A comparison of local phosphorescence detection and fluid dynamic gauging methods for studying the removal of cohesive fouling layers: Effect of layer roughness

The performance of industrial cleaning in place (CIP) procedures is critically important for food manufacture. CIP has yet to be optimised for many processes, in part since the mechanisms involved in cleaning are not fully understood. Laboratory tests have an important role in guiding industrial trials, and this paper introduces and compares two experimental techniques developed for studying CIP mechanisms: local phosphorescence detection (LPD), and scanning fluid dynamic gauging (sFDG). To illustrate the comparison, each technique is used to investigate the influence of soil topology on the cleaning of pre-gelatinised starch-based layers from 316 stainless steel substrates by aqueous NaOH solutions at ambient temperature. The roughness of the soil surface is varied by incorporating zinc sulphide particles with different particle size distributions (range 1 - 80 μm) into the starch suspensions. The soil roughness increased with the use of larger particles, increasing the 3D arithmetic mean roughness (Sa) of the dry layers (range 0.37 - 3.33 μm). Rough layers were cleaned more readily than those containing small inclusions, with a good correlation between the cleaning rates observed during LPD and FDG measurements. The LPD technique, which is an instrumented CIP test, gives a better indication of the cleaning time, while sFDG measurements provide further insight into the removal mechanisms.NOTICE: this is the author's version of a work that was accepted for publication in Food Bioproducts Processing. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Food Bioproducts Proc., 92, 46-53 DOI http://dx.doi.org/10.1016/j.fbp.2013.07.01

Oct4 differentially regulates chromatin opening and enhancer transcription in pluripotent stem cells

The transcription factor Oct4 is essential for the maintenance and induction of stem cell pluripotency, but its functional roles are not fully understood. Here, we investigate the functions of Oct4 by depleting and subsequently recovering it in mouse embryonic stem cells (ESCs) and conducting a time-resolved multiomics analysis. Oct4 depletion leads to an immediate loss of its binding to enhancers, accompanied by a decrease in mRNA synthesis from its target genes that are part of the transcriptional network that maintains pluripotency. Gradual decrease of Oct4 binding to enhancers does not immediately change the chromatin accessibility but reduces transcription of enhancers. Conversely, partial recovery of Oct4 expression results in a rapid increase in chromatin accessibility, whereas enhancer transcription does not fully recover. These results indicate different concentration-dependent activities of Oct4. Whereas normal ESC levels of Oct4 are required for transcription of pluripotency enhancers, low levels of Oct4 are sufficient to retain chromatin accessibility, likely together with other factors such as Sox2

Natural formation of chloro- and bromoacetone in salt lakes of Western Australia

Western Australia is a semi-/arid region known for saline lakes with a wide range of geochemical parameters (pH 2.5-7.1, Cl- 10-200 g L-1. This study reports on the haloacetones chloro- and bromoacetone in air over 6 salt lake shorelines. Significant emissions of chloroacetone (up to 0.2 µmol m-2 h-1) and bromoacetone (up to 1. 5 µmol m-2 h-1) were detected, and a photochemical box model was employed to evaluate the contribution of their atmospheric formation from the olefinic hydrocarbons propene and methacrolein in the gas phase. The measured concentrations could not explain the photochemical halogenation reaction, indicating a strong hitherto unknown source of haloacetones. Aqueous-phase reactions of haloacetones, investigated in the laboratory using humic acid in concentrated salt solutions, were identified as alternative formation pathway by liquid-phase reactions, acid catalyzed enolization of ketones, and subsequent halogenation. In order to verify this mechanism, we made measurements of the Henry's law constants, rate constants for hydrolysis and nucleophilic exchange with chloride, UV-spectra and quantum yields for the photolysis of bromoacetone and 1,1-dibromoacetone in the aqueous phase. We suggest that heterogeneous processes induced by humic substances in the quasi-liquid layer of the salt crust, particle surfaces and the lake water are the predominating pathways for the formation of the observed haloacetones

Human haematopoietic stem cells express Oct4 pseudogenes and lack the ability to initiate Oct4 promoter-driven gene expression

The transcription factor Oct4 is well defined as a key regulator of embryonic stem (ES) cell pluripotency. In recent years, the role of Oct4 has purportedly extended to the self renewal and maintenance of multipotency in adult stem cell (ASC) populations. This profile has arisen mainly from reports utilising reverse transcription-polymerase chain reaction (RT-PCR) based methodologies and has since come under scrutiny following the discovery that many developmental genes have multiple pseudogenes associated with them. Six known pseudogenes exist for Oct4, all of which exhibit very high sequence homology (three >97%), and for this reason the generation of artefacts may have contributed to false identification of Oct4 in somatic cell populations. While ASC lack a molecular blueprint of transcription factors proposed to be involved with 'stemness' as described for ES cells, it is not unreasonable to assume that similar gene patterns may exist. The focus of this work was to corroborate reports that Oct4 is involved in the regulation of ASC self-renewal and differentiation, using a combination of methodologies to rule out pseudogene interference. Haematopoietic stem cells (HSC) derived from human umbilical cord blood (UCB) and various differentiated cell lines underwent RT-PCR, product sequencing and transfection studies using an Oct4 promoter-driven reporter. In summary, only the positive control expressed Oct4, with all other cell types expressing a variety of Oct4 pseudogenes. Somatic cells were incapable of utilising an exogenous Oct4 promoter construct, leading to the conclusion that Oct4 does not appear involved in the multipotency of human HSC from UCB

Linear mtDNA fragments and unusual mtDNA rearrangements associated with pathological deficiency of MGME1 exonuclease.

MGME1, also known as Ddk1 or C20orf72, is a mitochondrial exonuclease found to be involved in the processing of mitochondrial DNA (mtDNA) during replication. Here, we present detailed insights on the role of MGME1 in mtDNA maintenance. Upon loss of MGME1, elongated 7S DNA species accumulate owing to incomplete processing of 5' ends. Moreover, an 11-kb linear mtDNA fragment spanning the entire major arc of the mitochondrial genome is generated. In contrast to control cells, where linear mtDNA molecules are detectable only after nuclease S1 treatment, the 11-kb fragment persists in MGME1-deficient cells. In parallel, we observed characteristic mtDNA duplications in the absence of MGME1. The fact that the breakpoints of these mtDNA rearrangements do not correspond to either classical deletions or the ends of the linear 11-kb fragment points to a role of MGME1 in processing mtDNA ends, possibly enabling their repair by homologous recombination. In agreement with its functional involvement in mtDNA maintenance, we show that MGME1 interacts with the mitochondrial replicase PolgA, suggesting that it is a constituent of the mitochondrial replisome, to which it provides an additional exonuclease activity. Thus, our results support the viewpoint that MGME1-mediated mtDNA processing is essential for faithful mitochondrial genome replication and might be required for intramolecular recombination of mtDNA

Influence of fly ash blending on hydration and physical behavior of Belite-Alite-Ye'elimite cements

A cement powder, composed of belite, alite and ye’elimite, was blended with 0, 15 and 30 wt% of fly ash and the resulting lended cements were further characterized. During hydration, the presence of fly ash caused the partial inhibition of both AFt degradation and belite reactivity, even after 180 days. The compressive strength of the corresponding mortars increased by increasing the fly ash content (68, 73 and 82 MPa for mortars with 0, 15 and 30 wt% of fly ash, respectively, at 180 curing days), mainly due to the diminishing porosity and pore size values. Although pozzolanic reaction has not been directly proved there are indirect evidences.This work is part of the Ph.D. of D. Londono-Zuluaga funded by Beca Colciencias 646—Doctorado en el exterior and Enlaza Mundos 2013 program grant. Cement and Building materials group (CEMATCO) from National University of Colombia is acknowledged for providing the calorimetric measurements. Funding from Spanish MINECO BIA2017-82391-R and I3 (IEDI-2016-0079) grants, co-funded by FEDER, are acknowledged

Lysine-based surfactants in nanovesicle formulations: the role of cationic charge position and hydrophobicity in in vitro cytotoxicity and intracellular delivery

Understanding nanomaterial interactions within cells is of increasing importance for assessing their toxicity and cellular transport. Here, we developed nanovesicles containing bioactive cationic lysine-based amphiphiles, and assessed whether these cationic compounds increase the likelihood of intracellular delivery and modulate toxicity. We found different cytotoxic responses among the formulations, depending on surfactant, cell line and endpoint assayed. The induction of mitochondrial dysfunction, oxidative stress and apoptosis were the general mechanisms underlying cytotoxicity. Fluorescence microscopy analysis demonstrated that nanovesicles were internalized by HeLa cells, and evidenced that their ability to release endocytosed materials into cell cytoplasm depends on the structural parameters of amphiphiles. The cationic charge position and hydrophobicity of surfactants determine the nanovesicle interactions within the cell and, thus, the resulting toxicity and intracellular behavior after cell uptake of the nanomaterial. The insights into some toxicity mechanisms of these new nanomaterials contribute to reducing the uncertainty surrounding their potential health hazards

Genome editing reveals a role for OCT4 in human embryogenesis.

Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.DW was supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme. NK was supported by the University of Oxford Clarendon Fund. AB was supported by a British Heart Foundation PhD Studentship (FS/11/77/39327). LV was supported by core grant funding from the Wellcome Trust and Medical Research Council (PSAG028). J-SK was supported by the Institute for Basic Science (IBS-R021-D1). Work in the KKN and JMAT labs was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK, the UK Medical Research Council, and the Wellcome Trust (FC001120 and FC001193)

The effect of Young's modulus on the neuronal differentiation of mouse embryonic stem cells

There is substantial evidence that cells produce a diverse response to changes in ECM stiffness depending on their identity. Our aim was to understand how stiffness impacts neuronal differentiation of embryonic stem cells (ESC's), and how this varies at three specific stages of the differentiation process. In this investigation, three effects of stiffness on cells were considered; attachment, expansion and phenotypic changes during differentiation. Stiffness was varied from 2 kPa to 18 kPa to finally 35 kPa. Attachment was found to decrease with increasing stiffness for both ESC's (with a 95% decrease on 35 kPa compared to 2 kPa) and neural precursors (with a 83% decrease on 35 kPa). The attachment of immature neurons was unaffected by stiffness. Expansion was independent of stiffness for all cell types, implying that the proliferation of cells during this differentiation process was independent of Young's modulus. Stiffness had no effect upon phenotypic changes during differentiation for mESC's and neural precursors. 2 kPa increased the proportion of cells that differentiated from immature into mature neurons. Taken together our findings imply that the impact of Young's modulus on attachment diminishes as neuronal cells become more mature. Conversely, the impact of Young's modulus on changes in phenotype increased as cells became more mature