SARS-CoV-2 accessory protein 7b forms homotetramers in detergent

A global pandemic is underway caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 genome, like its predecessor SARS-CoV, contains open reading frames that encode accessory proteins involved in virus-host interactions active during infection and which likely contribute to pathogenesis. One of these accessory proteins is 7b, with only 44 (SARS-CoV) and 43 (SARS-CoV-2) residues. It has one predicted transmembrane domain fully conserved, which suggests a functional role, whereas most variability is contained in the predicted cytoplasmic C-terminus. In SARS-CoV, 7b protein is expressed in infected cells, and the transmembrane domain was necessary and sufficient for Golgi localization. Also, anti-p7b antibodies have been found in the sera of SARS-CoV convalescent patients. In the present study, we have investigated the hypothesis that SARS-2 7b protein forms oligomers with ion channel activity. We show that in both SARS viruses 7b is almost completely α-helical and has a single transmembrane domain. In SDS, 7b forms various oligomers, from monomers to tetramers, but only monomers when exposed to reductants. Combination of SDS gel electrophoresis and analytical ultracentrifugation (AUC) in both equilibrium and velocity modes suggests a dimer-tetramer equilibrium, but a monomer–dimer–tetramer equilibrium in the presence of reductant. This data suggests that although disulfide-linked dimers may be present, they are not essential to form tetramers. Inclusion of pentamers or higher oligomers in the SARS-2 7b model were detrimental to fit quality. Preliminary models of this association was generated with AlphaFold2, and two alternative models were exposed to a molecular dynamics simulation in presence of a model lipid membrane. However, neither of the two models provided any evident pathway for ions. To confirm this, SARS-2 p7b was studied using Planar Bilayer Electrophysiology. Addition of p7b to model membranes produced occasional membrane permeabilization, but this was not consistent with bona fide ion channels made of a tetrameric assembly of α-helices

In Vitro and Ectopic In Vivo Studies toward the Utilization of Rapidly Isolated Human Nasal Chondrocytes for Single-Stage Arthroscopic Cartilage Regeneration Therapy

Nasal chondrocytes (NCs) have a higher and more reproducible chondrogenic capacity than articular chondrocytes, and the engineered cartilage tissue they generate in vitro has been demonstrated to be safe in clinical applications. Here, we aimed at determining the feasibility for a single-stage application of NCs for cartilage regeneration under minimally invasive settings. In particular, we assessed whether NCs isolated using a short collagenase digestion protocol retain their potential to proliferate and chondro-differentiate within an injectable, swiftly cross-linked and matrix-metalloproteinase (MMP)-degradable polyethylene glycol (PEG) gel enriched with human platelet lysate (hPL). NC-hPL-PEG gels were additionally tested for their capacity to generate cartilage tissue in vivo and to integrate into cartilage/bone compartments of human osteochondral plugs upon ectopic subcutaneous implantation into nude mice. NCs isolated with a rapid protocol and embedded in PEG gels with hPL at low cell density were capable of efficiently proliferating and of generating tissue rich in glycosaminoglycans and collagen II. NC-hPL-PEG gels developed into hyaline-like cartilage tissues upon ectopic in vivo implantation and integrated with surrounding native cartilage and bone tissues. The delivery of NCs in PEG gels containing hPL is a feasible strategy for cartilage repair and now requires further validation in orthotopic in vivo models. Keywords: cartilage regeneration; autologous chondrocyte implantation; nasal chondrocytes; single-stage; arthroscopy; tissue engineering; polyethylene glycol; hydrogel; platelet lysat

Amnion Cells in Tailored Hydrogels Deposit Human Amnion Native Extracellular Matrix

Fetal therapies regularly result in iatrogenic preterm premature rupture of the fetal membranes (iPPROM), which is associated with preterm birth. Biomaterials that promote the healing of traumatized fetal membranes (FMs) may prevent iPPROM-associated preterm births, addressing this unmet clinical need. Here, a fully defined synthetic poly(ethylene glycol) (PEG) hydrogel is developed to study the healing functions of human amnion-derived mesenchymal stromal cells (hAMCs) in 3D cultures. A pipeline to analyze extracellular matrix (ECM) proteins deposited by hAMCs in PEG hydrogels is established involving label-free quantification of mass-spectrometry data. Owing to the contaminant-free PEG hydrogels and a short fetal bovine serum (FBS)-free culture period, 128 ECM proteins, of which 97 are present in the native amnion, are identified. Upon stimulation with platelet-derived growth factor BB (PDGF-BB), a cell proliferation and migration inducing factor, hAMCs remodel their surroundings and deposit ECM proteins pericellularly. Among the most abundantly deposited amnion proteins, transforming growth factor β-induced protein ig-h3 (TGFβi), a very distinctive amnion protein that is involved in the wound healing cascade, is identified. These data support the potential of PDGF-BB to promote the repair of traumatized FMs and encourage its use for the engineering of biomaterials for FM healing, to ultimately prevent iPPROM

Use of the barbed suture (V-loc ™) in the laparoscopic gastroyeyunal by-pass: experience in 354 intervened patients

Introduction: The barbed suture is a device developed in recent years to simplify the intracorporeal suture and improve safety in laparoscopic surgery. We describe our experience in the use of V-Loc ™ in Laparoscopic Roux-en-Y gastric bypass (LRYGB). This suture has been used for the closure of enterotomy after mechanical lateral J-J anastomosis (bile limb-alimentary limb), for the closure of the mesenteric defect and, occasionally, for the closure of the Petersen space. It is presented as an observational study from 2012 to 2019 with the results of 354 patients undergoing bypass. Objective: To evaluate the safety of this type of suture by demonstrating the absence of leakage, bleeding, stenosis or other complications associated with its use in bariatric laparoscopic surgery. Material and Methods: Between June 2012 and July 2019, a total of 746 bariatric surgeries were performed in our unit. Of all of them, 354 corresponded to bypass in which barbed suture (V-Loc ™ 3-0 6 ”15cm, non-absorbable Polybutester (PBT), Covidien ™) was used in different phases of surgery. The results of the series are analyzed retrospectively. Results: Of the 354 surgical procedures performed, only one case underwent urgent laparoscopic examination within the next 24 hours after surgery due to a defect in the closure of enterotomy in the J-J anastomosis. There were no short-term or longterm postoperative complications in the rest of the patients operated on. Conclusion: The use of V-Loc ™ is safe, effective and reproducible applied to bariatric surgery, especially LGYB

Entornos llenos de Matemáticas: el caso de la Sagrada Familia, en Barcelona

En general, las matemáticas están presentes en multitud de elementos de nuestro entorno, de una manera más o menos explícita. En el caso de edificios como la Sagrada Familia, hay elementos matemáticos evidentes y otros cuya interpretación parece reservada a un reducido grupo de personas eruditas. Con este taller el Grup Vilatzara quiere mostrar que todos tenemos la posibilidad de comprender (y admirar) los secretos guardados en piedra en este templo de Antoni Gaudí. Nos centraremos en cuatro aspectos: poliedros estrellados, simbolismo numérico, superficies regladas y diseño del entorno del templo. Las personas asistentes podrán construir algunos de estos elementos. A su vez, deseamos animar al profesorado a utilizar las muestras artísticas de todo tipo que se encuentren en su entorno para ayudar a desarrollar las competencias matemáticas de su alumnado. Observar el entrono con mirada matemática nos permite descubrir estos recursos para el aula

Impact of Novel Technologies on Virgin Olive Oil Processing, Consumer Acceptance, and the Valorization of Olive Mill Wastes

There is a growing consumer preference for high quality extra virgin olive oil (EVOO) with health‐promoting and sensory properties that are associated with a higher content of phenolic and volatile compounds. To meet this demand, several novel and emerging technologies are being under study to be applied in EVOO production. This review provides an update of the effect of emerging technologies (pulsed electric fields, high pressure, ultrasound, and microwave treatment), compared to traditional EVOO extraction, on yield, quality, and/or content of some minor compounds and bioactive components, including phenolic compounds, tocopherols, chlorophyll, and carotenoids. In addition, the consumer acceptability of EVOO is discussed. Finally, the application of these emerging technologies in the valorization of olive mill wastes, whose generation is of concern due to its environmental impact, is also addressed

Chromatin establishes an immature version of neuronal protocadherin selection during the naive-to-primed conversion of pluripotent stem cells.

In the mammalian genome, the clustered protocadherin (cPCDH) locus provides a paradigm for stochastic gene expression with the potential to generate a unique cPCDH combination in every neuron. Here we report a chromatin-based mechanism that emerges during the transition from the naive to the primed states of cell pluripotency and reduces, by orders of magnitude, the combinatorial potential in the human cPCDH locus. This mechanism selectively increases the frequency of stochastic selection of a small subset of cPCDH genes after neuronal differentiation in monolayers, 10-month-old cortical organoids and engrafted cells in the spinal cords of rats. Signs of these frequent selections can be observed in the brain throughout fetal development and disappear after birth, except in conditions of delayed maturation such as Down's syndrome. We therefore propose that a pattern of limited cPCDH-gene expression diversity is maintained while human neurons still retain fetal-like levels of maturation

Fast axonal transport of the proteasome complex depends on membrane interaction and molecular motor function

Protein degradation by the ubiquitin-proteasome system in neurons depends on the correct delivery of the proteasome complex. In neurodegenerative diseases, aggregation and accumulation of proteins in axons link transport defects with degradation impairments; however, the transport properties of proteasomes remain unknown. Here, using in vivo experiments, we reveal the fast anterograde transport of assembled and functional 26S proteasome complexes. A high-resolution tracking system to follow fluorescent proteasomes revealed three types of motion: actively driven proteasome axonal transport, diffusive behavior in a viscoelastic axonema and proteasome-confined motion. We show that active proteasome transport depends on motor function because knockdown of the KIF5B motor subunit resulted in impairment of the anterograde proteasome flux and the density of segmental velocities. Finally, we reveal that neuronal proteasomes interact with intracellular membranes and identify the coordinated transport of fluorescent proteasomes with synaptic precursor vesicles, Golgi-derived vesicles, lysosomes and mitochondria. Taken together, our results reveal fast axonal transport as a new mechanism of proteasome delivery that depends on membrane cargo ‘hitch-hiking’ and the function of molecular motors. We further hypothesize that defects in proteasome transport could promote abnormal protein clearance in neurodegenerative diseases.Fil: Otero, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Alloatti, Matías. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Cromberg, Lucas Eneas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Almenar Queralt, Angels. University of California at San Diego; Estados UnidosFil: Encalada, Sandra E.. University of California at San Diego; Estados UnidosFil: Pozo Devoto, Victorio Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Bruno, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Goldstein, Lawrence S. B.. University of California at San Diego; Estados UnidosFil: Falzone, Tomas Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentin

3D magnetotelluric modeling using high-order tetrahedral Nédélec elements on massively parallel computing platforms

We present a routine for 3D magnetotelluric (MT) modeling based upon high-order edge finite element method (HEFEM), tailored and unstructured tetrahedral meshes, and high-performance computing (HPC). This implementation extends the PETGEM modeller capabilities, initially developed for active-source electromagnetic methods in frequency-domain. We assess the accuracy, robustness, and performance of the code using a set of reference models developed by the MT community in well-known reported workshops. The scale and geological properties of these 3D MT setups are challenging, making them ideal for addressing a rigorous validation. Our numerical assessment proves that this new algorithm can produce the expected solutions for arbitrarily 3D MT models. Also, our extensive experimental results reveal four main insights: (1) high-order discretizations in conjunction with tailored meshes can offer excellent accuracy; (2) a rigorous mesh design based on the skin-depth principle can be beneficial for the solution of the 3D MT problem in terms of numerical accuracy and run-time; (3) high-order polynomial basis functions achieve better speed-up and parallel efficiency ratios than low-order polynomial basis functions on cutting-edge HPC platforms; (4) a triple helix approach based on HEFEM, tailored meshes, and HPC can be extremely competitive for the solution of realistic and complex 3D MT models and geophysical electromagnetics in general

Synthetic lethal interaction of cetuximab with MEK1/2 inhibition in NRAS-mutant metastatic colorectal cancer

KRAS mutations are an established predictor of lack of response to EGFR-targeted therapies in patients with metastatic colorectal cancer (mCRC). However, little is known about the role of the rarer NRAS mutations as a mechanism of primary resistance to the anti-EGFR monoclonal antibody cetuximab in wild-type KRAS mCRC. Using isogenic mCRC cells with a heterozygous knock-in of the NRAS activating mutation Q61K, we aimed to elucidate the mechanism(s) by which mutant NRAS blocks cetuximab from inhibiting mCRC growth. NRASQ61K/+ cells were refractory to cetuximab-induced growth inhibition. Pathway-oriented proteome profiling revealed that cetuximab-unresponsive ERK1/2 phosphorylation was the sole biomarker distinguishing cetuximab-refractory NRASQ61K/+ from cetuximab-sensitive NRAS+/+ cells. We therefore employed four representative MEK1/2 inhibitors (binimetinib, trametinib, selumetinib, and pimasertib) to evaluate the therapeutic value of MEK/ERK signaling in cetuximab-refractory NRAS mutation-induced mCRC. Co-treatment with an ineffective dose of cetuximab augmented, up to more than 1,300-fold, the cytotoxic effects of pimasertib against NRASQ61K/+ cells. Simultaneous combination of MEK1/2 inhibitors with cetuximab resulted in extremely high and dose-dependent synthetic lethal effects, which were executed, at least in part, by exacerbated apoptotic cell death. Dynamic monitoring of real-time cell growth rates confirmed that cetuximab synergistically sensitized NRASQ61K/+ cellsto MEK1/2 inhibition. Our discovery of a synthetic lethal interaction of cetuximab in combination with MEK1/2 inhibition for the NRAS mutant subgroup of mCRC underscores the importance of therapeutic intervention both in the MEK-ERK and EGFR pathways to achieve maximal therapeutic efficacy against NRAS-mutant mCRC tumors