Twenty-eight bacteriophages infecting the local host Bacillus pumilus BL-8 were isolated, purified, and characterized. Nine genomes were sequenced, of which six were annotated and are the first of this host submitted to the public record. The 28 phages were divided into two groups by sequence and morphological similarity, yielding 27 cluster BpA phages and 1 cluster BpB phage, which is a BL-8 prophage. Most of the BpA phages have a host range restricted to distantly related strains, B. pumilus and B. simplex, reflecting the complexities of Bacillus taxonomy. Despite isolation over wide geographic and temporal space, the six cluster BpA phages share most of their 23 functionally annotated protein features and show a high degree of sequence similarity, which is unique among phages of the Bacillus genera. This is the first report of B. pumilus phages since 1981.

The Mediator complex is a key regulator of gene transcription and several studies demonstrated altered expressions of particular subunits in diverse human diseases, especially cancer. However a systematic study deciphering the transcriptional expression of the Mediator across different cancer entities is still lacking.We therefore performed a comprehensive in silico cancer vs. benign analysis of the Mediator complex subunits (MEDs) for 20 tumor entities using Oncomine datasets. The transcriptional expression profiles across almost all cancer entities showed differentially expressed MEDs as compared to benign tissue. Differential expression of MED8 in renal cell carcinoma (RCC) and MED12 in lung cancer (LCa) were validated and further investigated by immunohistochemical staining on tissue microarrays containing large numbers of specimen. MED8 in clear cell RCC (ccRCC) associated with shorter survival and advanced TNM stage and showed higher expression in metastatic than primary tumors. In vitro, siRNA mediated MED8 knockdown significantly impaired proliferation and motility in ccRCC cell lines, hinting at a role for MED8 to serve as a novel therapeutic target in ccRCC. Taken together, our Mediator complex transcriptome proved to be a valid tool for identifying cancer-related shifts in Mediator complex composition, revealing that MEDs do exhibit cancer specific transcriptional expression profiles.

BackgroundLung cancer is one of the most common malignant neoplasms worldwide and has a high mortality rate. To enable individualized therapy regimens, a better understanding of the molecular tumor biology has still to be elucidated. The expression of the cell surface protein CD24 has already been claimed to be associated with shorter patient survival in non-small cell lung cancer (NSCLC), however, the prognostic value and applicability of CD24 immunostaining in paraffin embedded tissue specimens has been questioned due to the recent acknowledgement of restricted epitope specificity of the commonly used antibody SN3b.MethodsA cohort of 137 primary NSCLC cases was immunostained with a novel CD24 antibody (clone SWA11), which specifically recognizes the CD24 protein core and the resulting expression data were compared with expression profiles based on the monoclonal antibody SN3b. Furthermore, expression data were correlated to clinico-pathological parameters. Univariate and multivariate survival analyses were conducted with Kaplan Meier estimates and Cox regression, respectively.ResultsCD24 positivity was found in 34 % resp. 21 % (SN3b) of NSCLC with a membranous and/or cytoplasmic staining pattern. Kaplan-Meier analyses revealed that membranous, but not cytoplasmic CD24 expression (clone SWA11) was associated with lympho-nodular spread and shorter overall survival times (both p < 0.05). CD24 expression established by SN3b antibodies did not reveal significant clinicopathological correlations with overall survival, neither for cytoplasmic nor membranous CD24 staining.ConclusionsMembranous CD24 immunoreactivity, as detected with antibody clone SWA11 may serve as a prognostic factor for lymphonodular spread and poorer overall survival. Furthermore, these results corroborate the importance of a careful distinction between membranous and cytoplasmic localisation, if CD24 is to be considered as a potential prognostic biomarker.

Distilled water plus 0.1% surfactant suspensions of spores of Aspergillus flavus and Aspergillus parasiticus were exposed to several radiation levels of cobalt-60 gamma rays. Spores of A. flavus isolate M-141 were exposed to radiation levels of approximately 16, 90 and 475 Krads and inoculated onto a sterile rice substrate which was then monitored for aflatoxin production. In this initial trial with A. flavus M-141, aflatoxins B1 and M production on rice increased as radiation dose increased. At the highest dose, this increase was more than 50 times higher than the non-irradiated controls. Spores of an aflatoxin G1-producing A. parasiticus isolate, M-1094, were exposed to 90, 215 and 430 Krads and resulted in increased production of aflatoxins G1, B1, and M. Aflatoxin production by M-1094 was highest at the low and medium dose levels. Irradiation of spores of this isolate with 430 Krads produced no observable spore germination or growth on rice and no detectable aflatoxin after 1 week of incubation at 27 C. A typical colonies from irradiated spores were selected and their mycotoxin production was determined. Increase in aflatoxin production by these strains, as compared to non-irradiated controls, was 67:1 for aflatoxin B1, 136:1 for B2, and 138:1 for M. This potential for greatly increased mycotoxin production must be considered when food is irradiated or when a high production of aflatoxins is desired.

Two aflatoxin-producing isolates of Aspergillus flavus were grown for 5 days on Wort media at 2, 7, 13, 18, 24, 29, 35, 41, 46, and 52 C. Maximal production of aflatoxins occurred at 24 C. Maximal growth of A. flavus isolates occurred at 29 and 35 C. The ratio of the production of aflatoxin B 1 to aflatoxin G 1 varied with temperature. Aflatoxin production was not related to growth rate of A. flavus ; one isolate at 41 C, at almost maximal growth of A. flavus , produced no aflatoxins. At 5 days, no aflatoxins were produced at temperatures lower than 18 C or higher than 35 C. Color of CHCl 3 extracts appeared to be directly correlated with aflatoxin concentrations. A. flavus isolates grown at 2, 7, and 41 C for 12 weeks produced no aflatoxins. At 13 C, both isolates produced aflatoxins in 3 weeks, and one isolate produced increasing amounts with time. The second isolate produced increasing amounts through 6 weeks, but at 12 weeks smaller amounts of aflatoxins were recovered than at 6 weeks.

Aflatoxicogenic isolates of Aspergillus were tested for their aflatoxin production after 8 weeks of growth on wort agar medium at high (41, 46 ± 1 C) and low (2, 7 ± 0.5 C) temperatures. Controls were grown at approximately 22 C, a temperature known to be favorable for aflatoxin production. There were two replications of each treatment. All replicate cultures of the 25 isolates grown at 22 C were positive for aflatoxin. Aflatoxins were not detected in wort agar when cultures were incubated at other test temperatures. It appears that both Aspergillus flavus and Aspergillus parasiticus will not produce aflatoxins when grown at ⩽ 7.5 C or at ⩾ 40 C.

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Experiments were conducted to determine the culture requirements that would insure a probability of high ochratoxin production with a given isolate. Experiments at ambient temperatures (72 ± 2°F) determined the levels of the two most readily controlled variables, time and moisture content, during the growth of a high toxin-producing isolate (M298) of Aspergillus ochraceus. Tests were conducted in 2.8 L - Fernbach flasks containing 100 g shredded wheat and inoculated with spores on a transfer needle. Amounts of ochratoxin A/flask were determined by comparison against standards on TLC after chloroform extraction. Statistical analysis showed a relation between ochratoxin yield, water levels, and incubation periods. A water level of 40–70 ml/flask with an incubation period of 19-21 days at ambient temperatures should give a near to maximum yield of ochratoxin A.