Archaea influence composition of endoscopically visible ileocolonic biofilms.

The gut microbiota has been implicated as a driver of irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Recently we described, mucosal biofilms, signifying alterations in microbiota composition and bile acid (BA) metabolism in IBS and ulcerative colitis (UC). Luminal oxygen concentration is a key factor in the gastrointestinal (GI) ecosystem and might be increased in IBS and UC. Here we analyzed the role of archaea as a marker for hypoxia in mucosal biofilms and GI homeostasis. The effects of archaea on microbiome composition and metabolites were analyzed via amplicon sequencing and untargeted metabolomics in 154 stool samples of IBS-, UC-patients and controls. Mucosal biofilms were collected in a subset of patients and examined for their bacterial, fungal and archaeal composition. Absence of archaea, specifically Methanobrevibacter, correlated with disrupted GI homeostasis including decreased microbial diversity, overgrowth of facultative anaerobes and conjugated secondary BA. IBS-D/-M was associated with absence of archaea. Presence of Methanobrevibacter correlated with Oscillospiraceae and epithelial short chain fatty acid metabolism and decreased levels of Ruminococcus gnavus. Absence of fecal Methanobrevibacter may indicate a less hypoxic GI environment, reduced fatty acid oxidation, overgrowth of facultative anaerobes and disrupted BA deconjugation. Archaea and Ruminococcus gnavus could distinguish distinct subtypes of mucosal biofilms. Further research on the connection between archaea, mucosal biofilms and small intestinal bacterial overgrowth should be performed.

Nfe2l2/NRF2 Deletion Attenuates Tumorigenesis and Increases Bacterial Diversity in a Mouse Model of Lynch Syndrome.

Lynch syndrome (LS) is the most prevalent heritable form of colorectal cancer. Its early onset and high lifetime risk for colorectal cancer emphasize the necessity for effective chemoprevention. NFE2L2 (NRF2) is often considered a potential druggable target, and many chemopreventive compounds induce NRF2. However, although NRF2 counteracts oxidative stress, it is also overexpressed in colorectal cancer and may promote tumorigenesis. In this study, we evaluated the role of NRF2 in the prevention of LS-associated neoplasia. We found increased levels of NRF2 in intestinal epithelia of mice with intestinal epithelium-specific Msh2 deletion (MSH2ΔIEC) compared with C57BL/6 (wild-type) mice, as well as an increase in downstream NRF2 targets NAD(P)H dehydrogenase (quinone 1) and glutamate-cysteine ligase catalytic subunit. Likewise, NRF2 levels were increased in human MSH2-deficient LS tumors compared with healthy human controls. In silico analysis of a publicly accessible RNA sequencing LS dataset also found an increase in downstream NRF2 targets. Upon crossing MSH2ΔIEC with Nrf2null (MSH2ΔIECNrf2null) mice, we unexpectedly found reduced tumorigenesis in MSH2ΔIECNrf2null mice compared with MSH2ΔIEC mice after 40 weeks, which occurred despite an increase in oxidative damage in MSH2ΔIECNrf2null mice. The loss of NRF2 impaired proliferation as seen by Ki67 intestinal staining and in organoid cultures. This was accompanied by diminished WNT/ß-catenin signaling, but apoptosis was unaffected. Microbial α-diversity increased over time with the loss of NRF2 based upon 16S rRNA gene amplicon sequencing of murine fecal samples. Altogether, we show that NRF2 protein levels are increased in MSH2 deficiency and associated neoplasia, but the loss of NRF2 attenuates tumorigenesis. Activation of NRF2 may not be a feasible strategy for chemoprevention in LS. Prevention Relevance: Patients with LS have an early onset and high lifetime risk for colorectal cancer. In this study, we show that NRF2 protein levels are increased in MSH2 deficiency and associated neoplasia, but the loss of NRF2 attenuates tumorigenesis. This suggests that NRF2 may not be a tumor suppressor in this specific context.

GLP-2 Improves Hepatic Inflammation and Fibrosis in Mdr2-/- Mice Via Activation of NR4a1/Nur77 in Hepatic Stellate Cells and Intestinal FXR Signaling.

BACKGROUND & AIMS: Glucagon-like peptide (GLP)-2 may exert antifibrotic effects on hepatic stellate cells (HSCs). Thus, we aimed to test whether application of the GLP-2 analogue teduglutide has hepatoprotective and antifibrotic effects in the Mdr2/Abcb4-/- mouse model of sclerosing cholangitis displaying hepatic inflammation and fibrosis. METHODS: Mdr2-/- mice were injected daily for 4 weeks with teduglutide followed by gene expression profiling (bulk liver; isolated HSCs) and immunohistochemistry. Activated HSCs (LX2 cells) and immortalized human hepatocytes and human intestinal organoids were treated with GLP-2. mRNA profiling by reverse transcription polymerase chain reaction and electrophoretic mobility shift assay using cytosolic and nuclear protein extracts was performed. RESULTS: Hepatic inflammation, fibrosis, and reactive cholangiocyte phenotype were improved in GLP-2-treated Mdr2-/- mice. Primary HSCs isolated from Mdr2-/- mice and LX2 cells exposed to GLP-2 in vitro displayed significantly increased mRNA expression levels of NR4a1/Nur77 (P < .05). Electrophoretic mobility shift assay revealed an increased nuclear NR4a1 binding after GLP-2 treatment in LX2 cells. Moreover, GLP-2 alleviated the Tgfß-mediated reduction of NR4a1 nuclear binding activity. In vivo, GLP-2 treatment of Mdr2-/- mice resulted in increased intrahepatic levels of muricholic acids (accordingly Cyp2c70 mRNA expression was significantly increased), and in reduced mRNA levels of Cyp7a1 and FXR. Serum Fgf15 levels were increased in Mdr2-/- mice treated with GLP-2. Accordingly, GLP-2 treatment of human intestinal organoids activated their FXR-FGF19 signaling axis. CONCLUSIONS: GLP-2 treatment increased NR4a1/Nur77 activation in HSCs, subsequently attenuating their activation. GLP-2 promoted intestinal Fxr-Fgf15/19 signaling resulting in reduced Cyp7a1 and increased Cyp2c70 expression in the liver, contributing to hepatoprotective and antifibrotic effects of GLP-2 in the Mdr2-/- mouse model.

Atypical enteropathogenic E. coli are associated with disease activity in ulcerative colitis.

With increasing urbanization and industrialization, the prevalence of inflammatory bowel diseases (IBDs) has steadily been rising over the past two decades. IBD involves flares of gastrointestinal (GI) inflammation accompanied by microbiota perturbations. However, microbial mechanisms that trigger such flares remain elusive. Here, we analyzed the association of the emerging pathogen atypical enteropathogenic E. coli (aEPEC) with IBD disease activity. The presence of diarrheagenic E. coli was assessed in stool samples from 630 IBD patients and 234 age- and sex-matched controls without GI symptoms. Microbiota was analyzed with 16S ribosomal RNA gene amplicon sequencing, and 57 clinical aEPEC isolates were subjected to whole-genome sequencing and in vitro pathogenicity experiments including biofilm formation, epithelial barrier function and the ability to induce pro-inflammatory signaling. The presence of aEPEC correlated with laboratory, clinical and endoscopic disease activity in ulcerative colitis (UC), as well as microbiota dysbiosis. In vitro, aEPEC strains induce epithelial p21-activated kinases, disrupt the epithelial barrier and display potent biofilm formation. The effector proteins espV and espG2 distinguish aEPEC cultured from UC and Crohn's disease patients, respectively. EspV-positive aEPEC harbor more virulence factors and have a higher pro-inflammatory potential, which is counteracted by 5-ASA. aEPEC may tip a fragile immune-microbiota homeostasis and thereby contribute to flares in UC. aEPEC isolates from UC patients display properties to disrupt the epithelial barrier and to induce pro-inflammatory signaling in vitro.

Loss of bile salt export pump aggravates lipopolysaccharide-induced liver injury in mice due to impaired hepatic endotoxin clearance.

BACKGROUND AND AIMS: Lipopolysaccharide (LPS) clearance is delayed in cholestatic liver diseases. While compromised clearance by Kupffer cells (KCs) is involved, the role of LPS uptake into hepatocytes and canalicular excretion remains unclear. APPROACH AND RESULTS: Wild-type (WT) and bile salt export pump (Bsep) knockout (KO) mice were challenged i.p. with LPS. Liver injury was assessed by serum biochemistry, histology, molecular inflammation markers, and immune cell infiltration. LPS concentrations were determined in liver tissue and bile. Subcellular kinetics of fluorescently labeled LPS was visualized by intravital two-photon microscopy, and the findings in Bsep KO mice were compared to common bile duct-ligated (BDL) and multidrug resistance protein 2 (Mdr2) KO mice. Changes in gut microbiota composition were evaluated by 16S ribosomal RNA gene amplicon sequencing analysis. Bsep KO mice developed more pronounced LPS-induced liver injury and inflammatory signaling, with subsequently enhanced production of proinflammatory cytokines and aggravated hepatic immune cell infiltration. After LPS administration, its concentrations were higher in liver but lower in bile of Bsep KO compared to WT mice. Intravital imaging of LPS showed a delayed clearance from sinusoidal blood with a basolateral uptake block into hepatocytes and reduced canalicular secretion. Moreover, LPS uptake into KCs was reduced. Similar findings with respect to hepatic LPS clearance were obtained in BDL and Mdr2 KO mice. Pretreatment with the microtubule inhibitor colchicine inhibited biliary excretion of LPS in WT mice, indicating that LPS clearance is microtubule-dependent. Microbiota analysis showed no change of the gut microbiome between WT and Bsep KO mice at baseline but major changes upon LPS challenge in WT mice. CONCLUSIONS: Absence of Bsep and cholestasis in general impair LPS clearance by a basolateral uptake block into hepatocytes and consequently less secretion into canaliculi. Impaired LPS removal aggravates hepatic inflammation in cholestasis.

Mucosal Biofilms Are an Endoscopic Feature of Irritable Bowel Syndrome and Ulcerative Colitis.

BACKGROUND & AIMS: Irritable bowel syndrome (IBS) and inflammatory bowel diseases result in a substantial reduction in quality of life and a considerable socioeconomic impact. In IBS, diagnosis and treatment options are limited, but evidence for involvement of the gut microbiome in disease pathophysiology is emerging. Here we analyzed the prevalence of endoscopically visible mucosal biofilms in gastrointestinal disease and associated changes in microbiome composition and metabolism. METHODS: The presence of mucosal biofilms was assessed in 1426 patients at 2 European university-based endoscopy centers. One-hundred and seventeen patients were selected for in-depth molecular and microscopic analysis using 16S ribosomal RNA gene amplicon-sequencing of colonic biopsies and fecal samples, confocal microscopy with deep learning-based image analysis, scanning electron microscopy, metabolomics, and in vitro biofilm formation assays. RESULTS: Biofilms were present in 57% of patients with IBS and 34% of patients with ulcerative colitis compared with 6% of controls (P < .001). These yellow-green adherent layers of the ileum and right-sided colon were microscopically confirmed to be dense bacterial biofilms. 16S-sequencing links the presence of biofilms to a dysbiotic gut microbiome, including overgrowth of Escherichia coli and Ruminococcus gnavus. R. gnavus isolates cultivated from patient biofilms also formed biofilms in vitro. Metabolomic analysis found an accumulation of bile acids within biofilms that correlated with fecal bile acid excretion, linking this phenotype with a mechanism of diarrhea. CONCLUSIONS: The presence of mucosal biofilms is an endoscopic feature in a subgroup of IBS and ulcerative colitis with disrupted bile acid metabolism and bacterial dysbiosis. They provide novel insight into the pathophysiology of IBS and ulcerative colitis, illustrating that biofilm can be seen as a tipping point in the development of dysbiosis and disease.

The food additive EDTA aggravates colitis and colon carcinogenesis in mouse models.

Inflammatory bowel disease is a group of conditions with rising incidence caused by genetic and environmental factors including diet. The chelator ethylenediaminetetraacetate (EDTA) is widely used by the food and pharmaceutical industry among numerous other applications, leading to a considerable environmental exposure. Numerous safety studies in healthy animals have revealed no relevant toxicity by EDTA. Here we show that, in the presence of intestinal inflammation, EDTA is surprisingly capable of massively exacerbating inflammation and even inducing colorectal carcinogenesis at doses that are presumed to be safe. This toxicity is evident in two biologically different mouse models of inflammatory bowel disease, the AOM/DSS and the IL10-/- model. The mechanism of this effect may be attributed to disruption of intercellular contacts as demonstrated by in vivo confocal endomicroscopy, electron microscopy and cell culture studies. Our findings add EDTA to the list of food additives that might be detrimental in the presence of intestinal inflammation, but the toxicity of which may have been missed by regulatory safety testing procedures that utilize only healthy models. We conclude that the current use of EDTA especially in food and pharmaceuticals should be reconsidered. Moreover, we suggest that intestinal inflammatory models should be implemented in the testing of food additives to account for the exposure of this primary organ to environmental and dietary stress.

A Novel PAK1-Notch1 Axis Regulates Crypt Homeostasis in Intestinal Inflammation.

BACKGROUND & AIMS: p21-activated kinase-1 (PAK1) belongs to a family of serine-threonine kinases and contributes to cellular pathways such as nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), and Wingless-related integration site(Wnt)/ß-catenin, all of which are involved in intestinal homeostasis. Overexpression of PAK1 is linked to inflammatory bowel disease as well as colitis-associated cancer (CAC), and similarly was observed in interleukin (IL)10 knockout (KO) mice, a model of colitis and CAC. Here, we tested the effects of PAK1 deletion on intestinal inflammation and carcinogenesis in IL10 KO mice. METHODS: IL10/PAK1 double-knockout (DKO) mice were generated and development of colitis and CAC was analyzed. Large intestines were measured and prepared for histology or RNA isolation. Swiss rolls were stained with H&E and periodic acid-Schiff. Co-immunoprecipitation and immunofluorescence were performed using intestinal organoids, SW480, and normal human colon epithelial cells 1CT. RESULTS: When compared with IL10 KO mice, DKOs showed longer colons and prolonged crypts, despite having higher inflammation and numbers of dysplasia. Crypt hyperproliferation was associated with Notch1 activation and diminished crypt differentiation, indicated by a reduction of goblet cells. Gene expression analysis indicated up-regulation of the Notch1 target hairy and enhancer of split-1 and the stem cell receptor leucin-rich repeat-containing G-protein-coupled receptor 5 in DKO mice. Interestingly, the stem cell marker olfactomedin-4 was present in colonic tissue. Increased ß-catenin messenger RNA and cytoplasmic accumulation indicated aberrant Wnt signaling. Co-localization and direct interaction of Notch1 and PAK1 was found in colon epithelial cells. Notch1 activation abrogated this effect whereas silencing of PAK1 led to Notch1 activation. CONCLUSIONS: PAK1 contributes to the regulation of crypt homeostasis under inflammatory conditions by controlling Notch1. This identifies a novel PAK1-Notch1 axis in intestinal pathophysiology of inflammatory bowel disease and CAC.

Crypt residing bacteria and proximal colonic carcinogenesis in a mouse model of Lynch syndrome.

Colorectal cancer is a multifactorial disease involving inherited DNA mutations, environmental factors, gut inflammation and intestinal microbiota. Certain germline mutations within the DNA mismatch repair system are associated with Lynch syndrome tumors including right-sided colorectal cancer with mucinous phenotype and presence of an inflammatory infiltrate. Such tumors are more often associated with bacterial biofilms, which may contribute to disease onset and progression. Inflammatory bowel diseases are also associated with colorectal cancer and intestinal dysbiosis. Herein we addressed the question, whether inflammation can aggravate colorectal cancer development under mismatch repair deficiency. MSH2loxP/loxP Vill-cre mice were crossed into the IL-10-/- background to study the importance of inflammation and mucosal bacteria as a driver of tumorigenesis in a Lynch syndrome mouse model. An increase in large bowel tumorigenesis was found in double knockout mice both under conventional housing and under specific pathogen-free conditions. This increase was mostly due to the development of proximal tumors, a hotspot for tumorigenesis in Lynch syndrome, and was associated with a higher degree of inflammation. Additionally, bacterial invasion into the mucus of tumor crypts was observed in the proximal tumors. Inflammation shifted fecal and mucosal microbiota composition and was associated with enrichment in Escherichia-Shigella as well as Akkermansia, Bacteroides and Parabacteroides genera in fecal samples. Tumor-bearing double knockout mice showed a similar enrichment for Escherichia-Shigella and Parabacteroides. Lactobacilli, Lachnospiraceae and Muribaculaceae family members were depleted upon inflammation. In summary, chronic inflammation aggravates colonic tumorigenesis under mismatch repair deficiency and is associated with a shift in microbiota composition.

Monoacylglycerol Lipase Inhibition Protects From Liver Injury in Mouse Models of Sclerosing Cholangitis.

BACKGROUND AND AIMS: Monoacylglycerol lipase (MGL) is the last enzymatic step in triglyceride degradation, hydrolyzing monoglycerides into glycerol and fatty acids (FAs) and converting 2-arachidonoylglycerol into arachidonic acid, thus providing ligands for nuclear receptors as key regulators of hepatic bile acid (BA)/lipid metabolism and inflammation. We aimed to explore the role of MGL in the development of cholestatic liver and bile duct injury in mouse models of sclerosing cholangitis, a disease so far lacking effective pharmacological therapy. APPROACH AND RESULTS: To this aim we analyzed the effects of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding to induce sclerosing cholangitis in wild-type (WT) and knockout (MGL-/- ) mice and tested pharmacological inhibition with JZL184 in the multidrug resistance protein 2 knockout (Mdr2-/- ) mouse model of sclerosing cholangitis. Cholestatic liver injury and fibrosis were assessed by serum biochemistry, liver histology, gene expression, and western blot characterization of BA and FA synthesis/transport. Moreover, intestinal FAs and fecal microbiome were analyzed. Transfection and silencing were performed in Caco2 cells. MGL-/- mice were protected from DDC-induced biliary fibrosis and inflammation with reduced serum liver enzymes and increased FA/BA metabolism and ß-oxidation. Notably, pharmacological (JZL184) inhibition of MGL ameliorated cholestatic injury in DDC-fed WT mice and protected Mdr2-/- mice from spontaneous liver injury, with improved liver enzymes, inflammation, and biliary fibrosis. In vitro experiments confirmed that silencing of MGL decreases prostaglandin E2 accumulation in the intestine and up-regulates peroxisome proliferator-activated receptors alpha and gamma activity, thus reducing inflammation. CONCLUSIONS: Collectively, our study unravels MGL as a metabolic target, demonstrating that MGL inhibition may be considered as potential therapy for sclerosing cholangitis.

Switching to a Healthy Diet Prevents the Detrimental Effects of Western Diet in a Colitis-Associated Colorectal Cancer Model.

Inflammatory bowel disease increases the odds of developing colitis-associated cancer. We hypothesized that Western-style diet (WD) aggravates azoxymethane (AOM)/dextran sulfate sodium salt (DSS)-induced colitis-associated tumorigenesis and that switching to the standard AIN93G diet will ameliorate disease symptoms even after cancer initiation. Female BALB/c mice received either WD (WD group) or standard AIN93G diet (AIN group) for the whole experimental period. After five weeks, the mice received 12.5 mg/kg AOM intraperitoneally, followed by three DSS cycles. In one group of mice, the WD was switched to AIN93G the day before starting the first DSS cycle (WD/AIN group). Feeding the WD during the whole experimental period aggravated colitis symptoms, shortened the colon (p < 0.05), changed microbiota composition and increased tumor promotion. On molecular level, the WD reduced proliferation (p < 0.05) and increased expression of the vitamin D catabolizing enzyme Cyp24a1 (p < 0.001). The switch to the AIN93G diet ameliorated this effect, reflected by longer colons, fewer (p < 0.05) and smaller (p < 0.01) aberrant colonic crypt foci, comparable with the AIN group. Our results show that switching to a healthy diet, even after cancer initiation is able to revert the deleterious effect of the WD and could be an effective preventive strategy to reduce colitis symptoms and prevent tumorigenesis.

Mesalamine and azathioprine modulate junctional complexes and restore epithelial barrier function in intestinal inflammation.

Disruption of mucosal structure and barrier function contribute to the pathogenesis of inflammatory bowel disease (IBD). Efficacy of therapy in IBD is based on endoscopic mucosal healing, which occurs by a dynamic interplay of epithelial cell regeneration, migration and differentiation. Both mesalamine (5-ASA) and azathioprine (AZTP) promote this process through mechanisms not clearly understood. We examined molecular pathways implicated in epithelial barrier function that were altered by 5-ASA and AZTP. Paracellular permeability induced by inflammatory mediators was mitigated by both compounds through restoration of cellular anchoring complexes. 5-ASA and AZTP induced rearrangement and membranous localization of junctional proteins and modulated genes involved in tight junctions. Intestinal organoids from wildtype-mice treated with TNF-α and IL-10- deficient-mice displayed impaired epithelial barrier with loss of membranous E-cadherin and reduced Desmoglein-2 expression. These effects were counteracted by 5-ASA and AZTP. Unlike AZTP that exhibited antiproliferative effects, 5-ASA promoted wound healing in colon epithelial cells. Both affected cellular senescence, cell cycle distribution and restricted cells in G1 or S phase without inducing apoptosis. This study provides mechanistic evidence that molecular actions of 5-ASA and AZTP on intestinal epithelia are fundamental in the resolution of barrier dysfunction.

Inorganic SnIP-Type Double Helices in Main-Group Chemistry.

Inspired by the synthesis of the first atomic-scale double-helix semiconductor SnIP, this study deals with the question of whether more atomistic, inorganic double-helix compounds are accessible. With the aid of quantum chemical calculations, we have identified 31 candidates by a homoatomic substitution in MXPn, varying the Group 14 M-element from Si to Pb, the Group 17 X-element from F to I and replacing the pnictide (Pn) phosphorus by arsenic. The double-helical structure of SnIP has been used as the starting model for all candidates and the electronic structure and vibrational spectra were determined within the framework of density functional theory (DFT). Varying the outer MX or the inner Pn helix led to the conclusion that iodide- and bromide-containing MXPn compounds show similar structures to SnIP. Here, the calculations indicate interesting effects for electronic band-gap tuning. For the highly polarized fluorides, a segregation of the helices to more complex MX substructures is predicted.

Inorganic Double Helices in Semiconducting SnIP.

SnIP is the first atomic-scale double helical semiconductor featuring a 1.86 eV bandgap, high structural and mechanical flexibility, and reasonable thermal stability up to 600 K. It is accessible on a gram scale and consists of a racemic mixture of right- and left-handed double helices composed by [SnI] and [P] helices. SnIP nanorods <20 nm in diameter can be accessed mechanically and chemically within minutes.

Polymorphism in Zintl Phases ACd4Pn3: Modulated Structures of NaCd4Pn3 with Pn = P, As.

NaCd4P3 and NaCd4As3 were synthesized via short-way transport using the corresponding elements and CdI2 as mineralizer. At room temperature, the two ß-polymorphs adopt the RbCd4As3 structure type which has been recently reported for alkali metal (A)-d(10) transition metal (T)-pnictides (Pn). The title compounds crystallize rhombohedrally in space group R3̅m at room temperature and show reversible phase transitions to incommensurately modulated α-polymorphs at lower temperatures. The low-temperature phases are monoclinic and can be described in space group Cm(α0γ)s with q vectors of q = (-0.04,0,0.34) for α-NaCd4P3 and q1 = (-0.02,0,0.34) for α-NaCd4As3. Thermal properties, Raman spectroscopy, and electronic structures have been determined. Both compounds are Zintl phases with band gaps of 1.05 eV for ß-NaCd4P3 and ∼0.4 eV for ß-NaCd4As3.

One-Dimensional [P15](-) Tubes in Layered Semiconducting AgP15.

AgP15 was synthesized from the elements via a short-way transport reaction following the mineralizer concept. The needle-shaped crystals were characterized by single-crystal and powder X-ray diffraction. It crystallizes triclinically in space group P1̅ with cell parameters of a = 6.937(1) Å, b = 9.000(1) Å, c = 11.103(2) Å, α = 99.95(1)°, ß = 99.61(1)°, and γ = 105.980(9)°. AgP15 exhibits a tubular phosphorus substructure related but neither isotypic nor isostructural to the alkaline phosphides MP15 (M = Li-Rb). The thermal properties, electronic structure, and experimental band gap of this new semiconductor have been determined. Finally, Raman spectra of AgP15 and selected alkaline-metal polyphosphides MP15 have been measured and interpreted. AgP15 represents the first transition-metal representative of this class of materials.