Effect of eicosapentaenoic acid, protein and amino acids on protein synthesis and degradation in skeletal muscle of cachectic mice

Atrophy of skeletal muscle reduces both the quality and quantity of life of patients with cancer cachexia. Loss of muscle mass is thought to arise from a reduction in protein synthesis combined with an enhanced rate of protein degradation, and few treatments are available to counteract this process. Eicosapentaenoic acid (EPA) has been shown to attenuate the enhanced protein degradation, but to have no effect on protein synthesis. This study examines the effect of EPA combined with a protein and amino-acid supplementation on protein synthesis and degradation in gastrocnemius muscle of mice bearing the cachexia-inducing MAC16 tumour. Muscles from cachectic mice showed an 80% reduction in protein synthesis and about a 50-fold increase in protein degradation compared with muscles from nontumour-bearing mice of the same age and weight. Treatment with EPA (1 g kg-1) daily reduced protein degradation by 88%, but had no effect on protein synthesis. Combination of EPA with casein (5.35 g kg-1) also had no effect on protein synthesis, but when combined with the amino acids leucine, arginine and methionine there was almost a doubling of protein synthesis. The addition of carbohydrate (10.7 g kg-1) to stimulate insulin release had no additional effect. The combination involving the amino acids produced almost a doubling of the ratio of protein synthesis to protein degradation in gastrocnemius muscle over that of EPA alone. No treatment had a significant effect on tumour growth rate, but the inclusion of amino acids had a more significant effect on weight loss induced by the MAC16 tumour than that of EPA alone. The results suggest that combination therapy of cancer cachexia involving both inhibition of the enhanced protein degradation and stimulation of the reduced protein synthesis may be more effective than either treatment alone. © 2004 Cancer Research UK.

(Table 2) Concentrations and D/L values for three amino acids in bulk foraminifera from ODP Sites 182-1126 and 182-1127

A number of studies have shown that methanogens are active in the presence of sulfate under some conditions. This phenomenon is especially exemplified in carbonate sediments of the southern Australian continental margin. Three sites cored during Ocean Drilling Program (ODP) Leg 182 in the Great Australian Bight have high concentrations of microbially-generated methane and hydrogen sulfide throughout almost 500 m of sediments. In these cores, the sulfate-reducing and methanogenic zones overlap completely; that is, the usual sulfate-methane transition zone is absent. Amino acid racemization data show that the gassy sediments consist of younger carbonates than the low-gas sites. High concentrations of the reduced gases also occur in two ODP sites on the margin of the Bahamas platform, both of which have similar sedimentary conditions to those of the high-gas sites of Leg 182. Co-generation of these reduced gases results from an unusual combination of conditions, including: (1) a thick Quaternary sequence of iron-poor carbonate sediments, (2) a sub-seafloor brine, and (3) moderate amounts of organic carbon. The probable explanation for the co-generation of hydrogen sulfide and methane in all these sites, as well as in other reported environments, is that methanogens are utilizing non-competitive substrates to produce methane within the sulfate-reducing zone. Taken together, these results form the basis of a new model for sulfate reduction and methanogenesis in marine sediments. The biogeochemical end-members of the model are: (1) minimal sulfate reduction, (2) complete sulfate reduction followed by methanogenesis, and (3) overlapping sulfate reduction and methanogenesis with no transition zone.

The impact of fluctuating light on the dinoflagellate Prorocentrum micans depends on NO3- and CO2 availability

Increasing atmospheric pCO2 and its dissolution into oceans leads to ocean acidification and warming, which reduces the thickness of upper mixing layer (UML) and upward nutrient supply from deeper layers. These events may alter the nutritional conditions and the light regime to which primary producers are exposed in the UML. In order to better understand the physiology behind the responses to the concomitant climate changes factors, we examined the impact of light fluctuation on the dinoflagellate Prorocentrum micans grown at low (1 µmol/L) or high (800 µmol/L) [NO3(-)] and at high (1000 µatm) or low (390 µatm, ambient) pCO2. The light regimes to which the algal cells were subjected were (1) constant light at a photon flux density (PFD) of either 100 (C100) or 500 (C500) µmol/m**2/s or (2) fluctuating light between 100 or 500 µmol photons/m**2/s with a frequency of either 15 (F15) or 60 (F60) min. Under continuous light, the initial portion of the light phase required the concomitant presence of high CO2 and NO3(-) concentrations for maximum growth. After exposure to light for 3h, high CO2 exerted a negative effect on growth and effective quantum yield of photosystem II (F'(v)/F'(m)). Fluctuating light ameliorated growth in the first period of illumination. In the second 3h of treatment, higher frequency (F15) of fluctuations afforded high growth rates, whereas the F60 treatment had detrimental consequences, especially when NO3(-) concentration was lower. F'(v)/F'(m) respondent differently from growth to fluctuating light: the fluorescence yield was always lower than at continuous light at 100 µmol/m**2/s, and always higher at 500 µmol/m**2/s. Our data show that the impact of atmospheric pCO2 increase on primary production of dinoflagellate depends on the availability of nitrate and the irradiance (intensity and the frequency of irradiance fluctuations) to which the cells are exposed. The impact of global change on oceanic primary producers would therefore be different in waters with different chemical and physical (mixing) properties.

Granulometry, geochemistry, amino acids, radiocarbon ages and paleomagnetics of samples from Heidemann Valley

A Pliocene (2.6-3.5 Ma) age is determined from glacial sediments studied in a 20m long, 4 m deep trench excavated in Heidemann Valley, Vestfold Hills, East Antarctica. The age determination is based on a combined study of amino acid racemization, diatoms, foraminifera, and magnetic polarity, and supports earlier estimates of the age of the sedimentary section; all are beyond 14C range. Four till units are recognized and documented, and 16 subunits are identified. All are ascribed to deposition during a Late Pliocene glaciation that was probably the last time the entire Vestfold Hills was covered by an enlarged East Antarctic Ice Sheet (EAIS). Evidence for other more recent glacial events of the 'Vestfold Glaciation' may have been due to lateral expansion of the Sorsdal Glacier and limited expansion of the icesheet margin during the Last Glacial Maximum rather than a major expansion of the EAIS. The deposit appears to correlate with a marine deposition event recorded in Ocean Drilling Program Site 1166 in Prydz Bay, possibly with the Bardin Bluffs Formation of the Prince Charles Mountains and with part of the time represented in the ANDRILL AND-1B core in the Ross Sea.

Carbon-supported ionic liquids as innovative adsorbents for CO2 separation from synthetic flue-gas

Fixed-bed thermodynamic CO2 adsorption tests were performed in model flue-gas onto Filtrasorb 400 and Nuchar RGC30 activated carbons (AC) functionalized with [Hmim][BF4] and [Emim][Gly] ionic liquids (IL). A comparative analysis of the CO2 capture results and N2 porosity characterization data evidenced that the use of [Hmim][BF4], a physical solvent for carbon dioxide, ended up into a worsening of the parent AC capture performance, due to a dominating pore blocking effect at all the operating temperatures. Conversely, the less sterically-hindered and amino acid-based [Emim][Gly] IL was effective in increasing the AC capture capacity at 353 K under milder impregnation conditions, the beneficial effect being attributed to both its chemical affinity towards CO2 and low pore volume reduction. The findings derived in this work outline interesting perspectives for the application of amino acid-based IL supported onto activated carbons for CO2 separation under post-combustion conditions, and future research efforts should be focused on the search for AC characterized by optimal pore size distribution and surface properties for IL functionalization.

Metabolic channelingof phe for lignin biosynthesis in maritime pine

METABOLIC CHANNELING OF PHE FOR LIGNIN BIOSYNTHESIS IN MARITIME PINE Jorge El-Azaz, Fernando de la Torre, Belén Pascual, Concepción Ávila and Francisco M. Cánovas Departamento de Biología Molecular y Bioquímica, Universidad de Málaga. Málaga, Spain Email: jelazaz@alu.uma.es The amino acid phenylalanine (Phe) is the main precursor of phenylpropanoids biosynthesis in plants. This vast family of Phederived compounds can represent up to 30% of captured photosynthetic carbon, playing essential roles in plants such as cell wall components, defense molecules, pigments and flavors. In addition to its physiological importance, phenylpropanoids and particularly lignin, a component of wood, are targets in plant biotechnology. The arogenate pathway has been proposed as the main pathway for Phe biosynthesis in plants (Maeda et al., 2010). The final step in Phe biosynthesis, catalyzed by the enzyme arogenate dehydratase (ADT), has been considered as a key regulatory point in Phe biosynthesis, due to its key branch position in the pathway, the multiple isoenzymes identified in plants and the existence of a feedback inhibition mechanism by Phe. So far, the regulatory mechanisms underlying ADT genes expression have been poorly characterized, although a strong regulation of the Phe metabolic flux should be expected depending on its alternative use for protein biosynthesis versus phenylpropanoid biosynthesis. This second fate involves a massive carbon flux compared to the first one. In this study we report our current research activities in the transcriptional regulation of ADT genes by MYB transcription factors in the conifer Pinus pinaster (maritime pine). The conifers channels massive amounts of photosynthetic carbon for phenylpropanoid biosynthesis during wood formation. We have identified the complete ADT gene family in maritime pine (El-Azaz et al., 2016) and a set of ADT isoforms specifically related with the lignification process. The potential control of transcription factors previously reported as key regulators in pine wood formation (Craven-Bartle et al., 2013) will be presented. Maeda et al. (2010) Plant Cell 22: 832-849. El-Azaz et al. (2016) The Plant Jounal. Accepted article, doi: 10.1111/tpj.13195 Craven-Bartle et al. (2013). The Plant Journal 74(5):755-766

In vitro and in vivo examination of the cell surface glycoprotein CDCP1

A number of reports have demonstrated the importance of the CUB domaincontaining protein 1 (CDCP1) in facilitating cancer progression in animal models and the potential of this protein as a prognostic marker in several malignancies. CDCP1 facilitates metastasis formation in animal models by negatively regulating anoikis, a type of apoptosis triggered by the loss of attachment signalling from cell-cell contacts or cell-extra cellular matrix (ECM) contacts. Due to the important role CDCP1 plays in cancer progression in model systems, it is considered a potential drug target to prevent the metastatic spread of cancers. CDCP1 is a highly glycosylated 836 amino acid cell surface protein. It has structural features potentially facilitating protein-protein interactions including 14 N-glycosylation sites, three CUB-like domains, 20 cysteine residues likely to be involved in disulfide bond formation and five intracellular tyrosine residues. CDCP1 interacts with a variety of proteins including Src family kinases (SFKs) and protein kinase C ä (PKCä). Efforts to understand the mechanisms regulating these interactions have largely focussed on three CDCP1 tyrosine residues Y734, Y743 and Y762. CDCP1-Y734 is the site where SFKs phosphorylate and bind to CDCP1 and mediate subsequent phosphorylation of CDCP1-Y743 and -Y762 which leads to binding of PKCä at CDCP1-Y762. The resulting trimeric protein complex of SFK•CDCP1•PKCä has been proposed to mediate an anti-apoptotic cell phenotype in vitro, and to promote metastasis in vivo. The effect of mutation of the three tyrosines on interactions of CDCP1 with SFKs and PKCä and the consequences on cell phenotype in vitro and in vivo have not been examined. CDCP1 has a predicted molecular weight of ~90 kDa but is usually detected as a protein which migrates at ~135 kDa by Western blot analysis due to its high degree of glycosylation. A low molecular weight form of CDCP1 (LMWCDCP1) of ~70 kDa has been found in a variety of cancer cell lines. The mechanisms leading to the generation of LMW-CDCP1 in vivo are not well understood but an involvement of proteases in this process has been proposed. Serine proteases including plasmin and trypsin are able to proteolytically process CDCP1. In addition, the recombinant protease domain of the serine protease matriptase is also able to cleave the recombinant extracellular portion of CDCP1. Whether matriptase is able to proteolytically process CDCP1 on the cell surface has not been examined. Importantly, proteolytic processing of CDCP1 by trypsin leads to phosphorylation of its cell surface-retained portion which suggests that this event leads to initiation of an intracellular signalling cascade. This project aimed to further examine the biology of CDCP1 with a main of focus on exploring the roles played by CDCP1 tyrosine residues. To achieve this HeLa cells stably expressing CDCP1 or the CDCP1 tyrosine mutants Y734F, Y743F and Y762F were generated. These cell lines were used to examine: • The roles of the tyrosine residues Y734, Y743 and Y762 in mediating interactions of CDCP1 with binding proteins and to examine the effect of the stable expression on HeLa cell morphology. • The ability of the serine protease matriptase to proteolytically process cell surface CDCP1 and to examine the consequences of this event on HeLa cell phenotype and cell signalling in vitro. • The importance of these residues in processes associated with cancer progression in vitro including adhesion, proliferation and migration. • The role of these residues on metastatic phenotype in vivo and the ability of a function-blocking anti-CDCP1 antibody to inhibit metastasis in the chicken embryo chorioallantoic membrane (CAM) assay. Interestingly, biochemical experiments carried out in this study revealed that mutation of certain CDCP1 tyrosine residues impacts on interactions of this protein with binding proteins. For example, binding of SFKs as well as PKCä to CDCP1 was markedly decreased in HeLa-CDCP1-Y734F cells, and binding of PKCä was also reduced in HeLa-CDCP1-Y762F cells. In contrast, HeLa-CDCP1-Y743F cells did not display altered interactions with CDCP1 binding proteins. Importantly, observed differences in interactions of CDCP1 with binding partners impacted on basal phosphorylation of CDCP1. It was found that HeLa-CDCP1, HeLa-CDCP1-Y743F and -Y762F displayed strong basal levels of CDCP1 phosphorylation. In contrast, HeLa-CDCP1-Y734F cells did not display CDCP1 phosphorylation but exhibited constitutive phosphorylation of focal adhesion kinase (FAK) at tyrosine 861. Significantly, subsequent investigations to examine this observation suggested that CDCP1-Y734 and FAK-Y861 are competitive substrates for SFK-mediated phosphorylation. It appeared that SFK-mediated phosphorylation of CDCP1- Y734 and FAK-Y861 is an equilibrium which shifts depending on the level of CDCP1 expression in HeLa cells. This suggests that the level of CDCP1 expression may act as a regulatory mechanism allowing cells to switch from a FAK-Y861 mediated pathway to a CDCP1-Y734 mediated pathway. This is the first time that a link between SFKs, CDCP1 and FAK has been demonstrated. One of the most interesting observations from this work was that CDCP1 altered HeLa cell morphology causing an elongated and fibroblastic-like appearance. Importantly, this morphological change depended on CDCP1- Y734. In addition, it was observed that this change in cell morphology was accompanied by increased phosphorylation of SFK-Y416. This suggests that interactions of SFKs with CDCP1-Y734 increases SFK activity since SFKY416 is critical in regulating kinase activity of these proteins. The essential role of SFKs in mediating CDCP1-induced HeLa cell morphological changes was demonstrated using the SFK-selective inhibitor SU6656. This inhibitor caused reversion of HeLa-CDCP1 cell morphology to an epithelial appearance characteristic of HeLa-vector cells. Significantly, in vitro studies revealed that certain CDCP1-mediated cell phenotypes are mediated by cellular pathways dependent on CDCP1 tyrosine residues whereas others are independent of these sites. For example, CDCP1 expression caused a marked increase in HeLa cell motility that was independent of CDCP1 tyrosine residues. In contrast, CDCP1- induced decrease in HeLa cell proliferation was most prominent in HeLa- CDCP1-Y762F cells, potentially indicating a role for this site in regulating proliferation in HeLa cells. Another cellular event which was identified to require phosphorylation of a particular CDCP1 tyrosine residue is adhesion to fibronectin. It was observed that the CDCP1-mediated strong decrease in adhesion to fibronectin is mostly restored in HeLa-CDCP1-Y743F cells. This suggests a possible role for CDCP1-Y743 in causing a CDCP1-mediated decrease in adhesion. Data from in vivo experiments indicated that HeLa-CDCP1-Y734F cells are more metastic than HeLa-CDCP1 cells in vivo. This indicates that interaction of CDCP1 with SFKs and PKCä may not be required for CDCP1-mediated metastasis formation of HeLa cells in vivo. The metastatic phenotype of these cells may be caused by signalling involving FAK since HeLa-CDCP1- Y734F cells are the only CDCP1 expressing cells displaying constitutive phosphorylation of FAK-Y861. HeLa-CDCP1-Y762F cells displayed a very low metastatic ability which suggests that this CDCP1 tyrosine residue is important in mediating a pro-metastatic phenotype in HeLa cells. More detailed exploration of cellular events occurring downstream of CDCP1-Y734 and -Y762 may provide important insights into the mechanisms altering the metastatic ability of CDCP1 expressing HeLa cells. Complementing the in vivo studies, anti-CDCP1 antibodies were employed to assess whether these antibodies are able to inhibit metastasis of CDCP1 and CDCP1 tyrosine mutants expressing HeLa cells. It was found that HeLa- CDCP1-Y734F cells were the only cell line which was markedly reduced in the ability to metastasise. In contrast, the ability of HeLa-CDCP1, HeLa- CDCP1-Y743F and -Y762F cells to metastasise in vivo was not inhibited. These data suggest a possible role of interactions of CDCP1 with SFKs, occurring at CDCP1-Y734, in preventing an anti-metastatic effect of anti- CDCP1 antibodies in vivo. The proposal that SFKs may play a role in regulating anti-metastatic effects of anti-CDCP1 antibodies was supported by another experiment where differences between HeLa-CDCP1 cells and CDCP1 expressing HeLa cells (HeLa-CDCP1-S) from collaborators at the Scripps Research Institute were examined. It was found that HeLa-CDCP1-S cells express different SFKs than CDCP1 expressing HeLa cells generated for this study. This is important since HeLa-CDCP1-S cells can be inhibited in their metastatic ability using anti-CDCP1 antibodies in vivo. Importantly, these data suggest that further examinations of the roles of SFKs in facilitating anti-metastatic effects of anti-CDCP1 antibodies may give insights into how CDCP1 can be blocked to prevent metastasis in vivo. This project also explored the ability of the serine protease matriptase to proteolytically process cell surface localised CDCP1 because it is unknown whether matriptase can cleave cell surface CDCP1 as it has been reported for other proteases such as trypsin and plasmin. Furthermore, the consequences of matriptase-mediated proteolysis on cell phenotype in vitro and cell signalling were examined since recent reports suggested that proteolysis of CDCP1 leads to its phosphorylation and may initiate cell signalling and consequently alter cell phenotype. It was found that matriptase is able to proteolytically process cell surface CDCP1 at low nanomolar concentrations which suggests that cleavage of CDCP1 by matriptase may facilitate the generation of LWM-CDCP1 in vivo. To examine whether matriptase-mediated proteolysis induced cell signalling anti-phospho Erk 1/2 Western blot analysis was performed as this pathway has previously been examined to study signalling in response to proteolytic processing of cell surface proteins. It was found that matriptase-mediated proteolysis in CDCP1 expressing HeLa cells initiated intracellular signalling via Erk 1/2. Interestingly, this increase in phosphorylation of Erk 1/2 was also observed in HeLa-vector cells. This suggested that initiation of cell signalling via Erk 1/2 phosphorylation as a result of matriptase-mediated proteolysis occurs by pathways independent of CDCP1. Subsequent investigations measuring the flux of free calcium ions and by using a protease-activated receptor 2 (PAR2) agonist peptide confirmed this hypothesis. These data suggested that matriptase-mediated proteolysis results in cell signalling via a pathway induced by the activation of PAR2 rather than by CDCP1. This indicates that induction of cell signalling in HeLa cells as a consequence of matriptase-mediated proteolysis occurs via signalling pathways which do not involve phosphorylation of Erk 1/2. Consequently, it appears that future attempts should focus on the examination of cellular pathways other than Erk 1/2 to elucidate cell signalling initiated by matriptase-mediated proteolytic processing of CDCP1. The data presented in this thesis has explored in vitro and in vivo aspects of the biology of CDCP1. The observations summarised above will permit the design of future studies to more precisely determine the role of CDCP1 and its binding partners in processes relevant to cancer progression. This may contribute to further defining CDCP1 as a target for cancer treatment.

Metabolic and ecological study of environmental pentose utilizing bacteria (E-PUB)

Lignocellulosic materials, such as sugar cane bagasse, a waste product of the sugarcane processing industry, agricultural residues and herbaceous crops, may serve as an abundant and comparatively cheap feedstock for largescale industrial fermentation, resulting in the production of marketable end-products. However, the complex structure of lignocellulosic materials, the presence of various hexose and pentose sugars in the hemicellulose component, and the presence of various compounds that inhibit the organisms selected for the fermentation process, all constitute barriers that add to the production costs and make full scale industrial production economically less feasible. The work presented in this thesis was conducted in order to screen microorganisms for ability to utilize pentose sugars derived from the sugar mill industrial waste. A large number of individual bacterial strains were investigated from hemi-cellulose rich material collected at the Proserpine and Maryborough sugar mills, notably soil samples from the mill sites. The research conducted to isolation of six pentose-capable Gram-positive organisms from the actinomycetes group by using pentose as a sole carbon source in the cultivation process. The isolates were identified as Corynebacterium glutamicum, Actinomyces odontolyticus, Nocardia elegans, and Propionibacterium freudenreichii all of which were isolated from the hemicellulose-enriched soil. Pentose degrading microbes are very rare in the environment, so this was a significant discovery. Previous research indicated that microbes could degrade pentose after genetic modification but the microbes discovered in this research were able to naturally utilize pentose. Six isolates, identified as four different genera, were investigated for their ability to utilize single sugars as substrates (glucose, xylose, arabinose or ribose), and also dual sugars as substrates (a hexose plus a pentose). The results demonstrated that C. glutamicum, A. odontolyticus, N. elegans, and P. freudenreichii were pentose-capable (able to grow using xylose or other pentose sugar), and also showed diauxie growth characteristics during the dual-sugar (glucose, in combination with xylose, arabinose or ribose) carbon source tests. In addition, it was shown that the isolates displayed very small differences in growth rates when grown on dual sugars as compared to single sugars, whether pentose or hexose in nature. The anabolic characteristics of C. glutamicum, A. odontolyticus, N. elegans and P. freudenreichii were subsequently investigated by qualitative analysis of their end-products, using high performance liquid chromatography (HPLC). All of the organisms produced arginine and cysteine after utilization of the pentose substrates alone. In addition, P. freudenreichii produced alanine and glycine. The end-product profile arising from culture with dual carbon sources was also tested. Interestingly, this time the product was different. All of them produced the amino acid glycine, when grown on a combination substrate-mix of glucose with xylose, and also glucose with arabinose. Only N. elegans was able to break down ribose, either singly or in combination with glucose, and the end-product of metabolism of the glucose plus ribose substrate combination was glutamic acid. The ecological analysis of microbial abundance in sugar mill waste was performed using denaturing gradient gel electrophoresis (DGGE) and also the metagenomic microarray PhyloChip method. Eleven solid samples and seven liquid samples were investigated. A very complex bacterial ecosystem was demonstrated in the seven liquid samples after testing with the PhyloChip method. It was also shown that bagasse leachate was the most different, compared to all of the other samples, by virtue of its richness in variety of taxa and the complexity of its bacterial community. The bacterial community in solid samples from Proserpine, Mackay and Maryborough sugar mills showed huge diversity. The information found from 16S rDNA sequencing results was that the bacterial genera Brevibacillus, Rhodospirillaceae, Bacillus, Vibrio and Pseudomonas were present in greatest abundance. In addition, Corynebacterium was also found in the soil samples. The metagenomic studies of the sugar mill samples demonstrate two important outcomes: firstly that the bagasse leachate, as potentially the most pentose-rich sample tested, had the most complex and diverse bacterial community; and secondly that the pentose-capable isolates that were initially discovered at the beginning of this study, were not amongst the most abundant taxonomic groups discovered in the sugar mill samples, and in fact were, as suspected, very rare. As a bioprospecting exercise, therefore, the study has discovered organisms that are naturally present, but in very small numbers, in the appropriate natural environment. This has implications for the industrial application of E-PUB, in that a seeding process using a starter culture will be necessary for industrial purposes, rather than simply assuming that natural fermentation might occur.

Preparative deracemization of unnatural amino acids

Unnatural amino acids are a growing class of intermediates required for pharmaceuticals, agrochemicals and other industrial products. However, no single method has proven sufficiently versatile to prepare these compounds broadly at scale. To address this need, we have developed a general chemoenzymatic process to prepare enantiomerically pure L- and D-amino acids in high yield by deracemization of racemic starting materials. This method involves the concerted action of an enantioselective oxidase biocatalyst and a non-selective chemical reducing agent to effect the stereoinversion of one enantiomer and can result in an enantiomeric excess of >99% from the starting racemate, and product yields of over 90%. This approach compares very favourably with resolution processes, which have a maximum single-pass yield of 50%. We have developed efficient methods to adapt the process towards new target compounds and to optimize key factors that influence process efficiency and offer competitive economics at scale.