Conjugative DNA Transfer From E. coli to Transformation-Resistant Lactobacilli

Lactic acid bacteria (LAB) belonging to the genus classically known as Lactobacillus, recently split into 25 different genera, include many relevant species for the food industry. The well-known properties of lactobacilli as probiotics make them an attractive model also for vaccines and therapeutic proteins delivery in humans. However, scarce tools are available to accomplish genetic modification of these organisms, and most are only suitable for laboratory strains. Here, we test bacterial conjugation as a new tool to introduce genetic modifications into many biotechnologically relevant laboratory and wild type lactobacilli. Using mobilizable shuttle plasmids from a donor Escherichia coli carrying either RP4 or R388 conjugative systems, we were able to get transconjugants to all tested Lactocaseibacillus casei strains, including many natural isolates, and to several other genera, including Lentilactobacillus parabuchneri, for which no transformation protocol has been reported. Transconjugants were confirmed by the presence of the oriT and 16S rRNA gene sequencing. Serendipitously, we also found transconjugants into researcher-contaminant Staphylococcus epidermidis. Conjugative DNA transfer from E. coli to S. aureus was previously described, but at very low frequencies. We have purified this recipient strain and used it in standard conjugation assays, confirming that both R388 and RP4 conjugative systems mediate mobilization of plasmids into S. epidermidis. This protocol could be assayed to introduce DNA into other Gram-positive microorganisms which are resistant to transformation.FUNDING: Work in ML lab was supported by the grant BIO2017-87190-R from the Spanish Ministry of Science and Innovation. Work in MÁ lab was funded by the Spanish State Research Agency (AEI) and the European Regional Development Fund (FEDER) (AGL2016-78708-R, AEI/FEDER, EU). DG-H was a recipient of a predoctoral appointment from the University of Cantabria. RM-C received an ErasmusC traineeship grant

Hyaenids, felids and canids as bone accumulators: Does the natural history of extant species support zooarchaeological inferences?

Mammalian carnivores may be important agents of prehistoric bone accumulations. Taphonomic analyses of bone assemblages used for specific assignment usually include information on feeding, breeding, denning and even defecating ecology of extant species. Here, we review literature for the Hyaenidae, Felidae and Canidae families of carnivores, focusing on the ecological and behavioural traits that are commonly used as criteria to assign bone accumulations to specific carnivores, and whether these correspond to the present behaviour and ecology of extant species. We found a total of 93 records where 12 species (9 extant species) of these families were considered as bone accumulators in archaeozoological sites. Hyaenidae was the group most often cited, followed by Felidae and Canidae. Crocuta crocuta was by far the species most often cited as a bone accumulator. Most bone deposits assigned to carnivores (84.9%) were found in underground cavities, and to a lesser extent in non-cave deposits (15.1%). The use assigned to the sites was mainly as a den (29.5%) or breeding den (29.5%), followed by prey depot (16.2%), feeding shelter (12.4%), and to a lesser extent a hunting place (7.6%), with some remarkable differences among families. Coprolites were also found in 53.8% of cases. The behaviour of present hyenas may be similar to that of prehistoric ones as they commonly use underground dens, defecate inside of them and frequently accumulate prey remains. On the other hand, even though present canids are more often recorded than felids using underground dens and accumulating prey, the latter are more often recorded as prehistoric bone accumulators than the former. The behaviour of only one present species of canid (V. vulpes) and other a felid (P. pardus) matches the one presumed for prehistoric individuals of such species in relation to bone and scat accumulation. The role of the remaining species as bone and scat accumulator agents in prehistoric sites remains questionable due to differences in their present behaviour. Therefore, many assignments of bone accumulation to specific carnivores are based on assumptions, which did not coincide with the present natural history of the species. Our review also highlights the absence of records of small species as prehistoric bone accumulators.We thank Cuauhtemoc Ch avez and Ana Carolina Srbek for their unpublished information on jaguars. HRV is a beneficiary of a PhD scholarship “Severo Ochoa” from the Regional Government of Principality of Asturias, and AMG was supported by the Predoctoral Fellowship PRE2018-086102

Estrategia aplicada a simulador de negocios Capstone

El siguiente documento tiene como objetivo describir, analizar y reflexionar sobre la gestión estratégica de una compañía dentro de la industria de sensores durante un tiempo de ocho años en un ambiente virtual, el cual es facilitado por Capsima a través de su aplicación de simulador de negocios Capstone. De igual forma, se presentan conceptos, teorías y estudios de empresas líderes sobre estrategias de negocios que permiten entender con mayor profundidad el tema

Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR–Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.This work has been supported by grant nos. PID2019-109087RB-I00 (to R.P.-J.) and RTI2018-101223-B-I00 and PID2021-127644OB-I00 (to L.M.) from the Spanish Ministry of Science and Innovation. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 964764 (to R.P.-J.). The content presented in this document represents the views of the authors, and the European Commission has no liability in respect to the content. We acknowledge financial support from the Spanish Foundation for the Promotion of Research of Amyotrophic Lateral Sclerosis. A.F. acknowledges Spanish Center for Biomedical Network Research on Rare Diseases (CIBERE) intramural funds (no. ER19P5AC756/2021). F.J.M.M. acknowledges research support by Conselleria d’Educació, Investigació, Cultura i Esport from Generalitat Valenciana, research project nos. PROMETEO/2017/129 and PROMETEO/2021/057. M.M. acknowledges funding from CIBERER (grant no. ER19P5AC728/2021). The work has received funding from the Regional Government of Madrid (grant no. B2017/BMD3721 to M.A.M.-P.) and from Instituto de Salud Carlos III, cofounded with the European Regional Development Fund ‘A way to make Europe’ within the National Plans for Scientific and Technical Research and Innovation 2017–2020 and 2021–2024 (nos. PI17/1659, PI20/0429 and IMP/00009; to M.A.M.-P. B.P.K. was supported by an MGH ECOR Howard M. Goodman Award and NIH P01 HL142494

CCL2/MCP-I Genotype-Phenotype Relationship in Latent Tuberculosis Infection

Among the known biomarkers, chemokines, secreted by activated macrophages and T cells, attract groups of immune cells to the site of infection and may determine the clinical outcome. Association studies of CCL-2/MCP-1 -2518 A/G functional SNP linked to high and low phenotypes with tuberculosis disease susceptibility have shown conflicting results in tuberculosis. Some of these differences could be due the variability of latent infection and recent exposure in the control groups. We have therefore carried out a detailed analysis of CCL-2 genotype SNP -2518 (A/G transition) with plasma CCL-2 levels and related these levels to tuberculin skin test positivity in asymptomatic community controls with no known exposure to tuberculosis and in recently exposed household contacts of pulmonary tuberculosis patients. TST positivity was linked to higher concentrations of plasma CCL2 (Mann Whitney U test; p = 0.004) and was more marked when the G allele was present in TST+ asymptomatic controls (A/G; p = 0.01). Recent exposure also had a significant effect on CCL-2 levels and was linked to the G allele (p = 0.007). Therefore association studies for susceptibility or protection from disease should take into consideration the PPD status as well as recent exposure of the controls group used for comparison. Our results also suggest a role for CCL-2 in maintaining the integrity of granuloma in asymptomatic individuals with latent infection in high TB burden settings. Therefore additional studies into the role of CCL-2 in disease reactivation and progression are warranted