Choline degradation in Paracoccus denitrificans: identification of sources of formaldehyde.

Paracoccus denitrificans is a facultative methylotroph that can grow on methanol and methylamine as sole sources of carbon and energy. Both are oxidized to formaldehyde and then to formate, so growth on C1 substrates induces the expression of genes encoding enzymes required for the oxidation of formaldehyde and formate. This induction involves a histidine kinase response regulator pair (FlhSR) that is likely triggered by formaldehyde. Catabolism of some complex organic substrates (e.g., choline and L-proline betaine) also generates formaldehyde. Thus, flhS and flhR mutants that fail to induce expression of the formaldehyde catabolic enzymes cannot grow on methanol, methylamine, and choline. Choline is oxidized to glycine via glycine betaine, dimethylglycine, and sarcosine. By exploring flhSR growth phenotypes and the activities of a promoter and enzyme known to be upregulated by formaldehyde, we identify the oxidative demethylations of glycine betaine, dimethylglycine, and sarcosine as sources of formaldehyde. Growth on glycine betaine, dimethylglycine, and sarcosine is accompanied by the production of up to three, two, and one equivalents of formaldehyde, respectively. Genetic evidence implicates two orthologous monooxygenases in the oxidation of glycine betaine. Interestingly, one of these appears to be a bifunctional enzyme that also oxidizes L-proline betaine (stachydrine). We present preliminary evidence to suggest that growth on L-proline betaine induces expression of a formaldehyde dehydrogenase distinct from the enzyme induced during growth on other formaldehyde-generating substrates.IMPORTANCEThe bacterial degradation of one-carbon compounds (methanol and methylamine) and some complex multi-carbon compounds (e.g., choline) generates formaldehyde. Formaldehyde is toxic and must be removed, which can be done by oxidation to formate and then to carbon dioxide. These oxidations provide a source of energy; in some species, the CO2 thus generated can be assimilated into biomass. Using the Gram-negative bacterium Paracoccus denitrificans as the experimental model, we infer that oxidation of choline to glycine generates up to three equivalents of formaldehyde, and we identify the three steps in the catabolic pathway that are responsible. Our work sheds further light on metabolic pathways that are likely important in a variety of environmental contexts.

DksA, ppGpp, and RegAB Regulate Nitrate Respiration in Paracoccus denitrificans.

The periplasmic (NAP) and membrane-associated (Nar) nitrate reductases of Paracoccus denitrificans are responsible for nitrate reduction under aerobic and anaerobic conditions, respectively. Expression of NAP is elevated in cells grown on a relatively reduced carbon and energy source (such as butyrate); it is believed that NAP contributes to redox homeostasis by coupling nitrate reduction to the disposal of excess reducing equivalents. Here, we show that deletion of either dksA1 (one of two dksA homologs in the P. denitrificans genome) or relA/spoT (encoding a bifunctional ppGpp synthetase and hydrolase) eliminates the butyrate-dependent increase in nap promoter and NAP enzyme activity. We conclude that ppGpp likely signals growth on a reduced substrate and, together with DksA1, mediates increased expression of the genes encoding NAP. Support for this model comes from the observation that nap promoter activity is increased in cultures exposed to a protein synthesis inhibitor that is known to trigger ppGpp synthesis in other organisms. We also show that, under anaerobic growth conditions, the redox-sensing RegAB two-component pair acts as a negative regulator of NAP expression and as a positive regulator of expression of the membrane-associated nitrate reductase Nar. The dksA1 and relA/spoT genes are conditionally synthetically lethal; the double mutant has a null phenotype for growth on butyrate and other reduced substrates while growing normally on succinate and citrate. We also show that the second dksA homolog (dksA2) and relA/spoT have roles in regulation of expression of the flavohemoglobin Hmp and in biofilm formation. IMPORTANCE Paracoccus denitrificans is a metabolically versatile Gram-negative bacterium that is used as a model for studies of respiratory metabolism. The organism can utilize nitrate as an electron acceptor for anaerobic respiration, reducing it to dinitrogen via nitrite, nitric oxide, and nitrous oxide. This pathway (known as denitrification) is important as a route for loss of fixed nitrogen from soil and as a source of the greenhouse gas nitrous oxide. Thus, it is important to understand those environmental and genetic factors that govern flux through the denitrification pathway. Here, we identify four proteins and a small molecule (ppGpp) which function as previously unknown regulators of expression of enzymes that reduce nitrate and oxidize nitric oxide.

Carer and staff perceptions of end-of-life care provision: case of a hospice-at-home service.

People requiring palliative care should have their needs met by services acting in accordance with their wishes. A hospice in the south of England provides such care via a 24/7 hospice at home service. This study aimed to establish how a nurse-led night service supported patients and family carers to remain at home and avoid hospital admissions. Semi-structured interviews were carried out with family carers (n=38) and hospice-at-home staff (n=9). Through night-time phone calls and visits, family carers felt supported by specialist hospice staff whereby only appropriate hospital admission was facilitated. Staff provided mediation between family carer and other services enabling more integrated care and support to remain at home. A hospice-at-home night service can prevent unnecessary hospital admissions and meet patient wishes through specialist care at home.

The Cost of Visit-based Home Care for up to Two Weeks in the Last Three Months of Life: APilot Study of Community Care Based at a Hospice-at-home Service in South East of England.

The cost of visit-based community care based around a 24/7 hospice-at-home (HatH) service in the last 3 months of life was assessed. Thirty families completed a health and social carediary of at-home visits over two-weeks following contact with the HatH night service. Diaries captured 333 days of care provision, averaging 11 diary days per family, 708 health care professional and carer visits, lasting 604 hours at a cost of £20,192 ($24,946). Hat H care, integrated with community support, seems an economic proposition but highlights the complexities of assessing cost of end of life care.

Sequential screening for lung cancer in a high-risk group: randomised controlled trial: LungSEARCH: a randomised controlled trial of Surveillance using sputum and imaging for the EARly detection of lung Cancer in a High-risk group.

BACKGROUND: Low-dose computed tomography (LDCT) screening detects early-stage lung cancer and reduces mortality. We proposed a sequential approach targeted to a high-risk group as a potentially efficient screening strategy. METHODS: LungSEARCH was a national multicentre randomised trial. Current/ex-smokers with mild/moderate chronic obstructive pulmonary disease (COPD) were allocated (1:1) to have 5 years surveillance or not. Screened participants provided annual sputum samples for cytology and cytometry, and if abnormal were offered annual LDCT and autofluorescence bronchoscopy (AFB). Those with normal sputum provided annual samples. The primary end-point was the percentage of lung cancers diagnosed at stage I/II (nonsmall cell) or limited disease (small cell). RESULTS: 1568 participants were randomised during 2007-2011 from 10 UK centres. 85.2% of those screened provided an adequate baseline sputum sample. There were 42 lung cancers among 785 screened individuals and 36 lung cancers among 783 controls. 54.8% (23 out of 42) of screened individuals versus 45.2% (14 out of 31) of controls with known staging were diagnosed with early-stage disease (one-sided p=0.24). Relative risk was 1.21 (95% CI 0.75-1.95) or 0.82 (95% CI 0.52-1.31) for early-stage or advanced cancers, respectively. Overall sensitivity for sputum (in those randomised to surveillance) was low (40.5%) with a cumulative false-positive rate (FPR) of 32.8%. 55% of cancers had normal sputum results throughout. Among sputum-positive individuals who had AFB, sensitivity was 45.5% and cumulative FPR was 39.5%; the corresponding measures for those who had LDCT were 100% and 16.1%, respectively. CONCLUSIONS: Our sequential strategy, using sputum cytology/cytometry to select high-risk individuals for AFB and LDCT, did not lead to a clear stage shift and did not improve the efficiency of lung cancer screening.

Research in progress--LungSEARCH: a randomised controlled trial of surveillance for the early detection of lung cancer in a high-risk group.

Low-dose CT screening for lung cancer is effective but expensive. Therefore, cheaper or more focused screening strategies may be required. LungSEARCH is a randomised prospective trial of 1568 high-risk individuals (ie, current or former moderate to heavy smokers with mild/moderate COPD) who undergo either annual sputum cytology/cytometry testing or no screening. Those with abnormal sputum then receive annual CT and fluorescent bronchoscopy for the remainder of 5 years, to identify early stage lung cancer. It is hoped that these simple initial tests could identify those requiring expensive CT scans, and the aim is to demonstrate a stage shift towards early stage cancers.Trial registration numbers ISRCTN: ISRCTN80745975, clinicaltrials.gov: NCT00512746.

Inefficient translation of nsrR constrains behaviour of the NsrR regulon in Escherichia coli.

The NsrR protein of Escherichia coli is a transcriptional repressor that contains an [Fe-S] cluster that is the binding site for nitric oxide (NO). Reaction of NsrR with NO leads to de-repression of its target genes, which include those encoding an NO scavenging flavohaemoglobin and the RIC (repair of iron centres) protein involved in the repair of NO-damaged [Fe-S] clusters. The nsrR gene is promoter proximal in a transcription unit with rnr, encoding the cold shock-inducible RNase R. Here, we show that nsrR is expressed from a strong promoter, but that its translation is extremely inefficient, leading to a low cellular NsrR concentration. Conversion of the nsrR start codon from the wild-type GUG to AUG increased the efficiency of translation (which, nevertheless, remained extremely low) and had measurable effects on the expression of some NsrR-regulated genes. We conclude that NsrR abundance in the cell is such that promoters with low-affinity NsrR binding sites may partially escape NsrR-mediated repression. Expression profiling confirmed that genes regulated by NsrR (whether directly or indirectly) tend to express lower mRNA levels when the nsrR start codon is AUG than when it is GUG. Transcriptomics data implicated the pyruvate oxidase gene poxB as a novel NsrR target, which we confirmed and showed to be due to read-through transcription from the upstream hcp-hcr genes. We also present evidence to suggest that NsrR is a regulator of the sufABCDSE genes, which encode the components of an [Fe-S] cluster biogenesis and repair system.

Cooperative and allosterically controlled nucleotide binding regulates the DNA binding activity of NrdR.

Ribonucleotide reductases (RNRs) are required for the synthesis of deoxyribonucleoside triphosphates (dNTPs) from ribonucleotides. In Escherichia coli, regulation of RNR expression is co-ordinated with the cell cycle, and involves several regulatory proteins. One of these, NrdR, has recently been shown to regulate all three nrd operons that encode RNR isoenzymes. Repression by NrdR is believed to be stimulated by elevated dNTPs, although there is no direct evidence for this model. Here, we sought to elucidate the mechanism by which NrdR regulates nrd expression according to the abundance of (d)NTPs. We determined that ATP and dATP bind to NrdR in a negatively cooperative fashion, such that neither can fully occupy the protein. Both nucleotides also appear to act as positive heterotropic effectors, since the binding of one stimulates binding of the other. Nucleotide binding stimulates self-association of NrdR, with tri- and diphosphates stimulating oligomerization more effectively than monophosphates. As-prepared NrdR contains (deoxy)nucleoside monophosphates, diphosphates and triphosphates, and its DNA binding activity is inhibited by triphosphates and diphosphates but not by monophosphates. We propose a model in which NrdR selectively binds (deoxy)nucleoside triphosphates, which are hydrolysed to their monophosphate counterparts in order to regulate DNA binding.

The European initiative for quality management in lung cancer care.

Lung cancer is the commonest cause of cancer-related death worldwide and poses a significant respiratory disease burden. Little is known about the provision of lung cancer care across Europe. The overall aim of the Task Force was to investigate current practice in lung cancer care across Europe. The Task Force undertook four projects: 1) a narrative literature search on quality management of lung cancer; 2) a survey of national and local infrastructure for lung cancer care in Europe; 3) a benchmarking project on the quality of (inter)national lung cancer guidelines in Europe; and 4) a feasibility study of prospective data collection in a pan-European setting. There is little peer-reviewed literature on quality management in lung cancer care. The survey revealed important differences in the infrastructure of lung cancer care in Europe. The European guidelines that were assessed displayed wide variation in content and scope, as well as methodological quality but at the same time there was relevant duplication. The feasibility study demonstrated that it is, in principle, feasible to collect prospective demographic and clinical data on patients with lung cancer. Legal obligations vary among countries. The European Initiative for Quality Management in Lung Cancer Care has provided the first comprehensive snapshot of lung cancer care in Europe.

Finely tuned regulation of the aromatic amine degradation pathway in Escherichia coli.

FeaR is an AraC family regulator that activates transcription of the tynA and feaB genes in Escherichia coli. TynA is a periplasmic topaquinone- and copper-containing amine oxidase, and FeaB is a cytosolic NAD-linked aldehyde dehydrogenase. Phenylethylamine, tyramine, and dopamine are oxidized by TynA to the corresponding aldehydes, releasing one equivalent of H2O2 and NH3. The aldehydes can be oxidized to carboxylic acids by FeaB, and (in the case of phenylacetate) can be further degraded to enter central metabolism. Thus, phenylethylamine can be used as a carbon and nitrogen source, while tyramine and dopamine can be used only as sources of nitrogen. Using genetic, biochemical and computational approaches, we show that the FeaR binding site is a TGNCA-N8-AAA motif that occurs in 2 copies in the tynA and feaB promoters. We show that the coactivator for FeaR is the product rather than the substrate of the TynA reaction. The feaR gene is upregulated by carbon or nitrogen limitation, which we propose reflects regulation of feaR by the cyclic AMP receptor protein (CRP) and the nitrogen assimilation control protein (NAC), respectively. In carbon-limited cells grown in the presence of a TynA substrate, tynA and feaB are induced, whereas in nitrogen-limited cells, only the tynA promoter is induced. We propose that tynA and feaB expression is finely tuned to provide the FeaB activity that is required for carbon source utilization and the TynA activity required for nitrogen and carbon source utilization.

Attitudes to participation in a lung cancer screening trial: a qualitative study.

BACKGROUND: Earlier diagnosis of lung cancer is key to reducing mortality. New evidence suggests that smokers have negative attitudes to screening and participation in lung cancer screening trials is poor (<1 in 6 of those eligible). Understanding participation is important since uptake in screening trials is likely to predict uptake in screening programmes. A qualitative study of people accepting and declining participation in the Lung-SEARCH screening trial was conducted. Two questions were addressed: Are the screening methods offered acceptable to patients? Why do some people take part and others decline? METHODS: The qualitative study used semi-structured interviews with 60 respondents from three groups: (a) trial participants providing an annual sputum sample; (b) trial participants with a sputum sample showing abnormal cytology and thus undergoing annual CT scanning and bronchoscopy; and (c) those declining trial participation. RESULTS: Most respondents (48/60, 80%) viewed sputum provision, CT scanning and bronchoscopy as largely acceptable. Those declining trial participation described fear of bronchoscopy, inconvenience of travelling to hospitals for screening investigations and perceived themselves as having low susceptibility to lung cancer or being too old to benefit. Patients declining participation discounted their risk from smoking and considered negative family histories and good health to be protective. Four typological behaviours emerged within those declining: 'too old to be bothered', 'worriers', 'fatalists' and 'avoiders'. CONCLUSION: Sputum provision, CT scanning and bronchoscopy are largely acceptable to those participating in a screening trial. However, the decision to participate or decline reflects a complex balance of factors including acceptability and convenience of screening methods, risk perception, altruism and self-interest. Improving practical and changing cognitive aspects of participation will be key to improving uptake of lung cancer screening.

Screening for lung cancer: is this the way forward?

While low-dose CT scans have been shown to detect greater numbers of early lung cancers than conventional CXR, the first randomized trial of CT versus CXR screening in more than 50 000 subjects has shown a 20% reduction in mortality with CT. There are several other randomized trials in progress. CT scanning may be a useful technique for identifying lung cancer at an earlier stage and may reduce mortality. However, before it can be used on a wider scale, issues such as overdiagnosis bias, cost-effectiveness, false positive findings of multiple noncalcified nodules and the willingness of the relevant population to accept CT scanning need to be evaluated. There is still very little information on the cost per life-year saved as a result of CT scanning, as the data to date is very imprecise. There is no evidence that screening programs influence smoking rates despite the inclusion of cessation programs in many trials. Furthermore, if CT screening is adopted, much work is needed to persuade individuals at high risk, mostly current or former heavy smokers with some airflow obstruction, to participate in a screening program.

An alternative route to nitric oxide resistance.

Vibrio fischeri is a bioluminescent bacterium that enters into a symbiosis with the bobtail squid Euprymna scolopes. The bacterium colonizes a specialized light organ, in which it generates light that might help the squid to hide its silhouette from animals beneath it. Previous studies have shown that the host nitric oxide (NO) synthase is active during colonization, suggesting that V. fischeri symbionts are exposed to NO. Thus, NO might play a role in regulating the symbiosis, a role possibly analogous to that of NO in the interaction between some pathogens and their hosts. One possibility is that NO helps to exclude other species from the light organ, in which case, the response of V. fischeri to NO is of considerable interest. In this issue of Molecular Microbiology, Dunn et al. report that V. fischeri produces an NO-inducible and NO-resistant alternative oxidase (Aox) that allows respiration to continue in the presence of NO concentrations that are inhibitory to the conventional respiratory oxidases. This is an important step towards a better understanding of the role that NO plays in the Vibrio-squid symbiosis, and provides the first indication of a physiological function for a bacterial homologue of the plant Aox.

Vibrio fischeri flavohaemoglobin protects against nitric oxide during initiation of the squid-Vibrio symbiosis.

Nitric oxide (NO) is implicated in a wide range of biological processes, including innate immunity against pathogens, signal transduction and protection against oxidative stress. However, its possible roles in beneficial host-microbe associations are less well recognized. During the early stages of the squid-vibrio symbiosis, the bacterial symbiont Vibrio fischeri encounters host-derived NO, which has been hypothesized to serve as a specificity determinant. We demonstrate here that the flavohaemoglobin, Hmp, of V. fischeri protects against NO, both in culture and during colonization of the squid host. Transcriptional analyses indicate that hmp expression is highly responsive to NO, principally through the repressor, NsrR. Hmp protects V. fischeri from NO inhibition of aerobic respiration, and removes NO under both oxic and anoxic conditions. A Δhmp mutant of V. fischeri initiates squid colonization less effectively than wild type, but is rescued by the presence of an NO synthase inhibitor. The hmp promoter is activated during the initial stage of colonization, during which the Δhmp strain fails to form normal-sized aggregates of colonizing cells. Taken together, these results suggest that the sensing of host-derived NO by NsrR, and the subsequent removal of NO by Hmp, influence aggregate size and, thereby, V. fischeri colonization efficiency.

A non-haem iron centre in the transcription factor NorR senses nitric oxide.

Nitric oxide (NO), synthesized in eukaryotes by the NO synthases, has multiple roles in signalling pathways and in protection against pathogens. Pathogenic microorganisms have apparently evolved defence mechanisms that counteract the effects of NO and related reactive nitrogen species. Regulatory proteins that sense NO mediate the primary response to NO and nitrosative stress. The only regulatory protein in enteric bacteria known to serve exclusively as an NO-responsive transcription factor is the enhancer binding protein NorR (refs 9, 10-11). In Escherichia coli, NorR activates the transcription of the norVW genes encoding a flavorubredoxin (FlRd) and an associated flavoprotein, respectively, which together have NADH-dependent NO reductase activity. The NO-responsive activity of NorR raises important questions concerning the mechanism of NO sensing. Here we show that the regulatory domain of NorR contains a mononuclear non-haem iron centre, which reversibly binds NO. Binding of NO stimulates the ATPase activity of NorR, enabling the activation of transcription by RNA polymerase. The mechanism of NorR reveals an unprecedented biological role for a non-haem mononitrosyl-iron complex in NO sensing.

Down-regulation of the Escherichia coli K-12 nrf promoter by binding of the NsrR nitric oxide-sensing transcription repressor to an upstream site.

FNR-dependent activation of the Escherichia coli K-12 nrf promoter is downregulated by the nitric oxide-sensitive NsrR protein together with the nucleoid-associated protein IHF, which bind to overlapping targets adjacent to the DNA site for FNR. The NsrR target is inactivated by mutation at the Salmonella enterica serovar Typhimurium nrf promoter.

NsrR targets in the Escherichia coli genome: new insights into DNA sequence requirements for binding and a role for NsrR in the regulation of motility.

The Escherichia coli NsrR protein is a nitric oxide-sensitive repressor of transcription. The NsrR-binding site is predicted to comprise two copies of an 11 bp motif arranged as an inverted repeat with 1 bp spacing. By mutagenesis we confirmed that both 11 bp motifs are required for maximal NsrR repression of the ytfE promoter. We used chromatin immunoprecipitation and microarray analysis (ChIP-chip) to show that NsrR binds to 62 sites close to the 5' ends of genes. Analysis of the ChIP-chip data suggested that a single 11 bp motif (with the consensus sequence AANATGCATTT) can function as an NsrR-binding site in vivo. NsrR binds to sites in the promoter regions of the fliAZY, fliLMNOPQR and mqsR-ygiT transcription units, which encode proteins involved in motility and biofilm development. Reporter fusion assays confirmed that NsrR negatively regulates the fliA and fliL promoters. A mutation in the predicted 11 bp NsrR-binding site in the fliA promoter impaired repression by NsrR and prevented detectable binding in vivo. Assays on soft-agar confirmed that NsrR is a negative regulator of motility in E. coli K12 and in a uropathogenic strain; surface attachment assays revealed decreased levels of attached growth in the absence of NsrR.

Lung cancer: progress in diagnosis, staging and therapy.

Lung cancer remains one of the greatest medical challenges with nearly 1.5 million new cases worldwide each year and a growing tobacco epidemic in the developing world. This review summarizes briefly the current status in growing areas of clinical research. The value of screening for early disease is not yet established and trials to see if mortality can be improved as a result are in progress. Better and more accurate staging will both streamline investigation and prove cost-effective once ultrasound-guided biopsy and aspiration of mediastinal nodes become universally accepted. This, allied to the new staging classification, will improve selection of cases for surgery, intensive multimodality therapy and for adjuvant treatment postoperatively. Much still needs to be done to refine staging as within a particular stage group, the outcome shows great variation. More information is needed on the genetic make-up in some groups of tumours and not just their size; that is, more biological data on tumour growth patterns are likely to be at least as discriminating. The place of the stem cell theory of tumorigenesis is also explored in this paper. Finally, targeted therapy for advanced non-small-cell lung cancer is highlighted as a development with early promise, but still much clarification is required, before it can be considered as a universal approach in late disease.

Regulators of bacterial responses to nitric oxide.

Nitric oxide (NO) is an intermediate of the respiratory pathway known as denitrification, and is a by-product of anaerobic nitrite respiration in the enteric Bacteria. Pathogens are also exposed to NO inside host phagocytes, and possibly in other host niches as well. In recent years it has become apparent that there are multiple regulatory systems in prokaryotes that mediate responses to NO exposure. Owing to its reactivity, NO also has the potential to perturb the activities of other regulatory proteins, which are not necessarily directly involved in the response to NO. This review describes the current state of understanding of regulatory systems that respond to NO. An emerging trend is the predominance of iron proteins among the known physiological NO sensors.