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Clinical applications of precision oncology require accurate tests that can distinguish true cancer-specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor–normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection.

Purpose: To determine the diagnostic yield and clinical impact of exome sequencing (ES) in patients with suspected monogenic kidney disease. Method(s): We performed clinically accredited singleton ES in a prospectively ascertained cohort of 204 patients assessed in multidisciplinary renal genetics clinics at four tertiary hospitals in Melbourne, Australia. Result(s): ES identified a molecular diagnosis in 80 (39%) patients, encompassing 35 distinct genetic disorders. Younger age at presentation was independently associated with an ES diagnosis (p < 0.001). Of those diagnosed, 31/80 (39%) had a change in their clinical diagnosis. ES diagnosis was considered to have contributed to management in 47/80 (59%), including negating the need for diagnostic renal biopsy in 10/80 (13%), changing surveillance in 35/80 (44%), and changing the treatment plan in 16/80 (20%). In cases with no change to management in the proband, the ES result had implications for the management of family members in 26/33 (79%). Cascade testing was subsequently offered to 40/80 families (50%). Conclusion(s): In this pragmatic pediatric and adult cohort with suspected monogenic kidney disease, ES had high diagnostic and clinical utility. Our findings, including predictors of positive diagnosis, can be used to guide clinical practice and health service design.Copyright © 2020, The Author(s).

Purpose: To determine the diagnostic yield and clinical impact of exome sequencing (ES) in patients with suspected monogenic kidney disease. Method(s): We performed clinically accredited singleton ES in a prospectively ascertained cohort of 204 patients assessed in multidisciplinary renal genetics clinics at four tertiary hospitals in Melbourne, Australia. Result(s): ES identified a molecular diagnosis in 80 (39%) patients, encompassing 35 distinct genetic disorders. Younger age at presentation was independently associated with an ES diagnosis (p < 0.001). Of those diagnosed, 31/80 (39%) had a change in their clinical diagnosis. ES diagnosis was considered to have contributed to management in 47/80 (59%), including negating the need for diagnostic renal biopsy in 10/80 (13%), changing surveillance in 35/80 (44%), and changing the treatment plan in 16/80 (20%). In cases with no change to management in the proband, the ES result had implications for the management of family members in 26/33 (79%). Cascade testing was subsequently offered to 40/80 families (50%). Conclusion(s): In this pragmatic pediatric and adult cohort with suspected monogenic kidney disease, ES had high diagnostic and clinical utility. Our findings, including predictors of positive diagnosis, can be used to guide clinical practice and health service design.Copyright © 2020, The Author(s).

In the clinical investigation of a family with debilitating centrofacial pruritus by exome sequencing, we have observed a clear segregation of the TRPM3 variant outlined, which is highly suggestive of a causal relationship.

Clinical applications of precision oncology require accurate tests that can distinguish true cancer-specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor–normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection.

AbstractBackgroundStrong associations between diet and maternal and child outcomes emphasise the importance of evidence-based care for women across preconception, antenatal and postnatal periods. A 2008 survey of Australian maternal health dietetic services documented critically low resourcing with considerable variation in staffing levels and models of care. This study repeated the survey to examine resourcing in Australian maternal health services.MethodsA cross-sectional online survey was emailed to publicly-funded Australian maternal health dietetic services in May 2018. Quantitative and qualitative variables collected across preconception to postnatal services (including diabetes) included; births per year (BPY), number of beds, staffing (full time equivalents; FTE), referral processes, and models of care. Results were collated in > 5000; 3500 and 5000; and < 3500 BPY.ResultsForty-three eligible surveys were received from seven states/territories. Dietetic staffing levels ranged from 0 to 4.0 FTE (> 5000 BPY), 0–2.8 FTE (3500–5000 BPY), and 0–2.0 FTE (< 3500 BPY). The offering of preconception, antenatal and postnatal services varied significantly between hospitals (format, staffing, referral processes, delivery models). Few sites reported service effectiveness monitoring and only one delivered gestational diabetes mellitus care according to nutrition practice guidelines. Low staffing levels and extensive service gaps, including lack of processes to deliver and evaluate services, were evident with major concerns expressed about the lack of capacity to provide evidence-based care.ConclusionsTen years after the initial survey and recommendations t

PURPOSE: To determine the diagnostic yield and clinical impact of exome sequencing (ES) in patients with suspected monogenic kidney disease. METHODS: We performed clinically accredited singleton ES in a prospectively ascertained cohort of 204 patients assessed in multidisciplinary renal genetics clinics at four tertiary hospitals in Melbourne, Australia. RESULTS: ES identified a molecular diagnosis in 80 (39%) patients, encompassing 35 distinct genetic disorders. Younger age at presentation was independently associated with an ES diagnosis (p < 0.001). Of those diagnosed, 31/80 (39%) had a change in their clinical diagnosis. ES diagnosis was considered to have contributed to management in 47/80 (59%), including negating the need for diagnostic renal biopsy in 10/80 (13%), changing surveillance in 35/80 (44%), and changing the treatment plan in 16/80 (20%). In cases with no change to management in the proband, the ES result had implications for the management of family members in 26/33 (79%). Cascade testing was subsequently offered to 40/80 families (50%). CONCLUSION: In this pragmatic pediatric and adult cohort with suspected monogenic kidney disease, ES had high diagnostic and clinical utility. Our findings, including predictors of positive diagnosis, can be used to guide clinical practice and health service design.

Newly emerging viruses often circulate as a heterogeneous swarm in wild animal reservoirs prior to their emergence in humans, and their antigenic identities are often unknown until an outbreak situation. The newly emerging severe acute respiratory syndrome coronavirus (SARS-CoV) and reemerging influenza virus cause disproportionate disease in the aged, who are also notoriously difficult to successfully vaccinate, likely due to immunosenescence. To protect against future emerging strains, vaccine platforms should induce broad cross-reactive immunity that is sufficient to protect from homologous and heterologous challenge in all ages. From initial studies, we hypothesized that attenuated Venezuelan equine encephalitis virus (VEE) replicon particle (VRP) vaccine glycoproteins mediated vaccine failure in the aged. We then compared the efficacies of vaccines bearing attenuated (VRP3014) or wild-type VEE glycoproteins (VRP3000) in young and aged mice within novel models of severe SARS-CoV pathogenesis. Aged animals receiving VRP3000-based vaccines were protected from SARS-CoV disease, while animals receiving the VRP3014-based vaccines were not. The superior protection for the aged observed with VRP3000-based vaccines was confirmed in a lethal influenza virus challenge model. While the VRP3000 vaccine's immune responses in the aged were sufficient to protect against lethal homologous and heterologous challenge, our data suggest that innate defects within the VRP 3014 platform mediate vaccine failure. Exploration into the mechanism(s) of successful vaccination in the immunosenescent should aid in the development of successful vaccine strategies for other viral diseases disproportionately affecting the elderly, like West Nile virus, influenza virus, norovirus, or other emerging viruses of the future. Copyright

Newly emerging viruses often circulate as a heterogeneous swarm in wild animal reservoirs prior to their emergence in humans, and their antigenic identities are often unknown until an outbreak situation. The newly emerging severe acute respiratory syndrome coronavirus (SARS-CoV) and reemerging influenza virus cause disproportionate disease in the aged, who are also notoriously difficult to successfully vaccinate, likely due to immunosenescence. To protect against future emerging strains, vaccine platforms should induce broad cross-reactive immunity that is sufficient to protect from homologous and heterologous challenge in all ages. From initial studies, we hypothesized that attenuated Venezuelan equine encephalitis virus (VEE) replicon particle (VRP) vaccine glycoproteins mediated vaccine failure in the aged. We then compared the efficacies of vaccines bearing attenuated (VRP3014) or wild-type VEE glycoproteins (VRP3000) in young and aged mice within novel models of severe SARS-CoV pathogenesis. Aged animals receiving VRP3000-based vaccines were protected from SARS-CoV disease, while animals receiving the VRP3014-based vaccines were not. The superior protection for the aged observed with VRP3000-based vaccines was confirmed in a lethal influenza virus challenge model. While the VRP3000 vaccine's immune responses in the aged were sufficient to protect against lethal homologous and heterologous challenge, our data suggest that innate defects within the VRP 3014 platform mediate vaccine failure. Exploration into the mechanism(s) of successful vaccination in the immunosenescent should aid in the development of successful vaccine strategies for other viral diseases disproportionately affecting the elderly, like West Nile virus, influenza virus, norovirus, or other emerging viruses of the future. Copyright

Background. Severe acute respiratory syndrome (SARS) emerged as a human disease in 2002. Detailed phylogenetic analysis and epidemiologic studies have suggested that the SARS Coronavirus (SARS-CoV) originated from animals. The spike (S) glycoprotein has been identified as a major target of protective immunity and contains ≥3 regions that are targeted by neutralizing antibodies in the S1 and S2 domains. We previously characterized a panel of neutralizing human monoclonal antibodies (MAbs), but the majority of epitopes recognized by the MAbs remain unknown. Methods. In the present study, we generated neutralization escape mutants and studied the effect of these neutralization escape mutations on human and animal receptor usage as well as on in vitro and in vivo fitness. Results. Distinct but partially overlapping sets of amino acids were identified that are critical to the binding of MAbs with differential neutralization profiles. We also identified possible interactions between the S1 and S2 domains of the SARS-CoV S glycoprotein. Finally, we showed that escape from neutralization usually attenuates SARS-CoV infection. Conclusions. These data provide a mechanism for overcoming neutralization escape by use of broadly crossreactive cocktails of cross-neutralizing MAbs that recognize residues within the receptor-binding domain that are critical for virus replication and virulence.

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002, and detailed phylogenetic and epidemiological analyses have suggested that it originated from animals. The spike (S) glycoprotein has been identified as a major component of protective immunity, and 23 different amino acid changes were noted during the expanding epidemic. Using a panel of SARS-CoV recombinants bearing the S glycoproteins from isolates representing the zoonotic and human early, middle, and late phases of the epidemic, we identified 23 monoclonal antibodies (MAbs) with neutralizing activity against one or multiple SARS-CoV spike variants and determined the presence of at least six distinct neutralizing profiles in the SARS-CoV S glycoprotein. Four of these MAbs showed cross-neutralizing activity against all human and zoonotic S variants in vitro, and at least three of these were mapped in distinct epitopes using escape mutants, structure analyses, and competition assays. These three MAbs (S109.8, S227.14, and S230.15) were tested for use in passive vaccination studies using lethal SARS-CoV challenge models for young and senescent mice with four different homologous and heterologous SARS-CoV S variants. Both S227.14 and S230.15 completely protected young and old mice from weight loss and virus replication in the lungs for all viruses tested, while S109.8 completely protected mice from weight loss and clinical signs in the presence of viral titers. We conclude that a single human MAb can confer broad protection against lethal challenge with multiple zoonotic and human SARS-CoV isolates, and we identify a robust cocktail formulation that targets distinct epitopes and minimizes the likely generation of escape mutants. Copyright

Zoonotic severe acute respiratory syndrome coronavirus (SARS-CoV) likely evolved to infect humans by a series of transmissio

The severe acute respiratory syndrome (SARS) epidemic was characterized by high mortality rates in the elderly. The molecular mechanisms that govern enhanced susceptibility of elderly populations are not known, and robust animal models are needed that recapitulate the increased pathogenic phenotype noted with increasing age. Using synthetic biology and reverse genetics, we describe the construction of a panel of isogenic SARS coronavirus (SARS-CoV) strains bearing variant spike glycoproteins that are representative of zoonotic strains found in palm civets and raccoon dogs, as well as isolates spanning the early, middle, and late phases of the SARS-CoV epidemic. The recombinant viruses replicated efficiently in cell culture and demonstrated variable sensitivities to neutralization with antibodies. The human but not the zoonotic variants replicated efficiently in human airway epithelial cultures, supporting earlier hypotheses that zoonotic isolates are less pathogenic in humans but can evolve into highly pathogenic strains. All viruses replicated efficiently, but none produced clinical disease or death in young animals. In contrast, severe clinical disease, diffuse alveolar damage, hyaline membrane formation, alveolitis, and death were noted in 12-month-old mice inoculated with the palm civet HC/SZ/61/03 strain or early-human-phase GZ02 variants but not with related middle- and late-ph

University of Michigan., Vol. 1, no. 2- issued as the University of Michigan official publication, v. 63, no. 74, Electronic serial mode of access: World Wide Web., Michigan quarterly review, Michigan quarterly review. : Vol XIV : Issue 3, (dlps) volume: act2080.0014.003, (dlps) article: act2080.0014.003:5, These pages may freely searched and displayed. Permission must be received for subsequent distribution in print or electronically. Please contact spo-help@umich.edu for more information.

University of Michigan., Vol. 1, no. 2- issued as the University of Michigan official publication, v. 63, no. 74, Electronic serial mode of access: World Wide Web., Michigan quarterly review, Michigan quarterly review. : Vol XIV : Issue 3, (dlps) volume: act2080.0014.003, (dlps) article: act2080.0014.003:5, These pages may freely searched and displayed. Permission must be received for subsequent distribution in print or electronically. Please contact spo-help@umich.edu for more information.

This program was conducted to develop the overall methodology, guidelines, procedures, and guidance documentation required to establish an Air Force EMR hardness program for air launched ordnance systems. [The handbook title is 'Management and Design Guidance Electromagnetic Radiation Hardness (EMR) for Air Launched Ordnance System', MIL-HDBK-335(USAF)].

For a wide range of materials, emission of electrons (EE), positive ions (PIE), and photons (phE) is observed accompanying fracture. Here we report on work concerning emission from adhesive failure. The systems studied include interfacial failure between epoxy--brittle materials, polybutadiene-glass, and pressure sensitive adhesives--polymers. When interfacial failure occurs, it produces highly excited surfaces with intense charge separation. De-excitation in the presence of surface charge produces intense, long lasting (several minutes) emission. Energy distribution curves for both EE and PIE decrease monotonically with a tail extending out to several hundred eV. For a few systems we compare EE and phE which show different decay characteristics.

The objective of this program was to evaluate current radiated emission and susceptibility measurement techniques to determine their applicability to performing measurements required under MIL-STD-462. To accomplish this objective, program efforts were directed to a review and comparison of the technical capabilities and limitations of current radiated emission and susceptibility measurement techniques and to the identification of voids and deficiencies in current measurement methodologies. From the results of this program, it is concluded that a number of measurement techniques offer the potential of serving as viable alternates to those measurement techniques currently employed to satisfy the measurement requirements of MIL-STD-462. Shortcomings in current measurement techniques include (1) deficiencies which prevent the maximum utilization of available measurement techniques, (2) deficiencies in reducing to practice those measurement techniques which have been identified as possible alternates to current techniques, and (3) deficiencies related to the lack of correlation of measurement results obtained with different techniques.

The emission of electrons and positive ions from materials undergoing fracture is investigated. We present a survey of charged particle emission from a number of materials including crystalline insulators, glass, graphite, polymers, and composites. Particular attention is given to fiber-reinforced epoxy systems which yield unique forms of charge emission. Energy distributions of the emitted particles are given for E-glass/epoxy strands, polybutadiene filled with glass beads, and mica. Evidence is presented that interfacial failure and charge separation play important roles in the observed emission. (Author)

This report presents the results of studies to develop the rationale for electromagnetic measurement techniques for use in the EMC evaluation of millimeter wave (MMW) communication-electronic equipment and systems. In the development of this rationale, seven basic tasks were performed. Under the first task, the EMC/EMI data requirements for MMW equipments and systems were established to provide a clear definition of the type of data needed. The second task was directed to the definition of appropriate measurement philosophy and techniques for obtaining the required data. Third task, the state-of-the-art in EMC/EMI measurement instrumentation and components for the 10-300 GHz frequency band was reviewed to identify the instrumentation which is available to implement the measurement techniques defined in the previous tasks. Fourth task involved an evaluation of the advantages and disadvantages of manual, semi-automated, and full automated, and fully automated measurement techniques to determine both the feasibility and cost-effectiveness of these three approaches to data collection and analysis. Fifth task, the likely errors associated with EMC/EMI measurements at MMW frequencies were identified. The sixth task involved selected experimental measurements to investigate the possible impact of surface wave phenomena on EMI coupling at MMW frequencies. (Author)

The report describes a study of methods for controlling the interference susceptibility of mixers in communications receivers. The effects of several mixer characteristics which influence spurious product formations are discussed, including temperature, bias, and impedance effects. The dependence of spurious response levels on local oscillator drive is also considered, as a function of both local oscillator level and waveshape. A number of mixing techniques which provide control of spurious response levels are presented. A frequency-modulated oscillator is shown for which accurate prediction of response levels can be realized. The use of negative feedback in reducing mixer-generated interference is demonstrated for a transistor mixer. The application of a square wave local oscillator signal is shown to be effective in reducing the levels of responses associated with harmonics of the input signal. (Author)

The report summarizes a thirteen month program concerned with the investigation of spurious product formations in conventional mixer circuits, and with the development of techniques whereby the levels of such formations could be predicted from limited measurements of mixer parameters. The interference characteristics of diode, pentode, triode, and transistor mixer types were investigated for numerous ordered arrangements of mixer dc parameters, circuit configurations, and signal injection levels. The results have demonstrated that changes in the mode of signal and/or local oscillator injection, local oscillator level, mixer load conditions, or mixer dc voltages produce changes in response levels which are apparently unpredictable, and that attempts to develop prediction models based on discrete mixer parameters may be impractical. Tests were conducted on a diode mixer which verify that multiple mix products can occur due to signal and/or local oscillator harmonic injection, and that the levels of such products are dependent upon the phase orientation of the injected signals. Tests were also conducted which show the effects of mixer bias voltage on response phase position. A spurious response prediction technique for diode mixers previously developed at Georgia Tech was investigated to determine if modifications and improvements could be made to reduce prediction errors, and if this technique could be successfully applied to other mixer types. Maximum-upper-bound prediction methods which might give worst-case approximations of spurious response levels were also investigated. Prediction results are presented which were derived from the actual prediction of numerous data sets on diode, pentode, triode, and transistor mixers. (Author), See also AD-633-743.

Spurious response formations in a 6AS6W pentode mixer and a 6C4 triode mixer were investigated for numerous ordered arrangements of mixer dc parameters, circuit configurations, and signal injection levels. Response levels were measured for different methods of signal and local oscillator injection, and for resistive and tuned load conditions. The results have demonstrated that changes in the mode of signal and/or local oscillator injection, local oscillator level, mixer load conditions, or mixer dc voltages produce changes in response levels that would be difficult to predict. A spurious response prediction technique for diode mixers previously developed at Georgia Tech was investigated to determine if modifications and improvements could be made to reduce prediction errors, and if this technique could be successfully applied to other mixer types. The results of this study are presented in the form of prediction errors derived from the actual prediction of diode and pentagrid mixer data. Maximum-upper-bound prediction methods are discussed, and practical examples of possible techniques are presented. (Author), See also AD-628 324.