Effects of gravel on the evaluation of soil organic carbon density in Pinus massoniana plantations.

Gravel (>2 mm) is one of the main parameters for estimating soil carbon pool. To assess the effects of gravel on soil bulk density (BD) and organic carbon density (SOCD) in Pinus massoniana plantations, we estimated the BD and SOCD at the 0-10, 10-20 and 20-40 cm soil depths of 131 plots under two different conditions, with and without removing gravel. The BD of each soil layer after removing gravel was 0.58-1.57, 0.60-1.67, and 0.59-1.75 g·cm-3, respectively, which was significantly lower than that before removing gravel. Gravel increased the BD by 6.5%-6.8%. The SOCD of each soil layer before removing gravel was 8.93-65.97, 7.63-59.08, and 8.79-94.53 t·hm-2, respectively, which was higher than that after removing gravel. Overall, by neglecting the effect of gravel, SOCD was overestimated by 4.9%-11.8%. As gravel content increased, the relative deviation in the estimated BD and SOCD among different methods increased. When the gravel content was higher than 20%, the estimated SOCD at soil layer of 0-40 cm showed a significant difference between neglecting gravel and removing gravel, with the former being 29.7%-47.4% higher than the latter. In conclusion, gravel markedly affected the estimations of BD and SOCD. It was recommended that SOCD should be estimated by the method that not only uses the BD after removing gravel but also considers gravel as a correction factor (especially when gravel content is above 20.0%) to avoid overestimation of soil carbon pool.

The Concentration of Non-structural Carbohydrates, N, and P in Quercus variabilis Does Not Decline Toward Its Northernmost Distribution Range Along a 1500 km Transect in China.

Understanding the mechanisms that determine plant distribution range is crucial for predicting climate-driven range shifts. Compared to altitudinal gradients, less attention has been paid to the mechanisms that determine latitudinal range limit. To test whether intrinsic resource limitation contributes to latitudinal range limits of woody species, we investigated the latitudinal variation in non-structural carbohydrates (NSC; i.e., total soluble sugar plus starch) and nutrients (nitrogen and phosphorus) in mature and juvenile Chinese cork oak (Quercus variabilis Blume) along a 1500 km north-south transect in China. During the growing season and dormant season, leaves, branches, and fine roots were collected from both mature and juvenile oaks in seven sites along the transect. Tissue concentration of NSCs, N, and P did not decrease with increasing latitude irrespective of sampling season and ontogenetic stage. Furthermore, higher levels of NSCs and N in tissues of juveniles relative to mature trees were found during the dormant season. Partial correlation analysis also revealed that during the dormant season, soluble sugar, NSC, the ratio of soluble sugar to starch, and tissue nitrogen concentration were correlated positively with latitude but negatively with precipitation and mean temperature of dormant season. Our results suggest that carbon or nutrient availability may not be the driving factors of the latitudinal range limit of the studied species. Further studies should be carried out at the community or ecosystem level with multiple species to additionally test the roles of factors such as regeneration, competition, and disturbance in determining a species' northern distribution limit.

[Latitudinal trends in foliar δ13C and δ15N of Quercus variabilis and their influencing factors.] / æ “çš®æ Žå¶ç‰‡Î´13C和δ15Nçš„çº¬å‘è¶‹åŠ¿åŠå ¶å½±å“å› å­.

We aimed to reveal the latitudinal trends in foliar δ13C and δ15N of Quercus variabilis, a widely distributed species in East Asia, associated with two ontogenetic stages (juvenile and mature trees) along a North-South transect (26°-40° N). The results showed that mature trees had higher foliar δ13C and δ15N values than juveniles. Foliar δ13C and δ15N values of trees with both ontogenetic stages were nonlinearly increased and decreased with latitude, respectively. No interaction between ontogenetic stage and latitude for the changes of foliar δ15N and δ13C indicated that both ontogenetic stages across the transect consistently responded to latitudinal environmental variations. Results from the random forest models indicated that foliar δ15N of Q. variabilis was mainly affected by soil nutrient contents, e.g., soil organic matter, phosphorus, nitrogen, whereas dominated factors for foliar δ13C were related to moisture, such as relative humidity, precipitation of growing season.

Roles of the genomic sequence surrounding the stem-loop structure in the junction region including the 3' terminus of open reading frame 1 in hepatitis E virus replication.

Hepatitis E virus (HEV), a member of the family Hepeviridae, causes both acute and chronic viral hepatitis. We have previously demonstrated that the stem-loop structure in the junction region (JR) of HEV genome plays a critical role in HEV replication. However, the function of the sequence bordering the JR, including the 3' terminus of open reading frame (ORF1), in HEV replication is unknown. In this study, a panel of HEV Renilla luciferase (Rluc) replicons containing various deletions at 5' or 3' termini of the JR was constructed to determine the effect of the deletions on HEV replication in Huh7 human liver cells. We showed that even a single nucleotide deletion at the 5' terminus of the JR abolished HEV replication, whereas deletions at the 3' terminus of the JR also decreased virus replication efficiency. Furthermore, we also constructed firefly luciferase and Rluc dual-reporter HEV replicons containing the 3' terminal ORF1 of various lengths and the JR inserted upstream of the Rluc reporter. A higher level of HEV replication was observed in cells transfected with replicons containing the 3' terminal ORF1 than that of the JR only replicon. We also showed that the ORF3 noncoding sequence along with the JR promoted a higher level of translation activity than that promoted by JR and the ORF2 noncoding sequence.

Recombinant Porcine Reproductive and Respiratory Syndrome Virus Expressing Membrane-Bound Interleukin-15 as an Immunomodulatory Adjuvant Enhances NK and γδ T Cell Responses and Confers Heterologous Protection.

Cytokines are often used as adjuvants to improve vaccine immunogenicity, since they are important in initiating and shaping the immune response. The available commercial modified live-attenuated vaccines (MLVs) against porcine reproductive and respiratory syndrome virus (PRRSV) are unable to mount sufficient heterologous protection, as they typically induce weak innate and inadequate T cell responses. In this study, we investigated the immunogenicity and vaccine efficacy of recombinant PRRSV MLVs incorporated with the porcine cytokine interleukin-15 (IL-15) or IL-18 gene fused to a glycosylphosphatidylinositol (GPI) modification signal that can anchor the cytokines to the cell membrane. We demonstrated that both cytokines were successfully expressed on the cell membrane of porcine alveolar macrophages after infection with recombinant MLVs. Pigs vaccinated with recombinant MLVs or the parental Suvaxyn MLV had significantly reduced lung lesions and viral RNA loads in the lungs after heterologous challenge with the PRRSV NADC20 strain. The recombinant MLVs SUV-IL-15 and SUV-IL-18 recovered the inhibition of the NK cell response seen with Suvaxyn MLV. The recombinant MLV SUV-IL-15 significantly increased the numbers of gamma interferon (IFN-γ)-producing cells in circulation at 49 days postvaccination (dpv), especially for IFN-γ-producing CD4- CD8+ T cells and γδ T cells, compared to the Suvaxyn MLV and SUV-IL-18. Additionally, MLV SUV-IL-15-vaccinated pigs also had elevated levels of γδ T cell responses observed at 7 dpv, 49 dpv, and 7 days postchallenge. These data demonstrate that the recombinant MLV expressing membrane-bound IL-15 enhances NK and T cell immune responses after vaccination and confers improved heterologous protection, although this was not statistically significant compared to the parental MLV.IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS) has arguably been the most economically important global swine disease, causing immense economic losses worldwide. The available commercial modified live-attenuated vaccines (MLVs) against PRRS virus (PRRSV) are generally effective against only homologous or closely related virus strains but are ineffective against heterologous strains, partially due to the insufficient immune response induced by the vaccine virus. To improve the immunogenicity of MLVs, in this study, we present a novel approach of using porcine IL-15 or IL-18 as an adjuvant by directly incorporating its encoding gene into a PRRSV MLV and expressing it as an adjuvant. Importantly, we directed the expression of the incorporated cytokines to the cell membrane surface by fusing the genes with a membrane-targeting signal from CD59. The recombinant MLV virus expressing the membrane-bound IL-15 cytokine greatly enhanced NK cell and γδ T cell responses and also conferred improved protection against heterologous challenge with the PRRSV NADC20 strain.

Substitution of amino acid residue V1213 in the helicase domain of the genotype 3 hepatitis E virus reduces virus replication.

BACKGROUND: Genotype 3 hepatitis E virus (HEV) infection is generally associated with mild disease. However, recently eight genotype 3 HEV isolates were identified from patients with severe hepatitis. Importantly, three mutations (S605P, I978V and V1213A) in these genotype 3 isolates were found to be typical of genotype 4 HEV, which is sometime associated with more severe hepatitis. Therefore in this study we seek to determine if these unique mutations contribute to enhanced virus replication and thus potentially severe disease. METHODS: In the lack of an efficient cell culture system to study the effect of mutations on HEV replication, we developed a genotype 3 HEV replicon with Renilla luciferase (Rluc) as reporter and subsequently used it to construct numerous mutants, including swMu-1 (V1213A), swMu-2 (Q1246H), swMu-3 (V1213A and Q1246H), swMu-4 (S605P and I978V), and swMu-5 (V1213A, S605P and I978V). RNA transcripts from mutant replicons were transfected into Huh7 S10-3 liver cells to measure the effect of mutations on HEV replication efficiency. RESULTS: The results showed that the V1213A mutant had the highest reduction in HEV replication efficiency than other mutants. The V1213A and S605P + I978V mutations have a cumulative, if not synergistic, effect on HEV replication. The Q1246H mutant decreased HEV replication compared to the wild-type HEV Rluc replicon but replicated better than the V1213A mutant. The amino acid residue V1213 favors the replication of both genotypes 3 and 4 HEV strains, but not genotype 1 HEV. CONCLUSION: The results suggested that the V1213A mutation reduced HEV replication, but is likely not associated with the reported severe hepatitis caused by genotype 3 HEV isolates containing this mutation.

Effects of drought on leaf carbon source and growth of European beech are modulated by soil type.

Drought potentially affects carbon balance and growth of trees, but little is known to what extent soil plays a role in the trade-off between carbon gain and growth investment. In the present study, we analyzed leaf non-structural carbohydrates (NSC) as an indicator of the balance of photosynthetic carbon gain and carbon use, as well as growth of European beech (Fagus sylvatica L.) saplings, which were grown on two different soil types (calcareous and acidic) in model ecosystems and subjected to a severe summer drought. Our results showed that drought led in general to increased total NSC concentrations and to decreased growth rate, and drought reduced shoot and stem growth of plants in acidic soil rather than in calcareous soil. This result indicated that soil type modulated the carbon trade-off between net leaf carbon gain and carbon investment to growth. In drought-stressed trees, leaf starch concentration and growth correlated negatively whereas soluble sugar:starch ratio and growth correlated positively, which may contribute to a better understanding of growth regulation under drought conditions. Our results emphasize the role of soil in determining the trade-off between the balance of carbon gain and carbon use on the leaf level and growth under stress (e.g. drought).

[Population structure and spatial pattern of Quercus variabilis among different geographical areas, China.] / ä¸åŒåœ°ç†åŒºåŸŸæ “çš®æ Žç§ç¾¤ç»“æž„åŠå ¶ç©ºé—´æ ¼å±€.

Age structures and spatial distribution patterns of Quercus variabilis populations were analyzed across geographical gradients (latitude, longitude and altitude) by using the size-grading method and the ratio of variance to mean. The results showed as following: Over the horizontal gradient, the northern, middle, southern and western populations of Q. variabilis exhibited an inverse-J shape, but the eastern populations declined. The spatial patterns of adult individuals were all clumped except the northern populations which were randomly distributed. Juveniles were clumped in the northern, middle and western populations, but were randomly distributed in the southern and eastern populations. The aggregation intensity of juveniles across latitude decreased with the order as the central, northern and southern populations, but as the central, southern and northern populations for adult individuals. The aggregation intensity of both juveniles and adults across longitude followed a decreased order as the central, western and eastern populations. Along the altitudinal gra-dient, the inverse-J type occurred only in the low- and middle-altitude populations, but populations in the high altitude declined. The juveniles in populations among altitude gradient all were clumped, but the adults were all clumped except the low-altitude populations which were randomly distributed. The aggregation intensities of both juveniles and adults were higher in the middle than the other altitudinal populations. Compared with adults, juveniles generally had higher aggregation intensities across various geographical gradients. Our results revealed that the age structure and spatial distribution pattern of Q. variabilis were mainly determined by environment variation across geographical gradients and the species' biological property, which supported the central-marginal hypothesis.

[Effects of nitrogen and phosphorus addition on leaf thermal tolerance in different provenances of Quercus variabilis]. / æ°®ç£·æ·»åŠ å¯¹æ “çš®æ Žä¸åŒåœ°ç†ç§æºå¹¼è‹—æ¸©åº¦è€æ€§çš„å½±å“.

To explore potential effects of soil nutrient on leaf thermal tolerance across geographical origins, five provenance, i.e., Chengbu of Hunan (CB), Zigui of Hubei (ZG), Neixiang of Henan (NX), Lincheng of Heibei (LC) and Pinggu of Beijing (PG), seedlings of Quercus variabilis were cultivated under nitrogen and phosphorus addition. Leaf thermal tolerance parameters (cold, heat and span), nutrient concentrations (nitrogen and phosphorus), nitrogen use efficiency (NUE) and phosphorus use efficiency (PUE), as well as non-structural carbohydrate (soluble sugar and starch) concentrations were analyzed. The results were shown as follows: Nutrient absorption and utilization efficiency varied between oak origins with no obvious geographical trends. PG had higher nutrient use efficiency (NUE and PUE), whereas NX had lower PUE under all treatments. CB had the highest PUE under phosphorus addition. Compared with the control, nutrient addition increased the cold tolerance of PG and LC, and to some extent, increased the heat tolerance of the three middle provenances (ZG, NX, LC). However, the thermal span was opposite to the cold tolerance, as nutrient addition decreased the thermal span of PG and LC but increased that of NX. The leaf cold tolerance had significantly positive correlation with soluble sugar concentration, while the heat tolerance negatively and positively correlated with leaf phosphorus and ratio of nitrogen to phosphorus, respectively. No significant correlation was found between leaf thermal span and leaf chemical substances. To sum up, nutrient use efficiency varying in provenances might be contributed by the original habitats and consequently presented with some local adaptation characteristics, which complicated the response of thermal tolerance to nutrient addition.

Defense pattern of Chinese cork oak across latitudinal gradients: influences of ontogeny, herbivory, climate and soil nutrients.

Knowledge of latitudinal patterns in plant defense and herbivory is crucial for understanding the mechanisms that govern ecosystem functioning and for predicting their responses to climate change. Using a widely distributed species in East Asia, Quercus variabilis, we aim to reveal defense patterns of trees with respect to ontogeny along latitudinal gradients. Six leaf chemical (total phenolics and total condensed tannin concentrations) and physical (cellulose, hemicellulose, lignin and dry mass concentration) defensive traits as well as leaf herbivory (% leaf area loss) were investigated in natural Chinese cork oak (Q. variabilis) forests across two ontogenetic stages (juvenile and mature trees) along a ~14°-latitudinal gradient. Our results showed that juveniles had higher herbivory values and a higher concentration of leaf chemical defense substances compared with mature trees across the latitudinal gradient. In addition, chemical defense and herbivory in both ontogenetic stages decreased with increasing latitude, which supports the latitudinal herbivory-defense hypothesis and optimal defense theory. The identified trade-offs between chemical and physical defense were primarily determined by environmental variation associated with the latitudinal gradient, with the climatic factors (annual precipitation, minimum temperature of the coldest month) largely contributing to the latitudinal defense pattern in both juvenile and mature oak trees.

The non-structural protein Nsp2TF of porcine reproductive and respiratory syndrome virus down-regulates the expression of Swine Leukocyte Antigen class I.

Porcine reproductive and respiratory syndrome virus (PRRSV) is arguably the most economically-important global swine pathogen. Here we demonstrated that PRRSV down-regulates Swine Leukocyte Antigen class I (SLA-I) expression in porcine alveolar macrophages, PK15-CD163 cells and monocyte-derived dendritic cells. To identify the viral protein(s) involved in SLA-I down-regulation, we tested all 22 PRRSV structural and non-structural proteins and identified that Nsp1α and Nsp2TF, and GP3 significantly down-regulated SLA-I expression with Nsp2TF showing the greatest effect. We further generated a panel of mutant viruses in which the Nsp2TF protein synthesis was abolished, and found that the two mutants with disrupted -2 ribosomal frameshifting elements and additional stop codons in the TF domain were unable to down-regulate SLA-I expression. Additionally we demonstrated that the last 68 amino acids of TF domain in Nsp2TF are critical for this function. Collectively, the results indicate a novel function of Nsp2TF in negative modulation of SLA-I expression.

Chimeric porcine reproductive and respiratory syndrome virus containing shuffled multiple envelope genes confers cross-protection in pigs.

The extensive genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) strains is a major obstacle for vaccine development. We previously demonstrated that chimeric PRRSVs in which a single envelope gene (ORF3, ORF4, ORF5 or ORF6) was shuffled via DNA shuffling had an improved heterologous cross-neutralizing ability. In this study, we incorporate all of the individually-shuffled envelope genes together in different combinations into an infectious clone backbone of PRRSV MLV Fostera(®) PRRS. Five viable progeny chimeric viruses were rescued, and their growth characteristics were characterized in vitro. In a pilot pig study, two chimeric viruses (FV-SPDS-VR2,FV-SPDS-VR5) were found to induce cross-neutralizing antibodies against heterologous strains. A subsequent vaccination/challenge study in 72 pigs revealed that chimeric virus FV-SPDS-VR2 and parental virus conferred partial cross-protection when challenged with heterologous strains NADC20 or MN184B. The results have important implications for future development of an effective PRRSV vaccine that confers heterologous protection.

Computer-aided codon-pairs deoptimization of the major envelope GP5 gene attenuates porcine reproductive and respiratory syndrome virus.

Synthetic attenuated virus engineering (SAVE) is an emerging technology that enables rapid attenuation of viruses. In this study, by using SAVE we demonstrated rapid attenuation of an arterivirus, porcine reproductive and respiratory syndrome virus (PRRSV). The major envelope GP5 gene of PRRSV was codon-pair deoptimized aided by a computer algorithm. The codon-pair deoptimized virus, designated as SAVE5 with a deoptimized GP5 gene, was successfully rescued in vitro. The SAVE5 virus replicated at a lower level in vitro with a significant decrease of GP5 protein expression compared to the wild-type PRRSV VR2385 virus. Pigs experimentally infected with the SAVE5 virus had significantly lower viremia level up to 14 days post-infection as well as significantly reduced gross and histological lung lesions when compared to wild-type PRRSV VR2385 virus-infected pigs, indicating the attenuation of the SAVE5 virus. This study proved the feasibility of rapidly attenuating PRRSV by SAVE.

Broadening the heterologous cross-neutralizing antibody inducing ability of porcine reproductive and respiratory syndrome virus by breeding the GP4 or M genes.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important swine pathogens, which causes reproductive failure in sows and respiratory disease in piglets. A major hurdle to control PRRSV is the ineffectiveness of the current vaccines to confer protection against heterologous strains. Since both GP4 and M genes of PRRSV induce neutralizing antibodies, in this study we molecularly bred PRRSV through DNA shuffling of the GP4 and M genes, separately, from six genetically different strains of PRRSV in an attempt to identify chimeras with improved heterologous cross-neutralizing capability. The shuffled GP4 and M genes libraries were each cloned into the backbone of PRRSV strain VR2385 infectious clone pIR-VR2385-CA. Three GP4-shuffled chimeras and five M-shuffled chimeras, each representing sequences from all six parental strains, were selected and further characterized in vitro and in pigs. These eight chimeric viruses showed similar levels of replication with their backbone strain VR2385 both in vitro and in vivo, indicating that the DNA shuffling of GP4 and M genes did not significantly impair the replication ability of these chimeras. Cross-neutralization test revealed that the GP4-shuffled chimera GP4TS14 induced significantly higher cross-neutralizing antibodies against heterologous strains FL-12 and NADC20, and similarly that the M-shuffled chimera MTS57 also induced significantly higher levels of cross-neutralizing antibodies against heterologous strains MN184B and NADC20, when compared with their backbone parental strain VR2385 in infected pigs. The results suggest that DNA shuffling of the GP4 or M genes from different parental viruses can broaden the cross-neutralizing antibody-inducing ability of the chimeric viruses against heterologous PRRSV strains. The study has important implications for future development of a broadly protective vaccine against PRRSV.

Attenuation of porcine reproductive and respiratory syndrome virus by molecular breeding of virus envelope genes from genetically divergent strains.

Molecular breeding via DNA shuffling can direct the evolution of viruses with desired traits. By using a positive-strand RNA virus, porcine reproductive and respiratory syndrome virus (PRRSV), as a model, rapid attenuation of the virus was achieved in this study by DNA shuffling of the viral envelope genes from multiple strains. The GP5 envelope genes of 7 genetically divergent PRRSV strains and the GP5-M genes of 6 different PRRSV strains were molecularly bred by DNA shuffling and iteration of the process, and the shuffled genes were cloned into the backbone of a DNA-launched PRRSV infectious clone. Two representative chimeric viruses, DS722 with shuffled GP5 genes and DS5M3 with shuffled GP5-M genes, were rescued and shown to replicate at a lower level and to form smaller plaques in vitro than their parental virus. An in vivo pathogenicity study revealed that pigs infected with the two chimeric viruses had significant reductions in viral-RNA loads in sera and lungs and in gross and microscopic lung lesions, indicating attenuation of the chimeric viruses. Furthermore, pigs vaccinated with the chimeric virus DS722, but not pigs vaccinated with DS5M3, still acquired protection against PRRSV challenge at a level similar to that of the parental virus. Therefore, this study reveals a unique approach through DNA shuffling of viral envelope genes to attenuate a positive-strand RNA virus. The results have important implications for future vaccine development and will generate broad general interest in the scientific community in rapidly attenuating other important human and veterinary viruses.

Complete genome sequence of hepatitis E virus from rabbits in the United States.

Hepatitis E virus (HEV) is a single-strand positive-sense RNA virus in the family Hepeviridae. The disease caused by HEV, hepatitis E, is an important public health problem in developing countries of Asia and Africa and is also endemic in many industrialized countries, including the United States. HEV has been identified from several other animal species in addition to humans, including the pig, chicken, mongoose, deer, rabbit, ferret, bat, and fish. Here we report the complete genome sequence of the first strain of HEV from rabbits in the United States. Sequence and phylogenetic analyses revealed that the U.S. rabbit HEV is a distant member of the zoonotic genotype 3 HEV, thus raising a concern for potential zoonotic human infection. A unique 90-nucleotide insertion within the X domain of the ORF1 was identified in the rabbit HEV, and this insertion may play a role in the species tropism of HEV.

DNA shuffling of the GP3 genes of porcine reproductive and respiratory syndrome virus (PRRSV) produces a chimeric virus with an improved cross-neutralizing ability against a heterologous PRRSV strain.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important swine pathogen. Here we applied the DNA shuffling approaches to molecularly breed the PRRSV GP3 gene, a neutralizing antibodies inducer, in an attempt to improve its heterologous cross-neutralizing ability. The GP3 genes of six different PRRSV strains were bred by traditional DNA shuffling. Additionally, synthetic DNA shuffling of the GP3 gene was also performed using degenerate oligonucleotides. The shuffled-GP3-libraries were cloned into the backbone of a DNA-launched PRRSV infectious clone pIR-VR2385-CA. Four traditional-shuffled chimeras each representing all 6 parental strains and four other synthetic-shuffled chimeras were successfully rescued. These chimeras displayed similar levels of replication both in vitro and in vivo, compared to the backbone parental virus, indicating that the GP3 shuffling did not impair the replication capability of the chimeras. One chimera GP3TS22 induced significantly higher levels of cross-neutralizing antibodies in pigs against a heterologous PRRSV strain FL-12.

New model of in-situ xenograft lymphangiogenesis by a human colonic adenocarcinoma cell line in nude mice.

OBJECTIVE: To explore a new model of in-situ xenograft lymphangiogenesis of human colonic adenocarcinomas in nude mice. METHOD: On the basis of establishing subcutaneous xenograft lymphangiogenesis model of human colonic adenocarcinoms, in-situ xenografts were established through the in situ growth of the HT-29 human colonic adenocarcinoma cell line in nude mice. The numbers of lymphangiogenic microvessels, the expression of lymphatic endothelial cell markers lymphatic vessel endothelial hyaloronic acid receptor-1 (LYVE-1), D2-40 and the lymphatic endothelial growth factors vascular endothelial growth factor-C (VEGF-C), -D (VEGF-D) and receptor-3 (VEGFR-3) were compared by immunohistochemical staining, Western bolt and quantitative RT-PCR in xenograft in-situ models. RESULTS: Some microlymphatics with thin walls, large and irregular or collapsed cavities and increased LMVD, with strong positive of LYVE-1, D2-40 in immunohistochemistry, were observed, identical with the morphological characteristics of lymphatic vessels and capillaries. Expression of LYVE-1 and D2-40 proteins and mRNAs were significantly higher in xenografts in-situ than in the negative control group (both P<0.01). Moreover, the expression of VEGF-C, VEGF-D and VEGFR-3 proteins and mRNAs were significantly higher in xenografts in-situ (both P<0.01), in conformity with the signal regulation of the VEGF-C,-D/VEGFR-3 axis of tumor lymphangiogenesis. CONCLUSIONS: In-situ xenografts of a human colonic adenocarcinoma cell line demonstrate tumor lymphangiogenesis. This novel in-situ animal model should be useful for further studying mechanisms of lymph node metastasis, drug intervention and anti-metastasis therapy in colorectal cancer.

Suppression of lymphangiogenesis in human lymphatic endothelial cells by simultaneously blocking VEGF-C and VEGF-D/VEGFR-3 with norcantharidin.

Lymph node metastasis of tumors is a crucial early step in the metastatic process. Tumor lymphangiogenesis plays an important role in promoting tumor metastasis to regional lymph nodes. Norcantharidin (NCTD) has been reported to possess potent anti-angiogenesis and antitumor properties in several cell lines and xenograft tumor models. However, its role in tumor-associated lymphangiogenesis and lymphatic metastasis remains unclear. Here, we investigated the effect of NCTD on proliferation, apoptosis, migration, invasion and the lymphatic tube formation, lymphangiogenesis, of human lymphatic endothelial cells (HLECs) in vitro by MTT, proliferation assay, Hoechst staining and flow cytometry, scraping line method, Matrigel invasion assay, inverted or fluorescence microscope and transmission electron microscope. Moreover, the underlying mechanisms, such as VEGF-C, VEGF-D, VEGFR-3 at protein and mRNA levels in lymphangiogenesis using 3-dimensional (3-D) culture of HLECs were measured by immunohistochemistry, western blotting and real-time polymerase chain reaction (RT-PCR). It was shown that NCTD inhibited proliferation, migration, invasion and lymphatic tube formation (forming-lymphatic and/or formed-lymphatic) of HLECs, induced HLEC apoptosis (all P<0.01) significantly, in a dose- and time-dependent manner (IC50 6.8 µg/ml); and downregulated the expression of VEGF-C, VEGF-D and VEGFR-3 at protein or/and mRNA levels (P<0.01) in HLEC lymphatic tube formation. Thus, we identified for the first time that NCTD inhibited HLEC lymphangiogenesis by simultaneously blocking VEGF-C and VEGF-D/VEGFR-3 in vitro. The present findings may be of importance to explore the therapeutical target or strategy of NCTD for tumor lymphangiogenesis and lymphatic metastasis.

Establishment of a DNA-launched infectious clone for a highly pneumovirulent strain of type 2 porcine reproductive and respiratory syndrome virus: identification and in vitro and in vivo characterization of a large spontaneous deletion in the nsp2 region.

A highly pneumovirulent strain of porcine reproductive and respiratory syndrome virus (PRRSV), ATCC VR2385, was isolated from a pig exhibiting typical PRRS in the early 90s. While passaging the virus in monkey kidney cells, we identified a large spontaneous deletion of a 435-bp in the nsp2 gene. To assess the biological significance of this spontaneous deletion, we first determined the full-length genomic sequence of this virus and established a DNA-launched infectious clone of the passage 14 virus containing the 435-bp nsp2 deletion (designated as pIR-VR2385-CA). The full-length viral genome engineered with two ribozyme elements at both ends was placed under the control of the eukaryotic CMV promoter. The infectious virus was successfully rescued from pIR-VR2385-CA DNA-transfected BHK-21 cells. To characterize the biological and pathological significance of this large nsp2 deletion, we subsequently constructed another DNA-launched infectious clone, pIR-VR2385-R, in which we restored the deleted 435-bp nsp2 sequence back to the pIR-VR2385-CA backbone. The growth characteristics of the two rescued viruses (VR2385-CA and VR2385-R) were compared, and the results showed that the VR2385-CA virus with the nsp2 deletion replicated more efficiently in vitro (1.0-1.5 log titer higher) than the VR2385-R virus with the restored nsp2 sequence but the VR2385-CA virus exhibited a significantly reduced serum viral RNA load in vivo. A comparative pathogenicity study in pigs (n=10) revealed that the nsp2 deletion had no effect on virus virulence, and the restored nsp2 sequence in the VR2385-R virus remains stable during virus replication in pigs. The results from this study indicates that the spontaneous nsp2 deletion plays a role for enhanced PRRSV replication in vitro but has no effect on the pathogenicity of the virus.