Genetic analysis of Phytophthora infestans populations in the Nordic European countries reveals high genetic variability

Late blight, caused by the oomycete Phytophthora infestans, is the most important disease of potato (Solanum tuberosum). The pathogen is highly adaptable and to get an overview of the genetic variation in the Nordic countries, Denmark, Finland, Norway and Sweden we have analyzed 200 isolates from different fields using nine simple-sequence repeat (SSR) markers. Forty-nine alleles were detected among the nine SSR loci and isolates from all four Nordic countries shared the most common alleles across the loci. In total 169 multilocus genotypes (based on seven loci) were identified among 191 isolates. The genotypic diversities, quantified by a normalized Shannon's diversity index (H(s)), were 0.95 for the four Nordic countries. The low F(ST) value of 0.04 indicates that the majority of variation is found within the four Nordic countries. The large number of genotypes and the frequency distribution of mating types (60% A1) support the hypothesis that sexual reproduction is contributing notably to the genetic variation of P. infestans in the Nordic countries.     

Identification of two imidazole binding sites and key residues for substrate specificity in human primary amine oxidase AOC3

Human membrane primary amine oxidase (hAOC3; also known as vascular adhesion protein-1, VAP-1) is expressed upon inflammation in most tissues, where its enzymatic activity plays a crucial role in leukocyte trafficking. We have determined two new structures of a soluble, proteolytically cleaved form of hAOC3 (sAOC3), which was extracted from human plasma. In the 2.6 ? sAOC3 structure, an imidazole molecule is hydrogen bonded to the topaquinone (TPQ) cofactor, which is in an inactive on-copper conformation, while in the 2.95 ? structure, an imidazole molecule is covalently bound to the active off-copper conformation of TPQ. A second imidazole bound by Tyr394 and Thr212 was identified in the substrate channel. We furthermore demonstrated that imidazole has an inhibitory role at high concentrations used in crystallization. A triple mutant (Met211Val/Tyr394Asn/Leu469Gly) of hAOC3 was previously reported to change substrate preferences toward those of hAOC2, another human copper-containing monoamine oxidase. We now mutated these three residues and Thr212 individually to study their distinct role in the substrate specificity of hAOC3. Using enzyme activity assays, the effect of the four single mutations was tested with four different substrates (methylamine, benzylamine, 2-phenylethylamine, and p-tyramine), and their binding modes were predicted by docking studies. As a result, Met211 and Leu469 were shown to be key residues for substrate specificity. The native structures of sAOC3 and the mutational data presented in this study will aid the design of hAOC3 specific inhibitors.     

Candidate Gene Analysis of Tooth Agenesis Identifies Novel Mutations in Six Genes and Suggests Significant Role for WNT and EDA Signaling and Allele Combinations

Failure to develop complete dentition, tooth agenesis, is a common developmental anomaly manifested most often as isolated but also as associated with many developmental syndromes. It typically affects third molars or one or few other permanent teeth but severe agenesis is also relatively prevalent. Here we report mutational analyses of seven candidate genes in a cohort of 127 probands with non-syndromic tooth agenesis. 82 lacked more than five permanent teeth excluding third molars, called as oligodontia. We identified 28 mutations, 17 of which were novel. Together with our previous reports, we have identified two mutations in MSX1, AXIN2 and EDARADD, five in PAX9, four in EDA and EDAR, and nine in WNT10A. They were observed in 58 probands (44%), with a mean number of missing teeth of 11.7 (range 4 to 34). Almost all of these probands had severe agenesis. Only few of the probands but several relatives with heterozygous genotypes of WNT10A or EDAR conformed to the common type of non-syndromic tooth agenesis, incisor-premolar hypodontia. Mutations in MSX1 and PAX9 affected predominantly posterior teeth, whereas both deciduous and permanent incisors were especially sensitive to mutations in EDA and EDAR. Many mutations in EDAR, EDARADD and WNT10A were present in several families. Biallelic or heterozygous genotypes of WNT10A were observed in 32 and hemizygous or heterozygous genotypes of EDA, EDAR or EDARADD in 22 probands. An EDARADD variant were in seven probands present together with variants in EDAR or WNT10A, suggesting combined phenotypic effects of alleles in distinct genes.     

The cerebrospinal fluid provides a proliferative niche for neural progenitor cells

Cortical development depends on the active integration of cell-autonomous and extrinsic cues, but the coordination of these processes is poorly understood. Here, we show that the apical complex protein Pals1 and Pten have opposing roles in localizing the Igf1R to the apical, ventricular domain of cerebral cortical progenitor cells. We found that the cerebrospinal fluid (CSF), which contacts this apical domain, has an age-dependent effect on proliferation, much of which is attributable to Igf2, but that CSF contains other signaling activities as well. CSF samples from patients with glioblastoma multiforme show elevated Igf2 and stimulate stem cell proliferation in an Igf2-dependent manner. Together, our findings demonstrate that the apical complex couples intrinsic and extrinsic signaling, enabling progenitors to sense and respond appropriately to diffusible CSF-borne signals distributed widely throughout the brain. The temporal control of CSF composition may have critical relevance to normal development and neuropathological conditions.     

Homing-associated molecules CD73 and VAP-1 as targets to prevent harmful inflammations and cancer spread

Homing-associated molecules are of fundamental importance for proper functioning of our immune system as they direct the cells to sites of inflammation to create an immune response. However, they are also responsible for harmful cell trafficking, which takes place in acute and chronic inflammations as well as in tumor progression and metastatic spread of cancer. Therefore, these molecules are potential targets for developing drugs to prevent harmful inflammation and metastases of cancer. In this review, we will discuss about the most recent advances in studies elucidating the role of two homing-associated ecto-enzymes in physiological and pathological cell trafficking and their use as drug targets.     

Northward invasion of the parasitic deer ked ( Lipoptena cervi), is there geographical variation in pupal size and development duration?

The deer ked (Lipoptena cervi) is a common ectoparasite of cervids. During the last decades the species has rapidly invaded in northern Europe, especially in Finland, towards the north and increased its prevalence on the moose population. Consequently, during this rapid invasion the deer ked has faced more severe climatic conditions. We studied whether pupal size (measured as pupal weight) and pupal development duration of the deer ked varies along historical invasion zones and temperature zones towards north in Finland. Moreover, we explored possible size- and gender-dependent variation in pupal development duration. We divided wild-collected pupae in respect to their origin in two ways: (1) temperature zones (from south-west to colder north-east) and (2) invasion history (from early to late establishment). We reared pupae in the controlled laboratory conditions in identical temperature and light conditions. Pupal size decreased towards north and the smaller pupae developed faster. However, the results do not show differences in pupal size or developmental characteristics between the invasion zones. This supports the idea of rapid developmental plasticity of the deer ked and that not the invasion history but the current temperature regime determines the life history of the deer ked when invading towards a colder environment.     

Characterization of the glyoxalase 1 gene <Emphasis Type="Italic">TcGLX1</Emphasis> in the metal hyperaccumulator plant <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis>

Stress tolerance is currently one of the major research topics in plant biology because of the challenges posed by changing climate and increasing demand to grow crop plants in marginal soils. Increased Zn tolerance and accumulation has been reported in tobacco expressing the glyoxalase 1-encoding gene from Brassica juncea. Previous studies in our laboratory showed some Zn tolerance-correlated differences in the levels of glyoxalase 1-like protein among accessions of Zn hyperaccumulator Thlaspi caerulescens. We have now isolated the corresponding gene (named here TcGLX1), including ca. 570 bp of core and proximal promoter region. The predicted protein contains three glyoxalase 1 motifs and several putative sites for post-translational modification. In silico analysis predicted a number of cis-acting elements related to stress. The expression of TcGLX1 was not responsive to Zn. There was no correlation between the levels of TcGLX1 expression and the degrees of Zn tolerance or accumulation among T. caerulescens accessions nor was there co-segregation of TcGLX1 expression with Zn tolerance or Zn accumulation among F3 lines derived from crosses between plants from accessions with contrasting phenotypes for these properties. No phenotype was observed in an A. thaliana T-DNA insertion line for the closest A. thaliana homolog of TcGLX1, ATGLX1. These results suggest that glyoxalase 1 or at least the particular isoform studied here is not a major determinant of Zn tolerance in the Zn hyperaccumulator plant T. caerulescens. In addition, ATGLX1 is not essential for normal Zn tolerance in the non-tolerant, non-accumulator plant A. thaliana. Possible explanations for the apparent discrepancy between this and previous studies are discussed.     

Development of ground vegetation biomass and nutrient pools in a clear-cut disc-plowed boreal forest

Nutrient leaching from forest substrate after clear-cutting and subsequent soil preparation is strongly influenced by the capacity of ground vegetation to sequester the released nutrients. We studied the rates and patterns of biomass and nutrient accumulation in ground vegetation growing on ridges, in furrows and on undisturbed surfaces for 2–5 years after disc-plowing in eastern Finland. The biomass of mosses on ridges remained significantly lower than that in furrows and on undisturbed surfaces. Field layer biomass on ridges and in furrows was significantly lower than on undisturbed surfaces throughout the study period. Field layer biomass increased more on ridges than in furrows. Root biomass on ridges and undisturbed surfaces was considerably higher than in furrows. Five years after disc-plowing, total biomass and nutrient pools for ridges (biomass 4,975 kg ha⁻¹, N 40 kg ha⁻¹, P 5 kg ha⁻¹, K 20 kg ha⁻¹ and Ca 18 kg ha⁻¹) and undisturbed surfaces (biomass 5,613 kg ha⁻¹, N 43 kg ha⁻¹, P 5 kg ha⁻¹, K 22 kg ha⁻¹ and Ca 18 kg ha⁻¹) were similar, but considerably lower for furrows (biomass 1,807 kg ha⁻¹, N 16 kg ha⁻¹, P 2 kg ha⁻¹, K 10 kg ha⁻¹ and Ca 6 kg ha⁻¹). Ridges covered 25% of the area, furrows 30 and 45% was undisturbed surfaces. Taking into account the proportion of each type of surface, values for the whole prepared clear-cut area were 4,312, 34, 4, 18 and 14 kg ha⁻¹ for biomass, N, P, K and Ca, respectively. Biomass and nutrient pools had not returned to uncut forest levels at the end of the 5-year study period. The results indicate that mosses and field layer vegetation respond differently to soil preparation, that the development of biomass on ridges, in furrows and on undisturbed surfaces proceeds at different rates, and that the biomass and nutrient uptake of ground vegetation remains below pre-site preparation levels for several years. However, ridges, which are known to be the most susceptible to leaching, revegetate rapidly. Responsible Editor: Tibor Kalapos.     

Impacts of Paternal Care and Seasonal Change on Offspring Survival: A Multiseason Experimental Study of a Caribbean Frog

Parental care is widespread among animals and many studies have investigated how environmental conditions influence parental care strategies. However, few studies have coupled observations of the frequency and form of parental care with manipulative studies on the function of care across multiple seasons. To address this gap, we made field observations and conducted male removal experiments in a glass frog with paternal egg attendance from the island of Tobago, West Indies. To test the hypothesis that seasonality impacts the frequency, form, and function of parental care, field observations and male removal experiments were undertaken three times, twice during the rainy season, and once in the dry season. We found seasonal differences in the frequency of egg attendance with males increasing the frequency of this behavior in the dry season, likely in response to a greater risk of clutch desiccation. We also found that clutches in which the male was removed had strongly reduced survivorship compared with controls and this pattern did not change seasonally. However, sources of mortality were strongly influenced by season. Arthropod predation was the primary source of mortality during the wet season, but in the dry season, desiccation was more important. Together, these results suggest that seasonal variation is both impacting care function as well as shaping the form that parental care takes.     

Pre-targeting and direct immunotargeting of liposomal drug carriers to ovarian carcinoma

Background

Epidermal growth factor receptor (EGFR) is overexpressed in many solid tumor types, such as ovarian carcinoma. Immunoliposome based drug targeting has shown promising results in drug delivery to the tumors. However, the ratio of tumor-to-normal tissue concentrations should be increased to minimize the adverse effects of cytostatic drugs.

Methodology/Principal Findings

We studied the EGFR-targeted doxorubicin immunoliposomes using pre-targeting and local intraperitoneal (i.p.) administration of the liposomes. This approach was used to increase drug delivery to tumors as compared to direct intravenous (i.v.) administration of liposomes. EGFR antibodies were attached on the surface of PEG coated liposomes using biotin-neutravidin binding. Receptor mediated cellular uptake and cytotoxic efficacy of EGFR-targeted liposomes were investigated in human ovarian adenocarcinoma (SKOV-3 and SKOV3.ip1) cells. In vivo distribution of the liposomes in mice was explored using direct and pre-targeting approaches and SPECT/CT imaging. Targeted liposomes showed efficient and specific receptor-mediated binding to ovarian carcinoma cells in vitro, but the difference in cytotoxicity between targeted and non-targeted liposomes remained small. The relatively low cytotoxic efficacy is probably due to insufficient doxorubicin release from the liposomes rather than lack of target binding. Tumor uptake of targeted liposomes in vivo was comparable to that of non-targeted liposomes after both direct and pre-targeting administration. For both EGFR-targeted and non-targeted liposomes, the i.p. administration increased liposome accumulation to the tumors compared to i.v. injections.

Conclusions/Significance

Intraperitoneal administration of liposomes may be a beneficial approach to treat the tumors in the abdominal cavity. The i.p. pre-targeting method warrants further studies as a potential approach in cancer therapy.