system due to significant changes in the alignment and recording systems. The complex set of component requirements, as well as significant space constraints in its intended location, makes the design of this aperture challenging. In addition, lessons learned from development of prior apertures mandate careful aperture metrology prior to first use. This paper discusses the PHA requirements, constraints, and the final design. The PHA design is complex due to size constraints, machining precision, assembly tolerances, and design requirements. When fully assembled, the aperture is a 15 mm × 15 mm × 200 mm tungsten and gold assembly. The PHA body is made from 2 layers of tungsten and 11 layers of gold. The gold layers include 4 layers containing penumbral openings, 4 layers containing pinholes and 3 spacer layers. In total, there are 64 individual, triangular pinholes with a field of view (FOV) of 200 μm and 6 penumbral apertures. Each pinhole is pointed to a slightly different location in the target plane, making the effective FOV of this PHA a 700 μm square in the target plane. The large FOV of the PHA reduces the alignment requirements both for the PHA and the target, allowing for alignment with a laser tracking system at NIF.

Differential effects of arsenic on intracellular free calcium levels and the proliferative response of murine mitogen-stimulated lymphocytes.

PubMed

Goytia-Acevedo, Raquel C; Cebrian, Mariano E; Calderon-Aranda, Emma S

2003-08-01

This study examined the effects of sodium arsenite treatment on free [Ca(2+)]i and cell death in mitogen-activated murine lymphocytes. The main findings of this study were that simultaneous sodium arsenite treatment inhibited PHA- but not Con A-induced T cell proliferation, induced a higher increase in free [Ca(2+)]i and an early increase in the proportion of dead cells in PHA than in Con A activated cells. Sodium arsenite pre-treatment reduced both PHA- and Con A-induced T-cell proliferation. Phorbol myristate ester (PMA) did not prevent the inhibitory effects of both sodium arsenite treatments, suggesting that sodium arsenite did not significantly decreased PKC activation or that its effects occurred on events parallel to PKC activation. Both PHA and Con A increased free [Ca(2+)]i after stimulation, yet the effect was more pronounced in mitogen-activated cells simultaneously treated with sodium arsenite and particularly in those activated with PHA. The increase in free [Ca(2+)]i was in agreement with the early cell death induced by sodium arsenite in PHA-activated cells, a finding consistent with the inhibitory effects on PHA-induced proliferation. Sodium arsenite-induced cell death occurred faster in PHA-activated cells. Further studies are needed to ascertain the relationships between the effects of sodium arsenite on free [Ca(2+)]i levels and the type of cell death induced by sodium arsenite and their relevance for the proliferative response of T cells.

Metabolic pathway engineering in cotton: Biosynthesis of polyhydroxybutyrate in fiber cells

PubMed Central

John, Maliyakal E.; Keller, Greg

1996-01-01

Alcaligenes eutrophus genes encoding the enzymes, β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB), and polyhydroxyalkanoate synthase (phaC) catalyze the production of aliphatic polyester poly-d-(−)-3-hydroxybutyrate (PHB) from acetyl-CoA. PHB is a thermoplastic polymer that may modify fiber properties when synthesized in cotton. Endogenous β-ketothiolase activity is present in cotton fibers. Hence cotton was transformed with engineered phaB and phaC genes by particle bombardment, and transgenic plants were selected based on marker gene, β-glucuronidase (GUS), expression. Fibers of 10 transgenic plants expressed phaB gene, while eight plants expressed both phaB and phaC genes. Electron microscopy examination of fibers expressing both genes indicated the presence of electron-lucent granules in the cytoplasm. High pressure liquid chromatography, gas chromatography, and mass spectrometry evidence suggested that the new polymer produced in transgenic fibers is PHB. Sixty-six percent of the PHB in fibers is in the molecular mass range of 0.6 × 106 to 1.8 × 106 Da. The presence of PHB granules in transgenic fibers resulted in measurable changes of thermal properties. The fibers exhibited better insulating characteristics. The rate of heat uptake and cooling was slower in transgenic fibers, resulting in higher heat capacity. These data show that metabolic pathway engineering in cotton may enhance fiber properties by incorporating new traits from other genetic sources. This is an important step toward producing new generation fibers for the textile industry. PMID:11038522

24 CFR 983.251 - How participants are selected.

Code of Federal Regulations, 2010 CFR

2010-04-01

.... (a) Who may receive PBV assistance? (1) The PHA may select families who are participants in the PHA's tenant-based voucher program and families who have applied for admission to the voucher program. (2... PHA may only select families determined eligible for admission at commencement of PBV assistance. (3...

Polyhydroxyalkanoates (PHA) Bioplastic Packaging Materials

DTIC Science & Technology

2010-05-01

FINAL REPORT Polyhydroxyalkanoates (PHA) Bioplastic Packaging Materials SERDP Project WP-1478 MAY 2010 Dr.Chris Schwier Metabolix...biopolymer, biodegradable, polyhydroxyalkanoate 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF...Acronyms and Definitions ASTM – American Society of Test Methods ISO – International Standardization Organization PHA – Polyhydroxyalkanoates

24 CFR 902.69 - PHA right of petition and appeal.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false PHA right of petition and appeal... appeal. (a) Appeal of troubled designation and petition for removal troubled designation. A PHA may: (1) Appeal its troubled designation (including designation as troubled with respect to its performance under...

24 CFR 982.151 - Annual contributions contract.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Contract and PHA Administration of Program § 982.151 Annual contributions contract. (a) Nature of ACC. (1) An annual contributions contract (ACC) is a written contract between HUD and a PHA. Under the ACC... owners and for the PHA administrative fee. The ACC specifies the maximum payment over the ACC term. The...

24 CFR 982.151 - Annual contributions contract.

Code of Federal Regulations, 2010 CFR

2010-04-01

... Contract and PHA Administration of Program § 982.151 Annual contributions contract. (a) Nature of ACC. (1) An annual contributions contract (ACC) is a written contract between HUD and a PHA. Under the ACC... owners and for the PHA administrative fee. The ACC specifies the maximum payment over the ACC term. The...

24 CFR 902.5 - Applicability.

Code of Federal Regulations, 2011 CFR

2011-04-01

...(s) for which they assumed management responsibilities. (2) ACC. The ACC makes a PHA legally... the PHA and not the RMC or AME is ultimately responsible to HUD under the ACC, the PHAS score of a PHA will be based on all of the projects covered by the ACC, including those with management operations...

Crystallization and initial X-ray analysis of polyhydroxyalkanoate granule-associated protein from Aeromonas hydrophila

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Minglian; Li, Zhenguo; Zheng, Wei

The phasin PhaP{sub Ah} from A. hydrophila strain 4AK4 was crystallized using the hanging-drop vapour-diffusion method. Polyhydroxyalkanoate (PHA) granule-associated proteins (phasins) were discovered in PHA-accumulating bacteria. They play a crucial role as a structural protein during initial PHA-granule formation and granule growth and also serve as interfaces for granule stabilization in vivo. The phasin PhaP{sub Ah} from Aeromonas hydrophila strain 4AK4 was crystallized using the hanging-drop vapour-diffusion method. Single crystals were cryocooled for X-ray diffraction analysis. The phasin crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 80.8, b = 108.9, c = 134.4 Å.

Improved detergent-based recovery of polyhydroxyalkanoates (PHAs).

PubMed

Yang, Yung-Hun; Brigham, Christopher; Willis, Laura; Rha, ChoKyun; Sinskey, Anthony

2011-05-01

Extracting polyhydroxyalkanoate (PHA) polymer from bacterial cells often involves harsh conditions, including use of environmentally harmful solvents. We evaluated different detergents under various conditions to extract PHA from Ralstonia eutropha and Escherichia coli cells. Most detergents tested recovered highly pure PHA polymer from cells in amounts that depended on the percentage of polymer present in the cell. Detergents such as linear alkylbenzene sulfonic acid (LAS-99) produced a high yield of high purity polymer, and less detergent was needed compared to the amount of SDS to produce comparable yields. LAS-99 also has the advantage of being biodegradable and environmentally safe. Chemical extraction of PHA with detergents could potentially minimize or eliminate the need to use harsh organic solvents, thus making industrial PHA production a cleaner technology process. © Springer Science+Business Media B.V. 2011

A phasin with extra talents: a polyhydroxyalkanoate granule-associated protein has chaperone activity.

PubMed

Mezzina, Mariela P; Wetzler, Diana E; de Almeida, Alejandra; Dinjaski, Nina; Prieto, M Auxiliadora; Pettinari, Maria Julia

2015-05-01

Phasins are proteins associated to intracellular polyhydroxyalkanoate granules that affect polymer accumulation and the number and size of the granules. Previous work demonstrated that a phasin from Azotobacter sp FA-8 (PhaPAz ) had an unexpected growth-promoting and stress-protecting effect in Escherichia coli, suggesting it could have chaperone-like activities. In this work, in vitro and in vivo experiments were performed in order to investigate this possibility. PhaPAz was shown to prevent in vitro thermal aggregation of the model protein citrate synthase and to facilitate the refolding process of this enzyme after chemical denaturation. Microscopy techniques were used to analyse the subcellular localization of PhaPAz in E. coli strains and to study the role of PhaPAz in in vivo protein folding and aggregation. PhaPAz was shown to colocalize with inclusion bodies of PD, a protein that aggregates when overexpressed. A reduction in the number of inclusion bodies of PD was observed when it was coexpressed with PhaPAz or with the known chaperone GroELS. These results demonstrate that PhaPAz has chaperone-like functions both in vitro and in vivo in E. coli recombinants, and suggests that phasins could have a general protective role in natural polyhydroxyalkanoate producers. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Polyhydroxyalkanoates in waste activated sludge enhances anaerobic methane production through improving biochemical methane potential instead of hydrolysis rate.

PubMed

Wang, Qilin; Sun, Jing; Zhang, Chang; Xie, Guo-Jun; Zhou, Xu; Qian, Jin; Yang, Guojing; Zeng, Guangming; Liu, Yiqi; Wang, Dongbo

2016-01-21

Anaerobic sludge digestion is the main technology for sludge reduction and stabilization prior to sludge disposal. Nevertheless, methane production from anaerobic digestion of waste activated sludge (WAS) is often restricted by the poor biochemical methane potential and slow hydrolysis rate of WAS. This work systematically investigated the effect of PHA levels of WAS on anaerobic methane production, using both experimental and mathematical modeling approaches. Biochemical methane potential tests showed that methane production increased with increased PHA levels in WAS. Model-based analysis suggested that the PHA-based method enhanced methane production by improving biochemical methane potential of WAS, with the highest enhancement being around 40% (from 192 to 274 L CH4/kg VS added; VS: volatile solid) when the PHA levels increased from 21 to 143 mg/g VS. In contrast, the hydrolysis rate (approximately 0.10 d(-1)) was not significantly affected by the PHA levels. Economic analysis suggested that the PHA-based method could save $1.2/PE/y (PE: population equivalent) in a typical wastewater treatment plant (WWTP). The PHA-based method can be easily integrated into the current WWTP to enhance methane production, thereby providing a strong support to the on-going paradigm shift in wastewater management from pollutant removal to resource recovery.

Polyhydroxyalkanoates in waste activated sludge enhances anaerobic methane production through improving biochemical methane potential instead of hydrolysis rate

PubMed Central

Wang, Qilin; Sun, Jing; Zhang, Chang; Xie, Guo-Jun; Zhou, Xu; Qian, Jin; Yang, Guojing; Zeng, Guangming; Liu, Yiqi; Wang, Dongbo

2016-01-01

Anaerobic sludge digestion is the main technology for sludge reduction and stabilization prior to sludge disposal. Nevertheless, methane production from anaerobic digestion of waste activated sludge (WAS) is often restricted by the poor biochemical methane potential and slow hydrolysis rate of WAS. This work systematically investigated the effect of PHA levels of WAS on anaerobic methane production, using both experimental and mathematical modeling approaches. Biochemical methane potential tests showed that methane production increased with increased PHA levels in WAS. Model-based analysis suggested that the PHA-based method enhanced methane production by improving biochemical methane potential of WAS, with the highest enhancement being around 40% (from 192 to 274 L CH4/kg VS added; VS: volatile solid) when the PHA levels increased from 21 to 143 mg/g VS. In contrast, the hydrolysis rate (approximately 0.10 d−1) was not significantly affected by the PHA levels. Economic analysis suggested that the PHA-based method could save $1.2/PE/y (PE: population equivalent) in a typical wastewater treatment plant (WWTP). The PHA-based method can be easily integrated into the current WWTP to enhance methane production, thereby providing a strong support to the on-going paradigm shift in wastewater management from pollutant removal to resource recovery. PMID:26791952

Biochemical characterization of a new type of intracellular PHB depolymerase from Rhodospirillum rubrum with high hydrolytic activity on native PHB granules.

PubMed

Sznajder, Anna; Jendrossek, Dieter

2011-03-01

A Rhodospirillum rubrum gene that is predicted to code for an extracellular poly(3-hydroxybutyrate) (PHB) depolymerase by the recently published polyhydroxyalkanoates (PHA) depolymerase engineering database was cloned. The gene product (PhaZ3( Rru )) was expressed in recombinant E. coli, purified and biochemically characterized. PhaZ3( Rru ) turned out, however, to share characteristics of intracellular PHB depolymerases and revealed a combination of properties that have not yet been described for other PHB depolymerases. A fusion of PhaZ3( Rru )with the enhanced cyan fluorescent protein was able to bind to PHB granules in vivo and supported the function as an intracellular PHB depolymerase. Purified PhaZ3( Rru ) was specific for short-chain-length polyhydroxyalkanoates (PHA(SCL)) and hydrolysed both untreated native PHB granules as well as trypsin-activated native PHB granules to a mixture of mono- and dimeric 3-hydroxybutyrate. Crystalline (denatured) PHB granules were not hydrolysed by PhayZ3( Rru ). Low concentrations of calcium or magnesium ions (1-5 mM) reversibly (EDTA) inhibited the enzyme. Our data suggest that PhaZ3( Rru ) is the representative of a new type of the growing number of intracellular PHB depolymerases.

Correlation between ultrafiltration rate and phase angle measured by BIA in chronic kidney disease patients on regular hemodialysis

NASA Astrophysics Data System (ADS)

Nasution, B. R.; Lubis, A. R.

2018-03-01

Chronic Kidney Disease (CKD) patients with regular hemodialysis have high rates of morbidity and mortality that may be related to the hemodynamic effects of rapid UFR and low PhA value. In this study, we investigated whether high UFR is associated with a low value of PhA thus indirectly affect the risk of morbidity and mortality. UFR and Bioelectrical Impedance Analysis (BIA) examination on 92 subjects were recorded shortly after HD and analyzed by using Pearson correlation test. Multivariate analysis was also conducted to identify several factors that can affect the value of Phase angle. The number of HD regular CKD patients with PhA<4 based on the division of the UFR (cc/kg/h) <10, 10-13, ≥ 13, respectively were3, 10 and 6, whereas patients with ≥ 4 PhA <10, 10-13, ≥ 13respectively were 60, 11, and 2. The results showed a significant relationship between UFR with PhA. In CKD patients with regular HD, UFR has aninverse relationship with the value of PhA. After multivariate analysis, the UFR and the etiology of HD are still significantly affect the value of PhA. UFR optimal value in patients with CKD with regular HD is <10 cc/kg/h.

Polyhydroxyalkanoates in waste activated sludge enhances anaerobic methane production through improving biochemical methane potential instead of hydrolysis rate

NASA Astrophysics Data System (ADS)

Wang, Qilin; Sun, Jing; Zhang, Chang; Xie, Guo-Jun; Zhou, Xu; Qian, Jin; Yang, Guojing; Zeng, Guangming; Liu, Yiqi; Wang, Dongbo

2016-01-01

Anaerobic sludge digestion is the main technology for sludge reduction and stabilization prior to sludge disposal. Nevertheless, methane production from anaerobic digestion of waste activated sludge (WAS) is often restricted by the poor biochemical methane potential and slow hydrolysis rate of WAS. This work systematically investigated the effect of PHA levels of WAS on anaerobic methane production, using both experimental and mathematical modeling approaches. Biochemical methane potential tests showed that methane production increased with increased PHA levels in WAS. Model-based analysis suggested that the PHA-based method enhanced methane production by improving biochemical methane potential of WAS, with the highest enhancement being around 40% (from 192 to 274 L CH4/kg VS added; VS: volatile solid) when the PHA levels increased from 21 to 143 mg/g VS. In contrast, the hydrolysis rate (approximately 0.10 d-1) was not significantly affected by the PHA levels. Economic analysis suggested that the PHA-based method could save $1.2/PE/y (PE: population equivalent) in a typical wastewater treatment plant (WWTP). The PHA-based method can be easily integrated into the current WWTP to enhance methane production, thereby providing a strong support to the on-going paradigm shift in wastewater management from pollutant removal to resource recovery.

Suppressed PHA Activation of T Lymphocytes in Simulated Microgravity Is Restored by Direct Activation of Protein Kinase C with Phorbol Ester

NASA Technical Reports Server (NTRS)

Cooper, David; Pellis, Neal R.

1997-01-01

Various aspects of spaceflight, including microgravity, cosmic radiation, and physiological stress, may perturb immune function. We sought to understand the impact of microgravity alone on the cellular mechanisms critical to immunity. We utilized clinostatic RWV bioreactors that simulate aspects of microgravity to analyze the response of human PBMC to polyclonal activation. PHA responsiveness in the RWV was almost completely diminished. IL-2 and IFN-gamma secretion was reduced whereas IL- 1 beta and IL-6 secretion was increased, suggesting that monocytes may not be as adversely affected by simulated microgravity as T cells. Activation marker expression (CD25, CD69, CD71) was significantly reduced in RWV cultures. Furthermore, addition of exogenous IL-2 to these cultures did not restore proliferation. Reduced cell-cell and cell-substratum interactions may play a role in the loss of PHA responsiveness. However, PHA activation in Teflon culture bags that limit cell-substratum interactions did not suppress PHA activation. Furthermore, increasing cell density and, therefore, cell-cell interactions in the RWV cultures did not help restore PHA activation. However, placing PBMC within small collagen beads did partially restore PHA responsiveness. Activation of both PBMC and purified T cells with PMA and ionomycin was unaffected by RWV culture, indicating that signaling mechanisms downstream of PKC activation and calcium flux are not sensitive to simulated microgravity. Furthermore, submitogenic doses of PMA alone but not ionomycin alone restored PHA responsiveness of PBMC in RWV culture. Thus, our data indicate that during polyclonal activation the signaling pathways upstream of PKC activation are sensitive to simulated microgravity.

Characterization of a Novel Subgroup of Extracellular Medium-Chain-Length Polyhydroxyalkanoate Depolymerases from Actinobacteria

PubMed Central

Gangoiti, Joana; Santos, Marta; Prieto, María Auxiliadora; de la Mata, Isabel; Llama, María J.

2012-01-01

Nineteen medium-chain-length (mcl) poly(3-hydroxyalkanoate) (PHA)-degrading microorganisms were isolated from natural sources. From them, seven Gram-positive and three Gram-negative bacteria were identified. The ability of these microorganisms to hydrolyze other biodegradable plastics, such as short-chain-length (scl) PHA, poly(ε-caprolactone) (PCL), poly(ethylene succinate) (PES), and poly(l-lactide) (PLA), has been studied. On the basis of the great ability to degrade different polyesters, Streptomyces roseolus SL3 was selected, and its extracellular depolymerase was biochemically characterized. The enzyme consisted of one polypeptide chain of 28 kDa with a pI value of 5.2. Its maximum activity was observed at pH 9.5 with chromogenic substrates. The purified enzyme hydrolyzed mcl PHA and PCL but not scl PHA, PES, and PLA. Moreover, the mcl PHA depolymerase can hydrolyze various substrates for esterases, such as tributyrin and p-nitrophenyl (pNP)-alkanoates, with its maximum activity being measured with pNP-octanoate. Interestingly, when poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate [11%]) was used as the substrate, the main hydrolysis product was the monomer (R)-3-hydroxyoctanoate. In addition, the genes of several Actinobacteria strains, including S. roseolus SL3, were identified on the basis of the peptide de novo sequencing of the Streptomyces venezuelae SO1 mcl PHA depolymerase by tandem mass spectrometry. These enzymes did not show significant similarity to mcl PHA depolymerases characterized previously. Our results suggest that these distinct enzymes might represent a new subgroup of mcl PHA depolymerases. PMID:22865072

Design of the polar neutron-imaging aperture for use at the National Ignition Facility

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fatherley, V. E., E-mail: vef@lanl.gov; Martinez, J. I.; Merrill, F. E.

2016-11-15

The installation of a neutron imaging diagnostic with a polar view at the National Ignition Facility (NIF) required design of a new aperture, an extended pinhole array (PHA). This PHA is different from the pinhole array for the existing equatorial system due to significant changes in the alignment and recording systems. The complex set of component requirements, as well as significant space constraints in its intended location, makes the design of this aperture challenging. In addition, lessons learned from development of prior apertures mandate careful aperture metrology prior to first use. This paper discusses the PHA requirements, constraints, and themore » final design. The PHA design is complex due to size constraints, machining precision, assembly tolerances, and design requirements. When fully assembled, the aperture is a 15 mm × 15 mm × 200 mm tungsten and gold assembly. The PHA body is made from 2 layers of tungsten and 11 layers of gold. The gold layers include 4 layers containing penumbral openings, 4 layers containing pinholes and 3 spacer layers. In total, there are 64 individual, triangular pinholes with a field of view (FOV) of 200 μm and 6 penumbral apertures. Each pinhole is pointed to a slightly different location in the target plane, making the effective FOV of this PHA a 700 μm square in the target plane. The large FOV of the PHA reduces the alignment requirements both for the PHA and the target, allowing for alignment with a laser tracking system at NIF.« less

Polyhydroxyalkanoate synthesis in transgenic plants as a new tool to study carbon flow through beta-oxidation.

PubMed

Mittendorf, V; Bongcam, V; Allenbach, L; Coullerez, G; Martini, N; Poirier, Y

1999-10-01

Transgenic plants producing peroxisomal polyhydroxy- alkanoate (PHA) from intermediates of fatty acid degradation were used to study carbon flow through the beta-oxidation cycle. Growth of transgenic plants in media containing fatty acids conjugated to Tween detergents resulted in an increased accumulation of PHA and incorporation into the polyester of monomers derived from the beta-oxidation of these fatty acids. Tween-laurate was a stronger inducer of beta-oxidation, as measured by acyl-CoA oxidase activity, and a more potent modulator of PHA quantity and monomer composition than Tween-oleate. Plants co-expressing a peroxisomal PHA synthase with a capryl-acyl carrier protein thioesterase from Cuphea lanceolata produced eightfold more PHA compared to plants expressing only the PHA synthase. PHA produced in double transgenic plants contained mainly saturated monomers ranging from 6 to 10 carbons, indicating an enhanced flow of capric acid towards beta-oxidation. Together, these results support the hypothesis that plant cells have mechanisms which sense levels of free or esterified unusual fatty acids, resulting in changes in the activity of the beta-oxidation cycle as well as removal and degradation of these unusual fatty acids through beta-oxidation. Such enhanced flow of fatty acids through beta-oxidation can be utilized to modulate the amount and composition of PHA produced in transgenic plants. Furthermore, synthesis of PHAs in plants can be used as a new tool to study the quality and relative quantity of the carbon flow through beta-oxidation as well as to analyse the degradation pathway of unusual fatty acids.

Optimization of biodegradable plastic production on sugar cane molasses in Enterobacter sp. SEL2

PubMed Central

Naheed, Nighat; Jamil, Nazia

2014-01-01

Contaminated environments have a large number of bacteria which can accumulate PHA as their energy reserves. Out of 54 isolated bacterial strains from three groups of contaminated sites 48 were found PHA positive. The sites were grouped on the basis of the type of carbon sources i.e. sugars, fatty acids and much diverse type. Strains MFD5, MFD11, UML3, USL2, SEL2, SEL3, SEL10 and PFW1 produced 69.9 ± 0.29, 75.27 ± 0.45, 65.43 ± 0.1, 72.54 ± 0.27, 76.61 ± 0.28, 61.81 ± 0.05, 71.16 ± 0.09 and 74.92 ± 0.5 percent of PHA to their constant cell weight (CCW) respectively in PHA detection media supplemented with 2% glucose. Molasses, whey, crumbs hydrolysate and palm oil were checked as inexpensive carbon sources. Molasses alone could supply the required nutrients for growth and PHA production. Strain SEL2 produced 47.36 ± 0.45% PHA using 2% molasses at 37 °C and pH 7.0. Upon production optimization the best accumulation (80.95 ± 0.01%) was observed in PHA detection media with 0.2% nitrogen source, 3% molasses, pH 5.0 and 37 °C by the strain SEL2. The overall effect of the presence of increased molasses concentration in the media was positive it increased the accumulation period till 72 h. Enterobacter sp. SEL2 (JF901810) is first time being reported for PHA production. PMID:25242924

Extensive Acclimation in Ectotherms Conceals Interspecific Variation in Thermal Tolerance Limits

PubMed Central

Pintor, Anna F. V.; Schwarzkopf, Lin; Krockenberger, Andrew K.

2016-01-01

Species’ tolerance limits determine their capacity to tolerate climatic extremes and limit their potential distributions. Interspecific variation in thermal tolerances is often proposed to indicate climatic vulnerability and is, therefore, the subject of many recent meta-studies on differential capacities of species from climatically different habitats to deal with climate change. Most studies on thermal tolerances do not acclimate animals or use inconsistent, and insufficient, acclimation times, limiting our knowledge of the shape, duration and extent of acclimation responses. Consequently patterns in thermal tolerances observed in meta-analyses, based on data from the literature are based on inconsistent, partial acclimation and true trends may be obscured. In this study we describe time-course of complete acclimation of critical thermal minima in the tropical ectotherm Carlia longipes and compare it to the average acclimation response of other reptiles, estimated from published data, to assess how much acclimation time may contribute to observed differences in thermal limits. Carlia longipes decreased their lower critical thermal limits by 2.4°C and completed 95% of acclimation in 17 weeks. Wild populations did not mirror this acclimation process over the winter. Other reptiles appear to decrease cold tolerance more quickly (95% in 7 weeks) and to a greater extent, with an estimated average acclimation response of 6.1°C. However, without data on tolerances after longer acclimation times available, our capacity to estimate final acclimation state is very limited. Based on the subset of data available for meta-analysis, much of the variation in cold tolerance observed in the literature can be attributed to acclimation time. Our results indicate that (i) acclimation responses can be slow and substantial, even in tropical species, and (ii) interspecific differences in acclimation speed and extent may obscure trends assessed in some meta-studies. Cold tolerances of wild animals are representative of cumulative responses to recent environments, while lengthy acclimation is necessary for controlled comparisons of physiological tolerances. Measures of inconsistent, intermediate acclimation states, as reported by many studies, represent neither the realised nor the potential tolerance in that population, are very likely underestimates of species’ physiological capacities and may consequently be of limited value. PMID:26990769

Extensive Acclimation in Ectotherms Conceals Interspecific Variation in Thermal Tolerance Limits.

PubMed

Pintor, Anna F V; Schwarzkopf, Lin; Krockenberger, Andrew K

2016-01-01

Species' tolerance limits determine their capacity to tolerate climatic extremes and limit their potential distributions. Interspecific variation in thermal tolerances is often proposed to indicate climatic vulnerability and is, therefore, the subject of many recent meta-studies on differential capacities of species from climatically different habitats to deal with climate change. Most studies on thermal tolerances do not acclimate animals or use inconsistent, and insufficient, acclimation times, limiting our knowledge of the shape, duration and extent of acclimation responses. Consequently patterns in thermal tolerances observed in meta-analyses, based on data from the literature are based on inconsistent, partial acclimation and true trends may be obscured. In this study we describe time-course of complete acclimation of critical thermal minima in the tropical ectotherm Carlia longipes and compare it to the average acclimation response of other reptiles, estimated from published data, to assess how much acclimation time may contribute to observed differences in thermal limits. Carlia longipes decreased their lower critical thermal limits by 2.4°C and completed 95% of acclimation in 17 weeks. Wild populations did not mirror this acclimation process over the winter. Other reptiles appear to decrease cold tolerance more quickly (95% in 7 weeks) and to a greater extent, with an estimated average acclimation response of 6.1°C. However, without data on tolerances after longer acclimation times available, our capacity to estimate final acclimation state is very limited. Based on the subset of data available for meta-analysis, much of the variation in cold tolerance observed in the literature can be attributed to acclimation time. Our results indicate that (i) acclimation responses can be slow and substantial, even in tropical species, and (ii) interspecific differences in acclimation speed and extent may obscure trends assessed in some meta-studies. Cold tolerances of wild animals are representative of cumulative responses to recent environments, while lengthy acclimation is necessary for controlled comparisons of physiological tolerances. Measures of inconsistent, intermediate acclimation states, as reported by many studies, represent neither the realised nor the potential tolerance in that population, are very likely underestimates of species' physiological capacities and may consequently be of limited value.

24 CFR 982.301 - Information when family is selected.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 24 Housing and Urban Development 4 2011-04-01 2011-04-01 false Information when family is selected....301 Information when family is selected. (a) PHA briefing of family. (1) When the PHA selects a family to participate in a tenant-based program, the PHA must give the family an oral briefing. The briefing...

Polyhydroxyalkanoate synthesis in plants

DOEpatents

Srienc, Friedrich; Somers, David A.; Hahn, J. J.; Eschenlauer, Arthur C.

2000-01-01

Novel transgenic plants and plant cells are capable of biosynthesis of polyhydroxyalkanoate (PHA). Heterologous enzymes involved in PHA biosynthesis, particularly PHA polymerase, are targeted to the peroxisome of a transgenic plant. Transgenic plant materials that biosynthesize short chain length monomer PHAs in the absence of heterologous .beta.-ketothiolase and acetoacetyl-CoA reductase are also disclosed.

24 CFR 966.4 - Lease requirements.

Code of Federal Regulations, 2011 CFR

2011-04-01

..., or court-awarded custody of a child. The family must request PHA approval to add any other family... foster child or a live-in aide may reside in the unit. The PHA may adopt reasonable policies concerning residence by a foster child or a live-in-aide, and defining the circumstances in which PHA consent will be...

24 CFR 982.303 - Term of voucher.

Code of Federal Regulations, 2010 CFR

2010-04-01

.... (a) Initial term. The initial term of a voucher must be at least 60 calendar days. The initial term... family one or more extensions of the initial voucher term in accordance with PHA policy as described in the PHA administrative plan. Any extension of the term is granted by PHA notice to the family. (2) If...

24 CFR 982.161 - Conflict of interest.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false Conflict of interest. 982.161... and PHA Administration of Program § 982.161 Conflict of interest. (a) Neither the PHA nor any of its... prospective interest to the PHA and HUD. (c) The conflict of interest prohibition under this section may be...

24 CFR 982.304 - Illegal discrimination: PHA assistance to family.

Code of Federal Regulations, 2010 CFR

2010-04-01

... assistance to family. 982.304 Section 982.304 Housing and Urban Development Regulations Relating to Housing... Leasing a Unit § 982.304 Illegal discrimination: PHA assistance to family. A family may claim that illegal... prevents the family from finding or leasing a suitable unit with assistance under the program. The PHA must...

24 CFR 982.157 - Budget and expenditure.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false Budget and expenditure. 982.157... and PHA Administration of Program § 982.157 Budget and expenditure. (a) Budget submission. Each PHA fiscal year, the PHA must submit its proposed budget for the program to HUD for approval at such time and...

24 CFR 982.554 - Informal review for applicant.

Code of Federal Regulations, 2010 CFR

2010-04-01

... obtain the informal review. (b) Informal review process. The PHA must give an applicant an opportunity...) The applicant must be given an opportunity to present written or oral objections to the PHA decision.... The PHA is not required to provide the applicant an opportunity for an informal review for any of the...

24 CFR 990.145 - Dwelling units with approved vacancies.

Code of Federal Regulations, 2011 CFR

2011-04-01

... work, and the construction is on schedule according to a HUD-approved PHA Annual Plan; or (ii) The unit... PHA Annual Plan, but the time period for placing the vacant unit under construction has not yet expired. The PHA shall place the vacant unit under construction within two federal fiscal years (FFYs...

Syntrophic association of termite gut bacterial symbionts with bifunctional characteristics of cellulose degrading and polyhydroxyalkanoate producing bacteria.

PubMed

Cibichakravarthy, Balasubramanian; Abinaya, Subramani; Prabagaran, Solai Ramatchandirane

2017-10-01

The guild between higher termites and their partnership with the diverse community of bacteria and archaea in their gut is a marvel evolutionary achievement. Sustained attempts were made worldwide with a quest for identifying viable important biological macromolecule polyhydroxyalkanoate (PHA) accumulating bacteria. Termite gut serve as a novel source for bacteria with dual properties like PHA production as well as cellulose degradation. Among 40 isolates cultivated, 32.5% turned positive for PCR based screening of PhaC gene. The 16S rRNA gene sequencing revealed that elite PHA producer and cellulose degrader which is phylogenetically affiliated to Bacillus cereus. The PHA production was maximized by employing different carbon and nitrogen sources along with altered pH and temperatures. GC-MS, FTIR and 1 HNMR analyses confirmed the presence of PHA and the thermal characterization was performed through TGA and DSC for the termite gut isolate. Our results indicated that the combined integrative approach using isolated strains from termite gut would be preferable choice in producing biomolecules from cellulosic materials. Copyright © 2017. Published by Elsevier B.V.

Sustainable multistage process for enhanced productivity of bioplastics from waste remediation through aerobic dynamic feeding strategy: Process integration for up-scaling.

PubMed

Amulya, K; Jukuri, Srinivas; Venkata Mohan, S

2015-01-01

Polyhydroxyalkanoates (PHA) production was evaluated in a multistage operation using food waste as a renewable feedstock. The first step involved the production of bio-hydrogen (bio-H2) via acidogenic fermentation. Volatile fatty acid (VFA) rich effluent from bio-H2 reactor was subsequently used for PHA production, which was carried out in two stages, Stage II (culture enrichment) and Stage III (PHA production). PHA-storing microorganisms were enriched in a sequencing batch reactor (SBR), operated at two different cycle lengths (CL-24; CL-12). Higher polymer recovery as well as VFA removal was achieved in CL-12 operation both in Stage II (16.3% dry cell weight (DCW); VFA removal, 84%) and Stage III (23.7% DCW; VFA removal, 88%). The PHA obtained was a co-polymer [P(3HB-co-3HV)] of PHB and PHV. The results obtained indicate that this integrated multistage process offers new opportunities to further leverage large scale PHA production with simultaneous waste remediation in the framework of biorefinery. Copyright © 2015 Elsevier Ltd. All rights reserved.

Immobilization of phenylalanine-dehydrogenase on nano-sized polytaurine: a new platform for application of nano-polymeric materials on enzymatic biosensing technology.

PubMed

Omidinia, Eskandar; Shadjou, Nasrin; Hasanzadeh, Mohammad

2014-09-01

A strategy of phenylalanine-dehydrogenase (PheDH) entrapment within the polytaurine matrix is demonstrated to probe the direct electrochemistry of phenylalanine (Pha). It was found that PheDH has been stably immobilized on glassy carbon electrode modified by polytaurine based on simple technique. Cyclic voltammetric study indicated that the oxidation process is irreversible and diffusion controlled. The number of exchanged electrons in the electro-oxidation process was obtained, and the data indicated that Pha is oxidized via one-electron steps. The results revealed that Pha promotes the rate of oxidation by increasing the peak current. The diffusion coefficient and electron-transfer coefficient of Pha were found to be 0.2×10(-6)cm(2)s(-1) and 0.467, respectively. A sensitive, simple and time-saving differential-pulse voltammetric procedure was developed for the analysis of Pha. The results show that by using the proposed method, Pha can be determined with a detection limit of 9 nM. Copyright © 2014 Elsevier B.V. All rights reserved.

Physiological plasticity of cardiorespiratory function in a eurythermal marine teleost, the longjaw mudsucker, Gillichthys mirabilis.

PubMed

Jayasundara, Nishad; Somero, George N

2013-06-01

An insufficient supply of oxygen under thermal stress is thought to define thermal optima and tolerance limits in teleost fish. When under thermal stress, cardiac function plays a crucial role in sustaining adequate oxygen supply for respiring tissues. Thus, adaptive phenotypic plasticity of cardiac performance may be critical for modifying thermal limits during temperature acclimation. Here we investigated effects of temperature acclimation on oxygen consumption, cardiac function and blood oxygen carrying capacity of a eurythermal goby fish, Gillichthys mirabilis, acclimated to 9, 19 and 26°C for 4 weeks. Acclimation did not alter resting metabolic rates or heart rates; no compensation of rates was observed at acclimation temperatures. However, under an acute heat ramp, warm-acclimated fish exhibited greater heat tolerance (CTmax=33.3, 37.1 and 38.9°C for 9°C-, 19°C- and 26°C-acclimated fish, respectively) and higher cardiac arrhythmia temperatures compared with 9°C-acclimated fish. Heart rates measured under an acute heat stress every week during 28 days of acclimation suggested that both maximum heart rates and temperature at onset of maximum heart rates changed over time with acclimation. Hemoglobin levels increased with acclimation temperature, from 35 g l(-1) in 9°C-acclimated fish to 60-80 g l(-1) in 19°C- and 26°C-acclimated fish. Oxygen consumption rates during recovery from acute heat stress showed post-stress elevation in 26°C-acclimated fish. These data, coupled with elevated resting metabolic rates and heart rates at warm temperatures, suggest a high energetic cost associated with warm acclimation in G. mirabilis. Furthermore, acclimatory capacity appears to be optimized at 19°C, a temperature shown by behavioral studies to be close to the species' preferred temperature.

Combination of Fluorescence In Situ Hybridization with Staining Techniques for Cell Viability and Accumulation of PHA and polyP in Microorganisms in Complex Microbial Systems

NASA Astrophysics Data System (ADS)

Nielsen, Jeppe Lund; Kragelund, Caroline; Nielsen, Per Halkjær

Fluorescence in situ hybridization (FISH) can be combined with a number of staining techniques to reveal the relationships between the microorganisms and their function in complex microbial systems with a single-cell resolution. In this chapter, we have focused on staining methods for intracellular storage compounds (polyhydroxyalkanoates, polyphosphate) and a measure for cell viability, reduction of the tetrazolium-based redox stain CTC. These protocols are optimized for the study of microorganisms in waste-water treatment (activated sludge and biofilms), but they may also be used with minor modifications in many other ecosystems.

Partnership for a Healthier America: Creating Change Through Private Sector Partnerships.

PubMed

Simon, Caitlin; Kocot, S Lawrence; Dietz, William H

2017-06-01

This review provides background on the formation of the Partnership for a Healthier America (PHA), that was created in conjunction with the Let's Move! initiative, and an overview of its work to date. To encourage industry to offer and promote healthier options, PHA partners with the private sector. Principles that guide PHA partnerships include ensuring that partnerships represent meaningful change, partners sign a legally binding contract and progress is monitored and publicly reported. Since 2010, PHA has established private sector partnerships in an effort to transform the marketplace to ensure that every child has the chance to grow up at a healthy weight. Many agreements between PHA and its industry partners align with the White House Task Force Report on Childhood Obesity. The reach and impact of over 200 partnerships attest to the success of this initiative.

Selective activation of α7 nicotinic acetylcholine receptor by PHA-543613 improves Aβ25-35-mediated cognitive deficits in mice.

PubMed

Sadigh-Eteghad, S; Talebi, M; Mahmoudi, J; Babri, S; Shanehbandi, D

2015-07-09

Agonists of α7 nicotinic acetylcholine receptors (nAChRs) are currently being considered as therapeutic approaches for managing cognitive deficits in Alzheimer's disease (AD). Present study was designed to evaluate the effect of α7 nAChR selective activation by PHA-543613 (PHA) on beta-amyloid (Aβ)25-35-mediated cognitive deficits in mice. For this purpose, PHA (1mg/kg, i.p.), a selective α7 nAChR agonist, and galantamine (Gal) (3mg/kg, s.c.), an acetylcholine-esterase inhibitor (AChEI) effects on α7 nAChR were tested in Aβ25-35-received (intracerebroventricular, 10 nmol) mice model of AD. Methyllycaconitine (MLA) (1mg/kg, i.p.), a α7 nAChR antagonist, was used for receptor blockage effects evaluation. Working and reference memory in animals was assessed by the Morris water maze (MWM) task. The mRNA and protein levels of α7 subunit were analyzed by real-time PCR and Western blotting, respectively. PHA and Gal, ameliorate Aβ-impaired working and reference memory. However, Gal had less effect than PHA in this regard. Pretreatment with MLA reverses both Gal and PHA effects in MWM. PHA and Gal treatment prevent Aβ-induced α7 subunit protein reduction, but Gal has lesser effect than PHA. This effect blocked by pretreatment with MLA. In neither the pretreatment nor treatment group, the mRNA levels of nAChR α7 subunit were significantly changed. Therefore, α7 nAChR activation, reduces Aβ-induced cognitive deficits and increases the α7 protein level and subsequent neuron survival. However, blockage of receptor, increases Aβ toxicity and cognitive impairment and reduces the α7 nAChR protein level and flowing neuroprotection. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Molecular analysis of two phytohemagglutinin genes and their expression in Phaseolus vulgaris cv. Pinto, a lectin-deficient cultivar of the bean

PubMed Central

Voelker, Toni A.; Staswick, Paul; Chrispeels, Maarten J.

1986-01-01

Phytohemagglutinin (PHA), the seed lectin of the common bean, Phaseolus vulgaris, is encoded by two highly homologous, tandemly linked genes, dlec1 and dlec2, which are coordinately expressed at high levels in developing cotyledons. Their respective transcripts translate into closely related polypeptides, PHA-E and PHA-L, constituents of the tetrameric lectin which accumulates at high levels in developing seeds. In the bean cultivar Pinto UI111, PHA-E is not detectable, and PHA-L accumulates at very reduced levels. To investigate the cause of the Pinto phenotype, we cloned and sequenced the two PHA genes of Pinto, called Pdlec1 and Pdlec2, and determined the abundance of their respective mRNAs in developing cotyledons. Both genes are more than 90% homologous to the normal PHA genes found in other cultivars. Pdlec1 carries a 1-bp frameshift mutation close to the 5' end of its coding sequence. Only very truncated polypeptides could be made from its mRNA. The gene Pdlec2 encodes a polypeptide, which resembles PHA-L and its predicted amino acid sequence agrees with the available Pinto PHA amino acid sequence data. Analysis of the mRNA of developing cotyledons revealed that the Pdlec1 message is reduced 600-fold, and Pdlec2 mRNA is reduced 20-fold with respect to mRNA levels in normal cultivars. A comparison of the sequences which are upstream from the coding sequence shows that Pdlec2 has a 100-bp deletion compared to the other genes (dlec1, dlec2 and Pdlec1). This deletion which contains a large tandem repeat may be responsible for the low level of expression of Pdlec2. The very low expression of Pdlec1 is as yet unexplained. ImagesFig. 5. PMID:16453730

Kinetics of prostaglandin E2 and thromboxane A2 synthesis and suppression of PHA-stimulated peripheral blood mononuclear leucocytes.

PubMed Central

Awara, W; Hillier, K; Jones, D

1986-01-01

The immunomodulatory effects of thromboxane A2 and prostaglandin E2 on peripheral blood mononuclear leucocytes stimulated with PHA in vitro, and the relationship of this to the time-course of their synthesis in culture, were investigated using prostaglandin E2, a thromboxane A2 synthesis inhibitor (UK37248), a thromboxane A2 mimic (U46619) and a thromboxane A2 receptor blocker (EP045). The inhibitory effect of prostaglandin E2 on PHA-induced human peripheral blood mononuclear leucocyte proliferation diminishes if the addition of PGE2 is delayed. If added 4 hr after a maximum concentration of PHA (5 micrograms/ml), the effect of PGE2 was reduced by 60%. If a submaximal concentration of PHA (1 microgram/ml) was used, the effect of PGE2 was not reduced if added 4 hr later but fell by about 60% after 16 hr. UK37248 moderately inhibited PHA-induced activation while substantially inhibiting thromboxane A2 synthesis and simultaneously enhancing PGE2 synthesis. The enhanced accumulation of PGE2 occurs while sensitivity to PGE2 is dropping. U46619, exogenously applied as a thromboxane A2 mimic, inhibited PHA-induced activation at concentrations that did not significantly alter PGE2 synthesis. EP045, which may modulate the effects of endogenous thromboxane A2 by blocking receptors, did not alter PHA-induced activation. We conclude that thromboxane A2 may have a role in inhibiting PHA-induced activation on the basis of the effect of U46619. However, this study highlights difficulties in utilizing prostaglandin and thromboxane receptor and synthesis inhibitors to examine their endogenous role in the modulation of mitogen-induced activation in vitro. If sensitivity to the purported endogenous substance is limited to the early stages of culture and if only low levels are synthesized at this early stage, then blocking drugs would have little effect. PMID:3468061

Effects of PHA-665752 and vemurafenib combination treatment on in vitro and murine xenograft growth of human colorectal cancer cells with BRAFV600E mutations.

PubMed

Zhi, Jie; Li, Zhongxin; Lv, Jian; Feng, Bo; Yang, Donghai; Xue, Liang; Zhao, Zhaolong; Zhang, Yanni; Wu, Jianhua; Jv, Yingchao; Jia, Yitao

2018-03-01

It remains unknown whether blockade of B-Raf proto-oncogene, serine/threonine kinase (BRAF) V600E signaling and MET proto-oncogene, receptor tyrosine kinase (c-Met) signaling is effective in suppressing the growth of human colorectal cancer (CRC) cells. The present study investigated the effects of the vemurafenib alone and in combination with c-Met inhibitor PHA-665752 on the growth of human CRC cells in vitro and in mouse xenografts. HT-29 and RKO CRC cell lines with BRAF V600E mutations and mice bearing HT-29 xenografts were treated with vemurafenib in the absence or presence of PHA-665752. Cell viability and cycle phase were respectively examined by using the MTT and flow cytometry assay. Immunohistochemistry was conducted to detect the protein expression levels of hepatocyte growth factor (HGF), phosphorylated (p)-c-Met, p-AKT serine/threonine kinase (AKT) and p-extracellular signal-regulated kinase (p-ERK). The MTT assay demonstrated that the growth of RKO and HT-29 cells was inhibited by PHA-665752 in a time- and dose-dependent manner (P<0.05), however no significant suppressive effects were observed with vemurafenib. Relative to the PHA-665752 or vemurafenib stand-alone treatment groups, the combination of PHA-665752 and vemurafenib had a significant inhibitory effect on the proliferation of CRC cell lines (P<0.05). The mean tumor volume in mice treated with vemurafenib in combination with PHA-665752 was significantly smaller compared with those treated with only vemurafenib or PHA-665752 (P<0.05). Flow cytometry assay revealed that the G0/G1 phase frequency was significantly increased in the combination group compared with any other treatment groups (P<0.05). Immunohistochemistry demonstrated that vemurafenib in combination with PHA-665752 effectively induced the expression of p-c-Met, p-AKT and p-ERK, however had no effect on HGF.

In-season heat stress compromises postharvest quality and low-temperature sweetening resistance in potato (Solanum tuberosum L.).

PubMed

Zommick, Daniel H; Knowles, Lisa O; Pavek, Mark J; Knowles, N Richard

2014-06-01

The effects of soil temperature during tuber development on physiological processes affecting retention of postharvest quality in low-temperature sweetening (LTS) resistant and susceptible potato cultivars were investigated. 'Premier Russet' (LTS resistant), AO02183-2 (LTS resistant) and 'Ranger Russet' (LTS susceptible) tubers were grown at 16 (ambient), 23 and 29 °C during bulking (111-164 DAP) and maturation (151-180 DAP). Bulking at 29 °C virtually eliminated yield despite vigorous vine growth. Tuber specific gravity decreased as soil temperature increased during bulking, but was not affected by temperature during maturation. Bulking at 23 °C and maturation at 29 °C induced higher reducing sugar levels in the proximal (basal) ends of tubers, resulting in non-uniform fry color at harvest, and abolished the LTS-resistant phenotype of 'Premier Russet' tubers. AO02183-2 tubers were more tolerant of heat for retention of LTS resistance. Higher bulking and maturation temperatures also accelerated LTS and loss of process quality of 'Ranger Russet' tubers, consistent with increased invertase and lower invertase inhibitor activities. During LTS, tuber respiration fell rapidly to a minimum as temperature decreased from 9 to 4 °C, followed by an increase to a maximum as tubers acclimated to 4 °C; respiration then declined over the remaining storage period. The magnitude of this cold-induced acclimation response correlated directly with the extent of buildup in sugars over the 24-day LTS period and thus reflected the effects of in-season heat stress on propensity of tubers to sweeten and lose process quality at 4 °C. While morphologically indistinguishable from control tubers, tubers grown at elevated temperature had different basal metabolic (respiration) rates at harvest and during cold acclimation, reduced dormancy during storage, greater increases in sucrose and reducing sugars and associated loss of process quality during LTS, and reduced ability to improve process quality through reconditioning. Breeding for retention of postharvest quality and LTS resistance should consider strategies for incorporating more robust tolerance to in-season heat stress.

The ultrastructural characterization of mitochondria-rich cells as a response to variations in salinity in two types of teleostean pseudobranch: milkfish (Chanos chanos) and Mozambique tilapia (Oreochromis mossambicus).

PubMed

Yang, Sheng-Hui; Tsai, Jeng-Dau; Kang, Chao-Kai; Yang, Wen-Kai; Kung, Hsiu-Ni; Lee, Tsung-Han

2017-03-01

The pseudobranchs of two euryhaline teleost species, the milkfish (Chanos chanos) and the Mozambique tilapia (Oreochromis mossambicus), were studied after acclimization to different salinities using optical and electron microscopy. The milkfish pseudobranch was the lamellae-free type, with separate lamellae along the filaments containing two groups of mitochondria (Mt)-rich cells: chloride cells (CCs) and pseudobranch type cells (PSCs). Conversely, the tilapia pseudobranch was the embedded type, covered with connective tissues and with only one group of Mt-rich PSCs. Chloride cells were identified according to the apical openings and branched tubular networks around randomly distributed and diversely shaped Mt. Pseudobranchs type cells, however, were characterized according to the orderly arrangement of parallel tubules around closely packed Mt; both the tubules and the Mt were distributed in the vascular side of the cell, but were absent from the apical region. Compared with those of seawater (SW)-acclimated milkfish, the pseudobranchial lamellae of freshwater (FW) specimens were longer on average, and the Mt of the CCs had fewer cristae, were less electron-dense, and were often vacuolated. The Mt in the PSCs of FW-acclimated milkfish and tilapia were larger and more electron-dense than those of their SW-acclimated counterparts; in addition, more tubules were found to aggregately surround the Mt and basolateral membranes in the PSCs of fish from the hypo-osmotic environment. Conversely, the PSCs of tilapia were periodic acid-Schiff (PAS)-positive, and Mt in PSCs were concentrated with more parallel arrays of the tubule system than those of milkfish. Therefore, salinity-dependent changes in the ultrastructures of PSCs suggest their potential role in energy metabolism of both lamellae-free and embedded pseudobranchs, whereas the PAS-positive staining characteristics suggest a role in releasing or storaging polysaccharides in the embedded pseudobranch. J. Morphol. 278:390-402, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Determinants and short-term physiological consequences of PHA immune response in lesser kestrel nestlings.

PubMed

Rodríguez, Airam; Broggi, Juli; Alcaide, Miguel; Negro, Juan José; Figuerola, Jordi

2014-08-01

Individual immune responses are likely affected by genetic, physiological, and environmental determinants. We studied the determinants and short-term consequences of Phytohaemagglutinin (PHA) induced immune response, a commonly used immune challenge eliciting both innate and acquired immunity, on lesser kestrel (Falco naumanni) nestlings in semi-captivity conditions and with a homogeneous diet composition. We conducted a repeated measures analyses of a set of blood parameters (carotenoids, triglycerides, β-hydroxybutyrate, cholesterol, uric acid, urea, total proteins, and total antioxidant capacity), metabolic (resting metabolic rate), genotypic (MHC class II B heterozygosity), and biometric (body mass) variables. PHA challenge did not affect the studied physiological parameters on a short-term basis (<12 hr), except plasma concentrations of triglycerides and carotenoids, which decreased and increased, respectively. Uric acid was the only physiological parameter correlated with the PHA induced immune response (skin swelling), but the change of body mass, cholesterol, total antioxidant capacity, and triglycerides between sessions (i.e., post-pre treatment) were also positively correlated to PHA response. No relationships were detected between MHC gene heterozygosity or resting metabolic rate and PHA response. Our results indicate that PHA response in lesser kestrel nestlings growing in optimal conditions does not imply a severe energetic cost 12 hr after challenge, but is condition-dependent as a rapid mobilization of carotenoids and decrease of triglycerides is elicited on a short-term basis. © 2014 Wiley Periodicals, Inc.

Characterization of Site-Specific Mutations in a Short-Chain-Length/Medium-Chain-Length Polyhydroxyalkanoate Synthase: In Vivo and In Vitro Studies of Enzymatic Activity and Substrate Specificity

PubMed Central

Chuah, Jo-Ann; Tomizawa, Satoshi; Yamada, Miwa; Tsuge, Takeharu; Doi, Yoshiharu

2013-01-01

Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaCCs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaCCs for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C4 and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases. PMID:23584780

Biosynthesis of poly-beta-hydroxyalkanoates by Sphingopyxis chilensis S37 and Wautersia sp. PZK cultured in cellulose pulp mill effluents containing 2,4,6-trichlorophenol.

PubMed

Tobella, Lorena M; Bunster, Marta; Pooley, Amalia; Becerra, José; Godoy, Felix; Martínez, Miguel A

2005-09-01

Poly-beta-hydroxyalkanoates (PHA) polymer is synthesized by different bacterial species. There has been considerable interest in the development and production of biodegradable polymers; however, the high cost of PHA production has restricted its applications. Kraft cellulose industry effluents containing 2,4,6-trichlorophenol (10 or 20 microg ml(-1)) were used by the bacteria Sphingopyxis chilensis S37 and Wautersia sp. PZK to synthesize PHA. In this condition, S. chilensis S37 was able to grow and degrade 2,4,6-trichlorophenol (ca. 60%) and 80% of these cells accumulated PHA. Wautersia PZK completely degraded 2,4,6-TCP and more than 90% of the cells accumulated PHA in 72 h. The PHA detection was performed by flow cytometry and polyester composition was characterized by gas chromatography-mass spectroscopy (GC-MS), indicating that these polymers are made by 3-hydroxybutyric acid and 3-hydroxyhexadecanoic acid for S37 and PZK strains, respectively. Results demonstrated that strains' growth and PHA production and composition are not modified in cellulose effluents with or without 2,4,6-TCP (10-20 microg ml(-1)). Therefore, our results indicate that S. chilensis S37 and Wautersia sp. PZK are able to degrade a toxic compound such as a 2,4,6-TCP and simultaneously produce a valuable biopolymer using low-value substrates.

Post-hypercapnic alkalosis is associated with ventilator dependence and increased ICU stay.

PubMed

Banga, Amit; Khilnani, G C

2009-12-01

Posthypercapnic alkalosis (PHA) is frequently overlooked as a complication of mechanical ventilation in patients with exacerbation of chronic obstructive pulmonary disease (COPD). The current study was conducted to determine the incidence, risk factors for development and effect on outcome of PHA. Eighty-four patients (62 +/- 11 years, range 42-78 years, M:F 58: 26) with exacerbation of COPD with underlying chronic hypercapnic respiratory failure requiring mechanical ventilation were included in a retrospective fashion. PHA was defined as static or rising serum bicarbonate levels, 72 hours or more after return of PaCO2 to baseline, with concurrent pH > 7.44. Development of PHA was noted in 17 patients (20.2%). Corticosteroid use >or=10 days during the hospital stay was an independent risk factor for development of PHA (Adjusted OR, 95% CI: 9.4, 1.6-55.3; P = 0.013). Development of PHA was associated with an increased incidence of ventilator dependence (64.7% vs. 37.3%, OR, 95% CI: 3.1, 1.1-9.4, P = 0.04) and duration of ICU stay (14.7 +/- 6.7 vs. 9.5 +/- 5.9, P = 0.01) but no increase in hospital mortality (43.3% vs. 41.2%, P = NS). It is concluded that PHA is a common complication in patients with exacerbation of COPD requiring mechanical ventilation and is associated with increased incidence of ventilator dependence and ICU stay.

Advances in cyanobacterial polyhydroxyalkanoates production.

PubMed

Singh, Akhilesh Kumar; Mallick, Nirupama

2017-11-01

Polyhydroxyalkanoates (PHAs) have received much attention in the current scenario due to their attractive material properties, namely biodegradability, biocompatibility, thermoplasticity, hydrophobicity, piezoelectricity and stereospecificity. All these properties make them highly competitive for various industrial applications similar to non-degradable conventional plastics. In PHA biosynthesis, PHA synthase acts as a natural catalyst for PHA polymerization process using the (R)-hydroxyacyl-CoA as substrate. Cyanobacteria can accumulate PHAs under photoautotrophic and/or mixotrophic growth conditions with organic substrates such as acetate, glucose, propionate, valerate, and so on. The natural incidence of PHA accumulation by the cyanobacteria is known since 1966. Nevertheless, PHA accumulation in cyanobacteria based on the cell biomass and volumetric productivity is critically lower than the heterotrophic bacteria. Consequently, cyanobacteria are nowadays not considered for commercial production of PHAs. Thus, strain improvements by genetic modification, new cultivation and harvesting techniques, advanced photobioreactor development, efficient and sustainable downstream processes, alternate economical carbon sources and usage of various metabolic inhibitors are suggested for enhancing cyanobacterial PHA accumulation. In addition, identification of transcriptional regulators like RNA polymerase sigma factor (SigE) and a response regulator (Rre37) together with the recent major scientific breakthrough on the existence of complete Krebs cycle in cyanobacteria would be helpful in taking PHA production from cyanobacteria to a new-fangled height in near future. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

24 CFR 982.553 - Denial of admission and termination of assistance for criminals and alcohol abusers.

Code of Federal Regulations, 2014 CFR

2014-04-01

... admission—(1) Prohibiting admission of drug criminals. (i) The PHA must prohibit admission to the program of... federally assisted housing for drug-related criminal activity. However, the PHA may admit the household if the PHA determines: (A) That the evicted household member who engaged in drug-related criminal...

24 CFR 982.553 - Denial of admission and termination of assistance for criminals and alcohol abusers.

Code of Federal Regulations, 2011 CFR

2011-04-01

... admission—(1) Prohibiting admission of drug criminals. (i) The PHA must prohibit admission to the program of... federally assisted housing for drug-related criminal activity. However, the PHA may admit the household if the PHA determines: (A) That the evicted household member who engaged in drug-related criminal...

24 CFR 982.553 - Denial of admission and termination of assistance for criminals and alcohol abusers.

Code of Federal Regulations, 2012 CFR

2012-04-01

... admission—(1) Prohibiting admission of drug criminals. (i) The PHA must prohibit admission to the program of... federally assisted housing for drug-related criminal activity. However, the PHA may admit the household if the PHA determines: (A) That the evicted household member who engaged in drug-related criminal...

76 FR 10135 - Public Housing Evaluation and Oversight: Changes to the Public Housing Assessment System (PHAS...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-02-23

... PHA to four key areas of a PHA's operations: (1) The physical condition of the PHA's properties; (2... and to require PHAs to be scored on performance based on evaluation of four indicators: physical... changes proposed to each of the four current PHAS indicators are as follows: Physical. The physical...

24 CFR 905.120 - Penalties for slow obligation or expenditure of CFP assistance.

Code of Federal Regulations, 2010 CFR

2010-04-01

..., DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT THE PUBLIC HOUSING CAPITAL FUND PROGRAM § 905.120 Penalties for... the PHA not later than September 30, 1999. (b) Exceptions to obligation requirement—(1) Extension... 12 months, based on: (i) The size of the PHA; (ii) The complexity of the capital program of the PHA...

Accumulation of Polyhydroxyalkanoic Acid Containing Large Amounts of Unsaturated Monomers in Pseudomonas fluorescens BM07 Utilizing Saccharides and Its Inhibition by 2-Bromooctanoic Acid

PubMed Central

Lee, Ho-Joo; Choi, Mun Hwan; Kim, Tae-Un; Yoon, Sung Chul

2001-01-01

A psychrotrophic bacterium, Pseudomonas fluorescens BM07, which is able to accumulate polyhydroxyalkanoic acid (PHA) containing large amounts of 3-hydroxy-cis-5-dodecenoate unit up to 35 mol% in the cell from unrelated substrates such as fructose, succinate, etc., was isolated from an activated sludge in a municipal wastewater treatment plant. When it was grown on heptanoic acid (C7) to hexadecanoic acid (C16) as the sole carbon source, the monomer compositional characteristics of the synthesized PHA were similar to those observed in other fluorescent pseudomonads belonging to rRNA homology group I. However, growth on stearic acid (C18) led to no PHA accumulation, but instead free stearic acid was stored in the cell. The existence of the linkage between fatty acid de novo synthesis and PHA synthesis was confirmed by using inhibitors such as acrylic acid and two other compounds, 2-bromooctanoic acid and 4-pentenoic acid, which are known to inhibit β-oxidation enzymes in animal cells. Acrylic acid completely inhibited PHA synthesis at a concentration of 4 mM in 40 mM octanoate-grown cells, but no inhibition of PHA synthesis occurred in 70 mM fructose-grown cells in the presence of 1 to 5 mM acrylic acid. 2-Bromooctanoic acid and 4-pentenoic acid were found to much inhibit PHA synthesis much more strongly in fructose-grown cells than in octanoate-grown cells over concentrations ranging from 1 to 5 mM. However, 2-bromooctanoic acid and 4-pentenoic acid did not inhibit cell growth at all in the fructose media. Especially, with the cells grown on fructose, 2-bromooctanoic acid exhibited a steep rise in the percent PHA synthesis inhibition over a small range of concentrations below 100 μM, a finding indicative of a very specific inhibition, whereas 4-pentenoic acid showed a broad, featureless concentration dependence, suggesting a rather nonspecific inhibition. The apparent inhibition constant Ki (the concentration for 50% inhibition of PHA synthesis) for 2-bromooctanoic acid was determined to be 60 μM, assuming a single-site binding of the inhibitor at a specific inhibition site. Thus, it seems likely that a coenzyme A thioester derivative of 2-bromooctanoic acid specifically inhibits an enzyme linking the two pathways, fatty acid de novo synthesis and PHA synthesis. We suggest that 2-bromooctanoic acid can substitute for the far more expensive (2,000 times) and cell-growth-inhibiting PHA synthesis inhibitor, cerulenin. PMID:11679314

Prestroke physical activity is associated with good functional outcome and arterial recanalization after stroke due to a large vessel occlusion.

PubMed

Ricciardi, Ana Clara; López-Cancio, Elena; Pérez de la Ossa, Natalia; Sobrino, Tomás; Hernández-Pérez, María; Gomis, Meritxell; Munuera, Josep; Muñoz, Lucía; Dorado, Laura; Millán, Mónica; Dávalos, Antonio; Arenillas, Juan F

2014-01-01

Although multiple studies and meta-analyses have consistently suggested that regular physical activity (PhA) is associated with a decreased stroke risk and recurrence, there is limited data on the possible preconditioning effect of prestroke PhA on stroke severity and prognosis. We aimed to study the association of prestroke PhA with different outcome variables in patients with acute ischemic stroke due to an anterior large vessel occlusion. The Prestroke Physical Activity and Functional Recovery in Patients with Ischemic Stroke and Arterial Occlusion trial is an observational and longitudinal study that included consecutive patients with acute ischemic stroke admitted to a single tertiary stroke center. Main inclusion criteria were: anterior circulation ischemic stroke within 12 h from symptom onset; presence of a confirmed anterior large vessel occlusion, and functional independence previous to stroke. Prestroke PhA was evaluated with the International Physical Activity Questionnaire and categorized into mild, moderate and high levels by means of metabolic equivalent (MET) minutes per week thresholds. The primary outcome measure was good functional outcome at 3 months (modified Rankin scale ≤2). Secondary outcomes were severity of stroke at admission, complete early recanalization, early dramatic neurological improvement and final infarct volume. During the study period, 159 patients fulfilled the above criteria. The mean age was 68 years, 62% were men and the baseline NIHSS score was 17. Patients with high levels of prestroke PhA were younger, had more frequently distal occlusions and had lower levels of blood glucose and fibrinogen at admission. After multivariate analysis, a high level of prestroke PhA was associated with a good functional outcome at 3 months. Regarding secondary outcome variables and after adjustment for relevant factors, a high level of prestroke PhA was independently associated with milder stroke severity at admission, early dramatic improvement, early arterial recanalization after intravenous thrombolysis and lower final infarct volume. The beneficial association of prestroke PhA with stroke outcomes was already present with a cutoff point of 1,000 MET min/week, a level of PhA easily achieved by walking 1 h/day during 5 days or by doing a vigorous aerobic activity 1 h/day twice a week. Prestroke PhA is independently associated with favorable stroke outcomes after a large vessel occlusion. Future research on the underlying mechanisms is needed to understand this neuroprotective effect of PhA. © 2014 S. Karger AG, Basel.

The PHA Test Reflects Acquired T-Cell Mediated Immunocompetence in Birds

PubMed Central

Tella, José L.; Lemus, Jesús A.; Carrete, Martina; Blanco, Guillermo

2008-01-01

Background cological immunology requires techniques to reliably measure immunocompetence in wild vertebrates. The PHA-skin test, involving subcutaneous injection of a mitogen (phytohemagglutinin, PHA) and measurement of subsequent swelling as a surrogate of T-cell mediated immunocompetence, has been the test of choice due to its practicality and ease of use in the field. However, mechanisms involved in local immunological and inflammatory processes provoked by PHA are poorly known, and its use and interpretation as an acquired immune response is currently debated. Methodology Here, we present experimental work using a variety of parrot species, to ascertain whether PHA exposure produces larger secondary than primary responses as expected if the test reflects acquired immunocompetence. Moreover, we simultaneously quantified T-lymphocyte subsets (CD4+, CD5+ and CD8+) and plasma proteins circulating in the bloodstream, potentially involved in the immunological and inflammatory processes, through flow cytometry and electrophoresis. Principal Findings Our results showed stronger responses after a second PHA injection, independent of species, time elapsed and changes in body mass of birds between first and second injections, thus supporting the adaptive nature of this immune response. Furthermore, the concomitant changes in the plasma concentrations of T-lymphocyte subsets and globulins indicate a causal link between the activation of the T-cell mediated immune system and local tissue swelling. Conclusions/Significance These findings justify the widespread use of the PHA-skin test as a reliable evaluator of acquired T-cell mediated immunocompetence in diverse biological disciplines. Further experimental research should be aimed at evaluating the relative role of innate immunocompetence in wild conditions, where the access to dietary proteins varies more than in captivity, and to ascertain how PHA responses relate to particular host-parasite interactions. PMID:18820730

Comparative proteome analysis reveals four novel polyhydroxybutyrate (PHB) granule-associated proteins in Ralstonia eutropha H16.

PubMed

Sznajder, Anna; Pfeiffer, Daniel; Jendrossek, Dieter

2015-03-01

Identification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated from Ralstonia eutropha but absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative α/β-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon with phaB2 (acetoacetyl-coenzyme A [CoA] reductase) and phaC2 (PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmed in vivo by fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Comparative Proteome Analysis Reveals Four Novel Polyhydroxybutyrate (PHB) Granule-Associated Proteins in Ralstonia eutropha H16

PubMed Central

Sznajder, Anna; Pfeiffer, Daniel

2014-01-01

Identification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated from Ralstonia eutropha but absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative α/β-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon with phaB2 (acetoacetyl-coenzyme A [CoA] reductase) and phaC2 (PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmed in vivo by fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism. PMID:25548058

Toxicity Assessment of Common Beans (Phaseolus vulgaris L.) Widely Consumed by Tunisian Population.

PubMed

Nciri, Nader; Cho, Namjun; El Mhamdi, Faiçal; Ben Ismail, Hanen; Ben Mansour, Abderraouf; Sassi, Fayçal Haj; Ben Aissa-Fennira, Fatma

2015-09-01

This research aimed at assessing the content and the functional properties of phytohemagglutinin (PHA) in different varieties of beans widely consumed in Tunisia through soaking, cooking, autoclaving, germination, and their combinations. This study was carried out on three varieties of white beans grown in different localities of Tunisia, namely Twila, Coco, and Beldia, as well as on imported and local canned beans. All bean samples underwent biochemical and immunological evaluation by employing several techniques such as indirect competitive enzyme-linked immunosorbent assay (ELISA), hemagglutinating assay, Ouchterlony double immunodiffusion, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Biochemical and immunological analyses indicated that raw dry beans contained a considerable amount of proteins and PHAs. ELISA demonstrated that soaking, either in plain water or in alkaline solution, caused an increase in the concentration of PHA. A slight increase of PHA was produced equally by germination during 4 days in all bean varieties. Cooking or autoclaving of presoaked beans resulted in a complete disappearance of PHA. ELISA test also proved that both imported and local canned beans contained fingerprints of PHA. Hemagglutination assays showed that not only cooked and autoclaved presoaked beans lacked the ability to agglutinate red blood cells but also autoclaved unsoaked beans did. In agar gel immunodiffusion using rabbit anti-PHA serum, raw, soaked, cooked unsoaked, and sprouted beans gave precipitin arc reactions, indicating that PHA existed in immunoreactive form in the tested seeds. SDS-PAGE electrophoretograms showed protein isolates of Twila and Beldia beans to have different profiles through soaking, cooking, and autoclaving processes. This work revealed that the combination of soaking and cooking/autoclaving was the best way in reducing PHA content and its activity in all bean varieties when compared with germination.

Validity of Monod kinetics at different sludge ages--peptone biodegradation under aerobic conditions.

PubMed

Orhon, Derin; Cokgor, Emine Ubay; Insel, Guclu; Karahan, Ozlem; Katipoglu, Tugce

2009-12-01

The study presented an evaluation of the effect of culture history (sludge age) on the growth kinetics of a mixed culture grown under aerobic conditions. It involved an experimental setup where a lab-scale sequencing batch reactor was operated at steady-state at two different sludge ages (theta(X)) of 2 and 10 days. The system sustained a mixed culture fed with a synthetic substrate mainly consisting of peptone. The initial concentration of substrate COD was selected around 500 mg COD/L. Polyhydroxyalkanoate (PHA) storage occurred to a limited extent, around 30 mg COD/L for theta(X)=10 days and 15 mg COD/L for theta(X)=2 days. Evaluation of the experimental data based on calibration of two different models provided consistent and reliable evidence for a variable Monod kinetics where the maximum specific growth rate, was assessed as 6.1/day for theta(X)=2 days and 4.1/day for theta(X)=10 days. A similar variability was also applicable for the hydrolysis and storage kinetics. The rate of storage was significantly lower than the levels reported in the literature, exhibiting the ability of the microorganisms to regulate their metabolic mechanisms for adjusting the rate of microbial growth and storage competing for the same substrate. This adjustment evidently resulted in case-specific, variable kinetics both for microbial growth and substrate storage.

Hypoxic acclimation leads to metabolic compensation after reoxygenation in Atlantic salmon yolk-sac alevins.

PubMed

Polymeropoulos, Elias T; Elliott, Nicholas G; Frappell, Peter B

2017-11-01

Hypoxia is common in aquatic environments and has substantial effects on development, metabolism and survival of aquatic organisms. To understand the physiological effects of hypoxia and its dependence on temperature, metabolic rate ( [Formula: see text] ) and cardiorespiratory function were studied in response to acute hypoxia (21→5kPa) at different measurement temperatures (T a ; 4, 8 and 12°C) in Salmo salar alevins that were incubated under normoxic conditions (P O 2 =21kPa) or following hypoxic acclimation (P O 2 =10kPa) as well as two different temperatures (4°C or 8°C). Hypoxic acclimation lead to a developmental delay manifested through slower yolk absorption. The general response to acute hypoxia was metabolic depression (~60%). Hypoxia acclimated alevins had higher [Formula: see text] s when measured in normoxia than alevins acclimated to normoxia. [Formula: see text] s were elevated to the same degree (~30% per 4°C change) irrespective of T a . Under severe, acute hypoxia (~5kPa) and irrespective of T a or acclimation, [Formula: see text] s were similar between most groups. This suggests that despite different acclimation regimes, O 2 transport was limited to the same degree. While cardiorespiratory function (heart-, ventilation rate) was unchanged in response to acute hypoxia after normoxic acclimation, hypoxic acclimation led to cardiorespiratory changes predominantly in severe hypoxia, indicating earlier onset and plasticity of cardiorespiratory control mechanisms. Although [Formula: see text] in normoxia was higher after hypoxic acclimation, at the respective acclimation P O 2 , [Formula: see text] was similar in normoxia and hypoxia acclimated alevins. This is indicative of metabolic compensation to an intrinsic [Formula: see text] at the acclimation condition in hypoxia-acclimated alevins after re-exposure to normoxia. Copyright © 2017 Elsevier Inc. All rights reserved.

Rapid Acclimation Ability Mediated by Transcriptome Changes in Reef-Building Corals.

PubMed

Bay, Rachael A; Palumbi, Stephen R

2015-05-15

Population response to environmental variation involves adaptation, acclimation, or both. For long-lived organisms, acclimation likely generates a faster response but is only effective if the rates and limits of acclimation match the dynamics of local environmental variation. In coral reef habitats, heat stress from extreme ocean warming can occur over several weeks, resulting in symbiont expulsion and widespread coral death. However, transcriptome regulation during short-term acclimation is not well understood. We examined acclimation during a 11-day experiment in the coral Acropora nana. We acclimated colonies to three regimes: ambient temperature (29 °C), increased stable temperature (31 °C), and variable temperature (29-33 °C), mimicking local heat stress conditions. Within 7-11 days, individuals acclimated to increased temperatures had higher tolerance to acute heat stress. Despite physiological changes, no gene expression changes occurred during acclimation before acute heat stress. However, we found strikingly different transcriptional responses to heat stress between acclimation treatments across 893 contigs. Across these contigs, corals acclimated to higher temperatures (31 °C or 29-33 °C) exhibited a muted stress response--the magnitude of expression change before and after heat stress was less than in 29 °C acclimated corals. Our results show that corals have a rapid phase of acclimation that substantially increases their heat resilience within 7 days and that alters their transcriptional response to heat stress. This is in addition to a previously observed longer term response, distinguishable by its shift in baseline expression, under nonstressful conditions. Such rapid acclimation may provide some protection for this species of coral against slow onset of warming ocean temperatures. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Rapid Acclimation Ability Mediated by Transcriptome Changes in Reef-Building Corals

PubMed Central

Bay, Rachael A.; Palumbi, Stephen R.

2015-01-01

Population response to environmental variation involves adaptation, acclimation, or both. For long-lived organisms, acclimation likely generates a faster response but is only effective if the rates and limits of acclimation match the dynamics of local environmental variation. In coral reef habitats, heat stress from extreme ocean warming can occur over several weeks, resulting in symbiont expulsion and widespread coral death. However, transcriptome regulation during short-term acclimation is not well understood. We examined acclimation during a 11-day experiment in the coral Acropora nana. We acclimated colonies to three regimes: ambient temperature (29 °C), increased stable temperature (31 °C), and variable temperature (29–33 °C), mimicking local heat stress conditions. Within 7–11 days, individuals acclimated to increased temperatures had higher tolerance to acute heat stress. Despite physiological changes, no gene expression changes occurred during acclimation before acute heat stress. However, we found strikingly different transcriptional responses to heat stress between acclimation treatments across 893 contigs. Across these contigs, corals acclimated to higher temperatures (31 °C or 29–33 °C) exhibited a muted stress response—the magnitude of expression change before and after heat stress was less than in 29 °C acclimated corals. Our results show that corals have a rapid phase of acclimation that substantially increases their heat resilience within 7 days and that alters their transcriptional response to heat stress. This is in addition to a previously observed longer term response, distinguishable by its shift in baseline expression, under nonstressful conditions. Such rapid acclimation may provide some protection for this species of coral against slow onset of warming ocean temperatures. PMID:25979751

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Cupriavidus necator from waste rapeseed oil using propanol as a precursor of 3-hydroxyvalerate.

PubMed

Obruca, Stanislav; Marova, Ivana; Snajdar, Ondrej; Mravcova, Ludmila; Svoboda, Zdenek

2010-12-01

Waste rapeseed oil is a useful substrate for polyhydroxyalkanoates (PHA) production employing Cupriavidus necator H16. In fed-batch mode, we obtained biomass and PHA yields of 138 and 105 g l(-1), respectively. Yield coefficient and volumetric productivity were 0.83 g PHA per g oil and 1.46 g l(-1) h(-1), respectively. Propanol at 1% (v/v) enhanced both PHA and biomass formation significantly and, furthermore, resulted in incorporation of 3-hydroxyvalerate units into PHA structure. Thus, propanol can be used as an effective precursor of 3-hydroxyvalarete for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer. During the fed-batch cultivation, propanol concentration was maintained at 1% which resulted in 8% content of 3-hydroxyvalerate in copolymer.

24 CFR 943.144 - What financial impact do operations of a subsidiary, affiliate, or joint venture have on a PHA?

Code of Federal Regulations, 2010 CFR

2010-04-01

... of a subsidiary, affiliate, or joint venture have on a PHA? 943.144 Section 943.144 Housing and Urban... CONSORTIA AND JOINT VENTURES Subsidiaries, Affiliates, Joint Ventures in Public Housing § 943.144 What financial impact do operations of a subsidiary, affiliate, or joint venture have on a PHA? Income generated...

24 CFR 903.2 - With respect to admissions, what must a PHA do to deconcentrate poverty in its developments and...

Code of Federal Regulations, 2012 CFR

2012-04-01

... must a PHA do to deconcentrate poverty in its developments and comply with fair housing requirements... URBAN DEVELOPMENT PUBLIC HOUSING AGENCY PLANS Deconcentration of Poverty and Fair Housing in Program Admissions § 903.2 With respect to admissions, what must a PHA do to deconcentrate poverty in its...

24 CFR 903.2 - With respect to admissions, what must a PHA do to deconcentrate poverty in its developments and...

Code of Federal Regulations, 2014 CFR

2014-04-01

... must a PHA do to deconcentrate poverty in its developments and comply with fair housing requirements... URBAN DEVELOPMENT PUBLIC HOUSING AGENCY PLANS Deconcentration of Poverty and Fair Housing in Program Admissions § 903.2 With respect to admissions, what must a PHA do to deconcentrate poverty in its...

24 CFR 903.2 - With respect to admissions, what must a PHA do to deconcentrate poverty in its developments and...

Code of Federal Regulations, 2013 CFR

2013-04-01

... must a PHA do to deconcentrate poverty in its developments and comply with fair housing requirements... URBAN DEVELOPMENT PUBLIC HOUSING AGENCY PLANS Deconcentration of Poverty and Fair Housing in Program Admissions § 903.2 With respect to admissions, what must a PHA do to deconcentrate poverty in its...

Response to phytohaemagglutinin of lymphocytes from mice treated with anti-lymphocyte globulin

PubMed Central

Tursi, A.; Greaves, M. F.; Torrigiani, G.; Playfair, J. H. L.; Roitt, I. M.

1969-01-01

Thymus, spleen, lymph node and peripheral blood lymphocytes taken from mice treated with anti-lymphocyte globulin (ALG) showed a greatly diminished response to PHA in vitro. Recovery of circulating lymphocyte levels preceded recovery of responsiveness to PHA. The latter could be prevented by reinjection of ALG or by thymectomy. Grafts were rejected within a period equal to the normal rejection time after PHA responsiveness had recovered to a value of approximately 20 per cent of the normal. Thus the effect of ALG on thymus dependent lymphocytes in mice can be monitored by assessing the PHA sensitivity of peripheral white blood cells. ImagesFIG. 1 PMID:4900922

Obtaining lower bounds from the progressive hedging algorithm for stochastic mixed-integer programs

DOE PAGES

Gade, Dinakar; Hackebeil, Gabriel; Ryan, Sarah M.; ...

2016-04-02

We present a method for computing lower bounds in the progressive hedging algorithm (PHA) for two-stage and multi-stage stochastic mixed-integer programs. Computing lower bounds in the PHA allows one to assess the quality of the solutions generated by the algorithm contemporaneously. The lower bounds can be computed in any iteration of the algorithm by using dual prices that are calculated during execution of the standard PHA. In conclusion, we report computational results on stochastic unit commitment and stochastic server location problem instances, and explore the relationship between key PHA parameters and the quality of the resulting lower bounds.

Growth kinetics, effect of carbon substrate in biosynthesis of mcl-PHA by Pseudomonas putida Bet001

PubMed Central

Gumel, A.M.; Annuar, M.S.M.; Heidelberg, T.

2014-01-01

Growth associated biosynthesis of medium chain length poly-3-hydroxyalkanoates (mcl-PHA) in Pseudomonas putida Bet001 isolated from palm oil mill effluent was studied. Models with substrate inhibition terms described well the kinetics of its growth. Selected fatty acids (C8:0 to C18:1) and ammonium were used as carbon and nitrogen sources during growth and PHA biosynthesis, resulting in PHA accumulation of about 50 to 69% (w/w) and PHA yields ranging from 10.12 g L−1 to 15.45 g L−1, respectively. The monomer composition of the PHA ranges from C4 to C14, and was strongly influenced by the type of carbon substrate fed. Interestingly, an odd carbon chain length (C7) monomer was also detected when C18:1 was fed. Polymer showed melting temperature (Tm) of 42.0 (± 0.2) °C, glass transition temperature (Tg) of −1.0 (± 0.2) °C and endothermic melting enthalpy of fusion (ΔHf) of 110.3 (± 0.1) J g−1. The molecular weight (Mw) range of the polymer was relatively narrow between 55 to 77 kDa. PMID:25242925

Growth kinetics, effect of carbon substrate in biosynthesis of mcl-PHA by Pseudomonas putida Bet001.

PubMed

Gumel, A M; Annuar, M S M; Heidelberg, T

2014-01-01

Growth associated biosynthesis of medium chain length poly-3-hydroxyalkanoates (mcl-PHA) in Pseudomonas putida Bet001 isolated from palm oil mill effluent was studied. Models with substrate inhibition terms described well the kinetics of its growth. Selected fatty acids (C8:0 to C18:1) and ammonium were used as carbon and nitrogen sources during growth and PHA biosynthesis, resulting in PHA accumulation of about 50 to 69% (w/w) and PHA yields ranging from 10.12 g L(-1) to 15.45 g L(-1), respectively. The monomer composition of the PHA ranges from C4 to C14, and was strongly influenced by the type of carbon substrate fed. Interestingly, an odd carbon chain length (C7) monomer was also detected when C18:1 was fed. Polymer showed melting temperature (T m) of 42.0 (± 0.2) °C, glass transition temperature (T g) of -1.0 (± 0.2) °C and endothermic melting enthalpy of fusion (ΔHf) of 110.3 (± 0.1) J g(-1). The molecular weight (M w) range of the polymer was relatively narrow between 55 to 77 kDa.

MET inhibitor PHA-665752 suppresses the hepatocyte growth factor-induced cell proliferation and radioresistance in nasopharyngeal carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Tongxin; Li, Qi; Sun, Quanquan

2014-06-20

Highlights: • We demonstrated that irradiation induced MET overexpression and activation. • The aberrant MET signal mediated by HGF induced proliferation and radioresistance of NPC cells. • MET inhibitor PHA-665752 effectively suppressed HGF induced cell proliferation and radioresistance in NPC cells. • PHA-665752 suppressed the three downstream pathway of HGF/MET signal in a dose-dependent manner. - Abstract: Although ionizing radiation (IR) has provided considerable improvements in nasopharyngeal carcinoma (NPC), in subsets of patients, radioresistance is still a major problem in the treatment. In this study, we demonstrated that irradiation induced MET overexpression and activation, and the aberrant MET signal mediatedmore » by hepatocyte growth factor (HGF) induced radioresistance. We also found that MET inhibitor PHA-665752 effectively suppressed HGF induced cell proliferation and radioresistance in NPC cells. Further investigation indicated that PHA-665752 suppressed the phosphorylation of the Akt, ERK1/2, and STAT3 proteins in a dose-dependent manner. Our data indicated that the combination of IR with a MET inhibitor, such as PHA-665752, might be a promising therapeutic strategy for NPC.« less

Exploring how organic matter controls structural transformations in natural aquatic nanocolloidal dispersions.

PubMed

King, Stephen M; Jarvie, Helen P

2012-07-03

The response of the dispersion nanostructure of surface river bed sediment to the controlled removal and readdition of natural organic matter (NOM), in the absence and presence of background electrolyte, was examined using the technique of small-angle neutron scattering (SANS). Partial NOM removal induced aggregation of the mineral particles, but more extensive NOM removal restored colloidal stability. When peat humic acid (PHA) was added to a NOM-deficient sediment concentration-related structural transformations were observed: at 255 mg/L PHA aggregation of the nanocolloid was actually enhanced, but at 380 mg/L PHA disaggregation and colloidal stability were promoted. The addition of 2 mM CaCl(2) induced mild aggregation in the native sediment but not in sediments with added PHA, suggesting that the native NOM and the PHA respond differently to changes in ionic strength. A first attempt at using SANS to directly characterize the thickness and coverage of an adsorbed PHA layer in a natural nanocolloid is also presented. The results are discussed in the context of a hierarchical aquatic colloidal nanostructure, and the implications for contemporary studies of the role of dissolved organic carbon (DOC) in sustaining the transport of colloidal iron in upland catchments.

Endogenous ethanol affects biopolyester molecular weight in recombinant Escherichia coli.

PubMed

Hiroe, Ayaka; Hyakutake, Manami; Thomson, Nicholas M; Sivaniah, Easan; Tsuge, Takeharu

2013-11-15

In biopolyester synthesis, polyhydroxyalkanoate (PHA) synthase (PhaC) catalyzes the polymerization of PHA in bacterial cells, followed by a chain transfer (CT) reaction in which the PHA polymer chain is transferred from PhaC to a CT agent. Accordingly, the frequency of CT reaction determines PHA molecular weight. Previous studies have shown that exogenous alcohols are effective CT agents. This study aimed to clarify the effect of endogenous ethanol as a CT agent for poly[(R)-3-hydroxybutyrate] [P(3HB)] synthesis in recombinant Escherichia coli, by comparing with that of exogenous ethanol. Ethanol supplementation to the culture medium reduced P(3HB) molecular weights by up to 56% due to ethanol-induced CT reaction. NMR analysis of P(3HB) polymers purified from the culture supplemented with (13)C-labeled ethanol showed the formation of a covalent bond between ethanol and P(3HB) chain at the carboxyl end. Cultivation without ethanol supplementation resulted in the reduction of P(3HB) molecular weight with increasing host-produced ethanol depending on culture aeration. On the other hand, production in recombinant BW25113(ΔadhE), an alcohol dehydrogenase deletion strain, resulted in a 77% increase in molecular weight. Analysis of five E. coli strains revealed that the estimated number of CT reactions was correlated with ethanol production. These results demonstrate that host-produced ethanol acts as an equally effective CT agent as exogenous ethanol, and the control of ethanol production is important to regulate the PHA molecular weight.

Fungal degradation of polyhydroxyalkanoates and a semiquantitative assay for screening their degradation by terrestrial fungi.

PubMed

Matavulj, M; Molitoris, H P

1992-12-01

The current problems with decreasing fossile resources and increasing environmental pollution by petrochemical-based plastics have stimulated investigations to find biosynthetic materials which are also biodegradable. Bacterial reserve materials such as polyhydroxyalkanoates (PHA) have been discovered to possess thermoplastic properties and can be synthesized from renewable resources. Poly-beta-hydroxybutyric acid (PHB) is at present the most promising PHA; and BIOPOL, its copolymer with poly-beta-hydroxy-valerate (PHV), is already industrially produced (ICI, UK), and used as packaging material (WELLA, FRG). According to the literature, PHA degradation has so far mainly been observed in bacteria; only under certain environmental conditions has fungal degradation of PHAs been indicated. Since fungi constitute an important part of microbial populations participating in degradation processes, a simple screening method for fungal degradation of BIOPOL, a PHA-based plastic, was developed. Several media with about 150 fungal strains from different terrestrial environments and belonging to different systematic and ecological groups were used. PHA depolymerization was tested on three PHB-based media, each with 0.1% BIOPOL or PHB homopolymer causing turbidity of the medium. The media contained either a comparatively low or high content of organic carbon (beside PHA) or were based on mineral medium with PHA as the principal source of carbon. The degradation activity was detectable due to formation of a clear halo around the colony (Petri plates) or a clear zone under the colony (test tubes).(ABSTRACT TRUNCATED AT 250 WORDS)

Correlations between phytohemagglutinin response and leukocyte profile, and bactericidal capacity in a wild rodent.

PubMed

Zhang, Zhiqiang; Zhao, Zhijun

2015-05-01

Phytohemagglutinin (PHA)-induced swelling is widely used to investigate cell-mediated and innate immunity across different vertebrate taxa. However, its physiological mechanism is still an open question due to the complexity of the involved immune components. In the present study, we measured the synchronous variations of PHA response, the proportion of different subtypes of leukocytes, as well as serum bactericidal capacity in circulation blood at 6, 12 and 24 h after PHA versus PBS injection in striped hamster, Cricetulus barabensis. First, the results showed that PHA responses reached a peak at 6 h postinjection, then sharply declined at 12 h and 24 h postinjection. Serum bactericidal capacity was higher at 6 h and 12 h than at 24 h. The proportion of different subtypes of leukocytes, as well as the ratio of neutrophils to lymphocytes did not display significant changes across different time points. Second, PHA response was positively correlated with the proportion of neutrophils and serum bactericidal capacity. The proportion of monocytes was negatively correlated with that of eosinophils and neutrophils. The proportion of basophils was negatively correlated with that of lymphocytes. Our results indicate that earlier enhanced PHA response is important for the striped hamster to cope with changing environmental conditions due to its small body mass, and the increased components of innate immunity in circulation blood may contribute to the enhancement of PHA swelling response. © 2015 International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and Wiley Publishing Asia Pty Ltd.

Controlling lipid oxidation via a biomimetic iron chelating active packaging material.

PubMed

Tian, Fang; Decker, Eric A; Goddard, Julie M

2013-12-18

Previously, a siderophore-mimetic metal chelating active packaging film was developed by grafting poly(hydroxamic acid) (PHA) from the surface of polypropylene (PP) films. The objective of the current work was to demonstrate the potential applicability of this PP-g-PHA film to control iron-promoted lipid oxidation in food emulsions. The iron chelating activity of this film was investigated, and the surface chemistry and color intensity of films were also analyzed after iron chelation. In comparison to the iron chelating activity in the free Fe(3+) solution, the PP-g-PHA film retained approximately 50 and 30% of its activity in nitrilotriacetic acid (NTA)/Fe(3+) and citric acid/Fe(3+) solutions, respectively (pH 5.0), indicating a strong chelating strength for iron. The ability of PP-g-PHA films to control lipid oxidation was demonstrated in a model emulsion system (pH 3.0). PP-g-PHA films performed even better than ethylenediaminetetraacetic acid (EDTA) in preventing the formation of volatile oxidation products. The particle size and ζ potential results of emulsions indicated that PP-g-PHA films had no adverse effects on the stability of the emulsion system. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) analysis suggested a non-migratory nature of the PP-g-PHA film surface. These results suggest that such biomimetic, non-migratory metal chelating active packaging films have commercial potential in protecting foods against iron-promoted lipid oxidation.

Biosynthesis of poly-beta-hydroxyalkanoate by Brevundimonas vesicularis LMG P-23615 and Sphingopyxis macrogoltabida LMG 17324 using acid-hydrolyzed sawdust as carbon source.

PubMed

Silva, Johanna A; Tobella, Lorena M; Becerra, José; Godoy, Félix; Martínez, Miguel A

2007-06-01

Poly-beta-hydroxyalkanoate (PHA) is a biodegradable polymer accumulated in intracellular granules by different bacterial species. Its physical and chemical properties are similar to those of petroleum-derived plastics. Material generated by the acid hydrolysis of wood was evaluated for use in the bacterial synthesis of PHA. Acid-hydrolyzed sawdust was prepared and adjusted to pH 7. Mineral salts with carbon:nitrogen (C:N) proportions of 100:1, 100:3.5, 100:10, 100:30, or 100:50 and trace elements were added and these solutions were inoculated with a bacterial strain Brevundimonas vesicularis LMG P-23615 or Sphingopyxis macrogoltabida LMG 17324. The percentage of cells accumulating PHA was evaluated by flow cytometry. The hydrolyzed sawdust composition was analyzed by gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC). The organic material (601.5 mg l(-1)) contained 112.5 mg l(-1) sugars. Over 96% of these sugars were consumed and more than 90% of the bacterial cells accumulated PHA. The 100:3.5 C:N proportion was optimal for growth and PHA synthesis, with yields ranging from 64% to 72% of the dry cell weight. The results suggest that acid-hydrolyzed sawdust can be used by bacteria as a carbon source for growth and PHA production. This forestry by sub-product offers a low-cost alternative for obtaining biodegradable plastics (e.g., PHA) synthesized by bacteria.

Effect of temperature acclimation on red blood cell oxygen affinity in Pacific bluefin tuna (Thunnus orientalis) and yellowfin tuna (Thunnus albacares).

PubMed

Lilly, Laura E; Bonaventura, Joseph; Lipnick, Michael S; Block, Barbara A

2015-03-01

Hemoglobin-oxygen (Hb-O2) binding properties are central to aerobic physiology, and must be optimized for an animal's aerobic requirements and environmental conditions, both of which can vary widely with seasonal changes or acutely with diving. In the case of tunas, the matter is further complicated by large regional temperature differences between tissues within the same animal. This study investigates the effects of thermal acclimation on red blood cell Hb-O2 binding in Pacific bluefin tuna (T. orientalis) and yellowfin tuna (T. albacares) maintained in captive tanks at acclimation temperatures of 17°, 20° and 24 °C. Oxygen binding properties of acclimated tuna isolated red blood cells were examined under varying experimental temperatures (15°-35 °C) and CO2 levels (0%, 0.5% and 1.5%). Results for Pacific bluefin tuna produced temperature-independence at 17 °C- and 20 °C-acclimation temperatures and significant reverse temperature-dependence at 24 °C-acclimation in the absence of CO2, with instances of reverse temperature-dependence in 17 °C- and 24 °C-acclimations at 0.5% and 1.5% CO2. In contrast, yellowfin tuna produced normal temperature-dependence at each acclimation temperature at 0% CO2, temperature-independence at 0.5% and 1.5% CO2, and significant reverse temperature-dependence at 17 °C-acclimation and 0.5% CO2. Thermal acclimation of Pacific bluefin tuna increased O2 binding affinity of the 17 °C-acclimation group, and produced a significantly steeper oxygen equilibrium curve slope (nH) at 24 °C-acclimation compared to the other acclimation temperatures. We discuss the potential implications of these findings below. Copyright © 2014 Elsevier Inc. All rights reserved.

Thermal preference, thermal resistance, and metabolic rate of juvenile Chinese pond turtles Mauremys reevesii acclimated to different temperatures.

PubMed

Xu, Wei; Dang, Wei; Geng, Jun; Lu, Hong-Liang

2015-10-01

The thermal acclimatory capacity of a particular species may determine its resilience to environmental change. Evaluating the physiological acclimatory responses of economically important species is useful for determining their optimal culture conditions. Here, juvenile Chinese three-keeled pond turtles (Mauremys reevesii) were acclimated to one of three different temperatures (17, 25 or 33°C) for four weeks to assess the effects of thermal acclimation on some physiological traits. Thermal acclimation significantly affected thermal resistance, but not thermal preference, of juvenile M. reevesii. Turtles acclimated to 17°C were less resistant to high temperatures than those acclimated to 25°C and 33°C. However, turtles increased resistance to low temperatures with decreasing acclimation temperature. The acclimation response ratio of the critical thermal minimum (CTMin) was lower than that of the critical thermal maximum (CTMax) for acclimation temperatures between 17 and 25°C, but slightly higher between 25 and 33°C. The thermal resistance range (i.e., the difference between CTMax and CTMin) was widest in turtles acclimated to the intermediate temperature (25°C), and narrowest in those acclimated to low temperature (17°C). The standard metabolic rate increased as body temperature and acclimation temperature increased, and the temperature quotient (Q10) between acclimation temperatures 17 and 25°C was higher than the Q10 between 25 and 33°C. Our results suggest that juvenile M. reevesii may have a greater resistance under mild thermal conditions resembling natural environments, and better physiological performance at relatively warm temperatures. Copyright © 2015 Elsevier Ltd. All rights reserved.

Voluntary Hospital Coalitions to Promote Patient Safety

DTIC Science & Technology

2005-01-01

Health and Accountability (PHA) as a comprehensive, voluntary patient safety program. With a focus on systemic prevention strategies, PHA fulfills...Prescribed by ANSI Std Z39-18 Advances in Patient Safety: Vol. 3 494 The Partnership for Health and Accountability Background PHA, officially...Assembly recognized PHA’s Accountability and Health Safety (A&HS) Committee as a testing ground for a unique voluntary patient safety initiative

24 CFR 884.120 - Responsibility for contract administration and defaults (private-owner/PHA projects).

Code of Federal Regulations, 2010 CFR

2010-04-01

... the Contract, subject to review and audit by HUD. (b) Defaults by PHA and/or owner. (1) The ACC and... assure that the obligations of the PHA to the Owner are carried out. (2) The ACC shall contain a... accordance with the terms of the ACC and the Contract. (3) The Contract shall contain a provision to the...

24 CFR 884.120 - Responsibility for contract administration and defaults (private-owner/PHA projects).

Code of Federal Regulations, 2011 CFR

2011-04-01

... the Contract, subject to review and audit by HUD. (b) Defaults by PHA and/or owner. (1) The ACC and... assure that the obligations of the PHA to the Owner are carried out. (2) The ACC shall contain a... accordance with the terms of the ACC and the Contract. (3) The Contract shall contain a provision to the...

Probiotic Lactobacillus plantarum 299v Does Not Counteract Unfavorable Phytohemagglutinin-Induced Changes in the Rat Intestinal Microbiota ▿

PubMed Central

Gross, Gabriele; Wildner, Jessica; Schonewille, Arjan; Rademaker, Jan L. W.; van der Meer, Roelof; Snel, Johannes

2008-01-01

Application of phytohemagglutinin (PHA) in weaning feed has been suggested to stimulate intestinal epithelium maturation. In this study, PHA strongly affected the fecal bacterial population structure of rats. Escherichia coli overgrowth was not prevented by probiotic mannose-adhering Lactobacillus plantarum 299v. Therefore, use of PHA in weaning feed deserves careful evaluation. PMID:18606805

Beyond feast and famine: Selecting a PHA accumulating photosynthetic mixed culture in a permanent feast regime.

PubMed

Fradinho, J C; Reis, M A M; Oehmen, A

2016-11-15

Currently, the feast and famine (FF) regime is the most widely applied strategy to select for polyhydroxyalkanoate (PHA) accumulating organisms in PHA production systems with mixed microbial cultures. As an alternative to the FF regime, this work studied the possibility of utilizing a permanent feast regime as a new operational strategy to select for PHA accumulating photosynthetic mixed cultures (PMCs). The PMC was selected in an illuminated environment and acetate was constantly present in the mixed liquor to guarantee a feast regime. During steady-state operation, the culture presented low PHA accumulation levels, likely due to low light availability, which resulted in most of the acetate being used for biomass growth (Y x/s of 0.64 ± 0.18 Cmol X/Cmol Acet). To confirm the light limitation on the PMC, SBR tests were conducted with higher light availability, at similar levels as would be expectable from natural sunlight. In this case, the Y x/s reduced to 0.11 ± 0.01 Cmol X/Cmol Acet and the culture presented a PHB production yield on acetate of 0.67 ± 0.01 Cmol PHB/Cmol Acet, leading to a maximum PHB content of 60%. Unlike other studied PMCs, the PMC was capable of simultaneous growth and PHB accumulation continuously throughout the cycle. Thus far, 60% PHA content is the maximum value ever reported for a PMC, a result that prospects the utilization of feast regimes as an alternative strategy for the selection of PHA accumulating PMCs. Furthermore, the PMC also presented high phosphate removal rates, delivering an effluent that complies with phosphate discharge limits. The advantages of selecting PMCs under a permanent feast regime are that no aeration inputs are required; it allows higher PHA contents and phosphate removal rates in comparison to FF-operated PMC systems; and it represents a novel means of integrating wastewater treatment with resource recovery in the form of PHA. Copyright © 2016 Elsevier Ltd. All rights reserved.

Low acclimation capacity of narrow-ranging thermal specialists exposes susceptibility to global climate change.

PubMed

Markle, Tricia M; Kozak, Kenneth H

2018-05-01

Thermal acclimation is hypothesized to offer a selective advantage in seasonal habitats and may underlie disparities in geographic range size among closely-related species with similar ecologies. Understanding this relationship is also critical for identifying species that are more sensitive to warming climates. Here, we study North American plethodontid salamanders to investigate whether acclimation ability is associated with species' latitudinal extents and the thermal range of the environments they inhabit. We quantified variation in thermal physiology by measuring standard metabolic rate (SMR) at different test and acclimation temperatures for 16 species of salamanders with varying latitudinal extents. A phylogenetically-controlled Markov chain Monte Carlo generalized linear mixed model (MCMCglmm) was then employed to determine whether there are differences in SMR between wide- and narrow-ranging species at different acclimation temperatures. In addition, we tested for a relationship between the acclimation ability of species and the environmental temperature ranges they inhabit. Further, we investigated if there is a trade-off between critical thermal maximum (CTMax) and thermal acclimation ability. MCMCglmm results show a significant difference in acclimation ability between wide and narrow-ranging temperate salamanders. Salamanders with wide latitudinal distributions maintain or slightly increase SMR when subjected to higher test and acclimation temperatures, whereas several narrow-ranging species show significant metabolic depression. We also found significant, positive relationships between acclimation ability and environmental thermal range, and between acclimation ability and CTMax. Wide-ranging salamander species exhibit a greater capacity for thermal acclimation than narrow-ranging species, suggesting that selection for acclimation ability may have been a key factor enabling geographic expansion into areas with greater thermal variability. Further, given that narrow-ranging salamanders are found to have both poor acclimation ability and lower tolerance to warm temperatures, they are likely to be more susceptible to environmental warming associated with anthropogenic climate change.

Changes in extreme cold tolerance, membrane composition and cardiac transcriptome during the first day of thermal acclimation in the porcelain crab Petrolisthes cinctipes.

PubMed

Ronges, Daria; Walsh, Jillian P; Sinclair, Brent J; Stillman, Jonathon H

2012-06-01

Intertidal zone organisms can experience transient freezing temperatures during winter low tides, but their extreme cold tolerance mechanisms are not known. Petrolisthes cinctipes is a temperate mid-high intertidal zone crab species that can experience wintertime habitat temperatures below the freezing point of seawater. We examined how cold tolerance changed during the initial phase of thermal acclimation to cold and warm temperatures, as well as the persistence of cold tolerance during long-term thermal acclimation. Thermal acclimation for as little as 6 h at 8°C enhanced cold tolerance during a 1 h exposure to -2°C relative to crabs acclimated to 18°C. Potential mechanisms for this enhanced tolerance were elucidated using cDNA microarrays to probe for differences in gene expression in cardiac tissue of warm- and cold-acclimated crabs during the first day of thermal acclimation. No changes in gene expression were detected until 12 h of thermal acclimation. Genes strongly upregulated in warm-acclimated crabs represented immune response and extracellular/intercellular processes, suggesting that warm-acclimated crabs had a generalized stress response and may have been remodelling tissues or altering intercellular processes. Genes strongly upregulated in cold-acclimated crabs included many that are involved in glucose production, suggesting that cold acclimation involves increasing intracellular glucose as a cryoprotectant. Structural cytoskeletal proteins were also strongly represented among the genes upregulated in only cold-acclimated crabs. There were no consistent changes in composition or the level of unsaturation of membrane phospholipid fatty acids with cold acclimation, which suggests that neither short- nor long-term changes in cold tolerance are mediated by changes in membrane fatty acid composition. Overall, our study demonstrates that initial changes in cold tolerance are likely not regulated by transcriptomic responses, but that gene-expression-related changes in homeostasis begin within 12 h, the length of a tidal cycle.

Improving Models of Photosynthetic Thermal Acclimation: Which Parameters are Most Important and How Many Should Be Modified?

NASA Astrophysics Data System (ADS)

Stinziano, J. R.; Way, D.; Bauerle, W.

2017-12-01

Photosynthetic temperature acclimation could strongly affect coupled vegetation-atmosphere feedbacks in the global carbon cycle, especially as the climate warms. Thermal acclimation of photosynthesis can be modelled as changes in the parameters describing the direct effect of temperature on photosynthetic capacity (activation energy, Ea; deactivation energy, Hd; entropy parameter, ΔS) or the basal value of photosynthetic capacity (i.e. photosynthetic capacity measured at 25 °C), however the impact of acclimating these parameters (individually or in combination) on vegetative carbon gain is relatively unexplored. Here we compare the ability of 66 photosynthetic temperature acclimation scenarios to improve predictions of a spatially explicit canopy carbon flux model, MAESTRA, for eddy covariance data from a loblolly pine forest. We show that: 1) incorporating seasonal temperature acclimation of basal photosynthetic capacity improves the model's ability to capture seasonal changes in carbon fluxes; 2) multifactor scenarios of photosynthetic temperature acclimation provide minimal (if any) improvement in model performance over single factor acclimation scenarios; 3) acclimation of enzyme activation energies should be restricted to the temperature ranges of the data from which the equations are derived; and 4) model performance is strongly affected by the choice of deactivation energy. We suggest that a renewed effort be made into understanding the thermal acclimation of enzyme activation and deactivation energies across broad temperature ranges to better understand the mechanisms underlying thermal photosynthetic acclimation.

Methods for the isolation of genes encoding novel PHB cycle enzymes from complex microbial communities.

PubMed

Nordeste, Ricardo F; Trainer, Maria A; Charles, Trevor C

2010-01-01

Development of different PHAs as alternatives to petrochemically derived plastics can be facilitated by mining metagenomic libraries for diverse PHA cycle genes that might be useful for synthesis of bioplastics. The specific phenotypes associated with mutations of the PHA synthesis pathway genes in Sinorhizobium meliloti allows for the use of powerful selection and screening tools to identify complementing novel PHA synthesis genes. Identification of novel genes through their function rather than sequence facilitates finding functional proteins that may otherwise have been excluded through sequence-only screening methodology. We present here methods that we have developed for the isolation of clones expressing novel PHA metabolism genes from metagenomic libraries.

Co-metabolism of substrates by Bacillus thuringiensis regulates polyhydroxyalkanoate co-polymer composition.

PubMed

Ray, Subhasree; Kalia, Vipin Chandra

2017-01-01

Polyhydroxyalkanoate (PHA) production by Bacillus thuringiensis EGU45 was studied by co-metabolism of crude glycerol (CG) (1%, v/v), glucose (0.05-0.5%, w/v) and propionic acid (0.05-0.5%, v/v) under batch (shake flask) culture conditions. Glycerol+PA combination resulted in 15-100mg/L PHA co-polymers with a HV content of 33-81mol%. The addition of NH 4 Cl (0.5%, w/v) to CG+PA enhanced PHA production by 1.55-fold, with a HV content of 58-70mol%. The time period of incubation of PA to the feed: CG+glucose was optimized to be 3h after initiation of fermentation. The PHA contents were found to be stable at 1900-2050mg/L up scaling from 0.4 to 2.0L feed material. Biochemical characterization through GC-MS of PHA co-polymer revealed the presence of 3-hydroxydecanoate (3-HDD), 3-hydroxyoctadecanoate (3HOD), 3-hydroxyhexadecanoate (3HHD). Copyright © 2016 Elsevier Ltd. All rights reserved.

Synthesis, Characterization and Application of Thermoresponsive Polyhydroxyalkanoate-graft-Poly(N-isopropylacrylamide).

PubMed

Ma, Yi-Ming; Wei, Dai-Xu; Yao, Hui; Wu, Lin-Ping; Chen, Guo-Qiang

2016-08-08

A thermoresponsive graft copolymer polyhydroxyalkanoate-g-poly(N-isopropylacrylamide) or short as PHA-g-PNIPAm, was successfully synthesized via a three-step reaction. First, PNIPAm oligomer with a trithiocarbonate-based chain transfer agent (CTA), short as PNIPAm-CTA, with designed polymerization degree was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Subsequently, the PNIPAm-CTA was treated with n-butylamine for aminolysis in order to obtain a pendant thiol group at the end of the chain (PNIPAm-SH). Finally, the PNIPAm-SH was grafted onto unsaturated P(3HDD-co-3H10U), a random copolymer of 3-hydroxydodecanoate (3HDD) and 3-hydroxy-10-undecylenate (3H10U), via a thiol-ene click reaction. Enhanced hydrophilicity and thermoresponsive property of the resulted PHA-g-PNIPAm were confirmed by water contact angle studies. The biocompatibility of PHA-g-PNIPAm was comparable to poly-3-hydroxybutyrate (PHB). The graft copolymer PHA-g-PNIPAm based on biopolyester PHA could be a promising material for biomedical applications.

Integrative Approach for Producing Hydrogen and Polyhydroxyalkanoate from Mixed Wastes of Biological Origin.

PubMed

Patel, Sanjay K S; Lee, Jung-Kul; Kalia, Vipin C

2016-09-01

In this study, an integrative approach to produce biohydrogen (H2) and polyhydroxyalkanoates (PHA) from the wastes of biological origin was investigated. A defined set of mixed cultures was used for hydrolysis and the hydrolysates were used to produce H2. The effluent from H2 production stage was used for PHA production. Under batch culture, a maximum of 62 l H2/kg of pure potato peels (Total solid, TS 2 %, w/v) and 54 l H2/kg of mixed biowastes (MBW1) was recorded. Using effluent from the H2 production stage of biowaste mixture (MBW1), Bacillus cereus EGU43 could produce 195 mg PHA/l and 15.6 % (w/w). Further, supplementation of GM-2 medium (0.1×) and glucose (0.5 %) in H2 production stage effluents, resulted in significant improvements of up to 11 and 41.7 % of PHA contents, respectively. An improvement of 3.9- and 17-fold in PHA yields as compared to with and without integrative H2 production from the MBW1 has been recorded. This integrative approach seems to be a suitable process to improve the yields of H2 and PHA by mixing biowastes.

Functional Stability Of A Mixed Microbial Consortia Producing PHA From Waste Carbon Sources

DOE Office of Scientific and Technical Information (OSTI.GOV)

David N. Thompson; Erik R. Coats; William A. Smith

2006-04-01

Polyhydroxyalkanoates (PHAs), naturally-occurring biological polyesters that are microbially synthesized from a myriad of carbon sources, can be utilized as biodegradable substitutes for petroleum-derived thermoplastics. However, current PHA commercialization schemes are limited by high feedstock costs, the requirement for aseptic reactors, and high separation and purification costs. Bacteria indigenous to municipal waste streams can accumulate large quantities of PHA under environmentally controlled conditions; hence, a potentially more environmentally-effective method of production would utilize these consortia to produce PHAs from inexpensive waste carbon sources. In this study, PHA production was accomplished in sequencing batch bioreactors utilizing mixed microbial consortia from municipal activatedmore » sludge as inoculum, in cultures grown on real wastewaters. PHA production averaged 85%, 53%, and 10% of the cell dry weight from methanol-enriched pulp-and-paper mill foul condensate, fermented municipal primary solids, and biodiesel wastewater, respectively. The PHA-producing microbial consortia were examined to explore the microbial community changes that occurred during reactor operations, employing denaturing gradient gel electrophoresis (DGGE) of 16S-rDNA from PCR-amplified DNA extracts. Distinctly different communities were observed both between and within wastewaters following enrichment. More importantly, stable functions were maintained despite the differing and contrasting microbial populations.« less

Immune reactions in acute viral hepatitis.

PubMed Central

Newble, D I; Holmes, K T; Wangel, A G; Forbes, I J

1975-01-01

Serial studies of PHA-induced lymphocyte transformation, serum autoantibodies, immunoglobulins and complement were performed in seventeen patients with hepatitis A and nine patients with hepatitis B. In both types of hepatitis PHA-induced transformation was markedly impaired during the 1st week after the onset of jaundice and there was less marked but prolonged impairment for a further period of 6-10 weeks. A group of eleven subjects with a previous history of hepatitis had values which were similar to those of healthy persons. Serum from patients with hepatitis A and hepatitis B contains an inhibitor of lymphocyte response to PHA. The inhibitor depresses the function of both patients' and normal lymphocytes and is only detectable during the acute phase of the illness. Washing lymphocytes free from autologous serum did not restore the PHA response to normal but the markedly impaired response present during the first 2 weeks of the illness was improved. A serum factor or factors may therefore be responsible for at least part of the impaired response of lymphocytes to PHA during the acute phase of hepatitis but does not appear to account for the more prolonged impairment of the PHA response. The protracted lymphocyte defect is possibly induced by hepatitis virus. The incidence of autoantibodies and the changes in immunoglobulin levels were similar to those reported by other workers. PMID:1204253

Impact of closed-system drug transfer device on exposure of environment and healthcare provider to cyclophosphamide in Japanese hospital.

PubMed

Miyake, Tomohiro; Iwamoto, Takuya; Tanimura, Manabu; Okuda, Masahiro

2013-12-01

In spite of current recommended safe handling procedures, the potential for the exposure of healthcare providers to hazardous drugs exists in the workplace. A reliance on biological safety cabinets to provide total protection against the exposure to hazardous drugs is insufficient. Preventing workplace contamination is the best strategy to minimize cytotoxic drug exposure in healthcare providers. This study was conducted to compare surface contamination and personnel exposure to cyclophosphamide before and after the implementation of a closed-system drug transfer device, PhaSeal, under the influence of cleaning according to the Japanese guidelines. Personnel exposure was evaluated by collecting 24 h urine samples from 4 pharmacists. Surface contamination was assessed by the wiping test. Four of 6 wipe samples collected before PhaSeal indicated a detectable level of cyclophosphamide. About 7 months after the initiation of PhaSeal, only one of 6 wipe samples indicated a detectable level of cyclophosphamide. Although all 4 employees who provided urine samples had positive results for the urinary excretion of cyclophosphamide before PhaSeal, these levels returned to minimal levels in 2 pharmacists after PhaSeal. In combination with the biological safety cabinet and cleaning according to the Japanese guidelines, PhaSeal further reduces surface contamination and healthcare providers exposure to cyclophosphamide to almost undetectable levels.

Evaluation of medium-chain-length polyhydroxyalkanoate production by Pseudomonas putida LS46 using biodiesel by-product streams.

PubMed

Fu, Jilagamazhi; Sharma, Umesh; Sparling, Richard; Cicek, Nazim; Levin, David B

2014-07-01

Medium-chain-length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida LS46 was analyzed in shake-flask-based batch reactions, using pure chemical-grade glycerol (PG), biodiesel-derived "waste" glycerol (WG), and biodiesel-derived "waste" free fatty acids (WFA). Cell growth, substrate consumption, mcl-PHA accumulation within the cells, and the monomer composition of the synthesized biopolymers were monitored. The patterns of mcl-PHA synthesis in P. putida LS46 cells grown on PG and WG were similar but differed from that of cells grown with WFA. Polymer accumulation in glycerol-based cultures was stimulated by nitrogen limitation and plateaued after 48 h in both PG and WG cultures, with a total accumulation of 17.9% cell dry mass and 16.3% cell dry mass, respectively. In contrast, mcl-PHA synthesis was independent of nitrogen concentration in P. putida LS46 cells cultured with WFA, which accumulated to 29% cell dry mass. In all cases, the mcl-PHAs synthesized consisted primarily of 3-hydroxyoctanoate (C(8)) and 3-hydroxydecanoate (C(10)). WG and WFA supported similar or greater cell growth and mcl-PHA accumulation than PG under the experimental conditions used. These results suggest that biodiesel by-product streams could be used as low-cost carbon sources for sustainable mcl-PHA production.

Biosynthesis of polyhydroxyalkanaotes by a novel facultatively anaerobic Vibrio sp. under marine conditions.

PubMed

Numata, Keiji; Doi, Yoshiharu

2012-06-01

Marine bacteria have recently attracted attention as potentially useful candidates for the production of practical materials from marine ecosystems, including the oceanic carbon dioxide cycle. The advantages of using marine bacteria for the biosynthesis of poly(hydroxyalkanoate) (PHA), one of the eco-friendly bioplastics, include avoiding contamination with bacteria that lack salt-water resistance, ability to use filtered seawater as a culture medium, and the potential for extracellular production of PHA, all of which would contribute to large-scale industrial production of PHA. A novel marine bacterium, Vibrio sp. strain KN01, was isolated and characterized in PHA productivity using various carbon sources under aerobic and aerobic-anaerobic marine conditions. The PHA contents of all the samples under the aerobic-anaerobic condition, especially when using soybean oil as the sole carbon source, were enhanced by limiting the amount of dissolved oxygen. The PHA accumulated using soybean oil as a sole carbon source under the aerobic-anaerobic condition contained 14% 3-hydroxypropionate (3HP) and 3% 5-hydroxyvalerate (5HV) units in addition to (R)-3-hydroxybutyrate (3HB) units and had a molecular weight of 42 × 10³ g/mol. The present result indicates that the activity of the beta-oxidation pathway under the aerobic-anaerobic condition is reduced due to a reduction in the amount of dissolved oxygen. These findings have potential for use in controlling the biosynthesis of long main-chain PHA by regulating the activity of the beta-oxidation pathway, which also could be regulated by varying the dissolved oxygen concentration.

Further characterization of the circulating cell in chronic lymphocytic leukemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schutz, E.F.; Davis, S.; Rubin, A.D.

Peripheral lymphocytes from normal individuals and from patients with chronic lymphocytic leukemia (CLL) were cultured in vitro for 1-7 days. The growth response to phytohemagglutinin (PHA) was quantitated by the incorporation of tritiated uridine into RNA nucleotide during a 2-hr pulse with the radioisotope. While the maximum response in PHA-stimulated normal cultures appeared at 2-3 days, CLL cultures required 5-7 days to develop their maximal response, which was 50 percent-60 percent of the normal magnitude. Dilution of the number of normally reactive lymphocytes by culturing them with totally unreactive, mitomycin-treated cells produced a normal 72-hr maximal response, no matter whatmore » proportion of unreactive cells was included in the PHA-stimulated cultures. In addition, the response of peripheral lymphocytes from patients with myeloblastic leukemia, where large numbers of unreactive myeloblasts diluted the normal small lymphocytes, a depressed reaction occurred at the anticipated 2-3 days. Nylon fiber-adherent lymphocytes consisting of 85 percent immunoglobulin (Ig)-bearing cells responded minimally to PHA, but showed no evidence of a delay. When isolated from CLL patients, both fiber-adherent cells (ig-bearing) as well as non-fiber-adherent (sheep erythrocyte-rosetting) cells responded to PHA in a delayed fashion. Similarly, a case of CLL, in which 93.5 percent of the circulating lymphocytes bore sheep red blood cell receptors, showed its peak response to PHA at 7 days. Therefore, using surface marker criteria considered characteristic of normal T cells and B cells, the delayed response to PHA on the part of CLL lymphocytes was independent of thymic or nonthymic origin.« less

Synthesis of Medium-Chain-Length Polyhydroxyalkanoate Homopolymers, Random Copolymers, and Block Copolymers by an Engineered Strain of Pseudomonas entomophila.

PubMed

Wang, Ying; Chung, Ahleum; Chen, Guo-Qiang

2017-04-01

Medium-chain-length polyhydroxyalkanoates (mcl-PHAs), widely used in medical area, are commonly synthesized by Pseudomonas spp. This study tries to use β-oxidation pathways engineered P. entomophila to achieve single source of a series of mcl-monomers for microbial production of PHA homopolymers. The effort is proven successful for the first time to obtain a wide range of mcl-PHA homopolymers from engineered P. entomophila LAC23 grown on various fatty acids, respectively, ranging from poly(3-hydroxyheptanoate) to poly(3-hydroxytetradecanoate). Effects of a PHA monomer chain length on thermal and crystallization properties including the changes of T m , T g , and T d5% are investigated. Additionally, strain LAC23 is used to synthesize random copolymers of 3-hydroxyoctanoate (3HO) and 3-hydroxydodecanoate (3HDD) or 3-hydroxytetradecanoates, their compositions could be controlled by adjusting the ratios of two related fatty acids. Meanwhile, block copolymer P(3HO)-b-P(3HDD) is synthesized by the same strain. It is found for the first time that even- and odd number mcl-PHA homopolymers have different physical properties. When the gene of the PHA synthase in the engineered P. entomophila is replaced by phaC from Aeromonas hydrophila 4AK4, poly(3-hydroxybutyrate-co-30 mol%-3-hydroxyhexanoate) is synthesized. Therefore, P. entomophila can be used to synthesize the whole range of PHA (C7-C14) homopolymers, random- and block copolymers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression of Aeromonas caviae polyhydroxyalkanoate synthase gene in Burkholderia sp. USM (JCM15050) enables the biosynthesis of SCL-MCL PHA from palm oil products.

PubMed

Chee, J-Y; Lau, N-S; Samian, M-R; Tsuge, T; Sudesh, K

2012-01-01

Burkholderia sp. USM (JCM15050) isolated from oil-polluted wastewater is capable of utilizing palm oil products and glycerol to synthesize poly(3-hydroxybutyrate) [P(3HB)]. To confer the ability to produce polymer containing 3-hydroxyhexanoate (3HHx), plasmid (pBBREE32d13) harbouring the polyhydroxyalkanoate (PHA) synthase gene of Aeromonas caviae (phaC(Ac)) was transformed into this strain.   The resulting transformant incorporated approximately 1 ± 0·3 mol% of 3HHx in the polymer when crude palm kernel oil (CPKO) or palm kernel acid oil was used as the sole carbon source. In addition, when the transformed strain was cultivated in the mixtures of CPKO and sodium valerate, PHA containing 69 mol% 3HB, 30 mol% 3-hydroxyvalerate and 1 mol% 3HHx monomers was produced. Batch feeding of carbon sources with 0·5% (v/v) CPKO at 0 h and 0·25% (w/v) sodium valerate at 36 h yielded 6 mol% of 3HHx monomer by controlled-feeding strategies. Burkholderia sp. USM (JCM15050) has the metabolic pathways to supply both the short-chain length (SCL) and medium-chain length (MCL) PHA monomers. By transforming the strain with the Aer. caviae PHA synthase with broader substrate specificity, SCL-MCL PHA was produced.   This is the first study demonstrating the ability of transformant Burkholderia to produce P(3HB-co-3HHx) from a single carbon source. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

Bio-oil upgrading strategies to improve PHA production from selected aerobic mixed cultures.

PubMed

Moita Fidalgo, Rita; Ortigueira, Joana; Freches, André; Pelica, João; Gonçalves, Magarida; Mendes, Benilde; Lemos, Paulo C

2014-06-25

Recent research on polyhydroxyalkanoates (PHAs) has focused on developing cost-effective production processes using low-value or industrial waste/surplus as substrate. One of such substrates is the liquid fraction resulting from pyrolysis processes, bio-oil. In this study, valorisation of bio-oil through PHA production was investigated. The impact of the complex bio-oil matrix on PHA production by an enriched mixed culture was examined. The performance of the direct utilization of pure bio-oil was compared with the utilization of three defined substrates contained in this bio-oil: acetate, glucose and xylose. When compared with acetate, bio-oil revealed lower capacity for polymer production as a result of a lower polymer yield on substrate and a lower PHA cell content. Two strategies for bio-oil upgrade were performed, anaerobic fermentation and vacuum distillation, and the resulting liquid streams were tested for polymer production. The first one was enriched in volatile fatty acids and the second one mainly on phenolic and long-chain fatty acids. PHA accumulation assays using the upgraded bio-oils attained polymer yields on substrate similar or higher than the one achieved with acetate, although with a lower PHA content. The capacity to use the enriched fractions for polymer production has yet to be optimized. The anaerobic digestion of bio-oil could also open-up the possibility to use the fermented bio-oil directly in the enrichment process of the mixed culture. This would increase the selective pressure toward an optimized PHA accumulating culture selection. Copyright © 2013 Elsevier B.V. All rights reserved.

Physical properties and morphology of electrospun composite fiber mats of polyhydroxyalkanoate containing nanoclay and tricalcium phosphate additives

NASA Astrophysics Data System (ADS)

Tanadchangsaeng, N.; Boonyagul, S.

2018-05-01

Recently, nanofiber research has gained substantial attention from scientists. In this study, the main component of the nanofiber sheet is polyhydroxyalkanoate (PHA) polymer, which is strong, ductile, flexible and adhesive to human skin. Two major additives of nanofiber sheet that we applied are nanoclay and tricalcium phosphate. The additives are generally synthetic substances that can be chemically synthesized and compatible with tissues body. Nanoclay has a low density, strong, durable to compressive strength and humidity. While, tricalcium phosphate is a calcium phosphate ceramic that is biocompatible to human tissue. From the reasons above, we proposed to choose both nanoclay and tricalcium phosphate for adding into PHA nanofibers for film formation. Thus, this study aims to investigate the morphological and mechanical properties of the fiber mat by using PHA added with various amount of nanoclay and tricalcium phosphate at 0.1%, 1% and 10% by weight, and fabricate nanofiber samples by electrospinning technique. The tested results of scanning electron microscope (SEM) morphology show that the fibers have a uniformed pattern. The PHA containing nanoclay of all additive contents exhibited micrometer diameter distributions, while PHA loaded with 1% tricalcium phosphate still had the nano-scale diameter range, and might be the optimum additive load for further nanometer medical applications. A tensile test was performed to determine the effect of nanoclay and tricalcium phosphate contents on the mechanical properties of the electrospun PHA films, and reflect the level of modularity. With nanoclay components being integrated into the polymer matrix, subsequent reduction in fiber crystallinity was occurred after addition of nanoclay with an increase of modulus value. The results confirmed that PHA fiber mat containing 1% nanoclay may have a potential for using as a rigid scaffold which bearing force loading in human organ system. Whereas, it can be indicated that PHA fiber mat containing 1% tricalcium phosphate might be employed as a flexible scaffold for biomedical materials application due to a high elongation at break value.

Effects of Low Phytanic Acid-Concentrated DHA on Activated Microglial Cells: Comparison with a Standard Phytanic Acid-Concentrated DHA.

PubMed

Ruiz-Roso, María Belén; Olivares-Álvaro, Elena; Quintela, José Carlos; Ballesteros, Sandra; Espinosa-Parrilla, Juan F; Ruiz-Roso, Baltasar; Lahera, Vicente; de Las Heras, Natalia; Martín-Fernández, Beatriz

2018-05-30

Docosahexaenoic acid (DHA, 22:6 n-3) is an essential omega-3 (ω-3) long chain polyunsaturated fatty acid of neuronal membranes involved in normal growth, development, and function. DHA has been proposed to reduce deleterious effects in neurodegenerative processes. Even though, some inconsistencies in findings from clinical and pre-clinical studies with DHA could be attributed to the presence of phytanic acid (PhA) in standard DHA treatments. Thus, the aim of our study was to analyze and compare the effects of a low PhA-concentrated DHA with a standard PhA-concentrated DHA under different neurotoxic conditions in BV-2 activated microglial cells. To this end, mouse microglial BV-2 cells were stimulated with either lipopolysaccharide (LPS) or hydrogen peroxide (H 2 O 2 ) and co-incubated with DHA 50 ppm of PhA (DHA (PhA:50)) or DHA 500 ppm of PhA (DHA (PhA:500)). Cell viability, superoxide anion (O 2 - ) production, Interleukin 6 (L-6), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), glutathione peroxidase (GtPx), glutathione reductase (GtRd), Caspase-3, and the brain-derived neurotrophic factor (BDNF) protein expression were explored. Low PhA-concentrated DHA protected against LPS or H 2 O 2 -induced cell viability reduction in BV-2 activated cells and O 2 - production reduction compared to DHA (PhA:500). Low PhA-concentrated DHA also decreased COX-2, IL-6, iNOS, GtPx, GtRd, and SOD-1 protein expression when compared to DHA (PhA:500). Furthermore, low PhA-concentrated DHA increased BDNF protein expression in comparison to DHA (PhA:500). The study provides data supporting the beneficial effect of low PhA-concentrated DHA in neurotoxic injury when compared to a standard PhA-concentrated DHA in activated microglia.

No effects of acclimation to heat on immune and hormonal responses to passive heating in healthy volunteers

NASA Astrophysics Data System (ADS)

Kanikowska, Dominika; Sato, Maki; Sugenoya, Junichi; Iwase, Satoshi; Shimizu, Yuuki; Nishimura, Naoki; Inukai, Yoko

2012-01-01

Heat acclimation results in whole body-adaptations that increase heat tolerance, and might also result in changed immune responses. We hypothesized that, after heat acclimation, tumor necrosis factor alpha, interleukin 6 and the lymphocyte count would be altered. Heat acclimation was induced in 6 healthy men by 100 min of heat exposure for 9 days. Heat exposure consisted of (1) 10 min of immersion up to chest-level in water at 42°C and (2) 90 min of passive heating by a warm blanket to maintain tympanic temperature at 37.5°C. The climatic chamber was maintained at 40°C and a relative humidity of 50%. Blood samples were analyzed before and after heat acclimation for natural killer (NK) cell activity, counts of lymphocytes B and T, before and after heat acclimation for peripheral blood morphology, interleukin 6, tumor necrosis factor alpha, and cortisol. A Japanese version of the profile of mood states questionnaire was also administered before and after acclimation. The concentrations of white blood cells, lymphocytes B and T, cortisol, interleukin 6, tumor necrosis factor alpha and NK cell activity showed no significant differences between pre- and post-acclimation, but there was a significantly lower platelet count after acclimation and, with the profile of mood states questionnaire, there was a significant rise in anger after acclimation. It is concluded that heat acclimation by passive heating does not induce alterations in immune or endocrine responses.

A comparison of the effects of two methods of acclimation of aerobic biodegradability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watson, H.M.

1993-11-01

The acclimation or adaptation of microorganisms to organic chemicals is an important factor influencing both the rate and the extent of biodegradation. In this study two acclimation procedures were evaluated in terms of their effectiveness in enhancing biodegradation, their relative ease of use in the laboratory, and the implications for biodegradability testing. In the single-flask procedure, microorganisms were acclimated for 2 to 7 d in a single acclimation flask at constant or increasing concentrations of the test chemical without transfer of microorganisms. In the second procedure, the enrichment procedure, microorganisms were acclimated in a series of flasks over a 21-dmore » period by making adaptive transfers to increasing concentrations of the test chemical. Acclimated microorganisms from each procedure were used as the source of inoculum for subsequent biodegradation tests in which carbon dioxide evolution was measured. Six chemicals were tested: quinoline, p-nitrophenol, N-methylaniline, N,N-dimethylaniline, acrylonitrile, and 2,2,4-trimethyl-1,3-pentanediol monoisobutyrate. Microorganisms acclimated in the single-flask procedure were much more effective than those acclimated in the enrichment procedure in degrading the test chemicals. The single-flask procedure is more convenient to use, and it permits monitoring of the time needed for acclimation. The results from these studies have implications for the methodology used in biodegradation test systems and suggest caution before adopting a multiple-flask, enrichment acclimation procedure before the performance of standardized tests for aerobic biodegradability.« less

Acclimation of CO2 Assimilation in Cotton Leaves to Water Stress and Salinity 1

PubMed Central

Plaut, Zvi; Federman, Evelyn

1991-01-01

Cotton (Gossypium hirsutum L. cv Acala SJ2) plants were exposed to three levels of osmotic or matric potentials. The first was obtained by salt and the latter by withholding irrigation water. Plants were acclimated to the two stress types by reducing the rate of stress development by a factor of 4 to 7. CO2 assimilation was then determined on acclimated and nonacclimated plants. The decrease of CO2 assimilation in salinity-exposed plants was significantly less in acclimated as compared with nonacclimated plants. Such a difference was not found under water stress at ambient CO2 partial pressure. The slopes of net CO2 assimilation versus intercellular CO2 partial pressure, for the initial linear portion of this relationship, were increased in plants acclimated to salinity of −0.3 and −0.6 megapascal but not in nonacclimated plants. In plants acclimated to water stress, this change in slopes was not significant. Leaf osmotic potential was reduced much more in acclimated than in nonacclimated plants, resulting in turgor maintenance even at −0.9 megapascal. In nonacclimated plants, turgor pressure reached zero at approximately −0.5 megapascal. The accumulation of Cl− and Na+ in the salinity-acclimated plants fully accounted for the decrease in leaf osmotic potential. The rise in concentration of organic solutes comprised only 5% of the total increase in solutes in salinity-acclimated and 10 to 20% in water-stress-acclimated plants. This acclimation was interpreted in light of the higher protein content per unit leaf area and the enhanced ribulose bisphosphate carboxylase activity. At saturating CO2 partial pressure, the declined inhibition in CO2 assimilation of stress-acclimated plants was found for both salinity and water stress. ImagesFigure 2 PMID:16668429

Heat- and humidity-induced plastic changes in body lipids and starvation resistance in the tropical fly Zaprionus indianus during wet and dry seasons.

PubMed

Girish, T N; Pradeep, B E; Parkash, Ravi

2018-05-04

Insects in tropical wet or dry seasons are likely to cope with starvation stress through plastic changes (developmental as well as adult acclimation) in energy metabolites. Control and experimental groups of Zaprionus indianus flies were reared under wet or dry conditions, but adults were acclimated at different thermal or humidity conditions. Adult flies of the control group were acclimated at 27°C and low (50%) or high (60%) relative humidity (RH). For experimental groups, adult flies were acclimated at 32°C for 1 to 6 days and under low (40%) or high (70%) RH. For humidity acclimation, adult flies were acclimated at 27°C but under low (40%) or high (70%) RH for 1 to 6 days. Plastic changes in experimental groups as compared with the control group (developmental as well as adult acclimation) revealed significant accumulation of body lipids owing to thermal or humidity acclimation of wet season flies, but low humidity acclimation did not change the level of body lipids in dry season flies. Starvation resistance and body lipids were higher in the males of dry season flies but in the females of wet season flies. Adults acclimated under different thermal or humidity conditions exhibited changes in the rate of utilization of body lipids, carbohydrates and proteins. Adult acclimation of wet or dry season flies revealed plastic changes in mean daily fecundity; and a reduction in fecundity under starvation. Thus, thermal or humidity acclimation of adults revealed plastic changes in energy metabolites to support starvation resistance of wet or dry season flies. © 2018. Published by The Company of Biologists Ltd.

Assessing the capacity of growth, survival, and acid adaptive response of Listeria monocytogenes during storage of various cheeses and subsequent simulated gastric digestion.

PubMed

Kapetanakou, Anastasia E; Gkerekou, Maria A; Vitzilaiou, Eirini S; Skandamis, Panagiotis N

2017-04-04

Different physicochemical and microbiological characteristics of cheeses may affect Listeria monocytogenes potential to grow, survive, or exhibit an acid adaptive response during storage and digestion. The objectives of the present study were to assess: i) the survival or growth potential of L.monocytogenes on various cheeses during storage, ii) the effect of initial indigenous microbiota on pathogen growth in comparison to expected growth curves retrieved by existing predictive models, and iii) the impact of habituation on/in cheeses surfaces on the subsequent acid resistance during simulated gastric digestion. Portions of cream (Cottage and Mascarpone), soft (Anthotyros, Camembert, Mastelo®, Manouri, Mozzarella, Ricotta), and semi-hard (Edam, Halloumi, Gouda) cheeses were inoculated with ca. 100CFU/g or cm 2 of L.monocytogenes and stored under vacuum or aerobic conditions at 7°C (n=4). The impact of varying (initial) levels of starter culture or indigenous spoilage microbiota on pathogen growth was evaluated by purchasing cheese packages on different dates in relation to production and expiration date (subsequently reflecting to different batches) mimicking a potential situation of cheese contamination with L.monocytogenes during retail display. Values of pH and a w were also monitored and used to simulate growth of L. monocytogenes by existing models and compare it with the observed data of the study. Survival in simulated gastric fluid (SGF) (pH1.5; HCl; max. 120min) was assessed at three time points during storage. Mascarpone, Ricotta, Mozzarella, Camembert, and Halloumi supported L.monocytogenes growth by 0.5-0.8logCFU/g or cm 2 per day, since low initial levels of total viable counts (TVC) (1.8-3.8logCFU/g or cm 2 ) and high pH/a w values (ca. 6.23-6.64/0.965-0.993) were recorded. On Cottage, Anthotyros, Manouri, Mastelo®, Edam, and Gouda, the pathogen survived at populations similar or lower than the inoculation level due to the high reported competition and/or low pH/a w during storage. L. monocytogenes growth was significantly suppressed (p<0.05) on samples purchased close to expiration date (bearing high TVC), compared to those close to production date, regardless of cheese. Cheeses which supported growth of L.monocytogenes enabled higher survival in gastric acidity along their shelf-life compared to cheeses which did not support growth. However, even in the latter cheeses (i.e., Cottage, Mastelo®, Gouda), total elimination of a persisting low initial contamination was not always achieved. Such findings may provide useful evidence for assessing the risk posed by various cheeses types in relation to their compliance with food safety regulations. Copyright © 2017 Elsevier B.V. All rights reserved.

In Vitro Interleukin-1 and 2 Production and Interleukin 2 Receptor Expression in the Rhesus Monkey

NASA Technical Reports Server (NTRS)

Schmitt, Didier A.; Sonnenfeld, Gerald; Husson, David; Tkaczuk, Jean; Andre, Eric; Schaffar, Laurance

1996-01-01

Anti-human monoclonal antibodies were used to detect and quantify interleukins-1 and 2 and interleukin-2 receptor expression in peripheral blood mononuclear cells from a rhesus monkey. Interleukin-1 production could be induced by phorbol esters (PMA) and was potentiated by phytohemagglutinin (PHA). Interleukin-2 secretion could also be induced by the combination of PHA and PMA, but only weakly with PHA alone. Interleukin-2 receptor expression was present in a subpopulation of unstimulated lymphocytes and could be enhanced by PHA or PMA. These data show once again that the rhesus monkey immune system is cross-reactive with the human one and that rhesus macaque could be a good model to study interleukin therapy.

Exogenous thyroid hormones regulate the activity of citrate synthase and cytochrome c oxidase in warm- but not cold-acclimated lake whitefish (Coregonus clupeaformis)

USGS Publications Warehouse

Zak, Megan A.; Regish, Amy M.; McCormick, Stephen; Manzon, Richard G.

2017-01-01

Thermal acclimation is known to elicit metabolic adjustments in ectotherms, but the cellular mechanisms and endocrine control of these shifts have not been fully elucidated. Here we examined the relationship between thermal acclimation, thyroid hormones and oxidative metabolism in juvenile lake whitefish. Impacts of thermal acclimation above (19 °C) or below (8 °C) the thermal optimum (13 °C) and exposure to exogenous thyroid hormone (60 µg T4/g body weight) were assessed by quantifying citrate synthase and cytochrome c oxidase activities in liver, red muscle, white muscle and heart. Warm acclimation decreased citrate synthase activity in liver and elevated both citrate synthase and cytochrome c oxidase activities in red muscle. In contrast, induction of hyperthyroidism in warm-acclimated fish stimulated a significant increase in liver citrate synthase and heart cytochrome c oxidase activities, and a decrease in the activity of both enzymes in red muscle. No change in citrate synthase or cytochrome c oxidase activities was observed following cold acclimation in either the presence or absence of exogenous thyroid hormones. Collectively, our results indicate that thyroid hormones influence the activity of oxidative enzymes more strongly in warm-acclimated than in cold-acclimated lake whitefish, and they may play a role in mediating metabolic adjustments observed during thermal acclimation.

Changes in Isozyme Profiles of Catalase, Peroxidase, and Glutathione Reductase during Acclimation to Chilling in Mesocotyls of Maize Seedlings.

PubMed Central

Anderson, M. D.; Prasad, T. K.; Stewart, C. R.

1995-01-01

The response of antioxidants to acclimation and chilling in various tissues of dark-grown maize (Zea mays L.) seedlings was examined in relation to chilling tolerance and protection from chilling-induced oxidative stress. Chilling caused an accumulation of H2O2 in both the coleoptile + leaf and the mesocotyl (but not roots), and acclimation prevented this accumulation. None of the antioxidant enzymes were significantly affected by acclimation or chilling in the coleoptile + leaf or root. However, elevated levels of glutathione in acclimated seedlings may contribute to an enhanced ability to scavenge H2O2 in the coleoptile + leaf. In the mesocotyl (visibly most susceptible to chilling), catalase3 was elevated in acclimated seedlings and may represent the first line of defense from mitochondria-generated H2O2. Nine of the most prominent peroxidase isozymes were induced by acclimation, two of which were located in the cell wall, suggesting a role in lignification. Lignin content was elevated in mesocotyls of acclimated seedlings, likely improving the mechanical strength of the mesocotyl. One cytosolic glutathione reductase isozyme was greatly decreased in acclimated seedlings, whereas two others were elevated, possibly resulting in improved effectiveness of the enzyme at low temperature. When taken together, these responses to acclimation illustrate the potential ways in which chilling tolerance may be improved in preemergent maize seedlings. PMID:12228666

Exogenous thyroid hormones regulate the activity of citrate synthase and cytochrome c oxidase in warm- but not cold-acclimated lake whitefish (Coregonus clupeaformis).

PubMed

Zak, Megan A; Regish, Amy M; McCormick, Stephen D; Manzon, Richard G

2017-06-01

Thermal acclimation is known to elicit metabolic adjustments in ectotherms, but the cellular mechanisms and endocrine control of these shifts have not been fully elucidated. Here we examined the relationship between thermal acclimation, thyroid hormones and oxidative metabolism in juvenile lake whitefish. Impacts of thermal acclimation above (19°C) or below (8°C) the thermal optimum (13°C) and exposure to exogenous thyroid hormone (60µg T 4 /g body weight) were assessed by quantifying citrate synthase and cytochrome c oxidase activities in liver, red muscle, white muscle and heart. Warm acclimation decreased citrate synthase activity in liver and elevated both citrate synthase and cytochrome c oxidase activities in red muscle. In contrast, induction of hyperthyroidism in warm-acclimated fish stimulated a significant increase in liver citrate synthase and heart cytochrome c oxidase activities, and a decrease in the activity of both enzymes in red muscle. No change in citrate synthase or cytochrome c oxidase activities was observed following cold acclimation in either the presence or absence of exogenous thyroid hormones. Collectively, our results indicate that thyroid hormones influence the activity of oxidative enzymes more strongly in warm-acclimated than in cold-acclimated lake whitefish, and they may play a role in mediating metabolic adjustments observed during thermal acclimation. Copyright © 2017 Elsevier Inc. All rights reserved.

Synthesis Gas (Syngas)-Derived Medium-Chain-Length Polyhydroxyalkanoate Synthesis in Engineered Rhodospirillum rubrum.

PubMed

Heinrich, Daniel; Raberg, Matthias; Fricke, Philipp; Kenny, Shane T; Morales-Gamez, Laura; Babu, Ramesh P; O'Connor, Kevin E; Steinbüchel, Alexander

2016-10-15

The purple nonsulfur alphaproteobacterium Rhodospirillum rubrum S1 was genetically engineered to synthesize a heteropolymer of mainly 3-hydroxydecanoic acid and 3-hydroxyoctanoic acid [P(3HD-co-3HO)] from CO- and CO 2 -containing artificial synthesis gas (syngas). For this, genes from Pseudomonas putida KT2440 coding for a 3-hydroxyacyl acyl carrier protein (ACP) thioesterase (phaG), a medium-chain-length (MCL) fatty acid coenzyme A (CoA) ligase (PP_0763), and an MCL polyhydroxyalkanoate (PHA) synthase (phaC1) were cloned and expressed under the control of the CO-inducible promoter P cooF from R. rubrum S1 in a PHA-negative mutant of R. rubrum P(3HD-co-3HO) was accumulated to up to 7.1% (wt/wt) of the cell dry weight by a recombinant mutant strain utilizing exclusively the provided gaseous feedstock syngas. In addition to an increased synthesis of these medium-chain-length PHAs (PHA MCL ), enhanced gene expression through the P cooF promoter also led to an increased molar fraction of 3HO in the synthesized copolymer compared with the P lac promoter, which regulated expression on the original vector. The recombinant strains were able to partially degrade the polymer, and the deletion of phaZ2, which codes for a PHA depolymerase most likely involved in intracellular PHA degradation, did not reduce mobilization of the accumulated polymer significantly. However, an amino acid exchange in the active site of PhaZ2 led to a slight increase in PHA MCL accumulation. The accumulated polymer was isolated; it exhibited a molecular mass of 124.3 kDa and a melting point of 49.6°C. With the metabolically engineered strains presented in this proof-of-principle study, we demonstrated the synthesis of elastomeric second-generation biopolymers from renewable feedstocks not competing with human nutrition. Polyhydroxyalkanoates (PHAs) are natural biodegradable polymers (biopolymers) showing properties similar to those of commonly produced petroleum-based nondegradable polymers. The utilization of cheap substrates for the microbial production of PHAs is crucial to lower production costs. Feedstock not competing with human nutrition is highly favorable. Syngas, a mixture of carbon monoxide, carbon dioxide, and hydrogen, can be obtained by pyrolysis of organic waste and can be utilized for PHA synthesis by several kinds of bacteria. Up to now, the biosynthesis of PHAs from syngas has been limited to short-chain-length PHAs, which results in a stiff and brittle material. In this study, the syngas-utilizing bacterium Rhodospirillum rubrum was genetically modified to synthesize a polymer which consisted of medium-chain-length constituents, resulting in a rubber-like material. This study reports the establishment of a microbial synthesis of these so-called medium-chain-length PHAs from syngas and therefore potentially extends the applications of syngas-derived PHAs. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Global changes in gene expression, assayed by microarray hybridization and quantitative RT-PCR, during acclimation of three Arabidopsis thaliana accessions to sub-zero temperatures after cold acclimation.

PubMed

Le, Mai Q; Pagter, Majken; Hincha, Dirk K

2015-01-01

During cold acclimation plants increase in freezing tolerance in response to low non-freezing temperatures. This is accompanied by many physiological, biochemical and molecular changes that have been extensively investigated. In addition, plants of many species, including Arabidopsis thaliana, become more freezing tolerant during exposure to mild, non-damaging sub-zero temperatures after cold acclimation. There is hardly any information available about the molecular basis of this adaptation. Here, we have used microarrays and a qRT-PCR primer platform covering 1,880 genes encoding transcription factors (TFs) to monitor changes in gene expression in the Arabidopsis accessions Columbia-0, Rschew and Tenela during the first 3 days of sub-zero acclimation at -3 °C. The results indicate that gene expression during sub-zero acclimation follows a tighly controlled time-course. Especially AP2/EREBP and WRKY TFs may be important regulators of sub-zero acclimation, although the CBF signal transduction pathway seems to be less important during sub-zero than during cold acclimation. Globally, we estimate that approximately 5% of all Arabidopsis genes are regulated during sub-zero acclimation. Particularly photosynthesis-related genes are down-regulated and genes belonging to the functional classes of cell wall biosynthesis, hormone metabolism and RNA regulation of transcription are up-regulated. Collectively, these data provide the first global analysis of gene expression during sub-zero acclimation and allow the identification of candidate genes for forward and reverse genetic studies into the molecular mechanisms of sub-zero acclimation.

Zn2+-induced changes at the root level account for the increased tolerance of acclimated tobacco plants

PubMed Central

Bazihizina, Nadia; Taiti, Cosimo; Marti, Lucia; Rodrigo-Moreno, Ana; Spinelli, Francesco; Giordano, Cristiana; Caparrotta, Stefania; Gori, Massimo; Azzarello, Elisa; Mancuso, Stefano

2014-01-01

Evidence suggests that heavy-metal tolerance can be induced in plants following pre-treatment with non-toxic metal concentrations, but the results are still controversial. In the present study, tobacco plants were exposed to increasing Zn2+ concentrations (up to 250 and/or 500 μM ZnSO4) with or without a 1-week acclimation period with 30 μM ZnSO4. Elevated Zn2+ was highly toxic for plants, and after 3 weeks of treatments there was a marked (≥50%) decline in plant growth in non-acclimated plants. Plant acclimation, on the other hand, increased plant dry mass and leaf area up to 1.6-fold compared with non-acclimated ones. In non-acclimated plants, the addition of 250 μM ZnSO4 led to transient membrane depolarization and stomatal closure within 24h from the addition of the stress; by contrast, the acclimation process was associated with an improved stomatal regulation and a superior ability to maintain a negative root membrane potential, with values on average 37% more negative compared with non-acclimated plants. The different response at the plasma-membrane level between acclimated and non-acclimated plants was associated with an enhanced vacuolar Zn2+ sequestration and up to 2-fold higher expression of the tobacco orthologue of the Arabidopsis thaliana MTP1 gene. Thus, the acclimation process elicited specific detoxification mechanisms in roots that enhanced Zn2+ compartmentalization in vacuoles, thereby improving root membrane functionality and stomatal regulation in leaves following elevated Zn2+ stress. PMID:24928985

A New Player in the Biorefineries Field: Phasin PhaP Enhances Tolerance to Solvents and Boosts Ethanol and 1,3-Propanediol Synthesis in Escherichia coli.

PubMed

Mezzina, Mariela P; Álvarez, Daniela S; Egoburo, Diego E; Díaz Peña, Rocío; Nikel, Pablo I; Pettinari, M Julia

2017-07-15

The microbial production of biofuels and other added-value chemicals is often limited by the intrinsic toxicity of these compounds. The phasin PhaP from the soil bacterium Azotobacter sp. strain FA8 is a polyhydroxyalkanoate granule-associated protein that protects recombinant Escherichia coli against several kinds of stress. PhaP enhances growth and poly(3-hydroxybutyrate) synthesis in polymer-producing recombinant strains and reduces the formation of inclusion bodies during overproduction of heterologous proteins. In this work, the heterologous expression of this phasin in E. coli was used as a strategy to increase tolerance to several biotechnologically relevant chemicals. PhaP was observed to enhance bacterial fitness in the presence of biofuels, such as ethanol and butanol, and other chemicals, such as 1,3-propanediol. The effect of PhaP was also studied in a groELS mutant strain, in which both GroELS and PhaP were observed to exert a beneficial effect that varied depending on the chemical tested. Lastly, the potential of PhaP and GroEL to enhance the accumulation of ethanol or 1,3-propanediol was analyzed in recombinant E. coli Strains that overexpressed either groEL or phaP had increased growth, reflected in a higher final biomass and product titer than the control strain. Taken together, these results add a novel application to the already multifaceted phasin protein group, suggesting that expression of these proteins or other chaperones can be used to improve the production of biofuels and other chemicals. IMPORTANCE This work has both basic and applied aspects. Our results demonstrate that a phasin with chaperone-like properties can increase bacterial tolerance to several biochemicals, providing further evidence of the diverse properties of these proteins. Additionally, both the PhaP phasin and the well-known chaperone GroEL were used to increase the biosynthesis of the biotechnologically relevant compounds ethanol and 1,3-propanediol in recombinant E. coli These findings open the road for the use of these proteins for the manipulation of bacterial strains to optimize the synthesis of diverse bioproducts from renewable carbon sources. Copyright © 2017 American Society for Microbiology.

Combining iGoogle and personal health records to create a prototype personal health application for diabetes self-management.

PubMed

Fonda, Stephanie J; Kedziora, Richard J; Vigersky, Robert A; Bursell, Sven-Erik

2010-05-01

The aim of this project is to create a prototype for a personal health application (PHA) for patients (i.e., consumers) with diabetes by employing a user-centered design process. This article describes the design process for and resulting architecture, workflow, and functionality of such a PHA. For the design process, we conducted focus groups with people who have diabetes (n = 21) to ascertain their needs for a PHA. We then developed a prototype in response to these needs, and through additional focus groups and step-by-step demonstrations for people with diabetes as well as healthcare providers, we obtained feedback about the prototype. The feedback led to changes in the PHA's presentation and function. Focus group participants said they wanted a tool that could give them timely, readily available information on how diabetes-related domains interact, how their behaviors affect them, and what to do next. Thus, the prototype PHA is Internet-based, retrieves data for diabetes self-management from a personal health record, displays those data using gadgets in the consumer's iGoogle page, and makes the data available to a decision-support component that provides lifestyle-oriented advice. Manipulation of the data enables consumers to anticipate the results of future actions and to see interrelationships. A user-centered design process resulted in a PHA that uses technology that is publicly available, employs a personal health record, and is Internet based. This PHA can provide the backbone for a decision support system that can bring together the cornerstones of diabetes self-management and integrate them into the life of the person with diabetes.

Phase angle assessment by bioelectrical impedance analysis and its predictive value for malnutrition risk in hospitalized geriatric patients.

PubMed

Varan, Hacer Dogan; Bolayir, Basak; Kara, Ozgur; Arik, Gunes; Kizilarslanoglu, Muhammet Cemal; Kilic, Mustafa Kemal; Sumer, Fatih; Kuyumcu, Mehmet Emin; Yesil, Yusuf; Yavuz, Burcu Balam; Halil, Meltem; Cankurtaran, Mustafa

2016-12-01

Phase angle (PhA) value determined by bioelectrical impedance analysis (BIA) is an indicator of cell membrane damage and body cell mass. Recent studies have shown that low PhA value is associated with increased nutritional risk in various group of patients. However, there have been only a few studies performed globally assessing the relationship between nutritional risk and PhA in hospitalized geriatric patients. The aim of the study is to evaluate the predictive value of the PhA for malnutrition risk in hospitalized geriatric patients. One hundred and twenty-two hospitalized geriatric patients were included in this cross-sectional study. Comprehensive geriatric assessment tests and BIA measurements were performed within the first 48 h after admission. Nutritional risk state of the patients was determined with NRS-2002. Phase angle values of the patients with malnutrition risk were compared with the patients that did not have the same risk. The independent variables for predicting malnutrition risk were determined. SPSS version 15 was utilized for the statistical analyzes. The patients with malnutrition risk had significantly lower phase angle values than the patients without malnutrition risk (p = 0.003). ROC curve analysis suggested that the optimum PhA cut-off point for malnutrition risk was 4.7° with 79.6 % sensitivity, 64.6 % specificity, 73.9 % positive predictive value, and 73.9 % negative predictive value. BMI, prealbumin, PhA, and Mini Mental State Examination Test scores were the independent variables for predicting malnutrition risk. PhA can be a useful, independent indicator for predicting malnutrition risk in hospitalized geriatric patients.

A Micro-Ark for Cells: Highly Open Porous Polyhydroxyalkanoate Microspheres as Injectable Scaffolds for Tissue Regeneration.

PubMed

Wei, Dai-Xu; Dao, Jin-Wei; Chen, Guo-Qiang

2018-06-19

To avoid large open surgery using scaffold transplants, small-sized cell carriers are employed to repair complexly shaped tissue defects. However, most cell carriers show poor cell adherences and viability. Therefore, polyhydroxyalkanoate (PHA), a natural biopolymer, is used to prepare highly open porous microspheres (OPMs) of 300-360 µm in diameter, combining the advantages of microspheres and scaffolds to serve as injectable carriers harboring proliferating stem cells. In addition to the convenient injection to a defected tissue, and in contrast to poor performances of OPMs made of polylactides (PLA OPMs) and traditional less porous hollow microspheres (PHA HMs), PHA OPMs present suitable surface pores of 10-60 µm and interconnected passages with an average size of 8.8 µm, leading to a high in vitro cell adhesion of 93.4%, continuous proliferation for 10 d and improved differentiation of human bone marrow mesenchymal stem cells (hMSCs). PHA OPMs also support stronger osteoblast-regeneration compared with traditional PHA HMs, PLA OPMs, commercial hyaluronic acid hydrogels, and carrier-free hMSCs in an ectopic bone-formation mouse model. PHA OPMs protect cells against stresses during injection, allowing more living cells to proliferate and migrate to damaged tissues. They function like a micro-Noah's Ark to safely transport cells to a defect tissue. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Production of copolymer, poly (hydroxybutyrate-co-hydroxyvalerate) by Halomonas campisalis MCM B-1027 using agro-wastes.

PubMed

Kulkarni, S O; Kanekar, P P; Jog, J P; Sarnaik, S S; Nilegaonkar, S S

2015-01-01

For cost effective production of PHA, agro-wastes like fruit peels, bagasse and deoiled cakes were screened as a sole source of carbon. Halomonas campisalis MCM B-1027, which was isolated from one of the extreme environment, i.e. Lonar Lake, India, was explored for the production of PHA using fruit peels and bagasse having fermentable sugars. Among the agro-wastes tested, 1% (v/v) aqueous extract of bagasse was found to be the optimum carbon source with 47% PHA production on dry cell weight basis. Significant amount of total sugars are utilized and converted into cell mass and PHA, e.g. 62% sugar utilized from bagasse extract, 84% from orange peel extract and 71% from banana peel extract as compared to 51% in case of maltose. Hence the cost of production would be positively reduced. The detailed characterization of PHA formed by H. campisalis using bagasse extract as sole carbon source revealed that the organism produces a copolymer of PHB-co-PHV (94.4:5.6) having molecular weight M(w) 1.394 × 10(6) and melting temperature 168.9 °C. Production of PHA by H. campisalis using aqueous extract of fruit peels and a copolymer PHB-co-PHV using aqueous extract of bagasse is presumably the first report. Copyright © 2014 Elsevier B.V. All rights reserved.

Green technology for conversion of food scraps to biodegradable thermoplastic polyhydroxyalkanoates.

PubMed

Du, Guocheng; Yu, Jian

2002-12-15

A new technology is developed and demonstrated that couples anaerobic digestion of food scraps with production of biodegradable thermoplastics, polyhydroxyalkanoates (PHAs). The food wastes were digested in an anaerobic reactor producing four major organic acids. The concentrations of acetic, propionic, butyric, and lactic acids reached 5.5, 1.8, 27.4, and 32.7 g/L, respectively. The fermentative acids were transferred through membranes via molecule diffusion into an air-bubbling reactor where the acids were utilized to produce PHAs in an enriched culture of Ralstonia eutropha. With a silicone rubber membrane, butyric acid and small amounts of acetic and propionic acids were transferred and used, producing a homopolymer PHA, poly(3-hydroxybutyrate). The dry cell weight and PHA content reached 11.3 g/L and 60.2% (w/w), respectively. With a dialysis membrane, the mass transfer rates of fermentative acids were enhanced, and the PHA production was significantly improved. The dry cell weight and its PHA content reached 22.7 g/L and 72.6% (w/w), respectively. The formed PHA was a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate (HV) with 2.8 mol % HV monomer unit. The polymer content (72.6% of dry cell mass) reported in this study is the highest one obtained from organic wastes and is comparable with the PHA content from pure glucose fermentation.

Construction of Halomonas bluephagenesis capable of high cell density growth for efficient PHA production.

PubMed

Ren, Yilin; Ling, Chen; Hajnal, Ivan; Wu, Qiong; Chen, Guo-Qiang

2018-05-01

High-cell-density cultivation is an effective way to improve the productivity of microbial fermentations and in turn reduce the cost of the final products, especially in the case of intracellular products. Halomonas bluephagenesis TD01 is a halophilic platform bacterium for the next generation of industrial biotechnology with a native PHA synthetic pathway, able to grow under non-sterile continuous fermentation conditions. A selection strategy for mutant strains that can grow to a high cell density was developed. Based on an error-prone DNA polymerase III ε subunit, a genome-wide random mutagenesis system was established and used in conjunction with an artificial high cell density culture environment during the selection process. A high-cell-density H. bluephagenesis TDHCD-R3 obtained after 3 rounds of selection showed an obvious enhancement of resistance to toxic metabolites including acetate, formate, lactate and ethanol compared to wild-type. H. bluephagenesis TDHCD-R3-8-3 constructed from H. bluephagenesis TDHCD-R3 by overexpressing an optimized phaCAB operon was able to grow to 15 g/L cell dry weight (CDW) containing 94% PHA in shake flask studies. H. bluephagenesis TDHCD-R3-8-3 was grown to more than 90 g/L CDW containing 79% PHA compared with only 81 g/L with 70% PHA by the wild type when incubated in a 7-L fermentor under the same conditions.

Purification and characterization of a 14-kilodalton protein that is bound to the surface of polyhydroxyalkanoic acid granules in Rhodococcus ruber.

PubMed Central

Pieper-Fürst, U.; Madkour, M. H.; Mayer, F.; Steinbüchel, A.

1994-01-01

The N-terminal amino acid sequence of the polyhydroxyalkanoic acid (PHA) granule-associated M(r)-15,500 protein of Rhodococcus ruber (the GA14 protein) was analyzed. The sequence revealed that the corresponding structural gene is represented by open reading frame 3, encoding a protein with a calculated M(r) of 14,175 which was recently localized downstream of the PHA synthase gene (U. Pieper and A. Steinbüchel, FEMS Microbiol. Lett. 96:73-80, 1992). A recombinant strain of Escherichia coli XL1-Blue carrying the hybrid plasmid (pSKXA10*) with open reading frame 3 overexpressed the GA14 protein. The GA14 protein was subsequently purified in a three-step procedure including chromatography on DEAE-Sephacel, phenyl-Sepharose CL-4B, and Superose 12. Determination of the molecular weight by gel filtration as well as electron microscopic studies indicates that a tetrameric structure of the recombinant, native GA14 protein is most likely. Immunoelectron microscopy demonstrated a localization of the GA14 protein at the periphery of PHA granules as well as close to the cell membrane in R. ruber. Investigations of PHA-leaky and PHA-negative mutants of R. ruber indicated that expression of the GA14 protein depended strongly on PHA synthesis. Images PMID:8021220

Phase angle as bioelectrical marker to identify elderly patients at risk of sarcopenia.

PubMed

Basile, Claudia; Della-Morte, David; Cacciatore, Francesco; Gargiulo, Gaetano; Galizia, Gianluigi; Roselli, Mario; Curcio, Francesco; Bonaduce, Domenico; Abete, Pasquale

2014-10-01

Several markers have been associated with sarcopenia in the elderly, including bioelectrical indices. Phase angle (PhA) is an impedance parameter and it has been suggested as an indicator of cellular death. Thus, the relationship between PhA and muscle mass and strength was investigated in 207 consecutively elderly participants (mean age 76.2±6.7years) admitted for multidimensional geriatric evaluation. Muscle strength by grip strength using a hand-held dynamometer and muscle mass was measured by bioimpedentiometer. PhA was calculated directly with its arctangent (resistance/reactance×180°/π). Linear relationship among muscular mass and strength and with clinical and biochemical parameters, including PhA at uni- and multivariate analysis were performed. Linear regression analysis demonstrated that lower level of PhA is associated with reduction in grip strength (y=3.16+0.08x; r=0.49; p<0.001), and even more, with muscle mass (y=3.04+0.25x; r=0.60; p<0001). Multivariate analysis confirms these relationships (grip strength β=0.245, p=0.031; muscular mass β=0.623, p<0.01). Thus, PhA is inversely related to muscle mass and strength in elderly subjects and it may be considered a good bioelectrical marker to identify elderly patients at risk of sarcopenia. Copyright © 2014 Elsevier Inc. All rights reserved.

Simple and rapid method for isolation and quantitation of polyhydroxyalkanoate by SDS-sonication treatment.

PubMed

Arikawa, Hisashi; Sato, Shunsuke; Fujiki, Tetsuya; Matsumoto, Keiji

2017-08-01

We developed a new method for isolation and quantitation of polyhydroxyalkanoate (PHA) from culture broth. In this method, the cells were sonicated in sodium dodecyl sulfate (SDS) solution and centrifuged to recover PHA. The recovered PHA was rinsed with deionized water and ethanol, and then weighed after drying. Hazardous chemicals such as chloroform, methanol, and sulfuric acid were not used, and no expensive analytical instruments were needed. We applied this method to Cupriavidus necator culture broths that included various amounts of poly(3-hydroxybutyrate) (PHB) or poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) from flasks and jar fermentors. The quantitation by this method was practical for use with a wide range of production amounts and PHA monomer compositions compared to the conventional whole-cell methanolysis method with gas chromatographic analysis, and besides, the recovered PHAs were adequately pure (≥96% purity). Therefore, this new method would be valuable not only for quantitation of PHA but also for preparation of samples to characterize their mechanical properties. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Suppressed PHA activation of T lymphocytes in simulated microgravity is restored by direct activation of protein kinase C

NASA Technical Reports Server (NTRS)

Cooper, D.; Pellis, N. R.; McIntire, L. V. (Principal Investigator)

1998-01-01

Utilizing clinostatic rotating wall vessel (RWV) bioreactors that simulate aspects of microgravity, we found phytohemagglutinin (PHA) responsiveness to be almost completely diminished. Activation marker expression was significantly reduced in RWV cultures. Furthermore, cytokine secretion profiles suggested that monocytes are not as adversely affected by simulated microgravity as T cells. Reduced cell-cell and cell-substratum interactions may play a role in the loss of PHA responsiveness because placing peripheral blood mononuclear cells (PBMC) within small collagen beads did partially restore PHA responsiveness. However, activation of purified T cells with cross-linked CD2/CD28 and CD3/CD28 antibody pairs was completely suppressed in the RWV, suggesting a defect in signal transduction. Activation of purified T cells with PMA and ionomycin was unaffected by RWV culture. Furthermore, sub-mitogenic doses of PMA alone but not ionomycin alone restored PHA responsiveness of PBMC in RWV culture. Thus our data indicate that during polyclonal activation the signaling pathways upstream of PKC activation are sensitive to simulated microgravity.

Crystallization and preliminary crystallographic analysis of poly(3-hydroxybutyrate) depolymerase from Bacillus thuringiensis.

PubMed

Wang, Yung Lin; Lin, Yi Ting; Chen, Chia Lin; Shaw, Gwo Chyuan; Liaw, Shwu Huey

2014-10-01

Poly[(R)-3-hydroxybutyrate] (PHB) is a microbial biopolymer that has been commercialized as biodegradable plastics. The key enzyme for the degradation is PHB depolymerase (PhaZ). A new intracellular PhaZ from Bacillus thuringiensis (BtPhaZ) has been screened for potential applications in polymer biodegradation. Recombinant BtPhaZ was crystallized using 25% polyethylene glycol 3350, 0.2 M ammonium acetate, 0.1 M bis-tris pH 6.5 at 288 K. The crystals belonged to space group P1, with unit-cell parameters a = 42.97, b = 83.23, c = 85.50 Å, α = 73.45, β = 82.83, γ = 83.49°. An X-ray diffraction data set was collected to 1.42 Å resolution with an Rmerge of 6.4%. Unexpectedly, a molecular-replacement solution was obtained using the crystal structure of Streptomyces lividans chloroperoxidase as a template, which shares 24% sequence identity to BtPhaZ. This is the first crystal structure of an intracellular poly(3-hydroxybutyrate) depolymerase.

Perspectives on the production of polyhydroxyalkanoates in biorefineries associated with the production of sugar and ethanol.

PubMed

Silva, Luiziana Ferreira; Taciro, Marilda Keico; Raicher, Gil; Piccoli, Rosane Aparecida Moniz; Mendonça, Thatiane Teixeira; Lopes, Mateus Schreiner Garcez; Gomez, José Gregório Cabrera

2014-11-01

Polyhydroxyalkanoates (PHA) are biodegradable and biocompatible bacterial thermoplastic polymers that can be obtained from renewable resources. The high impact of the carbon source in the final cost of this polymer has been one of the major limiting factors for PHA production and agricultural residues, mainly lignocellulosic materials, have gained attention to overcome this problem. In Brazil, production of 2nd generation ethanol from the glucose fraction, derived from sugarcane bagasse hydrolysate has been studied. The huge amounts of remaining xylose will create an opportunity for the development of other bioprocesses, generating new products to be introduced into a biorefinery model. Although PHA production from sucrose integrated to a 1G ethanol and sugar mill has been proposed in the past, the integration of the process of 2G ethanol in the context of a biorefinery will provide enormous amounts of xylose, which could be applied to produce PHA, establishing a second-generation of PHA production process. Those aspects and perspectives are presented in this article. Copyright © 2014 Elsevier B.V. All rights reserved.

Heat and cold acclimation in helium-cold hypothermia in the hamster.

NASA Technical Reports Server (NTRS)

Musacchia, X. J.

1972-01-01

A study was made of the effects of acclimation of hamsters to high (34-35 C) and low (4-5 C) temperatures for periods up to 6 weeks on the induction of hypothermia in hamsters. Hypothermia was achieved by exposing hamsters to a helox mixture of 80% helium and 20% oxygen at 0 C. Hypothermic induction was most rapid (2-3 hr) in heat-acclimated hamsters and slowest (6-12 hr) in cold-acclimated hamsters. The induction period was intermediate (5-8 hr) in room temperature nonacclimated animals (controls). Survival time in hypothermia was relatable to previous temperature acclimations. The hypothesis that thermogenesis in cold-acclimated hamsters would accentuate resistance to induction of hypothermia was substantiated.

Deconditioning-induced exercise responses as influenced by heat acclimation

NASA Technical Reports Server (NTRS)

Shvartz, E.; Bhattacharya, A.; Sperinde, S. J.; Brock, P. J.; Sciaraffa, D.; Haines, R. F.; Greenleaf, J. E.

1979-01-01

A study to determine the effect of heat acclimation and physical training in temperate conditions on changes in exercise tolerance following water-immersion deconditioning is presented. Five young men were tested on a bicycle ergometer before and after heat acclimation and after water immersion. The subjects and the experimental procedure, heat acclimation and exercise training, water immersion, and exercise tolerance are discussed. Heat acclimation resulted in the usual decreases in exercise heart rate and rectal temperature and an increase in sweat rate. Water immersion resulted in substantial diuresis despite water consumed. The results show that heat acclimation provides an effective method of preventing the adverse effects of water-immersion deconditioning on exercise tolerance.

Acclimation to low level exposure of copper in Bufo arenarum embryos: linkage of effects to tissue residues.

PubMed

Herkovits, Jorge; Pérez-Coll, Cristina Silvia

2007-06-01

The acclimation possibilities to copper in Bufo arenarum embryos was evaluated by means of three different low level copper exposure conditions during 14 days. By the end of the acclimation period the copper content in control embryos was 1.04 +/- 0.09 microg g(-1) (wet weight) while in all the acclimated embryos a reduction of about 25% of copper was found. Thus copper content could be considered as a biomarker of low level exposure conditions. Batches of 10 embryos (by triplicate) from each acclimation condition were challenged with three different toxic concentrations of copper. As a general pattern, the acclimation protocol to copper exerted a transient beneficial effect on the survival of the Bufo arenarum embryos. The acclimation phenomenon could be related to the selection of pollution tolerant organisms within an adaptive process and therefore the persistence of information within an ecological system following a toxicological stressor.

Successful treatment with pazopanib plus PD-1 inhibitor and RAK cells for advanced primary hepatic angiosarcoma: a case report.

PubMed

Qiao, Yu; Yang, Jihong; Liu, Lili; Zeng, Yixin; Ma, Jie; Jia, Jing; Zhang, Li; Li, Xiaoguang; Wu, Peihong; Wang, Wenchao; Liu, Dongge; Chen, Huan; Zhao, Yunbo; Xi, Huan; Wang, Yao

2018-02-21

Primary hepatic angiosarcoma (PHA) is a rare and aggressive solid tumor, with high rates of local recurrence and distant metastasis, and poor prognosis. There are no established treatment guidelines for PHA. A 78-year-old asymptomatic man with PHA that was successfully treated with pazopanib plus PD-1 inhibitor and RetroNectin-activated killer cells (RAK cells). After one month of treatment, there was a clear reduction in the size and number of the liver metastases; and after nearly 15 months, most of the lesions were stable, no new lesions had developed, and the side effect of treatment was minor. Pazopanib, PD-1 inhibitor and RAK cells could serve as a potential option for the treatment of advanced PHA.

Changes in ABA and gene expression in cold-acclimated sugar maple.

PubMed

Bertrand, A; Robitaille, G; Castonguay, Y; Nadeau, P; Boutin, R

1997-01-01

To determine if cold acclimation of sugar maple (Acer saccharum Marsh.) is associated with specific changes in gene expression under natural hardening conditions, we compared bud and root translatable mRNAs of potted maple seedlings after cold acclimation under natural conditions and following spring dehardening. Cold-hardened roots and buds were sampled in January when tissues reached their maximum hardiness. Freezing tolerance, expressed as the lethal temperature for 50% of the tissues (LT(50)), was estimated at -17 degrees C for roots, and at lower than -36 degrees C for buds. Approximately ten transcripts were specifically synthesized in cold-acclimated buds, or were more abundant in cold-acclimated buds than in unhardened buds. Cold hardening was also associated with changes in translation. At least five translation products were more abundant in cold-acclimated buds and roots compared with unhardened tissues. Abscisic acid (ABA) concentration increased approximately tenfold in the xylem sap following winter acclimation, and the maximum concentration was reached just before maximal acclimation. We discuss the potential involvement of ABA in the observed modification of gene expression during cold hardening.

Benefits of thermal acclimation in a tropical aquatic ectotherm, the Arafura filesnake, Acrochordus arafurae.

PubMed

Bruton, Melissa J; Cramp, Rebecca L; Franklin, Craig E

2012-05-01

The presumption that organisms benefit from thermal acclimation has been widely debated in the literature. The ability to thermally acclimate to offset temperature effects on physiological function is prevalent in ectotherms that are unable to thermoregulate year-round to maintain performance. In this study we examined the physiological and behavioural consequences of long-term exposure to different water temperatures in the aquatic snake Acrochordus arafurae. We hypothesised that long dives would benefit this species by reducing the likelihood of avian predation. To achieve longer dives at high temperatures, we predicted that thermal acclimation of A. arafurae would reduce metabolic rate and increase use of aquatic respiration. Acrochordus arafurae were held at 24 or 32°C for 3 months before dive duration and physiological factors were assessed (at both 24 and 32°C). Although filesnakes demonstrated thermal acclimation of metabolic rate, use of aquatic respiration was thermally independent and did not acclimate. Mean dive duration did not differ between the acclimation groups at either temperature; however, warm-acclimated animals increased maximum and modal dive duration, demonstrating a longer dive duration capacity. Our study established that A. arafurae is capable of thermal acclimation and this confers a benefit to the diving abilities of this snake.

Zebrafish take their cue from temperature but not photoperiod for the seasonal plasticity of thermal performance.

PubMed

Condon, Catriona H; Chenoweth, Stephen F; Wilson, Robbie S

2010-11-01

Organisms adjust to seasonal variability in the environment by responding to cues that indicate environmental change. As most studies of seasonal phenotypic plasticity test only the effect of a single environmental cue, how animals may integrate information from multiple cues to fine-tune plastic responses remains largely unknown. We examined the interaction between correlated (seasonally matching) and conflicting (seasonally opposite) temperature and photoperiod cues on the acclimation of performance traits in male zebrafish, Danio rerio. We acclimated fish for 8 weeks and then tested the change in thermal dependence of maximum burst swimming and feeding rate between 8 and 38°C. We predicted that correlated environmental cues should induce a greater acclimation response than uncorrelated cues. However, we found that only temperature was important for the seasonal acclimation of performance traits in zebrafish. Thermal acclimation shifted the thermal performance curve of both traits. For maximum burst swimming, performance increased for each group near the acclimation temperature and reduced in environments that were far from their acclimation temperature. The feeding rate of cold-acclimated zebrafish was reduced across the test temperature range compared with that of warm-acclimated fish. Our study is the first that has found no effect of the covariation between temperature and photoperiod acclimation cues on locomotor performance in fishes. Our results support the intuitive idea that photoperiod may be a less important seasonal cue for animals living at lower latitudes.

Effects of prolonged acclimation to cold on the extra--and intracellular acid-base status in the land snail Helix lucorum (L.).

PubMed

Staikou, A; Stiakakis, M; Michaelidis, B

2001-01-01

The aim of this study was to examine the effect of prolonged acclimation to cold on the acid-base status of extra- and intracellular fluids in the land snail Helix lucorum. For this purpose, acid-base parameters in the hemolymph and tissues were determined. In addition, the buffer values of hemolymph and tissues were determined in order to examine whether they change in the snails during acclimation to cold. According to the results presented, there is an inverse pH-temperature relationship in the hemolymph within the first day of acclimation, which is consistent with alphastat regulation. The Pco2 decreased, and pH in the hemolymph (pH(e)) increased by 0.32 U within the first day of acclimation to cold, which corresponds to a change of 0.013 U degrees C(-1). After the first day of acclimation, Pco2 increased in the hemolymph, resulting in a significant drop in pH(e) by 90 d of acclimation to cold. Acclimation of snails to low temperatures did not change the buffer value of the hemolymph. Also, intracellular pH (pH(i)) and intracellular buffer values remained stable during acclimation to cold for prolonged periods. The latter results in conjunction with those obtained by the in vitro determination of the passive component of intracellular fluids indicate an active regulation of pH(i) in H. lucorum during acclimation to cold.

Comparative transcriptome analysis on the alteration of gene expression in ayu (Plecoglossus altivelis) larvae associated with salinity change.

PubMed

Lu, Xin-Jiang; Zhang, Hao; Yang, Guan-Jun; Li, Ming-Yun; Chen, Jiong

2016-05-18

Ayu (Plecoglossus altivelis) fish, which are an amphidromous species distributed in East Asia, live in brackish water (BW) during their larval stage and in fresh water (FW) during their adult stage. In this study, we found that FW-acclimated ayu larvae exhibited a slower growth ratio compared with that of BW-acclimated larvae. However, the mechanism underlying FW acclimation on growth suppression is poorly known. We employed transcriptome analysis to investigate the differential gene expression of FW acclimation by RNA sequencing. We identified 158 upregulated and 139 downregulated transcripts in FW-acclimated ayu larvae compared with that in BW-acclimated larvae. As determined by Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway mapping, functional annotation of the genes covered diverse biological functions and processes, and included neuroendocrinology, osmotic regulation, energy metabolism, and the cytoskeleton. Transcriptional expression of several differentially expressed genes in response to FW acclimation was further confirmed by real-time quantitative PCR. In accordance with transcriptome analysis, iodothyronine deiodinase (ID), pro-opiomelanocortin (POMC), betaine-homocysteine S-methyltransferase 1(BHMT), fructose-bisphosphate aldolase B (aldolase B), tyrosine aminotransferase (TAT), and Na(+)-K(+) ATPase (NKA) were upregulated after FW acclimation. Furthermore, the mRNA expressions of b-type natriuretic peptide (BNP) and transgelin were downregulated after FW acclimation. Our data indicate that FW acclimation reduced the growth rate of ayu larvae, which might result from the expression alteration of genes related to endocrine hormones, energy metabolism, and direct osmoregulation.

Do mitochondrial properties explain intraspecific variation in thermal tolerance?

PubMed

Fangue, Nann A; Richards, Jeffrey G; Schulte, Patricia M

2009-02-01

As global temperatures rise, there is a growing need to understand the physiological mechanisms that determine an organism's thermal niche. Here, we test the hypothesis that increases in mitochondrial capacity with cold acclimation and adaptation are associated with decreases in thermal tolerance using two subspecies of killifish (Fundulus heteroclitus) that differ in thermal niche. We assessed whole-organism metabolic rate, mitochondrial amount and mitochondrial function in killifish acclimated to several temperatures. Mitochondrial enzyme activities and mRNA levels were greater in fish from the northern subspecies, particularly in cold-acclimated fish, suggesting that the putatively cold-adapted northern subspecies has a greater capacity for increases in mitochondrial amount in response to cold acclimation. When tested at the fish's acclimation temperature, maximum ADP-stimulated (State III) rates of mitochondrial oxygen consumption in vitro were greater in cold-acclimated northern fish than in southern fish but did not differ between subspecies at higher acclimation temperatures. Whole-organism metabolic rate was greater in fish of the northern subspecies at all acclimation temperatures. Cold acclimation also changed the response of mitochondrial respiration to acute temperature challenge. Mitochondrial oxygen consumption was greater in cold-acclimated northern fish than in southern fish at low test temperatures, but the opposite was true at high test temperatures. These differences were reflected in whole-organism oxygen consumption. Our data indicate that the plasticity of mitochondrial function and amount differs between killifish subspecies, with the less high-temperature tolerant, and putatively cold adapted, northern subspecies having greater ability to increase mitochondrial capacity in the cold. However, there were few differences in mitochondrial properties between subspecies at warm acclimation temperatures, despite differences in both whole-organism oxygen consumption and thermal tolerance at these temperatures.

Consequences of thermal acclimation for the mating behaviour and swimming performance of female mosquito fish.

PubMed

Wilson, Robbie S; Condon, Catriona H L; Johnston, Ian A

2007-11-29

The mating system of eastern mosquito fish (Gambusia holbrooki) is dominated by male sexual coercion, where all matings are forced and females never appear to cooperate and actively avoid all attempts. Previous research has shown that male G. holbrooki offer a model system for examining the benefits of reversible thermal acclimation for reproductive success, but examining the benefits to female avoidance behaviour has been difficult. In this study, we examined the ability of non-male-deprived female G. holbrooki to avoid forced-coercive matings following acclimation to either 18 or 30 degrees C for six weeks (12h light:12h dark photoperiod). Thermal acclimation of burst and sustained swimming performance was also assessed, as these traits are likely to underlie their ability to avoid forced matings. There was no influence of thermal acclimation on the burst swimming performance of female G. holbrooki over the range 18-30 degrees C; however, sustained swimming performance was significantly lower in the warm- than the cool-acclimation group. For mating behaviour, we tested the hypothesis that acclimation would enhance the ability of female G. holbrooki to avoid forced matings at their host acclimation temperature relative to females acclimated to another environment. However, our hypothesis was not supported. The rate of copulations was almost three times greater for females acclimated to 30 degrees C than 18 degrees C when tested at 30 degrees C, indicating that they possess the ability to alter their avoidance behaviour to 'allow' more copulations in some environments. Coupled with previous studies, female G. holbrooki appear to have greater control on the outcome of coercive mating attempts than previously considered and can alter their propensity to receive forced matings following thermal acclimation. The significance of this change in female mating-avoidance behaviours with thermal acclimation remains to be explored.

Mitochondrial acclimation capacities to ocean warming and acidification are limited in the antarctic Nototheniid Fish, Notothenia rossii and Lepidonotothen squamifrons.

PubMed

Strobel, Anneli; Graeve, Martin; Poertner, Hans O; Mark, Felix C

2013-01-01

Antarctic notothenioid fish are characterized by their evolutionary adaptation to the cold, thermostable Southern Ocean, which is associated with unique physiological adaptations to withstand the cold and reduce energetic requirements but also entails limited compensation capacities to environmental change. This study compares the capacities of mitochondrial acclimation to ocean warming and acidification between the Antarctic nototheniid Notothenia rossii and the sub-Antarctic Lepidonotothen squamifrons, which share a similar ecology, but different habitat temperatures. After acclimation of L. squamifrons to 9°C and N. rossii to 7°C (normocapnic/hypercapnic, 0.2 kPa CO2/2000 ppm CO2) for 4-6 weeks, we compared the capacities of their mitochondrial respiratory complexes I (CI) and II (CII), their P/O ratios (phosphorylation efficiency), proton leak capacities and mitochondrial membrane fatty acid compositions. Our results reveal reduced CII respiration rates in warm-acclimated L. squamifrons and cold hypercapnia-acclimated N. rossii. Generally, L. squamifrons displayed a greater ability to increase CI contribution during acute warming and after warm-acclimation than N. rossii. Membrane unsaturation was not altered by warm or hypercapnia-acclimation in both species, but membrane fatty acids of warm-acclimated L. squamifrons were less saturated than in warm normocapnia-/hypercapnia-acclimated N. rossii. Proton leak capacities were not affected by warm or hypercapnia-acclimation of N. rossii. We conclude that an acclimatory response of mitochondrial capacities may include higher thermal plasticity of CI supported by enhanced utilization of anaplerotic substrates (via oxidative decarboxylation reactions) feeding into the citrate cycle. L. squamifrons possesses higher relative CI plasticities than N. rossii, which may facilitate the usage of energy efficient NADH-related substrates under conditions of elevated energy demand, possibly induced by ocean warming and acidification. The observed adjustments of electron transport system complexes with a higher flux through CI under warming and acidification suggest a metabolic acclimation potential of the sub-Antarctic L. squamifrons, but only limited acclimation capacities for N. rossii.

Foliar carbon dynamics of piñon and juniper in response to experimental drought and heat

NASA Astrophysics Data System (ADS)

Collins, A.; Ryan, M. G.; Adams, H. D.; Dickman, L. T.; Garcia-Forner, N.; Grossiord, C.; Powers, H. H.; Sevanto, S.; McDowell, N. G.

2015-12-01

Plant respiration (R) is generally well-coupled with temperature and in the absence of thermal acclimation, respiration is expected to increase as climate change brings higher temperatures. Increased drought is also predicted for future climate, which could drive respiration higher if the carbon (C) cost to maintain tissues (Rm) or grow increases, or lower if substrate or other factors become limiting. We examined the effects of temperature and drought on R as well as photosynthesis, growth, and carbohydrate storage of mature individuals of two co-dominant tree species. Three mature, in-situ piñon (Pinus edulis) and juniper (Juniperus monosperma) trees were assigned to each of the following treatments: +4.8 °C; 45% reduced precipitation; a combination of both (heat + drought); along with ambient control and treatment controls. Rm measured prior to foliar and twig growth was far more sensitive to drought in piñon, and heat in juniper. Total respiration (Rt, R not partitioned) acclimated to temperature in piñon such that elevated temperature had minimal impacts on Rt; however, juniper exhibited higher Rt with elevated temperature, thus juniper did not display any thermal acclimation. Rt in both species was weakly associated with temperature, but strongly correlated with pre-dawn water potential, photosynthetic assimilation (A) rates, and in piñon, foliar carbohydrates. For both species, heat caused far more days where A-R was negative than did drought. The consequences of drought alone and heat alone in piñon included higher Rt per unit growth, indicating that each abiotic stress forces a greater allocation of Rt to maintenance costs, and both drought + heat in combination results in far fewer days that foliar carbohydrates could sustain R in both species. Notably, the much higher A and R of juniper than piñon is consistent with predicted superior carbon budget regulation of juniper than piñon during drought; however, juniper's lack of temperature acclimation in contrast to piñon suggests climate warming may have a greater deleterious impact on juniper carbon balance than piñon.

Effects of acclimation temperature on thermal tolerance, locomotion performance and respiratory metabolism in Acheta domesticus L. (Orthoptera: Gryllidae).

PubMed

Lachenicht, M W; Clusella-Trullas, S; Boardman, L; Le Roux, C; Terblanche, J S

2010-07-01

The effects of acclimation temperature on insect thermal performance curves are generally poorly understood but significant for understanding responses to future climate variation and the evolution of these reaction norms. Here, in Acheta domesticus, we examine the physiological effects of 7-9 days acclimation to temperatures 4 degrees C above and below optimum growth temperature of 29 degrees C (i.e. 25, 29, 33 degrees C) for traits of resistance to thermal extremes, temperature-dependence of locomotion performance (jumping distance and running speed) and temperature-dependence of respiratory metabolism. We also examine the effects of acclimation on mitochondrial cytochrome c oxidase (CCO) enzyme activity. Chill coma recovery time (CRRT) was significantly reduced from 38 to 13min with acclimation at 33-25 degrees C, respectively. Heat knockdown resistance was less responsive than CCRT to acclimation, with no significant effects of acclimation detected for heat knockdown times (25 degrees C: 18.25, 29 degrees C: 18.07, 33 degrees C: 25.5min). Thermal optima for running speed were higher (39.4-40.6 degrees C) than those for jumping performance (25.6-30.9 degrees C). Acclimation temperature affected jumping distance but not running speed (general linear model, p=0.0075) although maximum performance (U(MAX)) and optimum temperature (T(OPT)) of the performance curves showed small or insignificant effects of acclimation temperature. However, these effects were sensitive to the method of analysis since analyses of T(OPT), U(MAX) and the temperature breadth (T(BR)) derived from non-linear curve-fitting approaches produced high inter-individual variation within acclimation groups and reduced variation between acclimation groups. Standard metabolic rate (SMR) was positively related to body mass and test temperature. Acclimation temperature significantly influenced the slope of the SMR-temperature reaction norms, whereas no variation in the intercept was found. The CCO enzyme activity remained unaffected by thermal acclimation. Finally, high temperature acclimation resulted in significant increases in mortality (60-70% at 33 degrees C vs. 20-30% at 25 and 29 degrees C). These results suggest that although A. domesticus may be able to cope with low temperature extremes to some degree through phenotypic plasticity, population declines with warmer mean temperatures of only a few degrees are likely owing to the limited plasticity of their performance curves. Copyright 2010 Elsevier Ltd. All rights reserved.

Leaf Respiratory Acclimation: Magnitude of Acclimation to the Long-term Warming in Tallgrass Prairie

NASA Astrophysics Data System (ADS)

Jung, C. G.; Peng, F.; Luo, Y.

2016-12-01

Plant respiration has a positive response with temperature; hence, the plant under warmer climate makes plant releases more CO2. However, plant leaf can acclimate to the warmer climate so that plant leaf respiratory acclimation contributes less positive feedback between climate warming and ecosystem CO2 release. In order to examine the feedback between ecosystem and evolution of carbon dioxide due to global warming, we conducted the experiment of warming and clipping as mimicking grazing effect in a tall grass prairie in central Oklahoma, US since November 1999. The warming plot's air and soil temperature show 1.1 °C and 2.3 °C higher than ambient, respectively. Since our experiment has been over 16 years, the plot's species compositions and plant richness have changed so far. Most species composition events occurred at the clipping plot; therefore, we selected the plants within unclipped plots to see whether plants that exposed long-term warming, play a role of thermal acclimation and how those major plant species across experimental site possess difference magnitude of acclimation. We have investigated five species, one legume, one forb, and three of C4 grass: Illinois bundle (Desmanthus illinoensis, C3), stiff goldenrod (Solidago rigida, C3), King Ranch bluestem (Bothriochloa ischaemum, C4), Indian grass (Sorghastrum nutans, C4), and Little bluestem (Schizachyrium scoparium, C4). Data has collected from May as the first month of growing season in our field site in 2016. In our results, measurements in +2 °C warming show strong acclimation across the species (185% ±41% s.e.m. among species). The strongest acclimation occurred by stiff goldenrod (309%). The lowest acclimation rate is 51% in Illinois bundle, as well as the partial acclimation. The other three C4 grass species have 188% acclimation rate (±37% s.e.m. among species). Whether different plant species have a different capability of acclimation or respond through different way as shown various magnitudes, our results provide strong evidence for plant leaf thermal acclimation and its actual degree in the experimental warmed tall grass prairie. Further analysis will distinguish the plant species into the different type of acclimation; furthermore, our results can contribute a precise prediction of terrestrial feedback.

A survey of the governance capacity of national public health associations to enhance population health.

PubMed

Chauvin, James; Shukla, Mahesh; Rice, James; Rispel, Laetitia

2016-03-11

National public health associations (PHAs) are key partners with governments and communities to improve, protect and promote the public's health. Governance and organizational capacity are among the key determinants of a PHA's effectiveness as an advocate for appropriate public health policies and practice. During 2014, the World Federation of Public Health Associations (WFPHA) conducted an on-line survey of its 82 PHA members, to identify the state of organizational governance of national public health associations, as well as the factors that influence optimal organizational governance. The survey consisted of 13 questions and focused on the main elements of organizational governance: cultivating accountability; engaging stakeholders; setting shared direction; stewarding resources; and, continuous governance enhancement. Four questions included a qualitative open-ended response for additional comments. The survey data were analyzed using Microsoft Excel. The qualitative data was analyzed using thematic content analysis Responses were received from 62 PHAs, constituting a 75.6 % response rate. The two most important factors that support governance effectiveness were a high degree of integrity and ethical behavior of the PHA's leaders (77 %) and the competence of people serving on the PHA's governing body (76 %). The lack of financial resources was considered as the most important factor that negatively affected organizational governance effectiveness (73 %). The lack of mentoring for future PHA leaders; ineffective or incompetent leadership; lack of understanding about good governance practices; and lack of accurate information for strategic planning were identified as factors influencing PHA governance effectiveness. Critical elements for PHA sustainability included diversity, gender-responsiveness and inclusive governance practices, and strategies to build the future generation of public health leaders. National PHA have a responsibility to put into place the practices and infrastructure that enhance organizational governance. This will enhance their ability to be effective advocates for policies and practices that enhance, protect and promote the public's health. The WFPHA has an important role to play in providing the technical assistance and financial resources to assist PHAs in attaining and sustaining a higher level of governance capacity.

The bioimpedance phase angle predicts low muscle strength, impaired quality of life, and increased mortality in old patients with cancer.

PubMed

Norman, Kristina; Wirth, Rainer; Neubauer, Maxi; Eckardt, Rahel; Stobäus, Nicole

2015-02-01

We investigated the impact of low phase angle (PhA) values on muscle strength, quality of life, symptom severity, and 1-year mortality in older cancer patients. Prospective study with 1-year follow-up. Cancer patients aged >60 years. PhA was derived from whole body impedance analysis. The fifth percentile of age-, sex-, and body mass index-stratified reference values were used as cut-off. Quality of life was determined with the European Organization of Research and Treatment in Cancer questionnaire, reflecting both several function scales and symptom severity. Muscle strength was assessed by hand grip strength, knee extension strength, and peak expiratory flow. 433 cancer patients, aged 60-95 years, were recruited. Patients with low PhA (n = 197) exhibited decreased muscle strength compared with patients with normal PhA (hand grip strength: 22 ± 8.6 vs 28.9 ± 8.9 kg, knee extension strength: 20.8 ± 11.8 vs 28.1 ± 14.9 kg, and peak expiratory flow: 301.1 ± 118 vs 401.7 ± 142.6 L/min, P < .001). Physical function, global health status, and role function from the European Organization of Research and Treatment in Cancer questionnaire were reduced, and most symptoms (fatigue, anorexia, pain, and dyspnea) increased in patients with low PhA (P < .001). In a risk-factor adjusted regression analysis, PhA emerged as independent predictor of physical function (ß:-0.538, P = .023), hand grip strength (ß:-4.684, P < .0001), knee extension strength (ß:-4.548, P = .035), and peak expiratory flow (ß:-66.836, P < .0001). Low PhA moreover predicted 1-year mortality in the Cox proportional hazards regression model, whereas grip strength was no longer significant. PhA below the fifth reference percentile is highly predictive of decreased muscle strength, impaired quality of life, and increased mortality in old patients with cancer and should be evaluated in routine assessment. Copyright © 2015 AMDA – The Society for Post-Acute and Long-Term Care Medicine. Published by Elsevier Inc. All rights reserved.

Differences in the metabolic response to temperature acclimation in nine-spined stickleback (Pungitius pungitius) populations from contrasting thermal environments.

PubMed

Bruneaux, Matthieu; Nikinmaa, Mikko; Laine, Veronika N; Lindström, Kai; Primmer, Craig R; Vasemägi, Anti

2014-12-01

Metabolic responses to temperature changes are crucial for maintaining the energy balance of an individual under seasonal temperature fluctuations. To understand how such responses differ in recently isolated populations (<11,000 years), we studied four Baltic populations of the nine-spined stickleback (Pungitius pungitius L.) from coastal locations (seasonal temperature range, 0-29°C) and from colder, more thermally stable spring-fed ponds (1-19°C). Salinity and predation pressure also differed between these locations. We acclimatized wild-caught fish to 6, 11, and 19°C in common garden conditions for 4-6 months and determined their aerobic scope and hepatosomatic index (HSI). The freshwater fish from the colder (2-14°C), predator-free pond population exhibited complete temperature compensation for their aerobic scope, whereas the coastal populations underwent metabolic rate reduction during the cold treatment. Coastal populations had higher HSI than the colder pond population at all temperatures, with cold acclimation accentuating this effect. The metabolic rates and HSI for freshwater fish from the pond with higher predation pressure were more similar to those of the coastal ones. Our results suggest that ontogenic effects and/or genetic differentiation are responsible for differential energy storage and metabolic responses between these populations. This work demonstrates the metabolic versatility of the nine-spined stickleback and the pertinence of an energetic framework to better understand potential local adaptations. It also demonstrates that instead of using a single acclimation temperature thermal reaction norms should be compared when studying individuals originating from different thermal environments in a common garden setting. © 2014 Wiley Periodicals, Inc.

The effect of cold acclimation on active ion transport in cricket ionoregulatory tissues.

PubMed

Des Marteaux, Lauren E; Khazraeenia, Soheila; Yerushalmi, Gil Y; Donini, Andrew; Li, Natalia G; Sinclair, Brent J

2018-02-01

Cold-acclimated insects defend ion and water transport function during cold exposure. We hypothesized that this is achieved via enhanced active transport. The Malpighian tubules and rectum are likely targets for such transport modifications, and recent transcriptomic studies indicate shifts in Na + -K + ATPase (NKA) and V-ATPase expression in these tissues following cold acclimation. Here we quantify the effect of cold acclimation (one week at 12°C) on active transport in the ionoregulatory organs of adult Gryllus pennsylvanicus field crickets. We compared primary urine production of warm- and cold-acclimated crickets in excised Malpighian tubules via Ramsay assay at a range of temperatures between 4 and 25°C. We then compared NKA and V-ATPase activities in Malpighian tubule and rectal homogenates from warm- and cold-acclimated crickets via NADH-linked photometric assays. Malpighian tubules of cold-acclimated crickets excreted fluid at lower rates at all temperatures compared to warm-acclimated crickets. This reduction in Malpighian tubule excretion rates may be attributed to increased NKA activity that we observed for cold-acclimated crickets, but V-ATPase activity was unchanged. Cold acclimation had no effect on rectal NKA activity at either 21°C or 6°C, and did not modify rectal V-ATPase activity. Our results suggest that an overall reduction, rather than enhancement of active transport in the Malpighian tubules allows crickets to maintain hemolymph water balance during cold exposure, and increased Malpighian tubule NKA activity may help to defend and/or re-establish ion homeostasis. Copyright © 2017 Elsevier Inc. All rights reserved.

Development and characterization of porous silver-incorporated hydroxyapatite ceramic for separation and elimination of microorganisms.

PubMed

Yang, Lei; Ning, Xiaoshan; Xiao, Qunfang; Chen, Kexin; Zhou, Heping

2007-04-01

A novel filter material for separating and eliminating microorganisms in water and gas was fabricated by incorporating silver ions into porous hydroxyapatite (HA) ceramics prepared by a starch additive technique. The porous ceramics reveal a microstructure of both large and small pores. Microorganism separating and eliminating properties of the porous silver-incorporated HA ceramics (PHA-Ag) were investigated by bacterial and viral filtration tests. The PHA-Ag demonstrated excellent separating and antibacterial effects on Escherichia coli and the mechanisms were studied. Adsorption of bacterial cells to the HA and the barricading effect of small pores contribute to the separating property of PHA-Ag, while the Ag+ ions equip the ceramics with antibacterial property. Furthermore, the PHA-Ag exhibited an observable virus-eliminating property and its probable mechanism was also discussed. (c) 2006 Wiley Periodicals, Inc.

Novel extracellular medium-chain-length polyhydroxyalkanoate depolymerase from Streptomyces exfoliatus K10 DSMZ 41693: a promising biocatalyst for the efficient degradation of natural and functionalized mcl-PHAs.

PubMed

Martínez, Virginia; de Santos, Patricia Gómez; García-Hidalgo, Javier; Hormigo, Daniel; Prieto, M Auxiliadora; Arroyo, Miguel; de la Mata, Isabel

2015-11-01

Cloning and biochemical characterization of a novel extracellular medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase from Streptomyces exfoliatus K10 DSMZ 41693 are described. The primary structure of the depolymerase (PhaZSex2) includes the lipase consensus sequence (serine-histidine-aspartic acid) which is known for serine hydrolases. Secondary structure analysis shows 7.9 % α-helix, 43.9 % β-sheet, 19.4 % β-turns, and 31.2 % random coil, suggesting that this enzyme belongs to the α/β hydrolase fold family, in agreement with other PHA depolymerases and lipases. The enzyme was efficiently produced as an extracellular active form in Rhodococcus and purified by two consecutive hydrophobic chromatographic steps. Matrix-assisted laser desorption-time-of-flight (MALDI-TOF) analysis of the purified enzyme revealed a monomer of 27.6 kDa with a midpoint transition temperature of 44.2 °C. Remarkably, the activity is significantly enhanced by low concentrations of nonionic and anionic detergents and thermal stability is improved by the presence of 10 % glycerol. PhaZSex2 is an endo-exohydrolase that cleaves both large and small PHA molecules, producing (R)-3-hydroxyoctanoic acid monomers as the main reaction product. Markedly, PhaZSex2 is able to degrade functionalized polymers containing thioester groups in the side chain (PHACOS), releasing functional thioester-based monomers and oligomers demonstrating the potentiality of this novel biocatalyst for the industrial production of enantiopure (R)-3-hydroxyalkanoic acids.

Development of Iron-Chelating Poly(ethylene terephthalate) Packaging for Inhibiting Lipid Oxidation in Oil-in-Water Emulsions.

PubMed

Johnson, David R; Tian, Fang; Roman, Maxine J; Decker, Eric A; Goddard, Julie M

2015-05-27

Foods such as bulk oils, salad dressings, and nutritionally fortified beverages that are susceptible to oxidative degradation are often packaged in poly(ethylene terephthalate) (PET) bottles with metal chelators added to the food to maintain product quality. In the present work, a metal-chelating active packaging material is designed and characterized, in which poly(hydroxamic acid) (PHA) metal-chelating moieties were grafted from the surface of PET. Biomimetic PHA groups were grafted in a two-step UV-initiated process without the use of a photoinitiator. Surface characterization of the films by attenuated total reflective Fourier transform infrared spectroscopy (ATR-FTIR) and scanning electron microscopy (SEM) suggested successful grafting and conversion of poly(hydroxyethyl acrylate) (PHEA) to PHA chelating moieties from the surface of PET. Colorimetric (ferrozine) and inductively coupled plasma mass spectroscopy (ICP-MS) assays demonstrated the ability of PET-g-PHA to chelate iron in a low-pH (3.0) environment containing a competitive metal chelator (citric acid). Lipid oxidation studies demonstrated the antioxidant activity of PET-g-PHA films in inhibiting iron-promoted oxidation in an acidified oil-in-water (O/W) emulsion model system (pH 3.0). Particle size and ζ-potential analysis indicated that the addition of PET-g-PHA films did not affect the physical stability of the emulsion system. This work suggests that biomimetic chelating moieties can be grafted from PET and effectively inhibit iron-promoted degradation reactions, enabling removal of metal-chelating additives from product formulations.

Correlating interleukin-12 stimulated interferon-γ production and the absence of ectodermal dysplasia and anhidrosis (EDA) in patients with mutations in NF-κB essential modulator (NEMO).

PubMed

Haverkamp, Margje H; Marciano, Beatriz E; Frucht, David M; Jain, Ashish; van de Vosse, Esther; Holland, Steven M

2014-05-01

Patients with hypomorphic mutations in Nuclear Factor-κB Essential Modulator (NEMO) are immunodeficient (ID) and most display ectodermal dysplasia and anhidrosis (EDA). We compared cytokine production by NEMO-ID patients with and without EDA. PBMCs of NEMO-ID patients, four with EDA carrying E315A, C417R, D311N and Q403X, and three without EDA carrying E315A, E311_L333del and R254G, were cultured with PHA, PHA plus IL-12p70, LPS, LPS plus IFN-γ, TNF and IL-1β. The production of various cytokines was measured in the supernatants. Fifty-nine healthy individuals served as controls. PBMCs of NEMO-ID patients without EDA produce subnormal amounts of IFN-γ after stimulation with PHA, but normal amounts of IFN-γ after PHA plus IL-12p70. In contrast, IFN-γ production by patients with EDA was low in both cases. Patients with EDA also generate lower PHA-stimulated IL-10 and IL-1β than controls, whereas the production of these cytokines by patients without EDA was normal. Responses of PBMCs in NEMO-ID patients with EDA to PHA with and without IL-12p70 appear less robust than in NEMO-ID patients without EDA. This possibly indicates a better preserved NEMO function in our patients without EDA.

Functional redundancy ensures performance robustness in 3-stage PHA-producing mixed cultures under variable feed operation.

PubMed

Carvalho, Gilda; Pedras, Inês; Karst, Soren M; Oliveira, Catarina S S; Duque, Anouk F; Nielsen, Per H; Reis, Maria A M

2018-01-25

Polyhydroxyalkanoates (PHA) are biopolymers that can be produced by mixed microbial cultures using wastes or industrial by-products, which represent an economical and environmental advantage over pure culture processes. The use of alternate feedstocks enables using seasonal by-products, providing that the process is resilient to transient conditions. The mixed microbial communities of a 3-stage PHA producing system fed initially with molasses and then cheese whey were investigated through amplicon sequencing of the 16S rRNA gene. The transition in feedstock resulted in an adaptation of the acidogenic community, where Actinobacteria dominated with sugarcane molasses (up to 93% of the operational taxonomic units) and Firmicutes, with cheese whey (up to 97%). The resulting fermentation products profile also changed, with a higher fraction of HV precursors obtained with molasses than cheese whey (7.1±0.5 and 1.7±0.7 gCOD/L, respectively). As for the PHA storing culture, the genera Azoarcus, Thauera and Paracoccus were enriched with fermented molasses (average 89% of Bacteria). Later, fermented cheese whey fostered a higher diversity, including some less characterised PHA-storers such as the genera Paenibacillus and Lysinibacillus. Although the microbial community structure was significantly affected by the feedstock shift, the acidogenic and PHA storing performance of the 3-stage system was very similar once a pseudo steady state was attained, showing that a reliable level of functional redundancy was attained in both mixed cultures. Copyright © 2017 Elsevier B.V. All rights reserved.

Acclimation to Low Level Exposure of Copper in Bufo arenarum Embryos: Linkage of Effects to Tissue Residues

PubMed Central

Herkovits, Jorge; Pérez-Coll, Cristina Silvia

2007-01-01

The acclimation possibilities to copper in Bufo arenarum embryos was evaluated by means of three different low level copper exposure conditions during 14 days. By the end of the acclimation period the copper content in control embryos was 1.04 ± 0.09 μg.g−1 (wet weight) while in all the acclimated embryos a reduction of about 25% of copper was found. Thus copper content could be considered as a biomarker of low level exposure conditions. Batches of 10 embryos (by triplicate) from each acclimation condition were challenged with three different toxic concentrations of copper. As a general pattern, the acclimation protocol to copper exerted a transient beneficial effect on the survival of the Bufo arenarum embryos. The acclimation phenomenon could be related to the selection of pollution tolerant organisms within an adaptive process and therefore the persistence of information within an ecological system following a toxicological stressor. PMID:17617681

Effect of culture residence time on substrate uptake and storage by a pure culture of Thiothrix (CT3 strain) under continuous or batch feeding.

PubMed

Valentino, Francesco; Beccari, Mario; Villano, Marianna; Tandoi, Valter; Majone, Mauro

2017-05-25

A pure culture of the filamentous bacterium Thiothrix, strain CT3, was aerobically cultured in a chemostat under continuous acetate feeding at three different culture residence times (RT 6, 12 or 22 d) and the same volumetric organic load rate (OLR 0.12gCOD/L/d). Cells cultured at decreasing RT in the chemostat had an increasing transient response to acetate spikes in batch tests. The maximum specific acetate removal rate increased from 25 to 185mgCOD/gCOD/h, corresponding to a 1.8 to 8.1 fold higher respective steady-state rate in the chemostat. The transient response was mainly due to acetate storage in the form of poly(3-hydroxybutyrate) (PHB), whereas no growth response was observed at any RT. Interestingly, even though the storage rate also decreased as the RT increased, the storage yield increased from 0.41 to 0.50 COD/COD. This finding does not support the traditional view that storage plays a more important role as the transient response increases. The transient response of the steady-state cells was much lower than in cells cultured under periodic feeding (at 6 d RT, from 82 to 247mgCOD/gCOD/h), with the latter cells showing both storage and growth responses. On the other hand, even though steady-state cells had no growth response and their storage rate was also less, steady-state cells showed a higher storage yield than cells cultured under dynamic feeding. This suggests that in Thiothrix strain CT3, the growth response is triggered by periodic feeding, whereas the storage response is a constitutive mechanism, independent from previous acclimation to transient conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

PHA665752, a small-molecule inhibitor of c-Met, inhibits hepatocyte growth factor-stimulated migration and proliferation of c-Met-positive neuroblastoma cells.

PubMed

Crosswell, Hal E; Dasgupta, Anindya; Alvarado, Carlos S; Watt, Tanya; Christensen, James G; De, Pradip; Durden, Donald L; Findley, Harry W

2009-11-25

c-Met is a tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF), and both c-Met and its ligand are expressed in a variety of tissues. C-Met/HGF/SF signaling is essential for normal embryogenesis, organogenesis, and tissue regeneration. Abnormal c-Met/HGF/SF signaling has been demonstrated in different tumors and linked to aggressive and metastatic tumor phenotypes. In vitro and in vivo studies have demonstrated inhibition of c-Met/HGF/SF signaling by the small-molecule inhibitor PHA665752. This study investigated c-Met and HGF expression in two neuroblastoma (NBL) cell lines and tumor tissue from patients with NBL, as well as the effects of PHA665752 on growth and motility of NBL cell lines. The effect of the tumor suppressor protein PTEN on migration and proliferation of tumor cells treated with PHA665752 was also evaluated. Expression of c-Met and HGF in NBL cell lines SH-EP and SH-SY5Y and primary tumor tissue was assessed by immunohistochemistry and quantitative RT-PCR. The effect of PHA665752 on c-Met/HGF signaling involved in NBL cell proliferation and migration was evaluated in c-Met-positive cells and c-Met-transfected cells. The transwell chemotaxis assay and the MTT assay were used to measure migration and proliferation/cell-survival of tumor cells, respectively. The PPAR-gamma agonist rosiglitazone was used to assess the effect of PTEN on PHA665752-induced inhibition of NBL cell proliferation/cell-survival and migration High c-Met expression was detected in SH-EP cells and primary tumors from patients with advanced-stage disease. C-Met/HGF signaling induced both migration and proliferation of SH-EP cells. Migration and proliferation/cell-survival were inhibited by PHA665752 in a dose-dependent manner. We also found that induced overexpression of PTEN following treatment with rosiglitazone significantly enhanced the inhibitory effect of PHA665752 on NBL-cell migration and proliferation. c-Met is highly expressed in most tumors from patients with advanced-stage, metastatic NBL. Furthermore, using the NBL cell line SH-EP as a model, PHA665752 was shown to inhibit cMet/HGF/SF signaling in vitro, suggesting c-Met inhibitors may have efficacy for blocking local progression and/or metastatic spread of c-Met-positive NBL in vivo. These are novel findings for this disease and suggest that further studies of agents targeting the c-Met/HGF axis in NBL are warranted.

Sweating responses during heat acclimation and moderate conditioning

NASA Technical Reports Server (NTRS)

Shvartz, E.; Bhattacharya, A.; Sperinde, S. J.; Brock, P. J.; Sciaraffa, D.; Van Beaumont, W.

1979-01-01

Experiments were conducted on ten young male subjects to determine sweating onset, distribution, and patterns as well as the relationships of these responses to body temperature during heat acclimation and moderate conditioning performed in temperate (24 C) conditions. The subjects are randomly assigned to two groups of five subjects each. The experimental period consisted of eight successive days of either graded exercise to exhaustion on a bicycle ergometer in heat (acclimation group) or in a temperate environment (control group). Major conclusions are that (1) acclimation and conditioning result in relatively more sweat rate on the limbs than on the torso, but that these changes are less related to body temperature than torso sweat rate; and (2) sweating sensitivity increases during acclimation and conditioning, but its contribution to heat acclimation is minor.

Metabolic Cost of the Activation of Immune Response in the Fish-Eating Myotis (Myotis vivesi): The Effects of Inflammation and the Acute Phase Response

PubMed Central

Otálora-Ardila, Aída; Herrera M., L. Gerardo; Flores-Martínez, José Juan; Welch, Kenneth C.

2016-01-01

Inflammation and activation of the acute phase response (APR) are energetically demanding processes that protect against pathogens. Phytohaemagglutinin (PHA) and lipopolysaccharide (LPS) are antigens commonly used to stimulate inflammation and the APR, respectively. We tested the hypothesis that the APR after an LPS challenge was energetically more costly than the inflammatory response after a PHA challenge in the fish-eating Myotis bat (Myotis vivesi). We measured resting metabolic rate (RMR) after bats were administered PHA and LPS. We also measured skin temperature (Tskin) after the LPS challenge and skin swelling after the PHA challenge. Injection of PHA elicited swelling that lasted for several days but changes in RMR and body mass were not significant. LPS injection produced a significant increase in Tskin and in RMR, and significant body mass loss. RMR after LPS injection increased by 140–185% and the total cost of the response was 6.50 kJ. Inflammation was an energetically low-cost process but the APR entailed a significant energetic investment. Examination of APR in other bats suggests that the way in which bats deal with infections might not be uniform. PMID:27792729

Metabolic Cost of the Activation of Immune Response in the Fish-Eating Myotis (Myotis vivesi): The Effects of Inflammation and the Acute Phase Response.

PubMed

Otálora-Ardila, Aída; Herrera M, L Gerardo; Flores-Martínez, José Juan; Welch, Kenneth C

2016-01-01

Inflammation and activation of the acute phase response (APR) are energetically demanding processes that protect against pathogens. Phytohaemagglutinin (PHA) and lipopolysaccharide (LPS) are antigens commonly used to stimulate inflammation and the APR, respectively. We tested the hypothesis that the APR after an LPS challenge was energetically more costly than the inflammatory response after a PHA challenge in the fish-eating Myotis bat (Myotis vivesi). We measured resting metabolic rate (RMR) after bats were administered PHA and LPS. We also measured skin temperature (Tskin) after the LPS challenge and skin swelling after the PHA challenge. Injection of PHA elicited swelling that lasted for several days but changes in RMR and body mass were not significant. LPS injection produced a significant increase in Tskin and in RMR, and significant body mass loss. RMR after LPS injection increased by 140-185% and the total cost of the response was 6.50 kJ. Inflammation was an energetically low-cost process but the APR entailed a significant energetic investment. Examination of APR in other bats suggests that the way in which bats deal with infections might not be uniform.

Isolation of a Gram-positive poly(3-hydroxybutyrate) (PHB)-degrading bacterium from compost, and cloning and characterization of a gene encoding PHB depolymerase of Bacillus megaterium N-18-25-9.

PubMed

Takaku, Hiroaki; Kimoto, Ayumi; Kodaira, Shoko; Nashimoto, Masayuki; Takagi, Masamichi

2006-11-01

A Gram-positive poly(3-hydroxybutyrate) (PHB)-degrading bacterial strain was isolated from compost. This organism, identified as Bacillus megaterium N-18-25-9, produced a clearing zone on opaque NB-PHB agar, indicating the presence of extracellular PHB depolymerase. A PHB depolymerase gene, PhaZ(Bm), of B. megaterium N-18-25-9 was cloned and sequenced, and the recombinant gene product was purified from Escherichia coli. The N-terminal half region of PhaZ(Bm) shared significant homologies with a catalytic domain of other PHB depolymerases. Although the C-terminal half region of PhaZ(Bm) showed no significant similarity with those of other PHB depolymerases, that region was necessary for the PHB depolymerase activity. Therefore, this enzyme's domain structure is unique among extracellular PHB depolymerase domain structures. The addition of PHB to the medium led to a sixfold increase in PhaZ(Bm) mRNA, while the presence of glucose repressed PhaZ(Bm) expression. The maximum activity was observed at pH 9.0 at 65 degrees C.

Syngas obtained by microwave pyrolysis of household wastes as feedstock for polyhydroxyalkanoate production in Rhodospirillum rubrum.

PubMed

Revelles, Olga; Beneroso, Daniel; Menéndez, J Angel; Arenillas, Ana; García, J Luis; Prieto, M Auxiliadora

2017-11-01

The massive production of urban and agricultural wastes has promoted a clear need for alternative processes of disposal and waste management. The potential use of municipal solid wastes (MSW) as feedstock for the production of polyhydroxyalkanoates (PHA) by a process known as syngas fermentation is considered herein as an attractive bio-economic strategy to reduce these wastes. In this work, we have evaluated the potential of Rhodospirillum rubrum as microbial cell factory for the synthesis of PHA from syngas produced by microwave pyrolysis of the MSW organic fraction from a European city (Seville). Growth rate, uptake rate, biomass yield and PHA production from syngas in R. rubrum have been analysed. The results revealed the strong robustness of this syngas fermentation where the purity of the syngas is not a critical constraint for PHA production. Microwave-induced pyrolysis is a tangible alternative to standard pyrolysis, because it can reduce cost in terms of energy and time as well as increase syngas production, providing a satisfactory PHA yield. © 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Comparative Quantitative Assessments of Salivary Ion Activity Product for Hydroxyapatite and Buffering Capacity in Children with Different Caries Experience.

PubMed

Anand, Siddharth; Masih, Updesh; Yeluri, Ramakrishna

If a relation exists between salivary I PHA , buffer capacity and caries experience, then this relationship could be used as screening chair side test for caries risk assessment. One hundred ninety seven children aged 4 to 6 years were examined. Data was collected by interview and clinical examination. They were divided into low, moderate and high caries experience group of 20 children each. Two ml of each sample was used to measure the pH value with pH meter. Regarding the buffering capacity, freshly prepared hydrochloric acid (HCl) was titrated into saliva and pH was recorded. The collected saliva samples were sent to Laboratory for measurement of calcium and phosphorus. I PHA was calculated and the negative logarithms of I PHA were used to determine the enamel solubility. The correlation between salivary I PHA , buffering capacity and caries experience were evaluated. There was a significant relation between pH, log I PHA and dental caries experience, it could be considered as a predictor of dental caries. pH measurement after HCl titration in saliva could be used as chair side screening test for the assessment of caries risk.

[Adenosine triphosphatase activity in the organs of the crab Hemigrapsus sanguineus, acclimated to sea water of different salinity].

PubMed

Busev, V M

1977-01-01

In crabs acclimated to low salinity, the activity of Na, K-ATPase from the gills increases; the activity also increases in the antennal glands after acclimation of the animals to high salinity. The activity of Na, K-ATPase in the abdominal ganglion and in the heart does not depend on the salinity to which crabs had been acclimated. Changes in the activity of Mg-ATPase in the gills and antennal glands associated with acclimation of crabs to sea water with different salinity correspond to those in the activity of Na, K-ATPase.

24 CFR 966.56 - Procedures governing the hearing.

Code of Federal Regulations, 2010 CFR

2010-04-01

... controvert evidence relied on by the PHA or project management, and to confront and cross-examine all witnesses upon whose testimony or information the PHA or project management relies; and (5) A decision based...

Coping When You're Newly Diagnosed

MedlinePlus

... review Conflict of Interest Disclosures for PHA’s medical leadership, visit: Disclosures Last reviewed: April 2012 Support About ... Rare Disorders (NORD) awarded PHA the Abbey S. Meyers Leadership Award in 2012 for outstanding service to PHA ...

Coping with PH over the Long Term

MedlinePlus

... review Conflict of Interest Disclosures for PHA’s medical leadership, visit: Disclosures Last reviewed: April 2012 Support About ... Rare Disorders (NORD) awarded PHA the Abbey S. Meyers Leadership Award in 2012 for outstanding service to PHA ...

24 CFR 884.225 - PHA reporting requirements. [Reserved

Code of Federal Regulations, 2010 CFR

2010-04-01

... reporting requirements. [Reserved] ... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false PHA reporting requirements. [Reserved] 884.225 Section 884.225 Housing and Urban Development Regulations Relating to Housing and Urban...

24 CFR 983.252 - PHA information for accepted family.

Code of Federal Regulations, 2013 CFR

2013-04-01

... URBAN DEVELOPMENT PROJECT-BASED VOUCHER (PBV) PROGRAM Occupancy § 983.252 PHA information for accepted... communication, in accordance with 24 CFR 8.6, in conducting the oral briefing and in providing the written...

24 CFR 983.252 - PHA information for accepted family.

Code of Federal Regulations, 2012 CFR

2012-04-01

... URBAN DEVELOPMENT PROJECT-BASED VOUCHER (PBV) PROGRAM Occupancy § 983.252 PHA information for accepted... communication, in accordance with 24 CFR 8.6, in conducting the oral briefing and in providing the written...

24 CFR 983.252 - PHA information for accepted family.

Code of Federal Regulations, 2014 CFR

2014-04-01

... URBAN DEVELOPMENT PROJECT-BASED VOUCHER (PBV) PROGRAM Occupancy § 983.252 PHA information for accepted... communication, in accordance with 24 CFR 8.6, in conducting the oral briefing and in providing the written...

Enhanced poly(3-hydroxybutyrate) production in transgenic tobacco BY-2 cells using engineered acetoacetyl-CoA reductase.

PubMed

Yokoo, Toshinori; Matsumoto, Ken'ichiro; Ooba, Takashi; Morimoto, Kenjiro; Taguchi, Seiichi

2015-01-01

Highly active mutant of NADPH-dependent acetoacetyl-CoA reductase (PhaB) was expressed in Nicotiana tabacum cv. Bright Yellow-2 cultured cells to produce poly(3-hydroxybutyrate) [P(3HB)]. The mutated PhaB increased P(3HB) content by three-fold over the control, indicating that the mutant was a versatile tool for P(3HB) production. Additionally, the PhaB-catalyzed reaction was suggested to be a rate-limiting step of P(3HB) biosynthesis in tobacco BY-2 cells.

The glutathione-dependent system of antioxidant defense is not modulated by temperature acclimation in muscle tissues from striped bass, Morone saxatilis.

PubMed

Grim, Jeffrey M; Simonik, Elizabeth A; Semones, Molly C; Kuhn, Donald E; Crockett, Elizabeth L

2013-02-01

Cold temperature generally induces an enhancement of oxidative capacities, a greater content of intracellular lipids, and a remodeling of lipids in biological membranes. These physiological responses may pose a heightened risk of lipid peroxidation (LPO), while warm temperature could result in greater risk of LPO since rates involving reactive oxygen species and LPO will be elevated. The current study examines responses of the glutathione system of antioxidant defense after temperature acclimation. We measured total glutathione (tGSH), and protein levels of GPx1, GPx4, and GST (cardiac and skeletal muscles), and enzymatic activity (skeletal muscle) of glutathione-dependent antioxidants (GPx, GPx4, and GST) in tissues from striped bass (Morone saxatilis) acclimated for six weeks to 7 °C or 25 °C. tGSH of cardiac muscle from cold-acclimated animals was 1.2-times higher than in warm-bodied counterparts, but unchanged with temperature acclimation in skeletal muscle. A second low molecular weight antioxidant, ascorbate was 1.4- and 1.5-times higher in cardiac and skeletal muscle, respectively in warm- than cold-acclimated animals. Despite 1.2-times higher oxidative capacities (as indicated by citrate synthase activity), in skeletal muscle from cold- versus warm-acclimated fish, levels and activities of antioxidant enzymes were similar between acclimation groups. Lipid peroxidation products (as indicated by TBARS), normalized to tissue wet weight, were more than 2-times higher in skeletal muscle from cold- than warm-acclimated animals, however, when normalized to phospholipid content there was no statistical difference between acclimation groups. Our results demonstrate that the physiological changes, associated with acclimation to low temperature in the eurythermal striped bass, are not accompanied by an enhanced antioxidant defense in the glutathione-dependent system. Copyright © 2012 Elsevier Inc. All rights reserved.

Exercise-heat acclimation in young and older trained cyclists.

PubMed

Best, Stuart; Thompson, Martin; Caillaud, Corinne; Holvik, Liv; Fatseas, George; Tammam, Amr

2014-11-01

The purpose of this study was to investigate the effect of age on the capacity to acclimatise to exercise-heat stress. This study hypothesised that age would not affect body temperature and heat loss effector responses to short-term exercise-heat acclimation in trained subjects. Seven young subjects (19-32 years) were matched with 7 older subjects (50-63 years). Subjects were highly trained but not specifically heat acclimated when they exercised for 60 min at 70%VO2max in hot-dry (35 °C, 40%RH) and thermoneutral (20 °C, 40%RH) conditions, pre and post 6 days of exercise-heat acclimation (70%VO2max, 35 °C, 40%RH). Rectal temperature (Tr), skin temperature (Tsk), heart rate (HR), cutaneous vascular conductance (CVC) and whole body sweat loss (Msw) were measured during each testing session and Tr and HR were measured during each acclimation session. Tr, Tsk, %HRmax, CVC and Msw were similar across age groups both pre and post heat acclimation. Following heat acclimation relative decreases and increases in Tr and Msw, respectively, were similar in both subject groups. There was a significant reduction in heart rate (%HRmax) and increase in final CVC following the acclimation programme in the young group (all p < 0.05) but not the older group. When comparing young and older well trained adults we found age affected the cardiovascular adaptation but not body temperature or whole body sweat loss to exercise-heat acclimation. These data suggest age does not affect the capacity to acclimatise to exercise-heat stress in highly trained adults undergoing short-term heat acclimation. Copyright © 2013 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.

Thermal acclimation to cold alters myosin content and contractile properties of rainbow smelt, Osmerus mordax, red muscle.

PubMed

Coughlin, David J; Shiels, Lisa P; Nuthakki, Seshuvardhan; Shuman, Jacie L

2016-06-01

Rainbow smelt (Osmerus mordax), a eurythermal fish, live in environments from -1.8 to 20°C, with some populations facing substantial annual variation in environmental temperature. These different temperature regimes pose distinct challenges to locomotion by smelt. Steady swimming performance, red muscle function and muscle myosin content were examined to assess the prediction that cold acclimation by smelt will lead to improved steady swimming performance and that any performance shift will be associated with changes in red muscle function and in its myosin heavy chain composition. Cold acclimated (4°C) smelt had a faster maximum steady swimming speed and swam with a higher tailbeat frequency than warm acclimated (10°C) smelt when tested at the same temperature (10°C). Muscle mechanics experiments demonstrated faster contractile properties in the cold acclimated fish when tested at 10°C. The red muscle of cold acclimated smelt had a shorter twitch times, a shorter relaxation times and a higher maximum shortening velocity. In addition, red muscle from cold acclimated fish displayed reduced thermal sensitivity to cold, maintaining higher force levels at 4°C compared to red muscle from warm acclimated fish. Immunohistochemistry suggests shifts in muscle myosin composition and a decrease in muscle cross-sectional area with cold acclimation. Dot blot analysis confirmed a shift in myosin content. Rainbow smelt do show a significant thermal acclimation response to cold. An examination of published values of maximum muscle shortening velocity in fishes suggests that smelt are particularly well suited to high levels of activity in very cold water. Copyright © 2016 Elsevier Inc. All rights reserved.

The Rate of Seasonal Changes in Temperature Alters Acclimation of Performance under Climate Change.

PubMed

Nilsson-Örtman, Viktor; Johansson, Frank

2017-12-01

How the ability to acclimate will impact individual performance and ecological interactions under climate change remains poorly understood. Theory predicts that the benefit an organism can gain from acclimating depends on the rate at which temperatures change relative to the time it takes to induce beneficial acclimation. Here, we present a conceptual model showing how slower seasonal changes under climate change can alter species' relative performance when they differ in acclimation rate and magnitude. To test predictions from theory, we performed a microcosm experiment where we reared a mid- and a high-latitude damselfly species alone or together under the rapid seasonality currently experienced at 62°N and the slower seasonality predicted for this latitude under climate change and measured larval growth and survival. To separate acclimation effects from fixed thermal responses, we simulated growth trajectories based on species' growth rates at constant temperatures and quantified how much and how fast species needed to acclimate to match the observed growth trajectories. Consistent with our predictions, the results showed that the midlatitude species had a greater capacity for acclimation than the high-latitude species. Furthermore, since acclimation occurred at a slower rate than seasonal temperature changes, the midlatitude species had a small growth advantage over the high-latitude species under the current seasonality but a greater growth advantage under the slower seasonality predicted for this latitude under climate change. In addition, the two species did not differ in survival under the current seasonality, but the midlatitude species had higher survival under the predicted climate change scenario, possibly because rates of cannibalism were lower when smaller heterospecifics were present. These findings highlight the need to incorporate acclimation rates in ecological models.

Branchial ionocyte organization and ion-transport protein expression in juvenile alewives acclimated to freshwater or seawater

USGS Publications Warehouse

Christensen, A.K.; Hiroi, J.; Schultz, E.T.; McCormick, S.D.

2012-01-01

The alewife (Alosa pseudoharengus) is a clupeid that undergoes larval and juvenile development in freshwater preceding marine habitation. The purpose of this study was to investigate osmoregulatory mechanisms in alewives that permit homeostasis in different salinities. To this end, we measured physiological, branchial biochemical and cellular responses in juvenile alewives acclimated to freshwater (0.5p.p.t.) or seawater (35.0p.p.t.). Plasma chloride concentration was higher in seawater-acclimated than freshwater-acclimated individuals (141mmoll -1 vs 134mmoll -1), but the hematocrit remained unchanged. In seawateracclimated individuals, branchial Na +/K +-ATPase (NKA) activity was higher by 75%. Western blot analysis indicated that the abundance of the NKA subunit and a Na+/K+/2Cl- cotransporter (NKCC1) were greater in seawater-acclimated individuals by 40% and 200%, respectively. NKA and NKCC1 were localized on the basolateral surface and tubular network of ionocytes in both acclimation groups. Immunohistochemical labeling for the cystic fibrosis transmembrane conductance regulator (CFTR) was restricted to the apical crypt of ionocytes in seawater-acclimated individuals, whereas sodium/hydrogen exchanger 3 (NHE3) labeling was present on the apical surface of ionocytes in both acclimation groups. Ionocytes were concentrated on the trailing edge of the gill filament, evenly distributed along the proximal 75% of the filamental axis and reduced distally. Ionocyte size and number on the gill filament were not affected by salinity; however, the number of lamellar ionocytes was significantly lower in seawater-acclimated fish. Confocal z-series reconstructions revealed that mature ionocytes in seawater-acclimated alewives occurred in multicellular complexes. These complexes might reduce paracellular Na + resistance, hence facilitating Na+ extrusion in hypo-osmoregulating juvenile alewives after seaward migration. ?? 2012. Published by The Company of Biologists Ltd.

Short-term acclimation to warmer temperatures accelerates leaf carbon exchange processes across plant types.

PubMed

Smith, Nicholas G; Dukes, Jeffrey S

2017-11-01

While temperature responses of photosynthesis and plant respiration are known to acclimate over time in many species, few studies have been designed to directly compare process-level differences in acclimation capacity among plant types. We assessed short-term (7 day) temperature acclimation of the maximum rate of Rubisco carboxylation (V cmax ), the maximum rate of electron transport (J max ), the maximum rate of phosphoenolpyruvate carboxylase carboxylation (V pmax ), and foliar dark respiration (R d ) in 22 plant species that varied in lifespan (annual and perennial), photosynthetic pathway (C 3 and C 4 ), and climate of origin (tropical and nontropical) grown under fertilized, well-watered conditions. In general, acclimation to warmer temperatures increased the rate of each process. The relative increase in different photosynthetic processes varied by plant type, with C 3 species tending to preferentially accelerate CO 2 -limited photosynthetic processes and respiration and C 4 species tending to preferentially accelerate light-limited photosynthetic processes under warmer conditions. R d acclimation to warmer temperatures caused a reduction in temperature sensitivity that resulted in slower rates at high leaf temperatures. R d acclimation was similar across plant types. These results suggest that temperature acclimation of the biochemical processes that underlie plant carbon exchange is common across different plant types, but that acclimation to warmer temperatures tends to have a relatively greater positive effect on the processes most limiting to carbon assimilation, which differ by plant type. The acclimation responses observed here suggest that warmer conditions should lead to increased rates of carbon assimilation when water and nutrients are not limiting. © 2017 John Wiley & Sons Ltd.

Alpha-7 nicotinic acetylcholine receptor agonist treatment reduces neuroinflammation, oxidative stress, and brain injury in mice with ischemic stroke and bone fracture.

PubMed

Han, Zhenying; Li, Li; Wang, Liang; Degos, Vincent; Maze, Mervyn; Su, Hua

2014-11-01

Bone fracture at the acute stage of stroke exacerbates stroke injury by increasing neuroinflammation. We hypothesize that activation of α-7 nicotinic acetylcholine receptor (α-7 nAchR) attenuates neuroinflammation and oxidative stress, and reduces brain injury in mice with bone fracture and stroke. Permanent middle cerebral artery occlusion (pMCAO) was performed in C57BL/6J mice followed by tibia fracture 1 day later. Mice were treated with 0.8 mg/kg PHA 568487 (PHA, α-7 nAchR-specific agonist), 6 mg/kg methyllycaconitine (α-7 nAchR antagonist), or saline 1 and 2 days after pMCAO. Behavior was tested 3 days after pMCAO. Neuronal injury, CD68(+) , M1 (pro-inflammatory) and M2 (anti-inflammatory) microglia/macrophages, phosphorylated p65 component of nuclear factor kappa b in microglia/macrophages, oxidative and anti-oxidant gene expression were quantified. Compared to saline-treated mice, PHA-treated mice performed better in behavioral tests, had fewer apoptotic neurons (NeuN(+) TUNEL(+) ), fewer CD68(+) and M1 macrophages, and more M2 macrophages. PHA increased anti-oxidant gene expression and decreased oxidative stress and phosphorylation of nuclear factor kappa b p65. Methyllycaconitine had the opposite effects. Our data indicate that α-7 nAchR agonist treatment reduces neuroinflammation and oxidative stress, which are associated with reduced brain injury in mice with ischemic stroke plus tibia fracture. Bone fracture at the acute stage of stroke exacerbates neuroinflammation, oxidative stress, and brain injury, and our study has shown that the α-7 nAchR agonist, PHA (PHA 568487), attenuates neuroinflammation, oxidative stress, and brain injury in mice with stroke and bone fracture. Hence, PHA could provide an opportunity to develop a new strategy to reduce brain injury in patients suffering from stroke and bone fracture. © 2014 International Society for Neurochemistry.

The conversion of BTEX compounds by single and defined mixed cultures to medium-chain-length polyhydroxyalkanoate.

PubMed

Nikodinovic, Jasmina; Kenny, Shane T; Babu, Ramesh P; Woods, Trevor; Blau, Werner J; O'Connor, Kevin E

2008-09-01

Here, we report the use of petrochemical aromatic hydrocarbons as a feedstock for the biotechnological conversion into valuable biodegradable plastic polymers--polyhydroxyalkanoates (PHAs). We assessed the ability of the known Pseudomonas putida species that are able to utilize benzene, toluene, ethylbenzene, p-xylene (BTEX) compounds as a sole carbon and energy source for their ability to produce PHA from the single substrates. P. putida F1 is able to accumulate medium-chain-length (mcl) PHA when supplied with toluene, benzene, or ethylbenzene. P. putida mt-2 accumulates mcl-PHA when supplied with toluene or p-xylene. The highest level of PHA accumulated by cultures in shake flask was 26% cell dry weight for P. putida mt-2 supplied with p-xylene. A synthetic mixture of benzene, toluene, ethylbenzene, p-xylene, and styrene (BTEXS) which mimics the aromatic fraction of mixed plastic pyrolysis oil was supplied to a defined mixed culture of P. putida F1, mt-2, and CA-3 in the shake flasks and fermentation experiments. PHA was accumulated to 24% and to 36% of the cell dry weight of the shake flask and fermentation grown cultures respectively. In addition a three-fold higher cell density was achieved with the mixed culture grown in the bioreactor compared to shake flask experiments. A run in the 5-l fermentor resulted in the utilization of 59.6 g (67.5 ml) of the BTEXS mixture and the production of 6 g of mcl-PHA. The monomer composition of PHA accumulated by the mixed culture was the same as that accumulated by single strains supplied with single substrates with 3-hydroxydecanoic acid occurring as the predominant monomer. The purified polymer was partially crystalline with an average molecular weight of 86.9 kDa. It has a thermal degradation temperature of 350 degrees C and a glass transition temperature of -48.5 degrees C.

Phase angle is related with inflammatory and oxidative stress biomarkers in older women.

PubMed

Tomeleri, Crisieli Maria; Cavaglieri, Cláudia Regina; de Souza, Mariana Ferreira; Cavalcante, Edilaine Fungari; Antunes, Melissa; Nabbuco, Hellen Clair Garcez; Venturini, Danielle; Barbosa, Decio Sabbatini; Silva, Analiza Mônica; Cyrino, Edilson Serpeloni

2018-02-01

The aim of this study was to examine the relation between phase angle (PhA) and inflammatory and oxidative stress biomarkers in older women. One hundred and fifty-five physically independent older women participated in this study (67.7±5.7years, 27.0±4.4kg/m 2 ). Inflammatory markers included interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and acute phase reactive protein (CRP). Oxidative stress biomarkers comprised superoxide dismutase (SOD), catalase (CAT), advanced oxidation protein products (AOPP), and total radical-trapping antioxidant potential (TRAP). A spectral bioelectrical impedance device was used to estimate resistance (R) and reactance (Xc) at frequency 50kHz, and subsequently PhA was calculated as arc-tangent (Xc/R)×180°/π. The covariates appendicular lean soft-tissue (ALST), trunk fat mass, and total body fat were determined by whole-body dual-energy X-ray absorptiometry. Linear regression analysis was conducted to further test if PhA is related with the dependent variables, after adjusting for potential covariates. After controlling for the potential covariates (age, trunk fat mass, ALST, and number of diseases) PhA exhibited a significant inverse relation with IL-6 (β=-0.97; P<0.01), TNF-α (β=-0.84; P<0.01), and CRP (β=-0.58; P<0.01). Conversely, PhA was significantly related to CAT (β=7.27; P<0.01), SOD (β=10.55; P<0.01) and TRAP (β=73.08; P<0.01). The AOPP did not demonstrate a significant correlation with PhA (P>0.05). Our findings show that PhA is a simple and relevant explanatory variable which is related inflammatory and stress oxidative markers in physically independent older women, regardless of age, number of diseases, and body composition. Copyright © 2017 Elsevier Inc. All rights reserved.

Production and characterization of microbial polyhydroxyalkanoates

USDA-ARS?s Scientific Manuscript database

Poyhydroxyalkanoates (PHA) are biologically-produced polyesters. These polymers are of great interest due to both their potential production from renewable substrates and their inherent biodegradability. Polyhydroxybutyrate (PHB) is the most common type of biological PHA. However, the material prop...

75 FR 81634 - Notice of Submission of Proposed Information Collection to OMB; Emergency Comment Request...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-28

... with program partners, and reviews of client files and administrative data collected by the PHA... interviews with PHA staff, interviews with program partners, and reviews of client files and administrative...

24 CFR 941.608 - Technical processing and approval.

Code of Federal Regulations, 2011 CFR

2011-04-01

... execution an ACC amendment and/or a grant agreement. If the PHA has already executed a front-end ACC amendment, HUD will send to the PHA for execution a special ACC amendment for the mixed-finance development...

24 CFR 943.115 - What programs are covered under this subpart?

Code of Federal Regulations, 2011 CFR

2011-04-01

... Annual Contributions Contract (ACC) with HUD; and (2) PHA administration of grants to the PHA in... to an ACC entered under the Request for Proposals (RFP) published May 19, 1999 (64 FR 27358); (2...

24 CFR 943.130 - What are the responsibilities of participating PHAs?

Code of Federal Regulations, 2010 CFR

2010-04-01

... in a consortium, each participating PHA remains responsible for its own obligations under its ACC... is a breach of the ACC with each of the participating PHAs, so each PHA is responsible for the...

24 CFR 941.305 - Technical processing and approval.

Code of Federal Regulations, 2011 CFR

2011-04-01

... shall notify the PHA in writing and shall forward to it for execution an ACC (or ACC amendment). If the PHA already has executed an ACC (or ACC amendment) for the entire reserved amount, HUD shall notify...

24 CFR 941.305 - Technical processing and approval.

Code of Federal Regulations, 2010 CFR

2010-04-01

... shall notify the PHA in writing and shall forward to it for execution an ACC (or ACC amendment). If the PHA already has executed an ACC (or ACC amendment) for the entire reserved amount, HUD shall notify...

24 CFR 943.115 - What programs are covered under this subpart?

Code of Federal Regulations, 2010 CFR

2010-04-01

... Annual Contributions Contract (ACC) with HUD; and (2) PHA administration of grants to the PHA in... to an ACC entered under the Request for Proposals (RFP) published May 19, 1999 (64 FR 27358); (2...

24 CFR 943.130 - What are the responsibilities of participating PHAs?

Code of Federal Regulations, 2011 CFR

2011-04-01

... in a consortium, each participating PHA remains responsible for its own obligations under its ACC... is a breach of the ACC with each of the participating PHAs, so each PHA is responsible for the...

24 CFR 941.608 - Technical processing and approval.

Code of Federal Regulations, 2010 CFR

2010-04-01

... execution an ACC amendment and/or a grant agreement. If the PHA has already executed a front-end ACC amendment, HUD will send to the PHA for execution a special ACC amendment for the mixed-finance development...

Effect of thermal acclimation on locomotor energetics and locomotor performance in a lungless salamander, Desmognathus ochrophaeus.

PubMed

Feder, M E

1986-03-01

To determine the effects of thermal acclimation upon locomotor performance and the rate of oxygen consumption (MO2) during activity, small (less than 3 g), lungless salamanders, Desmognathus ochrophaeus Cope, were acclimated to three temperatures (5, 13 and 21 degrees C) and exercised at various controlled speeds within an exercise wheel while their MO2 was measured. MO2 increased with speed at low speeds (less than 14 cm min-1). Although animals could sustain greater speeds, MO2 did not increase further. These small, exclusively skin-breathing salamanders could increase their MO2 9-11 times during exercise and could sustain nearly half of the oxygen flux expected across a similar surface area of the mammalian lung. However, their maximum aerobic speed was remarkably slow (14 cm min-1) and their net cost of transport remarkably large (15-17 ml O2 g-1 km-1). Thermal acclimation affected MO2 during activity, the maximum sustainable speed and locomotor stamina in different ways. During exercise at 13 degrees C, cold-acclimated animals had a significantly greater MO2 than warm-acclimated animals, but did not differ in stamina or the maximum sustainable speed. During exercise at 21 degrees C, cold acclimation did not affect the MO2 significantly, but it decreased the stamina and increased the rate of lactate accumulation. Thus, these results suggest that thermal acclimation of the MO2 is not tightly coupled to thermal acclimation of locomotor performance in salamanders.

Low temperature tolerance, cold hardening and acclimation in tadpoles of the neotropical túngara frog (Engystomops pustulosus).

PubMed

Vo, Pacific; Gridi-Papp, Marcos

2017-05-01

Many frogs from temperate climates can tolerate low temperatures and increase their thermal tolerance through hardening and acclimation. Most tropical frogs, on the other hand, fail to acclimate to low temperatures. This lack of acclimation ability is potentially due to lack of selection pressure for acclimation because cold weather is less common in the tropics. We tested the generality of this pattern by characterizing the critical temperature minimum (CTMin), hardening, and acclimation responses of túngara frogs (Engystomops pustulosus). These frogs belong to a family with unknown thermal ecology. They are found in a tropical habitat with a highly constant temperature regime. The CTMin of the tadpoles was on average 12.5°C. Pre-metamorphic tadpoles hardened by 1.18°C, while metamorphic tadpoles hardened by 0.36°C. When raised at 21°C, tadpoles acclimated expanding their cold tolerance by 1.3°C in relation to larvae raised at 28°C. These results indicate that the túngara frog has a greatly reduced cold tolerance when compared to species from temperate climates, but it responds to cold temperatures with hardening and acclimation comparable to those of temperate-zone species. Cold tolerance increased with body length but cold hardening was more extensive in pre-metamorphic tadpoles than in metamorphic ones. This study shows that lack of acclimation ability is not general to the physiology of tropical anurans. Copyright © 2017 Elsevier Ltd. All rights reserved.

Combined effects of temperature acclimation and cadmium exposure on mitochondrial function in eastern oysters Crassostrea virginica gmelin (Bivalvia: Ostreidae).

PubMed

Cherkasov, Anton S; Ringwood, Amy H; Sokolova, Inna M

2006-09-01

Cadmium and temperature have strong impacts on the metabolic physiology of aquatic organisms. To analyze the combined impact of these two stressors on aerobic capacity, effects of Cd exposure (50 microg/L) on mitochondrial function were studied in oysters (Crassostrea virginica) acclimated to 12 and 20 degrees C in winter and to 20 and 28 degrees C in fall. Cadmium exposure had different effects on mitochondrial bioenergetics of oysters depending on the acclimation temperature. In oysters acclimated to 12 degrees C, Cd exposure resulted in elevated intrinsic rates of mitochondrial oxidation, whereas at 28 degrees C, a rapid and pronounced decrease of mitochondrial oxidative capacity was found in Cd-exposed oysters. At the intermediate acclimation temperature (20 degrees C), effects of Cd exposure on intrinsic rates of mitochondrial oxidation were negligible. Degree of coupling significantly decreased in mitochondria from 28 degrees C-acclimated oysters but not in that from 12 degrees C- or 20 degrees C-acclimated oysters. Acclimation at elevated temperatures also increased sensitivity of oyster mitochondria to extramitochondrial Cd. Variation in mitochondrial membrane potential explained 41% of the observed variation in mitochondrial adenosine triphosphate synthesis and proton leak between different acclimation groups of oysters. Temperature-dependent sensitivity of metabolic physiology to Cd has significant implications for toxicity testing and for extrapolation of laboratory studies to field populations of aquatic poikilotherms, indicating the importance of taking into account the thermal regime of the environment.

Cold resistance depends on acclimation and behavioral caste in a temperate ant

NASA Astrophysics Data System (ADS)

Modlmeier, Andreas P.; Pamminger, Tobias; Foitzik, Susanne; Scharf, Inon

2012-10-01

Adjusting to low temperatures is important for animals living in cold environments. We studied the chill-coma recovery time in temperate ant workers ( Temnothorax nylanderi) from colonies collected in autumn and spring in Germany. We experimentally acclimated these ant colonies to cold temperatures followed by warm temperatures. As expected, cold-acclimated workers recovered faster from freezing temperatures, but subsequent heat acclimation did not change the short recovery times observed after cold acclimation. Hence, either heat acclimation improves cold tolerance, possibly as a general response to stress, or at least it does not negate enhanced cold tolerance following cold acclimation. Colonies collected in spring showed similar cold tolerance levels to cold-acclimated colonies in the laboratory. Next, we compared the chill-coma recovery time of different worker castes and found that exterior workers recovered faster than interior workers. This difference may be related to their more frequent exposure to cold, higher activity level, or distinct physiology. Interior workers were also heavier and showed a higher gaster-to-head ratio and thorax ratio compared to exterior workers. An obvious difference between exterior and interior workers is activity level, but we found no link between activity and cold tolerance. This suggests that physiology rather than behavioral differences could cause the increased cold tolerance of exterior workers. Our study reveals the importance of acclimation for cold tolerance under natural and standardized conditions and demonstrates differences in cold tolerance and body dimensions in monomorphic behavioral castes of an ant.

Enhanced polyhydroxyalkanoate production from organic wastes via process control.

PubMed

Vargas, Alejandro; Montaño, Liliana; Amaya, Rodolfo

2014-03-01

This work explores the use of a model-based control scheme to enhance the productivity of polyhroxyalkanoate (PHA) production in a mixed culture two-stage system fed with synthetic wastewater. The controller supplies pulses of substrate while regulating the dissolved oxygen (DO) concentration and uses the data to fit a dynamic mathematical model, which in turn is used to predict the time until the next pulse addition. Experiments in a bench scale system first determined the optimal DO set-point and initial substrate concentration. Then the proposed feedback control strategy was compared with a simpler empiric algorithm. The results show that a substrate conversion rate of 1.370±0.598mgPHA/mgCOD/d was achieved. The proposed strategy can also indicate when to stop the accumulation of PHA upon saturation, which occurred with a PHA content of 71.0±7.2wt.%. Copyright © 2014 Elsevier Ltd. All rights reserved.

Consolidated bioprocessing for production of polyhydroxyalkanotes from red algae Gelidium amansii.

PubMed

Sawant, Shailesh S; Salunke, Bipinchandra K; Kim, Beom Soo

2018-04-01

Noncompetitive carbon sources such as algae are unconventional and promising raw material for sustainable biofuel production. The capability of one marine bacterium, Saccharophagus degradans 2-40 to degrade red seaweed Gelidium amansii for production of polyhydroxyalkanoates (PHA) was evaluated in this study. S. degradans can readily attach to algae, degrade algal carbohydrates, and utilize that material as main carbon source. Minimal media containing 8g/L G. amansii were used for the growth of S. degradans. The PHA content obtained was 17-27% of dry cell weight by pure culture of S. degradans and co-culture of S. degradans and Bacillus cereus, a contaminant found with S. degradans cultures. The PHA type was found to be poly(3-hydroxybutyrate) by gas chromatography and Fourier transform-infrared spectroscopy. This work demonstrates PHA production through consolidated bioprocessing of insoluble, untreated red algae by bacterial pure culture and co-culture. Copyright © 2017 Elsevier B.V. All rights reserved.

Bioelectrical Impedance Vector Analysis and Muscular Fitness in Healthy Men

PubMed Central

Rodríguez-Rodríguez, Fernando; Cristi-Montero, Carlos; González-Ruíz, Katherine; Correa-Bautista, Jorge Enrique; Ramírez-Vélez, Robinson

2016-01-01

Muscle strength can define the general muscular fitness (MF) measurable through hand-grip strength (HG), which is a factor that relates to the health of people of different ages. In this study we evaluated the muscle strength together with a bioimpedance electric analysis in 223 healthy Colombian adult subjects. The bioelectrical impedance vector analysis (BIVA) was conducted to determine the resistance (R), reactance (Xc) and phase angle (PhA). We classified the subjects into three groups (for tertiles), obtaining lower values of R and Xc in subjects with lower HG, plus a high correlation between PhA and HG. An increase in the level of PhA is associated with a high level of MF in a sample of healthy Latin American adult men. The BIVA’s parameters and PhA are a potentially effective preventive measure to be integrated into routine screening in the clinical setting. PMID:27384579

Evaluation of enzyme-linked immunosorbent and alternative assays for detection of HIV antibodies using panels of Brazilian sera.

PubMed

Ivo-Dos-Santos, J; Mello, D L; Couto-Fernandez, J C; Passos, R M; Dias-Carneiro, L A; Castilho, E A; Galvão-Castro, B

1990-01-01

Sera from 472 Brazilian subjects, confirmed to be either positive or negative for HIV antibodies and comprising the total clinical spectrum of HIV infection, were utilized in the evaluation of six commercially available enzyme-linked immunosorbent assays (ELISA), as well as of four alternative assays, namely indirect immunofluorescence (IIF), passive hemagglutination (PHA), dot blot and Karpas AIDS cell test. The sensitivities ranged from 100% (Abbott and Roche ELISA) to 84.2% (PHA) and the specificities ranged from 99.3% (IIF) to 80.2% (PHA). The sensitivity and specificity of the PHA and the sensitivity of the Karpas cell test were significantly lower than those of the other tests. Although the IFF and dot blot had good sensitivities and specificities, the six ELISA were more attractive than those tests when other parameters such as ease of reading and duration of assay were considered.

A techno-economic analysis of polyhydroxyalkanoate and hydrogen production from syngas fermentation of gasified biomass.

PubMed

Choi, DongWon; Chipman, David C; Bents, Scott C; Brown, Robert C

2010-02-01

A techno-economic analysis was conducted to investigate the feasibility of a gasification-based hybrid biorefinery producing both hydrogen gas and polyhydroxyalkanoates (PHA), biodegradable polymer materials that can be an attractive substitute for conventional petrochemical plastics. The biorefinery considered used switchgrass as a feedstock and converted that raw material through thermochemical methods into syngas, a gaseous mixture composed mainly of hydrogen and carbon monoxide. The syngas was then fermented using Rhodospirillum rubrum, a purple non-sulfur bacterium, to produce PHA and to enrich hydrogen in the syngas. Total daily production of the biorefinery was assumed to be 12 Mg of PHA and 50 Mg of hydrogen gas. Grassroots capital for the biorefinery was estimated to be $55 million, with annual operating costs at $6.7 million. With a market value of $2.00/kg assumed for the hydrogen, the cost of producing PHA was determined to be $1.65/kg.

Physiological-biochemical properties and the ability to synthesize polyhydroxyalkanoates of the glucose-utilizing strain of the hydrogen bacterium Ralstonia eutropha B8562.

PubMed

Volova, T G; Trusova, M Y; Kalacheva, G S; Kozhevnicov, I V

2006-11-01

Physiological-biochemical, genetic, and cultural properties of the glucose-utilizing mutant strain Ralstonia eutropha B8562 have been compared with those of its parent strain R. eutropha B5786. It has been shown that growth characteristics of the strain cultured on glucose as the sole carbon and energy source are comparable with those of the parent strain. Strain B8562 is characterized by high polyhydroxyalkanoate (PHA) yields on different carbon sources (CO(2), fructose, and glucose). PHA accumulation in the strain batch cultured on glucose under nitrogen deficiency reaches 90 %. The major monomer in the PHA is beta-hydroxybutyric acid (more than 99 mol %); the identified minor components are beta-hydroxyvaleric acid (0.25-0.72 mol %) and beta-hydroxyhexanoic acid (0.08-1.5 mol %). The strain is a promising PHA producer on available sugar-containing media with glucose.

Proteomic analysis of Oenococcus oeni freeze-dried culture to assess the importance of cell acclimation to conduct malolactic fermentation in wine.

PubMed

Cecconi, Daniela; Milli, Alberto; Rinalducci, Sara; Zolla, Lello; Zapparoli, Giacomo

2009-09-01

Cultures of Oenococcus oeni, the most important malolactic bacterium, are used to induce malolactic fermentation in wine. Survival assays in two different wines confirmed that cells acclimated for 24 h in half-strength wine-like medium (acclimation medium) enhanced the malolactic performances. To investigate the effect of the pre-incubation phase on cell physiology, a proteomic study was carried out. Total protein extracts of acclimated and non-acclimated cell cultures (control) were analyzed by 2-D-PAGE. A total of 20 out of approximately 400 spots varied significantly. All the spots were identified by MS analysis and most of them were proteins involved in metabolism, transcription/translation processes and stress response. The results revealed the different physiological status between non-acclimated and acclimated cells explaining, in part, their different behavior in wine. Regulation of stress proteins such as heat and cold shock proteins was involved. Moreover, the availability of sugars and amino acids (even if at low concentration) in acclimation medium determined a modulation of energy metabolism enhancing the resistance to stressful conditions (as those that cells find in wine when inoculated). Finally, this proteomic study increased knowledge concerning the physiological changes in freeze-dried culture occurring with pre-inoculation procedures.

Global convergence in leaf respiration from estimates of thermal acclimation across time and space.

PubMed

Vanderwel, Mark C; Slot, Martijn; Lichstein, Jeremy W; Reich, Peter B; Kattge, Jens; Atkin, Owen K; Bloomfield, Keith J; Tjoelker, Mark G; Kitajima, Kaoru

2015-09-01

Recent compilations of experimental and observational data have documented global temperature-dependent patterns of variation in leaf dark respiration (R), but it remains unclear whether local adjustments in respiration over time (through thermal acclimation) are consistent with the patterns in R found across geographical temperature gradients. We integrated results from two global empirical syntheses into a simple temperature-dependent respiration framework to compare the measured effects of respiration acclimation-over-time and variation-across-space to one another, and to a null model in which acclimation is ignored. Using these models, we projected the influence of thermal acclimation on: seasonal variation in R; spatial variation in mean annual R across a global temperature gradient; and future increases in R under climate change. The measured strength of acclimation-over-time produces differences in annual R across spatial temperature gradients that agree well with global variation-across-space. Our models further project that acclimation effects could potentially halve increases in R (compared with the null model) as the climate warms over the 21st Century. Convergence in global temperature-dependent patterns of R indicates that physiological adjustments arising from thermal acclimation are capable of explaining observed variation in leaf respiration at ambient growth temperatures across the globe. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Proteomics and transcriptomics of broccoli subjected to exogenously supplied and transgenic senescence-induced cytokinin for amelioration of postharvest yellowing.

PubMed

Liu, Mao-Sen; Li, Hui-Chun; Lai, Ying-Mi; Lo, Hsiao-Feng; Chen, Long-Fang O

2013-11-20

Previously, we investigated transgenic broccoli harboring senescence-associated-gene (SAG) promoter-triggered isopentenyltransferase (ipt), which encodes the key enzyme for cytokinin (CK) synthesis and mimics the action of exogenous supplied CK in delaying postharvest senescence of broccoli. Here, we used proteomics and transcriptomics to compare the mechanisms of ipt-transgenic and N(6)-benzylaminopurine (BA) CK treatment of broccoli during postharvest storage. The 2 treatments conferred common and distinct mechanisms. BA treatment decreased the quantity of proteins involved in energy and carbohydrate metabolism and amino acid metabolism, and ipt-transgenic treatment increased that of stress-related proteins and molecular chaperones and slightly affected levels of carbohydrate metabolism proteins. Both treatments regulated genes involved in CK signaling, sugar transport, energy and carbohydrate metabolism, amino acid metabolism and lipid metabolism, although ipt-transgenic treatment to a lesser extent. BA treatment induced genes encoding molecular chaperones, whereas ipt-transgenic treatment induced stress-related genes for cellular protection during storage. Both BA and ipt-transgenic treatments acted antagonistically on ethylene functions. We propose a long-term acclimation of metabolism and protection systems with ipt-transgenic treatment of broccoli and short-term modulation of metabolism and establishment of a protection system with both BA and ipt-transgenic treatments in delaying senescence of broccoli florets. Transgenic broccoli harboring senescence-associated-gene (SAG) promoter-triggered isopentenyltransferase (ipt), which encodes the key enzyme for cytokinin (CK) synthesis and N(6)-benzylaminopurine (BA) CK treated broccoli both showed retardation of postharvest senescence during storage. The mechanisms underlying the two treatments were compared. The combination of proteomic and transcriptomic evidences revealed that the 2 treatments conferred common and distinct mechanisms in delaying senescence of broccoli florets. We propose a long-term acclimation of metabolism and protection systems with ipt-transgenic treatment of broccoli and short-term modulation of metabolism and establishment of a protection system with both BA and ipt-transgenic treatments in delaying senescence of broccoli florets. This article is part of a Special Issue entitled: Translational Plant Proteomics. Copyright © 2013 Elsevier B.V. All rights reserved.

Differential phosphorylation of azidothymidine, dideoxycytidine, and dideoxyinosine in resting and activated peripheral blood mononuclear cells.

PubMed Central

Gao, W Y; Shirasaka, T; Johns, D G; Broder, S; Mitsuya, H

1993-01-01

The antiviral activity of azidothymidine (AZT), dideoxycytidine (ddC), and dideoxyinosine (ddI) against HIV-1 was comparatively evaluated in PHA-stimulated PBM. The mean drug concentration which yielded 50% p24 Gag negative cultures were substantially different: 0.06, 0.2, and 6 microM for AZT, ddC, and ddI, respectively. We found that AZT was preferentially phosphorylated to its triphosphate (TP) form in PHA-PBM rather than unstimulated, resting PBM (R-PBM), producing 10- to 17-fold higher ratios of AZTTP/dTTP in PHA-PBM than in R-PBM. The phosphorylation of ddC and ddI to their TP forms was, however, much less efficient in PHA-PBM, resulting in approximately 5-fold and approximately 15-fold lower ratios of ddCTP/dCTP and ddATP/dATP, respectively, in PHA-PBM than in R-PBM. The comparative order of PHA-induced increase in cellular enzyme activities examined was: thymidine kinase > uridine kinase > deoxycytidine kinase > adenosine kinase > 5'-nucleotidase. We conclude that AZT, ddC, and ddI exert disproportionate antiviral effects depending on the activation state of the target cells, i.e., ddI and ddC exert antiviral activity more favorably in resting cells than in activated cells, while AZT preferentially protects activated cells against HIV infection. Considering that HIV-1 proviral DNA synthesis in resting lymphocytes is reportedly initiated at levels comparable with those of activated lymphocytes, the current data should have practical relevance in the design of anti-HIV chemotherapy, particularly combination chemotherapy. PMID:8387546

Modeling the competition between PHA-producing and non-PHA-producing bacteria in feast-famine SBR and staged CSTR systems.

PubMed

Marang, Leonie; van Loosdrecht, Mark C M; Kleerebezem, Robbert

2015-12-01

Although the enrichment of specialized microbial cultures for the production of polyhydroxyalkanoates (PHA) is generally performed in sequencing batch reactors (SBRs), the required feast-famine conditions can also be established using two or more continuous stirred-tank reactors (CSTRs) in series with partial biomass recirculation. The use of CSTRs offers several advantages, but will result in distributed residence times and a less strict separation between feast and famine conditions. The aim of this study was to investigate the impact of the reactor configuration, and various process and biomass-specific parameters, on the enrichment of PHA-producing bacteria. A set of mathematical models was developed to predict the growth of Plasticicumulans acidivorans-as a model PHA producer-in competition with a non-storing heterotroph. A macroscopic model considering lumped biomass and an agent-based model considering individual cells were created to study the effect of residence time distribution and the resulting distributed bacterial states. The simulations showed that in the 2-stage CSTR system the selective pressure for PHA-producing bacteria is significantly lower than in the SBR, and strongly affected by the chosen feast-famine ratio. This is the result of substrate competition based on both the maximum specific substrate uptake rate and substrate affinity. Although the macroscopic model overestimates the selective pressure in the 2-stage CSTR system, it provides a quick and fairly good impression of the reactor performance and the impact of process and biomass-specific parameters. © 2015 Wiley Periodicals, Inc.

Enhanced Incorporation of 3-Hydroxy-4-Methylvalerate Unit into Biosynthetic Polyhydroxyalkanoate Using Leucine as a Precursor

PubMed Central

2011-01-01

Ralstonia eutropha PHB-4 expressing Pseudomonas sp. 61-3 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Ps) synthesizes PHA copolymer containing 3-hydroxybutyrate (3HB) and a small amount (0.5 mol%) of 3-hydroxy-4-methylvalerate (3H4MV) from fructose as a carbon source. In this study, enhanced incorporation of 3H4MV into PHA was investigated using branched amino acid leucine as a precursor of 3H4MV. Leucine has the same carbon backbone as 3H4MV and is expected to be a natural and self-producible precursor. We found that the incorporation of 3H4MV was enhanced by the supplementation of excess amount (10 g/L) of leucine in the culture medium. This finding indicates that 3H4MV can be derived from leucine. To increase metabolic flux to leucine biosynthesis in the host strain by eliminating the feedback inhibition, the cells were subjected to N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis and leucine analog resistant mutants were generated. The mutants showed statistically higher 3H4MV fraction than the parent strain without supplementing leucine. Additionally, by supplying excess amount of leucine, the mutants synthesized 3HB-based PHA copolymer containing 3.1 mol% 3H4MV and 1.2 mol% 3-hydroxyvalerate (3HV) as minor constituents, which significantly affected the thermal properties of the copolymer. This study demonstrates that it is possible to enhance the monomer supply of 3H4MV into PHA by manipulating leucine metabolism. PMID:21906338

Cocoa procyanidins and human cytokine transcription and secretion.

PubMed

Mao, T; Van De Water, J; Keen, C L; Schmitz, H H; Gershwin, M E

2000-08-01

We examined whether cocoa, in its isolated procyanidin fractions (monomer through decamer), would modulate cytokine production at the levels of transcription and protein secretion in both resting and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). In resting cells, interleukin (IL)-1beta and IL-4 gene expression from cocoa-treated cells varied markedly among the subjects tested. However, at the protein level, the larger fractions (pentamer through decamer) stimulated a dramatic increase in IL-1beta concentration (up to ninefold) with increasing degree of polymerization. Similarly, these larger fractions augmented IL-4 concentration by as much as 2 pg/ml, whereas the control displayed levels nearly undetectable. In the presence of PHA, gene expression also seemed to be most affected by the larger procyanidin fractions. The pentameric through decameric fractions increased IL-1 beta expression by 7-19% compared with PHA control, whereas the hexameric through decameric fractions significantly inhibited PHA-induced IL-4 transcription in the range of 71-86%. This observation at the transcription level for IL-1 beta was reflected at the protein level in PHA-stimulated PBMC. Significant reductions in mitogen-induced IL-4 production were also seen at the protein level with the hexamer, heptamer and octamer. Individual oligomeric cocoa fractions were unstimulatory for IL-2 in resting PBMC. However, when induced with PHA, the pentamer, hexamer and heptamer fractions caused a 61-73% inhibition in IL-2 gene expression. This study offers additional data for the consideration of the health benefits of dietary polyphenols from a wide variety of foods, including those benefits associated specifically with cocoa and chocolate consumption.

A feasibility pilot using a mobile personal health assistant (PHA) app to assist stroke patient and caregiver communication after hospital discharge.

PubMed

Siegel, Jason; Edwards, Emily; Mooney, Lesia; Smith, Christina; Peel, J Brent; Dole, Adam; Maler, Paul; Freeman, W David

2016-01-01

Recent advancements have lowered national acute stroke mortality, yet posthospital care and readmission rates remain challenges. A personal health assistant (PHA) may help manage the spectrum of posthospital care. We hypothesized that a PHA application (app) would be associated with high poststroke patient care satisfaction and might prevent hospital readmission. This is a case series of acute stroke patients admitted to a single, tertiary care, comprehensive stroke center (Mayo Clinic, Jacksonville, Florida) who were offered a personal health assistance through a smart phone app. Patients were screened based on having a cerebrovascular event and the ability to use a necessary device. All patients received the standard poststroke discharge protocol, the PHA app, and the 30-day Likert scale survey. We screened 21 patients and enrolled 3 (14%) before premature financial closure. Two of the 3 patients rated the app highly, and the third patient had not started using it. Of the ineligible patients, 4 had no device, 3 declined enrollment, and 2 were not able to use the device. One of the 2 patients who used the PHA app was readmitted for new stroke symptoms. Both patients who used the app were very satisfied with the PHA and their posthospital care coordination. This study had an enrollment rate of about 14% due to various factors, including limited access or utilization of necessary technology. Though limited by final patient sample size and early termination from funding, this study provides useful information about developing future mobile health apps for acute stroke patients.

Microbial production of polyhydroxyalkanoate block copolymer by recombinant Pseudomonas putida.

PubMed

Li, Shi Yan; Dong, Cui Ling; Wang, Shen Yu; Ye, Hai Mu; Chen, Guo-Qiang

2011-04-01

Polyhydroxyalkanoate (PHA) synthesis genes phaPCJ(Ac) cloned from Aeromonas caviae were transformed into Pseudomonas putida KTOY06ΔC, a mutant of P. putida KT2442, resulting in the ability of the recombinant P. putida KTOY06ΔC (phaPCJ(A.c)) to produce a short-chain-length and medium-chain-length PHA block copolymer consisting of poly-3-hydroxybutyrate (PHB) as one block and random copolymer of 3-hydroxyvalerate (3HV) and 3-hydroxyheptanoate (3HHp) as another block. The novel block polymer was studied by differential scanning calorimetry (DSC), nuclear magnetic resonance, and rheology measurements. DSC studies showed the polymer to possess two glass transition temperatures (T(g)), one melting temperature (T(m)) and one cool crystallization temperature (T(c)). Rheology studies clearly indicated a polymer chain re-arrangement in the copolymer; these studies confirmed the polymer to be a block copolymer, with over 70 mol% homopolymer (PHB) of 3-hydroxybutyrate (3HB) as one block and around 30 mol% random copolymers of 3HV and 3HHp as the second block. The block copolymer was shown to have the highest tensile strength and Young's modulus compared with a random copolymer with similar ratio and a blend of homopolymers PHB and PHVHHp with similar ratio. Compared with other commercially available PHA including PHB, PHBV, PHBHHx, and P3HB4HB, the short-chain- and medium-chain-length block copolymer PHB-b-PHVHHp showed differences in terms of mechanical properties and should draw more attentions from the PHA research community. © Springer-Verlag 2010

Phosphoprotein SAK1 is a regulator of acclimation to singlet oxygen in Chlamydomonas reinhardtii.

PubMed

Wakao, Setsuko; Chin, Brian L; Ledford, Heidi K; Dent, Rachel M; Casero, David; Pellegrini, Matteo; Merchant, Sabeeha S; Niyogi, Krishna K

2014-05-23

Singlet oxygen is a highly toxic and inevitable byproduct of oxygenic photosynthesis. The unicellular green alga Chlamydomonas reinhardtii is capable of acclimating specifically to singlet oxygen stress, but the retrograde signaling pathway from the chloroplast to the nucleus mediating this response is unknown. Here we describe a mutant, singlet oxygen acclimation knocked-out 1 (sak1), that lacks the acclimation response to singlet oxygen. Analysis of genome-wide changes in RNA abundance during acclimation to singlet oxygen revealed that SAK1 is a key regulator of the gene expression response during acclimation. The SAK1 gene encodes an uncharacterized protein with a domain conserved among chlorophytes and present in some bZIP transcription factors. The SAK1 protein is located in the cytosol, and it is induced and phosphorylated upon exposure to singlet oxygen, suggesting that it is a critical intermediate component of the retrograde signal transduction pathway leading to singlet oxygen acclimation.DOI: http://dx.doi.org/10.7554/eLife.02286.001. Copyright © 2014, Wakao et al.

The impact of anode acclimation strategy on microbial electrolysis cell treating hydrogen fermentation effluent.

PubMed

Li, Xiaohu; Zhang, Ruizhe; Qian, Yawei; Angelidaki, Irini; Zhang, Yifeng

2017-07-01

The impact of different anode acclimation methods for enhancing hydrogen production in microbial electrolysis cell (MEC) was investigated in this study. The anodes were first acclimated in microbial fuel cells using acetate, butyrate and corn stalk fermentation effluent (CSFE) as substrate before moving into MECs, respectively. Subsequently, CSFE was used as feedstock in all the three MECs. The maximum hydrogen yield with the anode pre-acclimated with butyrate (5.21±0.24L H 2 /L CSFE) was higher than that pre-acclimated with acetate (4.22±0.19L H 2 /L CSFE) and CSFE (4.55±0.14L H 2 /L CSFE). The current density (480±11A/m 3 ) and hydrogen production rate (4.52±0.13m 3 /m 3 /d) with the anode pre-acclimated with butyrate were also higher that another two reactors. These results demonstrated that the anode biofilm pre-acclimated with butyrate has significant advantages in CSFE treatment and could improve the performance of hydrogen production in MEC. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tracing the evolution of degraders in activated sludge during the sludge’s acclimation to a xenobiotic organic

NASA Astrophysics Data System (ADS)

Chong, N. M.; Fan, C. H.; Yang, Y. C.

2017-01-01

The molecular biology method of high-throughput pyrosequencing was employed to examine the change of activated sludge community structures during the process in which activated sludge was acclimated to and degraded a target xenobiotic. The sample xenobiotic organic compound used as the activated sludge acclimation target was the herbicide 2,4-dichlorphenoxyacetic acid (2,4-D). Indigenous activated sludge microorganisms were acclimated to 2,4-D as the sole carbon source in both the batch and the continuous-flow reaction modes. Sludge masses at multiple time points during the course of acclimation were subjected to pyrosequencing targeting the microorganisms’ 16S rRNA genes. With the bacterial 16S rRNA sequencing results the genera that increased in abundance were checked with degradative pathway databases or literature to confirm that they are commonly seen as potent degraders of 2,4-D. From this systematic examination of degrader changes at time points during activated sludge acclimation and degradation of the target xenobiotic, the trend of degrader evolution in activated sludge over the sludge’s acclimation process to a xenobiotic was traced.

Response of microbial community structure to pre-acclimation strategies in microbial fuel cells for domestic wastewater treatment.

PubMed

Park, Younghyun; Cho, Hyunwoo; Yu, Jaechul; Min, Booki; Kim, Hong Suck; Kim, Byung Goon; Lee, Taeho

2017-06-01

Microbial community structures and performance of air-cathode microbial fuel cells (MFCs) inoculated with activated sludge from domestic wastewater were investigated to evaluate the effects of three substrate pre-acclimation strategies: 1, serial pre-acclimation with acetate and glucose before supplying domestic wastewater; 2, one step pre-acclimation with acetate before supplying domestic wastewater; and 3, direct supply of domestic wastewater without any pre-acclimation. Strategy 1 showed much higher current generation (1.4mA) and Coulombic efficiency (33.5%) than strategies 2 (0.7mA and 9.4%) and 3 (0.9mA and 10.3%). Pyrosequencing showed that microbial communities were significantly affected by pre-acclimation strategy. Although Proteobacteria was the dominant phylum with all strategies, Actinobacteria was abundant when MFCs were pre-acclimated with glucose after acetate. Not only anode-respiring bacteria (ARB) in the genus Geobacter but also non-ARB belonging to the family Anaerolinaceae seemed to play important roles in air-cathode MFCs to produce electricity from domestic wastewater. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effects of fasting on maximum thermogenesis in temperature-acclimated rats

NASA Astrophysics Data System (ADS)

Wang, L. C. H.

1981-09-01

To further investigate the limiting effect of substrates on maximum thermogenesis in acute cold exposure, the present study examined the prevalence of this effect at different thermogenic capabilities consequent to cold- or warm-acclimation. Male Sprague-Dawley rats (n=11) were acclimated to 6, 16 and 26‡C, in succession, their thermogenic capabilities after each acclimation temperature were measured under helium-oxygen (21% oxygen, balance helium) at -10‡C after overnight fasting or feeding. Regardless of feeding conditions, both maximum and total heat production were significantly greater in 6>16>26‡C-acclimated conditions. In the fed state, the total heat production was significantly greater than that in the fasted state at all acclimating temperatures but the maximum thermogenesis was significant greater only in the 6 and 16‡C-acclimated states. The results indicate that the limiting effect of substrates on maximum and total thermogenesis is independent of the magnitude of thermogenic capability, suggesting a substrate-dependent component in restricting the effective expression of existing aerobic metabolic capability even under severe stress.

24 CFR 968.240 - HUD review of PHA performance.

Code of Federal Regulations, 2011 CFR

2011-04-01

... with HUD requirements, the adequacy of a PHA's inspections as evidenced by the quality of work, and the timeliness of the work. HUD's review may be conducted either in-office or on-site. Where conducted in-office...

Preliminary acclimation strategies for successful startup in conventional biofilters.

PubMed

Elías, Ana; Barona, Astrid; Gallastegi, Gorka; Rojo, Naiara; Gurtubay, Luis; Ibarra-Berastegi, Gabriel

2010-08-01

The question of how to obtain the best inocula for conventional biofilters arises when an acclimation/adaptation procedure is to be applied. Bearing in mind that no standardized procedure for acclimating inocula exists, certain preliminary strategies for obtaining an active inoculum from wastewater treatment sludge are proposed in this work. Toluene was the contaminant to be degraded. Concerning the prior separation of sludge phases, no obvious advantage was found in separating the supernatant phase of the sludge before acclimation. As far as a continuous or discontinuous acclimation mode is concerned, the latter is recommended for rapidly obtaining acclimated sludge samples by operating the system for no longer than 1 month. The continuous mode rendered similar degradation rates, although it required longer operating time. Nevertheless, the great advantage of the continuous system lay in the absence of daily maintenance and the ready availability of the activated sample.

Effects of egg storage on hatchability, chick quality, performance and immunocompetence parameters of broiler chickens.

PubMed

Goliomytis, Michael; Tsipouzian, Theofania; Hager-Theodorides, Ariadne L

2015-09-01

Pre-incubation egg storage is a necessity for the poultry industry. This study evaluated the effects of pre-incubation storage length of broiler eggs on hatchability, 1-day-old chick quality, subsequent performance, and immunocompetence. To this end, a total of 360 hatching eggs were stored for 4, 12, or 16 d prior to incubation. Hatchability and chick quality were assessed at hatch, and growth performance and immunocompetence parameters were assessed during a 35 d rearing period. Hatchability of set and fertile eggs, and embryonic mortality, were not affected by egg storage. On the contrary, 1-day-old chick BW and length were linearly negatively correlated with egg storage length (P-linear<0.05). Nevertheless, BW corrected for egg weight prior to setting was unaffected, and corrected chick length was positively affected by storage length. One-day-old chick Tona score, navel quality, and post-hatch growth performance (BW at 7 and 35 d, cumulative feed intake, and feed conversion ratio at 35 d) were unaffected by egg storage (P, P-linear>0.05). Lymphoid organ weights at 2 and 35 d, the titre of maternal anti-NDV antibodies, most of the thymocyte subpopulations defined by CD3, CD4, and CD8 cell surface expression in the thymus of 2-d-old chicks, cellular responses to the PHA skin test, humoral responses to primary SRBC, and NDV immunizations were also not influenced by length of storage (P, P-linear>0.05). On the contrary, the length of egg storage was found to negatively influence the abundance of CD3+CD4-CD8- thymocytes that represent the majority of γδ-T cells in the thymus of 2-day-old chicks, as well as the humoral response to booster NDV immunization of the birds. In brief, pre-incubation storage of broiler hatching eggs for up to 16 d did not affect most developmental and growth parameters investigated, except for BW and length at hatch. Egg storage was found to suppress some aspects of the immunocompetence of the birds, particularly aspects of acquired immunity. © 2015 Poultry Science Association Inc.

Characterization and identification of the proteins bound to two types of polyhydroxyalkanoate granules in Pseudomonas sp. 61-3.

PubMed

Hokamura, Ayaka; Fujino, Kanako; Isoda, Yoshiko; Arizono, Koji; Shiratsuchi, Hideki; Matsusaki, Hiromi

2015-01-01

Pseudomonas sp. 61-3 accumulates two types of polyhydroxyalkanoates (PHAs), poly(3-hydroxybutyrate) [P(3HB)], and poly(3HB-co-3-hydroxyalkanoates) [P(3HB-co-3HA)], and some proteins associated with their PHA granules have been identified. To date, PhaFPs (GA36) and PhaIPs (GA18) were identified from P(3HB-co-3HA) granules. In this study, the gene encoding GA24 associated with P(3HB) granule was identified as phbPPs. PhbPPs was composed of 192 amino acids with a calculated molecular mass of 20.4 kDa and was assumed to be a phasin. phbFPs gene and unknown ORF were also found on phb locus. PhbFPs was anticipated to be the transcriptional repressor of phbPPs gene. PhbPPs was bound to the P(3HB-co-3HA) granules with 3HB composition of more than 87 mol%, and PhaIPs and PhaFPs were bound to the P(3HB-co-3HA) granules with 3HA (C6-C12) composition of more than 13 mol% in the producing cells, suggesting that localization of these proteins is attributed to the monomer compositions of the copolymers.

Purification of target proteins from intracellular inclusions mediated by intein cleavable polyhydroxyalkanoate synthase fusions.

PubMed

Du, Jinping; Rehm, Bernd H A

2017-11-02

Recombinant protein production and purification from Escherichia coli is often accompanied with expensive and complicated procedures, especially for therapeutic proteins. Here it was demonstrated that, by using an intein cleavable polyhydroxyalkanoate synthase fusion, recombinant proteins can be first produced and sequestered on a natural resin, the polyhydroxyalkanoate (PHA) inclusions, then separated from contaminating host proteins via simple PHA bead isolation steps, and finally purified by specific release into the soluble fraction induced by a pH reduction. By translationally fusing a target protein to PHA synthase using a self-cleaving intein as linker, intracellular production of PHA beads was achieved. Upon isolation of respective PHA beads the soluble pure target protein was released by a simple pH shift to 6. The utility of this approach was exemplified by producing six target proteins, including Aequorea victoria green fluorescent protein (GFP), Mycobacterium tuberculosis vaccine candidate Rv1626, the immunoglobulin G (IgG) binding ZZ domain of protein A derived from Staphylococcus aureus, human tumor necrosis factor alpha (TNFα), human granulocyte colony-stimulating factor (G-CSF), and human interferon alpha 2b (IFNα2b). Here a new method for production and purification of a tag-less protein was developed through intein cleavable polyhydroxyalkanoate synthase fusion. Pure target protein could be easily obtained without laborious downstream processing.

Isolation and Characterization of PHA-Producing Bacteria from Propylene Oxide Saponification Wastewater Residual Sludge.

PubMed

Li, Ruirui; Gu, Pengfei; Fan, Xiangyu; Shen, Junyu; Wu, Yulian; Huang, Lixuan; Li, Qiang

2018-03-21

A polyhydroxyalkanoate (PHA)-producing strain was isolated from propylene oxide (PO) saponification wastewater activated sludge and was identified as Brevundimonas vesicularis UJN1 through 16S rDNA sequencing and Biolog microbiological identification. Single-factor and response surface methodology experiments were used to optimize the culture medium and conditions. The optimal C/N ratio was 100/1.04, and the optimal carbon and nitrogen sources were sucrose (10 g/L) and NH 4 Cl (0.104 g/L) respectively. The optimal culture conditions consisted of initial pH of 6.7 and an incubation temperature of 33.4 °C for 48 h, with 15% inoculum and 100 mL medium at an agitation rate of 180 rpm. The PHA concentration reached 34.1% of the cell dry weight and increased three times compared with that before optimization. The only report of PHA-producing bacteria by Brevundimonas vesicularis showed that the conversion rate of PHAs using glucose as the optimal carbon source was 1.67%. In our research, the conversion rate of PHAs with sucrose as the optimal carbon source was 3.05%, and PHA production using sucrose as the carbon source was much cheaper than that using glucose as the carbon source.

High levels of E4-PHA-reactive oligosaccharides: potential as marker for cells with characteristics of hepatic progenitor cells.

PubMed

Sasaki, Nozomi; Moriwaki, Kenta; Uozumi, Naofumi; Noda, Katsuhisa; Taniguchi, Naoyuki; Kameyama, Akihiko; Narimatsu, Hisashi; Takeishi, Shunsaku; Yamada, Masao; Koyama, Nobuto; Miyoshi, Eiji

2009-12-01

Oligosaccharides serve as markers of the cell surface and have been used as certain kinds of tumor markers. In the present study, we established a simple method for isolating hepatic progenitor cells using a lectin, which recognizes a characteristic oligosaccharide structure. Rat liver epithelial (RLE) cells, which have been established as a hepatic stem-like cell, were used to identify characteristic oligosaccharide structures on hepatic stem cells. As a result from lectin micro array, several types of lectin including E4-PHA were identified to bind RLE cells specifically. Furthermore, lectin blot and lectin flow cytometry analyses showed that binding to E(4)-PHA lectin was significantly increased in RLE cells, compared to hepatocytes, and hepatoma cells. The induction of differentiation into a hepatocyte lineage of RLE cells by treatment with Oncostatin M and dexamethasone resulted in a decrease in E(4)-PHA binding. Using an E(4)-PHA column, we succeeded in isolating hepatic stem cells from LEC (Long-Evans with cinnamon coat color) rat livers with fluminant hepatitis. The characteristics of the established cells were similar to RLE cells and had a potential of proliferating in rat liver. These results suggest that oligosaccharides can serve as a novel marker for the isolation of the hepatic progenitor cells.

Trait Acclimation Mitigates Mortality Risks of Tropical Canopy Trees under Global Warming.

PubMed

Sterck, Frank; Anten, Niels P R; Schieving, Feike; Zuidema, Pieter A

2016-01-01

There is a heated debate about the effect of global change on tropical forests. Many scientists predict large-scale tree mortality while others point to mitigating roles of CO2 fertilization and - the notoriously unknown - physiological trait acclimation of trees. In this opinion article we provided a first quantification of the potential of trait acclimation to mitigate the negative effects of warming on tropical canopy tree growth and survival. We applied a physiological tree growth model that incorporates trait acclimation through an optimization approach. Our model estimated the maximum effect of acclimation when trees optimize traits that are strongly plastic on a week to annual time scale (leaf photosynthetic capacity, total leaf area, stem sapwood area) to maximize carbon gain. We simulated tree carbon gain for temperatures (25-35°C) and ambient CO2 concentrations (390-800 ppm) predicted for the 21st century. Full trait acclimation increased simulated carbon gain by up to 10-20% and the maximum tolerated temperature by up to 2°C, thus reducing risks of tree death under predicted warming. Functional trait acclimation may thus increase the resilience of tropical trees to warming, but cannot prevent tree death during extremely hot and dry years at current CO2 levels. We call for incorporating trait acclimation in field and experimental studies of plant functional traits, and in models that predict responses of tropical forests to climate change.

Thermal plasticity of diving behavior, aquatic respiration, and locomotor performance in the Mary River turtle Elusor macrurus.

PubMed

Clark, Natalie J; Gordos, Matthew A; Franklin, Craig E

2008-01-01

Locomotion is a common measure of performance used in studies of thermal acclimation because of its correlation with predator escape and prey capture. However, for sedentary animals such as freshwater turtles, we propose that diving behavior may be a more ecologically relevant measure of performance. Increasing dive duration in hatchling turtles reduces predator exposure and therefore functions as an ecological benefit. Diving behavior is thermally dependent, and in some species of freshwater turtles, it is also reliant on aquatic respiration. This study examined the influence of thermal acclimation on diving behavior, aquatic respiration, and locomotor performance in the endangered, bimodally respiring Mary River turtle Elusor macrurus. Diving behavior was found to partially acclimate at 17 degrees C, with turtles acclimated to a cold temperature (17 degrees C) having a significantly longer dive duration than hatchlings acclimated to a warm temperature (28 degrees C). This increase in dive duration at 17 degrees C was not a result of physiological alterations in metabolic rate but was due instead to an increase in aquatic oxygen consumption. Increasing aquatic oxygen consumption permitted cold-acclimated hatchlings to remain submerged for significantly longer periods, with one turtle undertaking a dive of over 2.5 d. When burst-swimming speed was used as the measure of performance, thermal acclimation was not detected. Overall, E. macrurus demonstrated a partial ability to acclimate to changes in environmental temperature.

Acclimation-dependent expression of heat shock protein 70 in Pacific abalone ( Haliotis discus hannai Ino) and its acute response to thermal exposure

NASA Astrophysics Data System (ADS)

Li, Jiaqi; He, Qingguo; Sun, Hui; Liu, Xiao

2012-01-01

Heat shock protein 70 (Hsp70) is one important member of heat shock protein (Hsp) family that is responsible for various stresses, especially thermal stress. Here we examined the response of Hsp70 gene to both chronic and acute thermal exposure in Pacific abalone ( Haliotis discus hannai Ino). For the chronic exposure, abalones were maintained at 8, 12, 20, and 30°C for four months and their mRNA levels were measured. The highest mRNA level of Hsp70 gene relative to actin gene was detected in the 30°C-acclimated group, followed by the 8°C-acclimated group and then the 12°C- and 20°C-acclimated groups. After the long-term acclimation, gills from each of the above acclimation groups were dissected and exposed to different temperatures between 8°C and 38°C for 30 min. Hsp70 expression in gills acclimated to different temperatures responded differentially to the same temperature exposure. The incubation temperature that induced maximum Hsp70 mRNA expression was higher in the higher temperature acclimation groups than lower temperature groups. Pacific abalones could alter the expression pattern of Hsp70 gene according to environmental thermal conditions, through which they deal with the stress of thermal variations.

Cold acclimation and cognitive performance: A review.

PubMed

Jones, Douglas M; Bailey, Stephen P; Roelands, Bart; Buono, Michael J; Meeusen, Romain

2017-12-01

Athletes, occupational workers, and military personnel experience cold temperatures through cold air exposure or cold water immersion, both of which impair cognitive performance. Prior work has shown that neurophysiological pathways may be sensitive to the effects of temperature acclimation and, therefore, cold acclimation may be a potential strategy to attenuate cold-induced cognitive impairments for populations that are frequently exposed to cold environments. This review provides an overview of studies that examine repeated cold stress, cold acclimation, and measurements of cognitive performance to determine whether or not cold acclimation provides beneficial protection against cold-induced cognitive performance decrements. Studies included in this review assessed cognitive measures of reaction time, attention, logical reasoning, information processing, and memory. Repeated cold stress, with or without evidence of cold acclimation, appears to offer no added benefit of improving cognitive performance. However, research in this area is greatly lacking and, therefore, it is difficult to draw any definitive conclusions regarding the use of cold acclimation to improve cognitive performance during subsequent cold exposures. Given the current state of minimal knowledge on this topic, athletes, occupational workers, and military commands looking to specifically enhance cognitive performance in cold environments would likely not be advised to spend the time and effort required to become acclimated to cold. However, as more knowledge becomes available in this area, recommendations may change. Copyright © 2017 Elsevier B.V. All rights reserved.

The effects of cold acclimation on photosynthetic apparatus and the expression of COR14b in four genotypes of barley (Hordeum vulgare) contrasting in their tolerance to freezing and high-light treatment in cold conditions.

PubMed

Rapacz, Marcin; Wolanin, Barbara; Hura, Katarzyna; Tyrka, Miroslaw

2008-04-01

Cold acclimation modifies the balance of the energy absorbed and metabolized in the dark processes of photosynthesis, which may affect the expression of cold-regulated (COR) genes. At the same time, a gradual acclimation to the relatively high light conditions is observed, thereby minimizing the potential for photo-oxidative damage. As a result, the resistance to photoinhibition in the cold has often been identified as a trait closely related to freezing tolerance. Using four barley genotypes that differentially express both traits, the effect of cold acclimation on freezing tolerance and high-light tolerance was studied together with the expression of COR14b, one of the best-characterized barley COR genes. Plants were cold acclimated for 2 weeks at 2 degrees C. Freezing tolerance was studied by means of electrolyte leakage. Changes in photosynthetic apparatus and high-light tolerance were monitored by means of chlorophyll fluorescence. Accumulation of COR14b and some proteins important in photosynthetic acclimation to cold were studied with western analysis. COR14b transcript accumulation during cold acclimation was assessed with real-time PCR. Cold acclimation increased both freezing tolerance and high-light tolerance, especially when plants were treated with high light after non-lethal freezing. In all plants, cold acclimation triggered the increase in photosynthetic capacity during high-light treatment. In two plants that were characterized by higher high-light tolerance but lower freezing tolerance, higher accumulation of COR14b transcript and protein was observed after 7 d and 14 d of cold acclimation, while a higher transient induction of COR14b expression was observed in freezing-tolerant plants during the first day of cold acclimation. High-light tolerant plants were also characterized with a higher level of PsbS accumulation and more efficient dissipation of excess light energy. Accumulation of COR14b in barley seems to be important for resistance to combined freezing and high-light tolerance, but not for freezing tolerance per se.

Thyroid hormone regulates muscle function during cold acclimation in zebrafish (Danio rerio).

PubMed

Little, Alexander G; Seebacher, Frank

2013-09-15

Thyroid hormone (TH) is a universal regulator of growth, development and metabolism during cold exposure in mammals. In zebrafish (Danio rerio), TH regulates locomotor performance and metabolism during cold acclimation. The influence of TH on locomotor performance may be via its effect on metabolism or, as has been shown in mammals, by modulating muscle phenotypes. Our aim was to determine whether TH influences muscle phenotypes in zebrafish, and whether this could explain changes in swimming capacity in response to thermal acclimation. We used propylthiouracil and iopanoic acid to induce hypothyroidism in zebrafish over a 3-week acclimation period to either 18 or 28°C. To verify that physiological changes following hypothyroid treatment were in fact due to the action of TH, we supplemented hypothyroid fish with 3,5-diiodothryronine (T2) or 3,5,3'-triiodothyronine (T3). Cold-acclimated fish had significantly greater sustained swimming performance (Ucrit) but not burst speed. Greater Ucrit was accompanied by increased tail beat frequency, but there was no change in tail beat amplitude. Hypothyroidism significantly decreased Ucrit and burst performance, as well as tail beat frequency and SERCA activity in cold-acclimated fish. However, myofibrillar ATPase activity increased in cold-acclimated hypothyroid fish. Hypothyroid treatment also decreased mRNA concentrations of myosin heavy chain fast isoforms and SERCA 1 isoform in cold-acclimated fish. SERCA 1 mRNA increased in warm-acclimated hypothyroid fish, and SERCA 3 mRNA decreased in both cold- and warm-acclimated hypothyroid fish. Supplementation with either T2 or T3 restored Ucrit, burst speed, tail beat frequency, SERCA activity and myosin heavy chain and SERCA 1 and 3 mRNA levels of hypothyroid fish back to control levels. We show that in addition to regulating development and metabolism in vertebrates, TH also regulates muscle physiology in ways that affect locomotor performance in fish. We suggest that the role of TH in modulating SERCA1 expression during cold exposure may have predisposed it to regulate endothermic thermogenesis.

Long-term water stress leads to acclimation of drought sensitivity of photosynthetic capacity in xeric but not riparian Eucalyptus species

PubMed Central

Zhou, Shuang-Xi; Medlyn, Belinda E.; Prentice, Iain Colin

2016-01-01

Background and Aims Experimental drought is well documented to induce a decline in photosynthetic capacity. However, if given time to acclimate to low water availability, the photosynthetic responses of plants to low soil moisture content may differ from those found in short-term experiments. This study aims to test whether plants acclimate to long-term water stress by modifying the functional relationships between photosynthetic traits and water stress, and whether species of contrasting habitat differ in their degree of acclimation. Methods Three Eucalyptus taxa from xeric and riparian habitats were compared with regard to their gas exchange responses under short- and long-term drought. Photosynthetic parameters were measured after 2 and 4 months of watering treatments, namely field capacity or partial drought. At 4 months, all plants were watered to field capacity, then watering was stopped. Further measurements were made during the subsequent ‘drying-down’, continuing until stomata were closed. Key Results Two months of partial drought consistently reduced assimilation rate, stomatal sensitivity parameters (g1), apparent maximum Rubisco activity (Vcmax′) and maximum electron transport rate (Jmax′). Eucalyptus occidentalis from the xeric habitat showed the smallest decline in Vcmax′ and Jmax′; however, after 4 months, Vcmax′ and Jmax′ had recovered. Species differed in their degree of Vcmax′ acclimation. Eucalyptus occidentalis showed significant acclimation of the pre-dawn leaf water potential at which the Vcmax′ and ‘true’ Vcmax (accounting for mesophyll conductance) declined most steeply during drying-down. Conclusions The findings indicate carbon loss under prolonged drought could be over-estimated without accounting for acclimation. In particular, (1) species from contrasting habitats differed in the magnitude of V′cmax reduction in short-term drought; (2) long-term drought allowed the possibility of acclimation, such that V′cmax reduction was mitigated; (3) xeric species showed a greater degree of V′cmax acclimation; and (4) photosynthetic acclimation involves hydraulic adjustments to reduce water loss while maintaining photosynthesis. PMID:26493470