Conditional gene regulation models demonstrate a pro-proliferative role for growth hormone receptor in prostate cancer.

BACKGROUND: Humans with inactivating mutations in growth hormone receptor (GHR) have lower rates of cancer, including prostate cancer. Similarly, mice with inactivating Ghr mutations are protected from prostatic intraepithelial neoplasia in the C3(1)/TAg prostate cancer model. However, gaps in clinical relevance in those models persist. The current study addresses these gaps and the ongoing role of Ghr in prostate cancer using loss-of-function and gain-of-function models. METHODS: Conditional Ghr inactivation was achieved in the C3(1)/TAg model by employing a tamoxifen-inducible Cre and a prostate-specific Cre. In parallel, a transgenic GH antagonist was also used. Pathology, proliferation, and gene expression of 6-month old mouse prostates were assessed. Analysis of The Cancer Genome Atlas data was conducted to identify GHR overexpression in a subset of human prostate cancers. Ghr overexpression was modeled in PTEN-P2 and TRAMP-C2 mouse prostate cancer cells using stable transfectants. The growth, proliferation, and gene expression effects of Ghr overexpression was assessed in vitro and in vivo. RESULTS: Loss-of-function for Ghr globally or in prostatic epithelial cells reduced proliferation and stratification of the prostatic epithelium in the C3(1)/TAg model. Genes and gene sets involved in the immune system and tumorigenesis, for example, were dysregulated upon global Ghr disruption. Analysis of The Cancer Genome Atlas revealed higher GHR expression in human prostate cancers with ERG-fusion genes or ETV1-fusion genes. Modeling the GHR overexpression observed in these human prostate cancers by overexpressing Ghr in mouse prostate cancer cells with mutant Pten or T-antigen driver genes increased proliferation of prostate cancer cells in vitro and in vivo. Ghr overexpression regulated the expression of multiple genes oppositely to Ghr loss-of-function models. CONCLUSIONS: Loss-of-function and gain-of-function Ghr models, including prostatic epithelial cell specific alterations in Ghr, altered proliferation, and gene expression. These data suggest that changes in GHR activity in human prostatic epithelial cells play a role in proliferation and gene regulation in prostate cancer, suggesting the potential for disrupting GH signaling, for example by the FDA approved GH antagonist pegvisomant, may be beneficial in treating prostate cancer.

A Method for Improving the Accuracy and Efficiency of Bacteriophage Genome Annotation.

Bacteriophages are the most numerous entities on Earth. The number of sequenced phage genomes is approximately 8000 and increasing rapidly. Sequencing of a genome is followed by annotation, where genes, start codons, and functions are putatively identified. The mainstays of phage genome annotation are auto-annotation programs such as Glimmer and GeneMark. Due to the relatively small size of phage genomes, many groups choose to manually curate auto-annotation results to increase accuracy. An additional benefit of manual curation of auto-annotated phage genomes is that the process is amenable to be performed by students, and has been shown to improve student recruitment to the sciences. However, despite its greater accuracy and pedagogical value, manual curation suffers from high labor cost, lack of standardization and a degree of subjectivity in decision making, and susceptibility to mistakes. Here, we present a method developed in our lab that is designed to produce accurate annotations while reducing subjectivity and providing a degree of standardization in decision-making. We show that our method produces genome annotations more accurate than auto-annotation programs while retaining the pedagogical benefits of manual genome curation.

Complete genome sequences of cluster F1 and cluster B1 Mycobacterium smegmatis phages Karhdo and Basato.

We present the complete genome sequences of Mycobacterium smegmatis phages Karhdo and Basato, isolated in Clark County, Nevada. The phages were isolated and annotated by students enrolled in undergraduate research courses over two semesters at the University of Nevada, Las Vegas.

Paenibacillus larvae and their phages; a community science approach to discovery and initial testing of prophylactic phage cocktails against American Foulbrood in New Zealand.

Background: American foulbrood (AFB) is a devastating disease of the European honey bee (Apis mellifera) and is found throughout the world. AFB is caused by the bacterium Paenibacillus larvae (P. larvae). Treatment with antibiotics is strictly forbidden in many regions, including New Zealand. Safe and natural prophylactic solutions to protect honey bees from AFB are needed. Bacteriophages are a well-studied alternative to antibiotics and have been shown to be effective against P. larvae in other countries. Methods: We employed a community science approach to obtaining samples from around New Zealand to discover novel bacteriophages. Standard isolation approaches were employed for both bacteria and bacteriophages. Host range testing was performed by agar overlay spot tests, and cocktail formulation and in vitro testing were performed in 96-well plate assays, followed by sub-sampling and CFU visualization on agar plates. Results: Herein, we describe the discovery and isolation of eight P. larvae bacterial isolates and 26 P. larvae bacteriophages that are novel and native to New Zealand. The phage genomes were sequenced and annotated, and their genomes were compared to extant sequenced P. larvae phage genomes. We test the host ranges of the bacteriophages and formulate cocktails to undertake in vitro testing on a set of representative bacterial strains. These results form the basis of a promising solution for protecting honey bees in New Zealand from AFB.

In Vitro Evolution to Increase the Titers of Difficult Bacteriophages: RAMP-UP Protocol.

Background: Bacteriophages are becoming increasingly important in the race to find alternatives to antibiotics. Unfortunately, bacteriophages that might otherwise be useful are sometimes discarded due to low titers making them unsuitable for downstream applications. Methods: Here, we present two distinct approaches used to experimentally evolve novel New Zealand Paenibacillus larvae bacteriophages. The first approach uses the traditional agar-overlay method, whereas the other was a 96-well plate liquid infection protocol that improved phage titers in as little as four days. We also used a mathematical model to probe the parameters and limits of the RAMP-UP approach to rapidly select mutants that improve bacteriophage titers. Results: Both experimental approaches resulted in an increase in plaque-forming units (PFU/mL). The liquid infection approach developed here, which we call RAMP-UP for Rapid Adaptive Mutation of Phage - UP, was significantly faster and simpler, and allowed us to evolve high titer bacteriophages in as little as four days. Titers were increased from 100-100,000-fold relative to their ancestors. The resultant titers were sufficient to extract and sequence DNA from these bacteriophages. An analysis of these phage genomes is provided. Conclusion: The RAMP-UP protocol is an effective method for experimentally evolving previously intractable bacteriophages in a high-throughput and expeditious manner.

Complete genome sequences of cluster G1 and cluster K4 Mycobacterium smegmatis phages omnicritical and Barkley26.

We present the complete genome sequence of two Actinobacteriophages, OmniCritical and Barkley26, isolated in Clark County, NV. Over two semesters, The University of Nevada, Las Vegas (UNLV) students isolated and purified phages and manually annotated the genomes. The courses follow the HHMI Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Sciences (SEA-PHAGES) curricula.

Formation of BCR oligomers provides a mechanism for B cell affinity discrimination.

B cells encounter antigen over a wide affinity range, from K(A)=10(5) M(-1) to K(A)=10(10) M(-1). The strength of B cell antigen receptor (BCR) signaling in response to antigen increases with affinity, a process known as "affinity discrimination". In this work, we use a computational simulation of B cell surface dynamics and membrane-proximal signaling to show that affinity discrimination can arise from the formation of BCR oligomers. It is known that BCRs form oligomers upon encountering antigen, and that the size and rate of formation of these oligomers both increase with affinity. In our simulation, we have introduced a requirement that only BCR-antigen complexes that are part of an oligomer can engage cytoplasmic signaling molecules such as Src-family kinases. Our simulation shows that as affinity increases, BCR signaling activity increases in addition to the number of collected antigen. Our results are also consistent with the existence of an experimentally-observed threshold affinity of activation at K(A)=10(5)-10(6) M(-1) (no signaling activity below this affinity value) and affinity discrimination ceiling of K(A)=10(10) M(-1) (no affinity discrimination above this affinity value). Comparison with experiments shows that the time scale of BCR oligomer formation predicted by our model (less than 10 s) is well within the time scale of experimentally observed association of BCR with Src-family kinases (10-20 s).

GH Action in Prostate Cancer Cells Promotes Proliferation, Limits Apoptosis, and Regulates Cancer-related Gene Expression.

Previous studies investigating the effects of blocking the growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis in prostate cancer found no effects of the growth hormone receptor (GHR) antagonist, pegvisomant, on the growth of grafted human prostate cancer cells in vivo. However, human GHR is not activated by mouse GH, so direct actions of GH on prostate cancer cells were not evaluated in this context. The present study addresses the species specificity of GH-GHR activity by investigating GH actions in prostate cancer cell lines derived from a mouse Pten-deletion model. In vitro cell growth was stimulated by GH and reduced by pegvisomant. These in vitro GH effects were mediated at least in part by the activation of JAK2 and STAT5. When Pten-mutant cells were grown as xenografts in mice, pegvisomant treatment dramatically reduced xenograft size, and this was accompanied by decreased proliferation and increased apoptosis. RNA sequencing of xenografts identified 1765 genes upregulated and 953 genes downregulated in response to pegvisomant, including many genes previously implicated as cancer drivers. Further evaluation of a selected subset of these genes via quantitative reverse transcription-polymerase chain reaction determined that some genes exhibited similar regulation by pegvisomant in prostate cancer cells whether treatment was in vivo or in vitro, indicating direct regulation by GH via GHR activation in prostate cancer cells, whereas other genes responded to pegvisomant only in vivo, suggesting indirect regulation by pegvisomant effects on the host endocrine environment. Similar results were observed for a prostate cancer cell line derived from the mouse transgenic adenocarcinoma of the mouse prostate (TRAMP) model.

Characterization of CRISPR Spacer and Protospacer Sequences in Paenibacillus larvae and Its Bacteriophages.

The bacterium Paenibacillus larvae is the causative agent of American foulbrood, the most devastating bacterial disease of honeybees. Because P. larvae is antibiotic resistant, phages that infect it are currently used as alternative treatments. However, the acquisition by P. larvae of CRISPR spacer sequences from the phages could be an obstacle to treatment efforts. We searched nine complete genomes of P. larvae strains and identified 714 CRISPR spacer sequences, of which 384 are unique. Of the four epidemiologically important P. larvae strains, three of these have fewer than 20 spacers, while one strain has over 150 spacers. Of the 384 unique spacers, 18 are found as protospacers in the genomes of 49 currently sequenced P. larvae phages. One P. larvae strain does not have any protospacers found in phages, while another has eight. Protospacer distribution in the phages is uneven, with two phages having up to four protospacers, while a third of phages have none. Some phages lack protospacers found in closely related phages due to point mutations, indicating a possible escape mechanism. This study serve a point of reference for future studies on the CRISPR-Cas system in P. larvae as well as for comparative studies of other phage-host systems.

Phage Commander, an Application for Rapid Gene Identification in Bacteriophage Genomes Using Multiple Programs.

The number of sequenced bacteriophage genomes is growing at an exponential rate. The majority of sequenced bacteriophage genomes are annotated by one or more of several freely available gene identification programs (Glimmer, GeneMark, RAST, Prodigal, etc.). No program has been shown to consistently outperform the others; thus, the choice of which program to use is not obvious. We present the Phage Commander application for rapid identification of bacteriophage genes using multiple gene identification programs. Phage Commander runs a bacteriophage genome sequence through nine gene identification programs (and an additional program for identification of tRNAs) and integrates the results within a single output table. Phage Commander also generates formatted output files for direct export to National Center for Biotechnology Information GenBank or genome visualization programs such as DNA Master. Users can select the threshold for which genes to export (genes identified by at least one program, genes identified by at least two programs, etc.). Phage Commander was benchmarked using eight high-quality bacteriophage genomes whose genes are backed by experimental data. Our results show that the most accurate annotations are obtained by exporting genes identified by at least two or three programs. Many groups opt to manually curate the annotations obtained from gene identification programs, and Phage Commander was designed to facilitate manual curation of genome annotations. Our benchmarking results show that manual curation does indeed produce more accurate annotations than any individual gene identification program. The authors thus recommend manually curating the output of Phage Commander to generate maximally accurate annotations. Phage Commander is currently being used in the corresponding author's bacteriophage genome annotation class and has reduced the labor cost and improved the quality of genome annotations.

Complete Genome Sequences of Cluster A6 and Cluster G1 Mycobacterium smegmatis Phages Hoot and Jolene.

We present the complete genome sequences of Mycobacterium smegmatis phages Hoot and Jolene, isolated in Las Vegas, NV. The phages were isolated and annotated by students enrolled in an undergraduate research course at the University of Nevada, Las Vegas. Hoot is a cluster A6 mycobacteriophage, while Jolene is in cluster G1.

Paenibacillus larvae bacteriophages: obscure past, promising future.

Paenibacillus larvae is a Gram-positive, spore-forming bacterium that is the causative agent of American foulbrood (AFB), the most devastating bacterial disease of the honeybee. P. larvae is antibiotic resistant, complicating treatment efforts. Bacteriophages that target P. larvae are rapidly emerging as a promising treatment. The first P. larvae phages were isolated in the 1950s, but as P. larvae was not antibiotic resistant at the time, interest in them remained scant. Interest in P. larvae phages has grown rapidly since the first P. larvae phage genome was sequenced in 2013. Since then, the number of sequenced P. larvae phage genomes has reached 48 and is set to grow further. All sequenced P. larvae phages encode a conserved N-acetylmuramoyl-l-alanine amidase that is responsible for cleaving the peptidoglycan cell wall of P. larvae. All P. larvae phages also encode either an integrase, excisionase or Cro/CI, indicating that they are temperate. In the last few years, several studies have been published on using P. larvae phages and the P. larvae phage amidase as treatments for AFB. Studies were conducted on infected larvae in vitro and also on hives in the field. The phages have a prophylactic effect, preventing infection, and also a curative effect, helping resolve infection. P. larvae phages have a narrow range, lysing only P. larvae, and are unable to lyse even related Paenibacillus species. P. larvae phages thus appear to be safe to use and effective as treatment for AFB, and interest in them in the coming years will continue to grow.

Complete Genome Sequences of Cluster P1 and Cluster C1 Mycobacterium smegmatis Phages Jung and Ronan.

We present the complete genome sequences of Mycobacterium smegmatis phages Jung and Ronan, isolated from soil in Las Vegas, Nevada. The phages were isolated and annotated by students enrolled in a course for undergraduate research experience (CURE). Jung is a cluster P1 mycobacteriophage, while Ronan is in cluster C1.

Diversity and Distribution of a Novel Genus of Hyperthermophilic Aquificae Viruses Encoding a Proof-Reading Family-A DNA Polymerase.

Despite the high abundance of Aquificae in many geothermal systems, these bacteria are difficult to culture and no viruses infecting members of this phylum have been isolated. Here, we describe the complete, circular dsDNA Uncultivated Virus Genome (UViG) of Thermocrinis Octopus Spring virus (TOSV), derived from metagenomic data, along with eight related UViGs representing three additional viral species. Despite low overall similarity among viruses from different hot springs, the genomes shared a high degree of synteny, and encoded numerous genes for nucleotide metabolism, including a PolA-type DNA polymerase polyprotein with likely accessory functions, a DNA Pol III sliding clamp, a thymidylate kinase, a DNA gyrase, a helicase, and a DNA methylase. Also present were conserved genes predicted to code for phage capsid, large and small subunits of terminase, portal protein, holin, and lytic transglycosylase, all consistent with a distant relatedness to cultivated Caudovirales. These viruses are predicted to infect Aquificae, as multiple CRISPR spacers matching the viral genomes were identified within the genomes and metagenomic contigs from these bacteria. Based on the predicted atypical bi-directional replication strategy, low sequence similarity to known viral genomes, and unique position in gene-sharing networks, we propose a new putative genus, "Pyrovirus," in the order Caudovirales.

Genomic diversity of bacteriophages infecting Microbacterium spp.

The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to ~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics.

Complete Genome Sequences of Mycobacterium smegmatis Phages NihilNomen and Carlyle, Isolated in Las Vegas, Nevada.

We present the complete genomes of the Mycobacterium smegmatis phages Carlyle and NihilNomen, isolated from soil in Las Vegas, Nevada. The phages were isolated and annotated by undergraduate students enrolled in the Phage Discovery course offered by the School of Life Sciences at the University of Nevada Las Vegas.

Complete Genome Sequences of Mycobacterium smegmatis Phages Chewbacca, Reptar3000, and Riparian, Isolated in Las Vegas, Nevada.

Here, we present the complete genome sequences of Mycobacterium smegmatis phages Chewbacca, Reptar3000, and Riparian, isolated from soil in Las Vegas, NV. The phages were isolated and annotated by undergraduate students enrolled in the Phage Discovery course offered by the School of Life Sciences at the University of Nevada, Las Vegas.

Monte Carlo study of single molecule diffusion can elucidate the mechanism of B cell synapse formation.

B cell receptors have been shown to cluster at the intercellular junction between a B cell and an antigen-presenting cell in the form of a segregated pattern of B cell receptor/antigen complexes known as an immunological synapse. We use random walk-based theoretical arguments and Monte Carlo simulations to study the effect of diffusion of surface-bound molecules on B cell synapse formation. Our results show that B cell synapse formation is optimal for a limited range of receptor-ligand complex diffusion coefficient values, typically one-to-two orders of magnitude lower than the diffusion coefficient of free receptors. Such lower mobility of receptor-ligand complexes can significantly affect the diffusion of a tagged receptor or ligand in an affinity dependent manner, as the binding/unbinding of such receptor or ligand molecules crucially depends on affinity. Our work shows how single molecule tracking experiments can be used to estimate the order of magnitude of the diffusion coefficient of receptor-ligand complexes, which is difficult to measure directly in experiments due to the finite lifetime of receptor-ligand bonds. We also show how such antigen movement data at the single molecule level can provide insight into the B cell synapse formation mechanism. Thus, our results can guide further single molecule tracking experiments to elucidate the synapse formation mechanism in B cells, and potentially in other immune cells.

Genomic Analysis of 48 Paenibacillus larvae Bacteriophages.

The antibiotic-resistant bacterium Paenibacillus larvae is the causative agent of American foulbrood (AFB), currently the most destructive bacterial disease in honeybees. Phages that infect P. larvae were isolated as early as the 1950s, but it is only in recent years that P. larvae phage genomes have been sequenced and annotated. In this study we analyze the genomes of all 48 currently sequenced P. larvae phage genomes and classify them into four clusters and a singleton. The majority of P. larvae phage genomes are in the 38⁻45 kbp range and use the cohesive ends (cos) DNA-packaging strategy, while a minority have genomes in the 50⁻55 kbp range that use the direct terminal repeat (DTR) DNA-packaging strategy. The DTR phages form a distinct cluster, while the cos phages form three clusters and a singleton. Putative functions were identified for about half of all phage proteins. Structural and assembly proteins are located at the front of the genome and tend to be conserved within clusters, whereas regulatory and replication proteins are located in the middle and rear of the genome and are not conserved, even within clusters. All P. larvae phage genomes contain a conserved N-acetylmuramoyl-l-alanine amidase that serves as an endolysin.

Complete Genome Sequences of Paenibacillus larvae Phages Halcyone, Heath, Scottie, and Unity from Las Vegas, Nevada.

We present the complete genome sequences of four phages that infect Paenibacillus larvae, the causative agent of American foulbrood disease in honeybees. The phages were isolated from beehives and beeswax products from Las Vegas, Nevada. The genomes are 50 to 55 kbp long and use the "direct terminal repeats" DNA-packaging strategy.