VP2 capsid domain of the H-1 parvovirus determines susceptibility of human cancer cells to H-1 viral infection.

Although H-1 parvovirus is used as an antitumor agent, not much is known about the relationship between its specific tropism and oncolytic activity. We hypothesize that VP2, a major capsid protein of H-1 virus, determines H-1-specific tropism. To assess this, we constructed chimeric H-1 viruses expressing Kilham rat virus (KRV) capsid proteins, in their complete or partial forms. Chimeric H-1 viruses (CH1, CH2 and CH3) containing the whole KRV VP2 domain could not induce cytolysis in HeLa, A549 and Panc-1 cells. However, the other chimeric H-1 viruses (CH4 and CH5) expressing a partial KRV VP2 domain induced cytolysis. Additionally, the significant cytopathic effect caused by CH4 and CH5 infection in HeLa cells resulted from preferential viral amplification via DNA replication, RNA transcription and protein synthesis. Modeling of VP2 capsid protein showed that two variable regions (VRs) (VR0 and VR2) of H-1 VP2 protein protrude outward, because of the insertion of extra amino-acid residues, as compared with those of KRV VP2 protein. This might explain the precedence of H-1 VP2 protein over KRV in determining oncolytic activity in human cancer cells. Taking these results together, we propose that the VP2 protein of oncolytic H-1 parvovirus determines its specific tropism in human cancer cells.

Risk-based long-term screening for hepatocellular carcinoma recurrence after living donor liver transplantation.

BACKGROUND: This study sought to establish an actual risk-based long-term screening protocol for hepatocellular carcinoma (HCC) recurrence after liver transplantation (OLT). METHODS: The study was a retrospective review of medical records from 334 HCC patients who underwent primary living donor OLT and followed up for at least 5 years. RESULTS: Overall 10-year patient survival rate was 67.5%, with a 4.8% perioperative mortality. HCC recurred in 68/318 (21.4%) surviving patients over a mean follow-up of 77 months. HCC recurrence was 20.7% at 5 and 22.2% at 10 years. Annual recurrence rates were 11.4%, 6.6%, and 2.0% during the first, second, and third years, respectively. Among patients within Milan criteria, the annual incidence of HCC recurrence was highest during the first 3 years; thereafter only 6 sporadic recurrences were observed during next 8 years. Among subjects beyond Milan criteria, recurrence was common during, but not after 3 years. In 43 patients (63.2%) increased alpha-fetoprotein (AFP) was an initial indication to perform further imaging studies to diagnosis recurrence, whereas they were detected incidentally on protocol screening imaging among another 25 patients (36.8%) in the absence of an AFP rise. There was a close correlation between pretransplant AFP level and AFP increase after HCC recurrence. CONCLUSIONS: Patients beyond the Milan criteria require frequent tumor marker tests and imaging studies over the first 3 years; and those within Milan criteria require 10-years to follow-up primarily with tumor marker tests.

Enteric methane emission, diet digestibility, and nitrogen excretion from beef heifers fed sainfoin or alfalfa.

Effects of plant-bound condensed tannin (CT)-containing sainfoin vs. CT-free alfalfa (or low-CT alfalfa-sainfoin mixture), plant stage of maturity, and their interaction on enteric methane (CH4) emissions, diet digestibility, and N excretion were studied, using 8 ruminally cannulated beef heifers in 2 sequential short-term experiments (Exp. 1 and 2). In Exp. 1, first growth legumes were harvested daily and offered fresh to heifers. Heifers were assigned to 100% sainfoin or 80% alfalfa:20% sainfoin (as-fed basis). Responses were measured at early (late vegetative to early bud; stage 2 to 3) and late (early flower; stage 5) stage of maturity. In Exp. 2, the same legumes were harvested from second growth (late bud; stage 4) and offered to heifers as hay; 100% sainfoin or 100% alfalfa. In both experiments, heifers were fed once daily at 1× maintenance. When fed as fresh forage (Exp. 1), sainfoin, compared with the alfalfa-sainfoin blend, had greater digestibility of OM (74.7 vs. 70.9%; P = 0.02), yet tended to have lower CP digestibility (73.2 vs. 77.1%; P = 0.059). There was no difference between fresh legumes for CH4 emissions [25.9 g/kg DMI ± 4.02 SE; 8.5% of gross energy intake (GEI) ± 1.26 SE; or 36.8 g/kg digested OM ± 1.75 SE]. The fresh legumes were more digestible at early, rather than at late, maturity and, consequently, enteric CH4 (27.4 vs. 24.4 g/kg DMI; P < 0.004; 8.9 vs. 8.1% GEI; P < 0.008) was greater at early, rather than at later, growth. When fed as hay (Exp. 2), sainfoin, compared with alfalfa, had greater digestibility of OM (60.5 vs. 50.3%; P = 0.007), lower digestibility of CP (64.2 vs. 68.8%; P = 0.004), yet there was no difference between the legume hays for CH4 emissions (22.4 g/kg DMI ± 1.29 SD and 7.1% GEI ± 0.40 SD). However, on the basis of OM digested, CH4 emissions were lower for sainfoin than alfalfa hay (44.3 vs. 59.0 g/kg; P = 0.008). Percentage of total N excretion in urine was less for sainfoin compared with alfalfa, both for fresh legumes in Exp. 1 (74 vs. 78%; P = 0.017) or hay in Exp. 2 (64 vs. 72%; P < 0.001), and increasing maturity lowered urinary N excretion. In conclusion, feeding CT-containing sainfoin partially shifted N excretion from urine to feces, but it had little impact on enteric CH4 emissions from beef cattle fed at maintenance as compared with feeding either 80% alfalfa:20% sainfoin (fresh forages) or 100% alfalfa (hay). Feeding fresh legumes harvested between the late vegetative to early bud stage, compared with harvested at the early flower stage, increased N excreted in urine as well as enteric CH4 emissions from beef cattle fed at maintenance.

Synergistic effects of A1896, T1653 and T1762/A1764 mutations in genotype c2 hepatitis B virus on development of hepatocellular carcinoma.

The effects of genomic changes in hepatitis B virus (HBV) on the occurrence of hepatocellular carcinoma (HCC) are still unclear, especially in relation to the genotype of HBV. In this study, we examined the effects of genomic changes in HBV of genotype C2 on the development of HCC. A total of 318 patients with HBV-associated HCC and 234 patients with chronic hepatitis B (CHB) were studied. All of HCC cases were diagnosed histologically and treated with surgical resection. The whole of the X, S, basal core promoter (BCP) and precore regions of the viral genome from sera or liver tissues were sequenced. All subjects had HBV of genotype C2. The prevalence of the T1653 mutation in the X region and the A1896 mutation in the precore region of HBV was significantly higher in the HCC group than in the control CHB group (22% vs 11%, P = 0.003; 50% vs 23%, P < 0.001, respectively). Moreover, the T1762/A1764 mutations in the BCP region in combination with either T1653 or A1896 were more common in the HCC compared with the CHB group (BCP+X1653: 18% vs 11%, P = 0.05; BCP+PC, 40% vs 15%, P < 0.001, respectively). In multivariate analysis, T1653 and A1896 were revealed to be independent risk factors for HCC development. G1896A in the precore region and C1653T mutation in the X region of genotype C2 HBV are important risk factors for HCC development. Also, the A1762T/G1764A double mutation may act in synergy with C1653T to increase the risk of HCC in patients chronically infected with HBV genotype C2.

Cancer upregulated gene 2, a novel oncogene, confers resistance to oncolytic vesicular stomatitis virus through STAT1-OASL2 signaling.

We have recently found a novel oncogene, named cancer upregulated gene 2 (CUG2), which activates Ras and mitogen-activated protein kinases (MAPKs), including ERK, JNK and p38 MAPK. Because activation of these signaling pathways has previously been shown to enhance cancer cell susceptibility to oncolysis by certain viruses, we examined whether vesicular stomatitis virus (VSV) could function as a potential therapeutic agent by efficiently inducing cytolysis in cells transformed by CUG2. Unexpectedly, NIH3T3 cells stably expressing CUG2 (NIH-CUG2) were resistant to VSV because of the activation of signal transducers and activators of transcription 1 (STAT1). The result was supported by evidence showing that suppression of STAT1 with short interference RNA (siRNA) renders cells susceptible to VSV. Furthermore, 2'-5' oligoadenylate synthetase-like (OASL) 2 was the most affected by STAT1 expression level among anti-viral proteins and furthermore suppression of OASL2 mRNA level caused NIH-CUG2 cells to succumb to VSV as seen in NIH-CUG2 cells treated with STAT1 siRNA. In addition, Colon26L5 carcinoma cells stably expressing CUG2 (Colon26L5-CUG2) exhibited resistance to VSV, whereas Colon26L5 stably expressing a control vector yielded to VSV infection. Moreover, Colon26L5-CUG2 cells stably suppressing STAT1 succumbed to VSV infection, resulting in apoptosis. Taken together, we propose that VSV treatment combined with the selective regulation of genes such as STAT1 and OASL2 will improve therapeutic outcomes for CUG2-overexpressing tumors.

Effects of partially replacing dietary starch with dry glycerol in a lactating cow diet on ruminal fermentation during continuous culture.

The effects of dry glycerol as a partial replacement for dietary starch in a lactating cow diet on ruminal fermentation and bacterial protein synthesis were evaluated using 4 single-flow, continuous-culture fermentors (ranging from 1,015 to 1,040 mL in volume). The basal lactating cow diet was formulated to have partial contents of dietary starch provided from a corn starch supplement [at 12.37% diet dry matter (DM)], which was partially or completely replaced by a dry glycerol product. Both the corn starch supplement and dry glycerol product contained 65% of pure corn starch or glycerol, respectively. The final inclusion rate for pure glycerol was at 0, 3, 5, or 8% of DM in the basal diet. The experiment was conducted using a 4 × 4 Latin square design with four 9-d periods, with the first 6 d for adaptation and last 3 d for sampling. Fermentors were inoculated with 1L of ruminal fluid and 25 g of ruminal digesta from a ruminally cannulated cow receiving a lactation total mixed ration (16% crude protein, 32% neutral detergent fiber, and 25% starch; DM basis). Each fermentor was fed 75 g of DM of its respective experimental diet daily in 3 equal portions (at 0800, 1400, and 2000 h). Liquid dilution rate of the fermentors was maintained at 10%/h and solids retention time was set at 24 h. Fermentation fluid and the effluent from each fermentor were sampled once daily (at 1330 h) from d 7 to 9 of each period and pooled by period. Postprandial ruminal fermentation was studied by sampling the fermentors hourly for 5 h after the 0800 h feeding on d 9 of each period. The total fermentation contents were harvested at the end of the period for estimations of bacterial protein synthesis. Replacing corn starch with dry glycerol linearly increased the proportions of propionate and valerate at the expense of acetate in the fermentation fluid measured daily or for the first 5h after feeding. Replacing corn starch with dry glycerol also linearly increased the digestibility of dietary neutral detergent fiber without a change on the flow or efficiency of bacterial protein synthesis during continuous culture. Results indicate that glycerol as a dry product can replace dietary starch as corn starch at a level of up to 8% of DM in the diet without negatively affecting ruminal fermentation and digestibility during continuous culture.

A fibrolytic enzyme additive for lactating Holstein cow diets: ruminal fermentation, rumen microbial populations, and enteric methane emissions.

The objective was to determine if supplementing a dairy cow diet with an exogenous fibrolytic enzyme additive (Econase RDE; AB Vista, Marlborough, Wiltshire, UK) altered fermentation, pH, and microbial populations in the rumen or enteric methane (CH(4)) emissions. In a companion study, this enzyme additive improved efficiency of fat-corrected milk production in a dose-dependent manner by up to 11% for early lactation dairy cows. Nine ruminally cannulated, lactating Holstein cows were used in a replicated 3 × 3 Latin square design with 21-d periods. Dietary treatments were 0 (control), 0.5 (low), and 1.0 (high) mL of enzyme/kg of total mixed ration dry matter. Rumen contents were collected on 2 d (d 15 and 19), ruminal pH was measured continuously for 6 d (d 13 to 18) by using an indwelling system, and enteric CH(4) production was measured for 3 d (d 16 to 18) using the sulfur hexafluoride tracer gas technique. The enzyme additive did not alter volatile fatty acids, NH(3), pH, or population densities of total protozoa, bacteria, and methanogens in ruminal fluid. However, population densities of certain bacteria, calculated as copy number of species-specific 16S-rRNA, were affected by enzyme treatment. Population density of Ruminobacter amylophilus was increased and that of Fibrobacter succinogenes tended to be increased by the high enzyme treatment. Selenomonas ruminantium tended to increase linearly with increasing levels of enzyme in the diet, although its population density was only numerically increased by the high enzyme treatment. Streptococcus bovis, however, tended to be decreased by the low enzyme treatment. Increasing the level of enzyme supplement in the diet also linearly increased enteric CH(4) production, even when adjusted for feed intake or milk production (19.3, 20.8, and 21.7 g of CH(4)/kg of dry matter intake or 12.9, 13.6, and 15.1g of CH(4)/kg of milk for the control, low, and high enzyme treatments, respectively). This shift in ruminal bacterial communities and higher CH(4) emissions could imply increased ruminal digestion of feed, which needs to be substantiated in longer term studies.

Relationship between rumen methanogens and methane production in dairy cows fed diets supplemented with a feed enzyme additive.

AIMS: To investigate the relationship between ruminal methanogen community and host enteric methane (CH(4) ) production in lactating dairy cows fed diets supplemented with an exogenous fibrolytic enzyme additive. METHODS AND RESULTS: Ecology of ruminal methanogens from dairy cows fed with or without exogenous fibrolytic enzymes was examined using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analyses and quantitative real-time PCR (qRT-PCR). The density of methanogens was not affected by the enzyme additive or sampling times, and no relationship was observed between the total methanogen population and CH(4) yield (as g per head per day or g kg(-1) DMI). The PCR-DGGE profiles consisted of 26 distinctive bands, with two bands similar to Methanogenic archaeon CH1270 negatively correlated, and one band similar to Methanobrevibacter gottschalkii strain HO positively correlated, with CH(4) yield. Three bands similar to Methanogenic archaeon CH1270 or Methanobrevibacter smithii ATCC 35061 appeared after enzyme was added. CONCLUSIONS: Supplementing a dairy cow diet with an exogenous fibrolytic enzyme additive increased CH(4) yield and altered the composition of the rumen methanogen community, but not the overall density of methanogens. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to identify the correlation between methanogen ecology and host CH(4) yield from lactating dairy cows.

Differing effects of 2 active dried yeast (Saccharomyces cerevisiae) strains on ruminal acidosis and methane production in nonlactating dairy cows.

Fifteen ruminally cannulated, nonlactating Holstein cows were used to measure the effects of 2 strains of Saccharomyces cerevisiae, fed as active dried yeasts, on ruminal pH and fermentation and enteric methane (CH(4)) emissions. Nonlactating cows were blocked by total duration (h) that their ruminal pH was below 5.8 during a 6-d pre-experimental period. Within each block, cows were randomly assigned to control (no yeast), yeast strain 1 (Levucell SC), or yeast strain 2 (a novel strain selected for enhanced in vitro fiber degradation), with both strains (Lallemand Animal Nutrition, Montréal, QC, Canada) providing 1 × 10(10) cfu/head per day. Cows were fed once daily a total mixed ration consisting of a 50:50 forage to concentrate ratio (dry matter basis). The yeast strains were dosed via the rumen cannula daily at the time of feeding. During the 35-d experiment, ruminal pH was measured continuously for 7 d (d 22 to 28) by using an indwelling system, and CH(4) gas was measured for 4 d (d 32 to 35) using the sulfur hexafluoride tracer gas technique (with halters and yokes). Rumen contents were sampled on 2 d (d 22 and 26) at 0, 3, and 6h after feeding. Dry matter intake, body weight, and apparent total-tract digestibility of nutrients were not affected by yeast feeding. Strain 2 decreased the average daily minimum (5.35 vs. 5.65 or 5.66), mean (5.98 vs. 6.24 or 6.34), and maximum ruminal pH (6.71 vs. 6.86 or 6.86), and prolonged the time that ruminal pH was below 5.8 (7.5 vs. 3.3 or 1.0 h/d) compared with the control or strain 1, respectively. The molar percentage of acetate was lower and that of propionate was greater in the ruminal fluid of cows receiving strain 2 compared with cows receiving no yeast or strain 1. Enteric CH(4) production adjusted for intake of dry matter or gross energy, however, did not differ between either yeast strain compared with the control but it tended to be reduced by 10% when strain 2 was compared with strain 1. The study shows that different strains of S. cerevisiae fed as active dried yeasts vary in their ability to modify the rumen fermentative pattern in nonlactating dairy cows. Because strain 2 tended (when compared with strain 1) to lower CH(4) emissions but increase the risk of acidosis, it may be prudent to further evaluate this strain in cattle fed high-forage diets, for which the risk of acidosis is low but CH(4) emissions are high.

Inclusion of flaxseed in hay- and barley silage diets increases alpha-linolenic acid in cow plasma independent of forage type.

Feeding flaxseed to cattle may be a means of increasing omega-3 fatty acid levels in ruminant products, but possible interactions with conserved forages have not been investigated. Twelve Holstein cows were used in a replicated 4 × 4 Latin Square experiment. Cows were fed one of four 50:50 forage:concentrate diets (DM basis): hay (hay control, HC), hay plus 15% ground flaxseed (hay-flaxseed, HF), barley silage (silage control, SC), and barley silage plus 15% ground flaxseed (silage-flaxseed, SF). Plasma concentrations of alpha-linolenic acid (ALA) did not differ between SC and HC diets. Flaxseed increased ALA (P < 0.05), but levels were not influenced by forage type. Flaxseed slightly increased 18:2n-6 (P < 0.05) and some n-6 and n-3 elongation and desaturation products, particularly arachidonic acid (ARA) and eicosapentaenoic acid (EPA). Flaxseed also increased C18:0 (P < 0.05) with this increase being greater (P < 0.01) for cows fed SF than HF. Feeding flaxseed also increased plasma C18:1-trans isomers (P < 0.01), predominantly vaccenic acid (VAA, 18:1-t11), with this increase being greater (P < 0.05) in cows fed HF than SF. Although conjugated linoleic acid (CLA) was increased (P < 0.001) with flaxseed it was not influenced by forage type (P = 0.06). Overall, feeding flaxseed increased plasma ALA, EPA, ARA and CLA independently of forage type. Feeding flaxseed with silage, however, resulted in more 18:0, while feeding flaxseed with hay resulted in greater accumulations of plasma 18:1-trans isomers mainly in the form of VAA.

Improved milk production efficiency in early lactation dairy cattle with dietary addition of a developmental fibrolytic enzyme additive.

A 3-part study was conducted to evaluate the effect of a developmental fibrolytic enzyme additive on the digestibility of selected forages and the production performance of early-lactation dairy cows. In part 1, 4 replicate 24-h batch culture in vitro incubations were conducted with alfalfa hay, alfalfa silage, and barley silage as substrates and ruminal fluid as the inoculum. A developmental fibrolytic enzyme additive (AB Vista, Marlborough, UK) was added at 5 doses: 0, 0.5, 1.0, 1.5, and 2.0 µL/g of forage dry matter (DM). After the 24-h incubation, DM, neutral detergent fiber (NDF), and acid detergent fiber (ADF) disappearance were determined. For alfalfa hay, DM, NDF, and ADF disappearance was greater at the highest dosage compared with no enzyme addition. Barley silage NDF and ADF and alfalfa silage NDF disappearance tended to be greater for the highest enzyme dosage compared with no enzyme addition. In part 2, 6 ruminally cannulated, lactating Holstein dairy cows were used to determine in situ degradation of alfalfa and barley silage, with (1.0 mL/kg of silage DM) and without added enzyme. Three cows received a control diet (no enzyme added) and the other 3 received an enzyme-supplemented (1.0 mL/kg of diet DM) diet. Enzyme addition after the 24h in situ incubation did not affect the disappearance of barley silage or alfalfa silage. In part 3, 60 early-lactation Holstein dairy cows were fed 1 of 3 diets for a 10-wk period: (1) control (CTL; no enzyme), (2) low enzyme (CTL treated with 0.5 mL of enzyme/kg of diet DM), and (3) high enzyme (CTL treated with 1.0 mL of enzyme/kg of diet DM). Adding enzyme to the diet had no effect on milk yield, but dry matter intake was lower for the high enzyme treatment and tended to be lower for the low enzyme treatment compared with CTL. Consequently, milk production efficiency (kg of 3.5% fat-corrected milk/kg of DM intake) linearly increased with increasing enzyme addition. Cows fed the low and high enzyme diets were 5.3 (not statistically significant) and 11.3% more efficient, respectively, compared with CTL cows. This developmental fibrolytic enzyme additive has the potential to increase fiber digestibility of forages, which could lead to greater milk production efficiency for dairy cows in early lactation.

CUG2, a novel oncogene confers reoviral replication through Ras and p38 signaling pathway.

As we have recently found a novel oncogene, the cancer-upregulated gene 2 (CUG2), which was elevated in a variety of tumor tissues such as the ovary, liver, lung and pancreas, we examined whether reovirus could efficiently induce cytolysis in cancer cells expressing CUG2 and thus be used as a potential cancer therapeutic agent. In this study, we describe experiments in which we use reovirus to treat NIH3T3 cells stably expressing either CUG2 (NIH-CUG2) or vector only (NIH-Vec). NIH-CUG2 cells readily support reoviral proliferation and undergo apoptosis, whereas NIH-Vec cells are highly resistant to reoviral infection and virus-induced apoptosis. This notable result may be explained by the observation that CUG2 expression inhibits PKR activation, leading to reoviral proliferation in nonpermissive NIH3T3 cells. Furthermore, reovirus infection results in almost complete regression of tumorgenic NIH-CUG2 cells in transplanted nude mice. As we found that CUG2 enhances activation of MAPK (ERK, JNK and p38), Src kinase and Ras, we examined whether CUG2 confers reoviral replication independent of the Ras or p38 MAPK signaling pathway. From these experiments we found that either inhibition of p38 MAPK or Ras blocks reoviral proliferation even in the presence of CUG2 but inhibition of ERK, JNK and Src kinase does not, indicating that activation of p38 MAPK and Ras has critical roles in reoviral replication in CUG2-expressing tumor cells. Accordingly, we propose that reovirus can be useful in the treatment of transformed cells expressing CUG2, which is commonly detected in various tumor tissues.

Down-regulation of HIF-1alpha by oncolytic reovirus infection independently of VHL and p53.

Many oncolytic viruses are currently being tested as potential cancer therapeutic agents. To be effective, these viruses must replicate and propagate efficiently through the tumor mass. However, it is possible that the hypoxia that characterizes many tumors may be an obstacle to viral therapy because of its inhibition of viral replication and propagation. We, therefore, decided to test how oncolytic reovirus and its target cells respond to hypoxia. We found that reovirus infection suppresses hypoxia inducible factor (HIF)-1alpha protein levels (but not transcript abundance) in colon cancer HCT116 cells under CoCl(2) or hypoxia. Reovirus infection was able to reduce HIF-1alpha levels in both von Hippel Lindau (VHL)-/- renal carcinoma A498 and p53-/- HCT116 cells, indicating that the decrease of HIF-1alpha mediated by reovirus requires neither VHL nor p53 proteins. However, treatment with the inhibitor MG132 restored HIF-1alpha levels, suggesting that reovirus-induced HIF-1alpha decrease needs proteosomal activity. A498 VHL-/- cells with constitutive expression of HIF-1alpha were relatively resistant to reovirus-induced apoptosis when compared with A498 VHL+/+ cells. However, we found that the use of YC-1 to target HIF-1alpha promoted reovirus-induced apoptosis in A498 VHL-/- cells. Accordingly, we propose that reovirus may be used together with YC-1 as a potential therapeutic agent against chemoresistant or radioresistant tumors that are hypoxic and show increased levels of HIF-1alpha.

Ruminal and blood responses to propylene glycol during frequent feeding.

The objective of the current experiment was to study the responses of ruminal and blood metabolites of Holstein dairy cows to propylene glycol (PG) under different methods of delivery during frequent feeding. By providing the same amount (200 mL or 200 g) of PG, delivery methods for PG were assessed: 1) control treatment: no PG; 2) dietary treatment: 200 g of PG as a dry product (65% purity; corresponded to 308 g of the dry product) mixed into the TMR; 3) oral-drench treatment: 200 mL of liquid PG (100% purity) orally drenched; and 4) rumen-drench treatment: 200 g of PG as a dry product drenched via the rumen cannula to mimic top dressing. Eight multiparous (lactation = 3 +/- 1.1 SD) ruminally cannulated Holstein dairy cows (DIM = 204 +/- 104.5 SD) were fed PG for 4 d (d 11 to 14) in a replicated 4 x 4 Latin square design with an experimental length of 14 d for each period. On the last day of each period, serial blood samples were removed from an indwelling catheter placed in the right jugular vein immediately before and for 4 h after PG administration. Cows were fed at 12x feeding/d for 2 d before entering the serial sampling period to minimize postprandial influences on blood metabolites. Ruminal content was also sampled hourly for 4 h on d 14. Milk was sampled from 2 consecutive milkings on d 13 during each period. Dry matter intake and milk yield were not affected by PG. Percentages of milk lactose were increased by PG delivered by all methods tested in the current experiment. Ruminal concentrations (as percentages of total volatile fatty acids) of acetate were decreased and concentrations of propionate and isovalerate were increased by PG, regardless of the delivery method; however, total volatile fatty acid concentration was not affected by PG. Ruminal concentrations of butyrate were decreased and concentrations of valerate were increased by PG drench, via either an oral or ruminal drench. The degree of reduction in butyrate concentration or increase in valerate concentration was affected by PG dose. Serum insulin peaked more rapidly and at a greater concentration for cows receiving PG via drenching, but not when PG was provided as a part of the TMR. Plasma glucose, however, tended to peak more rapidly at a greater concentration for cows receiving PG, regardless of the delivery method. Propylene glycol for the amount drenched (orally or ruminally) or fed (incorporated into the ration) shifted ruminal fermentation toward a more glucogenic environment. Drenching demonstrated a better efficacy than feeding PG because of the amount of PG that was available to the animal at the time of sampling. Effects of drenching dry PG into the rumen were comparable with orally drenching liquid PG.

Effects of dietary forage level and monensin on lactation performance, digestibility and fecal excretion of nutrients, and efficiency of feed nitrogen utilization of Holstein dairy cows.

Two experiments (Exp. 1 and 2) were conducted using a 4 x 4 Latin square design with 2 replications (n = 8) to evaluate effects of feeding Holstein dairy cows a total mixed ration containing 50 or 60% of ration dry matter (DM) from forages with or without supplementation of monensin. In Exp. 1, alfalfa silage (AS) was used as the major forage (55% forage DM), and corn silage (CS; 45% forage DM) was used to make up the rest of the forage portion of diets (55AS:45CS). In Exp. 2, CS was used as the major forage (70% forage DM) and alfalfa hay (AH; 30% forage DM) was used to make up the rest of the forage portion of diets (70CS:30AH). Experimental diets were arranged in a 2 x 2 factorial with 50 or 60% ration DM from forages and monensin supplemented at 0 or 300 mg/cow daily. In Exp. 1 (55AS:45CS), feeding 60% forage diets decreased DM intake (DMI; 27.3 vs. 29.6 kg/d) but maintained the same levels of milk (45.8 vs. 47.0 kg/d) compared with 50% forage diets. The efficiency of converting feed to milk or 3.5% fat-corrected milk was greater for cows fed 60% compared with 50% forage diets (1.7 vs. 1.6 kg milk or 3.5% fat-corrected milk/kg of DMI, respectively). Increasing dietary forage level from 50 to 60% of ration DM increased milk fat percentage (3.4 to 3.5%); however, adding monensin to the 60% forage diet inhibited the increase in milk fat percentage. Feeding 60% forage diets decreased feed cost, but this decrease ($0.5/head per day) in feed cost did not affect income over feed cost. Feeding 60% forage diets decreased fecal excretion of DM (10.6 to 9.6 kg/d) and nitrogen (N; 354 to 324 g/d) and improved apparent digestibility of neutral detergent fiber from 43 to 49% and apparent efficiency of feed N utilization from 32.3 to 35.9% compared with 50% forage diets. In Exp. 2 (70CS:30AH), feeding 60% forage diets decreased DMI from 29.6 to 28.2 kg but maintained the same level of milk (41.1 vs. 40.8 kg/d) and therefore increased the efficiency of converting feed to milk (1.46 vs. 1.38 kg milk/kg DMI) compared with 50% forage diets. Daily feed cost for feeding 60% forage diets was $0.3/head lower than for the 50% forage diets. Fecal excretion of DM (10.3 vs. 11.5 kg/d) was lower and fecal excretion of N (299 vs. 328 g/d) tended to be lower for 60% compared with 50% forage diets. Results from these 2 experiments suggest that a 60% forage diet consisting of either AS or CS as the major forage can be fed to high producing Holstein dairy cows without affecting milk production while improving or maintaining the efficiency of converting feed to milk and the apparent efficiency of utilization of feed N. Cows receiving a 60% forage diet had a similar or improved digestibility of nutrients with a similar or reduced fecal excretion of nutrients. Effects of monensin under the conditions of the current experiments were minimal.

Effects of rumen-protected choline and dry propylene glycol on feed intake and blood parameters for Holstein dairy cows in early lactation.

A 6 x 6 Latin square design was used to test 3 sets of comparisons simultaneously to study response in dry matter intake, milk yield, and blood parameters to propylene glycol (PG) supplementation delivered by 2 methods [incorporating PG into the total mixed ration (TMR) vs. top dressing; comparison I]; individual or combined dietary choline and PG supplementation as a 2 x 2 factorial (comparison II); or increasing amounts of dietary choline (comparison III). Six multiparous (lactation number = 1.5 +/- 0.8 SD) Holstein dairy cows were at 41 d in milk (+/- 9 SD) at the start of the experiment. Propylene glycol used was a dry product containing 65% PG, and choline was a rumen-protected choline product (RPC; estimated to be 50% rumen-protected) containing 50% choline chloride. In comparison I, treatments compared were 1) control: no PG; 2) PG-TMR: 250 g/d of dry PG (corresponding to 162.5 g/d of PG) incorporated into the TMR; and 3) PG-top dress: 250 g/d of dry PG top-dressed onto the TMR. In comparison II, treatments compared were 1) control: no PG and no RPC; 2) PG: 250 g/d of dry PG incorporated into the TMR; 3) RPC: 50 g/d of RPC top-dressed onto the TMR; and 4) PG+RPC: combination of treatments 2 and 3. In comparison III, treatments compared were 0, 25, and 50 g/d of RPC top-dressed onto the TMR. Each experimental period lasted 10 d with 9 d of adaptation followed by 1 d of serial blood sampling. Dry matter intake and milk yield were recorded daily. During the serial blood sampling, jugular blood was sampled every 20 min for the first 4 h and at 8 and 12 h after treatment administration. Results obtained from comparison I showed that feeding 250 g/d of PG as a dry product decreased plasma beta-hydroxybutyrate (BHBA) concentration (mean +/- SEM) from 701 +/- 81 (control) to 564 +/- 76 micromol/L without affecting serum insulin, plasma glucose, or plasma nonesterified fatty acid concentrations. Top-dressing PG decreased plasma BHBA concentrations more than by incorporating it into the TMR [527 vs. 601 micromol/L (+/- 81 pooled SEM)]. Results obtained from comparison II showed that supplementing choline as RPC, PG, or both had no effect on dry matter intake, milk yield, or any of the blood parameters measured. Results obtained from comparison III showed that milk yield tended to increase linearly with increasing amounts of dietary choline as RPC. We concluded that feeding PG as a dry product reduced plasma BHBA concentration but top-dressing PG was more efficient at reducing plasma BHBA level than incorporating PG into the TMR. Dietary choline as RPC tended to increase milk yield linearly. However, a combined effect of dietary PG and choline was not evident and therefore not beneficial.

Expression of HBX, an oncoprotein of hepatitis B virus, blocks reoviral oncolysis of hepatocellular carcinoma cells.

Although reovirus has been used in tests as a potential cancer therapeutic agent against a variety of cancer cells, its application to hepatocellular carcinoma cells, in which the hepatitis B virus (HBV) X (HBX) protein of HBV plays a primary role, has not yet been explored. Here, we describe experiments in which we use reovirus to treat Chang liver carcinoma cells expressing either a vector only (Chang-vec) or a vector encoding HBX protein (Chang-HBX). Although Chang-vec cells readily support reoviral proliferation and undergo apoptosis, Chang-HBX cells are highly resistant to reoviral infection and virus-induced apoptosis, even though HBX protein induces activation of Ras and inactivation of PKR, which are normally thought to enhance reoviral oncolysis. The resistance of Chang-HBX cells to reovirus may instead be explained by HBX-induced downregulation of death receptor 5 and activation of Stat1. Phosphorylated Stat1 activates interferon (IFN)-stimulated regulatory element (ISRE)- and IFN-gamma-activated sequence (GAS)-mediated transcription, leading to the production of IFN-beta, whereas the reduced expression of Stat1 with its siRNA results in a decrease in IFN-beta production, by which Chang-HBX cells eventually succumb to reovirus infection. This result further indicates that HBX induces the establishment of an antiviral state through Stat1 activation. Thus, it appears that active Ras does not override the antiviral effect mediated by the activation of Stat1. Accordingly, we report that HBX, an oncoprotein of HBV, can prevent reoviral oncolysis of hepatocellular carcinoma. This suggests there may be limits to the practical application of reovirus in the treatment of human cancers already expressing other oncoviral proteins.

Effects of feeding dry propylene glycol to early postpartum Holstein dairy cows on production and blood parameters.

In all, 18 multiparous and 19 primiparous Holstein dairy cows were used in a completely randomized design with restrictions to evaluate the effects of feeding propylene glycol (PG) as a dry product, via two delivery methods, on production and blood parameters. PG treatments were administered from parturition through 21 days postpartum. Treatments were: (i) control, no PG; (ii) top dress, 162.5 g PG/day by top dressing onto the total mixed ration (TMR) and; (iii) mixing, 162.5 g PG/day as a part of the TMR by incorporating it into the TMR. PG used was a dry product which contained 65% pure PG and 35% silicon dioxide as the dry carrier. Coccygeal blood was sampled on 4, 7, 14 and 21 days in milk (±1.50 pooled s.d.). Supplementation of dry PG by top dressing onto, or incorporating into, the TMR had no effects on average dry matter intake, milk yield and composition, serum insulin, serum and plasma metabolites and milk ketones. Concentrations of urine ketones tended (P = 0.10) to be reduced by PG supplementation from 41.5 to 15.2 mg/dl. Supplementation of PG tended (P = 0.07) to decrease the incidence for subclinical ketosis from 39% to 24% and 13% for cows fed a TMR supplemented with no dry PG, with dry PG as a top dress and dry PG as a part of the TMR, respectively. It is concluded that supplementing PG as a dry product via incorporating into the TMR is as effective as when used as a top dress, based on the efficacies of both delivery methods to numerically reduce urine ketones concentrations and, therefore, the incidence for subclinical ketosis during the first 21 days of lactation. However, it should be noted that the number of cows used in the current study was minimal, and more cows are needed to confirm the efficacy of supplementing PG as a dry product on reducing the prevalence of subclinical ketosis in dairy cows during the first month of lactation.

Cancer patients' awareness of clinical trials, perceptions on the benefit and willingness to participate: Korean perspectives.

To understand patients' perceptions of clinical trials (CTs) is the principal step in the enrollment of patients to CTs. However, these perceptions in eastern countries are very rare. From 12 February 2007 to 13 April 2007, we consecutively distributed the questionnaire to 842 cancer patients who initiated a first cycle of chemotherapy regardless of each treatment step in the Seoul National University Hospital. Younger age, higher educational degree, higher economic status, and possession of private cancer insurance were related with significantly higher awareness of CTs (P=0.001, P=0.006, P=0.002, and P=0.009, respectively). However, unlike awareness, perceptions on benefits of CTs were not changed according to age, educational degree, and economic status (P=0.709, P=0.920, and P=0.847, respectively). Willingness was also not changed according to age, educational degree, economic status, and private cancer insurance (P=0.381, P=0.775, P=0.887, and P=0.392, respectively). Instead, males and heavily treated patients had more positive perceptions on benefits (P=0.002 and P=0.001, respectively) and more willingness to participate in CTs (OR=1.17, 1.14-2.75: OR=1.59, 1.01-2.51, respectively). In summary, cancer patients' awareness of CTs, perceptions on the benefit in CTs, and willingness to participate are differently influenced by diverse medical and social conditions. This information would be very helpful for investigators to properly conduct CTs in eastern cancer patients.