Unravelling the metabolic landscape of cutaneous melanoma: Insights from single-cell sequencing analysis and machine learning for prognostic assessment of lactate metabolism.

This manuscript presents a comprehensive investigation into the role of lactate metabolism-related genes as potential prognostic markers in skin cutaneous melanoma (SKCM). Bulk-transcriptome data from The Cancer Genome Atlas (TCGA) and GSE19234, GSE22153, and GSE65904 cohorts from GEO database were processed and harmonized to mitigate batch effects. Lactate metabolism scores were assigned to individual cells using the 'AUCell' package. Weighted Co-expression Network Analysis (WGCNA) was employed to identify gene modules correlated with lactate metabolism. Machine learning algorithms were applied to construct a prognostic model, and its performance was evaluated in multiple cohorts. Immune correlation, mutation analysis, and enrichment analysis were conducted to further characterize the prognostic model's biological implications. Finally, the function of key gene NDUFS7 was verified by cell experiments. Machine learning resulted in an optimal prognostic model, demonstrating significant prognostic value across various cohorts. In the different cohorts, the high-risk group showed a poor prognosis. Immune analysis indicated differences in immune cell infiltration and checkpoint gene expression between risk groups. Mutation analysis identified genes with high mutation loads in SKCM. Enrichment analysis unveiled enriched pathways and biological processes in high-risk SKCM patients. NDUFS7 was found to be a hub gene in the protein-protein interaction network. After the expression of NDUFS7 was reduced by siRNA knockdown, CCK-8, colony formation, transwell and wound healing tests showed that the activity, proliferation and migration of A375 and WM115 cell lines were significantly decreased. This study offers insights into the prognostic significance of lactate metabolism-related genes in SKCM.

In Vivo Assessment of Astrocyte Reactivity in Patients with Progressive Supranuclear Palsy.

OBJECTIVE: Although astrocytic pathology is a pathological hallmark of progressive supranuclear palsy (PSP), its pathophysiological role remains unclear. This study aimed to assess astrocyte reactivity in vivo in patients with PSP. Furthermore, we investigated alterations in brain lactate levels and their relationship with astrocyte reactivity. METHODS: We included 30 patients with PSP-Richardson syndrome and 30 healthy controls; in patients, tau deposition was confirmed through 18F-florzolotau positron emission tomography. Myo-inositol, an astroglial marker, and lactate were quantified in the anterior cingulate cortex through magnetic resonance spectroscopy. We measured plasma biomarkers, including glial fibrillary acidic protein as another astrocytic marker. The anterior cingulate cortex was histologically assessed in postmortem samples of another 3 patients with PSP with comparable disease durations. RESULTS: The levels of myo-inositol and plasma glial fibrillary acidic protein were significantly higher in patients than those in healthy controls (p < 0.05); these increases were significantly associated with PSP rating scale and cognitive function scores (p < 0.05). The lactate level was high in patients, and correlated significantly with high myo-inositol levels. Histological analysis of the anterior cingulate cortex in patients revealed reactive astrocytes, despite mild tau deposition, and no marked synaptic loss. INTERPRETATION: We discovered high levels of astrocyte biomarkers in patients with PSP, suggesting astrocyte reactivity. The association between myo-inositol and lactate levels suggests a link between reactive astrocytes and brain energy metabolism changes. Our results indicate that astrocyte reactivity in the anterior cingulate cortex precedes pronounced tau pathology and neurodegenerative processes in that region, and affects brain function in PSP. ANN NEUROL 2024;96:247-261.

Modelling and assessment of glucose-lactate kinetics in youth with overweight, obesity and metabolic dysfunction-associated steatotic liver disease: A pilot study.

AIM: In this study, we investigated glucose and lactate kinetics during a 75 g oral glucose tolerance test (OGTT) in 23 overweight and obese adolescents and assessed putative differences among participants with and without metabolic dysfunction-associated steatotic liver disease (MASLD). METHODS: We enrolled 23 young people (six girls) with obesity [body mass index 33 (29-37)]. Glucose-lactate kinetics parameters (disposal glucose insulin sensitivity, SID; fraction of glucose converted into lactate, fr; fractional lactate clearance rate, kL) and lactate production rate (LPR) were estimated using the oral glucose-lactate minimal model. MASLD presence was assessed using the proton density fat fraction. We analysed glucose, lactate and LPR time to peak, peak values and area under the curve and evaluated differences using the Wilcoxon test. MASLD and no-MASLD participants were compared using the Mann-Whitney test. Correlations between parameters were assessed using the Spearman correlation coefficient (ρ). We also tested the performance of two (4 or 3 h OGTT) protocols in estimating oral glucose-lactate minimal model and LPR parameters. RESULTS: Glucose peaks 30 min earlier than lactate (p = .0019). This pattern was present in the no-MASLD group (p < .001). LPR peaks 30 min later in the MASLD group (p = .02). LPR and kL were higher in MASLD, suggesting higher glycolysis and lactate utilization. SID and fr correlate significantly (ρ = -0.55, p = .008). SID and fr were also correlated with the body mass index, (ρ = -0.45, p = .04; and ρ = 0.45; p = .03). The protocol duration did not influence the estimates of the parameters. DISCUSSION: Youth with MASLD showed a delayed glucose metabolism, possibly because of greater utilization of the underlying substrates. A 3-h OGTT may be used to assess lactate metabolism effectively.

Lactic acid fermented microalgae and cyanobacteria as a new source of lipid reducing compounds: assessment through zebrafish Nile red fat metabolism assay and untargeted metabolomics.

Obesity is one of the most important threats to human health. Besides existing pharmacological or clinical interventions, novel effective and largely available solutions are still necessary. Among diverse natural resources, microalgae are well known for their complexity in the production of novel secondary metabolites. At the same time, lactic acid bacteria (LAB) are known for their capacity to metabolize, through fermentation, different matrices, and consequently to modify or produce new compounds with potential bioactivity. This work aimed to study the production of fermented microalgae and cyanobacteria, and to analyse their extracts in the zebrafish Nile red fat metabolism assay. Three microalgal species (Chlorella vulgaris, Chlorococcum sp. and Arthrospira platensis) were fermented with seven strains of LAB from 4 species (Lacticaseibacillus rhamnosus, Lacticaseibacillus casei, Lactobacillus delbrueckii bulgaricus and Lacticaseibacillus paracasei), derived from the UPCCO - University of Parma Culture Collection, Parma, Italy). All the selected strains were able to ferment the selected species of microalgae, and the most suitable substrate for LAB growth was Arthrospira platensis. Extracts from fermented Chlorella vulgaris and Chlorococcum sp. reduced significantly the neutral lipid reservoirs, which was not observed without fermentations. The strongest lipid reducing effect was obtained with Arthrospira platensis fermented with Lactobacillus delbrueckii bulgaricus 1932. Untargeted metabolomics identified some compound families, which could be related to the observed bioactivity, namely fatty acids, fatty amides, triterpene saponins, chlorophyll derivatives and purine nucleotides. This work opens up the possibility of developing novel functional foods or food supplements based on microalgae, since lactic acid fermentation enhanced the production of bioactive compounds with lipid reducing activities.

Brain energy metabolism is optimized to minimize the cost of enzyme synthesis and transport.

The energy metabolism of the brain is poorly understood partly due to the complex morphology of neurons and fluctuations in ATP demand over time. To investigate this, we used metabolic models that estimate enzyme usage per pathway, enzyme utilization over time, and enzyme transportation to evaluate how these parameters and processes affect ATP costs for enzyme synthesis and transportation. Our models show that the total enzyme maintenance energy expenditure of the human body depends on how glycolysis and mitochondrial respiration are distributed both across and within cell types in the brain. We suggest that brain metabolism is optimized to minimize the ATP maintenance cost by distributing the different ATP generation pathways in an advantageous way across cell types and potentially also across synapses within the same cell. Our models support this hypothesis by predicting export of lactate from both neurons and astrocytes during peak ATP demand, reproducing results from experimental measurements reported in the literature. Furthermore, our models provide potential explanation for parts of the astrocyte-neuron lactate shuttle theory, which is recapitulated under some conditions in the brain, while contradicting other aspects of the theory. We conclude that enzyme usage per pathway, enzyme utilization over time, and enzyme transportation are important factors for defining the optimal distribution of ATP production pathways, opening a broad avenue to explore in brain metabolism.

Assessment of Genomic and Metabolic Characteristics of Cholesterol-Reducing and GABA Producer Limosilactobacillus fermentum AGA52 Isolated from Lactic Acid Fermented Shalgam Based on "In Silico" and "In Vitro" Approaches.

This study aimed to characterize the genomic and metabolic properties of a novel Lb. fermentum strain AGA52 which was isolated from a lactic acid fermented beverage called "shalgam." The genome size of AGA52 was 2,001,184 bp, which is predicted to carry 2024 genes, including 50 tRNAs, 3 rRNAs, 3 ncRNAs, 15 CRISPR repeats, 14 CRISPR spacers, and 1 CRISPR array. The genome has a GC content of 51.82% including 95 predicted pseudogenes, 56 complete or partial transposases, and 2 intact prophages. The similarity of the clusters of orthologous groups (COG) was analyzed by comparison with the other Lb. fermentum strains. The detected resistome on the genome of AGA52 was found to be intrinsic originated. Besides, it has been determined that AGA52 has an obligate heterofermentative carbohydrate metabolism due to the absence of the 1-phosphofructokinase (pfK) enzyme. Furthermore, the strain is found to have a better antioxidant capacity and to be tolerant to gastrointestinal simulated conditions. It was also observed that the AGA52 has antimicrobial activity against Yersinia enterocolitica ATCC9610, Bacillus cereus ATCC33019, Salmonella enterica sv. Typhimurium, Escherichia coli O157:h7 ATCC43897, Listeria monocytogenes ATCC7644, Klebsiella pneumoniae ATCC13883, and Proteus vulgaris ATCC8427. Additionally, AGA52 exhibited 42.74 ± 4.82% adherence to HT29 cells. Cholesterol assimilation (33.9 ± 0.005%) and GABA production capacities were also confirmed by "in silico" and "in vitro." Overall, the investigation of genomic and metabolic features of the AGA52 revealed that is a potential psychobiotic and probiotic dietary supplement candidate and can bring functional benefits to the host.

Genome and fermentation analyses of Enterococcus faecalis DB-5 isolated from Japanese Mandarin orange: An assessment of potential application in lactic acid production.

Enterococcus faecalis strain DB-5 is a lactic acid bacterium newly isolated from the Japanese mandarin orange (mikan). The DB-5 strain produces organic acid from various carbohydrate sources including glycerol and starch. To gain deeper insights into its potential application in lactic acid fermentation (LAF), the genome and fermentation analyses of E. faecalis DB-5 were performed. Whole genome sequencing was carried out using the DNBSEQ platform. After trimming and assembly, the total size of the assembled genome was revealed to be 3,048,630 bp, distributed into 63 contigs with an N50 value of 203,673. The genome has 37.2% GC content, 2928 coding DNA sequences, and 54 putative RNA genes. The DB-5 strain harbored two l-lactate dehydrogenases (L-LDHs), both of which conserved the catalytic domain sequences. The optical purity measurement showed that strain DB-5 is homofermentative and produced only l-lactic acid (LA), which correlated with genome-based pathway analysis. To confirm its LA productivity at high temperatures, open repeated batch fermentation was performed at 45 °C using sucrose as a carbon source. The volumetric LA productivity of DB-5 was averaged at 3.66 g L-1 h-1 for 24 h during the 3rd to 11th fermentation cycles. E. faecalis DB-5 could efficiently convert around 94% of sucrose to LA throughout the fermentation cycles at 45 °C. These genomic characteristics and fermentation properties of E. faecalis DB-5 provide beneficial information for a deeper understanding of the functional properties of future high-temperature LAFs from biomass resources.

Assessment of focal renal ischemia-reperfusion injury in a porcine model using hyperpolarized [1-13 C]pyruvate MRI.

PURPOSE: Ischemic injury in the kidney is a common pathophysiological event associated with both acute kidney injury and chronic kidney disease; however, regional ischemia-reperfusion as seen in thromboembolic renal disease is often undetectable and thus subclinical. Here, we assessed the metabolic alterations following subclinical focal ischemia-reperfusion injury with hyperpolarized [1-13 C]pyruvate MRI in a porcine model. METHODS: Five pigs were subjected to 60 min of focal kidney ischemia. After 90 min of reperfusion, a multiparametric proton MRI protocol was performed on a clinical 3T scanner system. Metabolism was evaluated using 13 C MRI following infusion of hyperpolarized [1-13 C]pyruvate. Ratios of pyruvate to its detectable metabolites (lactate, bicarbonate, and alanine) were used to quantify metabolism. RESULTS: The focal ischemia-reperfusion injury resulted in injured areas with a mean size of 0.971 cm3 (±1.019). Compared with the contralateral kidney, the injured areas demonstrated restricted diffusion (1269 ± 83.59 × 10-6 mm2 /s vs. 1530 ± 52.73 × 10-6 mm2 /s; p = 0.006) and decreased perfusion (158.8 ± 29.4 mL/100 mL/min vs. 274 ± 63.1 mL/100 mL/min; p = 0.014). In the metabolic assessment, the injured areas displayed increased lactate/pyruvate ratios compared with the entire ipsilateral and the contralateral kidney (0.35 ± 0.13 vs. 0.27 ± 0.1 vs. 0.25 ± 0.1; p = 0.0086). Alanine/pyruvate ratio was unaltered, and we were unable to quantify bicarbonate due to low signal. CONCLUSION: MRI with hyperpolarized [1-13 C]pyruvate in a clinical setup is capable of detecting the acute, subtle, focal metabolic changes following ischemia. This may prove to be a valuable future addition to the renal MRI suite.

Assessment of Metabolic Flexibility by Substrate Oxidation Responses and Blood Lactate in Women Expressing Varying Levels of Aerobic Fitness and Body Fat.

ABSTRACT: Waldman, HS, Bryant, AR, Knight, SN, Killen, LG, Davis, BA, Robinson, MA, and O'Neal, EK. Assessment of metabolic flexibility by substrate oxidation responses and blood lactate in women expressing varying levels of aerobic fitness and body fat. J Strength Cond Res 37(3): 581-588, 2023-Collection of substrate oxidation responses during exercise is proposed as a noninvasive means for assessing metabolic flexibility in male subjects. However, because of hormonal and metabolic differences between sexes, this method may not be applicable to female subjects. This study assessed metabolic flexibility through indirect calorimetry across female subjects with different maximal oxidative capacities. Thirty-eight (18-45 years) eumenorrheic female subjects were stratified ( p < 0.05) based on V̇ o2 peak (mL·kg -1 ·min -1 ) into (1) endurance-trained (ET, n = 12, 42.6 ± 5.3), (2) recreationally active (RA, n = 13, 32.3 ± 1.6), or (3) overweight female subjects (OW, n = 13, 21.0 ± 4.0). Subjects completed the same 5-stage graded exercise test with intensities of 30, 45, 60, 75, and 90 W. Lactate [La - ], carbohydrate (CHOox), and fat (FATox) oxidation rates were assessed during the last min of each 5-minute stage. Subjects then cycled to exhaustion to determine V̇ o2 peak. Endurance-trained and RA female subjects expressed significantly ( p ≤ 0.05) higher absolute rates and rates scaled to fat-free mass of CHOox and FATox compared with OW female subjects during multiple stages. [La - ] failed to consistently differentiate the 3 groups with higher [La - ] for OW only found during stage 4; however, RER differed by 0.09 units or more at each stage for OW vs. ET. It seems that RER was more sensitive to cohort characteristics than [La - ] contrasting recent findings in male cohorts. In conclusion, indirect calorimetry is a practical and noninvasive method for assessing metabolic flexibility in eumenorrheic female subjects of varying aerobic fitness levels.

Macroalgal biomass as a potential resource for lactic acid fermentation.

Lactic acid is an essential platform chemical with various applications in the chemicals, food, pharmaceutical, and cosmetic industries. Currently, the demand for lactic acid is driven by the role of lactic acid as the starting material for the production of bioplastic polylactide. Microbial fermentation for lactic acid production is favored due to the production of enantiomerically pure lactic acid required for polylactide synthesis, as opposed to the racemic mixture obtained via chemical synthesis. The utilization of first-generation feedstock for commercial lactic acid production is challenged by feedstock costs and sustainability issues. Macroalgae are photosynthetic benthic aquatic plants that contribute tremendously towards carbon capture with subsequent carbon-rich biomass production. Macroalgae are commercially cultivated to extract hydrocolloids, and recent studies have focused on applying biomass as a fermentation feedstock. This review provides comprehensive information on the design and development of sustainable and cost-effective, algae-based lactic acid production. The central carbon regulation in lactic acid bacteria and the metabolism of seaweed-derived sugars are described. An exhaustive compilation of lactic acid fermentation of macroalgae hydrolysates revealed that lactic acid bacteria can effectively ferment the mixture of sugars present in the hydrolysate with comparable yields. The environmental impacts and economic prospects of macroalgal lactic acid are analyzed. Valorization of the vast amounts of spent macroalgal biomass residue post hydrocolloid extraction in a biorefinery is a viable strategy for cost-effective lactic acid production.

Metabolic Assessment of Human Induced Pluripotent Stem Cells-Derived Astrocytes and Fetal Primary Astrocytes: Lactate and Glucose Turnover.

Astrocytes represent one of the main cell types in the brain and play a crucial role in brain functions, including supplying the energy demand for neurons. Moreover, they are important regulators of metabolite levels. Glucose uptake and lactate production are some of the main observable metabolic actions of astrocytes. To gain insight into these processes, it is essential to establish scalable and functional sources for in vitro studies of astrocytes. In this study, we compared the metabolic turnover of glucose and lactate in astrocytes derived from human induced pluripotent stem cell (hiPSC)-derived Astrocytes (hiAstrocytes) as a scalable astrocyte source to human fetal astrocytes (HFAs). Using a user-friendly, commercial flow-based biosensor, we could verify that hiAstrocytes are as glycogenic as their fetal counterparts, but their normalized metabolic turnover is lower. Specifically, under identical culture conditions in a defined media, HFAs have 2.3 times higher levels of lactate production compared to hiAstrocytes. In terms of glucose, HFAs have 2.1 times higher consumption levels than hiAstrocytes at 24 h. Still, as we describe their glycogenic phenotype, our study demonstrates the use of hiAstrocytes and flow-based biosensors for metabolic studies of astrocyte function.

Assessment of higher-order singular value decomposition denoising methods on dynamic hyperpolarized [1-13C]pyruvate MRI data from patients with glioma.

BACKGROUND: Real-time metabolic conversion of intravenously-injected hyperpolarized [1-13C]pyruvate to [1-13C]lactate and [13C]bicarbonate in the brain can be measured using dynamic hyperpolarized carbon-13 (HP-13C) MRI. However, voxel-wise evaluation of metabolism in patients with glioma is challenged by the limited signal-to-noise ratio (SNR) of downstream 13C metabolites, especially within lesions. The purpose of this study was to evaluate the ability of higher-order singular value decomposition (HOSVD) denoising methods to enhance dynamic HP [1-13C]pyruvate MRI data acquired from patients with glioma. METHODS: Dynamic HP-13C MRI were acquired from 14 patients with glioma. The effects of two HOSVD denoising techniques, tensor rank truncation-image enhancement (TRI) and global-local HOSVD (GL-HOSVD), on the SNR and kinetic modeling were analyzed in [1-13C]lactate data with simulated noise that matched the levels of [13C]bicarbonate signals. Both methods were then evaluated in patient data based on their ability to improve [1-13C]pyruvate, [1-13C]lactate and [13C]bicarbonate SNR. The effects of denoising on voxel-wise kinetic modeling of kPL and kPB was also evaluated. The number of voxels with reliable kinetic modeling of pyruvate-to-lactate (kPL) and pyruvate-to-bicarbonate (kPB) conversion rates within regions of interest (ROIs) before and after denoising was then compared. RESULTS: Both denoising methods improved metabolite SNR and regional signal coverage. In patient data, the average increase in peak dynamic metabolite SNR was 2-fold using TRI and 4-5 folds using GL-HOSVD denoising compared to acquired data. Denoising reduced kPL modeling errors from a native average of 23% to 16% (TRI) and 15% (GL-HOSVD); and kPB error from 42% to 34% (TRI) and 37% (GL-HOSVD) (values were averaged voxelwise over all datasets). In contrast-enhancing lesions, the average number of voxels demonstrating within-tolerance kPL modeling error relative to the total voxels increased from 48% in the original data to 84% (TRI) and 90% (GL-HOSVD), while the number of voxels showing within-tolerance kPB modeling error increased from 0% to 15% (TRI) and 8% (GL-HOSVD). CONCLUSION: Post-processing denoising methods significantly improved the SNR of dynamic HP-13C imaging data, resulting in a greater number of voxels satisfying minimum SNR criteria and maximum kinetic modeling errors in tumor lesions. This enhancement can aid in the voxel-wise analysis of HP-13C data and thereby improve monitoring of metabolic changes in patients with glioma following treatment.

Assessment of organic acid salts on fermentation quality, aerobic stability, and in vitro rumen digestibility of total mixed ration silage.

The work aimed to investigate the effects of four organic acid salts on fermentation quality, aerobic stability, and in vitro rumen digestibility of total mixed ration (TMR) silage prepared with citric acid residue, wet brewers' grains, and Napier grass. The TMR was ensiled with the following: (1) no additives (control), (2) 0.1% sodium benzoate (SB), (3) 0.1% potassium sorbate (PS), (4) 0.5% sodium diacetate (SDA), (5) 0.5% calcium propionate (CAP) on a fresh weight basis. All silos (10 L) were opened after 60 days of ensiling to determine fermentation profiles and in vitro rumen digestibility, and then were subjected to a 9-day aerobic stability test. Four organic acid salts significantly (p < 0.05) increased dry matter contents, lactic acid bacteria count, and decreased ethanol content and yeast count compared with the control. The SDA and CAP significantly (p < 0.05) increased water-soluble carbohydrates, lactic acid, and crude protein contents, and decreased pH, ammonia nitrogen, neutral detergent fiber, and hemicellulose contents compared with other TMR silages after 60 days of ensiling. Organic acid salts significantly (p < 0.05) prolonged the hours of aerobic stability and significantly (p < 0.05) increased cumulative gas production and potential gas production compared with the control. The treatments of SDA and CAP significantly (p < 0.05) improved aerobic stability as indicated by higher (p < 0.05) lactic acid and water-soluble carbohydrates contents, and lower (p < 0.05) pH, ammonia nitrogen, ethanol contents, and yeast count compared with the control. The treatments of SDA and CAP significantly (p < 0.05) increased in vitro rumen parameters, as indicated by higher (p < 0.05) in vitro digestibility of dry matter, crude protein, and neutral detergent fiber after 60 days of ensiling. Overall, these results indicated that the addition of SDA and CAP could ensure the good fermentation quality and improve aerobic stability of TMR silages. By comprehensive consideration, CAP was recommended for improving fermentation quality, aerobic stability, and in vitro rumen digestibility of TMR silages prepared with wet brewers' grains, citric acid residue, and Napier grass.

L-lactate as an indicator for cellular metabolic status: An easy and cost-effective colorimetric L-lactate assay.

BACKGROUND: In recent times, the study of metabolic pathways has become inevitable and predominant for a variety of research fields as cancer biology and immunology. L-lactate as a product of anaerobic glycolysis has shown to be an important indicator of the cellular metabolic status and can be associated with diverse cellular effects. For this reason, L-lactate assay kits are of high demand when metabolic effects need to be considered. Nevertheless, commercially available kits are not affordable if multiple samples must be evaluated. PRINCIPAL FINDING: In this work, we develop an easy and cost-effective colorimetric assay for quantification of L-lactate suitable for cells with low or high L-lactate production based on LDH activity and suitable for 96 well-plate format. Using different metabolic regulators, we demonstrate the capacity of the assay to detect and quantify L-lactate from the supernatant of HeLa cancer cell line. Furthermore, we validate the assay against a commercially available kit by demonstrating no significant difference between both assays. Finally, we show that the assay is capable of quantifying L-lactate in primary cells such as hPBMCs that were stimulated with toll-like receptor ligands and treated with different metabolic regulators. CONCLUSION: We herein present an easy custom assay that is suitable for cells with low and high L-lactate production at very low cost compared to commercially available kits. These advantages of the custom assay can simplify the research in the field of metabolism and related fields.

Clinical assessment of liver metabolism during hypothermic oxygenated machine perfusion using microdialysis.

BACKGROUND: While growing evidence supports the use of hypothermic oxygenated machine perfusion (HOPE) in liver transplantation, its effects on liver metabolism are still incompletely understood. METHODS: To assess liver metabolism during HOPE using microdialysis (MD), we conducted an open-label, observational pilot study on 10 consecutive grafts treated with dual-HOPE (D-HOPE). Microdialysate and perfusate levels of glucose, lactate, pyruvate, glutamate, and flavin mononucleotide (FMN) were measured during back table preparation and D-HOPE and correlated to graft function and patient outcome. RESULTS: Median (IQR) MD and D-HOPE time was 228 (210, 245) and 116 (103, 143) min. Three grafts developed early allograft dysfunction (EAD), with one requiring retransplantation. During D-HOPE, MD glucose and lactate levels increased (ANOVA = 9.88 [p = 0.01] and 3.71 [p = 0.08]). Their 2nd-hour levels were higher in EAD group and positively correlated with L-GrAFT score. 2nd-hour MD glucose and lactate were also positively correlated with cold ischemia time, macrovesicular steatosis, weight gain during D-HOPE, and perfusate FMN. These correlations were not apparent when perfusate levels were considered. In contrast, MD FMN levels invariably dropped steeply after D-HOPE start, whereas perfusate FMN was higher in dysfunctioning grafts. CONCLUSION: MD glucose and lactate during D-HOPE are markers of hepatocellular injury and could represent additional elements of the viability assessment.

A proof of concept study on real-time LiMAx CYP1A2 liver function assessment of donor grafts during normothermic machine perfusion.

No single reliable parameter exists to assess liver graft function of extended criteria donors during ex-vivo normothermic machine perfusion (NMP). The liver maximum capacity (LiMAx) test is a clinically validated cytochromal breath test, measuring liver function based on 13CO2 production. As an innovative concept, we aimed to integrate the LiMAx breath test with NMP to assess organ function. Eleven human livers were perfused using NMP. After one hour of stabilization, LiMAx testing was performed. Injury markers (ALT, AST, miR-122, FMN, and Suzuki-score) and lactate clearance were measured and related to LiMAx values. LiMAx values ranged between 111 and 1838 µg/kg/h, and performing consecutive LiMAx tests during longer NMP was feasible. No correlation was found between LiMAx value and miR-122 and FMN levels in the perfusate. However, a significant inverse correlation was found between LiMAx value and histological injury (Suzuki-score, R = - 0.874, P < 0.001), AST (R = - 0.812, P = 0.004) and ALT (R = - 0.687, P = 0.028). Furthermore, a significant correlation was found with lactate clearance (R = 0.683, P = 0.043). We demonstrate, as proof of principle, that liver function during NMP can be quantified using the LiMAx test, illustrating a positive correlation with traditional injury markers. This new breath-test application separates livers with adequate cytochromal liver function from inadequate ones and may support decision-making in the safe utilization of extended criteria donor grafts.

Cell Energy Budget Platform for Multiparametric Assessment of Cell and Tissue Metabolism.

Specific bioenergetic signature reports on the current metabolic state of the cell, which may be affected by metabolic rearrangement, dysfunction or dysregulation of relevant signaling pathways, altered physiological condition or energy stress. A combined analysis of respiration , glycolytic flux, Krebs cycle activity, ATP levels, and total biomass allows informative initial assessment. Such simple, high-throughput, multiparametric methodology, called cell energy budget (CEB ) platform, is presented here and demonstrated with particular cell and tissue models. The CEB uses a commercial fluorescent lanthanide probe pH-Xtra™ to measure extracellular acidification (ECA) associated with lactate (L-ECA) and combined lactate/CO2 (T-ECA), a phosphorescent probe MitoXpress®-Xtra to measure oxygen consumption rate (OCR), a bioluminescent ATP kit, and an absorbance-based total protein assay. All the assays are performed on a standard multi-label reader. Using the same readouts, the CEB approach can be extended to more detailed mechanistic studies, by targeting specific pathways in cell bioenergetics and measuring other cellular parameters, such as NAD(P)H, Ca2+, mitochondrial pH, membrane potential, redox state, with conventional fluorescent or luminescent probes.

Assessment of Tissue Perfusion Using the Peripheral Perfusion Index and Lactate Clearance in Shock in Pediatric Patients.

BACKGROUND: Pediatric shock has a high mortality rate because many of the early clinical signs are subtle and have poor sensitivity and specificity. Pediatric shock was categorized either: compensated with normal blood pressure, poor skin perfusion (CRT >2 s, mottled, cool peripheries, peripheral cyanosis), weak peripheral pulse, age specific tachycardia, tachypnoea, and oliguria or decompensated with hypotension (SBP < 70 + (2× age in years) mm Hg and decreased mental status. The perfusion index is a non-invasive method for assessing peripheral perfusion and may be a useful marker for identifying shock early in pediatric patients. OBJECTIVE: This prospective cohort study (November 2019 to August 2020) evaluated whether the perfusion index, lactate, and/or lactate clearance could predict mortality among pediatric shock patients. METHODS: Fifty children (68% male) with shock underwent assessments at presentation to the emergency room to evaluate their heart rate, blood pressure, capillary refill time, central venous pressure, perfusion index, cardiac index, systemic vascular resistance, central venous oxygen saturation, and lactate clearance. RESULTS: The perfusion index range was 0.03 to 2.2 and ≤0.18 as the cut-off for mortality prediction providing 74% sensitivity and 78% specificity. The serum lactate concentration range was 0 to 16 mmol/L and >5.7 mmol/L as the cut-off for mortality prediction provided 70% sensitivity and 96% specificity at presentation to the emergency room. The lactate clearance range was 3% to 75% and >10% as the cut-off for survival prediction after resuscitation and at 6 h later. CONCLUSION: Perfusion index (PI), lactate, and lactate clearance provided comparable sensitivity and specificity for predicting outcomes among pediatric patients with shock Therefore, we suggest that the PI is an inexpensive, rapid, and non-invasive tool that can be used to predict illness severity and mortality in busy pediatric intensive care units and emergency departments. This tool may guide better patient triage and an earlier diagnosis of shock in this setting.

Genomic-, phenotypic-, and toxicity-based safety assessment and probiotic potency of Bacillus coagulans IDCC 1201 isolated from green malt.

Probiotics are beneficial microorganisms, and the evaluation of their safety for human use in the food industry has become critical. This study examines the safety of Bacillus coagulans IDCC 1201 isolated from green malt by analyzing its genomic and phenotypic characteristics and determining its toxicity. The presence of antibiotic resistance and toxigenic genes and gene transferability were investigated using whole-genome analysis. The strain's hemolytic and enzyme activities, minimum inhibitory concentrations of antibiotics, and biogenic amine and D-lactate production were also examined. Furthermore, the principal properties of B. coagulans IDCC 1201 as probiotics, such as resistance to abiotic stress and intestinal adhesion, were studied. The whole-genome analysis demonstrated that B. coagulans IDCC 1201 had no antibiotic resistance or toxigenic genes; the strain was susceptible to the nine antibiotics proposed by the European Food Safety Authority. Moreover, this strain lacked hemolytic and ß-glucuronidase activities. Additionally, it was confirmed that B. coagulans IDCC 1201 produced undesirable metabolites, including biogenic amines or D-lactate, at a safe level. Finally, the strain exhibited functional potential as a probiotic in terms of abiotic tolerance, such as bile tolerance and intestinal adhesion in in vitro experiments. In conclusion, B. coagulans IDCC 1201 can be considered as a safe probiotic with regard to human health.

Non-invasive assessment of telomere maintenance mechanisms in brain tumors.

Telomere maintenance is a universal hallmark of cancer. Most tumors including low-grade oligodendrogliomas use telomerase reverse transcriptase (TERT) expression for telomere maintenance while astrocytomas use the alternative lengthening of telomeres (ALT) pathway. Although TERT and ALT are hallmarks of tumor proliferation and attractive therapeutic targets, translational methods of imaging TERT and ALT are lacking. Here we show that TERT and ALT are associated with unique 1H-magnetic resonance spectroscopy (MRS)-detectable metabolic signatures in genetically-engineered and patient-derived glioma models and patient biopsies. Importantly, we have leveraged this information to mechanistically validate hyperpolarized [1-13C]-alanine flux to pyruvate as an imaging biomarker of ALT status and hyperpolarized [1-13C]-alanine flux to lactate as an imaging biomarker of TERT status in low-grade gliomas. Collectively, we have identified metabolic biomarkers of TERT and ALT status that provide a way of integrating critical oncogenic information into non-invasive imaging modalities that can improve tumor diagnosis and treatment response monitoring.