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Purpose A large proportion of the European Union’s tomato crop is discarded during harvesting and there is a valorisation potential to recover proteins from this waste. Methods Cherry tomatoes were segregated into three separate components: juice, pomace (peels and skins), and seeds. The peels and skins, and seeds were separately hydrolyzed with carbohydrases to determine whether protein recovery could be increased. In addition, a strategy to fractionate the seeds was developed using sequential washing of milled tomato seeds, followed by low-speed centrifugation to remove the denser seed hulls and to collect the protein rich kernels remaining in suspension. Results The protein content of the seeds was highest with 27.4% while the peels and skins contained 7.6%. Carbohydrase mediated hydrolysis resulted in a minor increase in protein recovery of 10% from seeds using Filta 02L (cellulase, xylanase and β-glucanase), and the quantity of protein recovered from peels and skins increased by 210% using Tail 157 (pectinase, hemicellulase). The strategy to separate the seeds into two fractions, revealed that a higher proportion of the fibre (65%) was associated with the hull fraction compared with the original seeds (47%). A significant proportion of the fibre in this fraction was composed of lignin although the protein contents between both fractions was similar ranging from 27.4 to 29.9%. Conclusions These results reveal that carbohydrases were quite effective in protein extraction from peels and skins, but not from seeds. An alternative strategy was developed to remove the seed hulls from the milled seeds and to collect a fraction containing protein and dietary fibre where oil could be removed at this stage. Graphical Abstract

Introduction: Fusosomes are viral vectors pseudotyped with modified paramyxovirus envelopes targeting specific cell types. A CD8-targeted fusosome delivering CD19CAR transgene has the potential to provide an off-the-shelf therapeutic approach to generate CD19-directed chimeric antigen receptor (CAR) T cells in patients after direct intravenous delivery or extracorporeal delivery (ECD), i.e., a short-term exposure of apheresis product to fusosome before re-infusion into the patient. Here, we demonstrate that an improved manufacturing process resulted in higher fusosome titer and quality, increasing the potency of CAR T generation from resting CD8 T cells. This potency is also observed when fusosomes are evaluated in a clinically relevant mock ECD approach. Methods: Fusosome titer was quantified by ddPCR directly for physical titer (vector genomes, GQA) or after infections of SupT1 cells for functional titer (IU/mL). Transduction of CD8 T cells was measured by flow cytometry and vector copy number analysis after short-term (1-4 hour) incubation of PBMCs with fusosome followed by wash and culture in resting or CD3/CD28-activated conditions. To assess CD19CAR T cell generation at a mock clinical setting and scale, a healthy donor was apheresed to collect PBMCs, fusosome was administered into the apheresis bag and PBMCs were sampled from the bag over a 2.5-hour time course for further analysis. In vivo anti-tumor efficacy of the fusosome was measured in immune-deficient NSG mice engrafted intravenously with Nalm6, then PBMCs followed by fusosome one day later. Tumor growth was monitored by bioluminescence imaging. Results: Fusosomes produced by the improved process had both a higher functional concentration (>14-fold higher IU/mL) and better infectivity (>10-fold higher IU/GQA) in comparison to fusosomes produced by the original process. Exposure of PBMCs to fusosomes produced by the improved process led to increased fusosome binding to CD8 T cells at high IU/PBMC doses and higher transduction efficiency in CD8 T cells at the same calculated dose per cell compared to fusosomes produced by the original process (>3-fold higher mean CAR+ percentage after 2-hour exposure to 2 IU/PBMC, n=8). Furthermore, fusosomes produced by the improved process generated higher frequencies of CAR+ CD8 T cells and resulted in increased ability to control tumor growth at lower doses in vivo in a Nalm6 model. Importantly, the ECD approach translated well to a mock clinical setting, resulting in successful generation of CAR T cells (12.6% of CD8 T cells after a 2.5-hour incubation). Conclusion: CD8-targeted fusosome encoding a CD19CAR transgene represents a potential novel therapeutic approach for cancer. This study demonstrates that improvements in the manufacturing process can significantly increase the potency of the fusosome in T cells, and we intend to move forward to clinical trials using this improved process. Citation Format: Jesse Green, Brian Granger, Ouwen Liang, Jesus Moreno, Andrew Tucker, Aesha Vakil, Reuben Burch, Nicholas Mozdzierz, Matthew Scott-Skandera, Stephanie Riofrio, Mike DeCosta, Amey Gaikwad, Sanket Phadke, Kaely Gallagher, Adam Charlton, Sarah Miller, Hosein Kouros-Mehr, Misha Shamashkin, Neal Van Hoeven, Kutlu Elpek, Terry Fry. Increased potency of CD8-targeted fusosomes enhances CAR gene delivery to resting primary T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB311.

PurposeA large proportion of the European Union’s tomato crop is discarded during harvesting and there is a valorisation potential to recover proteins from this waste. MethodsCherry tomatoes were segregated into three separate components: juice, pomace (peels and skins), and seeds. The peels and skins, and seeds were separately hydrolyzed with carbohydrases to determine whether protein recovery could be increased. In addition, a strategy to fractionate the seeds using sequential washing of milled tomato seeds followed by low-speed centrifugation to remove the denser seed hulls and to collect the protein rich kernels remaining in suspension. ResultsThe protein content of the seeds was highest with 27.4% while the peels and skins contained 7.6%. Carbohydrase mediated hydrolysis revealed a minor increase in protein recovery from seeds by 10% using Filta 02L (cellulase, xylananse and β-glucanase), and the quantity of protein recovered from peels and skins increased by 210% using Tail 157 (pectinase, hemicellulase). The strategy to separate the seeds into two fractions, revealed that a higher proportion of the fibre (65%) was associated with the hull fraction compared with the original seeds (47%). A significant proportion of the fibre in this fraction was composed lignin although the protein contents between both fractions was similar ranging from 27.4% to 29.9%. ConclusionsThese results reveal that carbohydrases were quite effective in protein extraction from peels and skins, but not from seeds. An alternative strategy was developed to remove the seed hulls from the milled seeds and to collect a crude protein fraction where the protein content could be further improved.

The conversion of raw fruits and vegetables, including tomatoes into processed food products creates side streams of residues that can place a burden on the environment. However, these processed residues are still rich in bioactive compounds and in an effort to valorize these materials in tomato by-product streams, the main aim of this study is to extract proteins and identify the main phenolic compounds present in tomato pomace (TP), peel and skins (TPS) by HPLC-DAD-ESI-QTOF. Forty different phenolic compounds were identified in the different tomato extracts, encompassing different groups of phenolic compounds, including derivatives of simple phenolic acid derivatives, hydroxycinnamoylquinic acid, flavones, flavonones, flavonol, and dihydrochalcone. In the crude protein extract (TPE) derived from tomatoes, most of these compounds were still present, confirming that valuable phenolic compounds were not degraded during food processing of these co-product streams. Moreover, phenolic compounds present in the tomato protein crude extract could provide a valuable contribution to the required daily intake of phenolics that are usually supplied by consuming fresh vegetables and fruits.

During research of a broadly neutralizing antibody (bNAb) for HIV-1 infection, site-specific clipping was observed during cell culture incubation. Protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), was supplemented to the cell culture feeding to mitigate clipping as one of the control strategies. It led to the need and development of a new assay to monitor the free AEBSF-related impurities during the purification process. In this work, a reversed-phase liquid chromatography (RPLC-UV) method was developed to measure the total concentration of AEBSF and its major degradant product, 4-(aminoethyl) benzenesulfonic acid (AEBS-OH). This quantitative approach involved hydrolysis pre-treatment to drive all AEBSF to AEBS-OH, a filtration step to remove large molecules, followed by RPLC-UV analysis. The method was qualified and shown to be capable of measuring AEBS-OH down to 0.5 μM with good accuracy and precision, which was then applied for process clearance studies. The results demonstrated that a Protein A purification step in conjunction with a mock ultrafiltration/diafiltration (UF/DF) step could remove AEBSF-related impurities below the detection level. Overall, this study is the first to provide a unique approach for monitoring the clearance of free AEBSF and its related degradant, AEBS-OH, in support of the bNAb research.

The application of solid-state fermentation offers an alternative to conventional, submerged approaches for a variety of bioconversion processes, including animal feeds, biofuels and fungal bioproducts. Optimizing solid-state fermentation under low moisture conditions could significantly impact the proportion of dry biomass that could be processed and improve the commercial viability of this approach, because of reduced input costs and higher yields of final products. Pleurotus erygnii that appeared to show tolerance to low moisture conditions was grown on saturated and desaturated wheat straw. Pleurotus erygnii showed insignificant fibre degradation although showed significantly lower biomass decomposition on desaturated wheat straw. Fibre decomposition by the fungus on wheat straw containing wheat bran showed marginally higher decomposition when saturated although there was no difference in biomass decomposition. The levels of delignification achieved were similar under different saturation conditions. It would appear that the fungus effectively decomposed fibre under low moisture conditions often resulting in lower biomass losses. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, a white rot fungus, Pleurotus erygnii, effectively decomposed fibre under low moisture conditions when grown on wheat straw at similar levels under higher moisture conditions. However, the addition of wheat bran to wheat straw created a heterogeneous system that appeared to allow P. erygnii to thrive under much lower moisture conditions although lower levels of fibre decomposition was obtained. These factors could influence the preparation of solid-state fermentation.

Graphical abstract, Highlights • In-line electroanalysis of d-glucose during bioethanol production. • Second-generation biosensor suitable for industrial process analytical technology. • Greater d-glucose yield from wheat straw refined under pressure. • d-Glucose productivity is 20–40 mg/(L h) over a 5 L scale., Mechanical pre-treatment (disc refining) of wheat straw, at both atmospheric and elevated pressure, is shown to be an efficient process to access fermentable monosaccharides, with the potential to integrate within the infrastructure of existing first-generation bioethanol plants. The mild, enzymatic degradation of this sustainable lignocellulosic biomass affords ca. 0.10–0.13 g/g (dry weight) of d-glucose quantifiable voltammetrically in real time, over a two hundred-fold range in experimental laboratory scales (25 mL to 5.0 L), with pressure disc refining of the wheat straw enabling almost twice the amount of d-glucose to be generated during the hydrolysis stage than experiments using atmospheric refining (0.06–0.09 g/g dry weight). Fermentation of the resulting hydrolysate affords 0.08–0.10 g/g (dry weight) of ethanol over similar scales, with ethanol productivity at ca. 37 mg/(L h). These results demonstrate that minimal cellulose decomposition occurs during pressure refining of wheat straw, in contrast to hemicellulose, and suggest that the development of green, mechanochemical processes for the scalable and cost-effective manufacture of second-generation bioethanol requires improved cellulose decomposition.

The valorization of olive pomace through the extraction of phenolic compounds at an industrial scale is influenced by several factors that can have a significant impact on the feasibility of this approach. These include the types and levels of phenolic compounds that are present, the impact that seasonal variation and cultivar type have on the phenolic compound content in both olive pomace and mill effluents and the technological approach used to process the olive crop. Chemical analysis of phenolic compounds was performed using an HPLC-diode-array detector (DAD)-qTOF system, resulting in the identification of 45 compounds in olive mill wastewater and pomace, where secoiridoids comprised 50&ndash, 60% of the total phenolic content. This study examined three different factors that could impact the phenolic compound content of these processing streams, including cultivar types typically grown on local farms in Slovenia, the type of downstream processing used and seasonality effects. Olive crop varieties sourced from local farms showed high variability, and the highest phenolic content was associated with the local variety &ldquo, Istrska Belica&rdquo, During processing, the phenolic content was on average approximately 50% higher during two-phase decanting compared to three-phase decanting and the type of compound present significantly different. An investigation into the seasonal effects revealed that the phenolic content was 20% higher during the 2019 growing season compared to 2018. A larger sample size over additional growing seasons is required to fully understand the annual variation in phenolic compound content. The methods and results used in this study provide a basis for further analysis of phenolic compounds present in the European Union&rsquo, s olive crop processing residues and will inform techno-economic modelling for the development of olive biorefineries in Slovenia.

The agricultural production of olives, rapeseed, tomatoes and citrus fruits within Europe is significant, resulting in a considerable amount of residual material. Rapeseed contains a high proportion of protein but the presence of anti-nutritional components, including glucosinolates, limits its use in food and feed applications. In contrast, the protein quantities associated with the other crop residues are much lower, although each of the residues could be separated into different constitutive parts where some have shown higher protein contents. A variety of different enzymatic based approaches to deconstruct crop residues have shown to be effective in increasing the yields of protein recovered. These studies show that valorisation of selected crop components could form the basis of a crop biorefinery process to capture proteins and other potentially useful compounds.

This preliminary study assesses a potential pre-treatment approach for agricultural residues, in order to improve enzyme access and cellulose digestibility that increased with increasing refining pressure. Wheat straw, an important European agri-residue, was chopped then refined at pilot-scale under different pressures (4–10 bar) and two refiner plate configurations. The most energy efficient runs used 0.94–0.96 kWh electricity; 8.9–11.0 MJ heat per kg dry matter fibre. A scaling factor specific to the machinery used in the trial suggested that wheat straw could be refined using approximately 160 kWh electricity and 980–1900 MJ heat per tonne DM yield at commercial-scale. Hemicellulose content in wheat straw at 31.8% decreased to the lowest level of 14.6% after refining at 10 bar. Pressurised disc refining did not appear to produce significant quantities of acetic acid, a key fermentation inhibitor, that could limit microbial fermentation.

Six types of lignin-carbohydrate complex (LCC) fractions were isolated from Eucalyptus. The acidic dioxane treatment applied significantly improved the yield of LCCs. The extraction conditions had a limited impact on the LCC structures and linkages. Characterization of the lignin-carbohydrate complex (LCC) structures and linkages promises to offer insight on plant cell wall chemistry. In this case, Eucalyptus LCCs were extracted by aqueous dioxane, and then precipitated sequentially by 70% ethanol, 100% ethanol, and acidic water (pH = 2). The composition and structure of the six LCC fractions obtained by selective precipitation were investigated by sugar analysis, molecular weight determination, and 2D HSQC NMR. It was found that the acidic (0.05-M HCl) dioxane treatment significantly improved the yield of LCCs (66.4% based on Klason lignin), which was higher than the neutral aqueous dioxane extraction, and the extraction condition showed limited impact on the LCC structures and linkages. In the fractionation process, the low-molecular-weight LCCs containing a high content of carbohydrates (60.3-63.2%) were first precipitated by 70% ethanol from the extractable solution. The phenyl glycoside (PhGlc) bonds (13.0-17.0 per 100Ar) and highly acetylated xylans were observed in the fractions recovered by the precipitation with 100% ethanol. On the other hand, such xylan-rich LCCs exhibited the highest frequency of β-O-4 linkages. The benzyl ether (BE) bonds were only detected in the fractions obtained by acidic water precipitation.

The removal of heavy metals ions from wastewater by an economic, high-effective, and environmentally friendly method is particularly important. In this study, an effective lignin-based bio-adsorbent (SAPL-1.5), which contained specific functional groups and spatial cross-linking structures, was synthesized through chemical modification. SAPL-1.5 was comprehensively characterized by 31P, 1H, 13C NMR, and elemental analysis as compared to the raw lignin. The results showed that the chemical reactivity of lignin was significantly improved after phenolation process, and the adsorption groups were successfully grafted onto lignin macromolecule. In addition, the influences of pH, SAPL-1.5 dosage, contact time, and initial Pb (II) concentration on the adsorption performance was systematically investigated. The highest adsorption capacity reached to 130.2 mg/g (Pb (II), 140 mg/L), and a removal efficiency of 100% was achieved (Pb (II), 20 mg/L). Moreover, the adsorption isotherm and adsorption kinetics indicated that the results were fitting well with the Langmuir and pseudo-second-order model, respectively. Furthermore, the removal efficiency of SAPL-1.5 for Pb (II) (20 mg/mL) still maintained over 85% after 5 cycles. Therefore, the lignin-based material obtained could be considered as a promising potential adsorbent with a low cost, high performance and reutilization for its application in the wastewater treatment process. It is believed that the lignin-based bio-sorbent can enlarge the lignin valorization in the current biorefinery process.

The fungal enzyme activities and breakdown of wood components may be influenced by the fibrous and structural composition across different wood species. White-rot fungi were inoculated onto wood chips of F. excelsior, A. pseudoplatanus or Q. robur and incubated for 28 days revealing that most fungi appeared to successfully colonize the different types of wood chips. Fibre analysis revealed that the fungi causing the highest mass losses in F. excelsior and A. pseudoplatanus were those that degraded more cellulose compared with hemicellulose or lignin. Fungal degradation leading to high mass losses of Q. robur was more complicated as lignin-degrading activities became more important. The structural composition in terms of the largest vessel sizes only showed an inverse correlation with remaining moisture content and not with mass loss or fibre degradation. These results provide an insight into fungal degradation of wood from three common tree species, and the link between the compositional characteristics of each wood type and the ease of degradation. This could have an impact on future biological pre-treatment strategies and valorisation approaches for forestry residues in integrated biorefineries.

Plastics accumulating in the environment, especially microplastics (defined as particles

Anionic, cationic, and nonionic surfactants with varying concentrations (0.2–1.2%) were introduced to neutralize beads on lignin nanofibers by decreasing the surface tension of spinning dopes. The surfactants used in this work were sodium dodecyl sulfate (SDS), N,N,N-trimethyl-1-dodecanaminium bromide (DTAB), and Triton X-100 (TX-100). The effects of viscosity, rheological properties, surface tension, and conductivity of the solutions on the morphology and physicochemical performances of fibers were investigated. As expected, the presence of certain amounts of surfactants eliminated the beads and resulted in the formation of smooth and bead-free fibers with small diameters. Furthermore, the gravimetric capacitance of the carbon mat with 1% SDS was slightly improved from 66.3 to 80.7 F g–1. The results suggested that the surfactants benefit from the electrospinning of lignin and allow for the control over nanofiber morphology without compromising their performance as supercapacitor electrodes.

A representative lignin was firstly prepared and characterized as well as an efficient lignin depolymerization process with highly controllable products was presented using Cs-substituted tungstophosphate (CsTP) cooperated with Raney Ni in the present study. The double enzymatic lignin (DEL) was depolymerized efficiently in the temperature range of 250–280 °C. The synergistic effects of Raney Ni with CsTP and temperature on the degradation of lignin were investigated by FT-IR, NMR, GPC, and GC-MS techniques. Under the optimal condition with tandem catalyst at 270 °C for 3 h, the yield of depolymerized DEL was over 70 wt% and phenolic monomers were over 20%. The weight average molecular weight of the lignin was reduced significantly from 15,770 to 1150 g/mol (for aqueous phase lignin) and about 420 g/mol (organic phase lignin). In addition, only a few syringols and cyclohexanols were obtained, indicating that this tandem catalyst facilitates the depolymerization and demethoxylation but prevents hydrogenation of benzene ring of lignin. More importantly, the formation of char was restrained effectively in this process.

This paper assesses the environmental impact of pilot-scale wood fibre production across a range of refining pressures using energy monitoring at an individual component level. Under optimal refining conditions (8 bar refining pressure; 15 µm plate gap width), electricity use accounted for 88% of greenhouse gas (GHG) emissions associated with the fibre production, and was the dominant input in almost all other impact categories. At an individual component level, the refiner, dryer fan and hot oil burner were the most significant parts of the process (representing 30, 20 and 16% of cradle-to-gate GHGs, respectively), while ancillary processes such as the air compressor (6.9%) and dust extraction (6.3%) also made notable contributions. The analysis suggests that energy efficiencies made with these key components may offer the best potential gains in terms of the environmental profile of pressurised refining, as long as these can be achieved without compromising yield or quality of the fibre produced.

Pressure refining, a pulp making process to separate fibres of lignocellulosic materials, deposits lignin granules on the surface of the fibres that could enable increased access to lignin degrading enzymes. Three different white rot fungi were grown on pressure refined (at 6 bar and 8 bar) and milled Miscanthus. Growth after 28 days showed highest biomass losses on milled Miscanthus compared to pressure refined Miscanthus. Ceriporiopsis subvermispora caused a significantly higher proportion of lignin removal when grown on 6 bar pressure refined Miscanthus compared to growth on 8 bar pressure refined Miscanthus and milled Miscanthus. RM22b followed a similar trend but Phlebiopsis gigantea SPLog6 did not. Conversely, C. subvermispora growing on pressure refined Miscanthus revealed that the proportion of cellulose increased. These results show that two of the three white rot fungi used in this study showed higher delignification on pressure refined Miscanthus than milled Miscanthus.

There are many factors influencing the current global interest in the biorefining of biomass feedstocks to produce a wide variety of extracts, fuels and chemicals. Identifying renewable sources of target molecules currently produced from fossil fuels is one of these, which will ultimately have a positive impact on climate change. Another driver is identifying potential uses for land masses, where a low GDP is affecting communities in those areas. From a UK perspective, a decline in total income generated from farming in Wales has had a detrimental impact on many communities right across the Principality. There is a considerable body of data to argue that with effective land use and the use of a range of enzymic and chemical processing technologies, the utilisation of lignocellulosic biomass as feedstock for a biorefining industry would result in both social and economic regeneration of these rural communities. In order to create a sustainable biorefining in Wales, alongside regeneration of the rural economy there is a requirement for expansion of the high technology skill base, in areas such as chemistry, biotechnology and engineering. The key to developing this sustainable and economically viable biorefining industry within Wales and ultimately in the UK is based on several technical issues which need to be addressed. These include ensuring that the feedstocks can be grown on marginal land which will not therefore compete with traditional food crops, the need to create local supply chains linking regions together, but which can also feed into the rapidly expanding networks of biomass-based industrial activity in other areas of the UK, and ensuring that the existing transport infrastructure can absorb this increase in activity. This paper will consider the options for large scale biorefining of high sugar perennial ryegrasses in Wales, as a model for producing sustainable, bulk quantities of chemicals, including biofuels.

The synthesis, electrochemical and spectroscopic properties of a series of thiophene-substituted 1,3-dithiole-2-ones is described. The derivatives Th-3,3, Th-2,2, Th-2,3, Th-3,3(2,2′-Me), and Th3,3-(2,5,2′,5′-Me), have been successfully polymerized by cyclic voltammetry. From the UV−visible spectra of the neutral films, it was determined that PTh-3,3, PTh-2,2 and PTh-2,3 have a bandgap of 2.04, 2.30, and 2.18 eV, respectively. The voltammetric response of Th-3,3(2,2′-Me) was noticeably different from the other polymers. The SNIFTIRS data suggested that Th-3,3(2,2′-Me) was formed by bonding via the β-positions of the thiophene ring, resulting in a poorly conductive polymer. The in situ solid-state modification of PTh-3,3 to produce a new TTF-derivatized polythiophene was carried out. The cyclic voltammetry of the modified polymer confirmed the inclusion of TTF into the backbone of the film.

The synthesis, electrochemical and spectroscopic properties of the new thiophene-substituted metal dithiolene complexes [Ni(b-3ted)2] (3) and [NBu4][Ni(b-3ted)2] ([NBu4][3]) are described [b-3ted = bis(3-thienyl)-1,2-ethylenedithiolene] and compared with new studies of the known compounds [Ni(b-2ted)2] (2) and [NBu4][Ni(b-2ted)2] ([NBu4][2]). X-Ray structures were determined for the neutral complexes 2 and 3 and both showed planar nickel dithiolene units with thiophene groups twisted out of the molecular plane, precluding close molecular π-stacking. Electrochemical investigations revealed two redox processes corresponding to interconversion between neutral, monoanionic and dianionic species for 3, analogous to 2. Oxidation of 0.1 mM [3]− in CH3CN was shown to give a green molecular film of 3 which, in contrast to as-prepared crystals of 3, showed significant conductivity. Electrochemical polymerisation of 3 carried out at different concentrations resulted in films; electrochemical, SNIFTIRS and UV-Vis results were consistent with incorporation of the intact metal dithiolene complex in these films. In contrast, electrochemical polymerisation of 2 gave a film charateristic of a polymerised thiophene but with no evidence of incorporation of the intact metal dithiolene complex. Copolymerisation of 3 with thiophene gave a gold-coloured electropolymerised film of significant conductivity that, upon dissolution in CH2Cl2, showed the characteristic low energy absorption of the Ni-dithiolene complex, confirming its incorporation. This copolymerisation approach offers a versatile route to the synthesis of metal dithiolene–polythiophene hybrid films.

Differences have been shown between ryegrass and fescue within the Festulolium subline introgression family for fructan synthesis, metabolism and polymer-size traits. It is well-established that there is considerable variation for water-soluble carbohydrate and fructan content within perennial ryegrass. However there is much still to be discovered about the fructan polymer pool in this species, especially in regard to its composition and regulation. It is postulated that similar considerable variation for polymer traits may exist, providing useful polymers for biorefining applications. Seasonal effects on fructan content together with fructan synthesis and polymer-size traits have been examined in diverse perennial ryegrass material comprising contrasting plants from a perennial ryegrass F2 mapping family and from populations produced by three rounds of phenotypic selection. Relationships with copy number variation in candidate genes have been investigated. There was little evidence of any variation in fructan metabolism across this diverse germplasm under these conditions that resulted in substantial differences in the complement of fructan polymers present in leaf tissue at high water-soluble carbohydrate concentrations. The importance of fructan synthesis during fructan accumulation was unclear as fructan content and polymer characteristics in intact plants during the growing season did not reflect the capacity for de novo synthesis. However, the retention of fructan in environmental conditions favouring high sink / low source demand may be an important component of the high sugar trait and the roles of breakdown and turnover are discussed.

The fructans found as storage carbohydrates in temperate forage grasses have a physiological role in regrowth and stress tolerance. They are also important for the nutritional value of fresh and preserved livestock feeds, and are potentially useful as feedstocks for biorefining. Seasonal variation in fructan content and the capacity for de novo fructan synthesis have been examined in a Festulolium monosomic substitution line family to investigate variation in the polymers produced by grasses in the ryegrass-fescue complex. There were significant differences between ryegrass and fescue. Fescue had low polymeric fructan content and a high oligomer/polymer ratio; synthesis of polymers longer than degree of polymerisation 6 (DP6) from oligomers was slow. However, extension of polymer length from DP10/DP20 upwards appeared to occur relatively freely, and, unlike ryegrass, fescue had a relatively even spread of polymer chain lengths above DP20. This included the presence of some very large polymers. Additionally fescue retained high concentrations of fructan, both polymeric and oligomeric, during conditions of low source/high sink demand. There were indications that major genes involved in the control of some of these traits might be located on fescue chromosome 3 opening the possibility to develop grasses optimised for specific applications.

Aim The objective of this work was to determine if the existence of ancient forests on cliffs was specific to the Niagara Escarpment, Canada, or part of a globally widespread pattern. Location Sixty-five cliff sites were visited in five countries in the temperate climatic zone, and trees were sampled for age and growth rate on forty-six of these. Methods Two hundred and twenty-four core samples or cross-sections were taken from trees on cliffs that varied in height, aspect, rock-type, and exposure. General observations were also made of regeneration of the tree species forming the mature canopy, and other habitat conditions. Results The evidence shows that ancient slow-growing forest occurs on most cliffs. Age and growth rate distributions were similar at all treed sites. Small-statured Thuja, Juniperus, or Taxus stems with age estimates in excess of 1000 years were found in the United States, the United Kingdom and France, and small Pinus and Quercus stems nearly 400 years in Germany. There was a high rate of recurrence of plants in the genera Polypodium, Asplenium, Cystopteris, Campanula, Rosa, Prunus, Hedera, and Sorbus. Most of the sites appear to be habitats of completely natural origin. Conclusions We conclude that ancient natural forest is a normal feature of cliffs, at least in the temperate zone.

The synthesis and solution electrochemical properties of two novel 3-thienyl substituted tetrathiafulvalene (TTF) donors 7 and 11 are described. The single-crystal X-ray structure of 7 is reported, which is characterized by the presence of short intermolecular SS contacts. Cyclic voltammetry of the derivatives indicates that two reversible redox waves, characteristic of TTF derivatives, are present. Attempts were made to electropolymerize the two derivatives at a platinum electrode in dichloromethane. Formation of a blue-coloured solid, characteristic of the thiophene radical cation species, was observed for one of the derivatives, but it was not possible to isolate a polymer on the working electrode. Preliminary electropolymerization studies on the TTF ‘half unit’ 6 are also reported.

Attempted oxidative photolytic conversion of tetrathiofulvalenes (I) to planar conjugated systems results in unexpected formation of the novel heterocyclic system (II).

Irradiation of tetrathienyltetrathiafulvalenes in an oxygen saturated solution gives tetrathienyl substituted 1,2,5,8-tetrathiecine-6,7-diones, via singlet oxygen generation followed by cycloaddition onto the TTF double bond and a final rearrangement process. The thermal and photochemical behaviour and reductive methylation of this new class of macro-heterocyclic system have also been investigated.

Recent developments in the synthesis of metal complexes of sulphur-donor ligands are discussed, together with an account of the preparation of the new 1,2-dithiolate ligand 6-methylene-1,4-dithiepin-2,3-dithiol (H 2 dpdt). The structure and electrochemical properties of [Bu 4 N][Cu(dpdt) 2 ] are described.

[Ni(dpdt) 2 ] (H 2 dpdt = 6-methylene-1,4-dithiepin-2,3-dithiol) has been prepared from [NBu 4 ][Ni(dpdt) 2 ], using both chemical and electrochemical methods. Electrical conduction studies indicate that the compound is a semiconductor (σ RT = 10 −5 S cm −1 ). The X-ray crystal structure of [Ni(dpdt) 2 ] is reported.

The synthesis and physical properties of a series of soluble, tetrakis(n-alkylthio)nickel bis(ethylene-1,2-dithiolene), [TTCn-Ni(edt)2] complexes (n= 4–11) is described. The room-temperature electrical conductivities of these materials in the undoped state are reported and the X-ray crystal structures of the n= 4 and 6 derivatives have been determined.

NH4Ni(mnt)2. H2O shows localized moments with antiferromagnetic coupling at room temperature. The antiferromagnetism persists down to 100K at which point there is an abrupt change in the slope of the susceptibility corresponding to an anomaly observed in the resistivity. Below 100K there is evidence for ferromagnetic coupling between the spins. The crystal has an orthorhombic space group Pnam, with a=12.096A, b=3. 918A, c=29. 216A, U=1384. 8A3 and Z = 4, and the crystal structure is highly anisotropic, in common with other charge transfer salts of this type. The anions form uniform stacks along the b-axis in which the molecules are tilted by 26° to the stacking axis. Results are presented here on the electrical and magnetic behaviour of this salt.

We report on the magnetic and transport properties of the organic salt NH 4 Ni(mnt) 2 H 2 O where (mnt) is the maleonitrile dithiolate ligand. This material is antiferromagnetic down to 100K, below which there is a transition to a paramagnetic state with ferromagnetic exchange. This is accompanied by an increase in resistivity by a factor of two which we consider indicative of a structural phase transition. Long range ferromagnetic order is seen below 4.9K.

The synthesis and electrochemical properties of several maleimide derivatives of 3-(2-aminoethyl)thiophene are described. Of the four soluble thiophene derivatives prepared only one, N -{3-(2-ethyl)thiophene}-3,4-dichloromaleimide, was capable of being polymerized electrochemically. The resulting polymer exhibited a conductivity of −5 S cm −1 . The influence that bulky maleimide groups have on preparing maleimide-substituted polythiophenes is discussed.

Where an affinity tag has served its purpose it may become desirable to remove it from the protein of interest. This chapter describes the removal of such fusion partners from the intended protein product by cleavage with site-specific endoproteases. Methods to achieve proteolytic cleavage of the fusion proteins are provided, along with techniques for optimising the yield of authentic product.

Where an affinity tag has served its purpose it may become desirable to remove it from the protein of interest. This chapter describes the removal of such fusion partners from the intended protein product by cleavage with site-specific endoproteases. Methods to achieve proteolytic cleavage of the fusion proteins are provided, along with techniques for optimising the yield of authentic product.

The preparation of a poly(maleimide-ether) containing pendent thiophene rings via the reaction of 2-(3-thienylmethyl)-1,3-N,N'-bis(3,4-dichloromaleimido) propane with 1,4-dihydroxybenzene is described. The novel 2-substituted-1,3-N,N'-bis(3,4-dichloromaleimido) propanes were obtained by allowing their corresponding diamine hydrochlorides to react with 3,4-dichloromaleic anhydride in acetic acid in the presence of sodium methoxide. The diamine hydrochlorides were prepared by the reduction of their respective diamides. The poly(maleimide-ether)s were fully characterized and 13C-NMR spectroscopy was used to confirm the microstructure of the materials. The properties of the thiophene substituted polymer are compared with model poly(maleimide-ether)s. Electrochemical studies indicated that the thiophene rings in the thiophene-substituted poly(maleimide-ether) were not electroactive. © 1992 John Wiley & Sons, Inc.

The synthesis, structure and spectroscopic properties of a new class of asymmetric nickel dithiolene complex containing pendent thiophene units [Ni(R2pipdt)(b-3ted)] are described {R = Bz (3), iPr (4); pipdt = piperazine-3,2-dithione; b-3ted = bis(3-thienyl)-1,2-ethylenedithiolene}. X-ray crystallography of both neutral species shows planar nickel dithiolene units and the thiophene groups twisted out of the molecular plane, with the R group having a large effect on the packing structure. DFT geometry calculations show good agreement with experimental data for 3 and 4 and confirm that the low energy absorption, assigned to a HOMO → LUMO transition, is inter-ligand charge-transfer in character. Solid state calculations show greater band dispersion in 4, which corresponds to a closer packing in the crystal lattice. Cyclic voltammetry of both complexes displays three reversible processes, corresponding to an interconversion between cationic, neutral, anionic and dianionic species, with tuning of the redox potentials by variation of the R group. Electrochemical polymerisation of 3 shows incorporation of intact dithiolene units into a polymer film with redox functionality similar to that of the monomer, whereas co-polymerisation of 3 with thiophene yields a highly conductive film with redox functionality similar to polythiophene and enhanced absorption across the near IR.

Immobilized metal affinity chromatography (IMAC) is a common place technique in modem protein purification. IMAC is distinct from most other affinity chromatography technologies in that it can operate on a native, unmodified protein without the need for a specialized affinity "tag" to facilitate binding. This can be particularly important where a protein of interest is to be separated from a complex mixture such as serum or an environmental isolate. Relying on the interaction of specific surface amino acids of the target protein and chelated metal ions, IMAC can provide powerful discrimination between small differences in protein sequence and structure. Additionally, IMAC supports have been demonstrated to function effectively as cation exchangers, allowing for two modes of purification with a single column. This chapter provides methodologies to perform IMAC in its most fundamental form, that of the interaction between histidine and immobilized metal ions, those that enable purification of proteins that lack surface histidines and the operation of IMAC supports in cation exchange mode.

Where an affinity tag has served its purpose, it may become desirable to remove it from the protein of interest. This chapter describes the removal of such fusion partners from the intended protein product by cleavage with site-specific endoproteases. Methods to achieve proteolytic cleavage of the fusion proteins are provided, along with techniques for optimizing the yield of authentic product.

Immobilized metal affinity chromatography (IMAC) is a common place technique in modem protein purification. IMAC is distinct from most other affinity chromatography technologies in that it can operate on a native, unmodified protein without the need for a specialized affinity "tag" to facilitate binding. This can be particularly important where a protein of interest is to be separated from a complex mixture such as serum or an environmental isolate. Relying on the interaction of specific surface amino acids of the target protein and chelated metal ions, IMAC can provide powerful discrimination between small differences in protein sequence and structure. Additionally, IMAC supports have been demonstrated to function effectively as cation exchangers, allowing for two modes of purification with a single column. This chapter provides methodologies to perform IMAC in its most fundamental form, that of the interaction between histidine and immobilized metal ions, those that enable purification of proteins that lack surface histidines and the operation of IMAC supports in cation exchange mode.