Danio rerio embryos on Prozac – Effects on the detoxification mechanism and embryo development

In the past decade the presence of psychopharmaceuticals, including fluoxetine (FLU), in the aquaticenvironment has been associated with the increasing trend in human consumption of these substances.Aquatic organisms are usually exposed to chronic low doses and, therefore, risk assessments shouldevaluate the effects of these compounds in non-target organisms. Teleost fish possess an array of activedefence mechanisms to cope with the deleterious effects of xenobiotics. These include ABC transporters,phase I and II of cellular detoxification and oxidative stress enzymes. Hence, the present study aimed atcharacterising the effect of FLU on embryo development of the model teleost zebrafish (Danio rerio) con-comitantly with changes in the detoxification mechanisms during early developmental phases. Embryoswere exposed to different concentrations of FLU (0.0015, 0.05, 0.1, 0.5 and 0.8-M) for 80 hours post fer-tilization. Development was screened and the impact in the transcription of key genes, i.e., abcb4, abcc1,abcc2, abcg2, cyp1a, cyp3a65, gst, sod, cat, ahr, pxr, ppar˛, pparˇ, ppar-, rxraa, rxrab, rxrbb, rxrga, rxrgb,raraa, rarab, rarga evaluated. In addition, accumulation assays were performed to measure the activity ofABC proteins and antioxidant enzymes (CAT and Cu/ZnSOD) after exposure to FLU. Embryo developmentwas disrupted at the lowest FLU concentration tested (0.0015 -M), which is in the range of concen-trations found in WWTP effluents. Embryos exposed to higher concentrations of FLU decreased Cu/ZnSOD, and increased CAT (0.0015 and 0.5 -M) enzymatic activity. Exposure to higher concentrations ofFLU decreased the expression of most genes belonging to the detoxification system and upregulated catat 0.0015 -M of FLU. Most of the tested concentrations downregulated ppar˛, pparˇ, ppar-, and raraa,rxraa, rxrab, rxrbb rxrgb and ahr gene expression while pxr was significantly up regulated at all testedconcentrations. In conclusion, this study shows that FLU can impact zebrafish embryo development, atconcentrations found in effluents of WWTPs, concomitantly with changes in antioxidant enzymes, andthe transcription of key genes involved in detoxification and development. These finding raises additionalconcerns supporting the need to monitor the presence of this compound in aquatic reservoirs

Discrimination and surveillance of infectious severe acute respiratory syndrome Coronavirus 2 in wastewater using cell culture and RT-qPCR

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA has been extensively detected in raw wastewater in studies exploring wastewater-based epidemiology (WBE) for early warning purposes. Nonetheless, only a few limited studies investigated the presence of SARS-CoV-2 in treated wastewaters to determine the potential health risks across the water cycle. The detection of SARS-CoV-2 has been done mostly by RT-qPCR and ddPCR, which only provides information on the presence of nucleic acids rather than information on potential infectivity. In this study, we set to develop and evaluate the use of viability RT-qPCR for the selective discrimination and surveillance of infectious SARS-CoV-2 in secondary-treated wastewater. Enzymatic (nuclease) and viability dye (Reagent D) pretreatments were applied to infer infectivity through RT-qPCR using porcine epidemic diarrhea virus (PEDV) as a CoV surrogate. Infectivity tests were first performed on PEDV purified RNA, then on infectious and heat-inactivated PEDV, and finally on heat inactivated PEDV spiked in concentrated secondary-treated wastewater. The two viability RT-qPCR methods were then applied to 27 secondary-treated wastewater samples positive for SARS-CoV-2 RNA at the outlet of five large urban wastewater treatment plants in Portugal. Reagent D pretreatment showed similar behavior to cell culture for heat-inactivated PEDV and both viability RT-qPCR methods performed comparably to VERO E6 cell culture for SARS-CoV-2 present in secondary-treated wastewater, eliminating completely the RT-qPCR signal. Our study demonstrated the lack of infectious SARS-CoV-2 viral particles on secondary-treated wastewater through the application of two pretreatment methods for the rapid inference of infectivity through RT-qPCR, showing their potential application in environmental screening. This study addressed a knowledge gap on the public health risks of SARS-CoV-2 across the water cycle.info:eu-repo/semantics/publishedVersio

Upstream and downstream process development of a Vero cell-based yellow fever vaccine

Yellow fever (YF) is a lethal viral disease that is endemic in some tropical regions of South America, Central America and Africa. An attenuated vaccine produced in embryonated eggs is available since the 1930’s and is known to be highly effective and safe. However, after large vaccination campaigns in the 2000’s, reports of rare, but serious adverse events have stimulated Biomanguinhos/FIOCRUZ, who produces the current attenuated 17DD vaccine, to develop a new, inactivated vaccine. Over the last years, through a partnership of the Federal University of Rio de Janeiro and Biomanguinhos/FIOCRUZ, both upstream and downstream processes were developed. The upstream process was established based on Vero cell cultivation on microcarriers in serum-free medium, using stirred-tank bioreactors. The first studies were carried out in spinner flasks to select the microcarrier type and the serum-free medium. Also, statistical DOE tools were used to study the infection step, varying the moiety of infection and the time of infection. This process was then scale-up to stirred-tank bioreactors and further optimized regarding microcarrier concentration, stepwise medium addition, dissolved oxygen level/sparging intensity, impeller configuration and time of harvest. The final upstream process that was established results in virus titers of 10^8 pfu/mL within a time frame 144h post inoculation of the cells in the bioreactor. The downstream process was designed priorizing chromatographic techniques, aiming at achieving high purity levels and extensive removal of process-related critical contaminants, such as DNA and host-cell proteins (HCP), as preconized by the regulatory authorities. For the capture step, both cation- and anion-exchange chromatographies were evaluated. A Q membrane adsorber process was selected and the best operational conditions in terms of pH, temperature, buffers and washing strategies were determined. For the second purification step, three techniques were evaluated: multimodal chromatography, ultrafiltration/diafiltration, and hydrophobic interaction chromatography using a HIC membrane adsorber. The multimodal resin showed the best results, and operational conditions of this step were further optimized. The final 2-step yellow-fever virus purification process resulted in an overall yield of 52% and residual HCP of 350 ppm (0.05%). Residual DNA was 1.2 ng per dose, considering the dose established based on animal studies, and is in agreement with the limit recommended by the World Health Organization (\u3c10 ng/dose). Electrophoretic analysis (SDS-PAGE) of the purified samples showed a band corresponding to 96% of identified proteins with molecular mass of 56 kDa, which is the expected mass for the virus envelope protein (E). Anti-E Western blot (WB) showed a single band, confirming the identity of the samples. No band was revealed in the anti-HCP blot, confirming the low HCP levels quantified. The developed process allows the production of a new, high-purity yellow-fever vaccine through a scalable technology, which is better suited than egg-based technology to meet emergency demands in case of epidemics and is useful in the current scenario of increasing worldwide demand YF vaccine

Dynamic culture of osteogenic cells in biomimetically coated poly(caprolactone) nanofibre mesh constructs

In our previous work, biomimetic calcium phosphate-coated poly(caprolactone) nanofibre meshes (BCP-NMs) were demonstrated to be more effective for supporting cell attachment and proliferation under static conditions, when compared with poly(caprolactone) nanofibre meshes (PCL-NMs). In many applications, in vitro cultivation of constructs using bioreactors that support efficient nutrition of cells has appeared as an important step toward the development of functional grafts. This work aimed at studying the effects of dynamic culture conditions and biomimetic coating on bone cells grown on the nanofibre meshes. BCP-NM and PCL-NM were seeded with osteoblast-like cells (MG63--human osteosarcoma-derived cell line). The cell-seeded constructs were cultured within a rotating bioreactor that simulated microgravity, at a fixed rotating speed, for different time periods, and then characterized. Cell morphology, viability, and phenotype were assessed. PCL-NM constructs presented a higher number of dead cells than BCP-NM constructs. Under dynamic conditions, the production of proteins associated with the extracellular matrix of bone was higher on BCP-NM constructs than in the PCL-NM ones, which indicates that coated samples may provide cells with a better environment for tissue growth. It is suggested that improved mass transfer in the bioreactor in combination with the appropriate substrate were decisive factors for this highly positive outcome for generating bone.This work was developed under the scope of the EU Project Network of Excellence "Expertissues'' (NMP3-CT-2004-500283) and supported by Alea jacta est Marie Curie Actions (MEST-CT-2004-008104). M. Alves da Silva would like to acknowledge the Portuguese Foundation for Science and Technology for her grant (SFRH-BD-28708-2006). Jose V. Araujo is grateful to S. Rathbone, H. Sura, I. Wimpenny, I. Dublon, G. Jones, and E. D. Pinho for useful technical discussions

Morphological Divergence among Progeny of Macroptilium lathyroides Accessions from the Semi-Arid Region of Pernambuco, Brazil

Macroptilium is a legume genus with approximately 20 species, usually annual or biennial, herbaceous and distributed mainly in the Americas. It is widely used as a forage resource in grasslands and usually fixes atmospheric N. Martins et al. (2001) indicated that half-sib family selection with progeny testing is the most common plant breeding method used in Brazil. In the scientific literature, however, there are few studies dedicated to Macroptilium spp. This study evaluated morphological divergence among Macroptilium spp. progeny from accessions collected in 4 counties located in the semi-arid region of Pernambuco State, NE Brazil

A wastewater-based epidemiology tool for COVID-19 surveillance in Portugal

Funding: Strategic funding of Fundação para a Ciência e a Tecnologia (FCT), Portugal, to cE3c and BioISI Research Units ( UIDB/00329/2020 and UIDB/04046/2020 ] is also gratefully acknowledged. This work was supported by Programa Operacional de Competitividade e Internacionalização (POCI) (FEDER component), Programa Operacional Regional de Lisboa , and Programa Operacional Regional do Norte (Project COVIDETECT, ref. 048467 ).The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater produced interest in its use for sentinel surveillance at a community level and as a complementary approach to syndromic surveillance. With this work, we set the foundations for wastewater-based epidemiology (WBE) in Portugal by monitoring the trends of SARS-CoV-2 RNA circulation in the community, on a nationwide perspective during different epidemiological phases of the pandemic. The Charité assays (E_Sarbecco, RdRP, and N_Sarbecco) were applied to monitor, over 32-weeks (April to December 2020), the dynamics of SARS-CoV-2 RNA at the inlet of five wastewater treatment plants (WWTP), which together serve more than two million people in Portugal. Raw wastewater from three Coronavirus disease 2019 (COVID-19) reference hospitals was also analyzed during this period. In total, more than 600 samples were tested. For the first weeks, detection of SARS-CoV-2 RNA was sporadic, with concentrations varying from 103 to 105 genome copies per liter (GC/L). Prevalence of SARS-CoV-2 RNA increased steeply by the end of May into late June, mainly in Lisboa e Vale do Tejo region (LVT), during the reopening phase. After the summer, with the reopening of schools in mid-September and return to partial face-to-face work, a pronounced increase of SARS-CoV-2 RNA in wastewater was detected. In the LVT area, SARS-CoV-2 RNA load agreed with reported trends in hotspots of infection. Synchrony between trends of SARS-CoV-2 RNA in raw wastewater and daily new COVID-19 cases highlights the value of WBE as a surveillance tool, particularly after the phasing out of the epidemiological curve and when hotspots of disease re-emerge in the population which might be difficult to spot based solely on syndromic surveillance and contact tracing. This is the first study crossing several epidemiological stages highlighting the long-term use of WBE for SARS-CoV-2.PostprintPeer reviewe

Death ideation in cancer patients: contributing factors

Advances in cancer research and therapy have improved prognosis and the quality of life of many patients. However, previous epidemiological studies in oncologic patients have shown an increased risk of suicide. Suicidal thoughts, relatively well known in those terminally ill, may be just as important for cancer patients who are survivors or are living with the disease. Nonetheless, there is a relative paucity of data about suicidality in this setting. The authors conducted a prospective observational study to identify death thoughts and to explore the factors associated with suicidal ideation in cancer patients. A sample of 130 patients referred for psychiatric consultation was obtained following informed consent and authorization from the local ethics committee. A semistructured interview assessed sociodemographic data, psychosocial support, and information regarding the cancer process and its treatment. Psychometric instruments were used to evaluate psychopathology, namely the Hospital Anxiety and Depression Scale, the Beck Hopelessness Scale, and the Beck Scale for Suicide Ideation. Psychiatric diagnoses were obtained through the application of the Mini International Neuropsychiatric Interview. Death ideation was identified in 34.6% of patients, yet only 10% had active suicidal thoughts. Risk of suicide was associated with female gender, a psychiatric diagnosis (major depressive disorder, panic disorder, or dysthymia), difficult interpersonal relationships, associated pain, high hopelessness, and depressive and anxiety symptoms. Although suicidal thoughts are frequent in cancer patients at different stages of disease, most are transitory. Risk factors for suicidal ideation have been identified, such as depression, hopelessness, uncontrolled pain, and difficult interpersonal relationships. Further assessment is necessary to identify those at higher risk of attempting suicide, and underlying psychiatric disorders should be vigorously treated

Exploring the correlations between epi indicators of COVID-19 and the concentration of pharmaceutical compounds in Wastewater Treatment Plants in Northern Portugal

The COVID-19 pandemic caused by the SARS-CoV-2 virus led to changes in the lifestyle and human behaviour, which resulted in different consumption patterns of some classes of pharmaceuticals including curative, symptom-relieving, and psychotropic drugs. The trends in the consumption of these compounds are related to their concentrations in wastewater systems, since incompletely metabolised drugs (or their metabolites back transformed into the parental form) may be detected and quantified by analytical methods. Pharmaceuticals are highly recalcitrant compounds and conventional activated sludge processes implemented in wastewater treatment plants (WWTP) are ineffective at degrading these substances. As a results, these compounds end up in waterways or accumulate in the sludge, being a serious concern given their potential effects on ecosystems and public health. Therefore, it is crucial to evaluate the presence of pharmaceuticals in water and sludge to assist in the search for more effective processes. In this work, eight pharmaceuticals from five therapeutic classes were analysed in wastewater and sludge samples collected in two WWTP located in the Northern Portugal, during the third COVID-19 epidemic wave in Portugal. The two WWTP demonstrated a similar pattern with respect to the concentration levels in that period. However, the drugs loads reaching each WWTP were dissimilar when normalising the concentrations to the inlet flow rate. Acetaminophen (ACET) was the compound detected at highest concentrations in aqueous samples of both WWTP (98. 516 g L1 in WWTP2 and 123. 506 g L1in WWTP1), indicating that this drug is extensively used without the need of a prescription, known of general public knowledge as an antipyretic and analgesic agent to treat pain and fever. The concentrations determined in the sludge samples were below 1.65 µg g1 in both WWTP, the highest value being found for azithromycin (AZT). This result may be justified by the physico-chemical characteristics of the compound that favour its adsorption to the sludge surface through ionic interactions. It was not possible to establish a clear relationship between the incidence of COVID-19 cases in the sewer catchment and the concentration of drugs detected in the same period. However, looking at the data obtained, the high incidence of COVID-19 in January 2021 is in line with the high concentration of drugs detected in the aqueous and sludge samples but prediction of drug load from viral load data was unfeasible.This study was supported by the Competitiveness and Internationalisation Operational Programme, Lisbon Regional Operational Programme and Algarve Regional Operational Programme with the support of FEDER, through the Incentive Scheme: research and development activities and investment in testing and optimisation (upscaling) infrastructures in the context of COVID-19, through the Project “SARS CONTROL: Evaluation of the impacts of SARS-CoV-2 on the urban water cycle and the downstream effects on Public Health" (Ref. 070076). Acknowledge is also due to the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, and by LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020. Strategic funding from FCT to cE3c and BioISI Research Units (UIDB/00329/2020 and UIDB/04046/2020) and to the Associate Laboratory CHANGE (LA/P/0121/2020) is also gratefully acknowledged. ARS holds an FCT grant SFRH/BD/131905/2017 and COVID/BD/151951/2021.ARLR and MFRP acknowledge the financial support from LA/P/0045/2020 (ALiCE), UIDB/50020/2020 and UIDP/50020/2020 (LSRE-LCM), funded by national funds through FCT/MCTES (PIDDAC). ARLR acknowledges FCT funding under DL57/2016 Transitory Norm Programme.info:eu-repo/semantics/publishedVersio

Encapsulation of Nanostructures in a Dielectric Matrix Providing Optical Enhancement in Ultrathin Solar Cells

The incorporation of nanostructures in optoelectronic devices for enhancing their optical performance is widely studied. However, several problems related to the processing complexity and the low performance of the nanostructures have hindered such actions in real-life devices. Herein, a novel way of introducing gold nanoparticles in a solar cell structure is proposed in which the nanostructures are encapsulated with a dielectric layer, shielding them from high temperatures and harsh growth processing conditions of the remaining device. Through optical simulations, an enhancement of the effective optical path length of approximately four times the nominal thickness of the absorber layer is verified with the new architecture. Furthermore, the proposed concept in a Cu(In,Ga)Se2 solar cell device is demonstrated, where the short-circuit current density is increased by 17.4%. The novel structure presented in this work is achieved by combining a bottom-up chemical approach of depositing the nanostructures with a top-down photolithographic process, which allows for an electrical contact.This work was funded in part by the Fundação para a Ciência e a Tecnologia (FCT) under Grants IF/00133/2015, PD/BD/142780/2018 and SFRH/BD/ 146776/2019. The authors also want to acknowledge the European Union’s Horizon 2020 Research and Innovation Programme through the ARCIGS-M project under Grant 720887, the Special Research Fund (BOF) of Hasselt University, the FCT through the project NovaCell (PTDC/CTM-CTM/28075/ 2017), and InovSolarCells (PTDC/FISMAC/29696/2017) co-funded by FCT and the ERDF through COMPETE2020. The authors also want to acknowledge Sandra Maya for the production of images used in this work.info:eu-repo/semantics/publishedVersio