Biomolecules

Article

20 pages, 1624 KiB

Open AccessEditor’s ChoiceArticle

(3α,5α)3-Hydroxypregnan-20-one (3α,5α-THP) Regulation of the HPA Axis in the Context of Different Stressors and Sex

by Giorgia Boero, Ryan E. Tyler, Todd K. O’Buckley, Irina Balan, Joyce Besheer and A. Leslie Morrow

Biomolecules 2022, 12(8), 1134; https://doi.org/10.3390/biom12081134 - 18 Aug 2022

Cited by 10 | Viewed by 1624

Abstract

Corticotropin-releasing factor (CRF) regulates the stress response in the hypothalamus and modulates neurotransmission across the brain through CRF receptors. Acute stress increases hypothalamic CRF and the GABAergic neurosteroid (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP). We previously showed that 3α,5α-THP regulation of CRF is sex and brain region [...] Read more.

Corticotropin-releasing factor (CRF) regulates the stress response in the hypothalamus and modulates neurotransmission across the brain through CRF receptors. Acute stress increases hypothalamic CRF and the GABAergic neurosteroid (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP). We previously showed that 3α,5α-THP regulation of CRF is sex and brain region dependent. In this study, we investigated 3α,5α-THP regulation of stress-induced hypothalamic CRF, CRF receptor type 1 (CRFR1), CRF binding protein (CRFBP), pro-opiomelanocortin (POMC), and glucocorticoid receptor (GR) by western blot and circulating corticosterone (CORT) by enzyme-linked immunosorbent assay (ELISA) in male and female Sprague Dawley rats. Tissue was collected after rats were injected with 3α,5α-THP (15 mg/kg, IP) or vehicle 15 min prior to 30 min of restraint stress (RS), or 10 min of forced swim stress (FSS) and 20 min recovery. The initial exposure to a stress stimulus increased circulating CORT levels in both males and females, but 3α,5α-THP attenuated the CORT response only in females after RS. 3α,5α-THP reduced GR levels in male and females, but differently between stressors. 3α,5α-THP decreased the CRF stress response after FSS in males and females, but after RS, only in female rats. 3α,5α-THP reduced the CRFR1, CRFBP, and POMC increases after RS and FSS in males, but in females only after FSS. Our results showed different stress responses following different types of stressors: 3α,5α-THP regulated the HPA axis at different levels, depending on sex. Full article

(This article belongs to the Special Issue Recent Advances in Steroid Research and Nervous System Function)

► Show Figures

Figure 1

28 pages, 3892 KiB

Open AccessEditor’s ChoiceArticle

A Comprehensive Study of the Microbiome, Resistome, and Physical and Chemical Characteristics of Chicken Waste from Intensive Farms

by Aleksandra Błażejewska, Magdalena Zalewska, Anna Grudniak and Magdalena Popowska

Biomolecules 2022, 12(8), 1132; https://doi.org/10.3390/biom12081132 - 17 Aug 2022

Cited by 4 | Viewed by 3976

Abstract

The application of chicken waste to farmland could be detrimental to public health. It may contribute to the dissemination of antibiotic-resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) from feces and their subsequent entry into the food chain. The present study analyzes the metagenome [...] Read more.

The application of chicken waste to farmland could be detrimental to public health. It may contribute to the dissemination of antibiotic-resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) from feces and their subsequent entry into the food chain. The present study analyzes the metagenome and resistome of chicken manure and litter obtained from a commercial chicken farm in Poland. ARB were isolated, identified, and screened for antibiogram fingerprints using standard microbiological and molecular methods. The physicochemical properties of the chicken waste were also determined. ARGs, integrons, and mobile genetic elements (MGE) in chicken waste were analyzed using high-throughput SmartChip qPCR. The results confirm the presence of many ARGs, probably located in MGE, which can be transferred to other bacteria. Potentially pathogenic or opportunistic microorganisms and phytopathogens were isolated. More than 50% of the isolated strains were classified as being multi-drug resistant, and the remainder were resistant to at least one antibiotic class; these pose a real risk of entering the groundwater and contaminating the surrounding environment. Our results indicate that while chicken manure can be sufficient sources of the nutrients essential for plant growth, its microbiological aspects make this material highly dangerous to the environment. Full article

(This article belongs to the Special Issue State-of-the-Art in Antibiotics Resistance)

► Show Figures

Graphical abstract

28 pages, 4791 KiB

Open AccessEditor’s ChoiceArticle

An Interpretable Machine-Learning Algorithm to Predict Disordered Protein Phase Separation Based on Biophysical Interactions

by Hao Cai, Robert M. Vernon and Julie D. Forman-Kay

Biomolecules 2022, 12(8), 1131; https://doi.org/10.3390/biom12081131 - 17 Aug 2022

Cited by 19 | Viewed by 3152

Abstract

Protein phase separation is increasingly understood to be an important mechanism of biological organization and biomaterial formation. Intrinsically disordered protein regions (IDRs) are often significant drivers of protein phase separation. A number of protein phase-separation-prediction algorithms are available, with many being specific for [...] Read more.

Protein phase separation is increasingly understood to be an important mechanism of biological organization and biomaterial formation. Intrinsically disordered protein regions (IDRs) are often significant drivers of protein phase separation. A number of protein phase-separation-prediction algorithms are available, with many being specific for particular classes of proteins and others providing results that are not amenable to the interpretation of the contributing biophysical interactions. Here, we describe LLPhyScore, a new predictor of IDR-driven phase separation, based on a broad set of physical interactions or features. LLPhyScore uses sequence-based statistics from the RCSB PDB database of folded structures for these interactions, and is trained on a manually curated set of phase-separation-driving proteins with different negative training sets including the PDB and human proteome. Competitive training for a variety of physical chemical interactions shows the greatest contribution of solvent contacts, disorder, hydrogen bonds, pi–pi contacts, and kinked beta-structures to the score, with electrostatics, cation–pi contacts, and the absence of a helical secondary structure also contributing. LLPhyScore has strong phase-separation-prediction recall statistics and enables a breakdown of the contribution from each physical feature to a sequence’s phase-separation propensity, while recognizing the interdependence of many of these features. The tool should be a valuable resource for guiding experiments and providing hypotheses for protein function in normal and pathological states, as well as for understanding how specificity emerges in defining individual biomolecular condensates. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

► Show Figures

Graphical abstract

11 pages, 2260 KiB

Open AccessEditor’s ChoiceArticle

by Amanda A. de Oliveira, Valentina O. Mendoza, Fernanda Priviero, R. Clinton Webb and Kenia P. Nunes

Biomolecules 2022, 12(8), 1125; https://doi.org/10.3390/biom12081125 - 16 Aug 2022

Cited by 4 | Viewed by 1939

Abstract

Aging impairs the expression of HSP70, an emergent player in vascular biology. However, it is unknown if age-related alterations in HSP70 are linked to a decline in arterial function. In this study, we test the hypothesis that the contributions of HSP70 to vascular [...] Read more.

Aging impairs the expression of HSP70, an emergent player in vascular biology. However, it is unknown if age-related alterations in HSP70 are linked to a decline in arterial function. In this study, we test the hypothesis that the contributions of HSP70 to vascular contraction are diminished in middle-aged animals. We determined the basal levels of HSP70 in the aorta of young and middle-aged Sprague Dawley male rats using Western blotting. Functional studies were performed in a wire myograph system. Force development in response to phenylephrine was assessed in the presence or absence of extracellular calcium (Ca²⁺), and in aortic rings treated or non-treated with an HSP70 inhibitor. Fluorescent probes were used to evaluate vascular oxidative stress and nitric oxide levels. We report that middle-aged rats have significantly lower levels of HSP70. Blockade of HSP70 attenuated vascular phasic and tonic contraction in isolated aortas. It appears that a functional HSP70 is required for proper Ca²⁺ handling as inhibition of this protein led to reduced force–displacement in response to Ca²⁺ dynamics. Furthermore, middle-aged aortic rings exposed to the HSP70 inhibitor display higher reactive oxygen species levels without changes in nitric oxide. In summary, we show that middle-aged animals have lower levels of HSP70 in aortas, which associates with an age-related decline in vascular responses to α-1 adrenergic stimulation. Full article

(This article belongs to the Special Issue HSP70: From Signaling Mechanisms to Therapeutics)

► Show Figures

Figure 1

13 pages, 2087 KiB

Open AccessEditor’s ChoiceArticle

A Setmelanotide-like Effect at MC4R Is Achieved by MC4R Dimer Separation

by Nanina Reininghaus, Sarah Paisdzior, Friederike Höpfner, Sabine Jyrch, Cigdem Cetindag, Patrick Scheerer, Peter Kühnen and Heike Biebermann

Biomolecules 2022, 12(8), 1119; https://doi.org/10.3390/biom12081119 - 15 Aug 2022

Cited by 6 | Viewed by 2897

Abstract

Melanocortin 4 receptor (MC4R) is part of the leptin-melanocortin pathway and plays an essential role in mediating energy homeostasis. Mutations in the MC4R are the most frequent monogenic cause for obesity. Due to increasing numbers of people with excess body weight, the MC4R [...] Read more.

Melanocortin 4 receptor (MC4R) is part of the leptin-melanocortin pathway and plays an essential role in mediating energy homeostasis. Mutations in the MC4R are the most frequent monogenic cause for obesity. Due to increasing numbers of people with excess body weight, the MC4R has become a target of interest in the search of treatment options. We have previously reported that the MC4R forms homodimers, affecting receptor G_s signaling properties. Recent studies introducing setmelanotide, a novel synthetic MC4R agonist, suggest a predominant role of the G_q/11 pathway regarding weight regulation. In this study, we analyzed effects of inhibiting homodimerization on G_q/11 signaling using previously reported MC4R/CB1R chimeras. NanoBRET^TM studies to determine protein–protein interaction were conducted, confirming decreased homodimerization capacities of chimeric receptors in HEK293 cells. G_q/11 signaling of chimeric receptors was analyzed using luciferase-based reporter gene (NFAT) assays. Results demonstrate an improvement of alpha-MSH-induced NFAT signaling of chimeras, reaching the level of setmelanotide signaling at wild-type MC4R (MC4R-WT). In summary, our study shows that inhibiting homodimerization has a setmelanotide-like effect on G_q/11 signaling, with chimeric receptors presenting increased potency compared to MC4R-WT. These findings indicate the potential of inhibiting MC4R homodimerization as a therapeutic target to treat obesity. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Compartmentalized GPCR Signaling)

► Show Figures

Figure 1

18 pages, 2697 KiB

Open AccessEditor’s ChoiceArticle

Translocation of Distinct Alpha Synuclein Species from the Nucleus to Neuronal Processes during Neuronal Differentiation

by Katharina Pieger, Verena Schmitt, Carina Gauer, Nadja Gießl, Iryna Prots, Beate Winner, Jürgen Winkler, Johann Helmut Brandstätter and Wei Xiang

Biomolecules 2022, 12(8), 1108; https://doi.org/10.3390/biom12081108 - 12 Aug 2022

Cited by 4 | Viewed by 2293

Abstract

Alpha synuclein (aSyn) and its aggregation are crucial for the neurodegeneration of Parkinson’s disease (PD). aSyn was initially described in the nucleus and presynaptic nerve terminals. However, the biology of nuclear aSyn and the link of aSyn between subcellular compartments are less understood. [...] Read more.

Alpha synuclein (aSyn) and its aggregation are crucial for the neurodegeneration of Parkinson’s disease (PD). aSyn was initially described in the nucleus and presynaptic nerve terminals. However, the biology of nuclear aSyn and the link of aSyn between subcellular compartments are less understood. Current knowledge suggests the existence of various aSyn species with distinct structural and biochemical properties. Here, we identified a C-terminal-targeting aSyn antibody (Nu-aSyn-C), which has a high immunoaffinity towards aSyn in the nucleus. Comparing the Nu-aSyn-C antibody to aSyn antibodies developed against phosphorylated or aggregated forms, we observed that nuclear aSyn differs from cytosolic aSyn by an increased phosphorylation and assembly level in proliferating cells. Employing Nu-aSyn-C, we characterized aSyn distribution during neuronal differentiation in midbrain dopaminergic neurons (mDANs) derived from human-induced pluripotent stem cells (hiPSCs) and Lund human mesencephalic cells, and in primary rat hippocampal neurons. We detected a specific translocation pattern of aSyn during neuronal differentiation from the nucleus to the soma and finally to neuronal processes. Interestingly, a remarkable shift of Nu-aSyn-C-positive species towards neurites was detected in hiPSC mDANs from a PD patient carrying aSyn gene duplication. Together, our results reveal distinct nuclear and cytosolic aSyn species that redistribute during neuronal differentiation—a process that is altered in PD-derived neurons. Full article

(This article belongs to the Special Issue Common Mechanisms in Alzheimer’s Disease and Other Neurodegenerative Disorders)

► Show Figures

Figure 1

11 pages, 2243 KiB

Open AccessEditor’s ChoiceArticle

Interaction of the Emerging Mycotoxins Beauvericin, Cyclopiazonic Acid, and Sterigmatocystin with Human Serum Albumin

by Eszter Fliszár-Nyúl, Zelma Faisal, Renáta Skaper, Beáta Lemli, Bayarsaikhan Bayartsetseg, Csaba Hetényi, Patrik Gömbös, András Szabó and Miklós Poór

Biomolecules 2022, 12(8), 1106; https://doi.org/10.3390/biom12081106 - 11 Aug 2022

Cited by 13 | Viewed by 1885

Abstract

Beauvericin (BEA), cyclopiazonic acid (CPA), and sterigmatocystin (STC) are emerging mycotoxins. They appear as contaminants in food and animal feed, leading to economic losses and health risks. Human serum albumin (HSA) forms stable complexes with certain mycotoxins, including ochratoxins, alternariol, citrinin, and zearalenone. [...] Read more.

Beauvericin (BEA), cyclopiazonic acid (CPA), and sterigmatocystin (STC) are emerging mycotoxins. They appear as contaminants in food and animal feed, leading to economic losses and health risks. Human serum albumin (HSA) forms stable complexes with certain mycotoxins, including ochratoxins, alternariol, citrinin, and zearalenone. HSA binding can influence the toxicokinetics of xenobiotics, and albumin can also be considered and applied as a relatively cheap affinity protein. Therefore, we examined the potential interactions of BEA, CPA, and STC with HSA employing fluorescence spectroscopy, ultracentrifugation, ultrafiltration, and molecular modeling. Spectroscopic and ultracentrifugation studies demonstrated the formation of low-affinity BEA–HSA (K_a ≈ 10³ L/mol) and moderately strong CPA–HSA and STC–HSA complexes (K_a ≈ 10⁴ L/mol). In ultrafiltration experiments, CPA slightly displaced each site marker (warfarin, naproxen, and camptothecin) tested, while BEA and STC did not affect significantly the albumin binding of these drugs. Modeling studies suggest that CPA occupies Sudlow’s site I, while STC binds to the Heme site (FA1) on HSA. Considering the interactions of CPA with the site markers, the CPA–HSA interaction may have toxicological importance. Full article

► Show Figures

Figure 1

9 pages, 759 KiB

Open AccessEditor’s ChoiceArticle

Plasma P-Tau181 for the Discrimination of Alzheimer’s Disease from Other Primary Dementing and/or Movement Disorders

by John S. Tzartos, Fotini Boufidou, Christos Stergiou, Jens Kuhle, Eline Willemse, Lina Palaiodimou, Ioanna Tsantzali, Eleni Sideri, Anastasios Bonakis, Sotirios Giannopoulos, Konstantinos I. Voumvourakis, Georgios Tsivgoulis, Socrates J. Tzartos, Elisabeth Kapaki and George P. Paraskevas

Biomolecules 2022, 12(8), 1099; https://doi.org/10.3390/biom12081099 - 10 Aug 2022

Cited by 4 | Viewed by 2039

Abstract

Blood phospho-tau181 may offer a useful biomarker for Alzheimer’s disease. However, the use of either serum or plasma phospho-tau181 and their diagnostic value are currently under intense investigation. In a pilot study, we measured both serum and plasma phospho-tau181 (pT181-Tau) by single molecule [...] Read more.

Blood phospho-tau181 may offer a useful biomarker for Alzheimer’s disease. However, the use of either serum or plasma phospho-tau181 and their diagnostic value are currently under intense investigation. In a pilot study, we measured both serum and plasma phospho-tau181 (pT181-Tau) by single molecule array (Simoa) in a group of patients with Alzheimer’s disease and a mixed group of patients with other primary dementing and/or movement disorders. Classical cerebrospinal fluid biomarkers were also measured. Plasma (but not serum) pT181-Tau showed a significant increase in Alzheimer’s disease and correlated significantly with cerebrospinal fluid amyloid and pT181-Tau. Receiver operating curve analysis revealed a significant discrimination of Alzheimer’s from non-Alzheimer’s disease patients, with an area under the curve of 0.83 and an excellent sensitivity but a moderate specificity. Plasma pT181-Tau is not an established diagnostic biomarker for Alzheimer’s disease, but it could become one in the future, or it may serve as a screening tool for specific cases of patients or presymptomatic subjects. Full article

(This article belongs to the Special Issue Biomolecules and Biomarkers in Head and Neck Medicine)

► Show Figures

Figure 1

18 pages, 2544 KiB

Open AccessEditor’s ChoiceArticle

Vitamin A Deficiency Alters the Phototransduction Machinery and Distinct Non-Vision-Specific Pathways in the Drosophila Eye Proteome

by Mukesh Kumar, Canan Has, Khanh Lam-Kamath, Sophie Ayciriex, Deepshe Dewett, Mhamed Bashir, Clara Poupault, Kai Schuhmann, Oskar Knittelfelder, Bharath Kumar Raghuraman, Robert Ahrends, Jens Rister and Andrej Shevchenko

Biomolecules 2022, 12(8), 1083; https://doi.org/10.3390/biom12081083 - 6 Aug 2022

Cited by 3 | Viewed by 3642

Abstract

The requirement of vitamin A for the synthesis of the visual chromophore and the light-sensing pigments has been studied in vertebrate and invertebrate model organisms. To identify the molecular mechanisms that orchestrate the ocular response to vitamin A deprivation, we took advantage of [...] Read more.

The requirement of vitamin A for the synthesis of the visual chromophore and the light-sensing pigments has been studied in vertebrate and invertebrate model organisms. To identify the molecular mechanisms that orchestrate the ocular response to vitamin A deprivation, we took advantage of the fact that Drosophila melanogaster predominantly requires vitamin A for vision, but not for development or survival. We analyzed the impacts of vitamin A deficiency on the morphology, the lipidome, and the proteome of the Drosophila eye. We found that chronic vitamin A deprivation damaged the light-sensing compartments and caused a dramatic loss of visual pigments, but also decreased the molar abundance of most phototransduction proteins that amplify and transduce the visual signal. Unexpectedly, vitamin A deficiency also decreased the abundances of specific subunits of mitochondrial TCA cycle and respiratory chain components but increased the levels of cuticle- and lens-related proteins. In contrast, we found no apparent effects of vitamin A deficiency on the ocular lipidome. In summary, chronic vitamin A deficiency decreases the levels of most components of the visual signaling pathway, but also affects molecular pathways that are not vision-specific and whose mechanistic connection to vitamin A remains to be elucidated. Full article

(This article belongs to the Special Issue Invertebrates as Emerging Model Organisms in Nutrition Research)

► Show Figures

Graphical abstract

20 pages, 3015 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Isolation, Characterization, and Autophagy Function of BECN1-Splicing Isoforms in Cancer Cells

by Chinmay Maheshwari, Chiara Vidoni, Rossella Titone, Andrea Castiglioni, Claudia Lora, Carlo Follo and Ciro Isidoro

Biomolecules 2022, 12(8), 1069; https://doi.org/10.3390/biom12081069 - 2 Aug 2022

Cited by 2 | Viewed by 2584

Abstract

Alternative splicing allows the synthesis of different protein variants starting from a single gene. Human Beclin 1 (BECN1) is a key autophagy regulator that acts as haploinsufficient tumor suppressor since its decreased expression correlates with tumorigenesis and poor prognosis in cancer [...] Read more.

Alternative splicing allows the synthesis of different protein variants starting from a single gene. Human Beclin 1 (BECN1) is a key autophagy regulator that acts as haploinsufficient tumor suppressor since its decreased expression correlates with tumorigenesis and poor prognosis in cancer patients. Recent studies show that BECN1 mRNA undergoes alternative splicing. Here, we report on the isolation and molecular and functional characterization of three BECN1 transcript variants (named BECN1-α, -β and -γ) in human cancer cells. In ovarian cancer NIHOVCAR3, these splicing variants were found along with the canonical wild-type. BECN1-α lacks 143 nucleotides at its C-terminus and corresponds to a variant previously described. BECN1-β and -γ lack the BCL2 homology 3 domain and other regions at their C-termini. Following overexpression in breast cancer cells MDA-MB231, we found that BECN1-α stimulates autophagy. Specifically, BECN1-α binds to Parkin and stimulates mitophagy. On the contrary, BECN1-β reduces autophagy with a dominant negative effect over the endogenous wild-type isoform. BECN1-γ maintains its ability to interact with the vacuolar protein sorting 34 and only has a slight effect on autophagy. It is possible that cancer cells utilize the alternative splicing of BECN1 for modulating autophagy and mitophagy in response to environmental stresses. Full article

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

► Show Figures

Figure 1

17 pages, 2729 KiB

Open AccessEditor’s ChoiceArticle

GraphSite: Ligand Binding Site Classification with Deep Graph Learning

by Wentao Shi, Manali Singha, Limeng Pu, Gopal Srivastava, Jagannathan Ramanujam and Michal Brylinski

Biomolecules 2022, 12(8), 1053; https://doi.org/10.3390/biom12081053 - 29 Jul 2022

Cited by 4 | Viewed by 3710

Abstract

The binding of small organic molecules to protein targets is fundamental to a wide array of cellular functions. It is also routinely exploited to develop new therapeutic strategies against a variety of diseases. On that account, the ability to effectively detect and classify [...] Read more.

The binding of small organic molecules to protein targets is fundamental to a wide array of cellular functions. It is also routinely exploited to develop new therapeutic strategies against a variety of diseases. On that account, the ability to effectively detect and classify ligand binding sites in proteins is of paramount importance to modern structure-based drug discovery. These complex and non-trivial tasks require sophisticated algorithms from the field of artificial intelligence to achieve a high prediction accuracy. In this communication, we describe GraphSite, a deep learning-based method utilizing a graph representation of local protein structures and a state-of-the-art graph neural network to classify ligand binding sites. Using neural weighted message passing layers to effectively capture the structural, physicochemical, and evolutionary characteristics of binding pockets mitigates model overfitting and improves the classification accuracy. Indeed, comprehensive cross-validation benchmarks against a large dataset of binding pockets belonging to 14 diverse functional classes demonstrate that GraphSite yields the class-weighted F1-score of 81.7%, outperforming other approaches such as molecular docking and binding site matching. Further, it also generalizes well to unseen data with the F1-score of 70.7%, which is the expected performance in real-world applications. We also discuss new directions to improve and extend GraphSite in the future. Full article

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

► Show Figures

Figure 1

13 pages, 2105 KiB

Open AccessEditor’s ChoiceArticle

Selenocysteine Machinery Primarily Supports TXNRD1 and GPX4 Functions and Together They Are Functionally Linked with SCD and PRDX6

by Didac Santesmasses and Vadim N. Gladyshev

Biomolecules 2022, 12(8), 1049; https://doi.org/10.3390/biom12081049 - 28 Jul 2022

Cited by 9 | Viewed by 2882

Abstract

The human genome has 25 genes coding for selenocysteine (Sec)-containing proteins, whose synthesis is supported by specialized Sec machinery proteins. Here, we carried out an analysis of the co-essentiality network to identify functional partners of selenoproteins and Sec machinery. One outstanding cluster included [...] Read more.

The human genome has 25 genes coding for selenocysteine (Sec)-containing proteins, whose synthesis is supported by specialized Sec machinery proteins. Here, we carried out an analysis of the co-essentiality network to identify functional partners of selenoproteins and Sec machinery. One outstanding cluster included all seven known Sec machinery proteins and two critical selenoproteins, GPX4 and TXNRD1. Additionally, these nine genes were further positively associated with PRDX6 and negatively with SCD, linking the latter two genes to the essential role of selenium. We analyzed the essentiality scores of gene knockouts in this cluster across one thousand cancer cell lines and found that Sec metabolism genes are strongly selective for a subset of primary tissues, suggesting that certain cancer cell lineages are particularly dependent on selenium. A separate outstanding cluster included selenophosphate synthetase SEPHS1, which was linked to a group of transcription factors, whereas the remaining selenoproteins were linked neither to these clusters nor among themselves. The data suggest that key components of Sec machinery have already been identified and that their primary role is to support the functions of GPX4 and TXNRD1, with further functional links to PRDX6 and SCD. Full article

(This article belongs to the Special Issue Selenocysteine: Synthesis, Function, and Evolution of the 21st Amino Acid)

► Show Figures

Figure 1

23 pages, 3787 KiB

Open AccessEditor’s ChoiceArticle

Astaxanthin Carotenoid Modulates Oxidative Stress in Adipose-Derived Stromal Cells Isolated from Equine Metabolic Syndrome Affected Horses by Targeting Mitochondrial Biogenesis

by Malwina Mularczyk, Nabila Bourebaba, Krzysztof Marycz and Lynda Bourebaba

Biomolecules 2022, 12(8), 1039; https://doi.org/10.3390/biom12081039 - 27 Jul 2022

Cited by 6 | Viewed by 3161

Abstract

Astaxanthin is gaining recognition as a natural bioactive component. This study aimed to test whether astaxanthin could protect adipose-derived stromal stem cells (ASCs) from apoptosis, mitochondrial dysfunction and oxidative stress. Phaffia rhodozyma was used to extract astaxanthin, whose biocompatibility was tested after 24, [...] Read more.

Astaxanthin is gaining recognition as a natural bioactive component. This study aimed to test whether astaxanthin could protect adipose-derived stromal stem cells (ASCs) from apoptosis, mitochondrial dysfunction and oxidative stress. Phaffia rhodozyma was used to extract astaxanthin, whose biocompatibility was tested after 24, 48 and 72 h of incubation with the cells; no harmful impact was found. ASCs were treated with optimal concentrations of astaxanthin. Several parameters were examined: cell viability, apoptosis, reactive oxygen levels, mitochondrial dynamics and metabolism, superoxide dismutase activity, and astaxanthin’s antioxidant capacity. A RT PCR analysis was performed after each test. The astaxanthin treatment significantly reduced apoptosis by modifying the normalized caspase activity of pro-apoptotic pathways (p21, p53, and Bax). Furthermore, by regulating the expression of related master factors SOD1, SOD2, PARKIN, PINK 1, and MFN 1, astaxanthin alleviated the oxidative stress and mitochondrial dynamics failure caused by EMS. Astaxanthin restored mitochondrial oxidative phosphorylation by stimulating markers associated with the OXPHOS machinery: COX4I1, COX4I2, UQCRC2, NDUFA9, and TFAM. Our results suggest that astaxanthin has the potential to open new possibilities for potential bio-drugs to control and suppress oxidative stress, thereby improving the overall metabolic status of equine ASCs suffering from metabolic syndrome. Full article

(This article belongs to the Topic Compounds with Medicinal Value)

► Show Figures

Graphical abstract

19 pages, 4435 KiB

Open AccessEditor’s ChoiceArticle

Lipid Readjustment in Yarrowia lipolytica Odd-Chain Fatty Acids Producing Strains

by Sonia Abreu, Young-Kyoung Park, Camilla Pires de Souza, Lea Vidal, Pierre Chaminade and Jean-Marc Nicaud

Biomolecules 2022, 12(8), 1026; https://doi.org/10.3390/biom12081026 - 25 Jul 2022

Cited by 3 | Viewed by 2670

Abstract

Yarrowia lipolytica is a promising oleaginous yeast for producing unusual lipids, such as odd-chain fatty acids (OCFA). Their diverse applications and low natural production make OCFA particularly interesting. In recent studies, inhibiting the catabolic pathway of precursor, boosting precursor pools, and optimizing substrate [...] Read more.

Yarrowia lipolytica is a promising oleaginous yeast for producing unusual lipids, such as odd-chain fatty acids (OCFA). Their diverse applications and low natural production make OCFA particularly interesting. In recent studies, inhibiting the catabolic pathway of precursor, boosting precursor pools, and optimizing substrate combination greatly improved the production of OCFA in Y. lipolytica. We explored the lipid readjustment of OCFA in engineered Y. lipolytica strains. NPLC-Corona-CAD^® evidenced a time-dependent overproduction of free fatty acids, diglycerides, and phosphatidylcholine (PC) in obese LP compared to obese L. Phosphatidylethanolamine (PE) and phosphatidylinositol, largely overproduced in obese LP at 72 h compared to obese L, vanished at 216 h. The fatty acyls (FAs) composition of glycero- and glycerophospholipids was determined by NPLC-APPI⁺-HRMS from in-source generated monoacylglycerol-like fragment ions. C18:1 and C17:1 were predominant acylglycerols in obese L and obese LP, respectively. Phosphatidic acid, PE, and PC exhibited similar FAs composition but differed in their molecular species distributions. Cardiolipin (CL) is known to contain mostly C18:2 FAs corresponding to the composition in obese L, 50% of C18:2, and 35% of C18:1. In obese LP, both FAs dropped to drop to 20%, and C17:1 were predominant, reaching 55%. We hypothesize that CL-modified composition in obese LPs may alter mitochondrial function and limit lipid production. Full article

(This article belongs to the Special Issue Mass Spectrometry in the Lipid Metabolism)

► Show Figures

Figure 1

16 pages, 2609 KiB

Open AccessEditor’s ChoiceArticle

Mitochondria Transfer from Adipose Stem Cells Improves the Developmental Potential of Cryopreserved Oocytes

by Udayanga Sanath Kankanam Gamage, Shu Hashimoto, Yuki Miyamoto, Tatsuya Nakano, Masaya Yamanaka, Akiko Koike, Manabu Satoh and Yoshiharu Morimoto

Biomolecules 2022, 12(7), 1008; https://doi.org/10.3390/biom12071008 - 21 Jul 2022

Cited by 8 | Viewed by 2653

Abstract

Although it is not a well-established technology, oocyte cryopreservation is becoming prevalent in assisted reproductive technologies in response to the growing demands of patients’ sociological and pathological conditions. Oocyte cryopreservation can adversely affect the developmental potential of oocytes by causing an increase in [...] Read more.

Although it is not a well-established technology, oocyte cryopreservation is becoming prevalent in assisted reproductive technologies in response to the growing demands of patients’ sociological and pathological conditions. Oocyte cryopreservation can adversely affect the developmental potential of oocytes by causing an increase in intracellular oxidative stresses and damage to the mitochondrial structure. In this study, we studied whether autologous adipose stem cell (ASC) mitochondria supplementation with vitrified and warmed oocytes could restore post-fertilization development that decreased due to mitochondrial damage following cryopreservation. ASC mitochondria showed similar morphology to oocytes’ mitochondria and had a higher ATP production capacity. The vitrified-warmed oocytes from juvenile mice were supplemented with ASC mitochondria at the same time as intracellular sperm injection (ICSI), after which we compared their developmental capacity and the mitochondria quality of 2-cell embryos. We found that, compared to their counterpart, mitochondria supplementation significantly improved development from 2-cell embryos to blastocysts (56.8% vs. 38.2%) and ATP production in 2-cell embryos (905.6 & 561.1 pmol), while reactive oxygen species levels were comparable. With these results, we propose that ASC mitochondria supplementation could restore the quality of cryopreserved oocytes and enhance the embryo developmental capacity, signifying another possible approach for mitochondrial transplantation therapy. Full article

(This article belongs to the Special Issue State-of-the-Art Molecular Reproduction in Japan)

► Show Figures

Figure 1

16 pages, 1682 KiB

Open AccessEditor’s ChoiceArticle

Calpain-Independent Intracellular Protease Activity Is Elevated in Excitotoxic Cortical Neurons Prior to Delayed Calcium Deregulation and Mitochondrial Dysfunction

by Brian M. Polster, Karla A. Mark, Rafael Arze and Derek Hudson

Biomolecules 2022, 12(7), 1004; https://doi.org/10.3390/biom12071004 - 20 Jul 2022

Cited by 1 | Viewed by 2322

Abstract

Glutamate excitotoxicity contributes to many neurodegenerative diseases. Excessive glutamate receptor-mediated calcium entry causes delayed calcium deregulation (DCD) that coincides with abrupt mitochondrial depolarization. We developed cA-TAT, a live-cell protease activity reporter based on a vimentin calpain cleavage site, to test whether glutamate increases [...] Read more.

Glutamate excitotoxicity contributes to many neurodegenerative diseases. Excessive glutamate receptor-mediated calcium entry causes delayed calcium deregulation (DCD) that coincides with abrupt mitochondrial depolarization. We developed cA-TAT, a live-cell protease activity reporter based on a vimentin calpain cleavage site, to test whether glutamate increases protease activity in neuronal cell bodies prior to DCD. Treatment of rat cortical neurons with excitotoxic (100 µM) glutamate increased the low baseline rate of intracellular cA-TAT proteolysis by approximately three-fold prior to DCD and by approximately seven-fold upon calcium deregulation. The glutamate-induced rate enhancement prior to DCD was suppressed by glutamate receptor antagonists, but not by calpain or proteasome inhibitors, whereas DCD-stimulated proteolysis was partly attenuated by the proteasome inhibitor MG132. Further suggesting that cA-TAT cleavage is calpain-independent, cA-TAT fluorescence was observed in immortalized Capn4 knockout fibroblasts lacking the regulatory calpain subunit. About half of the neurons lost calcium homeostasis within two hours of a transient, 20 min glutamate receptor stimulation. These neurons had a significantly (49%) higher mean baseline cA-TAT proteolysis rate than those maintaining calcium homeostasis, suggesting that the unknown protease(s) cleaving cA-TAT may influence DCD susceptibility. Overall, the results indicate that excitotoxic glutamate triggers the activation of calpain-independent neuronal protease activity prior to the simultaneous loss of calcium homeostasis and mitochondrial bioenergetic function. Full article

(This article belongs to the Special Issue Mitochondria and Central Nervous System Disorders)

► Show Figures

Graphical abstract

22 pages, 3957 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

by Hayet Bouzid, Feryel Soualmia, Katerina Oikonomopoulou, Antoninus Soosaipillai, Francine Walker, Khaoula Louati, Rea Lo Dico, Marc Pocard, Chahrazade El Amri, Natalia A. Ignatenko and Dalila Darmoul

Biomolecules 2022, 12(7), 1003; https://doi.org/10.3390/biom12071003 - 19 Jul 2022

Cited by 9 | Viewed by 2724

Abstract

Kallikrein-related peptidases (KLKs) are implicated in many cancer-related processes. KLK6, one of the 15 KLK family members, is a promising biomarker for diagnosis of many cancers and has been associated with poor prognosis of colorectal cancer (CRC) patients. Herein, we evaluated the expression [...] Read more.

Kallikrein-related peptidases (KLKs) are implicated in many cancer-related processes. KLK6, one of the 15 KLK family members, is a promising biomarker for diagnosis of many cancers and has been associated with poor prognosis of colorectal cancer (CRC) patients. Herein, we evaluated the expression and cellular functions of KLK6 in colon cancer-derived cell lines and in clinical samples from CRC patients. We showed that, although many KLKs transcripts are upregulated in colon cancer-derived cell lines, KLK6, KLK10, and KLK11 are the most highly secreted proteins. KLK6 induced calcium flux in HT29 cells by activation and internalization of protease-activated receptor 2 (PAR2). Furthermore, KLK6 induced extracellular signal–regulated kinases 1 and 2 (ERK1/2) phosphorylation. KLK6 suppression in HCT-116 colon cancer cells decreased the colony formation, increased cell adhesion to extracellular matrix proteins, and reduced spheroid formation and compaction. Immunohistochemistry (IHC) analysis demonstrated ectopic expression of KLK6 in human colon adenocarcinomas but not in normal epithelia. Importantly, high levels of KLK6 protein were detected in the ascites of CRC patients with peritoneal metastasis, but not in benign ascites. These data indicate that KLK6 overexpression is associated with aggressive CRC, and may be applied to differentiate between benign and malignant ascites. Full article

(This article belongs to the Special Issue Role of Proteases in Cancer)

► Show Figures

Figure 1

22 pages, 5889 KiB

Open AccessEditor’s ChoiceArticle

Development of a Multi-Enzymatic Approach for the Modification of Biopolymers with Ferulic Acid

by Archontoula Giannakopoulou, Georgia Tsapara, Anastassios N. Troganis, Panagiota Koralli, Christos L. Chochos, Angeliki C. Polydera, Petros Katapodis, Nektaria-Marianthi Barkoula and Haralambos Stamatis

Biomolecules 2022, 12(7), 992; https://doi.org/10.3390/biom12070992 - 17 Jul 2022

Cited by 3 | Viewed by 3137

Abstract

A series of polymers, including chitosan (CS), carboxymethylcellulose (CMC) and a chitosan–gelatin (CS–GEL) hybrid polymer, were functionalized with ferulic acid (FA) derived from the enzymatic treatment of arabinoxylan through the synergistic action of two enzymes, namely, xylanase and feruloyl esterase. Subsequently, the ferulic [...] Read more.

A series of polymers, including chitosan (CS), carboxymethylcellulose (CMC) and a chitosan–gelatin (CS–GEL) hybrid polymer, were functionalized with ferulic acid (FA) derived from the enzymatic treatment of arabinoxylan through the synergistic action of two enzymes, namely, xylanase and feruloyl esterase. Subsequently, the ferulic acid served as the substrate for laccase from Agaricus bisporus (AbL) in order to enzymatically functionalize the above-mentioned polymers. The successful grafting of the oxidized ferulic acid products onto the different polymers was confirmed through ultraviolet–visible (UV–Vis) spectroscopy, attenuated total reflectance (ATR) spectroscopy, scanning electron microscopy (SEM) and nuclear magnetic resonance (NMR) spectroscopy. Additionally, an enhancement of the antioxidant properties of the functionalized polymers was observed according to the DDPH and ABTS protocols. Finally, the modified polymers exhibited strong antimicrobial activity against bacterial populations of Escherichia coli BL21DE3 strain, suggesting their potential application in pharmaceutical, cosmeceutical and food industries. Full article

(This article belongs to the Special Issue Recent Advances in the Enzymatic Synthesis of Phenolic Compounds Derivatives)

► Show Figures

Graphical abstract

21 pages, 2847 KiB

Open AccessEditor’s ChoiceArticle

Analysis of Alternative mRNA Splicing in Vemurafenib-Resistant Melanoma Cells

by Honey Bokharaie, Walter Kolch and Aleksandar Krstic

Biomolecules 2022, 12(7), 993; https://doi.org/10.3390/biom12070993 - 17 Jul 2022

Cited by 5 | Viewed by 3427

Abstract

Alternative mRNA splicing is common in cancers. In BRAF V600E-mutated malignant melanoma, a frequent mechanism of acquired resistance to BRAF inhibitors involves alternative splicing (AS) of BRAF. The resulting shortened BRAF protein constitutively dimerizes and conveys drug resistance. Here, we have analysed AS [...] Read more.

Alternative mRNA splicing is common in cancers. In BRAF V600E-mutated malignant melanoma, a frequent mechanism of acquired resistance to BRAF inhibitors involves alternative splicing (AS) of BRAF. The resulting shortened BRAF protein constitutively dimerizes and conveys drug resistance. Here, we have analysed AS in SK-MEL-239 melanoma cells and a BRAF inhibitor (vemurafenib)-resistant derivative that expresses an AS, shortened BRAF V600E transcript. Transcriptome analysis showed differential expression of spliceosome components between the two cell lines. As there is no consensus approach to analysing AS events, we used and compared four common AS softwares based on different principles, DEXSeq, rMATS, ASpli, and LeafCutter. Two of them correctly identified the BRAF V600E AS in the vemurafenib-resistant cells. Only 12 AS events were identified by all four softwares. Testing the AS predictions experimentally showed that these overlapping predictions are highly accurate. Interestingly, they identified AS caused alterations in the expression of melanin synthesis and cell migration genes in the vemurafenib-resistant cells. This analysis shows that combining different AS analysis approaches produces reliable results and meaningful, biologically testable hypotheses. Full article

(This article belongs to the Section Bioinformatics and Systems Biology)

► Show Figures

Figure 1

24 pages, 5103 KiB

Open AccessEditor’s ChoiceArticle

Cloning and Characterization of Drosophila melanogaster Juvenile Hormone Epoxide Hydrolases (JHEH) and Their Promoters

by Dov Borovsky, Hilde Breyssens, Esther Buytaert, Tom Peeters, Carole Laroye, Karolien Stoffels and Pierre Rougé

Biomolecules 2022, 12(7), 991; https://doi.org/10.3390/biom12070991 - 16 Jul 2022

Cited by 3 | Viewed by 2571

Abstract

Juvenile hormone epoxide hydrolase (JHEH) plays an important role in the metabolism of JH III in insects. To study the control of JHEH in female Drosophila melanogaster, JHEH 1, 2 and 3 cDNAs were cloned and sequenced. Northern blot analyses showed that [...] Read more.

Juvenile hormone epoxide hydrolase (JHEH) plays an important role in the metabolism of JH III in insects. To study the control of JHEH in female Drosophila melanogaster, JHEH 1, 2 and 3 cDNAs were cloned and sequenced. Northern blot analyses showed that the three transcripts are expressed in the head thorax, the gut, the ovaries and the fat body of females. Molecular modeling shows that the enzyme is a homodimer that binds juvenile hormone III acid (JH IIIA) at the catalytic groove better than JH III. Analyses of the three JHEH promoters and expressing short promoter sequences behind a reporter gene (lacZ) in D. melanogaster cell culture identified a JHEH 3 promoter sequence (626 bp) that is 10- and 25-fold more active than the most active promoter sequences of JHEH 2 and JHEH 1, respectively. A transcription factor (TF) Sp1 that is involved in the activation of JHEH 3 promoter sequence was identified. Knocking down Sp1 using dsRNA inhibited the transcriptional activity of this promoter in transfected D. melanogaster cells and JH III and 20HE downregulated the JHEH 3 promoter. On the other hand, JH IIIA and farnesoic acid did not affect the promoter, indicating that JH IIIA is JHEH’s preferred substrate. A transgenic D. melanogaster expressing a highly activated JHEH 3 promoter behind a lacZ reporter gene showed promoter transcriptional activity in many D. melanogaster tissues. Full article

(This article belongs to the Special Issue Contributions of Promoters to the Physiology, Biochemistry and Molecular Biology Processes of Insects)

► Show Figures

Graphical abstract

23 pages, 2695 KiB

Open AccessEditor’s ChoiceArticle

Cell Confluence Modulates TRPV4 Channel Activity in Response to Hypoxia

by Solène Barbeau, Alexandre Joushomme, Yann Chappe, Guillaume Cardouat, Isabelle Baudrimont, Véronique Freund-Michel, Christelle Guibert, Roger Marthan, Patrick Berger, Pierre Vacher, Yann Percherancier, Jean-François Quignard and Thomas Ducret

Biomolecules 2022, 12(7), 954; https://doi.org/10.3390/biom12070954 - 7 Jul 2022

Cited by 3 | Viewed by 2466

Abstract

Transient receptor potential vanilloid 4 (TRPV4) is a polymodal Ca²⁺-permeable channel involved in various hypoxia-sensitive pathophysiological phenomena. Different tools are available to study channel activity, requiring cells to be cultured at specific optimal densities. In the present study, we examined if [...] Read more.

Transient receptor potential vanilloid 4 (TRPV4) is a polymodal Ca²⁺-permeable channel involved in various hypoxia-sensitive pathophysiological phenomena. Different tools are available to study channel activity, requiring cells to be cultured at specific optimal densities. In the present study, we examined if cell density may influence the effect of hypoxia on TRPV4 activity. Transiently TRPV4-transfected HEK293T cells were seeded at low or high densities corresponding to non-confluent or confluent cells, respectively, on the day of experiments, and cultured under in vitro normoxia or hypoxia. TRPV4-mediated cytosolic Ca²⁺ responses, single-channel currents, and Ca²⁺ influx through the channel were measured using Ca²⁺ imaging/microspectrofluorimetric assay, patch-clamp, and Bioluminescence Resonance Energy Transfer (BRET), respectively. TRPV4 plasma membrane translocation was studied using confocal microscopy, biotinylation of cell surface proteins, and BRET. Our results show that hypoxia exposure has a differential effect on TRPV4 activation depending on cell confluence. At low confluence levels, TRPV4 response is increased in hypoxia, whereas at high confluence levels, TRPV4 response is strongly inhibited, due to channel internalization. Thus, cell density appears to be a crucial parameter for TRPV4 channel activity. Full article

(This article belongs to the Special Issue Physiological Cell Culture)

► Show Figures

Figure 1

16 pages, 7465 KiB

Open AccessEditor’s ChoiceArticle

Acute Effects of Focused Ultrasound-Induced Blood-Brain Barrier Opening on Anti-Pyroglu3 Abeta Antibody Delivery and Immune Responses

by Praveen Bathini, Tao Sun, Mathias Schenk, Stephan Schilling, Nathan J. McDannold and Cynthia A. Lemere

Biomolecules 2022, 12(7), 951; https://doi.org/10.3390/biom12070951 - 6 Jul 2022

Cited by 10 | Viewed by 3012

Abstract

Alzheimer’s Disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid plaques and hyperphosphorylated tau in the brain. Currently, therapeutic agents targeting amyloid appear promising for AD, however, delivery to the CNS is limited due to the blood-brain-barrier (BBB). Focused ultrasound [...] Read more.

Alzheimer’s Disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid plaques and hyperphosphorylated tau in the brain. Currently, therapeutic agents targeting amyloid appear promising for AD, however, delivery to the CNS is limited due to the blood-brain-barrier (BBB). Focused ultrasound (FUS) is a method to induce a temporary opening of the BBB to enhance the delivery of therapeutic agents to the CNS. In this study, we evaluated the acute effects of FUS and whether the use of FUS-induced BBB opening enhances the delivery of 07/2a mAb, an anti-pyroglutamate-3 Aβ antibody, in aged 24 mo-old APP/PS1dE9 transgenic mice. FUS was performed either unilaterally or bilaterally with mAb infusion and the short-term effect was analyzed 4 h and 72 h post-treatment. Quantitative analysis by ELISA showed a 5–6-fold increase in 07/2a mAb levels in the brain at both time points and an increased brain-to-blood ratio of the antibody. Immunohistochemistry demonstrated an increase in IgG2a mAb detection particularly in the cortex, enhanced immunoreactivity of resident Iba1+ and phagocytic CD68+ microglial cells, and a transient increase in the infiltration of Ly6G+ immune cells. Cerebral microbleeds were not altered in the unilaterally or bilaterally sonicated hemispheres. Overall, this study shows the potential of FUS therapy for the enhanced delivery of CNS therapeutics. Full article

(This article belongs to the Special Issue Common Mechanisms in Alzheimer’s Disease and Other Neurodegenerative Disorders)

► Show Figures

Figure 1

16 pages, 2123 KiB

Open AccessEditor’s ChoiceArticle

A Novel Analytical Approach to Assessing Sorption of Trace Organic Compounds into Micro- and Nanoplastic Particles

by Julia Reichel, Johanna Graßmann, Oliver Knoop, Thomas Letzel and Jörg E. Drewes

Biomolecules 2022, 12(7), 953; https://doi.org/10.3390/biom12070953 - 6 Jul 2022

Cited by 2 | Viewed by 2387

Abstract

Assessing the sorption of trace organic compounds (TOrCs) into micro- and nanoplastic particles has traditionally been performed using an aqueous phase analysis or solvent extractions from the particle. Using thermal extraction/desorption–gas chromatography/mass spectrometry (TD-Pyr-GC/MS) offers a possibility to analyze the TOrCs directly from [...] Read more.

Assessing the sorption of trace organic compounds (TOrCs) into micro- and nanoplastic particles has traditionally been performed using an aqueous phase analysis or solvent extractions from the particle. Using thermal extraction/desorption–gas chromatography/mass spectrometry (TD-Pyr-GC/MS) offers a possibility to analyze the TOrCs directly from the particle without a long sample preparation. In this study, a combination of two analytical methods is demonstrated. First, the aqueous phase is quantified for TOrC concentrations using Gerstel Twister^® and TD-GC/MS. Subsequently, the TOrCs on the particles are analyzed. Different polymer types and sizes (polymethyl methacrylate (PMMA), 48 µm; polyethylene (PE), 48 µm; polystyrene (PS), 41 µm; and PS, 78 nm) were analyzed for three selected TOrCs (phenanthrene, triclosan, and α-cypermethrin). The results revealed that, over a period of 48 h, the highest and fastest sorption occurred for PS 78 nm particles. This was confirmed with a theoretical calculation of the particle surface area. It was also shown for the first time that direct quantification of TOrCs from PS 78 nm nanoparticles is possible. Furthermore, in a mixed solute solution, the three selected TOrCs were sorbed onto the particles simultaneously. Full article

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

► Show Figures

Figure 1

13 pages, 1818 KiB

Open AccessEditor’s ChoiceArticle

NMR Reveals Specific Tracts within the Intrinsically Disordered Regions of the SARS-CoV-2 Nucleocapsid Protein Involved in RNA Encountering

by Letizia Pontoriero, Marco Schiavina, Sophie M. Korn, Andreas Schlundt, Roberta Pierattelli and Isabella C. Felli

Biomolecules 2022, 12(7), 929; https://doi.org/10.3390/biom12070929 - 2 Jul 2022

Cited by 14 | Viewed by 2713

Abstract

The SARS-CoV-2 nucleocapsid (N) protein is crucial for the highly organized packaging and transcription of the genomic RNA. Studying atomic details of the role of its intrinsically disordered regions (IDRs) in RNA recognition is challenging due to the absence of structure and to [...] Read more.

The SARS-CoV-2 nucleocapsid (N) protein is crucial for the highly organized packaging and transcription of the genomic RNA. Studying atomic details of the role of its intrinsically disordered regions (IDRs) in RNA recognition is challenging due to the absence of structure and to the repetitive nature of their primary sequence. IDRs are known to act in concert with the folded domains of N and here we use NMR spectroscopy to identify the priming events of N interacting with a regulatory SARS-CoV-2 RNA element. ¹³C-detected NMR experiments, acquired simultaneously to ¹H detected ones, provide information on the two IDRs flanking the N-terminal RNA binding domain (NTD) within the N-terminal region of the protein (NTR, 1–248). We identify specific tracts of the IDRs that most rapidly sense and engage with RNA, and thus provide an atom-resolved picture of the interplay between the folded and disordered regions of N during RNA interaction. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

► Show Figures

Graphical abstract

22 pages, 4840 KiB

Open AccessEditor’s ChoiceArticle

Data Size and Quality Matter: Generating Physically-Realistic Distance Maps of Protein Tertiary Structures

by Fardina Fathmiul Alam and Amarda Shehu

Biomolecules 2022, 12(7), 908; https://doi.org/10.3390/biom12070908 - 29 Jun 2022

Cited by 5 | Viewed by 3067

Abstract

With the debut of AlphaFold2, we now can get a highly-accurate view of a reasonable equilibrium tertiary structure of a protein molecule. Yet, a single-structure view is insufficient and does not account for the high structural plasticity of protein molecules. Obtaining a multi-structure [...] Read more.

With the debut of AlphaFold2, we now can get a highly-accurate view of a reasonable equilibrium tertiary structure of a protein molecule. Yet, a single-structure view is insufficient and does not account for the high structural plasticity of protein molecules. Obtaining a multi-structure view of a protein molecule continues to be an outstanding challenge in computational structural biology. In tandem with methods formulated under the umbrella of stochastic optimization, we are now seeing rapid advances in the capabilities of methods based on deep learning. In recent work, we advance the capability of these models to learn from experimentally-available tertiary structures of protein molecules of varying lengths. In this work, we elucidate the important role of the composition of the training dataset on the neural network’s ability to learn key local and distal patterns in tertiary structures. To make such patterns visible to the network, we utilize a contact map-based representation of protein tertiary structure. We show interesting relationships between data size, quality, and composition on the ability of latent variable models to learn key patterns of tertiary structure. In addition, we present a disentangled latent variable model which improves upon the state-of-the-art variable autoencoder-based model in key, physically-realistic structural patterns. We believe this work opens up further avenues of research on deep learning-based models for computing multi-structure views of protein molecules. Full article

(This article belongs to the Special Issue Protein Modeling and Simulation: Selected articles from the Computational Structural Bioinformatics Workshop 2021)

► Show Figures

Figure 1

15 pages, 3586 KiB

Open AccessEditor’s ChoiceArticle

Characterization of Endothelium-Dependent Relaxation in the Saphenous Artery and Its Caudal Branches in Young and Old Adult Sprague Dawley Rats

by Andrea N. Wang, Graham M. Fraser and John J. McGuire

Biomolecules 2022, 12(7), 889; https://doi.org/10.3390/biom12070889 - 25 Jun 2022

Cited by 2 | Viewed by 3071

Abstract

Ageing is associated with reduced endothelium-derived nitric oxide (NO) production in the femoral artery of Sprague Dawley (SD) rats. In the current study, we examined endothelium-dependent relaxation (EDR) in the saphenous artery and its caudal branches. We used acetylcholine and the Proteinase-Activated receptor-2 [...] Read more.

Ageing is associated with reduced endothelium-derived nitric oxide (NO) production in the femoral artery of Sprague Dawley (SD) rats. In the current study, we examined endothelium-dependent relaxation (EDR) in the saphenous artery and its caudal branches. We used acetylcholine and the Proteinase-Activated receptor-2 (PAR2)-specific agonist (2fLIGRLO) with nitroarginine methylester (L-NAME) to assess EDR in two groups of male SD rats (age in weeks: young, 10–12; old, 27–29). Acetylcholine and 2fLIGRLO were potent NO-dependent relaxant agents in all arteries. For all arteries, EDR by acetylcholine decreased significantly in old compared to young SD rats. Interestingly, PAR2-induced EDR of proximal saphenous artery segments and caudal branches decreased significantly in old compared to young, but did not differ for the in-between middle and distal ends of the saphenous artery. L-NAME treatment increased subsequent contractions of proximal and middle segments of saphenous arteries by phenylephrine and U46619 in young, but not in old, SD rats. We conclude the SD saphenous artery and caudal branches exhibit regional characteristics that differ in response to specific EDR agonists, endothelial NO synthase inhibitor, and changes to endothelium function with increased age, which are, in part, attributed to decreased sensitivity of vascular smooth muscle to the gaseous transmitter NO. Full article

(This article belongs to the Special Issue Gaseous Transmitters and Cardiovascular System)

► Show Figures

Figure 1

33 pages, 4624 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

A Novel and Selective Dopamine Transporter Inhibitor, (S)-MK-26, Promotes Hippocampal Synaptic Plasticity and Restores Effort-Related Motivational Dysfunctions

by Shima Kouhnavardi, Alev Ecevitoglu, Vladimir Dragačević, Fabrizio Sanna, Edgar Arias-Sandoval, Predrag Kalaba, Michael Kirchhofer, Jana Lubec, Marco Niello, Marion Holy, Martin Zehl, Matthias Pillwein, Judith Wackerlig, Rita Murau, Andrea Mohrmann, Kathryn R. Beard, Harald H. Sitte, Ernst Urban, Claudia Sagheddu, Marco Pistis, Roberto Plasenzotti, John D. Salamone, Thierry Langer, Gert Lubec and Francisco J. Monje Show full author list Hide full author list

Biomolecules 2022, 12(7), 881; https://doi.org/10.3390/biom12070881 - 24 Jun 2022

Cited by 15 | Viewed by 4444

Abstract

Dopamine (DA), the most abundant human brain catecholaminergic neurotransmitter, modulates key behavioral and neurological processes in young and senescent brains, including motricity, sleep, attention, emotion, learning and memory, and social and reward-seeking behaviors. The DA transporter (DAT) regulates transsynaptic DA levels, influencing all [...] Read more.

Dopamine (DA), the most abundant human brain catecholaminergic neurotransmitter, modulates key behavioral and neurological processes in young and senescent brains, including motricity, sleep, attention, emotion, learning and memory, and social and reward-seeking behaviors. The DA transporter (DAT) regulates transsynaptic DA levels, influencing all these processes. Compounds targeting DAT (e.g., cocaine and amphetamines) were historically used to shape mood and cognition, but these substances typically lead to severe negative side effects (tolerance, abuse, addiction, and dependence). DA/DAT signaling dysfunctions are associated with neuropsychiatric and progressive brain disorders, including Parkinson’s and Alzheimer diseases, drug addiction and dementia, resulting in devastating personal and familial concerns and high socioeconomic costs worldwide. The development of low-side-effect, new/selective medicaments with reduced abuse-liability and which ameliorate DA/DAT-related dysfunctions is therefore crucial in the fields of medicine and healthcare. Using the rat as experimental animal model, the present work describes the synthesis and pharmacological profile of (S)-MK-26, a new modafinil analogue with markedly improved potency and selectivity for DAT over parent drug. Ex vivo electrophysiology revealed significantly augmented hippocampal long-term synaptic potentiation upon acute, intraperitoneally delivered (S)-MK-26 treatment, whereas in vivo experiments in the hole-board test showed only lesser effects on reference memory performance in aged rats. However, in effort-related FR5/chow and PROG/chow feeding choice experiments, (S)-MK-26 treatment reversed the depression-like behavior induced by the dopamine-depleting drug tetrabenazine (TBZ) and increased the selection of high-effort alternatives. Moreover, in in vivo microdialysis experiments, (S)-MK-26 significantly increased extracellular DA levels in the prefrontal cortex and in nucleus accumbens core and shell. These studies highlight (S)-MK-26 as a potent enhancer of transsynaptic DA and promoter of synaptic plasticity, with predominant beneficial effects on effort-related behaviors, thus proposing therapeutic potentials for (S)-MK-26 in the treatment of low-effort exertion and motivational dysfunctions characteristic of depression and aging-related disorders. Full article

► Show Figures

Graphical abstract

20 pages, 2949 KiB

Open AccessEditor’s ChoiceArticle

Development of a Biotechnology Platform for the Fast-Growing Cyanobacterium Synechococcus sp. PCC 11901

by Lauren A. Mills, José Ángel Moreno-Cabezuelo, Artur Włodarczyk, Angelo J. Victoria, Rebeca Mejías, Anja Nenninger, Simon Moxon, Paolo Bombelli, Tiago T. Selão, Alistair J. McCormick and David J. Lea-Smith

Biomolecules 2022, 12(7), 872; https://doi.org/10.3390/biom12070872 - 23 Jun 2022

Cited by 9 | Viewed by 5334

Abstract

Synechococcus sp. PCC 11901 reportedly demonstrates the highest, most sustained growth of any known cyanobacterium under optimized conditions. Due to its recent discovery, our knowledge of its biology, including the factors underlying sustained, fast growth, is limited. Furthermore, tools specific for genetic manipulation [...] Read more.

Synechococcus sp. PCC 11901 reportedly demonstrates the highest, most sustained growth of any known cyanobacterium under optimized conditions. Due to its recent discovery, our knowledge of its biology, including the factors underlying sustained, fast growth, is limited. Furthermore, tools specific for genetic manipulation of PCC 11901 are not established. Here, we demonstrate that PCC 11901 shows faster growth than other model cyanobacteria, including the fast-growing species Synechococcuselongatus UTEX 2973, under optimal growth conditions for UTEX 2973. Comparative genomics between PCC 11901 and Synechocystis sp. PCC 6803 reveal conservation of most metabolic pathways but PCC 11901 has a simplified electron transport chain and reduced light harvesting complex. This may underlie its superior light use, reduced photoinhibition, and higher photosynthetic and respiratory rates. To aid biotechnology applications, we developed a vitamin B₁₂ auxotrophic mutant but were unable to generate unmarked knockouts using two negative selectable markers, suggesting that recombinase- or CRISPR-based approaches may be required for repeated genetic manipulation. Overall, this study establishes PCC 11901 as one of the most promising species currently available for cyanobacterial biotechnology and provides a useful set of bioinformatics tools and strains for advancing this field, in addition to insights into the factors underlying its fast growth phenotype. Full article

► Show Figures

Figure 1

14 pages, 2185 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Early Signs of Molecular Defects in iPSC-Derived Neural Stems Cells from Patients with Familial Parkinson’s Disease

by Elissavet Akrioti, Timokratis Karamitros, Panagiotis Gkaravelas, Georgia Kouroupi, Rebecca Matsas and Era Taoufik

Biomolecules 2022, 12(7), 876; https://doi.org/10.3390/biom12070876 - 23 Jun 2022

Cited by 5 | Viewed by 3747

Abstract

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, classically associated with extensive loss of dopaminergic neurons of the substantia nigra pars compacta. The hallmark of the disease is the accumulation of pathogenic conformations of the presynaptic protein, α-synuclein (αSyn), and the [...] Read more.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, classically associated with extensive loss of dopaminergic neurons of the substantia nigra pars compacta. The hallmark of the disease is the accumulation of pathogenic conformations of the presynaptic protein, α-synuclein (αSyn), and the formation of intraneuronal protein aggregate inclusions. Neurodegeneration of dopamine neurons leads to a prominent dopaminergic deficiency in the basal ganglia, responsible for motor disturbances. However, it is now recognized that the disease involves more widespread neuronal dysfunction, leading to early and late non-motor symptoms. The development of in vitro systems based on the differentiation of human-induced pluripotent stem cells provides us the unique opportunity to monitor alterations at the cellular and molecular level throughout the differentiation procedure and identify perturbations that occur early, even at the neuronal precursor stage. Here we aim to identify whether p.A53T-αSyn induced disturbances at the molecular level are already present in neural precursors. Towards this, we present data from transcriptomics analysis of control and p.A53T-αSyn NPCs showing altered expression in transcripts involved in axon guidance, adhesion, synaptogenesis, ion transport, and metabolism. The comparative analysis with the transcriptomics profile of p.A53T-αSyn neurons shows both distinct and overlapping pathways leading to neurodegeneration while meta-analysis with transcriptomics data from both neurodegenerative and neurodevelopmental disorders reveals that p.A53T-pathology has a significant overlap with the latter category. This is the first study showing that molecular dysregulation initiates early at the p.A53T-αSyn NPC level, suggesting that synucleinopathies may have a neurodevelopmental component. Full article

(This article belongs to the Special Issue Recent Advances in α-Synuclein Neurobiology in Health and Disease)

► Show Figures

Figure 1

16 pages, 4049 KiB

Open AccessEditor’s ChoiceArticle

Midazolam as a Probe for Heterotropic Drug-Drug Interactions Mediated by CYP3A4

by Ilia G. Denisov, Yelena V. Grinkova, Mark A. McLean, Tyler Camp and Stephen G. Sligar

Biomolecules 2022, 12(6), 853; https://doi.org/10.3390/biom12060853 - 20 Jun 2022

Cited by 8 | Viewed by 2793

Abstract

Human cytochrome P450 CYP3A4 is involved in the processing of more than 35% of current pharmaceuticals and therefore is responsible for multiple drug-drug interactions (DDI). In order to develop a method for the detection and prediction of the possible involvement of new drug [...] Read more.

Human cytochrome P450 CYP3A4 is involved in the processing of more than 35% of current pharmaceuticals and therefore is responsible for multiple drug-drug interactions (DDI). In order to develop a method for the detection and prediction of the possible involvement of new drug candidates in CYP3A4-mediated DDI, we evaluated the application of midazolam (MDZ) as a probe substrate. MDZ is hydroxylated by CYP3A4 in two positions: 1-hydroxy MDZ formed at lower substrate concentrations, and up to 35% of 4-hydroxy MDZ at high concentrations. The ratio of the formation rates of these two products (the site of metabolism ratio, SOM) was used as a measure of allosteric heterotropic interactions caused by effector molecules using CYP3A4 incorporated in lipid nanodiscs. The extent of the changes in the SOM in the presence of effectors is determined by chemical structure and is concentration-dependent. MD simulations of CYP3A4 in the lipid bilayer suggest that experimental results can be explained by the movement of the F-F’ loop and concomitant changes in the shape and volume of the substrate-binding pocket. As a result of PGS binding at the allosteric site, several residues directly contacting MDZ move away from the substrate molecule, enabling the repositioning of the latter for minor product formation. Full article

(This article belongs to the Special Issue New Insights into Cytochrome P450s)

► Show Figures

Figure 1

14 pages, 2356 KiB

Open AccessEditor’s ChoiceArticle

Growth-Suppressive and Apoptosis-Inducing Effects of Tetrandrine in SW872 Human Malignant Liposarcoma Cells via Activation of Caspase-9, Down-Regulation of XIAP and STAT-3, and ER Stress

by Mohammed Samsuzzaman and Byeong-Churl Jang

Biomolecules 2022, 12(6), 843; https://doi.org/10.3390/biom12060843 - 17 Jun 2022

Cited by 5 | Viewed by 2104

Abstract

Liposarcoma is a rare and heterogeneous soft tissue malignant tumor and has a significant impact on mortality with a poor prognosis. To date, there is no effective treatment for liposarcoma, whereas surgical resection is only the gold treatment with numerous adverse effects. Here [...] Read more.

Liposarcoma is a rare and heterogeneous soft tissue malignant tumor and has a significant impact on mortality with a poor prognosis. To date, there is no effective treatment for liposarcoma, whereas surgical resection is only the gold treatment with numerous adverse effects. Here we investigated whether tetrandrine inhibits the growth of SW872 human malignant liposarcoma cells. Of note, tetrandrine at 10 μM vastly inhibited growth and induced apoptosis, as evidenced by increased nuclear DNA fragmentation and sub-G1 population of SW872 cells. Mechanistically, treatment with tetrandrine led to activation of caspase-9/3 in SW872 cells, and z-VAD-fmk, a pan-caspase inhibitor, attenuated the tetrandrine-induced apoptosis and growth suppression in SW872 cells. In addition, tetrandrine treatment resulted in down-regulation of XIAP andSTAT-3 in SW872 cells, and importantly knockdown of STAT-3 caused a significant reduction of the cell survival. Tetrandrine also had abilities to up-regulate not only the expression of GRP78 and ATF-4 but also the phosphorylation of eIF-2α in SW872 cells. In summary, these results demonstrated that tetrandrine has strong growth-suppressive and apoptosis-inducing effects on SW872 cells, which are mediated through control of the intrinsic caspase pathway, down-regulation of XIAP and STAT-3, and triggering ER stress. Full article

(This article belongs to the Collection Feature Papers in Chemical Biology)

► Show Figures

Figure 1

24 pages, 4142 KiB

Open AccessEditor’s ChoiceArticle

Non-Equilibrium Protein Folding and Activation by ATP-Driven Chaperones

by Huafeng Xu

Biomolecules 2022, 12(6), 832; https://doi.org/10.3390/biom12060832 - 15 Jun 2022

Cited by 3 | Viewed by 3644

Abstract

Recent experimental studies suggest that ATP-driven molecular chaperones can stabilize protein substrates in their native structures out of thermal equilibrium. The mechanism of such non-equilibrium protein folding is an open question. Based on available structural and biochemical evidence, I propose here a unifying [...] Read more.

Recent experimental studies suggest that ATP-driven molecular chaperones can stabilize protein substrates in their native structures out of thermal equilibrium. The mechanism of such non-equilibrium protein folding is an open question. Based on available structural and biochemical evidence, I propose here a unifying principle that underlies the conversion of chemical energy from ATP hydrolysis to the conformational free energy associated with protein folding and activation. I demonstrate that non-equilibrium folding requires the chaperones to break at least one of four symmetry conditions. The Hsp70 and Hsp90 chaperones each break a different subset of these symmetries and thus they use different mechanisms for non-equilibrium protein folding. I derive an upper bound on the non-equilibrium elevation of the native concentration, which implies that non-equilibrium folding only occurs in slow-folding proteins that adopt an unstable intermediate conformation in binding to ATP-driven chaperones. Contrary to the long-held view of Anfinsen’s hypothesis that proteins fold to their conformational free energy minima, my results predict that some proteins may fold into thermodynamically unstable native structures with the assistance of ATP-driven chaperones, and that the native structures of some chaperone-dependent proteins may be shaped by their chaperone-mediated folding pathways. Full article

(This article belongs to the Special Issue Hsp90 Structure, Mechanism and Disease)

► Show Figures

Figure 1

16 pages, 3725 KiB

Open AccessEditor’s ChoiceArticle

Selective Upregulation of SIRT1 Expression in Retinal Ganglion Cells by AAV-Mediated Gene Delivery Increases Neuronal Cell Survival and Alleviates Axon Demyelination Associated with Optic Neuritis

by Ahmara G. Ross, Brahim Chaqour, Devin S. McDougald, Kimberly E. Dine, Thu T. Duong, Ryan E. Shindler, Jipeng Yue, Tehui Liu and Kenneth S. Shindler

Biomolecules 2022, 12(6), 830; https://doi.org/10.3390/biom12060830 - 14 Jun 2022

Cited by 13 | Viewed by 3241

Abstract

Optic neuritis (ON), the most common ocular manifestation of multiple sclerosis, is an autoimmune inflammatory demyelinating disease also characterized by degeneration of retinal ganglion cells (RGCs) and their axons, which commonly leads to visual impairment despite attempted treatments. Although ON disease etiology is [...] Read more.

Optic neuritis (ON), the most common ocular manifestation of multiple sclerosis, is an autoimmune inflammatory demyelinating disease also characterized by degeneration of retinal ganglion cells (RGCs) and their axons, which commonly leads to visual impairment despite attempted treatments. Although ON disease etiology is not known, changes in the redox system and exacerbated optic nerve inflammation play a major role in the pathogenesis of the disease. Silent information regulator 1 (sirtuin-1/SIRT1) is a ubiquitously expressed NAD⁺-dependent deacetylase, which functions to reduce/prevent both oxidative stress and inflammation in various tissues. Non-specific upregulation of SIRT1 by pharmacologic and genetic approaches attenuates RGC loss in experimental ON. Herein, we hypothesized that targeted expression of SIRT1 selectively in RGCs using an adeno-associated virus (AAV) vector as a delivery vehicle is an effective approach to reducing neurodegeneration and preserving vision in ON. We tested this hypothesis through intravitreal injection of AAV7m8.SNCG.SIRT1, an AAV2-derived vector optimized for highly efficient SIRT1 transgene transfer and protein expression into RGCs in mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis that recapitulates optic neuritis RGC loss and axon demyelination. Our data show that EAE mice injected with a control vehicle exhibit progressive alteration of visual function reflected by decreasing optokinetic response (OKR) scores, whereas comparatively, AAV7m8.SNCG.SIRT1-injected EAE mice maintain higher OKR scores, suggesting that SIRT1 reduces the visual deficit imparted by EAE. Consistent with this, RGC survival determined by immunolabeling is increased and axon demyelination is decreased in the AAV7m8.SNCG.SIRT1 RGC-injected group of EAE mice compared to the mouse EAE counterpart injected with a vehicle or with control vector AAV7m8.SNCG.eGFP. However, immune cell infiltration of the optic nerve is not significantly different among all EAE groups of mice injected with either vehicle or AAV7m8.SNCG.SIRT1. We conclude that despite minimally affecting the inflammatory response in the optic nerve, AAV7m8-mediated SIRT1 transfer into RGCs has a neuroprotective potential against RGC loss, axon demyelination and vison deficits associated with EAE. Together, these data suggest that SIRT1 exerts direct effects on RGC survival and function. Full article

(This article belongs to the Special Issue Molecular and Cellular Mechanisms of Optic Neuropathies)

► Show Figures

Figure 1

21 pages, 4098 KiB

Open AccessEditor’s ChoiceArticle

Occurrence of Lymphangiogenesis in Peripheral Nerve Autografts Contrasts Schwann Cell-Induced Apoptosis of Lymphatic Endothelial Cells In Vitro

by Carina Hromada, Jaana Hartmann, Johannes Oesterreicher, Anton Stoiber, Anna Daerr, Barbara Schädl, Eleni Priglinger, Andreas H. Teuschl-Woller, Wolfgang Holnthoner, Johannes Heinzel and David Hercher

Biomolecules 2022, 12(6), 820; https://doi.org/10.3390/biom12060820 - 12 Jun 2022

Cited by 8 | Viewed by 2974

Abstract

Peripheral nerve injuries pose a major clinical concern world-wide, and functional recovery after segmental peripheral nerve injury is often unsatisfactory, even in cases of autografting. Although it is well established that angiogenesis plays a pivotal role during nerve regeneration, the influence of lymphangiogenesis [...] Read more.

Peripheral nerve injuries pose a major clinical concern world-wide, and functional recovery after segmental peripheral nerve injury is often unsatisfactory, even in cases of autografting. Although it is well established that angiogenesis plays a pivotal role during nerve regeneration, the influence of lymphangiogenesis is strongly under-investigated. In this study, we analyzed the presence of lymphatic vasculature in healthy and regenerated murine peripheral nerves, revealing that nerve autografts contained increased numbers of lymphatic vessels after segmental damage. This led us to elucidate the interaction between lymphatic endothelial cells (LECs) and Schwann cells (SCs) in vitro. We show that SC and LEC secretomes did not influence the respective other cell types’ migration and proliferation in 2D scratch assay experiments. Furthermore, we successfully created lymphatic microvascular structures in SC-embedded 3D fibrin hydrogels, in the presence of supporting cells; whereas SCs seemed to exert anti-lymphangiogenic effects when cultured with LECs alone. Here, we describe, for the first time, increased lymphangiogenesis after peripheral nerve injury and repair. Furthermore, our findings indicate a potential lymph-repellent property of SCs, thereby providing a possible explanation for the lack of lymphatic vessels in the healthy endoneurium. Our results highlight the importance of elucidating the molecular mechanisms of SC–LEC interaction. Full article

(This article belongs to the Special Issue Recent Advances in Schwann Cells)

► Show Figures

Figure 1

9 pages, 3284 KiB

Open AccessEditor’s ChoiceArticle

Efficient Viral Transduction in Fetal and Adult Human Inner Ear Explants with AAV9-PHP.B Vectors

by Edward S. A. van Beelen, Wouter H. van der Valk, Thijs O. Verhagen, John C. M. J. de Groot, Margot A. Madison, Wijs Shadmanfar, Erik F. Hensen, Jeroen C. Jansen, Peter Paul G. van Benthem, Jeffrey R. Holt and Heiko Locher

Biomolecules 2022, 12(6), 816; https://doi.org/10.3390/biom12060816 - 10 Jun 2022

Cited by 5 | Viewed by 2635

Abstract

Numerous studies have shown the recovery of auditory function in mouse models of genetic hearing loss following AAV gene therapy, yet translation to the clinic has not yet been demonstrated. One limitation has been the lack of human inner ear cell lines or [...] Read more.

Numerous studies have shown the recovery of auditory function in mouse models of genetic hearing loss following AAV gene therapy, yet translation to the clinic has not yet been demonstrated. One limitation has been the lack of human inner ear cell lines or tissues for validating viral gene therapies. Cultured human inner ear tissue could help confirm viral tropism and efficacy for driving exogenous gene expression in targeted cell types, establish promoter efficacy and perhaps selectivity for targeted cells, confirm the expression of therapeutic constructs and the subcellular localization of therapeutic proteins, and address the potential cellular toxicity of vectors or exogenous constructs. To begin to address these questions, we developed an explant culture method using native human inner ear tissue excised at either fetal or adult stages. Inner ear sensory epithelia were cultured for four days and exposed to vectors encoding enhanced green fluorescent protein (eGFP). We focused on the synthetic AAV9-PHP.B capsid, which has been demonstrated to be efficient for driving eGFP expression in the sensory hair cells of mouse and non-human primate inner ears. We report that AAV9-PHP.B also drives eGFP expression in fetal cochlear hair cells and in fetal and adult vestibular hair cells in explants of human inner ear sensory epithelia, which suggests that both the experimental paradigm and the viral capsid may be valuable for translation to clinical application. Full article

(This article belongs to the Special Issue Inner Ear Therapeutics)

► Show Figures

Figure 1

19 pages, 3172 KiB

Open AccessEditor’s ChoiceArticle

Exploring Silk Sericin for Diabetic Wounds: An In Situ-Forming Hydrogel to Protect against Oxidative Stress and Improve Tissue Healing and Regeneration

by Sara Baptista-Silva, Beatriz G. Bernardes, Sandra Borges, Ilda Rodrigues, Rui Fernandes, Susana Gomes-Guerreiro, Marta Teixeira Pinto, Manuela Pintado, Raquel Soares, Raquel Costa and Ana Leite Oliveira

Biomolecules 2022, 12(6), 801; https://doi.org/10.3390/biom12060801 - 8 Jun 2022

Cited by 14 | Viewed by 4002

Abstract

Chronic wounds are one of the most frequent complications that are associated with diabetes mellitus. The overproduction of reactive oxygen species (ROS) is a key factor in the delayed healing of a chronic wound. In the present work, we develop a novel in [...] Read more.

Chronic wounds are one of the most frequent complications that are associated with diabetes mellitus. The overproduction of reactive oxygen species (ROS) is a key factor in the delayed healing of a chronic wound. In the present work, we develop a novel in situ-forming silk sericin-based hydrogel (SSH) that is produced by a simple methodology using horseradish peroxidase (HRP) crosslinking as an advanced dressing for wound healing. The antioxidant and angiogenic effects were assessed in vitro and in vivo after in situ application using an excisional wound-healing model in a genetically-induced diabetic db/db mice and though the chick embryo choriollantoic membrane (CAM) assay, respectively. Wounds in diabetic db/db mice that were treated with SSH closed with reduced granulation tissue, decreased wound edge distance, and wound thickness, when compared to Tegaderm, a dressing that is commonly used in the clinic. The hydrogel also promoted a deposition of collagen fibers with smaller diameter which may have had a boost effect in re-epithelialization. SSH treatment slightly induced two important endogenous antioxidant defenses, superoxide dismutase and catalase. A CAM assay made it possible to observe that SSH led to an increase in the number of newly formed vessels without inducing an inflammatory reaction. The present hydrogel may result in a multi-purpose technology with angiogenic, antioxidant, and anti-inflammatory properties, while advancing efficient and organized tissue regeneration. Full article

► Show Figures

Graphical abstract

11 pages, 1655 KiB

Open AccessEditor’s ChoiceArticle

Characterization of DAG Binding to TRPC Channels by Target-Dependent cis–trans Isomerization of OptoDArG

by Hazel Erkan-Candag, Denis Krivic, Mathias A. F. Gsell, Mina Aleksanyan, Thomas Stockner, Rumiana Dimova, Oleksandra Tiapko and Klaus Groschner

Biomolecules 2022, 12(6), 799; https://doi.org/10.3390/biom12060799 - 7 Jun 2022

Cited by 6 | Viewed by 2228

Abstract

Azobenzene-based photochromic lipids are valuable probes for the analysis of ion channel–lipid interactions. Rapid photoisomerization of these molecules enables the analysis of lipid gating kinetics and provides information on lipid sensing. Thermal relaxation of the metastable cis conformation to the trans conformation of [...] Read more.

Azobenzene-based photochromic lipids are valuable probes for the analysis of ion channel–lipid interactions. Rapid photoisomerization of these molecules enables the analysis of lipid gating kinetics and provides information on lipid sensing. Thermal relaxation of the metastable cis conformation to the trans conformation of azobenzene photolipids is rather slow in the dark and may be modified by ligand–protein interactions. Cis photolipid-induced changes in pure lipid membranes as visualized from the morphological response of giant unilamellar vesicles indicated that thermal cis–trans isomerization of both PhoDAG-1 and OptoDArG is essentially slow in the lipid bilayer environment. While the currents activated by cis PhoDAG remained stable upon termination of UV light exposure (dark, UV-OFF), cis OptoDArG-induced TRPC3/6/7 activity displayed a striking isoform-dependent exponential decay. The deactivation kinetics of cis OptoDArG-induced currents in the dark was sensitive to mutations in the L2 lipid coordination site of TRPC channels. We conclude that the binding of cis OptoDArG to TRPC channels promotes transition of cis OptoDArG to the trans conformation. This process is suggested to provide valuable information on DAG–ion channel interactions and may enable highly selective photopharmacological interventions. Full article

(This article belongs to the Special Issue Lipid-Gating and Lipid-Protein Interactions in Ion Channels)

► Show Figures

Figure 1

17 pages, 1813 KiB

Open AccessEditor’s ChoiceArticle

Biochemical and Thermodynamic Studies on a Novel Thermotolerant GH10 Xylanase from Bacillus safensis

by Panayiotis D. Glekas, Styliani Kalantzi, Anargiros Dalios, Dimitris G. Hatzinikolaou and Diomi Mamma

Biomolecules 2022, 12(6), 790; https://doi.org/10.3390/biom12060790 - 6 Jun 2022

Cited by 10 | Viewed by 2234

Abstract

Xylanases have a broad range of applications in agro-industrial processes. In this study, we report on the discovery and characterization of a new thermotolerant GH10 xylanase from Bacillus safensis, designated as BsXyn10. The xylanase gene (bsxyn10) was cloned from Bacillus [...] Read more.

Xylanases have a broad range of applications in agro-industrial processes. In this study, we report on the discovery and characterization of a new thermotolerant GH10 xylanase from Bacillus safensis, designated as BsXyn10. The xylanase gene (bsxyn10) was cloned from Bacillus safensis and expressed in Escherichia coli. The reduced molecular mass of BsXyn10 was 48 kDa upon SDS-PAGE. Bsxyn10 was optimally active at pH 7.0 and 60 °C, stable over a broad range of pH (5.0–8.0), and also revealed tolerance toward different modulators (metal cations, EDTA). The enzyme was active toward various xylans with no activity on the glucose-based polysaccharides. K_M, v_max, and k_cat for oat spelt xylan hydrolysis were found to be 1.96 g·L⁻¹, 58.6 μmole·min⁻¹·(mg protein)⁻¹, and 49 s⁻¹, respectively. Thermodynamic parameters for oat spelt xylan hydrolysis at 60 °C were ΔS* = −61.9 J·mol⁻¹·K⁻¹, ΔH* = 37.0 kJ·mol⁻¹ and ΔG* = 57.6 kJ·mol⁻¹. BsXyn10 retained high levels of activity at temperatures up to 60 °C. The thermodynamic parameters (ΔH*_D, ΔG*_D, ΔS*_D) for the thermal deactivation of BsXyn10 at a temperature range of 40–80 °C were: 192.5 ≤ ΔH*_D ≤ 192.8 kJ·mol⁻¹, 262.1 ≤ ΔS*_D ≤ 265.8 J·mol⁻¹·K⁻¹, and 99.9 ≤ ΔG*_D ≤ 109.6 kJ·mol⁻¹. The BsXyn10-treated oat spelt xylan manifested the catalytic release of xylooligosaccharides of 2–6 DP, suggesting that BsXyn10 represents a promising candidate biocatalyst appropriate for several biotechnological applications. Full article

(This article belongs to the Section Enzymology)

► Show Figures

Figure 1

15 pages, 1570 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Myristic Acid Supplementation Aggravates High Fat Diet-Induced Adipose Inflammation and Systemic Insulin Resistance in Mice

by Viswanathan Saraswathi, Narendra Kumar, Weilun Ai, Thiyagarajan Gopal, Saumya Bhatt, Edward N. Harris, Geoffrey A. Talmon and Cyrus V. Desouza

Biomolecules 2022, 12(6), 739; https://doi.org/10.3390/biom12060739 - 24 May 2022

Cited by 12 | Viewed by 3671

Abstract

Saturated fatty acids (SFAs) are considered to be detrimental to human health. One of the SFAs, myristic acid (MA), is known to exert a hypercholesterolemic effect in mice as well as humans. However, its effects on altering adipose tissue (AT) inflammation and systemic [...] Read more.

Saturated fatty acids (SFAs) are considered to be detrimental to human health. One of the SFAs, myristic acid (MA), is known to exert a hypercholesterolemic effect in mice as well as humans. However, its effects on altering adipose tissue (AT) inflammation and systemic insulin resistance (IR) in obesity are still unclear. Here, we sought to determine the effects of a high fat (HF) diet supplemented with MA on obesity-associated metabolic disorders in mice. Wild-type C57BL/6 mice were fed a HF diet in the presence or absence of 3% MA for 12 weeks. Plasma lipids, plasma adipokines, AT inflammation, systemic IR, glucose homeostasis, and hepatic steatosis were assessed. The body weight and visceral adipose tissue (VAT) mass were significantly higher in mice receiving the HF+MA diet compared to HF diet-fed controls. Plasma total cholesterol levels were marginally increased in HF+MA-fed mice compared to controls. Fasting blood glucose was comparable between HF and HF+MA-fed mice. Interestingly, the plasma insulin and HOMA-IR index, a measure of insulin resistance, were significantly higher in HF+MA-fed mice compared to HF controls. Macrophage and inflammatory markers were significantly elevated in the AT and AT-derived stromal vascular cells upon MA feeding. Moreover, the level of circulating resistin, an adipokine promoting insulin resistance, was significantly higher in HF+MA-fed mice compared with HF controls. The insulin tolerance test revealed that the IR was higher in mice receiving the MA supplementation compared to HF controls. Moreover, the glucose tolerance test showed impairment in systemic glucose homeostasis in MA-fed mice. Analyses of liver samples showed a trend towards an increase in liver TG upon MA feeding. However, markers of oxidative stress and inflammation were reduced in the liver of mice fed an MA diet compared to controls. Taken together, our data suggest that chronic administration of MA in diet exacerbates obesity-associated insulin resistance and this effect is mediated in part, via increased AT inflammation and increased secretion of resistin. Full article

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

► Show Figures

Figure 1

16 pages, 3889 KiB

Open AccessEditor’s ChoiceArticle

Impairment of the Retinal Endothelial Cell Barrier Induced by Long-Term Treatment with VEGF-A₁₆₅ No Longer Depends on the Growth Factor’s Presence

by Heidrun L. Deissler, Matus Rehak and Armin Wolf

Biomolecules 2022, 12(5), 734; https://doi.org/10.3390/biom12050734 - 23 May 2022

Cited by 2 | Viewed by 2213

Abstract

As responses of immortalized endothelial cells of the bovine retina (iBREC) to VEGF-A₁₆₅ depend on exposure time to the growth factor, we investigated changes evident after long-term treatment for nine days. The cell index of iBREC cultivated on gold electrodes—determined as a [...] Read more.

As responses of immortalized endothelial cells of the bovine retina (iBREC) to VEGF-A₁₆₅ depend on exposure time to the growth factor, we investigated changes evident after long-term treatment for nine days. The cell index of iBREC cultivated on gold electrodes—determined as a measure of permeability—was persistently reduced by exposure to the growth factor. Late after addition of VEGF-A₁₆₅ protein levels of claudin-1 and CD49e were significantly lower, those of CD29 significantly higher, and the plasmalemma vesicle associated protein was no longer detected. Nuclear levels of β-catenin were only elevated on day two. Extracellular levels of VEGF-A—measured by ELISA—were very low. Similar to the binding of the growth factor by brolucizumab, inhibition of VEGFR2 by tyrosine kinase inhibitors tivozanib or nintedanib led to complete, although transient, recovery of the low cell index when added early, though was inefficient when added three or six days later. Additional inhibition of other receptor tyrosine kinases by nintedanib was similarly unsuccessful, but additional blocking of c-kit by tivozanib led to sustained recovery of the low cell index, an effect observed only when the inhibitor was added early. From these data, we conclude that several days after the addition of VEGF-A₁₆₅ to iBREC, barrier dysfunction is mainly sustained by increased paracellular flow and impaired adhesion. Even more important, these changes are most likely no longer VEGF-A-controlled. Full article

(This article belongs to the Special Issue Cell and Organ Cultures for Studying Retinal Diseases)

► Show Figures

Figure 1

17 pages, 2074 KiB

Open AccessEditor’s ChoiceArticle

Reduced Platelet MAO-B Activity Is Associated with Psychotic, Positive, and Depressive Symptoms in PTSD

by Senka Repovecki, Gordana Nedic Erjavec, Suzana Uzun, Lucija Tudor, Matea Nikolac Perkovic, Marcela Konjevod, Oliver Kozumplik, Dubravka Svob Strac, Zrnka Kovacic Petrovic, Ninoslav Mimica and Nela Pivac

Biomolecules 2022, 12(5), 736; https://doi.org/10.3390/biom12050736 - 23 May 2022

Viewed by 2980

Abstract

Post-traumatic stress disorder (PTSD) is a trauma-related disorder. Platelet monoamine oxidase (MAO-B) is a peripheral biomarker associated with various symptoms in different psychopathologies, but its role in PTSD or different symptoms in PTSD is not clear. This study elucidated the association between platelet [...] Read more.

Post-traumatic stress disorder (PTSD) is a trauma-related disorder. Platelet monoamine oxidase (MAO-B) is a peripheral biomarker associated with various symptoms in different psychopathologies, but its role in PTSD or different symptoms in PTSD is not clear. This study elucidated the association between platelet MAO-B activity and clinical symptoms occurring in PTSD. Platelet MAO-B activity was determined in 1053 male Caucasian subjects: 559 war veterans with PTSD (DSM-5 criteria), 62 combat exposed veterans who did not develop PTSD, and 432 non-combat exposed healthy controls. Clinical symptoms in PTSD were determined using CAPS and PANSS. Platelet MAO-B activity, controlled for the effect of smoking, was significantly increased in PTSD with severe versus mild and moderate traumatic symptoms, and was significantly decreased in PTSD subjects with severe versus mild positive, psychotic, and depressive symptoms. This finding was further confirmed with reduced platelet MAO-B activity in PTSD veterans with severe versus mild individual items of the PANSS-depressed, PANSS-psychotic, and PANSS-positive subscales. Altered platelet MAO-B activity, controlled for the possible confounders, was associated with the development and severity of different symptoms occurring in PTSD. These findings confirmed the role of platelet MAO-B activity as a peripheral marker of various psychopathological symptoms. Full article

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

► Show Figures

Figure 1

20 pages, 4302 KiB

Open AccessEditor’s ChoiceArticle

Anti-Inflammatory Effects of GM1 Ganglioside on Endotoxin-Induced Uveitis in Rats

by Tzu-Heng Weng, Chang-Chih Ke and Yuahn-Sieh Huang

Biomolecules 2022, 12(5), 727; https://doi.org/10.3390/biom12050727 - 21 May 2022

Cited by 3 | Viewed by 2961

Abstract

Exogenous ganglioside GM1 has been reported to exert an immunomodulatory effect. We investigated the anti-inflammatory effect of GM1 ganglioside on endotoxin-induced uveitis (EIU) in rats and RAW 264.7 macrophages. Methods: EIU was induced in Lewis rats by administering a subcutaneous injection of lipopolysaccharide [...] Read more.

Exogenous ganglioside GM1 has been reported to exert an immunomodulatory effect. We investigated the anti-inflammatory effect of GM1 ganglioside on endotoxin-induced uveitis (EIU) in rats and RAW 264.7 macrophages. Methods: EIU was induced in Lewis rats by administering a subcutaneous injection of lipopolysaccharide (LPS). GM1 was injected intraperitoneally for three consecutive days prior to the LPS injection. Twenty-four hours after the LPS injection, the integrity of the blood-aqueous barrier was evaluated by determining the protein concentration and number of infiltrating cells in the aqueous humor (AqH). Immunohistochemical and Western blot analyses of the iris-ciliary body (ICB) were performed to evaluate the effect of GM1 on the LPS-induced expression of cyclooxygenase-2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1). The effect of GM1 on proinflammatory mediators and signaling cascades was examined in LPS-stimulated RAW 264.7 cells using Western blotting and immunofluorescence staining to further clarify the underlying anti-inflammatory mechanism. Results: GM1 significantly reduced the protein concentration and number of infiltrating cells in the AqH of rats with EIU. GM1 also decreased the LPS-induced expression of the ICAM-1 and COX-2 proteins in the ICB. In RAW 264.7 cells, GM1 inhibited the proinflammatory mediators induced by LPS, including inducible nitric oxide synthase (iNOS), COX-2, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), and this inhibitory effect was potentially mediated by suppressing reactive oxygen species (ROS)-mediated activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs). Conclusions: Based on this study, GM1 may be a potential anti-inflammatory agent for ocular inflammatory diseases. Full article

(This article belongs to the Topic Compounds with Medicinal Value)

► Show Figures

Figure 1

15 pages, 1374 KiB

Open AccessEditor’s ChoiceArticle

DYT-PRKRA Mutation P222L Enhances PACT’s Stimulatory Activity on Type I Interferon Induction

by Lauren S. Vaughn, Kenneth Frederick, Samuel B. Burnett, Nutan Sharma, D. Cristopher Bragg, Sarah Camargos, Francisco Cardoso and Rekha C. Patel

Biomolecules 2022, 12(5), 713; https://doi.org/10.3390/biom12050713 - 17 May 2022

Cited by 2 | Viewed by 3080

Abstract

DYT-PRKRA (dystonia 16 or DYT-PRKRA) is caused by mutations in the PRKRA gene that encodes PACT, the protein activator of interferon (IFN)-induced double-stranded (ds) RNA-activated protein kinase (PKR). PACT participates in several cellular pathways, of which its role as a [...] Read more.

DYT-PRKRA (dystonia 16 or DYT-PRKRA) is caused by mutations in the PRKRA gene that encodes PACT, the protein activator of interferon (IFN)-induced double-stranded (ds) RNA-activated protein kinase (PKR). PACT participates in several cellular pathways, of which its role as a PKR activator protein during integrated stress response (ISR) is the best characterized. Previously, we have established that the DYT-PRKRA mutations cause enhanced activation of PKR during ISR to sensitize DYT-PRKRA cells to apoptosis. In this study, we evaluate if the most prevalent substitution mutation reported in DYT-PRKRA patients alters PACT’s functional role in induction of type I IFNs via the retinoic acid-inducible gene I (RIG-I) signaling. Our results indicate that the P222L mutation augments PACT’s ability to induce IFN β in response to dsRNA and the basal expression of IFN β and IFN-stimulated genes (ISGs) is higher in DYT-PRKRA patient cells compared to cells from the unaffected controls. Additionally, IFN β and ISGs are also induced at higher levels in DYT-PRKRA cells in response to dsRNA. These results offer a new avenue for investigations directed towards understanding the underlying molecular pathomechanisms in DYT-PRKRA. Full article

(This article belongs to the Special Issue Inflammatory Pathways in Neuro-Muscular Degeneration, Metabolic Syndromes, Cancer and Infection)

► Show Figures

Graphical abstract

9 pages, 1483 KiB

Open AccessEditor’s ChoiceArticle

Fluorescent Anti-CEA Nanobody for Rapid Tumor-Targeting and Imaging in Mouse Models of Pancreatic Cancer

by Thinzar M. Lwin, Michael A. Turner, Hiroto Nishino, Siamak Amirfakhri, Sophie Hernot, Robert M. Hoffman and Michael Bouvet

Biomolecules 2022, 12(5), 711; https://doi.org/10.3390/biom12050711 - 16 May 2022

Cited by 6 | Viewed by 3032

Abstract

Tumor-specific targeting with fluorescent probes can enhance contrast for identification of cancer during surgical resection and visualize otherwise invisible tumor margins. Nanobodies are the smallest naturally-occurring antigen-binding molecules with rapid pharmacokinetics. The present work demonstrates the efficacy of a fluorescent anti-CEA nanobody conjugated [...] Read more.

Tumor-specific targeting with fluorescent probes can enhance contrast for identification of cancer during surgical resection and visualize otherwise invisible tumor margins. Nanobodies are the smallest naturally-occurring antigen-binding molecules with rapid pharmacokinetics. The present work demonstrates the efficacy of a fluorescent anti-CEA nanobody conjugated to an IR800 dye to target and label patient derived pancreatic cancer xenografts. After intravenous administration, the probe rapidly localized to the pancreatic cancer tumors within an hour and had a tumor-to-background ratio of 2.0 by 3 h. The fluorescence signal was durable over a prolonged period of time. With the rapid kinetics afforded by fluorescent nanobodies, both targeting and imaging can be performed on the same day as surgery. Full article

► Show Figures

Figure 1

14 pages, 1758 KiB

Open AccessEditor’s ChoiceArticle

Gal-2 Increases H3K4me³ and H3K9ac in Trophoblasts and Preeclampsia

by Laura Hahn, Sarah Meister, Mareike Mannewitz, Susanne Beyer, Stefanie Corradini, Uwe Hasbargen, Sven Mahner, Udo Jeschke, Thomas Kolben and Alexander Burges

Biomolecules 2022, 12(5), 707; https://doi.org/10.3390/biom12050707 - 15 May 2022

Cited by 2 | Viewed by 2272

Abstract

Preeclampsia (PE) is a severe pregnancy disorder with a pathophysiology not yet completely understood and without curative therapy. The histone modifications H3K4me³ and H3K9ac, as well as galectin-2 (Gal-2), are known to be decreased in PE. To gain a better understanding of [...] Read more.

Preeclampsia (PE) is a severe pregnancy disorder with a pathophysiology not yet completely understood and without curative therapy. The histone modifications H3K4me³ and H3K9ac, as well as galectin-2 (Gal-2), are known to be decreased in PE. To gain a better understanding of the development of PE, the influence of Gal-2 on histone modification in trophoblasts and in syncytialisation was investigated. Immunohistochemical stains of 13 PE and 13 control placentas were correlated, followed by cell culture experiments. An analysis of H3K4me³ and H3K9ac was conducted, as well as cell fusion staining with E-cadherin and β-catenin—both after incubation with Gal-2. The expression of H3K4me³ and H3K9ac correlated significantly with the expression of Gal-2. Furthermore, we detected an increase in H3K4me³ and H3K9ac after the addition of Gal-2 to BeWo/HVT cells. Moreover, there was increased fusion of HVT cells after incubation with Gal-2. Gal-2 is associated with the histone modifications H3K4me³ and H3K9ac in trophoblasts. Furthermore, syncytialisation increased after incubation with Gal-2. Therefore, we postulate that Gal-2 stimulates syncytialisation, possibly mediated by H3K4me³ and H3K9ac. Since Gal-2, as well as H3K4me³ and H3K9ac, are decreased in PE, the induction of Gal-2 might be a promising therapeutic target. Full article

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

► Show Figures

Figure 1

19 pages, 3710 KiB

Open AccessEditor’s ChoiceArticle

CAPTURE of the Human U2 snRNA Genes Expands the Repertoire of Associated Factors

by Joana Guiro, Mathias Fagbemi, Michael Tellier, Justyna Zaborowska, Stephanie Barker, Marjorie Fournier and Shona Murphy

Biomolecules 2022, 12(5), 704; https://doi.org/10.3390/biom12050704 - 14 May 2022

Cited by 1 | Viewed by 3359

Abstract

In order to identify factors involved in transcription of human snRNA genes and 3′ end processing of the transcripts, we have carried out CRISPR affinity purification in situ of regulatory elements (CAPTURE), which is deadCas9-mediated pull-down, of the tandemly repeated U2 snRNA genes [...] Read more.

In order to identify factors involved in transcription of human snRNA genes and 3′ end processing of the transcripts, we have carried out CRISPR affinity purification in situ of regulatory elements (CAPTURE), which is deadCas9-mediated pull-down, of the tandemly repeated U2 snRNA genes in human cells. CAPTURE enriched many factors expected to be associated with these human snRNA genes including RNA polymerase II (pol II), Cyclin-Dependent Kinase 7 (CDK7), Negative Elongation Factor (NELF), Suppressor of Ty 5 (SPT5), Mediator 23 (MED23) and several subunits of the Integrator Complex. Suppressor of Ty 6 (SPT6); Cyclin K, the partner of Cyclin-Dependent Kinase 12 (CDK12) and Cyclin-Dependent Kinase 13 (CDK13); and SWI/SNF chromatin remodelling complex-associated SWI/SNF-related, Matrix-associated, Regulator of Chromatin (SMRC) factors were also enriched. Several polyadenylation factors, including Cleavage and Polyadenylation Specificity Factor 1 (CPSF1), Cleavage Stimulation Factors 1 and 2 (CSTF1,and CSTF2) were enriched by U2 gene CAPTURE. We have already shown by chromatin immunoprecipitation (ChIP) that CSTF2—and Pcf11 and Ssu72, which are also polyadenylation factors—are associated with the human U1 and U2 genes. ChIP-seq and ChIP-qPCR confirm the association of SPT6, Cyclin K, and CDK12 with the U2 genes. In addition, knockdown of SPT6 causes loss of subunit 3 of the Integrator Complex (INTS3) from the U2 genes, indicating a functional role in snRNA gene expression. CAPTURE has therefore expanded the repertoire of transcription and RNA processing factors associated with these genes and helped to identify a functional role for SPT6. Full article

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

► Show Figures

Figure 1

29 pages, 6616 KiB

Open AccessEditor’s ChoiceArticle

Automated—Mechanical Procedure Compared to Gentle Enzymatic Tissue Dissociation in Cell Function Studies

by Mariele Montanari, Sabrina Burattini, Caterina Ciacci, Patrizia Ambrogini, Silvia Carloni, Walter Balduini, Daniele Lopez, Giovanna Panza, Stefano Papa and Barbara Canonico

Biomolecules 2022, 12(5), 701; https://doi.org/10.3390/biom12050701 - 14 May 2022

Cited by 8 | Viewed by 6815

Abstract

The first step to obtain a cellular suspension from tissues is the disaggregation procedure. The cell suspension method has to provide a representative sample of the different cellular subpopulations and to maximize the number of viable functional cells. Here, we analyzed specific cell [...] Read more.

The first step to obtain a cellular suspension from tissues is the disaggregation procedure. The cell suspension method has to provide a representative sample of the different cellular subpopulations and to maximize the number of viable functional cells. Here, we analyzed specific cell functions in cell suspensions from several rat tissues obtained by two different methods, automated–mechanical and enzymatic disaggregation. Flow cytometric, confocal, and ultrastructural (TEM) analyses were applied to the spleen, testis, liver and other tissues. Samples were treated by an enzymatic trypsin solution or processed by the Medimachine II (MMII). The automated–mechanical and enzymatic disaggregation procedures have shown to work similarly in some tissues, which displayed comparable amounts of apoptotic/necrotic cells. However, cells obtained by the enzyme-free Medimachine II protocols show a better preservation lysosome and mitochondria labeling, whereas the enzymatic gentle dissociation appears to constantly induce a lower amount of intracellular ROS; nevertheless, lightly increased ROS can be recognized as a complimentary signal to promote cell survival. Therefore, MMII represents a simple, fast, and standardized method for tissue processing, which allows to minimize bias arising from the operator’s ability. Our study points out technical issues to be adopted for specific organs and tissues to obtain functional cells. Full article

(This article belongs to the Collection Feature Papers in Chemical Biology)

► Show Figures

Figure 1

15 pages, 6939 KiB

Open AccessEditor’s ChoiceArticle

Extracellular Vesicles from Steatotic Hepatocytes Provoke Pro-Fibrotic Responses in Cultured Stellate Cells

by Maria Teresa Koenen, Elisa Fabiana Brandt, Dawid Marcin Kaczor, Tim Caspers, Alexandra Catharina Anna Heinzmann, Petra Fischer, Daniel Heinrichs, Theresa Hildegard Wirtz, Christian Trautwein, Rory R Koenen and Marie-Luise Berres

Biomolecules 2022, 12(5), 698; https://doi.org/10.3390/biom12050698 - 13 May 2022

Cited by 4 | Viewed by 2838

Abstract

Hepatic steatosis and chronic hepatocyte damage ultimately lead to liver fibrosis. Key pathophysiological steps are the activation and transdifferentiation of hepatic stellate cells. We assessed the interplay between hepatocytes and hepatic stellate cells under normal and steatotic conditions. We hypothesized that hepatocyte-derived extracellular [...] Read more.

Hepatic steatosis and chronic hepatocyte damage ultimately lead to liver fibrosis. Key pathophysiological steps are the activation and transdifferentiation of hepatic stellate cells. We assessed the interplay between hepatocytes and hepatic stellate cells under normal and steatotic conditions. We hypothesized that hepatocyte-derived extracellular vesicles (EVs) modify the phenotype of stellate cells. By high speed centrifugation, EVs were isolated from conditioned media of the hepatocellular carcinoma cell line HepG2 under baseline conditions (C-EVs) or after induction of steatosis by linoleic and oleic acids for 24 h (FA-EVs). Migration of the human stellate cell line TWNT4 and of primary human stellate cells towards the respective EVs and sera of MAFLD patients were investigated using Boyden chambers. Phenotype alterations after incubation with EVs were determined by qRT-PCR, Western blotting and immunofluorescence staining. HepG2 cells released more EVs after treatment with fatty acids. Chemotactic migration of TWNT4 and primary hepatic stellate cells was increased, specifically towards FA-EVs. Prolonged incubation of TWNT4 cells with FA-EVs induced expression of proliferation markers and a myofibroblast-like phenotype. Though the expression of the collagen type 1

α

1 gene did not change after FA-EV treatment, expression of the myofibroblast markers, e.g.,

α

-smooth-muscle-cell actin and TIMP1, was significantly increased. We conclude that EVs from steatotic hepatocytes can influence the behavior, phenotypes and expression levels of remodeling markers of stellate cells and guides their directed migration. These findings imply EVs as operational, intercellular communicators in the pathophysiology of steatosis-associated liver fibrosis and might represent a novel diagnostic parameter and therapeutic target. Full article

(This article belongs to the Special Issue Recent Advances in Cellular and Molecular Mechanisms of Cardiovascular and Metabolic Diseases)

► Show Figures

Figure 1

14 pages, 3155 KiB

Open AccessEditor’s ChoiceArticle

Design, Synthesis, and Antisickling Investigation of a Nitric Oxide-Releasing Prodrug of 5HMF for the Treatment of Sickle Cell Disease

by Rana T. Alhashimi, Mohini S. Ghatge, Akua K. Donkor, Tanvi M. Deshpande, Nancy Anabaraonye, Dina Alramadhani, Richmond Danso-Danquah, Boshi Huang, Yan Zhang, Faik N. Musayev, Osheiza Abdulmalik and Martin K. Safo

Biomolecules 2022, 12(5), 696; https://doi.org/10.3390/biom12050696 - 12 May 2022

Cited by 4 | Viewed by 2217

Abstract

5-hydroxyfurfural (5HMF), an allosteric effector of hemoglobin (Hb) with an ability to increase Hb affinity for oxygen has been studied extensively for its antisickling effect in vitro and in vivo, and in humans for the treatment of sickle cell disease (SCD). One of [...] Read more.

5-hydroxyfurfural (5HMF), an allosteric effector of hemoglobin (Hb) with an ability to increase Hb affinity for oxygen has been studied extensively for its antisickling effect in vitro and in vivo, and in humans for the treatment of sickle cell disease (SCD). One of the downstream pathophysiologies of SCD is nitric oxide (NO) deficiency, therefore increasing NO (bio)availability is known to mitigate the severity of SCD symptoms. We report the synthesis of an NO-releasing prodrug of 5HMF (5HMF-NO), which in vivo, is expected to be bio-transformed into 5HMF and NO, with concomitant therapeutic activities. In vitro studies showed that when incubated with whole blood, 5HMF-NO releases NO, as anticipated. When incubated with sickle blood, 5HMF-NO formed Schiff base adduct with Hb, increased Hb affinity for oxygen, and prevented hypoxia-induced erythrocyte sickling, which at 1 mM concentration were 16%, 10% and 27%, respectively, compared to 21%, 18% and 21% for 5HMF. Crystal structures of 5HMF-NO with Hb showed 5HMF-NO bound to unliganded (deoxygenated) Hb, while the hydrolyzed product, 5HMF bound to liganded (carbonmonoxy-ligated) Hb. Our findings from this proof-of-concept study suggest that the incorporation of NO donor group to 5HMF and analogous molecules could be a novel beneficial strategy to treat SCD and warrants further detailed in vivo studies. Full article

► Show Figures

Figure 1

20 pages, 2779 KiB

Open AccessEditor’s ChoiceArticle

Randomizing of Oligopeptide Conformations by Nearest Neighbor Interactions between Amino Acid Residues

by Reinhard Schweitzer-Stenner, Bridget Milorey and Harald Schwalbe

Biomolecules 2022, 12(5), 684; https://doi.org/10.3390/biom12050684 - 11 May 2022

Cited by 4 | Viewed by 2068

Abstract

Flory’s random coil model assumes that conformational fluctuations of amino acid residues in unfolded poly(oligo)peptides and proteins are uncorrelated (isolated pair hypothesis, IPH). This implies that conformational energies, entropies and solvation free energies are all additive. Nearly 25 years ago, analyses of coil [...] Read more.

Flory’s random coil model assumes that conformational fluctuations of amino acid residues in unfolded poly(oligo)peptides and proteins are uncorrelated (isolated pair hypothesis, IPH). This implies that conformational energies, entropies and solvation free energies are all additive. Nearly 25 years ago, analyses of coil libraries cast some doubt on this notion, in that they revealed that aromatic, but also β-branched side chains, could change the ³J(H^NH^Cα) coupling of their neighbors. Since then, multiple bioinformatical, computational and experimental studies have revealed that conformational propensities of amino acids in unfolded peptides and proteins depend on their nearest neighbors. We used recently reported and newly obtained Ramachandran plots of tetra- and pentapeptides with non-terminal homo- and heterosequences of amino acid residues to quantitatively determine nearest neighbor coupling between them with a Ising type model. Results reveal that, depending on the choice of amino acid residue pairs, nearest neighbor interactions either stabilize or destabilize pairs of polyproline II and β-strand conformations. This leads to a redistribution of population between these conformations and a reduction in conformational entropy. Interactions between residues in polyproline II and turn(helix)-forming conformations seem to be cooperative in most cases, but the respective interaction parameters are subject to large statistical errors. Full article

(This article belongs to the Collection Feature Papers in Molecular Structure and Dynamics)

► Show Figures

Figure 1

Journal Menu

Journal Browser

Editor’s Choice Articles

Article

Further Information

Guidelines

MDPI Initiatives

Follow MDPI