Complete replication of hepatitis C virus in cell culture

Many aspects of the hepatitis C virus (HCV) life cycle have not been reproduced in cell culture, which has slowed research progress on this important human pathogen. Here, we describe a full-length HCV genome that replicates and produces virus particles that are infectious in cell culture (HCVcc). Replication of HCVcc was robust, producing nearly 10(5) infectious units per milliliter within 48 hours. Virus particles were filterable and neutralized with a monoclonal antibody against the viral glycoprotein E2. Viral entry was dependent on cellular expression of a putative HCV receptor, CD81. HCVcc replication was inhibited by interferon-alpha and by several HCV-specific antiviral compounds, suggesting that this in vitro system will aid in the search for improved antivirals.     

发达国家畜禽废物处理技术与立法

源于集约化畜禽养殖场的大量粪便及废水导致的环境污染问题越来越受到人们的关注,发达国家十分重视畜禽废物的处理技术研究及控制由其引起污染的立法工作。该文在分析畜禽废物管理系统的构成、约束及目标基础上,对发达国家近年来处理技术的发展进行综述,包括还田利用、好氧与厌氧生物处理、存贮、收集与运输、提取新产品及优化模型与决策支持系统等方面,最后对有关畜禽废物管理方面的法规做了介绍。     

Bioaccumulation of Total Mercury and Monomethylmercury in the Earthworm Eisenia Fetida

Bioaccumulation factors (BAFs) for inorganic mercury in earthworms are usually < 1; however, factors up to ～10 have been reported. Little information is available concerning the bioaccumulation of organic mercury in earthworms from actual contaminated soils and thus there has been uncertainty in the risk characterization phase of ecological risk assessments of mercury-contaminated sites. This study was initiated to determine the rate of uptake and bioaccumulation of total mercury (T-Hg) and monomethylmercury (MMHg) in Eisenia fetida from soils which have been contaminated with mercury for approximately 30 years. The study consisted of a 28-day uptake phase in three mercury-contaminated soils and one soil with background concentrations of mercury followed by a 14-day depuration phase in background soil only. Total mercury concentrations in the study soils ranged from 85 to 11,542 μg kg^?1 dry weight soil; MMHg concentrations ranged from 1.12 to 7.35 μg kg^?1 dry weight soil. Time to 90% steady states for T-Hg ranged from 36 to 42 days. A steady state did not occur for any of the MMHg exposures during the 42-day study; estimated time to 90% steady state varied from 97 to 192 days. BAFs for T-Hg ranged from 0.6 to 3.3. BAFs for MMHg ranged from 175 to 249. The BAFs for T-Hg and MMHg were larger in earthworms exposed to the lower contaminated soils and smaller in the higher mercury-contaminated soils. The absolute concentrations of T-Hg and MMHg bioaccumulated in E. fetida, however, were higher in the earthworms exposed to the higher mercury soils and lower in the less mercury-contaminated soils.     

Evaluating homogeneity of LL601 rice in commercial lots using quantitative real-time PCR

Homogeneity analysis was performed on four distinctive commercial lots, derived from the 2006 rice harvest in the United States. Lots that had previously been tested and suspected to have some level of LL601 were selected to determine lot homogeneity. LL601 infiltration in the lots was low and estimated to contain <0.01% (sigma = 0.026), 0.014% (sigma = 0.020), 0.054% (sigma = 0.043), and 0.074% (sigma = 0.031) LL601. Lots were analyzed statistically as a one-way classification, or one-factor experiment, to assess the presence of strata within the lot. A p value of 0.05 or lower is needed to declare statistical significance and would suggest significant differences among the samples. The data revealed p values ranging between 0.105 and 0.607. The calculated p values for all lots were greater than the critical value of 0.05. Samples taken from different locations throughout these four commercial lots did not show statistically significant stratifications within the lot.     

Chemical and mineral control of soil carbon turnover in abandoned tropical pastures

We investigated changes in soil carbon (C) cycling with reforestation across a long-term, replicated chronosequence of tropical secondary forests regrowing on abandoned pastures. We applied CP MAS ¹³C NMR spectroscopy and radiocarbon modeling to soil density fractions from the top 10 cm to track changes in C chemistry and turnover during secondary forest establishment on former pastures. Our results showed that inter-aggregate, unattached, particulate organic C (free light fraction) and particulate C located inside soil aggregates (occluded light fraction) represent distinct soil C pools with different chemical composition and turnover rates. The signal intensity of the O-alkyl region, primarily representing carbohydrates, decreased, and alkyl C, attributed to recalcitrant waxy compounds and microbially resynthesized lipids, increased from plant litter to soil organic matter and with incorporation into soil aggregates. The alkyl/O-alkyl ratio, a common index of humification, was higher in the occluded than in the free light fraction. Greater variability in the chemical makeup of the occluded light fraction suggests that it represents material in varied stages of decomposition. Mean residence times (¹⁴C-based) of the free light fraction were significantly shorter (4 ± 1 years) than for the heavy fraction. We report two scenarios for the occluded light fraction, one fast-cycling in which the occluded and free light fractions have similar turnover rates, and one slow-cycling, in which the occluded light fraction resembles the heavy fraction. Mean residence times of the occluded light fraction and heavy fraction in active pastures and 10-year old secondary forests in the earliest stage of succession were longer than in older secondary forests and primary forests. This is likely due to a preferential loss of physically unprotected C of more labile composition in the pastures and in the youngest successional forests, resulting in an increase in the dominance of slow-cycling C pools. Soil carbon turnover rates of the mineral-associated C in secondary forests recovering from abandoned pasture resembled those of primary forests in as little as 20 years of succession.     

Extensive large colon resection in the pony. I. Surgical procedures and clinical results

The effect of resection of 75% (length measurement) of the large colon in ponies was studied. Ten ponies in good physical condition were divided into two groups: group I consisted of the six experimental ponies and group II of four control ponies. Preoperatively and postoperatively for five months, ten clinicopathological parameters were determined: body weight, venous blood pH, plasma bicarbonate, total plasma protein concentration, serum electrolyte values (sodium, potassium, chloride), and fecal osmolarity. Subjective assessment of attitude and appetite revealed no difference between the groups or within groups at any time. Resection of a major portion of the large colon did not significantly influence the clinicopathological parameters evaluated. The hematocrit of the ponies in group I did decrease during the first postoperative month and this was attributed to whole blood loss associated with the resection procedures. The results of this investigation indicated that resection of 75% of the large colon in ponies is compatible with life.     

An immunogenomics approach to understanding periparturient immunosuppression and mastitis susceptibility in dairy cows

Studies comparing in vivo and in vitro functional capacities of leukocytes from non-parturient and periparturient dairy cows have provided substantial evidence that systemic and local mammary immune defenses are deficient around parturition. This evidence has lead to the reasonable hypothesis that immune deficiency underlies the heightened mastitis susceptibility of periparturient cows. Nutrition and vaccine studies substantiate this hypothesis, showing that dietary antioxidant supplementation and rigorous immunization regimes can bolster innate and humoral immunity to the point that mastitis severity and time for return to normal milk production are reduced. However, completely effective resolution of this significant production disease has not been achieved because so little is understood about its complex etiology. In particular, we possess almost no knowledge of how or why immune cells responding to parturient physiology end up with deficient functional capacities. Fluctuations in reproductive steroid hormones and chronic shifts in neuroendocrine hormones with roles in nutrient partitioning and appetite control may affect the expression of critical leukocyte genes in periparturient dairy cows. A thorough understanding of leukocyte biology during periparturition would seem a critical goal for future development of effective mastitis prevention strategies. Recently, our group has begun to use cDNA microarray technology to explore bovine leukocyte RNA for global gene expression changes occurring around parturition. We are working within the context of a hypothesis that the physiology of parturition negatively affects expression of critical genes in blood leukocytes. In the current study we initiated hypothesis testing using leukocyte RNA from a high producing Holstein cow collected at 14 days prepartum and 6 hours postpartum to interrogate a cDNA microarray spotted with > 700 cDNAs representing unique bovine leukocyte genes. This analysis revealed 18 genes with > or = 1.2-fold higher expression 14 days prepartum than 6 hours postpartum. BLASTN analysis of these genes revealed only one that can be considered a classical immune response gene. All other repressed genes were either unknown or putatively identified as encoding key proteins involved in normal growth and metabolism of cells. Given the critical roles of these repressed genes in normal cell functions, we may have identified good candidates to pursue with respect to periparturient immunosuppression and mastitis susceptibility.     

Pre-translational regulation of neutrophil L-selectin in glucocorticoid-challenged cattle.

L-selectin (CD62L) gene expression in neutrophils is commonly referred to as "constitutive" because circulating neutrophils require a constant supply of this adhesion molecule for continuous trafficking into peripheral tissues. Under normal circumstances, marginating blood neutrophils and neutrophils that become activated for migration into infected tissues rapidly shed surface CD62L that is ligated to the vascular endothelium. However, this does not shut down CD62L gene expression because these cells continue to express surface CD62L. In contrast, glucocorticoid challenges resulting from stress and hormone injections result in gradual and chronic down-regulation of CD62L on the surface of blood neutrophils. Rather than being associated with migration, this type of CD62L down-regulation associates with pronounced neutrophilia and increased susceptibility to infections. Nothing is currently known about glucocorticoid regulation of CD62L expression in neutrophils. In other cell systems, however, this steroid hormone binds to cytoplasmic glucocorticoid receptors (GR) that influence expression of glucocorticoid-responsive genes at multiple pre-translational levels. Thus, the hypothesis of the present study was that glucocorticoid challenge suppresses CD62L mRNA expression in blood neutrophils. Suppressed CD62L gene expression might help explain the chronic down-regulation of surface CD62L in neutrophils and accompanying neutrophilia. The main objectives of the study were to monitor neutrophil CD62L mRNA abundance before and during subtle and severe glucocorticoid challenges and to determine if CD62L mRNA expression correlates with degree of glucocorticoid challenge. Parturient dairy cows and dexamethasone-treated steers were used as models of subtle and severe (respectively) glucocorticoid challenges. Data presented from both models support the hypothesis and show for the first time that glucocorticoids regulate neutrophil CD62L at a pre-translational level. Results also showed that inhibited CD62L mRNA expression correlated precisely with down-regulated surface expression of CD62L on neutrophils and peak neutrophilia during severe glucocorticoid challenge. Therefore, results of this study indicate that bovine neutrophils are highly sensitive to the blood environment, displaying full capacity to alter CD62L gene expression and trafficking patterns in response to changing glucocorticoid levels. This may serve animals well when heightened inflammatory responses begin to lead to tissue damage, but may be detrimental to overall health if animals are exposed to opportunistic pathogens while stressed or undergoing glucocorticoid therapy. Although this study did not elucidate how glucocorticoids inhibit neutrophil CD62L mRNA expression, presented data implicate GR as possibly being involved because neutrophils from cattle in both models expressed GR mRNA. Further in vitro studies using purified populations of neutrophils will be required to determine if GR is directly involved in glucocorticoid regulation of CD62L gene expression and, if so, at what level.     

Influences of Follicular Size on Parthenogenetic Activation and in Vitro Heat Shock on the Cytoskeleton in Cattle Oocytes

The availability of cow ovaries from the slaughterhouse has been very limited in Taiwan. To maximize the use of cow ovaries for research purposes, whole ovary dissection was performed and the developmental competence of the oocytes derived from different sizes of follicles was assessed by the rates of in vitro maturation (IVM) and parthenogenetic activation of the oocytes in Experiment 1 (Exp 1). Cumulus-oocyte complexes (COCs) derived from small (1-2 mm) and large (3-8 mm) follicles were subjected to standard IVM culture for 24 h. Mature oocytes were selected and then parthenogenetically activated using A23187 (5 microm, 5 min) or thimerosal (200 microm, 10 min) alone or combined with 6-dimethylaminopurine (2.5 mm and 3.5 h, respectively). Activation rates of the oocytes, neither from the large nor small follicles, were affected by different activation treatments (single or combined stimuli). Whereas maturation rates for the oocytes from large follicles were superior to those from small follicles in both the single (59% vs 45%) and combined treatments (76% vs 40%; p < 0.05). To understand how prolonged heat shock (HS) influences cytoskeletal configurations of mature bovine oocytes, in Experiment 2 (Exp 2), matured oocytes derived from large follicles were randomly allocated to different durations of HS treatments at 41.5 degrees C for 0 (C0h, control, n = 12), 1 (HS1h, n = 28), 2 (HS2h, n = 31), and 4 h (HS4h, n = 30). An additional control group was cultured for 4 h without HS (38.5 degrees C, 4 h, n = 35). Alterations in nuclear structures, microtubules (MTs), and microfilaments (MFs) of the oocytes were examined. Abnormalities in the chromosomes, spindle MTs and the percentages of oocytes with cytoplasmic MTs increased with time of HS treatment. The intensity of the MF distribution in the HS oocytes was also altered. Significant changes in the cytoskeleton after HS may be associated with the reduced development under hyperthermia and, perhaps, with the low pregnancy rates of the animals during hot seasons.