Construction of a Tn5 mutant library in Xanthomonas cucurbitae

The health and yield of important crop plants around the world is a significant concern when faced with food scarcity in the generations to come. The Xanthomonas bacterial genus can infect more than 400 different plant species including economically important crops such as rice and soy. In Illinois, Xanthomonas cucurbitae can infect pumpkin and winter squash and cause bacterial spot disease (Figure 1) resulting in up to 100% yield loss of pumpkin crops. The disease caused by this pathogen has become one of the most significant threats to production of pumpkins in Illinois, the U.S. and the world. There are several known virulence factors in the Xanthomonas genus that induce pathogenic response in host plants. These include the hypersensitive response and pathogenicity (hrp) gene cluster and quorum-sensing (QS) pathways. Much of the genome of X. cucurbitae, however, is unknown. We aim to identify and understand the function of novel virulence genes in the X. cucurbitae genome by generating a transposonmediated mutant library and analyzing mutants for genes involved in QS response and hrp activity by using a reporter plasmid to allow for screening of the transposon’s effects

Evaluating the Efficiency in the Application of Transformation and CRISPR/Cas9 Gene-editing Technique on Pumpkins

With the simplicity of a unique genome engineering mechanism, CRISPR/Cas9 gene-editing technique has amazed scholars with its effectiveness and efficiency in manipulating gene sequences.[1] As this advanced technique develops, its applications on different species arise as prominent subjects yet to be determined. Due to the great economic value of pumpkins and the need for examining CRISPR/Cas9 gene-editing efficiency, Casperita pumpkin (Cucurbita pepo) is chosen as the subject to be investigated on. Through introducing CRISPR/Cas9 system —for modifying phytoene desaturase (PDS) gene— into pumpkin seeds with Agrobacterium-mediated transformation, we regenerate transgenic pumpkins and expect to observe albino leaves in the transformed plants. By identifying mutated pumpkins and analyzing genotyping data, the efficiency in the application of transformation and CRISPR/Cas9 gene-editing technique on pumpkins can be established. Utilizing the findings, we aim to make contribution in developing an effective, promising gene-editing practice for pumpkin and maximizing its benefits in agriculture

Molecular characterization of differences between the tomato immune receptors flagellin sensing 3 and flagellin sensing 2

Plants mount defense responses by recognizing indicators of pathogen invasion, including microbe-associated molecular patterns (MAMPs). Flagellin, from the bacterial pathogen Pseudomonas syringae pv. tomato (Pst), contains two MAMPs, flg22 and flgII-28, that are recognized by tomato (Solanum lycopersicum) receptors Flagellin sensing2 (Fls2) and Fls3, respectively, but to what degree each receptor contributes to immunity and whether they promote immune responses using the same molecular mechanisms are unknown. Here, we characterized CRISPR/Cas9-generated Fls2 and Fls3 tomato mutants and found that the two receptors contribute equally to disease resistance both on the leaf surface and in the apoplast. However, we observed striking differences in certain host responses mediated by the two receptors. Compared to Fls2, Fls3 mediated a more sustained production of reactive oxygen species and an increase in transcript abundance of 44 tomato genes, with two genes serving as specific reporters for the Fls3 pathway. Fls3 had greater in vitro kinase activity than Fls2 and could transphosphorylate a substrate. Using chimeric Fls2/Fls3 proteins, we found no evidence that a single receptor domain is responsible for the Fls3-sustained reactive oxygen species, suggesting involvement of multiple structural features or a nullified function of the chimeric construct. This work reveals differences in certain immunity outputs between Fls2 and Fls3, suggesting that they might use distinct molecular mechanisms to activate pattern-triggered immunity in response to flagellin-derived MAMPs.Fil: Roberts, Robyn. Boyce Thompson Institute for Plant Research; Estados UnidosFil: Liu, Alexander E.. Boyce Thompson Institute for Plant Research; Estados UnidosFil: Wan, Lingwei. Boyce Thompson Institute for Plant Research; Estados UnidosFil: Geiger, Annie M.. Boyce Thompson Institute for Plant Research; Estados UnidosFil: Hind, Sarah R.. Boyce Thompson Institute for Plant Research; Estados UnidosFil: Rosli, Hernan Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; ArgentinaFil: Martin, Gregory B.. Boyce Thompson Institute for Plant Research; Estados Unidos. Cornell University; Estados Unido

A preventative lifestyle intervention for older adults (lifestyle matters): a randomised controlled trial

to test whether an occupation-based lifestyle intervention can sustain and improve the mental well-being of adults aged 65 years or over compared to usual care, using an individually randomised controlled trial. 288 independently living adults aged 65 years or over, with normal cognition, were recruited from two UK sites between December 2011 and November 2015. lifestyle Matters is a National Institute for Health and Care Excellence recommended multi-component preventive intervention designed to improve the mental well-being of community living older people at risk of decline. It involves weekly group sessions over 4 months and one to one sessions. the primary outcome was mental well-being at 6 months (mental health (MH) dimension of the SF-36). Secondary outcomes included physical health dimensions of the SF-36, extent of depression (PHQ-9), quality of life (EQ-5D) and loneliness (de Jong Gierveld Loneliness Scale), assessed at 6 and 24 months. data on 262 (intervention = 136; usual care = 126) participants were analysed using intention to treat analysis. Mean SF-36 MH scores at 6 months differed by 2.3 points (95 CI: -1.3 to 5.9; P = 0.209) after adjustments. analysis shows little evidence of clinical or cost-effectiveness in the recruited population with analysis of the primary outcome revealing that the study participants were mentally well at baseline. The results pose questions regarding how preventive interventions to promote well-being in older adults can be effectively targeted in the absence of proactive mechanisms to identify those who at risk of decline. ISRCTN67209155

Will opposites attract? Similarities and differences in students' perceptions of the stereotype profiles of other health and social care professional groups

The extent to which health and social care (HSC) students hold stereotypical views of other HSC professional groups is of great potential importance to team working in health care. This paper explores students' perceptions of different HSC professional groups at the beginning of their university programmes. Findings are presented from an analysis of baseline data collected as part of the New Generation Project longitudinal cohort study which is assessing the impact of interprofessional education over time on a range of variables including stereotyping. Questionnaires were administered to a cohort of over 1200 students from 10 different HSC professional groups entering their first year of university. Stereotypes were measured using a tool adapted from Barnes et al. (2000) designed to elicit stereotype ratings on a range of nine characteristics. The findings confirm that students arrive at university with an established and consistent set of stereotypes about other health and social care professional groups. Stereotypical profiles were compiled for each professional group indicating the distinctive characteristics of the groups as well as the similarities and differences between groups. Midwives, social workers and nurses were rated most highly on interpersonal skills and on being a team player whilst doctors were rated most highly on academic ability. Doctors, midwives and social workers were perceived as having the strongest leadership role, whilst doctors were also rated most highly on decision making. All professions were rated highly on confidence and professional competence and, with the exception of social workers, on practical skills. A comparison of profiles for each professional group reveals that, for example, pharmacists and doctors were perceived as having very similar characteristics as were social workers, midwives and nurses. However, the profiles of nurses and doctors were perceived to be very different. The implications of these similarities and differences are discussed in terms of their potential impact on interprofessional interactions, role boundaries and team working

Bolaamphiphile Analogues of 12-bis-THA Cl2 Are Potent Antimicrobial Therapeutics with Distinct Mechanisms of Action against Bacterial, Mycobacterial, and Fungal Pathogens

12-Bis-THA Cl2 [12,12'-(dodecane-1,12-diyl)-bis-(9-amino-1,2,3,4-tetrahydroacridinium) chloride] is a cationic bolalipid adapted from dequalinium chloride (DQC), a bactericidal anti-infective indicated for bacterial vaginosis (BV). Here, we used a structure-activity-relationship study to show that the factors that determine effective killing of bacterial, fungal, and mycobacterial pathogens differ, to generate new analogues with a broader spectrum of activity, and to identify synergistic relationships, most notably with aminoglycosides against Acinetobacter baumannii and Pseudomonas aeruginosa, where the bactericidal killing rate was substantially increased. Like DQC, 12-bis-THA Cl2 and its analogues accumulate within bacteria and fungi. More hydrophobic analogues with larger headgroups show reduced potential for DNA binding but increased and broader spectrum antibacterial activity. In contrast, analogues with less bulky headgroups and stronger DNA binding affinity were more active against Candida spp. Shortening the interconnecting chain, from the most lipophilic twelve-carbon chain to six, improved the selectivity index against Mycobacterium tuberculosis in vitro, but only the longer chain analogue was therapeutic in a Galleria mellonella infection model, with the shorter chain analogue exacerbating the infection. In vivo therapy of Escherichia coli ATCC 25922 and epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA-15) infections in Galleria mellonella was also achieved with longer-chain analogues, as was therapy for an A. baumannii 17978 burn wound infection with a synergistic combination of bolaamphiphile and gentamicin. The present study shows how this class of bolalipids may be adapted further to enable a wider range of potential applications. IMPORTANCE While we face an acute threat from antibiotic resistant bacteria and a lack of new classes of antibiotic, there are many effective antimicrobials which have limited application due to concerns regarding their toxicity and which could be more useful if such risks are reduced or eliminated. We modified a bolalipid antiseptic used in throat lozenges to see if it could be made more effective against some of the highest-priority bacteria and less toxic. We found that structural modifications that rendered the lipid more toxic against human cells made it less toxic in infection models and we could effectively treat caterpillars infected with either Mycobacterium tuberculosis, methicillin resistant Staphylococcus aureus, or Acinetobacter baumannii. The study provides a rationale for further adaptation toward diversifying the range of indications in which this class of antimicrobial may be used

Qualitative meta-synthesis of user experience of computerised therapy for depression and anxiety

Objective: Computerised therapies play an integral role in efforts to improve access to psychological treatment for patients with depression and anxiety. However, despite recognised problems with uptake, there has been a lack of investigation into the barriers and facilitators of engagement. We aimed to systematically review and synthesise findings from qualitative studies of computerised therapies, in order to identify factors impacting on engagement. Method: Systematic review and meta-synthesis of qualitative studies of user experiences of computer delivered therapy for depression and/or anxiety. Results: 8 studies were included in the review. All except one were of desktop based cognitive behavioural treatments. Black and minority ethnic and older participants were underrepresented, and only one study addressed users with a comorbid physical health problem. Through synthesis, we identified two key overarching concepts, regarding the need for treatments to be sensitive to the individual, and the dialectal nature of user experience, with different degrees of support and anonymity experienced as both positive and negative. We propose that these factors can be conceptually understood as the ‘non-specific’ or ‘common’ factors of computerised therapy, analogous to but distinct from the common factors of traditional face-to-face therapies. Conclusion: Experience of computerised therapy could be improved through personalisation and sensitisation of content to individual users, recognising the need for users to experience a sense of ‘self’ in the treatment which is currently absent. Exploiting the common factors of computerised therapy, through enhancing perceived connection and collaboration, could offer a way of reconciling tensions due to the dialectal nature of user experience. Future research should explore whether the findings are generalisable to other patient groups, to other delivery formats (such as mobile technology) and other treatment modalities beyond cognitive behaviour therapy. The proposed model could aid the development of enhancements to current packages to improve uptake and support engagement

Bolaamphiphile analogues of 12-bis-THA Cl2 are potent antimicrobial therapeutics with distinct mechanisms of action against bacterial, mycobacterial, and fungal pathogens

12-Bis-THA Cl2 [12,12′-(dodecane-1,12-diyl)-bis-(9-amino-1,2,3,4-tetrahydroacridinium) chloride] is a cationic bolalipid adapted from dequalinium chloride (DQC), a bactericidal anti-infective indicated for bacterial vaginosis (BV). Here, we used a structure-activity-relationship study to show that the factors that determine effective killing of bacterial, fungal, and mycobacterial pathogens differ, to generate new analogues with a broader spectrum of activity, and to identify synergistic relationships, most notably with aminoglycosides against Acinetobacter baumannii and Pseudomonas aeruginosa, where the bactericidal killing rate was substantially increased. Like DQC, 12-bis-THA Cl2 and its analogues accumulate within bacteria and fungi. More hydrophobic analogues with larger headgroups show reduced potential for DNA binding but increased and broader spectrum antibacterial activity. In contrast, analogues with less bulky headgroups and stronger DNA binding affinity were more active against Candida spp. Shortening the interconnecting chain, from the most lipophilic twelve-carbon chain to six, improved the selectivity index against Mycobacterium tuberculosis in vitro, but only the longer chain analogue was therapeutic in a Galleria mellonella infection model, with the shorter chain analogue exacerbating the infection. In vivo therapy of Escherichia coli ATCC 25922 and epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA-15) infections in Galleria mellonella was also achieved with longer-chain analogues, as was therapy for an A. baumannii 17978 burn wound infection with a synergistic combination of bolaamphiphile and gentamicin. The present study shows how this class of bolalipids may be adapted further to enable a wider range of potential applications

Association of Forced Vital Capacity with the Developmental Gene <i>NCOR2</i>

Background Forced Vital Capacity (FVC) is an important predictor of all-cause mortality in the absence of chronic respiratory conditions. Epidemiological evidence highlights the role of early life factors on adult FVC, pointing to environmental exposures and genes affecting lung development as risk factors for low FVC later in life. Although highly heritable, a small number of genes have been found associated with FVC, and we aimed at identifying further genetic variants by focusing on lung development genes. Methods Per-allele effects of 24,728 SNPs in 403 genes involved in lung development were tested in 7,749 adults from three studies (NFBC1966, ECRHS, EGEA). The most significant SNP for the top 25 genes was followed-up in 46,103 adults (CHARGE and SpiroMeta consortia) and 5,062 chi

Association of Variants in the SPTLC1 Gene With Juvenile Amyotrophic Lateral Sclerosis

Importance: Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of ALS characterized by age of symptom onset less than 25 years and a variable presentation.Objective: To identify the genetic variants associated with juvenile ALS.Design, Setting, and Participants: In this multicenter family-based genetic study, trio whole-exome sequencing was performed to identify the disease-associated gene in a case series of unrelated patients diagnosed with juvenile ALS and severe growth retardation. The patients and their family members were enrolled at academic hospitals and a government research facility between March 1, 2016, and March 13, 2020, and were observed until October 1, 2020. Whole-exome sequencing was also performed in a series of patients with juvenile ALS. A total of 66 patients with juvenile ALS and 6258 adult patients with ALS participated in the study. Patients were selected for the study based on their diagnosis, and all eligible participants were enrolled in the study. None of the participants had a family history of neurological disorders, suggesting de novo variants as the underlying genetic mechanism.Main Outcomes and Measures: De novo variants present only in the index case and not in unaffected family members.Results: Trio whole-exome sequencing was performed in 3 patients diagnosed with juvenile ALS and their parents. An additional 63 patients with juvenile ALS and 6258 adult patients with ALS were subsequently screened for variants in the SPTLC1 gene. De novo variants in SPTLC1 (p.Ala20Ser in 2 patients and p.Ser331Tyr in 1 patient) were identified in 3 unrelated patients diagnosed with juvenile ALS and failure to thrive. A fourth variant (p.Leu39del) was identified in a patient with juvenile ALS where parental DNA was unavailable. Variants in this gene have been previously shown to be associated with autosomal-dominant hereditary sensory autonomic neuropathy, type 1A, by disrupting an essential enzyme complex in the sphingolipid synthesis pathway.Conclusions and Relevance: These data broaden the phenotype associated with SPTLC1 and suggest that patients presenting with juvenile ALS should be screened for variants in this gene.</p