What's Changed in 75 Years of RadRes? - An Australian Perspective on Selected Topics.

Several scientific themes are reviewed in the context of the 75-year period relevant to this special platinum issue of Radiation Research. Two criteria have been considered in selecting the scientific themes. One is the exposure of the associated research activity in the annual meetings of the Radiation Research Society (RRS) and in the publications of the Society's Journal, thus reflecting the interest of members of RRS. The second criteria is a focus on contributions from Australian members of RRS. The first theme is the contribution of radiobiology to radiation oncology, featuring two prominent Australian radiation oncologists, the late Rod Withers and his younger colleague, Lester Peters. Two other themes are also linked to radiation oncology; preclinical research aimed at developing experimental radiotherapy modalities, namely microbeam radiotherapy (MRT) and Auger endoradiotherapy. The latter has a long history, in contrast to MRT, especially in Australia, given that the associated medical beamline at the Australian Synchrotron in Melbourne only opened in 2011. Another theme is DNA repair, which has a trajectory parallel to the 75-year period of interest, given the birth of molecular biology in the 1950s. The low-dose radiobiology theme has a similar timeline, predominantly prompted by the nuclear era, which is also connected to the radioprotector theme, although radioprotectors also have a long-established potential utility in cancer radiotherapy. Finally, two themes are associated with biodosimetry. One is the micronucleus assay, highlighting the pioneering contribution from Michael Fenech in Adelaide, South Australia, and the other is the γ-H2AX assay and its widespread clinical applications.

Generation of Rapid and High-Quality Serum by Recombinant Prothrombin Activator Ecarin (RAPClot™).

We recently reported the potential application of recombinant prothrombin activator ecarin (RAPClot™) in blood diagnostics. In a new study, we describe RAPClot™ as an additive to develop a novel blood collection prototype tube that produces the highest quality serum for accurate biochemical analyte determination. The drying process of the RAPClot™ tube generated minimal effect on the enzymatic activity of the prothrombin activator. According to the bioassays of thrombin activity and plasma clotting, γ-radiation (>25 kGy) resulted in a 30-40% loss of the enzymatic activity of the RAPClot™ tubes. However, a visual blood clotting assay revealed that the γ-radiation-sterilized RAPClot™ tubes showed a high capacity for clotting high-dose heparinized blood (8 U/mL) within 5 min. This was confirmed using Thrombelastography (TEG), indicating full clotting efficiency under anticoagulant conditions. The storage of the RAPClot™ tubes at room temperature (RT) for greater than 12 months resulted in the retention of efficient and effective clotting activity for heparinized blood in 342 s. Furthermore, the enzymatic activity of the RAPClot™ tubes sterilized with an electron-beam (EB) was significantly greater than that with γ-radiation. The EB-sterilized RAPClot™ tubes stored at RT for 251 days retained over 70% enzyme activity and clotted the heparinized blood in 340 s after 682 days. Preliminary clinical studies revealed in the two trials that 5 common analytes (K, Glu, lactate dehydrogenase (LD), Fe, and Phos) or 33 analytes determined in the second study in the γ-sterilized RAPClot™ tubes were similar to those in commercial tubes. In conclusion, the findings indicate that the novel RAPClot™ blood collection prototype tube has a significant advantage over current serum or lithium heparin plasma tubes for routine use in measuring biochemical analytes, confirming a promising application of RAPClot™ in clinical medicine.

Ataxia Telangiectasia patient-derived neuronal and brain organoid models reveal mitochondrial dysfunction and oxidative stress.

Ataxia Telangiectasia (AT) is a rare disorder caused by mutations in the ATM gene and results in progressive neurodegeneration for reasons that remain poorly understood. In addition to its central role in nuclear DNA repair, ATM operates outside the nucleus to regulate metabolism, redox homeostasis and mitochondrial function. However, a systematic investigation into how and when loss of ATM affects these parameters in relevant human neuronal models of AT was lacking. We therefore used cortical neurons and brain organoids from AT-patient iPSC and gene corrected isogenic controls to reveal levels of mitochondrial dysfunction, oxidative stress, and senescence that vary with developmental maturity. Transcriptome analyses identified disruptions in regulatory networks related to mitochondrial function and maintenance, including alterations in the PARP/SIRT signalling axis and dysregulation of key mitophagy and mitochondrial fission-fusion processes. We further show that antioxidants reduce ROS and restore neurite branching in AT neuronal cultures, and ameliorate impaired neuronal activity in AT brain organoids. We conclude that progressive mitochondrial dysfunction and aberrant ROS production are important contributors to neurodegeneration in AT and are strongly linked to ATM's role in mitochondrial homeostasis regulation.

Potential Application of Recombinant Snake Prothrombin Activator Ecarin in Blood Diagnostics.

We describe here the purification and cloning of a codon-optimized form of the snake prothrombin activator ecarin from the saw scaled viper (Echis carinatus) expressed in mammalian cells. Expression of recombinant ecarin (rEcarin) was carried out in human embryonic kidney cells (HEK) cells under conditions for the development and performance of a novel and scalable recombinant snake ecarin to industry standards. Clotting performance of the rEcarin was established in recalcified citrated whole blood, plasma, and fresh whole blood and found to be comparable to native ecarin (N-Ecarin). Furthermore, hemolysis was observed with N-Ecarin at relatively high doses in both recalcified citrated and fresh whole blood, while clotting was not observed with rEcarin, providing an important advantage for the recombinant form. In addition, rEcarin effectively clotted both recalcified citrated whole blood and fresh whole blood containing different anticoagulants including heparin, warfarin, dabigatran, Fondaparinux, rivaroxaban and apixaban, forming firm clots in the blood collection tubes. These results demonstrate that rEcarin efficiently clots normal blood as well as blood spiked with high concentrations of anticoagulants and has great potential as an additive to blood collection tubes to produce high quality serum for analyte analysis in diagnostic medicine.

Snake Venom Hydrogels as a Rapid Hemostatic Agent for Uncontrolled Bleeding.

Uncontrolled bleeding from traumatic injury remains the leading cause of preventable death with loss of balance between blood clotting (coagulation) and blood clot breakdown (fibrinolysis). A major limitation of existing hemostatic agents is that they require a functioning clotting system to control the bleeding and are largely based on gauze delivery scaffolds. Herein, a novel rapid wound sealant, composed of two recombinant snake venom proteins, the procoagulant ecarin, to rapidly initiate blood clotting and the antifibrinolytic textilinin, to prevent blood clot breakdown within a synthetic thermoresponsive hydrogel scaffold is developed. In vitro, it is demonstrated that clotting is rapidly initiated with only nanomolar concentrations of venom protein and clot breakdown is effectively inhibited by textilinin. A stable clot is formed within 60 s compared to normal clot formation in 8 min. In vivo studies reveal that the snake venom hydrogel rapidly controls warfarin-induced bleeding, reducing the bleed volume from 48% to 12% and has demonstrated immune compatibility. A new class of hemostatic agents that achieve formation of rapid and stable blood clots even in the presence of blood thinners is demonstrated here.

The hallmarks of aging in Ataxia-Telangiectasia.

Ataxia-telangiectasia (A-T) is caused by absence of the catalytic activity of ATM, a protein kinase that plays a central role in the DNA damage response, many branches of cellular metabolism, redox and mitochondrial homeostasis, and cell cycle regulation. A-T is a complex disorder characterized mainly by progressive cerebellar degeneration, immunodeficiency, radiation sensitivity, genome instability, and predisposition to cancer. It is increasingly recognized that the premature aging component of A-T is an important driver of this disease, and A-T is therefore an attractive model to study the aging process. This review outlines the current state of knowledge pertaining to the molecular and cellular signatures of aging in A-T and proposes how these new insights can guide novel therapeutic approaches for A-T.

miRNA-34c-5p targets Fra-1 to inhibit pulmonary fibrosis induced by silica through p53 and PTEN/PI3K/Akt signaling pathway.

Silica dust particles are representative of air pollution and long-term inhalation of silicon-containing dust through the respiratory tract can cause pulmonary fibrosis. Epithelial-mesenchymal transformation (EMT) plays an important role in the development of fibrosis. This process can relax cell-cell adhesion complexes and enhance cell migration and invasion properties of these cells. Dysregulation of microRNA-34c (miR-34c) is highly correlated with organ fibrosis including pulmonary fibrosis. In this study, we found that miR-34c-5p could alleviate the occurrence and development of silica-mediated EMT. Fos-related antigen 1 was identified as a functional target of miR-34c-5p by bioinformatics analysis and the dual luciferase gene reporting assay. Importantly, chemically induced up-regulation of hsa-miR-34c-5p correlated inversely with the expression of Fra-1 and further exploration found that the miR-34c-5p/Fra-1 axis inhibits the activation of the phosphatase and tensin homolog deleted on chromosome 10/phosphatidylinositol-4,5-bisphosphate3-kinase/protein kinase B (PTEN/PI3K/AKT) signaling pathway. In addition, through interaction with PTEN/p53 it inhibits the proliferation and migration of human bronchial epithelial cells stimulated by silica, and promotes cell apoptosis, thereby preventing EMT. This finding provides a promising biomarker for the diagnosis and prognosis of pulmonary fibrosis. Furthermore, overexpression of miR-34c-5p represents a potential therapeutic approach.

Metabolic Stress and Mitochondrial Dysfunction in Ataxia-Telangiectasia.

The ataxia-telangiectasia mutated (ATM) protein kinase is, as the name implies, mutated in the human genetic disorder ataxia-telangiectasia (A-T). This protein has its "finger in many pies", being responsible for the phosphorylation of many thousands of proteins in different signaling pathways in its role in protecting the cell against a variety of different forms of stress that threaten to perturb cellular homeostasis. The classical role of ATM is the protection against DNA damage, but it is evident that it also plays a key role in maintaining cell homeostasis in the face of oxidative and other forms of non-DNA damaging stress. The presence of ATM is not only in the nucleus to cope with damage to DNA, but also in association with other organelles in the cytoplasm, which suggests a greater protective role. This review attempts to address this greater role of ATM in protecting the cell against both external and endogenous damage.

Integrated genome and transcriptome analyses reveal the mechanism of genome instability in ataxia with oculomotor apraxia 2.

Mutations in the SETX gene, which encodes Senataxin, are associated with the progressive neurodegenerative diseases ataxia with oculomotor apraxia 2 (AOA2) and amyotrophic lateral sclerosis 4 (ALS4). To identify the causal defect in AOA2, patient-derived cells and SETX knockouts (human and mouse) were analyzed using integrated genomic and transcriptomic approaches. A genome-wide increase in chromosome instability (gains and losses) within genes and at chromosome fragile sites was observed, resulting in changes to gene-expression profiles. Transcription stress near promoters correlated with high GCskew and the accumulation of R-loops at promoter-proximal regions, which localized with chromosomal regions where gains and losses were observed. In the absence of Senataxin, the Cockayne syndrome protein CSB was required for the recruitment of the transcription-coupled repair endonucleases (XPG and XPF) and RAD52 recombination protein to target and resolve transcription bubbles containing R-loops, leading to genomic instability. These results show that transcription stress is an important contributor to SETX mutation-associated chromosome fragility and AOA2.

Microcrystalline silica particles induce inflammatory response via pyroptosis in primary human respiratory epithelial cells.

The mechanism of the sterile inflammatory response in the respiratory tract induced by exposure to sterile particles has not been fully elucidated. The aim of our study is to explore the earlier events in initiating inflammatory response at molecular and cellular level in primary cultured human airway epithelial cells (AEC) exposed to silica particles in order to provide information for earlier diagnosis and prevention of silica particle-induced toxicity as well as possible information on the genesis of silicosis. We isolated primary AEC from three healthy adults and treated them with silica particles at different concentrations for 48 h. We found evidence for silica-induced inflammasome activation by the co-localization of Caspase-1 and NLRP3, as well as increased levels of IL-1ß and IL-18. Lactate dehydrogenase and NucGreen analysis proved the occurrence of pyroptosis. High throughput mRNA sequencing showed that the inflammatory response and NF-κB signaling pathways were significantly enriched in gene ontology and Kyoto encyclopedia of genes and genomes analysis, and pyroptosis-related genes were up-regulated. The miR-455-3p and five lncRNAs (LOC105375913, NEAT1, LOC105375181, LOC100506098, and LOC105369370) were verified as key factors related to the mechanism by ceRNA network analysis. LOC105375913 was first discovered to be associated with inflammation in AEC. These data suggest that microcrystalline silica can induce significant inflammation and pyroptosis in human primary AEC through NLRP3 inflammasome pathway and NF-κB signaling pathway at both the gene and protein levels, and the possible mechanism could be miR-455-3p mediated ceRNA hypothesis. Our data provide a method for the studies of the respiratory toxicity of fine particulate matter and the pathogenesis of early silicosis. The miR-455-3p and five lncRNAs related ceRNA network might be the toxicity mechanism of microcrystalline silica particles to AEC.

DNA damage rather than type I IFN signaling is the primary mediator of neural dysfunction in Aicardi-Goutières syndrome after RNASEH2 disruption.

Mutations in genes that function in nucleic metabolism have been shown to be linked to Aicardi-Goutières syndrome. In this issue of Neuron, Aditi et al. (2021) provide evidence that DNA damage-dependent signaling rather than type I interferon signaling underlies neurodegeneration in this class of neurodevelopmental/neuroinflammatory disease.

Ataxia Telangiectasia iPSC line generated from a patient olfactory biopsy identifies novel disease-causing mutations.

Ataxia Telangiectasia is a rare autosomal recessive disorder caused by a mutated ATM gene. The most debilitating symptom of Ataxia Telangiectasia is the progressive neurodegeneration of the cerebellum, though the molecular mechanisms driving this degeneration remains unclear. Here we describe the generation and validation of an induced pluripotent stem cell (iPSC) line from an olfactory biopsy from a patient with Ataxia Telangiectasia. Sequencing identified two previously unreported disease-causing mutations in the ATM gene. This line can be used to generate 2D and 3D patient-specific neuronal models enabling investigations into the mechanisms underlying neurodegeneration.

Metformin Attenuates Silica-Induced Pulmonary Fibrosis by Activating Autophagy via the AMPK-mTOR Signaling Pathway.

Long-term exposure to crystalline silica particles leads to silicosis characterized by persistent inflammation and progressive fibrosis in the lung. So far, there is no specific treatment to cure the disease other than supportive care. In this study, we examined the effects of metformin, a prescribed drug for type || diabetes on silicosis and explored the possible mechanisms in an established rat silicosis model in vivo, and an in vitro co-cultured model containing human macrophages cells (THP-1) and human bronchial epithelial cells (HBEC). Our results showed that metformin significantly alleviated the inflammation and fibrosis of lung tissues of rats exposed to silica particles. Metformin significantly reduced silica particle-induced inflammatory cytokines including transforming growth factor-ß1 (TGF-ß1), tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in rat lung tissue and HBEC culture supernatant. The protein levels of Vimentin and α-smooth muscle actin (α-SMA) were significantly decreased by metfomin while expression level of E-cadherin (E-Cad) increased. Besides, metformin increased the expression levels of phosphorylated adenosine 5'-monophosphate (AMP)-activated protein kinase (p-AMPK), microtubule-associated protein (MAP) light chain 3B (LC3B) and Beclin1 proteins, and reduced levels of phosphorylated mammalian target of rapamycin (p-mTOR) and p62 proteins in vivo and in vitro. These results suggest that metformin could inhibit silica-induced pulmonary fibrosis by activating autophagy through the AMPK-mTOR pathway.

Hesperetin attenuates silica-induced lung injury by reducing oxidative damage and inflammatory response.

Oxidative stress and the inflammatory response are two important mechanisms of silica-induced lung injury. Hesperetin (HSP) is a natural flavonoid compound that is found in citrus fruits and has been indicated to exhibit strong antioxidant and anti-inflammatory properties. The current study evaluated the protective effect of HSP on lung injury in rats exposed to silica. The results indicated that the degree of alveolitis and pulmonary fibrosis in the HSP-treated group was significantly decreased compared with the silica model group. The content of hydroxyproline (HYP) was also revealed to decrease overall in the HSP treated group compared with the silica model group, indicating that the degree of pulmonary fibrosis was decreased compared with the silica model group. The present study also demonstrated that HSP reduced oxidation levels of malondialdehyde (MDA) and increased the activities of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-PX). Total antioxidant capacity (T-AOC) was also increased following HSP treatment, indicating that HSP can alleviate oxidative stress in the lung tissue of silica-exposed rats. In addition, HSP was revealed to inhibit the synthesis and secretion of fibrogenic factor TGF-ß1, reduce the production of pro-inflammatory cytokines IL-1ß, IL-4, TNF-α and increase the levels of anti-inflammatory factors IFN-γ and IL-10. The current study demonstrated that HSP can effectively attenuate silica-induced lung injury by reducing oxidative damage and the inflammatory response.

The spectrum of ATM gene mutations in Iranian patients with ataxia-telangiectasia.

BACKGROUND: Ataxia-telangiectasia (A-T) is a rare genetic disorder characterized by a distinct range of clinical manifestations, including progressive ataxia, immunodeficiency, and radiosensitivity. METHODS: Clinical data, laboratory results, and genetic data were collected from forty-three A-T patients. Whole-exome sequencing and Sanger sequencing were done for the patients clinically diagnosed as suffering from A-T. Based on the phenotype severity of the disease, patients were divided into severe and mild subgroups. RESULTS: The median (IQR) age of diagnosis in this cohort was 5 (3-7) years, and various types of clinical manifestations, including fever (P =.005), lower respiratory tract infection (P = .033), diarrhea (P = .014), and hepatosplenomegaly (P = .032), were significantly higher among patients diagnosed with the severe phenotype. Our results showed a correlation between phenotype severity and mutation type. The chance of having severe phenotype in patients who have severe mutations, including frameshift and nonsense, was 7.3 times higher than in patients who were categorized in the mild genotype group (odds ratio = 7.3, P = .006). Thirty-four types of mutations including 9 novel mutations were observed in our study. CONCLUSION: Molecular analysis provides the opportunity for accurate diagnosis and timely management in A-T patients with chronic progressive disease, especially infections and the risk of malignancies. This study characterizes for the first time the broad spectrum of mutations and phenotypes in Iranian A-T patients, which is required for carrier detection and reducing the burden of disease in the future using the patients' families and for the public healthcare system.

Impaired endoplasmic reticulum-mitochondrial signaling in ataxia-telangiectasia.

There is evidence that ATM mutated in ataxia-telangiectasia (A-T) plays a key role in protecting against mitochondrial dysfunction, the mechanism for which remains unresolved. We demonstrate here that ATM-deficient cells are exquisitely sensitive to nutrient deprivation, which can be explained by defective cross talk between the endoplasmic reticulum (ER) and the mitochondrion. Tethering between these two organelles in response to stress was reduced in cells lacking ATM, and consistent with this, Ca2+ release and transfer between ER and mitochondria was reduced dramatically when compared with control cells. The impact of this on mitochondrial function was evident from an increase in oxygen consumption rates and a defect in mitophagy in ATM-deficient cells. Our findings reveal that ER-mitochondrial connectivity through IP3R1-GRP75-VDAC1, to maintain Ca2+ homeostasis, as well as an abnormality in mitochondrial fusion defective in response to nutrient stress, can account for at least part of the mitochondrial dysfunction observed in A-T cells.

Emodin attenuates silica-induced lung injury by inhibition of inflammation, apoptosis and epithelial-mesenchymal transition.

Silicosis is a fatal pulmonary disease caused by the inhalation of silica dust, and characterized by inflammation and fibrosis of the lung, with no effective treatment to date. Here we investigate the effect of emodin, an anthraquinone derivative isolated from rhubarb using a mouse silicosis model and in vitro cultured human macrophages and alveolar epithelial cells. Results from histological examination indicated that emodin reduced the degree of alveolitis and fibrosis in the lungs of mice exposed to silica particles. We also demonstrated that emodin effectively inhibited the phosphorylation of Smad3 and NF-κB and reduced the levels of inflammatory factors in the lung tissue of mice treated with silica particles. In addition, we found that emodin inhibited apoptosis and demonstrated an anti-fibrotic effect by down-regulating the pro-apoptotic protein Bax and up-regulating the anti-apoptotic protein Bcl-2. Furthermore, emodin increased E-cadherin levels, reduced the expression of Vimentin, α-SMA and Col-I, as well as pro-inflammatory factors TGF-ß1, TNF-α and IL-1ß in vivo and in vitro. These results suggested that emodin can regulate epithelial-mesenchymal transition (EMT) through the inhibition of the TGF-ß1/Smad3 signaling pathway and the NF-κB signaling pathway to prevent alveolar inflammation and apoptotic process. Overall, this study showed that emodin can alleviate pulmonary fibrosis in silicosis through regulating the inflammatory response and fibrotic process at multiple levels.

Regulation of RNA degradation pathways during the lipopolysaccharide response in Macrophages.

The innate immune response to LPS is highly dynamic yet tightly regulated. The majority of studies of gene expression have focussed on transcription. However, it is also important to understand how post-transcriptional pathways are regulated in response to inflammatory stimuli as the rate of RNA degradation relative to new transcription is important for overall expression. RNA decay pathways include nonsense-mediated decay, the RNA decay exosome, P-body localized deadenylation, decapping and degradation, and AU-rich element targeted decay mediated by tristetraprolin. Here, bone marrow-derived Mϕs were treated with LPS over a time course of 0, 2, 6, and 24 h and the transcriptional profiles were analyzed by RNA sequencing. The data show that components of RNA degradation pathways are regulated during an LPS response. Processing body associated decapping enzyme DCP2 and regulatory subunit DCP1A, and 5' exonuclease XRN1 and sequence specific RNA decay pathways were upregulated. Nonsense mediated decay was also increased in response to LPS induced signaling, initially by increased activation and at later timepoints at the mRNA and protein levels. This leads to increased nonsense mediated decay efficiency across the 24 h following LPS treatment. These findings suggest that LPS activation of Mϕs results in targeted regulation of RNA degradation pathways in order to change how subsets of mRNAs are degraded during an inflammatory response.

Understanding the mechanisms of silica nanoparticles for nanomedicine.

As a consequence of recent progression in biomedicine and nanotechnology, nanomedicine has emerged rapidly as a new discipline with extensive application of nanomaterials in biology, medicine, and pharmacology. Among the various nanomaterials, silica nanoparticles (SNPs) are particularly promising in nanomedicine applications due to their large specific surface area, adjustable pore size, facile surface modification, and excellent biocompatibility. This paper reviews the synthesis of SNPs and their recent usage in drug delivery, biomedical imaging, photodynamic and photothermal therapy, and other applications. In addition, the possible adverse effects of SNPs in nanomedicine applications are reviewed from reported in vitro and in vivo studies. Finally, the potential opportunities and challenges for the future use of SNPs are discussed. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Emerging Technologies.