Time-correlated single photon counting FLIM: some considerations for physiologists

Recent developments in cellular imaging now permit the minimally invasive study of protein interactions in living cells. These advances are of enormous interest to cell biologists, as proteins rarely act in isolation, but rather in concert with others in forming cellular machinery. Up until recently, all protein interactions had to be determined in vitro using biochemical approaches. This biochemical legacy has provided cell biologists with the basis to test defined protein-protein interactions not only inside cells, but now also with spatial resolution. More recent developments in TCSPC imaging are now also driving towards being able to determine protein interaction rates with similar spatial resolution, and together, these experimental advances allow investigators to perform biochemical experiments inside living cells. Here, we discuss some findings we have made along the way which may be useful for physiologists to consider.     

Characteristics of CFRP retrofitted hollow brick infill walls of reinforced concrete frames

The mechanical characteristics of infill walls retrofitted with carbon fiber reinforced polymer (CFRP) sheets are really important for the realistic prediction of seismic performance of the vulnerable reinforced concrete (RC) frames retrofitted through CFRP strengthened infill walls. In this study, 36 hollow brick wall specimens were tested either under uniaxial compression or diagonal tension before and after retrofitting externally with CFRP sheets. The test parameters are the dimensions of the walls, the orientation of holes of bricks, the type of mortar, the amount of CFRP sheets and the details of strengthening application. At the end of the tests, a significant contribution of CFRP sheets on the mechanical characteristics of hollow brick walls was observed in terms of several important structural design parameters such as Young and shears moduli, axial and shears strengths as well as the deformation capacity. Finally, the strength and deformability characteristics of the walls and frames retrofitted with CFRP sheets were predicted analytically. The predictions were in good agreement with the experimental data.     

Biological activities of the essential oil and methanolic extract of Micromeria fruticosa (L) Druce ssp serpyllifolia (Bieb) PH Davis plants from the eastern Anatolia region of Turkey

The in vitro antimicrobial and antioxidant activities of the essential oil and methanolic extract of Micromeria fruticosa ssp serpyllifolia as well as the composition of the essential oil were examined. The essential oil exhibited activity against 14 bacteria, three fungi and a yeast, with minimal inhibitory concentration (MIC) values ranging from 31.25 to 125 µl ml^?1, whilst the methanolic extract was inactive. Antioxidant activity was measured by two methods, namely scavenging of the free radical DPPH and inhibition of linoleic acid oxidation. The methanolic extract exhibited significant antioxidant activity in both assays, providing 50% inhibition at 70.9 ± 0.5 µg ml^?1 concentration in the DPPH assay and inhibiting linoleic acid oxidation to 59% at 2 mg ml^?1 concentration, whilst the essential oil showed activity only at higher concentrations. The gallic acid equivalent total phenolic content of the methanolic extract was found to be 55.2 ± 2.00 µg mg^?1 dry weight extract (5.5% w/w). The chemical composition of the hydrodistilled essential oil was analysed by means of GC/MS. Twenty‐nine constituents were identified, the main ones being piperitenone (50.61%) and pulegone (29.19%). Copyright © 2004 Society of Chemical Industry     

Identification of meat species by TaqMan-based real-time PCR assay

In this study, a convenient, sensitive and specific real-time PCR assay was described for the species identification and their quantification in raw and cooked meat products. Specific primers and TaqMan probes were designed on the mitochondrial ND2, ND5 and ATP 6-8 genes for donkey, pork and horse, respectively, and the performance of the method was tested. In the results, no cross-reaction was observed between the donkey and pork species specific primer-probe systems and non-target species (bovine, ovine, chicken and turkey). Only one cross reaction was observed between the horse species specific primer-probe set and 100 ng pork DNA at the ct 33.01 level (corresponding to 0.01 ng horse DNA). The real-time quantitative assay used in this study allowed the detection of as little as 0.0001 ng template DNA from pure meat for each species investigated and experimental meat mixtures. In conclusion, it can be suggested that the TaqMan probe assay used in this research might be a rapid and sensitive method for the routine meat species identifications studies in raw or cooked meat products.     

Using three-phase aquatic microcosms to assess fates and impacts of chemicals in microbial communities

A method for assessing the biogeochemical fate and effects of toxic and other chemicals has been developed using a three-phase aquatic microcosm (TPAM) having sediment, water and gas phases to represent aquatic environments. Mass balances of critical variables indicate that microcosm responses are sensitive to heavy metals and nutrients. TPAM benthic dissolved oxygen consumption was typical of natural environments. Trace additions of Zn, Cr, Cd, Pb and Hg depressed the productivity of TPAM's using Lake Powell sediments. The aquatic chemistry of zinc and biological uptake accounted for its distribution. The method has broad application for conveniently and inexpensively evaluating a large number of substances under a variety of ecological conditions.     

Purification and characterization of an alkaline pectin lyase produced by a newly isolated Brevibacillus borstelensis (P35) and its applications in fruit juice and oil extraction

An alkaline pectin lyase (PNL) (EC 4.2.2.10) secreted by Brevibacillus borstelensis P35 (GenBank Number: FJ417406) was purified using ammonium sulfate fractionation, anion exchange chromatography on DEAE-cellulose and gel filtration chromatography on Sephadex G-150. The pH and temperature optima of the enzyme were found to be 8.0 and 60 °C. The enzyme does not loose activity up to 60 °C if exposed for 1 h. The values of K _m and V _max of the enzyme were 0.625 mg/mL and 126.32 s^?1, respectively. The molecular weight was found to be 36 ± 01 kDa. The presence of 10 mM concentration of Ca²⁺, Cu²⁺, Mn²⁺, Mg²⁺, Zn²⁺, Hg²⁺, Fe²⁺ and EDTA, l-cystein, ascorbic acid significantly enhanced the PNL of the purified enzyme. In the course of the laboratory trials, it was demonstrated that PNL from B. borstelensis (P35) could be successfully applied to the production and clarification of fruit juice and oil extraction.     

Isolation of some luteolin derivatives from Mentha longifolia (L.) Hudson subsp. longifolia and determination of their genotoxic potencies

This study was designed to evaluate the mutagenic and antimutagenic activities of luteolin derivatives (luteolin 7-O-glucoside, luteolin 7-O-rutinoside and luteolin 7-O-glucuronide) isolated from Mentha longifolia (L.) Huds. subsp. longifolia by using Ames Salmonella test (TA 1535 and TA1537 strains). In the antimutagenicity assays, luteolin 7-O-glucoside, luteolin 7-O-rutinoside and luteolin 7-O-glucuronide showed antimutagenic effects on TA1537 and TA1535 strains. The highest inhibition rates for luteolin 7-O-glucoside, luteolin 7-O-rutinoside and luteolin 7-O-glucuronide on TA1537 strain were 84.03%, 87.63% and 67.77%, respectively. Similarly, in the antimutagenicity assays performed with the TA1535 strain, the inhibition rates for luteolin 7-O-glucoside and luteolin 7-O-rutinoside were 23.86% and 23.76% respectively. Our findings showed that the antimutagenic properties of luteolin derivatives on TA1537 and TA1535 strains have been found to be structure dependent. The clarification of differences in antimutagenic potency of these luteolin derivatives based on their structures has been demonstrated in this study.     

Isolation of some active compounds from Origanum vulgare L. ssp. vulgare and determination of their genotoxic potentials

Origanum vulgare L. (oregano) is a flavoring herb widely used around the world. In the present study, crude extract of O. vulgare L. (oregano) and its petroleum ether, chloroform, ethyl acetate, n-butanol, water fractions were prepared in order to isolate and investigate antimutagenic compounds from the active extract through the bacterial genotoxicity assay guided fractionation procedures. The methanol extract and its n-butanol fraction showed significant antimutagenic activity. In the end of sub-fractionation process of the n-butanol extract, luteolin 7-O-glucuronide and luteolin 7-O-xyloside were isolated. These compounds showed significant antimutagenic activity against 9-Aminoacridine and N-Methyl-N′-nitro-N-nitrosoguanidine mutagenicity. The inhibition rates ranged from 22.1% (luteolin 7-O-xyloside: Salmonella typhimurium TA1537 – 0.4 mM/plate) to 67.8% (luteolin 7-O-glucuronide: S. typhimurium TA1537 – 0.8 μM/plate). In conclusion, the results revealed that luteolin 7-O-glucuronide and luteolin 7-O-xyloside are two of the antimutagenic compounds of O. vulgare L. ssp. vulgare.     

Combination of culture-dependent and culture-independent molecular methods for the determination of lactic microbiota in sucuk

In this study, the culture-dependent and culture-independent molecular methods were used for the identification of lactic acid bacteria (LAB) in sucuk a Turkish fermented dry sausage. On the one hand, the PCR-DGGE method targetting the V1 and V3 regions of 16S DNA was applied to DNA that was directly extracted from sucuk samples. On the other hand, rep-PCR fingerprinting was performed for the primary differentiation and grouping of the isolates, and the results were confirmed by sequencing of the 16S rDNA and 16S-23S rDNA intergenic spacer region. As a result of the PCR-DGGE analysis of all the samples, total 8 different lactic acid bacteria were identified, and Lactobacillus sakei, Lactobacillus curvatus and Weissella viridescens were the dominant microbiota among these bacteria. The culture-dependent approach indicated that the majority of the strains belonged to the Lactobacillus genera including Lb. sakei, Lactobacillus plantarum, Lb. curvatus, Lactobacillus brevis, Lactobacillus farciminis and Lactobacillus alimentarius. However, Leuconostoc and Weisella were also detected as minor genera. Again, Lactococcus piscium, Weissella halotolerans, Staphylococcus succinus and the comigrated Staphylococcus piscifermentans/Staphylococcus condimenti/Staphylococcus carnosus group were detected only with the culture-independent method while Lb. plantarum, Leuconostoc mesenteroides and Leuconostoc citreum were identified only by using the culture-dependent method. In the results, it was concluded that the combination of culture-dependent and culture-independent methods was necessary for reliable and detailed investigation of LAB communities in fermented food products.     

Biosensing Applications of Modified Core–Shell Magnetic Nanoparticles

Fe₃O₄ magnetic nanoparticles (MNPs) were synthesized and silanized to form a core–shell (Fe₃O₄–SiO₂) structure. Afterwards, surface modification with amino silane was carried out to produce amino groups on the MNPs for the biomolecule immobilization. In order to test the performance of amino functional MNPs as immobilization platform in biosensing applications, glucose oxidase was immobilized on the surface via glutaraldehyde. Obtained Bio-MNPs were then fixed onto the carbon paste electrode by the aid of magnetic force and used as the working electrode during the amperometric measurements at −0.7 V versus Ag/AgCl. After optimization of some parameters affecting the biosensor performance, analytical characterization was carried out. Linearity was found in the range of 0.25–2.0 mM glucose and defined by the equation of y = 8.366x + 1.819, (R ² = 0.996). Proposed biosensor was then applied for the glucose analysis in various beverages. Finally, data were compared with a commercial enzyme assay kit based on spectrophotometric Trinder reaction as a reference method.