Increase In Free Iron Research Articles

Golgi protein ACBD3 downregulation sensitizes cells to ferroptosis.

Ferroptosis, a form of cell death driven by iron-dependent lipid peroxidation, is emerging as a promising target in cancer therapy. It is regulated by a network of molecules and pathways that modulate lipid metabolism, iron homeostasis and redox balance, and related processes. However, there are still numerous regulatory molecules intricately involved in ferroptosis that remain to be identified. Here, we indicated that suppression of Golgi protein acyl-coenzyme A binding domain A containing 3 (ACBD3) increased the sensitivity of Henrieta Lacks and PANC1 cells to ferroptosis. ACBD3 knockdown increases labile iron levels by promoting ferritinophagy. This increase in free iron, coupled with reduced levels of glutathione peroxidase 4 due to ACBD3 knockdown, leads to the accumulation of reactive oxygen species and lipid peroxides. Moreover, ACBD3 knockdown also results in elevated levels of polyunsaturated fatty acid-containing glycerophospholipids through mechanisms that remain to be elucidated. Furthermore, inhibition of ferrtinophagy in ACBD3 downregulated cells by knocking down the nuclear receptor co-activator 4 or Bafilomycin A1 treatment impeded ferroptosis. Collectively, our findings highlight the pivotal role of ACBD3 in governing cellular resistance to ferroptosis and suggest that pharmacological manipulation of ACBD3 levels is a promising strategy for cancer therapy.

Inhibition of HSPA8 by rifampicin contributes to ferroptosis via enhancing autophagy.

Rifampicin is the most common pathogenic factor in anti-tuberculosis drug-induced liver injury (AT-DILI), the mechanisms that it promotes hepatocyte damage in AT-DILI are not yet to be thoroughly elucidated. In this study, we investigated the potential molecular mechanisms for ferroptosis involving rifampicin hepatotoxicity. Animal and cell injury models of rifampicin were constructed, and the toxicity of rifampicin was assessed by physicochemical staining and cell viability assay. Next, flow cytometry was employed to detect changes in ferroptosis-related markers, and Western blotting was used to detect protein expression. Then, the important role of autophagy and ferroptosis was verified with small molecule compound intervention. We found that ferritinophagy-induced ferroptosis participates in the toxicity of rifampicin, and the mechanism is that rifampicin precisely activates high-throughput autophagy, which leads to the massive degradation of ferritin and the increase of free iron. Moreover, rifampicin exhibited conspicuous inhibition of Human 71 kDa heat shock cognate protein (HSPA8) that is intimately associated with Microtubule-associated protein light chain 3 isoform B (LC3B) expression, in turn, HSPA8 inducer attenuated intracellular autophagy flux. Of note, inducing HSPA8 or inhibition of autophagy and ferroptosis considerably relieved the hepatotoxicity of rifampicin in mouse model. The present study highlights the crucial roles of the HSPA8 and autophagy in ferroptotic cell death driving by rifampicin, particularly illumines multiple promising regulatory nodes for therapeutic interventions in diseases involving AT-DILI.

Lactoferrin Binding to SARS-CoV-2 Spike Glycoprotein Blocks Pseudoviral Entry and Relieves Iron Protein Dysregulation in Several In Vitro Models

SARS-CoV-2 causes COVID-19, a predominantly pulmonary disease characterized by a burst of pro-inflammatory cytokines and an increase in free iron. The viral glycoprotein Spike mediates fusion to the host cell membrane, but its role as a virulence factor is largely unknown. Recently, the antiviral activity of lactoferrin against SARS-CoV-2 was demonstrated in vitro and shown to occur via binding to cell surface receptors, and its putative interaction with Spike was suggested by in silico analyses. We investigated the anti-SARS-CoV-2 activity of bovine and human lactoferrins in epithelial and macrophagic cells using a Spike-decorated pseudovirus. Lactoferrin inhibited pseudoviral fusion and counteracted the deleterious effects of Spike on iron and inflammatory homeostasis by restoring basal levels of iron-handling proteins and of proinflammatory cytokines IL-1β and IL-6. Using pull-down assays, we experimentally proved for the first time that lactoferrin binds to Spike, immediately suggesting a mechanism for the observed effects. The contribution of transferrin receptor 1 to Spike-mediated cell fusion was also experimentally demonstrated. In silico analyses showed that lactoferrin interacts with transferrin receptor 1, suggesting a multifaceted mechanism of action for lactoferrin. Our results give hope for the use of bovine lactoferrin, already available as a nutraceutical, as an adjuvant to standard therapies in COVID-19.

Regulation of iron homeostasis by peroxide-sensitive CatR, a Fur-family regulator in Streptomyces coelicolor.

CatR, a peroxide-sensing transcriptional repressor of Fur family, can de-repress the transcription of the catA gene encoding catalase upon peroxide stress in Streptomyces coelicolor. Since CatR-regulated genes other than catA and its own gene catR have not been identified in detail, the understanding of the role of CatR regulon is very limited. In this study, we performed transcriptomic analysis to identify genes influenced by both ΔcatR mutation and hydrogen peroxide treatment. Through ChIP-qPCR and other analyses, a new consensus sequence was found in CatR-responsive promoter region of catR gene and catA operon for direct regulation. In addition, vtlA (SCO2027) and SCO4983 were identified as new members of the CatR regulon. Expression levels of iron uptake genes were reduced by hydrogen peroxide and a DmdR1 binding sequence was identified in promoters of these genes. The increase in free iron by hydrogen peroxide was thought to suppress the iron import system by DmdR1. A putative exporter protein VtlA regulated by CatR appeared to reduce intracellular iron to prevent oxidative stress. The name vtlA (VIT1-like transporter) was proposed for iron homeostasis related gene SCO2027.

RESTORATION OF ERYTHROCYTE MORPHOLOGY AFTER CRYOPRESERVATION

During the erythrocyte transfusion, a significant part of the cells (~ 25%) is utilized by the spleen and liver in a day due to the deformations and damage to cell membranes that resulted from the hypothermic storage or cryopreservation. An increase in free iron in the blood during the decomposition of red blood cells leads to the stimulation of oxidative stress and inflammation, as well as to dysfunction of the main organs. The primary indicator of damage to the membranes of erythrocytes can be detected through the changes in their morphology. The work investigated the morphological characteristics of erythrocytes after cryopreservation in a medium containing PEG-1500 and 1,2-PD. Erythrocytes washed after thawing and suspended in an isotonic NaCl solution are represented by echinocytes, at the same time, when placed into a solution with albumin, the cells demonstrate the restoration of their discoid shape. According to the well-established concept, the regulation of the shape of erythrocytes is determined by a change in the conformation of the anionic carrier, which is formed by the main integral membrane protein, the protein of band 3. The study has shown that the treatment of erythrocytes with DIDS, which leads to the fixation of the conformation of the carrier with the transport site facing the outer side of the membrane, does not change the morphomodulatory effect of albumin. At the same time, with a decrease in the medium pH, when the albumin charge approaches zero, its effect is eliminated. The results obtained suggest that, upon freezing-thawing of erythrocytes, the deterioration of hydrophobic interactions of transmembrane segments of the protein of band 3 is accompanied by a change in the balance of electrostatic interactions. Probably, the action of albumin is carried out due to the restoration and stabilization of the indicated balance of membranes, charactestic of the typical eruthrocyte discoid shape. Restoration of the morphology of cryopreserved erythrocytes before the transfusion can help reduce cell deformation, improve systemic hemodynamics and prevent the development of negative clinical consequences.

Iron Dyshomeostasis Participated in Rat Hippocampus Toxicity Caused by Aluminum Chloride.

Aluminum (Al) alters iron regulatory factors content and leads to the changes in iron-related proteins causing iron accumulation. But limited evidence ascertains this hypothesis. Therefore, our experiment was conducted and four groups of male Wistar rats were orally administrated of 0, 50, 150, and 450mg/kg BW/d aluminum chloride (AlCl3) for 90days by drinking water, respectively. The cognitive function, pathological lesion of hippocampus, oxidative stress, as well as iron-related proteins and iron regulatory factors expression were detected. The results showed that AlCl3 remarkably induced the oxidative stress and pathological lesion in the hippocampus and impaired the learning-memory ability. The contents of Al and iron increased in all AlCl3-exposed groups. Meanwhile, the increased divalent metal transporter 1 (DMT1) expression enhanced iron import and the decreased ferroportin 1 (Fpn1) expression reduced iron export in AlCl3-exposed groups. The iron accumulated and ferritin heavy chains (Fth) expression decreased in all AlCl3-exposed groups led to an increase in free iron. The study also showed that iron regulatory factor iron regulatory protein 2 (IRP2) was decreased and hepcidin was increased in AlCl3-exposed groups. The results indicated that AlCl3 induces iron dyshomeostasis presenting as iron accumulation, the disordered expression of iron import, export, store, and regulatory proteins in rat hippocampus accompanied with oxidative stress, pathological lesion, and impaired learning-memory ability.

Biliverdin reductase deficiency triggers an endothelial-to-mesenchymal transition in human endothelial cells

Endothelial dysfunction accompanied by the loss of endothelial cell phenotype plays an essential role in cardiovascular diseases. Here, we report that knockdown of biliverdin reductase (BVR), the enzyme of the heme degradation pathway converting biliverdin to bilirubin, shifts endothelial phenotype of the primary human aortic endothelial cells (HAECs) to mesenchymal-like one. It is reflected by the loss of endothelial markers and angiogenic response, with concomitant acquiring of mesenchymal markers, increased migratory capacity and metalloproteinase activity. BVR-deficiency induces the activity of Nrf2 transcription factor and increases heme oxygenase-1 (HO-1) level, which is accompanied by the reduction of cellular heme content, increase in a free iron fraction and oxidative stress. Accordingly, the phenotype of BVR-deficient cells can be mimicked by hemin or iron overload. Depletion of HO-1 in BVR-deficient ECs abrogates the increase in intracellular free iron and oxidative stress, preventing the loss of endothelial markers. Treatment of BVR-deficient cells with bilirubin does not rescue the endothelial phenotype of HAECs. Unlike BLVRA mRNA level, the expression of HMOX1, HMOX1:BLVRA ratio and HO-1 protein level positively correlate with abdominal aortic aneurysm size in clinical samples. Collectively, the non-enzymatic activity of BVR contributes to the maintenance of healthy endothelial phenotype through the prevention of HO-1-dependent iron-overload, oxidative stress and subsequent endothelial-to-mesenchymal transition (EndMT).

Intense exercise increases protein oxidation in spleen and liver of mice.

Studies have indicated that sports anemia is mainly associated with intravascular hemolysis induced by exercise. We hypothesized that such exercise-induced hemolysis leads to oxidative damage due to an increase in free iron caused by hematocyte destruction. Thirty-one male ICR mice were randomly divided into 3 groups: a rested control group, an intense-exercise group, and a group rested for 24 hours after intense exercise. The serum haptoglobin level of the intense-exercise group decreased compared with that of the rested control group, suggesting hemolysis. Tissue iron and protein carbonyl levels in the liver were increased after exercise, and the protein carbonyl level in the spleen on the day after exercise was significantly increased compared with that of the resting state. These results suggest that the spleen and liver, where extravascular hemolysis occurs, were subjected to oxidative modification by the free iron, which was released from large numbers of hemocytes that were destroyed due to the intense exercise.

Hierro e infección fúngica invasiva

Iron is an essential factor for both the growth and virulence of most of microorganisms. As a part of the innate (or nutritional) immune system, mammals have developed different mechanisms to store and transport this element in order to limit free iron bioavailability. To survive in this hostile environment, pathogenic fungi have specific uptake systems for host iron sources, one of the most important of which is based on the synthesis of siderophores-soluble, low-molecular-mass, high-affinity iron chelators. The increase in free iron that results from iron-overload conditions is a well-established risk factor for invasive fungal infection (IFI) such as mucormycosis or aspergillosis. Therefore, iron chelation may be an appealing therapeutic option for these infections. Nevertheless, deferoxamine –the first approved iron chelator– paradoxically increases the incidence of IFI, as it serves as a xeno-siderophore to Mucorales. On the contrary, the new oral iron chelators (deferiprone and deferasirox) have shown to exert a deleterious effect on fungal growth both in vitro and in animal models. The present review focuses on the role of iron metabolism in the pathogenesis of IFI and summarises the preclinical data, as well as the limited clinical experience so far, in the use of new iron chelators as treatment for mucormycosis and invasive aspergillosis.

Impact of Iron Indices, Mitochondrial Oxidative Capacity, Oxidative Stress and Inflammatory Markers on Insulin Resistance and Secretion: A Pathophysiologic Perspective

Objective: To elucidate how iron deficiency anemia influence the natural history of diabetes mellitus and to study the effect of iron indices, mitochondrial oxidative capacity and oxidative stress on insulin resistance and secretion in established cases of type 2 diabetes. Research design and methods: A comparative cross-sectional study was conducted in 40 healthy controls, 40 iron deficiency anemia (IDA), 40 type 2 diabetes without iron deficiency anemia and 40 type 2 diabetes with iron deficiency patients. All the Patients were BMI, age and sex matched. Glycemic indices, fasting serum insulin levels, iron indices, inflammatory markers, Total Antioxidation Capacity (TAOC), Mitochondrial Oxidative Capacity (MOC) were analyzed. Results: In iron deficiency anemia there was decrease in MOC, TAOC and HOMA B (p<0.001). Iron was positively correlated with MOC which in turn was positively correlated with HOMA B. In type 2 diabetes there was increase in free iron, ferritin, transferrin, TNF-alpha and HOMA IR (p<0.001) and decrease in TAOC, MOC and HOMA B. In type 2 diabetes with IDA there was decrease in iron, ferritin HOMA-IR (p<0.001), TNF- α (p<0.05) and increase in IL-6(p<0.05) as compared to type 2 diabetes without IDA. Conclusion: Iron deficiency anemia is associated with decreased mitochondrial oxidative capacity and insulin secretion with no change in insulin sensitivity. This prompts us to question whether chronic IDA predisposes to early development of diabetes especially where both diabetes and IDA are highly prevalent. Both diabetes and iron deficiency anemia can together worsen the oxidative stress and the pathophysiology of diabetes mellitus despite improvement in insulin resistance.

Heme oxygenase‐1 induction in rat spleen following aniline exposure

Splenic toxicity of aniline is characterized by vascular congestion, hyperplasia, fibrosis and development of a variety of sarcomas in rats. However, mechanisms underlying splenic toxicity are not well understood. Previously we observed that aniline exposure causes oxidative damage to the spleen. To further explore the involvement of oxidative mechanism in aniline toxicity, we evaluated the role of heme oxygenase‐1 (HO‐1), which catalyzes heme degradation leading to the formation of biliverdin, carbon monoxide and iron. Male SD rats were given 1 mmol/kg/day aniline in water by gavage for 1 or 7 days, while the controls received water only. HO‐1 quantitation by ELISA showed a 2.6 fold increase in spleens of rats treated with aniline for 7 days. These increases were supported by Western blot analysis (~2.5 fold increase in HO‐1 expression). No changes were observed in animals exposed for 1 day. Immunohistochemical analysis showed greater staining for HO‐1 in the red pulp areas of spleen from rats treated for 7 days. Increase in HO‐1 expression was associated with 1.9 fold increase in ferritin, 2.7 fold increase in total iron and, more importantly, a 3.5 fold increase in free iron in spleens of rats exposed for 7 days. It is evident from our data that increased HO‐1 expression and its association with iron accumulation could be an important pro‐oxidant mechanism leading to oxidative damage in the spleen. Supported by ES006476.

Relationship between free iron and glycated hemoglobin in uncontrolled type 2 diabetes patients associated with complications.

Free iron in serum has been found in several disease conditions including diabetes. In the present work, we studied the relationship between free iron, fasting blood glucose (FBG) and glycated haemoglobin (HbA(1c)). Study was carried out on 50 type 2 diabetes cases under poor glycemic control associated with complications, 53 type 2 diabetes cases under good glycemic control and 40 healthy controls. We estimated free iron, both ferrous (Fe(+2)) and ferric (Fe(+3)) form, protein thiols, lipid hydroperoxides, FBG, HbA1c and serum ferritin levels in serum. There was a significant increase in free iron in Fe(+3) state (p <0.01), HbA(1c) (p<0.01), serum ferritin (p<0.01), lipid hydroperoxides (p<0.01) and significant decrease in protein thiols (<0.01) in diabetes cases under poor glycemic control compared to diabetes cases under good glycemic control and healthy controls. Free iron correlated positively with HbA(1c) (p<0.01). Poor glycemic control and increase in glycation of haemoglobin is contributing to the increase in free iron pool which is known to increase oxidant generation.

Does excess iron play a role in breast carcinogenesis? an unresolved hypothesis

Free iron is a pro-oxidant and can induce oxidative stress and DNA damage. The carcinogenicity of iron has been demonstrated in animal models, and epidemiologic studies have shown associations with several human cancers. However, a possible role of excess body iron stores or of elevated iron intake in breast carcinogenesis has received little attention epidemiologically. We propose that iron overload and the disruption of iron homeostasis with a resulting increase in free iron may contribute to the development of breast cancer, and we summarize the relevant evidence from mechanistic studies, animal experiments, and studies in humans. Over time a high intake of iron can lead to iron overload. Furthermore, body iron stores increase in women following menopause. Reactive oxygen species produced by normal aerobic cellular metabolism can lead to the release of free iron from ferritin. In the presence of superoxide radical and hydrogen peroxide, stored ferric iron (Fe(3+)) is reduced to ferrous iron (Fe(2+)), which catalyzes the formation of the hydroxyl radical (*OH). *OH in turn can promote lipid peroxidation, mutagenesis, DNA strand breaks, oncogene activation, and tumor suppressor inhibition, increasing the risk of breast cancer. In addition to its independent role as a proxidant, high levels of free iron may potentiate the effects of estradiol, ethanol, and ionizing radiation - three established risk factors for breast cancer. In order to identify the role of iron in breast carcinogenesis, improved biomarkers of body iron stores are needed, as are cohort studies which assess heme iron intake. Ultimately, it is important to determine whether iron levels in the breast and iron-induced pathology are higher in women who go on to develop breast cancer compared to women who do not.

Depolarization-Associated Iron Release with Abrupt Reduction in Pulmonary Endothelial Shear StressIn Situ

This study evaluated the roles of endothelial cell membrane potential and reactive oxygen species (ROS) in the increase of tissue free iron during lung ischemia. Oxygenated ischemia was produced in the isolated rat lung by discontinuing perfusion while ventilation with O2 was maintained. We have shown previously that tissue oxygenation is maintained in this model of ischemia and that biochemical changes are the result of an abrupt reduction in endothelial shear stress. With 1 hr oxygenated ischemia, generation of ROS, evaluated by oxidation of dichlorodihydrofluorescein (H2DCF) to fluorescent dichlorofluorescein, increased 8.0-fold, lung thiobarbituric acid reactive substances (TBARS) increased 3.4-fold, and lung protein carbonyl content increased 2.4-fold. Lung tissue free iron, measured in the lung homogenate with a fluorescent desferrioxamine derivative, increased 4.0-fold during ischemia. Pretreatment of lungs with thapsigargin abolished the increase in free iron with ischemia indicating that this effect is dependent on Ca2+ release from intracellular stores. Perfusion of lungs with high (25 mM) K+ to depolarize the endothelium also led to a significant increase in tissue free iron. Pretreatment of lungs with 35 microM cromakalim, a K+-channel agonist, significantly inhibited both ischemia-induced tissue oxidant injury and the increase in free iron with ischemia or with high K+ perfusion. A similar increase in free iron was observed when lungs were ventilated with either O2 or N2 during the ischemic period or were pre-perfused with an inhibitor of ROS production (diphenyleneiodonium). These results indicate that ROS generation is not required for ischemia-mediated iron release. Thus, ROS generation and iron release with ischemia are independent although both are subsequent to endothelial cell membrane depolarization.

Ferritin Oxidation in Vitro: Implication of Iron Release and Degradation by the 20S Proteasome

Ferritin, the major iron storage protein in mammalian cells, was treated with various concentrations of different oxidants: xanthine/xanthine oxidase, Sin-1 (3-morpholinosydnonimine, purchased from Alexis, Grunberg, Germany), DEA-NO (Diethylamine NONOate, purchased from Calblochem-Novabiochem, Schwalbach, Germany), and hydrogen peroxide. The proteolytic susceptibility towards the isolated 20S proteasome of untreated ferritin and oxidized ferritin was measured in parallel with the iron liberated by these oxidants. With increasing hydrogen peroxide, Sin-1, and xanthine oxidase concentrations, the measured proteasomal degradation of ferritin also increased. At higher oxidant concentrations, however, the proteolytic susceptibility began to decrease. The oxidation of ferritin by DEA-NO was accompanied by a lesser increase of proteolytic susceptibility in comparison with the effects of the other oxidants. Addition of DEA-NO to Sin-1 suppressed the increase in proteolytic susceptibility of ferritin, whereas adding xanthine/xanthine oxidase had no additional effect. Iron was liberated readily from ferritin as a result of the oxidation process, although the increase in proteolytic susceptibility was not always correlated to the iron release. In fact, the degradation of oxidatively damaged ferritin was not accompanied by a further increase of free iron. Therefore, we conclude that the proteasome is a secondary antioxidative defense system that degrades only nonfunctional ferritin.

The labile iron pool in hepatocytes: prooxidant-induced increase in free iron precedes oxidative cell injury.

The labile iron pool (LIP) represents the nonferritin-bound, redox-active iron that has been implicated in oxidative stress and cell injury. Here we examined whether alterations in LIP can be detected in cultured murine hepatocytes and whether increases in LIP are related to the oxidative damage inflicted by the redox cycling drug nitrofurantoin (NFT). Early changes in LIP were monitored with the metal-sensitive fluorescent probe calcein (CA), the fluorescence of which is quenched on binding to iron. Short-term exposure (<1 h) to NFT reduced the CA fluorescence signal by 30%, indicating that the amount of LIP-associated iron had increased. Prolonged exposure (2 h) to NFT caused oxidative cell injury. The addition of the cell-permeable ferrous iron chelator 2,2'-bipyridyl not only prevented the quenching of CA fluorescence but also partially protected from NFT toxicity. It is concluded that reductive stress-induced increase in LIP is an essential event that precedes oxidative cell damage in intact hepatocytes.

Induction of Ferritin Synthesis in Cells Infected with Mengo Virus

We have recently identified ferritin as a cellular protein particle whose synthesis is stimulated in mouse or human cells infected by the picornavirus Mengo. Immunoprecipitation of the particle from infected murine L929 cells showed a 4- and 6-fold increase in the intracellular concentrations of H and L apoferritin subunits, respectively. This differential expression altered the H/L subunit ratio from 3.0 in uninfected cells to 2.2 in Mengo virus-infected cells. The induction is not due to an increase in transcription of the apoferritin L and H genes, nor is it due to an increase in stability of the apoferritin mRNAs. At the level of translation, the iron regulatory protein (IRP) remained intact, with similar amounts being detected in uninfected and infected cells. The Mengo virus RNA genome does not compete with the iron regulatory element (IRE) for the binding of IRP, and sequence analysis confirmed that there are no IREs in the virus RNA. The IRE binding activity of IRP in infected cells decreased approximately 30% compared with uninfected cells. The decrease in binding activity could be overcome by the addition of Desferal (deferoxamine mesylate; CIBA) an intracellular iron chelator, which suggests that virus infection causes an increase in intracellular free iron. Electron paramagnetic resonance (EPR) studies have confirmed the increase in free iron in Mengo virus infected cells. The permeability of cells for iron does not change in virus infected cells, suggesting that the induction of ferritin by Mengo virus is due to a change in the form of intracellular iron from a bound to a free state.

Iron in brain function and dysfunction with emphasis on Parkinson's disease.

Metals such as lead, zinc, copper, aluminum and manganese have been implicated in neuropsychiatric disorders. However, until fairly recently the role of iron in brain function was rather obscure, because little attention was paid to its metabolism in the brain. It is now apparent that maintenance of brain iron homoeostasis is important for the normal functioning of his organ. Most of the studies have been directed towards the cognitive and attentional deficit resulting from nutritional iron deficiency. Evidence so far suggests subsensitivity of striatal dopamine neurotransmission. By contrast the selective increase in free iron in the substantia nigra pars compacta of parkinsonian brains is thought to initiate oxidative stress, from iron-induced liberation of cytotoxic oxygen free radicals. Such radicals are known to promote membrane fluidity, alteration in cellular calcium homoeostasis, lipid peroxidation and finally cell death in systemic organs. Evidence supporting similar processes being responsible for nigrostriatal dopamine neuron degeneration in Parkinson's disease is now becoming available. Such possibilities afford the development of neuroprotective drugs as a means to retard the progression of this disorder. These include other selective monoamine oxidase B inhibitors, iron chelators with the ability to cross the blood-brain barrier, selective calcium channel antagonists and mitochondrial electron transport system protectors.