Biology subjects

Bennett, R.

Publications and source records attributed to Bennett, R..

12 recordsLinked to original sources

bioRxiv2026

A chromosome-level genome assembly of Thecaphora frezzii, cause of peanut smut, reveals the largest genome among the true smut fungi

Peanut smut, caused by the fungus Thecaphora frezzii, is a significant disease of peanuts in Argentina. Infected plants have seeds replaced by a mass of dark teliospores, reducing yield and seed quality. To prevent the spread of the pathogen, several countries have limited import of raw peanuts from Argentina, a major grower and exporter. Following successful in vitro culture of the fungus in its haploid stage, we produced a chromosome-level genome assembly of the species for the first time. We compare this genome with those of 49 other species of true smut fungi, or Ustilaginomycetes, including species of medical, agricultural, and industrial importance, some of which are known as pathogens and others only as saprotrophic yeasts. At almost 39 Mb, T. frezzii has the largest genome of the smut fungi sequenced to date and the highest repetitive content. While it shares some core effectors with species of the distantly related and better studied Ustilago and related fungi, predicted effectors only found in T. frezzii or in Thecaphora suggest unique infection strategies. Comparisons among the 50 smut genomes also show that the 14 smut fungi observed only as yeasts share genomic traits such as low repeat content and generally smaller genomes. This supports the hypothesis that some smut fungi are adapted to saprotrophic growth as yeasts. The high-quality, annotated genome for T. frezzii will be a valuable resource for investigating the population dynamics and evolution of an economically important pathogen, as well as illuminating an understudied clade of smut fungi. Article summaryPeanut smut is a destructive and costly disease of peanuts in Argentina. For the first time, a high quality, annotated genome is presented for the causal agent, Thecaphora frezzii. This fungus has the largest and most repetitive genome of the true smut fungi, thus prompting comparison with 49 other species of smut fungi with available genomes, including non-pathogenic ones. While it shares some likely pathogenicity factors with well-studied smuts, it has many unique genes, a trait reflective of its evolutionary distance and likely novel mechanism for infection. This important genomic resource will benefit research regarding the evolution and adaption of T. frezzii, the development of diagnostic tools that enable rapid detection of it, and the study of smut fungi broadly.

genomics↗

bioRxiv2024

An epigenetic clock for Xenopus tropicalis reveals age-associated sites enriched in H4K20me3-marked heterochromatin

DNA methylation clocks have been widely used for accurate age prediction, but most studies have been carried out on mammals. Here we present a clock for the aquatic frog Xenopus tropicalis, a widely used model organism in developmental biology and genomics. The clock, trained on targeted bisulfite sequencing data, achieved a cross-validation median absolute error of 0.70 years. To construct the clock, we collected DNA methylation data from 192 frogs at genomic regions containing CpG sites previously shown to have age-associated methylation in Xenopus. We found that CpG sites with methylation levels significantly increasing or decreasing with age are enriched in heterochromatic regions marked with H4K20me3 and H3K9me3. CpG sites with positively age-associated methylation levels are enriched in bivalent chromatin and gene bodies with H3K36me3, and tend to be proximal to lowly expressed genes. These epigenetic features of aging are similar to those found in mammals, suggesting evolutionary conservation of epigenetic aging mechanisms. Our clock enables future aging biology experiments that leverage the unique properties of amphibians.

genomics↗

bioRxiv2024

GENCODE: massively expanding the lncRNA catalog through capture long-read RNA sequencing

Accurate and complete gene annotations are indispensable for understanding how genome sequences encode biological functions. For more than twenty years, the GENCODE consortium has developed reference annotations for the human and mouse genomes, becoming a foundation for biomedical and genomics communities worldwide. Nevertheless, collections of important yet poorly-understood gene classes like long non-coding RNAs (lncRNAs) remain incomplete and scattered across multiple, uncoordinated catalogs. To address this, GENCODE has undertaken the most comprehensive lncRNA annotation effort to date. This is founded on the manually supervised computational annotation of full-length targeted long-read sequencing, on matched embryonic and adult tissues, of orthologous regions in human and mouse. Altogether 17,931 human genes (140,268 transcripts) and 22,784 mouse genes (136,169 transcripts) have been added to the GENCODE catalog representing a 2-fold and 6-fold growth in transcripts, respectively - the greatest increase in the number of annotated human genes since the sequencing of the human genome. Our targeted design assigned human-mouse orthologs at a rate beyond previous studies, tripling the number of human disease-associated lncRNAs that have mouse orthologs. Novel lncRNA genes consistently exhibit biological signals of functionality, and they greatly enhance the functional interpretability of the human genome. While poorly expressed in bulk RNA-Seq samples, many of them are highly expressed in specific cell populations, maybe even contributing to cell-type determination. The expanded GENCODE lncRNA annotations mark a critical step toward deciphering the human and mouse genomes.

genomics↗

bioRxiv2024

Expression-based selection identifies a microglia-tropic AAV capsid for direct and CSF routes of administration in mice

Microglia are critical innate immune cells of the brain. In vivo targeting of microglia using gene-delivery systems is crucial for studying brain physiology and developing gene therapies for neurodegenerative diseases and other brain disorders such as NeuroAIDS. Historically, microglia have been extremely resistant to transduction by viral vectors, including adeno-associated virus (AAV) vectors. Recently, there has been some progress demonstrating the feasibility and potential of using AAV to transduce microglia after direct intraparenchymal vector injection. Data suggests that combining specific AAV capsids with microglia-specific gene expression cassettes to reduce neuron off-targeting will be key. However, no groups have developed AAV capsids for microglia transduction after intracerebroventricular (ICV) injection. The ICV route of administration has advantages such as increased brain biodistribution while avoiding issues related to systemic injection. Here, we performed an in vivo selection using an AAV peptide display library that enables recovery of capsids that mediate transgene expression in microglia. Using this approach, we identified a capsid, MC5, which mediated enhanced transduction of microglia after ICV injection compared to AAV9. Furthermore, MC5 enhanced both the efficiency (85%) and specificity (93%) of transduction compared to a recently described evolved AAV9 capsid for microglia targeting after direct injection into the brain parenchyma. Exploration of the use of MC5 in a mouse models of Alzheimers disease revealed transduced microglia surrounding and within plaques. Overall, our results demonstrate that the MC5 capsid is a useful gene transfer tool to target microglia in vivo by direct and ICV routes of administration.

bioengineering↗

bioRxiv2024

Molecular profiling of frontal and occipital subcortical white matter hyperintensities in Alzheimer's disease

White matter hyperintensities (WMHs) are commonly detected on T2-weighted magnetic resonance imaging (MRI) scans, occurring in both typical aging and Alzheimers disease. Despite their frequent appearance and their association with cognitive decline, the molecular factors contributing to WMHs remain unclear. In this study, we investigated the transcriptomic profiles of two commonly affected brain regions with coincident AD pathology--frontal subcortical white matter (frontal-WM) and occipital subcortical white matter (occipital-WM)--and compared with age-matched healthy controls. Through RNA-sequencing in frontal- and occipital-WM bulk tissues, we identified an upregulation of genes associated with brain vasculature function in AD white matter. To further elucidate vasculature-specific transcriptomic features, we performed RNA-seq analysis on blood vessels isolated from these white matter regions, which revealed an upregulation of genes related to protein folding pathways. Finally, comparing gene expression profiles between AD individuals with high-versus low-WMH burden showed an increased expression of pathways associated with immune function. Taken together, our study characterizes the diverse molecular profiles of white matter changes in AD compared to normal aging and provides new mechanistic insights processes underlying AD-related WMHs.

neuroscience↗

bioRxiv2024

Brain Vasculature Accumulates Tau and Is Spatially Related to Tau Tangle Pathology in Alzheimer's Disease

Insoluble pathogenic proteins accumulate along blood vessels in conditions of cerebral amyloid angiopathy (CAA), exerting a toxic effect on vascular cells and impacting cerebral homeostasis. In this work we provide new evidence from three-dimensional human brain histology that tau protein, the main component of neurofibrillary tangles, can similarly accumulate along brain vascular segments. We quantitatively assessed n=6 Alzheimers disease (AD), and n=6 normal aging control brains and saw that tau-positive blood vessel segments were present in all AD cases. Tau-positive vessels are enriched for tau at levels higher than the surrounding tissue and appear to affect arterioles across cortical layers (I-V). Further, vessels isolated from these AD tissues were enriched for N-terminal tau and tau phosphorylated at T181 and T217. Importantly, tau-positive vessels are associated with local areas of increased tau neurofibrillary tangles. This suggests that accumulation of tau around blood vessels may reflect a local clearance failure. In sum, these data indicate tau, like amyloid beta, accumulates along blood vessels and may exert a significant influence on vasculature in the setting of AD.

neuroscience↗

bioRxiv2022

SituSeq: An offline protocol for rapid and remote Nanopore amplicon sequence analysis

Microbiome analysis through 16S rRNA gene sequencing is a crucial tool for understanding the microbial ecology of any habitat or ecosystem. However, workflows require large equipment, stable internet, and extensive computing power such that most of the work is performed far away from sample collection in both space and time. Performing amplicon sequencing and analysis at sample collection would have positive implications in many instances including remote fieldwork and point-of-care medical diagnoses. Here we present SituSeq, an offline and portable workflow for the sequencing and analysis of 16S rRNA gene amplicons using the Nanopore MinION and a standard laptop computer. SituSeq was validated using the same environmental DNA to sequence Nanopore 16S rRNA gene amplicons, Illumina 16S rRNA gene amplicons, and Illumina metagenomes. Comparisons revealed consistent community composition, ecological trends, and sequence identity across platforms. Correlation between the abundance of phyla in Illumina and Nanopore data sets was high (Pearsons r = 0.9), and over 70% of Illumina 16S rRNA gene sequences matched a Nanopore sequence with greater than 97% sequence identity. On board a research vessel on the open ocean, SituSeq was used to analyze amplicon sequences from deep sea sediments less than two hours after sequencing, and eight hours after sample collection. The rapidly available results informed decisions about subsequent sampling in near real-time while the offshore expedition was still underway. SituSeq is a portable and robust workflow that helps to bring the power of microbial genomics and diagnostics to many more researchers and situations.

microbiology↗

bioRxiv2022

Cellular Heterogeneity of Pluripotent Stem Cell Derived Cardiomyocyte Grafts is Mechanistically Linked to Treatable Arrhythmias

BackgroundExciting pre-clinical data have confirmed that human pluripotent stem cell derived cardiomyocytes (PSC-CMs) can remuscularise the injured or diseased heart, with several clinical trials now in planning or recruitment stages worldwide. However, ventricular arrhythmias are a predictable complication following engraftment of intramyocardially injected PSC-CMs. Therefore, there is an urgent unmet need to gain mechanistic insights and treatment strategies to control or prevent these engraftment arrhythmias (EAs). MethodsWe used a porcine model of myocardial infarction and PSC-CM transplantation to investigate efficacy of pharmacologic and catheter based anti-arrhythmic strategies in mitigating EAs. Furthermore, cell doses were robustly phenotyped using single cell ribonucleic acid sequencing and high parameter flow cytometry to identify cellular characteristics predictive of arrhythmogenesis. ResultsCombination therapy with amiodarone and ivabradine significantly reduced EA rate and burden following PSC-CM transplantation. Catheter ablation was also a feasible and effective treatment strategy which could be considered in the case of pharmacologically refractory arrhythmias. In addition, we show that EAs are mechanistically linked to cellular heterogeneity in the input PSC-CM and resultant graft. Specifically, we identify atrial and pacemaker-like cardiomyocytes as culprit arrhythmogenic subpopulations. We further describe two unique surface marker signatures, SIRPA+/CD90-/CD200+ and SIRPA+/CD90-/CD200-, which identify arrhythmogenic and non-arrhythmogenic cardiomyocytes respectively. ConclusionOur data deepens mechanistic understanding of EAs and suggests that modifications to current PSC-CM production and/or selection protocols could ameliorate this problem. We further show that current clinical pharmacologic and interventional anti-arrhythmic strategies can control and potentially abolish these arrhythmias, an important safety consideration given several impending clinical trials.

cell biology↗

bioRxiv2022

Age associated DNA methylation changes in Xenopus frogs

Age-associated changes in DNA methylation have been widely established and characterized in mammals but not yet extended to other species. We present clear evidence that the aquatic vertebrate species Xenopus tropicalis displays clear patterns of age-associated changes in DNA methylation. This observation allows us to leverage the unique resources available for Xenopus to study how DNA methylation relates to other hallmarks of aging. We have generated whole genome bisulfite sequencing (WGBS) profiles in nine samples representing young, mature and old adults and characterize the gene- and chromosome-scale changes. Moreover, we selected a few thousands CpG sites to expand the findings in a larger cohort of individuals through targeted DNA methylation analysis to produce a cost effective epigenetic clock assay.

systems biology↗

bioRxiv2018

Signal sequences in the genome of Mononegavirales regulate the generation of copy-back defective viral genomes

Defective viral genomes of the copy-back type (cbDVGs) are the primary initiators of the antiviral immune response during infection with respiratory syncytial virus (RSV) both in vitro and in vivo. However, the mechanism governing cbDVG generation remains unknown, thereby limiting our ability to manipulate cbDVG content in order to modulate the host response to infection. Here we report a specific genomic signal that mediates the generation of RSV cbDVGs. Using a customized bioinformatics tool, we identified regions in the RSV genome frequently used to generate cbDVGs during infection. We then created a minigenome system to validate the function of one of these sequences and to determine if specific nucleotides were essential for cbDVG generation at that position. Further, we created a recombinant virus that selectively produced a specific cbDVG based on variations introduced in this sequence. The identified sequence was also found as a common site for cbDVG generation during natural RSV infections, and common cbDVGs generated at this sequence were found among samples from various infected patients. These data demonstrate that sequences encoded in the viral genome are critical determinants of the location of cbDVG generation and, therefore, this is not a stochastic process. Most importantly, these findings open the possibility of genetically manipulating cbDVG formation to modulate infection outcome. Author summaryCopy-back defective viral genomes (cbDVGs) regulate infection and pathogenesis of Mononegavirales. cbDVG are believed to arise from random errors that occur during virus replication and the predominant hypothesis is that the viral polymerase is the main driver of cbDVG generation. Here we describe a specific genomic sequence in the RSV genome that is necessary for the generation of a large proportion of the cbDVG population present during infection. We identified specific nucleotides that when modified altered cbDVG generation at this position, and we created a recombinant virus that selectively produced cbDVGs based on mutations in this sequence. These data demonstrate that the generation of RSV cbDVGs is regulated by specific viral sequences and that these sequences can be manipulated to alter the content and quality of cbDVG generated during infection.

microbiology↗

bioRxiv2018

Multiple laboratory mouse reference genomes define strain specific haplotypes and novel functional loci

The most commonly employed mammalian model organism is the laboratory mouse. A wide variety of genetically diverse inbred mouse strains, representing distinct physiological states, disease susceptibilities, and biological mechanisms have been developed over the last century. We report full length draft de novo genome assemblies for 16 of the most widely used inbred strains and reveal for the first time extensive strain-specific haplotype variation. We identify and characterise 2,567 regions on the current Genome Reference Consortium mouse reference genome exhibiting the greatest sequence diversity between strains. These regions are enriched for genes involved in defence and immunity, and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. Several immune related loci, some in previously identified QTLs for disease response have novel haplotypes not present in the reference that may explain the phenotype. We used these genomes to improve the mouse reference genome resulting in the completion of 10 new gene structures, and 62 new coding loci were added to the reference genome annotation. Notably this high quality collection of genomes revealed a previously unannotated gene (Efcab3-like) encoding 5,874 amino acids, one of the largest known in the rodent lineage. Interestingly, Efcab3-like-/- mice exhibit severe size anomalies in four regions of the brain suggesting a mechanism of Efcab3-like regulating brain development.

genomics↗

bioRxiv2016

DNA damage in 3D constricted migration or after lamin-A depletion in 2D: shared mechanisms of repair factor mis-localization under nuclear stress

Cells that migrate through small, rigid pores and that have normal levels of the nuclear structure protein lamin-A exhibit an increase in DNA damage, which is also observed with lamin-A depletion in diseases such as cancer and with many lamin-A mutations. Here we show nuclear envelope rupture is a shared feature that increases in standard culture after lamin-A knockdown, which causes nuclear loss of multiple DNA repair factors and increased DNA damage. Some repair factors are merely mis-localized to cytoplasm whereas others are partially depleted unless rescued by lamin-A expression. Compared to standard cultures on rigid glass coverslips, the growth of lamin-A low cells on soft matrix relaxes cytoskeletal stress on the nucleus, suppresses the mis-localization of DNA repair factors, and minimizes DNA damage nearly to wildtype levels. Conversely, constricted migration of the lamin-A low cells causes abnormally high levels of DNA damage, consistent with sustained loss of repair factors. The findings add insight into why monogenic progeroid syndromes that often associate with increased DNA damage and predominantly impact cells in stiff tissues result from mutations only in lamin-A or DNA repair factors.

cell biology↗