The mouse splenocyte assay, an in vivo/in vitro system for biological monitoring: studies with X-rays, fission neutrons and bleomycin.

A modified mouse splenocyte culture system was standardized after testing different mitogens (i.e., phytohemagglutinin (PHA), concanavalin A (Con A)). The mitotic index was determined for comparison between different mitogens. Following selection of appropriate mitogen (PHA 16, Flow), a series of experiments were conducted to evaluate the application of a cytokinesis-block for scoring micronuclei and assays for chromosomal aberrations produced by treatment in G0 and G2 for the purposes of biological dosimetry following in vivo and/or in vitro exposure to X-rays, fission neutrons and bleomycin. In the X-irradiation studies, the frequencies of micronuclei and chromosomal aberrations (i.e., dicentrics and rings) increased in a dose-dependent manner. These data could be fitted to a linear-quadratic model. No difference was observed between irradiation in vivo and in vitro, suggesting that measurement of dicentrics and micronuclei in vitro after X-irradiation can be used as an in vivo dosimeter. Following in vivo irradiation with 1 MeV fission neutrons and in vitro culturing of mouse splenocytes, linear dose-response curves were obtained for induction of micronuclei and chromosomal aberrations. The lethal effects of neutrons were shown to be significantly greater than for a similar dose of X-rays. The relative biological effectiveness (RBE) was 6-8 in a dose range of 0.25-3 Gy for radiation-induced asymmetrical exchanges (dicentrics and rings), and about 8 for micronuclei in a dose range of 0.25-2 Gy. Furthermore, the induction of chromosomal aberrations by bleomycin was investigated in mouse G0 splenocytes (in vitro) and compared with X-ray data. Following bleomycin treatment (2 h) a similar pattern of dose-response curve was obtained as with X-rays. In this context a bleomycin rad equivalent of 20 micrograms/ml = 0.50 Gy was estimated.     

Crucisoma bernardi n.g., n.sp. (Crustacea: Copepoda) parasitic in a deep-sea mollusc Cuspidaria filatovae Bernard, 1979 (Bivalvia: Anomalodesmata)

Summary Crucisoma bernardi, a copepod parasitizing a deepsea mollusc, Cuspidaria filatovae (Bivalvia: Anomalodesmata) off the coast of Oregon, is described and illustrated. A definitely poecilostomatoid copepod, belonging to a new genus, this species is treated by the author as incertae sedis, although there are many reasons why it should provide grounds for the erection of a new family. ac]19810127     

CONTAMINATED LEVEL AND RISK ASSESSMENT OF PAES IN THE UPPER REACHES OF THE YANGTZE RIVER北大核心CSCD

PAEs have been proved to be one of the major organic pollutants. The present study determined the level of PAEs using surface water samples from the upper reaches of the Yangtze River. The results showed that DEHP contributed the most to PAEs pollution, followed by DBP. Risk Quotients of PAEs were used for preliminary screening, and DEHP and BBP were identified as potential risk factors with RQ of 310 and 70.7. The MOS10 of DEHP and BBP were 1.40 and 1.32×105, respectively, indicating that BBP may pose little risk to aquatic organisms. The potential risk of DEHP was further analyzed with joint probabilistic curves. Among different biological groups, fish and zoobenthos were two major groups sensitive to DEHP-induced damage under current concentrations with 99.4% and 98.3% for 5% species of each group. While among the toxicity endpoints, reproduction may be more sensitive than others. The 5% and 10% aquatic species were suffered from reproductive damages by 100% and 97.8%, respectively. Overall, our results indicated that DEHP in the surface water of the upper Yangtze River may pose potential risk to aquatic organisms, especially on their reproduction. Therefore, more concerns should be paid in species protection and environmental management. © 2019, Institute of Hydrobiology, Chinese Academy of Sciences. All rights reserved.     

Discovery of Power-Law Growth in the Self-Renewal of Heterogeneous Glioma Stem Cell Populations

Background

Accumulating evidence indicates that cancer stem cells (CSCs) drive tumorigenesis. This suggests that CSCs should make ideal therapeutic targets. However, because CSC populations in tumors appear heterogeneous, it remains unclear how CSCs might be effectively targeted. To investigate the mechanisms by which CSC populations maintain heterogeneity during self-renewal, we established a glioma sphere (GS) forming model, to generate a population in which glioma stem cells (GSCs) become enriched. We hypothesized, based on the clonal evolution concept, that with each passage in culture, heterogeneous clonal sublines of GSs are generated that progressively show increased proliferative ability.

Methodology/Principal Findings

To test this hypothesis, we determined whether, with each passage, glioma neurosphere culture generated from four different glioma cell lines become progressively proliferative (i.e., enriched in large spheres). Rather than monitoring self-renewal, we measured heterogeneity based on neurosphere clone sizes (#cells/clone). Log-log plots of distributions of clone sizes yielded a good fit (r>0.90) to a straight line (log(% total clones) = k*log(#cells/clone)) indicating that the system follows a power-law (y = x^k) with a specific degree exponent (k = −1.42). Repeated passaging of the total GS population showed that the same power-law was maintained over six passages (CV = −1.01 to −1.17). Surprisingly, passage of either isolated small or large subclones generated fully heterogeneous populations that retained the original power-law-dependent heterogeneity. The anti-GSC agent Temozolomide, which is well known as a standard therapy for glioblastoma multiforme (GBM), suppressed the self-renewal of clones, but it never disrupted the power-law behavior of a GS population.

Conclusions/Significance

Although the data above did not support the stated hypothesis, they did strongly suggest a novel mechanism that underlies CSC heterogeneity. They indicate that power-law growth governs the self-renewal of heterogeneous glioma stem cell populations. That the data always fit a power-law suggests that: (i) clone sizes follow continuous, non-random, and scale-free hierarchy; (ii) precise biologic rules that reflect self-organizing emergent behaviors govern the generation of neurospheres. That the power-law behavior and the original GS heterogeneity are maintained over multiple passages indicates that these rules are invariant. These self-organizing mechanisms very likely underlie tumor heterogeneity during tumor growth. Discovery of this power-law behavior provides a mechanism that could be targeted in the development of new, more effective, anti-cancer agents.     

High pressure effect on photosynthetic properties of green plant leaves

We investigated the effects of high pressure treatment on green plant leaves of two species, red rose (Rosa rubiginosa L.) and silver birch male (Betula pendula Roth). Both species were treated with pressure up to 100 bar in order to explore stress reactions, including desirable or undesirable metabolites in plant. When increasing the pressure, chlorophyll (Chl) fluorescence maximum shifts to the wavelength of about 680 nm for both red rose and silver birch, with shift rates–0.062 nm/bar and–0.082 nm/bar, respectively. High pressure induces the changes of the position for the second fluorescence maximum at approximately 730 nm in both species with the same shift rate–0.083 nm/bar. When increasing pressure the change of the photosynthetic apparatus efficiency decreases for both plant species slowly and nonlinearly. High-pressure treatments irreversibly damaged the leaf tissue and at this way induced changes of Chl fluorescence and photosynthetic efficiency.     

Residence time distribution of liquid phase in an external-loop airlift bioreactor

The residence time distribution analysis was used to investigated the flow behaviour in an external-loop airlift bioreactor regarded as a single unit and discriminating its different sections. The experimental results were fitted according to plug flow with superimposed axial dispersion and tank-in-series models, which have proved that it is reasonable to assume plug flow with axial dispersion in the overall reactor, in riser and downcomer sections, as well, while the gas separator should be considered as a perfectly mixed zone. Also, the whole reactor could be replaced with 105-30 zones with perfect mixing in series, while its separate zones, that is the riser with 104-27, the downcomer with 115-35 and the gas separator with 25-5 perfectly mixed zones in series, respectively, depending on gas superficial velocity, A_D/A_R ratio and the liquid feed rate.List of Symbols A _D cross sectional area of downcomer (m²) - A _R cross sectional area of riser (m²) - A ₁ A ₂ length of connecting pipes (m) - Bo Bodenstein number (Bo=vL·L/D _ax (-) - C concentration (kg m^–3) - C residence time distribution function - C ₀ coefficientEquation (12) - C _r dimensionless concentration - D _D diameter of downcomer (m) - D _R diameter of riser column (m) - D _ax axial dispersion coefficient (m²s^–1) - H _d height of gas-liquid dispersion (m) - H _L height of clear liquid (m) - i number of complete circulations - L length of path (m) - m order of moments - N _eq number of perfectly mixed zones in series - n _c circulating number - Q _c recirculating liquid flow rate (m³ s^–1) - q _F liquid feed flow rate (m³s^–1) - Q _G gas flow rate (m³s^–1) - Q _T total liquid flow rate (m³s^–1) - r recycle factor - s exponent inEquation (12) regarded as logarithmic decrement of the oscillating part of RTD curve - t time (s) - t _C circulation time (s) - t _s mean residence time (s) - t ₉₉ time necessary to remove 99% of the tracer concentration (s) - V _A volume of connecting pipes (m³) - V _D volume of downcomer (m³) - V _L liquid volume in reactor (m³) - V _R volume of riser (m³) - V _LD linear liquid velocity in downcomer (m s^–1) - V _LR linear liquid velocity in riser (m s^–1) - V _SLD superficial liquid velocity in downcomer (m s^–1) - V _SLR superficial liquid velocity in riser (m s^–1) - x independent variable inEquation (1) - ¯x mean value of x - z axial coordinate - _GR gas holdup in riser - _m(x) central moment of m order - ² variance - dimensionless time     

Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycle

Orange carotenoid protein (OCP), responsible for the photoprotection of the cyanobacterial photosynthetic apparatus under excessive light conditions, undergoes significant rearrangements upon photoconversion and transits from the stable orange to the signaling red state. This is thought to involve a 12-Å translocation of the carotenoid cofactor and separation of the N- and C-terminal protein domains. Despite clear recent progress, the detailed mechanism of the OCP photoconversion and associated photoprotection remains elusive. Here, we labeled the OCP of Synechocystis with tetramethylrhodamine-maleimide (TMR) and obtained a photoactive OCP-TMR complex, the fluorescence of which was highly sensitive to the protein state, showing unprecedented contrast between the orange and red states and reflecting changes in protein conformation and the distances from TMR to the carotenoid throughout the photocycle. The OCP-TMR complex was sensitive to the light intensity, temperature, and viscosity of the solvent. Based on the observed Förster resonance energy transfer, we determined that upon photoconversion, the distance between TMR (donor) bound to a cysteine in the C-terminal domain and the carotenoid (acceptor) increased by 18 Å, with simultaneous translocation of the carotenoid into the N-terminal domain. Time-resolved fluorescence anisotropy revealed a significant decrease of the OCP rotation rate in the red state, indicating that the light-triggered conversion of the protein is accompanied by an increase of its hydrodynamic radius. Thus, our results support the idea of significant structural rearrangements of OCP, providing, to our knowledge, new insights into the structural rearrangements of OCP throughout the photocycle and a completely novel approach to the study of its photocycle and non-photochemical quenching. We suggest that this approach can be generally applied to other photoactive proteins.     

Mutation mechanism of chlorophyll-less barley mutant <Emphasis Type="Italic">NYB</Emphasis>

NYB is chlorophyll-less barley mutant, which is controlled by a recessive nuclear gene. The mutation mechanism is revealed. The activities of enzymes transforming 5-aminolevulinic acid into protochlorophyllide were the same in both NYB and the wild type (WT), but the activity of the protochlorophyllide oxidoreductase (POR) in WT was much higher than that of NYB. Most of the photosystem 2 apoproteins were present in both WT and NYB, suggesting that the capability of protein synthesis was probably fully preserved in the mutant. Thus chlorophyll (Chl) biosynthesis in NYB was hampered at conversion form protochlorophyllide (Pchlide) into chlorophyllide. The open reading frame of porB gene in NYB was inserted with a 95 bp fragment, which included a stop codon. The NYB mutant is a very useful material for studies of Chl biosynthesis, chloroplast signalling, and structure of light-harvesting POR-Pchlide complex (LHPP).