Biomolecules

17 pages, 2885 KiB

Open AccessArticle

Isoallopregnanolone Inhibits Estrus Cycle-Dependent Aggressive Behavior

by Torbjörn Bäckström, Sara K. S. Bengtsson, Jessica Sjöstedt, Evgenya Malinina, Maja Johansson, Gianna Ragagnin, Karin Ekberg and Per Lundgren

Biomolecules 2023, 13(6), 1017; https://doi.org/10.3390/biom13061017 - 20 Jun 2023

Cited by 3 | Viewed by 1769

Abstract

Among female rats, some individuals show estrus cycle-dependent irritability/aggressive behaviors, and these individual rats may be used as a model for premenstrual dysphoric disorder (PMDD). We wanted to investigate if these behaviors are related to the estrus cycle phase containing moderately increased levels [...] Read more.

Among female rats, some individuals show estrus cycle-dependent irritability/aggressive behaviors, and these individual rats may be used as a model for premenstrual dysphoric disorder (PMDD). We wanted to investigate if these behaviors are related to the estrus cycle phase containing moderately increased levels of positive GABA-A receptor-modulating steroids (steroid-PAM), especially allopregnanolone (ALLO), and if the adverse behavior can be antagonized. The electrophysiology studies in this paper show that isoallopregnanolone (ISO) is a GABA-A-modulating steroid antagonist (GAMSA), meaning that ISO can antagonize the agonistic effects of positive GABA-A receptor-modulating steroids in both α1β2γ2L and α4β3δ GABA-A receptor subtypes. In this study, we also investigated whether ISO could antagonize the estrus cycle-dependent aggressive behaviors in female Wistar rats using a resident–intruder test. Our results confirmed previous reports of estrus cycle-dependent behaviors in that 42% of the tested rats showed higher levels of irritability/aggression at diestrus compared to those at estrus. Furthermore, we found that, during the treatment with ISO, the aggressive behavior at diestrus was alleviated to a level comparable to that of estrus. We noticed an 89% reduction in the increase in aggressive behavior at diestrus compared to that at estrus. Vehicle treatment in the same animals showed a minimal effect on the diestrus-related aggressive behavior. In conclusion, we showed that ISO can antagonize Steroid-PAM both in α1β2γ2L and α4β3δ GABA-A receptor subtypes and inhibit estrus cycle-dependent aggressive behavior. Full article

(This article belongs to the Special Issue Role of Neuroactive Steroids in Health and Disease)

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19 pages, 2331 KiB

Open AccessReview

Flexible Attachment and Detachment of Centromeres and Telomeres to and from Chromosomes

by Riku Kuse and Kojiro Ishii

Biomolecules 2023, 13(6), 1016; https://doi.org/10.3390/biom13061016 - 20 Jun 2023

Viewed by 1935

Abstract

Accurate transmission of genomic information across multiple cell divisions and generations, without any losses or errors, is fundamental to all living organisms. To achieve this goal, eukaryotes devised chromosomes. Eukaryotic genomes are represented by multiple linear chromosomes in the nucleus, each carrying a [...] Read more.

Accurate transmission of genomic information across multiple cell divisions and generations, without any losses or errors, is fundamental to all living organisms. To achieve this goal, eukaryotes devised chromosomes. Eukaryotic genomes are represented by multiple linear chromosomes in the nucleus, each carrying a centromere in the middle, a telomere at both ends, and multiple origins of replication along the chromosome arms. Although all three of these DNA elements are indispensable for chromosome function, centromeres and telomeres possess the potential to detach from the original chromosome and attach to new chromosomal positions, as evident from the events of telomere fusion, centromere inactivation, telomere healing, and neocentromere formation. These events seem to occur spontaneously in nature but have not yet been elucidated clearly, because they are relatively infrequent and sometimes detrimental. To address this issue, experimental setups have been developed using model organisms such as yeast. In this article, we review some of the key experiments that provide clues as to the extent to which these paradoxical and elusive features of chromosomally indispensable elements may become valuable in the natural context. Full article

(This article belongs to the Special Issue Yeast Models for Gene Regulation)

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8 pages, 793 KiB

Open AccessEditorial

Per Aspera ad Chaos: Vladimir Uversky’s Odyssey through the Strange World of Intrinsically Disordered Proteins

by Prakash Kulkarni, Stefania Brocca, A. Keith Dunker and Sonia Longhi

Biomolecules 2023, 13(6), 1015; https://doi.org/10.3390/biom13061015 - 19 Jun 2023

Viewed by 1891

Abstract

Until the late 1990s, we believed that protein function required a unique, well-defined 3D structure encrypted in the amino acid sequence [...] 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)

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13 pages, 3491 KiB

Open AccessArticle

Identification of a Favorable Prognostic Subgroup in Oral Squamous Cell Carcinoma: Characterization of ITGB4/PD-L1^high with CD8/PD-1^high

by Si-Rui Ma, Jian-Feng Liu, Rong Jia, Wei-Wei Deng and Jun Jia

Biomolecules 2023, 13(6), 1014; https://doi.org/10.3390/biom13061014 - 19 Jun 2023

Cited by 4 | Viewed by 1837

Abstract

Integrin β4 (ITGB4) is a member of the integrin family, which plays a crucial role in mediating cell adhesion to the extracellular matrix. Recent studies have demonstrated that ITGB4 is involved in tumorigenesis and metastasis during the development of cancer. However, the role [...] Read more.

Integrin β4 (ITGB4) is a member of the integrin family, which plays a crucial role in mediating cell adhesion to the extracellular matrix. Recent studies have demonstrated that ITGB4 is involved in tumorigenesis and metastasis during the development of cancer. However, the role of ITGB4 in oral squamous cell carcinoma (OSCC) remains unclear. A Multiplex immunohistochemistry (OPAL™, mIHC) assay was employed to stain ITGB4, ALDH1, PD-L1, cytokeratin (CK), CD8 and PD-1 in a human OSCC tissue microarray, containing 26 normal oral epithelium samples, 21 oral epithelium dysplasia samples and 76 OSCC samples. The expression pattern and clinicopathological characteristics of ITGB4 were analyzed and compared with those of PD-1, PD-L1, ALDH1 and CD8. The correlation between subgroups of tumor cells, including ITGB4⁺PD-L1⁺ and ITGB4⁺ALDH1⁺, and subgroups of T cells, including CD8⁺ and CD8⁺PD-1⁺, was evaluated using two-tailed Pearson’s statistics. A Kaplan–Meier curve was built, and a log-rank test was performed to analyze the survival rate of different subgroups. The mIHC staining results show that ITGB4 was mostly expressed in the tumor cells, with a significant increase in the OSCC specimens compared with normal oral epithelium and oral epithelium dysplasia. The paired analysis, conducted between the OSCC tumor tissue and normal paracancer mucosa, confirmed the results. The study further revealed that ITGB4⁺PD-L1⁺ cancer cells, but not ITGB4⁺ALDH1⁺ cancer cells, were significantly associated with the infiltration of CD8⁺ T cells (positivity p = 0.005, positive number p = 0.03). Additionally, ITGB4⁺PD-L1⁺ tumor cells were positively correlated with CD8⁺PD-1⁺ T cells (positivity p = 0.02, positive number p = 0.03). Most intriguingly, the subgroup of ITGB4/PD-L1^high with CD8/PD-1^high displayed the best prognosis compared with the other considered subgroups. The results show that the expression of ITGB4 was increased in OSCC compared with normal oral mucosa. Furthermore, a specific subgroup with high levels of expression of ITGB4/PD-L1 and CD8/PD-1 was found to have a relatively better prognosis compared with the other subgroups. Ultimately, this study sheds light on the potential role of ITGB4 in OSCC and provides a basis for further investigation. Full article

(This article belongs to the Section Molecular Biomarkers)

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17 pages, 2255 KiB

Open AccessArticle

Monoamine Oxidase A Contributes to Serotonin—But Not Norepinephrine-Dependent Damage of Rat Ventricular Myocytes

by Jonas Knittel, Nadja Itani, Rolf Schreckenberg, Jacqueline Heger, Susanne Rohrbach, Rainer Schulz and Klaus-Dieter Schlüter

Biomolecules 2023, 13(6), 1013; https://doi.org/10.3390/biom13061013 - 19 Jun 2023

Cited by 2 | Viewed by 1926

Abstract

Serotonin effects on cardiac hypertrophy, senescence, and failure are dependent either on activation of specific receptors or serotonin uptake and serotonin degradation by monoamine oxidases (MAOs). Receptor-dependent effects are specific for serotonin, but MAO-dependent effects are nonspecific as MAOs also metabolize other substrates [...] Read more.

Serotonin effects on cardiac hypertrophy, senescence, and failure are dependent either on activation of specific receptors or serotonin uptake and serotonin degradation by monoamine oxidases (MAOs). Receptor-dependent effects are specific for serotonin, but MAO-dependent effects are nonspecific as MAOs also metabolize other substrates such as catecholamines. Our study evaluates the role of MAO-A in serotonin- and norepinephrine-dependent cell damage. Experiments were performed in vivo to study the regulation of MAOA and MAOB expression and in vitro on isolated cultured adult rat ventricular cardiomyocytes (cultured for 24 h) to study the function of MAO-A. MAOA but not MAOB expression increased in maladaptive hypertrophic stages. Serotonin and norepinephrine induced morphologic cell damage (loss of rod-shaped cell structure). However, MAO-A inhibition suppressed serotonin-dependent but not norepinephrine-dependent damages. Serotonin but not norepinephrine caused a reduction in cell shortening in nondamaged cells. Serotonin induced mitochondria-dependent oxidative stress. In vivo, MAOA was induced during aging and hypertension but the expression of the corresponding serotonin uptake receptor (SLC6A4) was reduced and enzymes that reduce either oxidative stress (CAT) or accumulation of 5-hydroxyindolacetaldehyde (ALDH2) were induced. In summary, the data show that MAO-A potentially affects cardiomyocytes’ function but that serotonin is not necessarily the native substrate. Full article

(This article belongs to the Special Issue Animal Models: Explore the Oxidative Stress Accumulation, Mitochondrial Dysfunction, and Aging Mechanism)

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16 pages, 3444 KiB

Open AccessArticle

Oligomerization and Adjuvant Activity of Peptides Derived from the VirB4-like ATPase of Clostridioides difficile

by Julya Sorokina, Irina Sokolova, Mariya Majorina, Anastasia Ungur, Vasiliy Troitskiy, Amir Tukhvatulin, Bogdan Melnik and Yury Belyi

Biomolecules 2023, 13(6), 1012; https://doi.org/10.3390/biom13061012 - 18 Jun 2023

Viewed by 1500

Abstract

In a previous study, we demonstrated that the Clostridioides difficile VirB4-like ATPase forms oligomers in vitro. In the current investigation, to study the observed phenomenon in more detail, we prepared a library of VirB4-derived peptides (delVirB4s) fused to a carrier maltose-binding protein (MBP). [...] Read more.

In a previous study, we demonstrated that the Clostridioides difficile VirB4-like ATPase forms oligomers in vitro. In the current investigation, to study the observed phenomenon in more detail, we prepared a library of VirB4-derived peptides (delVirB4s) fused to a carrier maltose-binding protein (MBP). Using gel chromatography and polyacrylamide gel electrophoresis, we found a set of overlapping fragments that contribute most significantly to protein aggregation, which were represented as water-soluble oligomers with molecular masses ranging from ~300 kD to several megadaltons. Membrane filtration experiments, sucrose gradient ultracentrifugation, and dynamic light scattering measurements indicated the size of the soluble complex to be 15–100 nm. It was sufficiently stable to withstand treatment with 1 M urea; however, it dissociated in a 6 M urea solution. As shown by the changes in GFP fluorescence and the circular dichroism spectra, the attachment of the delVirB4 peptide significantly altered the structure of the partner MBP. The immunization of mice with the hybrid consisting of the selected VirB4-derived peptide and MBP, GST, or GFP resulted in increased production of specific antibodies compared to the peptide-free carrier proteins, suggesting significant adjuvant activity of the VirB4 fragment. This feature could be useful for the development of new vaccines, especially in the case of “weak” antigens that are unable to elicit a strong immune response by themselves. Full article

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

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18 pages, 4942 KiB

Open AccessArticle

Dual-Step Controlled Release of Berberine Hydrochloride from the Trans-Scale Hybrids of Nanofibers and Microparticles

by Jianfeng Zhou, Yelin Dai, Junhao Fu, Chao Yan, Deng-Guang Yu and Tao Yi

Biomolecules 2023, 13(6), 1011; https://doi.org/10.3390/biom13061011 - 18 Jun 2023

Cited by 29 | Viewed by 2294

Abstract

In this nano era, nanomaterials and nanostructures are popular in developing novel functional materials. However, the combinations of materials at micro and macro scales can open new routes for developing novel trans-scale products with improved or even new functional performances. In this work, [...] Read more.

In this nano era, nanomaterials and nanostructures are popular in developing novel functional materials. However, the combinations of materials at micro and macro scales can open new routes for developing novel trans-scale products with improved or even new functional performances. In this work, a brand-new hybrid, containing both nanofibers and microparticles, was fabricated using a sequential electrohydrodynamic atomization (EHDA) process. Firstly, the microparticles loaded with drug (berberine hydrochloride, BH) molecules in the cellulose acetate (CA) were fabricated using a solution electrospraying process. Later, these microparticles were suspended into a co-dissolved solution that contained BH and a hydrophilic polymer (polypyrrolidone, PVP) and were co-electrospun into the nanofiber/microparticle hybrids. The EHDA processes were recorded, and the resultant trans-scale products showed a typical hybrid topography, with microparticles distributed all over the nanofibers, which was demonstrated by SEM assessments. FTIR and XRD demonstrated that the components within the hybrids were presented in an amorphous state and had fine compatibility with each other. In vitro dissolution tests verified that the hybrids were able to provide the designed dual-step drug release profiles, a combination of the fast release step of BH from the hydrophilic PVP nanofibers through an erosion mechanism and the sustained release step of BH from the insoluble CA microparticles via a typical Fickian diffusion mechanism. The present protocols pave a new way for developing trans-scale functional materials. Full article

(This article belongs to the Special Issue Novel Materials for Biomedical Applications: 2nd Edition)

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14 pages, 1619 KiB

Open AccessFeature PaperReview

Sperm and Oocyte Chromosomal Abnormalities

by Osamu Samura, Yoshiharu Nakaoka and Norio Miharu

Biomolecules 2023, 13(6), 1010; https://doi.org/10.3390/biom13061010 - 17 Jun 2023

Cited by 4 | Viewed by 4562

Abstract

Gametogenesis, the process of producing gametes, differs significantly between oocytes and sperm. Most oocytes have chromosomal aneuploidies, indicating that chromosomal aberrations in miscarried and newborn infants are of oocyte origin. Conversely, most structural anomalies are of sperm origin. A prolonged meiotic period caused [...] Read more.

Gametogenesis, the process of producing gametes, differs significantly between oocytes and sperm. Most oocytes have chromosomal aneuploidies, indicating that chromosomal aberrations in miscarried and newborn infants are of oocyte origin. Conversely, most structural anomalies are of sperm origin. A prolonged meiotic period caused by increasing female age is responsible for an increased number of chromosomal aberrations. Sperm chromosomes are difficult to analyze because they cannot be evaluated using somatic cell chromosome analysis methods. Nevertheless, researchers have developed methods for chromosome analysis of sperm using the fluorescence in situ hybridization method, hamster eggs, and mouse eggs, allowing for the cytogenetic evaluation of individual sperm. Reproductive medicine has allowed men with severe spermatogenic defects or chromosomal abnormalities to have children. However, using these techniques to achieve successful pregnancies results in higher rates of miscarriages and embryos with chromosomal abnormalities. This raises questions regarding which cases should undergo sperm chromosome analysis and how the results should be interpreted. Here, we reviewed clinical trials that have been reported on oocyte and sperm chromosome analyses. Examination of chromosomal abnormalities in gametes is critical in assisted reproductive technology. Therefore, it is necessary to continue to study the mechanism underlying gametic chromosomal abnormalities. Full article

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

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15 pages, 13255 KiB

Open AccessArticle

Three-Dimensional Histological Characterization of the Placental Vasculature Using Light Sheet Microscopy

by Lennart Freise, Rose Yinghan Behncke, Hanna Helene Allerkamp, Tim Henrik Sandermann, Ngoc Hai Chu, Eva Maria Funk, Lukas Jonathan Hondrich, Alina Riedel, Christian Witzel, Nils Rouven Hansmeier, Magdalena Danyel, Alexandra Gellhaus, Ralf Dechend and René Hägerling

Biomolecules 2023, 13(6), 1009; https://doi.org/10.3390/biom13061009 - 17 Jun 2023

Viewed by 2485

Abstract

The placenta is the first embryonic organ, representing the connection between the embryo and the mother, and is therefore necessary for the embryo’s growth and survival. To meet the ever-growing need for nutrient and gas exchange, the maternal spiral arteries undergo extensive remodeling, [...] Read more.

The placenta is the first embryonic organ, representing the connection between the embryo and the mother, and is therefore necessary for the embryo’s growth and survival. To meet the ever-growing need for nutrient and gas exchange, the maternal spiral arteries undergo extensive remodeling, thus increasing the uteroplacental blood flow by 16-fold. However, the insufficient remodeling of the spiral arteries can lead to severe pregnancy-associated disorders, including but not limited to pre-eclampsia. Insufficient endovascular trophoblast invasion plays a key role in the manifestation of pre-eclampsia; however, the underlying processes are complex and still unknown. Classical histopathology is based on two-dimensional section microscopy, which lacks a volumetric representation of the vascular remodeling process. To further characterize the uteroplacental vascularization, a detailed, non-destructive, and subcellular visualization is beneficial. In this study, we use light sheet microscopy for optical sectioning, thus establishing a method to obtain a three-dimensional visualization of the vascular system in the placenta. By introducing a volumetric visualization method of the placenta, we could establish a powerful tool to deeply investigate the heterogeneity of the spiral arteries during the remodeling process, evaluate the state-of-the-art treatment options, effects on vascularization, and, ultimately, reveal new insights into the underlying pathology of pre-eclampsia. Full article

(This article belongs to the Special Issue Lymphatic Vessel Disease - New Insights into Vascular Anatomy, Cell Biology, Diagnostics and Treatment)

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15 pages, 1888 KiB

Open AccessEditor’s ChoiceArticle

Dynamics of Urinary Extracellular DNA in Urosepsis

by Michaela Mihaľová, Nadja Šupčíková, Alexandra Gaál Kovalčíková, Ján Breza, Jr., Ľubomíra Tóthová, Peter Celec and Ján Breza, Sr.

Biomolecules 2023, 13(6), 1008; https://doi.org/10.3390/biom13061008 - 17 Jun 2023

Cited by 3 | Viewed by 2039

Abstract

Extracellular DNA (ecDNA) is a promising candidate marker for the early diagnosis and monitoring of urinary tract infections (UTIs). The aim of our study is to describe the dynamics of ecDNA in the plasma and urine of patients with urosepsis as well as [...] Read more.

Extracellular DNA (ecDNA) is a promising candidate marker for the early diagnosis and monitoring of urinary tract infections (UTIs). The aim of our study is to describe the dynamics of ecDNA in the plasma and urine of patients with urosepsis as well as in a mouse model of UTI. Samples of blood and urine were collected from adult patients with UTIs and obstructive uropathy (n = 36) during the first 3 days at the hospital and during a follow-up. Bacterial burden and urinary ecDNA were evaluated in a mouse UTI model (n = 26) at baseline; 24, 48, and 72 h after UTI induction; and 7 days after UTI induction. The plasma ecDNA did not change during urosepsis, but the plasma DNase activity increased significantly at the follow-up. The urinary ecDNA decreased significantly during hospitalization and remained low until the follow-up (90% lower vs. admission). No change was seen in the urinary DNase activity. C-reactive protein (CRP) and procalcitonin are positively correlated with plasma and urinary ecDNA. A UTI caused sepsis in 23% of mice. The urinary ecDNA decreased by three-fold and remained low until day 7 post-infection. Urinary bacterial burden is correlated with urinary ecDNA. Urinary ecDNA is a potential non-invasive marker for monitoring the effects of treatment during urosepsis and is related to UTI progression in the experimental animal model. Full article

(This article belongs to the Special Issue Biomarkers in Renal Diseases)

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31 pages, 5418 KiB

Open AccessReview

Vibrational Spectroscopy of Phytochromes

by Peter Hildebrandt

Biomolecules 2023, 13(6), 1007; https://doi.org/10.3390/biom13061007 - 17 Jun 2023

Cited by 4 | Viewed by 2220

Abstract

Phytochromes are biological photoswitches that translate light into physiological functions. Spectroscopic techniques are essential tools for molecular research into these photoreceptors. This review is directed at summarizing how resonance Raman and IR spectroscopy contributed to an understanding of the structure, dynamics, and reaction [...] Read more.

Phytochromes are biological photoswitches that translate light into physiological functions. Spectroscopic techniques are essential tools for molecular research into these photoreceptors. This review is directed at summarizing how resonance Raman and IR spectroscopy contributed to an understanding of the structure, dynamics, and reaction mechanism of phytochromes, outlining the substantial experimental and theoretical challenges and describing the strategies to master them. It is shown that the potential of the various vibrational spectroscopic techniques can be most efficiently exploited using integral approaches via a combination of theoretical methods as well as other experimental techniques. Full article

(This article belongs to the Special Issue New Advances in Phytochromes)

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33 pages, 1676 KiB

Open AccessReview

The Role of Trace Elements and Minerals in Osteoporosis: A Review of Epidemiological and Laboratory Findings

by Anatoly V. Skalny, Michael Aschner, Ekaterina V. Silina, Victor A. Stupin, Oleg N. Zaitsev, Tatiana I. Sotnikova, Serafima Ia. Tazina, Feng Zhang, Xiong Guo and Alexey A. Tinkov

Biomolecules 2023, 13(6), 1006; https://doi.org/10.3390/biom13061006 - 17 Jun 2023

Cited by 15 | Viewed by 4729

Abstract

The objective of the present study was to review recent epidemiological and clinical data on the association between selected minerals and trace elements and osteoporosis, as well as to discuss the molecular mechanisms underlying these associations. We have performed a search in the [...] Read more.

The objective of the present study was to review recent epidemiological and clinical data on the association between selected minerals and trace elements and osteoporosis, as well as to discuss the molecular mechanisms underlying these associations. We have performed a search in the PubMed-Medline and Google Scholar databases using the MeSH terms “osteoporosis”, “osteogenesis”, “osteoblast”, “osteoclast”, and “osteocyte” in association with the names of particular trace elements and minerals through 21 March 2023. The data demonstrate that physiological and nutritional levels of trace elements and minerals promote osteogenic differentiation through the up-regulation of BMP-2 and Wnt/β-catenin signaling, as well as other pathways. miRNA and epigenetic effects were also involved in the regulation of the osteogenic effects of trace minerals. The antiresorptive effect of trace elements and minerals was associated with the inhibition of osteoclastogenesis. At the same time, the effect of trace elements and minerals on bone health appeared to be dose-dependent with low doses promoting an osteogenic effect, whereas high doses exerted opposite effects which promoted bone resorption and impaired bone formation. Concomitant with the results of the laboratory studies, several clinical trials and epidemiological studies demonstrated that supplementation with Zn, Mg, F, and Sr may improve bone quality, thus inducing antiosteoporotic effects. Full article

(This article belongs to the Special Issue Biochemistry in Medicine—Honorary Special Issue Commemorating Research and Work of Professors Jan O. Aaseth and Jan Alexander)

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13 pages, 870 KiB

Open AccessReview

β3 Receptor Signaling in Pregnant Human Myometrium Suggests a Role for β3 Agonists as Tocolytics

by Iain L. O. Buxton, Hazik Asif and Scott D. Barnett

Biomolecules 2023, 13(6), 1005; https://doi.org/10.3390/biom13061005 - 17 Jun 2023

Cited by 3 | Viewed by 2080

Abstract

Preterm labor leading to preterm birth is the leading cause of infant morbidity and mortality. At the present time, nothing can reliably halt labor once it begins. The knowledge that agonists of the β2 adrenergic receptor relax airway smooth muscle and are effective [...] Read more.

Preterm labor leading to preterm birth is the leading cause of infant morbidity and mortality. At the present time, nothing can reliably halt labor once it begins. The knowledge that agonists of the β2 adrenergic receptor relax airway smooth muscle and are effective in the treatment of asthma led to the notion that β2 mimetics would prevent preterm birth by relaxing uterine smooth muscle. The activation of cAMP-dependent protein kinase by β2 receptors is unable to provide meaningful tocolysis. The failure of β2 agonists such as ritodrine and terbutaline to prevent preterm birth suggests that the regulation of uterine smooth muscle is disparate from that of airway. Other smooth muscle quiescent-mediating molecules, such as nitric oxide, relax vascular smooth muscle in a cGMP-protein kinase G-dependent manner; however, nitric oxide activation of protein kinase G fails to explain the relaxation of the myometrium to nitric oxide. Moreover, nitric oxide-mediated relaxation is blunted in preterm labor, and thus, for this reason and because of the fall in maternal blood pressure, nitric oxide cannot be employed as a tocolytic. The β3 adrenergic receptor-mediated relaxation of the human myometrium is claimed to be cAMP-dependent protein kinase-dependent. This is scientifically displeasing given the failure of β2 agonists as tocolytics and suggests a non-canonical signaling role for β3AR in myometrium. The addition of the β3 agonist mirabegron to pregnant human myometrial strips in the tissue bath relaxes oxytocin-induced contractions. Mirabegron stimulates nitric oxide production in myometrial microvascular endothelial cells, and the relaxation of uterine tissue in vitro is partially blocked by the addition of the endothelial nitric oxide synthase blocker Nω-Nitro-L-arginine. Recent data suggest that both endothelial and smooth muscle cells respond to β3 stimulation and contribute to relaxation through disparate signaling pathways. The repurposing of approved medications such as mirabegron (Mybetriq™) tested in human myometrium as uterine tocolytics can advance the prevention of preterm birth. Full article

(This article belongs to the Special Issue Advances in β3-Adrenoceptor)

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17 pages, 1356 KiB

Open AccessEditor’s ChoiceReview

Aldosterone: Essential for Life but Damaging to the Vascular Endothelium

by Michael Crompton, Laura J. Skinner, Simon C. Satchell and Matthew J. Butler

Biomolecules 2023, 13(6), 1004; https://doi.org/10.3390/biom13061004 - 17 Jun 2023

Cited by 10 | Viewed by 4790

Abstract

The renin angiotensin aldosterone system is a key regulator of blood pressure. Aldosterone is the final effector of this pathway, acting predominantly via mineralocorticoid receptors. Aldosterone facilitates the conservation of sodium and, with it, water and acts as a powerful stimulus for potassium [...] Read more.

The renin angiotensin aldosterone system is a key regulator of blood pressure. Aldosterone is the final effector of this pathway, acting predominantly via mineralocorticoid receptors. Aldosterone facilitates the conservation of sodium and, with it, water and acts as a powerful stimulus for potassium excretion. However, evidence for the pathological impact of excess mineralocorticoid receptor stimulation is increasing. Here, we discussed how in the heart, hyperaldosteronism is associated with fibrosis, cardiac dysfunction, and maladaptive hypertrophy. In the kidney, aldosterone was shown to cause proteinuria and fibrosis and may contribute to the progression of kidney disease. More recently, studies suggested that aldosterone excess damaged endothelial cells. Here, we reviewed how damage to the endothelial glycocalyx may contribute to this process. The endothelial glycocalyx is a heterogenous, negatively charged layer on the luminal surface of cells. Aldosterone exposure alters this layer. The resulting structural changes reduced endothelial reactivity in response to protective shear stress, altered permeability, and increased immune cell trafficking. Finally, we reviewed current therapeutic strategies for limiting endothelial damage and suggested that preventing glycocalyx remodelling in response to aldosterone exposure may provide a novel strategy, free from the serious adverse effect of hyperkalaemia seen in response to mineralocorticoid blockade. Full article

(This article belongs to the Special Issue Molecular Aspect of Cardiovascular Risk Factors)

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17 pages, 8841 KiB

Open AccessArticle

The Comparative Invasiveness of Endometriotic Cell Lines to Breast and Endometrial Cancer Cell Lines

by Katherine Ellis and Rachael Wood

Biomolecules 2023, 13(6), 1003; https://doi.org/10.3390/biom13061003 - 17 Jun 2023

Cited by 3 | Viewed by 2996

Abstract

Endometriosis is an invasive condition that affects 10% of women (and people assigned as female at birth) worldwide. The purpose of this study was to characterize the relative invasiveness of three available endometriotic cell lines (EEC12Z, iEc-ESCs, tHESCs) to cancer cell lines (MDA-MB-231, [...] Read more.

Endometriosis is an invasive condition that affects 10% of women (and people assigned as female at birth) worldwide. The purpose of this study was to characterize the relative invasiveness of three available endometriotic cell lines (EEC12Z, iEc-ESCs, tHESCs) to cancer cell lines (MDA-MB-231, SW1353 and EM-E6/E7/TERT) and assess whether the relative invasiveness was consistent across different invasion assays. All cell lines were subjected to transwell, spheroid drop, and spheroid-gel invasion assays, and stained for vimentin, cytokeratin, E-Cadherin and N-Cadherin to assess changes in expression. In all assays, endometriotic cell lines showed comparable invasiveness to the cancer cell lines used in this study, with no significant differences in invasiveness identified. EEC12Z cells that had invaded within the assay periods showed declines in E-Cadherin expression compared to cells that had not invaded within the assay period, without significant changes in N-Cadherin expression, which may support the hypothesis that an epithelial-to-mesenchymal transition is an influence on the invasiveness shown by this peritoneal endometriosis cell line. Full article

(This article belongs to the Special Issue Molecular and Cell Biology in Endometriosis and Endometrial Cancer)

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18 pages, 5385 KiB

Open AccessReview

Emerging Extracellular Molecular Targets for Innovative Pharmacological Approaches to Resistant Mtb Infection

by Alice Italia, Mohammed Monsoor Shaik and Francesco Peri

Biomolecules 2023, 13(6), 999; https://doi.org/10.3390/biom13060999 - 16 Jun 2023

Cited by 4 | Viewed by 2229

Abstract

Emerging pharmacological strategies that target major virulence factors of antibiotic-resistant Mycobacterium tuberculosis (Mtb) are presented and discussed. This review is divided into three parts corresponding to structures and functions important for Mtb pathogenicity: the cell wall, the lipoarabinomannan, and the secretory proteins. Within [...] Read more.

Emerging pharmacological strategies that target major virulence factors of antibiotic-resistant Mycobacterium tuberculosis (Mtb) are presented and discussed. This review is divided into three parts corresponding to structures and functions important for Mtb pathogenicity: the cell wall, the lipoarabinomannan, and the secretory proteins. Within the cell wall, we further focus on three biopolymeric sub-components: mycolic acids, arabinogalactan, and peptidoglycan. We present a comprehensive overview of drugs and drug candidates that target cell walls, envelopes, and secretory systems. An understanding at a molecular level of Mtb pathogenesis is provided, and potential future directions in therapeutic strategies are suggested to access new drugs to combat the growing global threat of antibiotic-resistant Mtb infection. Full article

(This article belongs to the Special Issue Virulence Factors in Mycobacterium tuberculosis Infection: Structural and Functional Studies)

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23 pages, 2364 KiB

Open AccessReview

Cell-Type-Specific Neuroproteomics of Synapses

by Yun Young Yim and Eric J. Nestler

Biomolecules 2023, 13(6), 998; https://doi.org/10.3390/biom13060998 - 16 Jun 2023

Cited by 1 | Viewed by 3480

Abstract

In the last two decades, our knowledge of synaptic proteomes and their relationship to normal brain function and neuropsychiatric disorders has been expanding rapidly through the use of more powerful neuroproteomic approaches. However, mass spectrometry (MS)-based neuroproteomic studies of synapses still require cell-type, [...] Read more.

In the last two decades, our knowledge of synaptic proteomes and their relationship to normal brain function and neuropsychiatric disorders has been expanding rapidly through the use of more powerful neuroproteomic approaches. However, mass spectrometry (MS)-based neuroproteomic studies of synapses still require cell-type, spatial, and temporal proteome information. With the advancement of sample preparation and MS techniques, we have just begun to identify and understand proteomes within a given cell type, subcellular compartment, and cell-type-specific synapse. Here, we review the progress and limitations of MS-based neuroproteomics of synapses in the mammalian CNS and highlight the recent applications of these approaches in studying neuropsychiatric disorders such as major depressive disorder and substance use disorders. Combining neuroproteomic findings with other omics studies can generate an in-depth, comprehensive map of synaptic proteomes and possibly identify new therapeutic targets and biomarkers for several central nervous system disorders. Full article

(This article belongs to the Special Issue Advances in Neuroproteomics)

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14 pages, 1992 KiB

Open AccessArticle

Identification of the Stapled α-Helical Peptide ATSP-7041 as a Substrate and Strong Inhibitor of OATP1B1 In Vitro

by Rika Ishikawa, Kosuke Saito, Takashi Misawa, Yosuke Demizu and Yoshiro Saito

Biomolecules 2023, 13(6), 1002; https://doi.org/10.3390/biom13061002 - 16 Jun 2023

Viewed by 2033

Abstract

ATSP-7041, a stapled α-helical peptide that inhibits murine double minute-2 (MDM2) and MDMX activities, is a promising modality targeting protein–protein interactions. As peptides of molecular weights over 1000 Da are not usually evaluated, data on the drug–drug interaction (DDI) potential of stapled α-helical [...] Read more.

ATSP-7041, a stapled α-helical peptide that inhibits murine double minute-2 (MDM2) and MDMX activities, is a promising modality targeting protein–protein interactions. As peptides of molecular weights over 1000 Da are not usually evaluated, data on the drug–drug interaction (DDI) potential of stapled α-helical peptides remain scarce. Here, we evaluate the interaction of ATSP-7041 with hepatic cytochrome P450s (CYPs; CYP1A2, CYP2C9, CYP2C19, CYP3A4, and CYP2D6) and transporters (organic anion transporting polypeptides (OATPs; OATP1B1 and OATP1B3), P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP)). ATSP-7041 demonstrated negligible metabolism in human liver S9 fraction and a limited inhibition of CYP activities in yeast microsomes or S9 fractions. On the contrary, a substantial uptake by OATPs in HEK 293 cells, a strong inhibition of OATP activities in the cells, and an inhibition of P-gp and BCRP activities in reversed membrane vesicles were observed for ATSP-7041. A recent report describes that ALRN-6924, an ATSP-7041 analog, inhibited OATP activities in vivo; therefore, we focused on the interaction between ATSP-7041 and OATP1B1 to demonstrate that ATSP-7041, as a higher molecular weight stapled peptide, is a substrate and strong inhibitor of OATP1B1 activity. Our findings demonstrated the possibility of transporter-mediated DDI potential by high molecular weight stapled peptides and the necessity of their evaluation for drug development. Full article

(This article belongs to the Collection Peptides, Proteins and Their Derivatives with Therapeutic Applications)

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13 pages, 2043 KiB

Open AccessArticle

OTUD7B Activates Wnt Signaling Pathway through the Interaction with LEF1

by Yuri Lee, Hai-long Piao and Jongchan Kim

Biomolecules 2023, 13(6), 1001; https://doi.org/10.3390/biom13061001 - 16 Jun 2023

Cited by 1 | Viewed by 1872

Abstract

The Wnt signaling pathway plays a critical role in regulating normal cellular processes, including proliferation, differentiation, and apoptosis. Dysregulation of Wnt signaling has been implicated in various human diseases, including cancer. β-catenin and LEF1 are key mediators of Wnt signaling, and their dysregulation [...] Read more.

The Wnt signaling pathway plays a critical role in regulating normal cellular processes, including proliferation, differentiation, and apoptosis. Dysregulation of Wnt signaling has been implicated in various human diseases, including cancer. β-catenin and LEF1 are key mediators of Wnt signaling, and their dysregulation is a hallmark of many cancer types. In this study, we aimed to identify the deubiquitinases (DUBs) that regulate the Wnt signaling pathway through the essential component LEF1. Screening candidate DUBs from the human DUB library, we discovered that OTUD7B interacts with LEF1 and activates Wnt signaling. OTUD7B and LEF1 interact with each other through the UBA and HMG domains, respectively. Furthermore, OTUD7B promotes the nuclear localization of LEF1, leading to an increased interaction with β-catenin in the nucleus while not noticeably affecting ubiquitination on LEF1. Using qPCR array analysis, we found that OTUD7B overexpression leads to an upregulation of 75% of the tested Wnt target genes compared to the control. These findings suggest that OTUD7B may serve as a potential therapeutic target in human diseases, including cancers where Wnt signaling is frequently dysregulated. Full article

(This article belongs to the Special Issue Deubiquitinating Enzymes in Health and Disease)

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18 pages, 8874 KiB

Open AccessArticle

Efflux Pump-Binding 4(3-Aminocyclobutyl)Pyrimidin-2-Amines Are Colloidal Aggregators

by Tania Szal, Shweta Singh Chauhan, Philipp Lewe, Fatima-Zahra Rachad, Marina Madre, Laura Paunina, Susanne Witt, Ramakrishnan Parthasarathi and Björn Windshügel

Biomolecules 2023, 13(6), 1000; https://doi.org/10.3390/biom13061000 - 16 Jun 2023

Cited by 1 | Viewed by 2282

Abstract

Efflux pumps are a relevant factor in antimicrobial resistance. In E. coli, the tripartite efflux pump AcrAB-TolC removes a chemically diverse set of antibiotics from the bacterium. Therefore, small molecules interfering with efflux pump function are considered adjuvants for improving antimicrobial therapies. [...] Read more.

Efflux pumps are a relevant factor in antimicrobial resistance. In E. coli, the tripartite efflux pump AcrAB-TolC removes a chemically diverse set of antibiotics from the bacterium. Therefore, small molecules interfering with efflux pump function are considered adjuvants for improving antimicrobial therapies. Several compounds targeting the periplasmic adapter protein AcrA and the efflux pump AcrB have been identified to act synergistically with different antibiotics. Among those, several 4(3-aminocyclobutyl)pyrimidin-2-amines have been shown to bind to both proteins. In this study, we intended to identify analogs of these substances with improved binding affinity to AcrA using virtual screening followed by experimental validation. While we succeeded in identifying several compounds showing a synergistic effect with erythromycin on E. coli, biophysical studies suggested that 4(3-aminocyclobutyl)pyrimidin-2-amines form colloidal aggregates that do not bind specifically to AcrA. Therefore, these substances are not suited for further development. Our study emphasizes the importance of implementing additional control experiments to identify aggregators among bioactive compounds. Full article

(This article belongs to the Section Molecular Biophysics)

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27 pages, 2635 KiB

Open AccessEditor’s ChoiceReview

Deciphering Plant-Insect-Microorganism Signals for Sustainable Crop Production

by Gareth Thomas, Quint Rusman, William R. Morrison III, Diego M. Magalhães, Jordan A. Dowell, Esther Ngumbi, Jonathan Osei-Owusu, Jessica Kansman, Alexander Gaffke, Kamala Jayanthi Pagadala Damodaram, Seong Jong Kim and Nurhayat Tabanca

Biomolecules 2023, 13(6), 997; https://doi.org/10.3390/biom13060997 - 15 Jun 2023

Cited by 11 | Viewed by 7730

Abstract

Agricultural crop productivity relies on the application of chemical pesticides to reduce pest and pathogen damage. However, chemical pesticides also pose a range of ecological, environmental and economic penalties. This includes the development of pesticide resistance by insect pests and pathogens, rendering pesticides [...] Read more.

Agricultural crop productivity relies on the application of chemical pesticides to reduce pest and pathogen damage. However, chemical pesticides also pose a range of ecological, environmental and economic penalties. This includes the development of pesticide resistance by insect pests and pathogens, rendering pesticides less effective. Alternative sustainable crop protection tools should therefore be considered. Semiochemicals are signalling molecules produced by organisms, including plants, microbes, and animals, which cause behavioural or developmental changes in receiving organisms. Manipulating semiochemicals could provide a more sustainable approach to the management of insect pests and pathogens across crops. Here, we review the role of semiochemicals in the interaction between plants, insects and microbes, including examples of how they have been applied to agricultural systems. We highlight future research priorities to be considered for semiochemicals to be credible alternatives to the application of chemical pesticides. Full article

(This article belongs to the Topic Advances in Chemical Ecology)

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27 pages, 8490 KiB

Open AccessEditor’s ChoiceArticle

The Ubiquitin-Proteasome System Participates in Sperm Surface Subproteome Remodeling during Boar Sperm Capacitation

by Michal Zigo, Karl Kerns and Peter Sutovsky

Biomolecules 2023, 13(6), 996; https://doi.org/10.3390/biom13060996 - 15 Jun 2023

Cited by 4 | Viewed by 3192

Abstract

Sperm capacitation is a complex process endowing biological and biochemical changes to a spermatozoon for a successful encounter with an oocyte. The present study focused on the role of the ubiquitin–proteasome system (UPS) in the remodeling of the sperm surface subproteome. The sperm [...] Read more.

Sperm capacitation is a complex process endowing biological and biochemical changes to a spermatozoon for a successful encounter with an oocyte. The present study focused on the role of the ubiquitin–proteasome system (UPS) in the remodeling of the sperm surface subproteome. The sperm surface subproteome from non-capacitated and in vitro capacitated (IVC) porcine spermatozoa, with and without proteasomal inhibition, was selectively isolated. The purified sperm surface subproteome was analyzed using high-resolution, quantitative liquid chromatography–mass spectrometry (LC-MS) in four replicates. We identified 1680 HUGO annotated proteins, out of which we found 91 to be at least 1.5× less abundant (p < 0.05) and 141 to be at least 1.5× more abundant (p < 0.05) on the surface of IVC spermatozoa. These proteins were associated with sperm capacitation, hyperactivation, metabolism, acrosomal exocytosis, and fertilization. Abundances of 14 proteins were found to be significantly different (p < 0.05), exceeding a 1.5-fold abundance between the proteasomally inhibited (100 µM MG132) and vehicle control (0.2% ethanol) groups. The proteins NIF3L1, CSE1L, NDUFB7, PGLS, PPP4C, STK39, and TPRG1L were found to be more abundant; while BPHL, GSN, GSPT1, PFDN4, STYXL1, TIMM10, and UBXN4 were found to be less abundant in proteasomally inhibited IVC spermatozoa. Despite the UPS having a narrow range of targets, it modulated sperm metabolism and binding by regulating susceptible surface proteins. Changes in CSE1L, PFDN4, and STK39 during in vitro capacitation were confirmed using immunocytochemistry, image-based flow cytometry, and Western blotting. The results confirmed the active participation of the UPS in the extensive sperm surface proteome remodeling that occurs during boar sperm capacitation. This work will help us to identify new pharmacological mechanisms to positively or negatively modulate sperm fertilizing ability in food animals and humans. Full article

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

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22 pages, 5410 KiB

Open AccessArticle

Blood Biomarkers in Takotsubo Syndrome Point to an Emerging Role for Inflammaging in Endothelial Pathophysiology

by Michiaki Nagai, Sergey Shityakov, Manuel Smetak, Hannah Jill Hunkler, Christian Bär, Nicolas Schlegel, Thomas Thum and Carola Yvette Förster

Biomolecules 2023, 13(6), 995; https://doi.org/10.3390/biom13060995 - 15 Jun 2023

Cited by 1 | Viewed by 2543

Abstract

Takotsubo syndrome (TTS), an acute cardiac condition characterized by transient wall motion abnormalities mostly of the left ventricle, results in difficulties in diagnosing patients. We set out to present a detailed blood analysis of TTS patients analyzing novel markers to understand the development [...] Read more.

Takotsubo syndrome (TTS), an acute cardiac condition characterized by transient wall motion abnormalities mostly of the left ventricle, results in difficulties in diagnosing patients. We set out to present a detailed blood analysis of TTS patients analyzing novel markers to understand the development of TTS. Significant differences in proinflammatory cytokine expression patterns and sex steroid and glucocorticoid receptor (GR) expression levels were observed in the TTS patient collected. Remarkably, the measured catecholamine serum concentrations determined from TTS patient blood could be shown to be two orders of magnitude lower than the levels determined from experimentally induced TTS in laboratory animals. Consequently, the exposure of endothelial cells and cardiomyocytes in vitro to such catecholamine concentrations did not damage the cellular integrity or function of either endothelial cells forming the blood–brain barrier, endothelial cells derived from myocardium, or cardiomyocytes in vitro. Computational analysis was able to link the identified blood markers, specifically, the proinflammatory cytokines and glucocorticoid receptor GR to microRNA (miR) relevant in the ontogeny of TTS (miR-15) and inflammation (miR-21, miR-146a), respectively. Amongst the well-described risk factors of TTS (older age, female sex), inflammaging-related pathways were identified to add to these relevant risk factors or prediagnostic markers of TTS. Full article

(This article belongs to the Special Issue Regulation of the Endothelial Cell Barrier)

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41 pages, 4112 KiB

Open AccessReview

(Patho)Physiology of Glycosylphosphatidylinositol-Anchored Proteins II: Intercellular Transfer of Matter (Inheritance?) That Matters

by Günter A. Müller and Timo D. Müller

Biomolecules 2023, 13(6), 994; https://doi.org/10.3390/biom13060994 - 15 Jun 2023

Cited by 4 | Viewed by 2398

Abstract

Glycosylphosphatidylinositol (GPI)-anchored proteins (APs) are anchored at the outer leaflet of the plasma membrane (PM) bilayer by covalent linkage to a typical glycolipid and expressed in all eukaryotic organisms so far studied. Lipolytic release from PMs into extracellular compartments and intercellular transfer are [...] Read more.

Glycosylphosphatidylinositol (GPI)-anchored proteins (APs) are anchored at the outer leaflet of the plasma membrane (PM) bilayer by covalent linkage to a typical glycolipid and expressed in all eukaryotic organisms so far studied. Lipolytic release from PMs into extracellular compartments and intercellular transfer are regarded as the main (patho)physiological roles exerted by GPI-APs. The intercellular transfer of GPI-APs relies on the complete GPI anchor and is mediated by extracellular vesicles such as microvesicles and exosomes and lipid-free homo- or heteromeric aggregates, and lipoprotein-like particles such as prostasomes and surfactant-like particles, or lipid-containing micelle-like complexes. In mammalian organisms, non-vesicular transfer is controlled by the distance between donor and acceptor cells/tissues; intrinsic conditions such as age, metabolic state, and stress; extrinsic factors such as GPI-binding proteins; hormones such as insulin; and drugs such as anti-diabetic sulfonylureas. It proceeds either “directly” upon close neighborhood or contact of donor and acceptor cells or “indirectly” as a consequence of the induced lipolytic release of GPI-APs from PMs. Those displace from the serum GPI-binding proteins GPI-APs, which have retained the complete anchor, and become assembled in aggregates or micelle-like complexes. Importantly, intercellular transfer of GPI-APs has been shown to induce specific phenotypes such as stimulation of lipid and glycogen synthesis, in cultured human adipocytes, blood cells, and induced pluripotent stem cells. As a consequence, intercellular transfer of GPI-APs should be regarded as non-genetic inheritance of (acquired) features between somatic cells which is based on the biogenesis and transmission of matter such as GPI-APs and “membrane landscapes”, rather than the replication and transmission of information such as DNA. Its operation in mammalian organisms remains to be clarified. Full article

(This article belongs to the Section Biomacromolecules: Proteins)

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15 pages, 3556 KiB

Open AccessArticle

Inhibition of the Mitochondrial Carnitine/Acylcarnitine Carrier by Itaconate through Irreversible Binding to Cysteine 136: Possible Pathophysiological Implications

by Nicola Giangregorio, Annamaria Tonazzi, Lara Console, Mariafrancesca Scalise and Cesare Indiveri

Biomolecules 2023, 13(6), 993; https://doi.org/10.3390/biom13060993 - 15 Jun 2023

Cited by 3 | Viewed by 1752

Abstract

Background: The carnitine/acylcarnitine carrier (CAC) represents the route of delivering acyl moieties to the mitochondrial matrix for accomplishing the fatty acid β-oxidation. The CAC has a couple of Cys residues (C136 and C155) most reactive toward ROS and redox signaling compounds such as [...] Read more.

Background: The carnitine/acylcarnitine carrier (CAC) represents the route of delivering acyl moieties to the mitochondrial matrix for accomplishing the fatty acid β-oxidation. The CAC has a couple of Cys residues (C136 and C155) most reactive toward ROS and redox signaling compounds such as GSH, NO, and H₂S. Among physiological compounds reacting with Cys, itaconate is produced during inflammation and represents the connection between oxidative metabolism and immune responses. The possible interaction between the CAC and itaconate has been investigated. Methods: the modulatory effects of itaconate on the transport activity of the native and recombinant CAC were tested using the proteoliposome experimental model together with site-directed mutagenesis and computational analysis. Results: Itaconate reacts with the CAC causing irreversible inhibition. Dose–response experiment performed with the native and recombinant protein showed IC₅₀ for itaconate of 11 ± 4.6 mM and 8.4 ± 2.9 mM, respectively. The IC₅₀ decreased to 3.8 ± 1.0 mM by lowering the pH from pH 7.0 to pH 6.5. Inhibition kinetics revealed a non-competitive type of inhibition. C136 is the main target of itaconate, as demonstrated by the increased IC₅₀ of mutants in which this Cys was substituted by Val. The central role of C136 was confirmed by covalent docking. Administration of dimethyl itaconate to HeLa cells inhibited the CAC transport activity, suggesting that itaconate could react with the CAC also in intact cells. Full article

(This article belongs to the Special Issue Advances in Mitochondrial Transport Research)

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14 pages, 4941 KiB

Open AccessArticle

Method of Monitoring 26S Proteasome in Cells Revealed the Crucial Role of PSMA3 C-Terminus in 26S Integrity

by Shirel Steinberger, Julia Adler and Yosef Shaul

Biomolecules 2023, 13(6), 992; https://doi.org/10.3390/biom13060992 - 15 Jun 2023

Viewed by 1753

Abstract

Proteasomes critically regulate proteostasis via protein degradation. Proteasomes are multi-subunit complexes composed of the 20S proteolytic core particle (20S CP) that, in association with one or two 19S regulatory particles (19S RPs), generates the 26S proteasome, which is the major proteasomal complex in [...] Read more.

Proteasomes critically regulate proteostasis via protein degradation. Proteasomes are multi-subunit complexes composed of the 20S proteolytic core particle (20S CP) that, in association with one or two 19S regulatory particles (19S RPs), generates the 26S proteasome, which is the major proteasomal complex in cells. Native gel protocols are used to investigate the 26S/20S ratio. However, a simple method for detecting these proteasome complexes in cells is missing. To this end, using CRISPR technology, we YFP-tagged the endogenous PSMB6 (β1) gene, a 20S CP subunit, and co-tagged endogenous PSMD6 (Rpn7), a 19S RP subunit, with the mScarlet fluorescent protein. We observed the colocalization of the YFP and mScarlet fluorescent proteins in the cells, with higher nuclear accumulation. Nuclear proteasomal granules are formed under osmotic stress, and all were positive for YFP and mScarlet. Previously, we have reported that PSMD1 knockdown, one of the 19 RP subunits, gives rise to a high level of “free” 20S CPs. Intriguingly, under this condition, the 20S-YFP remained nuclear, whereas the PSMD6-mScarlet was mostly in cytoplasm, demonstrating the distinct subcellular distribution of uncapped 20S CPs. Lately, we have shown that the PSMA3 (α7) C-terminus, a 20S CP subunit, binds multiple intrinsically disordered proteins (IDPs). Remarkably, the truncation of the PSMA3 C-terminus is phenotypically reminiscent of PSMD1 knockdown. These data suggest that the PSMA3 C-terminal region is critical for 26S proteasome integrity. Full article

(This article belongs to the Section Cellular Biochemistry)

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17 pages, 5453 KiB

Open AccessArticle

High PD-L1 Expression Correlates with an Immunosuppressive Tumour Immune Microenvironment and Worse Prognosis in ALK-Rearranged Non-Small Cell Lung Cancer

by Xia Tian, Yalun Li, Qin Huang, Hao Zeng, Qi Wei and Panwen Tian

Biomolecules 2023, 13(6), 991; https://doi.org/10.3390/biom13060991 - 15 Jun 2023

Cited by 5 | Viewed by 2201

Abstract

High tumour programmed cell death-ligand 1 (PD-L1) expression is associated with poor progression-free survival (PFS) after tyrosine kinase inhibitor (TKI) therapy in ALK-rearranged non-small cell lung cancer (NSCLC). However, the characteristics of the tumour microenvironment (TME) and their prognostic values in ALK [...] Read more.

High tumour programmed cell death-ligand 1 (PD-L1) expression is associated with poor progression-free survival (PFS) after tyrosine kinase inhibitor (TKI) therapy in ALK-rearranged non-small cell lung cancer (NSCLC). However, the characteristics of the tumour microenvironment (TME) and their prognostic values in ALK-rearranged NSCLC are unknown. Here, we collected tumour tissues from pretreated ALK-rearranged NSCLC patients, immunohistochemical staining was used to assess PD-L1 expression, and tumour-infiltrating immune cells were determined via multiplex immunofluorescence staining (mIF). Our data showed that the median values of PFS for the high PD-L1 group and low PD-L1 group who received ALK-TKI treatment were 4.4 and 16.4 months, respectively (p = 0.008). The median overall survival (OS) of the two groups was 24.0 months and not reached, respectively (p = 0.021). Via univariate and multivariate analyses, a high PD-L1 expression and a worse ECOG PS were determined to be independent prognostic factors of OS (HR = 3.35, 95% CI: 1.23–9.11, p = 0.018; HR = 6.42, 95% CI: 1.45–28.44, p = 0.014, respectively). In addition, the high PD-L1 group had increased Tregs and exhausted CD8+ T cells in both the tumour and stroma (all p < 0.05). High PD-L1 expression was an adverse predictive and prognostic biomarker for ALK-rearranged NSCLC. The characteristics of the TME in patients with high PD-L1 expression were shown to have an immunosuppressive status. Full article

(This article belongs to the Special Issue The Role of Biomolecules in Tumor Microenvironment)

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11 pages, 4740 KiB

Open AccessArticle

Graphene Oxide-Coated Patterned Silk Fibroin Films Promote Cell Adhesion and Induce Cardiomyogenic Differentiation of Human Mesenchymal Stem Cells

by Jie Wang, Yi Wu, Yecheng Wang, Yajun Shuai, Zongpu Xu, Quan Wan, Yuyin Chen and Mingying Yang

Biomolecules 2023, 13(6), 990; https://doi.org/10.3390/biom13060990 - 14 Jun 2023

Cited by 4 | Viewed by 1706

Abstract

Cardiac tissue engineering is a promising strategy for the treatment of myocardial damage. Mesenchymal stem cells (MSCs) are extensively used in tissue engineering. However, transformation of MSCs into cardiac myocytes is still a challenge. Furthermore, weak adhesion of MSCs to substrates often results [...] Read more.

Cardiac tissue engineering is a promising strategy for the treatment of myocardial damage. Mesenchymal stem cells (MSCs) are extensively used in tissue engineering. However, transformation of MSCs into cardiac myocytes is still a challenge. Furthermore, weak adhesion of MSCs to substrates often results in poor cell viability. Here, we designed a composite matrix based on silk fibroin (SF) and graphene oxide (GO) for improving the cell adhesion and directing the differentiation of MSCs into cardiac myocytes. Specifically, patterned SF films were first produced by soft lithographic. After being treated by air plasma, GO nanosheets could be successfully coated on the patterned SF films to construct the desired matrix (P-GSF). The resultant P-GSF films presented a nano-topographic surface characterized by linear grooves interlaced with GO ridges. The P-GSF films exhibited high protein absorption and suitable mechanical strength. Furthermore, the P-GSF films accelerated the early cell adhesion and directed the growth orientation of MSCs. RT-PCR results and immunofluorescence imaging demonstrated that the P-GSF films significantly improved the cardiomyogenic differentiation of MSCs. This work indicates that patterned SF films coated with GO are promising matrix in the field of myocardial repair tissue engineering. Full article

(This article belongs to the Topic Development and Application of Novel Biomaterials for Tissue Engineering)

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35 pages, 16698 KiB

Open AccessArticle

Volatile Phases Derived from Serum, DC, or MLC Culture Supernatants to Deduce a VOC-Based Diagnostic Profiling Strategy for Leukemic Diseases

by Tobias Baudrexler, Tobias Boeselt, Lin Li, Sophia Bohlscheid, Ursel Boas, Christoph Schmid, Andreas Rank, Jörg Schmohl, Rembert Koczulla and Helga Maria Schmetzer

Biomolecules 2023, 13(6), 989; https://doi.org/10.3390/biom13060989 - 14 Jun 2023

Cited by 2 | Viewed by 2213

Abstract

Volatile organic compounds (VOCs) reflect the metabolism in healthy and pathological conditions, and can be collected easily in a noninvasive manner. They are directly measured using electronical nose (eNose), and may qualify as a systemic tool to monitor biomarkers related to disease. Myeloid [...] Read more.

Volatile organic compounds (VOCs) reflect the metabolism in healthy and pathological conditions, and can be collected easily in a noninvasive manner. They are directly measured using electronical nose (eNose), and may qualify as a systemic tool to monitor biomarkers related to disease. Myeloid leukemic blasts can be transformed into leukemia-derived dendritic cells (DC_leu) able to improve (anti-leukemic) immune responses. To profile immunological changes in healthy and acute myeloid leukemic (AML) patients’ ex vivo cell cultures, we correlated the cell biological data with the profiles of cell culture supernatant-derived VOCs. DC/DC_leu from leukemic or healthy whole blood (WB) were generated without (Control) or with immunomodulatory Kit M (Granulocyte macrophage-colony-stimulating-factor (GM-CSF) + prostaglandin E₁ (PGE₁)) in dendritic cell cultures (DC culture). Kit-pretreated/not pretreated WB was used to stimulate T cell-enriched immunoreactive cells in mixed lymphocyte cultures (MLC culture). Leukemia-specific adaptive and innate immune cells were detected with a degranulation assay (Deg) and an intracellular cytokine assay (InCyt). Anti-leukemic cytotoxicity was explored with a cytotoxicity fluorolysis assay (CTX). VOCs collected from serum or DC- and MLC culture supernatants (with vs. without Kit M pretreatment and before vs. after culture) were measured using eNose. Compared to the Control (without treatment), Kit M-pretreated leukemic and healthy WB gave rise to higher frequencies of mature (leukemia-derived) DC subtypes of activated and (memory) T cells after MLC. Moreover, antigen (leukemia)-specific cells of several lines (innate and adaptive immunity cells) were induced, giving rise to blast-lysing cells. The eNose could significantly distinguish between healthy and leukemic patients’ serum, DC and MLC culture supernatant-derived volatile phases and could significantly separate several supernatant (with vs. without Kit M treatment, cultured vs. uncultured)-derived VOCs within subgroups (healthy DC or leukemic DC, or healthy MLC or leukemic MLC supernatants). Interestingly, the eNose could indicate a Kit M- and culture-associated effect. The eNose may be a prospective option for the deduction of a VOC-based profiling strategy using serum or cell culture supernatants and could be a useful diagnostic tool to recognize or qualify AML disease. Full article

(This article belongs to the Section Molecular Biomarkers)

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29 pages, 4388 KiB

Open AccessReview

Pharmaceuticals Promoting Premature Termination Codon Readthrough: Progress in Development

by Shan Li, Juan Li, Wenjing Shi, Ziyan Nie, Shasha Zhang, Fengdie Ma, Jun Hu, Jianjun Chen, Peiqiang Li and Xiaodong Xie

Biomolecules 2023, 13(6), 988; https://doi.org/10.3390/biom13060988 - 14 Jun 2023

Cited by 9 | Viewed by 4059

Abstract

Around 11% of all known gene lesions causing human genetic diseases are nonsense mutations that introduce a premature stop codon (PTC) into the protein-coding gene sequence. Drug-induced PTC readthrough is a promising therapeutic strategy for treating hereditary diseases caused by nonsense mutations. To [...] Read more.

Around 11% of all known gene lesions causing human genetic diseases are nonsense mutations that introduce a premature stop codon (PTC) into the protein-coding gene sequence. Drug-induced PTC readthrough is a promising therapeutic strategy for treating hereditary diseases caused by nonsense mutations. To date, it has been found that more than 50 small-molecular compounds can promote PTC readthrough, known as translational readthrough-inducing drugs (TRIDs), and can be divided into two major categories: aminoglycosides and non-aminoglycosides. This review summarizes the pharmacodynamics and clinical application potential of the main TRIDs discovered so far, especially some newly discovered TRIDs in the past decade. The discovery of these TRIDs brings hope for treating nonsense mutations in various genetic diseases. Further research is still needed to deeply understand the mechanism of eukaryotic cell termination and drug-induced PTC readthrough so that patients can achieve the greatest benefit from the various TRID treatments. Full article

(This article belongs to the Section Molecular Genetics)

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