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Flow cytometry is an effective and widely used tool for determination of ploidy in fish, but it is not always possible to access the fresh samples for analysis. We investigated the potential for extended storage of fish tissue with sterlet and tench as representative species of Chondrostei and Teleostei, using blood and fin of subadult/adult specimens and tail of larvae. Thirteen procedures for extending storage, selected for rapidity and simplicity in both field and laboratory conditions, were tested for each tissue sample. Flow cytometry was applied to fresh tissue immediately after sampling and to tissue subjected to experimental protocols, always along with species‐specific standard, after 1, 5, and 10 days storage at 0–4°C or freezing at −80°C. The fluorochrome 4′,6‐diamidine‐2′‐phenylindole dihydrochloride was used with excitation/emission maximum 358/461 nm. Based on the measurability of stored samples, evaluation of directly measured coefficients of variation of their DNA peaks and the changes in fluorescence intensity compared to fresh tissue, optimal procedures for extended storage of the selected tissue types of the model species are suggested. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

Circulating tumour nucleic acids (ctNAs) are released from tumours cells and can be detected in blood samples, providing a way to track tumors without requiring a tissue sample. This "liquid biopsy" approach has the potential to replace invasive, painful, and costly tissue biopsies in cancer diagnosis and management. However, a very sensitive and specific approach is required to detect relatively low amounts of mutant sequences linked to cancer because they are masked by the high levels of wild-type sequences. This review discusses high-performance nucleic acid biosensors for ctNA analysis in patient samples. We compare sequencing- and amplification-based methods to next-generation sensors for ctDNA and ctRNA (including microRNA) profiling, such as electrochemical methods, surface plasmon resonance, Raman spectroscopy, and microfluidics and dielectrophoresis-based assays. We present an overview of the analytical sensitivity and accuracy of these methods as well as the biological and technical challenges they present.

Fish species substitution and fraud has become a worldwide economic issue in the seafood industry. In this study, an ultraviolet-visible (UV-Vis) spectroscopy-based method was developed for the identification of fish samples. Sixty fish samples from twelve commonly consumed fish species in China were analyzed as models to testify the protocol. The obtained results show that UV-Vis spectroscopy combined with chemometric analysis, such as principal component analysis (PCA), can accurately distinguish two fish species by boiling fish tissue sample in trifluoroacetic acid (TFA) solution for 2 min and analyzing the resultant samples using a UV-Vis spectrometer. The developed strategy was successfully applied to the classification and identification of fish samples on the market. It is a promising strategy that can be applied to the classification and authenticity testing of closely related fish species in order to detect and recognize fish substitution.

Elemental and molecular imaging play a crucial role in understanding disease pathogenesis. To accurately correlate elemental and molecular markers, it is desirable to perform sequential elemental and molecular imaging on a single-tissue section. However, very little is known about the impact of performing these measurements in sequence. In this work, we highlight some of the challenges and successes associated with performing elemental mapping in sequence with mass spectrometry imaging. Specifically, the feasibility of molecular mapping using the mass spectrometry imaging (MSI) techniques matrix-assisted laser desorption ionization (MALDI) and desorption electrospray ionization (DESI) in sequence with the elemental mapping technique particle-induced X-ray emission (PIXE) is explored. Challenges for integration include substrate compatibility, as well as delocalization and spectral changes. We demonstrate that while sequential imaging comes with some compromises, sequential DESI-PIXE imaging is sufficient to correlate sulfur, iron, and lipid markers in a single tissue section at the 50 μm scale.

Food adulteration is a serious concern faced by the importers of various food products across the globe. In this study, a simple, sensitive and robust method for detecting pork in processed/complex food samples using next-generation DNA sequencing (NGS) technology is described. The experimentation involves a generalized library preparation kit for performing shotgun sequencing of the genomic DNA irrespective of its intactness. The method was applied on different complex food samples containing pork along with other species (up to twelve) as well as without pork to test the specificity of the method. The DNA sequences were mapped with the online NCBI nucleotide database for their identification followed by a calculation of the relative abundance of the reads. The adulteration of pork was correctly identified in the analyzed samples. Although the relative abundance of pork DNA reads could not make a precise quantitative relevance with the contributed amount of the tissue sample, yet this method has the potential to determine extremely low as well as high contents of adulterating/contaminating species in complex food products.

Porous graphitized carbon (PGC) based chromatography achieves high-resolution separation of glycan structures released from glycoproteins. This approach is especially valuable when resolving structurally similar isomers and for discovery of novel and/or sample-specific glycan structures. However, the implementation of PGC-based separations in glycomics studies has been limited because system-independent retention values have not been established to normalize technical variation. To address this limitation, this study combined the use of hydrolyzed dextran as an internal standard and Skyline software for post-acquisition normalization to reduce retention time and peak area technical variation in PGC-based glycan analyses. This approach allowed assignment of system-independent retention values that are applicable to typical PGC-based glycan separations and supported the construction of a library containing >300 PGC-separated glycan structures with normalized glucose unit (GU) retention values. To enable the automation of this normalization method, a spectral MS/MS library was developed of the dextran ladder, achieving confident discrimination against isomeric glycans. The utility of this approach is demonstrated in two ways. First, to inform the search space for bioinformatically predicted but unobserved glycan structures, predictive models for two structural modifications, core-fucosylation and bisecting GlcNAc, were developed based on the GU library. Second, the applicability of this method for the analysis of complex biological samples is evidenced by the ability to discriminate between cell culture and tissue sample types by the normalized intensity of N-glycan structures alone. Overall, the methods and data described here are expected to support the future development of more automated approaches to glycan identification and quantitation.

Quantification of the amount of cryoprotective agent (CPA) in a tissue is an essential step in the design of successful cryopreservation protocols. This chapter details two inexpensive methods to measure cryoprotective agent permeation into tissues as functions of time. One of the methods to measure the CPA permeation is to permeate a series of tissue samples from a surrounding solution at a specified concentration of CPA, each sample for a different amount of time, and then to quantitate the amount of CPA that was taken up in the tissue during that time period. The quantification is performed by equilibrating the permeated tissue with a surrounding solution and then measuring the osmolality of the solution to determine the amounts of CPAs that have come out of each tissue sample corresponding to each permeation time. An alternative method to measuring the CPA permeation as a function of time, which requires fewer tissue samples, is to measure the CPA efflux as a function of time. In the efflux method, a CPA-permeated tissue sample is placed in a surrounding solution, and solution samples are taken at different time points throughout the efflux to quantitate how much CPA has left the tissue by each time point.

File main pdf file (CRRC of dispersed-settled spheroidal cells.pdf) describes experimental results and their interpretation for a study of tissue relevance of analyses of dispersed-settled tissues cells by Cytometry of Reaction Rate Constant (CRRC). CRRC uses time-lapse fluorescence microscopy to measure a rate constant of a catalytic reaction in individual cells and, thus, facilitate accurate size determination for cell subpopulations with distinct efficiencies of this reaction. Practical CRRC requires that a tissue sample be disintegrated into a suspension of dispersed cells and these cells settle on the support surface before being analyzed by CRRC. We call such cells “dispersed-settled” to distinguish them from cells cultured as a monolayer. Studies of the dispersed-settled cells can be tissue-relevant only if the cells maintain their 3D tissue state during the multi-hour CRRC procedure. Here we propose an approach for assessing tissue relevance of the CRRC-based analysis of the dispersed-settled cells. Our approach utilizes cultured multicellular spheroids as a 3D cell model and cultured cell monolayers as a 2D cell model. The CRRC results of the dispersed-settled cells derived from spheroids are compared to those of spheroids and monolayers in order to find if the dispersed-settled cells are representative of the spheroids. To demonstrate its practical use, we applied this approach to a cellular reaction of multi-drug-resistance (MRD) transport which was followed by extrusion of a fluorescent substrate from the cells. The approach proved to be reliable and revealed long-term maintenance of MDR transport in the dispersed-settled cells obtained from cultured ovarian cancer spheroids. Accordingly, CRRC can be used to determine accurately the size of a cell subpopulation with an elevated level of MDR transport in tumor samples, which makes CRRC a suitable method for the development of MDR-based predictors of chemoresistance. The proposed spheroid-based approach for validation of CRRC is applicable to other types of cellular reactions, and, thus, will be an indispensable tool for transforming CRRC from an experimental technique into practical analytical tool. Additional (zip) files contain supporting images, kinetic traces, and histograms. Their detailed descriptions are provided in the main pdf file.

Quantification of human cells may be performed using quantitative polymerase chain reaction (qPCR). In preclinical studies, the human Alu sequence is widely used as biomarker for human DNA. However, because the Alu gene is shared by primates, its use is limited to non-primate studies. The biodistribution of human cells in primates is also necessary for translational studies. Therefore, we aimed to design a novel, human-specific primer/probe that enables the quantification of human cells in primates and other animal models. A novel primer/probe set was successfully designed based on highly repetitive LINE1 sequences. qPCR efficiency (94.95–99.21%) and linearity of calibration curves (r2 = 0.996–0.999) were confirmed in tissue homogenates of cynomolgus monkey. The lower limit of detection was 10 cells per 15-mg tissue sample, a sensitivity that is equivalent to existing Alu primers/probes. The set was also effective in other animal models such as mice, rabbits, pigs, and common marmosets. To our knowledge, this is the first study describing the successful design of a human-specific qPCR primer/probe for human cell quantification in various animals, including non-human primates, using LINE1 sequence. The excellent selectivity, sensitivity, and versatility of the LINE1 primers/probes make it a promising quantification tool in preclinical biodistribution studies.

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

A significant and common problem in analytical chemistry is determining if a sample belongs to a specific class, e.g., establishing if a food product is genuine or counterfeit or a tissue sample is benign or malignant. This problem is termed one-class classification (class modeling). Problematic with class modeling is determining which one-class classifier to use followed by the challenge of optimizing the chosen classifier (identifying the best tuning parameter value(s)). With spectroscopic data, two other conundrums arise: which data preprocessing method(s) and spectral region(s) to use. Presented in this paper is a hybrid fusion process that can combine nonoptimized classifiers across multiple instruments, preprocessing methods, and measurements. Instead of optimizing classifiers, a window of tuning parameters is used for each classifier. The flexible fusion method of sum of ranking differences (SRD) is applied to combine all assessment values. Defining the best SRD ranking value (threshold) for determining class membership is the one tuning parameter value needed. However, this SRD ranking value is automatically optimized by using a receiver operator characteristic (ROC) curve. The approach is demonstrated on two analytical data sets. The first is a beer authentication sample set measured on five instruments: near-infrared, mid infrared (MIR), ultraviolet, visible, and thermogravimetric. Three different fusion protocols of all five instruments are demonstrated. The second data set is MIR spectra of strawberry puree with two categories: strawberry puree and nonstrawberry puree. Fusing nonoptimized classifiers provides reliable classifications relative to accuracy, sensitivity, and specificity.

Liver plays a major role in metabolism and acts as a source of energy for the body by storing glycogen. With the growing interest and investigation in the biological effects in recent years, it is important and necessary to develop accurate and comparable analytical methods for elements in bio-samples. It has, however, been 10 years since the tissue sample (bovine liver) of CCQM-K49 key comparison. The purpose of CCQM-K145 is to ensure the comparable and traceable measurement results for essential and toxic elements such as P, S, Zn, Mn, Ni, Mo, Sr, Cr, Co, Pb, As and Hg in bovine liver among NMIs and other designated measurement bodies worldwide. The comparison was agreed by IAWG as 6th IAWG Benchmarking Exercise with Zn and Ni as exemplary elements at the meeting in Korea in the early October 2016. The results of CCQM-K145 are expected to cover the measurement capability and support CMCs claiming for inorganic elements in the similar biological tissue materials and food samples. 30 NMIs and DIs registered in CCQM-K145. With respect to the methodology, a variety of techniques such as IDMS, ICP-OES, ICP-MS(non-ID), AAS and NAA were adopted by the participants. For Zn, Ni, Sr, Pb and Hg measurements, most participants chose ID-ICP-MS method, which showed the better performance in terms of consistency and reliability of the measurement results. In aspect of the traceability for the measurement results in CCQM-K145, most participants used their own (in house) CRMs or other NMI's CRMs to guarantee trace to SI unit. Most participants used similar matrix CRMs for quality control or method validation. Base on different statistic way to calculate the reference mass fraction values and associated uncertainties for each measurand, removal of the suspected extreme values, and discussion at the IAWG meetings, the median values are proposed as the KCRV for Zn, Ni, Mn, Mo, Cr, Pb and Hg; the arithmetic mean values are proposed as the KCRV for P, S, Sr, Co and As. In general, the performances of the majority of CCQM-K145 participants are very good, illustrating their measurement capabilities for Zn, Ni, P, S, Mn, Mo, Sr, Cr, As, Co, Pb and Hg in a complex biological tissue matrix. Bovine liver contains many kinds of nutrients and microelements, it can be regarded as a typical representative material of biological tissue and food. In CCQM-K145, the analytes involved alkali metals and transition elements, metalloids / semi-metals and non metals with a range of mass fraction from mg/g to μg/kg. CCQM-K145 also tested the ability of NMIs/DIs to determine elements that were easy to be lost and polluted, and interfered significantly. The chemical pretreatment methods of samples used in the comparison is suitable for general food and biological matrix samples. A variety of measurement methods used in the comparison represent the main instrumental technology for elemental analysis. Therefore, for supporting CMC claim, CCQM-K145 is readily applicable to measurement of more elements in a wide range of biological materials (including liquids and solids) and meat products. Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

Subsequent to conducting the Chromosome-Centric Human Proteome Project, we have focused on human testis-enriched missing proteins (MPs) since 2015. For protein coverage to be enhanced, a multiprotease strategy was used for separation of samples by 10% SDS-PAGE. For the separating efficiency to be improved, a high-pH reverse phase (RP) separation strategy was applied to fractionate complex samples in this study. A total of 11,558 proteins was identified, which is the largest proteome data set for single human tissue sample so far. On the basis of this large-scale data set, we verified 14 MPs (PE2) in neXtProt (2018-01) after spectrum quality analysis, isobaric post-translational modification, and single amino acid variant filtering, and synthesized peptide matching. Tissue expression analysis showed that 3 of 14 MPs were testis-specific proteins. Functional analysis showed that 10 of 14 MPs were closely related to liver tumor, liver carcinoma, and hepatocellular carcinoma. Another 100 MPs were listed as candidates but required additional verification information. All MS data sets have been deposited into the ProteomeXchange with the identifier PXD009737.

Pretreatment is a critical step in the analysis of samples with complex matrices such as most food samples. Time-saving pre-processing methods are always desirable. Aimed at obtaining a rapid method for seafood authenticity analysis, several one-step methods were compared to pretreat fish muscle tissues for obtaining an optimal pretreatment. The acid processing method relies on boiling tissue sample in 0.1 M trifluoroacetic acid (TFA) for 5 min, exhibiting advantages of being reproducible, efficient, and capable of realizing a good spectral resolution when analyzing the resultant samples by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). When combined this acid method with similarity coefficient-based analysis for their mass spectra, different fish samples can be distinguished. Two single-blind seafood samples from market were authenticated. The acid method combined with comparison of mass spectral pattern greatly simplifies the seafood identification analysis and can promisingly be applied in super-fast food authenticity testing.

Background & Aim To date, one of the main bottle necks to the industrialisation and commercialisation of advanced therapy medicinal products (ATMPs) is both the access to, and the variation in, the cellular starting material. While there are existing pathways in place for the collection of liquid tissues (apheresis, blood, bone marrow, cord) there is no established pathway for the in situ collection of solid tissues following surgical removal (see concept pathway map Figure 1). Due to the in-built variability in the type, size and composition of solid tissues, particularly tumour tissue, it's essential that the processes used to collect and stabilise the tissue provides suitable yields of viable relevant cells. Enabling a standardised and reproducible tissue collection pathway to minimise further starting material variation due to the collection process is therefore crucial to the industrial scale-up of resultant ATMP production. Additionally, the manufacture of ATMPs from solid tissues currently utilises a fresh-in, fresh-out approach which relies on pre-planned manufacture and distribution of each individual product. However, this approach results in inflexible scheduling at every stage of the process and is not a strategy that can be implemented for the production of ATMPs on an industrial scale. Methods, Results & Conclusion As such, our concept has focused on the design and development of a pathway capable of reproducibly collecting and disaggregating solid tissue samples, of various origin and sizes to produce viable cell suspensions that can be stabilised at source. Furthermore, following the disaggregation of the tissue samples, it's possible to directly cryopreserve the generated cell suspension to minimise time between harvest of the tissue sample and stable storage, ultimately to enable ATMP manufacture in the future and disconnect the starting material collection procedure and the upstream manufacturing steps to aid the industrialisation of these therapies.

Porous graphitized carbon (PGC) based chromatography achieves high-resolution separation of glycan structures released from glycoproteins. This approach is especially valuable when resolving structurally similar isomers and for discovery of novel and/or sample-specific glycan structures. However, the implementation of PGC-based separations in glycomics studies has been limited because system-independent retention values have not been established to normalize technical variation. To address this limitation, this study combined the use of hydrolyzed dextran as an internal standard and Skyline software for post-acquisition normalization to reduce retention time and peak area technical variation in PGC-based glycan analyses. This approach allowed assignment of system-independent retention values that are applicable to typical PGC-based glycan separations and supported the construction of a library containing >300 PGC-separated glycan structures with normalized glucose unit (GU) retention values. To enable the automation of this normalization method, a spectral MS/MS library was developed of the dextran ladder, achieving confident discrimination against isomeric glycans. The utility of this approach is demonstrated in two ways. First, to inform the search space for bioinformatically predicted but unobserved glycan structures, predictive models for two structural modifications, core-fucosylation and bisecting GlcNAc, were developed based on the GU library. Second, the applicability of this method for the analysis of complex biological samples is evidenced by the ability to discriminate between cell culture and tissue sample types by the normalized intensity of N-glycan structures alone. Overall, the methods and data described here are expected to support the future development of more automated approaches to glycan identification and quantitation.

We have developed Digital Spatial Profiling (DSP), a non-destructive method for high-plex spatial profiling of proteins and RNA, using oligonucleotide detection technologies with unlimited multiplexing capability. The key breakthroughs underlying DSP are threefold: (1) multiplexed readout of proteins/RNA using oligo-tags; (2) oligo-tags attached to affinity reagents (antibodies/RNA probes) through a photocleavable (PC) linker; (3) photocleaving light projected onto the tissue sample to release PC-oligos in any spatial pattern. Here we show precise analyte reproducibility, validation, and cellular resolution using DSP. We also demonstrate biological proof-of-concept using lymphoid, colorectal tumor, and autoimmune tissue as models to profile immune cell populations, stroma, and cancer cells to identify factors specific for the diseased microenvironment. DSP utilizes the unlimited multiplexing capability of modern genomic approaches, while simultaneously providing spatial context of protein and RNA to examine biological questions based on analyte location and distribution.

Immunofluorescent staining (IF) uses antigen-antibody complexes tagged with fluorochromes to observe the expression of proteins within a tissue sample. Multiple groups have described optimized methods to visualize several proteins simultaneously within the same tissue section using immunofluorescence in both mouse and human FFPE tissues. Our group routinely uses an optimized protocol described here to examine nuclear receptor expression in experimental samples from conditional knockout in vivo studies.

NMR-based metabolomics has shown promise in the diagnosis of diseases as it enables identification and quantification of metabolic biomarkers. Using high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy, metabolic profiles from intact tissue specimens can be obtained with high spectral resolution. In addition, HR-MAS NMR requires minimal sample preparation and the sample is kept intact for subsequent analyses. In this chapter, we describe a typical protocol for NMR-based metabolomics of tissue samples. We cover all major steps ranging from tissue sample collection to determination of biomarkers, including experimental precautions taken to ensure reproducible and reliable reporting of data in the area of clinical application.

Investigation of hyperpolarized substrate metabolism has been showing utility in real-time determination of in-cell and in vivo enzymatic activities. Intracellular reaction rates may vary during the course of a measurement, even on the very short time scales of visibility on hyperpolarized MR, due to many factors such as the availability of the substrate and co-factors in the intracellular space. Despite this potential variation, the kinetic analysis of hyperpolarized signals typically assumes that the same rate constant (and in many cases, the same rate) applies throughout the course of the reaction as observed via the build-up and decay of the hyperpolarized signals. We demonstrate here an acquisition approach that can null the need for such an assumption and enable the detection of instantaneous changes in the rate of the reaction during an ex vivo hyperpolarized investigation, (i.e. in the course of the decay of one hyperpolarized substrate dose administered to a viable tissue sample ex vivo). This approach utilizes hyperpolarized product selective saturating-excitation pulses. Similar pulses have been previously utilized in vivo for spectroscopic imaging. However, we show here favorable consequences to kinetic rate determinations in the preparations used. We implement this acquisition strategy for studies on perfused tissue slices and develop a theory that explains why this particular approach enables the determination of changes in enzymatic rates that are monitored via the chemical conversions of hyperpolarized substrates. Real-time changes in intracellular reaction rates are demonstrated in perfused brain, liver, and xenograft breast cancer tissue slices and provide another potential differentiation parameter for tissue characterization.

Rationale Desorption electrospray ionization (DESI) imaging is a fast analytical technique used to assess spatially resolved biological processes over unmodified sample surfaces. Although DESI profiling experiments have demonstrated that the properties of the sample surface significantly affect the outcomes of DESI analyses, the potential implications of these phenomena in imaging applications have not yet been explored extensively. Methods The distribution of endogenous and exogenous organic acids in pith and out pith region of grapevine stems was investigated by using DESI imaging, ion chromatography and direct infusion methods. Several common normalization strategies to account for the surface effect, including TIC normalization, addition of the internal standard in the spray solvent and deposition of the standard over the sample surface, were critically evaluated. Results DESI imaging results show that, in our case, the measured distributions of these small organic acids are not consistent with their 'true' localizations within the tissues. Furthermore, our results indicate that the common normalization strategies are not able to completely compensate for the observed surface effect. Conclusions Variations in the tissue surface properties across the tissue sample can greatly affect the semi-quantitative detection of organic acids. Attention should be paid when interpreting DESI imaging results and an independent analytical validation step is important in untargeted DESI imaging investigations. Copyright © 2016 John Wiley & Sons, Ltd.

Untargeted metabolomics attempts to acquire a comprehensive and reproducible set of small-molecule metabolites in biological systems. However, metabolite extraction method significantly affects the quality of metabolomics data. In the present study, we calculated the number of peaks (NP) and coefficient of variation (CV) to reflect metabolome coverage and reproducibility in untargeted NMR-based metabolic profiling of tissue samples in rats under different methanol/chloroform/water (MCW) extraction conditions. Different MCW extractions expectedly generated diverse characteristics of metabolome. Moreover, the classic MCW method revealed tissue-specific differences in the NP and CV values. To obtain high-quality metabolomics data, therefore, we used mixture design methods to optimize the MCW extraction strategy by maximizing the NP value and minimizing the CV value in each tissue sample. Results show that the optimal formulations of MCW extraction were 2:2:8 (ml/mg tissue) for brain sample, 2:4:6 (ml/mg tissue) for heart sample, 1.3:2:8.7 (ml/mg tissue) for liver sample, 4:2:6 (ml/mg tissue) for kidney sample, 2:3:7 (ml/mg tissue) for muscle sample, and 2:4:6 (ml/mg tissue) for pancreas sample. Therefore, these findings demonstrate that different tissue samples need a specific optimal extraction condition for balancing metabolome coverage and reproducibility in the untargeted metabolomics study. Mixture design method is an effective tool to optimize metabolite extraction strategy for tissue samples. Graphical abstract ᅟ.

Improved speed and sensitivity of mass spectrometry allow the simultaneous quantification of high numbers of proteins from increasingly smaller quantities of tissue sample. Quantitative data of the proteome is highly valuable for providing unbiased information on, for example, protein expression changes related to disease or identifying related biomarkers. In brain diseases the affected area can be small and pathogenic events can be related to a specific cell type in an otherwise heterogeneous tissue type. An emerging approach dedicated to analyzing this type of samples is laser micro-dissection (LMD) combined with LC-MS/MS into a single workflow. In this chapter, we describe different options for isolating tissue suitable for LC-MS/MS analysis.

Stable isotope dilution-selective reaction monitoring-mass spectrometry (SID-SRM-MS) has been widely used for the absolute quantitative analysis of proteins. However, when performing the large-scale absolute quantification of proteins from a more complex tissue sample, such as mouse liver, in addition to a high-throughput approach for the preparation and calibration of large amounts of stable-isotope-labelled internal standards, a more powerful separation method prior to SRM analysis is also urgently needed. To address these challenges, a high-throughput absolute quantification strategy based on an improved two-dimensional reversed-phase (2D RP) separation and quantification concatemer (QconCAT) approach is presented in this study. This strategy can be used to perform the simultaneous quantification of hundreds of proteins from mouse liver within one week of total MS measurement time. By using calibrated synthesised peptides from the protein glutathione S-transferase (GST), large amounts of GST-tagged QconCAT internal standards corresponding to hundreds of proteins can be accurately and rapidly quantified. Additionally, using an improved 2D RP separation method, a mixture containing a digested sample and QconCAT standards can be efficiently separated and absolutely quantified. When a maximum gradient of 72 min is employed in the first LC dimension, resulting in 72 fractions, identification and absolute quantification experiments for all fractions can be completed within one week of total MS measurement time. The quantification approach developed here can further extend the dynamic range and increase the analytical sensitivity of SRM analysis of complex tissue samples, thereby helping to increase the coverage of absolute quantification in a whole proteome.

Purpose To evaluate which common post-processing method applied to gradient-echo DSC-MRI data, acquired with a single gadolinium injection and low flip-angle, most accurately reflects microvascular histopathology for patients with de novo, treatment-naive glioblastoma multiforme (GBM). Materials and Methods Seventy-two tissue samples were collected from 35 patients with treatment-naive GBM. Sample locations were co-registered to preoperative gradient-echo dynamic susceptibility contrast (DSC) MRI acquired with 35° flip-angle and 0.1 mmol/kg gadolinium. Estimates of blood volume and leakiness at each sample location were calculated using four common postprocessing methods (leakage-corrected nonlinear gamma-variate, non-parametric, scaled MR-signal, and unscaled MR-signal). Tissue sample microvascular morphology was characterized using Factor VIII immunohistochemical analysis. A random-effects regression model, adjusted for repeated measures and contrast-enhancement (CE), identified whether MR parameter estimates significantly predicted IHC findings. Results Elevated blood volume estimates from nonlinear and non-parametric methods significantly predicted increased microvascular hyperplasia. Abnormal microvasculature existed beyond the CE-lesion and was significantly reflected by increased blood volume from nonlinear, non-parametric, and scaled MR-signal analysis. Conclusion This study provides histopathological support for both non-parametric and nonlinear post-processing of low flip-angle DSC-MRI for characterizing microvascular hyperplasia within GBM. Non-parametric analysis with a single gadolinium injection may be a particularly useful strategy clinically, as it requires less computational expense and limits gadolinium exposure. J. Magn. Reson. Imaging 2013;38:388–400. © 2013 Wiley Periodicals, Inc.

Carcinoma of the gallbladder (CaGB) is a common health problem in Northern India. Exact causative factors are still obscure. Dietary habits are also known to be a major factor in the gallbladder carcinogenesis. Mustard oil is mostly used as cooking media, which is adulterated by sanguinarine, diethylnitrosamine and repeated frying. We tried to find out the association of mustard oil as cooking media with CaGB. Twenty patients each of CaGB (group I) and cholelithiasis (group II) were included in the study. Sanguinarine and diethylnitrosamine (DEN) were extracted from the tissue and blood samples from both groups. Mean and standard error of mean of the concentration of the sanguinarine and DEN were calculated. Mann–Whitney U test was applied to test the level of significance between the two groups. The mean concentration of tissue sanguinarine in both groups (I and II) was 195.18 ng/mg and 24.05 ng/mg, respectively, and the difference was statistically highly significant (p

A simple enzymatic screening method has been developed to detect whether a tissue sample has been preserved with formalin or with ethanol only because such a method is a useful tool for predicting the quality of genetic test results. The method is based on enzymatic digestion at 55°C at neutral pH. The screening method shows that only ethanol-preserved tissue samples are dissolved, whereas formalin-preserved samples remain undissolved. The method was developed by the incorporation of laboratory rats preserved under controlled conditions in either 4% neutral buffered formalin or 96% ethanol. The method was subsequently tested on wild-living preserved specimens and an archived specimen. The protease enzyme used was Savinase® 16 L, Type EX from Novozymes A/S. The enzymatic screening test demands only simple laboratory equipment. The method is useful for natural history collections in museums where DNA analyses of archived specimens are performed. Wasted time and resources can be avoided through the detection of formalin-fixed specimens because these specimens yield low-quality, damaged DNA. In addition to the screening method, it is shown that formalin-preserved specimens can be macerated by enzymatic digestion under alkaline conditions at 55°C.

Screening of tissue samples for glycogen is very important in assessing the ageing, but also the state of health of the tissue. Therefore, two needle stochastic sensors based on maltodextrins presenting different dextrose equivalence (DE) MD-m (DE 13.0-17.0), and MD-M (DE 16.5-19.5) immobilized in diamond paste (obtained from synthetic diamond and paraffin oil) were designed and characterized. These stochastic sensors were used reliable for both qualitative and quantitative analysis for the assay of glycogen in tissue samples with limits of determination as low as 1fmol L-1.

Objective. To estimate the genetic diversity of the Anadara tuberculosa en five mangrove swams of Tumaco, Nariño, Colombia using as a mitocondrial molecular marker the cytochromo oxidase sub-unit I (COI). Materials and methods. A total of 50 individuals were collected from the San Jorge, La Tiburonera, El Pajal, La Playa y Bajito Vaquería mangrove swamps, randomly selecting 10 specimens of each zone. The tissue sample was worked with absolute alcohol at ambient temperature in microtubes. DNA was extracted, and the mitocondrial DNA was amplified using the PCR technique (polymerase chain reaction). The amplified and quantified products of PCR were sequenced on both sides (Macrogen). Each one of the obtained sequences was edited and aligned. Later, the parameters of genetic diversity (haplotypical and nucleotidical) were measured, and the analysis of distribution between frequency pairs (Mistmach distribution) was elaborated. Finally, the analysis of nucleotidic variation and population structure (AMOVA) was completed. Results. The amplified product gene weighed 710 bp. The haplotypical diversity reported for all the populations was high (0.683±0.060) and the reported nucleotídical diversity was low for all the populations (0.040±0.020). The AMOVA results indicate that the variance amongst populations is low (4.20%) and that the variance within populations is high (95.80%). Conclusions. The studied populations are not structured and although there is a decrease of natural banks, the genetic diversity is high., RESUMENObjetivo. Estimar la diversidad genética de Anadara tuberculosa en cinco manglares de Tumaco Nariño, Colombia utilizando como marcador molecular mitocondrial la subunidad I de la citocromo oxidasa (COI). Materiales y métodos. Se colectaron en total 50 individuos de los manglares San Jorge, La Tiburonera, El Pajal, La Playa y Bajito Vaquería, tomando 10 ejemplares al azar de cada extrajo y amplificó el ADN mitocondrial mediante la técnica de PCR (Polymerase Chain Reaction). Los productos de PCR amplificados y cuantificados se secuenciaron por ambos lados (Macrogen). Una vez se obtuvo las secuencias, se editó y alineo cada secuencia. Posteriormente, se midió los parámetros de diversidad genética (haplotípica y nucleotídica) y se elaboró el análisis de distribución entre pares de frecuencias (Mistmach distribution). Finalmente se efectuó el análisis de variación nucleotídica y la estructura poblacional (AMOVA). Resultados. El gen amplificado tuvo una longitud de 710 pb. La diversidad haplotípica reportada para todas las poblaciones fue alta (0.683±0.060) y la diversidad nucleotídica reportada fue baja para todas las poblaciones (0.040±0.020). Los resultados del AMOVA indican que la varianza entre poblaciones es baja (4.20%) y la varianza dentro de las poblaciones es alta (95.80%). Conclusiones. Las poblaciones estudiadas no se encuentran estructuradas y a pesar de la disminución de los bancos naturales de las poblaciones de Anadara tuberculosa, se estima que la diversidad genética es alta.

Two NMR experiments are designed for selective excitation of spin I =5/2 nuclei that exhibit residual quadrupolar splittings. The I =5/2 Jeener–Broekaert experiment is preferred to the four-quantum filtration experiment as it is shown to be a more sensitive technique in experimental practice. Both techniques are applied to 17 O-enriched water in biological systems. The occurrence of water which displays a residual 17 O quadrupolar splitting is demonstrated for the first time in a model biological system and an excised tissue sample. The resulting 17 O NMR spectra are shown to have the characteristics predicted in computer-simulated I =5/2 NMR spectra.

In this work, an analytical protocol based on on-line matrix solid-phase dispersion (MSPD) ultra-fast LC–MS/MS was successfully established, for rapid determination of chloramphenicol (CAP) present in soft-shelled turtle tissues. Trace level of CAP was analysed and separated on a core-shell C18 silica column in 2 min. The performance of the on-line MSPD-LC–MS/MS method was evaluated using multi-reaction monitoring (MRM) quantification mode. The calibration curve was linear over the range of 0.01–100 ng mL−1. The mean values of relative standard deviation of intra- and inter-day ranging from 2.05% to 8.33% and from 3.05% to 10.17% were obtained, respectively. In the four fortified levels (1, 5, 7.5 and 10 ng), recoveries of CAP ranged from 92.05% to 98.07% with RSDs ⩽ 4.20%. The proposed method has been applied successfully to the analysis of CAP in soft-shelled turtle tissue sample, which demonstrates that this new instrumental coupling is ultra-fast, sensitive, reliable and environmental friendly.

Metabolism has an essential role in biological systems. Identification and quantitation of the compounds in the metabolome is defined as metabolic profiling, and it is applied to define metabolic changes related to genetic differences, environmental influences and disease or drug perturbations. Chromatography-mass spectrometry (MS) platforms are frequently used to provide the sensitive and reproducible detection of hundreds to thousands of metabolites in a single biofluid or tissue sample. Here we describe the experimental workflow for long-term and large-scale metabolomic studies involving thousands of human samples with data acquired for multiple analytical batches over many months and years. Protocols for serum- and plasma-based metabolic profiling applying gas chromatography-MS (GC-MS) and ultraperformance liquid chromatography-MS (UPLC-MS) are described. These include biofluid collection, sample preparation, data acquisition, data pre-processing and quality assurance. Methods for quality control-based robust LOESS signal correction to provide signal correction and integration of data from multiple analytical batches are also described.

At its 25th meeting the ECVAM Scientific Advisory Committee (ESAC) unanimously endorsed that the SkinEthic Reconstructed Human Epidermis (RHE) model could be used for distinguishing between corrosive and non-corrosive chemicals within the context of the Organisation Economic for Co-operation and Development (OECD) test guideline, TG 431 (ESAC 16-17 November 2006). Both test method development and multi-center study were performed using 0.63 cm(2) RHE tissue samples. The purpose of the present study was to demonstrate that similar results could be obtained using the validated test method adapted to 0.5cm(2) RHE tissue samples. Test method adaptation only consisted in applying a reduced volume of test substance (40 microL instead of 50 microL for liquids and 20 microL water+20mg test substance instead of 25 microL water+25mg test substance for solids) and a reduced propan-2-ol extraction volume (1.5 mL instead of 2 mL) during the MTT reduction assay. The test method was assessed with 25 representative test substances of different chemical classes. Among the latter, the 12 OECD reference test substances (6 corrosives and 6 non-corrosives) were evaluated and showed to be similarly classified as in vivo. More generally, the SkinEthic skin corrosion test adapted to 0.5 cm(2) RHE tissue samples fully complies with the OECD performance and reproducibility requirements with the 25 test substances.

Imaging MS is a powerful technique that combines the chemical and spatial analysis of surfaces. It allows spatial localization of multiple different compounds that are recorded in parallel without the need of a label. It is currently one of the rapidly developing techniques in the proteomics toolbox. Different complementary imaging MS methods, i.e. MALDI and secondary ion MS imaging for direct tissue analysis, can be applied on exactly the same tissue sample. This allows the identification of small molecules, peptides and proteins present on the same sample surface. Sample preparation is crucial to obtain high quality, reliable and reproducible complementary molecular images. It is essential to optimize the conditions for each step in the sample preparation protocol, ranging from sample collection and storage to surface modification. In this article, we review and discuss the importance of correct sample treatment in case of MALDI and secondary ion MS imaging experiments and describe the experimental requirements for optimal sample preparation.

INTRODUCTION: Haemangioma is the most common tumour of infancy. Its biological behaviour is one of a benign nature and characterised by a typical biphasic growth pattern. After an initial proliferation during the child's first year of life an involution takes place and usually lasts until the first decade. The expression of cellular and extracellular markers as well as cytokines follows its biphasic growth. In-vitro models reported so far have provided new insights in the tumour's biology and have identified possible targets for pharmacological agents. PURPOSE: Our study should enhance the functionality of current in-vitro models in establishing a suspension model of isolated proliferating haemangioma vascular endothelial cells (HVECs). MATERIAL AND METHODS: Tissue samples isolated from four haemangiomas (H1-H4) during the proliferation phase were cultivated in four different ways: H1 was cultivated in an MCDB-131 medium whereas for the H2 samples EGM2-MV medium was used. In the H3 series dispase enabled an immediate isolation of pure HVECs from the tissue sample which were cultivated under EMG2-MV medium. In the similarly cultivated H4 series an isolation of cultivated HVECs was enabled by the use of magnetic bead separation which finally led to a suspension model of isolated HVECs. RESULTS: In all four series HVECs were cultivated successfully. Depending on the selected medium different growth rates were observed. The use of dispase and the magnetic bead separation technique resulted in an isolation of cultivated HVECs which was documented in haematoxylin/eosin staining as well as under CD-31 immunohistochemistry. CONCLUSIONS: Research on tumour-specific processes as well as possible pharmacological targets necessitates a stable in-vitro model of proliferating HVECs. For our purposes, the described suspension model of HVECs fulfills the requirements of further research on antiproliferation and anti-angiogenesis.

The lipid content and total mercury concentration were measured in whole tissue composites of all edible tissues of farmed southern bluefin tuna (Thunnus maccoyii) and each of the marketed tissue cuts of these fish (akami, chu-toro, o-toro). Despite differences in fish size, condition factor and culture time, the mercury concentrations of tissue cuts and composite samples were found to decrease with increasing lipid content at a consistent rate of −0.00476 Hg (mg/kg)/% lipid within each fish. Consequently, lipid accumulation appears to have a dilution effect on mercury already associated with fish tissues. The increased affinity of lipid for certain tissue cuts (o-toro) over that of others (e.g. akami), results in cross carcass variation in the mercury concentration of fish muscular tissue with clear implications for mercury advisory statements – the tissue sample collected for analysis is critical.

Raman spectroscopy (RS) of Nasopharyngeal carcinoma (NPC) tissue contained various biomedicine features. These features indicated molecular-level information of tissue at different carcinoma development-level. This study suggested an automatic and quick method for the NPC Raman spectra classification at different stages by multivariate statistical analysis. In the RS measurement, high quality Raman spectra was acquired from each NPC tissue sample in two groups: one group consisted of 30 NPC patients at the early stages (I-II), another group was 46 NPC patients at the advanced stages (III-IV). Moreover, tentative diagnostic algorithms based on principle components analysis (PCA) and support vector machine (SVM) were employed to classify the multivariate data of Raman spectra effectively. The classification performance (sensitivities and specificities were 70% (21/30) and 91% (42/46)) was achieved by the PCA-SVM in conjunction with leave-one-out cross validation method. In this beneficial study, the RS technique in conjunction with PCA-SVM provided a great clinical potential for rapid NPC stage diagnosis.

The objectives of the present study were to assess heavy metal cadmium (Cd), lead (Pb) and organochlorine pesticide concentrations in tissues of red deer (Cervus elaphus) and wild boar (Sus scrofa) from nine hunting areas and to evaluate related risk factors for the host animal. Over a period of 2 years, a total of 1055 and 210 masseters, 424 and 201 livers, 642 and 152 kidneys were collected from wild boar and red deer, respectively, and concentrations of Cd, Pb and organochlorine pesticides were determined. Comparing the two species, Cd concentration in the kidney (3.72 mg/kg), liver (0.67 mg/kg) and muscle (0.02 mg/kg) of wild boar was found to be significantly higher than in the organs of red deer (1.02 mg/kg in the kidneys, 0.07 mg/kg in the liver and 0.006 mg/kg in muscle). Mean Pb concentrations were found to be similar in both animals, with 0.39, 0.52 and 2.60 mg/kg detected in the wild boar kidney, liver and muscle, respectively, and 0.24, 0.21 and 2.04 mg/kg in the respective organs of the red deer. No difference in concentrations were found based on age class, location of tissue sample or contaminant in the case of wild boar. By contrast, a significantly lower Cd concentration was found in the kidney of the young red deer. The search for organochlorine pesticides in both red deer and wild boar produced negative results with values below the limits of detection. Due to the high levels of renal Cd and muscle Pb detected in wild boar and red deer, further research needs to be carried out in an effort to identify the source of contamination and preserve the health of animals and humans.

Metabolomics and peptidomics are systems biology approaches in which broad populations of molecular species produced in a cell or tissue sample are identified and quantified. These two molecular populations, metabolites and peptides, can be extracted from tissues in a similar fashion, and we therefore have here developed an integrated platform for their extraction and characterization. This was accomplished by liquid-liquid extraction of peptides and metabolites from tissue samples and online strong cation exchange (SCX) separation to allow characterization of each population individually. The platform was validated both by a mixed set of purified standards and by an analysis of splenic tissue from SIV-infected macaques, showing both good reproducibility in chromatography, with relative standard deviation (RSD) of hold time less than 0.4%, and clear separation of charge state, with ∼ 95% of molecular features in SCX separated runs at charge states of +1 or +2. Finally, we used this platform to analyze the physiological response to infection in the spleen, showing that the spleen contains an abundance of hemoglobin-derived peptides, which do not appear to change in response to infection, and that there appears to be a large and variable metabolic response to infection. We therefore present a method for peptidomic and metabolomic profiling which is simple, robust, and easy to implement.

A simple and sensitive HPLC method has been developed for the simultaneous determination of enrofloxacin (ENR) and ciprofloxacin (CIP) in pig tissue using difloxacin (DIF) as internal standard. Tissue sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1 m), followed by extraction with trichloromethane. Fluoroquinolones were separated on a reversed-phase column and eluted with aqueous buffer solution-acetonitrile (80:20, v/v). The concentrations of CIP, ENR and DIF eluted from the column, with retention times of 2.20, 2.73 and 4.38 min, respectively, were monitored by fluorescence detection at λex 276 and λem 442 nm. The detection and quantitation limit were 8 and 25 ng/g, respectively, for both compounds. Standard curves were linearly related to concentration in the range 25–400 ng/g. The consequences of the introduction of minor reasonable variations (ruggedness studies) have also been analysed. Finally, the measurement of the tissue levels of ENR and CIP in the pig tissues after oral administration confirmed the utility of the proposed method. Copyright © 2004 John Wiley & Sons, Ltd.

Changes in regional cerebral glucose metabolism were investigated for varying levels of tissue oxygenation using a dynamic positron autoradiography technique. While incubating fresh rat brain slices with [18F]FDG in an oxygenated solution, serial images of the tissue slices were obtained over a time period of up to 300 min and archived onto over 20 phosphorous imaging plate exposures. In order to properly create time activity curves of the uptake levels, images of the individual tissue samples were automatically located, digitally extracted, and registered with the later images of the same tissue samples. After applying image processing techniques for aligning tissue sample images, time activity curves were extracted for individual substructures in the rat brain and quantitative results were reported using Patlak plots. Since the levels of oxygenation can be controlled for these experiments, [18F]FDG uptakes can be reported representing states of hypoxia, pseudoischemia, and reoxygenation. The image processing techniques developed for this application have enabled more experiments and tissue samples to be acquired and analyzed than would otherwise be possible using manual ROI techniques. The objective spatial registration of tissue samples and automated extraction of data has increased the analysis accuracy and decreased the operator error associated with the interactive handling of the image data. This supports improved kinetic modeling of FDG uptake in animal studies, and can be used for more accurate dosimetry calculations in humans.

During cancer surgery, surgeons often must wait for pathologists to analyze a removed tissue sample to confirm they’ve gotten all the cancer. Researchers have previously shown that analyzing tissue with mass spectrometry during surgerycan help speed this process. But those surgical mass spec approaches rely on methods that often damage the tissue or are suitable only for analyzing removed samples. Livia S. Eberlinof the University of Texas, Austin, and coworkers have now come up with a biocompatible device that uses discrete water droplets to nondestructively obtain molecular profiles of cancer tissue (Sci. Transl. Med. 2017, DOI: 10.1126/scitranslmed.aan3968). The handheld device delivers a water droplet to the tissue surface, where it gently extracts metabolites, lipids, and proteins. Afterward, a plastic tube carries the water droplet to an attached mass spectrometer. The researchers used the device to sample 253 tissue pieces from a variety of cancers, finding that the mass spectra allow

A simple, rapid, reproducible and sensitive method based on HPLC with ultraviolet detection was developed for the determination of methimazole (MMI) in animal tissues and plasma samples. Under the optimum experimental conditions, the calibration curves for MMI were linear in the tested range 0.5-20 mg kg(-1) tissue sample (mg l(-1) plasma) with correlation coefficients better than 0.99. The performance of the proposed method was tested for the determination of MMI levels in brain, liver, thyroid gland and plasma of MMI-treated hens, as well as in their eggs and embryos. The proposed method reduces time and simplifies the sample preparation procedure.

A method was developed and validated for determination and quantitation of tilmicosin residues in swine, cattle, and sheep edible tissues, as well as chicken fat, skin, and muscle over a concentration range of 0.025 μg/g–20 μg/g. For chicken kidney and liver, the method was validated over a range of 0.060 μg/g–20 μg/g. The tissue sample was extracted with methanol and a C18 cartridge was used for solid-phase extraction cleanup. A reversed phase gradient liquid chromatographic method with detection at 280 nm was used to separate the tilmicosin from matrix components in 30 min run time. The limit of quantitation (LOQ) of the method was 0.025 μg/g for all tested tissues except chicken kidney and liver, for which the LOQ was 0.06 μg/g. Average recoveries for tissue samples ranged from 73 to 98%. Relative standard deviation values ranged from 0.6 to 14.7%.

Three replicate trials were conducted to determine the influence of raw breast meat color and pH on subsequent cooked meat color and pH. In each trial, approximately 50 breast fillets were obtained from a commercial processing plant based on being either normal, lighter than normal, or darker than normal. Color (L* = lightness, a* = redness, and b* = yellowness) of each fillet was determined in triplicate on the underside surface of the fillet (to avoid scalding effects), and the pH was determined on a tissue sample removed from the posterior portion of each fillet. Fillets were then cooked in steam at 98 C for 20 min and cooled to room temperature, and a second sample was removed from the posterior section for cooked meat pH. Cooked meat color was measured on an exposed surface, to avoid cooking-related discoloration. The data were subjected to linear regression analysis to determine the relationship between raw and cooked values. Results indicated a significant linear relationship between raw and cooked values for each color parameter as well as pH. Model R2 values were 0.43, 0.40, 0.64, and 0.78 for L*, a*, b*, and pH, respectively. There were also significant linear relationships between raw meat L* and raw muscle pH (R2 = 0.59) as well as cooked meat L* and raw meat pH (R2 = 0.36). These results indicate that raw breast meat color and pH affect cooked breast meat color and pH but that cooking reduces the degree of color variation. Moreover, cooked meat lightness is more closely associated with raw breast meat pH than with cooked meat pH.

Fermentation of a marine fungal species obtained from a tissue sample of a marine sponge collected in Indonesia in October 1996, yielded the novel hexaketide compounds iso-cladospolide B (1); seco-patulolide C (2); the 12-membered macrolides, pandangolide 1 (3) and pandangolide 2 (4); and the known terrestrial fungal metabolite, cladospolide B (5).

A partially automated technique for the isolation and characterization of N-linked sugar chains from glycoproteins of crude tissue samples is established. The N-linked sugar chains from the acetone-extracted tissues are made free by a process of hydrazinolysis and subsequently N-acetylated by GlycoPrep 1000 (Oxford Glycosystems). These free sugar chains are further converted to pyridylamino derivatives by GlycoTag (Takara). Characterization of these sugar chains is achieved by a combination of HPLC columns using a highly sensitive fluorescence detector at femtomole levels. Tissue sample can be successfully pyridylaminated and analyzed to give highly reproducible results with consistent yield, requiring fewer purification steps, minimum skills, and less time. Moreover, fixed tissues can also be analyzed employing this technique, giving a similar sugar chain pattern compared to normal tissue samples. Using this method we show that the pattern of N-linked sugar chains present in human sera or in one small region of brain is strikingly similar among the different individuals. However, the absence of a highlighted peak in one of the samples suggests this method can be extrapolated to identify changes, if any, associated with disorders such as inflammation or cancer. Furthermore, this two-dimensional display of sugar chains would discover the function-specific molecules as we see in proteins.

2-DE is a powerful protein analytical tool whose major strengths include semiglobal quantitation and charge separation of complex protein mixtures, enabling the analysis of differential protein expression, and variable post-translational modification. One of 2-DE's limitations relates to its limited dynamic range and consequently the number of proteins expressed that can be analyzed on a single gel. In an attempt to improve the yield of detectable proteins during sample preparation, we applied a novel extraction technique called pressure cycling technology.

Mealiness is a quality characteristic that is used to describe fruit which is soft and which causes a dry feeling in the mouth during mastication. The purpose of this work was to visualise the difference between fresh and mealy apples and to quantify those differences. Three commercially important Belgian apple cultivars, `Jonagold', `Cox's Orange Pippin' and `Boskoop' apples ( Malus domestica Borkh.) were used in the experiments. Mealy apples were produced by holding apples at 20°C and 95% RH for periods of time depending on the cultivar and the required mealiness level. Images were made with a light microscope. Cell parameters such as area and perimeter, and two roundness parameters were measured. `Cox's Orange Pippin' and `Boskoop' apples could be separated into the two groups: fresh and mealy, using the four calculated cell parameters. The number of broken and intact cells at the rupture surface of a fractured tissue sample after tensile loading was also calculated. After one week's storage of fruit in mealiness-inducing conditions, the amount of broken cells at the surface of a fractured sample was significantly lower than in fresh apples.

For more than 25 years, the United States Transuranium and Uranium Registries (USTUR) and the Dosimetry Registry of the Mayak Industrial Association (DRMIA) of the Russian Federation have, each independently, collected tissues at autopsy from workers with potential or confirmed body burdens of actinide elements resulting from occupational exposures. Tissues, thus obtained, were radiochemically analyzed for actinides for the purpose of evaluating the biokinetics of these elements in the human body. Scientists of these two organizations have recently begun a collaborative research program to compare, combine and analyze the data to verify or refine biokinetic models needed for radiation dosimetry.