Coxiella burnetii: Intranasal immunizations and axenic media adaptations

Coxiella burnetii is an obligate intracellular pathogen and the causative agent of the zoonotic illness Q fever. With an infectious dose of 1-10 organisms, C. burnetii is one of the most infectious bacteria known. This low infectious dose coupled with its environmental stability and aerosol route of infection has placed it on the Centers for Disease Control and Prevention's Class B Select Agent List, making a vaccine against C. burnetii infection particularly desirable. Formulations of killed whole-cell C. burnetii virulent strains has been shown to be effective when administered intramuscularly, intraperitoneally, and sub-cutaneously, but has not been assessed using an intranasal route of inoculation that would also bring to bear the mucosal immune response. We hypothesized that intranasal immunization would be protective against subsequent challenge with live virulent C. burnetii. Using a guinea pig model, intranasal immunization with either a mixture of killed whole-cell C. burnetii Nine Mile phase I combined with double mutant lethal toxin (DmLT) adjuvant, or a mock immunization of PBS-DmLT. Booster immunizations were administered at 14 and 28 days. Sera was drawn for IgG titers prior to, and at 14, 28, and 56 days following intranasal immunization. Analysis of sera indicates that a robust humoral response is elicited following intranasal immunization. Additionally, immunized guinea pigs that were subsequently challenged with virulent C. burnetii NMI did not develop significant clinical symptoms as measured by fever and weight loss when compared to animals receiving the mock immunization. Studies defining minimal immunization requirements, potential hypersensitivity response, and protective antigen profile using this physiologically relevant exposure route will aid us in defining a protective response to this unusual aerosol acquired pathogen.For the first 80 years following its discovery, C. burnetii could only be grown in a system providing live cells within which it would replicate. The relatively recent advent of cell free media has been a boon for C. burnetii research on many fronts. We noticed when infecting cultured cells using C. burnetii that had been repeatedly passaged in cell free defined acidified citrate cysteine media (ACCM-D), that the infectivity rates were lower than expected based on the number of genomes used to initiate the infection relative to C. burnetii that had been derived from infected cells. Infectious assays comparing C. burnetii from passages 1, 3, 5 and 10 determined that by passage 10 the organism's relative ability to infect cultured cells had decreased 2-logs. We hypothesized that molecular changes were occurring in these early passages that related to the ability of the bacteria to infect or grow within host cells at their normal rate. We initiated a "Reverse Evolution" approach to study the molecular changes that occur following serial passages in axenic media to determine what occurs when the stressors associated with C. burnetii's typically intracellular lifestyle are removed. We found that the concentration of DotA and IcmX, two structural proteins of the Type IVB Secretion System, which is required for intracellular growth, were significantly reduced by passage 10. Mass spec analysis of passages 1, 3, and 5 also demonstrate changes between protein levels in subsequent passages analyzed. Expanded mass spec and RNAseq analysis to include passage 10, along with immunoblot analysis and genome sequencing of both early and longer-term passages will elucidate molecular mechanisms required for C. burnetii's intracellular survival and growth

Comparative Transcriptomics and Genomics from Continuous Axenic Media Growth Identifies

Coxiella burnetii (Cb) is an obligate intracellular pathogen in nature and the causative agent of acute Q fever as well as chronic diseases. In an effort to identify genes and proteins crucial to their normal intracellular growth lifestyle, we applied a Reverse evolution approach where the avirulent Nine Mile Phase II strain of Cb was grown for 67 passages in chemically defined ACCM-D media and gene expression patterns and genome integrity from various passages was compared to passage number one following intracellular growth. Transcriptomic analysis identified a marked downregulation of the structural components of the type 4B secretion system (T4BSS), the general secretory (sec) pathway, as well as 14 out of 118 previously identified genes encoding effector proteins. Additional downregulated pathogenicity determinants genes included several chaperones, LPS, and peptidoglycan biosynthesis. A general marked downregulation of central metabolic pathways was also observed, which was balanced by a marked upregulation of genes encoding transporters. This pattern reflected the richness of the media and diminishing anabolic and ATP-generation needs. Finally, genomic sequencing and comparative genomic analysis demonstrated an extremely low level of mutation across passages, despite the observed Cb gene expression changes following acclimation to axenic media

Navigating to new frontiers in behavioral neuroscience: traditional neuropsychological tests predict human performance on a rodent-inspired radial-arm maze

We constructed an 11-arm, walk-through, human radial-arm maze (HRAM) as a translational instrument to compare existing methodology in the areas of rodent and human learning and memory research. The HRAM, utilized here, serves as an intermediary test between the classic rat radial-arm maze (RAM) and standard human neuropsychological and cognitive tests. We show that the HRAM is a useful instrument to examine working memory ability, explore the relationships between rodent and human memory and cognition models, and evaluate factors that contribute to human navigational ability. One-hundred-and-fifty-seven participants were tested on the HRAM, and scores were compared to performance on a standard cognitive battery focused on episodic memory, working memory capacity, and visuospatial ability. We found that errors on the HRAM increased as working memory demand became elevated, similar to the pattern typically seen in rodents, and that for this task, performance appears similar to Miller's classic description of a processing-inclusive human working memory capacity of 7 ± 2 items. Regression analysis revealed that measures of working memory capacity and visuospatial ability accounted for a large proportion of variance in HRAM scores, while measures of episodic memory and general intelligence did not serve as significant predictors of HRAM performance. We present the HRAM as a novel instrument for measuring navigational behavior in humans, as is traditionally done in basic science studies evaluating rodent learning and memory, thus providing a useful tool to help connect and translate between human and rodent models of cognitive functioning

Evaluation of polygenic risk scores for breast and ovarian cancer risk prediction in BRCA1 and BRCA2 mutation carriers

Background: Genome-wide association studies (GWAS) have identified 94 common single-nucleotide polymorphisms (SNPs) associated with breast cancer (BC) risk and 18 associated with ovarian cancer (OC) risk. Several of these are also associated with risk of BC or OC for women who carry a pathogenic mutation in the high-risk BC and OC genes BRCA1 or BRCA2. The combined effects of these variants on BC or OC risk for BRCA1 and BRCA2 mutation carriers have not yet been assessed while their clinical management could benefit from improved personalized risk estimates. Methods: We constructed polygenic risk scores (PRS) using BC and OC susceptibility SNPs identified through population-based GWAS: for BC (overall, estrogen receptor [ER]-positive, and ER-negative) and for OC. Using data from 15 252 female BRCA1 and 8211 BRCA2 carriers, the association of each PRS with BC or OC risk was evaluated using a weighted cohort approach, with time to diagnosis as the outcome and estimation of the hazard ratios (HRs) per standard deviation increase in the PRS. Results: The PRS for ER-negative BC displayed the strongest association with BC risk in BRCA1 carriers (HR = 1.27, 95% confidence interval [CI] = 1.23 to 1.31, P = 8.2 x 10(53)). In BRCA2 carriers, the strongest association with BC risk was seen for the overall BC PRS (HR = 1.22, 95% CI = 1.17 to 1.28, P = 7.2 x 10(-20)). The OC PRS was strongly associated with OC risk for both BRCA1 and BRCA2 carriers. These translate to differences in absolute risks (more than 10% in each case) between the top and bottom deciles of the PRS distribution; for example, the OC risk was 6% by age 80 years for BRCA2 carriers at the 10th percentile of the OC PRS compared with 19% risk for those at the 90th percentile of PRS. Conclusions: BC and OC PRS are predictive of cancer risk in BRCA1 and BRCA2 carriers. Incorporation of the PRS into risk prediction models has promise to better inform decisions on cancer risk management

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts

Spatial Organization and Molecular Correlation of Tumor-Infiltrating Lymphocytes Using Deep Learning on Pathology Images

Beyond sample curation and basic pathologic characterization, the digitized H&E-stained images of TCGA samples remain underutilized. To highlight this resource, we present mappings of tumorinfiltrating lymphocytes (TILs) based on H&E images from 13 TCGA tumor types. These TIL maps are derived through computational staining using a convolutional neural network trained to classify patches of images. Affinity propagation revealed local spatial structure in TIL patterns and correlation with overall survival. TIL map structural patterns were grouped using standard histopathological parameters. These patterns are enriched in particular T cell subpopulations derived from molecular measures. TIL densities and spatial structure were differentially enriched among tumor types, immune subtypes, and tumor molecular subtypes, implying that spatial infiltrate state could reflect particular tumor cell aberration states. Obtaining spatial lymphocytic patterns linked to the rich genomic characterization of TCGA samples demonstrates one use for the TCGA image archives with insights into the tumor-immune microenvironment

Reward-Related Dorsal Striatal Activity Differences between Former and Current Cocaine Dependent Individuals during an Interactive Competitive Game

Cocaine addiction is characterized by impulsivity, impaired social relationships, and abnormal mesocorticolimbic reward processing, but their interrelationships relative to stages of cocaine addiction are unclear. We assessed blood-oxygenation-level dependent (BOLD) signal in ventral and dorsal striatum during functional magnetic resonance imaging (fMRI) in current (CCD; n = 30) and former (FCD; n = 28) cocaine dependent subjects as well as healthy control (HC; n = 31) subjects while playing an interactive competitive Domino game involving risk-taking and reward/punishment processing. Out-of-scanner impulsivity-related measures were also collected. Although both FCD and CCD subjects scored significantly higher on impulsivity-related measures than did HC subjects, only FCD subjects had differences in striatal activation, specifically showing hypoactivation during their response to gains versus losses in right dorsal caudate, a brain region linked to habituation, cocaine craving and addiction maintenance. Right caudate activity in FCD subjects also correlated negatively with impulsivity-related measures of self-reported compulsivity and sensitivity to reward. These findings suggest that remitted cocaine dependence is associated with striatal dysfunction during social reward processing in a manner linked to compulsivity and reward sensitivity measures. Future research should investigate the extent to which such differences might reflect underlying vulnerabilities linked to cocaine-using propensities (e.g., relapses)

Finding Diagnostically Useful Patterns in Quantitative Phenotypic Data.

Trio-based whole-exome sequence (WES) data have established confident genetic diagnoses in ∼40% of previously undiagnosed individuals recruited to the Deciphering Developmental Disorders (DDD) study. Here we aim to use the breadth of phenotypic information recorded in DDD to augment diagnosis and disease variant discovery in probands. Median Euclidean distances (mEuD) were employed as a simple measure of similarity of quantitative phenotypic data within sets of ≥10 individuals with plausibly causative de novo mutations (DNM) in 28 different developmental disorder genes. 13/28 (46.4%) showed significant similarity for growth or developmental milestone metrics, 10/28 (35.7%) showed similarity in HPO term usage, and 12/28 (43%) showed no phenotypic similarity. Pairwise comparisons of individuals with high-impact inherited variants to the 32 individuals with causative DNM in ANKRD11 using only growth z-scores highlighted 5 likely causative inherited variants and two unrecognized DNM resulting in an 18% diagnostic uplift for this gene. Using an independent approach, naive Bayes classification of growth and developmental data produced reasonably discriminative models for the 24 DNM genes with sufficiently complete data. An unsupervised naive Bayes classification of 6,993 probands with WES data and sufficient phenotypic information defined 23 in silico syndromes (ISSs) and was used to test a "phenotype first" approach to the discovery of causative genotypes using WES variants strictly filtered on allele frequency, mutation consequence, and evidence of constraint in humans. This highlighted heterozygous de novo nonsynonymous variants in SPTBN2 as causative in three DDD probands