Interobserver agreement of skeletal muscle mass measurement on head and neck CT imaging at the level of the third cervical vertebra.

OBJECTIVES: Skeletal muscle mass (SMM) is most often assessed in cancer patients on abdominal computed tomography (CT) imaging at the level of the third lumbar vertebra (L3). Abdominal CT imaging is not routinely performed in head and neck cancer (HNC) patients. Recently, a novel method to assess SMM on a single transversal CT slice at the level of the third cervical vertebra (C3) was published. The objective of this study was to assess the robustness of this novel C3 measurement method in terms of interobserver agreement. PATIENTS AND METHODS: Patients diagnosed with locally advanced head and neck squamous cell carcinoma (LA-HNSCC) at our center between 2007 and 2011 were evaluated. Fifty-four patients with were randomly selected for analysis. Six observers independently measured the cross-sectional muscle area (CSMA) at the level of C3 using a predefined, written protocol as instruction. Interobserver agreement was assessed using intraclass correlation coefficients (ICCs), a Bland-Altman plot and Fleiss' kappa (κ). RESULTS: The agreement in vertebra selection between all observers was excellent (Fleiss' κ: 0.96). There was a substantial agreement between all observers in single slice selection (Fleiss' κ: 0.61). For all CSMA measurements, ICCs were excellent (0.763-0.969; all p < 0.001). The Bland-Altman plot showed good agreement between measurements, with narrow limits of agreement. CONCLUSION: Interobserver agreement for SMM measurement at the level of C3 was excellent. Assessment of SMM at the level of C3 is easy and robust and can performed on routinely available imaging in HNC patients.

Strain diversity and phage resistance in complex dairy starter cultures.

The compositional stability of the complex Gouda cheese starter culture Ur is thought to be influenced by diversity in phage resistance of highly related strains that co-exist together with bacteriophages. To analyze the role of bacteriophages in maintaining culture diversity at the level of genetic lineages, simple blends of Lactococcus lactis strains were made and subsequently propagated for 152 generations in the absence and presence of selected bacteriophages. We first screened 102 single-colony isolates (strains) from the complex cheese starter for resistance to bacteriophages isolated from this starter. The collection of isolates represents all lactococcal genetic lineages present in the culture. Large differences were found in bacteriophage resistance among strains belonging to the same genetic lineage and among strains from different lineages. The blends of strains were designed such that 3 genetic lineages were represented by strains with different levels of phage resistance. The relative abundance of the lineages in blends with phages was not stable throughout propagation, leading to continuous changes in composition up to 152 generations. The individual resistance of strains to phage predation was confirmed as one of the factors influencing starter culture diversity. Furthermore, loss of proteolytic activity of initially proteolytic strains was found. Reconstituted blends with only 4 strains with a variable degree of phage resistance showed complex behavior during prolonged propagation.

Enhanced stress resistance of Bifidobacterium breve NRBB57 by induction of stress proteins at near-zero growth rates.

Bifidobacterium breve is a common habitant of the human gut and is used as probiotic in functional foods. B. breve has to cope with multiple stress conditions encountered during processing and passage through the human gut, including high temperature, low pH and exposure to oxygen. Additionally, during industrial processing and in the gut, B. breve could encounter nutrient limitation resulting in reduced growth rates that can trigger adaptive stress responses. For this reason, it is important to develop culture methods that elicit resistance to multiple stresses (robustness) encountered by the bacteria. To investigate the impact of caloric restriction on robustness of the probiotic B. breve NRBB57, this strain was grown in lactose-limited chemostat cultures and in retentostat for 21 days, at growth rates ranging from 0.4 h-1 to 0.00081 h-1. Proteomes of cells harvested at different growth rates were correlated to acid, hydrogen peroxide and heat stress survival capacity. Comparative proteome analysis showed that retentostat-grown cells had significantly increased abundance of a variety of stress proteins involved in protein quality maintenance and DNA repair (DnaJ, Hsp90, FtsH, ClpB, ClpP1, ClpC, GroES, RuvB, RecA), as well as proteins involved in oxidative stress defence (peroxiredoxin, ferredoxin, thioredoxin peroxidase, glutaredoxin and thioredoxin reductase). Exposure to three different stress conditions, 45 °C, pH 3, and 10 mM H2O2, showed highest stress resistance of retentostat cells sampled at week 2 and week 3 grown at 0.0018 and 0.00081 h-1. Our findings show that cultivation at near-zero growth rates induces higher abundance of stress defence proteins contributing to the robustness of B. breve NRBB57, thereby offering an approach that may support its production and functionality.

Validation of skeletal muscle mass assessment at the level of the third cervical vertebra in patients with head and neck cancer.

BACKGROUND: Low skeletal muscle mass (SMM) is associated with adverse outcomes. SMM is often assessed at the third lumbar vertebra (L3) on abdominal imaging. Abdominal imaging is not routinely performed in patients with head and neck cancer (HNC). We aim to validate SMM measurement at the level of the third cervical vertebra (C3) on head and neck imaging. MATERIAL AND METHODS: Patients with pre-treatment whole-body computed tomography (CT) between 2010 and 2018 were included. Cross-sectional muscle area (CSMA) was manually delineated at the level of C3 and L3. Correlation coefficients and intraclass correlation coefficients (ICCs) were calculated. Cohen's kappa was used to assess the reliability of identifying a patient with low SMM. RESULTS: Two hundred patients were included. Correlation between CSMA at the level of C3 and L3 was good (r = 0.75, p < 0.01). Using a multivariate formula to estimate CSMA at L3, including gender, age, and weight, correlation improved (r = 0.82, p < 0.01). The agreement between estimated and actual CSMA at L3 was good (ICC 0.78, p < 0.01). There was moderate agreement in the identification of patients with low SMM based on the estimated lumbar skeletal muscle mass index (LSMI) and actual LSMI (Cohen's κ: 0.57, 95%CI 0.45-0.69). CONCLUSIONS: CSMA at C3 correlates well with CSMA at L3. There is moderate agreement in the identification of patients with low SMM based on the estimated lumbar SMI (based on measurement at C3) and actual LSMI.

Development of a minimal growth medium for Lactobacillus plantarum.

AIM: A medium with minimal requirements for the growth of Lactobacillus plantarum WCFS was developed. The composition of the minimal medium was compared to a genome-scale metabolic model of L. plantarum. METHODS AND RESULTS: By repetitive single omission experiments, two minimal media were developed: PMM5 (true minimal medium) and PMM7 [a pseudominimal medium, supporting proper biomass formation of 350 mg l(-1) dry weight (DW)]. The specific growth rate of L. plantarum on PMM7 was found to be 50% and 63% lower when compared to growth on established growth media (chemically defined medium and MRS, respectively). Using a genome-scale metabolic model of L. plantarum, it was predicted that PMM5 and PMM7 would not support the growth of L. plantarum. This is because the biosynthesis of para-aminobenzoic acid (pABA) was predicted to be essential for growth. The discrepancy in simulated growth and experimental growth on PMM7 was further investigated for pABA; a molecule which plays an important role in folate production. The growth performance and folate production were determined on PMM7 in the presence and absence of pABA. It was found that a 12,000-fold reduction in folate pools exerted no influence on formation of biomass or growth rate of L. plantarum cultures when grown in the absence of pABA. CONCLUSION: Largely reduced folate production pools do not have an effect on the growth of L. plantarum, showing that L. plantarum makes folate in a large excess. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments illustrate the importance of combining genome-scale metabolic models with growth experiments on minimal media.

A novel bacteriocin with a YGNGV motif from vegetable-associated Enterococcus mundtii: full characterization and interaction with target organisms.

A novel broad-spectrum antimicrobial peptide produced by vegetable-associated Enterococcus mundtii was purified and characterized, and designated mundticin. To our knowledge, this is the first report on bacteriocin production by this organism. The elucidation of the full primary amino acid sequence of mundticin (KYYGNGVSCNKKGCSVDWGKAIGIIGNNSAANLATGGAAGWSK) revealed that this antimicrobial peptide belongs to the class IIa bacteriocins of lactic acid bacteria which share a highly conserved N-terminal 'YGNGV' motif. Data obtained by computer modelling indicated an oblique orientation of the alpha-helical regions of mundticin and homologous class IIa bacteriocins at a hydrophobic-hydrophilic interface, which may play a role in the destabilization of phospholipid bilayers. The average mass of mundticin, as determined by electron spray mass spectrometry, was found to be 4287.21+/-0.59 Da. With respect to its biological activity, mundticin was shown to inhibit the growth of Listeria monocytogenes, Clostridium botulinum and a variety of lactic acid bacteria. Moreover, it was demonstrated to have a bactericidal effect on L. monocytogenes as a result of the dissipation of the membrane potential, and a loss of intracellular ATP in absence of ATP leakage. Its good solubility in water, and its stability over a wide pH and temperature range indicate the potential of this broad spectrum bacteriocin as a natural preservation agent for foods.

Antioxidative properties of Lactobacillus sake upon exposure to elevated oxygen concentrations.

The ability of bacteria to overcome oxidative stress is related to the levels and types of antioxidative mechanisms which they possess. In this study, the antioxidative properties in Lactobacillus sake strains from different food origins were determined at low temperature (8 degrees C) and upon exposure to oxygen levels between 20 and 90% O(2). The L. sake strains tested grew well at 8 degrees C and in the presence of 20% O(2), however, most of the strains could not grow at O(2) levels as high as 50 and/or 90%. Cell-free extracts of all strains possessed certain levels of hydroxyl radical scavenging, metal chelating and reducing capacities essential for growth of cells at ambient O(2). At elevated O(2) concentrations, a high H(2)O(2) splitting capacity and low specific rates of H(2)O(2) production were demonstrated in the O(2)-insensitive strain L. sake NCFB 2813, which could grow at elevated O(2) conditions. Although H(2)O(2) was generated in the O(2)-sensitive L. sake DSM 6333 at levels which were not directly toxic to the cells (<0.2 mM), we can conclude that its removal is essential for cell protection at elevated O(2) conditions.

Influence of carvacrol on growth and toxin production by Bacillus cereus.

The natural antimicrobial compound carvacrol was investigated for its effect on diarrheal toxin production by Bacillus cereus. Carvacrol (0-0.06 mg/ml) reduced the viable count and the maximal specific growth rate (mumax) of B. cereus in BHI broth. The total amount of protein was not affected by carvacrol. However, a sharp decrease (80%) in diarrheal toxin production was observed in the presence of 0.06 mg/ml carvacrol. Carvacrol also inhibited toxin production in soup, but approximately 50-fold higher concentrations were needed to achieve the same effect as in broth. From this study it can be concluded that carvacrol can be added to food products at doses below the MIC value, thereby reducing the risk of toxin production by B. cereus and increasing the safety of the products.

Antimicrobial activity of carvacrol toward Bacillus cereus on rice.

The antimicrobial activity of carvacrol, a compound present in the essential oil fraction of oreganum and thyme, toward the foodborne pathogen Bacillus cereus on rice was studied. Carvacrol showed a dose-related inhibition of growth of the pathogen. Concentrations of 0.15 mg/g and higher inhibited the growth and the extent of inhibition depended on the initial inoculum size. To decrease the input of carvacrol on the taste and flavor of the product, a combined treatment with the structure analog cymene was tested. Due to the smell and taste of carvacrol at high concentrations, carvacrol was combined with cymene, a natural antimicrobial compound with a similar structure. A synergistic effect was observed when 0.30 mg/g carvacrol was combined with 0.27 mg/g cymene. Finally it was demonstrated that a common taste enhancer like soya sauce also increased the antimicrobial action of carvacrol toward B. cereus. The antimicrobial activity of carvacrol with cymene or soya sauce was influenced by the addition of NaCl.

Influence of food matrix on inactivation of Bacillus cereus by combinations of nisin, pulsed electric field treatment, and carvacrol.

Carvacrol was used as a third preservative factor to enhance further the synergy between nisin and pulsed electric field (PEF) treatment against vegetative cells of Bacillus cereus. When applied simultaneously with nisin (0.04 microg/ml), carvacrol (0.5 mM) enhanced the synergy found between nisin and PEF treatment (16.7 kV/cm, 30 pulses) in potassium-N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES) buffer. The influence of food ingredients on bactericidal activity was tested using skimmed milk that was diluted to 20% with sterile demineralized water. The efficacy of PEF treatment was not affected by the presence of proteins, and results found in HEPES buffer correlated well with results in milk (20%). Nisin showed less activity against B. cereus in milk. Carvacrol was not able to enhance the synergy between nisin and PEF treatment in milk, unless used in high concentrations (1.2 mM). This concentration in itself did not influence the viable count. Carvacrol did act synergistically with PEF treatment in milk, however not in HEPES buffer. This synergy was not influenced by proteins in milk, as 5% milk still allows synergy between carvacrol and PEF treatment to the same extent as 20% milk.

Production of aroma compounds in lactic fermentations.

This review describes recent scientific research on the production of aroma compounds by lactic acid bacteria (LAB) in fermented food products. We discuss the various precursor molecules for the formation of aroma compounds in connection with the metabolic pathways involved. The roles of nonmetabolic properties such as cell lysis are also described in relation to aroma formation. Finally, we provide an overview of the literature on methods to steer and control aroma formation by LAB in mixed culture fermentations. We demonstrate that the technological progress made recently in high-throughput analysis methods has been driving the development of new approaches to understand, control, and steer aroma formation in (dairy) fermentation processes. This currently entails proposing new rules for designing stable, high-performance mixed cultures constituting a selection of strains, which in concert and on the basis of their individual predicted gene contents deliver the required functionalities.

Functional genomics for food fermentation processes.

This review describes recent scientific and technological drivers of food fermentation research. In addition, a number of practical implications of the results of this development will be highlighted. The first part of the manuscript elaborates on the message that genome sequence information gives us an unprecedented view on the biodiversity of microbes in food fermentation. This information can be made applicable for tailoring relevant characteristics of food products through fermentation. The second part deals with the integration of genome sequence data into metabolic models and the use of these models for a number of topics that are relevant for food fermentation processes. The final part will be about metagenomics approaches to reveal the complexity and understand the functionality of undefined complex microbial consortia used in a diverse range of food fermentation processes.

Combined action of nisin and carvacrol on Bacillus cereus and Listeria monocytogenes.

Nisin, a small antimicrobial protein, was tested for its bactericidal action against Listeria monocytogenes and Bacillus cereus and a typical biphasic reduction of the viable count was observed. The reduction was most fast during the first 10 min of exposure, while the viable count remained stable in the last part of the exposure period. Bacillus cereus was more sensitive towards nisin than L. monocytogenes and the inhibitory effect of nisin was stronger towards cells cultivated and exposed at 8 degrees C than towards cells cultivated and exposed at 20 degrees C. Combining nisin with sublethal doses of carvacrol resulted in an increased reduction in the viable count of both organisms, indicating synergy between nisin and carvacrol. Addition of lysozyme as a third preservative factor increased the synergistic effect between nisin and carvone, especially in the last part of the exposure period.

Relationship between utilization of proline and proline-containing peptides and growth of Lactococcus lactis.

Proline, which is the most abundant residue in beta-casein, stimulates growth of Lactococcus lactis in a proline-requiring strain (Lactococcus lactis subsp. cremoris Wg2) and in a proline-prototrophic strain (Lactococcus lactis subsp. lactis ML3). Both strains lack a proline-specific uptake system, and free proline can enter the cell only by passive diffusion across the cytoplasmic membrane. On the other hand, lactococci can actively take up proline-containing peptides via the lactococcal di- and tripeptide transport system, and these peptides are the major source of proline. Consequently, lactococcal growth on amino acid-based media is highly stimulated by the addition of proline-containing di- and tripeptides. Growth of L. lactis subsp. lactis ML3 on chemically defined media supplemented with casein does not appear proline limited. Addition of dipeptides (including proline-containing peptides) severely inhibits growth on a casein-containing medium, which indicates that the specific growth rate is determined by the balanced supply of different di- or tripeptides which compete for the same di- and tripeptide transport system.

Anaerobic Nitrate Respiration by Erwinia carotovora subsp. atroseptica during Potato Tuber Invasion.

The in planta induction of anaerobic nitrate respiration by Erwinia carotovora subsp. atroseptica in relation to the in situ oxygen status in soft rotting potato tubers has been investigated. In vitro experiments have shown that nitrate was required for the induction of respiratory nitrate reductase activity in E. carotovora. In addition, oxygen was found to repress this activity. Expression of respiratory nitrate reductase was found in E. carotovora cells extracted from soft rotting potato tuber tissue. However, the rate of nitrite production in these cells was approximately 70-fold lower than the rate recorded in fully induced anaerobic cultures. Oxygen measurements in soft rotting potato tubers indicated that the invading bacteria encounter the lowest oxygen concentration at the interphase between healthy and macerated tissue. Consequently, growth of bacteria present in this specific zone will be stimulated by nitrate which is present in sufficient amounts in tuber tissue. A high nitrate content of the tuber will most likely facilitate the proliferation of E. carotovora in the tuber tissue.

Kinetic properties of a phosphate-bond-driven glutamate-glutamine transport system in Streptococcus lactis and Streptococcus cremoris.

In Streptococcus lactis ML3 and Streptococcus cremoris Wg2 the uptake of glutamate and glutamine is mediated by the same transport system, which has a 30-fold higher affinity for glutamine than for glutamate at pH 6.0. The apparent affinity constant for transport (KT) of glutamine is 2.5 +/- 0.3 microM, independent of the extracellular pH. The KTS for glutamate uptake are 3.5, 11.2, 77, and 1200 microM at pH 4.0, 5.1, 6.0, and 7.0, respectively. Recalculation of the affinity constants based on the concentration of glutamic acid in the solution yield KTS of 1.8 +/- 0.5 microM independent of the external pH, indicating that the protonated form of glutamate, i.e., glutamic acid, and glutamine are the transported species. The maximal rates of glutamate and glutamine uptake are independent of the extracellular pH as long as the intracellular pH is kept constant, despite large differences in the magnitude and composition of the components of the proton motive force. Uptake of glutamate and glutamine requires the synthesis of ATP either from glycolysis or from arginine metabolism and appears to be essentially unidirectional. Cells are able to maintain glutamate concentration gradients exceeding 4 X 10(3) for several hours even in the absence of metabolic energy. The t1/2s of glutamate efflux are 2, 12, and greater than 30 h at pH 5.0, 6.0, and 7.0, respectively. After the addition of lactose as energy source, the rate of glutamine uptake and the level of ATP are both very sensitive to arsenate. When the intracellular pH is kept constant, both parameters decrease approximately in parallel (between 0.2 and 1.0 mM ATP) with increasing concentrations of the inhibitor. These results suggest that the accumulation of glutamate and glutamine is energized by ATP or an equivalent energy-rich phosphorylated intermediate and not by the the proton motive force.

Peptide uptake is essential for growth of Lactococcus lactis on the milk protein casein.

The chlorated dipeptide L-alanyl-beta-chloro-L-alanine (diACA) is very toxic for Lactococcus lactis. Spontaneous mutants resistant to the dipeptide were isolated from plates. The presence and activities of cell wall-associated proteinase, different peptidases in cell extracts, amino acid transport systems, and di- and oligopeptide transport systems were examined and compared in a diACA-resistant mutant and the wild type. Only the rates of di- and tripeptide transport were found to be significantly reduced in the diACA-resistant mutant of L. lactis ML3. Since all other characteristics of this mutant were comparable to those of the wild type, the diACA-resistant mutant is most likely deficient in di- and tripeptide transport. Uptake of di- and tripeptides by L. lactis ML3 was found to be mainly mediated by one peptide transport system. The peptide transport-deficient mutant was found to be unable to grow on a chemically defined medium supplemented with casein as the sole nitrogen source, whereas growth could be restored by the addition of amino acids. These results indicate that peptide transport in L. lactis ML3 is an essential component in the process of casein utilization during growth in milk.

Mechanism and energetics of dipeptide transport in membrane vesicles of Lactococcus lactis.

Alanyl-alpha-glutamate transport has been studied in Lactococcus lactis ML3 cells and in membrane vesicles fused with liposomes containing beefheart cytochrome c oxidase as a proton-motive-force-generating system. The uptake of Ala-Glu observed in de-energized cells can be stimulated 26-fold upon addition of lactose. No intracellular dipeptide pool could be detected in intact cells. In fused membranes, a 40-fold accumulation of Ala-Glu was observed in response to a proton motive force. Addition of ionophores and uncouplers resulted in a rapid efflux of the accumulated dipeptide, indicating that Ala-Glu accumulation is directly coupled to the proton motive force as a driving force. Ala-Glu uptake is an electrogenic process and the dipeptide is transported in symport with two protons. In both fused membranes and intact cells the same affinity constant (0.70 mM) for Ala-Glu uptake was found. Accumulated Ala-Glu is exchangeable with externally added alanyl-glutamate, glutamyl-glutamate, and leucyl-leucine, while no exchange occurred upon addition of the amino acid glutamate or alanine. These results indicate that the Ala-Glu transport system has a broad substrate specificity.