The vast bacteriophage population harbors an immense reservoir of genetic information. Almost 2000 phage genomes have been sequenced from phages infecting hosts in the phylum Actinobacteria, and analysis of these genomes reveals substantial diversity, pervasive mosaicism, and novel mechanisms for phage replication and lysogeny. Here, we describe the isolation and genomic characterization of 46 phages from environmental samples at various geographic locations in the U.S. infecting a single Arthrobacter sp. strain. These phages include representatives of all three virion morphologies, and Jasmine is the first sequenced podovirus of an actinobacterial host. The phages also span considerable sequence diversity, and can be grouped into 10 clusters according to their nucleotide diversity, and two singletons each with no close relatives. However, the clusters/singletons appear to be genomically well separated from each other, and relatively few genes are shared between clusters. Genome size varies from among the smallest of siphoviral phages (15,319 bp) to over 70 kbp, and G+C contents range from 45–68%, compared to 63.4% for the host genome. Although temperate phages are common among other actinobacterial hosts, these Arthrobacter phages are primarily lytic, and only the singleton Galaxy is likely temperate.

Beef feedyards produce nitrous oxide (N2O), a potent greenhouse gas. Limited research has evaluated the processes that produce feedyard N2O, and how rainfall and temperature impact N2O losses. Manure in feedyard pens develops into a complex ecosystem of microbes, extracellular enzymes, feces, and urine, with varying H2O content. This study aimed to improve understanding of feedyard N cycling under differing environmental conditions by incubation of manure in simulated feedyard pens using large chambers under laboratory conditions. We hypothesized that nitrification was the primary source of feedyard N2O, with interactions among temperature, H2O content, and manure properties. Emissions of N2O were monitored with a real–time N2O analyzer. Manure samples were taken at intervals for analyses of physicochemical properties, denitrification enzyme activity (DEA), and nitrification activity (NA). Due to equipment limitations, there was only one chamber per temperature tested. Correlation was poor among N2O emissions and rates of DEA and NA. However, significant relationships were found among key manure characteristics, such as ammonia/ammonium and nitrate/nitrite concentrations, manure dry matter, redox status, and temperature. These data suggest that most N2O was derived from denitrification in the top 5 cm of the manure pack. Further study is warranted to identify the processes involved in flushes of N2O emitted immediately after rainfall, possibly due to abiotic chemical reactions that release N2O sequestered in manure pores.

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The RNA binding protein RBFOX2 is linked to heart and skeletal muscle diseases; yet, RBFOX2-regulated RNA networks have not been systematically identified.Although RBFOX2 has a well-known function in alternative splicing (AS), it is unclear whether RBFOX2 has other roles in RNA metabolism that affect gene expression and function. Utilizing state of the art techniques Poly(A)-ClickSeq (PAC-seq) and nanopore cDNA sequencing, we revealed a new role for RBFOX2 in fine tuning alternative polyadenylation (APA) of pre-mRNAs in myoblasts. We found that depletion of RBFOX2 altered expression of mitochondrial genes. We identified the mitochondrial gene Slc25a4 gene that transports ATP/ADP across inner mitochondrial membrane as a target of RBFOX2. Dissecting how RBFOX2 affects Slc25a4 APA uncovered that RBFOX2 binding motifs near the distal polyadenylation site (PAS) are critical for expression of Slc25a4. Consistent with changes in expression of mitochondrial genes, loss of RBFOX2 altered mitochondrial membrane potential and induced mitochondrial swelling. Our results unveiled a novel role for RBFOX2 in maintaining APA decisions and expression of mitochondrial genes in myoblasts relevant to heart diseases. Keywords: alternative polyadenylation/mitochondria/nanopore sequencing/ poly(A) sequencing/RBFOX2 Non-standard Abbreviations and Acronyms: AS alternative splicing PAC-seq poly(A)-ClickSeq APA alternative polyadenylation KD knock down 3´UTR 3´untranslated region PAS poly(A)-site PAC poly(A)-cluster DPAC differential-poly(A)-clustering dPAS distal poly(A)-site pPAS proximal poly(A)-site ANT1 (Slc25a4) ATP/ADP translocator (mitochondrial) MFN1 Mitofusin

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The nucleus accumbens shell (NAcSh) and its afferent and efferent neuronal projections control key aspects of motivation for cocaine. A recently described regulator of γ-aminobutyric acid (GABA) projections from the dorsal raphe nucleus (DRN) to the NAcSh (DRN → NAcSh) is the neuropeptide neuromedin U (NMU). Here, we find that systemic administration of NMU decreases breakpoint for cocaine on a progressive ratio schedule of reinforcement in male rats. Employing a retrograde adeno-associated virus (AAV), we found that RNAi-mediated knockdown of the NMU receptor 2 (NMUR2) in afferent DRN projections to the NAcSh increases the breakpoint for cocaine. Our previous studies demonstrated that NMU regulates GABA release in the NAcSh, and our current investigation found that systemic NMU administration suppresses cocaine-evoked GABA release in the NAcSh and increases phosphorylated c-Fos expression in neurons projecting from the NAcSh to the ventral pallidum (VP). To further probe the impact of NMU/NMUR2 on neuroanatomical pathways regulating motivation for cocaine, we employed multi-viral transsynaptic studies. Using a combination of rabies virus and retrograde AAV helper virus, we mapped the impact of NMU across three distinct brain regions simultaneously and found a direct connection of GABAergic DRN neurons to the NAcSh → VP pathway. Together, these data reveal that NMU/NMUR2 modulates a direct connection within the GABAergic DRN → NAcSh → VP circuit that diminishes breakpoints for cocaine. These findings importantly advance our understanding of the neurochemical underpinnings of pathway-specific regulation of neurocircuitry that may regulate cocaine self-administration, providing a unique therapeutic perspective.

Modern molecular techniques enable characterization of the microbial biome in livestock manure, from which there is particular concern over emission of greenhouse gases. This study evaluated how sampling depth, time, temperature, and artificial rainfall affected microbial community structure in feedyard manure, and relationships between the manure biome and known parameters related to nitrous oxide (N 2 O) emissions. In three large incubation chambers, maintained at different temperatures that received two applications of artificial rainfall, we evaluated manure microbiome composition and abundance of N 2 O-producing enzymes (nirK and nirS) using quantitative polymerase chain reaction (qPCR). These data were used with previously published data from the same study on N 2 O emissions and assessment of manure physicochemical properties, denitrification enzyme activity (DEA), and nitrification activity (NA). Microbiome composition was Firmicutes (50%), followed by 32% Actinobacteria, 11% Proteobacteria, 5% Bacteroidetes, 1% Chloroflexi, and small populations (<0.5%) of Planctomycetes, Deinococcus-Thermus, Gemmatimonadetes, Verrucomicrobia, Tenericutes, and other organisms. Average bacterial populations varied largely as a function of sampling depth and time. Firmicutes increases tended to coincide with high N 2 O emissions. Overall, the largest change observed was increased Proteobacteria at 5-10 cm, where relative abundance increased from 10% (17.2 ˚C) to 24% (46.2 ˚C) over time and with increased temperature. Firmicutes and Actinobacteria predominated the microbial community of manure, but favorable conditions may lead to increases in Bacteroidetes, Proteobacteria, and Chloroflexi, which could influence N cycling and N 2 O emissions from feedyards.

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