Generation of iPSCs from identical twin, one affected by LHON and one unaffected, both carrying a combination of two mitochondrial variants: m.14484 T>C and m.10680G>A.

Leber hereditary optic neuropathy (LHON) is one of the most common mitochondrial illness, causing retinal ganglion cell degeneration and central vision loss. It stems from point mutations in mitochondrial DNA (mtDNA), with key mutations being m.3460G > A, m.11778G > A, and m.14484 T > C. Fibroblasts from identical twins, sharing m.14484 T > C and m.10680G > A variants each with 70 % heteroplasmy, were used to generate iPSC lines. Remarkably, one twin, a LHON patient, displayed symptoms, while the other, a carrier, remained asymptomatic. These iPSCs offer a valuable tool for studying factors influencing disease penetrance and unravelling the role of m.10680G > A, which is still debated.

Genetic variants affecting NQO1 protein levels impact the efficacy of idebenone treatment in Leber hereditary optic neuropathy.

Idebenone, the only approved treatment for Leber hereditary optic neuropathy (LHON), promotes recovery of visual function in up to 50% of patients, but we can neither predict nor understand the non-responders. Idebenone is reduced by the cytosolic NAD(P)H oxidoreductase I (NQO1) and directly shuttles electrons to respiratory complex III, bypassing complex I affected in LHON. We show here that two polymorphic variants drastically reduce NQO1 protein levels when homozygous or compound heterozygous. This hampers idebenone reduction. In its oxidized form, idebenone inhibits complex I, decreasing respiratory function in cells. By retrospectively analyzing a large cohort of idebenone-treated LHON patients, classified by their response to therapy, we show that patients with homozygous or compound heterozygous NQO1 variants have the poorest therapy response, particularly if carrying the m.3460G>A/MT-ND1 LHON mutation. These results suggest consideration of patient NQO1 genotype and mitochondrial DNA mutation in the context of idebenone therapy.

Comparison of sperm preparation methods to improve the recovery of mature spermatozoa in sub-fertile males.

The integrity of chromatin in the spermatozoon is essential for reproductive outcome. The aim of this study was to evaluate the most effective and cost-effective method to reduce the percentage of spermatozoa with defects in chromatin decondensation for use in assisted reproductive technologies (ART) procedures. Sperm samples from 15 sub-fertile males were examined at CFA Naples to determine the sperm decondensation index (SDI), using the aniline blue test, before and after preparation, comparing density gradients with two different swim-up approaches. All three techniques led to a reduction in decondensed spermatozoa with no statistical difference (P > 0.05) between the control and the treated sperm. In contrast, we found a highly significant decrease in SDI (P < 0.01) after the two swim-up methods in all the samples, confirming the efficacy of these methods in lowering the percentage of chromatin compaction damage. There was no statistical difference between the two swim-up methods, however swim-up from the pellet led to improved count, motility and the percentage of normal condensed spermatozoa. We suggest that swim-up from the pellet be used in ART on sub-fertile males, both to reduce cell stress by multiple centrifugation and improve the recovery rate of mature spermatozoa.

A structured physical activity program in an adolescent population with overweight or obesity: a prospective interventional study.

Obesity is a significant health problem, with increasing involvement of young population worldwide. The aim of this study was to evaluate the effects of 2 different types of physical exercise (resistance vs. combined aerobic-resistance) on cardiovascular and anthropometric profile of a sample of sedentary adolescents with overweight and obesity. After undergoing clinical, cardiovascular and anthropometric-metabolic evaluation (T0), subjects with overweight and obesity were randomized to a 6-month resistance or combined aerobic-resistance training program. Clinical, cardiovascular and anthropometric-metabolic evaluations were repeated after 6 months of training (T1) and after 3 months of detraining (T2). Thirty adolescents with overweight/obesity were enrolled and 20 subjects completed training program. A significant improvement in body composition was detected after 6 months, with a reduction of body mass index (32.1 [30.5 to 34.4] vs. 31.1 [29.6 to 33.4] kg/m2, p = 0.02) and adipose tissue (45.5 [41.1 to 49.7] vs. 41.6 [37.0 to 49.2] kg, p < 0.01). A reduction in diastolic blood pressure (75.5 ± 8.9 vs. 68.2 ± 6.4 mm Hg, p = 0.02) and pulse wave velocity (5.7 [5.1 to 5.9] vs. 5.2 [4.7 to 5.7] m/s, p = 0.04) was also observed. Persistence of the effect on the most important parameters was observed also after detraining period. In conclusion, regular physical exercise induces positive metabolic and cardiovascular effects, persisting even after brief discontinuation. Novelty: Physical exercise induces positive effect on cardiovascular risk profile. Positive effects persist also after brief discontinuation. Physical exercise reduces early signs of autonomic disfunction.

Comparison of nurse attended and unattended automated office blood pressure with conventional measurement techniques in clinical practice.

Accuracy in blood pressure measurement is critical for proper hypertension diagnosis and treatment in clinical practice. Automated office blood pressure (AOBP) can simplify the measurement process, reducing human error and minimizing the white-coat effect in the unattended mode. The aim of this study was to compare AOBP, both unattended and nurse attended, with conventional office and out-of-office blood pressure measurement techniques. Four different methods of blood pressure measurement were performed in a cohort of hypertensive patients: conventional office blood pressure (OBP), unattended automated office blood pressure (uAOBP), nurse attended automated office blood pressure (nAOBP), and home blood pressure monitoring (HBPM). uAOBP and nAOBP were conducted with the same rigorous standardized procedure. We enrolled 118 consecutive patients. nAOBP values were slightly higher than uAOBP ones (respectively 132.8/73.3 ± 19.4/12.9 and 129.2/71.1 ± 19.0/12.3 mmHg), even if the difference was influenced by order of execution of AOBP measurement. nAOBP was significantly lower than HBPM and OBP (mean values 135.2/80.9 ± 16.6/8.1 and 140.9/84.6 ± 18.7/10.8 mmHg, respectively). AOBP, either attended or unattended, provides lower values than conventional OBP. uAOBP and nAOBP values showed small differences, even if they are not completely interchangeable. This evidence reflects a lower white-coat effect, even in nurse attended technique, but is also due to a lower measurement error through the application of a rigorous standardized protocol.

Knowledge on arterial hypertension in general population: Results from a community pharmacy screening program.

BACKGROUNDS AND AIMS: Hypertension is a risk factor for renal, cardiovascular and cerebrovascular diseases. It is responsible for a large proportion of overall morbidity and mortality every year. Hypertension-mediated organ damage is largely not reversible. For these reasons, prevention has primary importance: sensibilization of population on hypertension-related consequences is essential for therapeutic adherence and reduction of unhealthy lifestyle behaviour. This study aimed to evaluate awareness about hypertension among community pharmacies customers. METHODS AND RESULTS: A questionnaire about hypertension was collected by 2731 customers from 94 community pharmacies in North West Italy, during a hypertension screening program. Hypertension awareness was unsatisfactory in a large proportion of the sample, with only 15% of subjects having an overall good level of knowledge. Furthermore, lower awareness was associated to higher blood pressure values (132/79 ± 19/11 mmHg vs 128/78 ± 18/10 mmHg, p < 0.001) and subjects resulted hypertensive or uncontrolled despite antihypertensive therapy, presented worse questionnaire scores (4.7 ± 1.9 vs 4.9 ± 2.0, p = 0.03). CONCLUSION: Knowledge about hypertension is largely unsatisfactory among population. Community pharmacies may play as a setting for health education and hypertension screening.

DAAM1 and PREP are involved in human spermatogenesis.

During differentiation of the male gamete, there is a massive remodelling in the shape and architecture of all the cells in the seminiferous epithelium. The cytoskeleton, as well as many associated proteins, plays a pivotal role in this process. To better characterise the factors involved, we analysed two proteins: the formin, dishevelled-associated activator of morphogenesis 1 (DAAM1), which participates in the regulation of actin polymerisation, and the protease, prolyl endopeptidase (PREP), engaged in microtubule-associated processes. In our previous studies we demonstrated their involvement in cytoskeletal dynamics necessary for correct postnatal development of the rat testis. Here, we used samples of testicular tissue obtained from infertile men by testicular sperm extraction and the spermatozoa of asthenoteratozoospermic patients. By western blot and immunofluorescent analysis, we found that DAAM1 and PREP expression and localisation were impaired in both the testis and spermatozoa, and in particular in the midpiece as well as in the principal and end-pieces of the flagella, as compared with spermatozoa of normospermic men. Our results provide new knowledge of the dynamics of spermatogenesis, raising the possibility of using DAAM1 and PREP as new markers of normal fertility.

First evidence of DAAM1 localization in mouse seminal vesicles and its possible involvement during regulated exocytosis.

Dishevelled-associated activator of morphogenesis 1 (DAAM1) is a protein belonging to the formin family, which regulates, together with the small GTPase RhoA, the nucleation and the assembly of actin fibres through Wnt-Dishevelled PCP pathway. Its role has been investigated in essential biological processes, such as cell polarity, movement and adhesion during morphogenesis and organogenesis. In this work, we studied the expression of DAAM1 mRNA and protein by PCR and Western blot analyses and its co-localization with actin in adult mouse seminal vesicles by immunofluorescence. We show that both proteins are cytoplasmic: actin is evident at cell-cell junctions and at cell cortex; DAAM1 had a more diffused localization, but is also prominent at the apical plasmatic membrane of epithelial cells. These findings support our hypothesis of a role of DAAM1 in cytoskeletal rearrangement that occurs during the exocytosis of secretory vesicles, and in particular concerning actin filaments. We were also able to detect DAAM1 and actin association in the smooth muscle cells that surround the epithelium too. In this case, we could only speculate the possible involvement of this formin in muscular cells in the maintenance and the regulation of the contractile structures. The present results strongly suggest that DAAM1 could have a pivotal role in vesicle exocytosis and in the physiology of mouse seminal vesicles.

Temporal and spatial expression of insulin-like peptide (insl5a and insl5b) paralog genes during the embryogenesis of Danio rerio.

Relaxin (RLN) and insulin (INSL)-like peptides are member of the INSL/RLN superfamily, which are encoded by seven genes in humans and can activate the G-protein coupled receptor RXFP 1-4. These peptides evolved from a common ancestor, RLN3-like gene. Two rounds of whole genome duplication (WGD) in early vertebrate evolution, together with an additional WGD in the teleost lineage, caused an expansion of RLN genes set in the genome of Danio rerio. In particular, six RLN genes are present: a single copy of rln and insl3 genes, and two paralogs for the rln3 gene (rln3a and rln3b), and the insl5 gene (insl5a and insl5b). We have already reported the presence of rln3a and rln3b genes in the developing zebrafish brain, as well as the expression of rln gene in the developing zebrafish brain and extraneural territories, such as thyroid gland and pancreas. Here, we report for the first time the expression of the two parologs genes for insl5, insl5a, and insl5b in D. rerio embryonic development. The corresponding transcripts of both the paralogs are present in all embryonic stages analyzed by RT-qPCR. In situ hybridization analyses showed a restricted signal in intestinal cells and the pancreatic region at 72 hpf for insl5a, while at 96 hpf both genes are expressed in specific intestinal cells. Furthermore, in adult zebrafish intestine tissue, in situ hybridation experiments showed that insl5a transcript is specifically localized in the goblet cells, while insl5b transcript is in enteroendocrine cells. These data revealed a high degree of gene expression pattern conservation for such genes in vertebrate evolution.