Comparison of Four Methods of RNA Extraction and cDNA Synthesis from The Venom of Peruvian Snakes of the Genus Bothrops of Clinical Importance.

RNA purification and cDNA synthesis represents the starting point for molecular analyses of snake venom proteins-enzymes. Usually, the sacrifice of snakes is necessary for venom gland extraction to identify protein-coding transcripts; however, the venom can be used as a source of transcripts. Although there are methods for obtaining RNA from venom, no comparative analysis has been conducted in the Bothrops genus. In the present study, we compared four commercial methods for RNA purification and cDNA synthesis from venom (liquid, lyophilized, or long-term storage) of four clinically relevant species of Peruvian Bothrops. Our results show that the TRIzol method presents the highest yield of RNA purified from venom (59 ± 11 ng/100 µL or 10 mg). The SuperScript First-Strand Synthesis System kit produced high amounts of cDNA (3.2 ± 1.2 ng cDNA/ng RNA), and the highest value was from combination with the Dynabeads mRNA DIRECT kit (4.8 ± 2.0 ng cDNA/ng RNA). The utility of cDNA was demonstrated with the amplification of six relevant toxins: thrombin-like enzymes, P-I and P-III metalloproteinases, acid and basic phospholipases A2, and disintegrins. To our knowledge, this is the first comparative study of RNA purification and cDNA synthesis methodologies from Bothrops genus venom.

Simultaneous identification of three clinically relevant peruvian pit vipers by multiplex loop-mediated isothermal amplification (mLAMP).

Previous knowledge about the taxonomic distribution of venomous snake species is very useful for epidemiological aspects of ophidism. Here, we sought to develop an assay for the differential identification of clinically relevant snakes in Peru: Bothrops atrox, Lachesis muta, and Crotalus durissus using a multiplex loop-mediated isothermal amplification (mLAMP) assay. For this, DNA was extracted from the shed snake skins and the mitochondrial genes Cytb, COI, and 12S rRNA were amplified and further sequenced, for the design of mLAMP reaction primers. For each snake species the forward and reverse primers, internal forward and reverse primers, and the loop primers were obtained, bearing the latter different fluorophores for product identification. Finally, the reaction was standardized in the presence of all primer sets, and an optimal amount of low molecular weight polyethyleneimine. The precipitated products were observed in a UV light transilluminator, finding a differential fluorescence according to the DNA used, with a detection limit to the naked eye in the range of 0.2-25 ng of DNA, within 30 min. This study is the first report on the use of mLAMP technology for the identification of venomous snakes.

Squalene in oil-based adjuvant improves the immunogenicity of SARS-CoV-2 RBD and confirms safety in animal models.

COVID-19 pandemic has accelerated the development of vaccines against its etiologic agent, SARS-CoV-2. However, the emergence of new variants of the virus lead to the generation of new alternatives to improve the current sub-unit vaccines in development. In the present report, the immunogenicity of the Spike RBD of SARS-CoV-2 formulated with an oil-in-water emulsion and a water-in-oil emulsion with squalene was evaluated in mice and hamsters. The RBD protein was expressed in insect cells and purified by chromatography until >95% purity. The protein was shown to have the appropriate folding as determined by ELISA and flow cytometry binding assays to its receptor, as well as by its detection by hamster immune anti-S1 sera under non-reducing conditions. In immunization assays, although the cellular immune response elicited by both adjuvants were similar, the formulation based in water-in-oil emulsion and squalene generated an earlier humoral response as determined by ELISA. Similarly, this formulation was able to stimulate neutralizing antibodies in hamsters. The vaccine candidate was shown to be safe, as demonstrated by the histopathological analysis in lungs, liver and kidney. These results have shown the potential of this formulation vaccine to be evaluated in a challenge against SARS-CoV-2 and determine its ability to confer protection.

Preclinical Assessment of IgY Antibodies Against Recombinant SARS-CoV-2 RBD Protein for Prophylaxis and Post-Infection Treatment of COVID-19.

Within the framework of the current COVID-19 pandemic, there is a race against time to find therapies for the outbreak to be controlled. Since vaccines are still tedious to develop and partially available for low-income countries, passive immunity based on egg-yolk antibodies (IgY) is presented as a suitable approach to preclude potential death of infected patients, based on its high specificity/avidity/production yield, cost-effective manufacture, and ease of administration. In the present study, IgY antibodies against a recombinant RBD protein of SARS-CoV-2 were produced in specific-pathogen-free chickens and purified from eggs using a biocompatible method. In vitro immunoreactivity was tested, finding high recognition and neutralization values. Safety was also demonstrated prior to efficacy evaluation, in which body weight, kinematics, and histopathological assessments of hamsters challenged with SARS-CoV-2 were performed, showing a protective effect administering IgY intranasally both as a prophylactic treatment or a post-infection treatment. The results of this study showed that intranasally delivered IgY has the potential to both aid in prevention and in overcoming COVID-19 infection, which should be very useful to control the advance of the current pandemic and the associated mortality.

Intranasal vaccination of hamsters with a Newcastle disease virus vector expressing the S1 subunit protects animals against SARS-CoV-2 disease.

The coronavirus disease-19 (COVID-19) pandemic has already claimed millions of lives and remains one of the major catastrophes in the recorded history. While mitigation and control strategies provide short term solutions, vaccines play critical roles in long term control of the disease. Recent emergence of potentially vaccine-resistant and novel variants necessitated testing and deployment of novel technologies that are safe, effective, stable, easy to administer, and inexpensive to produce. Here we developed three recombinant Newcastle disease virus (rNDV) vectored vaccines and assessed their immunogenicity, safety, and protective efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in mice and hamsters. Intranasal administration of rNDV-based vaccine candidates elicited high levels of neutralizing antibodies. Importantly, the nasally administrated vaccine prevented lung damage, and significantly reduced viral load in the respiratory tract of vaccinated animal which was compounded by profound humoral immune responses. Taken together, the presented NDV-based vaccine candidates fully protected animals against SARS-CoV-2 challenge and warrants evaluation in a Phase I human clinical trial as a promising tool in the fight against COVID-19.

Consenso Colombiano para la prevención, diagnóstico y manejo de condiciones trombóticas en pacientes adultos con COVID-19: aplicando el Marco de la Evidencia a la Decisión (EtD) GRADE / Colombian consensus for the prevention, diagnosis and treatment of thrombotic conditions in adult patients with COVID-19: applying GRADE Evidence to Decision (EtD) Frameworks

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Abstract Recent studies have reported the occurrence of thrombotic phenomena or coagulopathy in patients with COVID-19. There are divergent positions regarding the prevention, diagnosis, and treatment of these phenomena, and current clinical practice is based solely on deductions by extension from retrospective studies, case series, observational studies, and international guidelines developed prior to the pandemic. In this context, the aim was to generate a group of recommendations on the prevention, diagnosis and management of thrombotic complications associated with COVID-19. Methods: A rapid guidance was carried out applying the GRADE Evidence to Decision (EtD) frameworks and an iterative participation system, with statistical and qualitative analysis. Results: 31 clinical recommendations were generated focused on: a) Coagulation tests in symptomatic adults with suspected infection or confirmed SARS CoV-2 infection; b) Thromboprophylaxis in adults diagnosed with COVID-19 (Risk scales, thromboprophylaxis for outpatient, in-hospital management, and duration of thromboprophylaxis after discharge from hospitalization), c) Diagnosis and treatment of thrombotic complications, and d) Management of people with previous indication of anticoagulant agents. Conclusions: Recommendations of this consensus guide clinical decision-making regarding the prevention, diagnosis, and treatment of thrombotic phenomena in patients with COVID-19, and represent an agreement that will help decrease the dispersion in clinical practices according to the challenge imposed by the pandemic.

Modification of the Surgical Strategy for the Dissection of the Recurrent Laryngeal Nerve Using Continuous Intraoperative Nerve Monitoring.

BACKGROUND: The aim of this study was to describe first experiences and changes in management using continuous intraoperative neuromonitoring (C-IONM) in thyroid and parathyroid surgery. METHOD: Retrospective analysis of patients who underwent surgery with C-IONM since 2012. Surgical maneuvers were modified when electrophysiologic events occurred. Patients with persistent loss of signal (LOS) underwent postoperative laryngoscopy. RESULTS: One hundred and one patients (of 1586 neck surgeries) were included and 19 had events: In 13 these were temporary (resolved before end of surgery) and led to intraoperative modifications in surgical approach; in all cases traction was released, and in 8, recurrent laryngeal nerve (RLN) approach was changed [superior approach (2), inferior approach (2), both (4)]. Six patients had persistent LOS (5.9%, present at end of procedure), with RLN palsy (RLNP) on postoperative day 1: In three, LOS occurred at electrode placement on the vagus nerve, leading to distal placement of the electrode allowing ipsilateral dissection under continuous monitoring; all three had complete recovery at 6 months. In the three other patients, LOS occurred on the RLN: one probable thermal, one traction lesion and one accidental section of the anterior RLN branch. The RLN recovered within 6 months in two patients, and in the third, RLNP persisted after 6 months (1/101 = 1%). CONCLUSION: C-IONM provides real-time evaluation of the RLN function, allowing for adaptation of surgical maneuvers to prevent RLNP. It seems particularly useful in difficult cases like redo neck surgery, invasive thyroid cancer and intrathoracic or large goiter. Care should be given at electrode placement on the vagus nerve.

Melancolía y desvanecimiento del yo en el poema “El húsar” (1953) de Álvaro Mutis / Melancholy and fading in Álvaro Mutis's poem El Húsar [The Hussar] (1953)

En este artículo proponemos una lectura analítica del problema del fading o desvanecimiento del yo como una característica de la melancolía en el poema “El húsar” (1953) del poeta colombiano Àlvaro Mutis. Planteamos para ello un análisis basado en la aplicación del método psicoanalítico propuesto por Jacques Lacan, el cual ofrece una perspectiva hermenéutica original en el estudio de la literatura. En esta lectura proponemos que existe un proceso de transformación progresivo de la figura del héroe a partir del cual las fronteras entre el sujeto y el objeto se van perdiendo. Este proceso se conoce como fading o desvanecimiento del yo según el cual el objeto y el sujeto se funden para dar paso al puro real de la muerte y la posterior desaparición del héroe mutisiano.

In this article we analyze in Álvaro Mutis's poem El húsar [The Hussar] (1953) the theoretical problem of fading of the I as a characteristic of melancholy theorized in Lacan´s psychoanalytical theory. We demonstrate how the psychoanalytical method could be applied to literary analysis as an original hermeneutical tool that offers a valuable approach in the study of literature. Our main argument in this article is that in the poem El húsar the hero's self is set in an ongoing process of fading where the frontier between subject and object tend to disappear. At the end of this semantic process the signifier loses its symbolic value and the real of death is what lasts.

Cet article a pour but d'examiner le problème du fading ou effacement du moi en tant que caractéristique de la mélancolie dans le poème "Le hussard" (1953) du poète colombien Álvaro Mutis. Pour ce faire, une lecture basée sur l'application de la méthode psychanalytique de Jacques Lacan est proposée. Cette analyse démontrera que la psychanalyse offre une perspective herméneutique originale dans l'étude de la littérature. Dans ce poème il existe un processus de transformation progressive du personnage du héros: les frontières du sujet et de l'objet se fondent dans un processus de fading dans lequel le signifiant perd graduellement sa valeur symbolique pour donner lieu au réel de la mort et à l'ultérieure disparition du héros.

Desempeño profesional, satisfacción y condiciones laborales de egresados de la Facultad de EnfermerÍa de la Universidad de Cartagena (Colombia) / Professional performance, satisfaction and working conditions of graduates of the School of Nursing of the University of Cartagena (Colômbia)

Resumen:

Objetivo: describir el desempeño profesional, satisfacción y condiciones laborales de los egresados de la Facultad de Enfermería de la Universidad de Cartagena (Colombia) del año 2003 al 2008. Método: estudio descriptivo. Población de 416 egresados y muestra de 222 egresados. Se utilizó una encuesta sociodemográfica, la escala SL-PC para medir condiciones laborales y la escala WES 10 para medir la satisfacción laboral. Fueron encuestados los empleadores para conocer la percepción sobre el desempeño.

Resultados: participaron 222 egresados, de los cuales el 57,9% (114) percibe su desempeño profesional como bueno. Así mismo, para un 57,1% (12) de los empleadores el desempeño del egresado es apropiado. La satisfacción laboral obtuvo un promedio de 30,54 de un máximo posible de 50 puntos. Quedó demostrado que los egresados trabajan en condiciones buenas, aunque trabajan un promedio de 45,67 horas semanales. Conclusión: a pesar de tener un buen desempeño, los egresados no están satisfechos laboralmente y las condiciones laborales no son las mejores.

Abstract:

Objective: To describe the professional performance, satisfaction and working conditions of graduates of the School of Nursing at the University of Cartagena from 2003 to 2008. Method: In a descriptive study (Population of 416 graduates, sample of 222 graduates), we used a demographic survey, the SL-PC scale to measure working conditions and WES scale 10 to measure job satisfaction. Employers were also surveyed to determine their perception of the performance. Results: A total of 222 graduates. Of these, 57.9% (114) perceived well their professional experience. Also, to 57.1% (12) employers graduate performance is appropriate. Job satisfaction scored an average of 30.54 out of a possible 50 points. It was found that graduates working in good conditions, although they working an average of 45.67 hours weekly. Conclusion: Despite scoring well, graduates are dissatisfied occupationally and working conditions are not the best. Key words: Professional groups, professional nursing practice, job satisfaction, nursing working conditions, nursing education.

Objetivo: Descrever o desempenho profissional, satisfação e condições de trabalho dos graduados da Escola de Enfermagem da Universidade de Cartagena de 2003 a 2008.

Método: Um estudo descritivo. População de 416 formandos. Mostra de 222 diplomados. Foi utilizado um questionário demográfico, a escala SL PC para medir as condições de trabalho e WES escala de 10 para medir a satisfação no trabalho. Empregadores foram entrevistados para determinar a percepção do desempenho. Resultado: Um total de 222 formandos. Destes, 57,9% (114), percebendo a profissional também. Além disso, a 57,1% (12) dos empregadores desempenho graduado é apropriado. A satisfação no trabalho apresentaram uma média de 30,54 de um possível 50 pontos. Verificou-se que os formados a trabalhar em boas condições, mas trabalhando uma média de 45,67 horas semanal. Conclusão: A pesar de ter marcado bem formados estão insatisfeitos profissionalmente e condições de trabalho não são as melhores.

Fibrotic lesions in the testis of bulls and relationship to semen quality.

Ultrasonography of the testes was done in bulls at three locations in western Canada (n=325) and one in Argentina (n=387) to determine the prevalence of fibrotic lesions and to examine the relationship between fibrotic lesions and location, age, breed, right compared with left testes, testis size and semen quality. Fibrotic lesions were common in the testes of bulls raised under intensive rearing conditions in western Canada as well as in the more extensive rearing conditions of Argentina. Fibrotic lesions appeared as early as 5-6 months of age and the number of cases continued to increase until at least 12-14 months of age. The severity of lesions increased in some cases during this period; however, it appears that the development of lesions occurred during a finite period of pubertal development. It is unlikely that the prevalence of lesions is influenced by breed, right compared with left testes or testis size. The cause of the lesions is unknown, but there was an association between the development of fibrotic lesions and an outbreak of BRSV disease in Argentina in one group of bulls. There was some indication that during the active process that leads to fibrosis spermatogenesis is adversely affected; however, the presence of a large number of fibrotic lesions that may occupy as much as 50% of the testis parenchyma did not preclude the production of a greater percentage of sperm with normal morphology.

Intraglandular injection of antibiotics for the treatment of vesicular adenitis in bulls.

Two experiments were designed to determine the efficacy of intraglandular antibiotic treatment in beef bulls. Experiment 1 was designed to evaluate the glandular tissue reaction to intraglandular antibiotic treatment. Experiment 2 was conducted to determine the efficacy intraglandular injection of antibiotics for the treatment of naturally occurring cases of vesicular adenitis. Healthy beef bulls (n=15), 2 and 3 years of age, were randomly allocated to three equal treatment groups to receive 10% of the daily recommended parenteral dose of penicillin, ceftiofur, or oxytetracycline in a volume of 6 mL injected directly into one of the vesicular glands. Ultrasonography was performed before, immediately after, and at 24, 48 and 168 h after intraglandular injection. The size and hardness of vesicular glands injected with oxytetracycline was greater (P<0.01) than those injected with ceftiofur. Ultrasonographic pixel intensity increased (P<0.01) after treatment with antibiotics, especially after treatment with oxytetracycline or penicillin. In Experiment 2, yearling beef bulls with clinical vesicular adenitis (n=14) were referred to the Western College of Veterinary Medicine for treatment. Eight bulls had unilateral and six had bilateral vesicular adenitis. The most common isolate was Arcanobacterium pyogenes. Corynebacterium pseudotuberculosis was isolated from one bull. Bulls were subjected to rectal palpation and ultrasonography of the vesicular glands, semen collection by electroejaculation, and intraglandular treatment with ceftiofur (n=13) and if necessary, a second intraglandular treatment of penicillin (n=6). One bull was treated only with an initial intraglandular injection of penicillin. Bulls were evaluated once a week over 6 weeks by palpation of the glands, and evaluation of semen. All bulls recovered from vesicular adenitis after 3-6 weeks. There was a difference in the amount of pus (P=0.042), leukocytes (P<0.001) and blood (P=0.003) present in ejaculates from before treatment to 3 weeks after treatment. Pixel intensities in ultrasonographic images of healthy or affected vesicular glands, whether treated or untreated, did not change over time. Intraglandular injection of ceftiofur in yearling bulls via the ischiorectal fossa was effective for treating vesicular adenitis.

Relationship between semen quality and pixel-intensity of testicular ultrasonograms after scrotal insulation in beef bulls.

The objective of this study was to determine the relationship between semen quality and testicular pixel-intensity derived from image analysis of ultrasonograms after scrotal insulation in bulls. In addition, the ability to predict semen quality based on testicular pixel-intensity was evaluated. Sixteen beef bulls were selected on the basis of satisfactory semen quality and normal testicular ultrasonogram appearance. Bulls were allocated into two groups for scrotal insulation for 4 days (group 1) or 8 days (group 2). Semen was collected and evaluated twice weekly and testicular ultrasonograms were evaluated once weekly for 8 weeks after removal of scrotal insulation. In general, the percentages of motile and morphologically normal spermatozoa decreased below pre-insulation levels from 1 to 5 weeks after scrotal insulation removal. Overall, group 1 had greater (P < 0.01) percentages of motile and normal spermatozoa than group 2. Mean testicular pixel-intensity (PI), and the number of pixels corresponding to the intensity that occurs most frequently (NP) decreased in the first 2-3 weeks after scrotal insulation, coincidently with the decrease in sperm motility and normal morphology. When the entire data set was evaluated, there was no association between testicular PI or NP with semen quality observed at the same week of ultrasound examinations. However, regression models indicated that testicular PI and NP accounted for 13-25% of the variation in sperm motility and morphology in ejaculates collected 2-4 weeks after ultrasound exam. Testicular PI and NP had moderate sensitivity and negative predictive values (64.5-82.6%), but low specificity and positive predictive values (33.3-61.2%) as predictors of satisfactory semen quality (> or = 60% motile spermatozoa and > or = 70% morphologically normal spermatozoa) for ejaculates collected 2-4 weeks after ultrasound exam. In conclusion, the deleterious effects of scrotal insulation on semen quality were dependent on the length of the period of insulation and were associated with changes in testicular ultrasonogram pixel-intensity. Testicular ultrasonogram pixel-intensity had a better association with future semen quality than with present semen quality and was a better predictor of unsatisfactory semen quality than satisfactory semen quality.

Use of internal artificial vaginas for breeding soundness evaluation in range bulls: an alternative for electroejaculation allowing observation of sex drive and mating ability.

The objective of this study was to test the efficacy of an inexpensive, reusable internal artificial vagina (IAV) developed for breeding soundness evaluation of range beef bulls. In addition, sexual behavior during semen collection by IAV was compared to behavior during pasture breeding. Breeding soundness exams (BSEs) were conducted on 165 bulls in two consecutive years (96 in Year 1 and 69 in Year 2). In Year 1, BSEs were done twice in all bulls, once by a conventional protocol using electroejaculation (EEJ), followed by the IAV method, one week later. In Year 2, all BSEs were done on one day; 69 bulls by the IAV method followed by EEJ in 21 bulls that failed to serve the IAV. For semen collection using an IAV, mount cows were restrained in breeding crates and an IAV was inserted into the vagina just beyond the depth of the vestibular sphincter. After each copulation, the IAV was replaced for the next bull to be tested. Semen collection by IAV was successful for all bulls that mounted and penetrated cows during the testing period (54.3 and 69.6% of the bulls served the cows with IAVs in Year 1 and 2, respectively). Semen was collected successfully by EEJ from all bulls in both years. Differences were observed between semen collection methods in semen volume and percentage of sperm staining alive; however, the differences were opposite in Year 1 and 2 and, therefore, were probably due to natural variations in time and within bull rather than the method of semen collection. Semen collection by IAV allowed the detection of problems that prevented copulation in 8 bulls (4.8%) that were determined to be satisfactory potential breeders when semen was collected by EEJ. In Year 1, breeding observations were made at pasture for 15 bulls that served, and 15 bulls that did not serve cows with an IAV. Bulls that did not serve the IAV during the test period had fewer mounts, attempts to mount, and completed services at pasture than bulls that had served the IAV, indicating that such bulls are likely to be less efficient breeders. In conclusion, IAVs might be used as an alternative to EEJ for breeding soundness evaluation in about 50-70% of range bulls. Semen collection by IAV also allows observation of sex drive and mating ability thus improves the ability to select sound breeding bulls.

Recomendaciones para el diagnóstico, tratamiento y prevención de la neumonía adquirida en la comunidad en adultos / Diagnosis, treatment and prevention recommendations for adult community-acquired pneumonia

Definición: la NAC es la infección aguda del parénquima pulmonar que se desarrolla fuera del ambiente hospitalario, se maniefiesta en las primeras 48 horas del ingreso al hospital o después de siete días de haber egresado. Cuadro clínico: Síntomas de una infección aguda del tracto respiratorio inferior: tos y por lo menos uno de los siguientes: expectoración mucopurulenta, dolor torácico de tipo pleurítico, disnea. Por lo menos un síntoma o signo sistémico: sensación de fiebre, suduración, dolores, malestar general y/o temperatura de 38.3°C O más. Infiltrado focal o asimétrico, en la radiografía del tórax, que no se haya observado o descrito previamente y para el cual no se tenga otra explicación. Al examen físico del tórax se puede encontrar o no anormalidades, generalmente localizadas o asimétricas, o signos de consolidación pulmonar. Epidemiología: la NAC es una enfermedad frecuente cuya incidencia varía de acuerdo con la edad, es mayor en los extremos de la vida (mayores de 65 años y menores de cinco) La neumonía es una enfermedad con un alto índice de morbilidad y mortalidad. La mortalidad general producida por la NAC oscila entre 1 por ciento y 25 por ciento, y depende de múltiples factores, entre los que destacan la severidad de la enfermedad y el german causante. Etiología: la posivilidad de identificar el german causante NAC, aun en las mejores condiciones, es relativamente baja (40 por ciento a 60 por ciento). Para el caso colombiano es importante destacar: La causa más frecuente es el S.pneumoniae. M. tuberculosis debe considerarse como causa de NAC en el país, aunque su frecuencia real se desconoce, pero pueden presentarse hasta en 20 por ciento de casos...(AU: Asociación Colombiana de Neumología y Cirugía de Tórax; Asociación Colombiana de Infectología; Asociación Colombiana de Medicina Interna)