-
Journal of Biomedical Optics Jun 2024To enable non-destructive longitudinal assessment of drug agents in intact tumor tissue without the use of disruptive probes, we have designed a label-free method to...
SIGNIFICANCE
To enable non-destructive longitudinal assessment of drug agents in intact tumor tissue without the use of disruptive probes, we have designed a label-free method to quantify the health of individual tumor cells in excised tumor tissue using multiphoton fluorescence lifetime imaging microscopy (MP-FLIM).
AIM
Using murine tumor fragments which preserve the native tumor microenvironment, we seek to demonstrate signals generated by the intrinsically fluorescent metabolic co-factors nicotinamide adenine dinucleotide phosphate [NAD(P)H] and flavin adenine dinucleotide (FAD) correlate with irreversible cascades leading to cell death.
APPROACH
We use MP-FLIM of NAD(P)H and FAD on tissues and confirm viability using standard apoptosis and live/dead (Caspase 3/7 and propidium iodide, respectively) assays.
RESULTS
Through a statistical approach, reproducible shifts in FLIM data, determined through phasor analysis, are shown to correlate with loss of cell viability. With this, we demonstrate that cell death achieved through either apoptosis/necrosis or necroptosis can be discriminated. In addition, specific responses to common chemotherapeutic treatment inducing cell death were detected.
CONCLUSIONS
These data demonstrate that MP-FLIM can detect and quantify cell viability without the use of potentially toxic dyes, thus enabling longitudinal multi-day studies assessing the effects of therapeutic agents on tumor fragments.
Topics: Animals; Mice; Cell Survival; Microscopy, Fluorescence, Multiphoton; Apoptosis; Flavin-Adenine Dinucleotide; NADP; Cell Line, Tumor; Optical Imaging
PubMed: 38881557
DOI: 10.1117/1.JBO.29.S2.S22709 -
Frontiers in Nutrition 2024This study investigated the efficacy of a mixed beet-based supplement (BEET) versus placebo (PL) in countering inflammation during recovery from 2.25 h of intensive...
OBJECTIVES
This study investigated the efficacy of a mixed beet-based supplement (BEET) versus placebo (PL) in countering inflammation during recovery from 2.25 h of intensive cycling in 20 male and female cyclists. A multi-omics approach was used that included untargeted proteomics and a targeted oxylipin panel.
METHODS
A randomized, placebo-controlled, double-blind, crossover design was used with two 2-week supplementation periods and a 2-week washout period. Supplementation periods were followed by a 2.25 h cycling bout at close to 70%VO. The BEET supplement provided 212 mg of nitrates per day, 200 mg caffeine from green tea extract, 44 mg vitamin C from Camu Camu berry, B-vitamins from quinoa sprouts (40% Daily Value for thiamin, riboflavin, niacin, and vitamin B6), and 2.5 g of a mushroom blend containing Cordyceps sinensis and Inonotus obliquus. Six blood samples were collected before and after supplementation (overnight fasted state), immediately post-exercise, and at 1.5 h-, 3 h-, and 24 h-post-exercise.
RESULTS
The 2.25 h cycling bout increased plasma levels of 41 of 67 oxylipins detected. BEET supplementation significantly increased plasma nitrate (NO ) and nitrite (NO ) (sum, NO + NO ) concentrations (interaction effect, < 0.001) and two anti-inflammatory oxylipins [18-hydroxyeicosapentaenoic acid (18-HEPE) and 4-hydroxy-docosahexanoic acid (4-HDoHE)]. The untargeted proteomics analysis identified 616 proteins (458 across all times points), and 2-way ANOVA revealed a cluster of 45 proteins that were decreased and a cluster of 21 that were increased in the BEET versus PL trials. Functional enrichment supported significant BEET-related reductions in inflammation-related proteins including several proteins related to complement activation, the acute phase response, and immune cell adhesion, migration, and differentiation.
DISCUSSION
Intake of a BEET-based supplement during a 2-week period was linked to higher plasma levels of NO + NO , elevated post-exercise levels of two anti-inflammatory oxylipins, and a significant decrease in a cluster of proteins involved in complement activation and inflammation. These data support that 2-weeks intake of nitrate from a mixed beet-based supplement moderated protein biomarkers of exercise-induced inflammation in athletes.
PubMed: 38873567
DOI: 10.3389/fnut.2024.1408804 -
Frontiers in Veterinary Science 2024Garlic skin (GAS) has been proven to improve the growth performance of fattening sheep. However, the mechanism by which GAS affects fattening sheep is not yet clear. The...
OBJECTIVE
Garlic skin (GAS) has been proven to improve the growth performance of fattening sheep. However, the mechanism by which GAS affects fattening sheep is not yet clear. The aim of this study is to investigate the effects of adding GAS to feed on the growth performance, rumen and fecal microbiota, serum and urine metabolism, and transcriptomics of rumen epithelial cells in fattening sheep.
METHODS
GAS with 80 g/kg dry matter (DM) was added to the diet of fattening sheep to study the effects of GAS on gut microbiota, serum and urine metabolism, and transcriptome of rumen epithelial tissue in fattening sheep. Twelve Hu sheep (body weights; BW, 23.0 ± 2.3 kg and ages 120 ± 3.5 d) were randomly divided into two groups. The CON group was the basal diet, while the GAS group was supplemented with GAS in the basal diet. The trial period was 10 weeks, with the first 2 weeks being the pre-trial period.
RESULTS
The daily average weight gain of fattening sheep in the GAS group was significantly higher than that in the CON group ( < 0.05), and the serum GSH-Px of the GAS group fattening sheep was significantly increased, while MDA was significantly reduced ( < 0.05). Based on the genus classification level, the addition of garlic peel in the diet changed the intestinal microbial composition, and the relative abundance was significantly upregulated by ( < 0.05), while significantly downregulated by , , and ( < 0.05). Metabolomics analysis found that there were 166 significantly different metabolites in serum and 68 significantly different metabolites in urine between the GAS and CON groups ( < 0.05). GAS had an impact on amino acid metabolism, pyrimidine metabolism, methane metabolism, riboflavin metabolism, and unsaturated fatty acid synthesis pathways ( < 0.05). Transcriptome sequencing showed that differentially expressed genes were mainly enriched in immune regulatory function, improving the health of fattening sheep.
CONCLUSION
Adding GAS can improve the energy metabolism and immune function of fattening sheep by altering gut microbiota, metabolome, and transcriptome, thereby improving the growth performance of fattening sheep.
PubMed: 38872796
DOI: 10.3389/fvets.2024.1409518 -
Frontiers in Bioengineering and... 2024Previous laboratory-scale studies have consistently shown that carbon-based conductive materials can notably improve the anaerobic digestion of food waste, typically...
Previous laboratory-scale studies have consistently shown that carbon-based conductive materials can notably improve the anaerobic digestion of food waste, typically employing reactors with regular capacity of 1-20 L. Furthermore, incorporating riboflavin-loaded conductive materials can further address the imbalance between fermentation and methanogenesis in anaerobic systems. However, there have been few reports on pilot-scale investigation. In this study, a 10 m of riboflavin modified carbon cloth was incorporated into a pilot-scale (2 m) food waste anaerobic reactor to improve its treatment efficiency. The study found that the addition of riboflavin-loaded carbon cloth can increase the maximum organic loading rate (OLR) by 40% of the pilot-scale reactor, compared to the system using carbon cloth without riboflavin loading, while ensuring efficient operation of the reaction system, effectively alleviating system acidification, sustaining methanogen activity, and increasing daily methane production by 25%. Analysis of the microbial community structure revealed that riboflavin-loaded carbon cloth enriched the methanogenic archaea in the genera of and , which are capable of extracellular direct interspecies electron transfer (DIET). And metabolic pathway analysis identified the methane production pathway, highly enriched on the reduction of acetic acid and CO at riboflavin-loaded carbon cloth sample. The expression levels of genes related to methane production via DIET pathway were also significantly upregulated. These results can provide important guidance for the practical application of food waste anaerobic digestion engineering.
PubMed: 38863495
DOI: 10.3389/fbioe.2024.1395810 -
Se Pu = Chinese Journal of... Jun 2024Mitochondria perform various metabolic processes that significantly affect cell differentiation, proliferation, signal transduction, and programmed cell death. The...
Mitochondria perform various metabolic processes that significantly affect cell differentiation, proliferation, signal transduction, and programmed cell death. The disruption of mitochondrial bioenergetic and metabolic functions is closely related to many disorders. The specific isolation and purification of intact, high-purity, and functional mitochondria are central to the understanding of their mechanism of action but remain challenging tasks. In this study, a mitochondrial penetrating peptide (MPP) with the sequence FrFKFrFK(Ac) was used as a mitochondrial recognition motif to construct a peptide-guided affinity separation material. The multiple aromatic phenylalanine (F) residues in this amphiphilic peptide can confer lipophilicity to the mitochondrial membrane, whereas the basic residues (D-arginine and lysine) render the MPP surface positively charged, thereby promoting the binding of negatively charged mitochondria. After the derivatization of the N terminal of MPP with an oligoglycine spacer, the peptide ligands were conjugated to matrix beads (MB) with surface aldehyde functional groups. Peptide functionalization was performed via a condensation reaction between the amino group in the peptide ligand and the aldehyde group on the beads. The generated Schiff bases were reduced, affording stable covalent bonds. The dense and stable functionalization of the beads with the mitochondria-targeting peptides was demonstrated using high performance liquid chromatography (HPLC), zeta potential assay, and scanning electron microscopy (SEM). The immobilization efficiency of the peptide ligands was 1.47 μmol/g, and the surface potential of MB@MPP was 11 mV. MB@MPP was used for the direct isolation of mitochondria after cell homogenization. As observed by SEM, mitochondria with a cross-sectional diameter of 500 nm were efficiently captured on the MB@MPP surface. Because the mitochondrial membrane potential is an important marker of mitochondrial function and the driving force behind the staining of mitochondria with Mito Tracker dyes, the specific binding and separation of fluorescent mitochondria from the cell samples revealed that the proposed MB@MPP-based isolation approach can keep mitochondria intact and retain their functions. Western blot assays were employed to characterize the protein markers of the mitochondria (citrate synthase (CS) and voltage-dependent anion channel protein (VDAC)) and cytoplasmic protein (vinculin), and examine the integrity and purity of the captured mitochondria. The results showed that the lysates released from MB@MPP had high CS and VDAC contents. By contrast, vinculin, which is highly abundant in whole-cell lysates, was barely detected in the lysates from MB@MPP. These results suggest that MB@MPP isolates mitochondria with high affinity, specificity, and antifouling ability by using the targeting peptide as the capture handle. A comparison with a commercial mitochondrial isolation kit demonstrated that MB@MPP can separate mitochondria with higher CS and VDAC abundance and purity. Given the superior separation performance of MB@MPP, the molecular profiles of the isolated mitochondria under stress were subjected to further analysis of their molecular profiles under stress. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established to detect tryptophan (Trp) and riboflavin in the mitochondria. Quantification was performed in multiple-reaction monitoring (MRM) mode. Owing to the high purity of the mitochondria, the Trp and riboflavin contents were determined to be 265 and 0.67 nmol/mg, respectively. The metabolic response of mitochondria to external stimuli was further examined using acadesine, an adenosine 5'-monophosphate (AMP)-activated protein kinase activator with a wide range of metabolic effects, to treat cells. After cell homogenization, MB@MPP was used to separate the mitochondria from the cell samples with and without acadesine treatment, followed by LC-MS/MS analysis. The quantification results demonstrated that acadesine induced a 14% upregulation of Trp content in the mitochondria. By contrast, the riboflavin content decreased to 0.48 nmol/mg, which is 72% of that in untreated mitochondria. The changes in Trp and riboflavin contents could influence their metabolic pathways and, thus, the levels of their metabolites, such as nicotinamide adenine dinucleotide, flavin mononucleotide, and flavin adenine dinucleotide, which are essential coenzymes in mitochondria. Peptide-functionalized affinity microbeads with high affinity and specificity for mitochondria are promising for the efficient isolation of high-quality mitochondria, and offer a useful tool for understanding the complicated functions and dynamics of this unique organelle.
Topics: Mitochondria; Peptides; Animals; Chromatography, Affinity
PubMed: 38845516
DOI: 10.3724/SP.J.1123.2024.01013 -
Poultry Science May 2024This investigation aimed to evaluate the impact of immersion (IM) riboflavin treatment on the hatchability, production efficiency, and carcass characteristics of...
This investigation aimed to evaluate the impact of immersion (IM) riboflavin treatment on the hatchability, production efficiency, and carcass characteristics of Japanese quail eggs. A total of 260 eggs of Japanese quail birds were used for hatching and were randomly divided into 4 treatments with 5 replicates (13 eggs/replicate) in a fully randomized design. Hatching eggs were immersed in riboflavin for 2 min before incubation. The experiment treatments were designed as follows: G1 control group with no treatment, G2 treated with 3 g/L vit. B2 (IM), G3 treated with 4 g/L vit. B2 (IM) and G4 were treated with 5 g/L vit. B2 (IM). After hatching, 128 Japanese quail chicks, aged 7 d, were randomly grouped into 4 treatment groups, with 32 birds in each group. When quails were given vitamin B2 via immersion, they demonstrated significant enhancements in live body weight, body weight gain, feed consumption, and feed conversion ratio at different stages compared to the control group. Compared to control and other groups, the carcass parameters of Japanese quails given a 4 g/L immersion solution showed a significant improvement (P < 0.05). Hatchability and fertility (%) were considerably raised by Vit.B2 treatments of 3, 4, and 5g; the group immersed in 5 g/L had the highest percentages compared to the other groups. Furthermore, treated chickens with all concentrations of vitamin B2 had significantly higher blood indices than the controls. During the exploratory phase (1-6 wk) of age, the highest returns were reported in G4 treated with 5g/L vit. B2 (IM). Treating Japanese quail eggs with different dosages of vitamin B2 by immersion may be recommended to improve their productive and reproductive performance, blood indices, carcass traits, and economic efficiency.
PubMed: 38838591
DOI: 10.1016/j.psj.2024.103858 -
Current Opinion in Virology Jun 2024Mucosal-associated invariant T (MAIT) cells are an unconventional T cell population that are highly abundant in humans. They possess a semi-invariant T cell receptor... (Review)
Review
Mucosal-associated invariant T (MAIT) cells are an unconventional T cell population that are highly abundant in humans. They possess a semi-invariant T cell receptor (TCR) that recognises microbial metabolites formed during riboflavin biosynthesis, presented on a nonpolymorphic MHC-like molecule MR1. MAIT cells possess an array of effector functions, including type 1, type 17, and tissue repair activity. Deployment of these functions depends on the stimuli they receive through their TCR and/or cytokine receptors. Strong cytokine signalling, such as in response to vaccination, can bypass TCR triggering and provokes a strong proinflammatory response. Although data are still emerging, multiple aspects of MAIT cell biology are associated with modulation of immunity induced by the coronavirus disease 2019 mRNA and adenovirus vector vaccines. In this review, we will address how MAIT cells may play a role in immunogenicity of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and how these cells can be harnessed as cellular adjuvants.
PubMed: 38838550
DOI: 10.1016/j.coviro.2024.101412 -
Frontiers in Veterinary Science 2024Locoweed is a poisonous plant widely present in grasslands around the world. Swainsonine (SW), an indole alkaloid that, is the main toxic component of the locoweed. To...
Locoweed is a poisonous plant widely present in grasslands around the world. Swainsonine (SW), an indole alkaloid that, is the main toxic component of the locoweed. To understand the mechanism of SW-induced toxicity and to delineate the metabolic profile of locoweed poisoning we performed the LC-MS/MS untargeted metabolomic study to analyze metabolites in SW-treated renal tubular epithelial cells (0.8 mg/mL, 12 h) and in order to identify the SW-induced metabolomic changes. The analysis identified 2,563 metabolites in positive ion mode and 1,990 metabolites in negative ion mode. Our results showed that the metabolites were mainly benzenoids, lipids and lipid-like molecules, nucleosides, nucleotides, and analogs, organic acids, and derivatives. The differential metabolites were primarily enriched in pathways involving bile secretion, primary bile acid biosynthesis, riboflavin metabolism, ferroptosis, drug metabolism-cytochrome P450, and primidine metabolism. We have screened out substances such as swainsonine, 3alpha,7alpha-Dihydroxy-5beta-cholestanate, 2-Hydroxyiminostilbene, and glycochenodeoxycholate, which may have the potential to serve as biomarkers for swainsonine poisoning. This study provides insights into the types of metabolomic alteration in renal tubular epithelial cells induced by swainsonine.
PubMed: 38835895
DOI: 10.3389/fvets.2024.1387853 -
Progress in Retinal and Eye Research Jun 2024Changes in the biomechanical and biochemical properties of the human cornea play an important role in the pathogenesis of ectatic diseases. A number of conditions in... (Review)
Review
Changes in the biomechanical and biochemical properties of the human cornea play an important role in the pathogenesis of ectatic diseases. A number of conditions in primarily acquired (keratoconus or pellucid marginal degeneration) or secondarily induced (iatrogenic keratectasia after refractive laser surgeries) ectatic disorders lead to decreased biomechanical stability. Corneal collagen cross-linking (CXL) represents a technique to slow or even halt the progression of ectatic pathologies. In this procedure, riboflavin is applied in combination with ultraviolet A radiation. This interaction induces the production of reactive oxygen species, which leads to the formation of additional covalent bonds between collagen molecules and subsequent biomechanical corneal strengthening. This procedure is so far the only method that partially interferes etiopathogenetically in the treatment of ectatic diseases that slows or stops the process of corneal destabilization, otherwise leading to the need for corneal transplantation. Besides, CXL process increases markedly resistance of collagenous matrix against digesting enzymes supporting its use in the treatment of corneal ulcers. Since the discovery of this therapeutic procedure and the first laboratory experiments, which confirmed the validity of this method, and the first clinical studies that proved the effectiveness and safety of the technique, it has been spread and adopted worldwide, even with further modifications. Making use of the Bunsen-Roscoe photochemical law it was possible to shorten the duration of this procedure in accelerated CXL and thus improve the clinical workflow and patient compliance while maintaining the efficacy and safety of the procedure. The indication spectrum of CXL can be further expanded by combining it with other vision-enhancing procedures such as individualized topographically-guided excimer ablation. Complementing both techniques will allow a patient with a biomechanically stable cornea to regularize it and improve visual acuity without the need for tissue transplantation, leading to a long-term improvement in quality of life.
PubMed: 38830532
DOI: 10.1016/j.preteyeres.2024.101276