The impact of seasonal calving systems with varying pasture allowance on Cheddar cheese composition, nutritional quality and ripening characteristics.

The objective of this study was to examine the impact of stage of lactation (early, mid and late) and proportion of pasture in the cows diet (high: GRS, medium: PMR and no: TMR) on the composition and quality of Cheddar cheese. Triplicate trials were carried out in each stage of lactation, and milk protein and fat contents were standardized for Cheddar cheese manufacture at pilot scale. As cheese milks were standardized for milk fat and protein contents, gross composition did not differ as a result of diet. Fatty acid profiles of GRS cheese were significantly different from TMR, while PMR profiles were less distinct and more similar to both GRS and TMR profiles, as illustrated by partial least squares discriminatory analysis. Fatty acids including CLA C18:2 cis-9, trans-11, C22:1 n-9 and C18:3 n-3 were most influential in this separation of profiles. Fatty acid profiling revealed that GRS derived cheese contained higher proportions of nutrients considered beneficial for human health including higher proportions of unsaturated fatty acids and omega-3 fatty acids. A biomarker model utilizing the proportions of 5 fatty acids was constructed and was effective at distinguishing between cheese of GRS, TMR and PMR feeding systems. Proportions of ρ-κ-casein, αs2-casein and αs1-casein in cheese also differed between diets while proportions of ρ-κ-casein, αs1-casein and ß-casein were lowest in late lactation cheese. The impact of diet was less influential compared with that of stage of lactation on the ripening characteristics of cheese. An index of primary proteolysis was highest in late lactation cheese. The peptides derived from the proteolysis of κ-casein and ß-casein and levels of secondary proteolysis, in particular, the proportions of 12 free amino acids were most influenced by stage of lactation. Overall this study demonstrated the effects of increasing pasture allowance and stage of lactation on the nutritional quality and ripening properties of Cheddar cheese.

Detailed analysis of metabolism reveals growth-rate-promoting interactions between Anaerostipes caccae and Bacteroides spp.

INTRODUCTION: Human gut microbiota species which are next-generation probiotics (NGPs) candidates are of high interest as they have shown the potential to treat intestinal inflammation and other diseases. Unfortunately, these species are often not robust enough for large-scale cultivation, especially in maintaining diversity in co-culture production. OBJECTIVES: In this study, we describe interactions between human gut microbiota species in the cultivation process with unique substrates. We also demonstrated that it is possible to change the species ratio in co-culture by changing the ratio of carbon sources. METHODS: We screened 25 different bacterial species based on their metabolic capabilities. After evaluating unique substrate possibilities, we chose Anaerostipes caccae (A. caccae), Bacteroides thetaiotaomicron (B. thetaiotaomicron), and Bacteroides vulgatus (B. vulgatus) as subjects for further study. D-sorbitol, D-xylose, and D-galacturonic acid were selected as substrates for A. caccae, B. thetaiotaomicron, and B. vulgatus respectively. All three species were cultivated as both monocultures and in co-cultures in serial batch fermentations in an isothermal microcalorimeter. RESULTS: Positive interactions were detected between the species in both co-cultures (A. caccae + B. thetaiotaomicron; A. caccae + B. vulgatus) resulting in higher heat production compared to the sum of the monocultures. The same positive cross-feeding interactions took place in larger-scale cultivation experiments. We confirmed acetate and lactate cross-feeding between A. caccae and B. thetaiotaomicron with flux balance analysis (FBA). CONCLUSION: Changing the ratio of the selected carbon sources in the medium changed the species ratio accordingly. Such robustness is the basis for developing more efficient industrial co-culture processes including the production of NGPs.

Synthesis of 6'-galactosyllactose, a deviant human milk oligosaccharide, with the aid of Candida antarctica lipase-B.

Research on human milk oligosaccharides (HMOs) has increased over the past decade showing great interest in their beneficial effects. Here we describe a method for the selective deacetylation using immobilised Candida antarctica lipase-B, Novozyme N435 (N435), of pyranose saccharides in organic media with the aim of simplifying and improving the pathways for the synthesis of HMOs. By first studying in depth the deacetylation reaction of peracetylated D-glucose two reaction conditions were found, which were used on different HMO building blocks, peracetylated saccharides and thioglycosides. D-Glucose based saccharides showed selectivity towards the fourth and the sixth position deacetylation. While α-anomer of peracetylated D-galactose remained unreactive and ß-anomer favoured the first position deacetylation. Peracetylated L-fucose, on the other hand, had no selectivity as the main product was fully unprotected L-fucose. Taking the peracetylated D-glucose deacetylation reaction product and selectively protecting the primary hydroxyl group in the sixth position left only the fourth position open for the glycosylation. Meanwhile, the deacetylation product of D-galactose thioglycoside, with the sixth position deacetylated, had both acceptor and donor capabilities. Using the two aforementioned products derived from the N435 deacetylation reactions a deviant HMO, 6'-galactosyllactose (6'-GL) was synthesised.

Development and Optimisation of HILIC-LC-MS Method for Determination of Carbohydrates in Fermentation Samples.

Saccharides are the most common carbon source for Streptococcus thermophilus, which is a widely used bacterium in the production of fermented dairy products. The performance of the strain is influenced by the consumption of different saccharides during fermentation. Therefore, a precise measurement of the concentrations of saccharides in the fermentation media is essential. An 18-min long method with limits of quantitation in the range of 0.159-0.704 mg/L and with 13C labelled internal standards employing hydrophilic interaction chromatography coupled to mass spectrometric detection-(HILIC-LC-MS) allowed for simultaneous quantification of five saccharides: fructose, glucose, galactose, sucrose, and lactose in the fermentation samples. The method included a four-step sample preparation protocol, which could be easily applied to high-throughput analysis. The developed method was validated and applied to the fermentation samples produced by Streptococcus thermophilus.

Cancer sniffer dogs: how can we translate this peculiarity in laboratory medicine? Results of a pilot study on gastrointestinal cancers.

BACKGROUND: Identification of cancer biomarkers to allow early diagnosis is an urgent need for many types of tumors, whose prognosis strongly depends on the stage of the disease. Canine olfactory testing for detecting cancer is an emerging field of investigation. As an alternative, here we propose to use GC-Olfactometry (GC/O), which enables the speeding up of targeted biomarker identification and analysis. A pilot study was conducted in order to determine odor-active compounds in urine that discriminate patients with gastrointestinal cancers from control samples (healthy people). METHODS: Headspace solid phase microextraction (HS-SPME)-GC/MS and GC-olfactometry (GC/O) analysis were performed on urine samples obtained from gastrointestinal cancer patients and healthy controls. RESULTS: In total, 91 key odor-active compounds were found in the urine samples. Although no odor-active biomarkers present were found in cancer carrier's urine, significant differences were discovered in the odor activities of 11 compounds in the urine of healthy and diseased people. Seven of above mentioned compounds were identified: thiophene, 2-methoxythiophene, dimethyl disulphide, 3-methyl-2-pentanone, 4-(or 5-)methyl-3-hexanone, 4-ethyl guaiacol and phenylacetic acid. The other four compounds remained unknown. CONCLUSIONS: GC/O has a big potential to identify compounds not detectable using untargeted GC/MS approach. This paves the way for further research aimed at improving and validating the performance of this technique so that the identified cancer-associated compounds may be introduced as biomarkers in clinical practice to support early cancer diagnosis.

Genome-wide investigation of mRNA lifetime determinants in Escherichia coli cells cultured at different growth rates.

BACKGROUND: Changes to mRNA lifetime adjust mRNA concentration, facilitating the adaptation of growth rate to changes in growth conditions. However, the mechanisms regulating mRNA lifetime are poorly understood at the genome-wide scale and have not been investigated in bacteria growing at different rates. RESULTS: We used linear covariance models and the best model selected according to the Akaike information criterion to identify and rank intrinsic and extrinsic general transcript parameters correlated with mRNA lifetime, using mRNA half-life datasets for E. coli, obtained at four growth rates. The principal parameter correlated with mRNA stability was mRNA concentration, the mRNAs most concentrated in the cells being the least stable. However, sequence-related features (codon adaptation index (CAI), ORF length, GC content, polycistronic mRNA), gene function and essentiality also affected mRNA lifetime at all growth rates. We also identified sequence motifs within the 5'UTRs potentially related to mRNA stability. Growth rate-dependent effects were confined to particular functional categories (e.g. carbohydrate and nucleotide metabolism). Finally, mRNA stability was less strongly correlated with the amount of protein produced than mRNA concentration and CAI. CONCLUSIONS: This study provides the most complete genome-wide analysis to date of the general factors correlated with mRNA lifetime in E. coli. We have generalized for the entire population of transcripts or excluded determinants previously defined as regulators of stability for some particular mRNAs and identified new, unexpected general indicators. These results will pave the way for discussions of the underlying mechanisms and their interaction with the growth physiology of bacteria.

Advanced continuous cultivation methods for systems microbiology.

Increasing the throughput of systems biology-based experimental characterization of in silico-designed strains has great potential for accelerating the development of cell factories. For this, analysis of metabolism in the steady state is essential as only this enables the unequivocal definition of the physiological state of cells, which is needed for the complete description and in silico reconstruction of their phenotypes. In this review, we show that for a systems microbiology approach, high-resolution characterization of metabolism in the steady state--growth space analysis (GSA)--can be achieved by using advanced continuous cultivation methods termed changestats. In changestats, an environmental parameter is continuously changed at a constant rate within one experiment whilst maintaining cells in the physiological steady state similar to chemostats. This increases the resolution and throughput of GSA compared with chemostats, and, moreover, enables following of the dynamics of metabolism and detection of metabolic switch-points and optimal growth conditions. We also describe the concept, challenge and necessary criteria of the systematic analysis of steady-state metabolism. Finally, we propose that such systematic characterization of the steady-state growth space of cells using changestats has value not only for fundamental studies of metabolism, but also for systems biology-based metabolic engineering of cell factories.

Excess of threonine compared with serine promotes threonine aldolase activity in Lactococcus lactis IL1403.

Lactococcus lactis is an important lactic acid starter for food production as well as a cell factory for production of food grade additives, among which natural flavour production is one of the main interests of food producers. Flavour production is associated with the degradation of amino acids and comprehensive studies are required to elucidate mechanisms behind these pathways. In this study using chemically defined medium, labelled substrate and steady-state cultivation, new data for the catabolism of threonine in Lc. lactis have been obtained. The biosynthesis of glycine in this organism is associated with the catabolic pathways of glucose and serine. Nevertheless, if threonine concentration in the growth environment exceeds that of serine, threonine becomes the main source for glycine biosynthesis and the utilization of serine decreases. Also, the conversion of threonine to glycine was initiated by a threonine aldolase and this was the principal pathway used for threonine degradation. As in Streptococcus thermophilus, serine hydroxymethyltransferase in Lc. lactis may possess a secondary activity as threonine aldolase. Other catabolic pathways of threonine (e.g. threonine dehydrogenase and threonine dehydratase) were not detected.

Protein turnover forms one of the highest maintenance costs in Lactococcus lactis.

Protein turnover plays an important role in cell metabolism by regulating metabolic fluxes. Furthermore, the energy costs for protein turnover have been estimated to account for up to a third of the total energy production during cell replication and hence may represent a major limiting factor in achieving either higher biomass or production yields. This work aimed to measure the specific growth rate (µ)-dependent abundance and turnover rate of individual proteins, estimate the ATP cost for protein production and turnover, and compare this with the total energy balance and other maintenance costs. The lactic acid bacteria model organism Lactococcus lactis was used to measure protein turnover rates at µ = 0.1 and 0.5 h(-1) in chemostat experiments. Individual turnover rates were measured for ~75% of the total proteome. On average, protein turnover increased by sevenfold with a fivefold increase in growth rate, whilst biomass yield increased by 35%. The median turnover rates found were higher than the specific growth rate of the bacterium, which suggests relatively high energy consumption for protein turnover. We found that protein turnover costs alone account for 38 and 47% of the total energy produced at µ = 0.1 and 0.5 h(-1), respectively, and gene ontology groups Energy metabolism and Translation dominated synthesis costs at both growth rates studied. These results reflect the complexity of metabolic changes that occur in response to changes in environmental conditions, and signify the trade-off between biomass yield and the need to produce ATP for maintenance processes.

Single-cell model of prokaryotic cell cycle.

One of the recognized prokaryotic cell cycle theories is Cooper-Helmstetter (CH) theory which relates start of DNA replication to particular (initiation) cell mass, cell growth and division. Different aspects of this theory have been extensively studied in the past. In the present study CH theory was applied at single cell level. Universal equations were derived for different cell parameters (cell mass and volume, surface area, DNA amount and content) depending on constructivist cell cycle parameters (unit mass, replication and division times, cell age, cell cycle duration) based on selected growth laws of cell mass (linear, exponential). The equations derived can be integrated into single-cell models for the analysis and design of bacterial cells.

Nutritional requirements and media development for Lactococcus lactis IL1403.

Lactic acid bacteria are extensively used in food technology and for the production of various compounds, but they are fastidious in nutrient requirements. In order to elucidate the role of each component precisely, defined multicomponent media are required. This study focuses on determining nutrient auxotrophies and minimizing media components (amino acids, vitamins, metal ions, buffers and additional compounds) for the cultivation of Lactococcus lactis subsp. lactis IL1403, using microtitre plates and test tubes. It was shown that glutamine and asparagine were the most important media components for achieving higher biomass yields while the branched-chain amino acids were necessary to increase specific growth rate. The amino acid and glucose ratio was reduced to achieve minimal residual concentration of amino acids in the medium after the growth of cells, whereas the specific growth rate and biomass yield of cells were not considerably affected. As the percentage of each consumed amino acid compared to initial amount is larger than measurement error, these optimized media are important for achieving more precise data about amino acid utilization and metabolism.

Coordinated activation of PTA-ACS and TCA cycles strongly reduces overflow metabolism of acetate in Escherichia coli.

Elimination of acetate overflow in aerobic cultivation of Escherichia coli would improve many bioprocesses as acetate accumulation in the growth environment leads to numerous negative effects, e.g. loss of carbon, inhibition of growth, target product synthesis, etc. Despite many years of studies, the mechanism and regulation of acetate overflow are still not completely understood. Therefore, we studied the growth of E. coli K-12 BW25113 and several of its mutant strains affecting acetate-related pathways using the continuous culture method accelerostat (A-stat) at various specific glucose consumption rates with the aim of diminishing acetate overflow. Absolute quantitative exo-metabolome and proteome analyses coupled to metabolic flux analysis enabled us to demonstrate that onset of acetate overflow can be postponed and acetate excretion strongly reduced in E. coli by coordinated activation of phosphotransacetylase-acetyl-CoA synthetase (PTA-ACS) and tricarboxylic acid (TCA) cycles. Fourfold reduction of acetate excretion (2 vs. 8 % from total carbon) at fastest growth compared to wild type was achieved by deleting the genes responsible for inactivation of acetyl-CoA synthetase protein (pka) and TCA cycle regulator arcA. The Δpka ΔarcA strain did not accumulate any other detrimental by-product besides acetate and showed identical µ max and only ~5 % lower biomass yield compared to wild type. We conclude that a fine-tuned coordination between increasing the recycling capabilities of acetate in the PTA-ACS node through a higher concentration of active acetate scavenging Acs protein and downstream metabolism throughput in the TCA cycle is necessary for diminishing overflow metabolism of acetate in E. coli and achieving higher target product production in bioprocesses.

Multiplying steady-state culture in multi-reactor system.

Cultivation of microorganisms in batch experiments is fast and economical but the conditions therein change constantly, rendering quantitative data interpretation difficult. By using chemostat with controlled environmental conditions the physiological state of microorganisms is fixed; however, the unavoidable stabilization phase makes continuous methods resource consuming. Material can be spared by using micro scale devices, which however have limited analysis and process control capabilities. Described herein are a method and a system combining the high throughput of batch with the controlled environment of continuous cultivations. Microorganisms were prepared in one bioreactor followed by culture distribution into a network of bioreactors and continuation of independent steady state experiments therein. Accelerostat cultivation with statistical analysis of growth parameters demonstrated non-compromised physiological state following distribution, thus the method effectively multiplied steady state culture of microorganisms. The theoretical efficiency of the system was evaluated in inhibitory compound analysis using repeated chemostat to chemostat transfers.

The Quantitative Measurement of Peptidoglycan Components Obtained from Acidic Hydrolysis in Gram-Positive and Gram-Negative Bacteria via Hydrophilic Interaction Liquid Chromatography Coupled with Mass Spectrometry.

The high throughput in genome sequencing and metabolic model (MM) reconstruction has democratised bioinformatics approaches such as flux balance analysis. Fluxes' prediction accuracy greatly relates to the deepness of the MM curation for a specific organism starting from the cell composition. One component is the cell wall, which is a functional barrier (cell shape, exchanges) with the environment. The bacterial cell wall (BCW), including its thickness, structure, and composition, has been extensively studied in Escherichia coli but poorly described for other organisms. The peptidoglycan (PG) layer composing the BCW is usually thinner in Gram- bacteria than in Gram+ bacteria. In both bacteria groups, PG is a polymeric mesh-like structure of amino acids and sugars, including N-acetylglucosamine, N-acetylmuramic acid, and amino acids. In this study, we propose a high-throughput method to characterise and quantify PG in Gram-positive and Gram-negative bacteria using acidic hydrolysis and hydrophilic interaction liquid chromatography coupled with mass spectrometry (HILIC-MS). The method showed a relatively short time frame (11 min analytical run), low inter- and intraday variability (3.2% and 4%, respectively), and high sensitivity and selectivity (limits of quantification in the sub mg/L range). The method was successfully applied on two Gram-negative bacteria (Escherichia coli K12 MG1655, Bacteroides thetaiotaomicron DSM 2079) and one Gram-positive bacterium (Streptococcus salivarius ssp. thermophilus DSM20259). The PG concentration ranged from 1.6% w/w to 14% w/w of the dry cell weight. The results were in good correlation with previously published results. With further development, the PG concentration provided by this newly developed method could reinforce the curation of MM.

Techno-Functional and Sensory Characterization of Commercial Plant Protein Powders.

Many new plant proteins are appearing on the market, but their properties are insufficiently characterized. Hence, we collected 24 commercial proteins from pea, oat, fava bean, chickpea, mung bean, potato, canola, soy, and wheat, including different batches, and assessed their techno-functional and sensory properties. Many powders had yellow, red, and brown color tones, but that of fava bean was the lightest. The native pH ranged from 6.0 to 7.7. The water solubility index was 28% on average, but after heat treatment the solubility typically increased. Soy isolate had by far the best water-holding capacity of 6.3 g (H2O) g-1, and canola had the highest oil-holding capacity of 2.8 g (oil) g-1. The foaming capacity and stability results were highly varied but typical to the raw material. The emulsification properties of all powders were similar. Upon heating, the highest viscosity and storage modulus were found in potato, canola, and mung bean. All powders had raw material flavor, were bitter and astringent, and undissolved particles were perceived in the mouth. Large differences in functionality were found between the batches of one pea powder. In conclusion, we emphasize the need for methodological standardization, but while respecting the conditions found in end applications like meat and dairy analogs.

Self-reproduction and doubling time limits of different cellular subsystems.

Ribosomes which can self-replicate themselves practically autonomously in beneficial physicochemical conditions have been recognized as the central organelles of cellular self-reproduction processes. The challenge of cell design is to understand and describe the rates and mechanisms of self-reproduction processes of cells as of coordinated functioning of ribosomes and the enzymatic networks of different functional complexity that support those ribosomes. We show that doubling times of proto-cells (ranging from simplest replicators up to those reaching the size of E. coli) increase rather with the number of different cell component species than with the total numbers of cell components. However, certain differences were observed between cell components in increasing the doubling times depending on the types of relationships between those cell components and ribosomes. Theoretical limits of doubling times of the self-reproducing proto-cells determined by the molecular parameters of cell components and cell processes were in the range between 6-40 min.

Characterisation of chemical, microbial and sensory profiles of commercial kombuchas.

The kombucha market is a fast-growing segment in the functional beverage category. The selection of kombuchas on the market varies between the traditional and flavoured kombuchas. Our research aimed to characterise the chemical, microbial, and sensory profiles of the commercial kombuchas. We analysed 16 kombuchas from 6 producers. The dominant metabolites were acetate, lactate, and ethanol, the last of which might put some kombuchas into the alcoholic beverage section in some countries. The metagenomic analyses demonstrated that LAB dominates in green tea, and AAB in black tea kombuchas. The main bacterial species were Komagataeibacter rhaeticus and Lactobacillus ssp, and yeast species Dekkera anomala and Dekkera bruxellensis. The sweet and sour balance correlated with acid concentrations. The free sorting task showed that commercial kombuchas clustered into three main categories "fruity and artificial flavour", herbal and tea notes", and "classical notes". Our research results showed the necessity of the definition of kombucha.

Stock culture heterogeneity rather than new mutational variation complicates short-term cell physiology studies of Escherichia coli K-12 MG1655 in continuous culture.

Nutrient-limited continuous cultures in chemostats have been used to study microbial cell physiology for over 60 years. Genome instability and genetic heterogeneity are possible uncontrolled factors in continuous cultivation experiments. We investigated these issues by using high-throughput (HT) DNA sequencing to characterize samples from different phases of a glucose-limited accelerostat (A-stat) experiment with Escherichia coli K-12 MG1655 and a duration regularly used in cell physiology studies (20 generations of continuous cultivation). Seven consensus mutations from the reference sequence and five subpopulations characterized by different mutations were detected in the HT-sequenced samples. This genetic heterogeneity was confirmed to result from the stock culture by Sanger sequencing. All the subpopulations in which allele frequencies increased (betA, cspG/cspH, glyA) during the experiment were also present at the end of replicate A-stats, indicating that no new subpopulations emerged during our experiments. The fact that ~31 % of the cells in our initial cultures obtained directly from a culture stock centre were mutants raises concerns that even if cultivations are started from single colonies, there is a significant chance of picking a mutant clone with an altered phenotype. Our results show that current HT DNA sequencing technology allows accurate subpopulation analysis and demonstrates that a glucose-limited E. coli K-12 MG1655 A-stat experiment with a duration of tens of generations is suitable for studying cell physiology and collecting quantitative data for metabolic modelling without interference from new mutations.

Multi-omics approach to study the growth efficiency and amino acid metabolism in Lactococcus lactis at various specific growth rates.

BACKGROUND: Lactococcus lactis is recognised as a safe (GRAS) microorganism and has hence gained interest in numerous biotechnological approaches. As it is fastidious for several amino acids, optimization of processes which involve this organism requires a thorough understanding of its metabolic regulations during multisubstrate growth. RESULTS: Using glucose limited continuous cultivations, specific growth rate dependent metabolism of L. lactis including utilization of amino acids was studied based on extracellular metabolome, global transcriptome and proteome analysis. A new growth medium was designed with reduced amino acid concentrations to increase precision of measurements of consumption of amino acids. Consumption patterns were calculated for all 20 amino acids and measured carbon balance showed good fit of the data at all growth rates studied. It was observed that metabolism of L. lactis became more efficient with rising specific growth rate in the range 0.10-0.60 h(-1), indicated by 30% increase in biomass yield based on glucose consumption, 50% increase in efficiency of nitrogen use for biomass synthesis, and 40% reduction in energy spilling. The latter was realized by decrease in the overall product formation and higher efficiency of incorporation of amino acids into biomass. L. lactis global transcriptome and proteome profiles showed good correlation supporting the general idea of transcription level control of bacterial metabolism, but the data indicated that substrate transport systems together with lower part of glycolysis in L. lactis were presumably under allosteric control. CONCLUSIONS: The current study demonstrates advantages of the usage of strictly controlled continuous cultivation methods combined with multi-omics approach for quantitative understanding of amino acid and energy metabolism of L. lactis which is a valuable new knowledge for development of balanced growth media, gene manipulations for desired product formation etc. Moreover, collected dataset is an excellent input for developing metabolic models.

Inhibition of pyruvate dehydrogenase kinase influence microbiota and metabolomic profile in pancreatic cancer xenograft mice.

OBJECTIVE: Despite recent advances in treatment options, pancreatic cancer remains the most deadly major cancer. Targeting metabolism represents an emerging anti-cancer strategy. RESULTS: Metagenomic 16S analysis was employed to explore the effect of Dichloroacetate (DCA) on the composition of the fecal microbiota and metabolomic profile was assessed on in vivo pancreatic cancer mouse xenograft model. Pancreatic cancer xenograft mice displayed a shift of microbiota' profile as compared to control mice without DCA treatment and a significant decrease of the purine bases inosine xanthine together with their metabolically-related compound hypoxanthine were observed in the DCA treated group as compared to the control group. Two aminoacids methionine and aspartic acid resulted decreased and increased respectively. DCA affects tumor environment and studies are needed in order to understand whether DCA supplementation could be supportive as synergistic approach to enhance the efficacy of existing cancer treatments in pancreatic cancer patients.