Stability of the intra- and extracellular toxins of Prymnesium parvum using a microalgal bioassay

Prymnesium parvum produces a variety of toxic compounds, which affect other algae, grazers and organisms at higher trophic levels. Here we provide the method for development of a sensitive algal bioassay using a microalgal target, Teleaulax acuta, to measure strain variability in P. parvum toxicity, as well as the temporal stability of both the intracellular and the extracellular lytic compounds of P. parvum. We show high strain variation in toxicities after 3 h incubation with LC₅₀s ranging from 24 to 223 × 10³ cells ml⁻¹. Most importantly we prove the necessity of testing physico-chemical properties of P. parvum toxins before attempting to isolate and characterize them. The extracellular toxin in the supernatant is highly unstable, and it loses significant lytic effects after 3 days despite storage at −20 °C and after only 24 h stored at 4 °C. However, when stored at −80 °C, lytic activity is more easily maintained. Reducing oxidation by storing the supernatant with no headspace in the vials significantly slowed loss of activity when stored at 4 °C. We show that the lytic activity of the intracellular toxins, when released by sonication, is not as high as the extracellular toxins, however the stability of the intracellular toxins when kept as a cell pellet at −20 °C is excellent, which proves this is a sufficient storage method for less than 3 months. Our results provide an ecologically appropriate algal bioassay to quantify lytic activity of P. parvum toxins and we have advanced our knowledge of how to handle and store the toxins from P. parvum so as to maintain biologically relevant toxicity.     

Application of 13C-[2] - and 13C-[1,2] acetate in metabolic labelling studies of yeast and insect cells

The advantage of using ¹³C-labelled glucose in metabolic studies is that it is an important carbon and energy source for almost all biotechnologically and medically important organisms. On the other hand, the disadvantage is its relatively high cost in the labelling experiments. Looking for cheaper alternatives we found that ¹³C-[2] acetate or ¹³C-[1,2] acetate is a prospective compound for such experiments. Acetate is well incorporated by many organisms, including mammalian and insect cell cultures as preferred source of acetyl-CoA. Our experimental results using ¹³C NMR demonstrated that acetate was efficiently incorporated into glutamate and alanine secreted by the insect cell culture. Using D-stat culture of Saccharomyces uvarum on glucose/¹³C-acetate mineral media we demonstrated that the labelling patterns of proteinogenic amino acids can be well predicted on the basis of specific substrate consumption rates using the modified scheme of yeast metabolism and stoichiometric modelling. According to this scheme aspartate and alanine in S. uvarum under the experimental conditions used is synthesised in the mitochondria. Synthesis of alanine in the mitochondria was also demonstrated for Spodoptera frugiperda. For both organisms malic enzyme was also operative. For S. uvarum it was shown that the activity of malic enzyme is sufficient for supporting the mitochondrial biosynthetic reactions with NADPH.     

MET inhibition enhances PARP inhibitor efficacy in castration-resistant prostate cancer by suppressing the ATM/ATR and PI3K/AKT pathways

Up to 30% of patients with metastatic castration-resistant prostate cancer (CRPC) patients carry altered DNA damage response genes, enabling the use of poly adenosine diphosphate–ribose polymerase (PARP) inhibitors in advanced CRPC. The proto-oncogene mesenchymal–epithelial transition (MET) is crucial in the migration, proliferation, and invasion of tumour cells. Aberrant expression of MET and its ligand hepatocyte growth factor is associated with drug resistance in cancer therapy. Here, we found that MET was highly expressed in human CRPC tissues and overexpressed in DU145 and PC3 cells in a drug concentration-dependent manner and is closely related to sensitivity to PARP inhibitors. Combining the PARP inhibitor olaparib with the MET inhibitor crizotinib synergistically inhibited CRPC cell growth both in vivo and in vitro. Further analysis of the underlying molecular mechanism underlying the MET suppression-induced drug sensitivity revealed that olaparib and crizotinib could together downregulate the ATM/ATR signaling pathway, inducing apoptosis by inhibiting the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway, enhancing the olaparib-induced antitumour effect in DU145 and PC3 cells. In conclusion, we demonstrated that MET inhibition enhances sensitivity of CRPC to PARP inhibitors by suppressing the ATM/ATR and PI3K/AKT pathways and provides a novel, targeted therapy regimen for CRPC.     

Effects of branched-chain amino acid supplement on knee peak torque and indicators of muscle damage following isokinetic exercise-induced delayed onset muscle soreness

[Purpose] This study aimed to investigate the effects of branched-chain amino acid (BCAA) supplement on delayed onset muscle soreness (DOMS) by analyzing the maximum muscle strength and indicators of muscle damage.[Methods] Twelve men with majors in physical education were assigned to the BCAA group and placebo group in a double-blinded design, and repeated measurements were conducted. DOMS was induced with an isokinetic exercise. Following BCAA administration, the changes in the knee extension peak torque, flexion peak torque, aspartate aminotransferase (AST), creatine kinase (CK), and lactate dehydrogenase (LDH) concentrations were analyzed. The maximum knee muscle strength was measured at the baseline (pre-D0) following BCAA administration for 5 days before exercise (-D5, -4D, -3D, -2D, -1D). In contrast, the post-treatment measurements (D3) were recorded after BCAA administration for 3 days (post-D0, D1, D2). Blood samples were obtained before (pre-D0), immediately after (post-D0), 24 h (D1), 48 h (D2), and 72 h (D3) after the exercise to analyze the indicators of muscle strength. BCAA was administered twice daily for 8 days (5 days and 3 days before inducing DOMS and during the experimental period, respectively).[Results] There was no difference in the flexion peak torque between the groups. However, the BCAA group showed a significantly higher extension peak torque at D3 (second isokinetic exercise), compared to the placebo group (p<.05). There was no difference in AST changes between the groups. Nonetheless, the CK and LDH were significantly reduced in the BCAA group, compared to the placebo group. There was no correlation between the extension peak torque and flexion peak torque. However, the CK and LDH increased proportionately in DOMS. Moreover, their concentrations significantly increased with a decreasing peak torque (p<.01).[Conclusion] An exercise-induced DOMS results in a decrease in the peak torque and a proportional increase in the CK and LDH concentrations. Moreover, the administration of BCAA inhibits the reduction of the extension peak torque and elevation of CK and LDH concentrations. Therefore, BCAA might be administered as a supplement to maintain the muscle strength and prevent muscle damage during vigorous exercises that may induce DOMS in sports settings.     

Analysis of Global and Site-Specific Radiation Damage in Cryo-EM

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A time course study on dose-response relationship between alcohol exposure and its effects on lipid profile and biomarkers of tissue damage

This present research investigated variations in lipid profiles and important biomarkers of tissue damage in response to graded concentrations of alcohol administration in male Wistar rats. Group A (control) received distilled water while group B, C and D received 30%, 40% and 50% (v/v) alcohol respectively. Five rats each from groups A-D were sacrificed after day(s) 1, 7, 14, 21 and 28 of administration. A significant increase was observed at day 28 for serum cholesterol by 79% (group B), 78% (group C) and 47% (group D) together with serum phospholipid 58% (group B), 50% (group C) and 92% (group D). Serum triacylglycerol increased by 71% (group B), 43% (group C) and 16% (group D) at day 21, while concentration of serum albumin decreased at day 28 by 40.9% (group B), 50.2% (group C), 53.3% (group D) respectively when compared with control (group A). Serum aminotransferases and alkaline phosphatase specific activities, as well as creatinine and uric acid concentration increased in a concentration-dependent manner, following alcohol administration. Though most of these effects induced by alcohol were time- and concentration-dependent, 40% alcohol appear to be more stable, giving results consistent with alcohol-induced damages, with minimal mortality. This study therefore further validated dyslipidemia and imbalance in clinical biomarkers as hallmarks of tissue damage induced by excessive alcohol consumption with an insight on the time- and concentration-response relationship between alcohol consumption and its toxicity.     

Identification and molecular characterization of <Emphasis Type="Italic">Theileria</Emphasis> sp. infecting red deer (<Emphasis Type="Italic">Cervus elaphus</Emphasis>) in northwestern Poland

Piroplasms from Theileria genus were detected in blood and spleen of red deer Cervus elaphus culled during the months of September 2004–January 2005 in northwestern Poland. The polymerase chain reaction revealed the presence of Theileria deoxyribonucleic acid in 88% (36 of 41) of the animals examined. Molecular characterization of the parasites based on large piece of 18S ribosomal ribonucleic acid gene containing hypervariable region V4 showed 99.9% similarity to two Theileria spp. sequences: Theileria sp. 3185/02 and Theileria capreoli BAB1158. Phylogenetic analysis confirmed that the three isolates cluster together with high bootstrap support. It is supposed that those pathogens can be classified as one group characteristic for the Eurasian continent, contrary to protozoon of Theileria from the T. cervi group, which are often found on the North American continent and can also infect the representatives of Cervidae. In conclusion, this study suggested that free-living C. elaphus in northwestern Poland are a competent reservoir of Theileria sp. ZS T04 C.e. parasites, although the vector of the piroplasms is still unknown.     

Regulation of Cellular Senescence by Polycomb Chromatin Modifiers through Distinct DNA Damage- and Histone Methylation-Dependent Pathways

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Effect of cooling rate and cryoprotectant concentration on intracellular ice formation of small abalone (Haliotis diversicolor) eggs

The intracellular ice formation (IIF) behavior of Haliotis diversicolor (small abalone) eggs is investigated in this study, in relation to controlling the cooling rate and the concentration of dimethyl sulfoxide (DMSO). The IIF phenomena are monitored under a self-developed thermoelectric cooling (TEC) cryomicroscope system which can achieve accurate temperature control without the use of liquid nitrogen. The accuracy of the isothermal and ramp control is within ±0.5 °C. The IIF results indicate that the IIF of small abalone eggs is well suppressed at cooling rates of 1.5, 3, 7 and 12 °C/min with 2.0, 2.5, 3.0 and 4.0 M DMSO in sea water. As 2.0 M DMSO in sea water is the minimum concentration that has sufficient IIF suppression, it is selected as the suspension solution for the cryopreservation of small abalone eggs in order to consider the solution’s toxicity effect. Moreover, IIF characteristics of the cumulative probability of IIF temperature distribution are shown to be well fitted by the Weibull probabilistic distribution. According to our IIF results and the Weibull distribution parameters, we conclude that cooling at 1.5 °C/min from 20 to −50 °C with 2.0 M DMSO in sea water is more feasible than other combinations of cooling rates and DMSO concentrations in our experiments. Applying this protocol and observing the subsequent osmotic activity, 48.8% of small abalone eggs are osmotically active after thawing. In addition, the higher the cooling rate, the less chance of osmotically active eggs. A separate fertility test experiment, with a cryopreservation protocol of 1.5 °C/min cooling rate and 2.0 M DMSO in sea water, achieves a hatching rate of 23.7%. This study is the first to characterize the IIF behavior of small abalone eggs in regard to the cooling rate and the DMSO concentration. The Weibull probabilistic model fitting in this study is an approach that can be applied by other researchers for effective cryopreservation variability estimation and analysis.