Aims
To evaluate the persistence of Mycobacterium avium subsp paratuberculosis (Myco. paratuberculosis), Salmonella enterica serotype Typhimurium (Salm.Typhimurium) and a commensal Escherichia coli ...(E. coli) isolate under the low pH and high organic acid (OA) conditions of ensiling of forages.
Methods and Results
Decay rates and the time required to obtain a 90% reduction in cell concentration were calculated following (i) exposure to buffered OA (pH 4·0, 5·0, 6·0 or 7·0) (ii) exposure to silage exudates and (iii) survival through ensiling of forage materials. Salm. Typhimurium had higher decay rates in silage exudates (−0·5601 day−1) than did E. coli (−0·1265 day−1), but both exhibited lower decay rates in silage than in OA or silage exudates. Myco. paratuberculosis showed no decrease in silage and decay rates in silage exudates were significantly lower (2–12 times) than for the other two organisms.
Conclusions
Escherichia coli, Salm. Typhimurium and Myco. paratuberculosis exhibit marked differences in response to acidity. All three organisms show acid resistance, but Myco. paratuberculosis in particular, if present in manure and applied to forage grasses, may survive the low pH and high OA of the ensilaging process; silage may therefore be a potential route of infection if ingested by a susceptible animal.
Significance and Impact of Study
This information contributes to the understanding of potential risks associated with silage preservation and contamination of livestock feed with manure‐borne pathogens.
Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of chronic myeloid leukemia in chronic phase (CML-CP). Unfortunately, 25% of TKI-naive patients and 50-90% of patients developing ...TKI-resistance carry CML clones expressing TKI-resistant BCR-ABL1 kinase mutants. We reported that CML-CP leukemia stem and progenitor cell populations accumulate high amounts of reactive oxygen species, which may result in accumulation of uracil derivatives in genomic DNA. Unfaithful and/or inefficient repair of these lesions generates TKI-resistant point mutations in BCR-ABL1 kinase. Using an array of specific substrates and inhibitors/blocking antibodies we found that uracil DNA glycosylase UNG2 were inhibited in BCR-ABL1-transformed cell lines and CD34(+) CML cells. The inhibitory effect was not accompanied by downregulation of nuclear expression and/or chromatin association of UNG2. The effect was BCR-ABL1 kinase-specific because several other fusion tyrosine kinases did not reduce UNG2 activity. Using UNG2-specific inhibitor UGI, we found that reduction of UNG2 activity increased the number of uracil derivatives in genomic DNA detected by modified comet assay and facilitated accumulation of ouabain-resistant point mutations in reporter gene Na(+)/K(+)ATPase. In conclusion, we postulate that BCR-ABL1 kinase-mediated inhibition of UNG2 contributes to accumulation of point mutations responsible for TKI resistance causing the disease relapse, and perhaps also other point mutations facilitating malignant progression of CML.
Currently, there is no bone fixation material that could be fully replaced by the competent recipient bone. The creeping substitution of the bone graft by the recipient bone is the result of its ...unique potential related to the presence of bone morphogenetic proteins (BMPs). However, the size of the human bone limits the use of allogenic implants for surgical (orthopedic) fixation.
The aim of this project was to develop a novel composite material for guided bone regeneration, consisting of human bone powder obtained from a tissue bank and a resorbable polymer (13 wt% of bone powder in a medical poly-l-lactide polymer). Such a biomaterial could possess osteoinductive properties and be used to manufacture bone fixation implants of different shapes and sizes.
The samples were obtained by tape casting and foils pressing, and subsequently radiation sterilized with a dose of 35 kGy. Two cell lines—normal mouse embryo fibroblasts (Balb 3T3/c) and human fetal osteoblasts (hFOB 1.19)—were cultured with the extracts of the biomaterials (MTT assay) or in indirect contact with the evaluated biomaterials (agar diffusion method). In addition, cell viability was evaluated after 5 days of incubation with biomaterial using ThinCert tissue culture inserts. Then, the following in vivo examinations were conducted: acute systemic toxicity, skin irritation and sensitization, and local effects after implantation.
The evaluated composite material showed a high degree of cytocompatibility and biocompatibility according to the International Standards.
The preclinical evaluation we performed on the new, polylactide-based allogenic biomaterial opens up possibilities to patent pending and advanced in vivo testing.
•According to EN 10993-5:2009 the tested biomaterial is not cytotoxic to 3T3 cells.•According to EN 10993-5:2009 the tested biomaterial is not cytotoxic to hFOB 1.19 cells.•There were no adverse effects on any of the animals according to EN 10993-5:2009.
Eight Holstein cows (4 primiparous and 4 multiparous) were used in a replicated 4×4 Latin square design to determine milk production response and N balance when diets had no NRC-predicted excess of ...rumen-undegradable protein (RUP) or rumen-degradable protein (RDP), 10% RUP excess, 10% RDP excess, or 10% excess of both RUP and RDP. Diets were fed as a total mixed ration with (dry matter basis) 25% alfalfa silage, 25% corn silage, 19 to 21% corn grain, and varying proportions of solvent soybean meal and expeller soybean meal as primary sources of supplemental RDP and RUP, respectively. Milk yield and dry matter intake (DMI) were recorded daily, and total collection of feces and urine was completed in the last 3 d of each 21-d period. Dietary crude protein averaged 17.5 and 18.5% for the recommended and excess RDP diets, respectively, and 17.3 and 18.4% for the recommended and excess RUP diets, respectively. When cows were fed excess RUP diets in the form of expeller soybean meal, DMI and milk production increased, but the opposite was true when the diets contained excess RDP in the form of solvent soybean meal. Milk composition was not affected by RDP, RUP, or by parity, and there were no parity×RDP interactions for any of the measurements. However, apparent digestibility of neutral detergent fiber, dry matter, and N increased in multiparous cows but not in primiparous cows because of excess RUP. The increase in the yield of milk N with excess RUP was not influenced by parity, but multiparous cows retained more of the additional N apparently absorbed, whereas primiparous cows excreted the additional apparently absorbed N in the urine. Overall, the difference in urinary N due to parity (70g/d) was about 4 times greater than the impact of dietary treatments (17g/d). Our results suggest that multiparous cows have either a much larger urea pool or a greater demand to restore body protein mobilized earlier in lactation compared with primiparous cows. Reduction in urinary N excretion in commercial dairy herds could be obtained by separately balancing rations for first and later lactations.
In this paper, we present cytotoxicity tests performed in a microanalytical hybrid system. The microchambers of cell culture were integrated with a concentration gradient generator (CGG), which ...created five different concentrations of evaluated agent in a single step. 5-Fluorouracil (5-FU) is one of the most important chemotherapeutic agents for cancer treatment, therefore it was tested in the microsystem. Independent tests of the cytotoxic effect of 5-FU were performed in this microsystem on different cell lines (human lung carcinoma cells – A549 and human colon carcinoma cells – HT-29). We also present the dependence of cell viability at different times of incubation with 5-FU. We proved that 5-FU has a stronger inhibition effect on A549 than on HT-29 cells. Moreover, the results obtained in the microfluidic system were compared with that of a macroscale. It can be concluded that the conditions of cultivation can affect the level of cytotoxicity of drugs, and the microsystem is a good example that could be used in this field of research.
The DNA methyltransferase (DNMT) inhibitors azacytidine and decitabine are the most successful epigenetic drugs to date and are still the most widely used as epigenetic modulators, even though their ...application for oncological diseases is restricted by their relative toxicity and poor chemical stability. Zebularine (1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one), a more stable and less toxic cytidine analog, is another inhibitor of DNMT with concomitant inhibitory activity towards cytidine deaminase. Unfortunately, there is no new information related to the possible clinical applications of zebularine. Although many new inhibitors of DNMT have been identified, none of them can so far replace azacytidine, decitabine and, to a lesser degree, zebularine. This review summarizes the current data and knowledge about azacytidine, decitabine and zebularine, and their role in present and possible future epigenetic cancer therapy. We also discuss the molecular modes of action of these agents with consideration of their different toxicities and demethylation profiles, reflecting their complex and partially overlapping biological effects.
Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of hematopoietic stem cells. At the molecular level, the disorder results from t(9;22)(q34;q11) reciprocal translocation between ...chromosomes, which leads to the formation of an oncogenic
gene fusion. Instead of progress in the understanding of the molecular etiology of CML and the development of novel therapeutic strategies, clinicians still face many challenges in the effective treatment of patients. In this review, we discuss the pathways of diagnosis and treatment of patients, as well as the problems appearing in the course of disease development. We also briefly refer to several aspects regarding the current knowledge on the molecular basis of CML and new potential therapeutic targets.
While light controls dormancy occurrence in various groups of organisms, it may play different roles in its initiation and termination. Seasonally changing photoperiod is frequently used to announce ...cyclical events and trigger diapause initiation in advance of seasonal changes. A similar role for photoperiod has often been uncritically extended to diapause termination. In the present study we investigated the role of photoperiod and light quantity in diapause termination of resting eggs of a freshwater crustacean. In a few tests, a cohort of resting eggs of Daphnia magna were incubated under various light conditions. A short-day photoperiod appeared as successful as a long day or artificial one in reactivating resting eggs. The hatching proportion was more related to total light energy applied than to its temporal pattern. The hatching proportion increased asymptotically from 0% reported after 6 h of light exposition or shorter, to 32% after 48 h of total light period of moderate intensity (55 μmol m−2 s−1). Longer light exposition did not increase hatching success considerably. A 15 times lower light intensity (3.5 vs. 55 μmol m−2 s−1) applied for a given period of time (96 h) resulted in a 3 times lower hatching success. Our results challenge the common opinion of the decisive role of photoperiod in diapause termination and support the alternative light-energy threshold hypothesis. It seems that the resting eggs of D. magna do not monitor seasonal changes of the photoperiod but reactivate after completion of the refractory period and accumulation of a certain amount of light energy dose.
Background: The development of colon cancer is probably angiogenesis-dependent. Recently, sulindac sulfide was shown to possess
anti-angiogenic activity. In the present work, the question of whether ...this activity reflects a specific interaction with
angiogenesis or is secondary to the effect of sulindac sulfide on the survival of endothelial cells was addressed. Materials
and Methods: Endothelial and normal mouse fibroblast cell lines were incubated with non-steroidal anti-inflammatory drugs
(NSAIDs), arachidonic acid (AA) and prostaglandin E 2 (PGE 2 ). Cell viability (survival), PGE 2 synthesis, cell cycle and apoptosis were measured. Western blotting and semi-quantitative RT-PCR multiplex methods verified
the changes in the levels of pro-apoptotic proteins and their expressions, respectively. Results: Sulindac sulfide and celecoxib
inhibited the survival of endothelial cells, whereas other NSAIDs were ineffective. In contrast to celecoxib, sulindac sulfide
did not affect the survival of normal fibroblast cells. Both agents inhibited the production of PGE 2 from AA and arrested the cell cycle in the S-phase. Moreover, sulindac sulfide activated caspases 3 and 8, decreased the
levels of Bax and Bid proteins, caused cleavage of PARP and increased the expressions of the bax and caspase 3 genes. Conclusion:
The results suggest that the anti-angiogenic activity of sulindac sulfide is secondary to the inhibition of endothelial cell
survival resulting from cell cycle arrest and apoptosis.
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