Universal DNA methylation age across mammalian tissues.

Aging, often considered a result of random cellular damage, can be accurately estimated using DNA methylation profiles, the foundation of pan-tissue epigenetic clocks. Here, we demonstrate the development of universal pan-mammalian clocks, using 11,754 methylation arrays from our Mammalian Methylation Consortium, which encompass 59 tissue types across 185 mammalian species. These predictive models estimate mammalian tissue age with high accuracy (r > 0.96). Age deviations correlate with human mortality risk, mouse somatotropic axis mutations and caloric restriction. We identified specific cytosines with methylation levels that change with age across numerous species. These sites, highly enriched in polycomb repressive complex 2-binding locations, are near genes implicated in mammalian development, cancer, obesity and longevity. Our findings offer new evidence suggesting that aging is evolutionarily conserved and intertwined with developmental processes across all mammals.

Pre-hospital stroke screening and notification of patients with reperfusion-eligible acute ischaemic stroke using modified Face Arm Speech Time test.

OBJECTIVES: To investigate the effects of pre-hospital stroke screening and notification on reperfusion therapy for patients with acute ischaemic stroke. METHODS: Pre-hospital stroke screening criteria were established based on a modified version of the Face Arm Speech Time (FAST) test. Screening was performed during ambulance transport by emergency medical service (EMS) personnel who completed a 2-hour training session on stroke screening. Temporal trends affecting acute ischaemic stroke investigation and intervention were compared before and after implementation of the pre-hospital screening. RESULTS: From July 2018 to October 2019, 298 patients with suspected stroke were screened by EMS personnel during ambulance transport prior to hospital arrival. Of these 298 patients, 213 fulfilled the screening criteria, 166 were diagnosed with acute stroke, and 32 received reperfusion therapy. The onset-to-door time was shortened by more than 1.5 hours (100.6 min vs 197.6 min, P<0.001). The door-to-computed tomography time (25.6 min vs 32.0 min, P=0.021), door-to-needle time (49.2 min vs 70.1 min, P=0.003), and door-to-groin puncture time for intra-arterial mechanical thrombectomy (126.7 min vs 168.6 min, P=0.04) were significantly shortened after implementation of the pre-hospital screening and notification, compared with historical control data of patients admitted from January 2018 to June 2018, before implementation of the screening system. CONCLUSION: Implementation of pre-hospital stroke screening using criteria based on a modified version of the FAST test, together with pre-arrival notification, significantly shortened the door-to-reperfusion therapy time for patients with ischaemic stroke. Pre-hospital stroke screening during ambulance transport by EMS personnel who complete a 2-hour focused training session is effective for identifying reperfusion-eligible patients with stroke.

Biodiesel production from wet municipal sludge: evaluation of in situ transesterification using xylene as a cosolvent.

This study proposes a method to produce biodiesel from wet wastewater sludge. Xylene was used as an alternative cosolvent to hexane for transesterification in order to enhance the biodiesel yield from wet wastewater sludge. The water present in the sludge could be separated during transesterification by employing xylene, which has a higher boiling point than water. Xylene enhanced the biodiesel yield up to 8.12%, which was 2.5 times higher than hexane. It was comparable to the maximum biodiesel yield of 9.68% obtained from dried sludge. Xylene could reduce either the reaction time or methanol consumption, when compared to hexane for a similar yield. The fatty acid methyl esters (FAMEs) content of the biodiesel increased approximately two fold by changing the cosolvent from hexane to xylene. The transesterification method using xylene as a cosolvent can be applied effectively and economically for biodiesel recovery from wet wastewater sludge without drying process.

The small molecule indirubin-3'-oxime activates Wnt/ß-catenin signaling and inhibits adipocyte differentiation and obesity.

OBJECTIVES: Activation of the Wnt/ß-catenin signaling pathway inhibits adipogenesis by maintaining preadipocytes in an undifferentiated state. We investigated the effect of indirubin-3'-oxime (I3O), which was screened as an activator of the Wnt/ß-catenin signaling, on inhibiting the preadipocyte differentiation in vitro and in vivo. METHODS: 3T3L1 preadipocytes were differentiated with 0, 4 or 20 µM of I3O. The I3O effect on adipocyte differentiation was observed by Oil-red-O staining. Activation of Wnt/ß-catenin signaling in I3O-treated 3T3L1 cells was shown using immunocytochemical and immunoblotting analyses for ß-catenin. The regulation of adipogenic markers was analyzed via real-time reverse transcription-PCR (RT-PCR) and immunoblotting analyses. For the in vivo study, mice were divided into five different dietary groups: chow diet, high-fat diet (HFD), HFD supplemented with I3O at 5, 25 and 100 mg kg(-1). After 8 weeks, adipose and liver tissues were excised from the mice and subject to morphometry, real-time RT-PCR, immunoblotting and histological or immunohistochemical analyses. In addition, adipokine and insulin concentrations in serum of the mice were accessed by enzyme-linked immunosorbent assay. RESULTS: Using a cell-based approach to screen a library of pharmacologically active small molecules, we identified I3O as a Wnt/ß-catenin pathway activator. I3O inhibited the differentiation of 3T3-L1 cells into mature adipocytes and decreased the expression of adipocyte markers, CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor γ, at both mRNA and protein levels. In vivo, I3O inhibited the development of obesity in HFD-fed mice by attenuating HFD-induced body weight gain and visceral fat accumulation without showing any significant toxicity. Factors associated with metabolic disorders such as hyperlipidemia and hyperglycemia were also improved by treatment of I3O. CONCLUSION: Activation of the Wnt/ß-catenin signaling pathway can be used as a therapeutic strategy for the treatment of obesity and metabolic syndrome and implicates I3O as a candidate anti-obesity agent.

Weight control attempts in underweight korean adults: Korea national health and nutrition examination survey, 2007-2010.

BACKGROUND: Underweight refers to the weight range in which health risk can increase, since the weight is lower than a healthy weight. Negative attitudes towards obesity and socio-cultural preference for thinness could induce even underweight persons to attempt weight control. This study was conducted to investigate factors related to weight control attempts in underweight Korean adults. METHODS: This was a cross-sectional study on 690 underweight adults aged 25 to 69 years using data from the Korea National Health and Nutrition Examination Survey, 2007-2010. Body image perception, weight control attempts during the past one year, various health behaviors, history of chronic diseases, and socioeconomic status were surveyed. RESULTS: Underweight women had a higher rate of weight control attempts than underweight men (25.4% vs. 8.1%, P < 0.001). Among underweight men, subjects with the highest physical activity level (odds ratio [OR], 7.75), subjects with physician-diagnosed history of chronic diseases (OR, 7.70), and subjects with non-manual jobs or other jobs (OR, 6.22; 12.39 with reference to manual workers) had a higher likelihood of weight control attempts. Among underweight women, subjects who did not perceive themselves as thin (OR, 4.71), subjects with the highest household income level (OR, 2.61), and unmarried subjects (OR, 2.08) had a higher likelihood of weight control attempts. CONCLUSION: This study shows that numbers of underweight Korean adults have tried to control weight, especially women. Seeing that there are gender differences in factors related to weight control attempts in underweight adults, gender should be considered in helping underweight adults to maintain a healthy weight.

Fusarium oxysporum Causing Wilt and Stem Rot in Chrysanthemum × morifolium in Korea.

The hardy garden mum Chrysanthemum, or "mum" (Chrysanthemum × morifolium Ram.), is a popular flowering herbaceous perennial that is commonly grown for fall sales. In October 2011, suspected wilt disease was observed in potted hardy garden mums (cv. Guiin) grown in greenhouses in Jinju, South Korea. Symptoms included unilateral chlorosis of leaves at the stem apex. Wilted leaves occurred initially on the most severely affected side of the plant, but as the disease progressed, the entire plant wilted and died. Black necrosis and vascular discoloration at the base of stems always developed. Five fungal isolates, successfully isolated from 10 infected stems on potato dextrose agar (PDA), yielded rapidly growing floccose to felt-like colonies, initially white, but turning peach colored. The microconidia were ellipsoid, ovoid, and cylindrical, and measured 3 to 12 × 1 to 3 µm. The macroconidia were falcate, lunate, and measured 8 to 30 × 2 to 4 µm, and had 1 to 5 septa. Pathogenicity was studied in inoculated, potted plants in a greenhouse. A representative isolate of the fungus was grown on PDA at 20°C for about 10 days before inoculation. To obtain conidial suspensions, 10 ml of sterile distilled water (SDW) was added to the culture plates and scraped with a paintbrush to dislodge conidia. The suspension from the culture plates was filtered through cheesecloth and diluted to 2 × 104 micro- and macroconidia/ml with SDW. Nine 3-month-old hardy garden mums were planted in 20-cm-diameter plastic pots containing fine sand. After 10 days, the roots were cut to a depth of 5 cm on two sides of each plant at a distance of 2 cm from the stems. Then, 10 ml of conidial suspension were poured into each pot above the cuts roots, followed by 20 ml 12 days later. Three mums treated with SDW served as controls. Plants were fertilized twice weekly with 100 ml/pot of a nutrient solution (1) that lowered the soil pH and enhanced wilt development. Thirty days after inoculation, all of the artificially inoculated plants had wilted. The control mums remained healthy. The fungus was successfully reisolated to complete Koch's postulates. On the basis of the morphological characters, the fungus was identified as Fusarium oxysporum (3). To identify the isolated fungus, the complete internal transcribe spacer (ITS) rDNA and translation elongation factor 1-alpha (EF1-α) sequences were amplified using the primers ITS1/ITS4 and EF1/EF2, respectively, and sequenced. The resulting sequences were deposited in GenBank (Accession Nos. KC491873 and KC491875). A BLAST search of ITS rDNA (544 bp) and EF1-α (712 bp) sequences against a database of fungal isolates found 100% and 99% similarity to those of F. oxysporum, respectively. Fusarium wilt caused by F. oxysporim on C. morifolium has been previously recorded in North America and India but, to our knowledge, this is the first report of F. oxysporum causing wilt in hardy garden mum in Korea (2). F. oxysporum isolates causing wilts are specific to certain hosts and even to host varieties or cultivars. Further work is required to determine to which forma specialis and race the pathogen belongs. References: (1) A. W. Engelhard and S. S. Woltz. Proc. Fla. State Hort. Soc. 84:351, 1971. (2) H. C. Huang et al. Plant Pathol. Bull. 1:57, 1992. (3) C. V. Subramanian. CMI Descriptions of Pathogenic Fungi and Bacteria. No. 217, 1970.

Marital Status and Health Behavior in Middle-aged Korean Adults.

BACKGROUND: Marital status is as an important sociodemographic variable for health studies. We assessed the association between marital status and health behavior in middle-aged Korean adults. METHODS: This is a cross-sectional study of 2,522 Korean middle-aged adults (1,049 men, 1,473 women) from the 2010 Korean National Health and Nutrition Examination Survey. The subjects were classified as living with a partner or living without a partner (never married, separated, widowed, and divorced). We assessed the relationship between marital status and five health behaviors (smoking, high-risk alcohol intake, regular exercise, regular breakfast consumption, and undergoing periodic health screening). RESULTS: Age, income level, educational level, and occupational classification were all significantly associated with marital status. The risk of undergoing health screening (odds ratio [OR], 0.53; 95% confidence interval [CI], 0.32 to 0.90) and having regular breakfast (OR, 0.50; 95% CI, 0.27 to 0.92) were significantly lower in men living without a partner than with a partner. Women living without a partner had a higher smoking risk (OR, 2.27; 95% CI, 1.09 to 4.73) and a higher risk of high-risk alcohol consumption (OR, 5.33; 95% CI, 1.65 to 17.24) than their counterparts. CONCLUSION: Korean middle-aged adults living with partners are more likely to have healthier behavior than living without a partner. The association between marital status and health behaviors differed by sex.

An Outbreak of Onion Center Rot Caused by Pantoea ananatis in Korea.

Symptoms typical of center rot of onion (Allium cepa L.) were observed in farmers' fields in spring of 2009 and 2010 in Hamyang, South Korea, at an incidence of 30 to 50% (five affected fields representing approximately 6 ha). The symptoms were identical to those reported from infected onions in Georgia in 1997 (1). Harvested bulbs of symptomatic plants had reddish, collapsed scales near the neck. Symptomatic bulb tissues were surface-sterilized by immersing sections of the tissue for 30 s in 1% NaOCl, then rinsing the sections with sterilized, distilled water. Tissues were then macerated in 1 ml of sterilized, distilled water in a 1.5 ml Eppendorf tube using a sterile scalpel. The macerated tissue was left to soak for 10 min, after which a 5 µl suspension from each section was streaked onto plates of nutrient agar (NA). Gram-negative, rod-shaped, yellow bacteria were consistently recovered on NA. Three bacterial isolates recovered were each facultative anaerobes and induced a hypersensitive reaction on tobacco leaves. The biochemical test, API 20E (Biomérieux, Marcy l'Etoile, France), was also used for identification. All three strains tested positive for ß-D-galactosidase, utilization of citrate, and production of acetoin, catalase, and indole. All three strains tested negative for ornithine decarboxylase, lysine decarboxylase, urease, and oxidase. All produced acid from arabinose, glucose, mannitol, and sorbitol; while none produced acid from melibiose, inositol, and rhamnose. These characteristics are consistent with those of P. ananatis (1,2). Five bulbs were each surface-disinfested with 70% ethanol, dried, and injected with 50 µl of the appropriate bacterial suspension containing ~108 CFU/ml, using a syringe. The bulbs were placed in plastic boxes with four sheets of wet paper towel to maintain the relative humidity at 100%, and incubated at 25°C for 2 weeks. Three onion bulbs treated similarly but injected with sterilized, distilled water served as replicates of the control treatment. After 1 week of incubation, inoculated onion bulbs developed a brown discoloration and decay of the internal, fleshy scales matching those observed in the original farmers' fields. The control onion bulbs remained asymptomatic. Bacteria reisolated from lesions in the fleshy bulb scales of the inoculated bulbs had the same characteristics as the original isolates inoculated, proving Koch's postulates. Bacteria were not reisolated from any of the control bulbs. To confirm identity of the isolated bacteria, 16S rRNA and recA genes were amplified with primers 27mF: 5'-AGAGTTTGATCMTGGCTCAG-3' and 1492mR: 5'-GGYTACCTTGTTACGACTT-3', and PAGRECA21: 5'-GGTGAAGACCGCTCAATGGA-3' and PAGRECA621: 5'-CACCGATACGGCGGATATCA-3', respectively (3). Amplification of the 16S rRNA gene generated a 1,506-bp consensus sequence (GenBank Accession No. JQ762264), and amplification of the recA gene generated a consensus sequence of 601 bp (JQ762265). The 16S rRNA and recA gene sequences shared 99% nucleotide identity with those of a P. ananatis strain in GenBank (DQ195523 and AY219004, respectively). Based on symptoms, biochemical tests, and molecular analyses, the bacterium responsible for the onion symptoms in Korea was identified as P. ananatis. To our knowledge, this is the first report of center rot of onion caused by P. ananatis in Korea. References: (1) R. D. Gitaitis and J. D. Gay. Plant Dis. 81:1096, 1997. (2) H. G. Truper and L. de Clari. Int. J. Syst. Bacteriol. 47:908, 1997. (3) A. Wensing et al. Appl. Environ. Microbiol. 76:6248, 2010.

First Report of Kalanchoe Leaf Scorch Caused by Stemphylium xanthosomatis in Korea.

Kalanchoe (Kalanchoe blossfeldiana Poelln.) is widely cultivated in Korea as an ornamental houseplant and succulent garden plant because of its ease of propagation, low water requirements, and wide variety of flower colors. In August 2010, suspected nursery-stage kalanchoe leaf scorch was found at a grower's greenhouses located in Gimhae, Korea. In some greenhouses, 20 to 30%, and occasionally as much as 50%, of the plants were affected. Symptoms on kalanchoe include browning of the leaf margins and yellowing or darkening of tissues between the main leaf veins. As the disease progresses, affected leaves dried up, turned brown, and became brittle. A velvety, blackish olive mold formed on the surface of the dead tissue, followed by plant defoliation. Fresh leaf specimens were collected from infected plants and the causal pathogen was purified with a single-spore isolation technique and transferred onto potato dextrose agar (PDA). Colonies on PDA developed a gray or grayish brown, hairy, velvety mycelium that was mostly immersed and also formed conidia. Conidia were pale to mid brown, oblong, smooth or verruculose, with three to five transverse and one to two longitudinal septa in two to three transverse divisions, and 32 to 55 × 11 to 18 µm. Conidiophores were pale to mid brown, solitary or in fascicles, unbranched or occasionally branched, straight or flexuous, more or less cylindrical but enlarged slightly at one to three apical percurrent proliferations, septate, and 80 to 300 × 2 to 5 µm. A representative isolate of the pathogen was inoculated on kalanchoe leaves for pathogenicity testing. Cultures grown on PDA were flooded with sterile distilled water and after rubbing with an artist's paintbrush with hair bristles, the resulting suspensions were filtered through sterile cheesecloth. Conidial suspensions were adjusted to 2.5 × 104 conidia/ml with sterile distilled water. The leaves of five 1-month-old potted plants were wounded by applying pressure with forceps having serrated teeth, bruising the tissue. Wounded plants were sprayed with a conidial suspension until runoff. Five plants sprayed with sterile distilled water served as controls. The plants were maintained for 48 h at 25°C in a humidity chamber with 100% relative humidity and were then moved to a greenhouse. Symptoms similar to those observed in the farmer's greenhouse developed on wounded leaves within 9 days. The causal pathogen was reisolated from the lesions to prove Koch's postulates. To confirm the identity of the fungus, the complete internal transcribed spacer (ITS) rDNA and glyceraldehyde 3-phosphate dehydrogenase (gpd) gene were amplified and sequenced (1). Amplification of the ITS region generated a 579-bp sequence (GenBank Accession No. HQ840713) and gpd was 558 bp (GenBank Accession No. JF776462). The ITS and gpd sequences were 100% similar to the sequences of Stemphylium xanthosomatis (GenBank Accession Nos. AF442804 and AF443903, respectively). On the basis of symptoms, mycological characteristics, pathogenicity, and molecular data, this fungus was identified as S. xanthosomatis. The type culture of the fungus is stored at the Korean Agricultural Culture Collection (KACC 45812), National Academy of Agricultural Science, Korea. To our knowledge, this is the first report of leaf scorch caused by S. xanthosomatis on kalanchoe in Korea. Reference: (1) M. P. S. Câmara et al. Mycologia 94:660, 2002.

First Report of Flyspeck Caused by Zygophiala wisconsinensis on Sweet Persimmon Fruit in Korea.

Sweet persimmon (Diospyros kaki L.), a fruit tree in the Ebenaceae, is cultivated widely in Korea and Japan, the leading producers worldwide (2). Sweet persimmon fruit with flyspeck symptoms were collected from orchards in the Jinju area of Korea in November 2010. The fruit had fungal clusters of black, round to ovoid, sclerotium-like fungal bodies with no visible evidence of a mycelial mat. Orchard inspections revealed that disease incidence ranged from 10 to 20% in the surveyed area (approximately 10 ha) in 2010. Flyspeck symptoms were observed on immature and mature fruit. Sweet persimmon fruit peels with flyspeck symptoms were removed, dried, and individual speck lesions transferred to potato dextrose agar (PDA) and cultured at 22°C in the dark. Fungal isolates were obtained from flyspeck colonies on 10 sweet persimmon fruit harvested from each of three orchards. Fungal isolates that grew from the lesions were identified based on a previous description (1). To confirm identity of the causal fungus, the complete internal transcribed spacer (ITS) rDNA sequence of a representative isolate was amplified and sequenced using primers ITS1 and ITS4 (4). The resulting 552-bp sequence was deposited in GenBank (Accession No. HQ698923). Comparison with ITS rDNA sequences showed 100% similarity with a sequence of Zygophiala wisconsinensis Batzer & Crous (GenBank Accession No. AY598855), which infects apple. To fulfill Koch's postulates, mature, intact sweet persimmon fruit were surface sterilized with 70% ethanol and dried. Three fungal isolates from this study were grown on PDA for 1 month. A colonized agar disc (5 mm in diameter) of each isolate was cut from the advancing margin of a colony with a sterilized cork borer, transferred to a 1.5-ml Eppendorf tube, and ground into a suspension of mycelial fragments and conidia in a blender with 1 ml of sterile, distilled water. The inoculum of each isolate was applied by swabbing a sweet persimmon fruit with the suspension. Three sweet persimmon fruit were inoculated per isolate. Three fruit were inoculated similarly with sterile, distilled water as the control treatment. After 1 month of incubation in a moist chamber at 22°C, the same fungal fruiting symptoms were reproduced as observed in the orchards, and the fungus was reisolated from these symptoms, but not from the control fruit, which were asymptomatic. On the basis of morphological characteristics of the fungal colonies, ITS sequence, and pathogenicity to persimmon fruit, the fungus was identified as Z. wisconsinensis (1). Flyspeck is readily isolated from sweet persimmon fruit in Korea and other sweet persimmon growing regions (3). The exposure of fruit to unusual weather conditions in Korea in recent years, including drought, and low-temperature and low-light situations in late spring, which are favorable for flyspeck, might be associated with an increase in occurrence of flyspeck on sweet persimmon fruit in Korea. To our knowledge, this is the first report of Z. wisconsinensis causing flyspeck on sweet persimmon in Korea. References: (1) J. C. Batzer et al. Mycologia 100:246, 2008. (2) FAOSTAT Database. Retrieved from http://faostat.fao.org/ , 2008. (3) H. Nasu and H. Kunoh. Plant Dis. 71:361, 1987. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, Inc., New York, 1990.

Nitrification inhibition by silver nanoparticles.

Nitrification inhibition by silver nanoparticles (nanosilver) was evaluated by extant respirometry using enriched nitrifying bacteria isolated from wastewater treatment plants. Silver nanoparticles were more toxic than silver ions or silver chloride colloids, all of which did not disrupt cell membrane integrity at 1 mg/L Ag. The toxicity of silver nanoparticles was reduced in the presence of various anions, especially sulfide. The results suggest that silver nanoparticles have the same behaviour of surface complexation as silver ions, and inhibition by nanosilver in wastewater treatment may be removed by reaction of silver nanoparticles with soluble sulfide species.

A context-aware fitness guide system for exercise optimization in U-health.

In this paper, exercise management systems have been introduced, which are generally used to optimize exercise. They create a proper exercise program via an exercise prescription based on the personal physical status of the user. However, exercise programs, generally created at intervals of two weeks to three months, are static and cannot reflect the user's exercise goals, which change dynamically. This paper proposes context-aware exercise architecture (CAEA), which provides an exercise program via a dynamic exercise prescription based on awareness of the user's status. We use sensors of a U-health environment and implement CAEA as an intelligent fitness guide (IFG) system. The IFG system selectively receives necessary parameters as input according to the user's exercise goals. Based on the changes in the user's exercise type, frequency, and intensity, the system creates an exercise program via an exercise optimization algorithm. In this paper, to show the exercise efficiency using the IFG system, we compared a noncontrol group to a control group. An eight-week study was performed comparing the changes of body weight in the two study groups. The study showed that the control group using the IFG system approached the desired body weight 2.57% more closely than the noncontrol group. Since IFG provides a real-time exercise program for users via an exercise optimization algorithm, it enables the user to perform effective and stable exercise according to the user's physical status.

Development of MCTS technique for 3-PM liquid scintillation counting.

We have developed a multi-channel time scaling method that is suitable for activity measurement of beta emitting nuclides by means of 3-PM Liquid Scintillation Counting, using non-extending dead times and linear amplifiers. Since it enables to obtain the accidental coincidences directly, the true values for both double and triple coincidences are determined by simply taking into account the correction due to dead times. The advantages of the method are demonstrated by studying the activity of 204Tl and 14C. The measured results were compared with those derived by using the mathematical formulae.

Cumulus cells apoptosis as an indicator to predict the quality of oocytes and the outcome of IVF-ET.

PURPOSE: Our purpose was to establish an evaluation system for oocyte quality based on the incidence of cumulus cells apoptosis and to examine the effect of coculture, using autologous cumulus cells, on the outcome of IVF-ET according to proliferative activities of helper cells and the incidence of cumulus cells apoptosis. METHODS: Cumulus cell masses were collected from 91 mature oocytes among 330 oocytes retrieved from a total of 34 IVF-ET cycles with tubal infertility and unexplained infertility. The incidence of apoptosis in cumulus cells was assessed by apoptosis detection kit fluorescein. On ovum pick up, 2nd day embryos were cocultured with autologous cumulus cells. Prior to coculture, in vitro proliferative activity of cumulus cells was evaluated. RESULTS: Cumulus cells from patient groups over 40 years old had a significantly increased apoptosis incidence, a lower fertilization rate, and the decreased number of oocytes retrieved compared to the other age groups (P < .05). The incidence of cumulus cells apoptosis was significantly lower when the number of oocytes retrieved was 5 or less (P < .05). Cumulus cells from fertilized oocytes (0.43 +/- 0.07%) and those from patients who became pregnant (0.44 +/- 0.11%) following IVF-ET showed a significantly lower incidence of apoptosis compared to those of unfertilized oocytes (1.80 +/- 0.35%; P < .001) and the nonpregnant group (0.81 +/- 0.10%; P < .05). Embryo quality also had a negative correlation with the incidence of cumulus cells apoptosis. Coculture of fertilized oocytes with cumulus cells with high proliferative activity resulted in improved rates of implantation and pregnancy compared to that with poor active cumulus cells. No significant difference was found between the in vitro proliferative activity of cumulus cells and the incidence of cumulus cells apoptosis (P < .063). CONCLUSIONS: The age of women might influence the incidence of apoptosis in cumulus cells, and the increased incidence of apoptosis is associated with the number of oocytes retrieved, the fertilization rate, and the pregnancy outcome following IVF-ET. These results suggest that the incidence of cumulus cells apoptosis can be used in predicting oocyte quality, outcome of IVF-ET, and age-related decline in fertility.

Influence of buffer composition and sample pretreatment on efficiency separation for monitoring neuropeptides in plasma using capillary electrophoresis.

More efficient and faster separation conditions for qualitative as well as quantitative analysis of neuropeptides in human plasma using capillary zone electrophoresis (CZE) have been developed. The analysis method for neuropeptides has been improved specifically to study thyroid hormone related neuropetides for the regulation of thyroid disease. In this study, we investigated the pretreatment methods, composition of the running buffer and rinsing procedures between runs in order to obtain more sensitive and faster separation of trace neuropeptides in plasma by CZE. The tested neuropeptides were somatostatin (SOMA), vasopressin (VP), neurotensin (NT), and thyrotropin-releasing hormone (TRH). Plasma samples were pretreated by deproteinization and solid-phase extraction method. The fraction of neuropeptides was reconstituted in 40% acetonitrile followed by ultrafiltration, and then analyzed by CZE. Resolution and sensitivity was improved using the separation buffer composition with 100 mM Tris-phosphate buffer (pH 2.0) while the sensitivity was further improved via a stacking method using the sample buffer of 40% acetonitrile. These sample pretreatment methods and buffer condition permit quantitative analysis on tested neuropeptides at the 20 ng/mL level. The rinsing procedures between runs using 90% ethanol dramatically shortened the rinsing time to 30 min.

Capillary electrophoresis of high-molecular chitosan: the natural carbohydrate biopolymer.

Due to its high resolving power and diverse application range, capillary electrophoresis (CE) has been successfully applied to the analysis of carbohydrates. In this paper, a method for the determination of high-molecular chitosan (Mr 200,000) using CE is presented. We studied the optimal condition of buffer pH and type, and column type for determination of chitosan. Optimal CE performance was found when employing 100 mM triethylamine (TEA)-phosphate buffer, pH 2.0 and untreated fused-silica capillary (50 microm x 27 cm) for the chitosan analysis. Under optimum conditions, excellent linear responses were obtained in the concentration range of 1.25-20 microM, with a linear correlation coefficient of 0.9983. The standard deviations of the migration time and peak area were found to be 2.5 and 6.4%, respectively. This method could be readily applied to chitosan determination in real biological samples and commercial products.

Relationships between concentrations of tumor necrosis factor-alpha and nitric oxide in follicular fluid and oocyte quality.

PURPOSE: Our objective was to explain a relationship between concentrations of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in follicular fluid, oocyte quality, and outcomes of in vitro fertilization--embryo transfer (IVF-ET). METHODS: The concentrations of TNF-alpha and NO were measured in 115 follicular fluid samples collected from 43 patients undergoing IVF-ET program, due to tubal obstruction, some with endometriosis (8 patients) or hydrosalpinx (5 patients). A correlation of these factors concentrations and the oocyte quality, the oocyte maturity, and infertility-associated diseases was analyzed. RESULTS: No correlation was found between concentrations of NO and TNF-alpha in follicular fluid. NO concentrations in follicular fluids were significantly higher in patients with endometriosis (P < 0.001) or hydrosalpinx (P < 0.01) compared to the patients with just tubal obstruction. Follicular NO concentration differences according to oocyte maturity and oocyte quality were not found. In contrast, TNF-alpha concentrations in follicular fluids were significantly higher in poor quality oocytes (P < 0.05) but were not associated with infertility-associated diseases, such as hydrosalphinx or endometriosis,and the oocyte maturity. No significant differences in follicular levels of NO and TNF-alpha as well as IVF-ET parameters of pregnant and nonpregnant groups were revealed. CONCLUSIONS: There is no significant correlation between the concentrations of NO and TNF-alpha in follicular fluid. NO levels in follicular fluid are altered in infertility-associated diseases. However, TNF-alpha levels but not NO levels influence oocyte quality. These results suggest that the production of NO and TNF-alpha in follicular fluid may be regulated via different pathways and can be tempered with infertility-associated diseases, thereby influencing oocyte quality locally.

Diverse effects of sphingosine on calcium mobilization and influx in differentiated HL-60 cells.

Sphingosine induces a biphasic increase in cytosolic-free Ca(2+)([Ca(2+)](i)) with an initial peak followed by a sustained increase in HL-60 cells differentiated into neutrophil-like cells. The initial peak is not affected by the presence of ethylene glycol bis (beta-aminoethyl ether) N, N, N', N-tetraacetic acid (EGTA) in the buffer and appears to be dependent on conversion of sphingosine to sphingosine -1-phosphate (S1P) by sphingosine kinase, since it is blocked by the presence of N, N-dimethylsphingosine (DMS), which, like sphingosine, causes a sustained increase itself. The sustained increase that is elicited by sphingosine or DMS is abolished by the presence of EGTA in the buffer. The sustained sphingosine-induced Ca(2+)influx does not appear due to Ca(2+)influx through store-operated Ca(2+)(SOC) channels, since the influx is not inhibited by SKF 96365, nor is it augmented by loperamide. In addition, sphingosine and DMS attenuate the Ca(2+)influx through SOC channels that occurs after depletion of intracellular stores by ATP or thapsigargin. Both the initial peak and the sustained increase in [Ca(2+)](i)elicited by sphingosine can be blocked by phorbol 12-myristate 13-acetate (PMA)-elicited activation of protein kinase C. Thus, in HL-60 cells sphingosine causes a mobilization of Ca(2+)from intracellular Ca(2+)stores, which requires conversion to S1P, while both sphingosine and DMS elicit a Ca(2+)influx through an undefined Ca(2+)channel and cause a blockade of SOC channels.

Capillary electrophoresis of nonprotein and protein amino acids without derivatization.

An efficient separation of eleven nonprotein amino acids (NPAAs) and three protein amino acids containing aromatic moieties was achieved by capillary electrophoresis without derivatization. The fourteen amino acids were well separated with a 100 mM sodium phosphate run buffer (pH 2.0) using a 57 cm fused-silica capillary (50 microm ID, 50 cm effective length) at 20 degrees C. With an electric field of 351 V/cm, the time needed for the separation was less than 20 min. Under optimum conditions, excellent linear responses were obtained in the concentration range of 5-100 microM, with the linear correlation coefficient ranging from 0.9785 or greater. The relative standard deviations of the migration times and the corrected peak areas were found to be 1.5-3.9% and 8.0-11.5%, respectively. In order to improve the limit of detection (LOD), simple stacking and large volume stacking using an EOF pump (LVSEP) methods were used. Improved LODs were about 300 nM in stacking and below 15 nM for five small NPAAs in LVSEP.