Formation of Mycobacterium abscessus colonies in cellular culture in an in vitro infection model.

Mycobacterium abscessus is one of the most important nontuberculous mycobacteria that cause lung diseases. In vitro infection models developed to analyze the immune response are frequently based on the addition of mycobacteria to mononuclear cells or neutrophils from peripheral blood. An important requirement of these assays is that most cells phagocytose mycobacteria, only accomplished by using large multiplicities of infection (1 or more bacteria per cell) which may not adequately reflect the inhalation of a few mycobacteria by the host. We propose modifications that try to mimic some of the conditions in which immune cells deal with mycobacteria. For the preparation of the inoculum mycobacteria are grown in solid media followed by preparation to a single cell suspension. Multiplicities of infection (number of bacteria per cell) are below 0.01. Serum-free cellular media is used to allow the growth of M. abscessus. After several days of incubation Bacterial Colonies in Cellular Culture (BCCC) develop, which are enumerated directly under an inverted microscope. These colonies may represent biofilm formation during chronic infections. •Low multiplicity of infection (below 0.01 bacteria per cell) reflects more realistically conditions encountered by immune cells in the lungs.•The surface of mycobacteria prepared for infection assays that are grown in solid media are less affected than that of mycobacteria grown in liquid media with detergents.•Colony formation in the infected cells may reflect the aggregation and biofilm formation in the lungs during chronic infection.

A putative bifunctional CPD/ (6-4) photolyase from the cyanobacteria Synechococcus sp. PCC 7335 is encoded by a UV-B inducible operon: New insights into the evolution of photolyases.

Photosynthetic organisms are continuously exposed to solar ultraviolet radiation-B (UV-B) because of their autotrophic lifestyle. UV-B provokes DNA damage, such as cyclobutane pyrimidine dimers (CPD) or pyrimidine (6-4) pyrimidone photoproducts (6-4 PPs). The cryptochrome/photolyase family (CPF) comprises flavoproteins that can bind damaged or undamaged DNA. Photolyases (PHRs) are enzymes that repair either CPDs or 6-4 PPs. A natural bifunctional CPD/(6-4)- PHR (PhrSph98) was recently isolated from the UV-resistant bacteria Sphingomonas sp. UV9. In this work, phylogenetic studies of bifunctional CPD/(6-4)- photolyases and their evolutionary relationship with other CPF members were performed. Amino acids involved in electron transfer and binding to FAD cofactor and DNA lesions were conserved in proteins from proteobacteria, planctomycete, bacteroidete, acidobacteria and cyanobacteria clades. Genome analysis revealed that the cyanobacteria Synechococcus sp. PCC 7335 encodes a two-gene assembly operon coding for a PHR and a bifunctional CPD/(6-4) PHR- like. Operon structure was validated by RT-qPCR analysis and the polycistronic transcript accumulated after 15 min of UV-B irradiation. Conservation of structure and evolution is discussed. This study provides evidence for a UV-B inducible PHR operon that encodes a CPD/(6-4)- photolyase homolog with a putative bifunctional role in the repair of CPDs and 6-4 PPs damages in oxygenic photosynthetic organisms.

Wolbachia Infection in Native Populations of the Invasive Tawny Crazy Ant Nylanderia fulva.

Antagonistic interactions can affect population growth and dispersal of an invasive species. Wolbachia are intracellular endosymbiont bacteria that infect arthropod and nematode hosts and are able to manipulate reproduction, which in some cases leads to cocladogenesis. Moreover, the presence of the strictly maternally transferred Wolbachia in a population can indirectly induce selective sweeps on the hosts' mitochondria. Ants have a Wolbachia infection rate of about 34%, which makes phylogenetic studies using mitochondrial markers vulnerable of being confounded by the effect of the endosymbiont. Nylanderia fulva is an invasive ant native to South America, considered a pest in the United States. Its distribution and biology are poorly known in its native range, and the taxonomic identity of this and its closely related species, Nylanderia pubens, has only recently been understood with the aid of molecular phylogenies. Aiming at estimating robust phylogenetic relationships of N. fulva in its native range, we investigated the presence and pattern of Wolbachia infection in populations of N. fulva from Argentina, part of its native range, to account for its possible effect on the host population structure. Using the ftsZ gene, 30 nests of N. fulva and four from sympatric Nylanderia species were screened for the presence of Wolbachia. We sequenced the MLST genes, the highly variable gene wsp, as well as glyQ, a novel target gene for which new primers were designed. Phylogeny of the ants was estimated using mtDNA (COI). We found supergroup A Wolbachia strains infecting 73% of N. fulva nests and two nests of Nylanderia sp. 1. Wolbachia phylogenetic tree inferred with MLST genes is partially congruent with the host phylogeny topology, with the exception of a lineage of strains shared by ants from different N. fulva clades. Furthermore, by comparing with Wolbachia sequences infecting other ants, we found that the strains infecting different N. fulva clades are not monophyletic. Our findings suggest there are three recent independent horizontally transmitted Wolbachia infections in N. fulva, and we found no evidence of influence of Wolbachia in the host mtDNA based phylogeny.

Selection and optimization of reference genes for RT-qPCR normalization: A case study in Solanum lycopersicum exposed to UV-B.

Quantitative RT- PCR is one of the most common methods to study gene expression in response to stress. Therefore, it is crucial to have suitable reference genes (RGs) for result normalization. Although several reports describe UV-B-modulated gene expression in Solanum lycopersicum, there are no suitable RGs identified until now. The aim of this work was to evaluate the suitability of seven traditional genes: actin (ACT), tubulin (TUB), ubiquitin (UBI), glyceraldehyde- 3 phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1α), phosphatase 2A catalytic subunit (PP2A) and GAGA binding transcriptional activator (GAGA); and two non-traditional genes: thioredoxin h1 (TRX h1) and UV-B RESISTANCE LOCUS 8 (UVR8), as candidate RGs for their potential use as reliable internal controls in leaves, stems and roots of tomato seedlings exposed to acute and chronic UV-B. The stability of these genes expression was evaluated using five statistical algorithms: geNorm, NormFinder, BestKeeper, Delta Ct and ANOVA. Considering the comprehensive stability ranking, we recommend ACT+TUB as the best pair of RGs for leaves, PP2A+GAPDH+TRX h1 for stems and TUB+UVR8 for roots. The reliability of the selected RGs for each tissue was verified amplifying tomato chalcone synthase 1 (CHS1) and cyclobutane pyrimidine dimer (CPD) photolyase (PHR1-LIKE). Under UV-B treatment, CHS1 was upregulated in leaves, stems and roots whereas PHR1-LIKE was only upregulated in leaves and stems. This interpretation differs when the most and least stable RGs are chosen. This is the first report regarding suitable RGs selection for accurate normalization of gene expression in tomato seedlings exposed to UV-B irradiation.

Reflexiones sobre la educación universitaria en pandemia. Experiencia en odontología / Reflections on university education in a pandemic. Experience in dentistry

¿Cuándo fue que nos despertamos y nos dimos cuenta de que estábamos en una escena de película? Algo impensado para nosotros, un aislamiento, nos hizo sentir más que nunca que somos seres sociales. ¿Y qué ocurrió con la educación en este contexto? El modo en que el ámbito universitario atravesó la pandemia y la inclusión de la tecnología en la enseñanza odontológica invitan a reflexionar sobre los cambios que tuvieron lugar y que llegaron para quedarse (AU)

That moment when we found ourselves in the middle of a movie scene. Something unexpected for us, the isolation showed us the importance for human beings to socialise. The changes experienced in education during the lockdown in 2020 with the inclusion of technology like internet in dental education, invites us to reflect about those changes and how they will affect dental education in the future (AU)

Supercolonial structure of invasive populations of the tawny crazy ant Nylanderia fulva in the US.

BACKGROUND: Social insects are among the most serious invasive pests in the world, particularly successful at monopolizing environmental resources to outcompete native species and achieve ecological dominance. The invasive success of some social insects is enhanced by their unicolonial structure, under which the presence of numerous queens and the lack of aggression against non-nestmates allow high worker densities, colony growth, and survival while eliminating intra-specific competition. In this study, we investigated the population genetics, colony structure and levels of aggression in the tawny crazy ant, Nylanderia fulva, which was recently introduced into the United States from South America. RESULTS: We found that this species experienced a genetic bottleneck during its invasion lowering its genetic diversity by 60%. Our results show that the introduction of N. fulva is associated with a shift in colony structure. This species exhibits a multicolonial organization in its native range, with colonies clearly separated from one another, whereas it displays a unicolonial system with no clear boundaries among nests in its invasive range. We uncovered an absence of genetic differentiation among populations across the entire invasive range, and a lack of aggressive behaviors towards conspecifics from different nests, even ones separated by several hundreds of kilometers. CONCLUSIONS: Overall, these results suggest that across its entire invasive range in the U.S.A., this species forms a single supercolony spreading more than 2000 km. In each invasive nest, we found several, up to hundreds, of reproductive queens, each being mated with a single male. The many reproductive queens per nests, together with the free movement of individuals between nests, leads to a relatedness coefficient among nestmate workers close to zero in introduced populations, calling into question the stability of this unicolonial system in which indirect fitness benefits to workers is apparently absent.

Spanish Society of Hospital Pharmacy position paper on biosimilar medicines.

Biological medicines nowadays have a great impact, as they offer treatment for  diverse diseases and suppose a high cost for health system. Biosimilar medicines contain a version of an active substance already authorized as an original  biotechnological medicine, whose patent has expired, and they comply with the  guidelines published by the European Medicines Agency. These guidelines, where biosimilarity criteria are established, guarantee comparability between biosimilar product and reference one. Biosimilars' authorization is carried out through a  centralized procedure based on clinical, non-clinical and quality studies. These  studies allow the extrapolation of indications, frequently, without carrying out additional analyses. In several European countries, switching between  original and biosimilar medicine is considered safe. In Spain, Pharmacy and Therapeutic Committee of hospitals, as consensus bodies among health professionals, are the most suitable bodies to establish the  interchangeability criteria in each center. Biosimilar drugs contribute to  sustainability and to improvement of the accessibility to medicines. Faced with  this situation, Spanish Society of Hospital Pharmacy considers interesting to  express its position about biosimilar medicines' strategies. Spanish Society of  Hospital Pharmacy, in September 2015, published an information note about biosimilar medicines, in which its role as medicines similar in quality,  safety and efficacy to the originals, but at lower cost, was highlighted. Likewise, it was stressed the role of hospital pharmacists within the Pharmacy  and Therapeutic Committee of hospitals, where their knowledge for the  selection, evaluation and use of medicines could be useful, in coordination and permanent collaboration with other units or clinical services of hospitals.

Los medicamentos biológicos actualmente presentan un gran impacto, ya que ofrecen tratamiento para diversas enfermedades y suponen un elevado  coste para el sistema sanitario. Los medicamentos biosimilares contienen una  versión de una sustancia activa ya autorizada como medicamento biotecnológico original, cuya patente ha caducado, y cumplen con las guías publicadas por la European Medicines Agency, que establecen los criterios de biosimilitud para garantizar la comparabilidad entre el producto biosimilar y el de referencia.  La autorización de los biosimilares se realiza mediante un procedimiento  centralizado, a través de estudios comparativos clínicos, no-clínicos y de calidad, que permiten la extrapolación de indicaciones, frecuentemente sin realizar  estudios adicionales. Algunos países europeos consideran seguro el intercambio  entre medicamento original y biosimilar. En España, las Comisiones de Farmacia y Terapéutica hospitalarias, como órganos de consenso entre  profesionales sanitarios, se consideran las más adecuadas para establecer los  criterios de intercambiabilidad en cada centro. Los biosimilares contribuyen a la  sostenibilidad y a la accesibilidad a los medicamentos. Ante esta situación, la  Sociedad Española de Farmacia Hospitalaria considera de interés expresar su  posicionamiento sobre las estrategias relacionadas con los medicamentos  biosimilares. La Sociedad Española de Farmacia Hospitalaria, en septiembre de  2015, publicó una nota informativa sobre los medicamentos biosimilares, en la  que destacó su similitud en calidad, seguridad y eficacia respecto a los  originales, pero con menor coste. Igualmente, se recalcó el papel del  farmacéutico hospitalario en las Comisiones de Farmacia y Terapéutica  hospitalarias, donde sus conocimientos son útiles para la selección, evaluación y  empleo de los medicamentos, en coordinación y colaboración permanente con  los demás servicios clínicos del hospital.

Teachers' Perceived Disruption at School and Related Variables from Teachers and School Functioning.

This study explores variables related to teachers' perception of disruption at school as a function of teachers (sense of personal accomplishment, professional disengagement and depersonalization and emotional exhaustion) and school (overall school management and quality of school rules) factors. Using a questionnaire regarding school climate, data from 4,055 teachers across 187 high schools were analyzed. Hierarchical linear modeling was applied and the results indicate that, taken separately, significant individual teacher predictors (Model 1) explain 26% (95% CI [.23, .29]) of the variability of the perceived disruption, especially depersonalization and emotional exhaustion. Contextual school variables (Model 2) explained 15% (95% CI [.12, .18]) of variance in teachers' perceived disruption, with a significant negative relationship with the quality of rules. Model 3 included the above factors plus interactions between the emotional exhaustion and depersonalization variables and school indicators (30% of variance explained; 95% CI [.26, .33]). The results indicated the existence of a moderating effect for the quality of school rules, so that fair and properly-applied rules in the school context may be associated with a decrease in the relationship between depersonalization and emotional exhaustion and perceived disruption.

[Survival of older patients starting hemodialysis in Chile]. / Análisis de sobrevida en pacientes de edad avanzada que inician hemodiálisis crónica en Servicio de Salud Chileno.

UNLABELLED: The proportion of older people with end stage renal disease is increasing. Their prognosis is characterized by a high mortality and poor quality of life. AIM: To analyze the survival of patients starting chronic hemodialysis (CHD) according to their age. MATERIAL AND METHODS: Patients admitted to CHD in the East Metropolitan Health Service of Santiago in a 2-year period were analyzed. Four age groups were created, separating patients older than 70 years in a special group. RESULTS: During the study period, 459 patients were admitted to CHD and were followed for an average of 27 months. The frequency of cardiovascular comorbidity, cancer, and chronic renal disease of unknown cause (attributed to nephrosclerosis) increased along with age. Mortality was higher at older ages. There was a significant association between starting CHD with a catheter, Charlson comorbidity index and increasing age with mortality. For those aged over 80 years, mortality at three months and one year was 25 and 43% respectively. CONCLUSIONS: Age, Charlson index and vascular access are predictors of mortality in older adults entering hemodialysis. This study suggests the importance of considering comorbidities, assessment by specialists and creating an arteriovenous fistula in this age group.

Análisis de sobrevida en pacientes de edad avanzada que inician hemodiálisis crónica en Servicio de Salud Chileno / Survival of older patients starting hemodialysis in Chile

The proportion of older people with end stage renal disease is increasing. Their prognosis is characterized by a high mortality and poor quality of life. Aim: To analyze the survival of patients starting chronic hemodialysis (CHD) according to their age. Material and Methods: Patients admitted to CHD in the East Metropolitan Health Service of Santiago in a 2-year period were analyzed. Four age groups were created, separating patients older than 70 years in a special group. Results: During the study period, 459 patients were admitted to CHD and were followed for an average of 27 months. The frequency of cardiovascular comorbidity, cancer, and chronic renal disease of unknown cause (attributed to nephrosclerosis) increased along with age. Mortality was higher at older ages. There was a significant association between starting CHD with a catheter, Charlson comorbidity index and increasing age with mortality. For those aged over 80 years, mortality at three months and one year was 25 and 43% respectively. Conclusions: Age, Charlson index and vascular access are predictors of mortality in older adults entering hemodialysis. This study suggests the importance of considering comorbidities, assessment by specialists and creating an arteriovenous fistula in this age group.

Fibrin(ogen)olytic and antiplatelet activities of a subtilisin-like protease from Solanum tuberosum (StSBTc-3).

Plant serine proteases have been widely used in food science and technology as well as in medicine. In this sense, several plant serine proteases have been proposed as potential anti-coagulants and anti-platelet agents. Previously, we have reported the purification and identification of a plant serine protease from Solanum tuberosum leaves. This potato enzyme, named as StSBTc-3, has a molecular weight of 72 kDa and it was characterized as a subtilisin like protease. In this work we determine and characterize the biochemical and medicinal properties of StSBTc-3. Results obtained show that, like the reported to other plant serine proteases, StSBTc-3 is able to degrade all chains of human fibrinogen and to produces fibrin clot lysis in a dose dependent manner. The enzyme efficiently hydrolyzes ß subunit followed by partially hydrolyzed α and γ subunits of human fibrinogen. Assays performed to determine StSBTc-3 substrate specificity using oxidized insulin ß-chain as substrate, show seven cleavage sites: Asn3-Gln4; Cys7-Gly8; Glu13-Ala14; Leu15-Tyr16; Tyr16-Leu17; Arg22-Gly23 and Phe25-Tyr26, all of them were previously reported for other serine proteases with fibrinogenolytic activity. The maximum StSBTc-3 fibrinogenolytic activity was determined at pH 8.0 and at 37 C. Additionally, we demonstrate that StSBTc-3 is able to inhibit platelet aggregation and is unable to exert cytotoxic activity on human erythrocytes in vitro at all concentrations assayed. These results suggest that StSBTc-3 could be evaluated as a new agent to be used in the treatment of thromboembolic disorders such as strokes, pulmonary embolism and deep vein thrombosis.

Isolation and characterization of a Solanum tuberosum subtilisin-like protein with caspase-3 activity (StSBTc-3).

Plant proteases with caspase-like enzymatic activity have been widely studied during the last decade. Previously, we have reported the presence and induction of caspase-3 like activity in the apoplast of potato leaves during Solanum tuberosum- Phytophthora infestans interaction. In this work we have purified and identified a potato extracellular protease with caspase-3 like enzymatic activity from potato leaves infected with P. infestans. Results obtained from the size exclusion chromatography show that the isolated protease is a monomeric enzyme with an estimated molecular weight of 70 kDa approximately. Purified protease was analyzed by MALDI-TOF MS, showing a 100% of sequence identity with the deduced amino acid sequence of a putative subtilisin-like protease from S. tuberosum (Solgenomics protein ID: PGSC0003DMP400018521). For this reason the isolated protease was named as StSBTc-3. This report constitutes the first evidence of isolation and identification of a plant subtilisin-like protease with caspase-3 like enzymatic activity. In order to elucidate the possible function of StSBTc-3 during plant pathogen interaction, we demonstrate that like animal caspase-3, StSBTc-3 is able to produce in vitro cytoplasm shrinkage in plant cells and to induce plant cell death. This result suggest that, StSBTc-3 could exert a caspase executer function during potato- P. infestans interaction, resulting in the restriction of the pathogen spread during plant-pathogen interaction.

DEVDase activity is induced in potato leaves during Phytophthora infestans infection.

Programmed cell death (PCD) occurs in plants, animals and several branches of unicellular eukaryotes as a part of developmental and/or defense processes. Caspase proteases are universal mediators of animal apoptosis, a type of PCD. In plants, there are not animal caspase homologs; therefore, the characterization of caspase-like activities is of considerable importance to our understanding of PCD in plants. Here we report for the first time the involvement of caspase-3-like activity in the resistance mechanism of potato to Phytophthora infestans infection. We showed that disease development in infected potato leaves is dependent of caspase-3-like activity. Unlike plant DEVDases previously reported, this DEVDase activity was sensitive to the serine protease inhibitor PMSF. As reported for other subtilisin- like proteases with caspase activity, potato DEVDase activity was mainly localized in the apoplast. We demonstrated that in total protein extract DEVDase activity accounts for a 60% of serine proteases; however, this percentage increases to 100% in the apoplast. Additionally, this caspase-3-like activity is constitutively expressed in the apoplast of potato leaves. Total DEVDase activity is induced only in potato cultivars with high field resistance to P. infestans. These results show that potato caspase-3-like protease could constitute a tool in the potato defense mechanisms resulting in partial resistance, although further assays would be necessary in order to elucidate its role.

Hydrophobic proteins secreted into the apoplast may contribute to resistance against Phytophthora infestans in potato.

During plant-pathogen interaction, oomycetes secrete effectors into the plant apoplast where they interact with host resistance proteins, which are accumulated after wounding or infection. Previous studies showed that the expression profile of pathogenesis related proteins is proportional to the resistance of different cultivars toward Phytophthora infestans infection. The aim of this work was to analyze the expression pattern of apoplastic hydrophobic proteins (AHPs), after 24 h of wounding or infection, in tubers from two potato cultivars with different resistance to P. infestans, Spunta (susceptible) and Innovator (resistant). Intercellular washing fluid (IWF) was extracted from tubers and chromatographed into a PepRPC™ HR5-5 column in FPLC eluted with a linear gradient of 75% acetonitrile. Then, AHPs were analyzed by SDS-PAGE and identified by MALDI-TOF-MS. Innovator cv. showed a higher basal AHP content compared to Spunta cv. In the latter, infection induced accumulation of patatins and protease inhibitors (PIs), whereas in Innovator cv. no changes in PIs accumulation were observed. In response to P. infestans infection, lipoxygenase, enolase, annexin p34 and glutarredoxin/cyclophilin were accumulated in both cultivars. These results suggest that the AHPs content may be related to the protection against the oomycete and with the degree of potato resistance to pathogens. Additionally, a considerable number of the proteins putatively identified lacked the signal peptide and, being SecretomeP positive, suggest unconventional protein secretion.