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Head and neck squamous cell carcinoma (HNSCC) is characterized by the frequent manifestation of DNA crosslink repair defects. We established novel expression-based DNA repair defect markers to determine the clinical impact of such repair defects. Using hypersensitivity to the DNA crosslinking agents, mitomycin C and olaparib, as proxies for functional DNA repair defects in a panel of 25 HNSCC cell lines, we applied machine learning to define gene expression models that predict repair defects. The expression profiles established predicted hypersensitivity to DNA-damaging agents and were associated with mutations in crosslink repair genes, as well as downregulation of DNA damage response and repair genes, in two independent datasets. The prognostic value of the repair defect prediction profiles was assessed in two retrospective cohorts with a total of 180 patients with advanced HPV-negative HNSCC, who were treated with cisplatin-based chemoradiotherapy. DNA repair defects, as predicted by the profiles, were associated with poor outcome in both patient cohorts. The poor prognosis association was particularly strong in normoxic tumor samples and was linked to an increased risk of distant metastasis. In vitro, only crosslink repair–defective HNSCC cell lines are highly migratory and invasive. This phenotype could also be induced in cells by inhibiting rad51 in repair competent and reduced by DNA-PK inhibition. In conclusion, DNA crosslink repair prediction expression profiles reveal a poor prognosis association in HNSCC.Significance:This study uses innovative machine learning-based approaches to derive models that predict the effect of DNA repair defects on treatment outcome in HNSCC.

Figure S1: Genetic alterations in the cell line panel. Figure S2: Strategy for internal cross validation of prediction performance. Figure S3: Cross validation at different drug sensitivity cutoffs. Figure S4: Mitomycin C and olaparib sensitivity in the CGP dataset. Figure S5: Relationship between model predictions and clinical parameters. Figure S6: Univariate analysis of variables used in multi-variate analyses. Figure S7: DNA repair defects and hypoxia share downstream effector pathways. Figure S8: Relation of DNA repair defects and cisplatin treatment. Figure S9: Prognostic value of the threeClass model ensemble. Figure S10. Multivariate analysis using disease stage as predictor. Figure S11: Rad51 inhibitor induced growth inhibition in DNA repair defective HNSCC cell lines. Figure S12: Inhibition of migration in scratch assays. Figure S13: Relationship between EMT and DNA repair defects.

Mutations in genes encoding proteins that are involved in mitochondrial heme synthesis, iron-sulfur cluster biogenesis, and mitochondrial protein synthesis have previously been implicated in the pathogenesis of the congenital sideroblastic anemias (CSAs). We recently described a syndromic form of CSA associated with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD). Here we demonstrate that SIFD is caused by biallelic mutations in TRNT1, the gene encoding the CCA-adding enzyme essential for maturation of both nuclear and mitochondrial transfer RNAs. Using budding yeast lacking the TRNT1 homolog, CCA1, we confirm that the patient-associated TRNT1 mutations result in partial loss of function of TRNT1 and lead to metabolic defects in both the mitochondria and cytosol, which can account for the phenotypic pleiotropy.

In addition to two genes (ENO1 and ENO2) known to code for enolase (EC4.2.1.11), the Saccharomyces cerevisiae genome contains three enolase-related regions (ERR1, ERR2 and ERR3) which could potentially encode proteins with enolase function. Here, we show that products of these genes (Err2p and Err3p) have secondary and quaternary structures similar to those of yeast enolase (Eno1p). In addition, Err2p and Err3p can convert 2-phosphoglycerate to phosphoenolpyruvate, with kinetic parameters similar to those of Eno1p, suggesting that these proteins could function as enolases in vivo. To address this possibility, we overexpressed the ERR2 and ERR3 genes individually in a double-null yeast strain lacking ENO1 and ENO2, and showed that either ERR2 or ERR3 could complement the growth defect in this strain when cells are grown in medium with glucose as the carbon source. Taken together, these data suggest that the ERR genes in Saccharomyces cerevisiae encode a protein that could function in glycolysis as enolase. The presence of these enolase-related regions in Saccharomyces cerevisiae and their absence in other related yeasts suggests that these genes may play some unique role in Saccharomyces cerevisiae. Further experiments will be required to determine whether these functions are related to glycolysis or other cellular processes.

A gene encoding ATP (CTP):tRNA nucleotidyltransferase (EC2.7.7.25) was isolated from Candida (Torulopsis) glabrata by complementation in Saccharomyces cerevisiae. The predicted amino acid sequence of the protein revealed a large region with high sequence similarity to members of the Class II group of the nucleotidyltransferase superfamily and an N-terminal region characteristic of a mitochondrial targeting sequence. The essential role of the carboxylates within the conserved DXD and RRD motifs was confirmed by mutagenesis. C. glabrata strains bearing truncated CCA1 genes that lacked sequences encoding the putative mitochondrial targeting peptide were unable to grow on non-fermentable carbon sources but were able to grow on a fermentable carbon source. These results suggest that, as in S. cerevisiae, the C. glabrata CCA-adding enzyme is a sorting isozyme that functions in multiple cellular compartments. Mapping of the 5'-ends of primary transcripts of CCA1 revealed multiple transcription start sites located both upstream of and between two in-frame start codons. When the cells were cultured on a non-fermentable carbon source the longer transcripts appeared more abundant, suggesting that the choice of transcription start sites was influenced by carbon source. The shorter transcripts, which lacked sequences encoding the mitochondrial targeting information, were more predominant in cells grown on glucose. These observations suggest that expression of CCA-adding isozymes in C. glabrata may be regulated. The DNA sequence has been assigned GenBank Accession No. AF098803.

A gene (KlCCA1) encoding ATP(CTP):tRNA specific tRNA nucleotidyltransferase (EC 2.7.7.25) was isolated from Kluyveromyces lactis by complementation of the Saccharomyces cerevisiae cca1-1 mutation. Sequencing of a 2665 bp EcoRI-SpeI restriction fragment revealed an open reading frame potentially encoding a protein of 489 amino acids with 57% sequence similarity to its S. cerevisiae homologue. Southern hybridization revealed a single copy of KlCCA1 in the K. lactis genome. KlCCA1 was able to complement both the mitochondrial and cytosolic defects in the cca1-1 mutant, suggesting that, as in S. cerevisiae, the K. lactis gene encodes a sorting isozyme that is targeted to mitochondria and the nucleus and/or cytosol. An altered KlCCA1 gene encoding a tRNA nucleotidyltransferase that lacked its first 35 amino acids was able to complement the nuclear/cytosolic but not the mitochondrial defect in the S. cerevisiae cca1-1 mutant, suggesting that the 35 amino-terminal amino acids are necessary for targeting to mitochondria but are not required for enzyme activity. Our results suggest that the mechanisms for production and distribution of mitochondrial and nuclear/cytosolic tRNA nucleotidyltransferase in K. lactis differ from those seen in S. cerevisiae.

We report that the temperature-sensitive (ts) phenotype in Saccharomyces cerevisiae associated with a variant tRNA nucleotidyltransferase containing an amino acid substitution at position 189 results from a reduced ability to incorporate AMP and CMP into tRNAs. We show that this defect can be compensated for by a second-site suppressor converting residue arginine 64 to tryptophan. The R64W substitution does not alter the structure or thermal stability of the enzyme dramatically but restores catalytic activity in vitro and suppresses the ts phenotype in vivo. R64 is found in motif A known to be involved in catalysis and nucleotide triphosphate binding while E189 lies within motif C previously thought only to connect the head and neck domains of the protein. Although mutagenesis experiments indicate that residues R64 and E189 do not interact directly, our data suggest a critical role for residue E189 in enzyme structure and function. Both R64 and E189 may contribute to the organization of the catalytic domain of the enzyme. These results, along with overexpression and deletion analyses, show that the ts phenotype of cca1-E189F does not arise from thermal instability of the variant tRNA nucleotidyltransferase but instead from the inability of a partially active enzyme to support growth only at higher temperatures.

ATP (CTP):tRNA nucleotidyltransferase (EC 2.7.7.25) was purified to apparent homogeneity from a crude extract of Lupinus albus seeds. Purification was accomplished using a multistep protocol including ammonium sulfate fractionation and chromatography on anion-exchange, hydroxylapatite and affinity columns. The lupin enzyme exhibited a pH optimum and salt and ion requirements that were similar to those of tRNA nucleotidyltransferases from other sources. Oligonucleotides, based on partial amino acid sequence of the purified protein, were used to isolate the corresponding cDNA. The cDNA potentially encodes a protein of 560 amino acids with a predicted molecular mass of 64 164 Da in good agreement with the apparent molecular mass of the pure protein determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The size and predicted amino acid sequence of the lupin enzyme are more similar to the enzyme from yeast than from Escherichia coli with some blocks of amino acid sequence conserved among all three enzymes. Functionality of the lupin cDNA was shown by complementation of a temperature-sensitive mutation in the yeast tRNA nucleotidyltransferase gene. While the lupin cDNA compensated for the nucleocytoplasmic defect in the yeast mutant it did not enable the mutant strain to grow at the non-permissive temperature on a non-fermentable carbon source.

The objective of this paper was to investigate the influence of environmental policies on dairy cattle breeding goals. Linear programming was used to model a dairy farm, including multiple restrictions on product output and production factor input. Management was optimized to maximize labour income. Effects of genetic improvement on labour income (economic values) were derived for dry-matter intake capacity, body weight and milk production traits (carrier, fat, and protein). Also effects of genetic improvement on nitrogen losses of the farm were computed. Basic economic values calculated were −0.18, 0.78, 13.81, −0.05 and 0 for carrier, fat, protein, body weight and dry-matter intake capacity, respectively. With more stringent environmental legislation, the economic value of fat increased (1.68) and the economic value of body weight decreased (−0.21), with constant economic values of carrier, protein and dry-matter intake capacity. Effects of genetic improvement on nitrogen losses of the farm varied considerably. Generally, a higher fat production per cow resulted in a reduction of nitrogen losses, and an increase in body weight increased nitrogen losses.

Base-induced cyclisations of 2-alkynylphenylhydroxamic acids occur by attack of nitrogen onto the alkyne group in either exo or endo fashions to give the corresponding isoindol-1(2H)-ones or 1(2H)-isoquinolinones depending upon the alkyne substituent.

Parotid secretory protein (PSP) (C20orf70) is a salivary protein of unknown function. The protein belongs to the palate, lung, and nasal epithelium clone (PLUNC) family of mucosal secretory proteins that are predicted to be structurally similar to lipid-binding and host-defense proteins including bactericidal/permeability-increasing protein and lipopolysaccharide-binding protein. However, the PLUNC proteins exhibit significant sequence variation and different biological functions have been proposed for different family members. This study tested the functional implications of the proposed similarity of PSP to the acute phase protein lipopolysaccharide-binding protein (LBP). PSP was identified in human saliva and was soluble in 70% ethanol, as shown for other PLUNC proteins. PSP binds lipopolysaccharide and can be eluted by non-ionic detergent, but not by urea or high salt. A synthetic PSP peptide, GL13NH2, which corresponds to a lipopolysaccharide-inhibiting peptide from LBP, inhibited the binding of lipopolysaccharide to both PSP and lipopolysaccharide-binding protein. Peptides from other regions of PSP and the control peptide polymyxin B showed no effect on the binding of PSP to lipopolysaccharide. GL13NH2 also inhibited lipopolysaccharide-stimulated secretion of tumor necrosis factor from macrophages. The other PSP peptides had no effect in this assay. PSP peptides had no or only minor effect on macrophage cell viability. These results indicate that PSP is a lipopolysaccharide-binding protein that is functionally related to LBP, as suggested by their predicted structural similarities.

Isolation distances to limit the risk of cross-pollination from transgenic to nontransgenic crops can severely limit the potential use of transgenic crops through a so-called 'domino effect' where a field of non-transgenic crops limits adoption of transgenic crops not only on plots in its direct vicinity, but also in plots further away as its neighbors are forced to grow the non-transgenic varieties, forcing their neighbors to grow the non-transgenic variety, and so on. The extent to which this effect takes place, however, may depend crucially on the type of farm. For example, dairy farms can use grassland as a buffer between transgenic and conventional maize plots. This article assesses the effects of isolation distances for transgenic maize in dairy farming. A spatially explicit farm model is applied to a region in the Southern Netherlands to identify to what extent a single farmer (who uses non-transgenic maize) can limit other farmers’ potential to grow transgenic maize. The main findings are that 50% or more of the farms in the study area will not affect the potential adoption of transgenic maize by growing conventional maize at all. This result even holds under distance measures of 800m, which is the largest distance implemented by member states of the European Union. When they do have such effects, isolation distances can reduce the benefits from transgenic maize by €5,000 - €6,000, for a considerable part through a domino effect. Large net benefits of transgenic maize may limit the spatial effects as farmers are more willing to relocate maize production to areas where transgenic maize is allowed.

OBJECTIVE To identify the cells expressing the M3 muscarinic receptor subtype in the lamina propria of the bladder. MATERIALS AND METHODS The bladders from five normal guinea pigs were isolated and fixed in 4% paraformaldehyde. Tissues sections (10 µm) were then cut and stained with antibodies to the type 3 muscarinic receptor (M3), the interstitial cell marker vimentin and the nonspecific nerve marker PGP 9.5. The specificity of the antibody to the M3 receptor was established using the complementary blocking peptide and Western blot analysis of human embryonic kidney (HEK) cells transfected to express the M3 receptor protein. RESULTS The M3 antibody pre-incubated with its blocking peptide showed no immunohistochemical staining. Investigating this antibody using HEK cells transfected to express the M3 receptor protein and control HEK cells showed a single band in the transfected cells and no band in the control cells. M3 receptor immunoreactivity (M3-IR) was detected primarily on a dense network of vimentin-positive (vim+) cells lying immediately below the urothelium, i.e. the suburothelial interstitial cells (Su-ICs). The M3-IR was punctate and appeared to be located on the cell surface. The diffuse network of cells in the remaining regions of the lamina propria showed no M3-IR. Few nerve fibres were associated with the M3-IR Su-ICs. The M3-IR Su-ICs were most numerous and prominent in the lateral wall. The number of M3-IR/vim+ cells diminished towards the bladder base and were absent in the bladder urethral junction. In the base and urethral junction there were vim+ cells that were not M3-IR. A population of umbrella cells in the lateral wall also showed weak punctate M3-IR. CONCLUSIONS Using a well-characterized M3 antibody, these results show for the first time that the M3 muscarinic receptor in the lamina propria is located specifically on the Su-ICs. The physiological role of these cells is unknown and consequently the significance of what appears to be a cholinergic signalling system is unclear. Previously published data showed that these cells respond to nitric oxide and atrial natriuretic peptide with an increase in cGMP and possibly prostaglandin. All of these observations, taken together, suggest that the Su-ICs receive multiple inputs and that they must be part of a complex signalling system in this region of the bladder wall.

Although the benefits of organic farming are already well known, the conversion to organic farming does not proceed as the Dutch government expected. In order to investigate the conversion decisions of Dutch arable farms, a discrete stochastic dynamic utility-efficient programming (DUEP) model is developed with special attention for yield and price risk of conventional, conversion and organic crops. The model maximizes the expected utility of the farmer depending on the farmer’s risk attitude. The DUEP model is an extension of a dynamic linear programming model that maximized the labour income of conversion from conventional to organic farming over a 10 year planning horizon. The DUEP model was used to model a typical farm for the central clay region in the Netherlands. The results show that for a risk-neutral farmer it is optimal to convert to organic farming. However, for a more risk-averse farmer it is only optimal to fully convert if policy incentives are applied such as taxes on pesticides or subsidies on conversion, or if the market for the organic products becomes more stable.

DOEL Onderzoek naar de variatie in oordelen tussen inspecteurs van de Inspectie voor de Gezondheidszorg (IGZ) in het systeem van gefaseerd toezicht op de verpleeghuiszorg en naar de relatie tussen het type oordeel en de aan- of afwezigheid van onderbouwing daarbij. OPZET Descriptief, kwantitatief, retrospectief. METHODE In totaal werden 4914 oordelen met bijbehorende onderbouwingen van 26 inspecteurs uit 182 toezichtrapporten geanalyseerd. De oordelen waren in 2005 en 2006 gegeven op 25 criteria voor verantwoorde zorg in verpleeghuizen. Om de inspecteurs en hun oordelen over verschillende instellingen te kunnen vergelijken, werd met covariantieanalyse statistisch gecorrigeerd voor instellingskenmerken. RESULTATEN Er waren statistisch significante verschillen in beoordeling tussen de inspecteurs. Na correctie voor instellingskenmerken bleven voor 14 van de 25 criteria significante verschillen bestaan. Tevens bleek de aanwezigheid van een onderbouwing bij een oordeel zowel afhankelijk te zijn van de inspecteur als van het gegeven oordeel. CONCLUSIE Beoordelaarsverschillen tussen inspecteurs spelen een rol in de tweede fase van het gefaseerd toezicht op de verpleeghuizen. De IGZ zal resultaten van dit onderzoek gebruiken om haar toezicht verder te ontwikkelen.

The challenge for agricultural policymakers and planners, particularly in the context of Rwanda with high population density and consequently food insecurity, is how to enable farmers to adopt new technology. It is known that adoption of new technology may vary among farm households because of socio-economic characteristics. This paper intends to typify farm households in Rwanda based on the exploration of factors explaining adoption of new technology. Ultimately, typical farms obtained from the typology will be used, later as basis to develop representative mathematical programming models. Multivariate statistical techniques offer the means of creating such typologies, particularly when an in-depth database is available. This multivariate analysis approach, combining principal component analysis (PCA) and cluster analysis (CA), has allowed us to identify clearly five typical farm households and their socio-economic characteristics explaining adoption of new technology.. Multivariate statistical techniques, such as PCA and CA, are great tools to envisage building mathematical programming models on the basis of typical farm households.

ATP(CTP) : tRNA nucleotidyltransferase catalyses the posttranscriptional addition of cytidine, cytidine and adenosine to the 3' ends of tRNAs. Previously, a temperature-sensitive phenotype in Saccharomyces cerevisiae resulting from a mutation in the CCA1 gene coding for this enzyme was identified. Here, we show that a single guanine-to-adenine transition in cca1-1 generates the temperature-sensitive phenotype. Alignment of the amino acid sequence of S. cerevisiae tRNA nucleotidyltransferase with other tRNA nucleotidyltransferases for which crystal structures have been solved suggests that the resulting Glu-to-Lys substitution is in a ss-turn connecting the structurally and functionally important head and neck domains of the protein. Proteins containing Gln, His or Phe at this position were constructed to further characterize the importance of this residue in enzyme structure and function. As with the Lys variant, the Phe and His variants generate a temperature-sensitive phenotype in isogenic yeast strains, further supporting the role of this position in maintaining the structure and function of this enzyme. Comparative biophysical and biochemical characterization of both the wild-type and variant proteins indicates that amino acid substitutions at this position can result in a structural change in the protein that reduces enzyme activity (both at the permissive and non-permissive temperatures), decreases the melting temperature of the protein and alters its stability at the non-permissive temperature (37 degrees C).

Introduction: Stepped care strategies are potentially effective to organise integrated care but unknown is whether they function well in practice. This paper evaluates the implementation of a stepped care programme for depression in primary care and secondary care. Theory and methods: We developed a stepped care algorithm for diagnostics and treatment of depression, supported by a liaison-consultation function. In a 2½ year study with pre-post design in a pilot region, adherence to the protocol was assessed by interviewing 28 caregivers of 235 patients with mild, moderate, or severe major depression. Consultation and referral patterns between primary and secondary care were analysed. Results: Adherence of general practitioners and consultant caregivers to the stepped care protocol proved to be 96%. The percentage of patients referred for depression to secondary care decreased significantly from 26% to 21% (p=0.0180). In the post-period more patients received treatment in primary care and requests for consultation became more concordant with the stepped care protocol. Conclusions: Implementation of a stepped care programme is feasible in a primary and secondary care setting and is associated with less referrals. Discussion: Further research on all subsequent treatment steps in a standardised stepped care protocol is needed.

‘Farming for Nature’, a relatively new policy instrument being tried out in the Netherlands, is evaluated. The concept has been designed to allow dairy farmers to improve nature conservation on their farms. Under the scheme, no manure, fertilizer, or feed – concentrates or roughage - may be imported into farm systems from external sources. The feasibility of such a self-sustaining system and the conditions required for it to deliver the desired results, are explored with a farm-based linear programming model known as FIONA (Farm based Integrated Optimization Model for Nature and Agriculture). The model is explained and applied to ‘de Langstraat’, a region in southern Netherlands. The results show that levels of production under the ‘Farming for Nature’ regime are dependent upon soil fertility and the proportion of land that is suitable for growing arable crops. If all available land on a dairy farm in the scheme is arable land, then high production levels of up to 7,500 kg milk per hectare can be realized. If only 30% of the farm area is suitable for arable crops, then only lower production levels, of about 6,600 kg milk per hectare can be realized. The scheme has positive ecological effects. Both nature and cultural landscape values may benefit significantly from the concept. Improvement in ecological terms however, carries a price in terms of agricultural income. An average dairy farm adopting the concept of ‘Farming for Nature’ experiences an income loss of approximately € 840 per hectare in the short-run (5-10 years). More important is the observation that the scale of such farms in the short-run might be too small to earn an attractive income for its workers, even when fully compensated according to European Union regulations.

‘Farming for Nature’, a relatively new policy instrument being tried out in the Netherlands, is evaluated. The concept has been designed to allow dairy farmers to improve nature conservation on their farms. Under the scheme, no manure, fertilizer, or feed – concentrates or roughage - may be imported into farm systems from external sources. The feasibility of such a self-sustaining system and the conditions required for it to deliver the desired results, are explored with a farm-based linear programming model known as FIONA (Farm based Integrated Optimization Model for Nature and Agriculture). The model is explained and applied to ‘de Langstraat’, a region in southern Netherlands. The results show that levels of production under the ‘Farming for Nature’ regime are dependent upon soil fertility and the proportion of land that is suitable for growing arable crops. If all available land on a dairy farm in the scheme is arable land, then high production levels of up to 7,500 kg milk per hectare can be realized. If only 30% of the farm area is suitable for arable crops, then only lower production levels, of about 6,600 kg milk per hectare can be realized. The scheme has positive ecological effects. Both nature and cultural landscape values may benefit significantly from the concept. Improvement in ecological terms however, carries a price in terms of agricultural income. An average dairy farm adopting the concept of ‘Farming for Nature’ experiences an income loss of approximately € 840 per hectare in the short-run (5-10 years). More important is the observation that the scale of such farms in the short-run might be too small to earn an attractive income for its workers, even when fully compensated according to European Union regulations.