Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences

Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There were no indications that the tomato DNA inserts conferred autonomous replication on the plasmids. Instead, Southern blot hybridization analysis of seven kanamycin resistant calli revealed the presence of at least one kanamycin resistance locus per transformant integrated in the tomato nuclear DNA. Generally one to three truncated plasmid copies were found integrated into the tomato nuclear DNA, often physically linked to each other. For one transformant we have been able to use the bacterial ampicillin resistance marker of the vector plasmid pUC9 to 'rescue' a recombinant plasmid from the tomato genome. Analysis of the foreign sequences included in the rescued plasmid showed that integration had occurred in a non-repetitive DNA region. Calf-thymus DNA, used as a carrier in transformation procedure, was found to be covalently linked to plasmid DNA sequences in the genomic DNA of one transformant. A model is presented describing the fate of exogenously added DNA during the transformation of a plant cell. The results are discussed in reference to the possibility of isolating DNA sequences responsible for autonomous replication in tomato.

Consensus for methods and outcomes in trials of oral morphine versus transmucosal diamorphine for breakthrough pain in children in the UK: the DIPPER study

Objectives: No randomised controlled trials have been conducted for breakthrough pain in paediatric palliative care and there are currently no standardised outcome measures. The DIPPER study aims to establish the feasibility of conducting a prospective randomised controlled trial comparing oral and transmucosal administration of opioids for breakthrough pain. The aim of the current study was to achieve consensus on design aspects for a small-scale prospective study to inform a future randomised controlled trial of oral morphine, the current first-line treatment, versus transmucosal diamorphine. / Methods: The nominal group technique was used to achieve consensus on best practice for mode of administration, dose regimen and a range of suitable pain intensity outcome measures for transmucosal diamorphine in children and young people with breakthrough pain. An expert panel of ten clinicians in paediatric palliative care and three parent representatives participated. Consensus was achieved when agreement was reached and no further comments from participants were forthcoming. / Results: The panel favoured the buccal route of administration, with dosing according to the recommendations in the Association for Paediatric Palliative Medicine formulary (fifth Edition, 2020). The verbal Numerical Rating Scale was selected to measure pain in children 8 years old and older, the Faces Pain Scale-Revised for children between 4 and 8 years old, and Face, Legs, Activity, Cry and Consolability (FLACC)/FLACC-Revised as the observational tools. / Conclusions: The nominal group technique allowed consensus to be reached for a small-scale, prospective, cohort study and provided information to inform the design of a randomised controlled trial

Universal Stress Proteins Are Important for Oxidative and Acid Stress Resistance and Growth of Listeria monocytogenes EGD-e In Vitro and In Vivo

Background: Pathogenic bacteria maintain a multifaceted apparatus to resist damage caused by external stimuli. As part of this, the universal stress protein A (UspA) and its homologues, initially discovered in Escherichia coli K-12 were shown to possess an important role in stress resistance and growth in several bacterial species. Methods and Findings: We conducted a study to assess the role of three homologous proteins containing the UspA domain in the facultative intracellular human pathogen Listeria monocytogenes under different stress conditions. The growth properties of three UspA deletion mutants (deltalmo0515, deltalmo1580 and deltalmo2673) were examined either following challenge with a sublethal concentration of hydrogen peroxide or under acidic conditions. We also examined their ability for intracellular survival within murine macrophages. Virulence and growth of usp mutants were further characterized in invertebrate and vertebrate infection models. Tolerance to acidic stress was clearly reduced in Δlmo1580 and deltalmo0515, while oxidative stress dramatically diminished growth in all mutants. Survival within macrophages was significantly decreased in deltalmo1580 and deltalmo2673 as compared to the wild-type strain. Viability of infected Galleria mellonella larvae was markedly higher when injected with deltalmo1580 or deltalmo2673 as compared to wild-type strain inoculation, indicating impaired virulence of bacteria lacking these usp genes. Finally, we observed severely restricted growth of all chromosomal deletion mutants in mice livers and spleens as compared to the load of wild-type bacteria following infection. Conclusion: This work provides distinct evidence that universal stress proteins are strongly involved in listerial stress response and survival under both in vitro and in vivo growth conditions

Proxy evidence for state-dependence of climate sensitivity in the Eocene greenhouse

Despite recent advances, the link between the evolution of atmospheric CO2 and climate during the Eocene greenhouse remains uncertain. In particular, modelling studies suggest that in order to achieve the global warmth that characterised the early Eocene, warmer climates must be more sensitive to CO2 forcing than colder climates. Here, we test this assertion in the geological record by combining a new high-resolution boron isotope-based CO2 record with novel estimates of Global Mean Temperature. We find that Equilibrium Climate Sensitivity (ECS) was indeed higher during the warmest intervals of the Eocene, agreeing well with recent model simulations, and declined through the Eocene as global climate cooled. These observations indicate that the canonical IPCC range of ECS (1.5 to 4.5 °C per doubling) is unlikely to be appropriate for high-CO2 warm climates of the past, and the state dependency of ECS may play an increasingly important role in determining the state of future climate as the Earth continues to warm

The present and future of serum diagnostic tests for testicular germ cell tumours.

Testicular germ cell tumours (GCTs) are the most common malignancy occurring in young adult men and the incidence of these tumours is increasing. Current research priorities in this field include improving overall survival for patients classified as being 'poor-risk' and reducing late effects of treatment for patients classified as 'good-risk'. Testicular GCTs are broadly classified into seminomas and nonseminomatous GCTs (NSGCTs). The conventional serum protein tumour markers α-fetoprotein (AFP), human chorionic gonadotrophin (hCG) and lactate dehydrogenase (LDH) show some utility in the management of testicular malignant GCT. However, AFP and hCG display limited sensitivity and specificity, being indicative of yolk sac tumour (AFP) and choriocarcinoma or syncytiotrophoblast (hCG) subtypes. Furthermore, LDH is a very nonspecific biomarker. Consequently, seminomas and NSGCTs comprising a pure embryonal carcinoma subtype are generally negative for these conventional markers. As a result, novel universal biomarkers for testicular malignant GCTs are required. MicroRNAs are short, non-protein-coding RNAs that show much general promise as biomarkers. MicroRNAs from two 'clusters', miR-371-373 and miR-302-367, are overexpressed in all malignant GCTs, regardless of age (adult or paediatric), site (gonadal or extragonadal) and subtype (seminomas, yolk sac tumours or embryonal carcinomas). A panel of four circulating microRNAs from these two clusters (miR-371a-3p, miR-372-3p, miR-373-3p and miR-367-3p) is highly sensitive and specific for the diagnosis of malignant GCT, including seminoma and embryonal carcinoma. In the future, circulating microRNAs might be useful in diagnosis, disease monitoring and prognostication of malignant testicular GCTs, which might also reduce reliance on serial CT scanning. For translation into clinical practice, important practical considerations now need addressing.The authors would like to acknowledge grant funding from CwCUK/GOSHCC (M.J.M. N.C. grant W1058), SPARKS (M.J.M. N.C. grant 11CAM01), CRUK (N.C. grant A13080) MRC (M.J.M. grant MC_EX_G0800464) and National Health Service funding to the Royal Marsden/Institute of Cancer Research National Institute for Health Research Biomedical Research Centre for Cancer (R.A.H.). The authors also thank the Max Williamson Fund, the Josh Carrick Foundation and The Perse Preparatory School, Cambridge for support.This is the author accepted manuscript. The final version is available fromNature Publishing Group via https://doi.org/10.1038/nrurol.2016.17

Blue and Red Light Modulates SigB-Dependent Gene Transcription, Swimming Motility and Invasiveness in Listeria monocytogenes

Background: In a number of gram-positive bacteria, including Listeria, the general stress response is regulated by the alternative sigma factor B (SigB). Common stressors which lead to the activation of SigB and the SigB-dependent regulon are high osmolarity, acid and several more. Recently is has been shown that also blue and red light activates SigB in Bacillus subtilis. Methodology/Principal Findings: By qRT-PCR we analyzed the transcriptional response of the pathogen L. monocytogenes to blue and red light in wild type bacteria and in isogenic deletion mutants for the putative blue-light receptor Lmo0799 and the stress sigma factor SigB. It was found that both blue (455 nm) and red (625 nm) light induced the transcription of sigB and SigB-dependent genes, this induction was completely abolished in the SigB mutant. The blue-light effect was largely dependent on Lmo0799, proving that this protein is a genuine blue-light receptor. The deletion of lmo0799 enhanced the red-light effect, the underlying mechanism as well as that of SigB activation by red light remains unknown. Blue light led to an increased transcription of the internalin A/B genes and of bacterial invasiveness for Caco-2 enterocytes. Exposure to blue light also strongly inhibited swimming motility of the bacteria in a Lmo0799- and SigB-dependent manner, red light had no effect there. Conclusions/Significance: Our data established that visible, in particular blue light is an important environmental signal with an impact on gene expression and physiology of the non-phototrophic bacterium L. monocytogenes. In natural environments these effects will result in sometimes random but potentially also cyclic fluctuations of gene activity, depending on the light conditions prevailing in the respective habitat