Study of erythrocyte sedimentation behavior by piezoelectric crystal impedance sensor

Based on the impedance characteristic of erythrocytes at high frequency, the response of piezoelectric crystal impedance (PCI) sensor in the erythrocyte suspension was derived and verified experimentally. A method of using PCI sensor to investigate erythrocyte aggregation-sedimentation phenomenon was proposed. From the frequency response of the PCI sensor, the erythrocyte aggregation time and sedimentation rate could be obtained during erythrocyte aggregation and sedimentation. With the present method, the effects of the erythrocyte deformability, the osmotic pressure and the coexisting macromolecules on the erythrocyte sedimentation rate were studied. The results show that the PCI sensor possesses some advantages, such as good sensitivity, simplicity of use and no thermal effect for the impedance study of erythrocyte aggregation and sedimentation.     

Full-scan high resolution accurate mass spectrometry (HRMS) in regulated bioanalysis: LC-HRMS for the quantitation of prednisone and prednisolone in human plasma

A liquid chromatography-full scan high resolution accurate mass spectrometry (LC-HRMS) method for quantifying prednisone and prednisolone in human plasma using a quadrupole time-of-flight mass spectrometer (Q-TOF) was developed. Plasma samples were extracted using a liquid-liquid extraction procedure. Full scan data were acquired in the TOF only mode and extracted ion chromatograms were generated post-acquisition with the exact masses of the analytes. The calibration range was 5-2500 ng/mL, with a Lower Limit of Quantitation (LLOQ) of 5 ng/mL. The assay accuracy was between 98.4% and 106.3%. The between-run (inter-day) and within-run (intra-day) precision were within 1.7% and 2.9%, respectively. The matrix effect was between 0.98 and 1.10 for the six different lots of human plasma evaluated. Pooled incurred samples were analyzed by the method and the results matched those obtained from an LC-MS/MS method. In addition, qualitative information on phospholipids, and other endogenous components were also extracted from the full-scan data acquired.     

Inhibition of Rhizoctonia by endospore forming bacteria indigenous to landscape planting beds

Endospore forming bacteria were collected from root samples of 35 genera of bedding plants growing in established commercial landscape beds in Central Florida. One hundred and twenty-nine bacterial strains associated with 14 species were identified using fatty acid analysis (Microbial Identification System, MIDI). All strains were evaluated for in vitro inhibition of damping off disease caused by the fungus Rhizoctonia solani. The strongest inhibition of Rhizoctonia by soluble exudates in cocultivation was observed with strains belonging to six species: B. cereus, B. pumilus, B. thuringiensis, L. sphaericus, B. amyloliquefaciens, and B. subtilis. Most strains that inhibited Rhizoctonia growth in cocultivation also inhibited growth via volatile compounds. All 129 strains were evaluated for ability to protect impatiens plants from subsequent challenge infection by Rhizoctonia solani. Certain strains of endospore forming bacteria also enhanced plant growth. It is apparent from this study that the Bacillus community associated with bedding plants in established planting beds produce soluble antifungal compounds, volatile antifungal compounds, and enhance plant growth. In developing biological control, it may be a more practical approach to promote or enhance a natural, multifaceted community of Bacillus strains within our planting beds.     

Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine

By using specific anti-ACAT-1 antibodies in immunodepletion studies, we previously found that ACAT-1, a 50-kDa protein, plays a major catalytic role in the adult human liver, adrenal glands, macrophages, and kidneys but not in the intestine. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in the intestine may be largely derived from a different ACAT protein. To test this hypothesis, we produced specific polyclonal anti-ACAT-2 antibodies that quantitatively immunodepleted human ACAT-2, a 46-kDa protein expressed in Chinese hamster ovary cells. In hepatocyte-like HepG2 cells, ACAT-1 comprises 85-90% of the total ACAT activity, with the remainder attributed to ACAT-2. In adult intestines, most of the ACAT activity can be immunodepleted by anti-ACAT-2. ACAT-1 and ACAT-2 do not form hetero-oligomeric complexes. In differentiating intestinal enterocyte-like Caco-2 cells, ACAT-2 protein content increases by 5-10-fold in 6 days, whereas ACAT-1 protein content remains relatively constant. In the small intestine, ACAT-2 is concentrated at the apices of the villi, whereas ACAT-1 is uniformly distributed along the villus-crypt axis. In the human liver, ACAT-1 is present in both fetal and adult hepatocytes. In contrast, ACAT-2 is evident in fetal but not adult hepatocytes. Our results collectively suggest that in humans, ACAT-2 performs significant catalytic roles in the fetal liver and in intestinal enterocytes.     

Forward chemical genetic screens in Arabidopsis identify genes that influence sensitivity to the phytotoxic compound sulfamethoxazole

The rise of high mountain chains is widely seen as one of the factors driving rapid diversification of land plants and the formation of biodiversity hotspots. Supporting evidence was reported for the impact of the rapid rise of the Andean mountains but this hypothesis has so far been less explored for the impact of the “roof of the world”. The formation of the Himalaya, and especially the rise of the Qinghai–Tibetan Plateau in the recent 20 million years, altered the monsoon regimes that dominate the current climates of South East Asia. Here, we infer the hypothesis that the rise of Himalaya had a strong impact on the plant diversity in the biodiversity hotspot of the Southwest Chinese Mountains. Our analyses of the diversification pattern of the derived fern genus Lepisorus recovered evidence for changes in plant diversity that correlated with the strengthening of South East Asian monsoon. Southwest China or Southwest China and Japan was recovered as the putative area of origin of Lepisorus and enhancing monsoon regime were found to shape the early diversification of the genus as well as subsequent radiations during the late Miocene and Pliocene. We report new evidence for a coincidence of plant diversification and changes of the climate caused by the uplift of the Himalaya. These results are discussed in the context of the impact of incomplete taxon sampling, uncertainty of divergence time estimates, and limitations of current methods used to assess diversification rates.     

Genetic and structural analysis of a virulence determinant in polyomavirus VP1.

The LID strain of polyomavirus differs from other laboratory strains in causing a rapidly lethal infection of newborn C3H/Bi mice. This virulent behavior of LID was attenuated by dilution, yet at sublethal doses LID was able to induce tumors at a high frequency, like its parent virus PTA. By constructing and assaying LID-PTA recombinant viruses and by DNA sequencing, the determinant of virulence in LID was mapped to the major viral capsid protein, VP1. The VP1s of LID and PTA differed at two positions: at 185, LID has phenylalanine and PTA has tyrosine, and at 296, LID has alanine and PTA has valine. Results obtained with viruses constructed by site-directed mutagenesis showed that alanine at position 296 is sufficient to confer a fully virulent phenotype regardless of which amino acid is at position 185. However, with valine at position 296, an effect of phenylalanine at position 185 is apparent, as this virus possesses an intermediate level of virulence. A crystal structure of polyomavirus complexed with 3'-sialyl lactose previously indicated van der Waals contacts between the side chain of valine 296 and the sialic acid ring (T. Stehle, Y. Yan, T. L. Benjamin, and S. C. Harrison, Nature [London] 369:160-163, 1994). When this interaction was modeled with alanine, these contacts were greatly reduced. Direct confirmation that the substitutions in VP1 affected receptor binding was obtained by studying virus hemagglutination behavior. The ensemble of results are discussed in terms of the idea that a lower affinity of the virus for its receptor can result in more rapid spread and increased pathogenicity.     

Inhibition by bromoestrogens of the effects of estradiol on apomorphine-induced climbing behavior

The chronic administration of estrogens to mice or rats will result in antidopaminergic effects. Apomorphine-induced climbing behavior in mice, the result of direct stimulation of dopamine receptors in the striatal and mesolimbic regions, is a simple animal model for examining these antidopaminergic effects of estrogens. Bromoestrogens, inhibitors of catechol estrogen formation, have been utilized in order to examine the role of estrogen metabolism in dopaminergic antagonism. Mice were pretreated for 3 days with 2-bromoestradiol, 4-bromoestradiol, or 2,4-dibromoestradiol dibenzoates alone or in combination with estradiol benzoate prior to apomorphine administration. The haloestrogens did not alter the climbing-induced responses elicited by apomorphine, whereas estradiol benzoate clearly attentuated the actions of apomorphine. Furthermore, the bromoestradiol dibenzoates were effective in reversing the effects of estradiol benzoate when the two steroids (estradiol benzoate and a bromoestrogen dibenzoate) were administered simultaneously during pretreatment. Thus, the bromoestrogens are able to inhibit the antidopaminergic effects of estradiol exhibited in the apomorphine-induced mouse climbing model.     

Nitric oxide storage and transport in cells are mediated by glutathione S-transferase P1-1 and multidrug resistance protein 1 via dinitrosyl iron complexes

Nitrogen monoxide (NO) plays a role in the cytotoxic mechanisms of activated macrophages against tumor cells by inducing iron release. We showed that NO-mediated iron efflux from cells required glutathione (GSH) (Watts, R. N., and Richardson, D. R. (2001) J. Biol. Chem. 276, 4724-4732) and that the GSH-conjugate transporter, multidrug resistance-associated protein 1 (MRP1), mediates this release potentially as a dinitrosyl-dithiol iron complex (DNIC; Watts, R. N., Hawkins, C., Ponka, P., and Richardson, D. R. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 7670-7675). Recently, glutathione S-transferase P1-1 (GST P1-1) was shown to bind DNICs as dinitrosyl-diglutathionyl iron complexes. Considering this and that GSTs and MRP1 form an integrated detoxification unit with chemotherapeutics, we assessed whether these proteins coordinately regulate storage and transport of DNICs as long lived NO intermediates. Cells transfected with GSTP1 (but not GSTA1 or GSTM1) significantly decreased NO-mediated 59Fe release from cells. This NO-mediated 59Fe efflux and the effect of GST P1-1 on preventing this were observed with NO-generating agents and also in cells transfected with inducible nitric oxide synthase. Notably, 59Fe accumulated in cells within GST P1-1-containing fractions, indicating an alteration in intracellular 59Fe distribution. Furthermore, electron paramagnetic resonance studies showed that MCF7-VP cells transfected with GSTP1 contain significantly greater levels of a unique DNIC signal. These investigations indicate that GST P1-1 acts to sequester NO as DNICs, reducing their transport out of the cell by MRP1. Cell proliferation studies demonstrated the importance of the combined effect of GST P1-1 and MRP1 in protecting cells from the cytotoxic effects of NO. Thus, the DNIC storage function of GST P1-1 and ability of MRP1 to efflux DNICs are vital in protection against NO cytotoxicity.     

Formalin-Inactivated EV71 Vaccine Candidate Induced Cross-Neutralizing Antibody against Subgenotypes B1, B4, B5 and C4A in Adult Volunteers

Background

Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia. No effective EV71 vaccine is available. A randomized and open-label phase I clinical study registered with ClinicalTrials.gov #NCT01268787, aims to evaluate the safety, reactogenicity and immunogenicity of a formalin-inactivated EV71 vaccine candidate (EV71vac) at 5- and 10-µg doses. In this study we report the cross-neutralizing antibody responses from each volunteer against different subgenotypes of EV71 and CVA16.

Methods

Sixty eligible healthy adults were recruited and vaccinated. Blood samples were obtained on day 0, 21 and 42 and tested against B1, B4, B5, C2, C4A, C4B and CVA16 for cross-neutralizing antibody responses.

Results

The immunogenicity of both 5- and 10- µg doses were found to be very similar. Approximately 45% of the participants had <8 pre-vaccination neutralization titers (Nt) against the B4 vaccine strain. After the first EV71vac immunization, 95% of vaccinees have >4-fold increase in Nt, but there was no further increase in Nt after the second dose. EV71vac induced very strong cross-neutralizing antibody responses in >85% of volunteers without pre-existing Nt against subgenotype B1, B5 and C4A. EV71vac elicited weak cross-neutralizing antibody responses (∼20% of participants) against a C4B and Coxsackie virus A16. Over 90% of vaccinated volunteers did not develop cross-neutralizing antibody responses (Nt<8) against a C2 strain. EV71vac can boost and significantly enhance the neutralizing antibody responses in volunteers who already had pre-vaccination antibodies against EV71 and/or CVA16.

Conclusion

EV71vac is efficient in eliciting cross-neutralizing antibody responses against EV71 subgenotypes B1, B4, B5, and C4A, and provides the rationale for its evaluation in phase II clinical trials.

Trial Registration

ClinicalTrials.gov __NCT01268787