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Human phenylalanine hydroxylase (hPAH) hydroxylates l -phenylalanine ( l -Phe) to l -tyrosine, a precursor for neurotransmitter biosynthesis. Phenylketonuria (PKU), caused by mutations in PAH that impair PAH function, leads to neurological impairment when untreated. Understanding the hPAH structural and regulatory properties is essential to outline PKU pathophysiological mechanisms. Each hPAH monomer comprises an N-terminal regulatory, a central catalytic and a C-terminal oligomerisation domain. To maintain physiological l -Phe levels, hPAH employs complex regulatory mechanisms. Resting PAH adopts an auto-inhibited conformation where regulatory domains block access to the active site. l -Phe-mediated allosteric activation induces a repositioning of the regulatory domains. Since a structure of activated wild-type hPAH is lacking, we addressed hPAH l -Phe-mediated conformational changes and report the first solution structure of the allosterically activated state. Our solution structures obtained by small-angle X-ray scattering support a tetramer with distorted P222 symmetry, where catalytic and oligomerisation domains form a core from which regulatory domains protrude, positioning themselves close to the active site entrance in the absence of l -Phe. Binding of l -Phe induces a large movement and dimerisation of regulatory domains, exposing the active site. Activated hPAH is more resistant to proteolytic cleavage and thermal denaturation, suggesting that the association of regulatory domains stabilises hPAH.

Ovarian cancer is the main cause of death from gynecological cancer, with its poor prognosis mainly related to late diagnosis and chemoresistance (acquired or intrinsic) to conventional alkylating and reactive oxygen species (ROS)-generating drugs. We and others reported that the availability of cysteine and glutathione (GSH) impacts the mechanisms of resistance to carboplatin in ovarian cancer. Different players in cysteine metabolism can be crucial in chemoresistance, such as the cystine/glutamate antiporter system Xc (xCT) and the H2S-synthesizing enzyme cystathionine β-synthase (CBS) in the pathway of cysteine catabolism. We hypothesized that, by disrupting cysteine metabolic flux, chemoresistance would be reverted. Since the xCT transporter is also able to take up selenium, we used selenium-containing chrysin (SeChry) as a plausible competitive inhibitor of xCT. For that, we tested the effects of SeChry on three different ovarian cancer cell lines (ES2, OVCAR3, and OVCAR8) and in two non-malignant cell lines (HaCaT and HK2). Results showed that, in addition to being highly cytotoxic, SeChry does not affect the uptake of cysteine, although it increases GSH depletion, indicating that SeChry might induce oxidative stress. However, enzymatic assays revealed an inhibitory effect of SeChry toward CBS, thus preventing production of the antioxidant H2S. Notably, our data showed that SeChry and folate-targeted polyurea dendrimer generation four (SeChry@PUREG4-FA) nanoparticles increased the specificity for SeChry delivery to ovarian cancer cells, reducing significantly the toxicity against non-malignant cells. Collectively, our data support SeChry@PUREG4-FA nanoparticles as a targeted strategy to improve ovarian cancer treatment, where GSH depletion and CBS inhibition underlie SeChry cytotoxicity.

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule. The enzymes 3-mercaptopyruvate sulfurtransferase (MST), partly localized in mitochondria, and the inner mitochondrial membrane-associated sulfide:quinone oxidoreductase (SQR), besides being respectively involved in the synthesis and catabolism of H2S, generate sulfane sulfur species such as persulfides and polysulfides, currently recognized as mediating some of the H2S biological effects. Reprogramming of H2S metabolism was reported to support cellular proliferation and energy metabolism in cancer cells. As oxidative stress is a cancer hallmark and N-acetylcysteine (NAC) was recently suggested to act as an antioxidant by increasing intracellular levels of sulfane sulfur species, here we evaluated the effect of prolonged exposure to NAC on the H2S metabolism of SW480 colon cancer cells. Cells exposed to NAC for 24 h displayed increased expression and activity of MST and SQR. Furthermore, NAC was shown to: (i) persist at detectable levels inside the cells exposed to the drug for up to 24 h and (ii) sustain H2S synthesis by human MST more effectively than cysteine, as shown working on the isolated recombinant enzyme. We conclude that prolonged exposure of colon cancer cells to NAC stimulates H2S metabolism and that NAC can serve as a substrate for human MST.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The ‘gasotransmitters’ hydrogen sulfide (H2S), nitric oxide (NO), and carbon monoxide (CO) act as second messengers in human physiology, mediating signal transduction via interaction with or chemical modification of protein targets, thereby regulating processes such as neurotransmission, blood flow, immunomodulation, or energy metabolism. Due to their broad reactivity and potential toxicity, the biosynthesis and breakdown of H2S, NO, and CO are tightly regulated. Growing evidence highlights the active role of gasotransmitters in their mutual cross-regulation. In human physiology, the transsulfuration enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are prominent H2S enzymatic sources. While CBS is known to be inhibited by NO and CO, little is known about CSE regulation by gasotransmitters. Herein, we investigated the effect of s-nitrosation on CSE catalytic activity. H2S production by recombinant human CSE was found to be inhibited by the physiological nitrosating agent s-nitrosoglutathione (GSNO), while reduced glutathione had no effect. GSNO-induced inhibition was partially reverted by ascorbate and accompanied by the disappearance of one solvent accessible protein thiol. By combining differential derivatization procedures and mass spectrometry-based analysis with functional assays, seven out of the ten protein cysteine residues, namely Cys84, Cys109, Cys137, Cys172, Cys229, Cys307, and Cys310, were identified as targets of s-nitrosation. By generating conservative Cys-to-Ser variants of the identified s-nitrosated cysteines, Cys137 was identified as most significantly contributing to the GSNO-mediated CSE inhibition. These results highlight a new mechanism of crosstalk between gasotransmitters.

Phenylketonuria (PKU) is a genetic disease caused by deficient activity of human phenylalanine hydroxylase (hPAH) that, when untreated, can lead to severe psychomotor impairment. Protein misfolding is recognized as the main underlying pathogenic mechanism of PKU. Therefore, the use of stabilizers of protein structure and/or activity is an attractive therapeutic strategy for this condition. Here, we report that 3-hydroxyquinolin-2(1H)-one derivatives can act as protectors of hPAH enzyme activity. Electron paramagnetic resonance spectroscopy demonstrated that the 3-hydroxyquinolin-2(1H)-one compounds affect the coordination of the non-heme ferric center at the enzyme active-site. Moreover, surface plasmon resonance studies showed that these stabilizing compounds can be outcompeted by the natural substrate l-phenylalanine. Two of the designed compounds functionally stabilized hPAH by maintaining protein activity. This effect was observed on the recombinant purified protein and in a cellular model. Besides interacting with the catalytic iron, one of the compounds also binds to the N-terminal regulatory domain, although to a different location from the allosteric l-Phe binding site, as supported by the solution structures obtained by small-angle X-ray scattering.

The stabilization of G-Quadruplex DNA structures by ligands is a promising strategy for telomerase inhibition in cancer therapy since this enzyme is responsible for the unlimited proliferation of cancer cells. To assess the potential of a compound as a telomerase inhibitor, selectivity for quadruplex over duplex DNA is a fundamental attribute, as the drug must be able to recognize quadruplex DNA in the presence of a large amount of duplex DNA, in the cellular nucleus. By using different spectroscopic techniques, such as ultraviolet-visible, fluorescence and circular dichroism, this work evaluates the potential of a series of multicharged phthalocyanines, bearing four or eight positive charges, as G-Quadruplex stabilizing ligands. This work led us to conclude that the existence of a balance between the number and position of the positive charges in the phthalocyanine structure is a fundamental attribute for its selectivity for G-Quadruplex structures over duplex DNA structures. Two of the studied phthalocyanines, one with four peripheral positive charges (ZnPc1) and the other with less exposed eight positive charges (ZnPc4) showed high selectivity and affinity for G-Quadruplex over duplex DNA structures and were able to accumulate in the nucleus of UM-UC-3 bladder cancer cells.

Background In humans the stress response is known to be modulated to a great extent by psychological factors, particularly by the predictability and the perceived control that the subject has of the stressor. This psychological dimension of the stress response has also been demonstrated in animals phylogenetically closer to humans (i.e. mammals). However, its occurrence in fish, which represent a divergent vertebrate evolutionary lineage from that of mammals, has not been established yet, and, if present, would indicate a deep evolutionary origin of these mechanisms across vertebrates. Moreover, the fact that psychological modulation of stress is implemented in mammals by a brain cortical top-down inhibitory control over subcortical stress-responsive structures, and the absence of a brain cortex in fish, has been used as an argument against the possibility of psychological stress in fish, with implications for the assessment of fish sentience and welfare. Here, we have investigated the occurrence of psychological stress in fish by assessing how stressor controllability modulates the stress response in European seabass ( Dicentrarchus labrax ). Results Fish were exposed to either a controllable or an uncontrollable stressor (i.e. possibility or impossibility to escape a signaled stressor). The effect of loss of control (possibility to escape followed by impossibility to escape) was also assessed. Both behavioral and circulating cortisol data indicates that the perception of control reduces the response to the stressor, when compared to the uncontrollable situation. Losing control had the most detrimental effect. The brain activity of the teleost homologues to the sensory cortex (Dld) and hippocampus (Dlv) parallels the uncontrolled and loss of control stressors, respectively, whereas the activity of the lateral septum (Vv) homologue responds in different ways depending on the gene marker of brain activity used. Conclusions These results suggest the psychological modulation of the stress response to be evolutionary conserved across vertebrates, despite being implemented by different brain circuits in mammals (pre-frontal cortex) and fish (Dld-Dlv).

Purpose This paper aims to explore how nurses stay engaged in their work with the impact of structural empowerment and civility. It delves deeper into how kind, empathetic and respectful behaviours (civility) among colleagues influence the link between structural empowerment and nurses’ engagement. Design/methodology/approach Data was collected from 580 nurses working in a Public Hospital in the metropolitan area of Lisbon. Self-report questionnaires measuring civility, structural empowerment and engagement were administered. Hayes’ PROCESS macro for mediation analysis in SPSS was used to test the hypothesised model. Findings Results demonstrated that civility and structural empowerment were positively associated (r = 0.491, p < 0.01) also showing a positive influence on nurses’ engagement (r = 0.492, p < 0.01; r = 0.485, p < 0.01, respectively). Civility was found to partially mediate the association between structural empowerment and engagement (ß = 0.315, 95% CI [0.222, 0.417], 5,000 bootstrap resamples). Practical implications Findings from this study may be used for health-care employees and organisations, implying that when nurses perceive themselves as structurally empowered at work within a respectful environment, they experience an enhanced sense of community and involvement in their organization. Originality/value To the best of the authors’ knowledge, this is the first attempt in exploring the relationship of combined workplace civility, structural empowerment and engagement in a sample of Portuguese nurses. Future research could substantially increase our understanding of how civility contributes to a positive workplace. Propósito Este artículo explora cómo las enfermeras se mantienen comprometidas (work engagement) en su trabajo con el impacto del empoderamiento estructural y la civilidad. Profundiza en cómo los comportamientos amables, empáticos y respetuosos (civilidad) entre colegas influyen en el vínculo entre el empoderamiento estructural y el work engagement de las enfermeras/os. Diseño/metodología/enfoque Se recogieron datos de 580 enfermeras que trabajaban en un hospital público del área metropolitana de Lisboa. Se administraron cuestionarios de autoinforme que medían la civilidad, el empoderamiento estructural y el work engagement. Se utilizó la macro PROCESS de Hayes para el análisis de mediación en SPSS con el fin de probar el modelo hipotetizado. Resultados Los resultados demostraron que la civilidad y el empoderamiento estructural estaban positivamente asociados (r = 0.491, p < 0.01) mostrando también una influencia positiva en el work engagement de las enfermeras (r = 0.492, p < 0.01; r = 0.485, p < 0.01, respectivamente). Se observó que la civilidad mediaba parcialmente la asociación entre el empoderamiento estructural y el work engagement (β = 0.315, 95% CI [0.222, 0.417], 5,000 resamples bootstrap). Implicaciones prácticas Los resultados de este estudio pueden ser utilizados por los empleados y las organizaciones sanitarias, ya que implican que cuando las enfermeras se perciben a sí mismas como estructuralmente empoderadas en el trabajo dentro de un entorno respetuoso, experimentan un mayor sentido de comunidad e implicación en su organización. Originalidad/valor Hasta donde sabemos, este es el primer intento de explorar la relación entre la civilidad en el lugar de trabajo, el empoderamiento estructural y el work engagement en una muestra de enfermeras portuguesas. Futuras investigaciones podrían aumentar sustancialmente nuestra comprensión de cómo el civismo contribuye a un lugar de trabajo positivo. Propósito Este documento explora a forma como os enfermeiras/os se mantêm empenhados e envolvidos (work engagement) no seu trabalho, tendo em conta o impacto do empoderamento estrutural e da civilidade. Aprofunda a forma como os comportamentos de gentileza, empatia e respeito (civilidade) entre colegas influenciam a relação entre o empoderamento estrutural e o work engagement dos enfermeiros. Desenho/metodologia/abordagem Foram recolhidos dados de 580 enfermeiros de um Hospital Público da área metropolitana de Lisboa. Foram aplicados questionários de auto-relato para avaliar a civilidade, o empoderamento estrutural e o work engagement. A macro PROCESS de Hayes para análise de mediação no SPSS foi utilizada para testar o modelo hipotético. Resultados Os resultados demonstraram que a civilidade e o empoderamento estrutural estavam positivamente associados (r = 0.491, p < 0.01), mostrando também uma influência positiva no work engagement dos enfermeiros (r = 0.492, p < 0.01; r = 0.485, p < 0.01, respetivamente). Verificou-se que a civilidade medeia, parcialmente, a associação entre a empoderamento estrutural e o work engagement (β = 0.315, 95% CI [0.222, 0.417], 5,000 resamples bootstrap). Implicações práticas Os resultados deste estudo podem ser uteis para os trabalhadores e organizações de cuidados de saúde, implicando que, quando os enfermeiros se consideram estruturalmente capacitados no trabalho num ambiente de respeito, experimentam um maior sentido de comunidade e envolvimento na sua organização. Originalidade/valor Até à data, esta é a primeira tentativa de explorar a relação entre civilidade no local de trabalho, empoderamento estrutural e work engagement numa amostra de enfermeiros portugueses. Futuras investigações poderão aumentar substancialmente a nossa compreensão de como a civilidade contribui para um ambiente laboral positivo.

Abstract Objective Pain intensity is the most commonly assessed domain in pain research and clinical settings. To facilitate cross-cultural research, knowledge regarding the psychometric properties of pain intensity measures in individuals from different countries is needed. However, the majority of this research has been conducted in English-speaking countries. Design Survey study. Setting University. Subjects Four hundred nineteen college students. Methods Participants were asked to complete four measures assessing average pain intensity: 1) the 0–10 numerical rating scale (NRS-11), 2) the 100-mm visual analog scale (VAS), 3) the four-point verbal rating scale (VRS-4), and 4) the Faces Pain Scale–Revised (FPS-R). Results The rates of incorrect completion of the four scales were uniformly low (range = 1–2%). The NRS-11 had the highest preference rate (31%), although a substantial number of participants also preferred each of the other three scales (range = 22–24%). The findings support the utility and construct validity of all four pain intensity scales in this Spanish-speaking sample. Conclusions When considered in light of research from other non-English-speaking samples indicating significant psychometric weaknesses for the NRS-11 and VAS and relative strengths of the FPS-R in some groups, the findings suggest that the FPS-R might be the most appropriate pain intensity scale to use when comparisons across populations from different countries is a goal. More research is needed to determine the extent to which demographic (i.e., age, education levels, socioeconomic status) vs cultural factors (i.e., country of origin) influence the reliability, validity, and utility of different pain measures.