Carnosol Induces ROS-Mediated Beclin1-Independent Autophagy and Apoptosis in Triple Negative Breast Cancer

Background

In this study we investigated the in vitro and in vivo anticancer effect of carnosol, a naturally occurring polyphenol, in triple negative breast cancer.

Results

We found that carnosol significantly inhibited the viability and colony growth induced G2 arrest in the triple negative MDA-MB-231. Blockade of the cell cycle was associated with increased p21/WAF1 expression and downregulation of p27. Interestingly, carnosol was found to induce beclin1-independent autophagy and apoptosis in MDA-MB-231 cells. The coexistence of both events, autophagy and apoptosis, was confirmed by electron micrography. Induction of autophagy was found to be an early event, detected within 3 h post-treatment, which subsequently led to apoptosis. Carnosol treatment also caused a dose-dependent increase in the levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2). Moreover, we show that carnosol induced DNA damage, reduced the mitochondrial potential and triggered the activation of the intrinsic and extrinsic apoptotic pathway. Furthermore, we found that carnosol induced a dose-dependent generation of reactive oxygen species (ROS) and inhibition of ROS by tiron, a ROS scavenger, blocked the induction of autophagy and apoptosis and attenuated DNA damage. To our knowledge, this is the first report to identify the induction of autophagy by carnosol.

Conclusion

In conclusion our findings provide strong evidence that carnosol may be an alternative therapeutic candidate against the aggressive form of breast cancer and hence deserves more exploration.     

Site-directed mutagenesis of an alkaline phytase: influencing specificity, activity and stability in acidic milieu

Site-directed mutagenesis of a thermostable alkaline phytase from Bacillus sp. MD2 was performed with an aim to increase its specific activity and activity and stability in an acidic environment. The mutation sites are distributed on the catalytic surface of the enzyme (P257R, E180N, E229V and S283R) and in the active site (K77R, K179R and E227S). Selection of the residues was based on the idea that acid active phytases are more positively charged around their catalytic surfaces. Thus, a decrease in the content of negatively charged residues or an increase in the positive charges in the catalytic region of an alkaline phytase was assumed to influence the enzyme activity and stability at low pH. Moreover, widening of the substrate-binding pocket is expected to improve the hydrolysis of substrates that are not efficiently hydrolysed by wild type alkaline phytase. Analysis of the phytase variants revealed that E229V and S283R mutants increased the specific activity by about 19% and 13%, respectively. Mutation of the active site residues K77R and K179R led to severe reduction in the specific activity of the enzyme. Analysis of the phytase mutant-phytate complexes revealed increase in hydrogen bonding between the enzyme and the substrate, which might retard the release of the product, resulting in decreased activity. On the other hand, the double mutant (K77R-K179R) phytase showed higher stability at low pH (pH 2.6-3.0). The E227S variant was optimally active at pH 5.5 (in contrast to the wild type enzyme that had an optimum pH of 6) and it exhibited higher stability in acidic condition. This mutant phytase, displayed over 80% of its initial activity after 3 h incubation at pH 2.6 while the wild type phytase retained only about 40% of its original activity. Moreover, the relative activity of this mutant phytase on calcium phytate, sodium pyrophosphate and p-nitro phenyl phosphate was higher than that of the wild type phytase.     

Beam Steering Plasma Reflectarray/Transmitarray Antennas

The aim of this paper is to design and analyze plasma reflectarray/transmitarray antennas which include investigation of performance parameters for these antennas in free space. The unit cell element for both arrays consists of a cubic glass box with fixed dimensions filled by argon gas. The plasma frequency of the argon gas is varied by changing the applied voltage at both ends of the glass box. This allows steering the reflected/transmitted wave to a certain direction. Full-wave simulations using the finite element method (FEM) and finite integral technique (FIT) are used to optimize and analyze different plasma reflectarray and plasma transmitarray antennas. The plasma reflectarray antenna is composed of 13?×?13 elements and covered an area of 208?×?208 mm². A circular horn is used to feed the antenna at a distance of 20.8 cm (focal-to-diameter ratio (F/D)?=?1). The reflectarray is designed at f?=?12 GHz. The radiation patterns of the plasma reflectarray for scanning angles of 10° to 70° angles are illustrated. For larger scanning angles, significant sidelobe levels are produced. The maximum value of beam scanning gain patterns is reduced. The half-power beamwidth (HPBW) is increased. The plasma transmitarray is composed of 9?×?9 cell elements and covered an area of 72?×?72 mm². The feed is a linearly polarized circular horn. The F/D ratio is set to 1. The transmitarray is operating at f?=?15 GHz. The radiation patterns of the plasma transmitarray for scanning angles of ?40° to +40° are demonstrated. Significant sidelobe levels are produced at large scanning angles.     

Empagliflozin on top of metformin treatment improves arterial function in patients with type 1 diabetes mellitus

Background

Deteriorated arterial function and high incidence of cardiovascular events characterise diabetes mellitus. Metformin and recent antidiabetic drugs, SGLT2 inhibitors, reduce cardiovascular events. We explored the possible effects of empagliflozin’s effect on top of metformin treatment on endothelial function and arterial stiffness parameters in type 1 diabetes mellitus (T1DM) patients.

Methods

Forty T1DM patients were randomised into three treatment groups: (1) empagliflozin (25 mg daily), (2) metformin (2000 mg daily) and (3) empagliflozin/metformin (25 mg daily and 2000 mg daily, respectively). The fourth group received placebo. Arterial function was assessed at inclusion and after 12 weeks treatment by: endothelial function [brachial artery flow-mediated dilation (FMD), reactive hyperaemia index (RHI)], arterial stiffness [pulse wave velocity (PWV) and common carotid artery stiffness (β-stiffness)]. For statistical analysis one-way analysis of variance with Bonferroni post-test was used.

Results

Empagliflozin on top of metformin treatment significantly improved endothelial function as did metformin after 12 weeks of treatment: FMD [2.6-fold (P?<?0.001) vs. 1.8-fold (P?<?0.05)] and RHI [1.4-fold (P?<?0.01) vs. 1.3-fold (P?<?0.05)]. Empagliflozin on top of metformin treatment was superior to metformin in improving arterial stiffness parameters; it significantly improved PWV and β-stiffness compared to metformin [by 15.8% (P?<?0.01) and by 36.6% (P?<?0.05), respectively]. Metformin alone did not influence arterial stiffness.

Conclusion

Empagliflozin on top of metformin treatment significantly improved arterial stiffness compared to metformin in T1DM patients. Endothelial function was similarly improved in all treatment groups. Empagliflozin seems to possess a specific capacity to decrease arterial stiffness, which could support its cardioprotective effects observed in large clinical studies.Trial registration Clinical trial registration: NCT03639545

     

Root-favoured biomass allocation improves growth and yield of field-grown rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) plants only when the shoot sink is expandable

We tested the hypothesis that high root/shoot (R/S) in rice improves plant growth and yield when the shoot sink is expandable, and that in a genotype with exaggerated R/S ratio, the shoot growth is not limited by root resources. This study involved the three rice genotypes, Giza 178, PM12, and Moroberekan with a range of R/S ratios and shoot sink sizes. Root regrowth after trimming or high- and low-nitrogen treatments revealed that Moroberekan has consistently high root-favoured biomass partitioning than Giza 178 or PM12. Increasing the R/S ratios by detillering improved the culm growth in Giza 178 and PM12 (by 43.4 and 17.7% of control, respectively) but not Moroberekan, indicating that PM12 was closer to achieving its growth potential than Giza 178 but Moroberekan was operating at maximal shoot growth potential because of high R/S ratio and small sink size. Under drought, shoot growth, gas exchange, and grain yield correlated strongly with R/S ratio and root length density (RLD) in the droughted but not the well-watered plants. We further hypothesized that R/S ratio of Moroberekan was in excess of shoot requirement for optimum growth. Crossing Moroberekan to PM12 generated three F1 hybrids with intermediate R/S ratios but higher growth, gas exchange, and yield than either parent. We conclude that increasing the R/S ratio improved growth and yield in PM12 but not Moroberekan, because the shoot sink size was expandable in PM12. Moreover, lower R/S ratios than that of Moroberekan could support higher shoot growth if shoot sink is expandable.     

A novel signal transduction pathway in Saccharomyces cerevisiae defined by Snf3-regulated expression of HXT6.

We show that cells deleted for SNF3, HXT1, HXT2, HXT3, HXT4, HXT6, and HXT7 do not take up glucose and cannot grow on media containing glucose as a sole carbon source. The expression of Hxt1, Hxt2, Hxt3, Hxt6, or Gal2 in these cells resulted in glucose transport and allowed growth on glucose media. In contrast, the expression of Snf3 failed to confer glucose uptake or growth on glucose. HXT6 is highly expressed on raffinose, low glucose, or nonfermentable carbon sources but is repressed in the presence of high concentrations of glucose. The maintenance of HXT6 glucose repression is strictly dependent on Snf3 and not on intracellular glucose. In snf3 delta cells expression of HXT6 is constitutive even when the entire repertoire of HXT genes is present and glucose uptake is abundant. In addition, glucose repression of HXT6 does not require glucose uptake by HXT1, HXT2, HXT3 or HXT4. We show that a signal transduction pathway defined by the Snf3-dependent hexose regulation of HXT6 is distinct from but also overlaps with general glucose regulation pathways in Saccharomyces cerevisiae. Finally, glucose repression of ADH2 and SUC2 is intact in snf3 delta hxt1 delta hxt2 delta hxt3 delta hxt4 delta hxt6 delta hxt7 delta gal2 cells, suggesting that the sensing and signaling mechanism for general glucose repression is independent from glucose uptake.     

Frameshift Suppression in SACCHAROMYCES CEREVISIAE. III. Isolation and Genetic Properties of Group III Suppressors

Suppressors of ICR-induced mutations that exhibit behavior similar to bacterial frameshift suppressors have been identified in the yeast Saccharomyces cerevisiae. The yeast suppressors have been divided into two groups. Previous evidence indicated that suppressors of one group (Group II: SUF1, SUF3, SUF4, SUF5 and SUF6) represent mutations in the structural genes for glycyl-tRNA's. Suppressors of the other group (Group III: SUF2 and SUF7) were less well characterized. Although they suppressed some ICR-revertible mutations, they failed to suppress Group II frameshift mutations. This communication provides a more thorough characterization of the Group III suppressors and describes the isolation and properties of four new suppressors in that group (SUF8, SUF9, SUF10 and suf11).——In our original study, Group III suppressors were isolated as revertants of the Group III mutations his4–712 and his4–713. All suppressors obtained as ICR-induced revertants of these mutations mapped at the SUF2 locus near the centromere of chromosome III. Suppressors mapping at other loci were obtained in this study by analyzing spontaneous and UV-induced revertants of the Group III mutations. SUF2 and SUF10 suppress both Group III his4 mutations, whereas SUF7, SUF8, SUF9 and suf11 suppress his4–713, but not his4–712. All of the suppressors except suf11 are dominant in diploids homozygous for his4-713. The suppressors fail to suppress representative UAA, UAG and UGA nonsense mutations.——SUF9 is linked to the centromere of chromosome VI, and SUF10 is linked to the centromere of chromosome XIV. A triploid mapping procedure was used to determine the chromosome locations of SUF7 and SUF8. Subsequent standard crosses revealed linkage of SUF7 to cdc5 on chromosome XIII and linkage of SUF8 to cdc12 and pet3 on chromosome VIII.     

Codon recognition during frameshift suppression in Saccharomyces cerevisiae.

A genetic approach has been used to establish the molecular basis of 4-base codon recognition by frameshift suppressor tRNA containing an extra nucleotide in the anticodon. We have isolated all possible base substitution mutations at the position 4 (N) in the 3'-CCCN-5' anticodon of a Saccharomyces cerevisiae frameshift suppressor glycine tRNA encoded by the SUF16 gene. Base substitutions at +1 frameshift sites in the his4 gene have also been obtained such that all possible 4-base 5'-GGGN-3' codons have been identified. By testing for suppression in different strains that collectively represent all 16 possible combinations of position 4 nucleotides, we show that frameshift suppression does not require position 4 base pairing. Nonetheless, position 4 interactions influence the efficiency of suppression. Our results suggest a model in which 4-base translocation of mRNA on the ribosome is directed primarily by the number of nucleotides in the anticodon loop, whereas the resulting efficiency of suppression is dependent on the nature of position 4 nucleotides.     

Experiences from the implementation of a biosafety system in Slovenia

The development and implementation of an effective national biosafety system is important for several key reasons: to ensure safe access to products of modern biotechnology, to build public confidence, to encourage the growth of domestic modern biotechnology, and to comply with international standards and agreements. There is no single best approach in the development and implementation of a national biosafety system and each country is faced with unique challenges. Slovenia is a small country and a new EU Member State. However, it has developed and implemented an efficient national biosafety system. The key elements of this system are administrative procedure, risk assessment, enforcement, and public participation and information.