Assessing the potential application of bacteria-based self-healing cementitious materials for enhancing durability of wastewater treatment infrastructure

Wastewater treatment plants (WWTPs) around the world are mainly built using concrete. The continuous exposure to wastewater affects the durability of concrete structures and requires costly maintenance or replacement. Concrete production and repair represents ∼8% of the global anthropogenic CO2 emissions due to the use of cement, thus contributing to climate change. Developing a more sustainable cementitious material is therefore required for this vital health infrastructure. In this study, the feasibility of using bacteria-based self-healing (BBSH) cementitious materials for WWTPs is assessed by exposing BBSH mortar prisms to a continuous municipal wastewater flow and comparing their self-healing capacity to equivalent mortar prisms exposed to tap water. Microscopy imaging, water-flow tests and micro-CT analyses were performed to evaluate the self-healing efficiency of the mortar prisms, while SEM-EDX and Raman spectroscopy were used to characterise the healing products. Our work represents the first systematic study of the healing potential of BBSH in mortar exposed to wastewater. The results indicate that the purposely added bacteria are able to induce calcium carbonate precipitation when exposed to wastewater conditions. Moreover, if additional sources of calcium and carbon are embedded within the cement matrix, the rich bacterial community inherently present in the wastewater is capable of inducing calcium carbonate precipitation, even if no bacteria are purposely added to the mortar. The results of this study offer promising avenues for the construction of more sustainable wastewater infrastructure, with the potential of significantly reducing costs and simplifying the production process of BBSH concretes for this specific application

Impact of retrotransposon protein L1 ORF1p expression on oncogenic pathways in hepatocellular carcinoma: the role of cytoplasmic PIN1 upregulation

BACKGROUND: Molecular characterisation of hepatocellular carcinoma (HCC) is central to the development of novel therapeutic strategies for the disease. We have previously demonstrated mutagenic consequences of Long-Interspersed Nuclear Element-1 (LINE1s/L1) retrotransposition. However, the role of L1 in HCC, besides somatic mutagenesis, is not well understood. METHODS: We analysed L1 expression in the TCGA-HCC RNAseq dataset (n = 372) and explored potential relationships between L1 expression and clinical features. The findings were confirmed by immunohistochemical (IHC) analysis of an independent human HCC cohort (n = 48) and functional mechanisms explored using in vitro and in vivo model systems. RESULTS: We observed positive associations between L1 and activated TGFβ-signalling, TP53 mutation, alpha-fetoprotein and tumour invasion. IHC confirmed a positive association between pSMAD3, a surrogate for TGFβ-signalling status, and L1 ORF1p (P < 0.0001, n = 32). Experimental modulation of L1 ORF1p levels revealed an influence of L1 ORF1p on key hepatocarcinogenesis-related pathways. Reduction in cell migration and invasive capacity was observed upon L1 ORF1 knockdown, both in vitro and in vivo. In particular, L1 ORF1p increased PIN1 cytoplasmic localisation. Blocking PIN1 activity abrogated L1 ORF1p-induced NF-κB-mediated inflammatory response genes while further activated TGFβ-signalling confirming differential alteration of PIN1 activity in cellular compartments by L1 ORF1p. DISCUSSION: Our data demonstrate a causal link between L1 ORF1p and key oncogenic pathways mediated by PIN1, presenting a novel therapeutic avenue

Regulation of CHK1 inhibitor resistance by a c-Rel and USP1 dependent pathway

Previously, we discovered that deletion of c-Rel in the Em-Myc mouse model of lymphoma results in earlier onset of disease, a finding that contrasted with the expected function of this NF-κB subunit in B-cell malignancies. Here we report that Em-Myc/cRel−/− cells have an unexpected and major defect in the CHK1 pathway. Total and phospho proteomic analysis revealed that Em-Myc/cRel−/− lymphomas highly resemble wild-type (WT) Em-Myc lymphomas treated with an acute dose of the CHK1 inhibitor (CHK1i) CCT244747. Further analysis demonstrated that this is a consequence of Em-Myc/cRel−/ − lymphomas having lost expression of CHK1 protein itself, an effect that also results in resistance to CCT244747 treatment in vivo. Similar down-regulation of CHK1 protein levels was also seen in CHK1i resistant U2OS osteosarcoma and Huh7 hepatocellular carcinoma cells. Further investigation revealed that the deubiquitinase USP1 regulates CHK1 proteolytic degradation and that its down-regulation in our model systems is responsible, at least in part, for these effects. We demonstrate that treating WT Em-Myc lymphoma cells with the USP1 inhibitor ML323 was highly effective at reducing tumour burden in vivo. Targeting USP1 activity may thus be an alternative therapeutic strategy in MYC-driven tumours

Regulation of CHK1 inhibitor resistance by a c-Rel and USP1 dependent pathway

AbstractWe previously discovered that deletion of c-Rel in the Eμ-Myc mouse model of lymphoma results in earlier onset of disease, a finding that contrasted with the expected function of this NF-κB subunit in B-cell malignancies. Here we report that c-rel -/- Eµ-Myc cells have an unexpected and major defect in the CHK1 pathway, with almost undetectable levels of CHK1 and CLSPN protein leading to therapeutic resistance to the highly specific CHK1 inhibitor (CHK1i) CCT244747. Similar downregulation of CHK1 levels was also seen in CCT244747 resistant U20S osteosarcoma cells. Further investigation revealed that downregulation of the deubiquitinase USP1 is responsible, at least in part, for these effects. Importantly, we demonstrate that c-rel -/- Eµ-Myc lymphoma cells survive though upregulation of compensatory PI3K/AKT pathway activity. Moreover, targeting this pathway with Pictilisib (GDC-0941) effectively killed c-rel -/- Eµ-Myc in vivo, while having no effect on wild type Eμ-Myc cells. This data reveals an NF-κB regulated pathway controlling CHK1 activity in cancer cells and identifies a potential mechanism for both acquiring and overcoming CHK1i resistance in cancer patients.</jats:p

Imaging the root–rhizosphere interface using micro computed tomography: quantifying void ratio and root volume ratio profiles

Root growth alters soil fabric and consequently its mechanical and physical properties. Recent studies show that roots induce compaction of soil in their immediate vicinity, a region that is central for plant health. However, high quality quantification of root influence on the soil fabric, able to inform computational models is lacking from the literature. This study quantifies the relationship between soil physical characteristics and root growth, giving special emphasis on how roots in early stage formation influence the physical architecture of the surrounding soil structure. High-resolution X-ray micro-Computed Tomography (µCT) is used to acquire three dimensional images of two homogeneously-packed samples. It is observed that the void ratio profile extending from the soil-root interface into the bulk soil is altered by root growth. The roots considerably modify the immediate soil physical characteristics by creating micro cracks at the soil-root interface and by increasing void ratio. This paper presents the mechanisms that led to the observed structure as well as some of the implications that it has in such a dynamic zone

Imaging the root–rhizosphere interface using micro computed tomography: quantifying void ratio and root volume ratio profiles

Root growth alters soil fabric and consequently its mechanical and physical properties. Recent studies show that roots induce compaction of soil in their immediate vicinity, a region that is central for plant health. However, high quality quantification of root influence on the soil fabric, able to inform computational models is lacking from the literature. This study quantifies the relationship between soil physical characteristics and root growth, giving special emphasis on how roots in early stage formation influence the physical architecture of the surrounding soil structure. High-resolution X-ray micro-Computed Tomography (µCT) is used to acquire three dimensional images of two homogeneously-packed samples. It is observed that the void ratio profile extending from the soil-root interface into the bulk soil is altered by root growth. The roots considerably modify the immediate soil physical characteristics by creating micro cracks at the soil-root interface and by increasing void ratio. This paper presents the mechanisms that led to the observed structure as well as some of the implications that it has in such a dynamic zone

How Do Roots Interact with Layered Soils?

Vegetation alters soil fabric by providing biological reinforcement and enhancing the overall mechanical behaviour of slopes, thereby controlling shallow mass movement. To predict the behaviour of vegetated slopes, parameters representing the root system structure, such as root distribution, length, orientation and diameter, should be considered in slope stability models. This study quantifies the relationship between soil physical characteristics and root growth, giving special emphasis on (1) how roots influence the physical architecture of the surrounding soil structure and (2) how soil structure influences the root growth. A systematic experimental study is carried out using high-resolution X-ray micro-computed tomography (&micro;CT) to observe the root behaviour in layered soil. In total, 2 samples are scanned over 15 days, enabling the acquisition of 10 sets of images. A machine learning algorithm for image segmentation is trained to act at 3 different training percentages, resulting in the processing of 30 sets of images, with the outcomes prompting a discussion on the size of the training data set. An automated in-house image processing algorithm is employed to quantify the void ratio and root volume ratio. This script enables post processing and image analysis of all 30 cases within few hours. This work investigates the effect of stratigraphy on root growth, along with the effect of image-segmentation parameters on soil constitutive properties

Non-invasive synchronous monitoring of neutrophil migration using whole body near-infrared fluorescence-based imaging

Abstract Advances in fluorescence imaging coupled with the generation of near infrared probes have significantly improved the capabilities of non-invasive, real-time imaging in whole animals. In this study we were able to overcome a limitation of in vivo fluorescence imaging and have established a dual cell tracking method where two different cell types can be monitored according to the spectral signature of the cell labelling fluorophore. Using a mouse model of acute liver injury, we have characterised the in vivo migration patterns of wild type and transgenic neutrophils with impaired chemotaxis. Here, we were able to demonstrate that IVIS provides a sensitive multiplexing technology to differentiate two different cell populations based on the spectral signature of the cell labelling fluorophores. This spectral unmixing methodology has the potential to uncover multidimensional cellular interactions involved in many diseases such as fibrosis and cancer. In vivo spectral un-mixing provides a useful tool for monitoring multiple biological process in real-time in the same animal

Upregulation of the PI3K/AKT and RHO/RAC/PAK signaling pathways in CHK1 inhibitor resistant Eμ-Myc lymphoma cells

The development of resistance and the activation of bypass pathway signalling represents a major problem for the clinical application of protein kinase inhibitors. While investigating the effect of either a c-Rel deletion or RelAT505A phosphosite knockin on the Eμ-Myc mouse model of B-cell lymphoma, we discovered that both NF-B subunit mutations resulted in CHK1 inhibitor resistance, arising from either loss or alteration of CHK1 activity respectively. However, since Eμ-Myc lymphomas depend on CHK1 activity to cope with high levels of DNA replication stress and consequent genomic instability, it was not clear how these mutant NF-B subunit lymphomas were able to survive. To understand these survival mechanisms and to identify potential compensatory bypass signalling pathways in these lymphomas, we applied a multi-omics strategy. With c-Rel-/- Eμ-Myc lymphomas we observed high levels of Phosphatidyl-inositol 3-kinase (PI3K) and AKT pathway activation. Moreover, treatment with the PI3K inhibitor Pictilisib (GDC-0941) selectively inhibited the growth of reimplanted c-Rel-/- and RelAT505A, but not wild type (WT) Eμ-Myc lymphomas. We also observed upregulation of a RHO/RAC pathway gene expression signature in both Eμ-Myc NF-B subunit mutation models. Further investigation demonstrated activation of the RHO/RAC effectors p21-activated kinase (PAK) 2. Here, the PAK inhibitor, PF-3758309 successfully overcame resistance of RelAT505A but not WT lymphomas. These findings demonstrate that upregulation of multiple bypass pathways occurs in CHK1 inhibitor resistant Eμ-Myc lymphomas. Consequently, drugs targeting these pathways could potentially be used as either second line or combinatorial therapies to aid the successful clinical application of CHK1 inhibitors