It\u27s Not What You Pay; It\u27s What It Costs You. A Geotechnical Engineering Case Study

The project consists of a middle school constructed on a previously undeveloped site. Site development required leveling a mountain ridge and perimeter tills up to 150 feet deep to create the relatively level 15 acre building site. Access to the site required a 1000 foot long side hill road from the adjacent main highway. Neither the owner nor the design team obtained geotechnical investigations of the site. As a result, they failed to consider the impact of the geologic setting on seepage, ground water flow, and slope stability. Near the end of construction, slope instabilities occurred in a cut section of the entrance road and one of the major embankment sections. The owner, the county school board, hired the writer to determine the cause of the instabilities and provide testimony in their ongoing litigation. This paper summarizes the site conditions and project history, describes the writer\u27s investigation, describes the dispute resolution processes, and presents two procedural lessons learned from the case: the importance of qualified professional geotechnical advice, and the inherent and sometimes unrecognized value of ADR procedures

Non-Volatile Memory Characteristics of Submicrometre Hall Structures Fabricated in Epitaxial Ferromagnetic MnAl Films on GaAs

Hall-effect structures with submicrometre linewidths (<0.3pm) have been fabricated in ferromagnetic thin films of Mn[sub 0.60]Al[sub 0.40] which are epitaxially grown on a GaAs substrate. The MnAl thin films exhibit a perpendicular remanent magnetisation and an extraordinary Hall effect with square hysteretic behaviour. The presence of two distinct stable readout states demonstrates the potential of using ultrasmall ferromagnetic volumes for electrically addressable, nonvolatile storage of digital information

The extraordinary Hall effect in coherent epitaxial tau (Mn,Ni)Al thin films on GaAs

Ultrathin coherent epitaxial films of ferromagnetic tau(Mn,Ni)0.60Al0.40 have been grown by molecular beam epitaxy on GaAs substrates. X-ray scattering and cross-sectional transmission electron microscopy measurements confirm that the c axis of the tetragonal tau unit cell is aligned normal to the (001) GaAs substrate. Measurements of the extraordinary Hall effect (EHE) show that the films are perpendicularly magnetized, exhibiting EHE resistivities saturating in the range of 3.3-7.1 muOMEGA-cm at room temperature. These values of EHE resistivity correspond to signals as large as +7 and -7 mV for the two magnetic states of the film with a measurement current of 1 mA. Switching between the two magnetic states is found to occur at distinct field values that depend on the previously applied maximum field. These observations suggest that the films are magnetically uniform. As such, tau(Mn,Ni)Al films may be an excellent medium for high-density storage of binary information

Gene transfection of HEK cells on supermacroporous polyacrylamide monoliths: a comparison of transient and stable recombinant protein expression in perfusion culture

Transient and continuous recombinant protein expression by HEK cells was evaluated in a perfused monolithic bioreactor. Highly porous synthetic cryogel scaffolds (10ml bed volume) were characterised by scanning electron microscopy and tested as cell substrates. Efficient seeding was achieved (94% inoculum retained, with 91-95% viability). Metabolite monitoring indicated continuous cell growth, and endpoint cell density was estimated by genomic DNA quantification to be 5.2x108, 1.1x109 and 3.5x1010 at day 10, 14 and 18. Culture of stably transfected cells allowed continuous production of the Drosophila cytokine Spätzle by the bioreactor at the same rate as in monolayer culture (total 1.2 mg at d18) and this protein was active. In transient transfection experiments more protein was produced per cell compared with monolayer culture. Confocal microscopy confirmed homogenous GFP expression after transient transfection within the bioreactor. Monolithic bioreactors are thus shown to be a flexible and powerful tool for manufacturing recombinant proteins

Next-generation cell line selection methodology leveraging data lakes, natural language generation and advanced data analytics

Cell line development is an essential stage in biopharmaceutical development that often lies on the critical path. Failure to fully characterise the lead clone during initial screening can lead to lengthy project delays during scale-up, which can potentially compromise commercial manufacturing success. In this study, we propose a novel cell line development methodology, referenced as CLD4, which involves four steps enabling autonomous data-driven selection of the lead clone. The first step involves the digitalisation of the process and storage of all available information within a structured data lake. The second step calculates a new metric referenced as the cell line manufacturability index (MICL) quantifying the performance of each clone by considering the selection criteria relevant to productivity, growth and product quality. The third step implements machine learning (ML) to identify any potential risks associated with process operation and relevant critical quality attributes (CQAs). The final step of CLD4 takes into account the available metadata and summaries all relevant statistics generated in steps 1–3 in an automated report utilising a natural language generation (NLG) algorithm. The CLD4 methodology was implemented to select the lead clone of a recombinant Chinese hamster ovary (CHO) cell line producing high levels of an antibody-peptide fusion with a known product quality issue related to end-point trisulfide bond (TSB) concentration. CLD4 identified sub-optimal process conditions leading to increased levels of trisulfide bond that would not be identified through conventional cell line development methodologies. CLD4 embodies the core principles of Industry 4.0 and demonstrates the benefits of increased digitalisation, data lake integration, predictive analytics and autonomous report generation to enable more informed decision making

Direct observation of topoisomerase IA gate dynamics

Type IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although type IA topoisomerases are assumed to accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the Escherichia coli type IA topoisomerases I and III. We found that after cleavage of single-stranded DNA, the protein gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. Key differences in the cleavage, ligation, and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allowed us to develop a mechanistic model of interactions between type IA topoisomerases and single-stranded DNA

Studying protein–protein affinity and immobilized ligand–protein affinity interactions using MS-based methods

This review discusses the most important current methods employing mass spectrometry (MS) analysis for the study of protein affinity interactions. The methods are discussed in depth with particular reference to MS-based approaches for analyzing protein–protein and protein–immobilized ligand interactions, analyzed either directly or indirectly. First, we introduce MS methods for the study of intact protein complexes in the gas phase. Next, pull-down methods for affinity-based analysis of protein–protein and protein–immobilized ligand interactions are discussed. Presently, this field of research is often called interactomics or interaction proteomics. A slightly different approach that will be discussed, chemical proteomics, allows one to analyze selectivity profiles of ligands for multiple drug targets and off-targets. Additionally, of particular interest is the use of surface plasmon resonance technologies coupled with MS for the study of protein interactions. The review addresses the principle of each of the methods with a focus on recent developments and the applicability to lead compound generation in drug discovery as well as the elucidation of protein interactions involved in cellular processes. The review focuses on the analysis of bioaffinity interactions of proteins with other proteins and with ligands, where the proteins are considered as the bioactives analyzed by MS