Filamentous structures in the cell envelope are associated with bacteroidetes gliding machinery.

Many bacteria belonging to the phylum Bacteroidetes move on solid surfaces, called gliding motility. In our previous study with the Bacteroidetes gliding bacterium Flavobacterium johnsoniae, we proposed a helical loop track model, where adhesive SprB filaments are propelled along a helical loop on the cell surface. In this study, we observed the gliding cell rotating counterclockwise about its axis when viewed from the rear to the advancing direction of the cell and revealed that one labeled SprB focus sometimes overtook and passed another SprB focus that was moving in the same direction. Several electron microscopic analyses revealed the presence of a possible multi-rail structure underneath the outer membrane, which was associated with SprB filaments and contained GldJ protein. These results provide insights into the mechanism of Bacteroidetes gliding motility, in which the SprB filaments are propelled along tracks that may form a multi-rail system underneath the outer membrane. The insights may give clues as to how the SprB filaments get their driving force.

Key Amino Acids for Transferase Activity of GDSL Lipases.

The Gly-Asp-Ser-Leu (GDSL) motif of esterase/lipase family proteins (GELPs) generally exhibit esterase activity, whereas transferase activity is markedly preferred in several GELPs, including the Tanacetum cinerariifolium GDSL lipase TciGLIP, which is responsible for the biosynthesis of the natural insecticide, pyrethrin I. This transferase activity is due to the substrate affinity regulated by the protein structure and these features are expected to be conserved in transferase activity-exhibiting GELPs (tr-GELPs). In this study, we identified two amino acid residues, [N/R]208 and D484, in GELP sequence alignments as candidate key residues for the transferase activity of tr-GELPs by two-entropy analysis. Molecular phylogenetic analysis demonstrated that each tr-GELP is located in the clusters for non-tr-GELPs, and most GELPs conserve at least one of the two residues. These results suggest that the two conserved residues are required for the acquisition of transferase activity in the GELP family. Furthermore, substrate docking analyses using ColabFold-generated structure models of both natives and each of the two amino acids-mutated TciGLIPs also revealed numerous docking models for the proper access of substrates to the active site, indicating crucial roles of these residues of TciGLIP in its transferase activity. This is the first report on essential residues in tr-GELPs for the transferase activity.

Draft Genome of Tanacetum Coccineum: Genomic Comparison of Closely Related Tanacetum-Family Plants.

The plant Tanacetum coccineum (painted daisy) is closely related to Tanacetum cinerariifolium (pyrethrum daisy). However, T. cinerariifolium produces large amounts of pyrethrins, a class of natural insecticides, whereas T. coccineum produces much smaller amounts of these compounds. Thus, comparative genomic analysis is expected to contribute a great deal to investigating the differences in biological defense systems, including pyrethrin biosynthesis. Here, we elucidated the 9.4 Gb draft genome of T. coccineum, consisting of 2,836,647 scaffolds and 103,680 genes. Comparative analyses of the draft genome of T. coccineum and that of T. cinerariifolium, generated in our previous study, revealed distinct features of T. coccineum genes. While the T. coccineum genome contains more numerous ribosome-inactivating protein (RIP)-encoding genes, the number of higher-toxicity type-II RIP-encoding genes is larger in T. cinerariifolium. Furthermore, the number of histidine kinases encoded by the T. coccineum genome is smaller than that of T. cinerariifolium, suggesting a biological correlation with pyrethrin biosynthesis. Moreover, the flanking regions of pyrethrin biosynthesis-related genes are also distinct between these two plants. These results provide clues to the elucidation of species-specific biodefense systems, including the regulatory mechanisms underlying pyrethrin production.

Insertional Inactivation and Gene Complementation of Prevotella intermedia Type IX Secretion System Reveals Its Indispensable Roles in Black Pigmentation, Hemagglutination, Protease Activity of Interpain A, and Biofilm Formation.

Prevotella intermedia, a Gram-negative oral anaerobic bacterium, is frequently isolated from the periodontal pockets of patients with chronic periodontitis. In recent years, the involvement of the bacterium in respiratory tract infections as well as in oral infections has been revealed. P. intermedia possesses several potent virulence factors, such as cysteine proteinase interpain A encoded by the inpA gene. The genome of P. intermedia carries genes of the type IX secretion system (T9SS), which enables the translocation of virulence factors across the outer membrane in several pathogens belonging to the phylum Bacteroidetes; however, it is still unclear whether the T9SS is functional in this microorganism. Recently, we performed targeted mutagenesis in the strain OMA14 of P. intermedia. Here, we successfully obtained mutants deficient in inpA and the T9SS component genes porK and porT. None of the mutants exhibited protease activity of interpain A. The porK and porT mutants, but not the inpA mutant, showed defects in colony pigmentation, hemagglutination, and biofilm formation. We also obtained a complemented strain for the porK gene that recovered all the above abilities. These results indicate that T9SS functions in P. intermedia and that interpain A is one of the T9SS cargo proteins. IMPORTANCE The virulence factors of periodontal pathogens such as Prevotella intermedia have not been elucidated. Using our established procedure, we succeeded in generating type IX secretion system mutants and gene complementation strains that might transfer virulence factors to the bacterial surface. The generated strains clearly indicate that T9SS in P. intermedia is essential for colonial pigmentation, hemagglutination, and biofilm formation. These results indicated that interpain A is a T9SS cargo protein.

Porphyromonas gingivalis Gingipains Induce Cyclooxygenase-2 Expression and Prostaglandin E2 Production via ERK1/2-Activated AP-1 (c-Jun/c-Fos) and IKK/NF-κB p65 Cascades.

Porphyromonas gingivalis is commonly known as one of the major pathogens contributing to periodontitis, and its persistent infection may increase the risk for the disease. The proinflammatory mediators, including IL-6, TNF-α, and cyclooxygenase-2 (COX-2)/PGE2, are closely associated with progression of periodontitis. In this study, we focused on the cysteine protease "gingipains," lysine-specific gingipain, arginine-specific gingipain (Rgp) A, and RgpB, produced by P. gingivalis, and used the wild-type strain and several gene-deletion mutants (rgpA, rgpB, kgp, and fimA) to elucidate the involvement of gingipains in COX-2 expression and PGE2 production. We infected human monocytes, which are THP-1 cells and primary monocytes, with these bacterial strains and found that gingipains were involved in induction of COX-2 expression and PGE2 production. We have shown that the protease activity of gingipains was crucial for these events by using gingipain inhibitors. Furthermore, activation of ERK1/2 and IκB kinase was required for gingipain-induced COX-2 expression/PGE2 production, and these kinases activated two transcription factors, c-Jun/c-Fos (AP-1) and NF-κB p65, respectively. In particular, these data suggest that gingipain-induced c-Fos expression via ERK is essential for AP-1 formation with c-Jun, and activation of AP-1 and NF-κB p65 plays a central role in COX-2 expression/PGE2 production. Thus, we show the (to our knowledge) novel finding that gingipains with the protease activity from P. gingivalis induce COX-2 expression and PGE2 production via activation of MEK/ERK/AP-1 and IκB kinase/NF-κB p65 in human monocytes. Hence it is likely that gingipains closely contribute to the inflammation of periodontal tissues.

Colony spreading of the gliding bacterium Flavobacterium johnsoniae in the absence of the motility adhesin SprB.

Colony spreading of Flavobacterium johnsoniae is shown to include gliding motility using the cell surface adhesin SprB, and is drastically affected by agar and glucose concentrations. Wild-type (WT) and ΔsprB mutant cells formed nonspreading colonies on soft agar, but spreading dendritic colonies on soft agar containing glucose. In the presence of glucose, an initial cell growth-dependent phase was followed by a secondary SprB-independent, gliding motility-dependent phase. The branching pattern of a ΔsprB colony was less complex than the pattern formed by the WT. Mesoscopic and microstructural information was obtained by atmospheric scanning electron microscopy (ASEM) and transmission EM, respectively. In the growth-dependent phase of WT colonies, dendritic tips spread rapidly by the movement of individual cells. In the following SprB-independent phase, leading tips were extended outwards by the movement of dynamic windmill-like rolling centers, and the lipoproteins were expressed more abundantly. Dark spots in WT cells during the growth-dependent spreading phase were not observed in the SprB-independent phase. Various mutations showed that the lipoproteins and the motility machinery were necessary for SprB-independent spreading. Overall, SprB-independent colony spreading is influenced by the lipoproteins, some of which are involved in the gliding machinery, and medium conditions, which together determine the nutrient-seeking behavior.

Transport and Polymerization of Porphyromonas gingivalis Type V Pili.

Adhesive pili (or fimbriae) in bacteria are classified into five types, among which type V pili have been most recently described. Type V pili differ from other pili types with respect to transport mechanism, structure, and pilin synthesis. Genes of type V pili are restricted to the phylum Bacteroidetes. Protein subunits that compose type V pili are transported to the cell surface as lipoprotein precursors and then polymerized into a pilus through a strand-exchange mechanism, which is demonstrated by several experiments, including palmitic acid labeling and Cys-Cys cross-linking analysis. Here, we describe the use of these methods to analyze type V pili.

PorA, a conserved C-terminal domain-containing protein, impacts the PorXY-SigP signaling of the type IX secretion system.

Porphyromonas gingivalis, a periodontal pathogen, translocates many virulence factors including the cysteine proteases referred to as gingipains to the cell surface via the type IX secretion system (T9SS). Expression of the T9SS component proteins is regulated by the tandem signaling of the PorXY two-component system and the ECF sigma factor SigP. However, the details of this regulatory pathway are still unknown. We found that one of the T9SS conserved C-terminal domain-containing proteins, PGN_0123, which we have designated PorA, is involved in regulating expression of genes encoding T9SS structural proteins and that PorA can be translocated onto the cell surface without the T9SS translocation machinery. X-ray crystallography revealed that PorA has a domain similar to the mannose-binding domain of Escherichia coli FimH, the tip protein of Type 1 pilus. Mutations in the cytoplasmic domain of the sensor kinase PorY conferred phenotypic recovery on the ΔporA mutant. The SigP sigma factor, which is activated by the PorXY two-component system, markedly decreased in the ΔporA mutant. These results strongly support a potential role for PorA in relaying a signal from the cell surface to the PorXY-SigP signaling pathway.

Effect of Porphyromonas gingivalis infection on gut dysbiosis and resultant arthritis exacerbation in mouse model.

BACKGROUND: Porphyromonas gingivalis (Pg) infection causes periodontal disease and exacerbates rheumatoid arthritis (RA). It is reported that inoculation of periodontopathogenic bacteria (i.e., Pg) can alter gut microbiota composition in the animal models. Gut microbiota dysbiosis in human has shown strong associations with systemic diseases, including RA, diabetes mellitus, and inflammatory bowel disease. Therefore, this study investigated dysbiosis-mediated arthritis by Pg oral inoculation in an experimental arthritis model mouse. METHODS: Pg inoculation in the oral cavity twice a week for 6 weeks was performed to induce periodontitis in SKG mice. Concomitantly, a single intraperitoneal (i.p.) injection of laminarin (LA) was administered to induce experimental arthritis (Pg-LA mouse). Citrullinated protein (CP) and IL-6 levels in serum as well as periodontal, intestinal, and joint tissues were measured by ELISA. Gut microbiota composition was determined by pyrosequencing the 16 s ribosomal RNA genes after DNA purification of mouse feces. Fecal microbiota transplantation (FMT) was performed by transferring Pg-LA-derived feces to normal SKG mice. The effects of Pg peptidylarginine deiminase (PgPAD) on the level of citrullinated proteins and arthritis progression were determined using a PgPAD knockout mutant. RESULTS: Periodontal alveolar bone loss and IL-6 in gingival tissue were induced by Pg oral infection, as well as severe joint destruction, increased arthritis scores (AS), and both IL-6 and CP productions in serum, joint, and intestinal tissues. Distribution of Deferribacteres and S24-7 was decreased, while CP was significantly increased in gingiva, joint, and intestinal tissues of Pg-inoculated experimental arthritis mice compared to experimental arthritis mice without Pg inoculation. Further, FMT from Pg-inoculated experimental arthritis mice reproduced donor gut microbiota and resulted in severe joint destruction with increased IL-6 and CP production in joint and intestinal tissues. The average AS of FMT from Pg-inoculated experimental arthritis was much higher than that of donor mouse. However, inoculation of the PgPAD knockout mutant inhibited the elevation of arthritis scores and ACPA level in serum and reduced CP amount in gingival, joint, and intestinal tissues compared to Pg wild-type inoculation. CONCLUSION: Pg oral infection affected gut microbiota dysbiosis and joint destruction via increased CP generation.

Type IX Secretion System Cargo Proteins Are Glycosylated at the C Terminus with a Novel Linking Sugar of the Wbp/Vim Pathway.

Porphyromonas gingivalis and Tannerella forsythia use the type IX secretion system to secrete cargo proteins to the cell surface where they are anchored via glycolipids. In P. gingivalis, the glycolipid is anionic lipopolysaccharide (A-LPS), of partially known structure. Modified cargo proteins were deglycosylated using trifluoromethanesulfonic acid and digested with trypsin or proteinase K. The residual modifications were then extensively analyzed by tandem mass spectrometry. The C terminus of each cargo protein was amide-bonded to a linking sugar whose structure was deduced to be 2-N-seryl, 3-N-acetylglucuronamide in P. gingivalis and 2-N-glycyl, 3-N-acetylmannuronic acid in T. forsythia The structures indicated the involvement of the Wbp pathway to produce 2,3-di-N-acetylglucuronic acid and a WbpS amidotransferase to produce the uronamide form of this sugar in P. gingivalis The wbpS gene was identified as PGN_1234 as its deletion resulted in the inability to produce the uronamide. In addition, the P. gingivalisvimA mutant which lacks A-LPS was successfully complemented by the T. forsythiavimA gene; however, the linking sugar was altered to include glycine rather than serine. After removal of the acetyl group at C-2 by the putative deacetylase, VimE, VimA presumably transfers the amino acid to complete the biosynthesis. The data explain all the enzyme activities required for the biosynthesis of the linking sugar accounting for six A-LPS-specific genes. The linking sugar is therefore the key compound that enables the attachment of cargo proteins in P. gingivalis and T. forsythia We propose to designate this novel linking sugar biosynthetic pathway the Wbp/Vim pathway.IMPORTANCEPorphyromonas gingivalis and Tannerella forsythia, two pathogens associated with severe gum disease, use the type IX secretion system (T9SS) to secrete and attach toxic arrays of virulence factor proteins to their cell surfaces. The proteins are tethered to the outer membrane via glycolipid anchors that have remained unidentified for more than 2 decades. In this study, the first sugar molecules (linking sugars) in these anchors are identified and found to be novel compounds. The novel biosynthetic pathway of these linking sugars is also elucidated. A diverse range of bacteria that do not have the T9SS were found to have the genes for this pathway, suggesting that they may synthesize similar linking sugars for utilization in different systems. Since the cell surface attachment of virulence factors is essential for virulence, these findings reveal new targets for the development of novel therapies.

Biogenesis of Type V pili.

Pili or fimbriae, which are filamentous structures present on the surface of bacteria, were purified from a periodontal pathogen, Porphyromonas gingivalis, in 1980s. The protein component of pili (stalk pilin), which is its major component, was named FimA; it has a molecular weight of approximately 41 kDa. Because the molecular weight of the pilin from P. gingivalis is twice that of pilins from other bacterial pili, the P. gingivalis Fim pili were suggested to be formed via a novel mechanism. In earlier studies, we reported that the FimA pilin is secreted on the cell surface as a lipoprotein precursor, and the subsequent N-terminal processing of the FimA precursor by arginine-specific proteases is necessary for Fim pili formation. The crystal structures of FimA and its related proteins were determined recently, which show that Fim pili are formed by a protease-mediated strand-exchange mechanism. The most recent study conducted by us, wherein we performed cryoelectron microscopy of the pilus structure, provided evidence in support of this mechanism. As the P. gingivalis Fim pili are formed through novel transport and assembly mechanisms, such pili are now designated as Type V pili. Surface lipoproteins, including the anchor pilin FimB of Fim pili that are present on the outer membrane, have been detected in certain Gram-negative bacteria. Here, we describe the assembly mechanisms of pili, including those of Type V and other pili, as well as the lipoprotein transport mechanisms.

PorM, a core component of bacterial type IX secretion system, forms a dimer with a unique kinked-rod shape.

Porphyromonas gingivalis, which is a major pathogen of the periodontal disease, secrets virulence factors such as gingipain proteases via the type IX secretion system (T9SS). T9SS consists of a trans-periplasmic core complex, the outer membrane translocon complex and the cell-surface complex attached on the outer membrane. PorM is a major component of the trans-periplasmic core complex and is believed to connect the outer membrane component with the inner membrane component. Recent structural studies have revealed that the periplasmic region of GldM, a PorM homolog of a gliding bacterium, consist of four domains and forms a dimer with a straight rod shape. However, only fragment structures are known for PorM. Moreover, one of the PorM fragment structure shows a kink. Here we show the structure of the entire structure of the periplasmic region of PorM (PorMp) at 3.7 Å resolution. PorMp is made up of four domains and forms a unique dimeric structure with an asymmetric, kinked-rod shape. The structure and the following mutational analysis revealed that R204 stabilizes the kink between the D1 and D2 domains and is essential for gingipains secretion, suggesting that the kinked structure of PorM is important for the functional T9SS formation.

Structure of polymerized type V pilin reveals assembly mechanism involving protease-mediated strand exchange.

Bacterial adhesion is a general strategy for host-microbe and microbe-microbe interactions. Adhesive pili are essential for colonization, biofilm formation, virulence and pathogenesis of many environmental and pathogenic bacteria1,2. Members of the class Bacteroidia have unique type V pili, assembled by protease-mediated polymerization3. Porphyromonas gingivalis is the main contributor to periodontal disease and its type V pili are a key factor for its virulence4. However, the structure of the polymerized pilus and its assembly mechanism are unknown. Here we show structures of polymerized and monomeric states of FimA stalk pilin from P. gingivalis, determined by cryo-electron microscopy and crystallography. The atomic model of assembled FimA shows that the C-terminal strand of a donor subunit is inserted into a groove in the ß-sheet of an acceptor subunit after N-terminal cleavage by the protease RgpB. The C terminus of the donor strand is essential for polymerization. We propose that type V pili assemble via a sequential polar assembly mechanism at the cell surface, involving protease-mediated strand exchange, employed by various Gram-negative species belonging to the class Bacteroidia. Our results reveal functional surfaces related to pathogenic properties of polymerized FimA. These insights may facilitate development of antibacterial drugs.

Tree of motility - A proposed history of motility systems in the tree of life.

Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms. This makes defining the breadth of motility nontrivial, because novel motilities may be driven by unknown mechanisms. Here, we classify the known motilities based on the unique classes of movement-producing protein architectures. Based on this criterion, the current total of independent motility systems stands at 18 types. In this perspective, we discuss these modes of motility relative to the latest phylogenetic Tree of Life and propose a history of motility. During the ~4 billion years since the emergence of life, motility arose in Bacteria with flagella and pili, and in Archaea with archaella. Newer modes of motility became possible in Eukarya with changes to the cell envelope. Presence or absence of a peptidoglycan layer, the acquisition of robust membrane dynamics, the enlargement of cells and environmental opportunities likely provided the context for the (co)evolution of novel types of motility.

Draft genome of Tanacetum cinerariifolium, the natural source of mosquito coil.

Pyrethrum (Tanacetum cinerariifolium), which is a perennial Asteraceae plant with white daisy-like flowers, is the original source of mosquito coils and is known for the biosynthesis of the pyrethrin class of natural insecticides. However, the molecular basis of the production of pyrethrins by T. cinerariifolium has yet to be fully elucidated. Here, we present the 7.1-Gb draft genome of T. cinerariifolium, consisting of 2,016,451 scaffolds and 60,080 genes predicted with high confidence. Notably, analyses of transposable elements (TEs) indicated that TEs occupy 33.84% of the genome sequence. Furthermore, TEs of the sire and oryco clades were found to be enriched in the T. cinerariifolium-specific evolutionary lineage, occupying a total of 13% of the genome sequence, a proportion approximately 8-fold higher than that in other plants. InterProScan analysis demonstrated that biodefense-related toxic proteins (e.g., ribosome inactivating proteins), signal transduction-related proteins (e.g., histidine kinases), and metabolic enzymes (e.g., lipoxygenases, acyl-CoA dehydrogenases/oxygenases, and P450s) are also highly enriched in the T. cinerariifolium genome. Molecular phylogenetic analysis detected a variety of enzymes with genus-specific multiplication, including both common enzymes and others that appear to be specific to pyrethrin biosynthesis. Together, these data identify possible novel components of the pyrethrin biosynthesis pathway and provide new insights into the unique genomic features of T. cinerariifolium.

Retraction Note: Temporal activation of anti- and pro-apoptotic factors in human gingival fibroblasts infected with the periodontal pathogen, Porphyromonas gingivalis: potential role of bacterial proteases in host signalling.

The Editor has retracted this article [1] following an investigation by the University of South Alabama which found.

Tenascin XB Is a Novel Diagnostic Marker for Malignant Mesothelioma.

BACKGROUND/AIM: Malignant mesothelioma (MM) is an aggressive tumor with poor prognosis. The establishment of a new diagnostic and therapeutic approach for MM is expected. This study investigated the diagnostic significance of tenascin XB (TNXB) for MM. MATERIALS AND METHODS: TNXB gene expression was found to be significantly higher in MM tumor tissues compared to paired normal tissues, as assessed by the Gene Expression Omnibus database. The inhibition of TNXB using small interfering RNAs suppressed the proliferation and colony formation of MM cells. Expression of TNXB and calretinin, a current diagnostic marker of MM, was evaluated by immunohistochemistry. RESULTS: The sensitivity and specificity of TNXB for MM were 80.0% and 69.5%, respectively. When the detection of TNXB was combined with that of calretinin, 83.3% of MM cases were detected. CONCLUSION: These findings suggest that TNXB is a novel diagnostic biomarker for MM. A combination of detecting TNXB and calretinin may be useful for the differential diagnosis of MM from lung adenocarcinoma.

PGN_0297 is an essential component of the type IX secretion system (T9SS) in Porphyromonas gingivalis: Tn-seq analysis for exhaustive identification of T9SS-related genes.

The type IX secretion system (T9SS) was originally discovered in Porphyromonas gingivalis, one of the pathogenic bacteria associated with periodontal disease and is now known to be present in many members of the phylum Bacteroidetes. The T9SS secretes a number of potent virulence factors, including the highly hydrolytic proteases called gingipains, across the outer membrane in P. gingivalis. To understand the entire machinery of T9SS, an exhaustive search for T9SS-related genes in P. gingivalis using the mariner family transposon (Tn) and Tn-seq analysis was performed. Seven hundred and two Tn insertion sites in Tn mutants with no colony pigmentation that is associated with Lys-gingipain (Kgp) defectiveness were determined, and it was found that the Tn was inserted in the kgp gene and 54 T9SS-related candidate genes. Thirty-three out of the 54 genes were already known as T9SS-related genes. Furthermore, deletion mutant analysis of the remaining 21 genes revealed that they were not related to the T9SS. The 33 T9SS-related genes include a gene for PGN_0297, which was found to be associated with the T9SS components PorK and PorN. A PGN_0297 gene deletion mutant was constructed, and it was found that the mutant showed no colony pigmentation, hemagglutination or gingipain activities, indicating that PGN_0297 was an essential component of the T9SS. The 33 genes did not include the six genes (gppX, omp17, porY, rfa, sigP and wzx) that were also reported as T9SS-related genes. gppX deletion and insertion mutants were constructed, and it was found that they did not show deficiency in the T9SS.

Probing heat generation during tensile plastic deformation of a bulk metallic glass at cryogenic temperature.

Despite significant research efforts, the deformation and failure mechanisms of metallic glasses remain not well understood. In the absence of periodic structure, these materials typically deform in highly localized, thin shear bands at ambient and low temperatures. This process usually leads to an abrupt fracture, hindering their wider use in structural applications. The dynamics and temperature effects on the formation and operation of those shear bands have been the focus of long-standing debate. Here, we use a new experimental approach based on localized boiling of liquid nitrogen by the heat generated in the shear bands to monitor the tensile plastic deformation of a bulk metallic glass submerged in a cryogenic bath. With the "nitrogen bubbles heat sensor", we could capture the heat dissipation along the primary shear banding plane and follow the dynamics of the shear band operation. The observation of nitrogen boiling on the surface of the deforming metallic glass gives direct evidence of temperature increase in the shear bands, even at cryogenic temperatures. An acceleration in bubble nucleation towards the end of the apparent plastic deformation suggests a change from steady-state to runaway shear and premonitions the fracture, allowing us to resolve the sequence of deformation and failure events.