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Although rhinovirus (RV) infections can cause asthma exacerbations and alter lower airway inflammation and physiology, it is unclear how important bronchial infection is to these processes. To study the kinetics, location, and frequency of RV appearance in lower airway tissues during an acute infection, immunohistochemistry and quantitative polymerase chain reaction analysis were used to analyze the presence of virus in cells from nasal lavage, sputum, bronchoalveolar lavage, bronchial brushings, and biopsy specimens from 19 subjects with an experimental RV serotype 16 (RV16) cold. RV was detected by polymerase chain reaction analysis on cells from nasal lavage and induced sputum samples from all subjects after RV16 inoculation, as well as in 5 of 19 bronchoalveolar lavage cell samples and in 5 of 18 bronchial biopsy specimens taken 4 days after virus inoculation. Immunohistochemistry detected RV16 in 39 and 36% of all biopsy and brushing samples taken 4 and 15 days, respectively, after inoculation. Infected cells were primarily distributed in discrete patches on the epithelium. These results confirm that infection of lower airway tissues is a frequent finding during a cold and further demonstrate a patchy distribution of infected cells, a pattern similar to that reported in upper airway tissues. [ABSTRACT FROM AUTHOR]

Background: Sleep is critical for healthy brain development and emotional wellbeing, especially during adolescence when sleep, behavior, and neurobiology are rapidly evolving. Theoretical reviews and empirical research have historically focused on how sleep influences mental health through its impact on higher-order brain systems. No studies have leveraged data-driven network neuroscience methods to uncover interpretable, brain-wide signatures of sleep duration in adolescence, their socio-environmental origins, or their consequences for cognition and mental health., Methods: Here, we implement graph theory and component-based predictive modeling to examine how a multimodal index of sleep duration is associated with intrinsic brain architecture in 3,173 youth (11-12 years) from the Adolescent Brain Cognitive Development SM Study., Results: We demonstrate that network integration/segregation exhibit a strong, generalizable multivariate association with sleep duration. We next identify a single component of brain architecture centered on a single network as the dominant contributor of this relationship. This component is characterized by increasing disconnection of a lower-order system - the somatomotor network - from other systems, with shorter sleep duration. Finally, greater somatomotor disconnection is associated with lower socioeconomic resources, longer screen times, reduced cognitive/academic performance, and elevated externalizing problems., Conclusions: These findings reveal a novel neural signature of shorter sleep in adolescence that is intertwined with environmental risk, cognition, and psychopathology. By robustly elucidating the key involvement of an understudied brain system in sleep, cognition, and psychopathology, this study can inform theoretical and translational research directions on sleep to promote neurobehavioral development and mental health during the adolescent transition.

AbstractBioelectrical impedance analysis (BIA) is gaining popularity in wildlife studies as a portable technology for immediate and nondestructive predictions of body composition components, such as fat-free and fat masses. Successful application of BIA for field-based research requires the identification and control of potential sources of error, as well as the creation of and adherence to a standardized protocol for measurement. The aim of our study was to determine sources of error and to provide a standardization protocol to improve measurement precision of BIA on juvenile green turtles ( Chelonia mydas ; n = 35 ). We assessed the effects of altered environmental temperature (20°C-30°C), postprandial state (2-72 h), and time out of the water (2 h) on five impedance parameters (resistance at infinite frequency [ R inf ], resistance at zero frequency [ R 0 ], resistance at 50 kHz [ R 50 ], phase angle at 50 kHz [PhA 50 ], and intracellular resistance [ R i ]) using a bioimpedance spectroscopy device. Technical reproducibility of measurements and interanimal variability were also assessed. We found an inverse exponential relationship between change in environmental temperature and impedance parameters R inf , R 0 , and R 50 . Postprandial state significantly increased R inf and R i 72 h after feeding. BIA measurements were reproducible within individual juvenile green turtles at temperatures from 20°C to 30°C. Significant variation in impedance values was found between animals at all temperatures, sampling times, and postprandial states, but the relative differences (%) were small in magnitude. Our study suggests that measurement precision is improved by measuring animals at consistent environmental temperatures close to their preferred thermal range. We propose a standardized protocol of measurement conditions to facilitate laboratory and field use of BIA for body composition assessment studies in turtles.

The canonical function of a bacterial sigma (σ) factor is to determine the gene specificity of the RNA polymerase (RNAP). In several diverse bacterial species, the σ 54 factor uniquely confers distinct functional and regulatory properties on the RNAP. A hallmark feature of the σ 54 -RNAP is the obligatory requirement for an activator ATPase to allow transcription initiation. Different activator ATPases couple diverse environmental cues to the σ 54 -RNAP to mediate adaptive changes in gene expression. Hence, the genes that rely upon σ 54 for their transcription have a wide range of different functions suggesting that the repertoire of functions performed by genes, directly or indirectly affected by σ 54 , is not yet exhaustive. By comparing the growth patterns of prototypical enteropathogenic, uropathogenic, and nonpathogenic Escherichia coli strains devoid of σ 54 , we uncovered that the absence of σ 54 results in two differently sized colonies that appear at different times specifically in the uropathogenic E. coli (UPEC) strain. Notably, UPEC bacteria devoid of individual activator ATPases of the σ 54 -RNAP do not phenocopy the σ 54 mutant strain. Thus, it seems that σ 54 's role as a determinant of uniform colony appearance in UPEC bacteria represents a putative non-canonical function of σ 54 in regulating genetic information flow. IMPORTANCE RNA synthesis is the first step of gene expression. The multisubunit RNA polymerase (RNAP) is the central enzyme responsible for RNA synthesis in bacteria. The dissociable sigma (σ) factor subunit directs the RNAP to different sets of genes to allow their expression in response to various cellular needs. Of the seven σ factors in Escherichia coli and related bacteria, σ 54 exists in a class of its own. This study has uncovered that σ 54 is a determinant of the uniform growth of uropathogenic E. coli on solid media. This finding suggests a role for this σ 54 in gene regulation that extends beyond its known function as an RNAP gene specificity factor.

Staphylococcus aureus ( S. aureus ) is an aggressive opportunistic pathogen of prominent virulence and antibiotic resistance. These characteristics are due in part to the accessory gene regulator ( agr ) quorum-sensing system, which allows for the rapid adaptation of S. aureus to environmental changes and thus promotes virulence and the development of pathogenesis. AgrA is the agr system response regulator that binds to the P2 and P3 promoters and upregulates agr expression. In this study, we reveal that S. aureus AgrA is modified by covalent binding of CoA (CoAlation) in response to oxidative or metabolic stress. The sites of CoAlation were mapped by liquid chromatography tandem mass spectrometry (LC-MS/MS) and revealed that oxidation-sensing Cys199 is modified by CoA. Surface plasmon resonance (SPR) analysis showed an inhibitory effect of CoAlation on the DNA-binding activity, as CoAlated AgrA had significantly lower affinity towards the P2 and P3 promoters than non-CoAlated AgrA. Overall, this study provides novel insights into the mode of transcriptional regulation in S. aureus and further elucidates the link between the quorum-sensing and oxidation-sensing roles of the agr system.

The initial adaptive responses to nutrient depletion in bacteria often occur at the level of gene expression. Hfq is an RNA-binding protein present in diverse bacterial lineages that contributes to many different aspects of RNA metabolism during gene expression. Using photoactivated localization microscopy and single-molecule tracking, we demonstrate that Hfq forms a distinct and reversible focus-like structure in Escherichia coli specifically experiencing long-term nitrogen starvation. Using the ability of T7 phage to replicate in nitrogen-starved bacteria as a biological probe of E. coli cell function during nitrogen starvation, we demonstrate that Hfq foci have a role in the adaptive response of E. coli to long-term nitrogen starvation. We further show that Hfq foci formation does not depend on gene expression once nitrogen starvation has set in and occurs indepen-dently of the transcription factor N-regulatory protein C, which activates the initial adaptive response to N starvation in E. coli These results serve as a paradigm to demonstrate that bacterial adaptation to long-term nutrient starvation can be spatiotemporally coordinated and can occur independently of de novo gene expression during starvation., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 McQuail et al.)

Bacteria initially respond to nutrient starvation by eliciting large-scale transcriptional changes. The accompanying changes in gene expression and metabolism allow the bacterial cells to effectively adapt to the nutrient-starved state. How the transcriptome subsequently changes as nutrient starvation ensues is not well understood. We used nitrogen (N) starvation as a model nutrient starvation condition to study the transcriptional changes in Escherichia coli experiencing long-term N starvation. The results reveal that the transcriptome of N-starved E. coli undergoes changes that are required to maximize chances of viability and to effectively recover growth when N starvation conditions become alleviated. We further reveal that, over time, N-starved E. coli cells rely on the degradation of allantoin for optimal growth recovery when N becomes replenished. This study provides insights into the temporally coordinated adaptive responses that occur in E. coli experiencing sustained N starvation. IMPORTANCE Bacteria in their natural environments seldom encounter conditions that support continuous growth. Hence, many bacteria spend the majority of their time in states of little or no growth due to starvation of essential nutrients. To cope with prolonged periods of nutrient starvation, bacteria have evolved several strategies, primarily manifesting themselves through changes in how the information in their genes is accessed. How these coping strategies change over time under nutrient starvation is not well understood, and this knowledge is important not only to broaden our understanding of bacterial cell function but also to potentially find ways to manage harmful bacteria. This study provides insights into how nitrogen-starved Escherichia coli bacteria rely on different genes during long-term nitrogen starvation., (Copyright © 2020 Switzer et al.)

Like eukaryotic and archaeal viruses, which coopt the host's cellular pathways for their replication, bacteriophages have evolved strategies to alter the metabolism of their bacterial host. SPO1 bacteriophage infection of Bacillus subtilis results in comprehensive remodeling of cellular processes, leading to conversion of the bacterial cell into a factory for phage progeny production. A cluster of 26 genes in the SPO1 genome, called the host takeover module, encodes for potentially cytotoxic proteins that specifically shut down various processes in the bacterial host, including transcription, DNA synthesis, and cell division. However, the properties and bacterial targets of many genes of the SPO1 host takeover module remain elusive. Through a systematic analysis of gene products encoded by the SPO1 host takeover module, here we identified eight gene products that attenuated B. subtilis growth. Of the eight phage gene products that attenuated bacterial growth, a 25-kDa protein called Gp53 was shown to interact with the AAA+ chaperone protein ClpC of the ClpCP protease of B. subtilis Our results further reveal that Gp53 is a phage-encoded adaptor-like protein that modulates the activity of the ClpCP protease to enable efficient SPO1 phage progeny development. In summary, our findings indicate that the bacterial ClpCP protease is the target of xenogeneic (dys)regulation by a SPO1 phage-derived factor and add Gp53 to the list of antibacterial products that target bacterial protein degradation and therefore may have utility for the development of novel antibacterial agents., (© 2019 Mulvenna et al.)

T7 development in Escherichia coli requires the inhibition of the housekeeping form of the bacterial RNA polymerase (RNAP), Eσ 70 , by two T7 proteins: Gp2 and Gp5.7. Although the biological role of Gp2 is well understood, that of Gp5.7 remains to be fully deciphered. Here, we present results from functional and structural analyses to reveal that Gp5.7 primarily serves to inhibit Eσ S , the predominant form of the RNAP in the stationary phase of growth, which accumulates in exponentially growing E. coli as a consequence of the buildup of guanosine pentaphosphate [(p)ppGpp] during T7 development. We further demonstrate a requirement of Gp5.7 for T7 development in E. coli cells in the stationary phase of growth. Our finding represents a paradigm for how some lytic phages have evolved distinct mechanisms to inhibit the bacterial transcription machinery to facilitate phage development in bacteria in the exponential and stationary phases of growth., Competing Interests: The authors declare no conflict of interest.

Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the bacterial RNA polymerase (RNAP) by the 7 kDa T7 protein Gp2. We describe the identification and functional and structural characterisation of a novel 7 kDa T7 protein, Gp5.7, which adopts a winged helix-turn-helix-like structure and specifically represses transcription initiation from host RNAP-dependent promoters on the phage genome via a mechanism that involves interaction with DNA and the bacterial RNAP. Whereas Gp2 is indispensable for T7 growth in E. coli, we show that Gp5.7 is required for optimal infection outcome. Our findings provide novel insights into how phages fine-tune the activity of the host transcription machinery to ensure both successful and efficient phage progeny development., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The Escherichia coli eukaryote-like serine/threonine kinase, encoded by yeaG, is expressed in response to diverse stresses, including nitrogen (N) starvation. A role for yeaG in bacterial stress response is unknown. Here we reveal for the first time that wild-type E. coli displays metabolic heterogeneity following sustained periods of N starvation, with the metabolically active population displaying compromised viability. In contrast, such heterogeneity in metabolic activity is not observed in an E. coli ∆yeaG mutant, which continues to exist as a single and metabolically active population and thus displays an overall compromised ability to survive sustained periods of N starvation. The mechanism by which yeaG acts, involves the transcriptional repression of two toxin/antitoxin modules, mqsR/mqsA and dinJ/yafQ. This, consequently, has a positive effect on the expression of rpoS, the master regulator of the general bacterial stress response. Overall, results indicate that yeaG is required to fully execute the rpoS-dependent gene expression program to allow E. coli to adapt to sustained N starvation and unravels a novel facet to the regulatory basis that underpins adaptive response to N stress.

Most DNA-binding bacterial transcription factors contact DNA through a recognition α-helix in their DNA-binding domains. An emerging class of DNA-binding transcription factors, predominantly found in pathogenic bacteria interact with the DNA via a relatively novel type of DNA-binding domain, called the LytTR domain, which mainly comprises β strands. Even though the crystal structure of the LytTR domain of the virulence gene transcription factor AgrA from Staphylococcus aureus bound to its cognate DNA sequence is available, the contribution of specific amino acid residues in the LytTR domain of AgrA to transcription activation remains elusive. Here, for the first time, we have systematically investigated the role of amino acid residues in transcription activation in a LytTR domain-containing transcription factor. Our analysis, which involves in vivo and in vitro analyses and molecular dynamics simulations of S. aureus AgrA identifies a highly conserved tyrosine residue, Y229, as a major amino acid determinant for maximal activation of transcription by AgrA and provides novel insights into structure-function relationships in S. aureus AgrA., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs.

Mutations in the park2 gene, encoding the RING-inBetweenRING-RING E3 ubiquitin ligase parkin, cause 50% of autosomal recessive juvenile Parkinsonism cases. More than 70 known pathogenic mutations occur throughout parkin, many of which cluster in the inhibitory amino-terminal ubiquitin-like domain, and the carboxy-terminal RING2 domain that is indispensable for ubiquitin transfer. A structural rationale showing how autosomal recessive juvenile Parkinsonism mutations alter parkin function is still lacking. Here we show that the structure of parkin RING2 is distinct from canonical RING E3 ligases and lacks key elements required for E2-conjugating enzyme recruitment. Several pathogenic mutations in RING2 alter the environment of a single surface-exposed catalytic cysteine to inhibit ubiquitination. Native parkin adopts a globular inhibited conformation in solution facilitated by the association of the ubiquitin-like domain with the RING-inBetweenRING-RING C-terminus. Autosomal recessive juvenile Parkinsonism mutations disrupt this conformation. Finally, parkin autoubiquitinates only in cis, providing a molecular explanation for the recessive nature of autosomal recessive juvenile Parkinsonism.

Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser(65). We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser(65). We further show that phosphorylation of Parkin at Ser(65) leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser(65) or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr(257), which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser(65) and/or PINK1 at Thr(257) represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD.

Parkin is an E3 ubiquitin ligase, mutations in which cause Autosomal Recessive Parkinson's Disease. Many studies aimed at understanding Parkin function, regulation and dysfunction are performed using N-terminal epitope tags. We report here that the use of small tags such as FLAG, cMyc and HA, influence the physical stability and activity of Parkin in and out of cells, perturbing the autoinhibited native state of Parkin, resulting in an active-for-autoubiquitination species.

Candida albicans is the most prevalent fungal pathogen in humans and a major source of life-threatening nosocomial infections. The Als (agglutinin-like sequence) glycoproteins are an important virulence factor for this fungus and have been associated with binding of host-cell surface proteins and small peptides of random sequence, the formation of biofilms and amyloid fibers. High-resolution structures of N-terminal Als adhesins (NT-Als; up to 314 amino acids) show that ligand recognition relies on a motif capable of binding flexible C termini of peptides in extended conformation. Central to this mechanism is an invariant lysine that recognizes the C-terminal carboxylate of ligands at the end of a deep-binding cavity. In addition to several protein-peptide interactions, a network of water molecules runs parallel to one side of the ligand and contributes to the recognition of diverse peptide sequences. These data establish NT-Als adhesins as a separate family of peptide-binding proteins and an unexpected adhesion system for primary, widespread protein-protein interactions at the Candida/host-cell interface.

Parkin is an E3-ubiquitin ligase belonging to the RBR (RING-InBetweenRING-RING family), and is involved in the neurodegenerative disorder Parkinson's disease. Autosomal recessive juvenile Parkinsonism, which is one of the most common familial forms of the disease, is directly linked to mutations in the parkin gene. However, the molecular mechanisms of Parkin dysfunction in the disease state remain to be established. We now demonstrate that the ubiquitin-like domain of Parkin functions to inhibit its autoubiquitination. Moreover pathogenic Parkin mutations disrupt this autoinhibition, resulting in a constitutively active molecule. In addition, we show that the mechanism of autoregulation involves ubiquitin binding by a C-terminal region of Parkin. Our observations provide important molecular insights into the underlying basis of Parkinson's disease, and in the regulation of RBR E3-ligase activity.

dUTPases are a ubiquitous family of enzymes that are essential for all organisms and catalyse the breakdown of 2-deoxyuridine triphosphate (dUTP). In Bacillus subtilis there are two homotrimeric dUTPases: a genomic and a prophage form. Here, the structures of the genomic dUTPase and of its complex with the substrate analogue dUpNHpp and calcium are described, both at 1.85 A resolution. The overall fold resembles that of previously solved trimeric dUTPases. The C-terminus, which contains one of the conserved sequence motifs, is disordered in both structures. The crystal of the complex contains six independent protomers which accommodate six dUpNHpp molecules, with three triphosphates in the trans conformation and the other three in the active gauche conformation. The structure of the complex confirms the role of several key residues that are involved in ligand binding and the position of the catalytic water. Asp82, which has previously been proposed to act as a general base, points away from the active site. In the complex Ser64 reorients in order to hydrogen bond the phosphate chain of the substrate. A novel feature has been identified: the position in the sequence of the ;Phe-lid', which packs against the uracil moiety, is adjacent to motif III, whereas in all other dUTPase structures the lid is in a conserved position in motif V of the flexible C-terminal arm. This requires a reconsideration of some aspects of the accepted mechanism.

Rhinovirus (RV) infections can alter lower airway physiology and inflammation, yet the characteristics of RV replication in lower airway cells are incompletely understood. An RV serotype 16 (RV16)-specific monoclonal antibody was identified. Immunohistochemistry and an infectious center assay were used to quantitate the infectivity of RV16 in primary bronchial and adenoidal epithelial cells. The proportion of infectible epithelial cells increased with the inoculum but did not exceed 10%. Analysis of bronchial tissue samples infected ex vivo demonstrated a small subset of RV-infected cells in the epithelial layer. These data confirm previous reports that RV infects only a small subset of epithelial cells in upper airway tissues and indicate that lower airway epithelial cells have a similar susceptibility to RV infection. In confirming that RV can infect cells in the lower airway, these results suggest that lower airway dysfunction occurs through this mechanism in susceptible persons.

Background: In our previously described primate renal allograft model, T cell ablation leads to long-term graft survival. The role of endothelial cell alteration in chronic rejection was examined in our model., Methods: Renal transplants were performed in rhesus monkeys using a T cell- depleting immunotoxin, FN18-CRM9. Sections from 10 rejected kidneys (5 acute and 7 chronic rejection) were examined after immunohistochemical staining for expression of endothelium-related proteins [von Willebrand factor (vWF), CD62P, and CD31], fibrinogen, and a macrophage marker (CD68). Glomerular staining for each antigen was graded on a semiquantitative scale., Results: Intense staining for vWF was consistently observed in glomerular endothelium, subendothelium, and mesangium in all kidneys removed due to chronic rejection. vWF staining was weak in kidneys showing acute rejection. The difference in glomerular staining was statistically significant. Staining for vWF in extraglomerular vessels was nearly identical in kidneys showing acute and chronic rejection. Expression of CD62P was increased in extraglomerular vessels in allografts with chronic rejection, but the glomeruli showed little or no staining. There was no significant difference in the glomerular staining for CD62P or CD31 in organs showing acute and chronic rejection. Fibrinogen staining of glomerular mesangium was seen in kidneys with chronic rejection. Macrophages (CD68+) infiltrating glomeruli were more numerous in kidneys showing chronic rejection., Conclusion: Increased glomerular deposition of vWF in renal allografts showing chronic rejection, without increased staining for CD62P or CD31, suggests increased constitutive secretion of vWF from endothelial cells as a component of the mechanism of chronic rejection in our model.

Objective: To determine the efficacy of adjuvant psychological therapy in patients with testicular cancer and to compare the characteristics and psychosocial outcomes of men who agreed to participate with those who declined to participate in a randomised trial of psychological intervention., Design: Newly diagnosed patients were asked to participate in a randomised trial of psychological support compared with standard medical care. Participants and non-participants completed self assessment questionnaires at baseline and at 2, 4 and 12 months., Setting: Testicular Tumour Unit of the Royal Marsden Hospital., Subjects: 73 of 184 (40%) eligible patients agreed to enter the randomised trial (participants) and 81 (44%) declined to participate but agreed to complete further assessments (non-participants). 30 patients wanted no further contact with the researchers., Outcome Measures: Hospital anxiety and depression scale, psychosocial adjustment to illness scale, Rotterdam symptom checklist, mental adjustment to cancer scale. Only scores on the hospital anxiety and depression scale are reported for evaluating treatment efficacy., Results: 111 of 184 (60%) eligible men declined to participate in the trial. Patients with stage I disease were most likely to refuse to participate. A patient was less likely to participate if he had low volume disease and was receiving no further treatment. Likelihood of participation was associated with stage of disease and with type of primary treatment (P < 0.001 for heterogeneity). Patients with early stage disease (P < 0.001) and fewer physical symptoms (P < 0.001) were less likely to participate. Psychosocial factors associated with participation included anxious preoccupation regarding disease (P = 0.01). There were no differences in outcome between participants and non-participants during follow up. Patients seemed to gain little benefit from adjuvant psychological therapy. At 2 months change from baseline favoured the treated group in the anxiety subscale (mean difference between groups -1.41 (95% confidence interval -2.86 to 0.03)). This was not sustained when adjusted for factors related to the disease. By 12 months change from baseline seemed to favour the control group (mean difference between groups 1.66 (-0.18 to 3.50))., Conclusions: Patients with testicular cancer seem to have considerable coping abilities. Those who declined to participate in the trial differed from those who participated. Those who agreed to participate may comprise the clinical group who perceive a need for psychological support. No evidence was found to indicate a need for routinely offering adjuvant psychological therapy.