In Vitro Detoxification of Aflatoxin B1, Deoxynivalenol,Fumonisins, T-2 Toxin and Zearalenone by Probiotic Bacteria from Genus Lactobacillus and Saccharomyces cerevisiae Yeast

The aim of the following research was to determine the detoxification properties of probiotic Lactobacillus sp. bacteria (12 strains) and S. cerevisiae yeast (6 strains) towards mycotoxins, such as aflatoxin B₁, deoxynivalenol, fumonisins, T-2 toxin and zearalenone, which pose as frequent feed contamination. The experiment involved analysing changes in concentration of mycotoxins in PBS solutions, after 6, 12 and 24 h of incubation with monocultures of tested microorganisms, measured by high-performance liquid chromatography (HPLC). We found that all strains detoxified the mycotoxins, with the highest reduction in concentration observed for the fumonisin B₁ and B₂ mixture, ranging between 62 and 77% for bacterial strains and 67–74% for yeast. By contrast, deoxynivalenol was the most resistant mycotoxin: its concentration was reduced by 19–39% by Lactobacillus sp. strains and 22–43% by yeast after 24 h of incubation. High detoxification rates for aflatoxin B₁, T-2 toxin and zearalenone were also observed, with concentration reduced on average by 60%, 61% and 57% by Lactobacillus, respectively, and 65%, 69% and 52% by yeast, respectively. The greatest extent of reduction in the concentration for all mycotoxins was observed after 6 h of incubation; however, a decrease in concentration was noted even after 24 h of incubation. Thus, the tested microorganisms can potentially be used as additives to decrease the concentrations of toxins in animal feed.

     

Changes in polyamine pattern mediates sex differentiation and unisexual flower development in monoecious cucumber (Cucumis sativus L.)

Changes in the levels of polyamines are associated with fundamental physiological processes such as embryogenesis, induction of flowering, fruit development and ripening, senescence, and responses to environmental stresses, but the role of polyamines in sex differentiation and unisexual flower development has not been deeply studied. To extend the knowledge on the regulatory mechanisms of flowering in monoecious plant (producing unisexual flowers), we investigated the morphogenesis and free polyamine levels in Cucumis sativus during sex differentiation and unisexual flower development in vitro using histocytological and biochemical methods. As shown in our study, floral development in vitro was undisturbed and flowers of both sexes were produced. Sex differentiation relied on preventing the development of generative organs of the opposite sex, as we observed carpel repression in male flowers and stamen repression in female flowers. Pollen viability was negatively correlated with female flower development on the same node. Biochemical analysis revealed increased accumulation of aliphatic amines (tri, tetra‐amines) in generative (flower buds and flowers) compare to vegetative (axillary buds and leaves) organs. Undifferentiated floral buds contained elevated levels of agmatine, cadaverine, spermidine and spermine. Sex differentiation was associated with significantly decreased levels of agmatine and cadaverine. Our results showed that female flowers contained higher levels of total polyamine than male flowers. The increased level of cadaverine was associated with macrogametogenesis and female flower maturation. Putrescine was important for male flower development. Such results support the hypothesis that aliphatic amines are involved in unisexual flower development.     

Effects of genetically modified human skin fibroblasts,stably overexpressing hepatocyte growth factor,on hepatic functions of cocultured C3A cells

Diseases leading to terminal hepatic failure are among the most common causes of death worldwide. Transplant of the whole organ is the only effective method to cure liver failure. Unfortunately, this treatment option is not available universally due to the serious shortage of donors. Thus, alternative methods have been developed that are aimed at prolonging the life of patients, including hepatic cells transplantation and bridging therapy based on hybrid bioartificial liver devices. Parenchymal liver cells are highly differentiated and perform many complex functions, such as detoxification and protein synthesis. Unfortunately, isolated hepatocytes display a rapid decline in viability and liver‐specific functions. A number of methods have been developed to maintain hepatocytes in their highly differentiated state in vitro, amongst them the most promising being 3D growth scaffolds and decellularized tissues or coculture with other cell types required for the heterotypic cell‐cell interactions. Here we present a novel approach to the hepatic cells culture based on the feeder layer cells genetically modified using lentiviral vector to stably produce additional amounts of hepatocyte growth factor and show the positive influence of these coculture conditions on the preservation of the hepatic functions of the liver parenchymal cells' model—C3A cells.     

Mutational analysis in podocin-associated hereditary nephrotic syndrome in Polish patients: founder effect in the Kashubian population

Hereditary nephrotic syndrome is caused by mutations in a number of different genes, the most common being NPHS2. The aim of the study was to identify the spectrum of NPHS2 mutations in Polish patients with the disease. A total of 141 children with steroid-resistant nephrotic syndrome (SRNS) were enrolled in the study. Mutational analysis included the entire coding sequence and intron boundaries of the NPHS2 gene. Restriction fragment length polymorphism (RFLP) and TaqMan genotyping assay were applied to detect selected NPHS2 sequence variants in 575 population-matched controls. Twenty patients (14 %) had homozygous or compound heterozygous NPHS2 mutations, the most frequent being c.1032delT found in 11 children and p.R138Q found in four patients. Carriers of the c.1032delT allele were exclusively found in the Pomeranian (Kashubian) region, suggesting a founder effect origin. The 14 % NPHS2 gene mutation detection rate is similar to that observed in other populations. The heterogeneity of mutations detected in the studied group confirms the requirement of genetic testing the entire NPHS2 coding sequence in Polish patients, with the exception of Kashubs, who should be initially screened for the c.1032delT deletion.     

The Comparison of the Effects of Three Physiotherapy Techniques on Hamstring Flexibility in Children: A Prospective,Randomized, Single-Blind Study

The aim of the study was to evaluate changes in hamstring flexibility in 120 asymptomatic children who participated in a 6-week program consisting of one physiotherapy session per week and daily home exercises. The recruitment criteria included age (10–13 years), no pain, injury or musculoskeletal disorder throughout the previous year, physical activity limited to school sport. Subjects were randomly assigned to one of the three groups: (1) post-isometric relaxation – PIR (n = 40), (2) static stretch combined with stabilizing exercises – SS (n = 40) and (3) stabilizing exercises – SE (n = 40). Hamstring flexibility was assessed with straight leg raise (SLR), popliteal angle (PA) and finger-to-floor (FTF) tests. The examinations were conducted by blinded observers twice, prior to the program and a week after the last session with the physiotherapist. Twenty-six children who did not participate in all six exercise sessions with physiotherapists were excluded from the analysis. The results obtained by 94 children were analyzed (PIR, n = 32; SS, n = 31; SE, n = 31). In the PIR and SS groups, a significant (P<0.01) increase in SLR, PA, FTF results was observed. In the SE group, a significant (P<0.001) increase was observed in the SLR but not in the PA and FTF (P>0.05). SLR result in the PIR and SS groups was significantly (P<0.001) higher than in the SE group. As far as PA results are concerned, a significant difference was observed only between the SS and SE groups (P = 0.014). There were no significant (P = 0.15) differences regarding FTF results between the three groups. Post-isometric muscle relaxation and static stretch with stabilizing exercises led to a similar increase in hamstring flexibility and trunk forward bend in healthy 10–13-year-old children. The exercises limited to straightening gluteus maximus improved the SLR result, but did not change the PA and FTF results.     

Characterization of a Small Auxin-Up RNA (SAUR)-Like Gene Involved in Arabidopsis thaliana Development

The root of Arabidopsis thaliana is used as a model system to unravel the molecular nature of cell elongation and its arrest. From a micro-array performed on roots that were treated with aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, a Small auxin-up RNA (SAUR)-like gene was found to be up regulated. As it appeared as the 76th gene in the family, it was named SAUR76. Root and leaf growth of overexpression lines ectopically expressing SAUR76 indicated the possible involvement of the gene in the division process. Using promoter::GUS and GFP lines strong expression was seen in endodermal and pericycle cells at the end of the elongation zone and during several stages of lateral root primordia development. ACC and IAA/NAA were able to induce a strong up regulation of the gene and changed the expression towards cortical and even epidermal cells at the beginning of the elongation zone. Confirmation of this up regulation of expression was delivered using qPCR, which also indicated that the expression quickly returned to normal levels when the inducing IAA-stimulus was removed, a behaviour also seen in other SAUR genes. Furthermore, confocal analysis of protein-GFP fusions localized the protein in the nucleus, cytoplasm and plasma membrane. SAUR76 expression was quantified in several mutants in ethylene and auxin-related pathways, which led to the conclusion that the expression of SAUR76 is mainly regulated by the increase in auxin that results from the addition of ACC, rather than by ACC itself.     

Predictive Factors of Late Venous Aortocoronary Graft Failure: Ultrastructural Studies

Background

Venous aortocoronary graft arterialization may precede a preterm occlusion in some coronary artery bypass grafting (CABG) patients. The aim of the present study was to identify ultrastructural variations in the saphenous vein wall that may have an impact on the development of venous graft disease in CABG patients.

Methods

The study involved 365 consecutive patients with a mean age of 62.9±9.4 years who underwent isolated CABG. The thickness and area of the whole venous wall, the tunica intima, the tunica media and the adventitia and the number and shape (length, thickness and length/thickness ratio) of the nuclei in the medial smooth muscle cells nuclei in the distal saphenous vein segments were evaluated by ultrastructural studies. Patients were followed up for 41 to 50 months (mean 45.1±5.1). Saphenous vein graft patency was assessed by follow-up coronary angiography. Logistic regression models were used to identify independent risk factors for late graft failure.

Results

In 71 patients significant lesions in the saphenous vein grafts were observed. The whole venous wall thickness (437.5 µm vs. 405.5 µm), tunica media thickness (257.2 µm vs. 211.5 µm), whole venous wall area (2.23 mm² vs. 2.02 mm²) and tunica media area (1.09 mm² vs. 0.93 mm²) were significantly larger for this group of patients than for those without graft disease. In the latter group more elongated smooth muscle cell nuclei (higher length/thickness ratio) were found in the tunica media of the saphenous vein segments. Thickening of the saphenous vein tunica media and chunky smooth muscle cell nuclei were identified as independent risk factors for graft disease development.

Conclusions

Saphenous vein tunica media hypertrophy (resulting in wall thickening) and chunky smooth muscle cell nuclei might predict the development of venous graft disease.     

Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5′-Terminal Region of p53 mRNA In Vitro and in the Living Cells

The p53 protein is a key player in cell response to stress events and cancer prevention. However, up-regulation of p53 that occurs during radiotherapy of some tumours results in radio-resistance of targeted cells. Recently, antisense oligonucleotides have been used to reduce the p53 level in tumour cells which facilitates their radiation-induced apoptosis. Here we describe the rational design of antisense oligomers directed against the 5′-terminal region of p53 mRNA aimed to inhibit the synthesis of p53 protein and its ΔNp53 isoform. A comprehensive analysis of the sites accessible to oligomer hybridization in this mRNA region was performed. Subsequently, translation efficiency from the initiation codons for both proteins in the presence of selected oligomers was determined in rabbit reticulocyte lysate and in MCF-7 cells. The antisense oligomers with 2′-OMe and LNA modifications were used to study the mechanism of their impact on translation. It turned out that the remaining RNase H activity of the lysate contributed to modulation of protein synthesis efficiency which was observed in the presence of antisense oligomers. A possibility of changing the ratio of the newly synthetized p53 and ΔNp53 in a controlled manner was revealed which is potentially very attractive considering the relationship between the functioning of these two proteins. Selected antisense oligonucleotides which were designed based on accessibility mapping of the 5′-terminal region of p53 mRNA were able to significantly reduce the level of p53 protein in MCF-7 cells. One of these oligomers might be used in the future as a support treatment in anticancer therapy.     

Rational design of the survivin/CDK4 complex by combining protein–protein docking and molecular dynamics simulations

Survivin, the smallest inhibitor of apoptosis protein (IAP), is a valid target for cancer research. It mediates both the apoptosis pathway and the cell cycle and has been proposed to form a complex with the cyclin-dependent kinase protein CDK4. The resulting complex transports CDK4 from the cytosol to the nucleus, where CDK4 participates in cell division. Survivin has been recognized as a node protein that interacts with several partners; disruption of the formed complexes can lead to new anticancer compounds. We propose a rational model of the survivin/CDK4 complex that fulfills the experimental evidence and that can be used for structure-based design of inhibitors modifying its interface recognition. In particular, the suggested complex involves the alpha helical domain of survivin and resembles the mode of binding of survivin in the survivin/borealin X-ray structure. The proposed model has been obtained by combining protein–protein docking, fractal-based shape complementarity, electrostatics studies and extensive molecular dynamics simulations.