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Functional foods and their by-products contain a wide range of bioactive components with an array of health benefits and were proposed to improve public health, well-being, and others. To achieve a circular economy, the processing and extraction of flavonoids, phenolic compounds, and others from functional food and agri-food wastes will require the use of environmentally friendly, sustainable, and a low-cost solution. Extraction methods that can eliminate the use of organic solvents, suitable for use in the laboratory and production of extracts will be covered. This will include subcritical water extraction (SBE), pressurized hot water extraction (PHWE), supercritical fluid extraction (SFE), and others. Based on the selected analytical methods, the determination of the marker or bioactive compounds and chemical fingerprints will provide the control measures to identify the batch-to-batch variation of the composition of the functional food products obtained. The combination of chemical standardization with antioxidant assay, such as DPPH and ABTS + will provide further information on the quality of the extracts. Lastly, to ascertain the biological and physiological relevance of the antioxidant properties of the target sample, treatment of the antioxidant compounds or extracts was carried out using cellular models, and validated using other experimental endpoints, such as metabolomics.

Bioactive compounds in functional foods, medicinal plants and others are considered attractive value-added molecules based on their wide range of bioactivity. It is clear that an important role is occupied by polyphenol, phenolic compounds and others. Urine is an effective biofluid to evaluate and monitor alterations in homeostasis and other processes related to metabolism. The current review provides a detailed description of the formation of urine in human body, various aspects relevant to sampling and analysis of urinary metabolites before presenting recent developments leveraging on metabolite profiling of urine. For the profiling of small molecules in urine, advancement of liquid chromatography mass tandem spectrometry (LC/MS/MS), establishment of standardized chemical fragmentation libraries, computational resources, data-analysis approaches with pattern recognition tools have made it an attractive option. The profiling of urinary metabolites gives an overview of the biomarkers associated with the diet and evaluates its biological effects. Metabolic pathways such as glycolysis, tricarboxylic acid cycle, amino acid metabolism, energy metabolism, purine metabolism and others can be evaluated. Finally, a combination of metabolite profiling with chemical standardization and bioassay in functional food and medicinal plants will likely lead to the identification of new biomarkers and novel biochemical insights.

Endothelial dysfunction is a critical initiating factor contributing to cardiovascular diseases, involving the gut microbiome-derived metabolite trimethylamine N-oxide (TMAO). This study aims to clarify the time-dependent molecular pathways by which TMAO mediates endothelial dysfunction through transcriptomics and metabolomics analyses in human microvascular endothelial cells (HMEC-1). Cell viability and reactive oxygen species (ROS) generation were also evaluated. TMAO treatment for either 24H or 48H induces reduced cell viability and enhanced oxidative stress. Interestingly, the molecular signatures were distinct between the two time-points. Specifically, few Gene Ontology biological processes (BPs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were modulated after a short (24H) compared to a long (48H) treatment. However, the KEGG signalling pathways namely "tumour necrosis factor (TNF)" and "cytokine-cytokine receptor interaction" were downregulated at 24H but activated at 48H. In addition, at 48H, BPs linked to inflammatory phenotypes were activated (confirming KEGG results), while BPs linked to extracellular matrix (ECM) structural organisation, endothelial cell proliferation, and collagen metabolism were repressed. Lastly, metabolic profiling showed that arachidonic acid, prostaglandins, and palmitic acid were enriched at 48H. This study demonstrates that TMAO induces distinct time-dependent molecular signatures involving inflammation and remodelling pathways, while pathways such as oxidative stress are also modulated, but in a non-time-dependent manner., (© 2023. The Author(s).)

The mangosteen ( Garcinia mangostana L.) pericarp is known to be rich in potent bioactive phytochemical compounds such as xanthones, which possess pharmacologically important antioxidant activity and beneficial cardiometabolic properties. Mangosteen pericarp is typically classified as unavoidable food waste and discarded, despite being rich in bioactive phytochemical compounds that therefore present an exciting opportunity for valorization. Thus, this study aims to extract phytochemical compounds from mangosteen pericarp using pressurized hot water extraction (PHWE) and determine its biological effects in endothelial cells using RNA sequencing. Liquid chromatography with MS/MS (LC/MSMS) and UV detection (LC/UV) was subsequently used to identify three key phytochemical compounds extracted from the mangosteen pericarp: α-Mangostin, γ-Mangostin, and Gartanin. Within the tested range of extraction temperatures by PHWE, our results demonstrated that an extraction temperature of 120 °C yielded the highest concentrations of α-Mangostin, γ-Mangostin, and Gartanin with a concomitant improvement in antioxidant capacity compared to other extraction temperatures. Using global transcriptomic profiling and bioinformatic analysis, the treatment of endothelial cells with mangosteen pericarp extracts (120 °C PHWE) for 48 h caused 408 genes to be differentially expressed. Furthermore, our results demonstrated that key biological processes related to "steroid biosynthesis and metabolism", likely involving the activation of the AMPK signaling pathway, were upregulated by mangosteen pericarp extract treatment. In conclusion, our study suggests a green extraction method to valorize phytochemical compounds from mangosteen pericarp as a natural product with potential beneficial effects on cardiometabolic health.

Orange peels are often discarded as food waste despite being a nutritious source of vitamins and antioxidants. These orange peel wastes (OPW) are produced in millions of tons globally every year; discarding them results in detrimental environmental and economical impacts. This paper discusses the application of 3D printing technology to effectively upcycle the OPW into edible, healthy snacks for consumption. We aimed to develop a method to enable OPW to formulate 3D-printable inks for direct ink writing (DIW). Using DIW 3D printing, we successfully created edible constructs of rheologically modified inks containing OPW. The formulated ink possessed an initial viscosity of 22.5 kPa.s, a yield stress of 377 Pa, and a storage modulus of 44.24 kPa. To validate the method, we conducted a biochemical analysis of the OPW at each stage of the fabrication process. This study suggested that our ink formulation and 3D printing process did not affect the content of bioflavonoids and antioxidants of the OPW. The cell viability test using human dermal microvascular endothelium (HMEC-1) suggested that the OPW did not exhibit cytotoxicity throughout the entire process of the ink manipulation. Overall, this study has highlighted a potential scenario to revalorize food waste into the food value chain using 3D printing toward more sustainable and circular food manufacturing and consumption., Competing Interests: The authors declare no conflict of interests., (Copyright:© 2023, Tan JD, Lee CP, Foo SY, et al.)

Background: The peptide hormone relaxin has potent anti-fibrotic and anti-inflammatory properties in various organs, including the kidneys. However, the protective effects of relaxin in the context of diabetic kidney complications remain controversial. Here, we aimed to evaluate the effects of relaxin treatment on key markers of kidney fibrosis, oxidative stress, and inflammation and their subsequent impact on bile acid metabolism in the streptozotocin-induced diabetes mouse model., Methods and Results: Male mice were randomly allocated to placebo-treated control, placebo-treated diabetes or relaxin-treated diabetes groups (0.5 mg/kg/d, final 2 weeks of diabetes). After 12 weeks of diabetes or sham, the kidney cortex was harvested for metabolomic and gene expression analyses. Diabetic mice exhibited significant hyperglycaemia and increased circulating levels of creatine, hypoxanthine and trimethylamine N-oxide in the plasma. This was accompanied by increased expression of key markers of oxidative stress (Txnip), inflammation (Ccl2 and Il6) and fibrosis (Col1a1, Mmp2 and Fn1) in the diabetic kidney cortex. Relaxin treatment for the final 2 weeks of diabetes significantly reduced these key markers of renal fibrosis, inflammation, and oxidative stress in diabetic mice. Furthermore, relaxin treatment significantly increased the levels of bile acid metabolites, deoxycholic acid and sodium glycodeoxycholic acid, which may in part contribute to the renoprotective action of relaxin in diabetes., Conclusion: In summary, this study shows the therapeutic potential of relaxin and that it may be used as an adjunctive treatment for diabetic kidney complications., Competing Interests: Conflict of interest statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Objectives: The purpose of this study is to evaluate if social media and Google search data can identify seasonal and geographic trends in the incidence of corneal ulcers in the United States., Methods: This is a case series of all corneal ulcer-related data collected from two major social media platforms and Google trends from US users between 2017 and 2021. Instagram and Twitter were searched for posts and hashtags related to "corneal ulcer." Web and image search volume of "corneal ulcer" were collected from Google trends ( https://trends.google.com ). Data were compared between seasons, defined by 3-month intervals, and chi-square tests were used to determine the statistical significance., Results: One hundred and sixty-five individuals (79% female) and 164 individuals (79% female) posted personal new corneal ulcer diagnoses on Twitter and Instagram, respectively. Summer resulted in the highest number of both Twitter (34%, P =0.07) and Instagram (33%, P =0.68) posts. Summer was also the most popular season for Google web and image searches of "corneal ulcer" (search volume average of 58.4 and 41.2, P =0.74 and P =0.01, respectively, with 100 being peak popularity). Across all platforms, the South was the most represented (32% Twitter, 38% Instagram, 32% Google Web, and 33% Google Images)., Conclusions: Our results indicate that social media and Google trends may reflect seasonal and geographic patterns of corneal ulcer incidence in the United States. However, further study with increased power is needed., Competing Interests: The authors have no funding or conflicts of interest to disclose, (Copyright © 2022 Contact Lens Association of Ophthalmologists.)

Orange peel waste (OPW) is known to contain an abundant amount of polyphenols compounds such as flavonoids, well-reported for their antioxidant and anti-inflammatory properties. While OPW is generally regarded as a food waste, the opportunity to extract bioactive compounds from these "wastes" arises due to their abundance, allowing the investigation of their potential effects on endothelial cells. Hence, this study aims to use a green extraction method and pressurized hot water extraction (PHWE) to extract bioactive compounds from OPW. Liquid chromatography with UV detection (LC/UV) and liquid chromatography mass spectrometry (LC/MS) were subsequently used to identify the bioactive compounds present. Through the optimization of the extraction temperature for PHWE, our results demonstrated that extraction temperatures of 60 °C and 80 °C yield distinct bioactive compounds and resulted in better antioxidant capacity compared to other extraction temperatures or organic solvent extraction. Despite having similar antioxidant capacity, their effects on endothelial cells were distinct. Specifically, treatment of endothelial cells with 60 °C OPW extracts inhibited TNFα-induced vascular inflammation and endothelial dysfunction in vitro, suggesting that OPW possess vasoprotective effects likely mediated by anti-inflammatory effects., Competing Interests: The authors have no conflict of interest to declare that are relevant to the content of this article.

The pulp of avocado (Persea Americana) is widely consumed as the primary food source, while the seed is often discarded as food waste. Increased consumption of avocado would inevitably results in production of waste by-products such as avocado seeds, hence the ability to extract phytochemicals from such waste, and upcycling to potential nutraceutical products is of great interest. The overall aim of this study is to explore avocado seeds as potential functional food through the combined use of a green extraction method, chemical standardization and pattern recognition tools, and biological characterization assays. Specifically, this study utilized an organic solvent-free extraction method, pressurized hot water extraction (PHWE) to extract phytochemicals from avocado seeds and liquid chromatography mass spectrometry (LCMS) was used to identify the phytochemicals present in the avocado seeds. Our results demonstrated that avocado seed extracts have antioxidant activity and inhibited oxidative stress-induced metabolomics changes in endothelial cells, suggesting that avocado seed extracts have vasoprotective actions., (© 2022. The Author(s).)

By the year 2050, it is estimated that the demand for palm oil is expected to reach an enormous amount of 240 Mt. With a huge demand in the future for palm oil, it is expected that oil palm by-products will rise with the increasing demand. This represents a golden opportunity for sustainable biohydrogen production using oil palm biomass and palm oil mill effluent (POME) as the renewable feedstock. Among the different biological methods for biohydrogen production, dark fermentation and photo-fermentation have been widely studied for their potential to produce biohydrogen by using various waste materials as feedstock, including POME and oil palm biomass. However, the complex structure of oil palm biomass and POME, such as the lignocellulosic composition, limits fermentable substrate available for conversion to biohydrogen. Therefore, proper pre-treatment and suitable process conditions are crucial for effective biohydrogen generation from these feedstocks. In this review, the characteristics of palm oil industrial waste, the process used for biohydrogen production using palm oil industrial waste, their pros and cons, and the influence of various factors have been discussed, as well as a comparison between studies in terms of types of reactors, pre-treatment strategies, the microbial culture used, and optimum operating condition have been presented. Through biological production, hydrogen production rates up to 52 L-H 2 /L-medium/h and 6 L-H 2 /L-medium/h for solid and liquid palm oil industrial waste, respectively, can be achieved. In short, the continuous supply of palm oil production by-product and relatively, the low cost of the biological method for hydrogen production indicates the potential source of renewable energy., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Annually, approximately 1.3 billion tons of food is lost worldwide, accounting for one-third of annual food production. Therefore, turning food waste into energy is of enormous environmental significance because of its sustainable nature. Nutrients and organic acids present in food waste can be used to produce (bio)products such as biohydrogen through biological processes. However, our understanding of the production of biohydrogen from food waste through photofermentation and dark fermentation is still restricted. This comprehensive study aims to review the potential of food waste for biohydrogen production using microbial mediators, including a brief overview of process parameters that affect the (bio)hydrogen production pathway., Competing Interests: Declaration of Interests The authors declare no conflicts of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

An increase in urbanization and industrialization has not only contributed to an improvement in the lifestyle of people, but it has also contributed to a surge in the generation of wastewater. To date, conventional physico-chemical and biological treatment methods are widely used for the treatment of wastewater. However, the efficient operation of these systems require substantial operation and maintenance costs, and the application of novel technologies for the treatment and disposal of sludge/residues. This review paper focuses on the application of different treatment options such as chemical, catalyst-based, thermochemical and biological processes for wastewater or sludge treatment and membrane-based technologies (i.e. pressure-driven and non-pressure driven) for the separation of the recovered products from wastewater and its residues. As evident from the literature, a wide variety of treatment and resource recovery options are possible, both from wastewater and its residues; however, the lack of planning and selecting the most appropriate design (treatment train) to scale up from pilot to the field scale has limited its practical application. The economic feasibility of the selected technologies was critically analyzed and the future research prospects of resource recovery from wastewater have been outlined in this review., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Abelmoschus esculentus L. Moench (okra) is a commonly consumed vegetable that consists of the seeds and peel component which are rich in polyphenolic compounds. The aim of this study is to utilize pressurized hot water extraction (PHWE) for the extraction of bioactive phytochemicals from different parts of okra. A single step PHWE was performed at various temperatures (60 °C, 80 °C, 100 °C and 120 °C) to determine which extraction temperature exhibits the optimum phytochemical profile, antioxidant and antidiabetic activities. The optimum temperature for PHWE extraction was determined at 80 °C and the biological activities of the different parts of okra (Inner Skin, Outer Skin and Seeds) were characterized using antioxidant (DPPH and ABTS), α-glucosidase and vasoprotective assays. Using PHWE, the different parts of okra displayed distinct phytochemical profiles, which consist of primarily polyphenolic compounds. The okra Seeds were shown to have the most antioxidant capacity and antidiabetic effects compared to other okra parts, likely to be attributed to their higher levels of polyphenolic compounds. Similarly, okra Seeds also reduced vascular inflammation by downregulating TNFα-stimulated VCAM-1 and SELE expression. Furthermore, metabolite profiling by LC/MS also provided evidence of the cytoprotective effect of okra Seeds in endothelial cells. Therefore, the use of PHWE may be an alternative approach for the environmentally friendly extraction and evaluation of plant extracts for functional food applications.

Background: CRS with HIPEC is a complex operation that has shown survival benefit in patients with a variety of primary and metastatic peritoneal surface malignancies. While optimal oncologic and perioperative outcomes have been defined by expert consensus and demonstrated at university-affiliated, academic centers, similar results have never been presented from a non-university-affiliated, community center in the literature to date., Methods: All cases of CRS with HIPEC performed at a non-university-affiliated, community center were retrospectively reviewed and analyzed. Oncologic and perioperative outcomes were compared Chicago Working Group benchmarks and with results from university-affiliated, academic centers recently published in high-impact-factor, peer-reviewed journals., Results: All 112 cases completed over 5 years were reviewed. 3 were excluded from analysis since they were palliative HIPEC procedures for distressing ascites-related symptoms only without CRS. A wide variety of tumors were treated. Average PCI was 18±9.1. Median PCI was 14. CC 0-1 was achieved in 89% of patients. Average length of stay was 11.6±9.3 days. Serious perioperative morbidity, defined as a Clavien-Dindo Grade III or IV complication, was observed in 22% of patients. The frequency of major complications decreased after the first year. There were no perioperative deaths., Conclusions: Optimal oncologic and perioperative outcomes of CRS and HIPEC are attainable at a non universityaffiliated, community center. A multidisciplinary team and high clinical volume are necessary to obtain these results., Competing Interests: Declaration of competing interest None of the authors of “Optimal Oncologic and Perioperative Outcomes of Cytoreductive Surgery with Hyperthermic Intraperitoneal Chemotherapy are Attainable at a Community Center” have any conflicts of interest to disclose. None of the authors have any conflicts of interest to disclose., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose of Review: The corneal epithelium is a crucial barrier against pathogens, and when disrupted in the setting of certain underlying risk factors such as neurotrophic keratopathy (NK), may result in persistent epithelial defects (PEDs) of the cornea. Management is challenging and may require a variety of different approaches ranging from conservative medical therapy to surgical intervention. The purpose of this review is to provide an update on current and potential future therapeutic options for PEDs and NK., Recent Findings: Recent research has yielded promising results for numerous novel therapies aimed at treating PEDs. Many of these attempt to stimulate healing at the cellular level, via signaling of corneal epithelial differentiation, migration, and proliferation. Considerable advances have also been made regarding medical and surgical promotion of corneal re-innervation and restoration of corneal sensitivity to directly address the underlying NK condition., Summary: Together with the current well established therapeutic options available for PEDs and NK, growing research on newer alternatives suggest increasing potential for both more effective and more convenient therapies for these difficult situations., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Quinoa is widely noted for its nutritional value. The seed is the main edible part of the plant and exists in at least three different colors: white, red and black. This study utilized a pressurized hot water extraction (PHWE) for the extraction of phytochemicals from quinoa. Chemical fingerprints with LC/UV and LC/MS using a targeted approach and pattern recognition tools were used to evaluate the quinoa extracts. The antioxidant properties for various types of quinoa were evaluated using DPPH assay, ABTS assay and the cytoprotective effect of quinoa extracts were investigated in HMEC-1 cell line. Distinctive chemical profiles obtained from black and red quinoa were well correlated with the antioxidant activities and cytoprotective effects. The combination of PHWE, chemical standardization with LC/UV and LC/MS, pattern recognition tools and biological assay provided an approach for the evaluation and eventual production of quinoa extracts for functional food.

Background/aims: The frequent lack of association between dry eye (DE) symptoms and signs leads to challenges in diagnosing and assessing the disease., Methods: Participants underwent ocular surface examinations to evaluate signs of disease and completed questionnaires to assess ocular symptoms, psychological status and medication use. To assess nociceptive system integrity, quantitative sensory testing (QST), including vibratory and thermal threshold measures and temporal summation of pain were obtained at the forearm and forehead. Correlations between DE discordance score (degree of discrepancy between symptom severity and DE signs) and patient characteristics were determined. Higher discordance scores indicated more symptoms than signs., Results: 326 patients participated (mean age: 62 years; SD: 10 years; 92% men). Age was negatively correlated with DE discordance score (Pearson r=-0.30, p<0.0005), while mental health indices were positively correlated. Chronic pain elsewhere in the body (ie, non-ocular pain conditions) and intensity ratings of prolonged aftersensations of pain evoked by noxious hot and cold stimuli were also significantly correlated with DE discordance score. Multiple linear regression demonstrated that post-traumatic stress disorder and non-ocular pain intensity were important predictors of DE discordance score, Dry Eye Questionnaire-5 and Ocular Surface Disease Index and that DE discordance was also sensitive to QST as well., Conclusions: The present study provides evidence that the degree of discordance between DE symptom report and measurable signs of ocular surface disease is associated with comorbidities related to clinical pain and to hyperalgesia as demonstrated with QST. Understanding the epidemiology of DE discordance can aid in interpreting the DE exam and individualising treatment., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Docosahexaenoic acid (DHA) is enriched in membrane phospholipids of the central nervous system (CNS) and has a role in aging and neuropsychiatric disorders. DHA is metabolized by the enzyme Alox15 to 17S-hydroxy-DHA, which is then converted to 7S-hydroperoxy,17S-hydroxy-DHA by a 5-lipoxygenase, and thence via epoxy intermediates to the anti-inflammatory molecule, resolvin D1 (RvD1 or 7S,8R,17S-trihydroxy-docosa-Z,9E,11E,13Z,15E,19Z-hexaenoic acid). In this study, we investigated the distribution and function of Alox15 in the CNS. RT-PCR of the CNS showed that the prefrontal cortex exhibits the highest Alox15 mRNA expression level, followed by the parietal association cortex and secondary auditory cortex, olfactory bulb, motor and somatosensory cortices, and the hippocampus. Western blot analysis was consistent with RT-PCR data, in that the prefrontal cortex, cerebral cortex, hippocampus, and olfactory bulb had high Alox15 protein expression. Immunohistochemistry showed moderate staining in the olfactory bulb, cerebral cortex, septum, striatum, cerebellar cortex, cochlear nuclei, spinal trigeminal nucleus, and dorsal horn of the spinal cord. Immuno-electron microscopy showed localization of Alox15 in dendrites, in the prefrontal cortex. Liquid chromatography mass spectrometry analysis showed significant decrease in resolvin D1 levels in the prefrontal cortex after inhibition or antisense knockdown of Alox15. Alox15 inhibition or antisense knockdown in the prefrontal cortex also blocked long-term potentiation of the hippocampo-prefrontal cortex pathway and increased errors in alternation, in the T-maze test. They indicate that Alox15 processing of DHA contributes to production of resolvin D1 and LTP at hippocampo-prefrontal cortical synapses and associated spatial working memory performance. Together, results provide evidence for a key role of anti-inflammatory molecules generated by Alox15 and DHA, such as resolvin D1, in memory. They suggest that neuroinflammatory brain disorders and chronic neurodegeneration may 'drain' anti-inflammatory molecules that are necessary for normal neuronal signaling, and compromise cognition.

In this study, extraction of immature fruits using an environmentally friendly pressurized hot water extraction (PHWE) method was compared with the traditional reflux method. Extracts were tested for their polyphenol content using the Folin-Ciocalteu assay and for their antioxidant activity using the oxygen radical absorbance capacity (ORAC) assay. The highest amount of polyphenol was extracted from grape (stem) using PHWE at 100°C, or reflux extraction. This was followed by reflux extraction of grape (fruit). The results were similar for the ORAC assay. All samples extracted using PHWE at 100°C showed cytoprotective activity against H 2 O 2 -induced oxidative stress in Crandell-Reese feline kidney (CRFK) cells. This study demonstrated that beneficial compounds can be extracted from immature fruits without the use of organic solvents. The utilization of beneficial compounds present in immature fruits can also contribute to the reduction in agriculture waste generated., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Importance: Dry eye syndrome is a common condition that affects millions of individuals. Many cross-sectional studies have evaluated risk factors for dry eye severity, but few have assessed risk factors or symptom progression over time., Objectives: To assess symptom progression in dry eye syndrome and determine risk factors associated with severe symptoms at 1 year., Design, Setting, and Participants: A longitudinal study was conducted from October 1, 2013, to April 30, 2015, among patients at the Miami Veterans Affairs Hospital with a wide variety of dry eye symptoms and signs (ranging from none to severe)., Main Outcomes and Measures: Change in dry eye symptom severity during 1 year, as assessed by responses to dry eye symptom questionnaires administered at the initial visit and 1 year later, as well as baseline risk factor analysis for severe dry eye symptoms at 1 year, defined as a Dry Eye Questionnaire 5 score of 12 or more., Results: Of the 120 patients (mean [SD] age, 64 [11] years; 109 male and 11 female), 26 of 58 (44.8%) with either no symptoms or mild or moderate symptoms at baseline progressed to more severe symptoms at 1 year, while 46 of 62 patients (74.2%) with severe symptoms at baseline reported that severe symptoms persisted at 1 year. Baseline ocular risk factors for severe dry eye symptoms at 1 year included more severe dry eye symptoms, ocular pain, and neuropathic pain-like ocular symptoms. Nonocular risk factors included sleep disturbances (eg, sleep apnea and insomnia), mental health status (eg, posttraumatic stress disorder and depression), nonocular pain, and medications (eg, anxiolytics and analgesics). In a multivariable analysis, the most significant risk factors were sleep apnea (odds ratio [OR], 3.80; 95% CI, 1.00-14.49; P = .05), Dry Eye Questionnaire 5 score (OR, 1.15; 95% CI, 1.02-1.30; P = .02), and posttraumatic stress disorder score (OR, 1.04; 95% CI, 1.01-1.08; P = .02)., Conclusions and Relevance: Patients with severe dry eye symptoms and ocular pain at baseline were more likely to have persistent severe dry eye symptoms on 1-year follow-up. Furthermore, nonocular risk factors that have been associated with dry eye cross-sectionally, such as psychiatric comorbidities and nonocular pain, were also associated with severe dry eye symptoms at 1 year. Although this cohort was limited to US veterans, which may not be generalizable to other populations, our results suggest that pain perception and severity are important when evaluating and managing dry eye.

The study of single cells has evolved over the past several years to include expression and genomic analysis of an increasing number of single cells. Several studies have demonstrated wide spread variation and heterogeneity within cell populations of similar phenotype. While the characterization of these populations will likely set the foundation for our understanding of genomic- and expression-based diversity, it will not be able to link the functional differences of a single cell to its underlying genomic structure and activity. Currently, it is difficult to perturb single cells in a controlled environment, monitor and measure the response due to perturbation, and link these response measurements to downstream genomic and transcriptomic analysis. In order to address this challenge, we developed a platform to integrate and miniaturize many of the experimental steps required to study single-cell function. The heart of this platform is an elastomer-based integrated fluidic circuit that uses fluidic logic to select and sequester specific single cells based on a phenotypic trait for downstream experimentation. Experiments with sequestered cells that have been performed include on-chip culture, exposure to various stimulants, and post-exposure image-based response analysis, followed by preparation of the mRNA transcriptome for massively parallel sequencing analysis. The flexible system embodies experimental design and execution that enable routine functional studies of single cells.

A laboratory-assembled surfactant-assisted pressurized liquid extraction system at room temperature was used for the extraction of glycyrrhizin (GLY) in Radix glycyrrhizae. Environmentally friendly saccharide fatty acid ester such as glucose oleic acid ester is proposed to replace chemical-based surfactants. As the chemical properties of the surfactant obtained were unknown initially, lipase-catalyzed synthesis and liquid chromatography with tandem mass spectrometry were used to ascertain the identity. Surfactant-assisted pressurized liquid extraction (PLE) was carried out dynamically and the extraction efficiencies of the proposed method using different concentration of glucose oleic acid ester were compared with sonication using an organic solvent (ethanol/water, 70:30). The extraction efficiencies of GLY in Radix glycyrrhizae using surfactant-assisted PLE was observed to be higher compared with sonication. The method precision was found to vary from 1.3 to 5.1% (relative standard deviation, RSD, n= 6) on different days. The new method demonstrated the possibility for the extraction to be carried out at room temperature for the production of botanical extracts., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Colon and pancreatic cancer are associated with significant thrombophilia. Colon and pancreas tumor cells have an increase in hemeoxygenase-1 (HO-1) activity, the endogenous enzyme responsible for carbon monoxide production. Given that carbon monoxide enhances plasmatic coagulation, we determined if patients undergoing resection of colon and pancreatic tumors had an increase in endogenous carbon monoxide and plasmatic hypercoagulability. Patients with colon (n = 17) and pancreatic (n = 10) tumors were studied. Carbon monoxide was determined by the measurement of carboxyhemoglobin (COHb). A thrombelastographic method to assess plasma coagulation kinetics and formation of carboxyhemefibrinogen (COHF) was utilized. Nonsmoking patients with colon and pancreatic tumors had abnormally increased COHb concentrations of 1.4 ± 0.9 and 1.9 ± 0.7%, respectively, indicative of HO-1 upregulation. Coagulation analyses comparing both tumor groups demonstrated no significant differences in any parameter; thus the data were combined for the tumor groups for comparison with 95% confidence interval values obtained from normal individuals (n = 30) plasma. Seventy percent of tumor patients had a velocity of clot formation greater than the 95% confidence interval value of normal individuals, with 53% of this hypercoagulable group also having COHF formation. Further, 67% of tumor patients had clot strength that exceeded the normal 95% confidence interval value, and 56% of this subgroup had COHF formation. Finally, 63% of all tumor patients had COHF formation. Future investigation of HO-1-derived carbon monoxide in the pathogenesis of colon and pancreatic tumor-related thrombophilia is warranted.

Gallstone disease is the most common cause of acute pancreatitis in the Western world. In most cases, gallstone pancreatitis is a mild and self-limiting disease, and patients may proceed without complications to cholecystectomy to prevent future recurrence. Severe disease occurs in about 20% of cases and is associated with significant mortality; meticulous management is critical. A thorough understanding of the disease process, diagnosis, severity stratification, and principles of management is essential to the appropriate care of patients presenting with this disease. This article reviews these topics with a focus on surgical management, including appropriate timing and choice of interventions., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Although cancer-mediated changes in hemostatic proteins unquestionably promote hypercoagulation, the effects of neoplasia on fibrinolysis in the circulation are less well defined. The goals of the present investigation were to determine if plasma obtained from patients with breast, lung, pancreas and colon cancer was less or more susceptible to lysis by tissue-type plasminogen activator (tPA) compared to plasma obtained from normal individuals. Archived plasma obtained from patients with breast (n = 18), colon/pancreas (n = 27) or lung (n = 19) was compared to normal individual plasma (n = 30) using a thrombelastographic assay that assessed fibrinolytic vulnerability to exogenously added tPA. Plasma samples were activated with tissue factor/celite, had tPA added, and had data collected until clot lysis occurred. Additional, similar samples had potato carboxypeptidase inhibitor added to assess the role played by thrombin-activatable fibrinolysis inhibitor in cancer-modulated fibrinolysis. Rather than inflicting a hypofibrinolytic state, the three groups of cancers demonstrated increased vulnerability to tPA (e.g. decreased time to lysis, increased speed of lysis, decreased clot lysis time). However, hypercoagulation manifested as increased speed of clot formation and strength compensated for enhanced fibrinolytic vulnerability, resulting in a clot residence time that was not different from normal individual thrombi. In sum, enhanced hypercoagulability associated with cancer was in part diminished by enhanced fibrinolytic vulnerability to tPA.

Chrysanthemum is a ubiquitous plant with many species and wide uses, and it is usually consumed as functional food. The main aim of this paper is to demonstrate that chromatographic fingerprints obtained from the HPLC/UV analysis of the pressurized hot water extraction (PHWE) extracts together with the aid of principal component analysis (PCA), allowed for the clustering of various chrysanthemums of different species and provenance. In addition, a parallel study of pressurized fluid extraction (PFE) with methanol was carried out for comparison. From the results, a clearer separation and clustering was obtained with the environmentally-benign water extracts compared with methanol extracts. This study shows that PHWE in combination with HPLC/UV and PCA can be used successfully as a green and effective approach for characterisation and quality control of ubiquitous functional food such as chrysanthemum., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Context: While perioperative mortality after pancreaticoduodenectomy is decreasing, key factors remain to be elucidated., Objective: The purpose of this study was to investigate inpatient mortality after pancreaticoduodenectomy in the Nationwide Inpatient Sample (NIS), a representative inpatient database in the USA., Methods: Patient discharge data (diagnostic and procedure codes) and hospital characteristics were investigated for years 2009 and 2010. The inclusion criteria were a procedure code for pancreaticoduodenectomy, elective procedure, and a pancreatic or peripancreatic cancer diagnosis. Chi-square test determined statistical significance. A logistic regression model for mortality was created from significant variables., Results: Two-thousand and 958 patients were identified with an average age of 65±12 years; 53% were male. The mean length of stay was 15±12 days with a mortality of 4% and a complication rate of 57%. Eighty-six percent of pancreaticoduodenectomy occurred in teaching hospitals. Pancreaticoduodenectomy performed in teaching hospitals in the first half of the academic year were associated with higher mortality than in the latter half (5.5% vs. 3.4%, P=0.005). On logistic regression analysis, non-surgical complications are the largest predictor of death (P<0.001) while operations in the latter half of the academic year are associated with decreased mortality (P<0.01)., Conclusions: The timing of pancreaticoduodenectomy for cancer remained more predictive of mortality than age or length of stay; only complications were more predictive of death than time of year. This suggests that there remains a clinically and statistically significant learning curve for trainees in identifying complications; further study is needed to prove that identification of complications leads to a decrease in mortality rate by taking corrective actions.

Background: The purpose of this study was to evaluate the effect of epidural analgesia use on postoperative complications in patients undergoing pancreaticoduodenectomy., Methods: This retrospective cohort study used the 2009 Nationwide Inpatient Sample from the Agency for Healthcare Research and Quality. Patients who underwent pancreaticoduodenectomy were grouped on the basis of whether they received epidural analgesia. The effect of epidural use on the composite end point of major complications including death was investigated using a generalized linear model., Results: Overall, 8,610 cases of pancreaticoduodenectomy occurred within the United States in 2009, and 11.0% of these patients received epidural analgesia. After controlling for various potential confounders, results of the multivariate regression indicated that epidural analgesia use was associated with lower odds of composite complications including death (odds ratio, .61; 95% confidence interval, .37-.99; P = .044)., Conclusions: In patients who underwent pancreaticoduodenectomy, epidural analgesia was associated with significantly lower postoperative composite complications., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We investigate in this study the approach of small bowel delineation that would best correlate with acute lower GI toxicity during adjuvant intensity-modulated radiation therapy (IMRT) for endometrial cancer in this study. Thirty-two endometrial cancer patients (FIGO IB-IVA) were treated with postoperative pelvic IMRT to 48.2 ± 6 3.1 Gy. The small bowel was delineated as separate loops, limited bowel space (BS), or an intestinal cavity (IC). The volume of the small bowel (VSB) in absolute volume or as the percentage of the total volume at various dose levels was obtained from the dose volume histograms (DVHs). Each patient's acute lower gastro-intestinal (GI) toxicity was assessed prospectively during the course of IMRT. After a median follow up of 19.6 months, the median survival, loco-regional control, progression-free-survival (PFS), and distant metastasis-free survival (DMFS) were 40.9 months, 81.2%, 62.5%, and 68.8%, respectively. Acute lower GI toxicity observed were of grade 0, 1, and 2 only: 34.4%, 31.2%, and 34.4%, respectively. The difference in %VSB with the small bowel delineated as IC at 45 Gy (%VSB(IC45)) between grade 2 and grade 0 acute lower GI toxicity reached statistical significance upon linear regression analysis ( p = 0.0347). Thus, the proportion of small bowel contoured as IC in the high dose region can potentially be an important predictor for acute lower GI toxicity during and after postoperative pelvic IMRT.

Aim: To evaluate the safety and effectiveness of our new cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemoperfusion (HIPEC) program., Methods: Retrospective review of patients with gastrointestinal malignancies who were suitable candidates for CRS and HIPEC between 12/1/2009 and 10/1/2010. All clinicopathologic data were reviewed with a special focus on the surgical outcome and the postoperative morbidity and mortality., Results: Fourteen patients were identified. Median age was 64 years; seven were female. The primary tumors were: colonic (29%), appendiceal (36%), peritoneal mesothelioma (14%), gastric (7%), adenocarcinoma of unknown primary (7%), and gastrointestinal stromal tumor (7%). Eleven patients (79%) received CRS/HIPEC, three for palliation. Three patients that did not undergo CRS/HIPEC had an average peritoneal cancer index (PCI) of 25. The eight patients that underwent curative CRS/HIPEC had an average PCI of 10 and a completeness of cytoreduction score of 0 (87.5%) or 1 (12.5%). Postoperative morbidity was 36%; the worst adverse event was Grade 3 ileus. Mortality rate was 0%., Conclusion: CRS with HIPEC is safe and feasible at tertiary institutions with fledgling programs. PCI is an accurate predictor of surgical outcomes.

Berberine, long used as a remedy in China and India for intestinal infections, has been discovered in recent years in western countries and is now being used to treat ailments ranging from urinary tract infections to diabetes and obesity. In order to study the effect of berberine more deeply, a combined metabolomic and metallomic approach was developed in this study using the hypercholesterolaemic rat model, which involved the use of proton nuclear magnetic resonance for the analysis of rat urine to achieve metabolic fingerprinting and inductively coupled plasma mass spectrometry for the analysis of rat blood serum to achieve metallomic fingerprinting. The results obtained indicated that major metabolic processes like Krebs cycle, cholesterol metabolism and osmoregulation in hypercholesterolaemic rats are perturbed upon berberine injection. In addition, the changes of some elements, such as V, Mn, Na and K, revealed in the metallomic study may contribute to the search of new biomarkers for hypercholesterolaemic disease. We concluded that both the metabolomic and metallomic profiles of berberine-treated hypercholesterolaemic rats were different from those of the control group and that the selected metabolites and elements could probably be applied as potential biomarkers for the understanding of the effect of berberine on biochemical process in the animal model. Such a multi-analytical approach will potentially provide an information-rich platform for the elucidation of effects of xenobiotics and drug efficacy studies.

Background: Minority groups with colorectal cancer have not experienced the decline in incidence and mortality that has been reported in whites. We sought to determine whether differences exist in treatment and survival between white and Native American patients with colorectal cancer because little has been written about this specific minority group., Study Design: The Surveillance Epidemiology and End Results (SEER) database for colorectal cancer was used to compare treatment and survival in whites (colon, n = 137,949; rectum, n = 46,843) and Native Americans (colon, n = 872; rectum, n = 316). Cox proportional hazards models were used to compare cancer-specific survival in Native Americans with whites, adjusted for stage, sex, age and year of diagnosis, socioeconomic status, and treatment., Results: Native Americans presented at younger ages than whites for both colon and rectal cancer (p < 0.001). They were diagnosed at more advanced stages of disease than whites for only colon cancer. No significant differences were detected in the proportion of patients recommended for surgery between the two groups, for either cancer at any stage (all p > 0.05). Native Americans with rectal cancer were more likely to receive radiation than whites (p < 0.001), but they received less sphincter-preserving surgery (60.0% vs 65.4%; p = 0.045). Native Americans with colon cancer fared significantly worse than whites (hazard ratio = 1.20; 95% CI = 1.08 to 1.34), but there is no difference in cancer-free survival between races for rectal cancer., Conclusions: Compared with whites, Native Americans with colon cancer have worse cancer-specific survival., (Published by Elsevier Inc.)

Significant increases in levels of cholesterol and cholesterol oxidation products are detected in the hippocampus undergoing degeneration after excitotoxicity induced by the potent glutamate analog, kainate (KA), but until now, it is unclear whether the cholesterol is in the free or esterified form. The present study was carried out to examine the expression of the enzyme involved in cholesteryl ester biosynthesis, acyl-coenzyme A: cholesterol acyltransferase (ACAT) and cholesteryl esters after KA excitotoxicity. A 1000-fold greater basal mRNA level of ACAT1 than ACAT2 was detected in the normal brain. ACAT1 mRNA and protein were upregulated in the hippocampus at 1 and 2 weeks after KA injections, at a time of glial reaction. Immunohistochemistry showed ACAT1 labeling of oligodendrocytes in the white matter and axon terminals in hippocampal CA fields of normal rats, and loss of staining in neurons but increased immunoreactivity of oligodendrocytes, in areas affected by KA. Gas chromatography-mass spectrometry analyses confirmed previous observations of a marked increase in level of total cholesterol and cholesterol oxidation products, whilst nuclear magnetic resonance spectroscopy showed significant increases in cholesteryl ester species in the degenerating hippocampus. Upregulation of ACAT1 expression was detected in OLN93 oligodendrocytes after KA treatment, and increased expression was prevented by an antioxidant or free radical scavenger in vitro. This suggests that ACAT1 expression may be induced by oxidative stress. Together, our results show elevated ACAT1 expression and increased cholesteryl esters after KA excitotoxicity. Further studies are necessary to determine a possible role of ACAT1 in acute and chronic neurodegenerative diseases., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background and Objectives: Many laparoscopic surgeons are now transitioning from standard multiple-port laparoscopic cholecystectomy to single-incision laparoscopic surgery (SILS) in an attempt to improve cosmetic outcomes and decrease postoperative morbidity. However, little has been published regarding the potential complications of SILS operations., Methods: We report the case of a patient undergoing SILS cholecystectomy who developed the complication of a large hepatic hematoma, resulting in significant postoperative morbidity, blood transfusion requirement, and reoperation., Results: After an in-depth internal review of the postoperative morbidity of this case, it appears that the causative factor may be instrument shaft torque on the liver surface., Conclusion: Single-incision laparoscopic surgery may pose significant and unique risks that warrant additional operative caution. Quantitative comparison of SILS to the gold-standard laparoscopic cholecystectomy is needed to further elucidate definitive benefits and complications of this novel technique.

Pressurized hot water extraction (PHWE) has become a popular green extraction method for different classes of compounds present in numerous kinds of matrices such as environmental, food and botanical samples. PHWE is also used in sample preparation to extract organic contaminants from foodstuff for food safety analysis and soils/sediments for environmental monitoring purposes. The main parameters which influence its extraction efficiency are namely the temperature, extraction time, flow rates and addition of modifiers/additives. Among these different parameters studied, temperature is described as the most important one. It is reported that the extraction of certain compounds is rather dependent on pressurized water with different applied temperature. Thus, the stability and reduced solubilities of certain compounds at elevated temperatures are highlighted in this review. With some modifications, a scaled-up PHWE could extract a higher amount of desirable compounds from solid and powdered samples such as plant and food materials. The PHWE extracts from plants are rich in chemical compounds or metabolites which can be a potential lead for drug discovery or development of disease-resistant food crops., (Copyright 2009 Elsevier B.V. All rights reserved.)

Colorectal cancer (CRC) arises as the consequence of progressive changes from normal epithelial cells through polyp to tumor, and thus is an useful model for studying metabolic shift. In the present study, we studied the metabolomic profiles using high analyte specific gas chromatography/mass spectrometry (GC/MS) and liquid chromatography tandem mass spectrometry (LC/MS/MS) to attain a systems-level view of the shift in metabolism in cells progressing along the path to CRC. Colonic tissues including tumor, polyps and adjacent matched normal mucosa from 26 patients with sporadic CRC from freshly isolated resections were used for this study. The metabolic profiles were obtained using GC/MS and LC/MS/MS. Our data suggest there was a distinct profile change of a wide range of metabolites from mucosa to tumor tissues. Various amino acids and lipids in the polyps and tumors were elevated, suggesting higher energy needs for increased cellular proliferation. In contrast, significant depletion of glucose and inositol in polyps revealed that glycolysis may be critical in early tumorigenesis. In addition, the accumulation of hypoxanthine and xanthine, and the decrease of uric acid concentration, suggest that the purine biosynthesis pathway could have been substituted by the salvage pathway in CRC. Further, there was a step-wise reduction of deoxycholic acid concentration from mucosa to tumors. It appears that to gain a growth advantage, cancer cells may adopt alternate metabolic pathways in tumorigenesis and this flexibility allows them to adapt and thrive in harsh environment.

Introduction: The aim of this study was to compare the knowledge and practices of household mosquito-breeding control measures between a dengue hotspot (HS) and a non-hotspot (NHS)., Materials and Methods: Eight hundred households were randomly sampled from HS and NHS areas, and an National Environment Agency (NEA) questionnaire was administered to heads of the households. Interviewers were blinded to the dengue status of households. We included subjects aged above 16 years, who were communicative and currently living in the household. Chi-square test was used to compare proportions and multiple logistic regression was used to adjust for socio-demographic differences between both areas., Results: The overall response rate was 59.0% (n = 472). There were significant differences in gender, educational level, employment status and housing type between HS and NHS (all P <0.05). NHS residents were less knowledgeable in 6 out of 8 NEA-recommended anti-mosquito breeding actions: changing water in vase/bowls [AOR (adjusted OR), 0.20; CI, 0.08-0.47; P <0.01], adding sand granular insecticide to water [AOR, 0.49; CI, 0.31-0.71; P <0.01], turning over pails when not in use [AOR, 0.39; CI, 0.17-0.89; P = 0.02], removing flower pot/plates [AOR, 0.35; CI, 0.18-0.67; P <0.01], removing water in flower pot/plates [AOR, 0.36; CI, 0.17-0.75; P <0.01] and putting insecticide in roof gutters [AOR 0.36; CI, 0.13-0.98; P = 0.04]. Hotspot residents reported better practice of only 2 out of 8 NEA-recommended mosquito-breeding control measures: changing water in vases or bowls on alternate days [AOR, 2.74; CI, 1.51-4.96; P <0.01] and removing water from flower pot plates on alternate days [AOR, 1.95; CI, 1.01-3.77; P = 0.05]., Conclusion: More HS residents were knowledgeable and reported practicing mosquito-breeding control measures compared to NHS residents. However, a knowledge-practice gap still existed.

Symphytum officinale L. (comfrey) is a medicinal plant commonly used in decoctions and aliments. Besides therapeutic bioactive compounds present in the herb, it is found to contain hepatotoxic pyrrolizidine alkaloids (PAs), such as lycopsamine and others. In the present study, PAs such as lycopsamine, echimidine and lasiocarpine were determined using electrospray liquid chromatography-mass spectrometry (LC-MS) with the method precision (relative standard deviation, RSD) <10%. Detection of lycopsamine, symviridine and their N-oxides could be confirmed with a newly developed method based on HPLC ion-trap and orbitrap MS with electrospray ionization interface. With LC-MS, quantitative analysis of lycopsamine in the botanical extract was carried out. The effect of extraction solvent was optimized by sonication and methanol: H(2)O (50:50) was selected. Then a rapid method based on pressurized hot water extraction (PHWE) was employed for the extraction of lycopsamine from comfrey followed by the comparison with heating under reflux with the RSD ranging from 2.49% to 19.32%. Our results showed a higher extraction efficiency for heating under reflux compared with PHWE. It was proposed that the lower extraction efficiency for PHWE was attributable to dissolved nitrogen from air which caused the reduction in the solubility of lycopsamine in the compressed hot solvent. In this study, quantitative analysis of PAs in comfrey was demonstrated. In addition, it was found that the use of subcritical water for extractions depended on the physical properties of the dissolved solutes and their tendency to degrade under the chosen extraction conditions.

Tussilago farfara (Kuan Donghua) is an important Chinese herbal medicine which has been shown to contain many bioactive compounds and widely used to relieve cough and resolve phlegm. However, besides therapeutic bioactive compounds, this herb has been found to contain toxic pyrrolizidine alkaloids (PAs), mainly senkirkine and traces of senecionine. In this report, conditions for microwave-assisted extraction (MAE) and pressurized hot water extraction (PHWE) were optimized for the extraction of the PAs. The results were compared against heating under reflux. It was found that the binary mixture of MeOH:H(2)O (1:1) acidified using HCl to pH 2-3 was the optimal solvent for the extraction of the PAs in the plant materials. Liquid chromatography (LC) with ultra-violet (UV) detection and electrospray ionization mass spectrometry (ESI-MS) in the positive mode was used for the determination and quantitation of senkirkine and senecionine in the botanical extract. The proposed extraction methods with LC/MS allow for the rapid detection of the major and the minor alkaloids in T. farfara in the presence of co-eluting peaks. With LC/MS, the quantitative analysis of PAs in the extract was done using internal standard calibration and the precision was found to vary from 0.6% to 5.4% on different days. The limits of detection (LODs) and limits of quantitation (LOQs) for MAE and PHWE were found to vary from 0.26 microg/g to 1.04 micro/g and 1.32 micro/g to 5.29 microg/g, respectively. The method precision of MAE and PHWE were found to vary from 3.7% to 10.4% on different days. The results showed that major and minor alkaloids extracted using MAE and PHWE were comparable to that by heating under reflux. Our data also showed that significant ion suppression was not observed in the analysis of senkirkine and senecionine in the botanical extracts with co-eluting peaks.

A method using 1H NMR and LC/MS with pattern recognition tools such as principal component analysis (PCA) and orthogonal projection to latent structure discriminant analysis (O-PLS-DA) was used to study the urinary metabolic profiles associated with an increase in urinary albumin in a general population. The normalized peak intensities obtained from 1H NMR and LC/MS with nonparametric two-tailed Mann-Whitney analysis was used for the identification of network of potential biomarkers corresponding to the increase of albumin in urine. The specificity of detecting the stated metabolites by 1H NMR and LC/MS was demonstrated. Our preliminary data obtained demonstrated that LC/MS may produce more distinctive metabolic profiles. For the patient group, changes in alanine, kyneurnic acid, and xanthurenic acid might be associated with changes in the tryptophan metabolism. At the same time, other metabolites that were involved in citric acid cycle, amino acid metabolism, and cellular functions were affected in the patient group. The proposed approach provided a comprehensive picture of the metabolic changes induced by the increase of protein in urine and demonstrated the advantages of using multiple diagnostic biomarkers. At the same time, the current work was demonstrated as a potential cost-effective solution of high-throughput analysis with pattern recognition tools as applied here in a real clinical situation.

A metabolomic study was performed to investigate the biochemical profiles of livers from male and female zebrafish (Danio rerio), using a multiple platform approach, incorporating 1H NMR, GC/MS and LC/MS. The reproducibility and reliability of the three methods were validated prior to the assays. Major biomolecules detected using one method were also cross examined using the other techniques. These metabolites included carbohydrates, lipids, amino acids detected using 1H NMR and GC/MS, and acetylcarnitine, choline and various phospholipids determined using 1H NMR and LC/MS. Our findings suggest that 1H NMR provided comprehensive information on glucose, amino acids, pyruvate and other smaller biochemical constituents of the zebrafish liver. On the other hand, GC/MS was able to assay cholesterol, saturated and unsaturated fatty acids, and LC/MS was ideal for the analysis of lipids/phospholipids. These techniques revealed that there are significant differences in the biochemical profiles of male and female zebrafish liver tissue extracts. Specifically, we noted that although there were no significant differences observed for the carbohydrate profile, the amino acid profile was rather different in male and female zebrafish liver. Furthermore, data from all three techniques revealed that although the saturated fatty acid profile was similar, the compositions of unsaturated fatty acids were different in the two phenotypes. The overall findings suggested that this multiplatform approach offers comprehensive coverage of a metabolome as well as provides valuable insight towards understanding the different biochemical profiles of a biosystem.

An approach that combined green-solvent methods of extraction with chromatographic chemical fingerprint and pattern recognition tools such as principal component analysis (PCA) was used to evaluate the quality of medicinal plants. Pressurized hot water extraction (PHWE) and microwave-assisted extraction (MAE) were used and their extraction efficiencies to extract two bioactive compounds, namely stevioside (SV) and rebaudioside A (RA), from Stevia rebaudiana Bertoni (SB) under different cultivation conditions were compared. The proposed methods showed that SV and RA could be extracted from SB using pure water under optimized conditions. The extraction efficiency of the methods was observed to be higher or comparable to heating under reflux with water. The method precision (RSD, n = 6) was found to vary from 1.91 to 2.86% for the two different methods on different days. Compared to PHWE, MAE has higher extraction efficiency with shorter extraction time. MAE was also found to extract more chemical constituents and provide distinctive chemical fingerprints for quality control purposes. Thus, a combination of MAE with chromatographic chemical fingerprints and PCA provided a simple and rapid approach for the comparison and classification of medicinal plants from different growth conditions. Hence, the current work highlighted the importance of extraction method in chemical fingerprinting for the classification of medicinal plants from different cultivation conditions with the aid of pattern recognition tools used.

Cytokinins ribosides such as kinetin riboside are a class of plant hormone that were first identified as factors that promote cell division and have since been implicated in many other aspects of plant growth and development. From the data obtained from cell cycle analysis with flow cytometry, the in vitro growth inhibition of human heptamoa, HepG2 cells with kinetin riboside was mediated by causing G2/M cell cycle arrest and cell death. At the same time, treatment with various doses of kinetin riboside in HepG2 cells did not result in a population of cells positive for the active caspase 3. Differentially expressed proteins in the mitochondria of HepG2 cells with cell death induced by kinetin riboside were investigated. Without the use of stable isotope labeling, the proposed method using LC/MSMS provided a rapid approach to study the differentially expressed proteins in the mitochondria due to the cell death induced by kinetin riboside in HepG2 cells. The ability of kinetin riboside to induce cell death and attenuate G1 to S transition is probably a consequence of its ability to interfere with several components in the mitochondria. Hence, it was proposed that the cell death caused by kinetin riboside in HepG2 cells affected a network of proteins involved in cell death and electron transport.