Biology subjects

Blackburn, J.

Publications and source records attributed to Blackburn, J..

18 recordsLinked to original sources

bioRxiv2026

NHE1 sustains leukemia-propagating capacity and mitochondrial fitness in T-cell acute lymphoblastic leukemia

Leukemia-propagating cells (LPCs) sustain T-cell acute lymphoblastic leukemia (T-ALL) and are thought to contribute to disease progression and relapse. However, the lack of reliable LPC markers has hindered efforts to identify their vulnerabilities. We screened 786 FDA-approved compounds using a zebrafish transplantation assay that directly measures leukemia-propagating activity. Amiloride markedly reduced functional LPC frequency (p < 0.0001) without significantly delaying leukemia onset at higher transplantation doses. Because amiloride inhibits the sodium-hydrogen exchanger NHE1, we investigated its role in T-ALL. NHE1 knockdown reduced self-renewal-associated colony formation by >70% human T-ALL cells, with only modest increases in apoptosis. Transcriptomic and proteomic analyses revealed extensive molecular remodeling, including reduced abundance of proteins involved in mitochondrial respiration and translation. Amiloride treatment and NHE1 knockdown impaired mitochondrial respiration and reduced mitochondrial content, while NHE1 knockdown also decreased mitochondrial membrane potential. Metabolomic profiling revealed alterations in TCA cycle intermediates and broader cellular metabolism. In mouse xenografts, NHE1 knockdown reduced leukemia involvement in the bone marrow and spleen despite similar overall leukemia progression and survival. Together, these findings implicate NHE1 as a previously unrecognized regulator of T-ALL leukemia-propagating activity, reveal unexpected contributions of this ion exchanger to mitochondrial fitness and leukemia involvement in hematopoietic tissues, and support further investigation of NHE1 as a potential therapeutic target in T-ALL.

cancer biology↗

bioRxiv2025

Disruption of the Wnt-antagonist Apc in the pituitary stem cells drives the development of adamantinomatous craniopharyngioma.

Adamantinomatous craniopharyngiomas (aCPs) are complex intracranial neoplasms that generally arise in the sellar and suprasellar region of the brain which affect the endocrine and nervous systems causing severe sequelae. Activating mutations resulting in degradation-resistant forms of {beta}-catenin (CTNNB1) have been shown to be the main driver for many of these neoplasms. However, the underlying genetic driver for a proportion of these tumours is still unknown. Using murine transgenic models, we show that genetic disruption of the Wnt-antagonist and tumour suppressor, adenomatous polyposis coli (Apc), within the pituitary progenitors/stem cells, leads to pituitary tumours that closely resemble human aCPs. These tumours present classic histopathological hallmarks of aCPs, such as large deposits of wet keratin, stellar-reticular-like cells, large cystic components and clusters of accumulating nucleo-cytoplasmic {beta}-catenin that are slow-dividing and exhibit a secretory phenotype. We show that a hypomorphic allele of Apc is sufficient for tumour development, indicating that disruption of Apc function can lead to aCP formation. Moreover, we identify that bi-allelic loss of Apc in the Sox2+ve pituitary stem cells is sufficient to initiate tumour formation, indicating that Sox2+ve stem cells are the cell origin of these Apc-driven aCPs. Transcriptomic analyses of early tumour-initiating cells revealed that Apc-driven clusters of accumulating {beta}-catenin undergo senescence-associated secretory phenotype (SASP), which is p21-mediated and results in secretion of inflammasome, angiosome and developmental growth factors. Our data unequivocally show a causal role for the disruption of the tumour suppressor Apc as a primary driver of aCPs independent of mutations in {beta}-catenin. We provide murine models representing a novel genetic subtype of human aCPs offering insights into aCP pathogenesis. Our work reinforces the importance of genetic testing for mutations in APC in patients with aCPs and identifies a potential need to screen patients with familial adenomatous polyposis (FAP) or spectrum of APC-pathogenic syndromes for aCPs in early life.

cancer biology↗

bioRxiv2025

Protein Kinase C δ: a critical hub regulating macrophage immunomodulatory functions during Mycobacterium tuberculosis infection

A host-modulating candidate gene involved in putative pathogen-killing pathways, with potential novel therapeutic intervention, Protein Kinase C - {delta} (PKC{delta}) has been recognized as a critical marker of inflammation with clinical and experimental evidence in recent years. Pulmonary microenvironment during Mtb infection is largely governed by lung resident macrophages, initiating innate and subsequent adaptive immune responses. We investigated the role of PKC{delta} in macrophages using a macrophage-specific PKC{delta} knockout mice model (LysMcrePKC{delta}flox/flox). PKC{delta} deficiency in macrophages triggers an early lymphocytic immune response, increases neutrophil recruitment, and reduces inflammatory macrophages in the lungs, leading to higher Mtb burden and exacerbated pathology. Experimental and omics analysis further revealed that dysregulation of antimicrobial effector functions is detrimental to macrophages ability to restrict bacterial growth in vitro. Importantly this defect was mitigated by exogenous GM-CSF supplementation and/or overexpressing PKC{delta} in macrophages. Thus, PKC{delta} plays a crucial role in immune modulation during Mtb infection with GM-CSF amongst several downstream pathways through which PKC{delta} exerts its regulatory effects. TeaserPKC{delta} is crucial for immune modulation during Mtb infection revealing macrophages as a potential axis of signaling.

immunology↗

bioRxiv2024

The conserved landscape of RNA modifications and transcript diversity across mammalian evolution

Gene expression programs underpin the development of shared phenotypes, yet the importance of transcript complexity in shaping mammalian evolution remains unclear. Here we present a comprehensive long-read direct RNA sequencing atlas of full-length transcripts and their m6A modifications across six tissues (hippocampus, frontal cortex, cerebellum, testes, skeletal muscle, and liver) in five mammals (human, mouse, rat, dog, and cow) and a non-mammalian out-group (chicken). Our analysis reveals that 29% of genes have multiple mammalian-conserved alternative transcripts, with 31% of these genes showing tissue-specific switching of the major transcript isoforms. We uncover extensive conservation of coordinated splicing events, primarily driven by tissue-specific mutually associated exon splicing, particularly in neural tissues and cytoskeletal genes. At the epitranscriptome level, we find that 14.2% of m6A RNA modifications are conserved across mammals, with 39% of analysed genes containing a conserved m6A RNA modification. Our work provides unprecedented insight into the evolution of transcript complexity and the epitranscriptome, highlighting their potential roles in shaping mammalian phenotypic diversity and providing a valuable resource for understanding post-transcriptional regulation across mammalian evolutionary time.

genomics↗

bioRxiv2023

Chronic striatal cholinergic interneuron excitation induces clinically-relevant dystonic behavior in mice

Dystonia is common, debilitating, often medically refractory, and difficult to diagnose. The gold standard for both clinical and mouse model dystonia evaluation is subjective assessment, ideally by expert consensus. However, this subjectivity makes translational quantification of clinically-relevant dystonia metrics across species nearly impossible. Many mouse models of genetic dystonias display abnormal striatal cholinergic interneuron excitation, but few display subjectively dystonic features. Therefore, whether striatal cholinergic interneuron pathology causes dystonia remains unknown. To address these critical limitations, we first demonstrate that objectively quantifiable leg adduction variability correlates with leg dystonia severity in people. We then show that chemogenetic excitation of striatal cholinergic interneurons in mice causes comparable leg adduction variability in mice. This clinically-relevant dystonic behavior in mice does not occur with acute excitation, but rather develops after 14 days of ongoing striatal cholinergic interneuron excitation. This requirement for prolonged excitation recapitulates the clinically observed phenomena of a delay between an inciting brain injury and subsequent dystonia manifestation and demonstrates a causative link between chronic striatal cholinergic interneuron excitation and clinically-relevant dystonic behavior in mice. Therefore, these results support targeting striatal ChIs for dystonia drug development and suggests early treatment in the window following injury but prior to dystonia onset. One Sentence SummaryChronic excitation of dorsal striatal cholinergic interneuron causes clinically-relevant dystonic phenotypes in mice

neuroscience↗

bioRxiv2023

Enamel proteins reveal biological sex and genetic variability within southern African Paranthropus

The evolutionary relationships among extinct African hominin taxa are highly debated and largely unresolved, due in part to a lack of molecular data. Even within taxa, it is not always clear, based on morphology alone, whether ranges of variation are due to sexual dimorphism versus potentially undescribed taxonomic diversity. For Paranthropus robustus, a Pleistocene hominin found only in South Africa, both phylogenetic relationships to other taxa 1,2 and the nature of intraspecific variation 3-6 are still disputed. Here we report the mass spectrometric (MS) sequencing of enamel proteomes from four ca. 2 million year (Ma) old dental specimens attributed morphologically to P. robustus, from the site of Swartkrans. The identification of AMELY-specific peptides and semi-quantitative MS data analysis enabled us to determine the biological sex of all the specimens. Our combined molecular and morphometric data also provide compelling evidence of a significant degree of variation within southern African Paranthropus, as previously suggested based on morphology alone 6. Finally, the molecular data also confirm the taxonomic placement of Paranthropus within the hominin clade. This study demonstrates the feasibility of recovering informative Early Pleistocene hominin enamel proteins from Africa. Crucially, it also shows how the analysis of these proteins can contribute to understanding whether hominin morphological variation is due to sexual dimorphism or to taxonomic differences. We anticipate that this approach can be widely applied to geologically-comparable sites within South Africa, and possibly more broadly across the continent.

evolutionary biology↗

bioRxiv2022

Auto-Immunoproteomics Analysis of COVID-19 ICU Patients Revealed Increased Levels of Autoantibodies Related to Male Reproductive System

The role of autoantibodies in coronavirus disease (COVID-19) complications is not yet fully understood. The current investigation screened two independent cohorts of 97 COVID-19 patients (Discovery (Disc) cohort from Qatar (n = 49) and Replication (Rep) cohort from New York (n = 48)) utilizing high-throughput KoRectly Expressed (KREX) immunome protein-array technology. Autoantibody responses to 57 proteins were significantly altered in the COVID-19 Disc cohort compared to healthy controls (P [≤] 0.05). The Rep cohort had altered autoantibody responses against 26 proteins compared to non-COVID-19 ICU patients that served as controls. Both cohorts showed substantial similarities (r2 = 0.73) and exhibited higher autoantibodies responses to numerous transcription factors, immunomodulatory proteins, and human disease markers. Analysis of the combined cohorts revealed elevated autoantibody responses against SPANXN4, STK25, ATF4, PRKD2, and CHMP3 proteins in COVID-19 patients. KREX analysis of the specific IgG autoantibody responses indicates that the targeted host proteins are supposedly increased in COVID-19 patients. The autoantigen-autoantibody response was cross-validated for SPANXN4 and STK25 proteins using Uniprot BLASTP and sequence alignment tools. SPANXN4 is essential for spermiogenesis and male fertility, which may predict a potential role for this protein in COVID-19 associated male reproductive tract complications and warrants further research. Significance StatementCoronavirus disease (COVID-19), caused by the SARS-CoV-2 virus, has emerged as a global pandemic with a high morbidity rate and multiorgan complications. It is observed that the host immune system contributes to the varied responses to COVID-19 pathogenesis. Autoantibodies, immune system proteins that mistakenly target the bodys own tissue, may underlie some of this variation. We screened total IgG autoantibody responses against 1,318 human proteins in two COVID-19 patient cohorts. We observed several novel markers in COVID-19 patients that are associated with male fertility, such as sperm protein SPANXN4, STK25, and the apoptotic factor ATF4. Particularly, elevated levels of autoantibodies against the testicular tissue-specific protein SPANXN4 offer significant evidence of anticipating the protein role in COVID-19 associated male reproductive complications.

immunology↗

bioRxiv2021

Nanopore sequencing methods detect cell-free DNA associated with minimal residual disease and central nervous system infiltration in pediatric Acute Lymphoblastic Leukemia

Acute Lymphoblastic Leukemia (ALL) patients that are positive for minimal residual disease (MRD) after therapy or have leukemic infiltration into the central nervous system (CNS) are considered high risk and receive intensive chemotherapy regimens. Current methods to diagnose MRD and CNS infiltration rely on detecting leukemic cells in patient samples using pathology, flow cytometry, or next-generation sequencing. However, leukemic blasts may persist in the patient but not be physically present in bone marrow biopsy or biofluid sample, leading to inaccurate or delayed patient diagnosis. We have developed a nanopore sequencing workflow to detect B-ALL-associated cell-free DNA (cfDNA) in blood and cerebrospinal fluid (CSF) samples. Quantitation of B-cell specific VDJ recombination events in cfDNA samples defined B-ALL clonal heterogeneity. This workflow allowed us to track the response of individual B-ALL clones throughout treatment. Detection of cfDNA also predicted the clinical diagnosis of MRD and CNS disease. Importantly, we identified patients diagnosed as CNS negative who had low B-cell derived cell-free DNA levels in their CSF sample that correlated with B-cell clones present in the bone marrow. These data suggest that cfDNA assays may be useful in detecting the presence of ALL in the patient even when blasts are not in the biofluid sample. Nanopore analysis of cell-free DNA is a simple, rapid, and inexpensive assay that can serve as a valuable complement to traditional clinical diagnostic approaches for ALL. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=50 SRC="FIGDIR/small/462067v2_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@1373bd3org.highwire.dtl.DTLVardef@138b2e3org.highwire.dtl.DTLVardef@168661forg.highwire.dtl.DTLVardef@1088626_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

Two transcriptionally distinct pathways drive female development in a reptile with both genetic and temperature dependent sex determination

How temperature determines sex remains unknown. A recent hypothesis proposes that conserved cellular mechanisms (calcium and redox; CaRe status) sense temperature and identify genes and regulatory pathways likely to be involved in driving sexual development. We take advantage of the unique sex determining system of the model organism, Pogona vitticeps, to assess predictions of this hypothesis. P. vitticeps has ZZ male: ZW female sex chromosomes whose influence can be overridden in genetic males by high temperatures, causing male-to-female sex reversal. We compare a developmental transcriptome series of ZWf females and temperature sex reversed ZZf females. We demonstrate that early developmental cascades differ dramatically between genetically driven and thermally driven females, later converging to produce a common outcome (ovaries). We show that genes proposed as regulators of thermosensitive sex determination play a role in temperature sex reversal. Our study greatly advances the search for the mechanisms by which temperature determines sex. Author SummaryIn many reptiles and fish, environment can determine, or influence, the sex of developing embryos. How this happens at a molecular level that has eluded resolution for half a century of intensive research. We studied the bearded dragon, a lizard that has sex chromosomes (ZZ male and ZW female), but in which that temperature can override ZZ sex chromosomes to cause male to female sex reversal. This provides an unparalleled opportunity to disentangle, in the same species, the biochemical pathways required to make a female by these two different routes. We sequenced the transcriptomes of gonads from developing ZZ reversed and normal ZW dragon embryos and discovered that different sets of genes are active in ovary development driven by genotype or temperature. Females whose sex was initiated by temperature showed a transcriptional profile consistent with the recently-proposed Calcium-Redox hypotheses of cellular temperature sensing. These findings are an important for understanding how the environment influences the development of sex, and more generally how the environment can epigenetically modify the action of genes.

genetics↗

bioRxiv2021

A relative-motion method for parsing spatio-temporal behaviour of dyads using GPS relocation data

O_LIIn this paper, we introduce a novel method for classifying and computing the frequencies of movement modes of intra and interspecific dyads, focusing in particular on distance-mediated approach, retreat, following and side by side movement modes. C_LIO_LIBesides distance, the method includes factors such as sex, age, time of day, or season that cause frequencies of movement modes to deviate from random. C_LIO_LIWe demonstrate and validate our method using both simulated and empirical data. Our simulated data were obtained from a relative-motion, biased random-walk (RM-BRW) model with attraction and repulsion circumferences. Our empirical data were GPS relocation time series collected from African elephants in Etosha National Park, Namibia. The simulated data were primarily used to validate our method while the empirical data analysis were used to illustrate the types of behavioral assessment that our methodology reveals. C_LIO_LIOur methodology facilitates automated, observer-bias-free analysis of the locomotive interactions of dyads using GPS relocation data, which is becoming increasingly ubiquitous as telemetry and related technologies improve. Our method should open up a whole new vista of behavioral-interaction type analyses to movement and behavioral ecologists. C_LI

ecology↗

bioRxiv2019

Protein Tyrosine Phosphatase 4A3 (PTP4A3) modulates Src signaling in T-cell Acute Lymphoblastic Leukemia to promote leukemia migration and progression

T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive blood cancer, and currently, there are no immunotherapies or molecularly targeted therapeutics available for treatment of this malignancy. The identification and characterization of genes and pathways that drive T-ALL progression is critical for development of new therapies for T-ALL. Here, we determined that Protein Tyrosine Phosphatase 4A3 (PTP4A3) plays a critical role in disease initiation and progression by promoting cell migration in T-ALL. PTP4A3 expression was upregulated in patient T-ALL samples at both the mRNA and protein levels compared to normal lymphocytes. Inhibition of PTP4A3 function with a small molecule inhibitor and knock-down of PTP4A3 expression using short-hairpin RNA (shRNA) in human T-ALL cells significantly impeded T-ALL cell migration capacity in vitro and reduced their ability to engraft and proliferate in vivo in xenograft mouse models. Additionally, PTP4A3 overexpression in a Myc-induced zebrafish T-ALL model significantly accelerated disease onset and shortened the time needed for cells to enter blood circulation. Reverse phase protein array (RPPA) revealed that manipulation of PTP4A3 expression levels in T-ALL cells directly affected the SRC signaling pathway, which plays a well-characterized role in migratory behavior of several cell types. Taken together, our study revealed that PTP4A3 is a key regulator of T-ALL migration via SRC signaling, and suggests that PTP4A3 plays an important role as an oncogenic driver in T-ALL.\n\nHighlightsO_LIA subset of T-cell Acute Lymphoblastic Leukemia (T-ALL) highly express the phosphatase PTP4A3\nC_LIO_LIPTP4A3 expression promotes leukemia development in zebrafish T-ALL models\nC_LIO_LILoss of PTP4A3 prevents T-ALL engraftment in mouse xenograft models\nC_LIO_LIKnock-down or small molecule inhibition of PTP4A3 prevents T-ALL migration in part via modulation of SRC signaling.\nC_LI

cancer biology↗

bioRxiv2019

MetaNovo: a probabilistic approach to peptide and polymorphism discovery in complex mass spectrometry datasets

BackgroundMicrobiome research is providing important new insights into the metabolic interactions of complex microbial ecosystems involved in fields as diverse as the pathogenesis of human diseases, agriculture and climate change. Poor correlations typically observed between RNA and protein expression datasets make it hard to accurately infer microbial protein synthesis from metagenomic data. Additionally, mass spectrometry-based metaproteomic analyses typically rely on focussed search libraries based on prior knowledge for protein identification that may not represent all the proteins present in a set of samples. Metagenomic 16S rRNA sequencing will only target the bacterial component, while whole genome sequencing is at best an indirect measure of expressed proteomes. We describe a novel approach, MetaNovo, that combines existing open-source software tools to perform scalable de novo sequence tag matching with a novel algorithm for probabilistic optimization of the entire UniProt knowledgebase to create tailored databases for target-decoy searches directly at the proteome level, enabling analyses without prior expectation of sample composition or metagenomic data generation, and compatible with standard downstream analysis pipelines. ResultsWe compared MetaNovo to published results from the MetaPro-IQ pipeline on 8 human mucosal-luminal interface samples, with comparable numbers of peptide and protein identifications, many shared peptide sequences and a similar bacterial taxonomic distribution compared to that found using a matched metagenome database - but simultaneously identified many more non-bacterial peptides than the previous approaches. MetaNovo was also benchmarked on samples of known microbial composition against matched metagenomic and whole genomic database workflows, yielding many more MS/MS identifications for the expected taxa, with improved taxonomic representation, while also highlighting previously described genome sequencing quality concerns for one of the organisms, and identifying a known sample contaminant without prior expectation. ConclusionsBy estimating taxonomic and peptide level information directly on microbiome samples from tandem mass spectrometry data, MetaNovo enables the simultaneous identification of peptides from all domains of life in metaproteome samples, bypassing the need for curated sequence search databases. We show that the MetaNovo approach to mass spectrometry metaproteomics is more accurate than current gold standard approaches of tailored or matched genomic database searches, can identify sample contaminants without prior expectation and yields insights into previously unidentified metaproteomic signals, building on the potential for complex mass spectrometry metaproteomic data to speak for itself. The pipeline source code is available on GitHub1 and documentation is provided to run the software as a singularity-compatible docker image available from the Docker Hub2.

bioinformatics↗

bioRxiv2019

Targeted, High-Resolution RNA Sequencing Of Non-Coding Genomic Regions Associated With Neuropsychiatric Functions

The human brain is one of the last frontiers of biomedical research. Genome-wide association studies (GWAS) have succeeded in identifying thousands of haplotype blocks associated with a range of neuropsychiatric traits, including disorders such as schizophrenia, Alzheimers and Parkinsons disease. However, the majority of single nucleotide polymorphisms (SNPs) that mark these haplotype blocks fall within non-coding regions of the genome, hindering their functional validation. While some of these GWAS loci may contain cis-acting regulatory DNA elements such as enhancers, we hypothesized that many are also transcribed into non-coding RNAs that are missing from publicly available transcriptome annotations. Here, we use targeted RNA capture ( RNA CaptureSeq) in combination with nanopore long-read cDNA sequencing to transcriptionally profile 1,023 haplotype blocks across the genome containing non-coding GWAS SNPs associated with neuropsychiatric traits, using post-mortem human brain tissue from three neurologically healthy donors. We find that the majority (62%) of targeted haplotype blocks, including 13% of intergenic blocks, are transcribed into novel, multi-exonic RNAs, most of which are not yet recorded in GENCODE annotations. We validated our findings with short-read RNA-seq, providing orthogonal confirmation of novel splice junctions and enabling a quantitative assessment of the long-read assemblies. Many novel transcripts are supported by independent evidence of transcription including cap analysis of gene expression (CAGE) data and epigenetic marks, and some show signs of potential functional roles. We present these transcriptomes as a preliminary atlas of non-coding transcription in human brain that can be used to connect neurological phenotypes with gene expression.

genomics↗

bioRxiv2019

Exploring Environmental Coverages of Species: A New Variable Selection Methodology for Rulesets from the Genetic Algorithm for Ruleset Prediction

Variable selection for, and determination of variable importance within, species distribution models (SDMs) remain an important area of research with continuing challenges. Most SDM algorithms provide normally exhaustive searches through variable space, however, selecting variables to include in models is a first challenge. The estimation of the explanatory power of variables and the selection of the most appropriate variable set within models can be a second challenge. Although some SDMs incorporate the variable selection rubric inside the algorithms, there is no integrated rubric to evaluate the variable importance in the Genetic Algorithm for Ruleset Production (GARP). Here, we designed a novel variable selection methodology based on the rulesets generated from a GARP experiment. The importance of the variables in a GARP experiment can be estimated based on the consideration of the prevalence of each environmental variable in the dominant presence rules of the best subset of models and its coverage. We tested the performance of this variable selection method based on simulated species with both weak and strong responses to simulated environmental covariates. The variable selection method generally performed well during the simulations with over 2/3 of the trials correctly identifying most covariates. We then predict the distribution of Bacillus anthracis (the bacterium that causes anthrax) in the continental United States (US) and apply our variable selection procedure as a real-world example. We found that the distribution of B. anthracis was primarily determined by organic content, soil pH, calcic vertisols, vegetation, sand fraction, elevation, and seasonality in temperature and moisture.

ecology↗

bioRxiv2018

High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes

High-throughput single-cell RNA-Sequencing is a powerful technique for gene expression profiling of complex and heterogeneous cellular populations such as the immune system. However, these methods only provide short-read sequence from one end of a cDNA template, making them poorly suited to the investigation of gene-regulatory events such as mRNA splicing, adaptive immune responses or somatic genome evolution. To address this challenge, we have developed a method that combines targeted long-read sequencing with short-read based transcriptome profiling of barcoded single cell libraries generated by droplet-based partitioning. We use Repertoire And Gene Expression sequencing (RAGE-seq) to accurately characterize full-length T cell (TCR) and B cell (BCR) receptor sequences and transcriptional profiles of more than 7,138 lymphocytes sampled from the primary tumour and draining lymph node of a breast cancer patient. With this method we show that somatic mutation, alternate splicing and clonal evolution of T and B lymphocytes can be tracked across these tissue compartments. Our results demonstrate that RAGE-Seq is an accessible and cost-effective method for high-throughput deep single cell profiling, applicable to a wide range of biological challenges.

genomics↗

bioRxiv2018

Chiral DNA sequences as commutable reference standards for clinical genomics

Chirality is a geometric property describing any object that is inequivalent to a mirror image of itself. Due to its 5-3 directionality, a DNA sequence is distinct from a mirrored sequence arranged in reverse nucleotide order, and is therefore chiral. A given sequence and its opposing chiral partner sequence share many properties, such as nucleotide composition and sequence entropy. Here we demonstrate that chiral DNA sequence pairs also perform equivalently during molecular and bioinformatic techniques that underpin modern genetic analysis, including PCR amplification, hybridization, whole-genome, target-enriched and nanopore sequencing, sequence alignment and variant detection. Given these shared properties, synthetic DNA sequences that directly mirror clinically relevant and/or analytically challenging regions of the human genome are ideal reference standards for clinical genomics. We show how the addition of chiral DNA standards to patient tumor samples can prevent false-positive and false-negative mutation detection and, thereby, improve diagnosis. Accordingly, we propose that chiral DNA standards can fulfill the unmet need for commutable internal reference standards in precision medicine.

genomics↗

bioRxiv2017

Spores and soil from six sides: interdisciplinarity and the environmental biology of anthrax (Bacillus anthracis)

Environmentally Transmitted Diseases Are Comparatively Poorly Understood And Managed, And Their Ecology Is Particularly Understudied. Here We Identify Challenges Of Studying Environmental Transmission And Persistence With A Six-Sided Interdisciplinary Review Of The Biology Of Anthrax (Bacillus Anthracis). Anthrax Is A Zoonotic Disease Capable Of Maintaining Infectious Spore Banks In Soil For Decades (Or Even Potentially Centuries), And The Mechanisms Of Its Environmental Persistence Have Been The Topic Of Significant Research And Controversy. Where Anthrax Is Endemic, It Plays An Important Ecological Role, Shaping The Dynamics Of Entire Herbivore Communities. The Complex Eco-Epidemiology Of Anthrax, And The Mysterious Biology Of Bacillus Anthracis During Its Environmental Stage, Have Necessitated An Interdisciplinary Approach To Pathogen Research. Here, We Illustrate Different Disciplinary Perspectives Through Key Advances Made By Researchers Working In Etosha National Park, A Long-Term Ecological Research Site In Namibia That Has Exemplified The Complexities Of AnthraxS Enzootic Process Over Decades Of Surveillance. In Etosha, The Role Of Scavengers And Alternate Routes (Waterborne Transmission And Flies) Has Proved Unimportant, Relative To The Long-Term Persistence Of Anthrax Spores In Soil And Their Infection Of Herbivore Hosts. Carcass Deposition Facilitates Green-Ups Of Vegetation To Attract Herbivores, Potentially Facilitated By Anthrax Spores Role In The Rhizosphere. The Underlying Seasonal Pattern Of Vegetation, And Herbivores Immune And Behavioral Responses To Anthrax Risk, Interact To Produce Regular \"Anthrax Seasons\" That Appear To Be A Stable Feature Of The Etosha Ecosystem. Through The Lens Of Microbiologists, Geneticists, Immunologists, Ecologists, Epidemiologists, And Clinicians, We Discuss How Anthrax Dynamics Are Shaped At The Smallest Scale By Population Genetics And Interactions Within The Bacterial Communities Up To The Broadest Scales Of Ecosystem Structure. We Illustrate The Benefits And Challenges Of This Interdisciplinary Approach To Disease Ecology, And Suggest Ways Anthrax Might Offer Insights Into The Biology Of Other Important Pathogens. Bacillus Anthracis, And The More Recently Emerged Bacillus Cereus Biovar Anthracis, Share Key Features With Other Environmentally-Transmitted Pathogens, Including Several Zoonoses And Panzootics Of Special Interest For Global Health And Conservation Efforts. Understanding The Dynamics Of Anthrax, And Developing Interdisciplinary Research Programs That Explore Environmental Persistence, Is A Critical Step Forward For Understanding These Emerging Threats.

ecology↗

bioRxiv2017

Universal Alternative Splicing Of Noncoding Exons

The human transcriptome is so large, diverse and dynamic that, even after a decade of investigation by RNA sequencing (RNA-Seq), we are yet to resolve its true dimensions. RNA-Seq suffers from an expression-dependent bias that impedes characterization of low-abundance transcripts. We performed targeted single-molecule and short-read RNA-Seq to survey the transcriptional landscape of a single human chromosome (Hsa21) at unprecedented resolution. Our analysis reaches the lower limits of the transcriptome, identifying a fundamental distinction between protein-coding and noncoding gene content: almost every noncoding exon undergoes alternative splicing, producing a seemingly limitless variety of isoforms. Analysis of syntenic regions of the mouse genome shows that few noncoding exons are shared between human and mouse, yet human splicing profiles are recapitulated on Hsa21 in mouse cells, indicative of regulation by a deeply conserved splicing code. We propose that noncoding exons are functionally modular, with alternative splicing generating an enormous repertoire of potential regulatory RNAs and a rich transcriptional reservoir for gene evolution.

genomics↗