Cyst-theca relationship and phylogenetic positions of Scrippsiella plana sp. nov. and S. spinifera (Peridiniales,Dinophyceae)

Species belonging to the dinophyte genus Scrippsiella are frequently reported in marine waters, but information on their distribution in brackish environments is limited. Here we describe a new species, S. plana, through incubation of non-calcified cysts from sediments collected in the South China Sea and Caspian Sea. The vegetative cells consist of a conical epitheca and a rounded hypotheca with the plate formula of Po, X, 4′, 3a, 7′′, 5C+t, 5S, 5′′′, 2′′′′. It differs from other Scrippsiella species by its flattened body in dorsoventral view and a small first anterior intercalary (1a) plate (half the size of plate 3a). Scrippsiella plana strains from the South China Sea and Caspian Sea share identical internal transcribed spacer (ITS) sequences, and show phenotypic plasticity and local adaptation in growth rate at various salinities, consistent with the environments in which they originated. In addition, two strains of S. spinifera were obtained by incubating ellipsoid cysts with calcareous spines from sediments collected along the Turkish and Hawaiian coast. They also share identical ITS sequences and differ from Duboscquodinium collinii (a parasite of tintinnids) only at two base pair positions (in the ITS2 region). Molecular phylogeny based on ITS and large subunit ribosomal DNA (LSU rDNA) sequences revealed that S. plana was nested within the Calciodinellum (CAL) clade and S. spinifera within the S. trochoidea (STR) clade. The phylogenetic position of ‘Peridinium’ wisconsinense is reported for the first time, which supports multiple transitions of the Peridiniales to freshwater.     

Quaternary structure, protein dynamics, and synaptic function of SAP97 controlled by L27 domain interactions

Single-particle electron microscopy (EM) combined with biochemical measurements revealed the molecular shape of SAP97 and a monomer-dimer transition that depended on the N-terminal L27 domain. Overexpression of SAP97 drove GluR1 to synapses, potentiated AMPA receptor (AMPAR) excitatory postsynaptic currents (EPSCs), and occluded LTP. Synaptic potentiation and GluR1 delivery were dissociable by L27 domain mutants that inhibit multimerization of SAP97. Loss of potentiation was correlated with faster turnover of monomeric SAP97 mutants in dendritic spines. We propose that L27-mediated interactions of SAP97 with itself or other proteins regulate the synaptic delivery of AMPARs. RNAi knockdown of endogenous PSD-95 depleted surface GluR1 and impaired AMPA EPSCs. In contrast, RNAi knockdown of endogenous SAP97 reduced surface expression of both GluR1 and GluR2 and inhibited both AMPA and NMDA EPSCs. Thus SAP97 has a broader role than its close relative, PSD-95, in the maintenance of synaptic function.     

A rapid novel strategy for screening of antibody phage libraries for production,purification, and functional characterization of amber stop codons containing single-chain antibody fragments

Phage display antibody (PDA) libraries, allows the rapid isolation and characterization of high specificity monoclonal antibodies for therapeutic and diagnostic applications. However, selection of positive binding clones from synthetic and semi-synthetic libraries has an inherent bias towards clones containing randomly generated amber stop codons, complicating the identification of high affinity binding antibodies. We screened Tomlinson I and J library against receptor binding domain (RBD) of SARS CoV2, eight clones which showed positive binding in phage ELISA, contained one or more amber stop codons in their single-chain antibody fragment (scFv) gene sequences. The presence of amber stop codons within the antibody sequence causes the premature termination of soluble form of scFv expression in nonsuppressor Escherichia coli strain. In the present study, we have used a novel strategy that allows soluble expression of scFvs having amber stop codon in their gene sequences (without phage PIII protein fusion), in the suppressor strain. This strategy of introduction of Ochre (TAA) codon at the junction of scFv and PIII gene, speeds up the initial screening process which is critical for selecting the right scFvs for further studies. Present strategy leads to the identification of a scFv, B8 that binds specifically with nanomolar affinity toward SARS CoV 2 RBD, which otherwise lost in terms of traditional methodology.     

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.     

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.     

The role of cotyledon metabolism in the establishment of quinoa (Chenopodium quinoa) seedlings growing under salinity

A growth chamber experiment was conducted to assess the effect of salinity on emergence, growth, water status, photosynthetic pigments, osmolyte accumulation, and ionic content of quinoa seedlings (Chenopodium quinoa). The aim was to test the hypothesis that quinoa seedlings are well adapted to grow under salinity due to their ability to adjust the metabolic functionality of their cotyledons. Seedlings were grown for 21 days at 250 mM NaCl from the start of the germination. Germination percentage and cotyledon area were not affected by salt whereas seedling height decreased 15%. FW increased in both control and salt-treated cotyledons, but the increase was higher under salinity. DW only increased in salt-treated cotyledons. The DW/FW ratio did not show significant differences between treatments. Relative water content, chlorophyll, carotenoids, lipids, and proteins were significantly lower under salinity. Total soluble sugars, sucrose and glucose concentrations were higher in salt-treated than in control cotyledons. Ion concentration showed a different distribution pattern. Na⁺ and Cl^? concentrations were higher under salinity, while an inverse result was observed for K⁺ concentration. Proline and glycinebetaine concentrations increased under salinity, but the increase was higher in the former than the latter. The osmoprotective role of proline, glycinebetaine, and soluble sugars is discussed.     

Cadmium induces changes in sucrose partitioning, invertase activities, and membrane functionality in roots of Rangpur lime (Citrus limonia L. Osbeck)

Cadmium (Cd) uptake effects on sucrose content, invertase activities, and plasma membrane functionality were investigated in Rangpur lime roots ( CITRUS LIMONIA L. Osbeck). Cadmium accumulation was significant in roots but not in shoots and leaves. Cadmium produced significant reduction in roots DW and increment in WC. Leaves and shoots did not show significant differences on both parameters. Sucrose content was higher in control roots than in Cd-exposed ones. Apoplastic sucrose content was much higher in Cd-exposed roots than in control ones. Cd-exposed roots showed a significant decrease in both cell wall-bound and cytoplasmic (neutral) invertase activities; while the vacuolar isoform did not show any change. Alterations in lipid composition and membrane fluidity of Cd-exposed roots were also observed. In Cd-exposed roots phospholipid and glycolipid contents decreased about 50 %, while sterols content was reduced about 22 %. Proton extrusion was inhibited by Cd. Lipid peroxidation and proton extrusion inhibition were also detected by histochemical analysis. This work's findings demonstrate that Cd affects sucrose partitioning and invertase activities in apoplastic and symplastic regions in Rangpur lime roots as well as the plasma membrane functionality and H (+)-ATPase activity.     

Computational modeling of the adsorption and •OH initiated photochemical and photocatalytic primary oxidation of nitrobenzene

The adsorption and primary oxidation step for the photodegradation of nitrobenzene (NB) have been studied computationally using MSINDO SCF MO method. The method performs efficiently for extended surface models such as Ti₃₆O₉₀H₃₆. Molecular dynamics simulations have revealed that NB is linked to TiO₂ surface at the titanium ion via the oxygen atoms of NO₂ group. In addition, the computed vibrational density of states for the adsorbed NB molecule is in reasonably good agreement with the available experimental data and theoretical results. In order to identify the primary photochemical and photocatalytic ^•OH initiated photooxidation intermediates, we have employed two different theoretical approaches, frontier orbital theory and Wheland localization theory. It has been found that the meta- hydroxynitrocyclohexadienyl radical is energetically more favored than para- and ortho-hydroxynitrocyclohexadienyl radicals for the photochemical photolysis, whereas in the case of photocatalysis, the ^•OH radical attack is unselective and all three possible isomers have comparable stabilities. Figure Minimum energy adsorption conformation of nitrobenzene onto TiO₂ (100) surface