The Yuma Territorial Prison Cemetery: Cold Cases of Grave Importance

Cemeteries, via grave markers and burial records, usually offer sufficiently scant data to enable a reconstruction of the communities they represent, but cemeteries of total institutions, here, the Yuma Territorial Prison, often yield even less data. With only the variables of ethnicity, sex, prisoner number, date of death, and cause of death, prison conditions were reconstructed for the 111 who died during the prison’s operation (1876-1909), and likely for the other 2,958 who were incarcerated there. First, prisoner number had a high, positive correlation with year of death, indicating that those who died in prison did not live long after incarceration. Further investigation found statistical dependence between the ethnicity of the prisoner and cause (and thus, manner) of death, with tuberculosis disproportionately effecting Hispanics and Native Americans, perhaps suggesting segregation by ethnicity. Hispanics were the only ones shot and killed attempting escape, though numerous escapes were attempted. Also statistically dependent was the decade of death by ethnicity, perhaps reflecting some ethnic sequence of incarceration. Finally, cause and manner of death, over time, were also dependent, likely reflecting deteriorating prison conditions. That so few variables can reveal so much refutes the adage that dead men tell no tales

Oligonucleotide sequences forming short self-complimentary hairpins can expedite the down-regulation of Coprinopsis cinerea genes

Gene silencing in fungi is often induced by dsRNA hairpin forming constructs the preparation of which can require multiple cloning steps. To simplify gene silencing in the filamentous fungi we have evaluated a high throughput cloning method for target sequences using the homobasidiomycete Coprinopsis cinerea, the GFP reporter and a commercially available vector system. The pSUPER RNAi System™, which was developed for mammalian experiments, exploits the human H1 Polymerase III (Pol III) RNA gene promoter and expedites cloning/expression of specific user-defined oligonucleotide sequences to form short self-complimentary hairpins. Transformation of C. cinerea with pSUPER constructs harboring specific oligonucleotides (19 nt stem length) enabled recovery of transformants with reduced transcripts of the GFP transgene, that were less fluorescent in protein assays and microscopic phenotypes. This technological advance should expedite functional genomic studies in C. cinerea and has wider potential for utility in other homobasidiomycete and filamentous fungi

Exploring fungal RiPPs from the perspective of chemical ecology

Since the initial detection, in 2007, of fungal ribosomally synthesised and post-translationally modified peptides (RiPPs), this group of natural products has undergone rapid expansion, with four separate classes now recognised: amatoxins/phallotoxins, borosins, dikaritins, and epichloëcyclins. Largely due to their historically anthropocentric employment in medicine and agriculture, novel fungal proteins and peptides are seldom investigated in relation to the fungus itself. Therefore, although the benefits these compounds confer to humans are often realised, their evolutionary advantage to the fungus, the reason for their continued production, is often obscure or ignored. This review sets out to summarise current knowledge on how these small peptide-derived products influence their producing species and surrounding biotic environment

Characterization of serine proteinase expression in agaricus bisporus and coprinopsis cinerea by using green fluorescent protein and the A. bisporus SPR1 Promoter

The Agaricus bisporus serine proteinase 1 (SPR1) appears to be significant in both mycelial nutrition and senescence of the fruiting body. We report on the construction of an SPR promoter::green fluorescent protein (GFP) fusion cassette, pGreen_hph1_SPR_GFP, for the investigation of temporal and developmental expression of SPR1 in homobasidiomycetes and to determine how expression is linked to physiological and environmental stimuli. Monitoring of A. bisporus pGreen_hph1_SPR_GFP transformants on media rich in ammonia or containing different nitrogen sources demonstrated that SPR1 is produced in response to available nitrogen. In A. bisporus fruiting bodies, GFP activity was localized to the stipe of postharvest senescing sporophores. pGreen_hph1_SPR_GFP was also transformed into the model basidiomycete Coprinopsis cinerea. Endogenous C. cinerea proteinase activity was profiled during liquid culture and fruiting body development. Maximum activity was observed in the mature cap, while activity dropped during autolysis. Analysis of the C. cinerea genome revealed seven genes showing significant homology to the A. bisporus SPR1 and SPR2 genes. These genes contain the aspartic acid, histidine, and serine residues common to serine proteinases. Analysis of the promoter regions revealed at least one CreA and several AreA regulatory motifs in all sequences. Fruiting was induced in C. cinerea dikaryons, and fluorescence was determined in different developmental stages. GFP expression was observed throughout the life cycle, demonstrating that serine proteinase can be active in all stages of C. cinerea fruiting body development. Serine proteinase expression (GFP fluorescence) was most concentrated during development of young tissue, which may be indicative of high protein turnover during cell differentiatio

A native promoter and inclusion of an intron is necessary for efficient expression of GFP or mRFP in Armillaria mellea

Armillaria mellea is a significant pathogen that causes Armillaria root disease on numerous hosts in forests, gardens and agricultural environments worldwide. Using a yeast-adapted pCAMBIA0380 Agrobacterium vector, we have constructed a series of vectors for transformation of A. mellea, assembled using yeast-based recombination methods. These have been designed to allow easy exchange of promoters and inclusion of introns. The vectors were first tested by transformation into basidiomycete Clitopilus passeckerianus to ascertain vector functionality then used to transform A. mellea. We show that heterologous promoters from the basidiomycetes Agaricus bisporus and Phanerochaete chrysosporium that were used successfully to control the hygromycin resistance cassette were not able to support expression of mRFP or GFP in A. mellea. The endogenous A. mellea gpd promoter delivered efficient expression, and we show that inclusion of an intron was also required for transgene expression. GFP and mRFP expression was stable in mycelia and fluorescence was visible in transgenic fruiting bodies and GFP was detectable in planta. Use of these vectors has been successful in giving expression of the fluorescent proteins GFP and mRFP in A. mellea, providing an additional molecular tool for this pathogen

Plant virus infections control stomatal development

Stomata are important regulators of carbon dioxide uptake and transpirational water loss. They also represent points of vulnerability as bacterial and fungal pathogens utilise this natural opening as an entry portal, and thus have an increasingly complex relationship. Unlike the situation with bacterial and fungal pathogens, we know very little about the role of stomata in viral infection. Here we report findings showing that viral infection influences stomatal development in two susceptible host systems (Nicotiana tabacum with TMV (Tobacco mosaic virus), and Arabidopsis thaliana with TVCV (Turnip vein-clearing virus)), but not in resistant host systems (Nicotiana glutinosa and Chenopodium quinoa with TMV). Virus infected plants had significantly lower stomatal indices in systemic leaves of susceptible systems; N. tabacum 9.8% reduction and A. thaliana 12.3% reduction, but not in the resistant hosts. Stomatal density in systemic leaves was also significantly reduced in virus infected A. thaliana by 19.6% but not in N. tabacum or the resistant systems. In addition, transpiration rate was significantly reduced in TMV infected N. tabacum

Endophytic Trichoderma spp. can protect strawberry and privet plants from infection by the fungus Armillaria mellea

Armillaria mellea is an important fungal pathogen worldwide, affecting a large number of hosts in the horticulture and forestry industries. Controlling A. mellea infection is expensive, labour intensive and time-consuming, so a new, environmentally friendly management solution is required. To this effect, endophytic Trichoderma species were studied as a potential protective agent for Armillaria root rot (ARR) in strawberry and privet plants. A collection of forty endophytic Trichoderma isolates were inoculated into strawberry (Fragaria × ananassa) plants and plant growth was monitored for two months, during which time Trichoderma treatment had no apparent effect. Trichoderma-colonised strawberry plants were then inoculated with A. mellea and after three months plants were assessed for A. mellea infection. There was considerable variation in ARR disease levels between plants inoculated with different Trichoderma spp. isolates, but seven isolates reduced ARR below the level of positive controls. These isolates were further tested for protective potential in Trichoderma-colonized privet (Ligustrum vulgare) plants where five Trichoderma spp. isolates, including two highly effective Trichoderma atrobrunneum isolates, were able to significantly reduce levels of disease. This study highlights the potential of plants pre-colonised with T. atrobrunneum for effective protection against A. mellea in two hosts from different plant families

Characterisation of the Temperature-dependent Dark Rate of Hamamatsu R7081-100 10" Photomultiplier Tubes

Dark noise is a dominant background in photomultiplier tubes (PMTs), which are commonly used in liquid-filled particle detectors for single-photon detection to see the results of particle interactions. A major contribution to dark noise is thermionic emission from the photocathode. The dark noise of Hamamatsu R7081-100 PMTs is characterised in a temperature and purity controlled water tank, with the thermionic emission contribution isolated. The results suggest that the intrinsic dark rate of PMTs does not depend on the medium, but does follow Richardson's law of thermionic emission. There are external contributions to the overall observed PMT count rate identified, but the intrinsic PMT dark rate in water matches that measured in air.Comment: 11 pages, 7 figures, 2 tables, prepared for submission to J. Instru