Search results for: Marino Zanetti

Authors: Andreas Aceranti, Guido Bighiani, Francesca Crotto, Marco Colorato, Stefania Zaghi, Marino Zanetti, Simonetta Vernocchi

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The management of eye parameters such as convergence, accommodation, and miosis is very complex; in fact, both the neurovegetative system and the complex Oculocephalgiria system come into play. We have found the effectiveness of the "highvelocity low amplitude" technique directed on C7-T1 (where the cilio-spinal nucleus of the budge is located) in improving the convergence parameter through the measurement of the point of maximum convergence. With this research, we set out to investigate whether the improvement obtained through the High Velocity Low Amplitude maneuver lasts over time, carrying out a pre-manipulation measurement, one immediately after manipulation and one month after manipulation. We took a population of 30 subjects with both refractive and non-refractive problems. Of the 30 patients tested, 27 gave a positive result after the High Velocity Low Amplitude maneuver, giving an improvement in the point of maximum convergence. After a month, we retested all 27 subjects: some further improved the result, others kept, and three subjects slightly lost the gain obtained. None of the re-tested patients returned to the point of maximum convergence starting pre-manipulation. This result opens the door to a multidisciplinary approach between ophthalmologists and osteopaths with the aim of addressing oculomotricity and convergence deficits that increasingly afflict our society due to the massive use of devices and for the conduct of life in closed and restricted environments.

Keywords: point of maximum convergence, HVLA, improvement in PPC, convergence

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Authors: Cristiano Giordani, Marco Diociaiuti, Cecilia Bombelli, Laura Zanetti-Polzi, Marcello Belfiore, Raoul Fioravanti, Gianfranco Macchia

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We investigated induced functional effects by evaluating Ca2+-influx in liposomes and cell viability in HT22-DIFF neurons. Only solutions rich in unstructured Prefibrillar-Oligomers (PFOs) were able, in the presence of Monosialoganglioside-GM1 (GM1), to induce Ca2+-influx and were also neurotoxic, suggesting a correlation between the two phenomena. Thus, in the presence of GM1, we investigated the protein conformation and liposome modification due to the interaction. Circular Dichroism showed that GM1 fostered the formation of β-structures and Energy Filtered-Transmission Electron Microscopy that PFOs formed “amyloid-channels” as reported for Aβ. We speculate that electrostatic forces occurring between the positive PFOs and negative GM1 drive the initial binding, while the hydrophobic profile of the flexible PFO is responsible for the subsequent pore formation. Conversely, the rigid β-structured mature/fibers (MFs) and proto-fibers (PFs) were unable to induce membrane damage and Ca2+- influx.

Keywords: amyloid proteins, neurotoxicity, lipid-rafts, GM1

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Authors: Lukasz Grzegorz Broda, Taiwo Oseni, Andrew Stranieri, Rodrigo Marino, Ronelle Welton, Mark Yates

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The oral health of residents in aged care centres in Australia is poor, contributing to infections, hospital admissions, and increased suffering. Although the use of electric toothbrushes has been deployed in many centres, smartbrushes that record and transmit information about brushing patterns and duration are not routinely deployed. Yet, the use of smartbrushes for aged care residents promises better oral care. Thus, a study aimed at investigating the appropriateness and suitability of a smartbrush for aged care residents is currently underway. Due to the peculiarity of the aged care setting, the incorporation of smartbrushes into residents’ care does require careful planning and design considerations. This paper describes an initial design process undertaken through the use of an actor to understand the important elements to be incorporated whilst installing a smartbrush for use in aged care settings. The design covers the configuration settings of the brush and app, including ergonomic factors related to brush and smartphone placement. A design science approach led to an installation re-design and a revised protocol for the planned study, the ultimate aim being to design installations to enhance perceived usefulness, ease of use, and attitudes towards the incorporation of smartbrushes for improving oral health care for aged care residents.

Keywords: smartbrush, applied computing, life and medical sciences, health informatics

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Authors: Ana Corberan-Vallet, Karen C. Florez, Ingrid C. Marino, Jose D. Bermudez

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In 2016, the Region of the Americas was declared free of measles, a viral disease that can cause severe health problems. However, since 2017, measles has reemerged in Venezuela and has subsequently reached neighboring countries. In 2018, twelve American countries reported confirmed cases of measles. Governmental and health authorities in Colombia, a country that shares the longest land boundary with Venezuela, are aware of the need for a strong response to restrict the expanse of the epidemic. In this work, we apply a Bayesian hierarchical Poisson model with an underlying cluster structure to describe disease incidence in Colombia. Concretely, the proposed methodology provides relative risk estimates at the department level and identifies clusters of disease, which facilitates the implementation of targeted public health interventions. Socio-demographic factors, such as the percentage of migrants, gross domestic product, and entry routes, are included in the model to better describe the incidence of disease. Since the model does not impose any spatial correlation at any level of the model hierarchy, it avoids the spatial confounding problem and provides a suitable framework to estimate the fixed-effect coefficients associated with spatially-structured covariates.

Keywords: Bayesian analysis, cluster identification, disease mapping, risk estimation

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Authors: German Marino Rivera

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Linear plantations are a common practice in several countries as living fences (LF) delimiting agroecosystems. They are composed of multipurpose perennial woods that provide assets, protection, and supply services. However, not much is known in some traditional communities like the Andean region, including the species composition and the social and ecological benefits of the species used. In the High Andean Colombian region, LF seems to be very typical and diverse. This study aimed to analyze the traditional knowledge about LF systems, including the species composition and their uses in rural communities of Alto Casanare, Colombia. Field measurements, interviews, guided tours, and species sampling were carried out in order to describe traditional practices and the species used in the LF systems. The use values were estimated through the Coefficient of Importance of the Species (CIS). A total of 26 farms engage in LF practices, covering an area of 9283.3 m. In these systems, 30 species were identified, belonging to 23 families. Alnus acuminata was the specie with the highest CIS. The species presented multipurpose uses for both economic and ecological purposes. The transmission of knowledge (TEK) about the used species is very heterogeneous among the farmers. Many species used were not documented, with reciprocal gaps between the literature and traditional species uses. Exchanging this information would increase the species' versatility, the socioeconomic aspects of these communities, increases the agrobiodiversity and ecological services provided by LF. The description of the TEK on LF provides a better understanding of the relationship of these communities with the natural resources, pointing out creative approaches to achieve local environment conservation in these agroecosystems and promoting socioeconomic development.

Keywords: ethnobotany, living fences, traditional communities, agroecology

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Authors: Alessandra Marino

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This paper thoroughly investigates the design characteristics necessary for intergenerational living and evaluates their applicability within the Italian social panorama in order to identify a model that can serve as a reference for subsequent regulatory adjustments of a new building typology. The applied methodology involves the collaboration of people with various background and architects, all representing the three main ages of life - childhood or youth, adulthood, seniority - through questionnaires aimed at researching the peculiar characteristics that contemporary intergenerational housing should include; the questionnaires are then compared with each other in order to identify any recurring patterns by age group and/or influenced by the specialist knowledge on the subject of the architects compared to the rest of the user sample. The results indicate that among specialist users in the field of architecture, young students identify home automation as the key to the inclusion of the weakest groups within the building, adult architects believe that the identification of intergenerational/community services within the building is the cornerstone, and senior architects focus on widespread spatial accessibility. At the same time, the results among non-specialist users do not identify a significantly diversified model by age group but are generally in agreement in the importance of separation between private environments and collective spaces. The interpretation of the results obtained leads to a compositional study of a new building typology with the future objective of channeling the subsequent outcomes within the regulatory adjustments of the sector.

Keywords: intergenerational living, social sustainability, health, lifestyle, well-being

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Authors: Arlett A. Rosado-Torres, Ismael Marino-Tapia

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Changes in benthic cover of coral dominated systems to macroalgae dominance are widely studied worldwide. Watershed pollutants are potentially as important as overfishing causing phase shift. In certain regions of the world most of the continental inputs are through submarine groundwater discharges (SGD), which can play a significant ecological role because the concentration of its nutrients is usually greater that the one found in surface seawater. These stressors have adversely affected coral reefs, particularly in the Caribbean. Measurements of benthic cover (with video tracing, through a Go Pro camera), reef roughness (acoustic estimates with an Acoustic Doppler Current Velocity profiler and a differential GPS), thermohaline conditions (conductivity-temperature-depth (CTD) instrument) and nutrient measurements were taken in different sites in the reef lagoon of Puerto Morelos, Q. Roo, Mexico including those with influence of SGD and without it. The results suggest a link between SGD, macroalgae cover and structural complexity. Punctual water samples and data series from a CTD Diver confirm the presence of the SGD. On the site where the SGD is, the macroalgae cover is larger than in the other sites. To establish a causal link between this phase shift and SGD, the DELFT 3D hydrodynamic model (FLOW and WAVE modules) was performed under different environmental conditions and discharge magnitudes. The model was validated using measurements of oceanographic instruments anchored in the lagoon and forereef. The SGD is consistently favoring macroalgae populations and affecting structural complexity of the reef.

Keywords: hydrodynamic model, macroalgae, nutrients, phase shift

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Authors: Yuban Sebastian Cuartas-Agudelo, Sandra Marcela López-Hincapié, Vivianna Alexandra Garrido-Altamar, María de los Ángeles Rodríguez-Gázquez, Camilo Ruiz-Mejía, Lina María Martínez-Sánchez, Gloria Inés Martínez-Domínguez, Luis Eduardo Contreras, Felipe Eduardo Marino-Isaza

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Introduction: Doping is a prohibited practice in competitive sports with potential adverse effects; therefore, it is crucial to describe the attitudes of athletes towards this behavior and to determine which o these increase the susceptibility to carry out this practice. Objective: To determine the attitude of high-performance athletes towards doping in a sports institute in the city of Medellin, Colombia. Methods: We performed a cross-sectional study during 2016, with a sample taken to convenience consisting of athletes over 18 years old enrolled in a sports institute of the city of Medellin (Colombia). The athletes filled by themselves the Petroczi and Aidman questionnaire: Performance Enhancement Attitude Scale (PEAS) adapted to the Spanish language by Morente-Sánchez et al. This scale has 17 items with likert answer options, with a score ranging from 1 to 6, with a higher score indicating a stronger tendency towards doping practices. Results: 112 athletes were included with an average age of 21.6 years old, a 60% of them were male and the most frequent sports were karate 17%, judo 12.5% and athletics 9.8%. The average score of the questionnaire was 35.5 points of a 102 possible points. The lowest score was obtained in the following items: Is Doping necessary 1,4 and Doping isn’t cheating, everyone does it 1,5. Conclusion: In our population, there is a low tendency towards doping practices.

Keywords: sports, doping in sports, athletic performance, attitude

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Authors: Gennaro Marino

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This paper concerns a case of a 10.75 inch diameter buried gas transmission line which was exposed to mine subsidence ground movements. The pipeline was buried about 4ft. below the surface with maximum operating pressure of 1440 psi. The mine subsidence movement was the result of long walling ore at a depth of approximately 1600 ft. As ore extraction progressed, the stress in the monitored pipeline worsened and was approaching unacceptable levels. The excessive pipe compression resulted when it was exposed to the compression zone of subsidence basin created by mining. The pipe stress reached a significant compressive level due to the extensive length of the pipe exposed to frictional ground-pipe slip resistance. The backfill ground movement slip resistance depends on normal stress around the pipe, the rate of slip, and the backfill characteristics. Normal stress depends on the burial depth of the backfill density and the lateral subsidence induced stress. The backfill in this site has a soil dry density of approximately 90 PCF. A suite of direct shear tests was conducted a residual friction angle of 36 was determined for the ambient backfill. These tests showed that the residual shearing resistance was reached within a fraction of an inch. The pipe was coated with fusion-bonded epoxy, so friction reduce factory of 0.6 can be considered. To relieve ground movement induced compressive stress, the line was uncovered. As more of the pipeline was exposed, the pipe abruptly bowed in the excavation. An analysis of this pipe formation which was performed is provided in this paper. Also discussed in this paper are ways to mitigate this pipe deformation or upheaval buckling from occurring. Keywords: Pipe Upheaval, Pipe Buckling, Ground subsidence, Buried Pipeline, Pipe Stress Mitigation.

Keywords: pipe upheaval, pipe buckling, ground subsidence, buried pipeline, pipe stress mitigation

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Authors: B. B. S. Pereira, M. O. Souza, L. P. Zanetti, L. C. S. Oliveira, J. R. P. Moreno, M. P. Souza, V. R. Colturato, C. M. Machado

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Background: The introduction of prophylactic or preemptive therapies has effectively decreased the CMV mortality rates after hematopoietic stem cell transplantation (HSCT). CMV antigenemia (pp65) or quantitative PCR are methods currently approved for CMV surveillance in pre-emptive strategies. Commercial assays are preferred as cut-off levels defined by in-house assays may vary among different protocols and in general show low reproducibility. Moreover, comparison of published data among different centers is only possible if international standards of quantification are included in the assays. Recently, the World Health Organization (WHO) established the first international standard for CMV detection. The real time PCR COBAS Ampliprep/ CobasTaqMan (CAP/CTM) (Roche®) was developed using the WHO standard for CMV quantification. However, the cut-off for the introduction of antiviral has not been determined yet. Methods: We conducted a retrospective study to determine: 1) the sensitivity and specificity of the new CMV CAP/CTM test in comparison with pp65 antigenemia to detect episodes of CMV infection/reactivation, and 2) the cut-off of viral load for introduction of ganciclovir (GCV). Pp65 antigenemia was performed and the corresponding plasma samples were stored at -20°C for further CMV detection by CAP/CTM. Comparison of tests was performed by kappa index. The appearance of positive antigenemia was considered the state variable to determine the cut-off of CMV viral load by ROC curve. Statistical analysis was performed using SPSS software version 19 (SPSS, Chicago, IL, USA.). Results: Thirty-eight patients were included and followed from August 2014 through May 2015. The antigenemia test detected 53 episodes of CMV infection in 34 patients (89.5%), while CAP/CTM detected 37 episodes in 33 patients (86.8%). AG and PCR results were compared in 431 samples and Kappa index was 30.9%. The median time for first AG detection was 42 (28-140) days, while CAP/CTM detected at a median of 7 days earlier (34 days, ranging from 7 to 110 days). The optimum cut-off value of CMV DNA was 34.25 IU/mL to detect positive antigenemia with 88.2% of sensibility, 100% of specificity and AUC of 0.91. This cut-off value is below the limit of detection and quantification of the equipment which is 56 IU/mL. According to CMV recurrence definition, 16 episodes of CMV recurrence were detected by antigenemia (47.1%) and 4 (12.1%) by CAP/CTM. The duration of viremia as detected by antigenemia was shorter (60.5% of the episodes lasted ≤ 7 days) in comparison to CAP/CTM (57.9% of the episodes lasting 15 days or more). This data suggests that the use of antigenemia to define the duration of GCV therapy might prompt early interruption of antiviral, which may favor CMV reactivation. The CAP/CTM PCR could possibly provide a safer information concerning the duration of GCV therapy. As prolonged treatment may increase the risk of toxicity, this hypothesis should be confirmed in prospective trials. Conclusions: Even though CAP/CTM by ROCHE showed great qualitative correlation with the antigenemia technique, the fully automated CAP/CTM did not demonstrate increased sensitivity. The cut-off value below the limit of detection and quantification may result in delayed introduction of pre-emptive therapy.

Keywords: antigenemia, CMV COBAS/TAQMAN, cytomegalovirus, antiviral cut-off

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Authors: Averalda van Graan, Joelaine Chetty, Malory Links, Agness Mwangwela, Sitilitha Masangwi, Dalitso Chimwala, Shiban Ghosh, Elizabeth Marino-Costello

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Food composition data is becoming increasingly important as dealing with food insecurity and malnutrition in its persistent form of under-nutrition is now coupled with increasing over-nutrition and its related ailments in the developing world, of which Malawi is not spared. In the absence of a food composition database (FCDB) inherent to our dietary patterns, efforts were made to develop a country-specific FCDB for nutrition practice, research, and programming. The main objective was to develop a multi-user, country-specific food composition database, and table from existing published and unpublished scientific literature. A multi-phased approach guided by the project framework was employed. Phase 1 comprised a scoping mission to assess the nutrition landscape for compilation activities. Phase 2 involved training of a compiler and data collection from various sources, primarily; institutional libraries, online databases, and food industry nutrient data. Phase 3 subsumed evaluation and compilation of data using FAO and IN FOODS standards and guidelines. Phase 4 concluded the process with quality assurance. 316 Malawian food items categorized into eight food groups for 42 components were captured. The majority were from the baby food group (27%), followed by a staple (22%) and animal (22%) food group. Fats and oils consisted the least number of food items (2%), followed by fruits (6%). Proximate values are well represented; however, the percent missing data is huge for some components, including Se 68%, I 75%, Vitamin A 42%, and lipid profile; saturated fat 53%, mono-saturated fat 59%, poly-saturated fat 59% and cholesterol 56%. A multi-phased approach following the project framework led to the development of the first Malawian FCDB and table. The table reflects inherent Malawian dietary patterns and nutritional concerns. The FCDB can be used by various professionals in nutrition and health. Rising over-nutrition, NCD, and changing diets challenge us for nutrient profiles of processed foods and complete lipid profiles.

Keywords: analytical data, dietary pattern, food composition data, multi-phased approach

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Authors: Glauco M. M. M. Lustosa, João Paulo C. Costa, Sonia M. Zanetti, Mario Cilense, Leinig Antônio Perazolli, Maria Aparecida Zaghete

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The contamination of the environment has been one of the biggest problems of our time, mostly due to developments of many industries. SnO2 is an n-type semiconductor with band gap about 3.5 eV and has its electrical conductivity dependent of type and amount of modifiers agents added into matrix ceramic during synthesis process, allowing applications as sensing of gaseous pollutants on ambient. The chemical synthesis by polymeric precursor method consists in a complexation reaction between tin ion and citric acid at 90 °C/2 hours and subsequently addition of ethyleneglycol for polymerization at 130 °C/2 hours. It also prepared polymeric resin of zinc, cobalt and niobium ions. Stoichiometric amounts of the solutions were mixed to obtain the systems (Zn, Nb)-SnO2 and (Co, Nb) SnO2 . The metal immobilization reduces its segregation during the calcination resulting in a crystalline oxide with high chemical homogeneity. The resin was pre-calcined at 300 °C/1 hour, milled in Atritor Mill at 500 rpm/1 hour, and then calcined at 600 °C/2 hours. X-Ray Diffraction (XDR) indicated formation of SnO2 -rutile phase (JCPDS card nº 41-1445). The characterization by Scanning Electron Microscope of High Resolution showed spherical ceramic powder nanostructured with 10-20 nm of diameter. 20 mg of SnO2 -based powder was kept in 20 ml of isopropyl alcohol and then taken to an electrophoretic deposition (EPD) system. The EPD method allows control the thickness films through the voltage or current applied in the electrophoretic cell and by the time used for deposition of ceramics particles. This procedure obtains films in a short time with low costs, bringing prospects for a new generation of smaller size devices with easy integration technology. In this research, films were obtained in an alumina substrate with interdigital electrodes after applying 2 kV during 5 and 10 minutes in cells containing alcoholic suspension of (Zn, Nb)-SnO2 and (Co, Nb) SnO2 of powders, forming a sensing layer. The substrate has designed integrated micro hotplates that provide an instantaneous and precise temperature control capability when a voltage is applied. The films were sintered at 900 and 1000 °C in a microwave oven of 770 W, adapted by the research group itself with a temperature controller. This sintering is a fast process with homogeneous heating rate which promotes controlled growth of grain size and also the diffusion of modifiers agents, inducing the creation of intrinsic defects which will change the electrical characteristics of SnO2 -based powders. This study has successfully demonstrated a microfabricated system with an integrated micro-hotplate for detection of CO and NO2 gas at different concentrations and temperature, with self-heating SnO2 - based nanoparticles films, being suitable for both industrial process monitoring and detection of low concentrations in buildings/residences in order to safeguard human health. The results indicate the possibility for development of gas sensors devices with low power consumption for integration in portable electronic equipment with fast analysis. Acknowledgments The authors thanks to the LMA-IQ for providing the FEG-SEM images, and the financial support of this project by the Brazilian research funding agencies CNPq, FAPESP 2014/11314-9 and CEPID/CDMF- FAPESP 2013/07296-2.

Keywords: chemical synthesis, electrophoretic deposition, self-heating, gas sensor

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Authors: Raj Chandran, Saul Datwyler, Jaime Marino, Daniel West, Karla Grasso, Adam Buss, Hina Syed, Zina Al Sahouri, Jennifer Yen, Krista Caudle, Beth McQuiston

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The Alinity i TBI test is Therapeutic Goods Administration (TGA) registered and is a panel of in vitro diagnostic chemiluminescent microparticle immunoassays for the measurement of glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase L1 (UCH-L1) in plasma and serum. The Alinity i TBI performance was evaluated in a multi-center pivotal study to demonstrate the capability to assist in determining the need for a CT scan of the head in adult subjects (age 18+) presenting with suspected mild TBI (traumatic brain injury) with a Glasgow Coma Scale score of 13 to 15. TBI has been recognized as an important cause of death and disability and is a growing public health problem. An estimated 69 million people globally experience a TBI annually1. Blood-based biomarkers such as glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase L1 (UCH-L1) have shown utility to predict acute traumatic intracranial injury on head CT scans after TBI. A pivotal study using prospectively collected archived (frozen) plasma specimens was conducted to establish the clinical performance of the TBI test on the Alinity i system. The specimens were originally collected in a prospective, multi-center clinical study. Testing of the specimens was performed at three clinical sites in the United States. Performance characteristics such as detection limits, imprecision, linearity, measuring interval, expected values, and interferences were established following Clinical and Laboratory Standards Institute (CLSI) guidance. Of the 1899 mild TBI subjects, 120 had positive head CT scan results; 116 of the 120 specimens had a positive TBI interpretation (Sensitivity 96.7%; 95% CI: 91.7%, 98.7%). Of the 1779 subjects with negative CT scan results, 713 had a negative TBI interpretation (Specificity 40.1%; 95% CI: 37.8, 42.4). The negative predictive value (NPV) of the test was 99.4% (713/717, 95% CI: 98.6%, 99.8%). The analytical measuring interval (AMI) extends from the limit of quantitation (LoQ) to the upper LoQ and is determined by the range that demonstrates acceptable performance for linearity, imprecision, and bias. The AMI is 6.1 to 42,000 pg/mL for GFAP and 26.3 to 25,000 pg/mL for UCH-L1. Overall, within-laboratory imprecision (20 day) ranged from 3.7 to 5.9% CV for GFAP and 3.0 to 6.0% CV for UCH-L1, when including lot and instrument variances. The Alinity i TBI clinical performance results demonstrated high sensitivity and high NPV, supporting the utility to assist in determining the need for a head CT scan in subjects presenting to the emergency department with suspected mild TBI. The GFAP and UCH-L1 assays show robust analytical performance across a broad concentration range of GFAP and UCH-L1 and may serve as a valuable tool to help evaluate TBI patients across the spectrum of mild to severe injury.

Keywords: biomarker, diagnostic, neurology, TBI

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Authors: Lizeth Fuentes-Mera, Vanessa Perez-Silos, Nidia K. Moncada-Saucedo, Alejandro Garcia-Ruiz, Alberto Camacho, Jorge Lara-Arias, Ivan Marino-Martinez, Victor Romero-Diaz, Adolfo Soto-Dominguez, Humberto Rodriguez-Rocha, Hang Lin, Victor Pena-Martinez

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Biphasic scaffolds in cartilage tissue engineering have been designed to influence not only the recapitulation of the osteochondral architecture but also to take advantage of the healing ability of bone to promote the implant integration with the surrounding tissue and then bone restoration and cartilage regeneration. This study reports the development and characterization of a biphasic scaffold based on the assembly of a cartilage phase constituted by fibroin biofunctionalized with bovine cartilage matrix; cellularized with differentiated pre-chondrocytes from adipose tissue stem cells (autologous) and well attached to a bone phase (bone bovine decellularized) to mimic the structure of the nature of native tissue and to promote the cartilage regeneration in a model of joint damage in pigs. Biphasic scaffolds were assembled by fibroin crystallization with methanol. The histological and ultrastructural architectures were evaluated by optical and scanning electron microscopy respectively. Mechanical tests were conducted to evaluate Young's modulus of the implant. For the biological evaluation, pre-chondrocytes were loaded onto the scaffolds and cellular adhesion, proliferation, and gene expression analysis of cartilage extracellular matrix components was performed. The scaffolds that were cellularized and matured for 10 days were implanted into critical 3 mm in diameter and 9-mm in depth osteochondral defects in a porcine model (n=4). Three treatments were applied per knee: Group 1: monophasic cellular scaffold (MS) (single chondral phase), group 2: biphasic scaffold, cellularized only in the chondral phase (BS1), group 3: BS cellularized in both bone and chondral phases (BS2). Simultaneously, a control without treatment was evaluated. After 4 weeks of surgery, integration and regeneration tissues were analyzed by x-rays, histology and immunohistochemistry evaluation. The mechanical assessment showed that the acellular biphasic composites exhibited Young's modulus of 805.01 kPa similar to native cartilage (400-800 kPa). In vitro biological studies revealed the chondroinductive ability of the biphasic implant, evidenced by an increase in sulfated glycosaminoglycan (GAGs) and type II collagen, both secreted by the chondrocytes cultured on the scaffold during 28 days. No evidence of adverse or inflammatory reactions was observed in the in vivo trial; however, In group 1, the defects were not reconstructed. In group 2 and 3 a good integration of the implant with the surrounding tissue was observed. Defects in group 2 were fulfilled by hyaline cartilage and normal bone. Group 3 defects showed fibrous repair tissue. In conclusion; our findings demonstrated the efficacy of biphasic and bioactive scaffold based on silk fibroin, which entwined chondroinductive features and biomechanical capability with appropriate integration with the surrounding tissue, representing a promising alternative for osteochondral tissue-engineering applications.

Keywords: biphasic scaffold, extracellular cartilage matrix, silk fibroin, osteochondral tissue engineering

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Authors: Leila Noori, Rosario Barone, Francesca Rappa, Antonella Marino Gammazza, Alessandra Maria Vitale, Giuseppe Donato Mangano, Giusy Sentiero, Filippo Macaluso, Kathryn H. Myburgh, Francesco Cappello, Federica Scalia

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The neuromuscular system controls, directs, and allows movement of the body through the action of neural circuits, which include motor neurons, sensory neurons, and skeletal muscle fibers. Protein homeostasis of the involved cytotypes appears crucial to maintain the correct and prolonged functions of the neuromuscular system, and both neuronal cells and skeletal muscle fibers express significant quantities of protein chaperones, the molecular machinery responsible to maintain the protein turnover. Genetic mutations or defective post-translational modifications of molecular chaperones (i.e., genetic or acquired chaperonopathies) may lead to neuromuscular disorders called as neurochaperonopathies. The limited knowledge of the effects of the defective chaperones on skeletal muscle fibers and neurons impedes the progression of therapeutic approaches. A distinct genetic variation of CCT5 gene encoding for the subunit 5 of the chaperonin CCT (Chaperonin Containing TCP1; also known as TRiC, TCP1 Ring Complex) was recently described associated with severe distal motor neuropathy by our team. In this study, we investigated the histopathological abnormalities of the skeletal muscle biopsy of the pediatric patient affected by the mutation Leu224Val in the CCT5 subunit. We provide molecular and structural features of the diseased skeletal muscle tissue that we believe may be useful to identify undiagnosed cases of this rare genetic disorder. We investigated the histological abnormalities of the affected tissue via hematoxylin and eosin staining. Then we used immunofluorescence and qPCR techniques to explore the expression and distribution of CCT5 in diseased and healthy skeletal muscle tissue. Immunofluorescence and immunohistochemistry assays were performed to study the sarcomeric and structural proteins of skeletal muscle, including actin, myosin, tubulin, troponin-T, telethonin, and titin. We performed Western blot to examine the protein expression of CCT5 and some heat shock proteins, Hsp90, Hsp60, Hsp27, and α-B crystallin, along with the main client proteins of the CCT5, actin, and tubulin. Our findings revealed muscular atrophy, abnormal morphology, and different sizes of muscle fibers in affected tissue. The swollen nuclei and wide interfiber spaces were seen. Expression of CCT5 had been decreased and showed a different distribution pattern in the affected tissue. Altered expression, distribution, and bandage pattern were detected by confocal microscopy for the interested muscular proteins in tissue from the patient compared to the healthy control. Protein levels of the studied Hsps normally located at the Z-disk were reduced. Western blot results showed increased levels of the actin and tubulin proteins in the diseased skeletal muscle biopsy compared to healthy tissue. Chaperones must be expressed at high levels in skeletal muscle to counteract various stressors such as mechanical, oxidative, and thermal crises; therefore, it seems relevant that defects of molecular chaperones may result in damaged skeletal muscle fibers. So far, several chaperones or cochaperones involved in neuromuscular disorders have been defined. Our study shows that alteration of the CCT5 subunit is associated with the damaged structure of skeletal muscle fibers and alterations of chaperone system components and paves the way to explore possible alternative substrates of chaperonin CCT. However, further studies are underway to investigate the CCT mechanisms of action to design applicable therapeutic strategies.

Keywords: molecular chaperones, neurochaperonopathy, neuromuscular system, protein homeostasis

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