Impact of miRNAs expression modulation on the methylation status of breast cancer stem cell-related genes

Purpose Altered miRNAs play a crucial role in the emergence of the breast cancer stem cell (BCSC) phenotype. The interplay between miRNAs and methylation enzymes has been documented. One of the most aggressive breast cancer cell lines, MDA-MB-231, has expressed much more DNMT3B than DNMT3A. This study aims to evaluate the ability of miR-203 restoration and miR-150 inhibition to regulate DNMT3B and DNMT3A to modify the methylation level of BCSC-associated genes. Methods MDA-MB-231 cells were transfected with miR-203 mimic or miR-150 inhibitor or DNMT3B siRNA, and downstream analysis was performed by flow cytometry, real-time PCR and Western blotting. Results DNMT3A and DNMT3B are regulated both by miR-203a-3p and miR-150-5p. Transfection with miR-203 mimic and miR-150 inhibitor significantly reduced the CD44+CD24− subpopulation and down-regulated the expression of CD44 mRNA by increasing promoter methylation levels. SiRNA knockdown of DNMT3B increased the CD44+CD24− subpopulation and the expression of CD44 and ALDH1A3 by decreasing methylation density. The inhibition of miR-150 down-regulated OCT3/4 and SOX2 expression without affecting methylation levels, while miR-203 restoration and miR-150 inhibition down-regulated NANOG expression by elevating the methylation level. A positive-feedback loop was found between miR-203 and its target DNMT3B, as restoring miR-203 suppressed DNMT3B, while knocking down DNMT3B up-regulated miR-203. The restoration of miR-203 and knockdown of DNMT3B decreased methylation levels and increased the expression of miR-141 and miR-200c. Conclusions The study concluded that miR-203 and miR-150 play a role in the regulation of genes involved in BCSC methylation, including other miRNAs, by targeting DNMT3B and DNMT3A (AU)

Impact of miRNAs expression modulation on the methylation status of breast cancer stem cell-related genes.

PURPOSE: Altered miRNAs play a crucial role in the emergence of the breast cancer stem cell (BCSC) phenotype. The interplay between miRNAs and methylation enzymes has been documented. One of the most aggressive breast cancer cell lines, MDA-MB-231, has expressed much more DNMT3B than DNMT3A. This study aims to evaluate the ability of miR-203 restoration and miR-150 inhibition to regulate DNMT3B and DNMT3A to modify the methylation level of BCSC-associated genes. METHODS: MDA-MB-231 cells were transfected with miR-203 mimic or miR-150 inhibitor or DNMT3B siRNA, and downstream analysis was performed by flow cytometry, real-time PCR and Western blotting. RESULTS: DNMT3A and DNMT3B are regulated both by miR-203a-3p and miR-150-5p. Transfection with miR-203 mimic and miR-150 inhibitor significantly reduced the CD44+CD24- subpopulation and down-regulated the expression of CD44 mRNA by increasing promoter methylation levels. SiRNA knockdown of DNMT3B increased the CD44+CD24- subpopulation and the expression of CD44 and ALDH1A3 by decreasing methylation density. The inhibition of miR-150 down-regulated OCT3/4 and SOX2 expression without affecting methylation levels, while miR-203 restoration and miR-150 inhibition down-regulated NANOG expression by elevating the methylation level. A positive-feedback loop was found between miR-203 and its target DNMT3B, as restoring miR-203 suppressed DNMT3B, while knocking down DNMT3B up-regulated miR-203. The restoration of miR-203 and knockdown of DNMT3B decreased methylation levels and increased the expression of miR-141 and miR-200c. CONCLUSIONS: The study concluded that miR-203 and miR-150 play a role in the regulation of genes involved in BCSC methylation, including other miRNAs, by targeting DNMT3B and DNMT3A.

Integrated catchment management for reducing pesticide levels in water: Engaging with stakeholders in East Anglia to tackle metaldehyde.

In the agriculture intensive eastern region of England, plant protection products are widely applied to protect crops such as wheat and oilseed rape from pests and diseases, thus creating a risk of reaching nearby water courses through surface runoff. The EU Drinking Water Directive sets a stringent limit of 0.1 µg/l and 0.5 µg/l for individual and total pesticides respectively in treated potable water. However, peak metaldehyde levels have been persistently detected in raw water and reducing them to these limits has proven challenging and costly, in particular when using conventional treatment. In line with the EU Water Framework Directive, a more suitable approach and one adopted by the local water company, Anglian Water Services Ltd., would require moving towards mitigating pollution at source, preferably through participative action with multiple stakeholders in the agricultural industry. Initial findings demonstrate the potential of product substitution for reducing metaldehyde levels in surface waters. Reviewing Anglian Water's "Slug it Out" trial, we discuss key learnings derived from their experiences and make recommendations about the potential of the catchment approach to address the wider pesticide challenge.

Effect of topical mometasone furoate on adenoidal lymphoid tissue: a light microscopic study.

BACKGROUND: Adenoid hypertrophy is a common cause of upper airway obstruction, and adenoidectomy is one of the most frequently performed operations in children. Topical nasal steroids can act directly on nasopharyngeal lymphoid tissue to decrease its reactive inflammatory changes and potentially reduce its size. OBJECTIVE: To study the light microscopic changes in adenoidal lymphoid tissue after one month of topical steroid use. METHODS: Twenty-six children with adenoid hypertrophy grade 3 scheduled for adenoidectomy were randomly divided into two equal groups: one group received mometasone furoate aqueous nasal spray (Nasonex) 100 mcg/day for four weeks, and a control group received nasal normal saline 0.9 per cent for four weeks. The removed adenoids were examined histopathologically. RESULTS: Adenoidal tissue from the mometasone group had less reactive germinal centres and less spongiosis compared to the control group. The latter showed proliferating, reactive, variable sized and shaped lymphoid follicles, with congested blood vessels in the interfollicular areas. CONCLUSION: The use of intranasal mometasone furoate aqueous nasal spray (Nasonex) for one month reduced adenoidal tissue reactive cellular changes and its vascularity. This is, however, a pilot study; a longer treatment period is needed to assess the effect of treatment on adenoidal size.

Oxidative hemolysis of erythrocytes induced by various vitamins.

Hemolytic effect of some water-soluble vitamins (niacin B5, pyridoxine B6, thiamine B1 and ascorbic and acid C) on erythrocytes was studied spectrophotometrically at relatively high concentration. The oxidation mechanism of hemoglobin was the same for the used vitamins. Vitamin C was the strongest hemolytic agent in comparison with the other vitamins, while vitamin B1 is the weakest one. The results were confirmed by studying the variation in conductivity of erythrocytes with temperature in the range 20-40°C for the used vitamins at a concentration of 2 mM and after two hours from adding each vitamin to the erythrocytes suspension. The conductivity measurements show that the conductivity for the used vitamins is lower than that for control (without adding vitamin) due to hemoglobin oxidation, also may be due to the electrical reorganization of the erythrocyte membrane after the interaction of the used vitamin with it. The obtained results insure the oxidizing effect of the used vitamins on hemoglobin and consequently their hemolytic effect on erythrocytes.

Isolation and characterization of histogranin, a natural peptide with NMDA receptor antagonist activity.

Histogranin, was co-purified with bombesin-like immunoreactive peptides from bovine adrenal medulla. Its structure, H-Met-Asn-Tyr-Ala-Leu-Lys-Gly-Gln-Gly-Arg-Thr-Leu-Tyr-Gly-Phe-COOH, was determined by gas-phase Edman degradation. It was in accordance with its amino acid composition and corresponded to a 15 amino acid fragment (fragment 86-100) of histone H4 with substitutions in positions 1 (Val), 2 (Val) and 7 (Arg). The peptide was synthesized by the solid-phase procedure and the synthetic product was identical to the natural peptide as determined by its retention time on three analytical high-performance liquid chromatography systems. An antibody was raised against synthetic [Ser1]histogranin and used to monitor the presence of histogranin in various rat tissues and subcellular fractions of bovine adrenal medulla. In rats, immunoreactive histogranin was mainly concentrated in the pituitary (5065 pmol/g) and the adrenal glands (268 pmol/g), but it was also present in other tissues including the brain (1.6 pmol/g) and blood plasma (24 fmol/ml). A neuropeptide function for the adrenal peptide was suggested by its relative high concentration in chromaffin granules (42 fmol/mg protein as compared with 1 fmol/mg protein in cytosol) and its release from perfused bovine adrenal glands. In rat brain membrane preparations, synthetic histogranin displaced the binding of [3H]CGP 39653, a specific ligand of N-methyl-D-aspartate (NMDA) receptor. The displacement curve was biphasic with IC50 of 0.6 and 3955 nM, representing 33% and 67% of the binding sites, respectively. Intracerebroventricular (i.c.v.) injection of the peptide (5-100 nmol) in mice produced a dose-dependent protection against NMDA (0.5-1.0 nmol) -induced convulsions but not against (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA, 0.25-2.0 nmol), kainate (0.25-0.75 nmol) and bicuculline (1-10 nmol)-induced convulsions. These results suggest that histogranin may be an endogenous modulator of NMDA receptor functions.

Design of potent and selective dynorphin A related peptides devoid of supraspinal motor effects in mice.

Dynorphin A-(1-13)-Tyr-Leu-Phe-Asn-Gly-Pro (Dyn Ia) was previously shown to be a highly potent and selective kappa opioid peptide. Four analogs of Dyn Ia are synthesized by the solid-phase procedure, introducing pseudo CH2NH linkage between positions 6 and 7 as follows: analog 1, [6 psi 7 (CH2NH)]Dyn Ia; analog 2, [6 psi 7 (CH2NH), D-Leu8]Dyn Ia; analog 3, [N(Me)-Tyr1, 6 psi 7 (CH2NH)]Dyn Ia; and analog 4, [N(Me)-Tyr1, 6 psi 7 (CH2NH), D-Leu8]Dyn Ia. The purified peptides are compared in vitro with Dyn Ia for their ability to compete with the binding of selective kappa, mu, and delta opioid ligands using membrane preparations of guinea pig cerebellum (kappa) and rat brain (mu and delta). The synthetic compounds are also compared in vivo in mice (intracerebroventricularly administered) for their analgesic activity against acetic acid induced writhing and their ability to produce motor dysfunction. All compounds display a high affinity (Ki = 0.5-1.8 nM) and a good selectivity for the kappa opioid receptor, and their rank order of potency on the kappa site (analog 2 > analog 1 > analog 3 > analog 4) closely parallels their potency (AD50 = 1.57-5 nmol/mouse) in inhibiting acetic acid induced writhing in mice (analog 2 > analog 1 > analog 4 > analog 3). On the other hand, all the synthetic analogs are less potent than Dyn Ia in producing motor effects, analog 2 being the least potent (CD50 = 15.4 nM as compared with 2.9 nM for Dyn Ia).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of different types of textile fabric on spermatogenesis: electrostatic potentials generated on the surface of the human scrotum by wearing different types of fabric.

This study was conducted to evaluate the effect of the electrostatic potentials generated on the surface of the scrotal area when different types of textile fabric were worn. Twenty-one healthy volunteers were divided into three equal groups. The first group was dressed in underpants made of 100% polyester, the second wore underpants of 100% cotton, and the third wore a 50/50% polyester/cotton mixture. With an electrostatic kilovoltmeter, the electrostatic potentials were measured 1 h after wearing the pants once during the day and a second time at night. The test was repeated 4 times, each on a separate day. No electrostatic potentials were detected on the cotton underpants. The polyester pants showed the highest potentials (mean 338.9 +/- 25 SD V/cm2), while the mixed polyester/cotton pants produced less than half that level (mean 148.3 +/- 16 SD V/cm2). The readings during the day were higher than those at night, probably due to the higher temperature during the day. This study could explain the cause of diminished spermatogenesis in dogs dressed in polyester pants. A new theory is put forward holding that the polyester underpants create an "electrostatic field" across the scrotal sac that disturbs the testicular and/or epididymal function.

Effect of different types of textile fabric on spermatogenesis. I. Electrostatic potentials generated on surface of human scrotum by wearing different types of fabric.

This paper comprises a study of the electrostatic potentials generated on the surface of the scrotal area when different types of textile fabric were worn. 21 healthy volunteers were divided into 3 equal groups. The first group was dressed in underpants made of 100% polyester, the second of 100% cotton, and the third of a 50:50% polyester:cotton mixture. With an electrostatic kilovoltmeter, the electrostatic potentials were measured 1 h after wearing the pants once at daytime and a second time at night. The test was repeated 4 times, each on a separate day. No electrostatic potentials were detected on the cotton underpants. The polyester pants showed the highest potentials (mean 338.9 +/- 25 SD Volt cm-2) while the mixed polyester:cotton pants produced less than half that level (mean 148.3 +/- 16 SD Volt cm-2). The readings at daytime were higher than at night, probably due to the higher temperature during the day. The accumulated electrostatic charges on the pants are due to the friction of the pants with the skin. As a result of friction, equal and opposite charges are accumulated on the skin surface and on the inner surface of the pants facing the skin. Eventually, an 'electrostatic field' is produced traversing the scrotal contents and may disturb the testicles and/or epididymis leading to disordered spermatogenesis.

Synthesis of allyl glycosides for conversion into neoglycoproteins bearing epitopes of mycobacterial glycolipid antigens.

Neoglycoproteins bearing key glycosyl substituents of several glycopeptidolipid antigens of pathogenic Mycobacterium species have been synthesized. Allyl glycosides of the terminal 6-deoxyhexose-containing units of the antigens were prepared, with appropriate ether and ester substituents in place. Ozonolysis of the allyl glycosides was then followed by reductive coupling with epsilon-amino groups of lysine residues in bovine serum albumin, using sodium cyanoborohydride at pH 7.8. The resulting neoglycoproteins emulated the antigenicity of the native molecule in several serological tests.