Association between Levels of Loneliness, Laboratory Measurements, and Behavioral Aspects in a Primary Care Setting in Crete, Greece.

This paper examines potential associations of loneliness with laboratory data and specific psychosocial and behavioral attitudes. The sample collection took place in an urban Primary Health Care unit between May and July 2023, consecutively, and once exclusion criteria were implemented. Participants were aged between 40 and 75 years. Routine laboratory test results upon study initiation and six months before were used. The University of California, Los Angeles (UCLA), Loneliness Scale (Version 3), blood glucose, serum lipids, Fibrosis-4 index, and Creatinine Clearance (CrCl) were assessed through hierarchical multiple logistic regression analysis. Based on full model (3rd) analysis, those who were engaged in an individual sport or activity or had contacts with more friends presented significantly lower odds for increased loneliness levels (odds ratio (OR): 0.28 [95% confidence interval (CI) 0.09-0.91], p = 0.034 and OR: 0.76 [95%CI 0.66-0.88], p < 0.001, respectively). The consumption of alcohol was associated with increased loneliness (OR: 5.55 [95%CI 1.42-21.63], p = 0.014). Elevated triglyceride levels were linked with moderate or no loneliness (OR: 0.20 [95%CI 0.05-0.83], p = 0.026), while an increased LDL/HDL atherosclerotic index was related to increased subjective loneliness (OR: 4.50 [95%CI 1.12-18.13], p = 0.035). The need for holistic approaches-involving primary care personnel-in understanding and addressing loneliness, recognizing its multifaceted nature as well as the diverse factors that contribute to this issue, is considered challenging.

Mining the ZINC database of natural products for specific, testosterone-like, OXER1 antagonists.

OXER1, the receptor for the oxidized arachidonic acid metabolite 5-oxo-ETE has been reported to play a significant role in inflammatory responses, being responsible for leucocyte chemotactic responses. Recently, we have identified OXER1 (GPR170) as a membrane receptor for androgens in prostate and breast cancer cells. Testosterone action via OXER1 induces specific Ca2+ release from intracellular organelles, modifies polymerized actin distribution induces apoptosis and decreases cancer cell migration. These actions are antagonized by 5-oxo-ETE. In addition, 5-oxo-ETE through a Gαi protein decreases cAMP, an action antagonized by testosterone. In this work, we mined the ZINC15 database, using QSAR, for natural compounds able to signal through Gαi and Gßγ simultaneously, mimicking testosterone actions, as well as for specific Gßγ interactors, inhibiting 5-oxo-ETE tumor promoting actions. We were able to identify four druggable Gαßγ and seven Gßγ specific OXER1 interactors. We further confirmed by bio-informatic methods their binding to the 5-oxo-ETE/testosterone binding groove of the receptor, their ADME properties and their possible interaction with other receptor and/or enzyme targets. Two compounds, ZINC04017374 (Naphthofluorescein) and ZINC08589130 (Puertogaline A) were purchased, tested in vitro and confirmed their OXER1 Gßγ and Gαßγ activity, respectively. The methodology followed is useful for a better understanding of the mechanism by which OXER1 mediates its actions, it has the potential to provide structural insights, in order to design small molecular specific interactors and ultimately design new anti-inflammatory and anti-cancer agents. Finally, the methodology may also be useful for identifying specific agonists/antagonists of other GPCRs.

A Mixture of Essential Oils from Three Cretan Aromatic Plants Inhibits SARS-CoV-2 Proliferation: A Proof-of-Concept Intervention Study in Ambulatory Patients.

INTRODUCTION: The need for effective therapeutic regimens for non-critically ill patients during the COVID-19 pandemic remained largely unmet. Previous work has shown that a combination of three aromatic plants' essential oils (CAPeo) (Thymbra capitata (L.) Cav., Origanum dictamnus L., Salvia fruticose Mill.) has remarkable in vitro antiviral activity. Given its properties, it was urgent to explore its potential in treating mild COVID-19 patients in primary care settings. METHODS: A total of 69 adult patients were included in a clinical proof-of-concept (PoC) intervention study. Family physicians implemented the observational study in two arms (intervention group and control group) during three study periods (IG2020, n=13, IG2021/22, n=25, and CG2021/22, n=31). The SARS-CoV-2 infection was confirmed by real-time PCR. The CAPeo mixture was administered daily for 14 days per os in the intervention group, while the control group received usual care. RESULTS: The PoC study found that the number and frequency of general symptoms, including general fatigue, weakness, fever, and myalgia, decreased following CAPeo administration. By Day 7, the average presence (number) of symptoms decreased in comparison with Day 1 in IG (4.7 to 1.4) as well as in CG (4.0 to 3.1), representing a significant decrease in the cumulative presence in IC (-3.3 vs. -0.9, p < 0.001; η2 = 0.20) on Day 7 and on Day 14 (-4.2 vs. -2.9, p = 0.027; η2 = 0.08). DISCUSSION/CONCLUSIONS: Our findings suggest that CAPeo possesses potent antiviral activity against SARS-CoV-2 in addition tο its effect against influenza A and B and human rhinovirus HRV14 strains. The early and effective impact on alleviating key symptoms of COVID-19 may suggest this mixture can act as a complementary natural agent for patients with mild COVID-19.

Promising Perspectives of the Antiproliferative GPER Inverse Agonist ERα17p in Breast Cancer.

The estrogen receptor α (ERα) corresponds to a large platform in charge of the recruitment of a panel of molecules, including steroids and related heterocyclic derivatives, oligonucleotides, peptides and proteins. Its 295-311 region is particularly targeted by post-translational modifications, suggesting that it could be crucial for the control of transcription. In addition to anionic phospholipids, the ERα 295-311 fragment interacts with Ca2+-calmodulin, the heat shock protein 70 (Hsp70), ERα and possibly importins. More recently, we have demonstrated that it is prone to interacting with the G-protein-coupled estrogen receptor (GPER). In light of these observations, the pharmacological profile of the corresponding peptide, namely ERα17p, has been explored in breast cancer cells. Remarkably, it exerts apoptosis through GPER and induces a significant decrease (more than 50%) of the size of triple-negative breast tumor xenografts in mice. Herein, we highlight not only the promising therapeutic perspectives in the use of the first peptidic GPER modulator ERα17p, but also the opportunity to modulate GPER for clinical purposes.

Corrigendum to "Recognition motifs for importin 4 [(L)PPRS(G/P)P] and importin 5 [KP(K/Y)LV] binding, identified by bio-informatic simulation and experimental in vitro validation" [Comput Struct Biotechnol J 20 (2022) 5952-5961].

[This corrects the article DOI: 10.1016/j.csbj.2022.10.015.].

Oxytocin preprotein and oxytocin receptor mRNA expression is altered in semen samples with abnormal semen parameters.

RESEARCH QUESTION: Are oxytocin preprotein and the oxytocin receptor expressed in human spermatozoa and is their mRNA expression different between normal semen samples and samples with at least one abnormal parameter? DESIGN: An in-vitro prospective study of 175 semen samples from Greek men, according to World Health Organization criteria, 2010. mRNA expression levels were compared between different categories of semen samples, classified according to their concentration, total number, motility and morphology. Immunohistochemistry was used to detect oxytocin preprotein and its receptor on spermatozoa smears. RESULTS: Compared with normal samples (normal motility and normal concentration), samples with at least one abnormal sperm parameter had statistically significantly lower oxytocin preprotein mRNA expression (P = 0.019) and higher oxytocin receptor mRNA expression levels (P < 0.001). Oligozoospermic samples had statistically significantly higher oxytocin preprotein mRNA expression levels (P = 0.002) and lower oxytocin receptor mRNA expression levels (P = 0.047). Asthenozoospermic samples had statistically significantly lower oxytocin preprotein mRNA expression levels (P < 0.001). Teratozoospermic samples had statistically significantly lower oxytocin preprotein mRNA expression levels (P = 0.049) and higher oxytocin receptor mRNA expression levels (P < 0.001). Oxytocin preprotein mRNA expression was positively associated with total progressive motility (P < 0.001) and negatively associated with the percentage of immotile spermatozoa (P = 0.001). Oxytocin receptor mRNA expression was negatively associated with the percentage of normal forms (P < 0.001). CONCLUSION: Oxytocin preprotein and oxytocin receptor mRNA expression in spermatozoa could be used as a novel and unbiased diagnostic tool for male infertility.

5-Oxo-ETE/OXER1: A Link between Tumor Cells and Macrophages Leading to Regulation of Migration.

Chronic inflammation is an important factor in the development of cancer. Macrophages found in tumors, known as tumor associated macrophages (TAMs), are key players in this process, promoting tumor growth through humoral and cellular mechanisms. 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), an arachidonic acid metabolite, has been described to possess a potent chemoattractant activity for human white blood cells (WBCs). The biological actions of 5-oxo-ETE are mediated through the GPCR 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid receptor (OXER1). In addition, we have previously reported OXER1 as one of the membrane androgen receptors with testosterone antagonizing 5-oxo-ETE's actions. OXER1 is highly expressed in inflammatory cells and many normal and cancer tissues and cells, including prostate and breast cancer, promoting cancer cell survival. In the present study we investigate the expression and role of OXER1 in WBCs, THP-1 monocytes, and THP-1 derived macrophages, as well as its possible role in the interaction between macrophages and cancer cells (DU-145 and T47D). We report that OXER1 is differentially expressed between WBCs and macrophages and that receptor expression is modified by LPS treatment. Our results show that testosterone and 5-oxo-ETE can act in an antagonistic way affecting Ca2+ movements, migration, and cytokines' expression in immune-related cells, in a differentiation-dependent manner. Finally, we report that 5-oxo-ETE, through OXER1, can attract macrophages to the tumor site while tumor cells' OXER1 activation in DU-145 prostate and T47D breast cancer cells, by macrophages, induces actin cytoskeletal changes and increases their migration.

Correction: Daskalaki et al. Early Postoperative Parathormone and Calcium as Prognostic Factors for Postoperative Hypocalcemia. J. Clin. Med. 2022, 11, 2389.

In the published article [...].

Recognition motifs for importin 4 [(L)PPRS(G/P)P] and importin 5 [KP(K/Y)LV] binding, identified by bio-informatic simulation and experimental in vitro validation.

Nuclear translocation of large proteins is mediated through karyopherins, carrier proteins recognizing specific motifs of cargo proteins, known as nuclear localization signals (NLS). However, only few NLS signals have been reported until now. In the present work, NLS signals for Importins 4 and 5 were identified through an unsupervised in silico approach, followed by experimental in vitro validation. The sequences LPPRS(G/P)P and KP(K/Y)LV were identified and are proposed as recognition motifs for Importins 4 and 5 binding, respectively. They are involved in the trafficking of important proteins into the nucleus. These sequences were validated in the breast cancer cell line T47D, which expresses both Importins 4 and 5. Elucidating the complex relationships of the nuclear transporters and their cargo proteins is very important in better understanding the mechanism of nuclear transport of proteins and laying the foundation for the development of novel therapeutics, targeting specific importins.

Importins involved in the nuclear transportation of steroid hormone receptors: In silico and in vitro data.

The nuclear receptor superfamily (NRS) consists of 48 receptors for lipophilic substances and is divided into 7 different subfamilies, with subfamily 3 comprising steroid hormone receptors. Several nuclear receptors usually bind their cognate ligands in the cytosol and the complex (mono- or dimerized) is transported to the nucleus, where it acts as a transcription initiating factor for a number of genes. The general structure of nuclear receptors consists of an N-terminal activating domain (A/B), important for the binding of activating or inhibitory co-factors, the DNA-binding domain (C), responsible for the association of the receptor-ligand-co-factor complex to the nucleus, the ligand-AF2 domain (E/F), where ligand binding occurs as well as that of ligand-dependent activating/inhibiting factors, and a flexible/non-structured domain (D), linking the DBD and LBD, called hinge region, on which a significant number of post-translational modifications occur. This hinge domain, for the sub-class of steroid receptors, is a non-structured domain and was reported as mainly responsible for the nuclear transport of steroid receptors, since it contains a specific amino acid sequence (Nuclear Localization Signal-NLS), recognized by importin α. In addition to the importin α/ß complex, a number of other importins have been discovered and reported to be responsible for the nuclear transport of a number of significant proteins; however, the corresponding recognition sequences for these importins have not been identified. Recently, we have reported the identification of the NLS sequences for importins 4, 5 and 7. In this work, we provide in silico data, followed by experimental in vitro validation, showing that these alternative importins are responsible for the nuclear transportation of steroid hormone receptors such as ERα, AR and PR, and therefore they may consist of alternative targets for the pharmacological manipulation of steroid hormone actions. Moreover, we provide additional in silico data for the hinge region of steroid hormone receptors which is highly enriched with NLS sequences for importins 4, 5 and 7, in addition to the recognition NLS for importin α/ß.

From Traditional Ethnopharmacology to Modern Natural Drug Discovery: A Methodology Discussion and Specific Examples.

Ethnopharmacology, through the description of the beneficial effects of plants, has provided an early framework for the therapeutic use of natural compounds. Natural products, either in their native form or after crude extraction of their active ingredients, have long been used by different populations and explored as invaluable sources for drug design. The transition from traditional ethnopharmacology to drug discovery has followed a straightforward path, assisted by the evolution of isolation and characterization methods, the increase in computational power, and the development of specific chemoinformatic methods. The deriving extensive exploitation of the natural product chemical space has led to the discovery of novel compounds with pharmaceutical properties, although this was not followed by an analogous increase in novel drugs. In this work, we discuss the evolution of ideas and methods, from traditional ethnopharmacology to in silico drug discovery, applied to natural products. We point out that, in the past, the starting point was the plant itself, identified by sustained ethnopharmacological research, with the active compound deriving after extensive analysis and testing. In contrast, in recent years, the active substance has been pinpointed by computational methods (in silico docking and molecular dynamics, network pharmacology), followed by the identification of the plant(s) containing the active ingredient, identified by existing or putative ethnopharmacological information. We further stress the potential pitfalls of recent in silico methods and discuss the absolute need for in vitro and in vivo validation as an absolute requirement. Finally, we present our contribution to natural products' drug discovery by discussing specific examples, applying the whole continuum of this rapidly evolving field. In detail, we report the isolation of novel antiviral compounds, based on natural products active against influenza and SARS-CoV-2 and novel substances active on a specific GPCR, OXER1.

Early Postoperative Parathormone and Calcium as Prognostic Factors for Postoperative Hypocalcemia.

BACKGROUND: Postoperative hypocalcemia is one of the most common complications after total thyroidectomy. Parathormone (PTH) and calcium levels, measured several hours after surgery, have been suggested as valuable markers for detecting patients at risk for post-thyroidectomy hypocalcemia. We aimed to determine if early post-surgery PTH and calcium levels can be used for the early identification of patients at risk for symptomatic hypocalcemia. METHODS: PTH and calcium were measured before surgery and at 10 min and 4 h post-thyroidectomy, in 77 patients. Performance characteristics of PTH and calcium levels and their post/pre-surgery ratios were calculated. RESULTS: Four-hour calcium was a sensitive (93.75%) but not specific (67.61%) indicator of patients at risk for symptomatic hypocalcemia. The 4-h/pre-surgery PTH ratio was the most accurate (90.81%) and the most specific (94.37%) test to identify patients at risk. Serum calcium at 4-h, 4-h/pre-surgery PTH ratio, and PTH at 10 min post-surgery had the higher diagnostic odds ratios (50.86, 32.85, and 29.04, respectively). The 4-h/pre-surgery PTH ratio also had the highest (0.694) Youden's J statistic. CONCLUSIONS: Low serum calcium levels 4 h after thyroidectomy and the 4-h/pre-surgery PTH ratio could be valuable additions to everyday clinical practice in post-thyroidectomy patients.

OXER1 mediates testosterone-induced calcium responses in prostate cancer cells.

In prostate cancer, calcium homeostasis plays a significant role in the disease's development and progression. Intracellular calcium changes are an important secondary signal, triggered by a variety of extracellular stimuli, that controls many cellular functions. One of the main events affecting calcium is androgen signaling. Indeed, via calcium changes, androgens regulate cell processes like cell growth, differentiation and motility. In the present work we explored the nature of the receptor involved in calcium response induced by membrane-acting testosterone in prostate cancer cells. We report that testosterone, independently of the presence of the classical androgen receptor, can rapidly increase intracellular calcium from calcium stores, through the oxoeicosanoid receptor 1 (OXER1) and a specific signaling cascade that triggers calcium release from the endoplasmic reticulum. These findings reveal for the first time the receptor involved in the rapid calcium changes induced by androgens. Moreover, they further support the notion that androgens, even in the absence of AR, can still exert specific effects that regulate cancer cell fate.

New Antagonists of the Membrane Androgen Receptor OXER1 from the ZINC Natural Product Database.

OXER1 (oxoeicosanoid receptor 1) was deorphanized in 1993 and found to be the specific receptor for the arachidonic acid metabolite 5-oxo-ETE. Recently, we have reported that androgen binds to this receptor also, being a membrane androgen receptor, triggering a number of its membrane-mediated actions (cell migration, apoptosis, cell proliferation, Ca2+ movements). In addition, our previous work suggested that a number of natural monomeric and oligomeric polyphenols interact with OXER1, acting similar to testosterone. Here, we interrogated the natural product chemical space and identified nine polyphenolic molecules with interesting in silico pharmacological activities as putative OXER1 antagonists. The molecule with the best pharmacokinetic-pharmacodynamic properties (ZINC15959779) was purchased and tested on OXER1, in prostate cancer cell cultures. It showed that it has actions similar to those of testosterone in inhibiting cAMP, while it had no action in intracellular Ca2+ mobilization or actin cytoskeleton rearrangement/migration. These results are discussed under the prism of structure-activity relationships and in silico models of the OXER1 binding groove. We suggest that these compounds, together with the previously reported (poly)phenolic compounds, can be lead structures for the exploration of the anti-inflammatory and antiproliferative effects of OXER1 antagonists.

Enhanced OXER1 expression is indispensable for human cancer cell migration.

OXER1 is a recently identified receptor, binding the arachidonic acid metabolic product 5-oxo-ETE, considered an inflammatory receptor, implicated in chemoattraction of circulating mononuclear cells, Ca2+ surge in neutrophils, inflammation and cancer. Recently, we have shown that OXER1 is also a membrane androgen receptor in various cancer tissues. It was reported that the presence of OXER1 in leucocytes and the production and release of 5-oxo-ETE by wounded tissues is a wound sensing mechanism, leading to lymphocyte attraction. In view of the similarity of hallmarks of cancer and wound healing, we have explored the role of OXER1 and its endogenous ligand in the control of cell migration of human cancer epithelial cells (DU-145, T47D and Hep3B), mimicking the activation/migration phase of healing. We show that OXER1 is up-regulated only at the leading edge of the wound and its expression is up-regulated by its ligand 5-oxo-ETE, in a time-related manner. Knock-down of OXER1 or inhibition of 5-oxo-ETE synthesis led to decreased migration of cells and a prolongation of healing, in culture prostate cancer cell monolayers, with a substantial modification of actin cytoskeleton and a decreased filopodia formation. Inhibition of cell migration is a phenomenon mediated by Gßγ OXER1 mediated actions. These results provide a novel mechanism of OXER1 implication in cancer progression and might be of value for the design of novel OXER1 antagonists.

Natural Polyphenols Inhibit the Dimerization of the SARS-CoV-2 Main Protease: The Case of Fortunellin and Its Structural Analogs.

3CL-Pro is the SARS-CoV-2 main protease (MPro). It acts as a homodimer to cleave the large polyprotein 1ab transcript into proteins that are necessary for viral growth and replication. 3CL-Pro has been one of the most studied SARS-CoV-2 proteins and a main target of therapeutics. A number of drug candidates have been reported, including natural products. Here, we employ elaborate computational methods to explore the dimerization of the 3CL-Pro protein, and we formulate a computational context to identify potential inhibitors of this process. We report that fortunellin (acacetin 7-O-neohesperidoside), a natural flavonoid O-glycoside, and its structural analogs are potent inhibitors of 3CL-Pro dimerization, inhibiting viral plaque formation in vitro. We thus propose a novel basis for the search of pharmaceuticals as well as dietary supplements in the fight against SARS-CoV-2 and COVID-19.

Glycosylation Modulates Plasma Membrane Trafficking of CD24 in Breast Cancer Cells.

In breast cancer, expression of Cluster of Differentiation 24 (CD24), a small GPI-anchored glycoprotein at the cell periphery, is associated with metastasis and immune escape, while its absence is associated with tumor-initiating capacity. Since the mechanism of CD24 sorting is unknown, we investigated the role of glycosylation in the subcellular localization of CD24. Expression and localization of wild type N36- and/or N52-mutated CD24 were analyzed using immunofluorescence in luminal (MCF-7) and basal B (MDA-MB-231 and Hs578T) breast cancer cells lines, as well as HEK293T cells. Endogenous and exogenously expressed wild type and mutated CD24 were found localized at the plasma membrane and the cytoplasm, but not the nucleoplasm. The cell lines showed different kinetics for the sorting of CD24 through the secretory/endocytic pathway. N-glycosylation, especially at N52, and its processing in the Golgi were critical for the sorting and expression of CD24 at the plasma membrane of HEK293T and basal B type cells, but not of MCF-7 cells. In conclusion, our study highlights the contribution of N-glycosylation for the subcellular localization of CD24. Aberrant N-glycosylation at N52 of CD24 could account for the lack of CD24 expression at the cell surface of basal B breast cancer cells.

ERα36-GPER1 Collaboration Inhibits TLR4/NFκB-Induced Pro-Inflammatory Activity in Breast Cancer Cells.

Inflammation is important for the initiation and progression of breast cancer. We have previously reported that in monocytes, estrogen regulates TLR4/NFκB-mediated inflammation via the interaction of the Erα isoform ERα36 with GPER1. We therefore investigated whether a similar mechanism is present in breast cancer epithelial cells, and the effect of ERα36 expression on the classic 66 kD ERα isoform (ERα66) functions. We report that estrogen inhibits LPS-induced NFκB activity and the expression of downstream molecules TNFα and IL-6. In the absence of ERα66, ERα36 and GPER1 are both indispensable for this effect. In the presence of ERα66, ERα36 or GPER1 knock-down partially inhibits NFκB-mediated inflammation. In both cases, ERα36 overexpression enhances the inhibitory effect of estrogen on inflammation. We also verify that ERα36 and GPER1 physically interact, especially after LPS treatment, and that GPER1 interacts directly with NFκB. When both ERα66 and ERα36 are expressed, the latter acts as an inhibitor of ERα66 via its binding to estrogen response elements. We also report that the activation of ERα36 leads to the inhibition of breast cancer cell proliferation. Our data support that ERα36 is an inhibitory estrogen receptor that, in collaboration with GPER1, inhibits NFκB-mediated inflammation and ERα66 actions in breast cancer cells.