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Vitamin D is a steroid hormone classically involved in the calcium metabolism and bone homeostasis. Recently, new and interesting aspects of vitamin D metabolism has been elucidated, namely the special role of the skin, the metabolic control of liver hydroxylase CYP2R1, the specificity of 1α-hydroxylase in different tissues and cell types and the genomic, non-genomic and epigenomic effects of vitamin D receptor, which will be addressed in the present review. Moreover, in the last decades, several extraskeletal effects which can be attributed to vitamin D have been shown. These beneficial effects will be here summarized, focusing on the immune system and cardiovascular system.

(1) Background: In recent years, numerous studies have highlighted the beneficial effects of extra virgin olive oil (EVOO) as an active ingredient against chronic diseases. The properties of EVOO are due to its peculiar composition, mainly to its rich content of polyphenols. In fact, polyphenols may contribute to counteract oxidative stress, which often accompanies chronic diseases. In this work, the antioxidant effects of high-value polyphenol oleocanthal (OC) and its main metabolites, tyrosol (Tyr) and oleocanthalic acid (OA), respectively, have been investigated along with their impact on cell viability. (2) Methods: OC, Tyr, and OA have been evaluated regarding antiradical properties in term of scavenging capacity towards biologically relevant reactive species, including O2●−, HOCl, and ROO●, as well as their antioxidant/antiradical capacity (FRAP, DPPH●, ABTS●+). Moreover, the ability to permeate the intestinal membrane was assessed by an intestinal co-culture model composed by Caco-2 and HT29-MTX cell lines. (3) Results: The capacity of OC and Tyr as radical oxygen species (ROS) scavengers, particularly regarding HOCl and O2●−, was clearly demonstrated. Furthermore, the ability to permeate the intestinal co-culture model was plainly proved by the good permeations (>50%) achieved by all compounds. (4) Conclusions: OC, OA, and Tyr revealed promising properties against oxidative diseases.

Probiotics are microorganisms with recognized beneficial properties that are used to improve host health. In particular, probiotics administered as spores, such as those belonging to the genera Bacillus and Alkalihalobacillus, are attracting great interest due to their high tolerance to gastrointestinal conditions. This in vitro study aimed to assess the probiotic attributes potentially contributing to the in vivo beneficial effects of a commercial spore-based probiotic formulation composed of four Alkalihalobacillus clausii strains. The tolerance and survival of the spores from the formulation in simulated gastrointestinal fluids, as well as their germination rate and adhesion to mucins, were analyzed. Furthermore, metabolic properties of spore-derived vegetative cells were assessed, including lactose degradation and biosynthesis of antioxidant enzymes (catalase and superoxide dismutase), group B vitamins (B2, B8, B9, and B12), short-chain fatty acids (acetate, propionate, and butyrate), and D-lactate. A. clausii spores were shown to survive in artificial gastric juice, adhere to mucins and germinate in vitro, and replicate in simulated intestinal fluid, suggesting their potential resilience in the gastrointestinal tract, where they can exert beneficial effects after germination. A. clausii was also able to produce beneficial enzymes and metabolites, including β-galactosidase, catalase, superoxide dismutase, group B vitamins, and short-chain fatty acids, but it was unable to produce D-lactic acid. Our findings highlight the probiotic properties and potential of such A. clausii strains in both their spore and vegetative forms, reinforcing the clinical relevance of this multi-strain spore-based formulation for enhancing intestinal health.

We took advantage of the fluorescent features of a serotonin-derived fluorophore to develop a simple and low-cost assay for copper in urine. The quenching-based fluorescence assay linearly responds within the concentration range of clinical interest in buffer and in artificial urine, showing very good reproducibility (CVav% = 4% and 3%) and low detection limits (16 ± 1 μg L−1 and 23 ± 1 μg L−1). The Cu2+ content was also estimated in human urine samples, showing excellent analytical performances (CVav% = 1%), with a limit of detection of 59 ± 3 μg L−1 and a limit of quantification of 97 ± 11 μg L−1, which are below the reference value for a pathological Cu2+ concentration. The assay was successfully validated through mass spectrometry measurements. To the best of our knowledge, this is the first example of copper ion detection exploiting the fluorescence quenching of a biopolymer, offering a potential diagnostic tool for copper-dependent diseases.

The human retinal pigment epithelium-specific 65-kDa protein (hRPE65) plays a crucial role within the retinoid visual cycle and several mutations affecting either its expression level or its enzymatic function are associated with inherited retinal diseases such as Retinitis Pigmentosa. The gene therapy product voretigene neparvovec (Luxturna) has been recently approved for treating hereditary retinal dystrophies; however, the treatment is currently accessible only to patients presenting confirmed biallelic mutations that severely impair hRPE65 function, and many reported hRPE65 missense mutations lack sufficient evidences for proving their pathogenicity. In this context, we developed a computational approach aimed at evaluating the potential pathogenic effect of hRPE65 missense variants located on the dimerisation domain of the protein. The protocol evaluates how mutations may affect folding and conformation stability of this protein region, potentially helping clinicians to evaluate the eligibility for gene therapy of patients diagnosed with this type of hRPE65 variant of uncertain significance.

Aims/hypothesisThe glucosuria induced by sodium–glucose cotransporter 2 (SGLT2) inhibition stimulates endogenous (hepatic) glucose production (EGP), blunting the decline in HbA1c. We hypothesised that, in response to glucosuria, a renal signal is generated and stimulates EGP. To examine the effect of acute administration of SGLT2 inhibitors on EGP, we studied non-diabetic individuals who had undergone renal transplant with and without removal of native kidneys.MethodsThis was a parallel, randomised, double-blind, placebo-controlled, single-centre study, designed to evaluate the effect of a single dose of dapagliflozin or placebo on EGP determined by stable-tracer technique. We recruited non-diabetic individuals who were 30–65 years old, with a BMI of 25–35 kg/m2 and stable body weight (±2 kg) over the preceding 3 months, and HbA1c <42 mmol/mol (6.0%). Participants had undergone renal transplant with and without removal of native kidneys and were on a stable dose of immunosuppressive medications. Participants received a single dose of dapagliflozin 10 mg or placebo on two separate days, at a 5- to 14-day interval, according to randomisation performed by our hospital pharmacy, which provided dapagliflozin and matching placebo, packaged in bulk bottles that were sequentially numbered. Both participants and investigators were blinded to group assignment.ResultsTwenty non-diabetic renal transplant patients (ten with residual native kidneys, ten with bilateral nephrectomy) participated in the study. Dapagliflozin induced greater glucosuria in individuals with residual native kidneys vs nephrectomised individuals (8.6 ± 1.1 vs 5.5 ± 0.5 g/6 h; p = 0.02; data not shown). During the 6 h study period, plasma glucose decreased only slightly and similarly in both groups, with no difference compared with placebo (data not shown). Following administration of placebo, there was a progressive time-related decline in EGP that was similar in both nephrectomised individuals and individuals with residual native kidneys. Following dapagliflozin administration, EGP declined in both groups, but the differences between the decrement in EGP with dapagliflozin and placebo in the group with bilateral nephrectomy (Δ = 1.11 ± 0.72 μmol min−1 kg−1) was significantly lower (p = 0.03) than in the residual native kidney group (Δ = 2.56 ± 0.33 μmol min−1 kg−1). In the population treated with dapagliflozin, urinary glucose excretion was correlated with EGP (r = 0.34, p < 0.05). Plasma insulin, C-peptide, glucagon, prehepatic insulin:glucagon ratio, lactate, alanine and pyruvate concentrations were similar following placebo and dapagliflozin treatment. β-Hydroxybutyrate increased with dapagliflozin treatment in the residual native kidney group, while a small increase was observed only at 360 min in the nephrectomy group. Plasma adrenaline (epinephrine) did not change after dapagliflozin and placebo treatment in either group. Following dapagliflozin administration, plasma noradrenaline (norepinephrine) increased slightly in the residual native kidney group and decreased in the nephrectomy group.Conclusions/interpretationIn nephrectomised individuals, the hepatic compensatory response to acute SGLT2 inhibitor-induced glucosuria was attenuated, as compared with individuals with residual native kidneys, suggesting that SGLT2 inhibitor-mediated stimulation of hepatic glucose production via efferent renal nerves occurs in an attempt to compensate for the urinary glucose loss (i.e. a renal–hepatic axis).Trial registrationClinicalTrials.gov NCT03168295FundingThis protocol was supported by Qatar National Research Fund (QNRF) Award No. NPRP 8-311-3-062 and NIH grant DK024092-38.Graphical abstract

N-acetylaspartate (NAA) is the second most abundant metabolite in the human brain. Quantifiable amounts of NAA are also present in the blood, but its role in the peripheral tissues is largely unknown. First, we determined the acute effects of insulin administration on NAA concentrations; second, we assessed whether circulating NAA levels associate with markers of central and peripheral insulin sensitivity. A total of 24 persons living with obesity and 19 healthy, lean controls, without neurological disorders, underwent a euglycemic hyperinsulinemic clamp combined with fluorodeoxyglucose positron emission tomography ([18F]FDG-PET) imaging of the brain, abdomen, and femoral area. Plasma concentrations of NAA were measured at baseline and ~2 h into the clamp using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS-MS). Glucose uptake (GU) rates were analysed using a fractional uptake rate. Serum acetate levels were also assessed using nuclear magnetic resonance (NMR) metabolomics. From baseline to steady-state, insulin levels increased from a mean level of 66 to 447 pmol/L (p < 0.0001). Over this period, circulating NAA concentrations decreased by 5% (p = 0.01), similarly in both groups. The change in NAA was inversely related with the change in plasma acetate (r = −0.36, p = 0.048). Circulating NAA was associated with waist–hip ratio (rho = −0.54, p = 0.0002), steady-state free fatty acids (rho = −0.44, p = 0.003), and directly with HDL cholesterol (rho = 0.54, p = 0.0002), adiponectin (rho = 0.48, p = 0.003), and whole-body insulin sensitivity (rho = 0.34, p = 0.03). Circulating NAA was directly related with skeletal muscle (rho = 0.42, p = 0.01) and visceral adipose tissue GU (rho = 0.41, p = 0.02). Insulin administration leads to a small decrease in circulating NAA levels, and NAA associates consistently with markers of insulin sensitivity. While plasma NAA may be relevant to aspects of whole-body homeostasis, mechanistic insights are needed.

hRPE65 is a fundamental enzyme of the retinoid visual cycle, and many missense mutations affecting its expression or function are associated with a wide range of diseases. Many hRPE65 missense mutations lack a clear pathogenicity classification or are labelled as VUS. In this context, we recently developed a protocol based on µs-long molecular dynamics simulations to study the potential pathogenic effect of hRPE65 missense mutations. In the present work, the structure-based protocol was integrated with a hRPE65-tailored consensus bioinformatics strategy, named ConPath, that showed high performance in predicting known pathogenic/benign hRPE65 missense mutations. The combined strategy was used to perform a multi-level evaluation of the potential pathogenicity of 13 different hRPE65 VUS, which were classified based on their likelihood of pathogenic effect. The obtained results provide information that may support the reclassification of these VUS and help clinicians evaluate the eligibility for gene therapy of patients diagnosed with such variants.

Background/Objectives: This study investigated susceptibility factors that may contribute to Post-Polio Syndrome (PPS) in elderly polio survivors. Methods: Serum immunoglobulin (Ig) levels, poliovirus neutralizing antibodies (PV NAb), and vitamin D status were evaluated in 80 PPS patients, 40 family members, and 89 healthy controls. Results: A significant number of PPS patients and their family members showed reduced levels of total IgG and/or IgA, and specific IgG subclasses, indicating a high prevalence of primary humoral immunodeficiencies within these groups. Despite these Ig deficits, PV NAb titers were similar across all groups, indicating high protection against poliovirus, likely due to vaccination campaigns with live virus in Italy and intense exposure to poliovirus, especially in long-term rehabilitation institutions. However, a small group of PPS subjects lacked neutralizing antibodies for specific poliovirus serotypes, suggesting more severe antibody deficiencies. Additionally, PPS subjects had a high prevalence of vitamin D deficiency, which likely increases their risk for osteoporosis/osteopenia and fractures. It is unclear if this deficiency was also present in their infancy, potentially enhancing their susceptibility to poliovirus. Conclusions: Overall, the findings indicate that genetic, immunological, or nutritional factors may increase individual susceptibility to the pathogenic effects of poliovirus. This study—limited to serum antibodies—highlights the complex relationship between immune status and long-term health in aging polio survivors. The results emphasize the need for potent poliovirus drugs and vaccines to help contain possible outbreaks but also—for poliomyelitis survivors—to avoid or mitigate the progression to PPS, the latest phase of this devastating disease.

The current guidelines for sweat chloride analysis identify the procedures for sweat collection, but not for chloride assay, which is usually performed by methods originally not aiming at the low concentrations of chloride found in sweat. To overcome this limitation, we set up, characterized, and adopted an original inductively coupled plasma mass spectrometry (ICP-MS) method for sweat chloride determination, which was designed for its easy use in a clinical laboratory. The method was linear in the range 8.5E−3 to 272.0E−3 mM, precision exhibited a relative standard deviation < 6%, and accuracy was in the range 99.7–103.8%. Limit of blank, limit of detection, and limit of quantitation were 2.1 mM, 3.2 mM, and 7.0 mM, respectively, which correspond to real concentrations injected into the mass spectrometer of 3.9E−3 mM for LOD and 8.5E−3 mM for LOQ. At first, the method was tested on 50 healthy volunteers who exhibited a mean chloride concentration of 15.7 mM (25–75th percentile 10.1–19.3 mM, range 2.8–37.4 mM); then, it was used to investigate two patients with suspected cystic fibrosis, who exhibited sweat chloride values of 65.6 mM and 81.2 mM, respectively. Moreover, the method was cross-validated by assaying 50 samples with chloride concentration values in the range 10–131 mM, by both ICP-MS and coulometric titration, which is the technology officially used in Tuscany for cystic fibrosis newborn screening. The reference analytical performances and the relatively low cost of ICP-MS, accompanied by the advantageous cost of a single sweat chloride assay, make this technology the best candidate to provide a top reference method for the quantification of chloride in sweat. The method that we propose was optimized and validated for sweat samples ≥ 75 mg, which is the minimum amount requested by the international protocols. However, the method sensitivity and, in addition, the possibility to reduce the sample dilution factor, make possible the quantification of chloride even in samples weighting < 75 mg that are discarded according to the current guidelines.