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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 -
Journal of Chromatographic Science Jun 2024The aim of this study was to develop and validate methods for the determination of vitamins B2, B9, E and A in serum using liquid chromatography with mass spectrometry...
The aim of this study was to develop and validate methods for the determination of vitamins B2, B9, E and A in serum using liquid chromatography with mass spectrometry (MS) detection. Vitamin analysis was performed using an ultra performance liquid chromatography combined with tandem MS. The compounds were separated on a BEH C18 RP column (2.1 × 100 mm, 1.7 μm) using a gradient elution with an analysis time of 10 min. Sample preparation included protein precipitation with ethanol. The concentration range in human serum was as follows: riboflavin 5-1000 nmol/L, folic acid 2.5-250 nmol/L, α-tocopherol 0.5-100 μmol/L and all-trans-retinol 25-2500 nmol/L. Accuracy and precision were validated according to Food and Drug Administration guidelines, with coefficients of variation ranging from 3.1-11.7% and recoveries from 94.4-107.5%. Routine monitoring of the complex range of vitamins in bariatric medicine is still not common. This is despite the fact that patients are at risk for glitch deficits, especially of a neurological nature. An analytical method that allows for the complex measurement of both water-soluble and fat-soluble vitamins is important and necessary for the clinical monitoring of bariatric patients. The method we have described could benefit both clinical practice and nutritional research.
PubMed: 38841803
DOI: 10.1093/chromsci/bmae035 -
Wei Sheng Yan Jiu = Journal of Hygiene... May 2024To establish an ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) method for simultaneous determination of 11 nutritional components(thiamine,...
OBJECTIVE
To establish an ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) method for simultaneous determination of 11 nutritional components(thiamine, riboflavin, nicotinamide, nicotinic acid, pantothenic acid, pyridoxine, pyridoxal, pyridoxamine, biotin, choline, L-carnitine) in liquid milk.
METHODS
Milk samples were shaken with 20 mmol/L ammonium formate solution and heated in a water bath at 100 ℃ for 30 min, then incubated with papain and acid phosphatase at 45 ℃ for 16 h, the lower liquid was collected after centrifugation for analysis. UPLC separation was performed on an ACQUITY~(TM) HSS T3(3.0 mm×150 mm, 1.8 μm) column, 2 mmol/L ammonium formate(containing 0.1% formic acid) solution and acetonitrile(containing 0.1% formic acid) were used as mobile phase. Quantitative detection was performed by internal standard method.
RESULTS
11 nutritional components can be effectively separated and detected in 12 min, and the linear correlation coefficients(R~2) were all above 0.995. The limits of detection(LODs) were between 0.05 and 0.50 μg/L, and the limits of quantification(LOQs) were between 0.20 and 1.25 μg/L. The recovery rates of three-level addition were 85.6%-119.3%, and the precision RSDs were between 3.68% and 7.82%(n=6). Based on the detection of 60 liquid milk samples from 5 different animals, it was found that the contents of 11 nutrients in liquid milk from different milk sources were significantly different, but pyridoxine could not be detected.
CONCLUSION
The method can quantitatively detect 11 water-soluble nutrients, including free and bound forms, by effective enzymolysis. It is sensitive, reproducible and can meet the needs of quantitative detection.
Topics: Milk; Tandem Mass Spectrometry; Animals; Chromatography, High Pressure Liquid; Niacinamide; Riboflavin; Nutrients; Pantothenic Acid; Cattle; Pyridoxine; Niacin; Carnitine
PubMed: 38839588
DOI: 10.19813/j.cnki.weishengyanjiu.2024.03.017 -
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 -
Journal of Chemical Information and... Jun 2024Lysine-specific demethylase 1 (LSD1), a highly sophisticated epigenetic regulator, orchestrates a range of critical cellular processes, holding promising therapeutic...
Lysine-specific demethylase 1 (LSD1), a highly sophisticated epigenetic regulator, orchestrates a range of critical cellular processes, holding promising therapeutic potential for treating diverse diseases. However, the clinical research progress targeting LSD1 is very slow. After 20 years of research, only one small-molecule drug, BEA-17, targeting the degradation of LSD1 and CoREST has been approved by the U.S. Food and Drug Administration. The primary reason for this may be the lack of abundant structural data regarding its intricate functions. To gain a deeper understanding of its conformational dynamics and guide the drug design process, we conducted molecular dynamics simulations to explore the conformational states of LSD1 in the apo state and under the influence of cofactors of flavin adenine dinucleotide (FAD) and CoREST. Our results showed that, across all states, the substrate binding pocket exhibited high flexibility, whereas the FAD binding pocket remained more stable. These distinct dynamical properties are essential for LSD1's ability to bind various substrates while maintaining efficient demethylation activity. Both pockets can be enlarged by merging with adjacent pockets, although only the substrate binding pocket can shrink into smaller pockets. These new pocket shapes can inform inhibitor design, particularly for selectively FAD-competitive inhibitors of LSD1, given the presence of numerous FAD-dependent enzymes in the human body. More interestingly, in the absence of FAD binding, the united substrate and FAD binding pocket are partitioned by the conserved residue of Tyr761, offering valuable insights for the design of inhibitors that disrupt the crucial steric role of Tyr761 and the redox role of FAD. Additionally, we identified pockets that positively or negatively correlate with the substrate and FAD binding pockets, which can be exploited for the design of allosteric or concurrent inhibitors. Our results reveal the intricate dynamical properties of LSD1 as well as multiple novel conformational states, which deepen our understanding of its sophisticated functions and aid in the rational design of new inhibitors.
Topics: Histone Demethylases; Flavin-Adenine Dinucleotide; Molecular Dynamics Simulation; Drug Design; Binding Sites; Humans; Enzyme Inhibitors; Substrate Specificity; Protein Conformation; Protein Binding
PubMed: 38837697
DOI: 10.1021/acs.jcim.4c00398 -
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 -
International Journal of Biological... Jun 2024Intervertebral disc degeneration arises from damage or degeneration of the nucleus pulposus (NP). In this study, we developed a photo-crosslinkable hydrogel...
Intervertebral disc degeneration arises from damage or degeneration of the nucleus pulposus (NP). In this study, we developed a photo-crosslinkable hydrogel incorporating FG4592 to support the growth and differentiation of bone-marrow-derived mesenchymal stem cells (BMSC). Initially, hyaluronic acid was modified with tyramine and combined with collagen to introduce riboflavin as a photo-crosslinker. This hydrogel transitioned from liquid to gel upon exposure to blue light in 3 min. The results showed that the hydrogel was biodegradable and had mechanical properties comparable to those of human NP tissues. Scanning electron microscopy after BMSC seeding in the hydrogel revealed an even distribution, and cells adhered to the collagen fibers in the hydrogel with minimal cell mortality. The effect of FG4592 on BMSC proliferation and differentiation was examined, revealing the capability of FG4592 to promote BMSC proliferation and direct differentiation resembling human NP cells. After cultivating BMSCs in the photo-crosslinked hydrogel, there was an upregulation in the expression of glycosaminoglycans, aggrecan, type II collagen, and keratin 19 proteins. Cross-species analyses of rat and human BMSCs revealed consistent results. For potential clinical applications, BMSC loaded with photo-crosslinked hydrogels can be injected into damaged intervertebral disc to facilitate NP regeneration.
PubMed: 38834125
DOI: 10.1016/j.ijbiomac.2024.132828 -
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 -
BJPsych Open Jun 2024Broad-spectrum micronutrients (minerals and vitamins) have shown benefit for treatment of depressive symptoms.
BACKGROUND
Broad-spectrum micronutrients (minerals and vitamins) have shown benefit for treatment of depressive symptoms.
AIMS
To determine whether additional micronutrients reduce symptoms of antenatal depression.
METHOD
Eighty-eight medication-free pregnant women at 12-24 weeks gestation, who scored ≥13 on the Edinburgh Postnatal Depression Scale (EPDS), were randomised 1:1 to micronutrients or active placebo (containing iodine and riboflavin), for 12 weeks. Micronutrient doses were generally between recommended dietary allowance and tolerable upper level. Primary outcomes (EPDS and Clinical Global Impression - Improvement Scale (CGI-I)) were analysed with constrained longitudinal data analysis.
RESULTS
Seventeen (19%) women dropped out, with no group differences, and four (4.5%) gave birth before trial completion. Both groups improved on the EPDS, with no group differences ( = 0.1018); 77.3% taking micronutrients and 72.7% taking placebos were considered recovered. However, the micronutrient group demonstrated significantly greater improvement, based on CGI-I clinician ratings, over time ( = 0.0196). The micronutrient group had significantly greater improvement on sleep and global assessment of functioning, and were more likely to identify themselves as 'much' to 'very much' improved (68.8%) compared with placebo (38.5%) (odds ratio 3.52, = 0.011; number needed to treat: 3). There were no significant group differences on treatment-emergent adverse events, including suicidal ideation. Homocysteine decreased significantly more in the micronutrient group. Presence of personality difficulties, history of psychiatric medication use and higher social support tended to increase micronutrient response compared with placebo.
CONCLUSIONS
This study highlights the benefits of active monitoring on antenatal depression, with added efficacy for overall functioning when taking micronutrients, with no evidence of harm. Trial replication with larger samples and clinically diagnosed depression are needed.
PubMed: 38828982
DOI: 10.1192/bjo.2024.706