Recombinant Escherichia coli as a gene delivery vector into airway epithelial cells

Abstract To transfer genes into airway epithelial cells, we have generated auxotrophic dap Escherichia coli BM2710 mutant that expresses the invasin of Yersinia pseudotuberculosis and the listeriolysin of Listeria monocytogenes. E. coli BM2710 harboring a plasmid carrying the gfp gene was incubated with immortalized normal or cystic fibrosis (CF) airway epithelial cells or with primary bronchial epithelial cells grown as an explant-outgrowth cell culture model. Approximately 2% of immortalized cells expressed GFP. Few primary cells were transfected that were always poorly differentiated and located at the edge of the outgrowth. This was consistent with the expression of h1-integrins only on these cells and with the required interaction for cell entry of E. coli expressing the invasin with h1-integrins. The subsequent intracellular trafficking of E. coli BM2710 studied by confocal and electronic microscopy showed that the E. coli-containing phagosomes rapidly matured into phagolysosomes. This is the first demonstration that recombinant bacteria are able to transfer genes into primary airway epithelial cells, provided that they are able to invade the cells

Reconstructing Gene Regulatory Networks That Control Hematopoietic Commitment.

Hematopoietic stem cells (HSCs) reside at the apex of the hematopoietic hierarchy, possessing the ability to self-renew and differentiate toward all mature blood lineages. Along with more specialized progenitor cells, HSCs have an essential role in maintaining a healthy blood system. Incorrect regulation of cell fate decisions in stem/progenitor cells can lead to an imbalance of mature blood cell populations-a situation seen in diseases such as leukemia. Transcription factors, acting as part of complex regulatory networks, are known to play an important role in regulating hematopoietic cell fate decisions. Yet, discovering the interactions present in these networks remains a big challenge. Here, we discuss a computational method that uses single-cell gene expression data to reconstruct Boolean gene regulatory network models and show how this technique can be applied to enhance our understanding of transcriptional regulation in hematopoiesis.Work in the author’s laboratory is supported by grants from the Wellcome, Bloodwise, Cancer Research UK, NIH-NIDDK and core support grants by the Wellcome to the Cambridge Institute for Medical Research and Wellcome & MRC Cambridge Stem Cell Institute. F.K.H. is a recipient of a Medical Research Council PhD Studentship

Mechanisms for the Intracellular Manipulation of Organelles by Conventional Electroporation

Conventional electroporation (EP) changes both the conductance and molecular permeability of the plasma membrane (PM) of cells and is a standard method for delivering both biologically active and probe molecules of a wide range of sizes into cells. However, the underlying mechanisms at the molecular and cellular levels remain controversial. Here we introduce a mathematical cell model that contains representative organelles (nucleus, endoplasmic reticulum, mitochondria) and includes a dynamic EP model, which describes formation, expansion, contraction, and destruction for the plasma and all organelle membranes. We show that conventional EP provides transient electrical pathways into the cell, sufficient to create significant intracellular fields. This emerging intracellular electrical field is a secondary effect due to EP and can cause transmembrane voltages at the organelles, which are large enough and long enough to gate organelle channels, and even sufficient, at some field strengths, for the poration of organelle membranes. This suggests an alternative to nanosecond pulsed electric fields for intracellular manipulations.National Science Foundation (U.S.) (NSF Graduate Research Fellowship)National Institutes of Health (U.S.) (grant No. R01-GM63857)Aegis Industries, Inc

Recent evolutions of gender, state feminism and care models in Latin America and Europe

Production of INCASI Project H2020-MSCA-RISE-2015 GA 691004This chapter presents and characterises the way in which, in the twenty-first century, after years of feminist struggles inside and outside of institutions, gender relations are organised in the different countries of the INCASI project (on the European side, Spain, Italy, Finland, France and the United Kingdom, on the side of the South American Southern Cone, Argentina, Brazil, Chile and Uruguay). It pays special attention to the implementation of feminist issues on political agendas, and in particular the assignment of women to unpaid care work-an aspect of the power continuum that we look to relate to other aspects. Gradually and for almost a century all countries in both continents have granted women the status of subjects, citizens and employees. However, the conditions, challenges and timelines of this process differ considerably from one continent to another, so they need to be addressed separately. The neoliberal era did not have the same impact in Europe as it did in South America (nor was it exactly the same between particular European countries or among South American ones)

The Level of DING Proteins Is Increased in HIV-Infected Patients: In Vitro and In Vivo Studies

DING proteins constitute an interesting family, owing to their intriguing and important activities. However, after a decade of research, little is known about these proteins. In humans, at least five different DING proteins have been identified, which were implicated in important biological processes and diseases, including HIV. Indeed, recent data from different research groups have highlighted the anti-HIV activity of some DING representatives. These proteins share the ability to inhibit the transcriptional step of HIV-1, a key step of the viral cycle that is not yet targeted by the current therapies. Since such proteins have been isolated from humans, we undertook a comprehensive study that focuses on the relationship between these proteins and HIV-infection in an infectious context. Hence, we developed a home-made ELISA for the quantification of the concentration of DING proteins in human serum. Using this method, we were able to determine the concentration of DING proteins in healthy and HIV-infected patients. Interestingly, we observed a significant increase of the concentration of DING proteins in non treated and treated HIV-infected patients compared to controls. In addition, cell cultures infected with HIV also show an increased expression of DING proteins, ruling out the possible role of antiretroviral treatment in the increase of the expression of DING proteins. In conclusion, results from this study show that the organism reacts to HIV-infection by an overexpression of DING proteins

Neuronal Deletion of Caspase 8 Protects against Brain Injury in Mouse Models of Controlled Cortical Impact and Kainic Acid-Induced Excitotoxicity

system. mice demonstrated superior survival, reduced seizure severity, less apoptosis, and reduced caspase 3 processing. Uninjured aged knockout mice showed improved learning and memory, implicating a possible role for caspase 8 in cognitive decline with aging.Neuron-specific deletion of caspase 8 reduces brain damage and improves post-traumatic functional outcomes, suggesting an important role for this caspase in pathophysiology of acute brain trauma

Insights into household transmission of SARS-CoV-2 from a population-based serological survey

Understanding the risk of infection from household- and community-exposures and the transmissibility of asymptomatic infections is critical to SARS-CoV-2 control. Limited previous evidence is based primarily on virologic testing, which disproportionately misses mild and asymptomatic infections. Serologic measures are more likely to capture all previously infected individuals. We apply household transmission models to data from a cross-sectional, household-based population serosurvey of 4,534 people ≥5 years from 2,267 households enrolled April-June 2020 in Geneva, Switzerland. We found that the risk of infection from exposure to a single infected household member aged ≥5 years (17.3%,13.7-21.7) was more than three-times that of extra-household exposures over the first pandemic wave (5.1%,4.5-5.8). Young children had a lower risk of infection from household members. Working-age adults had the highest extra-household infection risk. Seropositive asymptomatic household members had 69.4% lower odds (95%CrI,31.8-88.8%) of infecting another household member compared to those reporting symptoms, accounting for 14.5% (95%CrI, 7.2-22.7%) of all household infections