Glioblastoma-infiltrating CD8+ T cells are predominantly a clonally expanded GZMK+ effector population.

Recent clinical trials have highlighted the limited efficacy of T cell-based immunotherapy in patients with glioblastoma (GBM). To better understand the characteristics of tumor-infiltrating lymphocytes (TIL) in GBM, we performed cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and single-cell RNA sequencing (scRNA-seq) with paired V(D)J sequencing, respectively, on TIL from two cohorts of patients totaling 15 patients with high grade glioma, including GBM or astrocytoma, IDH mutant, grade 4 (G4A). Analysis of the CD8+ TIL landscape reveals an enrichment of clonally expanded GZMK+ effector T cells in the tumor compared to matched blood, which was validated at the protein level. Furthermore, integration with other cancer types highlights the lack of a canonically exhausted CD8+ T cell population in GBM TIL. These data suggest that GZMK+ effector T cells represent an important T cell subset within the GBM microenvironment and which may harbor potential therapeutic implications.

Single-cell multi-omic analysis of the vestibular schwannoma ecosystem uncovers a nerve injury-like state.

Vestibular schwannomas (VS) are benign tumors that lead to significant neurologic and otologic morbidity. How VS heterogeneity and the tumor microenvironment (TME) contribute to VS pathogenesis remains poorly understood. In this study, we perform scRNA-seq on 15 VS, with paired scATAC-seq (n = 6) and exome sequencing (n = 12). We identify diverse Schwann cell (SC), stromal, and immune populations in the VS TME and find that repair-like and MHC-II antigen-presenting SCs are associated with myeloid cell infiltrate, implicating a nerve injury-like process. Deconvolution analysis of RNA-expression data from 175 tumors reveals Injury-like tumors are associated with larger tumor size, and scATAC-seq identifies transcription factors associated with nerve repair SCs from Injury-like tumors. Ligand-receptor analysis and in vitro experiments suggest that Injury-like VS-SCs recruit myeloid cells via CSF1 signaling. Our study indicates that Injury-like SCs may cause tumor growth via myeloid cell recruitment and identifies molecular pathways that may be therapeutically targeted.

Impact of CD4 T cells on intratumoral CD8 T-cell exhaustion and responsiveness to PD-1 blockade therapy in mouse brain tumors.

BACKGROUND: Glioblastoma is a fatal disease despite aggressive multimodal therapy. PD-1 blockade, a therapy that reinvigorates hypofunctional exhausted CD8 T cells (Tex) in many malignancies, has not shown efficacy in glioblastoma. Loss of CD4 T cells can lead to an exhausted CD8 T-cell phenotype, and terminally exhausted CD8 T cells (Tex term) do not respond to PD-1 blockade. GL261 and CT2A are complementary orthotopic models of glioblastoma. GL261 has a functional CD4 T-cell compartment and is responsive to PD-1 blockade; notably, CD4 depletion abrogates this survival benefit. CT2A is composed of dysfunctional CD4 T cells and is PD-1 blockade unresponsive. We leverage these models to understand the impact of CD4 T cells on CD8 T-cell exhaustion and PD-1 blockade sensitivity in glioblastoma. METHODS: Single-cell RNA sequencing was performed on flow sorted tumor-infiltrating lymphocytes from female C57/BL6 mice implanted with each model, with and without PD-1 blockade therapy. CD8+ and CD4+ T cells were identified and separately analyzed. Survival analyses were performed comparing PD-1 blockade therapy, CD40 agonist or combinatorial therapy. RESULTS: The CD8 T-cell compartment of the models is composed of heterogenous CD8 Tex subsets, including progenitor exhausted CD8 T cells (Tex prog), intermediate Tex, proliferating Tex, and Tex term. GL261 is enriched with the PD-1 responsive Tex prog subset relative to the CT2A and CD4-depleted GL261 models, which are composed predominantly of the PD-1 blockade refractory Tex term subset. Analysis of the CD4 T-cell compartments revealed that the CT2A microenvironment is enriched with a suppressive Treg subset and an effector CD4 T-cell subset that expresses an inhibitory interferon-stimulated (Isc) signature. Finally, we demonstrate that addition of CD40 agonist to PD-1 blockade therapy improves survival in CT2A tumor-bearing mice. CONCLUSIONS: Here, we describe that dysfunctional CD4 T cells are associated with terminal CD8 T-cell exhaustion, suggesting CD4 T cells impact PD-1 blockade efficacy by controlling the severity of exhaustion. Given that CD4 lymphopenia is frequently observed in patients with glioblastoma, this may represent a basis for resistance to PD-1 blockade. We demonstrate that CD40 agonism may circumvent a dysfunctional CD4 compartment to improve PD-1 blockade responsiveness, supporting a novel synergistic immunotherapeutic approach.

Single-cell profiling of human dura and meningioma reveals cellular meningeal landscape and insights into meningioma immune response.

BACKGROUND: Recent investigations of the meninges have highlighted the importance of the dura layer in central nervous system immune surveillance beyond a purely structural role. However, our understanding of the meninges largely stems from the use of pre-clinical models rather than human samples. METHODS: Single-cell RNA sequencing of seven non-tumor-associated human dura samples and six primary meningioma tumor samples (4 matched and 2 non-matched) was performed. Cell type identities, gene expression profiles, and T cell receptor expression were analyzed. Copy number variant (CNV) analysis was performed to identify putative tumor cells and analyze intratumoral CNV heterogeneity. Immunohistochemistry and imaging mass cytometry was performed on selected samples to validate protein expression and reveal spatial localization of select protein markers. RESULTS: In this study, we use single-cell RNA sequencing to perform the first characterization of both non-tumor-associated human dura and primary meningioma samples. First, we reveal a complex immune microenvironment in human dura that is transcriptionally distinct from that of meningioma. In addition, we characterize a functionally diverse and heterogenous landscape of non-immune cells including endothelial cells and fibroblasts. Through imaging mass cytometry, we highlight the spatial relationship among immune cell types and vasculature in non-tumor-associated dura. Utilizing T cell receptor sequencing, we show significant TCR overlap between matched dura and meningioma samples. Finally, we report copy number variant heterogeneity within our meningioma samples. CONCLUSIONS: Our comprehensive investigation of both the immune and non-immune cellular landscapes of human dura and meningioma at single-cell resolution builds upon previously published data in murine models and provides new insight into previously uncharacterized roles of human dura.

Hematopoietic cell transplantation donor-derived memory-like NK cells functionally persist after transfer into patients with leukemia.

Natural killer (NK) cells are innate lymphoid cells that eliminate cancer cells, produce cytokines, and are being investigated as a nascent cellular immunotherapy. Impaired NK cell function, expansion, and persistence remain key challenges for optimal clinical translation. One promising strategy to overcome these challenges is cytokine-induced memory-like (ML) differentiation, whereby NK cells acquire enhanced antitumor function after stimulation with interleukin-12 (IL-12), IL-15, and IL-18. Here, reduced-intensity conditioning (RIC) for HLA-haploidentical hematopoietic cell transplantation (HCT) was augmented with same-donor ML NK cells on day +7 and 3 weeks of N-803 (IL-15 superagonist) to treat patients with relapsed/refractory acute myeloid leukemia (AML) in a clinical trial (NCT02782546). In 15 patients, donor ML NK cells were well tolerated, and 87% of patients achieved a composite complete response at day +28, which corresponded with clearing high-risk mutations, including TP53 variants. NK cells were the major blood lymphocytes for 2 months after HCT with 1104-fold expansion (over 1 to 2 weeks). Phenotypic and transcriptional analyses identified donor ML NK cells as distinct from conventional NK cells and showed that ML NK cells persisted for over 2 months. ML NK cells expressed CD16, CD57, and high granzyme B and perforin, along with a unique transcription factor profile. ML NK cells differentiated in patients had enhanced ex vivo function compared to conventional NK cells from both patients and healthy donors. Overall, same-donor ML NK cell therapy with 3 weeks of N-803 support safely augmented RIC haplo-HCT for AML.

Genetic and Transcriptional Contributions to Relapse in Normal Karyotype Acute Myeloid Leukemia.

To better understand clonal and transcriptional adaptations after relapse in patients with acute myeloid leukemia (AML), we collected presentation and relapse samples from six normal karyotype AML cases. We performed enhanced whole-genome sequencing to characterize clonal evolution, and deep-coverage single-cell RNA sequencing on the same samples, which yielded 142,642 high-quality cells for analysis. Identifying expressed mutations in individual cells enabled us to discriminate between normal and AML cells, to identify coordinated changes in the genome and transcriptome, and to identify subclone-specific cell states. We quantified the coevolution of genetic and transcriptional heterogeneity during AML progression, and found that transcriptional changes were significantly correlated with genetic changes. However, transcriptional adaptation sometimes occurred independently, suggesting that clonal evolution does not represent all relevant biological changes. In three cases, we identified cells at diagnosis that likely seeded the relapse. Finally, these data revealed a conserved relapse-enriched leukemic cell state bearing markers of stemness, quiescence, and adhesion. SIGNIFICANCE: These data enabled us to identify a relapse-enriched leukemic cell state with distinct transcriptional properties. Detailed case-by-case analyses elucidated the complex ways in which the AML genome, transcriptome, and immune microenvironment interact to evade chemotherapy. These analyses provide a blueprint for evaluating these factors in larger cohorts.This article is highlighted in the In This Issue feature, p. 1.

Characteristics of publicly available skin cancer image datasets: a systematic review.

Publicly available skin image datasets are increasingly used to develop machine learning algorithms for skin cancer diagnosis. However, the total number of datasets and their respective content is currently unclear. This systematic review aimed to identify and evaluate all publicly available skin image datasets used for skin cancer diagnosis by exploring their characteristics, data access requirements, and associated image metadata. A combined MEDLINE, Google, and Google Dataset search identified 21 open access datasets containing 106 950 skin lesion images, 17 open access atlases, eight regulated access datasets, and three regulated access atlases. Images and accompanying data from open access datasets were evaluated by two independent reviewers. Among the 14 datasets that reported country of origin, most (11 [79%]) originated from Europe, North America, and Oceania exclusively. Most datasets (19 [91%]) contained dermoscopic images or macroscopic photographs only. Clinical information was available regarding age for 81 662 images (76·4%), sex for 82 848 (77·5%), and body site for 79 561 (74·4%). Subject ethnicity data were available for 1415 images (1·3%), and Fitzpatrick skin type data for 2236 (2·1%). There was limited and variable reporting of characteristics and metadata among datasets, with substantial under-representation of darker skin types. This is the first systematic review to characterise publicly available skin image datasets, highlighting limited applicability to real-life clinical settings and restricted population representation, precluding generalisability. Quality standards for characteristics and metadata reporting for skin image datasets are needed.

Clinician-driven artificial intelligence in ophthalmology: resources enabling democratization.

PURPOSE OF REVIEW: This article aims to discuss the current state of resources enabling the democratization of artificial intelligence (AI) in ophthalmology. RECENT FINDINGS: Open datasets, efficient labeling techniques, code-free automated machine learning (AutoML) and cloud-based platforms for deployment are resources that enable clinicians with scarce resources to drive their own AI projects. SUMMARY: Clinicians are the use-case experts who are best suited to drive AI projects tackling patient-relevant outcome measures. Taken together, open datasets, efficient labeling techniques, code-free AutoML and cloud platforms break the barriers for clinician-driven AI. As AI becomes increasingly democratized through such tools, clinicians and patients stand to benefit greatly.

A global review of publicly available datasets for ophthalmological imaging: barriers to access, usability, and generalisability.

Health data that are publicly available are valuable resources for digital health research. Several public datasets containing ophthalmological imaging have been frequently used in machine learning research; however, the total number of datasets containing ophthalmological health information and their respective content is unclear. This Review aimed to identify all publicly available ophthalmological imaging datasets, detail their accessibility, describe which diseases and populations are represented, and report on the completeness of the associated metadata. With the use of MEDLINE, Google's search engine, and Google Dataset Search, we identified 94 open access datasets containing 507 724 images and 125 videos from 122 364 patients. Most datasets originated from Asia, North America, and Europe. Disease populations were unevenly represented, with glaucoma, diabetic retinopathy, and age-related macular degeneration disproportionately overrepresented in comparison with other eye diseases. The reporting of basic demographic characteristics such as age, sex, and ethnicity was poor, even at the aggregate level. This Review provides greater visibility for ophthalmological datasets that are publicly available as powerful resources for research. Our paper also exposes an increasing divide in the representation of different population and disease groups in health data repositories. The improved reporting of metadata would enable researchers to access the most appropriate datasets for their needs and maximise the potential of such resources.

Prospective Phase 2 Study of Radiation Therapy Dose and Volume De-escalation for Elective Neck Treatment of Oropharyngeal and Laryngeal Cancer.

PURPOSE: The required elective nodal dose and volumes for head and neck intensity modulated radiation therapy have largely been extrapolated from conventional radiation therapy fields. In this prospective, dual-center, phase 2 study, we investigated the efficacy and tolerability of reduced elective nodal volume and dose in oropharyngeal and laryngeal squamous cell carcinoma. METHODS AND MATERIALS: Patients with newly diagnosed squamous cell carcinoma of the oropharynx and larynx were eligible for enrollment. Each lymph node was characterized as involved or suspicious based on imaging criteria. For oropharynx cancer, only involved and immediately adjacent stations were treated to 40 Gy in 20 fractions. In larynx patients, at least bilateral levels II and III were treated to 40 Gy, with level IV treated only if level III was involved. Involved and suspicious nodes were then boosted with 30 Gy and 24 Gy in 15 fractions, respectively. Concurrent chemotherapy was required for stage T3N0-1 and IVA/B patients. The primary endpoint of the study was solitary elective volume recurrence, with secondary endpoints including patterns of failure and patient-reported outcomes. RESULTS: A total of 72 (51 oropharynx, 21 larynx) patients completed treatment on this trial from January 2017 through November 2018. The stages at presentations were 5, 17, and 50 stage I-II, III, and IV, respectively, with 90% treated with chemoradiation therapy. At a median follow-up of 24.7 months for surviving patients, there have been no solitary elective nodal recurrences. Seven patients developed a nodal recurrence, 5 of which were in-field and 2 were elective with synchronous in-field recurrence. Patient-reported outcomes assessment at 1 year showed superior or equivalent outcomes compared with baseline, except for saliva and taste measures. CONCLUSIONS: The results of this trial suggest that elective dose and volume reduction is oncologically sound for oropharyngeal and laryngeal cancer treated with intensity modulated radiation therapy, with promising quality-of-life outcomes.

Association between type 2 diabetes mellitus & TCF7L2 gene variants in the Emirati population: Genetics of diabetes in the United Arab Emirates.

OBJECTIVE: Type 2 diabetes mellitus (T2DM) has a multifactorial etiology involving a complex interplay between genes and the environment. The prevalence of T2DM among the countries of the Gulf Corporation Council (GCC), including the United Arab Emirates (UAE), ranks among the top 15 in the world. A number of studies have shown an increase in T2DM risk for the "TT" genotype at the rs4506565 and rs12255372 Single Nucleotide Polymorphisms (SNP) of the TCF7L2 gene. However, the association between TCF7L2 and T2DM still needs to be investigated in the UAE population. Therefore, this study analyzed the potential associations with rs4506565 and rs12255372 in UAE subjects. METHODS: For this case-control study, T2DM patients (n = 890) and healthy subjects (n = 686) were genotyped using a Taqman Real-Time PCR assay. Statistical analysis was performed with the resulting data using the R (version 3.3.1) and STATA (version 13) software packages. RESULTS: The rs12255372 SNP was significantly associated with T2DM (OR = 1.16, 95% CI = 1.00-1.34; P = .042). However, no significant association was found for the rs4506565 SNP (P = .120). After gender stratification, a significant association was found for both SNPs in males (Prs4506565 = .009 and Prs12255372 = .021). Interestingly, we found the interaction between the SNP rs4506565 with gender alone (P = .032) and in conjunction with BMI and age (P = .036) confers associations with T2DM. CONCLUSIONS: These findings suggest that the genetic variants of the TCF7L2 gene are associated with an increased susceptibility to T2DM, especially in Emirati males. Our study also highlights the impact of biological and environmental risk factors including age, BMI, and gender on the genetic susceptibility to T2DM.

MU-PseUDeep: A deep learning method for prediction of pseudouridine sites.

Pseudouridine synthase binds to uridine sites and catalyzes the conversion of uridine to pseudouridine (Ψ). This binding takes place in a specific context and in the conformation of nucleotides. Most machine-learning methods for Ψ site classification use nucleotide frequency as a feature, which may not fully depict the relevant conformation around a Ψ site. Using the power of deep learning and raw sequence, as well as secondary structure features, our tool MU-PseUDeep is designed to capture both the sequence and secondary structure context, which inputs the raw RNA sequence and the predicted secondary structure to two sets of convolutional neural networks. It has shown considerable improvement in Ψ site prediction over existing tools, XG-PseU, PseUI, and iRNA-PseU for both balanced and imbalanced datasets. To the best of our knowledge, this is the most accurate tool for Ψ site prediction. We also used MU-PseUDeep to scan the human transcriptome, which shows that the genes with predicted Ψ sites are enriched in nucleotide and protein binding, as well as in neurodegeneration pathways. The tool is open source, available at https://github.com/smk5g5/MU-PseUDeep.

Tetralogy of Fallot: morphological variations and implications for surgical repair.

OBJECTIVES: Tetralogy of Fallot is characterized by anterocephalad deviation of the outlet septum, along with abnormal septoparietal trabeculations, which lead to subpulmonary infundibular stenosis. Archives of retained hearts are an important resource for improving our understanding of congenital heart defects and their morphological variability. This study aims to define variations in aortic override, coronary arterial patterns and ventricular septal defects in tetralogy of Fallot as observed in a morphological archive, highlighting implications for surgical management. METHODS: The Birmingham Children's Hospital archive contains 211 hearts with tetralogy of Fallot, of which 164 were analysed [69 (42.1%) unrepaired and 95 (57.9%) operated specimens]. A detailed morphological and geometric analysis was performed using a rigorous 5-layer review process. RESULTS: Anomalies were observed in the orifices, origins and course of the coronary arteries: 20 hearts (13.0%) had more than 2 orifices and 3 hearts (1.9%) had a single orifice. In 7 hearts (4.3%), a coronary artery crossed the right ventricular outflow tract. The extent of aortic override ranged from 31.0% to 100% (median of 59.5%). The ventricular septal defect was most often perimembranous (139, 84.8%), but we also found muscular (14, 8.5%), atrioventricular (7, 4.3%) and doubly committed juxta-arterial (2, 1.2%) variants. CONCLUSIONS: Anatomical variations are common and can impact surgical management. Anomalous coronary arteries may require a conduit rather than a transannular patch. Variability in aortic override determines the size of patch used to baffle blood to the aorta. The type of ventricular septal defect affects patch closure and the risk of postoperative conduction defects.

Divergent cytosine DNA methylation patterns in single-cell, soybean root hairs.

Chromatin modifications, such as cytosine methylation of DNA, play a significant role in mediating gene expression in plants, which affects growth, development, and cell differentiation. As root hairs are single-cell extensions of the root epidermis and the primary organs for water uptake and nutrients, we sought to use root hairs as a single-cell model system to measure the impact of environmental stress. We measured changes in cytosine DNA methylation in single-cell root hairs as compared with multicellular stripped roots, as well as in response to heat stress. Differentially methylated regions (DMRs) in each methylation context showed very distinct methylation patterns between cell types and in response to heat stress. Intriguingly, at normal temperature, root hairs were more hypermethylated than were stripped roots. However, in response to heat stress, both root hairs and stripped roots showed hypomethylation in each context, especially in the CHH context. Moreover, expression analysis of mRNA from similar tissues and treatments identified some associations between DMRs, genes and transposons. Taken together, the data indicate that changes in DNA methylation are directly or indirectly associated with expression of genes and transposons within the context of either specific tissues/cells or stress (heat).

PGen: large-scale genomic variations analysis workflow and browser in SoyKB.

BACKGROUND: With the advances in next-generation sequencing (NGS) technology and significant reductions in sequencing costs, it is now possible to sequence large collections of germplasm in crops for detecting genome-scale genetic variations and to apply the knowledge towards improvements in traits. To efficiently facilitate large-scale NGS resequencing data analysis of genomic variations, we have developed "PGen", an integrated and optimized workflow using the Extreme Science and Engineering Discovery Environment (XSEDE) high-performance computing (HPC) virtual system, iPlant cloud data storage resources and Pegasus workflow management system (Pegasus-WMS). The workflow allows users to identify single nucleotide polymorphisms (SNPs) and insertion-deletions (indels), perform SNP annotations and conduct copy number variation analyses on multiple resequencing datasets in a user-friendly and seamless way. RESULTS: We have developed both a Linux version in GitHub ( https://github.com/pegasus-isi/PGen-GenomicVariations-Workflow ) and a web-based implementation of the PGen workflow integrated within the Soybean Knowledge Base (SoyKB), ( http://soykb.org/Pegasus/index.php ). Using PGen, we identified 10,218,140 single-nucleotide polymorphisms (SNPs) and 1,398,982 indels from analysis of 106 soybean lines sequenced at 15X coverage. 297,245 non-synonymous SNPs and 3330 copy number variation (CNV) regions were identified from this analysis. SNPs identified using PGen from additional soybean resequencing projects adding to 500+ soybean germplasm lines in total have been integrated. These SNPs are being utilized for trait improvement using genotype to phenotype prediction approaches developed in-house. In order to browse and access NGS data easily, we have also developed an NGS resequencing data browser ( http://soykb.org/NGS_Resequence/NGS_index.php ) within SoyKB to provide easy access to SNP and downstream analysis results for soybean researchers. CONCLUSION: PGen workflow has been optimized for the most efficient analysis of soybean data using thorough testing and validation. This research serves as an example of best practices for development of genomics data analysis workflows by integrating remote HPC resources and efficient data management with ease of use for biological users. PGen workflow can also be easily customized for analysis of data in other species.

Evaluation of genetic variation among Brazilian soybean cultivars through genome resequencing.

BACKGROUND: Soybean [Glycine max (L.) Merrill] is one of the most important legumes cultivated worldwide, and Brazil is one of the main producers of this crop. Since the sequencing of its reference genome, interest in structural and allelic variations of cultivated and wild soybean germplasm has grown. To investigate the genetics of the Brazilian soybean germplasm, we selected soybean cultivars based on the year of commercialization, geographical region and maturity group and resequenced their genomes. RESULTS: We resequenced the genomes of 28 Brazilian soybean cultivars with an average genome coverage of 14.8X. A total of 5,835,185 single nucleotide polymorphisms (SNPs) and 1,329,844 InDels were identified across the 20 soybean chromosomes, with 541,762 SNPs, 98,922 InDels and 1,093 CNVs that were exclusive to the 28 Brazilian cultivars. In addition, 668 allelic variations of 327 genes were shared among all of the Brazilian cultivars, including genes related to DNA-dependent transcription-elongation, photosynthesis, ATP synthesis-coupled electron transport, cellular respiration, and precursors of metabolite generation and energy. A very homogeneous structure was also observed for the Brazilian soybean germplasm, and we observed 41 regions putatively influenced by positive selection. Finally, we detected 3,880 regions with copy-number variations (CNVs) that could help to explain the divergence among the accessions evaluated. CONCLUSIONS: The large number of allelic and structural variations identified in this study can be used in marker-assisted selection programs to detect unique SNPs for cultivar fingerprinting. The results presented here suggest that despite the diversification of modern Brazilian cultivars, the soybean germplasm remains very narrow because of the large number of genome regions that exhibit low diversity. These results emphasize the need to introduce new alleles to increase the genetic diversity of the Brazilian germplasm.

Effect of lipo-chitooligosaccharide on early growth of C4 grass seedlings.

Although lipo-chitooligosaccharides (LCOs) are important signal molecules for plant-symbiont interactions, a number of reports suggest that LCOs can directly impact plant growth and development, separate from any role in plant symbioses. In order to investigate this more closely, maize and Setaria seedlings were treated with LCO and their growth was evaluated. The data indicate that LCO treatment significantly enhanced root growth. RNA-seq transcriptomic analysis of LCO-treated maize roots identified a number of genes whose expression was significantly affected by the treatment. Among these genes, some LCO-up-regulated genes are likely involved in root growth promotion. Interestingly, some stress-related genes were down-regulated after LCO treatment, which might indicate reallocation of resources from defense responses to plant growth. The promoter activity of several LCO-up-regulated genes using a ß-glucuronidase reporter system was further analysed. The results showed that the promoters were activated by LCO treatment. The data indicate that LCO can directly impact maize root growth and gene expression.

Epigenome-wide inheritance of cytosine methylation variants in a recombinant inbred population.

Cytosine DNA methylation is one avenue for passing information through cell divisions. Here, we present epigenomic analyses of soybean recombinant inbred lines (RILs) and their parents. Identification of differentially methylated regions (DMRs) revealed that DMRs mostly cosegregated with the genotype from which they were derived, but examples of the uncoupling of genotype and epigenotype were identified. Linkage mapping of methylation states assessed from whole-genome bisulfite sequencing of 83 RILs uncovered widespread evidence for local methylQTL. This epigenomics approach provides a comprehensive study of the patterns and heritability of methylation variants in a complex genetic population over multiple generations, paving the way for understanding how methylation variants contribute to phenotypic variation.

Distinctive left-sided distribution of adrenergic-derived cells in the adult mouse heart.

Adrenaline and noradrenaline are produced within the heart from neuronal and non-neuronal sources. These adrenergic hormones have profound effects on cardiovascular development and function, yet relatively little information is available about the specific tissue distribution of adrenergic cells within the adult heart. The purpose of the present study was to define the anatomical localization of cells derived from an adrenergic lineage within the adult heart. To accomplish this, we performed genetic fate-mapping experiments where mice with the cre-recombinase (Cre) gene inserted into the phenylethanolamine-n-methyltransferase (Pnmt) locus were cross-mated with homozygous Rosa26 reporter (R26R) mice. Because Pnmt serves as a marker gene for adrenergic cells, offspring from these matings express the ß-galactosidase (ßGAL) reporter gene in cells of an adrenergic lineage. ßGAL expression was found throughout the adult mouse heart, but was predominantly (89%) located in the left atrium (LA) and ventricle (LV) (p<0.001 compared to RA and RV), where many of these cells appeared to have cardiomyocyte-like morphological and structural characteristics. The staining pattern in the LA was diffuse, but the LV free wall displayed intermittent non-random staining that extended from the apex to the base of the heart, including heavy staining of the anterior papillary muscle along its perimeter. Three-dimensional computer-aided reconstruction of XGAL+ staining revealed distribution throughout the LA and LV, with specific finger-like projections apparent near the mid and apical regions of the LV free wall. These data indicate that adrenergic-derived cells display distinctive left-sided distribution patterns in the adult mouse heart.

Tracking multiple occluding people by localizing on multiple scene planes.

Occlusion and lack of visibility in crowded and cluttered scenes make it difficult to track individual people correctly and consistently, particularly in a single view. We present a multi-view approach to solving this problem. In our approach we neither detect nor track objects from any single camera or camera pair; rather evidence is gathered from all the cameras into a synergistic framework and detection and tracking results are propagated back to each view. Unlike other multi-view approaches that require fully calibrated views our approach is purely image-based and uses only 2D constructs. To this end we develop a planar homographic occupancy constraint that fuses foreground likelihood information from multiple views, to resolve occlusions and localize people on a reference scene plane. For greater robustness this process is extended to multiple planes parallel to the reference plane in the framework of plane to plane homologies. Our fusion methodology also models scene clutter using the Schmieder and Weathersby clutter measure, which acts as a confidence prior, to assign higher fusion weight to views with lesser clutter. Detection and tracking are performed simultaneously by graph cuts segmentation of tracks in the space-time occupancy likelihood data. Experimental results with detailed qualitative and quantitative analysis, are demonstrated in challenging multi-view, crowded scenes.