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International Journal of Hyperthermia :... Sep 2017Isolated limb perfusion (ILP) is a treatment option for patients with in-transit metastases of malignant melanoma in the extremities, as well as locally advanced... (Clinical Trial)
Clinical Trial
INTRODUCTION
Isolated limb perfusion (ILP) is a treatment option for patients with in-transit metastases of malignant melanoma in the extremities, as well as locally advanced sarcoma. ILP allows for a delivery of high-dose chemotherapy to an isolated extremity with minimal systemic toxicity. However, local toxicity like oedema, blistering, nerve damage and compartment syndrome can occur. Myoglobin measurements have been used as a screening method to predict the most severe cases of local toxicity. The aim was to investigate if myoglobin is a predictive factor for local toxicity after ILP in patients with melanoma in-transit metastases.
METHODS
One hundred and ninety-three patients were treated for the first time with ILP for in-transit metastases between 2001 and 2015. Myoglobin was measured once the first hours after the perfusion (POD0), and for the first five post-operative days (POD1-5). Local toxicity was graded according to Wieberdink, and grouped in mild (I and II), moderate (III), and severe (IV and V). Wieberdink-groups were compared with myoglobin measurements, and myoglobin measurements were compared between gender, perfusion time, perfusion temperature and cannulated vessels.
RESULTS
There is no statistically significant difference in myoglobin serum levels during the first five days post perfusion between patients suffering from mild, moderate or severe local toxicity. There is no difference between toxicity groups when it comes to distribution of sex, tumour size, or tumour numbers.
CONCLUSION
Levels of myoglobin do not predict local toxicity for patients with melanoma in-transit metastases treated with ILP when measured during the first five post-operative days.
Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents, Alkylating; Chemotherapy, Cancer, Regional Perfusion; Extremities; Female; Humans; Male; Melanoma; Melphalan; Middle Aged; Myoglobin; Skin Neoplasms; Young Adult
PubMed: 28540796
DOI: 10.1080/02656736.2017.1309082 -
Genes Jul 2022The visual appearance of the fish fillet is a significant determinant of consumers' purchase decisions. Depending on the rainbow trout diet, a uniform bright white or...
The visual appearance of the fish fillet is a significant determinant of consumers' purchase decisions. Depending on the rainbow trout diet, a uniform bright white or reddish/pink fillet color is desirable. Factors affecting fillet color are complex, ranging from the ability of live fish to accumulate carotenoids in the muscle to preharvest environmental conditions, early postmortem muscle metabolism, and storage conditions. Identifying genetic markers of fillet color is a desirable goal but a challenging task for the aquaculture industry. This study used weighted, single-step GWAS to explore the genetic basis of fillet color variation in rainbow trout. We identified several SNP windows explaining up to 3.5%, 2.5%, and 1.6% of the additive genetic variance for fillet redness, yellowness, and whiteness, respectively. SNPs are located within genes implicated in carotenoid metabolism (β,β-carotene 15,15'-dioxygenase, retinol dehydrogenase) and myoglobin homeostasis (ATP synthase subunit β, mitochondrial ()). These genes are involved in processes that influence muscle pigmentation and postmortem flesh coloration. Other identified genes are involved in the maintenance of muscle structural integrity (kelch protein 41b (), collagen α-1(XXVIII) chain (), and cathepsin K ()) and protection against lipid oxidation (peroxiredoxin, superoxide dismutase 2 (), sestrin-1, Ubiquitin carboxyl-terminal hydrolase-10 ()). A-to-G single-nucleotide polymorphism in β,β-carotene 15,15'-dioxygenase, and result in isoleucine-to-valine and proline-to-leucine non-synonymous amino acid substitutions, respectively. Our observation confirms that fillet color is a complex trait regulated by many genes involved in carotenoid metabolism, myoglobin homeostasis, protection against lipid oxidation, and maintenance of muscle structural integrity. The significant SNPs identified in this study could be prioritized via genomic selection in breeding programs to improve fillet color in rainbow trout.
Topics: Animals; Carotenoids; Genome-Wide Association Study; Lipids; Myoglobin; Oncorhynchus mykiss; beta-Carotene 15,15'-Monooxygenase
PubMed: 35893068
DOI: 10.3390/genes13081331 -
Transgenic Research Dec 2023Previous studies using myoglobin (Mb) knockout mice and knockdown zebrafish have presented conflicting results about in vivo phenotypes resulting from the loss of this...
Previous studies using myoglobin (Mb) knockout mice and knockdown zebrafish have presented conflicting results about in vivo phenotypes resulting from the loss of this conserved and highly expressed protein, and therefore a new well-characterized knockout model is warranted. We here describe the generation of three distinct zebrafish mb knockout lines using the CRISPR/Cas system. None of the three lines exhibited any morphological phenotypes, changes in length, or lethality during embryonic and larval development. The adult homozygous knockout mb(Auzf13.2) zebrafish line were absent of Mb protein, had an almost complete degradation of mb mRNA, and showed no changes in viability, length, or heart size. Furthermore, transcriptomic analysis of adult heart tissue showed that mb knockout did not cause altered expression of other genes. Lastly, no off-targeting was observed in 36 screened loci. In conclusion, we have generated three mb knockout lines with indistinguishable phenotypes during embryonic and larval development and validated one of these lines, mb(Auzf13.2), to have no signs of genetic compensation or off-target effects in the adult heart. These findings suggests that the mb(Auzf13.2) shows promise as a candidate for investigating the biological role of Mb in zebrafish.
Topics: Animals; Mice; Zebrafish; Myoglobin; Zebrafish Proteins; CRISPR-Cas Systems; Phenotype; Gene Knockout Techniques
PubMed: 37847464
DOI: 10.1007/s11248-023-00369-3 -
Journal of the American Chemical Society Jul 2016Enzymes in the sulfur network generate the signaling molecule, hydrogen sulfide (H2S), from the amino acids cysteine and homocysteine. Since it is toxic at elevated...
Enzymes in the sulfur network generate the signaling molecule, hydrogen sulfide (H2S), from the amino acids cysteine and homocysteine. Since it is toxic at elevated concentrations, cells are equipped to clear H2S. A canonical sulfide oxidation pathway operates in mitochondria, converting H2S to thiosulfate and sulfate. We have recently discovered the ability of ferric hemoglobin to oxidize sulfide to thiosulfate and iron-bound hydropolysulfides. In this study, we report that myoglobin exhibits a similar capacity for sulfide oxidation. We have trapped and characterized iron-bound sulfur intermediates using cryo-mass spectrometry and X-ray absorption spectroscopy. Further support for the postulated intermediates in the chemically challenging conversion of H2S to thiosulfate and iron-bound catenated sulfur products is provided by EPR and resonance Raman spectroscopy in addition to density functional theory computational results. We speculate that the unusual sensitivity of skeletal muscle cytochrome c oxidase to sulfide poisoning in ethylmalonic encephalopathy, resulting from the deficiency in a mitochondrial sulfide oxidation enzyme, might be due to the concentration of H2S by myoglobin in this tissue.
Topics: Animals; Horses; Hydrogen Sulfide; Iron; Kinetics; Myoglobin; Oxidation-Reduction; Protein Binding
PubMed: 27310035
DOI: 10.1021/jacs.6b03456 -
Scientific Reports Oct 2022Some of the best nucleating agents in nature are ice-nucleating proteins, which boost ice growth better than any other material. They can induce immersion freezing of...
Some of the best nucleating agents in nature are ice-nucleating proteins, which boost ice growth better than any other material. They can induce immersion freezing of supercooled water only a few degrees below 0 °C. An open question is whether this ability also extends to the deposition mode, i.e., to water vapor. In this work, we used three proteins, apoferritin, InaZ (ice nucleation active protein Z), and myoglobin, of which the first two are classified as ice-nucleating proteins for the immersion freezing mode. We studied the ice nucleation ability of these proteins by differential scanning calorimetry (immersion freezing) and by environmental scanning electron microscopy (deposition freezing). Our data show that InaZ crystallizes water directly from the vapor phase, while apoferritin first condenses water in the supercooled state, and subsequently crystallizes it, just as myoglobin, which is unable to nucleate ice.
Topics: Apoferritins; Calorimetry; Freezing; Ice; Microscopy, Electron; Myoglobin; Steam
PubMed: 36192511
DOI: 10.1038/s41598-022-20430-1 -
Journal of Biological Physics Dec 2021Hans Frauenfelder's discovery of conformational substates in studies of myoglobin carbon monoxide geminate rebinding kinetics at cryogenic temperatures (Austin RH,... (Review)
Review
Hans Frauenfelder's discovery of conformational substates in studies of myoglobin carbon monoxide geminate rebinding kinetics at cryogenic temperatures (Austin RH, Beeson KW, Eisenstein L, Frauenfelder H, & Gunsalus IC (1975) Dynamics of Ligand Binding to Myoglobin. Biochemistry 14(24):5355-5373) followed by his introduction of energy landscape theory with Peter Wolynes (Frauenfelder H, Sligar SG, & Wolynes PG (1991) The Energy Landscapes and Motions of Proteins. Science 254(5038):1598-1603) marked the beginning of a new era in the physics and physical chemistry of proteins. Their work played a major role in demonstrating the power and importance of dynamics and of Kramers reaction rate theory for understanding protein function. The biggest impact of energy landscape theory has been in the protein folding field, which is well-known and has been documented in numerous articles and reviews, including a recent one of my own (Eaton WA (2021) Modern Kinetics and Mechanism of Protein Folding: a Retrospective. J. Phys. Chem. B. 125(14):3452-3467). Here I will describe the much less well-known impact of their modern view of proteins on both experimental and theoretical studies of hemoglobin kinetics and function. I will first describe how Frauenfelder's experiments motivated and influenced my own research on myoglobin, which were key ingredients to my work on understanding hemoglobin.
Topics: Hemoglobins; Kinetics; Myoglobin; Physics; Protein Conformation; Retrospective Studies
PubMed: 34762226
DOI: 10.1007/s10867-021-09588-3 -
Stem Cell Research & Therapy May 2022The metabolic phenotype of stem cells is increasingly recognized as a hallmark of their pluripotency with mitochondrial and oxygen-related metabolism playing a not...
BACKGROUND
The metabolic phenotype of stem cells is increasingly recognized as a hallmark of their pluripotency with mitochondrial and oxygen-related metabolism playing a not completely defined role in this context. In a previous study, we reported the ectopic expression of myoglobin (MB) in bone marrow-derived hematopoietic stem/progenitor cells. Here, we have extended the analysis to mesenchymal stem cells (MSCs) isolated from different tissues.
METHODS
MSCs were isolated from human placental membrane, mammary adipose tissue and dental pulp and subjected to RT-PCR, Western blotting and mass spectrometry to investigate the expression of MB. A combination of metabolic flux analysis and cyto-imaging was used to profile the metabolic phenotype and the mitochondria dynamics in the different MSCs.
RESULTS
As for the hematopoietic stem/progenitor cells, the expression of Mb was largely driven by an alternative transcript with the protein occurring both in the monomer and in the dimer forms as confirmed by mass spectrometry analysis. Comparing the metabolic fluxes between neonatal placental membrane-derived and adult mammary adipose tissue-derived MSCs, we showed a significantly more active bioenergetics profile in the former that correlated with a larger co-localization of myoglobin with the mitochondrial compartment. Differences in the structure of the mitochondrial network as well as in the expression of factors controlling the organelle dynamics were also observed between neonatal and adult mesenchymal stem cells. Finally, the expression of myoglobin was found to be strongly reduced following osteogenic differentiation of dental pulp-derived MSCs, while it was upregulated following reprogramming of human fibroblasts to induce pluripotent stem cells.
CONCLUSIONS
Ectopic expression of myoglobin in tissues other than muscle raises the question of understanding its function therein. Properties in addition to the canonical oxygen storage/delivery have been uncovered. Finding of Mb expressed via an alternative gene transcript in the context of different stem cells with metabolic phenotypes, its loss during differentiation and recovery in iPSCs suggest a hitherto unappreciated role of Mb in controlling the balance between aerobic metabolism and pluripotency. Understanding how Mb contributes through modulation of the mitochondrial physiology to the stem cell biology paves the way to novel perspectives in regenerative medicine as well as in cancer stem cell therapy.
Topics: Cell Differentiation; Female; Hematopoietic Stem Cells; Humans; Mesenchymal Stem Cells; Myoglobin; Osteogenesis; Oxygen; Placenta; Pregnancy
PubMed: 35598009
DOI: 10.1186/s13287-022-02880-6 -
Nutrients Jul 2023This study investigated the acute effects of natural antioxidants, derived from yeast fermentation containing glutathione and dietary vitamin C supplementation, on... (Randomized Controlled Trial)
Randomized Controlled Trial
Enhancing Supplemental Effects of Acute Natural Antioxidant Derived from Yeast Fermentation and Vitamin C on Sports Performance in Triathlon Athletes: A Randomized, Double-Blinded, Placebo-Controlled, Crossover Trial.
This study investigated the acute effects of natural antioxidants, derived from yeast fermentation containing glutathione and dietary vitamin C supplementation, on metabolic function, skeletal muscle oxygenation, cardiac function, and antioxidant function during submaximal exercise in middle-aged triathlon athletes. Twelve participants (aged 49.42 ± 5.9 years) completed 90 min submaximal cycling trials corresponding to 70% maximal oxygen uptake with either vitamin C and glutathione (VitC+Glu), vitamin C (VitC), glutathione (Glu) supplementation, or placebo. Metabolic function (minute ventilation, oxygen uptake, carbon dioxide output [VCO], respiratory exchange ratio [RER], oxygen pulse [Opulse], carbohydrate oxidation, fat oxidation, and energy expenditure), skeletal muscle oxygenation (oxidized hemoglobin and myoglobin in skeletal muscle tissue, total hemoglobin and myoglobin in skeletal muscle tissue [tHb]), cardiac function (heart rate [HR], stroke volume [SV], cardiac output, end-diastolic volume, end-systolic volume, and ejection fraction), and antioxidant function parameters (blood lactate, superoxide dismutase, catalase, glutathione peroxidases, glutathione [GSH], diacron reactive oxygen metabolite [dROM], and biological antioxidant potential [BAP]) were measured during submaximal exercise and recovery. VCO, RER, HR, blood lactate after exercise, and dROM were significantly lower, and Opulse, tHb, and BAP were significantly higher for VitC+Glu than for the other trials ( < 0.05). In conclusion, combined vitamin C and glutathione supplementation was more effective in improving metabolic function, skeletal oxygenation, cardiac function, and antioxidant function during prolonged submaximal exercise in middle-aged triathletes.
Topics: Humans; Middle Aged; Antioxidants; Ascorbic Acid; Saccharomyces cerevisiae; Cross-Over Studies; Fermentation; Myoglobin; Vitamins; Athletic Performance; Glutathione; Muscle, Skeletal; Athletes; Oxygen; Lactates; Dietary Supplements
PubMed: 37571262
DOI: 10.3390/nu15153324 -
Microbial Cell Factories Mar 2023Heme proteins, such as hemoglobin, horseradish peroxidase and cytochrome P450 (CYP) enzyme, are highly versatile and have widespread applications in the fields of food,...
BACKGROUND
Heme proteins, such as hemoglobin, horseradish peroxidase and cytochrome P450 (CYP) enzyme, are highly versatile and have widespread applications in the fields of food, healthcare, medical and biological analysis. As a cofactor, heme availability plays a pivotal role in proper folding and function of heme proteins. However, the functional production of heme proteins is usually challenging mainly due to the insufficient supply of intracellular heme.
RESULTS
Here, a versatile high-heme-producing Escherichia coli chassis was constructed for the efficient production of various high-value heme proteins. Initially, a heme-producing Komagataella phaffii strain was developed by reinforcing the C4 pathway-based heme synthetic route. Nevertheless, the analytical results revealed that most of the red compounds generated by the engineered K. phaffii strain were intermediates of heme synthesis which were unable to activate heme proteins. Subsequently, E. coli strain was selected as the host to develop heme-producing chassis. To fine-tune the C5 pathway-based heme synthetic route in E. coli, fifty-two recombinant strains harboring different combinations of heme synthesis genes were constructed. A high-heme-producing mutant Ec-M13 was obtained with negligible accumulation of intermediates. Then, the functional expression of three types of heme proteins including one dye-decolorizing peroxidase (Dyp), six oxygen-transport proteins (hemoglobin, myoglobin and leghemoglobin) and three CYP153A subfamily CYP enzymes was evaluated in Ec-M13. As expected, the assembly efficiencies of heme-bound Dyp and oxygen-transport proteins expressed in Ec-M13 were increased by 42.3-107.0% compared to those expressed in wild-type strain. The activities of Dyp and CYP enzymes were also significantly improved when expressed in Ec-M13. Finally, the whole-cell biocatalysts harboring three CYP enzymes were employed for nonanedioic acid production. High supply of intracellular heme could enhance the nonanedioic acid production by 1.8- to 6.5-fold.
CONCLUSION
High intracellular heme production was achieved in engineered E. coli without significant accumulation of heme synthesis intermediates. Functional expression of Dyp, hemoglobin, myoglobin, leghemoglobin and CYP enzymes was confirmed. Enhanced assembly efficiencies and activities of these heme proteins were observed. This work provides valuable guidance for constructing high-heme-producing cell factories. The developed mutant Ec-M13 could be employed as a versatile platform for the functional production of difficult-to-express heme proteins.
Topics: Escherichia coli; Myoglobin; Leghemoglobin; Carrier Proteins; Heme; Oxygen; Escherichia coli Proteins
PubMed: 36978060
DOI: 10.1186/s12934-023-02067-5 -
Angewandte Chemie (International Ed. in... Jul 2018Functionalized indoles are recurrent motifs in bioactive natural products and pharmaceuticals. While transition metal-catalyzed carbene transfer has provided an...
Functionalized indoles are recurrent motifs in bioactive natural products and pharmaceuticals. While transition metal-catalyzed carbene transfer has provided an attractive route to afford C3-functionalized indoles, these protocols are viable only in the presence of N-protected indoles, owing to competition from the more facile N-H insertion reaction. Herein, a biocatalytic strategy for enabling the direct C-H functionalization of unprotected indoles is reported. Engineered variants of myoglobin provide efficient biocatalysts for this reaction, which has no precedents in the biological world, enabling the transformation of a broad range of indoles in the presence of ethyl α-diazoacetate to give the corresponding C3-functionalized derivatives in high conversion yields and excellent chemoselectivity. This strategy could be exploited to develop a concise chemoenzymatic route to afford the nonsteroidal anti-inflammatory drug indomethacin.
Topics: Biocatalysis; Catalysis; Indoles; Molecular Structure; Myoglobin
PubMed: 29905974
DOI: 10.1002/anie.201804779