Total rupture of Achilles tendon induces inflammatory response and glial activation on the spinal cord of mice.

Rupture of Achilles tendon is a common accident affecting professional and recreational athletes. Acute and chronic pain are symptoms commonly observed in patients with rupture. However, few studies have investigated whether Achilles tendon rupture is able to promote disorders in the central nervous system (CNS). Therefore, the current study aimed to evaluate nociceptive alterations and inflammatory response in the L5 lumbar segment of Balb/c mice spinal cord after Achilles tendon rupture. We found increased algesia in the paw of the ruptured group on the 7th and 14th days post-tenotomy compared with the control group. This phenomenon was accompanied by overexpression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase-2 (NOS-2) as well as hyperactivation of astrocytes and microglia in nociceptive areas of L5 spinal cord as evidenced by intense GFAP and IBA-1 immunostaining, respectively. Biochemical studies also demonstrated increased levels of nitrite in the L5 spinal cord of tenotomized animals compared with the control group. Thus, we have demonstrated for the first time that total rupture of the Achilles tendon induced inflammatory response and nitrergic and glial activation in the CNS in the L5 spinal cord region.

Total rupture of Achilles tendon induces inflammatory response and glial activation on the spinal cord of mice

Rupture of Achilles tendon is a common accident affecting professional and recreational athletes. Acute and chronic pain are symptoms commonly observed in patients with rupture. However, few studies have investigated whether Achilles tendon rupture is able to promote disorders in the central nervous system (CNS). Therefore, the current study aimed to evaluate nociceptive alterations and inflammatory response in the L5 lumbar segment of Balb/c mice spinal cord after Achilles tendon rupture. We found increased algesia in the paw of the ruptured group on the 7th and 14th days post-tenotomy compared with the control group. This phenomenon was accompanied by overexpression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase-2 (NOS-2) as well as hyperactivation of astrocytes and microglia in nociceptive areas of L5 spinal cord as evidenced by intense GFAP and IBA-1 immunostaining, respectively. Biochemical studies also demonstrated increased levels of nitrite in the L5 spinal cord of tenotomized animals compared with the control group. Thus, we have demonstrated for the first time that total rupture of the Achilles tendon induced inflammatory response and nitrergic and glial activation in the CNS in the L5 spinal cord region.

[Setting up Objective Structured Clinical Examination (OSCE) in ophthalmology]. / Mise en place des examens cliniques objectifs structurés (ECOS) facultaires en ophtalmologie.

Obective Structured Clinical Examinations (OSCE) are a reproducible and objective way to evaluate medical students and have been used for many years in English-speaking countries, Canada and Switzerland. They evaluate candidates more on the basis of their practical skills, know-how and interpersonal skills than on their theoretical knowledge. From a nationally validated, limited list of typical clinical situations, stations are set up by the teaching team with standardized patients played by actors, designed to test a variety of problem solving, technical, diagnostic, therapeutic, communication, examination, and history taking skills, possibly with simulation tools. Setting up a station, as well as creating an OSCE cycle with several stations through which the candidates rotate, requires significant preparation prior to the examination: creating the station scenario with precise instruction sheets for the candidates, simulated patients and evaluators, multiple stages of proofreading, verifying the required equipment and adapting rating scales. OSCEs seek to evaluate students "objectively," as they are the only variable in this type of examination, in which the scripts, materials and rating scales have been standardized to limit subjectivity. This examination method is a flagship measure of the reform of the second cycle of French medical studies. OSCEs are now part of the testing modalities for the 2021-2022 academic year and will be integrated into the National Dematerialized Examination (NDE) starting in May 2023. They may also be useful in validating the achievements of students and residents in various stages of training, as well as in continuing medical education (CME). We present herein the key elements of these new evaluation tools and their practical applications in the evaluation of students in ophthalmology.

Caffeine modulates the intercalation of drugs on DNA: A study at the single molecule level.

We use optical tweezers to characterize the ability of Caffeine (Caf) to modulate the intercalation of drugs into the DNA double-helix at the single molecule level. When previously bound to the double-helix, Caf hinders ethidium bromide (EtBr) intercalation, decreasing its effective equilibrium binding constant with DNA. The dominant mechanism of such singular ability is a direct binding of Caf to the intercalating drugs in solution, which decreases the effective concentration of such compounds available to interact with DNA. When EtBr intercalation into the DNA double-helix occurs firstly, on the other hand, the measured cooperativity between Caf molecules interacting with DNA can be modulated, a feature also correlated to the Caf-EtBr interaction in solution. The results achieved here unveil many peculiarities about the details of such interactions at the molecular level and provide new insights on the use of Caf in therapeutic applications.

Hydroxychloroquine Exhibits a Strong Complex Interaction with DNA: Unraveling the Mechanism of Action.

In the past months, the use of the drug hydroxychloroquine has considerably increased in many countries, associated with a proposed treatment for the COVID-19 disease. Although there is no conclusive evidence about the efficacy of the drug for this purpose, surprisingly there are no conclusive studies in the literature concerning its mechanism of action inside cells, which is related to its interaction with nucleic acids. Here, we performed a robust characterization of the interaction between hydroxychloroquine and double-stranded DNA using single-molecule force spectroscopy and gel electrophoresis. Two different binding modes were identified, namely, minor groove binding for low drug concentrations and intercalation for high drug concentrations, and the sets of binding parameters were determined for each of these modes. Such results have unraveled in detail the molecular mechanism of action of the drug as a DNA ligand.

On the use of Europium (Eu) for designing new metal-based anticancer drugs.

Europium oxide (Eu2O3) was used to evaluate the affinity of this rare earth element for interacting with double-stranded (ds) DNA molecules. To perform the study, we used single molecule force spectroscopy with optical tweezers and gel electrophoresis assays. Force spectroscopy experiments show that Eu2O3 presents a strong interaction with dsDNA, and the binding is independent on the ionic strength used in the surrounding environment. Among the main characteristics of the interaction, Eu2O3 tends to bind in a cooperative way, forming bound clusters of ∼ 3 molecules, and presents a high equilibrium association binding constant on the order of 105 M-1. In addition, gel electrophoresis confirm the weak electrostatic character of the interaction and explicit show that Eu2O3 does not interfere on drug intercalation into the double-helix. Such results demonstrate the potential of europium for interacting with nucleic acids and strongly suggest that this rare earth element may be considered for the design of new metal-based anticancer drugs in the future.

Silicon microparticles as handles for optical tweezers experiments.

We study the behavior of silicon microparticles in a 1064 nm Gaussian-beam optical tweezers, showing that this semiconductor can be used to perform different types of optical manipulation experiments. Depending on the focal position and the laser power used, the particles can present an oscillatory dynamics in the tweezers or can be stably 3D-trapped with a trap stiffness that allows the application of femtoNewton forces with accuracy. A new, to the best of our knowledge, interpretation based on the photoexcitation of electrons in the valence band is proposed to explain the oscillations, and the quantities associated with such dynamics (e.g., amplitude, period, etc.) were characterized as a function of relevant parameters to optical tweezers setups.

Effects of caffeine on the structure and conformation of DNA: A force spectroscopy study.

Here, we use single molecule force spectroscopy performed with optical tweezers in order to investigate the interaction between Caffeine and the DNA molecule for various different concentrations of the alkaloid and under two distinct ionic strengths of the surrounding buffer. We were able to determine the mechanical changes induced on the double-helix structure due to Caffeine binding, the binding mode and the binding parameters of the interaction. The results obtained show that Caffeine binds to DNA by outside the double-helix with a higher affinity at lower ionic strengths. On the other hand, a considerable cooperativity was found only for sufficient high ionic strengths, suggesting that Caffeine may binding forming dimers and/or trimers along the double-helix under this condition. Finally, it was also shown that Caffeine stabilizes the DNA double-helix upon binding, preventing force-induced DNA melting.

The role of interleukins in vitiligo: a systematic review.

Vitiligo is a disorder of the skin that causes depigmentation and asymptomatic macules whose exact cause is still unclear. Although its aetiology is not fully elucidated, the main theory of its pathomechanism is that it is associated with the autoimmune process. There is few summarized information about the role of inflammatory mediators, as interleukins, in vitiligo, so our aim was to present a systematic review of the role of interleukins in vitiligo, focusing on interleukins. In this review, we included all studies assessing interleukin levels in vitiligo patients conducted up to June 2017. Quality assessment of these studies was performed using the Newcastle-Ottawa Scale (NOS). The interleukins mainly involved were IL-2, IL-4, IL-6, IL-10 and IL-17. The studies highlight the crucial role of IL-17 in the onset and progression of the disease, and its synergistic action with IL-2, IL-6 and IL-33. Dysregulated levels of the interleukins were also correlated with the stage of disease, the affected skin surface area, and indicated as the main factor for lymphocyte infiltration found in depigmented regions. These findings illustrate the growing need for new therapies targeting vitiligo and further research into the role of interleukins as an area of particular interest.

Bovine oocyte membrane permeability and cryosurvival: Effects of different cryoprotectants and calcium in the vitrification media.

The cryopreservation process must be improved to enhance oocyte cryosurvival and functionality. Two protocols with different cryoprotectants (CPAs), containing either ethylene glycol (EG), dimethyl sulfoxide (DMSO) and sucrose (EGDMSO) or 1,2-propanediol and sucrose (PrOH) were evaluated. In both protocols, calcium (Ca2+) free or -containing base media were tested. Oocytes were subjected to vitrification or only exposed to CPAs without immersion in liquid nitrogen. Oocyte's viability, cortical granules location and competence for development after fertilization were assessed. Finally, fatty acid composition and membrane permeability of oocytes exposed to CPAs were analyzed. Independently of Ca2+ concentration in the vitrification media, the development rates were higher in oocytes vitrified with EGDMSO protocols (p = 0.0005). After warming, higher cleavage rates were obtained in EGDMSO + Ca2+ compared to the PrOH without Ca2+ protocol (p = 0.02). Oocytes exposed to PrOH without Ca2+ presented lower cleavage rates compared to control (p = 0.04). An enhanced premature zona hardening in vitrified oocytes as well as lower concentrations of the fatty acids c11:18:1 and 20:4n-6 in cumulus oocyte complexes exposed to PrOH protocols were identified. The oocytes minimum volume and permeability were affected by the exposure to PrOH and Ca2+ (p ≤ 0.007). In conclusion, the most effective protocol for bovine oocytes cryopreservation combines EG and DMSO, independently of Ca2+ concentration in the media. A higher toxicity and an incomplete depletion of water during PrOH loading may hamper oocyte viability. The type of CPAs and Ca2+ interfered differentially on oocyte pathways to functionality, and this should be considered when choosing a cryopreservation protocol.

The accuracy of PiCCO® in measuring cardiac output in patients under therapeutic hypothermia: Comparison with transthoracic echocardiography / La exactitud de PiCCO ® para medir el gasto cardíaco en pacientes bajo hipotermia terapéutica: comparación con ecocardiografía transtorácica

Background: Invasive cardiac monitoring using thermodilution methods such as PiCCO® is widely used in critically ill patients and provides a wide range of hemodynamic variables, including cardiac output (CO). However, in post-cardiac arrest patients subjected to therapeutic hypothermia, the low body temperature possibly could interfere with the technique. Transthoracic Doppler echocardiography (ECHO) has long proved its accuracy in estimating CO, and is not influenced by temperature changes. Objective: To assess the accuracy of PiCCO® in measuring CO in patients under therapeutic hypothermia, compared with ECHO. Design and patients: Thirty paired COECHO/COPiCCO measurements were analyzed in 15 patients subjected to hypothermia after cardiac arrest. Eighteen paired measurements were obtained at under 36°C and 12 at ≥36°C. A value of 0.5l/min was considered the maximum accepted difference between the COECHO and COPiCCO values. Results: Under conditions of normothermia (≥36°C), the mean difference between COECHO and COPiCCO was 0.030 l/min, with limits of agreement (−0.22, 0.28) - all of the measurements differing by less than 0.5 l/min. In situations of hypothermia (<36°C), the mean difference in CO measurements was −0.426 l/min, with limits of agreement (−1.60, 0.75), and only 44% (8/18) of the paired measurements fell within the interval (−0.5, 0.5). The calculated temperature cut-off point maximizing specificity was 35.95°C: above this temperature, specificity was 100%, with a false-positive rate of 0%. Conclusions: The results clearly show clinically relevant discordance between COECHO and COPiCCO at temperatures of <36°C, demonstrating the inaccuracy of PiCCO® for cardiac output measurements in hypothermic patients (AU)

Introducción : La monitorización invasiva cardiaca mediante métodos de termodilución, como PiCCO®, es ampliamente utilizada en pacientes críticamente enfermos y proporciona una gran variedad de variables hemodinámicas, como el gasto cardiaco (GC). No obstante, en los pacientes post-paro cardíaco bajo hipotermia terapéutica, la baja temperatura corporal podría interferir con la técnica. La ecocardiografía doppler transtorácica (ECHO) ha demostrado su exactitud en la estimación del GC y no está influenciada por los cambios de temperatura. Objetivo: El objetivo del presente estudio fue evaluar la exactitud de PiCCO® para medir el GC en pacientes bajo hipotermia terapéutica, en comparación con ECHO. Diseño y pacientes: Se analizaron 30 pares de mediciones GC_ECHO/GC_PiCCO en 15 pacientes sometidos a hipotermia después de un paro cardíaco. La máxima diferencia aceptada entre los valores de GC_ECHO y GC_PiCCO se consideró 18 mediciones pareadas se realizaron a menos de 36°C y 12 a ≥36°C. 0,5L/min. Resultados: En la normotermia (≥36°C), la diferencia media entre GC_ECHO y GC_PiCCO fue de 0,030L/min, con límites de concordancia (-0,22; 0,28), todas las medidas difieren menos de 0,5L/min. En la hipotermia (<36°C), la diferencia media de las mediciones fue -0,426L/min con límites de concordancia (-1,60; 0,75) y solo el 44% de las mediciones cayeron en el intervalo (-0,5; 0,5). El límite de temperatura calculado que maximiza la especificidad fue 35,95°C, por encima del cual la especificidad fue del 100% y la tasa de falsos positivos del 0%. Conclusiones: Los resultados muestran claramente una discordancia clínicamente relevante entre GC_ECHO y GC_PiCCO en temperatura <36°C, lo que revela la inexactitud de PiCCO® para las mediciones del gasto cardíaco en pacientes hipotérmicos (AU)

Biophysical characterization of the DNA interaction with the biogenic polyamine putrescine: A single molecule study.

We have performed a biophysical characterization, at single molecule level, of the interaction between the DNA molecule and the biogenic polyamine putrescine. By using force spectroscopy, we were able to monitor the complexes formation as putrescine is added to the sample, determining the mechanical properties of such complexes and the physicochemical (binding) parameters of the interaction for three different ionic strengths. In particular, it was shown that the behavior of the equilibrium binding constant as a function of the counterion concentration deviates from the prediction of the Record-Lohman model. The measured constants were (1.3 ± 0.2) × 105 M- 1 for [Na] = 150 mM, (2.1 ± 0.2) × 105 M- 1 for [Na] = 10 mM, and (2.2 ± 0.3) × 105 M- 1 for [Na] = 1 mM. The cooperativity degree of the binding reaction, on the other hand, increases with the ionic strength. From these analysis, the DNA-putrescine binding mechanisms are inferred, and a comparison with results reported for ordinary bivalent ions like magnesium is performed. Such study provides new insights on the general behavior of the DNA interactions with biogenic polyamines.

The accuracy of PiCCO® in measuring cardiac output in patients under therapeutic hypothermia: Comparison with transthoracic echocardiography.

BACKGROUND: Invasive cardiac monitoring using thermodilution methods such as PiCCO® is widely used in critically ill patients and provides a wide range of hemodynamic variables, including cardiac output (CO). However, in post-cardiac arrest patients subjected to therapeutic hypothermia, the low body temperature possibly could interfere with the technique. Transthoracic Doppler echocardiography (ECHO) has long proved its accuracy in estimating CO, and is not influenced by temperature changes. OBJECTIVE: To assess the accuracy of PiCCO® in measuring CO in patients under therapeutic hypothermia, compared with ECHO. DESIGN AND PATIENTS: Thirty paired COECHO/COPiCCO measurements were analyzed in 15 patients subjected to hypothermia after cardiac arrest. Eighteen paired measurements were obtained at under 36°C and 12 at ≥36°C. A value of 0.5l/min was considered the maximum accepted difference between the COECHO and COPiCCO values. RESULTS: Under conditions of normothermia (≥36°C), the mean difference between COECHO and COPiCCO was 0.030 l/min, with limits of agreement (-0.22, 0.28) - all of the measurements differing by less than 0.5 l/min. In situations of hypothermia (<36°C), the mean difference in CO measurements was -0.426 l/min, with limits of agreement (-1.60, 0.75), and only 44% (8/18) of the paired measurements fell within the interval (-0.5, 0.5). The calculated temperature cut-off point maximizing specificity was 35.95°C: above this temperature, specificity was 100%, with a false-positive rate of 0%. CONCLUSIONS: The results clearly show clinically relevant discordance between COECHO and COPiCCO at temperatures of <36°C, demonstrating the inaccuracy of PiCCO® for cardiac output measurements in hypothermic patients.

Full-length genomic and molecular characterization of Canine parvovirus in dogs from North of Brazil.

With the objective of characterizing Canine parvovirus (CPV) from some suspected fecal samples of dogs collected from the Veterinarian Hospital in Belém city, five positive samples were found by PCR assay and an update molecular characterization was provided of the CPV-2 circulation in Belém. Through sequencing of the complete DNA sequences (NS1, NS2, VP1, and VP2 genes), the CPV-2 strain was identified as CPV-2b (Asn426Asp) circulating in Belém. The CPV-2b strain with a different change at the position Tyr324Leu was detected in all samples assessed and thus reported for the first time for the scientific community. Phylogenetic analysis indicated that Belém CPV-2b and CPV-2a strains would be related to a cluster with samples after the 1990s, suggesting that CPV-2b in Belém originated from CPV-2a circulating in Brazil after the 1990s. Potential recombination events were analyzed using RDP4 and SplitsTree4; therefore, results suggest that CPV-2 sequences here described were not potentially recombination events. Continuous monitoring and molecular characterization of CPV-2 samples are needed not only to identify possible genetic and antigenic changes that may interfere with the effectiveness of vaccines but also to bring a better understanding of the mechanisms that drive the evolution of CPV-2 in Brazil.

Higher membrane fluidity mediates the increased subcutaneous fatty acid content in pigs fed reduced protein diets.

The production of pork with moderate amounts of intramuscular fat (IMF) without an increase in subcutaneous fat is highly desirable for the meat industry. Several studies indicate that dietary protein reduction during the growing-finishing period of pigs enhances IMF content, but its consequence on carcass fat deposition is still contradictory. In this study, we hypothesized that the effects of reduced protein diets (RPD), corrected or not with the limiting amino acid lysine, on subcutaneous fat deposition from pigs with distinct genotypes are mediated by adipose membranes biophysical properties. In total, 36 crossbred (Large White×Landrace×Pietrain - a lean genotype) and purebred (Alentejana breed - a fatty genotype) male pigs were randomly assigned to the control group, the RPD group or the reduced protein diet equilibrated for lysine (RPDL) group, allowing a 2×3 factorial arrangement (n=6). Backfat thickness and total fatty acid content were higher in Alentejana relative to crossbred pigs. Although dietary treatments did not change backfat thickness, RPD and RPDL increased total fatty acids content of subcutaneous fat. In order to understand this effect, adipose tissue membranes isolated from pig's subcutaneous fat were assayed for glycerol permeability and fluidity, using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-(trimethylamino)-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) probes. The glycerol transport across adipose membranes was not mediated by aquaglyceroporins and remained unchanged across dietary groups. Regardless of lysine correction, RPD increased membrane fluidity at the hydrocarbon region (lower DPH fluorescence anisotropy) in both genotypes of pigs. This result was associated with a lower ratio between oleic acid and linoleic acid on membrane's fatty acid composition. Adipose membrane's cholesterol content was independent from genotype and diet. Taken together, the present study shows that dietary protein reduction is successful in maintaining backfat thickness, although a negative side effect was observed on total fatty acids in subcutaneous fat, which may be due to changes in the fluidity of adipose membranes.

Atypical hemolytic uremic syndrome in intensive care: Case report in an adult / Síndrome hemolítico urémico atípico en cuidados intensivos: caso clínico de un adulto

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