Ten-year survival outcomes after radical nephroureterectomy with a risk-stratified approach using prior diagnostic ureteroscopy: a single-institution observational retrospective cohort study.

OBJECTIVES: To evaluate the long-term oncological outcomes of patients with upper tract urothelial carcinoma (UTUC) undergoing radical nephroureterectomy (RNU) and the impact of diagnostic ureteroscopy (URS) on survival outcomes. MATERIALS AND METHODS: A retrospective analysis of all consecutive patients undergoing RNU for suspected UTUC at a UK tertiary referral centre from a prospectively maintained database was conducted. The primary outcome measures were 5- and 10-year cancer-specific survival (CSS). The secondary outcomes were: overall survival (OS), recurrence-free survival (RFS), impact of prior diagnostic URS on OS, CSS and intravesical RFS (intravesical-RFS), and predictors of intravesical recurrence. Statistical analysis was performed in R using the 'survminer' and 'survival' packages. The Kaplan-Meier method was used to calculate survival functions and these were expressed in graphical form. Uni-/multivariate survival analyses were performed using the Cox proportional hazard regression model. Statistical significance in this study was set at P < 0.05. RESULTS: A total of 422 patients underwent RNU with confirmed UTUC. The median (interquartile range) follow-up of patients with confirmed UTUC was 9.2 (5.6-12.7) years. The 5- and 10-year CSS rates were 70.5% (95% confidence interval [CI] 65.9-74.9) and 67.1% (95% CI 62.4-71.6), respectively. OS (HR 1.04 [95% CI 0.78-1.38]; P = 0.46) and CSS (HR 0.96 [95% CI 0.68-1.34]; P = 0.81) were similar in the diagnostic URS and the direct RNU cohorts. intravesical RFS was superior for the direct RNU cohort (HR 1.94 [95% CI 1.19-3.17]; P = 0.008). In multivariate analysis, prior URS, T2 stage, proximal ureter tumour and bladder cancer history were predictors of metachronous bladder recurrence. CONCLUSION: This single-centre retrospective cohort study reports the long-term oncological outcomes of RNU with a median follow-up of 9.2 years, serving as a reference standard in counselling patients undergoing RNU. Stage and grade of the RNU specimen were the only two studied factors that appeared to adversely impact long-term CSS and OS. Our results suggest that the risk of intravesical recurrence is increased nearly twofold in patients who have undergone diagnostic URS prior to RNU. Prior URS, however, does not appear to adversely impact long-term CSS and OS. The authors suggest that a risk-stratified approach be adopted, wherein diagnostic URS is offered only in equivocal cases.

Heterotrophic Foraminifera Capable of Inorganic Nitrogen Assimilation.

Nitrogen availability often limits biological productivity in marine systems, where inorganic nitrogen, such as ammonium is assimilated into the food web by bacteria and photoautotrophic eukaryotes. Recently, ammonium assimilation was observed in kleptoplast-containing protists of the phylum foraminifera, possibly via the glutamine synthetase/glutamate synthase (GS/GOGAT) assimilation pathway imported with the kleptoplasts. However, it is not known if the ubiquitous and diverse heterotrophic protists have an innate ability for ammonium assimilation. Using stable isotope incubations (15N-ammonium and 13C-bicarbonate) and combining transmission electron microscopy (TEM) with quantitative nanoscale secondary ion mass spectrometry (NanoSIMS) imaging, we investigated the uptake and assimilation of dissolved inorganic ammonium by two heterotrophic foraminifera; a non-kleptoplastic benthic species, Ammonia sp., and a planktonic species, Globigerina bulloides. These species are heterotrophic and not capable of photosynthesis. Accordingly, they did not assimilate 13C-bicarbonate. However, both species assimilated dissolved 15N-ammonium and incorporated it into organelles of direct importance for ontogenetic growth and development of the cell. These observations demonstrate that at least some heterotrophic protists have an innate cellular mechanism for inorganic ammonium assimilation, highlighting a newly discovered pathway for dissolved inorganic nitrogen (DIN) assimilation within the marine microbial loop.

Metabarcoding Insights Into the Trophic Behavior and Identity of Intertidal Benthic Foraminifera.

Foraminifera are ubiquitous marine protists with an important role in the benthic carbon cycle. However, morphological observations often fail to resolve their exact taxonomic placement and there is a lack of field studies on their particular trophic preferences. Here, we propose the application of metabarcoding as a tool for the elucidation of the in situ feeding behavior of benthic foraminifera, while also allowing the correct taxonomic assignment of the feeder, using the V9 region of the 18S (small subunit; SSU) rRNA gene. Living foraminiferal specimens were collected from two intertidal mudflats of the Wadden Sea and DNA was extracted from foraminiferal individuals and from the surrounding sediments. Molecular analysis allowed us to confirm that our foraminiferal specimens belong to three genetic types: Ammonia sp. T6, Elphidium sp. S5 and Haynesina sp. S16. Foraminiferal intracellular eukaryote communities reflected to an extent those of the surrounding sediments but at different relative abundances. Unlike sediment eukaryote communities, which were largely determined by the sampling site, foraminiferal intracellular eukaryote communities were driven by foraminiferal species, followed by sediment depth. Our data suggests that Ammonia sp. T6 can predate on metazoan classes, whereas Elphidium sp. S5 and Haynesina sp. S16 are more likely to ingest diatoms. These observations, alongside the use of metabarcoding in similar ecological studies, significantly contribute to our overall understanding of the ecological roles of these protists in intertidal benthic environments and their position and function in the benthic food webs.

16S rRNA gene metabarcoding and TEM reveals different ecological strategies within the genus Neogloboquadrina (planktonic foraminifer).

Uncovering the complexities of trophic and metabolic interactions among microorganisms is essential for the understanding of marine biogeochemical cycling and modelling climate-driven ecosystem shifts. High-throughput DNA sequencing methods provide valuable tools for examining these complex interactions, although this remains challenging, as many microorganisms are difficult to isolate, identify and culture. We use two species of planktonic foraminifera from the climatically susceptible, palaeoceanographically important genus Neogloboquadrina, as ideal test microorganisms for the application of 16S rRNA gene metabarcoding. Neogloboquadrina dutertrei and Neogloboquadrina incompta were collected from the California Current and subjected to either 16S rRNA gene metabarcoding, fluorescence microscopy, or transmission electron microscopy (TEM) to investigate their species-specific trophic interactions and potential symbiotic associations. 53-99% of 16S rRNA gene sequences recovered from two specimens of N. dutertrei were assigned to a single operational taxonomic unit (OTU) from a chloroplast of the phylum Stramenopile. TEM observations confirmed the presence of numerous intact coccoid algae within the host cell, consistent with algal symbionts. Based on sequence data and observed ultrastructure, we taxonomically assign the putative algal symbionts to Pelagophyceae and not Chrysophyceae, as previously reported in this species. In addition, our data shows that N. dutertrei feeds on protists within particulate organic matter (POM), but not on bacteria as a major food source. In total contrast, of OTUs recovered from three N. incompta specimens, 83-95% were assigned to bacterial classes Alteromonadales and Vibrionales of the order Gammaproteobacteria. TEM demonstrates that these bacteria are a food source, not putative symbionts. Contrary to the current view that non-spinose foraminifera are predominantly herbivorous, neither N. dutertrei nor N. incompta contained significant numbers of phytoplankton OTUs. We present an alternative view of their trophic interactions and discuss these results within the context of modelling global planktonic foraminiferal abundances in response to high-latitude climate change.

Quantitative PCR Profiling of Escherichia coli in Livestock Feces Reveals Increased Population Resilience Relative to Culturable Counts under Temperature Extremes.

The relationship between culturable counts (CFU) and quantitative PCR (qPCR) cell equivalent counts of Escherichia coli in dairy feces exposed to different environmental conditions and temperature extremes was investigated. Fecal samples were collected in summer and winter from dairy cowpats held under two treatments: field-exposed versus polytunnel-protected. A significant correlation in quantified E. coli was recorded between the qPCR and culture-based methods (r = 0.82). Evaluation of the persistence profiles of E. coli over time revealed no significant difference in the E. coli numbers determined as either CFU or gene copies during the summer for the field-exposed cowpats, whereas significantly higher counts were observed by qPCR for the polytunnel-protected cowpats, which were exposed to higher ambient temperatures. In winter, the qPCR returned significantly higher counts of E. coli for the field-exposed cowpats, thus representing a reversal of the findings from the summer sampling campaign. Results from this study suggest that with increasing time post-defecation and with the onset of challenging environmental conditions, such as extremes in temperature, culture-based counts begin to underestimate the true resilience of viable E. coli populations in livestock feces. This is important not only in the long term as the Earth changes in response to climate-change drivers but also in the short term during spells of extremely cold or hot weather.

A New Integrated Approach to Taxonomy: The Fusion of Molecular and Morphological Systematics with Type Material in Benthic Foraminifera.

A robust and consistent taxonomy underpins the use of fossil material in palaeoenvironmental research and long-term assessment of biodiversity. This study presents a new integrated taxonomic protocol for benthic foraminifera by unequivocally reconciling the traditional taxonomic name to a specific genetic type. To implement this protocol, a fragment of the small subunit ribosomal RNA (SSU rRNA) gene is used in combination with 16 quantitative morphometric variables to fully characterise the benthic foraminiferal species concept of Elphidium williamsoni Haynes, 1973. A combination of live contemporary topotypic specimens, original type specimens and specimens of genetic outliers were utilised in this study. Through a series of multivariate statistical tests we illustrate that genetically characterised topotype specimens are morphologically congruent with both the holotype and paratype specimens of E. williamsoni Haynes, 1973. We present the first clear link between morphologically characterised type material and the unique SSU rRNA genetic type of E. williamsoni. This example provides a standard framework for the benthic foraminifera which bridges the current discontinuity between molecular and morphological lines of evidence, allowing integration with the traditional Linnaean roots of nomenclature to offer a new prospect for taxonomic stability.

Denitrifying alphaproteobacteria from the Arabian Sea that express nosZ, the gene encoding nitrous oxide reductase, in oxic and suboxic waters.

Marine ecosystems are significant sources of the powerful greenhouse gas nitrous oxide (N2O). A by-product of nitrification and an intermediate in the denitrification pathway, N2O is formed primarily in oxygen-deficient waters and sediments. We describe the isolation of a group of alphaproteobacteria from the suboxic waters of the Arabian Sea that are phylogenetically affiliated with Labrenzia spp. and other denitrifiers. Quantitative PCR assays revealed that these organisms were very broadly distributed in this semienclosed ocean basin. Their biogeographical range extended from the productive, upwelling region off the Omani shelf to the clear, oligotrophic waters that are found much further south and also included the mesotrophic waters overlying the oxygen minimum zone (OMZ) in the northeastern sector of the Arabian Sea. These organisms actively expressed NosZ (N2O reductase, the terminal step in the denitrification pathway) within the OMZ, an established region of pelagic denitrification. They were found in greatest numbers outside the OMZ, however, and nosZ mRNAs were also readily detected near the base of the upper mixed layer in nutrient-poor, oxic regions. Our findings provide firm molecular evidence of a potential sink for N2O within well-ventilated, oceanic surface waters in this biogeochemically important region. We show that the Labrenzia-like denitrifiers and their close relatives are habitual colonizers of the pseudobenthic environment provided by Trichodesmium spp. We develop the conjecture that the O2-depleted microzones that occur within the colonies of these filamentous, diazotrophic cyanobacteria might provide unexpected niches for the reduction of nitrogen oxides in tropical and subtropical surface waters.

Transcriptionally active heterotrophic diazotrophs are widespread in the upper water column of the Arabian Sea.

Pelagic nitrogen fixation makes an important contribution to the fixed nitrogen budget of the world's oceans. Filamentous and unicellular cyanobacteria are significant players in this process but less is known of the potential activity of heterotrophic diazotrophs, although they are present and can be quite numerous in the nitrogen-deplete surface waters of the tropical and sub-tropical oceans. In this study we focused on the potential activity of several clades of heterotrophic nitrogen-fixers identified by phylogenetic analysis of 44 non-Trichodesmium-related, nifH (encoding the Fe-subunit of nitrogenase) clones from the Arabian Sea. Specific Northern slot blot protocols were developed to quantify nifH mRNAs from each clade and showed that two groups of Gammaproteobacteria, including the previously characterized UMB clade, and a third, novel phylotype affiliated with cluster III anaerobes, were actively expressing nitrogenase in the equatorial waters of this region. Transcripts (nifH mRNAs) from the latter clade were particularly abundant and were also detected in the suboxic waters of the oxygen minimum zone further north. Like the gammaproteobacterial groups, nifH expression by these organisms appeared to be insensitive to combined nitrogen concentrations and was readily detected in the nutrient-replete waters below the upper mixed layer as well as at shallower depths.

Short-term interventions on wards fail to reduce preanalytical errors: results of two prospective controlled trials.

BACKGROUND: Preventing laboratory errors promotes patient safety and reduces the cost of unnecessary processing. The aim of this study was to test the effectiveness of two short-term interventions at reducing errors in the preanalytical stage of laboratory testing. METHODS: Error data were reviewed from inpatient wards at Bradford Royal Infirmary (BRI), Leeds General Infirmary (LGI) and St James' University Hospital (SJUH) for 22 weeks. Two separate interventions lasted for two weeks. The outcome measures were inadequate tube and form labelling, incorrect tube selection and insufficient sample volume. Posters targeting these errors were created and displayed on inpatient wards in SJUH (n = 48). BRI and LGI were control hospitals. Qualitative interviews were held with clinical staff to raise awareness of common errors, give advice and discuss error reduction (n = 37). Ten weeks later, screensavers warning against labelling errors were displayed (LGI and SJUH). Quantitative error data, routinely collected by the laboratory, were used for analysis. RESULTS: There was no change in error rate or type at the intervention site(s) compared with the control(s). There were 7058 reported errors across three sites, of which 6623 were errors targeted by the interventions. The overall error rate remained stable on all three sites (analysis of variance, P = 1.0). When interviewing clinical staff, 29% thought that equipment was the main contributing factor to errors while 23% struggled with tube selection. CONCLUSIONS: Despite enthusiasm on the part of the ward-based staff, both short-term interventions had no significant impact on preanalytical error rates. Most errors are due to human factors. These may be reduced with the introduction of an electronic ordering system.

Lack of control of nitrite assimilation by ammonium in an oceanic picocyanobacterium, Synechococcus sp. strain WH 8103.

In cyanobacteria, the transcriptional activator NtcA is involved in global nitrogen control and, in the absence of ammonium, regulates the expression of genes involved in the assimilation of alternative nitrogen sources. The oceanic picocyanobacterium Synechococcus sp. strain WH 8103 harbors a copy of ntcA, but in the present study, we show that unlike other marine cyanobacteria that have been investigated, this strain is capable of coassimilating nitrite when grown in the presence of ammonium. Transcript levels for the genes encoding the nitrate/nitrite-bispecific permease NrtP and nitrate reductase (NarB) were substantially down-regulated by ammonium, whereas the abundances of nitrite reductase (NirA) transcripts were similar in nitrite- and ammonium-grown cells. The growth of Synechococcus sp. strain WH 8103 in medium containing both ammonium and nitrite resulted in only minor changes in the expression profile in comparison to that of nitrite-grown cells with the exception that the gene encoding the high-affinity ammonium transporter Amt1 was down-regulated to the levels seen in ammonium-grown cells. Whereas the expression of nrtP, narB, and amt1 appears to be NtcA dependent in this marine cyanobacterium, the transcription and expression of nirA appear not to be. The ability to coassimilate nitrite and reduced-nitrogen sources like ammonium may be an adaptive trait that enables oceanic strains like Synechococcus sp. strain WH 8103 to exploit the low nitrite concentrations found in oceanic surface waters that are not available to their principal and more numerous competitor, Prochlorococcus.

Spatial distribution and transcriptional activity of an uncultured clade of planktonic diazotrophic gamma-proteobacteria in the Arabian sea.

The spatial distribution of an uncultured clade of marine diazotrophic gamma-proteobacteria in the Arabian Sea was investigated by the development of a specific primer pair to amplify an internal fragment of nifH by PCR. These organisms were most readily detected in highly oligotrophic surface waters but could also be found in deeper waters below the nutricline. nifH transcripts originating from this clade were detected in oligotrophic surface waters and, in addition, in the deeper and the more productive near-coastal waters. The nifH sequences most closely related to the unidentified marine bacterial group are from environmental clones amplified from the Atlantic and Pacific Oceans. These findings suggest that these gamma-proteobacteria are widespread and likely to be an important component of the heterotrophic diazotrophic microbial community of the tropical and subtropical oceans.

Nitrate/nitrite assimilation system of the marine picoplanktonic cyanobacterium Synechococcus sp. strain WH 8103: effect of nitrogen source and availability on gene expression.

The genes encoding the structural components of the nitrate/nitrite assimilation system of the oceanic cyanobacterium Synechococcus sp. strain WH 8103 were cloned and characterized. The genes encoding nitrate reductase (narB) and nitrite reductase (nirA) are clustered on the chromosome but are organized in separate transcriptional units. Upstream of narB is a homologue of nrtP that encodes a nitrate/nitrite-bispecific permease rather than the components of an ABC-type nitrate transporter found in freshwater cyanobacteria. Unusually, neither nirA nor ntcA (encoding a positive transcription factor of genes subject to nitrogen control) were found to be tightly regulated by ammonium. Furthermore, transcription of glnA (encoding glutamine synthetase) is up-regulated in ammonium-grown cells, highlighting significant differences in nitrogen control in this cyanobacterium. Nitrogen depletion led to the transient up-regulation of ntcA, nirA, nrtP, narB, and glnA in what appears to be an NtcA-dependent manner. The NtcA-like promoters found upstream of nirA, nrtP, and narB all differ in sequence from the canonical NtcA promoter established for other cyanobacteria, and in the case of nirA, the NtcA-like promoter was functional only in cells deprived of combined nitrogen. The ecological implications of these findings are discussed in the context of the oligotrophic nature of oceanic surface waters in which Synechococcus spp. thrive.

Constipation: causes and cures.

Constipation is a common but poorly understood problem. Within the UK it is estimated that three million GP consultations relate to constipation every year. It is a problem that could affect any person at any time, yet it is often preventable. There is no accepted definition for constipation, however, and it is open to individual interpretation. Some may describe constipation as passing hard stools, others may describe it as infrequent defaecation. Constipation can affect a person's physical, psychological and social wellbeing. Nurses are in a key position to help with this problem, although a multidisciplinary approach is needed if treatment is to be successful.