Fermentation of pseudocereals quinoa, canihua, and amaranth to improve mineral accessibility through degradation of phytate.

BACKGROUND: Pseudocereals are nutrient-rich grains with high mineral content but also phytate content. Phytate is a mineral absorption inhibitor. The study's aim was to evaluate phytate degradation during spontaneous fermentation and during Lactobacillus plantarum 299v® fermentation of quinoa, canihua, and amaranth grains and flours. It also aimed to evaluate the accessibility of iron, zinc, and calcium and to estimate their bioavailability before and after the fermentation of flours with starter culture. Lactic acid, pH, phytate, and mineral content were analyzed during fermentation. RESULTS: Higher phytate degradation was found during the fermentation of flours (64-93%) than during that of grains (12-51%). Results suggest that phytate degradation was mainly due to endogenous phytase activity in different pseudocereals rather than the phytase produced by added microorganisms. The addition of Lactobacillus plantarum 299v® resulted in a higher level of lactic acid (76.8-82.4 g kg-1 DM) during fermentation, and a relatively quicker reduction in pH to 4 than in spontaneous fermentation. Mineral accessibility was increased (1.7-4.6-fold) and phytate : mineral molar ratios were reduced (1.5-4.2-fold) in agreement with phytate degradation (1.8-4.2-fold) in fermented flours. The reduced molar ratios were still above the threshold value for the improved estimated mineral bioavailability of mainly iron. CONCLUSION: Fermentation proved to be effective for degrading phytate in pseudocereal flours, but less so in grains. Fermentation with Lactobacillus plantarum 299v® improved mineral accessibility and estimated bioavailability in flours. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effect of fermentation and dry roasting on the nutritional quality and sensory attributes of quinoa.

BACKGROUND: Quinoa is a pseudocereal with relatively high content of proteins and minerals that also contains mineral inhibitors such as phytate. The aim of the present study was to evaluate lactic acid fermentation and dry roasting on the nutritional quality and sensory attributes of quinoa. Various processes were evaluated, and quinoa grains were dry-roasted, milled, and fermented, either with or without the addition of wheat phytase or activated quinoa phytase (added as back-slop starter), for 10 hr. In other processes, raw quinoa flour was fermented for 10 hr or 4 hr and dry-roasted. Hedonic sensory evaluation was then performed to evaluate the acceptability of the fermented flours prepared as porridges. RESULTS: The combined dry roasting and fermentation processes significantly (p < .05) degraded phytate between 30% and 73% from initial content. The most effective process was fermentation of raw quinoa flour followed by dry roasting, which improved the estimated zinc and iron bioavailability. Particularly, estimated zinc bioavailability improved from low (Phy:Zn 25.4, Phy·Zn:Ca 295) to moderate (Phy:Zn 7.14, Phy·Zn:Ca 81.5). Phytate degradation was mainly attributed to the activation of endogenous phytase during fermentation. Dry roasting was effective in improving the sensory attributes of the fermented quinoa flour. Porridge made with raw quinoa flour fermented for 4 hr and dry-roasted was more favorable to overall acceptability than that which was fermented for 10 hr and dry-roasted. CONCLUSION: Fermentation of quinoa flour for 4 hr followed by dry roasting was successful in improving both nutritional and sensory attributes of the final product.

Circulating Linoleic Acid is Associated with Improved Glucose Tolerance in Women after Gestational Diabetes.

Women with previously diagnosed gestational diabetes mellitus (GDM) are at increased risk of type-2-diabetes mellitus (T2D). We aimed to establish links between glucose tolerance (GT) and serum fatty acid (FA) profile in the transition from GDM to T2D. Six years after GDM, 221 women were grouped as having normal GT (NGT), impaired GT (IGT), or T2D based on oral GT test results. Fasting serum FAs were profiled, anthropometric measures taken, and dietary intake determined. Linoleic acid (LA) was significantly higher in NGT women (p < 0.001) compared with IGT and T2D, and emerged as a strong predictor of low glucose and insulin levels, independently of BMI. Self-reported vegetable oil consumption correlated with LA serum levels and glucose levels. Delta-6-, delta-9-, and stearoyl-CoA-desaturase activities were associated with decreased GT, and delta-5-desaturase activities with increased GT. In a subgroup of women at high risk of diabetes, low LA and high palmitic acid levels were seen in those that developed T2D, with no differences in other FAs or metabolic measurements. Results suggest that proportions of LA and palmitic acid are of particular interest in the transition from GDM to T2D. Interconversions between individual FAs regulated by desaturases appear to be relevant to glucose metabolism.

Effect of Blood Collection Tube Type and Time to Processing on the Enumeration and High-Content Characterization of Circulating Tumor Cells Using the High-Definition Single-Cell Assay.

CONTEXT: - As circulating tumor cell (CTC) assays gain clinical relevance, it is essential to address preanalytic variability and to develop standard operating procedures for sample handling in order to successfully implement genomically informed, precision health care. OBJECTIVE: - To evaluate the effects of blood collection tube (BCT) type and time-to-assay (TTA) on the enumeration and high-content characterization of CTCs by using the high-definition single-cell assay (HD-SCA). DESIGN: - Blood samples of patients with early- and advanced-stage breast cancer were collected into cell-free DNA (CfDNA), EDTA, acid-citrate-dextrose solution, and heparin BCTs. Time-to-assay was evaluated at 24 and 72 hours, representing the fastest possible and more routine domestic shipping intervals, respectively. RESULTS: - We detected the highest CTC levels and the lowest levels of negative events in CfDNA BCT at 24 hours. At 72 hours in this BCT, all CTC subpopulations were decreased with the larger effect observed in high-definition CTCs and cytokeratin-positive cells smaller than white blood cells. Overall cell retention was also optimal in CfDNA BCT at 24 hours. Whole-genome copy number variation profiles were generated from single cells isolated from all BCT types and TTAs. Cells from CfDNA BCT at 24-hour TTA exhibited the least noise. CONCLUSIONS: - Circulating tumor cells can be identified and characterized under a variety of collection, handling, and processing conditions, but the highest quality can be achieved with optimized conditions. We quantified performance differences of the HD-SCA for specific preanalytic variables that may be used as a guide to develop best practices for implementation into patient care and/or research biorepository processes.

Correction: Formation of reactive aldehydes (MDA, HHE, HNE) during the digestion of cod liver oil: comparison of human and porcine in vitro digestion models.

Correction for 'Formation of reactive aldehydes (MDA, HHE, HNE) during the digestion of cod liver oil: comparison of human and porcine in vitro digestion models' by Cecilia Tullberg et al., Food Funct., 2016, 7, 1401-1412.

Reexamining the Association of Body Mass Index With Overall Survival Outcomes After Liver Transplantation.

BACKGROUND: Several studies have shown that obese patients undergoing liver transplantation (LT) have an increased risk of mortality regardless of Model of End Stage Liver Disease (MELD) scores. The purpose of this study is to identify the range of body mass index (BMI) at LT associated with the lowest risks of posttransplant mortality by MELD category. METHODS: A retrospective cohort of patients aged 18 years or older from the Organ Procurement and Transplantation Network database undergoing LT between February 27, 2002, and December 31, 2013, was identified and followed up through March 14, 2014. Patients' MELD score at the time of transplantation was categorized into 10 or lower (MELD1), 11 to 18 (MELD2), 19 to 24 (MELD3), and 25 or higher (MELD4). Multivariable adjusted Cox proportional hazard analyses were conducted. RESULTS: Among 48 226 patients in the analytic cohort (14.8% were in MELD1, 33.7% were in MELD2, 19.6% were in MELD3, and 32.0% were in MELD4), 25% died with mean follow-up of 1371 days. For MELD1, patient BMI ranging from 30 to 33 was associated with a better survival outcome than BMI less than 30 or 33 or greater; for MELD2, BMI ranging from 28 to 37 had a better survival outcome than BMI less than 28 or 37 or greater; for MELD3, the survival outcome improved with an increasing BMI; for MELD4, the survival outcome was not associated with patient BMI. CONCLUSIONS: This study provides evidence that obesity in LT patients is not necessarily associated with higher posttransplantation mortality and highlights the importance of the interaction between BMI and MELD category to determine their survival likelihood.

A temperature-dependent physiologically based model for the invasive apple snail Pomacea canaliculata.

In order to set priorities in management of costly and ecosystem-damaging species, policymakers and managers need accurate predictions not only about where a specific invader may establish but also about its potential abundance at different geographical scales. This is because density or biomass per unit area of an invasive species is a key predictor of the magnitude of environmental and economic impact in the invaded habitat. Here, we present a physiologically based demographic model describing and explaining the population dynamics of a widespread freshwater invader, the golden apple snail Pomacea canaliculata, which is causing severe environmental and economic impacts in invaded wetlands and rice fields in Southeastern Asia and has also been introduced to North America and Europe. The model is based on bio-demographic functions for mortality, development and fecundity rates that are driven by water temperature for the aquatic stages (juveniles and adults) and by air temperature for the aerial egg masses. Our model has been validated against data on the current distribution in South America and Japan, and produced consistent and realistic patterns of reproduction, growth, maturation and mortality under different scenarios in accordance to what is known from real P. canaliculata populations in different regions and climates. The model further shows that P. canaliculata will use two different reproductive strategies (semelparity and iteroparity) within the potential area of establishment, a plasticity that may explain the high invasiveness of this species across a wide range of habitats with different climates. Our results also suggest that densities, and thus the magnitude of environmental and agricultural damage, will be largely different in locations with distinct climatic regimes within the potential area of establishment. We suggest that physiologically based demographic modelling of invasive species will become a valuable tool for invasive species managers.

Racial disparity in life expectancies and life years lost associated with multiple obesity-related chronic conditions.

OBJECTIVE: This study investigated racial disparity in life expectancies (LEs) and life years lost (LYL) associated with multiple obesity-related chronic conditions (OCCs). METHODS: Data from the Medical Expenditure Panel Survey, 2008-2012, were used. Four OCCs were studied: diabetes, hypertension, coronary heart disease (CHD), and stroke. LE for each subpopulation was simulated by Markov modelling. LYL associated with a disease for a subpopulation was computed by taking the difference between LEs for members of that subpopulation without disease and LEs for members of that subpopulation who had that disease. Racial disparities were measured in the absolute differences in LE and LYL between black women/men and white women/men. RESULTS: Black individuals had higher risks of developing diabetes, hypertension, and stroke. This disparity in LE between white and black participants was largest in men age 40 to 49 with at least stroke: black men lived 3.12 years shorter than white men. The disparity in LYL between white and black participants was largest in women age 70 to 79 with at least CHD: black women had 1.98 years more LYL than white women. CONCLUSIONS: Racial disparity exists in incident disease and mortality risks, LEs, and LYL associated with multiple OCCs. Efforts targeting subpopulations with large disparities are required to reduce disparities in the burden of multiple OCCs.

The Burden of Obesity on Diabetes in the United States: Medical Expenditure Panel Survey, 2008 to 2012.

BACKGROUND: Diabetes is one of the most prevalent and costly chronic diseases in the United States. OBJECTIVES: To analyze the risk of developing diabetes and the annual cost of diabetes for a US general population. METHODS: Data from the Medical Expenditure Panel Survey, 2008 to 2012, were used to analyze 1) probabilities of developing diabetes and 2) annual total health care expenditures for diabetics. The age-, sex-, race-, and body mass index (BMI)-specific risks of developing diabetes were estimated by fitting an exponential survival function to age at first diabetes diagnosis. Annual health care expenditures were estimated using a generalized linear model with log-link and gamma variance function. Complex sampling designs in the Medical Expenditure Panel Survey were adjusted for. All dollar values are presented in 2012 US dollars. RESULTS: We observed a more than 6 times increase in diabetes risks for class III obese (BMI ≥ 40 kg/m2) individuals compared with normal-weight individuals. Using age 50 years as an example, we found a more than 3 times increase in annual health care expenditures for those with diabetes ($13,581) compared with those without diabetes ($3,954). Compared with normal-weight (18.5 ≤ BMI < 25 kg/m2) individuals, class II obese (35 ≤ BMI < 40 kg/m2) and class III obese (BMI ≥ 40 kg/m2) individuals incurred an annual marginal cost of $628 and $756, respectively. The annual health care expenditure differentials between those with and without diabetes of age 50 years were the highest for individuals with class II ($12,907) and class III ($9,703) obesity. CONCLUSIONS: This article highlights the importance of obesity on diabetes burden. Our results suggested that obesity, in particular, class II and class III (i.e., BMI ≥ 35 kg/m2) obesity, is associated with a substantial increase in the risk of developing diabetes and imposes a large economic burden.

Obesity and the Transformation of Monoclonal Gammopathy of Undetermined Significance to Multiple Myeloma: A Population-Based Cohort Study.

Background: Multiple myeloma (MM) is one of the most common hematologic malignancies in the United States and is consistently preceded by monoclonal gammopathy of undetermined significance (MGUS). This study investigates the role of obesity in the progression of MGUS to MM. Methods: A retrospective identified cohort of patients in the US Veterans Health Administration database diagnosed with MGUS between October 1, 1999, and December 31, 2009, was followed through August 6, 2013. Patient-level clinical data were reviewed to verify MM diagnosis, if any. Survival analyses utilizing interval-censored data were used to investigate the risk of progression of MGUS to MM. Statistical tests were two-sided. Results: The analytic cohort consisted of 7878 MGUS patients with a median follow-up of 68 months. Within the cohort, 39.8% were overweight and 33.8% were obese; 64.1% were of white race. During follow-up, 329 MGUS patients (4.2%) progressed to MM: 72 (3.5%) normal-weight patients (median follow-up = 61.9 months), 144 (4.6%) overweight patients (median follow-up = 69.1 months), and 113 (4.3%) obese patients (median follow-up = 70.6 months). In the multivariable analysis, overweight (hazard ratio [HR] = 1.55, 95% confidence interval [CI] = 1.16 to 2.06) and obesity (HR = 1.98, 95% CI = 1.47 to 2.68) were associated with an increased risk of transformation of MGUS to MM. Moreover, black race was associated with a higher risk of MM (HR = 1.98, 95% CI = 1.55 to 2.54). Conclusions: Obesity and black race are risk factors for transformation of MGUS to MM. Future clinical trials should examine whether weight loss is a way to prevent the progression to MM in MGUS patients.

Herring and chicken/pork meals lead to differences in plasma levels of TCA intermediates and arginine metabolites in overweight and obese men and women.

SCOPE: What effect does replacing chicken or pork with herring as the main dietary source of protein have on the human plasma metabolome? METHOD AND RESULTS: A randomised crossover trial with 15 healthy obese men and women (age 24-70 years). Subjects were randomly assigned to four weeks of herring diet or a reference diet of chicken and lean pork, five meals per week, followed by a washout and the other intervention arm. Fasting blood serum metabolites were analysed at 0, 2 and 4 weeks for eleven subjects with available samples, using GC-MS based metabolomics. The herring diet decreased plasma citrate, fumarate, isocitrate, glycolate, oxalate, agmatine and methyhistidine and increased asparagine, ornithine, glutamine and the hexosamine glucosamine. Modelling found that the tricarboxylic acid cycle, glyoxylate, and arginine metabolism were affected by the intervention. The effect on arginine metabolism was supported by an increase in blood nitric oxide in males on the herring diet. CONCLUSION: The results suggest that eating herring instead of chicken and lean pork leads to important metabolic effects, particularly on energy and amino acid metabolism. Our findings support the hypothesis that there are metabolic effects of herring intake unrelated to the long chain n-3 polyunsaturated fatty acid content.

Associations of individual characteristics and lifestyle factors with metabolism of di-2-ethylhexyl phthalate in NHANES 2001-2012.

BACKGROUND: Previous studies suggest that a higher ratio of primary to secondary metabolites of di-2-ethylhexyl phthalate (DEHP), reflective of a slower DEHP conversion rate, is associated with a greater physiologic effect. We examined associations of several individual characteristics and lifestyle factors with the ratio of mono-2-ethylhexyl phthalate to mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHP:MEHHP) and %MEHP (the ratio of MEHP to the sum of the secondary metabolites). METHODS: We used the data from the National Health and Nutrition Examination Survey, 2001-2012. The study included adults with BMI<30 and no diabetes. Pregnant women were excluded. We examined associations of age, race, gender, Body Mass Index, smoking, alcohol and caffeine consumption, medication use, cancer history, and menopausal status and postmenopausal hormone use (in women) with MEHP:MEHHP and %MEHP using multivariable linear regression. The values for %MEHP were log-transformed in the analysis. RESULTS: In multivariable analysis, non-Caucasian individuals had higher %MEHP (non-Hispanic Blacks: ß=0.114, 95% Confidence interval [CI]: 0.050, 0.177; Hispanic: ß=0.089, 95% CI: 0.024, 0.154; other race: ß=0.126, 95% CI: 0.033, 0.219). Age was inversely associated with MEHP:MEHHP (ß=-0.001, 95% CI: -0.002, -0.001) and %MEHP (ß=-0.006, 95% CI: -0.008, -0.004). Overweight individuals had lower MEHP: MEHHP and lower %MEHP (ß=-0.035, 95% CI: 0.062, -0.008 and ß=-0.104, 95% CI: -0.162, -0.046, respectively). Alcohol consumption was inversely associated with %MEHP among men (p-trend=0.03). CONCLUSIONS: Individual and lifestyle characteristics are associated with differences in DEHP metabolism. Understanding underlying biological mechanisms could help to identify individuals at a greater risk of adverse effects from DEHP exposure.

Formation of reactive aldehydes (MDA, HHE, HNE) during the digestion of cod liver oil: comparison of human and porcine in vitro digestion models.

In this work, we investigated lipid oxidation of cod liver oil during gastrointestinal (GI) digestion using two types of in vitro digestion models. In the first type of model, we used human GI juices, while we used digestive enzymes and bile from porcine origin in the second type of model. Human and porcine models were matched with respect to factors important for lipolysis, using a standardized digestion protocol. The digests were analysed for reactive oxidation products: malondialdehyde (MDA), 4-hydroxy-trans-2-nonenal (HNE), and 4-hydroxy-trans-2-hexenal (HHE) by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS), and for free fatty acids (FFA) obtained during the digestion by gas chromatography-mass spectrometry (GC-MS). The formation of the oxidation products MDA, HHE, and HNE was low during the gastric digestion, however, it increased during the duodenal digestion. The formation of the oxidation products reached higher levels when digestive juices of human origin were used (60 µM of MDA, 9.8 µM of HHE, and 0.36 µM of HNE) [corrected] compared to when using enzymes and bile of porcine origin (0.96, and 1.6 µM of MDA; 0.16, and 0.23 µM of HHE; 0.026, [corrected] and 0.005 µM of HNE, respectively, in porcine models I and II). In all models, FFA release was only detected during the intestinal step, and reached up to 31% of total fatty acids (FA). The findings in this work may be of importance when designing oxidation oriented lipid digestion studies.

Association between metformin use and progression of monoclonal gammopathy of undetermined significance to multiple myeloma in US veterans with diabetes mellitus: a population-based retrospective cohort study.

BACKGROUND: Multiple myeloma is one of the most common haematological malignancies in the USA and is consistently preceded by monoclonal gammopathy of undetermined significance (MGUS). We aimed to assess the association between metformin use and progression of MGUS to multiple myeloma. METHODS: We did a retrospective cohort study of patients registered in the US Veterans Health Administration database and diagnosed with MGUS between Oct 1, 1999, and Dec 31, 2009. We included patients (aged >18 years) with at least one International Classification of Diseases (9th revision) code for diabetes mellitus and one treatment for their diabetes before MGUS diagnosis. We reviewed patient-level clinical data to verify diagnoses and extract any available data for size of baseline M-protein and type of MGUS. We defined metformin users as patients with diabetes who were given metformin consistently for 4 years after their diabetes diagnosis and before multiple myeloma development, death, or censorship. Our primary outcome was time from MGUS diagnosis to multiple myeloma diagnosis. We used Kaplan-Meier curves and Cox models to analyse the association between metformin use and MGUS progression. FINDINGS: We obtained data for 3287 patients, of whom 2003 (61%) were included in the final analytical cohort. Median follow-up was 69 months (IQR 49­96). 463 (23%) participants were metformin users and 1540 (77%) participants were non-users. 13 (3%) metformin users progressed to multiple myeloma compared with 74 (5%) non-users. After adjustment, metformin use was associated with a reduced risk of progression to multiple myeloma (hazard ratio 0·47, 95% CI 0·25­0·87). INTERPRETATION: For patients with diabetes diagnosed with MGUS, metformin use for 4 years or longer was associated with a reduced risk of progression of MGUS to multiple myeloma. Prospective studies are needed to establish whether this association is causal and whether these results can be extrapolated to non-diabetic individuals. FUNDING: Barnes-Jewish Hospital Foundation, National Institutes of Health, Agency for Healthcare Research and Quality, American Cancer Society.

A fluorescence spectroscopy assay for real-time monitoring of enzyme immobilization into mesoporous silica particles.

Mesoporous silica particles are used as support material for immobilization of enzymes. Here we investigated a fluorescence-based assay for real-time monitoring of the immobilization of lipase, bovine serum albumin, and glucose oxidase into micrometer-sized mesoporous silica particles. The proteins are labeled with the dye epicocconone, and the interaction with the particles is observed as an increase in emission intensity of the protein-dye conjugates that can be quantified if correcting for a comparatively slow photobleaching. The immobilization occurs in tens of minutes to hours depending on particle concentration and type of protein. In the limit of excess particles over proteins, the formation of the particle-protein complexes can be described by a single exponential growth for all three investigated proteins, and the fitted pseudo-first-order rate constant increases linearly with particle concentration for each protein type. The derived second-order rate constant k varies with the protein hydrodynamic radius according to k∼RH(-4.70±0.01), indicating that the rate-limiting step at high particle concentrations is not the diffusional encounter between proteins and particles but rather the entry into the pores, consistent with the hydrodynamic radii of the three proteins being smaller but comparable to the pore radius of the particles.

Quantification of total fatty acids in microalgae: comparison of extraction and transesterification methods.

Determination of microalgaes' fatty acid content is often done with chloroform and methanol according to the Bligh and Dyer extraction, though faster methods exist. A number of comparisons between the Bligh and Dyer and faster methods have resulted in contradicting data, possibly due to differences in algae used and the different versions of the Bligh and Dyer method applied. Here, various forms of direct-transesterification (D-TE) and two-step transesterification (2-TE), including three versions developed in our lab, are compared with the original Bligh and Dyer (Can J Biochem Physiol 37: 911-917, 1959) extraction and two modifications thereof (Lee et al. J AOAC Int 79:487-492, 1996, and our own acidified version) on microalgae with different cell walls: Isochrysis galbana, Nannochloropsis oculata, and Phaeodactylum tricornutum. In total, fatty acid extracts from 11 methods were separated and quantified by gas chromatography with mass spectrometry. Results show that, for N. oculata and P. tricornutum, methods based on chloroform-methanol underestimated the fatty acid content compared with the 2-TE and D-TE methods, which gave similar results. Moreover, D-TE methods are faster than chloroform-methanol methods and use chemicals that are less toxic. Of the D-TE methods, the ones using hydrochloric acid and sulfuric acid recovered the most fatty acids, while boron trifluoride recovered slightly less. The main qualitative difference between the fatty acids recovered was that the chloroform-methanol methods recovered less saturated fatty acids in P. tricornutum.

A new approach to measuring vitamin D in human adipose tissue using time-of-flight secondary ion mass spectrometry: a pilot study.

Circulating concentrations of vitamin D, 25(OH)D, and 1,25(OH)2D are lower in obese than lean individuals, but little is known about the adipose tissue content of these molecules. The aim of this study was to explore the possibility to use time-of-flight secondary ion mass spectrometry (TOF-SIMS) to measure vitamin D and its metabolites in fat tissue in obese and lean subjects. Abdominal subcutaneous adipose tissue (SAT) biopsies were obtained from three lean and three obese women, and paired biopsies SAT and visceral adipose tissue (VAT) were obtained from three obese subjects during gastric bypass surgery. TOF-SIMS was used to measure vitamin D3, 25(OH)D3, and 1,25(OH)2D3 in adipose tissue. We found that vitamin D3, 25(OH)D3, and 1,25(OH)2D3 in adipose tissue can be measured with TOF-SIMS. In adipose tissue, vitamin D3 and its metabolites were located in adipocyte lipid droplets. The content of vitamin D3 (P=0.006) and 25(OH)D3 (P=0.018) were lower in SAT in obese compared with lean women. TOF-SIMS has the potential to semi-quantitatively measure vitamin D metabolites in adipose tissue, and offers a possibility to compare vitamin D levels in different depots and groups of individuals. It also gives the opportunity to explore the localization of vitamin D metabolites at a cellular level.

Peptide-membrane interactions of arginine-tryptophan peptides probed using quartz crystal microbalance with dissipation monitoring.

Membrane-active peptides include peptides that can cross cellular membranes and deliver macromolecular cargo as well as peptides that inhibit bacterial growth. Some of these peptides can act as both transporters and antibacterial agents. It is desirable to combine the knowledge from these two different fields of membrane-active peptides into design of new peptides with tailored actions, as transporters of cargo or as antibacterial substances, targeting specific membranes. We have previously shown that the position of the amino acid tryptophan in the peptide sequence of three arginine-tryptophan peptides affects their uptake and intracellular localization in live mammalian cells, as well as their ability to inhibit bacterial growth. Here, we use quartz crystal microbalance with dissipation monitoring to assess the induced changes caused by binding of the three peptides to supported model membranes composed of POPC, POPC/POPG, POPC/POPG/cholesterol or POPC/lactosyl PE. Our results indicate that the tryptophan position in the peptide sequence affects the way these peptides interact with the different model membranes and that the presence of cholesterol in particular seems to affect the membrane interaction of the peptide with an even distribution of tryptophans in the peptide sequence. These results give mechanistic insight into the function of these peptides and may aid in the design of membrane-active peptides with specified cellular targets and actions.

Enzymes immobilized in mesoporous silica: a physical-chemical perspective.

Mesoporous materials as support for immobilized enzymes have been explored extensively during the last two decades, primarily not only for biocatalysis applications, but also for biosensing, biofuels and enzyme-controlled drug delivery. The activity of the immobilized enzymes inside the pores is often different compared to that of the free enzymes, and an important challenge is to understand how the immobilization affects the enzymes in order to design immobilization conditions that lead to optimal enzyme activity. This review summarizes methods that can be used to understand how material properties can be linked to changes in enzyme activity. Real-time monitoring of the immobilization process and techniques that demonstrate that the enzymes are located inside the pores is discussed by contrasting them to the common practice of indirectly measuring the depletion of the protein concentration or enzyme activity in the surrounding bulk phase. We propose that pore filling (pore volume fraction occupied by proteins) is the best standard for comparing the amount of immobilized enzymes at the molecular level, and present equations to calculate pore filling from the more commonly reported immobilized mass. Methods to detect changes in enzyme structure upon immobilization and to study the microenvironment inside the pores are discussed in detail. Combining the knowledge generated from these methodologies should aid in rationally designing biocatalyst based on enzymes immobilized in mesoporous materials.

Membrane interaction and secondary structure of de novo designed arginine-and tryptophan peptides with dual function.

Cell-penetrating peptides and antimicrobial peptides are two classes of positively charged membrane active peptides with several properties in common. The challenge is to combine knowledge about the membrane interaction mechanisms and structural properties of the two classes to design peptides with membrane-specific actions, useful either as transporters of cargo or as antibacterial substances. Membrane active peptides are commonly rich in arginine and tryptophan. We have previously designed a series of arg/trp peptides and investigated how the position and number of tryptophans affect cellular uptake. Here we explore the antimicrobial properties and the interaction with lipid model membranes of these peptides, using minimal inhibitory concentrations assay (MIC), circular dichroism (CD) and linear dichroism (LD). The results show that the arg/trp peptides inhibit the growth of the two gram positive strains Staphylococcus aureus and Staphylococcus pyogenes, with some individual variations depending on the position of the tryptophans. No inhibition of the gram negative strains Proteus mirabilis or Pseudomonas aeruginosa was noticed. CD indicated that when bound to lipid vesicles one of the peptides forms an α-helical like structure, whereas the other five exhibited rather random coiled structures. LD indicated that all six peptides were somehow aligned parallel with the membrane surface. Our results do not reveal any obvious connection between membrane interaction and antimicrobial effect for the studied peptides. By contrast cell-penetrating properties can be coupled to both the secondary structure and the degree of order of the peptides.