Evolution of a biological thermocouple by adaptation of cytochrome c oxidase in a subterrestrial metazoan.

In this study we report a naturally evolved temperature-sensing electrical regulator in the cytochrome c oxidase of the Devil Worm, Halicephalobus mephisto. This extremophile metazoan was isolated 1.3 km underground in a South African goldmine, where it adapted to heat and potentially to hypoxia, making its mitochondrial sequence a likely target of adaptational change. We obtained the full mitochondrial genome sequence of this organism, and show through dN/dS analysis statistically robust evidence of positive selection in H. mephisto cytochrome c oxidase subunits. Seventeen of these positively-selected amino acid substitutions were localized in proximity to the H- and K-pathway proton channels of the complex. Surprisingly, the H. mephisto cytochrome c oxidase proton pump completely shuts down at low temperatures (20°C) leading to approximately a 4.8-fold reduction in the transmembrane proton gradient voltage (ΔΨm) compared to optimal temperature (37°C). Direct measurement of oxygen consumption found a corresponding 4.7-fold drop at 20°C compared to 37°C. Correspondingly, the lifecycle of H. mephisto takes four-fold longer at the low temperature compared to higher. This elegant evolutionary adaptation creates a finely-tuned mitochondrial temperature sensor, allowing this ectothermic organism to maximize its reproductive success in varying environmental temperatures. Our study shows that evolutionary innovation may remodel core metabolism to make it more accurately map onto environmental variation.

Unrealized potential from smaller institutions: Four strategies for advancing STEM diversity.

Diversity within science, technology, engineering, and mathematics (STEM) remains disturbingly low. Relative to larger, highly funded universities, smaller schools harbor more diverse student demographics and more limited resources. Here, we propose four strategies leveraging the unique advantages of smaller institutions to advance underrepresented scholars along STEM pathways.

A Transcriptomic Pipeline Adapted for Genomic Sequence Discovery of Germline-Restricted Sequence in Zebra Finch, Taeniopygia guttata.

Songbirds have an unusual genomic element which is only found in their germline cells, known as the germline-restricted chromosome (GRC). Because germ cells contain both GRC and non-GRC (or A-chromosome) sequences, confidently identifying the GRC-derived elements from genome assemblies has proven difficult. Here, we introduce a new application of a transcriptomic method for GRC sequence identification. By adapting the Stringtie/Ballgown pipeline to use somatic and germline DNA reads, we find that the ratio of fragments per kilobase per million mapped reads can be used to confidently assign contigs to the GRC. Using this comparative coverage analysis, we successfully identify 733 contigs as high confidence GRC sequences (720 newly identified in this study) and 51 contigs which were validated using quantitative polymerase chain reaction. We also identified two new GRC genes, one hypothetical protein and one gene encoding an RNase H-like domain, and placed 16 previously identified but unplaced genes onto their host contigs. With the current focus on sequencing GRCs from different songbirds, our work adds to the genomic toolkit to identify GRC elements, and we provide a detailed protocol and GitHub repository at https://github.com/brachtlab/Comparative_Coverage_Analysis (last accessed May 12, 2021).

Direct Detection of DNA and RNA on Carbon Fiber Microelectrodes Using Fast-Scan Cyclic Voltammetry.

DNA and RNA have been measured with many techniques but often with relatively long analysis times. In this study, we utilize fast-scan cyclic voltammetry (FSCV) for the subsecond codetection of adenine, guanine, and cytosine, first as free nucleosides, and then within custom synthesized oligos, plasmid DNA, and RNA from the nematode Caenorhabditis elegans. Previous studies have shown the detection of adenosine and guanosine with FSCV with high spatiotemporal resolution, while we have extended the assay to include cytidine and adenine, guanine, and cytosine in RNA and single- and double-stranded DNA (ssDNA and dSDNA). We find that FSCV testing has a higher sensitivity and yields higher peak oxidative currents when detecting shorter oligonucleotides and ssDNA samples at equivalent nucleobase concentrations. This is consistent with an electrostatic repulsion from negatively charged oxide groups on the surface of the carbon fiber microelectrode (CFME), the negative holding potential, and the negatively charged phosphate backbone. Moreover, as opposed to dsDNA, ssDNA nucleobases are not hydrogen-bonded to one another and thus are free to adsorb onto the surface of the carbon electrode. We also demonstrate that the simultaneous determination of nucleobases is not masked even in biologically complex serum samples. This is the first report demonstrating that FSCV, when used with CFMEs, is able to codetect nucleobases when polymerized into DNA or RNA and could potentially pave the way for future uses in clinical, diagnostic, or research applications.

Regional sequence expansion or collapse in heterozygous genome assemblies.

High levels of heterozygosity present a unique genome assembly challenge and can adversely impact downstream analyses, yet is common in sequencing datasets obtained from non-model organisms. Here we show that by re-assembling a heterozygous dataset with variant parameters and different assembly algorithms, we are able to generate assemblies whose protein annotations are statistically enriched for specific gene ontology categories. While total assembly length was not significantly affected by assembly methodologies tested, the assemblies generated varied widely in fragmentation level and we show local assembly collapse or expansion underlying the enrichment or depletion of specific protein functional groups. We show that these statistically significant deviations in gene ontology groups can occur in seemingly high-quality assemblies, and result from difficult-to-detect local sequence expansion or contractions. Given the unpredictable interplay between assembly algorithm, parameter, and biological sequence data heterozygosity, we highlight the need for better measures of assembly quality than N50 value, including methods for assessing local expansion and collapse.

Epigenetic Regulation of Neuregulin-1 Tunes White Adipose Stem Cell Differentiation.

Expansion of subcutaneous adipose tissue by differentiation of new adipocytes has been linked to improvements in metabolic health. However, an expandability limit has been observed wherein new adipocytes cannot be produced, the existing adipocytes become enlarged (hypertrophic) and lipids spill over into ectopic sites. Inappropriate ectopic storage of these surplus lipids in liver, muscle, and visceral depots has been linked with metabolic dysfunction. Here we show that Neuregulin-1 (NRG1) serves as a regulator of adipogenic differentiation in subcutaneous primary human stem cells. We further demonstrate that DNA methylation modulates NRG1 expression in these cells, and a 3-day exposure of stem cells to a recombinant NRG1 peptide fragment is sufficient to reprogram adipogenic cellular differentiation to higher levels. These results define a novel molecular adipogenic rheostat with potential implications for the expansion of adipose tissue in vivo.

The role of estrogens in the adipose tissue milieu.

One of the leading causes for the development of adverse metabolic effects, including type 2 diabetes, dyslipidemia, and cardiovascular diseases, is the accumulation of excess body weight, often measured by body mass index (BMI). Although BMI, calculated using weight and height, is the standard measure used to determine body adiposity in clinical and public health guidelines, an inherent limitation is that BMI does not distinguish where in the body adiposity is deposited. Central obesity, characterized by greater accumulation of adiposity in the abdominal region, has been associated with a higher risk of mortality, independent of BMI. Importantly, one of the determinants of body fat distribution is sex hormones. Both estrogens and androgens appear to directly and indirectly influence body fat distribution. Our review will focus specifically on the role of estrogens and their influence in determining body fat distribution and overall health of adipose tissues, and the role of epigenetic mechanisms in regulating the production and function of estrogens.

The genome of a subterrestrial nematode reveals adaptations to heat.

The nematode Halicephalobus mephisto was originally discovered inhabiting a deep terrestrial aquifer 1.3 km underground. H. mephisto can thrive under conditions of abiotic stress including heat and minimal oxygen, where it feeds on a community of both chemolithotrophic and heterotrophic prokaryotes in an unusual ecosystem isolated from the surface biosphere. Here we report the comprehensive genome and transcriptome of this organism, identifying a signature of adaptation: an expanded repertoire of 70 kilodalton heat-shock proteins (Hsp70) and avrRpt2 induced gene 1 (AIG1) proteins. The expanded Hsp70 genes are transcriptionally induced upon growth under heat stress, and we find that positive selection is detectable in several members of this family. We further show that AIG1 may have been acquired by horizontal gene transfer (HGT) from a rhizobial fungus. Over one-third of the genes of H. mephisto are novel, highlighting the divergence of this nematode from other sequenced organisms. This work sheds light on the genomic basis of heat tolerance in a complete subterrestrial eukaryotic genome.

Stress Adapted Mollusca and Nematoda Exhibit Convergently Expanded Hsp70 and AIG1 Gene Families.

We recently sequenced the genome of the first subterrestrial metazoan, the nematode Halicephalobus mephisto. A central finding was a dramatic expansion of genes encoding avrRpt2 induced gene (AIG1), and 70 kDa heat shock (Hsp70) domains. While the role of Hsp70 in thermotolerance is well established, the contribution of AIG1 is much more poorly characterized, though in plants some members of this family are heat-induced. Hypothesizing that this dual domain expansion may constitute a general biosignature of thermal stress adaptation, here we examine a number of genomes, finding that expansion of both AIG1 and Hsp70 is common in bivalves. Phylogenetic analysis reveals that the bivalve-specific Hsp70 protein expansion groups with H. mephisto sequences. Our identification of the same gene expansions in bivalves and a nematode implies that this biosignature may be a general stress adaptation strategy for protostomes, particularly those organisms that cannot escape their stressful environments. We hypothesize that the two families play largely complementary mechanistic roles, with Hsp70 directly refolding heat-denatured proteins while AIG1 promotes cellular and organismal survival by inhibiting apoptosis.

Programmed genome rearrangements in Oxytricha produce transcriptionally active extrachromosomal circular DNA.

Extrachromosomal circular DNA (eccDNA) is both a driver of eukaryotic genome instability and a product of programmed genome rearrangements, but its extent had not been surveyed in Oxytricha, a ciliate with elaborate DNA elimination and translocation during development. Here, we captured rearrangement-specific circular DNA molecules across the genome to gain insight into its processes of programmed genome rearrangement. We recovered thousands of circularly excised Tc1/mariner-type transposable elements and high confidence non-repetitive germline-limited loci. We verified their bona fide circular topology using circular DNA deep-sequencing, 2D gel electrophoresis and inverse polymerase chain reaction. In contrast to the precise circular excision of transposable elements, we report widespread heterogeneity in the circular excision of non-repetitive germline-limited loci. We also demonstrate that circular DNAs are transcribed in Oxytricha, producing rearrangement-specific long non-coding RNAs. The programmed formation of thousands of eccDNA molecules makes Oxytricha a model system for studying nucleic acid topology. It also suggests involvement of eccDNA in programmed genome rearrangement.

Identification of a DNA N6-Adenine Methyltransferase Complex and Its Impact on Chromatin Organization.

DNA N6-adenine methylation (6mA) has recently been described in diverse eukaryotes, spanning unicellular organisms to metazoa. Here, we report a DNA 6mA methyltransferase complex in ciliates, termed MTA1c. It consists of two MT-A70 proteins and two homeobox-like DNA-binding proteins and specifically methylates dsDNA. Disruption of the catalytic subunit, MTA1, in the ciliate Oxytricha leads to genome-wide loss of 6mA and abolishment of the consensus ApT dimethylated motif. Mutants fail to complete the sexual cycle, which normally coincides with peak MTA1 expression. We investigate the impact of 6mA on nucleosome occupancy in vitro by reconstructing complete, full-length Oxytricha chromosomes harboring 6mA in native or ectopic positions. We show that 6mA directly disfavors nucleosomes in vitro in a local, quantitative manner, independent of DNA sequence. Furthermore, the chromatin remodeler ACF can overcome this effect. Our study identifies a diverged DNA N6-adenine methyltransferase and defines the role of 6mA in chromatin organization.

Rapid molecular detection of macrolide resistance.

BACKGROUND: Emerging antimicrobial resistance is a significant threat to human health. However, methods for rapidly diagnosing antimicrobial resistance generally require multi-day culture-based assays. Macrolide efflux gene A, mef(A), provides resistance against erythromycin and azithromycin and is known to be laterally transferred among a wide range of bacterial species. METHODS: We use Recombinase Polymerase Assay (RPA) to detect the antimicrobial resistance gene mef(A) from raw lysates without nucleic acid purification. To validate these results we performed broth dilution assays to assess antimicrobial resistance to erythromycin and ampicillin (a negative control). RESULTS: We validate the detection of mef(A) in raw lysates of Streptococcus pyogenes, S. pneumoniae, S. salivarius, and Enterococcus faecium bacterial lysates within 7-10 min of assay time. We show that detection of mef(A) accurately predicts real antimicrobial resistance assessed by traditional culture methods, and that the assay is robust to high levels of spiked-in non-specific nucleic acid contaminant. The assay was unaffected by single-nucleotide polymorphisms within divergent mef(A) gene sequences, strengthening its utility as a robust diagnostic tool. CONCLUSIONS: This finding opens the door to implementation of rapid genomic diagnostics in a clinical setting, while providing researchers a rapid, cost-effective tool to track antibiotic resistance in both pathogens and commensal strains.

Novel Sequence Discovery by Subtractive Genomics.

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a larger genomic context. Subtractive genomics enables a researcher to isolate a target sequence of interest (T) by comprehensive sequencing and subtracting out known genetic elements (reference, R). The method can be used to identify novel sequences such as mitochondria, chloroplasts, viruses, or germline restricted chromosomes, and is particularly useful when T cannot be easily isolated from R. Beginning with the comprehensive genomic data (R + T), the method uses Basic Local Alignment Search Tool (BLAST) against a reference sequence, or sequences, to remove the matching known sequences (R), leaving behind the target (T). For subtraction to work best, R should be a relatively complete draft that is missing T. Since sequences remaining after subtraction are tested through quantitative Polymerase Chain Reaction (qPCR), R does not need to be complete for the method to work. Here we link computational steps with experimental steps into a cycle that can be iterated as needed, sequentially removing multiple reference sequences and refining the search for T. The advantage of subtractive genomics is that a completely novel target sequence can be identified even in cases in which physical purification is difficult, impossible, or expensive. A drawback of the method is finding a suitable reference for subtraction and obtaining T-positive and negative samples for qPCR testing. We describe our implementation of the method in the identification of the first gene from the germline-restricted chromosome of zebra finch. In that case computational filtering involved three references (R), sequentially removed over three cycles: an incomplete genomic assembly, raw genomic data, and transcriptomic data.

Capture of complete ciliate chromosomes in single sequencing reads reveals widespread chromosome isoforms.

BACKGROUND: Whole-genome shotgun sequencing, which stitches together millions of short sequencing reads into a single genome, ushered in the era of modern genomics and led to a rapid expansion of the number of genome sequences available. Nevertheless, assembly of short reads remains difficult, resulting in fragmented genome sequences. Ultimately, only a sequencing technology capable of capturing complete chromosomes in a single run could resolve all ambiguities. Even "third generation" sequencing technologies produce reads far shorter than most eukaryotic chromosomes. However, the ciliate Oxytricha trifallax has a somatic genome with thousands of chromosomes averaging only 3.2 kbp, making it an ideal candidate for exploring the benefits of sequencing whole chromosomes without assembly. RESULTS: We used single-molecule real-time sequencing to capture thousands of complete chromosomes in single reads and to update the published Oxytricha trifallax JRB310 genome assembly. In this version, over 50% of the completed chromosomes with two telomeres derive from single reads. The improved assembly includes over 12,000 new chromosome isoforms, and demonstrates that somatic chromosomes derive from variable rearrangements between somatic segments encoded up to 191,000 base pairs away. However, while long reads reduce the need for assembly, a hybrid approach that supplements long-read sequencing with short reads for error correction produced the most complete and accurate assembly, overall. CONCLUSIONS: This assembly provides the first example of complete eukaryotic chromosomes captured by single sequencing reads and demonstrates that traditional approaches to genome assembly can mask considerable structural variation.

Therapeutic applications of adipose-derived stem cells in cardiovascular disease.

Cardiovascular disease (CVD) is the number one cause of death globally, and new therapeutic techniques outside of traditional pharmaceutical and surgical interventions are currently being developed. At the forefront is stem cell-centered therapy, with adipose derived stem cells (ADSCs), an adult stem population, providing significant clinical promise. When introduced into damaged heart tissue, ADSCs promote cardiac regeneration by a variety of mechanisms including differentiation into new cardiomyocytes and secretion of paracrine factors acting on endogenous cardiac cells. We discuss the application of ADSCs, their biochemical capabilities, availability, ease of extraction, clinical trial results, and areas of concern. The multipotent capacity of ADSCs along with their ability to secrete factors promoting cell survival and regeneration, along with their immunosuppressive capacity, make them an extremely promising approach in the field of CVD therapy.

Discovery of a stem-like multipotent cell fate.

Adipose derived stem cells (ASCs) can be obtained from lipoaspirates and induced in vitro to differentiate into bone, cartilage, and fat. Using this powerful model system we show that after in vitro adipose differentiation a population of cells retain stem-like qualities including multipotency. They are lipid (-), retain the ability to propagate, express two known stem cell markers, and maintain the capacity for trilineage differentiation into chondrocytes, adipocytes, and osteoblasts. However, these cells are not traditional stem cells because gene expression analysis showed an overall expression profile similar to that of adipocytes. In addition to broadening our understanding of cellular multipotency, our work may be particularly relevant to obesity-associated metabolic disorders. The adipose expandability hypothesis proposes that inability to differentiate new adipocytes is a primary cause of metabolic syndrome in obesity, including diabetes and cardiovascular disease. Here we have defined a differentiation-resistant stem-like multipotent cell population that may be involved in regulation of adipose expandability in vivo and may therefore play key roles in the comorbidities of obesity.

Discovery of the First Germline-Restricted Gene by Subtractive Transcriptomic Analysis in the Zebra Finch, Taeniopygia guttata.

Developmentally programmed genome rearrangements are rare in vertebrates, but have been reported in scattered lineages including the bandicoot, hagfish, lamprey, and zebra finch (Taeniopygia guttata) [1]. In the finch, a well-studied animal model for neuroendocrinology and vocal learning [2], one such programmed genome rearrangement involves a germline-restricted chromosome, or GRC, which is found in germlines of both sexes but eliminated from mature sperm [3, 4]. Transmitted only through the oocyte, it displays uniparental female-driven inheritance, and early in embryonic development is apparently eliminated from all somatic tissue in both sexes [3, 4]. The GRC comprises the longest finch chromosome at over 120 million base pairs [3], and previously the only known GRC-derived sequence was repetitive and non-coding [5]. Because the zebra finch genome project was sourced from male muscle (somatic) tissue [6], the remaining genomic sequence and protein-coding content of the GRC remain unknown. Here we report the first protein-coding gene from the GRC: a member of the α-soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein (α-SNAP) family hitherto missing from zebra finch gene annotations. In addition to the GRC-encoded α-SNAP, we find an additional paralogous α-SNAP residing in the somatic genome (a somatolog)-making the zebra finch the first example in which α-SNAP is not a single-copy gene. We show divergent, sex-biased expression for the paralogs and also that positive selection is detectable across the bird α-SNAP lineage, including the GRC-encoded α-SNAP. This study presents the identification and evolutionary characterization of the first protein-coding GRC gene in any organism.

Thousands of RNA-cached copies of whole chromosomes are present in the ciliate Oxytricha during development.

The ciliate Oxytricha trifallax maintains two genomes: a germline genome that is active only during sexual conjugation and a transcriptionally active, somatic genome that derives from the germline via extensive sequence reduction and rearrangement. Previously, we found that long noncoding (lnc) RNA "templates"-telomere-containing, RNA-cached copies of mature chromosomes-provide the information to program the rearrangement process. Here we used a modified RNA-seq approach to conduct the first genome-wide search for endogenous, telomere-to-telomere RNA transcripts. We find that during development, Oxytricha produces long noncoding RNA copies for over 10,000 of its 16,000 somatic chromosomes, consistent with a model in which Oxytricha transmits an RNA-cached copy of its somatic genome to the sexual progeny. Both the primary sequence and expression profile of a somatic chromosome influence the temporal distribution and abundance of individual template RNAs. This suggests that Oxytricha may undergo multiple rounds of DNA rearrangement during development. These observations implicate a complex set of thousands of long RNA molecules in the wiring and maintenance of a highly elaborate somatic genome architecture.

Chromosome fusions triggered by noncoding RNA.

Chromosomal fusions are common in normal and cancer cells and can produce aberrant gene products that promote transformation. The mechanisms driving these fusions are poorly understood, but recurrent fusions are widespread. This suggests an underlying mechanism, and some authors have proposed a possible role for RNA in this process. The unicellular eukaryote Oxytricha trifallax displays an exorbitant capacity for natural genome editing, when it rewrites its germline genome to form a somatic epigenome. This developmental process provides a powerful model system to directly test the influence of small noncoding RNAs on chromosome fusion events during somatic differentiation. Here we show that small RNAs are capable of inducing chromosome fusions in 4 distinct cases (out of 4 tested), including one fusion of 3 chromosomes. We further show that these RNA-mediated chromosome fusions are heritable over multiple sexual generations and that transmission of the acquired fusion is associated with endogenous production of novel piRNA molecules that target the fused junction. We also demonstrate the capacity of a long noncoding RNA (lncRNA) to induce chromosome fusion of 2 distal germline loci. These results underscore the ability of short-lived, aberrant RNAs to act as drivers of chromosome fusion events that can be stably transmitted to future generations.

The architecture of a scrambled genome reveals massive levels of genomic rearrangement during development.

Programmed DNA rearrangements in the single-celled eukaryote Oxytricha trifallax completely rewire its germline into a somatic nucleus during development. This elaborate, RNA-mediated pathway eliminates noncoding DNA sequences that interrupt gene loci and reorganizes the remaining fragments by inversions and permutations to produce functional genes. Here, we report the Oxytricha germline genome and compare it to the somatic genome to present a global view of its massive scale of genome rearrangements. The remarkably encrypted genome architecture contains >3,500 scrambled genes, as well as >800 predicted germline-limited genes expressed, and some posttranslationally modified, during genome rearrangements. Gene segments for different somatic loci often interweave with each other. Single gene segments can contribute to multiple, distinct somatic loci. Terminal precursor segments from neighboring somatic loci map extremely close to each other, often overlapping. This genome assembly provides a draft of a scrambled genome and a powerful model for studies of genome rearrangement.