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Introduction Identifying predictors of bacterial and viral pathogens in sputum from patients with acute exacerbations of chronic obstructive pulmonary disease (COPD) may help direct management. Methods We used data from a trial evaluating a C-reactive protein (CRP) point of care guided approach to managing COPD exacerbations in primary care. We used regression analyses to identify baseline clinical features, including CRP value in those randomized to testing, associated with bacterial, viral or mixed infections, defined by the presence of bacterial and viral pathogens in sputum, detected by culture or polymerase chain reaction (PCR), respectively. Results Of 386 participants with baseline sputum samples, 79 (20.5%), 123 (31.9%), and 91 (23.6%) had bacterial, viral/atypical, and mixed bacterial/viral/atypical pathogens identified, respectively. Increasing sputum purulence assessed by color chart was associated with increased odds of finding bacterial and mixed (bacterial and viral/atypical) pathogens in sputum (area under the ROC curve (AUROC) for bacterial pathogens =0.739 (95% CI: 0.670, 0.808)). Elevated CRP was associated with increased odds of finding bacterial pathogens and mixed pathogens but did not significantly increase the AUROC for predicting bacterial pathogens over sputum color alone (AUROC for combination of sputum color and CRP = 0.776 (95% CI: 0.708, 0.843), p for comparison of models = 0.053). We found no association between the presence of sputum pathogens and other clinical or demographic features. Conclusion Sputum purulence was the best predictor of sputum bacterial pathogens and mixed bacterial viral/atypical pathogens in patients with COPD exacerbations in our study. Elevated CRP was associated with bacterial pathogens but did not add to the predictive value of sputum purulence.

Background Care homes are an increasingly important sector of care. Care home residents are particularly vulnerable to infections and are often prescribed antibiotics, driving antibiotic resistance. Probiotics may be a cheap and safe way to reduce antibiotic use. Efficacy and possible mechanisms of action are yet to be rigorously evaluated in this group. Objective The objective was to evaluate efficacy and explore mechanisms of action of a daily oral probiotic combination in reducing antibiotic use and infections in care home residents. Design This was a multicentre, parallel, individually randomised, placebo-controlled, double-blind trial, with qualitative evaluation and mechanistic studies. Setting A total of 310 care home residents were randomised from 23 UK care homes (from December 2016 to May 2018). Participants The participants were care home residents aged ≥ 65 years who were willing and able to give informed consent or, if they lacked capacity to consent, had a consultee to advise about participation on their behalf. Intervention A daily capsule containing an oral probiotic combination of Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB-12 (n = 155) or matched placebo (n = 155) for up to 1 year. Main outcome measures The primary outcome was cumulative systemic antibiotic administration days for all-cause infections. Secondary outcomes included incidence and duration of infections, antibiotic-associated diarrhoea, quality of life, hospitalisations and the detection of resistant Enterobacterales cultured from stool samples (not exclusively). Methods Participants were randomised (1 : 1) to receive capsules containing probiotic or matched placebo. Minimisation was implemented for recruiting care home and care home resident sex. Care home residents were followed up for 12 months with a review by a research nurse at 3 months and at 6–12 months post randomisation. Care home residents, consultees, care home staff and all members of the trial team, including assessors and statisticians, were blinded to group allocation. Results Care home residents who were randomised to probiotic had a mean 12.9 cumulative systemic antibiotic administration days (standard error 1.49 days) (n = 152) and care home residents randomised to placebo had a mean 12.0 cumulative systemic antibiotic administration days (standard error 1.50 days) (n = 153) (adjusted incidence rate ratio = 1.13, 95% confidence interval 0.79 to 1.63; p = 0.495). There was no evidence of any beneficial effects on incidence and duration of infections, antibiotic-associated diarrhoea, quality of life, hospitalisations, the detection of resistant Enterobacterales cultured from stool samples or other secondary outcomes. There was no evidence that this probiotic combination improved blood immune cell numbers, subtypes or responses to seasonal influenza vaccination. Conclusions Care home residents did not benefit from daily consumption of a combination of the probiotics Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB-12 to reduce antibiotic consumption. Limitations Limitations included the following: truncated follow-up of some participants; higher than expected probiotics in stool samples at baseline; fewer events than expected meant that study power may have been lower than anticipated; standard infection-related definitions were not used; and findings are not necessarily generalisable because effects may be strain specific and could vary according to patient population. Future work Future work could involve further rigorous efficacy, mechanisms and effectiveness trials of other probiotics in other population groups and settings regarding antibiotic use and susceptibility to and recovery from infections, in which potential harms should be carefully studied. Trial registration Current Controlled Trials ISRCTN16392920. Funding This project was funded by the Efficacy and Mechanism Evaluation (EME) programme, a MRC and NIHR partnership. This will be published in full in Efficacy and Mechanism Evaluation; Vol. 8, No. 7. See the NIHR Journals Library website for further project information.

Antimicrobial resistance in neonatal sepsis is rising, yet mechanisms of resistance that often spread between species via mobile genetic elements, ultimately limiting treatments in low- and middle-income countries (LMICs), are poorly characterized. The Burden of Antibiotic Resistance in Neonates from Developing Societies (BARNARDS) network was initiated to characterize the cause and burden of antimicrobial resistance in neonatal sepsis for seven LMICs in Africa and South Asia. A total of 36,285 neonates were enrolled in the BARNARDS study between November 2015 and December 2017, of whom 2,483 were diagnosed with culture-confirmed sepsis. Klebsiella pneumoniae (n = 258) was the main cause of neonatal sepsis, with Serratia marcescens (n = 151), Klebsiella michiganensis (n = 117), Escherichia coli (n = 75) and Enterobacter cloacae complex (n = 57) also detected. We present whole-genome sequencing, antimicrobial susceptibility and clinical data for 916 out of 1,038 neonatal sepsis isolates (97 isolates were not recovered from initial isolation at local sites). Enterobacterales (K. pneumoniae, E. coli and E. cloacae) harboured multiple cephalosporin and carbapenem resistance genes. All isolated pathogens were resistant to multiple antibiotic classes, including those used to treat neonatal sepsis. Intraspecies diversity of K. pneumoniae and E. coli indicated that multiple antibiotic-resistant lineages cause neonatal sepsis. Our results will underpin research towards better treatments for neonatal sepsis in LMICs., Genomic and clinical analysis of 916 bacterial isolates from neonates with sepsis in seven low- and middle-income countries (the BARNARDS study) reveals that the main species present were antimicrobial-resistant Klebsiella, Escherichia coli and Enterobacter.

BackgroundC-reactive protein (CRP) point-of-care testing can reduce antibiotic use in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) in primary care, without compromising patient care. Further safe reductions may be possible.AimTo investigate the associations between presenting features and antibiotic prescribing in patients with AECOPD in primary care.Design and settingSecondary analysis of a randomised controlled trial of participants presenting with AECOPD in primary care (the PACE trial).MethodClinicians collected participants’ demographic features, comorbid illnesses, clinical signs, and symptoms. Antibiotic prescribing decisions were made after participants were randomised to receive a point-of-care CRP measurement or usual care. Multivariable regression models were fitted to explore the association between patient and clinical features and antibiotic prescribing, and extended to further explore any interactions with CRP measurement category (CRP not measured, CRP ResultsA total of 649 participants from 86 general practices across England and Wales were included. Odds of antibiotic prescribing were higher in the presence of clinician-recorded crackles (adjusted odds ratio [AOR] = 5.22, 95% confidence interval [CI] = 3.24 to 8.41), wheeze (AOR = 1.64, 95% CI = 1.07 to 2.52), diminished vesicular breathing (AOR = 2.95, 95% CI = 1.70 to 5.10), or clinician-reported evidence of consolidation (AOR = 34.40, 95% CI = 2.84 to 417.27). Increased age was associated with lower odds of antibiotic prescribing (AOR per additional year increase = 0.98, 95% CI = 0.95 to 1.00), as was the presence of heart failure (AOR = 0.32, 95% CI = 0.12 to 0.85).ConclusionSeveral demographic features and clinical signs and symptoms are associated with antibiotic prescribing in AECOPD. Diagnostic and prognostic value of these features may help identify further safe reductions.

IntroductionChronic lung disease of prematurity (CLD), also known as bronchopulmonary dysplasia (BPD), is a cause of significant respiratory morbidity in childhood and beyond. Coupled with lung immaturity, infections (especially by Ureaplasma spp) are implicated in the pathogenesis of CLD through promotion of pulmonary inflammation. Azithromycin, which is a highly effective against Ureaplasma spp also has potent anti-inflammatory properties. Thus, azithromycin therapy may improve respiratory outcomes by targeting infective and inflammatory pathways. Previous trials using macrolides have not been sufficiently powered to definitively assess CLD rates. To address this, the azithromycin therapy for chronic lung disease of prematurity (AZTEC) trial aims to determine if a 10-day early course of intravenous azithromycin improves rates of survival without CLD when compared with placebo with an appropriately powered study.Methods and analysis796 infants born at less than 30 weeks’ gestational age who require at least 2 hours of continuous respiratory support within the first 72 hours following birth are being enrolled by neonatal units in the UK. They are being randomised to receive a double-blind, once daily dose of intravenous azithromycin (20 mg/kg for 3 days, followed by 10 mg/kg for a further 7 days), or placebo. CLD is being assessed at 36 weeks’ PMA. Whether colonisation with Ureaplasma spp prior to randomisation modifies the treatment effect of azithromycin compared with placebo will also be investigated. Secondary outcomes include necrotising enterocolitis, intraventricular/cerebral haemorrhage, retinopathy of prematurity and nosocomial infections, development of antibiotic resistance and adverse reactions will be monitored.Ethics and disseminationEthics permission has been granted by Wales Research Ethics Committee 2 (Ref 18/WA/0199), and regulatory permission by the Medicines and Healthcare Products Regulatory Agency (Clinical Trials Authorisation reference 21323/0050/001–0001). The study is registered on ISRCTN (ISRCTN11650227). The study is overseen by an independent Data Monitoring Committee and an independent Trial Steering Committee. We shall disseminate our findings via national and international peer-reviewed journals, and conferences. A summary of the findings will also be posted on the trial website.

Importance Probiotics are frequently used by residents in care homes (residential homes or nursing homes that provide residents with 24-hour support for personal care or nursing care), although the evidence on whether probiotics prevent infections and reduce antibiotic use in these settings is limited. Objective To determine whether a daily oral probiotic combination of Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp lactis BB-12 compared with placebo reduces antibiotic administration in care home residents. Design, Setting, and Participants Placebo-controlled randomized clinical trial of 310 care home residents, aged 65 years and older, recruited from 23 care homes in the United Kingdom between December 2016 and May 2018, with last follow-up on October 31, 2018. Interventions Study participants were randomized to receive a daily capsule containing a probiotic combination of Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp lactis BB-12 (total cell count per capsule, 1.3 × 1010 to 1.6 × 1010) (n = 155), or daily matched placebo (n = 155), for up to 1 year. Main Outcomes and Measures The primary outcome was cumulative antibiotic administration days for all-cause infections measured from randomization for up to 1 year. Results Among 310 randomized care home residents (mean age, 85.3 years; 66.8% women), 195 (62.9%) remained alive and completed the trial. Participant diary data (daily data including study product use, antibiotic administration, and signs of infection) were available for 98.7% randomized to the probiotic group and 97.4% randomized to placebo. Care home residents randomized to the probiotic group had a mean of 12.9 cumulative systemic antibiotic administration days (95% CI, 0 to 18.05), and residents randomized to placebo had a mean of 12.0 days (95% CI, 0 to 16.95) (absolute difference, 0.9 days [95% CI, –3.25 to 5.05]; adjusted incidence rate ratio, 1.13 [95% CI, 0.79 to 1.63]; P = .50). A total of 120 care home residents experienced 283 adverse events (150 adverse events in the probiotic group and 133 in the placebo group). Hospitalizations accounted for 94 of the events in probiotic group and 78 events in the placebo group, and deaths accounted for 33 of the events in the probiotic group and 32 of the events in the placebo group. Conclusions and Relevance Among care home residents in the United Kingdom, a daily dose of a probiotic combination of Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp lactis BB-12 did not significantly reduce antibiotic administration for all-cause infections. These findings do not support the use of probiotics in this setting. Trial Registration ISRCTN Identifier:16392920

BackgroundAntibiotics are prescribed to >70% of patients presenting in primary care with an acute exacerbation of chronic obstructive pulmonary disease (AECOPD). The PACE randomised controlled trial found that a C-reactive protein point-of-care test (CRP-POCT) management strategy for AECOPD in primary care resulted in a 20% reduction in patient-reported antibiotic consumption over 4 weeks.AimTo understand perceptions of the value of CRP-POCT for guiding antibiotic prescribing for AECOPD; explore possible mechanisms, mediators, and pathways to effects; and identify potential barriers and facilitators to implementation from the perspectives of patients and clinicians.Design and settingQualitative process evaluation in UK general practices.MethodSemi-structured telephone interviews with 20 patients presenting with an AECOPD and 20 primary care staff, purposively sampled from the PACE study. Interviews were audio-recorded, transcribed, and analysed using framework analysis.ResultsPatients and clinicians felt that CRP-POCT was useful in guiding clinicians’ antibiotic prescribing decisions for AECOPD, and were positive about introduction of the test in routine care. The CRP-POCT enhanced clinician confidence in antibiotic prescribing decisions, reduced decisional ambiguity, and facilitated communication with patients. Some clinicians thought the CRP-POCT should be routinely used in consultations for AECOPD; others favoured use only when there was decisional uncertainty. CRP-POCT cartridge preparation time and cost were potential barriers to implementation.ConclusionCRP-POCT-guided antibiotic prescribing for AECOPD had high acceptability, but commissioning arrangements and further simplification of the CRP-POCT need attention to facilitate implementation in routine practice.

Background Most patients presenting with acute exacerbations of chronic obstructive pulmonary disease (AECOPD) in primary care are prescribed antibiotics, but these may not be beneficial, and they can cause side effects and increase the risk of subsequent resistant infections. Point-of-care tests (POCTs) could safely reduce inappropriate antibiotic prescribing and antimicrobial resistance. Objective To determine whether or not the use of a C-reactive protein (CRP) POCT to guide prescribing decisions for AECOPD reduces antibiotic consumption without having a negative impact on chronic obstructive pulmonary disease (COPD) health status and is cost-effective. Design A multicentre, parallel-arm, randomised controlled open trial with an embedded process, and a health economic evaluation. Setting General practices in Wales and England. A UK NHS perspective was used for the economic analysis. Participants Adults (aged ≥ 40 years) with a primary care diagnosis of COPD, presenting with an AECOPD (with at least one of increased dyspnoea, increased sputum volume and increased sputum purulence) of between 24 hours’ and 21 days’ duration. Intervention CRP POCTs to guide antibiotic prescribing decisions for AECOPD, compared with usual care (no CRP POCT), using remote online randomisation. Main outcome measures Patient-reported antibiotic consumption for AECOPD within 4 weeks post randomisation and COPD health status as measured with the Clinical COPD Questionnaire (CCQ) at 2 weeks. For the economic evaluation, patient-reported resource use and the EuroQol-5 Dimensions were included. Results In total, 653 participants were randomised from 86 general practices. Three withdrew consent and one was randomised in error, leaving 324 participants in the usual-care arm and 325 participants in the CRP POCT arm. Antibiotics were consumed for AECOPD by 212 out of 274 participants (77.4%) and 150 out of 263 participants (57.0%) in the usual-care and CRP POCT arm, respectively [adjusted odds ratio 0.31, 95% confidence interval (CI) 0.20 to 0.47]. The CCQ analysis comprised 282 and 281 participants in the usual-care and CRP POCT arms, respectively, and the adjusted mean CCQ score difference at 2 weeks was 0.19 points (two-sided 90% CI –0.33 to –0.05 points). The upper limit of the CI did not contain the prespecified non-inferiority margin of 0.3. The total cost from a NHS perspective at 4 weeks was £17.59 per patient higher in the CRP POCT arm (95% CI –£34.80 to £69.98; p = 0.408). The mean incremental cost-effectiveness ratios were £222 per 1% reduction in antibiotic consumption compared with usual care at 4 weeks and £15,251 per quality-adjusted life-year gained at 6 months with no significant changes in sensitivity analyses. Patients and clinicians were generally supportive of including CRP POCT in the assessment of AECOPD. Conclusions A CRP POCT diagnostic strategy achieved meaningful reductions in patient-reported antibiotic consumption without impairing COPD health status or increasing costs. There were no associated harms and both patients and clinicians valued the diagnostic strategy. Future work Implementation studies that also build on our qualitative findings could help determine the effect of this intervention over the longer term. Trial registration Current Controlled Trials ISRCTN24346473. Funding This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 24, No. 15. See the NIHR Journals Library website for further project information.

IntroductionCare home residents are at increased risk of infections and antibiotic prescription. Reduced antibiotic use from fewer infections would improve quality of life. TheProbiotics toReduceInfections iN CarEhome reSidentS(PRINCESS) trial aims to determine the efficacy and investigate mechanisms of daily probiotics on antibiotic use and incidence of infections in care home residents.Methods and analysisPRINCESS is a double-blind, individually randomised, placebo-controlled trial that will assess the effect of a daily oral probiotic combination ofLactobacillus rhamnosus,GG (LGG) andBifidobacterium animalissubsp.lactis, BB-12 (BB-12) on cumulative antibiotic administration days (CAADs) (primary outcome) for infection in up to 330 care home residents aged ≥65 years over up to 12 months. Secondary outcomes include:Infection:Total number of days of antibiotic administration for each infection type (respiratory tract infection, urinary tract infection, gastrointestinal infection, unexplained fever and other); number, site, duration of infection; estimation of incidence and duration of diarrhoea and antibiotic-associated diarrhoea;Stool microbiology:Clostridium difficileinfection; Gram-negative Enterobacteriaceae and vancomycin-resistant enterococci; LGG and BB-12.Oral microbiology: Candidaspp.Health and well-being:Self and/or proxy health-related quality of life EQ5D (5 L); self-and/or proxy-reported ICEpop CAPability measure for older people.Hospitalisations:number and duration of all-cause hospital stays.Mortality:deaths.Mechanistic immunology outcomes:influenza vaccine efficacy (haemagglutination inhibition assay and antibody titres); full blood count and immune cell phenotypes, plasma cytokines and chemokines; cytokine and chemokine response in whole blood stimulatedex vivoby toll-like receptor 2 and 4 agonists; monocyte and neutrophil phagocytosis ofEscherichia coli; serum vitamin D.Ethics and disseminationEthics approval is from the Wales Research Ethics Committee 3. Findings will be disseminated through peer-reviewed journals and conferences; results will be of interest to patient and policy stakeholders.Trial registration numberISRCTN16392920; Pre-results.

Background: C-reactive protein point of care tests (CRP-POCTs) for reducing unnecessary antibiotic use without harming patients with acute exacerbations of COPD (AECOPD) in primary care have not been evaluated in randomized trials. Methods: Patients consulting at 86 general practices in England and Wales with AECOPD were randomized to undergo CRP-POCT guided management or usual management without a CRP-POCT. The co-primary outcomes were patient-reported antibiotic consumption for AECOPD within four weeks (superiority), and COPD health status measured by the Clinical COPD Questionnaire (CCQ) at two weeks (non-inferiority). Results: 653 participants were randomized. Antibiotics were consumed by 57.0% CRP-POCT and 77.4% usual care participants (adjusted odds ratio = 0.31, 95% CI: 0.20 to 0.47). Adjusted mean CCQ score difference at two-weeks was -0.19 points lower (better) for CRP-POCT participants (two-sided 90% CI: -0.33 to -0.05). CRP-POCT participants were prescribed 22% fewer antibiotics at initial consultation (47.7% vs 69.7%, AOR = 0.31, 95% CI: 0.21 to 0.45), and 21% fewer antibiotics over four-week follow-up (59.1% vs 79.7%, AOR = 0.30, 95% CI: 0.20 to 0.46). There were no meaningful between-group differences in secondary clinical, microbiological, condition specific quality of life, or service utilization outcomes. Conclusions: Primary care physician use of CRP-POCT guided management of AECOPD in primary care achieves meaningful reductions in patient reported antibiotic consumption and clinician antibiotic prescribing without a detrimental effect on patient-reported specific health status.

The reverse transcriptase and endogenous DNA product synthesized by virus-like particles in the cytoplasm of human leukemic cells have been studied for their genetic relatedness to homologous components obtained from several animal RNA tumor viruses. The human reverse transcriptase activity was inhibited by antibodies prepared against reverse transcriptase from some animal RNA tumor viruses. The DNA molecules synthesized endogenously by the human cytoplasmic particle in the presence of actinomycin D, using the reverse transcriptase enzyme and RNA template residing in the particle, hybridized to 70S RNA purified from certain animal RNA tumor viruses. Both the human reverse transcriptase and DNA product are closely related to homologues from primate type-C viruses, more distantly related to those from murine type-C viruses, and essentially unrelated to similar structures from feline or avian type-C viruses. They are not related to type-B RNA tumor viruses. The results demonstrate that the components from the human leukemic cells are viral (type-C) and primate in nature.

mRNA and protein expression of the Th2 cytokines IL-4 and IL-5 from human lung were examined during the first 4 hr following IgE-mediated triggering, a time representative of the evolving late-phase reaction (LPR). Lung explants were incubated for 16 hr at 37 degrees C in culture media alone or with added dexamethasone (10(-6) M), washed, and then challenged with buffer or anti-IgE (3 micrograms/ml). Using RNase protection assays, in 16/16 individual lungs IL-5 mRNA expression was observed at 4 hr following anti-IgE and at no points following buffer challenge. Fragments released 1129 +/- 499 ng of IL-5/g wet wt over a 24-hr period (mean +/- SEM, n = 5). Neither IL-4 transcripts nor protein were detected in any anti-IgE challenges. Both the IgE-mediated IL-5 mRNA and protein responses were below the limits of detection following dexamethasone preincubation, suggesting a mechanism for the potent inhibitory effects of these agents observed in the LPR.

HERV-K is a 50-copy, human endogenous, class 1 retroviral element that contains some polycistrons with gag, pol, and env open reading frames. Although expression of HERV-K proviruses has been shown in cultured human cell lines, expression of these elements has not been shown in human blood leukocytes. Using both reverse transcriptase-polymerase chain reaction and ribonuclease protection techniques, we show HERV-K pol gene expression in human blood leukocytes. Expression in blood leukocytes from 7 normal individuals was from a variety of different HERV-K proviruses, while restricted expression was observed in blood cells of 5 leukemia patients and 3 polycythemia vera patients. Evidence is presented suggesting that the restricted expression in leukemia blood cells is a result of gene regulation, not gene amplification.

We have synthesized a novel heteropolymer double-stranded RNA (dsRNA) molecule of defined length and strandedness (dsRNA309) and evaluated its ability to induce cytokine gene expression, activate dsRNA-dependent enzymes, and inhibit both tumor cell growth and virus replication. Unlike the conventionally studied synthetic homopolymer dsRNAs, polyinosinic acid:polycytidylic acid (poly(I-C)) and its mismatched analogue polyinosinic:polycytidylic, uridylic acid (poly(I-C12,U), dsRNA309 possessed restricted biological activity. dsRNA309 was unable to inhibit tumor cell growth or efficiently induce cytokine (i.e. interferon-beta and interleukin-1 alpha) gene expression. However, dsRNA309 was able to inhibit virus replication and activate dsRNA-dependent intracellular enzymes, 2'-5' oligoadenylate synthetase (2'-5' A synthetase) and the dsRNA-activated inhibitor kinase in in vitro assay systems. Overall, dsRNA309 provided a means for examining the mechanisms governing the dsRNA-regulated antiviral and antiproliferative responses, and studies with dsRNA309 demonstrated that the ability of a synthetic dsRNA to activate dsRNA-dependent intracellular enzymes does not necessarily predict the same gene inducing capacity.

Estuarine microphytobenthos are frequently exposed to excessively high irradiances. Photoinhibition in microalgae is prevented by various photophysiological responses. We describe here the role of the xanthophyll pigments in photoacclimation. The pigment composition of the microphytobenthos was studied in three European estuaries (Barrow, Ireland; Eden, UK; Tagus, Portugal). Using HPLC-analyses, microscale changes in biomass and pigment composition were monitored over short (hourly) and long (seasonal) time scales. In the Barrow estuary, the biomass of microphytobenthos (measured as chlorophyll a) increased significantly in the top 400-500 µm of the sediment surface within 1 h of emersion; simultaneously, the xanthophyll pool size (diadinoxanthin plus diatoxanthin, dd + dt) almost doubled. A more gradual conversion of dd into dt was observed, with the dt:dd ratio increasing from 0.1 at the start of emersion to 0.3 after 3 h emersion. Similar trends in the dt:dd ratio were observed in the surface sediments of the Eden and the Tagus estuaries. Higher ratios were recorded in the Tagus estuary, which may be explained by higher incident irradiance. In addition, seasonal studies carried out in the Eden and Tagus estuaries showed that the xanthophyll pool size increased by 10% in the summer months. The pool size was highest in the Tagus estuary. Concurrently, high values for the de-epoxidation state were recorded, with values for dt/(dt + dd) 0.35 recorded in the summer. At the Eden, the ratio never exceeded 0.3. The de-epoxidation state was higher in winter than in summer, which was ascribed to the low winter temperatures. During a vertical migration study, a negative relationship between chlorophyll a and the de-epoxidation state was observed. It is suggested that this relationship originates from 'micro-migration' within the biofilm. Migration within the euphotic zone may provide an alternative means for cells to escape photodamage. In this paper, we propose that both xanthophyll cycling and 'micro'-migration play an important role in photoacclimation and it appears that these processes operate in parallel to regulate the photosynthetic response.

Molecular basis of hybridization and effect of different conditions, such as probe size, salt concentration, temperature, detergent concentration, time of washing, sample preparation and sample purity, on hybridization is discussed. Different formats of hybridization such as in solution hybridization, solid phase hybridization, and methods for probe preparation are presented. Polymerase chain reaction for the amplification of target and alternative methods for signal amplification are introduced in this review.

Large DNA palindromes form sporadically in many eukaryotic and prokaryotic genomes and are often associated with amplified genes. The presence of a short inverted repeat sequence near a DNA double-strand break has been implicated in the formation of large palindromes in a variety of organisms. Previously we have established that in Saccharomyces cerevisae a linear DNA palindrome is efficiently formed from a single-copy circular plasmid when a DNA double-strand break is introduced next to a short inverted repeat sequence. In this study we address whether the linear palindromes form by an intermolecular reaction (that is, a reaction between two identical fragments in a head-to-head arrangement) or by an unusual intramolecular reaction, as it apparently does in other examples of palindrome formation. Our evidence supports a model in which palindromes are primarily formed by an intermolecular reaction involving homologous recombination of short inverted repeat sequences. We have also extended our investigation into the requirement for DNA double-strand break repair genes in palindrome formation. We have found that a deletion of the RAD52 gene significantly reduces palindrome formation by intermolecular recombination and that deletions of two other genes in the RAD52-epistasis group (RAD51 and MRE11) have little or no effect on palindrome formation. In addition, palindrome formation is dramatically reduced by a deletion of the nucleotide excision repair gene RAD1.

Using a new strategy, capture-RT-PCR, bcrabl fusion mRNA sequences were specifically and sensitively detected in samples of whole blood from leukemia patients with the Philadelphia chromosome. Sample processing required only mixing blood with the chaotropic salt, GuSCN, and mRNA was captured during a short incubation of prepared blood with an affinity membrane. Immobilized mRNA sequences were amplified without elution. No radioisotopes or Southern transfer were needed.

Messenger RNA (mRNA) sequences were amplified from whole blood prepared in guanidine thiocyanate (GuSCN) after capture of the mRNA with an affinity membrane.

We have previously demonstrated the presence of a reverse transcriptase-like enzyme in retroviral particles from patients with essential thrombocythemia, polycythemia vera, and chronic myelogenous leukemia. It was subsequently shown that the human genome contains 50 copies of HERV-K. HERV-K is a human endogenous class I retroviral element that contains gag, pol, and env open reading frames. Using both reverse transcriptase-polymerase chain reaction and ribonuclease protection assays, it is demonstrated that the HERV-K pol is expressed in human blood leukocytes. The data indicates that this expression is restricted in CML white cells and is the result of gene regulation.

The study of double-stranded RNA (dsRNA) encompasses a variety of fields. Basic research in this area has contributed to a greater mechanistic understanding of gene induction, tumor cell growth arrest, the establishment of antiviral states, and immunomodulation. Because of the possible clinical value of these molecules, physicians are now exploring the use of synthetic dsRNA to treat patients with cancer, HIV-1 disease, and immune dysfunction. Continued studies of the mechanisms of action of dsRNA are likely to suggest an even wider scope of clinical applications.

Knowledge of the molecular biology of cancer and other diseases has grown enormously in the past decade. The role certain genes play in disease states is clearer, creating new opportunities for diagnostic applications of molecular biological techniques in clinical medicine. The measurement of an abundance of certain nucleic acids could be valuable as a diagnostic or prognostic test. For example, aberrant expression of growth-promoting genes has correlated with the type and severity of certain neoplasias. Also, the determination of an abundance of exogenous sequences, arising from viruses or other pathogens, can be useful for evaluating virus load and activity during infection. Changes in the levels of these sequences could provide a means to determine the efficacy of therapy. Nucleic acid sequences can be measured by molecular hybridization using complementary nucleic acids as probes. Strategies for performing quantitative molecular hybridization, and their application in the clinic, are discussed.

Gene expression at the mRNA level is likely to be a rapidly responding intermediate biomarker for use as a surrogate endpoint in Phase II clinical trials of breast cancer. Using a technology called “capture-RT-PCR,” we have been able to show ectopic int-2 mRNA expression in 7/9 breast tumors, including two fibroadenomas. The level of expression varied, high expression being observed in three tumors. Frequent expression of int-2 in breast tumors is contrary to published reports, possibly reflecting differences in technology. int-2 expression in fibroadenomas suggests that the marker may precede malignancy. Conversely, if fibroadenomas are not premalignant, int-2 expression may be gratuitous. These alternatives need to be tested in a clinical trial. Human endogenous retrovirus K (HERV-K) env gene expression was detected in only 1/7 breast tumors. Expression of this retroviral gene is more restricted than int-2 expression. c-myc was expressed in all 10 tumors studied, albeit at widely varying levels. Expression of prad1 and erbB-2 are under study. Gene expression will be compared with gene amplification. It is anticipated that a capture-RT-PCR assay simultaneously signifying levels of expression of several oncogenes/anti-oncogenes will be a most informative surrogate endpoint biomarker. In particular, chaotropic salt methods that simplify sample preparation and mRNA isolation and reduce these combined steps to a 10 minute procedure will be presented.

Hybridisation of RNA from a baboon endogenous type C RNA virus to DNA from tissues of leukaemic patients indicates that a virus of this type is horizontally transmitted among humans. DNA from several patients with leukaemia hybridised 70% of the hybridisable RNA from baboon endogenous type C RNA virus (BaEV) and yielded hybrids of high tm, whereas DNA from normal human tissues hybridised only 23% of the BaEV RNA, and the tm of these hybrids was lower.

RNA purified from two related RNA tumor viruses, one isolated from a baboon, Papio anubis, and the second from cultured blood leukocytes of a patient with acute myelogenous leukemia, was labeled with 125I and hybridized to DNA from different primates. RNA from both viruses showed maximum sequence homology with genes in baboons and little homology with genes of humans. The results confirm earlier suggestions that both viruses originated by transcription of baboon virogenes, and that one was transmitted to humans in nature. Hybridization of the viral RNA to cell DNA followed complicated kinetic patterns, indicating the presence of both repeated and infrequent virogene elements. This conclusion was verified in experiments using varied DNA:RNA ratios. It is proposed that virogenes, though composed of genes repeated 10 times or more, consist of some sequences more preferentially conserved than others. The non-uniformity of virogene sequence conservation limits the use of viral probes in studies concerning certain aspects of virogene evolution.

A highly repeated DNA was isolated from the West African baboon (Papio papio) as a 343-base-pair fragment after digestion of total baboon DNA with the restriction endonuclease BamHI. The DNA sequence of this fragment was obtained by chemical cleavage methods and is compared with the DNA sequence of related highly repeated primate DNAs from African green monkey (Cercopithecus aethiops) and man. The 343-base-pair baboon repeat consists of two related but nonidentical wings of 172 and 171 base pairs, respectively. The baboon 172-base-pair wing shares more homology with the African green monkey 172-base-pair repeat than with the baboon 171-base-pair wing. Comparison with the previously published monkey and human DNA sequences indicates that: (i) All the DNA sequences apparently arose from a common ancestral sequence. (ii) Evolution of the primate DNA sequences can be explained by a model involving unequal crossovers at specific points within the repeated DNA, possibly mediated by the sequence 5'-AAGG-3' 3'-TTCC-5' or its invert 5'-GGAA-3' 3'-CCTT-5'. (iii) There are alternating domains of conserved and divergent DNA sequences within each greater than 170-base-pair wing sequence. Taken together, the DNA sequences of these primates suggest a model whereby highly repeated DNAs are established and evolve as a consequence of unequal nonrandom exchanges of DNA duplexes. These exchanges may be mediated by short repeated nucleotide sequences and involve exchanges within and between the greater than 170-base-pair wings.

Cultured peripheral blood leukocytes from a woman (patient HL23) with acute myelogenous leukemia produced type-C RNA tumor viruses (HL23V). The viruses were analyzed by molecular hybridization experiments after transmission to five secondary cell culture lines. Using the criteria of molecular hybridization, we concluded that all of the transmitted virus isolates have nucleotide sequences related to the genome of simian sarcoma virus (SiSV). In addition, in agreement with data reported elsewhere, some of the transmitted viruses also have nucleotide sequences related to those of the baboon endogenous virus (BaEV). We also used molecular hybridization to ascertain whether both viruses could have originated from the patient HL23. Utilizing [3H] cDNA complementary to RNA from the separated BaEV-related component of HL23V and hybridizing this cDNA to DNA from tissues of the patient, we detected sequences related to BaEV in DNA obtained from the patient's spleen. These BaEV DNA sequences were also detectable when 125I-labeled RNA from BaEV was used as a probe. In agreement with earlier results, however, no SiSV-related sequences were detectable in the DNA of her tissues. Cytoplasmic viral-like particles, which had a buoyant density of 1.15-1.2 g/ml and were capable of synthesizing cDNA in association with a 35S RNA in vitro, were also found in the patient's fresh uncultured leukemic blood cells. cDNA synthesized by the cytoplasmic particles contained some sequences that hybridized to RNA from SiSV and, in addition, some that hybridized to RNA from BaEV. The cDNA also hybridized significantly to DNA isolated from the spleen of patient HL23 and to cytoplasmic RNA from the patient's leukocytes. These molecular hybridization results with nucleic acids obtained from the fresh blood cells of the patient, combined with the repeated isolation of similar viruses from different blood and bone marrow samples from the same patient, suggest that the virus come directly from the leukemic cell samples. The finding of BaEV-related DNA proviral sequences in the spleen of the patient strongly supports this interpretation. The failure so far to find a complete SiSV-related provirus is perplexing, but could be attributable to the existence of such a provirus in DNA of only a small population of cells in most leukemic patient.

RNA- and reverse transcriptase (R.T.)-containing viruses have been etiologically related to leukemia in several species of animals. There is no convincing report of isolation of a similar virus from man. In the past few years, however, biochemical evidence for three components thought to be characteristic of type C viruses has been detected in human acute leukemic cells. This includes the following: 1) An enzyme with physical and biochemical properties so far indistinguishable from viral R.T. has been purified from these cells. It was also recently shown that this enzyme is immunologically closely related to R.T. purified from type C primate RNA tumor viruses, much less to murine virus R.T., and not at all to avian or feline. 2) RNA has been detected in these cells with some nucleotide sequence relatedness to the genomic RNA of some murine and primate type-C viruses. 3) The DNA product of the endogenous R.T. of human leukemic cells has nucleotide sequences related to murine and especially primate type C sarcoma viruses. Thus, certain virus-like components exist in human leukemic cells in a functional form within ribonucleoprotein particles which resemble animal type C viruses. However, if they represent virus, they are defective—not released under ordinary conditions—and a causal relationship to human neoplasia, if any, has not been shown and may be impossible to prove.

The results of molecular hybridization experiments with high-molecular-weight RNA isolated from RNA tumor viruses and DNA from normal cells suggest that RNA tumor virus genomes originate from cell genes. Some RNA tumor viruses (here called class 1) appear to have been generated in recent times in that their RNA is closely related in nucleotide sequence to certain cell genes (class 1 genes). A second class of RNA tumor viruses (here called class 2) is more distantly related to genomic information of normal cells. Structural properties of the RNA of RNA tumor viruses lead us to propose that the tumor virus RNA is originated when RNA transcripts of class 1 genes are processed by a mechanism we call "paraprocessing." We postulate that RNA paraprocessing is normally used only at particular times during differentiation and is characterized by the cytoplasmic appearance of high-molecular-weight RNA chains containing terminal polyadenylic acid (200 residues). Paraprocessing of class 1 gene transcripts in committed or differentiated cells is considered to be aberrant in transcription that can lead to the generation of an RNA tumor virus genome. If the paraprocessed class 1 gene transcript codes for a reverse transcriptase, replication of the RNA becomes possible. Transfer of the replicating RNA to a new cell can result in genetic change such that the virus genome mutates, differing from the original progenitor genes. We propose that this genetic change causes class 1 viruses to become class 2. These ideas are applied to evidence concerning the biology of infection of RNA tumor viruses and concerning the involvement of RNA tumor viruses in human cancer. Genetic change can also occur during the origination of an RNA tumor virus genome by repeated reverse transcription and recombination (45) or by genetic alteration of particularly changeable cell genes ("hot spots") (43).

For increased clinical applications of nucleic acid probes to gene diagnosis, current procedures must be modified to become more amenable to the rapid processing of many samples with as few manipulations as possible. Here we summarize progress in the development of a strategy for performing molecular hybridization directly in lysate of biological samples dissolved in solutions containing the chaotropic agent, guanidine thiocyanate. Hybrids are purified by a process referred to as "reversible target capture," in which specific nucleic acid sequences are rapidly purified from crude lysate. We illustrate the use of this strategy to assay HIV-1 RNA and to rapidly purify HIV-1 RNA before enzymatic amplification by the polymerase chain reaction method.

A C-type virus isolated from long term cultures of myeloid cells from a patient with acute myelogenous leukaemia is infectious for a wide variety of cells. The establishment of chronically infected cells enabled us to characterise the virus by biological, immunological, and biochemical tests. The virus is closely related to the simian sarcoma-associated virus isolated from a woolly monkey fibrosarcoma.

[ 3 H]DNA copies of avian, feline, murine, and primate RNA tumor virus genomes were synthesized in vitro by an RNA-dependent DNA polymerase reaction. These DNAs were hybridized to 60-70S RNA that had been purified from the viruses. The amount of the [ 3 H]DNA hybridized yielded a measure of the genetic relatedness among the DNA preparations synthesized by the viruses. When many combinations of DNA and RNA were analyzed, the pattern of hybridization showed in some cases that the DNA copies of the viral RNA were related to each other in the same way that the natural hosts of the viruses are phylogenetically related. This pattern was observed only among the RNA leukemia viruses. The sarcoma component in sarcoma-leukemia viruses from rats and primates appeared to be unusually closely related. The mouse mammary carcinoma virus and two unclassified viruses (MPMV and Visna) appeared to be genetically distinct. A similar analysis of DNA synthesized by an RNA-dependent DNA polymerase associated with a viral-like particle obtained from the cytoplasm of human leukemic white blood cells demonstrated that this DNA occupied a space in the affinity pattern of leukemia viruses which is expected of a nucleic acid from a primate-type-C RNA tumor virus. This observation strengthens earlier evidence that components of RNA tumor viruses are associated with human leukemia.

Phosphoproteins of control and IFN-β treated human bladder carcinoma cells (RT4) were labelled in vitro with [ 32 P]-ATP and analyzed by polyacrylamide gel electrophoresis. Cells treated with antiproliferative doses of IFN had reduced levels of phosphorylated 60 Kd and 40 Kd proteins. IFN also induced within 24 hours the modification of a low molecular weight phosphoprotein doublet in the 22–24,000 molecular weight range. The ability to phosphorylate high molecular weight proteins by the in vitro procedures was generally depressed by IFN treatment. There was a dramatic shift in the phosphorylation of alkali stable phosphoamino acids associated with proteins in the 43–50,000 molecular weight range in IFN-treated cells. Preliminary studies indicate that at least some of the IFN-induced modifications of cellular phosphoproteins may result from transcriptional control of specific oncogenes.

Quick-blot, a method for selectively immobilizing either mRNA or DNA on nitrocellulose, is described in detail. Essential elements of the procedure for immobilizing DNA include tissue lysis, proteinase K treatment, solubilization of nucleic acids in hot 12.2 molal NaI, passage through a nitrocellulose filter, and acetylation of residual protein with acetic anhydride. Advantages include speed, quantitative recovery, low background, and elimination of the usual baking step. Essential elements of the procedure for selectively immobilizing mRNA include dissolving cells in Brij-35 and desoxycholate, proteinase K treatment, solubilizing nucleic acids in room temperature 12.2 molal NaI, filtration through nitrocellulose, and acetylation of residual protein. Advantages include selective immobilization of mRNA but not tRNA, rRNA, or DNA, and the maintenance of biological activity of the immobilized mRNA. Control experiments to optimize the procedures and examples of their application are shown.

A set of probes was designed for the quantitation of HIV-1 RNA in infected cells by a molecular hybridization procedure called reversible target capture. Reversible target capture is analogous to sandwich hybridization, except that the link between hybrid complexes and the affinity support was reversible, allowing for repeated capture of hybrids on, and release from, fresh affinity support. Repeated cycles of capture resulted in a high degree of purification of hybrids from unreacted probe, thereby greatly reducing assay noise and increasing assay sensitivity. Probes against the HIV-1 pol gene were chosen because their target sequences were highly conserved among HIV-1 isolates, while being divergent enough to provide discrimination from other human T cell tropic viruses. Subpicogram quantities of HIV-1 pol gene RNA were measured with signal:noise ratios of over 10. Hybridization signal increased with increasing target RNA with a proportionality constant of 1.

Gene diagnosis is defined and evaluated in light of existing methods of clinical diagnosis. Gene diagnosis for diseases caused by somatic or inherited mutations is more powerful than existing clinical methods, but the state of this art is too cumbersome and expensive for routine diagnosis. Gene diagnosis for infectious disease is more specific than existing detection methods, but usually is not yet as sensitive as culture or as advanced. The format for both forms of gene diagnosis will evolve to liquid hybridization followed by a “dot” format for evaluating results.

DNA synthesis is supported by small particles from platelets of patients with myeloproliferative disorders. In vitro synthesis consists of the addition of a few deoxynucleotides to a low molecular weight RNA primer. Specific bases are preferred in the first 10 nucleotides and oligodeoxynucleotides of defined length are synthesized. A reverse transcription mechanism for this activity is suggested.

Platelets from 28 patients with the myeloproliferative diseases (MPD) polycythaemia vera (9), essential thrombocythaemia (6), myelofibrosis with myeloid metaplasia (5) and chronic myelogenous leukaemia (8) were examined for an RNA-dependent DNA polymerase activity using standardized conditions permitting highly reproducible quantitation. Low levels of activity were detected in platelets of normal individuals, but platelets of nearly all MPD patients (25/28) possessed higher levels. The polymerase activity correlated with diagnosis (P = 0.001) and did not correlate with platelet counts (P greater than 0.2). Quantitation of this RNA-dependent DNA polymerase activity may be a useful parameter in the diagnosis of myeloproliferative disorders.

A repeat sequence 682 base pairs (bp) long produced by cleavage of human DNA with Xba I restriction enzyme is composed of four tandemly arranged subunits with lengths of 171, 170, 171, and 170 bp each. The sequence organization of the 682 bp Xba I repeat bears a striking resemblance to other complex satellite DNAs of primates, including the Eco RI human alpha satellite family which also occurs as a 170 bp repeat. The Eco RI tetramer and the 682 bp Xba I repeat show a sequence divergence of 21%. The 682 bp Xba I repeat sequence is restricted to humans and is only distantly related to the previously reported 340 bp Xba human repeated DNA sequence. These finding are consistent with the concept of occasional amplifications of members or groups of members of alpha satellite DNA during human evolution. Amplifications apparently occurred after humans, apes and gibbons diverged from Old World monkeys (Eco RI satellite), after humans and apes diverged from gibbons (340 bp Xba I satellite) and after humans diverged from the great apes (682 bp Xba I satellite).

A seven-year-old male gibbon ape (Hylobates lar) with malignant lymphoma and leukemia was obtained from a colony in Bermuda. Tissues obtained at necropsy were infiltrated with tumor cells to varying degrees, the most extensively involved being the kidneys, ureters, liver, lymph nodes, heart, salivary glands, and mesentery. These cells showed typical lymphoblast morphology. Type-C virus particles were abundant in the plasma and were detectable in all tissues tested. Intracytoplasmic virus was seen in some nonleukemic parenchymal cells. However, budding virus particles were observed only from the leukemic peripheral blood lymphoblasts. Tissue extracts contained viral p30, reverse transcriptase, viral RNA, and proviral DNA in amounts which generally correlated with the degree of leukemic cell infiltration. Viral markers were particularly abundant in tissue from the region of the oral cavity. Proviral DNA was found in all tissues except muscle and brain, which contained little or no leukemic cell infiltration. Replicating virus was transmitted to heterologous cells in tissue culture from many tissues, including muscle and brain. This virus, called GaLVH was classified as a member of the gibbon ape leukemia virus-simian sarcoma (simian sarcoma-associated) virus group by comparative tests of the antigenic relatedness of p30 and reverse transcriptase, and by nucleotide sequence homology of RNA from this virus and from other members of this group. These results: (a) highlight the extraordinary replication of this type of virus in gibbons; (b) further implicate this virus group in the etiology of spontaneous gibbon ape leukemia; (c) suggest that the oral route is a likely mode of virus transmission; and (d) fail to provide evidence for a genetic reservoir of virus outside of the tumor cells.

Peripheral blood mononuclear cells from HIV-1-infected persons were mixed with 5 M guanidine thiocyanate, and HIV-1-specific probes were hybridized with target RNA directly in the lysate. No RNA purification was needed. Hybrids were purified by repeated capture on superparamagnetic beads coated with oligo(dT) in a device-assisted format, a procedure termed "reversible target capture." Blood mononuclear cells from 70 ARC and AIDS patients were examined and found to have an average of 1.8 x 10(5) molecules of HIV-1 RNA per 2 x 10(6) cells.

Media from cells producing RNA tumor viruses, when treated with sodium dodecyl sulfate and polyvinyl sulfate, yield 70 S RNA as the major species binding oligo(dT)-cellulose. The procedure described for purifying 70 S RNA requires no special equipment and is suitable for rapidly processing large quantities of media or for purifying RNA from commercially available virus, with a 5- to 10-fold higher yield than was obtained using existing methods.

Synthesis of coat protein of bacteriophage f2 in infected cells, as well as in the cell-free system, is initiated with an amino-terminal formyl-methionine. The formyl-methionine is removed when the nascent chain is 40 to 60 amino acids long.

An improved method for the formation of DNA—RNA hybrids is described. The procedure involves immobilizing denatured DNA on nitrocellulose membrane filters, hybridizing complementary RNA to the membrane-fixed DNA, and eliminating RNA “noise”. Denatured DNA and hybridized RNA remain on the filter throughout the procedure. Unpaired RNA is removed by washing, and RNA complexed over short regions is eliminated by RNase treatment. Many samples can be easily handled, permitting kinetic and saturation studies. Large amounts-of DNA can be loaded on a filter without interfering with the efficiency of the annealing reaction. Moreover, the “noise” can be depressed to a level (0·003% of the input RNA) permitting the identification of small regions of DNA complementary to a given RNA species. The results are quantitatively more certain than annealing in liquid, since the competing DNA renaturation reaction is suppressed.

A mutant of E. coli, Ts68b, selected as unable to grow at 43 C, is unable to synthesize proteins at 43 C, though it can carry out this function at 30 C. This defect is shown to be in the protein-synthetic rather than the RNA-synthetic machinery by an analysis of the response of the strain to infection with the RNA bacteriophage f2. An analysis of the capacity for RNA synthesis and the polyribosome content of these cells at 44 C indicates that the defect resides in the elongation step of protein synthesis. No defects could be detected in vitro. The results are discussed in light of similar data on other mutants and in relation to the general approach of analyzing complex mutants selected with ill-defined phenotypes.

Upon a temperature shift from 30 C to 42 to 44 C, Escherichia coli experiences a transient slowdown in rate of optical density increase, uridine incorporation, and amino acid incorporation. The data show that the primary target for thermal inactivation is a molecule(s) required for the initiation of protein synthesis. The effect on ribonucleic acid synthesis is a secondary effect.