<Emphasis Type="Italic">Castanea sativa</Emphasis>: genotype-dependent recovery from chestnut blight

In Lovran (coastal Croatia), a unique forest/orchard of evenly mixed grafted marrons and naturally growing nongrafted sweet chestnut trees exists. This old chestnut population has been devastated by chestnut blight, caused by an aggressive introduced pathogenic fungus, Cryphonectria parasitica. However, initial observations indicated recovery of naturally growing chestnut trees in that area, mediated by Cryphonectria-associated hypovirus (Cryphonectria hypovirus 1 (CHV-1)). Such recovery was not observed on grafted trees. Genotyping both, we confirmed the clonal origin of the grafted ones—marrons. No significant difference was observed between fungal strains isolated from naturally growing trees and the ones from marrons regarding fungal vegetative compatibility types or the prevalence of CHV-1. A strong correlation was observed between the types of canker: active/deep-expanding versus healing callus or superficial necrosis and the absence or presence of CHV-1 in the fungal isolates, sampled from naturally growing trees (Spearman rho 0.686, p value 7.81?×?10^?5, Kendall tau 0.686, p value 5.18?×?10^?7). Such correlation was not observed on marrons (Spearman rho 0.236, p value 0.235, Kendall tau 0.236, p value 0.084), because, unexpectedly, active/deep-expanding cankers were often associated with hypovirulent fungal isolates. These data indicate that the lack or unequal distribution of naturally occurring hypovirulence were not the cause of substantial marron decay in Lovran. Ecological and age-dependant differences were ruled out because all sampled trees are growing in close proximity and are of similar age. The results imply that the marron genotype is especially vulnerable and its ability to recover is limited even when the hypovirulent strain of the fungus is present in the canker.     

Analysis of heterogeneity and instability of stable mAb-expressing CHO cells

Screening and isolation of high expression mammalian cell lines for production of recombinant proteins for the clinic is a resource-intensive and time-consuming procedure due to the substantial variation in expression levels of recombinant protein expression amongst transfected cells. Several investigators have reported instability in expression titers early in cell line development and in cell banks. However, in most cases the exact molecular mechanisms of instability remain unknown. In this study we used a fluorescence-activated cell sorting (FACS) based mAb staining method to enable the detection and selective gating of cells with vastly different recombinant expression levels present in transfected pools. Expression diversity and changes within transfected populations were detected and isolated in real time during cell line development. Molecular genetic analysis on the isolated clones revealed an unsuspected rearrangement of the heavy chain in the non-expressing clones. Implications of the genetic rearrangements as well as the use of the FACS method as a tool to improve cell line development to detect expression heterogeneity in pools and to investigate root cause for the molecular genetics of expression instability will be discussed.     

Biotransformation of Phenylacetonitrile to 2-Hydroxyphenylacetic Acid by Marine Fungi

Marine fungi belonging to the genera Aspergillus, Penicillium, Cladosporium, and Bionectria catalyzed the biotransformation of phenylacetonitrile to 2-hydroxyphenylacetic acid. Eight marine fungi, selected and cultured with phenylacetonitrile in liquid mineral medium, catalyzed it quantitative biotransformation to 2-hydroxyphenylacetic acid. In this study, the nitrile group was firstly hydrolysed, and then, the aromatic ring was hydroxylated, producing 2-hydroxyphenylacetic acid with 51 % yield isolated. In addition, the 4-fluorophenylacetonitrile was exclusively biotransformed to 4-fluorophenylacetic acid by Aspergillus sydowii Ce19 (yield?=?51 %). The enzymatic biotransformation of nitriles is not trivial, and here, we describe an efficient method for production of phenylacetic acids in mild conditions.     

Mitochondrial genomes of the genus Ephydatia Lamouroux, 1816: can palindromic elements be used in species-level studies?

Poriferan mitochondrial DNA (mtDNA), especially large intergenic regions, is a target for the insertion of repetitive hairpin-forming elements. These elements are responsible for the large mt genome size differences observed even among closely related sponge taxa. In this study, we present the new, nearly complete, mt genome sequence of Ephydatia fluviatilis and compare it with previously published mt genomes of freshwater sponges. Special emphasis was placed on comparison with the closely related species Ephydatia muelleri, thereby comparing the only two species of the genus Ephydatia on the western Balkan Peninsula. In particular, we analyzed repetitive palindromic elements within the mitochondrial intergenic regions. The genomic distribution of these repetitive elements was analyzed and their potential role in the evolution of mt genomes discussed. We show here that palindromic elements are widespread through the whole mt genome, including the protein coding genes, thus introducing genetic variability into mt genomes.     

Polymorphisms of interleukin-4, -10 and 12B genes and diabetic retinopathy

In diabetic retinopathy (DR) and other angiogenesis-associated diseases, increased levels of cytokines, inflammatory cells, and angiogenic factors are present. We investigated the hypothesis that rs2243250 polymorphism of the interleukin 4 (IL-4) gene or rs1800896 polymorphism of the interleukin 10 (IL-10) gene, and rs3212227 polymorphism of the 3’ untranslated region (3’ UTR) of the interleukin-12 p40 gene (IL12B) may be associated with the development of proliferative diabetic retinopathy (PDR) in Caucasians with type 2 diabetes (DM2). This cross sectional case — control study included 189 patients with PDR and 187 patients with type 2 diabetes without PDR. Polymorphisms rs1800896 of the IL-10 gene, rs2243250 of the IL-4 gene, and rs3212227 of IL12B gene were analyzed by ARMS -PCR and RFLP -PCR methods. Multivariate analysis demonstrated the GG genotype of the rs1800896 polymorphism of the IL-10 gene to be associated with increased risk for PDR (OR=1.99; 95% CI=1.11–3.57; P=0.02), whereas the TT genotype of the rs2243250 polymorphism of the IL-4 gene and the AA genotype of the rs3212227 polymorphism of the IL-12 gene were not independent risk factors for PDR. Our findings suggest that the genetic variations at the IL-10 promoter gene might be a genetic risk factor for PDR in Caucasians with type 2 diabetes.     

Cytopathology and diagnostics of Warthin's tumour

Warthin's tumour (WT) is a benign epithelial salivary tumour, one type of salivary adenoma. Histologically, WT is structured of two components, epithelial tissue that often lines cystic formations and lymphoid tissue in the tumour stroma. FNA is a reliable diagnostic approach in the diagnosis of salivary gland lesions allowing a highly accurate categorization of benign tumour‐like lesions, benign tumours and malignant tumours. In the proposed Milan reporting system of salivary gland lesions, WT is categorized in the IVA group of benign neoplasms. Accurate cytological diagnosis is straightforward when three characteristic components are present: oncocytes, either isolated or associated in clusters, lymphocytes and lymphoid cells and often an inflammatory/necrotic‐like substance. Also, specific features of scintigraphy and radiological imaging contribute to the diagnosis of WT. WT is categorized according to Seifert G. et al in 4 types, depending on the proportions of the epithelial component and lymphoid stroma. Differential cytopathological and pathohistological diagnosis include other salivary gland lesions with lymphoid, oncocytic epithelial and cystic components. In some cases, such as the metaplastic WT variant, there are additional cytopathological and histological diagnostic difficulties. Moreover, bilateral, multicentric or multiple and infrequently seen extra‐salivary localizations of WT are associated with further cytopathological diagnostic difficulties. Also, a rare possibility of malignant transformation of the epithelial or lymphoid component of WT as well as possible association with other primary tumours remains a challenge in accurate cytopathological and histological diagnosis of WT.     

UV-radiation, apoptosis and skin

Apoptosis or programmed cell death is a key function in regulating skin development, homeostasis and tumorigenesis. The epidermis is exposed to various external stimuli and one of the most important is UV radiation. The UVA and UVB spectra differ in their biological effects and in their depth of penetration through the skin layers. UVB rays are absorbed directly by DNA which results in its damage. UVA can also cause DNA damage but primarily by the generation of reactive oxygen species. By eliminating photodamaged cells, apoptosis has an important function in the prevention of epidermal carcinogenesis. UV-induced apoptosis is a complex event involving different pathways. These include: 1. activation of the tumour suppressor gene p53; 2. triggering of cell death receptors directly by UV or by autocrine release of death ligands; 3. mitochondrial damage and cytochrome C release. The extrinsic pathway through death receptors such as fibroblast-associated, tumour necrosis factor receptor and TNF related apoptosis inducing ligand receptor activate caspase cascade. The intrinsic or mitochondrial pathway of apoptosis is regulated by the Bcl-2 family of proteins, anti-apoptotic (Bcl-2, Bcl-xl, Bcl-w) and the pro-apoptotic (Bax, Bak, Bid). The balance between the pro-apoptotic and anti-apoptotic proteins determines cell survival or death. We discuss recent findings in the molecular mechanisms of UV induced apoptosis.