Prospects for High-Elevation Radio Detection of >100 PeV Tau Neutrinos

Tau neutrinos are expected to comprise roughly one third of both the astrophysical and cosmogenic neutrino flux, but currently the flavor ratio is poorly constrained and the expected flux at energies above $10^{17}$ eV is low. We present a detector concept aimed at measuring the diffuse flux of tau neutrinos in this energy range via a high-elevation mountaintop detector using the radio technique. The detector searches for radio signals from upgoing air showers generated by Earth-skimming tau neutrinos. Signals from several antennas in a compact array are coherently summed at the trigger level, permitting not only directional masking of anthropogenic backgrounds, but also a low trigger threshold. This design takes advantage of both the large viewing area available at high-elevation sites and the nearly full duty cycle available to radio instruments. We present trade studies that consider the station elevation, frequency band, number of antennas in the array, and the trigger threshold to develop a highly efficient station design. Such a mountaintop detector can achieve a factor of ten improvement in acceptance over existing instruments with 100 independent stations. With 1000 stations and three years of observation, it can achieve a sensitivity to an integrated $\mathcal{E}^{-2}$ flux of $<10^{-9}$ GeV cm$^{-2}$ sr$^{-1}$ s$^{-1}$, in the range of the expected flux of all-flavor cosmogenic neutrinos assuming a pure iron cosmic-ray composition.Comment: 26 pages, 11 figure

Assessments of the Deepwater Horizon oil spill impact on Gulf coast microbial communities

Peer reviewe

The RHO-1 RhoGTPase Modulates Fertility and Multiple Behaviors in Adult C. elegans

The Rho family of small GTPases are essential during early embryonic development making it difficult to study their functions in adult animals. Using inducible transgenes expressing either a constitutively active version of the single C. elegans Rho ortholog, RHO-1, or an inhibitor of endogenous Rho (C3 transferase), we demonstrate multiple defects caused by altering Rho signaling in adult C. elegans. Changes in RHO-1 signaling in cholinergic neurons affected locomotion, pharyngeal pumping and fecundity. Changes in RHO-1 signaling outside the cholinergic neurons resulted in defective defecation, ovulation, and changes in C. elegans body morphology. Finally both increased and decreased RHO-1 signaling in adults resulted in death within hours. The multiple post-developmental roles for Rho in C. elegans demonstrate that RhoA signaling pathways continue to be used post-developmentally and the resulting phenotypes provide an opportunity to further study post-developmental Rho signaling pathways using genetic screens

Nucleic acid-guided genome defense systems from bacteria and archaea

Bacterial and archaeal genomes are under constant threat by genetic invaders. The need to maintain genomic and cellular integrity has driven the evolution of numerous and diverse genome defense systems. A common theme in prokaryotic defense strategies is interference of foreign DNA and RNA on the sequence level. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems confer adaptive immunity to previously encountered genetic invaders. Guided by short RNAs, the main effectors of CRISPR-Cas systems are sequence specific nucleases that catalyze degradation of exogenous nucleic acids. At the center of a similar method of genome defense to CRISPR-Cas systems, but operating through non-homologous proteins and pathways, prokaryotic Argonaute proteins (pAgos) have been proposed as sequence specific defense systems. However, our mechanistic knowledge of both CRISPR-Cas and pAgo systems stems from a small subset of the total genetic diversity of these systems. Here, we address this limited understanding through analysis of new CRISPR-Cas and pAgo systems, as well as describe novel activity for previously identified members.CRISPR-Cas9 has rapidly been adopted as a programmable platform for sequence-specific DNA targeting with endless applications in gene editing, genome-wide screening, and transcriptional control. Current applications draw upon the biochemical activities of a few common Cas9 enzymes. The study of diverse homologs has potential to yield novel Cas9 proteins with desired traits such as increased efficiency or specificity. Surveying a vast metagenomic database, we report the first Cas9 from archaea, expanding the occurrence of CRISPR-Cas9 systems to a new domain of life. DNA targeting is a hallmark of CRISPR-Cas9 systems. Engineering SpyCas9 to bind and target RNA has been difficult and suffers from reduced efficiency. We sought to identify Cas9 homologs with a natural ability to target RNA molecules. Using in vitro purification and biochemical assays, we discovered Cas9 enzymes that efficiently cleave RNA. Furthermore, we show that this activity can be harnessed to reduce phage infection and mediate gene repression in vivo, expanding the toolkit of CRISPR-Cas nucleases.Analogous to CRISPR-Cas systems, Argonaute proteins are well known, RNA-guided nucleases that operate in eukaryotic RNA-interference. Motivated by initial observations that Argonaute homologs in prokaryotes constitute a nucleic acid-guided genome defense system, we studied the physiological role and biochemical activities of a novel clade of pAgos. We offer the first experimental evidence of complex formation between natural, two-piece Argonaute proteins. Preliminary in vivo observations implicate this split-pAgo in maintaining motility under conditions induced by introduction of an exogenous plasmid. Together with our studies on CRISPR-Cas systems, our work highlights unexpected functional diversity across divergent nucleic acid-guided genome defense systems

Nucleic acid-guided genome defense systems from bacteria and archaea

Bacterial and archaeal genomes are under constant threat by genetic invaders. The need to maintain genomic and cellular integrity has driven the evolution of numerous and diverse genome defense systems. A common theme in prokaryotic defense strategies is interference of foreign DNA and RNA on the sequence level. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems confer adaptive immunity to previously encountered genetic invaders. Guided by short RNAs, the main effectors of CRISPR-Cas systems are sequence specific nucleases that catalyze degradation of exogenous nucleic acids. At the center of a similar method of genome defense to CRISPR-Cas systems, but operating through non-homologous proteins and pathways, prokaryotic Argonaute proteins (pAgos) have been proposed as sequence specific defense systems. However, our mechanistic knowledge of both CRISPR-Cas and pAgo systems stems from a small subset of the total genetic diversity of these systems. Here, we address this limited understanding through analysis of new CRISPR-Cas and pAgo systems, as well as describe novel activity for previously identified members.CRISPR-Cas9 has rapidly been adopted as a programmable platform for sequence-specific DNA targeting with endless applications in gene editing, genome-wide screening, and transcriptional control. Current applications draw upon the biochemical activities of a few common Cas9 enzymes. The study of diverse homologs has potential to yield novel Cas9 proteins with desired traits such as increased efficiency or specificity. Surveying a vast metagenomic database, we report the first Cas9 from archaea, expanding the occurrence of CRISPR-Cas9 systems to a new domain of life. DNA targeting is a hallmark of CRISPR-Cas9 systems. Engineering SpyCas9 to bind and target RNA has been difficult and suffers from reduced efficiency. We sought to identify Cas9 homologs with a natural ability to target RNA molecules. Using in vitro purification and biochemical assays, we discovered Cas9 enzymes that efficiently cleave RNA. Furthermore, we show that this activity can be harnessed to reduce phage infection and mediate gene repression in vivo, expanding the toolkit of CRISPR-Cas nucleases.Analogous to CRISPR-Cas systems, Argonaute proteins are well known, RNA-guided nucleases that operate in eukaryotic RNA-interference. Motivated by initial observations that Argonaute homologs in prokaryotes constitute a nucleic acid-guided genome defense system, we studied the physiological role and biochemical activities of a novel clade of pAgos. We offer the first experimental evidence of complex formation between natural, two-piece Argonaute proteins. Preliminary in vivo observations implicate this split-pAgo in maintaining motility under conditions induced by introduction of an exogenous plasmid. Together with our studies on CRISPR-Cas systems, our work highlights unexpected functional diversity across divergent nucleic acid-guided genome defense systems

RNA-dependent RNA targeting by CRISPR-Cas9.

Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo. We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. These results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications

Metatranscriptomics reveals the molecular mechanism of large granule formation in granular anammox reactor

Granules enriched with anammox bacteria are essential in enhancing the treatment of ammonia-rich wastewater, but little is known about how anammox bacteria grow and multiply inside granules. Here, we combined metatranscriptomics, quantitative PCR and 16S rRNA gene sequencing to study the changes in community composition, metabolic gene content and gene expression in a granular anammox reactor with the objective of understanding the molecular mechanism of anammox growth and multiplication that led to formation of large granules. Size distribution analysis revealed the spatial distribution of granules in which large granules having higher abundance of anammox bacteria (genus Brocadia) dominated the bottom biomass. Metatranscriptomics analysis detected all the essential transcripts for anammox metabolism. During the later stage of reactor operation, higher expression of ammonia and nitrite transport proteins and key metabolic enzymes mainly in the bottom large granules facilitated anammox bacteria activity. The high activity resulted in higher growth and multiplication of anammox bacteria and expanded the size of the granules. This conceptual model for large granule formation proposed here may assist in the future design of anammox processes for mainstream wastewater treatment.BT/Environmental Biotechnolog

A bacterial Argonaute with noncanonical guide RNA specificity.

Eukaryotic Argonaute proteins induce gene silencing by small RNA-guided recognition and cleavage of mRNA targets. Although structural similarities between human and prokaryotic Argonautes are consistent with shared mechanistic properties, sequence and structure-based alignments suggested that Argonautes encoded within CRISPR-cas [clustered regularly interspaced short palindromic repeats (CRISPR)-associated] bacterial immunity operons have divergent activities. We show here that the CRISPR-associated Marinitoga piezophila Argonaute (MpAgo) protein cleaves single-stranded target sequences using 5'-hydroxylated guide RNAs rather than the 5'-phosphorylated guides used by all known Argonautes. The 2.0-Å resolution crystal structure of an MpAgo-RNA complex reveals a guide strand binding site comprising residues that block 5' phosphate interactions. Using structure-based sequence alignment, we were able to identify other putative MpAgo-like proteins, all of which are encoded within CRISPR-cas loci. Taken together, our data suggest the evolution of an Argonaute subclass with noncanonical specificity for a 5'-hydroxylated guide