Biomolecules

Article

19 pages, 3251 KiB

Open AccessEditor’s ChoiceArticle

Enhanced L-β-Aminoisobutyric Acid Is Involved in the Pathophysiology of Effectiveness for Treatment-Resistant Schizophrenia and Adverse Reactions of Clozapine

by Kouji Fukuyama, Eishi Motomura and Motohiro Okada

Biomolecules 2023, 13(5), 862; https://doi.org/10.3390/biom13050862 - 19 May 2023

Cited by 4 | Viewed by 2009

Abstract

Clozapine is an effective antipsychotic for the treatment of antipsychotic-resistant schizophrenia; however, specific types of A/B adverse effects and clozapine-discontinuation syndromes are also well known. To date, both the critical mechanisms of clinical actions (effective for antipsychotic-resistant schizophrenia) and the adverse effects of [...] Read more.

Clozapine is an effective antipsychotic for the treatment of antipsychotic-resistant schizophrenia; however, specific types of A/B adverse effects and clozapine-discontinuation syndromes are also well known. To date, both the critical mechanisms of clinical actions (effective for antipsychotic-resistant schizophrenia) and the adverse effects of clozapine remain to be elucidated. Recently, we demonstrated that clozapine increased the synthesis of L-β-aminoisobutyric acid (L-BAIBA) in the hypothalamus. L-BAIBA is an activator of the adenosine monophosphate-activated protein kinase (AMPK), glycine receptor, GABA_A receptor, and GABA_B receptor (GABA_B-R). These targets of L-BAIBA overlap as potential targets other than the monoamine receptors of clozapine. However, the direct binding of clozapine to these aminoacidic transmitter/modulator receptors remains to be clarified. Therefore, to explore the contribution of increased L-BAIBA on the clinical action of clozapine, this study determined the effects of clozapine and L-BAIBA on tripartite synaptic transmission, including GABA_B-R and the group-III metabotropic glutamate receptor (III-mGluR) using cultured astrocytes, as well as on the thalamocortical hyper-glutamatergic transmission induced by impaired glutamate/NMDA receptors using microdialysis. Clozapine increased astroglial L-BAIBA synthesis in time/concentration-dependent manners. Increased L-BAIBA synthesis was observed until 3 days after clozapine discontinuation. Clozapine did not directly bind III-mGluR or GABA_B-R, whereas L-BAIBA activated these receptors in the astrocytes. Local administration of MK801 into the reticular thalamic nucleus (RTN) increased L-glutamate release in the medial frontal cortex (mPFC) (MK801-evoked L-glutamate release). Local administration of L-BAIBA into the mPFC suppressed MK801-evoked L-glutamate release. These actions of L-BAIBA were inhibited by antagonists of III-mGluR and GABA_B-R, similar to clozapine. These in vitro and in vivo analyses suggest that increased frontal L-BAIBA signaling likely plays an important role in the pharmacological actions of clozapine, such as improving the effectiveness of treating treatment-resistant schizophrenia and several clozapine discontinuation syndromes via the activation of III-mGluR and GABA_B-R in the mPFC. Full article

(This article belongs to the Special Issue Glutamate and Glutamate Receptors in Health and Diseases)

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12 pages, 1454 KiB

Open AccessEditor’s ChoiceArticle

Role of the MDR Efflux Pump AcrAB in Epithelial Cell Invasion by Shigella flexneri

by Marco Coluccia, Aude Béranger, Rita Trirocco, Giulia Fanelli, Francesco Zanzi, Bianca Colonna, Milena Grossi, Gianni Prosseda and Martina Pasqua

Biomolecules 2023, 13(5), 823; https://doi.org/10.3390/biom13050823 - 11 May 2023

Cited by 4 | Viewed by 2120

Abstract

The tripartite complex AcrAB-TolC is the major RND pump in Escherichia coli and other Enterobacteriaceae, including Shigella, the etiological agent of bacillary dysentery. In addition to conferring resistance to many classes of antibiotics, AcrAB plays a role in the pathogenesis and virulence [...] Read more.

The tripartite complex AcrAB-TolC is the major RND pump in Escherichia coli and other Enterobacteriaceae, including Shigella, the etiological agent of bacillary dysentery. In addition to conferring resistance to many classes of antibiotics, AcrAB plays a role in the pathogenesis and virulence of several bacterial pathogens. Here, we report data demonstrating that AcrAB specifically contributes to Shigella flexneri invasion of epithelial cells. We found that deletion of both acrA and acrB genes causes reduced survival of S. flexneri M90T strain within Caco-2 epithelial cells and prevents cell-to-cell spread of the bacteria. Infections with single deletion mutant strains indicate that both AcrA and AcrB favor the viability of the intracellular bacteria. Finally, we were able to further confirm the requirement of the AcrB transporter activity for intraepithelial survival by using a specific EP inhibitor. Overall, the data from the present study expand the role of the AcrAB pump to an important human pathogen, such as Shigella, and add insights into the mechanism governing the Shigella infection process. Full article

(This article belongs to the Special Issue Theme Issue Honoring Scientist Louis Pasteur on His 200th Birthday)

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19 pages, 4128 KiB

Open AccessEditor’s ChoiceArticle

Evolution of the Immunoglobulin Isotypes—Variations of Biophysical Properties among Animal Classes

by Nancy D. Pomarici, Roberta Cacciato, Janik Kokot, Monica L. Fernández-Quintero and Klaus R. Liedl

Biomolecules 2023, 13(5), 801; https://doi.org/10.3390/biom13050801 - 8 May 2023

Cited by 3 | Viewed by 2644

Abstract

The adaptive immune system arose around 500 million years ago in jawed fish, and, since then, it has mediated the immune defense against pathogens in all vertebrates. Antibodies play a central role in the immune reaction, recognizing and attacking external invaders. During the [...] Read more.

The adaptive immune system arose around 500 million years ago in jawed fish, and, since then, it has mediated the immune defense against pathogens in all vertebrates. Antibodies play a central role in the immune reaction, recognizing and attacking external invaders. During the evolutionary process, several immunoglobulin isotypes emerged, each having a characteristic structural organization and dedicated function. In this work, we investigate the evolution of the immunoglobulin isotypes, in order to highlight the relevant features that were preserved over time and the parts that, instead, mutated. The residues that are coupled in the evolution process are often involved in intra- or interdomain interactions, meaning that they are fundamental to maintaining the immunoglobulin fold and to ensuring interactions with other domains. The explosive growth of available sequences allows us to point out the evolutionary conserved residues and compare the biophysical properties among different animal classes and isotypes. Our study offers a general overview of the evolution of immunoglobulin isotypes and advances the knowledge of their characteristic biophysical properties, as a first step in guiding protein design from evolution. Full article

(This article belongs to the Section Biomacromolecules: Proteins)

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21 pages, 3287 KiB

Open AccessEditor’s ChoiceArticle

Mice Mutated in the Third Fibronectin Domain of L1 Show Enhanced Hippocampal Neuronal Cell Death, Astrogliosis and Alterations in Behavior

by Ludovica Congiu, Viviana Granato, Igor Jakovcevski, Ralf Kleene, Luciana Fernandes, Sandra Freitag, Matthias Kneussel, Melitta Schachner and Gabriele Loers

Biomolecules 2023, 13(5), 776; https://doi.org/10.3390/biom13050776 - 29 Apr 2023

Cited by 2 | Viewed by 1898

Abstract

Adhesion molecules play major roles in cell proliferation, migration, survival, neurite outgrowth and synapse formation during nervous system development and in adulthood. The neural cell adhesion molecule L1 contributes to these functions during development and in synapse formation and synaptic plasticity after trauma [...] Read more.

Adhesion molecules play major roles in cell proliferation, migration, survival, neurite outgrowth and synapse formation during nervous system development and in adulthood. The neural cell adhesion molecule L1 contributes to these functions during development and in synapse formation and synaptic plasticity after trauma in adulthood. Mutations of L1 in humans result in L1 syndrome, which is associated with mild-to-severe brain malformations and mental disabilities. Furthermore, mutations in the extracellular domain were shown to cause a severe phenotype more often than mutations in the intracellular domain. To explore the outcome of a mutation in the extracellular domain, we generated mice with disruption of the dibasic sequences RK and KR that localize to position ₈₅₈RKHSKR₈₆₃ in the third fibronectin type III domain of murine L1. These mice exhibit alterations in exploratory behavior and enhanced marble burying activity. Mutant mice display higher numbers of caspase 3-positive neurons, a reduced number of principle neurons in the hippocampus, and an enhanced number of glial cells. Experiments suggest that disruption of the dibasic sequence in L1 results in subtle impairments in brain structure and functions leading to obsessive-like behavior in males and reduced anxiety in females. Full article

(This article belongs to the Special Issue More than Just Adhesion: Recognition Molecules, a Theme Issue Honoring Professor Melitta Schachner)

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17 pages, 6737 KiB

Open AccessEditor’s ChoiceArticle

Unexpected Distribution of Chitin and Chitin Synthase across Soft-Bodied Cnidarians

by Lauren E. Vandepas, Michael G. Tassia, Kenneth M. Halanych and Chris T. Amemiya

Biomolecules 2023, 13(5), 777; https://doi.org/10.3390/biom13050777 - 29 Apr 2023

Cited by 4 | Viewed by 2517

Abstract

Cnidarians are commonly recognized as sea jellies, corals, or complex colonies such as the Portuguese man-of-war. While some cnidarians possess rigid internal calcareous skeletons (e.g., corals), many are soft-bodied. Intriguingly, genes coding for the chitin-biosynthetic enzyme, chitin synthase (CHS), were recently identified [...] Read more.

Cnidarians are commonly recognized as sea jellies, corals, or complex colonies such as the Portuguese man-of-war. While some cnidarians possess rigid internal calcareous skeletons (e.g., corals), many are soft-bodied. Intriguingly, genes coding for the chitin-biosynthetic enzyme, chitin synthase (CHS), were recently identified in the model anemone Nematostella vectensis, a species lacking hard structures. Here we report the prevalence and diversity of CHS across Cnidaria and show that cnidarian chitin synthase genes display diverse protein domain organizations. We found that CHS is expressed in cnidarian species and/or developmental stages with no reported chitinous or rigid morphological structures. Chitin affinity histochemistry indicates that chitin is present in soft tissues of some scyphozoan and hydrozoan medusae. To further elucidate the biology of chitin in cnidarian soft tissues, we focused on CHS expression in N. vectensis. Spatial expression data show that three CHS orthologs are differentially expressed in Nematostella embryos and larvae during development, suggesting that chitin has an integral role in the biology of this species. Understanding how a non-bilaterian lineage such as Cnidaria employs chitin may provide new insight into hitherto unknown functions of polysaccharides in animals, as well as their role in the evolution of biological novelty. Full article

(This article belongs to the Collection Feature Papers in Biochemistry)

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13 pages, 1831 KiB

Open AccessEditor’s ChoiceArticle

Glucose Transporter Glut1-Dependent Metabolic Reprogramming Regulates Lipopolysaccharide-Induced Inflammation in RAW264.7 Macrophages

by Alex Cornwell, Hubert Ziółkowski and Alireza Badiei

Biomolecules 2023, 13(5), 770; https://doi.org/10.3390/biom13050770 - 29 Apr 2023

Cited by 4 | Viewed by 2967

Abstract

This study investigated the critical role of Glut1-mediated glucose metabolism in the inflammatory response of macrophages, which are energy-intensive cells within the innate immune system. Inflammation leads to increased Glut1 expression, ensuring sufficient glucose uptake to support macrophage functions. We demonstrated that using [...] Read more.

This study investigated the critical role of Glut1-mediated glucose metabolism in the inflammatory response of macrophages, which are energy-intensive cells within the innate immune system. Inflammation leads to increased Glut1 expression, ensuring sufficient glucose uptake to support macrophage functions. We demonstrated that using siRNA to knock down Glut1 reduces the expression of various pro-inflammatory cytokines and markers, such as IL-6, iNOS, MHC II/CD40, reactive oxygen species, and the hydrogen sulfide (H₂S)-producing enzyme cystathionine γ-lyase (CSE). Glut1 activates a pro-inflammatory profile through a nuclear factor (NF)-κB, while silencing Glut1 can prevent lipopolysaccharide (LPS)-induced IκB degradation, blocking NF-κB activation. Glut1’s role in autophagy, an essential process for macrophage functions such as antigen presentation, phagocytosis, and cytokine secretion, was also measured. The findings show that LPS stimulation decreases autophagosome formation, but Glut1 knockdown reverses this effect, increasing autophagy beyond control levels. The study highlights Glut1’s importance in macrophage immune responses and its regulation of apoptosis during LPS stimulation. Knocking down Glut1 negatively impacts cell viability and mitochondrial intrinsic pathway signaling. These findings collectively suggest that targeting macrophage glucose metabolism through Glut1 could potentially serve as a target for controlling inflammation. Full article

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17 pages, 5074 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Physico-Chemical Changes Induced by Gamma Irradiation on Some Structural Protein Extracts

by Maria Stanca, Carmen Gaidau, Traian Zaharescu, George-Alin Balan, Iulia Matei, Aurica Precupas, Anca Ruxandra Leonties and Gabriela Ionita

Biomolecules 2023, 13(5), 774; https://doi.org/10.3390/biom13050774 - 29 Apr 2023

Cited by 6 | Viewed by 1898

Abstract

In this study, the effect of gamma irradiation (10 kGy) on proteins extracted from animal hide, scales, and wool was evidenced by calorimetric (μDSC) and spectroscopic (IR, circular dichroism, and EPR) methods. Keratin was obtained from sheep wool, collagen and bovine gelatin from [...] Read more.

In this study, the effect of gamma irradiation (10 kGy) on proteins extracted from animal hide, scales, and wool was evidenced by calorimetric (μDSC) and spectroscopic (IR, circular dichroism, and EPR) methods. Keratin was obtained from sheep wool, collagen and bovine gelatin from bovine hide, and fish gelatin from fish scales. The μDSC experiments evidenced that gamma irradiation influences the thermal stability of these proteins differently. The thermal stability of keratin decreases, while a resistance to thermal denaturation was noticed for collagen and gelatins after gamma irradiation. The analysis of the IR spectra demonstrated that gamma irradiation determines changes in the vibrational modes of the amide groups that are associated with protein denaturation, most meaningfully in the case of keratin. As evidenced by circular dichroism for all proteins considered, exposure to gamma radiation produces changes in the secondary structure that are more significant than those produced by UV irradiation. Riboflavin has different effects on the secondary structure of the investigated proteins, a stabilizing effect for keratin and fish gelatin and a destabilizing effect for bovine gelatin, observed in both irradiated and non-irradiated samples. The EPR spectroscopy evidences the presence, in the gamma-irradiated samples, of free radicals centered on oxygen, and the increase in their EPR signals over time due to the presence of riboflavin. Full article

(This article belongs to the Special Issue Biomarkers of Oxidative and Radical Stress)

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15 pages, 3389 KiB

Open AccessEditor’s ChoiceArticle

Liposomal DQ in Combination with Copper Inhibits ARID1A Mutant Ovarian Cancer Growth

by Xuejia Kang, Qi Wang, Siqi Wu, Chuanyu Wang, Manjusha Annaji, Chung-Hui Huang, Jianzhong Shen, Pengyu Chen and R. Jayachandra Babu

Biomolecules 2023, 13(5), 744; https://doi.org/10.3390/biom13050744 - 25 Apr 2023

Cited by 1 | Viewed by 2135

Abstract

Therapeutic strategies for ARID1A-mutant ovarian cancers are limited. Higher basal reactive oxygen species (ROS) and lower basal glutathione (GSH) empower the aggressive proliferation ability and strong metastatic property of OCCCs, indicated by the increased marker of epithelial-mesenchymal transition (EMT) and serving the immunosuppressive [...] Read more.

Therapeutic strategies for ARID1A-mutant ovarian cancers are limited. Higher basal reactive oxygen species (ROS) and lower basal glutathione (GSH) empower the aggressive proliferation ability and strong metastatic property of OCCCs, indicated by the increased marker of epithelial-mesenchymal transition (EMT) and serving the immunosuppressive microenvironment. However, the aberrant redox homeostasis also empowers the sensitivity of DQ-Lipo/Cu in a mutant cell line. DQ, a carbamodithioic acid derivative, generates dithiocarbamate (DDC) in response to ROS, and the chelation of Cu and DDC further generates ROS and provides a ROS cascade. Besides, quinone methide (QM) released by DQ targets the vulnerability of GSH; this effect, plus the increase of ROS, destroys the redox homeostasis and causes cancer cell death. Also importantly, the formed Cu(DDC)₂ is a potent cytotoxic anti-cancer drug that successfully induces immunogenic cell death (ICD). The synergistic effect of EMT regulation and ICD will contribute to managing cancer metastasis and possible drug resistance. In summary, our DQ-Lipo/Cu shows promising inhibitory effects in cancer proliferation, EMT markers, and “heat” the immune response. Full article

(This article belongs to the Section Molecular Medicine)

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17 pages, 2389 KiB

Open AccessEditor’s ChoiceArticle

Plasmalogens: Free Radical Reactivity and Identification of Trans Isomers Relevant to Biological Membranes

by Carla Ferreri, Alessandra Ferocino, Gessica Batani, Chryssostomos Chatgilialoglu, Vanda Randi, Maria Vittoria Riontino, Fabrizio Vetica and Anna Sansone

Biomolecules 2023, 13(5), 730; https://doi.org/10.3390/biom13050730 - 24 Apr 2023

Cited by 7 | Viewed by 2918

Abstract

Plasmalogens are membrane phospholipids with two fatty acid hydrocarbon chains linked to L-glycerol, one containing a characteristic cis-vinyl ether function and the other one being a polyunsaturated fatty acid (PUFA) residue linked through an acyl function. All double bonds in these structures display [...] Read more.

Plasmalogens are membrane phospholipids with two fatty acid hydrocarbon chains linked to L-glycerol, one containing a characteristic cis-vinyl ether function and the other one being a polyunsaturated fatty acid (PUFA) residue linked through an acyl function. All double bonds in these structures display the cis geometrical configuration due to desaturase enzymatic activity and they are known to be involved in the peroxidation process, whereas the reactivity through cis-trans double bond isomerization has not yet been identified. Using 1-(1Z-octadecenyl)-2-arachidonoyl-sn-glycero-3-phosphocholine (C18 plasm-20:4 PC) as a representative molecule, we showed that the cis-trans isomerization can occur at both plasmalogen unsaturated moieties, and the product has characteristic analytical signatures useful for omics applications. Using plasmalogen-containing liposomes and red blood cell (RBC) ghosts under biomimetic Fenton-like conditions, in the presence or absence of thiols, peroxidation, and isomerization processes were found to occur with different reaction outcomes due to the particular liposome compositions. These results allow gaining a full scenario of plasmalogen reactivity under free radical conditions. Moreover, clarification of the plasmalogen reactivity under acidic and alkaline conditions was carried out, identifying the best protocol for RBC membrane fatty acid analysis due to their plasmalogen content of 15–20%. These results are important for lipidomic applications and for achieving a full scenario of radical stress in living organisms. Full article

(This article belongs to the Special Issue Biomarkers of Oxidative and Radical Stress)

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29 pages, 4603 KiB

Open AccessEditor’s ChoiceArticle

Structural and Biochemical Characterization of Mycobacterium tuberculosis Zinc SufU-SufS Complex

by Ingie Elchennawi, Philippe Carpentier, Christelle Caux, Marine Ponge and Sandrine Ollagnier de Choudens

Biomolecules 2023, 13(5), 732; https://doi.org/10.3390/biom13050732 - 24 Apr 2023

Cited by 4 | Viewed by 3792

Abstract

Iron-sulfur (Fe-S) clusters are inorganic prosthetic groups in proteins composed exclusively of iron and inorganic sulfide. These cofactors are required in a wide range of critical cellular pathways. Iron-sulfur clusters do not form spontaneously in vivo; several proteins are required to mobilize sulfur [...] Read more.

Iron-sulfur (Fe-S) clusters are inorganic prosthetic groups in proteins composed exclusively of iron and inorganic sulfide. These cofactors are required in a wide range of critical cellular pathways. Iron-sulfur clusters do not form spontaneously in vivo; several proteins are required to mobilize sulfur and iron, assemble and traffic-nascent clusters. Bacteria have developed several Fe-S assembly systems, such as the ISC, NIF, and SUF systems. Interestingly, in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), the SUF machinery is the primary Fe-S biogenesis system. This operon is essential for the viability of Mtb under normal growth conditions, and the genes it contains are known to be vulnerable, revealing the Mtb SUF system as an interesting target in the fight against tuberculosis. In the present study, two proteins of the Mtb SUF system were characterized for the first time: Rv1464(sufS) and Rv1465(sufU). The results presented reveal how these two proteins work together and thus provide insights into Fe-S biogenesis/metabolism by this pathogen. Combining biochemistry and structural approaches, we showed that Rv1464 is a type II cysteine-desulfurase enzyme and that Rv1465 is a zinc-dependent protein interacting with Rv1464. Endowed with a sulfurtransferase activity, Rv1465 significantly enhances the cysteine-desulfurase activity of Rv1464 by transferring the sulfur atom from persulfide on Rv1464 to its conserved Cys40 residue. The zinc ion is important for the sulfur transfer reaction between SufS and SufU, and His354 in SufS plays an essential role in this reaction. Finally, we showed that Mtb SufS-SufU is more resistant to oxidative stress than E. coli SufS-SufE and that the presence of zinc in SufU is likely responsible for this improved resistance. This study on Rv1464 and Rv1465 will help guide the design of future anti-tuberculosis agents. Full article

(This article belongs to the Special Issue Biomolecule-Metal Ion Interaction)

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15 pages, 1638 KiB

Open AccessEditor’s ChoiceArticle

Adenosine A_2A Receptors Shut Down Adenosine A₁ Receptor-Mediated Presynaptic Inhibition to Promote Implementation of Hippocampal Long-Term Potentiation

by Cátia R. Lopes, Francisco Q. Gonçalves, Simão Olaio, Angelo R. Tomé, Rodrigo A. Cunha and João Pedro Lopes

Biomolecules 2023, 13(4), 715; https://doi.org/10.3390/biom13040715 - 21 Apr 2023

Cited by 7 | Viewed by 2058

Abstract

Adenosine operates a modulation system fine-tuning the efficiency of synaptic transmission and plasticity through A₁ and A_2A receptors (A₁R, A_2AR), respectively. Supramaximal activation of A₁R can block hippocampal synaptic transmission, and the tonic engagement of [...] Read more.

Adenosine operates a modulation system fine-tuning the efficiency of synaptic transmission and plasticity through A₁ and A_2A receptors (A₁R, A_2AR), respectively. Supramaximal activation of A₁R can block hippocampal synaptic transmission, and the tonic engagement of A₁R-mediated inhibition is increased with increased frequency of nerve stimulation. This is compatible with an activity-dependent increase in extracellular adenosine in hippocampal excitatory synapses, which can reach levels sufficient to block synaptic transmission. We now report that A_2AR activation decreases A₁R-medated inhibition of synaptic transmission, with particular relevance during high-frequency-induced long-term potentiation (LTP). Thus, whereas the A₁R antagonist DPCPX (50 nM) was devoid of effects on LTP magnitude, the addition of an A_2AR antagonist SCH58261 (50 nM) allowed a facilitatory effect of DPCPX on LTP to be revealed. Additionally, the activation of A_2AR with CGS21680 (30 nM) decreased the potency of the A₁R agonist CPA (6–60 nM) to inhibit hippocampal synaptic transmission in a manner prevented by SCH58261. These observations show that A_2AR play a key role in dampening A₁R during high-frequency induction of hippocampal LTP. This provides a new framework for understanding how the powerful adenosine A₁R-mediated inhibition of excitatory transmission can be controlled to allow the implementation of hippocampal LTP. Full article

(This article belongs to the Special Issue Pharmacology of Purinergic Receptors—Honorary Special Issue Dedicated to Prof. Pier Andrea Borea)

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21 pages, 4159 KiB

Open AccessEditor’s ChoiceArticle

A Soluble Platelet-Derived Growth Factor Receptor-β Originates via Pre-mRNA Splicing in the Healthy Brain and Is Upregulated during Hypoxia and Aging

by Laura Beth Payne, Hanaa Abdelazim, Maruf Hoque, Audra Barnes, Zuzana Mironovova, Caroline E. Willi, Jordan Darden, Clifton Houk, Meghan W. Sedovy, Scott R. Johnstone and John C. Chappell

Biomolecules 2023, 13(4), 711; https://doi.org/10.3390/biom13040711 - 21 Apr 2023

Cited by 1 | Viewed by 3013

Abstract

The platelet-derived growth factor-BB (PDGF-BB) pathway provides critical regulation of cerebrovascular pericytes, orchestrating their investment and retention within the brain microcirculation. Dysregulated PDGF Receptor-beta (PDGFRβ) signaling can lead to pericyte defects that compromise blood-brain barrier (BBB) integrity and cerebral perfusion, impairing neuronal activity [...] Read more.

The platelet-derived growth factor-BB (PDGF-BB) pathway provides critical regulation of cerebrovascular pericytes, orchestrating their investment and retention within the brain microcirculation. Dysregulated PDGF Receptor-beta (PDGFRβ) signaling can lead to pericyte defects that compromise blood-brain barrier (BBB) integrity and cerebral perfusion, impairing neuronal activity and viability, which fuels cognitive and memory deficits. Receptor tyrosine kinases such as PDGF-BB and vascular endothelial growth factor-A (VEGF-A) are often modulated by soluble isoforms of cognate receptors that establish signaling activity within a physiological range. Soluble PDGFRβ (sPDGFRβ) isoforms have been reported to form by enzymatic cleavage from cerebrovascular mural cells, and pericytes in particular, largely under pathological conditions. However, pre-mRNA alternative splicing has not been widely explored as a possible mechanism for generating sPDGFRβ variants, and specifically during tissue homeostasis. Here, we found sPDGFRβ protein in the murine brain and other tissues under normal, physiological conditions. Utilizing brain samples for follow-on analysis, we identified mRNA sequences corresponding to sPDGFRβ isoforms, which facilitated construction of predicted protein structures and related amino acid sequences. Human cell lines yielded comparable sequences and protein model predictions. Retention of ligand binding capacity was confirmed for sPDGFRβ by co-immunoprecipitation. Visualizing fluorescently labeled sPDGFRβ transcripts revealed a spatial distribution corresponding to murine brain pericytes alongside cerebrovascular endothelium. Soluble PDGFRβ protein was detected throughout the brain parenchyma in distinct regions, such as along the lateral ventricles, with signals also found more broadly adjacent to cerebral microvessels consistent with pericyte labeling. To better understand how sPDGFRβ variants might be regulated, we found elevated transcript and protein levels in the murine brain with age, and acute hypoxia increased sPDGFRβ variant transcripts in a cell-based model of intact vessels. Our findings indicate that soluble isoforms of PDGFRβ likely arise from pre-mRNA alternative splicing, in addition to enzymatic cleavage mechanisms, and these variants exist under normal physiological conditions. Follow-on studies will be needed to establish potential roles for sPDGFRβ in regulating PDGF-BB signaling to maintain pericyte quiescence, BBB integrity, and cerebral perfusion—critical processes underlying neuronal health and function, and in turn, memory and cognition. Full article

(This article belongs to the Special Issue The Role of Vascular Dysfunction in Neuronal Degeneration and Cognitive Impairment)

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21 pages, 5694 KiB

Open AccessEditor’s ChoiceArticle

Tyrosinase Magnetic Cross-Linked Enzyme Aggregates: Biocatalytic Study in Deep Eutectic Solvent Aqueous Solutions

by Myrto G. Bellou, Michaela Patila, Renia Fotiadou, Konstantinos Spyrou, Feng Yan, Petra Rudolf, Dimitrios P. Gournis and Haralambos Stamatis

Biomolecules 2023, 13(4), 643; https://doi.org/10.3390/biom13040643 - 3 Apr 2023

Cited by 3 | Viewed by 9918

Abstract

In the field of biocatalysis, the implementation of sustainable processes such as enzyme immobilization or employment of environmentally friendly solvents, like Deep Eutectic Solvents (DESs) are of paramount importance. In this work, tyrosinase was extracted from fresh mushrooms and used in a carrier-free [...] Read more.

In the field of biocatalysis, the implementation of sustainable processes such as enzyme immobilization or employment of environmentally friendly solvents, like Deep Eutectic Solvents (DESs) are of paramount importance. In this work, tyrosinase was extracted from fresh mushrooms and used in a carrier-free immobilization towards the preparation of both non-magnetic and magnetic cross-linked enzyme aggregates (CLEAs). The prepared biocatalyst was characterized and the biocatalytic and structural traits of free tyrosinase and tyrosinase magnetic CLEAs (mCLEAs) were evaluated in numerous DES aqueous solutions. The results showed that the nature and the concentration of the DESs used as co-solvents significantly affected the catalytic activity and stability of tyrosinase, while the immobilization enhanced the activity of the enzyme in comparison with the non-immobilized enzyme up to 3.6-fold. The biocatalyst retained the 100% of its initial activity after storage at −20 °C for 1 year and the 90% of its activity after 5 repeated cycles. Tyrosinase mCLEAs were further applied in the homogeneous modification of chitosan with caffeic acid in the presence of DES. The biocatalyst demonstrated great ability in the functionalization of chitosan with caffeic acid in the presence of 10% v/v DES [Bet:Gly (1:3)], enhancing the antioxidant activity of the films. Full article

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10 pages, 2839 KiB

Open AccessEditor’s ChoiceArticle

CRISPR/Cas9 Mediated Fluorescent Tagging of Caenorhabditis elegans SPE-38 Reveals a Complete Localization Pattern in Live Spermatozoa

by Yamei Zuo, Xue Mei and Andrew Singson

Biomolecules 2023, 13(4), 623; https://doi.org/10.3390/biom13040623 - 30 Mar 2023

Cited by 1 | Viewed by 1930

Abstract

The Caenorhabditis elegans spe-38 gene encodes a four-pass transmembrane molecule that is required in sperm for fertilization. In previous work, the localization of the SPE-38 protein was examined using polyclonal antibodies on spermatids and mature amoeboid spermatozoa. SPE-38 is localized to unfused membranous organelles [...] Read more.

The Caenorhabditis elegans spe-38 gene encodes a four-pass transmembrane molecule that is required in sperm for fertilization. In previous work, the localization of the SPE-38 protein was examined using polyclonal antibodies on spermatids and mature amoeboid spermatozoa. SPE-38 is localized to unfused membranous organelles (MOs) in nonmotile spermatids. Different fixation conditions revealed that SPE-38 either localized to fused MOs and the cell body plasma membrane or the pseudopod plasma membrane of mature sperm. To address this localization paradox in mature sperm, CRISPR/Cas9 genome editing was used to tag endogenous SPE-38 with fluorescent wrmScarlet-I. Homozygous male and hermaphrodite worms encoding SPE-38::wrmScarlet-I were fertile indicating the fluorescent tag does not interfere with SPE-38 function during sperm activation or fertilization. We found that SPE-38::wrmScarlet-I localized to MOs in spermatids consistent with previous antibody localization. In mature and motile spermatozoa we found SPE-38::wrmScarlet-I in fused MOs, the cell body plasma membrane, and the pseudopod plasma membrane. We conclude that the localization pattern observed with SPE-38::wrmScarlet-I represents the complete distribution of SPE-38 in mature spermatozoa and this localization pattern is consistent with a hypothesized role of SPE-38 directly in sperm-egg binding and/or fusion. Full article

(This article belongs to the Special Issue Gametogenesis and Gamete Interaction)

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14 pages, 1901 KiB

Open AccessEditor’s ChoiceArticle

Blood–Brain Barrier Integrity Is Perturbed in a Mecp2-Null Mouse Model of Rett Syndrome

by Giuseppe Pepe, Salvatore Fioriniello, Federico Marracino, Luca Capocci, Vittorio Maglione, Maurizio D’Esposito, Alba Di Pardo and Floriana Della Ragione

Biomolecules 2023, 13(4), 606; https://doi.org/10.3390/biom13040606 - 28 Mar 2023

Cited by 4 | Viewed by 3018

Abstract

Rett syndrome (RTT, online MIM 312750) is a devastating neurodevelopmental disorder characterized by motor and cognitive disabilities. It is mainly caused by pathogenetic variants in the X-linked MECP2 gene, encoding an epigenetic factor crucial for brain functioning. Despite intensive studies, the RTT pathogenetic [...] Read more.

Rett syndrome (RTT, online MIM 312750) is a devastating neurodevelopmental disorder characterized by motor and cognitive disabilities. It is mainly caused by pathogenetic variants in the X-linked MECP2 gene, encoding an epigenetic factor crucial for brain functioning. Despite intensive studies, the RTT pathogenetic mechanism remains to be fully elucidated. Impaired vascular function has been previously reported in RTT mouse models; however, whether an altered brain vascular homeostasis and the subsequent blood–brain barrier (BBB) breakdown occur in RTT and contribute to the disease-related cognitive impairment is still unknown. Interestingly, in symptomatic Mecp2-null (Mecp2^-/y, Mecp2^tm1.1Bird) mice, we found enhanced BBB permeability associated with an aberrant expression of the tight junction proteins Ocln and Cldn-5 in different brain areas, in terms of both transcript and protein levels. Additionally, Mecp2-null mice showed an altered expression of different genes encoding factors with a role in the BBB structure and function, such as Cldn3, Cldn12, Mpdz, Jam2, and Aqp4. With this study, we provide the first evidence of impaired BBB integrity in RTT and highlight a potential new molecular hallmark of the disease that might open new perspectives for the setting-up of novel therapeutic strategies. Full article

(This article belongs to the Special Issue Neurodevelopmental Disorders: Linking Genetics and Epigenetics to Disease-Related Pathways)

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15 pages, 2600 KiB

Open AccessEditor’s ChoiceArticle

Triphenyltin(IV) Carboxylates with Exceptionally High Cytotoxicity against Different Breast Cancer Cell Lines

by Ivana Predarska, Mohamad Saoud, Ibrahim Morgan, Peter Lönnecke, Goran N. Kaluđerović and Evamarie Hey-Hawkins

Biomolecules 2023, 13(4), 595; https://doi.org/10.3390/biom13040595 - 26 Mar 2023

Cited by 11 | Viewed by 2164

Abstract

Organotin(IV) carboxylates are a class of compounds explored as alternatives to platinum-containing chemotherapeutics due to propitious in vitro and in vivo results, and distinct mechanisms of action. In this study, triphenyltin(IV) derivatives of non-steroidal anti-inflammatory drugs (indomethacin (HIND) and flurbiprofen (HFBP)) are synthesized [...] Read more.

Organotin(IV) carboxylates are a class of compounds explored as alternatives to platinum-containing chemotherapeutics due to propitious in vitro and in vivo results, and distinct mechanisms of action. In this study, triphenyltin(IV) derivatives of non-steroidal anti-inflammatory drugs (indomethacin (HIND) and flurbiprofen (HFBP)) are synthesized and characterized, namely [Ph₃Sn(IND)] and [Ph₃Sn(FBP)]. The crystal structure of [Ph₃Sn(IND)] reveals penta-coordination of the central tin atom with almost perfect trigonal bipyramidal geometry with phenyl groups in the equatorial positions and two axially located oxygen atoms belonging to two distinct carboxylato (IND) ligands leading to formation of a coordination polymer with bridging carboxylato ligands. Employing MTT and CV probes, the antiproliferative effects of both organotin(IV) complexes, indomethacin, and flurbiprofen were evaluated on different breast carcinoma cells (BT-474, MDA-MB-468, MCF-7 and HCC1937). [Ph₃Sn(IND)] and [Ph₃Sn(FBP)], unlike the inactive ligand precursors, were found extremely active towards all examined cell lines, demonstrating IC₅₀ concentrations in the range of 0.076–0.200 µM. Flow cytometry was employed to examine the mode of action showing that neither apoptotic nor autophagic mechanisms were triggered within the first 48 h of treatment. However, both tin(IV) complexes inhibited cell proliferation potentially related to the dramatic reduction in NO production, resulting from downregulation of nitric oxide synthase (iNOS) enzyme expression. Full article

(This article belongs to the Special Issue Recent Advances in Anti-tumor Metal Complexes and Drug Delivery Systems)

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28 pages, 6466 KiB

Open AccessEditor’s ChoiceArticle

Genome and Genetic Engineering of the House Cricket (Acheta domesticus): A Resource for Sustainable Agriculture

by Aaron T. Dossey, Brenda Oppert, Fu-Chyun Chu, Marcé D. Lorenzen, Brian Scheffler, Sheron Simpson, Sergey Koren, J. Spencer Johnston, Kosuke Kataoka and Keigo Ide

Biomolecules 2023, 13(4), 589; https://doi.org/10.3390/biom13040589 - 24 Mar 2023

Cited by 10 | Viewed by 8016

Abstract

Background: The house cricket, Acheta domesticus, is one of the most farmed insects worldwide and the foundation of an emerging industry using insects as a sustainable food source. Edible insects present a promising alternative for protein production amid a plethora of reports [...] Read more.

Background: The house cricket, Acheta domesticus, is one of the most farmed insects worldwide and the foundation of an emerging industry using insects as a sustainable food source. Edible insects present a promising alternative for protein production amid a plethora of reports on climate change and biodiversity loss largely driven by agriculture. As with other crops, genetic resources are needed to improve crickets for food and other applications. Methods: We present the first high quality annotated genome assembly of A. domesticus from long read data and scaffolded to chromosome level, providing information needed for genetic manipulation. Results: Gene groups related to immunity were annotated and will be useful for improving value to insect farmers. Metagenome scaffolds in the A. domesticus assembly, including Invertebrate Iridescent Virus 6 (IIV6), were submitted as host-associated sequences. We demonstrate both CRISPR/Cas9-mediated knock-in and knock-out of A. domesticus and discuss implications for the food, pharmaceutical, and other industries. RNAi was demonstrated to disrupt the function of the vermilion eye-color gene producing a useful white-eye biomarker phenotype. Conclusions: We are utilizing these data to develop technologies for downstream commercial applications, including more nutritious and disease-resistant crickets, as well as lines producing valuable bioproducts, such as vaccines and antibiotics. Full article

(This article belongs to the Special Issue The Genomics Era: From Reference Genomes to Pan-Genomic Graphs)

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15 pages, 1519 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Comparison of Biomolecular Condensate Localization and Protein Phase Separation Predictors

by Erich R. Kuechler, Alex Huang, Jennifer M. Bui, Thibault Mayor and Jörg Gsponer

Biomolecules 2023, 13(3), 527; https://doi.org/10.3390/biom13030527 - 13 Mar 2023

Cited by 4 | Viewed by 3012

Abstract

Research in the field of biochemistry and cellular biology has entered a new phase due to the discovery of phase separation driving the formation of biomolecular condensates, or membraneless organelles, in cells. The implications of this novel principle of cellular organization are vast [...] Read more.

Research in the field of biochemistry and cellular biology has entered a new phase due to the discovery of phase separation driving the formation of biomolecular condensates, or membraneless organelles, in cells. The implications of this novel principle of cellular organization are vast and can be applied at multiple scales, spawning exciting research questions in numerous directions. Of fundamental importance are the molecular mechanisms that underly biomolecular condensate formation within cells and whether insights gained into these mechanisms provide a gateway for accurate predictions of protein phase behavior. Within the last six years, a significant number of predictors for protein phase separation and condensate localization have emerged. Herein, we compare a collection of state-of-the-art predictors on different tasks related to protein phase behavior. We show that the tested methods achieve high AUCs in the identification of biomolecular condensate drivers and scaffolds, as well as in the identification of proteins able to phase separate in vitro. However, our benchmark tests reveal that their performance is poorer when used to predict protein segments that are involved in phase separation or to classify amino acid substitutions as phase-separation-promoting or -inhibiting mutations. Our results suggest that the phenomenological approach used by most predictors is insufficient to fully grasp the complexity of the phenomenon within biological contexts and make reliable predictions related to protein phase behavior at the residue level. Full article

(This article belongs to the Collection Feature Papers in Bioinformatics and Systems Biology Section)

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12 pages, 3894 KiB

Open AccessEditor’s ChoiceArticle

Kawain Inhibits Urinary Bladder Carcinogenesis through Epigenetic Inhibition of LSD1 and Upregulation of H3K4 Methylation

by Xia Xu, Xuejiao Tian, Liankun Song, Jun Xie, Joseph C. Liao, Joshua J. Meeks, Xue-Ru Wu, Greg E. Gin, Beverly Wang, Edward Uchio and Xiaolin Zi

Biomolecules 2023, 13(3), 521; https://doi.org/10.3390/biom13030521 - 13 Mar 2023

Cited by 2 | Viewed by 2046

Abstract

Epidemiological evidence suggests that kava (Piper methysticum Forst) drinks may reduce the risk of cancer in South Pacific Island smokers. However, little is known about the anti-carcinogenic effects of kava on tobacco smoking-related bladder cancer and its underlying mechanisms. Here we [...] Read more.

Epidemiological evidence suggests that kava (Piper methysticum Forst) drinks may reduce the risk of cancer in South Pacific Island smokers. However, little is known about the anti-carcinogenic effects of kava on tobacco smoking-related bladder cancer and its underlying mechanisms. Here we show that dietary feeding of kawain (a major active component in kava root extracts) to mice either before or after hydroxy butyl(butyl) nitrosamine (OH-BBN) carcinogen exposure slows down urinary bladder carcinogenesis and prolongs the survival of the OH-BBN-exposed mice. OH-BBN-induced bladder tumors exhibit significantly increased expression of lysine-specific demethylase 1 (LSD1), accompanied by decreased levels of H3K4 mono-methylation compared to normal bladder epithelium, whereas dietary kawain reverses the effects of OH-BBN on H3K4 mono-methylation. Human bladder cancer tumor tissues at different pathological grades also show significantly increased expression of LSD1 and decreased levels of H3K4 mono-methylation compared to normal urothelium. In addition, kava root extracts and the kavalactones kawain and methysticin all increase the levels of H3K4 mono- and di-methylation, leading to inhibitory effects on cell migration. Taken together, our results suggest that modification of histone lysine methylation may represent a new approach to bladder cancer prevention and treatment and that kavalactones may be promising agents for bladder cancer interception in both current and former smokers. Full article

(This article belongs to the Special Issue Dietary Phytochemicals - Mediated Cellular Signalling, Epigenetics/Epigenomics and Cancer Chemoprevention)

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14 pages, 1975 KiB

Open AccessEditor’s ChoiceArticle

Dysregulation of AMPA Receptor Trafficking and Intracellular Vesicular Sorting in the Prefrontal Cortex of Dopamine Transporter Knock-Out Rats

by Giorgia Targa, Francesca Mottarlini, Beatrice Rizzi, Damiana Leo, Lucia Caffino and Fabio Fumagalli

Biomolecules 2023, 13(3), 516; https://doi.org/10.3390/biom13030516 - 11 Mar 2023

Cited by 2 | Viewed by 1973

Abstract

Dopamine (DA) and glutamate interact, influencing neural excitability and promoting synaptic plasticity. However, little is known regarding the molecular mechanisms underlying this crosstalk. Since perturbation of DA-AMPA receptor interaction might sustain pathological conditions, the major aim of our work was to evaluate the [...] Read more.

Dopamine (DA) and glutamate interact, influencing neural excitability and promoting synaptic plasticity. However, little is known regarding the molecular mechanisms underlying this crosstalk. Since perturbation of DA-AMPA receptor interaction might sustain pathological conditions, the major aim of our work was to evaluate the effect of the hyperactive DA system on the AMPA subunit composition, trafficking, and membrane localization in the prefrontal cortex (PFC). Taking advantage of dopamine transporter knock-out (DAT^−/−) rats, we found that DA overactivity reduced the translation of cortical AMPA receptors and their localization at both synaptic and extra-synaptic sites through, at least in part, altered intracellular vesicular sorting. Moreover, the reduced expression of AMPA receptor-specific anchoring proteins and structural markers, such as Neuroligin-1 and nCadherin, likely indicate a pattern of synaptic instability. Overall, these data reveal that a condition of hyperdopaminergia markedly alters the homeostatic plasticity of AMPA receptors, suggesting a general destabilization and depotentiation of the AMPA-mediated glutamatergic neurotransmission in the PFC. This effect might be functionally relevant for disorders characterized by elevated dopaminergic activity. Full article

(This article belongs to the Special Issue Biogenic Amines, Their Receptors and Transporters in Health and Disease: Commemorative Issue in Honor of Professor Marc G. Caron (1946–2022))

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23 pages, 2458 KiB

Open AccessEditor’s ChoiceArticle

How Clustered DNA Damage Can Change the Electronic Properties of ds-DNA—Differences between GAG, GA^OXOG, and ^OXOGA^OXOG

by Boleslaw Karwowski

Biomolecules 2023, 13(3), 517; https://doi.org/10.3390/biom13030517 - 11 Mar 2023

Cited by 5 | Viewed by 1501

Abstract

Every 24 h, roughly 3 × 10¹⁷ incidences of DNA damage are generated in the human body as a result of intra- or extra-cellular factors. The structure of the formed lesions is identical to that formed during radio- or chemotherapy. Increases in [...] Read more.

Every 24 h, roughly 3 × 10¹⁷ incidences of DNA damage are generated in the human body as a result of intra- or extra-cellular factors. The structure of the formed lesions is identical to that formed during radio- or chemotherapy. Increases in the clustered DNA damage (CDL) level during anticancer treatment have been observed compared to those found in untreated normal tissues. 7,8-dihydro-8-oxo-2′-deoxyguanosine (^OXOG) has been recognized as the most common lesion. In these studies, the influence of ^OXOG, as an isolated (oligo-^OG) or clustered DNA lesion (oligo-^OG^OG), on charge transfer has been analyzed in comparison to native oligo-G. DNA lesion repair depends on the damage recognition step, probably via charge transfer. Here the electronic properties of short ds-oligonucleotides were calculated and analyzed at the M062x/6-31++G** level of theory in a non-equilibrated and equilibrated solvent state. The rate constant of hole and electron transfer according to Marcus’ theory was also discussed. These studies elucidated that ^OXOG constitutes the sink for migrated radical cations. However, in the case of oligo-^OG^OG containing a 5′-^OXOGA^XOXG-3′ sequence, the 3′-End ^OXOG becomes predisposed to electron-hole accumulation contrary to the undamaged GAG fragment. Moreover, it was found that the 5′-End ^OXOG present in an ^OXOGA^OXOG fragment adopts a higher adiabatic ionization potential than the 2′-deoxyguanosine of an undamaged analog if both ds-oligos are present in a cationic form. Because increases in CDL formation have been observed during radio- or chemotherapy, understanding their role in the above processes can be crucial for the efficiency and safety of medical cancer treatment. Full article

(This article belongs to the Special Issue DNA Damage and DNA Repair in Cancer)

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15 pages, 2180 KiB

Open AccessEditor’s ChoiceArticle

Solid-State Preparation and Characterization of 2-Hydroxypropylcyclodextrins-Iodine Complexes as Stable Iodophors

by Sandro Dattilo, Fabiola Spitaleri, Danilo Aleo, Maria Grazia Saita and Angela Patti

Biomolecules 2023, 13(3), 474; https://doi.org/10.3390/biom13030474 - 3 Mar 2023

Cited by 9 | Viewed by 3041

Abstract

The use of iodine as antiseptic poses some issues related to its low water solubility and high volatility. Stable solid iodine-containing formulations are highly advisable and currently limited to the povidone-iodine complex. In this study, complexes of molecular iodine with 2-hydroxypropyl α-, β- [...] Read more.

The use of iodine as antiseptic poses some issues related to its low water solubility and high volatility. Stable solid iodine-containing formulations are highly advisable and currently limited to the povidone-iodine complex. In this study, complexes of molecular iodine with 2-hydroxypropyl α-, β- and γ-cyclodextrins were considered water-soluble iodophors and prepared in a solid state by using three different methods (liquid-assisted grinding, co-evaporation and sealed heating). The obtained solids were evaluated for their iodine content and stability over time in different conditions using a fully validated UV method. The assessment of the actual formation of an inclusion complex in a solid state was carried out by thermal analysis, and the presence of iodine was further confirmed by SEM/EDX and XPS analyses. High levels of iodine content (8.3–10.8%) were obtained with all the tested cyclodextrins, and some influence was exerted by the employed preparation method. Potential use as solid iodophors can be envisaged for these iodine complexes, among which those with 2-hydroxypropyl-α-cyclodextrin were found the most stable, regardless of the preparation technique. The three prepared cyclodextrin–iodine complexes proved effective as bactericides against S. epidermidis. Full article

(This article belongs to the Special Issue Cyclodextrin-Based Drug Release and Drug Delivery Systems)

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13 pages, 954 KiB

Open AccessEditor’s ChoiceArticle

Insulin Resistance Is Associated with an Unfavorable Serum Lipoprotein Lipid Profile in Women with Newly Diagnosed Gestational Diabetes

by Mikael Huhtala, Tapani Rönnemaa and Kristiina Tertti

Biomolecules 2023, 13(3), 470; https://doi.org/10.3390/biom13030470 - 3 Mar 2023

Cited by 5 | Viewed by 2063

Abstract

Background: Gestational diabetes (GDM) is associated with various degrees of insulin resistance—a feature related to increased risk of adverse perinatal outcomes. We aimed to determine the previously poorly investigated associations between maternal insulin resistance and serum fasting metabolome at the time of GDM [...] Read more.

Background: Gestational diabetes (GDM) is associated with various degrees of insulin resistance—a feature related to increased risk of adverse perinatal outcomes. We aimed to determine the previously poorly investigated associations between maternal insulin resistance and serum fasting metabolome at the time of GDM diagnosis. Methods: Serum lipoprotein and amino acid profile was analyzed in 300 subjects with newly diagnosed GDM using a validated nuclear magnetic resonance spectroscopy protocol. Associations between insulin resistance (homeostasis model assessment of insulin resistance, HOMA2-IR) and serum metabolites were examined with linear regression. Results: We found insulin resistance to be associated with a distinct lipid pattern: increased concentration of VLDL triglycerides and phospholipids and total triglycerides. VLDL size was positively related and LDL and HDL sizes were inversely related to insulin resistance. Of fatty acids, increased total fatty acids, relative increase in saturated and monounsaturated fatty acids, and relative decrease in polyunsaturated and omega fatty acids were related to maternal insulin resistance. Conclusions: In newly diagnosed GDM, the association between maternal insulin resistance and serum lipoprotein profile was largely as described in type 2 diabetes. Lifestyle interventions aiming to decrease insulin resistance from early pregnancy could benefit pregnancy outcomes via more advantageous lipid metabolism. Full article

(This article belongs to the Section Molecular Medicine)

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18 pages, 4933 KiB

Open AccessEditor’s ChoiceArticle

Novel scFv against Notch Ligand JAG1 Suitable for Development of Cell Therapies toward JAG1-Positive Tumors

by Gabriela Silva, Ana F. Rodrigues, Susana Ferreira, Carolina Matos, Rute P. Eleutério, Gonçalo Marques, Khrystyna Kucheryava, Ana R. Lemos, Pedro M. F. Sousa, Rute Castro, Ana Barbas, Daniel Simão and Paula M. Alves

Biomolecules 2023, 13(3), 459; https://doi.org/10.3390/biom13030459 - 2 Mar 2023

Cited by 3 | Viewed by 2722

Abstract

The Notch signaling ligand JAG1 is overexpressed in various aggressive tumors and is associated with poor clinical prognosis. Hence, therapies targeting oncogenic JAG1 hold great potential for the treatment of certain tumors. Here, we report the identification of specific anti-JAG1 single-chain variable fragments [...] Read more.

The Notch signaling ligand JAG1 is overexpressed in various aggressive tumors and is associated with poor clinical prognosis. Hence, therapies targeting oncogenic JAG1 hold great potential for the treatment of certain tumors. Here, we report the identification of specific anti-JAG1 single-chain variable fragments (scFvs), one of them endowing chimeric antigen receptor (CAR) T cells with cytotoxicity against JAG1-positive cells. Anti-JAG1 scFvs were identified from human phage display libraries, reformatted into full-length monoclonal antibodies (Abs), and produced in mammalian cells. The characterization of these Abs identified two specific anti-JAG1 Abs (J1.B5 and J1.F1) with nanomolar affinities. Cloning the respective scFv sequences in our second- and third-generation CAR backbones resulted in six anti-JAG1 CAR constructs, which were screened for JAG1-mediated T-cell activation in Jurkat T cells in coculture assays with JAG1-positive cell lines. Studies in primary T cells demonstrated that one CAR harboring the J1.B5 scFv significantly induced effective T-cell activation in the presence of JAG1-positive, but not in JAG1-knockout, cancer cells, and enabled specific killing of JAG1-positive cells. Thus, this new anti-JAG1 scFv represents a promising candidate for the development of cell therapies against JAG1-positive tumors. Full article

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13 pages, 3375 KiB

Open AccessEditor’s ChoiceArticle

Protein Design Using Physics Informed Neural Networks

by Sara Ibrahim Omar, Chen Keasar, Ariel J. Ben-Sasson and Eldad Haber

Biomolecules 2023, 13(3), 457; https://doi.org/10.3390/biom13030457 - 1 Mar 2023

Cited by 3 | Viewed by 3185

Abstract

The inverse protein folding problem, also known as protein sequence design, seeks to predict an amino acid sequence that folds into a specific structure and performs a specific function. Recent advancements in machine learning techniques have been successful in generating functional sequences, outperforming [...] Read more.

The inverse protein folding problem, also known as protein sequence design, seeks to predict an amino acid sequence that folds into a specific structure and performs a specific function. Recent advancements in machine learning techniques have been successful in generating functional sequences, outperforming previous energy function-based methods. However, these machine learning methods are limited in their interoperability and robustness, especially when designing proteins that must function under non-ambient conditions, such as high temperature, extreme pH, or in various ionic solvents. To address this issue, we propose a new Physics-Informed Neural Networks (PINNs)-based protein sequence design approach. Our approach combines all-atom molecular dynamics simulations, a PINNs MD surrogate model, and a relaxation of binary programming to solve the protein design task while optimizing both energy and the structural stability of proteins. We demonstrate the effectiveness of our design framework in designing proteins that can function under non-ambient conditions. Full article

(This article belongs to the Special Issue Advances in Drug Design and Development for Human Therapeutics Using Artificial Intelligence II)

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16 pages, 2074 KiB

Open AccessEditor’s ChoiceArticle

LC-MS/MS-Based Proteomics Approach for the Identification of Candidate Serum Biomarkers in Patients with Narcolepsy Type 1

by Akeem Sanni, Mona Goli, Jingfu Zhao, Junyao Wang, Chloe Barsa, Samer El Hayek, Farid Talih, Bartolo Lanuzza, Firas Kobeissy, Giuseppe Plazzi, Monica Moresco, Stefania Mondello, Raffaele Ferri and Yehia Mechref

Biomolecules 2023, 13(3), 420; https://doi.org/10.3390/biom13030420 - 23 Feb 2023

Cited by 9 | Viewed by 4752

Abstract

Narcolepsy type 1 (NT1) is the most common type of narcolepsy known to be caused by the loss of specific neurons responsible for producing peptide neurotransmitters (orexins/hypocretins), resulting in a sleep-wake cycle disorder. It is characterized by its association with cataplexy and abnormalities [...] Read more.

Narcolepsy type 1 (NT1) is the most common type of narcolepsy known to be caused by the loss of specific neurons responsible for producing peptide neurotransmitters (orexins/hypocretins), resulting in a sleep-wake cycle disorder. It is characterized by its association with cataplexy and abnormalities in rapid eye movement. To date, no cure has been established for this life-threatening condition. Misdiagnosis of NT1 is also quite common, although it is not exceedingly rare. Therefore, successfully identifying candidate serum biomarkers for NT1 would be a head start for accurate diagnosis and development of therapeutics for this disorder. This study aims to identify such potential serum biomarkers. A depletion protocol was employed for 27 human serum samples (16 NT1 and 11 healthy controls), followed by applying LC-MS/MS bottom-up proteomics analysis, then LC-PRM-MS for validation. The comparison of the proteome profiles of the low-abundant proteins in the samples was then investigated based on age, sex, sample groups, and the presence of the Human Leukocyte Antigen (HLA) DQB1*0602 allele. The results were tracked to gene expression studies as well as system biology to identify key proteins and understand their relationship in the pathogenesis of NT1. Our results revealed 36 proteins significantly and differentially expressed. Among the impaired pathways and bioprocesses, the complement activation pathway is impaired by six of the differentially expressed proteins (DEPs). They are coded by the genes C2, CFB, C5, C1R, C1S, and MASP1, while 11 DEPs are involved in Acute Phase Response Signaling (APRS), which are coded by the genes FN1, AMBP, APOH, CFB, CP, ITIH2, C5, C2, F2, C1, and ITIH4. The combined AUCs of the downregulated and upregulated DEPs are 0.95 and 0.76, respectively. Overall, this study reveals potential serum-protein biomarkers of NT1 and explains the possible correlation between the biomarkers and pathophysiological effects, as well as important biochemical pathways involved in NT1. Full article

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23 pages, 2110 KiB

Open AccessEditor’s ChoiceArticle

Using GPT-3 to Build a Lexicon of Drugs of Abuse Synonyms for Social Media Pharmacovigilance

by Kristy A. Carpenter and Russ B. Altman

Biomolecules 2023, 13(2), 387; https://doi.org/10.3390/biom13020387 - 18 Feb 2023

Cited by 8 | Viewed by 4770

Abstract

Drug abuse is a serious problem in the United States, with over 90,000 drug overdose deaths nationally in 2020. A key step in combating drug abuse is detecting, monitoring, and characterizing its trends over time and location, also known as pharmacovigilance. While federal [...] Read more.

Drug abuse is a serious problem in the United States, with over 90,000 drug overdose deaths nationally in 2020. A key step in combating drug abuse is detecting, monitoring, and characterizing its trends over time and location, also known as pharmacovigilance. While federal reporting systems accomplish this to a degree, they often have high latency and incomplete coverage. Social-media-based pharmacovigilance has zero latency, is easily accessible and unfiltered, and benefits from drug users being willing to share their experiences online pseudo-anonymously. However, unlike highly structured official data sources, social media text is rife with misspellings and slang, making automated analysis difficult. Generative Pretrained Transformer 3 (GPT-3) is a large autoregressive language model specialized for few-shot learning that was trained on text from the entire internet. We demonstrate that GPT-3 can be used to generate slang and common misspellings of terms for drugs of abuse. We repeatedly queried GPT-3 for synonyms of drugs of abuse and filtered the generated terms using automated Google searches and cross-references to known drug names. When generated terms for alprazolam were manually labeled, we found that our method produced 269 synonyms for alprazolam, 221 of which were new discoveries not included in an existing drug lexicon for social media. We repeated this process for 98 drugs of abuse, of which 22 are widely-discussed drugs of abuse, building a lexicon of colloquial drug synonyms that can be used for pharmacovigilance on social media. Full article

(This article belongs to the Special Issue Biomolecular Data Science—in Honor of Professor Philip E. Bourne)

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17 pages, 9451 KiB

Open AccessEditor’s ChoiceArticle

Commonly Used Therapeutics Associated with Changes in Arousal Inhibit GABA_AR Activation

by Anling Kaplan, Abigail I. Nash, Amanda A. H. Freeman, Lauren G. Lewicki, David B. Rye, Lynn Marie Trotti, Asher L. Brandt and Andrew Jenkins

Biomolecules 2023, 13(2), 365; https://doi.org/10.3390/biom13020365 - 15 Feb 2023

Cited by 2 | Viewed by 2768

Abstract

GABA_A receptor-positive modulators are well-known to induce sedation, sleep, and general anesthesia. Conversely, GABA_A receptor negative allosteric modulators (GABA_ARNAMs) can increase arousal and induce seizures. Motivated by our studies with patients with hypersomnia, and our discovery that two GABA [...] Read more.

GABA_A receptor-positive modulators are well-known to induce sedation, sleep, and general anesthesia. Conversely, GABA_A receptor negative allosteric modulators (GABA_ARNAMs) can increase arousal and induce seizures. Motivated by our studies with patients with hypersomnia, and our discovery that two GABA_ARNAMs can restore the Excitation/Inhibition (E/I) balance in vitro and arousal in vivo, we chose to screen 11 compounds that have been reported to modulate arousal, to see if they shared a GABA-related mechanism. We determined modulation with both conventional and microfluidic patch clamp methods. We found that receptor activation was variably modulated by all 11 compounds: Rifampicin (RIF), Metronidazole (MET), Minocycline (MIN), Erythromycin (ERY), Ofloxacin (OFX), Chloroquine (CQ), Hydroxychloroquine sulfate (HCQ), Flumazenil (FLZ), Pentylenetetrazol (PTZ), (-)-Epigallocatechin Gallate (EGCG), and clarithromycin (CLR). The computational modeling of modulator–receptor interactions predicted drug action at canonical binding sites and novel orphan sites on the receptor. Our findings suggest that multiple avenues of investigation are now open to investigate large and brain-penetrant molecules for the treatment of patients with diminished CNS E/I balance. Full article

(This article belongs to the Special Issue GABA(A) Receptors: Structure and Function)

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20 pages, 44518 KiB

Open AccessEditor’s ChoiceArticle

Mechanistic Analysis of CCP1 in Generating ΔC2 α-Tubulin in Mammalian Cells and Photoreceptor Neurons

by Takashi Hotta, Alexandra Plemmons, Margo Gebbie, Trevor A. Ziehm, Teresa Lynne Blasius, Craig Johnson, Kristen J. Verhey, Jillian N. Pearring and Ryoma Ohi

Biomolecules 2023, 13(2), 357; https://doi.org/10.3390/biom13020357 - 12 Feb 2023

Cited by 2 | Viewed by 2222

Abstract

An important post-translational modification (PTM) of α-tubulin is the removal of amino acids from its C-terminus. Removal of the C-terminal tyrosine residue yields detyrosinated α-tubulin, and subsequent removal of the penultimate glutamate residue produces ΔC2-α-tubulin. These PTMs alter the ability of the α-tubulin [...] Read more.

An important post-translational modification (PTM) of α-tubulin is the removal of amino acids from its C-terminus. Removal of the C-terminal tyrosine residue yields detyrosinated α-tubulin, and subsequent removal of the penultimate glutamate residue produces ΔC2-α-tubulin. These PTMs alter the ability of the α-tubulin C-terminal tail to interact with effector proteins and are thereby thought to change microtubule dynamics, stability, and organization. The peptidase(s) that produces ΔC2-α-tubulin in a physiological context remains unclear. Here, we take advantage of the observation that ΔC2-α-tubulin accumulates to high levels in cells lacking tubulin tyrosine ligase (TTL) to screen for cytosolic carboxypeptidases (CCPs) that generate ΔC2-α-tubulin. We identify CCP1 as the sole peptidase that produces ΔC2-α-tubulin in TTLΔ HeLa cells. Interestingly, we find that the levels of ΔC2-α-tubulin are only modestly reduced in photoreceptors of ccp1^−/− mice, indicating that other peptidases act synergistically with CCP1 to produce ΔC2-α-tubulin in post-mitotic cells. Moreover, the production of ΔC2-α-tubulin appears to be under tight spatial control in the photoreceptor cilium: ΔC2-α-tubulin persists in the connecting cilium of ccp1^−/− but is depleted in the distal portion of the photoreceptor. This work establishes the groundwork to pinpoint the function of ΔC2-α-tubulin in proliferating and post-mitotic mammalian cells. Full article

(This article belongs to the Special Issue Molecular Functions of Microtubules)

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21 pages, 2296 KiB

Open AccessEditor’s ChoiceArticle

Targeting of the Interleukin-13 Receptor (IL-13R)α2 Expressing Prostate Cancer by a Novel Hybrid Lytic Peptide

by Riaz Jannoo, Zhidao Xia, Paula E. Row and Venkateswarlu Kanamarlapudi

Biomolecules 2023, 13(2), 356; https://doi.org/10.3390/biom13020356 - 12 Feb 2023

Cited by 3 | Viewed by 1941

Abstract

The IL-13Rα2 cell surface receptor is highly expressed in tumours such as prostate cancer. In this report, we evaluated the hypothesis that prostate cancer cells with enhanced IL-13Rα2 expression are a suitable target for the hybrid lytic peptide (Pep-1-Phor21) peptide, which is generated [...] Read more.

The IL-13Rα2 cell surface receptor is highly expressed in tumours such as prostate cancer. In this report, we evaluated the hypothesis that prostate cancer cells with enhanced IL-13Rα2 expression are a suitable target for the hybrid lytic peptide (Pep-1-Phor21) peptide, which is generated by fusing the IL-13Rα2 specific ligand (Pep-1) and a cell membrane disrupting lytic peptide (Phor21). The expression of IL-13Rα2 mRNA and protein in prostate cancer tissues and cell lines was assessed via real-time PCR (RT-PCR) and immunoblotting. The effect of Pep-1-Phor21 on the viability of prostate cancer cells grown in monolayers (2D) and microtissue spheroids (3D) was assessed via CellTox green cytotoxic assay. IL-13Rα2 expression and Pep-1-Phor21-mediated killing were also determined in the cells treated with epigenetic regulators (Trichostatin A (TSA) and 5-aza-2 deoxycytidine (5-Aza-dC)). The hybrid lytic peptide cytotoxic activity correlated with the expression of IL-13Rα2 in prostate cancer cell lines cultured as monolayers (2D) or 3D spheroids. In addition, TSA or 5-Aza-dC treatment of prostate cancer cells, particularly those with low expression of IL-13Rα2, enhanced the cells’ sensitivity to the lytic peptide by increasing IL-13Rα2 expression. These results demonstrate that the Pep-1-Phor21 hybrid lytic peptide has potent and selective anticancer properties against IL-13Rα2-expressing prostate cancer cells. Full article

(This article belongs to the Special Issue Functional Peptides and Their Interactions: From Molecules to Systems)

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18 pages, 2619 KiB

Open AccessEditor’s ChoiceArticle

Novel Green Fluorescent Polyamines to Analyze ATP13A2 and ATP13A3 Activity in the Mammalian Polyamine Transport System

by Marine Houdou, Nathalie Jacobs, Jonathan Coene, Mujahid Azfar, Roeland Vanhoutte, Chris Van den Haute, Jan Eggermont, Veronique Daniëls, Steven H. L. Verhelst and Peter Vangheluwe

Biomolecules 2023, 13(2), 337; https://doi.org/10.3390/biom13020337 - 9 Feb 2023

Cited by 8 | Viewed by 3205

Abstract

Cells acquire polyamines putrescine (PUT), spermidine (SPD) and spermine (SPM) via the complementary actions of polyamine uptake and synthesis pathways. The endosomal P_5B-type ATPases ATP13A2 and ATP13A3 emerge as major determinants of mammalian polyamine uptake. Our biochemical evidence shows that fluorescently [...] Read more.

Cells acquire polyamines putrescine (PUT), spermidine (SPD) and spermine (SPM) via the complementary actions of polyamine uptake and synthesis pathways. The endosomal P_5B-type ATPases ATP13A2 and ATP13A3 emerge as major determinants of mammalian polyamine uptake. Our biochemical evidence shows that fluorescently labeled polyamines are genuine substrates of ATP13A2. They can be used to measure polyamine uptake in ATP13A2- and ATP13A3-dependent cell models resembling radiolabeled polyamine uptake. We further report that ATP13A3 enables faster and stronger cellular polyamine uptake than does ATP13A2. We also compared the uptake of new green fluorescent PUT, SPD and SPM analogs using different coupling strategies (amide, triazole or isothiocyanate) and fluorophores (symmetrical BODIPY, BODIPY-FL and FITC). ATP13A2 promotes the uptake of various SPD and SPM analogs, whereas ATP13A3 mainly stimulates the uptake of PUT and SPD conjugates. However, the polyamine linker and coupling position on the fluorophore impacts the transport capacity, whereas replacing the fluorophore affects polyamine selectivity. The highest uptake in ATP13A2 or ATP13A3 cells is observed with BODIPY-FL-amide conjugated to SPD, whereas BODIPY-PUT analogs are specifically taken up via ATP13A3. We found that P_5B-type ATPase isoforms transport fluorescently labeled polyamine analogs with a distinct structure–activity relationship (SAR), suggesting that isoform-specific polyamine probes can be designed. Full article

(This article belongs to the Special Issue Polyamine Metabolism and Function)

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15 pages, 4316 KiB

Open AccessEditor’s ChoiceArticle

Time- and Sex-Dependent Effects of Fingolimod Treatment in a Mouse Model of Alzheimer’s Disease

by Pablo Bascuñana, Mirjam Brackhan, Luisa Möhle, Jingyun Wu, Thomas Brüning, Ivan Eiriz, Baiba Jansone and Jens Pahnke

Biomolecules 2023, 13(2), 331; https://doi.org/10.3390/biom13020331 - 9 Feb 2023

Cited by 7 | Viewed by 2513

Abstract

Alzheimer’s disease (AD) is the most common cause of dementia. Fingolimod has previously shown beneficial effects in different animal models of AD. However, it has shown contradictory effects when it has been applied at early disease stages. Our objective was to evaluate fingolimod [...] Read more.

Alzheimer’s disease (AD) is the most common cause of dementia. Fingolimod has previously shown beneficial effects in different animal models of AD. However, it has shown contradictory effects when it has been applied at early disease stages. Our objective was to evaluate fingolimod in two different treatment paradigms. To address this aim, we treated male and female APP-transgenic mice for 50 days, starting either before plaque deposition at 50 days of age (early) or at 125 days of age (late). To evaluate the effects, we investigated the neuroinflammatory and glial markers, the Aβ load, and the concentration of the brain-derived neurotrophic factor (BDNF). We found a reduced Aβ load only in male animals in the late treatment paradigm. These animals also showed reduced microglia activation and reduced IL-1β. No other treatment group showed any difference in comparison to the controls. On the other hand, we detected a linear correlation between BDNF and the brain Aβ concentrations. The fingolimod treatment has shown beneficial effects in AD models, but the outcome depends on the neuroinflammatory state at the start of the treatment. Thus, according to our data, a fingolimod treatment would be effective after the onset of the first AD symptoms, mainly affecting the neuroinflammatory reaction to the ongoing Aβ deposition. Full article

(This article belongs to the Special Issue New Advances in the Molecular Mechanism and Target Therapy in Alzheimer’s Diseases)

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19 pages, 2650 KiB

Open AccessEditor’s ChoiceArticle

Do Amino Acid Antiporters Have Asymmetric Substrate Specificity?

by Gregory Gauthier-Coles, Stephen J. Fairweather, Angelika Bröer and Stefan Bröer

Biomolecules 2023, 13(2), 301; https://doi.org/10.3390/biom13020301 - 6 Feb 2023

Cited by 5 | Viewed by 1909

Abstract

Amino acid antiporters mediate the 1:1 exchange of groups of amino acids. Whether substrate specificity can be different for the inward and outward facing conformation has not been investigated systematically, although examples of asymmetric transport have been reported. Here we used LC–MS to [...] Read more.

Amino acid antiporters mediate the 1:1 exchange of groups of amino acids. Whether substrate specificity can be different for the inward and outward facing conformation has not been investigated systematically, although examples of asymmetric transport have been reported. Here we used LC–MS to detect the movement of ¹²C- and ¹³C-labelled amino acid mixtures across the plasma membrane of Xenopus laevis oocytes expressing a variety of amino acid antiporters. Differences of substrate specificity between transporter paralogs were readily observed using this method. Our results suggest that antiporters are largely symmetric, equalizing the pools of their substrate amino acids. Exceptions are the antiporters y⁺LAT1 and y⁺LAT2 where neutral amino acids are co-transported with Na⁺ ions, favouring their import. For the antiporters ASCT1 and ASCT2 glycine acted as a selective influx substrate, while proline was a selective influx substrate of ASCT1. These data show that antiporters can display non-canonical modes of transport. Full article

(This article belongs to the Special Issue Recent Advances in Amino Acid Transporters)

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19 pages, 2829 KiB

Open AccessEditor’s ChoiceArticle

Copper Binding and Redox Activity of α-Synuclein in Membrane-Like Environment

by Chiara Bacchella, Francesca Camponeschi, Paulina Kolkowska, Arian Kola, Isabella Tessari, Maria Camilla Baratto, Marco Bisaglia, Enrico Monzani, Luigi Bubacco, Stefano Mangani, Luigi Casella, Simone Dell’Acqua and Daniela Valensin

Biomolecules 2023, 13(2), 287; https://doi.org/10.3390/biom13020287 - 3 Feb 2023

Cited by 6 | Viewed by 2332

Abstract

α-Synuclein (αSyn) constitutes the main protein component of Lewy bodies, which are the pathologic hallmark in Parkinson’s disease. αSyn is unstructured in solution but the interaction of αSyn with lipid membrane modulates its conformation by inducing an α-helical structure of the N-terminal [...] Read more.

α-Synuclein (αSyn) constitutes the main protein component of Lewy bodies, which are the pathologic hallmark in Parkinson’s disease. αSyn is unstructured in solution but the interaction of αSyn with lipid membrane modulates its conformation by inducing an α-helical structure of the N-terminal region. In addition, the interaction with metal ions can trigger αSyn conformation upon binding and/or through the metal-promoted generation of reactive oxygen species which lead to a cascade of structural alterations. For these reasons, the ternary interaction between αSyn, copper, and membranes needs to be elucidated in detail. Here, we investigated the structural properties of copper-αSyn binding through NMR, EPR, and XAS analyses, with particular emphasis on copper(I) coordination since the reduced state is particularly relevant for oxygen activation chemistry. The analysis was performed in different membrane model systems, such as micellar sodium dodecyl sulfate (SDS) and unilamellar vesicles, comparing the binding of full-length αSyn and N-terminal peptide fragments. The presence of membrane-like environments induced the formation of a copper:αSyn = 1:2 complex where Cu⁺ was bound to the Met1 and Met5 residues of two helical peptide chains. In this coordination, Cu⁺ is stabilized and is unreactive in the presence of O₂ in catechol substrate oxidation. Full article

(This article belongs to the Special Issue Synuclein Proteins II)

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18 pages, 5401 KiB

Open AccessEditor’s ChoiceArticle

Oxaliplatin-Induced Damage to the Gastric Innervation: Role in Nausea and Vomiting

by Ahmed A. Rahman, Philenkosini Masango, Rhian Stavely, Paul Bertrand, Amanda Page and Kulmira Nurgali

Biomolecules 2023, 13(2), 276; https://doi.org/10.3390/biom13020276 - 1 Feb 2023

Cited by 4 | Viewed by 2051

Abstract

Nausea and vomiting are common gastrointestinal side effects of oxaliplatin chemotherapy used for the treatment of colorectal cancer. However, the mechanism underlying oxaliplatin-induced nausea and vomiting is unknown. The stomach is involved in the emetic reflex but no study investigated the effects of [...] Read more.

Nausea and vomiting are common gastrointestinal side effects of oxaliplatin chemotherapy used for the treatment of colorectal cancer. However, the mechanism underlying oxaliplatin-induced nausea and vomiting is unknown. The stomach is involved in the emetic reflex but no study investigated the effects of oxaliplatin treatment on the stomach. In this study, the in vivo effects of oxaliplatin treatment on eating behaviour, stomach content, intrinsic gastric neuronal population, extrinsic innervation to the stomach, levels of mucosal serotonin (5-hydroxytryptamine, 5-HT), and parasympathetic vagal efferent nerve activity were analysed. Chronic systemic oxaliplatin treatment in mice resulted in pica, indicated by increased kaolin consumption and a reduction in body weight. Oxaliplatin treatment significantly increased the stomach weight and content. The total number of myenteric and nitric oxide synthase-immunoreactive neurons as well as the density of sympathetic, parasympathetic, and sensory fibres in the stomach were decreased significantly with oxaliplatin treatment. Oxaliplatin treatment significantly increased the levels in mucosal 5-HT and the number of enterochromaffin-like cells. Chronic oxaliplatin treatment also caused a significant increase in the vagal efferent nerve activity. The findings of this study indicate that oxaliplatin exposure has adverse effects on multiple components of gastric innervation, which could be responsible for pica and gastric dysmotility. Full article

(This article belongs to the Special Issue Enteric Nervous System: Normal Functions and Enteric Neuropathies)

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17 pages, 5756 KiB

Open AccessEditor’s ChoiceArticle

Oligodendrocytes Prune Axons Containing α-Synuclein Aggregates In Vivo: Lewy Neurites as Precursors of Glial Cytoplasmic Inclusions in Multiple System Atrophy?

by Francesco De Nuccio, Marianna Kashyrina, Francesca Serinelli, Florent Laferrière, Dario Domenico Lofrumento, Francesca De Giorgi and François Ichas

Biomolecules 2023, 13(2), 269; https://doi.org/10.3390/biom13020269 - 1 Feb 2023

Cited by 7 | Viewed by 7917

Abstract

α-Synucleinopathies are spreading neurodegenerative disorders characterized by the intracellular accumulation of insoluble aggregates populated by α-Synuclein (α-Syn) fibrils. In Parkinson’s disease (PD) and dementia with Lewy bodies, intraneuronal α-Syn aggregates are referred to as Lewy bodies in the somata and as Lewy neurites [...] Read more.

α-Synucleinopathies are spreading neurodegenerative disorders characterized by the intracellular accumulation of insoluble aggregates populated by α-Synuclein (α-Syn) fibrils. In Parkinson’s disease (PD) and dementia with Lewy bodies, intraneuronal α-Syn aggregates are referred to as Lewy bodies in the somata and as Lewy neurites in the neuronal processes. In multiple system atrophy (MSA) α-Syn aggregates are also found within mature oligodendrocytes (OLs) where they form Glial Cytoplasmic Inclusions (GCIs). However, the origin of GCIs remains enigmatic: (i) mature OLs do not express α-Syn, precluding the seeding and the buildup of inclusions and (ii) the artificial overexpression of α-Syn in OLs of transgenic mice results in a burden of soluble phosphorylated α-Syn but fails to form α-Syn fibrils. In contrast, mass spectrometry of α-Syn fibrillar aggregates from MSA patients points to the neuronal origin of the proteins intimately associated with the fibrils within the GCIs. This suggests that GCIs are preassembled in neurons and only secondarily incorporated into OLs. Interestingly, we recently isolated a synthetic human α-Syn fibril strain (1B fibrils) capable of seeding a type of neuronal inclusion observed early and specifically during MSA. Our goal was thus to investigate whether the neuronal α-Syn pathology seeded by 1B fibrils could eventually be transmitted to OLs to form GCIs in vivo. After confirming that mature OLs did not express α-Syn to detectable levels in the adult mouse brain, a series of mice received unilateral intra-striatal injections of 1B fibrils. The resulting α-Syn pathology was visualized using phospho-S129 α-Syn immunoreactivity (pSyn). We found that even though 1B fibrils were injected unilaterally, many pSyn-positive neuronal somas were present in layer V of the contralateral perirhinal cortex after 6 weeks. This suggested a fast retrograde spread of the pathology along the axons of crossing cortico-striatal neurons. We thus scrutinized the posterior limb of the anterior commissure, i.e., the myelinated interhemispheric tract containing the axons of these neurons: we indeed observed numerous pSyn-positive linear Lewy Neurites oriented parallel to the commissural axis, corresponding to axonal segments filled with aggregated α-Syn, with no obvious signs of OL α-Syn pathology at this stage. After 6 months however, the commissural Lewy neurites were no longer parallel but fragmented, curled up, sometimes squeezed in-between two consecutive OLs in interfascicular strands, or even engulfed inside OL perikarya, thus forming GCIs. We conclude that the 1B fibril strain can rapidly induce an α-Syn pathology typical of MSA in mice, in which the appearance of GCIs results from the pruning of diseased axonal segments containing aggregated α-Syn. Full article

(This article belongs to the Special Issue α-Synuclein Amyloids & Parkinson’s Disease: On Growth, Spread, and Neurodegeneration 2.0)

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13 pages, 2560 KiB

Open AccessEditor’s ChoiceArticle

Identification of Inhibitors of Tubulin Polymerization Using a CRISPR-Edited Cell Line with Endogenous Fluorescent Tagging of β-Tubulin and Histone H1

by Harutyun Khachatryan, Bartlomiej Olszowy, Carlos A. Barrero, John Gordon and Oscar Perez-Leal

Biomolecules 2023, 13(2), 249; https://doi.org/10.3390/biom13020249 - 29 Jan 2023

Cited by 7 | Viewed by 7455

Abstract

Tubulin is a protein that plays a critical role in maintaining cellular structure and facilitating cell division. Inhibiting tubulin polymerization has been shown to be an effective strategy for inhibiting the proliferation of cancer cells. In the past, identifying compounds that could inhibit [...] Read more.

Tubulin is a protein that plays a critical role in maintaining cellular structure and facilitating cell division. Inhibiting tubulin polymerization has been shown to be an effective strategy for inhibiting the proliferation of cancer cells. In the past, identifying compounds that could inhibit tubulin polymerization has required the use of in vitro assays utilizing purified tubulin or immunofluorescence of fixed cells. This study presents a novel approach for identifying tubulin polymerization inhibitors using a CRISPR-edited cell line that expresses fluorescently tagged β-tubulin and a nuclear protein, enabling the visualization of tubulin polymerization dynamics via high-content imaging analysis (HCI). The cells were treated with known tubulin polymerization inhibitors, colchicine, and vincristine, and the resulting phenotypic changes indicative of tubulin polymerization inhibition were confirmed using HCI. Furthermore, a library of 429 kinase inhibitors was screened, resulting in the identification of three compounds (ON-01910, HMN-214, and KX2-391) that inhibit tubulin polymerization. Live cell tracking analysis confirmed that compound treatment leads to rapid tubulin depolymerization. These findings suggest that CRISPR-edited cells with fluorescently tagged endogenous β-tubulin can be utilized to screen large compound libraries containing diverse chemical families for the identification of novel tubulin polymerization inhibitors. Full article

(This article belongs to the Section Molecular Biology)

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14 pages, 2704 KiB

Open AccessEditor’s ChoiceArticle

Metabolic Pathway Analysis: Advantages and Pitfalls for the Functional Interpretation of Metabolomics and Lipidomics Data

by Sofia Tsouka and Mojgan Masoodi

Biomolecules 2023, 13(2), 244; https://doi.org/10.3390/biom13020244 - 27 Jan 2023

Cited by 8 | Viewed by 4742

Abstract

Over the past decades, pathway analysis has become one of the most commonly used approaches for the functional interpretation of metabolomics data. Although the approach is widely used, it is not well standardized and the impact of different methodologies on the functional outcome [...] Read more.

Over the past decades, pathway analysis has become one of the most commonly used approaches for the functional interpretation of metabolomics data. Although the approach is widely used, it is not well standardized and the impact of different methodologies on the functional outcome is not well understood. Using four publicly available datasets, we investigated two main aspects of topological pathway analysis, namely the consideration of non-human native enzymatic reactions (e.g., from microbiota) and the interconnectivity of individual pathways. The exclusion of non-human native reactions led to detached and poorly represented reaction networks and to loss of information. The consideration of connectivity between pathways led to better emphasis of certain central metabolites in the network; however, it occasionally overemphasized the hub compounds. We proposed and examined a penalization scheme to diminish the effect of such compounds in the pathway evaluation. In order to compare and assess the results between different methodologies, we also performed over-representation analysis of the same datasets. We believe that our findings will raise awareness on both the capabilities and shortcomings of the currently used pathway analysis practices in metabolomics. Additionally, it will provide insights on various methodologies and strategies that should be considered for the analysis and interpretation of metabolomics data. Full article

(This article belongs to the Collection Metabolomics and Integrated Multi-Omics in Health and Disease)

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16 pages, 3321 KiB

Open AccessEditor’s ChoiceArticle

The Role of the Hydrogen Bond Network in Maintaining Heme Pocket Stability and Protein Function Specificity of C. diphtheriae Coproheme Decarboxylase

by Federico Sebastiani, Chiara Baroni, Gaurav Patil, Andrea Dali, Maurizio Becucci, Stefan Hofbauer and Giulietta Smulevich

Biomolecules 2023, 13(2), 235; https://doi.org/10.3390/biom13020235 - 25 Jan 2023

Cited by 5 | Viewed by 4081

Abstract

Monoderm bacteria accumulate heme b via the coproporphyrin-dependent biosynthesis pathway. In the final step, in the presence of two molecules of H₂O₂, the propionate groups of coproheme at positions 2 and 4 are decarboxylated to form vinyl groups by [...] Read more.

Monoderm bacteria accumulate heme b via the coproporphyrin-dependent biosynthesis pathway. In the final step, in the presence of two molecules of H₂O₂, the propionate groups of coproheme at positions 2 and 4 are decarboxylated to form vinyl groups by coproheme decarboxylase (ChdC), in a stepwise process. Decarboxylation of propionate 2 produces an intermediate that rotates by 90° inside the protein pocket, bringing propionate 4 near the catalytic tyrosine, to allow the second decarboxylation step. The active site of ChdCs is stabilized by an extensive H-bond network involving water molecules, specific amino acid residues, and the propionate groups of the porphyrin. To evaluate the role of these H-bonds in the pocket stability and enzyme functionality, we characterized, via resonance Raman and electronic absorption spectroscopies, single and double mutants of the actinobacterial pathogen Corynebacterium diphtheriae ChdC complexed with coproheme and heme b. The selective elimination of the H-bond interactions between propionates 2, 4, 6, and 7 and the polar residues of the pocket allowed us to establish the role of each H-bond in the catalytic reaction and to follow the changes in the interactions from the substrate to the product. Full article

(This article belongs to the Special Issue Hemoproteins and Proteolytic Enzymes: Variations on the Theme of Functional Modulation: A Themed Issue in Honor of Professor Massimo Coletta)

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15 pages, 3130 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Hazard Assessment of Polystyrene Nanoplastics in Primary Human Nasal Epithelial Cells, Focusing on the Autophagic Effects

by Balasubramanyam Annangi, Aliro Villacorta, Montserrat López-Mesas, Victor Fuentes-Cebrian, Ricard Marcos and Alba Hernández

Biomolecules 2023, 13(2), 220; https://doi.org/10.3390/biom13020220 - 23 Jan 2023

Cited by 21 | Viewed by 3003

Abstract

The human health risks posed by micro/nanoplastics (MNPLs), as emerging pollutants of environmental/health concern, need to be urgently addressed as part of a needed hazard assessment. The routes of MNPL exposure in humans could mainly come from oral, inhalation, or dermal means. Among [...] Read more.

The human health risks posed by micro/nanoplastics (MNPLs), as emerging pollutants of environmental/health concern, need to be urgently addressed as part of a needed hazard assessment. The routes of MNPL exposure in humans could mainly come from oral, inhalation, or dermal means. Among them, inhalation exposure to MNPLs is the least studied area, even though their widespread presence in the air is dramatically increasing. In this context, this study focused on the potential hazard of polystyrene nanoplastics (PSNPLs with sizes 50 and 500 nm) in human primary nasal epithelial cells (HNEpCs), with the first line of cells acting as a physical and immune barrier in the respiratory system. Primarily, cellular internalization was evaluated by utilizing laboratory-labeled fluorescence PSNPLs with iDye, a commercial, pink-colored dye, using confocal microscopy, and found PSNPLs to be significantly internalized by HNEpCs. After, various cellular effects, such as the induction of intracellular reactive oxygen species (iROS), the loss of mitochondrial membrane potential (MMP), and the modulation of the autophagy pathway in the form of the accumulation of autophagosomes (LC3-II) and p62 markers (a ubiquitin involved in the clearance of cell debris), were evaluated after cell exposure. The data demonstrated significant increases in iROS, a decrease in MMP, as well as a greater accumulation of LC3-II and p62 in the presence of PSNPLs. Notably, the autophagic effects did indicate the implications of PSNPLs in defective or insufficient autophagy. This is the first study showing the autophagy pathway as a possible target for PSNPL-induced adverse effects in HNEpCs. When taken together, this study proved the cellular effects of PSNPLs in HNEpCs and adds value to the existing studies as a part of the respiratory risk assessment of MNPLs. Full article

(This article belongs to the Special Issue How Environmental Plastic Particles and Their Adsorbed Contaminants Interact with Biomolecules?)

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13 pages, 2207 KiB

Open AccessEditor’s ChoiceArticle

Aquaporin-1 and Aquaporin-4 Expression in Ependyma, Choroid Plexus and Surrounding Transition Zones in the Human Brain

by Ronja Bihlmaier, Felix Deffner, Ulrich Mattheus, Peter H. Neckel, Bernhard Hirt and Andreas F. Mack

Biomolecules 2023, 13(2), 212; https://doi.org/10.3390/biom13020212 - 22 Jan 2023

Cited by 6 | Viewed by 2610

Abstract

The choroid plexus (CP) is a structure in the brain ventricles that produces the main part of the cerebrospinal fluid (CSF). It is covered with specialized cells which show epithelial characteristics and are the site of the blood–CSF barrier. These cells form a [...] Read more.

The choroid plexus (CP) is a structure in the brain ventricles that produces the main part of the cerebrospinal fluid (CSF). It is covered with specialized cells which show epithelial characteristics and are the site of the blood–CSF barrier. These cells form a contiguous cell sheet with ventricle-lining ependymal cells which are known to express aquaporin-4 (AQP4). In contrast, CP epithelial cells express aquaporin-1 (AQP1) apically. We investigated the expression patterns of aquaporins in the CP-ependyma transition from human body donors using immunofluorescence and electron microscopy. Ependymal cells and subependymal astrocytes at the base of the CP showed a particularly high AQP4 immunoreactivity. Astrocytic processes formed a dense meshwork or glial plate around the blood vessels entering the CP. Interestingly, some of these astrocytic processes were in direct contact with the CP stroma, which contains fenestrated blood vessels, separated only by a basal lamina. Electron microscopy confirmed the continuity of the subastrocytic basal lamina with the CP epithelium. We also probed for components of the AQP4 anchoring dystrophin–dystroglycan complex. Immunolabeling for dystrophin and AQP4 showed an overlapping staining pattern in the glial plate but not in previously reported AQP4-positive CP epithelial cells. In contrast, dystroglycan expression was associated with laminin staining in the glial plate and the CP epithelium. This suggests different mechanisms for AQP4 anchoring in the cell membrane. The high AQP4 density in the connecting glial plate might facilitate the transport of water in and out of the CP stroma and could possibly serve as a drainage and clearing pathway for metabolites. Full article

(This article belongs to the Special Issue Aquaporins in the CNS–in Honor of the 30th Anniversary of the Discovery of Aquaporins)

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21 pages, 2060 KiB

Open AccessEditor’s ChoiceArticle

Updated Virophage Taxonomy and Distinction from Polinton-like Viruses

by Simon Roux, Matthias G. Fischer, Thomas Hackl, Laura A. Katz, Frederik Schulz and Natalya Yutin

Biomolecules 2023, 13(2), 204; https://doi.org/10.3390/biom13020204 - 19 Jan 2023

Cited by 15 | Viewed by 4885

Abstract

Virophages are small dsDNA viruses that hijack the machinery of giant viruses during the co-infection of a protist (i.e., microeukaryotic) host and represent an exceptional case of “hyperparasitism” in the viral world. While only a handful of virophages have been isolated, a vast [...] Read more.

Virophages are small dsDNA viruses that hijack the machinery of giant viruses during the co-infection of a protist (i.e., microeukaryotic) host and represent an exceptional case of “hyperparasitism” in the viral world. While only a handful of virophages have been isolated, a vast diversity of virophage-like sequences have been uncovered from diverse metagenomes. Their wide ecological distribution, idiosyncratic infection and replication strategy, ability to integrate into protist and giant virus genomes and potential role in antiviral defense have made virophages a topic of broad interest. However, one limitation for further studies is the lack of clarity regarding the nomenclature and taxonomy of this group of viruses. Specifically, virophages have been linked in the literature to other “virophage-like” mobile genetic elements and viruses, including polinton-like viruses (PLVs), but there are no formal demarcation criteria and proper nomenclature for either group, i.e., virophage or PLVs. Here, as part of the ICTV Virophage Study Group, we leverage a large set of genomes gathered from published datasets as well as newly generated protist genomes to propose delineation criteria and classification methods at multiple taxonomic ranks for virophages ‘sensu stricto’, i.e., genomes related to the prototype isolates Sputnik and mavirus. Based on a combination of comparative genomics and phylogenetic analyses, we show that this group of virophages forms a cohesive taxon that we propose to establish at the class level and suggest a subdivision into four orders and seven families with distinctive ecogenomic features. Finally, to illustrate how the proposed delineation criteria and classification method would be used, we apply these to two recently published datasets, which we show include both virophages and other virophage-related elements. Overall, we see this proposed classification as a necessary first step to provide a robust taxonomic framework in this area of the virosphere, which will need to be expanded in the future to cover other virophage-related viruses such as PLVs. Full article

(This article belongs to the Special Issue Virology 130 Years After Ivanovsky—A Theme Issue Commemorating the Discovery of Viruses by Dmitri Ivanovsky)

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13 pages, 2656 KiB

Open AccessEditor’s ChoiceArticle

Chronic Exposure to Low-Molecular-Weight Polycyclic Aromatic Hydrocarbons Promotes Lipid Accumulation and Metabolic Inflammation

by Asia Bright, Fenfen Li, Miranda Movahed, Hang Shi and Bingzhong Xue

Biomolecules 2023, 13(2), 196; https://doi.org/10.3390/biom13020196 - 18 Jan 2023

Cited by 19 | Viewed by 2345

Abstract

2-naphthol is a low-molecular-weight (LMW) polycyclic aromatic hydrocarbon (PAH) and air pollutant associated with childhood obesity. There has been a recent emergence of studies on the consequences of PAHs on human health. Current epidemiological reports suggest LMW-PAHs may contribute to obesity incidences in [...] Read more.

2-naphthol is a low-molecular-weight (LMW) polycyclic aromatic hydrocarbon (PAH) and air pollutant associated with childhood obesity. There has been a recent emergence of studies on the consequences of PAHs on human health. Current epidemiological reports suggest LMW-PAHs may contribute to obesity incidences in children, yet most studies focus on high-molecular-weight PAHs. This study explores 2-naphthol’s impact on obesity and obesity-associated metabolic disorders. To investigate 2-naphthol’s effect on lipid metabolism and inflammation, we employed 3T3-L1 and BAT1 cell lines to model white and brown adipocytes, respectively, alongside a murine macrophage cell line (RAW264.7). We found that 2-naphthol increased the expression of key adipogenic and lipogenic genes while decreasing lipolytic gene expression in chronically treated 3T3-L1 and BAT1 adipocytes. In addition, chronic 2-naphthol treatment also suppressed adrenergic-stimulated thermogenic gene expression in BAT1 brown adipocytes. In consistence, an increase in lipid accumulation was demonstrated in BODIPY and Oil Red O-stained adipocytes. Additionally, 3T3-L1 adipocytes and RAW264.7 macrophages chronically exposed to 2-naphthol showed upregulated mRNA expression of major inflammatory cytokines (e.g., tumor necrosis factor α (Tnfα), interleukin-1β (Il-1β), and Il-6). In summary, chronic exposure to 2-naphthol stimulates lipid accumulation in adipocytes and inflammation in adipocytes and macrophages. These findings support previous research that demonstrates 2-naphthol has obesogenic potential. Full article

(This article belongs to the Collection Molecular Mechanisms of Obesity, Diabetes, Inflammation and Aging)

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15 pages, 1926 KiB

Open AccessEditor’s ChoiceArticle

Glycosylated Lipopeptides—Synthesis and Evaluation of Antimicrobial Activity and Cytotoxicity

by Karol Sikora, Marta Bauer, Sylwia Bartoszewska, Damian Neubauer and Wojciech Kamysz

Biomolecules 2023, 13(1), 172; https://doi.org/10.3390/biom13010172 - 13 Jan 2023

Cited by 1 | Viewed by 2256

Abstract

Ultrashort cationic lipopeptides (USCLs) are promising antimicrobial agents that may be used to combat pathogens such as bacteria and fungi. USCLs consist of a few basic amino acid residues and at least one lipid moiety, usually a fatty acid chain. Generally, USCLs are [...] Read more.

Ultrashort cationic lipopeptides (USCLs) are promising antimicrobial agents that may be used to combat pathogens such as bacteria and fungi. USCLs consist of a few basic amino acid residues and at least one lipid moiety, usually a fatty acid chain. Generally, USCLs are potent antimicrobials but their major shortcoming is a relatively high cytotoxicity and hemolytic activity. Glycopeptide antibiotics (e.g. vancomycin) are essential in combating bacterial infections and are popular in medicinal practice. However, literature concerning the effect of glycosylation of peptides on their antimicrobial activity is rather scarce. For the first time, this study highlights the effect of USCLs glycosylation on in vitro biological activity. The aim of this study was to evaluate the impact of glycosylation of a series of USCLs on antimicrobial activity, cytotoxicity and hemolytic activity. Straight-chain fatty acids (C14, C16, C18) were attached to the N-terminal amino group of tripeptides—SRR-NH₂, RSR-NH₂ and RRS-NH₂. Two groups of the lipopeptides were synthetized, the first with unmodified L-serine (USCLs) and the other with L-serine O-glycosylated by N-acetyl-β-d-glucosamine to produce new class of glycosylated ultrashort cationic lipopeptide (gUSCLs). Both USCLs and gUSCLs were tested against planktonic and biofilm cultures of ESKAPE strains (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) and Candida glabrata, and hemolytic activity on human erythrocytes and cytotoxicity against the HaCaT cell line was examined. Generally, USCLs and gUSCLs proved to be active against all the tested strains. The highest activity displayed was by lipopeptides containing the C18 fatty acid. Antimicrobial, hemolytic and cytotoxic activities were mainly correlated with amino acid sequence (position of serine/glycosylated serine) and hydrophobicity of molecule and were found to be highly strain-dependent. In general, glycosylation did not guarantee an increased antimicrobial activity or a decreased hemolytic and cytotoxic activities. However, in some cases, gUSCLs proved to be superior to their USCLs analogs. The most pronounced differences were found for peptides with C18 fatty acid and serine at the first and second position against both planktonic cells and biofilm of C. glabrata, as well as the second and third position against S. aureus. It is noteworthy that gUSCLs were also more active against biofilm than were USCLs. Full article

(This article belongs to the Special Issue Nature Inspired Peptides in Medical Sciences)

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12 pages, 2632 KiB

Open AccessEditor’s ChoiceArticle

Sub-Millisecond Photoinduced Dynamics of Free and EL222-Bound FMN by Stimulated Raman and Visible Absorption Spectroscopies

by Yingliang Liu, Aditya S. Chaudhari, Aditi Chatterjee, Prokopis C. Andrikopoulos, Alessandra Picchiotti, Mateusz Rebarz, Miroslav Kloz, Victor A. Lorenz-Fonfria, Bohdan Schneider and Gustavo Fuertes

Biomolecules 2023, 13(1), 161; https://doi.org/10.3390/biom13010161 - 12 Jan 2023

Cited by 2 | Viewed by 2650

Abstract

Time-resolved femtosecond-stimulated Raman spectroscopy (FSRS) provides valuable information on the structural dynamics of biomolecules. However, FSRS has been applied mainly up to the nanoseconds regime and above 700 cm⁻¹, which covers only part of the spectrum of biologically relevant time scales [...] Read more.

Time-resolved femtosecond-stimulated Raman spectroscopy (FSRS) provides valuable information on the structural dynamics of biomolecules. However, FSRS has been applied mainly up to the nanoseconds regime and above 700 cm⁻¹, which covers only part of the spectrum of biologically relevant time scales and Raman shifts. Here we report on a broadband (~200–2200 cm⁻¹) dual transient visible absorption (visTA)/FSRS set-up that can accommodate time delays from a few femtoseconds to several hundreds of microseconds after illumination with an actinic pump. The extended time scale and wavenumber range allowed us to monitor the complete excited-state dynamics of the biological chromophore flavin mononucleotide (FMN), both free in solution and embedded in two variants of the bacterial light-oxygen-voltage (LOV) photoreceptor EL222. The observed lifetimes and intermediate states (singlet, triplet, and adduct) are in agreement with previous time-resolved infrared spectroscopy experiments. Importantly, we found evidence for additional dynamical events, particularly upon analysis of the low-frequency Raman region below 1000 cm⁻¹. We show that fs-to-sub-ms visTA/FSRS with a broad wavenumber range is a useful tool to characterize short-lived conformationally excited states in flavoproteins and potentially other light-responsive proteins. Full article

(This article belongs to the Collection Feature Papers in Molecular Biophysics Section)

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15 pages, 3374 KiB

Open AccessEditor’s ChoiceArticle

Combining Semi-Targeted Metabolomics and Machine Learning to Identify Metabolic Alterations in the Serum and Urine of Hospitalized Patients with COVID-19

by Gerard Baiges-Gaya, Simona Iftimie, Helena Castañé, Elisabet Rodríguez-Tomàs, Andrea Jiménez-Franco, Ana F. López-Azcona, Antoni Castro, Jordi Camps and Jorge Joven

Biomolecules 2023, 13(1), 163; https://doi.org/10.3390/biom13010163 - 12 Jan 2023

Cited by 10 | Viewed by 2740

Abstract

Viral infections cause metabolic dysregulation in the infected organism. The present study used metabolomics techniques and machine learning algorithms to retrospectively analyze the alterations of a broad panel of metabolites in the serum and urine of a cohort of 126 patients hospitalized with [...] Read more.

Viral infections cause metabolic dysregulation in the infected organism. The present study used metabolomics techniques and machine learning algorithms to retrospectively analyze the alterations of a broad panel of metabolites in the serum and urine of a cohort of 126 patients hospitalized with COVID-19. Results were compared with those of 50 healthy subjects and 45 COVID-19-negative patients but with bacterial infectious diseases. Metabolites were analyzed by gas chromatography coupled to quadrupole time-of-flight mass spectrometry. The main metabolites altered in the sera of COVID-19 patients were those of pentose glucuronate interconversion, ascorbate and fructose metabolism, nucleotide sugars, and nucleotide and amino acid metabolism. Alterations in serum maltose, mannonic acid, xylitol, or glyceric acid metabolites segregated positive patients from the control group with high diagnostic accuracy, while succinic acid segregated positive patients from those with other disparate infectious diseases. Increased lauric acid concentrations were associated with the severity of infection and death. Urine analyses could not discriminate between groups. Targeted metabolomics and machine learning algorithms facilitated the exploration of the metabolic alterations underlying COVID-19 infection, and to identify the potential biomarkers for the diagnosis and prognosis of the disease. Full article

(This article belongs to the Special Issue Metabolic Pathways and COVID-19: Mechanisms and Clinical Implications)

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17 pages, 26388 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Improving Protein–Ligand Interaction Modeling with cryo-EM Data, Templates, and Deep Learning in 2021 Ligand Model Challenge

by Nabin Giri and Jianlin Cheng

Biomolecules 2023, 13(1), 132; https://doi.org/10.3390/biom13010132 - 9 Jan 2023

Cited by 12 | Viewed by 3830

Abstract

Elucidating protein–ligand interaction is crucial for studying the function of proteins and compounds in an organism and critical for drug discovery and design. The problem of protein–ligand interaction is traditionally tackled by molecular docking and simulation, which is based on physical forces and [...] Read more.

Elucidating protein–ligand interaction is crucial for studying the function of proteins and compounds in an organism and critical for drug discovery and design. The problem of protein–ligand interaction is traditionally tackled by molecular docking and simulation, which is based on physical forces and statistical potentials and cannot effectively leverage cryo-EM data and existing protein structural information in the protein–ligand modeling process. In this work, we developed a deep learning bioinformatics pipeline (DeepProLigand) to predict protein–ligand interactions from cryo-EM density maps of proteins and ligands. DeepProLigand first uses a deep learning method to predict the structure of proteins from cryo-EM maps, which is averaged with a reference (template) structure of the proteins to produce a combined structure to add ligands. The ligands are then identified and added into the structure to generate a protein–ligand complex structure, which is further refined. The method based on the deep learning prediction and template-based modeling was blindly tested in the 2021 EMDataResource Ligand Challenge and was ranked first in fitting ligands to cryo-EM density maps. These results demonstrate that the deep learning bioinformatics approach is a promising direction for modeling protein–ligand interactions on cryo-EM data using prior structural information. Full article

(This article belongs to the Collection Feature Papers in Bioinformatics and Systems Biology Section)

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20 pages, 3979 KiB

Open AccessEditor’s ChoiceArticle

Selenium-Substituted Monomethine Cyanine Dyes as Selective G-Quadruplex Spectroscopic Probes with Theranostic Potential

by Ivana Fabijanić, Atanas Kurutos, Ana Tomašić Paić, Vanja Tadić, Fadhil S. Kamounah, Lucija Horvat, Anamaria Brozovic, Ivo Crnolatac and Marijana Radić Stojković

Biomolecules 2023, 13(1), 128; https://doi.org/10.3390/biom13010128 - 7 Jan 2023

Cited by 4 | Viewed by 3131

Abstract

The binding interactions of six ligands, neutral and monocationic asymmetric monomethine cyanine dyes comprising benzoselenazolyl moiety with duplex DNA and RNA and G-quadruplex structures were evaluated using fluorescence, UV/Vis (thermal melting) and circular dichroism (CD) spectroscopy. The main objective was to assess the [...] Read more.

The binding interactions of six ligands, neutral and monocationic asymmetric monomethine cyanine dyes comprising benzoselenazolyl moiety with duplex DNA and RNA and G-quadruplex structures were evaluated using fluorescence, UV/Vis (thermal melting) and circular dichroism (CD) spectroscopy. The main objective was to assess the impact of different substituents (methyl vs. sulfopropyl vs. thiopropyl/thioethyl) on the nitrogen atom of the benzothiazolyl chromophore on various nucleic acid structures. The monomethine cyanine dyes with methyl substituents showed a 100-fold selectivity for G-quadruplex versus duplex DNA. Study results indicate that cyanines bind with G-quadruplex via end π-π stacking interactions and possible additional interactions with nucleobases/phosphate backbone of grooves or loop bases. Cyanine with thioethyl substituent distinguishes duplex DNA and RNA and G-quadruplex structures by distinctly varying ICD signals. Furthermore, cell viability assay reveals the submicromolar activity of cyanines with methyl substituents against all tested human cancer cell lines. Confocal microscopy analysis shows preferential accumulation of cyanines with sulfopropyl and thioethyl substituents in mitochondria and indicates localization of cyanines with methyl in nucleus, particularly nucleolus. This confirms the potential of examined cyanines as theranostic agents, possessing both fluorescent properties and cell viability inhibitory effect. Full article

(This article belongs to the Special Issue Polynucleotides)

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15 pages, 3524 KiB

Open AccessEditor’s ChoiceArticle

The Placentas of Women Who Suffer an Episode of Psychosis during Pregnancy Have Increased Lipid Peroxidation with Evidence of Ferroptosis

by Miguel A. Ortega, Oscar Fraile-Martinez, Cielo García-Montero, Rosa M. Funes Moñux, Sonia Rodriguez-Martín, Coral Bravo, Juan A. De Leon-Luis, Jose V. Saz, Miguel A. Saez, Luis G. Guijarro, Guillermo Lahera, Fernando Mora, Sonia Fernandez-Rojo, Javier Quintero, Jorge Monserrat, Natalio García-Honduvilla, Julia Bujan, Melchor Alvarez-Mon and Miguel Angel Alvarez-Mon

Biomolecules 2023, 13(1), 120; https://doi.org/10.3390/biom13010120 - 6 Jan 2023

Cited by 14 | Viewed by 2887

Abstract

Psychosis is a complex entity characterized by psychological, behavioral, and motor alterations resulting in a loss of contact with reality. Although it is not common, pregnancy can be a period in which a first episode of psychosis can manifest, entailing detrimental consequences for [...] Read more.

Psychosis is a complex entity characterized by psychological, behavioral, and motor alterations resulting in a loss of contact with reality. Although it is not common, pregnancy can be a period in which a first episode of psychosis can manifest, entailing detrimental consequences for both the fetus and the mother. The pathophysiological basis and study of maternofetal wellbeing need to be further elucidated. Lipid peroxidation and ferroptosis are two phenomena that are tightly linked to the placental dysfunction commonly observed in different complications of pregnancy. In the present study, we aim to explore the histopathological and gene expression of different markers of lipid peroxidation and ferroptosis in the placentas of women who underwent a first episode of psychosis during their pregnancy (n = 22). The aim is to then compare them with healthy pregnant women (n = 20). In order to achieve this goal, iron deposits were studied using Prussian Blue staining. In addition, the protein/gene expression of a transferrin receptor (TFRC), as well as an acyl-CoA synthetase long-chain family member 4 (ACSL-4), arachidonate lipoxygenase-5 (ALOX-5), malondialdehyde (MDA), and glutathione peroxidase 4 (GPX4) were all analyzed through gene expression (RT-qPCR) and immunohistochemical procedures. Our results demonstrate an increased presence of iron deposits that are accompanied by a further expression of TFRC, ACSL-4, ALOX-5, MDA, and GPX4—all of which are observed in the placenta tissue of women who have suffered from a first episode of psychosis. Therefore, in our study, a histopathological increase in lipid peroxidation and ferroptosis markers in the affected women is suggested. However, further studies are needed in order to validate our results and to establish possible consequences for the reported alterations. Full article

(This article belongs to the Special Issue Placental-Related Disorders of Pregnancy)

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21 pages, 2757 KiB

Open AccessEditor’s ChoiceArticle

Effects of Chronic Caffeine Consumption on Synaptic Function, Metabolism and Adenosine Modulation in Different Brain Areas

by Cátia R. Lopes, Andreia Oliveira, Ingride Gaspar, Matilde S. Rodrigues, Joana Santos, Eszter Szabó, Henrique B. Silva, Ângelo R. Tomé, Paula M. Canas, Paula Agostinho, Rui A. Carvalho, Rodrigo A. Cunha, Ana Patrícia Simões, João Pedro Lopes and Samira G. Ferreira

Biomolecules 2023, 13(1), 106; https://doi.org/10.3390/biom13010106 - 4 Jan 2023

Cited by 8 | Viewed by 6372

Abstract

Adenosine receptors mainly control synaptic function, and excessive activation of adenosine receptors may worsen the onset of many neurological disorders. Accordingly, the regular intake of moderate doses of caffeine antagonizes adenosine receptors and affords robust neuroprotection. Although caffeine intake alters brain functional connectivity [...] Read more.

Adenosine receptors mainly control synaptic function, and excessive activation of adenosine receptors may worsen the onset of many neurological disorders. Accordingly, the regular intake of moderate doses of caffeine antagonizes adenosine receptors and affords robust neuroprotection. Although caffeine intake alters brain functional connectivity and multi-omics analyses indicate that caffeine intake modifies synaptic and metabolic processes, it is unclear how caffeine intake affects behavior, synaptic plasticity and its modulation by adenosine. We now report that male mice drinking caffeinated water (0.3 g/L) for 2 weeks were behaviorally indistinguishable (locomotion, mood, memory) from control mice (drinking water) and displayed superimposable synaptic plasticity (long-term potentiation) in different brain areas (hippocampus, prefrontal cortex, amygdala). Moreover, there was a general preservation of the efficiency of adenosine A₁ and A_2A receptors to control synaptic transmission and plasticity, although there was a tendency for lower levels of endogenous adenosine ensuring A₁ receptor-mediated inhibition. In spite of similar behavioral and neurophysiological function, caffeine intake increased the energy charge and redox state of cortical synaptosomes. This increased metabolic competence likely involved a putative increase in the glycolytic rate in synapses and a prospective greater astrocyte–synapse lactate shuttling. It was concluded that caffeine intake does not trigger evident alterations of behavior or of synaptic plasticity but increases the metabolic competence of synapses, which might be related with the previously described better ability of animals consuming caffeine to cope with deleterious stimuli triggering brain dysfunction. Full article

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