Lentivirus pre-packed with Cas9 protein for safer gene editing.

The CRISPR/Cas9 system provides an easy way to edit specific site/s in the genome and thus offers tremendous opportunity for human gene therapy for a wide range of diseases. However, one major concern is off-target effects, particularly with long-term expression of Cas9 nuclease when traditional expression methods such as via plasmid/viral vectors are used. To overcome this limitation, we pre-packaged Cas9 protein (Cas9P LV) in lentiviral particles for transient exposure and showed its effectiveness for gene disruption in cells, including primary T cells expressing specific single guide RNAs (sgRNAs). We then constructed an 'all in one virus' to express sgRNAs in association with pre-packaged Cas9 protein (sgRNA/Cas9P LV). We successfully edited CCR5 in TZM-bl cells by this approach. Using an sgRNA-targeting HIV long terminal repeat, we also were able to disrupt HIV provirus in the J-LAT model of viral latency. Moreover, we also found that pre-packaging Cas9 protein in LV particle reduced off-target editing of chromosome 4:-29134166 locus by CCR5 sgRNA, compared with continued expression from the vector. These results show that sgRNA/Cas9P LV can be used as a safer approach for human gene therapy applications.

Models for Estimating Genetic Parameters of Milk Production Traits Using Random Regression Models in Korean Holstein Cattle.

The objectives of the study were to estimate genetic parameters for milk production traits of Holstein cattle using random regression models (RRMs), and to compare the goodness of fit of various RRMs with homogeneous and heterogeneous residual variances. A total of 126,980 test-day milk production records of the first parity Holstein cows between 2007 and 2014 from the Dairy Cattle Improvement Center of National Agricultural Cooperative Federation in South Korea were used. These records included milk yield (MILK), fat yield (FAT), protein yield (PROT), and solids-not-fat yield (SNF). The statistical models included random effects of genetic and permanent environments using Legendre polynomials (LP) of the third to fifth order (L3-L5), fixed effects of herd-test day, year-season at calving, and a fixed regression for the test-day record (third to fifth order). The residual variances in the models were either homogeneous (HOM) or heterogeneous (15 classes, HET15; 60 classes, HET60). A total of nine models (3 orders of polynomials×3 types of residual variance) including L3-HOM, L3-HET15, L3-HET60, L4-HOM, L4-HET15, L4-HET60, L5-HOM, L5-HET15, and L5-HET60 were compared using Akaike information criteria (AIC) and/or Schwarz Bayesian information criteria (BIC) statistics to identify the model(s) of best fit for their respective traits. The lowest BIC value was observed for the models L5-HET15 (MILK; PROT; SNF) and L4-HET15 (FAT), which fit the best. In general, the BIC values of HET15 models for a particular polynomial order was lower than that of the HET60 model in most cases. This implies that the orders of LP and types of residual variances affect the goodness of models. Also, the heterogeneity of residual variances should be considered for the test-day analysis. The heritability estimates of from the best fitted models ranged from 0.08 to 0.15 for MILK, 0.06 to 0.14 for FAT, 0.08 to 0.12 for PROT, and 0.07 to 0.13 for SNF according to days in milk of first lactation. Genetic variances for studied traits tended to decrease during the earlier stages of lactation, which were followed by increases in the middle and decreases further at the end of lactation. With regards to the fitness of the models and the differential genetic parameters across the lactation stages, we could estimate genetic parameters more accurately from RRMs than from lactation models. Therefore, we suggest using RRMs in place of lactation models to make national dairy cattle genetic evaluations for milk production traits in Korea.

A genome-wide analysis of the ultimate pH in swine.

Meat pH is an important factor influencing meat quality traits in swine. This study evaluated a large number of genetic variants that covered all of the swine chromosomal regions. Approximately 68,000 single nucleotide polymorphisms (SNPs), found on Illumina Porcine SNP chips, were tested for associations with meat pH values. A genome-wide association study (GWAS) found that 19 SNPs on Sus scrofa chromosome 4 were significantly associated with pH. Two major candidate genomic regions were defined: a 1.08-Mb region (at nucleotide 30118313 to 31207050) contained 10 significant SNPs, based on an effect value of 5.0; and a 2.7-Mb genomic region (at nucleotide 73293076 to 76023681) contained 9 significant SNPs. Three putative genes--PKHD1L1, VCPIP1, and LOC102166532--were identified by GWAS near significant SNPs. These genes may account for variations in pH levels. Three pseudogenes and two non-coding RNAs were also detected by GWAS analysis. Estimations of expected and observed P values for pH revealed significant departures from the null hypothesis. A total of 9 haplotype blocks (HB) were constructed: HBs 1, 3, and 5 showed significant effects on pH24 and pH45, whereas an association was not confirmed between pH24 and HBs 4, 6, and 8. Findings from this study indicate that the three genes identified may influence pH of pig meat.

Estimation of Genetic Associations between Production and Meat Quality Traits in Duroc Pigs.

Data collected from 690 purebred Duroc pigs from 2009 to 2012 were used to estimate the heritability, and genetic and phenotypic correlations between production and meat quality traits. Variance components were obtained through the restricted maximum likelihood procedure using Wombat and SAS version 9.0. Animals were raised under the same management in five different breeding farms. The average daily gain, loin muscle area (LMA), backfat thickness (BF), and lean percent (LP) were measured as production traits. Meat quality traits included pH, cooking loss, lightness (L*), redness (a*), yellowness (b*), marbling score (MS), moisture content (MC), water holding capacity (WHC), and shear force. The results showed that the heritability estimates for meat quality traits varied largely from 0.19 to 0.79. Production traits were moderate to highly heritable from 0.41 to 0.73. Genotypically, the BF was positively correlated (p<0.05) with MC (0.786), WHC (0.904), and pH (0.328) but negatively correlated with shear force (-0.533). The results of genetic correlations indicated that selection for less BF could decrease pH, moisture content, and WHC and increase the shear force of meat. Additionally, a significant positive correlation was recorded between average daily gain and WHC, which indicates pork from faster-growing animals has higher WHC. Furthermore, selection for larger LMA and LP could increase MS and lightness color of meat. The meat quality and production traits could be improved simultaneously if desired. Hence, to avoid further deterioration of pork characteristics, appropriate selection of traits should be considered.

Estimation of Genetic Parameters for Somatic Cell Scores of Holsteins Using Multi-trait Lactation Models in Korea.

The study was conducted to analyze the genetic parameters of somatic cell score (SCS) of Holstein cows, which is an important indicator to udder health. Test-day records of somatic cell counts (SCC) of 305-day lactation design from first to fifth lactations were collected on Holsteins in Korea during 2000 to 2012. Records of animals within 18 to 42 months, 30 to 54 months, 42 to 66 months, 54 to 78 months, and 66 to 90 months of age at the first, second, third, fourth and fifth parities were analyzed, respectively. Somatic cell scores were calculated, and adjusted for lactation production stages by Wilmink's function. Lactation averages of SCS (LSCS1 through LSCS5) were derived by further adjustments of each test-day SCS for five age groups in particular lactations. Two datasets were prepared through restrictions on number of sires/herd and dams/herd, progenies/sire, and number of parities/cow to reduce data size and attain better relationships among animals. All LSCS traits were treated as individual trait and, analyzed through multiple-trait sire models and single trait animal models via VCE 6.0 software package. Herd-year was fitted as a random effect. Age at calving was regressed as a fixed covariate. The mean LSCS of five lactations were between 3.507 and 4.322 that corresponded to a SCC range between 71,000 and 125,000 cells/mL; with coefficient of variation from 28.2% to 29.9%. Heritability estimates from sire models were within the range of 0.10 to 0.16 for all LSCS. Heritability was the highest at lactation 2 from both datasets (0.14/0.16) and lowest at lactation 5 (0.11/0.10) using sire model. Heritabilities from single trait animal model analyses were slightly higher than sire models. Genetic correlations between LSCS traits were strong (0.62 to 0.99). Very strong associations (0.96 to 0.99) were present between successive records of later lactations. Phenotypic correlations were relatively weaker (<0.55). All correlations became weaker at distant lactations. The estimated breeding values (EBVs) of LSCS traits were somewhat similar over the years for a particular lactation, but increased with lactation number increment. The lowest EBV in first lactation indicated that selection for SCS (mastitis resistance) might be better with later lactation records. It is expected that results obtained from these multi-trait lactation model analyses, being the first large scale SCS data analysis in Korea, would create a good starting step for application of advanced statistical tools for future genomic studies focusing on selection for mastitis resistance in Holsteins of Korea.

Curcumin decreases oleic acid-induced lipid accumulation via AMPK phosphorylation in hepatocarcinoma cells.

BACKGROUND AND OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is one of the most common metabolic syndromes and is characterized by the accumulation of hepatic triglycerides (TG), which result from an imbalance between uptake, synthesis, export, and oxidation of fatty acids. Curcumin is a polyphenol derived from the herbal remedy and dietary spice turmeric, was found to prevent obesity and diabetes in mouse models. However, a hypolipidemic effect of curcumin in oleic acid- induced hepatocarcinoma cells has not been reported. In this study, we examined the effect of curcumin on reducing lipid accumulation in hepatic cells. MATERIALS AND METHODS: Hepatocytes were treated with oleic acid (OA) containing with or without curcumin to observe the lipid accumulation by Oil Red O stain. We also tested the effects of curcumin on triglycerides (TG) and total cholesterol (TC) in HepG2 cells. Western blot and reverse transcription polymerase chain reaction (RT-PCR) was used to measure sterol regulatory element binding proteins-1 (SREBP-1), fatty acid synthase (FAS), peroxisome proliferator-activated receptor (PPAR)-α, and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) expression. RESULTS: Curcumin suppressed OA-induced lipid accumulation and TG and TC levels. Also, curcumin decreased hepatic lipogenesis such as SREBP-1, and FAS. Besides, we also found out the antioxidative effect of curcumin by increasing the expression of PPARα. Curcumin increased AMPK phosphorylation in hepatocytes. CONCLUSIONS: These results indicated that curcumin has the same ability to activate AMPK and then reduce SREBP-1, and FAS expression, finally leading to inhibit hepatic lipogenesis and hepatic antioxidative ability. In this report, we found curcumin exerted a regulatory effect on lipid accumulation by decreasing lipogenesis in hepatocyte. Therefore, curcumin extract may be active in the prevention of fatty liver.

DPSC-Derived Extracellular Vesicles Promote Rat Jawbone Regeneration.

Repair and functional reconstruction of large jawbone defects remain one of the challenges in the field of head and neck surgery. The recent progress in tissue engineering technologies and stem cell biology has significantly promoted the development of regenerative reconstruction of jawbone defects. The multiple trophic activities of extracellular vesicles (EVs) produced by mesenchymal stem cells (MSCs) may play a critical role in their therapeutic effects. Accumulating evidence has shown the promise of dental pulp stem cells (DPSCs) in bone regeneration, but less is known about the regenerative effects of DPSC-EVs on jawbone defects. The purpose of this study is to explore the osteogenic effects of DPSC-EVs on jawbone marrow-derived MSCs (JB-MSCs) in vitro and their osteoinductive effects in a mandibular bone defect model in rats. Our results showed that JB-MSCs could efficiently uptake DPSC-EVs, which in turn significantly promoted the expression of osteogenic genes, such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteocalcin (OCN), as well as the osteogenic differentiation capability of JB-MSCs. Meanwhile, we found that the pro-osteogenic effect in vitro induced by DPSC-EVs was comparable to that induced by BMP-2 (bone morphogenetic protein 2), currently the only Food and Drug Administration-approved osteoinductive growth factor. In vivo, animals that were locally treated with DPSC-EVs laden with a commercially available collagen membrane exhibited a relatively fast wound closure and increased new bone density at the mandible defects. Our results provide evidence for the osteogenic and osteoinductive effects of DPSC-EVs on jawbone regeneration. Due to the accessibility, rapid proliferation, and osteogenic propensity of DPSCs, DPSC-EVs may represent a safe cell-free therapeutic approach for craniofacial bone regeneration.

Anti-inflammatory effects of Hylomecon hylomeconoides in RAW 264.7 cells.

BACKGROUND AND OBJECTIVES: Papaveraceae serve as a rich source of various alkaloids which have anti-inflammatory effect. MATERIALS AND METHODS: In this study, we investigated the effect of Hylomecon hylomeconoides ethanol extract (HHE) on lipopolysaccharide (LPS)-induced NO and interleukin-6 (IL-6) production in RAW 264.7 cells. RESULTS: HHE inhibited LPS-induced NO and IL-6 production. Moreover, HHE suppressed the phosphorylation of ERK1/2 and p38 in LPS-induced RAW 264.7 in a dose-dependent manner. Furthermore, major constituents, dihydrosanguinarine and 6-methoxydihydrosanguinarine, of the chloroform-soluble extract were analyzed. CONCLUSIONS: Taken together, the results of this study indicate that the anti-inflammatory effects of HHE may occur via the inhibition of NO and IL-6 expression through the down-regulation of MAP kinase (ERK1/2, p38) phosphorylation in RAW 264.7 cells.

Antibacterial activity of bark of Alnus pendula against methicillin-resistant Staphylococcus aureus.

BACKGROUND AND OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) infections are a rapidly growing health problem around the globe. Recently, there has been considerable interest in the use of plant materials as an alternative method to control pathogenic microorganisms. In this study we evaluated the antibacterial activity of bark of Alnus pendula against MRSA. MATERIALS AND METHODS: The MIC determination was done using the microdilution broth method and bacterial growth was determined by measuring optical density using spectrophotometer. RESULTS: Alnus pendula bark EtOH extract and fractions (F-1, -2, -3 and -4) were investigated against MRSA. The most active fractions (F-3 and F-4) led to the isolation of oregonin (ORE) and hirsutanone (HIR). These compounds were active against MRSA strains with minimum inhibitory concentrations (MICs) ranging from 31.25 to 250 microg/ml MIC and 2 MIC of HIR completely inhibited the growth of MRSA. CONCLUSIONS: The bark EtOH extract of Alnus Pendula has potent antibacterial activity against MRSA.

Effect of alcohol fermented feed on lactating performance, blood metabolites, milk Fatty Acid profile and cholesterol content in holstein lactating cows.

A feeding experiment with 40 lactating Holstein cows and 4 dietary treatments was conducted to investigate supplementation with different levels of alcohol fermented feed to the TMR on lactating performance, blood metabolites, milk fatty acid profile and cholesterol concentration of blood and milk. Forty Holstein lactating cows (106±24 d post-partum; mean±SD) were distributed into four groups and randomly assigned to one of four treatments with each containing 10 cows per treatment. The treatment supplemented with TMR (DM basis) as the control (CON), and CON mixed with alcohol-fermented feeds (AFF) at a level of 5%, 10% and 15% of the TMR as T1, T2 and T3, respectively. Dry matter intake and milk yield were not affected by supplementation of AFF. An increased 4% FCM in the milk occurred in cows fed T3 diet compared with CON, while T1 and T2 diets decreased 4% FCM in a dose dependent manner. Supplementation of AFF increased the concentration of albumin, total protein (TP), ammonia, and high density lipoprotein-cholesterol in serum compared with CON. In contrast, supplementation with AFF clearly decreased concentration of blood urea nitrogen (BUN) and total cholesterol (TC) compare with CON. AFF supplementation increased the proportion of C18:1n9 and C18:2n6 compared to CON. A decrease in the concentration of saturated fatty acid (SFA) for T1, T2 and T3 resulted in an increased unsaturated fatty acid (USFA) to SFA ratio compared to CON. Concentration of cholesterol in milk fat was reduced in proportion to the supplemental level of AFF. Feeding a diet supplemented with a moderate level AFF to lactating cows could be a way to alter the feed efficiency and fatty acid profile of milk by increasing potentially human consumer healthy fatty acid without detrimental effects on feed intake and milk production. A substantially decreased cholesterol proportion in milk induced by supplementation AFF suggests that alcohol fermented feed may improve milk cholesterol levels without any negative effects in lactating cows.

Characterization of the low-pathogenic H5N1 avian influenza virus in South Korea.

The low-pathogenic avian influenza (LPAI) virus can serve as a progenitor of the highly pathogenic avian influenza virus, so it is important to monitor the LPAI virus as well as the highly pathogenic avian influenza virus. The Korean LPAI H5N1 virus, A/wild duck/Kr/CSM4-12/09 (H5N1) [Wd/CSM4-12/09], was first isolated from feces of the wild duck in South Korea. Genetic analysis showed that 7 genes of Wd/CSM 4-12/09 clustered in eastern Asia and that the neuraminidase (NA) gene of this isolate was closely related to European LPAI viruses. The Korean LPAI H5N1 virus has the highest similarity with the Japanese LPAI H5N1 virus, A/mallard/Hokkaido/24/09 (H5N1), in 6 genes [polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1), polymerase acidic protein (PA), hemagglutinin (HA), NA, and nonstructural (NS) genes]. The Korean LPAI H5N1 virus did not replicate in experimentally infected chickens, whereas it replicated in ducks and mice without preadaptation. This study shows that the first Korean LPAI H5N1 reassortment, which occurred between influenza viruses from wild migratory birds in Eurasia, has contributed to the increased diversity of the viral gene pool in eastern Asia; this has the potential to change the host range and to allow the virus to evolve into forms with increased pathogenicity.

Genetic analyses of avian influenza viruses in Mongolia, 2007 to 2009, and their relationships with Korean isolates from domestic poultry and wild birds.

The present study was conducted to monitor wild birds based on the concern that they could disseminate avian influenza virus (AIV) between Mongolia and Korea, which shares the same migratory flyway. Of 1,528 fecal samples analyzed, 21 low-pathogenic AIV were isolated from 2007 to 2009. Nineteen AIV-positive fecal samples were identified as Anseriformes by DNA bar coding. The most frequently isolated subtype was H3 (61.9%), and the most prevalent hemagglutinin/neuraminidase combination was H3N8 (52.4%). Phylogenetic analysis was performed to assess their genetic relationships with those of domestic poultry and wild birds in Korea. The H3 and H7 surface genes belonged to the Eurasian lineage and clustered together in a group with Korean wild birds and poultry. Most N8 genes clustered phylogenetically with viruses isolated in Eurasia, whereas 1 of the Mongolian viruses and some Korean viruses belonged to the North American lineage. The polymerase acidic protein of the internal gene was not distinguishable from the H5N1 highly pathogenic AIV of the goose/Guangdong/1/1996 (Gs/Gd)-like virus. Our study suggests that Mongolian AIV isolates have evolved with genetically multiple genotypes and are closely related to those of AIV in poultry as well as in wild birds in Korea.

Antimicrobial activity of the constituents of Smallanthus sonchifolius leaves against methicillin-resistant Staphylococcus aureus.

BACKGROUND AND OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) has been a serious problem as its infection is associated with higher mortality and increase cost worldwide. In the present study, the antibacterial activity of enhydrin, polymatin B, allo-schkuhriolide from the leaves of Smallanthus sonchifolius was investigated. MATERIAL AND METHODS: Enhydrin, polymatin B, allo-schkuhriolide from the leaves of Smallanthus sonchifolius were tested for antimicrobial activity using micro dilution broth method against 2 strains of ATCC 33591, ATCC 25923 and 15 strains of clinical isolates MRSA. RESULTS: The antibacterial activity of Smallanthus sonchifolius can safely be attributed to enhydrin as polymatin B, and allo-schkuhriolide are not showing any activity against Staphylococcus aureus strains. The enhydrin showed good antibacterial activity against all tested strains (MIC = 125-500 microg/ml). DISCUSSION: These results suggest that only enhydrin can be considered as an antibacterial drug against MRSA.

Survival rate of H5N1 highly pathogenic avian influenza viruses at different temperatures.

The survival rate of Korean H5N1 highly pathogenic avian influenza (HPAI) viruses was investigated at different temperatures under the laboratory conditions. The estimated survival days for a starting viral concentration of 10(6.5) 50% egg infectious dose/0.1 mL were 930, 1,042, and 3,213 d at 4 degrees C; 226, 232, and 293 d at 20 degrees C; and 51, 55, and 58 d at 30 degrees C for A/chicken/Korea/ES/03, A/chicken/Korea/IS/06, and A/chicken/Korea/Gimje/08 (Gimje/08) viruses, respectively. The stability of the Gimje/08 virus was statistically significant compared with the other 2 viruses except for the data between Gimje/08 and A/chicken/Korea/IS/06 virus at 30 degrees C. This result indicated that the survival rate of 3 Korean HPAI viruses is different at various temperatures, which might have partially influenced the large scale of HPAI outbreak in Korea in 2008.

Serum ammonia as an early predictor of in-hospital mortality in patients with glufosinate poisoning.

BACKGROUND: The mortality rate associated with human glufosinate poisoning is high. We evaluated the usefulness of serum ammonia and sequential organ failure assessment (SOFA) and acute physiology and chronic health evaluation II (APACHE II) scores for early prediction of in-hospital mortality in glufosinate ammonium poisoning. METHODS: A prospectively collected pesticide poisoning registry at a single academic medical center was retrospectively analyzed. Data were collected from consecutive patients diagnosed with glufosinate ammonium poisoning between May 2007 and February 2018. The initial serum ammonia level was defined as the highest serum ammonia level measured within 12 h after emergency department (ED) arrival. The SOFA and APACHE II scores were calculated using data obtained within the first 12 h after ED arrival. The patients were divided into survivor and nonsurvivor groups by in-hospital death status. RESULTS: In total, 110 patients were included. Ten patients (9.1%) died in the hospital despite treatment. Median initial serum ammonia level was significantly higher in the nonsurvivor group than in the survivor group (219 (range: 158-792) versus 100.5 (range: 25-317) µg/dL, p < 0.001). Median SOFA and APACHE II scores in the survivor and nonsurvivor groups were 2 (range: 0-10) versus 5 (range: 1-8) (p = 0.044) and 7 (range: 0-28) versus 16 (range: 8-22) (p = 0.001), respectively. In the multiple logistic regression analysis, the initial serum ammonia level was the only independent predictor (cutoff value: 151 µg/dL). CONCLUSION: An initial serum ammonia level >151 µg/dL was an independent early predictor of in-hospital mortality in glufosinate ammonium poisoning.

Porogen effect on characteristics of banana pith carbon and the sorption of dichlorophenols.

Banana pith was used as precursor material to prepare carbon with and without porogens. Characterization of the carbons showed higher BET surface area (1285 m2/g) for ZnCl2-treated carbon, comparatively. Adsorption experiments were conducted to study the removal of 2,4-dichlorophenol (DCP) from aqueous solutions using the carbons under varying experimental conditions. Decrease in pH increased the percentage removal. All the carbons studied showed greater percentage of DCP removal with decrease in the initial concentration of DCP. Kinetic studies showed that the adsorption of DCP on the carbons was a rapid process. Nonlinear forms of pseudo-first-order and pseudo-second-order models were used to fit the experimental data. Among these the pseudo-first-order model described the data with high correlation coefficients and low percentage error values. Four nonlinear isotherm models including the Langmuir, Freundlich, Toth, and Sips were used to analyze the experimental DCP isotherms under different pH (2-4) conditions. Adsorption capacities (Qmax) from the Langmuir model were found to be 129.4, 67.7, and 49.9 mg/g for ZnCl2-treated, KOH-treated, and porogen-free carbon, respectively, at pH 2. From desorption studies it seemed that chemisorption played a major role in the adsorption process. The results indicated that ZnCl2-treated carbon could effectively remove phenols from wastewater.

Organic micro-pollutant removal in liquid-phase using carbonized silk cotton hull.

Phenolic compounds constitute one of the major pollutants in the modern world. Although many physical and chemical treatment technologies for their removal exist, most of them are economically not feasible. The present study was aimed at using silk cotton hull, a potent agricultural waste as an adsobent for removal of 2,4-dichlorophenol (2,4-DCP), which was used as a model phenolic compound. The process parameters were investigated and optimized conditions were determined. The equilibrium time was found to be 60 and 80 min for 10 and 20 mg/L and 100 min for 30 and 40 mg/L 2,4-DCP concentrations, respectively. Among the kinetic models applied, pseudo-second order model fitted well. The maximum adsorption capacity was 16.0 mg/g by Langmuir isotherm. Acidic pH was found favorable for the adsorption of 2,4-DCP. Studies on pH effect and desorption seemed to show that chemisorption played a major role in the adsorption process. In thermodynamic study, the change in entropy (DeltaS0) and heat of adsorption (DeltaH0) of silk cotton hull carbon (SCHC) was estimated as 14.01 J/(mol x K) and 3.04 kJ/mol, respectively. SCHC as adsorbent for removal of 2,4-DCP from aqueous solution, is effective, inexpensive, indigenous, reusable, has low treatment time and is easily available in large quantities as waste there by significantly lowers the cost of wastewater treatment.

Effects of homologous and heterologous neuraminidase vaccines in chickens against H5N1 highly pathogenic avian influenza.

The 2004 Asian H5N1 epizootic outbreak indicates the urgent need for vaccines against highly pathogenic avian influenza (HPAI) virus. The manufacture of inactivated whole-virus vaccines from HPAI viruses by traditional methods is not feasible for safety reasons as well as technical issues. The low pathogenic avian influenza A/wild bird feces/CSM2/02 (H5N3) virus was used as a heterologous neuraminidase vaccine, and HPAI A/CK/Korea/ES/03 (H5N1) virus was used as a homologous neuraminidase vaccine. Protection efficacy of both vaccines was evaluated by clinical signs, mortality rates, and virus shedding from oropharynx and cloaca of vaccinated chickens after challenge with HPAI A/CK/Korea/ES/03 (H5N1) virus. One dose of 128 hemagglutinin (HA) homologous H5N1 vaccine induced 100% protection in mortality and prevented viral shedding completely after lethal dose virus challenge, whereas one dose of 64 HA unit of heterologous H5N3 vaccine only induced 50% protection in mortality, and it did not prevent viral shedding. However, two doses at a 3-wk interval of 64 HA unit of heterologous H5N3 vaccine as well as one dose of 1024 HA unit of heterologous H5N3 vaccine induced 100% survival rate and could prevent viral shedding completely. Furthermore, we could differentiate the sera of infected birds from those of vaccinated birds by indirect immunofluorescent antibody test. These results suggest that heterologous neuraminidase H5N3 vaccine could be a useful tool for the control of H5N1 HPAI epidemic in poultry.

Highly pathogenic avian influenza in magpies (Pica pica sericea) in South Korea.

Highly pathogenic avian influenza (HPAI) is an extremely infectious, systemic viral disease of birds that produces high mortality and morbidity. HPAI was diagnosed in the three dead magpies (Pica pica sericea) submitted to the National Veterinary Research and Quarantine Service. At necropsy, the prominent lesions were multifocal or coalescing necrosis of the pancreas with enlargement of the livers and spleens. Microscopically, there were severely necrotizing pancreatitis and lymphocytic meningoencephalitis. Influenza viral antigen was also detected in areas closely associated with histologic lesions. Avian influenza virus was isolated from cecal tonsils and feces of the magpies. The isolated virus was identified as a highly pathogenic H5N1, with hemagglutinin proteolytic cleavage site deduced amino acid sequence of QREKRKKR/GLFGAIAG. To determine the pathogenicity of the isolate, eight 6-wk-old specific-pathogen-free chickens were inoculated intravenously with the virus, and all birds died within 24 hr after inoculation. This is the first report of HPAI in magpies.

Domain and genomic sequence analysis of bdellin-KL, a leech-derived trypsin-plasmin inhibitor.

Bdellin-KL is a trypsin-plasmin inhibitor from Hirudo nipponia, whose N-terminal sequence was identified as a non-classical Kazal-type. A cDNA clone encoding the inhibitor was isolated by reverse transcription-PCR and 5' rapid amplification of cDNA ends. The cDNA showed an open reading frame of 155 amino acids comprising one signal peptide and two separated domains. The C-terminal domain consists of distinct internal repeats, including HHEE and HHDD. The bdellin-KL sequence, from the constructed genomic library of Korean leech, was determined for the 2109 bases comprising the open reading frame and flanking regions (3' and 5'). The promoter region contains potential regulatory sequence motifs, including TATA, CAAT, and GC boxes. To characterize the properties of each domain, an N-terminal fragment was prepared by limited proteolysis of the intact protein. The inhibitory activity of the region was as potent as that of the intact protein. This suggests that the compact domain plays an important part in the inhibitory action of bdellin-KL. The C-terminal domain was revealed to have binding affinity to ions such as Ca(2+), Zn(2+), Fe(3+), and Fe(2+) without an influence on the inhibitory activity. This study demonstrates that bdellin-KL may be a novel bifunctional protein with two distinct domains.