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To efficiently and accurately quantify the interactions of bacteria with mammalian cells, a reliable fluorescence microscopy assay was developed. Bacteria were engineered to become rapidly and stably fluorescent using Green Fluorescent Protein (GFP) expressed from an inducible Tet promoter. Upon application of the fluorescent bacteria onto a monolayer, extracellular bacteria could be discriminated from intracellular bacteria by antibody staining and microscopy. All bacteria could be detected by GFP expression. External bacteria stained orange, whereas internalised bacteria did not. Internalised bacteria could thus be discriminated from external bacteria by virtue of being green but not orange fluorescent. Image acquisition and counting of various fluorophore-stained entities were accomplished with a high-content screening platform. This allowed for semi-automated and accurate counting of intracellular and extracellular bacteria.

A major virulence determinant of swine vesicular disease virus (SVDV), an Enterovirus that causes an acute vesicular disease, has been mapped to residue 20 of the 2A protease. The SVDV 2A protease cleaves the 1D-2A junction in the viral polyprotein, induces cleavage of translation initiation factor eIF4GI, and stimulates the activity of enterovirus internal ribosome entry sites (IRESs). The 2A protease from an attenuated strain of SVDV (Ile at residue 20) is significantly defective at inducing cleavage of eIF4GI and the activation of IRES-dependent translation compared to the 2A protease from a pathogenic strain (J1/73, Arg at residue 20), but the two proteases have similar 1D-2A cleavage activities (Y. Sakoda, N. Ross-Smith, T. Inoue, and G. J. Belsham, J. Virol. 75:10643-10650, 2001). Residue 20 has now been modified to every possible amino acid, and the activities of each mutant 2A protease has been analyzed. Selected mutants were reconstructed into full-length SVDV cDNA, and viruses were rescued. The rate of virus growth in cultured swine kidney cells reflected the efficiency of 2A protease activity. In experimentally infected pigs, all four of the mutant viruses tested displayed much-reduced virulence compared to the J1/73 virus but a significant, albeit reduced, level of viral replication and excretion was detected. Direct sequencing of cDNA derived from samples taken early and late in infection indicated that a gradual selection-reversion to a more efficient protease occurred. The data indicated that extensive sequence change and selection may introduce a severe bottleneck in virus replication, leading to a decreased viral load and reduced or no clinical disease.

To investigate whether foot-and-mouth disease virus (FMDV) RNA loads in oesophageal–pharyngeal fluid (OP-fluid) in the early course of infection is related to the outcome of virus persistence, viral RNA in OP-fluid samples from cattle experimentally infected with FMDV type O was quantitatively analysed by using a quantitative real-time RT-PCR. Viral RNA was detected within 24 h post-infection (p.i.) in all infected animals. Rapid virus replication led to peak levels of viral RNA load by 30–53 h p.i., and then the load declined at various rates. In some animals (n=12, so-called non-carriers) viral RNA became undetectable between 7 and 18 days p.i. In contrast, in persistently infected animals (n=12, so-called carriers) viral RNA persisted in OP-fluid samples at detectable levels beyond 28 days p.i. Analysis of early viral decay/clearance and virus clearance half-life in OP-fluid samples showed that the extent of reduction of viral RNA in OP-fluid samples immediately following peak levels is a critical determinant of the outcome of FMDV persistence.

The axons in the parahippocampal white matter (PWM) region that includes the perforant pathway relay multimodal sensory information, important for memory function, from the entorhinal cortex to the hippocampus. Previous work suggests that the integrity of the PWM shows changes in individuals with amnestic mild cognitive impairment and is further compromised as Alzheimer’s disease progresses. The present study was undertaken to determine the effects of healthy aging on macro-and micro-structural alterations in the PWM. The study characterized in vivo white matter changes in the parahippocampal region that includes the perforant pathway in cognitively healthy young (YNG, n = 21) compared to cognitively healthy older (OLD, n = 21) individuals using volumetry, diffusion tensor imaging (DTI) and tractography. Results demonstrated a significant reduction in PWM volume in old participants, with further indications of reduced integrity of remaining white matter fibers. In logistic regressions, PWM volume, memory performance and DTI indices of PWM integrity were significant indicator variables for differentiating the young and old participants. Taken together, these findings suggest that age-related alterations do occur in the PWM region and may contribute to the normal decline in memory function seen in healthy aging by degrading information flow to the hippocampus.

The electron transfer properties of the complex, ReO(ECD), where H 2 ECD is N , N ′-1,2-ethylenediylbis- L -cysteine, diethyl ester, are reported. The results of this study will demonstrate that ReO(ECD) has an unusual reductive electron transfer mechanism, in that the addition of one electron to this complex results in the loss of H /sd from the ligand. This corresponds to the net loss of a proton (H + ) from the complex and hence the product of the reduction is the same as that obtained by the reaction of a base with ReO(ECD) to form [ReO(ECD)] − .

Foot-and-mouth disease virus (FMDV) can be spread by direct animal-to-animal contact, indirect contact facilitated by contaminated materials or by airborne spread. The rate of spread and the incubation period, as well as the severity of disease, depends on many variables including the dose received, the route of introduction, the virus strain, the animal species and the conditions under which the animals are kept. Quantitative data related to these variables are needed if model predictions are to be used in practical disease control. This experimental study quantifies the risk of transmission of FMDV in pigs exposed by contact, sheep exposed by indirect contact with pigs and sheep exposed to airborne FMDV. Groups of pigs were inoculated with the FMDV O UKG 34/2001 strain and susceptible pigs were then exposed to the inoculated animals at different stages of the infection cycle. The mean incubation period in the susceptible pigs ranged from 1 to 10 days. The length of the incubation period, severity of clinical disease and efficiency of spread were related to dose (i.e. infectiousness of source and intensity of contact). Low intensity transmission increased the proportion of subclinical or abortive infections. Local conditions are important in the efficiency and speed of transmission of FMDV. The results of the experiments described above suggest that transmission is frequency dependent rather than density dependent. The sheep experiments provided further evidence that development of infection and clinical disease is dependent upon local conditions. Dose, infectiousness, intensity of contact and local factors are thus important determinants for the outcome of an initial outbreak and must be truthfully accounted for in mathematical models of epidemiological spread.

This paper provides a quantitative description of the early infectious process in pigs experimentally infected with foot-and-mouth disease virus (FMDV), obtained by dose-dependent, time course studies of viral load in serum. Pigs were inoculated by the intravenous or intradermal/subcutaneous route with FMDV and housed together in groups or individually. The effects of dose, inoculation route and exposure intensity on the replication of FMDV in vivo and the development of disease were studied. It was shown that the higher the dose, the shorter was the time to the start of active viraemia and to the onset of clinical signs. Exposure intensity and housing conditions influenced the viral dynamics of FMDV. Increasing the exposure intensity, by increasing the number of infected pigs housed together, had the effect of synchronizing the infection and reducing the variance in the start of active viraemia. Increasing the number of pigs housed together also increased the interaction between the pigs and the activity of individual pigs, which had the effect of shortening the time to the onset of clinical signs such as vesicle formation. Intradermal inoculation was more effective than intravenous inoculation for transmitting FMDV to pigs, resulting in shorter times to the start of active viraemia and in higher clinical scores.

Serotonergic neuronal networks are important for food intake and body weight regulation. Dexfenfluramine (dF), a serotonin releaser and reuptake inhibitor, was used to investigate changes in food intake, body weight development, energy expenditure, respiratory quotient, and substrate oxidation rates for 12 days. Rats, which had been made obese by early postnatal overfeeding, received an energy-controlled mash diet and water ad lib and were intraperitoneally injected daily with either saline, 5 or 10 mg dF/kg. Compared to controls, food intake, body weight development, and energy expenditure were decreased in a dose-dependent manner, especially during the first 6 days. Lipid oxidation was increased while oxidation of carbohydrates was decreased. Pair-feeding experiments over 2 days revealed that this was not solely a result of diminished food intake but also an additional metabolic effect of dF, different from its anorectic effect. At the end of these experiments, plasma glucose and liver glycogen were unchanged after dF, but plasma free fatty acids were significantly decreased. Insulin-sensitivity was probably improved, indicated by decreased insulin levels and increases in muscle glycogen contents and activities of muscle pyruvate kinase. Liver-glutamine and contents of valine, leucine, and isoleucine in the muscle were significantly decreased after dF-treatment, the latter indicating a diminished proteolysis. The plasma tryptophan/large neutral amino acids ratio of the dF-rats was unchanged but that of the paired-fed rats was changed, despite similar changes in food intake. It is concluded that both increased oxidation of endogenous fat and reduced food intake could mediate the body weight reducing effect of dF.

Although nuclear transfer (NT) has successfully produced cloned piglets, the development to blastocyst and to term is still low. Activation of the NT embryos is one of the key factors to improve the developmental ability of porcine NT embryos. Electric pulses as well as chemicals have been used to activate porcine NT embryos. This study was conducted to investigate the effect of continued activation following fusion pulses on in vitro development of porcine NT Embryos. Oocytes derived from a local abattoir were matured for 42 to 44h and enucleated. Ear skin cells were obtained from a 4-day-old transgenic pig transduced with eGFP recombinant retrovirus. Enucleated oocytes were reconstructed and cultured in PZM-3 in a gas atmosphere of 5% CO2 in air. Cleavage and blastocyst developmental rates were assessed under a stereomicroscope on Day 3 or 6. Blastocysts were stained with 5μg of Hoechst 33342 and total cell number was determined with an epifluorescent microscope. In Experiment 1, oocytes were activated with two 1.2kV/cm for 30μs (E) in 0.3M mannitol supplemented with either 0.1 or 1.0mM Ca2+. In each treatment, activated oocytes were divided into three groups. The first group was control (E). Other two groups were exposed to either ionomycin and 6-DMAP (E+I+D) or 6-DMAP (E+D) immediately after the electric pulses. In Experiment 2, fusion was conducted by using 1.0mM Ca2+ in the fusion medium. Fused NT embryos were divided into three treatments. NT embryos were fused and activated simultaneously with electric pulse as a control (C); the second group was treated with 6-DMAP immediately after fusion treatment (D0); and the third group was treated with 6-DMAP at 20min (D20) after fusion. In experiment 1, for 0.1mM Ca2+, developmental rates to the blastocyst stage for E, E+I+D or E+D were 12.5, 26.7 and 22.5%, respectively. For 1.0mM Ca2+, developmental rates to the blastocyst stage were 11.4, 28.3 and 35.6%, respectively. The activated oocytes treated with 6-DMAP following the electric pulses by using 1.0mM Ca2+ in fusion medium had higher (P