A study of selected factors affecting efficacy of compost tea against several fungal pathogens of potato.

AIM: To investigate selected factors of two nonaerated compost teas (NCT) and mechanisms that influence the restriction of several fungal potato pathogens. METHODS AND RESULTS: Two NCTs, made from either commercial compost, (CCT) or vineyard compost (VCT), were tested for their ability to suppress potato pathogens. The VCT was more suppressive than CCT to mycelial growth of Alternaria solani and Rhizoctonia solani isolate 299, but not for R. solani isolate 422. Metagenomic studies of microbial diversity revealed that the CCT had higher fungal and bacterial diversity and richness than the VCT. Use of CCT significantly reduced lesion area of Alternaria alternata on detached leaves, however, a gum adjuvant did not lead to significantly greater control. Scanning microscopy showed that the spatial distribution of microbes from the CCT was altered with gum addition, to resemble what may have been a microbial biofilm. CONCLUSION: We confirmed that each NCT could suppress the mycelial growth of selected potato pathogens in culture, and CCT reduced A. alternata lesions on detached leaves. Factors including concentration, microbial communities and physio-chemical properties could not be consistently linked to NCT efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: This study particularly highlights the application of scanning microscopy to study the interaction between pathogens and putative NCT microbes on foliar surfaces. This adds insight to mechanisms of NCT efficacy, along with physico-chemical and microbial characterization of the teas. This study shows the potential for the use of NCTs as a crop protection tool of low-cost which could be of particular benefit in smallholder agriculture.

Diverse microbial communities in non-aerated compost teas suppress bacterial wilt.

Non-aerated compost teas (NCTs) are water extracts of composted organic materials and are used to suppress soil borne and foliar disease in many pathosystems. Greenhouse trials were used to test the effectiveness of NCTs to suppress potato bacterial wilt caused by Ralstonia solanacearum on plants grown in soils inoculated with a virulent isolate of the pathogen (biovar II). NCTs prepared from matured compost sources: agricultural waste (AWCT), vermicompost (VCT) and solid municipal waste (SMWCT) were evaluated at three initial application times (7 days before inoculation, at time of inoculation and 7 days after inoculation) prior to weekly applications, in a randomized complete-block design. AWCT applied initially at the time of inoculation resulted in the greatest disease suppression, with the disease severity index 2.5-fold less than the non-treated plants and the "area under the disease progress curve" (AUDPC) 3.2-fold less. VCT and SMWCT were less suppressive than AWCT regardless of initial application time. Next generation sequencing of the v4 region of 16S rRNA gene and the internal transcribed spacer region (ITS1) revealed that diversity and composition of the bacterial and fungal communities across the NCTs varied significantly. Dominant bacterial phyla such as Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, Verrucomicrobia, Chloroflexi, Planctomycetes, Acidobacteria, and a fungal phylum Ascomycota were detected in all NCTs. AWCT had optimum physico-chemical measurements with higher bacterial Shannon diversity indices (H) and fungal richness (S) than the other treatments. We conclude that bacterial wilt of potatoes grown in controlled conditions can be suppressed by a non-aerated compost tea with a high microbial diversity when applied at planting and weekly thereafter.

Predictive model for the growth of spoilage bacteria on modified atmosphere packaged Atlantic salmon produced in Australia.

Most existing models for the spoilage of modified atmosphere packed Atlantic salmon are based on the growth of the spoilage organism Photobacterium phosphoreum. However, there is evidence that this organism is not the specific spoilage organism on salmon produced and packaged in Australia. We developed a predictive model for the growth of bacteria in Australian-produced Atlantic salmon stored under modified atmosphere conditions (30-98% carbon dioxide in nitrogen) at refrigeration temperatures (0-10 °C). As expected, both higher levels of carbon dioxide and lower temperatures decreased the observed growth rates of the total population. A Belehrádek-type model for growth rate fitted the data best with an acceptably low root mean square error. At low temperatures (∼0 °C) the growth rates in this study were similar to those predicted by other models but at higher temperatures (∼10 °C) the growth rates were significantly lower in the current study.

Dynamics of seawater bacterial communities in a shellfish hatchery.

Bacterial disease is a significant issue for larviculture of several species of shellfish, including oysters. One source of bacteria is the seawater used throughout the hatchery. In this study carried out at a commercial oyster hatchery in Tasmania, Australia, the diversity of the bacterial community and its relationship with larval production outcomes were studied over a 2-year period using terminal restriction fragment length polymorphism and tag-encoded pyrosequencing. The bacterial communities were very diverse, dominated by the Alphaproteobacteria, Gammaproteobacteria, Flavobacteria and Cyanobacteria. The communities were highly variable on scales of days, weeks and seasons. The difference between the intake seawater and treated clean seawater used in the hatchery was smaller than the observed temporal differences in the seawater throughout the year. No clear correlation was observed between production outcomes and the overall bacterial community structure. However, one group of Cyanobacterial sequences was more abundant when mass mortality events occurred than when healthy spat were produced although they were always present.

Microbial communities on Australian modified atmosphere packaged Atlantic salmon.

The role of specific spoilage organisms (SSO) in products such as Atlantic salmon has been well documented. However, little is known about what other micro-organisms are present and these organisms may indirectly influence spoilage by their interactions with the SS0. We used a combination of culture-based and DNA-based methods to explore the microbial communities found on Atlantic salmon fillets packed in a modified atmosphere of carbon dioxide and nitrogen. After 15 days the communities were dominated by Shewanella spp. or Carnobacterium spp. and a variety of other genera were present in smaller numbers. Variability in the microbial community composition in packages processed on the same day was also observed. This was mostly due to differences in the presence of minor members of the community including species from genera such as Iodobacter, Serratia, Morganella and Yersinia. The combination of culture-based and culture-independent methods provided greater insight into the development of microbial communities on Atlantic salmon than would have been possible using only one method. This work highlights the potential importance of lactic acid bacteria (LAB) in fresh Atlantic salmon stored under modified atmosphere conditions.

Use of near infrared spectroscopy to predict microbial numbers on Atlantic salmon.

The potential of a near infrared spectroscopy (NIR) method to detect as well as predict microbial spoilage on Atlantic salmon (Salmo salar) was investigated. Principal component analysis (PCA) of the NIR spectra showed clear separation between the fresh salmon fillets and those stored for nine days at 4°C indicating that NIR could detect spoilage. A partial least squares regression (PLS) prediction model for total aerobic plate counts after nine days was established using the NIR spectra collected when the fish was fresh to predict the number of bacteria that would be present nine days later. The calibration equation was good (R(2) = 0.95 and RMSE = 0.12 log cfu/g) although the error of the validation curve was larger (R(2) = 0.64 and RMSE = 0.32 log cfu/g). These results indicate that with further model development, it may be possible to use NIR to predict bacterial numbers, and hence shelf-life, in Atlantic salmon and other seafood.

Clues to the pathogenesis of familial colorectal cancer.

A predisposition to colorectal cancer is shown to be linked to markers on chromosome 2 in some families. Molecular features of "familial" cancers were compared with those of sporadic colon cancers. Neither the familial nor sporadic cancers showed loss of heterozygosity for chromosome 2 markers, and the incidence of mutations in KRAS, P53, and APC was similar in the two groups of tumors. Most of the familial cancers, however, had widespread alterations in short repeated DNA sequences, suggesting that numerous replication errors had occurred during tumor development. Thirteen percent of sporadic cancers had identical abnormalities and these cancers shared biologic properties with the familial cases. These data suggest a mechanism for familial tumorigenesis different from that mediated by classic tumor suppressor genes.

Prohibitin, a protein downregulated by androgens, represses androgen receptor activity.

Prohibitin (PHB) is a cell cycle regulatory protein, known to repress E2F1-mediated gene activation via recruitment of transcriptional regulatory factors such as retinoblastoma and histone deacetylase 1 (HDAC1). We previously identified PHB as a target protein of androgen signaling in prostate cancer cells and showed that downregulation of PHB is required for androgen-induced cell cycle entry in these cells. We now present evidence that PHB, which has 54% homology at the protein level to the oestrogen receptor corepressor REA (repressor of oestrogen receptor activity), can repress androgen receptor (AR)-mediated transcription and androgen-dependent cell growth. Depletion of endogenous PHB resulted in an increase in expression of the androgen-regulated prostate-specific antigen gene. The repression appears to be specific to androgen and closely related receptors, as it is also evident for the glucocorticoid and progesterone, but not oestrogen, receptors. In spite of interaction of PHB with HDAC1, HDAC activity is not required for this repression. Although AR and PHB could be co-immunoprecipitated, no direct interaction was detectable, suggesting that PHB forms part of a repressive complex with the AR. Competition with the co-activator SRC1 further suggests that formation of a complex with AR, PHB and other cofactors is the mechanism by which repression is achieved. It appears then that repression of AR activity is one mechanism by which PHB inhibits androgen-dependent growth of prostate cells. Further, this study implies that the AR itself could, by mediating downregulation of a corepressor, be involved in the progression of prostate tumours to the hormone refractory stage.

Impact of primary tumour stage on survival in dogs with solitary lung tumours.

OBJECTIVES: The objective of this study was to determine simple prognostic criteria for differentiation of canine solitary lung tumour cases into those that will and will not benefit from thoracic surgery. METHODS: This was a retrospective study using the records of cases presented to Davies Veterinary Specialists, Hitchin, UK, from December 1998 to December 2005. Survival analyses were performed using the Kaplan-Meier and logrank methods. Potentially significant variables were evaluated by multivariate Cox analysis. RESULTS: Forty-two patients met the inclusion criteria. Primary tumour stage T1, absence of neoplastic lymph nodes and metastases, and papillary tumour type were statistically significant favourable prognostic indicators on univariate analysis. Multivariate analysis attributed significance to primary tumour stage T1 and papillary type only. Median survival times were 555 days for T1N0M0 tumours of papillary type and 72 days for the remainder. CLINICAL SIGNIFICANCE: Survival time following surgery in dogs with primary lung tumours was poor except in clinical stage T1N0M0 cases. These data support use of clinical techniques to dichotomise cases as T1N0M0 or other, improving decision making in thoracic surgery. These data validate initiation of prospective studies examining the role of chemotherapy in the management of advanced cases.

Overexpression of CEBPbeta correlates with decreased TFF1 in gastric cancer.

CCAAT element binding protein beta (C/EBPbeta) is an important regulator of cell growth, differentiation and in promoting tumor invasiveness. C/EBPbeta is located on chromosome 20q, which is amplified in many solid tumors including gastric cancers (GC). We sought to characterize the status of C/EBPbeta expression in GCs, which was recently found to repres TFF1 gene. Microarray analysis revealed overexpression of C/EBPbeta in 25 of 27 (93%) GC when compared to 12 normal gastric tissue samples. RT-PCR analysis confirmed the overexpression of C/EBPbeta transcripts in 54 of 59 (91%) GC. In total, 15 of 18 gastric tumors exhibited at least fivefold higher C/EBPbeta transcript levels compared to their corresponding adjacent normal gastric tissue samples. Moreover, immunohistochemistry analysis demonstrated increased nuclear staining of C/EBPbeta in 10 of 13 GC and at least fourfold overexpression of C/EBPbeta in three primary GC compared to adjacent normal gastric tissue. Furthermore, a striking correlation of decreased TFF1 expression with increased C/EBPbeta was observed in the gastric tumors studied. Microarray analysis demonstrated a loss of TFF1 expression in all 27 GC cases examined, of which 25 exhibited high C/EBPbeta expression compared to normal gastric tissue. RT-PCR analysis revealed loss of TFF1 expression in 56 of 59 gastric tumors in which 54 of these tumors exhibited overexpression of C/EBPbeta. Immunohistochemical analysis revealed overexpression of C/EBPbeta in 10 of 13 gastric tumors that exhibited low expression of TFF1 at the protein level. Thus, overexpression of the transcription factor C/EBPbeta in the majority of GCs is a novel finding.

Inactivation of Smad4 in gastric carcinomas.

Allelic loss of chromosome 18q has been noted in intestinal type gastric adenocarcinomas. Smad4 is a gene located at 18q that was recently cloned in humans and found to be significantly altered in pancreatic cancers. We sought to determine whether Smad4 genetic alterations played a significant role in gastric tumorigenesis by studying 35 gastric adenocarcinomas of all histopathological types and pathological stages. Microdissected specimens were used for mutational analysis of Smad4 at the nucleotide level, including the entire coding region and intron/exon boundaries. Allelic imbalance was also analyzed at the Smad4 locus using two nearby microsatellite markers. One case of apparent biallelic inactivation of Smad4 was found in our study of 35 gastric carcinomas. A nonsense point mutation at codon 334 was demonstrated, which, similar to other Smad4 mutations, is predicted to truncate the conserved COOH-terminal domain of this protein. This Smad4 C to T transition mutation was proven to be somatically acquired. Allelic loss was also noted on chromosome 18q at a marker near Smad4 in this mutated gastric cancer, apparently producing complete inactivation of Smad4 in this tumor. Significant 18q allelic loss (56% of 34 informative cases) was noted in our gastric carcinomas using microsatellite markers near the Smad4 locus, regardless of histological subtype or pathological stage. Additionally, three cases of microsatellite instability were observed. Thus, Smad4 inactivation was noted in our gastric carcinomas; however, this event was rare. The frequent loss of chromosomal arm 18q observed in gastric cancers suggests the presence of other tumor suppressor genes in this region that are involved in gastric tumorigenesis. Further studies are needed to identify these other targets of inactivation during gastric cancer development.

Allelotype of pancreatic adenocarcinoma.

Knowledge of the patterns of allelic loss has been useful in identifying the spectrum of the tumor suppressor genes involved in various tumor types. Such analyses in pancreatic carcinoma have been difficult due to the characteristic host desmoplastic reaction to the neoplasm. We have assembled the first allelotype of pancreatic adenocarcinoma, a survey for allelic loss among each chromosomal arm, using seven cryostat-dissected neoplasms. The fractional allelic loss in these seven neoplasms was 0.18, a value similar to that seen previously in colorectal carcinoma. Alleles of chromosome 18q (lost in five of six informative tumors) and of chromosome 17p (lost in four of five informative tumors) were commonly affected. Neither APC mutations (33 neoplasms), allelic shifts of dinucleotide repeats (26 neoplasms), nor immunohistochemical evidence of retinoblastoma protein underexpression (7 neoplasms) were found. Further evaluation of allelic loss in pancreatic cancer would benefit from improved methods for the analysis of lost genetic material which overcome the problems posed by the high admixture of nonneoplastic stromal and inflammatory cells in these tumors.

Molecular determinants of dysplasia in colorectal lesions.

One hallmark of malignant potential is dysplasia, the disruption of normal morphology. While it is generally recognized that cancer is the result of a series of genetic changes, the relationship of these alterations and their timing to the advent of dysplasia remains obscure. To address this issue, 54 small benign colorectal lesions of various malignant potential were analyzed for APC and K-RAS mutations, two alterations which have been implicated in the early stages of colorectal tumorigenesis. APC mutations were closely associated with dysplasia. In contrast, K-RAS mutations were found to be remarkably common in small nondysplastic lesions which apparently have a limited potential to progress to larger tumors. These results provide evidence that the nature and order of genetic changes can have a specific impact on both tumor morphology (e.g., dysplasia) and the likelihood of tumor progression.

Consistent genetic alterations in xenografts of proximal stomach and gastro-esophageal junction adenocarcinomas.

The genetic alterations underlying the development of gastric and gastro-esophageal carcinoma remain largely undefined. DNA copy number changes were determined by comparative genomic hybridization in eight xenografts of proximal gastric and gastro-esophageal junction adenocarcinomas of the intestinal type. All tumors exhibited DNA copy number changes, with a total of 139 changes detected (range, 11-24 per tumor; mean = 17), indicating numerous and widespread alterations within these cancers. Gains (65%) in DNA copy number were more frequent than losses (35%). Our most striking finding was gain (all eight cases) or high-level amplification (four cases) in 20q, with a minimal common overlapping region at 20q13. Other frequent gains were observed at 6p, 7q, and 17q (six cases each) and at 1q, 2q, and 8q (five cases each). Frequent losses were observed at 4q and 5q (six cases each) and at 9p (five cases). No differences in DNA copy number changes were seen in tumors arising from the gastro-esophageal junction compared to those of the proximal stomach. The presence of common and consistent DNA copy number changes in these tumors implicate a number of chromosomal regions that may harbor important genes that are involved in tumorigenesis of the proximal stomach and gastro-esophageal junction.

Allelotype of gastric adenocarcinoma.

Gastric adenocarcinoma is a leading cause of cancer mortality world-wide. Yet, the underlying molecular events important in the development of this cancer are largely undefined. Thus, we performed a comprehensive survey for allelic loss on our panel of xenografted human gastric carcinomas. Contaminating normal stromal cells of primary cancers often limit mutational analyses. Xenografted samples of our gastric carcinomas provided optimally enriched tumors for neoplasia that clearly and sensitively demonstrated genetic alterations. Additionally, total absence of allelic signals in these xenografted samples confirmed true loss of alleles rather than just allelic imbalance. Analysis of at least two highly polymorphic microsatellite markers per nonacrocentric chromosomal arm in our xenografted human gastric carcinomas demonstrated significant loss of heterozygosity well above background levels at 3p, 4p, 5q, 8p, 9p, 13q, 17p, and 18q. Several of these loci represent novel findings of significant loss in gastric cancers. On chromosome 17p, p53 is known to be inactivated either by mutation or deletion in a majority of gastric carcinomas. The critical target(s) of inactivation in gastric cancers at these other loci remain to be characterized.

Molecular classification of human carcinomas by use of gene expression signatures.

Classification of human tumors according to their primary anatomical site of origin is fundamental for the optimal treatment of patients with cancer. Here we describe the use of large-scale RNA profiling and supervised machine learning algorithms to construct a first-generation molecular classification scheme for carcinomas of the prostate, breast, lung, ovary, colorectum, kidney, liver, pancreas, bladder/ureter, and gastroesophagus, which collectively account for approximately 70% of all cancer-related deaths in the United States. The classification scheme was based on identifying gene subsets whose expression typifies each cancer class, and we quantified the extent to which these genes are characteristic of a specific tumor type by accurately and confidently predicting the anatomical site of tumor origin for 90% of 175 carcinomas, including 9 of 12 metastatic lesions. The predictor gene subsets include those whose expression is typical of specific types of normal epithelial differentiation, as well as other genes whose expression is elevated in cancer. This study demonstrates the feasibility of predicting the tissue origin of a carcinoma in the context of multiple cancer classes.

Identification of germ-line E-cadherin mutations in gastric cancer families of European origin.

E-cadherin germ-line mutations have recently been described as a molecular basis for early-onset familial gastric cancer in Maori kindred. We screened 18 gastric cancer families of European origin for germ-line mutations to determine the proportion in which E-cadherin mutations occur and the clinical characteristics of the affected families. Truncating mutations were identified in three kindred with familial diffuse gastric cancer. In these families, the age of onset of gastric cancer was variable, the penetrance was incomplete, and one kindred contained individuals with cancers at other sites. Here, we show that a proportion of diffuse gastric cancer families of European origin have germ-line E-cadherin mutations; however, these mutations are absent in intestinal gastric cancer families.

Hypermethylation of the hMLH1 gene promoter in human gastric cancers with microsatellite instability.

Human gastric carcinoma shows a higher prevalence of microsatellite instability (MSI) than does any other type of sporadic human cancer. The reasons for this high frequency of MSI are not yet known. In contrast to endometrial and colorectal carcinoma, mutations of the DNA mismatch repair (MMR) genes hMLH1 or hMSH2 have not been described in gastric carcinoma. However, hypermethylation of the hMLH1 MMR gene promoter is quite common in MSI-positive endometrial and colorectal cancers. This hypermethylation has been associated with hMLH1 transcriptional blockade, which is reversible with demethylation, suggesting that an epigenetic mechanism underlies hMLH1 gene inactivation and MMR deficiency. Therefore, we studied the prevalence of hMLH1 promoter hypermethylation in a total of 65 gastric tumors: 18 with frequent MSI (MSI-H), 8 with infrequent MSI (MSI-L), and 39 that were MSI negative. We found a striking association between hMLH1 promoter hypermethylation and MSI; of 18 MSI-H tumors, 14 (77.8%) showed hypermethylation, whereas 6 of 8 MSI-L tumors (75%) were hypermethylated at hMLH1. In contrast, only 1 of 39 (2.6%) MSI-negative tumors demonstrated hMLH1 hypermethylation (P<0.0001 for MSI-H or MSI-L versus MSI-negative). Moreover, hypermethylated cancers demonstrated diminished expression of hMLH1 protein by both immunohistochemistry and Western blotting, whereas nonhypermethylated tumors expressed abundant hMLH1 protein. These data indicate that hypermethylation of hMLH1 is strongly associated with MSI in gastric cancers and suggest an epigenetic mechanism by which defective MMR occurs in this group of cancers.

Frequent mutation of the E2F-4 cell cycle gene in primary human gastrointestinal tumors.

The E2F group of transcription factors transactivates genes that promote progression through the G1-S transition of the cell cycle. Members of the retinoblastoma (Rb) family of proteins bind to E2Fs and inhibit this function. E2F-4, one example of the E2F group, functions as an oncogene when transfected into nontransformed cells in vitro. On the other hand, mice that are homozygously lacking a normal E2F-1 gene develop cancers, consistent with a tumor-suppressive role for this gene. The exact function of E2Fs has thus been unclear; moreover, direct involvement of this gene in primary human tumorigenesis has not been shown. We, therefore, investigated mutation within the E2F-4 coding region in 16 primary gastric adenocarcinomas, 12 ulcerative colitis-associated neoplasms, 46 sporadic colorectal carcinomas, 9 endometrial cancers, and 3 prostatic carcinomas. We limited our investigation to the serine repeat within E2F-4, reasoning that this tract might be altered in genetically unstable tumors (replication error-positive, or RER+). All tumors were RER+, with the exception of a control group of 15 RER- sporadic colorectal carcinomas. PCR with incorporation of [32P]dCTP was performed using primers flanking the serine trinucleotide (AGC) repeat. Twenty-two of 59 gastrointestinal tumors (37%) contained E2F-4 mutations; these comprised 5 of 16 gastric tumors (31%), 4 of 12 ulcerative colitis-associated neoplasms (33%, including 1 dysplastic lesion), and 13 of 31 sporadic colorectal cancers (42%). No mutation was present in any of the endometrial, prostate, or RER- colorectal tumors. Of note, homozygous mutations occurred in three cases, and two of seven informative patients showed loss of one E2F-4 allele in their tumors. Furthermore, the RER+ sporadic colorectal tumors were evaluated at trinucleotide repeats within the genes for N-cadherin and B-catenin; no tumors demonstrated mutation of these genes. These data suggest that E2F-4 is a target of defective DNA repair in these tumors.