Intracellular cGMP increase is not involved in thyroid cancer cell death

Introduction: Type 5 phosphodiesterase (PDE5) inhibitors (PDE5i) lead to intracellular cyclic-guanosine monophosphate (cGMP) increase and are used for clinical treatment of erectile dysfunction. Studies found that cGMP may up/downregulate the growth of certain endocrine tumor cells, suggesting that PDE5i could impact cancer risk. Aim: We evaluated if PDE5i may modulate thyroid cancer cell growth in vitro. Materials and methods: We used malignant (K1) and benign (Nthy-ori 3-1) thyroid cell lines, as well as the COS7 cells as a reference model. Cells were treated 0-24 h with the PDE5i vardenafil or the cGMP analog 8-br-cGMP (nM-μM range). cGMP levels and caspase 3 cleavage were evaluated by BRET, in cGMP or caspase 3 biosensor-expressing cells. Phosphorylation of the proliferation-associated extracellularly-regulated kinases 1 and 2 (ERK1/2) was evaluated by Western blotting, while nuclear fragmentation by DAPI staining. Cell viability was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: Both vardenafil and 8-br-cGMP effectively induced dose-dependent cGMP BRET signals (p≤0.05) in all the cell lines. However, no differences in caspase 3 activation occurred comparing PDE5i-treated vs untreated cells, at all concentrations and time-points tested (p>0.05). These results match those obtained upon cell treatment with 8-br-cGMP, which failed in inducing caspase 3 cleavage in all the cell lines (p>0.05). Moreover, they reflect the lack of nuclear fragmentation. Interestingly, the modulation of intracellular cGMP levels with vardenafil or the analog did not impact cell viability of both malignant and benign thyroid tumor cell lines, nor the phosphorylation of ERK1/2 (p>0.05). Conclusions: This study demonstrates that increased cGMP levels are not linked to cell viability or death in K1 and Nthy-ori 3-1 cell lines, suggesting that PDE5i do not impact the growth of thyroid cancer cells. Since different results were previously published, further investigations are recommended to clarify the impact of PDE5i on thyroid cancer cells

Separate Evaluation of Nonlinearity-Due Q Penalties in Long-Haul Very Dense WDM Optical Systems

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Effect of Optical Filtering on 20-Gbit/s RZ-DQPSK Transmission over 2000 km in a 64-Channel DWDM System

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Genetic signature of differentiated thyroid carcinoma susceptibility: a machine learning approach

To identify a peculiar genetic combination predisposing to differentiated thyroid carcinoma (DTC), we selected a set of single-nucleotide polymorphisms (SNPs) associated with DTC risk, considering polygenic risk score (PRS), Bayesian statistics, and a machine learning (ML) classifier to describe cases and controls in 3 different datasets. Dataset 1 (649 DTC, 431 controls) has been previously genotyped in a genome-wide association study (GWAS) on Italian DTC. Dataset 2 (234 DTC, 101 controls) and dataset 3 (404 DTC, 392 controls) were genotyped. Associations of 171 SNPs reported to predispose to DTC in candidate studies were extracted from the GWAS of dataset 1, followed by replication of SNPs associated with DTC risk (P<0.05) in dataset 2. The reliability of the identified SNPs was confirmed by PRS and Bayesian statistics after merging the three datasets. SNPs were used to describe the case/control state of individuals by ML classifier. Starting from 171 SNPs associated with DTC, 15 were positive in both the datasets 1 and 2. Using these markers, PRS revealed that individuals in the fifth quintile had a 7-fold increased risk of DTC than those in the first. Bayesian inference confirmed that the selected 15 SNPs differentiate cases from controls. Results were corroborated by ML, finding a maximum AUC of about 0.7. A restricted selection of only 15 DTC-associated SNPs is able to describe the inner genetic structure of Italian individuals and ML allows a fair prediction of case or control status based solely on the individual genetic background

Short-Term Exposure to Bisphenol A Does Not Impact Gonadal Cell Steroidogenesis In Vitro

: Bisphenol A (BPA) is a ubiquitous, synthetic chemical proven to induce reproductive disorders in both men and women. The available studies investigated the effects of BPA on male and female steroidogenesis following long-term exposure to the compound at relatively high environmental concentrations. However, the impact of short-term exposure to BPA on reproduction is poorly studied. We evaluated if 8 and 24 h exposure to 1 nM and 1 µM BPA perturbs luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e., the mouse tumour Leydig cell line mLTC1, and human primary granulosa lutein cells (hGLC). Cell signalling studies were performed using a homogeneous time-resolved fluorescence (HTRF) assay and Western blotting, while gene expression analysis was carried out using real-time PCR. Immunostainings and an immunoassay were used for intracellular protein expression and steroidogenesis analyses, respectively. The presence of BPA leads to no significant changes in gonadotropin-induced cAMP accumulation, alongside phosphorylation of downstream molecules, such as ERK1/2, CREB and p38 MAPK, in both the cell models. BPA did not impact STARD1, CYP11A1 and CYP19A1 gene expression in hGLC, nor Stard1 and Cyp17a1 expression in mLTC1 treated with LH/hCG. Additionally, the StAR protein expression was unchanged upon exposure to BPA. Progesterone and oestradiol levels in the culture medium, measured by hGLC, as well as the testosterone and progesterone levels in the culture medium, measured by mLTC1, did not change in the presence of BPA combined with LH/hCG. These data suggest that short-term exposure to environmental concentrations of BPA does not compromise the LH/hCG-induced steroidogenic potential of either human granulosa or mouse Leydig cells

Perinatal care in SARS-CoV-2 infected women: the lesson learnt from a national prospective cohort study during the pandemic in Italy

Background: Despite the growing importance given to ensuring high-quality childbirth, perinatal good practices have been rapidly disrupted by SARS-CoV-2 pandemic. This study aimed at describing the childbirth care provided to infected women during two years of COVID-19 emergency in Italy. Methods: A prospective cohort study enrolling all women who gave birth with a confirmed SARS-CoV-2 infection within 7 days from hospital admission in the 218 maternity units active in Italy during the periods February 25, 2020-June 30, 2021, and January 1-May 31, 2022. Perinatal care was assessed by evaluating the prevalence of the following indicators during the pandemic: presence of a labour companion; skin-to-skin; no mother-child separation at birth; rooming-in; breastfeeding. Logistic regression models including women' socio-demographic, obstetric and medical characteristics, were used to assess the association between the adherence to perinatal practices and different pandemic phases. Results: During the study period, 5,360 SARS-CoV-2 positive women were enrolled. Overall, among those who had a vaginal delivery (n = 3,574; 66.8%), 37.5% had a labour companion, 70.5% of newborns were not separated from their mothers at birth, 88.1% were roomed-in, and 88.0% breastfed. These four indicators showed similar variations in the study period with a negative peak between September 2020 and January 2021 and a gradual increase during the Alpha and Omicron waves. Skin-to-skin (mean value 66.2%) had its lowest level at the beginning of the pandemic and gradually increased throughout the study period. Among women who had a caesarean section (n = 1,777; 33.2%), all the indicators showed notably worse outcomes with similar variations in the study period. Multiple logistic regression analyses confirm the observed variations during the pandemic and show a lower adherence to good practices in southern regions and in maternity units with a higher annual number of births. Conclusions: Despite the rising trend in the studied indicators, we observed concerning substandard childbirth care during the SARS-CoV-2 pandemic. Continued efforts are necessary to underscore the significance of the experience of care as a vital component in enhancing the quality of family-centred care policies

Pleotropism of gonadotropin action

LE STUDIUM Multidisciplinary Journal. This is the final version of the article, which has been published in final form at http://www.lestudium-ias.com/content/pleotropism-gonadotropin-action-0International audienceEvidence exists that the gonadotropins LH and FSH can substitute to each other under certain circumstances, in addition to the fact that they can act together in granulosa cells. The aim of this study is to investigate how the two human gonadotropins influence each other in granulosa cells expressing both receptors, or by co-culturing cells expressing either the LHCGR or the FSHR (as a model granulosa/theca interaction). Plasmids encoding the c-myc-tagged-LHCGR and the FLAG-tagged FSHR under the control of an inducible coumermycin-responsive or doxycycline-responsive promoter, respectively were produced. These plasmids were used to permanently transfect human granulosa cell-derived KGN cells and HEK293 cells. The following cell lines were obtained and partially characterized: #1 c-myc-tagged-LHCGR-KGN; #2 FLAG-tagged FSHR_HEK293; #3 FLAG-tagged FSHR-KGN; #4 Double, c-myc-tagged-LHCGR and FLAG-tagged FSHR-KGN. After induction of receptor expression, the cell lines #1 and #2 and #3 responded to hCG and FSH stimulation, respectively by producing cAMP. Receptor expression was demonstrated by RT-PCR and flow cytometry. The characterization of the cell line #4 is ongoing. These cell lines are now available for the study of cell signaling and steroid synthesis, as well as in silico modeling, to gain insight into the dynamics of the intertwined cell response to FSH and LH in granulosa cells. These experiments will continue in parallel in both laboratories involved. Our cell lines represent new, very valuable instruments for the study of molecular pharmacology of FSH and LH, in order to improve infertility treatment, (multi)follicular growth for assisted reproduction, ovulation and spermatogenesis