A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprin

In the past, protease-substrate finding proved to be rather haphazard and was executed by in vitro cleavage assays using singly selected targets. In the present study, we report the first protease proteomic approach applied to meprin, an astacin-like metalloendopeptidase, to determine physiological substrates in a cell-based system of Madin-Darby canine kidney epithelial cells. A simple 2D IEF/SDS/PAGE-based image analysis procedure was designed to find candidate substrates in conditioned media of Madin-Darby canine kidney cells expressing meprin in zymogen or in active form. The method enabled the discovery of hitherto unknown meprin substrates with shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments upon LC-MS/MS analysis. Of 22 (17 nonredundant) candidate substrates identified, the proteolytic processing of vinculin, lysyl oxidase, collagen type V and annexin A1 was analysed by means of immunoblotting validation experiments. The classification of substrates into functional groups may propose new functions for meprins in the regulation of cell homeostasis and the extracellular environment, and in innate immunity, respectively.

Adherence to host extracellular matrix and serum components by Enterococcus faecium isolates of diverse origin.

Enterococcus faecium has emerged as an important cause of nosocomial infections over the last two decades. We recently demonstrated collagen type I (CI) as a common adherence target for some E. faecium isolates and a significant correlation was found to exist between acm-mediated CI adherence and clinical origin. Here, we evaluated 60 diverse E. faecium isolates for their adherence to up to 15 immobilized host extracellular matrix and serum components. Adherence phenotypes were most commonly observed to fibronectin (Fn) (20% of the 60 isolates), fibrinogen (17%) and laminin (Ln) (13%), while only one or two of the isolates adhered to collagen type V (CV), transferrin or lactoferrin and none to the other host components tested. Adherence to Fn and Ln was almost exclusively restricted to clinical isolates, especially the endocarditis-enriched nosocomial genogroup clonal complex 17 (CC17). Thus, the ability to adhere to Fn and Ln, in addition to CI, may have contributed to the emergence and adaptation of E. faecium, in particular CC17, as a nosocomial pathogen.

Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium.

Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about the pathogenic determinants of this organism. We have previously identified a cell-wall-anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad-spectrum binding to extracellular matrix proteins. Here, we analysed the draft genome of strain TX0016 for potential microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Genome-based bioinformatics identified 22 predicted cell-wall-anchored E. faecium surface proteins (Fms), of which 15 (including Acm) had characteristics typical of MSCRAMMs, including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one [Fms10; redesignated second collagen adhesin of E. faecium (Scm)] revealed that recombinant Scm(65) (A- and B-domains) and Scm(36) (A-domain) bound to collagen type V efficiently in a concentration-dependent manner, bound considerably less to collagen type I and fibrinogen, and differed from Acm in their binding specificities to collagen types IV and V. Results from far-UV circular dichroism measurements of recombinant Scm(36) and of Acm(37) indicated that these proteins were rich in beta-sheets, supporting our folding predictions. Whole-cell ELISA and FACS analyses unambiguously demonstrated surface expression of Scm in most E. faecium isolates. Strikingly, 11 of the 15 predicted MSCRAMMs clustered in four loci, each with a class C sortase gene; nine of these showed similarity to Enterococcus faecalis Ebp pilus subunits and also contained motifs essential for pilus assembly. Antibodies against one of the predicted major pilus proteins, Fms9 (redesignated EbpC(fm)), detected a 'ladder' pattern of high-molecular-mass protein bands in a Western blot analysis of cell surface extracts from E. faecium, suggesting that EbpC(fm) is polymerized into a pilus structure. Further analysis of the transcripts of the corresponding gene cluster indicated that fms1 (ebpA(fm)), fms5 (ebpB(fm)) and ebpC(fm) are co-transcribed, a result consistent with those for pilus-encoding gene clusters of other Gram-positive bacteria. All 15 genes occurred frequently in 30 clinically derived diverse E. faecium isolates tested. The common occurrence of MSCRAMM- and pilus-encoding genes and the presence of a second collagen-binding protein may have important implications for our understanding of this emerging pathogen.

Terrestrial and submarine evidence for the extent and timing of the Last Glacial Maximum and the onset of deglaciation on the maritime-Antarctic and sub-Antarctic islands

This paper is the maritime and sub–Antarctic contribution to the Scientific Committee for Antarctic Research (SCAR) Past Antarctic Ice Sheet Dynamics (PAIS) community Antarctic Ice Sheet reconstruction. The overarching aim for all sectors of Antarctica was to reconstruct the Last Glacial Maximum (LGM) ice sheet extent and thickness, and map the subsequent deglaciation in a series of 5000 year time slices. However, our review of the literature found surprisingly few high quality chronological constraints on changing glacier extents on these timescales in the maritime and sub–Antarctic sector. Therefore, in this paper we focus on an assessment of the terrestrial and offshore evidence for the LGM ice extent, establishing minimum ages for the onset of deglaciation, and separating evidence of deglaciation from LGM limits from those associated with later Holocene glacier fluctuations. Evidence included geomorphological descriptions of glacial landscapes, radiocarbon dated basal peat and lake sediment deposits, cosmogenic isotope ages of glacial features and molecular biological data. We propose a classification of the glacial history of the maritime and sub–Antarctic islands based on this assembled evidence. These include: (Type I) islands which accumulated little or no LGM ice; (Type II) islands with a limited LGM ice extent but evidence of extensive earlier continental shelf glaciations; (Type III) seamounts and volcanoes unlikely to have accumulated significant LGM ice cover; (Type IV) islands on shallow shelves with both terrestrial and submarine evidence of LGM (and/or earlier) ice expansion; (Type V) Islands north of the Antarctic Polar Front with terrestrial evidence of LGM ice expansion; and (Type VI) islands with no data. Finally, we review the climatological and geomorphological settings that separate the glaciological history of the islands within this classification scheme.

Strain typing and antimicrobial susceptibility of methicillin-resistant coagulase-positive staphylococcal species in dogs and people associated with dogs in Thailand

AIMS This study was to investigate and to characterize methicillin-resistant coagulase-positive staphylococci (MRCoPS) harboring in dogs and people associated with dogs in Thailand. METHODS AND RESULTS Staphylococci were collected from 100 dogs, 100 dog owners, 200 small animal veterinarians and 100 people without pet association. Species of MRCoPS were identified phenotypically and genotypically. Molecular characteristics were determined by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and SCCmec typing, and antimicrobial susceptibility was assayed by broth microdilution and by microarray analysis for resistance genes. Methicillin-resistant Staphylococcus pseudintermedius (MRSP), methicillin-resistant Staphylococcus schleiferi subsp. coagulans (MRSSc) and methicillin-resistant Staphylococcus aureus (MRSA) were isolated from dogs (45, 17 and 1%, respectively), veterinarians (8, 2 and 1·5%, respectively) and dog owners (3, 2 and 0%, respectively). Seventeen sequence types (STs) were identified among 83 MRSP isolates which specifically carried SCCmec V, II-III, ΨSCCmec57395 and three uncharacterized SCCmec types. MRSP ST 45, 68 and novel STs including 169, 178, 181 and 183 were shared among canine and human isolates. Most of MRSA ST398 and MRSSc carried SCCmec type V. The MRCoPS commonly displayed multiple resistances to tested antimicrobials and carried various resistance genes. CONCLUSION Variety of MRCoPS, especially new MRSP clones, distributed in dogs and people in Thailand. SIGNIFICANCE AND IMPACT OF THE STUDY The existence of MRCoPS circulating between dogs and humans in Thailand provides indirect evidence of interspecies transmission and represents a potential public health hazard.

A combined biomaterial and cellular approach for annulus fibrosus rupture repair

Recurrent intervertebral disc (IVD) herniation and degenerative disc disease have been identified as the most important factors contributing to persistent pain and disability after surgical discectomy. An annulus fibrosus (AF) closure device that provides immediate closure of the AF rupture, restores disc height, reduces further disc degeneration and enhances self-repair capacities is an unmet clinical need. In this study, a poly(trimethylene carbonate) (PTMC) scaffold seeded with human bone marrow derived mesenchymal stromal cells (MSCs) and covered with a poly(ester-urethane) (PU) membrane was assessed for AF rupture repair in a bovine organ culture annulotomy model under dynamic load for 14 days. PTMC scaffolds combined with the sutured PU membrane restored disc height of annulotomized discs and prevented herniation of nucleus pulposus (NP) tissue. Implanted MSCs showed an up-regulated gene expression of type V collagen, a potential AF marker, indicating in situ differentiation capability. Furthermore, MSCs delivered within PTMC scaffolds induced an up-regulation of anabolic gene expression and down-regulation of catabolic gene expression in adjacent native disc tissue. In conclusion, the combined biomaterial and cellular approach has the potential to hinder herniation of NP tissue, stabilize disc height, and positively modulate cell phenotype of native disc tissue.

Permian manganese nodules near Dillon, Montana

Concentrically ringed manganese nodules, similar in form to many found on modern ocean and sea floors, occur in a very fine grained argillaceous sandstone bed of the Permian Park City Formation near Dillon, Montana. They are enriched in many rare elements and contain us much as 2.5 percent zinc, l.3 percent nickel, and 0.22 percent cobalt. The manganese minerals are chalcophanite and todorokite. The nodules probably formed in a shallow marine oxidizing environment on the western side of the Permian sedimentary basin. The occurrence of an appreciable amount of fluorite in the bed suggests that the water was saline.

Analysis of ferromanganiferous sedimentary rocks from Timor

Seven manganese nodules, eight ferromanganiferous shales from the Cretaceous Wai Bua Formation of Timor, and a pelagic limestone with four ferromanganese enriched layers from the Middle Eocene of Timor have been analysed. The nodules are compared with modern deep-sea nodules, and the ferromanganiferous shales are contrasted with relatively shallow marine manganiferous shales. The conclusion is reached that these rocks from Timor were probably deposited in a bathypelagic environment. There is a total absence of any indication that volcanic material has contributed to these deposits. The chemical composition of the ferromanganiferous rocks are discussed and some indications of biogenic influences are noted. The Middle Eocene pelagic limestone is compared with a similar modern sediment described from the Easter Island Rise in the Pacific.

Chemistry on manganese nodules from the Pacific Ocean

Three manganese nodules from the Pacific Ocean have been analysed for 35 elements by using mainly spectrophotometric and spectrographic methods. Cu, Co, Ni, Zn, and Pb were found in amounts approaching 1 %, which far exceeds their average concentrations in igneous rocks. On the other hand, elements having readily hydrolysable ions, such as Ga, Sc, Zr, Y, La and Ti, are present only in amounts comparable with their concentrations in igneous rocks. Sb, Bit Be, and Cr were not detected. The hydrochloric acid-insoluble fraction of nodules is practically free of the heavy metals that are characteristic of the acid-soluble fraction; it consists principally of clay minerals, together with lesser amounts of quartz, apatite, biotite and sodium and potassium felspars.

Chemistry of volcanic ash from Celebes and Sulu Sea Basins

During Leg 124, off the Philippines, volcanic material was recovered in deep-sea sediments dating from the late Oligocene in the Celebes Sea Basin, and from the early Miocene in the Sulu Sea Basin. Chemical and petrological studies of fallout ash deposits are used to characterize volcanic pulses and to determine their possible origin. All of the glass and mineral compositions belong to medium-K and high-K calc-alkaline arc-related magmatic suites including high-Al basalts, pyroxene-hornblende andesites, dacites, and rhyolites. Late Oligocene and early Miocene products may have originated from the Sunda arc or from the Sabah-Zamboanga old Sulu arc. Late early Miocene Sulu Sea tuffs originated from the Cagayan arc, whereas early late Miocene fallout ashes are attributed to the Sulu arc. A complex magmatic production is distinguished in the Plio-Quaternary with three sequences of basic to acidic lava suites. Early Pliocene strata registered an important activity in both Celebes Sea and Sulu Sea areas, from the newly born Sangihe arc (low-alumina andesite series) and from the Sulu, Zamboanga, and Negros arcs (high-alumina basalt series and high-K andesite series). In the late Pliocene and the early Pleistocene, renewal of activity affects the Sangihe-Cotobato arc as well as the Sulu and Negros arcs (same magmatic distinctions). The last volcanic pulse took place in the late Pleistocene with revival of all the present arc systems.

Marine diatoms in deep sea sediments

Long-term evolution is thought to take opportunities that arise as a consequence of mass extinction (as argued, for example, by Gould, 2002) and the following biotic recovery, but there is absolutely no evidence for this being the case. However, our study shows that eutrophication by oceanic mixing also played a part in the enhancement of several evolutionary events amongst marine organisms, and these results could indicate that the rates of oceanic biodiversification may be slowed if upwelling becomes weakened by future global warming. This paper defines three distinct evolutionary events of resting spores of the marine diatom genus Chaetoceros, to reconstruct past upwelling through the analysis of several DSDP, ODP and land-based successions from the North, South and equatorial Pacific as well as the Atlantic Ocean during the past 40 million years. The Atlantic Chaetoceros Explosion (ACE) event occurred across the E/O boundary in the North Atlantic, and is characterized by resting spore diversification that occurred as a consequence of the onset of upwelling following changes in thermohaline circulation through global cooling in the early Oligocene. Pacific Chaetoceros Explosion events-1 and -2 (PACE-1 and PACE-2) are characterized by relatively higher occurrences of iron input following the Himalayan uplift and aridification at 8.5 Ma and ca. 2.5 Ma in the North Pacific region. These events not only enhanced the diversification and increased abundance of primary producers, including that of Chaetoceros, other diatoms and seaweeds, but also stimulated the evolution of zooplankton and larger predators, such as copepods and marine mammals, which ate these phytoplankton and plants. Current thinking suggests new evolutionary niches open up after a mass extinction, but our study finds that eutrophication can also stimulate evolutionary diversification. Moreover, in the opposite fashion, our results show that as thermohaline circulation abates, global warming progresses and the ocean surface becomes warmer, many marine organisms will be affected by the environmental degradation.

Geochemistry of marin ash layers and epiclastic rocks from the Kerguelen Plateau

Epiclastic volcanogenic rocks recovered from the Kerguelen Plateau during Ocean Drilling Program Legs 119 and 120 comprise (pre-)Cenomanian(?) claystones (52 m thick, Site 750); a Turonian(?) basaltic pebble conglomerate (1.2 m thick, Site 748; Danian mass flows (45 m thick, Site 747); and volcanogenic debris flows of Quaternary age at Site 736 (clastic apron of Kerguelen Island). Pyroclastic rocks comprise numerous Oligocene to Quaternary marine ash layers. The epiclastic sediments with transitional mid-ocean-ridge basalt (T-MORB) origin indicate weathering (Site 750) and erosion (Site 747) of Early Cretaceous T-MORB from a then-emergent Kerguelen Plateau, connected to Late Cretaceous tectonic events. The basal pebble conglomerate of Site 748 has an oceanic-island basalt (OIB) composition and denotes erosion and reworking of seamount to oceanic-island-type volcanic sources. The vitric- to crystal-rich marine ash layers are a few centimeters thick, have rather uniform grain sizes around 60 ± 40 µm, and are a result of Plinian eruptions. Crystal-poor silicic vitric ashes may also represent co-ignimbrite ashes. The ash layers have bimodal, basaltic, and silicic compositions with a few intermediate shards. The basaltic ashes are evolved high-titanium T-MORB; a few grains in a silicic pumice lapilli layer have a low-titanium basaltic composition. The silicic ashes comprise trachytic and rhyolitic glass shards belonging to a high-K series, except for a few low-K glasses admixed to a basaltic ash layer. Feldspar and clinopyroxene compositions fit the glass chemistry: high-Ti tholeiite-basaltic glasses have Plagioclase of An40-80 and pigeonite to augite clinopyroxene compositions. Silicic ashes have K-rich anorthoclase and minor Plagioclase around An20 and ferriaugitic to hedenbergitic clinopyroxene compositions. The line of magmatic evolution for the glass shards is not compatible with simple two-end member (high-Ti T-MORB and high-K rhyolite) mixing, but favors successive Ca-Mg-Fe pyroxene, Ti magnetite, and apatite fractionation, and K-rich alkali feldspar fractionation in trachytic magmas to yield rhyolitic compositions. Plagioclase fractionation occurs throughout. This qualitative model is in basic accordance with the observed mineral assemblage. However, as the time span for explosive volcanism spans >30 m.y., this basic model cannot comply with fractional crystallization in a single magma reservoir. The ash layers resulted from highly explosive eruptions on Kerguelen and, with less probability, Heard islands since the Oligocene. The explosive history starts with widespread Oligocene basaltic ash layers that indicate sea-level or subaerial volcanism on the Northern Kerguelen Plateau. After a hiatus of 24 m.y.(?), explosive magmatic activity was vigorously renewed in the late Miocene with more silicic eruptions. A peak in explosive activity is inferred for the Pliocene-Pleistocene. The composition and evolution of Kerguelen Plateau ash layers resemble those from other hotspot-induced, oceanic-island realms such as Iceland and Jan Mayen in the North Atlantic, and the Canary Islands archipelago in the Central Atlantic.

K-Ar ages of basalts from islands of the Franz Josef Land archipelago

New K-Ar age determinations of basalt samples from three drill holes and outcrops on the Franz Josef Land suggest that flood volcanism throughout the archipelago fits in a very narrow age interval (116±5 Ma). For 95% of the samples we studied, age scatter is within analytical uncertainty. New data on basaltic bulk-rock, trace element, and REE compositions point to mantle plume affinity for Early Cretaceous magmatism on the Franz Josef Land, which preceded the onset of seafloor spreading in the Canada Basin.

Chemiscal composition of basalts from DSDP Legs 69 and 70 Holes

Chemical compositions and 1-atm. phase relations were determined for basalts drilled from Holes 501, 504A, 504B, 505, and 505B on Legs 68, 69, and 70 of the Deep Sea Drilling Project. Chemical, experimental, and petrographic data indicate that these basalts are moderately evolved (Mg' values from 0.60 to 0.70), with olivine plus Plagioclase and often clinopyroxene on the liquidus. Chemical stratigraphy was used to infer that sequential influxes of magma into a differentiating magma chamber or separate flows from different magma chambers or both had occurred. Two major types of basalt were found to be inter layered: Group M, a rarely occurring type with major element chemistry and magmaphile element abundances within the range of the majority of ocean-floor basalts (TiO2 = 1.3%, Na2O 2.5%, Zr = 103 ppm, Nb = 2.5 ppm, and Y = 31 ppm); and Group D, a highly unusual series of basalt compositions that exhibit much lower magmaphile element abundances (TiO2 = 0.75-1.2%, Na2O = 1.7-2.3%, Zr = 34-60 ppm, Nb = 0.5-1.2 ppm, and Y = 16-27 ppm). The liquidus temperatures of the Group D basalts are high (1230- 1260°C) compared with those of other ocean-floor basalts of similar Mg' values. They have high CaO/Na2O ratios (5-8) and are calculated to be in equilibrium with unusually calcic Plagioclase (An78-84). The two basalt groups cannot be related by fractionation processes. However, constant Zr/Nb ratios (>40) for the two groups suggest a single mantle source, with differences in magmaphile element abundances and other element ratios (e.g., Zr/Ti, Zr/Y, Ce/Yb) arising through sequential melting of the same source. Magmas similar to Group D, if mixed with more typical mid-ocean-ridge basalt (MORB) magmas in shallow magma chambers, could provide a source for the highly calcic Plagioclase phenocrysts that appear in more common (i.e., less depleted) phyric ocean-floor basalts.

Petrology of basalts from DSDP Hole 83-504B

Basalts from Hole 504B, Leg 83, exhibit remarkable uniformity in major and trace element composition throughout the 1075.5 m of basement drilled. The majority of the basalts, Group D', have unusual compositions relative to normal (Type I) mid-ocean ridge basalts (MORB). These basalts have relatively high mg values (0.60-0.70) and CaO abundances (11.7-13.7%; Ca/Al = 0.78-0.89), but exhibit a marked depletion in compatible trace elements (Cr and Ni); moderately incompatible trace elements (Zr, Y, Ti, etc.); and highly incompatible trace elements (Nb, LREE, etc.). Petrographic and compositional data indicate that most of these basalts are evolved, having fractionated significant amounts of plagioclase, olivine, and clinopyroxene. Melting experiments on similar basalt compositions from the upper portion of Hole 504B (Leg 70; Autio and Rhodes, 1983) indicate that the basalts are co-saturated with olivine and plagioclase and often clinopyroxene on the 1-atm. liquidus. Two rarely occurring groups, M' and T, are compositionally distinct from Group D' basalts. Group T is strongly depleted in all magmaphile elements except the highly incompatible ones (Nb, La, etc.), while Group M' has moderate concentrations of both moderately and highly incompatible trace elements and is similar to Type I MORB. Groups M' and T cannot be related to Group D' nor to each other by crystal fractionation, crystal accumulation, or magma mixing. The large differences in magmaphile element ratios (Zr/Nb, La/Yb) among these three chemical groups may be accounted for by complex melting models and/or local heterogeneity of the mantle beneath the Costa Rica Ridge. Xenocrysts and xenoliths of plagioclase and clinopyroxene similar in texture and mineral composition to crystals in coarse-grained basalts from the lower portion of the hole are common in Hole 504B basalts. These suggest that addition of solid components either from conduit or magma chamber walls has occurred and may be a common source of disequilibrium crystals in these basalts. However, mixing of plagioclase-laden depleted melts (similar to the Costa Rica Ridge Zone basalts) with normal MORB magmas could provide an alternate source for some refractory plagioclase crystals found out of equilibrium in many phyric MORB. The uniformity of major element compositions in Hole 504B basalts affords an ideal situation for investigating the effects of alteration on some major and trace elements in oceanic basalts. Alteration observed in whole-rock samples records primarily two events - a high-temperature and a low-temperature phase. High-temperature phases include: chlorite, talc, albite, actinolite, sphene, quartz, and pyrite. The low-temperature phases include smectite (saponite), epistilbite or laumontite, and minor calcite. Laumontite may actually straddle the gap between the low- and high-temperature mineral assemblages. Alteration is restricted primarily to partial replacement of primary phases. Metamorphic grade, in general, increases from the top to the bottom of Hole 504B (Legs 69, 70, and 83) as seen in the change from a smectiteto- chlorite-dominated secondary mineral assemblage. However, a systematic progression for the interval recovered during Leg 83 is not apparent. Rather, the extent of alteration appears to be a function of the initial texture and fracture density. Variations in whole-rock major and trace element concentrations cannot be attributed convincingly to any differences in alteration observed. Compositional characteristics of the secondary minerals indicated that extensive remobilization of elements has not occurred; local redistribution is suggested in most cases. Thus, the major and trace element signature of these basalts remains effectively the same as the original composition prior to alteration.