Purification of high molecular-weight antibacterial proteins of insect pathogenic Brevibacillus laterosporus isolates

Brevibacillus laterosporus (Bl) is a Gram-positive and spore-forming bacterium belonging to the Brevibacillus brevis phylogenetic cluster. Globally, insect pathogenic strains of the bacterium have been isolated, characterised, and some activities patented. Two isolates, Bl 1821L and Bl 1951, exhibiting pathogenicity against the diamondback moth and mosquitoes, are under development as a biopesticide in New Zealand. However, due to the suspected activity of putative antibacterial proteins (ABPs), the endemic isolates often grow erratically. Various purification methods including size exclusion chromatography, sucrose density gradient centrifugation, polyethylene glycol precipitation, and ammonium sulphate precipitation employed in this study enabled the isolation of two putative antibacterial proteins of ~30 kD and ~48 kD from Bl 1821L and one putative antibacterial protein of ~30 kD from Bl 1951. Purification of the uninduced cultures of Bl 1821L and Bl 1951 also yielded the protein bands of ~30 kD and ~48 kD on SDS-PAGE which indicated their spontaneous induction. Disc diffusion assay was used to determine the antagonistic activities of the putative ABPs. Subsequent transmission electron microscope (TEM) examination of purified putative antibacterial protein-containing solution showed the presence of encapsulin (~30 kD) and polysheath (~48 kD) like structures. Although only the ~30 kD protein was purified from Bl 1951, both structures were seen in this strain under TEM. Furthermore, while assessing the antibacterial activity of some fractions of Bl 1951 against Bl 1821L in size exclusion chromatography method, population of Bl 1821L persister cells was noted. Overall, this work added a wealth of knowledge for the purification of the HMW proteins (bacteriocins) of the Gram-positive bacteria including Bl

Isolation, purification, and characterisation of a phage tail-like bacteriocin from the insect pathogenic bacterium Brevibacillus laterosporus

The Gram-positive and spore-forming bacterium Brevibacillus laterosporus (Bl) belongs to the Brevibacillus brevis phylogenetic cluster. Isolates of the species have demonstrated pesticidal potency against a wide range of invertebrate pests and plant diseases. Two New Zealand isolates, Bl 1821L and Bl 1951, are under development as biopesticides for control of diamondback moth and other pests. However, due to the often-restricted growth of these endemic isolates, production can be an issue. Based on the previous work, it was hypothesised that the putative phages might be involved. During investigations of the cause of the disrupted growth, electron micrographs of crude lysate of Bl 1821L showed the presence of phages’ tail-like structures. A soft agar overlay method with PEG 8000 precipitation was used to differentiate between the antagonistic activity of the putative phage and phage tail-like structures (bacteriocins). Assay tests authenticated the absence of putative phage activity. Using the same method, broad-spectrum antibacterial activity of Bl 1821L lysate against several Gram-positive bacteria was found. SDS-PAGE of sucrose density gradient purified and 10 kD MWCO concentrated lysate showed a prominent protein band of ∼48 kD, and transmission electron microscopy revealed the presence of polysheath-like structures. N-terminal sequencing of the ∼48 kD protein mapped to a gene with weak predicted amino acid homology to a Bacillus PBSX phage-like element xkdK, the translated product of which shared >90% amino acid similarity to the phage tail-sheath protein of another Bl published genome, LMG15441. Bioinformatic analysis also identified an xkdK homolog in the Bl 1951 genome. However, genome comparison of the region around the xkdK gene between Bl 1821L and Bl 1951 found differences including two glycine rich protein encoding genes which contain imperfect repeats (1700 bp) in Bl 1951, while a putative phage region resides in the analogous Bl 1821L region. Although comparative analysis of the genomic organisation of Bl 1821L and Bl 1951 PBSX-like region with the defective phages PBSX, PBSZ, and PBP 180 of Bacillus subtilis isolates 168 and W23, and Bacillus phage PBP180 revealed low amino acids similarity, the genes encode similar functional proteins in similar arrangements, including phage tail-sheath (XkdK), tail (XkdO), holin (XhlB), and N-acetylmuramoyl-L-alanine (XlyA). AMPA analysis identified a bactericidal stretch of 13 amino acids in the ∼48 kD sequenced protein of Bl 1821L. Antagonistic activity of the purified ∼48 kD phage tail-like protein in the assays differed remarkably from the crude lysate by causing a decrease of 34.2% in the number of viable cells of Bl 1951, 18 h after treatment as compared to the control. Overall, the identified inducible phage tail-like particle is likely to have implications for the in vitro growth of the insect pathogenic isolate Bl 1821L

Generational Differences in Faculty and Student Comfort with Technology Use

Background: Navigating through online education courses continues to be a struggle for some nursing students. At the same time, integrating technology into online courses can be difficult for nursing faculty. Purpose: The purpose of this study was to assess faculty technology integration practices, student attitudes about technology use, and generational differences related to faculty and student technology use. Methods: A descriptive cross-sectional survey design was used to obtain data for this study. Results: Integration of technology into online courses and student attitudes about technology use were not significantly different by generation. Faculty and students from the Baby Boomer and Generation X reported less comfort using technology and higher levels of anxiety using technology than did individuals from Generation Y. Conclusion: Significant generational variations were not noted in relation to technology integration into courses and overall student attitudes about technology in this study, but differences were noted in relation to comfort with use of technology and anxiety when using technology. Student learning outcomes and satisfaction with learning may be influenced by the student\u27s comfort using technology and faculty\u27s confidence in integrating and using technology to provide online instruction

You are what you eat: Fungal metabolites and host plant affect the susceptibility of diamondback moth to entomopathogenic fungi

Background: Beauveria are entomopathogenic fungi of a broad range of arthropod pests. Many strains of Beauveria have been developed and marketed as biopesticides. Beauveria species are well-suited as the active ingredient within biopesticides because of their ease of mass production, ability to kill a wide range of pest species, consistency in different conditions, and safety with respect to human health. However, the efficacy of these biopesticides can be variable under field conditions. Two under-researched areas, which may limit the deployment of Beauveria-based biopesticides, are the type and amount of insecticidal compounds produced by these fungi and the influence of diet on the susceptibility of specific insect pests to these entomopathogens. Methods: To understand and remedy this weakness, we investigated the effect of insect diet and Beauveria-derived toxins on the susceptibility of diamondback moth larvae to Beauveria infection. Two New Zealand-derived fungal isolates, B. pseudobassiana I12 Damo and B. bassiana CTL20, previously identified with high virulence towards diamondback moth larvae, were selected for this study. Larvae of diamondback moth were fed on four different plant diets, based on different types of Brassicaceae, namely broccoli, cabbage, cauliflower, and radish, before their susceptibility to the two isolates of Beauveria was assessed. A second experiment assessed secondary metabolites produced from three genetically diverse isolates of Beauveria for their virulence towards diamondback moth larvae. Results: Diamondback moth larvae fed on broccoli were more susceptible to infection by B. pseudobassiana while larvae fed on radish were more susceptible to infection by B. bassiana. Furthermore, the supernatant from an isolate of B. pseudobassiana resulted in 55% and 65% mortality for half and full-strength culture filtrates, respectively, while the filtrates from two other Beauveria isolates, including a B. bassiana isolate, killed less than 50% of larvae. This study demonstrated different levels of susceptibility of the insects raised on different plant diets and the potential use of metabolites produced by Beauveria isolates in addition to their conidia

Purification of high-molecular-weight antibacterial proteins of insect pathogenic Brevibacillus laterosporus isolates

Brevibacillus laterosporus (Bl) is a Gram-positive and spore-forming bacterium belonging to the Brevibacillus brevis phylogenetic cluster. Globally, insect pathogenic strains of the bacterium have been isolated, characterised, and some activities have been patented. Two isolates, Bl 1821L and Bl 1951, exhibiting pathogenicity against the diamondback moth and mosquitoes, are under development as a biopesticide in New Zealand. However, due to the suspected activity of putative antibacterial proteins (ABPs), the endemic isolates often grow erratically. Various purification methods, including size exclusion chromatography, sucrose density gradient centrifugation, polyethylene glycol precipitation, and ammonium sulphate precipitation employed in this study, enabled the isolation of two putative antibacterial proteins of ∼30 and ∼48 kD from Bl 1821L and one putative antibacterial protein of ∼30 kD from Bl 1951. Purification of the uninduced cultures of Bl 1821L and Bl 1951 also yielded protein bands of ∼30 and ∼48 kD on SDS-PAGE, which indicated their spontaneous induction. A disc diffusion assay was used to determine the antagonistic activities of the putative ABPs. Subsequent transmission electron microscope (TEM) examination of a purified putative antibacterial protein-containing solution showed the presence of encapsulin (∼30 kD) and polysheath (∼48 kD)-like structures. Although only the ∼30 kD protein was purified from Bl 1951, both structures were seen in this strain under TEM. Furthermore, while assessing the antibacterial activity of some fractions of Bl 1951 against Bl 1821L in the size exclusion chromatography method, the population of Bl 1821L persister cells was noted. Overall, this work added a wealth of knowledge about the purification of the high-molecular-weight (HMW) proteins (bacteriocins) of Gram-positive bacteria including Bl

Risk, Unexpected Uncertainty, and Estimation Uncertainty: Bayesian Learning in Unstable Settings

Recently, evidence has emerged that humans approach learning using Bayesian updating rather than (model-free) reinforcement algorithms in a six-arm restless bandit problem. Here, we investigate what this implies for human appreciation of uncertainty. In our task, a Bayesian learner distinguishes three equally salient levels of uncertainty. First, the Bayesian perceives irreducible uncertainty or risk: even knowing the payoff probabilities of a given arm, the outcome remains uncertain. Second, there is (parameter) estimation uncertainty or ambiguity: payoff probabilities are unknown and need to be estimated. Third, the outcome probabilities of the arms change: the sudden jumps are referred to as unexpected uncertainty. We document how the three levels of uncertainty evolved during the course of our experiment and how it affected the learning rate. We then zoom in on estimation uncertainty, which has been suggested to be a driving force in exploration, in spite of evidence of widespread aversion to ambiguity. Our data corroborate the latter. We discuss neural evidence that foreshadowed the ability of humans to distinguish between the three levels of uncertainty. Finally, we investigate the boundaries of human capacity to implement Bayesian learning. We repeat the experiment with different instructions, reflecting varying levels of structural uncertainty. Under this fourth notion of uncertainty, choices were no better explained by Bayesian updating than by (model-free) reinforcement learning. Exit questionnaires revealed that participants remained unaware of the presence of unexpected uncertainty and failed to acquire the right model with which to implement Bayesian updating

Genetic characterization and phylogeography of the wild boar Sus scrofa introduced into Uruguay

The European wild boar Sus scrofa was first introduced into Uruguay, in southern South America during the early decades of the last century. Subsequently, and starting from founder populations, its range spread throughout the country and into the neighbouring Brazilian state Rio Grande do Sul. Due to the subsequent negative impact, it was officially declared a national pest. The main aim in the present study was to provide a more comprehensive scenario of wild boar differentiation in Uruguay, by using mtDNA markers to access the genetic characterization of populations at present undergoing rapid expansion. A high level of haplotype diversity, intermediate levels of nucleotide diversity and considerable population differentiation, were detected among sampled localities throughout major watercourses and catchment dams countrywide. Phylogenetic analysis revealed the existence of two different phylogroups, thereby reflecting two deliberate introduction events forming distantly genetic lineages in local wild boar populations. Our analysis lends support to the hypothesis that the invasive potential of populations emerge from introgressive hybridization with domestic pigs. On taking into account the appreciable differentiation and reduced migration between locales in wild boar populations, management strategies could be effective if each population were to be considered as a single management unit

Genomic Hypomethylation in the Human Germline Associates with Selective Structural Mutability in the Human Genome

The hotspots of structural polymorphisms and structural mutability in the human genome remain to be explained mechanistically. We examine associations of structural mutability with germline DNA methylation and with non-allelic homologous recombination (NAHR) mediated by low-copy repeats (LCRs). Combined evidence from four human sperm methylome maps, human genome evolution, structural polymorphisms in the human population, and previous genomic and disease studies consistently points to a strong association of germline hypomethylation and genomic instability. Specifically, methylation deserts, the ∼1% fraction of the human genome with the lowest methylation in the germline, show a tenfold enrichment for structural rearrangements that occurred in the human genome since the branching of chimpanzee and are highly enriched for fast-evolving loci that regulate tissue-specific gene expression. Analysis of copy number variants (CNVs) from 400 human samples identified using a custom-designed array comparative genomic hybridization (aCGH) chip, combined with publicly available structural variation data, indicates that association of structural mutability with germline hypomethylation is comparable in magnitude to the association of structural mutability with LCR–mediated NAHR. Moreover, rare CNVs occurring in the genomes of individuals diagnosed with schizophrenia, bipolar disorder, and developmental delay and de novo CNVs occurring in those diagnosed with autism are significantly more concentrated within hypomethylated regions. These findings suggest a new connection between the epigenome, selective mutability, evolution, and human disease

Who Eats Whom in a Pool? A Comparative Study of Prey Selectivity by Predatory Aquatic Insects

Predatory aquatic insects are a diverse group comprising top predators in small fishless water bodies. Knowledge of their diet composition is fragmentary, which hinders the understanding of mechanisms maintaining their high local diversity and of their impacts on local food web structure and dynamics. We conducted multiple-choice predation experiments using nine common species of predatory aquatic insects, including adult and larval Coleoptera, adult Heteroptera and larval Odonata, and complemented them with literature survey of similar experiments. All predators in our experiments fed selectively on the seven prey species offered, and vulnerability to predation varied strongly between the prey. The predators most often preferred dipteran larvae; previous studies further reported preferences for cladocerans. Diet overlaps between all predator pairs and predator overlaps between all prey pairs were non-zero. Modularity analysis separated all primarily nectonic predator and prey species from two groups of large and small benthic predators and their prey. These results, together with limited evidence from the literature, suggest a highly interconnected food web with several modules, in which similarly sized predators from the same microhabitat are likely to compete strongly for resources in the field (observed Pianka’s diet overlap indices >0.85). Our experiments further imply that ontogenetic diet shifts are common in predatory aquatic insects, although we observed higher diet overlaps than previously reported. Hence, individuals may or may not shift between food web modules during ontogeny

Lineage of origin in rhabdomyosarcoma informs pharmacological response

Lineage or cell of origin of cancers is often unknown and thus is not a consideration in therapeutic approaches. Alveolar rhabdomyosarcoma (aRMS) is an aggressive childhood cancer for which the cell of origin remains debated. We used conditional genetic mouse models of aRMS to activate the pathognomonic Pax3:Foxo1 fusion oncogene and inactivate p53 in several stages of prenatal and postnatal muscle development. We reveal that lineage of origin significantly influences tumor histomorphology and sensitivity to targeted therapeutics. Furthermore, we uncovered differential transcriptional regulation of the Pax3:Foxo1 locus by tumor lineage of origin, which led us to identify the histone deacetylase inhibitor entinostat as a pharmacological agent for the potential conversion of Pax3:Foxo1-positive aRMS to a state akin to fusion-negative RMS through direct transcriptional suppression of Pax3:Foxo1.Stem Cell and Regenerative Biolog