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Abstract IP3 receptor (IP3R)-mediated Ca2+ transfer at the mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) drives mitochondrial Ca2+ uptake and oxidative metabolism and is linked to different pathologies, including Parkinson’s disease (PD). The dependence of Ca2+ transfer efficiency on the ER-mitochondria distance remains unexplored. Employing molecular rulers that stabilize ER-mitochondrial distances at 5 nm resolution, and using genetically encoded Ca2+ indicators targeting the ER lumen and the sub-mitochondrial compartments, we now show that a distance of ~20 nm is optimal for Ca2+ transfer and mitochondrial oxidative metabolism due to enrichment of IP3R at MERCS. In human iPSC-derived astrocytes from PD patients, 20 nm MERCS were specifically reduced, which correlated with a reduction of mitochondrial Ca2+ uptake. Stabilization of the ER-mitochondrial interaction at 20 nm, but not at 10 nm, fully rescued mitochondrial Ca2+ uptake in PD astrocytes. Our work determines with precision the optimal distance for Ca2+ flux between ER and mitochondria and suggests a new paradigm for fine control over mitochondrial function.

AbstractObjective To evaluate the effects of alpha lipoic acid (ALA) on hormonal and metabolic parameters in a group of overweight/obese Polycystic Ovary Syndrome (PCOS) patients.Methods This was a retrospective study in which thirty-two overweight/obese patients with PCOS (n = 32) not requiring hormonal treatment were selected from the database of the ambulatory clinic of the Gynecological Endocrinology Center at the University of Modena and Reggio Emilia, Italy. The hormonal profile, routine exams and insulin and C-peptide response to oral glucose tolerance test (OGTT) were evaluated before and after 12 weeks of complementary treatment with ALA (400 mg/day). Hepatic Insulin Extraction (HIE) index was also calculated.Results ALA administration significantly improved insulin sensitivity and decreased ALT and AST plasma levels in all subjects, though no changes were observed on reproductive hormones. When PCOS patients were subdivided according to the presence or absence of familial diabetes background, the higher effects of ALA were observed in the former group that showed AST and ALT reduction and greater HIE index decrease.Conclusion ALA administration improved insulin sensitivity in overweight/obese PCOS patients, especially in those with familial predisposition to diabetes. ALA administration improved both peripheral sensitivity to insulin and liver clearance of insulin. Such effects potentially decrease the risk of nonalcoholic fat liver disease and diabetes in PCOS patients.

Background: Combined oral contraceptives (COCs) work mostly by preventing the pre-ovulatory gonadotropin surge, but the action of COCs on spontaneous episodic and GnRH (gonadotropin-releasing hormone)-induced LH (luteinizing hormone) release has been poorly evaluated. Oral contraceptives are known to act on the spontaneous hypothalamic–pituitary functions reducing both GnRH and gonadotropin release and blocking ovulation. Aim: To evaluate spontaneous and GnRH-induced LH release during both phases of the menstrual cycle or under the use of the contraceptive pill. Methods: A group of 12 women, subdivided into two groups, volunteered for the study. Group A (n = 6, controls) received no treatments, while Group B (n = 6) received a 21 + 7 combination of ethinyl-estradiol (EE) 30 µg + drospirenone (DRSP) 3 mg. Both groups were evaluated twice: Group A during follicular and luteal phases, Group B during pill assumption and during the suspension interval, performing a pulsatility test, GnRH stimulation test, and hormonal parameters evaluation. Spontaneous and GnRH-induced secretory pulses were evaluated, as well as the instantaneous secretory rate (ISR). Results: COC treatment lowered LH and FSH (follicle stimulating hormone) levels significantly if compared to the follicular phase of spontaneous cycles. During the suspension interval, hormone levels rapidly rose and became comparable to those of the follicular phase of the control group. The LH pulse frequency under COC administration during the suspension interval was similar to that observed during the follicular phase (2.6 ± 0.3 pulses/180 min and 2.3 ± 0.2 pulses/180 min, respectively). The GnRH-induced LH peaks were greater in amplitude and duration than those observed after ISR computation in both groups. The GnRH-induced LH release during the luteal phase of the control subjects was higher than in the follicular phase (51.2 ± 12.3 mIU/mL and 14.9 ± 1.8 mIU/mL, respectively). Conversely, subjects under COC showed a GnRH-induced LH response similar during COC and during the suspension interval. Conclusions: Our data support that the EE + DRSP preparation acts on both spontaneous pulsatile release and GnRH-induced LH release during the withdrawal period of the treatment, and that after 5–7 days from the treatment suspension, steroidal secretion from the ovary is resumed, such as that of androgens. This suggests that in hyperandrogenic patients, a suspension interval as short as 4 days might be clinically better.

Macrophages, key players in the innate immune system, showcase remarkable adaptability. Derived from monocytes, these phagocytic cells excel in engulfing and digesting pathogens and foreign substances as well as contributing to antigen presentation, initiating and regulating adaptive immunity. Macrophages are highly plastic, and the microenvironment can shaper their phenotype leading to numerous distinct polarized subsets, exemplified by the two ends of the spectrum: M1 (classical activation, inflammatory) and M2 (alternative activation, anti-inflammatory). RNA sequencing (RNA-Seq) has revolutionized molecular biology, offering a comprehensive view of transcriptomes. Unlike microarrays, RNA-Seq detects known and novel transcripts, alternative splicing, and rare transcripts, providing a deeper understanding of genome complexity. Despite the decreasing costs of RNA-Seq, data consolidation remains limited, hindering noise reduction and the identification of authentic signatures. Macrophages polarization is routinely ascertained by qPCR to evaluate those genes known to be characteristic of M1 or M2 skewing. Yet, the choice of these genes is literature- and experience-based, lacking therefore a systematic approach. This manuscript builds on the significant increase in deposited RNA-Seq datasets to determine an unbiased and robust murine M1 and M2 polarization profile. We now provide a consolidated list of global M1 differentially expressed genes (i.e. robustly modulated by IFN-γ, LPS, and LPS+ IFN-γ) as well as consolidated lists of genes modulated by each stimulus (IFN-γ, LPS, LPS+ IFN-γ, and IL-4).

The debate about contraception has become increasingly important as more and more people seek safe and effective contraception. More than 1 billion women of reproductive age worldwide need a method of family planning, and wellbeing, socio-economic status, culture, religion and more influence the reasons why a woman may ask for contraception. Different contraceptive methods exist, ranging from ‘natural methods’ (fertility awareness-based methods - FABMs) to barrier methods and hormonal contraceptives (HCs). Each method works on a different principle, with different effectiveness.FABMs and HCs are usually pitted against each other, although it’s difficult to really compare them. FABMs are a valid alternative for women who cannot or do not want to use hormone therapy, although they may have a high failure rate if not used appropriately and require specific training. HCs are commonly used to address various clinical situations, although concerns about their possible side effects are still widespread. However, many data show that the appropriate use of HC has a low rate of adverse events, mainly related to personal predisposition.The aim of this review is to summarize the information on the efficacy and safety of FABMs and HCs to help clinicians and women choose the best contraceptive method for their needs.

Abstract Oxaliplatin (OHP)-induced peripheral neurotoxicity (OIPN), one of the major dose-limiting side effects of colorectal cancer treatment, is characterized by both acute and chronic syndromes. Acute exposure to low dose OHP on dorsal root ganglion (DRG) neurons is able to induce an increase in intracellular calcium and proton concentration, thus influencing ion channels activity and neuronal excitability. The Na+/H+ exchanger isoform-1 (NHE1) is a plasma membrane protein that plays a pivotal role in intracellular pH (pHi) homeostasis in many cell types, including nociceptors. Here we show that OHP has early effects on NHE1 activity in cultured mouse DRG neurons: the mean rate of pHi recovery was strongly reduced compared to vehicle-treated controls, reaching levels similar to those obtained in the presence of cariporide (Car), a specific NHE1 antagonist. The effect of OHP on NHE1 activity was sensitive to FK506, a specific calcineurin (CaN) inhibitor. Lastly, molecular analyses revealed transcriptional downregulation of NHE1 both in vitro, in mouse primary DRG neurons, and in vivo, in an OIPN rat model. Altogether, these data suggest that OHP-induced intracellular acidification of DRG neurons largely depends on CaN-mediated NHE1 inhibition, revealing new mechanisms that OHP could exert to alter neuronal excitability, and providing novel druggable targets.

Background Nicotinamide phosphoribosyltransferase (NAMPT) is a key intracellular enzyme that participates in nicotinamide adenine dinucleotide (NAD) homeostasis as well as a released cytokine (eNAMPT) that is elevated in inflammatory conditions and in cancer. In patients with breast cancer, circulating eNAMPT is elevated and its plasma levels correlate with prognosis and staging. In light of this, we investigated the contribution of eNAMPT in triple negative mammary carcinoma progression by investigating the effect of its neutralization via a specific neutralizing monoclonal antibody (C269).Methods We used female BALB/c mice injected with 4T1 clone 5 cells and female C57BL6 injected with EO771 cells, evaluating tumoral size, spleen weight and number of metastases. We injected two times a week the anti-eNAMPT neutralizing antibody and we sacrificed the mice after 28 days. Harvested tumors were analyzed by histopathology, flow cytometry, western blot, immunohistochemistry, immunofluorescence and RNA sequencing to define tumor characteristics (isolating tumor infiltrating lymphocytes and tumoral cells) and to investigate the molecular mechanisms behind the observed phenotype. Moreover, we dissected the functional relationship between T cells and tumoral cells using three-dimensional (3D) co-cultures.Results The neutralization of eNAMPT with C269 led to decreased tumor size and reduced number of lung metastases. RNA sequencing and functional assays showed that eNAMPT controlled T-cell response via the programmed death-ligand 1/programmed cell death protein 1 (PD-L1/PD-1) axis and its neutralization led to a restoration of antitumoral immune responses. In particular, eNAMPT neutralization was able to activate CD8+IFNγ+GrzB+ T cells, reducing the immunosuppressive phenotype of T regulatory cells.Conclusions These studies indicate for the first time eNAMPT as a novel immunotherapeutic target for triple negative breast cancer.

All cells require sustained intracellular energy flux, which is driven by redox chemistry at the subcellular level. NAD+, its phosphorylated variant NAD(P)+, and its reduced forms NAD(P)/NAD(P)H are all redox cofactors with key roles in energy metabolism and are substrates for several NAD-consuming enzymes (e.g. poly(ADP-ribose) polymerases, sirtuins, and others). The nicotinamide salvage pathway, constituted by nicotinamide mononucleotide adenylyltransferase (NMNAT) and nicotinamide phosphoribosyltransferase (NAMPT), mainly replenishes NAD+ in eukaryotes. However, unlike NMNAT1, NAMPT is not known to be a nuclear protein, prompting the question of how the nuclear NAD+ pool is maintained and how it is replenished upon NAD+ consumption. In the present work, using human and murine cells; immunoprecipitation, pulldown, and surface plasmon resonance assays; and immunofluorescence, small-angle X-ray scattering, and MS-based analyses, we report that GAPDH and NAMPT form a stable complex that is essential for nuclear translocation of NAMPT. This translocation furnishes NMN to replenish NAD+ to compensate for the activation of NAD-consuming enzymes by stressful stimuli induced by exposure to H2O2 or S-nitrosoglutathione and DNA damage inducers. These results indicate that by forming a complex with GAPDH, NAMPT can translocate to the nucleus and thereby sustain the stress-induced NMN/NAD+ salvage pathway.

Research into non-hormonal, alternative therapies is necessary for women for whom menopausal hormone therapy is contraindicated or for women who do not wish to take hormones. This review focuses on one such non-hormonal option, namely, purified and specific cytoplasmic pollen extract, or PureCyTonin®. This extract has been evaluated in several preclinical and clinical studies, where it demonstrated its value as a safe and non-estrogenic alternative for menopause. This review presents the beneficial effects of PureCyTonin® in the treatment of menopausal symptoms (e.g. hot flushes) in healthy women, as well as in premenstrual syndrome. We discuss the mechanism of action of PureCyTonin®, an SSRI-'like' therapy. The lack of estrogenic effect demonstrated in preclinical studies suggests that PureCyTonin® may also be a suitable option for the management of menopausal symptoms in women with breast cancer.

Abstract Deregulation of protein synthesis and ER stress/unfolded protein response (ER stress/UPR) have been reported in astrocytes. However, the relationships between protein synthesis deregulation and ER stress/UPR, as well as their role in the altered homeostatic support of Alzheimer’s disease (AD) astrocytes remain poorly understood. Previously, we reported that in astrocytic cell lines from 3xTg-AD mice (3Tg-iAstro) protein synthesis was impaired and ER-mitochondria distance was reduced. Here we show that impaired protein synthesis in 3Tg-iAstro is associated with an increase of p-eIF2α and downregulation of GADD34. Although mRNA levels of ER stress/UPR markers were increased two-three-fold, we found neither activation of PERK nor downstream induction of ATF4 protein. Strikingly, the overexpression of a synthetic ER-mitochondrial linker (EML) resulted in a reduced protein synthesis and augmented p-eIF2α without any effect on ER stress/UPR marker genes. In vivo, in hippocampi of 3xTg-AD mice, reduced protein synthesis, increased p-eIF2α and downregulated GADD34 protein were found, while no increase of p-PERK or ATF4 proteins was observed, suggesting that in AD astrocytes, both in vitro and in vivo, phosphorylation of eIF2α and impairment of protein synthesis are PERK-independent. Next, we investigated the ability of 3xTg-AD astrocytes to support metabolism and function of other cells of the central nervous system. Astrocyte-conditioned medium (ACM) from 3Tg-iAstro cells significantly reduced protein synthesis rate in primary hippocampal neurons. When added as a part of pericyte/endothelial cell (EC)/astrocyte 3D co-culture, 3Tg-iAstro, but not WT-iAstro, severely impaired formation and ramification of tubules, the effect, replicated by EML overexpression in WT-iAstro cells. Finally, a chemical chaperone 4-phenylbutyric acid (4-PBA) rescued protein synthesis, p-eIF2α levels in 3Tg-iAstro cells and tubulogenesis in pericyte/EC/3Tg-iAstro co-culture. Collectively, our results suggest that a PERK-independent, p-eIF2α-associated impairment of protein synthesis compromises astrocytic homeostatic functions, and this may be caused by the altered ER-mitochondria interaction.

Background: Advances in cancer medicines have resulted in tangible health impacts, but the magnitude of benefits of approved cancer medicines could vary greatly. Health Technology Assessment (HTA) is a multidisciplinary process used to inform resource allocation through a systematic value assessment of health technology. This paper reviews the challenges in conducting HTA for cancer medicines arising from oncology trial designs and uncertainties of safety-efficacy data. Methods: Multiple databases (PubMed, Scopus and Google Scholar) and grey literature (public health agencies and governmental reports) were searched to inform this policy narrative review. Results: A lack of robust efficacy-safety data from clinical trials and other relevant sources of evidence has made HTA for cancer medicines challenging. The approval of cancer medicines through expedited pathways has increased in recent years, in which surrogate endpoints or biomarkers for patient selection have been widely used. Using these surrogate endpoints has created uncertainties in translating surrogate measures into patient-centric clinically (survival and quality of life) and economically (cost-effectiveness and budget impact) meaningful outcomes, with potential effects on diverting scarce health resources to low-value or detrimental interventions. Potential solutions include policy harmonization between regulatory and HTA authorities, commitment to generating robust post-marketing efficacy-safety data, managing uncertainties through risk-sharing agreements, and using value frameworks. Conclusion: A lack of robust efficacy-safety data is a central problem for conducting HTA of cancer medicines, potentially resulting in misinformed resource allocation.

Background: Polycystic ovary syndrome (PCOS) is a frequent reproductive disease characterized by hyperandrogenism, oligo /anovulation, and polycystic aspects at ultrasound. In these last years, a body of evidence disclosed the frequent occurrence in PCOS patients of insulin resistance (IR) and compensatory hyperinsulinemia. Aim: To evaluate whether any relationship exists between IR, compensatory hyperinsulinemia and familial predisposition to diabetes. Methods: A group of overweight/obese PCOS patients (n = 84) was selected from our Clinic database according to the Rotterdam criteria and the following parameters were extracted from the database: insulin, C Peptide, aspartate amino transferase (AST), alanine amino transferase (ALT), HOMA (Homeostasis Model Assessment) index, total cholesterol, LDL (Low Density Lypoprotein), HDL (High Density Lypoprotein), and body mass index (BMI). The presence and absence of diabetes among first grade relatives (parents and/or grandparents) were also considered. The Hepatic Insulin Extraction (HIE) index was computed as a ratio between insulin and C-Peptide plasma levels. Results: PCOS patients with familial diabetes showed higher levels of ALT, AST, HOMA index, and HIE. Baseline insulin levels above 12 μU/mL were more frequently observed in PCOS with familial diabetes. HIE index, ALT, and AST were higher in these latter PCOS patients than in PCOS without diabetic first grade relatives, sustaining the hypothesis of an impaired liver clearance of insulin in the case of familial diabetes. Conclusions: According to our study, the presence of anamnestic evidence of familial diabetes together with baseline levels of insulin higher that 12 µIU/mL and elevated transaminase levels should be considered as a consistent clinical suspect of liver impairment that might trigger compensatory hyperinsulinemia and lead to NAFLD and liver steatosis.

BackgroundNicotinamide phosphoribosyltransferase (NAMPT) and nicotinic acid phosphoribosyl transferase (NAPRT) are key intracellular enzymes that participate in the biosynthesis on NAD but have also been shown to be released as proinflammatory cytokines. A number of reports have shown that circulating NAMPT is increased in serum of patients with inflammatory disorders, including inflammatory bowel diseases (IBD), while nothing is known regarding circulating NAPRT and the presence of both cytokines in IBD patient stools. In the present study, we evaluated eNAMPT and eNAPRT levels in a large cohort of IBD patients not on biological therapy and in a subset that then was prescribed biologics.MethodsWe conducted a retro-perspective study on 180 patients, of which 111 underwent subsequent biological treatment (adalimumab, vedolizumab, and ustekinumab). We analyzed eNAMPT and eNAPRT concentrations in serum and faces of IBD patients, correlating them with response to biologics.ResultsWe now report that eNAMPT and eNAPRT are significantly increased in both serum and stools of IBD patients. NAMPT and NAPRT levels correlate with disease severity, with C reactive protein and with serum IL-6 levels. Importantly, levels of NAMPT in patients starting treatment with adalimumab correlate with response failure at three months: patients with levels above 4 ng/ml were significantly less likely to obtain benefit. Serum NAMPT as a biomarker of response yields a sensitivity of 91% and a specificity of 100%.ConclusionThe present work strongly suggests that a prospective trial evaluating eNAMPT and eNAPRT levels in relation to response to biologicals in IBD should be initiated.

Abstract Background and aim Evidence on determinants of prices for orphan medicines is scarce and not available for Italy. The aim of this paper is to provide an evidence on variables affecting the annual treatment cost of orphan drugs in Italy, testing the hypothesis of a negative correlation with the dimension of the target population and a positive correlation with the added therapeutic value of the drug and the quality of the evidence of pivotal studies. Methods Drugs with a European orphan designation reimbursed in Italy in the last 6 years (2014–2019) were considered. Univariate, cluster analysis and multiple regression models were used to investigate the correlation between the annual treatment cost and, as explanatory variables, the dimension of the target population, the existence of Randomized Clinical Trials as a proxy of the quality of the pivotal studies, the added therapeutic value. Results In the univariate analysis prevalence and added therapeutic value, as expected, have a negative and positive correlation with cost respectively. The correlation with RCT is not significant. In the multivariate model, coefficients for prevalence and added value are confirmed but for the latter are not significant anymore. We also found, through an interaction analysis, that the existence of an RCT has a positive impact on annual treatment cost when the target population is very small. Conclusions Our results suggest that value arguments and sustainability (dimension of the target population and its impact on budget impact) issues are considered for orphan drugs pricing: the role played by sustainability is systematically supported by our results. A more transparent and reproducible price negotiation process for orphan drugs is needed in Italy. This paper has contributed to highlight the implicit drivers of this process.

Menopause is the permanent cessation of menstrual cycles following the loss of ovarian follicular activity. Quality of life of postmenopausal woman is the result of a series of psychobiological transformations, that see in the reduction of sex hormones and steroids the etiopathogenetic determinant moment. Symptoms of menopause range from somatic side such as metabolic changes, increased cardiovascular disease, irregular vaginal bleeding, urogenital symptoms, vaginal dryness, osteoporosis and risk of bones fractures to changes of central nervous system as vasomotor symptoms, sleep disruption, mood changes, migraine, sexual dysfunctions. It is fundamental to know the mechanisms underlying changes in the central nervous system during menopause, related to hypoestrogenism, to be able to create appropriate target therapy for patients, improving their quality of life. In fact, the central nervous system is now one of the major targets of sex steroids that cannot be achieved disregard when dealing with the problem of choice of a particular type of MHT.

During the Symposium “Systems Medicine. Integration models in clinical practice and new therapeutic solutions”, "The Milan Declaration 2022 – New goals for Medicine" – which outlines the current and future social and health scenarios that make necessary to define a new paradigm of Medicine – was approved by the speakers (Prof. Giuseppe Bellelli, Prof. Sergio Bernasconi, Prof. Gianni Bona, Prof. Mario Clerici, Prof. Giuseppe De Benedittis, Dr. Marco Del Prete, Prof. Fabio Esposito, Prof. Vassilios Fanos, Prof. Alessandro Genazzani, Prof. Paolo Inghilleri, Prof. Davide Lauro, Prof. Jeanette Maier, Prof. Stefano Masiero, Prof. Marco Matucci Cerinic, Prof. Alberto Migliore, Prof. Emilio Minelli, Prof. Andrea Modesti, Prof. Claudio Molinari, Prof. Valter Santilli, Prof. Umberto Solimene). The Symposium was held in Milan, Italy, at the University of Milan, on 5 May 2022 under the auspices of: World Health Organization (WHO) Collaborating Center for Integrative Medicine, P.R.M. Academy (International Academy of Physiological Regulating Medicine), FEMTEC (Worldwide Federation of Hydrotherapy and Climatotherapy), under the patronage of: Italian Ministry of Health, FNOMCeO (Italian Federation of the Associations of Surgeons and Dentists).