Biomolecules, Volume 14, Issue 1 (January 2024) – 138 articles

The intracellular and intercellular flux of calcium ions represents an ancient and universal mode of signaling that regulates an extensive array of cellular processes. Evidence for the central role of calcium signaling includes various techniques that allow the visualization of calcium activity in living cells. While extensively investigated in mature cells, calcium activity is equally important in developing cells, particularly the embryonic nervous system where it has been implicated in a wide variety array of determinative events. However, unlike in mature cells, where the calcium dynamics display regular, predictable patterns, calcium activity in developing systems is far more sporadic, irregular, and diverse. This renders the ability to assess calcium activity in a consistent manner extremely challenging, challenges reflected in the diversity of methods employed to analyze calcium activity in neural development. Here we review the wide array of calcium detection and analysis methods used across studies, limiting the extent to which they can be comparatively analyzed. The goal is to provide investigators not only with an overview of calcium activity analysis techniques currently available, but also to offer suggestions for future work and standardization to enable informative comparative evaluations of this fundamental and important process in neural development. Full article

Kidney fibrosis, diffused into the interstitium, vessels, and glomerulus, is the main pathologic feature associated with loss of renal function and chronic kidney disease (CKD). Fibrosis may be triggered in kidney diseases by different genetic and molecular insults. However, several studies have shown that fibrosis can be linked to oxidative stress and mitochondrial dysfunction in CKD. In this review, we will focus on three pathways that link oxidative stress and kidney fibrosis, namely: (i) hyperglycemia and mitochondrial energy imbalance, (ii) the mineralocorticoid signaling pathway, and (iii) the hypoxia-inducible factor (HIF) pathway. We selected these pathways because they are targeted by available medications capable of reducing kidney fibrosis, such as sodium-glucose cotransporter-2 (SGLT2) inhibitors, non-steroidal mineralocorticoid receptor antagonists (MRAs), and HIF-1alpha-prolyl hydroxylase inhibitors. These drugs have shown a reduction in oxidative stress in the kidney and a reduced collagen deposition across different CKD subtypes. However, there is still a long and winding road to a clear understanding of the anti-fibrotic effects of these compounds in humans, due to the inherent practical and ethical difficulties in obtaining sequential kidney biopsies and the lack of specific fibrosis biomarkers measurable in easily accessible matrices like urine. In this narrative review, we will describe these three pathways, their interconnections, and their link to and activity in oxidative stress and kidney fibrosis. Full article

Apoptosis, or programmed cell death, is a fundamental process that maintains tissue homeostasis, eliminates damaged or infected cells, and plays a crucial role in various biological phenomena. The deregulation of apoptosis is involved in many human diseases, including cancer. One of the emerging players in the intricate regulatory network of apoptosis is apoptosis inhibitor 5 (API5), also called AAC-11 (anti-apoptosis clone 11) or FIF (fibroblast growth factor-2 interacting factor). While it may not have yet the same level of notoriety as some other cancer-associated proteins, API5 has garnered increasing attention in the cancer field in recent years, as elevated API5 levels are often associated with aggressive tumor behavior, resistance to therapy, and poor patient prognosis. This review aims to shed light on the multifaceted functions and regulatory mechanisms of API5 in cell fate decisions as well as its interest as therapeutic target in cancer. Full article

Na⁺,K⁺-ATPase actively extrudes three cytoplasmic Na⁺ ions in exchange for two extracellular K⁺ ions for each ATP hydrolyzed. The atomic structure with bound Na⁺ identifies three Na⁺ sites, named I, II, and III. It has been proposed that site III is the first to be occupied and site II last, when Na⁺ binds from the cytoplasmic side. It is usually assumed that the occupation of all three Na⁺ sites is obligatory for the activation of phosphoryl transfer from ATP. To obtain more insight into the individual roles of the ion-binding sites, we have analyzed a series of seven mutants with substitution of the critical ion-binding residue Ser777, which is a shared ligand between Na⁺ sites I and III. Surprisingly, mutants with large and bulky substituents expected to prevent or profoundly disturb Na⁺ access to sites I and III retain the ability to form a phosphoenzyme from ATP, even with increased apparent Na⁺ affinity. This indicates that Na⁺ binding solely at site II is sufficient to promote phosphorylation. These mutations appear to lock the membrane sector into an E₁-like configuration, allowing Na⁺ but not K⁺ to bind at site II, while the cytoplasmic sector undergoes conformational changes uncoupled from the membrane sector. Full article

Lampedusa, the largest island of the Pelagie archipelago, Sicily, Italy, has proven to be a rich source of plants and shrubs used in folk medicine. These plants, often native to the island, have been very poorly investigated for their phytochemical composition and biological potential to be translated into pharmacological applications. To start achieving this purpose, a specimen of Limonium lopadusanum, a plant native to Lampedusa, was investigated for the first time. This manuscript reports the results of a preliminary biological assay, focused on antimicrobial activity, carried out using the plant organic extracts, and the isolation and chemical and biological characterization of the secondary metabolites obtained. Thus 3-hydroxy-4-methoxybenzoic acid methyl ester (syn: methyl isovanillate, (1), methyl syringate (2), pinoresinol (3), erythrinassinate C (4) and tyrosol palmitate (5) were isolated. Their antimicrobial activity was tested on several strains and compound 4 showed promising antibacterial activity against Enterococcus faecalis. Thus, this metabolite has antibiotic potential against the drug-resistant opportunistic pathogen E. faecalis. Full article

Infectious bronchitis (IB) is a highly infectious viral disease of chickens which causes significant economic losses in the poultry industry worldwide. An effective vaccine against IB is urgently needed to provide both biosafety and high-efficiency immune protection. In this study, the S1 protein of the infectious bronchitis virus was delivered by a recombinant attenuated Salmonella typhimurium vector to form the vaccine candidate χ11246(pYA4545-S1). S. typhimurium χ11246 carried a sifA⁻ mutation with regulated delayed systems, striking a balance between host safety and immunogenicity. Here, we demonstrated that S1 protein is highly expressed in HD11 cells. Immunization with χ11246(pYA4545-S1) induced the production of antibody and cytokine, leading to an effective immune response against IB. Oral immunization with χ11246(pYA4545-S1) provided 72%, 56%, and 56% protection in the lacrimal gland, trachea, and cloaca against infectious bronchitis virus infection, respectively. Furthermore, it significantly reduced histopathological lesions in chickens. Together, this study provides a new idea for the prevention of IB. Full article

Synonymous mutations result from the degeneracy of the genetic code. Most amino acids are encoded by two or more codons, and mutations that change a codon to another synonymous codon do not change the amino acid in the gene product. Historically, such mutations have been considered silent because they were assumed to have no to very little impact. However, research in the last few decades has produced several examples where synonymous mutations play important roles. These include optimizing expression by enhancing translation initiation and accelerating or decelerating translation elongation via codon usage and mRNA secondary structures, stabilizing mRNA molecules and preventing their breakdown before translation, and faulty protein folding or increased degradation due to enhanced ubiquitination and suboptimal secretion of proteins into the appropriate cell compartments. Some consequences of synonymous mutations, such as mRNA stability, can lead to different outcomes in prokaryotes and eukaryotes. Despite these examples, the significance of synonymous mutations in evolution and in causing disease in comparison to nonsynonymous mutations that do change amino acid residues in proteins remains controversial. Whether the molecular mechanisms described by which synonymous mutations affect organisms can be generalized remains poorly understood and warrants future research in this area. Full article

Telomere repeats protect linear chromosomes from degradation, and telomerase has a prominent role in their maintenance. Telomerase has telomere-independent effects on cell proliferation, DNA replication, differentiation, and tumorigenesis. TERT (telomerase reverse transcriptase enzyme), the catalytic subunit of telomerase, is required for enzyme activity. TERT promoter mutation and methylation are strongly associated with increased telomerase activation in cancer cells. TERT levels and telomerase activity are downregulated in stem cells during differentiation. The link between differentiation and telomerase can provide a valuable tool for the study of the epigenetic regulation of TERT. Oxygen levels can affect cellular behaviors including proliferation, metabolic activity, stemness, and differentiation. The role of oxygen in driving TERT promoter modifications in embryonic stem cells (ESCs) is poorly understood. We adopted a monolayer ESC differentiation model to explore the role of physiological oxygen (physoxia) in the epigenetic regulation of telomerase and TERT. We further hypothesized that DNMTs played a role in physoxia-driven epigenetic modification. ESCs were cultured in either air or a 2% O₂ environment. Physoxia culture increased the proliferation rate and stemness of the ESCs and induced a slower onset of differentiation than in ambient air. As anticipated, downregulated TERT expression correlated with reduced telomerase activity during differentiation. Consistent with the slower onset of differentiation in physoxia, the TERT expression and telomerase activity were elevated in comparison to the air-oxygen-cultured ESCs. The TERT promoter methylation levels increased during differentiation in ambient air to a greater extent than in physoxia. The chemical inhibition of DNMT3B reduced TERT promoter methylation and was associated with increased TERT gene and telomerase activity during differentiation. DNMT3B ChIP (Chromatin immunoprecipitation) demonstrated that downregulated TERT expression and increased proximal promoter methylation were associated with DNMT3B promoter binding. In conclusion, we have demonstrated that DNMT3B directly associates with TERT promoter, is associated with differentiation-linked TERT downregulation, and displays oxygen sensitivity. Taken together, these findings help identify novel aspects of telomerase regulation that may play a role in better understanding developmental regulation and potential targets for therapeutic intervention. Full article

The existing literature supports the anti-inflammatory, antioxidant, and antiviral capacities of the polyphenol extracts derived from Geranium sanguineum L. These extracts exhibit potential in hindering viral replication by inhibiting enzymes like DNA polymerase and reverse transcriptase. The antiviral properties of G. sanguineum L. seem to complement its immunomodulatory effects, contributing to infection resolution. While preclinical studies on G. sanguineum L. suggest its potential effectiveness against COVID-19, there is still a lack of clinical evidence. Therefore, the polyphenols extracted from this herb warrant further investigation as a potential alternative for preventing and treating COVID-19 infections. Full article

Hydrogen sulfide (H₂S) and polysulfides (H₂S_n, n ≥ 2) produced by enzymes play a role as signalling molecules regulating neurotransmission, vascular tone, cytoprotection, inflammation, oxygen sensing, and energy formation. H₂S_n, which have additional sulfur atoms to H₂S, and other S-sulfurated molecules such as cysteine persulfide and S-sulfurated cysteine residues of proteins, are produced by enzymes including 3-mercaptopyruvate sulfurtransferase (3MST). H₂S_n are also generated by the chemical interaction of H₂S with NO, or to a lesser extent with H₂O₂. S-sulfuration (S-sulfhydration) has been proposed as a mode of action of H₂S and H₂S_n to regulate the activity of target molecules. Recently, we found that H₂S/H₂S₂ regulate the release of neurotransmitters, such as GABA, glutamate, and D-serine, a co-agonist of N-methyl-D-aspartate (NMDA) receptors. H₂S facilitates the induction of hippocampal long-term potentiation, a synaptic model of memory formation, by enhancing the activity of NMDA receptors, while H₂S₂ achieves this by activating transient receptor potential ankyrin 1 (TRPA1) channels in astrocytes, potentially leading to the activation of nearby neurons. The recent findings show the other aspects of TRPA1 channels—that is, the regulation of the levels of sulfur-containing molecules and their metabolizing enzymes. Disturbance of the signalling by H₂S/H₂S_n has been demonstrated to be involved in various diseases, including cognitive and psychiatric diseases. The physiological and pathophysiological roles of these molecules will be discussed. Full article

Questions about which reactive oxygen species (ROS) or reactive nitrogen species (RNS) can escape from the mitochondria and activate signals must be addressed. In this study, two parameters, the calculated dipole moment (debye, D) and permeability coefficient (Pm) (cm s⁻¹), are listed for hydrogen peroxide (H₂O₂), hydroxyl radical (•OH), superoxide (O₂^•−), hydroperoxyl radical (HO₂•), nitric oxide (•NO), nitrogen dioxide (•NO₂), peroxynitrite (ONOO⁻), and peroxynitrous acid (ONOOH) in comparison to those for water (H₂O). O₂^•− is generated from the mitochondrial electron transport chain (ETC), and several other ROS and RNS can be generated subsequently. The candidates which pass through the mitochondrial membrane include ROS with a small number of dipoles, i.e., H₂O₂, HO₂•, ONOOH, •OH, and •NO. The results show that the dipole moment of •NO₂ is 0.35 D, indicating permeability; however, •NO₂ can be eliminated quickly. The dipole moments of •OH (1.67 D) and ONOOH (1.77 D) indicate that they might be permeable. This study also suggests that the mitochondria play a central role in protecting against further oxidative stress in cells. The amounts, the long half-life, the diffusion distance, the Pm, the one-electron reduction potential, the pK_a, and the rate constants for the reaction with ascorbate and glutathione are listed for various ROS/RNS, •OH, singlet oxygen (¹O₂), H₂O₂, O₂^•−, HO₂•, •NO, •NO₂, ONOO⁻, and ONOOH, and compared with those for H₂O and oxygen (O₂). Molecules with negative electrical charges cannot directly diffuse through the phospholipid bilayer of the mitochondrial membranes. Short-lived molecules, such as •OH, would be difficult to contribute to intracellular signaling. Finally, HO₂• and ONOOH were selected as candidates for the ROS/RNS that pass through the mitochondrial membrane. Full article

Notch signaling is conserved in C. elegans, Drosophila, and mammals. Among the four NOTCH genes in humans, NOTCH1, NOTCH2, and NOTCH3 are known to cause monogenic hereditary disorders. Most NOTCH-related disorders are congenital and caused by a gain or loss of Notch signaling activity. In contrast, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) caused by NOTCH3 is adult-onset and considered to be caused by accumulation of the mutant NOTCH3 extracellular domain (N3ECD) and, possibly, by an impairment in Notch signaling. Pathophysiological processes following mutant N3ECD accumulation have been intensively investigated; however, the process leading to N3ECD accumulation and its association with canonical NOTCH3 signaling remain unknown. We reviewed the progress in clarifying the pathophysiological process involving mutant NOTCH3. Full article

(1) Background: Eosinophilia has traditionally been linked to eosinophilic asthma, for which it is the gold-standard prognostic biomarker. However, the association between eosinophilia and the presence of other diseases and comorbidities is yet unclear. (2) Methods: For this retrospective study, we reviewed the electronic medical records of 49,909 subjects with blood eosinophilia to gather data on the presence of asthma, COPD, sleep apnea, tuberculosis, dyslipidemia, hypertension, and other cardiovascular diseases and severe CRSwNP among these subjects. Demographic features including age, sex, and smoking habits were collected, as well as the number of hospitalizations and emergency department visits. T-tests, ANOVA, Fisher test, and logistic regression models were used. (3) Results: For all age groups studied, eosinophilia was significantly more prevalent among asthmatic subjects than nonasthmatics, especially in patients also presenting CRSwNP, hypertension, and dyslipidemia. The likelihood of developing asthma, COPD, and CRSwNP, and hospitalization, was increased when BEC was above 600 eosinophils/μL. The association between asthma, CRSwNP, and BEC was corroborated by multiple logistic regressions models. (4) Conclusions: We demonstrated the association of having over 600 blood eosinophils/μL with a higher number of hospitalizations and comorbidities (CRSwNP and COPD), which proves that BEC is a highly useful parameter to consider in subjects who present blood eosinophilia. Full article

Recombinant human erythropoietin (EPO) is a biopharmaceutical frequently used in the treatment of anemia. It is a heavily glycosylated protein with a diverse and complex glycome. EPO N-glycosylation influences important pharmacological parameters, prominently serum half-life. Therefore, EPO N-glycosylation analysis is of the utmost importance in terms of controlling critical quality attributes. In this work, we performed an interlaboratory study of glycoanalytical techniques for profiling and in-depth characterization, namely (1) hydrophilic interaction liquid chromatography with fluorescence detection after 2-aminobenzamide labeling (HILIC-FLD(2AB)) and optional weak anion exchange chromatography (WAX) fractionation and exoglycosidase digestion, (2) HILIC-FLD after procainamide labeling (PROC) optionally coupled to electrospray ionization-MS and (3) matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-MS). All techniques showed good precision and were able to differentiate the unique N-glycosylation profiles of the various EPO preparations. HILIC-FLD showed higher precision, while MALDI-TOF-MS covered the most analytes. However, HILIC-FLD differentiated isomeric N-glycans, i.e., N-acetyllactosamine repeats and O-acetylation regioisomers. For routine profiling, HILIC-FLD methods are more accessible and cover isomerism in major structures, while MALDI-MS covers more minor analytes with an attractively high throughput. For in-depth characterization, MALDI-MS and HILIC-FLD(2AB)/WAX give a similar amount of orthogonal information. HILIC-FLD(PROC)-MS is attractive for covering isomerism of major structures with a significantly less extensive workflow compared to HILIC-FLD(2AB)/WAX. Full article

Organisms are often subjected to conditions that promote cellular stress. Cell responses to stress include the activation of pathways to defend against and recover from the stress, or the initiation of programmed cell death to eliminate the damaged cells. One of the processes that can be triggered under stress is the transcription and variation in the number of copies of satellite DNA sequences (satDNA), which are involved in response mechanisms. Satellite DNAs are highly repetitive tandem sequences, mainly located in the centromeric and pericentromeric regions of eukaryotic chromosomes, where they form the constitutive heterochromatin. Satellite non-coding RNAs (satncRNAs) are important regulators of cell processes, and their deregulation has been associated with disease. Also, these transcripts have been associated with stress-response mechanisms in varied eukaryotic species. This review intends to explore the role of satncRNAs when cells are subjected to adverse conditions. Studying satDNA transcription under various stress conditions and deepening our understanding of where and how these sequences are involved could be a key factor in uncovering important facts about the functions of these sequences. Full article

BOB1, a mammalian lymphocyte-specific transcriptional coactivator of the transcription factors OCT1 and OCT2 (OCT1/2), plays important roles in normal immune responses, autoimmunity, and hematologic malignancies. The issue of a DNA sequence preference change imposed by BOB1 was raised more than two decades ago but remains unresolved. In this paper, using the EMSA–SELEX–Seq approach, we have reassessed the intrinsic ability of BOB1 to modulate the specificity of DNA recognition by OCT1 and OCT2. Our results have reaffirmed previous conclusions regarding BOB1 selectivity towards the dimer configuration of OCT1/2. However, they suggest that the monomeric configuration of these factors, assembled on the classical octamer ATGCAAAT and related motifs, are the primary targets of BOB1. Our data further specify the DNA sequence preference imposed by BOB1 and predict the probability of ternary complex formation. These results provide an additional insight into the action of BOB1—an essential immune regulator and a promising molecular target for the treatment of autoimmune diseases and hematologic malignancies. Full article

Voretigene neparvovec (VN) is the first available gene therapy for patients with biallelic RPE65-mediated inherited retinal dystrophy who have sufficient viable retinal cells. PERCEIVE is an ongoing, post-authorization, prospective, multicenter, registry-based observational study and is the largest study assessing the real-world, long-term safety and effectiveness of VN. Here, we present the outcomes of 103 patients treated with VN according to local prescribing information. The mean (SD) age was 19.5 (10.85) years, 52 (50.5%) were female, and the mean (SD) duration of the follow up was 0.8 (0.64) years (maximum: 2.3 years). Thirty-five patients (34%) experienced ocular treatment-emergent adverse events (TEAEs), most frequently related to chorioretinal atrophy (n = 13 [12.6%]). Eighteen patients (17.5%; 24 eyes [13.1%]) experienced ocular TEAEs of special interest, including intraocular inflammation and/or infection related to the procedure (n = 7). The mean (SD) changes from baseline in full-field light-sensitivity threshold testing (white light) at month 1, month 6, year 1, and year 2 were −16.59 (13.48) dB (51 eyes), −18.24 (14.62) dB (42 eyes), −15.84 (14.10) dB (10 eyes), and −13.67 (22.62) dB (13 eyes), respectively. The change in visual acuity from baseline was not clinically significant. Overall, the outcomes of the PERCEIVE study are consistent with the findings of VN pivotal clinical trials. Full article

Galectin-1 (Gal-1) is an evolutionarily conserved sugar-binding protein found in intra- and extracellular spaces. Extracellularly, it binds to glycoconjugates with β-galactoside(s) and functions in various biological phenomena, including immunity, cancer, and differentiation. Under extracellular oxidative conditions, Gal-1 undergoes oxidative inactivation, losing its sugar-binding ability, although it exhibits sugar-independent functions. An age-related decrease in serum Gal-1 levels correlates with decreasing bone mass, and Gal-1 knockout promotes osteoclastic bone resorption and suppresses bone formation. However, the effect of extracellular Gal-1 on osteoclast differentiation remains unclear. Herein, we investigated the effects of extracellular Gal-1 on osteoclastogenesis in human peripheral blood mononuclear cells (PBMCs) and mouse macrophage RAW264 cells. Recombinant Gal-1 suppressed the macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand-dependent osteoclast formation, actin ring formation, and bone-resorption activity of human PBMCs. Similar results were obtained for RAW264 cells. Gal-1 knockdown increased osteoclast-like cell formation, suggesting that it affected differentiation in an autocrine-like manner. Oxidized Gal-1 slightly affected differentiation, and in the presence of lactose, the differentiation inhibitory effect of galectin-1 was not observed. These findings suggest that extracellular Gal-1 inhibits osteoclast differentiation in a β-galactoside-dependent manner, and an age-related decrease in serum Gal-1 levels may contribute to reduced osteoclast activity and decreasing bone mass. Full article

Plant-derived diterpene synthases (PdiTPSs) play a critical role in the formation of structurally and functionally diverse diterpenoids. However, the specificity or functional-related features of PdiTPSs are not well understood. For a more profound insight, we collected, constructed, and curated 199 functionally characterized PdiTPSs and their corresponding 3D structures. The complex correlations among their sequences, domains, structures, and corresponding products were comprehensively analyzed. Ultimately, our focus narrowed to the geometric arrangement of local structures. We found that local structural alignment can rapidly localize product-specific residues that have been validated by mutagenesis experiments. Based on the 3D motifs derived from the residues around the substrate, we successfully searched diterpene synthases (diTPSs) from the predicted terpene synthases and newly characterized PdiTPSs, suggesting that the identified 3D motifs can serve as distinctive signatures in diTPSs (I and II class). Local structural analysis revealed the PdiTPSs with more conserved amino acid residues show features unique to class I and class II, whereas those with fewer conserved amino acid residues typically exhibit product diversity and specificity. These results provide an attractive method for discovering novel or functionally equivalent enzymes and probing the product specificity in cases where enzyme characterization is limited. Full article

Human noroviruses (HuNoVs) are a major cause of acute gastroenteritis, contributing significantly to annual foodborne illness cases. However, studying these viruses has been challenging due to limitations in tissue culture techniques for over four decades. Tulane virus (TV) has emerged as a crucial surrogate for HuNoVs due to its close resemblance in amino acid composition and the availability of a robust cell culture system. Initially isolated from rhesus macaques in 2008, TV represents a novel Calicivirus belonging to the Recovirus genus. Its significance lies in sharing the same host cell receptor, histo-blood group antigen (HBGA), as HuNoVs. In this study, we introduce, through cryo-electron microscopy (cryo-EM), the structure of a specific TV variant (the 9-6-17 TV) that has notably lost its ability to bind to its receptor, B-type HBGA—a finding confirmed using an enzyme-linked immunosorbent assay (ELISA). These results offer a profound insight into the genetic modifications occurring in TV that are necessary for adaptation to cell culture environments. This research significantly contributes to advancing our understanding of the genetic changes that are pivotal to successful adaptation, shedding light on fundamental aspects of Calicivirus evolution. Full article

Neurodegenerative disorders, such as Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease, are identified and characterized by the progressive loss of neurons and neuronal dysfunction, resulting in cognitive and motor impairment. Recent research has shown the importance of PTMs, such as phosphorylation, acetylation, methylation, ubiquitination, sumoylation, nitration, truncation, O-GlcNAcylation, and hydroxylation, in the progression of neurodegenerative disorders. PTMs can alter protein structure and function, affecting protein stability, localization, interactions, and enzymatic activity. Aberrant PTMs can lead to protein misfolding and aggregation, impaired degradation, and clearance, and ultimately, to neuronal dysfunction and death. The main objective of this review is to provide an overview of the PTMs involved in neurodegeneration, their underlying mechanisms, methods to isolate PTMs, and the potential therapeutic targets for these disorders. The PTMs discussed in this article include tau phosphorylation, α-synuclein and Huntingtin ubiquitination, histone acetylation and methylation, and RNA modifications. Understanding the role of PTMs in neurodegenerative diseases may provide new therapeutic strategies for these devastating disorders. Full article

With the rise in antimicrobial resistance, there is an urgent need for new classes of antibiotic with which to treat infectious disease. Marinomycin, a polyene antibiotic from a marine microbe, has been shown capable of killing methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF), as well as having promising activity against melanoma. An attractive solution to the photoprotection of this antibiotic has been demonstrated. Here, we report the identification and analysis of the marinomycin biosynthetic gene cluster (BGC), and the biosynthetic assembly of the macrolide. The marinomycin BGC presents a challenge in heterologous expression due to its large size and high GC content, rendering the cluster prone to rearrangement. We demonstrate the transformation of Streptomyces lividans using a construct containing the cluster, and the heterologous expression of the encoded biosynthetic machinery and production of marinomycin B. Full article

Background: While extensive research highlighted the involvement of metabolism and immune cells in female reproductive diseases, causality remains unestablished. Methods: Instrumental variables for 486 circulating metabolites (N = 7824) and 731 immunophenotypes (N = 3757) were derived from a genome-wide association study (GWAS) meta-analysis. FinnGen contributed data on 14 female reproductive disorders. A bidirectional two-sample Mendelian randomization study was performed to determine the relationships between exposures and outcomes. The robustness of results, potential heterogeneity, and horizontal pleiotropy were examined through sensitivity analysis. Results: High levels of mannose were found to be causally associated with increased risks of gestational diabetes (GDM) (OR [95% CI], 6.02 [2.85–12.73], p = 2.55 × 10⁻⁶). A genetically predicted elevation in the relative count of circulating CD28⁻CD25⁺⁺CD8⁺ T cells was causally related to increased female infertility risk (OR [95% CI], 1.26 [1.14–1.40], p = 1.07 × 10⁻⁵), whereas a high absolute count of NKT cells reduced the risk of ectopic pregnancy (OR [95% CI], 0.87 [0.82–0.93], p = 5.94 × 10⁻⁶). These results remained consistent in sensitivity analyses. Conclusions: Our study supports mannose as a promising GDM biomarker and intervention target by integrating metabolomics and genomics. Full article

Lung organoids display a tissue-specific functional phenomenon and mimic the features of the original organ. They can reflect the properties of the cells, such as morphology, polarity, proliferation rate, gene expression, and genomic profile. Alveolar type 2 (AT2) cells have a stem cell potential in the adult lung. They produce and secrete pulmonary surfactant and proliferate to restore the epithelium after damage. Therefore, AT2 cells are used to generate alveolar organoids and can recapitulate distal lung structures. Also, AT2 cells in human-induced pluripotent stem cell (iPSC)-derived alveolospheres express surfactant proteins and other factors, indicating their application as suitable models for studying cell–cell interactions. Recently, they have been utilized to define mechanisms of disease development, such as COVID-19, lung cancer, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease. In this review, we show lung organoid applications in various pulmonary diseases, drug screening, and personalized medicine. In addition, stem cell-based therapeutics and approaches relevant to lung repair were highlighted. We also described the signaling pathways and epigenetic regulation of lung regeneration. It is critical to identify novel regulators of alveolar organoid generations to promote lung repair in pulmonary diseases. Full article

The expression of drug efflux pump ABCB1/P-glycoprotein (P-gp), a transmembrane protein belonging to the ATP-binding cassette superfamily, is a leading cause of multidrug resistance (MDR). We previously curated a dataset of structurally diverse and selective inhibitors of ABCB1 to develop a pharmacophore model that was used to identify four novel compounds, which we showed to be potent and efficacious inhibitors of ABCB1. Here, we dock the inhibitors into a model structure of the human transporter and use molecular dynamics (MD) simulations to report the conformational dynamics of human ABCB1 induced by the binding of the inhibitors. The binding hypotheses are compared to the wider curated dataset and those previously reported in the literature. Protein–ligand interactions and MD simulations are in good agreement and, combined with LipE profiling, statistical and pharmacokinetic analyses, are indicative of potent and selective inhibition of ABCB1. Full article

Alternative lengthening of telomeres (ALT) is a homologous recombination-based pathway utilized by 10–15% of cancer cells that allows cells to maintain their telomeres in the absence of telomerase. This pathway was originally discovered in the yeast Saccharomyces cerevisiae and, for decades, yeast has served as a robust model to study ALT. Using yeast as a model, two types of ALT (RAD51-dependent and RAD51-independent) have been described. Studies in yeast have provided the phenotypic characterization of ALT survivors, descriptions of the proteins involved, and implicated break-induced replication (BIR) as the mechanism responsible for ALT. Nevertheless, many questions have remained, and answering them has required the development of new quantitative methods. In this review we discuss the historic aspects of the ALT investigation in yeast as well as new approaches to investigating ALT, including ultra-long sequencing, computational modeling, and the use of population genetics. We discuss how employing new methods contributes to our current understanding of the ALT mechanism and how they may expand our understanding of ALT in the future. Full article

Treatment of aging rats for 6 months with ladostigil (1 mg/kg/day) prevented a decline in recognition and spatial memory and suppressed the overexpression of gene-encoding pro-inflammatory cytokines, TNFα, IL1β, and IL6 in the brain and microglial cultures. Primary cultures of mouse microglia stimulated by lipopolysaccharides (LPS, 0.75 µg/mL) and benzoyl ATPs (BzATP) were used to determine the concentration of ladostigil that reduces the secretion of these cytokine proteins. Ladostigil (1 × 10⁻¹¹ M), a concentration compatible with the blood of aging rats in, prevented memory decline and reduced secretion of IL1β and IL6 by ≈50%. RNA sequencing analysis showed that BzATP/LPS upregulated 25 genes, including early-growth response protein 1, (Egr1) which increased in the brain of subjects with neurodegenerative diseases. Ladostigil significantly decreased Egr1 gene expression and levels of the protein in the nucleus and increased TNF alpha-induced protein 3 (TNFaIP3), which suppresses cytokine release, in the microglial cytoplasm. Restoration of the aberrant signaling of these proteins in ATP/LPS-activated microglia in vivo might explain the prevention by ladostigil of the morphological and inflammatory changes in the brain of aging rats. Full article

Nitric oxide (NO), synthesized by endothelial nitric oxide synthase (eNOS), plays a critical role in blood pressure regulation. Genome-wide association studies have identified genetic susceptibility loci for hypertension in human lymphocyte-specific protein 1 (LSP1) gene. LSP1 is recognized as modulator of leukocyte extravasation, and endothelial permeability, however, the role of LSP1 in regulation of NO signaling within endothelial cells (ECs) remains unknown. The present study investigated the role of LSP1 in the regulation of eNOS expression and activity utilizing human macrovascular ECs in vitro and LSP1 knockout (KO) mice. In ECs, specific CRISPR-Cas9 genomic editing deleted LSP1 and caused downregulation of eNOS expression. LSP1 gain-of-function through adenovirus-mediated gene transfer was associated with enhanced expression of eNOS. Co-immunoprecipitation and confocal fluorescence microscopy revealed that eNOS and LSP1 formed a protein complex under basal conditions in ECs. Furthermore, LSP1 deficiency in mice promoted significant upregulation and instability of eNOS. Utilizing a mass-spectrometry-based bottom-up proteomics approach, we identified novel truncated forms of eNOS in immunoprecipitates from LSP1 KO aortae. Our experimental data suggest an important role of endothelial LSP1 in regulation of eNOS expression and activity within human ECs and murine vascular tissues. Full article

Hydroxy fatty acids (HFAs) constitute a class of lipids, distinguished by the presence of a hydroxyl on a long aliphatic chain. This study aims to expand our insights into HFA bioactivities, while also introducing new methods for asymmetrically synthesizing unsaturated and saturated HFAs. Simultaneously, a procedure previously established by us was adapted to generate new HFA regioisomers. An organocatalytic step was employed for the synthesis of chiral terminal epoxides, which either by alkynylation or by Grignard reagents resulted in unsaturated or saturated chiral secondary alcohols and, ultimately, HFAs. 7-(S)-Hydroxyoleic acid (7SHOA), 7-(S)-hydroxypalmitoleic acid (7SHPOA) and 7-(R)- and (S)-hydroxymargaric acids (7HMAs) were synthesized for the first time and, together with regioisomers of (R)- and (S)-hydroxypalmitic acids (HPAs) and hydroxystearic acids (HSAs), whose biological activity has not been tested so far, were studied for their antiproliferative activities. The unsaturation of the long chain, as well as an odd-numbered (C17) fatty acid chain, led to reduced activity, while the new 6-(S)-HPA regioisomer was identified as exhibiting potent antiproliferative activity in A549 cells. 6SHPA induced acetylation of histone 3 in A549 cells, without affecting acetylated α-tubulin levels, suggesting the selective inhibition of histone deacetylase (HDAC) class I enzymes, and was found to inhibit signal transducer and activator of transcription 3 (STAT3) expression. Full article

This study identified proteomic changes in the seeds of two tolerant (SB-DT3 and SB-DT2) and two sensitive (Merlot and Stampede) common bean genotypes in response to terminal drought stress. Differentially expressed proteins (DEPs) were abundant in the susceptible genotype compared to the tolerant line. DEPs associated with starch biosynthesis, protein–chromophore linkage, and photosynthesis were identified in both genotypes, while a few DEPs and enriched biological pathways exhibited genotype-specific differences. The tolerant genotypes uniquely showed DEPs related to sugar metabolism and plant signaling, while the sensitive genotypes displayed more DEPs involved in plant–pathogen interaction, proteasome function, and carbohydrate metabolism. DEPs linked with chaperone and signal transduction were significantly altered between both genotypes. In summary, our proteomic analysis revealed both conserved and genotype-specific DEPs that could be used as targets in selective breeding and developing drought-tolerant common bean genotypes. Full article