Rapid optimization of chromatography operating conditions using a nano- liter scale column on a microfluidic chip with integrated pneumatic valves and optical sensors

Purification of monoclonal antibodies (mAbs) is traditionally achieved by chromatographic separations, which are very robust but require time-consuming optimization on a case-by-case, particularly if a non-affinity step is used. In this context, multimodal chromatography has been explored as a versatile and cost-effective alternative to the established affinity step employed for capturing mAbs. However, selective capture/polishing of a target mAb using such multimodal ligands comes with the need for extensive and time-consuming optimization, due to the multitude of interactions that can be simultaneously promoted in the ligand. In this work, we developed a novel microfluidic platform comprising multimodal chromatography beads inside micro-columns for rapid screening of operating conditions. Sequential liquid insertion in the device was achieved by using integrated pneumatic valves and the chromatographic assays were combined with a signal acquisition module for on-chip fluorescence measurements. Please click Additional Files below to see the full abstract

Immobilization of L-asparaginase towards surface-modified carbon nanotubes

L-asparaginase (ASNase, EC 3.5.1.1) is an enzyme that catalyzes L-asparagine hydrolysis into L-aspartic acid and ammonia and is mainly applied in pharmaceutical and food industries [1]. The ASNase currently commercialized for pharmaceutical purposes is produced from two main bacterial sources: recombinant Escherichia coli and Erwinia chrysanthemi. However, some disadvantages are associated with its free form, such as the shorter half-life [2]. Immobilization of ASNase has been proposed as an efficient approach to overcome this limitation [3]. In this work, a straightforward method, including the functionalization of multi-walled carbon nanotubes (MWCNTs) through a hydrothermal oxidation treatment with nitric acid, and the immobilization of ASNase by adsorption over pristine and modified MWCNTs was investigated. Different operation conditions, including pH, contact time, ASNase/MWCNT mass ratio, and the operational stability of the immobilized ASNase were evaluated. The characterization of the ASNase-MWCNT bioconjugate was addressed using different techniques, namely Transmission Electron Microscopy (TEM), Thermogravimetric analysis (TGA), and Raman spectroscopy. Functionalized MWCNTs showed promising results, with an immobilization yield and a relative recovered activity of commercial ASNase above 95%, under the optimized adsorption conditions (pH 8, 60 min of contact and 1.5´10–3 g.mL-1of ASNase). The ASNase-MWCNT bioconjugate also showed improved enzyme operational stability (6 consecutive reaction cycles without activity loss), proving its suitability for application in industrial processes.publishe

Immobilization of L-asparaginase towards surface-modified carbon nanotubes

L-asparaginase (LA) is an enzyme that catalyzes L-asparagine hydrolysis into L-aspartic acid and ammonia and is mainly applied in pharmaceutical and food industries. The LA currently commercialized for pharmaceutical purposes is produced from two main bacterial sources: recombinant Escherichia coli and Erwinia chrysanthemi. However, some disadvantages are associated with its free form, such as the shorter half-life. Immobilization of LA has been proposed as an efficient approach to overcome this limitation. In this work, a straightforward method, including the functionalization of multi-walled carbon nanotubes (MWCNTs) through a hydrothermal oxidation treatment and the immobilization of LA by adsorption over pristine and modified MWCNTs was investigated. Different operation conditions, including pH, contact time, ASNase/MWCNT mass ratio, and the operational stability of the immobilized LA, were evaluated. The characterization of the LA-MWCNT bioconjugate was addressed using different techniques, namely Transmission Electron Microscopy (TEM), Thermogravimetric analysis (TGA), and Raman spectroscopy. Functionalized MWCNTs showed promising results, with an immobilization yield and a relative recovered activity of commercial LA above 95%, under the optimized adsorption conditions (pH 8, 60 min of contact, and 1.510–3 g.mL-1 of LA). The LA-MWCNT bioconjugate also showed improved enzyme operational stability (6 consecutive reaction cycles without activity loss), proving its suitability for application in industrial processes.publishe

Superior operational stability of immobilized L-asparaginase over surface-modified carbon nanotubes

L-asparaginase (ASNase, EC 3.5.1.1) is an enzyme that catalyzes the L-asparagine hydrolysis into L-aspartic acid and ammonia, being mainly applied in pharmaceutical and food industries. However, some disadvantages are associated with its free form, such as the ASNase short half-life, which may be overcome by enzyme immobilization. In this work, the immobilization of ASNase by adsorption over pristine and modified multi-walled carbon nanotubes (MWCNTs) was investigated, the latter corresponding to functionalized MWCNTs through a hydrothermal oxidation treatment. Different operating conditions, including pH, contact time and ASNase/MWCNT mass ratio, as well as the operational stability of the immobilized ASNase, were evaluated. For comparison purposes, data regarding the ASNase immobilization with pristine MWCNT was detailed. The characterization of the ASNase-MWCNT bioconjugate was addressed using different techniques, namely Transmission Electron Microscopy (TEM), Thermogravimetric Analysis (TGA) and Raman spectroscopy. Functionalized MWCNTs showed promising results, with an immobilization yield and a relative recovered activity of commercial ASNase above 95% under the optimized adsorption conditions (pH 8, 60 min of contact and 1.5 × 10-3 g mL-1 of ASNase). The ASNase-MWCNT bioconjugate also showed improved enzyme operational stability (6 consecutive reaction cycles without activity loss), paving the way for its use in industrial processes.publishe

Assessment of the general public's knowledge about rheumatic diseases: evidence from a Portuguese population-based survey

Background. To identify incorrect beliefs and common knowledge about rheumatic diseases in the general population. Methods. Participants were selected during the follow-up of a representative cohort of adult population of Porto, Portugal; 1626 participants completed a questionnaire that included general knowledge items about rheumatic diseases. Discrete and continuous latent variable models were used to identify knowledge flaws and the target groups. Odds ratios (OR) estimated by multinomial logistic regression, and 95% confidence intervals (95%CI) were computed to evaluate magnitude of associations. Results. A continuous latent variable model identified two dimensions: one related to general beliefs (latent 1) and another concerning characteristics, treatment and impact of rheumatic diseases (latent 2). A 3-class latent variable model refined these results: the first class presented the lowest probabilities of correct answer for items associated with the first latent (mean of 39%), and the second class presented the lowest probabilities of correct answer for items with the second latent (mean of 62%). The third class showed the highest probability of a correct answer for almost all the items (mean of 79%). The age and sex standardized prevalence of the classes was 25.7%, 30.8% and 43.5%. Taking class 2 as reference, class 1 was positively associated with the presence of rheumatic diseases (OR = 2.79; CI95% = (2.10-3.70)), with females (OR = 1.28 CI95% = (0.99-1.67)) and older individuals (OR = 1.04; CI95% = (1.03-1.05)), and was negatively associated with education (OR = 0.84; CI95% = (0.81-0.86)); class 3 was positively associated with education (OR = 1.03; CI95% = (1.00-1.05)) and the presence of rheumatic diseases (OR = 1.29; CI95% = (0.97-1.70)). Conclusions. There are several knowledge flaws about rheumatic diseases in the general public. One out of four participants considered false general beliefs as true and approximately 30% did not have detailed knowledge on rheumatic disease. Higher education and the presence of disease contributed positively to the overall knowledge. These results suggest some degree of effectiveness of patient education, either conducted by health professionals or self-driven. © 2010 Severo et al; licensee BioMed Central Ltd

Osteoarthritis medical labelling and health-related quality of life in the general population

BACKGROUND: Osteoarthritis is the most common chronic joint disease. In the absence of an effective medical treatment and due to the chronic nature of this condition, an osteoarthritis medical diagnosis may finally result in decreased health-related quality of life. Therefore, the aim of this study was to measure the impact of the osteoarthritis medical labelling on physical and mental health-related quality of life. METHODS: Subjects (n = 1132, 58.7% women) were approached as participants of an urban population-based cohort (EPIPorto). Self-reported information on previous diagnosis of knee, hip or hand osteoarthritis was obtained and rheumatologists established knee, hip or hand osteoarthritis clinical diagnosis in symptomatic individuals. Physical and mental dimensions of health-related quality of life were evaluated using the self-administered Medical Outcomes Study: 36-Item Short Form Survey. Crude and adjusted linear regression coefficients (beta) and the corresponding 95% confidence intervals (95% CI) were computed to estimate the associations between being labelled as an osteoarthritis case and health-related quality of life. RESULTS: Regardless of disease medical labelling, individuals with osteoarthritis scored significantly lower physical health-related quality of life when compared to those without joint disease (knee(unexposed): beta = −5.3, 95% CI: −7.6, −3.1; knee(exposed): beta = −6.0, 95% CI: −8.4, −3.7; hip(unexposed): beta = −6.0, 95% CI: −9.8, −2.3; hip(exposed): beta = −11.0, 95% CI: −15.6, −6.4; hand(unexposed): beta = −4.3, 95% CI: −6.5, −2.0; hand(exposed): beta = −4.3, 95% CI: −6.6, −2.1). The same was not observed regarding mental health-related quality of life. Among subjects with clinically confirmed osteoarthritis, the medical labelling of this joint disease was not significantly associated to health-related quality of life. CONCLUSIONS: The labelling of knee, hip and hand osteoarthritis diagnosis may not add specific benefit to osteoarthritis patients in terms of its capability to improve health-related quality of life

Análise da aplicabilidade do custeio-meta na etapa de concepção de empreendimentos habitacionais de interesse social

Custeio-Meta é uma estratégia desenvolvida pela indústria para melhorar sistematicamente a qualidade do produto, entregando maior valor ao consumidor, respeitando o referencial de preço do mercado e mantendo estrito controle dos custos. Estudos recentes têm apontado caminhos para a adaptação dessa estratégia para a construção civil, apesar das características ímpares da produção de edifícios. Este trabalho apresenta uma análise da aplicabilidade do Custeio-Meta na etapa de concepção de empreendimentos habitacionais de interesse social. Foi realizado um estudo de caso no Programa de Arrendamento Residencial, no qual existe o envolvimento da empresa construtora desde as primeiras fases de planejamento do empreendimento. Também foram utilizados dados secundários gerados em dois estudos realizados anteriormente sobre a cadeia de negócios de empreendimentos e o processo de desenvolvimento do produto neste Programa. Os resultados sugerem que esse contexto propicia a aplicação do custeio-meta para que as necessidades dos clientes de tais empreendimentos, as quais normalmente não são totalmente atendidas devido ao teto estabelecido para financiamento, sejam melhor contempladas. Ao final são propostas diretrizes para melhorias no processo de projeto de forma a propiciar a aplicação do custeio-meta nesse contexto

Ação inseticida do extrato de Derris amazonica Killip para Cerotoma arcuatus Olivier (Coleoptera: Chrysomelidae

A abundância e o potencial inseticida de Derris amazonica e a necessidade de controle de Cerotoma arcuatus Olivier (Coleoptera: Chrysomelidae) na cultura do feijão-caupi (Vigna unguiculata L. Walp) estimularam a realização desta pesquisa, que objetivou avaliar a ação inseticida do extrato de D. amazonica a adultos de C. arcuatus em condições de laboratório. Os bioensaios testaram as vias de intoxicação por ingestão de folhas contaminadas, contato com superfície contaminada e aplicação tópica, com delineamento experimental inteiramente casualizado, com quatro repetições. Os valores de mortalidade e consumo foliar dos insetos foram submetidos à análise de regressão, sendo utilizada a análise de Probit para determinação das CL50, da DL50 e dos TL50. O extrato de D. amazonica, contendo 3,7% de rotenona, foi tóxico para adultos de C. arcuatus via ingestão de folhas contaminadas (CL50=15,14 µL do extrato.mL-1 de água), superfície contaminada (CL50=0,45 µL do extrato.cm-2) e aplicação tópica (DL50=1,44 µL do extrato.g-1 do inseto). Mortalidades de adultos de C. arcuatus superiores a 80% e os menores tempos letais médios foram obtidos na concentração de 5% (v v-1) do extrato em todos os bioensaios. O consumo foliar de adultos de C. arcuatus foi inversamente proporcional a concentração do extrato quando expostos por via de ingestão foliar ou aplicação tópica, sendo inclusive observada inibição da alimentação dos indivíduos. O extrato de D. amazonica é tóxico para C. arcuatus e inibe a alimentação dos insetos a partir da concentração de 1% (v v-1).The abundance and insecticidal potential of Derris amazonica in addition to need of controlling Cerotoma arcuatus for bean crop stimulated this research. The objective of this work was to evaluate insecticide action of the extract of D. amazonica to adults of C. arcuatus in laboratory conditions. The bioassays were carried out using three distend methodologies: leaf intake, contact in treated surface (filter paper) and topical application. A completed randomized experimental design was used with four replications. Mortality values and leaf consumption of the insects were subjected to regression analyses, being the Probit analyses used to determine of the i.e., LC50, LT50 and LD50. The extract of D. amazonica containing 3.7% of rotenone was toxic to adults C. arcuatus when exposed to treated leaves (LC50 = 15.14 µl.mL-1), treated surface (LC50 = 0.45 µl.cm-2) and subjected to topical exposure (LD50 = 1.44 µl.g-1). In all bioassays the adults mortality was higher than 80% with lower median lethal times obtained with 5% (v.v-1) concentrations of the extract. Leaf consumption by adults C. arcuatus was inversely proportional to the concentration of the extract when exposed by leaf intake or topical application, also being observed inhibition of feeding individuals. The extract of D. amazonica is toxic to C. arcuatus and inhibits the feeding of insects from the concentration of 1% (v v-1)

SARS-CoV-2 introductions and early dynamics of the epidemic in Portugal

Genomic surveillance of SARS-CoV-2 in Portugal was rapidly implemented by the National Institute of Health in the early stages of the COVID-19 epidemic, in collaboration with more than 50 laboratories distributed nationwide. Methods By applying recent phylodynamic models that allow integration of individual-based travel history, we reconstructed and characterized the spatio-temporal dynamics of SARSCoV-2 introductions and early dissemination in Portugal. Results We detected at least 277 independent SARS-CoV-2 introductions, mostly from European countries (namely the United Kingdom, Spain, France, Italy, and Switzerland), which were consistent with the countries with the highest connectivity with Portugal. Although most introductions were estimated to have occurred during early March 2020, it is likely that SARS-CoV-2 was silently circulating in Portugal throughout February, before the first cases were confirmed. Conclusions Here we conclude that the earlier implementation of measures could have minimized the number of introductions and subsequent virus expansion in Portugal. This study lays the foundation for genomic epidemiology of SARS-CoV-2 in Portugal, and highlights the need for systematic and geographically-representative genomic surveillance.We gratefully acknowledge to Sara Hill and Nuno Faria (University of Oxford) and Joshua Quick and Nick Loman (University of Birmingham) for kindly providing us with the initial sets of Artic Network primers for NGS; Rafael Mamede (MRamirez team, IMM, Lisbon) for developing and sharing a bioinformatics script for sequence curation (https://github.com/rfm-targa/BioinfUtils); Philippe Lemey (KU Leuven) for providing guidance on the implementation of the phylodynamic models; Joshua L. Cherry (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health) for providing guidance with the subsampling strategies; and all authors, originating and submitting laboratories who have contributed genome data on GISAID (https://www.gisaid.org/) on which part of this research is based. The opinions expressed in this article are those of the authors and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. This study is co-funded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study come from the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio