International Journal of Molecular Sciences

12 pages, 2681 KiB

Open AccessArticle

Strong Metal Support Effect of Pt/g-C₃N₄ Photocatalysts for Boosting Photothermal Synergistic Degradation of Benzene

by Zhongcheng Huang, Xiaorong Cai, Shaohong Zang, Yixin Li, Dandan Zheng and Fuying Li

Int. J. Mol. Sci. 2023, 24(7), 6872; https://doi.org/10.3390/ijms24076872 - 6 Apr 2023

Cited by 1 | Viewed by 2134

Catalysis is the most efficient and economical method for treating volatile organic pollutants (VOCs). Among the many materials that are used in engineering, platinized carbon nitride (Pt/g-C₃N₄) is an efficient and multifunctional catalyst which has strong light absorption and [...] Read more.

Catalysis is the most efficient and economical method for treating volatile organic pollutants (VOCs). Among the many materials that are used in engineering, platinized carbon nitride (Pt/g-C₃N₄) is an efficient and multifunctional catalyst which has strong light absorption and mass transfer capabilities, which enable it to be used in photocatalysis, thermal catalysis and photothermal synergistic catalysis for the degradation of benzene. In this work, Pt/g-C₃N₄ was prepared by four precursors for the photothermal synergistic catalytic degradation of benzene, which show different activities, and many tests were carried out to explore the possible reasons for the discrepancy. Among them, the Pt/g-C₃N₄ prepared from dicyanamide showed the highest activity and could convert benzene (300 ppm, 20 mL·min⁻¹) completely at 162 °C under solar light and 173 °C under visible light. The reaction temperature was reduced by nearly half compared to the traditional thermal catalytic degradation of benzene at about 300 °C. Full article

(This article belongs to the Special Issue Molecular Aspects in Catalytic Materials for Pollution Elimination and Green Chemistry 2.0)

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(a) XRD spectra, (b) FT-IR spectra of PDA, PMA, PUR and PCA samples. Full article ">Figure 2
The morphology and atomic structure of Pt/CN. (a) block CN, (b) nanosheet CN, (c–e) TEM image, (f) EDX mapping of PDA. Full article ">Figure 3
XPS spectra of (a) survey, (b) Pt 4f, (c) C 1s, (d) N 1s, (e) BET patterns, (f) thermalgravimetric curves of PDA. Full article ">Figure 4
(a) Conversion rate, (b) mineralization rate of benzene by PDA, PCA, PMA and PUR under 150 °C and solar light conditions, (c) conversion rate, (d) mineralization rate of benzene for PDA under only heat, visible-light photothermal or solar light photothermal conditions, (e) conversion and mineralization rate of benzene for PDA under visible light or solar light photothermal conditions, (f) conversion/mineralization rate of four-cycle experiments of PDA under 162 °C and solar light conditions. Full article ">Figure 5
(a) DRS patterns, (b) photoluminescence spectra, (c) EIS Nyquist plots, (d) Photocurrent responses of PDA, PCA, PUR and PMA samples, EPR spectra of PDA for the detection of (e) DMPO–·OH, (f) DMPO–·O2−. Full article ">Scheme 1
The mechanism of photothermal catalytic reaction by PDA. Full article ">

15 pages, 2348 KiB

Open AccessArticle

Enhancement of the Antioxidant Effect of Natural Products on the Proliferation of Caco-2 Cells Produced by Fish Protein Hydrolysates and Collagen

by Mercedes Taroncher, Yelko Rodríguez-Carrasco, Francisco J. Barba and María-José Ruiz

Int. J. Mol. Sci. 2023, 24(7), 6871; https://doi.org/10.3390/ijms24076871 - 6 Apr 2023

Cited by 5 | Viewed by 1752

Abstract

A large amount of fish side streams are produced each year, promoting huge economic and environmental problems. In order to address this issue, a potential alternative is to isolate the high-added-value compounds with beneficial properties on human health. The objectives of this study [...] Read more.

A large amount of fish side streams are produced each year, promoting huge economic and environmental problems. In order to address this issue, a potential alternative is to isolate the high-added-value compounds with beneficial properties on human health. The objectives of this study were to determine the effect of hydrolyzed fish protein and collagen samples on cell proliferation, as well as to determine the specific influence of minerals and metals on this effect and whether dietary antioxidants can enhance cell proliferation. The results of hydrolyzed fish protein and collagen samples showed negative effects on Caco-2 cell proliferation at the highest concentrations tested. Moreover, the pre-treatment of these hydrolyzates with vitamin C and E, quercetin and resveratrol increased the proliferation of bioaccessible fractions of hydrolyzated fish protein and collagen samples compared to the bioaccessible fractions without pre-treatment. The highest mineral concentrations were found for P, Ca and Mg. The metals found in the pure hydrolyzates were As, Cd, Hg and Pb; however, they appeared at almost undetectable levels in bioavailable fractions. It can be concluded that the consumption of hydrolyzates of fish by-products is an interesting strategy for complying with EFSA recommendations regarding fish consumption while at the same time reducing fish waste. Full article

(This article belongs to the Special Issue Natural Compounds and Oxidative Stress)

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Effect of bioaccessible fractions of product 1 (A), product 2 (B), product 3 (C) and collagen (D) on cell proliferation in Caco-2 cells after 24 h of exposure at increasing concentrations from 0.05 to 0.5 mg/mL as assessed by the MTT assay. Values are expressed as mean ± SEM (n = 3). Values in the same figure with different superscript letters are significantly different (p < 0.05). CRL: control. Full article ">Figure 2
Protective effect of vit C (200 and 400 µM) on the bioaccessible fractions (0.1 and 0.2 mg/mL) of product 1 (A), product 2 (B), product 3 (C) and collagen (D) in Caco-2 cells after 24 h of exposure as assessed by the MTT assay. Values are expressed as mean ± SEM (n = 3). Values in the same figure with different superscript letters are significantly different (p < 0.05). CRL: control. Full article ">Figure 3
Protective effect of vit E (10 and 25 µM) on the bioaccessible fractions (0.1 and 0.2 mg/mL) of product 1 (A), product 2 (B), product 3 (C) and collagen (D) in Caco-2 cells after 24 h of exposure as assessed by the MTT assay. Values are expressed as mean ± SEM (n = 3). Values in the same figure with different superscript letters are significantly different (p < 0.05). CRL: control. Full article ">Figure 4
Protective effect of QUE (5 and 10 µM) on the bioaccessible fractions (0.1 and 0.2 mg/mL) of product 1 (A), product 2 (B), product 3 (C) and collagen (D) in Caco-2 cells after 24 h of exposure as assessed by the MTT assay. Values are expressed as mean ± SEM (n = 3). Values in the same figure with different superscript letters are significantly different (p < 0.05). CRL: control; QUE: quercetin. Full article ">Figure 5
Protective effect of RSV (10 and 25 µM) on the bioaccessible fractions (0.1 and 0.2 mg/mL) of product 1 (A), product 2 (B), product 3 (C) and collagen (D) in Caco-2 cells after 24 h of exposure as assessed by the MTT assay. Values are expressed as mean ± SEM (n = 3). Values in the same figure with different superscript letters are significantly different (p < 0.05). CRL: control; RSV: resveratrol. Full article ">

19 pages, 3207 KiB

Open AccessArticle

Nonsteroidal Anti-Inflammatory Drug Conjugated with Gadolinium (III) Complex as an Anti-Inflammatory MRI Agent

by Bokyung Sung, Hee-Kyung Kim, Ah-Rum Baek, Byeong-Woo Yang, Yeoun-Hee Kim, Garam Choi, Hyun-Jin Park, Minsup Kim, Jongmin Lee and Yongmin Chang

Int. J. Mol. Sci. 2023, 24(7), 6870; https://doi.org/10.3390/ijms24076870 - 6 Apr 2023

Cited by 2 | Viewed by 2071

Abstract

Studies have been actively conducted to ensure that gadolinium-based contrast agents for magnetic resonance imaging (MRI) are accompanied by various biological functions. A new example is the anti-inflammatory theragnostic MRI agent to target inflammatory mediators for imaging diagnosis and to treat inflammatory diseases [...] Read more.

Studies have been actively conducted to ensure that gadolinium-based contrast agents for magnetic resonance imaging (MRI) are accompanied by various biological functions. A new example is the anti-inflammatory theragnostic MRI agent to target inflammatory mediators for imaging diagnosis and to treat inflammatory diseases simultaneously. We designed, synthesized, and characterized a Gd complex of 1,4,7-tris(carboxymethylaza) cyclododecane-10-azaacetylamide (DO3A) conjugated with a nonsteroidal anti-inflammatory drug (NSAID) that exerts the innate therapeutic effect of NSAIDs and is also applicable in MRI diagnostics. Gd-DO3A-fen (0.1 mmol/kg) was intravenously injected into the turpentine oil-induced mouse model, with Gd-DO3A-BT as a control group. In the in vivo MRI experiment, the contrast-to-noise ratio (CNR) was higher and persisted longer than that with Gd-DO3A-BT; specifically, the CNR difference was almost five times at 2 h after injection. Gd-DO3A-fen had a binding affinity (K_a) of 6.68 × 10⁶ M⁻¹ for the COX-2 enzyme, which was 2.1-fold higher than that of fenbufen, the original NSAID. In vivo evaluation of anti-inflammatory activity was performed in two animal models. In the turpentine oil-induced model, the mRNA expression levels of inflammatory parameters such as COX-2, TNF-α, IL-1β, and IL-6 were reduced, and in the carrageenan-induced edema model, swelling was suppressed by 72% and there was a 2.88-fold inhibition compared with the saline group. Correlation analysis between in vitro, in silico, and in vivo studies revealed that Gd-DO3A-fen acts as an anti-inflammatory theragnostic agent by directly binding to COX-2. Full article

(This article belongs to the Special Issue Development of Biologically Active Compounds – Design, Synthesis and Application)

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Figure 1
Diagram of Gd-DO3A-fen. (a) Representation of the mechanism of action of Gd-DO3A-fen. (b) The synthetic procedure of the anti-inflammatory MRI CAs Gd-DO3A-fen. Full article ">Figure 2
In vivo assessment in the inflammation-induced mouse model. (a–c) T1-weighted MR images were acquired before and after the administration of Gd-DO3A-fen and Gd-DO3A-BT (0.1 mmol/kg) by 1.5-T MRI. (a) Anatomical axial images of the inflammation site are displayed in gray scale and (b) CNR profile is shown as a function of time. Values are expressed as mean ± S.D. (n = 3). (c) Color-map at maximum-enhanced time. (d) Histogram of signal intensity values in the inflammatory region. The blue bars represent the signal intensity distribution for preinjection data, and the red bars represent the data obtained at 2 h postinjection of Gd-DO3A-fen (left) and Gd-DO3A-BT (right). Each line and value on the graph represent the average values for each distribution. (e) The inflammation-induced mouse model was sacrificed after the administration of Gd-DO3A-fen to quantify the in vivo biodistribution at each time point (n = 4). The results show the amount of Gd (mmol) in each tissue as a percentage (%) of total Gd administration dose (0.1 mmol/kg). The amount of Gd (mmol) in different tissues was determined by ICP-AES. GB, gallbladder; intest, intestines; inflam, inflamed tissue. Full article ">Figure 3
The binding sites and binding affinity of COX-2 for Gd-DO3A-fen and the NSAID. (a) The drug-binding site of COX-2 consists of the main hydrophobic and secondary pockets. (b) The docking results for fenbufen. This drug was bound to Arg 513, and Trp 387 deep in the hydrophobic pocket, with ionic and π–π stacking interactions. (c) The docking results for Gd-DO3A-fen. The binding conformation of Gd-DO3A-fen was associated with polar interactions with surface protein and π–π stacking interactions in the hydrophobic pocket. (d) COX-2-binding affinity of fenbufen in UV–vis spectra. Samples were prepared in DMSO. COX-2 was dissolved in Tris-HCl buffer. (e) COX-2-binding affinity of Gd-DO3A-fen in UV–vis spectra. Samples were prepared in Tris-HCl (1:9, pH 7.0) buffer. (d,e) The absorbance of fenbufen and Gd-DO3A-fen were decreased by the addition of COX-2 enzyme. The inset graphs represent the fitting curve for the calculation of the binding constant (Ka). Full article ">Figure 4
Antiinflammatory effect of drugs on PA-induced inflammation in C2C12 cells and in a turpentine oil-induced inflammation mouse model. (a) Effect of drugs on the viability of C2C12 cells (*** p < 0.001 vs. nontreated control; n = 3). (b) The protein levels of COX-2 analyzed in total cell lysates by Western blotting. The bar graph represents the average densitometry values relative to β-actin (*** p < 0.001 vs. nontreated control, ### p < 0.001 vs. PA, ††† p < 0.001 vs. fenbufen; n = 3). (c) Comparison of the mRNA expression levels of the proinflammatory enzyme and cytokines in the inflammation-induced mouse model injected with turpentine oil. The saline group was intravenously injected saline instead of drug in turpentine oil-induced model. The control group was injected with saline instead of turpentine oil. The bar graph represents the relative ratio of saline (** p < 0.05, *** p < 0.001 vs. nontreated control, # p < 0.01, ## p < 0.05, ### p < 0.001 vs. PA, † p < 0.01 vs. fenbufen; n = 3). Full article ">Figure 5
The inhibitory effect of Gd-DO3A-fen in the carrageenan-induced rat hind paw edema rat model. (a) The procedure for monitoring the anti-inflammatory effect of Gd-DO3A-fen on paw edema. The edema volume was measured before injection and at 1, 2, 3, 4, 5, and 6 h after the injection of carrageenan. (b) Images showing the rat paw before injection and at 5 h after the injection of carrageenan, respectively: carrageenan, carrageenan + saline, carrageenan + Gd-DO3A-fen. Yellow arrows indicate the carrageenan-induced rat paw. (c) Percentage swelling observed in the carrageenan-induced paw edema. (d) Comparison of % swelling of Gd-DO3A-fen group and that of saline group at 5 h after injection. (e) Percentage inhibition of paw edema by Gd-DO3A-fen and saline. (f) Comparison of % inhibition of Gd-DO3A-fen group and that of saline group at 5 h after injection. All values represent mean ± S.E.M. (n = 4 per group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. saline group). Full article ">

25 pages, 4413 KiB

Open AccessArticle

The Role of Tyr-His-Trp Triad and Water Molecule Near the N1-Atom of 2-Hydroperoxycoelenterazine in Bioluminescence of Hydromedusan Photoproteins: Structural and Mutagenesis Study

by Pavel V. Natashin, Ludmila P. Burakova, Margarita I. Kovaleva, Mikhail B. Shevtsov, Daria A. Dmitrieva, Elena V. Eremeeva, Svetlana V. Markova, Alexey V. Mishin, Valentin I. Borshchevskiy and Eugene S. Vysotski

Int. J. Mol. Sci. 2023, 24(7), 6869; https://doi.org/10.3390/ijms24076869 - 6 Apr 2023

Cited by 2 | Viewed by 2177

Abstract

Hydromedusan photoproteins responsible for the bioluminescence of a variety of marine jellyfish and hydroids are a unique biochemical system recognized as a stable enzyme-substrate complex consisting of apoprotein and preoxygenated coelenterazine, which is tightly bound in the protein inner cavity. The binding of [...] Read more.

Hydromedusan photoproteins responsible for the bioluminescence of a variety of marine jellyfish and hydroids are a unique biochemical system recognized as a stable enzyme-substrate complex consisting of apoprotein and preoxygenated coelenterazine, which is tightly bound in the protein inner cavity. The binding of calcium ions to the photoprotein molecule is only required to initiate the light emission reaction. Although numerous experimental and theoretical studies on the bioluminescence of these photoproteins were performed, many features of their functioning are yet unclear. In particular, which ionic state of dioxetanone intermediate decomposes to yield a coelenteramide in an excited state and the role of the water molecule residing in a proximity to the N1 atom of 2-hydroperoxycoelenterazine in the bioluminescence reaction are still under discussion. With the aim to elucidate the function of this water molecule as well as to pinpoint the amino acid residues presumably involved in the protonation of the primarily formed dioxetanone anion, we constructed a set of single and double obelin and aequorin mutants with substitutions of His, Trp, Tyr, and Ser to residues with different properties of side chains and investigated their bioluminescence properties (specific activity, bioluminescence spectra, stopped-flow kinetics, and fluorescence spectra of Ca²⁺-discharged photoproteins). Moreover, we determined the spatial structure of the obelin mutant with a substitution of His64, the key residue of the presumable proton transfer, to Phe. On the ground of the bioluminescence properties of the obelin and aequorin mutants as well as the spatial structures of the obelin mutants with the replacements of His64 and Tyr138, the conclusion was made that, in fact, His residue of the Tyr-His-Trp triad and the water molecule perform the “catalytic function” by transferring the proton from solvent to the dioxetanone anion to generate its neutral ionic state in complex with water, as only the decomposition of this form of dioxetanone can provide the highest light output in the light-emitting reaction of the hydromedusan photoproteins. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 4.0)

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Two-dimensional drawing of the hydrogen bonding network in the binding cavities of active (A) and Ca2+-discharged (B) wild-type obelins. Molecular surface representation of “proton channel” in active (C) and Ca2+-discharged (D) wild-type obelins. Amino acid residues and the molecules of 2-hydroperoxycoelenterazine and coelenteramide are shown as stick models. Water molecules are represented as red balls. Hydrogen bonds are shown as dashed lines. Partially represented surface is gray colored. (E) Sequence alignment of obelin from O. longissima [<a href="#B37-ijms-24-06869" class="html-bibr">37</a>] and aequorin from A. victoria [<a href="#B38-ijms-24-06869" class="html-bibr">38</a>]. The residues in obelin and aequorin mutated in this study are shown in blue. The Ca2+ binding loops are highlighted in gray. The helices are indicated by capital letters A–H based on the wild-type obelin structure (PDB code 1QV0). Aequorin numeration starts from Val, shown with an arrow, according to the original numbering of the amino acid sequence determined for the natural aequorin and aequorin structure (PDB code 1EJ3) [<a href="#B1-ijms-24-06869" class="html-bibr">1</a>,<a href="#B22-ijms-24-06869" class="html-bibr">22</a>]. Full article ">Figure 2
Normalized bioluminescence and fluorescence spectra of obelin (A,C), aequorin (B,D) and their mutants. Full article ">Figure 3
High-speed (A,B) and low-speed (C,D) stopped-flow plots of the bioluminescence signals of obelin (A,C), aequorin (B,D), and their mutants. The curves are individual shots. Full article ">Figure 4
Crystal structure of OL_H64F mutant. (A) Overall structure of OL_H64F mutant. The helices are marked by letters A–H. (B) Superimposition of OL_H64F mutant (blue, PDB 8C6O, chain A), wild-type obelin (red, PDB 1QV0), and OL_Y138F mutant (green, PDB 4MRX). Full article ">Figure 5
Two-dimensional representation of the hydrogen bond network. (A) OL_H64F mutant (PDB 8C6O, chain A), (B) wild-type obelin (PDB 1QV0), (C) active OL_Y138F mutant (PDB: 4MRX), and (D) Ca2+-discharged OL_Y138F mutant (PDB 4MRY). Mutated residues H64(F64) and Y138(F138) are shown in blue and red, respectively. Hydrogen bonds are shown as dashed lines, distances between atoms are shown as arrows, and “W” stands for a water molecule. Distances are given in Å. Full article ">Figure 6
Stereoview of the superimposition of 2-hydroperoxycoelenterazine molecules with the residues residing near their N1 atoms: (A) OL_H64F mutant (gray) vs. wild-type obelin (cyan) and (B) OL_H64F mutant (gray) vs. OL_Y138F mutant (green). Hydrogen bonds are shown as dashed lines. Oxygen and nitrogen atoms are colored with red and blue, respectively. Full article ">Figure 7
Dioxetanone derivatives that might be formed in bioluminescent reaction of hydromedusan photoproteins. Full article ">Scheme 1
Proposed reaction scheme for photoprotein bioluminescence [<a href="#B47-ijms-24-06869" class="html-bibr">47</a>]. X—an intermediate state; Y*—the product, coelenteramide, in the excited state; Y—coelenteramide in the ground state. Full article ">

11 pages, 3087 KiB

Open AccessArticle

Germination and Growth of Plasma-Treated Maize Seeds Planted in Fields and Exposed to Realistic Environmental Conditions

by Nina Recek, Rok Zaplotnik, Alenka Vesel, Gregor Primc, Peter Gselman, Miran Mozetič and Matej Holc

Int. J. Mol. Sci. 2023, 24(7), 6868; https://doi.org/10.3390/ijms24076868 - 6 Apr 2023

Cited by 1 | Viewed by 1615

Abstract

In this study, we applied an inductively coupled, radio frequency oxygen plasma to maize seeds and investigated its effects on seedling emergence, plant number at tasseling, and crop yield of maize in realistic field conditions. Maize seeds of seven different hybrids were treated [...] Read more.

In this study, we applied an inductively coupled, radio frequency oxygen plasma to maize seeds and investigated its effects on seedling emergence, plant number at tasseling, and crop yield of maize in realistic field conditions. Maize seeds of seven different hybrids were treated over two harvest years. In addition to plasma-treated seeds, a control sample, fungicide-treated seeds, an eco-layer, and a plasma and eco-layer combination, were planted. Seedling emergence, plant number at tasseling (plants/m²), and yield (kg/ha), were recorded. In the first harvest year, results were negatively affected by the presence of an insect pest. In the second harvest year, plant number and yield results were more uniform. In both years, for two and three hybrids, respectively, the highest yield arose from plants from plasma-treated seeds, but the differences were only partially significant. Considering our results, plasma treatment of maize seeds appears to have a positive effect on the yield of the plant. Full article

(This article belongs to the Special Issue Advances in Molecular Plant Sciences)

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Number of maize plants in two vegetative growth stages of maize in the harvest year 2020: (a) at seedling emergence; and (b) at tasseling time. The error bars represent standard error. Different lowercase letters above data points represent statistically significant differences (p < 0.05; post hoc Tukey’s range test) between treatments. Full article ">Figure 2
Correlation between the number of emerged seedlings (May) and the number of maize plants at tasseling (August) for the harvest year 2020. Full article ">Figure 3
Number of maize plants in two vegetative growth stages of maize in the harvest year 2021: (a) at seedling emergence; and (b) at tasseling time. The error bars represent standard error. Different lowercase letters above data points represent statistically significant differences (p < 0.05; post hoc Tukey’s range test) between treatments. Full article ">Figure 4
Correlation between the number of emerged seedlings (May) and the number of maize plants at tasseling (August) for the harvest year 2021. Full article ">Figure 5
(a) Maize yield; and (b) correlation between number of plants at tasseling time and maize yield in the harvest year 2020. The error bars represent standard error. Different lowercase letters above data points represent statistically significant differences (p < 0.05; post hoc Tukey’s range test) between treatments. Full article ">Figure 6
(a) Maize yield; and (b) correlation between the number of plants at tasseling time and maize yield in the harvest year 2021. The error bars represent standard error. Different lowercase letters above data points represent statistically significant differences (p < 0.05; post hoc Tukey’s range test) between treatments. Full article ">Figure 7
Average daily temperatures and precipitation for each month in years 2020 and 2021 measured on the nearest permanent weather station. Full article ">Figure 8
Schematic representation of industrial plasma reactor used to treat maize. Full article ">

22 pages, 1529 KiB

Open AccessReview

Imbalance of Essential Metals in Traumatic Brain Injury and Its Possible Link with Disorders of Consciousness

by Rosanna Squitti, Giuseppe Reale, Vincenzo Tondolo, Daniela Crescenti, Sonia Bellini, Marco Moci, Pietro Caliandro, Luca Padua and Mauro Rongioletti

Int. J. Mol. Sci. 2023, 24(7), 6867; https://doi.org/10.3390/ijms24076867 - 6 Apr 2023

Cited by 5 | Viewed by 2798

Abstract

Dysfunction of the complex cerebral networks underlying wakefulness and awareness is responsible for Disorders of Consciousness (DoC). Traumatic Brain Injury (TBI) is a common cause of DoC, and it is responsible for a multi-dimensional pathological cascade that affects the proper functioning of the [...] Read more.

Dysfunction of the complex cerebral networks underlying wakefulness and awareness is responsible for Disorders of Consciousness (DoC). Traumatic Brain Injury (TBI) is a common cause of DoC, and it is responsible for a multi-dimensional pathological cascade that affects the proper functioning of the brainstem and brain consciousness pathways. Iron (Fe), Zinc (Zn), and Copper (Cu) have a role in the neurophysiology of both the ascending reticular activating system, a multi-neurotransmitter network located in the brainstem that is crucial for consciousness, and several brain regions. We aimed to summarize the role of these essential metals in TBI and its possible link with consciousness alterations. We found that TBI alters many neuronal molecular mechanisms involving essential metals, causing neurodegeneration, neural apoptosis, synaptic dysfunction, oxidative stress, and inflammation. This final pattern resembles that described for Alzheimer’s disease (AD) and other neurological and psychiatric diseases. Furthermore, we found that amantadine, zolpidem, and transcranial direct current stimulation (tDCS)—the most used treatments for DoC recovery—seem to have an effect on essential metals-related pathways and that Zn might be a promising new therapeutic approach. This review summarizes the neurophysiology of essential metals in the brain structures of consciousness and focuses on the mechanisms underlying their imbalance following TBI, suggesting their possible role in DoC. The scenario supports further studies aimed at getting a deeper insight into metals’ role in DoC, in order to evaluate metal-based drugs, such as metal complexes and metal chelating agents, as potential therapeutic options. Full article

(This article belongs to the Special Issue Latest Review Papers in Molecular Pathology, Diagnostics, and Therapeutics 2023)

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Figure 1
Mesocircuit—Frontoparietal Model. The diagram shows the interrelationship between the forebrain mesocircuit, the frontoparietal network, and the ascending reticular activating system. The function of each system is reported in italics. Cx: cortex. Full article ">Figure 2
Physiological and pathological neuromodulation of Zn-containing pre- and postsynaptic neurons. Most vesicular Zn co-localizes with glutamate in subsets of glutamatergic zinc-enriched neurons, and it is also contained in the synaptic vesicles of subpopulations of glycinergic and GABAergic neurons. Zn2+-level regulation between cellular compartments, organelles, and extracellular space is ensured by ZIP and ZnT protein families, and by metallothioneins (MTs), which buffer cytoplasmic Zn2+, functioning as a temporary store for cellular Zn2+. In the presynaptic terminals, Zn2+ is transported into presynaptic vesicles by the Zn transporter ZnT3. During synaptic transmission, free Zn is released in the synaptic cleft, where it may be recycled back into the presynaptic boutons by ZIP/Zn2+ transporters or modulate excitatory (NMDA, AMPA) and inhibitory (GABA, glycine) amino acid receptors of the postsynaptic terminal; Zn can inhibit GABAAR and NMDAR, and potentiate/inhibit AMPAR and GlyR at low/high concentrations, respectively. Extracellular Zn can also alter the excitability of neurons through effects on voltage-gated ion channels (e.g., VGCC), affecting ions’ influx and neurotransmitter release. Ion channels and AMPAR/KAR allow synaptically released Zn to enter presynaptic and postsynaptic neurons to modulate intracellular Zn signaling functions. Excessive Zn2+ accumulation inside postsynaptic cells, as per excitotoxic stimulation, can lead to a series of toxic effects involving mitochondrial dysfunction and ROS/NOS production, eventually leading to oxidative damage to proteins and DNA, neuronal apoptosis, and/or necrosis. Glu, glutamate; GABAAR, GABA A receptor; NMDAR, NMDA receptor; AMPAR, AMPA receptor; GlyR, glycine receptor; VGCC, voltage-gated calcium channel; KAR, kainate receptor. Full article ">Figure 3
Model of Beta amyloid (Aβ), glutamate, oxidative stress, and ionic dyshomeostasis in neurodegenerative processes associated with Traumatic Brian Injury (TBI) and Alzheimer’s disease (AD). Aβ, alpha synuclein (α-syn), and hyper-phosphorylated Tau are among the most frequently reported molecules upregulated in TBI and are also closely related to AD. Experimental models of TBI [<a href="#B58-ijms-24-06867" class="html-bibr">58</a>] show that Cu concentrations were increased in the ipsilateral cortex adjacent, but not closest, to the impact zone only 28 days after the injury, and it may be cause for concern in relation to the potential chronic oxidative stress toxicity based on Cu abnormal metabolism, as has been observed in neurodegenerative disorders and more specifically for AD. The model proposed to highlight the main Cu toxic mechanisms that can be triggered by TBI in the long term and in AD. In a complex scenario, Aβ, oxidative stress, excitotoxicity, and Cu2+ dyshomeostasis act in concert to promote synaptic dysfunction and neuronal loss. Upon the production of excessive glutamate levels (1), Ca2+ ions enter the cell through the NMDA receptor (2) and (3) induce Cu-ATPase7A/B (ATP7A/B) translocation at synapses where vesicular Cu is released in the synaptic cleft. The released Cu2+ (in concentrations up to 100 µmol/L) may inhibit the NMDA receptor, thereby protecting neurons from glutamatergic excitotoxicity (4), or catalyze Fenton-type and Haber–Weiss reactions, thereby generating reactive oxygen species (ROS) (5). Enhanced ROS generation can damage proteins, lipids, and nucleic acids, eventually leading to cell death (5). Ca2+ overload can increase superoxide anion (O2•−) production from mitochondria (6), and nitric oxide (NO) generation via Ca2+-dependent activation of NO synthase (NOS) (7). Reactive oxygen and nitrosative (RNS) species mobilize Cu2+ from metallothionein 3 (MT-3) (8), leading to increased intracellular toxic Cu2+ concentrations (9) and promoting mitochondrial dysfunction, as well as release of pro-apoptotic factors (10). ROS-driven Cu2+ mobilization can further aggravate oxidative stress and initiate Aβ oligomerization (11). Altered trafficking of APP and/or elevated Aβ oligomer secretion can generate an intracellular Cu2+ deficiency, thereby causing oxidative stress by the loss of SOD-1 function (12). Aβ, α-synuclein, and PrP increased after TBI can modulate neurotransmission as [Cu2+] buffers within the synaptic cleft or amplify the vicious cycle by increasing oxidative stress (13). Furthermore, glutamate-driven mitochondrial Ca2+ overload can mobilize Cu2+ from these organelles (14). Excess Non-Cp Cu in the bloodstream is a source for the buildup of labile Cu2+ into the intermembrane space of mitochondria (15), promoting the ATPase7A/B translocation of Cu2+ into vesicles of the trans-Golgi network and endoplasmic reticulum (ER) (16). These processes, working intracellularly at the level of synaptic spines, in the synaptic cleft, and in the neurovascular unit (ref. [<a href="#B8-ijms-24-06867" class="html-bibr">8</a>]), can facilitate synaptic dysfunction, neuronal deafferentation, and ultimately brain cell death. Full article ">

16 pages, 1234 KiB

Open AccessArticle

The Nitric Oxide (NO) Donor Molsidomine Counteract Social Withdrawal and Cognition Deficits Induced by Blockade of the NMDA Receptor in the Rat

by Lamprini Katsanou, Evangelia Fragkiadaki, Sotirios Kampouris, Anastasia Konstanta, Aikaterini Vontzou and Nikolaos Pitsikas

Int. J. Mol. Sci. 2023, 24(7), 6866; https://doi.org/10.3390/ijms24076866 - 6 Apr 2023

Cited by 2 | Viewed by 1865

Abstract

The deficiency of the gaseous molecule nitric oxide (NO) seems to be critically involved in the pathogenesis of schizophrenia. Thus, molecules that can normalize NO levels, as are NO donors, might be of utility for the medication of this psychiatric disease. The aim [...] Read more.

The deficiency of the gaseous molecule nitric oxide (NO) seems to be critically involved in the pathogenesis of schizophrenia. Thus, molecules that can normalize NO levels, as are NO donors, might be of utility for the medication of this psychiatric disease. The aim of the present study was to detect the ability of the NO donor molsidomine to reduce schizophrenia-like impairments produced by the blockade of the N-methyl-D-aspartate (NMDA) receptor in rats. Molsidomine’s ability to attenuate social withdrawal and spatial recognition memory deficits induced by the NMDA receptor antagonist ketamine were assessed using the social interaction and the object location test, respectively. Further, the efficacy of the combination of sub-effective doses of molsidomine with sub-effective doses of the atypical antipsychotic clozapine in alleviating non-spatial recognition memory deficits was evaluated utilizing the object recognition task. Molsidomine (2 and 4 mg/kg) attenuated social withdrawal and spatial recognition memory deficits induced by ketamine. Co-administration of inactive doses of molsidomine (1 mg/kg) and clozapine (0.1 mg/kg) counteracted delay-dependent and ketamine-induced non-spatial recognition memory deficits. The current findings suggest that molsidomine is sensitive to glutamate hypofunction since it attenuated behavioral impairments in animal models mimicking the negative symptoms and cognitive deficits of schizophrenia. Additionally, the present results support the potential of molsidomine as an adjunctive drug for the therapy of schizophrenia. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Schizophrenia and Novel Targets 2.0)

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Figure 1
Social interaction test. The histogram represents the mean ± S.E.M of 8 pairs of rats per treatment group. (A) Social interaction levels expressed by different groups of rats. * p < 0.05 vs. all the other groups; + p < 0.05 vs. the vehicle + vehicle, vehicle + molsidomine 2 mg/kg and vehicle + molsidomine 4 mg/kg groups. (B) Locomotor activity expressed by different groups of rats during the social interaction test. Full article ">Figure 2
Object location task. The histogram represents the mean ± S.E.M of 8 rats per treatment group. The 1-h ITI was used. (A) Discrimination index D performance expressed by different groups of rats during T2. * p < 0.05 vs. all the other groups. (B) Total exploration times. Full article ">Figure 3
Object recognition task. The histograms represent the mean ± S.E.M of 8 rats per treatment group. The 24-h ITI was used. (A) Discrimination index D performance expressed by different groups of rats during T2. * p < 0.05 vs. all the other groups. (B) Total exploration times. Full article ">Figure 4
Object recognition task. The histograms represent the mean ± S.E.M of 8 rats per treatment group. The 1-h ITI was used. (A) Discrimination index D performance expressed by different groups of rats during T2. * p < 0.05 vs. all the other groups. (B) Total exploration times. Full article ">

26 pages, 6225 KiB

Open AccessArticle

Novel Insights into circRNA Saga Coming from Spermatozoa and Epididymis of HFD Mice

by Francesco Manfrevola, Teresa Chioccarelli, Vincenza Grazia Mele, Veronica Porreca, Monica Mattia, Donatella Cimini, Antonella D’Agostino, Gilda Cobellis, Silvia Fasano, Chiara Schiraldi, Rosanna Chianese and Riccardo Pierantoni

Int. J. Mol. Sci. 2023, 24(7), 6865; https://doi.org/10.3390/ijms24076865 - 6 Apr 2023

Cited by 4 | Viewed by 2178

Abstract

Obesity is a pathophysiological disorder associated with adiposity accumulation, oxidative stress, and chronic inflammation state that is progressively increasing in younger population worldwide, negatively affecting male reproductive skills. An emerging topic in the field of male reproduction is circRNAs, covalently closed RNA molecules [...] Read more.

Obesity is a pathophysiological disorder associated with adiposity accumulation, oxidative stress, and chronic inflammation state that is progressively increasing in younger population worldwide, negatively affecting male reproductive skills. An emerging topic in the field of male reproduction is circRNAs, covalently closed RNA molecules produced by backsplicing, actively involved in a successful spermatogenesis and in establishing high-quality sperm parameters. However, a direct correlation between obesity and impaired circRNA cargo in spermatozoa (SPZ) remains unclear. In the current work, using C57BL6/J male mice fed with a high-fat diet (HFD, 60% fat) as experimental model of oxidative stress, we investigated the impact of HFD on sperm morphology and motility as well as on spermatic circRNAs. We performed a complete dataset of spermatic circRNA content by a microarray strategy, and differentially expressed (DE)-circRNAs were identified. Using a circRNA/miRNA/target network (ceRNET) analysis, we identified circRNAs potentially involved in oxidative stress and sperm motility pathways. Interestingly, we demonstrated an enhanced skill of HFD sperm in backsplicing activity together with an inefficient epididymal circRNA biogenesis. Fused protein in sarcoma (FUS) and its ability to recruit quaking (QKI) could be involved in orchestrating such mechanism. Full article

(This article belongs to the Special Issue Novel Insights into the Biology of Spermatozoa 2.0)

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Figure 1
Characterization of HFD testis and sperm parameters. (A,B) H&E staining of testis (A) and cauda SPZ (B) collected from CTRL (n = 6) and HFD (n = 6) mice. (A) In testis sections, germ cell lumen infiltration was indicated by black arrowheads; scale bar: 100 μm. (B) Anomalous sperm heads were indicated by black arrowheads; scale bar: 50 μm; scale bar inset: 10 μm. (C) Percentage of anomalous sperm heads in CTRL and HFD cauda SPZ; data were reported as the percentage of anomalous sperm heads/total SPZ. Sperm viability (D) and motility (E) assay in CTRL and HFD cauda SPZ; data were expressed as the percentage of nonviable/total SPZ and motile/live SPZ, respectively, and reported as mean ± SEM; ** p < 0.01. Full article ">Figure 2
Overview of circRNA expression in mouse SPZ. (A) The distribution of up- and downregulated circRNAs in HFD (n = 3) compared with CTRL (n = 3) SPZ among a total of 9838 circRNAs. (B) The proportion of different types of circRNAs among all predicted circRNAs. (C) Chromosomal distribution of SPZ-derived circRNAs on strand + and strand –, according to their host gene location. Full article ">Figure 3
Differential expression of circRNAs between CTRL and HFD SPZ. (A) The distribution of up- and downregulated DE-circRNAs, among a total of 109 DE-circRNAs, in HFD compared with CTRL SPZ. (B) Hierarchical clustering analysis of DE-circRNAs in CTRL SPZ (samples CTRL-1, CTRL-2, CTRL-3) and HFD SPZ (samples HFD-1, HFD-2, HFD-3); the expression values (fold change > 1.5, p < 0.05) were represented in different colors, indicating expression levels above and below the median expression level across all samples. (C) The volcano plot was constructed using fold change and p-values; in detail, the values on X and Y axes are log2 (FC = fold change) and –log10 (p-values), respectively. Red points in the volcano plot represent the DE-circRNAs with statistical significance. (D) The distribution of up- and downregulated DE-circRNAs in the mouse genome, according to their host gene location. Full article ">Figure 4
Validation of circRNA microarray results in testis, epididymis, and SPZ from CTRL and HFD mice. (A) Expression analysis of 9 circRNAs upregulated in HFD compared with CTRL-derived SPZ. (B) Expression analysis of 9 circRNAs downregulated in HFD compared with CTRL-derived SPZ. qRT-PCR data are normalized using cyclophilin and ribosomal protein S18 (RPS18) for SPZ and tissues, respectively, expressed as fold expression (nfe) and reported as mean value ± S.E.M. **: p < 0.01; *: p < 0.05. Full article ">Figure 5
Characterization of backsplicing machinery in SPZ and epididymis of CTRL and HFD mice. (A) Immunofluorescence analysis of FUS protein in caput and cauda SPZ of CTRL and HFD mice. White arrowheads and white asterisks represent FUS localization (RED) in sperm head and tail, respectively. Nuclei were labeled with DAPI (blue). Scale bar: 5 µm. (B) Western blot analysis of FUS protein (75 KDa) in caput and cauda SPZ of CTRL and HFD mice (n = 5 different samples for each experimental group in triplicate). Signals were quantified by densitometry analysis and normalized to Ponceau Red (Pon.S). Data were expressed in OD values and reported as mean ± SEM. Experimental groups with statistically significant differences (p < 0.01) were indicated with different letters. (C) The enrichment levels of circADAM10 in the products of RIP assay (FUS-IP compared with IgG-IP) in caput and cauda SPZ of CTRL and HFD mice detected by qRT-PCR. Data are reported as mean ± SEM from three independent experiments. ** p < 0.01. (D) Immunocytochemistry of FUS in Bouin’s fixed caput and cauda epididymis sections (7 μm) of CTRL and HFD mice (n = 5 different samples for each experimental group in triplicate). The FUS protein localization in principal cells was indicated by black arrows. Scale bar: 50 μm; inset: H&E staining of Bouin’s fixed caput and cauda epididymis sections (7 μm) of CTRL and HFD mice; scale bar: 20 μm. (E) Western blot analysis of FUS protein in caput and cauda epididymis of CTRL and HFD mice. Signals were quantified by densitometry analysis and normalized to Ponceau Red (Pon.S). Data were expressed in OD values and reported as mean ± SEM. Experimental groups with statistically significant differences (p < 0.01) were indicated with different letters. (F) The enrichment levels of circPCSK6 in the products of RIP assay (FUS-IP compared with IgG-IP) in caput and cauda epididymis of CTRL and HFD mice detected by qRT-PCR. Data are reported as mean ± SEM from three independent experiments. ** p < 0.01. (G) Western blot analysis of QKI protein (40 KDa) in caput and cauda epididymis of CTRL and HFD mice. Signals were quantified by densitometry analysis and normalized to Ponceau Red (Pon.S). Data were expressed in OD values and reported as mean ± SEM. Experimental groups with statistically significant differences (p < 0.01) were indicated with different letters. (H) IP in caput and cauda epididymis of CTRL and HFD mice. Total proteins collected were immunoprecipitated using FUS antibody. Protein interaction between FUS and QKI was detected by Western blot analysis. Full article ">Figure 6
In vitro experiment of vesicle shuttle: from CTRL epididymis to HFD SPZ. (A) CeRNET of one circRNA downregulated in HFD SPZ. CircSMAD2 tethers a group of miRNAs as targets, all involved in sperm motility pathways. Networks were built using Cytoscape. Hexagonal and rectangular symbols represent circRNAs and miRNAs, respectively. The arrow indicates the tethering activity of circRNAs toward miRNAs, while the dotted arrow indicates the pathways upstream of the miRNAs. (B,C) qRT-PCR analysis of circSMAD2 (B) and PLAG1-mRNA (C) in CTRL cauda SPZ and HFD cauda SPZ in vitro coincubated with: PBS, CTRL-ELF, and CTRL-ELF pretreated with anti-CD9 antibody (n = 6 for each experimental group). qRT-PCR data were normalized using cyclophilin, expressed as fold expression (nfe) relative to CTRL SPZ and reported as mean value ± S.E.M. Experimental groups with statistically significant differences (p < 0.01) were indicated with different letters. (D) Sperm motility assay in CTRL cauda SPZ and HFD cauda SPZ in vitro coincubated with: PBS, CTRL-ELF, and CTRL-ELF pretreated with anti-CD9 antibody (n = 6 for each experimental group); data were expressed as the percentage of motile/live SPZ and reported as mean ± SEM. Experimental groups with statistically significant differences (p < 0.01) were indicated with different letters. Full article ">Figure 7
Schematic representation of experimental design. Full article ">

12 pages, 1994 KiB

Open AccessArticle

Conformational Changes of Acyl Carrier Protein Switch the Chain Length Preference of Acyl-ACP Thioesterase ChFatB2

by Tianxiang Yang, Yunlong Yang, Ming Yang, Jiangang Ren, Changying Xue, Yanbin Feng and Song Xue

Int. J. Mol. Sci. 2023, 24(7), 6864; https://doi.org/10.3390/ijms24076864 - 6 Apr 2023

Viewed by 1987

Abstract

Microbial fatty acids are synthesized by Type II fatty acid synthase and could be tailored by acyl-ACP thioesterase. With the prospects of medium-chain fatty-acid-derivative biofuels, the selectivity of thioesterase has been studied to control the fatty acid product chain length. Here, we report [...] Read more.

Microbial fatty acids are synthesized by Type II fatty acid synthase and could be tailored by acyl-ACP thioesterase. With the prospects of medium-chain fatty-acid-derivative biofuels, the selectivity of thioesterase has been studied to control the fatty acid product chain length. Here, we report an alternative approach by manipulating the acyl carrier protein portion of acyl-ACP to switch the chain length propensity of the thioesterase. It was demonstrated that ChFatB2 from Cuphea hookeriana preferred C10-ACP to C8-ACP with ACP from E. coli, while converting preference to C8-ACP with ACP from Cuphea lanceolate. Circular dichroism (CD) results indicated that the C8-EcACP encountered a 34.4% α-helix increment compared to C10-EcACP, which resulted in an approximate binding affinity decrease in ChFatB2 compared to C10-EcACP. Similarly, the C10-ClACP2 suffered a 45% decrease in helix content compared to C8–ClACP2, and the conformational changes resulted in an 18% binding affinity decline with ChFatB2 compared with C10-ClACP2. In brief, the study demonstrates that the ACP portion of acyl-ACP contributes to the selectivity of acyl-ACP thioesterase, and the conformational changes of EcACP and ClACP2 switch the chain length preference of ChFatB2 between C8 and C10. The result provides fundamentals for the directed synthesis of medium-chain fatty acids based on regulating the conformational changes of ACPs. Full article

(This article belongs to the Section Biochemistry)

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Comparisons of the structures and sequences of ACPs. (A) Structure overlay of EcACP by apo form (PDB entry 1T8K, colored gray) and C7-acylation form (PDB entry 2FAD, colored cyan). (B) Structure overlay of EcACP by C7-acylation form and C10-acylation form (PDB entry 2FAE, colored purple). (C) Structure overlay of SoACP by C10-acylation form (PDB entry 2FVF colored salmon) and C18:0-acylation form (PDB entry 2FVA, colored yellow). (D) Overlay of SoACP (PDB entry 2FVE, colored green) and ClACP2 (colored pink). (E) Overlay of EcACP by apo form and ClACP2. (F) Sequence comparison of EcACP, SoACP, and ClACP2. The red boxes show the 100% conserved residues; residues having similar properties are shown in blue boxes. Full article ">Figure 2
Relative activity of ChFatB2 with acyl-ACPs. (A) Relative activity of ChFatB2 with C8-EcACP and C8-ClACP2 as the substrate. (B) Relative activity of ChFatB2 with C10-EcACP and C10-ClACP2 as the substrate. Full article ">Figure 3
Circular dichroism spectra of the ACPs (A) holo-, C8-, and C10-EcACP from 190 to 260 nm wavelength. (B) holo-, C8-, and C10-ClACP2 from 190 to 260 nm wavelength. The mean residue ellipticity (θ) in degrees cm2 dmol−1. Full article ">Figure 4
Melting temperature curves of ACP. (A) The thermal-induced melting temperature curves of holo-, C8- and C10-EcACP. (B) The thermal-induced melting temperature curves of holo-, C8- and C10-ClACP2. Full article ">Figure 5
Binding affinity of ChFatB2 with the ACPs determined by liquid crystals assay. (A,B) show the optical micrographs of a thin layer of liquid crystal under polarized microscope. The liquid crystals were sandwiched between a DMOAP-coated glass slide and a DMOAP-coated glass slide with circular domain of immobilized ChFatB2 and EC-holo-ACP, C8-, and C10-EcACP (A), and immobilized ChFatB2 and holo-ClACP2, C8-ClACP2, and C10-ClACP2 (B). (C,D) are the grayscale values of the optical images of (A,B) obtained by Image J software. The analysis was repeated three times (#1, #2, and #3). Full article ">

13 pages, 982 KiB

Open AccessCommunication

Redox Proteomic Profile of Tirapazamine-Resistant Murine Hepatoma Cells

by Aušra Nemeikaitė-Čėnienė, Per Haberkant, Dalius Kučiauskas, Frank Stein and Narimantas Čėnas

Int. J. Mol. Sci. 2023, 24(7), 6863; https://doi.org/10.3390/ijms24076863 - 6 Apr 2023

Viewed by 2051

Abstract

3-Amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine, TPZ) and other heteroaromatic N-oxides (ArN→O) exhibit tumoricidal, antibacterial, and antiprotozoal activities. Their action is attributed to the enzymatic single-electron reduction to free radicals that initiate the prooxidant processes. In order to clarify the mechanisms of aerobic mammalian cytotoxicity of [...] Read more.

3-Amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine, TPZ) and other heteroaromatic N-oxides (ArN→O) exhibit tumoricidal, antibacterial, and antiprotozoal activities. Their action is attributed to the enzymatic single-electron reduction to free radicals that initiate the prooxidant processes. In order to clarify the mechanisms of aerobic mammalian cytotoxicity of ArN→O, we derived a TPZ-resistant subline of murine hepatoma MH22a cells (resistance index, 5.64). The quantitative proteomic of wild-type and TPZ-resistant cells revealed 5818 proteins, of which 237 were up- and 184 down-regulated. The expression of the antioxidant enzymes aldehyde- and alcohol dehydrogenases, carbonyl reductases, catalase, and glutathione reductase was increased 1.6–5.2 times, whereas the changes in the expression of glutathione peroxidase, superoxide dismutase, thioredoxin reductase, and peroxiredoxins were less pronounced. The expression of xenobiotics conjugating glutathione-S-transferases was increased by 1.6–2.6 times. On the other hand, the expression of NADPH:cytochrome P450 reductase was responsible for the single-electron reduction in TPZ and for the 2.1-fold decrease. These data support the fact that the main mechanism of action of TPZ under aerobic conditions is oxidative stress. The unchanged expression of intranuclear antioxidant proteins peroxiredoxin, glutaredoxin, and glutathione peroxidase, and a modest increase in the expression of DNA damage repair proteins, tend to support non-site-specific but not intranuclear oxidative stress as a main factor of TPZ aerobic cytotoxicity. Full article

(This article belongs to the Section Biochemistry)

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13 pages, 2000 KiB

Open AccessBrief Report

Doxorubicin–Mediated miR–433 Expression on Exosomes Promotes Bystander Senescence in Multiple Myeloma Cells in a DDR–Independent Manner

by Elisabetta Vulpis, Lorenzo Cuollo, Cristiana Borrelli, Fabrizio Antonangeli, Laura Masuelli, Marco Cippitelli, Cinzia Fionda, Giulio Caracciolo, Maria Teresa Petrucci, Angela Santoni, Alessandra Zingoni and Alessandra Soriani

Int. J. Mol. Sci. 2023, 24(7), 6862; https://doi.org/10.3390/ijms24076862 - 6 Apr 2023

Cited by 4 | Viewed by 2148

Abstract

The success of senescence-based anticancer therapies relies on their anti-proliferative power and on their ability to trigger anti-tumor immune responses. Indeed, genotoxic drug-induced senescence increases the expression of NK cell-activating ligands on multiple myeloma (MM) cells, boosting NK cell recognition and effector functions. [...] Read more.

The success of senescence-based anticancer therapies relies on their anti-proliferative power and on their ability to trigger anti-tumor immune responses. Indeed, genotoxic drug-induced senescence increases the expression of NK cell-activating ligands on multiple myeloma (MM) cells, boosting NK cell recognition and effector functions. Senescent cells undergo morphological change and context-dependent functional diversification, acquiring the ability to secrete a vast pool of molecules termed the senescence-associated secretory phenotype (SASP), which affects neighboring cells. Recently, exosomes have been recognized as SASP factors, contributing to modulating a variety of cell functions. In particular, evidence suggests a key role for exosomal microRNAs in influencing many hallmarks of cancer. Herein, we demonstrate that doxorubicin treatment of MM cells leads to the enrichment of miR-433 into exosomes, which in turn induces bystander senescence. Our analysis reveals that the establishment of the senescent phenotype on neighboring MM cells is p53- and p21-independent and is related to CDK-6 down-regulation. Notably, miR-433-dependent senescence does not induce the up-regulation of activating ligands on MM cells. Altogether, our findings highlight the possibility of miR-433-enriched exosomes to reinforce doxorubicin-mediated cellular senescence. Full article

(This article belongs to the Special Issue Cellular Senescence in Physiological and Pathological Processes 2.0)

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DOX-treated SKO-007(J3) multiple myeloma cells release exosomes able to induce a senescence phenotype. (A) SKO-007(J3) cells were incubated with doxorubicin (DOX) (0.05 μM) for 48 h and left for an additional 24 h in the absence of the drug. Exosomes were then isolated from the supernatants and characterized by transmission electron microscopy. A representative picture of SKO-007(J3)-derived exosomes is shown. Scale bar, 100 nm. (B) A representative experiment of exosome size distribution by Dynamic Light Scattering (DLS) m (nm scale) is shown. (C) Exosomes released by untreated and DOX-treated SKO-007(J3) were quantified by Bradford assay, normalized for 1 million cells and plotted as fold increase between untreated and DOX-treated cells. The mean of five independent experiments is shown. (D) SKO-007(J3) cells were incubated with exosomes (20 μg/mL) derived from untreated, DOX-treated and MEL-treated SKO-007(J3) cells. After 5 days, cells were washed and then incubated 1 h with bafilomycin A1 (100 nM) to induce lysosomal alkalinization, followed by 1 h incubation with C12FDG (33 μM). SA-β-galhigh senescent cells were analyzed by flow cytometry. Data are expressed as a fold increase in respect to untreated control cells and are the result of five different experiments. (E) MM cells were treated with a higher number of exosomes (50 μg/mL) for 24 h and analyzed by flow cytometry for the presence of senescent cells. A representative experiment is shown. (F) In parallel, SKO-007(J3) cells were fixed and incubated overnight at 37 °C without CO2 with SA-β-gal stain solution (“Senescence-associated β-galactosidase staining”). Senescent cells were identified as blue-stained cells and counted using microscopy. Magnification 200×. Results are representative of one of two independent experiments. (G) SKO-007(J3) cells were left untreated (T0) or treated for 48 h (T48) with exo-NT and exo-DOX, live cells were then counted by Trypan Blue staining using light microscopy. (H) SKO-007(J3) cells were treated with exo-NT and exo-DOX (50 μg/mL) for 24 and 48 h; the percentage of dead cells was measured by flow cytometry using Zombie Green. SA-β-gal activity was measured at 48 h to confirm the induction of senescence in the same experiment. A representative experiment is shown. Statistical analysis was performed with Student’s t-test (* p < 0.05, unpaired t-test, ** p < 0.01, unpaired t-test). Full article ">Figure 2
miRNA profiling of MM cell-derived exosomes and parental SKO-007(J3) cells. Total RNA was isolated from both SKO–007(J3) cells and SKO-007(J3)-derived exosomes and analyzed for microRNA expression profiling using Megaplex Pool cards A and B, as described in Materials and Methods. The Venn diagram was created using an online program (<a href="http://www.bioinformatics.lu/venn.php" target="_blank">http://www.bioinformatics.lu/venn.php</a>, accessed on 1 April 2023), and the data are reported in the table. In the left column of the table, the miRNAs expressed only in SKO-007(J3) cells are shown, the central column displays the miRNAs present in both SKO-007(J3) cells and SKO-007(J3)-derived exosomes and the right column shows those expressed only in SKO-007(J3)-derived exosomes. The results of the representative experiments are shown. Full article ">Figure 3
Exosomes derived from DOX-treated MM cells carry miR-433. (A) SKO-007(J3) cells were left untreated or treated with DOX and MEL, as described above. The expression of mir-433 was evaluated on both SKO-007(J3)-derived exosomes and SKO-007(J3) cells by real-time PCR. U6 and miR-48 were used to normalize miR-433 expression. (B) SKO-007(J3) cells were treated as described above and the expression level of miR-433 on the exosomes was verified by real-time PCR. The average of four different experiments is shown. Statistical analysis was performed using Student’s t-test (** p < 0.01, unpaired t-test, *** p < 0.001, unpaired t-test). (C) Bone marrow mononuclear cells (BMMCs) derived from 4 MM patients were left untreated or treated with DOX (0.05 μM) for 48 h. RNA was extracted from the exosomes (isolated from supernatants) and analyzed for the expression of miR-433 by real-time PCR (normalized with U6). Full article ">Figure 4
Exosomal transfer of miR-433 can promote a senescent phenotype through CDK6 down-modulation on MM cells. (A) Lentiviral-vectors overexpressing miR-433 or miR-CTR fused in-frame with mCherry were used to infect SKO-007(J3) cells. Cells were washed with PBS and visualized by fluorescence microscopy to determine the presence of mCherry-positive cells. (B) The expression of mir-433 was evaluated on infected SKO-007(J3) cells and their derived exosomes by real-time PCR 6 days post-infection. A representative experiment is shown. (C) Exosomes isolated from SKO-007(J3) cells infected with the lentiviral constructs were incubated with MM cells; SA-β-gal activity on MM cells was measured using flow cytometry upon 48 h of incubation. (D) The average of three different experiments is shown (incubation time of 6 days). Statistical analysis was performed with Student’s t-test (* p < 0.05, unpaired t-test). (E) Western blot analysis of p53, phospho-p53 (Ser15), p21 and γH2AX in SKO-007(J3) cells treated with exo-miR-CTR and exo-miR-433 for 48 and 72 h. p85 was used as protein loading control. A representative Western blot is shown. Irrelevant lanes were removed between lanes (dotted line). (F) SKO-007(J3) cells were treated with exo-miR-CTR and exo-miR-433 for the indicated times. Cell lysates were immunoblotted with anti-CDK6 or with anti-p85, used as loading control. A representative Western blot is shown. Irrelevant lanes were removed between lanes (dotted line). Full article ">

19 pages, 5011 KiB

Open AccessReview

Recent Advances in Manganese-Based Materials for Electrolytic Water Splitting

by Jing Hu, Yuru Zhou, Yinan Liu, Zhichao Xu and Haijin Li

Int. J. Mol. Sci. 2023, 24(7), 6861; https://doi.org/10.3390/ijms24076861 - 6 Apr 2023

Cited by 7 | Viewed by 3530

Abstract

Developing earth-abundant and highly effective electrocatalysts for electrocatalytic water splitting is a prerequisite for the upcoming hydrogen energy society. Recently, manganese-based materials have been one of the most promising candidates to replace noble metal catalysts due to their natural abundance, low cost, adjustable [...] Read more.

Developing earth-abundant and highly effective electrocatalysts for electrocatalytic water splitting is a prerequisite for the upcoming hydrogen energy society. Recently, manganese-based materials have been one of the most promising candidates to replace noble metal catalysts due to their natural abundance, low cost, adjustable electronic properties, and excellent chemical stability. Although some achievements have been made in the past decades, their performance is still far lower than that of Pt. Therefore, further research is needed to improve the performance of manganese-based catalytic materials. In this review, we summarize the research progress on the application of manganese-based materials as catalysts for electrolytic water splitting. We first introduce the mechanism of electrocatalytic water decomposition using a manganese-based electrocatalyst. We then thoroughly discuss the optimization strategy used to enhance the catalytic activity of manganese-based electrocatalysts, including doping and defect engineering, interface engineering, and phase engineering. Finally, we present several future design opportunities for highly efficient manganese-based electrocatalysts. Full article

(This article belongs to the Special Issue Electrochemical Behavior and Application of Advanced Electrode Materials)

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15 pages, 3122 KiB

Open AccessArticle

Electrochemical Behavior of Reduced Graphene Oxide Supported Germanium Oxide, Germanium Nitride, and Germanium Phosphide as Lithium-Ion Battery Anodes Obtained from Highly Soluble Germanium Oxide

by Alexey A. Mikhaylov, Alexander G. Medvedev, Dmitry A. Grishanov, Timur M. Fazliev, Vasilii Chernyshev, Elena A. Mel’nik, Tatiana A. Tripol’skaya, Ovadia Lev and Petr V. Prikhodchenko

Int. J. Mol. Sci. 2023, 24(7), 6860; https://doi.org/10.3390/ijms24076860 - 6 Apr 2023

Cited by 5 | Viewed by 2546

Abstract

Germanium and germanium-based compounds are widely used in microelectronics, optics, solar cells, and sensors. Recently, germanium and its oxides, nitrides, and phosphides have been studied as active electrode materials in lithium- and sodium-ion battery anodes. Herein, the newly introduced highly soluble germanium oxide [...] Read more.

Germanium and germanium-based compounds are widely used in microelectronics, optics, solar cells, and sensors. Recently, germanium and its oxides, nitrides, and phosphides have been studied as active electrode materials in lithium- and sodium-ion battery anodes. Herein, the newly introduced highly soluble germanium oxide (HSGO) was used as a versatile precursor for germanium-based functional materials. In the first stage, a germanium-dioxide-reduced graphene oxide (rGO) composite was obtained by complete precipitation of GeO₂ nanoparticles on the GO from an aqueous solution of HSGO and subsequent thermal treatment in argon at low temperature. The composition of the composite, GeO₂-rGO (20 to 80 wt.% of crystalline phase), was able to be accurately determined by the HSGO to GO ratio in the initial solution since complete deposition and precipitation were achieved. The chemical activity of germanium dioxide nanoparticles deposited on reduced graphene oxide was shown by conversion to rGO-supported germanium nitride and phosphide phases. The GeP-rGO and Ge₃N₄-rGO composites with different morphologies were prepared in this study for the first time. As a test case, composite materials with different loadings of GeO₂, GeP, and Ge₃N₄ were evaluated as lithium-ion battery anodes. Reversible conversion–alloying was demonstrated in all cases, and for the low-germanium loading range (20 wt.%), almost theoretical charge capacity based on the germanium content was attained at 100 mA g⁻¹ (i.e., 2595 vs. 2465 mAh g⁻¹ for Ge₃N₄ and 1790 vs. 1850 mAh g⁻¹ for GeP). The germanium oxide was less efficiently exploited due to its lower conversion reversibility. Full article

(This article belongs to the Special Issue State-of-the-Art Materials Science in Russia—2nd Edition)

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Powder X-ray diffractograms of GeO2-rGO-20 (a), GeO2-rGO-50 (b), GeO2-rGO-80 (c), Ge3N4-rGO-20 (d), Ge3N4-rGO-50 (e), Ge3N4-rGO-80 (f), GeP-rGO-20 (g), GeP-rGO-50 (h), and GeP-rGO-80 (i) powders. Vertical bars below represent the standard diffraction data for corresponding crystalline germanium compounds. Full article ">Figure 2
SEM images of GeO2-rGO-80 (a,b), Ge3N4-rGO-80 (c,d), and GeP-rGO-80 (e,f). Full article ">Figure 3
Cycling voltammetry of GeO2-rGO-80 (a), Ge3N4-rGO-80 (b), and GeP-rGO-80 (c) at 0.1 mV s−1. First and second charge−discharge curves for GeO2-rGO-80 (d), Ge3N4-rGO-80 (e), and GeP-rGO-80 (f) at 100 mA g−1. Full article ">Figure 4
Cycling voltammetry of GeO2-rGO-80 (a), Ge3N4-rGO-80 (b), and GeP-rGO-80 (c) at 0.1 mV s−1. First and second charge−discharge curves for GeO2-rGO-80 (d), Ge3N4-rGO-80 (e), and GeP-rGO-80 (f) at 100 mA g−1. Full article ">

13 pages, 5244 KiB

Open AccessArticle

Folic Acid Ameliorates Renal Injury in Experimental Obstructive Nephropathy: Role of Glycine N-Methyltransferase

by Ko-Lin Kuo, Chin-Wei Chiang, Yi-Ming Arthur Chen, Chih-Chin Yu and Tzong-Shyuan Lee

Int. J. Mol. Sci. 2023, 24(7), 6859; https://doi.org/10.3390/ijms24076859 - 6 Apr 2023

Cited by 1 | Viewed by 2267

Abstract

Folic acid exerts both anti-inflammatory and antifibrotic effects. Glycine N-methyltransferase (GNMT), the major folic acid-binding protein in the liver, is a crucial enzyme that regulates the cellular methylation process by maintaining S-adenosylmethionine levels. However, as yet neither the therapeutic effects of folic acid [...] Read more.

Folic acid exerts both anti-inflammatory and antifibrotic effects. Glycine N-methyltransferase (GNMT), the major folic acid-binding protein in the liver, is a crucial enzyme that regulates the cellular methylation process by maintaining S-adenosylmethionine levels. However, as yet neither the therapeutic effects of folic acid in renal fibrosis nor whether GNMT is involved in these folic acid-associated mechanisms has been investigated. First, the expression of GNMT was examined in human kidneys with or without obstructive nephropathy. Later, wild-type and GNMT knockout (GNMT^−/−) mice were subjected to unilateral ureteral obstruction (UUO) and then treated with either folic acid or vehicle for 14 days. Renal tubular injury, inflammation, fibrosis, and autophagy were evaluated by histological analysis and Western blotting. We observed increased expression of GNMT in humans with obstructive nephropathy. Furthermore, UUO significantly increased the expression of GNMT in mice; in addition, it caused renal injury as well as the development of both hydronephrosis and tubular injury. These were all alleviated by folic acid treatment. In contrast, GNMT^−/− mice exhibited exacerbated UUO-induced renal injury, but the protective effect of folic acid was not observed in GNMT^−/− mice. We propose a novel role for folic acid in the treatment of renal fibrosis, which indicates that GNMT may be a therapeutic target. Full article

(This article belongs to the Special Issue New Insights into the Molecular Mechanisms of Kidney Injury and Repair)

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GNMT expression is upregulated in patients with obstructive nephropathy. The expression of GNMT in the renal interstitium of kidney tissues was determined by immunohistochemistry. The data are presented as means ± standard errors. * p < 0.05 versus patients without obstructive nephropathy. Full article ">Figure 2
UUO upregulated GNMT expression in wild-type mice. (A) GNMT level in WT mice 3, 7 and 14 days after undergoing UUO surgery. The protein level of GNMT was analyzed by Western blotting. GAPDH was used as a loading control. (B) WT mice were observed for 14 days after undergoing UUO surgery. The protein expression of GNMT was measured by immunofluorescence. Neg: negative control. Magnification: 200×. Scale bar: 50 μm. The data are presented as means ± standard errors. * p < 0.05 versus the sham group. Full article ">Figure 3
Effect of folic acid on UUO-induced hydronephrosis in WT and GNMT−/− mice. (A) WT and GNMT−/− mice underwent UUO and received folic acid for 14 days. At the end of the experiment, mice were euthanized by CO2 and the kidneys were then photographed. (B) Both sham and UUO-treated kidneys were harvested and weighed. The development of hydronephrosis was evaluated via normalization to body weight. The data are presented as means ± standard errors. * p < 0.05 versus the sham group, # p < 0.05 versus non-folic acid-treated mice, $ p < 0.05 versus WT mice. Full article ">Figure 4
Effect of folic acid on UUO-induced renal tubular injury, leukocyte infiltration, and interstitial fibrosis in WT and GNMT−/− mice. Paraffin-embedded kidney specimens were cut into 8 µm sections for histological examination. Magnification: 200×. Scale bar: 50 µm. (A) Representative hematoxylin and eosin staining of renal tubular injury and leukocyte infiltration of kidney tissue. Both the degree of (B) renal tubular injury and (C) leukocyte infiltration were recorded. (D) Masson’s trichrome staining of collagen deposition in kidney tissue. (E) The degree of fibrotic area was recorded. (F) The average tubular injury, leukocyte infiltration, and fibrotic area scores were used as disease activity indices to indicate the degree of renal injury. The data are presented as means ± standard errors. * p < 0.05 versus the sham group, # p < 0.05 versus non-folic acid-treated mice, $ p < 0.05 versus WT mice. Full article ">Figure 5
Effect of folic acid on UUO-induced renal inflammation and leukocyte infiltration in WT and GNMT−/− mice. (A,B) Tissue extracts from kidneys were analyzed via immunoblotting with antibodies against the adhesion molecules ICAM-1 and VCAM-1, as well as inflammatory markers of iNOS. (C,D) Tissue extracts from kidneys were also analyzed via immunoblotting with antibodies against leukocyte markers, including F4/80, CD3, and MPO. * p < 0.05 versus the sham group, # p < 0.05 versus non-folic acid-treated mice, $ p < 0.05 versus WT mice. Full article ">Figure 6
Effect of folic acid on UUO-induced expression of fibrosis-related proteins in WT and GNMT−/− mice. (A,B) Tissue extracts from kidneys were analyzed by immunoblotting with antibodies against the fibrosis-related proteins TGF-β, α-SMA, COL1A1, and COL4A2; GAPDH was used as a loading control. * p < 0.05 versus the sham group, # p < 0.05 versus non-folic acid-treated mice, $ p < 0.05 versus WT mice. Full article ">Figure 7
Effect of folic acid on UUO-induced autophagy and its regulatory pathway in WT and GNMT−/− mice. (A–C) Tissues extracted from kidneys were analyzed via immunoblotting with antibodies against the autophagy markers LC-3 and p62; GAPDH was used as a loading control. (D–G) Tissues extracted from kidneys were analyzed via immunoblotting with antibodies against autophagy-related signaling pathway components: these included phosphorylated Akt, mTOR, and p70s6k, as well as total Akt, mTOR, and p70s6k. * p < 0.05 versus the sham group of WT mice, # p < 0.05 versus non-folic acid-treated UUO WT mice, $ p < 0.05 versus folic acid-treated UUO WT mice. Full article ">Figure 8
Schematic representation of the protective effect of folic acid on experimental obstructive nephropathy. UUO-induced obstructive nephropathy leads to the development of renal tubular injury, leukocyte infiltration, interstitial fibrosis, and hydronephrosis via an increase in autophagy. Administration of folic acid reduces UUO-induced renal injury by decreasing the level of autophagy in a GNMT-dependent manner. Full article ">

19 pages, 1215 KiB

Open AccessReview

Inclisiran—A Revolutionary Addition to a Cholesterol-Lowering Therapy

by Adrianna Dec, Aleksandra Niemiec, Eliza Wojciechowska, Mateusz Maligłówka, Łukasz Bułdak, Aleksandra Bołdys and Bogusław Okopień

Int. J. Mol. Sci. 2023, 24(7), 6858; https://doi.org/10.3390/ijms24076858 - 6 Apr 2023

Cited by 8 | Viewed by 2821

Abstract

Hypercholesterolemia plays a crucial role in the development of atherosclerosis, but it remains an undertreated and underdiagnosed disease. Taking into consideration the high prevalence of lipid disorders, long duration of the asymptomatic course of the disease, life-threatening complications resulting from inaccurate therapy, and [...] Read more.

Hypercholesterolemia plays a crucial role in the development of atherosclerosis, but it remains an undertreated and underdiagnosed disease. Taking into consideration the high prevalence of lipid disorders, long duration of the asymptomatic course of the disease, life-threatening complications resulting from inaccurate therapy, and stringent treatment goals concerning LDL cholesterol level in the prevention of cardiovascular events, novel lipid-lowering therapies have been introduced in the last few years. In this article, a drug belonging to the group of small interfering RNA (siRNA) called inclisiran is described. It is a novel molecule that increases the number of LDL receptors (LDLRs) on the surface of hepatic cells by preventing the formation of proprotein convertase subtilisin/kexin type 9 (PCSK9) responsible for the degradation of LDLRs. With great potential for lowering plasma LDL cholesterol level, high liver specificity, comfortable dosing regimen, and good tolerance without significant adverse effects, it could play an important part in future hypolipemic therapies. Full article

(This article belongs to the Special Issue Cardiovascular Diseases—a Focus on Atherosclerosis, Its Prophylaxis, Complications and Recent Advancements in Therapies 2.0)

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16 pages, 1142 KiB

Open AccessCommunication

The Effect of Metformin and Carbohydrate-Controlled Diet on DNA Methylation and Gene Expression in the Endometrium of Women with Polycystic Ovary Syndrome

by Elizabeth García-Gómez, Yadira Inés Gómez-Viais, Martin Mizael Cruz-Aranda, Luis Daniel Martínez-Razo, Christian Reyes-Mayoral, Lizeth Ibarra-González, Araceli Montoya-Estrada, Mauricio Osorio-Caballero, Otilia Perichart-Perera, Ignacio Camacho-Arroyo, Marco Cerbón, Enrique Reyes-Muñoz and Edgar Ricardo Vázquez-Martínez

Int. J. Mol. Sci. 2023, 24(7), 6857; https://doi.org/10.3390/ijms24076857 - 6 Apr 2023

Cited by 2 | Viewed by 3044

Abstract

Polycystic ovary syndrome (PCOS) is an endocrine disease associated with infertility and metabolic disorders in reproductive-aged women. In this study, we evaluated the expression of eight genes related to endometrial function and their DNA methylation levels in the endometrium of PCOS patients and [...] Read more.

Polycystic ovary syndrome (PCOS) is an endocrine disease associated with infertility and metabolic disorders in reproductive-aged women. In this study, we evaluated the expression of eight genes related to endometrial function and their DNA methylation levels in the endometrium of PCOS patients and women without the disease (control group). In addition, eight of the PCOS patients underwent intervention with metformin (1500 mg/day) and a carbohydrate-controlled diet (type and quantity) for three months. Clinical and metabolic parameters were determined, and RT-qPCR and MeDIP-qPCR were used to evaluate gene expression and DNA methylation levels, respectively. Decreased expression levels of HOXA10, GAB1, and SLC2A4 genes and increased DNA methylation levels of the HOXA10 promoter were found in the endometrium of PCOS patients compared to controls. After metformin and nutritional intervention, some metabolic and clinical variables improved in PCOS patients. This intervention was associated with increased expression of HOXA10, ESR1, GAB1, and SLC2A4 genes and reduced DNA methylation levels of the HOXA10 promoter in the endometrium of PCOS women. Our preliminary findings suggest that metformin and a carbohydrate-controlled diet improve endometrial function in PCOS patients, partly by modulating DNA methylation of the HOXA10 gene promoter and the expression of genes implicated in endometrial receptivity and insulin signaling. Full article

(This article belongs to the Special Issue New Insight to Polycystic Ovarian Syndrome)

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Expression of genes involved in endometrium function and insulin signaling in patients diagnosed with PCOS. Relative expression levels of HOXA10 (A), PAX6 (B), ESR1 (C), ESR2 (D), IGFBP1 (E), GAB1 (F), SLC2A4 (G), and ISR1 (H) are shown for healthy women (CONTROL) and PCOS patients. Data were obtained using the ΔΔCt method, normalizing mRNA levels with ACTB. Results are expressed as mean ± SE. * p ≤ 0.05, *** p ≤ 0.001. Full article ">Figure 2
Content of DNA methylation at global markers and promoters of genes related to endometrium function in PCOS patients and controls. The relative levels of 5-methylcytosine (5mC) enrichment on H19 gene (A), LINE-1 (B), and promoters of genes HOXA10 (C), PAX6 (D), and ESR1 (E) are shown for healthy women (CONTROL) and PCOS patients without treatment (PCOS). Data were obtained using MeDIP-qPCR and analyzed with the ΔΔCt method, normalizing with IgG enrichment. Results are expressed as mean ± SE. * p ≤ 0.05 vs. PCOS. Full article ">Figure 3
Effect of intervention with metformin and a carbohydrate-controlled diet on the expression of HOXA10, ESR1, GAB1, and SLC2A4 genes in patients diagnosed with PCOS. Relative expression levels of HOXA10 (A), ESR1 (B), GAB1 (C), and SLC2A4 (D) are shown for PCOS patients without treatment (PCOS) and after intervention (PCOS + MET). Data were obtained using the ΔΔCt method, normalizing mRNA levels with ACTB. Results are expressed as mean ± SE. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs. PCOS. Full article ">Figure 4
Effect of intervention with metformin and a carbohydrate-controlled diet on the content of DNA methylation at the promoter of HOXA10 gene in PCOS patients. The relative level of 5-methylcytosine (5mC) enrichment on the promoter of HOXA10 is shown for PCOS patients without treatment (PCOS) and after intervention (PCOS + MET). Data were obtained using MeDIP-qPCR and analyzed with the ΔΔCt method, normalizing with IgG enrichment. Results are expressed as mean ± SE. * p ≤ 0.05 vs. PCOS. Full article ">

18 pages, 5293 KiB

Open AccessReview

Computer-Assisted Design of Peptide-Based Radiotracers

by Vincenzo Patamia, Chiara Zagni, Ilaria Brullo, Erika Saccullo, Alessandro Coco, Giuseppe Floresta and Antonio Rescifina

Int. J. Mol. Sci. 2023, 24(7), 6856; https://doi.org/10.3390/ijms24076856 - 6 Apr 2023

Cited by 5 | Viewed by 2102

Abstract

In medical imaging, techniques such as magnetic resonance imaging, contrast-enhanced computerized tomography, positron emission tomography (PET), and single-photon emission computed tomography (SPECT) are extensively available and routinely used for disease diagnosis. PET probes with peptide-based targeting are typically composed of small peptides especially [...] Read more.

In medical imaging, techniques such as magnetic resonance imaging, contrast-enhanced computerized tomography, positron emission tomography (PET), and single-photon emission computed tomography (SPECT) are extensively available and routinely used for disease diagnosis. PET probes with peptide-based targeting are typically composed of small peptides especially developed to have high affinity and specificity for a range of cellular and tissue targets. These probes’ key benefits include being less expensive than traditional antibody-based PET tracers and having an effective chemical modification process that allows them to be radiolabeled with almost any radionuclide, making them highly appealing for clinical usage. Currently, as with every pharmaceutical design, the use of in silico strategies is steadily growing in this field, even though it is not part of the standard toolkit used during radiopharmaceutical design. This review describes the recent applications of computational design approaches in the design of novel peptide-based radiopharmaceuticals. Full article

(This article belongs to the Special Issue Latest Review Papers in Molecular Informatics 2023)

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13 pages, 3282 KiB

Open AccessArticle

RanBP1: A Potential Therapeutic Target for Cancer Stem Cells in Lung Cancer and Glioma

by Yeon-Jee Kahm, In-Gyu Kim and Rae-Kwon Kim

Int. J. Mol. Sci. 2023, 24(7), 6855; https://doi.org/10.3390/ijms24076855 - 6 Apr 2023

Cited by 3 | Viewed by 2063

Abstract

Cancer stem cells (CSCs) are known to be one of the factors that make cancer treatment difficult. Many researchers are thus conducting research to efficiently destroy CSCs. Therefore, we sought to suggest a new target that can efficiently suppress CSCs. In this study, [...] Read more.

Cancer stem cells (CSCs) are known to be one of the factors that make cancer treatment difficult. Many researchers are thus conducting research to efficiently destroy CSCs. Therefore, we sought to suggest a new target that can efficiently suppress CSCs. In this study, we observed a high expression of Ran-binding protein 1 (RanBP1) in lung cancer stem cells (LCSCs) and glioma stem cells (GSCs). Upregulated RanBP1 expression is strongly associated with the expression of CSC marker proteins and CSC regulators. In addition, an elevated RanBP1 expression is strongly associated with a poor patient prognosis. CSCs have the ability to resist radiation, and RanBP1 regulates this ability. RanBP1 also affects the metastasis-associated epithelial–mesenchymal transition (EMT) phenomenon. EMT marker proteins and regulatory proteins are affected by RanBP1 expression, and cell motility was regulated according to RanBP1 expression. The cancer microenvironment influences cancer growth, metastasis, and cancer treatment. RanBP1 can modulate the cancer microenvironment by regulating the cytokine IL-18. Secreted IL-18 acts on cancer cells and promotes cancer malignancy. Our results reveal, for the first time, that RanBP1 is an important regulator in LCSCs and GSCs, suggesting that it holds potential for use as a potential therapeutic target. Full article

(This article belongs to the Special Issue Novel Biological Molecules for Cancer Treatments 2.0)

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High expression of RanBP1 in A549 cells. (A) Comparison of the gene expression of CSC markers in A549 and H460 cells. GAPDH is used as a loading control. (B) Comparison of the expression of CSC marker proteins in A549 cells and H460 cells. β-actin is used as a loading control. WB: Western blotting. (C) Comparison of CSC marker protein expression through an ICC assay. Full article ">Figure 2
RanBP1 regulates the properties of CSCs in lung cancer cells. (A) A549 cells are transfected with si-RNA targeting RanBP1, and the sphere formation ability is analyzed. (B) RanBP1 gene inhibition using si-RNA followed by single cell assay. Experiments are performed in duplicate five times. (C) Limiting dilution assay is performed on 96-well plates. Here, 1, 50, 100, 150, and 200 cells per well are seeded. Results are confirmed 10 days after cell seeding. (D) Expression analysis of CSC marker proteins ALDH1A1, ALDH1A3, and CD44 according to the expression of RanBP1. (E) Western blot expression analysis of the CSC regulatory proteins SOX2, Oct-4, and Nanog. A549 cells are transfected with si-RNA targeting RanBP1. (F) Immunocytochemical analysis of CSC marker proteins using si-RanBP1-treated A549 cells. An antibody against the CSC marker protein is used as the primary antibody, and an antibody labeled with Alexa Fluor 488 is used as the secondary antibody. (G) Colony formation assay to view clonogenesis with A549 cells transfected with siRNA. Cells are irradiated with a 3Gy dose after 24 h. After 10 days of culture, colonies are stained with crystal violet. Error bars represent mean ± SD. Triplicate samples. * p < 0.05, ** p < 0.0005 versus control. Scale bar = 50 μm. Full article ">Figure 3
EMT is regulated by RanBP1 in lung cancer cells. (A) Western blot analysis of EMT marker proteins E-cadherin, N-cadherin, and Vimentin. A549 cells are transfected with si-RNA targeting RanBP1. (B) The EMT regulatory proteins Snail, Slug, TWIST, and ZEB1 are also analyzed by Western blot. The expression of RanBP1 is suppressed using si-RNA. (C) Immunocytochemical analysis of EMT marker proteins after transfection with si-RanBP1 in A549 cells. Primary antibodies targeting each marker protein are used, and secondary antibodies are labeled with Alexa Fluor 488. (D) Migration and invasion assay of cells by si-RNA transfected A549 cells. (E) Wound healing assay using RanBP1 knockdown A549 cells. Error bars represent mean ± SD. Triplicate samples. * p < 0.0005, ** p < 0.001 versus control. Scale bar = 50 μm. Full article ">Figure 4
Cytokines regulated by RanBP1 in lung cancer. (A) Identification of secreted factors regulated by RanBP1 using cytokine arrays. A549 cells are used, and the expression of RanBP1 is suppressed using si-RNA. (B) Comparison of the gene expression of cytokines regulated by RanBP1. (C) Analysis of cytokines regulated by RanBP1 using WB. (D) Comparison of the sphere formation ability after each neutralizing antibody treatment. The p value is less than 0.0001 versus the IgG Ab value. (E) Comparison of migration and invasion ability of cells after each neutralizing antibody treatment. Experiments performed in triplicate. (F) CSC marker protein analysis in A549 after treatment with IL-18 neutralizing antibody. (G) Confirmation of the expression levels of EMT marker proteins after treatment with the IL-18 neutralizing antibody. (H) Expression analysis of RanBP1 regulated by IL-18 using neutralizing antibodies. Error bars represent mean ± SD. Triplicate samples. * p < 0.05, ** p < 0.001, versus control. Scale bar = 50 μm. Full article ">Figure 5
Characteristics of CSC and EMT events regulated by RanBP1 in glioma. (A) Comparison of the expression of GSC marker proteins by RanBP1 gene suppression in GSCs. U87 cells are differentiated into GSCs using TM. (B) Comparison of the marker protein expression according to the expression of RanBP1 using ICC in GSCs. (C) Comparison of differences in sphere formation according to the expression level of RanBP1 in GSCs. Experiments performed in triplicate. (D) Comparison of the EMT marker protein expression after RanBP1 gene suppression using si-RNA. (E) Confirmation of EMT marker protein expression according to the expression of RanBP1 using ICC. (F) Analysis of cell migration and invasion ability after the suppression of RanBP1 expression using si-RNA. (G) Analysis of regulation of IL-18 expression by RanBP1 in GSCs. Error bars represent mean ± SD. Triplicate samples. * p < 0.001, ** p < 0.0005, versus control. Scale bar = 50 μm. Full article ">

17 pages, 4832 KiB

Open AccessArticle

Delivery of Doxorubicin by Ferric Ion-Modified Mesoporous Polydopamine Nanoparticles and Anticancer Activity against HCT-116 Cells In Vitro

by Mengwen Guo, Junhong Ling, Xinyi Xu and Xiaokun Ouyang

Int. J. Mol. Sci. 2023, 24(7), 6854; https://doi.org/10.3390/ijms24076854 - 6 Apr 2023

Cited by 6 | Viewed by 2340

Abstract

In clinical cancer research, photothermal therapy is one of the most effective ways to increase sensitivity to chemotherapy. Here, we present a simple and effective method for developing a nanotherapeutic agent for chemotherapy combined with photothermal therapy. The nanotherapeutic agent mesoporous polydopamine-Fe(III)-doxorubicin-hyaluronic acid [...] Read more.

In clinical cancer research, photothermal therapy is one of the most effective ways to increase sensitivity to chemotherapy. Here, we present a simple and effective method for developing a nanotherapeutic agent for chemotherapy combined with photothermal therapy. The nanotherapeutic agent mesoporous polydopamine-Fe(III)-doxorubicin-hyaluronic acid (MPDA-Fe(III)-DOX-HA) was composed of mesoporous polydopamine modified by ferric ions and loaded with the anticancer drug doxorubicin (DOX), as well as an outer layer coating of hyaluronic acid. The pore size of the mesoporous polydopamine was larger than that of the common polydopamine nanoparticles, and the particle size of MPDA-Fe(III)-DOX-HA nanoparticles was 179 ± 19 nm. With the presence of ferric ions, the heat generation effect of the MPDA-Fe(III)-DOX-HA nanoparticles in the near-infrared light at 808 nm was enhanced. In addition, the experimental findings revealed that the active targeting of hyaluronic acid to tumor cells mitigated the toxicity of DOX on normal cells. Furthermore, under 808 nm illumination, the MPDA-Fe(III)-DOX-HA nanoparticles demonstrated potent cytotoxicity to HCT-116 cells, indicating a good anti-tumor effect in vitro. Therefore, the system developed in this work merits further investigation as a potential nanotherapeutic platform for photothermal treatment of cancer. Full article

(This article belongs to the Special Issue Functional Nanomaterials for Healthcare)

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17 pages, 768 KiB

Open AccessReview

Neuroendocrine Neoplasms of the Gastrointestinal Tract versus Neuroendocrine Neoplasms of the Gynaecological Tract—Comparison of the Risk Factors and Non-Surgical Treatment Efficacy

by Anna Lorenz, Sebastian Lenkiewicz, Mateusz Kozłowski, Sebastian Kwiatkowski and Aneta Cymbaluk-Płoska

Int. J. Mol. Sci. 2023, 24(7), 6853; https://doi.org/10.3390/ijms24076853 - 6 Apr 2023

Viewed by 1651

Abstract

Neuroendocrine tumours of the gastrointestinal tract are rare. The incidence has increased in recent years due to improvements in diagnostic methods for detecting these lesions. These tumours have a poor prognosis, especially when detected at an advanced stage. The basis of the treatment [...] Read more.

Neuroendocrine tumours of the gastrointestinal tract are rare. The incidence has increased in recent years due to improvements in diagnostic methods for detecting these lesions. These tumours have a poor prognosis, especially when detected at an advanced stage. The basis of the treatment is resection, and non-surgical treatments are also standard in the treatment process. The situation is similar in even rarer neuroendocrine tumours of the reproductive tract, which are associated with an equally poor prognosis. In this article, we focus on learning about the risk factors (including genetic mutations) that increase the risk of the disease and comparing the effectiveness of non-surgical treatments—chemotherapy, radiotherapy, peptide receptor radionuclide therapy, somatostatin analogues, and immunotherapy. The efficacy of these treatments varies, and immunotherapy appears to be a promising form of treatment; however, this requires further research. Full article

(This article belongs to the Special Issue Gynecological Cancer 2023)

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4 pages, 182 KiB

Open AccessEditorial

The Cellular Response to DNA Damage: From DNA Repair to Polyploidy and Beyond

by Razmik Mirzayans

Int. J. Mol. Sci. 2023, 24(7), 6852; https://doi.org/10.3390/ijms24076852 - 6 Apr 2023

Cited by 1 | Viewed by 1377

Abstract

A major challenge in treating patients with solid tumors is posed by intratumor heterogeneity, with different sub-populations of cancer cells within the same tumor exhibiting therapy resistance through different biological processes [...] Full article

(This article belongs to the Special Issue The Cellular Response to DNA Damage 2.0: From DNA Repair to Polyploidy and Beyond)

25 pages, 9097 KiB

Open AccessArticle

Design, Synthesis and Biological Evaluation of 6-(Imidazo[1,2-a]pyridin-6-yl)quinazoline Derivatives as Anticancer Agents via PI3Kα Inhibition

by Mei Li, Daoping Wang, Qing Li, Fang Luo, Ting Zhong, Hongshan Wu, Liang Xiong, Meitao Yuan, Mingzhi Su and Yanhua Fan

Int. J. Mol. Sci. 2023, 24(7), 6851; https://doi.org/10.3390/ijms24076851 - 6 Apr 2023

Cited by 4 | Viewed by 2283

Abstract

Aberrant expression of the phosphatidylinositol 3-kinase (PI3K) signalling pathway is often associated with tumourigenesis, progression and poor prognosis. Hence, PI3K inhibitors have attracted significant interest for the treatment of cancer. In this study, a series of new 6-(imidazo[1,2-a]pyridin-6-yl)quinazoline derivatives were designed, synthesized and [...] Read more.

Aberrant expression of the phosphatidylinositol 3-kinase (PI3K) signalling pathway is often associated with tumourigenesis, progression and poor prognosis. Hence, PI3K inhibitors have attracted significant interest for the treatment of cancer. In this study, a series of new 6-(imidazo[1,2-a]pyridin-6-yl)quinazoline derivatives were designed, synthesized and characterized by ¹H NMR, ¹³C NMR and HRMS spectra analyses. In the in vitro anticancer assay, most of the synthetic compounds showed submicromolar inhibitory activity against various tumour cell lines, among which 13k is the most potent compound with IC₅₀ values ranging from 0.09 μΜ to 0.43 μΜ against all the tested cell lines. Moreover, 13k induced cell cycle arrest at G2/M phase and cell apoptosis of HCC827 cells by inhibition of PI3Kα with an IC₅₀ value of 1.94 nM. These results suggested that compound 13k might serve as a lead compound for the development of PI3Kα inhibitor. Full article

(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)

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Design strategies for target compounds. Full article ">Figure 2
The time-dependent activity of 13k on HCC827 cells. Cells were treated with 13k (0.03 to 0.50 µM) for 24 h to 72 h, and the survival rates were detected via MTT assay. Full article ">Figure 3
Compound 13k inhibited the expression of PI3K and its downstream related proteins (A–E). Expression of PI3K-related proteins was analysed by immunoblotting after treatment of cells with different concentrations of compound 13k (0, 0.08, 0.16 and 0.32 μM) for 48 h. Expression of the associated proteins was analysed using Image J. Each bar data are expressed as mean ± SD from three parallel experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control). Full article ">Figure 4
Effect of compound 13k on the MAPK signalling pathway (A–D). Expression of related proteins was analysed by immunoblotting after treatment of cells with different concentrations of compound 13k (0, 0.08, 0.16 and 0.32 μM) for 48 h. Expression of the associated proteins was analysed using Image J. Each bar Data are expressed as mean ± SD from three parallel experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control). Full article ">Figure 5
Molecular docking model of compound 13k with PI3Kα. (A) Docking of 13k to the active site of PI3Kα (PDB code: 4ZOP); (B) 13k docked in the ATP-binding pocket of PI3Kα; (C) 2D binding model of 13k and PI3Kα. The image was observed with BIOVIA Discovery Studio Visualizer 4.5. Full article ">Figure 6
Effect of 13k on the cell cycle of HCC827. (A) Compound 13k alters the distribution of the cell cycle. Cells were treated with compound 13k for 48 h, stained with propidium iodide mixed with RNase, incubated for 30 min at room temperature and protected from light and analysed by flow cytometry. ‘Ctrl’ refers to the control without the addition of compound 13k. (B) Quantitative histograms of the different phases of the cell cycle. (C–G) Western blot analysis of protein expression associated with G2/M phase. Changes in the corresponding proteins were quantified using Image j. Each bar represents the mean ± SD (n = 3) and was considered statistically significant when compared to the corresponding control values at * p < 0.05, ** p < 0.01 and *** p < 0.001. Full article ">Figure 7
Compound 13k induces apoptosis in HCC827 cells. (A) Apoptosis as well as nuclear morphology was measured by Hoechst 33342 staining after treatment of cells with compound 13k, scale bar = 250 μM. (B) Apoptosis was quantified by flow cytometry using Annexin V-FITC/PI double staining. ‘Ctrl’ refers to the control without the addition of compound 13k. (C–F) Western blot analysis was used to measure the regulation of apoptosis-associated proteins, using Image J for analysis. Each data is expressed as the mean ± SD of three parallel experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control). Full article ">Figure 8
Effect of 13k on HCC827 spheroid formation. HCC827 cells were seeded in ultralow attachment 96-well U bottom plates (40,000 cells/well) to generate tumour spheroids and treated with 5 fold of IC50 concentrations of 13k for the spheroid assay. After initiation, the spheroids were treated with 13k at the indicated concentrations every 3 days. After 12 days, pictures were taken with a ZEISS LSM 900 Airyscan 2 confocal laser scanning microscopy. ‘Ctrl’ refers to the control without the addition of compound 13k. Full article ">Scheme 1
Preparation of 7a–7p reagents and conditions: (i) DIPEA, POCl3, Toluene, 80 °C, 4 h; (ii) isopropanol, 60 °C, 2 h; (iii) K2CO3, Pd(dppf)Cl2, 1,4-dioxacyclohexane/H2O, 100 °C, 5 h. Full article ">Scheme 2
Preparation of 10a–10u reagents and conditions: (iv) NaHCO3, EtOH, 80 °C, 4 h. Full article ">Scheme 3
Preparation of 13a–13k reagents and conditions: (i) NaHCO3, EtOH, 80 °C, 4 h; (ii) K2CO3, Pd(dppf)Cl2, 1,4-dioxacyclohexane/H2O, 100 °C, 5 h. Full article ">

9 pages, 233 KiB

Open AccessCommunication

A Method and Formula for the Quantitative Analysis of the Total Bioactivity of Natural Products

by Shintu Mathew, Ritesh Raju, Xian Zhou, Francis Bodkin, Suresh Govindaraghavan and Gerald Münch

Int. J. Mol. Sci. 2023, 24(7), 6850; https://doi.org/10.3390/ijms24076850 - 6 Apr 2023

Cited by 4 | Viewed by 2369

Abstract

Identification of bioactive natural products from plants starts with the screening of extracts for a desired bioactivity such as antimicrobial, antifungal, anti-cancer, anti-inflammatory, or neuroprotective. When the bioactivity shows sufficient potency, the plant material is subjected to bio-activity-guided fractionation, which involves, e.g., sequential [...] Read more.

Identification of bioactive natural products from plants starts with the screening of extracts for a desired bioactivity such as antimicrobial, antifungal, anti-cancer, anti-inflammatory, or neuroprotective. When the bioactivity shows sufficient potency, the plant material is subjected to bio-activity-guided fractionation, which involves, e.g., sequential extraction followed by chromatographic separation, including HPLC. The bioactive compounds are then structurally identified by high-resolution mass spectrometry and nuclear magnetic resonance (NMR). One of the questions that come up during the purification process is how much of the bioactivity originally present in the crude extract is preserved during the purification process. If this is the case, it is interesting to investigate if the loss of total bioactivity is caused by the loss of material during purification or by the degradation or evaporation of potent compounds. A further possibility would be the loss of synergy between compounds present in the mixture, which disappears when the compounds are separated. In this publication, a novel formula is introduced that allows researchers to calculate total bioactivity in biological samples using experimental data from our research into the discovery of anti-inflammatory compounds from Backhousia myrtifolia (Grey Myrtle). The results presented show that a raw ethanolic extract retains slightly more bioactivity than the sum of all sequential extracts per gram of starting material and that—despite a large loss of material during HPLC purification—the total bioactivity in all purified fractions is retained, which is indicative of rather an additive than a synergistic principle. Full article

(This article belongs to the Collection Feature Papers in Molecular Pathology, Diagnostics, and Therapeutics)

25 pages, 21045 KiB

Open AccessArticle

POLD1 as a Prognostic Biomarker Correlated with Cell Proliferation and Immune Infiltration in Clear Cell Renal Cell Carcinoma

by Junjie Tian, Cheng Cheng, Jianguo Gao, Guanghou Fu, Zhijie Xu, Xiaoyi Chen, Yunfei Wu and Baiye Jin

Int. J. Mol. Sci. 2023, 24(7), 6849; https://doi.org/10.3390/ijms24076849 - 6 Apr 2023

Cited by 4 | Viewed by 2629

Abstract

DNA polymerase delta 1 catalytic subunit (POLD1) plays a vital role in genomic copy with high fidelity and DNA damage repair processes. However, the prognostic value of POLD1 and its relationship with tumor immunity in clear cell renal cell carcinoma (ccRCC) remains to [...] Read more.

DNA polymerase delta 1 catalytic subunit (POLD1) plays a vital role in genomic copy with high fidelity and DNA damage repair processes. However, the prognostic value of POLD1 and its relationship with tumor immunity in clear cell renal cell carcinoma (ccRCC) remains to be further explored. Transcriptional data sets and clinical information were obtained from the TCGA, ICGC, and GEO databases. Differentially expressed genes (DEGs) were derived from the comparison between the low and high POLD1 expression groups in the TCGA–KIRC cohort. KEGG and gene ontology (GO) analyses were performed for those DEGs to explore the potential influence of POLD1 on the biological behaviors of ccRCC. The prognostic clinical value and mutational characteristics of patients were described and analyzed according to the POLD1 expression levels. TIMER and TISIDB databases were utilized to comprehensively investigate the potential relevance between the POLD1 levels and the status of the immune cells, as well as the tumor infiltration of immune cells. In addition, RT-qPCR, Western blot, immunohistochemistry and several functional and animal experiments were performed for clinical, in vitro and in vivo validation. POLD1 was highly expressed in a variety of tumors including ccRCC, and further verified in a validation cohort of 60 ccRCC samples and in vitro cell line experiments. POLD1 expression levels in the ccRCC samples were associated with various clinical characteristics including pathologic tumor stage and histologic grade. ccRCC patients with high POLD1 expression have poor clinical outcomes and exhibit a higher rate of somatic mutations than those with low POLD1 expression. Cox regression analysis also showed that POLD1 could act as a potential independent prognostic biomarker. The DEGs associated with POLD1 were significantly enriched in the immunity-related pathways. Moreover, further immune infiltration analysis indicated that high POLD1 expression was associated with high NK CD56bright cells, Treg cells, and myeloid-derived suppressor cells’ (MDSCs) infiltration scores, as well as their marker gene sets of immune cell status. Meanwhile, POLD1 exhibited resistance to various drugs when highly expressed. Finally, the knockdown of POLD1 inhibited the proliferation and migration, and promoted the apoptosis of ccRCC cells in vitro and in vivo, as well as influenced the activation of oncogenic signaling. Our current study demonstrated that POLD1 is a potential prognostic biomarker for ccRCC patients. It might create a tumor immunosuppressive microenvironment and inhibit the susceptibility to ferroptosis leading to a poor prognosis. Full article

(This article belongs to the Section Molecular Genetics and Genomics)

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10 pages, 2013 KiB

Open AccessArticle

Genetic Variation among Heterodera schachtii Populations Coincided with Differences in Invasion and Propagation in Roots of a Set of Cruciferous Plants

by Rasha Haj Nuaima and Holger Heuer

Int. J. Mol. Sci. 2023, 24(7), 6848; https://doi.org/10.3390/ijms24076848 - 6 Apr 2023

Cited by 4 | Viewed by 1387

Abstract

Genes of host plants and parasitic nematodes govern the plant–nematode interaction. The biological receptors and parasitism effectors are variable among plant species and nematode populations, respectively. In the present study, hatch testing and bioassays on cabbage, oilseed radish, and mustard were conducted to [...] Read more.

Genes of host plants and parasitic nematodes govern the plant–nematode interaction. The biological receptors and parasitism effectors are variable among plant species and nematode populations, respectively. In the present study, hatch testing and bioassays on cabbage, oilseed radish, and mustard were conducted to compare the biological characteristics among six populations of the beet cyst nematode Heterodera schachtii. Genetic patterns of the vap1 gene for the studied populations were distinct as shown by denaturing the gradient gel electrophoresis of PCR-amplified gene fragments. Concurrently, significant differences in the hatching rates, number of penetrated J2 in roots, and eggs/cyst ratios among the six nematode populations for the three cruciferous species were observed. In conclusion, analyzing the population genetic structure of H. schachtii plays a pivotal role in illustrating the variability in the plant–nematode interaction among its populations and plant species, which in its role leads to developing nematode management depending on plant resistance. Full article

(This article belongs to the Special Issue Plant–Nematode Interactions)

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Figure 1
Egg hatching percentages of H. schachtii populations in zinc chloride solution over eight weeks. Each color represents the percentage ratio of J2 collected in the corresponding week to the egg content of 500 cysts. The percentage above each column refers to the total hatch percentage for each population during the eight weeks. Full article ">Figure 2
Numbers of second-stage juveniles (J2) from six populations of H. schachtii that penetrated roots of the susceptible cabbage (Brassica oleracea L., cv. Storema) or susceptible oilseed radish (Raphanus sativus L., cv. Siletina) or resistant white mustard (Sinapis alba L., cv. Serval). In total, 450 J2 were added to a seven-day seedling grown in the loess substrate and incubated under greenhouse conditions (20/16 °C for a 16/8 h day/night cycle) for seven days. The counting was for J2 in the roots, stained by acid fuchsin. (a,b,c,d,e,f) compare the mean numbers of J2 among populations that penetrated the same plant species. (A,B,C) compare the mean numbers of J2 of the same population that penetrated the three plant species. Different letters indicate significant differences (Wilcox’s test, p < 0.05, n = 8). Full article ">Figure 3
Numbers of eggs per propagated cyst of six populations of H. schachtii reared for two nematode generations on the susceptible cabbage (Brassica oleracea L., cv. Storema) or susceptible oilseed radish (Raphanus sativus L., cv. Siletina) or resistant white mustard (Sinapis alba L., cv. Serval). For each two-week seedling, 450 J2 were added and incubated under greenhouse conditions (20/16 °C for a 16/8 h day/night cycle) for three months. The counting was for eggs contained in the cysts extracted from the loess substrate by floatation on MgSO4 solution (1.28 specific density). (a,b,c,d,e) compare the mean numbers of egg/cyst ratio among populations reared on the same plant species. (A,B,C) compare the mean numbers of egg/cyst ratio of the same population reared on the three plant species. Different letters indicate significant differences (Wilcox’s test, p < 0.05, n = 8). Full article ">Figure 4
UPGMA clustering based on the Jaccard similarity coefficient, showing the genetic differences in vap1 patterns among six populations of H. schachtii based on DGGE profile analysis. Each color represents vap1 profiles from one population. Bootstrap values refer to the similarity percentage of vap1 patterns within and among populations. Full article ">

19 pages, 4012 KiB

Open AccessArticle

The Influence of Edaphic Factors on DNA Damage and Repair in Wild Wheat Triticum dicoccoides Körn. (Poaceae, Triticeae)

by Olga Raskina, Boris Shklyar and Eviatar Nevo

Int. J. Mol. Sci. 2023, 24(7), 6847; https://doi.org/10.3390/ijms24076847 - 6 Apr 2023

Cited by 1 | Viewed by 2064

Abstract

A complex DNA repair network maintains genome integrity and genetic stability. In this study, the influence of edaphic factors on DNA damage and repair in wild wheat Triticum dicoccoides was addressed. Plants inhabiting two abutting microsites with dry terra rossa and humid basalt [...] Read more.

A complex DNA repair network maintains genome integrity and genetic stability. In this study, the influence of edaphic factors on DNA damage and repair in wild wheat Triticum dicoccoides was addressed. Plants inhabiting two abutting microsites with dry terra rossa and humid basalt soils were studied. The relative expression level of seven genes involved in DNA repair pathways—RAD51, BRCA1, LigIV, KU70, MLH1, MSH2, and MRE11—was assessed using quantitative real-time PCR (qPCR). Immunolocalization of RAD51, LigIV, γH2AX, RNA Polymerase II, and DNA-RNA hybrid [S9.6] (R-loops) in somatic interphase nuclei and metaphase chromosomes was carried out in parallel. The results showed a lower expression level of genes involved in DNA repair and a higher number of DNA double-strand breaks (DSBs) in interphase nuclei in plants growing in terra rossa soil compared with plants in basalt soil. Further, the number of DSBs and R-loops in metaphase chromosomes was also greater in plants growing on terra rossa soil. Finally, RAD51 and LigIV foci on chromosomes indicate ongoing DSB repair during the M-phase via the Homologous Recombination and Non-Homologous End Joining pathways. Together, these results show the impact of edaphic factors on DNA damage and repair in the wheat genome adapted to contrasting environments. Full article

(This article belongs to the Collection Advances in Cytomolecular Organisation of the Nuclear Genome)

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15 pages, 1296 KiB

Open AccessReview

Uses of Papaya Leaf and Seaweed Supplementations for Controlling Glucose Homeostasis in Diabetes

by Benard B. Nyakundi and Jinzeng Yang

Int. J. Mol. Sci. 2023, 24(7), 6846; https://doi.org/10.3390/ijms24076846 - 6 Apr 2023

Cited by 6 | Viewed by 4573

Abstract

Studies from laboratory animal models and complementary medical practices have implied that nutrients from special plants or herbs contain antidiabetic, antioxidant, anti-obese, anti-hypertensive, and anti-inflammatory properties. Seaweed and tropical papaya, which are widely available in Asian and Pacific countries, have been used as [...] Read more.

Studies from laboratory animal models and complementary medical practices have implied that nutrients from special plants or herbs contain antidiabetic, antioxidant, anti-obese, anti-hypertensive, and anti-inflammatory properties. Seaweed and tropical papaya, which are widely available in Asian and Pacific countries, have been used as home remedies for centuries. The bioactive extracts from these plants contain vitamins A, C, B and E complexes, as well as polysaccharides, phenolic compounds, essential fatty acids, flavonoids, saponins, fucoidan, and phlorotannin. In this review, the authors examine the pathogenesis of diabetes characterized by hyperglycemia due to the dysregulation of glucose homeostasis, antidiabetic/antihyperglycemic seaweed or/and papaya derived bioactive phytochemicals and their proposed mechanisms of action in the management of Type 2 Diabetes Mellitus (T2DM). The authors also propose combining papaya and seaweed to enhance their antidiabetic effects, leveraging the advantages of herb-to-herb combination. Papaya and seaweed have demonstrated antidiabetic effects through in vitro assays, cellular models, and animal studies despite the limited clinical trials. Nutraceuticals with antidiabetic effects, such as secondary metabolites isolated from seaweed and papaya, could be combined for a synergistic effect on T2DM management. However, the application of these compounds in their purified or mixed forms require further scientific studies to evaluate their efficacy against diabetes-related complications, such as hyperlipidemia, elevated free radicals, pro-inflammatory molecules, insulin insensitivity, and the degeneration of pancreatic beta cells. Full article

(This article belongs to the Special Issue Bioactive Compounds in Metabolic Syndrome)

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31 pages, 11986 KiB

Open AccessArticle

Identification of Orbivirus Non-Structural Protein 5 (NS5), Its Role and Interaction with RNA/DNA in Infected Cells

by Fauziah Mohd Jaafar, Baptiste Monsion, Peter P. C. Mertens and Houssam Attoui

Int. J. Mol. Sci. 2023, 24(7), 6845; https://doi.org/10.3390/ijms24076845 - 6 Apr 2023

Cited by 5 | Viewed by 2589

Abstract

Bioinformatic analyses have predicted that orbiviruses encode an additional, small non-structural protein (NS5) from a secondary open reading frame on genome segment 10. However, this protein has not previously been detected in infected mammalian or insect cells. NS5-specific antibodies were generated in mice [...] Read more.

Bioinformatic analyses have predicted that orbiviruses encode an additional, small non-structural protein (NS5) from a secondary open reading frame on genome segment 10. However, this protein has not previously been detected in infected mammalian or insect cells. NS5-specific antibodies were generated in mice and were used to identify NS5 synthesised in orbivirus-infected BSR cells or cells transfected with NS5 expression plasmids. Confocal microscopy shows that although NS5 accumulates in the nucleus, particularly in the nucleolus, which becomes disrupted, it also appears in the cell cytoplasm, co-localising with mitochondria. NS5 helps to prevent the degradation of ribosomal RNAs during infection and reduces host-cell protein synthesis However, it helps to extend cell viability by supporting viral protein synthesis and virus replication. Pulldown studies showed that NS5 binds to ssRNAs and supercoiled DNAs and demonstrates interactions with ZBP1, suggesting that it modulates host-cell responses. Full article

(This article belongs to the Collection Feature Papers in “Molecular Biology”)

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18 pages, 5955 KiB

Open AccessArticle

Salvianolic-Acid-B-Loaded HA Self-Healing Hydrogel Promotes Diabetic Wound Healing through Promotion of Anti-Inflammation and Angiogenesis

by Guoying Zhou, Jiayan Zhu, Liang Jin, Jing Chen, Ruojiao Xu, Yali Zhao, Tingzi Yan and Haitong Wan

Int. J. Mol. Sci. 2023, 24(7), 6844; https://doi.org/10.3390/ijms24076844 - 6 Apr 2023

Cited by 12 | Viewed by 3119

Abstract

Inflammatory dysfunction and angiogenesis inhibition are two main factors leading to the delayed healing of diabetic wounds. Hydrogels with anti-inflammatory and angiogenesis-promoting effects have been considered as promising wound care materials. Herein, a salvianolic acid B (SAB)-loaded hyaluronic acid (HA) self-healing hydrogel (HA/SAB) [...] Read more.

Inflammatory dysfunction and angiogenesis inhibition are two main factors leading to the delayed healing of diabetic wounds. Hydrogels with anti-inflammatory and angiogenesis-promoting effects have been considered as promising wound care materials. Herein, a salvianolic acid B (SAB)-loaded hyaluronic acid (HA) self-healing hydrogel (HA/SAB) with anti-inflammatory and pro-angiogenesis capacities for diabetic wound healing is reported. The HA hydrogel was prepared via the covalent cross-linking of aldehyde groups in oxidized HA (OHA) and hydrazide groups in adipic dihydrazide (ADH)-modified HA (HA-ADH) with the formation of reversible acylhydrazone bonds. The obtained HA hydrogel exhibited multiple favorable properties such as porous structures, excellent self-healing properties, a sustainable release capacity of SAB, as well as excellent cytocompatibility. In addition, the effects of the SAB-loaded HA self-healing hydrogel were investigated via a full-thickness skin defect model using diabetic rats. The HA/SAB hydrogel showed enhanced skin regeneration effects with accelerated wound closure, shorter remaining dermal space length, thicker granulation tissue formation, and more collagen deposition. Furthermore, reduced inflammatory response and enhanced vascularization were found with HA/SAB2.5 hydrogel-treated wounds, indicating that the hydrogel promotes diabetic wound healing through the promotion of anti-inflammation and angiogenesis. Our results suggest that the fabricated SAB-loaded HA self-healing hydrogel is promising as a wound dressing for the treatment of diabetic wounds. Full article

(This article belongs to the Special Issue Advanced Therapies and Functional Materials for Wound Healing)

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Figure 1
Characterization of the basic properties of the HA hydrogel. (A) Synthesis of adipic dihydrazide (ADH)-modified HA (HA-ADH) and oxidized HA (OHA). (B) FT-IR spectra of HA, ADH, OHA, and HA/SAB2.5 hydrogel. (C) 1H NMR spectrum of HA, ADH, and HA-ADH. (D) Photographs of the injectable (i), moldable (ii), and self-healing (iii) properties of the HA hydrogel. (E) Schematic illustration of the HA hydrogel formation and self-healing properties. Full article ">Figure 2
(A) SEM images of the blank HA and salvianolic acid B (SAB)-loaded HA hydrogels (HA/SAB1, HA/SAB2.5, and HA/SAB5) (Scale bar = 100 μm). (B) Pore size distribution of the blank HA, HA/SAB1, HA/SAB2.5, and HA/SAB5 hydrogels (n = 20). (C) Storage modulus (G’) and loss modulus (G’’) of the HA hydrogel cross-linked by adipic dihydrazide (ADH)-modified HA (HA-ADH) and oxidized HA (OHA) at different times at 1% strain and 10 rad/s angular frequency. (D,E) Strain sweep of the HA hydrogel with strain from 0.1% to 1000% at an angular frequency of 10 rad/s. (F) Angular frequency sweep of the HA hydrogel from 0.1 to 10 rad/s at a constant strain of 1%. (G) Self-healing property of the blank HA and HA/SAB2.5 hydrogels at continuous step strain test of 1% and 300% strain every 200 s at an angular frequency of 10 rad/s. (H) Swelling and degradation behaviors of the blank HA, HA/SAB1, HA/SAB2.5, and HA/SAB5 hydrogels in PBS at 37 °C. (I) SAB release profile of the HA/SAB hydrogels in PBS at 37 °C. Data represent mean ± SD, n = 3. Full article ">Figure 3
Cytocompatibility of the blank HA and salvianolic acid B (SAB)-loaded HA hydrogels (HA/SAB1, HA/SAB2.5, and HA/SAB5) in vitro. Cytotoxicity analysis of the HA hydrogels with CCK-8 assay (A) and LIVE/DEAD staining assay (B) using NIH/3T3 cells on day 1 and 3. (Scale bar = 200 μm). Data represent mean ± SD, n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001. Full article ">Figure 4
The mRNA expression level of (A) IL-1β and (B) ccr7 in (LPS + IFN-γ)-stimulated THP-1-derived macrophages following treatment of salvianolic acid B (SAB, 100 μM). (C) The mRNA expression level of CD206 in IL-4-stimulated THP-1-derived macrophages following treatment of SAB (100 μM). Data represent mean ± SD, n = 3; * p < 0.05; *** p < 0.001. Full article ">Figure 5
(A) Schematic of the establishment, treatment, and assessment of diabetic wound model. (B) Representative photographs of diabetic wounds on day 0, 3, 7, and 14 for control, blank HA, and salvianolic acid B (SAB)-loaded HA hydrogel (HA/SAB2.5). (C) Schematic diagram of the wound healed by different treatments during 14 days. (D) Quantitative data of wound healing rate on day 3, 7, and 14 following treatment of blank HA hydrogel and HA/SAB2.5 hydrogel, n = 6. (E,F) The mRNA expression level of IL-1β and IL-10 in wound sites on day 7 following treatment of blank HA hydrogel and HA/SAB2.5 hydrogel, n = 3. Data represent mean ± SD; * p < 0.05; ** p < 0.01; *** p < 0.001. Full article ">Figure 6
Histological evaluation of regenerated skin. (A) Representative images of H&E staining (A) (scale bar = 1 mm; red arrow: wound site length; black arrow: granulation tissue) and Masson trichrome staining (B) (scale bar = 1 mm/200 μm) for control, blank HA, and salvianolic acid B (SAB)-loaded HA hydrogel (HA/SAB2.5) on day 14. (C–E) Quantification of the wound site length, granulation tissue thickness, and collagen deposition of wound sites on day 14. Data represent mean ± SD, n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001. Full article ">Figure 7
HA hydrogels promoted angiogenesis during wound healing process. (A) Immunofluorescence staining images of α-SMA (green), CD31 (red) and DAPI (blue) on day 14 post wounding for control, blank HA, and salvianolic acid B (SAB)-loaded HA hydrogel (HA/SAB2.5) (scale bar = 100 μm). (B) Mean intensity of CD31-positive cells in wounds. (C) Quantitative graph of blood vessel density on day 14 corresponding to α-SMA-positive staining in diabetic wounds. Data represent mean ± SD, n = 3; *** p < 0.001. Full article ">Figure 8
Salvianolic acid B-loaded HA self-healing hydrogel promotes diabetic wound healing through promotion of anti-inflammation and angiogenesis. Red arrow: HA/SAB hydrogel increased the expression of IL-10, CD31 and α-SMA. Blue arrow: HA/SAB hydrogel decreased the expression of IL-1β and ccr7. Red circular: wound sites established on the back of diabetic rats. Blue circular: wound sites treated with hydrogels. Full article ">

17 pages, 9565 KiB

Open AccessArticle

Engineering a HER2-CAR-NK Cell Secreting Soluble Programmed Cell Death Protein with Superior Antitumor Efficacy

by Wenjiao Xia, Jiaxin Chen, Wenqing Hou, Junsheng Chen, Ying Xiong, Hongyan Li, Xin Qi, Hui Xu, Zuoquan Xie, Mingfeng Li, Xiaomin Zhang and Jing Li

Int. J. Mol. Sci. 2023, 24(7), 6843; https://doi.org/10.3390/ijms24076843 - 6 Apr 2023

Cited by 9 | Viewed by 3317

Abstract

A new therapy strategy for relapsing patients who have received trastuzumab treatment urgently needs to be explored. HER2-specific chimeric antigen receptor (CAR)-expressing NK cells are being rapidly developed for solid tumor therapy, as they have many advantages over HER2-CAR-T cells. Endogenous soluble PD-1 [...] Read more.

A new therapy strategy for relapsing patients who have received trastuzumab treatment urgently needs to be explored. HER2-specific chimeric antigen receptor (CAR)-expressing NK cells are being rapidly developed for solid tumor therapy, as they have many advantages over HER2-CAR-T cells. Endogenous soluble PD-1 (sPD-1) from the PD-1 extracellular domain blocks PD-1/PD-L1 interaction to promote cancer immunology. Herein, we engineered a new HER2-CAR-NK cell that co-expresses sPD-1 (designed as sPD-1-CAR-NK cells) and assessed its cytotoxic activities toward various cancer cells, activation of immunity and sPD-1 release in vitro and in mouse models bearing breast cancer cells with high HER2 expression, with or without trastuzumab resistance. We demonstrated that sPD-1-CAR-NK cells were able to release bioactive sPD-1, thereby enhancing the cytolytic activities of HER2-CAR-NK cells against HER2 and PD-L1 highly expressing target cells accompanied by increases in the secretion of perforin, granzyme B and IFN-γ. In vivo, sPD-1-CAR-NK cells had superior immunological anticancer efficacy compared to HER2-CAR-NK cells, and they had advantages over HER2-CAR-NK cells in the intraperitoneal injection of sPD-1. Moreover, the infiltration and activation of NK and T cells into tumor tissue were increased in mice with sPD-1-CAR-NK cells. There was no significant change in the body temperature, organ tissue and body weight in all groups except for the group with the PD-1 injection. Together, these data indicate that HER2-specific sPD-1-CAR-NK cells can transport sPD-1 into cancer tissues with high HER2 expression, further improving the efficacy of HER-CAR-NK cells without obvious side effects. sPD-1-CAR-NK is a promising cytotherapeutic agent for patients bearing HER2-positive breast cancer, including those with trastuzumab resistance. Full article

(This article belongs to the Special Issue The Discovery, Synthesis and Development of Cancer Therapeutic Agents)

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