Recent advances in the contribution of glucosinolates degradation products to cruciferous foods odor: factors that influence degradation pathways and odor attributes.

As one of the most important vegetables and oils consumed globally, cruciferous foods are appreciated for their high nutritional value. However, there is no comprehensive knowledge to sufficiently unravel the "flavor mystery" of cruciferous foods. The present review provides a comprehensive literature on the recent advances regarding the contribution of glucosinolates (GSL) degradation products to cruciferous foods odor, which focuses on key GSL degradation products contributing to distinct odor of cruciferous foods (Brassica oleracea, Brassica rapa, Brassica napus, Brassica juncea, Raphanus sativus), and key factors affecting GSL degradation pathways (i.e., enzyme-induced degradation, thermal-induced degradation, chemical-induced degradation, microwave-induced degradation) during different processing and cooking. A total of 93 volatile GSL degradation products (i.e., 36 nitriles, 33 isothiocyanates, 3 thiocyanates, 5 epithionitriles, and 16 sulfides) and 29 GSL (i.e., 20 aliphatic, 5 aromatic, and 4 indolic) were found in generalized cruciferous foods. Remarkably, cruciferous foods have a distinctive pungent, spicy, pickled, sulfur, and vegetable odor. In general, isothiocyanates are mostly present in enzyme-induced degradation of GSL and are therefore often enriched in fresh-cut or low-temperature, short-time cooked cruciferous foods. In contrast, nitriles are mainly derived from thermal-induced degradation of GSL, and are thus often enriched in high-temperature, long-time cooked cruciferous foods.

Processing and cooking can cause degradation of glucosinolates and formation of volatiles.Structure­odor relationship of glucosinolates degradation products is discussed.Nitriles, isothiocyanates, and sulfides play an important role in cruciferous foods odor.Both enzyme- and thermal-induced degradation of glucosinolates is strongly pH-dependent.

Effects of irradiation on the aging and sensory quality of navel orange distilled spirits.

BACKGROUND: 60 Co-γ irradiation can simulate the effects of aging and enhance the flavor of distilled spirits. The present study aimed to investigate the effects of 0, 2, 4, 6, 8 and 10 kGy 60 Co-γ irradiation doses on the key aroma components in newly produced navel orange distilled spirits and thus determine the mechanism of their aging distilled spirits. RESULTS: The identification of aroma compounds demonstrated that ethyl hexanoate, d-limonene, ethyl octanoate, 3-methyl-1-butanol and linalool are the key aroma compounds in navel orange distilled spirits, which were increased except for linalool with irradiation doses of 2-6 kGy. Irradiation treatment simulated the effects of the aging of navel orange distilled spirits by promoting the content of total acids, total esters and aldehydes. Irradiation doses of 2-6 kGy increased the aroma intensity of navel orange distilled spirits, reaching an optimum at 6 kGy. However, irradiation doses as high as 8 and 10 kGy decreased the content of esters in navel orange distilled spirits, which led to a deterioration of the spirit flavor. CONCLUSION: Low doses of 60 Co-γ irradiation can simulate the effects of the aging by increasing the content of key aromatic compounds in navel orange distilled spirits. © 2023 Society of Chemical Industry.

Purification and characterization of limonin D-ring lactone hydrolase from sweet orange (Citrus sinensis (L.) Osbeck) seeds.

BACKGROUND: Citrus products often suffer from delayed bitterness, which is generated from the conversion of non-bitter precursors (limonoate A-ring lactone, LARL) to limonin under the catalysis of limonin D-ring lactone hydrolase (LDLH). In this study, LDLH was isolated and purified from sweet orange seeds, and a rapid and accurate high-performance liquid chromatography method to quantify LARL was developed and applied to analyze the activity and enzymatic properties of purified LDLH. RESULTS: Purified LDLH (25.22 U mg-1) showed bands of 245 kDa and 17.5 kDa molecular weights in native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate PAGE analysis respectively. After a 24 h incubation under strongly acidic (pH 3) or strongly alkaline (pH 9) conditions, LDLH still retained approximately 100% activity. Moreover, LDLH activity was not impaired by thermal treatment at 50 °C for 120 min. Enzyme inhibition assays showed that LDLH was inactivated only after ethylenediaminetetraacetic acid treatment, and other enzyme inhibitors showed no significant effect on its activity. In addition, the LDLH activity of calcium ion (Ca2+) intervention was 108% of that in the blank group, and that of zinc ion (Zn2+) intervention was 71%. CONCLUSION: LDLH purified in this study was a multimer containing 17.5 kDa monomer with a wide pH tolerance range (pH 3-9) and excellent thermal stability. Moreover, LDLH might be a metallopeptidase, and its activity was stimulated by Ca2+ and significantly inhibited by Zn2+. These findings improve our understanding of LDLH and provide some important implications for reducing the bitterness in citrus products in the future. © 2024 Society of Chemical Industry.

Identification of lysine isobutyrylation as a new histone modification mark.

Short-chain acylations of lysine residues in eukaryotic proteins are recognized as essential posttranslational chemical modifications (PTMs) that regulate cellular processes from transcription, cell cycle, metabolism, to signal transduction. Lysine butyrylation was initially discovered as a normal straight chain butyrylation (Knbu). Here we report its structural isomer, branched chain butyrylation, i.e. lysine isobutyrylation (Kibu), existing as a new PTM on nuclear histones. Uniquely, isobutyryl-CoA is derived from valine catabolism and branched chain fatty acid oxidation which is distinct from the metabolism of n-butyryl-CoA. Several histone acetyltransferases were found to possess lysine isobutyryltransferase activity in vitro, especially p300 and HAT1. Transfection and western blot experiments showed that p300 regulated histone isobutyrylation levels in the cell. We resolved the X-ray crystal structures of HAT1 in complex with isobutyryl-CoA that gleaned an atomic level insight into HAT-catalyzed isobutyrylation. RNA-Seq profiling revealed that isobutyrate greatly affected the expression of genes associated with many pivotal biological pathways. Together, our findings identify Kibu as a novel chemical modification mark in histones and suggest its extensive role in regulating epigenetics and cellular physiology.

Citrus juice off-flavor during different processing and storage: Review of odorants, formation pathways, and analytical techniques.

As the most widespread juice produced and consumed globally, citrus juice (mandarin juice, orange juice, and grapefruit juice) is appreciated for its attractive and distinct aroma. While the decrease of characteristic aroma-active compounds and the formation of off-flavor compounds are easy to occur in processing and storage conditions. This review provides a comprehensive literature of recent research and discovery on citrus juice off-flavor, primarily focusing on off-flavor compounds induced during processing and storage (i.e., thermal, storage, light, oxygen, package, fruit maturity, diseases, centrifugal pretreatment, and debittering process), formation pathways (i.e., terpene acid-catalyzed hydration, caramelization reaction, Maillard reaction, Strecker degradation, and other oxidative degradation) of the off-flavor compounds, effective inhibitor pathway to off-flavor (i.e., electrical treatments, high pressure processing, microwave processing, ultrasound processing, and chemical treatment), as well as odor assessment techniques based on molecular sensory science. The possible precursors (terpenes, sulfur-containing amino acids, carbohydrates, carotenoids, vitamins, and phenolic acids) of citrus juice off-flavor are listed and are also proposed. This review intends to unravel the regularities of aroma variations and even off-flavor formation of citrus juice during processing and storage. Future aroma analysis techniques will evolve toward a colorimetric sensor array for odor visualization to obtain a "marker" of off-flavor in citrus juice.

(1) Processing and storage can cause the degradation of nutrients in citrus juice and the formation of off-flavor compounds.(2) Terpene degradation products, sulfur-containing compounds, phenols, acids, and furans are contributed to citrus juice off-flavor.(3) Nonthermal techniques such as electrical treatments, high pressure, microwave, and ultrasound processing is beneficial to preservation of the original aroma and sensory qualities of citrus juice.(4) Potential off-flavor compounds (especially trace level) explored by molecular sensory science also significantly impact the aroma of citrus juice.

Serum markers of pre-eclampsia identified on proteomics.

AIM: Pre-eclampsia (PE) is a disorder of pregnancy associated with maternal and fetal mortality and morbidity. The aim of the present study was to use proteomics to identify biomarkers of, and elucidate the pathogenesis of, PE. METHODS: Serum samples were analyzed using peptide ligand library beads (PLLB) on liquid chromatography-mass spectrometry/mass spectrometry. Retinol-binding protein 4 (RBP4) was used as the target protein for further validation on enzyme-linked immunosorbent assay, immunohistochemistry and real-time polymerase chain reaction. Transwell invasion assay was used to evaluate whether RBP4 affects the invasive ability of trophoblast tumor cells. RESULTS: Twenty upregulated and 17 downregulated proteins were differentially expressed between severe PE patients and healthy pregnant women. Those proteins were further classified according to molecular function and biological process according to the gene ontology terms. RBP4 concentration was significantly lower in women with severe PE than in those with healthy pregnancy. CONCLUSIONS: RBP4 is able to function as biomarker to distinguish severe PE from normal pregnancy. More importantly, these results may shed light on the role of RPB4 in the pathogenesis in PE. Further studies are required to validate these results, and determine the precise role of RBP4 in the pathogenesis of PE.

Anisotropic materials based on carbohydrate polymers: A review of fabrication strategies, properties, and applications.

Anisotropic structures exist in almost all living organisms to endow them with superior properties and physiological functionalities. However, conventional artificial materials possess unordered isotropic structures, resulting in limited functions and applications. The development of anisotropic structures on carbohydrates is reported to have an impact on their properties and applications. In this review, various alignment strategies for carbohydrates (i.e., cellulose, chitin and alginate) from bottom-up to top-down strategies are discussed, including the rapidly developed innovative technologies such as shear-induced orientation through extrusion-based 3D/4D printing, magnetic-assisted alignment, and electric-induced alignment. The unique properties and wide applications of anisotropic carbohydrate materials across different fields, from biomedical, biosensors, smart actuators, soft conductive materials, to thermal management are also summarized. Finally, recommendations on the selection of fabrication strategies are given. The major challenge lies in the construction of long-range hierarchical alignment with high orientation degree and precise control over complicated architectures. With the future development of hierarchical alignment strategies, alignment control techniques, and alignment mechanism elucidation, the potential of anisotropic carbohydrate materials for scalable manufacture and clinical applications will be fully realized.

Characterizing and decoding the key odor-active compounds in fresh, pasteurized, and high pressure processing sea buckthorn (Hippophae rhamnoides L.) juice.

The non-thermal and thermal effects on aroma of sea buckthorn juice have rarely been investigated. In this study, 57 odor compounds were identified in fresh sea buckthorn juice (FSBJ), high pressure processing sea buckthorn juice (HSBJ), and pasteurized sea buckthorn juice (PSBJ), including 29 esters, 8 aldehydes, 1 ketone, 5 alcohols, 5 acids, 6 terpenoids, and 3 others. Ethyl 2-methylbutanoate, ethyl 3-methylbutanoate, ethyl hexanoate, and ethyl 2-hydroxy-3-methylbutanoate with flavor dilution factors ranging from 729 to 59,049 contributed to the fruity odors of FSBJ and HSBJ. Besides, the formation of off-odor compounds including hexanal, nonanal, furfural, 3-methylbutanoic acid, and dimethyl disulfide with odor activity values ≥ 1, imparts fatty, roasted, sweaty, and cooked odor in PSBJ. The variations of vitamin C and reducing sugar are significantly associated with changes in odor-active compounds during pasteurized processing. These findings provide new insights that high pressure processing minimizes the adverse effects of pasteurization.

Structural characteristics and gel properties of pectin from citrus physiological premature fruit drop.

This study is the first to extract and characterize pectin from citrus physiological premature fruit drop. The extraction yield of pectin reached 4.4 % by acid hydrolysis method. The degree of methoxy-esterification (DM) of citrus physiological premature fruit drop pectin (CPDP) was 15.27 %, indicating it was low-methoxylated pectin (LMP). The monosaccharide composition and molar mass test results showed CPDP was a highly branched macromolecular polysaccharide (ß: 0.02, Mw: 2.006 × 105 g/mol) with rich rhamnogalacturonan I domain (50.40 %) and long arabinose and galactose side chain (32.02 %). Based on the fact that CPDP is LMP, Ca2+ was used to induce CPDP to form gels. Textural and rheological tests showed that the gel strength and storage modulus of CPDP were higher than commercial citrus pectin (CP) used in this paper due to the lower DM and rich neutral sugar side chains of CPDP. Scanning electron microscope (SEM) results showed CPDP had stable gel network structure.

Inactivation and reactivation of the mitochondrial α-ketoglutarate dehydrogenase complex.

Reduced brain metabolism is an invariant feature of Alzheimer Disease (AD) that is highly correlated to the decline in brain functions. Decreased activities of key tricarboxylic acid cycle (TCA) cycle enzymes may underlie this abnormality and are highly correlated to the clinical state of the patient. The activity of the α-ketoglutarate dehydrogenase complex (KGDHC), an arguably rate-limiting enzyme of the TCA cycle, declines with AD, but the mechanism of inactivation and whether it can be reversed remains unknown. KGDHC consists of multiple copies of three subunits. KGDHC is sensitive to oxidative stress, which is pervasive in AD brain. The present studies tested the mechanism for the peroxynitrite-induced inactivation and subsequent reactivation of purified and cellular KGDHC. Peroxynitrite inhibited purified KGDHC activity in a dose-dependent manner and reduced subunit immunoreactivity and increased nitrotyrosine immunoreactivity. Nano-LC-MS/MS showed that the inactivation was related to nitration of specific tyrosine residues in the three subunits. GSH diminished the nitrotyrosine immunoreactivity of peroxynitrite-treated KGDHC, restored the activity and the immunoreactivity for KGDHC. Nano-LC-MS/MS showed this was related to de-nitration of specific tyrosine residues, suggesting KGDHC may have a denitrase activity. Treatment of N2a cells with peroxynitrite for 5 min followed by recovery of cells for 24 h reduced KGDHC activity and increased nitrotyrosine immunoreactivity. Increasing cellular GSH in peroxynitrite-treated cells rescued KGDHC activity to the control level. The results suggest that restoring KGDHC activity is possible and may be a useful therapeutic approach in neurodegenerative diseases.

Comparative Evaluation on the Bioaccessibility of Citrus Fruit Carotenoids In Vitro Based on Different Intake Patterns.

The intake pattern has a great impact on the bioaccessibility of carotenoids from citrus fruit. Here, we compared the bioaccessibility of carotenoids from fresh citrus fruit (FC), fresh citrus juice (FCJ), and not-from-concentrate citrus juice (NCJ) and analyzed the influencing factors. The results demonstrated that particle size, viscosity, and some active components of the samples during digestion are potential factors affecting the bioaccessibility of carotenoids. The total carotenoid bioaccessibility of NCJ (31.45 ± 2.58%) was significantly higher than that of FC (8.11 ± 0.43%) and FCJ (12.43 ± 0.49%). This work demonstrates that NCJ is an appropriate intake pattern to improve the bioaccessibility of carotenoids from citrus fruit. The findings also suggest that adjustment of food intake patterns is an effective way to improve the digestion and absorption of nutrients.

Combined high-throughput and fractionation approaches reveal changes of polysaccharides in blueberry skin cell walls during fermentation for wine production.

There have been rare reports about the structure/composition of polymers in blueberry skin and their changes during fermentation for wine production. In this study, the compositional changes occurring in blueberry skin during fermentation were tracked by a combination of cell wall analysis techniques including infra-red spectroscopy, monosaccharide analysis, and comprehensive microarray polymer profiling (CoMPP). The cross-corroborating data revealed that blueberry skin cell wall is particularly rich in xyloglucan. Chemical fractionation analysis indicated that the KOH soluble fraction is a dominant fraction in fermented blueberry skin. Interestingly, the KOH soluble fraction contained abundant epitopes associated with pectin branch chains, indicating tight binding of some enzyme-resistant pectin polymers to hemicellulose. This study provides important implications for the development of effective strategies to extract beneficial substances (such as aromatics, tannins and pigments) from berry tissues during processing.

Protective effect of orange essential oil on the formation of non-alcoholic fatty liver disease caused by high-fat diet.

The purpose of this study was to investigate the protective effect of sniffing orange essential oil (OEO) on the formation of non-alcoholic fatty liver disease (NAFLD) caused by a high-fat diet. The results confirmed that sniffing OEO could reduce obesity caused by a high-fat diet (HFD) by reducing the levels of triglycerides (TGs), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C). In addition, the observation of liver tissue sections showed that sniffing OEO could reduce lipid accumulation in liver cells. Further analysis by western blot analysis showed that OEO treatment made the expression levels of acetyl-CoA carboxylase (ACC) and Cytochrome P450 2E1 (CYP2E1) down-regulated and the expression levels of peroxisome proliferator-activated receptor-α (PPAR-α) and carnitine palmitoyltransferase-1 (CPT-1) up-regulated. These results indicate that the treatment of sniffing OEO could enhance the antioxidant capacity of mice and reduce liver damage caused by a high-fat diet. Furthermore, sniffing OEO could inhibit lipid synthesis and oxidative stress stimulated by a high-fat diet. Overall, OEO treatment had a certain protective effect on NAFLD-related diseases caused by a high-fat diet. Therefore, aromatherapy may be introduced as a treatment of long-term chronic diseases.

The difference of the impacts of surgical approaches on cellular immunity in patients with uterine malignancies: a comparative study of laparoscopy and laparotomy surgery.

OBJECTIVE: To explore the impact of laparoscopy and laparotomy surgery on cellular immunity in patients with malignant uterine tumors. METHODS: Thirty-eight women with uterine malignancies were enrolled in a prospective nonrandomized cohort study. Either laparoscopy or laparotomy was performed according to the patients' choice. The frequency of CD3+, CD4+, CD8+ T cells and natural killer cells derived from peripheral venous blood was evaluated by flow cytometry. RESULTS: (1) Postoperatively, there was a decrease in the number of lymphocyte counts, especially after laparotomy, on the first postoperative day (p < 0.01). (2) Compared with preoperative levels, the frequencies of CD3+ and CD4+ cells and the CD4+/CD8+ ratio were declined both in the laparoscopy and laparotomy groups on postoperative day 1 (p < 0.01). (3) The frequencies of CD3+ and CD4+ cells and the ratio of CD4+ to CD8+ cells were less depressed in the laparoscopy group on the first postoperative day (p < 0.05). (4) The frequency of natural killer cells increased, both in the laparoscopy and laparotomy groups on the first postoperative day (p < 0.01), but there were no significant differences between the two groups (p > 0.05). CONCLUSION: Cellular immunity was temporally depressed in patients with uterine malignancy after surgical treatment, but laparoscopic surgery depressed the immunity less than laparotomy.

A chemical probe of CARM1 alters epigenetic plasticity against breast cancer cell invasion.

CARM1 is a cancer-relevant protein arginine methyltransferase that regulates many aspects of transcription. Its pharmacological inhibition is a promising anti-cancer strategy. Here SKI-73 (6a in this work) is presented as a CARM1 chemical probe with pro-drug properties. SKI-73 (6a) can rapidly penetrate cell membranes and then be processed into active inhibitors, which are retained intracellularly with 10-fold enrichment for several days. These compounds were characterized for their potency, selectivity, modes of action, and on-target engagement. SKI-73 (6a) recapitulates the effect of CARM1 knockout against breast cancer cell invasion. Single-cell RNA-seq analysis revealed that the SKI-73(6a)-associated reduction of invasiveness acts by altering epigenetic plasticity and suppressing the invasion-prone subpopulation. Interestingly, SKI-73 (6a) and CARM1 knockout alter the epigenetic plasticity with remarkable difference, suggesting distinct modes of action for small-molecule and genetic perturbations. We therefore discovered a CARM1-addiction mechanism of cancer metastasis and developed a chemical probe to target this process.

Drugs that are small molecules have the potential to block the individual proteins that drive the spread of cancer, but their design is a challenge. This is because they need to get inside the cell and find their target without binding to other proteins on the way. However, small molecule drugs often have an electric charge, which makes it hard for them to cross the cell membrane. Additionally, most proteins are not completely unique, making it harder for the drugs to find the correct target. CARM1 is a protein that plays a role in the spread of breast cancer cells, and scientists are currently looking for a small molecule that will inhibit its action. The group of enzymes that CARM1 belongs to act by taking a small chemical group, called a methyl group, from a molecule called SAM, and transferring it to proteins that switch genes on and off. In the case of CARM1, this changes cell behavior by turning on genes involved in cell movement. Genetically modifying cells so they will not produce any CARM1 stops the spread of breast cancer cells, but developing a drug with the same effects has proved difficult. Existing drugs that can inhibit CARM1 in a test tube struggle to get inside cells and to distinguish between CARM1 and its related enzymes. Now, Cai et al. have modified and tested a CARM1 inhibitor to address these problems, and find out how these small molecules work. At its core, the inhibitor has a structure very similar to a SAM molecule, so it can fit into the SAM binding pocket of CARM1 and its related enzymes. To stop the inhibitor from binding to other proteins, Cai et al. made small changes to its structure until it only interacted with CARM1.Then, to get the inhibitor inside breast cancer cells, Cai et al. cloaked its charged area with a chemical shield, allowing it to cross the cell membrane. Inside the cell, the chemical shield broke away, allowing the inhibitor to attach to CARM1. Analysis of cells showed that this inhibition only affected the cancer cells most likely to spread. Blocking CARM1 switched off genes involved in cell movement and stopped cancer cells from travelling through 3D gels. This work is a step towards making a drug that can block CARM1 in cancer cells, but there is still further work to be done. The next stages will be to test whether the new inhibitor works in other types of cancer cells, in living animals, and in human patient samples.

RBP4 regulates trophoblastic cell proliferation and invasion via the PI3K/AKT signaling pathway.

Insufficient trophoblast invasion is associated with preeclampsia (PE) development. Retinol-binding protein 4 (RBP4) is important for regulating cell differentiation, migration and invasion. The aim of the present study was to determine RBP4 expression and function in the human placenta and to examine the underlying mechanisms. In the present study, RBP4 expression was determined in serum samples from 35 pregnant women with PE and 30 healthy pregnant women using enzyme-linked immunosorbent assays. Cell proliferation was assessed by Cell Counting Kit-8 assays, and cell invasion was examined with transwell assays. RBP4 concentrations were significantly lower in the PE group when compared with the control group. RBP4 overexpression enhanced HTR8/SVneo cell proliferation and invasion, and the levels of phosphorylated (p-) phosphoinositide 3-kinase (PI3K) and p-protein kinase B (AKT) in HTR8/SVneo cells. RBP4 knockdown significantly inhibited HTR8/SVneo cell proliferation and invasion, and repressed the expression of matrix metalloproteinases. In addition, RBP4 knockdown significantly reduced the levels of p-PI3K and p-AKT in HTR8/SVneo cells. Taken together, the results of the present study demonstrated that RBP4 overexpression increased HTR8/SVneo cell proliferation and invasion by suppressing PI3K/AKT signaling and RBP4 knockdown induced the opposite effects.

Proteomics analysis for finding serum markers of ovarian cancer.

A combination of peptide ligand library beads (PLLB) and 1D gel liquid chromatography-mass spectrometry/mass spectrometry (1DGel-LC-MS/MS) was employed to analyze serum samples from patients with ovarian cancer and from healthy controls. Proteomic analysis identified 1200 serum proteins, among which 57 proteins were upregulated and 10 were downregulated in the sera from cancer patients. Retinol binding protein 4 (RBP4) is highly upregulated in the ovarian cancer serum samples. ELISA was employed to measure plasma concentrations of RBP4 in 80 samples from ovarian cancer patients, healthy individuals, myoma patients, and patients with benign ovarian tumor, respectively. The plasma concentrations of RBP4 ranging from 76.91 to 120.08 ng/mL with the mean value 89.13 ± 1.67 ng/mL in ovarian cancer patients are significantly higher than those in healthy individuals (10.85 ± 2.38 ng/mL). Results were further confirmed with immunohistochemistry, demonstrating that RBP4 expression levels in normal ovarian tissue were lower than those in ovarian cancer tissues. Our results suggested that RBP4 is a potential biomarker for diagnostic of screening ovarian cancer.

Transthyretin as a novel candidate biomarker for preeclampsia.

Preeclampsia (PE) is considered to be a potentially fatal complication during pregnancy. However, no effective laboratory assessment has been developed to enable early diagnosis and monitoring of PE. The present study aimed to identify differentially expressed transthyretin (TTR) during severe PE and evaluate TTR as a possible biomarker of this disease. TTR levels were determined in the different gestational weeks of normal pregnancy (before 20 weeks, n=41; after 20 weeks, n=39) using enzyme-linked immunosorbent assay (ELISA). TTR concentrations in pregnant females with severe PE (n=43) were compared with those in healthy matched control subjects (n=37) using western blot analysis and ELISA. The median TTR concentration during severe PE in each month of gestation was significantly lower than the concentrations recorded during normal pregnancy. TTR levels in females with severe PE were significantly downregulated compared with the control subjects (P<0.001; area under the curve, 0.834-0.967). Thus, TTR may be used as a potential biomarker of PE.

Quantitative analysis of differentially expressed saliva proteins in human immunodeficiency virus type 1 (HIV-1) infected individuals.

In the present study, we have established a new methodology to analyze saliva proteins from HIV-1-seropositive patients before highly active antiretroviral therapy (HAART) and seronegative controls. A total of 593 and 601 proteins were identified in the pooled saliva samples from 5 HIV-1 subjects and 5 controls, respectively. Forty-one proteins were found to be differentially expressed. Bioinformatic analysis of differentially expressed salivary proteins showed an increase of antimicrobial proteins and decrease of protease inhibitors upon HIV-1 infection. To validate some of these differentially expressed proteins, a high-throughput quantitation method was established to determine concentrations of 10 salivary proteins in 40 individual saliva samples from 20 seropositive patients before HAART and 20 seronegative subjects. This method was based on limited protein separation within the zone of the stacking gel of the 1D SDS PAGE and using isotope-coded synthetic peptides as internal standards. The results demonstrated that a combination of protein profiling and targeted quantitation is an efficient method to identify and validate differentially expressed salivary proteins. Expression levels of members of the calcium-binding S100 protein family and deleted in malignant brain tumors 1 protein (DMBT1) were up-regulated while that of Mucin 5B was down-regulated in HIV-1 seropositive saliva samples, which may provide new perspectives for monitoring HIV-infection and understanding the mechanism of HIV-1 infectivity.