Biology subjects

Sankaran, S.

Publications and source records attributed to Sankaran, S..

28 records · Page 2Linked to original sources

bioRxiv2022

Light-regulated pro-angiogenic engineered living materials

I.Despite their promise, the application of growth factors in regenerative medicine is limited by their poor stability in the body, high costs of production/storage and need for localized and tightly controlled delivery to minimize adverse side effects. In this study, a unique strategy to overcome these limitations is explored based on engineered living materials (ELMs). These are an emerging class of composite materials, which contain live microorganisms that can be engineered to produce and secrete proteins in response to external stimuli. Herein, the development of an ELM that light-responsively releases a pro-angiogenic protein is described. This is achieved by optogenetically engineering bacteria to synthesize and secrete a fusion protein containing a vascular endothelial growth factor peptidomimetic linked to a collagen-binding domain. The bacteria are securely encapsulated in bilayer hydrogel constructs that support bacterial functionality but prevent their escape from the ELM. The possibility to switch protein release ON and OFF with light and to tune the amount released with different light intensities is demonstrated. Finally, it is shown that the released protein is active through its ability to bind to collagen and promote angiogenic network formation in human vascular endothelial cell cultures, indicating the regenerative potential of these ELMs.

bioengineering↗

bioRxiv2022

Optical Metabolic Imaging Uncovers Sex- and Diet-dependent Lipid Changes in Aging Drosophila Brain

Aging is associated with progressive declines in physiological integrity and functions alongside increases in vulnerability to develop a number of diseases. The brain regulates sensory and motor functions as well as endocrine functions, and age-associated changes in brain are likely prerequisite for the organismal aging. Lipid metabolism has been associated with brain aging, which could be easily intervened by diets and lifestyles. However, the underlying mechanism through which brain lipid metabolism is regulated by diet during aging is elusive. Using stimulated Raman scattering (SRS) imaging combined with deuterium water (D2O) labeling, we visualized that lipid metabolic activities were changed by diet manipulation in aging Drosophila brain. Furthermore, we illuminated that insulin/IGF-1 signaling (IIS) pathway mediates the transformation of brain lipid metabolic changes in both an aging- and a diet-dependent manner. The lipid droplets (LDs) in the brain gradually became inert in both activities of lipid synthesis and mobilization with aging. High sugar diets enhanced the metabolic activity through promoting lipogenesis while dietary restriction increased the metabolic activity in both lipogenesis and lipolysis in brain LDs. However, these effects were impaired in both chico1/+ and dfoxo Drosophila mutants. We also observed that old chico1/+ brains maintained high metabolic activities, whilst the aged dfoxo brains acted exactly the opposite. More interestingly, the sexual dimorphism in brain lipid metabolism was impaired under diet regulation in both chico1/+ and dfoxo mutants. Locally reduced IIS activity in glial cells can mimic the systemic changes in systematic IIS mutants to maintain lipogenesis and lipolysis in aged brains, providing mechanistic insight into the anti-aging effects of IIS pathway. Our results highlight the manipulation of glia-specific IIS activity as a promising strategy in anti-aging treatments.

bioengineering↗

bioRxiv2022

Encapsulation of bacteria in bilayer Pluronic thin film hydrogels: a safe format for engineered living materials

In engineered living materials (ELMs) non-living matrices encapsulate microorganisms to acquire capabilities like sensing or biosynthesis. The confinement of the organisms to the matrix and the prevention of overgrowth and escape during the lifetime of the material is necessary for the application of ELMs into real devices. In this study, a bilayer thin film hydrogel of Pluronic F127 and Pluronic F127 acrylate polymers supported on a solid substrate is introduced. The inner hydrogel layer contains genetically engineered bacteria and supports their growth, while the outer layer acts as an envelope and does not allow leakage of the living organisms outside of the film for at least 15 days. Due to the flat and transparent nature of the construct, the thin layer is suited for microscopy and spectroscopy-based analyses. The composition and properties of the inner and outer layer are adjusted independently to fulfil viability and confinement requirements. We demonstrate that bacterial growth and light-induced protein production are possible in the inner layer and their extent is influenced by the crosslinking degree of the used hydrogel. Bacteria inside the hydrogel are viable long term, they can act as lactate-sensors and remain active after storage in phosphate buffer at room temperature for at least 3 weeks. The versatility of bilayer bacteria thin-films is attractive for fundamental studies and for the development of application-oriented ELMs.

bioengineering↗

bioRxiv2022

In vitro evaluation of immune responses to bacterial hydrogels for the development of living therapeutic materials

In living therapeutic materials, organisms genetically programmed to produce and deliver drugs are encapsulated in porous matrices or hydrogels acting as physical barriers between the therapeutic organisms and the host cells. The therapeutic potential of such constructs has been highlighted in in vitro studies, but the translation to in vivo scenarios requires evaluation of the immune response to the presence of the encapsulated, living organisms. In this study, we investigate the responses of human peripheral blood mononuclear cells (PBMCs) exposed to a living therapeutic material consisting of engineered E. coli encapsulated in Pluronic F127-based hydrogels. The release of inflammation-related cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, TNF and IFN{gamma}) and cytotoxic proteins (granzyme A, granzyme B, perforin, granulysin, sFas, and sFasL) in response to the bacterial hydrogels, as well as the subsets of natural killer cells and T cells after exposure to the bacterial hydrogel for up to three days were examined. In direct contact with PBMCs, both E. coli and its endotoxin-free variant, ClearColi, induce apoptosis of the immune cells and trigger IL-6 release from the surviving cells. However, we found that encapsulation of the bacteria in Pluronic F127 diacrylate hydrogels considerably lowers their immunogenicity and practically abolishes apoptosis triggered by ClearColi. In comparison with E. coli, free and hydrogel-encapsulated ClearColi induced significantly lower levels of NK cell differentiation into the more cytolytic CD16dim subset. Our results demonstrate that ClearColi-encapsulated hydrogels generate low immunogenic response and are suitable candidates for the development of living therapeutic materials for in vivo testing to assess a potential clinical use. Nevertheless, we also observed a stronger immune response in pro-inflammatory PBMCs, possibly from donors with underlying infections. This suggests that including anti-inflammatory measures in living therapeutic material designs could be beneficial for such recipients.

immunology↗

bioRxiv2022

Gibson Assembly-based direct cloning of plasmid DNA in Lactiplantibacillus plantarum WCSF1

Lactobacilli are gram-positive bacteria that are growing in importance for the healthcare industry and genetically engineering them as living therapeutics is highly sought after. However, progress in this field is hindered since most strains are difficult to genetically manipulate, partly due since their complex and thick cell walls limit our capability to transform them with exogenous DNA. To overcome this, large amounts of DNA (>1 g) are normally required to successfully transform these bacteria. An intermediate host, like E. coli, is often used to amplify recombinant DNA to such amounts although this approach poses unwanted drawbacks such as increase in plasmid size, different methylation patterns and limitation of introducing only genes compatible with the intermediate host. In this work, we have developed a direct cloning method based on Gibson assembly and PCR to amplify the DNA to sufficient quantities for successful transformation in L. plantarum WCFS1. This advantage of this method is demonstrated in terms of shorter experimental duration and the possibility to introduce a gene incompatible with E. coli into L. plantarum WCFS1.

synthetic biology↗

bioRxiv2022

Novel genetic modules encoding high-level antibiotic-free protein expression in probiotic lactobacilli

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, that 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 tunable 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.

synthetic biology↗

bioRxiv2022

Benzopyrene biodegradation by multiple and individual mesophilic bacteria in axenic conditions and in soil samples

Thus far, only a handful of bacterial strains that can independently degrade and utilize benzo[a]pyrene (BaP) as the sole carbon source have been isolated and characterized. Here, three new bacterial strains, JBZ1A, JBZ2B, and JBZ5E, were isolated from contaminated soil and, using 16S rRNA sequencing, were identified as Bradyrhizobium japonicum, Micrococcus luteus, and Bacillus cereus, respectively. The growth ability of each individual strain and a consortium of all strains in the presence of BaP (4-400 {micro}mol{middle dot}l-1, pH 7, 37{degrees}C) was identified by the doubling time (dt). The results illustrated that dt decreased with increasing BaP concentrations for individual strain and the consortium. The optimum growth conditions of the consortium were 37{degrees}C, 0.5% NaCl (w/v), and pH 7. Under these conditions, the degradation rate was 1.06 {micro}mol{middle dot}l-1{middle dot}day-1, whereas that of individual strains ranged 0.9-0.38 {micro}mol{middle dot}l-1{middle dot}day-1. B. cereus had the strongest contribution to the consortiums activity, with a degradation rate of 0.9 {micro}mol{middle dot}l-1{middle dot}day-1. The consortium could also remove BaP spiked with soil, but at a lower rate (0.01 {micro}mol.l-1.day-1). High-performance liquid chromatography-high-resolution tandem mass spectrometry permitted the detection of the metabolites of these strains, and a biodegradation pathway has been proposed.

microbiology↗

bioRxiv2022

Regulating bacterial behavior within hydrogels of tunable viscoelasticity

Engineered living materials (ELMs) are a new class of materials in which living organism incorporated into diffusive matrices uptake a fundamental role in materials composition and function. Understanding how the spatial confinement in 3D affects the behavior of the embedded cells is crucial to design and predict ELMs function, regulate and minimize their environmental impact and facilitate their translation into applied materials. This study investigates the growth and metabolic activity of bacteria within an associative hydrogel network (Pluronic-based) with mechanical properties that can be tuned by introducing a variable degree of acrylate crosslinks. Individual bacteria distributed in the hydrogel matrix at low density form functional colonies whose size is controlled by the extent of permanent crosslinks. With increasing stiffness and decreasing plasticity of the matrix, a decrease in colony volumes and an increase in their sphericity is observed. Protein production surprisingly follows a different pattern with higher production yields occurring in networks with intermediate permanent crosslinking degrees. These results demonstrate that, bacterial mechanosensitivity can be used to control and regulate the composition and function of ELMs by thoughtful design of the encapsulating matrix, and by following design criteria with interesting similarities to those developed for 3D culture of mammalian cells.

bioengineering↗

bioRxiv2019

Exogenous supply of Hsp47 triggers fibrillar collagen deposition in skin cell cultures in vitro

BackgroundCollagen is a structural protein that provides mechanical stability and defined architectures to skin. In collagen-based skin disorders like Epidermolysis bullosa, EDS the ability to offer such stability is lost either due to mutations in collagens or defect in the chaperones involved in collagen assembly, which leads to chronic wounds, skin fragility, and blisters. Existing approaches to study and develop therapy against such conditions are the use of small molecules like 4-phenylbutyrate (4-PBA) or growth factors like TGF-{beta}. However, these approaches are not collagen specific resulting in unsolicited responses. Therefore, a collagen specific booster is required to guide the correct folding and deposition of collagen in a highly regulated manner. Hsp47 is a chaperone with a major role in collagen biosynthesis. Expression levels of Hsp47 correlate with collagen production. This article explores the stimulation of collagen deposition by exogenously supplied Hsp47 (collagen specific chaperone) in skin cells, including specific collagen subtypes quantification.\n\nResultsHere we quantify the collagen deposition level and the type of deposited collagens by different cell types from skin tissue (fibroblasts NHDF, L929 and MEF, keratinocytes HaCat and endothelial cells HDMEC) after Hsp47 stimulation. We find upregulated deposition of fibrillar collagen subtypes I, III and V after Hsp47 delivery. Network collagen IV deposition was enhanced in HaCat and HDMECs and fibril-associated collagen XII were not affected by the increased Hsp47 intracellular levels. The deposition levels of fibrillar collagen were cell-dependent i.e. Hsp47-stimulated fibroblasts deposited significantly higher amount of fibrillar collagen than Hsp47-stimulated HaCat and HDMECs.\n\nConclusionsA 3-fold enhancement of collagen deposition was observed in fibroblasts upon repeated dosage of Hsp47 within the first 6 days of culture. Our results provide fundamental understanding towards the idea of using Hsp47 as therapeutic protein to treat collagen disorders.

cell biology↗

bioRxiv2017

Patchiness and scale-free correlations: characterising criticality in ecosystems.

I.O_LIIn diverse ecosystems, organisms cluster together in such a manner that the frequency distribution of cluster sizes is a power-law function. Spatially-explicit models of ecosystems suggest that loss of such power-law clustering may indicate loss of ecosystem resilience. Hence, it is hypothesised that spatial clustering properties in ecosystems - which can be readily measured using remotely-sensed high-resolution data - can help infer proximity to ecosystem thresholds and may even provide early warning signals of ecosystem collapse. Recent empirical and simulation studies, however, dont find consistent relationships between spatial clustering and ecosystem resilience. Furthermore, how spatial clustering metrics relate to other well-known early warning signals of ecosystems collapse, specifically the phenomenon of critical slowing down (CSD), remains unclear. C_LIO_LIWe synthesize the literature on cluster sizes in empirical and theoretical studies that show how local interactions (especially, positive feedback) among organisms can cause power-law clustering. In addition, we analyse a minimal spatial model of ecosystem transitions that allows us to disentangle the role of environmental stressor and positive feedback on spatial patterns and ecosystem resilience. C_LIO_LIOur literature synthesis reveals that empirically observed power-law clustering in ecosystems is parsimoniously explained by local positive feedback. Our synthesis together with model analysis demonstrates that, depending on the strength of positive feedback, emergence of power-law clustering can occur at any distance from the critical threshold of ecosystem collapse. In fact, we find that for systems with strong positive feedbacks, which are most likely to exhibit abrupt transitions, loss of power-law clustering may not even occur prior to ecosystem thresholds. We also argue that cluster-size distributions are unrelated to the phenomenon of CSD. C_LIO_LIWe demonstrate that, due to CSD, a power-law feature does occur near critical thresholds but in a different quantity; specifically, a power-law decay of spatial correlations of ecosystem state. C_LIO_LIWe conclude that loss of power-law clustering cannot be used as a reliable indicator of ecosystem resilience. Our synthesis and model analyses highlights links between local positive feedback, emergent spatial properties and how they may be used to interpret ecosystem resilience. C_LI

ecology↗