Quality control of cacao beans is a significant issue in the chocolate industry. In this report, we describe how moisture damage to cacao beans alters the volatile chemical signature of the beans in a way that can be tracked quantitatively over time. The chemical signature of the beans is monitored via sampling the headspace of the vapor above a given bean sample. Headspace vapor sampled with solid-phase micro-extraction (SPME) was detected and analyzed with comprehensive two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GC × GC–TOFMS). Cacao beans from six geographical origins (Costa Rica, Ghana, Ivory Coast, Venezuela, Ecuador, and Panama) were analyzed. Twenty-nine analytes that change in concentration levels via the time-dependent moisture damage process were measured using chemometric software. Biomarker analytes that were independent of geographical origin were found. Furthermore, prediction algorithms were used to demonstrate that moisture damage could be verified before there were visible signs of mold by analyzing subsets of the 29 analytes. Thus, a quantitative approach to quality screening related to the identification of moisture damage in the absence of visible mold is presented.

BACKGROUND:Cocoa is a rich source of flavonoids that, among other functions, can act as antioxidants. In living systems, the production of reactive oxygen species (ROS) activate an array of intracellular cascades, including mitogen-activated protein kinases (MAPK), that are closely associated with cell death or survival pathways.AIM OF THE STUDY:To ascertain the role of a cocoa extract and its main flavonoid, (-)-epicatechin, in an in vitro model of oxidative stress induced in a neuronal cell line.METHODS:We analyzed ROS production by fluorometry (dichlorofluorescein assay), and activation of MAPK pathways including extracellular signal-regulated kinases 1/2 (ERK 1/2), c-Jun N-terminal kinase (JNK), and p-38, by Western blot analysis.RESULTS:Cells incubated with cocoa extract or (-)-epicatechin, reduced ROS production in a dose-dependent manner, reaching 35% inhibition. pJNK and p38, involved in apoptosis, were down-modulated by cocoa extract and (-)-epicatechin with p38 inhibition reaching up to 70%.CONCLUSIONS:Our results show that cocoa extract and (-)-epicatechin may exert a neuroprotective action by reducing ROS production and modulating MAPK activation.

Although the most common use of DNA microarrays is gene expression profiling, microarrays are also used for many other applications, including genotyping, resequencing, SNP analysis, and DNA methylation assays. Here we describe genotyping arrays for Influenza A subtype identification and for upper respiratory pathogen diagnostics using standard hybridization techniques and we also describe resequencing, SNP, and methylation assays using an enzyme-based strategy [25, 26].

A method to analyze volatile compounds from cacao beans has been developed and evaluated. The method utilizes solid phase micro extraction (SPME) sampling followed by comprehensive 2-D (GC x GC) coupled with TOFMS. For the SPME procedure, a polydimethyl siloxane/divinyl benzene (PDMS/DVB) fiber was implemented. Cacao beans from four geographical origins were studied under two storage conditions, either dry or high moisture. A given cacao bean sample was sealed in a SPME vial and heated for 15 min. Extraction temperatures of 45, 60, 80, and 100 degrees C were analyzed and an optimal extraction temperature of 60 degrees C was determined. Many peaks were found to change as a function of storage conditions with Fisher Ratio analysis. Four representative compounds were identified and quantified (on a relative basis): acetic acid, nonanal, tetramethyl pyrazine, and trimethyl pyrazine. Acetic acid and nonanal were elevated in samples without evident mold on the bean surface, while the two pyrazines were elevated when mold was evident on the bean surface. The results for these comparisons, indicate that metabolism at the bean surface plays a role in the concentration of analytes, and can be readily determined using this analytical technology and methodology.

Microarray assays typically involve multistage sample processing and fluidic handling, which are generally labor-intensive and time-consuming. Automation of these processes would improve robustness, reduce run-to-run and operator-to-operator variation, and reduce costs. In this chapter, a fully integrated and self-contained microfluidic biochip device that has been developed to automate the fluidic handling steps for microarray-based gene expression or genotyping analysis is presented. The device consists of a semiconductor-based CustomArray (R) chip with 12,000 features and a microfluidic cartridge. The CustomArray was manufactured using a semiconductor-based in situ synthesis technology. The microfluidic cartridge consists of microfluidic pumps, mixers, valves, fluid channels, and reagent storage chambers. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Electrochemical micropumps were integrated in the cartridge to provide pumping of liquid solutions. A micromixing technique based on gas bubbling generated by electrochemical micropumps was developed. Low-cost check valves were implemented in the cartridge to prevent cross-talk of the stored reagents. Gene expression study of the human leukemia cell line (K562) and genotyping detection and sequencing of influenza A subtypes have been demonstrated using this integrated biochip platform. For gene expression assays, the microfluidic CustomArray device detected sample RNAs with a concentration as low as 0.375 pM. Detection was quantitative over more than three orders of magnitude. Experiment also showed that chip-to-chip variability was low indicating that the integrated microfluidic devices eliminate manual fluidic handling steps that can be a significant source of variability in genomic analysis. The genotyping results showed that the device identified influenza A hemagglutinin and neuraminidase subtypes and sequenced portions of both genes, demonstrating the potential of integrated microfluidic and microarray technology for multiple virus detection. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluidic handling steps and allows microarray-based DNA analysis in a rapid and automated fashion.

The consumption of high-cacao-content chocolate has been associated with positive health benefits ascribed to flavanol [corrected] antioxidants derived from the ground, fermented cocoa seeds of Theobroma cacao. However, flavanols [corrected] impart a bitter, astringent flavor to foodstuffs, frequently masked in chocolates and confections by aggressive processing and adulteration with other flavors. Recent reports have implied that not all varieties of dark chocolate are created equally, and significant caveats exist regarding its potential health benefits. It is perhaps not surprising that extensive processing, dilution, and the addition of flavor modifiers may improve the palatability of chocolate, but could have negative nutritional and clinical benefits. This article examines the chemical composition of chocolate and the clinical data associated with the consumption of flavonoid-rich cocoa. We review the steps in chocolate manufacturing that directly affect the antioxidant levels in chocolate products, and the caveats associated with claims of health benefits from the consumption of dark chocolate.

DNA microarray technology has become one of the most promising analytical tools in molecular biology. It has been widely used for studying mRNA levels and examining gene expression in biological samples. It is becoming a powerful tool in the arena of diagnostics and personalized medicine. In this chapter, we present a fully integrated and self-contained microfluidic biochip device that has been developed to automate the fluidic handling steps required to carry out microarray-based gene expression or genotyping analysis. The device consists of a semiconductor-based CustomArray™ chip with 12,000 features and a microfluidic cartridge. The CustomArray™ was manufactured using a semiconductor-based in situ synthesis technology. The oligonucleotides were synthesized on an array of electrodes on a semiconductor chip using phosphoramidite chemistry under electrochemical control. The microfluidic cartridge consists of microfluidic pumps, mixers, valves, fluid channels and reagent storage chambers. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Electrochemical micropumps were integrated in the cartridge to provide pumping of liquid solutions. A micromixing technique based on gas bubbling generated by electrochemical micropumps was developed. Low-cost check valves were implemented in the cartridge to prevent cross talk of the stored reagents. Gene expression study of the human leukemia cell line (K562) and genotyping detection and sequencing of influenza A subtypes have been demonstrated using this integrated biochip platform. For gene expression assays, the microfluidic CustomArray™ device detected sample RNAs with a concentration as low as 0.375 pM. Detection was quantitative over more than three orders of magnitude. Experiment also showed that chip-to-chip variability was low indicating that the integrated microfluidic devices eliminate manual fluidic handling steps that can be a significant source of variability in genomic analysis. The genotyping results showed that the device identified influenza A hemagglutinin and neuraminidase subtypes and sequenced portions of both genes, demonstrating the potential of integrated microfluidic and microarray technology for multiple virus detection. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluidic handling steps and allows microarray-based DNA analysis in a rapid and automated fashion.

Bacterial and viral upper respiratory infections (URI) produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD) on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae) and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluinza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format.

ABSTRACT In the face of concerns over an influenza pandemic, identification of virulent influenza A virus isolates must be obtained quickly for effective responses. Rapid subtype identification, however, is difficult even in well-equipped virology laboratories or is unobtainable in the field under more austere conditions. Here we describe a genome assay and microarray design that can be used to rapidly identify influenza A virus hemagglutinin subtypes 1 through 15 and neuraminidase subtypes 1 through 9. Also described is an array-based enzymatic assay that can be used to sequence portions of both genes or any other sequence of interest.

Microarray technology is a tremendously powerful method for simultaneously monitoring the expression of thousands of species of nucleic acids, usually cellular mRNA, producing a high-resolution representation of the genes encoded or expressed in a cell. As such, microarray technology has great potential for impacting research and clinical approaches to treatment. However, this complex technology has been challenging to apply as a result of difficulties discerning biologic variation from technologic issues, therefore slowing the application of the technology to human diagnostics. Nevertheless, significant advances in microarray technology, improvements that avoid potential pitfalls, and a wider spectrum of application are making this technology easier to apply. Indeed, microarray technology has provided valuable insights into mechanisms involving gene regulation and expression in Alzheimer disease, and it remains a powerful tool to identify biomarkers for disease diagnosis. Ultimately, the most robust markers will enable the application of more specific treatments particular to disease stages or subcategories. Currently, no widely applicable molecular test is available to identify those at risk for developing Alzheimer disease or those who have early markers of pathology but show discernible cognitive impairment. The progression of this technology will lead to earlier detection of the disease through enhanced understanding of disease onset and progression.

There is now considerable evidence for the aberrant re–expression of a series of cell cycle–related proteins in specific vulnerable neuronal populations in Alzheimer disease (AD). That a mitotic cell cycle–related mechanism may play an important role in disease pathogenesis is highlighted by the earlier occurrence of cell cycle proteins compared to abnormal tau in AD. That cell cycle proteins are representative of a true cell cycle, rather than being an epiphenomena of other processes, is evident from recent evidence showing that, in AD and other neurodegenerative diseases, there is a true mitotic alteration that leads to DNA replication (i.e., S phase). These findings led us to develop a novel hypothesis that neurodegeneration in AD, like cancer, is a disease of inappropriate cell cycle control. In support of this notion, this study shows that the expression of a powerful cell cycle inducer, MYC, drives primary neurons to re–enter the cell cycle and, moreover, that oncogene–induced cell cycle re–entry leads to tau phosphorylation. Specifically, we used adenoviral–mediated expression of c–myc and ras oncogenes to drive postmitotic primary cortical neurons into the cell cycle. Neurons infected with adenoviruses expressing c–myc and ras re–enter the cell cycle as evidenced by increasing DNA content and the expression of cyclin B1, a marker for the G2/M phase of the cell cycle. Importantly, however, and directly related to disease pathogenesis, such infected neurons also display increased levels of two specific tau changes recognized by TG3, a phospho–tau specific antibody and Alz50, a conformational epitope considered a key marker of early AD pathology. Based on these results, we propose that early elements of AD neuropathology arise due to inappropriate re–entry into the cell cycle. As such, this neuronal cell model may be extremely valuable for not only characterizing cell cycle related changes as they pertain to AD but also for the development of novel therapeutic strategies.

Evidence showing the ectopic re-expression of cell cycle-related proteins in specific vulnerable neuronal populations in Alzheimer disease led us to formulate the hypothesis that neurodegeneration, like cancer, is a disease of inappropriate cell cycle control. To test this notion, we used adenoviral-mediated expression of c-myc and ras oncogenes to drive postmitotic primary cortical neurons into the cell cycle. Cell cycle re-entry in neurons was associated with increased DNA content, as determined using BrdU and DAPI, and the re-expression of cyclin B1, a marker for the G2/M phase of the cell cycle. Importantly, we also found that cell cycle re-entry in primary neurons leads to tau phosphorylation and conformational changes similar to that seen in Alzheimer disease. This study establishes that the cell cycle can be instigated in normally quiescent neuronal cells and results in a phenotype that shares features of degenerative neurons in Alzheimer disease. As such, our neuronal cell model may be extremely valuable for the development of novel therapeutic strategies.

MicroRNAs (miRNAs) are an extensive class of noncoding genes that regulate gene expression through posttranscriptional repression. Given the potential for large viral genomes to encode these transcripts, we examined the human cytomegalovirus AD169 genome for miRNAs using a bioinformatics approach. We identified 406 potential stem-loops, of which 110 were conserved between chimpanzee cytomegalovirus and several strains of human cytomegalovirus. Of these conserved stem-loops, 13 exhibited a significant score using the MiRscan algorithm. Examination of total RNA from human cytomegalovirus-infected cells demonstrated that 5 of the 13 predicted miRNAs were expressed during infection. These studies demonstrate that human cytomegalovirus encodes multiple conserved miRNAs and suggest that human cytomegalovirus may utilize an miRNA strategy to regulate cellular and viral gene function.

Adult neurons are generally accepted to be in a quiescent, nonproliferative state. However, it is becoming increasingly apparent that, in Alzheimer's disease (AD), alterations in cell cycle machinery, suggesting an attempt to reenter cell cycle, relate temporally and topographically to degenerating neurons. These findings, together with the fact that neurons lack the necessary components for completion of mitosis, have led to the notion that an ill‐regulated attempt to reenter the cell cycle is associated with disease pathogenesis and, ultimately, neuronal degeneration. To understand better the role of such cell cycle abnormalities in AD, we undertook a study of CIP‐1‐associated regulator of cyclin B (CARB), a protein that associates with two key proteins, p21 and cyclin B, involved in cellular checkpoints in the cell cycle. Our results show that there are increases in CARB localized to intraneuronal neurofibrillary tangles and granulovacuolar degeneration in susceptible hippocampal and cortical neurons in AD. By marked contrast, CARB is found only at background levels in these neuronal populations in nondiseased age‐matched controls. Our data not only provide another line of evidence indicative of cell cycle abnormalities in neurons in AD but also lend further credence to the hypothesis that susceptible neurons may be arrested at the G2/M phase of the cell cycle before they die. Therefore, therapeutics targeted toward initiators of the cell cycle are likely to prove of great efficacy for the treatment of AD. © 2004 Wiley‐Liss, Inc.

In response to DNA damage and genotoxic stress, the p53 tumor suppressor triggers either cell cycle arrest or apoptosis. The G(2) arrest after damage is, in part, mediated by the p53 target, 14-3-3final sigma (final sigma). Colorectal tumor cells lacking final sigma are exquisitely sensitive to DNA damage. Here we analyzed the mechanism of this sensitivity in final sigma(-/-) as compared with final sigma(+/+) human colorectal tumor cells. Exposure to adriamycin resulted in rapid apoptosis only in final sigma(-/-) cells. This was further characterized by caspase-3 activation, p21(CIP1) cleavage, and CDK2 activation. Moreover, Bax was rapidly translocated out of the cytoplasm, and cytochrome c was released in final sigma(-/-) cells. Transient adenovirus-mediated reconstitution of final sigma in the final sigma(-/-) cells led to effective rescue of this phenotype and protected cells against apoptosis. The association of final sigma, Bax, and CDK1 in protein complexes may be the basis for this antiapoptotic mechanism. In conclusion, final sigma not only enforces the p53-dependent G(2) arrest but also delays the apoptotic signal transduction.