Cyanine dyes for the detection of double stranded DNA

Twenty three novel cyanine dyes have been applied as fluorescent stains for the detection of nucleic acids in agarose gel electrophoresis. Significant fluorescence enhancement of these dyes in the presence of double stranded DNA was observed. Five dyes offered superior sensitivity in the detection and quantification of DNA, over Ethidium Bromide, the most commonly used nucleic acid stain.     

Inhibition of α-Synuclein Fibrillization by Dopamine Is Mediated by Interactions with Five C-Terminal Residues and with E83 in the NAC Region

The interplay between dopamine and α-synuclein (AS) plays a central role in Parkinson''s disease (PD). PD results primarily from a severe and selective devastation of dopaminergic neurons in substantia nigra pars compacta. The neuropathological hallmark of the disease is the presence of intraneuronal proteinaceous inclusions known as Lewy bodies within the surviving neurons, enriched in filamentous AS. In vitro, dopamine inhibits AS fibril formation, but the molecular determinants of this inhibition remain obscure. Here we use molecular dynamic (MD) simulations to investigate the binding of dopamine and several of its derivatives onto conformers representative of an NMR ensemble of AS structures in aqueous solution. Within the limitations inherent to MD simulations of unstructured proteins, our calculations suggest that the ligands bind to the ¹²⁵YEMPS¹²⁹ region, consistent with experimental findings. The ligands are further stabilized by long-range electrostatic interactions with glutamate 83 (E83) in the NAC region. These results suggest that by forming these interactions with AS, dopamine may affect AS aggregation and fibrillization properties. To test this hypothesis, we investigated in vitro the effects of dopamine on the aggregation of mutants designed to alter or abolish these interactions. We found that point mutations in the ¹²⁵YEMPS¹²⁹ region do not affect AS aggregation, which is consistent with the fact that dopamine interacts non-specifically with this region. In contrast, and consistent with our modeling studies, the replacement of glutamate by alanine at position 83 (E83A) abolishes the ability of dopamine to inhibit AS fibrillization.     

Three new species of Zerconidae (Acari: Mesostigmata) from Turkey

Three new species, Zercon afyonensis sp. n., Zercon karacamehmeti sp. n. and Zercon soguticus sp. n., are described and illustrated based on samples collected in the Inner Aegean Region. The similarities and differences between the related species are discussed.

http://www.zoobank.org/urn:lsid:zoobank.org:pub:3FA1701A-A243-4E45-8AE9-FCB733CC56A6     

(Bio)fouling of Polymeric Membranes: Atomic Force Microscope Study

Atomic force microscopy (AFM), in conjunction with colloid probe, coated colloid probe and cell probe techniques, has been used to measure directly the adhesive force between a polystyrene sphere (diameter 11 μm), protein bovine serum albumin (BSA) and a yeast cell, and two different membranes. These were polymeric ultrafiltration membranes of similar MWCO (4000 Da) but of different materials (ES 404 and XP 117, PCI Membrane Systems Ltd (UK)). The colloid probe was created by immobilising a polystyrene sphere onto a tipless V‐shaped AFM cantilever. The coated probe was made by adsorbing BSA on a 5 μm silica colloid, while immobilising a single yeast cell on such a tipless cantilever created the cell probe. Measurements were made in 10^–2 M NaCl solution. It was found for polystyrene, protein and cell systems that the adhesive force at the ES 404 membrane was greater than that at the XP 117 membrane. The paper shows that the colloid probe, coated colloid probe and cell probe techniques can provide useful means of directly quantifying the adhesion of both inorganic and biological materials to membrane surfaces.     

Biological control of soil-pathogenic nematodes infecting mungbean using Pseudomonas fluorescens

The addition of organic matters to soil has been explored as an alternative means of nematode control under field conditions. Several oil-seed cakes of neem (Azadirachta indica), castor (Ricinus communis), groundnut (Arachis hypogeae), linseed (Linum usitatissimum) and sunflower (Helianthus annuus) were found to be highly effective in reducing the multiplication of soil-pathogenic nematodes Meloidogyne incognita, Rotylenchulus reniformis, Tylenchorhynchus brassicae, etc. The plant growth parameters such as plant weight, per cent pollen fertility, number of pods per plant, root-nodulation and chlorophyll content of mungbean increased significantly. The multiplication rate of nematodes and number of root-galls were less in the presence of Pseudomonas fluorescens as compared to its absence. Damage caused by the nematodes was further reduced when P. fluorescens was added along with the oil-seed cakes. Neem cake was found most effective in combination with P. fluorescens.     

Talazoparib nanoparticles for overcoming multidrug resistance in triple-negative breast cancer

Herein, we investigated efflux pumps-mediated talazoparib-resistance in the treatment of triple-negative breast cancer (TNBC). Furthermore, we produced a novel talazoparib-solid lipid nanoparticles (SLNs) and then explored in vitro therapeutic efficacy of talazoparib-SLNs to overcome talazoparib-resistance in TNBC cells. Talazoparib-SLNs formulation was produced and then characterized. Calcein and Rho-123 were used to analyze the functional activity of drug efflux pumps in these cells. Additionally, RT-PCR, western blot and immunofluorescence analysis were used to detect the messenger RNA, and protein expression level, and cellular localization of the multidrug resistance (MDR1), breast cancer resistance protein (BCRP), and MRP1. We found that talazoparib efflux was mediated by BCRP and MRP1 pumps in TNBC cells. Talazoparib-SLNs could significantly enhance therapeutic efficacy of talazoparib. Furthermore, talazoparib-SLNs were more effective in the suppression of MDR1, BCRP, and MRP1 gene and protein expression levels than talazoparib. Consequently, this study suggests that talazoparib-SLNs formulation represents a promising therapeutic carrier to reverse MDR-mediated resistance in TNBC.     

The Adiponectin variants contribute to the genetic background of type 2 diabetes in Turkish population

Adiponectin, an adipose tissue specific protein encoded by the Adiponectin gene, modulates insulin sensitivity and plays an important role in regulating energy homeostasis. Many studies have shown that single nucleotide polymorphisms (SNPs) in the Adiponectin gene are associated with low plasma Adiponectin levels, insulin resistance and an increased risk of type 2 diabetes mellitus. The aim of the present study was to evaluate the contribution of the Adiponectin gene polymorphisms in genetic background of type 2 diabetes in a Turkish population. In total, 169 unrelated and non-obese diabetic patients and 119 age- and BMI-matched non-diabetic individuals with no family history of diabetes were enrolled in this study. We detected a significant association between type 2 diabetes and two SNPs: SNP − 11391G > A, which is located in the promoter region of the Adiponectin gene, and SNP + 276G > T, which is found in intron 2 of the gene (P < 0.05). The silence SNP G15G (+ 45T > G) in exon 1 and SNP + 349A > G in intron 2 also showed a weak association with type 2 diabetes (P = 0.06 and P = 0.07, respectively), while SNPs − 3971A > G in intron 1 and Y111H, R112C and H241P in exon 3 showed no association (P > 0.05). In conclusion, these findings suggest that Adiponectin gene polymorphisms might be effective on susceptibility for type 2 diabetes development which emerged from the interactions between multiple genes, variants and environmental factors.     

Human chorionic gonadotropin (hCG) prevents the transformed phenotypes induced by 17 β-estradiol in human breast epithelial cells

Human chorionic gonadotropin (hCG), a hormone produced during pregnancy, can elicit life-long refractoriness to carcinogenesis by differentiation of the breast epithelium. Human breast epithelial cells MCF-10F form tubules in collagen, mimicking the normal ductules. We have shown that 17 β-estradiol (E2) alter the ductulogenic pattern of these cells. The effect of the recombinant hCG (rhCG) in vitro was evaluated on the transformation of MCF-10F induced by E2. MCF-10F cells were treated with 70 nM E2 alone or in combination with 50 IU/ml rhCG during 2 weeks, while the controls were treated with DMSO (the solvent in which E2 was dissolved) or rhCG alone. At the end of treatment, the cells were plated in type I collagen matrix (3D-cultures) for detecting 2 main phenotypes of cell transformation, namely the loss of ductulogenic capacity and the formation of solid masses. Although E2 significantly increased solid mass formation, this effect was prevented when MCF-10F cells were treated with E2 in combination with rhCG. Furthermore, E2 increased the main duct width (p < 0.001), and caused a disruption of the luminal architecture, whereas rhCG increased the length of the tubules (p < 0.001) and produced tertiary branching. In conclusion, rhCG was able to abrogate the transforming abilities of estradiol, and had the differentiating property by increasing the branching of the tubules formed by breast epithelial cells in collagen. These results further support our hypothesis, known as the terminal differentiation hypothesis of breast cancer prevention, that predicts that hCG treatment results in protection from tumorigenic changes by the loss of susceptible stem cells 1 through a differentiation to refractory stem cells 2 and increase differentiation of the mammary gland.     

<Emphasis Type="Italic">FHIT</Emphasis> Gene Sequence Variants and Reduced Fhit Protein Expression in Glioblastoma Multiforme

Molecular studies have an important role in the elucidation of the mechanisms involved in Glioblastoma multiforme (GBM) development. The occurrence of FHIT gene alterations, which has an important role in different cancers, has not yet been studied well in GBM. We aimed to investigate the occurrence of alterations of FHIT gene sequence and protein expression in the GBMs. Sequence alterations in exons 5–9 of the FHIT gene were screened in 63 GBMs using the single-strand conformational polymorphism method, followed by DNA sequencing. Additionally, the level of Fhit protein expression in tissues of 48 tumors was assessed by immunohistochemistry (IHC). In our investigation, FHIT gene alterations in the coding region were detected in 11 of the 63 GBM cases (17.5%). Two different sequence variants were determined: one novel missense variant (G→C transition at codon 49) and one previously described silent alteration (C→T transition at codon 88). Using web-based programs, such as SIFT and ESEfinder, it was determined that both alterations might have caused significant modification on protein function. In addition, we identified a previously reported an intronic polymorphism (T→A transition at IVS8-17) in 47.5% of cases as a similar rate (45%) in the control group. Moreover, it was observed that Fhit protein expression was reduced in 87.5% of tumors. In conclusion, the reduction or loss of Fhit protein expression by genetic alterations or epigenetic mechanisms in GBM might be associated with brain tumorigenesis.     

Ecological Genetic Divergence of the Fungal Pathogen Didymella rabiei on Sympatric Wild and Domesticated Cicer spp. (Chickpea)

For millennia, chickpea (Cicer arietinum) has been grown in the Levant sympatrically with wild Cicer species. Chickpea is traditionally spring-sown, while its wild relatives germinate in the autumn and develop in the winter. It has been hypothesized that the human-directed shift of domesticated chickpea to summer production was an attempt to escape the devastating Ascochyta disease caused by Didymella rabiei. We estimated genetic divergence between D. rabiei isolates sampled from wild Cicer judaicum and domesticated C. arietinum and the potential role of temperature adaptation in this divergence. Neutral genetic markers showed strong differentiation between pathogen samples from the two hosts. Isolates from domesticated chickpea demonstrated increased adaptation to higher temperatures when grown in vitro compared with isolates from the wild host. The distribution of temperature responses among progeny from crosses of isolates from C. judaicum with isolates from C. arietinum was continuous, suggesting polygenic control of this trait. In vivo inoculations of host plants indicated that pathogenic fitness of the native isolates was higher than that of their hybrid progeny. The results indicate that there is a potential for adaptation to higher temperatures; however, the chances for formation of hybrids which are capable of parasitizing both hosts over a broad temperature range are low. We hypothesize that this pathogenic fitness cost is due to breakdown of coadapted gene complexes controlling pathogenic fitness on each host and may be responsible for maintenance of genetic differentiation between the pathogen demes.Environmental heterogeneity and genetic variability in host populations are major factors distinguishing natural from agricultural habitats. These differences exert powerful selective forces on plants and their pathogens, shaping the biology of pathosystems, epidemiological patterns, and pathogenic fitness (11, 21). Plant pathogens are dependent upon the abiotic environment as well as on their host plants and are subjected to strong selective forces exerted by their hosts. This process is shaped especially (but not exclusively) by genetic variation at loci controlling differential host specificity, which may ultimately be an important driver in speciation (37, 48, 49).The Neolithic revolution and the adoption of farming have had a large impact on plant communities as well as their related pathogens (11, 34, 57). The long-term interplay between plant pathogens and their hosts and the resulting evolutionary trajectories may have different patterns in natural plant communities as compared to agro-ecosystems (12). One striking observation is that pathogens of natural plant populations, although prevalent, rarely cause the destruction of their hosts (21). Therefore, investigations of the epidemiological and biological differences between pathogen populations from wild and domesticated origins are of fundamental interest and are highly relevant to understanding disease patterns, parasite evolution, and host resistance in agricultural systems. Such studies are expected to be especially fruitful in the centers of origin of crop species, because these regions are generally considered to be pathogen centers of origin as well (40, 57).Throughout West Asia, wild cereals and legumes and their domesticated derivatives have been growing sympatrically since the beginning of Near Eastern farming systems (41, 61). Domesticated chickpea, Cicer arietinum L, is grown sympatrically with a number of annual and perennial Cicer relatives, including the immediate wild progenitor of domesticated chickpea, C. reticulatum Ladiz (39, 58). Following the Neolithic agricultural revolution in southeastern Turkey (41), the Near Eastern crop package spread in all directions throughout the east Mediterranean and reached the southern Levant within 1 millennium (2, 3). This “passage” of the cultigens, from their core region in southeast Turkey into the southern Levant, traversed populations of many of their wild progenitors and more distantly related wild relatives (e.g., wild barley, wild emmer wheat, wild bitter vetch, wild lentils, and wild peas), (2, 3). Presumably, these natural populations were infested by pathogens capable of infecting the domesticated forms (2, 20, 24).Domesticated chickpea differs from the Near Eastern founder crops in its seasonal growth pattern. While most founder crops have retained the autumnal germination/spring maturation cycle like their wild relatives, domesticated chickpea is a spring-sown crop, germinating and developing up to 4 months later than its wild relatives (1, 3). This shift of life cycle is puzzling since water availability in the Levant is a major yield-limiting factor and autumn-sown crops enjoy a substantial yield benefit. It has been recently hypothesized that this shift was driven by the extreme vulnerability of chickpea to Ascochyta blight during the rainy season and was the only means to secure stable yields in ancient times (3). Didymella rabiei (Kovachevski) var. Arx. (Anamorph: Ascochyta rabiei (Pass) Labr.) is one of the most destructive diseases of domesticated chickpea, affecting all above-ground parts of the plant. Secondary spread of D. rabiei conidia occurs through rain splash, and epidemic intensity is governed by rain frequency and quantity. As Ascochyta blight epidemics proceed, foci of diseased plants become visible. Unlike other Ascochyta diseases of legumes and Septoria diseases of cereals, Ascochyta blight of chickpea may cause total yield loss under the appropriate environmental conditions (43). Autumn-sown chickpea is severely affected by Ascochyta blight because the crop growth period coincides with the rainy season and optimum environmental conditions for pathogen development and spread (3, 56).Unlike the often massive stands of wild cereals, C. reticulatum has a very narrow and fragmented distribution (2, 8, 38). However, other wild annual Cicer taxa are more common across the region and can be found in close proximity to the domesticated crop (1, 8). In the southern Levant, domesticated chickpea is grown sympatrically, often just few meters apart from C. judaicum (27). C. judaicum grows in patchy distributions in stony/rocky habitats in Israel and neighboring territories, mostly in sites with annual precipitation of >480 mm and altitude of <900 m (6). Unlike C. judaicum, modern chickpea cropping in Israel spans large tracts of land employing a 5-year rotation in individual fields. Recently, D. rabiei isolates sampled from C. judaicum and isolates sampled from C. arietinum were studied and found to be better adapted to their respective original host than to the other Cicer species (26, 27). In addition, in vitro hyphal growth rate experiments exposed an adaptation to higher temperatures among isolates originating from C. arietinum compared to isolates from C. judaicum (26). Given that the natural growing season of C. judaicum occurs during the Levantine winter and that chickpea is a traditional spring-sown crop in the region, it is likely that the apparent adaptation to higher temperatures of D. rabiei isolates from domesticated chickpea may represent an ecological shift following the introduction of summer cropping practices in the Near East (3). These sympatric wild and domesticated pathosystems of Cicer/Ascochyta represent a unique opportunity for studying the genetic basis of the pathogen''s ecological adaptation and its association with pathogenic fitness. Such a system may also help to determine the role of ecological factors and pathogenic fitness in pathogenic divergence and the evolutionary relationships among pathogen populations in natural and human-directed agro-ecosystems (57).In this context, our underlying hypotheses were as follows: (i) isolates sampled from C. arietinum and C. judaicum are conspecific but represent genetically distinct populations; (ii) the temperature growth response of D. rabiei isolates from C. judaicum and C. arietinum has a heritable genetic basis; (iii) the temperature growth response plays an important role in the ongoing pathogen divergence process and, therefore, it is expected to have high heritability values; and (iv) the existence of two sympatric D. rabiei populations (demes) requires the action of one or more genetic isolation mechanisms. In accord with the above hypotheses, the aims of this study were (i) to assess the genetic differentiation between D. rabiei isolates originating from C. judaicum versus C. arietinum, (ii) to determine the genetic basis of temperature response and estimate its heritability, and (iii) to assess the relationship between temperature adaptation and pathogenic fitness among progeny from crosses between D. rabiei isolates from C. judaicum and C. arietinum on the two original hosts.