Development of a bifunctional chelating agent containing isothiocyanate residue for one step F-18 labeling of peptides and application for RGD labeling

We report herein a novel isothiocyanate active ligand for fluorine-18 labeling prepared by four step synthesis. It can be conjugated to a target molecule containing an amino functional group under weak basic conditions by way of thiourea bond formation. We explored the application of synthesized ligand by conjugating to well known α_vβ₃ integrin targeting peptide, c(RGDyK). The conjugated peptide showed good radiochemical yield and efficiency with an excellent radiochemical purity (97.1 ± 1.2%) in a short reaction time (10 min). Labeled peptide showed excellent in vitro and in vivo stability (>95%). α_vβ₃ integrin specific tumor uptake was observed both in biodistribution and small animal microPET studies on α_vβ₃-positive U87MG (human glioma cells) xenograft bearing mice. In general, successful application of synthesized ligand for labeling of RGD peptide could facilitate the possibility of using this ligand for labeling peptides containing an amino functional group.     

不同结构类型糖类物质的荧光标记策略

为阐明糖链结构与功能的关系,寻找高灵敏度和高分离度的微量分析方法对糖 类物质的分析至关重要.近几年来,荧光标记方法作为糖类物质的色谱定量及辅助结 构分析的最佳选择,日益受到人们的广泛关注.荧光标记法分为柱前标记和柱后标记 两种,且灵敏度高、分离度好、有多种标记试剂可供选择.本文根据不同糖类物质的 结构特点,分别对中性糖和氨基糖以及含有唾液酸、糖醛酸、硫酸基的酸性糖的荧光 标记方法及其应用进行了系统的综述,并对近年来报道的荧光标记试剂的特点和反应 机理进行了比较和总结,以期为糖类物质微量分析方法的研究提供参考.     

Synthesis and evaluation of two new bifunctional carboxymethylated tetraazamacrocyclic chelating agents for protein labeling with indium-111

The synthesis of two new N- and C-functionalized tetraazamacrocyclic ligands intended to be covalently linked to biomolecules like monoclonal antibodies and to bind the gamma-emitting isotope indium-111 in a thermodynamically and/or kinetically inert way is described. 12-(p-Nitrobenzyl)-1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetraa cetic acid (L1) was synthesized by means of bimolecular cyclization with the appropriate malonic acid diethyl ester and triethylenetetraamine, followed by reduction with diborane and alkylation of the cyclic tetraamine with bromoacetic acid. The corresponding triscarboxymethylated ligand L2 was made by statistical alkylation of the tetraamine. Both ligands fulfill the criteria for antibody labeling using the bifunctional chelate approach, namely fast chelate formation, high radiochemical yield, and high stability under physiological conditions. Surprisingly the heptadentate ligand L2 confers higher stability to In3+ and exhibits faster complex formation than octadentate L1. 13C NMR spectra in solution indicate that the difference in stability is not due to incomplete coordination of all four carboxylate groups in In-L1.     

Bioburden assessment of banked bone used for allografts

Allograft bone is commonly used in reconstructive orthopaedic surgery and needs to be assessed for bioburden before transplant. The Microbiology Department of the South Eastern Area Laboratory Services (SEALS), located at the St. George Hospital, Sydney, has provided this service to the New South Wales (NSW) Bone Bank. This study reviewed the organisms isolated from femoral head allografts of living donors from the NSW Bone Bank over a 7-year period. It was found that growth was reported from 4.9% of samples with the predominant organism being coagulase-negative staphylococci. This review will focus on the micro-organisms isolated, the interaction of the laboratory with the bone bank, the relevance of the bioburden assessment in the overall quality process and patient safety.     

Synthesis and biological studies of nociceptin derivatives containing the DTPA chelating group for further labeling with therapeutic radionuclides

Nociceptin is an endogenous anti-opiate heptadecapeptide primarily interacting with the nociceptin (NOP) receptor. This neuropeptide-receptor system is involved in pain regulation, tolerance to and dependence on opiates as well as many other physiological and pathophysiological events. The role and mechanisms of nociceptin in pathological conditions is not clearly known yet. In an attempt to have a radiopharmaceutical labeled either with 99mTc or (111)In, we incorporated diethylenetriaminepentaacetic acid (DTPA) as chelator into the structure of [Arg14,Lys15]nociceptin(1-17)-NH2 at the epsilon-amino group of Lys15. Such a radiopeptide may be useful in imaging for diagnostical purposes. Preparation of the peptide ligands was carried out by solid phase synthesis. Two peptides containing DTPA were obtained and purified. The products were [Arg14,Lys(DTPA)15]nociceptin(1-17)-NH2 and its cross-linked dimer on the basis of mass spectrometric analysis. In (115)In3+ binding experiments the conjugates exhibited preserved indium ion chelating properties, indicating the potential use of radiolabeled DTPA-nociceptin derivatives as radiopharmaceutical. Biological properties of these compounds were studied in rat brain membrane preparations by radioligand binding, functional biochemical [35S]GTPgammaS binding assays and mouse vas deferens (MVD) bioassay. Besides the similar in vitro binding characteristics to nociceptin receptor, both of the DTPA-chelated compounds were more potent and efficient than nociceptin in functional biochemical and mouse vas deferens bioassays. Our further aim is to radiolabel these compounds in order to get a radiopharmaceutical which can be used diagnostically.     

Fluorescein diacetate used as a vital stain for labeling living pollen tubes

Snapdragon and tobacco pollen treated with fluorescein diacetate (FDA, 10 μg/ml) for 40 min could germinate and grew well in FDA-free medium. The pollen tubes showed bright fluorescence of the protoplasm when they were living. Thus the FDA method can be used, in addition to its previous usage for viability test of cells, also for vital staining of pollen tubes, and may be valuable to the study of other living cells as well.     

Fluorescent labeling of the carbohydrate moieties of human chorionic gonadotropin and alpha 1-acid glycoprotein

A method for covalent attachment of a fluorescent molecule to the carbohydrate moieties of glycoproteins is described. The glycoproteins were oxidized with periodate under mild conditions selective for sialic acid (Van Lenten, L. and Ashwell, G. (1971) J. Biol. Chem. 246, 1889--1894). The resulting aldehydes were condensed with either dansylhydrazine, dansylethylenediamine, or fluoresceinamine followed by reduction with NaCNBH3 and NaBH4. Conjugates prepared with dansylhydrazine were found to be insufficiently stable for spectroscopic analysis, whereas the primary amines produced stable conjugates whose fluorescence polarization (P) was constant for several hours at 37 degrees C. The degree of labeling correlated roughly with the sialic acid contents of the vaious glycoproteins. Very little covalent incorporation was observed with albumin (which is devoid of carbohydrate) or with asialo alpha 1-acid glycoprotein. Exclusion chromatography in the presence of a dissociating agent was sometimes required to remove significant amounts of noncovalently adsorbed dye. Fluorescent-labeled alpha subunits of human chorionic gonadotropin were shown to recombine normally with native beta subunits. However, the labeling procedure appeared to compromise the ability of the beta subunits to recombine. Electrophoretic analysis produced evidence of covalent cross-linking between subunits following periodate oxidation of the intact gonadotropin. The possibility that primary amine groups of the protein compete with added fluorescent amines for reaction with periodate-generated aldehydes is discussed.     

A crustal scarcity indicator for long-term global elemental resource assessment in LCA

The International Journal of Life Cycle Assessment - How to assess impacts of mineral resources is much discussed in life cycle assessment (LCA). We see a need for, and a lack of, a mineral...     

Life cycle assessment evaluation of green product labeling systems for residential construction

Purpose

Life cycle assessment (LCA) is a tool that can be utilized to holistically evaluate novel trends in the construction industry and the associated environmental impacts. Green labels are awarded by several organizations based on single or multiple attributes. The use of multi-criteria labels is a good start to the labeling process as opposed to single criteria labels that ignore a majority of impacts from products. Life cycle thinking, in theory, has the potential to improve the environmental impacts of labeling systems. However, LCA databases currently are lacking in detailed information about products or sometimes provide conflicting information.

Method

This study compares generic and green-labeled carpets, paints, and linoleum flooring using the Building for Environmental and Economic Sustainability (BEES) LCA database. The results from these comparisons are not intuitive and are contradictory in several impact categories with respect to the greenness of the product. Other data sources such as environmental product declarations and ecoinvent are also compared with the BEES data to compare the results and display the disparity in the databases.

Results

This study shows that partial LCAs focused on the production and transportation phase help in identifying improvements in the product itself and improving the manufacturing process but the results are uncertain and dependent upon the source or database. Inconsistencies in the data and missing categories add to the ambiguity in LCA results.

Conclusions

While life cycle thinking in concept can improve the green labeling systems available, LCA data is lacking. Therefore, LCA data and tools need to improve to support and enable market trends.     

Comparison of fluorescent tag DNA labeling methods used for expression analysis by DNA microarrays

Gene expression profiling by DNA microarrays has found wide application in many fields of biomedical research. The protocols for this technique are not yet standardized, and for each given step in microarray analysis a number of different protocols are in use. As a consequence, results obtained in different laboratories can be difficult to compare. Of particular importance in this respect are the methods for the preparation of fluorescent cDNA probes that should quantitatively reflect the abundance of different mRNAs in the two samples to be compared. Here we systematically evaluate and compare five different published and/or commercial principles for the synthesis offluorescently labeled probes for microarray analysis (direct labeling, 77 RNA polymerase amplification, aminoallyl labeling, hapten-antibody enzymatic labeling, and 3-D multi-labeled structures). We show that individual labeling methods can significantly influence the expression pattern obtained in a microarray experiment and discuss the respective benefits and limitations of each method.     

Stability of elemental content correlates with plant resistance to soil impoverishment

Plant and Soil - We investigated whether plant resistance to soil impoverishment would depend on their flexibility in taking up nutrients and on maintaining elemental stoichiometry. We mixed sand...     

Evaluation of five different cDNA labeling methods for microarrays using spike controls

Background

Several different cDNA labeling methods have been developed for microarray based gene expression analysis. We have examined the accuracy and reproducibility of such five commercially available methods in detection of predetermined ratio values from target spike mRNAs (A. thaliana) in a background of total RNA. The five different labeling methods were: direct labeling (CyScribe), indirect labeling (FairPlay? – aminoallyl), two protocols with dendrimer technology (3DNA^® Array 50? and 3DNA^® submicro?), and hapten-antibody enzymatic labeling (Micromax? TSA?). Ten spike controls were mixed to give expected Cy5/Cy3 ratios in the range 0.125 to 6.0. The amounts of total RNA used in the labeling reactions ranged from 5 – 50 μg.

Results

The 3DNA array 50 and CyScribe labeling methods performed best with respect to relative deviation from the expected values (16% and 17% respectively). These two methods also displayed the best overall accuracy and reproducibility. The FairPlay method had the lowest total experimental variation (22%), but the estimated values were consistently higher than the expected values (36%). TSA had both the largest experimental variation and the largest deviation from the expected values (45% and 48% respectively).

Conclusion

We demonstrate the usefulness of spike controls in validation and comparison of cDNA labeling methods for microarray experiments.

     

Life cycle assessment of waste strategies for used lubricating oil

Purpose

The study aimed to evaluate the environmental impacts of used lubricating oil (ULO) recovery in the largest oil consumer country in Africa, Egypt. The main questions were: What are the impacts of the different waste management strategies for the recovery of used lubricating oil and which waste management strategy is more eco-friendly?

Methods

Life cycle assessment (LCA) was employed to model the environmental impacts of the two waste management approaches for used lubricating oil recovery in Egypt: recycling by re-firing and recovery by co-firing. The model was applied to assess the impacts of one of the largest ULO recovery units in the Middle East and North Africa (MENA) region and the only operating unit in Egypt. The following impact categories were included: global warming potential (GWP), acidification potential (AP), eutrophication potential (EP), carcinogens potential (CP), ecotoxicity potential (ETP), respiratory inorganic formation potential (RIFP), respiratory organic formation potential (ROFP), radiation potential (RP), ozone layer depletion (OLD), mineral depletion (MD), land use (LU) and fossil fuel depletion (FFD).

Results and discussion

Results indicated that recycling by re-refining strategy is more environment-friendly. De-asphalting, de-aromatization and de-waxing processes are the main processes that affect the environmental impacts of lubricating oil production in both strategies, due to the use of hazard materials and toxic solvents in these processes. Fuel gas and fuel oil used as a fuel in the refinery and power units are the main contributors affecting the environmental impacts in case of recycling by re-refining strategy. The highest impacts were detected on FFD, followed by RIFP, GWP, AP, EP, ETP and CP in both strategies; no impacts were detected on RP, OLD and MD.

Conclusions

It can be concluded that recycling by re-refining of ULO is the more eco-friendly approach. This strategy is more energy conservative, saves a diminishing fossil fuel resource and reduces burdens on the environment. ULO containing high percentages of additive remnants such as viscosity index improvers and pour point depressants which represents a valuable resource and its proper management should be given the most attention.

     

Complexation of labile aluminium species by chelating resins Iontosorb--a new method for Al environmental risk assessment

The utilization of chelating ion-exchange by the method based on binding strength and kinetic discrimination for aluminium fractionation was studied. Two chelating cellulose resins, Iontosorb Oxin (IO) and Iontosorb Salicyl (IS), were used for the determination of quickly reacting labile aluminium species. The possibilities of aluminium fractionation on these chelating resins were investigated by a solid phase extraction technique. The study of the pH (2.5-6.0) influence on the Al complexation by both resins indicates that at low pH the IS has lower sorption capacity but better adsorptive kinetic properties than IO. The optimal resin complexation time for reactive Al species was experimentally found after aluminium sorption study at pH 4.0 in synthetic solutions containing some inorganic and organic ligands, which simulate the composition of analysed acid soil and water samples. The negative influence of sulphate and iron on the Al complexation by IS resin was found and investigated. The flame atomic absorption spectrometry was used for the aluminium quantification.     

Rapid identification of bio-molecules applied for detection of biosecurity agents using rolling circle amplification

Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification). The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans) and spores (Bacillus atrophaeus) released in field.     

Fluorescent labeling of nisin Z and assessment of anti-listerial action

Biomolecule labeling by fluorescent markers has emerged as an innovative methodology for bio-analytical purposes in food microbiology, medicine and pharmaceutics due to the great advantages of this method such as precision, wide detection limits, and in vivo recognition. Fluorescent nisin Z was synthesized by linking the carboxyl group and amino group of nisin Z and 5-aminoacetamido fluorescein (AAA-flu). This new structure was fully characterized by mass spectrometry with a molecular weight of 3717.3 Da. Intracellular K⁺ leakage and transmembrane electrical potential (Δψ) were used to evaluate the antibacterial action of the labeled molecule against three listerial strains and demonstrated that nisin Z endured the labeling process without any activity loss. In vivo activity of labeled nisin was observed by confocal laser microscope which revealed its localization at the septum of listerial cell division site where the membrane-bound cell wall precursor lipid II is maximal. Fluorescent nisin Z showed its great potential as a tool to study antibacterial mechanism of action of nisin in biological systems.     

ULS: a versatile method of labeling nucleic acids for FISH based on a monofunctional reaction of cisplatin derivatives with guanine moieties

The broad extension of an existing chemical DNA labeling technique for molecular cytogenetics is described. Called the Universal Linkage System (ULS(TM)), it is based on the capability of monoreactive cisplatin derivatives to react at the N7 position of guanine moieties in DNA. Simple repetitive probes, cosmids, PACs, and chromosome-specific painting probes were labeled by ULS and used in a series of multicolor fluorescence in situ hybridization experiments on interphase and metaphase cells. It is demonstrated that ULS-labeled probes, in general, perform as well as the more conventional enzymatically labeled probes. The advantage of ULS labeling over enzymatic labeling techniques is that it is a fast and simple procedure, and that the labeling can easily be scaled up for bulk probe synthesis. In addition, with ULS labeling it is possible to label degraded DNA, a situation in which enzymatic labeling is known to perform unsatisfactorily.     

Comparison of different labeling methods for the production of labeled target DNA for microarray hybridization

Different labeling methods were studied to compare various approaches to the preparation of labeled target DNA for microarray experiments. The methods under investigation included a post-PCR labeling method using the Klenow fragment and a DecaLabel DNA labeling kit, the use of a Cy3-labeled forward primer in the PCR, generating either double-stranded or single-stranded PCR products, and the incorporation of Cy3-labeled dCTPs in the PCR. A microarray that had already been designed and used for the detection of microorganisms in compost was used in the study. PCR products from the organisms Burkholderia cepacia and Staphylococcus aureus were used in the comparison study, and the signals from the probes for these organisms analyzed. The highest signals were obtained when using the post-PCR labeling method, although with this method, more non-specific hybridizations were found. Single-stranded PCR products that had been labeled by the incorporation of a Cy3-labeled forward primer in the PCR were found to give the next highest signals upon hybridization for a majority of the tested probes, with less non-specific hybridizations. Hybridization with double-stranded PCR product labeled with a Cy3-labeled forward primer, or labeled by the incorporation of Cy3-labeled dCTPs resulted in acceptable signal to noise ratios for all probes except the UNIV 1389a and Burkholderia genus probes, both located toward the 3' end of the 16S rRNA gene. The comparison of the different DNA labeling methods revealed that labeling via the Cy3-forward primer approach is the most appropriate of the studied methods for the preparation of labeled target DNA for our purposes.     

Variations in T-RFLP profiles with differing chemistries of fluorescent dyes used for labeling the PCR primers

Culture independent molecular methods have emerged as indispensable tools for studying microbial community structure and dynamics in natural habitats, since they allow a closer look at microbial diversity that is not reflected by culturing techniques. Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis is one of the informative and widely used techniques for such studies. However, the method has a few limitations to predict microbial community structure with significant accuracy. One of the major limitations is variation in real Terminal Restriction Fragment (TRF) length and observed TRF length. In the present study we report the generation of TRF length variations using different fluorescent dyes to label the PCR primers. T-RFLP profiles generated from primers labeled with different dyes varied significantly and led to inconsistent microbial species identification. Occurrence of such variations can have serious consequences on interpretation of the T-RFLP profiles from environmental samples representing complex microbial community. Therefore, in a T-RFLP study, the primers and labeling dye system should be carefully evaluated and optimized for an individual community under investigation. Further, it would be recommended to establish a target gene library in parallel with T-RFLP analysis to facilitate the accurate prediction of microbial community structure.     

Stability of Bacillus pumilus spore strips used for monitoring radiation sterilization.

Dried Bacillus pumilus spore strips had a stable D-value of 0.17 to 0.18 Mrad after 1 year at 5 or 25 C, but resistance was lost at 35 C. Refrigeration did not have an adverse effect on resistance to irradiation. The presence of water at the time of initial preparation increased this resistance to 0.23 to 0.25 Mrad. Proper drying is essential to achieve reproducible results in sterility tests using USP-type biological indicators.