Low-Molecular-Mass Penicillin Binding Protein 6b (DacD) Is Required for Efficient GOB-18 Metallo--Lactamase Biogenesis in Salmonella enterica and Escherichia coli

Metallo--lactamases (MBLs) are Zn2-containing secretory enzymes of clinical relevance, whose final folding and metal ion assembly steps in Gram-negative bacteria occur after secretion of the apo form to the periplasmic space. In the search of periplasmic factors assisting MBL biogenesis, we found that dacD null ( dacD) mutants of Salmonella enterica and Escherichia coli expressing the preGOB-18 MBL gene from plasmids showed significantly reduced resistance to cefotaxime and concomitant lower accumulation of GOB-18 in the periplasm. This reduced accumulation of GOB-18 resulted from increased accessibility to proteolytic attack in the periplasm, suggesting that the lack of DacD negatively affects the stability of secreted apo MBL forms. Moreover, dacD mutants of S. enterica and E. coli showed an altered ability to develop biofilm growth. DacD is a widely distributed low-molecular-mass (LMM) penicillin binding protein (PBP6b) endowed with low DD-carboxypeptidase activity whose functions are still obscure. Our results indicate roles for DacD in assisting biogenesis of particular secretory macromolecules in Gram-negative bacteria and represent to our knowledge the first reported phenotypes for bacterial mutants lacking this LMM PBP.Fil: Brambilla, Luciano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Moran Barrio, Jorgelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Viale, Alejandro Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Expression of the Escherichia coli ompW colicin S4 receptor gene is regulated by temperature and modulated by the H-NS and StpA nucleoid-associated proteins

The OmpW family consists of a ubiquitous group of small outer membrane (OM) β-barrel proteins of Gram-negative bacteria with proposed roles in environmental adaptation but poorly understood mechanisms of expression. We report here that Escherichia coli K-12 OmpW contents are drastically modified by temperature changes compatible with the leap from the environment to warm-blooded hosts and/or vice versa. Thus, while OmpW is present in the OM of bacteria grown at 37 °C, it sharply disappears at 23 °C with the concomitant acquisition of colicin S4 resistance by the cells. ompW::lacZY fusions indicated that temperature regulation operates at the level of transcription, being ompW expression almost abolished at 23 °C as compared to 37 °C. Moreover, E. coli Δhns mutants lacking H-NS showed reductions in ompW transcription and OmpW contents at 37 °C, indicating positive modulatory roles for this nucleoid-structuring protein in ompW expression. Also, ΔhnsΔstpA double mutants simultaneously lacking H-NS and its paralog StpA showed more severe reductions in ompW expression at 37 °C, resulting in the complete loss of OmpW. The overall results indicate that OmpW contents in E. coli are regulated by both temperature and H-NS and reinforce OmpW functions in bacterial adaptation to warm-blooded hostsFil: Brambilla, Luciano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Moran Barrio, Jorgelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Viale, Alejandro Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Does therapeutic drug monitoring (TDM) of trough concentrations suffice for optimizing preemptive therapy with ganciclovir of cytomegalovirus infections in non-renal solid organ transplant recipients?

Objectives: The aim of this study is to explore the relationship between ganciclovir exposure and clinical efficacy and/or safety in non-renal solid organ transplant (SOT) recipients receiving preemptive therapy with ganciclovir/valganciclovir and undergoing therapeutic drug monitoring (TDM)-guided dosing optimization. Methods: Non-renal SOT recipients admitted to IRCCS Azienda Ospedaliero-Universitaria of Bologna receiving preemptive therapy with ganciclovir or valganciclovir for active cytomegalovirus (CMV) infection and who underwent at least one TDM were included. Desired ganciclovir Cmin range was set at 1-3 mg/L, and average ganciclovir trough concentrations (Cmin ) were calculated for each patient. Reduced CMV viral load below the lower limit of quantification (LLQ) at 30 days and occurrence of myelotoxicity were selected as the primary outcome. Univariate analysis was performed by comparing patients with average Cmin below or above 1 or 3 mg/L. Receiver operating characteristic (ROC) curve analysis was performed to identify the average ganciclovir Cmin cut-off predictive for clinical efficacy or toxicity. Results: Twenty-nine out of 89 retrieved patients met the inclusion criteria, with a median (interquartile [IQR]) baseline CMV viral load of 27,163 copies/mL (IQR 13 159.75-151 340.25 copies/mL). Reduced CMV viral load below the LLQ at 30 days was found in 17 patients (58.6%). No difference was found in the primary outcome between patients showing average Cmin below or above 1 mg/L (100.0% vs. 53.8%; p = .25) and/or 3 mg/L (65.2% vs. 33.3%; p = .20). ROC analysis did not allow to identify an average Cmin cut-off predictive of clinical efficacy or toxicity. Conclusions: No clear relationship between ganciclovir Cmin and neither CMV eradication nor safety issues was identified

Search of brain-enriched proteins in salivary extracellular vesicles for their use as mental disease biomarkers: A pilot study of the neuronal glycoprotein M6a

Background: Mental disorders affect millions of people worldwide. Their etiology is complex and the fact that the main effects occur in the brain hampers biochemical diagnosis. Therefore, biomarker finding in peripheral fluids such as serum or saliva is desirable. Here, we searched for biomarkers in salivary extracellular vesicles (EVs). Then, we focused on the protein M6a, related to neuronal connectivity and associated with several mood disorders to study its usefulness in saliva for the diagnosis of depression and anxiety. Methods: Biomarker candidates were searched by proteomic analysis of human salivary EVs. M6a presence in salivary EVs was validated by transmission electron microscopy and Western blot. M6a levels were measured by ELISA in saliva samples of 88 individuals classified as control, depressed or anxious. Results: We identified ten protein candidates in salivary EVs: OLIG2, PMP2, CNP, CAMK2A, SLC25A22, MLLT11, HTR2A, MAPT, ATP2B2 and M6a, all associated with emotional disorders. Salivary M6a levels positively correlated with the scores for the perceived stress scale in individuals diagnosed with depression. Furthermore, saliva samples of depressed patients treated with serotonin reuptake inhibitors (SSRI) or benzodiazepines differed in their M6a levels with respect to untreated patients. Limitations: The main limitation of this study lies in the low number of patients involved, which warrants replication. Conclusions: Salivary EVs contain promising biomarker candidates for mental disorders. Further studies will help validate them for their potential use in diagnosis. Our results lead us to propose M6a as a workable molecule to take into account as a possible stress biomarker.Fil: Monteleone, Melisa Carolina. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Billi, Silvia Cristina. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Viale, Luciano. Gobierno de la Provincia de Buenos Aires. Hospital Interzonal Especializado de Agudos y Cronicos San Juan de Dios.; Argentina. Universidad Nacional de San Martín; ArgentinaFil: Catoira, Natalia P.. Gobierno de la Provincia de Buenos Aires. Hospital Interzonal Especializado de Agudos y Cronicos San Juan de Dios.; ArgentinaFil: Frasch, Alberto Carlos C.. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Brocco, Marcela Adriana. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentin

South American Expert Roundtable : increasing adaptive governance capacity for coping with unintended side effects of digital transformation

This paper presents the main messages of a South American expert roundtable (ERT) on the unintended side effects (unseens) of digital transformation. The input of the ERT comprised 39 propositions from 20 experts representing 11 different perspectives. The two-day ERT discussed the main drivers and challenges as well as vulnerabilities or unseens and provided suggestions for: (i) the mechanisms underlying major unseens; (ii) understanding possible ways in which rebound effects of digital transformation may become the subject of overarching research in three main categories of impact: development factors, society, and individuals; and (iii) a set of potential action domains for transdisciplinary follow-up processes, including a case study in Brazil. A content analysis of the propositions and related mechanisms provided insights in the genesis of unseens by identifying 15 interrelated causal mechanisms related to critical issues/concerns. Additionally, a cluster analysis (CLA) was applied to structure the challenges and critical developments in South America. The discussion elaborated the genesis, dynamics, and impacts of (groups of) unseens such as the digital divide (that affects most countries that are not included in the development of digital business, management, production, etc. tools) or the challenge of restructuring small- and medium-sized enterprises (whose service is digitally substituted by digital devices). We identify specific issues and effects (for most South American countries) such as lack of governmental structure, challenging geographical structures (e.g., inclusion in high-performance transmission power), or the digital readiness of (wide parts) of society. One scientific contribution of the paper is related to the presented methodology that provides insights into the phenomena, the causal chains underlying “wanted/positive” and “unwanted/negative” effects, and the processes and mechanisms of societal changes caused by digitalization

Relationship between immune response to SARS-CoV2 vaccines and development of breakthrough infection in solid organ transplant recipients: the CONTRAST cohort

Background: SARS-CoV-2 vaccination in solid organ transplant (SOT) is associated with poorer antibody response (AbR) compared to non-SOT recipients. However, its impact on the risk of breakthrough infection (BI) should yet be assessed. Methods: Single-center prospective longitudinal cohort study enrolling adult SOT recipients who received SARS-CoV2 vaccination during 1-year period from February 2021, and followed-up to April 30th 2022. Patients were tested for AbR at multiple timepoints. Primary endpoint was BI (laboratory confirmed SARS-CoV2 infection ≥14 days after 2nd dose). Immunization (positive AbR) was considered an intermediate state between vaccination and BI. Probabilities of being in vaccination, immunization and BI states were obtained for each type of graft and vaccination sequence with multistate survival analysis, then multivariable logistic regression was performed to analyse the risk of BI in AbR levels. Results: 614 SOT (275 kidney, 163 liver, 137 heart, 39 lung) recipients were included. Most patients (84.7%) received three vaccine doses, the first two consisted of BNT162b2 and mRNA-1273 in 73.5% and 26.5% of cases, respectively; while at the third dose mRNA-1273 was administered in 59.8% of patients. Overall, 75.4% of patients reached immunization and 18.4% developed BI. Heart transplant recipients showed lowest probability of immunization (0.418) and highest of BI (0.323), all-mRNA-1273 vaccine-sequence showed higher probability of immunization (0.732) and lowest of BI (0.098). Risk of BI was higher for non-high-level AbR, younger age and shorter time from transplant. Conclusions: SOT patients with non-high-level AbR, shorter time from transplantation, and heart recipients are at highest risk of BI

Programa de participación ciudadana para el relevamiento de impactos por eventos meteorológicos extremos en Córdoba, Argentina

Córdoba, Argentina es una región del planeta, especificada por tormentas severas con gran desarrollo vertical, vientos intensos, descargas eléctricas, granizo de gran tamaño y abundante, y lluvias torrenciales, ocasionando en algunos casos, inundaciones rápidas y repentinas. La caracterización de los impactos por granizo resulta dificultosa sin datos en terreno. El enfoque de la participación ciudadana en el relevamiento de información espacial de eventos extremos, mediante aplicaciones móviles y redes sociales, ha mostrado ser de utilidad para su caracterización y monitoreo. En este sentido, desde 2018, se ha desarrollado el programa COSECHEROS, inicialmente de granizos Córdoba que hoy es de Eventos Meteorológicos Extremos, que tiene la finalidad de relevar estos fenómenos extremos en un trabajo colaborativo entre los ciudadanos voluntarios y los investigadores. Para la implementación del programa en zonas rurales, el programa se articula con iniciativas en territorio, destinadas a integrar esfuerzos entre organismos de ciencia y técnica, técnicos agropecuarios, productores y tomadores de decisiones locales, en Mesas Agro-climáticas y Ambientales (MACA). En este marco, se realizaron diferentes actividades de formación tales como talleres y conferencias, con participación interinstitucional, multi e interdisciplinar (biólogo, arquitecto, médico, nutricionistas, estudiantes, asociación de productoras/es y gobiernos locales). En el presente trabajo se describirán las actividades realizadas con el fin de divulgar el programa y los resultados de su implementación en la MACA de Río Segundo y Pilar, en el caso de dos eventos meteorológicos extremos de granizo; uno de ellos, en el que el relevamiento de campo se realizó en forma absolutamente manual y tradicional y sólo se registraron los datos meteorológicos y de sensores remotos desde el programa y su aplicación y un segundo evento ( un año después- abril de 2022) en el que los datos fueron registrados en la APP y cotejados con el trabajo de campo.EEA ManfrediFil: Arena, Lucía. Observatorio Hidrometeorológico de Córdoba; ArgentinaFil: Pons, Diego Hernan. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Manfredi; ArgentinaFil: Pons, Diego Hernan. Universidad Nacional de Córdoba. Instituto de Altos Estudios Espaciales “Mario Gulich”; ArgentinaFil: Pons, Diego Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Instituto de Altos Estudios Espaciales “Mario Gulich”; ArgentinaFil: Giobellina, Beatriz Liliana. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Manfredi. Observatorio de Agricultura Urbana y Periurbana y Agroecología (O-AUPA); ArgentinaFil: Osiecki Tomás. Observatorio Hidrometeorológico de Córdoba; ArgentinaFil: Silva, Alejandro Ismael. Observatorio Hidrometeorológico de Córdoba; ArgentinaFil: Lighezzolo, Andres. Universidad Nacional de Córdoba. Unidad de desarrollos y Soluciones Ambientales - Comisión Nacional de Actividades Espaciales. Observatorio Hidrometeorológico de la Provincia de Córdoba. Laboratorio de Hidraúlica; ArgentinaFil: Narmona, Luis Rogelio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Manfredi. Observatorio de Agricultura Urbana y Periurbana y Agroecología (O-AUPA); ArgentinaFil: Narmona, Luis Rogelio. Consejo Nacional de Investigaciones Científicas y Técnicas. Observatorio de Agricultura Urbana y Periurbana y Agroecología (O-AUPA); ArgentinaFil: Arrascaeta, Ana Mercedes. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Manfredi; ArgentinaFil: Garello, Adriana Del Lourdes. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Manfredi. Agencia de Extensión Rural Oncativo; ArgentinaFil: Eandi, Mariana. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Grupo de Epidemiología Ambiental del Cáncer en Córdoba; ArgentinaFil: Dezzotti, Luciana. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Grupo de Epidemiología Ambiental del Cáncer en Córdoba; ArgentinaFil: Butinof, Mariana. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Grupo de Epidemiología Ambiental del Cáncer en Córdoba; ArgentinaFil: Bustos, Daniela. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Grupo de Epidemiología Ambiental del Cáncer en Córdoba; ArgentinaFil: Romero Asis, Melisa. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Grupo de Epidemiología Ambiental del Cáncer en Córdoba; ArgentinaFil: Aparicio, Lourdes. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Grupo de Epidemiología Ambiental del Cáncer en Córdoba; ArgentinaFil: Viale, María Virginia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Fisiología y Recursos Genéticos Vegetales; ArgentinaFil: Quinteros, Mario Gustavo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Manfredi. Agencia de Extensión Rural Córdoba; ArgentinaFil: Occhionero, Federico. Universidad Nacional de Córdoba. Facultad de Matemática, Astronomía, Física y Computación; ArgentinaFil: Cortés, Luciano. Gobierno de Córdoba. Ministerio de Agricultura y Ganadería; ArgentinaFil: Bisio, Cali. Gobierno de Córdoba. Ministerio de Agricultura y Ganadería; Argentin

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field