Selective specificity of calcium-binding proteins calbindin and calretinin expression in the magnocellular neurosecretory hypothalamic nuclei of tortoises and turtles

We have studied the distribution of calcium-binding proteins in the magnocellular neurosecretory nuclei of nonapeptidergic neurosecretory nuclei of the preoptic–hypothalamic complex in a tortoise (Testudo horsfieldi) and a pond turtle (Emys orbicularis) using immunohistochemistry. We have found that different types of cells in the paraventricular and supraoptic nuclei predominantly express calbindin and, to a lesser extent, calretinin, but not parvalbumin. The selective calbindin/calretinin control of the neurohormone secretion in these hypothalamic nuclei is an evolutionary conservative feature typical of reptiles and mammals.     

A novel concentration and viability detection method for <Emphasis Type="Italic">Brettanomyces</Emphasis> using the Cellometer image cytometry

Brettanomyces spp. can present unique cell morphologies comprised of excessive pseudohyphae and budding, leading to difficulties in enumerating cells. The current cell counting methods include manual counting of methylene blue-stained yeasts or measuring optical densities using a spectrophotometer. However, manual counting can be time-consuming and has high operator-dependent variations due to subjectivity. Optical density measurement can also introduce uncertainties where instead of individual cells counted, an average of a cell population is measured. In contrast, by utilizing the fluorescence capability of an image cytometer to detect acridine orange and propidium iodide viability dyes, individual cell nuclei can be counted directly in the pseudohyphae chains, which can improve the accuracy and efficiency of cell counting, as well as eliminating the subjectivity from manual counting. In this work, two experiments were performed to demonstrate the capability of Cellometer image cytometer to monitor Brettanomyces concentrations, viabilities, and budding/pseudohyphae percentages. First, a yeast propagation experiment was conducted to optimize software counting parameters for monitoring the growth of Brettanomyces clausenii, Brettanomyces bruxellensis, and Brettanomyces lambicus, which showed increasing cell concentrations, and varying pseudohyphae percentages. The pseudohyphae formed during propagation were counted either as multiple nuclei or a single multi-nuclei organism, where the results of counting the yeast as a single multi-nuclei organism were directly compared to manual counting. Second, a yeast fermentation experiment was conducted to demonstrate that the proposed image cytometric analysis method can monitor the growth pattern of B. lambicus and B. clausenii during beer fermentation. The results from both experiments displayed different growth patterns, viability, and budding/pseudohyphae percentages for each Brettanomyces species. The proposed Cellometer image cytometry method can improve efficiency and eliminate operator-dependent variations of cell counting compared with the traditional methods, which can potentially improve the quality of beverage products employing Brettanomyces yeasts.     

Ayurvedic <Emphasis Type="Italic">Amalaki Rasayana</Emphasis> promotes improved stress tolerance and thus has anti-aging effects in <Emphasis Type="BoldItalic">Drosophila melanogaster</Emphasis>

Amalaki Rasayana (AR) is a common Ayurvedic herbal formulation of Phyllanthus emblica fruits and some other ingredients, and is used for general good health and healthy aging. We reported it to improve life history traits and to suppress neurodegeneration as well as induced apoptosis in Drosophila. The present study examines responses of Drosophila reared on AR-supplemented food to crowding, thermal or oxidative stresses. Wild-type larvae/flies reared on AR-supplemented food survived the various cell stresses much better than those reared on control food. AR-fed mutant park ¹³ or DJ-1β ^Delta93 (Parkinson’s disease model) larvae/flies, however, showed only partial or no protection, respectively, against paraquat-induced oxidative stress, indicating essentiality of DJ-1β for AR-mediated oxidative stress tolerance. AR feeding reduced the accumulation of reactive oxygen species (ROS) and lipid peroxidation even in aged (35-day-old) wild-type flies while enhancing superoxide dismutase (SOD) activity. We show that while Hsp70 or Hsp83 expression under normal or stress conditions was not affected by AR feeding, Hsp27 levels were elevated in AR-fed wild-type control as well as heat-shocked larvae. Therefore, besides the known anti-oxidant activity of Phyllanthus emblica fruits, dietary AR also enhances cellular levels of Hsp27. Our in vivo study on a model organism shows that AR feeding significantly improves tolerance to a variety of cell stresses through reduced ROS and lipid peroxidation on the one hand, and enhanced SOD activity and Hsp27 on the other. The resulting better homeostasis improves life span and quality of organism’s life.     

Informative Value of Temperatures in Different Areas of the Human Body for Correcting Body Thermal Imbalance during Extravehicular Activities

Identification of noninvasive and informative sites on the body reflecting the development of body thermal imbalance during extravehicular activities (EVAs) is highly important for enhancing astronaut safety. Temperature changes were evaluated on several areas of the head (the mastoid fossa (T _mf ), the forehead (T _fo ), and the cheek (T _ch )) and on the fingers (T _fing ). Subjects were dressed in a multicompartment liquid cooling/warming garment. Studies I and II consisted of different combinations of hood versus garment cooling or warming imposed across stages; studies III and IV involved sagittally divided cooling or warming regimes with the hood worn (study III) or with the head uncovered (study IV). In studies I and II, T _mf significantly (P < 0.05) differed between stages 2, when the head was cooled and the rest of the body heated, and 3, when the head was heated and the rest of the body cooled. The T _mf changes were consistent with the thermal conditions imposed on the head but not reflective of the developing body heat deficit. In study III, the T _mf at stages 2 and 3 on the right or the left followed the thermal conditions on the ipsilateral side of the body (P < 0.01). In study IV, T _fing showed no significant differences across stages. In studies I–IV, T _fing showed consistent changes across stages (P < 0.05), reflecting the developing body heat deficit. In all studies, there were no significant differences in rectal temperature (T _re ) across stages. T _mf and temperatures at other head skin sites did not respond in accordance with the actual intensity of a heat or cold flux from the garment and were not reflective of the overall development of body thermal imbalance. T _fing was a more adequate indicator of initial thermal destabilization and provided information that would be useful for monitoring the thermal balance and comfort during EVAs.     

Morphological analyses and variation in carbohydrate content during the maturation of somatic embryos of <Emphasis Type="Italic">Carica papaya</Emphasis>

Efficient protocols for somatic embryogenesis of papaya (Carica papaya L.) have great potential for selecting elite hybrid genotypes. Addition of polyethylene glycol (PEG), a nonplasmolyzing osmotic agent, to a maturation medium increases the production of somatic embryos in C. papaya. To study the effects of PEG on somatic embryogenesis of C. papaya, we analyzed somatic embryo development and carbohydrate profile changes during maturation treatments with PEG (6%) or without PEG (control). PEG treatment (6%) increased the number of normal mature somatic embryos followed by somatic plantlet production. In both control and PEG treatments, pro-embryogenic differentiation to the cotyledonary stage was observed and was significantly higher with PEG treatment. Histomorphological analysis of embryonic cultures with PEG revealed meristematic centers containing small isodiametric cells with dense cytoplasm and evident nuclei. Concomitant with the increase in the differentiation of somatic embryos in PEG cultures, there was an increase in the endogenous content of sucrose and starch, which appears to be related to a rising demand for energy, a key point in the conversion of C. papaya somatic embryos. The endogenous carbohydrate profile may be a valuable parameter for developing optimized protocols for the maturation of somatic embryos in papaya.     

Investigation of stored wheat mycoflora,reporting the<Emphasis Type="Italic">Fusarium</Emphasis> cf.<Emphasis Type="Italic">langsethiae</Emphasis> in three provinces of Iran during 2007

Wheat is the most important cereal produced in Iran. A mycological survey was carried out for the first time, on the stored wheat samples in Tehran, East Azarbayejan and Mazandaran provinces in 2007. Exogenous and endogenous fungi, were isolated by the method of flotation with Malachite green agar (MGA 0.25) and Freeze blotter techniques respectively. In this study, 46 species belonging to 23 different genera were isolated.Cladosporium spp. (57.1–89.2%) andAlternaria spp. (82.4–100%) species were the predominant fungal species identified as endogenous mycoflora. The predominant exogenous fungi werePenicillium spp. (78.4–92.8%) andAspergillus spp. (71.4–85.7%) species.Fusarium proliferatum was the most prevalent species ofFusarium isolates.Aspergillus niger (39.4%) andAspergillus flavus (36.7%) were the predominantAspergillus species identified as exogenous mycoflora.Aspergillus flavus (26.6%) was the predominantAspergillus species identified as endogenous mycoflora. Flotation method with MGA 0.25 recommended for isolating of hyaline fungi from wheat cereals. In this study one isolate fromFusarium species was isolated on the basis of morphology and ribosomal internal transcribed spacer classified asFusarium langsethiae but on the basis of partial translation elongation factor-1alpha gene grouped withFusarium sporotrichioides. To our knowledge, this is the first report aboutF. cf.langsethiae in Iran and Asia.     

Molecular identification of Kiss/GPR54 and function analysis with mRNA expression profiles exposure to 17α-ethinylestradiol in rare minnow <Emphasis Type="Italic">Gobiocypris rarus</Emphasis>

17α-ethinylestradiol (EE2) is a widely existed endocrine disrupting chemical in water environment. Kisspeptins act as indispensable regulators through GPR54 in the hypothalamic–pituitary–gonadal (HPG) axis. This study aimed to provide further understanding of the effect of EE2 on HPG axis. Molecular cloning and tissue distribution of kiss genes and GPR54s were performed in Gobiocypris rarus. The mRNA expression profiles of kiss1, kiss2, GPR54s and GnRHs were detected in G. rarus brain and/or gonad following 3- and 6-days EE2 (1, 5, 25 and 125 ng/L) exposure. Results showed that kiss genes and GPR54s were highly expressed in brain and gonad. Both kiss1 and kiss2 were increased in female brain and suppressed in male brain following EE2 exposure. GnRHs were inhibited in a concentration-dependent manner in male brain following 3-days EE2 exposure. In gonad, GPR54b was almost suppressed in all of EE2 concentrations. The present findings suggest that EE2 impacts the genes expression of Kiss/GPR54-GnRH system in G. rarus, thereby probably disturbing the neuroendocrine homeostasis.     

Planar aromaticity of <Emphasis Type="Italic">D</Emphasis><Subscript><Emphasis Type="Italic">N</Emphasis><Emphasis Type="Italic">h</Emphasis></Subscript>-symmetrical systems as a perturbed two-dimensional (2D) rigid rotor

Energy levels for D _{N h} -symmetrical cyclic polyenes with planar aromaticity were obtained using two-dimensional (2D) rigid rotor as the zeroth approximation. The addition of nuclei was simulated by the cosine-type potential and treated at the first-order perturbation theory. The result is qualitatively equivalent to that obtained using Hückel’s molecular orbital theory. As an addition, the energetic shift between σ and π orbitals is obtained using the model of electron oscillating around the center of a positively charged ring.     

Thermal Preference of the Bush Cricket <Emphasis Type="Italic">Isophya rizeensis</Emphasis>; Testing the Effect of Countergradient Selection

Thermal preference is one of the most crucial components of behavioral thermoregulation in ectotherms, and documenting the adaptation of thermal preference carries great importance for studying the evolution of thermal biology. However there are not many studies focusing on the adaptation of thermal preference in elevational and latitudinal gradients. Isophya rizeensis is a color polymorphic bush cricket species endemic to the mountainous region of northeastern Turkey. Populations of this species are distributed in a wide elevational range between 350 and 2300 m. In this study, we hypothesized that the thermal preference of Isophya rizeensis might follow a countergradient variation where crickets from higher altitudes have higher temperature preferences compared to crickets from lower altitudes. To test this hypothesis, thermal preference values (T _pref ) of crickets from three altitudes groups (low, middle and high) were measured with a thermal gradient experiment. Additionally, body temperatures (T _b ) and environmental temperatures (T _a ) were measured in field. Deviation values of T _b and T _a from T _pref were calculated to investigate the extent of thermoregulation. As Isophya rizeensis is color polymorphic species where morphology pattern changes from lighter to darker types with increasing altitude we also tested whether coloration has any effect on temperature excess (T _ex ) and thermoregulation. Thermal preference values did not differ significantly between three groups and also colouration does not influence the extent of thermoregulation in this species. These results indicate that there is not sufficient evidence for the existence of a countergradient selection related with thermal behavior. However, the deviation of body (D _b ) and environmental (D _a ) temperatures suggest that at higher altitudes thermoregulation might be more efficient than lower altitudes.     

Dragonflies (Odonata) of thermal springs in Central Asia

Sixteen species of dragonflies were recorded in 12 thermal springs of Central Asia. Of these, Ischnura forcipata, I. pumilio, Orthetrum anceps, O. brunneum, and Sympetrum haritonovi were widely distributed in hydrothermal waters. In the high mountains of Pamir, dragonflies can develop only in hot springs. The maximum altitude recorded for the habitat of O. brunneum was 3950 m a.s.l.     

Phylogenetic in situ/ex situ analysis of a sulfur mat microbial community from a thermal sulfide spring in the north Caucasus

A phylogenetic in situ/ex situ analysis of a sulfur mat formed by colorless filamentous sulfur bacteria in a thermal sulfide spring (northern spur of the main Caucasian ridge) was carried out. Nine phylotypes were revealed in the mat. Thiothrix sp. and Sphaerotilus sp. were the dominant phylotypes (66.3% and 26.3%, respectively). The 16S rRNA gene nucleotide sequence of Sphaerotilus sp. phylotype from the clone library was identical to the sequences of the seven Sphaerotilus strains isolated from the same source. A very high degree of similarity of Sphaerotilus strains revealed by ERIC-PCR fingerprints indicated little or no population diversity of this species in the mat. Thiothrix phylotype from the clone library and two Thiothrix strains isolated from the same mat sample differed in one to three nucleotides of 16S rRNA genes; this is an indication of this organism’s population variability in the mat. 16S rRNA genes of the strains and clones of Thiothrix sp. exhibited the highest similarity (ca. 99%) with Thiothrix unzii; the strains and clones of Sphaerotilus had 99% similarity with the type species Sphaerotilus natans (the only species of this genus) and therefore can be assigned to this species. The minor seven components belong to the phylotypes from the Proteobacteria (3%), as well as the Chlorobia, Cyanobacteria, Clostridia, and Bacteroidetes phylogenetic groups, each of them constituting not more than 1%. Intracellular accumulation of elemental sulfur by Sphaerotilus similar to other filamentous sulfur bacteria was demonstrated for the first time (both in the population of the sulfur spring and in cultures with sulfide). Although mass growth of Sphaerotilus and Thiothrix is typical of bacterial populations of anthropogenic ecosystems (the activated sludge of treatment facilities), stable communities of these bacteria have not been previously found in the sulfur mats or “threads” of natural sulfide springs.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of oleaginous yeast <Emphasis Type="Italic">Lipomyces</Emphasis> species

Interest in using renewable sources of carbon, especially lignocellulosic biomass, for the production of hydrocarbon fuels and chemicals has fueled interest in exploring various organisms capable of producing hydrocarbon biofuels and chemicals or their precursors. The oleaginous (oil-producing) yeast Lipomyces starkeyi is the subject of active research regarding the production of triacylglycerides as hydrocarbon fuel precursors using a variety of carbohydrate and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements through the development and use of the tools of synthetic biology for this oleaginous species. The first step in establishment of synthetic biology tools for an organism is the development of effective and reliable transformation methods with suitable selectable marker genes and demonstration of the utility of the genetic elements needed for expression of introduced genes or deletion of endogenous genes. Chemical-based methods of transformation have been published but suffer from low efficiency. To address these problems, Agrobacterium-mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. In this study, Agrobacterium-mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species. The deletion of the peroxisomal biogenesis factor 10 gene was also demonstrated in L. starkeyi. In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial β-glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1α promoter was also stably expressed in six different Lipomyces species. The results from this study demonstrate that Agrobacterium-mediated transformation is a reliable and effective genetic tool for homologous recombination and expression of heterologous genes in L. starkeyi and other Lipomyces species.     

Modeling the effects of extracellular potassium on bursting properties in pre-Bötzinger complex neurons

There are many types of neurons that intrinsically generate rhythmic bursting activity, even when isolated, and these neurons underlie several specific motor behaviors. Rhythmic neurons that drive the inspiratory phase of respiration are located in the medullary pre-Bötzinger Complex (pre-BötC). However, it is not known if their rhythmic bursting is the result of intrinsic mechanisms or synaptic interactions. In many cases, for bursting to occur, the excitability of these neurons needs to be elevated. This excitation is provided in vitro (e.g. in slices), by increasing extracellular potassium concentration (K _out ) well beyond physiologic levels. Elevated K _out shifts the reversal potentials for all potassium currents including the potassium component of leakage to higher values. However, how an increase in K _out , and the resultant changes in potassium currents, induce bursting activity, have yet to be established. Moreover, it is not known if the endogenous bursting induced in vitro is representative of neural behavior in vivo. Our modeling study examines the interplay between K _out , excitability, and selected currents, as they relate to endogenous rhythmic bursting. Starting with a Hodgkin-Huxley formalization of a pre-BötC neuron, a potassium ion component was incorporated into the leakage current, and model behaviors were investigated at varying concentrations of K _out . Our simulations show that endogenous bursting activity, evoked in vitro by elevation of K _out , is the result of a specific relationship between the leakage and voltage-dependent, delayed rectifier potassium currents, which may not be observed at physiological levels of extracellular potassium.     

Identification of extremely halophilic archaea associated with adult <Emphasis Type="Italic">Artemia urmiana</Emphasis>

The brine shrimp, Artemia is the dominant macrozooplankton present in many hypersaline environments. Artemia urmiana is the only macroscopic organism in Urmia Salt Lake (Iran), and the high salinity of the lake makes it a suitable environment for halophilic archaea too. Because of common environment for Artemia and extreme halophiles; this investigation is concentrated on studying the relationship between Artemia and halophilic archaea in Urmia Lake. In this study first the procedure of arhaea isolation was done. Then, isolated strains were sub-cultured and DNA was extracted and amplified by PCR using specific primers for amplifying archaeal 16S rRNA. The amplified archeal DNA fragments were purified, and sequenced. 16S rRNA sequences were compared to known sequences using the NCBI BLAST program. Sequences relating to Halorubrum, Haloarcula and Halobacterium species were identified in Urmia Salt Lake water and Artemia adults and the phylogenetic tree of different species was constructed. Only Halorubrum species were present in association with Artemia. They belong to Halobacteriaceae family of archeae which are isolated from different salt lakes in different parts of world and we could show their existence in adult Artemia, another organism living in hypersaline enviroments.