The Proteasomal Deubiquitinating Enzyme PSMD14 Regulates Macroautophagy by Controlling Golgi-to-ER Retrograde Transport

Ubiquitination regulates several biological processes, however the role of specific members of the ubiquitinome on intracellular membrane trafficking is not yet fully understood. Here, we search for ubiquitin-related genes implicated in protein membrane trafficking performing a High-Content siRNA Screening including 1187 genes of the human “ubiquitinome” using amyloid precursor protein (APP) as a reporter. We identified the deubiquitinating enzyme PSMD14, a subunit of the 19S regulatory particle of the proteasome, specific for K63-Ub chains in cells, as a novel regulator of Golgi-to-endoplasmic reticulum (ER) retrograde transport. Silencing or pharmacological inhibition of PSMD14 with Capzimin (CZM) caused a robust increase in APP levels at the Golgi apparatus and the swelling of this organelle. We showed that this phenotype is the result of rapid inhibition of Golgi-to-ER retrograde transport, a pathway implicated in the early steps of the autophagosomal formation. Indeed, we observed that inhibition of PSMD14 with CZM acts as a potent blocker of macroautophagy by a mechanism related to the retention of Atg9A and Rab1A at the Golgi apparatus. As pharmacological inhibition of the proteolytic core of the 20S proteasome did not recapitulate these effects, we concluded that PSMD14, and the K63-Ub chains, act as a crucial regulatory factor for macroautophagy by controlling Golgi-to-ER retrograde transport

Genetic characterization of Shiga toxin-producing Escherichia coli O26:H11 strains isolated from animal, food, and clinical samples

The Shiga-toxin producing Escherichia coli (STEC) may cause serious illness in human. Here we analyze O26:H11 strains known to be among the most reported STEC strains causing human infections. Genetic characterization of strains isolated from animal, food, and clinical specimens in Argentina showed that most carried either stx1a or stx2a subtypes. Interestingly, stx2a-positive O26:H11 rarely isolated from cattle in other countries showed to be an important proportion of O26:H11 strains circulating in cattle and food in our region. Seventeen percent of the isolates harbored more than one gene associated with antimicrobial resistance. In addition to stx, all strains contained the virulence genes eae-β, tir, efa, iha, espB, cif, espA, espF, espJ, nleA, nleB, nleC, and iss; and all except one contained ehxA, espP, and cba genes. On the other hand, toxB and espI genes were exclusively observed in stx2-positive isolates, whereas katP was only found in stx1a-positive isolates. Our results show that O26:H11 STEC strains circulating in Argentina, including those isolated from humans, cattle, and meat products, present a high pathogenic potential, and evidence that cattle can be a reservoir of O26:H11 strains harboring stx2a.Fil: Krüger, Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tandil. Centro de Investigacion Veterinaria de Tandil; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Lucchesi, Paula Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tandil. Centro de Investigacion Veterinaria de Tandil; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Sanso, Andrea Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tandil. Centro de Investigacion Veterinaria de Tandil; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Etcheverría, Analía Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tandil. Centro de Investigacion Veterinaria de Tandil; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Bustamante, Ana Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tandil. Centro de Investigacion Veterinaria de Tandil; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Burgán, Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tandil. Centro de Investigacion Veterinaria de Tandil; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Fernandez, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tandil. Centro de Investigacion Veterinaria de Tandil; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Fernandez, Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tandil. Centro de Investigacion Veterinaria de Tandil; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Leotta, Gerardo Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Genética Veterinaria "Ingeniero Fernando Noel Dulout"; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; ArgentinaFil: Friedrich, Alexander W.. University of Groningen; Países BajosFil: Padola, Nora L.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tandil. Centro de Investigacion Veterinaria de Tandil; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Rossen, John W. A.. University of Groningen; Países Bajo

Abundance of unknots in various models of polymer loops

A veritable zoo of different knots is seen in the ensemble of looped polymer chains, whether created computationally or observed in vitro. At short loop lengths, the spectrum of knots is dominated by the trivial knot (unknot). The fractional abundance of this topological state in the ensemble of all conformations of the loop of $N$ segments follows a decaying exponential form, $\sim \exp (-N/N_0)$, where $N_0$ marks the crossover from a mostly unknotted (ie topologically simple) to a mostly knotted (ie topologically complex) ensemble. In the present work we use computational simulation to look closer into the variation of $N_0$ for a variety of polymer models. Among models examined, $N_0$ is smallest (about 240) for the model with all segments of the same length, it is somewhat larger (305) for Gaussian distributed segments, and can be very large (up to many thousands) when the segment length distribution has a fat power law tail.Comment: 13 pages, 6 color figure

Capturing the essence of folding and functions of biomolecules using Coarse-Grained Models

The distances over which biological molecules and their complexes can function range from a few nanometres, in the case of folded structures, to millimetres, for example during chromosome organization. Describing phenomena that cover such diverse length, and also time scales, requires models that capture the underlying physics for the particular length scale of interest. Theoretical ideas, in particular, concepts from polymer physics, have guided the development of coarse-grained models to study folding of DNA, RNA, and proteins. More recently, such models and their variants have been applied to the functions of biological nanomachines. Simulations using coarse-grained models are now poised to address a wide range of problems in biology.Comment: 37 pages, 8 figure

Additional value of screening for minor genes and copy number variants in hypertrophic cardiomyopathy

Introduction: Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited heart disease. Next-generation sequencing (NGS) is the preferred genetic test, but the diagnostic value of screening for minor and candidate genes, and the role of copy number variants (CNVs) deserves further evaluation. Methods: Three hundred and eighty-seven consecutive unrelated patients with HCM were screened for genetic variants in the 5 most frequent genes (MYBPC3, MYH7, TNNT2, TNNI3 and TPM1) using Sanger sequencing (N = 84) or NGS (N = 303). In the NGS cohort we analyzed 20 additional minor or candidate genes, and applied a proprietary bioinformatics algorithm for detecting CNVs. Additionally, the rate and classification of TTN variants in HCM were compared with 427 patients without structural heart disease. Results: The percentage of patients with pathogenic/likely pathogenic (P/LP) variants in the main genes was 33.3%, without significant differences between the Sanger sequencing and NGS cohorts. The screening for 20 additional genes revealed LP variants in ACTC1, MYL2, MYL3, TNNC1, GLA and PRKAG2 in 12 patients. This approach resulted in more inconclusive tests (36.0% vs. 9.6%, p<0.001), mostly due to variants of unknown significance (VUS) in TTN. The detection rate of rare variants in TTN was not significantly different to that found in the group of patients without structural heart disease. In the NGS cohort, 4 patients (1.3%) had pathogenic CNVs: 2 deletions in MYBPC3 and 2 deletions involving the complete coding region of PLN. Conclusions: A small percentage of HCM cases without point mutations in the 5 main genes are explained by P/LP variants in minor or candidate genes and CNVs. Screening for variants in TTN in HCM patients drastically increases the number of inconclusive tests, and shows a rate of VUS that is similar to patients without structural heart disease, suggesting that this gene should not be analyzed for clinical purposes in HCM

Single-molecule experiments in biological physics: methods and applications

I review single-molecule experiments (SME) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SME it is possible to: manipulate molecules one at a time and measure distributions describing molecular properties; characterize the kinetics of biomolecular reactions and; detect molecular intermediates. SME provide the additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SME it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level emphasizing the importance of SME to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SME from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOT), magnetic tweezers (MT), biomembrane force probe (BFP) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation), proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SME to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.Comment: Latex, 60 pages, 12 figures, Topical Review for J. Phys. C (Cond. Matt

Sur8, a determinant protein in colorectal cancer tumor progression

Resumen del trabajo presentado en el 43rd Annual Meeting of the SEBBM, celebrado en Barcelona (España) del 19 al 21 de julio de 2021.Colorectal cancer (CRC) has the highest incidence rate in the Spanish population. The most important challenge consists on the discovery of efficient disease treatments, due to high mortality rates in highly developed stages. Sur8 is a scaffold protein that positively modulates ERK signaling pathway, which has a major role in the progression and metastasis in colorectal cancer. The main goals of our research are to determine the role that Sur8 plays in the development and progression of CRC and to analyze its possible therapeutic potential. For this purpose, our group has developed an inducible conditional mouse model msur8f/fVillinCreERT2. In order to determine Sur8 action in the colonic tissue, we have developed organoids from the colon epithelium of healthy mice and have analyzed gene expression pattern by an RNAseq approach. Sur8 KO affects oncogenic CRC transcription factors expression, as well as the modulation of some Wnt pathway regulators. In regard to miRNA data, we have observed deregulation of miRNAs related to CRC in Sur8 KO organoids. To determine the role that Sur8 plays in the development and progression of CRC, we have subjected our inducible conditional mice to chemical carcinogenesis and we have observed that Sur8 KO males display less and smaller tumors and do not present any adenocarcinoma. In addition, we have carried out Sur8 silencing in human CRC cell lines by infection with constitutive shRNA lentiviruses. We have observed that Sur8 silencing produces decreases of cell tumor proliferation, and reduction of p-ERK levels. Finally, we are evaluating the effects of putative therapeutic agents against Sur8 in human CRC cell lines. Concretely, we are testing Celastrol, which has been described that binds and blocks the action of Sur8 in vitro. We have observed that Celastrol treatment diminishes the cell tumor proliferation in this model. Altogether, our results indicate that Sur8 may have a determinant role in CRC progression and that Sur8 could be a potential molecular target for the design of novel strategies against CRC

Influence of structural and magnetic properties in the heating performance of multicore bioferrofluids

Biomedical applications of superparamagnetic iron oxide particles have been of interest for quite a number of years. Recent developments show that multifunctionality can be efficiently achieved using polymers to coat the particles and to provide anchoring elements to their surface. This leads to the formation of nanobeads with a reduced number of particles trapped by the polymeric structure. While the magnetothermic behavior of isolated nanoparticles has been a subject of interest over the past several years, multicore magnetic nanobeads have thus far not received the same attention. The influence of structural and magnetic properties in the hyperthermia performance of a series of magnetic fluids designed for biomedical purposes is studied here. The fluids are made of maghemite multicore polymeric beads, with variable nanoparticle size and hydrodynamic size, dispersed in a buffer solution. The specific loss power (SLP) was measured from 5 to 100 kHz with a field intensity of 21.8 kA/m. SLP increases with increasing magnetic core size, reaching 32 W/g Fe2O3 at 100 kHz for 16.2 nm. Within the framework of the linear response theory, a graphical construction is proposed to describe the interplay of both size distributions and magnetic properties in the heating performance of such fluids in a given frequency range. Furthermore, a numerical model is developed to calculate the spare contribution of Neel and Brown relaxation mechanisms to SLP, which gives a fair reproduction of the experimental data

Prevention and control practices against Sars-Cov2 infection in the peruvian population

Objetive: To describe the prevention and control practices for the infection to SARS-COV2 in the Peruvian population. Material and Methods: Observational descriptive study. We evaluated a non-probabilistic sample of adult residents in some departments of Peru. Preventive practices were evaluated in people without a history of COVID-19 and control practices in people who had suffered it. Results: We evaluated 3630 Peruvians (mean age 25.4 ± 9.5), of that 3231 don't have a history of COVID-19 and 399 who had suffered it. The prevention and control practices that were realized often or always, with more frequencies, was the use of a mask when they go out home (97.9% vs 87.7), cover their nose or mouth when they sneeze (95.4% vs 89.9%), save the distance to other people in the street (91.4% vs 74.7%), wash their hands when they came home (92.5% vs 88.7%), and disinfect the objects and personal places (82.6% vs 77.4%). The 22.1% and 83.7%, the 59.7% and 80.2, and the 8.0% and 16.8% consumed some type of medicine, medicinal plant, and chlorine dioxide to prevent and control the infection, respectively. Conclusion: In general, less than 50% of the participants performed prevention and control practices against COVID-19 often or always.Revisión por pare