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.     

Enterohemolysin production is associated with a temperate bacteriophage in Escherichia coli serogroup O26 strains.

A temperate bacteriophage that determines the expression of enterohemolysin was isolated from Escherichia coli O26 strain C3888. The genetic determinant associated with enterohemolysin production (E-Hly determinant) was cloned from EcoRI-digested bacteriophage DNA in vector plasmid pUC8. pUC8 recombinant plasmid pEO19 carries a 3.7-kb EcoRI insert of phage DNA, and enterohemolysin was expressed in E. coli K-12 after transformation. Hemolysin-negative derivatives of pEO19 were generated by transposon mutagenesis with Tn1725. By subcloning, the phage E-Hly determinant was assigned to a 2,150-bp piece of DNA which is flanked by EcoRI and AccI restriction sites. The enterohemolysin-producing recombinant strains and wild-type strain C3888 express a 60-kDa protein which was detected in the bacterial outer membrane by Western immunoblotting. Biologically active enterohemolysin was detected only in bacteria grown to the stationary phase, and the hemolysin was not released into the culture medium. Lysis of erythrocytes was inhibited by 30 mM dextran 4, which functions as an osmotic protectant without destroying the enterohemolysin itself.     

The Interplay between Chondrocyte Redifferentiation Pellet Size and Oxygen Concentration

Chondrocytes dedifferentiate during ex vivo expansion on 2-dimensional surfaces. Aggregation of the expanded cells into 3-dimensional pellets, in the presence of induction factors, facilitates their redifferentiation and restoration of the chondrogenic phenotype. Typically 1×10⁵–5×10⁵ chondrocytes are aggregated, resulting in “macro” pellets having diameters ranging from 1–2 mm. These macropellets are commonly used to study redifferentiation, and recently macropellets of autologous chondrocytes have been implanted directly into articular cartilage defects to facilitate their repair. However, diffusion of metabolites over the 1–2 mm pellet length-scales is inefficient, resulting in radial tissue heterogeneity. Herein we demonstrate that the aggregation of 2×10⁵ human chondrocytes into micropellets of 166 cells each, rather than into larger single macropellets, enhances chondrogenic redifferentiation. In this study, we describe the development of a cost effective fabrication strategy to manufacture a microwell surface for the large-scale production of micropellets. The thousands of micropellets were manufactured using the microwell platform, which is an array of 360×360 µm microwells cast into polydimethylsiloxane (PDMS), that has been surface modified with an electrostatic multilayer of hyaluronic acid and chitosan to enhance micropellet formation. Such surface modification was essential to prevent chondrocyte spreading on the PDMS. Sulfated glycosaminoglycan (sGAG) production and collagen II gene expression in chondrocyte micropellets increased significantly relative to macropellet controls, and redifferentiation was enhanced in both macro and micropellets with the provision of a hypoxic atmosphere (2% O₂). Once micropellet formation had been optimized, we demonstrated that micropellets could be assembled into larger cartilage tissues. Our results indicate that micropellet amalgamation efficiency is inversely related to the time cultured as discreet microtissues. In summary, we describe a micropellet production platform that represents an efficient tool for studying chondrocyte redifferentiation and demonstrate that the micropellets could be assembled into larger tissues, potentially useful in cartilage defect repair.     

Association of nicotinamide-N-methyltransferase (NNMT) gene rs694539 variant with bipolar disorder

Here we report the association of the rs694539 variant of nicotinamide-N-methyltransferase gene with bipolar disorder in a case–control study of 95 bipolar disorder patients and 201 healthy controls (χ² = 13.382, P = 0.001). With the polymerase chain reaction restriction fragment length polymorphism method we developed we were able to show the association for the first time. This new finding may provide evidence to understand the mechanism of the disease.     

Acute adriamycin-induced cardiotoxicity is exacerbated by angiotension II

Adriamycin (ADR) increases the production of reactive oxygen species (ROS), which diminishes mitochondrial function. Angiotensin-II stimulates mitochondrial ROS generation. The aim of the study was to examine whether angiotensin converting enzyme (ACE) or renin inhibitors protect against ADR-induced mitochondrial function impairment. Rats were divided into five groups as control, ADR, co-treatment ADR with captopril, co-treatment ADR with aliskiren, co-treatment ADR with both captopril and aliskiren. Left ventricular function and blood pressures were assessed at the end of treatment period. Mitochondrial membrane potential (MMP) and ATP levels were determined. ADR treatment decreased the left ventricular pressure and increased the left ventricular end-diastolic pressure. ADR decreased MMP and ATP levels in myocyte mitochondria due to increasing oxidative stress. ADR decreased MMP and ATP levels due to increased oxidative stress in the heart. Inhibitors of ACE and renin caused the elevation of the decreased of MMP and ATP levels. The pathologic changes in electrocardiogram, blood pressure and left ventricular function were decreased by inhibition of Ang-II production. We concluded that inhibitors of angiotensin II are effective against ADR cardiotoxicity via the restoration of MMP and ATP production and prevention of mitochondrial damage in vivo.     

Molecular modelling and vibrational investigations of ammonium-based ionic liquid (CLTOAB)

CLTOAB is an ammonium-based ionic liquid composed of ε-Caprolactam (CL) C₆H₁₁NO and tetraoctylammonium bromide (TOAB) (C₃₂H₆₈BrN). In this study, experimental IR and Raman spectra of CLTOAB ionic liquid together with the computational results of the compound have been reported. The optimized geometry, vibrational frequencies, IR intensities and Raman activities of the CLTOAB were calculated using the wb97xd and B3LYP density functional methods combined with the 6-31G(d,p) basis set using Gaussian 03 program. The complete assignment of the bands was performed based on the potential energy distributions (PED%). The HOMO and LUMO analysis is used to determine the charge transfer within the molecule. The Gauge-including atomic orbital ¹H-NMR and ¹³C-NMR chemical shifts calculations were carried out and compared with the experimental data. Furthermore to evaluate interaction between CLTOAB and DNA, molecular docking study was carried out.

Communicated by Ramaswamy H. Sarma     

Packed Bed Bioreactor for the Isolation and Expansion of Placental-Derived Mesenchymal Stromal Cells

Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm²) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm² format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs.     

Purification and Characterisation of Rat Kidney Glutathione Reductase

Glutathione reductase [GR, E.C.1.8.1.7] catalyses NADPH dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH). Thus, it is the crucial enzyme to maintain high [GSH]/[GSSG] ratio and physiological redox status in cells. Kidney and liver tissues were considered as a rich source of GR. In this study, rat kidney GR was purified and some of its properties were investigated. The enzyme was purified 2,356 fold with a yield of 16% by using heat-denaturation and Sephadex G25 gel filtration, 2′,5′-ADP Agarose 4B, PBE94 column chromatographies. The purified enzyme had a specific activity (Vm) of 250 U/mg protein and the ratio of absorbances at wavelengths of A ₂₇₃/A _463, A ₂₈₀/A ₄₆₀, A ₃₆₅/A ₄₆₀, and A ₃₇₉/A ₄₆₃, were 7.1, 6.8, 1.2 and 1.0, respectively. Each mol of GR subunit bound 0.97 mol of FAD. NADH was used as a coenzyme by rat kidney GR but with a lower efficiency (32.7%) than NADPH. Its subunit molecular weight was estimated as 53 kDa. An optimum pH of 6.5 and optimum temperature of 65 °C were found for rat kidney GR. Its activation energy (Ea) and temperature coefficient (Q₁₀) were calculated as 7.02 kcal/mol and 1.42, respectively. The Km_(NADPH) and kcat/Km _(NADPH) values were found to be 15.3 ± 1.4 μM and 1.68 × 10⁷ M⁻¹ s⁻¹ for the concentration range of 10-200 μM NADPH and when GSSG is the variable substrate, the Km_(GSSG) and the kcat/Km_(GSSG) values of 53.1 ± 3.4 μM and 4.85 × 10⁶ M⁻¹ s⁻¹ were calculated for the concentration range of 20–1,200 μM GSSG.     

Purification,biochemical characterization and gene sequencing of a thermostable raw starch digesting α-amylase from Geobacillus thermoleovorans subsp. stromboliensis subsp. nov.

This study reports the purification and biochemical characterization of a raw starch-digesting α-amylase from Geobacillus thermoleovorans subsp. stromboliensis subsp. nov. (strain Pizzo^T). The molecular weight was estimated to be 58 kDa by SDS–PAGE. The enzyme was highly active over a wide range of pH from 4.0–10.0. The optimum temperature of the enzyme was 70°C. It showed extreme thermostability in the presence of Ca²⁺, retaining 50% of its initial activity after 90 h at 70°C. The enzyme efficiently hydrolyzed 20% (w/v) of raw starches, concentration normally used in starch industries. The α-amylase showed an high stability in presence of many organic solvents. In particular the residual activity was of 73% in presence of 15% (v/v) ethyl alcohol, which corresponds to ethanol yield in yeast fermentation process. By analyzing its complete amyA gene sequence (1,542 bp), the enzyme was proposed to be a new α-amylase.