Propolis Enhances 5-Fluorouracil Mediated Antitumor Efficacy and Reduces Side Effects in Colorectal Cancer: An in Vitro and in Vivo Study

In this study, we investigated the combined treatment of 5-fluorouracil (5-FU) and Anatolian propolis extract (PE) on colorectal cancer (CRC)using in vitro and in vivo studies. We exposed luciferase-transfected (Lovo-Luc CRC) cells and healthy colon cells (CCD-18Co) to varying concentrations of 5-FU and PE to assess their genotoxic, apoptotic, and cytotoxic effects, as well as their intracellular reactive oxygen species (iROS) levels. We also developed a xenograft model in nude mice and evaluated the anti-tumor effects of PE and 5-FU using various methods. Our findings showed that the combination of PE and 5-FU had selectivity against cancer cells, particularly at higher doses, and enhanced the anti-tumor effectiveness of 5-FU against colon CRC. The results suggest that PE can reduce side effects and increase the effectiveness of 5-FU through iROS generation in a dose-dependent manner.     

Protective effect of quercetin on some hematological parameters in rats exposed to cadmium

ABSTRACT

We investigated the effects of quercetin (Q) on some hematological parameters and determined the percentage of alpha-naphthyl acetate esterase (ANAE) positive lymphocytes in rats that had been exposed to cadmium (Cd). Thirty male Wistar albino rats were divided into four groups: control (C), quercetin (Q), cadmium (Cd) and Q + Cd (CdQ). Blood samples were taken to assess erythrocytes (RBC), leukocytes (WBC), hemoglobin levels (Hb), hematocrit values (Hct), platelets (PLT), alpha-naphthyl acetate esterase (ANAE) positive lymphocytes. RBC, Hb, Hct; the number of PLT significantly decreased in the Cd group. To the contrary, these parameters were increased significantly in the CdQ group compared to the Cd group. Although we found a significant increase in total WBC count and neutrophil percentage, the number of lymphocytes decreased in the Cd group compared to the other three groups. Also, the percentage of peripheral blood ANAE positive lymphocytes decreased significantly in the Cd group (p < 0.05). Q exhibits positive effects on some hematological characteristics and the percentage of ANAE positive lymphocyte in cases of acute CD toxicity.     

Staining human lymphocytes and onion root cell nuclei with madder root

We performed staining experiments on cells using natural dyes and different mordants using techniques that are used for wool and silk dyeing. The natural dye sources were madder root, daisy, corn cockle and yellow weed. Ferrous sulfate, copper sulfate, potassium tartrate, urea, potassium aluminum sulfate and potassium dichromate were used as mordants. Distilled water, distilled water plus ethanol, heptane, and distilled water plus methanol were used as solvents. All dye-mordant-solvent combinations were studied at pH 2.4, 3.2 and 4.2. The generic staining procedure was to boil 5–10 onion roots or stimulated human lymphocyte (SHL) preparations in a dye bath on a hot plate. Cells were examined at every half hour. For multicolor staining, madder-dyed lymphocytes were decolorized, then stained with Giemsa. The AgNOR technique was performed following the decolorization of Giemsa stained lymphocytes. Good results were obtained for both onion root cells and lymphocytes that were boiled for 3 h in a dye bath that included 4 g madder root, 4 g ferrous sulfate as mordant in 50 ml of 1:1 (v/v) methanol:distilled water. The pH was adjusted to 4.2 with 6 ml acetic acid. We conclude that madder root has potential as an alternative dye for staining biological materials.     

Calcium hypochlorite on mouse embryonic fibroblast cells (NIH3T3) in vitro cytotoxicity and genotoxicity: MTT and comet assay

Molecular Biology Reports - Antimicrobial irrigation solutions are widely used under clinical settings. Their effect on dental tissue is a subject of recent research, which aims for a safer...     

Clock gene PERIOD3 polymorphism is associated with susceptibility to Graves’ disease but not to Hashimoto’s thyroiditis

ABSTRACT

Circadian disruption has been linked with immune-related morbidities including autoimmune diseases. PERIOD3 (PER3) clock gene is a key player in the mammalian circadian system. This study evaluated the possible association of PER3 rs2797685 (G/A) polymorphism and susceptibility of autoimmune thyroid diseases (AITD) and assessed if this SNP contributes to disease characteristics and serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). The PER3 rs2797685 (G/A) polymorphism was assessed in 125 patients with AITD [Graves’ disease (GD), 69; Hashimoto’s thyroiditis (HT), 56] and 115 unrelated healthy controls. Subjects carrying at least one variant allele of PER3 rs2797685 (GA+AA) had increased risk for GD (OR 1.9, 95% CI 1–3.61, p= .05). There were no differences in the frequencies of genotypes and alleles of the PER3 rs2797685 polymorphism between HT patients and control subjects. No association was observed between genotypes of the studied SNP and any of the disease characteristics in GD and HT patients. The GA+AA genotype of PER3 rs2797685 was associated with lower levels of IL-6 in patients with Graves’ disease. There were no differences between genotypes of the studied SNP regarding TNF-α levels in GD, HT or control groups. In conclusion, this study provides the first evidence for a genetic association between GD and the PER3 gene, highlighting the possible relevance of polymorphisms in clock genes in the etiopathogenesis of AITD. However, functional studies to identify the underlying molecular mechanisms of this association are needed to translate these findings to clinical applications.     

Follow‐up of atypia and follicular lesions of undetermined significance in thyroid fine needle aspiration cytology

N. Dincer, S. Balci, A. Yazgan, G. Guney, R. Ersoy, B. Cakir and G. Guler
Follow‐up of atypia and follicular lesions of undetermined significance in thyroid fine needle aspiration cytology Objective: To report our experience of atypia of undetermined significance (AUS)/follicular lesion of undetermined significance (FLUS) rate and outcome. Methods: Among 7658 patients with 19 569 nodules, 524 (2.7%) nodules were diagnosed as AUS/FLUS on fine needle aspiration (FNA). After exclusion of patients with simultaneous nodules that were suspicious for follicular neoplasm or malignancy or that were malignant, 368 (4.8%) patients were diagnosed as AUS/FLUS. The outcome of 146 patients who had undergone surgery or repeated fine needle aspirate at the time of preparation of this study was evaluated. The original FNAs were matched to repeated FNAs and thyroidectomy or diagnostic lobectomy specimens. Results: Seventy‐two (19.6%) of the 368 patients had directly undergone surgery, either a lobectomy or a thyroidectomy: of these, 27 (37.5%) had neoplastic nodules (21 were malignant). Seventy‐four (20.1%) of the 368 patients had repeat FNA. On second FNA, 47 of 74 (63.5%) were benign, three were suspicious for follicular neoplasm, one was malignant and 23 (31.1%) were non‐diagnostic. Four patients had a third FNA: two were AUS/FLUS, one was malignant and one non‐diagnostic. One patient had a fourth FNA, which was diagnosed as AUS/FLUS. Sixteen (21.6%) of 74 patients with repeat FNA had surgery: three of these had neoplastic nodules (two were malignant). Overall, 88 of the 368 (23.9%) patients had a thyroidectomy of which 30 (34.1%) were neoplastic and 23 (26.1%) malignant. The neoplastic rate for patients who were once diagnosed with AUS/FLUS was 8.2% and the malignancy rate 6.3%. The malignancy rate for patients on follow‐up at the time we prepared the study was 15.7% (23/146); 222 remained on follow‐up without surgery or repeat FNA or were managed elsewhere. Conclusions: Although in this category repeat FNA is expected rather than excision, we suggest evaluation of all AUS/FLUS patients in multidisciplinary meetings to decide management and recommend follow‐up of all patients with this diagnosis.     

Cloning,purification and characterization of a cellulase-free xylanase from <Emphasis Type="Italic">Geobacillus thermodenitrificans</Emphasis> AK53

Geobacillus thermodenitrificans AK53 xyl gene encoding xylanase was isolated, cloned and expressed in Escherichia coli. After purifying recombinant xylanase from G. thermodenitrificans AK53 (GthAK53Xyl) to homogeneity by ammonium sulfate precipitation and ion exchange chromatography, biochemical properties of the enzyme were determined. The kinetic studies for GthAK53Xyl showed K_M value to be 4.34 mg/mL (for D-xylose) and V_max value to be 2028.9 μmoles mg^–1 min^–1. The optimal temperature and pH for enzyme activity were found out to be 70°C and 5.0, respectively. The expressed protein showed the highest sequence similarity with the xylanases of G. thermodenitrificans JK1 (JN209933) and G. thermodenitrificans T-2 (EU599644). Metal cations Mg²⁺ and Mn²⁺ were found to be required for the enzyme activity, however, Co²⁺, Hg²⁺, Fe²⁺ and Cu²⁺ ions caused inhibitor effect on it. GthAK53Xyl had no cellulolytic activity and degraded xylan in an endo-fashion. The action of the enzyme on xylan from oat spelt produced xylobiose and xylopentose. The reported results are suggestive of a xylanase exhibiting desirable kinetics, stability parameters and metal resistance required for the efficient production of xylobiose at industrial scale.     

Mass Spectrometric Quantification of Histone Post-translational Modifications by a Hybrid Chemical Labeling Method

Mass spectrometry is a powerful alternative to antibody-based methods for the analysis of histone post-translational modifications (marks). A key development in this approach was the deliberate propionylation of histones to improve sequence coverage across the lysine-rich and hydrophilic tails that bear most modifications. Several marks continue to be problematic however, particularly di- and tri-methylated lysine 4 of histone H3 which we found to be subject to substantial and selective losses during sample preparation and liquid chromatography-mass spectrometry. We developed a new method employing a “one-pot” hybrid chemical derivatization of histones, whereby an initial conversion of free lysines to their propionylated forms under mild aqueous conditions is followed by trypsin digestion and labeling of new peptide N termini with phenyl isocyanate. High resolution mass spectrometry was used to collect qualitative and quantitative data, and a novel web-based software application (Fishtones) was developed for viewing and quantifying histone marks in the resulting data sets. Recoveries of 53 methyl, acetyl, and phosphoryl marks on histone H3.1 were improved by an average of threefold overall, and over 50-fold for H3K4 di- and tri-methyl marks. The power of this workflow for epigenetic research and drug discovery was demonstrated by measuring quantitative changes in H3K4 trimethylation induced by small molecule inhibitors of lysine demethylases and siRNA knockdown of epigenetic modifiers ASH2L and WDR5.The field of Epigenetics has become important in drug discovery as many diseases have been linked to aberrations in chromatin and changes of histone post-translational modifications (PTMs)¹ (1, 2). The core histones (H2A, H2B, H3, and H4 and their variants) undergo a multitude of PTMs. Some, like lysine acetylation, lysine mono-, di-, and trimethlyation, and serine/threonine phosphorylation are well documented, with over 100 distinct, albeit generally low abundance, modifications reported for H3 alone (3). Mass spectrometry provides an alternative to antibody-based methods for detecting and quantifying histone PTMs, as the latter are prone to problems of specificity and epitope occlusion (4, 5). The most commonly applied approach to date is known as “bottom-up” mass spectrometry and involves an initial processing of the histones into smaller peptides (6). A key development in histone PTM analysis was the deliberate chemical modification of histone tail lysines by propionic anhydride, preventing digestion of these Lys- and Arg-rich domains into peptides too short or hydrophilic to be detected in reverse-phase liquid chromatography-mass spectrometry experiments (7–9).Despite this advance, some marks like H3K4 di- and tri-methylation remain problematic; in several examples from the recent literature the H3K4me3 mark is detected either only by means of specifically targeted methods (5), with larger quantitative variation than other marks (10), or not reported among detected marks at all (3, 11–13). Alternative approaches include top-down or middle-down mass spectrometry, in which entire histones, or large segments thereof are analyzed directly (14–16), but these techniques still suffer from relatively poor sensitivity in comparison to bottom-up workflows, and must contend with the full combinatorial complexity of histone PTMs (17).The H3K4me3 mark is of low natural abundance, having a very restricted genomic localization strongly associated with active gene promotors and enhancers (18, 19), and aberrant activities of writers and erasers of that mark are associated with a variety of diseases (1, 2). Difficulties in its quantitation thus hinder the investigation of both fundamental biology and the discovery of lifesaving drugs. We therefore undertook a re-evaluation of the bottom-up histone PTM workflow, streamlining sample preparation and investigating sources of bias or sample loss. Alternatives to the standard propionylation technique were also explored, resulting in a new hybrid chemical modification workflow yielding across-the-board improvements in recovery of peptides from the N-terminal tail of histone H3, and dramatically improved detection of hydrophilic peptides with marks like H3K4me2/me3.