Communicating computational workflows in a regulatory environment.

The volume of nucleic acid sequence data has exploded recently, amplifying the challenge of transforming data into meaningful information. Processing data can require an increasingly complex ecosystem of customized tools, which increases difficulty in communicating analyses in an understandable way yet is of sufficient detail to enable informed decisions or repeats. This can be of particular interest to institutions and companies communicating computations in a regulatory environment. BioCompute Objects (BCOs; an instance of pipeline documentation that conforms to the IEEE 2791-2020 standard) were developed as a standardized mechanism for analysis reporting. A suite of BCOs is presented, representing interconnected elements of a computation modeled after those that might be found in a regulatory submission but are shared publicly - in this case a pipeline designed to identify viral contaminants in biological manufacturing, such as for vaccines.

Transformation of Metallic Ti to TiH2 Phase in the Ti/MgH2 Composite and Its Influence on the Hydrogen Storage Behavior of MgH2.

The current work explores the in-situ formation of TiH2 additive in a Ti/MgH2 nanocomposite system. Mild mechanical milling leaves Ti chemically unchanged, while formation of stable TiH2-x occurs upon strong mechanical milling. TiH2-x further transforms to TiH2 upon recycling the powder (dehydrogenation and subsequent hydrogenation) and lowers the activation energy of MgH2 to 89.4 kJ (mol H2 )-1 [Ea of as-received MgH2 is 153 kJ (mol H2 )-1 ]. This work also reiterates that metallic Ti additive mixed MgH2 requires strong mechanical milling for better H2 ab/de-sorption performance. The current observations support the view that lattice strain may be an important factor in the catalysis of additives incorporated MgH2 hydrogen storage systems.

Considerations for Optimization of High-Throughput Sequencing Bioinformatics Pipelines for Virus Detection.

High-throughput sequencing (HTS) has demonstrated capabilities for broad virus detection based upon discovery of known and novel viruses in a variety of samples, including clinical, environmental, and biological. An important goal for HTS applications in biologics is to establish parameter settings that can afford adequate sensitivity at an acceptable computational cost (computation time, computer memory, storage, expense or/and efficiency), at critical steps in the bioinformatics pipeline, including initial data quality assessment, trimming/cleaning, and assembly (to reduce data volume and increase likelihood of appropriate sequence identification). Additionally, the quality and reliability of the results depend on the availability of a complete and curated viral database for obtaining accurate results; selection of sequence alignment programs and their configuration, that retains specificity for broad virus detection with reduced false-positive signals; removal of host sequences without loss of endogenous viral sequences of interest; and use of a meaningful reporting format, which can retain critical information of the analysis for presentation of readily interpretable data and actionable results. Furthermore, after alignment, both automated and manual evaluation may be needed to verify the results and help assign a potential risk level to residual, unmapped reads. We hope that the collective considerations discussed in this paper aid toward optimization of data analysis pipelines for virus detection by HTS.

Structure and Electrical-Transport Relations in Ba(Zr,Pr)O3-δ Perovskites.

Members of the perovskite solid solution BaZr1-xPrxO3-δ (0.2 ≤ x ≤ 0.8) with potential high-temperature electrochemical applications were synthesized via mechanical activation and high-temperature annealing at 1250 °C. Structural properties were examined by Rietveld analysis of neutron powder diffraction and Raman spectroscopy at room temperature, indicating rhombohedral symmetry (space group R3̅c) for members x = 0.2 and 0.4 and orthorhombic symmetry (Imma) for x = 0.6 and 0.8. The sequence of phase transitions for the complete solid solution from BaZrO3 to BaPrO3 is Pm3̅m → R3̅c → Imma → Pnma. The structural data indicate that Pr principally exists as Pr4+ on the B site and that oxygen content increases with higher Pr content. Electrical-conductivity measurements in the temperature range of 250-900 °C in dry and humidified (pH2O ≈ 0.03 atm) N2 and O2 atmospheres revealed an increase of total conductivity by over 2 orders of magnitude in dry conditions from x = 0.2 to x = 0.8 (σ ≈ 0.08 S cm-1 at 920 °C in dry O2 for x = 0.8). The conductivity for Pr contents x > 0.2 is attributable to positively charged electronic carriers, whereas for x = 0.2 transport in dry conditions is n-type. The change in conduction mechanism with composition is proposed to arise from the compensation regime for minor amounts of BaO loss changing from predominantly partitioning of Pr on the A site to vacancy formation with increasing Pr content. Conductivity is lower in wet conditions for x > 0.2 indicating that the positive defects are, to a large extent, charge compensated by less mobile protonic species. In contrast, the transport mechanism of the Zr-rich composition (x = 0.2), with much lower electronic conductivity, is essentially independent of moisture content.

Site Redistribution, Partial Frozen-in Defect Chemistry, and Electrical Properties of Ba1-x(Zr,Pr)O3-δ.

Changes in nominal composition of the perovskite (ABO3) solid solution Ba1-x(Zr,Pr)O3-δ and adjusted firing conditions at very high temperatures were used to induce structural changes involving site redistribution and frozen-in point defects, as revealed by Raman and photoluminescence spectroscopies. Complementary magnetic measurements allowed quantification of the reduced content of Pr. Weak dependence of oxygen stoichiometry with temperature was obtained by coulometric titration at temperatures below 1000 °C, consistent with a somewhat complex partial frozen-in defect chemistry. Electrical conductivity measurements combined with transport number and Seebeck coefficient measurements showed prevailing electronic transport and also indicated trends expected for partial frozen-in conditions. Nominal Ba deficiency and controlled firing at very high temperatures allows adjustment of structure and partial frozen-in defect chemistry, opening the way to engineer relevant properties for high-temperature electrochemical applications.

Formation of Mgx Nby Ox+y through the Mechanochemical Reaction of MgH2 and Nb2O5, and Its Effect on the Hydrogen-Storage Behavior of MgH2.

The present study aims to understand the catalysis of the MgH2 -Nb2 O5 hydrogen storage system. To clarify the chemical interaction between MgH2 and Nb2 O5 , the mechanochemical reaction products of a composite mixture of MgH2 +0.167 Nb2 O5 was monitored at different time intervals (2, 5, 15, 30, and 45 min, as well as 1, 2, 5, 10, 15, 20, 25, and 30 h). The study confirms the formation of catalytically active Nb-doped MgO nanoparticles (typically Mgx Nby Ox+y , with a crystallite size of 4-8 nm) by transforming reactants through an intermediate phase typified by Mgm-x Nb2n-y O5n-(x+y) . The initially formed Mgx Nby Ox+y product is shown to be Nb rich, with the concentration of Mg increasing upon increasing milling time. The nanoscale end-product Mgx Nby Ox+y closely resembles the crystallographic features of MgO, but with at least a 1-4 % higher unit cell volume. Unlike MgO, which is known to passivate the surfaces in MgH2 system, the Nb-dissolved MgO effectively mediates the Mg-H2 sorption reaction in the system. We believe that this observation will lead to new developments in the area of catalysis for metal-gas interactions.

Summary of the advanced virus detection technologies users group efforts-2013.

An informal consortium of scientists from the biopharmaceutical industry, academia, and government agencies, including regulators, coalesced during 2013 to further explore advanced virus detection technologies or appropriate applications for characterization and evaluation of biologicals. As a Parenteral Drug Association task force, the Users Group came to focus on four key work areas that required better understanding and data generation: (1) evaluation of sample preparation and processing steps for different sample types, (2) determination of method sensitivity by performing spike recovery studies using selected virus stocks, (3) development of a reliable and comprehensive viral sequence database, and (4) evaluation of bioinformatic analysis pipelines, with primary focus on next-generation DNA sequencing platforms. The year of monthly working meetings and additional subgroup discussions culminated in the 2013 PDA/FDA Advanced Technologies for Virus Detection in the Evaluation of Biologicals Conference held November 13-14, 2013 in Bethesda, MD, which provided a forum for group participants and others for data sharing and knowledge exchange. The Users Group has continued its efforts during 2014 as a PDA Interest Group with increased participation from technology developers and contract research organizations.

Modeling an approach to define sensitivity of viral detection in sample matrices-examples with microarray readout.

Advances in viral detection technologies have the potential to increase the safety assurance of medicines produced in biological production systems. However, taking full advantage of these technological advances in the regulated testing environment will require protocols for standardization and performance testing. The most essential performance characteristics of detection methods for these applications include sensitivity, breadth of detection, and consistency. We have evaluated an approach to establishing the suitability of advanced nucleic-acid-based detection systems for characterization of cell substrates and production cultures. This approach is based on selecting relevant challenge viruses, their preparation and characterization, their application to a sample preparation workflow, and quantitation of their recovery by an independent means as well as the advanced detection readout. This approach helps us evaluate the suitability of the workflow for handling diverse sample matrices across manufacturing platforms for vaccines and other biological products, and also suggests a means by which technology users, developers, and regulators may consider the critical performance attributes of novel detection technologies. We have applied this workflow to a novel microarray-based viral detection system in collaboration with Lawrence Livermore National Laboratory. This system is appealing because of the rapid turnaround of results compared to some other advanced detection technologies.

Defining a sample preparation workflow for advanced virus detection and understanding sensitivity by next-generation sequencing.

The application of next-generation sequencing (also known as deep sequencing or massively parallel sequencing) for adventitious agent detection is an evolving field that is steadily gaining acceptance in the biopharmaceutical industry. In order for this technology to be successfully applied, a robust method that can isolate viral nucleic acids from a variety of biological samples (such as host cell substrates, cell-free culture fluids, viral vaccine harvests, and animal-derived raw materials) must be established by demonstrating recovery of model virus spikes. In this report, we implement the sample preparation workflow developed by Feng et. al. and assess the sensitivity of virus detection in a next-generation sequencing readout using the Illumina MiSeq platform. We describe a theoretical model to estimate the detection of a target virus in a cell lysate or viral vaccine harvest sample. We show that nuclease treatment can be used for samples that contain a high background of non-relevant nucleic acids (e.g., host cell DNA) in order to effectively increase the sensitivity of sequencing target viruses and reduce the complexity of data analysis. Finally, we demonstrate that at defined spike levels, nucleic acids from a panel of model viruses spiked into representative cell lysate and viral vaccine harvest samples can be confidently recovered by next-generation sequencing.

Systematic evaluation of in vitro and in vivo adventitious virus assays for the detection of viral contamination of cell banks and biological products.

Viral vaccines and the cell substrates used to manufacture them are subjected to tests for adventitious agents, including viruses, contaminate. Some of the compendial methods (in vivo and in vitro in cell culture) were established in the mid-20th century. These methods have not been subjected to current assay validation, as new methods would need to be. This study was undertaken to provide insight into the breadth (selectivity) and sensitivity (limit of detection) of the routine methods, two such validation parameters. Sixteen viral stocks were prepared and characterized. These stocks were tested in serial dilutions by the routine methods to establish which viruses were detected by which methods and above what limit of detection. Sixteen out of sixteen viruses were detected in vitro, though one (bovine viral diarrhea virus) required special conditions to detect and another (rubella virus) was detected with low sensitivity. Many were detected at levels below 1 TCID50 or PFU (titers were established on the production cell line in most cases). In contrast, in vivo, only 6/11 viruses were detected, and 4 of these were detected only at amounts one or more logs above 1 TCID50 or PFU. Only influenza virus and vesicular stomatitis virus were detected at lower amounts in vivo than in vitro. Given the call to reduce, refine, or replace (3Rs) the use of animals in product safety testing and the emergence of new technologies for the detection of viruses, a re-examination of the current adventitious virus testing strategies seems warranted. Suggested pathways forward are offered.

Characterization of virus-like particles in GARDASIL® by cryo transmission electron microscopy.

Cryo-transmission electron microscopy (cryoTEM) is a powerful characterization method for assessing the structural properties of biopharmaceutical nanoparticles, including Virus Like Particle-based vaccines. We demonstrate the method using the Human Papilloma Virus (HPV) VLPs in GARDASIL®. CryoTEM, coupled to automated data collection and analysis, was used to acquire images of the particles in their hydrated state, determine their morphological characteristics, and confirm the integrity of the particles when absorbed to aluminum adjuvant. In addition, we determined the three-dimensional structure of the VLPs, both alone and when interacting with neutralizing antibodies. Two modes of binding of two different neutralizing antibodies were apparent; for HPV type 11 saturated with H11.B2, 72 potential Fab binding sites were observed at the center of each capsomer, whereas for HPV 16 interacting with H16.V5, it appears that 60 pentamers (each neighboring 6 other pentamers) bind five Fabs per pentamer, for the total of 300 potential Fab binding sites per VLP.

Haplotype analysis of the 185delAG BRCA1 mutation in ethnically diverse populations.

The 185delAG* BRCA1 mutation is encountered primarily in Jewish Ashkenazi and Iraqi individuals, and sporadically in non-Jews. Previous studies estimated that this is a founder mutation in Jewish mutation carriers that arose before the dispersion of Jews in the Diaspora ~2500 years ago. The aim of this study was to assess the haplotype in ethnically diverse 185delAG* BRCA1 mutation carriers, and to estimate the age at which the mutation arose. Ethnically diverse Jewish and non-Jewish 185delAG*BRCA1 mutation carriers and their relatives were genotyped using 15 microsatellite markers and three SNPs spanning 12.5 MB, encompassing the BRCA1 gene locus. Estimation of mutation age was based on a subset of 11 markers spanning a region of ~5 MB, using a previously developed algorithm applying the maximum likelihood method. Overall, 188 participants (154 carriers and 34 noncarriers) from 115 families were included: Ashkenazi, Iraq, Kuchin-Indians, Syria, Turkey, Iran, Tunisia, Bulgaria, non-Jewish English, non-Jewish Malaysian, and Hispanics. Haplotype analysis indicated that the 185delAG mutation arose 750-1500 years ago. In Ashkenazim, it is a founder mutation that arose 61 generations ago, and with a small group of founder mutations was introduced into the Hispanic population (conversos) ~650 years ago, and into the Iraqi-Jewish community ~450 years ago. The 185delAG mutation in the non-Jewish populations in Malaysia and the UK arose at least twice independently. We conclude that the 185delAG* BRCA1 mutation resides on a common haplotype among Ashkenazi Jews, and arose about 61 generations ago and arose independently at least twice in non-Jews.

Prevalence and type of BRCA mutations in Hispanics undergoing genetic cancer risk assessment in the southwestern United States: a report from the Clinical Cancer Genetics Community Research Network.

PURPOSE: To determine the prevalence and type of BRCA1 and BRCA2 (BRCA) mutations among Hispanics in the Southwestern United States and their potential impact on genetic cancer risk assessment (GCRA). PATIENTS AND METHODS: Hispanics (n = 746) with a personal or family history of breast and/or ovarian cancer were enrolled in an institutional review board-approved registry and received GCRA and BRCA testing within a consortium of 14 clinics. Population-based Hispanic breast cancer cases (n = 492) enrolled in the Northern California Breast Cancer Family Registry, negative by sequencing for BRCA mutations, were analyzed for the presence of the BRCA1 ex9-12del large rearrangement. RESULTS: Deleterious BRCA mutations were detected in 189 (25%) of 746 familial clinic patients (124 BRCA1, 65 BRCA2); 21 (11%) of 189 were large rearrangement mutations, of which 62% (13 of 21) were BRCA1 ex9-12del. Nine recurrent mutations accounted for 53% of the total. Among these, BRCA1 ex9-12del seems to be a Mexican founder mutation and represents 10% to 12% of all BRCA1 mutations in clinic- and population-based cohorts in the United States. CONCLUSION: BRCA mutations were prevalent in the largest study of Hispanic breast and/or ovarian cancer families in the United States to date, and a significant proportion were large rearrangement mutations. The high frequency of large rearrangement mutations warrants screening in every case. We document the first Mexican founder mutation (BRCA1 ex9-12del), which, along with other recurrent mutations, suggests the potential for a cost-effective panel approach to ancestry-informed GCRA.

Temperature dependence of the Henry's law constant for hydrogen storage in NaA zeolites: a Monte Carlo simulation study.

Grand canonical Monte Carlo simulations of hydrogen adsorption in zeolites NaA were carried out for a wide range of temperatures between 77 and 300 K and pressures up to 180 MPa. A potential model was used that comprised of three main interactions: van der Waals, coulombic and induced polarization by the electric field in the system. The computed average number of adsorbed molecules per unit cell was compared with available results and found to be in agreement in the regime of moderate to high pressures. The particle insertion method was used to calculate the Henry coefficient for this model and its dependence on temperature.

Ingredients for success: a familial cancer clinic in an oncology practice setting.

PURPOSE: Genetic cancer risk assessment (GCRA) has become increasingly important in clinical cancer care. Almost all published information on genetic risk assessment has come from academic institutions. However, a majority of patients with cancer are seen in the community practice setting. METHODS: We describe the evolution of a community oncology practice GCRA clinic. RESULTS: Over a 10-year period, 445 patients were seen for a possible genetic cancer syndrome. This included 325 patients with family history of breast or ovarian cancer, 92 patients with family history of colorectal cancer or polyposis, and 28 families with another familial cancer predisposition. Fifty-three unique families with a genetic mutation were identified. CONCLUSION: A GCRA clinic can be incorporated into an oncology practice setting and can enhance the standard of care for the entire community. We present data reflecting a 10-year experience with such a clinic and provide recommendations for establishing a successful one.

Paraneoplastic jaw dystonia and laryngospasm with antineuronal nuclear autoantibody type 2 (anti-Ri).

BACKGROUND: Opsoclonus-myoclonus syndrome and breast carcinoma were initially described as neurologic and oncologic accompaniments of antineuronal nuclear autoantibody type 2 (ANNA-2, also known as anti-Ri). However, the neurologic spectrum of ANNA-2 autoimmunity is broader, includes a syndrome of jaw dystonia and laryngospasm, and can be accompanied by lung carcinoma. OBJECTIVE: To describe clinically (with a video) ANNA-2-associated jaw dystonia and laryngospasm, its pathologic correlates, and therapeutic outcomes. DESIGN: Retrospective case series with prospective clinical follow-up. SETTING: Mayo Clinic's Neuroimmunology Laboratory, Rochester, Minnesota. PATIENTS: Consecutive patients with ANNA-2 seropositivity identified since January 1, 1990. MAIN OUTCOME METHODS: Clinical (in 9 patients) and neuropathologic (in 2 patients) findings were reviewed. RESULTS: Of 48 patients with ANNA-2 seropositivity, 9 (19%) had multifocal neurologic manifestations that included jaw dystonia and laryngospasm. Among 6 patients with jaw dystonia, 5 had severely impaired nutrition, causing profound weight loss. Five patients had documented laryngospasm, which contributed to 1 patient's death. Neuropathologic examination revealed diffuse infiltration by CD8(+) T lymphocytes, with axonal loss and gliosis in brainstem and descending spinal cord tracts. Some patients improved symptomatically after immunosuppressant or cytotoxic therapies; 1 patient improved after treatment with botulinum toxin. One patient who underwent tracheostomy because of recurrent laryngospasm was alive and well longer than 3 years after symptom onset. CONCLUSIONS: Jaw dystonia and laryngospasm are common accompaniments of ANNA-2 autoimmunity and are associated with significant morbidity. We propose that selective damage to antigen-containing inhibitory fibers innervating bulbar motor nuclei by CD8(+) T lymphocytes (histopathologically observed infiltrating brainstem reticular formation) is the proximal cause of this syndrome. Early and aggressive therapy offers the prospect of neurologic improvement or stabilization.

Interaction between nanoparticles and cytokine proteins: impact on protein and particle functionality.

There is increased use of nanomaterials in many applications due to their unique properties, such as their high surface area and surface reactivity. However, the potential health effects to workers, consumers and the environment exposed to nanoparticles (NPs) is unknown. The aim of this study was to investigate whether NPs which may enter the body could adsorb proteins and whether this interaction affects both the particle and the protein function. The cytokines IL-8 and TNF-alpha were adsorbed significantly more by 14 nm carbon black (CB) compared with a similar dose of 260 nm CB. Uncoated 14 nm CB particles produced a significant increase in intracellular calcium [Ca(2 + )](i) which was greater than a similar mass dose of 260 nm CB. The 260 nm CB produced an increase in ICAM-1 expression in A549 epithelial cells at a comparable dose of 14 nm CB, and after coating with TNF-alpha 260 nm CB produced significantly more ICAM-1 expression compared with control cells. TNF-alpha bound to 14 nm CB induced a level of ICAM-1 expression that was no greater than the control level, suggesting that the TNF-alpha activity may be inhibited. These results suggest that NP-protein interaction results both in a decrease in protein function and particle activity in the cellular assays tested and this is currently being investigated.

Estimating a weighted average of stratum-specific parameters.

This article investigates estimators of a weighted average of stratum-specific univariate parameters and compares them in terms of a design-based estimate of mean-squared error (MSE). The research is motivated by a stratified survey sample of Florida Medicaid beneficiaries, in which the parameters are population stratum means and the weights are known and determined by the population sampling frame. Assuming heterogeneous parameters, it is common to estimate the weighted average with the weighted sum of sample stratum means; under homogeneity, one ignores the known weights in favor of precision weighting. Adaptive estimators arise from random effects models for the parameters. We propose adaptive estimators motivated from these random effects models, but we compare their design-based performance. We further propose selecting the tuning parameter to minimize a design-based estimate of mean-squared error. This differs from the model-based approach of selecting the tuning parameter to accurately represent the heterogeneity of stratum means. Our design-based approach effectively downweights strata with small weights in the assessment of homogeneity, which can lead to a smaller MSE. We compare the standard random effects model with identically distributed parameters to a novel alternative, which models the variances of the parameters as inversely proportional to the known weights. We also present theoretical and computational details for estimators based on a general class of random effects models. The methods are applied to estimate average satisfaction with health plan and care among Florida beneficiaries just prior to Medicaid reform.

Assessing the viability of a clumpy mnn9 strain of Saccharomyces cerevisiae used in the manufacture of recombinant pharmaceutical proteins.

Demonstration of the viability of cryopreserved cell bank used to make a biopharmaceutical product is an important indicator of the ability to consistently manufacture over a long period of time, and is mandated in regulatory guidances. A mnn9 strain of Saccharomyces cerevisiae, chosen for its inability to hypermannosylate vaccine antigens, has a clumpy growth tendency due to the inactivation of the gene MNN9 (wild-type), complicating the interpretation of conventional viability measurements useful for single cells. Therefore, two growth-based measurements as well as staining by a membrane-impermeable dye were examined for their ability to reflect changes in viability of a clumpy mnn9 (defective) strain. The cell clumps proved to be stable to mixing, and variability of agar-plate-based viable counts (VC) of undisrupted suspensions of this clumpy mnn9 strain was consistent with variability observed for cell banks of a non-clumpy MNN9 strain. Both the VC and the growth times in an oxygen-sensing broth-based microplate assay corresponded well with shake-flask growth times for a set of stressed and unstressed samples, although the correlation was highest between the two broth-based systems. Counts of trypan-blue-stained cells within clumps also increased with time of stress, suggesting that this method could be adapted as a simple index of viability as well.

Characterization of the gene encoding glutamate dehydrogenase ( gdhA) from the ruminal bacterium Ruminococcus flavefaciens FD-1.

The gene encoding glutamate dehydrogenase ( gdhA) in the ruminal bacterium Ruminococcus flavefaciens FD-1 was cloned. A degenerate primer based on the N-terminal amino acid sequence of the purified protein was used in conjunction with genome walking to obtain the complete ORF of 1,365 bp, capable of encoding a polypeptide of 455 amino acid residues. The translated ORF contained the amino acid motifs characteristic of the subfamily GDH S_50(I) small glutamate dehydrogenases, including the catalytic site, and matched the originally deduced N-terminal amino acid sequence. BLAST search yielded high scores with other GdhA sequences from a variety of organisms, the closest match being with the GdhA sequence of Corynebacterium glutamicum (63% amino acid identity). Classification of the GdhA enzyme from R. flavefaciens FD-1 as a GDH S_50(I) subfamily member was further supported by phylogenetic analysis. The transcript size determined by Northern blot analysis was in good agreement with the putative regulatory region of the gene and confirmed its monocistronic structure. R. flavefaciens GdhA activity appears to be regulated primarily at the level of transcription. Brief exposure to 20 mM NH(4)Cl prior to extraction did not alter the level of activity. Transcriptional regulation, studied with quantitative real-time RT-PCR, demonstrated a three-fold increase of the gdhA transcript concentration in ammonia-limited cells in comparison with an excess of ammonia in the medium. This is in agreement with the enzyme activity data obtained under ammonia- and carbon-limited growth conditions.