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.

Acute effects of a chewable beetroot-based supplement on cognitive performance: a double-blind randomized placebo-controlled crossover clinical trial.

BACKGROUND: Dietary nitrate (NO3-) has been shown to be useful as an ergogenic aid with potential applications in health and disease (e.g., blood pressure control). However, there is no consensus about the effects of dietary NO3- or beetroot (BR) juice supplementation on cognitive function. OBJECTIVE: The aim of this study was to evaluate the effects of a single dose of a chewable BR-based supplement on cognitive performance. METHODS: A double-blind randomized placebo-controlled two-period crossover clinical trial was carried out based on the extension of the CONSORT guidelines for randomized crossover trials. A total of 44 participants (24 F; 20 M; 32.7 [12.5] years; 66.3 [9.0] kg; 170 [9.2] cm; 22.8 [1.4] kg/m2) were randomly allocated to receive first either four BR-based chewable tablets (BR-CT) containing 3 g of a Beta vulgaris extract (RedNite®) or four tablets of a placebo (maltodextrin). A 4-day washout period was used before crossover. Ninety minutes after ingestion of the treatments, a neuropsychological testing battery was administered in each period. The trial was registered at clinicaltrials.gov NCT05509075. RESULTS: Significant improvements with moderate effect size were found on memory consolidation at the short and long term only after BR-CT supplementation via the Rey Auditory Verbal Learning Test immediate (+ 20.69%) and delayed (+ 12.34%) recalls. Likewise, enhancement on both frontal lobe functions (+ 2.57%) and cognitive flexibility (+ 11.16%) were detected after BR-CT. There was no significant change (p < 0.05) on verbal memory of short-term digits, working memory and information processing speed. Mixed results were found on mood and anxiety through the Beck Depression Inventory-II (BDI-II) and the State-Trait Anxiety Inventory (STAI-Y1 and STAI-Y2); however, sequence and period effects were seen on STAI-Y2. CONCLUSIONS: The acute administration of a chewable BR-based supplement improves certain aspects of cognitive function in healthy females and males, particularly memory capacity and frontal skills.

Observational Study on a Large Italian Population with Lipedema: Biochemical and Hormonal Profile, Anatomical and Clinical Evaluation, Self-Reported History.

We analyzed the medical condition of 360 women affected by lipedema of the lower limbs in stages 1, 2, and 3. The data were assessed for the whole population and compared between different clinical stages, distinguishing between obese and non-obese patients. The most frequent clinical signs were pain when pinching the skin, subcutaneous nodules, and patellar fat pads. The most frequently painful site of the lower limbs was the medial lower third of the thigh. The pain score obtained on lower limb points increased progressively with the clinical stage. In all points evaluated, the thickness of the subcutaneous tissue increased with the clinical stage. Analyzing the data on the lower medial third of the leg and considering only patients with type 3 lipedema, the difference between stages was statistically significant after correction for age and BMI. We found higher levels of C-reactive protein at more severe clinical stages, and the difference was significant after correction for age and BMI between the stages. Overall, the prevalence of alterations of glucose metabolism was 34%, with a progressive increase in prevalence with the clinical stage. The most frequent comorbidities were vitamin D insufficiency, chronic venous disease, allergies, dyslipidemia, headache, and depression of mood. Interestingly, in comparison with the general population, we found higher prevalence of chronic autoimmune thyroiditis and polycystic ovary syndrome. Finally, the clinical stage and the involvement of the upper limbs or obesity suggest a worse clinical, anthropometric, and endocrine-metabolic profile.

Comparing Nutritional Values and Bioactivity of Kefir from Different Types of Animal Milk.

The growing interest in fermented dairy products is due to their health-promoting properties. The use of milk kefir grains as a starter culture made it possible to obtain a product with a better nutritional and biological profile depending on the type of milk. Cow, buffalo, camel, donkey, goat, and sheep milk kefirs were prepared, and the changes in sugar, protein, and phenol content, fatty acid composition, including conjugated linoleic acids (CLAs), as well as antioxidant activity, determined by ABTS and FRAP assays, were evaluated and compared. The protein content of cow, buffalo, donkey, and sheep milk increased after 24 h of fermentation. The fatty acid profile showed a better concentration of saturated and unsaturated lipids in all fermented milks, except buffalo milk. The highest content of beneficial fatty acids, such as oleic, linoleic, and C18:2 conjugated linoleic acid, was found in the cow and sheep samples. All samples showed a better antioxidant capacity, goat milk having the highest value, with no correlation to the total phenolic content, which was highest in the buffalo sample (260.40 ± 5.50 µg GAE/mL). These findings suggested that microorganisms living symbiotically in kefir grains utilize nutrients from different types of milk with varying efficiency.

SARS CoV-2 spike protein-guided exosome isolation facilitates detection of potential miRNA biomarkers in COVID-19 infections.

OBJECTIVES: Nearly three years into the pandemic, SARS-CoV-2 infections are occurring in vaccinated and naturally infected populations. While humoral and cellular responses in COVID-19 are being characterized, novel immune biomarkers also being identified. Recently, an increase in angiotensin-converting enzyme 2 expressing (aka, ACE2 positive) circulating exosomes (ExoACE2) were identified in the plasma of COVID-19 patients (El-Shennawy et al.). In this pilot study, we describe a method to characterize the exosome-associated microRNA (exo-miRNA) signature in ACE2-positive and ACE2-negative exosomal populations (non-ExoACE2). METHODS: We performed a sorting protocol using the recombinant biotin-conjugated SARS CoV-2 spike protein containing the receptor binding domain (RBD) on plasma samples from six patients. Following purification, exo-miRNA were characterized for ACE2-positive and ACE2-negative exosome subpopulations by RT-PCR. RESULTS: We identified differential expression of several miRNA. Specifically let-7g-5p and hsa-miR-4454+miR-7975 were upregulated, while hsa-miR-208a-3p and has-miR-323-3p were downregulated in ExoACE2 vs. non-ExoACE2. CONCLUSIONS: The SARS CoV-2 spike-protein guided exosome isolation permits isolation of ExoACE2 exosomes. Such purification facilitates detailed characterization of potential biomarkers (e.g. exo-miRNA) for COVID-19 patients. This method could be used for future studies to further the understanding mechanisms of host response against SARS CoV-2.

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.

The use of Lactobacillus casei DG® prevents symptomatic episodes and reduces the antibiotic use in patients affected by chronic bacterial prostatitis: results from a phase IV study.

PURPOSE: To evaluate the efficacy of Lactobacillus paracasei CNCM I-1572 (L. casei DG®) in both prevention of symptomatic recurrences and improvement of quality of life in patients with chronic bacterial prostatitis (CBP). METHODS: Patients with CBP attending a single Urological Institution were enrolled in this phase IV study. At enrollment, all patients were treated with antibiotics in agreement with EAU guidelines and then were treated with L. casei DG® (2 capsules/day for 3 months). Clinical and microbiological analyses were carried out before (enrollment, T0) and 6 months (T2) after the treatment. Both safety and adherence to the treatment were evaluated 3 months (T1) after the enrollment. NIH Chronic Prostatitis Symptom Index (CPSI), International Prostate Symptom Score (IPSS) and Quality of Well-Being (QoL) questionnaires were used. The outcome measures were the rate of symptomatic recurrence, changes in questionnaire symptom scores and the reduction of antibiotic use. RESULTS: Eighty-four patients were included. At T2, 61 patients (72.6%) reported a clinical improvement of symptoms with a return to their clinical status before symptoms. A time dependent improvement in clinical symptoms with significant changes in NIH-CPSI, IPSS and QoL (mean difference T2 vs T0: 16.5 ± 3.58; - 11.0 ± 4.32; + 0.3 ± 0.09; p < 0.001), was reported. We recorded that L. casei DG® treatment induced a statistically significant decrease in both (p < 0.001) symptomatic recurrence [1.9/3 months vs 0.5/3 months] and antibiotic use [- 7938 UDD]. No clinically relevant adverse effects were reported. CONCLUSIONS: L. casei DG® prevents symptomatic recurrences and improves the quality of life in patients with CBP, reducing the antibiotic use.

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).

Sarcopenia: Etiology, Nutritional Approaches, and miRNAs.

Sarcopenia, an age-related decline in skeletal muscle mass and function, dramatically affects the quality of life. Although there is a consensus that sarcopenia is a multifactorial syndrome, the etiology and underlying mechanisms are not yet delineated. Moreover, research about nutritional interventions to prevent the development of sarcopenia is mainly focused on the amount and quality of protein intake. The impact of several nutrition strategies that consider timing of food intake, anti-inflammatory nutrients, metabolic control, and the role of mitochondrial function on the progression of sarcopenia is not fully understood. This narrative review summarizes the metabolic background of this phenomenon and proposes an integral nutritional approach (including dietary supplements such as creatine monohydrate) to target potential molecular pathways that may affect reduce or ameliorate the adverse effects of sarcopenia. Lastly, miRNAs, in particular those produced by skeletal muscle (MyomiR), might represent a valid tool to evaluate sarcopenia progression as a potential rapid and early biomarker for diagnosis and characterization.

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.

Nano-hydrogel embedded with quercetin and oleic acid as a new formulation in the treatment of diabetic foot ulcer: A pilot study.

Wound healing, especially diabetic ones, is a relevant clinical problem, so it is not surprising that surgical procedures are often needed. To overcome invasive procedures, several strategies with drugs or natural compound are used. Recently, in an experimental study, we described an increase in keratinocyte proliferation after their exposition to quercetin plus oleic acid. In the present clinical study, we evaluated both the clinical efficacy and the safety of nano-hydrogel embedded with quercetin and oleic acid in the treatment of lower limb skin wound in patients with diabetes mellitus (DM). Fifty-six DM patients (28 men and 28 women, mean age 61.7 ± 9.2 years) unsuccessfully treated with mechanical compression were enrolled and randomised to receive an add on treatment with hyaluronic acid (0.2%) or nano-hydrogel embedded with quercetin and oleic acid. The treatment with nano-hydrogel embedded with quercetin and oleic acid significantly (P < .01) reduced the wound healing time, in comparison to hyaluronic acid (0.2%) without developing of adverse drug reactions, suggesting that this formulation could be used in the management of wound healing even if other clinical trials must be performed in order to validate this observation.

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.

Influence of all-trans retinoic acid on sperm metabolism and oxidative stress: Its involvement in the physiopathology of varicocele-associated male infertility.

The mechanisms by which varicocele affects fertility remain undetermined. Vitamin A (all-trans retinoic acid [ATRA]) is required for fertility and normal spermatogenesis; however, the mechanisms driving its action are not defined yet. Previously, we demonstrated in varicocele sperm a reduced RARα expression and that ATRA influence sperm performance. To further define vitamin A significance in male gamete and in the physiopathology of varicocele, we tested for the first time ATRA action on human sperm metabolism and antioxidant defense systems. Evaluating triglycerides content and lipase activity, in normal sperm ATRA had a lipid lowering effect, which was not observed in varicocele sperm. The modulation of the glucose-6-phosphate dehydrogenase activity, concomitantly with a reduction of the glucose content, highlight an ATRA role on glucose metabolism. ATRA induced the superoxide dismutase (SOD) and glutathione transferase activities, while it reduced the malondialdehyde and reactive oxygen species (ROS) production both in healthy and varicocele sperm. Interestingly, SOD1 and SOD2 have been localized in the acrosome and midpiece, glutathione- S-transferase omega 2 (GSTO2) in the acrosome, equatorial, and subacrosomial regions. SOD1, SOD2, and GSTO2 levels were significantly lower in varicocele with respect to healthy sperm. Herein, we discovered that ATRA treatment was able to reprogram sperm metabolism toward that of the capacitation status. The retinol protected human sperm from ROS damage enhancing the antioxidant enzymes activity, providing evidence toward the efficacy of vitamin A as therapeutic tool in improving sperm quality. These novel findings further confirm the importance of vitamin A in male fertility adding new insights into the retinoids complex biological framework.

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.

Estradiol via estrogen receptor beta influences ROS levels through the transcriptional regulation of SIRT3 in human seminoma TCam-2 cells.

Human testis, gonocytes, and adult germ cells mainly express estrogen receptor beta, and estrogen receptor beta loss is associated with advanced tumor stage; however, the molecular mechanisms of estrogen receptor beta-protective effects are still to be defined. Herein, we provide evidence that in human seminoma TCam-2 cells, E2 through estrogen receptor beta upregulates the mitochondrial deacetylase sirtuin-3 at protein and messenger RNA levels. Specifically, E2 increases sirtuin-3 expression through a transcriptional mechanism due to the occupancy of sirtuin-3 promoter by estrogen receptor beta, together with the transcription factor Sp1 as evidenced by Chip reChIp assay. This complex binds to a GC cluster located between -128 bp/+1 bp and is fundamental for E2 effects, as demonstrated by Sp1 small interfering RNA studies. Beside, after 24 h, E2 stimulus significantly increased activities of superoxide dismutase and catalase to scavenge reactive oxygen species produced by 30 min of E2 stimulus. In summary, this article indicates a novel functional interplay between estrogen receptor beta and sirtuin-3 counteracting reactive oxygen species production in TCam-2 cells. Our findings thus show that an important tumor-suppressive pathway through estrogen receptor beta is target of E2, actually proposing a distinctive protecting action against seminoma. Future studies may lead to additional strategies for the current therapy of seminoma.