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Abstract The tetraoctylammonium bromide (TOAB)-stabilized gold nanoparticles have been successfully fabricated. After an annealing of the as-synthesized nanoparticles at 300 °C for 30 min, the coalescence behavior of gold nanoparticles has been investigated using high-resolution transmission electron microscopy in detail. Two types of coalescence, one being an ordered combination of two or more particles in appropriate orientations through twinning, and the other being an ordered combination of two small particles with facets through a common lattice plane, have been observed.

Multiple myeloma is a treatable, but currently incurable, hematological malignancy of plasma cells characterized by diverse and complex tumor genetics for which precision medicine approaches to treatment are lacking. The Multiple Myeloma Research Foundation's Relating Clinical Outcomes in Multiple Myeloma to Personal Assessment of Genetic Profile study ( NCT01454297 ) is a longitudinal, observational clinical study of newly diagnosed patients with multiple myeloma (n = 1,143) where tumor samples are characterized using whole-genome sequencing, whole-exome sequencing and RNA sequencing at diagnosis and progression, and clinical data are collected every 3 months. Analyses of the baseline cohort identified genes that are the target of recurrent gain-of-function and loss-of-function events. Consensus clustering identified 8 and 12 unique copy number and expression subtypes of myeloma, respectively, identifying high-risk genetic subtypes and elucidating many of the molecular underpinnings of these unique biological groups. Analysis of serial samples showed that 25.5% of patients transition to a high-risk expression subtype at progression. We observed robust expression of immunotherapy targets in this subtype, suggesting a potential therapeutic option., (© 2024. The Author(s).)

The emergence of single nucleus RNA sequencing (snRNA-seq) offers to revolutionize the study of Alzheimer's disease (AD). Integration with complementary multiomics data such as genetics, proteomics and clinical data provides powerful opportunities to link cell subpopulations and molecular networks with a broader disease-relevant context. We report snRNA-seq profiles from superior frontal gyrus samples from 101 well characterized subjects from the Banner Brain and Body Donation Program in combination with whole genome sequences. We report findings that link common AD risk variants with CR1 expression in oligodendrocytes as well as alterations in hematological parameters. We observed an AD-associated CD83(+) microglial subtype with unique molecular networks and which is associated with immunoglobulin IgG4 production in the transverse colon. Our major observations were replicated in two additional, independent snRNA-seq data sets. These findings illustrate the power of multi-tissue molecular profiling to contextualize snRNA-seq brain transcriptomics and reveal disease biology., (© 2024. The Author(s).)

In recent years, the development of sensor and wearable technologies have led to their increased adoption in clinical and health monitoring settings. One area that is in early, but promising, stages of development is the use of biosensors for therapeutic drug monitoring (TDM). Traditionally, TDM could only be performed in certified laboratories and was used in specific scenarios to optimize drug dosage based on measurement of plasma/blood drug concentrations. Although TDM has been typically pursued in settings involving medications that are challenging to manage, the basic approach is useful for characterizing drug activity. TDM is based on the idea that there is likely a clear relationship between plasma/blood drug concentration (or concentration in other matrices) and clinical efficacy. However, these relationships may vary across individuals and may be affected by genetic factors, comorbidities, lifestyle, and diet. TDM technologies will be valuable for enabling precision medicine strategies to determine the clinical efficacy of drugs in individuals, as well as optimizing personalized dosing, especially since therapeutic windows may vary inter-individually. In this mini-review, we discuss emerging TDM technologies and their applications, and factors that influence TDM including drug interactions, polypharmacy, and supplement use. We also discuss how using TDM within single subject (N-of-1) and aggregated N-of-1 clinical trial designs provides opportunities to better capture drug response and activity at the individual level. Individualized TDM solutions have the potential to help optimize treatment selection and dosing regimens so that the right drug and right dose may be matched to the right person and in the right context., Competing Interests: Author WL was a paid consultant of Net/Bio, Inc. Authors LG, NS, and SS are founders of Net/Bio, Inc. with equity. Authors MS and BB-J are unpaid consultants., (Copyright © 2024 Liang, Beaulieu-Jones, Smalley, Snyder, Goetz and Schork.)

Purpose: Hyperthermic intraperitoneal chemotherapy (HIPEC) with cisplatin confers a survival benefit in epithelial ovarian cancer (EOC) but is associated with renal toxicity. Sodium thiosulfate (ST) is used for nephroprotection for HIPEC with cisplatin, but standard HIPEC practices vary., Methods: A prospective, nonrandomized, clinical trial evaluated safety outcomes of HIPEC with cisplatin 75 mg/m 2 during cytoreductive surgery (CRS) in patients with EOC (n = 34) and endometrial cancer (n = 6). Twenty-one patients received no ST (nST), and 19 received ST. Adverse events (AEs) were reported according to CTCAE v.5.0. Serum creatinine (Cr) was collected preoperatively and postoperatively (Days 5-8). Progression-free survival (PFS) was followed. Normal peritoneum was biopsied before and after HIPEC for whole transcriptomic sequencing to identify RNAseq signatures correlating with AEs., Results: Forty patients had HIPEC at the time of interval or secondary CRS. Renal toxicities in the nST group were 33% any grade AE and 9% grade 3 AEs. The ST group demonstrated no renal AEs. Median postoperative Cr in the nST group was 1.1 mg/dL and 0.5 mg/dL in the ST group (p = 0.0001). Median change in Cr from preoperative to postoperative levels were + 53% (nST) compared with - 9.6% (ST) (p = 0.003). PFS did not differ between the ST and nST groups in primary or recurrent EOC patients. Renal AEs were associated with downregulation of metabolic pathways and upregulation of immune pathways., Conclusions: ST significantly reduces acute renal toxicity associated with HIPEC with cisplatin in ovarian cancer patients. As nephrotoxicity is high in HIPEC with cisplatin, nephroprotective agents should be considered., (© 2023. The Author(s).)

Background: Small bowel carcinoids are insidious tumors that are often metastatic when diagnosed. Limited mutation landscape studies of carcinoids indicate that these tumors have a relatively low mutational burden. The development of targeted therapies will depend upon the identification of mutations that drive the pathogenesis and metastasis of carcinoid tumors. Methods: Whole exome and RNA sequencing of 5 matched sets of normal tissue, primary small intestine carcinoid tumors, and liver metastases were investigated. Germline and somatic variants included: single nucleotide variants (SNVs), insertions/deletions (indels), structural variants, and copy number alterations (CNAs). The functional impact of mutations was predicted using Ensembl Variant Effect Predictor. Results: Large-scale CNAs were observed including the loss of chromosome 18 in all 5 metastases and 3/5 primary tumors. Certain somatic SNVs were metastasis-specific; including mutations in ATRX , CDKN1B , MXRA5 (leading to the activation of a cryptic splice site and loss of mRNA), SMARCA2 , and the loss of UBE4B. Additional mutations in ATRX , and splice site loss of PYGL , leading to intron retention observed in primary and metastatic tumors. Conclusions: We observed novel mutations in primary/metastatic carcinoid tumor pairs, and some have been observed in other types of neuroendocrine tumors. We confirmed a previously observed loss of chromosome 18 and CDKN1B. Transcriptome sequencing added relevant information that would not have been appreciated with DNA sequencing alone. The detection of several splicing mutations on the DNA level and their consequences at the RNA level suggests that RNA splicing aberrations may be an important mechanism underlying carcinoid tumors., Competing Interests: Competing interests: Mackenzie Postel: There is no conflict of interest Sourat Darabi: Consulting and advisory work: Oncolens, Bayer, BostonGene James Howe: There is no conflict of interest Winnie Liang: Consultant for Net/Bio David W. Craig: There is no conflict of interest Michael J Demeure: Consulting or Advisory Role: Loxo/Lilly, Aadi, Orphagen Pharmaceuticals, Bayer, TD2, Theralink, OnCusp Therapeutics, Pfizer; Consultant Relationships: TransMed7, Boehringer-Ingelheim, (Copyright: © 2023 Postel MD et al.)

Ozone (O 3 ) is an air pollutant that primarily damages the lungs, but growing evidence supports the idea that O 3 also harms the brain; acute exposure to O 3 has been linked to central nervous system (CNS) symptoms such as depressed mood and sickness behaviors. However, the mechanisms by which O 3 inhalation causes neurobehavioral changes are limited. One hypothesis is that factors in the circulation bridge communication between the lungs and brain following O 3 exposure. In this study, our goals were to characterize neurobehavioral endpoints of O 3 exposure as they relate to markers of systemic and pulmonary inflammation, with a particular focus on serum amyloid A (SAA) and kynurenine as candidate mediators of O 3 behavioral effects. We evaluated O 3 -induced dose-, time- and sex-dependent changes in pulmonary inflammation, circulating SAA and kynurenine and its metabolic enzymes, and sickness and depressive-like behaviors in Balb/c and CD-1 mice. We found that 3 parts per million (ppm) O 3 , but not 2 or 1 ppm O 3 , increased circulating SAA and lung inflammation, which were resolved by 48 h and was worse in females. We also found that indoleamine 2,3-dioxygenase ( Ido1 ) mRNA expression was increased in the brain and spleen 24 h after 3 ppm O 3 and that kynurenine was increased in blood. Sickness and depressive-like behaviors were observed at all O 3 doses (1-3 ppm), suggesting that behavioral responses to O 3 can occur independently of increased SAA or neutrophils in the lungs. Using SAA knockout mice, we found that SAA did not contribute to O 3 -induced pulmonary damage or inflammation, systemic increases in kynurenine post-O 3 , or depressive-like behavior but did contribute to weight loss. Together, these findings indicate that acute O 3 exposure induces transient symptoms of sickness and depressive-like behaviors that may occur in the presence or absence of overt pulmonary neutrophilia and systemic increases of SAA. SAA does not appear to contribute to pulmonary inflammation induced by O 3 , although it may contribute to other aspects of sickness behavior, as reflected by a modest effect on weight loss.

Studies on humans that exploit contemporary data-intensive, high-throughput 'omic' assay technologies, such as genomics, transcriptomics, proteomics and metabolomics, have unequivocally revealed that humans differ greatly at the molecular level. These differences, which are compounded by each individual's distinct behavioral and environmental exposures, impact individual responses to health interventions such as diet and drugs. Questions about the best way to tailor health interventions to individuals based on their nuanced genomic, physiologic, behavioral, etc. profiles have motivated the current emphasis on 'precision' medicine. This review's purpose is to describe how the design and execution of N-of-1 (or personalized) multivariate clinical trials can advance the field. Such trials focus on individual responses to health interventions from a whole-person perspective, leverage emerging health monitoring technologies, and can be used to address the most relevant questions in the precision medicine era. This includes how to validate biomarkers that may indicate appropriate activity of an intervention as well as how to identify likely beneficial interventions for an individual. We also argue that multivariate N-of-1 and aggregated N-of-1 trials are ideal vehicles for advancing biomedical and translational science in the precision medicine era since the insights gained from them can not only shed light on how to treat or prevent diseases generally, but also provide insight into how to provide real-time care to the very individuals who are seeking attention for their health concerns in the first place., Competing Interests: Competing interest. N.J.S., S.S. and L.H.G. are founders of net. Bio, a company focusing on pursuing novel clinical protocols to ensure that individuals benefit from health interventions of all sorts. B.B.-J. and W.S.L. are paid consultants for net. Bio.

The development of effective treatments to prevent and slow Alzheimer's disease (AD) pathogenesis is needed in order to tackle the steady increase in the global prevalence of AD. This challenge is complicated by the need to identify key health shifts that precede the onset of AD and cognitive decline as these represent windows of opportunity for intervening and preventing disease. Such shifts may be captured through the measurement of biomarkers that reflect the health of the individual, in particular those that reflect brain age and biological age. Brain age biomarkers provide a composite view of the health of the brain based on neuroanatomical analyses, while biological age biomarkers, which encompass the epigenetic clock, provide a measurement of the overall health state of an individual based on DNA methylation analysis. Acceleration of brain and biological ages is associated with changes in cognitive function, as well as neuropathological markers of AD. In this mini-review, we discuss brain age and biological age research in the context of cognitive decline and AD. While more research is needed, studies show that brain and biological aging trajectories are variable across individuals and that such trajectories are non-linear at older ages. Longitudinal monitoring of these biomarkers may be valuable for enabling earlier identification of divergent pathological trajectories toward AD and providing insight into points for intervention., Competing Interests: Author WL was a paid consultant of NetBio, Inc. Authors LG and NS were founders of NetBio, Inc. with equity., (Copyright © 2022 Liang, Goetz and Schork.)

The posterior cingulate cortex, a key hub of the default mode network, underlies autobiographical memory retrieval and displays hypometabolic changes early in Alzheimer disease. To obtain an unbiased understanding of the molecular pathobiology of the aged posterior cingulate cortex, we performed RNA sequencing (RNA-seq) on tissue obtained from 26 participants of the Rush Religious Orders Study (11 males/15 females; aged 76-96 years) with a pre-mortem clinical diagnosis of no cognitive impairment and post-mortem neurofibrillary tangle Braak Stages I/II, III, and IV. Transcriptomic data were gathered using next-generation sequencing of RNA extracted from posterior cingulate cortex generating an average of 60 million paired reads per subject. Normalized expression of RNA-seq data was calculated using a global gene annotation and a microRNA profile. Differential expression (DESeq2, edgeR) using Braak staging as the comparison structure isolated genes for dimensional scaling, associative network building and functional clustering. Curated genes were correlated with the Mini-Mental State Examination and semantic, working and episodic memory, visuospatial ability, and a composite Global Cognitive Score. Regulatory mechanisms were determined by co-expression networks with microRNAs and an overlap of transcription factor binding sites. Analysis revealed 750 genes and 12 microRNAs significantly differentially expressed between Braak Stages I/II and III/IV and an associated six groups of transcription factor binding sites. Inputting significantly different gene/network data into a functional annotation clustering model revealed elevated presynaptic, postsynaptic and ATP-related expression in Braak Stages III and IV compared with Stages I/II, suggesting these pathways are integral for cognitive resilience seen in unimpaired elderly subjects. Principal component analysis and Kruskal-Wallis testing did not associate Braak stage with cognitive function. However, Spearman correlations between genes and cognitive test scores followed by network analysis revealed upregulation of classes of synaptic genes positively associated with performance on the visuospatial perceptual orientation domain. Upregulation of key synaptic genes suggests a role for these transcripts and associated synaptic pathways in cognitive resilience seen in elders despite Alzheimer disease pathology and dementia., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Purpose: Anesthetics are required for procedures that deliver drugs/biologics, infectious/inflammatory agents, and toxicants directly to the lungs. However, the possible confounding effects of anesthesia on lung inflammation and injury are underreported. Here, we evaluated the effects of two commonly used anesthetic regimens on lung inflammatory responses to ozone in mice., Methods: We tested the effects of brief isoflurane (Iso) or ketamine/xylazine/atipamezole (K/X/A) anesthesia prior to ozone exposure (4 h, 3 ppm) on lung inflammatory responses in mice. Anesthesia regimens modeled those used for non-surgical intratracheal instillations and were administered 1-2 h or 24 h prior to initiating ozone exposure., Results: We found that Iso given 1-2 h prior to ozone inhibited inflammatory responses in the lung, and this effect was absent when Iso was given 23-24 h prior to ozone. In contrast, K/X/A given 1-2 h prior to ozone increased lung and systemic inflammation., Conclusion: Our results highlight the need to comprehensively evaluate anesthesia as an experimental variable in the assessment of lung inflammation in response to ozone and other inflammatory stimuli., (© 2022. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Purpose: Hyperthermic intraperitoneal chemotherapy (HIPEC) confers a survival benefit in epithelial ovarian cancer (EOC) and in preclinical models. However, the molecular changes induced by HIPEC have not been corroborated in humans., Patients and Methods: A feasibility trial evaluated clinical and safety outcomes of HIPEC with cisplatin during optimal cytoreductive surgery (CRS) in patients with EOC diagnosed with stage III, IV, or recurrent EOC. Pre- and post-HIPEC biopsies were comprehensively profiled with genomic and transcriptomic sequencing to identify mutational and RNAseq signatures correlating with response; the tumor microenvironment was profiled to identify potential immune biomarkers; and transcriptional signatures of tumors and normal samples before and after HIPEC were compared to investigate HIPEC-induced acute transcriptional changes., Results: Thirty-five patients had HIPEC at the time of optimal CRS; all patients had optimal CRS. The median progression-free survival (PFS) was 24.7 months for primary patients and 22.4 for recurrent patients. There were no grade 4 or 5 adverse events. Anemia was the most common grade 3 adverse event (43%). Hierarchical cluster analyses identified distinct transcriptomic signatures of good versus poor responders to HIPEC correlating with a PFS of 29.9 versus 7.3 months, respectively. Among good responders, significant HIPEC-induced molecular changes included immune pathway upregulation and DNA repair pathway downregulation. Within cancer islands, % programmed cell death protein 1 expression in CD8+ T cells significantly increased after HIPEC. An exceptional responder (PFS 58 months) demonstrated the highest programmed cell death protein 1 increase. Heat shock proteins comprised the top differentially upregulated genes in HIPEC-treated tumors., Conclusion: Distinct transcriptomic signatures identify responders to HIPEC, and preclinical model findings are confirmed for the first time in a human cohort., Competing Interests: Ernest S. HanResearch Funding: Vergent Bioscience (Inst) Mustafa RaoofOpen Payments Link: https://openpaymentsdata.cms.gov/physician/3634542 Saul J. PricemanStock and Other Ownership Interests: Imugene Ltd, Adicet BioConsulting or Advisory Role: Imugene Ltd, Adicet Bio, MustangBio, BayerResearch Funding: Imugene Ltd, Carisma TherapeuticsPatents, Royalties, Other Intellectual Property: CAR Therapeutic, Oncolytic Virus Therapeutic Jeff F. LinHonoraria: C-SATSConsulting or Advisory Role: Aspira Women's HealthTravel, Accommodations, Expenses: Intuitive Surgical, GlaxoSmithKline Mehdi KebriaHonoraria: GlaxoSmithKlineSpeakers' Bureau: GlaxoSmithKline Nora RuelConsulting or Advisory Role: EPHOS Bioscience Benjamin I. PazEmployment: City of HopeStock and Other Ownership Interests: Bio-Path Holdings Inc Mark T. WakabayashiEmployment: RegeneronStock and Other Ownership Interests: Regeneron Mihaela C. CristeaHonoraria: AstraZeneca, AbbVieConsulting or Advisory Role: AstraZeneca, AbbVieSpeakers' Bureau: AstraZenecaNo other potential conflicts of interest were reported.

The RNA-binding protein HuD (a.k.a., ELAVL4) is involved in neuronal development and synaptic plasticity mechanisms, including addiction-related processes such as cocaine conditioned-place preference (CPP) and food reward. The most studied function of this protein is mRNA stabilization; however, we have recently shown that HuD also regulates the levels of circular RNAs (circRNAs) in neurons. To examine the role of HuD in the control of coding and non-coding RNA networks associated with substance use, we identified sets of differentially expressed mRNAs, circRNAs and miRNAs in the striatum of HuD knockout (KO) mice. Our findings indicate that significantly downregulated mRNAs are enriched in biological pathways related to cell morphology and behavior. Furthermore, deletion of HuD altered the levels of 15 miRNAs associated with drug seeking. Using these sets of data, we predicted that a large number of upregulated miRNAs form competing endogenous RNA (ceRNA) networks with circRNAs and mRNAs associated with the neuronal development and synaptic plasticity proteins LSAMP and MARK3. Additionally, several downregulated miRNAs form ceRNA networks with mRNAs and circRNAs from MEF2D, PIK3R3, PTRPM and other neuronal proteins. Together, our results indicate that HuD regulates ceRNA networks controlling the levels of mRNAs associated with neuronal differentiation and synaptic physiology.

The lack of samples for generating standardized DNA datasets for setting up a sequencing pipeline or benchmarking the performance of different algorithms limits the implementation and uptake of cancer genomics. Here, we describe reference call sets obtained from paired tumor-normal genomic DNA (gDNA) samples derived from a breast cancer cell line-which is highly heterogeneous, with an aneuploid genome, and enriched in somatic alterations-and a matched lymphoblastoid cell line. We partially validated both somatic mutations and germline variants in these call sets via whole-exome sequencing (WES) with different sequencing platforms and targeted sequencing with >2,000-fold coverage, spanning 82% of genomic regions with high confidence. Although the gDNA reference samples are not representative of primary cancer cells from a clinical sample, when setting up a sequencing pipeline, they not only minimize potential biases from technologies, assays and informatics but also provide a unique resource for benchmarking 'tumor-only' or 'matched tumor-normal' analyses., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

The robust detection of disease-associated splice events from RNAseq data is challenging due to the potential confounding effect of gene expression levels and the often limited number of patients with relevant RNAseq data. Here we present a novel statistical approach to splicing outlier detection and differential splicing analysis. Our approach tests for differences in the percentages of sequence reads representing local splice events. We describe a software package called Bisbee which can predict the protein-level effect of splice alterations, a key feature lacking in many other splicing analysis resources. We leverage Bisbee's prediction of protein level effects as a benchmark of its capabilities using matched sets of RNAseq and mass spectrometry data from normal tissues. Bisbee exhibits improved sensitivity and specificity over existing approaches and can be used to identify tissue-specific splice variants whose protein-level expression can be confirmed by mass spectrometry. We also applied Bisbee to assess evidence for a pathogenic splicing variant contributing to a rare disease and to identify tumor-specific splice isoforms associated with an oncogenic mutation. Bisbee was able to rediscover previously validated results in both of these cases and also identify common tumor-associated splice isoforms replicated in two independent melanoma datasets.

Although combination BRAF and MEK inhibitors are highly effective for the 40-50% of cutaneous metastatic melanomas harboring BRAFV600 mutations, targeted agents have been ineffective for BRAFV600wild-type (wt) metastatic melanomas. The SU2C Genomics-Enabled Medicine for Melanoma Trial utilized a Simon two-stage optimal design to assess whether comprehensive genomic profiling improves selection of molecular-based therapies for BRAFV600wt metastatic melanoma patients who had progressed on standard-of-care therapy, which may include immunotherapy. Of the response-evaluable patients, binimetinib was selected for 20 patients randomized to the genomics-enabled arm, and nine were treated on the alternate treatment arm. Response rates for 27 patients treated with targeted recommendations included one (4%) partial response, 18 (67%) with stable disease, and eight (30%) with progressive disease. Post-trial genomic and protein pathway activation mapping identified additional drug classes that may be considered for future studies. Our results highlight the complexity and heterogeneity of metastatic melanomas, as well as how the lack of response in this trial may be associated with limitations including monotherapy drug selection and the dearth of available single and combination molecularly-driven therapies to treat BRAFV600wt metastatic melanomas., Competing Interests: These commercial affiliations do not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Vascular complications at the puncture site is a common complication after femoral artery puncture. It will not only affect the postoperative effect and patient comfort, but also may endanger the life of the patient. The effective compression hemostasis methods at the puncture site can improve the comfort of the patient, shorten the hospital stay, and reduce the burden on the medical staff. The purpose of this research is to evaluate the effectiveness and safety of different compression methods after femoral artery puncture., Methods: We will include all relevant randomized controlled trials by searching major Chinese and English databases and clinical trial registration platforms. Use Cochrane Collaboration's Risk of bias tool for bias risk analysis. Use the Grades of Recommendation, Assessment, Development, and Evaluation to assess the quality of evidence. Data analysis will be performed using Stata (V.15.0) and WinBUGS (V.1.4.3)., Results: Five hundred ninety-seven records were obtained by searching the database but no records were obtained by other means. After removing duplicate records, 377 records remain. We excluded 103 records through abstract and title, leaving 274 full-text articles., Conclusion: This study will compare the application effects of different compression methods after femoral artery puncture. We hope that this study will help guide clinical decision-making and provide evidence for the management of patients after femoral artery puncture., Protocol Registration Number: INPLASY2020120094., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Emergence and intercontinental spread of highly pathogenic avian influenza A (HPAI) H5Nx virus clade 2.3.4.4 has resulted in substantial economic losses to the poultry industry in Asia, Europe, and North America. The long-distance migratory birds have been suggested to play a major role in the global spread of avian influenza viruses during this wave of panzootic outbreaks since 2013. Poultry farm epidemics caused by multiple introduction of different HPAI novel subtypes of clade 2.3.4.4 viruses also occurred in Taiwan between 2015 and 2017. The mandatory and active surveillance detected H5N3 and H5N6 circulation in 2015 and 2017, respectively, while H5N2 and H5N8 were persistently identified in poultry farms since their first arrival in 2015. This study intended to assess the importance of various ecological factors contributed to the persistence of HPAI during three consecutive years. We used satellite technology to identify the location of waterfowl flocks. Four risk factors consistently showed strong association with the spatial clustering of H5N2 and H5N8 circulations during 2015 and 2017, including high poultry farm density (aOR:17.46, 95%CI: 5.91-74.86 and 8.23, 95% CI: 2.12-54.86 in 2015 and 2017, respectively), poultry heterogeneity index (aOR of 12.28, 95%CI: 5.02-31.14 and 2.79, 95%CI: 1.00-7.69, in 2015 and 2017, respectively), non-registered waterfowl flock density (aOR: 6.8, 95%CI: 3.41-14.46 and 9.17, 95%CI: 3.73-26.20, in 2015 and 2017, respectively) and higher percentage of cropping land coverage (aOR of 1.36, 95%CI: 1.10-1.69 and 1.04, 95%CI: 1.02-1.07, in 2015 and 2017, respectively). Our study highlights the application of remote sensing and clustering analysis for the identification and characterization of environmental factors in facilitating and contributing to the persistent circulation of certain subtypes of H5Nx in poultry farms in Taiwan., Competing Interests: The authors have declared that no competing interests exist.

Circular RNAs (circRNAs) are evolutionarily conserved RNA species that are formed when exons "back-splice" to each other. Current computational algorithms to detect these back-splicing junctions produce divergent results, and hence there is a need for a method to distinguish true-positive circRNAs. To this end, we developed Assembly based CircRNA Validator (ACValidator) for in silico verification of circRNAs. ACValidator extracts reads from a user-defined window on either side of a circRNA junction and assembles them to generate contigs. These contigs are aligned against the circRNA sequence to find contigs spanning the back-spliced junction. When evaluated on simulated datasets, ACValidator achieved over ∼80% sensitivity on datasets with an average of 10 circRNA-supporting reads and with read lengths of at least 100 bp. In experimental datasets, ACValidator produced higher verification percentages for samples treated with ribonuclease R compared to nontreated samples. Our workflow is applicable to non-polyA-selected RNAseq datasets and can also be used as a candidate selection strategy for prioritizing experimental validations. All workflow scripts are freely accessible on our GitHub page https://github.com/tgen/ACValidator along with detailed instructions to set up and run ACValidator., (© The Author(s) 2020. Published by Oxford University Press.)

Background: Tumor heterogeneity underlies resistance and disease progression in glioblastoma (GBM), and tumors most commonly recur adjacent to the surgical resection margins in contrast non-enhancing (NE) regions. To date, no targeted therapies have meaningfully altered overall patient survival in the up-front setting. The aim of this study was to characterize intratumoral heterogeneity in recurrent GBM using bulk samples from primary resection and recurrent samples taken from contrast-enhancing (EN) and contrast NE regions., Methods: Whole exome and RNA sequencing were performed on matched bulk primary and multiple recurrent EN and NE tumor samples from 16 GBM patients who received standard of care treatment alone or in combination with investigational clinical trial regimens., Results: Private mutations emerge across multi-region sampling in recurrent tumors. Genomic clonal analysis revealed increased enrichment in gene alterations regulating the G2M checkpoint, Kras signaling, Wnt signaling, and DNA repair in recurrent disease. Subsequent functional studies identified augmented PI3K/AKT transcriptional and protein activity throughout progression, validated by phospho-protein levels. Moreover, a mesenchymal transcriptional signature was observed in recurrent EN regions, which differed from the proneural signature in recurrent NE regions., Conclusions: Subclonal populations observed within bulk resected primary GBMs transcriptionally evolve across tumor recurrence (EN and NE regions) and exhibit aberrant gene expression of common signaling pathways that persist despite standard or targeted therapy. Our findings provide evidence that there are both adaptive and clonally mediated dependencies of GBM on key pathways, such as the PI3K/AKT axis, for survival across recurrences., (© The Author(s) 2020. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Background: Molecular analysis has revealed four subtypes of pancreatic ductal adenocarcinoma (PDAC). One subtype identified for the presence of DNA damage repair deficiency can be targeted therapeutically with the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib. We performed a single institution retrospective analysis of treatment response in patients with PDAC treated with olaparib who have DNA damage repair deficiency mutations., Subjects, Materials, and Methods: Patients with germline or somatic mutations involving the DNA repair pathway were identified and treated with olaparib. The primary objective was to examine the objective response rate (ORR). The secondary objectives were assessing tolerability, overall survival, and change in cancer antigen 19-9. Quantitative texture analysis (QTA) was evaluated from CT scans to explore imaging biomarkers., Results: Thirteen individuals with metastatic PDAC were treated with Olaparib. The ORR to Olaparib was 23%. Median overall survival (OS) was 16.47 months. Four of seven patients with BRCA mutations had an effect on RAD51 binding, with a median OS of 24.60 months. Exploratory analysis of index lesions using QTA revealed correlations between lesion texture and OS (hepatic lesion tumor texture correlation coefficient [CC], 0.683, p = .042) and time on olaparib (primary pancreatic lesion tumor texture CC, 0.778, p = .023)., Conclusion: In individuals with metastatic PDAC who have mutations involved in DNA repair, Olaparib may provide clinical benefit. BRCA mutations affecting RAD51 binding domains translated to improved median OS. QTA of individual tumors may allow for additional information that predicts outcomes to treatment with PARP inhibitors., Implications for Practice: Pursuing germline and somatic DNA sequencing in individuals with pancreatic ductal adenocarcinoma may yield abnormalities in DNA repair pathways. These individuals may receive benefit with poly (ADP-ribose) polymerase (PARP) inhibition. Radiomics and deep sequencing analysis may yet uncover additional information that may predict outcome to treatment with PARP inhibitors., (© AlphaMed Press 2019.)

Glioma is recognized to be a highly heterogeneous CNS malignancy, whose diverse cellular composition and cellular interactions have not been well characterized. To gain new clinical- and biological-insights into the genetically-bifurcated IDH1 mutant (mt) vs wildtype (wt) forms of glioma, we integrated data from protein, genomic and MR imaging from 20 treatment-naïve glioma cases and 16 recurrent GBM cases. Multiplexed immunofluorescence (MxIF) was used to generate single cell data for 43 protein markers representing all cancer hallmarks, Genomic sequencing (exome and RNA (normal and tumor) and magnetic resonance imaging (MRI) quantitative features (protocols were T1-post, FLAIR and ADC) from whole tumor, peritumoral edema and enhancing core vs equivalent normal region were also collected from patients. Based on MxIF analysis, 85,767 cells (glioma cases) and 56,304 cells (GBM cases) were used to generate cell-level data for 24 biomarkers. K-means clustering was used to generate 7 distinct groups of cells with divergent biomarker profiles and deconvolution was used to assign RNA data into three classes. Spatial and molecular heterogeneity metrics were generated for the cell data. All features were compared between IDH mt and IDHwt patients and were finally combined to provide a holistic/integrated comparison. Protein expression by hallmark was generally lower in the IDHmt vs wt patients. Molecular and spatial heterogeneity scores for angiogenesis and cell invasion also differed between IDHmt and wt gliomas irrespective of prior treatment and tumor grade; these differences also persisted in the MR imaging features of peritumoral edema and contrast enhancement volumes. A coherent picture of enhanced angiogenesis in IDHwt tumors was derived from multiple platforms (genomic, proteomic and imaging) and scales from individual proteins to cell clusters and heterogeneity, as well as bulk tumor RNA and imaging features. Longer overall survival for IDH1mt glioma patients may reflect mutation-driven alterations in cellular, molecular, and spatial heterogeneity which manifest in discernable radiological manifestations., Competing Interests: The authors have declared that no competing interests exist.

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein interactions, and regulation of parental gene transcription. In classical next generation RNA sequencing (RNA-seq), circRNAs are typically overlooked as a result of poly-A selection during construction of mRNA libraries, or are found at very low abundance, and are therefore difficult to isolate and detect. Here, a circRNA library construction protocol was optimized by comparing library preparation kits, pre-treatment options and various total RNA input amounts. Two commercially available whole transcriptome library preparation kits, with and without RNase R pre-treatment, and using variable amounts of total RNA input (1 to 4 µg), were tested. Lastly, multiple tissue types; including liver, lung, lymph node, and pancreas; as well as multiple brain regions; including the cerebellum, inferior parietal lobe, middle temporal gyrus, occipital cortex, and superior frontal gyrus; were compared to evaluate circRNA abundance across tissue types. Analysis of the generated RNA-seq data using six different circRNA detection tools (find_circ, CIRI, Mapsplice, KNIFE, DCC, and CIRCexplorer) revealed that a stranded total RNA library preparation kit with RNase R pre-treatment and 4 µg RNA input is the optimal method for identifying the highest relative number of circRNAs. Consistent with previous findings, the highest enrichment of circRNAs was observed in brain tissues compared to other tissue types.

Importance: Pediatric cancers are epigenetic diseases; therefore, considering tumor gene expression information is necessary for a complete understanding of the tumorigenic processes., Objective: To evaluate the feasibility and utility of incorporating comparative gene expression information into the precision medicine framework for difficult-to-treat pediatric and young adult patients with cancer., Design, Setting, and Participants: This cohort study was conducted as a consortium between the University of California, Santa Cruz (UCSC) Treehouse Childhood Cancer Initiative and clinical genomic trials. RNA sequencing (RNA-Seq) data were obtained from the following 4 clinical sites and analyzed at UCSC: British Columbia Children's Hospital (n = 31), Lucile Packard Children's Hospital at Stanford University (n = 80), CHOC Children's Hospital and Hyundai Cancer Institute (n = 46), and the Pacific Pediatric Neuro-Oncology Consortium (n = 24). The study dates were January 1, 2016, to March 22, 2017., Exposures: Participants underwent tumor RNA-Seq profiling as part of 4 separate clinical trials at partner hospitals. The UCSC either downloaded RNA-Seq data from a partner institution for analysis in the cloud or provided a Docker pipeline that performed the same analysis at a partner institution. The UCSC then compared each participant's tumor RNA-Seq profile with more than 11 000 uniformly analyzed tumor profiles from pediatric and young adult patients with cancer, downloaded from public data repositories. These comparisons were used to identify genes and pathways that are significantly overexpressed in each patient's tumor. Results of the UCSC analysis were presented to clinical partners., Main Outcomes and Measures: Feasibility of a third-party institution (UCSC Treehouse Childhood Cancer Initiative) to obtain tumor RNA-Seq data from patients, conduct comparative analysis, and present analysis results to clinicians; and proportion of patients for whom comparative tumor gene expression analysis provided useful clinical and biological information., Results: Among 144 samples from children and young adults (median age at diagnosis, 9 years; range, 0-26 years; 72 of 118 [61.0%] male [26 patients sex unknown]) with a relapsed, refractory, or rare cancer treated on precision medicine protocols, RNA-Seq-derived gene expression was potentially useful for 99 of 144 samples (68.8%) compared with DNA mutation information that was potentially useful for only 34 of 74 samples (45.9%)., Conclusions and Relevance: This study's findings suggest that tumor RNA-Seq comparisons may be feasible and highlight the potential clinical utility of incorporating such comparisons into the clinical genomic interpretation framework for difficult-to-treat pediatric and young adult patients with cancer. The study also highlights for the first time to date the potential clinical utility of harmonized publicly available genomic data sets.

Purpose: Naturally occurring primary canine lung cancers share clinicopathologic features with human lung cancers in never-smokers, but the genetic underpinnings of canine lung cancer are unknown. We have charted the genomic landscape of canine lung cancer and performed functional characterization of novel, recurrent HER2 (ERBB2) mutations occurring in canine pulmonary adenocarcinoma (cPAC)., Experimental Design: We performed multiplatform genomic sequencing of 88 primary canine lung tumors or cell lines. Additionally, in cPAC cell lines, we performed functional characterization of HER2 signaling and evaluated mutation-dependent HER2 inhibitor drug dose-response., Results: We discovered somatic, coding HER2 point mutations in 38% of cPACs (28/74), but none in adenosquamous (cPASC, 0/11) or squamous cell (cPSCC, 0/3) carcinomas. The majority (93%) of HER2 mutations were hotspot V659E transmembrane domain (TMD) mutations comparable to activating mutations at this same site in human cancer. Other HER2 mutations were located in the extracellular domain and TMD. HER2 V659E was detected in the plasma of 33% (2/6) of dogs with localized HER2 V659E tumors. HER2 V659E cPAC cell lines displayed constitutive phosphorylation of AKT and significantly higher sensitivity to the HER2 inhibitors lapatinib and neratinib relative to HER2 -wild-type cell lines (IC 50 < 200 nmol/L in HER2 V659E vs. IC 50 > 2,500 nmol/L in HER2 WT )., Conclusions: This study creates a foundation for molecular understanding of and drug development for canine lung cancer. These data also establish molecular contexts for comparative studies in dogs and humans of low mutation burden, never-smoker lung cancer, and mutant HER2 function and inhibition., (©2019 American Association for Cancer Research.)

This clinical trial evaluated whether whole exome sequencing (WES) and RNA sequencing (RNAseq) of paired normal and tumor tissues could be incorporated into a personalized treatment plan for newly diagnosed patients (<25 years of age) with diffuse intrinsic pontine glioma (DIPG). Additionally, whole genome sequencing (WGS) was compared to WES to determine if WGS would further inform treatment decisions, and whether circulating tumor DNA (ctDNA) could detect the H3K27M mutation to allow assessment of therapy response. Patients were selected across three Pacific Pediatric Neuro-Oncology Consortium member institutions between September 2014 and January 2016. WES and RNAseq were performed at diagnosis and recurrence when possible in a CLIA-certified laboratory. Patient-derived cell line development was attempted for each subject. Collection of blood for ctDNA was done prior to treatment and with each MRI. A specialized tumor board generated a treatment recommendation including up to four FDA-approved agents based upon the genomic alterations detected. A treatment plan was successfully issued within 21 business days from tissue collection for all 15 subjects, with 14 of the 15 subjects fulfilling the feasibility criteria. WGS results did not significantly deviate from WES-based therapy recommendations; however, WGS data provided further insight into tumor evolution and fidelity of patient-derived cell models. Detection of the H3F3A or HIST1H3B K27M (H3K27M) mutation using ctDNA was successful in 92% of H3K27M mutant cases. A personalized treatment recommendation for DIPG can be rendered within a multicenter setting using comprehensive next-generation sequencing technology in a clinically relevant timeframe., (© 2019 UICC.)

Malignant melanoma (MM) exhibits a high propensity for central nervous system dissemination with ~50% of metastatic MM patients developing brain metastases (BM). Targeted therapies and immune checkpoint inhibitors have improved overall survival for MM patients with BM. However, responses are usually of short duration and new agents that effectively penetrate the blood brain barrier (BBB) are needed. Here, we report a MM patient with BM who experienced an exceptional response to E6201, an ATP-competitive MEK1 inhibitor, on a Phase 1 study, with ongoing near-complete response and overall survival extending beyond 8 years. Whole exome and transcriptome sequencing revealed a high mutational burden tumor (22 mutations/Megabase) with homozygous BRAF V600E mutation. Correlative preclinical studies demonstrated broad activity for E6201 across BRAF V600E mutant melanoma cell lines and effective BBB penetration in vivo. Together, these results suggest that E6201 may represent a potential new treatment option for BRAF-mutant MM patients with BM.

Oncoviral infection is responsible for 12%-15% of cancer in humans. Convergent evidence from epidemiology, pathology, and oncology suggests that new viral etiologies for cancers remain to be discovered. Oncoviral profiles can be obtained from cancer genome sequencing data; however, widespread viral sequence contamination and noncausal viruses complicate the process of identifying genuine oncoviruses. Here, we propose a novel strategy to address these challenges by performing virome-wide screening of early-stage clonal viral integrations. To implement this strategy, we developed VIcaller, a novel platform for identifying viral integrations that are derived from any characterized viruses and shared by a large proportion of tumor cells using whole-genome sequencing (WGS) data. The sensitivity and precision were confirmed with simulated and benchmark cancer data sets. By applying this platform to cancer WGS data sets with proven or speculated viral etiology, we newly identified or confirmed clonal integrations of hepatitis B virus (HBV), human papillomavirus (HPV), Epstein-Barr virus (EBV), and BK Virus (BKV), suggesting the involvement of these viruses in early stages of tumorigenesis in affected tumors, such as HBV in TERT and KMT2B (also known as MLL4 ) gene loci in liver cancer, HPV and BKV in bladder cancer, and EBV in non-Hodgkin's lymphoma. We also showed the capacity of VIcaller to identify integrations from some uncharacterized viruses. This is the first study to systematically investigate the strategy and method of virome-wide screening of clonal integrations to identify oncoviruses. Searching clonal viral integrations with our platform has the capacity to identify virus-caused cancers and discover cancer viral etiologies., (© 2019 Chen et al.; Published by Cold Spring Harbor Laboratory Press.)

Archival tumor samples represent a rich resource of annotated specimens for translational genomics research. However, standard variant calling approaches require a matched normal sample from the same individual, which is often not available in the retrospective setting, making it difficult to distinguish between true somatic variants and individual-specific germline variants. Archival sections often contain adjacent normal tissue, but this tissue can include infiltrating tumor cells. As existing comparative somatic variant callers are designed to exclude variants present in the normal sample, a novel approach is required to leverage adjacent normal tissue with infiltrating tumor cells for somatic variant calling. Here we present lumosVar 2.0, a software package designed to jointly analyze multiple samples from the same patient, built upon our previous single sample tumor only variant caller lumosVar 1.0. The approach assumes that the allelic fraction of somatic variants and germline variants follow different patterns as tumor content and copy number state change. lumosVar 2.0 estimates allele specific copy number and tumor sample fractions from the data, and uses a to model to determine expected allelic fractions for somatic and germline variants and to classify variants accordingly. To evaluate the utility of lumosVar 2.0 to jointly call somatic variants with tumor and adjacent normal samples, we used a glioblastoma dataset with matched high and low tumor content and germline whole exome sequencing data (for true somatic variants) available for each patient. Both sensitivity and positive predictive value were improved when analyzing the high tumor and low tumor samples jointly compared to analyzing the samples individually or in-silico pooling of the two samples. Finally, we applied this approach to a set of breast and prostate archival tumor samples for which tumor blocks containing adjacent normal tissue were available for sequencing. Joint analysis using lumosVar 2.0 detected several variants, including known cancer hotspot mutations that were not detected by standard somatic variant calling tools using the adjacent tissue as presumed normal reference. Together, these results demonstrate the utility of leveraging paired tissue samples to improve somatic variant calling when a constitutional sample is not available.

Background: The genomic landscape of Hodgkin lymphoma (HL) has been difficult to characterize due to the paucity of neoplastic cells and an abundant microenvironment. Such characterization is needed in order to improve treatment strategies., Materials and Methods: We performed comprehensive genomic profiling (CGP) using targeted next-generation sequencing on archival formalin-fixed paraffin embedded tumor samples from 63 patients to analyze the landscape of HL., Results: CGP was successful for 49/63 archival specimens (78%), and revealed aberrations impacting genes including B2M , TP53 , and XPO1 (E571). Of the 34 patients for whom total mutation burden (TMB; mutations/megabase [Mb]) was assessed, 5 (15%) had high TMB (≥20 mutations/Mb), 18 (53%) had intermediate TMB (6-19 mutations/Mb), and 11 (32%) had low TMB (≤5 mutations/Mb). We next tested 13 patients' plasma cell-free DNA with droplet digital polymerase chain reaction for the presence of XPO1 E571 mutation, which was confirmed in the plasma of 31% of patients. In three patients with serially collected plasma samples, XPO1 E571K allelic frequency changes corresponded with changes in tumor size on conventional radiographic imaging., Conclusion: The study demonstrates that comprehensive genomic profiling of archival Hodgkin lymphoma tumor samples is feasible and leads to the identification of genes that are recurrently mutated and that Hodgkin lymphoma has increased mutation burden in the majority of samples analyzed. Furthermore, tracking of XPO1 E571 mutant allele frequency in a subset of patients may also represent a potential disease-monitoring strategy and warrants further investigation., Implications for Practice: This study provides the first evidence that comprehensive genomic profiling can be performed to map the genomic landscape of Hodgkin lymphoma and that a subpopulation of patients has mutations in TP53 , B2M , XPO1 , and other genes. It was found that 15% of patients have high mutation burden, which, in cancers such as melanoma, may indicate sensitivity to immune checkpoint inhibitors, and may thus be explored for Hodgkin lymphoma. Lastly, this work demonstrates that changes in the mutant allele frequency of XPO1 in serially collected plasma cell-free DNA samples correspond with treatment outcomes measured with conventional radiographic imaging., Competing Interests: Disclosures of potential conflicts of interest may be found at the end of this article., (© AlphaMed Press 2018.)

Within the last decade, active research on circular RNAs (circRNAs) has dramatically improved our understanding of the expression and function of these non-coding RNAs. While several mechanisms for circRNA function have been proposed, including sequestration of microRNAs and regulation of cellular proteins, studies provide evidence that circRNAs can regulate transcription and may also serve as biomarkers. Due to the heterogeneous nature of the brain, and the dynamic transcriptional mechanisms that support neurobiological pathways, the influence of circRNAs is potentially extensive. Understanding how circRNAs contribute to key neurological pathways will fill gaps in our understanding of brain function and provide valuable insight into novel therapeutic approaches to treat neurological diseases. Here, we review recent research on circRNA expression in the brain, describe the proposed functions of circRNAs, and evaluate the role of circRNAs in neurological diseases.

Chordoma is a rare, orphan cancer arising from embryonal precursors of bone. Surgery and radiotherapy (RT) provide excellent local control, often at the price of significant morbidity because of the structures involved and the need for relatively high doses of RT; however, recurrence remains high. Although our understanding of the genetic changes that occur in chordoma is evolving rapidly, this knowledge has yet to translate into treatments. We performed comprehensive DNA (paired tumor/normal whole-exome and shallow whole-genome) and RNA (tumor whole-transcriptome) next-generation sequencing analyses of archival sacral and clivus chordoma specimens. Incorporation of transcriptomic data enabled the identification of gene overexpression and expressed DNA alterations, thus providing additional support for potential therapeutic targets. In three patients, we identified alterations that may be amenable to off-label FDA-approved treatments for other tumor types. These alterations include FGFR1 overexpression (ponatinib, pazopanib) and copy-number duplication of CDK4 (palbociclib) and ERBB3 (gefitinib). In a third patient, germline DNA demonstrated predicted pathogenic changes in CHEK2 and ATM , which may have predisposed the patient to developing chordoma at a young age and may also be associated with potential sensitivity to PARP inhibitors because of homologous recombination repair deficiency. Last, in the fourth patient, a missense mutation in IGF1R was identified, suggesting potential activity for investigational anti-IGF1R strategies. Our findings demonstrate that chordoma patients present with aberrations in overlapping pathways. These results provide support for targeting the IGF1R/FGFR/EGFR and CDK4/6 pathways as treatment strategies for chordoma patients. This study underscores the value of comprehensive genomic and transcriptomic analysis in the development of rational, individualized treatment plans for chordoma., (© 2018 Liang et al.; Published by Cold Spring Harbor Laboratory Press.)

Purpose: Pediatric diffuse midline glioma (DMG) are highly malignant tumors with poor clinical outcomes. Over 70% of patients with DMG harbor the histone 3 p.K27M (H3K27M) mutation, which correlates with a poorer clinical outcome, and is also used as a criterion for enrollment in clinical trials. Because complete surgical resection of DMG is not an option, biopsy at presentation is feasible, but rebiopsy at time of progression is rare. While imaging and clinical-based disease monitoring is the standard of care, molecular-based longitudinal characterization of these tumors is almost nonexistent. To overcome these hurdles, we examined whether liquid biopsy allows measurement of disease response to precision therapy., Experimental Design: We established a sensitive and specific methodology that detects major driver mutations associated with pediatric DMGs using droplet digital PCR ( n = 48 subjects, n = 110 specimens). Quantification of circulating tumor DNA (ctDNA) for H3K27M was used for longitudinal assessment of disease response compared with centrally reviewed MRI data., Results: H3K27M was identified in cerebrospinal fluid (CSF) and plasma in 88% of patients with DMG, with CSF being the most enriched for ctDNA. We demonstrated the feasibility of multiplexing for detection of H3K27M, and additional driver mutations in patient's tumor and matched CSF, maximizing the utility of a single source of liquid biome. A significant decrease in H3K27M plasma ctDNA agreed with MRI assessment of tumor response to radiotherapy in 83% (10/12) of patients., Conclusions: Our liquid biopsy approach provides a molecularly based tool for tumor characterization, and is the first to indicate clinical utility of ctDNA for longitudinal surveillance of DMGs., (©2018 American Association for Cancer Research.)

Objectives: To analyze the potential influences of propofol on the oxidative stress of H 2 O 2 -induced human esophageal squamous cell carcinoma (ESCC) Eca109 cell through mediating the Wnt/β-catenin signaling pathway., Materials and Methods: Eca109 cells were classified into 5 groups: Control group, H 2 O 2 group, Propofol + H 2 O 2 group, Dkk1 (Dickkopf-1, Wnt/β-catenin pathway antagonist) + H 2 O 2 group, and Propofol + LiCl (Lithium chloride, Wnt/β-catenin pathway agonist) + H 2 O 2 group. Western blotting was performed to determine the protein expressions, flow cytometry to measure the content of ROS, immunofluorescence staining to detect the oxidative DNA damage, as well as MTT, AnnexinV-FITC/PI, Wound-healing, and Transwell assays to test the biological characteristics of Eca109 cells., Results: H 2 O 2 resulted in the increased nuclear and cytoplasmatic expression of β-catenin, reduced p-GSK3β expression, up-regulated ROS content, and induced oxidative DNA damage in Eca109 cells. Moreover, Eca109 cells treated with H 2 O 2 alone had enhanced cell proliferation and metastasis but decreased cell apoptosis, as compared with those without any treatment; meanwhile, the declined Cyt C, Bax, and cleaved caspase-3, as well as the elevated Bcl-2 were also observed in Eca109 cells in the H 2 O 2 group, which were reversed by Propofol or Dkk1. Moreover, Propofol could inhibit the effect of LiCl on activating the Wnt/β-catenin signaling pathway in H 2 O 2 -induced Eca109 cells., Conclusion: Propofol elicits protective effects to inhibit H 2 O 2 -induced proliferation and metastasis and promote apoptosis of Eca109 cells via blocking the Wnt/β-catenin pathway, offering a possible therapeutic modality for ESCC., Competing Interests: The authors declare that there are no conflicts of interest.

Increasing evidence indicates that sirtuin 3 (Sirt3) has neuroprotective effects in regulating oxidative stress and energy metabolism, both of which are involved in the pathogenesis of Alzheimer's disease (AD). However, it is unclear whether Sirt3 is associated with cognitive performance and pathological changes in AD. We conducted a case-control study of the postmortem brains of AD (n = 16), mild cognitive impairment (n = 13), and age- and education-matched cognitively normal (CN, n = 11) subjects. We measured the mRNA and protein levels of Sirt3 and assessed their association with cognitive performance and AD pathology. In an ex vivo model of cortical neurons from transgenic mice that carry human tau protein, we modified Sirt3 expression by genetic knockdown and knock-in to investigate the cause-effect relationship between Sirt3 and tau. Sirt3 levels were reduced in the entorhinal cortex, the middle temporal gyrus, and the superior frontal gyrus of AD subjects compared to those of CN. This reduction was associated with poorer test scores of neuropsychological evaluation and the severity of tau pathology. Further study with genetic manipulation of Sirt3 revealed that amyloid-β increased levels of total tau acetylated tau through its modulation of Sirt3. These data suggest that reduction of Sirt3 is critically involved in pathogenesis of AD.

Alzheimer's disease (AD) is manifested by regional cerebral hypometabolism. Sirtuin 3 (Sirt3) is localized in mitochondria and regulates cellular metabolism, but the role of Sirt3 in AD-related hypometabolism remains elusive. We used expression profiling and weighted gene co-expression network analysis (WGCNA) to analyze cortical neurons from a transgenic mouse model of AD ( APPSwInd ). Based on WGCNA results, we measured NAD + level, NAD + / NADH ratio, Sirt3 protein level and its deacetylation activity, and ATP production across both in vivo and in vitro models. To investigate the effect of Sirt3 on amyloid-β (Aβ)-induced mitochondria damage, we knocked down and over-expressed Sirt3 in hippocampal cells. WGCNA revealed Sirt3 as a key player in Aβ-related hypometabolism. In APP mice, the NAD + level, NAD + / NADH ratio, Sirt3 protein level and activity, and ATP production were all reduced compared to the control. As a result, learning and memory performance were impaired in 9-month-old APP mice compared to wild type controls. Using hippocampal HT22 cells model, Sirt3 overexpression increased Sirt3 deacetylation activity, rescued mitochondria function, and salvaged ATP production, which were damaged by Aβ. Sirt3 plays an important role in regulating Aβ-induced cerebral hypometabolism. This study suggests a potential direction for AD therapy.

Lipopolysaccharide (LPS) is a stimulator of the innate immune system and is routinely used in animal models to study blood-brain barrier (BBB) dysfunction under inflammatory conditions. It is appreciated that both humans and mice have sexually dimorphic immune responses, which could influence the brain's response to a systemic inflammatory insult. Mouse strain is also an important factor that can contribute to pathophysiological responses to inflammatory stimuli. Therefore, we aimed to test whether BBB disruption and the associated cytokine profiles in response to LPS differed in male and female mice from two mouse strains most commonly used in blood-brain barrier studies: CD-1 and C57BL6/J (C57). Mice were treated with saline, a single injection of 0.3, or 3mg/kg LPS, or three injections of 3mg/kg LPS, and studied 28 hours after the first LPS injection. To assay BBB disruption, we utilized the tracer 99mTc-DTPA. A 23-plex panel of cytokines was assayed in brain and blood of the same cohort of mice, which allowed us to compare differences in the levels of individual cytokines as well as correlations among cytokines and 99mTc-DTPA uptake. We found that only the three-injection dose of LPS induced significant BBB disruption in all sexes and strains. The treatment, strain, and sex, as well as treatment-by- strain and treatment-by-sex interactions significantly contributed to the variance. The mean brain/serum ratios of 99mTc-DTPA in the three-injection LPS group were ranked CD-1 male < CD-1 female < C57 male < C57 female. There were significant sex and strain differences in cytokine profiles in brain and blood, and pro-inflammatory cytokines and chemokines in brain were most strongly correlated with 99mTc-DTPA brain/serum ratios., Competing Interests: The authors have declared that no competing interests exist.

Investigators have long suspected that pathogenic microbes might contribute to the onset and progression of Alzheimer's disease (AD) although definitive evidence has not been presented. Whether such findings represent a causal contribution, or reflect opportunistic passengers of neurodegeneration, is also difficult to resolve. We constructed multiscale networks of the late-onset AD-associated virome, integrating genomic, transcriptomic, proteomic, and histopathological data across four brain regions from human post-mortem tissue. We observed increased human herpesvirus 6A (HHV-6A) and human herpesvirus 7 (HHV-7) from subjects with AD compared with controls. These results were replicated in two additional, independent and geographically dispersed cohorts. We observed regulatory relationships linking viral abundance and modulators of APP metabolism, including induction of APBB2, APPBP2, BIN1, BACE1, CLU, PICALM, and PSEN1 by HHV-6A. This study elucidates networks linking molecular, clinical, and neuropathological features with viral activity and is consistent with viral activity constituting a general feature of AD., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Introduction: Our laboratories have demonstrated that accumulation of oligomeric amyloid β (OAβ) in neurons is an essential step leading to OAβ-mediated mitochondrial dysfunction., Methods: Alzheimer's disease (AD) and matching control hippocampal neurons, astrocytes, and microglia were isolated by laser-captured microdissection from the same subjects, followed by whole-transcriptome sequencing. Complementary in vitro work was performed in OAβ-treated differentiated SH-SY5Y, followed by the use of a novel CoQ 10 analogue for protection. This compound is believed to be effective both in suppressing reactive oxygen species and also functioning in mitochondrial electron transport., Results: We report decreases in the same mitochondrial-encoded mRNAs in Alzheimer's disease laser-captured CA1 neurons and in OAβ-treated SH-SY5Y cells, but not in laser-captured microglia and astrocytes. Pretreatment with a novel CoQ 10 analogue, protects neuronal mitochondria from OAβ-induced mitochondrial changes., Discussion: Similarity of expression changes in neurons from Alzheimer's disease brain and neuronal cells treated with OAβ, and the effect of a CoQ 10 analogue on the latter, suggests a pretreatment option to prevent OAβ toxicity, long before the damage is apparent., (Copyright © 2018 the Alzheimer's Association. Published by Elsevier Inc. All rights reserved.)

Background: Circular RNAs (circRNAs) are a novel class of endogenous, non-coding RNAs that form covalently closed continuous loops and that are both highly conserved and abundant in the mammalian brain. A role for circRNAs in sponging microRNAs (miRNAs) has been proposed, but the circRNA-miRNA-mRNA interaction networks in human brain cells have not been defined. Therefore, we identified circRNAs in RNA sequencing data previously generated from astrocytes microdissected from the posterior cingulate (PC) of Alzheimer's disease (AD) patients (N = 10) and healthy elderly controls (N = 10) using four circRNA prediction algorithms - CIRI, CIRCexplorer, find&#95;circ and KNIFE., Results: Overall, utilizing these four tools, we identified a union of 4438 unique circRNAs across all samples, of which 70.3% were derived from exonic regions. Notably, the widely reported CDR1as circRNA was detected in all samples across both groups by find&#95;circ. Given the putative miRNA regulatory function of circRNAs, we identified potential miRNA targets of circRNAs, and further, delineated circRNA-miRNA-mRNA networks using in silico methods. Pathway analysis of the genes regulated by these miRNAs identified significantly enriched immune response pathways, which is consistent with the known function of astrocytes as immune sensors in the brain., Conclusions: In this study, we performed circRNA detection on cell-specific transcriptomic data and identified potential circRNA-miRNA-mRNA regulatory networks in PC astrocytes. Given the known function of astrocytes in cerebral innate immunity and our identification of significantly enriched immune response pathways, the circRNAs we identified may be associated with such key functions. While we did not detect recurrent differentially expressed circRNAs in the context of healthy controls or AD, we report for the first time circRNAs and their potential regulatory impact in a cell-specific and region-specific manner in aged subjects. These predicted regulatory network and pathway analyses may help provide new insights into transcriptional regulation in the brain.

Pre-analytical factors can significantly affect circulating cell-free DNA (cfDNA) analysis. However, there are few robust methods to rapidly assess sample quality and the impact of pre-analytical processing. To address this gap and to evaluate effects of DNA extraction methods and blood collection tubes on cfDNA yield and fragment size, we developed a multiplexed droplet digital PCR (ddPCR) assay with 5 short and 4 long amplicons targeting single copy genomic loci. Using this assay, we compared 7 cfDNA extraction kits and found cfDNA yield and fragment size vary significantly. We also compared 3 blood collection protocols using plasma samples from 23 healthy volunteers (EDTA tubes processed within 1 hour and Cell-free DNA Blood Collection Tubes processed within 24 and 72 hours) and found no significant differences in cfDNA yield, fragment size and background noise between these protocols. In 219 clinical samples, cfDNA fragments were shorter in plasma samples processed immediately after venipuncture compared to archived samples, suggesting contribution of background DNA by lysed peripheral blood cells. In summary, we have described a multiplexed ddPCR assay to assess quality of cfDNA samples prior to downstream molecular analyses and we have evaluated potential sources of pre-analytical variation in cfDNA studies.

Purpose: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive ovarian cancer in young women that is universally driven by loss of the SWI/SNF ATPase subunits SMARCA4 and SMARCA2. A great need exists for effective targeted therapies for SCCOHT. Experimental Design: To identify underlying therapeutic vulnerabilities in SCCOHT, we conducted high-throughput siRNA and drug screens. Complementary proteomics approaches profiled kinases inhibited by ponatinib. Ponatinib was tested for efficacy in two patient-derived xenograft (PDX) models and one cell-line xenograft model of SCCOHT. Results: The receptor tyrosine kinase (RTK) family was enriched in siRNA screen hits, with FGFRs and PDGFRs being overlapping hits between drug and siRNA screens. Of multiple potent drug classes in SCCOHT cell lines, RTK inhibitors were only one of two classes with selectivity in SCCOHT relative to three SWI/SNF wild-type ovarian cancer cell lines. We further identified ponatinib as the most effective clinically approved RTK inhibitor. Reexpression of SMARCA4 was shown to confer a 1.7-fold increase in resistance to ponatinib. Subsequent proteomic assessment of ponatinib target modulation in SCCOHT cell models confirmed inhibition of nine known ponatinib target kinases alongside 77 noncanonical ponatinib targets in SCCOHT. Finally, ponatinib delayed tumor doubling time 4-fold in SCCOHT-1 xenografts while reducing final tumor volumes in SCCOHT PDX models by 58.6% and 42.5%. Conclusions: Ponatinib is an effective agent for SMARCA4 -mutant SCCOHT in both in vitro and in vivo preclinical models through its inhibition of multiple kinases. Clinical investigation of this FDA-approved oncology drug in SCCOHT is warranted. Clin Cancer Res; 24(8); 1932-43. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Expression array data from dozens of laboratories, including our own, show significant changes in expression of many genes in Alzheimer's disease (AD) patients compared with normal controls. These data typically rely on brain homogenates, and information about transcripts specific to microglia and other central nervous system (CNS) cell types, which far outnumber microglia-specific transcripts, is lost. We therefore used single-cell laser capture methods to assess the full range of microglia-specific expression changes that occur in different brain regions (substantia nigra and hippocampus CA1) and disease states (AD, Parkinson's disease, and normal controls). Two novel pathways, neuronal repair and viral processing were identified. Based on KEGG analysis (Kyoto Encyclopedia of Genes and Genomes, a collection of biological pathways), one of the most significant viruses was hepatitis B virus (HBV) (false discovery rate < 0.00000001). Immunohistochemical analysis using HBV-core antibody in HBV-positive control, amnestic mild cognitive impairment, and HBV-positive AD cases show increased HBV immunoreactivity as disease pathology increases. These results are the first, to our knowledge, to show regional differences in human microglia. In addition, these data reveal new functions for microglia and suggest a novel risk factor for AD., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Purpose: Glioblastoma is an aggressive and molecularly heterogeneous cancer with few effective treatment options. We hypothesized that next-generation sequencing can be used to guide treatment recommendations within a clinically acceptable time frame following surgery for patients with recurrent glioblastoma. Experimental Design: We conducted a prospective genomics-informed feasibility trial in adults with recurrent and progressive glioblastoma. Following surgical resection, genome-wide tumor/normal exome sequencing and tumor RNA sequencing were performed to identify molecular targets for potential matched therapy. A multidisciplinary molecular tumor board issued treatment recommendations based on the genomic results, blood-brain barrier penetration of the indicated therapies, drug-drug interactions, and drug safety profiles. Feasibility of generating genomics-informed treatment recommendations within 35 days of surgery was assessed. Results: Of the 20 patients enrolled in the study, 16 patients had sufficient tumor tissue for analysis. Exome sequencing was completed for all patients, and RNA sequencing was completed for 14 patients. Treatment recommendations were provided within the study's feasibility time frame for 15 of 16 (94%) patients. Seven patients received treatment based on the tumor board recommendations. Two patients reached 12-month progression-free survival, both adhering to treatments based on the molecular profiling results. One patient remained on treatment and progression free 21 months after surgery, 3 times longer than the patient's previous time to progression. Analysis of matched nonenhancing tissue from 12 patients revealed overlapping as well as novel putatively actionable genomic alterations. Conclusions: Use of genome-wide molecular profiling is feasible and can be informative for guiding real-time, central nervous system-penetrant, genomics-informed treatment recommendations for patients with recurrent glioblastoma. Clin Cancer Res; 24(2); 295-305. ©2017 AACR See related commentary by Wick and Kessler, p. 256 ., (©2017 American Association for Cancer Research.)

[This corrects the article DOI: 10.1371/journal.pone.0177814.].

Whole exome sequencing (WES) is a DNA sequencing strategy that provides a survey of base substitutions across coding genomic locations and other regions of interest. As the coding portion of the genome encompasses only 1-2% of the entire genome, this approach represents a more cost-effective strategy to detect DNA alterations that may alter protein function, compared to whole genome sequencing. Although the research community has and is currently delineating the functional implications of sequence changes in noncoding regions of the genome, WES is a currently available assay that provides valuable information for both discovery research and precision medicine applications. In this chapter, we present a WES library preparation protocol using the KAPA Hyper Prep Kit with Agilent SureSelect Human All Exon V5+UTR probes that demonstrates high DNA-to-library conversion efficiency for sequencing on the Illumina HiSeq platform.

With the rapid evolution of genomics technologies over the past decade, whole genome sequencing (WGS) has become an increasingly accessible tool in biomedical research. WGS applications include analysis of genomic DNA from single individuals, multiple related family members, and tumor/normal samples from the same patient in the context of oncology. A number of different modalities are available for performing WGS; this chapter focuses on wet lab library construction procedures for complex short insert WGS libraries using the KAPA Hyper Prep Kit (Kapa Biosystems), and includes a discussion of appropriate quality control measures for sequencing on the Illumina HiSeq2000 platform. Additional modifications to the protocol for long insert WGS library construction, to assess structural alterations and copy number changes, are also described.

Multiple Myeloma (MM) is a plasma cell malignancy with significantly greater incidence and mortality rates among African Americans (AA) compared to Caucasians (CA). The overall goal of this study is to elucidate differences in molecular alterations in MM as a function of self-reported race and genetic ancestry. Our study utilized somatic whole exome, RNA-sequencing, and correlated clinical data from 718 MM patients from the Multiple Myeloma Research Foundation CoMMpass study Interim Analysis 9. Somatic mutational analyses based upon self-reported race corrected for ancestry revealed significant differences in mutation frequency between groups. Of interest, BCL7A, BRWD3, and AUTS2 demonstrate significantly higher mutation frequencies among AA cases. These genes are all involved in translocations in B-cell malignancies. Moreover, we detected a significant difference in mutation frequency of TP53 and IRF4 with frequencies higher among CA cases. Our study provides rationale for interrogating diverse tumor cohorts to best understand tumor genomics across populations.