Space flight experiments with nonterrestrial minerals and bioorganic molecules

Meteorites probably played a central role in the evolution of life. Due to the structure, they tend to adsorb organic compounds and catalyze a variety of organic reactions critical to scenarios of life’s origins. We have shown experimentally that extraterrestrial minerals can catalyze the formation of peptides and nucleotides, thus, lending support to the RNA world hypothesis. This theory proposes that life based on RNA preceded the modern life, which is based on DNA and proteins. The present study was performed onboard different Russian space stations (Bion, Cosmos, and Mir) with various duration, altitude, and radiation conditions. Irradiation of solid samples, free or admixed with certain minerals, is the major task of future space flight experiments, planned for performing onboard Russian space satellite Bion-10M. Radiation behavior of bioorganic substances (BOS) on the Earth’s orbit could have truly applied significance (formation of uridine dimers).     

Life history focus on a malaria parasite: linked traits and variation among genetic clones

Life history theory has long been a major campaign in evolutionary ecology, but has typically focused only on animals and plants. Life history research on single-celled eukaryotic protists such as malaria parasites (Plasmodium) will offer new insights into the theory’s general utility as well as the parasite’s basic biology. For example, parasitologists have described the Plasmodium life cycle and cell types in exquisite detail, with little discussion of evolutionary issues such as developmental links between traits. We measured 10 life history traits of replicate single-genotype experimental Plasmodium mexicanum infections in its natural lizard host to identify groups of linked traits. These 10 traits formed 4 trait groups: “Rate/Peak” merges measures of growth rate and maximum parasitemia of infections; “Timing” combines time to patency and maximum parasitemia; “Growth Shape” describes the fit to an exponential growth curve; and “Sex Ratio” includes only the gametocyte sex ratio. Parasite genotype (clone) showed no effect on the life history trait groups, with the exception of gametocyte sex ratio. Therefore, variation in most life history traits among infections appears to be driven by environmental (individual host) effects. The findings support the model that life history traits are often linked by developmental constraints. Understanding why life history traits of Plasmodium are linked in this way would offer a new window into the evolution of the parasites, and also should inform public health efforts to control infection prevalence.     

The role of the cortex in indentation experiments of animal cells

We present a model useful for interpretation of indentation experiments on animal cells. We use finite element modeling for a thorough representation of the complex structure of an animal cell. In our model, the crucial constituent is the cell cortex—a rigid layer of cytoplasmic proteins present on the inner side of the cell membrane. It plays a vital role in the mechanical interactions between cells. The cell cortex is modeled by a three-dimensional solid to reflect its bending stiffness. This approach allows us to interpret the results of the indentation measurements and extract the mechanical properties of the individual elements of the cell structure. During the simulations, we scan a broad range of parameters such as cortex thickness and Young’s modulus, cytoplasm Young’s modulus, and indenter radius, which define cell properties and experimental conditions. Finally, we propose a simple closed-form formula that approximates the simulated results with satisfactory accuracy. Our formula is as easy to use as Hertz's function to extract cell properties from the measurement, yet it considers the cell’s inner structure, including cell cortex, cytoplasm, and nucleus.

     

Nitrogen deprivation of microalgae: effect on cell size,cell wall thickness,cell strength,and resistance to mechanical disruption

Nitrogen deprivation (N-deprivation) is a proven strategy for inducing triacylglyceride accumulation in microalgae. However, its effect on the physical properties of cells and subsequently on product recovery processes is relatively unknown. In this study, the effect of N-deprivation on the cell size, cell wall thickness, and mechanical strength of three microalgae was investigated. As determined by analysis of micrographs from transmission electron microscopy, the average cell size and cell wall thickness for N-deprived Nannochloropsis sp. and Chlorococcum sp. were ca. 25% greater than the N-replete cells, and 20 and 70% greater, respectively, for N-deprived Chlorella sp. The average Young’s modulus of N-deprived Chlorococcum sp. cells was estimated using atomic force microscopy to be 775 kPa; 30% greater than the N-replete population. Although statistically significant, these microstructural changes did not appear to affect the overall susceptibility of cells to mechanical rupture by high pressure homogenisation. This is important as it suggests that subjecting these microalgae to nitrogen starvation to accumulate lipids does not adversely affect the recovery of intracellular lipids.     

A membrane model from implicit elasticity theory: application to visceral pleura

A Fungean solid is derived for membranous materials as a body defined by isotropic response functions whose mathematical structure is that of a Hookean solid where the elastic constants are replaced by functions of state derived from an implicit, thermodynamic, internal energy function. The theory utilizes Biot’s (Lond Edinb Dublin Philos Mag J Sci 27:468–489, 1939) definitions for stress and strain that, in one-dimension, are the stress/strain measures adopted by Fung (Am J Physiol 28:1532–1544, 1967) when he postulated what is now known as Fung’s law. Our Fungean membrane model is parameterized against a biaxial data set acquired from a porcine pleural membrane subjected to three, sequential, proportional, planar extensions. These data support an isotropic/deviatoric split in the stress and strain-rate hypothesized by our theory. These data also demonstrate that the material response is highly nonlinear but, otherwise, mechanically isotropic. These data are described reasonably well by our otherwise simple, four-parameter, material model.     

Direct measurement of the melting temperature of supported DNA by electrochemical method

The development of biosensors based on DNA hybridization requires a more precise knowledge of the thermodynamics of the hybridization at a solid interface. In particular, the selectivity of hybridization can be affected by a lot of parameters such as the single-strand (ss)DNA density, the pH, the ionic strength or the temperature. The melting temperature, T_m, is in part a function of the ionic strength and of the temperature and therefore provides a useful variable in the control of the selectivity and sensitivity of a DNA chip. The electrochemical technique has been used to determine the T_m values when the probe is tethered by a DNA self-assembled monolayer (SAM). We have built a special thin layer cell, which allows the recording of the cyclic voltammogram under controlled temperature conditions. T_m has been determined by recording the thermogram (current versus temperature) of a redox indicator on a double-stranded hybrid (dsDNA) modified electrode and comparison with the corresponding ssDNA response. T_m of supported DNA varies linearly with the ionic strength. The stability of the SAMs has been considered and comparison between T_m in solution and on a solid support has been discussed.     

Thermal tolerance of Cyrtobagous salviniae: a biocontrol agent in a changing world

The weevil Cyrtobagous salviniae Calder & Sands 1985 (Family: Curculionidae) is a highly effective biocontrol agent for the invasive water fern Salvinia molesta D.S. Mitchell (Family: Salviniaceae). The life histories of both organisms are affected by temperature, making the potential impacts of climate change on efficacy of control an important area in which comprehensive understanding is required. Here we use warming tolerance (WT) and low temperature tolerance (LTT) as measures of C. salviniae’s sensitivity to climate warming, calculated across South Africa using critical thermal limits, lethal temperature limits and mean maximum (Tmax) and minimum (Tmin) environmental temperatures under present climatic conditions and two future periods (2040s and 2080s). From the present climate to the 2080s the WTs of C. salviniae decrease and LTTs increase indicating C. salviniae may face greater constraints on survival as Tmax nears the upper thermal limits, but increased population persistence over cool months as Tmin increases.     

Synthesis, characterization, X-ray crystallography, and cytotoxicity of a cymantrene keto carboxylic acid for IR labelling of bioactive peptides on a solid support

Cym-CO-CH₂-CH₂-COOH was prepared in good yield by Friedel-Crafts reaction of cymantrene (Cym, CpMn(CO)₃) with succinic anhydride for the IR labelling of peptides and fully characterized, including an X-ray structure analysis (monoclinic space group P2(1)/n, a = 5.727(3) Å, b = 19.865(9) Å, c = 10.518(5) Å, β = 91.211(9)°). The compound was isolated in pure form without the need for chromatographic work-up and subsequently used for solution-phase synthesis of a bioconjugate with phenylalanine methyl ester to allow a complete spectroscopic characterization of this model system. The cymantrene keto carboxylic acid also turned out to be a very robust marker in automated microwave-assisted solid phase peptide synthesis (SPPS). [Leu⁵]-enkephalin (Tyr-Gly-Gly-Phe-Leu) was prepared on a Wang resin and labelled with the cymantrene derivative on the solid support under microwave irradiation in all steps. The metal-carbonyl marker stayed intact during cleavage from the resin with concentrated trifluoroacetic acid. After simple precipitation and lyophilization, the cymantrene-enkephalin bioconjugate could be obtained in analytically pure form without the need of HPLC purification. As required, the compound is non-cytotoxic against MCF-7 cells at up to 100 μM. This protocol thus allows one to introduce organometallic IR spectroscopic labels to peptides in a very straightforward way.     

Comparative study of the effects of salinity and UV radiation on metabolism and morphology of the red macroalga <Emphasis Type="Italic">Acanthophora spicifera</Emphasis> (Rhodophyta,Ceramiales)

Increase of harmful radiation to the Earth’s surface due to ozone depletion results in higher exposure to harmful ultraviolet- B radiation (UV), while fluctuations in seawater salinity may alter water density, ionic concentration, nutrient uptake, and osmotic pressure. This study evaluated the effects of salinity and UV on metabolism and morphology of Acanthophora spicifera (M.Vahl) Børgesen. Water with 30 and 37 psu [g(salt) kg^–1(sea water)] was used for experiments during 7 d of exposure to UV (3 h per day). We demonstrated that UV treatment predisposed, irrespective of salinity, A. spicifera to a decrease in its growth rate and cell viability, as well as affected its morphological parameters. After exposure to PAR + UVA + UVB (PAB), samples showed structural changes and damage, such as increasing cell wall thickness and chloroplast disruption. Our results indicate that UV led to dramatic metabolic changes and cellular imbalances, but more remarkable changes were seen in samples exposed to high salinity.     

Sharing a joke: The effects of a similar sense of humor on affiliation and altruism

Cooperation requires that individuals are able to identify, and preferentially associate with, others who have compatible preferences and the shared background knowledge needed to solve interpersonal coordination problems. This body of shared knowledge constitute a substantial proportion of what is called ‘culture’. It has been argued that, for this reason, individuals prefer to associate with others who share their culture, and also that shared appreciation of humor provides a particularly effective means of identifying others with the relevant preferences and knowledge. The present experiment uses a ‘dummy rating procedure’ to compare the effects of sharing an appreciation of non-humorous (first lines of novels) and humorous (jokes) cultural stimuli on interpersonal affiliation, altruism and assessment. The results show that the degree of shared appreciation for both sets of stimuli had a positive effect on Affiliation; only humorous stimuli had an effect on Altruism; and neither effected the Assessment of others' personal traits. Thus, the results support the general theory that shared culture promotes affiliation, and provide evidence of the special role of humor in interpersonal relations.     

On a naturalist theory of health: a critique

This paper examines the most influential naturalist theory of health, Christopher Boorse’s ‘biostatistical theory’ (BST). I argue that the BST is an unsuitable candidate for the rôle that Boorse has cast it to play, namely, to underpin medicine with a theoretical, value-free science of health and disease. Following the literature, I distinguish between “real” changes and “mere Cambridge changes” in terms of the difference between an individual’s intrinsic and relational properties and argue that the framework of the BST essentially implies a Cambridge-change criterion. The examination reveals that this implicit criterion commits the BST to the troubling view that an individual could go from being diseased to healthy, or vice versa, without any physiological change in that individual. Two problems follow: (1) the current framework of the BST is ill-equipped to formally embrace Cambridge changes and (2) it is theoretically dubious. The arguments advanced here are not limited to the BST; I suggest they extend to any naturalist claim to underpin medical practice with a value-free theory of health and disease defined in terms of an evolutionary view of biological fitness.     

Food selection and predation risk in the Andean white-eared opossum (Didelphis pernigra Allen, 1900) in a suburban area of Bogotá, Colombia

Urbanization is spreading throughout the Andes, and despite it there is still wildlife which survives in these novel environments throughout Colombia and elsewhere. The Andean white-eared opossum (Didelphis pernigra Allen, 1900) can tolerate moderate levels of urbanization in the Andes highlands, and we evaluated its food selection based on optimal patch use theory using foraging stations to measure giving-up densities in a suburban area in Bogotá (∼2600 m a.s.l.). We offered the opossums equicaloric mixtures of protein-rich and sucrose-rich foods, and they preferred the sucrose mixture. However, the magnitude of this preference was influenced by temporal and spatial effects. We suggest that in environments such as the high Andes, where air temperature regularly drops below 10 °C, the opossums prefer foods that provide energy easily. We also assessed the opossums’ perceived risk of predation when domestic dogs (Canis lupus familiaris) are present by sprinkling dog urine in cotton swabs next to foraging stations; we expected opossums to forage more when the predator’s signal was not present. Dog urine indicated a risk for opossum’s at only one of six foraging stations, suggesting a weak predator’s signal effect that depends on location. Both food preferences and perception of risk by the opossum were affected by the spatial heterogeneity of the urban environment. Thus, microhabitat management at the study site, which is part of a recently created reserve to preserve Bogotá’s natural capital, appears to be key to conserve the opossums and their ecological functions in highly disturbed areas of the Andes.     

The role of Apodemus mice and Microtus voles in the diet of the Tengmalm’s owl in Central Europe

Based on a long-term dataset (1999–2010), we investigated how the availability of main prey affects the breeding density and food ecology of the Tengmalm’s owl (Aegolius funereus) in the Czech Republic. In particular, we assessed the role of Microtus voles and Apodemus mice in the diet, based on the main predictions of the optimal diet theory that the diet composition depends on the availability of the main prey. We found that (i) the Tengmalm’s owl exhibited no numerical response to the availability of Microtus voles and Apodemus mice in the field; (ii) the availability of Apodemus mice in the field positively affected their proportion in the diet (26 %), and despite a high proportion of Microtus voles in the owls’ diet (47 %), no relationship was found between their availability in the field and proportion in the diet; (iii) the proportion of Apodemus mice was negatively correlated to the proportion of Microtus voles, Sorex shrews and birds in the diet, but no similar relationship was detected for Microtus voles; (iv) the reproductive output of Tengmalm’s owls was positively correlated to the proportion of Apodemus mice in the diet, as well as to Apodemus mice and Microtus vole availability in the field; and (v) diet diversity and diet overlap were not significantly affected by the abundance of Apodemus mice and Microtus voles. Therefore, the validity of these main optimal diet theory predictions was not confirmed, especially for Microtus vole prey, due to an opportunistic choice between Apodemus mice and Microtus voles. We suggest that the reproductive output of nocturnal raptors in Central Europe may be less dependent on Microtus vole supply than that of their northern counterparts.     

Physiological mechanisms of frost tolerance: Possible role of protein in plant adaptation to cold

Studies performed on winter rape plants(Brassica nnpus var.oleifera, cv. ‘Gór-czański’) revealed that cold treatment affected the cell membranes and led to the temporary increase in electrolytic leakage from a tissue. This was followed by the marked decrease of the electrolytic leakage in the course of hardening. Changes in membrane properties were accompanied by the promotion of soluble protein accumulation. Inhibition of protein accumulation by the cycloheximide treatment brought about wilting of plants under cold conditions. Possible role of soluble protein in protection of cells against secondary water stress caused by the coldinduced changes in membrane properties is suggested. Cold-induced changes in the electrophoretic pattern of soluble protein are described and discussed.     

Towards a better understanding of Lactobacillus rhamnosus GG - host interactions

Background

An industrial approach to protein production demands maximization of cloned gene expression, balanced with the recombinant host’s viability. Expression of toxic genes from thermophiles poses particular difficulties due to high GC content, mRNA secondary structures, rare codon usage and impairing the host’s coding plasmid replication. TaqII belongs to a family of bifunctional enzymes, which are a fusion of the restriction endonuclease (REase) and methyltransferase (MTase) activities in a single polypeptide. The family contains thermostable REases with distinct specificities: TspGWI, TaqII, Tth111II/TthHB27I, TspDTI and TsoI and a few enzymes found in mesophiles. While not being isoschizomers, the enzymes exhibit amino acid (aa) sequence homologies, having molecular sizes of ~120 kDa share common modular architecture, resemble Type-I enzymes, cleave DNA 11/9 nt from the recognition sites, their activity is affected by S-adenosylmethionine (SAM).

Results

We describe the taqIIRM gene design, cloning and expression of the prototype TaqII. The enzyme amount in natural hosts is extremely low. To improve expression of the taqIIRM gene in Escherichia coli (E. coli), we designed and cloned a fully synthetic, low GC content, low mRNA secondary structure taqIIRM, codon-optimized gene under a bacteriophage lambda (λ) P _R promoter. Codon usage based on a modified ‘one amino acid–one codon’ strategy, weighted towards low GC content codons, resulted in approximately 10-fold higher expression of the synthetic gene. 718 codons of total 1105 were changed, comprising 65% of the taqIIRM gene. The reason for we choose a less effective strategy rather than a resulting in high expression yields ‘codon randomization’ strategy, was intentional, sub-optimal TaqII in vivo production, in order to decrease the high ‘toxicity’ of the REase-MTase protein.

Conclusions

Recombinant wt and synthetic taqIIRM gene were cloned and expressed in E. coli. The modified ‘one amino acid–one codon’ method tuned for thermophile-coded genes was applied to obtain overexpression of the ‘toxic’ taqIIRM gene. The method appears suited for industrial production of thermostable ‘toxic’ enzymes in E. coli. This novel variant of the method biased toward increasing a gene’s AT content may provide economic benefits for industrial applications.     

Fine-scale distributions of carnivores in a logging concession in Sarawak,Malaysian Borneo

Coarse-scale patterns of distribution and abundance of species are the outcome of processes occurring at finer spatial scales, hence the conservation of species depends on understanding their fine-scale ecology. For Bornean carnivores, little is known about fine-scale predictors of species occurrence and it is assumed that the two main threats to wildlife on Borneo, habitat disturbance and hunting, also impact their occurrence. To increase our understanding of the Borneo carnivore community, we deployed 60 cameras in a logging concession in northern Sarawak, Malaysian Borneo, where different landscape covariates, both natural and anthropogenic, were present. We built single-species occupancy models to investigate fine-scale carnivore occupancy. Overall, forest disturbance had a negative effect on Hose’s civet (Diplogale hosei), banded civet (Hemigalus derbyanus) and yellow-throated marten (Martes flavigula). Further, banded civet had greater occupancy probabilities in more remote areas. Logging roads had the most diverse effect on carnivore occupancy, with Hose’s civet and masked palm civet (Paguma larvata) negatively affected by roads, whereas Malay civet (Viverra tangalunga), short-tailed mongoose (Herpestes brachyurus) and leopard cat (Prionailurus bengalensis) showed higher occupancy closer to roads. Canopy height, canopy closure, number of trees with holes (cavities) and distance to nearest village also affected occupancy, though the directions of these effects varied among species. Our results highlight the need to collect often overlooked habitat variables: moss cover and ‘kerangas’ (tropical heath forest) were the most important variables predicting occurrence of Hose’s civet. The preservation of such forest conditions may be crucial for the long-term conservation of this little-known species. Our results confirm that logged forests, when left to regenerate, can host diverse carnivore communities on Borneo, provided less disturbed habitat is available nearby, though human access needs to be controlled. We recommend easy-to-implement forest management strategies including maintaining forest next to logging roads; preserving fruiting trees and trees with cavities, both standing and fallen; and blocks of remote, less disturbed, mid- to high-elevation forest with understorey vegetation.     

Continuous production of acetic acid using immobilized acetobacter aceti in a three-phase fluidized bed bioreactor

Continuous operation of a three-phase fluidized bed bioreactor using immobilized cells showed that both immobilized and suspended cells contributed to the production of acetic acid. Unlike the rapid decrease in the productivity at dilution rates above 0.25 h⁻¹ in the free cell fermentation, the productivity was little affected by the dilution rate in the immobilized cell fermentation. Theoretical models were proposed for the continuous process. The models approximately agreed with the experimental results. Experimental results and/or theoretical calculations based on the kinetic models showed that suspended cells were important in the production of acetic acid if the solid holdup was small or if gel radius was large. Theoretical calculations showed that an optimal solid holdup or gel size existed at higher dilution rates because of the k_La dependence on solid holdup and particle diameter.     

Optogenetic delivery of trophic signals in a genetic model of Parkinson’s disease

Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1^B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson’s disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair.     

Co-Encapsulation of Doxorubicin With Galactoxyloglucan Nanoparticles for Intracellular Tumor-Targeted Delivery in Murine Ascites and Solid Tumors

Doxorubicin (Dox) treatment is limited by severe toxicity and frequent episodes of treatment failure. To minimize adverse events and improve drug delivery efficiently and specifically in cancer cells, encapsulation of Dox with naturally obtained galactoxyloglucan polysaccharide (PST001), isolated from Tamarindus indica was attempted. Thus formed PST-Dox nanoparticles induced apoptosis and exhibited significant cytotoxicity in murine ascites cell lines, Dalton’s lymphoma ascites and Ehrlich’s ascites carcinoma. The mechanism contributing to the augmented cytotoxicity of nanoconjugates at lower doses was validated by measuring the Dox intracellular uptake in human colon, leukemic and breast cancer cell lines. PST-Dox nanoparticles showed rapid internalization of Dox into cancer cells within a short period of incubation. Further, in vivo efficacy was tested in comparison to the parent counterparts - PST001 and Dox, in ascites and solid tumor syngraft mice models. Treatment of ascites tumors with PST-Dox nanoparticles significantly reduced the tumor volume, viable tumor cell count, and increased survival and percentage life span in the early, established and prophylactic phases of the disease. Administration of nanoparticles through intratumoral route delivered more robust antitumor response than the intraperitoneal route in solid malignancies. Thus, the results indicate that PST-Dox nanoparticles have greater potential compared to the Dox as targeted drug delivery nanocarriers for loco regional cancer chemotherapy applications.     

A Behavioural Ecology Approach to Assessing the Effect of Alien Vegetation on a Threatened Giant Millipede

The importance of taking into account behavioural ecology when assessing the impacts of habitat deterioration on threatened species is evaluated. The Seychelles giant millipede (SGM), Sechelleptus seychellarum, is IUCN Red-Listed as “Vulnerable”. Here, we studied the effect of alien bamboo and coconut on the behavioural ecology of this species on Cousine Island, Seychelles. Coconut did not affect SGM density, but negatively affected its foraging behaviour, whereas bamboo negatively affected both its density and foraging behaviour. The SGM showed feeding preferences for Pisonia grandis and Ficus sp. leaf litter types. Alien bamboo and coconut pose a varied threat to the SGM, and their removal and replacement by indigenous forest species (e.g. P. grandis and Ficus sp.) should form part of an island’s restoration programme.