International Journal of Molecular Sciences

12 pages, 4021 KiB

Open AccessCase Report

Acute Erythroid Leukemia Post-Chemo-Radiotherapy and Autologous Stem Cell Transplantation Due to Multiple Myeloma: Tracing the Paths to Leukemic Transformation

by Gábor Méhes, Attila Mokánszki, Anikó Ujfalusi, Zsuzsa Hevessy, Zsófia Miltényi, Lajos Gergely and Judit Bedekovics

Int. J. Mol. Sci. 2024, 25(14), 8003; https://doi.org/10.3390/ijms25148003 - 22 Jul 2024

Viewed by 1160

Abstract

The clinical impact of therapy-related acute leukemias is increasing with the extension of cancer-related survival; however, the origins remain largely unknown. Acute erythroleukemia (AEL), a rare unfavorable type of myeloid neoplasia, may also develop secondary to cytotoxic therapy. The disorder is featured by [...] Read more.

The clinical impact of therapy-related acute leukemias is increasing with the extension of cancer-related survival; however, the origins remain largely unknown. Acute erythroleukemia (AEL), a rare unfavorable type of myeloid neoplasia, may also develop secondary to cytotoxic therapy. The disorder is featured by specific genetic alterations, most importantly multi-allelic mutations of the TP53 gene. While AEL might appear as a part of the therapy-related MDS/AML, spectrum information regarding the genetic complexity and progression is largely missing. We present two AEL cases arising after cytotoxic therapy and melphalan-based myeloablation/autologous peripheral stem cell transplantation due to multiple myeloma (MM). As stated, multiple pathogenic TP53 variants were present unrelated to preexisting MM, in parallel with uninvolved/wild-type hemopoiesis. Potential mechanisms of leukemic transformation are discussed, which include (1) preexisting preneoplastic hemopoietic stem cells (HSC) serving as the common origin for both MM and AEL, (2) the generation and intramedullary survival of p53-deficient post-chemotherapy HSCs, (3) reinoculation of mobilized autologous TP53 mutated HSCs, and (4) melphalan treatment-related late-onset myelodysplasia/leukemia with newly acquired TP53 mutations. Full article

(This article belongs to the Special Issue Genomic Oncology and Medicine in Leukemia: From Molecular Pathogenesis to Novel Therapeutic Approaches)

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Bone marrow biopsy morphology (Case 1). Key features are hypercellular parenchyma with maturation defect and up to 80% of early erythroid precursors by conventional HE staining (top left), suppression of the myeloid lineage and a lack of terminal granulopoieses (in red, NASD histochemistry, (top right)), dysplastic megakaryocytes ((bottom left), CD61 IHC), and a few mature plasmocytes ((bottom right), CD138 IHC) (×400 virtual magnification, scale bar = 100 µm). Full article ">Figure 2
Bone marrow IHC showing limited CD34 labeling (up to 5%, with blast morphology) (top left) and approximately 15% CD117+ cells, mostly with proerythroblast morphology (top right). The great majority of cells (80%) presented with CD71 expression (bottom left); up to 30% of the cells presented with E-cadherin positivity and proerythroblast morphology (bottom right) (×400 virtual magnification, scale bar = 100 µm). Full article ">Figure 3
p53 labeling was restricted to the erythroid lineage. CD34/p53 double-IHC presenting generally with CD34 (violet)-negative and p53-positive (brown) blast cells (top), CD71/p53 double-IHC displaying CD71+ (violet) erythroblasts with and without p53 labeling (brown) (bottom). Strong nuclear p53 positivity refers to mutant TP53 status in approximately 50% of the erythroblasts (×400 virtual magnification, scale bar = 100 µm). Full article ">Figure 4
Chromosome karyotyping presented individual subclones with two unrelated chromosome 17p alterations from the same bone marrow sample with AEL diagnosis (Case 1). Subclone 1 was highlighted by the karyotype 45,XY,-7,add(17)(p13.?3),-18,-21,+2mar[5] (left); one of the marker chromosomes is a potential derivate of chromosome 18 (not further analyzed), in contrast to subclone 2, with the karyotype 45,XY,-5,-7,add(11)(p15),del(17)(p13.?1),+mar[3] (right). A significant portion of dividing cells presented with the normal karyotype 46,XY[12]. Full article ">Figure 5
Bone marrow chromosome analysis presented the complex karyotype of 51,XY,+Y,del(5)(q12q3?5),+10,+11,+19,-20,+21,+mar[20] at the time of the AEL diagnosis (Case 2). Full article ">Figure 6
Potential evolution leukemic transformation and the development of pCT AEL following HDM-APSCT due MM. Chemo-radiotherapy and delayed APSCT were effective in treating MM in both presented cases (green line illustrates declining myeloma tumor burden). Secondary AEL showing characteristic TP53 mutations in immature erythroblasts as a unique feature could be the result of the transforming effect of VTD chemotherapy, with or without residual mutant HSCs, or of the HDM therapy (red line). Clonal aberrations in prior treatments, as well as in the pheresis product, could not be observed via NGS; thus, HDM-induced genotoxicity is favored as the most likely mechanism of leukemogenesis. (symbolic appearance of preleukemic/leukemic TP53 mutated myeloid clone in red, disappearance of the myeloma clone in green, dashed line represents lack of evident involvement in the presented case studies). Full article ">

15 pages, 8324 KiB

Open AccessArticle

Overexpression of BnaA10.WRKY75 Decreases Cadmium and Salt Tolerance via Increasing ROS Accumulation in Arabidopsis and Brassica napus L.

by Xiaoke Ping, Qianjun Ye, Mei Yan, Jia Wang, Taiyuan Zhang, Sheng Chen, Kadambot H. M. Siddique, Wallace A. Cowling, Jiana Li and Liezhao Liu

Int. J. Mol. Sci. 2024, 25(14), 8002; https://doi.org/10.3390/ijms25148002 - 22 Jul 2024

Cited by 3 | Viewed by 1052

Abstract

Soil is indispensable for agricultural production but has been seriously polluted by cadmium and salt in recent years. Many crops are suffering from this, including rapeseed, the third largest global oilseed crop. However, genes simultaneously related to both cadmium and salt stress have [...] Read more.

Soil is indispensable for agricultural production but has been seriously polluted by cadmium and salt in recent years. Many crops are suffering from this, including rapeseed, the third largest global oilseed crop. However, genes simultaneously related to both cadmium and salt stress have not been extensively reported yet. In this study, BnaA10.WRKY75 was screened from previous RNA-seq data related to cadmium and salt stress and further analyses including sequence comparison, GUS staining, transformation and qRT-PCR were conducted to confirm its function. GUS staining and qRT-PCR results indicated BnaA10.WRKY75 was induced by CdCl₂ and NaCl treatment. Sequence analysis suggested BnaA10.WRKY75 belongs to Group IIc of the WRKY gene family and transient expression assay showed it was a nuclear localized transcription factor. BnaA10.WRKY75-overexpressing Arabidopsis and rapeseed plants accumulated more H₂O₂ and O₂⁻ and were more sensitive to CdCl₂ and NaCl treatment compared with untransformed plants, which may be caused by the downregulation of BnaC03.CAT2. Our study reported that BnaA10.WRKY75 increases sensitivity to cadmium and salt stress by disrupting the balance of reactive oxygen species both in Arabidopsis and rapeseed. The results support the further understanding of the mechanisms underlying cadmium and salt tolerance and provide BnaA10.WRKY75 as a valuable gene for rapeseed abiotic stress breeding. Full article

(This article belongs to the Special Issue The Gene, Genomics, and Molecular Breeding in Cruciferae Plants (2nd Edition))

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13 pages, 2712 KiB

Open AccessArticle

The REPLUMLESS Transcription Factor Controls the Expression of the RECEPTOR-LIKE CYTOPLASMIC KINASE VI_A2 Gene Involved in Shoot and Fruit Patterning of Arabidopsis thaliana

by Erzsébet Kenesi, Orsolya Beöthy-Fehér, Réka Szőllősi, Ildikó Domonkos, Ildikó Valkai and Attila Fehér

Int. J. Mol. Sci. 2024, 25(14), 8001; https://doi.org/10.3390/ijms25148001 - 22 Jul 2024

Viewed by 696

Abstract

The promoter of the RECEPTOR-LIKE CYTOPLASMIC KINASE VI_A2 (RLCK VI_A2) gene contains nine binding sites for the REPLUMLESS (RPL) transcription factor. In agreement, the expression of the kinase gene was strongly downregulated in the rpl-4 mutant. Comparing phenotypes of loss-of-function mutants, [...] Read more.

The promoter of the RECEPTOR-LIKE CYTOPLASMIC KINASE VI_A2 (RLCK VI_A2) gene contains nine binding sites for the REPLUMLESS (RPL) transcription factor. In agreement, the expression of the kinase gene was strongly downregulated in the rpl-4 mutant. Comparing phenotypes of loss-of-function mutants, it was revealed that both genes are involved in stem growth, phyllotaxis, organization of the vascular tissues, and the replum, highlighting potential functional interactions. The expression of the RLCKVI_A2 gene from the constitutive 35S promoter could not complement the rpl-4 phenotypes but exhibited a dominant positive effect on stem growth and affected vascular differentiation and organization. The results also indicated that the number of vascular bundles is regulated independently from stem thickness. Although our study cannot demonstrate a direct link between the RPL and RLVKVI_A2 genes, it highlights the significance of the proper developmental regulation of the RLCKVI_A2 promoter for balanced stem development. Full article

(This article belongs to the Special Issue Modern Plant Cell Biotechnology: From Genes to Structure, 2nd Edition)

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Comparison of the expression of the At2G18890 (RLCKVI_A2) promoter driving the GUS marker gene in wild-type (A,C,E,G,I,K,M) and rpl-4 mutant (B,D,F,H,J,L,N) genetic backgrounds. Full article ">Figure 2
The phyllotactic pattern of siliques of wild-type (wt) (A,D,G), rpl-4 (B,E,H), and rlckvi_a2 (C,F,I) mutants without (A–C) and with (D–I) ectopic expression (OX1 and OX2) of the RLCKVI_A2 gene under the control of the 35S promoter, respectively, is shown. The divergence angles of two successive siliques on the stem were determined for a minimum of ten T4 generation plants at the developmental stage 8 [<a href="#B28-ijms-25-08001" class="html-bibr">28</a>]. The distribution of measured angles falling into the indicated angle size categories is shown according to [<a href="#B29-ijms-25-08001" class="html-bibr">29</a>]. Full article ">Figure 3
The replum of wild-type (wt), rpl-4, and rlckvi_a2 fruits. Scanning electron microscopy of the fruit surface (A–C) and cross-sections (D–F) verified a thinner visible (indicated by ] in (A–C)) and smaller inner (D–G) replum for the rpl-4 mutant compared to the other two lines. Overlaying cross-sections for three replums per line shows that the inner replum of the rlckvi_a2 mutant is larger than that of the wild-type (G), although the visible replum of these two lines is about the same size (A,C). Full article ">Figure 4
Stem thickness and vascular organization of wild-type (wt), rlckvi_a2, and rpl-4 mutant plants. Cross-sections of the inflorescence stems of the three investigated lines (A) were analyzed for stem cross-sectional area (B), the number of vascular bundles per stem (C), xylem size ((D); see dashed lines in (G)), the number of xylem vessels per xylem (E), and the size of xylem vessels (F). Stem parameters were determined for ten plants per line. Xylem parameters were measured for all xylems in four plants per line. The distribution of the measured values is represented as a box plot for each line. Significant differences from the wild-type were determined by Student’s t-test (p < 0.05 = *; p < 0.01 = **). The organization of vascular bundles is exemplified in (G). vb—vascular bundle; xv—xylem vessel; pc—arc of procambium. Full article ">Figure 5
Effect of ectopic RLCKVI_A2 expression on stem thickness and the vasculature. Stem cross-sectional area (A,D,G); the number of vascular bundles per stem (B,E,H); and the average size of xylems (C,F,I) were determined for wild-type (wt; (A–C)), rpl-4 (D–F), or rlckvi_a2 (G–I) plants without and with overexpression (OX1 and OX2) of the 35S:RLCKVI_A2 gene. Ten plants per line were measured. The distribution of the measured values is represented as a box plot for each line. The data of the overexpressor lines (OX1 and OX2) were compared to their respective control using Student’s t-test (p < 0.01 = **). Full article ">Figure 6
Stem cross-sections of wild-type (wt) and mutant plants (rpl-4 or rlckvi_a2) without and with overexpression (OX1 and OX2) of the 35S:RLCKVI_A2 gene. Red asterisks indicate closely placed/clustered vascular bundles. Full article ">

25 pages, 7766 KiB

Open AccessArticle

A Systematic Hierarchical Virtual Screening Model for RhlR Inhibitors Based on PCA, Pharmacophore, Docking, and Molecular Dynamics

by Jiarui Du, Jiahao Li, Juqi Wen, Jun Liu, Haichuan Xiao, Antian Zhang, Dongdong Yang, Pinghua Sun, Haibo Zhou and Jun Xu

Int. J. Mol. Sci. 2024, 25(14), 8000; https://doi.org/10.3390/ijms25148000 - 22 Jul 2024

Viewed by 882

Abstract

RhlR plays a key role in the quorum sensing of Pseudomonas aeruginosa. The current structure–activity relationship (SAR) studies of RhlR inhibitors mainly focus on elucidating the functional groups. Based on a systematic review of previous research on RhlR inhibitors, this study aims [...] Read more.

RhlR plays a key role in the quorum sensing of Pseudomonas aeruginosa. The current structure–activity relationship (SAR) studies of RhlR inhibitors mainly focus on elucidating the functional groups. Based on a systematic review of previous research on RhlR inhibitors, this study aims to establish a systematic, hierarchical screening model for RhlR inhibitors. We initially established a database and utilized principal component analysis (PCA) to categorize the inhibitors into two classes. Based on the training set, pharmacophore models were established to elucidate the structural characteristics of ligands. Subsequently, molecular docking, molecular dynamics simulations, and the calculation of binding free energy and strain energy were performed to validate the crucial interactions between ligands and receptors. Then, the screening criteria for RhlR inhibitors were established hierarchically based on ligand structure characteristics, ligand–receptor interaction, and receptor affinity. Test sets were finally employed to validate the hierarchical virtual screening model by comparing it with the current SAR studies of RhlR inhibitors. The hierarchical screening model was confirmed to possess higher accuracy and a true positive rate, which holds promise for subsequent screening and the discovery of active RhlR inhibitors. Full article

(This article belongs to the Section Molecular Informatics)

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18 pages, 758 KiB

Open AccessReview

Opioid Use and Gut Dysbiosis in Cancer Pain Patients

by Flaminia Coluzzi, Maria Sole Scerpa, Chiara Loffredo, Marina Borro, Joseph V. Pergolizzi, Jo Ann LeQuang, Elisa Alessandri, Maurizio Simmaco and Monica Rocco

Int. J. Mol. Sci. 2024, 25(14), 7999; https://doi.org/10.3390/ijms25147999 - 22 Jul 2024

Viewed by 1067

Abstract

Opioids are commonly used for the management of severe chronic cancer pain. Their well-known pharmacological effects on the gastrointestinal system, particularly opioid-induced constipation (OIC), are the most common limiting factors in the optimization of analgesia, and have led to the wide use of [...] Read more.

Opioids are commonly used for the management of severe chronic cancer pain. Their well-known pharmacological effects on the gastrointestinal system, particularly opioid-induced constipation (OIC), are the most common limiting factors in the optimization of analgesia, and have led to the wide use of laxatives and/or peripherally acting mu-opioid receptor antagonists (PAMORAs). A growing interest has been recently recorded in the possible effects of opioid treatment on the gut microbiota. Preclinical and clinical data, as presented in this review, showed that alterations of the gut microbiota play a role in modulating opioid-mediated analgesia and tolerability, including constipation. Moreover, due to the bidirectional crosstalk between gut bacteria and the central nervous system, gut dysbiosis may be crucial in modulating opioid reward and addictive behavior. The microbiota may also modulate pain regulation and tolerance, by activating microglial cells and inducing the release of inflammatory cytokines and chemokines, which sustain neuroinflammation. In the subset of cancer patients, the clinical meaning of opioid-induced gut dysbiosis, particularly its possible interference with the efficacy of chemotherapy and immunotherapy, is still unclear. Gut dysbiosis could be a new target for treatment in cancer patients. Restoring the physiological amount of specific gut bacteria may represent a promising therapeutic option for managing gastrointestinal symptoms and optimizing analgesia for cancer patients using opioids. Full article

(This article belongs to the Section Biochemistry)

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21 pages, 6015 KiB

Open AccessArticle

AdNAC20 Regulates Lignin and Coumarin Biosynthesis in the Roots of Angelica dahurica var. formosana

by Wenjie Qu, Wenjuan Huang, Chen Chen, Jinsong Chen, Lin Zhao, Yijie Jiang, Xuan Du, Renlang Liu, Yinyin Chen, Kai Hou, Dongbei Xu and Wei Wu

Int. J. Mol. Sci. 2024, 25(14), 7998; https://doi.org/10.3390/ijms25147998 - 22 Jul 2024

Viewed by 802

Abstract

Angelica dahurica var. formosana (ADF), which belongs to the Umbelliferae family, is one of the original plants of herbal raw material Angelicae Dahuricae Radix. ADF roots represent an enormous biomass resource convertible for disease treatment and bioproducts. But, early bolting of [...] Read more.

Angelica dahurica var. formosana (ADF), which belongs to the Umbelliferae family, is one of the original plants of herbal raw material Angelicae Dahuricae Radix. ADF roots represent an enormous biomass resource convertible for disease treatment and bioproducts. But, early bolting of ADF resulted in lignification and a decrease in the coumarin content in the root, and roots lignification restricts its coumarin for commercial utility. Although there have been attempts to regulate the synthesis ratio of lignin and coumarin through biotechnology to increase the coumarin content in ADF and further enhance its commercial value, optimizing the biosynthesis of lignin and coumarin remains challenging. Based on gene expression analysis and phylogenetic tree profiling, AdNAC20 as the target for genetic engineering of lignin and coumarin biosynthesis in ADF was selected in this study. Early-bolting ADF had significantly greater degrees of root lignification and lower coumarin contents than that of the normal plants. In this study, overexpression of AdNAC20 gene plants were created using transgenic technology, while independent homozygous transgenic lines with precise site mutation of AdNAC20 were created using CRISPR/Cas9 technology. The overexpressing transgenic ADF plants showed a 9.28% decrease in total coumarin content and a significant 12.28% increase in lignin content, while knockout mutant plants showed a 16.3% increase in total coumarin content and a 33.48% decrease in lignin content. Furthermore, 29,671 differentially expressed genes (DEGs) were obtained by comparative transcriptomics of OE-NAC20, KO-NAC20, and WT of ADF. A schematic diagram of the gene network interacting with AdNAC20 during the early-bolting process of ADF was constructed by DEG analysis. AdNAC20 was predicted to directly regulate the transcription of several genes with SNBE-like motifs in their promoter, such as MYB46, C3H, and CCoAOMT. In this study, AdNAC20 was shown to play a dual pathway function that positively enhanced lignin formation but negatively controlled coumarin formation. And the heterologous expression of the AdNAC20 gene at Arabidopsis thaliana proved that the AdNAC20 gene also plays an important role in the process of bolting and flowering. Full article

(This article belongs to the Special Issue Biosynthesis and Regulatory Mechanism of Secondary Metabolites in Medicinal Plants: 2nd Edition)

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22 pages, 3610 KiB

Open AccessArticle

Functional Activity of Isoform 2 of Human eRF1

by Alexey Shuvalov, Alexandr Klishin, Nikita Biziaev, Ekaterina Shuvalova and Elena Alkalaeva

Int. J. Mol. Sci. 2024, 25(14), 7997; https://doi.org/10.3390/ijms25147997 - 22 Jul 2024

Viewed by 1467

Abstract

Eukaryotic release factor eRF1, encoded by the ETF1 gene, recognizes stop codons and induces peptide release during translation termination. ETF1 produces several different transcripts as a result of alternative splicing, from which two eRF1 isoforms can be formed. Isoform 1 codes well-studied canonical [...] Read more.

Eukaryotic release factor eRF1, encoded by the ETF1 gene, recognizes stop codons and induces peptide release during translation termination. ETF1 produces several different transcripts as a result of alternative splicing, from which two eRF1 isoforms can be formed. Isoform 1 codes well-studied canonical eRF1, and isoform 2 is 33 amino acid residues shorter than isoform 1 and completely unstudied. Using a reconstituted mammalian in vitro translation system, we showed that the isoform 2 of human eRF1 is also involved in translation. We showed that eRF1iso2 can interact with the ribosomal subunits and pre-termination complex. However, its codon recognition and peptide release activities have decreased. Additionally, eRF1 isoform 2 exhibits unipotency to UGA. We found that eRF1 isoform 2 interacts with eRF3a but stimulated its GTPase activity significantly worse than the main isoform eRF1. Additionally, we studied the eRF1 isoform 2 effect on stop codon readthrough and translation in a cell-free translation system. We observed that eRF1 isoform 2 suppressed stop codon readthrough of the uORFs and decreased the efficiency of translation of long coding sequences. Based on these data, we assumed that human eRF1 isoform 2 can be involved in the regulation of translation termination. Moreover, our data support previously stated hypotheses that the GTS loop is important for the multipotency of eRF1 to all stop codons. Whereas helix α1 of the N-domain eRF1 is proposed to be involved in conformational rearrangements of eRF1 in the A-site of the ribosome that occur after GTP hydrolysis by eRF3, which ensure hydrolysis of peptidyl-tRNA at the P site of the ribosome. Full article

(This article belongs to the Special Issue Structure and Function of Ribosomal Proteins 2024)

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Expression of isoforms of human eRF1. (A) Amino acid sequence and structure comparison of isoforms of human eRF1. Isoform 2 of eRF1 is truncated for the first 33 amino acid residues, which include a α1-helix and important for stop codon recognition GTS motif. PDB: 1DT9. (B) Visualization of data on ribosomal profiling of human eRF1 transcripts (ENST00000360541–iso1; ENST00000499810–iso2) in the Trips-viz browser (URL (accessed on 26 May 2024): <a href="https://trips.ucc.ie/" target="_blank">https://trips.ucc.ie/</a>) [<a href="#B45-ijms-25-07997" class="html-bibr">45</a>,<a href="#B46-ijms-25-07997" class="html-bibr">46</a>]. Combined data show translation of the first 33 codons, unique to isoform 1, while the expression level of isoform 2 cannot be assessed. However, analysis of ribosomal profiling of human embryonic stem (hES) cells shows a reduced density of ribosome distribution in the first 33 codons and a significantly higher level of representation of ribosomal complexes on the common sequence for two isoforms. Full article ">Figure 2
eRF1iso2 bound to different ribosomes. Western blot analysis of ribosomal subunits isolated from placenta, HeLa, RRL, and Krebs-2 lysate. Antibodies raised against eRF1 (sc-365686) were used for detection. Ctrl—mixture of recombinant eRF1iso1 and eRF1iso2. The first and second lanes contain recombinant eRF1iso1 and eRF1iso2, 0.005 and 0.0025 pmol, respectively. All other control lanes (6, 10, 13) contain 0.005 pmol of recombinant eRF1iso1 and eRF1iso2. Full article ">Figure 3
eRF1iso2 binding with purified 80S ribosomes and ribosomal subunits. Western blot analysis of 80S ribosomes resolved by sucrose density gradients and 40S and 60S ribosomal subunits after incubation with eRF1iso1 or eRF1iso2 in the presence and absence of eRF3a. Antibodies raised against eRF1, eRF3a, rpL9, rpS15 were used for detection. The fractions of the SDG are indicated above the Western blots; fraction 1 corresponds to the top of the gradient and fraction 14 to the bottom. Blue lines indicate the junction of Western blot images after Protein Marker lane cutting, which was performed to facilitate comparison of matched samples (raw data available). 80S ribosomes were assembled from 40S and 60S subunits purified from RRL. Full article ">Figure 4
eRF1iso2 binding with the preTC. Western blot analysis of SDG of the preTC after incubation with eRF1iso1 or eRF1iso2 in the presence of eRF3a and GTP or GDPCP. Antibodies raised against eRF1, eRF3a, and rpL9 were used for detection. The fractions of the SDG are indicated above the Western blots; fraction 1 corresponds to the top of the gradient and fraction 14 to the bottom. Full article ">Figure 5
Functional activity of eRF1iso2 in translation termination. (A) Termination complex formation efficiency by eRF1iso1 and eRF1iso2 in the presence and absence of eRF3a. Toe-print analysis of TC formed at UAA stop codon in the various concentrations of eRF1 isoforms (n = 3). (B) Hydrolysis of peptidyl-tRNA induced by eRF1iso1, eRF1iso2, and eRF3a. Termiluc assay on the preTCs assembled at UAA, UAG, and UGA stop codons. v0 is the initial termination rate (RLU/min) (n = 3). (C) GTPase activity of eRF3a in the presence of eRF1iso1 and eRF1iso2 (n = 3). The error bars represent the standard deviation. Background activity measured in the presence of all components except eRF1 was subtracted. Asterisks indicate statistically significant differences (*, p < 0.05; **; p < 0.01; ***, p < 0.001). Full article ">Figure 6
Binding of eRF1iso2 with eRF3a in solution. (A) Representative figure of the binding of eRF1iso1 and eRF1iso2 to eRF3a determined by pull-down of His-SUMO-eRF3a in the presence of eRF1 isoforms. Protein samples before loading onto the Ni-NTA resin (ctrl) or after cleavage by Ulp1 protease were analyzed by SDS electrophoresis. All experiments were carried out in three replicates. (B) The intensities of the eRF1 bands were normalized to the intensity of the eRF3a band. Histogram data are presented as mean relative intensity ± standard error of the mean. The difference was considered significant when p value (two-tailed t-test) was less than 0.05. n.s., non-significant difference (p ≥ 0.05). Full article ">Figure 7
Effect of eRF1iso2 on the stop codon readthrough. (A) Effect of excess of eRF1 isoforms on the stop codon readthrough in HEK293 lysate on Fluc reporter mRNAs with different PTCs. (B) Effect of excess of eRF1 isoforms on the stop codon readthrough in HeLa lysate on the Nluc reporter mRNAs with different leader sequences. All experiments were carried out in three replicates. Histogram data are presented as mean relative intensity (readthrough, %) ± standard error of the mean. The difference was considered significant when p value (two-tailed t-test) was less than 0.05 (*). n.s., non-significant difference (p ≥ 0.05). Asterisks indicate statistically significant differences (*, p < 0.05; **; p < 0.01; ***, p < 0.001). Full article ">Figure 8
Effect of eRF1 isoforms on translation. (A) Translation of Nluc mRNAs with various CDS lengths in the presence of eRF1 isoforms in HeLa lysate. (B) Translation of Fluc mRNA in the presence of eRF1 isoforms in HEK293 lysate. (C) Translation of Nluc mRNAs with the additional sense codons in the presence of eRF1 isoforms in HEK293 lysate. 5x UXX–repeat of 5 identical codons, each of UGG, UCG, UUG, UCU, or UUA. All experiments were carried out in three replicates. Histogram data are presented as mean relative intensity (relative translation efficiency, %) ± standard error of the mean. The difference was considered significant when p value (two-tailed t-test) was less than 0.05 (*). Asterisks indicate statistically significant differences (*, p < 0.05; **; p < 0.01; ***, p < 0.001; ****, p < 0.0001). Full article ">Figure 9
Effect of additional factors on translation termination induced by eRF1iso2. (A) Hydrolysis of peptidyl-tRNA induced by eRF1iso2 in the presence of PDCD4, eIF3j, DDX19, Nsp1, eRF3a, and PABP. Termiluc assay on the preTCs assembled at UAA stop codon. v0 is the initial termination rate (RLU/min) (n = 3). The red color corresponds to eRF1iso2 data, and the blue color corresponds to eRF1iso1 data. (B) Hydrolysis of peptidyl-tRNA induced by eRF1iso1 and eRF1iso2 in the presence of PABP and eRF3a. Termi-luc assay on the preTCs assembled at UAA (n = 3). The red color corresponds to eRF1iso2 data, and the blue color corresponds to eRF1iso1 data. (C) GTPase activity of eRF3a in the presence of eRF1iso1, eRF1iso2, and PABP (n = 3). Background activity measured in the presence of all components except eRF1 was subtracted. The error bars represent the standard deviation. n.s., non-significant difference (p ≥ 0.05). Asterisks indicate statistically significant differences (*, p < 0.05; **; p < 0.01; ****, p < 0.0001). Full article ">

29 pages, 17140 KiB

Open AccessArticle

The Integrated Bioinformatic Approach Reveals the Prognostic Significance of LRP1 Expression in Ovarian Cancer

by Tesfaye Wolde, Vipul Bhardwaj, Md. Reyad-ul-Ferdous, Peiwu Qin and Vijay Pandey

Int. J. Mol. Sci. 2024, 25(14), 7996; https://doi.org/10.3390/ijms25147996 - 22 Jul 2024

Viewed by 1520

Abstract

A hyperactive tumour microenvironment (TME) drives unrestricted cancer cell survival, drug resistance, and metastasis in ovarian carcinoma (OC). However, therapeutic targets within the TME for OC remain elusive, and efficient methods to quantify TME activity are still limited. Herein, we employed an integrated [...] Read more.

A hyperactive tumour microenvironment (TME) drives unrestricted cancer cell survival, drug resistance, and metastasis in ovarian carcinoma (OC). However, therapeutic targets within the TME for OC remain elusive, and efficient methods to quantify TME activity are still limited. Herein, we employed an integrated bioinformatics approach to determine which immune-related genes (IRGs) modulate the TME and further assess their potential theragnostic (therapeutic + diagnostic) significance in OC progression. Using a robust approach, we developed a predictive risk model to retrospectively examine the clinicopathological parameters of OC patients from The Cancer Genome Atlas (TCGA) database. The validity of the prognostic model was confirmed with data from the International Cancer Genome Consortium (ICGC) cohort. Our approach identified nine IRGs, AKT2, FGF7, FOS, IL27RA, LRP1, OBP2A, PAEP, PDGFRA, and PI3, that form a prognostic model in OC progression, distinguishing patients with significantly better clinical outcomes in the low-risk group. We validated this model as an independent prognostic indicator and demonstrated enhanced prognostic significance when used alongside clinical nomograms for accurate prediction. Elevated LRP1 expression, which indicates poor prognosis in bladder cancer (BLCA), OC, low-grade gliomas (LGG), and glioblastoma (GBM), was also associated with immune infiltration in several other cancers. Significant correlations with immune checkpoint genes (ICGs) highlight the potential importance of LRP1 as a biomarker and therapeutic target. Furthermore, gene set enrichment analysis highlighted LRP1’s involvement in metabolism-related pathways, supporting its prognostic and therapeutic relevance also in BLCA, OC, low-grade gliomas (LGG), GBM, kidney cancer, OC, BLCA, kidney renal clear cell carcinoma (KIRC), stomach adenocarcinoma (STAD), and stomach and oesophageal carcinoma (STES). Our study has generated a novel signature of nine IRGs within the TME across cancers, that could serve as potential prognostic predictors and provide a valuable resource to improve the prognosis of OC. Full article

(This article belongs to the Special Issue New Insights in Translational Bioinformatics)

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27 pages, 2669 KiB

Open AccessArticle

Transcriptomic Analyses Reveal That Coffea arabica and Coffea canephora Have More Complex Responses under Combined Heat and Drought than under Individual Stressors

by Isabel Marques, Isabel Fernandes, Octávio S. Paulo, Dora Batista, Fernando C. Lidon, Ana P. Rodrigues, Fábio L. Partelli, Fábio M. DaMatta, Ana I. Ribeiro-Barros and José C. Ramalho

Int. J. Mol. Sci. 2024, 25(14), 7995; https://doi.org/10.3390/ijms25147995 - 22 Jul 2024

Cited by 1 | Viewed by 947

Abstract

Increasing exposure to unfavorable temperatures and water deficit imposes major constraints on most crops worldwide. Despite several studies regarding coffee responses to abiotic stresses, transcriptome modulation due to simultaneous stresses remains poorly understood. This study unravels transcriptomic responses under the combined action of [...] Read more.

Increasing exposure to unfavorable temperatures and water deficit imposes major constraints on most crops worldwide. Despite several studies regarding coffee responses to abiotic stresses, transcriptome modulation due to simultaneous stresses remains poorly understood. This study unravels transcriptomic responses under the combined action of drought and temperature in leaves from the two most traded species: Coffea canephora cv. Conilon Clone 153 (CL153) and C. arabica cv. Icatu. Substantial transcriptomic changes were found, especially in response to the combination of stresses that cannot be explained by an additive effect. A large number of genes were involved in stress responses, with photosynthesis and other physiologically related genes usually being negatively affected. In both genotypes, genes encoding for protective proteins, such as dehydrins and heat shock proteins, were positively regulated. Transcription factors (TFs), including MADS-box genes, were down-regulated, although responses were genotype-dependent. In contrast to Icatu, only a few drought- and heat-responsive DEGs were recorded in CL153, which also reacted more significantly in terms of the number of DEGs and enriched GO terms, suggesting a high ability to cope with stresses. This research provides novel insights into the molecular mechanisms underlying leaf Coffea responses to drought and heat, revealing their influence on gene expression. Full article

(This article belongs to the Special Issue Plants Responses to Climate Change)

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Total number of expressed genes in Coffea arabica cv. Icatu and C. canephora cv. Conilon Clone 153 (CL153) plants grown under well-watered (WW; light colors) and control temperature (25 °C; blue) conditions before gradual exposure to severe water deficit (SWD; dark colors). Afterward, WW and SWD plants were additionally exposed to increased temperatures of 37 °C (green) and 42 °C (orange), followed by a 2-week recovery period (REC14, yellow) with full rewatering and a temperature of 25 °C. Full article ">Figure 2
Differentially expressed genes (DEGs) relative to initial control conditions (25 °C, WW, light colors) in Coffea arabica cv. Icatu and C. canephora cv. Conilon Clone 153 (CL153) plants after gradual exposure to severe water deficit (SWD, dark colors) and to increased temperatures of 37 °C (green) and 42 °C (red), followed by a 2-week recovery period recovery (REC14, yellow) with full rewatering and a temperature of 25 °C. Intersections between 37 °C and 42 °C (gray), 42 °C and REC14 (pink), 37 °C and REC14 (ashy), and 42 °C and REC14 (violet) are shown. Full article ">Figure 3
Proportion of significantly up- (red) and down-regulated (blue) DEGs associated with antioxidant activities, lipid metabolism, photosynthesis, and respiration in Icatu (A) and CL153 (B). Treatments are as explained in <a href="#ijms-25-07995-f002" class="html-fig">Figure 2</a>. Full article ">Figure 4
Over-representation analysis of Gene Ontology (GO) terms performed with gProfiler against functional annotations in Icatu (A) and CL153 (B). GO terms are grouped by main category—Biological Process (BP), Molecular Function (MF), and Cellular Component (CC). Counts (size) indicate the number of DEGs annotated with each GO term, and dots are colored by the adjusted p-value (red: up-regulated DEGs; blue: down-regulated DEG). Treatments are as explained in <a href="#ijms-25-07995-f002" class="html-fig">Figure 2</a>. Full article ">Figure 5
Correlation results between RNA-seq data and qRT–PCR expression levels considering the following selected genes: PP2C51, protein phosphatase 2C 51-like; LEADC3, late embryogenesis abundant protein Dc3-like; DH1a, dehydrin DH1a; SUS2, sucrose synthase 2-like; PIP2-2, aquaporin PIP2-2-like; XTH6, xyloglucan endotransglucosylase/hydrolase protein 6; GOLS2, galactinol synthase 2-like; CuSOD1, superoxide dismutase [Cu-Zn]; APXChl, chloroplast ascorbate peroxidase. Full article ">

20 pages, 2526 KiB

Open AccessReview

An Overview on the Adhesion Mechanisms of Typical Aquatic Organisms and the Applications of Biomimetic Adhesives in Aquatic Environments

by Jiani Liu, Junyi Song, Ling Zeng and Biru Hu

Int. J. Mol. Sci. 2024, 25(14), 7994; https://doi.org/10.3390/ijms25147994 - 22 Jul 2024

Cited by 1 | Viewed by 1312

Abstract

Water molecules pose a significant obstacle to conventional adhesive materials. Nevertheless, some marine organisms can secrete bioadhesives with remarkable adhesion properties. For instance, mussels resist sea waves using byssal threads, sandcastle worms secrete sandcastle glue to construct shelters, and barnacles adhere to various [...] Read more.

Water molecules pose a significant obstacle to conventional adhesive materials. Nevertheless, some marine organisms can secrete bioadhesives with remarkable adhesion properties. For instance, mussels resist sea waves using byssal threads, sandcastle worms secrete sandcastle glue to construct shelters, and barnacles adhere to various surfaces using their barnacle cement. This work initially elucidates the process of underwater adhesion and the microstructure of bioadhesives in these three exemplary marine organisms. The formation of bioadhesive microstructures is intimately related to the aquatic environment. Subsequently, the adhesion mechanisms employed by mussel byssal threads, sandcastle glue, and barnacle cement are demonstrated at the molecular level. The comprehension of adhesion mechanisms has promoted various biomimetic adhesive systems: DOPA-based biomimetic adhesives inspired by the chemical composition of mussel byssal proteins; polyelectrolyte hydrogels enlightened by sandcastle glue and phase transitions; and novel biomimetic adhesives derived from the multiple interactions and nanofiber-like structures within barnacle cement. Underwater biomimetic adhesion continues to encounter multifaceted challenges despite notable advancements. Hence, this work examines the current challenges confronting underwater biomimetic adhesion in the last part, which provides novel perspectives and directions for future research. Full article

(This article belongs to the Special Issue Biomimetic Materials Applied in the Analytical and Biomedical Fields)

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Microscopic images of mussel byssal threads, sandcastle glue, and barnacle cement (A–C). Macroscopic and microscopic structures of mussel byssal threads. (A) Mussel byssal threads adhere to the surface of organic glass. (B) μ-CT images of iodine-stained mussel byssal threads on the ventral surface show the internal gland and groove structures near the middle and distal depressions of the foot, respectively. (C) Schematic diagram of a single byssal thread, with scanning electron microscope images showing the morphology of the cuticle (scale bar: 3 μm), core (scale bar: 5 μm), and plaque (scale bar: 50 μm) [<a href="#B30-ijms-25-07994" class="html-bibr">30</a>]. Copyright 2004, Wiley-VCH. (D–G) Morphology of sandcastle glue. (D) Laboratory conditions: Sandcastle worms construct shelters using zirconia beads. (E,F) Fine gaps among particles inhale sandcastle glue to form concave capillary bridges. (G) Scanning electron microscope images of fractured glue after freeze-drying [<a href="#B46-ijms-25-07994" class="html-bibr">46</a>]. Copyright 2013, American Chemical Society. (H–K) Nanofiber structure of barnacle cement. (H) Thick and opaque barnacle cement. (I) Scanning electron microscopy reveals abundant small nanofibers within (H). (J) Barnacles are placed on a substrate containing numerous glass beads for one day. (K) Abundant barnacle secretions are found among glass beads [<a href="#B55-ijms-25-07994" class="html-bibr">55</a>]. Copyright 2017, Elsevier. Full article ">Figure 2
Key aspects of adhesives in mussel plaques, sandcastle worm glue, and barnacle cement: (A) Cohesion and adhesion principles of mussel byssal proteins. (B) Underwater adhesion process and mechanism of sandcastle worms [<a href="#B7-ijms-25-07994" class="html-bibr">7</a>]. (C) Distribution and amino acid preferences of barnacle cement proteins. Full article ">

18 pages, 1710 KiB

Open AccessArticle

Mouse Model of Parkinson’s Disease with Bilateral Dorsal Striatum Lesion with 6-Hydroxydopamine Exhibits Cognitive Apathy-like Behavior

by Masato Okitsu, Masayo Fujita, Yuki Moriya, Hiroko Kotajima-Murakami, Soichiro Ide, Rika Kojima, Kazunari Sekiyama, Kazushi Takahashi and Kazutaka Ikeda

Int. J. Mol. Sci. 2024, 25(14), 7993; https://doi.org/10.3390/ijms25147993 - 22 Jul 2024

Viewed by 1016

Abstract

Among the symptoms of Parkinson’s disease (PD), apathy comprises a set of behavioral, affective, and cognitive features that can be classified into several subtypes. However, the pathophysiology and brain regions that are involved in these different apathy subtypes are still poorly characterized. We [...] Read more.

Among the symptoms of Parkinson’s disease (PD), apathy comprises a set of behavioral, affective, and cognitive features that can be classified into several subtypes. However, the pathophysiology and brain regions that are involved in these different apathy subtypes are still poorly characterized. We examined which subtype of apathy is elicited in a mouse model of PD with 6-hydroxydopamine (6-OHDA) lesions and the behavioral symptoms that are exhibited. Male C57/BL6J mice were allocated to sham (n = 8) and 6-OHDA (n = 13) groups and locally injected with saline or 4 µg 6-OHDA bilaterally in the dorsal striatum. We then conducted motor performance tests and apathy-related behavioral experiments. We then pathologically evaluated tyrosine hydroxylase (TH) immunostaining. The 6-OHDA group exhibited significant impairments in motor function. In the behavioral tests of apathy, significant differences were observed between the sham and 6-OHDA groups in the hole-board test and novelty-suppressed feeding test. The 6-OHDA group exhibited impairments in inanimate novel object preference, whereas social preference was maintained in the three-chamber test. The number of TH+ pixels in the caudate putamen and substantia nigra compacta was significantly reduced in the 6-OHDA group. The present mouse model of PD predominantly showed dorsal striatum dopaminergic neuronal loss and a decrease in novelty seeking as a symptom that is related to the cognitive apathy component. Full article

(This article belongs to the Special Issue Animal Research Model for Neurological Diseases)

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16 pages, 498 KiB

Open AccessReview

Biomarkers in the Diagnosis and Prediction of Medication Response in Depression and the Role of Nutraceuticals

by Cristina Beer, Fiona Rae, Annalese Semmler and Joanne Voisey

Int. J. Mol. Sci. 2024, 25(14), 7992; https://doi.org/10.3390/ijms25147992 - 22 Jul 2024

Viewed by 1028

Abstract

Depression continues to be a significant and growing public health concern. In clinical practice, it involves a clinical diagnosis. There is currently no defined or agreed upon biomarker/s for depression that can be readily tested. A biomarker is defined as a biological indicator [...] Read more.

Depression continues to be a significant and growing public health concern. In clinical practice, it involves a clinical diagnosis. There is currently no defined or agreed upon biomarker/s for depression that can be readily tested. A biomarker is defined as a biological indicator of normal physiological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention that can be objectively measured and evaluated. Thus, as there is no such marker for depression, there is no objective measure of depression in clinical practice. The discovery of such a biomarker/s would greatly assist clinical practice and potentially lead to an earlier diagnosis of depression and therefore treatment. A biomarker for depression may also assist in determining response to medication. This is of particular importance as not all patients prescribed with medication will respond, which is referred to as medication resistance. The advent of pharmacogenomics in recent years holds promise to target treatment in depression, particularly in cases of medication resistance. The role of pharmacogenomics in routine depression management within clinical practice remains to be fully established. Equally so, the use of pharmaceutical grade nutrients known as nutraceuticals in the treatment of depression in the clinical practice setting is largely unknown, albeit frequently self-prescribed by patients. Whether nutraceuticals have a role in not only depression treatment but also in potentially modifying the biomarkers of depression has yet to be proven. The aim of this review is to highlight the potential biomarkers for the diagnosis, prediction, and medication response of depression. Full article

(This article belongs to the Section Molecular Pharmacology)

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14 pages, 4396 KiB

Open AccessArticle

Theoretical and Experimental Study on Carbodiimide Formation

by Marcell Dániel Csécsi, Virág Kondor, Edina Reizer, Renáta Zsanett Boros, Péter Tóth, László Farkas, Béla Fiser, Zoltán Mucsi, Miklós Nagy and Béla Viskolcz

Int. J. Mol. Sci. 2024, 25(14), 7991; https://doi.org/10.3390/ijms25147991 - 22 Jul 2024

Viewed by 1294

Abstract

Carbodiimides are important crosslinkers in organic synthesis and are used in the isocyanate industry as modifier additives. Therefore, the understanding of their formation is of high importance. In this work, we present a theoretical B3LYP/6-31G(d) and SMD solvent model and experimental investigation of [...] Read more.

Carbodiimides are important crosslinkers in organic synthesis and are used in the isocyanate industry as modifier additives. Therefore, the understanding of their formation is of high importance. In this work, we present a theoretical B3LYP/6-31G(d) and SMD solvent model and experimental investigation of the formation of diphenylcarbodiimide (CDI) from phenyl isocyanate using a phosphorus-based catalyst (MPPO) in ortho-dichlorobenzene (ODCB) solvent. Kinetic experiments were based on the volumetric quantitation of CO₂ evolved, at different temperatures between 40 and 80 °C. Based on DFT calculations, we managed to construct a more detailed reaction mechanism compared to previous studies which is supported by experimental results. DFT calculations revealed that the mechanism is composed of two main parts, and the rate determining step of the first part, controlling the CO₂ formation, is the first transition state with a 52.9 kJ mol⁻¹ enthalpy barrier. The experimental activation energy was obtained from the Arrhenius plot (ln k vs. 1/T) using the observed second-order kinetics, and the obtained 55.8 ± 2.1 kJ mol⁻¹ was in excellent agreement with the computational one, validating the complete mechanism, giving a better understanding of carbodiimide production from isocyanates. Full article

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

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26 pages, 2404 KiB

Open AccessReview

Glioma and Peptidergic Systems: Oncogenic and Anticancer Peptides

by Manuel Lisardo Sánchez, Arturo Mangas and Rafael Coveñas

Int. J. Mol. Sci. 2024, 25(14), 7990; https://doi.org/10.3390/ijms25147990 - 22 Jul 2024

Cited by 2 | Viewed by 929

Abstract

Glioma cells overexpress different peptide receptors that are useful for research, diagnosis, management, and treatment of the disease. Oncogenic peptides favor the proliferation, migration, and invasion of glioma cells, as well as angiogenesis, whereas anticancer peptides exert antiproliferative, antimigration, and anti-angiogenic effects against [...] Read more.

Glioma cells overexpress different peptide receptors that are useful for research, diagnosis, management, and treatment of the disease. Oncogenic peptides favor the proliferation, migration, and invasion of glioma cells, as well as angiogenesis, whereas anticancer peptides exert antiproliferative, antimigration, and anti-angiogenic effects against gliomas. Other peptides exert a dual effect on gliomas, that is, both proliferative and antiproliferative actions. Peptidergic systems are therapeutic targets, as peptide receptor antagonists/peptides or peptide receptor agonists can be administered to treat gliomas. Other anticancer strategies exerting beneficial effects against gliomas are discussed herein, and future research lines to be developed for gliomas are also suggested. Despite the large amount of data supporting the involvement of peptides in glioma progression, no anticancer drugs targeting peptidergic systems are currently available in clinical practice to treat gliomas. Full article

(This article belongs to the Special Issue Current Research on Cancer Biology and Therapeutics: 2nd Edition)

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22 pages, 3560 KiB

Open AccessArticle

Phytochemical Analysis and Antioxidant and Antifungal Activities of Powders, Methanol Extracts, and Essential Oils from Rosmarinus officinalis L. and Thymus ciliatus Desf. Benth.

by Noui Hendel, Djamel Sarri, Madani Sarri, Edoardo Napoli, Antonio Palumbo Piccionello and Giuseppe Ruberto

Int. J. Mol. Sci. 2024, 25(14), 7989; https://doi.org/10.3390/ijms25147989 - 22 Jul 2024

Cited by 3 | Viewed by 1374

Abstract

Chemical residues in food pose health risks such as cancer and liver issues. This has driven the search for safer natural alternatives to synthetic fungicides and preservatives. The aim of this study was to characterize the chemical composition of the essential oils (EO), [...] Read more.

Chemical residues in food pose health risks such as cancer and liver issues. This has driven the search for safer natural alternatives to synthetic fungicides and preservatives. The aim of this study was to characterize the chemical composition of the essential oils (EO), determine the polyphenolic contents, and evaluate the in vitro antioxidant and antifungal activities of methanol extracts (ME), essential oils (EO), and powders from Rosmarinus officinalis L. (rosemary) and Thymus ciliatus (Desf) Benth. (thyme) from the M’sila region, Algeria. The chemical composition of the EOs was determined by GC-MS. R. officinalis EO was composed of 31 components, mainly camphor (41.22%), camphene (18.14%), and α-pinene (17.49%); T. ciliatus EO was composed of 58 components, mainly, in percentage, α-pinene (22.18), myrcene (13.13), β-pinene (7.73), β-caryophyllene (10.21), and germacrene D (9.90). The total phenols and flavonoids were determined spectrophotometrically, and the rosemary ME was found to possess the highest polyphenolic content (127.1 ± 2.40 µg GAE/mg), while the thyme ME had the highest flavonoid content (48.01 ± 0.99 µg QE/mg). The antioxidant activity was assessed using three methods: rosemary ME was the most potent, followed by DPPH (IC₅₀ = 13.43 ± 0.14 µg/mL), β-carotene/linoleic acid (IC₅₀ = 39.01 ± 2.16 μg/mL), and reducing power (EC₅₀ = 15.03 ± 1.43 µg/mL). Antifungal activity was assessed for 32 pathogenic and foodborne fungi. Four methods were applied to the solid medium. Incorporating the powdered plant into the culture medium (at 10%) reduced the fungal growth to greater than 50% in 21.88% and 6.25% of all fungal isolates, for R. officinalis and T. ciliatus, respectively. The ME, applied by the well diffusion method (0.1 g/mL), was less effective. Different concentrations of EO were tested. Incorporating the EO into the culture medium (1500 μL/L) inhibited 50% of the molds to levels of 50 and 75% for R. officinalis and T. ciliatus, respectively, with the complete inhibition of four fungi. Fumigated EO (15 μL) inhibited 65% of the molds to levels of 65 and 81.25% for R. officinalis and T. ciliatus, respectively, with the complete inhibition of five fungi. There was little to no sporulation in conjunction with the inhibition. Our results revealed some of the potential of the studied plants to fight foodborne molds and presented their promising characteristics as a source of alternatives to chemical pesticides and synthetic preservatives. Further studies are needed to find adequate application techniques in the food safety area. Full article

(This article belongs to the Special Issue Novel Molecular Research on Natural Bioactive Compounds in Human Health)

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Effect of powders and methanol extracts of R. officinalis and T. ciliatus on the radial growth of tested molds grown on Potato Dextrose Agar (PDA). (A) Powder (10%, w/v); (B) methanol extract (0.1 g/mL). The data are represented as the average ± SD (n = 3). Different letters indicate significant differences (p < 0.05) between the two tested plants on each mold, according to Sidak’s multiple comparisons test. Full article ">Figure 2
Effect of the powdered plant on the radial growth of some tested fungi; 1—A. ochraceus, 2—A. parasiticus, 3—B. aclada, 4—F. oxysporum, 5—P. expansum; (a) control, (b) PDA medium supplemented with R. officinalis, and (c) PDA medium supplemented with T. ciliatus. Full article ">Figure 3
Effect of the plant ME on the radial growth of some tested fungi; 1—P. digitatum, 2—F. graminearum, 3—A. alternata, 4—F. oxysporum, 5—F. proliferatum; (a) control, (b) PDA medium supplemented with R. officinalis, (c) PDA medium supplemented with T. ciliatus. Full article ">Figure 4
Effect of EOs of R. officinalis and T. ciliatus on the radial growth of the tested molds grown on PDA by direct contact method of (A) 500, (B) 1000, and (C) 1500 μL/L. The data are represented as the average ± SD (n = 3). Different letters indicate significant differences (p < 0.05) between the two tested plants on each mold, according to Sidak’s multiple comparisons test. Full article ">Figure 5
Effect of EOs of R. officinalis and T. ciliatus on the radial growth of the tested molds grown on PDA by direct contact method of (A) 5, (B) 10, and (C) 15 μL. The data are represented as the average ± SD (n = 3). Different letters indicate significant differences (p < 0.05) between the two tested plants on each mold, according to Sidak’s multiple comparisons test. Full article ">Figure 6
Effect of direct contact with EO on the radial growth of (1) M. suaveolens, (2) F. culmorum, (3) P. griseofulvum, and (4) P. expansum; a: Control; b,c: mold exposed to concentrations of 1000 and 1500 µL/mL of R. officinalis EO, respectively; d–f: mold exposed to concentrations of 500, 1000, and 1500 µL/mL of T. ciliatus EO, respectively. Full article ">Figure 7
Effect of EO fumigation on the radial growth of (1) B. cinerea, (2) A. alternata, (3) F. graminearum, and (4) A. flavus; a: Control; b,c: mold exposed to fumigation of 10 and 15 µL of R. officinalis EO, respectively; d–f: mold exposed to fumigation of 5, 10, and 15 µL of T. ciliatus EO, respectively. Full article ">

17 pages, 4354 KiB

Open AccessReview

Impact of Different Anti-Hyperglycaemic Treatments on Bone Turnover Markers and Bone Mineral Density in Type 2 Diabetes Mellitus Patients: A Systematic Review and Meta-Analysis

by Md Sadman Sakib Saadi, Rajib Das, Adhithya Mullath Ullas, Diane E. Powell, Emma Wilson, Ioanna Myrtziou, Chadi Rakieh and Ioannis Kanakis

Int. J. Mol. Sci. 2024, 25(14), 7988; https://doi.org/10.3390/ijms25147988 - 22 Jul 2024

Viewed by 1606

Abstract

Diabetic bone disease (DBD) is a frequent complication in patients with type 2 diabetes mellitus (T2DM), characterised by altered bone mineral density (BMD) and bone turnover marker (BTMs) levels. The impact of different anti-diabetic medications on the skeleton remains unclear, and studies have [...] Read more.

Diabetic bone disease (DBD) is a frequent complication in patients with type 2 diabetes mellitus (T2DM), characterised by altered bone mineral density (BMD) and bone turnover marker (BTMs) levels. The impact of different anti-diabetic medications on the skeleton remains unclear, and studies have reported conflicting results; thus, the need for a comprehensive systematic review is of paramount importance. A systematic search was conducted in PubMed and the Cochrane Library. The primary outcomes assessed were changes in BMD in relation to different anatomical sites and BTMs, including mainly P1NP and CTX as well as OPG, OCN, B-ALP and RANK-L. Risk of bias was evaluated using the JADAD score. The meta-analysis of 19 randomised controlled trials comprising 4914 patients showed that anti-diabetic medications overall increased BMD at the lumbar spine (SMD: 0.93, 95% CI [0.13, 1.73], p = 0.02), femoral neck (SMD: 1.10, 95% CI [0.47, 1.74], p = 0.0007) and in total hip (SMD: 0.33, 95% CI [−0.25, 0.92], p = 0.27) in comparison with placebo, but when compared with metformin, the overall effect favoured metformin over other treatments (SMD: −0.23, 95% CI [−0.39, −0.07], p = 0.004). GLP-1 receptor agonists and insulin analogues seem to improve BMD compared to placebo, while SGLT2 inhibitors and thiazolidinediones (TZDs) showed no significant effect, although studies’ number cannot lead to safe conclusions. For BTMs, TZDs significantly increased P1NP levels compared to placebo. However, no significant differences were observed for CTX, B-ALP, OCN, OPG, and RANK-L between anti-diabetic drugs and metformin or placebo. High heterogeneity and diverse follow-up durations among studies were evident, which obscures the validity of the results. This review highlights the variable effects of anti-diabetic drugs on DBD in T2DM patients, emphasising the need for long-term trials with robust designs to better understand these relationships and inform clinical decisions. Full article

(This article belongs to the Special Issue Diabetic Bone Disease: New Insights into Molecular Mechanisms and Therapeutic Approaches)

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16 pages, 5376 KiB

Open AccessArticle

Transcriptome-Wide Identification of m⁶A Writers, Erasers and Readers and Their Expression Profiles under Various Biotic and Abiotic Stresses in Pinus massoniana Lamb.

by Sheng Yao, Yidan Song, Xiang Cheng, Dengbao Wang, Qianzi Li, Jingjing Zhang, Qingyang Chen, Qiong Yu and Kongshu Ji

Int. J. Mol. Sci. 2024, 25(14), 7987; https://doi.org/10.3390/ijms25147987 - 22 Jul 2024

Viewed by 871

Abstract

N⁶-methyladenosine (m⁶A) RNA modification is the most prevalent form of RNA methylation and plays a crucial role in plant development. However, our understanding of m⁶A modification in Masson pine (Pinus massoniana Lamb.) remains limited. In this [...] Read more.

N⁶-methyladenosine (m⁶A) RNA modification is the most prevalent form of RNA methylation and plays a crucial role in plant development. However, our understanding of m⁶A modification in Masson pine (Pinus massoniana Lamb.) remains limited. In this study, a complete analysis of m⁶A writers, erasers, and readers in Masson pine was performed, and 22 m⁶A regulatory genes were identified in total, including 7 m⁶A writers, 7 m⁶A erases, and 8 readers. Phylogenetic analysis revealed that all m⁶A regulators involved in Masson pine could be classified into three distinct groups based on their domains and motifs. The tissue expression analysis revealed that the m⁶A regulatory gene may exert a significant influence on the development of reproductive organs and leaves in Masson pine. Moreover, the results from stress and hormone expression analysis indicated that the m⁶A regulatory gene in Masson pine might be involved in drought stress response, ABA-signaling-pathway activation, as well as resistance to Monochamus alternatus. This study provided valuable and anticipated insights into the regulatory genes of m⁶A modification and their potential epigenetic regulatory mechanisms in Masson pine. Full article

(This article belongs to the Section Molecular Plant Sciences)

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36 pages, 8923 KiB

Open AccessArticle

Discovery of Cell-Permeable Allosteric Inhibitors of Liver Pyruvate Kinase: Design and Synthesis of Sulfone-Based Urolithins

by Shazia Iqbal, Md. Zahidul Islam, Sajda Ashraf, Woonghee Kim, Amal A. AL-Sharabi, Mehmet Ozcan, Essam Hanashalshahaby, Cheng Zhang, Mathias Uhlén, Jan Boren, Hasan Turkez and Adil Mardinoglu

Int. J. Mol. Sci. 2024, 25(14), 7986; https://doi.org/10.3390/ijms25147986 - 22 Jul 2024

Viewed by 1101

Abstract

Metabolic dysfunction-associated fatty liver disease (MAFLD) presents a significant global health challenge, characterized by the accumulation of liver fat and impacting a considerable portion of the worldwide population. Despite its widespread occurrence, effective treatments for MAFLD are limited. The liver-specific isoform of pyruvate [...] Read more.

Metabolic dysfunction-associated fatty liver disease (MAFLD) presents a significant global health challenge, characterized by the accumulation of liver fat and impacting a considerable portion of the worldwide population. Despite its widespread occurrence, effective treatments for MAFLD are limited. The liver-specific isoform of pyruvate kinase (PKL) has been identified as a promising target for developing MAFLD therapies. Urolithin C, an allosteric inhibitor of PKL, has shown potential in preliminary studies. Expanding upon this groundwork, our study delved into delineating the structure-activity relationship of urolithin C via the synthesis of sulfone-based urolithin analogs. Our results highlight that incorporating a sulfone moiety leads to substantial PKL inhibition, with additional catechol moieties further enhancing this effect. Despite modest improvements in liver cell lines, there was a significant increase in inhibition observed in HepG2 cell lysates. Specifically, compounds 15d, 9d, 15e, 18a, 12d, and 15a displayed promising IC₅₀ values ranging from 4.3 µM to 18.7 µM. Notably, compound 15e not only demonstrated a decrease in PKL activity and triacylglycerol (TAG) content but also showed efficient cellular uptake. These findings position compound 15e as a promising candidate for pharmacological MAFLD treatment, warranting further research and studies. Full article

(This article belongs to the Section Biochemistry)

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19 pages, 10535 KiB

Open AccessArticle

Ribosome Pausing Negatively Regulates Protein Translation in Maize Seedlings during Dark-to-Light Transitions

by Mingming Hou, Wei Fan, Deyi Zhong, Xing Dai, Quan Wang, Wanfei Liu and Shengben Li

Int. J. Mol. Sci. 2024, 25(14), 7985; https://doi.org/10.3390/ijms25147985 - 22 Jul 2024

Viewed by 963

Abstract

Regulation of translation is a crucial step in gene expression. Developmental signals and environmental stimuli dynamically regulate translation via upstream small open reading frames (uORFs) and ribosome pausing. Recent studies have revealed many plant genes that are specifically regulated by uORF translation following [...] Read more.

Regulation of translation is a crucial step in gene expression. Developmental signals and environmental stimuli dynamically regulate translation via upstream small open reading frames (uORFs) and ribosome pausing. Recent studies have revealed many plant genes that are specifically regulated by uORF translation following changes in growth conditions, but ribosome-pausing events are less well understood. In this study, we performed ribosome profiling (Ribo-seq) of etiolated maize (Zea mays) seedlings exposed to light for different durations, revealing hundreds of genes specifically regulated at the translation level during the early period of light exposure. We identified over 400 ribosome-pausing events in the dark that were rapidly released after illumination. These results suggested that ribosome pausing negatively regulates translation from specific genes, a conclusion that was supported by a non-targeted proteomics analysis. Importantly, we identified a conserved nucleotide motif downstream of the pausing sites. Our results elucidate the role of ribosome pausing in the control of gene expression in plants; the identification of the cis-element at the pausing sites provides insight into the mechanisms behind translation regulation and potential targets for artificial control of plant translation. Full article

(This article belongs to the Special Issue New Insights in Translational Bioinformatics)

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Genomic distribution, length, and 3 nt periodicity of identified ribosome-protected fragments (RPFs): (A) Distribution of RPFs across different features of the maize genome. Annotated coding sequences (CDSs), upstream open reading frame (uORF), downstream ORF (dORF), overlapped ORF, and novel coding regions are indicated with different colors. The numbers out and in parentheses indicate the gene number and the percentage of total reads, respectively. (B) Meta-gene analysis of the 29-nucleotide (nt) RPFs near the annotated translation start and stop sites in the maize genome. The red, blue, and orange bars represent the three possible open reading frames. E, P, and A indicate the aminoacyl-tRNA entry site, the P-site (peptidyl-tRNA formation site), and the E-site (uncharged tRNA exit site) in ribosomes, respectively. The numbers in the drawn ribosomes indicate the number of nucleotides protected by ribosomes upstream of the start codon and downstream of the stop codon. Full article ">Figure 2
Translational regulation mainly occurs in the early stages of light exposure. (A–E) Differentially translational regulated genes (DTGs) following different durations of light exposure ((A), 0–0.5 h; (B), 0.5–1 h; (C), 1–2 h; (D), 2–4 h; (E), 0.5–4 h). Upregulated and downregulated DTGs are indicated with red and blue dots, respectively. (F) Venn diagrams showing the extent of overlap between differentially expressed genes (DEGs) and DTGs responsive to light exposure. DTGs and DEGs are indicated by the blue and yellow circles, respectively. The numbers represent the specific and common DEGs and DTGs. Full article ">Figure 3
Expression patterns of photomorphogenesis-related transcripts at different time points: (A–D) Scatterplots of the fold-changes in RNA or RPF abundance following various durations of light exposure: (A), 0–0.5 h; (B), 0.5–1 h; (C), 1–2 h; (D), 2–4 h. All values are log2-normalized fold-changes between the listed time points. (E–H) Scatterplots of the translation efficiency (TE) following various durations of light exposure: (E), 0–0.5 h; (F), 0.5–1 h; (G), 1–2 h; (H), 2–4 h. (I–L) Scatterplots of log2-normalized fold-changes in TE or RPF abundance following various durations of light exposure: (I), 0–0.5 h; (J), 0.5–1 h; (K), 1–2 h; (L), 2–4 h. Positive and negative regulators of photomorphogenesis are indicated with red and blue triangles, respectively. Chloroplast transcripts are indicated in green rectangles. Upregulated, downregulated, and unchanged transcripts are marked with pink, cyan, and gray dots, respectively. Full article ">Figure 4
Genome-wide light-responsive ribosome-pausing events in maize-etiolated seedlings: (A) Venn diagram showing the extent of overlap between significant ribosome-pausing events identified at each of the time points of illumination. The numbers indicate pausing events specific to each sample or common to different samples. (B) Clustering analysis of transcripts showing ribosome-pausing events defining five clusters. The color scale indicates the strength of ribosome pausing. (C) Gene ontology (GO) term enrichment analysis (biology processes) of the genes whose transcripts belong to one of the five clusters defined above. The size of the circles indicates the number of genes; the color indicates the P value. Full article ">Figure 5
Ribosome pausing negatively regulates translation in maize. (A) The Wilcoxon test was used to assess significant differences in TE for transcripts showing ribosome pausing at different time points of light exposure. Significant mark * for p value < 0.05. (B) Distribution and coverage of RPFs along five randomly chosen transcripts showing ribosome pausing at different time points of light exposure. β-tubulin 6b is a non-pausing control. (C) Translation intensity (TI) for transcripts with high pausing scores at 0 or 2 h into light exposure. Significant mark *** for p value < 0.001. (D) Protein abundance, based on mass spectrometry analysis, translated from transcripts with high pausing scores at 0 or 2 h into light exposure. Significant mark *** for p value < 0.001. Full article ">Figure 6
Sequence features of ribosome-pausing sites: (A) Meta-analysis showing the distribution of ribosome-pausing sites along different regions of maize transcripts. UTR, untranslated region. (B) Sequence logo of the region upstream of ribosome-pausing sites. The height of each letter indicates their probability at the corresponding positions. The positions along the x-axis are relative to the ribosome-pausing sites. The –1 position indicates the upstream 1 nt to the ribosome-pausing site. (C) Metaplot of GC content around ribosome-pausing sites (blue) and random RPFs (green). The GC content is over 500 bp on either side of the ribosome-pausing sites. (D) GC content over full-length transcripts with ribosome pausing and random transcripts. The lengths of transcripts have been normalized. Full article ">Figure 7
(A) A repeated CGC motif appears in the region downstream of ribosome-pausing sites. The height of each letter indicates the probability at the corresponding position. The positions along the x-axis are the length of the motif. (B) Maximum ribosome-pausing scores between transcripts with or without CGC motifs in all ribosome-paused transcripts. Significant mark *** for p value < 0.001. (C) Predicted secondary structure of the CGC motif. The possibilities of base-pairing are indicated as a gradient from blue (0%) to red (100%). (D) GO term enrichment analysis of genes whose transcripts contain the CGC motif. Full article ">

14 pages, 1518 KiB

Open AccessBrief Report

Investigating Expression Dynamics of miR-21 and miR-10b in Glioblastoma Cells In Vitro: Insights into Responses to Hypoxia and Secretion Mechanisms

by Hanna Charbit and Iris Lavon

Int. J. Mol. Sci. 2024, 25(14), 7984; https://doi.org/10.3390/ijms25147984 - 22 Jul 2024

Cited by 1 | Viewed by 939

Abstract

Glioblastoma poses significant challenges in oncology, with bevacizumab showing promise as an antiangiogenic treatment but with limited efficacy. microRNAs (miRNAs) 10b and 21 have emerged as potential biomarkers for bevacizumab response in glioblastoma patients. This study delves into the expression dynamics of miR-21 [...] Read more.

Glioblastoma poses significant challenges in oncology, with bevacizumab showing promise as an antiangiogenic treatment but with limited efficacy. microRNAs (miRNAs) 10b and 21 have emerged as potential biomarkers for bevacizumab response in glioblastoma patients. This study delves into the expression dynamics of miR-21 and miR-10b in response to hypoxia and explores their circulation mechanisms. In vitro experiments exposed glioma cells (A172, U87MG, U251) and human umbilical vein endothelial cells (HUVEC) to hypoxic conditions (1% oxygen) for 24 h, revealing heightened levels of miR-10b and miR-21 in glioblastoma cells. Manipulating miR-10b expression in U87MG, demonstrating a significant decrease in VEGF alpha (VEGFA) following miR-10b overexpression under hypoxic conditions. Size exclusion chromatography illustrated a notable shift towards miR-21 and miR-10b exosomal packaging during hypoxia. A proposed model suggests that effective bevacizumab treatment reduces VEGFA levels, heightening hypoxia and subsequently upregulating miR-21 and miR-10b expression. These miRNAs, released via exosomes, might impact various cellular processes, with miR-10b notably contributing to VEGFA level reduction. However, post-treatment increases in miR-10b and miR-21 could potentially restore cells to normoxic conditions through the downregulation of VEGF. This study highlights the intricate feedback loop involving miR-10b, miR-21, and VEGFA in glioblastoma treatment, underscoring the necessity for personalized therapeutic strategies. Further research should explore clinical implications for personalized glioma treatments. Full article

(This article belongs to the Special Issue The Occurrence, Evolution and Treatment of Glioblastoma: Second Edition)

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Impact of hypoxic conditions on miR-10b and miR-21 expression in glioblastoma and endothelial cell line. A172 (black bars), U87MG (gray bars), and U251 (light gray bars) glioblastoma cell lines, along with the endothelial cell line HUVECs (white bars), were exposed to 1% oxygen for 24 h, followed by RNA extraction for qPCR analysis. Quantification of both miRNAs was determined in each cell line relative to the respective cell line cultured under normoxic conditions. The asterisk indicates a significant increase under hypoxic conditions compared to normoxic conditions, with (*) denoting p < 0.05 and (**) denoting p < 0.01. Full article ">Figure 2
Relative quantification of VEGFA in U87MG cells following the overexpression of miR-10b under normoxic and hypoxic conditions. U87MG glioblastoma cells were transfected with various plasmids as indicated on the x-axis and incubated under either normoxic conditions (black bars) or hypoxic conditions (light gray bars). VEGFA expression was assessed by qPCR after 24 h of incubation. The asterisk indicates a significant expression following hypoxia induction in cells containing the empty plasmid compared to cells in normoxic conditions. Additionally, it denotes a significant expression under hypoxia in cells transfected with the miR-10b plasmid compared to cells transfected with the empty plasmid. Furthermore, it indicates a significant expression in cells under hypoxia co-transfected with the plasmid containing miR-10b and its competitive inhibitor compared to cells transfected with miR-10b alone. (*) represents p < 0.05 and (**) represents p < 0.01. Full article ">Figure 3
Quantification of miR-10b and miR-21 in size chromatography fractions from U87MG cell culture medium. U87MG cells were cultured under normoxic or hypoxic conditions for 24 h (as indicated), and their medium was processed using size exclusion columns. qPCR was then used to measure miR-10b (gray columns) and miR-21 (light gray columns) levels in each fraction. Exosomes, identified as large particles, are eluted in fractions 7–11, while proteins and molecules with a diameter smaller than 75 nm are eluted in fractions 12–20. The asterisk indicates a significant miR-10b and miR-21 quantification under normoxic conditions, particularly in the later eluted fractions (fractions 12–20), compared to the earlier eluted fractions (fractions 7–11). Additionally, it denotes a significant miRNA quantification following 24 h of hypoxia, specifically within the exosomal fractions (fractions 7–11) compared to the same fractions under normoxic conditions. (**) represents p < 0.01. Full article ">

21 pages, 6272 KiB

Open AccessArticle

Variation of Cyclodextrin (CD) Complexation with Biogenic Amine Tyramine: Pseudopolymorphs of β-CD Inclusion vs. α-CD Exclusion, Deep Atomistic Insights

by Thammarat Aree

Int. J. Mol. Sci. 2024, 25(14), 7983; https://doi.org/10.3390/ijms25147983 - 22 Jul 2024

Viewed by 777

Abstract

Tyramine (TRM) is a biogenic catecholamine neurotransmitter, which can trigger migraines and hypertension. TRM accumulated in foods is reduced and detected using additive cyclodextrins (CDs) while their association characteristics remain unclear. Here, single-crystal X-ray diffraction and density functional theory (DFT) calculation have been [...] Read more.

Tyramine (TRM) is a biogenic catecholamine neurotransmitter, which can trigger migraines and hypertension. TRM accumulated in foods is reduced and detected using additive cyclodextrins (CDs) while their association characteristics remain unclear. Here, single-crystal X-ray diffraction and density functional theory (DFT) calculation have been performed, demonstrating the elusive pseudopolymorphs in β-CD inclusion complexes with TRM base/HCl, β-CD·0.5TRM·7.6H₂O (1) and β-CD·TRM HCl·4H₂O (2) and the rare α-CD·0.5(TRM HCl)·10H₂O (3) exclusion complex. Both 1 and 2 share the common inclusion mode with similar TRM structures in the round and elliptical β-CD cavities, belong to the monoclinic space group P2₁, and have similar herringbone packing structures. Furthermore, 3 differs from 2, as the smaller twofold symmetry-related, round α-CD prefers an exclusion complex with the twofold disordered TRM–H⁺ sites. In the orthorhombic P2₁2₁2 lattice, α-CDs are packed in a channel-type structure, where the column-like cavity is occupied by disordered water sites. DFT results indicate that β-CD remains elliptical to suitably accommodate TRM, yielding an energetically favorable inclusion complex, which is significantly contributed by the β-CD deformation, and the inclusion complex of α-CD with the TRM aminoethyl side chain is also energetically favorable compared to the exclusion mode. This study suggests the CD implications for food safety and drug/bioactive formulation and delivery. Full article

(This article belongs to the Special Issue A Commemorative Issue in Honor of József Szejtli: Advances in Cyclodextrin Chemistry and Its Applications)

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20 pages, 5554 KiB

Open AccessArticle

Identification and Validation of New DNA-PKcs Inhibitors through High-Throughput Virtual Screening and Experimental Verification

by Liujiang Dai, Pengfei Yu, Hongjie Fan, Wei Xia, Yaopeng Zhao, Pengfei Zhang, John Z. H. Zhang, Haiping Zhang and Yang Chen

Int. J. Mol. Sci. 2024, 25(14), 7982; https://doi.org/10.3390/ijms25147982 - 22 Jul 2024

Cited by 1 | Viewed by 1210

Abstract

DNA-PKcs is a crucial protein target involved in DNA repair and response pathways, with its abnormal activity closely associated with the occurrence and progression of various cancers. In this study, we employed a deep learning-based screening and molecular dynamics (MD) simulation-based pipeline, identifying [...] Read more.

DNA-PKcs is a crucial protein target involved in DNA repair and response pathways, with its abnormal activity closely associated with the occurrence and progression of various cancers. In this study, we employed a deep learning-based screening and molecular dynamics (MD) simulation-based pipeline, identifying eight candidates for DNA-PKcs targets. Subsequent experiments revealed the effective inhibition of DNA-PKcs-mediated cell proliferation by three small molecules (5025-0002, M769-1095, and V008-1080). These molecules exhibited anticancer activity with IC₅₀ (inhibitory concentration at 50%) values of 152.6 μM, 30.71 μM, and 74.84 μM, respectively. Notably, V008-1080 enhanced homology-directed repair (HDR) mediated by CRISPR/Cas9 while inhibiting non-homologous end joining (NHEJ) efficiency. Further investigations into the structure-activity relationships unveiled the binding sites and critical interactions between these small molecules and DNA-PKcs. This is the first application of DeepBindGCN_RG in a real drug screening task, and the successful discovery of a novel DNA-PKcs inhibitor demonstrates its efficiency as a core component in the screening pipeline. Moreover, this study provides important insights for exploring novel anticancer therapeutics and advancing the development of gene editing techniques by targeting DNA-PKcs. Full article

(This article belongs to the Special Issue Molecular Dynamics Simulation and Computer-Aided Screening of Natural Compounds)

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The virtual screening procedure integrates DeepBindGCN models with other methods to identify highly reliable drug candidates for DNA-PKcs. (a) The screening inhibitors against the Chemdiv dataset for DNA-PKcs using a combination of DeepBindGCN_BC/RG, Schrödinger docking, MD simulation, and experimental methods. (b) The last frame from the 40 ns pocket MD simulation of the three identified active compounds, showing both 3D and 2D interaction details. Full article ">Figure 2
The calculated free energy landscape from metadynamics simulation for those candidates with favorable binding with DNA-PKcs. (a) The calculated free energy landscape for DNA-PKcs with candidates 4290-0112, 5025-0002, 5795-0108, 7238-1541, and 8601-0106. (b) The calculated free energy landscape for DNA-PKcs with candidates C163-0038, C163-0039, C163-0087, C200-6885, and C684-0025. (c) The calculated free energy landscape for DNA-PKcs with candidates E208-0020, G744-0225, L102-0385, M769-1095 and S431-0991. (d) The calculated free energy landscape for DNA-PKcs with candidates SA50-0140, V001-2119, V008-1080 and V014-8131. Full article ">Figure 3
The RMSD value and number of hydrogen bonds of selected compounds with DNA-PKcs during the MD simulation. (a) The RMSD value over the 40 ns MD simulation for the eight selected protein–compound complexes. (b) The number of hydrogen bonds between DNA-PKcs and the eight selected compounds. Full article ">Figure 4
DNA-PKcs inhibitors induce proliferation inhibition in 786-O RCC cells. (a,b) 786-O RCC cells were treated with M3814, other small molecules (10 μM, 100 μM) or vehicle control (0.1% DMSO) for applied time; cell inhibition was analyzed by CCK8 assay. (c–o) the cell viability IC50 values of different small molecules against 786-O cells. All p-values were obtained by comparing to the DMSO group at the same concentration. ns denotes not significant, * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001, **** denotes p < 0.0001. Full article ">Figure 5
Evaluation of HDR-mediated gene targeting efficiency by treatment with different small molecules. (a) Schematics of the donor plasmid and targeting strategy for HDR-mediated knock-in of the ires-GFP reporter at GAPDH 3-UTR. (b,c) FACS analysis of HEK293T cells treated with different small molecules or vehicles showing HDR-mediated integration of ires-GFP in the presence of RNP mixture and ires-GFP donor. The cells were co-transfected with donor/RNP by nucleofection and analyzed two days post transfection. Full article ">Figure 6
Evaluation of NHEJ efficiency in HEK293T cells treated with different small molecules using modified TLR reporter. (a) Schematic depicting the outcome after the induction of a site-specific double-strand break (DSB). If the break undergoes NHEJ mediated repair, eGFP will be translated out of frame and iRFP will be expressed, producing far-red fluorescent cells. (b) HEK293T cells overexpressing the TLR reporter (BFP positive) were nucleofected with Cas9 and sgRNA plasmids, then treated with different small molecules or vehicle. NHEJ efficiency was evaluated by detecting iRFP expression 48 h post-treatment using a flow cytometer. Full article ">Figure 7
Interaction analysis of DNA-PKcs with newly discovered and known active compounds with docked conformation. (A) Compound 5025-0002 binding to the DNA-PKcs pocket, displaying both 3D detailed interactions above and 2D representation below. (B) Interaction between M769-1095 and DNA-PKcs pocket. (C) Interaction between V008-1080 and DNA-PKcs pocket. (D) Interaction between M3814 and DNA-PKcs pocket. (E) Interaction between NU7441 and DNA-PKcs pocket. Residues in all 3D visualizations are color-coded by B-Factor in PyMOL, with distances for crucial interactions (depicted with dotted lines) measured. The 2D diagrams employ colors and symbols as standardized by the Schrödinger 2D interaction plots. Full article ">

12 pages, 2259 KiB

Open AccessArticle

A Comparative Investigation of the Pulmonary Vasodilating Effects of Inhaled NO Gas Therapy and Inhalation of a New Drug Formulation Containing a NO Donor Metabolite (SIN-1A)

by Attila Oláh, Bálint András Barta, Mihály Ruppert, Alex Ali Sayour, Dávid Nagy, Tímea Bálint, Georgina Viktória Nagy, István Puskás, Lajos Szente, Levente Szőcs, Tamás Sohajda, Endre Zima, Béla Merkely and Tamás Radovits

Int. J. Mol. Sci. 2024, 25(14), 7981; https://doi.org/10.3390/ijms25147981 - 22 Jul 2024

Viewed by 916

Abstract

Numerous research projects focused on the management of acute pulmonary hypertension as Coronavirus Disease 2019 (COVID-19) might lead to hypoxia-induced pulmonary vasoconstriction related to acute respiratory distress syndrome. For that reason, inhalative therapeutic options have been the subject of several clinical trials. In [...] Read more.

Numerous research projects focused on the management of acute pulmonary hypertension as Coronavirus Disease 2019 (COVID-19) might lead to hypoxia-induced pulmonary vasoconstriction related to acute respiratory distress syndrome. For that reason, inhalative therapeutic options have been the subject of several clinical trials. In this experimental study, we aimed to examine the hemodynamic impact of the inhalation of the SIN-1A formulation (N-nitroso-N-morpholino-amino-acetonitrile, the unstable active metabolite of molsidomine, stabilized by a cyclodextrin derivative) in a porcine model of acute pulmonary hypertension. Landrace pigs were divided into the following experimental groups: iNO (inhaled nitric oxide, n = 3), SIN-1A-5 (5 mg, n = 3), and SIN-1A-10 (10 mg, n = 3). Parallel insertion of a PiCCO system and a pulmonary artery catheter (Swan-Ganz) was performed for continuous hemodynamic monitoring. The impact of iNO (15 min) and SIN-1A inhalation (30 min) was investigated under physiologic conditions and U46619-induced acute pulmonary hypertension. Mean pulmonary arterial pressure (PAP) was reduced transiently by both substances. SIN-1A-10 had a comparable impact compared to iNO after U46619-induced pulmonary hypertension. PAP and PVR decreased significantly (changes in PAP: −30.1% iNO, −22.1% SIN-1A-5, −31.2% SIN-1A-10). While iNO therapy did not alter the mean arterial pressure (MAP) and systemic vascular resistance (SVR), SIN-1A administration resulted in decreased MAP and SVR values. Consequently, the PVR/SVR ratio was markedly reduced in the iNO group, while SIN-1A did not alter this parameter. The pulmonary vasodilatory impact of inhaled SIN-1A was shown to be dose-dependent. A larger dose of SIN-1A (10 mg) resulted in decreased PAP and PVR in a similar manner to the gold standard iNO therapy. Inhalation of the nebulized solution of the new SIN-1A formulation (stabilized by a cyclodextrin derivative) might be a valuable, effective option where iNO therapy is not available due to dosing difficulties or availability. Full article

(This article belongs to the Special Issue A Commemorative Issue in Honor of József Szejtli: Advances in Cyclodextrin Chemistry and Its Applications)

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Pharmacodynamics of inhaled nitric oxide (iNO) and inhaled SIN-1A formulation at different doses (SIN-1A-5 5 mg, SIN-1A-10 10 mg). PAP: pulmonary artery pressure. Full article ">Figure 2
Alterations in systemic and pulmonary circulation during administration of inhaled nitric oxide (iNO) and inhaled SIN-1A formulation at different doses (SIN-1-5 5 mg, SIN-1-10 10 mg). PAP: pulmonary artery pressure, PVR: pulmonary vascular resistance, SAP: systemic arterial pressure, SVR: systemic vascular resistance. * p < 0.05 vs. iNO baseline vs. baseline + inhalation or U46619 vs. U46619 + inhalation. # p < 0.05 vs. SIN-1A-5 baseline vs. baseline + inhalation or U46619 vs. U46619 + inhalation. ## p < 0.05 vs. SIN-1A-10 baseline vs. baseline + inhalation or U46619 vs. U46619 + inhalation. Full article ">Figure 3
Experimental setting: preparation steps, hemodynamic monitoring, and induction of acute pulmonary hypertension (PH). Dosing of inhaled nitric oxide (iNO) and inhaled SIN-1A formulation at different doses (SIN-1A-5 5 mg, SIN-1A-10 10 mg inhalation). CO: cardiac output, HR: heart rate, PAP: pulmonary artery pressure, PCWP: pulmonary wedge pressure, SAP: systemic arterial pressure. iNO, n = 3; SIN-1A 5 mg (SIN-1A-5), n = 3; SIN-1A 10 mg (SIN-1A-10), n = 3. Full article ">Figure 4
Metabolism and NO release of molsidomine. * is a chemical common sign. Full article ">

11 pages, 1145 KiB

Open AccessArticle

Hypersensitivity of Intrinsically Photosensitive Retinal Ganglion Cells in Migraine Induces Cortical Spreading Depression

by Eiichiro Nagata, Motoharu Takao, Haruki Toriumi, Mari Suzuki, Natsuko Fujii, Saori Kohara, Akio Tsuda, Taira Nakayama, Ayana Kadokura and Manaka Hadano

Int. J. Mol. Sci. 2024, 25(14), 7980; https://doi.org/10.3390/ijms25147980 - 22 Jul 2024

Viewed by 1047

Abstract

Migraine is a complex disorder characterized by episodes of moderate-to-severe, often unilateral headaches and generally accompanied by nausea, vomiting, and increased sensitivity to light (photophobia), sound (phonophobia), and smell (hyperosmia). Photophobia is considered the most bothersome symptom of migraine attacks. Although the underlying [...] Read more.

Migraine is a complex disorder characterized by episodes of moderate-to-severe, often unilateral headaches and generally accompanied by nausea, vomiting, and increased sensitivity to light (photophobia), sound (phonophobia), and smell (hyperosmia). Photophobia is considered the most bothersome symptom of migraine attacks. Although the underlying mechanism remains unclear, the intrinsically photosensitive retinal ganglion cells (ipRGCs) are considered to be involved in photophobia associated with migraine. In this study, we investigated the association between the sensitivity of ipRGCs and migraines and cortical spreading depression (CSD), which may trigger migraine attacks. The pupillary responses closely associated with the function of ipRGCs in patients with migraine who were irradiated with lights were evaluated. Blue (486 nm) light irradiation elicited a response from ipRGCs; however, red light (560 nm) had no such effect. Melanopsin, a photosensitive protein, phototransduces in ipRGCs following blue light stimulation. Hypersensitivity of ipRGCs was observed in patients with migraine. CSD was more easily induced with blue light than with incandescent light using a mouse CSD model. Moreover, CSD was suppressed, even in the presence of blue light, after injecting opsinamide, a melanopsin inhibitor. The hypersensitivity of ipRGCs in patients with migraine may induce CSD, resulting in migraine attacks. Full article

(This article belongs to the Special Issue Novel Therapeutic Approaches for Migraine Headaches)

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The representative alterations in pupil diameters in patients with migraine and controls. The upper panels show the alterations in pupil diameter in the controls, whereas the lower panels show the alterations in pupil diameter in the patients with migraine. Irradiation with light induced miosis (downward direction), whereas discontinuation of irradiation resulted in mydriasis (upper direction). When irradiation with blue-LED light was discontinued, the time required for patients with migraine to return to their baseline pupil diameter was longer than that in controls. Diagonal boxes: red-LED light irradiation time; black boxes: blue-LED light irradiation time; LED: light-emitting diode. Full article ">Figure 2
The attenuation rate of pupils in patients with migraine and controls. (a) The changes in pupil diameter. The pupil constricts when irradiated with light (miosis: down direction). The pupil dilates when irradiation is discontinued (mydriasis: upper direction). The time required for the pupil diameter to return to baseline after pupil miosis was divided into two parts: the first half and the second half. (b) Under blue-LED light irradiation, the attenuation rate was significantly increased during both the first and second halves in the patients with migraine compared with that in controls. However, no significant difference was observed between the two groups under red-LED irradiation. Attenuation rate = pupil contraction integral OFF (0–0.17.5 s)/contraction integral ON (20 s). LED: light-emitting diode. Full article ">Figure 3
Representative recording of CSD induction in mice. The upper graph shows the changes in direct current (DC) potential, whereas the lower graph shows the changes in regional cerebral blood flow (rCBF). Black inverted triangle, KCl addition; white triangle, opsinamide intraperitoneal injection; white circle, incandescent light is on; diagonal circle, blue LED light is on. The DC potential decreased when cortical spreading depression (CSD) was induced; at the same time, CBF increased. CSD was induced using 0.225 M KCl after 15 min in a dark room. Under incandescent light, CSD was induced using 0.2 M KCl. Under blue LED light, CSD was induced using 0.175 M KCl. Moreover, when opsinamide was injected intraperitoneally into the mice, the CSD threshold increased under blue LED light. LED: light-emitting diode. Full article ">Figure 4
Summary of the CSD threshold related to KCl concentrations. The cortical spreading depression (CSD) threshold under blue LED light was lower than that under incandescent light irradiation. The addition of opsinamide under these conditions increased the CSD threshold. LED: light-emitting diode. Full article ">Figure 5
A diagram depicting the experimental setting for inducing CSD. CSD, cortical spreading depression; rCBF: regional cerebral blood flow. Full article ">

23 pages, 2244 KiB

Open AccessReview

Glioma Stem Cells as Promoter of Glioma Progression: A Systematic Review of Molecular Pathways and Targeted Therapies

by Edoardo Agosti, Sara Antonietti, Tamara Ius, Marco Maria Fontanella, Marco Zeppieri and Pier Paolo Panciani

Int. J. Mol. Sci. 2024, 25(14), 7979; https://doi.org/10.3390/ijms25147979 - 22 Jul 2024

Cited by 2 | Viewed by 1881

Abstract

Gliomas’ aggressive nature and resistance to therapy make them a major problem in oncology. Gliomas continue to have dismal prognoses despite significant advancements in medical science, and traditional treatments like surgery, radiation (RT), and chemotherapy (CT) frequently prove to be ineffective. After glioma [...] Read more.

Gliomas’ aggressive nature and resistance to therapy make them a major problem in oncology. Gliomas continue to have dismal prognoses despite significant advancements in medical science, and traditional treatments like surgery, radiation (RT), and chemotherapy (CT) frequently prove to be ineffective. After glioma stem cells (GSCs) were discovered, the traditional view of gliomas as homogeneous masses changed. GSCs are essential for tumor growth, treatment resistance, and recurrence. These cells’ distinct capacities for differentiation and self-renewal are changing our knowledge of the biology of gliomas. This systematic literature review aims to uncover the molecular mechanisms driving glioma progression associated with GSCs. The systematic review adhered to PRISMA guidelines, with a thorough literature search conducted on PubMed, Ovid MED-LINE, and Ovid EMBASE. The first literature search was performed on 1 March 2024, and the search was updated on 15 May 2024. Employing MeSH terms and Boolean operators, the search focused on molecular mechanisms associated with GCSs-mediated glioma progression. Inclusion criteria encompassed English language studies, preclinical studies, and clinical trials. A number of 957 papers were initially identified, of which 65 studies spanning from 2005 to 2024 were finally included in the review. The main GSC model distribution is arranged in decreasing order of frequency: U87: 20 studies (32.0%); U251: 13 studies (20.0%); A172: 4 studies (6.2%); and T98G: 2 studies (3.17%). From most to least frequent, the distribution of the primary GSC pathway is as follows: Notch: 8 studies (12.3%); STAT3: 6 studies (9.2%); Wnt/β-catenin: 6 studies (9.2%); HIF: 5 studies (7.7%); and PI3K/AKT: 4 studies (6.2%). The distribution of molecular effects, from most to least common, is as follows: inhibition of differentiation: 22 studies (33.8%); increased proliferation: 18 studies (27.7%); enhanced invasive ability: 15 studies (23.1%); increased self-renewal: 5 studies (7.7%); and inhibition of apoptosis: 3 studies (4.6%). This work highlights GSC heterogeneity and the dynamic interplay within the glioblastoma microenvironment, underscoring the need for a tailored approach. A few key pathways influencing GSC behavior are JAK/STAT3, PI3K/AKT, Wnt/β-catenin, and Notch. Therapy may target these pathways. This research urges more study to fill in knowledge gaps in the biology of GSCs and translate findings into useful treatment approaches that could improve GBM patient outcomes. Full article

(This article belongs to the Special Issue Molecular Biology of Cancer—Implications for Diagnosis and Treatment: 2nd Edition)

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14 pages, 6340 KiB

Open AccessArticle

Computational Insights into Reproductive Toxicity: Clustering, Mechanism Analysis, and Predictive Models

by Huizi Cui, Qizheng He, Wannan Li, Yuying Duan and Weiwei Han

Int. J. Mol. Sci. 2024, 25(14), 7978; https://doi.org/10.3390/ijms25147978 - 22 Jul 2024

Viewed by 1105

Abstract

Reproductive toxicity poses significant risks to fertility and progeny health, making its identification in pharmaceutical compounds crucial. In this study, we conducted a comprehensive in silico investigation of reproductive toxic molecules, identifying three distinct categories represented by Dimethylhydantoin, Phenol, and Dicyclohexyl phthalate. Our [...] Read more.

Reproductive toxicity poses significant risks to fertility and progeny health, making its identification in pharmaceutical compounds crucial. In this study, we conducted a comprehensive in silico investigation of reproductive toxic molecules, identifying three distinct categories represented by Dimethylhydantoin, Phenol, and Dicyclohexyl phthalate. Our analysis included physicochemical properties, target prediction, and KEGG and GO pathway analyses, revealing diverse and complex mechanisms of toxicity. Given the complexity of these mechanisms, traditional molecule-target research approaches proved insufficient. Support Vector Machines (SVMs) combined with molecular descriptors achieved an accuracy of 0.85 in the test dataset, while our custom deep learning model, integrating molecular SMILES and graphs, achieved an accuracy of 0.88 in the test dataset. These models effectively predicted reproductive toxicity, highlighting the potential of computational methods in pharmaceutical safety evaluation. Our study provides a robust framework for utilizing computational methods to enhance the safety evaluation of potential pharmaceutical compounds. Full article

(This article belongs to the Special Issue Machine Learning Applications in Bioinformatics and Biomedicine: 2nd Edition)

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An overview of the study workflow. Full article ">Figure 2
(A) Chemical space of reproductive toxic and non-toxic molecules. (B) Clustering results for reproductive toxic molecules. Full article ">Figure 3
Physicochemical properties of three representative molecules. (A) Dimethylhydantoin. (B) Phenol. (C) Dicyclohexyl phthalate. Full article ">Figure 4
Distribution of top 50 targets for the three representative molecules. (A) Dimethylhydantoin. (B) Phenol. (C) Dicyclohexyl phthalate. (D) Intersection of targets. Full article ">Figure 5
Analysis of representative molecule Dimethylhydantoin. (A) GO analysis. (B) KEGG analysis. Full article ">Figure 6
Analysis of representative molecule Phenol. (A) GO analysis. (B) KEGG analysis. Full article ">Figure 7
Analysis of representative molecule Dicyclohexyl phthalate. (A) GO analysis. (B) KEGG analysis. Full article ">Figure 8
The architecture of the deep learning model. Full article ">

13 pages, 2057 KiB

Open AccessArticle

Exploring the Effects of an Alfalfa Leaf-Derived Adsorbent on Microbial Community, Ileal Morphology, Barrier Function, and Immunity in Turkey Poults during Chronic Aflatoxin B₁ Exposure

by María de Jesús Nava-Ramírez, Jing Liu, Juan Omar Hernández-Ramírez, Xochitl Hernandez-Velasco, Juan D. Latorre, Alma Vázquez-Durán, Guolong Zhang, Roberto Senas-Cuesta, Sergio Gómez-Rosales, Andressa Stein, Billy M. Hargis, Guillermo Téllez-Isaías, Abraham Méndez-Albores and Jesús A. Maguey-González

Int. J. Mol. Sci. 2024, 25(14), 7977; https://doi.org/10.3390/ijms25147977 - 22 Jul 2024

Viewed by 939

Abstract

This article follows-up on our recently published work, which evaluated the impact of the addition of an alfalfa leaf-derived adsorbent in the aflatoxin B₁ (AFB₁)-contaminated diet in regard to the production parameters, blood cell count, serum biochemistry, liver enzymes, and [...] Read more.

This article follows-up on our recently published work, which evaluated the impact of the addition of an alfalfa leaf-derived adsorbent in the aflatoxin B₁ (AFB₁)-contaminated diet in regard to the production parameters, blood cell count, serum biochemistry, liver enzymes, and liver histology of turkey poults. This paper presents complementary results on microbial community, ileal morphology, barrier function, and immunity. For this purpose, 350 1-day-old female turkey poults were randomly distributed into five groups: (1) Control, AFB₁-free diet; (2) AF, AFB₁-contaminated diet at 250 ng/g; (3) alfalfa, AFB₁-free diet + 0.5% (w/w) adsorbent; (4) alfalfa + AF, AFB₁-contaminated diet at 250 ng/g + 0.5% (w/w) adsorbent; and (5) YCW + AF, AFB₁-contaminated diet at 250 ng/g + 0.5% (w/w) commercial yeast cell wall-based adsorbent (reference group). In general, in the AF group, the growth of opportunistic pathogens was promoted, which lead to gut dysbacteriosis, mainly influenced by Streptococcus lutetiensis. Conversely, a significant increase in beneficial bacteria (Faecalibacterium and Coprococcus catus) was promoted by the addition of the plant-based adsorbent. Moreover, the AF group had the lowest villus height and a compromised barrier function, as evidenced by a significant (p < 0.05) increase in fluorescein isothiocyanate dextran (FITC-d), but these negative effects were almost reversed by the addition of the alfalfa adsorbent. Furthermore, the AF + YCW and alfalfa + AF groups exhibited a significant increase in the cutaneous basophil hypersensitivity response compared to the rest of the experimental groups. Taken together, these results pointed out that the alfalfa counteracts the adverse effects of AFB₁ in poults, facilitating the colonization of beneficial bacteria and improving the barrier function of the turkey poults. Full article

(This article belongs to the Section Molecular Microbiology)

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16 pages, 4092 KiB

Open AccessArticle

Evaluation of SARS-CoV-2-Specific IgY Antibodies: Production, Reactivity, and Neutralizing Capability against Virus Variants

by Jacob Schön, Andrea Aebischer, Nico Joël Halwe, Lorenz Ulrich, Donata Hoffmann, Sven Reiche, Martin Beer and Christian Grund

Int. J. Mol. Sci. 2024, 25(14), 7976; https://doi.org/10.3390/ijms25147976 - 21 Jul 2024

Viewed by 1334

Abstract

The emergence of SARS-CoV-2 in late 2019 initiated a global pandemic, which led to a need for effective therapeutics and diagnostic tools, including virus-specific antibodies. Here, we investigate different antigen preparations to produce SARS-CoV-2-specific and virus-neutralizing antibodies in chickens (n = 3/antigen) and [...] Read more.

The emergence of SARS-CoV-2 in late 2019 initiated a global pandemic, which led to a need for effective therapeutics and diagnostic tools, including virus-specific antibodies. Here, we investigate different antigen preparations to produce SARS-CoV-2-specific and virus-neutralizing antibodies in chickens (n = 3/antigen) and rabbits (n = 2/antigen), exploring, in particular, egg yolk for large-scale production of immunoglobulin Y (IgY). Reactivity profiles of IgY preparations from chicken sera and yolk and rabbit sera were tested in parallel. We compared three types of antigens based on ancestral SARS-CoV-2: an inactivated whole-virus preparation, an S1 spike-protein subunit (S1 antigen) and a receptor-binding domain (RBD antigen, amino acids 319–519) coated on lumazine synthase (LS) particles using SpyCather/SpyTag technology. The RBD antigen proved to be the most efficient immunogen, and the resulting chicken IgY antibodies derived from serum or yolk, displayed strong reactivity with ELISA and indirect immunofluorescence and broad neutralizing activity against SARS-CoV-2 variants, including Omicron BA.1 and BA.5. Preliminary in vivo studies using RBD–lumazine synthase yolk preparations in a hamster model showed that local application was well tolerated and not harmful. However, despite the in vitro neutralizing capacity, this antibody preparation did not show protective effect. Further studies on galenic properties seem to be necessary. The RBD–lumazine antigen proved to be suitable for producing SARS-CoV-2 specific antibodies that can be applied to such therapeutic approaches and as reference reagents for SARS-CoV-2 diagnostics, including virus neutralization assays. Full article

(This article belongs to the Special Issue COVID-19 Pandemic: Therapeutic Strategies and Vaccines: 2nd Edition)

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Immune response of chickens and rabbits following immunization with three different SARS-CoV-2 antigen preparations, the RBD antigen (SARS-CoV-2 receptor-binding-domain coupled to LS-particles), the S1 antigen (S1 subunit of the SARS-CoV-2 spike protein coupled to LS-particles) and an inactivated SARS-CoV-2 full-virus preparation. (A) The immunization and sampling scheme for the different antigens. (B) All sera (dilution factor 1:100) from immunized chickens and reference rabbits were tested by RBD-specific ELISA, and samples from animals immunized with the full-virus preparation were also tested with N-specific ELISA. The mean with SEM of individual animals is depicted for the sera. (C) Yolk samples of the chicken were analyzed by RBD-specific ELISA and for the whole-virus group with the N-specific ELISA, in addition. The single values for the weekly yolk-pool samples are shown. Full article ">Figure 2
Reactivity of antibody preparations from chickens immunized with either the RBD antigen (SARS-CoV-2 receptor-binding-domain coupled to LS-particles), the S1 antigen (S1 subunit of the SARS-CoV-2 spike protein coupled to LS-particles) or an inactivated SARS-CoV-2 full-virus preparation tested in immunofluorescence assay. (A) Serum and (B) yolk preparations from chickens are reactive with RBD antigen on immunofluorescence plates showing fluorescence signal of antibody preparations raised against recombinant RBD, S1 or whole-virus preparations. Bar 20 µm. (C) Yolk from the RBD group was also positive against the Delta and Omicron BA.1 variant. Bar 100 µm. Full article ">Figure 3
SARS-CoV-2 live-virus neutralization titers (VNT100 (titer-neutralizing 100 TCID50)) of sera and yolk samples from either the RBD antigen (SARS-CoV-2 receptor-binding-domain coupled to LS-particles), the S1 antigen (S1 subunit of the SARS-CoV-2 spike protein coupled to LS-particles) or an inactivated SARS-CoV-2 full-virus preparation-immunized chickens and rabbits. The course of the neutralizing immune response of chickens in (A) sera (mean with SEM) and (B) yolk samples (individual values) tested against the sequence-homologous WT variant. Neutralizing reactivity of (C) chicken and rabbit sera (mean with SEM) and (D) yolk preparations (individual values) were additionally tested against Alpha, Beta, Delta and Omicron BA.1 and BA.5 variants. Full article ">Figure 4
Intranasal application of RBD-yolk preparation in a prophylactic, therapeutic or post-exposure experimental setup in Syrian hamsters. (A) Experimental setup. (B) Relative body weight. (C) Virus genome (genome copies per mL) in nasal washing samples and (D) 5 dpc organ samples. (E) Infectious virus quantified in conchae, lung (cranial) and nasal washing samples of 3 dpc. Statistical differences were calculated by two-way ANOVA with Tukey´s multiple comparison test. Full article ">

41 pages, 1964 KiB

Open AccessReview

Histidine Phosphorylation: Protein Kinases and Phosphatases

by Jia Ning, Margaux Sala, Jeffrey Reina, Rajasree Kalagiri, Tony Hunter and Brandon S. McCullough

Int. J. Mol. Sci. 2024, 25(14), 7975; https://doi.org/10.3390/ijms25147975 - 21 Jul 2024

Cited by 1 | Viewed by 1790

Abstract

Phosphohistidine (pHis) is a reversible protein post-translational modification (PTM) that is currently poorly understood. The P-N bond in pHis is heat and acid-sensitive, making it more challenging to study than the canonical phosphoamino acids pSer, pThr, and pTyr. As advancements in the development [...] Read more.

Phosphohistidine (pHis) is a reversible protein post-translational modification (PTM) that is currently poorly understood. The P-N bond in pHis is heat and acid-sensitive, making it more challenging to study than the canonical phosphoamino acids pSer, pThr, and pTyr. As advancements in the development of tools to study pHis have been made, the roles of pHis in cells are slowly being revealed. To date, a handful of enzymes responsible for controlling this modification have been identified, including the histidine kinases NME1 and NME2, as well as the phosphohistidine phosphatases PHPT1, LHPP, and PGAM5. These tools have also identified the substrates of these enzymes, granting new insights into previously unknown regulatory mechanisms. Here, we discuss the cellular function of pHis and how it is regulated on known pHis-containing proteins, as well as cellular mechanisms that regulate the activity of the pHis kinases and phosphatases themselves. We further discuss the role of the pHis kinases and phosphatases as potential tumor promoters or suppressors. Finally, we give an overview of various tools and methods currently used to study pHis biology. Given their breadth of functions, unraveling the role of pHis in mammalian systems promises radical new insights into existing and unexplored areas of cell biology. Full article

(This article belongs to the Special Issue Selected Papers from the 13th International NME Conference: NME/NDPK/AWD 2023)

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Comparison of NME family members: (A) Evolutionary tree of the sequence alignment of NME1–10 based on their amino acid sequences obtained from UniProt. (B) Main differences between Group I and Group II family members. Image created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>. Full article ">Figure 2
Known subcellular localization of NME family members in eukaryotic cells. Image created with <a href="http://Biorender.com" target="_blank">Biorender.com</a>. Full article ">Figure 3
Histidine protein kinases and phosphatases in eukaryotic cells. NME2 and PHPT1 reversibly dephosphorylate His358 and His711 of the ion channel proteins KCa3.1 and TRPV5, respectively. TRPC4 is another ion channel protein histidine dephosphorylated by PHPT1 on His912. NME2 and PHPT1 phosphorylate and dephosphorylate His266 of the G beta subunit of G-protein coupled receptors (GPCR). ACLY is a cell metabolism protein reversibly phosphorylated on His760 by NME1 and PHPT1. Succinyl-CoA synthetase alpha (SCSα) and ALDOC are two other metabolic enzymes that are phosphorylated by NME1 on His299 and Asp319, respectively. NME1 can also phosphorylate the kinase suppressor of Ras (KSR) on Ser392 and Annexin A1. LHPP dephosphorylates NME1 and NME2 on His118, and PGAM5 dephosphorylates NME2 on His118. Image created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>. Full article ">Figure 4
Effects of PTMs and natural inhibitors on oligomerization, structure, and enzyme activity of NME1. NME1 hexamers are shown in each panel. For clarity, only two monomers are shown in colors (green and blue), and the rest are all in grey. Cysteines are represented by spheres, while the Kpn loop (residues 109–114) in chains A (the green chain) is in red. A 6-triangle cartoon model is shown at the bottom-right corner of each panel to show the topology of the oligomers, where red lines represent inter- or intra-molecular disulfides, and yellow crosses represent non-covalent CoA inhibitors: (A) Native state (PDB 2HVD). (B) Oxidized state (PDB 4ENO). (C) A model of dissociated NME1 adapted from panel B (PDB 4ENO). (D) CoA-bound NME1 (PDB 7ZTK). (E) A covalent CoAylation is modeled onto C109 (PDB 2HVD). Full article ">

67 pages, 2808 KiB

Open AccessReview

Circulating Liquid Biopsy Biomarkers in Glioblastoma: Advances and Challenges

by Attila A. Seyhan

Int. J. Mol. Sci. 2024, 25(14), 7974; https://doi.org/10.3390/ijms25147974 - 21 Jul 2024

Cited by 1 | Viewed by 2477

Abstract

Gliomas, particularly glioblastoma (GBM), represent the most prevalent and aggressive tumors of the central nervous system (CNS). Despite recent treatment advancements, patient survival rates remain low. The diagnosis of GBM traditionally relies on neuroimaging methods such as magnetic resonance imaging (MRI) or computed [...] Read more.

Gliomas, particularly glioblastoma (GBM), represent the most prevalent and aggressive tumors of the central nervous system (CNS). Despite recent treatment advancements, patient survival rates remain low. The diagnosis of GBM traditionally relies on neuroimaging methods such as magnetic resonance imaging (MRI) or computed tomography (CT) scans and postoperative confirmation via histopathological and molecular analysis. Imaging techniques struggle to differentiate between tumor progression and treatment-related changes, leading to potential misinterpretation and treatment delays. Similarly, tissue biopsies, while informative, are invasive and not suitable for monitoring ongoing treatments. These challenges have led to the emergence of liquid biopsy, particularly through blood samples, as a promising alternative for GBM diagnosis and monitoring. Presently, blood and cerebrospinal fluid (CSF) sampling offers a minimally invasive means of obtaining tumor-related information to guide therapy. The idea that blood or any biofluid tests can be used to screen many cancer types has huge potential. Tumors release various components into the bloodstream or other biofluids, including cell-free nucleic acids such as microRNAs (miRNAs), circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), proteins, extracellular vesicles (EVs) or exosomes, metabolites, and other factors. These factors have been shown to cross the blood-brain barrier (BBB), presenting an opportunity for the minimally invasive monitoring of GBM as well as for the real-time assessment of distinct genetic, epigenetic, transcriptomic, proteomic, and metabolomic changes associated with brain tumors. Despite their potential, the clinical utility of liquid biopsy-based circulating biomarkers is somewhat constrained by limitations such as the absence of standardized methodologies for blood or CSF collection, analyte extraction, analysis methods, and small cohort sizes. Additionally, tissue biopsies offer more precise insights into tumor morphology and the microenvironment. Therefore, the objective of a liquid biopsy should be to complement and enhance the diagnostic accuracy and monitoring of GBM patients by providing additional information alongside traditional tissue biopsies. Moreover, utilizing a combination of diverse biomarker types may enhance clinical effectiveness compared to solely relying on one biomarker category, potentially improving diagnostic sensitivity and specificity and addressing some of the existing limitations associated with liquid biomarkers for GBM. This review presents an overview of the latest research on circulating biomarkers found in GBM blood or CSF samples, discusses their potential as diagnostic, predictive, and prognostic indicators, and discusses associated challenges and future perspectives. Full article

(This article belongs to the Special Issue Circulating Non-coding RNAs as Diagnostic and Prognostic Markers of Human Diseases: 2nd Edition)

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Figure 1
Updated WHO classification of tumors of the CNS. The 2021 WHO classification of CNS tumors introduced significant changes, such as limiting the diagnosis of GBM to only IDH wild type tumors, reclassifying previously diagnosed IDH-mutated GBMs as astrocytomas, IDH-mutated, and grade 4, and requiring the presence of IDH mutations for tumors to be classified as astrocytomas or oligodendrogliomas. For the abbreviations, go to the abbreviations list at the end of the text. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a> (accessed on 6 March 2023). Full article ">Figure 2
Examples of biomarkers measured in circulation, along with the advantages and disadvantages of liquid biopsy versus tumor tissue biopsy, are shown. This schematic representation illustrates circulating biomarkers released from the tumor into the bloodstream through the partially disrupted BBB. These biomarkers may also be directly secreted into the CSF. In patients with GBM, a compromised BBB allows circulating biomarkers such as ctDNAs, miRNAs, EVs, CTCs, proteins, and metabolites to enter the bloodstream or CSF. These biomarkers can be collected through blood or CSF draws and subsequently analyzed. The illustration provides a breakdown of tumoral components within the circulatory system. Various analytical methods, including PCR, qRT-PCR, NGS, WGS, immunoaffinity capture, ELISA, mass spectrometry, chemiluminescent immunoassay, and density gradient centrifugation, have been used to detect circulating analytes. Each circulating analyte can be assessed for tumor-specific changes such as various types of mutations, epigenetic modifications, DNA fragmentation patterns, nucleosome patterning, chromosomal aberrations, and the presence, absence, or changes in levels of ctDNAs, miRNAs (and other noncoding RNAs as well as mRNAs), CTCs, proteins, cytokines, metabolites, EVs, or exosomes, along with post-translational modifications. Each type of biomarker detection method, whether blood- or CSF-based or tissue-based, has unique advantages and disadvantages in diagnosing and monitoring GBM patients. Abbreviations: BBB, blood–brain barrier; CSF, cerebrospinal fluid; CTCs, circulating tumor cells; ctDNA, circulating tumor DNA; DNA, deoxyribonucleic acid; EVs, extracellular vesicles; NGS, next-generation sequencing; PCR, polymerase chain reaction; RNA, ribonucleic acid. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a> (accessed on 11 July 2024). Full article ">Figure 3
Comparison of examples of liquid biopsy techniques, highlighting their capabilities, shortcomings, and available technologies. Abbreviations: CNA, copy number alterations; CTC, circulating tumor cells; ctDNA, circulating tumor DNA; DNA, deoxyribonucleic acid; ddPCR, droplet digital PCR; miRNA, microRNA; NGS, next-generation sequencing; PCR, polymerase chain reaction; RNA, ribonucleic acid. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a> (accessed on 11 July 2024). Full article ">

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Int. J. Mol. Sci., Volume 25, Issue 14 (July-2 2024) – 502 articles

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