The Multiplicity of Argonaute Complexes in Mammalian Cells.

Argonautes (AGOs) are a highly conserved family of proteins found in most eukaryotes and involved in mechanisms of gene regulation, both at the transcriptional and post-transcriptional level. Among other functions, AGO proteins associate with microRNAs (miRNAs) to mediate the post-transcriptional repression of protein-coding genes. In this process, AGOs associate with members of the trinucleotide repeat containing 6 protein (TNRC6) family to form the core of the RNA-induced silencing complex (RISC), the effector machinery that mediates miRNA function. However, the description of the exact composition of the RISC has been a challenging task due to the fact the AGO's interactome is dynamically regulated in a cell type- and condition-specific manner. Here, we summarize some of the most significant studies that have identified AGO complexes in mammalian cells, as well as the approaches used to characterize them. Finally, we discuss possible opportunities to exploit what we have learned on the properties of the RISC to develop novel anti-cancer therapies. SIGNIFICANCE STATEMENT: The RNA-induced silencing complex (RISC) is the molecular machinery that mediates miRNA function in mammals. Studies over the past two decades have shed light on important biochemical and functional properties of this complex. However, many aspects of this complex await further elucidation, mostly due to technical limitations that have hindered full characterization. Here, we summarize some of the most significant studies on the mammalian RISC and discuss possible sources of biases in the approaches used to characterize it.

Role of the antioxidant defence system and telomerase in arsenic-induced genomic instability.

Arsenic (AS) is a reactive oxygen species (ROS)-inducer carcinogen, whose mode of action is still unclear. To defend against ROS, cells use enzymatic and non-enzymatic antioxidants, such as superoxide dismutase (SOD) and catalase. Failure of antioxidant systems (AXS) can result in dicentric chromosomes formation as well as telomere associations for the reduced activity of telomerase. In order to clarify the long-term effects of a past AS exposure, we evaluated the efficiency of the AXS and the telomerase activity in the progeny of arsenite-treated cells named ASO (arsenic shake-off) cells, previously obtained from arsenite-treated V79 cells and selected by shake-off. Despite SOD1 expression level correlated to the level of ROS observed over time, no changes of the relative amount of antioxidant activities were observed in ASO cells. Moreover, we found that clones characterised by low levels of SOD1 and high levels of ROS acquired a transformed phenotype. Treatment with 5-azacytidine determined an increase of SOD1 expression in a clone and decrease in one other, suggesting that aberrant DNA methylation may be responsible for the abnormal expression of SOD 1 or SOD1 inhibitor genes in different clones. TRAP assay results showed that the progeny of arsenite-treated cells were characterised by a time-dependent decrease of telomerase activity. Integrated results suggest that the increases of ROS levels are accompanied by defective telomerase activity. Finally, we propose that cells escaping the arsenite-induced death perpetuated the memory of past exposure via ROS likely because antioxidant and telomerase activity impairment and ultimately acquire a transformed phenotype.

Effects of tadalafil treatment combined with physical activity in patients with low onset hypogonadism: results from a not-randomized single arm phase 2 study.

PURPOSE: To investigate a possible relation between penile Doppler ultrasound examination (PDUE) parameters and efficacy of chronic therapy with tadalafil (TAD) combined with a protocol of aerobic physical activity (PA) in patients with late onset hypogonadism (LOH). METHODS: The study evaluated 30 patients consecutively enrolled with LOH and erectile dysfunction which present contraindication to hormonal replacement therapy for concomitant prostate disease. These patients were subjected to a combined protocol with phosphodiesterase V selective inhibitors (TAD 5 mg daily) and aerobic PA. RESULTS: After three months, we observed significant improvements in erectile function [IIEF-5, median (IQR) = 13.0 (7.0-18.0) versus 6.0 (5.0-6.75); p < 0.01] and of the main metabolic [homeostatic model assessment index, median (IQR) = 2.5 (1.62-3.37) versus 3.0 (2.0-3.75); p < 0.01; body mass index, median (IQR) = 27.0 (24.0-28.75) versus 27.5 (24.0-29.5)] and vascular parameters [peak systolic velocity, median (IQR) = 29.5 (24.25-31.0) versus 28.0 (23.0-24.25); acceleration time, median (IQR) = 114 (105.25-134.0) versus 115.0 (106.5-134.0)], assessed by PDUE. CONCLUSION: PA in association with phosphodiesterase V inhibitors could compensate the effects of hypogonadism on erectile function and facilitate the clinical response to these drugs even in the absence of adequate serum concentrations of total testosterone.

Lowering the quantification limit of the QubitTM RNA HS assay using RNA spike-in.

BACKGROUND: RNA quantification is often a prerequisite for most RNA analyses such as RNA sequencing. However, the relatively low sensitivity and large sample consumption of traditional RNA quantification methods such as UV spectrophotometry and even the much more sensitive fluorescence-based RNA quantification assays, such as the Qubit™ RNA HS Assay, are often inadequate for measuring minute levels of RNA isolated from limited cell and tissue samples and biofluids. Thus, there is a pressing need for a more sensitive method to reliably and robustly detect trace levels of RNA without interference from DNA. METHODS: To improve the quantification limit of the Qubit™ RNA HS Assay, we spiked-in a known quantity of RNA to achieve the minimum reading required by the assay. Samples containing trace amounts of RNA were then added to the spike-in and measured as a reading increase over RNA spike-in baseline. We determined the accuracy and precision of reading increases between 1 and 20 pg/µL as well as RNA-specificity in this range, and compared to those of RiboGreen(®), another sensitive fluorescence-based RNA quantification assay. We then applied Qubit™ Assay with RNA spike-in to quantify plasma RNA samples. RESULTS: RNA spike-in improved the quantification limit of the Qubit™ RNA HS Assay 5-fold, from 25 pg/µL down to 5 pg/µL while maintaining high specificity to RNA. This enabled quantification of RNA with original concentration as low as 55.6 pg/µL compared to 250 pg/µL for the standard assay and decreased sample consumption from 5 to 1 ng. Plasma RNA samples that were not measurable by the Qubit™ RNA HS Assay were measurable by our modified method. CONCLUSIONS: The Qubit™ RNA HS Assay with RNA spike-in is able to quantify RNA with high specificity at 5-fold lower concentration and uses 5-fold less sample quantity than the standard Qubit™ Assay.

Regulation of the Fanconi anemia pathway by a CUE ubiquitin-binding domain in the FANCD2 protein.

The Fanconi anemia (FA)-BRCA pathway is critical for the repair of DNA interstrand crosslinks (ICLs) and the maintenance of chromosome stability. A key step in FA-BRCA pathway activation is the covalent attachment of monoubiquitin to FANCD2 and FANCI. Monoubiquitinated FANCD2 and FANCI localize in chromatin-associated nuclear foci where they interact with several well-characterized DNA repair proteins. Importantly, very little is known about the structure, function, and regulation of FANCD2. Herein, we describe the identification and characterization of a CUE (coupling of ubiquitin conjugation to endoplasmic reticulum degradation) ubiquitin-binding domain (UBD) in FANCD2, and demonstrate that the CUE domain mediates noncovalent binding to ubiquitin in vitro. We show that although mutation of the CUE domain destabilizes FANCD2, the protein remains competent for DNA damage-inducible monoubiquitination and phosphorylation. Importantly, we demonstrate that the CUE domain is required for interaction with FANCI, retention of monoubiquitinated FANCD2, and FANCI in chromatin, and for efficient ICL repair. Our results suggest a model by which heterodimerization of monoubiquitinated FANCD2 and FANCI in chromatin is mediated in part through a noncovalent interaction between the FANCD2 CUE domain and monoubiquitin covalently attached to FANCI, and that this interaction shields monoubiquitinated FANCD2 from polyubiquitination and proteasomal degradation.

p21 promotes error-free replication-coupled DNA double-strand break repair.

p21 is a well-established regulator of cell cycle progression. The role of p21 in DNA repair, however, remains poorly characterized. Here, we describe a critical role of p21 in a replication-coupled DNA double-strand break (DSB) repair that is mechanistically distinct from its cell cycle checkpoint function. We demonstrate that p21-deficient cells exhibit elevated chromatid-type aberrations, including gaps and breaks, dicentrics and radial formations, following exposure to several DSB-inducing agents. p21-/- cells also exhibit an increased DNA damage-inducible DNA-PKCS S2056 phosphorylation, indicative of elevated non-homologous DNA end joining. Concomitantly, p21-/- cells are defective in replication-coupled homologous recombination (HR), exhibiting decreased sister chromatid exchanges and HR-dependent repair as determined using a crosslinked GFP reporter assay. Importantly, we establish that the DSB hypersensitivity of p21-/- cells is associated with increased cyclin-dependent kinase (CDK)-dependent BRCA2 S3291 phosphorylation and MRE11 nuclear foci formation and can be rescued by inhibition of CDK or MRE11 nuclease activity. Collectively, our results uncover a novel mechanism by which p21 regulates the fidelity of replication-coupled DSB repair and the maintenance of chromosome stability distinct from its role in the G1-S phase checkpoint.

Abnormal mitotic spindle assembly and cytokinesis induced by D-Limonene in cultured mammalian cells.

D-Limonene is found widely in citrus and many other plant species; it is a major constituent of many essential oils and is used as a solvent for commercial purposes. With the discovery of its chemotherapeutic properties against cancer, it is important to investigate the biological effects of the exposure to D-Limonene and elucidate its, as yet unknown, mechanism of action. We reported here that D-Limonene is toxic in V79 Chinese hamster cells in a dose-dependent manner. Moreover, to determine the cellular target of D-Limonene, we performed morphological observations and immunocytochemical analysis and we showed that this drug has a direct effect on dividing cells preventing assembly of mitotic spindle microtubules. This affects both chromosome segregation and cytokinesis, resulting in aneuploidy that in turn can lead to cell death or genomic instability.

Endogenous retrotransposons cause catastrophic deoxyribonucleic acid damage in human trophoblasts.

OBJECTIVE: To determine the mechanistic role of mobile genetic elements in causing widespread DNA damage in primary human trophoblasts. DESIGN: Experimental ex vivo study. SETTING: Hospital-affiliated University. PATIENT(S): Trophoblasts from a patient with unexplained recurrent pregnancy loss and patients with spontaneous and elective abortions (n = 10). INTERVENTION(S): Biochemical and genetic analysis and modification of primary human trophoblasts. MAIN OUTCOME MEASURE(S): To phenotype and systematically evaluate the underlying pathogenic mechanism for elevated DNA damage observed in trophoblasts derived from a patient with unexplained recurrent pregnancy loss, transcervical embryoscopy, G-band karyotyping, RNA sequencing, quantitative polymerase chain reaction, immunoblotting, biochemical and siRNA assays, and whole-genome sequencing were performed. RESULT(S): Transcervical embryoscopy revealed a severely dysmorphic embryo that was euploid on G-band karyotyping. RNA sequencing was notable for markedly elevated LINE-1 expression, confirmed with quantitative polymerase chain reaction, and that resulted in elevated expression of LINE-1-encoded proteins, as shown by immunoblotting. Immunofluorescence, biochemical and genetic approaches demonstrated that overexpression of LINE-1 caused reversible widespread genomic damage and apoptosis. CONCLUSION(S): Derepression of LINE-1 elements in early trophoblasts results in reversible but widespread DNA damage.

Nutritional epigenomic and DNA-damage modulation effect of natural stilbenoids.

The aim of the present work is the evaluation of biological effects of natural stilbenoids found in Vitis vinifera, with a focus on their activity as epigenetic modulators. In the present study, resveratrol, pterostilbene and for the first time their dimers (±)-trans-δ-viniferin, (±)-trans-pterostilbene dehydrodimer were evaluated in Caco-2 and HepG-2 cell lines as potential epigenetic modulators. Stilbenoids were added in a Caco-2 cell culture as a model of the intestinal epithelial barrier and in the HepG-2 as a model of hepatic environment, to verify their dose-dependent toxicity, ability to interact with DNA, and epigenomic action. Resveratrol, pterostilbene, and (±)-trans-pterostilbene dehydrodimer were found to have no toxic effects at tested concentration and were effective in reversing arsenic damage in Caco-2 cell lines. (±)-trans-δ-viniferin showed epigenomic activity, but further studies are needed to clarify its mode of action.

Indicaxanthin Induces Autophagy in Intestinal Epithelial Cancer Cells by Epigenetic Mechanisms Involving DNA Methylation.

Autophagy is an evolutionarily conserved process critical in maintaining cellular homeostasis. Recently, the anticancer potential of autophagy inducers, including phytochemicals, was suggested. Indicaxanthin is a betalain pigment found in prickly pear fruit with antiproliferative and pro-apoptotic activities in colorectal cancer cells associated with epigenetic changes in selected methylation-silenced oncosuppressor genes. Here, we demonstrate that indicaxanthin induces the up-regulation of the autophagic markers LC3-II and Beclin1, and increases autophagolysosome production in Caco-2 cells. Methylomic studies showed that the indicaxanthin-induced pro-autophagic activity was associated with epigenetic changes. In addition to acting as a hypermethylating agent at the genomic level, indicaxanthin also induced significant differential methylation in 39 out of 47 autophagy-related genes, particularly those involved in the late stages of autophagy. Furthermore, in silico molecular modelling studies suggested a direct interaction of indicaxanthin with Bcl-2, which, in turn, influenced the function of Beclin1, a key autophagy regulator. External effectors, including food components, may modulate the epigenetic signature of cancer cells. This study demonstrates, for the first time, the pro-autophagic potential of indicaxanthin in human colorectal cancer cells associated with epigenetic changes and contributes to outlining its potential healthy effect in the pathophysiology of the gastrointestinal tract.

Genomic instability induced by α-pinene in Chinese hamster cell line.

Here, we report the effects of exposure of mammalian cells to α-pinene, a bicyclic monoterpene used in insecticides, solvents and perfumes. Morphological analysis, performed in V79-Cl3 cells exposed for 1 h to increasing concentrations (25 up to 50 µM) of α-pinene, indicated a statistically significant increase in micronucleated and multinucleated cell frequencies; apoptotic cells were seen at 40 and 50 µM. This monoterpene caused genomic instability by interfering with mitotic process; in fact, 50% of cells (versus 19% of control cells) showed irregular mitosis with multipolar or incorrectly localised spindles. Cytogenetic analysis demonstrated high-frequency hypodiploid metaphases as well as endoreduplicated cells and chromosome breaks. Clastogenic damage was prevalent over aneuploidogenic damage as demonstrated by the higher proportion of kinetochore-negative micronuclei. Alkaline comet confirmed that monoterpene exposure caused DNA lesions in a concentration-dependent manner. This damage probably arose by increased reactive oxygen species (ROS) production. In order to assess the generation of ROS, the cells were incubated with CM-H(2)DCFDA and then analysed by flow cytometry. Results demonstrated an increase in fluorescence intensity after α-pinene treatment indicating increased oxidative stress. On the whole, these findings strongly suggest that α-pinene is able to compromise genome stability preferentially through mitotic alterations and to damage DNA through ROS production.

The analysis of alcohol consumption during the severe acute respiratory syndrome Coronavirus 2 Italian lockdown.

BACKGROUND: The SARS-CoV-2 lockdown resulted in deep changes of lifestyles, promoting in many people the onset of psychological symptoms generally associated with drug and alcohol abuse. The aim of this study was to assess the variation of alcohol drinking habits in a sample of Italian citizens during lockdown and to identify the psychosocial factors surrounding it. METHODS: An online anonymous questionnaire was created and submitted from April 9 to April 28, 2020. Questions were related to personal psychosocial details and alcohol drinking habits during the lockdown, including Alcohol Use Disorders Identification Test (AUDIT C) questions. RESULTS: On a total of 1234 surveys the increase of both anxiety and fear was largely detected (63% and 61% respectively). The 18% increased alcohol consumption during the lockdown and it showed a significant correlation with anxiety and fear experienced (both P<0.001). The relative risk for 7 to 9 and more than 10 drinks per day consumption were directly linked to these symptoms (P<0.001). The most involved categories of participants showed this harmful association were self-employed workers and participants who live alone, subject aged 30-50 with high level of instruction or students, and not occupied people in the age range 18-19 (all P<0.001). Additionally, the subset of the study population that showed low alcohol consumption before the lockdown has demonstrated the worsening of alcohol assumption during the quarantine (P<0.0001). CONCLUSIONS: Several psychosocial factors are involved in determining the increase of alcohol consumption during lockdown and need the healthcare support to avoid awful impact on human life.

Alcoholic Consumption of Young Italians During the SARS-CoV-2 Pandemic.

BACKGROUND: The international health emergency caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which, at the end of 2019, hit the world, forced the governments of all countries to adopt stringent restrictive measures to contain the spread of the virus. Several studies have revealed worsening levels of anxiety, depression and perceived stress related to these restrictions and the resulting lifestyle changes. Some studies have also confirmed the presence of a relationship between SARS-CoV-2-related emotional distress and drinking behavior. Indeed, is a wellknown fact that alcohol consumption is one of the behavioral strategies used to reduce negative emotional states. However, it was documented that young people developed different responses to alcohol use during the pandemic than adults. OBJECTIVE: The aim of this work was to investigate the consumption habits of young Italians and how the consumption and purchase of alcoholic beverages have changed following the pandemic. New ways of drinking alcohol were also interesting to observe, such as online. METHODS: Young people between 18 and 35 years old were subjected to an anonymous questionnaire of 22 questions on the adoption of forms of behavior at risk through alcohol consumption, the quantity and occasions of preferential consumption, and on the methods and quantities of alcoholic beverage purchase, before and during the SARS-CoV-2 pandemic. The subjects who declared themselves "non-drinkers" were not included in the statistical survey. RESULTS: About 33% of the enrolled "drinkers" (268/823), adopted risky forms of alcoholic behavior. Males reported a higher average habit of drinking wine or alcohol (M = 1.9953 ± 1.39743, F = 1.7373 ± 1.36688, p <0.005); an increased frequency of drinking (M = 2.3025 ± 0.80610 F = 2.0494 ± 0.75043 p <0.001); a higher average number of drinks consumed (M = 1.5182 ± 0.85646, F = 1.2618 ± 0.53292, p <0.001) and binge drinking to the greatest extent (M = 1.1933 ± 0.96522 F = 0.8176 ± 0.85446 p <0.001). Education and employment were significantly correlated with the frequency of alcohol consumption (r = 0.107 p <0.005 and r = 0.120 p = 0.001 respectively). Subjects reported buying alcoholic beverages during the pandemic with a frequency of "less than once a month" (N = 291, 35.36%) and mainly in shops (N = 556, 67.56%), while before the pandemic they mainly bought alcohol once a week (N = 431, 52.37%) and predominantly in bars / clubs (N = 619, 75.21%). New ways of drinking alcohol such as online drinking, have not been significantly identified. CONCLUSION: A change in alcohol consumed and alcohol purchased before and during the SARSCoV- 2 pandemic was revealed.

Efficacy and Safety of Varenicline for Smoking Cessation in Patients With Type 2 Diabetes: A Randomized Clinical Trial.

Importance: Evidence of effective smoking cessation interventions in patients with diabetes is limited. The unique behavioral and metabolic characteristics of smokers with type 2 diabetes warrants a randomized clinical trial of the smoking cessation drug varenicline. Objective: To evaluate the efficacy and safety of varenicline in patients with type 2 diabetes with an intention to quit smoking. Design, Setting, and Participants: This multicenter, double-blind, placebo-controlled randomized clinical trial recruited patients from 6 outpatient clinics in 5 hospitals in Catania, Italy. Patients with type 2 diabetes, who were smoking at least 10 cigarettes a day, and who intended to quit smoking were screened for eligibility. Eligible patients were randomized to either varenicline or placebo treatment. The trial consisted of a 12-week treatment phase followed by a 40-week follow-up, nontreatment phase. Intention-to-treat data analysis was performed from December 2020 to April 2021. Interventions: Varenicline, 1 mg, twice daily or matched placebo administered for 12 weeks. Patients in both treatment groups also received smoking cessation counseling. Main Outcomes and Measures: The primary efficacy end point of the study was the continuous abstinence rate (CAR) at weeks 9 to 24. Secondary efficacy end points were the CAR at weeks 9 to 12 and weeks 9 to 52 as well as 7-day point prevalence of abstinence at weeks 12, 24, and 52. Results: A total of 300 patients (mean [SD] age, 57.4 [0.8] years; 117 men [78.0%] in varenicline group and 119 men [79.3%] in placebo group) were randomized to receive varenicline (n = 150) or placebo (n = 150). The CAR at weeks 9 to 24 was significantly higher for the varenicline than placebo group (24.0% vs 6.0%; odds ratio [OR], 4.95; 95% CI, 2.29-10.70; P < .001). The CARs at weeks 9 to 12 (31.3% vs 7.3%; OR, 5.77; 95% CI, 2.85-11.66; P < .001) and weeks 9 to 52 (18.7% vs 5.3%; OR, 4.07; 95% CI, 1.79-9.27; P < .001) as well as the 7-day point prevalence of abstinence at weeks 12, 24, and 52 were also significantly higher for the varenicline vs placebo group. The most frequent adverse events occurring in the varenicline group compared with the placebo group were nausea (41 [27.3%] vs 17 [11.4%]), insomnia (29 [19.4%] vs 19 [12.7%]), abnormal dreams (19 [12.7%] vs 5 [3.4%]), anxiety (17 [11.4%] vs 11 [7.3%]), and irritability (14 [9.4%] vs 8 [5.4%]). Serious adverse events were infrequent in both groups and not treatment-related. Conclusions and Relevance: Results of this trial showed that inclusion of varenicline in a smoking cessation program is efficacious in achieving long-term abstinence without serious adverse events. Varenicline should be routinely used in diabetes education programs to help patients with type 2 diabetes stop smoking. Trial Registration: ClinicalTrials.gov Identifier: NCT01387425.

The Lesson from the First Italian Lockdown: Impacts on Anxiety and Depressive Symptoms and Sleep Quality in Patients with Remission of Inflammatory Bowel Disease.

BACKGROUND: During the COVID-19 pandemic in Italy, decisions were taken to adopt restrictive legislative measures, such as the first half of the 2020 lockdown. In those months, patients with inflammatory bowel disease experienced social isolation and reduced access to health care. OBJECTIVE: We aimed to evaluate, in this condition, the presence of remission subgroups that were most impacted by the lockdown. METHODS: During the first Italian lockdown, we recruited patients with remission of inflammatory bowel disease by administering an online questionnaire including patient demographics, the Beck Anxiety Questionnaire Inventory, the Beck Depression Inventory questionnaire, and the Pittsburg Sleep Quality Index, all validated standardized questionnaires for anxiety symptom levels, depression, and sleep quality. RESULTS: Our results showed how female patients (p<0.0001) with Crohn's disease (p<0.001) experienced worse levels of anxiety symptoms. Female patients (p<0.0001) between 50 and 60 years of age (p=0.013) with Crohn's disease (p=0.047) experienced worse levels of depressive symptoms. Females also experienced significantly worse sleep levels (p<0.001). We found a correlation between the number of sleeping hours (p<0.001) and the time taken to fall asleep (p<0.001) and the Beck Anxiety Questionnaire Inventory,which showed a linear worsening of the number of minutes it took to fall asleep, and the Beck Depression Inventory questionnaire. CONCLUSION: Among patients with remission of inflammatory bowel disease, female patients, patients with Crohn's disease, and people aged between 50 and 70 years should be considered for screening for anxiety and depression disorders and an assessment of sleep quality.

International randomised controlled trial evaluating metabolic syndrome in type 2 diabetic cigarette smokers following switching to combustion-free nicotine delivery systems: the DIASMOKE protocol.

INTRODUCTION: Reducing exposure to cigarette smoke is an imperative for public health and for patients with diabetes. Increasingly, combustion-free nicotine delivery systems (C-F NDS) such as e-cigarettes and heated tobacco products are substituting conventional cigarettes and accelerating the downward trends in smoking prevalence. However, there is limited information about the long-term health impact in patients with diabetes who use C-F NDS. This randomised trial of type 2 diabetic cigarette smokers will test the hypothesis that following a switch from conventional cigarettes to C-F NDS a measurable improvement in metabolic syndrome (MetS) factors will be shown over the course of 2 years. METHODS AND ANALYSIS: The study is multicentre and thus will take place in five locations in four countries in an ambulatory setting. A total of 576 patients with diabetes will be randomised (1:2 ratio) to either a control arm (Study Arm A), in which they will be offered referral to smoking cessation programmes or to an intervention arm (Study Arm B) assigned to C-F NDS use. Participants will be at least 23 years old and of any gender. Patient recruitment will start in February 2021 and is expected to be completed by December 2021. Primary outcome measures include fasting plasma glucose, blood pressure, triglycerides, high-density lipoprotein and waist circumference, while secondary feature absolute change in the sum of the individual factors of MetS and change in each individual factor of MetS measured at each study time point. ETHICS AND DISSEMINATION: The approval of research ethics committee (REC) regarding the trial protocol, informed consent forms and other relevant documents is required to commence the study. Substantial amendments to the study protocol cannot be implemented until the REC grants a favourable opinion. The results of the study are intended to be published as articles in high quality peer-reviewed journals and disseminated through conference papers. TRIAL REGISTRATION NUMBER: NCT04231838. Pre-results stage.

Spermatic and ultrasound characterization of young diabetic patients.

AIM: Different authors showed clear correlations between diabetic disease and male reproductive damage (es. rate of nuclear DNA fragmentation, mithocondrial DNA mutations, increased of enzymatic glication products, etc...). The aim of this observational study carried out on a selected group of diabetic patients (average age 36) with primary infertility was to determine reactive oxygen species (ROS) production in sperm in connection with duration of disease, glicemic control and seminal vescicular emptying in the post ejaculatory. METHODS: All diabetic patients enrolled (20) were submitted to two consecutive spermiograms, ROS sperm analysis and transrectal ultrasound evaluation before and after ejaculation, performed according to standard conventional methods. RESULTS: Diabetic patients with better glicometabolic compensation (HBAI C < 7%) and duration of disease <5 years showed spermatic rate of ROS production significantly lower regarding the group with worse glicemic control and greater duration of disease. Diabetic patients with altered vescicular emptying in the post ejaculatory showed spermatic rate of ROS production significantly higher regarding patients with normal vescicular emptying. CONCLUSION: The degree of oxidative stress in sperm of diabetic patients follows the course of the other chronic complications, getting worse in connection with duration of disease and glicemic control. Altered vescicular emptying in the post ejaculatory could be an important mechanism for initiation of this higher response.

Direct toxicity of insulin on the human placenta and protection by metformin.

OBJECTIVE: To study the effects of insulin and metformin on primary trophoblasts from early pregnancies. DESIGN: Experimental in vitro study. SETTING: Academic research institute. PATIENT(S): Trophoblasts from healthy patients undergoing first trimester elective termination of pregnancy and primary lung fibroblasts (IMR-90). INTERVENTION(S): Culture and treatment with insulin and metformin of primary trophoblasts and primary lung fibroblasts (IMR-90). MAIN OUTCOME MEASURE(S): DNA damage measured by expression of γ-H2AX with immunofluorescence and Western blot. Apoptosis measured by expression of cleaved caspase-3 by Western blot. Cell survival measured by cell proliferation assay. RESULT(S): Culture of purified primary trophoblast cells in the presence of insulin at levels as low as 1 nM resulted in a 386% increase in the number of cell with elevated γ-H2AX expression, a 66% reduction in cell survival and a marked increase of cleaved caspase-3 expression. Pretreatment of trophoblasts with therapeutic doses of metformin prevented the detrimental effects of insulin. Treatment with insulin and/or metformin had no effects on primary fibroblasts. CONCLUSION(S): Elevated insulin levels are directly toxic to first trimester trophoblasts and result in increased DNA damage, apoptosis, and decreased cell survival. These effects are prevented by metformin. Trophoblast cells from early pregnancy are uniquely vulnerable to elevated levels of insulin. These findings, if confirmed in vivo, suggest that there may be a role for insulin resistance screening before attempting pregnancy and for focusing on prevention of hyperinsulinemia during early pregnancy.

Gut Microbiota in Alzheimer's Disease, Depression, and Type 2 Diabetes Mellitus: The Role of Oxidative Stress.

Gut microbiota consists of over 100 trillion microorganisms including at least 1000 different species of bacteria and is crucially involved in physiological and pathophysiological processes occurring in the host. An imbalanced gastrointestinal ecosystem (dysbiosis) seems to be a contributor to the development and maintenance of several diseases, such as Alzheimer's disease, depression, and type 2 diabetes mellitus. Interestingly, the three disorders are frequently associated as demonstrated by the high comorbidity rates. In this review, we introduce gut microbiota and its role in both normal and pathological processes; then, we discuss the importance of the gut-brain axis as well as the role of oxidative stress and inflammation as mediators of the pathological processes in which dysbiosis is involved. Specific sections pertain the role of the altered gut microbiota in the pathogenesis of Alzheimer's disease, depression, and type 2 diabetes mellitus. The therapeutic implications of microbiota manipulation are briefly discussed. Finally, a conclusion comments on the possible role of dysbiosis as a common pathogenetic contributor (via oxidative stress and inflammation) shared by the three disorders.