The mutational dynamics of the SARS-CoV-2 virus in serial passages in vitro

Since its outbreak in 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) keeps surprising the medical community by evolving diverse immune escape mutations in a rapid and effective manner. To gain deeper insight into mutation frequency and dynamics, we isolated ten ancestral strains of SARS-CoV-2 and performed consecutive serial incubation in ten replications in a suitable and common cell line and subsequently analysed them using RT-qPCR and whole genome sequencing. Along those lines we hoped to gain fundamental insights into the evolutionary capacity of SARS-CoV-2 in vitro. Our results identified a series of adaptive genetic changes, ranging from unique convergent substitutional mutations and hitherto undescribed insertions. The region coding for spike proved to be a mutational hotspot, evolving a number of mutational changes including the already known substitutions at positions S:484 and S:501. We discussed the evolution of all specific adaptations as well as possible reasons for the seemingly inhomogeneous potential of SARS-CoV-2 in the adaptation to cell culture. The combination of serial passage in vitro with whole genome sequencing uncovers the immense mutational potential of some SARS-CoV-2 strains. The observed genetic changes of SARS-CoV-2 in vitro could not be explained solely by selectively neutral mutations but possibly resulted from the action of directional selection accumulating favourable genetic changes in the evolving variants, along the path of increasing potency of the strain. Competition among a high number of quasi-species in the SARS-CoV-2 in vitro population gene pool may reinforce directional selection and boost the speed of evolutionary change.     

Piecing together the transport pathway of aliphatic glucosinolates

Glucosinolates are sulfur-rich secondary metabolites characteristic of the Brassicales order. Transport of glucosinolates was suggested more than 30 years ago through a number of studies which indicated that glucosinolates are produced in maternal tissue and subsequently transported to the seed. These observations laid the foundation for numerous studies on glucosinolate transport which have provided a wealth of information on biochemical properties of glucosinolate transport, source–sink relationships between organs and on the transport routes of glucosinolates. However, most of the conclusions and hypotheses proposed in these studies have not been discussed in context of each other to provide a complete overview of the current state of knowledge on glucosinolate transport. In this review, we are thus piecing together the glucosinolate pathway by presenting and critically analyzing all data on glucosinolate research. Furthermore, the data on glucosinolate transport is considered in the light of the newest findings on glucosinolate synthesis and distribution. The aim is to provide a comprehensive and updated set of hypotheses which may prove useful in directing future research on glucosinolate transport.     

Nerve growth factor: Cellular localization and regulation of synthesis

1. The role of nerve growth factor (NGF) as a retrograde messenger between peripheral target tissues and innervating sympathetic and neural crest-derived sensory neurons is supported by the observations that (a) the interruption of retrograde axonal transport has the same effects as the neutralization of endogenous NGF by anti-NGF antibodies and (b) the close correlation between the density of innervation by fibers of NGF-responsive neurons and the levels of NGF and mRNANGF in their target organs. 2. In situ hybridization experiments have demonstrated that a great variety of cells in the projection field or NGF-responsive neurons is synthesizing NGF, among them epithelial cells, smooth muscle cells, fibroblasts, and Schwann cells. 3. The temporal correlation between the growth of trigeminal sensory fibers into the whisker pad of the mouse and the commencement of NGF synthesis initially suggested a causal relationship between these two events. However, in chick embryos rendered aneural by prior removal of the neural tube or the neural crest, it was shown that the onset of NGF synthesis in the periphery is independent of neurons, and is controlled by an endogenous "clock" whose regulatory mechanism remains to be established. 4. A comparison between NGF synthesis in the nonneuronal cells of the newborn rat sciatic nerve and that in the adult sciatic nerve after lesion provided evidence for the important regulatory role played by a secretory product of activated macrophages. The identity of this product is currently under investigation.     

Detection of S-Phase Cells in Smear Cytology Using in Vitro Bromodeoxyuridine Labeling

A technique Is described for rapid detection of S-pha?e cells of tumor tissues in smear specimens using bromodeoxyuridine (BrdU) immunostaining. Mouse NR-S1 tumors and human tumor specimens were prepared for smear cytology after incubation in RPMI 1640 culture medium containing 200 μM BrdU at 37 °C under 3 atm for 1 hr. Samples were fixed in 70% ethanol for 30 min and used immediately or air dried for 30 min. Samples were then denatured in either 4 N HC1 or 0.07 N NaOH to prepare partially single-stranded DMA. Fixation with air drying for 30 min followed by 30 min in 70% ethanol and 1 min denaturation with 0.07 N NaOH resulted in satisfactory staining quality. Cultured tumor specimens were processed for routine paraffin sections after smears were made for cytology. The labeling indices of the smear specimens and of the paraffin sections gave similar results. This technique should be useful in evaluating the cell proliferative potential of tumor tissue in smear cytology without processing paraffin sections.     

Function of Pax1 and Pax9 in the sclerotome of medaka fish

We examined the expression and functions of Pax1 and Pax9 in a teleost fish, the medaka Oryzias latipes. While Pax1 and Pax9 show distinct expression in the sclerotome in amniotes, we could not detect the differential expression of Pax1 and Pax9 in the developing sclerotome of the medaka. Furthermore, unlike the mouse, in which Pax1 is essential for development of the vertebral body, and where the neural arch is formed independent of either Pax1 or Pax9, our morpholino knockdown experiments revealed that both Pax1 and Pax9 are indispensable for the development of the vertebral body and neural arch. Therefore, we conclude that after gene duplication, Pax1 and Pax9 subfunctionalize their roles in the sclerotome independently in teleosts and amniotes. In Stage-30 embryo, Pax9 was strongly expressed in the posterior mesoderm, as was also observed for mouse Pax9. Since this expression was not detected for Pax1 in the mouse or fish, this new expression in the posterior mesoderm likely evolved in Pax9 of ancestral vertebrates after gene duplication. Two-month-old fish injected with Pax9 morpholino oligonucleotide showed abnormal morphology in the tail hypural skeletal element, which may have been related to this expression.     

柞树体内营养元素的垂直分布

本文对柞树的皮、干、根及树冠各层的营养元素,按照不同的高度和层次进行了分析,阐明了这些营养元素在柞树各器官中的垂直分布规律,同时提出了有些营养元素在树皮、树干中的分布存在一个“过渡区”,为进一步研究柞树林的养分动态机理提供了依据。     

The impact of climate change on the future distribution of priority crop wild relatives in Indonesia and implications for conservation planning

The analysis of climate change impact is essential to include in conservation planning of crop wild relatives (CWR) to provide the guideline for adequate long-term protection under unpredictable future environmental conditions. These resources play an important role in sustaining the future of food security, but the evidence shows that they are threatened by climate change. The current analyses show that five taxa were predicted to have contraction of more than 30 % of their current ranges: Artocarpus sepicanus (based on RCP 4.5 in both no dispersal and unlimited dispersal scenario and RCP 8.5 in no dispersal scenario by 2050), Ficus oleifolia (RCP 4.5 5 in both no dispersal and unlimited dispersal scenario by 2080), Cocos nucifera and Dioscorea alata (RCP 8.5 in both no dispersal and unlimited dispersal scenario by 2050), and Ficus chartacea (RCP 8.5 in both no dispersal and unlimited dispersal scenario by 2050 and 2080). It shows that the climate change impact is species-specific. Representative Concentration Pathways (RCP) of greenhouse gas (GHG) emission and dispersal scenarios influence the prediction models, and the actual future distribution range of species falls in between those scenarios. Climate refugia, holdout populations, and non-analogue community assemblages were identified based on the Protected Areas (PAs) network. PAs capacity is considered an important element in implementing a conservation strategy for the priority CWR. In areas where PAs are isolated and have less possibility to build corridors to connect each other, such as in Java, unlimited dispersal scenarios are unlikely to be achieved and assisted dispersal is suggested. The holdout populations should be the priority target for the ex situ collection. Therefore, by considering the climate refugia, PAs capacity and holdout populations, the goal of keeping high genetic variations for the long-term conservation of CWR in Indonesia can be achieved.     

Bioavailable cadmium during the bioremediation of phenanthrene-contaminated soils using the diffusive gradients in thin-film technique

AIMS: To study the impact of fungal bioremediation of phenanthrene on trace cadmium solid-solution fluxes and solution phase concentration. METHODS AND RESULTS: The bioremediation of phenanthrene in soils was performed using the fungus Penicillium frequentans. Metal behaviour was evaluated by the techniques of diffusive gradient in thin-films (DGT) and filtration. Fluxes of cadmium (Cd) show a significant (P < 0.002) increase after the start of bioremediation, indicating that the bioremediation process itself releases significant amount of Cd into solution from the soil solid-phase. Unlike DGT devices, the solution concentration from filtration shows a clear bimodal distribution. We postulate that the initial action of the fungi is most likely to breakdown the surface of the solid phase to smaller, 'solution-phase' material (<0.45 microm) leading to a peak in Cd concentration in solution. CONCLUSIONS: Phenanthrene removal from soils by bioremediation ironically results in the mobilization of another toxic pollutant (Cd). SIGNIFICANCE AND IMPACT OF THE STUDY: Bioremediation of organic pollutants in contaminated soil will likely lead to large increases in the mobilization of toxic metals, increasing metal bio-uptake and incorporation into the wider food chain. Bioremediation strategies need to account for this behaviour and further research is required both to understand the generality of this behaviour and the operative mechanisms.     

Stabilization of yeast hexokinase A by polyol osmolytes: correlation with the physicochemical properties of aqueous solutions

Osmolytes of the polyol series are known to accumulate in biological systems under stress and stabilize the structures of a wide variety of proteins. While increased surface tension of aqueous solutions has been considered an important factor in protein stabilization effect, glycerol is an exception, lowering the surface tension of water. To clarify this anomalous effect, the effect of a series of polyols on the thermal stability of a highly thermolabile two domain protein yeast hexokinase A has been investigated by differential scanning calorimetry and by monitoring loss in the biological activity of the enzyme as a function of time. A larger increase in the T(m) of domain 1 compared with that of domain 2, varying linearly with the number of hydroxyl groups in polyols, has been observed, sorbitol being the best stabilizer against both thermal as well as urea denaturation. Polyols help retain the activity of the enzyme considerably and a good correlation of the increase in T(m) (DeltaT(m)) and the retention of activity with the increase in the surface tension of polyol solutions, except glycerol, which breaks this trend, has been observed. However, the DeltaT(m) values show a linear correlation with apparent molal heat capacity and volume of aqueous polyol solutions including glycerol. These results suggest that while bulk solution properties contribute significantly to protein stabilization, interfacial properties are not always a good indicator of the stabilizing effect. A subtle balance of various weak binding and exclusion effects of the osmolytes mediated by water further regulates the stabilizing effect. Understanding these aspects is critical in the rational design of stable protein formulations.     

Relative influence of habitat heterogeneity, climate, human disturbance, and spatial structure on vertebrate species richness in Spain

Many factors affect the distribution of species richness. This study examines the relative influence of habitat heterogeneity, climate, human disturbance, and spatial structure on the species-richness distribution of terrestrial vertebrates (amphibians, reptiles, birds and mammals) in mainland Spain. The results indicate that spatial structure and environment exert similar influences on species richness. For all four taxa, species richness increases southward and northward, being lower in the center of the country, when controlled for other variables. This may be the result of a peninsular effect, as found in other studies, and reflect the importance of historical events on species richness in the Iberian Peninsula. Climate is more important than habitat heterogeneity in determining species richness. Temperature is positively correlated with amphibian, reptile, and bird species richness, while mammalian species richness is highest at intermediate temperatures. This effect is stronger in ectotherms than among endotherms, perhaps reflecting physiological differences. Precipitation positively correlates with bird and mammalian species richness, but has no effect on ectotherm species richness. Amphibian species richness increases with altitudinal range, and bird species richness with habitat diversity. Human population density is positively correlated with bird and mammalian species richness, but does not affect ectotherm species richness, while amphibian and bird species richness is highest at moderate levels of human land alteration (farmland). However, unexplained variance remains, and we discuss that the effects of environmental variables on species richness may vary geographically, causing different effects to be obscured on a national scale, diminishing the explanatory power of environmental variables.