Detection methods of Cyprinid herpesvirus 2 infection in silver crucian carp (Carassius auratus gibelio) via a pORF72 monoclonal antibody.

Cyprinid herpesvirus 2 (CyHV-2) is the main pathogen responsible for causing haematopoietic necrosis disease in Carassius auratus gibelio. Although many nucleic acid-based diagnostic methods have been applied, no stable and sensitive immunological diagnostic approaches have been reported. In this study, to detect CyHV-2 in clinical samples using immunological methods, recombinant ORF72 protein (pORF72), encoded by the CyHV-2 ORF72 gene, was used as a capture antigen to identify blood and tissues infected with CyHV-2. First, ORF72 gene was amplified from the CyHV-2 genome and cloned into a PGEX-4t-3 expression vector to produce pORF72 in Escherichia coli. The purified pORF72 was used as an immunogen to prepare monoclonal antibodies. The Western blotting assays revealed that the monoclonal antibody could specifically identify the pORF72. Furthermore, an immunohistochemical protocol and a blood smear method were established to detect CyHV-2 in carps. The results indicate that the monoclonal antibody against pORF72 could be utilized as an effective detection tool for haematopoietic necrosis disease in Carassius auratus gibelio.

[Efficiency of integrin αvß3 inhibitor Cilengitide in acute cerebral ischemia in rats].

OBJECTIVE: To explore the effect of the integrin αvß3 inhibitor Cilengitide on the blood brain barrier (BBB) permeability, brain edema, neuronal cell apoptosis and the relation with the vascular endothelial growth factor (VEGF)expression in acute cerebral ischemia rats. METHODS: A rat focal cerebral ischemia/reperfusion model was established by middle cerebral artery occlusion. Rats with middle cerebral artery occlusion, in accordance with the random number table, were divided into four groups: (1) the rats in Cilengitide group A (n=30) were treated with Cilengitide at a dose of 100 µg/kg; (2) the rats in Cilengitide group B (n=28) were treated with Cilengitide at a dose of 200 µg/kg; (3) the rats in sham group (n=31), without inserting thread into middle cerebral artery, were treated with normal saline; (4) the rats in control group (n=27) were treated with normal saline.All rats were treated with Cilengitide or saline 1 hour after infarction, given reperfusion 2 hours after infarction and were sacrificed 22 hours after reperfusion.The brain-water content was measured by dry/wet weight method. The permeability of BBB was measured by quantifying Evans Blue. The infarction volume was measured by 2, 3, 5-tripheyl tetrazolium Chloride (TTC) staining. Expression level of VEGF, P-Flk, Cleaved-Caspase-3 was measured by immunohistochemistry and Western blot, respectively.The neuronal cell apoptosis was evaluated by terminal deoxynucleotidyl transferased UTP nick end labeling (TUNEL). RESULTS: Compared with Control group, treatment groups with cilengitide at the dose of 100 µg/kg and 200 µg/kg reduced brain-water content [(80.8±1.1)% vs (84.8±1.4)%, (81.0±1.4)% vs (84.8±1.4)%, P<0.05], reduced exudation of Evans blue[(9.2±1.1) µg/g vs (12.2±0.8) µg/g, (8.6±0.6) µg/g vs (12.2±0.8) g/g, P<0.05], reduced infarction volume[(31.9±4.9) mm(3) vs(43.0±2.2) mm(3), (29.2±3.5) mm(3) vs(43.0±2.2) mm(3), P<0.05] , reduced neuronal cell apoptosis [(36±4)vs(69±6)、(35±3)vs (69±6), P<0.05]. Compared with sham group, Cilengitide group A and Cilengitide group B had lower brain-water content, permeability of BBB, infarction volume, expression level of VEGF, P-Flk, Cleaved-Caspase-3 and neuronal cell apoptosis (P<0.05). When Cilengitide group A was compared with Cilengitide group B, there were no significant differences in brain-water content, permeability of BBB, infarction volume, expression level of VEGF, P-Flk, Cleaved-Caspase-3 and neuronal cell apoptosis (P>0.05). CONCLUSION: The integrin αvß3 inhibitor Cilengitide improves outcomes in the MCAO model by preserving the blood-brain barrier, attenuating brain edema and inhibiting neuronal cell apoptosis, which may occur in a VEGF-and VEGF-receptor-dependent manner, with the same efficacy between Cilengitide 100 µg/kg and 200 µg/kg after 23 hours treatment.

Identification and pathogenicity of Vibrio parahaemolyticus isolates and immune responses of Penaeus (Litopenaeus) vannamei (Boone).

Five different Vibrio parahaemolyticus strains (SH8, SH108, SH58, AH5 and GD10) isolated from the hepatopancreas of moribund shrimp in farms of mainland China were identified and capable of inducing massive mortality of Penaeus (Litopenaeus) vannamei. The immersion challenge results with five isolates indicated variance of virulence, while only GD10 caused massive sloughing of tubule epithelial cells which was recognized as the most significant symptom of AHPND. Differences in immune responses were detected of P. vannamei during 48 h post-infection (p.i.) by injection or immersion challenge with V. parahaemolyticus (SH8, SH108 and GD10) isolates. When injected SH8 and SH108 isolates, the expression of lysozyme (LSZ) showing statistically significant upregulation at 16 and 48 h p.i. and that of Toll-like receptors (TLR) showed statistically significant upregulation at 48 h p.i. When immersion challenge with the GD10 isolate, TLR were upregulated after 8 h p.i. challenge with 10(4)  cfu mL(-1) ; however, LSZ was downregulated when challenged with 10(3)  cfu mL(-1) . The results suggested that LSZ and TLR serve as crucial molecular markers of innate immunity in shrimp against V. parahaemolyticus infection. LSZ is a vital marker for acute bacterial infection, while TLR serves as a crucial marker for chronic infection.

Molecular cloning and immune responsive expression of a ribonuclease III orthologue involved in RNA interference, dicer, in grass carp Ctenopharyngodon idella.

In this study, the dicer gene (designated as cidicer) was identified and characterized from grass carp Ctenopharyngodon idella. The complementary DNA (cDNA) of cidicer contained an open reading frame (ORF) of 5646 nucleotides (nts) encoding a putative protein of 1881 amino acids (aa). The deduced Dicer protein contained all known functional domains identified in other organisms. Tissue tropism analysis indicated that cidicer is abundantly expressed in brain, gill, head kidney, liver, spleen, heart, muscle and intestine. In the C. idella kidney (CIK) cells, messenger RNA (mRNA) expression of cidicer was significantly up-regulated at 24 h (6·36-fold, P < 0·01) after grass carp reovirus (GCRV) infection, and its transcriptional expression level was also transiently induced to a high level (6·54-fold, P < 0·01) at 2 h post-stimulation of synthetic double-stranded polyinosinic-polycytidylic potassium salt [poly(I:C)]. In vivo analysis further showed that the expression of cidicer mRNA in the liver was induced to a significantly high level at 12 h (8·46-fold, P < 0·01), and then dropped to normal level at 72 h post-challenge with GCRV. The transcriptional expression pattern of cidicer in the spleen tissue was similar to that of liver tissue upon GCRV challenge. These results collectively implied that the identified cidicer was an inducible gene responding to viral infection both in vitro and in vivo, and the data would shed light on the interaction between RNA interference (RNAi) antiviral pathway and aquareovirus infection.

Early embryonic blood cells collect antigens and induce immunotolerance in the hatched chicken.

Earlier experimental data in our laboratory showed that introduction of an exogenous protein into early chicken embryonic blood leads to immunotolerance of hatched chicken to that protein. However, the underlying mechanism is yet unknown. In the present study, we show that the blood cells collecting circulating antigen might contribute to the establishment of immunotolerance. In this experiment, most of the chicken embryo blood cells took up injected fluorescein isothiocyanate-BSA at approximately embryonic d 3. At the same stage, 1 microL of embryo blood was taken out and incubated with BSA. After being loaded with BSA in vitro and washed, these cells were injected back into the original embryo. The BSA-specific lymphocytes were depleted in chickens whose early embryo cells had been loaded with BSA, as evidenced by a significant decrease in anti-BSA antibody after challenge with BSA when the chickens were 3 wk old. In addition, by direct injection of BSA to embryonic d 3 embryo blood, the hatched chickens had decreased amounts of anti-trinitrophenol antibody after the chickens were challenged with trinitrophenol-BSA, indicating that the helper function of BSA-specific T cells was impaired. In conclusion, these observations suggest that some early embryo blood cells possibly collect and store antigen for the establishment of self-tolerance before the maturation of B and T cells.

Early evidence of the genus Homo in East Asia.

The timing and route of the earliest dispersal from Africa to Eastern Asia are contentious topics in the study of early human evolution because Asian hominin fossil sites with precise age constraints are very limited. Here we report new high-resolution magnetostratigraphic results that place stringent age controls on excavated hominin incisors and stone tools from the Yuanmou Basin, southwest China. The hominin-bearing layer resides in a reverse polarity magnetozone just above the upper boundary of the Olduvai subchron, yielding an estimated age of 1.7Ma. The finding represents the age of the earliest documented presence of Homo, with affinities to Homo erectus, in mainland East Asia. This age estimate is roughly the same as for H. erectus in island Southeast Asia and immediately prior to the oldest archaeological evidence in northeast Asia. Mammalian fauna and pollen obtained directly from the hominin site indicate that the Yuanmou hominins lived in a varied habitat of open vegetation with patches of bushland and forest on an alluvial fan close to a lake or swamp. The age and location are consistent with a rapid southern migration route of initial hominin populations into Eastern Asia.

The prevalence of PD in a nutritionally deficient rural population in China.

OBJECTIVES: In most reports, the prevalence of PD in mainland China is lower than in western populations. To estimate PD prevalence in China, we performed a cross-sectional study in a rural population in Linxian County, China. PRIMARY OUTCOMES: Clinical diagnosis of PD. RESULTS: Among the 16,488 participants examined, the overall age- and gender-adjusted prevalence rate of PD was 522/100,000 (95% CI: 477-567) assuming no cases of PD would be found among those younger than 50 years of age. The gender-adjusted prevalence rates were 103 (95% CI: 83-123), 621 (95% CI: 572-670), 902 (95% CI: 843-961), and 1744 (95% CI: 1662-1826) per 100,000 in age groups 50-59, 60-69, 70-79, and 80 and above, respectively. CONCLUSIONS: The estimated prevalence of PD in Linxian, China is higher than most of those reported from other areas in China, and similar to those reported from non-Asian populations.

Poly(lactide-co-glycolide)/hydroxyapatite delivery of BMP-2-producing cells: a regional gene therapy approach to bone regeneration.

Currently, functional treatment of fracture non-unions and bone loss remains a significant challenge in the field of orthopaedic surgery. Tissue engineering of bone has emerged as a new treatment alternative in bone repair and regeneration. Our approach is to combine a polymeric matrix with a cellular vehicle for delivery of bone morphogenetic protein-2 (BMP-2), constructed through retroviral gene transfer. The objective of this study is to develop an osteoinductive, tissue-engineered bone replacement system by culturing BMP-2-producing cells on an osteoconductive, biodegradable, polymeric-ceramic matrix. The hypothesis is that retroviral gene transfer can be used effectively in combination with a biodegradable matrix to promote bone formation. First, we examined the in vitro attachment and growth of transfected BMP-producing cells on a PLAGA-HA scaffold. Second, the bioactivity of the produced BMP in vitro was evaluated using a mouse model. It was found that the polymer-ceramic scaffold supported BMP-2 production, allowing the attachment and growth of retroviral transfected, BMP-2-producing cells. In vivo, the scaffold successfully functioned as a delivery vehicle for bioactive BMP-2, as it induced heterotopic bone formation in a SCID mouse model.

Quantitative ultrastructure of synapses on functionally identified primary afferent neurons in the cat trigeminal mesencephalic nucleus.

Though a number of studies have reported the presence of synapses on neurons in the trigeminal mesencephalic nucleus (Vmes), there have been no quantitative studies of either the density of innervation, or the ultrastructure, of the synapses on single, physiologically identified neurons in this nucleus. In this study we recorded from single neurons in the Vmes, identified them as being either muscle spindle afferents (MS) or periodontal ligament mechanoreceptor afferents (PL), and then labeled the neurons by intra-axonal injection of horseradish peroxidase (HRP). The material was first processed to reveal the HRP activity, following which ultrathin sections through the labeled somata were cut and examined under the electron microscope. Complete serial reconstructions were made through the soma of one MS neuron and one PL neuron, and the contacts on the neurons reconstructed. Boutons were found on the soma, spines, appendages and the axon hillock and the initial segment of the axon. The numbers of boutons terminating on the two neurons were 198 (PL) and 424 (MS), giving a packing density of 4.4 and 10.7 boutons respectively (i.e., number of boutons/100 micron 2 of the postsynaptic membrane). Boutons could be separated into two types on the basis of their vesicles: those containing clear, round vesicles (i.e., S-type) and those containing a mixture of round, oval and flattened vesicles (P-type). Ninety-five (PL neuron) and 99% (MS neuron) of terminals on the two neurons were P-type. All the S-type boutons and 80% of the P-type boutons formed asymmetric synaptic contacts while 10% of the P-type boutons made symmetric contacts. Quantitative measurements of the P-type boutons on the labeled neurons, in which the data of MS and PL neurons were pooled, revealed that bouton volume was highly correlated with bouton surface area, active zone number, total active zone area, vesicle number, and mitochondrial volume. However, comparing the quantitative measurements of the P-type boutons with those of previously reported vibrissa afferent terminals and their associated axon terminals revealed that all the parameters were smaller for the P-type boutons (on Vmes neurons) than those of the vibrissa afferent terminals but similar to those of axon terminals presynaptic to the vibrissa afferents. Taken together, our results emphasize the wide scope for synaptic interactions in the Vmes and suggest that it may be more fruitful to view the Vmes as an integrating center.

[Insulin and hyperosmotic glucose solution external used for treating pressure sore].

OBJECTIVE: To observe the efficacy of insulin and hyperosmotic glucose solution for treating pressure sore. METHODS: Using moist burn ointment as the control drug, comparisons were made between the two groups in terms of average healing time. RESULTS: In trial group the average healing time of II degree pressure sore was 11.6 +/- 2.7 days, while in the control group 12.9 +/- 3.4 days, The difference was not statistically significant. The average healing time of III degree pressure sore in trial group was significantly shorter than that of the control group (22.3 +/- 4.3 days vs 24.8 +/- 3.9 days, P < 0.05). CONCLUSION: Insulin and hyperosmotic glucose solution is effective in treating pressure sore.

[Alteration of the expression of rat cardiac Galphaq/11 and Gialpha2 proteins during endothelin-1 pre-treatment].

The present study was undertaken to explore the mechanism of G protein-mediated signal transduction pathway during endothelin-1 (ET-1) pre-treatment and ischemic preconditioning (IP). Rats were divided into four groups: ET-1, IP, ischaemia-reperfusion (IR) and control groups. ET-1 pre-treatment model was prepared by administrating 0.5 nmol/(L.kg) ET-1 into rat left ventricle, whereas IP model was prepared by ligating the left coronary artery for 5 min followed by 30 min reperfusion. All the animals were subjected to 60 min regional ischaemia and 30 min reperfusion alternately and then parameters of ventricular arrhythmia and expression of cardiac Galphaq/11 and Gialpha2 were measured. The results showed that the scores of ventricular arrhythmia decreased significantly in both ET-1 and IP treated groups as compared with IR group. In comparison with control group, Galphaq/11 increased by 77.8% (P<0.05) and 110.6% (P<0.01) in IP and ET-1 group respectively. Gialpha2 showed no significant difference in IP group, while it decreased by 31.0% (P<0.01) in ET-1 group. In conclusion, activation of G alphaq/11 may be related to the protecting mechanism of ET-1 pre-treatment and IP, whereas Gialpha2 may only play a role in ET-1 pre-treatment.

Modelling of cardiac-related changes in lung resistivity measured with EITS.

Resistivity data from 9.6 kHZ to 1.2 MHz were recorded from eight normal subjects using an electrical impedance tomographic spectroscopy (EITS) system and then averaged to a mean cardiac cycle using the ECG gating technique. The Cole-Cole model, that is, extracellular resistance R connected in parallel with intracellular resistance S and membrane capacitance C in series, with a distribution parameter a, was applied to model the frequency characteristics and to produce parametric images. During systole, SC and RC were found to decrease and FR increase. The changes in R/S were not consistent among the subjects. We estimated the peak changes in R, S and C to be -2.5%, -3.3% and -7.6% respectively. The results can be explained by considering the blood vessels as spheres of different sizes with blood inside them. The decrease in R during systole might be caused by the increased blood content in relatively large vessels, whereas that in S by the increased blood volume in relatively small vessels. The capacitance of blood is normally smaller than that of lung tissue, whereas FR blood is higher than that of lung tissue. Hence, as blood content increases, C should decrease and FR increase.