Endothelial Heterogeneity in the Response to Autophagy Drives Small Vessel Muscularization in Pulmonary Hypertension.

BACKGROUND: Endothelial cell (EC) apoptosis and proliferation of apoptosis-resistant cells is a hallmark of pulmonary hypertension (PH). Yet, why some ECs die and others proliferate and how this contributes to vascular remodeling is unclear. We hypothesized that this differential response may: (1) relate to different EC subsets, namely pulmonary artery (PAECs) versus microvascular ECs (MVECs); (2) be attributable to autophagic activation in both EC subtypes; and (3) cause replacement of MVECs by PAECs with subsequent distal vessel muscularization. METHODS: EC subset responses to chronic hypoxia were assessed by single-cell RNA sequencing of murine lungs. Proliferative versus apoptotic responses, activation, and role of autophagy were assessed in human and rat PAECs and MVECs, and in precision-cut lung slices of wild-type mice or mice with endothelial deficiency in the autophagy gene Atg7 (Atg7EN-KO). Abundance of PAECs versus MVECs in precapillary microvessels was assessed in lung tissue from patients with PH and animal models on the basis of structural or surface markers. RESULTS: In vitro and in vivo, PAECs proliferated in response to hypoxia, whereas MVECs underwent apoptosis. Single-cell RNA sequencing analyses support these findings in that hypoxia induced an antiapoptotic, proliferative phenotype in arterial ECs, whereas capillary ECs showed a propensity for cell death. These distinct responses were prevented in hypoxic Atg7EN-KO mice or after ATG7 silencing, yet replicated by autophagy stimulation. In lung tissue from mice, rats, or patients with PH, the abundance of PAECs in precapillary arterioles was increased, and that of MVECs reduced relative to controls, indicating replacement of microvascular by macrovascular ECs. EC replacement was prevented by genetic or pharmacological inhibition of autophagy in vivo. Conditioned medium from hypoxic PAECs yet not MVECs promoted pulmonary artery smooth muscle cell proliferation and migration in a platelet-derived growth factor-dependent manner. Autophagy inhibition attenuated PH development and distal vessel muscularization in preclinical models. CONCLUSIONS: Autophagic activation by hypoxia induces in parallel PAEC proliferation and MVEC apoptosis. These differential responses cause a progressive replacement of MVECs by PAECs in precapillary pulmonary arterioles, thus providing a macrovascular context that in turn promotes pulmonary artery smooth muscle cell proliferation and migration, ultimately driving distal vessel muscularization and the development of PH.

Ileal mucus viscoelastic properties differ in Crohn's disease.

Crohn's disease (CD) is an inflammatory bowel disease that can affect any part of the gastrointestinal tract, frequently involving the terminal ileum. While colonic mucus alterations in CD patients have been described, terminal ileal mucus and its mechanobiological properties have been neglected. Our study is the first of its kind to decipher the viscoelastic and network properties of ileal mucus. With that aim, oscillatory rheological shear measurements based on an airway mucus protocol that was thoroughly validated for ileal mucus were performed. Our pilot study analyzed terminal ileum mucus from controls (n = 14) and CD patients (n = 14). Mucus network structure was visualized by scanning electron microscopy. Interestingly, a statistically significant increase in viscoelasticity as well as a decrease in mesh size was observed in ileal mucus from CD patients compared to controls. Furthermore, rheological data were analyzed in relation to study participants' clinical characteristics, revealing a noteworthy trend between non-smokers and smokers. In conclusion, this study provides the first data on the viscoelastic properties and structure of human ileal mucus in the healthy state and Crohn's disease, demonstrating significant alterations between groups and highlighting the need for further research on mucus and its effect on the underlying epithelial barrier.

Characterization of Commercially Available Human Primary Alveolar Epithelial Cells.

In vitro lung research requires appropriate cell culture models that adequately mimic in vivo structure and function. Previously, researchers extensively used commercially available and easily expandable A549 and NCI-H441 cells, which replicate some but not all features of alveolar epithelial cells. Specifically, these cells are often restricted by terminally altered expression while lacking important alveolar epithelial characteristics. Of late, human primary alveolar epithelial cells (hPAEpCs) have become commercially available but are so far poorly specified. Here, we applied a comprehensive set of technologies to characterize their morphology, surface marker expression, transcriptomic profile, and functional properties. At optimized seeding numbers of 7,500 cells per square centimeter and growth at a gas-liquid interface, hPAEpCs formed regular monolayers with tight junctions and amiloride-sensitive transepithelial ion transport. Electron microscopy revealed lamellar body and microvilli formation characteristic for alveolar type II cells. Protein and single-cell transcriptomic analyses revealed expression of alveolar type I and type II cell markers; yet, transcriptomic data failed to detect NKX2-1, an important transcriptional regulator of alveolar cell differentiation. With increasing passage number, hPAEpCs transdifferentiated toward alveolar-basal intermediates characterized as SFTPC-, KRT8high, and KRT5- cells. In spite of marked changes in the transcriptome as a function of passaging, Uniform Manifold Approximation and Projection plots did not reveal major shifts in cell clusters, and epithelial permeability was unaffected. The present work delineates optimized culture conditions, cellular characteristics, and functional properties of commercially available hPAEpCs. hPAEpCs may provide a useful model system for studies on drug delivery, barrier function, and transepithelial ion transport in vitro.

Uterine scars after caesarean delivery: From histology to the molecular and ultrastructural level.

Uterine rupture during a trial of labor after caesarean delivery (CD) is a serious complication for mother and fetus. The lack of knowledge on histological features and molecular pathways of uterine wound healing has hindered research in this area from evolving over time. We analysed collagen content and turnover in uterine scars on a histological, molecular and ultrastructural level. Therefore, tissue samples from the lower uterine segment were obtained during CD from 16 pregnant women with at least one previous CD, from 16 pregnant women without previous CD, and from 16 non-pregnant premenopausal women after hysterectomy for a benign disease. Histomorphometrical collagen quantification showed, that the collagen content of the scar area in uterine wall specimens after previous CD was significantly higher than in the unscarred myometrium of the same women and the control groups. Quantitative real-time PCR of uterine scar tissue from FFPE samples delineated by laser microdissection yielded a significantly higher COL3A1 expression and a significantly lower COL1A2/COL3A1 ratio in scarred uteri than in samples from unscarred uteri. Histological collagen content and the expression of COL1A2 and COL3A1 were positively correlated, while COL1A2/COL3A1 ratio was negatively correlated with the histological collagen content. Transmission electron microscopy revealed a destroyed myometrial ultrastructure in uterine scars with increased collagen density. We conclude that the high collagen content in uterine scars results from an ongoing overexpression of collagen I and III. This is a proof of concept to enable further analyses of specific factors that mediate uterine wound healing.

The unremarkable alveolar epithelial glycocalyx: a thorium dioxide-based electron microscopic comparison after heparinase or pneumolysin treatment.

Recent investigations analyzed in depth the biochemical and biophysical properties of the endothelial glycocalyx. In comparison, this complex cell-covering structure is largely understudied in alveolar epithelial cells. To better characterize the alveolar glycocalyx ultrastructure, unaffected versus injured human lung tissue explants and mouse lungs were analyzed by transmission electron microscopy. Lung tissue was treated with either heparinase (HEP), known to shed glycocalyx components, or pneumolysin (PLY), the exotoxin of Streptococcus pneumoniae not investigated for structural glycocalyx effects so far. Cationic colloidal thorium dioxide (cThO2) particles were used for glycocalyx glycosaminoglycan visualization. The level of cThO2 particles orthogonal to apical cell membranes (≙ stained glycosaminoglycan height) of alveolar epithelial type I (AEI) and type II (AEII) cells was stereologically measured. In addition, cThO2 particle density was studied by dual-axis electron tomography (≙ stained glycosaminoglycan density in three dimensions). For untreated samples, the average cThO2 particle level was ≈ 18 nm for human AEI, ≈ 17 nm for mouse AEI, ≈ 44 nm for human AEII and ≈ 35 nm for mouse AEII. Both treatments, HEP and PLY, resulted in a significant reduction of cThO2 particle levels on human and mouse AEI and AEII. Moreover, a HEP- and PLY-associated reduction in cThO2 particle density was observed. The present study provides quantitative data on the differential glycocalyx distribution on AEI and AEII based on cThO2 and demonstrates alveolar glycocalyx shedding in response to HEP or PLY resulting in a structural reduction in both glycosaminoglycan height and density. Future studies should elucidate the underlying alveolar epithelial cell type-specific distribution of glycocalyx subcomponents for better functional understanding.

Assessing and improving the validity of COVID-19 autopsy studies - A multicentre approach to establish essential standards for immunohistochemical and ultrastructural analyses.

BACKGROUND: Autopsy studies have provided valuable insights into the pathophysiology of COVID-19. Controversies remain about whether the clinical presentation is due to direct organ damage by SARS-CoV-2 or secondary effects, such as overshooting immune response. SARS-CoV-2 detection in tissues by RT-qPCR and immunohistochemistry (IHC) or electron microscopy (EM) can help answer these questions, but a comprehensive evaluation of these applications is missing. METHODS: We assessed publications using IHC and EM for SARS-CoV-2 detection in autopsy tissues. We systematically evaluated commercially available antibodies against the SARS-CoV-2 proteins in cultured cell lines and COVID-19 autopsy tissues. In a multicentre study, we evaluated specificity, reproducibility, and inter-observer variability of SARS-CoV-2 IHC. We correlated RT-qPCR viral tissue loads with semiquantitative IHC scoring. We used qualitative and quantitative EM analyses to refine criteria for ultrastructural identification of SARS-CoV-2. FINDINGS: Publications show high variability in detection and interpretation of SARS-CoV-2 abundance in autopsy tissues by IHC or EM. We show that IHC using antibodies against SARS-CoV-2 nucleocapsid yields the highest sensitivity and specificity. We found a positive correlation between presence of viral proteins by IHC and RT-qPCR-determined SARS-CoV-2 viral RNA load (N= 35; r=-0.83, p-value <0.0001). For EM, we refined criteria for virus identification and provide recommendations for optimized sampling and analysis. 135 of 144 publications misinterpret cellular structures as virus using EM or show only insufficient data. We provide publicly accessible digitized EM sections as a reference and for training purposes. INTERPRETATION: Since detection of SARS-CoV-2 in human autopsy tissues by IHC and EM is difficult and frequently incorrect, we propose criteria for a re-evaluation of available data and guidance for further investigations of direct organ effects by SARS-CoV-2. FUNDING: German Federal Ministry of Health, German Federal Ministry of Education and Research, Berlin University Alliance, German Research Foundation, German Center for Infectious Research.

Skeletal muscle provides the immunological micro-milieu for specific plasma cells in anti-synthetase syndrome-associated myositis.

Anti-synthetase syndrome (ASyS)-associated myositis is a major subgroup of the idiopathic inflammatory myopathies (IIM) and is characterized by disease chronicity with musculoskeletal, dermatological and pulmonary manifestations. One of eight autoantibodies against the aminoacyl-transferase RNA synthetases (ARS) is detectable in the serum of affected patients. However, disease-specific therapeutic approaches have not yet been established.To obtain a deeper understanding of the underlying pathogenesis and to identify putative therapeutic targets, we comparatively investigated the most common forms of ASyS associated with anti-PL-7, anti-PL-12 and anti-Jo-1. Our cohort consisted of 80 ASyS patients as well as healthy controls (n = 40), diseased controls (n = 40) and non-diseased controls (n = 20). We detected a reduced extent of necrosis and regeneration in muscle biopsies from PL-12+ patients compared to Jo-1+ patients, while PL-7+ patients had higher capillary dropout in biopsies of skeletal muscle. Aside from these subtle alterations, no significant differences between ASyS subgroups were observed. Interestingly, a tissue-specific subpopulation of CD138+ plasma cells and CXCL12+/CXCL13+CD20+ B cells common to ASyS myositis were identified. These cells were localized in the endomysium associated with alkaline phosphatase+ activated mesenchymal fibroblasts and CD68+MHC-II+CD169+ macrophages. An MHC-I+ and MHC-II+ MxA negative type II interferon-driven milieu of myofiber activation, topographically restricted to the perifascicular area and the adjacent perimysium, as well as perimysial clusters of T follicular helper cells defined an extra-medullary immunological niche for plasma cells and activated B cells. Consistent with this, proteomic analyses of muscle tissues from ASyS patients demonstrated alterations in antigen processing and presentation. In-depth immunological analyses of peripheral blood supported a B-cell/plasma-cell-driven pathology with a shift towards immature B cells, an increase of B-cell-related cytokines and chemokines, and activation of the complement system. We hypothesize that a B-cell-driven pathology with the presence and persistence of a specific subtype of plasma cells in the skeletal muscle is crucially involved in the self-perpetuating chronicity of ASyS myositis. This work provides the conceptual framework for the application of plasma-cell-targeting therapies in ASyS myositis.

The ultrastructural heterogeneity of lung surfactant revealed by serial section electron tomography: insights into the 3-D architecture of human tubular myelin.

Weibel's hypothetical three-dimensional (3-D) model in 1966 provided first ultrastructural details into tubular myelin (TM), a unique, complex surfactant subtype found in the hypophase of the alveolar lining layer. Although initial descriptions by electron microscopy (EM) were already published in the 1950s, a uniform morphological differentiation from other intra-alveolar surfactant subtypes is still missing and potential structure-function relationships remain enigmatic. Technical developments in volume EM methods now allow a more detailed reinvestigation, to address unanswered ultrastructural questions, we analyzed ultrathin sections of humanized SP-A1/SP-A2 coexpressing mouse and human lung samples by conventional transmission EM. We combined these two-dimensional (2-D) information with 3-D analysis of single- and dual-axis electron tomography of serial sections for high z-resolution (in a range of a few nanometers) and extended volumes of up to 1 µm total z-information, this study reveals that TM constitutes a heterogeneous surfactant organization mainly comprised of distorted parallel membrane planes with local intersections, which are distributed all over the TM substructure. These intersecting membrane planes form, among other various polygons, the well-known 2-D "lattice", respectively 3-D quadratic tubules, which in many analyzed spots of human alveoli appear to be less abundant than also observed nonconcentric 3-D lamellae, the additional application of serial section electron tomography to conventional transmission EM demonstrates a high heterogeneity of TM membrane networks, which indicates dynamic transformations between its substructures. Our method provides an ideal basis for further in and ex vivo structural analyses of surfactant under various conditions at nanometer scale.

SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis.

COVID-19-induced "acute respiratory distress syndrome" (ARDS) is associated with prolonged respiratory failure and high mortality, but the mechanistic basis of lung injury remains incompletely understood. Here, we analyze pulmonary immune responses and lung pathology in two cohorts of patients with COVID-19 ARDS using functional single-cell genomics, immunohistology, and electron microscopy. We describe an accumulation of CD163-expressing monocyte-derived macrophages that acquired a profibrotic transcriptional phenotype during COVID-19 ARDS. Gene set enrichment and computational data integration revealed a significant similarity between COVID-19-associated macrophages and profibrotic macrophage populations identified in idiopathic pulmonary fibrosis. COVID-19 ARDS was associated with clinical, radiographic, histopathological, and ultrastructural hallmarks of pulmonary fibrosis. Exposure of human monocytes to SARS-CoV-2, but not influenza A virus or viral RNA analogs, was sufficient to induce a similar profibrotic phenotype in vitro. In conclusion, we demonstrate that SARS-CoV-2 triggers profibrotic macrophage responses and pronounced fibroproliferative ARDS.

On Top of the Alveolar Epithelium: Surfactant and the Glycocalyx.

Gas exchange in the lung takes place via the air-blood barrier in the septal walls of alveoli. The tissue elements that oxygen molecules have to cross are the alveolar epithelium, the interstitium and the capillary endothelium. The epithelium that lines the alveolar surface is covered by a thin and continuous liquid lining layer. Pulmonary surfactant acts at this air-liquid interface. By virtue of its biophysical and immunomodulatory functions, surfactant keeps alveoli open, dry and clean. What needs to be added to this picture is the glycocalyx of the alveolar epithelium. Here, we briefly review what is known about this glycocalyx and how it can be visualized using electron microscopy. The application of colloidal thorium dioxide as a staining agent reveals differences in the staining pattern between type I and type II alveolar epithelial cells and shows close associations of the glycocalyx with intraalveolar surfactant subtypes such as tubular myelin. These morphological findings indicate that specific spatial interactions between components of the surfactant system and those of the alveolar epithelial glycocalyx exist which may contribute to the maintenance of alveolar homeostasis, in particular to alveolar micromechanics, to the functional integrity of the air-blood barrier, to the regulation of the thickness and viscosity of the alveolar lining layer, and to the defence against inhaled pathogens. Exploring the alveolar epithelial glycocalyx in conjunction with the surfactant system opens novel physiological perspectives of potential clinical relevance for future research.

Prevention of DNA Replication Stress by CHK1 Leads to Chemoresistance Despite a DNA Repair Defect in Homologous Recombination in Breast Cancer.

Chromosomal instability not only has a negative effect on survival in triple-negative breast cancer, but also on the well treatable subgroup of luminal A tumors. This suggests a general mechanism independent of subtypes. Increased chromosomal instability (CIN) in triple-negative breast cancer (TNBC) is attributed to a defect in the DNA repair pathway homologous recombination. Homologous recombination (HR) prevents genomic instability by repair and protection of replication. It is unclear whether genetic alterations actually lead to a repair defect or whether superior signaling pathways are of greater importance. Previous studies focused exclusively on the repair function of HR. Here, we show that the regulation of HR by the intra-S-phase damage response at the replication is of overriding importance. A damage response activated by Ataxia telangiectasia and Rad3 related-checkpoint kinase 1 (ATR-CHK1) can prevent replication stress and leads to resistance formation. CHK1 thus has a preferred role over HR in preventing replication stress in TNBC. The signaling cascade ATR-CHK1 can compensate for a double-strand break repair error and lead to resistance of HR-deficient tumors. Established methods for the identification of HR-deficient tumors for Poly(ADP-Ribose)-Polymerase 1 (PARP1) inhibitor therapies should be extended to include analysis of candidates for intra-S phase damage response.

Metabolic Glycoengineering Enables the Ultrastructural Visualization of Sialic Acids in the Glycocalyx of the Alveolar Epithelial Cell Line hAELVi.

The glycocalyx-a plethora of sugars forming a dense layer that covers the cell membrane-is commonly found on the epithelial surface of lumen forming tissue. New glycocalyx specific properties have been defined for various organs in the last decade. However, in the lung alveolar epithelium, its structure and functions remain almost completely unexplored. This is partly due to the lack of physiologically relevant, cost effective in vitro models. As the glycocalyx is an essential but neglected part of the alveolar epithelial barrier, understanding its properties holds the promise to enhance the pulmonary administration of drugs and delivery of nanoparticles. Here, using air-liquid-interface (ALI) cell culture, we focus on combining metabolic glycoengineering with glycan specific electron and confocal microscopy to visualize the glycocalyx of a recently immortalized human alveolar epithelial cell line (hAELVi). For this purpose, we applied different bioorthogonal labeling approaches to visualize sialic acid-an amino sugar that provides negative charge to the lung epithelial glycocalyx-using both fluorescence and gold-nanoparticle labeling. Further, we compared mild chemical fixing/freeze substitution and standard cytochemical electron microscopy embedding protocols for their capacity of contrasting the glycocalyx. In our study, we established hAELVi cells as a convenient model for investigating human alveolar epithelial glycocalyx. Transmission electron microscopy revealed hAELVi cells to develop ultrastructural features reminiscent of alveolar epithelial type II cells (ATII). Further, we visualized extracellular uni- and multilamellar membranous structures in direct proximity to the glycocalyx at ultrastructural level, indicating putative interactions. The lamellar membranes were able to form structures of higher organization, and we report sialic acid to be present within those. In conclusion, combining metabolite specific glycoengineering with ultrastructural localization presents an innovative method with high potential to depict the molecular distribution of individual components of the alveolar epithelial glycocalyx and its interaction partners.

Clustered DNA damage concentrated in particle trajectories causes persistent large-scale rearrangements in chromatin architecture.

BACKGROUND AND PURPOSE: High linear-energy-transfer (LET) irradiation (IR) is characterized by unique depth-dose distribution and advantageous biologic effectiveness compared to low-LET-IR, offering promising alternatives for radio-resistant tumors in clinical oncology. While low-LET-IR induces single DNA lesions such as double-strand breaks (DSBs), localized energy deposition along high-LET particle trajectories induces clustered DNA lesions that are more challenging to repair. During DNA damage response (DDR) 53BP1 and ATM are required for Kap1-dependent chromatin relaxation, thereby sustaining heterochromatic DSB repair. Here, spatiotemporal dynamics of chromatin restructuring were visualized during DDR after high-LET and low-LET-IR. MATERIAL AND METHODS: Human fibroblasts were irradiated with high-LET carbon/calcium ions or low-LET photons. At 0.1 h, 0.5 h, 5 h and 24 h post-IR fluorophore- and gold-labeled repair factors (53BP1, pATM, pKAP-1, pKu70) were visualized by immunofluorescence and transmission electron microscopy, to monitor formation and repair of DNA damage in chromatin ultrastructure. To track chromatin remodeling at damage sites, decondensed regions (DCR) were delineated based on local chromatin concentration densities. RESULTS: Low-LET-IR induced single DNA lesions throughout the nucleus, but nearly all DSBs were efficiently rejoined without visible chromatin decompaction. High-LET-IR induced clustered DNA damage and triggered profound changes in chromatin structure along particle trajectories. In DCR multiple heterochromatic DSBs exhibited delayed repair despite cooperative activity of 53BP1, pATM, pKap-1. These closely localized DSBs may disturb efficient repair and subsequent chromatin restoration, thereby affecting large-scale genome organization. CONCLUSION: Clustered damage concentrated in particle trajectories causes persistent rearrangements in chromatin architecture, which may affect structural and functional organization of cell nuclei.

Hair Follicle Stem Cell Faith Is Dependent on Chromatin Remodeling Capacity Following Low-Dose Radiation.

The main function of the skin, to protect against the environment, is supported by the activity of different stem cell populations. The main focus of this study was elucidating the coping mechanisms of stem cells against the stimulation of constant exposure to genotoxic stresses, both endogenous and exogenous, to ensure long-term function. Investigation of various mouse strains, differing in their DNA repair capacity, enables us to clarify fractionated low-dose irradiation (LDR)-induced consequences for different stem cell populations of the murine hair follicle (HF) in their physiological stem cell niche. Using microscopic techniques combined with flow cytometry, we could show that LDR induces accumulation of persisting; pKu70-independent 53BP1-foci ("chromatin-alterations") in heterochromatic regions of the HF stem cells (HFSCs). These remaining chromatin-alterations result in varying stem cell consequences. CD34-positive HFSCs react by ataxia telangiectasia mutated-dependent, premature senescence, which correlates with global chromatin compaction, whereby apoptosis is prevented by the activity of DNA-dependent protein kinase catalytic subunit. However, distinctively highly damaged HFSCs seem to be sorted out of the niche by differentiation, transferring their chromatin-alterations to more proliferative G protein-coupled receptor 5-positive stem cells. Consequentially, the loss of basal HFSCs is compensated by increased proliferation within the stem cell pool. Despite the initial success of these mechanisms in stem cell population maintenance, the combined effect of the chromatin-alterations and the modification in stem cell pool composition may lead to downstream long-term functional loss of tissue or organs. Stem Cells 2018;36:574-588.

Clustered double-strand breaks in heterochromatin perturb DNA repair after high linear energy transfer irradiation.

BACKGROUND AND PURPOSE: High linear energy transfer (LET) radiotherapy offers superior dose conformity and biological effectiveness compared with low-LET radiotherapy, representing a promising alternative for radioresistant tumours. A prevailing hypothesis is that energy deposition along the high-LET particle trajectories induces DNA lesions that are more complex and clustered and therefore more challenging to repair. The precise molecular mechanisms underlying the differences in radiobiological effects between high-LET and low-LET radiotherapies remain unclear. MATERIAL AND METHODS: Human fibroblasts were irradiated with high-LET carbon ions or low-LET photons. At 0.5h and 5h post exposure, the DNA-damage pattern in the chromatin ultrastructure was visualised using gold-labelled DNA-repair factors. The induction and repair of single-strand breaks, double-strand breaks (DSBs), and clustered lesions were analysed in combination with terminal dUTP nick-end labelling of DNA breaks. RESULTS: High-LET irradiation induced clustered lesions with multiple DSBs along ion trajectories predominantly in heterochromatic regions. The cluster size increased over time, suggesting inefficient DSB repair. Low-LET irradiation induced many isolated DSBs throughout the nucleus, most of which were efficiently rejoined. CONCLUSIONS: The clustering of DSBs in heterochromatin following high-LET irradiation perturbs efficient DNA repair, leading to greater biological effectiveness of high-LET irradiation versus that of low-LET irradiation.