A Rapid and Inexpensive PCR Test for Mastitis Diagnosis Based on NGS Data.

Mastitis is a common mammary gland disease of dairy cattle caused by a wide range of organisms including bacteria, fungi and algae. Mastitis contributes to economic losses of dairy farms due to reduced yield and poor quality of milk. Since the correct identification of pathogens responsible for the development of mastitis is crucial to the success of treatment, it is necessary to develop a quick and accurate test to distinguish the main pathogens causing this disease. In this paper, we describe the development of a test based on the multiplex polymerase chain reaction (PCR) method allowing for the identification of Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Staphylococcus aureus. When creating our test, we relied on the results from new generation sequencing (NGS) for accurate determination of species affiliation. The multiplex PCR test was verified on 100 strains including veterinary samples, ATCC and Polish Collection of Microorganisms (PCM) reference strains. The obtained results indicate that this test is accurate and displays high specificity. It may serve as a valuable molecular tool for the detection of major mastitis pathogens.

Cell Cycle Status Influences Resistance to Apoptosis Induced by Oxidative Stress in Human Breast Cancer Cells, Which Is Accompanied by Modulation of Autophagy.

Cancer cells are characterised by uncontrolled cell proliferation; however, some of them can temporarily arrest their cell cycle at the G0 or G1 phase, which could contribute to tumour heterogeneity and drug resistance. The cell cycle status plays a critical role in chemosensitivity; however, the influence of G0- and G1-arrest has not been elucidated. To study the cell cycle arrest-mediated resistance, we used MCF-7 cells and generated three populations of cells: (1) cells arrested in the G0-like phase, (2) cells that resumed the cell cycle after the G0-like phase and (3) cells arrested in early G1 with a history of G0-like arrest. We observed that both the G0-like- and the G1-arrested cells acquired resistance to apoptosis induced by oxidative stress, accompanied by a decreased intracellular reactive oxygen species and DNA damage. This effect was associated with increased autophagy, likely facilitating their survival at DNA damage insult. The cell cycle reinitiation restored a sensitivity to oxidative stress typical for cells with a non-modulated cell cycle, with a concomitant decrease in autophagy. Our results support the need for further research on the resistance of G0- and G1-arrested cancer cells to DNA-damaging agents and present autophagy as a candidate for targeting in anticancer treatment.

New Aspect of Composition and Biological Properties of Glechoma hederacea L. Herb: Detailed Phytochemical Analysis and Evaluation of Antioxidant, Anticoagulant Activity and Toxicity in Selected Human Cells and Plasma In Vitro.

It is known that phenolic compounds can alleviate the negative impact of oxidative stress and modulate hemostasis. However, the effect of extracts and phenolics from Glechoma hederacea L. on the biomarkers of these processes is not well documented. The aim of our study was to investigate the in vitro protective effects of one extract and three fractions (20, 60, and 85% fraction) from G. hederacea L. on oxidative stress and hemostasis. Phytochemical analysis showed that aerial parts of G. hederacea L. are rich in both phenolic acids (such as rosmarinic acid, neochlorogenic acid, and chlorogenic acid) and flavonoids (mainly rutin and glycoside derivatives of apigenin, quercetin, and luteolin). We observed that the 85% fraction (at three concentrations: 5, 10, and 50 µg/mL) inhibited protein carbonylation. Moreover, the extract and 85% fraction (at the concentration of 50 µg/mL) could reduce lipid peroxidation. All fractions and the extract were very effective at decreasing H2O2-induced DNA damage in PBM cells. The 85% fraction had the strongest protective potential against DNA oxidative damage. We also observed that the extract and fractions decreased PBM cell viability to a maximum of 65% after 24 h incubation. Our results indicate that the 85% fraction showed the strongest antioxidant potential. The main component of the 85% fraction was apigenin (26.17 ± 1.44 mg/g), which is most likely responsible for its strong antioxidant properties.

Antioxidant Activity of Ruthenium Cyclopentadienyl Complexes Bearing Succinimidato and Phthalimidato Ligands.

In these studies, we investigated the antioxidant activity of three ruthenium cyclopentadienyl complexes bearing different imidato ligands: (η5-cyclopentadienyl)Ru(CO)2-N-methoxysuccinimidato (1), (η5-cyclopentadienyl)Ru(CO)2-N-ethoxysuccinimidato (2), and (η5-cyclopentadienyl)Ru(CO)2-N-phthalimidato (3). We studied the effects of ruthenium complexes 1-3 at a low concentration of 50 µM on the viability and the cell cycle of peripheral blood mononuclear cells (PBMCs) and HL-60 leukemic cells exposed to oxidative stress induced by hydrogen peroxide (H2O2). Moreover, we examined the influence of these complexes on DNA oxidative damage, the level of reactive oxygen species (ROS), and superoxide dismutase (SOD) activity. We have observed that ruthenium complexes 1-3 increase the viability of both normal and cancer cells decreased by H2O2 and also alter the HL-60 cell cycle arrested by H2O2 in the sub-G1 phase. In addition, we have shown that ruthenium complexes reduce the levels of ROS and oxidative DNA damage in both cell types. They also restore SOD activity reduced by H2O2. Our results indicate that ruthenium complexes 1-3 bearing succinimidato and phthalimidato ligands have antioxidant activity without cytotoxic effect at low concentrations. For this reason, the ruthenium complexes studied by us should be considered interesting molecules with clinical potential that require further detailed research.

Effect of Kaempferol and Its Glycoside Derivatives on Antioxidant Status of HL-60 Cells Treated with Etoposide.

Kaempferol is a well-known antioxidant found in many plants and plant-based foods. In plants, kaempferol is present mainly in the form of glycoside derivatives. In this work, we focused on determining the effect of kaempferol and its glycoside derivatives on the expression level of genes related to the reduction of oxidative stress-NFE2L2, NQO1, SOD1, SOD2, and HO-1; the enzymatic activity of superoxide dismutases; and the level of glutathione. We used HL-60 acute promyelocytic leukemia cells, which were incubated with the anticancer drug etoposide and kaempferol or one of its three glycoside derivatives isolated from the aerial parts of Lens culinaris Medik.-kaempferol 3-O-[(6-O-E-caffeoyl)-ß-d-glucopyranosyl-(1→2)]-ß-d-galactopyranoside-7-O-ß-d-glucuropyranoside (P2), kaempferol 3-O-[(6-O-E-p-coumaroyl)-ß-d-glucopyranosyl-(1→2)]-ß-d-galactopyranoside-7-O-ß-d-glucuropyranoside (P5), and kaempferol 3-O-[(6-O-E-feruloyl)-ß-d-glucopyranosyl-(1→2)]-ß-d-galactopyranoside-7-O-ß-d-glucuropyranoside (P7). We showed that none of the tested compounds affected NFE2L2 gene expression. Co-incubation with etoposide (1 µM) and kaempferol (10 and 50 µg/mL) leads to an increase in the expression of the HO-1 (9.49 and 9.33-fold at 10 µg/mL and 50 µg/mL, respectively), SOD1 (1.68-fold at 10 µg/mL), SOD2 (1.72-fold at 10-50 µg/mL), and NQO1 (1.84-fold at 50 µg/mL) genes in comparison to cells treated only with etoposide. The effect of kaempferol derivatives on gene expression differs depending on the derivative. All tested polyphenols increased the SOD activity in cells co-incubated with etoposide. We observed that the co-incubation of HL-60 cells with etoposide and kaempferol or derivative P7 increases the level of total glutathione in these cells. Taken together, our observations suggest that the antioxidant activity of kaempferol is related to the activation of antioxidant genes and proteins. Moreover, we observed that glycoside derivatives can have a different effect on the antioxidant cellular systems than kaempferol.

Multifunctional compounds in the extract from mature seeds of Vicia faba var. minor: Phytochemical profiling, antioxidant activity and cellular safety in human selected blood cells in in vitro trials.

BACKGROUND: The field bean (Vicia faba) is a valuable fodder plant of the Fabaceae family, grown as a main crop for its seed yield. Its phytochemical profile is characterized by the presence of a range of compounds with various biological activities. PURPOSE: The present study investigates the phytochemical profile of the extract from mature seeds of Vicia faba var. minor and examines its impact on preventing oxidative damage to various lipids, protein and DNA molecules in vitro. METHODS: Human plasma was treated with H2O2/Fe (an OH. donor) to induce oxidative damage to lipids and proteins, and the plant extract was then added. As oxidative stress may influence the biological activity of plasma, e.g. coagulation, and influence cardiovascular disease, the study also examined the effect of the plant extract on coagulation and monoamine oxidase activity (MAO, EC 1.4.3.4). RESULTS: The tested extract exerted a protective effect on plasma lipids and proteins treated with H2O2/Fe. However, while it appears to effectively protect the DNA in peripheral blood mononuclear cells from oxidative damage, it did not induce changes in the coagulation process, and significantly reduced MAO activity when applied at 1, 5, and 10 µg/mL. It is possible that the observed antioxidant potential may be due to the complex chemical composition of the extract: the phytochemical profile demonstrated a range of phenolic compounds, including catechins. CONCLUSION: Our findings indicate that extract from mature seeds of V. faba var. minor may be a promising source of antioxidants in multiple applications, including diseases associated with oxidative stress; however, more studies based on in vitro and in vivo models are needed to determine its biological properties.

Natural Polyphenols as Modulators of Etoposide Anti-Cancer Activity.

Polyphenols are naturally occurring compounds found in abundance in fruits and vegetables. Their health-promoting properties and their use in the prevention and treatment of many human diseases, including cancer, have been known for years. Many anti-cancer drugs are derived from these natural compounds. Etoposide, which is a semi-synthetic derivative of podophyllotoxin, a non-alkaloid lignan isolated from the dried roots and rhizomes of Podophyllum peltatum or Podophyllum emodi (Berberidaceae), is an example of such a compound. In this review, we present data on the effects of polyphenols on the anti-cancer activity of etoposide in in vitro and in vivo studies.

Kaempferol and Its Glycoside Derivatives as Modulators of Etoposide Activity in HL-60 Cells.

Kaempferol is a polyphenol found in a variety of plants. Kaempferol exerts antitumor properties by affecting proliferation and apoptosis of cancer cells. We investigated whether kaempferol and its glycoside derivatives-kaempferol 3-O-[(6-O-E-caffeoyl)-ß-D-glucopyranosyl-(1→2)]-ß-D-galactopyranoside-7-O-ß-D-glucuropyranoside (P2), kaempferol 3-O-[(6-O-E-p-coumaroyl)-ß-D-glucopyranosyl-(1→2)]-ß-D-galactopyranoside-7-O-ß-D-glucuropyranoside (P5) and kaempferol 3-O-[(6-O-E-feruloyl)-ß-D-glucopyranosyl-(1→2)]-ß-D-galactopyranoside-7-O-ß-D-glucuropyranoside (P7), isolated from aerial parts of Lens culinaris Medik.-affect the antitumor activity of etoposide in human promyelocytic leukemia (HL-60) cells. We analyzed the effect of kaempferol and its derivatives on cytotoxicity, DNA damage, apoptosis, cell cycle progression and free radicals induced by etoposide. We demonstrated that kaempferol increases the sensitivity of HL-60 cells to etoposide but does not affect apoptosis induced by this drug. Kaempferol also reduces the level of free radicals generated by etoposide. Unlike kaempferol, some of its derivatives reduce the apoptosis of HL-60 cells (P2 and P7) and increase the level of free radicals (P2 and P5) induced by etoposide. Our results indicate that kaempferol and its glycoside derivatives can modulate the activity of etoposide in HL-60 cells and affect its antitumor efficacy in this way. Kaempferol derivatives may have the opposite effect on the action of etoposide in HL-60 cells compared to kaempferol.

Multidirectional effects of saponin fraction isolated from the leaves of sea buckthorn Elaeagnus rhamnoides (L.) A. Nelson.

Many studies show that saponins isolated from various plants have a cytotoxic effect on cancer cells inducing apoptosis and autophagy. On the other hand, saponins also exhibit a number of beneficial properties, such as antioxidant properties. Thus, saponins can be considered both in terms of their therapeutic and protective effects during anticancer treatment. In this study, we investigated the effect of the saponin fraction isolated from sea buckthorn (Elaeagnus rhamnoides (L.) A. Nelson) leaves on the viability of HL-60 cancer cells using resazurin assay and its ability to induction of apoptosis with Annexin V-FITC and propidium iodide (PI) double staining. Moreover, we studied its effect on the oxidative stress induced by H2O2, and anti-platelet and anticoagulant potential in whole blood using T-TAS, a microchip-based flow chamber system. We observed that the saponin fraction significantly decreased the viability of HL-60 cells at the concentration above 50 µg/mL and induced apoptosis at the concentration of 100 µg/mL. Moreover, we observed that saponin fraction used at lower concentrations, such as 0.5 and 1 µg/mL, stimulated HL-60 cells and increased their viability. The saponin fraction also decreased the level of free radicals and reduced oxidative DNA damage measured by the comet assay. However, at high concentration of oxidant H2O2 equal 5 mM, we noticed that the saponin fraction at 50 µg/mL increased the level of free radicals in HL-60 cells. We also demonstrated anticoagulant potential of the saponin fraction at the concentration of 50 µg/mL. Our results indicate that the saponin fraction obtained from sea buckthorn leaves can show both chemotherapeutic and chemoprotective potential.

DNA damage and antioxidant properties of CORM-2 in normal and cancer cells.

In this study, we compared the effect of tricarbonyldichlororuthenium (II) dimer (CORM-2) and its CO-depleted molecule (iCORM-2) on human peripheral blood mononuclear cells (PBMCs) and human promyelocytic leukemia HL-60 cells. We determined cell viability, DNA damage and DNA repair kinetics. We also studied the effect of both compounds on DNA oxidative damage, free radical level and HO-1 gene expression. We showed that at low concentrations both CORM-2 and iCORM-2 stimulate PBMCs viability. After 24-h incubation, CORM-2 and iCORM-2, at the concentration of 100 µM, reduce the viability of both PBMCs and HL-60 cells. We also demonstrated that CORM-2 and iCORM-2, in the 0.01-100 µM concentration range, cause DNA damage such as strand breaks and alkaline labile sites. DNA damage was repaired efficiently only in HL-60 cells. CORM-2 significantly reduces oxidative stress induced by 1 mM H2O2 in normal and cancer cells. On the contrary, iCORM-2 in HL-60 cells increases the level of free radicals in the presence of 1 and 5 mM H2O2. We also revealed that both CORM-2 and iCORM-2 induce HO-1 gene expression. However, CORM-2 induces this gene to a greater extent than iCORM-2, especially in HL-60 cells at 100 µM. Finally, we showed that CORM-2 and iCORM-2 reduce H2O2-induced DNA oxidative damage. Furthermore, CORM-2 proved to be a compound with stronger antioxidant properties than iCORM-2. Our results suggest that both active CORM-2 and inactive iCORM-2 exert biological effects such as cyto- and genotoxicity, antioxidant properties and the ability to induce the HO-1 gene. The released CO as well as iCORM-2 can be responsible for these effects.

Photoactive CO-releasing complexes containing iron - genotoxicity and ability in HO-1 gene induction in HL-60 cells.

This paper presents the results of research on the biological properties of two photoactive CO-releasing molecules containing iron, i.e. (η5-C5H5)Fe(CO)2(η1-N-maleimidato) (complex A) and (η5-C5H5)Fe(CO)2(η1-N-succinimidato) (complex B). We studied their cytotoxicity, genotoxicity and the ability of inducing the HO-1 gene in HL-60 cells. We also investigated the kinetics of DNA damage repair induced by complexes A and B. We demonstrated that complex B was not toxic to HL-60 cells in high doses (above 100 µM). The ability to induce DNA damage was higher for complex A. Importantly, there was no difference in irradiated and non-irradiated cells for both complexes. DNA damage induced by complex B was repaired efficiently, while the repair of DNA damage induced by complex A was disturbed. Complex B had a minor effect on HO-1 gene expression (less than 2-fold induction), while complex A had induced HO-1 gene expression to a great extent (over 17-fold for 10 µM) - similarly in irradiated and non-irradiated HL-60 cells. The results of our research indicate that the ability of both complexes to damage DNA and to upregulate HO-1 gene expression is not related to the release of CO. Further research is needed to test whether these compounds can be considered as potential CO carriers in humans.

Kaempferol derivatives isolated from Lens culinaris Medik. reduce DNA damage induced by etoposide in peripheral blood mononuclear cells.

Bioactive compounds isolated from plants are considered to be attractive candidates for cancer therapy. In this study, we examined the effect of kaempferol, its derivatives, the polyphenol fraction (PF) and an extract (EX) isolated from the aerial parts of Lens culinaris Medik. on DNA damage induced by etoposide in human cells. We also studied the effect of these compounds and their combinations on cell viability. The studies were conducted on HL-60 cells and human peripheral blood mononuclear cells (PBMCs). We used the comet assay in the alkaline version to evaluate DNA damage. To examine cell viability we applied the trypan blue exclusion assay. We demonstrated that kaempferol glycoside derivatives isolated from the aerial parts of Lens culinaris Medik. reduce DNA damage induced by etoposide in PBMCs, but do not have an impact on DNA damage in HL-60 cells. We also showed that kaempferol induces DNA damage in HL-60 cells and leads to an increase of DNA damage provoked by etoposide. Our data suggest that kaempferol derivatives can be further explored as a potential agent protecting normal cells against DNA damage induced by etoposide. Moreover, kaempferol's ability to induce DNA damage in cancer cells and to increase DNA damage caused by etoposide may be useful in designing and improving anticancer therapies.

Downregulation of PARP1 transcription by CDK4/6 inhibitors sensitizes human lung cancer cells to anticancer drug-induced death by impairing OGG1-dependent base excision repair.

Hallmarks of cancer cells include uncontrolled growth and rapid proliferation; thus, cyclin-dependent kinases are a therapeutic target for cancer treatment. Treating non-small lung cancer cells with sublethal concentrations of the CDK4/6 inhibitors, ribociclib (LEE011) and palbociclib (PD0332991), which are approved by the FDA for anticancer therapies, caused cell cycle arrest in the G1 phase and suppression of poly(ADP-ribose) polymerase 1 (PARP1) transcription by inducing recruitment of the RB1-E2F1-HDAC1-EZH2 repressive complex to the PARP1 promoter. Downregulation of PARP1 made cancer cells vulnerable to death triggered by the anticancer drugs (WP631 and etoposide) and H2O2. All agents brought about redox imbalance and DNA strand breaks. The lack of PARP1 and poly(ADP-ribosyl)ation impaired the 8-oxoguanine glycosylase (OGG1)-dependent base excision DNA repair pathway, which is critical for maintaining the viability of cells treated with CDK4/6 inhibitors during oxidative stress. Upon G1 arrest of PARP1 overexpressing cells, OGG1 formed an immunoprecipitable complex with PARP1. Similar to cells with downregulated PARP1 expression, inhibition of PARP1 or OGG1 in PARP1 overexpressing cells resulted in DNA damage and decreased viability. Thus, PARP1 and OGG1 act in the same regulatory pathway, and PARP1 activity is required for OGG1-mediated repair of oxidative DNA damage in G1-arrested cells. In conclusion, the action of CDK4/6 inhibitors is not limited to the inhibition of cell growth. CDK4/6 inhibitors also lead to accumulation of DNA damage by repressing PARP1 in oxidatively stressed cells. Thus, CDK4/6 inhibitors sensitize G1-arrested cells to anticancer drugs, since these cells require PARP1-OGG1 functional interaction for cell survival.