Consequences of Prenatal Stress on Appetite Control and the Energy Expenditure Pathway

Established research has illustrated that moderate exposure to stress in the womb influences both adult phonotype and genotype for several physiological pathways, especially in males. Proposed explanations include adaptions made by the fetus resulting from a limited supply of nutrients, referred to as the thrifty phenotype. In this study, we examine this fetal programming effect on the appetite control and energy expenditure pathways in prenatally stressed adult male offspring. Subjects were male rats born from time-mated female rats exposed to unpredictable, variable prenatal stress (UVPS) throughout gestation. An analysis of the adult male rat offspring genetic expression of epididymal fat pads and the plasma concentrations of hormones involved in appetite control and energy expenditure pathways showed a significantly diminished expression of leptin and adiponectin compared to unstressed controls. Leptin and adiponectin are both major hormones involved in the appetite control and energy expenditure pathways, with leptin regulating energy balance due to its function as an inhibitor of hunger, and adiponectin modulating glucose levels and fatty acid breakdown. We observed higher leptin concentrations within the prenatally stressed male plasma, and lower expression of leptin (OB) and adiponectin (ADIPOQ) genes from the epididymal fat pads. We suggest that elevated leptin in the plasma elicited a negative feedback effect on OB expression levels, decreasing their quantification compared to control animals. Further analysis will include plasma quantification of insulin and glucose, as well as expression of ghrelin, a peptide which acts on the central nervous system and the bodys perception of hunger

Clades of huge phages from across Earth's ecosystems.

Bacteriophages typically have small genomes1 and depend on their bacterial hosts for replication2. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is-to our knowledge-the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR-Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR-Cas systems of phages have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phages may repurpose bacterial CRISPR-Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth's ecosystems

A Fish By Any Other Name Would Taste as Sweet: Applied Ethics of Transgenic Fish Commercialization

Modern aquaculture has grown into a vital industry over the past half-century, such that it now supplies half of all the fish we consume. Nevertheless, it has been found to cause significant economic, environmental, and health problems, while commercial fishing has led to the decline in wild fish stocks. In response to this dilemma and the growing demand, AquaBounty Technologies has created a genetically modified “AquAdvantage” Atlantic salmon using foreign genetic elements from the ocean pout and Chinook Pacific salmon, in hopes to improve their fish farming efficiency. These modifications allow the AquAdvantage salmon to grow twice as fast as their domesticated counterparts and four times as fast as their wild brethren, The recent approval for commercialization of the AquAdvantage Salmon as the first genetically modified animal by the Food and Drug Administration (FDA) in the US market has sparked substantial controversy, with no small number of people urging for a moratorium or ban on Genetically Modified Organisms (GMOs) altogether. The significance of this approval cannot be overstated, as it sets a regulatory precedent for other pending commercializations of genetically engineered animals and future axioms of molecular and synthetic biology. In this article, I identify and evaluate some of the leading arguments for and against the adoption of GM salmon on store shelves, and this product’s position in terms of deep ecology, the precautionary principle, virtue ethics, and non-anthropocentrism. I rely on this pluralistic presentation to ensure that the key aspects are recognized, and that possible consequences are assessed from a plurality of positions to avoid a one-eyed perception of the topic and allow judgements to be made on a rational and informed basis, all ethical concerns considered

Bogs, Bugs, Borgs, and Bacteriophages: Metagenomic and Biochemical Insights into the Enigmatic World of Extrachromosomal Genetic Elements

As a Ph.D. Candidate and National Science Foundation Predoctoral Fellow at the University of California, Berkeley, working in the labs of Dr. Jillian Banfield and Dr. Jennifer Doudna, I have dedicated my Ph.D. to the discovery and investigation of novel extrachromosomal elements and tools for biotechnological applications through a combination of genomics and biochemistry.The first chapter of this thesis uncovers 10 new clades of the largest bacteriophages ever found across many ecosystems worldwide, with genome sizes rivaling those of the smallest bacteria. We found that the phages are not only equipped with a wide variety of features typically associated with life and cellular organisms such as ribosomal proteins, tRNA synthetases and initiation and elongation factors, but also some of the viruses intriguingly utilize alternative genetic codes to translate their proteins. Notably, I discovered that the huge phage genomes encode CRISPR-Cas systems that may be used for inter-viral warfare. Some of these are miniature, previously undescribed CRISPR-Cas systems that are about half of the size of Cas9. This work was published in Nature. The second chapter describes the analysis and testing of one of the novel phage CRISPR-Cas systems, CRISPR-CasΦ, that we have shown can indeed exclude mobile elements such as plasmids from infecting the same host cell despite their small size, and can be applicable for programmable genome editing in bacterial, plant, and mammalian cells as the most compact functional CRISPR-Cas systems to date, potentially circumventing cell delivery barriers exhibited with CRISPR-Cas9 gene editing. Intriguingly, the CRISPR-CasΦ system exhibited a previously undescribed consolidation of chemistries in a Cas nuclease as the RuvC active site mediated both double-stranded DNA cleavage and RNA processing in a metal-dependent manner. This work was published in Science. The third chapter examines the discovery of enigmatic giant linear extrachromosomal elements, which we refer to as “Borgs”, inhabiting archaea. These elements that are about 1 Mbp long were recovered from multiple environments and may play a previously unrecognized role in controlling greenhouse gas emissions. Their genomes are represented in 2 uneven replichores, with inverted repeats >1.5kbp long on either end and dozens of tandem repeats throughout their genomes. They contain no obvious hallmarks of previously reported viruses or plasmids, and ~80% of their genes consist of novel and uncharacterized proteins. Our analysis of horizontal gene transfer suggests that many ribosomal, metabolic, and extracellular electron transfer genes and operons recently transferred from their hosts, including the nif operon for Nitrogen fixation and the MCR complex which was recently proposed to be involved in oxidation of methane. Evidence also suggests recent recombination events between different Borgs presumably within the same host cell. This work is currently in review at Nature. The fourth chapter describes an open-science effort for robust viral discovery computational pipelines driven by the COVID-19 pandemic. Working with a truly collaborative global team of bioinformaticians, this work describes the discovery of over 100,000 species of viruses to which I have contributed novel huge phage genomes. This manuscript was published in Nature. The final chapter examines the discovery of thousands of viruses encoding CRISPR-Cas systems, many of which target competing cryptic mobile elements that are predicted to infect the same bacterial hosts. From genome-resolved metagenomics and bioinformatics-enabled phylogenetic insights to biochemistry, structural biology, and eukaryotic genome editing, I describe hundreds of novel hypercompact and divergent CRISPR-Cas systems, with special consideration towards the novel Casλ family. Casλ possesses an aberrant RNA structure reminiscent of a naturally-occurring sgRNA and processes its own crRNA at the 3’ end, unlike any previously described single-RNA CRISPR-Cas system. The tertiary structure determined via cryo-EM reveals the machinery for PAM recognition, hybrid assembly, and DNA cleavage. RNA-targeting systems on viruses lack crucial residues or accessory proteins that would, in their bacterial counterparts, result in acute abortive infection, suggesting a potential strategy for phage systems to maintain host viability while preventing superinfection. In addition to their streamlined nature that is advantageous for cellular delivery, hypercompact phage systems can produce efficient genome editing in endogenous genes in mammalian and plant cells on par with, or in some cases, exceeding gold-standard Cas12a editing, demonstrating significant utility for biotechnological applications.Overall, this dissertation describes the use of a combination of bioinformatics and biochemistry to shed light on gigantic bacterial viruses, the proteins they encode on their genomes, and elements such as Borgs which we are only beginning to understand. Huge phages and Borgs represent little-known biology, the platforms for which are distinct from previously known systems, and significantly broaden our overall understanding of “non-living” selfish genetic entities. The metagenomic discovery and biochemical and structural characterization of hypercompact CRISPR-Cas systems in addition to analyses of their genome editing utility in eukaryotic cells pave the road for efficacious delivery of treatments to human cells in the near future

Candidate Phyla Radiation Roizmanbacteria From Hot Springs Have Novel and Unexpectedly Abundant CRISPR-Cas Systems.

The Candidate Phyla Radiation (CPR) comprises a huge group of bacteria that have small genomes that rarely encode CRISPR-Cas systems for phage defense. Consequently, questions remain about their mechanisms of phage resistance and the nature of phage that infect them. The compact CRISPR-CasY system (Cas12d) with potential value in genome editing was first discovered in these organisms. Relatively few CasY sequences have been reported to date, and little is known about the function and activity of these systems in the natural environment. Here, we conducted a genome-resolved metagenomic investigation of hot spring microbiomes and recovered CRISPR systems mostly from Roizmanbacteria that involve CasY proteins that are divergent from published sequences. Within population diversity in the spacer set indicates current in situ diversification of most of the loci. In addition to CasY, some Roizmanbacteria genomes also encode large type I-B and/or III-A systems that, based on spacer targeting, are used in phage defense. CRISPR targeting identified three phage represented by complete genomes and a prophage, which are the first reported for bacteria of the Microgenomates superphylum. Interestingly, one phage encodes a Cas4-like protein, a scenario that has been suggested to drive acquisition of self-targeting spacers. Consistent with this, the Roizmanbacteria population that it infects has a CRISPR locus that includes self-targeting spacers and a fragmented CasY gene (fCasY). Despite gene fragmentation, the PAM sequence is the same as that of other CasY reported in this study. Fragmentation of CasY may avoid the lethality of self-targeting spacers. However, the spacers may still have some biological role, possibly in genome regulation. The findings expand our understanding of CasY diversity, and more broadly, CRISPR-Cas systems and phage of CPR bacteria

Structural coordination between active sites of a CRISPR reverse transcriptase-integrase complex.

CRISPR-Cas systems provide adaptive immunity in bacteria and archaea, beginning with integration of foreign sequences into the host CRISPR genomic locus and followed by transcription and maturation of CRISPR RNAs (crRNAs). In some CRISPR systems, a reverse transcriptase (RT) fusion to the Cas1 integrase and Cas6 maturase creates a single protein that enables concerted sequence integration and crRNA production. To elucidate how the RT-integrase organizes distinct enzymatic activities, we present the cryo-EM structure of a Cas6-RT-Cas1-Cas2 CRISPR integrase complex. The structure reveals a heterohexamer in which the RT directly contacts the integrase and maturase domains, suggesting functional coordination between all three active sites. Together with biochemical experiments, our data support a model of sequential enzymatic activities that enable CRISPR sequence acquisition from RNA and DNA substrates. These findings highlight an expanded capacity of some CRISPR systems to acquire diverse sequences that direct CRISPR-mediated interference

Genome editing in plants using the compact editor CasΦ

Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) systems have been developed as important tools for plant genome engineering. Here, we demonstrate that the hypercompact CasΦ nuclease is able to generate stably inherited gene edits in Arabidopsis, and that CasΦ guide RNAs can be expressed with either the Pol-III U6 promoter or a Pol-II promoter together with ribozyme mediated RNA processing. Using the Arabidopsis fwa epiallele, we show that CasΦ displays higher editing efficiency when the target locus is not DNA methylated, suggesting that CasΦ is sensitive to chromatin environment. Importantly, two CasΦ protein variants, vCasΦ and nCasΦ, both showed much higher editing efficiency relative to the wild-type CasΦ enzyme. Consistently, vCasΦ and nCasΦ yielded offspring plants with inherited edits at much higher rates compared to WTCasΦ. Extensive genomic analysis of gene edited plants showed no off-target editing, suggesting that CasΦ is highly specific. The hypercompact size, T-rich minimal protospacer adjacent motif (PAM), and wide range of working temperatures make CasΦ an excellent supplement to existing plant genome editing systems

Structural coordination between active sites of a CRISPR reverse transcriptase-integrase complex.

CRISPR-Cas systems provide adaptive immunity in bacteria and archaea, beginning with integration of foreign sequences into the host CRISPR genomic locus and followed by transcription and maturation of CRISPR RNAs (crRNAs). In some CRISPR systems, a reverse transcriptase (RT) fusion to the Cas1 integrase and Cas6 maturase creates a single protein that enables concerted sequence integration and crRNA production. To elucidate how the RT-integrase organizes distinct enzymatic activities, we present the cryo-EM structure of a Cas6-RT-Cas1-Cas2 CRISPR integrase complex. The structure reveals a heterohexamer in which the RT directly contacts the integrase and maturase domains, suggesting functional coordination between all three active sites. Together with biochemical experiments, our data support a model of sequential enzymatic activities that enable CRISPR sequence acquisition from RNA and DNA substrates. These findings highlight an expanded capacity of some CRISPR systems to acquire diverse sequences that direct CRISPR-mediated interference