Relative Optical Density Research Articles

Decline of antibody response in indirect ELISA tests during the periparturient period caused diagnostic gaps in Coxiella burnetii and BVDV serology in pluriparous cows within a Holstein dairy herd

In cattle, indirect enzyme-linked immunosorbent assay (ELISA) tests are commonly used in serological routine diagnostics. In a longitudinal study design, changes in relative optical density (OD) from drying-off until week 11 after calving were analyzed in blood and milk samples from pluriparous dairy cows (n=21) using a commercial indirect anti-C. burnetii ELISA test. In a second part of this study, changes over time were evaluated in blood and milk samples of 11 of these cows in an indirect ELISA detecting anti-Bovine viral diarrhea virus (BVDV) antibodies. Regarding the cows which were still in the herd at the time of calving, blood serological qualitative changes from positive to negative or indeterminate were demonstrated in 7/20 cows (35%) for the anti-C. burnetii indirect ELISA and in 2/10 cows (20%) for the anti-BVDV indirect ELISA, respectively, during the period from 14days ante partum to calving. Relative OD in the anti-C. burnetii and the anti-BVDV indirect ELISA tests followed basically similar courses over time. In blood serum, relative OD initially increased after drying-off, before a drop around calving was observed. After the colostrum period, relative OD in blood serum showed an increase until week 11 of lactation. In colostrum samples, relative OD levels were higher than in milk samples obtained one day before drying-off. After parturition, relative OD in milk decreased until week 6 of lactation in the anti-C. burnetii indirect ELISA and until week 11 in the anti-BVDV indirect ELISA, respectively. In conclusion, blood serological investigations in periparturient dairy cows using indirect ELISA kits should be avoided.

Kynurenic Acid Inhibits the Electrical Stimulation Induced Elevated Pituitary Adenylate Cyclase-Activating Polypeptide Expression in the TNC.

Migraine is a primary headache of imprecisely known mechanism, but activation of the trigeminovascular system (TS) appears to be essential during the attack. Intensive research has recently focused on pituitary adenylate cyclase-activating polypeptide (PACAP) and the kynurenine systems as potential pathogenic factors. We investigated the link between these important mediators and the effects of kynurenic acid (KYNA) and its synthetic analog (KYNA-a) on PACAP expression in the rat trigeminal nucleus caudalis (TNC) in a TS stimulation model related to migraine mechanisms. Adult male Sprague-Dawley rats were pretreated with KYNA, KYNA-a, the NMDA receptor antagonist MK-801, or saline (vehicle). Next, the trigeminal ganglion (TRG) was electrically stimulated, the animals were transcardially perfused following 180 min, and the TNC was removed. In the TNC samples, 38 amino acid form of PACAP (PACAP1-38)-like radioimmunoactivity was measured by radioimmunoassay, the relative optical density of preproPACAP was assessed by Western blot analysis, and PACAP1-38 mRNA was detected by real-time PCR. Electrical TRG stimulation resulted in significant increases of PACAP1-38-LI, preproPACAP, and PACAP1-38 mRNA in the TNC. These increases were prevented by the pretreatments with KYNA, KYNA-a, and MK-801. This is the first study to provide evidence for a direct link between PACAP and the kynurenine system during TS activation.

DEPENDENCE OF FLUORESCENT CHARACTERISTICS OF NANOCOMPOSITES ON THE BASIS OF DYE MOLECULES AND SILVER NANOPARTICLES ON THE OPTICAL DENSITY OF COMPONENTS

It is known that the addition of plazmon metal nanoparticles to the lasant can be used to improve the radiative characteristics of the medium. However, the problem of the influence of the component ratio on the intensity of the nanocomposites fluorescence, which is relevant from applied point of view, has not been well investigated and requires additional studies. In this paper, the fluorescence of the solutions of the Rhodamine 6G and Rhodamine C dyes at different excitation wavelengths when there are silver nanoparticles is investigated. The effect of the mixture components concentration on the fluorescence amplification coefficient of the dye molecules is studied. It is shown that one can consider the relative optical density of the mixture components, which is the ratio of the optical density of the nanoadditive to the optical density of the dye at the excitation wavelength, as a generalized parameter that has influence on the fluorescent characteristics of nanocomposites. The relative optical density is maximized with increase in the concentration of nanoparticles as well as with decrease of the concentration of the dye or in the case of excitation by radiation with the spectrum closest to the maximum of plasmon resonance of nanoparticles. In this case, the increase in the fluorescence amplification coefficient is observed. If the pumping spectrum is far from the wavelength of maximum of the plasmon resonance, the concentration of the nanoparticles is low, and the dye concentration is high, then the relative optical density is small, the fluorescence gain becomes insignificant, and even its quenching may occur. The results of these studies allow us to formulate a general approach to assessing the effect of the components ratio of mixtures composed of dye molecules and metallic nanoparticles on the fluorescence intensity of fluorophore molecules.

Long-term antibacterial performances of biodegradable polylactic acid materials with direct absorption of antibiotic agents.

In this study, polylactic acid (PLA) disks with antibacterial performances were prepared using 3D printing technology combined with direct adsorption of the antibiotic agents in solution baths. The effects of the layer thicknesses for the building of the 3D printing PLA disks and the amounts of antibiotic agents absorbed onto the sample surfaces on their antibacterial activities were investigated. The antibiotic agent release profiles from the samples surface into the buffer solution show that the antibacterial performances of these samples can reach up to 28 days. With a decrease in the concentration of antibiotic agent in the solution bath, the amount of antibiotic agent adsorbed on the sample surfaces also decreases, but their antibacterial performances can still maintain at least 7 days. In the bioactivity tests of the various organisms, the release amount of antibiotic agent from the sample can inhibit E. coli and S. aureus for over 80% up to 28 days. In the antibacterial activity tests, a PLA disk with suitable antibiotic agents covering its surface has a good inhibitory effect on the growth ability of S. aureus of less than 50% in six hours.

Effect of intracerebellar microinjection of cdc2-siRNA on the coordination of mice with Niemann-Pick disease type C

Objective To observe the effect of inhibiting the abnormal activation of cdc2 gene on the coordination of mice with Niemann-Pick disease type C(NPC). Methods Recombinant adeno-associated virus( rAAV) encoding cdc2-siRNA was packaged, and then was injected into the cerebellum of 2 weeks old npc-/- mice. Footprint test and vertical screen test were performed to assess the coordination of mice at the age of 8 weeks. Purkinje cells visualized by HE staining in cerebellum were counted, and the phosphorylation of microtubule-associated protein Tau recognized by PHF-1 antibody was detected by immunoblotting technology. Results (1) Footprint test showed that the stride length in cdc2-siRNA npc-/- group((4.92±0.31) cm) was markedly longer than that in empty vector npc-/- group((4.05±0.19) cm)(P<0.05). (2)Vertical screen test showed that the latency to turn head upwards or reach the upper edge of the screen in cdc2-siRNA npc-/- group((26.01±1.82) s, (50.93±1.98) s) was significantly shorter than that in the empty vector npc-/- group((31.96±3.47)s, (56.89±2.97)s), respectively(P<0.05 for all comparisons). (3)The number of Purkinje cells in cerebellum was dramatically increased in cdc2-siRNA npc-/- group(11.0±2.5) compared with the empty vector npc-/- group(5.1±2.2)(P<0.05). (4)The relative optical densities of cdc2 and phosphorylated Tau immunoreactive bands in cdc2-siRNA npc-/- group(1.42±0.22, 0.95±0.31)were significantly lower than those in the empty vector npc-/- group(2.11±0.29, 2.61±0.62), respectively(P<0.05 for all comparisons). Conclusion Inhibiting the abnormal activation of cdc2 gene can improve the coordination of npc-/- mice by ameliorating Purkinje cell’s loss and reducing the hyperphosphorylation of Tau in cerebellum. Key words: Niemann-Pick disease type C; Coordination; Cell division cycle 2; Purkinje cell

The impact of antiandrogen flutamide on the hypoxia inducible factor 1a and vascular endothelial growth factor A gene and protein expression in the pig placenta during late pregnancy.

In contrast to estradiol action, little is known about androgen signaling in placental development. The purpose of this study was to evaluate the impact of diminished androgen action on hypoxia inducible factor 1a (HIF-1a) and vascular endothelial growth factor A (VEGFA) protein expression as well as their mRNAs in the structures of fetal and maternal parts of porcine placenta during late pregnancy. Pregnant pigs were injected daily with antiandrogen flutamide, at a dose of 50 mg/kg body weight at different stages of pregnancy: between gestational days 83-89 (90 dpc) and 101-107 (108 dpc). Control groups (90 dpc or 108 dpc) were treated with vehicle (corn oil). One day after the last injection animals were sacrificed and tissues were collected. Tissue samples were frozen for mRNA isolation or fixed for immu-nohistochemistry (IHC). The expression of HIF-1a and VEGFA were investigated by real-time PCR and IHC. Flutamide treatment caused changes in both HIF-1a and VEGFA mRNA levels only in the placentas of the 90 dpc group. Relative optical density analysis showed decreased HIF-1a and increased VEGFA protein expression in the placentas obtained from flutamide-treated 108 dpc group while no differences were observed in the 90 dpc group. Experimentally induced androgen deficiency in pigs deregulates the expression of some genes important for placental blood circulation. We suggest that androgens are involved in the control of expression of HIF-1a and VEGFA in porcine placenta during late pregnancy.

The protective effect of Ginsenoside Rg1 on aging mouse pancreas damage induced by D-galactose.

The protective effect and mechanism of Ginsenoside Rg1 on aging mouse pancreas damaged by D-galactose (D-gal)-induced was studied. Two-month-old male C57BL/6J mice were randomly divided into three groups of 10 mice per group. The D-gal group of mice received hypodermic injection of D-gal (120 mg/kg/day) for 42 days; the Rg1+D-gal group of mice receiving D-gal + intraperitoneal injection Rg1 (40 mg/kg/day) for 27 days from the 16th day of D-gal replication; and the naïve group that constituted the normal control mice receiving the same dose of saline instead of the drug. The related indicators were tested on the second day after modeling and administration. Fasting blood glucose (FBG), oral glucose tolerance test (OGTT) and fasting insulin level were measured by taking peripheral blood. Samples of pancreas were weighed and visceral index was calculated. Paraffin sections were prepared. H&E staining sections were produced to observe pancreatic tissue morphology. Immunohistochemical staining was used to observe advanced glycation end products (AGEs) and integral optical density (IOD) of stained positive tissue in pancreas. Ultrathin slices were used to observe ultrastructural change of pancreatic tissue. Frozen sections were prepared to test the relative optical density of positive cells that were stained by senescence-associated β-galactosidase (SA-β-gal) in pancreatic tissue. Superoxide dismutase (SOD), malonaldehyde (MDA) and total antioxidant capacity (T-AOC) were detected by preparing pancreas tissue homogenates. Compared with the control group, Rg1+D-gal mice had significantly decreased pancreatic wet weight and visceral index and significantly lower FBG; OGTT for 30 and 120 min. There was no significant difference of the blood sugar level between the groups. The area under the curve and the number and size of the nucleated cells within islet were markedly reduced. In addition, SA-β-gal-positive particles in pancreas tissue intracytoplasmic cells significantly decreased and relative optical density also reduced. The IOD of AGEs in pancreas tissue and MDA content decreased. SOD and T-AOC activity significantly increased. Ginsenoside Rg1 can be effective antagonistic structure and function of the pancreas injury induced by D-gal. The mechanism may be associated with reducing oxidative damage.

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation.

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or in buffer (washed platelets), by the use of one or a combination of agonists. The use of washed platelets separated from their plasma environment and in the absence of anticoagulants allows for studying intrinsic platelet properties. Among the large panel of agonists, arachidonic acid (AA), adenosine di-phosphate (ADP), thrombin and collagen are the most frequently used. The aggregation response is quantified by measuring the relative optical density (OD) over time of platelet suspension under continuous stirring. Platelets in homogeneous suspension limit the passage of light after the addition of an agonist, platelet shape change occurs producing a small transitory increase in OD. Following this initial activation step, platelet clots form gradually, allowing the passage of light through the suspension as a result of decreased OD. The aggregation process is ultimately expressed as a percentage, compared to the OD of platelet-poor plasma or buffer. Rigorous calibration is thus essential at the beginning of each experiment. As a general rule: calibration to 0% is set by measuring the OD of a non-stimulated platelet suspension while measuring the OD of the suspension medium containing no platelets represents a value of 100%. Platelet aggregation is generally visualized as a real-time aggregation curve. Turbidimetry is one of the most commonly used laboratory techniques for the investigation of platelet function and is considered as the historical gold standard and used for the development of new pharmaceutical agents aimed at inhibiting platelet aggregation. Here, we describe detailed protocols for 1) preparation of human washed platelets and 2) turbidimetric analysis of collagen-induced aggregation of human washed platelets pretreated with the food dye Brilliant Blue FCF that was recently identified as an inhibitor of Pannexin1 (Panx1) channels.

Establishment of a Lateral Flow Colloidal Gold Immunoassay Strip for the Rapid Detection of Soybean Allergen β-Conglycinin

A rapid lateral flow colloidal gold immunoassay strip was established in a sandwich format with the colloidal gold-labelled mouse anti-β-conglycinin monoclonal antibody (mAb) and the rabbit anti-β-conglycinin polyclonal antibody (pAb) in order to specifically identify a soybean allergen, β-conglycinin. Soybean and soy derivatives have become ubiquitous in many vegetarian and meat-based food products, and as a result, dietary avoidance has become difficult. Therefore, soybean avoidance is challenging for individuals with soybean allergy. In our study, the selected mAb showed high affinity to the target and no cross-reactivity with other soybean allergens. Based on the sandwich immunoassay principle, the rabbit pAb was applied in the test line with high titre. The assay could be accomplished within 10 min without sophisticated procedures. The limit of detection of the test strip was calculated at 1.66 mg/kg in powdered milk using an optical density scanner that measures relative optical density. The assay showed high specificity for β-conglycinin, with no cross-reactions with other food allergens. The efficacy of the lateral flow test strip to detect β-conglycinin in powdered milk samples ranged between 80.8 and 89.2% with relative standard deviations of less than 7.2%. Therefore, the test strip is useful as a rapid and reliable detection method for β-conglycinin in powdered milk. In addition, this assay is suitable for monitoring β-conglycinin in other processed foods when samples are appropriately extracted.

Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) pre-exposure ensures follicle integrity during in vitro culture of ovarian tissue but not during cryopreservation in the domestic cat model.

Temporary and reversible downregulation of metabolism may improve the survival of tissues exposed to non-physiological conditions during transport, in vitro culture, and cryopreservation. The objectives of the study were to (1) optimize the concentration and duration of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP-a mitochondrial uncoupling agent) exposures for biopsies of domestic cat ovarian tissue and (2) examine the effects of FCCP pre-exposures on follicle integrity after tissue culture and/or cryopreservation. Biopsies of cat ovarian tissue were first treated with various concentrations of FCCP (0, 10, 40, or 200nM) for 10 or 120min to determine the most suitable pre-exposure conditions. Based on these results, tissues were pre-exposed to 200nM FCCP for 120min for the subsequent studies on culture and cryopreservation. In all experiments and for each treatment group, tissue activity and integrity were measured by mitochondrial membrane potential (relative optical density of rhodamine 123 fluorescence), follicular viability (calcein assay), follicular morphology (histology), granulosa cell proliferation (Ki-67 immunostaining), and follicular density. Ovarian tissues incubated with 200nM FCCP for 120min led to the lowest mitochondrial activity (1.17 ± 0.09; P < 0.05) compared to control group (0nM; 1.30 ± 0.12) while maintaining a constant percentage of viable follicles (75.3 ± 7.8%) similar to the control group (71.8 ± 11.7%; P > 0.05). After 2days of in vitro culture, percentage of viable follicles (78.8 ± 8.9%) in similar pre-exposure conditions was higher (P < 0.05) than in the absence of FCCP (61.2 ± 12.0%) with percentages of morphologically normal follicles (57.6 ± 17.3%) not different from the fresh tissue (70.2 ± 7.1%; P > 0.05). Interestingly, percentages of cellular proliferation and follicular density were unaltered by the FCCP exposures. Based on the indicators mentioned above, the FCCP-treated tissue fragments did not have a better follicle integrity after freezing and thawing. Pre-exposure to 200nM FCCP during 120min protects and enhances the follicle integrity in cat ovarian tissue during short-term in vitro culture. However, FCCP does not appear to exert a beneficial or detrimental effect during ovarian tissue cryopreservation.

Semi-Quantitative Analysis of Brain MR Imaging in 76 Cases of Neonatal Indirect Hyperbilirubinemia

Background: Neonatal hyperbilirubinemia is indeed common. However, severe nerve injuries and bilirubin encephalopathy are rare and only occur in the unusual cases of extreme hyperbilirubinemia. Objectives: To investigate brain magnetic resonance imaging (MRI) changes and their correlations with perinatal predisposing factors in neonates with indirect hyperbilirubinemia, via regions of interest (ROIs) analysis. Methods: Seventy-six neonates with a gestational age of ≥35 weeks diagnosed with neonatal indirect hyperbilirubinemia or bilirubin encephalopathy all underwent brain MRI during hospitalization. Depending on peak total serum bilirubin (TSB), they were assigned to group A ( μmol/L), C (≥42 μmol/L μmol/L), or D (≥428 μmol/L). The globus pallidus and the white matter around the anterior horn of the lateral ventricle and posterior horn of the lateral ventricle were selected as the ROIs. Average optical densities (AODs) of the ROIs and the cerebrospinal fluid region were measured. The ratio between the AODs was designated as the relative optical density (ROD), and used to determine relative signal intensity. Results: RODs of the globus pallidus were significantly lower in group D than in all other groups. TSB and the ratio of TSB to serum albumin concentration (B/A) was significantly negatively correlated with ROD in theglobus pallidus. Corrected age was significantly negatively correlated with the ROD of the ROIs. Conclusions: Semi-quantitative image analysis is a feasible method to assess conventional brain MRI for neonatal indirect hyperbilirubinemia. The signal intensity of the globus pallidus in T1-weighted images was significantly correlated with TSB level and B/A.

Protective effect of Angelica sinensis polysaccharides on subacute renal damages induced by D-galactose in mice and its mechanism

To explore the protective effect of Angelica sinensis polysaccharides(ASP) on subacute renal damages induced by D-galactose in mice and its mechanism. Male C57BL/6J mice were randomly divided into 3 groups, with 10 mice in each group. The D-galactose model group was subcutaneously injected with D-galactose (120 mg x kg(-1)), qd x 42; the ASP + D-galactose model group was intraperitoneally injected with ASP since the 8th day of the replication of the D-galactose model, qd x 35; and the normal control group was subcutaneously injected with saline at the same dose and time. On the 2nd day of after the injection, the peripheral blood was collected to measure the content of BUN, Crea, UA, Cys-C; paraffin sections were made to observe the renal histomorphology by HE staining; senescence-associated β-g-alactosidase (SA-β-Gal) stain was used to observe the relative optical density (ROD) in renal tissues; transmission electron microscopy was assayed to observe the renal ultrastructure; the renal tissue homogenate was prepared to measure the content of SOD, GSH-PX, MDA; the content of AGEs and 8-OH-dG were measured by ELISA. According to the result, compared with the D-galactose model group, the ASP + D-galactose model group showed obviously decreases in the content of BUN, Crea, UA, Cysc, AGES, 8-OH-dG, the number of hardening renal corpuscle, renal capsular space and renal tubular lumen, ROD of SA-β-Gal staining positive kidney cells, mesangial cells, basement membrane thickness, podocyte secondary processes fusion and MDA and increases in the number of normal renal corpuscle, ribosome and rough endoplasmic reticulum in podocytes, the activity of SOD and GSH-PX. In Conclusion, A. sinensis polysaccharides can antagonize kidney subacute damages induced by D-galactose in mice. Its protective mechanism may be correlated with the inhibition of the oxidative stress injury.

Evaluation of Objective Vitritis Grading Method Using Optical Coherence Tomography: Influence of Phakic Status and Previous Vitrectomy

To evaluate a proposed method for objective measurement of vitreous inflammation using a spectral-domain optical coherence tomography (SD OCT) device in a large cohort of uveitis eyes, including pseudophakic eyes and vitrectomized eyes. Retrospective, observational cohort study. One hundred five uveitis eyes (105 patients) with different vitreous haze score grades according to standardized protocols and corresponding SD OCT images (Cirrus HD-OCT; Carl Zeiss Meditec, Dublin, California, USA) were included. Clinical data recorded included phakic status, previous vitreoretinal surgery, and anterior chamber (AC) cells and flare. SD OCT images were analyzed using custom software that provided absolute measurements of vitreous (VIT) and retinal pigment epithelium (RPE) signal intensities, which were compared to generate a relative optical density ratio with arbitrary units (VIT/RPE-relative intensity) and compared to VHS. VIT/RPE-relative intensity showed a significant positive correlation with vitreous haze score (r = 0.535, P < .001) that remained significant after adjusting for factors governing media clarity, such as AC cells, AC flare, and phakic status (R(2)-adjusted = 0.424, P < .001). Significant differences were also observed between the different vitreous haze score groups (P < .001). Preliminary observation did not observe differences in VIT/RPE-relative intensity values between phakic and pseudophakic eyes (0.3522 vs 0.3577, P = .48) and between nonvitrectomized and vitrectomized eyes (0.3540 vs 0.3580, P = .52), overall and respectively for each vitreous haze score subgroup. VIT/RPE-relative intensity values provide objective measurements of vitreous inflammation employing an SD OCT device. Phakic status and previous vitrectomy surgery do not appear to influence these values, although these preliminary findings need validation in future studies.

Measurements of Neuronal Soma Size and Estimated Peptide Concentrations in Addition to Cell Abundance Offer a Higher Resolution of Seasonal and Reproductive Influences of GnRH-I and GnIH in European Starlings.

Hypothalamic neuropeptides involved in vertebrate reproduction, gonadotropin releasing hormone (GnRH-I) and gonadotropin-inhibitory hormone (GnIH), can vary in the abundance of immunoreactive cells as a function of the reproductive status and nest box occupation of European starlings (Sturnus vulgaris). While using the abundance of cells as an indicator of the activity of neurohormones is informative, incorporating information on cell size (readily observed using immunohistochemistry) can offer a more detailed understanding of environmentally-mediated changes in hormonal dynamics. In this study, we tested the hypothesis that the size of cells' somas and the estimated concentration of peptides in cells immunoreactive (ir) for GnRH-I and GnIH would vary throughout the breeding season and as a function of nest-box status (resident or not). In the absence of a direct assay of protein, we estimated an index of the concentration of hypothalamic peptides via the relative optical density (i.e., the difference between the mean optical density and the optical density of background staining). In support of our hypothesis, we found that GnRH-I- and GnIH-ir soma size and peptide concentration changed both in males and females throughout the breeding season. Somas were largest and estimated peptide concentration was highest mid-season when compared with earlier in the season or to the non-breeding period. For nest-box residents, GnIH-ir soma size and peptide concentration were higher during the middle of the breeding season than earlier in the breeding season, although residence in the nest box was not related to GnRH-I-ir variables. Our results confirm that previously reported changes in cell abundance mimic changes we see in GnRH-I and GnIH-ir soma size and our proxy for peptide concentration. However, investigating changes in the soma of GnRH-I-ir cells revealed a peak in size during the middle of the breeding season, a change not evident when solely examining data on the abundance of cells. We also report that GnRH-I- and GnIH-ir soma size and our proxy for peptide concentration positively co-varied with each other and, in males, were positively correlated with testosterone. In summary, we offer a higher resolution of understanding of the function of GnRH-I and GnIH during the breeding period of European starlings.

The free-radical scavenger edaravone accelerates thrombolysis with alteplase in an experimental thrombosis model

Background and PurposeReperfusion injury after thrombolytic therapy can have adverse neurologic effects. The free-radical scavenger edaravone is used in combination with the recombinant tissue plasminogen activator alteplase to treat acute ischemic stroke. However, basic investigations of this combination use remain inadequate. Here, we used an in vivo model to investigate the effects of edaravone on alteplase-induced thrombolysis. MethodsThrombolysis was evaluated by using a He-Ne-laser–induced thrombosis model in rat mesenteric microvessels. Changes in thrombus volume were analyzed with the image analysis software Image-Pro Plus (Media Cybernetics, USA).There were three experimental groups (placebo, alteplase 0.6mg/kg, alteplase 0.6mg/kg+edaravone 10.5mg/kg). Sequential changes (0 to 60min) in thrombus volume were compared by using a relative optical density method that we had used previously. ResultsIn the placebo group, the thrombus volume at 60min, reflecting the extent of thrombolysis, was 97.2%±5.7% of the initial value. In the alteplase group, thrombus volume decreased to 70.7%±4.1% (P<0.01) after 20min and 14.2%±6.6% after 60min. In the alteplase+edaravone group, thrombus volume decreased to 66.9%±7.2% (P<0.001) after 10min and 10.9%±2.3% after 60min. ConclusionsThese results support the hypothesis that edaravone accelerates thrombolysis by alteplase.

Vascular endothelial growth factor 165-transfected adipose-derived mesenchymal stem cells promote vascularization-assisted fat transplantation

Objective: To investigate the effect of vascular endothelial growth factor 165 (VEGF165) and adipose-derived mesenchymal stem cells (ASCs) in promoting the survival of fat grafts, and to provide new methods and theoretical evidence for increasing the survival rate of autologous fat particle grafts. Methods: The VEGF165 gene was recombined with the target fragment, and the recombinant gene was introduced into adenovirus pAdEasy-1 system; the virus was then packaged and the titer was detected. The control group received the same processing. ASCs were cultured and subcultured, and then identified with immunohistochemistry and adipogenic differentiation assay. The subsequent experiments were performed in three groups: the VEGF165 gene-virus group, blank virus group, and control group. After the viral solution was transfected into the ASCs, the viral transfection efficiency was detected using a tracing factor, EGFP. The expression of VEGF165 mRNA and protein in the transfected cells were determined. The proliferation of ASCs in each group was detected with the MTT assay. Results: (1) Recombinant adenoviral vector was constructed successfully in the two groups and the packaging was identified. The viral titer was 2.0 × 108 pfu/ml and 1.9 × 108 pfu/ml, which was in line with the requirements of the subsequent transfection experiments. (2) Immunohistochemistry and adipogenic differentiation results showed that the culture of ASCs was successful, and the cultured cells could serve as seed cells in this experiment. (3) The RT-PCR analysis showed that the relative optical density of VEGF165 mRNA expression was 0.76 ± 0.05 in the experimental group, and there were statistically significant differences compared with the values obtained for the other two groups (P < 0.05). (4) The western blot analysis showed that the relative optical density of VEGF165 protein expression in the experimental group was significantly higher than that in the other two groups (P < 0.05). (5) The proliferation of ASCs was significantly enhanced after transfection in the experimental group, relative to the other two groups (P < 0.05). This evidence indicated that VEGF165 significantly promoted the proliferation of ASCs. Conclusion: After transfection with the VEGF165-adenoviral vector, ASCs demonstrate sustained expression of the target protein and obviously promote the proliferation of ASCs, which lay the foundation for the in vitro experiments on transplantation of VEGF165 combined with ASCs, for the treatment of tissue defects.

Development of a multiplex flow-through immunoaffinity chromatography test for the on-site screening of 14 sulfonamide and 13 quinolone residues in milk

In this paper, a rapid and sensitive multiplex flow-through immunoaffinity chromatography test (FTIACT) was developed for the on-site screening of 14 sulfonamide and 13 quinolone residues in milk. The developed FTIACT method combines the purification, preconcentration and immunochemical detection of multiple antibiotics on the sepharose gel test layers. The use of liposome-encapsulated quantum dots (LQDs) with the FTIACT method exhibited the best results, with limits of detection (LODs) of 1 and 0.5ng/mL for the sulfonamides (SAs) and quinolones (QNs), respectively, through qualitative analysis (visual detection by the naked eye). In order to achieve low detection limit, the color intensity of the images were converted into relative optical density values to enable a quantitative evaluation. Quantitative analysis of the samples enabled the detection of SAs (0.13ng/mL) and QNs (0.062ng/mL) in spiked milk samples. The FTIACT described in this work shows promise as a multiplex immunoassay for the qualitative and quantitative screening of multiple chemical residues in milk.

Computed tomographic assessment of healing of long bone defect in rats after implantation in its cavity of osteoplastic material based on β-tricalcium phosphate

Objective: with using of the computed tomography to investigate compact bone (diaphysis) of femurs of the rats after implantation of osteoplastic material Chronos™, to improve knowledge about the dynamics and the rate of its biodegradation and integration, as well as the site of density in defect area and adjacent maternal bone, and evaluate postoperative complications. Methods: The experiment was conducted on 30 white laboratory male rats (8 months, weight (250 ± 10) g). Under ketamine anesthesia in the middle third of the femoral shaft using a portable drill spherical cutter at low speed with cooling we created defect with diameter of 2.5 mm toward a medullary canal and filled it with osteoplastic material Chronos™ (Synthes, Switzerland) without any rigid fixation. After 15, 30, 60, 120 and 180 days after surgery we investigated injured femurs implanted with the stuff on the 16-slice spiral computed tomograph «TOSHIBA Activion». Results: There was absence of any signs of bone rarefaction of the maternal bone at all stages of observation. 15 days after implantation structure of Chronos™ was maintained but since a 30-day there were detected some signs of resorption of osteoplastic material and their subsequent increase, and its replacement by bone tissue. Relatively high optical density of regenerate was established on the 15th day of the experiment and its equation with the absolute optical density of maternal bone, and even its predominance to 120–180 days. In the last period of the experiment it was established approximation of the relative optical density of regenerate to 100 % while the absence of restoring the original shape and integrity of bone so far as in the cortical bone hardly noticeable deepening or newly formed tough endosteal callus still remained.

Evaluation of a semi-quantitative ELISA for IgA antibody against Epstein–Barr virus capsid antigen in the serological diagnosis of nasopharyngeal carcinoma

This study was designed to establish an ELISA method, as well as the cut-off value, for IgA against Epstein-Barr virus (EBV) viral capsid antigen (VCA), as a screening assay for nasopharyngeal carcinoma (NPC) in southern China. In addition, the correlation between relative optical density (rOD) values from ELISA and titers from the immunoenzymatic assay (IEA) was also evaluated. Two hundred and fifty-eight NPC cases, 33 non-NPC head and neck cancer patients, and 1156 healthy controls were recruited for this study. VCA-IgA and early antigen (EA)-IgA were measured by ELISA kits and IEA in parallel. The total precision of the VCA-IgA ELISA achieved a level of <13.0% coefficient of variation. An rOD value of 1.60 for the VCA-IgA ELISA was determined as the cut-off point for southern China, and the sensitivity and specificity for NPC diagnosis when using this cut-off value were 93.0% and 92.4%, respectively. The area under the receiver operating characteristic curve (ROC-AUC) value was 0.969. The correlation coefficient between titers and rOD values was 0.957. rOD values were correlated with NPC overall stage and lymph node involvement. The cut-off level established in our study could be used to facilitate more accurate diagnosis of NPC in southern China. The rOD value might be an index for NPC prognosis, since it shows a good correlation with the titer from IEA.

The preventive effect of grape seed extract on artificial enamel caries progression in a microbial biofilm-induced caries model

ObjectivesThe aim of this study was to evaluate the effect of grape seed extract (GSE) on enamel caries lesion formation in an in vitro Streptococcus mutans biofilm model. MethodsEnamel fragments were prepared from bovine incisors and divided into six treatment groups (n=12): inoculated Brain Heart Infusion with 1% sucrose (BHIS), 1mg/mL GSE, 2mg/mL GSE, 3mg/mL GSE, 10ppm fluoride as NaF, and uninoculated BHIS. For biofilm formation, tooth fragments were incubated anaerobically in polystyrene 6-well tissue culture plates containing BHIS, the respective agents, and S. mutans (1×105CFU/mL) for 24h at 37°C. Culture medium was replaced with fresh BHIS and respective agents daily over a 7-day period. Following caries lesion formation, lesion depth (LD) and relative optical density (ROD) were determined by polarized light microscopy (PLM) and confocal laser scanning microscopy (CLSM), respectively, to evaluate lesion progression. ResultsLDs of the 2mg/mL GSE group (122.86±13.41μm) and the 3mg/mL GSE group (111.92±11.39μm) were significantly smaller than those of the 1mg/mL GSE (198.33±17.70μm) and control groups (210.86±15.50μm) (p<0.05). Compared with the 2mg/mL and 3mg/mL groups, the control and 1mg/mL GSE groups showed significantly lower ROD values when depth was less than 200μm, indicating greater mineral loss. ConclusionsDose-dependent GSE inhibits in vitro enamel caries formation due to its ability to suppress growth of S. mutans and the formation of biofilm. Clinical significanceGrape seed extract may be a novel virulence-targeted natural antimicrobial agent for caries prevention.