Follicular metabolic alterations are associated with obesity in mares and can be mitigated by dietary supplementation.

Obesity is a growing concern in human and equine populations, predisposing to metabolic pathologies and reproductive disturbances. Cellular lipid accumulation and mitochondrial dysfunction play an important role in the pathologic consequences of obesity, which may be mitigated by dietary interventions targeting these processes. We hypothesized that obesity in the mare promotes follicular lipid accumulation and altered mitochondrial function of oocytes and granulosa cells, potentially contributing to impaired fertility in this population. We also predicted that these effects could be mitigated by dietary supplementation with a combination of targeted nutrients to improve follicular cell metabolism. Twenty mares were grouped as: Normal Weight [NW, n = 6, body condition score (BCS) 5.7 ± 0.3], Obese (OB, n = 7, BCS 7.7 ± 0.2), and Obese Diet Supplemented (OBD, n = 7, BCS 7.7 ± 0.2), and fed specific feed regimens for ≥ 6 weeks before sampling. Granulosa cells, follicular fluid, and cumulus-oocyte complexes were collected from follicles ≥ 35 mm during estrus and after induction of maturation. Obesity promoted several mitochondrial metabolic disturbances in granulosa cells, reduced L-carnitine availability in the follicle, promoted lipid accumulation in cumulus cells and oocytes, and increased basal oocyte metabolism. Diet supplementation of a complex nutrient mixture mitigated most of the metabolic changes in the follicles of obese mares, resulting in parameters similar to NW mares. In conclusion, obesity disturbs the equine ovarian follicle by promoting lipid accumulation and altering mitochondrial function. These effects may be partially mitigated with targeted nutritional intervention, thereby potentially improving fertility outcomes in the obese female.

Fire Impacts on the Soil Metabolome and Organic Matter Biodegradability.

Global wildfire activity has increased since the 1970s and is projected to intensify throughout the 21st century. Wildfires change the composition and biodegradability of soil organic matter (SOM) which contains nutrients that fuel microbial metabolism. Though persistent forms of SOM often increase postfire, the response of more biodegradable SOM remains unclear. Here we simulated severe wildfires through a controlled "pyrocosm" approach to identify biodegradable sources of SOM and characterize the soil metabolome immediately postfire. Using microbial amplicon (16S/ITS) sequencing and gas chromatography-mass spectrometry, heterotrophic microbes (Actinobacteria, Firmicutes, and Protobacteria) and specific metabolites (glycine, protocatechuate, citric cycle intermediates) were enriched in burned soils, indicating that burned soils contain a variety of substrates that support microbial metabolism. Molecular formulas assigned by 21 T Fourier transform ion cyclotron resonance mass spectrometry showed that SOM in burned soil was lower in molecular weight and featured 20 to 43% more nitrogen-containing molecular formulas than unburned soil. We also measured higher water extractable organic carbon concentrations and higher CO2 efflux in burned soils. The observed enrichment of biodegradable SOM and microbial heterotrophs demonstrates the resilience of these soils to severe burning, providing important implications for postfire soil microbial and plant recolonization and ecosystem recovery.

Urine and Dried Blood Spots From Children and Pregnant Women Reveal Phytochemicals, Amino Acids, and Carnitine Metabolites as Cowpea Consumption Biomarkers.

SCOPE: Legumes consumption has been proven to promote health across the lifespan; cowpeas have demonstrated efficacy in combating childhood malnutrition and growth faltering, with an estimated malnutrition prevalence of 35.6% of children in Ghana. This cowpea feeding study aimed to identify a suite of metabolic consumption biomarkers in children and adults. METHODS AND RESULTS: Urine and dried blood spots (DBS) from 24 children (9-21 months) and 21 pregnant women (>18 years) in Northern Ghana are collected before and after dose-escalated consumption of four cowpea varieties for 15 days. Untargeted metabolomics identified significant increases in amino acids, phytochemicals, and lipids. The carnitine metabolism pathway is represented by 137 urine and 43 DBS metabolites, with significant changes to tiglylcarnitine and acetylcarnitine. Additional noteworthy candidate biomarkers are mansouramycin C, N-acetylalliin, proline betaine, N2, N5-diacetylornithine, S-methylcysteine, S-methylcysteine sulfoxide, and cis-urocanate. S-methylcysteine and S-methylcysteine sulfoxide are targeted and quantified in urine. CONCLUSION: This feeding study for cowpea biomarkers supports the utility of a suite of key metabolites classified as amino acids, lipids, and phytochemicals for dietary legume and cowpea-specific food exposures of global health importance.

Current Practices in LC-MS Untargeted Metabolomics: A Scoping Review on the Use of Pooled Quality Control Samples.

Untargeted metabolomics is an analytical approach with numerous applications serving as an effective metabolic phenotyping platform to characterize small molecules within a biological system. Data quality can be challenging to evaluate and demonstrate in metabolomics experiments. This has driven the use of pooled quality control (QC) samples for monitoring and, if necessary, correcting for analytical variance introduced during sample preparation and data acquisition stages. Described herein is a scoping literature review detailing the use of pooled QC samples in published untargeted liquid chromatography-mass spectrometry (LC-MS) based metabolomics studies. A literature query was performed, the list of papers was filtered, and suitable articles were randomly sampled. In total, 109 papers were each reviewed by at least five reviewers, answering predefined questions surrounding the use of pooled quality control samples. The results of the review indicate that use of pooled QC samples has been relatively widely adopted by the metabolomics community and that it is used at a similar frequency across biological taxa and sample types in both small- and large-scale studies. However, while many studies generated and analyzed pooled QC samples, relatively few reported the use of pooled QC samples to improve data quality. This demonstrates a clear opportunity for the field to more frequently utilize pooled QC samples for quality reporting, feature filtering, analytical drift correction, and metabolite annotation. Additionally, our survey approach enabled us to assess the ambiguity in the reporting of the methods used to describe the generation and use of pooled QC samples. This analysis indicates that many details of the QC framework are missing or unclear, limiting the reader's ability to determine which QC steps have been taken. Collectively, these results capture the current state of pooled QC sample usage and highlight existing strengths and deficiencies as they are applied in untargeted LC-MS metabolomics.

Secreted metabolome of porcine blastocysts encapsulated within in vitro 3D alginate hydrogel culture systems undergoing morphological changes provides insights into specific mechanisms involved in the initiation of porcine conceptus elongation.

CONTEXT: The exact mechanisms regulating the initiation of porcine conceptus elongation are not known due to the complexity of the uterine environment. AIMS: To identify contributing factors for initiation of conceptus elongation in vitro , this study evaluated differential metabolite abundance within media following culture of blastocysts within unmodified alginate (ALG) or Arg-Gly-Asp (RGD)-modified alginate hydrogel culture systems. METHODS: Blastocysts were harvested from pregnant gilts, encapsulated within ALG or RGD or as non-encapsulated control blastocysts (CONT), and cultured. At the termination of 96h culture, media were separated into blastocyst media groups: non-encapsulated control blastocysts (CONT); ALG and RGD blastocysts with no morphological change (ALG- and RGD-); ALG and RGD blastocysts with morphological changes (ALG+ and RGD+) and evaluated for non-targeted metabolomic profiling by liquid chromatography (LC)-mass spectrometry (MS) techniques and gas chromatography-(GC-MS). KEY RESULTS: Analysis of variance identified 280 (LC-MS) and 1 (GC-MS) compounds that differed (P <0.05), of which 134 (LC-MS) and 1 (GC-MS) were annotated. Metabolites abundance between ALG+ vs ALG-, RGD+ vs RGD-, and RGD+ vs ALG+ were further investigated to identify potential differences in metabolic processes during the initiation of elongation. CONCLUSIONS: This study identified changes in phospholipid, glycosphingolipid, lipid signalling, and amino acid metabolic processes as potential RGD-independent mechanisms of elongation and identified changes in lysophosphatidylcholine and sphingolipid secretions during RGD-mediated elongation. IMPLICATIONS: These results illustrate changes in phospholipid and sphingolipid metabolic processes and secretions may act as mediators of the RGD-integrin adhesion that promotes porcine conceptus elongation.

Metabolomic Profiles of Multidrug-Resistant Salmonella Typhimurium from Humans, Bovine, and Porcine Hosts.

Antimicrobial resistance (AMR) is a global public health threat, yet tools for detecting resistance patterns are limited and require advanced molecular methods. Metabolomic approaches produce metabolite profiles and help provide scientific evidence of differences in metabolite expressions between Salmonella Typhimurium from various hosts. This research aimed to evaluate the metabolomic profiles of S. Typhimurium associated with AMR and it compares profiles across various hosts. Three samples, each from bovine, porcine, and humans (total n = 9), were selectively chosen from an existing library to compare these nine isolates cultured under no drug exposure to the same isolates cultured in the presence of the antimicrobial drug panel ACSSuT (ampicillin, chloramphenicol, streptomycin, sulfisoxazole, tetracycline). This was followed by metabolomic profiling using UPLC and GC-mass spectrometry. The results indicated that the metabolite regulation was affected by antibiotic exposure, irrespective of the host species. When exposed to antibiotics, 59.69% and 40.31% of metabolites had increased and decreased expressions, respectively. The most significantly regulated metabolic pathway was aminoacyl-tRNA biosynthesis, which demonstrated increased expressions of serine, aspartate, alanine, and citric acid. Metabolites that showed decreased expressions included glutamate and pyruvate. This pathway and associated metabolites have known AMR associations and could be targeted for new drug discoveries and diagnostic methods.

Non-targeted metabolomics of cooked cowpea (Vigna unguiculata) and pigeon pea (Cajanus cajan) from Ghana using two distinct and complementary analytical platforms.

Legumes are global staple foods with multiple human health properties that merit detailed composition analysis in cooked forms. This study analyzed cowpea [Vigna unguiculata] (three varieties: Dagbantuya, Sangyi, and Tukara), pigeon pea [Cajanus cajan], and common bean [Phaseolus vulgaris] using two distinct ultra-performance liquid chromatography mass spectrometry (UPLC-MS) platforms and analytical workflows. Comparisons between cowpea and pigeon pea consumed in Ghana, and common bean (navy bean) from USA, revealed 75 metabolites that differentiated cowpeas. Metabolite fold-change comparisons resulted in 142 metabolites with significantly higher abundance in cowpea, and 154 higher in abundance from pigeon pea. 3-(all-trans-nonaprenyl)benzene-1,2-diol, N-tetracosanoylphytosphingosine, and sitoindoside II are novel identifications in cowpea, with notably higher abundance than other legumes tested. Cowpea variety specific markers were tonkinelin (Dagbantuya), pheophytin A (Sangyi), and linoleoyl ethanolamide (Tukara). This study identified novel cowpea and pigeon pea food metabolites that warrant continued investigation as bioactive food components following consumption in people.

Quantitative analysis of short-chain fatty acids in human plasma and serum by GC-MS.

Short-chain fatty acids (SCFAs) are volatile fatty acids produced by gut microbial fermentation of dietary nondigestible carbohydrates. Acetate, propionate, and butyrate SCFA measures are important to clinical and nutritional studies for their established roles in promoting healthy immune and gut function. Additionally, circulating SCFAs may influence the metabolism and allied function of additional tissues and organs. The accurate quantification of SCFAs in plasma/serum is critical to understanding the biological role of SCFAs. The low concentrations of circulating SCFAs and their volatile nature present challenges for quantitative analysis. Herein, we report a sensitive method for SCFA quantification via extraction with methyl tert-butyl ether after plasma/serum acidification. The organic extract of SCFAs is injected directly with separation and detection using a polar GC column coupled to mass spectrometry. The solvent-to-sample ratio, plasma volume, and amount of HCl needed for SCFA protonation were optimized. Method validation shows good within-day and inter-day repeatability. The limit of detection was 0.3-0.6 µg/mL for acetate and 0.03-0.12 µg/mL for propionate and butyrate. Successful application of this method on clinical plasma and serum samples was demonstrated in six datasets. By simplifying the sample preparation procedure, the present method reduces the risk of contamination, lowers the cost of analysis, increases throughput, and offers the potential for automated sample preparation.

Oocyte metabolic function, lipid composition, and developmental potential are altered by diet in older mares.

Dietary supplementation is the most feasible method to improve oocyte function and developmental potential in vivo. During three experiments, oocytes were collected from maturing, dominant follicles of older mares to determine whether short-term dietary supplements can alter oocyte metabolic function, lipid composition, and developmental potential. Over approximately 8 weeks, control mares were fed hay (CON) or hay and grain products (COB). Treated mares received supplements designed for equine wellness and gastrointestinal health, flaxseed oil, and a proprietary blend of fatty acid and antioxidant support (reproductive support supplement (RSS)) intended to increase antioxidant activity and lipid oxidation. RSS was modified for individual experiments with additional antioxidants or altered concentrations of n-3 to n-6 fatty acids. Oocytes from mares supplemented with RSS when compared to COB had higher basal oxygen consumption, indicative of higher aerobic metabolism, and proportionately more aerobic to anaerobic metabolism. In the second experiment, oocytes collected from the same mares prior to (CON) and after approximately 8 weeks of RSS supplementation had significantly reduced oocyte lipid abundance. In the final experiment, COB was compared to RSS supplementation, including RSS modified to proportionately reduce n-3 fatty acids and increase n-6 fatty acids. The ability of sperm-injected oocytes to develop into blastocysts was higher for RSS, regardless of fatty acid content, than for COB. We demonstrated that short-term diet supplementation can directly affect oocyte function in older mares, resulting in oocytes with increased metabolic activity, reduced lipid content, and increased developmental potential.

Selection for seed size has uneven effects on specialized metabolite abundance in oat (Avena sativa L.).

Plant breeding strategies to optimize metabolite profiles are necessary to develop health-promoting food crops. In oats (Avena sativa L.), seed metabolites are of interest for their antioxidant properties, yet have not been a direct target of selection in breeding. In a diverse oat germplasm panel spanning a century of breeding, we investigated the degree of variation of these specialized metabolites and how it has been molded by selection for other traits, like yield components. We also ask if these patterns of variation persist in modern breeding pools. Integrating genomic, transcriptomic, metabolomic, and phenotypic analyses for three types of seed specialized metabolites-avenanthramides, avenacins, and avenacosides-we found reduced heritable genetic variation in modern germplasm compared with diverse germplasm, in part due to increased seed size associated with more intensive breeding. Specifically, we found that abundance of avenanthramides increases with seed size, but additional variation is attributable to expression of biosynthetic enzymes. In contrast, avenacoside abundance decreases with seed size and plant breeding intensity. In addition, these different specialized metabolites do not share large-effect loci. Overall, we show that increased seed size associated with intensive plant breeding has uneven effects on the oat seed metabolome, but variation also exists independently of seed size to use in plant breeding. This work broadly contributes to our understanding of how plant breeding has influenced plant traits and tradeoffs between traits (like growth and defense) and the genetic bases of these shifts.

The Detection of Vancomycin in Sweat: A Next-Generation Digital Surrogate Marker for Antibiotic Tissue Penetration: A Pilot Study.

BACKGROUND: Assuring adequate antibiotic tissue concentrations at the point of infection, especially in skin and soft tissue infections, is pivotal for an effective treatment and cure. Despite the global issue, a reliable AB monitoring test is missing. Inadequate antibiotic treatment leads to the development of antimicrobial resistances and toxic side effects. ß-lactam antibiotics were already detected in sweat of patients treated with the respective antibiotics intravenously before. With the emergence of smartphone-based biosensors to analyse sweat on the spot of need, next-generation molecular digital biomarkers will be increasingly available for a non-invasive pharmacotherapy monitoring. OBJECTIVE: Here, we investigated if the glycopeptide antibiotic vancomycin is detectable in sweat samples of in-patients treated with intravenous vancomycin. METHODS: Eccrine sweat samples were collected using the Macroduct Sweat Collector®. Along every sweat sample, a blood sample was taken. Bio-fluid analysis was performed by Ultra-high Pressure Liquid Chromatograph-Tandem Quadrupole Mass Spectrometry coupled with tandem mass spectrometry. RESULTS: A total of 5 patients were included. Results demonstrate that vancomycin was detected in 5 out of 5 sweat samples. Specifically, vancomycin concentrations ranged from 0.011 to 0.118 mg/L in sweat and from 4.7 to 8.5 mg/L in blood. CONCLUSION: Our results serve as proof-of-concept that vancomycin is detectable in eccrine sweat and may serve as a surrogate marker for antibiotic tissue penetration. A targeted vancomycin treatment is crucial in patients with repetitive need for antibiotics and a variable antibiotic distribution such as in peripheral artery disease to optimize treatment effectiveness. If combined with on-skin smartphone-based biosensors and smartphone applications, the detection of antibiotic concentrations in sweat might enable a first digital, on-spot, lab-independent and non-invasive therapeutic drug monitoring in skin and soft tissue infections.

Application of Predicted Collisional Cross Section to Metabolome Databases to Probabilistically Describe the Current and Future Ion Mobility Mass Spectrometry.

Metabolomics is a powerful phenotyping platform with potential for high-throughput analyses. The primary technology for metabolite profiling is mass spectrometry. In recent years, the coupling of mass spectrometry with ion mobility spectrometry (IMS) has offered the promise of faster analysis time and greater resolving power. Our understanding of the potential impact of IMS on the field of metabolomics is limited by availability of comprehensive experimental data. In this analysis, we use a probabilistic approach to enumerate the strengths and limitations, the present and future, of this technology. This is accomplished through use of "model" metabolomes, predicted physicochemical properties, and probabilistic descriptions of resolving power. This analysis advances our understanding of the importance of orthogonality in resolving (separation) dimensions, describes the impact of the metabolome composition on resolution demands, and offers a system resolution landscape that may serve to guide practitioners in the coming years.

Equine maternal aging affects oocyte lipid content, metabolic function and developmental potential.

Advanced maternal age is associated with a decline in fertility and oocyte quality. We used novel metabolic microsensors to assess effects of mare age on single oocyte and embryo metabolic function, which has not yet been similarly investigated in mammalian species. We hypothesized that equine maternal aging affects the metabolic function of oocytes and in vitro-produced early embryos, oocyte mitochondrial DNA (mtDNA) copy number, and relative abundance of metabolites involved in energy metabolism in oocytes and cumulus cells. Samples were collected from preovulatory follicles from young (≤14 years) and old (≥20 years) mares. Relative abundance of metabolites in metaphase II oocytes (MII) and their respective cumulus cells, detected by liquid and gas chromatography coupled to mass spectrometry, revealed that free fatty acids were less abundant in oocytes and more abundant in cumulus cells from old vs young mares. Quantification of aerobic and anaerobic metabolism, respectively measured as oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in a microchamber containing oxygen and pH microsensors, demonstrated reduced metabolic function and capacity in oocytes and day-2 embryos originating from oocytes of old when compared to young mares. In mature oocytes, mtDNA was quantified by real-time PCR and was not different between the age groups and not indicative of mitochondrial function. Significantly more sperm-injected oocytes from young than old mares resulted in blastocysts. Our results demonstrate a decline in oocyte and embryo metabolic activity that potentially contributes to the impaired developmental competence and fertility in aged females.

Multicohort Metabolomics Analysis Discloses 9-Decenoylcarnitine to Be Associated With Incident Atrial Fibrillation.

Background The molecular mechanisms involved in atrial fibrillation are not well known. We used plasma metabolomics to investigate if we could identify novel biomarkers and pathophysiological pathways of incident atrial fibrillation. Methods and Results We identified 200 endogenous metabolites in plasma/serum by nontargeted ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry in 3 independent population-based samples (TwinGene, n=1935, mean age 68, 43% females; PIVUS [Prospective Investigation of the Vasculature in Uppsala Seniors], n=897, mean age 70, 51% females; and ULSAM [Uppsala Longitudinal Study of Adult Men], n=1118, mean age 71, all males), with available data on incident atrial fibrillation during 10 to 12 years of follow-up. A meta-analysis of ULSAM and PIVUS was used as a discovery sample and TwinGene was used for validation. In PIVUS, we also investigated associations between metabolites of interest and echocardiographic indices of myocardial geometry and function. Genome-wide association studies were performed in all 3 cohorts for metabolites of interest. In the meta-analysis of PIVUS and ULSAM with 430 incident cases, 4 metabolites were associated with incident atrial fibrillation at a false discovery rate <5%. Of those, only 9-decenoylcarnitine was associated with incident atrial fibrillation and replicated in the TwinGene sample (288 cases) following adjustment for traditional risk factors (hazard ratio, 1.24 per unit; 95% CI, 1.06-1.45, P=0.0061). A meta-analysis of all 3 cohorts disclosed another 4 significant metabolites. In PIVUS, 9-decenoylcarnitine was related to left atrium size and left ventricular mass. A Mendelian randomization analysis did not suggest a causal role of 9-decenoylcarnitine in atrial fibrillation. Conclusions A nontargeted metabolomics analysis disclosed 1 novel replicated biomarker for atrial fibrillation, 9-Decenoylcarnitine, but this acetylcarnitine is likely not causally related to atrial fibrillation.

Studying Charge Migration Fragmentation of Sodiated Precursor Ions in Collision-Induced Dissociation at the Library Scale.

Interpretation of fragmentation mass spectra depends on our knowledge of collision-induced dissociation mechanisms. Computational methods for the annotation of fragmentation mechanisms operate within the boundaries of recognized fragmentation pathways. The prevalence of charge migration fragmentation (CMF) in sodiated ion fragmentation spectra, which produces nonsodiated fragment ions, is unknown. Here, we investigated the extent of CMF in the fragmentation spectra of sodiated precursors by mining the NIST17 spectral library using a diagnostic mass difference. Our results showed that a substantial amount of fragment ions in sodiated precursor spectra are derived from CMF, indicating that this fragmentation mechanism should be commonly considered by computational methods for compound annotation.

Non-targeted urine metabolomics and associations with prevalent and incident type 2 diabetes.

Better risk prediction and new molecular targets are key priorities in type 2 diabetes (T2D) research. Little is known about the role of the urine metabolome in predicting the risk of T2D. We aimed to use non-targeted urine metabolomics to discover biomarkers and improve risk prediction for T2D. Urine samples from two community cohorts of 1,424 adults were analyzed by ultra-performance liquid chromatography/mass spectrometry (UPLC-MS). In a discovery/replication design, three out of 62 annotated metabolites were associated with prevalent T2D, notably lower urine levels of 3-hydroxyundecanoyl-carnitine. In participants without diabetes at baseline, LASSO regression in the training set selected six metabolites that improved prediction of T2D beyond established risk factors risk over up to 12 years' follow-up in the test sample, from C-statistic 0.866 to 0.892. Our results in one of the largest non-targeted urinary metabolomics study to date demonstrate the role of the urine metabolome in identifying at-risk persons for T2D and suggest urine 3-hydroxyundecanoyl-carnitine as a biomarker candidate.

Identification of Plasma Glycosphingolipids as Potential Biomarkers for Prostate Cancer (PCa) Status.

Prostate cancer (PCa) is the most common male cancer and the second leading cause of cancer death in United States men. Controversy continues over the effectiveness of prostate-specific antigen (PSA) for distinguishing aggressive from indolent PCa. There is a critical need for more specific and sensitive biomarkers to detect and distinguish low- versus high-risk PCa cases. Discovery metabolomics were performed utilizing ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) on plasma samples from 159 men with treatment naïve prostate cancer participating in the North Carolina-Louisiana PCa Project to determine if there were metabolites associated with aggressive PCa. Thirty-five identifiable plasma small molecules were associated with PCa aggressiveness, 15 of which were sphingolipids; nine common molecules were present in both African-American and European-American men. The molecules most associated with PCa aggressiveness were glycosphingolipids; levels of trihexosylceramide and tetrahexosylceramide were most closely associated with high-aggressive PCa. The Cancer Genome Atlas was queried to determine gene alterations within glycosphingolipid metabolism that are associated with PCa and other cancers. Genes that encode enzymes associated with the metabolism of glycosphingolipids were altered in 12% of PCa and >30% of lung, uterine, and ovarian cancers. These data suggest that the identified plasma (glyco)sphingolipids should be further validated for their association with aggressive PCa, suggesting that specific sphingolipids may be included in a diagnostic signature for PCa.

Non-invasive Drug Monitoring of ß-Lactam Antibiotics Using Sweat Analysis-A Pilot Study.

Background: Antimicrobial resistance is a major challenge in treating infectious diseases. Therapeutic drug monitoring (TDM) can optimize and personalize antibiotic treatment. Previously, antibiotic concentrations in tissues were extrapolated from skin blister studies, but sweat analyses for TDM have not been conducted. Objective: To investigate the potential of sweat analysis as a non-invasive, rapid, and potential bedside TDM method. Methods: We analyzed sweat and blood samples from 13 in-house patients treated with intravenous cefepime, imipenem, or flucloxacillin. For cefepime treatment, full pharmacokinetic sampling was performed (five subsequent sweat samples every 2 h) using ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry. The ClinicalTrials.gov registration number is NCT03678142. Results: In this study, we demonstrated for the first time that flucloxacillin, imipenem, and cefepime are detectable in sweat. Antibiotic concentration changes over time demonstrated comparable (age-adjusted) dynamics in the blood and sweat of patients treated with cefepime. Patients treated with standard flucloxacillin dosage showed the highest mean antibiotic concentration in sweat. Conclusions: Our results provide a proof-of-concept that sweat analysis could potentially serve as a non-invasive, rapid, and reliable method to measure antibiotic concentration and as a surrogate marker for tissue penetration. If combined with smart biosensors, sweat analysis may potentially serve as the first lab-independent, non-invasive antibiotic TDM method.

A novel culture medium with reduced nutrient concentrations supports the development and viability of mouse embryos.

Further refinement of culture media is needed to improve the quality of embryos generated in vitro. Previous results from our laboratory demonstrated that uptake of nutrients by the embryo is significantly less than what is supplied in traditional culture media. Our objective was to determine the impact of reduced nutrient concentrations in culture medium on mouse embryo development, metabolism, and quality as a possible platform for next generation medium formulation. Concentrations of carbohydrates, amino acids, and vitamins could be reduced by 50% with no detrimental effects, but blastocyst development was impaired at 25% of standard nutrient provision (reduced nutrient medium; RN). Addition of pyruvate and L-lactate (+PL) to RN at 50% of standard concentrations restored blastocyst development, hatching, and cell number. In addition, blastocysts produced in RN + PL contained more ICM cells and ATP than blastocysts cultured in our control (100% nutrient) medium; however, metabolic activity was altered. Similarly, embryos produced in the RN medium with elevated (50% control) concentrations of pyruvate and lactate in the first step medium and EAA and Glu in the second step medium were competent to implant and develop into fetuses at a similar rate as embryos produced in the control medium. This novel approach to culture medium formulation could help define the optimal nutrient requirements of embryos in culture and provide a means of shifting metabolic activity towards the utilization of specific metabolic pathways that may be beneficial for embryo viability.