The putative Escherichia coli dehydrogenase YjhC metabolises two dehydrated forms of N-acetylneuraminate produced by some sialidases

Homologues of the putative dehydrogenase YjhC are found in operons involved in the metabolism of N-acetylneuraminate (Neu5Ac) or related compounds. We observed that purified recombinant YjhC forms Neu5Ac from two dehydrated forms of this compound, 2,7-anhydro-N-acetylneuraminate (2,7-AN) and 2-deoxy-2,3-didehydro-N-acetylneuraminate (2,3-EN) that are produced during the degradation of sialoconjugates by some sialidases. The conversion of 2,7-AN into Neu5Ac is reversible and reaches its equilibrium when the ratio of 2,7-AN to Neu5Ac is ≈1/6. The conversion of 2,3-EN is irreversible, leading to a mixture of Neu5Ac and 2,7-AN. NMR analysis of the reaction catalysed by YjhC on 2,3-EN indicated that Neu5Ac was produced as the α-anomer. All conversions require NAD+ as a cofactor, which is regenerated in the reaction. They appear to involve the formation of keto (presumably 4-keto) intermediates of 2,7-AN, 2,3-EN and Neu5Ac, which were detected by liquid chromatography-mass spectrometry (LC-MS). The proposed reaction mechanism is reminiscent of the one catalysed by family 4 β-glycosidases, which also use NAD+ as a cofactor. Both 2,7-AN and 2,3-EN support the growth of Escherichia coli provided the repressor NanR, which negatively controls the expression of the yjhBC operons, has been inactivated. Inactivation of either YjhC or YjhB in NanR-deficient cells prevents the growth on 2,7-AN and 2,3-EN. This confirms the role of YjhC in 2,7-AN and 2,3-EN metabolism and indicates that transport of 2,7-AN and 2,3-EN is carried out by YjhB, which is homologous to the Neu5Ac transporter NanT

Interpreting intraplate tectonics for seismic hazard : a UK historical perspective

It is notoriously difficult to construct seismic source models for probabilistic seismic hazard assessment in intraplate areas on the basis of geological information, and many practitioners have given up the task in favour of purely seismicity-based models. This risks losing potentially valuable information in regions where the earthquake catalogue is short compared to the seismic cycle. It is interesting to survey how attitudes to this issue have evolved over the past 30 years. This paper takes the UK as an example, and traces the evolution of seismic source models through generations of hazard studies. It is found that in the UK, while the earliest studies did not consider regional tectonics in any way, there has been a gradual evolution towards more tectonically based models. Experience in other countries, of course, may differ

Extending displacement-based earthquake loss assessment (DBELA) for the computation of fragility curves

This paper presents a new procedure to derive fragility functions for populations of buildings that relies on the displacement-based earthquake loss assessment (DBELA) methodology. In the method proposed herein, thousands of synthetic buildings have been produced considering the probabilistic distribution describing the variability in geometrical and material properties. Then, their nonlinear capacity has been estimated using the DBELA method and their response against a large set of ground motion records has been estimated. Global limit states are used to estimate the distribution of buildings in each damage state for different levels of ground motion, and a regression algorithm is applied to derive fragility functions for each limit state. The proposed methodology is demonstrated for the case of ductile and non-ductile Turkish reinforced concrete frames with masonry infills

Seismic hazard and risk due to induced earthquakes at a shale gas site

Hydraulic fracturing of the first shale gas well at Preston New Road (PNR), Blackpool, United Kingdom, in late 2018, marked the end of a 7 yr United Kingdom‐wide moratorium on fracking. Despite a strict traffic‐light system being in place, seismic events up to ML 2.9 were induced. The ML 2.9 event was accompanied by reports of damage and was assigned European Macroseismic Scale 1998 (EMS‐98) intensity VI by the British Geological Survey. The moratorium was subsequently reinstated in late 2019. The study here presents a pseudo‐probabilistic seismic risk analysis and is applied to the larger of the induced events at PNR, in addition to hypothetical larger events. Initially, site characterization analysis is undertaken using direct and indirect methods. These analyses show low‐velocity deposits dominate the region (⁠VS30¯¯¯¯¯¯=227  m/s⁠). We test existing ground‐motion prediction equations using spatially dependent VS30 to determine applicability to the recorded waveform data and produce a referenced empirical model. Predicting median and 84th percentile peak ground velocity fields, we subsequently determine macroseismic intensities. Epicentral intensities of IV, IV–V, and VI–VII are predicted for the observed ML 2.9, and hypothetical ML 3.5 and 4.5 scenarios, respectively. A probabilistic analysis of damage is performed for 3500 ground‐motion realizations (⁠2.1≤ML≤4.5⁠) using the OpenQuake‐engine, with nonlinear dynamic analysis undertaken to define building fragility. Based on these analyses, the onset of cosmetic damage (DS1) in terms of median risk is observed for the ML 2.9 event. Mean modeled occurrences of DS1 and DS2 (minor structural damage), 75 and 10 instances, respectively, are consistent with reported damage (DS1:97, DS2:50). Significant occurrences (⁠median≥30 buildings) of DS2, DS3, and DS4 (minor to major structural damage) are likely for ML 3.5, 4.0, and 4.5 events, respectively. However, by comparing reported damage with modeled damage due to the ML 2.9 event and considering the fact that low macroseismic intensities (EMS‐98 <4⁠) are often not reported by the public, we conclude that the previously assigned intensity of VI is too high, with V being more appropriate

Ground-motion networks in the Groningen field: usability and consistency of surface recordings

Several strong-motion networks have been installed in the Groningen gas field in the Netherlands to record ground motions associated with induced earthquakes. There are now more than 450 permanent surface accelerographs plus a mobile array of 450 instruments, which, in addition to many instrumented boreholes, yield a wealth of data. The database of recordings has been of fundamental importance to the development of ground-motion models that form a key element of the seismic hazard and risk estimations for the field. In order to maximise the benefit that can be derived from these recordings, this study evaluates the usability of the recordings from the different networks, in general terms and specifically with regards to the frequency ranges with acceptable signal-to-noise ratios. The study also explores the consistency among the recordings from the different networks, highlighting in particular how a configuration error was identified and resolved. The largest accelerograph network consists of instruments housed in buildings around the field, frequently installed on the lower parts of walls rather than on the floor. A series of experiments were conducted, using additional instruments installed adjacent to these buildings and replicating the installation configuration in full-scale shake table tests, to identify the degree to which structural response contaminated the recordings. The general finding of these efforts was that for PGV and oscillator periods above 0.1 s, the response spectral ordinates from these recordings can be used with confidence

Hazard-consistent response spectra in the Region of Murcia (Southeast Spain): comparison to earthquake-resistant provisions

Hazard-consistent ground-motion characterisations of three representative sites located in the Region of Murcia (southeast Spain) are presented. This is the area where the last three damaging events in Spain occurred and there is a significant amount of data for comparing them with seismic hazard estimates and earthquake-resistant provisions. Results of a probabilistic seismic hazard analysis are used to derive uniform hazard spectra (UHS) for the 475-year return period, on rock and soil conditions. Hazard deaggregation shows that the largest hazard contributions are due to small, local events for short-period target motions and to moderate, more distant events for long-period target motions. For each target motion and site considered, the associated specific response spectra (SRS) are obtained. It is shown that the combination of two SRS, for short- and long-period ground motions respectively, provides a good approximation to the UHS at each site. The UHS are compared to design response spectra contained in current Spanish and European seismic codes for the 475-year return period. For the three sites analysed, only the Eurocode 8 (EC8) type 2 spectrum captures the basic shape of the UHS (and not the EC8 type 1, as could be expected a priori). An alternative response spectrum, anchored at short- and long-period accelerations, is tested, providing a close match to the UHS spectra at the three sites. Results underline the important contribution of the frequent, low-to-moderate earthquakes that characterize the seismicity of this area to seismic hazard (at the 475-year return period)

A consensus research agenda for optimising nasal drug delivery

Nasal drug delivery has specific challenges which are distinct from oral inhalation, alongside which it is often considered. The next generation of nasal products will be required to deliver new classes of molecule, e.g. vaccines, biologics and drugs with action in the brain or sinuses, to local and systemic therapeutic targets. Innovations and new tools/knowledge are required to design products to deliver these therapeutic agents to the right target at the right time in the right patients. We report the outcomes of an expert meeting convened to consider gaps in knowledge and unmet research needs in terms of (i) formulation and devices, (ii) meaningful product characterization and modeling, (iii) opportunities to modify absorption and clearance. Important research questions were identified in the areas of device and formulation innovation, critical quality attributes for different nasal products, development of nasal casts for drug deposition studies, improved experimental models, the use of simulations and nasal delivery in special populations. We offer these questions as a stimulus to research and suggest that they might be addressed most effectively by collaborative research endeavors

The metalloprotein YhcH is an anomerase providing N-acetylneuraminate aldolase with the open form of its substrate

N-acetylneuraminate (Neu5Ac), an abundant sugar present in glycans in vertebrates and some bacteria, can be used as an energy source by several prokaryotes, including Escherichia coli. In solution, more than 99% of Neu5Ac is in cyclic form (≈92% beta-anomer and ≈7% alpha-anomer), whereas &lt;0.5% is in the open form. The aldolase that initiates Neu5Ac metabolism in E. coli, NanA, has been reported to act on the alphaanomer. Surprisingly, when we performed this reaction at pH 6 to minimize spontaneous anomerization, we found NanA and its human homolog NPL preferentially metabolize the open form of this substrate. We tested whether the E. coli Neu5Ac anomerase NanM could promote turnover, finding it stimulated the utilization of both beta and alpha-anomers by NanA in vitro. However, NanM is localized in the periplasmic space and cannot facilitate Neu5Ac metabolism by NanA in the cytoplasm in vivo. We discovered that YhcH, a cytoplasmic protein encoded by many Neu5Ac catabolic operons and belonging to a protein family of unknown function (DUF386), also facilitated Neu5Ac utilization by NanA and NPL and displayed Neu5Ac anomerase activity in vitro. YhcH contains Zn, and its accelerating effect on the aldolase reaction was inhibited by metal chelators. Remarkably, several transition metals accelerated Neu5Ac anomerization in the absence of enzyme. Experiments with E. coli mutants indicated that YhcH expression provides a selective advantage for growth on Neu5Ac. In conclusion, YhcH plays the unprecedented role of providing an aldolase with the preferred unstable open form of its substrate

Mesenchymal cells reactivate Snail1 expression to drive three-dimensional invasion programs

Epithelial–mesenchymal transition (EMT) is required for mesodermal differentiation during development. The zinc-finger transcription factor, Snail1, can trigger EMT and is sufficient to transcriptionally reprogram epithelial cells toward a mesenchymal phenotype during neoplasia and fibrosis. Whether Snail1 also regulates the behavior of terminally differentiated mesenchymal cells remains unexplored. Using a Snai1 conditional knockout model, we now identify Snail1 as a regulator of normal mesenchymal cell function. Snail1 expression in normal fibroblasts can be induced by agonists known to promote proliferation and invasion in vivo. When challenged within a tissue-like, three-dimensional extracellular matrix, Snail1-deficient fibroblasts exhibit global alterations in gene expression, which include defects in membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invasive activity. Snail1-deficient fibroblasts explanted atop the live chick chorioallantoic membrane lack tissue-invasive potential and fail to induce angiogenesis. These findings establish key functions for the EMT regulator Snail1 after terminal differentiation of mesenchymal cells

MicroRNA miR-34 Inhibits Human Pancreatic Cancer Tumor-Initiating Cells

This is the published version, also available here: http://dx.doi.org/10.1371/journal.pone.0006816.Background MicroRNAs (miRNAs) have been implicated in cancer initiation and progression via their ability to affect expression of genes and proteins that regulate cell proliferation and/or cell death. Transcription of the three miRNA miR-34 family members was recently found to be directly regulated by p53. Among the target proteins regulated by miR-34 are Notch pathway proteins and Bcl-2, suggesting the possibility of a role for miR-34 in the maintenance and survival of cancer stem cells. Methodology/Principal Findings We examined the roles of miR-34 in p53-mutant human pancreatic cancer cell lines MiaPaCa2 and BxPC3, and the potential link to pancreatic cancer stem cells. Restoration of miR-34 expression in the pancreatic cancer cells by either transfection of miR-34 mimics or infection with lentiviral miR-34-MIF downregulated Bcl-2 and Notch1/2. miR-34 restoration significantly inhibited clonogenic cell growth and invasion, induced apoptosis and G1 and G2/M arrest in cell cycle, and sensitized the cells to chemotherapy and radiation. We identified that CD44+/CD133+ MiaPaCa2 cells are enriched with tumorsphere-forming and tumor-initiating cells or cancer stem/progenitor cells with high levels of Notch/Bcl-2 and loss of miR-34. More significantly, miR-34 restoration led to an 87% reduction of the tumor-initiating cell population, accompanied by significant inhibition of tumorsphere growth in vitro and tumor formation in vivo. Conclusions/Significance Our results demonstrate that miR-34 may restore, at least in part, the tumor suppressing function of the p53 in p53-deficient human pancreatic cancer cells. Our data support the view that miR-34 may be involved in pancreatic cancer stem cell self-renewal, potentially via the direct modulation of downstream targets Bcl-2 and Notch, implying that miR-34 may play an important role in pancreatic cancer stem cell self-renewal and/or cell fate determination. Restoration of miR-34 may hold significant promise as a novel molecular therapy for human pancreatic cancer with loss of p53–miR34, potentially via inhibiting pancreatic cancer stem cells