Quantitative detection of SARS-CoV-2 RNA in nasopharyngeal samples from infected patients with mild disease.

Diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) cases is based on the count of real-time reverse transcription-plymerase chain reaction (RT-PCR) positive people. Viral load by real-time RT-PCR has been suggested as a biomarker of the SARS-CoV-2 infection. However, the association of viral load and severity of the disease is not yet resolved. Nasopharyngeal samples from 458 patients were tested by RT-PCR for SARS-CoV-2 diagnosis. Relative quantitation was made by the comparative threshold cycle (ΔΔCt ) formula between ORF1ab viral and RNase P housekeeping genes. Absolute viral load was calculate using a reference positive control. Most prevalent clinical signs were cough (75.8%), myalgia (66.7%), and fever (48.5%). Hypertension (18.2%), neurological diseases (15.1%), and asthma and hypothyroidism (12.1%) were most frequent comorbidities. Fever, either as an exclusive symptom or combined with others, was associated with high viral loads ( 2-∆∆Ct range, 35.65-155.16; 4.25-4.89 log10 RNA copies/test]). During the first week after onset of symptoms in mild patients up to 60 years-old was detected the peak of viral load. Children under 10 years old have a high viral load (313.84; 2.50) in the first 2 days postinfection with a sharp decline thereafter. Cases between 10 and 49 years old mostly showed low and moderate viral load during the first 2 days postinfection (range, 0.03 to 17.24; -1.50 to 1.24). Patients over 60 years old have high viral load up to the second week after the onset of symptoms (range, 25.32-155.42; 1.40-2.19), indicating the longer presence of the virus in them. These findings suggest the viral load in nasopharyngeal swabs would help to monitor the SARS-CoV-2 infection in mild coronavirus disease 2019 cases.

Phylogenetic analysis of the first four SARS-CoV-2 cases in Chile.

The current pandemic caused by the new coronavirus is a worldwide public health concern. To aboard this emergency, and like never before, scientific groups around the world have been working in a fast and coordinated way to get the maximum of information about this virus when it has been almost 3 months since the first cases were detected in Wuhan province in China. The complete genome sequences of around 450 isolates are available, and studies about similarities and differences among them and with the close related viruses that caused similar epidemics in this century. In this work, we studied the complete genome of the first four cases of the new coronavirus disease in Chile, from patients who traveled to Europe and Southeast Asia. Our findings reveal at least two different viral variants entries to Chilean territory, coming from Europe and Asia. We also sub-classified the isolates into variants according to punctual mutations in the genome. Our work contributes to global information about transmission dynamics and the importance to take control measures to stop the spread of the infection.

Identification of orally-bioavailable antagonists of the TRPV4 ion-channel.

Antagonists of the TRPV4 receptor were identified using a focused screen, followed by a limited optimization program. The leading compounds obtained from this exercise, RN-1665 23 and RN-9893 26, showed moderate oral bioavailability when dosed to rats. The lead molecule, RN-9893 26, inhibited human, rat and murine variants of TRPV4, and showed excellent selectivity over related TRP receptors, such as TRPV1, TRPV3 and TRPM8. The overall profile for RN-9893 may permit its use as a proof-of-concept probe for in vivo applications.

Epidemiological and clinical differences of confirmed and discarded Mpox cases on the 2022 Chilean outbreak.

Objectives: To improve Mpox diagnosis by identifying distinctive symptoms in confirmed vs discarded cases due to outdated case definition. Methods: This is a case-control study conducted using data from the Institute of Public Health database, encompassing all suspected cases analyzed by real-time polymerase chain reaction between June 1 and September 30, 2022. We calculated means, frequencies, performed Fisher's test, and computed odds ratios (OR) using RStudio and Microsoft Excel. Results: Among 1415 suspected Mpox cases, 87% were men aged 30-39 with exanthema. Confirmed cases had higher rates of lymphadenopathy (31% vs 12%), fever (42% vs 29%), myalgia (35% vs 25%), and specific lesions: pustules (36% vs 27%), scabs (25% vs 17%), and umbilicated lesions (24% vs 7%) (P <0.05). Key risk factors for Mpox included contact with a positive case (OR 2.33), multiple sexual partners (OR 3.52), and male gender (OR 29.93). Conclusion: The symptomatology of confirmed Mpox cases closely aligns with that reported in the current outbreak. The primary risk factors identified include prior contact with another positive case, having multiple sexual partners, and male gender. However, to facilitate a more complete analysis, more evidence needs to be investigated.

Geographical Distribution of Genetic Variants and Lineages of SARS-CoV-2 in Chile.

The pandemic caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a worldwide public health concern. First confined in China and then disseminated widely across Europe and America, SARS-CoV-2 has impacted and moved the scientific community around the world to working in a fast and coordinated way to collect all possible information about this virus and generate new strategies and protocols to try to stop the infection. During March 2020, more than 16,000 full viral genomes have been shared in public databases that allow the construction of genetic landscapes for tracking and monitoring the viral advances over time and study the genomic variations present in geographic regions. In this work, we present the occurrence of genetic variants and lineages of SARS-CoV-2 in Chile during March to April 2020. Complete genome analysis of 141 viral samples from different regions of Chile revealed a predominance of variant D614G like in Europe and the USA and the major presence of lineage B.1. These findings could help take control measures due to the similarity of the viral variants present in Chile, compared with other countries, and monitor the dynamic change of virus variants in the country.

Identification and characterization of novel TRPV4 modulators.

TRPV4, a close relative of the vanilloid receptor TRPV1, is activated by diverse modalities such as endogenous lipid ligands, hypotonicity, protein kinases and, possibly, mechanical inputs. While its multiple roles in vivo are being explored with KO mice and selective agonists, there is a dearth of selective antagonists available to examine TRPV4 function. Herein we detail the use of a focused library of commercial compounds in order to identify RN-1747 and RN-1734, a pair of structurally related small molecules endowed with TRPV4 agonist and antagonist properties, respectively. Their activities against human, rat and mouse TRPV4 were characterized using electrophysiology and intracellular calcium influx. Significantly, antagonist RN-1734 was observed to completely inhibit both ligand- and hypotonicity-activated TRPV4. In addition, RN-1734 was found to be selective for TRPV4 in a TRP selectivity panel including TRPV1, TRPV3 and TRPM8, and could thus be a valuable pharmacological probe for TRPV4 studies.

Residues of the human nuclear vitamin D receptor that form hydrogen bonding interactions with the three hydroxyl groups of 1alpha,25-dihydroxyvitamin D3.

Most of the biological effects of 1,25-dihydroxyvitamin D(3) (hormone D) are mediated through the nuclear vitamin D receptor (VDR). Hormone binding induces conformational changes in VDR that enable the receptor to activate gene transcription. It is known that residues S237 and R274 form hydrogen bonds with the 1-hydroxyl group of hormone D, while residues Y143 and S278, and residues H305 and H397 form hydrogen bonds with the 3-hydroxyl and the 25-hydroxyl groups of the hormone. A series of VDR mutations were constructed (S237A, R274A, R274Q, Y143F, Y143A, S278A, H305A, and H397F; double mutants: S237A/R274A, Y143F/S278A, Y143A/S278A, and H305A/H397F). The relative binding affinities of the wild-type and variant VDRs were assessed. All of the mutants except H397F resulted in lower binding affinity compared to wild-type VDR. Binding to hormone was barely detectable in Y143F, H305A, and H305A/H397F mutants, and undetectable in mutants R274A, R274Q, Y143A, S237A/R274A, and Y143A/S278A, indicating the importance of these residues. Ability to activate gene transcription was also assessed. All of the VDR mutants, except the single mutant S278A, required higher doses of hormone D for half-maximal response. Defining the role of hormone D-VDR binding will lead to a better understanding of the vitamin D signal transduction pathway.

Estaciones de aprendizaje para abordar concepciones de fisiología y morfología sobre el concepto de célula / Learning stations to analyze physiology and morphology perceptions of the cell concept

RESUMEN: El objetivo del presente estudio fue determinar la influencia de la implementación y aplicación de la metodología de enseñanza estaciones de aprendizaje, sobre un aprendizaje activo de la fisiología y morfología de la célula. Se trabajó con 90 estudiantes de enseñanza secundaria, distribuidos en un grupo control y otro experimental, los cuales recibieron clases tradicionales y la aplicación de la metodología estaciones de aprendizaje, respectivamente. Los datos fueron recolectados en base a test de concepciones y conocimientos, más encuesta de valoración. El análisis de los datos reveló que las estaciones de aprendizaje son una metodología de enseñanza efectiva en el aprendizaje de la célula, ya que permiten mejorar significativamente las concepciones previas de los estudiantes, los cuales valoran de manera positiva y significativa la metodología de la que fueron partícipes.

SUMMARY: The aim of this study was to determine the influence of the implementation and application of the learning stations teaching methodology on active learning of the physiology and morphology of the cell. We worked with 90 high school students, distributed in a control group and an experimental group, who received traditional classes and the application of the learning stations methodology, respectively. The data were collected based on a conception and knowledge test, plus an evaluation survey. The analysis of the data revealed that the learning stations are an effective teaching methodology in learning of the cell, since they allow to improve prior perceptions of the students, who significantly and positively value the methodology in which they participated.

Effect of 25-hydroxyl group orientation on biological activity and binding to the 1alpha,25-dihydroxy vitamin D3 receptor.

The hormonal form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1,25D), generates many biological actions by interactions with its nuclear receptor (VDR). The presence of a carbon-25 hydroxyl group is necessary for optimizing binding to the VDR. To examine the effect of spatial orientation of the 25-hydroxyl, two pairs of 22,23-allene sidechain analogs were studied. The 22R orientation in analogs HR (52+/-2%) and LA (154+/-19%) resulted in higher affinity binding than the 22S orientation of analogs HQ (21+/-3%) and LB (3.5+/-1.3%; 1,25D=100%). Limited trypsin proteolysis showed that 22R analogs induced VDR conformational changes better able to protect VDR from digestion than 22S analogs. 22R analogs were also able to induce gene transcription at 10-100-fold lower concentrations than 1,25D; 22S analogs were less effective. Analog LA was at least 10-fold more potent than 1,25D at inducing differentiation, while the other analogs were less potent. None of the analogs were as potent as 1,25D in promoting in vivo intestinal calcium absorption or bone calcium mobilization. LA was the most potent of the analogs but required 20-30-fold higher doses than 1,25D. The 25-hydroxyl orientation combined with the 16,17-ene functionality of analog LA enhances its ability to interact with VDR and induce biological actions.

Role of residues 143 and 278 of the human nuclear Vitamin D receptor in the full-length and Delta165-215 deletion mutant.

Most of the actions of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] are mediated by binding to the Vitamin D nuclear receptor (VDR). The crystal structure of a deletion mutant (Delta165-215) of the VDR ligand-binding domain (LBD) bound to 1,25(OH)(2)D(3) indicates that amino acid residues tyrosine-143 and serine-278 form hydrogen bonding interactions with the 3-hydroxyl group of 1,25(OH)(2)D(3). Studies of VDR and three mutants (Y143F, S278A, and Y143F/S278A) did not indicate any differences in the binding affinity between the variant receptors and the wild-type receptor. This might indicate that the 3-hydroxyl group binds differently to the full-length VDR than the to deletion mutant. To further investigate, four deletion VDR mutants were constructed: VDR(Delta165-215), VDR(Delta165-215) (Y143F), VDR(Delta165-215) (S278A), VDR(Delta165-215) (Y143F/S278A). There were no significant differences in binding affinity between the wild-type receptor and the deletion mutants except for VDR(Delta165-215) (Y143F/S278A). In gene activation assays, VDR constructs with the single mutation Y143F and the double mutation Y143F/S278A, but not the single mutation S278A required higher doses of 1,25(OH)(2)D(3) for half-maximal response. This suggests that there are some minor structural and functional differences between the wild-type VDR and the Delta165-215 deletion mutant and that Y143 residue is more important for receptor function than residue S278.

Aplasia medular asociada a hepatitis: reporte de un caso / Hepatitis associated aplastic anemia

La aplasia medular es una enfermedad infrecuente caracterizada por pancitopenia en contexto de una médula ósea hipocelular. La aplasia medular asociada a hepatitis (HAAA) se considera una variante de la aplasia medular severa que se desarrolla luego de un episodio agudo de hepatitis. Se presenta con mayor frecuencia en adolescentes y adultos jóvenes de sexo masculino y tiene peor pronóstico que la aplasia medular por otras causas. La hepatitis es una enfermedad frecuente en la población pediátrica, sin embargo la HAAAcorresponde a una complicación extraña y grave, por lo que es fundamental para el médico general y especialista, sospechar esta asociación e iniciar una estrecha vigilancia y tratamiento. Se expone el caso de un adolescente de 13 años que es derivado hacia el Hospital Dr. Gustavo Fricke por cuadro de hepatitis, el que posteriormente desarrolla pancitopenia franca de rápida instalación.

Aplastic anemia is an infrequent disease characterized by pancytopenia with hypocellular bone marrow. Hepatitis associated aplastic anemia (HAAA) is considered to be a variant of severe aplastic anemia following an episode of acute hepatitis. It is more commonly seen in young adult or adolescent males and its prognosis is poorer than that of other aplastic anemias. Hepatitis is common in the pediatric population, however hepatitis associated aplastic anemia is a strange and serious complication and it is of utmost importance that the general doctor and the specialist suspect the association in order to monitor patients closely and treat opportunely. We present the case of a 13 year old adolescent with hepatitis referred to Hospital Gustavo Fricke who afterwards presented sudden serious pancytopenia.

Producción y evaluación in vitro de IgY contra Streptococcus mutans / In vitro production and evaluation of anti-Streptococcus mutans IgY

La caries dental es una enfermedad infecciosa de alcance mundial, producida por Streptococcus mutans. Una estrategia para combatir la bacteria es previniendo su adherencia al esmalte dental mediante el uso de inmunoglobulinas de yema de huevo (IgY). En este trabajo se desarrollaron IgY contra S. mutans y se determinó su reactividad a fin de evaluar su potencial para prevenir la caries dental. Los anticuerpos fueron producidos inmunizando gallinas con un liófilo de la bacteria. Se recolectaron los huevos pre y post inmunes y se purificaron las IgY por precipitación con PEG 6000-cloroformo. El mayor nivel de IgY se obtuvo en el día 42 postinmunización, medido por un ensayo ELISA y la reactividad se evaluó por Western Blot, observándose el reconocimiento de bandas específicas entre 41 y 150 kDa que corresponden a proteínas implicadas en la adherencia a la superficie dental. Por la prueba MABA, se observó reactividad cruzada con S. salivarius. Observamos la aglutinación e inhibición del crecimiento (MIC) de S. mutans in vitro por acción de las IgY. Los resultados demuestran el potencial de estos anticuerpos para ser aplicados en ensayos de inmunización pasiva en nuestro país como una alternativa para prevenir la caries dental.

Dental caries is a worldwide infectious disease produce by Streptococcus mutants. A strategy to combat this bacterium consists in preventing its adherence to tooth enamel through the use egg yolk immunoglobulin (IgY). In this study we developed anti-S.mutans IgY and determined its reactivity to evaluate its potential for preventing dental caries. The antibodies were produced by immunizing hens with lyophilized bacteria. Pre and post immunization eggs were collected and their IgY was purified by precipitation with PEG 6000-chloroform. The highest IgY level was obtained on the 42nd day post-immunization, as measured by an ELISA test. Reactivity was determined by Western Blot, showing the recognition of specific bands between 41 and 150 kDa, which correspond to proteins involved in the adherence to dental surfaces. A MABA test showed cross-reactivity with S. salivarum. IgY activity was shown by in vitro agglutination and growth inhibition (MIC) of S. mutans. These results show the potential of these antibodies for application in passive immunization trials as an alternative to prevent dental caries.