Coupling Peptide-Based Encapsulation of Enzymes with Bacteria for Paraoxon Bioremediation.

The catalytic efficiency of enzymes can be harnessed as an environmentally friendly solution for decontaminating various xenobiotics and toxins. However, for some xenobiotics, several enzymatic steps are needed to obtain nontoxic products. Another challenge is the low durability and stability of many native enzymes in their purified form. Herein, we coupled peptide-based encapsulation of bacterial phosphotriesterase with soil-originated bacteria, Arthrobacter sp. 4Hß as an efficient system capable of biodegradation of paraoxon, a neurotoxin pesticide. Specifically, recombinantly expressed and purified methyl parathion hydrolase (MPH), with high hydrolytic activity toward paraoxon, was encapsulated within peptide nanofibrils, resulting in increased shelf life and retaining ∼50% activity after 132 days since purification. Next, the addition of Arthrobacter sp. 4Hß, capable of degrading para-nitrophenol (PNP), the hydrolysis product of paraoxon, which is still toxic, resulted in nondetectable levels of PNP. These results present an efficient one-pot system that can be further developed as an environmentally friendly solution, coupling purified enzymes and native bacteria, for pesticide bioremediation. We further suggest that this system can be tailored for different xenobiotics by encapsulating the rate-limiting key enzymes followed by their combination with environmental bacteria that can use the enzymatic step products for full degradation without the need to engineer synthetic bacteria.

Data-driven digital health technologies in the remote clinical care of diabetic foot ulcers: a scoping review.

Background: The availability and effectiveness of Digital Health Technologies (DHTs) to support clinicians, empower patients, and generate economic savings for national healthcare systems are growing rapidly. Of particular promise is the capacity of DHTs to autonomously facilitate remote monitoring and treatment. Diabetic Foot Ulcers (DFUs) are characterised by high rates of infection, amputation, mortality, and healthcare costs. With clinical outcomes contingent on activities that can be readily monitored, DFUs present a promising focus for the application of remote DHTs. Objective: This scoping review has been conducted as a first step toward ascertaining fthe data-related challenges and opportunities for the development of more comprehensive, integrated, and individualised sense/act DHTs. We review the latest developments in the application of DHTs to the remote care of DFUs. We cover the types of DHTs in development and their features, technological readiness, and scope of clinical testing. Eligibility criteria: Only peer-reviewed original experimental and observational studies, case series and qualitative studies were included in literature searches. All reviews and manuscripts presenting pre-trial prototype technologies were excluded. Methods: An initial search of three databases (Web of Science, MEDLINE, and Scopus) generated 1,925 English-language papers for screening. 388 papers were assessed as eligible for full-text screening by the review team. 81 manuscripts were found to meet the eligibility criteria. Results: Only 19% of studies incorporated multiple DHTs. We categorised 56% of studies as 'Treatment-Manual', i.e. studies involving technologies aimed at treatment requiring manual data generation, and 26% as 'Prevention-Autonomous', i.e. studies of technologies generating data autonomously through wearable sensors aimed at ulcer prevention through patient behavioural change. Only 10% of studies involved more ambitious 'Treatment-Autonomous' interventions. We found that studies generally reported high levels of patient adherence and satisfaction. Conclusions: Our findings point to a major potential role for DHTs in remote personalised medical management of DFUs. However, larger studies are required to assess their impact. Here, we see opportunities for developing much larger, more comprehensive, and integrated monitoring and decision support systems with the potential to address the disease in a more complete context by capturing and integrating data from multiple sources from subjective and objective measurements.

Fluorescence-Activated Cell Sorting and Quantitative Real-Time PCR to Reveal VEGF-Expressing Macrophage Populations in the Zebrafish Larvae.

The transparent, genetically tractable zebrafish is increasingly recognized as a useful model to both live image and uncover mechanistic insight into cell interactions governing tissue homeostasis, pathology, and regeneration. Here, we describe a protocol for the isolation of macrophages from zebrafish wounds using fluorescence-activated cell sorting (FACS), and the identification of specific pro-angiogenic macrophage populations that express high levels of vascular endothelial growth factor (vegf) using quantitative real-time PCR (qPCR). The cell dissociation and FACS sorting techniques have been optimized for immune cells and successfully used to isolate other fluorescently marked populations within the wound such as neutrophils and endothelial cells. More broadly, this protocol can be easily adapted to other contexts where identification of pro-angiogenic immune cells is transformative for understanding, from development to pathologies such as infection, cancer, and diabetes.

Mining Marine Metagenomes Revealed a Quorum-Quenching Lactonase with Improved Biochemical Properties That Inhibits the Food Spoilage Bacterium Pseudomonas fluorescens.

The marine environment presents great potential as a source of microorganisms that possess novel enzymes with unique activities and biochemical properties. Examples of such are the quorum-quenching (QQ) enzymes that hydrolyze bacterial quorum-sensing (QS) signaling molecules, such as N-acyl-homoserine lactones (AHLs). QS is a form of cell-to-cell communication that enables bacteria to synchronize gene expression in correlation with population density. Searching marine metagenomes for sequences homologous to an AHL lactonase from the phosphotriesterase-like lactonase (PLL) family, we identified new putative AHL lactonases (sharing 30 to 40% amino acid identity to a thermostable PLL member). Phylogenetic analysis indicated that these putative AHL lactonases comprise a new clade of marine enzymes in the PLL family. Following recombinant expression and purification, we verified the AHL lactonase activity for one of these proteins, named moLRP (marine-originated lactonase-related protein). This enzyme presented greater activity and stability at a broad range of temperatures and pH, tolerance to high salinity levels (up to 5 M NaCl), and higher durability in bacterial culture, compared to another PLL member, parathion hydrolase (PPH). The addition of purified moLRP to cultures of Pseudomonas fluorescens inhibited its extracellular protease activity, expression of the protease encoding gene, biofilm formation, and the sedimentation process in milk-based medium. These findings suggest that moLRP is adapted to the marine environment and can potentially serve as an effective QQ enzyme, inhibiting the QS process in Gram-negative bacteria involved in food spoilage. IMPORTANCE Our results emphasize the potential of sequence and structure-based identification of new QQ enzymes from environmental metagenomes, such as from the ocean, with improved stability or activity. The findings also suggest that purified QQ enzymes can present new strategies against food spoilage, in addition to their recognized involvement in inhibiting bacterial pathogen virulence factors. Future studies on the delivery and safety of enzymatic QQ strategy against bacterial food spoilage should be performed.

Endothelial Heterogeneity in Development and Wound Healing.

The vasculature is comprised of endothelial cells that are heterogeneous in nature. From tissue resident progenitors to mature differentiated endothelial cells, the diversity of these populations allows for the formation, maintenance, and regeneration of the vascular system in development and disease, particularly during situations of wound healing. Additionally, the de-differentiation and plasticity of different endothelial cells, especially their capacity to undergo endothelial to mesenchymal transition, has also garnered significant interest due to its implication in disease progression, with emphasis on scarring and fibrosis. In this review, we will pinpoint the seminal discoveries defining the phenotype and mechanisms of endothelial heterogeneity in development and disease, with a specific focus only on wound healing.

Macrophage regulation of angiogenesis in health and disease.

Macrophages are primarily known as phagocytic innate immune cells, but are, in fact, highly dynamic multi-taskers that interact with many different tissue types and have regulatory roles in development, homeostasis, tissue repair, and disease. In all of these scenarios angiogenesis is pivotal and macrophages appear to play a key role in guiding both blood vessel sprouting and remodelling wherever that occurs. Recent studies have explored these processes in a diverse range of models utilising the complementary strengths of rodent, fish and tissue culture studies to unravel the mechanisms underlying these interactions and regulatory functions. Here we discuss how macrophages regulate angiogenesis and its resolution as embryonic tissues grow, as well as their parallel and different functions in repairing wounds and in pathologies, with a focus on chronic wounds and cancer.

Live-imaging of endothelial Erk activity reveals dynamic and sequential signalling events during regenerative angiogenesis.

The formation of new blood vessel networks occurs via angiogenesis during development, tissue repair, and disease. Angiogenesis is regulated by intracellular endothelial signalling pathways, induced downstream of vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). A major challenge in understanding angiogenesis is interpreting how signalling events occur dynamically within endothelial cell populations during sprouting, proliferation, and migration. Extracellular signal-regulated kinase (Erk) is a central downstream effector of Vegf-signalling and reports the signalling that drives angiogenesis. We generated a vascular Erk biosensor transgenic line in zebrafish using a kinase translocation reporter that allows live-imaging of Erk-signalling dynamics. We demonstrate the utility of this line to live-image Erk activity during physiologically relevant angiogenic events. Further, we reveal dynamic and sequential endothelial cell Erk-signalling events following blood vessel wounding. Initial signalling is dependent upon Ca2+ in the earliest responding endothelial cells, but is independent of Vegfr-signalling and local inflammation. The sustained regenerative response, however, involves a Vegfr-dependent mechanism that initiates concomitantly with the wound inflammatory response. This work reveals a highly dynamic sequence of signalling events in regenerative angiogenesis and validates a new resource for the study of vascular Erk-signalling in real-time.

Directed Enzyme Evolution and Encapsulation in Peptide Nanospheres of Quorum Quenching Lactonase as an Antibacterial Treatment against Plant Pathogen.

The need to increase agricultural yield has led to an extensive use of antibiotics against plant pathogens, which has resulted in the emergence of resistant strains. Therefore, there is an increasing demand for new methods, preferably with lower chances of developing resistant strains and a lower risk to the environment or public health. Many Gram-negative bacterial pathogens use quorum sensing, a population-density-dependent regulatory mechanism, to monitor the secretion of N-acyl-homoserine lactones (AHLs) and pathogenicity. Therefore, quorum sensing represents an attractive antivirulence target. AHL lactonases hydrolyze AHLs and have potential antibacterial properties; however, their use is limited by thermal instability and durability, or low activity. Here, we demonstrate that an AHL lactonase from the phosphotriesterase-like lactonase family exhibits high activity with the AHL secreted from the plant pathogen Erwinia amylovora and attenuates infection in planta. Using directed enzyme evolution, we were able to increase the enzyme's temperature resistance (T50, the temperature at which 50% of the activity is retained) by 8 °C. Then, by performing enzyme encapsulation in nanospherical capsules composed of tertbutoxycarbonyl-Phe-Phe-OH peptide, the shelf life was extended for more than 5 weeks. Furthermore, the encapsulated and free mutant were able to significantly inhibit up to 70% blossom's infection in the field, achieving the same efficacy as seen with antibiotics commonly used today to treat the plant pathogen. We conclude that specific AHL lactonase can inhibit E. amylovora infection in the field, as it degrades the AHL secreted by this plant pathogen. The combination of directed enzyme evolution and peptide nanostructure encapsulation significantly improved the thermal resistance and shelf life of the enzyme, respectively, increasing its potential in future development as antibacterial treatment.

In Vitro Coculture Assays of Angiogenesis.

During angiogenesis, endothelial cells must undergo a coordinated set of morphological changes in order to form a new vessel. There is a need for endothelial cells to communicate with each other in order to take up different identities in the sprout and to migrate collectively as a connected chord. Endothelial cells must also interact with a wide range of other cells that contribute to vessel formation. In ischemic disease, hypoxic cells in tissue will generate proangiogenic signals that promote and guide angiogenesis. In solid tumors, this function is co-opted by tumor cells, which make a complex range of interactions with endothelial cells, even integrating into the walls of vessels. In vessel repair, cells from the immune system contribute to the promotion and remodeling of new vessels. The coculture angiogenesis assay is a long-term in vitro protocol that uses fibroblasts to secrete and condition an artificial stromal matrix for tubules to grow through. We show here how the assay can be easily adapted to include additional cell types, facilitating the study of cellular interactions during neovascularization.

Live imaging the foreign body response in zebrafish reveals how dampening inflammation reduces fibrosis.

Implanting biomaterials in tissues leads to inflammation and a foreign body response (FBR), which can result in rejection. Here, we live image the FBR triggered by surgical suture implantation in a translucent zebrafish model and compare with an acute wound response. We observe inflammation extending from the suture margins, correlating with subsequent avascular and fibrotic encapsulation zones: sutures that induce more inflammation result in increased zones of avascularity and fibrosis. Moreover, we capture macrophages as they fuse to become multinucleate foreign body giant cells (FBGCs) adjacent to the most pro-inflammatory sutures. Genetic and pharmacological dampening of the inflammatory response minimises the FBR (including FBGC generation) and normalises the status of the tissue surrounding these sutures. This model of FBR in adult zebrafish allows us to live image the process and to modulate it in ways that may lead us towards new strategies to ameliorate and circumvent FBR in humans.This article has an associated First Person interview with the first author of the paper.

Fine-scale substrate heterogeneity does not affect arthropod communities on green roofs.

Green roofs, which are roofs with growing substrate and vegetation, can provide habitat for arthropods in cities. Maintaining a diversity of arthropods in an urban environment can enhance the functions they fill, such as pest control and soil development. Theory suggests that the creation of a heterogeneous environment on green roofs would enhance arthropod diversity. Several studies have examined how arthropod diversity can be enhanced on green roofs, and particularly whether substrate properties affect the arthropod community, but a gap remains in identifying the effect of substrate heterogeneity within a green roof on the arthropod community. In this paper, it is hypothesized that creating heterogeneity in the substrate would directly affect the diversity and abundance of some arthropod taxa, and indirectly increase arthropod diversity through increased plant diversity. These hypotheses were tested using green roof plots in four treatments of substrate heterogeneity: (1) homogeneous dispersion; (2) mineral heterogeneity-with increased tuff concentration in subplots; (3) organic heterogeneity-with decreased compost concentrations in subplots; (4) both mineral and organic heterogeneity. Each of the four treatments was replicated twice on each of three roofs (six replicates per treatment) in a Mediterranean region. There was no effect of substrate heterogeneity on arthropod diversity, abundance, or community composition, but there were differences in arthropod communities among roofs. This suggests that the location of a green roof, which can differ in local climatic conditions, can have a strong effect on the composition of the arthropod community. Thus, arthropod diversity may be promoted by building green roofs in a variety of locations throughout a city, even if the roof construction is similar on all roofs.

Cancer cell adaptation to hypoxia involves a HIF-GPRC5A-YAP axis.

Hypoxia is a hallmark of solid tumours and a key physiological feature distinguishing cancer from normal tissue. However, a major challenge remains in identifying tractable molecular targets that hypoxic cancer cells depend on for survival. Here, we used SILAC-based proteomics to identify the orphan G protein-coupled receptor GPRC5A as a novel hypoxia-induced protein that functions to protect cancer cells from apoptosis during oxygen deprivation. Using genetic approaches in vitro and in vivo, we reveal HIFs as direct activators of GPRC5A transcription. Furthermore, we find that GPRC5A is upregulated in the colonic epithelium of patients with mesenteric ischaemia, and in colorectal cancers high GPRC5A correlates with hypoxia gene signatures and poor clinical outcomes. Mechanistically, we show that GPRC5A enables hypoxic cell survival by activating the Hippo pathway effector YAP and its anti-apoptotic target gene BCL2L1 Importantly, we show that the apoptosis induced by GPRC5A depletion in hypoxia can be rescued by constitutively active YAP. Our study identifies a novel HIF-GPRC5A-YAP axis as a critical mediator of the hypoxia-induced adaptive response and a potential target for cancer therapy.

Live imaging of wound angiogenesis reveals macrophage orchestrated vessel sprouting and regression.

Wound angiogenesis is an integral part of tissue repair and is impaired in many pathologies of healing. Here, we investigate the cellular interactions between innate immune cells and endothelial cells at wounds that drive neoangiogenic sprouting in real time and in vivo Our studies in mouse and zebrafish wounds indicate that macrophages are drawn to wound blood vessels soon after injury and are intimately associated throughout the repair process and that macrophage ablation results in impaired neoangiogenesis. Macrophages also positively influence wound angiogenesis by driving resolution of anti-angiogenic wound neutrophils. Experimental manipulation of the wound environment to specifically alter macrophage activation state dramatically influences subsequent blood vessel sprouting, with premature dampening of tumour necrosis factor-α expression leading to impaired neoangiogenesis. Complementary human tissue culture studies indicate that inflammatory macrophages associate with endothelial cells and are sufficient to drive vessel sprouting via vascular endothelial growth factor signalling. Subsequently, macrophages also play a role in blood vessel regression during the resolution phase of wound repair, and their absence, or shifted activation state, impairs appropriate vessel clearance.

Host-Biomaterial Interactions in Zebrafish.

Host-biomaterial interactions are critical determinants of the success or failure of an implant. However, detailed understanding of this process is limited due to a lack of dynamic tools for in vivo analyses. Here we characterize host-biomaterial interactions in zebrafish (Danio rerio), which are optically translucent and genetically tractable. Histological and immunohistochemical analyses following polypropylene suture implantation into adult zebrafish showed prolonged elevation of immune cell recruitment and collagen deposition, resembling a foreign body response. Live in vivo analysis showed that adsorption of the immunomodulatory cytokine interleukin-10 to a polystyrene microparticle, microinjected into transgenic larval zebrafish, inhibited neutrophil recruitment after 24 h compared to control microparticles, with no change in macrophage recruitment. This study illustrates that zebrafish are useful to investigate host-biomaterial interactions and have potential for high-throughput analysis of novel immunomodulatory biomaterials.

Muscle Stem Cells Undergo Extensive Clonal Drift during Tissue Growth via Meox1-Mediated Induction of G2 Cell-Cycle Arrest.

Organ growth requires a careful balance between stem cell self-renewal and lineage commitment to ensure proper tissue expansion. The cellular and molecular mechanisms that mediate this balance are unresolved in most organs, including skeletal muscle. Here we identify a long-lived stem cell pool that mediates growth of the zebrafish myotome. This population exhibits extensive clonal drift, shifting from random deployment of stem cells during development to reliance on a small number of dominant clones to fuel the vast majority of muscle growth. This clonal drift requires Meox1, a homeobox protein that directly inhibits the cell-cycle checkpoint gene ccnb1. Meox1 initiates G2 cell-cycle arrest within muscle stem cells, and disrupting this G2 arrest causes premature lineage commitment and the resulting defects in muscle growth. These findings reveal that distinct regulatory mechanisms orchestrate stem cell dynamics during organ growth, beyond the G0/G1 cell-cycle inhibition traditionally associated with maintaining tissue-resident stem cells.

Asymmetric division of clonal muscle stem cells coordinates muscle regeneration in vivo.

Skeletal muscle is an example of a tissue that deploys a self-renewing stem cell, the satellite cell, to effect regeneration. Recent in vitro studies have highlighted a role for asymmetric divisions in renewing rare "immortal" stem cells and generating a clonal population of differentiation-competent myoblasts. However, this model currently lacks in vivo validation. We define a zebrafish muscle stem cell population analogous to the mammalian satellite cell and image the entire process of muscle regeneration from injury to fiber replacement in vivo. This analysis reveals complex interactions between satellite cells and both injured and uninjured fibers and provides in vivo evidence for the asymmetric division of satellite cells driving both self-renewal and regeneration via a clonally restricted progenitor pool.

Skeletal myogenesis in the zebrafish and its implications for muscle disease modelling.

Current evidence indicates that post-embryonic muscle growth and regeneration in amniotes is mediated almost entirely by stem cells derived from muscle progenitor cells (MPCs), known as satellite cells. Exhaustion and impairment of satellite cell activity is involved in the severe muscle loss associated with degenerative muscle diseases such as Muscular Dystrophies and is the main cause of age-associated muscle wasting. Understanding the molecular and cellular basis of satellite cell function in muscle generation and regeneration (myogenesis) is critical to the broader goal of developing treatments that may ameliorate such conditions. Considerable knowledge exists regarding the embryonic stages of amniote myogenesis. Much less is known about how post-embryonic amniote myogenesis proceeds, how adult myogenesis relates to embryonic myogenesis on a cellular or genetic level. Of the studies focusing on post-embryonic amniote myogenesis, most are post-mortem and in vitro analyses, precluding the understanding of cellular behaviours and genetic mechanisms in an undisturbed in vivo setting. Zebrafish are optically clear throughout much of their post-embryonic development, facilitating their use in live imaging of cellular processes. Zebrafish also possess a compartment of MPCs, which appear similar to satellite cells and persist throughout the post-embryonic development of the fish, permitting their use in examining the contribution of these cells to muscle tissue growth and regeneration.

Haematopoietic stem cell induction by somite-derived endothelial cells controlled by meox1.

Haematopoietic stem cells (HSCs) are self-renewing stem cells capable of replenishing all blood lineages. In all vertebrate embryos that have been studied, definitive HSCs are generated initially within the dorsal aorta (DA) of the embryonic vasculature by a series of poorly understood inductive events. Previous studies have identified that signalling relayed from adjacent somites coordinates HSC induction, but the nature of this signal has remained elusive. Here we reveal that somite specification of HSCs occurs via the deployment of a specific endothelial precursor population, which arises within a sub-compartment of the zebrafish somite that we have defined as the endotome. Endothelial cells of the endotome are specified within the nascent somite by the activity of the homeobox gene meox1. Specified endotomal cells consequently migrate and colonize the DA, where they induce HSC formation through the deployment of chemokine signalling activated in these cells during endotome formation. Loss of meox1 activity expands the endotome at the expense of a second somitic cell type, the muscle precursors of the dermomyotomal equivalent in zebrafish, the external cell layer. The resulting increase in endotome-derived cells that migrate to colonize the DA generates a dramatic increase in chemokine-dependent HSC induction. This study reveals the molecular basis for a novel somite lineage restriction mechanism and defines a new paradigm in induction of definitive HSCs.

A myogenic precursor cell that could contribute to regeneration in zebrafish and its similarity to the satellite cell.

The cellular basis for mammalian muscle regeneration has been an area of intense investigation over recent decades. The consensus is that a specialized self-renewing stem cell, termed the satellite cell, plays a major role during the process of regeneration in amniotes. How broadly this mechanism is deployed within the vertebrate phylogeny remains an open question. A lack of information on the role of cells analogous to the satellite cell in other vertebrate systems is even more unexpected given the fact that satellite cells were first designated in frogs. An intriguing aspect of this debate is that a number of amphibia and many fish species exhibit epimorphic regenerative processes in specific tissues, whereby regeneration occurs by the dedifferentiation of the damaged tissue, without deploying specialized stem cell populations analogous to satellite cells. Hence, it is feasible that a cellular process completely distinct from that deployed during mammalian muscle regeneration could operate in species capable of epimorphic regeneration. In this minireview, we examine the evidence for the broad phylogenetic distribution of satellite cells. We conclude that, in the vertebrates examined so far, epimorphosis does not appear to be deployed during muscle regeneration, and that analogous cells expressing similar marker genes to satellite cells appear to be deployed during the regenerative process. However, the functional definition of these cells as self-renewing muscle stem cells remains a final hurdle to the definition of the satellite cell as a generic vertebrate cell type.

Development and evolution of the muscles of the pelvic fin.

Locomotor strategies in terrestrial tetrapods have evolved from the utilisation of sinusoidal contractions of axial musculature, evident in ancestral fish species, to the reliance on powerful and complex limb muscles to provide propulsive force. Within tetrapods, a hindlimb-dominant locomotor strategy predominates, and its evolution is considered critical for the evident success of the tetrapod transition onto land. Here, we determine the developmental mechanisms of pelvic fin muscle formation in living fish species at critical points within the vertebrate phylogeny and reveal a stepwise modification from a primitive to a more derived mode of pelvic fin muscle formation. A distinct process generates pelvic fin muscle in bony fishes that incorporates both primitive and derived characteristics of vertebrate appendicular muscle formation. We propose that the adoption of the fully derived mode of hindlimb muscle formation from this bimodal character state is an evolutionary innovation that was critical to the success of the tetrapod transition.