Radiosensitization of HNSCC cells by EGFR inhibition depends on the induction of cell cycle arrests

The increase in cellular radiosensitivity by EGF receptor (EGFR) inhibition has been shown to be attributable to the induction of a G1-arrest in p53-proficient cells. Because EGFR targeting in combination with radiotherapy is used to treat head and neck squamous cell carcinomas (HNSCC) which are predominantly p53 mutated, we tested the effects of EGFR targeting on cellular radiosensitivity, proliferation, apoptosis, DNA repair and cell cycle control using a large panel of HNSCC cell lines. In these experiments EGFR targeting inhibited signal transduction, blocked proliferation and induced radiosensitization but only in some cell lines and only under normal (pre-plating) conditions. This sensitization was not associated with impaired DNA repair (53BP1 foci) or induction of apoptosis. However, it was associated with the induction of a lasting G2-arrest. Both, the radiosensitization and the G2-arrest were abrogated if the cells were re-stimulated (delayed plating) with actually no radiosensitization being detectable in any of the 14 tested cell lines. Therefore we conclude that EGFR targeting can induce a reversible G2 arrest in p53 deficient HNSCC cells, which does not consequently result in a robust cellular radiosensitization. Together with recent animal and clinical studies our data indicate that EGFR inhibition is no effective strategy to increase the radiosensitivity of HNSCC cells.</p

Cytosolic phosphoenolpyruvate carboxykinase deficiency : Expanding the clinical phenotype and novel laboratory findings

Cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) deficiency due to the homozygous PCK1 variant has recently been associated with childhood-onset hypoglycemia with a recognizable pattern of abnormal urine organic acids. In this study, 21 children and 3 adult patients with genetically confirmed PEPCK-C deficiency were diagnosed during the years 2016 to 2019 and the available biochemical and clinical data were collected. All patients were ethnic Finns. Most patients (22 out of 24) had a previously published homozygous PCK1 variant c.925G>A. Two patients had a novel compound heterozygous PCK1 variant c.925G>A and c.716C>T. The laboratory results showed abnormal urine organic acid profile with increased tricarboxylic acid cycle intermediates and inadequate ketone body production during hypoglycemia. The hypoglycemic episodes manifested predominantly in the morning. Infections, fasting or poor food intake, heavy exercise, alcohol consumption, and breastfeeding were identified as triggering factors. Five patients presented with neonatal hypoglycemia. Hypoglycemic seizures occurred in half of the patients (12 out of 24). The first hypoglycemic episode often occurred at the age of 1-2 years, but it sometimes presented at a later age, and could re-occur during school age or adulthood. This study adds to the laboratory data on PEPCK-C deficiency, confirming the recognizable urine organic acid pattern and identifying deficient ketogenesis as a novel laboratory finding. The phenotype is expanded suggesting that the risk of hypoglycemia may continue into adulthood if predisposing factors are present.Peer reviewe

Coulomb implosion of tetrabromothiophene observed under multiphoton ionization by free-electron-laser soft-x-ray pulses

Soft-x-ray free-electron-laser pulses were used to create highly charged molecular tetrabromothiophene species by sequential multiphoton ionization from bromine 3d orbitals. The experiment was performed at the SACLA facility in Japan and the products of molecular dissociation were analyzed by means of multicoincidence momentum-resolved ion time-of-flight spectroscopy. Total charge states up to +13 atomic units were produced, creating a particular dissociation pattern for the carbon ions, a Coulomb implosion, due to the concerted forces by the surrounding heavy bromine ions. This behavior was explored both experimentally and by numerical molecular-dynamics simulations and the fingerprints of the Coulomb implosion were identified in both. In simulations, Coulomb implosion was predicted to be highly sensitive to the initial (thermal) motion of the atoms and, after including vibrational motion for several temperatures, good general agreement between the experiment and simulations was found. The agreement with the experiment was further improved by adding charge dynamics to the simulation, according to our point-charge dynamics model with empirical rate constants

Activation of epidermal growth factor receptor is required for Chlamydia trachomatis development

Background Chlamydia trachomatis (C. trachomatis) is a clinically significant human pathogen and one of the leading causative agents of sexually transmitted diseases. As obligate intracellular bacteria, C. trachomatis has evolved strategies to redirect the host’s signaling and resources for its own survival and propagation. Despite the clinical notoriety of Chlamydia infections, the molecular interactions between C. trachomatis and its host cell proteins remain elusive. Results In this study, we focused on the involvement of the host cell epidermal growth factor receptor (EGFR) in C. trachomatis attachment and development. A combination of molecular approaches, pharmacological agents and cell lines were used to demonstrate distinct functional requirements of EGFR in C. trachomatisinfection. We show that C. trachomatis increases the phosphorylation of EGFR and of its downstream effectors PLCγ1, Akt and STAT5. While both EGFR and platelet-derived growth factor receptor-β (PDGFRβ) are partially involved in bacterial attachment to the host cell surface, it is only the knockdown of EGFR and not PDGFRβ that affects the formation of C. trachomatis inclusions in the host cells. Inhibition of EGFR results in small immature inclusions, and prevents C. trachomatis-induced intracellular calcium mobilization and the assembly of the characteristic F-actin ring at the inclusion periphery. By using complementary approaches, we demonstrate that the coordinated regulation of both calcium mobilization and F-actin assembly by EGFR are necessary for maturation of chlamydial inclusion within the host cells. A particularly important finding of this study is the co-localization of EGFR with the F-actin at the periphery of C. trachomatis inclusion where it may function to nucleate the assembly of signaling protein complexes for cytoskeletal remodeling required for C. trachomatisdevelopment. Conclusion Cumulatively, the data reported here connect the function of EGFR to C. trachomatis attachment and development in the host cells, and this could lead to new venues for targeting C. trachomatis infections and associated diseases

Mixed-method research approaches within non-governmental programmes to improve maternal and child health in Zimbabwe

Non-governmental organisations (NGOs) are key stakeholders in public health programmes in low resource settings. Despite known evidence-based interventions for improving maternal and child health (MNCH) and HIV outcomes, there remains an inequitable distribution of preventable and treatable maternal and infant morbidity and mortality within and between countries and regions globally. Through a scoping review and two published case studies, the aim of this research paper style thesis was to explore and critically reflect on how the use of theories, models and frameworks (TMFs) and mixed-method research approaches within NGO-implemented programmes can yield improved estimates of service uptake, clinical outcomes, and understanding of reasons for and consequences of failure to uptake evidence-based prevention of mother to child transmission (PMTCT) of HIV and MNCH services in Zimbabwe. Scoping review findings demonstrate that NGOs are central actors in the HIV response as catalysts of change through advocacy and activism, direct service delivery providers and technical partners in low resource settings. Limited evidence of application of scientific methods by NGOs through transdisciplinary research collaborations indicate missed opportunities for strengthening evidence-based practice in context. In the first case study, an observational, multisite, cohort study used sampling-based methods to actively trace a random sample of 371 HIV positive mothers in Mashonaland East Province Zimbabwe with no documented uptake of recommended Early Infant Diagnosis (EID) HIV testing for their HIV-exposed infants. Corrected estimates of EID uptake following tracing almost doubled, from 31.2% of infants with documented EID in routine records to a cumulative incidence of EID with death as a competing risk of 60.0% (95% CI: 58.7% to 61.3%) after contact tracing. Findings highlight significant underreporting of both infant HIV testing and mortality in routine data sources. The second case study explored the lived experience of ‘non-adherers’ to recommended HIV/MNCH services. A qualitative study among women with a recent non-institutional birth was conducted with 71 women (30 in-depth interviews; 41 in focus group discussions) who had delivered at home in the previous 6 months in Mashonaland Central Province, Zimbabwe. Narratives of women’s birth and postpartum experiences following a home delivery highlighted a coalescing of structural inequality and vulnerability at the time of delivery which prevented access to institutional delivery. Social norms for ‘facility delivery for all’ produced through health promotion initiatives to increase skilled attendance at birth, created unintended social consequences (shaming, blaming) and punishments (fines, reduced quality of healthcare) for home delivery which introduced additional risk to women at the time of delivery and in the postnatal period. Women’s narratives underscore the importance of understanding social norms, contextual vulnerabilities and potential unintended consequences of health policy translation on the most vulnerable who are unable to comply to recommended public health practice. Embedding mixed-method research within routine NGO programmes in this thesis yielded improved estimates of service uptake and outcomes; provided additional insights into the reasons for suboptimal uptake of PMTCT/MNCH services among rural women in Zimbabwe; and, led to the expansion of existing TMFs. The scoping review and case study findings provided critical insights to inform adaptation of NGO programme strategies that have improved programme performance and been adopted into national policy and programmes at scale in Zimbabwe. Transdisciplinary programme science embedded within NGO 7 programmes has the potential to optimise coverage and effectiveness of evidence-based interventions that are equitable and contextually relevant

Fate of the teratogenic and carcinogenic ochratoxin A in human perfused placenta

Ochratoxin A (OTA) is one of the most frequent mycotoxins detected in human blood worldwide. Apart from its well known nephrotoxicity, OTA-induced teratogenicity and carcinogenicity proven in animals are potential effects also in humans. Pregnant women have been exposed to this food contaminant via dietary exposure in a continuous and widespread manner. Although the transplacental transfer of OTA has been demonstrated in laboratory animals and the presence of OTA in human fetal samples has been reported, little is known about the role of human placenta in OTA toxicokinetics. In this study, human perfused placenta was used to reveal the actual placental toxicokinetics of OTA using concentrations found in serum of pregnant women. Moreover, the effect of protein concentration and biological significance of placental transporters on the OTA transfer in human placenta were also determined. Our study is the first to pursue the transfer of OTA through perfused human placenta. The transfer of OTA through term human placenta was barely detectable in all perfusions. Inhibitors of neither ABCG2 nor ABCC2 increased the transport of OTA to fetal circulation in placental perfusion, and thus these transporters apparently do not have biological significance in inhibiting transplacental transfer of OTA. Human albumin has inhibited OTA transfer through a tight monolayer of BeWo b30 cells. Finding from this study clearly contradict the existing epidemiological studies reporting higher OTA levels in fetal than in maternal circulation in vivo. © 2011 Elsevier Ireland Ltd.link_to_subscribed_fulltex

The xenoestrogens, bisphenol A and para-nonylphenol, decrease the expression of the ABCG2 transporter protein in human term placental explant cultures.

Many endogenous and xenobiotic compounds are substrates and regulators of human placental ABC transporters. ABCG2 is protecting fetus against foreign chemicals. Environmental xenoestrogens, like bisphenol A (BPA) and p-nonylphenol (p-NP), mimic natural estrogens and can affect hormonal systems. Effects of BPA, p-NP, DES (diethylstilbestrol) and estradiol (E2), on ABCG2 expression were studied using human first trimester and term placental explants. Role of estrogen receptors (ER) in the effects of chemicals was studied by ER antagonist. Term placenta expressed less ABCG2 protein. In term placentas BPA (p < 0.05), p-NP (p < 0.01) and E2 (p < 0.05) decreased the ABCG2 protein expression after 48 h exposure while after 24 h exposure, only E2 decreased the expression (p < 0.05). The chemicals did not affect ABCG2 in first trimester placentas. The ER antagonist affected differently the responses of chemicals. In conclusion, environmental xenoestrogens downregulate placental ABCG2 protein expression depending on gestational age

Quantitative estimation of mercury intake by toxicokinetic modelling based on total mercury levels in humans

Mercury is a toxic metal that can be disseminated into the environment from both natural and anthropogenic sources. Human exposure to the metal stems mainly from food, and more particularly from the consumption of fish and other seafoods. Examining dietary exposure and measuring mercury levels in body tissues are two ways of estimating exposure to mercury. In this study, we utilized a modelling system consisting of three linear toxicokinetic models for describing the fate of methyl mercury, inorganic mercury, and metallic mercury in the body, in order to estimate daily intake of mercury as measured through total mercury concentrations in the blood. We then compared the results stemming from our modelling system to those of the detailed semi-quantitative food frequency questionnaire (FFQ) of the Norwegian Fish and Game (NFG) Study, a project that focused on dietary mercury exposure. The results indicate that toxicokinetic modelling based on blood levels gave higher daily intake values of mercury compared to those of the FFQ. Furthermore, the former had a wider range of estimates than the latter. The properties of the toxicokinetic model or limitations in the dietary exposure assessment could be posited as reasons for the differences between the respective methods. Moreover, the results may have been influenced by sources of mercury exposure that cannot be described as dietary, such as amalgam filling