Examining Relationships Between Groundwater Nitrate Concentrations in Drinking Water and Landscape Characteristics to Understand Health Risks.

Nitrate ingested from drinking water has been linked to adverse health outcomes (e.g., cancer, birth defects) at levels as low as ∼2 mg/L NO3-N, far below the regulatory limits of 10 mg/L. In many areas, groundwater is a common drinking water source and may contain elevated nitrate, but limited data on the patterns and concentrations are available. Using an extensive regulatory data set of over 100,000 nitrate drinking water well samples, we developed new maps of groundwater nitrate concentrations from 76,724 wells in Michigan's Lower Peninsula, USA for the 2006-2015 period. Kriging, a geostatistical method, was used to interpolate concentrations and quantify probability of exceeding relevant thresholds (>0.4 [common detection limit], >2 mg/L NO3-N). We summarized this probability in small watersheds (∼80 km2) to identify correlated variables using the machine learning method classification and regression trees (CARTs). We found 79% of wells had concentrations below the detection limit in this analysis (<0.4 mg/L NO3-N). In the shallow aquifer (focus of study), 13% of wells exceeded 2 mg/L NO3-N and 2% exceeded the EPA maximum contaminant level of 10 mg/L. CART explained 40%-45% of variation in each model and identified three categories of critical correlated variables: source (high agricultural nitrogen inputs), vulnerable soil conditions (low soil organic carbon and high hydraulic conductivity), and transport mechanisms (high aquifer recharge). These findings add to the body of literature seeking to identify communities at risk of elevated nitrate and study associated adverse health outcomes.

Towards genomic selection for facial eczema disease tolerance in the New Zealand sheep industry.

Pithomycotoxicosis, more commonly known as facial eczema (FE), is a liver disease that occurs predominantly in New Zealand because of its toxigenic Pithomyces chartarum strains. The first reported case was in sheep in 1887. Since the 1930s, a number of studies have been conducted in an attempt to mitigate the problems FE has on the sheep and dairy industries. The research in these studies included work on fungicide and biological control of the saprophytic fungus, use of different pasture plants to inhibit fungal growth, stock management with respect to pasture fungal spore counts and the use of zinc prophylaxis on animals. The finding that there was a genetic basis in FE sensitivity in sheep prompted research for a genetic approach to mitigation in the form of a diagnostic DNA test for susceptibility to the disease. Recently, we have used the Illumina OvineSNP50 BeadChip to develop a genome-enabled prediction approach to screen for FE-tolerant sheep. Our current best genomic prediction for FE is for the Romney breed and has an accuracy of 0.38. This prediction accuracy is not as high as the individual accuracy gained by an artificial challenge test (0.64). However, it has the advantage of being a non-invasive test and can be provided as part of genomic testing for other traits at minimal cost.

Quantitative trait loci for live animal and carcass composition traits in Jersey and Limousin back-cross cattle finished on pasture or feedlot.

A quantitative trait locus (QTL) study was carried out in two countries, recording live animal and carcass composition traits. Back-cross calves (385 heifers and 398 steers) were generated, with Jersey and Limousin breed backgrounds. The New Zealand cattle were reared on pasture to carcass weights averaging 229 kg, whilst the Australian cattle were reared on grass and finished on grain (for at least 180 days) to carcass weights averaging 335 kg. From 11 live animal traits and 31 carcass composition traits respectively, 5 and 22 QTL were detected in combined-sire analyses, which were significant (P < 0.05) on a genome-wise basis. Fourteen significant traits for carcass composition QTL were on chromosome 2 and these were traits associated with muscling and fatness. This chromosome carried a variant myostatin allele (F94L), segregating from the Limousin ancestry. Despite very different cattle management systems between the two countries, the two populations had a large number of QTL in common. Of the 18 traits which were common to both countries, and which had significant QTL at the genome-wise level, eight were significant in both countries.

Resuming previously valued activities post-stroke: who or what helps?

PURPOSE: In this article, we aim to develop the understanding of what helps or hinders resumption of valued activities up to 12-months post-stroke. METHOD: As part of a longitudinal study, semi-structured interviews were conducted with 19 people with stroke and eight informal carers 12-months post-stroke. Interviews covered ongoing effects of stroke, experience of trying to resume activities highlighted as important pre-stroke and factors that influenced progress. Interviews were transcribed, coded and analysed in depth to explore this aspect of the experience of living with stroke. RESULTS: Valued activities discussed related to employment; domestic and social roles including driving; hobbies, sports and socialising. Outcomes for individuals were influenced by: aspects of physical or cognitive disability; environmental factors; the adaptability of the individual; support from others and professional help. Inability to resume activities impacted on people's sense of self and quality of life, but some tolerated change and presented themselves as adaptable. CONCLUSIONS: This study indicates a long-term role for rehabilitation services such as: identifying the significance of different types of activities; providing access to support and treatment for debilitating symptoms such as fatigue and dizziness; addressing patients' emotional and behavioural responses to their condition; working with patients' wider social networks and where appropriate, supporting adaptation to a changed way of life.

Going home to get on with life: patients and carers experiences of being discharged from hospital following a stroke.

PURPOSE: In this paper we aim to develop the understanding of what constitutes a 'good' or 'poor' experience in relation to the transition from hospital to home following a stroke. METHOD: Semi-structured interviews were carried out with 20 people and 13 carers within one month of being discharged from hospital following a stroke. Interviews covered views of mobility recovery and support from therapy and services. Interviews were transcribed verbatim, coded and analysed in depth in order to explore the discharge process. RESULTS: Participants described models of recovery, which involved a sense of momentum and getting on with their life. Discharge was successful if: (i) This sense of momentum was maintained, (ii) they felt supported, and (iii) they felt informed about what was happening. Discharge was seen as difficult when: (a) Momentum was perceived to be lost, (b) people did not feel supported, or (c) they felt in the dark about the plans or their recovery. CONCLUSIONS: The discharge experience could be improved by healthcare professionals understanding and exploring patients' individual models of recovery. This would allow professionals to: (a) Access patients concerns, (b) develop programmes addressing these, (c) correct misinterpretations, (d) keep people fully informed, and (e) share and validate the experience, to reduce their sense of isolation.

Predicting people with stroke at risk of falls.

BACKGROUND: falls are common following a stroke, but knowledge about predicting future fallers is lacking. OBJECTIVE: to identify, at discharge from hospital, those who are most at risk of repeated falls. METHODS: consecutively hospitalised people with stroke (independently mobile prior to stroke and with intact gross cognitive function) were recruited. Subjects completed a battery of tests (balance, function, mood and attention) within 2 weeks of leaving hospital and at 12 months post hospital discharge. RESULTS: 122 participants (mean age 70.2 years) were recruited. Fall status at 12 months was available for 115 participants and of those, 63 [55%; 95% confidence interval (CI) 46-64] experienced one or more falls, 48 (42%; 95% CI 33-51) experienced repeated falls, and 62 (54%) experienced near-falls. All variables available at discharge were screened as potential predictors of falling. Six variables emerged [near-falling in hospital, Rivermead leg and trunk score, Rivermead upper limb score, Berg Balance score, mean functional reach, and the Nottingham extended activities of daily living (NEADL) score]. A score of near-falls in hospital and upper limb function was the best predictor with 70% specificity and 60% sensitivity. CONCLUSION: participants who were unstable (near-falls) in hospital with poor upper limb function (unable to save themselves) were most at risk of falls.

cDNA microarray analysis reveals that antioxidant and immune genes are upregulated during involution of the bovine mammary gland.

We have used cDNA microarray analysis to identify genes that play a role in bovine mammary involution. Involution was induced by termination of milking, and alveolar tissue was collected from 48 nonpregnant Friesian cows in mid lactation sacrificed at 0, 6, 12, 18, 24, 36, 72, and 192 h (n = 6/group) postmilking. The most highly upregulated genes were those associated with oxidative stress. Quantitative real-time reverse-transcription PCR analysis confirmed that mRNA expression of spermidine/spermine N(1)-acetyltransferase was increased by 24 h, superoxide dismutase 2 and metallothionein 1A by 36 h, and glutathione peroxidase by 72 h postmilking. The mRNA expression of the host defense proteins lactoferrin and lingual antimicrobial peptide were increased by 192 h postmilking. A dramatic increase in the protein expression of lactoferrin by 192 h postmilking was also detected by Western analysis. Decreased mRNA expression of the milk protein genes alpha(S1)-, beta-, and kappa-casein, and alpha-lactalbumin were early events in the process of involution occurring within 24 to 36 h postmilking, whereas beta-lactoglobulin mRNA was decreased by 192 h postmilking. Decreases in alpha-lactalbumin and beta-lactoglobulin protein levels in alveolar tissue occurred by 24 and 192 h postmilking, respectively, and the cell survival factors beta1-integrin and focal adhesion kinase were decreased by 72 and 192 h postmilking, respectively. The results demonstrate that in the bovine mammary gland, decreased milk protein gene expression and cell survival signaling are associated with multiple protective responses to oxidative stress that occur before the induction of immune responses and mammary epithelial cell apoptosis during involution.

The influence of attention deficits on functional recovery post stroke during the first 12 months after discharge from hospital.

BACKGROUND: Attention deficits have been linked to poor recovery after stroke and may predict outcome. We explored the influence of attention on functional recovery post stroke in the first 12 months after discharge from hospital. METHODS: People with stroke completed measures of attention, balance, mobility and activities of daily living (ADL) ability at the point of discharge from hospital, and 6 and 12 months later. We used correlational analysis and stepwise linear regression to explore potential predictors of outcome. RESULTS: We recruited 122 men and women, mean age 70 years. At discharge, 56 (51%) had deficits of divided attention, 45 (37%) of sustained attention, 43 (36%) of auditory selective attention and 41 (37%) had visual selective attention deficits. Attention at discharge correlated with mobility, balance and ADL outcomes 12 months later. After controlling for the level of the outcome at discharge, correlations remained significant in only five of the 12 relationships. Stepwise linear regression revealed that the outcome measured at discharge, days until discharge and number of medications were better predictors of outcome: in no case was an attention variable at discharge selected as a predictor of outcome at 12 months. CONCLUSIONS: Although attention and function correlated significantly, this correlation was reduced after controlling for functional ability at discharge. Furthermore, side of lesion and the attention variables were not demonstrated as important predictors of outcome 12 months later.

Interference between balance, gait and cognitive task performance among people with stroke living in the community.

PURPOSE: To explore differences in cognitive-motor interference between people with stroke and controls when performing functional tasks and to compare dual task performance of stroke fallers and non-fallers. METHOD: Thirty-six people with stroke (mean age 66.5, SD 11.8, mean time since onset 16 months, range 7 - 56) and 24 controls (mean age 62.3, SD 11.61) performed balance and gait tasks in isolation and in conjunction with a cognitive task (remembering a seven item-shopping list). Three-dimensional movement analysis was used to assess anterior posterior (AP) and lateral (ML) sway; 5 m walk time, stride length and velocity. RESULTS: In the single task condition, people with stroke had greater AP sway, reduced velocity and stride length and a longer 5 m walk time than controls (p < 0.01). In the dual task condition, sway reduced and gait slowed in both groups (p < 0.01 for AP sway, stride length, velocity, walk time); only the increase in walk time was greater in people with stroke than in the controls (F = 4.2, p = 0.046). Cognitive performance was maintained during the balance trials but deteriorated during the dual task gait trials in people with stroke (p = 0.017). Similar trends were noted for fallers and non-fallers with stroke: Only group effects for stride length and velocity reached significance (p < 0.05) and only the reduction in stride length was significantly greater among fallers than non-fallers (F = 12.3, p = 0.001). CONCLUSIONS: People with stroke and controls employed similar strategies during the simultaneous performance of simple functional and silent cognitive tasks and maintained postural stability. Increased walk time and decreased cognitive recall were greater for people with stroke and reduced stride length distinguished fallers from non-fallers.

Stops walking when talking as a predictor of falls in people with stroke living in the community.

OBJECTIVE: To test "Stops walking when talking" (SWWT) as a predictor of falls among people with stroke living in the community. METHODS: People with stroke were identified through hospital records. Mobility, ADL (activities of daily living) ability, mental state, mood, and SWWT were assessed in a single session. Participants were followed prospectively for six months, using falls diaries and regular telephone calls. RESULTS: Sixty three participants (36 men, 27 women; mean (SD) age 68.4 (10.6)) were recruited. Four subjects had a brainstem lesion, 30 had right hemisphere, and 29 left hemisphere infarctions. Mean time since onset of stroke was 20 months (range 2-72). Twenty six subjects stopped walking when a conversation was started and 16 of them fell during the six month follow up period (11 experienced repeated falls). For all fallers (>or=1) the positive predictive value of SWWT was 62% (16/26), the negative predictive value 62% (23/37), specificity 70% (23/33) and sensitivity 53% (16/30). For repeat fallers (>or=2) the positive predictive value of SWWT was 42% (11/26), the negative predictive value 89% (33/37), specificity 69% (33/48) and sensitivity 73% (11/15). Those who stopped walking were significantly more disabled (p<0.001)-that is, they were more dependent in activities of daily living, had worse gross function as well as worse upper and lower limb function, and had depression (p = 0.012). CONCLUSIONS: The specificity of the SWWT test was lower but sensitivity was higher than previously reported. Although the SWWT test was easy to use, its clinical usefulness as a single indicator of fall risk in identifying those community dwelling people with stroke most at risk of falls and in need of therapeutic intervention is questionable.

People with stroke living in the community: Attention deficits, balance, ADL ability and falls.

PURPOSE: To describe levels of attention deficits among people with stroke living in the community and explore relationships between attention, balance, function and falls. METHOD: Forty-eight mobile community-dwelling people with stroke (30 men, 18 women, mean age 68.4 +/- 11.2) were recruited to this cross-sectional investigation through General Practitioners. Twenty-six participants had a right, 21 a left hemisphere infarction and one had a brain stem lesion; mean time since stroke was 46 months (range five to 204). Participants' were interviewed about fall-events; attention, balance and function were assessed using standardised tests. RESULTS: Visual inattention was identified in five participants (10%), deficits of sustained attention in 15 (31%), auditory selective attention in nine (19%), visual selective attention in 17 (35%) and divided attention deficits in 21 participants (43%). Sustained and divided attention scores correlated with balance, ADL ability and fall-status (p < 0.01). The balance and function of subjects with normal attention were better than those with abnormal scores (p < 0.01). Analysis of variance revealed differences between repeat-fallers and non-fallers with no near-falls for divided attention, balance and ADL ability (p < 0.01). CONCLUSIONS: Attention deficits were common among this sample; sustained and divided attention deficits correlated with functional impairments and falls, highlighting that attention deficits might contribute to accident prone behaviour and falling.

Evaluation of single-nucleotide polymorphisms in CAPN1 for association with meat tenderness in cattle.

Micromolar calcium activated neutral protease (CAPN1) was evaluated as a candidate gene for a quantitative trait locus (QTL) on BTA29 affecting meat tenderness by characterization of nucleotide sequence variation in the gene. Single-nucleotide polymorphisms (SNP) were identified by sequencing all 22 exons and 19 of the 21 introns in two sires (Piedmontese x Angus located at the U.S. Meat Animal Research Center in Clay Center, NE; Jersey x Limousin located at AgResearch in New Zealand) of independent resource populations previously shown to be segregating meat tenderness QTL on BTA29. The majority of the 38 SNP were found in introns or were synonymous substitutions in the coding regions, with two exceptions. Exons 14 and 9 contained SNP that were predicted to alter the protein sequence by the substitution of isoleucine for valine in Domain III of the protein, and alanine for glycine in Domain II of the protein. The resource populations were genotyped for these two SNP in addition to six intronic polymorphisms and two silent substitutions. Analysis of genotypes and shear force values in both populations revealed a difference between paternal CAPN1 alleles in which the allele encoding isoleucine at position 530 and glycine at position 316 associated with decreased meat tenderness (increased shear force values) relative to the allele encoding valine at position 530 and alanine at position 316 (P < 0.05). The association of maternal alleles with meat tenderness phenotypes is consistent with the hypothesis of CAPN1 as the gene underlying the QTL effect in two independent resource populations and presents the possibility of using these markers for selective breeding to reduce the numbers of animals with unfavorable meat tenderness traits.

Crystal structure of an aldehyde reductase Y50F mutant-NADP complex and its implications for substrate binding.

Pig aldehyde reductase containing the active site mutation tyrosine(50) to phenylalanine has been crystallized in the presence of the cofactor NADP(H) to a resolution of 2.2 A. This structure clearly shows loss of the tyrosine hydroxyl group and no other significant perturbations compared with previously determined structures. The mutant binds cofactor (both oxidized and reduced) more tightly than the wild-type enzyme but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone, as determined by fluorescence titrations. Numerous attempts at preparing a ternary complex with a range of small aldehyde substrates were unsuccessful. This result, in addition to the inability of the mutant protein to bind the inhibitor, provides strong evidence for the proposal that the tyrosine hydroxyl group is essential for substrate binding in addition to catalysis.

The crystal structure of an aldehyde reductase Y50F mutant-NADP complex and its implications for substrate binding.

In order to understand more fully the structural features of aldo-keto reductases (AKRs) that determine their substrate specificities it would be desirable to obtain crystal structures of an AKR with a substrate at the active site. Unfortunately the reaction mechanism does not allow a binary complex between enzyme and substrate and to date ternary complexes of enzyme, NADP(H) and substrate or product have not been achieved. Previous crystal structures, in conjunction with numerous kinetic and theoretical analyses, have led to the general acceptance of the active site tyrosine as the general acid-base catalytic residue in the enzyme. This view is supported by the generation of an enzymatically inactive site-directed mutant (tyrosine-48 to phenylalanine) in human aldose reductase [AKR1B1]. However, crystallization of this mutant was unsuccessful. We have attempted to generate a trapped cofactor/substrate complex in pig aldehyde reductase [AKR1A2] using a tyrosine 50 to phenylalanine site-directed mutant. We have been successful in the generation of the first high resolution binary AKR-Y50F:NADP(H) crystal structure, but we were unable to generate any ternary complexes. The binary complex was refined to 2.2A and shows a clear lack of density due to the missing hydroxyl group. Other residues in the active site are not significantly perturbed when compared to other available reductase structures. The mutant binds cofactor (both oxidized and reduced) more tightly but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone as determined by fluorescence titrations. Attempts at substrate addition to the active site, either by cocrystallization or by soaking, were all unsuccessful using pyridine-3-aldehyde, 4-carboxybenzaldehyde, succinic semialdehyde, methylglyoxal, and other substrates. The lack of ternary complex formation, combined with the significant differences in the binding of barbitone provides some experimental proof of the proposal that the hydroxyl group on the active site tyrosine is essential for substrate binding in addition to its major role in catalysis. We propose that the initial event in catalysis is the binding of the oxygen moiety of the carbonyl-group of the substrate through hydrogen bonding to the tyrosine hydroxyl group.

Crystal structure of CHO reductase, a member of the aldo-keto reductase superfamily.

Chinese hamster ovary (CHO) reductase is an enzyme belonging to the aldo-keto reductase (AKR) superfamily that is induced by the aldehyde-containing protease inhibitor ALLN (Inoue, Sharma, Schimke, et al., J Biol Chem 1993;268: 5894). It shows 70% sequence identity to human aldose reductase (Hyndman, Takenoshita, Vera, et al., J Biol Chem 1997;272:13286), which is a target for drug design because of its implication in diabetic complications. We have determined the crystal structure of CHO reductase complexed with nicotinamide adenine dinucleotide phosphate (NADP)+ to 2.4 A resolution. Similar to aldose reductase and other AKRs, CHO reductase is an alpha/beta TIM barrel enzyme with cofactor bound in an extended conformation. All key residues involved in cofactor binding are conserved with respect to other AKR members. CHO reductase shows a high degree of sequence identity (91%) with another AKR member, FR-1 (mouse fibroblast growth factor-regulated protein), especially around the variable C-terminal end of the protein and has a similar substrate binding pocket that is larger than that of aldose reductase. However, there are distinct differences that can account for differences in substrate specificity. Trp111, which lies horizontal to the substrate pocket in all other AKR members is perpendicular in CHO reductase and is accompanied by movement of Leu300. This coupled with movement of loops A, B, and C away from the active site region accounts for the ability of CHO reductase to bind larger substrates. The position of Trp219 is significantly altered with respect to aldose reductase and appears to release Cys298 from steric constraints. These studies show that AKRs such as CHO reductase are excellent models for examining the effects of subtle changes in amino acid sequence and alignment on binding and catalysis.

Sequence and expression levels in human tissues of a new member of the aldo-keto reductase family.

We have isolated a human cDNA clone from small intestine that represents a new member of the aldo-keto reductase family. This new member showed 70% identity at the protein level to human aldose reductase and around 80% identity to other Chinese hamster and mouse reductases. The expression pattern shows that this message is located primarily in the adrenal gland, thus suggesting an involvement in steroid metabolism. It is also strongly expressed in the intestinal tract and has been called human small intestine reductase.

Cloning, sequencing, and enzymatic activity of an inducible aldo-keto reductase from Chinese hamster ovary cells.

Treatment of Chinese hamster ovary (CHO) cells by the aldehyde containing calpain inhibitor I resulted in the induction of a 35-kDa protein that was partially sequenced and shown to be a member of the aldo-keto reductase superfamily (Inoue, S., Sharma, R. C., Schimke, R. T., and Simoni, R. D. (1993) J. Biol. Chem. 268, 5894-5898). Using rapid amplification of cDNA ends polymerase chain reaction, we have sequenced the cDNA for this protein (CHO reductase). This enzyme is a new member of the aldo-keto reductase superfamily and shows greatest amino acid sequence identity to mouse fibroblast growth factor-regulated protein and mouse vas deferens protein (92 and 80% sequence identity, respectively). The enzyme exhibits about 70% sequence identity with the aldose reductases (ALR2; EC 1.1.1.21) and about 47% with the aldehyde reductases (ALR1; EC 1.1.1.2). Northern analysis showed that it is induced in preference to either ALR1 or ALR2 and RNase protection assays showed gene expression in bladder, testis, jejunum, and ovary in descending order of expression. The cDNA for this inducible reductase was cloned into the pET16b vector and expressed in BL21(DE3) cells. Expressed CHO reductase showed kinetic properties distinct from either ALR1 or ALR2 including the ability to metabolize ketones. This protein joins a growing number of inducible aldo-keto reductases that may play a role in cellular regulation and protection.

Software to determine optimal oligonucleotide sequences based on hybridization simulation data.

In the design of oligonucleotide sequences for targeting DNA or RNA sequences, it can be difficult to identify sequences that will hybridize only to the intended target. The term "sequence-specific" or "sequence-nonspecific" is often used to describe the interactions of an oligonucleotide with a mixture of DNA or RNA. Our new computer program, HYBsimulator (formerly OligoProbe DesignStation), creates a set of candidate oligonucleotides from a target gene. For each of the candidate oligonucleotides, a large sequence database is searched for sequences that will hybridize to the oligonucleotide. This is referred to as computer hybridization simulation (CHS). Using the nearest-neighbor model, the HYBsimulator takes into account mismatches in hybridization and calculates the melting temperature (Tm) or free energy for hybridization to all sequences in a database. The specificity of each oligonucleotide is then quantified by the number of genes that may hybridize and the predicted Tms or free energies of hybridization to those genes. The CHS data are used to select oligonucleotides based on their specificity with respect to a database.