Transposable element landscapes in aging Drosophila.

Genetic mechanisms that repress transposable elements (TEs) in young animals decline during aging, as reflected by increased TE expression in aged animals. Does increased TE expression during aging lead to more genomic TE copies in older animals? To address this question, we quantified TE Landscapes (TLs) via whole genome sequencing of young and aged Drosophila strains of wild-type and mutant backgrounds. We quantified TLs in whole flies and dissected brains and validated the feasibility of our approach in detecting new TE insertions in aging Drosophila genomes when small RNA and RNA interference (RNAi) pathways are compromised. We also describe improved sequencing methods to quantify extra-chromosomal DNA circles (eccDNAs) in Drosophila as an additional source of TE copies that accumulate during aging. Lastly, to combat the natural progression of aging-associated TE expression, we show that knocking down PAF1, a conserved transcription elongation factor that antagonizes RNAi pathways, may bolster suppression of TEs during aging and extend lifespan. Our study suggests that in addition to a possible influence by different genetic backgrounds, small RNA and RNAi mechanisms may mitigate genomic TL expansion despite the increase in TE transcripts during aging.

A mosquito small RNA genomics resource reveals dynamic evolution and host responses to viruses and transposons.

Although mosquitoes are major transmission vectors for pathogenic arboviruses, viral infection has little impact on mosquito health. This immunity is caused in part by mosquito RNA interference (RNAi) pathways that generate antiviral small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). RNAi also maintains genome integrity by potently repressing mosquito transposon activity in the germline and soma. However, viral and transposon small RNA regulatory pathways have not been systematically examined together in mosquitoes. Therefore, we developed an integrated mosquito small RNA genomics (MSRG) resource that analyzes the transposon and virus small RNA profiles in mosquito cell cultures and somatic and gonadal tissues across four medically important mosquito species. Our resource captures both somatic and gonadal small RNA expression profiles within mosquito cell cultures, and we report the evolutionary dynamics of a novel Mosquito-Conserved piRNA Cluster Locus (MCpiRCL) made up of satellite DNA repeats. In the larger culicine mosquito genomes we detected highly regular periodicity in piRNA biogenesis patterns coinciding with the expansion of Piwi pathway genes. Finally, our resource enables detection of cross talk between piRNA and siRNA populations in mosquito cells during a response to virus infection. The MSRG resource will aid efforts to dissect and combat the capacity of mosquitoes to tolerate and spread arboviruses.

Challenges and recommendations to improve the installability and archival stability of omics computational tools.

Developing new software tools for analysis of large-scale biological data is a key component of advancing modern biomedical research. Scientific reproduction of published findings requires running computational tools on data generated by such studies, yet little attention is presently allocated to the installability and archival stability of computational software tools. Scientific journals require data and code sharing, but none currently require authors to guarantee the continuing functionality of newly published tools. We have estimated the archival stability of computational biology software tools by performing an empirical analysis of the internet presence for 36,702 omics software resources published from 2005 to 2017. We found that almost 28% of all resources are currently not accessible through uniform resource locators (URLs) published in the paper they first appeared in. Among the 98 software tools selected for our installability test, 51% were deemed "easy to install," and 28% of the tools failed to be installed at all because of problems in the implementation. Moreover, for papers introducing new software, we found that the number of citations significantly increased when authors provided an easy installation process. We propose for incorporation into journal policy several practical solutions for increasing the widespread installability and archival stability of published bioinformatics software.

An integrated map of structural variation in 2,504 human genomes.

Structural variants are implicated in numerous diseases and make up the majority of varying nucleotides among human genomes. Here we describe an integrated set of eight structural variant classes comprising both balanced and unbalanced variants, which we constructed using short-read DNA sequencing data and statistically phased onto haplotype blocks in 26 human populations. Analysing this set, we identify numerous gene-intersecting structural variants exhibiting population stratification and describe naturally occurring homozygous gene knockouts that suggest the dispensability of a variety of human genes. We demonstrate that structural variants are enriched on haplotypes identified by genome-wide association studies and exhibit enrichment for expression quantitative trait loci. Additionally, we uncover appreciable levels of structural variant complexity at different scales, including genic loci subject to clusters of repeated rearrangement and complex structural variants with multiple breakpoints likely to have formed through individual mutational events. Our catalogue will enhance future studies into structural variant demography, functional impact and disease association.

The genomic landscape of polymorphic human nuclear mitochondrial insertions.

The transfer of mitochondrial genetic material into the nuclear genomes of eukaryotes is a well-established phenomenon that has been previously limited to the study of static reference genomes. The recent advancement of high throughput sequencing has enabled an expanded exploration into the diversity of polymorphic nuclear mitochondrial insertions (NumtS) within human populations. We have developed an approach to discover and genotype novel Numt insertions using whole genome, paired-end sequencing data. We have applied this method to a thousand individuals in 20 populations from the 1000 Genomes Project and other datasets and identified 141 new sites of Numt insertions, extending our current knowledge of existing NumtS by almost 20%. We find that recent Numt insertions are derived from throughout the mitochondrial genome, including the D-loop, and have integration biases that differ in some respects from previous studies on older, fixed NumtS in the reference genome. We determined the complete inserted sequence for a subset of these events and have identified a number of nearly full-length mitochondrial genome insertions into nuclear chromosomes. We further define their age and origin of insertion and present an analysis of their potential impact to ongoing studies of mitochondrial heteroplasmy and disease.

Myc Dysregulation in Activated Macrophages Initiates Iron-Mediated Lipid Peroxidation that Fuels Type I Interferon and Compromises TB Resistance.

A quarter of human population is infected with Mycobacterium tuberculosis, but less than 10% of those infected develop clinical, mostly pulmonary, TB. To dissect mechanisms of susceptibility in immunocompetent individuals, we developed a genetically defined sst1-susceptible mouse model that uniquely reproduces a defining feature of human TB: development of necrotic lung lesions after infection with virulent Mtb. In this study, we explored the connectivity of the sst1-regulated pathways during prolonged macrophage activation with TNF. We determined that the aberrant response of the sst1-susceptible macrophages to TNF was primarily driven by conflicting Myc and antioxidant response pathways that resulted in a coordinated failure to properly sequester intracellular iron and activate ferroptosis inhibitor enzymes. Consequently, iron-mediated lipid peroxidation fueled IFNß superinduction and sustained the Type I Interferon (IFN-I) pathway hyperactivity that locked the sst1-susceptible macrophages in a state of unresolving stress and compromised their resistance to Mtb. The accumulation of the aberrantly activated, stressed, macrophages within granuloma microenvironment led to the local failure of anti-tuberculosis immunity and tissue necrosis. Our findings suggest a novel link between metabolic dysregulation in macrophages and susceptibility to TB, offering insights into potential therapeutic targets aimed at modulating macrophage function and improving TB control.

Global genomics of Aedes aegypti unveils widespread and novel infectious viruses capable of triggering a small RNA response.

The mosquito Aedes aegypti is a prominent vector for arboviruses, but the breadth of mosquito viruses that infects this specie is not fully understood. In the broadest global survey to date of over 200 Ae. aegypti small RNA samples, we detected viral small interfering RNAs (siRNAs) and Piwi interacting RNAs (piRNAs) arising from mosquito viruses. We confirmed that most academic laboratory colonies of Ae. aegypti lack persisting viruses, yet two commercial strains were infected by a novel tombus-like virus. Ae. aegypti from North to South American locations were also teeming with multiple insect viruses, with Anphevirus and a bunyavirus displaying geographical boundaries from the viral small RNA patterns. Asian Ae. aegypti small RNA patterns indicate infections by similar mosquito viruses from the Americas and reveal the first wild example of dengue virus infection generating viral small RNAs. African Ae. aegypti also contained various viral small RNAs including novel viruses only found in these African substrains. Intriguingly, viral long RNA patterns can differ from small RNA patterns, indicative of viral transcripts evading the mosquitoes' RNA interference (RNAi) machinery. To determine whether the viruses we discovered via small RNA sequencing were replicating and transmissible, we infected C6/36 and Aag2 cells with Ae. aegypti homogenates. Through blind passaging, we generated cell lines stably infected by these mosquito viruses which then generated abundant viral siRNAs and piRNAs that resemble the native mosquito viral small RNA patterns. This mosquito small RNA genomics approach augments surveillance approaches for emerging infectious diseases.

Extending and Running the Mosquito Small RNA Genomics Resource Pipeline.

The Mosquito Small RNA Genomics (MSRG) resource is a repository of analyses on the small RNA transcriptomes of mosquito cell cultures and somatic and gonadal tissues. This resource allows for comparing the regulation dynamics of small RNAs generated from transposons and viruses across mosquito species. This chapter covers the procedures to set up the MSRG resource pipeline as a new installation by detailing the necessary collection of genome reference and annotation files and lists of microRNAs (miRNAs) hairpin sequences, transposon repeats consensus sequences, and virus genome sequences. Proper execution of the MSRG resource pipeline yields outputs amenable to biologists to further analyze with desktop and spreadsheet software to gain insights into the balance between arthropod endogenous small RNA populations and the proportions of virus-derived small RNAs that include Piwi-interacting RNAs (piRNAs) and endogenous small interfering RNAs (siRNAs).

Transcriptomic and small RNA response to Mayaro virus infection in Anopheles stephensi mosquitoes.

Mayaro virus (MAYV) is an arboviral pathogen in the genus Alphavirus that is circulating in South America with potential to spread to naïve regions. MAYV is also one of the few viruses with the ability to be transmitted by mosquitoes in the genus Anopheles, as well as the typical arboviral transmitting mosquitoes in the genus Aedes. Few studies have investigated the infection response of Anopheles mosquitoes. In this study we detail the transcriptomic and small RNA responses of An. stephensi to infection with MAYV via infectious bloodmeal at 2, 7, and 14 days post infection (dpi). 487 unique transcripts were significantly regulated, 78 putative novel miRNAs were identified, and an siRNA response is observed targeting the MAYV genome. Gene ontology analysis of transcripts regulated at each timepoint shows a number of proteases regulated at 2 and 7 dpi, potentially representative of Toll or melanization pathway activation, and repression of pathways related to autophagy and apoptosis at 14 dpi. These findings provide a basic understanding of the infection response of An. stephensi to MAYV and help to identify host factors which might be useful to target to inhibit viral replication in Anopheles mosquitoes.

Dengue Virus-2 Infection Affects Fecundity and Elicits Specific Transcriptional Changes in the Ovaries of Aedes aegypti Mosquitoes.

Dengue fever (DF), caused by the dengue virus (DENV), is the most burdensome arboviral disease in the world, with an estimated 400 million infections each year. The Aedes aegypti mosquito is the main vector of DENV and transmits several other human pathogens, including Zika, yellow fever, and chikungunya viruses. Previous studies have shown that the pathogen infection of mosquitoes can alter reproductive fitness, revealing specific vector-pathogen interactions that are key determinants of vector competence. However, only a handful of studies have examined the effect of DENV infection in A. aegypti, showing a reduction in lifespan and fecundity over multiple blood meals. To provide a more comprehensive analysis of the impact of DENV infection on egg laying and fecundity, we assessed egg laying timing in DENV-2 blood-fed mosquitoes (infected group) compared to mock blood-fed mosquitoes (control group). We confirmed a significant decrease in fecundity during the first gonadotrophic cycle. To further investigate this phenotype and the underlying DENV-2 infection-dependent changes in gene expression, we conducted a transcriptomic analysis for differentially expressed genes in the ovaries of A. aegypti infected with DENV-2 vs. mock-infected mosquitoes. This analysis reveals several DENV-2-regulated genes; among them, we identified a group of 12 metabolic genes that we validated using reverse transcription-quantitative PCR (RT-qPCR). Interestingly, two genes found to be upregulated in DENV-infected mosquito ovaries exhibited an antiviral role for DENV-2 in an Aedes cell line. Altogether, this study offers useful insights into the virus-vector interface, highlighting the importance of gene expression changes in the mosquito's ovary during DENV-2 infection in the first gonadotrophic  cycle,  triggering  antiviral  responses  that  may  possibly  interfere  with mosquito reproduction. This information is extremely relevant for further investigation of A. aegypti's ability to tolerate viruses since virally infected mosquitoes in nature constitute a powerful source of supporting viruses during intra-epidemic periods, causing a huge burden on the public health system.

Characterization of nuclear mitochondrial insertions in the whole genomes of primates.

The transfer and integration of whole and partial mitochondrial genomes into the nuclear genomes of eukaryotes is an ongoing process that has facilitated the transfer of genes and contributed to the evolution of various cellular pathways. Many previous studies have explored the impact of these insertions, referred to as NumtS, but have focused primarily on older events that have become fixed and are therefore present in all individual genomes for a given species. We previously developed an approach to identify novel Numt polymorphisms from next-generation sequence data and applied it to thousands of human genomes. Here, we extend this analysis to 79 individuals of other great ape species including chimpanzee, bonobo, gorilla, orang-utan and also an old world monkey, macaque. We show that recent Numt insertions are prevalent in each species though at different apparent rates, with chimpanzees exhibiting a significant increase in both polymorphic and fixed Numt sequences as compared to other great apes. We further assessed positional effects in each species in terms of evolutionary time and rate of insertion and identified putative hotspots on chromosome 5 for Numt integration, providing insight into both recent polymorphic and older fixed reference NumtS in great apes in comparison to human events.

Interactions between the gut microbiome and host gene regulation in cystic fibrosis.

BACKGROUND: Cystic fibrosis is the most common autosomal recessive genetic disease in Caucasians. It is caused by mutations in the CFTR gene, leading to poor hydration of mucus and impairment of the respiratory, digestive, and reproductive organ functions. Advancements in medical care have led to markedly increased longevity of patients with cystic fibrosis, but new complications have emerged, such as early onset of colorectal cancer. Although the pathogenesis of colorectal cancer in cystic fibrosis remains unclear, altered host-microbe interactions might play a critical role. To investigate this, we characterized changes in the microbiome and host gene expression in the colonic mucosa of cystic fibrosis patients relative to healthy controls, and identified host gene-microbiome interactions in the colon of cystic fibrosis patients. METHODS: We performed RNA-seq on colonic mucosa samples from cystic fibrosis patients and healthy controls to determine differentially expressed host genes. We also performed 16S rRNA sequencing to characterize the colonic mucosal microbiome and identify gut microbes that are differentially abundant between patients and healthy controls. Lastly, we modeled associations between relative abundances of specific bacterial taxa in the gut mucosa and host gene expression. RESULTS: We find that 1543 genes, including CFTR, show differential expression in the colon of cystic fibrosis patients compared to healthy controls. These genes are enriched with functions related to gastrointestinal and colorectal cancer, such as metastasis of colorectal cancer, tumor suppression, p53, and mTOR signaling pathways. In addition, patients with cystic fibrosis show decreased gut microbial diversity, decreased abundance of butyrate producing bacteria, such as Ruminococcaceae and Butyricimonas, and increased abundance of other taxa, such as Actinobacteria and Clostridium. An integrative analysis identified colorectal cancer-related genes, including LCN2 and DUOX2, for which gene expression is correlated with the abundance of colorectal cancer-associated bacteria, such as Ruminococcaceae and Veillonella. CONCLUSIONS: In addition to characterizing host gene expression and mucosal microbiome in cystic fibrosis patients, our study explored the potential role of host-microbe interactions in the etiology of colorectal cancer in cystic fibrosis. Our results provide biomarkers that may potentially serve as targets for stratifying risk of colorectal cancer in patients with cystic fibrosis.

Extraction of high molecular weight DNA from microbial mats.

Due to the presence of inhibitors such as extracellular polymeric substances (EPSs) and salts, most microbial mat studies have relied on harsh methods of direct DNA extraction that result in DNA fragments too small for large-insert vector cloning. High molecular weight (HMW) DNA is crucial in functional metagenomic studies, because large fragments present greater access to genes of interest. Here we report improved methodologies for extracting HMW DNA from EPS-rich hypersaline microbial mats. The protocol uses a combination of microbial cell separation with mechanical and chemical methods for DNA extraction and purification followed by precipitation with polyethylene glycol (PEG). The protocol yields >2 µg HMW DNA (>48 kb) per gram of mat sample, with A260:280 ratios >1.7. In addition, 16S rRNA gene analysis using denaturing gradient gel electrophoresis and pyrosequencing showed that this protocol extracts representative DNA from microbial mat communities and results in higher overall calculated diversity indices compared with three other standard methods of DNA extraction. Our results show the importance of validating the DNA extraction methods used in metagenomic studies to ensure optimal recovery of microbial richness.