Genome-wide methylation profile following prenatal and postnatal dietary omega-3 fatty acid supplementation in pigs.

The objective of this study was to determine how prenatal and postnatal dietary omega-3 fatty acids alter white blood cell (leukocyte) DNA methylation of offspring. Fifteen gilts (n = 5 per treatment) were selected from one of three treatments: (i) control diet throughout gestation, lactation and nursery phase (CON); (ii) algal omega-3 fatty acid supplementation enriched in EPA and DHA (Gromega™ ) fed throughout gestation, lactation and nursery phase (Cn3); or (iii) Gromega™ supplementation maternally, during gestation and lactation only, and control diet during the nursery phase (Mn3). At 11 weeks of age and after 8 weeks of post-weaning nursery feeding, buffy coat genomic DNA was subjected to methyl CpG binding protein sequencing. The methylation enriched profile mapped to 26% of the porcine genome. On chromosome 4, a 27.7-kb differentially methylated region downstream of RUNX1T1 was hypomethylated in the Mn3 and Cn3 groups by 91.6% and 85.0% respectively compared to CON pigs. Conversely, hypermethylation was detected in intergenic regions of chromosomes 4 and 12. Regulatory impact factor and differential hubbing methods were used to identify pathways that were coordinately regulated by methylation due to feeding EPA and DHA during pregnancy. Despite limited ability to detect differential methylation, we describe methods that allow the identification of coordinated epigenetic regulation that could not otherwise be detected from subtle single locus changes in methylation. These data provide evidence of novel epigenetic regulation by maternal and early life supplementation of omega-3 fatty acids that may have implications to growth and inflammatory processes.

Senile hair graying: H2O2-mediated oxidative stress affects human hair color by blunting methionine sulfoxide repair.

Senile graying of human hair has been the subject of intense research since ancient times. Reactive oxygen species have been implicated in hair follicle melanocyte apoptosis and DNA damage. Here we show for the first time by FT-Raman spectroscopy in vivo that human gray/white scalp hair shafts accumulate hydrogen peroxide (H(2)O(2)) in millimolar concentrations. Moreover, we demonstrate almost absent catalase and methionine sulfoxide reductase A and B protein expression via immunofluorescence and Western blot in association with a functional loss of methionine sulfoxide (Met-S=O) repair in the entire gray hair follicle. Accordingly, Met-S=O formation of Met residues, including Met 374 in the active site of tyrosinase, the key enzyme in melanogenesis, limits enzyme functionality, as evidenced by FT-Raman spectroscopy, computer simulation, and enzyme kinetics, which leads to gradual loss of hair color. Notably, under in vitro conditions, Met oxidation can be prevented by L-methionine. In summary, our data feed the long-voiced, but insufficiently proven, concept of H(2)O(2)-induced oxidative damage in the entire human hair follicle, inclusive of the hair shaft, as a key element in senile hair graying, which does not exclusively affect follicle melanocytes. This new insight could open new strategies for intervention and reversal of the hair graying process.

The Ca2+-binding capacity of epidermal furin is disrupted by H2O2-mediated oxidation in vitiligo.

The Ca(2+)-dependent precursor convertase furin is abundantly expressed in epidermal keratinocytes and melanocytes. In this context, it is noteworthy that proopiomelanocortin (POMC) cleavage is also processed by furin, leading to ACTH, beta-lipotropin, and beta-endorphin. All prohormone convertases including furin are regulated by Ca(2+). Because numerous epidermal peptides and enzymes are affected by H(2)O(2)-mediated oxidation, including the POMC-derived peptides alpha-MSH and beta-endorphin as shown in the epidermis of patients with vitiligo, we here asked the question of whether furin could also be a possible target for this oxidation mechanism by using immunofluorescence, RT-PCR, Western blotting, Ca(2+)-binding studies, and computer modeling. Our results demonstrate significantly decreased in situ immunoreactivity of furin in the epidermis of patients with progressive vitiligo (n = 10), suggesting H(2)O(2)-mediated oxidation. This was confirmed by (45)Ca(2+)-binding studies with human recombinant furin identifying the loss of one Ca(2+)-binding site from the enzyme after oxidation with H(2)O(2). Computer simulation supported alteration of one of the two Ca(2+)-binding sites on furin. Taken together, our results implicate that the Ca(2+)-dependent proteolytic activity of this convertase is targeted by H(2)O(2), which in turn could contribute to the reduced epidermal expression of the POMC-derived peptides alpha-MSH and beta-endorphin as documented earlier in patients with vitiligo.

Effects of dietary enrichment with a marine oil-based n-3 LCPUFA supplement in sows with predicted birth weight phenotypes on growth performance and carcass quality of offspring.

Effects of a marine oil-based n-3 LCPUFA supplement (mLCPUFA) fed from weaning until the end of the next lactation to sows with a predicted low litter birth weight (LBW) phenotype on growth performance and carcass quality of litters born to these sows were studied, based on the hypothesis that LBW litters would benefit most from mLCPUFA supplementation. Sows were allocated to be fed either standard corn/soybean meal-based gestation and lactation diets (CON), or the same diets enriched with 0.5% of the mLCPUFA supplement at the expense of corn. The growth performance from birth until slaughter of the litters with the lowest average birth weight in each treatment (n=24 per treatment) is reported in this paper. At weaning, each litter was split between two nursery pens with three to six pigs per pen. At the end of the 5-week nursery period, two barrows and two gilts from each litter that had individual birth weights closest to their litter average birth weight, were moved to experimental grow-finish pens (barn A), where they were housed as two pigs per pen, sorted by sex within litter. Remaining pigs in each litter were moved to another grow-finish barn (barn B) and kept in mixed-sex pens of up to 10 littermates. After 8 weeks, one of the two pigs in each pen in barn A was relocated to the pens holding their respective littermates in barn B. The remaining barrows and gilts were individually housed in the pens in barn A until slaughter. Maternal mLCPUFA supplementation increased docosahexaenoic acid (DHA) concentration in the brain, liver and Semitendinosus muscle of stillborn pigs (P<0.01), did not affect eicosapentaenoic acid and DHA concentrations in sow serum at the end of lactation, and did not affect average daily gain, average daily feed intake or feed utilization efficiency of the offspring. BW was higher (P<0.01) in the second half of the grow-finish phase in pigs from mLCPUFA sows compared with controls in barn A, where space and competition for feed was minimal, but not barn B. Carcass quality was not affected by treatment for pigs from barn A, but maternal mLCPUFA supplementation negatively affected carcass quality in pigs from barn B. Collectively, these results suggest that nutritional supplementation of sows can have lasting effects on litter development, but that feeding mLCPUFA to sows during gestation and lactation was not effective in improving growth rates or carcass quality of LBW litters.

Effects of dietary enrichment with a marine oil-based n-3 LCPUFA supplement in sows with predicted birth weight phenotypes on birth litter quality and growth performance to weaning.

The effects of a marine oil-based n-3 long-chain polyunsaturated fatty acid (mLCPUFA) supplement fed to the sow from weaning, through the rebreeding period, during gestation and until end of lactation on litter characteristics from birth until weaning were studied in sows with known litter birth weight phenotypes. It was hypothesized that low birth weight (LBW) litters would benefit more from mLCPUFA supplementation than high birth weight litters. A total of 163 sows (mean parity=4.9 ± 0.9) were rebred after weaning. Sows were pair-matched by parity and litter average birth weight of the previous three litters. Within pairs, sows were allocated to be fed either standard corn/soyabean meal-based gestation and lactation diets (CON), or the same diets enriched with 0.5% of the mLCPUFA supplement at the expense of corn. Each litter between 9 and 16 total pigs born was classified as LBW or medium/high average birth weight (MHBW) litter and there was a significant correlation (P<0.001) between litter average birth weight of the current and previous litters within sows (r=0.49). Sow serum was harvested at day 113 of gestation for determination of immunoglobulin G (IgG) concentrations. The number of pigs born total and alive were lower (P=0.01) in mLCPUFA than CON sows, whereas the number of stillborn and mummified pigs were similar between treatments. Number of stillborns (trend) and mummies (P<0.01) were higher in LBW than MHBW litters. Tissue weights and brain : tissue weight ratios were similar between treatments, but LBW litters had decreased tissue weights and increased brain : tissue weight ratios compared with MHBW litters. Placental weight was lower (P=0.01) in LBW than MHBW litters, but was not different between treatments. Average and total litter weight at day 1 was similar between treatments. mLCPUFA increased weaning weight (P=0.08) and average daily gain (P<0.05) in MHBW litters, but not in LBW litters. Pre-weaning mortality was similar between treatments, but was higher (P<0.01) in LBW than MHBW litters. IgG concentration in sow serum was similar between treatments and litter birth weight categories. In conclusion, litter birth weight phenotype was repeatable within sows and LBW litters showed the benchmarks of intra-uterine growth retardation (lower placental weight and brain sparing effects). As maternal mLCPUFA supplementation decreased litter size overall, only improved litter growth rate until weaning in MHBW litters, and did not affect pre-weaning mortality, maternal mLCPUFA supplementation was not an effective strategy in our study for mitigating negative effects of a LBW litter phenotype.

Abnormal brainstem auditory evoked potentials and neuropathology in "locked-in" syndrome.

Brainstem auditory evoked potentials (BAEPs) were studied in a "locked-in" syndrome resulting from multiple occlusions of paramedian pontine arteries. Abnormality of BAEPs (first recorded 10 days after infarction) indicated that brainstem damage extended beyond the basis pontis, where, typically, interruption of corticospinal pathways results in the "locked-in" syndrome. Later, partial BAEP recovery suggested that the technique can be used to distinguish between permanent and transient brainstem involvement. Autopsy examination revealed close correspondence between permanent brainstem damage and persistent BAEP abnormalities. Persistent abnormal prolongation of III-V conduction time and aberration of wave IV were associated with damage near the lateral lemniscus contralateral to the stimulated ear.

Characterization of the potential antioxidant and pro-oxidant actions of some neuroleptic drugs.

It has been suggested in the literature that neuroleptic drugs may be able to exert antioxidant and/or pro-oxidant actions in vivo. The feasibility of this was tested by measuring the ability of chlorpromazine, prochlorperazine, metoclopramide, methotrimeprazine and haloperidol to scavenge biologically relevant oxygen-derived species in vitro. None of the drugs reacted with superoxide radical at a significant rate. Chlorpromazine, prochlorperazine, metoclopramide and methotrimeprazine were very powerful scavengers of hydroxyl radicals, reacting at almost a diffusion-controlled rate. Chlorpromazine showed some ability to inhibit iron ion-dependent hydroxyl radical formation. Chlorpromazine, methotrimeprazine, promethazine and prochlorperazine were powerful inhibitors of iron ion-dependent liposomal lipid peroxidation, scavengers of organic peroxyl radicals and inhibitors of haem protein/hydrogen peroxide-dependent peroxidation of arachidonic acid. Chlorpromazine, prochlorperazine, metoclopramide, methotrimeprazine and haloperidol were powerful scavengers of hypochlorous acid. Haloperidol showed no ability to inhibit lipid peroxidation or to scavenge peroxyl radicals, and reproducibly increased lipid peroxidation catalysed by haem proteins, in both the presence and absence of hydrogen peroxide. The relevance of these in vitro observations to events in vivo is discussed.

Histopathological and microanalytical study of zirconium dioxide and barium sulphate in bone cement.

AIMS: To report the appearances of zirconium dioxide and barium sulphate in interface membranes, synovium, and other tissues around joint prostheses. METHODS: Histological sections from 23 specimens were reviewed by light microscopy and polarisation. Scanning electron microscopy and x ray microanalysis were performed on routinely processed paraffin wax sections. RESULTS: Polyethylene, metals, and polymethylmethacrylate cement debris were easily recognisable. Almost all the cement remnants contained either zirconium dioxide or barium sulphate, confirmed by microanalysis. The contrast media had characteristic light microscopic appearances. Zirconium was identified in macrophages away from cement remnants. CONCLUSION: The presence of radiographic contrast media in tissues around prosthetic joints is common but not widely recognised.

Imaging factors for xeroradiography of the extremities.

A biological test object has been used to determine optimum imaging factors for xeroradiography of the extremities. Three observers independently demonstrated a clear preference for positive-mode imaging.

The mammographic demonstration of malignant spicules.

A specimen of breast tissue containing a carcinoma with multiple spiculations was used as a test object and examined by a range of mammographic techniques. The images were compared with a photograph of the cleared specimen. A quantitative method of evaluating the performance of each technique is described and used to compare these images. Xeroradiography clearly excelled other imaging methods.

Bone and joint infection in a renal unit.

Between 1980 and 1984 nine adult patients in the renal unit of Guy's Hospital developed bone and joint infection. The commonest site of infection was the spine. In this series two patients died, a mortality of 22%. The purpose of this paper is to illustrate the pitfalls in the diagnosis and management of bone and joint infection in patients with renal failure and renal transplants.

Pseudomalignant heterotopic ossification ("myositis ossificans"). Recurrence after excision with subsequent resorption.

An eight-year-old boy presented with massive pseudomalignant heterotopic ossification around the upper femur. The mass was completely excised because of severe pain, systemic illness and a flexion contracture at the hip. Symptomatic improvement was swift, but two weeks later the mass had recurred and was even more extensive. During the subsequent 18 months of conservative management he has been free of pain and there has been progressive resorption and remodelling of the heterotopic bone. There is now no limitation of physical activity and movement at the hip is full.

End-stage avascular necrosis of bone in renal transplant patients. The natural history.

We report the results of conservative treatment of stage III and stage IV avascular necrosis of bone (AVN) affecting the hip or knee in renal transplant patients. Twenty-nine patients were followed for a mean period of five years. Conservative management was successful in controlling symptoms in 40% of those with AVN of the hip and in 70% of those with AVN of the knee. Knowledge of the natural history of AVN is important because of the long survival times after renal transplantation.

The blood supply of the greater trochanter.

We perfused 16 human femora with a 50% barium sulphate suspension and studied the intra-osseous vessels by microfocal radiography and histology. There were few anastomoses between the vessels of the greater trochanter and those of the adjacent cancellous bone of the shaft. Ischaemia of the trochanter may contribute to nonunion after trochanteric osteotomy.

Fungal osteomyelitis of the foot. A report of an unusual case.

Mucormycosis is an opportunistic infection that very occasionally causes osteomyelitis and avascular necrosis of bone. The infection may prove fatal if not diagnosed promptly. If early treatment is instituted the prognosis is good.

Early avascular necrosis of the femoral head. Report of a case and review of the literature.

The clinical and pathological findings in a case of early avascular necrosis of the femoral head following renal transplantation are described. Regions of subchondral bone distant from the principal lesions showed increased numbers of empty osteocytic lacunae. This has been quantified and it is suggested that a loss of osteocytes is perhaps one of the earliest lesions leading to established avascular necrosis.

The femoral head in osteonecrosis. A quantitative study of osteocyte population.

The occurrence of osteonecrosis following renal transplantation is well recognised but its pathogenesis remains unknown. We have quantified the number of empty osteocytic lacunae in the subchondral bone of femoral heads from a control group of patients, and compared these with femoral heads from a group of renal transplant recipients without evidence of overt osteonecrosis. There is a significant increase in empty osteocytic lacunae in renal transplant patients. We conclude that loss of osteocytes precedes other manifestations of osteonecrosis.

Effect of menhaden fish oil supplementation and lipopolysaccharide exposure on nursery pigs. I. Effects on the immune axis when fed diets containing spray-dried plasma.

The objective of the present study was to evaluate the potential immunological benefit of adding menhaden fish oil to the diet of weaned pigs. Twenty-four crossbred male pigs were weaned at approximately 18 days of age and placed on a complex nursery diet containing 30% lactose and 7% plasma protein with 6% corn oil as the fat source (Cont, n=12) or with 5% menhaden fish oil and 1% corn oil as the fat source (MFO, n=12) for a period of 15 days. Body weights did not differ (P>0.78) between dietary groups either at the beginning or end of the 15 days feeding period. On day 15, all pigs were non-surgically fitted with an indwelling jugular catheter. On d 16, pigs received an i.v. injection of either saline (n=6/dietary group) or lipopolysaccharide (LPS; 150 microg/kg body weight; n=6/dietary group) and blood samples were collected at 30 min intervals for a period of 5h. Serum was harvested and stored at -80 degrees C for analysis of cortisol (CS), corticosteroid-binding globulin (CBG), tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). There was no significant effect of diet on basal concentrations (Time 0) of any of the blood parameters analyzed. A Time x Treatment x Diet interaction (P<0.03) was observed for serum CS such that those pigs which consumed the MFO diet followed by LPS treatment had a reduced CS response as compared to the LPS-treated pigs on the Cont diet. A Time x Treatment interaction (P<0.01) was observed for serum CBG such that LPS treatment reduced circulating CBG as compared to the saline-treated pigs. Time x Treatment x Diet interactions were also observed for serum concentrations of TNF-alpha (P=0.084) and IFN-gamma (P=0.022) such that both the TNF-alpha and IFN-gamma response to the LPS challenge was lower in those pigs receiving the MFO diet as compared to the LPS-treated pigs on the Cont diet. Overall, serum CS was negatively correlated with the CBG response (r=-0.40, P<0.001), however, the strongest negative correlation was observed in the LPS-treated pigs which consumed the MFO diet (r=-0.63, P<0.001). While further studies are needed to evaluate the immunological response of including MFO in the nursery pig diet, the present study demonstrates that supplementation with MFO does indeed alter the immunological response to an LPS challenge.

Early weaning to reduce tissue mobilization in lactating sows and milk supplementation to enhance pig weaning weight during extreme heat stress.

This study was conducted to determine the effect of reduced lactation length and supplemental milk replacer (MR) during high ambient temperatures. Thirty nine primiparous and 100 multiparous sows (PIC, Franklin, KY, C-22) were used in a 2 x 2 x 2 factorial arrangement of treatments. Treatments consisted of two lactation room temperatures (21 degrees C [TN] and 32 degrees C [HOT]), two lactation lengths (14 or 19 d), and two parity groups (primiparous, multiparous). Pigs were either: 1) sow-reared to 19 d or 2) sow-reared to 14 d, and then reared to 19 d with MR after sow removal. All sows were fed the same diet (1.07% lysine, 3,366 kcal of ME/kg). Sows were weighed and ultrasound for backfat thickness (BF) and longissimus muscle area (LMA) within 6 h after farrowing and at the time of sow removal (d 14 or 19). Pigs were individually weighed at weaning (d 19) and after a 47-d nursery period (d 66). Heat stress increased sow weight loss (-13.35 kg, P < 0.01) and decreased sow feed intake (4.63 kg/d, P < 0.01) during lactation compared with sows in TN (+4.5 kg and 7.5 kg/d, respectively). Early weaning (d 14) during heat stress decreased maternal weight loss (-10.1 vs. -16.6 kg, P < 0.01). Primiparous sows lost more BF in both environments (-2.60 vs. -1.56 mm, P < 0.05), and both parity groups lost more BF (-3.35 vs. -2.3 mm, P < 0.10) and LMA (-1.82 vs. -0.77 cm2, P < 0.05) when lactating for 19 d in the HOT environment than those lactating for 14 d. Pigs nursing primiparous and multiparous sows in the HOT environment and provided MR had heavier individual 19-d weights (7.37 and 8.12 kg/ pig, respectively) than those nursing to 19 d (5.57 and 6.04 kg/pig, P < 0.01). Milk replacer decreased the difference normally observed in 19-d weights between primiparous and multiparous sow-reared pigs in TN. Pigs fed MR in both environments and nursing multiparous sows had improved weight gains in the nursery compared with pigs nursing sows to 19 d (428 vs. 406 g/d, respectively; P < 0.01), or reared by primiparous sows (444 vs. 390 g/d , respectively; P < 0.01). Sow weaning on d 14 in the HOT environment decreased the wean-to-estrus interval in primiparous sows (22.8 vs. 9.2 d, P < 0.10). This study shows the benefit of early weaning in combination with milk replacer to preserve the sow and to restore pig weaning weights and nursery end weights under heat stress.

Phosphorus bioavailability and digestibility of normal and genetically modified low-phytate corn for pigs.

We conducted two studies to determine the bioavailability and apparent digestibility of P in a low-phytate corn hybrid (.28% total P, .10% phytate P) genetically modified to be homozygous for the 1pa1-1 allele and a nearly isogenic corn hybrid (normal) (.25% total P, .20% phytate P). Additionally, we conducted an in vitro assay involving a peptic and pancreatin digestion to estimate P availability. The first study used 50 individually penned pigs (initial body weight 9 kg) and 10 treatments in a randomized complete block design. A cornstarch-soybean meal basal diet (.6% Ca, .2% P) was used. Treatments consisted of the basal diet and the basal diet plus .05, .10, or .15% P from monosodium phosphate (MSP), low-phytate corn, or normal corn. After a 35-d feeding period, pigs were killed to collect the fourth metacarpal for measurements of ash and breaking load. Breaking load was regressed on added P intake, and the bioavailability of P was determined by the slope ratio method. The bioavailabilities of P (relative to MSP) for low-phytate and normal corn were 62 and 9%, respectively. These were similar to the determined in vitro values of 57 and 11% for low-phytate and normal corn, respectively. In the second study, 20 pigs (initial BW 20 kg) were used in a randomized complete block design with a 2 x 2 factorial arrangement of treatments. Two corn lines (low-phytate and normal) and two levels of supplemental P (0 and .2%) from dicalcium phosphate were used. Diets with no added P were formulated to contain .9% lysine, .6% Ca, and .34% P. Apparent nutrient digestibilities were calculated from total collection of urine and feces for 5 d. There were no differences among treatments for energy and nitrogen digestibility. Pigs fed low-phytate corn with no added P had increased digestibility and retention of P and reduced total P excretion (P < .05). We conclude that low-phytate corn contains at least five times as much available P as normal corn. The use oflow-phytate corn greatly reduced the amount of P excreted by the pig and increased the N:P ratio in the manure.