Woody (lignocellulosic) plant biomass is an abundant renewable feedstock, rich in polysaccharides that are bound into an insoluble fiber composite with lignin. Marine crustacean woodborers of the ...genus Limnoria are among the few animals that can survive on a diet of this recalcitrant material without relying on gut resident microbiota. Analysis of fecal pellets revealed that Limnoria targets hexose-containing polysaccharides (mainly cellulose, and also glucomannans), corresponding with the abundance of cellulases in their digestive system, but xylans and lignin are largely unconsumed. We show that the limnoriid respiratory protein, hemocyanin, is abundant in the hindgut where wood is digested, that incubation of wood with hemocyanin markedly enhances its digestibility by cellulases, and that it modifies lignin. We propose that this activity of hemocyanins is instrumental to the ability of Limnoria to feed on wood in the absence of gut symbionts. These findings may hold potential for innovations in lignocellulose biorefining.
ObjectivesFrailty is highly prevalent in haemodialysis (HD) patients, leading to poor outcomes. This study aimed to determine whether a randomised controlled trial (RCT) of intradialytic exercise is ...feasible for frail HD patients, and explore how the intervention may be tailored to their needs.DesignMixed-methods feasibility.Setting and participantsPrevalent adult HD patients of the CYCLE-HD trial with a Clinical Frailty Scale Score of 4–7 (vulnerable to severely frail) were eligible for the feasibility study.InterventionsParticipants in the exercise group undertook 6 months of three times per week, progressive, moderate intensity intradialytic cycling (IDC).OutcomesPrimary outcomes were related to feasibility. Secondary outcomes were falls incidence measured from baseline to 1 year following intervention completion, and exercise capacity, physical function, physical activity and patient-reported outcomes measured at baseline and 6 months. Acceptability of trial procedures and the intervention were explored via diaries and interviews with n=25 frail HD patients who both participated in (n=13, 52%), and declined (n=12, 48%), the trial.Results124 (30%) patients were eligible, and of these 64 (52%) consented with 51 (80%) subsequently completing a baseline assessment. n=24 (71% male; 59±13 years) dialysed during shifts randomly assigned to exercise and n=27 (81% male; 65±11 years) shifts assigned to usual care. n=6 (12%) were lost to follow-up. The exercise group completed 74% of sessions. 27%–89% of secondary outcome data were missing. Frail HD patients outlined several ways to enhance trial procedures. Maintaining ability to undertake activities of daily living and social participation were outcomes of primary importance. Participants desired a varied exercise programme.ConclusionsA definitive RCT is feasible, however a comprehensive exercise programme may be more efficacious than IDC in this population.Trial registration numbersISRCTN11299707; ISRCTN12840463.
Phenolic acid decarboxylase from
Bacillus subtilis
(
Bs
PAD) catalyses the decarboxylation of phenolic acids such as coumaric acid to give vinyl phenols, which are of interest as possible polymer ...precursors and flavour/fragrance compounds. The structure of the Tyr19Ala mutant of
Bs
PAD has been solved in complex with coumaric acid. In the active site, the substrate carboxylate is bound by Tyr11 and Tyr13, and the phenolic hydroxyl by the NE atom of Arg41. A comparison of the mutant complex with the wild-type apoenzyme reveals that the β1-β2 loop, running from Tyr11 to Ala19, closes over the active site in the presence of substrate, shielding it from bulk solvent. The complex structure, in conjunction with an activity study of point mutants of
Bs
PAD, provides support for a mechanism for PADs, proposed by Mancheño and co-workers for the homologue from
Lactobacillus plantarum
Proteins
, 2010,
78
, 1662-1676. In this mechanism, a quinone methide intermediate results from deprotonation of the phenolic hydroxyl of the substrate by Glu64, assisted by Arg41. Decarboxylation of the substrate is effected through binding of the carboxylate by Tyr11 and Tyr13, the latter being brought into contact with the substrate as a result of the movement of the β1-β2 loop on substrate binding.
Mutational analysis, coupled with a complex structure, reveals catalytic roles for Tyr11 and Tyr13 in carboxylate recognition and for Arg41 in binding of the phenolic hydroxyl of the substrate.
Transportation accounts for a large proportion of global CO2 emissions, estimated at 22% of all anthropogenically produced CO2 in 2010, with most of this coming from the use of liquid transportation ...fuels. Second generation biofuels offer a sustainable opportunity to decarbonise this sector of global energy use. To realise the potential of second generation biofuels more efficient methods of deconstructing their lignocellulosic starting material must be developed. Understanding how the marine wood borer, Limnoria quadripunctata, is able to do this biochemically without the assistance of microbial symbionts may inspire new techniques to achieve this in an industrial setting. Whilst the anatomy of Limnoria has been well studied, and the transcriptome of its hepatopancreas (HP), a secretory organ of the digestive system, has been previously elucidated, little is known about how the animal is able to derive nutrients from its diet of wood without the aid of microbial symbionts. In this work a proteomic study of the HP and gut tissue of Limnoria was carried out and the data generated analysed in concert with that of the HP transcriptome to identify proteins which may be involved in lignocellulose digestion in the animal. In this way the glycosyl hydrolases, hemocyanins (HCs), ferritins and leucine rich repeat proteins were identified as having the potential to be involved in lignocellulose digestion. Work was undertaken to characterise the function of HC proteins in Limnoria. Reverse transcriptase quantitative polymerase chain reaction analysis showed all five Limnoria HC genes to be solely expressed in the HP, whilst Western blot and proteomic examination showed HC protein to be present in the HP and gut, as well as the rest of the body. This indicates that HCs are transported post translation to the gut, the site where ingested wood particles are segregated and presumably digested. In vivo experiments suggested that a phenoloxidase enzyme activity was associated with the production of peroxides in the gut of Limnoria. Using a soluble extract of Limnoria tissue a di-phenoloxidase (DPO) enzyme activity was demonstrated which it was possible to inhibit using a phenoloxidase inhibitor. Mass spectrometry analysis implicated HC protein as the species responsible for this activity. Multiple sequence alignment showed that the Limnoria HC sequences possess the conserved features associated with the ability to carry out a DPO enzyme activity described in HC proteins from other arthropod species. Attempts were made to heterologously express Limnoria HC proteins in a number of systems with limited success being achieved using the ArcticExpress strain of Escherichia coli. Partially pure heterologous protein produced in this way was able to show a DPO enzyme activity which was inhibited by a phenoloxidase inhibitor. Mass spectrometry analysis implicates HC as the protein species responsible for this activity. Based on the findings of this work it seems possible that the hemocyanin proteins of Limnoria aid the animal in digesting wood by contributing to the formation of reactive peroxide compounds in its gut which attack lignocellulose. This process may also contribute towards the sterility of the gut by creating an inhospitable environment for microbial colonisation. Further work is now required to determine the mechanisms by which it occurs.
Transportation accounts for a large proportion of global CO2 emissions, estimated at 22% of all anthropogenically produced CO2 in 2010, with most of this coming from the use of liquid transportation ...fuels. Second generation biofuels offer a sustainable opportunity to decarbonise this sector of global energy use. To realise the potential of second generation biofuels more efficient methods of deconstructing their lignocellulosic starting material must be developed. Understanding how the marine wood borer, Limnoria quadripunctata, is able to do this biochemically without the assistance of microbial symbionts may inspire new techniques to achieve this in an industrial setting. Whilst the anatomy of Limnoria has been well studied, and the transcriptome of its hepatopancreas (HP), a secretory organ of the digestive system, has been previously elucidated, little is known about how the animal is able to derive nutrients from its diet of wood without the aid of microbial symbionts. In this work a proteomic study of the HP and gut tissue of Limnoria was carried out and the data generated analysed in concert with that of the HP transcriptome to identify proteins which may be involved in lignocellulose digestion in the animal. In this way the glycosyl hydrolases, hemocyanins (HCs), ferritins and leucine rich repeat proteins were identified as having the potential to be involved in lignocellulose digestion. Work was undertaken to characterise the function of HC proteins in Limnoria. Reverse transcriptase quantitative polymerase chain reaction analysis showed all five Limnoria HC genes to be solely expressed in the HP, whilst Western blot and proteomic examination showed HC protein to be present in the HP and gut, as well as the rest of the body. This indicates that HCs are transported post translation to the gut, the site where ingested wood particles are segregated and presumably digested. In vivo experiments suggested that a phenoloxidase enzyme activity was associated with the production of peroxides in the gut of Limnoria. Using a soluble extract of Limnoria tissue a di-phenoloxidase (DPO) enzyme activity was demonstrated which it was possible to inhibit using a phenoloxidase inhibitor. Mass spectrometry analysis implicated HC protein as the species responsible for this activity. Multiple sequence alignment showed that the Limnoria HC sequences possess the conserved features associated with the ability to carry out a DPO enzyme activity described in HC proteins from other arthropod species. Attempts were made to heterologously express Limnoria HC proteins in a number of systems with limited success being achieved using the ArcticExpress strain of Escherichia coli. Partially pure heterologous protein produced in this way was able to show a DPO enzyme activity which was inhibited by a phenoloxidase inhibitor. Mass spectrometry analysis implicates HC as the protein species responsible for this activity. Based on the findings of this work it seems possible that the hemocyanin proteins of Limnoria aid the animal in digesting wood by contributing to the formation of reactive peroxide compounds in its gut which attack lignocellulose. This process may also contribute towards the sterility of the gut by creating an inhospitable environment for microbial colonisation. Further work is now required to determine the mechanisms by which it occurs.
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