Promoter knock-in: a novel rational method for the fine tuning of genes

<p>Abstract</p> <p>Background</p> <p>Metabolic engineering aims at channeling the metabolic fluxes towards a desired compound. An important strategy to achieve this is the modification of the expression level of specific genes. Several methods for the modification or the replacement of promoters have been proposed, but most of them involve time-consuming screening steps. We describe here a novel optimized method for the insertion of constitutive promoters (referred to as "promoter knock-in") whose strength can be compared with the native promoter by applying a promoter strength predictive (PSP) model.</p> <p>Results</p> <p>Our method was successfully applied to fine tune the <it>ppc </it>gene of <it>Escherichia coli</it>. While developing the promoter knock-in methodology, we showed the importance of conserving the natural leader region containing the ribosome binding site (RBS) of the gene of interest and of eliminating upstream regulatory elements (transcription factor binding sites). The gene expression was down regulated instead of up regulated when the natural RBS was not conserved and when the upstream regulatory elements were eliminated. Next, three different promoter knock-ins were created for the <it>ppc </it>gene selecting three different artificial promoters. The measured constitutive expression of the <it>ppc </it>gene in these knock-ins reflected the relative strength of the different promoters as predicted by the PSP model. The applicability of our PSP model and promoter knock-in methodology was further demonstrated by showing that the constitutivity and the relative levels of expression were independent of the genetic background (comparing wild-type and mutant <it>E. coli </it>strains). No differences were observed during scaling up from shake flask to bioreactor-scale, confirming that the obtained expression was independent of environmental conditions.</p> <p>Conclusion</p> <p>We are proposing a novel methodology for obtaining appropriate levels of expression of genes of interest, based on the prediction of the relative strength of selected synthetic promoters combined with an optimized promoter knock-in strategy. The obtained expression levels are independent of the genetic background and scale conditions. The method constitutes therefore a valuable addition to the genetic toolbox for the metabolic engineering of <it>E. coli</it>.</p

Enabling NATO’s Collective Defense: Critical Infrastructure Security and Resiliency (NATO COE-DAT Handbook 1)

In 2014 NATO’s Center of Excellence-Defence Against Terrorism (COE-DAT) launched the inaugural course on “Critical Infrastructure Protection Against Terrorist Attacks.” As this course garnered increased attendance and interest, the core lecturer team felt the need to update the course in critical infrastructure (CI) taking into account the shift from an emphasis on “protection” of CI assets to “security and resiliency.” What was lacking in the fields of academe, emergency management, and the industry practitioner community was a handbook that leveraged the collective subject matter expertise of the core lecturer team, a handbook that could serve to educate government leaders, state and private-sector owners and operators of critical infrastructure, academicians, and policymakers in NATO and partner countries. Enabling NATO’s Collective Defense: Critical Infrastructure Security and Resiliency is the culmination of such an effort, the first major collaborative research project under a Memorandum of Understanding between the US Army War College Strategic Studies Institute (SSI), and NATO COE-DAT. The research project began in October 2020 with a series of four workshops hosted by SSI. The draft chapters for the book were completed in late January 2022. Little did the research team envision the Russian invasion of Ukraine in February this year. The Russian occupation of the Zaporizhzhya nuclear power plant, successive missile attacks against Ukraine’s electric generation and distribution facilities, rail transport, and cyberattacks against almost every sector of the country’s critical infrastructure have been on world display. Russian use of its gas supplies as a means of economic warfare against Europe—designed to undermine NATO unity and support for Ukraine—is another timely example of why adversaries, nation-states, and terrorists alike target critical infrastructure. Hence, the need for public-private sector partnerships to secure that infrastructure and build the resiliency to sustain it when attacked. Ukraine also highlights the need for NATO allies to understand where vulnerabilities exist in host nation infrastructure that will undermine collective defense and give more urgency to redressing and mitigating those fissures.https://press.armywarcollege.edu/monographs/1951/thumbnail.jp

Neurodegeneration of the retina in mouse models of Alzheimer’s disease: what can we learn from the retina?

Alzheimer’s disease (AD) is an age-related progressive neurodegenerative disease commonly found among elderly. In addition to cognitive and behavioral deficits, vision abnormalities are prevalent in AD patients. Recent studies investigating retinal changes in AD double-transgenic mice have shown altered processing of amyloid precursor protein and accumulation of β-amyloid peptides in neurons of retinal ganglion cell layer (RGCL) and inner nuclear layer (INL). Apoptotic cells were also detected in the RGCL. Thus, the pathophysiological changes of retinas in AD patients are possibly resembled by AD transgenic models. The retina is a simple model of the brain in the sense that some pathological changes and therapeutic strategies from the retina may be observed or applicable to the brain. Furthermore, it is also possible to advance our understanding of pathological mechanisms in other retinal degenerative diseases. Therefore, studying AD-related retinal degeneration is a promising way for the investigation on (1) AD pathologies and therapies that would eventually benefit the brain and (2) cellular mechanisms in other retinal degenerations such as glaucoma and age-related macular degeneration. This review will highlight the efforts on retinal degenerative research using AD transgenic mouse models

[Photo d'un dessin de combats avec autos-canons, liée à : Le tour du monde en guerre des autos-canons belges : 1915 - 1918]

Le dessin représente des soldats dans une tranchée, appuyés par des auto-canons, face à une attaque de fantassins. Marcel Thiry a publié dans le journal Le Soir "six articles qui sont devenus six chapitres" de l'ouvrage paru chez De Rache, selon son Avant-propos dans cet ouvrage (cfr MLA 7406 ou MLA 18710). Ce dessin y a été publié (p. 44) avec, en légende, les prénoms des illustrateurs