International Journal of Molecular Sciences

19 pages, 4648 KiB

Open AccessArticle

Unveiling the Anti-Cancer Potential of Onoceranoid Triterpenes from Lansium domesticum Corr. cv. kokosan: An In Silico Study against Estrogen Receptor Alpha

by Ari Hardianto, Sarah Syifa Mardetia, Wanda Destiarani, Yudha Prawira Budiman, Dikdik Kurnia and Tri Mayanti

Int. J. Mol. Sci. 2023, 24(19), 15033; https://doi.org/10.3390/ijms241915033 - 9 Oct 2023

Cited by 1 | Viewed by 1452

Abstract

Breast cancer is a significant global concern, with tamoxifen, the standard treatment, raising long-term safety issues due to side effects. In this study, we evaluated the potential of five onoceranoid triterpenes from Lansium domesticum Corr. cv. kokosan against estrogen receptor alpha (ERα) using [...] Read more.

Breast cancer is a significant global concern, with tamoxifen, the standard treatment, raising long-term safety issues due to side effects. In this study, we evaluated the potential of five onoceranoid triterpenes from Lansium domesticum Corr. cv. kokosan against estrogen receptor alpha (ERα) using in silico techniques. Utilizing molecular docking, Lipinski’s rule of five, in silico ADMET, and molecular dynamics simulations, we assessed the potency of five onoceranoid triterpenes against ERα. Molecular docking indicated competitive binding energies for these triterpenes relative to the active form of tamoxifen (4OHT) and estradiol, an ERα native ligand. Three triterpenes met drug-likeness criteria with favorable ADMET profiles. Notably, 2 demonstrated superior binding affinity in molecular dynamics simulations, outperforming estradiol, closely followed by 3 and 4. Hierarchical clustering on principal components (HCPC) and the spatial distribution of contact surface area (CSA) analyses suggest that these triterpenes, especially 2, may act as antagonist ligands akin to 4OHT. These findings highlight the potential of onoceranoid triterpenes in treating ERα-related breast cancer. Full article

(This article belongs to the Special Issue Advances in Molecular Modeling, Docking and Simulations of Protein Structure)

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28 pages, 9231 KiB

Open AccessArticle

Multi-Omics Profiling of Human Endothelial Cells from the Coronary Artery and Internal Thoracic Artery Reveals Molecular but Not Functional Heterogeneity

by Alexey Frolov, Arseniy Lobov, Marsel Kabilov, Bozhana Zainullina, Alexey Tupikin, Daria Shishkova, Victoria Markova, Anna Sinitskaya, Evgeny Grigoriev, Yulia Markova and Anton Kutikhin

Int. J. Mol. Sci. 2023, 24(19), 15032; https://doi.org/10.3390/ijms241915032 - 9 Oct 2023

Cited by 2 | Viewed by 1852

Abstract

Major adverse cardiovascular events occurring upon coronary artery bypass graft surgery are typically accompanied by endothelial dysfunction. Total arterial revascularisation, which employs both left and right internal thoracic arteries instead of the saphenous vein to create a bypass, is associated with better mid- [...] Read more.

Major adverse cardiovascular events occurring upon coronary artery bypass graft surgery are typically accompanied by endothelial dysfunction. Total arterial revascularisation, which employs both left and right internal thoracic arteries instead of the saphenous vein to create a bypass, is associated with better mid- and long-term outcomes. We suggested that molecular profiles of human coronary artery endothelial cells (HCAECs) and human internal mammary artery endothelial cells (HITAECs) are coherent in terms of transcriptomic and proteomic signatures, which were then investigated by RNA sequencing and ultra-high performance liquid chromatography-mass spectrometry, respectively. Both HCAECs and HITAECs overexpressed molecules responsible for the synthesis of extracellular matrix (ECM) components, basement membrane assembly, cell-ECM adhesion, organisation of intercellular junctions, and secretion of extracellular vesicles. HCAECs were characterised by higher enrichment with molecular signatures of basement membrane construction, collagen biosynthesis and folding, and formation of intercellular junctions, whilst HITAECs were notable for augmented pro-inflammatory signaling, intensive synthesis of proteins and nitrogen compounds, and enhanced ribosome biogenesis. Despite HCAECs and HITAECs showing a certain degree of molecular heterogeneity, no specific markers at the protein level have been identified. Coherence of differentially expressed molecular categories in HCAECs and HITAECs suggests synergistic interactions between these ECs in a bypass surgery scenario. Full article

(This article belongs to the Special Issue Atherosclerosis 2.0: From Molecular Mechanisms, Pathophysiology to Novel Therapeutic Approaches)

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Figure 1
Volcano plot showing the distribution of proteins in the proteome of human coronary artery endothelial cells (HCAECs) and human internal thoracic artery endothelial cells (HITAECs). Gray points depict the proteins with log2 fold change < 1 and FDR-corrected p value > 0.05. Green points depict the proteins with log2 fold change > 1 and FDR-corrected p value > 0.05. Blue points depict the proteins with log2 fold change < 1 and FDR-corrected p value < 0.05. Red points depict the proteins with log2 fold change > 1 and FDR-corrected p value < 0.05 (DEPs). Full article ">Figure 2
Volcano plot showing the distribution of transcripts in the transcriptome of human coronary artery endothelial cells (HCAECs) and human internal thoracic artery endothelial cells (HITAECs) under laminar flow. Gray points depict the genes with log2 fold change < 1 and FDR-corrected p value > 0.05. Green points depict the genes with log2 fold change > 1 and FDR-corrected p value > 0.05. Blue points depict the genes with log2 fold change < 1 and FDR-corrected p value < 0.05. Red points depict the genes with log2 fold change > 1 and FDR-corrected p value < 0.05 (DEGs). Full article ">Figure 3
Volcano plot showing the distribution of transcripts in the transcriptome of human coronary artery endothelial cells (HCAECs) and human internal thoracic artery endothelial cells (HITAECs) at static cell culture conditions. Gray points depict the genes with log2 fold change < 1 and FDR-corrected p value > 0.05. Green points depict the genes with log2 fold change > 1 and FDR-corrected p value > 0.05. Blue points depict the genes with log2 fold change < 1 and FDR-corrected p value < 0.05. Red points depict the genes with log2 fold change > 1 and FDR-corrected p value < 0.05 (DEGs). Full article ">Figure 4
Fluorescent Western blotting for cell adhesion molecules (VCAM1 and ICAM1), transcription factors of endothelial-to-mesenchymal transition (Snail and Slug, TWIST1, and ZEB1), mechanosensitive transcription factors (KLF2, KLF4, and NRF2), endothelial nitric oxide synthase eNOS, and loading control (GAPDH and CD31) in human coronary artery endothelial cells (HCAECs) and human internal thoracic artery endothelial cells (HITAECs) cultured at static conditions. (A) VCAM1 (pro-inflammatory cell adhesion molecule, green)/GAPDH (loading control, red), fluorescent Western blot (top), and total protein staining confirming an equal protein loading (bottom); (B) ICAM1 (pro-inflammatory cell adhesion molecule, green)/CD31 (loading control, red), fluorescent Western blot (top), and total protein staining confirming an equal protein loading (bottom); (C) Snail and Slug (endothelial-to-mesenchymal transition transcription factor, green)/CD31 (loading control, red), fluorescent Western blot (top), and total protein staining confirming an equal protein loading (bottom); (D) TWIST1 (endothelial-to-mesenchymal transition transcription factor, green)/ZEB1 (another endothelial-to-mesenchymal transition transcription factor, red), fluorescent Western blot (top), and total protein staining confirming an equal protein loading (bottom); (E) TWIST1 (endothelial-to-mesenchymal transition transcription factor, green)/KLF2 (atheroprotective mechanosensitive transcription factor, red), fluorescent Western blot (top), and total protein staining confirming an equal protein loading (bottom); (F) KLF4 (atheroprotective mechanosensitive transcription factor, green)/eNOS (endothelial nitric oxide synthase, red), fluorescent Western blot (top), and total protein staining confirming an equal protein loading (bottom); (G) NRF2 (atheroprotective mechanosensitive transcription factor, green)/GAPDH (loading control, red), fluorescent Western blot (top), and total protein staining confirming an equal protein loading (bottom). Each band within the groups represent a protein lysate from one experiment (n = 3 experiments in total). Total protein normalisation was conducted by Fast Green FCF staining of the membranes after the fluorescent imaging to ensure the equal protein loading at all blots (in addition to loading controls such as GAPDH or CD31). Fluorescent ladder (L) and molecular weight signatures (kDa) are provided to the left of the HCAECs and HITAECs protein bands. Ratios of 1:100 and 1:200 are dilutions of the antibody against TWIST1, highlighted to show low expression of this protein in the quiescent ECs. Full article ">Figure 5
Bioinformatic analysis of protein–protein interactions between primary human coronary endothelial cells (HCAECs) and human internal thoracic artery endothelial cells (HITAECs). (A) Overview of protein–protein interactions between HCAECs (blue colour) and HITAECs (red colour); (B) annotation of the interacting proteins related to the specific molecular terms (yellow colour) in comparison with unannotated overview (the main cluster of interacting proteins is demarcated by red contour); and (C) annotations of clustered interacting proteins in selected molecular terms: integrin-mediated cell adhesion (demarcated by blue contour), basement membrane (demarcated by violet contour), and elastic fiber formation (demarcated by green contour). Full article ">Figure 6
Bioinformatic analysis of gene–gene interactions between primary human coronary endothelial cells (HCAECs) and human internal thoracic artery endothelial cells (HITAECs) cultured under laminar flow. (A) Overview of gene–gene interactions between HCAECs (blue colour) and HITAECs (red colour) cultured under laminar flow; (B) annotation of the interacting genes related to the specific molecular terms (yellow colour) in comparison with unannotated overview (the main cluster of interacting genes is demarcated by red contour); and (C) annotations of clustered interacting genes in selected molecular terms: sprouting angiogenesis (demarcated by blue contour) and elastic fiber formation (demarcated by green contour). Full article ">Figure 7
Bioinformatic analysis of gene–gene interactions between primary human coronary endothelial cells (HCAECs) and human internal thoracic artery endothelial cells (HITAECs) cultured at static conditions. (A) Overview of gene–gene interactions between HCAECs (blue colour) and HITAECs (red colour) cultured under laminar flow; (B) annotation of the interacting genes related to the specific molecular terms (yellow colour) in comparison with unannotated overview; and (C) annotations of clustered interacting genes in selected molecular terms: angiogenesis (demarcated by blue contour) and stimulation of guanylate cyclase by nitric oxide (demarcated by green contour). Full article ">

25 pages, 12701 KiB

Open AccessArticle

Comparative Chloroplast Genomics of 21 Species in Zingiberales with Implications for Their Phylogenetic Relationships and Molecular Dating

by Dong-Mei Li, Hai-Lin Liu, Yan-Gu Pan, Bo Yu, Dan Huang and Gen-Fa Zhu

Int. J. Mol. Sci. 2023, 24(19), 15031; https://doi.org/10.3390/ijms241915031 - 9 Oct 2023

Cited by 2 | Viewed by 1370

Abstract

Zingiberales includes eight families and more than 2600 species, with many species having important economic and ecological value. However, the backbone phylogenetic relationships of Zingiberales still remain controversial, as demonstrated in previous studies, and molecular dating based on chloroplast genomes has not been [...] Read more.

Zingiberales includes eight families and more than 2600 species, with many species having important economic and ecological value. However, the backbone phylogenetic relationships of Zingiberales still remain controversial, as demonstrated in previous studies, and molecular dating based on chloroplast genomes has not been comprehensively studied for the whole order. Herein, 22 complete chloroplast genomes from 21 species in Zingiberales were sequenced, assembled, and analyzed. These 22 genomes displayed typical quadripartite structures, which ranged from 161,303 bp to 163,979 bp in length and contained 111–112 different genes. The genome structures, gene contents, simple sequence repeats, long repeats, and codon usage were highly conserved, with slight differences among these genomes. Further comparative analysis of the 111 complete chloroplast genomes of Zingiberales, including 22 newly sequenced ones and the remaining ones from the national center for biotechnology information (NCBI) database, identified three highly divergent regions comprising ccsA, psaC, and psaC-ndhE. Maximum likelihood and Bayesian inference phylogenetic analyses based on chloroplast genome sequences found identical topological structures and identified a strongly supported backbone of phylogenetic relationships. Cannaceae was sister to Marantaceae, forming a clade that was collectively sister to the clade of (Costaceae, Zingiberaceae) with strong support (bootstrap (BS) = 100%, and posterior probability (PP) = 0.99–1.0); Heliconiaceae was sister to the clade of (Lowiaceae, Strelitziaceae), then collectively sister to Musaceae with strong support (BS = 94–100%, and PP = 0.93–1.0); the clade of ((Cannaceae, Marantaceae), (Costaceae, Zingiberaceae)) was sister to the clade of (Musaceae, (Heliconiaceae, (Lowiaceae, Strelitziaceae))) with robust support (BS = 100%, and PP = 1.0). The results of divergence time estimation of Zingiberales indicated that the crown node of Zingiberales occurred approximately 85.0 Mya (95% highest posterior density (HPD) = 81.6–89.3 million years ago (Mya)), with major family-level lineages becoming from 46.8 to 80.5 Mya. These findings proved that chloroplast genomes could contribute to the study of phylogenetic relationships and molecular dating in Zingiberales, as well as provide potential molecular markers for further taxonomic and phylogenetic studies of Zingiberales. Full article

(This article belongs to the Collection Feature Papers in Molecular Genetics and Genomics)

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13 pages, 2561 KiB

Open AccessArticle

The Relationship between Clock Genes, Sirtuin 1, and Mitochondrial Activity in Head and Neck Squamous Cell Cancer: Effects of Melatonin Treatment

by César Rodríguez-Santana, Alba López-Rodríguez, Laura Martinez-Ruiz, Javier Florido, Olga Cela, Nazzareno Capitanio, Yolanda Ramírez-Casas, Darío Acuña-Castroviejo and Germaine Escames

Int. J. Mol. Sci. 2023, 24(19), 15030; https://doi.org/10.3390/ijms241915030 - 9 Oct 2023

Cited by 2 | Viewed by 1547

Abstract

The circadian clock is a regulatory system, with a periodicity of approximately 24 h, which generates rhythmic changes in many physiological processes, including mitochondrial activity. Increasing evidence links chronodisruption with aberrant functionality in clock gene expression, resulting in multiple diseases such as cancer. [...] Read more.

The circadian clock is a regulatory system, with a periodicity of approximately 24 h, which generates rhythmic changes in many physiological processes, including mitochondrial activity. Increasing evidence links chronodisruption with aberrant functionality in clock gene expression, resulting in multiple diseases such as cancer. Melatonin, whose production and secretion oscillates according to the light–dark cycle, is the principal regulator of clock gene expression. In addition, the oncostatic effects of melatonin correlate with an increase in mitochondrial activity. However, the direct links between circadian clock gene expression, mitochondrial activity, and the antiproliferative effects of melatonin in cancers, including head and neck squamous cell carcinoma (HNSCC), remain largely unknown. In this study, we analyzed the effects of melatonin on HNSCC cell lines (Cal-27 and SCC9), which were treated with 500 and 1000 µM melatonin. We found that the antiproliferative effect of melatonin is not mediated by the Bmal1 clock gene. Additionally, high doses of melatonin were observed to result in resynchronization of oscillatory circadian rhythm genes (Per2 and Sirt1). Surprisingly, the resynchronizing effect of melatonin on Per2 and Sirt1 did not produce alterations in the oscillation of mitochondrial respiratory activity. These results increase our understanding of the possible antiproliferative mechanisms in melatonin in the treatment of head and neck squamous cell carcinoma and suggest that its antiproliferative effects are independent of clock genes but are directly related to mitochondrial activity. Full article

(This article belongs to the Special Issue Molecular Pathology and Novel Therapies for Head and Neck Cancer)

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Figure 1
Relative expression of clock genes in HNSCC cell lines Cal-27 (A,B) and SCC9 (C,D): (A,C) Bmal1 and (B,D) Per2 after serum shock, control, and aMT (500 and 1000 µM) treatments; n = 3–6 independent experiments. The best cosinor fit is shown as a continuous line at a time of 48 h. Full article ">Figure 2
Relative expression of Bmal1 in Cal-27 cells with 10 nM aMT (A) and 100 µM aMT (B) treatments; n = 3–4 independent experiments. The best cosinor fit is shown as a continuous line at a time of 48 h. Full article ">Figure 3
Relative expression of Sirt1 in HNSCC cell lines Cal-27 (A) and SCC9 (B) after serum shock, control, and aMT (500 and 1000 µM) treatments; n = 3–6 independent experiments. The best cosinor fit is shown as a continuous line at a time of 48 h. Full article ">Figure 4
Measurement of mitochondrial respiratory activity in intact Cal-27 cells after serum shock, control, and aMT (500 and 1000 µM) treatments. OCR refers to the resting condition (i.e., endogenous substrate-sustained respiration); n = 3–6 independent experiments. The best cosinor fit is shown as a continuous line. Full article ">Figure 5
Measurement of mitochondrial respiratory activity in intact Cal-27 cells after serum shock, in control and aMT 1000 µM. OCR refers to resting the condition (i.e., endogenous substrate-sustained respiration); n = 3 independent experiments. The best cosinor fit is shown as a continuous line. Full article ">Figure 6
Effect of melatonin on Bmal1-silenced HNSCC cell lines Cal-27 (left) and SCC-9 (right). (A,B) Histogram showing the Bmal1 transcript level attained by q-RT-PCR in HNSCC cells transfected with siRNA-Bmal1. (C,D) The effect of melatonin on cell proliferation assessed by CyQuant assay. Data are presented as the mean ± SEM of n = 3 independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. mock control; # p < 0.05, ### p < 0.001 vs. siRNA-Bmal1 control. Full article ">

19 pages, 8161 KiB

Open AccessArticle

The MADF-BESS Protein CP60 Is Recruited to Insulators via CP190 and Has Redundant Functions in Drosophila

by Larisa Melnikova, Varvara Molodina, Valentin Babosha, Margarita Kostyuchenko, Pavel Georgiev and Anton Golovnin

Int. J. Mol. Sci. 2023, 24(19), 15029; https://doi.org/10.3390/ijms241915029 - 9 Oct 2023

Cited by 2 | Viewed by 1186

Abstract

Drosophila CP190 and CP60 are transcription factors that are associated with centrosomes during mitosis. CP190 is an essential transcription factor and preferentially binds to housekeeping gene promoters and insulators through interactions with architectural proteins, including Su(Hw) and dCTCF. CP60 belongs to a family [...] Read more.

Drosophila CP190 and CP60 are transcription factors that are associated with centrosomes during mitosis. CP190 is an essential transcription factor and preferentially binds to housekeeping gene promoters and insulators through interactions with architectural proteins, including Su(Hw) and dCTCF. CP60 belongs to a family of transcription factors that contain the N-terminal MADF domain and the C-terminal BESS domain, which is characterized by the ability to homodimerize. In this study, we show that the conserved CP60 region adjacent to MADF is responsible for interacting with CP190. In contrast to the well-characterized MADF-BESS transcriptional activator Adf-1, CP60 is recruited to most chromatin sites through its interaction with CP190, and the MADF domain is likely involved in protein–protein interactions but not in DNA binding. The deletion of the Map60 gene showed that CP60 is not an essential protein, despite the strong and ubiquitous expression of CP60 at all stages of Drosophila development. Although CP60 is a stable component of the Su(Hw) insulator complex, the inactivation of CP60 does not affect the enhancer-blocking activity of the Su(Hw)-dependent gypsy insulator. Overall, our results indicate that CP60 has an important but redundant function in transcriptional regulation as a partner of the CP190 protein. Full article

(This article belongs to the Special Issue Molecular Genetics of Drosophila Development)

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CP60 is a component of the Su(Hw) insulator complex: (A) Schematic presentation of the Su(Hw) insulator complex. The previously described interactions [<a href="#B58-ijms-24-15029" class="html-bibr">58</a>] between the components of the complex are shown. The CP190 domains are shown as yellow ovals, with four zinc fingers shown as yellow boxes; the Mod(mdg4)-67.2 domains are shown as green ovals; Su(Hw) domains are shown as lilac boxes. Domain abbreviations: CID—CP190 interacting domain; Ac—C-terminal acidic domain; Zn—zinc-finger domain; LZ—leucine zipper; BTB—BTB/POZ domain; Q—glutamine-rich region; DD—dimerization domain; FLYWCH—FLYWCH-type zinc finger; SID—Su(Hw) interacting domain; D—asparagine-rich domain; M—the microtubule- and centrosome-associated domain; E—glutamine-rich C-terminal domain. Bold capital letters indicate the Su(Hw) binding site. (B) Nuclear extract from Drosophila S2 cells was immunoprecipitated with antibodies against CP60, and the immunoprecipitates (IP) were analyzed via Western blotting for the presence of Su(Hw), Mod(mdg4)-67.2, and CP190 proteins. Input is the input fraction (1% of the lysate used for immunoprecipitation); output is the supernatant after immunoprecipitation; IP is the immunoprecipitate; PI is immunoprecipitation with nonspecific IgG. (C) Nuclear extract from Drosophila S2 cells was immunoprecipitated with antibodies against Su(Hw), Mod(mdg4)-67.2, or CP190, and the immunoprecipitates (IP) were analyzed via Western blotting for the presence of CP60 protein. The uncropped images for (B,C) are shown in <a href="#app1-ijms-24-15029" class="html-app">Supplementary Materials Figure S2</a>. (D) Identification of direct interactions between CP60 and other components of the Su(Hw) insulator complex using the yeast two-hybrid assay. The results are summarized in the table with + and—signs referring to a strong interaction or no interaction, respectively. Interactions with pGBT9 or pGAD424 vectors were used as the negative control. Interactions between CP190 and Mod(mdg4)-67.2 with Su(Hw) were used as a positive control. Full article ">Figure 2
CP190 is responsible for CP60 recruitment to the Su(Hw) chromatin sites: (A) Western blot analysis of RNAi efficiency; wt—S2 cells without treatment; proteins indicated below the line (RNAi) were knocked out by RNAi; antibodies for staining are listed on the left of the panel. Anti-tubulin antibodies (αTub) were used as a loading control. The uncropped images are shown in <a href="#app1-ijms-24-15029" class="html-app">Supplementary Materials Figure S3</a>. (B–E) ChIP-qPCR analysis of binding (B) Su(Hw), (C) CP190, (D) CP60, and (E) Mod(mdg4)-67.2 proteins to the selected Su(Hw) sites in wild-type (wt) S2 cells and after RNAi inactivation of each protein. The ras64B coding region (Ras) was used as a control that does not contain Su(Hw) binding sites. IgG—immunoprecipitation with nonspecific IgG. The percentage recovery of immunoprecipitated DNA (Y axis) was calculated relative to the amount of input DNA. Error bars indicate SDs of quadruplicate PCR measurements from two independent biological samples of chromatin. Asterisks indicate significance levels: * p < 0.05 and ** p < 0.01 (Student’s t-test). Dots on the bar plots indicate the values of individual experiments. Full article ">Figure 3
Analysis of the in vitro association between Su(Hw) or CP60 and Su(Hw) motifs: (A) Schematic representation of the tested Su(Hw) binding sites. The red squares show the localization of the Su(Hw) motifs. (B) The Adf-1 binding site from bxd PRE and purified Adf-1 protein were used in EMSA as the positive control. DNA fragments without protein are marked with a minus (−) sign. The triangle represents a threefold increase in Adf-1 concentration from 0.05 µg to 0.15 µg. (C) EMSA of the binding of Su(Hw) (0.05 µg) and CP60 (0.05 µg) proteins to DNA fragments 50 A, 62 D, 66 E, and 87 E. The Adf-1 binding site was used as the negative control for the Su(Hw) or CP60 proteins. (D) EMSA of the binding of CP60 and Su(Hw) proteins to the gypsy insulator (gypsy) and two copies of the minimal 125 bp 1A2 region (1A2 125 × 2), and 250 bp (1A2 250) and 450 bp (1A2 450) regions of 1A2 insulator site. The amount of Su(Hw) protein was increased threefold from 0.05 µg to 0.15 µg. Other designations are as in (B). (E) Cooperation of Su(Hw), CP190, and CP60 in the binding to the gypsy insulator. The combination of proteins (0.05 µg) is indicated above in the panel. The absence of the corresponding protein in the EMSA probe is marked as a minus (−) sign. (F) EMSA supershift by CP60 antibodies and different combinations of Su(Hw) insulator proteins (0.05 µg). Ab—CP60 antibodies were added to the probe. Other designations as in (E). Full article ">Figure 4
Mapping domains of CP60 that are responsible for homodimerization and interaction with CP190 in the yeast two-hybrid assay. The scale at the top of the figure is in amino acids. A schematic representation of the CP60 protein is shown with deletion margins on the left. The MADF domain is shown as a green rectangle. Conserved regions 1–3 are represented by red rectangles. The fourth C-terminal conserved region is shown as a blue rectangle. The interactions are summarized in columns on the right-hand side with + and − referring to the presence and absence of interactions, respectively. Full article ">Figure 5
Multiple alignment analysis of the CP60 C-terminal conserved region (440–441 aa) sequence with different known BESS domains. Alignment regions are shown as colored boxes or outlines that enclose one or more residue symbols. The residue coloring corresponds to the Clustal X color code (<a href="#app1-ijms-24-15029" class="html-app">Supplementary Table S1</a>). Full article ">Figure 6
Functional analysis of the Map60 gene: (A) CRISPR/Cas9 deletion of the Map60 gene. The CP60 coding regions are shown as orange boxes. White rectangles represent the 5′ and 3′UTR. CRISPR targets are shown as vertical bars. Primer sequences used in the CRISPR/Cas9 genome editing are given in <a href="#app1-ijms-24-15029" class="html-app">Supplementary Table S2</a>. The Red reporter (magenta box), controlled by the 3xP3 promoter (black line), was used for the selection of the Map60 deletion. The attP and lox sites were used for genome manipulation and are shown as a green box and vertical yellow arrows, respectively. SV40 terminator is shown as a blue box. The two black arrows represent primers 1 and 2, which were used to test the obtained mutations using PCR. (B) PCR analysis of genomic DNA from Map60Δ1 lines after CRE/loxP excision of marker dsRed cassette (ΔdsRed). The molecular weight in bp is shown on the right. (C) Western blot analysis (8% SDS PAGE) of protein extracts prepared as described previously [<a href="#B72-ijms-24-15029" class="html-bibr">72</a>] from adult three-day-old males of the wild-type (wt) line and three lines homozygous for Map60Δ1. The membrane was sequentially stained with tested polyclonal rat antibodies against CP60 (αCP60) and antibodies against tubulin (αTub) as loading control. The molecular weight in kDa is shown on the left. The uncropped images are shown in <a href="#app1-ijms-24-15029" class="html-app">Supplementary Materials Figure S7</a>. (D) Polytene chromosomes from the salivary glands of third-instar y2w1118ct6 (wt) and y2w1118ct6; Map60Δ1-1/Map60Δ1-1 (Map60Δ1-1) larvae costained with rat anti-CP60 antibodies (green), rabbit anti-CP190 antibodies (red), and DAPI (blue) Scale bars, 10 μm. (E) ChIP-qPCR analysis of Su(Hw), CP190, and CP60 binding to Su(Hw) sites (description in <a href="#ijms-24-15029-f002" class="html-fig">Figure 2</a>) in wt and Map60Δ1-1 (Δ1) lines. The ras64B coding region (Ras) that did not contain Su(Hw) binding sites was used as a control. IgG—immunoprecipitation with nonspecific IgG. The percentage recovery of immunoprecipitated DNA (Y axis) was calculated relative to the amount of input DNA. Error bars indicate SDs of quadruplicate PCR measurements from two independent biological samples of chromatin. Significance levels: p < 0.05 (Student’s t-test). Dots on the bar plots indicate the values of individual experiments. Full article ">Figure 7
Testing the genome-wide distribution of CP60 and its colocalization with the components of the Su(Hw) complex. Polytene chromosomes from the salivary glands of third-instar (A) y2w1118ct6 (wt) and (B) y2w1118ct6; Cp1902/Cp1903 (Cp1902/Cp1903) larvae costained with rat anti-CP60 antibodies (green), rabbit anti-CP190 or anti-Su(Hw) antibodies (red), and DAPI (blue). Scale bars, 10 μm. Full article ">Figure 8
CP190 is responsible for CP60 recruitment to dCTCF/CP190 and BEAF/CP190 sites. ChIP-qPCR analysis of binding of dCTCF, CP190, and CP60 to the (A) dCTCF (Fab3, Fab4, Fab8, and MCP)- and Pita (Fab7)-dependent sites of the Bitorax complex. (B) BEAF-dependent sites from promoter regions of the aurora, cg3281, and janA genes. (C) Promoter and regulatory regions from the Bitorax complex are not bound by CP190 but are enriched in isoforms of the Mod(mdg4) protein. The ras64B coding region (Ras) was used as a control devoid of dCTCF/CP190- and BEAF/CP190 binding sites. IgG—immunoprecipitation with nonspecific IgG. The percentage recovery of immunoprecipitated DNA (Y axis) was calculated relative to the amount of input DNA. Error bars indicate SDs of quadruplicate PCR measurements from two independent biological samples of chromatin. Significance levels: p < 0.01 (Student’s t-test). Dots on the bar plots indicate the values of individual experiments. Full article ">

15 pages, 2173 KiB

Open AccessReview

The Complex Network of ADP-Ribosylation and DNA Repair: Emerging Insights and Implications for Cancer Therapy

by Ziyuan Li, Aiqin Luo and Bingteng Xie

Int. J. Mol. Sci. 2023, 24(19), 15028; https://doi.org/10.3390/ijms241915028 - 9 Oct 2023

Cited by 1 | Viewed by 1782

Abstract

ADP-ribosylation is a post-translational modification of proteins that plays a key role in various cellular processes, including DNA repair. Recently, significant progress has been made in understanding the mechanism and function of ADP-ribosylation in DNA repair. ADP-ribosylation can regulate the recruitment and activity [...] Read more.

ADP-ribosylation is a post-translational modification of proteins that plays a key role in various cellular processes, including DNA repair. Recently, significant progress has been made in understanding the mechanism and function of ADP-ribosylation in DNA repair. ADP-ribosylation can regulate the recruitment and activity of DNA repair proteins by facilitating protein–protein interactions and regulating protein conformations. Moreover, ADP-ribosylation can influence additional post-translational modifications (PTMs) of proteins involved in DNA repair, such as ubiquitination, methylation, acetylation, phosphorylation, and SUMOylation. The interaction between ADP-ribosylation and these additional PTMs can fine-tune the activity of DNA repair proteins and ensure the proper execution of the DNA repair process. In addition, PARP inhibitors have been developed as a promising cancer therapeutic strategy by exploiting the dependence of certain cancer types on the PARP-mediated DNA repair pathway. In this paper, we review the progress of ADP-ribosylation in DNA repair, discuss the crosstalk of ADP-ribosylation with additional PTMs in DNA repair, and summarize the progress of PARP inhibitors in cancer therapy. Full article

(This article belongs to the Special Issue Molecular Mechanism of DNA Replication and Repair, 2nd Edition )

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36 pages, 9847 KiB

Open AccessArticle

Imidazo-Pyrazole-Loaded Palmitic Acid and Polystyrene-Based Nanoparticles: Synthesis, Characterization and Antiproliferative Activity on Chemo-Resistant Human Neuroblastoma Cells

by Giulia Elda Valenti, Barbara Marengo, Marco Milanese, Guendalina Zuccari, Chiara Brullo, Cinzia Domenicotti and Silvana Alfei

Int. J. Mol. Sci. 2023, 24(19), 15027; https://doi.org/10.3390/ijms241915027 - 9 Oct 2023

Cited by 1 | Viewed by 1395

Abstract

Neuroblastoma (NB) is a childhood cancer, commonly treated with drugs, such as etoposide (ETO), whose efficacy is limited by the onset of resistance. Here, aiming at identifying new treatments for chemo-resistant NB, the effects of two synthesized imidazo-pyrazoles (IMPs) (4G and 4I [...] Read more.

Neuroblastoma (NB) is a childhood cancer, commonly treated with drugs, such as etoposide (ETO), whose efficacy is limited by the onset of resistance. Here, aiming at identifying new treatments for chemo-resistant NB, the effects of two synthesized imidazo-pyrazoles (IMPs) (4G and 4I) were investigated on ETO-sensitive (HTLA-230) and ETO-resistant (HTLA-ER) NB cells, detecting 4I as the more promising compound, that demonstrated IC₅₀ values lower than those of ETO on HTLA ER. Therefore, to further improve the activity of 4I, we developed 4I-loaded palmitic acid (PA) and polystyrene-based (P5) cationic nanoparticles (P5PA-4I NPs) with high drug loading (21%) and encapsulation efficiency (97%), by a single oil-in-water emulsification technique. Biocompatible PA was adopted as an emulsion stabilizer, while synthesized P5 acted as an encapsulating agent, solubilizer and hydrophilic–lipophilic balance (HLB) improver. Optic microscopy and cytofluorimetric analyses were performed to investigate the micromorphology, size and complexity distributions of P5PA-4I NPs, which were also structurally characterized by chemometric-assisted Fourier transform infrared spectroscopy (FTIR). Potentiometric titrations allowed us to estimate the milliequivalents of PA and basic nitrogen atoms present in NPs. P5PA-4I NPs afforded dispersions in water with excellent buffer capacity, essential to escape lysosomal degradation and promote long residence time inside cells. They were chemically stable in an aqueous medium for at least 40 days, while in dynamic light scattering (DLS) analyses, P5PA-4I showed a mean hydrodynamic diameter of 541 nm, small polydispersity (0.194), and low positive zeta potentials (+8.39 mV), assuring low haemolytic toxicity. Biological experiments on NB cells, demonstrated that P5PA-4I NPs induced ROS-dependent cytotoxic effects significantly higher than those of pristine 4I, showing a major efficacy compared to ETO in reducing cell viability in HTLA-ER cells. Collectively, this 4I-based nano-formulation could represent a new promising macromolecular platform to develop a new delivery system able to increase the cytotoxicity of the anticancer drugs. Full article

(This article belongs to the Special Issue New Advances on Photosensitiser or Inhibitors Molecules for Cancer Treatment)

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27 pages, 4838 KiB

Open AccessArticle

Development of Novel Class of Phenylpyrazolo[3,4-d]pyrimidine-Based Analogs with Potent Anticancer Activity and Multitarget Enzyme Inhibition Supported by Docking Studies

by Ahmed K. B. Aljohani, Waheed Ali Zaki El Zaloa, Mohamed Alswah, Mohamed A. Seleem, Mohamed M. Elsebaei, Ashraf H. Bayoumi, Ahmed M. El-Morsy, Mohammed Almaghrabi, Aeshah A. Awaji, Ali Hammad, Marwa Alsulaimany and Hany E. A. Ahmed

Int. J. Mol. Sci. 2023, 24(19), 15026; https://doi.org/10.3390/ijms241915026 - 9 Oct 2023

Cited by 2 | Viewed by 1443

Abstract

Phenylpyrazolo[3,4-d]pyrimidine is considered a milestone scaffold known to possess various biological activities such as antiparasitic, antifungal, antimicrobial, and antiproliferative activities. In addition, the urgent need for selective and potent novel anticancer agents represents a major route in the drug discovery process. [...] Read more.

Phenylpyrazolo[3,4-d]pyrimidine is considered a milestone scaffold known to possess various biological activities such as antiparasitic, antifungal, antimicrobial, and antiproliferative activities. In addition, the urgent need for selective and potent novel anticancer agents represents a major route in the drug discovery process. Herein, new aryl analogs were synthesized and evaluated for their anticancer effects on a panel of cancer cell lines: MCF-7, HCT116, and HePG-2. Some of these compounds showed potent cytotoxicity, with variable degrees of potency and cell line selectivity in antiproliferative assays with low resistance. As the analogs carry the pyrazolopyrimidine scaffold, which looks structurally very similar to tyrosine and receptor kinase inhibitors, the potent compounds were evaluated for their inhibitory effects on three essential cancer targets: EGFR^WT, EGFR^T790M, VGFR2, and Top-II. The data obtained revealed that most of these compounds were potent, with variable degrees of target selectivity and dual EGFR/VGFR2 inhibitors at the IC₅₀ value range, i.e., 0.3–24 µM. Among these, compound 5i was the most potent non-selective dual EGFR/VGFR2 inhibitor, with inhibitory concentrations of 0.3 and 7.60 µM, respectively. When 5i was tested in an MCF-7 model, it effectively inhibited tumor growth, strongly induced cancer cell apoptosis, inhibited cell migration, and suppressed cell cycle progression leading to DNA fragmentation. Molecular docking studies were performed to explore the binding mode and mechanism of such compounds on protein targets and mapped with reference ligands. The results of our studies indicate that the newly discovered phenylpyrazolo[3,4-d]pyrimidine-based multitarget inhibitors have significant potential for anticancer treatment. Full article

(This article belongs to the Special Issue Natural Products and Synthetic Compounds for Drug Development)

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20 pages, 8177 KiB

Open AccessArticle

The Co-Expression Pattern of Calcium-Binding Proteins with γ-Aminobutyric Acid and Glutamate Transporters in the Amygdala of the Guinea Pig: Evidence for Glutamatergic Subpopulations

by Daniel Kalinowski, Krystyna Bogus-Nowakowska, Anna Kozłowska and Maciej Równiak

Int. J. Mol. Sci. 2023, 24(19), 15025; https://doi.org/10.3390/ijms241915025 - 9 Oct 2023

Viewed by 1438

Abstract

The amygdala has large populations of neurons utilizing specific calcium-binding proteins such as parvalbumin (PV), calbindin (CB), or calretinin (CR). They are considered specialized subsets of γ-aminobutyric acid (GABA) interneurons; however, many of these cells are devoid of GABA or glutamate decarboxylase. The [...] Read more.

The amygdala has large populations of neurons utilizing specific calcium-binding proteins such as parvalbumin (PV), calbindin (CB), or calretinin (CR). They are considered specialized subsets of γ-aminobutyric acid (GABA) interneurons; however, many of these cells are devoid of GABA or glutamate decarboxylase. The neurotransmitters used by GABA-immunonegative cells are still unknown, but it is suggested that a part may use glutamate. Thus, this study investigates in the amygdala of the guinea pig relationships between PV, CB, or CR-containing cells and GABA transporter (VGAT) or glutamate transporter type 2 (VGLUT2), markers of GABAergic and glutamatergic neurons, respectively. The results show that although most neurons using PV, CB, and CR co-expressed VGAT, each of these populations also had a fraction of VGLUT2 co-expressing cells. For almost all neurons using PV (~90%) co-expressed VGAT, while ~1.5% of them had VGLUT2. The proportion of neurons using CB and VGAT was smaller than that for PV (~80%), while the percentage of cells with VGLUT2 was larger (~4.5%). Finally, only half of the neurons using CR (~53%) co-expressed VGAT, while ~3.5% of them had VGLUT2. In conclusion, the populations of neurons co-expressing PV, CB, and CR are in the amygdala, primarily GABAergic. However, at least a fraction of neurons in each of them co-express VGLUT2, suggesting that these cells may use glutamate. Moreover, the number of PV-, CB-, and CR-containing neurons that may use glutamate is probably larger as they can utilize VGLUT1 or VGLUT3, which are also present in the amygdala. Full article

(This article belongs to the Section Molecular Neurobiology)

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(Modified) All figures were published in Równiak et al. [<a href="#B27-ijms-24-15025" class="html-bibr">27</a>]. Brightfield photomicrographs highlighting the patterns of parvalbumin (PV, A–A″), calbindin (CB, B–B″), and calretinin (CR, C–C″) immunoreactivity in the amygdala of the male guinea pig. The scale bar applies to all microphotographs, and it corresponds to the length of 2000 μm (A–C) and 200 μm (A′–C′, A″–C″). LAdl–lateral nucleus, lateral part, LAm–lateral nucleus, medial part, LAvl–lateral nucleus, ventral part, BLa–basomedial nucleus, anterior part, BMa–basomedial nucleus, anterior part, MEd–medial nucleus, dorsal part, CEm–central nucleus, medial part, CEl–central nucleus, lateral part, COP–cortical nucleus, PAC–piriform-amygdalar area. Noted that nuclei are indicated by the dotted line. Full article ">Figure 2
The volume density of vesicular GABA transporter (VGAT, A) and vesicular glutamate transporter type 2 (VGLUT2, B) in the “cortex-like” and “striatum-pallidum-like” amygdala of the guinea pig (n = 5). Note significant differences between “cortex-like” and “stratum-pallidum-like” nuclei (A1–A4). Data are expressed as box-and-whiskers plots, with the “box” depicting the median and the 25th and 75th quartiles and the “whiskers” showing the 5th and 95th percentile. * (p ≤ 0.05), ** (p ≤ 0.01), and *** (p ≤ 0.001) indicate statistically significant differences between studied nuclei analyzed by Student’s t-test, whereas ns means none statistically significant. Elements–immunoreactive somata, fibers, and neuropil. LA—lateral nucleus, BL—basolateral nucleus, BM—basomedial nucleus, CO—cortical nucleus, CE—central nucleus, ME—medial nucleus. Full article ">Figure 3
Representative color photomicrographs illustrating immunoreactivity of vesicular GABA transporter (VGAT, A–C) in the amygdala of guinea pig. The scale bar applies to all microphotographs, and it corresponds to the length of 200 μm. LA—lateral nucleus, BL—basolateral nucleus, BM—basomedial nucleus as a “cortex-like” amygdala, CE—central nucleus, ME—medial nucleus as a “striatum-pallidum-like” amygdala. Noted that nuclei are indicated by the dotted line. Full article ">Figure 4
Representative color photomicrographs illustrating the co-expression pattern of PV (A–A‴), CB (B–B‴), and CR (C–C‴) neurons with VGAT in the basolateral (A–A‴), basomedial (B–B‴) and cortical (C–C‴) nuclei as a “cortex-like” amygdala of the guinea pig. Arrows indicate double-labeled cells. Boxed regions in (A″–C″) are shown at a greater magnification in (A‴–C‴). The scale bar applies to all microphotographs, and it corresponds to the length of 50 μm. Full article ">Figure 5
Representative color photomicrographs illustrating the co-expression pattern of CB (A–A‴) and CR (B–B‴) neurons with VGAT in the medial nucleus (A–B‴) as a “striatum-pallidum-like” amygdala of the guinea pig. Arrows indicate double-labeled cells. Boxed regions in (A″,B″) are shown at a greater magnification in (A‴,B‴). The scale bar applies to all microphotographs, and it corresponds to the length of 50 μm. Full article ">Figure 6
Representative color photomicrographs illustrating the co-expression pattern of PV (A–A‴), CB (B–B‴), and CR (C–C‴) neurons with VGLUT2 in the basolateral (A–A‴,C–C‴) and cortical (B–B‴) nuclei as a “cortex-like” amygdala of the guinea pig. Arrows indicate double-labeled cells. Boxed regions in (A″–C″) are shown at a greater magnification in (A‴–C‴). The scale bar applies to all microphotographs, and it corresponds to the length of 50 μm. Full article ">Figure 7
Representative color photomicrographs illustrating the co-expression pattern of CB (A–A‴) and CR (B–B‴) neurons with VGLUT2 in the central (A–A‴) and medial (B–B‴) nuclei as a “striatum-pallidum-like” amygdala of the guinea pig. Arrows indicate double-labeled cells. Boxed regions in (A″,B″) are shown at a greater magnification in (A‴,B‴). The scale bar applies to all microphotographs, and it corresponds to the length of 50 μm. Full article ">

21 pages, 3592 KiB

Open AccessArticle

Multiple Sclerosis-Associated Gut Microbiome in the Israeli Diverse Populations: Associations with Ethnicity, Gender, Disability Status, Vitamin D Levels, and Mediterranean Diet

by Zehavit Nitzan, Elsebeth Staun-Ram, Anat Volkowich and Ariel Miller

Int. J. Mol. Sci. 2023, 24(19), 15024; https://doi.org/10.3390/ijms241915024 - 9 Oct 2023

Cited by 2 | Viewed by 1611

Abstract

Microbiome dysbiosis is increasingly being recognized as implicated in immune-mediated disorders including multiple sclerosis (MS). The microbiome is modulated by genetic and environmental factors including lifestyle, diet, and drug intake. This study aimed to characterize the MS-associated gut microbiome in the Israeli populations [...] Read more.

Microbiome dysbiosis is increasingly being recognized as implicated in immune-mediated disorders including multiple sclerosis (MS). The microbiome is modulated by genetic and environmental factors including lifestyle, diet, and drug intake. This study aimed to characterize the MS-associated gut microbiome in the Israeli populations and to identify associations with demographic, dietary, and clinical features. The microbiota from 57 treatment-naive patients with MS (PwMS) and 43 age- and gender-matched healthy controls (HCs) was sequenced and abundance compared. Associations between differential microbes with demographic or clinical characteristics, as well as diet and nutrient intake, were assessed. While there was no difference in α- or β-diversity of the microbiome, we identified 40 microbes from different taxonomic levels that differ in abundance between PwMS and HCs, including Barnesiella, Collinsella, Egerthella, Mitsuokella, Olsenella Romboutsia, and Succinivibrio, all enhanced in PwMS, while several members of Lacnospira were reduced. Additional MS-differential microbes specific to ethnicity were identified. Several MS-specific microbial patterns were associated with gender, vitamin D level, Mediterranean diet, nutrient intake, or disability status. Thus, PwMS have altered microbiota composition, with distinctive patterns related to geographic locations and population. Microbiome dysbiosis seem to be implicated in disease progression, gender-related differences, and vitamin D-mediated immunological effects recognized in MS. Dietary interventions may be beneficial in restoring a “healthy microbiota” as part of applying comprehensive personalized therapeutic strategies for PwMS. Full article

(This article belongs to the Special Issue Molecular Mechanism in Multiple Sclerosis and Related Disorders)

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General microbiome composition. (A): Relative composition at the class level. (B): Alpha-diversity between MS and HC calculated by Shannon index at the OTU level. (C): Beta-diversity between MS and HC calculated by Bray–Curtis dissimilarity at the OTU level. MS—multiple sclerosis. Full article ">Figure 2
Differential abundant taxa between PwMS and HCs. (A)—Representative graphs of taxa with significant difference in relative abundance, as determined by at least two out of three tools (DESeq2, MetagenomeSeq, and EdgeR) at different taxonomic levels (FDR < 0.1). Each black dot represents the relative abundance of a participant sample. (B)—Linear discriminant analysis (LDA) score of significant different taxa as determined by LDA effect size (LefSE) between PwMS and HCs at different taxonomic levels (p < 0.05). (C)—Venn diagram presenting the overlap of differentially abundant taxa identified in the analysis of all samples, Jewish participants or Arab participants. HC—healthy control, MS—multiple sclerosis. Full article ">Figure 3
Associations between MS-differentially abundant microbiota, EDSS, vitamin D, and gender. (A)—Representative graphs of MS-differentially abundant taxa, which significantly correlates with EDSS (Spearman correlation, adjusted for BMI). (B)—Representative graphs of MS-differentially abundant taxa, which significantly correlates with serum vitamin D level shown as continuous or divided into 3 groups: <50 nMol/L—vitamin D insufficiency, 50–75 nMol/L—vitamin D mild deficiency, >75 nMol/L—vitamin D sufficient (Spearman correlation, adjusted for BMI). (C)—Representative graphs of MS-differentially abundant taxa enriched in PwMS (I) or reduced in PwMS (II), which differ significantly between females and males. Figure presents FDR calculated from EdgeR, * FDR from DeSeq2. Each black dot represents the relative abundance of a participant sample. Full article ">Figure 4
Correlations between MS-differentially abundant microbiota and dietary data. (A)—The distribution of adherence to a Mediterranean diet (MDS) in PwMS and HCs (Kruskal–Wallis test). (B)—Alpha-diversity Shannon index and beta-diversity Bray–Curtis dissimilarity according to high, low, or intermediate MDS (OTU level). (C)—Representative graphs of differentially abundant taxa, enriched in PwMS (I) or reduced in PwMS (II) which significantly correlates with MDS (Spearman correlation). MDS shown as continuous or divided into 3 groups (MDS low (1–6 points), intermediate (7–11 points), high (2–17 points). MDS—Mediterranean diet score. Full article ">Figure 5
Dietary recommendations. Potential beneficial dietary recommendations for modulation and “repair” of altered microbiota abundance in PwMS, based upon correlations between nutrients and MS-differentially abundant taxa. Full article ">Figure 6
Predicted functional pathways. (A). Main KEGG metabolism pathways in the MS and healthy control cohorts (B). KOs discriminating between PwMS and HCs according to LEfSe at FDR < 0.1. KEGG—Kyoto Encyclopedia of Genes and Genomes, LEfSe: linear discriminant analysis effect size. Full article ">

26 pages, 2408 KiB

Open AccessReview

Aggregation, Transmission, and Toxicity of the Microtubule-Associated Protein Tau: A Complex Comprehension

by Jiaxin Hu, Wenchi Sha, Shuangshuang Yuan, Jiarui Wu and Yunpeng Huang

Int. J. Mol. Sci. 2023, 24(19), 15023; https://doi.org/10.3390/ijms241915023 - 9 Oct 2023

Cited by 5 | Viewed by 2466

Abstract

The microtubule-associated protein tau is an intrinsically disordered protein containing a few short and transient secondary structures. Tau physiologically associates with microtubules (MTs) for its stabilization and detaches from MTs to regulate its dynamics. Under pathological conditions, tau is abnormally modified, detaches from [...] Read more.

The microtubule-associated protein tau is an intrinsically disordered protein containing a few short and transient secondary structures. Tau physiologically associates with microtubules (MTs) for its stabilization and detaches from MTs to regulate its dynamics. Under pathological conditions, tau is abnormally modified, detaches from MTs, and forms protein aggregates in neuronal and glial cells. Tau protein aggregates can be found in a number of devastating neurodegenerative diseases known as “tauopathies”, such as Alzheimer’s disease (AD), frontotemporal dementia (FTD), corticobasal degeneration (CBD), etc. However, it is still unclear how the tau protein is compacted into ordered protein aggregates, and the toxicity of the aggregates is still debated. Fortunately, there has been considerable progress in the study of tau in recent years, particularly in the understanding of the intercellular transmission of pathological tau species, the structure of tau aggregates, and the conformational change events in the tau polymerization process. In this review, we summarize the concepts of tau protein aggregation and discuss the views on tau protein transmission and toxicity. Full article

(This article belongs to the Section Molecular Biology)

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Tau isoforms. Six isoforms of microtubule-associated protein tau (MAPT) are generated through alternative splicing. In the human brain, splicing of exons 2 and 3 results in three isoforms with 2, 1, or no N-terminus insertion of 29 amino acids (referred to as 2N, 1N, and 0N). Each isoform contains a microtubule binding domain encoded by exon 10 that consists of either three repeats (3R) or four repeats (4R), resulting in the genesis of six isoforms (2N4R, 1N4R, 0N4R, 2N3R, 1N3R, and 0N3R). Full article ">Figure 2
A stepwise model elucidating the process of tau aggregation. The microtubule-associated protein tau is physiologically associated with the tubulin heterodimer to maintain the stability of microtubules. Following post-translational modifications (PTMs) such as phosphorylation, tau undergoes dissociation from the tubulin heterodimer. The dissociation of tau monomers and the naturally unfolded “paper clip” tau species undergo a series of mis-sorting processes, resulting in the dendritic and somatic mislocalization of tau. This further facilitates PTMs and liquid–liquid phase separation (LLPS). The presence of PTMs and LLPS further facilitates the conformational changes in Tau, thereby exposing its aggregation-prone motifs. The intermolecular interface of tau facilitates the nucleation and formation of tau dimers and trimers, which serve as seeds to promote gradual polymerization through the extension of aggregates by the addition of tau monomers, ultimately resulting in the formation of oligomers and filaments that accompany conformational changes. Full article ">Figure 3
Post-translational modifications (PTMs) occur on the tau protein. The amino acid sequence of the longest tau isoform, 2N4R, is presented here. Distinctive colored dots are utilized to represent various PTMs, while the amino acids associated with tau pathology are highlighted in yellow. Full article ">Figure 4
Tau transmission. The transmission of tau is initiated with the release of pathological tau species, including monomers, dimers, oligomers, and NFTs. This process is facilitated by (1) the direct translocation across the plasma membrane, (2) membranous organelle-based unconventional secretion (MOBS), (3) shedding of microvesicles derived from the plasma membrane, and (4) nanotube-mediated transfer. The extracellular tau and tau-containing vesicles can be internalized by (5) translocation across the membrane, (6) micropinocytosis, and (7) endocytosis. Subsequently, they can be (8) released into the recipient cells, (9) seeding the endogenous tau to undergo conformational change and polymerization. Full article ">Figure 5
The toxic effects of tau aggregates. The deleterious effects of Tau aggregates encompass a variety of biological processes, including destabilization of microtubules and cytoskeletons, impairment of axonal transport and synaptic plasticity, disruption of mitochondrial function and proteostasis, hyperactivation of microglia, and induction of neuronal cell death, among others. Full article ">

15 pages, 7155 KiB

Open AccessArticle

Robust AMBER Force Field Parameters for Glutathionylated Cysteines

by Zineb Elftmaoui and Emmanuelle Bignon

Int. J. Mol. Sci. 2023, 24(19), 15022; https://doi.org/10.3390/ijms241915022 - 9 Oct 2023

Viewed by 1033

Abstract

S-glutathionylation is an oxidative post-translational modification, which is involved in the regulation of many cell signaling pathways. Increasing amounts of studies show that it is crucial in cell homeostasis and deregulated in several pathologies. However, the effect of S-glutathionylation on proteins’ structure and [...] Read more.

S-glutathionylation is an oxidative post-translational modification, which is involved in the regulation of many cell signaling pathways. Increasing amounts of studies show that it is crucial in cell homeostasis and deregulated in several pathologies. However, the effect of S-glutathionylation on proteins’ structure and activity is poorly understood, and a drastic lack of structural information at the atomic scale remains. Studies based on the use of molecular dynamics simulations, which can provide important information about modification-induced modulation of proteins’ structure and function, are also sparse, and there is no benchmarked force field parameters for this modified cysteine. In this contribution, we provide robust AMBER parameters for S-glutathionylation, which we tested extensively against experimental data through a total of 33

μ

s molecular dynamics simulations. We show that our parameter set efficiently describes the global and local structural properties of S-glutathionylated proteins. These data provide the community with an important tool to foster new investigations into the effect of S-glutathionylation on protein dynamics and function, in a common effort to unravel the structural mechanisms underlying its critical role in cellular processes. Full article

(This article belongs to the Section Physical Chemistry and Chemical Physics)

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(A) Structure of a glutathionylated cysteine. The glycine, cysteine, and glutamate residues within the glutathionylation moiety (SSG) are labeled in grey. The target cysteine backbone atoms are displayed as if embedded in a protein (without capping). (B) A few examples of proteins regulated by S-glutathionylation, with respect to their function [<a href="#B7-ijms-24-15022" class="html-bibr">7</a>,<a href="#B18-ijms-24-15022" class="html-bibr">18</a>,<a href="#B19-ijms-24-15022" class="html-bibr">19</a>]. Full article ">Figure 2
Boxplots of the RMSD values in Å of each system, considering all atoms (top) or the residues’ backbone atoms (bottom). RMSD values for dimers are shown by monomer (Mono 1 or Mono 2). The black lines inside each boxplot display the median values. The average and standard deviation values are written above each box. Full article ">Figure 3
Representative structures of the two main conformations of hLyso as calculated from the cluster analysis of MD ensembles. The structures of the crystal reference and from the AF and crys simulations are displayed in gray, cyan, and blue, respectively. The percentage of occurrence of each cluster (primary on the left and secondary on the right) is also given. Full article ">Figure 4
Boxplots of the S-glutathionylated cysteine (CSG) RMSD values in Å for each system. RMSD values for dimers are shown by monomer (Mono 1 or Mono 2). The black lines inside each boxplot display the median values. Average and standard deviation values are written above each box. Full article ">Figure 5
Times series of CSG RMSD values in Å of the three replicates for AF simulations of AtGrxC5, hGSTO2, PtGSTL1, PtGSTL3, PtGrx12, PtGSTF1, ScGrx2, and TvGSTO2C, which show better trends. Plots for the dimeric systems (hGSTO2, PtGSTF1, and TvGSTO2C) are paired in the columns, with CSG1 and CSG2 referring to Monomers 1 and 2, respectively. Full article ">Figure 6
(A) Representative structures of the major conformations of the CSG in AtGrxC5, TvGSTO2C Monomer 1, PtGSTL3, hLyso, ScTrx1, and PtTrxL2.1 as calculated from the cluster analysis of MD ensembles. Structures of the crystal reference and from the AF and crys simulations are displayed in gray, cyan, and blue, respectively. The percentage of occurrence of the clusters is also given. (B) Times series of the CSG RMSD values in Å of the three replicates for the AF (left) and crys (right) simulations of hLyso, ScTrx1, and PtTrxL2.1. Full article ">Figure 7
(A) CSG structure, highlighting the side chain atoms used to monitor hydrogen bonds. (B–D) Distribution of distances corresponding to the native interactions identified in the reference structures of hLyso, PtTrxL2.1, and ScTrx1, respectively. Both distributions for AF (cyan) and crys (blue) simulations are shown. Cation–<math display="inline"><semantics> <mi>π</mi> </semantics></math> interactions involving Y63 in hLyso are monitored as distances between CSG.N1 and the center of mass of the aromatic ring heavy atoms of Y63 (Y63.Ph). Full article ">

60 pages, 22427 KiB

Open AccessReview

Graphitic Carbon Nitride/Zinc Oxide-Based Z-Scheme and S-Scheme Heterojunction Photocatalysts for the Photodegradation of Organic Pollutants

by Gopal Panthi and Mira Park

Int. J. Mol. Sci. 2023, 24(19), 15021; https://doi.org/10.3390/ijms241915021 - 9 Oct 2023

Cited by 5 | Viewed by 2416

Abstract

Graphitic carbon nitride (g-C₃N₄), a metal-free polymer semiconductor, has been recognized as an attractive photocatalytic material for environmental remediation because of its low band gap, high thermal and photostability, chemical inertness, non-toxicity, low cost, biocompatibility, and optical and electrical [...] Read more.

Graphitic carbon nitride (g-C₃N₄), a metal-free polymer semiconductor, has been recognized as an attractive photocatalytic material for environmental remediation because of its low band gap, high thermal and photostability, chemical inertness, non-toxicity, low cost, biocompatibility, and optical and electrical efficiency. However, g-C₃N₄ has been reported to suffer from many difficulties in photocatalytic applications, such as a low specific surface area, inadequate visible-light utilization, and a high charge recombination rate. To overcome these difficulties, the formation of g-C₃N₄ heterojunctions by coupling with metal oxides has triggered tremendous interest in recent years. In this regard, zinc oxide (ZnO) is being largely explored as a self-driven semiconductor photocatalyst to form heterojunctions with g-C₃N₄, as ZnO possesses unique and fascinating properties, including high quantum efficiency, high electron mobility, cost-effectiveness, environmental friendliness, and a simple synthetic procedure. The synergistic effect of its properties, such as adsorption and photogenerated charge separation, was found to enhance the photocatalytic activity of heterojunctions. Hence, this review aims to compile the strategies for fabricating g-C₃N₄/ZnO-based Z-scheme and S-scheme heterojunction photocatalytic systems with enhanced performance and overall stability for the photodegradation of organic pollutants. Furthermore, with reference to the reported system, the photocatalytic mechanism of g-C₃N₄/ZnO-based heterojunction photocatalysts and their charge-transfer pathways on the interface surface are highlighted. Full article

(This article belongs to the Special Issue Catalysts: Design, Synthesis, and Molecular Applications)

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20 pages, 4935 KiB

Open AccessArticle

3β-Hydroxy-12-oleanen-27-oic Acid Exerts an Antiproliferative Effect on Human Colon Carcinoma HCT116 Cells via Targeting FDFT1

by Jue Tu, Xiang Meng, Juanjuan Wang, Ziyi Han, Zuoting Yu and Hongxiang Sun

Int. J. Mol. Sci. 2023, 24(19), 15020; https://doi.org/10.3390/ijms241915020 - 9 Oct 2023

Cited by 2 | Viewed by 1578

Abstract

3β-hydroxy-12-oleanen-27-oic acid (ATA), a cytotoxic oleanane triterpenoid with C14-COOH isolated from the rhizome of Astilbe chinensis, has been previously proven to possess antitumor activity and may be a promising antitumor agent. However, its molecular mechanisms of antitumor action were still [...] Read more.

3β-hydroxy-12-oleanen-27-oic acid (ATA), a cytotoxic oleanane triterpenoid with C14-COOH isolated from the rhizome of Astilbe chinensis, has been previously proven to possess antitumor activity and may be a promising antitumor agent. However, its molecular mechanisms of antitumor action were still unclear. This study explored the underlying mechanisms of cytotoxicity and potential target of ATA against human colorectal cancer HCT116 cells via integrative analysis of transcriptomics and network pharmacology in combination with in vitro and in vivo experimental validations. ATA significantly inhibited the proliferation of HCT116 cells in a concentration- and time-dependent manner and induced the cell cycle arrest at the G0/G1 phase, apoptosis, autophagy, and ferroptosis. Transcriptomic analysis manifested that ATA regulated mRNA expression of the genes related to cell proliferation, cell cycle, and cell death in HCT116 cells. The integrated analysis of transcriptomics, network pharmacology, and molecular docking revealed that ATA exerted cytotoxic activity via interactions with FDFT1, PPARA, and PPARG. Furthermore, FDFT1 was verified to be an upstream key target mediating the antiproliferative effect of ATA against HCT116 cells. Of note, ATA remarkably suppressed the growth of HCT116 xenografts in nude mice and displayed an apparent attenuation of FDFT1 in tumor tissues accompanied by the alteration of the biomarkers of autophagy, cell cycle, apoptosis, and ferroptosis. These results demonstrate that ATA exerted in vitro and in vivo antiproliferative effects against HCT116 cells through inducing cell apoptosis, autophagy, and ferroptosis via targeting FDFT1. Full article

(This article belongs to the Section Molecular Immunology)

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19 pages, 673 KiB

Open AccessReview

Novel Approaches to Treatment of Acute Myeloid Leukemia Relapse Post Allogeneic Stem Cell Transplantation

by Carmine Liberatore and Mauro Di Ianni

Int. J. Mol. Sci. 2023, 24(19), 15019; https://doi.org/10.3390/ijms241915019 - 9 Oct 2023

Cited by 3 | Viewed by 2690

Abstract

The management of patients with acute myeloid leukemia (AML) relapsed post allogeneic hematopoietic stem cell transplantation (HSCT) remains a clinical challenge. Intensive treatment approaches are limited by severe toxicities in the early post-transplantation period. Therefore, hypomethylating agents (HMAs) have become the standard therapeutic [...] Read more.

The management of patients with acute myeloid leukemia (AML) relapsed post allogeneic hematopoietic stem cell transplantation (HSCT) remains a clinical challenge. Intensive treatment approaches are limited by severe toxicities in the early post-transplantation period. Therefore, hypomethylating agents (HMAs) have become the standard therapeutic approach due to favorable tolerability. Moreover, HMAs serve as a backbone for additional anti-leukemic agents. Despite discordant results, the addition of donor lymphocytes infusions (DLI) generally granted improved outcomes with manageable GvHD incidence. The recent introduction of novel targeted drugs in AML gives the opportunity to add a third element to salvage regimens. Those patients harboring targetable mutations might benefit from IDH1/2 inhibitors Ivosidenib and Enasidenib as well as FLT3 inhibitors Sorafenib and Gilteritinib in combination with HMA and DLI. Conversely, patients lacking targetable mutations actually benefit from the addition of Venetoclax. A second HSCT remains a valid option, especially for fit patients and for those who achieve a complete disease response with salvage regimens. Overall, across studies, higher response rates and longer survival were observed in cases of pre-emptive intervention for molecular relapse. Future perspectives currently rely on the development of adoptive immunotherapeutic strategies mainly represented by CAR-T cells. Full article

(This article belongs to the Special Issue Pathophysiology to Novel Therapeutic Approaches for Leukemia)

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17 pages, 3809 KiB

Open AccessCommunication

The Effects of Combined Exposure to Bisphenols and Perfluoroalkyls on Human Perinatal Stem Cells and the Potential Implications for Health Outcomes

by Andrea Di Credico, Giulia Gaggi, Ines Bucci, Barbara Ghinassi and Angela Di Baldassarre

Int. J. Mol. Sci. 2023, 24(19), 15018; https://doi.org/10.3390/ijms241915018 - 9 Oct 2023

Cited by 3 | Viewed by 1317

Abstract

The present study investigates the impact of two endocrine disruptors, namely Bisphenols (BPs) and Perfluoroalkyls (PFs), on human stem cells. These chemicals leach from plastic, and when ingested through contaminated food and water, they interfere with endogenous hormone signaling, causing various diseases. While [...] Read more.

The present study investigates the impact of two endocrine disruptors, namely Bisphenols (BPs) and Perfluoroalkyls (PFs), on human stem cells. These chemicals leach from plastic, and when ingested through contaminated food and water, they interfere with endogenous hormone signaling, causing various diseases. While the ability of BPs and PFs to cross the placental barrier and accumulate in fetal serum has been documented, the exact consequences for human development require further elucidation. The present research work explored the effects of combined exposure to BPs (BPA or BPS) and PFs (PFOS and PFOA) on human placenta (fetal membrane mesenchymal stromal cells, hFM-MSCs) and amniotic fluid (hAFSCs)-derived stem cells. The effects of the xenobiotics were assessed by analyzing cell proliferation, mitochondrial functionality, and the expression of genes involved in pluripotency and epigenetic regulation, which are crucial for early human development. Our findings demonstrate that antenatal exposure to BPs and/or PFs may alter the biological characteristics of perinatal stem cells and fetal epigenome, with potential implications for health outcomes at birth and in adulthood. Further research is necessary to comprehend the full extent of these effects and their long-term consequences. Full article

(This article belongs to the Special Issue Endocrine Disruptors Exposure and Human Health)

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11 pages, 3887 KiB

Open AccessArticle

Enhancing the Heterologous Expression of a Thermophilic Endoglucanase and Its Cost-Effective Production in Pichia pastoris Using Multiple Strategies

by Wuling Dai, Haofan Dong, Zhaokun Zhang, Xin Wu, Tongtong Bao, Le Gao and Xiaoyi Chen

Int. J. Mol. Sci. 2023, 24(19), 15017; https://doi.org/10.3390/ijms241915017 - 9 Oct 2023

Cited by 1 | Viewed by 1525

Abstract

Although Pichia pastoris was successfully used for heterologous gene expression for more than twenty years, many factors influencing protein expression remain unclear. Here, we optimized the expression of a thermophilic endoglucanase from Thermothielavioides terrestris (TtCel45A) for cost-effective production in Pichia pastoris. To [...] Read more.

Although Pichia pastoris was successfully used for heterologous gene expression for more than twenty years, many factors influencing protein expression remain unclear. Here, we optimized the expression of a thermophilic endoglucanase from Thermothielavioides terrestris (TtCel45A) for cost-effective production in Pichia pastoris. To achieve this, we established a multifactorial regulation strategy that involved selecting a genome-editing system, utilizing neutral loci, incorporating multiple copies of the heterologous expression cassette, and optimizing high-density fermentation for the co-production of single-cell protein (SCP). Notably, even though all neutral sites were used, there was still a slight difference in the enzymatic activity of heterologously expressed TtCel45A. Interestingly, the optimal gene copy number for the chromosomal expression of TtCel45A was found to be three, indicating limitations in translational capacity, post-translational processing, and secretion, ultimately impacting protein yields in P. pastoris. We suggest that multiple parameters might influence a kinetic competition between protein elongation and mRNA degradation. During high-density fermentation, the highest protein concentration and endoglucanase activity of TtCel45A with three copies reached 15.8 g/L and 9640 IU/mL, respectively. At the same time, the remaining SCP of P. pastoris exhibited a crude protein and amino acid content of up to 59.32% and 46.98%, respectively. These findings suggested that SCP from P. pastoris holds great promise as a sustainable and cost-effective alternative for meeting the global protein demand, while also enabling the production of thermophilic TtCel45A in a single industrial process. Full article

(This article belongs to the Special Issue Microbial Enzymes for Biotechnological Applications)

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Figure 1
Overall structure of TtCel45A from Thermothielavioides terrestris. (a) The three-dimensional structure of Cel45A from Thermothielavioides terrestris. (b) Six disulfide bonds in the homology model of TtCel45a. Full article ">Figure 2
Percent of cells successfully transformed with the GFP expression cassette using (a) CRISPR-Cas9 and (b) homologous recombination, as detected via flow cytometry. Full article ">Figure 3
Comparison of TtCel45A expression in transformants with different copy numbers of the genomically integrated expression cassette. (a) Comparison of endoglucanase activity of transformants with different copy numbers. “*” for p < 0.05, “**” for p < 0.001, and “ns” for no significance. (b) SDS-PAGE of TtCel45A expression in transformants with different copy numbers. Red marker for 72KDa. Full article ">Figure 4
Analysis of fed-batch fermentation and nutritional content of P. pastoris biomass after fermentation. (a) Fed-batch fermentation of P. pastoris in a 5 L bioreactor. (b) Analysis of endoglucanase activity during fermentation. (c) Amino acid profile of single-cell protein from P. pastoris co-produced in fermentation. (d) High-cell-density fermentation of TtCel45A in a 5 L bioreactor. Full article ">

19 pages, 3403 KiB

Open AccessReview

Autophagy as a Target for Non-Immune Intrinsic Functions of Programmed Cell Death-Ligand 1 in Cancer

by Blanca Estela García-Pérez, Christian Pérez-Torres, Shantal Lizbeth Baltierra-Uribe, Juan Castillo-Cruz and Nayeli Shantal Castrejón-Jiménez

Int. J. Mol. Sci. 2023, 24(19), 15016; https://doi.org/10.3390/ijms241915016 - 9 Oct 2023

Cited by 5 | Viewed by 1818

Abstract

Autophagy is a catabolic process that is essential to the maintenance of homeostasis through the cellular recycling of damaged organelles or misfolded proteins, which sustains energy balance. Additionally, autophagy plays a dual role in modulating the development and progression of cancer and inducing [...] Read more.

Autophagy is a catabolic process that is essential to the maintenance of homeostasis through the cellular recycling of damaged organelles or misfolded proteins, which sustains energy balance. Additionally, autophagy plays a dual role in modulating the development and progression of cancer and inducing a survival strategy in tumoral cells. Programmed cell death-ligand 1 (PD-L1) modulates the immune response and is responsible for maintaining self-tolerance. Because tumor cells exploit the PD-L1–PD-1 interaction to subvert the immune response, immunotherapy has been developed based on the use of PD-L1-blocking antibodies. Recent evidence has suggested a bidirectional regulation between autophagy and PD-L1 molecule expression in tumor cells. Moreover, the research into the intrinsic properties of PD-L1 has highlighted new functions that are advantageous to tumor cells. The relationship between autophagy and PD-L1 is complex and still not fully understood; its effects can be context-dependent and might differ between tumoral cells. This review refines our understanding of the non-immune intrinsic functions of PD-L1 and its potential influence on autophagy, how these could allow the survival of tumor cells, and what this means for the efficacy of anti-PD-L1 therapeutic strategies. Full article

(This article belongs to the Special Issue Recent Research on Autophagy)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Schematic representation of PD-L1. PD-L1 is a transmembrane glycoprotein comprising 290 amino acids, grouped into two domains (IgV and IgC), followed by a short flexible stem. It has a hydrophobic transmembrane (TM) domain, as well as a small intracellular (IC) domain of 30 amino acids. The 3D image shown is from the Protein Data Bank and is available at <a href="https://www.rcsb.org/structure/3bis" target="_blank">https://www.rcsb.org/structure/3bis</a> (accessed on 21 September 2023). Full article ">Figure 2
The main mechanisms that modulate PD-L1 expression in cancer cells: In the tumor microenvironment, several signals can induce PD-L1 overexpression through the activation of downstream effectors. (1) Hypoxia-inducible factor 1α (HIF-1α), which can also be induced by other stimuli, such as growth factors, has been associated with the expression of PD-L1. (2) Some chemotherapeutic agents, such as paclitaxel or cisplatin, through the phosphorylation of ERK ½, induce the expression of PD-L1; however, the complete mechanisms behind this event remain unknown. (3) The activation of mTOR through various signals, such as growth factors, triggers signaling cascades that induce the expression of PD-L1, which is decreased when the mTOR inhibitor rapamycin is used. (4) Cytokines, such as IFN-γ, induce PD-L1 expression through the JAK/STAT pathway. (5) The stimulation of some TLRs induces the activation of canonical pathways to regulate PD-L1 overexpression. Green arrows mean activation vias, red block lines mean inhibition pathways, dotted arrows mean nuclear translocation, black arrow means decreasing. Full article ">Figure 3
A schematic representation of the bidirectional autophagy/PD-L1 relationship: The right side shows the main findings related to the effects of the activation (A) or inhibition (B) of autophagy on PD-L1 expression. The left side represents the effects of the intrinsic non-immune functions of PD-L1 and the known downstream molecules that culminate in the disruption (C) or activation (D) of autophagy. Green arrows mean activation vias, red block lines mean inhibition pathways, red dotted line means hypothetical pathway, black arrows aiming up mean increasing, black arrows aiming down mean decreasing. Full article ">

11 pages, 2454 KiB

Open AccessArticle

Proenkephalin Levels and Its Determinants in Patients with End-Stage Kidney Disease Treated with Hemodialysis and Peritoneal Dialysis

by Wiktoria Grycuk, Zuzanna Jakubowska and Jolanta Małyszko

Int. J. Mol. Sci. 2023, 24(19), 15015; https://doi.org/10.3390/ijms241915015 - 9 Oct 2023

Cited by 1 | Viewed by 1027

Abstract

Recently, proenkephalin A (PENK A) has been shown to reflect glomerular dysfunction and to predict new-onset acute kidney injury and heart failure. While previous studies have investigated PENK A as a biomarker in individuals with preserved renal function, PENK A concentration in patients [...] Read more.

Recently, proenkephalin A (PENK A) has been shown to reflect glomerular dysfunction and to predict new-onset acute kidney injury and heart failure. While previous studies have investigated PENK A as a biomarker in individuals with preserved renal function, PENK A concentration in patients with end-stage kidney disease (ESKD) was not investigated. Plasma PENK A concentration was assessed in 88 patients with ESKD treated with hemodialysis (HD) or peritoneal dialysis (PD), and its associations with kidney function and heart failure indicators were investigated. In HD patients, the difference in PENK A levels before and after hemodialysis, was measured and further assessed for an association with the type of HD membrane used. PENK A levels did not differ significantly between HD and PD patients. In HD patients, the median PENK A concentration was significantly higher before than after hemodialysis (1.368 vs. 2.061, p = 0.003). No correlation was found between PENK A level and urea (p = 0.192), eGFR (p = 0.922), dialysis vintage (p = 0.637), and residual urine output (p = 0.784). Heart failure (p = 0.961), EF (p = 0.361), and NT-proBNP (p = 0.949) were not associated with increased PENK A concentration. PENK A does not reflect renal function and cardiac status in patients with ESKD. Further research is required to establish the clinical utility of the new biomarker in patients with impaired kidney function. Full article

(This article belongs to the Special Issue Renal Dysfunction, Uremic Compounds, and Other Factors 2.0)

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Figure 1
Graphs showing the relationship between PENK A levels and kidney function indicators. No correlation was found between proenkephalin A and (a) eGFR (p = 0.922) and (b) urea (p = 0.192); (c) there were no significant differences in PENK A levels depending on the residual urine output (p = 0.784) and (d) dialysis vintage (p = 0.067). Full article ">Figure 1 Cont.
Graphs showing the relationship between PENK A levels and kidney function indicators. No correlation was found between proenkephalin A and (a) eGFR (p = 0.922) and (b) urea (p = 0.192); (c) there were no significant differences in PENK A levels depending on the residual urine output (p = 0.784) and (d) dialysis vintage (p = 0.067). Full article ">Figure 2
(a) No correlation was found between PENK A level and NT–proBNP. The comparison between groups showed no significant differences in PENK A levels depending on (b) the range of EF (p = 0.361) or (c) presence of HF (p = 0.961). Full article ">Figure 2 Cont.
(a) No correlation was found between PENK A level and NT–proBNP. The comparison between groups showed no significant differences in PENK A levels depending on (b) the range of EF (p = 0.361) or (c) presence of HF (p = 0.961). Full article ">Figure 3
Figure presenting PENK A levels in patients treated with high-flux (boxplot on the left) and low-flux (boxplot on the right) hemodialysis membranes. PENK A concentration was significantly higher in patients using low-flux membrane vs. patients using high-flux membranes, both before hemodialysis (p = 0.003) and afterwards (p = 0.003): (a) PENK A levels in samples drawn directly before hemodialysis; (b) PENK A levels after hemodialysis. Full article ">Figure 4
Figure presenting the distribution of residual urine output (mL/24 h) in patients treated with high-flux (green) and low-flux (blue) hemodialysis. No significant differences in residual diuresis were observed between the high-flux and low-flux groups (p = 0.793). Full article ">

13 pages, 2944 KiB

Open AccessArticle

Integrated Multi-Omics Analyses Reveal That Autophagy-Mediated Cellular Metabolism Is Required for the Initiation of Pollen Germination

by Xuemei Zhou, Qiuyu Zhang, Yuliang Zhao, Shanshan Ding and Guang-Hui Yu

Int. J. Mol. Sci. 2023, 24(19), 15014; https://doi.org/10.3390/ijms241915014 - 9 Oct 2023

Viewed by 1042

Abstract

Autophagy is an evolutionarily conserved mechanism for degrading and recycling various cellular components, functioning in both normal development and stress conditions. This process is tightly regulated by a set of autophagy-related (ATG) proteins, including ATG2 in the ATG9 cycling system and ATG5 in [...] Read more.

Autophagy is an evolutionarily conserved mechanism for degrading and recycling various cellular components, functioning in both normal development and stress conditions. This process is tightly regulated by a set of autophagy-related (ATG) proteins, including ATG2 in the ATG9 cycling system and ATG5 in the ATG12 conjugation system. Our recent research demonstrated that autophagy-mediated compartmental cytoplasmic deletion is essential for pollen germination. However, the precise mechanisms through which autophagy regulates pollen germination, ensuring its fertility, remain largely unknown. Here, we applied multi-omics analyses, including transcriptomic and metabolomic approaches, to investigate the downstream pathways of autophagy in the process of pollen germination. Although ATG2 and ATG5 play similar roles in regulating pollen germination, high-throughput transcriptomic analysis reveals that silencing ATG5 has a greater impact on the transcriptome than silencing ATG2. Cross-comparisons of transcriptome and proteome analysis reveal that gene expression at the mRNA level and protein level is differentially affected by autophagy. Furthermore, high-throughput metabolomics analysis demonstrates that pathways related to amino acid metabolism and aminoacyl-tRNA biosynthesis were affected by both ATG2 and ATG5 silencing. Collectively, our multi-omics analyses reveal the central role of autophagy in cellular metabolism, which is critical for initiating pollen germination and ensuring pollen fertility. Full article

(This article belongs to the Special Issue Recent Research on Autophagy)

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Figure 1
Global transcriptome analysis of WT, ATG2-silenced, and ATG5-silenced pollen (A). Overlap analysis of detected genes in three independent biological replicates. (B). Principal component analysis of the transcriptomes of WT, ATG2-silenced, and ATG5-silenced pollen. (C). Clustering analysis the transcriptome of WT, ATG2-silenced, and ATG5-silenced pollen according to the Pearson’s correlation efficient. Full article ">Figure 2
Identification of differentially expressed genes between WT and ATG-silenced pollen. (A). Volcano plots displaying differentially expressed genes between ATG2-silenced and WT pollen or ATG5-silenced and WT pollen. Red dots indicate upregulated genes, and blue dots indicate downregulated genes. (B). FPKM values of ATG2 and ATG5 in WT and their respective RNAi lines. (C). Number of differentially expressed genes between ATG2-silenced and WT pollen or ATG5-silenced and WT pollen. Fold changes are indicated with the distinct colors. Student’s t test, ** p < 0.05. Full article ">Figure 3
Identification of downstream pathways of autophagy in the process of pollen germination (A,B). Overlap analysis of upregulated genes (A) and downregulated genes (B) between ATG2/WT and ATG5/WT. (C,D). Heat maps showing the expression levels of the commonly downregulated (C) and commonly upregulated genes (D) in ATG2-silenced and ATG5-silenced pollen. (E,F). GO enrichment analysis of the commonly downregulated (E) and upregulated genes (F) in ATG2-silenced and ATG5-silenced pollen. Full article ">Figure 4
Comparisons between differentially regulated transcripts and proteins in ATG2-silenced and ATG5-silenced pollen (A,B). Overlap analysis of differentially regulated transcripts and proteins in ATG2-silenced (A) and ATG5-silenced (B) pollen. (C,D). Heat maps showing differentially expressed genes that consistently change at mRNA and protein levels in ATG2-silenced (C) and ATG5-silenced (D) pollen. (E,F). Heat maps showing genes that are differentially regulated at both mRNA and protein levels in ATG2-silenced (E) and ATG5-silenced (F) pollen. Full article ">Figure 5
Silencing ATGs results in the decreased metabolite levels in pollen (A). OPLS-DA of metabolite levels. The ellipse represents the Hotelling T2 with 95% confidence. (B,C). Volcano plots showing metabolites with different abundances between WT and ATG-silenced pollen. Blue dots indicate downregulated metabolites in ATG-silenced pollen, while pink dots indicate upregulated metabolites in ATG-silenced pollen. (t-test, p < 0.05). (D). Venn diagrams showing the overlaps of metabolites with significantly different contents in ATG-silenced pollen. Full article ">Figure 6
Significant impact of ATG downregulation on downstream metabolic pathways (A). Heat map depicting the relative metabolite contents. (B,C). Metabolic pathways regulated by ATG2 (B) or ATG5 (C). Full article ">

26 pages, 5591 KiB

Open AccessArticle

Combined Omics Approaches Reveal Distinct Mechanisms of Resistance and/or Susceptibility in Sugar Beet Double Haploid Genotypes at Early Stages of Beet Curly Top Virus Infection

by Paul J. Galewski, Rajtilak Majumdar, Matthew D. Lebar, Carl A. Strausbaugh and Imad A. Eujayl

Int. J. Mol. Sci. 2023, 24(19), 15013; https://doi.org/10.3390/ijms241915013 - 9 Oct 2023

Viewed by 1620

Abstract

Sugar beet is susceptible to Beet curly top virus (BCTV), which significantly reduces yield and sugar production in the semi-arid growing regions worldwide. Sources of genetic resistance to BCTV is limited and control depends upon insecticide seed treatments with neonicotinoids. Through double haploid [...] Read more.

Sugar beet is susceptible to Beet curly top virus (BCTV), which significantly reduces yield and sugar production in the semi-arid growing regions worldwide. Sources of genetic resistance to BCTV is limited and control depends upon insecticide seed treatments with neonicotinoids. Through double haploid production and genetic selection, BCTV resistant breeding lines have been developed. Using BCTV resistant (R) [KDH13; Line 13 and KDH4-9; Line 4] and susceptible (S) [KDH19-17; Line 19] lines, beet leafhopper mediated natural infection, mRNA/sRNA sequencing, and metabolite analyses, potential mechanisms of resistance against the virus and vector were identified. At early infection stages (2- and 6-days post inoculation), examples of differentially expressed genes highly up-regulated in the ‘R’ lines (vs. ‘S’) included EL10Ac5g10437 (inhibitor of trypsin and hageman factor), EL10Ac6g14635 (jasmonate-induced protein), EL10Ac3g06016 (ribosome related), EL10Ac2g02812 (probable prolyl 4-hydroxylase 10), etc. Pathway enrichment analysis showed differentially expressed genes were predominantly involved with peroxisome, amino acids metabolism, fatty acid degradation, amino/nucleotide sugar metabolism, etc. Metabolite analysis revealed significantly higher amounts of specific isoflavonoid O-glycosides, flavonoid 8-C glycosides, triterpenoid, and iridoid-O-glycosides in the leaves of the ‘R’ lines (vs. ‘S’). These data suggest that a combination of transcriptional regulation and production of putative antiviral metabolites might contribute to BCTV resistance. In addition, genome divergence among BCTV strains differentially affects the production of small non-coding RNAs (sncRNAs) and small peptides which may potentially affect pathogenicity and disease symptom development. Full article

(This article belongs to the Special Issue Advances and New Perspectives in Plant-Microbe Interactions 2.0)

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Figure 1
Heatmaps showing a subset of differentially expressed (DE) transcripts (p < 0.01) at (A) 2 days post inoculation (dpi), and (B) 6 dpi in the leaves of Beet curly top virus susceptible (Line 19; S) and resistant (Line 13 and Line 4; R) sugar beet lines. Data are Mean ± SE of 4–5 biological replicates and p < 0.01 between ‘S’ and ‘R’ lines; I = infected]. Full article ">Figure 2
Gene ontology (GO) of differentially expressed sugar beet genes in the leaves of Beet curly top virus infected sugar beet plants. (A) 2 dpi and (B) 6 dpi. Data are mean of 3 biological replicates. Full article ">Figure 3
Pathway enrichment of differentially expressed sugar beet genes in the leaves of Beet curly top virus infected plants. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of sugar beet genes in (A) 2 dpi and (B) 6 dpi. Data are mean of 4–5 biological replicates. Full article ">Figure 4
Weighted gene co-expression network analysis (WGCNA) of sugar beet genes in the leaves infected with or without Beet curly top virus (BCTV) show distinct clustering pattern in the BCTV susceptible and resistant sugar beet lines. (A) Gene cluster dendrogram, (B) Heatmap showing network of differentially expressed genes, and (C) module-sample relationship. Data are Mean of 4–5 biological replicates. Full article ">Figure 5
Heatmap of metabolites in the leaves of BCTV susceptible (Line 19; S) and resistant (Line 13 and Line 4; R) sugar beet lines at 2 d and 6 d in control (C; uninfected) and infected (I) samples. Data are Mean ± SE of 4–6 biological replicates. Full article ">Figure 6
Specific metabolites highly abundant in the leaves of Beet curly top virus (BCTV) susceptible [Line 19; S (represented in blue color)] and resistant [Line 13 (represented in green color) and Line 4 (represented in pink color); R] sugar beet lines at 2 d and 6 d in control (C; uninfected) and infected (I) samples. (A) vitexin 2″-glucoside, (B) 9-methoxy-7-[4-[3 4 5-trihydroxy-6-[[3 4 5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxymethyl]oxan-2-yl]oxyphenyl]-[1 3]dioxolo [4 5-g]chromen-8-one, (C) 9-methoxy-7-[4-[(2S 3R 4R 5S 6R)-3 4 5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyphenyl]-[1 3]dioxolo [4 5-g]chromen-8-one, (D) Agnuside, (E) 2-Phenylethyl 2-O-[(2S 3R 4R)-3 4-dihydroxy-4-(hydroxymethyl)tetrahydro-2-furanyl]-beta-D-glucopyranoside, and (F) ursolic acid. Data are Mean ± SE of 4–6 biological replicates; * p < 0.05 between ‘S’ and ‘R’ lines at a specific time point within uninfected control (C) or infected (I) samples. Full article ">Figure 7
Beet curly top virus (BCTV) strain specific distribution and relative abundance of virus-derived small non-coding RNAs during sugar beet infection. The solid gray blocks and colored blocks denote main open reading frames (ORFs) and predicted small ORFs, respectively. The outer track, which is gray, denotes viral derived small non-coding RNAs, while the colored portion represents small non-coding RNA core sequences showing sequence complementarity to putative sugar beet target genes [V3: movement protein; V2: SS-DS DNA regulator; V1: capsid protein; C3: replication enhancer; C2: pathogenesis enhancement protein; C1: rolling circle replication initiator protein; C4: cell cycle regulator]. Full article ">Figure 8
Divergence with respect to functional elements in the genomes of four Beet curly top virus (BCTV) strains. Higher values of FST at specific locations in the genomes are associated with greater divergence as evident by the peaks [small ORFs: small Open Reading Frames; small non-coding RNA: sncRNA; V3: movement protein; V2: SS-DS DNA regulator; V1: capsid protein; C3: replication enhancer; C2: pathogenesis enhancement protein; C1: rolling circle replication initiator protein; C4: cell cycle regulator]. Full article ">

16 pages, 1500 KiB

Open AccessArticle

Synthesis and Antiviral and Antitumor Activities of Novel 18β-Glycyrrhetinic Acid Derivatives

by Bo-Wen Pan, Liang-Liang Zheng, Yang Shi, Zhang-Chao Dong, Ting-Ting Feng, Jian Yang, Ying Wei and Ying Zhou

Int. J. Mol. Sci. 2023, 24(19), 15012; https://doi.org/10.3390/ijms241915012 - 9 Oct 2023

Cited by 1 | Viewed by 1293

Abstract

A series of novel derivatives of 18β-glycyrrhetinic acid (GA) were synthesized by introducing aromatic or heterocyclic structures to extend the side chain, thereby enhancing their interaction with amino acid residues in the active pocket of the target protein. These compounds were [...] Read more.

A series of novel derivatives of 18β-glycyrrhetinic acid (GA) were synthesized by introducing aromatic or heterocyclic structures to extend the side chain, thereby enhancing their interaction with amino acid residues in the active pocket of the target protein. These compounds were structurally characterized using ¹H NMR, ¹³C NMR, and HRMS. The compounds were subsequently evaluated for their inhibitory effects on HIV-1 protease and cell viability in the human cancer cell lines K562 and HeLa and the mouse cancer cell line CT26. Towards HIV-1 protease, compounds 28 and 32, which featured the introduction of heterocyclic moieties at the C3 position of GA, exhibited the highest inhibition, with inhibition rates of 76% and 70.5%, respectively, at 1 mg/mL concentration. Further molecular docking suggests that a 3-substituted polar moiety would be likely to enhance the inhibitory activity against HIV-1 protease. As for the anti-proliferative activities of the GA derivatives, incorporation of a thiazole heterocycle at the C3- position in compound 29 significantly enhanced the effect against K562 cells with an IC₅₀ value of 8.86 ± 0.93 µM. The introduction of electron-withdrawing substituents on the C3-substituted phenyl ring augmented the anti-proliferative activity against Hela and CT26 cells. Compound 13 exhibited the highest inhibitory activity against Hela cells with an IC₅₀ value of 9.89 ± 0.86 µM, whereas compound 7 exerted the strongest inhibition against CT26 cells with an IC₅₀ value of 4.54 ± 0.37 µM. These findings suggest that further modification of GA is a promising path for developing potent novel anti-HIV and anticancer therapeutics. Full article

(This article belongs to the Special Issue Antiviral Drug Targets: Structure, Function, and Drug Design 2.0)

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20 pages, 3094 KiB

Open AccessArticle

Blood-Based Transcriptomic Biomarkers Are Predictive of Neurodegeneration Rather Than Alzheimer’s Disease

by Artur Shvetcov, Shannon Thomson, Jessica Spathos, Ann-Na Cho, Heather M. Wilkins, Shea J. Andrews, Fabien Delerue, Timothy A. Couttas, Jasmeen Kaur Issar, Finula Isik, Simranpreet Kaur, Eleanor Drummond, Carol Dobson-Stone, Shantel L. Duffy, Natasha M. Rogers, Daniel Catchpoole, Wendy A. Gold, Russell H. Swerdlow, David A. Brown and Caitlin A. Finney

Int. J. Mol. Sci. 2023, 24(19), 15011; https://doi.org/10.3390/ijms241915011 - 9 Oct 2023

Cited by 1 | Viewed by 1829

Abstract

Alzheimer’s disease (AD) is a growing global health crisis affecting millions and incurring substantial economic costs. However, clinical diagnosis remains challenging, with misdiagnoses and underdiagnoses being prevalent. There is an increased focus on putative, blood-based biomarkers that may be useful for the diagnosis [...] Read more.

Alzheimer’s disease (AD) is a growing global health crisis affecting millions and incurring substantial economic costs. However, clinical diagnosis remains challenging, with misdiagnoses and underdiagnoses being prevalent. There is an increased focus on putative, blood-based biomarkers that may be useful for the diagnosis as well as early detection of AD. In the present study, we used an unbiased combination of machine learning and functional network analyses to identify blood gene biomarker candidates in AD. Using supervised machine learning, we also determined whether these candidates were indeed unique to AD or whether they were indicative of other neurodegenerative diseases, such as Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS). Our analyses showed that genes involved in spliceosome assembly, RNA binding, transcription, protein synthesis, mitoribosomes, and NADH dehydrogenase were the best-performing genes for identifying AD patients relative to cognitively healthy controls. This transcriptomic signature, however, was not unique to AD, and subsequent machine learning showed that this signature could also predict PD and ALS relative to controls without neurodegenerative disease. Combined, our results suggest that mRNA from whole blood can indeed be used to screen for patients with neurodegeneration but may be less effective in diagnosing the specific neurodegenerative disease. Full article

(This article belongs to the Special Issue Molecular Aspects of the Neurodegenerative Brain Diseases)

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Figure 1
Principal component analysis (PCA) of AD reference dataset, GSE97760. Full article ">Figure 2
K-means clustering of top 1000 dysregulated genes in GSE97760 identified using principal component analysis. (A) Principal component (PC) 1. Red n = 255, yellow n = 271, green n = 250, and blue n = 224 genes. (B) PC2. Red n = 239, yellow n = 216, green n = 261, and blue n = 284 genes. Gene names are listed in <a href="#app1-ijms-24-15011" class="html-app">Supplementary Tables S1 and S2</a>. Full article ">Figure 3
Receiver operating characteristic (ROC) curve of the random forest model’s performance for PC1 gene expression cluster in (A) dataset A, GSE63061, and (B) dataset B, GSE63060. AUC: area under the curve. Full article ">Figure 4
Receiver operating characteristic (ROC) curve of the random forest model’s performance for PC2 metabolic process cluster in (A) dataset A, GSE63061, and (B) dataset B, GSE63060. AUC: area under the curve. Full article ">Figure 5
Feature selection of the top-performing central gene nodes using recursive feature elimination (RFE) for (A) PC1, gene expression cluster, and (B) PC2, metabolic process cluster. The blue dot indicates peak performance of the model where features were identified from. (C,D) The eight top-performing genes for the (C) PC1 gene expression cluster and (D) PC2 metabolic process cluster. Full article ">Figure 6
Flow chart of the methodological pipeline. Abbreviations: PC: principal component; ND: neurodegenerative diseases. Full article ">

41 pages, 1260 KiB

Open AccessArticle

A Catalog of Coding Sequence Variations in Salivary Proteins’ Genes Occurring during Recent Human Evolution

by Lorena Di Pietro, Mozhgan Boroumand, Wanda Lattanzi, Barbara Manconi, Martina Salvati, Tiziana Cabras, Alessandra Olianas, Laura Flore, Simone Serrao, Carla M. Calò, Paolo Francalacci, Ornella Parolini and Massimo Castagnola

Int. J. Mol. Sci. 2023, 24(19), 15010; https://doi.org/10.3390/ijms241915010 - 9 Oct 2023

Cited by 1 | Viewed by 1269

Abstract

Saliva houses over 2000 proteins and peptides with poorly clarified functions, including proline-rich proteins, statherin, P-B peptides, histatins, cystatins, and amylases. Their genes are poorly conserved across related species, reflecting an evolutionary adaptation. We searched the nucleotide substitutions fixed in these salivary proteins’ [...] Read more.

Saliva houses over 2000 proteins and peptides with poorly clarified functions, including proline-rich proteins, statherin, P-B peptides, histatins, cystatins, and amylases. Their genes are poorly conserved across related species, reflecting an evolutionary adaptation. We searched the nucleotide substitutions fixed in these salivary proteins’ gene loci in modern humans compared with ancient hominins. We mapped 3472 sequence variants/nucleotide substitutions in coding, noncoding, and 5′-3′ untranslated regions. Despite most of the detected variations being within noncoding regions, the frequency of coding variations was far higher than the general rate found throughout the genome. Among the various missense substitutions, specific substitutions detected in PRB1 and PRB2 genes were responsible for the introduction/abrogation of consensus sequences recognized by convertase enzymes that cleave the protein precursors. Overall, these changes that occurred during the recent human evolution might have generated novel functional features and/or different expression ratios among the various components of the salivary proteome. This may have influenced the homeostasis of the oral cavity environment, possibly conditioning the eating habits of modern humans. However, fixed nucleotide changes in modern humans represented only 7.3% of all the substitutions reported in this study, and no signs of evolutionary pressure or adaptative introgression from archaic hominins were found on the tested genes. Full article

(This article belongs to the Special Issue Recent Advances in Salivary Gland and Their Function 2.0)

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Schematic representation of basic proline-rich genes and encoded proteins: PRB1 (A), PRB2 (B), PRB3 (C), PRB4 (D). For each protein, the genetic allelic variants (S, small; M, medium; L, large; and VL, very large) are shown on the left-sided column; the resulting alternative proteoforms are shown on the right-sided column as blocks, with the corresponding symbol on top. Vertical dashed lines indicate the pro-protein convertase cleavage sites with corresponding Arg (R) residues’ positions. The P enclosed in a circle denotes phosphorylation sites; aminoacidic substitutions are shown for selected isoforms. See text for additional details. Full article ">Figure 2
Schematic representation of acidic proline-rich proteins (A) and cystatins (B). For each protein, the genetic allelic variants (S, small; M, medium; L, large; and VL, very large) are shown on the left-sided column; the resulting alternative proteoforms are shown on the right-sided column as blocks with corresponding symbols on top. All cystatin alternative proteoforms feature two disulfide bridges (indicated by brackets between Cys), oxidation (ox), and phosphorylation (P) sites. Vertical dashed lines indicate the pro-protein convertase cleavage sites with corresponding Arg (R) residues’ positions. The P enclosed in a circle denotes phosphorylation sites; ox: oxidation sites; p-E: N-terminal pyroglutamic acid; aminoacidic substitutions are shown for selected isoforms. See text for additional details. Full article ">Figure 3
Nucleotide substitutions in salivary protein genes. The pie chart shows the type and number of 3472 nucleotide substitutions across the 17 tested salivary genes. In particular, the 428 substitutions found in coding regions included 307 nonsynonymous changes across all the 17 genes tested. See text for additional details. Full article ">Figure 4
Predicted archaic hominins’ PRB-1 (panel (a)) and PRB-2 (panels (b–d)) protein variants. Full article ">

23 pages, 7622 KiB

Open AccessArticle

Keras/TensorFlow in Drug Design for Immunity Disorders

by Paulina Dragan, Kavita Joshi, Alessandro Atzei and Dorota Latek

Int. J. Mol. Sci. 2023, 24(19), 15009; https://doi.org/10.3390/ijms241915009 - 9 Oct 2023

Cited by 1 | Viewed by 1770

Abstract

Homeostasis of the host immune system is regulated by white blood cells with a variety of cell surface receptors for cytokines. Chemotactic cytokines (chemokines) activate their receptors to evoke the chemotaxis of immune cells in homeostatic migrations or inflammatory conditions towards inflamed tissue [...] Read more.

Homeostasis of the host immune system is regulated by white blood cells with a variety of cell surface receptors for cytokines. Chemotactic cytokines (chemokines) activate their receptors to evoke the chemotaxis of immune cells in homeostatic migrations or inflammatory conditions towards inflamed tissue or pathogens. Dysregulation of the immune system leading to disorders such as allergies, autoimmune diseases, or cancer requires efficient, fast-acting drugs to minimize the long-term effects of chronic inflammation. Here, we performed structure-based virtual screening (SBVS) assisted by the Keras/TensorFlow neural network (NN) to find novel compound scaffolds acting on three chemokine receptors: CCR2, CCR3, and one CXC receptor, CXCR3. Keras/TensorFlow NN was used here not as a typically used binary classifier but as an efficient multi-class classifier that can discard not only inactive compounds but also low- or medium-activity compounds. Several compounds proposed by SBVS and NN were tested in 100 ns all-atom molecular dynamics simulations to confirm their binding affinity. To improve the basic binding affinity of the compounds, new chemical modifications were proposed. The modified compounds were compared with known antagonists of these three chemokine receptors. Known CXCR3 compounds were among the top predicted compounds; thus, the benefits of using Keras/TensorFlow in drug discovery have been shown in addition to structure-based approaches. Furthermore, we showed that Keras/TensorFlow NN can accurately predict the receptor subtype selectivity of compounds, for which SBVS often fails. We cross-tested chemokine receptor datasets retrieved from ChEMBL and curated datasets for cannabinoid receptors. The NN model trained on the cannabinoid receptor datasets retrieved from ChEMBL was the most accurate in the receptor subtype selectivity prediction. Among NN models trained on the chemokine receptor datasets, the CXCR3 model showed the highest accuracy in differentiating the receptor subtype for a given compound dataset. Full article

(This article belongs to the Special Issue G Protein-Coupled Receptors 2023)

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The validation of the inactive-state CXCR3 model through an analysis of micro- and macroswitches. The inactive-state model of CXCR3 (blue-to-red) was superposed on active-state chemokine receptor structures (gray): 7O7F (CCR5) [<a href="#B88-ijms-24-15009" class="html-bibr">88</a>] for TM6 and the tryptophan toggle switch W6.48 with F6.44 from the PIF motif [<a href="#B89-ijms-24-15009" class="html-bibr">89</a>], and 6WWZ (CCR6) for comparison of the tyrosine toggle switch Y7.53 and the ionic lock including R3.50 from the DRY motif. The residues have been labeled using the Ballesteros–Weinstein numbering system [<a href="#B90-ijms-24-15009" class="html-bibr">90</a>]. Full article ">Figure 2
Results of the MD simulations for CCR2 for four different compounds proposed by virtual screening. (Top) The interactions between the receptor and the ligand obtained after 100 ns of the simulation. The receptor was shown in the red-to-blue color scheme; yellow dashed lines—hydrogen bonds; blue dashed lines—pi-pi stacking. The residues have been labeled using Ballesteros–Weinstein numbering system [<a href="#B90-ijms-24-15009" class="html-bibr">90</a>]. (Bottom) The RMSD plots obtained for each of the ligands over the 100 ns simulation, as well as the average RMSD with its fluctuation range. Full article ">Figure 3
Results of the MD simulations for CCR3 for five proposed compounds. (Top) The interactions between the receptor and the ligand obtained after 100 ns of the simulation. The receptor was shown in the red-to-blue color scheme; yellow dashed lines—hydrogen bonds; blue dashed lines—pi-pi stacking; green dashed lines—pi-cation; purple dashed lines—salt bridges. The residues have been labeled using Ballesteros–Weinstein numbering system [<a href="#B90-ijms-24-15009" class="html-bibr">90</a>]. (Bottom) The RMSD plots obtained for each of the ligands over the 100 ns simulation, as well as the average RMSD with its fluctuation range. Full article ">Figure 4
Results of the MD simulations for CXCR3 for five proposed compounds. (Top) The interactions between the receptor and the ligand obtained for 100 ns of the simulation. The receptor was shown in the red-to-blue color scheme; yellow dashed lines—hydrogen bonds; blue dashed lines—pi-pi stacking. The residues have been labeled using Ballesteros–Weinstein numbering system [<a href="#B90-ijms-24-15009" class="html-bibr">90</a>]. (Bottom) The RMSD computed for each of the ligands over the 100 ns simulation, as well as the average RMSD with its fluctuation range. Full article ">

11 pages, 2781 KiB

Open AccessArticle

Fast and High-Efficiency Synthesis of Capsanthin in Pepper by Transient Expression of Geminivirus

by Zhimin Lin, Muhammad Moaaz Ali, Xiaoyan Yi, Lijuan Zhang and Shaojuan Wang

Int. J. Mol. Sci. 2023, 24(19), 15008; https://doi.org/10.3390/ijms241915008 - 9 Oct 2023

Cited by 1 | Viewed by 1219

Abstract

The color of the chili fruit is an important factor that determines the quality of the chili, as red chilies are more popular among consumers. The accumulation of capsanthin is the main cause of reddening of the chili fruit. Capsanthin is an important [...] Read more.

The color of the chili fruit is an important factor that determines the quality of the chili, as red chilies are more popular among consumers. The accumulation of capsanthin is the main cause of reddening of the chili fruit. Capsanthin is an important metabolite in carotenoid metabolism, and its production level is closely linked to the expression of the genes for capsanthin/capsorubin synthase (CCS) and carotenoid hydroxylase (CrtZ). We reported for the first time that the synthesis of capsanthin in chili was enhanced by using a geminivirus (Bean Yellow Dwarf Virus). By expressing heterologous β-carotenoid hydroxylase (CrtZ) and β-carotenoid ketolase (CrtW) using codon optimization, the transcription level of the CCS gene and endogenous CrtZ was directly increased. This leads to the accumulation of a huge amount of capsanthin in a very short period of time. Our results provide a platform for the rapid enhancement of endogenous CCS activity and capsanthin production using geminivirus in plants. Full article

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

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Visualization of GFP expression in N. benthamiana plants under UV light. (a). Normal tobacco plants under UV light; (b). GFP protein expression on tobacco leaves 3 days after injection under UV light. I, inoculated leaves; U, uninoculated leaves. Full article ">Figure 2
The CrtW and CrtZ genes are expressed in a geminivirus expression system under the CAMV-35S promoter and the strong rbcs promoter. (A) The p1300BR vector was used for CrtW gene expression; (B) the p1300BZ vector was used for CrtZ gene expression. Full article ">Figure 3
The CrtZ and CrtW genes promote the ripening of the pepper while increasing the expression of the CCS gene. (A) The phenotypic observation of p1300BZ and P1300BR, with Mock as a control; (B) RT-qPCR expression analysis of the CCS genes in p1300BR, p1300BZ, and Mock; (C) RT-qPCR analysis of the expression of the genes CrtW, CrtZ0, LCYB, PSY in p1300BR and Mock; (D) RT-qPCR analysis of the expression of the genes CrtZ, CrtZ0, LCYB, PSY in p1300BZ and Mock. All gene expression analyses were performed using ubiquitin as an internal reference. Full article ">Figure 4
Expression of CrtW gene significantly enhances capsanthin production in pepper in a geminivirus system. (A) Color comparison of three different pepper powders in cell lysate, including Mock, p1300BZ, and p1300BR; (B) carotenoid production of zeaxanthin, violaxanthin, capsorubin, capsanthin, and antheraxanthin in Mock and p1300BR; (C) values were calculated from the peak area of the HPLC chromatogram. Full article ">Figure 5
Synthesis pattern of capsanthin under a geminivirus expression system. CrtW—β-carotenoid ketolase; CrtY—lycopene β-cyclase; CrtZ—β-carotenoid hydroxylase; CCS—capsanthin/capsorubin synthase; ZEP—zeaxanthin epoxidase. Full article ">

22 pages, 3832 KiB

Open AccessArticle

PNA6, a Lactosyl Analogue of Angiotensin-(1-7), Reverses Pain Induced in Murine Models of Inflammation, Chemotherapy-Induced Peripheral Neuropathy, and Metastatic Bone Disease

by Maha I. Sulaiman, Wafaa Alabsi, Lajos Szabo, Meredith Hay, Robin Polt, Tally M. Largent-Milnes and Todd W. Vanderah

Int. J. Mol. Sci. 2023, 24(19), 15007; https://doi.org/10.3390/ijms241915007 - 9 Oct 2023

Viewed by 1332

Abstract

Pain is the most significant impairment and debilitating challenge for patients with bone metastasis. Therefore, the primary objective of current therapy is to mitigate and prevent the persistence of pain. Thus, cancer-induced bone pain is described as a multifaceted form of discomfort encompassing [...] Read more.

Pain is the most significant impairment and debilitating challenge for patients with bone metastasis. Therefore, the primary objective of current therapy is to mitigate and prevent the persistence of pain. Thus, cancer-induced bone pain is described as a multifaceted form of discomfort encompassing both inflammatory and neuropathic elements. We have developed a novel non-addictive pain therapeutic, PNA6, that is a derivative of the peptide Angiotensin-(1-7) and binds the Mas receptor to decrease inflammation-related cancer pain. In the present study, we provide evidence that PNA6 attenuates inflammatory, chemotherapy-induced peripheral neuropathy (CIPN) and cancer pain confined to the long bones, exhibiting longer-lasting efficacious therapeutic effects. PNA6, Asp-Arg-Val-Tyr-Ile-His-Ser-(O-β-Lact)-amide, was successfully synthesized using solid phase peptide synthesis (SPPS). PNA6 significantly reversed inflammatory pain induced by 2% carrageenan in mice. A second murine model of platinum drug-induced painful peripheral neuropathy was established using oxaliplatin. Mice in the oxaliplatin-vehicle treatment groups demonstrated significant mechanical allodynia compared to the oxaliplatin-PNA6 treatment group mice. In a third study modeling a complex pain state, E0771 breast adenocarcinoma cells were implanted into the femur of female C57BL/6J wild-type mice to induce cancer-induced bone pain (CIBP). Both acute and chronic dosing of PNA6 significantly reduced the spontaneous pain behaviors associated with CIBP. These data suggest that PNA6 is a viable lead candidate for treating chronic inflammatory and complex neuropathic pain. Full article

(This article belongs to the Special Issue New Advance on Molecular Targets for the Treatment of Pain)

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34 pages, 3227 KiB

Open AccessReview

Insulin Receptor Isoforms and Insulin Growth Factor-like Receptors: Implications in Cell Signaling, Carcinogenesis, and Chemoresistance

by Mariam Ahmed Galal, Samhar Samer Alouch, Buthainah Saad Alsultan, Huda Dahman, Nouf Abdullah Alyabis, Sarah Ammar Alammar and Ahmad Aljada

Int. J. Mol. Sci. 2023, 24(19), 15006; https://doi.org/10.3390/ijms241915006 - 9 Oct 2023

Cited by 6 | Viewed by 2476

Abstract

This comprehensive review thoroughly explores the intricate involvement of insulin receptor (IR) isoforms and insulin-like growth factor receptors (IGFRs) in the context of the insulin and insulin-like growth factor (IGF) signaling (IIS) pathway. This elaborate system encompasses ligands, receptors, and binding proteins, giving [...] Read more.

This comprehensive review thoroughly explores the intricate involvement of insulin receptor (IR) isoforms and insulin-like growth factor receptors (IGFRs) in the context of the insulin and insulin-like growth factor (IGF) signaling (IIS) pathway. This elaborate system encompasses ligands, receptors, and binding proteins, giving rise to a wide array of functions, including aspects such as carcinogenesis and chemoresistance. Detailed genetic analysis of IR and IGFR structures highlights their distinct isoforms, which arise from alternative splicing and exhibit diverse affinities for ligands. Notably, the overexpression of the IR-A isoform is linked to cancer stemness, tumor development, and resistance to targeted therapies. Similarly, elevated IGFR expression accelerates tumor progression and fosters chemoresistance. The review underscores the intricate interplay between IRs and IGFRs, contributing to resistance against anti-IGFR drugs. Consequently, the dual targeting of both receptors could present a more effective strategy for surmounting chemoresistance. To conclude, this review brings to light the pivotal roles played by IRs and IGFRs in cellular signaling, carcinogenesis, and therapy resistance. By precisely modulating these receptors and their complex signaling pathways, the potential emerges for developing enhanced anti-cancer interventions, ultimately leading to improved patient outcomes. Full article

(This article belongs to the Special Issue The Role of the IGF Axis in Disease, 3nd Edition)

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The formation of IR isoforms: (1) Gene expression: INSR gene encoding IR is transcribed from DNA in the nucleus. (2) Pre-mRNA splicing involving introns’ removal and exclusion/inclusion of E11 to form mature mRNA molecules that encode either IR-A or IR-B isoforms; this process is regulated by specific splicing factors that bind to cis-acting elements in the pre-mRNA. (3) mRNA export from the nucleus to the cytoplasm through nuclear pores to serve as templates for protein synthesis. (4) mRNA Translation for IR synthesis. (5) Post-translational modifications (PTMs) in the endoplasmic reticulum and Golgi apparatus, including glycosylation, disulfide bond formation, and proteolytic cleavage. (6) Protein trafficking and secretion: mature IR isoforms are transported from the ER to the plasma membrane, where they are inserted and anchored by transmembrane domains. This process occurs through vesicular transport and fusion with the plasma membrane. Once inserted into the plasma membrane, IR isoforms are available for ligand binding and downstream signaling. Full article ">Figure 2
Structure of IR receptors. (A) Illustration showing different IR domains encoded by the 22 exons. IR has two main subunits: α and β. The α subunit contains 5 main domains, L1 (AA 28–174) CR (AA 182–339), and L2 (AA 340–497), and 2 Fibronectin subunits: FnIII-1 (residue 624–726) and FnIII-2 (757–842). The two α-subunits are linked by a disulfide bond between the two Cys 524 in the first FnIII domain. One to three of the triplet Cys at 682, 683, and 685 in the insert within the second FnIII domain are also involved in α-α disulfide bridges. There is a single disulfide bridge between α and β subunits between Cys 647 in the insert domain and Cys 872. The β subunit details are explained in the main text. Teal arrow shows 6 O-glycosylations, whereas the pink arrows imply the N-glycosylation. (B) IR-B amino acid sequence colored to identify each domain sequence presented in (A). (C) 3D structure of IR-B showing the Λ-shaped structure when no ligand is bound to it. (D) Structural differences between IR-A and IR-B. JM: Juxtamembrane, TK: Tyrosine Kinase. The 3D models were created using Swiss Model. Full article ">Figure 3
Hypothetical mechanisms of IR receptor activation upon ligand binding as proposed in the literature. (A) Ward et al. [<a href="#B40-ijms-24-15006" class="html-bibr">40</a>]; (B) Lee et al. [<a href="#B99-ijms-24-15006" class="html-bibr">99</a>]; (C) Kavran et al. [<a href="#B100-ijms-24-15006" class="html-bibr">100</a>]; (D) Maruyama et al. [<a href="#B97-ijms-24-15006" class="html-bibr">97</a>]. Full article ">Figure 4
An illustration of the downstream pathways upon activation of IR and IGF-IR in physiological conditions. Specific differences between the receptors’ signaling pathways are explained in the text. Red circles are phosphorylation; green lines are excitatory signals; red lines are inhibitory signals. Full article ">

19 pages, 6727 KiB

Open AccessArticle

Genome-Wide Evolutionary Characterization and Expression Analysis of Major Latex Protein (MLP) Family Genes in Tomato

by Zhengliang Sun, Liangzhe Meng, Yuhe Yao, Yanhong Zhang, Baohui Cheng and Yan Liang

Int. J. Mol. Sci. 2023, 24(19), 15005; https://doi.org/10.3390/ijms241915005 - 9 Oct 2023

Cited by 2 | Viewed by 1397

Abstract

Major latex proteins (MLPs) play a key role in plant response to abiotic and biotic stresses. However, little is known about this gene family in tomatoes (Solanum lycopersicum). In this paper, we perform a genome-wide evolutionary characterization and gene expression analysis [...] Read more.

Major latex proteins (MLPs) play a key role in plant response to abiotic and biotic stresses. However, little is known about this gene family in tomatoes (Solanum lycopersicum). In this paper, we perform a genome-wide evolutionary characterization and gene expression analysis of the MLP family in tomatoes. We found a total of 34 SlMLP members in the tomato genome, which are heterogeneously distributed on eight chromosomes. The phylogenetic analysis of the SlMLP family unveiled their evolutionary relationships and possible functions. Furthermore, the tissue-specific expression analysis revealed that the tomato MLP members possess distinct biological functions. Crucially, multiple cis-regulatory elements associated with stress, hormone, light, and growth responses were identified in the promoter regions of these SlMLP genes, suggesting that SlMLPs are potentially involved in plant growth, development, and various stress responses. Subcellular localization demonstrated that SlMLP1, SlMLP3, and SlMLP17 are localized in the cytoplasm. In conclusion, these findings lay a foundation for further dissecting the functions of tomato SlMLP genes and exploring the evolutionary relationships of MLP homologs in different plants. Full article

(This article belongs to the Special Issue Plant Stress-Induced Responses and Tolerance Mechanisms at Biochemical, Cellular, Physiological and Molecular Levels)

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19 pages, 5873 KiB

Open AccessArticle

An Investigation of the JAZ Family and the CwMYC2-like Protein to Reveal Their Regulation Roles in the MeJA-Induced Biosynthesis of β-Elemene in Curcuma wenyujin

by Yuyang Liu, Shiyi Wu, Kaer Lan, Qian Wang, Tingyu Ye, Huanan Jin, Tianyuan Hu, Tian Xie, Qiuhui Wei and Xiaopu Yin

Int. J. Mol. Sci. 2023, 24(19), 15004; https://doi.org/10.3390/ijms241915004 - 9 Oct 2023

Cited by 3 | Viewed by 1224

Abstract

β-Elemene (C₁₅H₂₄), a sesquiterpenoid compound isolated from the volatile oil of Curcuma wenyujin, has been proven to be effective for multiple cancers and is widely used in clinical treatment. Unfortunately, the β-elemene content in C. wenyujin is very [...] Read more.

β-Elemene (C₁₅H₂₄), a sesquiterpenoid compound isolated from the volatile oil of Curcuma wenyujin, has been proven to be effective for multiple cancers and is widely used in clinical treatment. Unfortunately, the β-elemene content in C. wenyujin is very low, which cannot meet market demands. Our previous research showed that methyl jasmonate (MeJA) induced the accumulation of β-elemene in C. wenyujin. However, the regulatory mechanism is unclear. In this study, 20 jasmonate ZIM-domain (JAZ) proteins in C. wenyujin were identified, which are the core regulatory factors of the JA signaling pathway. Then, the conservative domains, motifs composition, and evolutionary relationships of CwJAZs were analyzed comprehensively and systematically. The interaction analysis indicated that CwJAZs can form homodimers or heterodimers. Fifteen out of twenty CwJAZs were significantly induced via MeJA treatment. As the master switch of the JA signaling pathway, the CwMYC2-like protein has also been identified and demonstrated to interact with CwJAZ2/3/4/5/7/15/17/20. Further research found that the overexpression of the CwMYC2-like gene increased the accumulation of β-elemene in C. wenyujin leaves. Simultaneously, the expressions of HMGR, HMGS, DXS, DXR, MCT, HDS, HDR, and FPPS related to β-elemene biosynthesis were also up-regulated by the CwMYC2-like protein. These results indicate that CwJAZs and the CwMYC2-like protein respond to the JA signal to regulate the biosynthesis of β-elemene in C. wenyujin. Full article

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

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