A naturally occurring G11S mutation in the 3C-like protease from the SARS-CoV-2 virus dramatically weakens the dimer interface.

The 3C-like protease (3CLpro ) is crucial to the replication of SARS-CoV-2, the causative agent of COVID-19, and is the target of several successful drugs including Paxlovid and Xocova. Nevertheless, the emergence of viral resistance underlines the need for alternative drug strategies. 3CLpro only functions as a homodimer, making the protein-protein interface an attractive drug target. Dimerization is partly mediated by a conserved glycine at position 11. However, some naturally occurring SARS-CoV-2 sequences contain a serine at this position, potentially disrupting the dimer. We have used concentration-dependent activity assays and mass spectrometry to show that indeed the G11S mutation reduces the stability of the dimer by 600-fold. This helps to set a quantitative benchmark for the minimum potency required of any future protein-protein interaction inhibitors targeting 3CLpro and raises interesting questions regarding how coronaviruses bearing such weakly dimerizing 3CLpro enzymes are capable of replication.

Cysteine-Mediated Green Synthesis of Copper Sulphide Nanoparticles: Biocompatibility Studies and Characterization as Counter Electrodes.

A one-pot green method for aqueous synthesis of fluorescent copper sulphide nanoparticles (NPs) was developed. The reaction was carried out in borax-citrate buffer at physiological pH, 37 °C, aerobic conditions and using Cu (II) and the biological thiol cysteine. NPs exhibit green fluorescence with a peak at 520 nm when excited at 410 nm and an absorbance peak at 410 nm. A size between 8-12 nm was determined by dynamic light scattering and transmission electron microscopy. An interplanar atomic distance of (3.5 ± 0.1) Å and a hexagonal chalcocite crystalline structure (ßCh) of Cu2S NPs were also determined (HR-TEM). Furthermore, FTIR analyses revealed a Cu-S bond and the presence of organic molecules on NPs. Regarding toxicity, fluorescent Cu2S NPs display high biocompatibility when tested in cell lines and bacterial strains. Electrocatalytic activity of Cu2S NPs as counter electrodes was evaluated, and the best value of charge transfer resistance (Rct) was obtained with FTO/Cu2S (four layers). Consequently, the performance of biomimetic Cu2S NPs as counter electrodes in photovoltaic devices constructed using different sensitizers (ruthenium dye or CdTe NPs) and electrolytes (S2-/Sn2- or I-/I3-) was successfully checked. Altogether, novel characteristics of copper sulfide NPs such as green, simple, and inexpensive production, spectroscopic properties, high biocompatibility, and particularly their electrochemical performance, validate its use in different biotechnological applications.

Carbohydrate esterase family 16 contains fungal hemicellulose acetyl esterases (HAEs) with varying specificity.

Acetyl esterases are an important component of the enzymatic machinery fungi use to degrade plant biomass and are classified in several Carbohydrate Esterase families of the CAZy classification system. Carbohydrate Esterase family 16 (CE16) is one of the more recently discovered CAZy families, but only a small number of its enzyme members have been characterized so far, revealing activity on xylan-derived oligosaccharides, as well as activity related to galactoglucomannan. The number of CE16 genes differs significantly in the genomes of filamentous fungi. In this study, four CE16 members were identified in the genome of Aspergillus niger NRRL3 and it was shown that they belong to three of the four phylogenetic Clades of CE16. Significant differences in expression profiles of the genes and substrate specificity of the enzymes were revealed, demonstrating the diversity within this family of enzymes. Detailed characterization of one of these four A. niger enzymes (HaeA) demonstrated activity on oligosaccharides obtained from acetylated glucuronoxylan, galactoglucomannan and xyloglucan, thus establishing this enzyme as a general hemicellulose acetyl esterase. Their broad substrate specificity makes these enzymes highly interesting for biotechnological applications in which deacetylation of polysaccharides is required.

Design, synthesis and in vitro evaluation of novel SARS-CoV-2 3CLpro covalent inhibitors.

Severe diseases such as the ongoing COVID-19 pandemic, as well as the previous SARS and MERS outbreaks, are the result of coronavirus infections and have demonstrated the urgent need for antiviral drugs to combat these deadly viruses. Due to its essential role in viral replication and function, 3CLpro (main coronaviruses cysteine-protease) has been identified as a promising target for the development of antiviral drugs. Previously reported SARS-CoV 3CLpro non-covalent inhibitors were used as a starting point for the development of covalent inhibitors of SARS-CoV-2 3CLpro. We report herein our efforts in the design and synthesis of submicromolar covalent inhibitors when the enzymatic activity of the viral protease was used as a screening platform.

Descripción histológica del aparato reproductor de avestruz hembra (Strutio camelus var. domesticus) / Histological description of the ostrich female reproductive system (Strutio camelus var. domesticus)

El auge de la producción intensiva del avestruz, comenzó en la década de los noventa impulsada por la calidad de su carne y potencialidad de sus subproductos. La raza empleada para producción por la calidad nutricional y sabor de su carne es el híbrido llamado African black (Struthio camelus var. domesticus). En cuanto a la reproducción, el avestruz hembra alcanza su madurez sexual a partir de los 2,5 años. Es importante considerar el aparato genital en aves de producción, ya que una alteración en él, puede generar deficiencias en la fertilidad que se traducen en un menor número de crías. El estudio histológico del aparato reproductor de la hembra será una herramienta más que permitirá resolver problemas reproductivos. Para este análisis se obtuvo muestras de los diferentes segmentos del aparato reproductor de 6 avestruces hembras en edad reproductiva y se procesaron de acuerdo a las técnicas histológicas de rutina. Los cortes fueron observados, fotografiados y analizados bajo microscopio de luz. Obtenidas las fotografías, se analizó comparativamente su morfología con la descrita en la gallina (Gallus gallus). El aparato reproductor de la hembra tiene la particularidad de tener desarrollado solo el ovario y oviducto izquierdo. El ovario es de gran tamaño y en forma de racimo, el cual varía según la estacionalidad. Presenta folículos primordiales, previtelogénicos, vitelogénicos y atrésicos. Los folículos vitelogénicos presentan células de la granulosa y de la teca interna y externa. El oviducto presenta de cefálico a caudal los siguientes segmentos: infundíbulo, magnum, istmo, útero y vagina, que desemboca en la cloaca a nivel del urodeo. En ellos hay pliegues de variada longitud, grosor y número que comprometen la mucosa y submucosa, con glándulas de secreción mucosa y serosa a excepción de la vagina. El análisis histológico comparativo, permitió establecer que la morfología del aparato reproductor de la hembra es semejante a la observada en la gallina con ciertas diferencias microscópicas (Gallus gallus).

In the 1990's, ostrich production reached a peak in our country, boosted by the special characteristics of its meat and the potential of the derivatives. The breed raised is a hybrid called African Black (Struthio camelus var. domesticus) which has a high quality meat in terms of nutritional factors and flavor. With regard to reproduction, the female ostrich reaches maturity at the age of 2.5 years. Genital organs are very important in fowl's production, because they can generate fertility deficiencies that, in turn can diminish brood number. Histological analysis allows a better understanding of the basic structure of the female's genital organs and is a helpful tool to resolve breeding problems. For this analysis samples were obtained from the different segments of the reproductive system of 6 female ostrich in reproductive age. These samples were processed using standard histological technique. Sections were observed, photographed and analyzed under the light-microscope. Photographs were compared with hen's samples. The ostrich female's reproductive system has the particularity of having just the left ovary and oviduct developed. The ovary has a big size and a cluster shape which varies from season to season. It presents paramount, previtellogenic, vitellogenic and atresic follicles. The vitellogenic follicles have granulosa cells and inner and external theca. The oviduct presents cephalocaudally: infundibulum, magnum, isthmus, uterus and vagina, flowing into the urodeum. It shows long pleats of different length and number, with drusen of mucose and serose secretion, except in the vagina. The comparative histological analysis allowed us to establish that the basic structure of the female reproductive system is similar to that of the hen (Gallus gallus).

Microarray analysis of the Escherichia coli response to CdTe-GSH Quantum Dots: understanding the bacterial toxicity of semiconductor nanoparticles.

BACKGROUND: Most semiconductor nanoparticles used in biomedical applications are made of heavy metals and involve synthetic methods that require organic solvents and high temperatures. This issue makes the development of water-soluble nanoparticles with lower toxicity a major topic of interest. In a previous work our group described a biomimetic method for the aqueous synthesis of CdTe-GSH Quantum Dots (QDs) using biomolecules present in cells as reducing and stabilizing agents. This protocol produces nanoparticles with good fluorescent properties and less toxicity than those synthesized by regular chemical methods. Nevertheless, biomimetic CdTe-GSH nanoparticles still display some toxicity, so it is important to know in detail the effects of these semiconductor nanoparticles on cells, their levels of toxicity and the strategies that cells develop to overcome it. RESULTS: In this work, the response of E. coli exposed to different sized-CdTe-GSH QDs synthesized by a biomimetic protocol was evaluated through transcriptomic, biochemical, microbiological and genetic approaches. It was determined that: i) red QDs (5 nm) display higher toxicity than green (3 nm), ii) QDs mainly induce expression of genes involved with Cd+2 stress (zntA and znuA) and tellurium does not contribute significantly to QDs-mediated toxicity since cells incorporate low levels of Te, iii) red QDs also induce genes related to oxidative stress response and membrane proteins, iv) Cd2+ release is higher in red QDs, and v) QDs render the cells more sensitive to polymyxin B. CONCLUSION: Based on the results obtained in this work, a general model of CdTe-GSH QDs toxicity in E. coli is proposed. Results indicate that bacterial toxicity of QDs is mainly associated with cadmium release, oxidative stress and loss of membrane integrity. The higher toxicity of red QDs is most probably due to higher cadmium content and release from the nanoparticle as compared to green QDs. Moreover, QDs-treated cells become more sensitive to polymyxin B making these biomimetic QDs candidates for adjuvant therapies against bacterial infections.

[Leser-Trélat sign associated with gastric cancer: report of one case]. / Signo de Leser-Trélat asociado a adenocarcinoma gástrico: caso clínico.

We report a 66-year-old male presenting with malaise, heartburn and pruritic seborrheic keratoses in both feet of sudden onset, suggesting a Leser-Trélat sign. An upper gastrointestinal endoscopy disclosed a gastric cancer. The patient was subjected to a total gastrectomy and during follow up, the skin lesions had disappeared.

Análisis Macroscópico y Microscópico del Desarrollo Embrionario y Fetal en el Gato (Felis catus), en Relación con el Desarrollo de la Vesícula Coriónica y de la Placenta / Macroscopic and Microscopic Analysis of the Embryonic and Fetal Growth in the Cat (Felis catus), in Relation to Chorionic Vesicle and Placental Development

La gata doméstica (Felis catus) presenta una gestación que dura, en promedio 62 + 5 días. Sin embargo, establecer la edad gestacional en forma más o menos precisa resulta difícil, ya que no existe un análisis que correlacione el tamaño de la vesícula coriónica; desarrollo de la placenta; desarrollo embrionario y Fetal durante la gestación. En este trabajo se utilizaron 12 úteros grávidos, provenientes de gatas mestizas entre 8 y 18 meses de edad, éstos fueron fijados en formol neutro al 10%. Para cada útero se determinó el número de vesículas coriónicas, de cada una se removieron el embrión, feto y la placenta, los que fueron medidos, tarados y fotografiados para su análisis morfológico. Las etapas del desarrollo embrionario y Fetal fueron establecidas conforme a las características y estructuras externas de los embriones y fetos, desde el inicio hasta el término de la gestación. A los 13 días de gestación se observó una gástrula tardía. Embriones somíticos, entre los 13 y 18 días. Embriones prefetales, entre los 18 y 28 días, y fetos desde el día 28 hasta el nacimiento. El amnios se cierra a los 17 días; la formación de la cara y cuello ocurre entre los 16 y 28 días, y de los miembros, entre los 17 y 28 días de gestación. A los 15 días de gestación comienza el latido cardiaco, momento en que se observa el tubo endocárdico. El tabicamiento del corazón se produce entre los 17 y 20 días. El tubo neural está cerrado a los 17 días de gestación. Todos los parámetros estudiados en los diferentes estadios del desarrollo en el gato están significativamente correlacionados (p < 0.0001).

The domestic cat (Felis catus) presents a gestation that lasts 62 ± 5 days. However, gestational age determination in a more precise way is difficult, as there are no analysis that correlate chorionic vesicle size; placental development; embryonic and Fetal development during gestation. In this work 12 gravid uteruses from hybrid cats between 8 and 18 months of age were studied; these were fixed in 10% neutral formaldehyde. For each uterus the chorionic vesicles number was determined. The embryos, fetuses and placenta were removed, measured, weighed and photographed for morphologic analysis. The embryonic and Fetal stages of development were determined according to the external characteristics and structures of the embryos or fetuses, from gastrulation to term. At 13 days of gestation a late gastrula was observed; between days 13 and 18, somitic embryos; between days 18 and 28, prefetal embryos; and fetuses from day 28 until birth. The amnion is closed by day 17; differentiation of the facial region occurs between days 16 and 28, while limb development takes place between days 17 and 28. At 15 days of gestation the heart begins to beat and the endocardial tube is observed. Septation of the heart occurs between days 17 and 20 and the neural tube is closed by day 17. All parameters studied in the different developmental stages in the cat are significantly correlated (p < 0.0001).

Obtaiment of Pudu (Pudu pudu) deer embryos by the somatic nuclear transfer technique

RESUMEN: Los objetivos de este trabajo fueron los de producir embriones de pudú, obtenidos por la transferencia de núcleos de fibroblastos de la oreja de pudú en ovocitos de un rumiante domésticos que es el bovino. Para posteriormente en un trabajo futuro proceder a la transferencia de embriones de pudú, al útero de hembras receptoras sincronizadas de otra especie. Se obtuvieron biopsias de 1 mm aproximadamente del borde externo de la orejas de dos ciervos pudu machos del jardín zoológico Buin-Zoo, Santiago de Chile. Las líneas celulares han sido establecidas y conservadas según los protocolos utilizados para las bovinos. Los ovocitos son obtenidos por punción del complejo cúmulos-ovocito (COC).desde ovarios de vacas recuperados del matadero. Cada ovocito es enucleado y fusionado con un fibroblasto aislado insertado bajo la zona pelúcida. La fusión de membranas celulares es obtenida por choques eléctricos. En cuanto a la cronología, observamos que al segundo día se forma una etapa de dos blastómeras, al tercer día mórulas de 8 a 16 células, y desde el cuarto día se ha diferenciado como blastocisto, el cuál al séptimo día termina por eclosionar de la zona pelúcida.La obtención de blastocistos embrionarios indica que es posible obtener embriones de pudú mediante clonaje heteroespecífico, aunque, el porcentaje de éxito obtenido es relativamente bajo. Queda aun por verificar la viabilidad de los embriones así obtenidos después de la transferencia in útero.

Attempts at Applying Cloning to the Conservation of Species in Danger of Extinction / Intentos de Aplicar la Clonación en la Conservación de Especies en Riesgo de Extinción

The somatic cloning by transfer of the nuclei of differentiated adult cells to previously enucleated oocytes is a promising technique for the production of embryos of high genetic value. The better mastering of somatic cloning gives us the possibility to produce embryos from endangered species. The huemul is an Andean native deer, that has been declared an endangered species, it holds a great patrimonial value and it is a Chilean national emblem. In Chile the huemul has the status of protected animal on thirteen Parks and National Reserves managed by Corporacion Nacional Forestal (CONAF). This protection, however, is considered insufficient due to the few geographical connections between the different protected areas. Furthermore, a great proportion of these areas are not subjected to use or they do not constitute adequate habitats. Many authors have proposed that the use of biotechnological methods in reproduction and assisted procreation may help conservational programs orientated to the protection of deer species threatened by extinction. All the anterior prompted us to initiate this study concerning the production of cloned huemul embryos.

El clonaje somático por transferencia del núcleo de células diferenciadas adultas a un ovocito, al que se le ha extraído el núcleo (enucleado), es una técnica prometedora para la producción de embriones de alto valor genético. El mejor dominio del clonaje somático da la posibilidad de producir embriones de especies amenazadas de extinción. El huemul es un ciervo andino autóctono, declarado como especie en peligro de extinción. tiene un gran valor patrimonial, y es emblema de la nación chilena. En este país, el huemul se encuentra protegido en trece Parques y Reservas Nacionales, manejadas por la Corporación Nacional Forestal (CONAF). Sin embargo, su protección se considera insuficiente debido a la baja conectividad entre las áreas protegidas y además, una gran proporción de estas áreas no son utilizadas o no constituyen un hábitat adecuado. Para las especies de cérvidos en vías de extinción el uso de biotecnología reproductiva y métodos de procreación asistida, según varios autores, pueden ayudar a los programas de conservación. Las técnicas clásicas de producción de embriones basados en superovulación, inseminación artificial y transferencia embrionaria,en los cérvidos, han resultado muy dificultosa. Esto, sumado a las características del huemul, que no permite su estabulación en cautiverio, nos ha movido a iniciar un estudio tendiente a la producción de embriones clonados de esta especie.

Clonación, producción de quimeras y células pluripotenciales / Clonation, production of chimeras ans pluripotencial cells

La Biología del Desarrollo y la Biotecnología han avanzado enormemente en los últimos años. Los estudios que se están realizando actualmente con nuevas técnicas moleculares, nos conducen a una nueva era en el diagnóstico y en el tratamiento de muchas enfermedades congénitas. Actualmente, se ha logrado aplicar los conocimientos derivados de ella, con incipiente éxito, en los aspectos productivos y también en la terapia de distintas enfermedades del Hombre y de otros animales y, por último, existen importantes perspectivas en la protección de especies amenazadas. En este Review, se analizan algunos de los aspectos técnicos más interesantes de la clonación, producción de quimeras y obtención de células pluripotenciales, como también los objetivos y proyecciones en el campo veterinario y de la salud animal y humana.

Histology of the jaw deformation in salmon of southern Chile (salmo salar)

In adult salmon of the sea centres in southern Chile, a jaw deformation (JD) has been identified. It affects the dental and hyomandibular bones, which bend ventrally up to 90° of their normal position. The deformation affects also the dental articular bone. This pathology is related to weight loss and increased mortality of the salmons. It was empirically postulated that a probable cause for this anomaly was food from vegetal origin in the diet of the fishes (which are carnivores) Therefore, the present work aims at comparing the biostructure of jaw bone of salmons fed either with vegetal (soja and gluten) formulation or animal formulation, mostly fish powder. Fifty specimens were analyzed from Puerto Montt, 35 having JD and 15 normal control. Samples were obtained in June, July and September 1999. (group 1) and March, Sept and October, 2000 (group 2). Group 1 was fed mostly with vegetal flour and group 2 with fish flour. Each group was subdivided in two subgroups, one of healthy animals and the other of fishes with JD. Jaw and articular bones were fixed in 10 per cent formaldehyde and 1 per cent glutaraldehyde and processed for histology (hematoxylin.eosin, Alcian blue, Masson trichrome) histochemistry (Syrius red and von Kossa) and scanning electron microscopy (SEM). The mandibular bone of group 1 with JD presented large amounts of osteoid tissue compared with its control. Collagen I disminishes and its architecture and composition changes, collagen III increases. No significant difference was found in calcium content between normal and JD fishes. SEM shows that the dental bone close to the joint in the fishes with JD displayed a disorganized structure and no trabecular formation, compared to controls, In group 2, these pathological findings were less evident, both macro- and microscopically. Results suggest that JD is of multifactorial origin; the primary cause can be a genetic or congenital alteration of the Jaw cartilage. There should be susceptibility for presentation of the defect in this group of fishes, but its expression is triggered by deficit of phosphorous of animal origin in food, so that this pathology is not seen in fishes with adequate nutrition.