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    Novel genetic modules encoding high-level antibiotic-free protein expression in probiotic lactobacilli
    (Oxford : Wiley-Blackwell, 2023) Dey, Sourik; Blanch‐Asensio, Marc; Balaji Kuttae, Sanjana; Sankaran, Shrikrishnan
    Lactobacilli are ubiquitous in nature, often beneficially associated with animals as commensals and probiotics, and are extensively used in food fermentation. Due to this close-knit association, there is considerable interest to engineer them for healthcare applications in both humans and animals, for which high-performance and versatile genetic parts are greatly desired. For the first time, we describe two genetic modules in Lactiplantibacillus plantarum that achieve high-level gene expression using plasmids that can be retained without antibiotics, bacteriocins or genomic manipulations. These include (i) a promoter, PtlpA, from a phylogenetically distant bacterium, Salmonella typhimurium, which drives up to 5-fold higher level of gene expression compared to previously reported promoters and (ii) multiple toxin-antitoxin systems as a self-contained and easy-to-implement plasmid retention strategy that facilitates the engineering of tuneable transient genetically modified organisms. These modules and the fundamental factors underlying their functionality that are described in this work will greatly contribute to expanding the genetic programmability of lactobacilli for healthcare applications.
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    Expanding the genetic programmability of Lactiplantibacillus plantarum
    (Oxford : Wiley-Blackwell, 2024) Blanch‐Asensio, Marc; Dey, Sourik; Tadimarri, Varun Sai; Sankaran, Shrikrishnan
    Lactobacilli are ubiquitous in nature and symbiotically provide health benefits for countless organisms including humans, animals and plants. They are vital for the fermented food industry and are being extensively explored for healthcare applications. For all these reasons, there is considerable interest in enhancing and controlling their capabilities through the engineering of genetic modules and circuits. One of the most robust and reliable microbial chassis for these synthetic biology applications is the widely used Lactiplantibacillus plantarum species. However, the genetic toolkit needed to advance its applicability remains poorly equipped. This mini-review highlights the genetic parts that have been discovered to achieve food-grade recombinant protein production and speculates on lessons learned from these studies for L. plantarum engineering. Furthermore, strategies to identify, create and optimize genetic parts for real-time regulation of gene expression and enhancement of biosafety are also suggested.
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    Human glabrous skin contains crystallized urea dendriform structures in the stratum corneum which affect the hydration levels
    (Oxford : Wiley-Blackwell, 2023) Infante, Victor Hugo Pacagnelli; Bennewitz, Roland; Kröger, Marius; Meinke, Martina C.; Darvin, Maxim E.
    Glabrous skin is hair-free skin with a high density of sweat glands, which is found on the palms, and soles of mammalians, covered with a thick stratum corneum. Dry hands are often an occupational problem which deserves attention from dermatologists. Urea is found in the skin as a component of the natural moisturizing factor and of sweat. We report the discovery of dendrimer structures of crystalized urea in the stratum corneum of palmar glabrous skin using laser scanning microscopy. The chemical and structural nature of the urea crystallites was investigated in vivo by non-invasive techniques. The relation of crystallization to skin hydration was explored. We analysed the index finger, small finger and tenar palmar area of 18 study participants using non-invasive optical methods, such as laser scanning microscopy, Raman microspectroscopy and two-photon tomography. Skin hydration was measured using corneometry. Crystalline urea structures were found in the stratum corneum of about two-thirds of the participants. Participants with a higher density of crystallized urea structures exhibited a lower skin hydration. The chemical nature and the crystalline structure of the urea were confirmed by Raman microspectroscopy and by second harmonic generated signals in two-photon tomography. The presence of urea dendrimer crystals in the glabrous skin seems to reduce the water binding capacity leading to dry hands. These findings highlight a new direction in understanding the mechanisms leading to dry hands and open opportunities for the development of better moisturizers and hand disinfection products and for diagnostic of dry skin.