Unusual Base Pair between Two 2-Thiouridines and Its Implication for Nonenzymatic RNA Copying.

2-Thiouridine (s2U) is a nucleobase modification that confers enhanced efficiency and fidelity both on modern tRNA codon translation and on nonenzymatic and ribozyme-catalyzed RNA copying. We have discovered an unusual base pair between two 2-thiouridines that stabilizes an RNA duplex to a degree that is comparable to that of a native A:U base pair. High-resolution crystal structures indicate similar base-pairing geometry and stacking interactions in duplexes containing s2U:s2U compared to those with U:U pairs. Notably, the C═O···H-N hydrogen bond in the U:U pair is replaced with a C═S···H-N hydrogen bond in the s2U:s2U base pair. The thermodynamic stability of the s2U:s2U base pair suggested that this self-pairing might lead to an increased error frequency during nonenzymatic RNA copying. However, competition experiments show that s2U:s2U base-pairing induces only a low level of misincorporation during nonenzymatic RNA template copying because the correct A:s2U base pair outcompetes the slightly weaker s2U:s2U base pair. In addition, even if an s2U is incorrectly incorporated, the addition of the next base is greatly hindered. This strong stalling effect would further increase the effective fidelity of nonenzymatic RNA copying with s2U. Our findings suggest that s2U may enhance the rate and extent of nonenzymatic copying with only a minimal cost in fidelity.

Providing environmentally sustainable nephrology care: focus in low- and middle-income countries.

Health care on a global scale significantly contributes to carbon emissions, with high-income countries being the primary culprits. Within health care, dialysis plays a significant role as a major source of emissions. Low- and middle-income countries have a high burden of kidney disease and are facing an increasing demand for dialysis. This reality presents multiple opportunities to plan for environmentally sustainable and quality kidney care. By placing a stronger emphasis on primary and secondary prevention of kidney disease and its progression, within the framework of universal health coverage, as well as empowering patients to enhance self-care, we can significantly reduce the need for costly and environmentally detrimental kidney replacement therapy. Mandating the adoption of lean and innovative low-carbon dialysis practices while also promoting the growth of kidney transplantation would enable low- and middle-income countries to take the lead in implementing environmentally friendly nephrology practices and reducing costs, thus optimizing sustainability and the well-being of individuals living with kidney disease.

Inequities in the global representation of sites participating in large, multicentre dialysis trials: a systematic review.

BACKGROUND: The number of dialysis recipients is growing worldwide, making it important that the full range of patient populations are represented in randomised trials. As trial recruitment has not previously been examined at a global level, we compared the location of trial sites recruiting to large multicentre randomised controlled trials (RCTs) in dialysis to the global distribution of dialysis recipients. METHODS: A systematic review (2007-2016) was conducted to identify RCTs enrolling ≥100 dialysis patients from ≥2 sites. The number and location of sites were extracted from manuscripts and trial registration. The proportion of sites from each International Society of Nephrology global region was divided by the proportion of the global dialysis population in that region to determine a 'representation index' (RI), where 1.0 indicated that the number of sites was proportionate to the number of dialysis recipients in that region. RESULTS: We identified 180 RCTs, recruiting from 6172 sites in 54 countries. Eastern and Central Europe had the highest RI at 2.45. Other well-represented regions were Western Europe (2.20), North America (2.06), and Russia and newly independent states (1.36). Africa had the lowest RI at 0.05, followed by South Asia (0.08), Latin America (0.15), Middle East (0.27), North-East Asia (0.41), and South-East Asia and Oceania (0.62). CONCLUSIONS: Regions of the world with growing numbers of dialysis patients are poorly represented in large, multicentre RCTs. Efforts to boost trial participation in these regions are required to ensure that generalisable and relevant information is available to local healthcare providers.

Ixodes scapularis tick distribution and infection rates in Ottawa, Ontario, 2017.

BACKGROUND: The incidence of Lyme disease has increased in many regions of Canada in recent years, including in Ottawa, Ontario. To date there has been limited active tick surveillance in the region. OBJECTIVES: To estimate both the distribution and density of Ixodes scapularis ticks in the city of Ottawa, and the infection rates of ticks with Borrelia burgdorferi (that causes Lyme disease) and other tick-borne pathogens. METHODS: Between June and October 2017, tick surveillance was conducted by drag sampling at 23 sites in Ottawa municipal parks, recreational trails and forests. Blacklegged ticks were tested for B. burgdorferi, Borrelia miyamotoi and Anaplasma phagocytophilum using quantitative polymerase chain reaction protocols. RESULTS: I. scapularis ticks were found in 16 of the 23 sites (70%). Recreational trails, conservation areas/forests and the provincial park within the city of Ottawa had significantly higher tick densities than municipal parks (p<0.01). Of the 194 adult and 26 nymphal I. scapularis tested, prevalence of infection was 29.5% for B. burgdorferi, 0.45% for B. miyamotoi and 0.91% for A. phagocytophilum. CONCLUSION: Almost 30% of I. scapularis ticks tested in suburban and rural areas of the city of Ottawa were infected with B. burgdorferi, known to cause Lyme disease. Other types of infection, known to cause anaplasmosis and tick-borne relapsing fever, were also detected, although were very rare. Conducting active tick surveillance at the local level may help to inform risk assessment and public health actions.

Genotypic and phenotypic characterization of Staphylococcus aureus causing persistent and nonpersistent subclinical bovine intramammary infections during lactation or the dry period.

Staphylococcus aureus is a significant pathogen frequently causing persistent intramammary infections (IMI) in dairy cows. We compared some genotypic and phenotypic characteristics of 285 strains collected from quarter milk samples from cows with persistent and nonpersistent subclinical IMI across Canada. Variable number of tandem repeats typing was used to infer the persistence of the same S. aureus strain in 3 consecutive quarter milk samples collected at intervals of 3 wk during lactation or before and after dry-off. All first isolates of the series were used as the representative strains from persistent IMI and were compared with nonpersistent strains for the presence of genes seg, sen, sec, and tst as well as by spa typing. Biofilm production in vitro and hld-RNAIII expression levels were also quantified. The gene seg was associated with a reduction in the likelihood of the bacteria to cause a persistent IMI during lactation. Strains persisting through the dry period produced significantly more biofilm in vitro than strains that do not persist after calving. Also, we showed that strains expressing more hld were more likely to be nonpersistent during either lactation or through the dry period. Three spa types were predominant (t529, t267, and a novel type: t13401). In the strains studied, the spa type tbl 2645 was the most frequent, and 97.0% of the strains of this spa type carried both sen and seg. Strains from the spa type tbl 2645 were less likely to cause a persistent IMI in the dry period. Most (86.7%) of the strains of the novel spa type (t13401) were negative for seg, sen, or both and produced significantly more biofilm in vitro than tbl 2645 and t267. The present study expanded our current knowledge on the genotypic and phenotypic traits of S. aureus strains recovered from persistent and nonpersistent IMI in Canada.

The importance of screening for serum free light chains in suspected cases of multiple myeloma and their impact on the kidney.

Multiple myeloma (MM) is the second most common haematological malignancy in the UK. We present a case series of three patients with light chain only myeloma who had normal serum protein electrophoretic studies at screening and were diagnosed using serum and urine free light chain assessment. This series reiterates the importance of thorough and robust screening for MM in patients presenting with renal disease. We review the up to date literature and we highlight the need to screen patients for MM with a combination of serum electrophoresis/immunofixation and either urinary or serum free light chain measurement and to maintain a high index of suspicion regardless of the presence or absence of proteinuria. We also discuss the emerging role of the serum free light chain assay.

Current redistribution and generation of kinetic energy in the stagnated Z pinch.

The structure of magnetic fields was investigated in stagnated wire-array Z pinches using a Faraday rotation diagnostic at the wavelength of 266 nm. The distribution of current in the pinch and trailing material was reconstructed. A significant part of current can switch from the main pinch to the trailing plasma preheated by x-ray radiation of the pinch. Secondary implosions of trailing plasma generate kinetic energy and provide enhanced heating and radiation of plasma at stagnation. Hot spots in wire-array Z pinches also provide enhanced radiation of the Z pinch. A collapse of a single hot spot radiates 1%-3% of x-ray energy of the Z pinch with a total contribution of hot spots of 10%-30%.

The effect of incomplete milking or nursing on milk production, blood metabolites, and immune functions of dairy cows.

During the transition from pregnancy to lactation, the sudden increase in nutrient demand for milk production causes metabolic perturbations and is associated with immunosuppression and a high incidence of metabolic and infectious diseases in high-yielding cows. In this study, we examined whether limiting milk harvest postpartum while maintaining milking stimulus could improve the metabolic status of cows without reducing overall milk production. Forty-seven Holstein cows were milked completely twice a day from calving (control); milked incompletely (about one-third of expected milk production was collected) twice a day until d 5 after calving (incomplete); or left to nurse their calf until d 5 and milked once a day from d 3 to d 5 (nursing). All cows were milked twice a day from d 6 to the end of the experiment (d 61). During the treatment period (d 1 to 5), milk production averaged 27.3 and 9.7 kg/d for the control and incomplete treatments, respectively. We observed no residual effect of treatment on milk production, which averaged 47.8, 45.7, and 46.4 kg/d for the control, incomplete, and nursing treatments, respectively, between wk 2 and 9. The dry matter intake of the cows was similar during and after treatment. From wk 2 to 9, milk protein and lactose percentage were not affected by treatments, but milk fat tended to be higher in control cows than in cows milked partially (incomplete + nursing). Blood concentrations of glucose and phosphorus were lower and concentrations of nonesterified fatty acids and ß-hydroxybutyrate were higher in control cows than in partially milked cows during the treatment period. The positive effects on glucose and ß-hydroxybutyrate remained significant up to d 28. Peripheral blood mononuclear cell (PBMC) proliferation and secretion of IL-4 were depressed during the postpartum period, and proliferation tended to be greater for cells incubated in serum from cows in the incomplete treatment on d 5 but lower on d 61. We observed no effect of treatments on polymorphonuclear neutrophilic leukocyte chemotaxis or phagocytosis. Proliferation and IL-4 secretion of PBMC were negatively correlated with concentration of serum nonesterified fatty acids. Reducing milk harvest postpartum while maintaining milking stimuli reduced metabolic stress without compromising productivity of high-yielding dairy cows.

Effect of dry period management on mammary gland function and its endocrine regulation in dairy cows.

The objective of this study was to evaluate the effect of shortening the dry period on the mammary gland and the hormonal regulation of its functions. Holstein cows (n = 18) were assigned to a short dry period (SDP; 35 d; n = 9) or a conventional dry period (CDP; 65 d; n = 9). All cows were fed the same diets, with the exception that, during the dry period, the SDP cows received only the pre-calving diet for 35 d, whereas the CDP cows were fed a high-fiber diet from 65 to 28 d before calving and then received the same pre-calving diet as the SDP cows. Mammary gland functional capacity was evaluated at 70 days in milk, and mammary biopsies were taken in early and midlactation. Dry period length averaged 64.3 ± 1.1 and 31.9 ± 1.0 d for the CDP and SDP cows, respectively. The SDP cows had a lower milk yield and a lower energy-corrected milk yield compared with the CDP cows. The SDP cows also had a lower dry matter intake from wk 5 to 20 of lactation and tended to have lower plasma concentrations of ß-hydroxybutyrate from wk 1 to 4. Prepartum serum progesterone and estradiol concentrations were unaffected by the dry period management. Serum growth hormone concentrations and milking-induced prolactin release were similar in both groups. However, during the period when the CDP cows were dry but the SDP cows were still being milked (wk -9 to -6), serum prolactin concentrations were higher in the SDP cows than in the CDP cows. The SDP cows had a lower milk BSA content than the CDP cows after the dry period and similar milk lactose concentrations, suggesting that their mammary tight junctions were closed following parturition and, therefore, that the later stage of their lactogenesis was not impaired by SDP management. In early and midlactation, mammary cell apoptosis and proliferation rates as well as mammary expression of genes involved in the function of this tissue were unaffected by the dry period management strategy. For cows in their second lactation, mammary gland functional capacity at 70 d in milk tended to be lower in the SDP cows. In conclusion, even though SDP management decreased milk production during the subsequent lactation, it did not affect mammary cell activity. Although direct evidence is still lacking, decreased mammary cell growth during the dry period is likely responsible for this negative effect. The higher prolactin concentrations in lactating cows during late gestation could be involved in this effect. More research is needed to test these hypotheses.

Hepatobiliary infections due to non-capsulated Haemophilus influenzae.

We present two cases of non-capsulated Haemophilus influenzae hepatobiliary infection and review the literature. Such cases are rare, and prior to routine immunization against H. influenzae serotype b invasive Haemophilus disease was largely caused by capsulated strains. The epidemiology of invasive Haemophilus infections has changed and the number of cases of intra-abdominal and hepatobiliary infection may be underestimated due to current microbiological processing practices.

Effect of milking frequency on lactation persistency and mammary gland remodeling in mid-lactation cows.

The objective of this study was to evaluate the effect of milking frequency on milk production and composition, mammary cell proliferation, apoptosis, and gene expression. For this investigation, 10 Holstein cows that were being milked twice a day in mid lactation were selected. To study the effect of differential milking, 2 quarters were milked once daily and the other 2 were milked thrice daily for 8wk. After that period, twice-daily milking was resumed for all quarters, and data were collected for an additional 6wk. Mammary gland biopsies were taken 1wk before differential milking (wk -1) and after 4 and 8wk of differential milking. Milk samples were collected weekly throughout the experiment. Once-daily milking resulted in an immediate reduction in milk yield, whereas thrice-daily milking resulted in an increase in milk yield. During differential milking, the daily milk yield of the quarters milked once daily declined by 0.54kg/wk, on average, but remained constant in the quarters milked thrice daily. Part of the difference in milk yield between the glands pairs persisted after twice-daily milking was reinitiated. In the quarters milked once daily, milk BSA concentration increased, indicating an increase in tight junction leakiness, and zymographic analysis of milk enzymes showed increased activity of several proteases. Reducing the milking frequency also increased mammary cell apoptosis and, surprisingly, mammary cell proliferation. Interestingly, milk concentrations of stanniocalcin-1 and insulin-like growth factor-I and mammary gland expression of several genes were also modulated by milking frequency. For example, expression of insulin-like growth factor I receptor was downregulated during once-daily milking. Last, expression of the short and long isoforms of the prolactin receptor and of CSN2 (beta-casein) were upregulated during thrice-daily milking. Taken together, these data suggest that milking frequency not only affects mammary gland remodeling and the expression of paracrine factors but also modulates hormone sensitivity.

Impact of postpartum milking frequency on the immune system and the blood metabolite concentration of dairy cows.

The transition from pregnancy to lactation is marked by metabolic, hormonal, and immunological changes that have an impact on the incidence of infectious and metabolic diseases. The aim of this study was to evaluate the effect on immune function and blood metabolite concentration of limiting milk production in early lactation to reduce negative energy balance. Twenty-two multiparous Holstein cows were milked either once a day (1x) or twice a day (2x) for the first week postpartum. All cows were milked twice daily for the rest of lactation. Blood concentrations of nonesterified fatty acids (NEFA), beta-hydroxybutyric acid (BHBA), calcium, bilirubin, urea, phosphorus, glucose, leptin, stanniocalcin-1, and 17beta-estradiol were determined in samples collected from 5 wk before scheduled calving to 5 wk after calving. Polymorphonuclear leukocytes (PMNL) were isolated from blood to conduct assays for chemotaxis, phagocytosis, and respiratory burst. Peripheral blood mononuclear cells (PBMC) were isolated to evaluate lymphocyte proliferation and cytokine production (tumor necrosis factor-alpha, IL-4, and interferon-gamma). Cows milked 1x produced 31% less milk than cows milked 2x during the first week of lactation. Over the following 13 wk of lactation, the milk production of cows milked 1x during the first week was 8.1% lower than for cows milked 2x. However, because the percentages of fat and protein were greater in the milk from 1x cows, the yields of milk components and energy-corrected milk were similar. Calving induced an increase in the concentrations of NEFA, BHBA, urea, and bilirubin. The increases in levels of NEFA and BHBA were greater in cows milked 2x than in cows milked 1x. During the same period, the serum glucose concentration decreased but remained greater in cows milked 1x. Serum calcium on d 4 and serum phosphorus on d 4 and 5 were greater in cows milked 1x. The differences between the 2 groups persisted beyond treatment until postpartum d 24 for NEFA and glucose and until postpartum d 14 for BHBA. After calving, the concentrations of leptin and stanniocalcin-1 decreased. During the first week postpartum, the decrease of leptin was less marked in cows milked 1x. The immune functions of PBMC and PMNL isolated from experimental cows and incubated using a standard medium did not show clear-cut peripartum immunosuppression. These variables were not significantly affected by the treatments, with the exception of interferon-gamma secretion, which was greater on d 5 and 14 in cows milked 1x. In conclusion, limiting milk production in early lactation had positive effects on metabolite concentration, but larger studies are necessary to establish if this could reduce disease incidence.

Local control of mammary involution: is stanniocalcin-1 involved?

There is considerable evidence to indicate the existence of local control of mammary gland involution, but the exact nature of this control has yet to be defined. Stanniocalcin-1 (STC-1) is a newly discovered mammalian hormone that seems involved in the lactation process and may be implicated in the control of involution. As a first step in investigating this hypothesis, the change in STC-1 levels in milk and serum was measured during drying off. Nine Holstein cows in late lactation were milked twice daily on half the gland, while the other half was left unmilked for a 14-d period. Milk and blood samples and mammary biopsies were taken on d -7, 1, 2, 7, and 14 relative to the onset of the nonmilking period. The concentrations of STC-1 in serum and milk were determined by RIA. The albumin concentration and proteinase activity of the milk were determined. Apoptosis of the mammary epithelium was quantified by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Finally, the effects of milk on cellular activity and apoptosis were tested in vitro on mammary epithelial cells by measuring the turnover of tetrazolium salts and by counting the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells. The drying off of 2 quarters increased the milk production of the quarters that were milked by 30%. Milk proteinase activity and BSA and STC-1 concentrations increased in the nonmilked quarters, but remained unchanged in the milked quarters. Moreover, at d 2, the apoptotic rate of the mammary cells was higher in the nonmilked quarters than in the milked quarters (0.22 +/- 0.04 vs. 0.07 +/- 0.04%, respectively). Finally, in vitro experimentation demonstrated that mammary epithelial cells cultured in the presence of milk from involuting quarters had 3-fold more apoptotic cells as compared with those cultured in milk from the milked quarters at d 14. The metabolic rate was reduced by 14.6% for milk from d 7 and 23.6% for milk from d 14. Interestingly, the metabolic rate was negatively correlated with the STC-1 concentration in milk (r = -0.65). This study shows for the first time that STC-1 in milk is increased following milk stasis, although its exact role in the involution process remains to be clarified.

Serum levels of stanniocalcin-1 in Holstein heifers and cows.

Stanniocalcin-1 is a hormone that possesses both paracrine and endocrine functions and has recently been identified in mammals. While paracrine functions have been determined for several organs, the role of circulating stanniocalcin-1 in cattle is still unclear but, observations in mice and cows suggest that stanniocalcin-1 plays a role in both gestation and lactation. The changes in serum stanniocalcin-1 content in different physiological states have never been evaluated in ruminants. We measured stanniocalcin-1 levels in sera from cattle ranging in age from post-weaned calves to 17-month-old heifers and in sera from cows during lactation and pregnancy. Our results indicate that the blood concentration of stanniocalcin-1 is increased by pregnancy, but not by lactation. The highest levels of stanniocalcin-1 were found in young calves and during the non-lactating period preceding calving. This suggests that stanniocalcin-1 is important for gestation and the preparation of the mammary gland for lactation.

Effect of stage of lactation and parity on mammary gland cell renewal.

Milk production is a function of the number and activity of mammary epithelial cells, regardless of stage of lactation. Milk yield is generally higher in multiparous cows than in primiparous cows, but persistency is usually greater in the latter group. We compared several measures related to metabolic activity, apoptosis, and endocrine control of mammary cell growth in 8 primiparous and 9 multiparous cows throughout lactation. Mammary gland biopsies were taken in early [10 d in milk (DIM)], peak (50 DIM), and late (250 DIM) lactation to evaluate gene expression and determine DNA and fatty acid synthase (FAS) content. Milk samples taken the day before the biopsies were used to detect protease activities and to determine stanniocalcin-1 (STC) concentrations. Blood samples served to measure insulin-like growth factor-1, prolactin, and STC concentrations. Milk yield was higher in multiparous cows than in primiparous cows at the 10 DIM (32.8 +/- 1.3 and 25.2 +/- 0.8 kg/d) and 50 DIM (38.0 +/- 1.2 and 29.8 +/- 1.1 kg/d), but it was the same for both groups at 250 DIM (23.9 +/- 1.5 and 23.8 +/- 1.1 kg/d). Except for stearoyl-coenzyme A desaturase, expression of genes related to milk synthesis was not affected by stage of lactation. However, gene expression of acetyl-coenzyme A carboxylase, beta-casein, and FAS was lower in early lactation in primiparous cows. Expression of both proapoptotic bax and antiapoptotic bcl-2 genes was higher in primiparous cows, whereas the bax-to-bcl-2 ratio was not changed. Mammary DNA concentration was higher in multiparous cows, as was the amount of FAS protein in early lactation. Two bands of protease activity were found in milk samples, and one of the bands had an apparent molecular weight similar to gelatinase A and was dependent on the stage of lactation. Serum insulin-like growth factor-1 increased with day of lactation and was higher in primiparous cows. Serum prolactin decreased in late lactation, but peak values were observed in early lactation for primiparous cows and peak lactation for multiparous cows. Milk STC content increased with advancing lactation. The results are consistent with a lower degree of differentiation and a greater capacity for cell renewal in the mammary gland of primiparous cows.

Sensitivity of Botrytis squamosa to Different Classes of Fungicides.

An automated quantitative (AQ) assay was compared with radial growth on solid media and with dry weight in liquid culture for assaying fungicide sensitivity in Botrytis squamosa, the causal agent of onion leaf blight. Five isolates of B. squamosa were assayed for sensitivity to mancozeb (Dithane DG) and iprodione (Rovral) at five concentrations (0.5, 1.0, 5.0, 10.0, and 50 ppm). For mancozeb, the correlations between 50% effective concentration (EC50) values obtained with the three assays were not significant; however for iprodione, correlations between EC50 values for AQ and radial growth and for AQ and dry weight were significant (r = 0.98 and 0.99, respectively). The AQ method was less time consuming and more reliable than the two standard assays. The AQ method was used to evaluate the sensitivity of 35 field isolates of B. squamosa to mancozeb (Dithane DG), iprodione (Rovral), vinclozolin (Ronilan DF), and chlorothalonil (Bravo 500). All isolates were sensitive to mancozeb (EC50 ranged from 3.36 to 12.97) and chlorothalonil (EC50 < 1.5 µg/ml), but four isolates were insensitive to both iprodione (EC50 ≥ 3.98 µg/ml) and vinclozolin (EC50 ≥ 17.49 µg/ml). The ratio of the EC50 values of the least-sensitive and the most-sensitive isolates of B. squamosa was 1.08, 3.86, 6.98, and 37.59 for chlorothalonil, mancozeb, iprodione, and vinclozolin, respectively. Cross-resistance was observed for the two dicarboximide fungicides, iprodione and vinclozolin, with a significant correlation (r = 0.94) in the sensitivity of the 35 isolates to these two fungicides.

Is the link between nutrients and foods understood? The case of fibre and folate.

OBJECTIVES: To assess and contrast awareness of the link between dietary fibre and folate and their major food sources (fruit, vegetables, bread and cereals). DESIGN: Mailed questionnaire investigating changes made to dietary intake of fibre, folate, fruit, vegetables, bread and cereals in the previous six months. SETTING: The survey was conducted between June and November 1998 in the Australian Capital Territory. SUBJECTS: One thousand one hundred and twenty-six adults randomly selected from the electoral roll. RESULTS: More women than men in both older (50+ years) and younger (18-49 years) age groups reported increasing their consumption of folate, fibre, fruit and vegetables in the prior six months. In contrast, more men than women reported increased consumption of bread, cereals, rice and pasta in the previous six months. For food categories and fibre, less than 4% of respondents were unsure about changes in these food habits. However, 26% of men and women were 'not sure' about changes to folate intake. Similar proportions of men and women (about 33%) reported consuming more fruit, vegetables or cereal-based foods over the prior six months, yet only 6% of these men and 14% of these women reported consuming more folate. In contrast, 44% of men and 51% of women who reported consuming more plant foods also reported consuming more dietary fibre. CONCLUSIONS: The results suggested that subjects, particularly the younger age group, had a poor understanding of the relationship between folate intake and its major food sources. The understanding of the relationship between fibre intake and its food sources appeared substantial, but confusion about specific food sources was still evident. These outcomes question the effectiveness of nutrition education used to date, particularly for the current priority of increasing folate intake in younger women in the new, 'health claims' environment.

Induction of humoral and cellular immune responses against the nucleocapsid of bovine viral diarrhea virus by an adenovirus vector with an inducible promoter.

A new recombinant adenovirus was constructed that expressed the nucleocapsid (C protein or p14) of the bovine viral diarrhea virus (BVDV) under the control of a tetracycline-regulatable promoter. Mice covaccinated with this recombinant adenovirus, accompanied by another recombinant adenovirus expressing the trans-activator protein, induced a strong humoral immune response to the BVDV/C protein as detected by ELISA. Splenocytes from mice immunized with the recombinant adenovirus showed a specific proliferation response to both genotypes (type 1 and 2) of BVDV. High levels of IFN-gamma were detected in the supernatant of murine mononuclear cells of mice immunized by the recombinant adenovirus when stimulated in vitro by both genotypes of BVDV. These results indicate that this recombinant adenovirus is highly immunogenic and stimulates both cellular and humoral immune responses against the nucleocapsid of BVDV.

Recombinant adenoviruses expressing the E2 protein of bovine viral diarrhea virus induce humoral and cellular immune responses.

The E2 protein of bovine viral diarrhea virus (BVDV) is a major viral glycoprotein and an attractive target for BVDV vaccines. Three replication defective recombinant adenoviruses expressing the BVDV/E2 protein (rAds/E2) were constructed. Two contain a constitutive promoter, and one an inducible promoter. All three recombinant adenoviruses induced very strong BVDV specific antibody responses in a mouse model as detected by enzyme-linked immunosorbant assay (ELISA) and neutralization tests. Induction of cellular immune responses was investigated in two recombinant adenoviruses with a constitutive promoter. The mononuclear cells from the immunized mice demonstrated a proliferative response after in vitro stimulation with an homologous BVDV strain, but only one of them induced the production of IFN-gamma.

Investigation of the immunological properties of the bovine viral diarrhea virus protein NS3 expressed by an adenovirus vector in mice.

Two replication-defective human adenovirus recombinants encoding the NS3 protein (p80) of bovine viral diarrhea virus (BVDV) under the control of a modified adenovirus major later promoter (BM5), rAdBM5/NS3, and human cytomegalovirus promoter (CMV5), rAdCMV5/NS3, were constructed. These two recombinant adenoviruses were tested for their expression of the NS3 protein in vitro in three different cell lines and also in vivo for the induction of BVDV-specific immune responses in mice. The recombinant adenoviruses containing two different promoters induced different levels of humoral responses to the NS3 protein. The rAdBM5/NS3 was used to vaccinate mice in order to evaluate the ability of the NS3 protein in the induction of cellular immune responses. The rAdBM5/NS3 did not cause a stimulation of cell proliferation but caused a very strong increase in production of IFN-gamma in murine mononuclear cells stimulated in vivo by BVDV strains of genotype 1 and 2.