Mónica Ferreira

Impairment of mice spermatogenesis by sodium arsenite

In order to assess the effect of arsenic on the male reproductive impairment in mice, 7-week-old animals were exposed to 7.5 mg sodium arsenite (NaAsO2)/kg body weight, during 35 days (one spermatogenic cycle). One group of animals was sacrificed after exposure, while another group received distilled water for an additional period of 35 days, in order to study the spermatoxic effect and the recovery of spermatogenesis. In mice sacrificed after NaAsO2 exposure, a decrease in testis/body weight ratio and reduction of tubular diameter were observed. Both groups of NaAsO2-exposed animals showed remarkable histopathological changes, such as sloughing of immature germ cells. Animals sacrificed after NaAsO2 exposure showed decreased sperm motility, increased abnormal sperm morphology and decreased sperm viability. The effects of NaAsO2 on sperm motility recovered to normal values after one spermatogenic cycle, while increased sperm abnormality was maintained. However, at this period, a decrease in acrosome integrity was detected. Concerning oxidative stress parameters, animals sacrificed after NaAsO2 exposure showed a decreased selenium-dependent glutathione peroxidase activity, which was not detected after the recovery. Conversely, at this period, total glutathione peroxidase activity increased in exposed animals. These results demonstrate the toxic effects of NaAsO2 on mice spermatogenesis and show the lack of recovery after one spermatogenic cycle.

Workshop- and Telephone-Based Interventions to Improve Adverse Drug Reaction Reporting

AbstractBackground: Spontaneous reporting of adverse drug reactions (ADRs) is the method most widely used by pharmacovigilance systems, with the principal limitation being the physician’s underreporting. Objective: This study sought to evaluate the results of workshop and telephone-interview interventions designed to improve the quantity and relevance of ADR reporting by physicians. Methods: A cluster-randomized controlled trial was conducted on 6579 physicians in northern Portugal in 2008. Following randomization, we allocated 1034 physicians to a telephone-interview intervention, 438 to a workshop intervention and the remaining 5107 to the control group. At the workshop, a real clinical case was presented and participants were then asked to report on it by completing the relevant form. In the telephone intervention, participants were asked (i) whether they had ever had any suspicion of ADRs; (ii) whether they had experienced any difficulties in reporting; (iii) whether they remembered the different methods that could be used for reporting purposes; and (iv) whether they attached importance to the individual physician’s role in reporting. We followed up physicians to assess ADR reporting rates to the Northern Pharmacovigilance Centre. In terms of relevance, adverse reactions were classified as serious or unexpected. Statistical analysis was performed on an intention-to-treat basis, and generalized linear mixed models were applied using the penalized quasi-likelihood method. The physicians studied were followed up over a period of 20 months. Results: Two hundred physicians underwent the educational intervention. Comparison with the control group showed that the workshop intervention increased the spontaneous ADR reporting rate by an average of 4-fold (relative risk [RR] 3.97; 95% CI 3.86, 4.08; p < 0.001) across the 20 months post-intervention. Telephone interviews, in contrast, proved less efficient since they led to no significant difference (p = 0.052) vis-à-vis the control group in ADR reporting (RR 1.02; 95% CI 1.00, 1.04). The effects of the interventions on the reporting rate of serious and high-causality ADRs indicated that the RRs associated with workshops were 6.84 (95% CI 6.69, 6.98; p<0.001) for serious ADRs and 3.58 (95% CI 3.51, 3.66; p<0.001) for high-causality ADRs. Conclusions: Whereas telephone interventions only increased spontaneous reporting in the first 4 months of follow-up, workshops significantly increased both the quantity and relevance of spontaneous ADR reporting for more than 1 year.

Identification and characterization of two distinct PPP1R2 isoforms in human spermatozoa

BackgroundProtein Ser/Thr Phosphatase PPP1CC2 is an alternatively spliced isoform of PPP1C that is highly enriched in testis and selectively expressed in sperm. Addition of the phosphatase inhibitor toxins okadaic acid or calyculin A to caput and caudal sperm triggers and stimulates motility, respectively. Thus, the endogenous mechanisms of phosphatase inhibition are fundamental for controlling sperm function and should be characterized. Preliminary results have shown a protein phosphatase inhibitor activity resembling PPP1R2 in bovine and primate spermatozoa.ResultsHere we show conclusively, for the first time, that PPP1R2 is present in sperm. In addition, we have also identified a novel protein, PPP1R2P3. The latter was previously thought to be an intron-less pseudogene. We show that the protein corresponding to the pseudogene is expressed. It has PPP1 inhibitory potency similar to PPP1R2. The potential phosphosites in PPP1R2 are substituted by non-phosphorylable residues, T73P and S87R, in PPP1R2P3. We also confirm that PPP1R2/PPP1R2P3 are phosphorylated at Ser121 and Ser122, and report a novel phosphorylation site, Ser127. Subfractionation of sperm structures show that PPP1CC2, PPP1R2/PPP1R2P3 are located in the head and tail structures.ConclusionsThe conclusive identification and localization of sperm PPP1R2 and PPP1R2P3 lays the basis for future studies on their roles in acrosome reaction, sperm motility and hyperactivation. An intriguing possibility is that a switch in PPP1CC2 inhibitory subunits could be the trigger for sperm motility in the epididymis and/or sperm hyperactivation in the female reproductive tract.

Workshop- and Telephone-Based Interventions to Improve Adverse Drug Reaction Reporting A Cluster-Randomized Trial in Portugal

BACKGROUND Spontaneous reporting of adverse drug reactions (ADRs) is the method most widely used by pharmacovigilance systems, with the principal limitation being the physicians underreporting. OBJECTIVE This study sought to evaluate the results of workshop and telephone-interview interventions designed to improve the quantity and relevance of ADR reporting by physicians. METHODS A cluster-randomized controlled trial was conducted on 6579 physicians in northern Portugal in 2008. Following randomization, we allocated 1034 physicians to a telephone-interview intervention, 438 to a workshop intervention and the remaining 5107 to the control group. At the workshop, a real clinical case was presented and participants were then asked to report on it by completing the relevant form. In the telephone intervention, participants were asked (i) whether they had ever had any suspicion of ADRs; (ii) whether they had experienced any difficulties in reporting; (iii) whether they remembered the different methods that could be used for reporting purposes; and (iv) whether they attached importance to the individual physicians role in reporting. We followed up physicians to assess ADR reporting rates to the Northern Pharmacovigilance Centre. In terms of relevance, adverse reactions were classified as serious or unexpected. Statistical analysis was performed on an intention-to-treat basis, and generalized linear mixed models were applied using the penalized quasi-likelihood method. The physicians studied were followed up over a period of 20 months. RESULTS Two hundred physicians underwent the educational intervention. Comparison with the control group showed that the workshop intervention increased the spontaneous ADR reporting rate by an average of 4-fold (relative risk [RR] 3.97; 95% CI 3.86, 4.08; p < 0.001) across the 20 months post-intervention. Telephone interviews, in contrast, proved less efficient since they led to no significant difference (p = 0.052) vis-à-vis the control group in ADR reporting (RR 1.02; 95% CI 1.00, 1.04). The effects of the interventions on the reporting rate of serious and high-causality ADRs indicated that the RRs associated with workshops were 6.84 (95% CI 6.69, 6.98; p < 0.001) for serious ADRs and 3.58 (95% CI 3.51, 3.66; p < 0.001) for high-causality ADRs. CONCLUSIONS Whereas telephone interventions only increased spontaneous reporting in the first 4 months of follow-up, workshops significantly increased both the quantity and relevance of spontaneous ADR reporting for more than 1 year.

Biogenesis and activity regulation of protein phosphatase 1

Protein phosphatase 1 (PP1) is expressed in all eukaryotic cells and catalyzes a substantial fraction of phosphoserine/threonine dephosphorylation reactions. It forms stable complexes with PP1-interacting proteins (PIPs) that guide the phosphatase throughout its life cycle and control its fate and function. The diversity of PIPs is huge (≈200 in vertebrates), and most of them combine short linear motifs to form large and unique interaction interfaces with PP1. Many PIPs have separate domains for PP1 anchoring, PP1 regulation, substrate recruitment and subcellular targeting, which enable them to direct associated PP1 to a specific subset of substrates and mediate acute activity control. Hence, PP1 functions as the catalytic subunit of a large number of multimeric holoenzymes, each with its own subset of substrates and mechanism(s) of regulation.

Correlation between Mitochondrial Reactive Oxygen and Severity of Atherosclerosis

Atherosclerosis has been associated with mitochondria dysfunction and damage. Our group demonstrated previously that hypercholesterolemic mice present increased mitochondrial reactive oxygen (mtROS) generation in several tissues and low NADPH/NADP+ ratio. Here, we investigated whether spontaneous atherosclerosis in these mice could be modulated by treatments that replenish or spare mitochondrial NADPH, named citrate supplementation, cholesterol synthesis inhibition, or both treatments simultaneously. Robust statistical analyses in pooled group data were performed in order to explain the variation of atherosclerosis lesion areas as related to the classic atherosclerosis risk factors such as plasma lipids, obesity, and oxidative stress, including liver mtROS. Using three distinct statistical tools (univariate correlation, adjusted correlation, and multiple regression) with increasing levels of stringency, we identified a novel significant association and a model that reliably predicts the extent of atherosclerosis due to variations in mtROS. Thus, results show that atherosclerosis lesion area is positively and independently correlated with liver mtROS production rates. Based on these findings, we propose that modulation of mitochondrial redox state influences the atherosclerosis extent.

Determinants of physician antibiotic prescribing behavior: a 3 year cohort study in Portugal

Abstract Objectives Antibiotic misprescription is a major driver of resistance, which is a worldwide public health problem. Therefore, our aim is to assess the influence of the determinants of physician prescribing on the quality of antibiotic use. Methods A 3 year cohort study including all primary-care physicians working in Portugal’s Central Regional Health Administration (n = 1094) was conducted. We assessed the determinants of prescribing using a pre-validated, personally addressed, reply-paid, self-administered questionnaire (sent four times to non-responders, between September 2011 and February 2012) designed to collect information on physicians’ attitudes to and knowledge of antibiotic prescribing as well as their socio-demographic and professional data. To evaluate antibiotic prescribing, we’ve calculated ESAC 12 quality indicators per physician per year, allowing us to stratify them as good or poor prescribers according to their performance on those indicators. Associations between determinants and outcomes were fitted with generalized linear mixed models. Results The overall response rate was 46.1%. Emergency activity (OR [95% CI] = 0.29 [0.16–0.54]; p < 0.05) and workload (number of patients seen per day: OR [95% CI] = 0.97 [0.94–1.00]; p < 0.05; number of patients seen per week in emergencies: OR [95% CI] = 0.98 [0.97–0.99]; p < 0.05) were both related to poor quality of antibiotic prescribing. Statistically significant odds ratios were also obtained for ignorance (IqOR [95% CI] = 2.14 [1.31–3.52]), complacency (1/IqOR [95% CI] = 1.19 [1.01–1.41]) and responsibility of others (1/IqOR [95% CI] = 1.78 [1.10–3.06]). Conclusions The above results serve to emphasize workload, working at emergency departments and physicians’ attitudes identified as critical factors affecting antibiotic prescribing. This provides new insights for clinicians, researchers and policy makers when it comes to developing and improving the clinical and economic outcomes of antibiotic use. Key limitations of the study included the difficulty of results extrapolation and the limitations of the stratification method based on the antibiotic prescribing quality indicators.

The deletion of the phosphatase regulator NIPP1 causes progenitor cell expansion in the adult liver

The Ppp1r8 gene encodes NIPP1, a nuclear interactor of protein phosphatase PP1. The deletion of NIPP1 is embryonic lethal at the gastrulation stage, which has hampered its functional characterization in adult tissues. Here, we describe the effects of a conditional deletion of NIPP1 in mouse liver epithelial cells. Ppp1r8−/− livers developed a ductular reaction, that is, bile‐duct hyperplasia with associated fibrosis. The increased proliferation of biliary epithelial cells was at least partially due to an expansion of the progenitor cell compartment that was independent of liver injury. Gene‐expression analysis confirmed an upregulation of progenitor cell markers in the liver knockout livers but showed no effect on the expression of liver‐injury associated regulators of cholangiocyte differentiation markers. Consistent with an inhibitory effect of NIPP1 on progenitor cell proliferation, Ppp1r8−/− livers displayed an increased sensitivity to diet‐supplemented 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine, which also causes bile‐duct hyperplasia through progenitor cell expansion. In contrast, the liver knockouts responded normally to injuries (partial hepatectomy, single CCl4 administration) that are restored through proliferation of differentiated parenchymal cells. Our data indicate that NIPP1 does not regulate the proliferation of hepatocytes but is a suppressor of biliary epithelial cell proliferation, including progenitor cells, in the adult liver. Stem Cells 2016;34:2256–2262

Brief Report: The Deletion of the Phosphatase Regulator NIPP1 Causes Progenitor Cell Expansion in the Adult Liver

The Ppp1r8 gene encodes NIPP1, a nuclear interactor of protein phosphatase PP1. The deletion of NIPP1 is embryonic lethal at the gastrulation stage, which has hampered its functional characterization in adult tissues. Here, we describe the effects of a conditional deletion of NIPP1 in mouse liver epithelial cells. Ppp1r8−/− livers developed a ductular reaction, that is, bile‐duct hyperplasia with associated fibrosis. The increased proliferation of biliary epithelial cells was at least partially due to an expansion of the progenitor cell compartment that was independent of liver injury. Gene‐expression analysis confirmed an upregulation of progenitor cell markers in the liver knockout livers but showed no effect on the expression of liver‐injury associated regulators of cholangiocyte differentiation markers. Consistent with an inhibitory effect of NIPP1 on progenitor cell proliferation, Ppp1r8−/− livers displayed an increased sensitivity to diet‐supplemented 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine, which also causes bile‐duct hyperplasia through progenitor cell expansion. In contrast, the liver knockouts responded normally to injuries (partial hepatectomy, single CCl4 administration) that are restored through proliferation of differentiated parenchymal cells. Our data indicate that NIPP1 does not regulate the proliferation of hepatocytes but is a suppressor of biliary epithelial cell proliferation, including progenitor cells, in the adult liver. Stem Cells 2016;34:2256–2262

The protein phosphatase 1 regulator NIPP1 is essential for mammalian spermatogenesis

NIPP1 is one of the major nuclear interactors of protein phosphatase PP1. The deletion of NIPP1 in mice is early embryonic lethal, which has precluded functional studies in adult tissues. Hence, we have generated an inducible NIPP1 knockout model using a tamoxifen-inducible Cre recombinase transgene. The inactivation of the NIPP1 encoding alleles (Ppp1r8) in adult mice occurred very efficiently in testis and resulted in a gradual loss of germ cells, culminating in a Sertoli-cell only phenotype. Before the overt development of this phenotype Ppp1r8−/− testis showed a decreased proliferation and survival capacity of cells of the spermatogenic lineage. A reduced proliferation was also detected after the tamoxifen-induced removal of NIPP1 from cultured testis slices and isolated germ cells enriched for undifferentiated spermatogonia, hinting at a testis-intrinsic defect. Consistent with the observed phenotype, RNA sequencing identified changes in the transcript levels of cell-cycle and apoptosis regulating genes in NIPP1-depleted testis. We conclude that NIPP1 is essential for mammalian spermatogenesis because it is indispensable for the proliferation and survival of progenitor germ cells, including (un)differentiated spermatogonia.