Multiplex Hybrid Antigen-Capture LC-MRM Quantification in Sera and Nasal Lining Fluid of AZD7442, a SARS-CoV-2-Targeting Antibody Combination.

AZD7442 (tixagevimab [AZD8895]/cilgavimab [AZD1061]) is a monoclonal antibody (mAb) combination in development for the prevention and treatment of coronavirus disease 2019. Traditionally, bioanalysis of mAbs is performed using ligand binding assays (LBAs), which offer sensitivity, robustness, and ease of implementation. However, LBAs frequently require generation of critical reagents that typically take several months. Instead, we developed a highly sensitive (5 ng/mL limit of quantification) method using a hybrid LBA-liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) approach for quantification of the two codosed antibodies in serum and nasal lining fluid (NLF), a rare matrix. The method was optimized by careful selection of multiple reaction monitoring, capture reagents, magnetic beads, chromatographic conditions, evaluations of selectivity, and matrix effect. The final assay used viral spike protein receptor-binding domain as capture reagent and signature proteotypic peptides from the complementarity-determining region of each mAb for detection. In contrast to other methods of similar/superior sensitivity, our approach did not require multidimensional separations and can be operated in an analytical flow regime, ensuring high throughput and robustness required for clinical analysis at scale. The sensitivity of this method significantly exceeds typical sensitivity of ∼100 ng/mL for analytical flow 1D LBA-LC-MS/MS methods for large macromolecules, such as antibodies. Furthermore, infection and vaccination status did not impact method performance, ensuring method robustness and applicability to a broad patient population. This report demonstrated the general applicability of the hybrid LBA-LC-MS/MS approach to platform quantification of antibodies with high sensitivity and reproducibility, with specialized extension to matrices of increasing interest, such as NLF.

Multifaceted Bioanalytical Methods for the Comprehensive Pharmacokinetic and Catabolic Assessment of MEDI3726, an Anti-Prostate-Specific Membrane Antigen Pyrrolobenzodiazepine Antibody-Drug Conjugate.

Complex biotherapeutic modalities, such as antibody-drug conjugates (ADC), present significant challenges for the comprehensive bioanalytical characterization of their pharmacokinetics (PK) and catabolism in both preclinical and clinical settings. Thus, the bioanalytical strategy for ADCs must be designed to address the specific structural elements of the protein scaffold, linker, and warhead. A typical bioanalytical strategy for ADCs involves quantification of the Total ADC, Total IgG, and Free Warhead concentrations. Herein, we present bioanalytical characterization of the PK and catabolism of a novel ADC. MEDI3726 targets prostate-specific membrane antigen (PMSA) and is comprised of a humanized IgG1 antibody site-specifically conjugated to tesirine (SG3249). The MEDI3726 protein scaffold lacks interchain disulfide bonds and has an average drug to antibody ratio (DAR) of 2. Based on the structural characteristics of MEDI3726, an array of 4 bioanalytical assays detecting 6 different surrogate analyte classes representing at least 14 unique species was developed, validated, and employed in support of a first-in-human clinical trial (NCT02991911). MEDI3726 requires the combination of heavy-light chain structure and conjugated warhead to selectively deliver the warhead to the target cells. Therefore, both heavy-light chain dissociation and the deconjugation of the warhead will affect the activity of MEDI3726. The concentration-time profiles of subjects dosed with MEDI3726 revealed catabolism of the protein scaffold manifested by the more rapid clearance of the Active ADC, while exhibiting minimal deconjugation of the pyrrolobenzodiazepine (PBD) warhead (SG3199).

Immunoaffinity LC-MS/MS to quantify a PEGylated anti-Factor D Fab biotherapeutic in cynomolgus monkey serum.

Aim: To develop a sensitive hybrid immunoaffinity LC-MS/MS monkey serum assay to quantify multiple components of anti-Factor D; a complex PEGylated Fab biotherapeutic explored as a therapy for age-related macular degeneration. Materials & methods: Immunoaffinity enrichment of PEGylated anti-Factor D Fab, including fully conjugated, partially conjugated and unconjugated (i.e., free) Fab species, using a capture reagent coupled to magnetic beads was performed. The surrogate peptides derived from the therapeutic Fab via trypsin digestion were measured to obtain the total Fab concentrations. Results & conclusion: The method demonstrated the ability to accurately quantify both PEGylated and unconjugated Fab species. It was successfully validated with a LLOQ at 25.0 ng/ml.

Combination of an AccQ•Tag-Ultra-Performance Liquid Chromatographic Method with Tandem Mass Spectrometry for the Analysis of Amino Acids.

Amino acid analysis is a powerful tool in life sciences. Current analytical methods used for the detection and quantitation of low abundance amino acids in complex samples face intrinsic challenges such as insufficient sensitivity, selectivity, and throughput. This chapter describes a protocol that makes use of AccQ•Tag chemical derivatization combined with the exceptional chromatographic resolution of ultra-performance liquid chromatography (UPLC) and the sensitivity and selectivity of tandem mass spectrometry (MS/MS). The method has been fully implemented and validated using different tandem quadrupole detectors and thoroughly tested for a variety of samples such as P. falciparum, human red blood cells, and Arabidopsis thaliana extracts. Compared to currently available methods for amino acid analysis, the AccQ•Tag UPLC-MS/MS method presented here provides enhanced sensitivity and reproducibility and offers excellent performance within a short analysis time and a broad dynamic range of analyte concentration. The focus of this chapter is the application of this improved protocol for the compositional amino acid analysis in Arabidopsis thaliana leaf extracts using the Xevo TQ for mass spectrometric detection.

Validation of a biotherapeutic immunoaffinity-LC-MS/MS assay in monkey serum: 'plug-and-play' across seven molecules.

BACKGROUND: Biotherapeutics development requires validated assays in biological matrices for pharmacokinetic assessment. Historically, ligand-binding assays have been the predominant platform available. Recently, alternative hybrid methods, combining ligand-binding analyte enrichment with LC-MS detection have emerged. Methodology & results: The validation of an immunoaffinity (IA)-LC-MS/MS method to quantify a monoclonal antibody biotherapeutic in cynomolgus monkey serum is described. This method includes immunoaffinity capture of the antibody in serum, followed by enzymatic digestion and detection of a framework peptide. Using similar method conditions, six additional biotherapeutic assays were readily validated in different nonhuman mammalian species, including mouse, rat and monkey. CONCLUSION: The immunoaffinity-LC-MS/MS assay validation results across seven antibody therapeutics, using comparable conditions, illustrate the 'plug-and-play' nature of the IA-LC-MS/MS mAb framework peptide assay format.

Colony size-frequency distribution of pocilloporid juvenile corals along a natural environmental gradient in the Red Sea.

Coral colony size-frequency distributions can be used to assess population responses to local environmental conditions and disturbances. In this study, we surveyed juvenile pocilloporids, herbivorous fish densities, and algal cover in the central and southern Saudi Arabian Red Sea. We sampled nine reefs with different disturbance histories along a north-south natural gradient of physicochemical conditions (higher salinity and wider temperature fluctuations in the north, and higher turbidity and productivity in the south). Since coral populations with negatively skewed size-frequency distributions have been associated with unfavorable environmental conditions, we expected to find more negative distributions in the southern Red Sea, where corals are potentially experiencing suboptimal conditions. Although juvenile coral and parrotfish densities differed significantly between the two regions, mean colony size and size-frequency distributions did not. Results suggest that pocilloporid colony size-frequency distribution may not be an accurate indicator of differences in biological or oceanographic conditions in the Red Sea.

¿Ha llegado el momento de considerar la inclusión de la medicina del viajero en la formación del médico en América Latina? / Is the moment to consider the inclusion of travel medicine in the training of phsysicians in Latin America?

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Invertebrate colonization on artificial substrates in a coral reef at Gorgona Island, Colombian Pacific Ocean / Colonización de invertebrados sobre sustratos artificiales en un arrecife de coral en la Isla Gorgona, Océano Pacífico colombiano

Habitat colonization is a fundamental process in marine population dinamics and community ecology. Marine invertebrate colonization of artificial hard substrates and its spatial variation was studied over the course of one year at La Azufrada coral reef, Gorgona Island, Colombia. Five sets of artificial plates (terracotta and ceramic) were deployed parallel to the bottom in each of three reef zones (backreef, flat and slope). Plates were recovered after 12 months of their installation. The community that developed on the artificial substrate plates was composed of 24 taxa, among which corals were remarkably absent. Species richness on plates did not differ between reef zones, type of plate material or plate surfaces. The resulting community was dominated by barnacles (Balanus trigonus) with a mean density of 26 787.8 (±47 301.0) individuals/m² (mean±1S.D.). The density of this species was significantly higher on plates deployed on the reef slope than on the reef flat or back reef, and was lower on the upper than on the lower or lateral surfaces of plates. The dominance of B. trigonus on artificial plates during this experiment suggests a possible synergistic effect between its reproductive traits and competitive ability for space, which prevents the successful recruitment of other reef organisms such as corals. Rev. Biol. Trop. 62 (Suppl. 1): 161-168. Epub 2014 February 01.

La colonización de hábitat disponible es un proceso fundamental de la ecología de poblaciones y comunidades de organismos marinos. La colonización sobre sustratos artificiales por invertebrados y su variación espacial fue estudiada por un año en el arrecife coralino de La Azufrada, Isla Gorgona (Colombia). Cinco juegos de baldosas (terracota y grés) fueron dispuestos en tres zonas arrecifales (trasarrecife, planicie y talud). Las baldosas fueron recuperadas y examinadas después de 12 meses posteriores a su instalación. La comunidad desarrollada estuvo compuesta por 24 taxa siendo notable la ausencia de corales. No se encontraron diferencias en la riqueza de especies entre las zonas arrecifales, los materiales utilizados y las superficies de las baldosas. Las baldosas fueron dominadas por cirripedios (Balanus trigonus) con una densidad de 26 787.8 (±47 301.0) ind/m² (media±1D.E). La densidad de esta especie fue significativamente más alta en el talud y menor en las superficies superiores. La dominancia de B. trigonus durante el experimento sugiere un posible efecto sinérgico entre sus características reproductivas y su habilidad competitiva por el espacio evitando que otros organismos, como los corales, recluten exitosamente.

Short-term assessment of the sediment deposition rate and water conditions during a rainy season on La Azufrada coral reef, Gorgona Island, Colombia / Evaluación a corto plazo de la tasa de deposición de sedimentos y las condiciones del agua durante una temporada de lluvias en el arrecife coralino La Azufrada, Isla Gorgona, Colombia

One of the major stresses on corals is the settlement of suspended sediment on their surfaces. This leads to the blocking of light, the covering of the coral mucus surface and an increased risk of disease. For this reason sediment deposition on a reef is considered a highly important variable in coral reef studies. With the use of sediment traps and oceanographic sensors, the sediment deposition rate and water conditions during a rainy season (April-May 2009) on a Tropical Eastern Pacific coral reef (La Azufrada) at Gorgona Island in Colombia were investigated. To quantify sediment deposition, sediment traps were established in nine stations along the coral reef (three stations per reef zone: backreef, flat and slope). To minimize disturbance by aquatic organisms in the sediment traps these were filled with hypersaline borax-buffered 10% formaline solution before their deployment; animals found in the filter contents were fixed and stored in a 4% formalin solution, frozen and identified in the laboratory. To determine the water conditions, discrete samples of water from 1 m and 10 m depths were collected using a Niskin bottle. Oceanographic variables (temperature, salinity and dissolved oxygen) as well as turbidity, chlorophyll and nutrient concentration (nitrite, nitrate and phosphorus) were measured in the samples from both depths. Vertical records of temperature and salinity were carried out with a Seabird-19 CTD nearest to La Azufrada and water transparency was measured using a Secchi disk. We found a mean trap collection rate of 23.30±4.34gm-2d-1 and did not detect a significant difference in the trap collection rate among reef zones. The mean temperature and salinity in the coral reef depth zone (0-10m layer) were 26.98±0.19°C and 32.60±0.52, respectively. Fourteen taxonomic groups of invertebrates were detected inside the sediment traps with bivalves and copepods being the most abundant and frequen. The findings presented here constitute the first report of both the potential sediment deposition rates and the water conditions of La Azufrada coral reef. Rev. Biol. Trop. 62 (Suppl. 1): 107-116. Epub 2014 February 01.

Uno de los principales factores de estrés en los corales es el asentamiento de sedimento suspendido sobre su superficie. Esto conlleva al bloqueo de la luz, el cubrimiento superficial del mucus coralino e incremento del riesgo de enfermedades. Por esta razón, la sedimentación en considerada como un variable de alta importancia en el estudio de los arrecifes coralinos. Por medio de trampas de sedimentación (en nueve estaciones: seis por zona arrecifal), este proceso fue estudiado en un arrecife coralino (La Azufrada) del Pacífico Oriental Tropical, localizado en Isla Gorgona, Colombia. En las trampas se encontró una tasa promedio de colecta de sedimento de 23.30±4.34gm-2d-1 a y no se detectaron diferencias significativas entre las zonas arrecifales. La temperatura promedio y salinidad de la columna de agua donde habitan corales en este arrecife (entre 0 y 10m) fue 26.98±0.19°C y 32.60±0.52, respectivamente. Catorce grupos taxonómicos de invertebrados fueron encontrados en las trampas de sedimentación. Los bivalvos y copépodos fueron los más abundantes y frecuentes. Los resultados presentados constituyen el primer reporte de la tasa de deposición de sedimentos y de las condiciones del agua en La Azufrada. Estos resultados representan una línea base para estudios posteriores, aunque se necesita más investigación sobre estos temas que son esenciales para el manejo de los arrecifes coralinos.

Temporal-spatial interaction between reactive oxygen species and abscisic acid regulates rapid systemic acclimation in plants.

Being sessile organisms, plants evolved sophisticated acclimation mechanisms to cope with abiotic challenges in their environment. These are activated at the initial site of exposure to stress, as well as in systemic tissues that have not been subjected to stress (termed systemic acquired acclimation [SAA]). Although SAA is thought to play a key role in plant survival during stress, little is known about the signaling mechanisms underlying it. Here, we report that SAA in plants requires at least two different signals: an autopropagating wave of reactive oxygen species (ROS) that rapidly spreads from the initial site of exposure to the entire plant and a stress-specific signal that conveys abiotic stress specificity. We further demonstrate that SAA is stress specific and that a temporal-spatial interaction between ROS and abscisic acid regulates rapid SAA to heat stress in plants. In addition, we demonstrate that the rapid ROS signal is associated with the propagation of electric signals in Arabidopsis thaliana. Our findings unravel some of the basic signaling mechanisms underlying SAA in plants and reveal that signaling events and transcriptome and metabolome reprogramming of systemic tissues in response to abiotic stress occur at a much faster rate than previously envisioned.

High-capacity peptide-centric platform to decode the proteomic response to brain injury.

Traumatic brain injury (TBI) is a progressive disease process underlain by dynamic and interactive biochemical mechanisms; thus, large-scale and unbiased assessments are needed to fully understand its highly complex pathobiology. Here, we report on a new high-capacity label-free proteomic platform to evaluate the post-TBI neuroproteome. Six orthogonal separation stages and data-independent MS were employed, affording reproducible quantitative assessment on 18 651 peptides across biological replicates. From these data 3587 peptides were statistically responsive to TBI of which 18% were post-translationally modified. Results revealed as many as 484 proteins in the post-TBI neuroproteome, which was fully nine times the number determined from our prior study of focal cortical injury. Yet, these data were generated using 25 times less brain tissue per animal relative to former methodology, permitting greater anatomical specificity and proper biological replication for increased statistical power. Exemplified by these data, we discuss benefits of peptide-centric differential analysis to more accurately infer novel biological findings testable in future hypothesis-driven research. The high-capacity label-free proteomic platform is designed for multi-factor studies aimed at expanding our knowledge on the molecular underpinnings of TBI and to develop better diagnostics and therapeutics.

Exposure of rats to environmental tobacco smoke during cerebellar development alters behavior and perturbs mitochondrial energetics.

BACKGROUND: Environmental tobacco smoke (ETS) exposure is linked to developmental deficits and disorders with known cerebellar involvement. However, direct biological effects and underlying neurochemical mechanisms remain unclear. OBJECTIVES: We sought to identify and evaluate underlying neurochemical change in the rat cerebellum with ETS exposure during critical period development. METHODS: We exposed rats to daily ETS (300, 100, and 0 µg/m3 total suspended particulate) from postnatal day 8 (PD8) to PD23 and then assayed the response at the behavioral, neuroproteomic, and cellular levels. RESULTS: Postnatal ETS exposure induced heightened locomotor response in a novel environment on par initially with amphetamine stimulation. The cerebellar mitochondrial subproteome was significantly perturbed in the ETS-exposed rats. Findings revealed a dose-dependent up-regulation of aerobic processes through the modification and increased translocation of Hk1 to the mitochondrion with corresponding heightened ATP synthase expression. ETS exposure also induced a dose-dependent increase in total Dnm1l mitochondrial fission factor; although more active membrane-bound Dnm1l was found at the lower dose. Dnm1l activation was associated with greater mitochondrial staining, particularly in the molecular layer, which was independent of stress-induced Bcl-2 family dynamics. Further, electron microscopy associated Dnm1l-mediated mitochondrial fission with increased biogenesis, rather than fragmentation. CONCLUSIONS: The critical postnatal period of cerebellar development is vulnerable to the effects of ETS exposure, resulting in altered behavior. The biological effect of ETS is underlain in part by a Dnm1l-mediated mitochondrial energetic response at a time of normally tight control. These findings represent a novel mechanism by which environmental exposure can impact neurodevelopment and function.

Combination of an AccQ·Tag-ultra performance liquid chromatographic method with tandem mass spectrometry for the analysis of amino acids.

Amino acid analysis is a powerful tool in life sciences. Current analytical methods used for the detection and quantitation of low abundance amino acids in complex samples face intrinsic challenges such as insufficient sensitivity, selectivity, and throughput. This chapter describes a protocol that makes use of AccQ∙Tag chemical derivatization combined with the exceptional chromatographic resolution of ultra performance liquid chromatography (UPLC), and the sensitivity and selectivity of tandem mass spectrometry (MS/MS). The method has been fully implemented and validated using different tandem quadrupole detectors, and thoroughly tested for a variety of samples such as Plasmodium falciparum, human red blood cells, and Arabidopsis thaliana extracts. Compared to currently available methods for amino acid analysis, the AccQ∙Tag UPLC-MS/MS method presented here provides enhanced sensitivity and reproducibility, and offers excellent performance within a short analysis time and a broad dynamic range of analyte concentration. The focus of this chapter is the application of this improved protocol for the compositional amino acid analysis in A. thaliana leaf extracts using the Xevo TQ for mass spectrometric detection.

Bioinformatics tools for cancer metabolomics.

It is well known that significant metabolic change take place as cells are transformed from normal to malignant. This review focuses on the use of different bioinformatics tools in cancer metabolomics studies. The article begins by describing different metabolomics technologies and data generation techniques. Overview of the data pre-processing techniques is provided and multivariate data analysis techniques are discussed and illustrated with case studies, including principal component analysis, clustering techniques, self-organizing maps, partial least squares, and discriminant function analysis. Also included is a discussion of available software packages.

Differential gene expression in normal and transformed human mammary epithelial cells in response to oxidative stress.

Oxidative stress plays a key role in breast carcinogenesis. To investigate whether normal and malignant breast epithelial cells differ in their responses to oxidative stress, we examined the global gene expression profiles of three cell types, representing cancer progression from a normal to a malignant stage, under oxidative stress. Normal human mammary epithelial cells (HMECs), an immortalized cell line (HMLER-1), and a tumorigenic cell line (HMLER-5) were exposed to increased levels of reactive oxygen species (ROS) by treatment with glucose oxidase. Functional analysis of the metabolic pathways enriched with differentially expressed genes demonstrated that normal and malignant breast epithelial cells diverge substantially in their response to oxidative stress. Whereas normal cells exhibit the up-regulation of antioxidant mechanisms, cancer cells are unresponsive to the ROS insult. However, the gene expression response of normal HMECs under oxidative stress is comparable to that of the malignant cells under normal conditions, indicating that altered redox status is persistent in breast cancer cells, which makes them resistant to increased generation of ROS. We discuss some of the possible adaptation mechanisms of breast cancer cells under persistent oxidative stress that differentiate them from normal mammary epithelial cells as regards the response to acute oxidative stress.

Plant metabolomics by GC-MS and differential analysis.

Metabolomics is a new genomics approach that aims at measuring all or a subset of metabolites in the cell. Several approaches to plant metabolomics are currently used in plant research. These include targeted analysis, metabolite profiling, and metabolic fingerprinting. Metabolic fingerprinting, unlike metabolite profiling, does not aim at separating or identifying all the metabolites present in the sample, but rather generates a fingerprint that characterizes a specific metabolic state of the plant system under investigation. This chapter describes the implementation of metabolic fingerprinting approach using gas chromatography coupled to mass spectrometry (GC-MS) and discriminant function analysis combined with genetic algorithm (GA-DFA). This approach enables the identification of specific metabolites that are biologically relevant, and which may go undetected if direct infusion-based fingerprinting approaches were used due to the sample complexity and matrix suppression effects.

Recent advances in the MS analysis of glycoproteins: Theoretical considerations.

Protein glycosylation is involved in a broad range of biological processes that regulate protein function and control cell fate. As aberrant glycosylation has been found to be implicated in numerous diseases, the study and large-scale characterization of protein glycosylation is of great interest not only to the biological and biomedical research community, but also to the pharmaceutical and biotechnology industry. Due to the complex chemical structure and differing chemical properties of the protein/peptide and glycan moieties, the analysis and structural characterization of glycoproteins has been proven to be a difficult task. Large-scale endeavors have been further limited by the dynamic outcome of the glycosylation process itself, and, occasionally, by the low abundance of glycoproteins in biological samples. Recent advances in MS instrumentation and progress in miniaturized technologies for sample handling, enrichment and separation, have resulted in robust and compelling analysis strategies that effectively address the challenges of the glycoproteome. This review summarizes the key steps that are involved in the development of efficient glycoproteomic analysis methods, and the latest innovations that led to successful strategies for the characterization of glycoproteins and their corresponding glycans. As a follow-up to this work, we review innovative capillary and microfluidic-MS workflows for the identification, sequencing and characterization of glycoconjugates.

Recent advances in the MS analysis of glycoproteins: Capillary and microfluidic workflows.

Recent developments in bioanalytical instrumentation, MS detection, and computational data analysis approaches have provided researchers with capabilities for interrogating the complex cellular glycoproteome, to help gain a better insight into the cellular and physiological processes that are associated with a disease and to facilitate the efforts centered on identifying disease-specific biomarkers. This review describes the progress achieved in the characterization of protein glycosylation by using advanced capillary and microfluidic MS technologies. The major steps involved in large-scale glycoproteomic analysis approaches are discussed, with special emphasis given to workflows that have evolved around complex MS detection functions. In addition, quantitative analysis strategies are assessed, and the bioinformatics aspects of glycoproteomic data processing are summarized. The developments in commercial and custom fabricated microfluidic front-end platforms to ESI- and MALDI-MS instrumentation, for addressing major challenges in carbohydrate analysis such as sensitivity, throughput, and ability to perform structural characterization, are further evaluated and illustrated with relevant examples.

Sensitive and rapid method for amino acid quantitation in malaria biological samples using AccQ.Tag ultra performance liquid chromatography-electrospray ionization-MS/MS with multiple reaction monitoring.

An AccQ*Tag ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (AccQ*Tag-UPLC-ESI-MS/MS) method for fast, reproducible, and sensitive amino acid quantitation in biological samples, particularly, the malaria parasite Plasmodium falciparum is presented. The Waters Acquity TQD UPLC/MS system equipped with a photodiode array (PDA) detector was used for amino acid separation and detection. The method was developed and validated using amino acid standard mixtures containing acidic, neutral, and basic amino acids. For MS analysis, the optimum cone voltage implemented, based on direct infusion analysis of a few selected AccQ*Tag amino acids with multiple reaction monitoring, varied from 29 to 39 V, whereas the collision energy varied from 15 to 35 V. Calibration curves were built using both internal and external standardization. Typically, a linear response for all amino acids was observed at concentration ranges of 3 x 10(-3)-25 pmol/muL. For some amino acids, concentration limits of detection were as low as 1.65 fmol. The coefficients of variation for retention times were within the range of 0.08-1.08%. The coefficients of variation for amino acid quantitation, determined from triplicate UPLC-MS/MS runs, were below 8% on the average. The developed AccQ*Tag-UPLC-ESI-MS/MS method revealed good technical and biological reproducibility when applied to P. falciparum and human red blood cells samples. This study should provide a valuable insight into the performance of UPLC-ESI-MS/MS for amino acid quantitation using AccQ*Tag derivatization.

Towards identifying the full set of genes expressed during cassava post-harvest physiological deterioration.

Storage roots of cassava (Manihot esculenta Crantz) exhibit a rapid post-harvest physiological deterioration (PPD) response that can occur within 24-72 h of harvest. PPD is an enzymatically mediated oxidative process with parallels to plant wound, senescence and defence responses. To characterise those genes that show significant change in expression during the PPD response we have used cDNA microarray technology to carry out a large-scale analysis of the cassava root transcriptome during the post-harvest period. We identified 72 non-redundant expressed sequence tags which showed altered regulation during the post-harvest period. Of these 63 were induced, whilst 9 were down-regulated. RNA blot analysis of selected genes was used to verify and extend the microarray data. Additional microarray hybridisation experiments allowed the identification of 21 root-specific and 24 root-wounding-specific sequences. Many of the up-regulated and PPD-specific expressed sequence tags were predicted to play a role in cellular processes including reactive oxygen species turnover, cell wall repair, programmed cell death, ion, water or metabolite transport, signal transduction or perception, stress response, metabolism and biosynthesis, and activation of protein synthesis.