Assessing a Fermented Whey Beverage Biofortified with Folate as a Potential Folate Source for Humans.

Folate, a vital water-soluble vitamin (B9), requires specific attention as its recommended daily intake frequently is not reached in countries without mandatory fortification. In this regard, biofortification with microorganisms like Bifidobacterium and Streptococcus offers a compelling approach for enhancing food with natural folates. A randomized, nonblinded, and monocentric human pilot study is conducted to assess the bioavailability of a folate-biofortified fermented whey beverage, comprising 3 intervention days and a controlled replenishment phase before and during the assay. Folate plasma concentration (5-CH3-H4folate) is determined using a stable isotope dilution assay and LC-MS/MS detection. Biokinetic parameters (cmax and tmax) are determined, and areas under the curve (AUC) normalized to the basal folate plasma concentration are calculated. An average bioavailability of 17.1% in relation to the 5-CH3-H4folate supplement, ranging from 0% to 39.8%, is obtained. These results reiterate the significance of additional research into folate bioavailability in general and dairy products. Further investigations are warranted into folate-binding proteins (FBP) and other potential limiting factors within the food and individual factors. In summary, biofortification via fermentation emerges as a promising avenue for enhancing the natural folate content in dairy and other food products.

Characterization of the key aroma compounds in different varieties of hops by application of the Sensomics approach.

Aroma is a crucial indicator of hop quality. This study analyzed the differences in aroma compound composition among six hop varieties from three regions: North America, Europe, and Asia. Descriptive analysis and sensomic approaches including gas chromatography-olfactometry/aroma extract dilution analysis, odour activity value calculation and aroma recombination were used for the detailed characterization and comparative analysis of hop aroma. A total of 55 aroma-active compounds were identified. Among them, linalool, geraniol, ß-myrcene, 2-undecanone, and methyl decanoate contributed significantly to hop aroma. Orthogonal partial least squares discriminant analysis revealed that, except for the SAAZ and XinYuan hops with some similarities in their aroma composition, the remaining hops exhibited unique aroma characteristics. A total of 16 compounds, including methyl 5-methylhexanoate and (E)-ß-farnesene, were identified as differentiating aroma compounds in the six hop samples. This study enriches the knowledge on hop flavour with different origins and provides valuable insights into its application.

A comparative study to determine the key aroma components of yogurt aroma types based on Sensomics and Flavoromics.

This study used Sensomics to examine four previously obtained yogurt aroma type profiles. 14 key aroma-active compounds were identified as significant contributors (p ≤ 0.05) in the four aroma types using gas chromatography-mass spectrometry-olfactometry (GC-MS/O), aroma extract dilution analysis (AEDA), odor activity values (OAV), and aroma recombination and omission experiments. The Sensomics and previous Flavoromics results were compared, showing that Flavoromics identified 10 indicator compounds for distinguishing aroma types. Eight were the same as the key aroma-active compounds identified via Sensomics, namely acetic acid, pentanoic acid, decanoic acid, 3-hydroxy-2-butanone, 2,3-pentanedione, acetaldehyde, δ-decalactone, and dimethyl sulfone. Sensomics revealed a prominent similarity between the categories of key aroma-active compounds of the four aroma types, with a higher sensory contribution. Flavoromics showed less overlapping between the indicator compounds, mainly related to the distinction between the four aroma types. Sensomics and Flavoromics serve distinct research objectives and should be selected according to the study subject.

Calculated Drug Concentrations in Currently Available Intravitreal Therapies: Determination of Dilution Factor and Deviation From Recommended Doses.

In ophthalmology, intravitreal therapies are currently not personalized/customized and are not adjusted to the individual vitreous volume. With reference to the recently published calculation formula for a more accurate estimation of the vitreous body, we determined the dose of intravitreal medication for different vitreous volumes and compared them with the average volume. Using the axial length of the eye, the formula for the vitreous volume exact (VIVEX) can provide a more accurate indication of the vitreous volume in individual cases than an assumed standard volume of 4 mL. The concentration of active substances in small eyes may be twice as high as that in normal-sized emmetropic eyes. In contrast, large eyes may show less than half of the recommended drug concentration. The calculated concentrations of the investigated intravitreal drugs in small and large eyeballs showed impressive differences with large deviations from the recommended doses. Further systematic studies should follow to find out whether this has any impact on the effectiveness or side effects of the injected drugs.

Construction of a Calibration Curve for Lycopene on a Liquid-Handling Platform─Wider Lessons for the Development of Automated Dilution Protocols.

Liquid-handling is a fundamental operation in synthetic biology─all protocols involve one or more liquid-handling operations. It is, therefore, crucial that this step be carefully automated in order to unlock the benefits of automation (e.g., higher throughput, higher replicability). In the paper, we present a study, conducted at the London Biofoundry at SynbiCITE, that approaches liquid-handling and its reliable automation from the standpoint of the construction of the calibration curve for lycopene in dimethyl sulfoxide (DMSO). The study has important practical industrial applications (e.g., lycopene is a carotenoid of industrial interest, DMSO is a popular extractant). The study was also an effective testbed for the automation of liquid-handling. It necessitated the development of flexible liquid-handling methods, which can be generalizable to other automated applications. In addition, because lycopene/DMSO is a difficult mix, it was capable of revealing issues with automated liquid-handling protocols and stress-testing them. An important component of the study is the constraint that, due to the omnipresence of liquid-handling steps, errors should be controlled to a high standard. It is important to avoid such errors propagating to other parts of the protocol. To achieve this, a practical framework based on regression was developed and utilized throughout the study to identify, assess, and monitor transfer errors. The paper concludes with recommendations regarding automation of liquid-handling, which are applicable to a large set of applications (not just to complex liquids such as lycopene in DMSO or calibration curves).

A semi-mechanistic aging model of Pb added to soil by a modified stable isotope dilution technique.

The aging of Pb added to soils has not been studied by the isotopic technology because of difficulties in determination of isotopically exchangeable Pb in soil, so that a set of 10 typical agricultural soils in China and a one-year aging experiment with the addition of water-soluble Pb to the soils were carried out. A modified stable isotope dilution technique to determine isotopically exchangeable Pb in soil was developed where 0.2 mM EDTA (ethylenediaminetetraacetic acid) as the extractant. When water-soluble Pb was added to soil, the isotopically exchangeable Pb (Eadd%, the percentage of isotopically exchangeable Pb to total Pb added to soil) initially decreased rapidly and gradually slowly. A semi-mechanistic aging model of Pb added to soils, including precipitation/nucleation (Y1), micropore diffusion (Y2), and organic matter encapsulation processes (Y3) was developed with the root mean square error 8.3% where Y1, Y2, and Y3 accounted for 0.02~26.9%, 1.4~21.8% and 3.8~11.3%, respectively, when the pH 4.0~8.0 and organic matter 2.0~6.0%. Soil pH was a vital factor affecting the aging rate. When the pH increased by 1 unit, the Eadd value decreased by approximately 9%. The model could be used to scale ecotoxicological data of Pb in soil generated in different aging times.

Evaluation of Gradient Diffusion in vitro Susceptibility Testing of Aerococcus urinae.

OBJECTIVES: Aerococcus urinae antimicrobial susceptibility testing (AST) can be performed via broth microdilution (BMD) with Mueller-Hinton broth supplemented with lysed horse blood (MHB-LHB). We sought to compare this to the commonly used gradient diffusion method. METHODS: We compared BMD with MHB-LHB to gradient diffusion via MH agar supplemented with sheep blood for 190 A. urinae isolates against sixteen antimicrobials. RESULTS: No antimicrobials demonstrated over 90% essential and categorical agreement (EA and CA) and less than 3% major and very major error rates (ME and VME). Trimethoprim-sulfamethoxazole (TMP-STX) demonstrated an 81% major error rate and ceftriaxone demonstrated a 76% very major error rate. Agar dilution (AD) with lysed horse blood was performed for TMP-STX against 94 isolates and found 100% susceptibility, consistent with previous studies. CONCLUSIONS: Our findings cannot support the routine use of gradient diffusion with MH agar supplemented with sheep blood for A. urinae in lieu of the CLSI method given its limitations in detecting resistant strains. Our results suggest A. urinae is usually susceptible to penicillin, linezolid, tetracycline, and vancomycin. Future studies should evaluate alternative testing methods for clinical microbiology laboratories.

Combination of Standard Addition and Isotope Dilution Mass Spectrometry for the Accurate Determination of Melamine and Cyanuric Acid in Infant Formula.

In the melamine scandals of the early 2000s, different companies of the dairy industry cheated their products by applying chemical substances to feign a higher content of nitrogen. However, this had a severe toxic impact on the kidney health of consumers. As a result, tremendous effort was put into the prevention of further harm to the public. In the present study, a fast-screening method for the determination of melamine and cyanuric acid in infant formula was developed. While a 1D-LC approach is faster and easier to set up, a 2D-LC approach allows for a more accurate result with better selectivity and sensitivity. For both instrumental approaches, the signal ratio of the isotopologues was crucial and had a dominant effect on the results and the measurement uncertainty. For this reason, the different contributions to the measurement uncertainty were determined experimentally using Matched Standard Addition-IDMS and compared to the Exact Matching Double IDMS.

Two novel Pleosporales species isolated from the bark of Acer saccharum.

During a survey of culturable microfungi from the bark of sugar maple (Acer saccharum), Atrocalyx glutinosus and Nigrograna rubescens, two novel species of Pleosporales (Dothideomycetes) were isolated from several locations in eastern Ontario, Canada. Formal species descriptions are presented based on unique colony phenotypes and micromorphological characteristics and supported using multi-locus molecular phylogenetic comparisons with similar species. Both A. glutinosus and N. rubescens produce pycnidial asexual morphs in culture. As their names imply, under specific culture conditions, A. glutinosus excretes large amounts of the glutinous polysaccharide pullulan and N. rubescens produces a dark red naphthoquinone pigment that diffuses in the culture medium. Citation: Mack JN, Sproule A, Shields SW, Seifert KA, Smith M, Overy DP (2024). Two novel Pleosporales species isolated from the bark of Acer saccharum . Fungal Systematics and Evolution 13: 1-14. doi: 10.3114/fuse.2024.13.01.

Evaluation of the Accuracy of Estimated Endpoint Titer of NOVA View in Indirect Immunofluorescent Antinuclear Antibody Testing.

For antinuclear antibody (ANA) screening, the gold standard method is an indirect immunofluorescence assay (IIFA) using HEp-2 cells, and a serial dilution test is needed to determine the endpoint titer. We aimed to evaluate the accuracy of the estimated endpoint titer (eEPT) by the NOVA View system, by comparing it with the EPT by the serial dilution method (dEPT). The endpoint titers of a total of 1518 ANA positive cases with five major patterns including speckled, homogeneous, centromere, nucleolar, and nuclear dots patterns were determined using both the estimation function and the serial dilution method by the NOVA View system. A significant correlation between the light intensity unit (LIU) values and dEPTs was identified in all five patterns with high ρ values, ranging from 0.666 to 0.832. However, the overall exact match rate between dEPT and eEPT was 22.1% (336/1518), with the ±one-titer match rate being highest in the centromere pattern (62.8%, 81/129), and lowest in the homogeneous pattern (37.6%, 200/532). This suggests that while LIU values correlate well with dEPT, there are discrepancies in numerical agreement. Most cases that did not show an exact match, showed one-to-three-titer overestimations by eEPT. Therefore, adjusting eEPT downward significantly improved the concordance rates with dEPTs. Further investigation for an appropriate cutoff of LIU values for determining eEPT should be performed for clinical application and contribution to the standardization of the ANA titer.

Diet and habitat as determinants of intestine length in fishes.

Fish biologists have long assumed a link between intestinal length and diet, and relative gut length or Zihler's index are often used to classify species into trophic groups. This has been done for specific fish taxa or specific ecosystems, but not for a global fish dataset. Here, we assess these relationships across a dataset of 468 fish species (254 marine, 191 freshwater, and 23 that occupy both habitats) in relation to body mass and fish length. Herbivores had significantly relatively stouter bodies and longer intestines than omni- and faunivores. Among faunivores, corallivores had longer intestines than invertivores, with piscivores having the shortest. There were no detectable differences between herbivore groups, possibly due to insufficient understanding of herbivorous fish diets. We propose that reasons for long intestines in fish include (i) difficult-to-digest items that require a symbiotic microbiome, and (ii) the dilution of easily digestible compounds with indigestible material (e.g., sand, wood, exoskeleton). Intestinal indices differed significantly between dietary groups, but there was substantial group overlap. Counter-intuitively, in the largest dataset, marine species had significantly shorter intestines than freshwater fish. These results put fish together with mammals as vertebrate taxa with clear convergence in intestine length in association with trophic level, in contrast to reptiles and birds, even if the peculiar feeding ecology of herbivorous fish is probably more varied than that of mammalian herbivores. Supplementary Information: The online version contains supplementary material available at 10.1007/s11160-024-09853-3.

Hyposalinity elicits physiological responses and alters intestinal microbiota in Korean rockfish Sebastes schlegelii.

Global warming significantly impacts aquatic ecosystems, with changes in the salt environment negatively affecting the physiological responses of fish. We investigated the impact of hyposalinity on the physiological responses and intestinal microbiota of Sebastes schlegelii under the context of increased freshwater influx due to climate change. We focused on the osmoregulatory capacity, oxidative stress responses, and alterations in the intestinal microbiome of S. schlegelii under low-salinity conditions. Our findings revealed compromised osmoregulatory capacity in S. schlegelii under low-salinity conditions, accompanied by the activation of oxidative stress responses, indicating physiological adaptations to cope with environmental stress. Specifically, changes in Na+/K+-ATPase (NKA) activity in gill tissues were associated with decreased osmoregulatory capacity. Furthermore, the analysis of the intestinal microbiome led to significant changes in microbial diversity. Exposure to low-salinity environments led to dysbiosis, with notable decreases in the relative abundance of Gammaproteobacteria at the class level and specific genera such as Enterovibrio, and Photobacterium. Conversely, Bacilli classes, along with genera like Mycoplasma, exhibited increased proportions in fish exposed to low-salinity conditions. These findings underscore the potential impact of environmental salinity changes on the adaptive capacity of fish species, particularly in the context of aquaculture. Moreover, they highlight the importance of considering both physiological and microbial responses in understanding the resilience of aquatic organisms to environmental stress. Additionally, they highlight the importance of intestinal microbiota analyses in understanding the immune system and disease management in fish.

The role of ruminant urine and faeces in the recycling of nutrients by forages.

This study addresses the effect of using animal excreta on the nutritional content of forages, focusing on macro- and micro-element concentrations (nitrogen; N, phosphorus; P, sulphur; S, copper; Cu, zinc; Zn, manganese; Mn, selenium; Se) from animal feed to excreta, soil, and plants. Data were collected from pot and field trials using separate applications of sheep or cattle urine and faeces. Key findings indicate that soil organic carbon (SOC) and the type of excreta significantly influences nutrient uptake by forages, with varied responses among the seven elements defined above. Although urine contributes fewer micronutrients compared to faeces (as applied at a natural volume/mass basis, respectively), it notably improves forage yield and micronutrient accumulation, thus potentially delivering positive consequences at the farm level regarding economic performance and soil fertility when swards upon clayey soil types receive said urine in temperate agro-climatic regions (i.e., South West England in the current context). In contrast, faeces application in isolation hinders Se and Mn uptake, once again potentially delivering unintended consequences such as micronutrient deficiencies in areas of high faeces deposition. As it is unlikely that (b)ovine grazing fields will receive either urine or faeces in isolation, we also explored combined applications of both excreta types which demonstrates synergistic effects on N, Cu, and Zn uptake, with either synergistic or dilution effects being observed for P and S, depending largely on SOC levels. Additionally, interactions between excreta types can result in dilution or antagonistic effects on Mn and Se uptake. Notably, high SOC combined with faeces reduces Mn and Se in forages, raising concerns for grazed ruminant systems under certain biotic situations, e.g., due to insufficient soil Se levels typically observed in UK pastures for livestock growth. These findings underscore the importance of considering SOC and excreta nutritional composition when designing forage management to optimize nutrient uptake. It should be noted that these findings have potential ramifications for broader studies of sustainable agriculture through system-scale analyses, as the granularity of results reported herein elucidate gaps in knowledge which could affect, both positively and negatively, the interpretation of model-based environmental impact assessments of cattle and sheep production (e.g., in the case of increased yields [beneficial] or the requirement of additional synthetic supplementation [detrimental]).

Isotope dilution with isotopically labeled biomass: An effective alternative for quantitative metabolomics.

BACKGROUND: State-of-the-art quantitative metabolomics relies on isotope dilution using internal standards (IS) derived from fully 13C labeled biomass. By spiking samples and external standards with known amounts of IS, the spike characterization demands are kept to a minimum. In fact, it is sufficient to experimentally assess the isotopic enrichment of the IS. This study develops the yeast derived IS toolbox further, (1) by characterizing the concentration levels of hydrophilic metabolites in a yeast fermentation batch and (2) by exploring the analytical figures of merit of one-point IS versus multipoint external calibration using IS, the established gold-standard for quantitative metabolomics. RESULTS: Independent reverse isotope dilution experiments using different chromatographic methods over a period of several months, delivered a list of 83 13C-labeled metabolites with fully characterized concentration and their uncertainty, covering 5 orders of magnitude, from the nanomolar to the low millimolar range. The 13C-labeled yeast-derived IS showed excellent intermediate stability with 92 % of molecules showing inter-method RSDs ≤30 % (75 % of molecules showed RSDs ≤15 %) over a timeframe of five months. One-point internal standardization with the characterized labeled biomass achieved figures of merit equivalent to multipoint calibrations for the majority of metabolites. SIGNIFICANCE: The proposed calibration workflow rationalizes time and standard expenditure and is particularly beneficial for laboratories dealing with wide-target assays and small analysis batches. The present assessment serves as a seminal study for further developments of the concept towards absolute quantification from archive high-resolution MS data of U13C-biomass-spiked samples and the implementation of quick biomass recalibration with each experiment, promising seamless transition between internal standards derived from different fermentation batches.

Maximum likelihood estimators for colony-forming units.

Measuring the abundance of microbes in a sample is a common procedure with a long history, but best practices are not well-conserved across microbiological fields. Serial dilution methods are commonly used to dilute bacterial cultures to produce countable numbers of colonies, and from these counts, to infer bacterial concentrations measured in colony-forming units (CFUs). The most common methods to generate data for CFU point estimates involve plating bacteria on (or in) a solid growth medium and counting their resulting colonies or counting the number of tubes at a given dilution that have growth. Traditionally, these types of data have been analyzed separately using different analytic methods. Here, we build a direct correspondence between these approaches, which allows one to extend the use of the most probable number method from the liquid tubes experiments, for which it was developed, to the growth plates by viewing colony-sized patches of a plate as equivalent to individual tubes. We also discuss how to combine measurements taken at different dilutions, and we review several ways of analyzing colony counts, including the Poisson and truncated Poisson methods. We test all point estimate methods computationally using simulated data. For all methods, we discuss their relevant error bounds, assumptions, strengths, and weaknesses. We provide an online calculator for these estimators.Estimation of the number of microbes in a sample is an important problem with a long history. Yet common practices, such as combining results from different measurements, remain sub-optimal. We provide a comparison of methods for estimating abundance of microbes and detail a mapping between different methods, which allows to extend their range of applicability. This mapping enables higher precision estimates of colony-forming units (CFUs) using the same data already collected for traditional CFU estimation methods. Furthermore, we provide recommendations for how to combine measurements of colony counts taken across dilutions, correcting several misconceptions in the literature.

Connecting genes to whole plants in dilution effect of target-site ALS inhibitor resistance of Schoenoplectiella juncoides (Roxb.) Lye (Cyperaceae).

This study focuses on dilution effect of target-site resistance (TSR) to acetolactate synthase (ALS) inhibitors in Schoenoplectiella juncoides, which harbors two ALS genes, ALS1 and ALS2. We assessed gene expression, enzyme activity, and whole-plant resistance profiles across four S. juncoides lines: the susceptible line, the parental resistant lines with a homozygous mutation in either ALS1 or ALS2, and the bred progeny line with homozygous mutations in both ALS1 and ALS2. Gene expression and enzyme function showed a proportional relationship that the expression ratios of ALS1 to ALS2, approximately 70:30, were consistent with the functional ratio predicted by the double-sigmoidal plateau positions observed in enzyme assays. However, at the whole-plant level, resistance did not correlate to the putative abundance of susceptible enzyme, but the parental lines showed similar resistance to each other despite different enzyme-level resistances. This suggests a non-proportional mechanism in the reflection of physiological enzymatic profiles to whole-plant resistance profiles. These findings highlight the complexity of herbicide resistance and the need for further research to understand the mechanisms that influence resistance outcomes. Understanding these relationships is essential for developing strategies to manage herbicide resistance effectively.

Determination of capsaicin at trace levels in different food, biological and environmental samples by quadruple isotope dilution-gas chromatography mass spectrometry after its preconcentration.

Despite the therapeutic properties of capsaicin for some diseases, it shows some side effects for human health. The goal of this study was to develop a precise and accurate analytical strategy for the trace determination of capsaicin in different food, biological and environmental samples including pepper, saliva and wastewater by gas chromatography-mass spectrometry (GC-MS) after spraying-based fine droplet formation-liquid phase microextraction (SFDF-LPME) and quadruple isotope dilution (ID4) method. Acetic anhydride was used as derivatizing agent, and the extraction method was used to enrich the analyte derivative to reach low detection limits. Under the optimum conditions, limit of detection (LOD) and limit of quantitation (LOQ) were determined to be 0.33 and 1.10 µg/kg, respectively. Percent recoveries calculated for SFDF-LPME-GC-MS method ranged between 84.1 and 131.7 %. After the application of ID4-SFDF-LPME-GC-MS method, percent recoveries were obtained in the range of 94.9 and 104.0 % (%RSD ≤ 2.8) for the selected samples. It is obvious that the isotope dilution-based method provided high accurate and precise results due to the elimination of errors during the derivatization, extraction and measurement steps.

A candidate reference measurement procedure for quantification of glycocholic acid in human serum based on isotope dilution liquid chromatography-tandem mass spectrometry.

Accurate measurement of serum glycocholic acid (GCA) is crucial for evaluating the activity of chronic hepatitis. Moreover, GCA is a novel identified biomarker for hepatocellular carcinoma. Although some laboratories have used the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure GCA in recent years, the problem of potential interference of GCA analogues has not been solved well yet. Neither reference measurement procedures nor reference materials for GCA have been listed in the Joint Committee for Traceability in Laboratory Medicine (JCTLM) database. For standardization of GCA, it is urgent to establish a candidate measurement procedure for GCA. In this study, a candidate reference measurement procedure for the quantification of GCA in human serum based on isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) by a two-step sample pretreatment of protein precipitation and MAX solid-phase extraction was developed and validated. GCA can be completely separated from its structural analogues with gradient elution in 9 min compared with short time gradients published in previous literature by Huang's group. Method validation indicated perfect quantitation precision with intra-day and inter-day values that were ≤1.30% and ≤1.80%, respectively. The method showed excellent linearity with high regression coefficients (R2 > 0.999) over a range of 0.92 ng/g-38.38 µg/g and perfect recoveries at three spiked levels (99.87-100.43%). No interference, matrix effect, and carryover were observed. Moreover, the cRMP was successfully applied to measure GCA in serum samples and compared with two immunoassays in a clinical laboratory. As a candidate reference method, this method can promote a GCA standardization program.

A high-accuracy measurement procedure for salbutamol, ractopamine, and clenbuterol in pork by isotope dilution-liquid chromatography/tandem mass spectrometry.

In-depth research into the precise evaluation of enzymatic digestion efficiency and the selection of a suitable deuterium-labelled internal standard remains a gap in the accurate determination of ß2-agonists in animal-derived food by isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LC-MS/MS). In this study, the enzymatic digestion conditions were optimized by monitoring the presence of ß2-agonist conjugates in positive samples, which proved to be reliable for ensuring complete enzymatic digestion. Comparative analysis of deuterium-labelled internal standards for salbutamol (SAL), ractopamine (RAC), and clenbuterol (CLB) revealed that CLB-D6 and SAL-D9 were less effective in compensating for matrix effects due to hydrogen­deuterium exchange during MS fragment formation. Consequently, SAL-D3, RAC-D3 and CLB-D9 were chosen for the implementation of ID-LC-MS/MS. The developed method demonstrates high accuracy and precision, with the average recoveries ranging from 93.8% to 107.3% with RSD <6.1%, which can provide higher-order measurement results for ß2-agonists in pork.