Exploration of plant genomes in the FLAGdb++ environment

Background

Efficient high throughput screening systems of useful mutants are prerequisite for study of plant functional genomics and lots of application fields. Advance in such screening tools, thanks to the development of analytic instruments. Direct analysis in real-time (DART)-mass spectrometry (MS) by ionization of complex materials at atmospheric pressure is a rapid, simple, high-resolution analytical technique. Here we describe a rapid, simple method for the genetic discrimination of intact Arabidopsis thaliana mutant seeds using metabolic profiling by DART-MS.

Results

To determine whether this DART-MS combined by multivariate analysis can perform genetic discrimination based on global metabolic profiling, intact Arabidopsis thaliana mutant seeds were subjected to DART-MS without any sample preparation. Partial least squares-discriminant analysis (PLS-DA) of DART-MS spectral data from intact seeds classified 14 different lines of seeds into two distinct groups: Columbia (Col-0) and Landsberg erecta (Ler) ecotype backgrounds. A hierarchical dendrogram based on partial least squares-discriminant analysis (PLS-DA) subdivided the Col-0 ecotype into two groups: mutant lines harboring defects in the phenylpropanoid biosynthetic pathway and mutants without these defects. These results indicated that metabolic profiling with DART-MS could discriminate intact Arabidopsis seeds at least ecotype level and metabolic pathway level within same ecotype.

Conclusion

The described DART-MS combined by multivariate analysis allows for rapid screening and metabolic characterization of lots of Arabidopsis mutant seeds without complex metabolic preparation steps. Moreover, potential novel metabolic markers can be detected and used to clarify the genetic relationship between Arabidopsis cultivars. Furthermore this technique can be applied to predict the novel gene function of metabolic mutants regardless of morphological phenotypes.     

Mutations in the juxtamembrane domain of c-KIT are associated with higher grade mast cell tumors in dogs

Mast cell tumors are among the most commonly seen tumors of the skin in dogs and are more highly aggressive than mast cell tumors of other species. Some breeds display a markedly higher incidence of mast cell tumor development than others and appear to have some genetic predisposition. Recently, mutations have been found in canine mast cell tumor tissues and cell lines within the juxtamembrane domain of the protooncogene c-KIT In previous studies utilizing a small number of cases, no association between the presence of a mutation and the breed of dog or grade of the tumor could be identified. An expanded study with a larger sample set was performed to explore this possibility. The juxtamembrane domain of c-KIT was amplified using the polymerase chain reaction from genomic DNA preparations of 88 paraffin-embedded mast cell tumors from selected breeds. Mutations, consisting of duplications and deletions, were found in 12 of the tumors. A significant association was found between the presence of a mutation and a higher grade of tumor but not between breed and grade or between breed and the presence of a mutation.     

A three‐dimensional cell culture system as an in vitro canine mammary carcinoma model for the expression of connective tissue modulators

In this study, derived complex carcinoma (CC) and simple carcinoma (SC) cell lines were established and cultured under two‐dimensional (2D) and three‐dimensional (3D) conditions. The 3D was performed in six‐well AlgiMatrix? (LifeTechnologies®, Carlsbad, CA, USA) scaffolds, resulting in spheroids sized 50–125 µm for CC and 175–200 µm for SC. Cell viability was demonstrated up to 14 days for both models. Epidermal growth factor receptor (EGFR) was expressed in CC and SC in both systems. However, higher mRNA and protein levels were observed in SC 2D and 3D systems when compared with CC (P < 0.005). The connective tissue modulators, metalloproteinases‐1, ‐2, ‐9 and ‐13 (MMPs), relaxin receptors 1 and 2 (RXR1 and RXR2) and E‐cadherin (CDH1) were quantitated. All were upregulated similarly when canine mammary tumour (CMT)‐derived cell lines were cultured under 3D AlgiMatrix, except CDH1 that was downregulated (P < 0.005). These results are promising towards the used of 3D system to increase a high throughput in vitro canine tumour model.     

Assessment of the efficacy of composite surgery for the treatment of dorsal displacement of the soft palate in a group of 53 racing Thoroughbreds (1990-1996)

REASONS FOR PERFORMING STUDY: There has been no objectively assessed case-control study of the efficacy of surgery to correct dorsal displacement of the soft palate (DDSP) previously reported. HYPOTHESIS: Composite surgery has a beneficial result on racing performance in horses affected with DDSP as compared to a matched control population. METHODS: Race records were obtained for 53 racing Thoroughbreds which underwent composite staphylectomy, sternothyrohyoideus myectomy and ventriculectomy for correction of idiopathic DDSP at the University of Bristol between 1990 and 1996. Each surgical case was matched for age, sex and training yard with 2 control horses. The racing performance, based on prize money won, of surgical cases and control horses were compared for 3 races run before and after the date of surgery. RESULTS: Ninety-two percent of the surgical cases returned to racing after surgery. There was a significant increase in earnings of the surgical group before and after surgery (P = 0.011), but there was no significant difference in earnings of the control group before and after the date of surgery (P = 0335). Sixty percent of the surgical group had higher earnings after surgery than before, compared to 40% of controls. When horses which underwent surgery were ranked relative to their 2 matched controls, surgical cases did not significantly change in rank (P = 033), whereas control horses significantly decreased in rank (P = 0.012). Additionally, horses within the surgical group were more likely (P < 0.01) to start in 3 post operative races than those in the control group. CONCLUSIONS AND POTENTIAL RELEVANCE: Composite surgery had a beneficial effect on racing performance of horses afflicted with idiopathic DDSP, and further studies to evaluate objectively the usefulness of other surgical techniques are warranted.     

Genetic Structure of Peruvian Populations of Phytophthora infestans

ABSTRACT Isolates of the late blight pathogen Phytophthora infestans (n = 327) from the central to southern Peruvian Andes were systematically collected in 1997 to 1999 and analyzed to determine the pathogen's population structure at its host's center of diversity. No isolates of the A2 mating type were detected. Cluster analysis of DNA fingerprinting data indicated that the collection consisted of five major groups that were interpreted to be clonal lineages. Two of the lineages (US-1 and EC-1) have been previously described, and three (PE-3, 5, and 6) are described here for the first time. Collections from three areas in the central Peruvian Andes, including two key sites used in an international potato breeding program, consisted of isolates of the EC-1 lineage, which has been reported to dominate the pathogen population in Andean countries to the north of Peru. The collections from Cusco and Puno were more diverse. More than one lineage was detected in 10 of the 20 fields sampled in Cusco. Data on virulence, metalaxyl sensitivity, and band data for allozymes, mitochondrial DNA, and ipiB1 suggested that PE-3 may have been produced through recombination events between US-1 and EC-1. Restriction fragment length polymorphism and amplified fragment length polymorphism marker data were not consistent with this hypothesis.     

Relationship between light,fruit and leaf mineral content with albinism incidence in strawberry (Fragaria x ananassa Duch.)

A study was conducted in strawberry to establish a relationship between shading, mineral nutrient of leaves and fruits with albinism incidence. Plants grown under shade produced albino fruits in higher proportion than those grown in open fields. Similarly, plants under shade produced smaller sized fruits and have lower fruit yield. Among cultivars, Etna had highest incidence of albinism (49.6%) and Sweet Charlie the lowest (14.4%). Dry matter content (%), concentration of five major nutrients, viz. N, P, K, Ca, Mg, S and N:Ca and K:Ca nutrient ratios did not differ significantly in the leaves of plants producing normal or albino fruits. However, in contrast, the concentration of K was notably higher (1.97 mg g⁻¹ fresh tissue weight) and that of Ca was lower (0.098 mg g⁻¹ fresh tissue weight) in albino fruits than normal ones. Consequently, the ratios of N:Ca (11.34) and K:Ca (20.08) were higher in albino fruits than normal ones. Cultivars also differed widely in respect to dry matter (%), mineral content and nutrient ratios. Thus, it appears that lower light intensity favours the development of albinism in strawberry, and it seems that calcium is not the basic cause of albinism, but increased vigour associated with overuse of N and K might be positively associated with it.     

Genetic analysis of human and swine influenza A viruses isolated in Northern Italy during 2010–2015

Influenza A virus (IAV) infection in swine plays an important role in the ecology of influenza viruses. The emergence of new IAVs comes through different mechanisms, with the genetic reassortment of genes between influenza viruses, also originating from different species, being common. We performed a genetic analysis on 179 IAV isolates from humans (n. 75) and pigs (n. 104) collected in Northern Italy between 2010 and 2015, to monitor the genetic exchange between human and swine IAVs. No cases of human infection with swine strains were noticed, but direct infections of swine with H1N1pdm09 strains were detected. Moreover, we pointed out a continuous circulation of H1N1pdm09 strains in swine populations evidenced by the introduction of internal genes of this subtype. These events contribute to generating new viral variants—possibly endowed with pandemic potential—and emphasize the importance of continuous surveillance at both animal and human level.     

Clinical and pathologic features of acute bovine liver disease in Australia

Acute bovine liver disease (ABLD) is a sporadic hepatic disease affecting cattle in southern Australia, characterized histologically by striking periportal hepatocellular necrosis. The cause of ABLD is unknown; however, the seasonality and acute presentation of outbreaks suggest mycotoxin involvement. We describe here the clinical and pathologic findings of ABLD in 45 naturally affected cattle from 13 outbreaks occurring from 2010 to 2019 in Victoria, Australia. Outbreaks occurred in herds located along the southern coastal plain of Victoria and were observed most frequently in lactating dairy cattle. Clinical signs commonly included a combination of mild photosensitization, progressive neurologic signs, and hypogalactia, which preceded death by ≤ 48 h. All affected animals had marked elevations in activities of glutamate dehydrogenase, aspartate aminotransferase, and gamma-glutamyl transferase. At autopsy, the most common lesions were serosal petechiae and/or gastrointestinal hemorrhage, and hepatomegaly with a pronounced hepatic reticular pattern. The principal histologic lesion was widespread—severe periportal hepatocellular coagulative necrosis and erythrocyte pooling—which often extended to massive necrosis. Lesions in other organs were uncommon. Our study of ABLD suggests involvement of a potent hepatotoxin that is either directly cytopathic or requires bioactivation by periportal-specific enzymes.