An Open One-Step RT-qPCR for SARS-CoV-2 detection.

The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, demonstrating sensitivity comparable to a commercial kit routinely employed in clinical settings for patient diagnosis. Further evaluation on 40 clinical samples (20 positive and 20 negative) confirmed its comparable diagnostic accuracy. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.

An Open One-Step RT-qPCR for SARS-CoV-2 detection.

The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, and the kit showed comparable sensitivity to approved commercial kits. The One-Step RT-qPCR was then tested on clinical samples and demonstrated similar performance to commercial kits in terms of positive and negative calls. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.

Antarctic Polyester Hydrolases Degrade Aliphatic and Aromatic Polyesters at Moderate Temperatures.

Polyethylene terephthalate (PET) is one of the most widely used synthetic plastics in the packaging industry, and consequently has become one of the main components of plastic waste found in the environment. However, several microorganisms have been described to encode enzymes that catalyze the depolymerization of PET. While most known PET hydrolases are thermophilic and require reaction temperatures between 60°C and 70°C for an efficient hydrolysis of PET, a partial hydrolysis of amorphous PET at lower temperatures by the polyester hydrolase IsPETase from the mesophilic bacterium Ideonella sakaiensis has also been reported. We show that polyester hydrolases from the Antarctic bacteria Moraxella sp. strain TA144 (Mors1) and Oleispira antarctica RB-8 (OaCut) were able to hydrolyze the aliphatic polyester polycaprolactone as well as the aromatic polyester PET at a reaction temperature of 25°C. Mors1 caused a weight loss of amorphous PET films and thus constitutes a PET-degrading psychrophilic enzyme. Comparative modeling of Mors1 showed that the amino acid composition of its active site resembled both thermophilic and mesophilic PET hydrolases. Lastly, bioinformatic analysis of Antarctic metagenomic samples demonstrated that members of the Moraxellaceae family carry candidate genes coding for further potential psychrophilic PET hydrolases. IMPORTANCE A myriad of consumer products contains polyethylene terephthalate (PET), a plastic that has accumulated as waste in the environment due to its long-term stability and poor waste management. One promising solution is the enzymatic biodegradation of PET, with most known enzymes only catalyzing this process at high temperatures. Here, we bioinformatically identified and biochemically characterized an enzyme from an Antarctic organism that degrades PET at 25°C with similar efficiency to the few PET-degrading enzymes active at moderate temperatures. Reasoning that Antarctica harbors other PET-degrading enzymes, we analyzed available data from Antarctic metagenomic samples and successfully identified other potential enzymes. Our findings contribute to increasing the repertoire of known PET-degrading enzymes that are currently being considered as biocatalysts for the biological recycling of plastic waste.

Homebrew reagents for low cost RT-LAMP.

RT-LAMP (reverse transcription - Loop-mediated isothermal amplification) has gained popularity for the detection of SARS-CoV-2. The high specificity, sensitivity, simple protocols and potential to deliver results without the use of expensive equipment has made it an attractive alternative to RT-PCR. However, the high cost per reaction, the centralized manufacturing of required reagents and their distribution under cold chain shipping limits RT-LAMP's applicability in low-income settings. The preparation of assays using homebrew enzymes and buffers has emerged worldwide as a response to these limitations and potential shortages. Here, we describe the production of Moloney murine leukemia virus (M-MLV) Reverse Transcriptase and BstLF DNA polymerase for the local implementation of RT-LAMP reactions at low cost. These reagents compared favorably to commercial kits and optimum concentrations were defined in order to reduce time to threshold, increase ON/OFF range and minimize enzyme quantities per reaction. As a validation, we tested the performance of these reagents in the detection of SARS-CoV-2 from RNA extracted from clinical nasopharyngeal samples, obtaining high agreement between RT-LAMP and RT-PCR clinical results. The in-house preparation of these reactions results in an order of magnitude reduction in costs, and thus we provide protocols and DNA to enable the replication of these tests at other locations. These results contribute to the global effort of developing open and low cost diagnostics that enable technological autonomy and distributed capacities in viral surveillance.

Homebrew reagents for low-cost RT-LAMP.

Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) has gained popularity for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The high specificity, sensitivity, simple protocols, and potential to deliver results without the use of expensive equipment has made it an attractive alternative to RT-PCR. However, the high cost per reaction, the centralized manufacturing of required reagents, and their distribution under cold chain shipping limit RT-LAMP's applicability in low-income settings. The preparation of assays using homebrew enzymes and buffers has emerged worldwide as a response to these limitations and potential shortages. Here, we describe the production of Moloney murine leukemia virus reverse transcriptase and BstLF DNA polymerase for the local implementation of RT-LAMP reactions at low cost. These reagents compared favorably to commercial kits, and optimum concentrations were defined in order to reduce time to threshold, increase ON/OFF range, and minimize enzyme quantities per reaction. As a validation, we tested the performance of these reagents in the detection of SARS-CoV-2 from RNA extracted from clinical nasopharyngeal samples, obtaining high agreement between RT-LAMP and RT-PCR clinical results. The in-house preparation of these reactions results in an order of magnitude reduction in costs; thus, we provide protocols and DNA to enable the replication of these tests at other locations. These results contribute to the global effort of developing open and low-cost diagnostics that enable technological autonomy and distributed capacities in viral surveillance.

Consideraciones clínicas para fonoaudiólogos en el tratamiento de personas con COVID-19 y traqueostomía. Parte I: Deglución / Clinical considerations for speech-language pathologists in the treatment of people with COVID-19 and tracheostomy. Part I: Swallowing

A la fecha de redacción de este artículo, más de 500 mil personas han sido afectadas por el virus SARS-CoV-2 en Chile, manifestando diferentes grados de la enfermedad COVID-19. Aquellas que sobrellevan condiciones más severas generan una condición que requiere soporte ventilatorio invasivo y tratamiento en unidades de cuidados intensivos, que de prolongarse en el tiempo deriva en la necesidad de una traqueostomía. A pesar de los beneficios que posee esta en la recuperación de personas con dificultades respiratorias, su implementación se asocia a alteraciones deglutorias que se suman a las generadas por COVID-19. Condición que supone un desafío para los/as fonoaudiólogos/as, quienes están expuestos/as al virus debido a su proceder en estructuras del tracto aerodigestivo y la realización de procedimientos potencialmente generadores de aerosol. El objetivo de este artículo es entregar orientaciones y herramientas clínicas para la intervención en la deglución de personas con traqueostomía y COVID-19. Estas emanan de un análisis pragmático de la evidencia disponible a la fecha, interpretadas bajo nuestra experiencia de atender a más de 561 personas con dicha condición. Se espera contribuir a la rehabilitación de la deglución en personas con COVID-19 y traqueostomía. Para ello se expone sobre las características de la deglución en esta población, su tratamiento, consideraciones para el uso de técnicas específicas, y orientaciones para la mejora de la calidad de vida mediante la mantención y/o recuperación de la funcionalidad deglutoria. Siempre bajo un esquema centrado en el cuidado y protección de las personas hospitalizadas y el equipo de salud.

At the time of writing this article, more than a million people have been affected by the SARS-CoV-2 virus in Chile, displaying different degrees of COVID-19 disease. Severe infections generate a condition that requires invasive ventilatory support and treatment in intensive care units, which, when extended in time, makes necessary conducting a tracheostomy. Despite its benefits for the recovery of patients with respiratory difficulties, it is linked to swallowing disorders that add to the problems generated by COVID-19. This represents a challenge for speech pathologists, who are potentially exposed to the virus because they work on structures of the aerodigestive tract and becuase they conduct procedures that may be aerosol-generating. The aim of this article is to provide guidance and clinical tools for swallowing-intervention in people with tracheostomies and COVID-19. Thees tools spring from a pragmatic analysis of the currently available evidence , interpreted based on our experience of caring more than561 infected patients. We hope to contribute to the rehabilitation of swallowing of patients with COVID-19 and a tracheostomy. The characteristics of swallowing in this population, its treatment, considerations for the use of specific techniques, and guidelines for improving the quality of life through the maintenance and/or recovery of swallowing functionality are discussed, focused caring and protecting hospitalized patients and the health team.

Consideraciones clínicas para fonoaudiólogos en el tratamiento de personas con COVID-19 y traqueostomía. Parte II: Mejorando la fonación para facilitar la comunicación / Clinical considerations for speech-language pathologists in the treatment of people with COVID-19 and tracheostomy. Part II: Improving phonation to facilitate communicatio

La enfermedad COVID-19 fue declarada pandemia por la Organización Mundial de la Salud. Su presentación más severa genera una condición que requiere tratamiento en unidades de cuidados intensivos, condición que al prolongarse en el tiempo requiere la implementación de una traqueostomía para facilitar la entrega de soporte ventilatorio invasivo. Si bien este dispositivo posee importantes ventajas que favorecen la recuperación y rehabilitación, también es cierto que genera diversas complicaciones en la comunicación de las personas, condición que se suma a los efectos propios del COVID-19 y la frecuente historia de intubación endotraqueal previa. El objetivo de este artículo es proveer orientaciones y herramientas clínicas para el tratamiento de la fonación para la comunicación en personas con traqueostomía y COVID-19. Se considera para ello las recomendaciones de la literatura existentes a la fecha, bajo un análisis pragmático y basado en nuestra experiencia de atender a más de 561 personas con esta condición. Se exponen las características de la comunicación en esta población, su tratamiento, consideraciones para el uso de técnicas específicas y orientaciones para la mejora de la calidad de vida. Siempre con un enfoque orientado al cuidado y protección de las/os usuarias/os y el equipo de salud, en particular fonoaudiólogas y fonoaudiólogos del país.

The COVID-19 disease was declared a pandemic by the World Health Organization. When most severe, it generates a condition that requires treatment in intensive care units, which, when extended in time, requires implementing of a tracheostomy to facilitate invasive ventilatory support. Although ventilatory support has important advantages that favor recovery and rehabilitation, it generates various complications for patients' communication, a condition that adds to the effects of COVID-19 and the frequent history of previous endotracheal intubation. The aim of this article is to provide guidance and clinical tools for the treatment of phonation to facilitate communication in people with tracheostomy and COVID-19. For this, the recommendations of the existing available literature are considered, under a pragmatic analysis and based on our experience of treating more than 561 infected patients. The characteristics of communication in this population, its treatment, considerations for the use of specific techniques and guidelines to improve quality of life are exposed. Always with an approach oriented to the care and protection of users and the health team, in particular speech-language pathologists in the country.

Egresos hospitalarios por cáncer urogenital en Chile: cuarta parte / Hospital discharges because of genitourinary cancer in Chile: fourth part

RESUMEN El objetivo del presente trabajo es actualizar el análisis de los egresos hospitalarios (EH) por cáncer genitourinario (CGU), específicamente cáncer de próstata, testículo, vejiga y riñón en nuestro país. Para estos efectos se obtuvieron os datos del Boletín de Egresos Hospitalarios del Ministerio de Salud (MINSAL) del año 2010 y 2015, utilizándose los códigos de la clasificación internacional de enfermedades de la Organización Mundial de la Salud (OMS CIE-10). Los distintos diagnósticos fueron caracterizados según su composición geográfica y demográfica, comparándose con la información publicada en los reportes anteriores.(AU)

ABSTRACT The aim of the study is to update the analysis of hospital discharges for genitourinary cancer, specifically prostatic, testicular, bladder and renal cancers in our country. For this purpose, data was obtained from the registers of the Chilean Ministry of Health (MINSAL) for the years 2010 and 2015, using the international classification codes of diseases from the World Health Organization (WHO ICD-10). The different diagnoses were characterized according to their geographic and demographic distribution, comparing them to the information published in the previous reports.(AU)

Model of the Ankyrin and SOCS Box Protein, ASB9, E3 Ligase Reveals a Mechanism for Dynamic Ubiquitin Transfer.

Cullin-RING E3 ligases (CRLs) are elongated and bowed protein complexes that transfer ubiquitin over 60 Å to proteins targeted for proteasome degradation. One such CRL contains the ankyrin repeat and SOCS box protein 9 (ASB9), which binds to and partially inhibits creatine kinase (CK). While current models for the ASB9-CK complex contain some known interface residues, the overall structure and precise interface of the ASB9-CK complex remains unknown. Through an integrative modeling approach, we report a third-generation model that reveals precisely the interface interactions and also fits the shape of the ASB9-CK complex as determined by small-angle X-ray scattering. We constructed an atomic model for the entire CK-targeting CRL to uncover dominant modes of motion that could permit ubiquitin transfer. Remarkably, only the correctly docked CK-containing E3 ligase and not incorrectly docked structures permitted close approach of ubiquitin to the CK substrate.

Three-Dimensional Domain Swapping Changes the Folding Mechanism of the Forkhead Domain of FoxP1.

The forkhead family of transcription factors (Fox) controls gene transcription during key processes such as regulation of metabolism, embryogenesis, and immunity. Structurally, Fox proteins feature a conserved DNA-binding domain known as forkhead. Interestingly, solved forkhead structures of members from the P subfamily (FoxP) show that they can oligomerize by three-dimensional domain swapping, whereby structural elements are exchanged between adjacent subunits, leading to an intertwined dimer. Recent evidence has largely stressed the biological relevance of domain swapping in FoxP, as several disease-causing mutations have been related to impairment of this process. Here, we explore the equilibrium folding and binding mechanism of the forkhead domain of wild-type FoxP1, and of two mutants that hinder DNA-binding (R53H) and domain swapping (A39P), using size-exclusion chromatography, circular dichroism, and hydrogen-deuterium exchange mass spectrometry. Our results show that domain swapping of FoxP1 occurs at micromolar protein concentrations within hours of incubation and is energetically favored, in contrast to classical domain-swapping proteins. Also, DNA-binding mutations do not significantly affect domain swapping. Remarkably, equilibrium unfolding of dimeric FoxP1 follows a three-state N2 ↔ 2I ↔ 2U folding mechanism in which dimer dissociation into a monomeric intermediate precedes protein unfolding, in contrast to the typical two-state model described for most domain-swapping proteins, whereas the A39P mutant follows a two-state N ↔ U folding mechanism consistent with the second transition observed for dimeric FoxP1. Also, the free-energy change of the N ↔ U in A39P FoxP1 is âˆ¼2 kcal⋅mol(-1) larger than the I ↔ U transition of both wild-type and R53H FoxP1. Finally, hydrogen-deuterium exchange mass spectrometry reveals that the intermediate strongly resembles the native state. Our results suggest that domain swapping in FoxP1 is at least partially linked to monomer folding stability and follows an unusual three-state folding mechanism, which might proceed via transient structural changes rather than requiring complete protein unfolding as do most domain-swapping proteins.