Vaginal microbiota diverges in sows with low and high reproductive performance after porcine reproductive and respiratory syndrome vaccination.

Previous studies have demonstrated evidence for a relationship between the vaginal microbiome and reproductive performance, suggesting the vaginal microbiota may serve as a tool to predict farrowing outcomes in commercial pigs. In this study, we compared the vaginal microbiome in sows with low and high farrowing performance and used it to classify animals with contrasting reproductive outcomes in commercial sows following immune challenge with porcine respiratory and reproductive syndrome (PRRS) vaccination. Eighteen microbes were differentially abundant (q-value < 0.05) between the Low and High farrowing performance groups. Among them, Campylobacter, Bacteroides, Porphyromonas, Lachnospiraceae unclassified, Prevotella, and Phascolarctobacterium were also selected in the discriminant and linear regression analyses, and could be used as potential biomarkers for reproductive outcomes. The correct classification rate in the two groups was 100%. In conclusion, this study demonstrates that vaginal microbiota collected after PRRS vaccination could be potentially used to classify sows into having low or high farrowing performance in commercial herds.

A comparison of accuracy validation methods for genomic and pedigree-based predictions of swine litter size traits using Large White and simulated data.

The objective of this study was to compare and determine the optimal validation method when comparing accuracy from single-step GBLUP (ssGBLUP) to traditional pedigree-based BLUP. Field data included six litter size traits. Simulated data included ten replicates designed to mimic the field data in order to determine the method that was closest to the true accuracy. Data were split into training and validation sets. The methods used were as follows: (i) theoretical accuracy derived from the prediction error variance (PEV) of the direct inverse (iLHS), (ii) approximated accuracies from the accf90(GS) program in the BLUPF90 family of programs (Approx), (iii) correlation between predictions and the single-step GEBVs from the full data set (GEBVFull ), (iv) correlation between predictions and the corrected phenotypes of females from the full data set (Yc ), (v) correlation from method iv divided by the square root of the heritability (Ych ) and (vi) correlation between sire predictions and the average of their daughters' corrected phenotypes (Ycs ). Accuracies from iLHS increased from 0.27 to 0.37 (37%) in the Large White. Approximation accuracies were very consistent and close in absolute value (0.41 to 0.43). Both iLHS and Approx were much less variable than the corrected phenotype methods (ranging from 0.04 to 0.27). On average, simulated data showed an increase in accuracy from 0.34 to 0.44 (29%) using ssGBLUP. Both iLHS and Ych approximated the increase well, 0.30 to 0.46 and 0.36 to 0.45, respectively. GEBVFull performed poorly in both data sets and is not recommended. Results suggest that for within-breed selection, theoretical accuracy using PEV was consistent and accurate. When direct inversion is infeasible to get the PEV, correlating predictions to the corrected phenotypes divided by the square root of heritability is adequate given a large enough validation data set.

A heuristic method to identify runs of homozygosity associated with reduced performance in livestock.

Although, for the most part, genome-wide metrics are currently used in managing livestock inbreeding, genomic data offer, in principle, the ability to identify functional inbreeding. Here, we present a heuristic method to identify haplotypes contained within a run of homozygosity (ROH) associated with reduced performance. Results are presented for simulated and swine data. The algorithm comprises 3 steps. Step 1 scans the genome based on marker windows of decreasing size and identifies ROH genotypes associated with an unfavorable phenotype. Within this stage, multiple aggregation steps reduce the haplotype to the smallest possible length. In step 2, the resulting regions are formally tested for significance with the use of a linear mixed model. Lastly, step 3 removes nested windows. The effect of the unfavorable haplotypes identified and their associated haplotype probabilities for a progeny of a given mating pair or an individual can be used to generate an inbreeding load matrix (ILM). Diagonals of ILM characterize the functional individual inbreeding load (IIL). We estimated the accuracy of predicting the phenotype based on IIL. We further compared the significance of the regression coefficient for IIL on phenotypes with genome-wide inbreeding metrics. We tested the algorithm using simulated scenarios (12 scenarios), combining different levels of linkage disequilibrium (LD) and number of loci impacting a quantitative trait. Additionally, we investigated 9 traits from 2 maternal purebred swine lines. In simulated data, as the LD in the population increased, the algorithm identified a greater proportion of the true unfavorable ROH effects. For example, the proportion of highly unfavorable true ROH effects identified rose from 32 to 41% for the low- to the high-LD scenario. In both simulated and real data, the haplotypes identified were contained within a much larger ROH (9.12-12.1 Mb). The IIL prediction accuracy was greater than 0 across all scenarios for simulated data (mean of 0.49 [95% confidence interval 0.47-0.52] for the high-LD scenario) and for nearly all swine traits (mean of 0.17 [SD 0.10]). On average, across simulated and swine data sets, the IIL regression coefficient was more closely related to progeny performance than any genome-wide inbreeding metric. A heuristic method was developed that identified ROH genotypes with reduced performance and characterized the combined effects of ROH genotypes within and across individuals.

The relationship between different measures of feed efficiency and feeding behavior traits in Duroc pigs.

Utilization of feed in livestock species consists of a wide range of biological processes, and therefore, its efficiency can be expressed in various ways, including direct measurement, such as daily feed intake, as well as indicator measures, such as feeding behavior. Measuring feed efficiency is important to the swine industry, and its accuracy can be enhanced by using automated feeding systems, which record feed intake and associated feeding behavior of individual animals. Each automated feeder space is often shared among several pigs and therefore raises concerns about social interactions among pen mates with regard to feeding behavior. The study herein used a data set of 14,901 Duroc boars with individual records on feed intake, feeding behavior, and other off-test traits. These traits were modeled with and without the random spatial effect of Pen_Room, a concatenation of room and pen, or random social interaction among pen mates. The nonheritable spatial effect of common Pen-Room was observed for traits directly measuring feed intake and accounted for up to 13% of the total phenotypic variance in the average daily feeding rate. The social interaction effect explained larger proportions of phenotypic variation in all the traits studied, with the highest being 59% for ADFI in the group of feeding behaviors, 73% for residual feed intake (RFI; RFI4 and RFI6) in the feed efficiency traits, and 69% for intramuscular fat percentage in the off-test traits. After accounting for the social interaction effect, residual BW gain and RFI and BW gain (RIG) were found to have the heritability of 0.38 and 0.18, respectively, and had strong genetic correlations with growth and off-test traits. Feeding behavior traits were found to be moderately heritable, ranging from 0.14 (ADFI) to 0.52 (average daily occupation time), and some of them were strongly correlated with feed efficiency measures; for example, there was a genetic correlation of 0.88 between ADFI and RFI6. Our work suggested that accounting for the social common pen effect was important for estimating genetic parameters of traits recorded by the automated feeding system. Residual BW gain and RIG appeared to be two robust measures of feed efficiency. Feeding behavior measures are worth further investigation as indicators of feed efficiency.

Genetic relationship between purebred and crossbred sow longevity.

BACKGROUND: The overall breeding objective for a nucleus swine selection program is to improve crossbred commercial performance. Most genetic improvement programs are based on an assumed high degree of positive relationship between purebred performance in a nucleus herd and their relatives' crossbred performance in a commercial herd. The objective of this study was to examine the relationship between purebred and crossbred sow longevity performance. Sow longevity was defined as a binary trait with a success occurring if a sow remained in the herd for a certain number of parities and including the cumulative number born alive as a measure of reproductive success. Heritabilities, genetic correlations, and phenotypic correlations were estimated using THRGIBBS1F90. RESULTS: Results indicated little to no genetic correlations between crossbred and purebred reproductive traits. This indicates that selection for longevity or lifetime performance at the nucleus level may not result in improved longevity and lifetime performance at the crossbred level. Early parity performance was highly correlated with lifetime performance indicating that an indicator trait at an early parity could be used to predict lifetime performance. This would allow a sow to have her own record for the selection trait before she has been removed from the herd. CONCLUSIONS: Results from this study aid in quantifying the relationship between purebred and crossbred performance and provide information for genetic companies to consider when developing a selection program where the objective is to improve crossbred sow performance. Utilizing crossbred records in a selection program would be the best way to improve crossbred sow productivity.

The use of multiple imputation for the accurate measurements of individual feed intake by electronic feeders.

Obtaining accurate individual feed intake records is the key first step in achieving genetic progress toward more efficient nutrient utilization in pigs. Feed intake records collected by electronic feeding systems contain errors (erroneous and abnormal values exceeding certain cutoff criteria), which are due to feeder malfunction or animal-feeder interaction. In this study, we examined the use of a novel data-editing strategy involving multiple imputation to minimize the impact of errors and missing values on the quality of feed intake data collected by an electronic feeding system. Accuracy of feed intake data adjustment obtained from the conventional linear mixed model (LMM) approach was compared with 2 alternative implementations of multiple imputation by chained equation, denoted as MI (multiple imputation) and MICE (multiple imputation by chained equation). The 3 methods were compared under 3 scenarios, where 5, 10, and 20% feed intake error rates were simulated. Each of the scenarios was replicated 5 times. Accuracy of the alternative error adjustment was measured as the correlation between the true daily feed intake (DFI; daily feed intake in the testing period) or true ADFI (the mean DFI across testing period) and the adjusted DFI or adjusted ADFI. In the editing process, error cutoff criteria are used to define if a feed intake visit contains errors. To investigate the possibility that the error cutoff criteria may affect any of the 3 methods, the simulation was repeated with 2 alternative error cutoff values. Multiple imputation methods outperformed the LMM approach in all scenarios with mean accuracies of 96.7, 93.5, and 90.2% obtained with MI and 96.8, 94.4, and 90.1% obtained with MICE compared with 91.0, 82.6, and 68.7% using LMM for DFI. Similar results were obtained for ADFI. Furthermore, multiple imputation methods consistently performed better than LMM regardless of the cutoff criteria applied to define errors. In conclusion, multiple imputation is proposed as a more accurate and flexible method for error adjustments in feed intake data collected by electronic feeders.

Using single-step genomic best linear unbiased predictor to enhance the mitigation of seasonal losses due to heat stress in pigs.

The purposes of this study were to analyze the impact of seasonal losses due to heat stress in pigs from different breeds raised in different environments and to evaluate the accuracy improvement from adding genomic information to genetic evaluations. Data were available for 2 different swine populations: purebred Duroc animals raised in Texas and North Carolina and commercial crosses of Duroc and F females (Landrace × Large White) raised in Missouri and North Carolina; pedigrees provided links for animals from different states. Pedigree information was available for 553,442 animals, of which 8,232 pure breeds were genotyped. Traits were BW at 170 d for purebred animals and HCW for crossbred animals. Analyses were done with an animal model as either single- or 2-trait models using phenotypes measured in different states as separate traits. Additionally, reaction norm models were fitted for 1 or 2 traits using heat load index as a covariable. Heat load was calculated as temperature-humidity index greater than 70 and was averaged over 30 d prior to data collection. Variance components were estimated with average information REML, and EBV and genomic EBV (GEBV) with BLUP or single-step genomic BLUP (ssGBLUP). Validation was assessed for 146 genotyped sires with progeny in the last generation. Accuracy was calculated as a correlation between EBV and GEBV using reduced data (all animals, except the last generation) and using complete data. Heritability estimates for purebred animals were similar across states (varying from 0.23 to 0.26), and reaction norm models did not show evidence of a heat stress effect. Genetic correlations between states for heat loads were always strong (>0.91). For crossbred animals, no differences in heritability were found in single- or 2-trait analysis (from 0.17 to 0.18), and genetic correlations between states were moderate (0.43). In the reaction norm for crossbreeds, heritabilities ranged from 0.15 to 0.30 and genetic correlations between heat loads were as weak as 0.36, with heat load ranging from 0 to 12. Accuracies with ssGBLUP were, on average, 25% greater than with BLUP. Accuracies were greater in 2-trait reaction norm models and at extreme heat load values. Impacts of seasonality are evident only for crossbred animals. Genomic information can help producers mitigate heat stress in swine by identifying superior sires that are more resistant to heat stress.

Variance component estimates for alternative litter size traits in swine.

Litter size at d 5 (LS5) has been shown to be an effective trait to increase total number born (TNB) while simultaneously decreasing preweaning mortality. The objective of this study was to determine the optimal litter size day for selection (i.e., other than d 5). Traits included TNB, number born alive (NBA), litter size at d 2, 5, 10, 30 (LS2, LS5, LS10, LS30, respectively), litter size at weaning (LSW), number weaned (NW), piglet mortality at d 30 (MortD30), and average piglet birth weight (BirthWt). Litter size traits were assigned to biological litters and treated as a trait of the sow. In contrast, NW was the number of piglets weaned by the nurse dam. Bivariate animal models included farm, year-season, and parity as fixed effects. Number born alive was fit as a covariate for BirthWt. Random effects included additive genetics and the permanent environment of the sow. Variance components were plotted for TNB, NBA, and LS2 to LS30 using univariate animal models to determine how variances changed over time. Additive genetic variance was minimized at d 7 in Large White and at d 14 in Landrace pigs. Total phenotypic variance for litter size traits decreased over the first 10 d and then stabilized. Heritability estimates increased between TNB and LS30. Genetic correlations between TNB, NBA, and LS2 to LS29 with LS30 plateaued within the first 10 d. A genetic correlation with LS30 of 0.95 was reached at d 4 for Large White and at d 8 for Landrace pigs. Heritability estimates ranged from 0.07 to 0.13 for litter size traits and MortD30. Birth weight had an h of 0.24 and 0.26 for Large White and Landrace pigs, respectively. Genetic correlations among LS30, LSW, and NW ranged from 0.97 to 1.00. In the Large White breed, genetic correlations between MortD30 with TNB and LS30 were 0.23 and -0.64, respectively. These correlations were 0.10 and -0.61 in the Landrace breed. A high genetic correlation of 0.98 and 0.97 was observed between LS10 and NW for Large White and Landrace breeds, respectively. This would indicate that NW could possibly be used as an effective maternal trait, given a low level of cross-fostering, to avoid back calculating litter size traits from piglet records. Litter size at d 10 would be a compromise between gain in litter size at weaning and minimizing the potentially negative effects of the nurse dam and direct additive genetics of the piglets, as they are expected to increase throughout lactation.

Feed intake, average daily gain, feed efficiency, and real-time ultrasound traits in Duroc pigs: II. Genomewide association.

Efficient use of feed resources has become a clear challenge for the U.S. pork industry as feed costs continue to be the largest variable expense. The availability of the Illumina Porcine60K BeadChip has greatly facilitated whole-genome association studies to identify chromosomal regions harboring genes influencing those traits. The current study aimed at identifying genomic regions associated with variation in feed efficiency and several production traits in a Duroc terminal sire population, including ADFI, ADG, feed conversion ratio, residual feed intake (RFI), real-time ultrasound back fat thickness (BF), ultrasound muscle depth, intramuscular fat content (IMF), birth weight (BW at birth), and weaning weight (BW at weaning). Single trait association analyses were performed using Bayes B models with 35,140 SNP on 18 autosomes after quality control. Significance of nonoverlapping 1-Mb length windows (n = 2,380) were tested across 3 QTL inference methods: posterior distribution of windows variances from Monte Carlo Markov Chain, naive Bayes factor, and nonparametric bootstrapping. Genes within the informative QTL regions for the traits were annotated. A region ranging from166 to 140 Mb (4-Mb length) on SSC 1, approximately 8 Mb upstream of the MC4R gene, was significantly associated with ADFI, ADG, and BF, where SOCS6 and DOK6 are proposed as the most likely candidate genes. Another region affecting BW at weaning was identified on SSC 4 (84-85 Mb), harboring genes previously found to influence both human and cattle height: PLAG1, CHCHD7, RDHE2 (or SDR16C5), MOS, RPS20, LYN, and PENK. No QTL were identified for RFI, IMF, and BW at birth. In conclusion, we have identified several genomic regions associated with traits affecting nutrient utilization that could be considered for future genomic prediction to improve feed utilization.

Feed intake, average daily gain, feed efficiency, and real-time ultrasound traits in Duroc pigs: I. Genetic parameter estimation and accuracy of genomic prediction.

The efficiency of producing salable products in the pork industry is largely determined by costs associated with feed and by the amount and quality of lean meat produced. The objectives of this paper were 1) to explore heritability and genetic correlations for growth, feed efficiency, and real-time ultrasound traits using both pedigree and marker information and 2) to assess accuracy of genomic prediction for those traits using Bayes A prediction models in a Duroc terminal sire population. Body weight at birth (BW at birth) and weaning (BW at weaning) and real-time ultrasound traits, including back fat thickness (BF), muscle depth (MD), and intramuscular fat content (IMF), were collected on the basis of farm protocol. Individual feed intake and serial BW records of 1,563 boars obtained from feed intake recording equipment (FIRE; Osborne Industries Inc., Osborne, KS) were edited to obtain growth, feed intake, and feed efficiency traits, including ADG, ADFI, feed conversion ratio (FCR), and residual feed intake (RFI). Correspondingly, 1,047 boars were genotyped using the Illumina PorcineSNP60 BeadChip. The remaining 516 boars, as an independent sample, were genotyped with a low-density GGP-Porcine BeadChip and imputed to 60K. Magnitudes of heritability from pedigree analysis were moderate for growth, feed intake, and ultrasound traits (ranging from 0.44 ± 0.11 for ADG to 0.58 ± 0.09 for BF); heritability estimates were 0.32 ± 0.09 for FCR but only 0.10 ± 0.05 for RFI. Comparatively, heritability estimates using marker information by Bayes A models were about half of those from pedigree analysis, suggesting "missing heritability." Moderate positive genetic correlations between growth and feed intake (0.32 ± 0.05) and back fat (0.22 ± 0.04), as well as negative genetic correlations between growth and feed efficiency traits (-0.21 ± 0.08, -0.05 ± 0.07), indicate selection solely on growth traits may lead to an undesirable increase in feed intake, back fat, and reduced feed efficiency. Genetic correlations among growth, feed intake, and FCR assessed by a multiple-trait Bayes A model resulted in increased genetic correlation between ADG and ADFI, a negative correlation between ADFI and FCR, and a positive correlation between ADG and FCR. Accuracies of genomic prediction for the traits investigated, ranging from 9.4% for RFI to 36.5% for BF, were reported that might provide new insight into pig breeding and future selection programs using genomic information.

Estimation of genetic parameters for birth weight, preweaning mortality, and hot carcass weight of crossbred pigs.

Genetic parameters for birth weight (BWT), preweaning mortality (PWM), and HCW were estimated for a crossbred pig population to determine if BWT could be used as an early predictor for later performances. Sire genetic effects for those traits were estimated to determine if early selection of purebred sires used in crossbreeding could be improved. Data were recorded from 1 commercial farm between 2008 and 2010. Data were from 24,376 crossbred pigs from Duroc sires and crossbred Large White × Landrace dams and included 24,376 BWT and PWM records and 13,029 HCW records. For the analysis, PWM was considered as a binary trait (0 for live or 1 for dead piglet at weaning). A multitrait threshold-linear animal model was used, with animal effect divided into sire genetic and dam effects; the dam effects included both genetic and environmental variation due to the absence of pedigree information for crossbred dams. Fixed effects were sex and parity for all traits, contemporary groups for BWT and HCW, and age at slaughter as a linear covariable for HCW. Random effects were sire additive genetic, dam, litter, and residual effects for all traits and contemporary group for PWM. Heritability estimates were 0.04 for BWT, 0.02 for PWM, and 0.12 for HCW. The ratio between sire genetic and total estimated variances was 0.01 for BWT and PWM and 0.03 for HCW. Dam and litter variances explained, respectively, 14% and 15% of total variance for BWT, 2% and 10% for PWM, and 3% and 8% for HCW. Genetic correlations were -0.52 between BWT and PWM, 0.55 between BWT and HCW, and -0.13 between PWM and HCW. Selection of purebred sires for higher BWT of crossbreds may slightly improve survival until weaning and final market weight at the commercial level.

A genome-wide association study of direct gestation length in US Holstein and Italian Brown populations.

Direct gestation length influences economically important traits in dairy cattle that are related to birth and peri-natal survival of the calf. The objective of this study was to identify single nucleotide polymorphisms (SNPs) that are significantly associated with direct gestation length through a genome-wide association study. Data used in the analysis included 7,308,194 cow gestation lengths from daughters of 4743 United States Holstein sires in the Cooperative Dairy DNA Repository population and 580,157 gestation lengths from 749 sires in the Italian Brown population. Association analysis included 36,768 and 35,082 SNPs spanning all autosomes for Holstein and Brown Swiss, respectively. Multiple shrinkage Bayesian was employed. Estimates of heritability for both populations were moderate, with values of 0.32 (±0.03) and 0.29 (±0.02) for Holstein and Brown Swiss, respectively. A panel of SNPs was identified, which included SNPs that have significant effects on direct gestation length, of which the strongest candidate region is located on chromosome 18. Two regions not previously linked to direct calving ease and calf survival were identified on chromosome 7 and 28, corresponding to regions that contain genes related to embryonic development and foetal development. SNPs were also identified in regions that have been previously mapped for calving difficulty and longevity. This study identifies target regions for the investigation of direct foetal effects, which are a significant factor in determining the ease of calving.

Genetic evaluations for measures of the milk-flow curve in the Italian Brown Swiss population.

The objective of this study was to estimate heritabilities and genetic correlations between milk-release parameters, somatic cell score, milk yield, and udder functional traits in the Italian Brown Swiss population. Data were available from 37,511 cows over a span of 12 yr (1997-2008) from 1,592 herds. Milking flows were recorded for each individual once during lactation. Three different analyses were performed to estimate variance components for all the traits of interest. The first analysis included single control data milk yield, somatic cell score, maximum milk flow, average milk flow, time of plateau, decreasing time, and total milking time, whereas the second analysis included milk-release parameters as well as total udder score, udder depth, and 305-d milk yield and somatic cell score as dependent variables. The third analysis included total milking time, 305-d milk yield and somatic cell score, total udder score, udder depth, and ratios of maximum milk flow over total milking time (R1), time of plateau (R2), and decreasing time (R3) to estimate the relationship between the shape of the milk-release curves and important milking traits. Results from the first and second analysis found similar heritabilities for milkability traits ranging from 0.05 to 0.41 with genetic correlations between production traits and flow traits ranging from low to moderate values. Positive genetic correlations were found among production, somatic cell score, and milkability traits. The third analysis showed that R1 had the greatest heritability of the ratio traits (0.37) with large genetic correlations with R2 and R3, a low correlation with 305-d somatic cell score, and no correlation with 305-d milk yield. Estimated responses to selection over 5 generations were also calculated using different indexes, which included either flow or ratio traits. The results of this study show that it is possible to use information collected through portable flowmeters to improve milkability traits. Using a set of variables or traits to describe the overall release of milk can be an advantageous selection strategy to decrease management costs while maintaining milk production.

Electron energy loss spectroscopy elucidates the elusive darkening of zinc potassium chromate in Georges Seurat's A Sunday on La Grande Jatte--1884.

The color darkening of selected brushstrokes of Georges Seurat's A Sunday on La Grande Jatte--1884 was noted as early as 1892. Artificial aging of model systems containing zinc yellow (K(2)O·4ZnCrO(4)·3H(2)O) mixed with linseed oil successfully replicated the phenomena observed on La Grande Jatte. Electron energy loss spectroscopy measurements performed on the uppermost few microns of thin sections of the paint layers (obtained with focused ion beam milling) determined that samples which turned olive green (aged at 90% RH, with SO(2) and light) contain mostly the altered species Cr(III) and that the ochre-yellow coloration of specimens aged at 50% RH with SO(2) and light is due to the presence of dichromate ions (Cr (VI)). The altered species are only present in the first few microns of paint and are likely poorly crystalline, so that they eluded other types of bulk and surface measurements, but they create a substantial color alteration at the surface of the paint films. A similar phenomenon was confirmed on a sample from La Grande Jatte, where Cr(III) and Cr(VI) dichromate ions were detected in the ochre-colored, altered brushstrokes of zinc yellow. Laboratory simulations demonstrated that the equilibrium between chromate and dichromate ions is reversible within the paint film and that the orange dichromate ions can be transformed back into bright yellow chromates by exposure to highly alkaline gases.

Q-Band resonance Raman investigation of turnip cytochrome f and Rhodobacter capsulatus cytochrome c1.

The results of a comprehensive Q-band resonance Raman investigation of cytochrome c1 and cytochrome f subunits of bc1 and b6f complexes are presented. Q-band excitation provides a particularly effective probe of the local heme environments of these species. The effects of protein conformation (particularly axial ligation) on heme structure and function were further investigated by comparison of spectra obtained from native subunits to those of a site directed c1 mutant (M183L) and various pH-dependent species of horse heart cytochrome c. In general, all species examined displayed variability in their axial amino acid ligation that suggests a good deal of flexibility in their hemepocket conformations. Surprisingly, the large scale protein rearrangements that accompany axial ligand replacement have little or no effect on macrocycle geometry in these species. This indicates the identity and/or conformation of the peptide linkage between the two cysteines that are covalently linked to the heme periphery may determine heme geometry.

Characterization of proteins utilized in the desulfurization of petroleum products by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF/MS) with delayed extraction is utilized in linear, reflected-ion and post-source decay (PSD) modes to directly characterize enzymes being developed for use in a petroleum desulfurization process. The DNA sequence for the genes isolated from Rhodococcus sp. strain IGTS8 that produce three of the four enzymes under study had been previously reported with a discrepancy in residue assignments for one of the enzymes, dsz-C. The use of proteolytic digests followed by MALDI/TOF/MS with delayed extraction in the reflected-ion mode provided sequence-specific information with mass accuracies exceeding 40 ppm over a range of masses and signal-to-noise values. Peptide mapping of >80% of the residues was accomplished for all four proteins. The use of PSD established the true sequence for dsz-C, resolving the discrepancy in the literature. A posttranslational loss of N-terminal methionine was observed for each of the four proteins in linear MALDI/MS and was reconfirmed by peptide mapping for three of the proteins.

Molecular mechanisms of biocatalytic desulfurization of fossil fuels.

The development of biocatalytic desulfurization of petroleum fractions may allow its use in place of conventional hydrodesulfurization (HDS). Dibenzothiophene (DBT) is representative of a broad range of sulfur heterocycles found in petroleum that are recalcitrant to desulfurization via HDS. Rhodococcus sp. strain IGTS8 has the ability to convert DBT to 2-hydroxybiphenyl (HBP) with the release of inorganic sulfur. The conversion of DBT to HBP is catalyzed by a multienzyme pathway consisting of two monooxygenases and a desulfinase. The final reaction catalyzed by the desulfinase appears to be the rate limiting step in the pathway. Each of the enzymes has been purified to homogeneity and their kinetic and physical properties studied. Neither monooxygenase has a tightly bound cofactor and each requires an NADH-FMN oxidoreductase for activity. An NADH-FMN oxidoreductase has been purified from Rhodococcus and is a protein of approximately 25,000 molecular weight with no apparent sequence homology to any other protein in the databases. We describe a unique sulfur acquisition system that Rhodococcus uses to obtain sulfur from very stable heterocyclic molecules.

Axial heme ligation in the cytochrome bc1 complexes of mitochondrial and photosynthetic membranes. A near-infrared magnetic circular dichroism and electron paramagnetic resonance study.

The combination of EPR and low-temperature near-IR magnetic circular dichroism spectroscopies have been used to investigate the axial ligation of the cytochromes in the cytochrome bc1 complexes from bovine heart mitochondria, Rhodobacter capsulatus, Rhodobacter sphaeroides, and Rhodospirillum rubrum, and the purified cytochromes c1 from bovine heart mitochondria, Rb. capsulatus and Rb. sphaeroides. The possibility of axial ligation of cytochrome c1 by the amino terminus of the polypeptide was also assessed by acetylating the N-terminus of Rb. capsulatus cytochrome c1 and comparing the properties of the acetylated and unmodified samples. The results are consistent with bis-histidine axial ligation for the high- and low-potential b-type cytochromes and histidine/methionine axial ligation for the c1-type cytochrome in the intact cytochrome bc1 complexes. Purified samples of cytochrome c1 are mixtures of two forms, one with histidine/methionine and the other with bis-histidine axial ligation. The form with bis-histidine axial ligation is also assembled in the M183L mutant of the Rb. capsulatus cyt bc1 complex in which the methionine residue coordinating cyt c1 is replaced by a leucine. The bis-histidine form appears to be an artifact of dissociation of cytochrome c1 from the cytochrome bc1 complex and is greatly enhanced particularly in the bacterial cytochromes c1 by sample handling and the addition of 50% (v/v) ethylene glycol or glycerol.

Effects of cold storage on the subsequent structure and function of microvenous autografts.

A study was undertaken to evaluate the structure and patency of cold stored rabbit femoral veins following reinsertion as autografts for 3 weeks. The periods of cold ischaemic storage were 1, 2, 3 and 4 weeks (n = 10/gp) and 6 and 10 weeks (n = 6/gp). All rabbits were subject to 3 operations under general anaesthesia. In the first, a 4 cm segment of left femoral vein was harvested and stored at 4 degrees C for the specified ischaemic interval. Following storage the graft was microsurgically reinserted at a second operation into the right femoral artery of the donor rabbit. Three weeks later, graft patency was assessed by surgical exploration and the graft processed for light and electron microscopy. Patency rates remained over 80% after 3 weeks in all groups except the 10 week storage group where only 1 of 6 (17%) grafts was patent at 3 weeks. In all groups normal vein structure was absent, being replaced by thin walled necrotic areas or by neointimal ingrowth. The excellent patency rates achieved indicate it is possible to cold preserve extra lengths of vein grafts harvested at initial operation for reuse should regrafting be necessary.

A look at adolescent treatment in a time of change.

Multiple changes in the authors' inpatient adolescent unit--including unit mergers, increased patient acuity, and shorter length of stay--created the need and opportunity to implement a new Behavioral Milieu Program with two significant components: A positive Behavioral Points System and 15 nurse-led groups. The authors describe this new program, which provides a safe and effective treatment modality and promotes each adolescent's sense of responsibility for his own behavior and life. Dramatic outcomes include decreased use of mechanical restraints by 56% in the first three months of implementation and by 82% in the second three months. This program has applications for inpatient units, community residences, and schools, and can even be adapted for home use by parents.