Differential Digestive Stability of Food-Derived microRNAs: The Case of miR-30c-5p and miR-92a-3p in Polyfloral Honey.

Dietary microRNAs (miRs) represent a new area in food science. Although they have been found in many foods, including honey, more research is needed about their stability and fate during digestion. Hence, this study aimed to analyze the digestive stability of two selected miRs in honey. We extracted miR-92a-3p and miR-30c-5p from pasteurized and unpasteurized forms of polyfloral honey using two different methods and kits: a column-based manual method and a phenol-free semi-automated magnetic-bead-based method. The latter option was used for the subsequent analysis of samples according to the INFOGEST static in vitro digestion protocol. Also, the honey samples were examined for exosome-like particles using dynamic light scattering. Although the expression levels of both miRs were significantly lower following intestinal digestion, we found a difference in the resilience of the miRs to gastrointestinal conditions, with miR-30c-5p being relatively stable compared to miR-92a-3p following digestion, regardless of the honey's pasteurization treatment. Moreover, there was marked heterogeneity in the extracellular vesicle profile of the pasteurized sample. We identified the presence of two broadly conserved miRs in honey: miR-92a-3p and miR-30c-5p. Despite honey exhibiting high digestibility, miR-92a-3p was less resilient than miR-30c-5p, demonstrating considerable resistance under gastrointestinal conditions. Although further research is needed, the results obtained from this study may represent a starting point for utilizing honey as a source of exogenous miRNAs for preventive strategies and more "natural" treatments.

Liver Damage and microRNAs: An Update.

One of the major organs in the body with multiple functions is the liver. It plays a central role in the transformation of macronutrients and clearance of chemicals and drugs. The serum biomarkers often used to indicate liver damage are not specifically for drug-induced liver injury (DILI) or liver injury caused by other xenobiotics, nor for viral infection. In this case, microRNAs (miRNAs) could play an exciting role as biomarkers of specific liver damage. In this review, we aimed to update the current literature on liver damage induced by drugs, as acute conditions and viral infections mediated by the hepatitis B virus (HBV) linked these two conditions to advanced research, with a focus on microRNAs as early biomarkers for liver damage. The undoubtable evidence that circulating miR-122 could be used as a human biomarker of DILI came from several studies in which a strong increase of it was linked with the status of liver function. In infancy, there is the possibility of an early miRNA detection for hepatitis B virus infection, but there are a lack of solid models for studying the HVB molecular mechanism of infection in detail, even if miRNAs do hold unrealized potential as biomarkers for early detection of hepatitis B virus infection mediated by HBV.

FFAR1/GPR40: One target, different binding sites, many agonists, no drugs, but a continuous and unprofitable tug-of-war between ligand lipophilicity, activity, and toxicity.

The progress made so far in the elucidation of the structure of free fatty acid receptor 1 (FFAR1) and its secondary and ternary complexes with partial and full allosteric ligands led to the discovery of various putative binding regions on the FFAR1 surface. Attempts to develop FFAR1 agonists culminated with the identification of TAK-875 (1), whose phase 3 clinical trials were terminated due to potential liver toxicity. In the search of safer agonists, numerous classes of new compounds were designed, synthesized, and tested. Chemical decoration of the scaffolds was rationalized to reach a good balance between lipophilicity, activity, and toxicity. Today, targeting FFAR1 with positive modulators represents an attractive pharmacological tool for the treatment of type 2 diabetes mellitus (T2DM), mainly because of the lack of hypoglycaemic side effects associated with several antidiabetic drugs currently available. Moreover, considering the involvement of FFAR1 in many physio-pathological processes, its agonists are also emerging as possible therapeutic tools for alleviating organ inflammation and fibrosis, as well as for the treatment of CNS disorders, such as Alzheimer's disease and dementia.

Pharmacophore-guided repurposing of fibrates and retinoids as GPR40 allosteric ligands with activity on insulin release.

A classical drug repurposing approach was applied to find new putative GPR40 allosteric binders. A two-step computational protocol was set up, based on an initial pharmacophoric-based virtual screening of the DrugBank database of known drugs, followed by docking simulations to confirm the interactions between the prioritised compounds and GPR40. The best-ranked entries showed binding poses comparable to that of TAK-875, a known allosteric agonist of GPR40. Three of them (tazarotenic acid, bezafibrate, and efaproxiral) affect insulin secretion in pancreatic INS-1 832/13 ß-cells with EC50 in the nanomolar concentration (5.73, 14.2, and 13.5 nM, respectively). Given the involvement of GPR40 in type 2 diabetes, the new GPR40 modulators represent a promising tool for therapeutic intervention towards this disease. The ability to affect GPR40 was further assessed in human breast cancer MCF-7 cells in which this receptor positively regulates growth activities (EC50 values were 5.6, 21, and 14 nM, respectively).

Effects of Long-Term Storage on Radical Scavenging Properties and Phenolic Content of Kombucha from Black Tea.

Kombucha is a fermented beverage. Its consumption has significantly increased during the last decades due to its perceived beneficial effects. For this reason, it has become a highly commercialized drink that is produced industrially. However, kombucha is still also a homemade beverage, and the parameters which, besides its organoleptic characteristics, define the duration of its potential beneficial properties over time, are poorly known. Therefore, this study aimed to determine the effect of 9-month storage at 4 °C with 30-day sampling on the pH, total phenolic, and flavonoid contents, free radical scavenging properties of kombucha fermented from black tea. Our results highlighted that, after four months, the phenolic content decreased significantly from the initial value of 234.1 ± 1.4 µg GAE mL-1 to 202.9 ± 2.1 µg GAE mL-1, as well its antioxidant capacity tested by two in vitro models, DPPH, and ABTS assays. Concomitantly, the pH value increased from 2.82 to 3.16. The novel findings of this pilot study revealed that kombucha from sugared black tea can be stored at refrigerator temperature for four months. After this period the antioxidant properties of kombucha are no longer retained.

Isolation and Purification of Glucans from an Italian Cultivar of Ziziphus jujuba Mill. and In Vitro Effect on Skin Repair.

Glucans possess a broad spectrum of biological activities. In this context, the present study was performed to isolate glucans from an Italian cultivar of Ziziphus jujuba Mill. at three different harvesting periods, in order to evaluate their effects on wound healing. The dry fruits were subjected to an alkaline extraction and then isolated glucans were purified by dialyzation. The crude and soluble samples were characterized by FT-IR and SEM analyses. Afterwards, total, α- and ß-glucan content was measured using an enzymatic procedure. The results highlighted that the glucan amount increased as the maturation proceeded as well as the ß-glucan percentage, which ranged from 48.2 at the first harvesting to 65.4 at the third harvesting. Furthermore, the effects of isolated glucans on the viability and migration of keratinocytes were evaluated using the in vitro MTT and scratch wound assays. The best proliferative effects on keratinocyte migration have been achieved with soluble glucans from third harvesting at 100 µM after 24 and 48 h (*** P < 0.001). The same treated group showed significant narrowing of the scratch area after 24 h and complete closure of the injury after 48 h. The findings highlighted the effectiveness of soluble glucans on regeneration of damaged skin.

Effect of Addition of Pectins from Jujubes (Ziziphus jujuba Mill.) on Vitamin C Production during Heterolactic Fermentation.

Soluble fibers, including pectins from apple and lemon, are commonly used as prebiotic and to prepare functional foods. The present study aimed to investigate the physicochemical and functional properties of pectins extracted from jujubes (Ziziphus jujuba Mill.). Pectins were extracted from jujubes at three stages of harvesting and characterized by FTIR and SEM analyses. Whole milk inoculated with kefir grains was supplemented by 0.25 mg·mL-1 of pectins. The pH value and vitamin C content were evaluated after 24 and 48 h of fermentation. Pectins from jujubes at the first harvesting stage (PJ1K) showed the lowest methoxylation degree. The addition of pectins enhanced the production of vitamin C during heterolactic process. This result was found to depend on jujube harvesting stage as PJ1K stimulated the growth of yeasts in kefir grains yielding to the highest amount of vitamin C (0.83 ± 0.01 µg·mL-1) compared to other samples (0.53-0.60 µg·mL-1) at 24 h. Lactic acid bacteria diminish pH rapidly with respect to control (4.13 ± 0.05), according to the stage of maturation, reducing its initial value by 38.3% in PJ1K. Besides being an excellent prebiotic, pectins from jujubes could be used to enrich kefir with vitamin C.

Quercetin-3-oleoyl derivatives as new GPR40 agonists: Molecular docking studies and functional evaluation.

The G-protein-coupled receptor 40 (GPR40) is an attractive molecular target for the treatment of type 2 diabetes mellitus. Previously, based on the natural oleic acid substrate, an exogenous ligand for this receptor, named AV1, was synthesized. In this context, here we validated the activity of AV1 as a full agonist, while the corresponding catechol analogue, named AV2, was investigated for the first time. The ligand-protein interaction between this new molecule and the receptor was highlighted in the lower portion of the GPR40 groove that generally accommodates DC260126. The functional assays performed have demonstrated that AV2 is a suitable GPR40 partial agonist, showing a therapeutic potential and representing a useful tool in the management of type 2 diabetes.

Quercetin, Epigallocatechin Gallate, Curcumin, and Resveratrol: From Dietary Sources to Human MicroRNA Modulation.

Epidemiologic studies suggest that dietary polyphenol intake is associated with a lower incidence of several non-communicable diseases. Although several foods contain complex mixtures of polyphenols, numerous factors can affect their content. Besides the well-known capability of these molecules to act as antioxidants, they are able to interact with cell-signaling pathways, modulating gene expression, influencing the activity of transcription factors, and modulating microRNAs. Here we deeply describe four polyphenols used as nutritional supplements: quercetin, resveratrol, epigallocatechin gallate (ECGC), and curcumin, summarizing the current knowledge about them, spanning from dietary sources to the epigenetic capabilities of these compounds on microRNA modulation.

9-cis Retinoic acid modulates myotrophin expression and its miR in physiological and pathophysiological cell models.

Functional studies indicate that essential cellular processes are controlled by Vitamin A derivatives. Among these the retinoic acid isoforms, all-trans- and 9-cis (9cRA), regulate the expression of various genes in both physiological and pathological conditions. Using several in vitro experimental models such as pancreatic ß-cells, pre-adipocytes and breast cancer cells with different phenotypes, we demonstrated the capability of 9cRA to modulate myotrophin (Mtpn) and miR-375 expressions. The 9cRA effect in pancreatic ß-cells line INS-1 832/13 point out a decreased expression of Mptn at both mRNA and protein levels associated to a concomitant increase of miR-375. We also studied the effect of this molecule on 3T3-L1 pre-adipocytes cells demonstrating a down-regulation of Mtpn and a dramatic increase of miR-375. Moreover, in the in vitro breast cancer model such as MDA-MB-231 and MCF-7 cells, 9cRA showed different effect on both Mtpn and miR-375 expression. In INS-1 832/13, 3T3-L1 pre-adipocytes and MCF-7 but not in MDA-MB-231, the effect of 9cRA on Mptn gene expression and its miR was under the control of RARs and RXRs receptors, as revealed by the exposure of these cell line to LE540 or HX603 receptor antagonists. In our findings 9cRA emerges has a hormone with a regulatory action on miR-375 that in most cases interfere with Mtpn expression.

Human kidney podocyte cell population as a novel biological target of nerve growth factor.

Human podocytes are highly specialized cells with a key role in kidney physiology. Alteration of their structure as a consequence of injury or developmental failure leads to severe renal diseases. Although several studies have tried to elucidate the molecular framework of this cellular system, the functional bases for the maintenance of podocytes in their specialized state to sustain kidney barrier filtration are not completely understood. In this study, the capability of podocytes to produce and secrete the nerve growth factor (NGF) has been demonstrated via a validated in vitro model. During the process of cell differentiation, NGF and its receptors are modulated in human podocytes just as NGF-responsive neurons. Blockade of NGF biological activity results in severe changes of cell morphology. Collectively, our results outline a novel function of the neurotrophin and add a new cellular target in the complex biological framework of NGF.

Apolipoprotein E and Alzheimer's Disease in Italian Population: Systematic Review and Meta-Analysis.

Objective: This meta-analysis with a systematic review was undertaken to assess the association between APOE allelic genotypes and the risk of Alzheimer's disease (AD) in the Italian population. Methods: The Web of Science, PubMed, and Scopus databases were searched until 15 November 2023. The odds ratio (OR) with a 95% confidence interval (CI) was calculated using fixed and random effect models, depending on the I2 statistic value. The systematic review and meta-analysis were conducted in agreement with the PRISMA guideline and registered with PROSPERO (CRD42023492580). Results: Our meta-analysis based on 15 studies revealed a higher risk of AD among Italian individuals carrying the APOE ε4 allele (OR = 3.60, 95% CI [2.90-4.47], p < 0.0001). The association of AD genotype APOE ε2ε4 (OR = 1.36, 95% CI [0.76-2.41], p = 0.29) was not statistically significant, while APOE ε3ε4 (OR = 3.43, 95% CI [2.95-3.99], p < 0.0001) has a high risk of AD development; the risk is more notably in the APOE ε4ε4 genotype (OR = 7.08, 95% CI [4.22-11.86], p < 0.0001). The APOE ε2 allele has a protective effect (APOE ε2 (OR = 0.47, 95% CI [0.29-0.74], p = 0.0013)), and similar results were achieved by APOE ε3 (OR = 0.49, 95% CI [0.37-0.65], p < 0.0001). Subgroup analysis of three areas of Italy (southern, northern, and center) revealed that that APOE ε4 allele was a risk factor with a higher OR in northern Italy (OR 4.22; 95% CI [3.46-5.16], p < 0.0001) compared to southern and center Italy (OR 3.02; 95% CI [2.28-4.01], p < 0.0001 and OR 3.97; 95% CI [1.37-11.56], p < 0.0001, respectively). As well, APOE ε4ε4 genotype carriers had a significantly higher OR in northern Italy (OR 9.69; 95% CI [4.94-18.99], p < 0.0001) compared to in southern and center Italy (OR 4.38; 95% CI [1.54-12.47], p < 0.0001 and OR 3.59; 95% CI [0.87-14.86], p < 0.0001, respectively). Conclusions: This systematic review with a meta-analysis of the Italian population on APOE alleles, genotyping, and AD incidence, highlights that individuals harboring APOE ε4 have a higher risk of developing AD compared to those with other alleles. It also supports the protective effect of the APOE ε2 allele against the progress of AD. The qualitative analysis on the complex genetic interactions influencing Alzheimer risk emphasizes the need for further research on genetic and environmental factors for effective prevention strategies.

FAM19A4 and hsa-miR124-2 Double Methylation as Screening for ASC-H- and CIN1 HPV-Positive Women.

The DNA methylation levels of host cell genes increase with the severity of the cervical intraepithelial neoplasia (CIN) grade and are very high in cervical cancer. Our study aims to evaluate FAM19A4 and hsa-miR124-2 methylation in Atypical Squamous cells with high-grade squamous intraepithelial lesions (ASC-H) and in CIN1, defined as low-grade squamous intraepithelial lesions (LSILs) by the Bethesda classification, as possible early warning biomarkers for managing women with high-risk HPV infections (hrHPV). FAM19A4 and hsa-miR124-2 methylation tests were conducted on fifty-six cervical screening samples from a subset of women aged 30-64 years old. Specimens were collected into ThinPrep PreservCyt Solution. Their HrHPV genotype and cytology diagnosis were known. A Qiasure (Qiagen) was used for FAM19A4 and hsa-miR124-2 methylation testing on bisulfite-converted DNA, according to the manufacturer's specifications. The reported results were hypermethylation-positive or -negative. We found that FAM194A4 and hsa-miR124-2 methylation was detected in 75% of ASC-H cases with a persistent infection of hrHPV. A total of 60% of CIN1 lesions were found to be positive for methylation, and 83.3% were when the cytology was CIN2/3. In addition, as a novelty of this pilot study, we found that combined FAM19A4 and hsa-miR124-2 methylation positivity rates (both methylated) were associated with the HPV genotypes 16, 18, and 59 and covered 22 and 25% of ASC-H and CIN1 cases, respectively. The methylation of these two genes, in combination with HPV genotyping, can be used as an early warning biomarker in the management and follow-up of women with ASC-H and CIN1 to avoid their progression to cervical cancer.

Determination of AGE-CML Level in Commercially Available Sports Bar in Italy.

INTRODUCTION: The process of bar creation involves improving the texture of the product to increase its palatability, which can be further induced by various physical or chemical changes during storage, such as sugar crystallization and molecular migrations in which Maillard's reaction occurs, forming the N-epsilon- (carboxymethyl) lysine (CML) adduct. In this study, we aimed to assess (the CML) adduct used in commercial bars today as meal substitutes or for athletic or sports purposes. The adduct CML is an advanced glycation end-product (AGEs) found in the human body (serum) and foods. It is the significant ligand for the Receptor for Advanced Glycation End Products (RAGE), resulting in chronic inflammation upon CML activation. Additionally, it aimed to assess the amount of AGEs-CML in various energy bars available on the Italian market. METHOD: CML OxiSelect ™ ELISA was used to assess the quantity of CML bars. The amount of AGE-CML was assessed in commercially available energy bars. RESULTS: According to the ELISA analysis, CML concentrations per g protein in all the tested energy bars varied from 138,42 to 1387,54 µg/gr per bar and from 461,41 to 3970,46 µg/gr per 100 gr of product, which depends on the quantity of protein. CONCLUSION: The amount per gram of protein is relatively uniform (with a variation of about 10%), and when compared to other foods, it is positioned in a medium-low range.

Localization of nerve growth factor (NGF) receptors in the mitochondrial compartment: characterization and putative role.

BACKGROUND: The neurotrophin NGF receptors trkA and p75NTR are expressed in the central and peripheral nervous system as well as in non-neuronal tissues; originally described to localize to the plasma membrane, recent studies have suggested other intracellular localizations for both NGF receptors. SCOPE OF REVIEW: In order to determine whether NGF receptors localize to the mitochondrial compartment mitochondria isolated from human kidney, rat tissues and a human podocyte as cell line before and after differentiation were used. MAJOR CONCLUSIONS: Our results demonstrate that NGF receptors are localized in the mitochondrial compartment of undifferentiated human podocytes and in all tissues analyzed including rat central nervous system. In mitochondria p75NTR, but not trkA, co-immunoprecipitates with the adenine nucleotide translocator (ANT) and the phosphodiesterase 4 isoform A5 (PDE4A5). Moreover, NGF, via trkA, protects isolated mitochondria of rat brain cortex from mitochondrial permeability transition induced by Ca(2+). GENERAL SIGNIFICANCE: Although NGF receptors have been described as mainly citoplasmatic so far, we proved evidence of their expression at the mitochondrial level and their interaction with specific proteins. Our results demonstrating the expression of NGF receptors in the mitochondria provide new insights into the role of NGF at subcellular level, in different areas of the organism, including CNS.

The Effect of Beta-Carotene on Cognitive Function: A Systematic Review.

ß-carotene is a powerful antioxidant and dietary precursor of vitamin A whose role in maintaining mental health and cognitive performance, either alone or in combination with other dietary compounds, has been a topic of recent research. However, its effectiveness is still unclear. This systematic review, conducted according to the PRISMA guideline and assisted by the MySLR platform, addressed this issue. A total of 16 eligible original research articles were identified. Dietary intake or ß-carotene serum levels were associated with improved measures of cognitive function in 7 out of 10 epidemiological studies included. In intervention studies, ß-carotene consumption alone did not promote better cognitive function in the short term, but only in a long-term intervention with a mean duration of 18 years. However, all but one intervention study suggested the beneficial effects of ß-carotene supplementation at doses ranging from 6 mg to 50 mg per day in combination with a multicomplex such as vitamin E, vitamin C, zinc, or selenium for a period of 16 weeks to 20 years. Despite the current limitations, the available evidence suggests a potential association between ß-carotene dietary/supplementary intake and the maintenance of cognitive function. The ß-carotene most probably does not act alone but in synergy with other micronutrients.

Special Issue "GPCRs: Ligands and beyond 2022".

The human genome encodes more than 800 different G protein-coupled receptors (GPCRs), uncovering their importance in human physiology [...].

The First Identification in Italy of SARS-CoV-2 Omicron BA.4 Harboring KSF141_del: A Genomic Comparison with Omicron Sub-Variants.

The rapid emergence and worldwide detection of the SARS-CoV-2 Omicron variant underscore the importance of robust genomic surveillance systems and prompt information sharing among global public health partners. The Omicron variant has rapidly replaced the Delta variant as a dominating SARS-CoV-2 variant because of natural selection, favoring the variant with higher infectivity and stronger vaccine breakthrough capability. The Omicron variant is also known as B.1.1.529. It has four sub-variants, indicated as BA.1, BA.2, BA.3 and BA.4. Among them, BA.1 is the currently prevailing sub-variant, and BA.2 has been found to be able to alarmingly re-infect patients initially infected by Omicron BA.1. The BA.3 sub-variant is a combination of mutations of BA.1 and BA.2, especially in the spike protein. Today, the BA.4 variant is emerging, which is herein described, and it was the first detected in Italy. Via bioinformatic analysis, we are reporting that the BA.4 that was identified harbors a new mutation, specifically a deletion in the ORF1ab gene, corresponding to KSF141_del in non-structural protein 1 (nsp1), a critical virulence factor able to suppress host translation. The bioinformatics comparison analysis with the other three sub-variants reveals that the deletion was not present before and was never reported until now. Therefore, we can speculate that Omicron BA.4 will become a new dominating "variant of concern" and may also break vaccine protection. Moreover, we show that other proteins are mutated in the BA.4. In particular, seven mutations are recognized in the nucleocapsid (N) protein, and the capability of five different types of rapid antigenic tests are used to identify it.

Olive Oil Lipophenols Induce Insulin Secretion in 832/13 ß-Cell Models.

Glycemic control is a mainstay of type 2 diabetes mellitus (T2DM) clinical management. Despite the continuous improvement in knowledge and progress in terms of treatment, the achievement of the physiologic metabolic profile is still an ongoing challenge in diabetic patients. Pancreatic ß-cell line INS-1 832/13 was used to assess the insulin secretagogue activity of hydroxytyrosyl oleate (HtyOle) and tyrosyl oleate (TyOle), two naturally occurring lipophenols deriving from the conjugation of oleic acid (OA) and hydroxytyrosol (Hty) or tyrosol (Ty), respectively. The insulin secretion was determined under a glucose-induced insulin secretion (GSIS) condition by the ELISA method. The potential involvement of G-protein-coupled receptor 40 (GPR40), also known as free fatty acid receptor 1 (FFAR1), was investigated by both molecular docking and functional pharmacological approaches. Herein, we demonstrated that HtyOle and TyOle exerted a facilitatory activity on insulin secretion under the GSIS condition. Moreover, we provided evidence that both lipophenols are natural modulators of FFAR1 receptor. From our results, the anti-diabetes properties associated with olive oil consumption can be partly explained by the HtyOle and TyOle effects.

Exosome microRNAs in Metabolic Syndrome as Tools for the Early Monitoring of Diabetes and Possible Therapeutic Options.

Exosomes are nano-sized extracellular vesicles produced and released by almost all cell types. They play an essential role in cell-cell communications by delivering cellular bioactive compounds such as functional proteins, metabolites, and nucleic acids, including microRNA, to recipient cells. Thus, they are involved in various physio-pathological conditions. Exosome-miRNAs are associated with numerous diseases, including type 2 diabetes, a complex multifactorial metabolic disorder linked to obesity. In addition, exosome-miRNAs are emerging as essential regulators in the progression of diabetes, principally for pancreatic ß-cell injury and insulin resistance. Here, we have clustered the recent findings concerning exosome-miRNAs associated with ß-cell dysfunction to provide a novel approach for the early diagnosis and therapy of diabetes.