Search results for: Cristina Theada Ferreira

Authors: Cristina Maria Mendoza Sanchez, Maria Angeles Navarro Perán

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Introduction: Nursing as a profession, from its initial formation and after its development in practice, has been built and identified mainly from its technical competence and professionalization within the positivist approach of the XIX century that provides a conception of the disease built on the basis of to the biomedical paradigm, where the care provided is more focused on the physiological processes and the disease than on the suffering person understood as a whole. The main issue that is in need of study here is a review of the nursing profession's history to get to know how the nursing profession was before the XIX century. It is unclear if there were organizations or people with knowledge about looking after others or if many people survived by chance. The holistic care, in which the appearance of the disease directly affects all its dimensions: physical, emotional, cognitive, social and spiritual. It is not a concept from the 21st century. It is common practice, most probably since established life in this world, with the final purpose of covering all these perspectives through quality care. Objective: In this paper, we describe and analyze the history of education in nursing learning in terms of reviewing and analysing theoretical foundations of clinical teaching and learning in nursing, with the final purpose of determining and describing the development of the nursing profession along the history. Method: We have done a descriptive systematic review study, doing a systematically searched of manuscripts and articles in the following health science databases: Pubmed, Scopus, Web of Science, Temperamentvm and CINAHL. The selection of articles has been made according to PRISMA criteria, doing a critical reading of the full text using the CASPe method. A compliment to this, we have read a range of historical and contemporary sources to support the review, such as manuals of Florence Nightingale and John of God as primary manuscripts to establish the origin of modern nursing and her professionalization. We have considered and applied ethical considerations of data processing. Results: After applying inclusion and exclusion criteria in our search, in Pubmed, Scopus, Web of Science, Temperamentvm and CINAHL, we have obtained 51 research articles. We have analyzed them in such a way that we have distinguished them by year of publication and the type of study. With the articles obtained, we can see the importance of our background as a profession before modern times in public health and as a review of our past to face challenges in the near future. Discussion: The important influence of key figures other than Nightingale has been overlooked and it emerges that nursing management and development of the professional body has a longer and more complex history than is generally accepted. Conclusions: There is a paucity of studies on the subject of the review to be able to extract very precise evidence and recommendations about nursing before modern times. But even so, as more representative data, an increase in research about nursing history has been observed. In light of the aspects analyzed, the need for new research in the history of nursing emerges from this perspective; in order to germinate studies of the historical construction of care before the XIX century and theories created then. We can assure that pieces of knowledge and ways of care were taught before the XIX century, but they were not called theories, as these concepts were created in modern times.

Keywords: nursing history, nursing theory, Saint John of God, Florence Nightingale, learning, nursing education

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Authors: Inês Amorim Leitão, Loes van Shaick, António Dinis Ferreira, Violette Geissen

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Plastic pollution is a growing concern worldwide: plastics are commercialized in large quantities and it takes a long time for them to degrade. When in the environment, plastic is fragmented into microplastics (<5mm), which have been found in all environmental compartments at different locations. Microplastics contribute to the environmental pollution in water, air and soil and are linked to human health problems. The progressive increase of population living in cities led to the aggravation of the pollution problem worldwide, especially in urban environments. Urban areas represent a strong source of pollution, through the roads, industrial production, wastewater, landfills, etc. It is expected that pollutants such as microplastics are transported diffusely from the sources through different pathways such as wind and rain. Therefore, it is very complex to quantify, control and treat these pollutants, designated current problematic issues by the European Commission. Green areas are pointed out by experts as natural filters for contaminants in cities, through their capacity of retention by vegetation. These spaces have thus the capacity to control the load of pollutants transported. This study investigates the spatial distribution of microplastics in urban soils of different land uses, their transport through atmospheric deposition, wind erosion, runoff and streams, as well as their deposition in vegetation like grass and tree leaves in urban environment. Coimbra, a medium large city located in the central Portugal, is the case-study. All the soil, sediments, water and vegetation samples were collected in Coimbra and were later analyzed in the Wageningen University & Research laboratory. Microplastics were extracted through the density separation using Sodium Phosphate as solution (~1.4 g cm−3) and filtration methods, visualized under a stereo microscope and identified using the u-FTIR method. Microplastic particles were found in all the different samples. In terms of soils, higher concentrations of microplastics were found in green parks, followed by landfills and industrial places, and the lowest concentrations in forests and pasture land-uses. Atmospheric deposition and streams after rainfall events seems to represent the strongest pathways of microplastics. Tree leaves can retain microplastics on their surfaces. Small leaves such as needle leaves seem to present higher amounts of microplastics per leaf area than bigger leaves. Rainfall episodes seem to reduce the concentration of microplastics on leaves surface, which suggests the wash of microplastics down to lower levels of the tree or to the soil. When in soil, different types of microplastics could be transported to the atmosphere through wind erosion. Grass seems to present high concentrations of microplastics, and the enlargement of the grass cover leads to a reduction of the amount of microplastics in soil, but also of the microplastics moved from the ground to the atmosphere by wind erosion. This study proof that vegetation can help to control the transport and dispersion of microplastics. In order to control the entry and the concentration of microplastics in the environment, especially in cities, it is essential to defining and evaluating nature-based land-use scenarios, considering the role of green urban areas in filtering small particles.

Keywords: microplastics, cities, sources, pathways, vegetation

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Authors: Fernanda Piculo, Giovana Vesentini, Gabriela Marini, Debora Cristina Damasceno, Angelica Mercia Pascon Barbosa, Marilza Vieira Cunha Rudge

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Introduction: Women with previous gestational diabetes mellitus (GDM) were significantly more likely to have urinary incontinence (UI) and pelvic floor muscle dysfunction compared to non-diabetic women two years after a cesarean section. Additional results demonstrated that induced diabetes causes detrimental effects on pregnant rat urethral muscle. These results indicate the need for exploration of the mechanistic role of a recovery factor in female UI. Chemokine ligand 7 (CCL7) was significantly over expressed in rat serum, urethral and vaginal tissues immediately following induction of stress UI in a rat model simulating birth trauma. CCL7 over expression has shown potency for stimulating targeted stem cell migration and provide a translational link (clinical measurement) which further provide opportunities for treatment. The aim of this study was to investigate the CCL7 levels profile in diabetic pregnant women with urinary incontinence during pregnancy over the first year postpartum. Methods: This study was conducted in the Perinatal Diabetes Research Center of the Botucatu Medical School/UNESP, and was approved by the Research Ethics Committee of the Institution (CAAE: 20639813.0.0000.5411). The diagnosis of GDM was established between 24th and 28th gestational weeks, by the 75 g-OGTT test according to ADA’s criteria. Urinary incontinence was defined according to the International Continence Society and the CCL7 levels was measured by ELISA (R&D Systems, Catalog Number DCC700). Two hundred twelve women were classified into four study groups: normoglycemic continent (NC), normoglycemic incontinent (NI), diabetic continent (DC) and diabetic incontinent (DI). They were evaluated at six-time-points: 12-18, 24-28 and 34-38 gestational weeks, 24-48 hours, 6 weeks and 6-12 months postpartum. Results: At 12-18 weeks, it was possible to consider only two groups, continent and incontinent, because at this early gestational period has not yet been the diagnosis of GDM. The group with GDM and UI (DI group) showed lower levels of CCL7 in all time points during pregnancy and postpartum, compared to normoglycemic groups (NC and NI), indicating that these women have not recovered from child birth induced UI during the 6-12 months postpartum compared to their controls, and that the progression of UI and/or lack of recovery throughout the first postpartum year can be related with lower levels of CCL7. Instead, serum CCL7 was significantly increased in the NC group. Taken together, these findings of overexpression of CCL7 in the NC group and decreased levels in the DI group, could confirm that diabetes delays the recovery from child birth induced UI, and that CCL7 could potentially be used as a serum marker of injury. Conclusion: This study demonstrates lower levels of CCL7 in the DI group during pregnancy and postpartum and suggests that the progression of UI in diabetic women and/or lack of recovery throughout the first postpartum year can be related with low levels of CCL7. This provides a translational potential where CCL7 measurement could be used as a surrogate for injury after delivery. Successful controlled CCL7 mediated stem cell homing to the lower urinary tract could one day introduce the potential for non-operative treatment or prevention of stress urinary incontinence.

Keywords: CCL7, gestational diabetes, pregnancy, urinary incontinence

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Authors: Rui Pedro de Sousa Guimarães Ferreira, Carlos Alberto Maia Domínguez

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Despite the ambitions to achieve climate neutrality by 2050, to fulfill the Paris Agreement's goals, the building and construction sector remains one of the most resource-intensive and greenhouse gas-emitting industries in the world, accounting for 40% of worldwide CO ₂ emissions. Over the past few decades, globalization and population growth have led to an exponential rise in demand in the housing market and, by extension, in the building industry. Considering this housing crisis, it is obvious that we will not stop building in the near future. However, the transition, which has already started, is challenging and complex because it calls for the worldwide participation of numerous organizations in altering how building systems, which have been a part of our everyday existence for over a century, are used. Wood is one of the alternatives that is most frequently used nowadays (under responsible forestry conditions) because of its physical qualities and, most importantly, because it produces fewer carbon emissions during manufacturing than steel or concrete. Furthermore, as wood retains its capacity to store CO ₂ after application and throughout the life of the building, working as a natural carbon filter, it helps to reduce greenhouse gas emissions. After a century-long focus on other materials, in the last few decades, technological advancements have made it possible to innovate systems centered around the use of wood. However, there are still some questions that require further exploration. It is necessary to standardize production and manufacturing processes based on prefabrication and modularization principles to achieve greater precision and optimization of the solutions, decreasing building time, prices, and waste from raw materials. In addition, this approach will make it possible to develop new architectural solutions to solve the rigidity and irreversibility of buildings, two of the most important issues facing housing today. Most current models are still created as inflexible, fixed, monofunctional structures that discourage any kind of regeneration, based on matrices that sustain the conventional family's traditional model and are founded on rigid, impenetrable compartmentalization. Adaptability and flexibility in housing are, and always have been, necessities and key components of architecture. People today need to constantly adapt to their surroundings and themselves because of the fast-paced, disposable, and quickly obsolescent nature of modern items. Migrations on a global scale, different kinds of co-housing, or even personal changes are some of the new questions that buildings have to answer. Designing with the reversibility of construction systems and materials in mind not only allows for the concept of "looping" in construction, with environmental advantages that enable the development of a circular economy in the sector but also unleashes multiple social benefits. In this sense, it is imperative to develop prefabricated and modular construction systems able to address the formalization of a reversible proposition that adjusts to the scale of time and its multiple reformulations, many of which are unpredictable. We must allow buildings to change, grow, or shrink over their lifetime, respecting their nature and, finally, the nature of the people living in them. It´s the ability to anticipate the unexpected, adapt to social factors, and take account of demographic shifts in society to stabilize communities, the foundation of real innovative sustainability.

Keywords: modular, timber, flexibility, housing

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Authors: Liz Carmem Silva-Pereira, Raffael Alencar Mesquita Rodrigues, Francisco Helton Mendes Barbosa, Emerson de Freitas Ferreira

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Due to the Covid-19 pandemic declared in March 2020 by the World Health Organization, the way of social coexistence across the planet was affected, especially in educational processes, from the implementation of the remote modality as a teaching strategy. This teaching-learning modality caused a change in the routine and learning of basic education students, which resulted in serious consequences for the return to face-to-face teaching in 2021. 2022, at the Federal Institute of Education, Science and Technology of Pará (IFPA) – Campus Paragominas had their training process severely affected, having studied the initial half of their training in the remote modality, which compromised the carrying out of practical classes, technical visits and field classes, essential for the student formation on the environmental technician. With the objective of promoting the recomposition of these students' learning after returning to the face-to-face modality, an educational strategy was developed in the last period of the course. As teaching methodologies were used for research as an educational principle, the integrative project and the parallel recovery action applied jointly, aiming at recomposing the basic knowledge of the natural sciences, together with the technical knowledge of the environmental area applied to the course. The project assisted 58 finalist students of the environmental technical course. A research instrument was elaborated with parameters of evaluation of the environmental quality for study in 19 collection points, in the Uraim River urban hydrographic basin, in the Paragominas City – Pará – Brazilian Amazon. Students were separated into groups under the professors' and laboratory assistants’ orientation, and in the field, they observed and evaluated the places' environmental conditions and collected physical data and water samples, which were taken to the chemistry and biology laboratories at Campus Paragominas for further analysis. With the results obtained, each group prepared a technical report on the environmental conditions of each evaluated point. This work methodology enabled the practical application of theoretical knowledge received in various disciplines during the remote teaching modality, contemplating the integration of knowledge, people, skills, and abilities for the best technical training of finalist students. At the activity end, the satisfaction of the involved students in the project was evaluated, through a form, with the signing of the informed consent term, using the Likert scale as an evaluation parameter. The results obtained in the satisfaction survey were: on the use of research projects within the disciplines attended, 82% of satisfaction was obtained; regarding the revision of contents in the execution of the project, 84% of satisfaction was obtained; regarding the acquired field experience, 76.9% of satisfaction was obtained, regarding the laboratory experience, 86.2% of satisfaction was obtained, and regarding the use of this methodology as parallel recovery, 71.8% was obtained of satisfaction. In addition to the excellent performance of students in acquiring knowledge, it was possible to remedy the deficiencies caused by the absence of practical classes, technical visits, and field classes, which occurred during the execution of the remote teaching modality, fulfilling the desired educational recomposition.

Keywords: integrative project, parallel recovery, research as an educational principle, teaching-learning

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Authors: Denise De Fátima Fernandes Barbosa, Tyane Mayara Ferreira Oliveira, Diego Jorge Maia Lima, Paula Renata Amorim Lessa, Ana Karina Bezerra Pinheiro, Cintia Gondim Pereira Calou, Glauberto Da Silva Quirino, Hellen Lívia Oliveira Catunda, Tatiana Gomes Guedes, Nicolau Da Costa

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The aim of this study is to evaluate the use of Digital Cervicography (DC) in the diagnosis of precancerous lesions related to Human Papillomavirus (HPV). Cross-sectional study with a quantitative approach, of evaluative type, held in a health unit linked to the Pro Dean of Extension of the Federal University of Ceará, in the period of July to August 2015 with a sample of 33 women. Data collecting was conducted through interviews with enforcement tool. Franco (2005) standardized the technique used for DC. Polymerase Chain Reaction (PCR) was performed to identify high-risk HPV genotypes. DC were evaluated and classified by 3 judges. The results of DC and PCR were classified as positive, negative or inconclusive. The data of the collecting instruments were compiled and analyzed by the software Statistical Package for Social Sciences (SPSS) with descriptive statistics and cross-references. Sociodemographic, sexual and reproductive variables were analyzed through absolute frequencies (N) and their respective percentage (%). Kappa coefficient (κ) was applied to determine the existence of agreement between the DC of reports among evaluators with PCR and also among the judges about the DC results. The Pearson's chi-square test was used for analysis of sociodemographic, sexual and reproductive variables with the PCR reports. It was considered statistically significant (p<0.05). Ethical aspects of research involving human beings were respected, according to 466/2012 Resolution. Regarding the socio-demographic profile, the most prevalent ages and equally were those belonging to the groups 21-30 and 41-50 years old (24.2%). The brown color was reported in excess (84.8%) and 96.9% out of them had completed primary and secondary school or studying. 51.5% were married, 72.7% Catholic, 54.5% employed and 48.5% with income between one and two minimum wages. As for the sexual and reproductive characteristics, prevailed heterosexual (93.9%) who did not use condoms during sexual intercourse (72.7%). 51.5% had a previous history of Sexually Transmitted Infection (STI), and HPV the most prevalent STI (76.5%). 57.6% did not use contraception, 78.8% underwent examination Cancer Prevention Uterus (PCCU) with shorter time interval or equal to one year, 72.7% had no cases of Cervical Cancer in the family, 63.6% were multiparous and 97% were not vaccinated against HPV. DC identified good level of agreement between raters (κ=0.542), had a specificity of 77.8% and sensitivity of 25% when compared their results with PCR. Only the variable race showed a statistically significant association with CRP (p=0.042). DC had 100% acceptance amongst women in the sample, revealing the possibility of other experiments in using this method so that it proves as a viable technique. The DC positivity criteria were developed by nurses and these professionals also perform PCCU in Brazil, which means that DC can be an important complementary diagnostic method for the appreciation of these professional’s quality of examinations.

Keywords: gynecological examination, human papillomavirus, nursing, papillomavirus infections, uterine lasmsneop

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Authors: Yasmin A. M. Ferreira, Ana C. K. Pelissari, Sofia De C. F. Vicente, Raquel M. Da S. Campos, Deborah C. L. Masquio, Lian Tock, Lila M. Oyama, Flavia C. Corgosinho, Valter T. Boldarine, Ana R. Dâmaso

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The prevalence of overweight and obesity is growing around the world and currently considered a global epidemic. Food and nutrition are essential requirements for promoting health and protecting non-communicable chronic diseases, such as obesity and cardiovascular disease. Specific dietary components may modulate the inflammation and oxidative stress in obese individuals. Few studies have investigated the dietary Inflammation Factor Rating (IFR) in obese adolescents. The IFR was developed to characterize an individual´s diet on anti- to pro-inflammatory score. This evaluation contributes to investigate the effects of inflammatory diet in metabolic profile in several individual conditions. Objectives: The present study aims to investigate the effects of a multidisciplinary weight loss therapy on inflammation factor rating and cardiometabolic risk in obese adolescents. Methods: A total of 26 volunteers (14-19 y.o) were recruited and submitted to 20 weeks interdisciplinary therapy allied to health education website- Ciclo do Emagrecimento®, including clinical, nutritional, psychological counseling and exercise training. The body weight was monitored weekly by self-report and photo. The adolescents answered a test to evaluate the knowledge of the topics covered in the videos. A 24h dietary record was applied at the baseline and after 20 weeks to assess the food intake and to calculate IFR. A negative IFR suggests that diet may have inflammatory effects and a positive IFR indicates an anti-inflammatory effect. Statistical analysis was performed using the program STATISTICA version 12.5 for Windows. The adopted significant value was α ≤ 5 %. Data normality was verified with the Kolmogorov Smirnov test. Data were expressed as mean±SD values. To analyze the effects of intervention it was applied test t. Pearson´s correlations test was performed. Results: After 20 weeks of treatment, body mass index (BMI), body weight, body fat (kg and %), abdominal and waist circumferences decreased significantly. The mean of high-density lipoprotein cholesterol (HDL-c) increased after the therapy. Moreover, it was found an improvement of inflammation factor rating from -427,27±322,47 to -297,15±240,01, suggesting beneficial effects of nutritional counselling. Considering the correlations analysis, it was found that pro-inflammatory diet is associated with increase in the BMI, very low-density lipoprotein cholesterol (VLDL), triglycerides, insulin and insulin resistance index (HOMA-IR); while an anti-inflammatory diet is associated with improvement of HDL-c and insulin sensitivity Check index (QUICKI). Conclusion: The 20-week blended multidisciplinary therapy was effective to reduce body weight, anthropometric circumferences and improve inflammatory markers in obese adolescents. In addition, our results showed that an increase in inflammatory profile diet is associated with cardiometabolic parameters, suggesting the relevance to stimulate anti-inflammatory diet habits as an effective strategy to treat and control of obesity and related comorbidities. Financial Support: FAPESP (2017/07372-1) and CNPq (409943/2016-9)

Keywords: cardiometabolic risk, inflammatory diet, multidisciplinary therapy, obesity

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Authors: Victor Mantilla, Romeu Vicente, António Figueiredo, Victor Ferreira, Sandra Sorte

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Integrating dynamic facades into contemporary building design is shaping a new era of energy efficiency and user comfort. These innovative facades, often constructed using lightweight construction systems and materials, offer an opportunity to have a responsive and adaptive nature to the dynamic behavior of the outdoor climate. Therefore, in regions characterized by high fluctuations in daily temperatures, the ability to adapt to environmental changes is of paramount importance and a challenge. This paper presents a thorough review of the state of the art on double-skin facades (DSF), focusing on lightweight solutions for the external envelope. Dynamic facades featuring elements like movable shading devices, phase change materials, and advanced control systems have revolutionized the built environment. They offer a promising path for reducing energy consumption while enhancing occupant well-being. Lightweight construction systems are increasingly becoming the choice for the constitution of these facade solutions, offering benefits such as reduced structural loads and reduced construction waste, improving overall sustainability. However, the performance of dynamic facades based on low thermal inertia solutions in climatic contexts with high thermal amplitude is still in need of research since their ability to adapt is traduced in variability/manipulation of the thermal transmittance coefficient (U-value). Emerging technologies can enable such a dynamic thermal behavior through innovative materials, changes in geometry and control to optimize the facade performance. These innovations will allow a facade system to respond to shifting outdoor temperature, relative humidity, wind, and solar radiation conditions, ensuring that energy efficiency and occupant comfort are both met/coupled. This review addresses the potential configuration of double-skin facades, particularly concerning their responsiveness to seasonal variations in temperature, with a specific focus on addressing the challenges posed by winter and summer conditions. Notably, the design of a dynamic facade is significantly shaped by several pivotal factors, including the choice of materials, geometric considerations, and the implementation of effective monitoring systems. Within the realm of double skin facades, various configurations are explored, encompassing exhaust air, supply air, and thermal buffering mechanisms. According to the review places a specific emphasis on the thermal dynamics at play, closely examining the impact of factors such as the color of the facade, the slat angle's dimensions, and the positioning and type of shading devices employed in these innovative architectural structures.This paper will synthesize the current research trends in this field, with the presentation of case studies and technological innovations with a comprehensive understanding of the cutting-edge solutions propelling the evolution of building envelopes in the face of climate change, namely focusing on double-skin lightweight solutions to create sustainable, adaptable, and responsive building envelopes. As indicated in the review, flexible and lightweight systems have broad applicability across all building sectors, and there is a growing recognition that retrofitting existing buildings may emerge as the predominant approach.

Keywords: adaptive, control systems, dynamic facades, energy efficiency, responsive, thermal comfort, thermal transmittance

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Authors: Lacramioara Ochiuz, Aurelia Vasile, Iulian Stoleriu, Cristina Ghiciuc, Maria Ignat

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Topical administration of chemotherapeutic agents (eg. carmustine, bexarotene, mechlorethamine etc.) in local treatment of cutaneous T-cell lymphoma (CTCL) is accompanied by multiple side effects, such as contact hypersensitivity, pruritus, skin atrophy or even secondary malignancies. A known method of reducing the side effects of anticancer agent is the development of modified drug release systems using drug incapsulation in biocompatible nanoporous inorganic matrices, such as mesoporous MCM-41 silica. Mesoporous MCM-41 silica is characterized by large specific surface, high pore volume, uniform porosity, and stable dispersion in aqueous medium, excellent biocompatibility, in vivo biodegradability and capacity to be functionalized with different organic groups. Therefore, MCM-41 is an attractive candidate for a wide range of biomedical applications, such as controlled drug release, bone regeneration, protein immobilization, enzymes, etc. The main advantage of this material lies in its ability to host a large amount of the active substance in uniform pore system with adjustable size in a mesoscopic range. Silanol groups allow surface controlled functionalization leading to control of drug loading and release. This study shows (I) the amino-grafting optimization of mesoporous MCM-41 silica matrix by means of co-condensation during synthesis and post-synthesis using APTES (3-aminopropyltriethoxysilane); (ii) loading the therapeutic agent (carmustine) obtaining a modified drug release systems; (iii) determining the profile of in vitro carmustine release from these systems; (iv) assessment of carmustine release kinetics by fitting on four mathematical models. Obtained powders have been described in terms of structure, texture, morphology thermogravimetric analysis. The concentration of the therapeutic agent in the dissolution medium has been determined by HPLC method. In vitro dissolution tests have been done using cell Enhancer in a 12 hours interval. Analysis of carmustine release kinetics from mesoporous systems was made by fitting to zero-order model, first-order model Higuchi model and Korsmeyer-Peppas model, respectively. Results showed that both types of highly ordered mesoporous silica (amino grafted by co-condensation process or post-synthesis) are thermally stable in aqueous medium. In what regards the degree of loading and efficiency of loading with the therapeutic agent, there has been noticed an increase of around 10% in case of co-condensation method application. This result shows that direct co-condensation leads to even distribution of amino groups on the pore walls while in case of post-synthesis grafting many amino groups are concentrated near the pore opening and/or on external surface. In vitro dissolution tests showed an extended carmustine release (more than 86% m/m) both from systems based on silica functionalized directly by co-condensation and after synthesis. Assessment of carmustine release kinetics revealed a release through diffusion from all studied systems as a result of fitting to Higuchi model. The results of this study proved that amino-functionalized mesoporous silica may be used as a matrix for optimizing the anti-cancer topical therapy by loading carmustine and developing prolonged-release systems.

Keywords: carmustine, silica, controlled, release

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Authors: Kazuhiro Tateda, Elisa Gonzalez, Shuhei Ito, Kirstin Heinrich, Kevin Sweetland, Pingping Zhang, Catia Ferreira, Michael Pride, Jennifer Moisi, Sharon Gray, Bennett Lee, Fred Angulo

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Clostridium difficile (C. difficile) is the most common cause of antibiotic-associated diarrhea and infectious diarrhea in healthcare settings. Japan has an aging population; the elderly are at increased risk of hospitalization, antibiotic use, and C. difficile infection (CDI). Little is known about the population-based incidence and disease burden of CDI in Japan although limited hospital-based studies have reported a lower incidence than the United States. To understand CDI disease burden in Japan, CLOUD (Clostridium difficile Infection Burden of Disease in Adults in Japan) was developed. CLOUD will derive population-based incidence estimates of the number of CDI cases per 100,000 population per year in Ota-ku (population 723,341), one of the districts in Tokyo, Japan. CLOUD will include approximately 14 of the 28 Ota-ku hospitals including Toho University Hospital, which is a 1,000 bed tertiary care teaching hospital. During the 12-month patient enrollment period, which is scheduled to begin in November 2018, Ota-ku residents > 50 years of age who are hospitalized at a participating hospital with diarrhea ( > 3 unformed stools (Bristol Stool Chart 5-7) in 24 hours) will be actively ascertained, consented, and enrolled by study surveillance staff. A stool specimen will be collected from enrolled patients and tested at a local reference laboratory (LSI Medience, Tokyo) using QUIK CHEK COMPLETE® (Abbott Laboratories). which simultaneously tests specimens for the presence of glutamate dehydrogenase (GDH) and C. difficile toxins A and B. A frozen stool specimen will also be sent to the Pfizer Laboratory (Pearl River, United States) for analysis using a two-step diagnostic testing algorithm that is based on detection of C. difficile strains/spores harboring toxin B gene by PCR followed by detection of free toxins (A and B) using a proprietary cell cytotoxicity neutralization assay (CCNA) developed by Pfizer. Positive specimens will be anaerobically cultured, and C. difficile isolates will be characterized by ribotyping and whole genomic sequencing. CDI patients enrolled in CLOUD will be contacted weekly for 90 days following diarrhea onset to describe clinical outcomes including recurrence, reinfection, and mortality, and patient reported economic, clinical and humanistic outcomes (e.g., health-related quality of life, worsening of comorbidities, and patient and caregiver work absenteeism). Studies will also be undertaken to fully characterize the catchment area to enable population-based estimates. The 12-month active ascertainment of CDI cases among hospitalized Ota-ku residents with diarrhea in CLOUD, and the characterization of the Ota-ku catchment area, including estimation of the proportion of all hospitalizations of Ota-ku residents that occur in the CLOUD-participating hospitals, will yield CDI population-based incidence estimates, which can be stratified by age groups, risk groups, and source (hospital-acquired or community-acquired). These incidence estimates will be extrapolated, following age standardization using national census data, to yield CDI disease burden estimates for Japan. CLOUD also serves as a model for studies in other countries that can use the CLOUD protocol to estimate CDI disease burden.

Keywords: Clostridium difficile, disease burden, epidemiology, study protocol

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Authors: Adriana Muñoz-Talavera, Ana Rosa Rincón-Sánchez, Abraham Escobedo-Moratilla, María Cristina Islas-Carbajal, Miguel Ángel Gómez-Lim

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The highest rates of diabetes incidence and prevalence worldwide will increase the number of diabetic patients requiring insulin or insulin analogues. Then, current production systems would not be sufficient to meet the future market demands. Therefore, developing efficient expression systems for insulin and insulin analogues are needed. In addition, insulin analogues with better pharmacokinetics and pharmacodynamics properties and without mitogenic potential will be required. SCI-57 (single chain insulin-57) is an insulin analogue having 10 times greater affinity to the insulin receptor, higher resistance to thermal degradation than insulin, native mitogenicity and biological effect. Plants as expression platforms have been used to produce recombinant proteins because of their advantages such as cost-effectiveness, posttranslational modifications, absence of human pathogens and high quality. Immunoglobulin production with a yield of 50% has been achieved by transient expression in Nicotiana benthamiana (Nb). The aim of this study is to produce SCI-57 by transient expression in Nb. Methodology: DNA sequence encoding SCI-57 was cloned in pICH31070. This construction was introduced into Agrobacterium tumefaciens by electroporation. The resulting strain was used to infiltrate leaves of Nb. In order to isolate SCI-57, leaves from transformed plants were incubated 3 hours with the extraction buffer therefore filtrated to remove solid material. The resultant protein solution was subjected to anion exchange chromatography on an FPLC system and ultrafiltration to purify SCI-57. Detection of SCI-57 was made by electrophoresis pattern (SDS-PAGE). Protein band was digested with trypsin and the peptides were analyzed by Liquid chromatography tandem-mass spectrometry (LC-MS/MS). A purified protein sample (20µM) was analyzed by ESI-Q-TOF-MS to obtain the ionization pattern and the exact molecular weight determination. Chromatography pattern and impurities detection were performed using RP-HPLC using recombinant insulin as standard. The identity of the SCI-57 was confirmed by anti-insulin ELISA. The total soluble protein concentration was quantified by Bradford assay. Results: The expression cassette was verified by restriction mapping (5393 bp fragment). The SDS-PAGE of crude leaf extract (CLE) of transformed plants, revealed a protein of about 6.4 kDa, non-present in CLE of untransformed plants. The LC-MS/MS results displayed one peptide with a high score that matches SCI-57 amino acid sequence in the sample, confirming the identity of SCI-57. From the purified SCI-57 sample (PSCI-57) the most intense charge state was 1069 m/z (+6) on the displayed ionization pattern corresponding to the molecular weight of SCI-57 (6412.6554 Da). The RP-HPLC of the PSCI-57 shows the presence of a peak with similar retention time (rt) and UV spectroscopic profile to the insulin standard (SCI-57 rt=12.96 and insulin rt=12.70 min). The collected SCI-57 peak had ELISA signal. The total protein amount in CLE from transformed plants was higher compared to untransformed plants. Conclusions: Our results suggest the feasibility to produce insulin analogue SCI-57 by transient expression in Nicotiana benthamiana. Further work is being undertaken to evaluate the biological activity by glucose uptake by insulin-sensitive and insulin-resistant murine and human cultured adipocytes.

Keywords: insulin analogue, mass spectrometry, Nicotiana benthamiana, transient expression

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Authors: Federica Verna, Alessandra Pautasso, Maria Caramelli, Cristiana Maurella, Walter Mignone, Cristina Casalone

Abstract:

Mosquito-Borne Diseases (MBDs) are dangerously increasing in prevalence, geographical distribution and severity, representing an emerging threat for both humans and animals. Interaction between multiple disciplines is needed for an effective early warning, surveillance and control of MBDs, according to the One Health concept. This work reports the integrated surveillance system enforced by IZSPLV in Piedmont, Liguria and Valle d’Aosta regions (Northwestern Italy) in order to control MDBs spread. Veterinary services and local human health authority are involved in an information network, to connect the surveillance of human clinical cases with entomological surveillance and veterinary monitoring in order to implement control measures in case of outbreak. A systematic entomological surveillance is carried out during the vector season using mosquitoes traps located in sites selected according to risk factors. Collected mosquitoes are counted, identified to species level by morphological standard classification keys and pooled by collection site, date and species with a maximum of 100 individuals. Pools are analyzed, after RNA extraction, by Real Time RT-PCR distinctive for West Nile Virus (WNV) Lineage 1 and Lineage 2, Real Time RT-PCR USUTU virus (USUV) and a traditional flavivirus End-point RT-PCR. Positive pools are sequenced and the related sequences employed to perform a basic local alignment search tool (BLAST) in the GenBank library. Positive samples are sent to the National Reference Centre for Animal Exotic Diseases (CESME, Teramo) for confirmation. With particular reference to WNV, after the confirmation, as provided by national legislation, control measures involving both local veterinary and human health services are activated: equine sera are randomly sampled within a 4 km radius from the positive collection sites and tested with ELISA kit and WNV NAT screening of blood donors is introduced. This surveillance network allowed to detect since 2011 USUV circulation in this area of Italy. WNV was detected in Piedmont and Liguria for the first time in 2014 in mosquitoes. During the 2015 vector season, we observed the expansion of its activity in Piedmont. The virus was detected in almost all Provinces both in mosquitoes (6 pools) and animals (19 equine sera, 4 birds). No blood bag tested resulted infected. The first neuroinvasive human case occurred too. Competent authorities should be aware of a potentially increased risk of MBDs activity during the 2016 vector season. This work shows that this surveillance network allowed to early detect the presence of MBDs in humans and animals, and provided useful information to public authorities, in order to apply control measures. Finally, an additional value of our diagnostic protocol is the ability to detect all viruses belonging to the Flaviviridae family, considering the emergence caused by other Flaviviruses in humans such as the recent Zika virus infection in South America. Italy has climatic and environmental features conducive to Zika virus transmission, the competent vector and many travellers from Brazil reported every year.

Keywords: integrated surveillance, mosquito borne disease, West Nile virus, Zika virus

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Authors: Irina Alexandra Paun, Mona Mihailescu, Marian Zamfirescu, Catalin Romeo Luculescu, Adriana Maria Acasandrei, Cosmin Catalin Mustaciosu, Roxana Cristina Popescu, Maria Dinescu

Abstract:

A major concern in bone tissue engineering is to develop complex 3D architectures that mimic the natural cells environment, facilitate the cells growth in a defined manner and allow the flow transport of nutrients and metabolic waste. In particular, porous structures of controlled pore size and positioning are indispensable for growing human-like bone structures. Another concern is to monitor both the structures and the seeded cells with high spatial resolution and without interfering with the cells natural environment. The present approach relies on laser-based technologies employed for fabricating porous biomimetic structures that support the growth of osteoblast-like cells and for their non-invasive 3D imaging. Specifically, the porous structures were built by two photon polymerization –direct writing (2PP_DW) of the commercially available photoresists IL-L780, using the Photonic Professional 3D lithography system. The structures consist of vertical tubes with micrometer-sized heights and diameters, in a honeycomb-like spatial arrangement. These were fabricated by irradiating the IP-L780 photoresist with focused laser pulses with wavelength centered at 780 nm, 120 fs pulse duration and 80 MHz repetition rate. The samples were precisely scanned in 3D by piezo stages. The coarse positioning was done by XY motorized stages. The scanning path was programmed through a writing language (GWL) script developed by Nanoscribe. Following laser irradiation, the unexposed regions of the photoresist were washed out by immersing the samples in the Propylene Glycol Monomethyl Ether Acetate (PGMEA). The porous structures were seeded with osteoblast like MG-63 cells and their osteogenic potential was tested in vitro. The cell-seeded structures were analyzed in 3D using the digital holographic microscopy technique (DHM). DHM is a marker free and high spatial resolution imaging tool, where the hologram acquisition is performed non-invasively i.e. without interfering with the cells natural environment. Following hologram recording, a digital algorithm provided a 3D image of the sample, as well as information about its refractive index, which is correlated with the intracellular content. The axial resolution of the images went down to the nanoscale, while the temporal scales ranged from milliseconds up to hours. The hologram did not involve sample scanning and the whole image was available in one frame recorded going over 200μm field of view. The digital holograms processing provided 3D quantitative information on the porous structures and allowed a quantitative analysis of the cellular response in respect to the porous architectures. The cellular shape and dimensions were found to be influenced by the underlying micro relief. Furthermore, the intracellular content gave evidence on the beneficial role of the porous structures in promoting osteoblast differentiation. In all, the proposed laser-based protocol emerges as a promising tool for the fabrication and non-invasive imaging of porous constructs for bone tissue engineering. Acknowledgments: This work was supported by a grant of the Romanian Authority for Scientific Research and Innovation, CNCS-UEFISCDI, project PN-II-RU-TE-2014-4-2534 (contract 97 from 01/10/2015) and by UEFISCDI PN-II-PT-PCCA no. 6/2012. A part of this work was performed in the CETAL laser facility, supported by the National Program PN 16 47 - LAPLAS IV.

Keywords: biomimetic, holography, laser, osteoblast, two photon polymerization

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