Bacterial blight of rice is an important disease in Asia and Africa. The pathogen, Xanthomonas oryzae pv. oryzae (Xoo), secretes one or more of six known transcription-activator-like effectors ...(TALes) that bind specific promoter sequences and induce, at minimum, one of the three host sucrose transporter genes SWEET11, SWEET13 and SWEET14, the expression of which is required for disease susceptibility. We used CRISPR-Cas9-mediated genome editing to introduce mutations in all three SWEET gene promoters. Editing was further informed by sequence analyses of TALe genes in 63 Xoo strains, which revealed multiple TALe variants for SWEET13 alleles. Mutations were also created in SWEET14, which is also targeted by two TALes from an African Xoo lineage. A total of five promoter mutations were simultaneously introduced into the rice line Kitaake and the elite mega varieties IR64 and Ciherang-Sub1. Paddy trials showed that genome-edited SWEET promoters endow rice lines with robust, broad-spectrum resistance.
Glycans introduce complexity to the proteins to which they are attached. These modifications vary during the progression of many diseases; thus, they serve as potential biomarkers for disease ...diagnosis and prognosis. The immense structural diversity of glycans makes glycosylation analysis and quantitation difficult. Fortunately, recent advances in analytical techniques provide the opportunity to quantify even low‐abundant glycopeptides and glycans derived from complex biological mixtures, allowing for the identification of glycosylation differences between healthy samples and those derived from disease states. Understanding the strengths and weaknesses of different quantitative glycomics analysis methods is important for selecting the best strategy to analyze glycosylation changes in any given set of clinical samples. To provide guidance towards selecting the proper approach, we discuss four widely used quantitative glycomics analysis platforms, including fluorescence‐based analysis of released N‐linked glycans and three different varieties of MS‐based analysis: liquid chromatography (LC)‐mass spectrometry (MS) analysis of glycopeptides, matrix‐assisted laser desorption ionization‐time of flight MS, and LC‐ESI‐MS analysis of released N‐linked glycans. These methods' strengths and weaknesses are compared, particularly associated with the figures of merit that are important for clinical biomarker studies, including: the initial sample requirements, the methods' throughput, sample preparation time, the number of species identified, the methods' utility for isomer separation and structural characterization, method‐related challenges associated with quantitation, repeatability, the expertise required, and the cost for each analysis. This review, therefore, provides unique guidance to researchers who endeavor to undertake a clinical glycomics analysis by offering insights on the available analysis technologies.
Blight-resistant rice lines are the most effective solution for bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo). Key resistance mechanisms involve SWEET genes as susceptibility ...factors. Bacterial transcription activator-like (TAL) effectors bind to effector-binding elements (EBEs) in SWEET gene promoters and induce SWEET genes. EBE variants that cannot be recognized by TAL effectors abrogate induction, causing resistance. Here we describe a diagnostic kit to enable analysis of bacterial blight in the field and identification of suitable resistant lines. Specifically, we include a SWEET promoter database, RT-PCR primers for detecting SWEET induction, engineered reporter rice lines to visualize SWEET protein accumulation and knock-out rice lines to identify virulence mechanisms in bacterial isolates. We also developed CRISPR-Cas9 genome-edited Kitaake rice to evaluate the efficacy of EBE mutations in resistance, software to predict the optimal resistance gene set for a specific geographic region, and two resistant 'mega' rice lines that will empower farmers to plant lines that are most likely to resist rice blight.
•Prolonged COVID-19 infection may occur in patients with primary immunodeficiency.•Antiviral treatment alone may be insufficient in patients lacking humoral immunity.•Antibody treatment, e.g. ...REGN-COV2, may aid in treating COVID-19 in these patients.•Prolonged immunosuppression may have unintended adverse effects.•Clinicians should consider early antibody treatment in those with immunodeficiency.
The role of antibodies in coronavirus disease 2019 (COVID-19) in patients with X-linked agammaglobulinaemia (XLA) has yet to be characterised and clinical courses observed in this cohort of patients have been heterogeneous. Whilst some exhibit spontaneous recovery, others have experienced a more protracted disease length. Previous reports have described successful use of convalescent plasma, however there is a paucity of information around the use of the REGN-COV2 antibody cocktail in these patients.
A patient with XLA was admitted to hospital with COVID-19 and remained persistently symptomatic with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) swab positivity despite treatment with Remdesivir and dexamethasone. Attempts at modulating the immune response with anakinra were unsuccessful. Consent for compassionate use of REGN-COV2 was obtained with administration taking place on day 87 of his illness. This was followed by a period of convalescence and SARS-CoV-2 nasopharyngeal swab negativity. As a consequence of prolonged immunosuppression, the patient developed pneumocystis pneumonia.
This case highlights the role of antibodies in clearing SARS-CoV-2 in a hypogammaglobulinaemic host and demonstrates the consequences of prolonged immunosuppression and delayed treatment. We propose that this may be of particular significance given the capacity of SARS-CoV-2 to develop advantageous mutations in a chronically infected host.
Glycomic-based approaches to discover potential biomarkers have shown great promise in their ability to distinguish between healthy and diseased individuals; these methods can identify when aberrant ...glycosylation is significant, but they cannot practically be adapted into widely implemented diagnostic assays because they are too complex, expensive, and low-throughput. We have developed a new strategy that addresses challenges associated with sample preparation, sample throughput, instrumentation needs, and data analysis to transfer the valuable knowledge provided by protein glycosylation into a clinical environment. Notably, the detection limits of the assay are in the single-digit picomole range. Proof of principle is demonstrated by quantifying the changes in the sialic acid content in fetuin. As the sialic acid content in proteins varies in a number of disease states, this example demonstrates the utility of the method for biomarker analysis. Furthermore, the developed method can be adapted to other biologically important saccharides, affording a broad array of quantitative glycomic analyses that are accessible in a high-throughput, plate-reader format. These studies enable glycomic-based biomarker discovery efforts to transition through the difficult landscape of developing a potential biomarker into a clinical assay.
Molecular diagnostics for crop diseases can enhance food security by enabling the rapid identification of threatening pathogens and providing critical information for the deployment of disease ...management strategies. Loop-mediated isothermal amplification (LAMP) is a PCR-based tool that allows the rapid, highly specific amplification of target DNA sequences at a single temperature and is thus ideal for field-level diagnosis of plant diseases. We developed primers highly specific for two globally important rice pathogens, Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight (BB) disease, and X. oryzae pv. oryzicola, the causal agent of bacterial leaf streak disease (BLS), for use in reliable, sensitive LAMP assays. In addition to pathovar distinction, two assays that differentiate X. oryzae pv. oryzae by African or Asian lineage were developed. Using these LAMP primer sets, the presence of each pathogen was detected from DNA and bacterial cells, as well as leaf and seed samples. Thresholds of detection for all assays were consistently 10(4) to 10(5) CFU ml(-1), while genomic DNA thresholds were between 1 pg and 10 fg. Use of the unique sequences combined with the LAMP assay provides a sensitive, accurate, rapid, simple, and inexpensive protocol to detect both BB and BLS pathogens.
Background The Heart Team ( HT ) comprises integrated interdisciplinary decision making. Current guidelines assign a Class Ic recommendation for an HT approach to complex coronary artery disease ( ...CAD ). However, there remains a paucity of data in regard to hard clinical end points. The aim was to determine characteristics and outcomes in patients with complex CAD following HT discussion. Methods and Results This observational study was conducted at St Thomas' Hospital (London, UK). Case mixture included unprotected left main, 2-vessel (including proximal left anterior descending artery) CAD , 3-vessel CAD , or anatomical and/or clinical equipoise. HT strategy was defined as optimal medical therapy ( OMT ) alone, OMT +percutaneous coronary intervention ( PCI ), or OMT +coronary artery bypass grafting. From April 2012 to 2013, 51 HT meetings were held and 398 cases were discussed. Patients tended to have multivessel CAD (74.1%), high SYNTAX (Synergy between PCI with Taxus and Cardiac Surgery) scores (median, 30; interquartile range, 23-39), and average age 69±11 years. Multinomial logistic regression analysis performed to determine variables associated with HT strategy demonstrated decreased likelihood of undergoing PCI compared with OMT in older patients with chronic kidney disease and peripheral vascular disease. The odds of undergoing coronary artery bypass grafting compared with OMT decreased in the presence of cardiogenic shock and left ventricular dysfunction and increased in younger patients with 3-vessel CAD . Three-year survival was 60.8% (84 of 137) in the OMT cohort, 84.3% (107 of 127) in the OMT + PCI cohort, and 90.2% in the OMT +coronary artery bypass grafting cohort (92 of 102). Conclusions In our experience, the HT approach involved a careful selection process resulting in appropriate patient-specific decision making and good long-term outcomes in patients with complex CAD .
•During the COVID-19 pandemic evidence and guidance in terms of therapeutics changed rapidly. In the UK, often this guidance was being implemented by prescribers who had been re-deployed from their ...usual areas of work, with less familiarity with local systems.•At the study organisation, following initial audits of adherence to guidance, an e-prescribing protocol was implemented to streamline and simplify the process of prescribing for patients with COVID-19 admitted to acute and general medical wards.•An improvement in adherence to guidance was demonstrated, particularly a substantial increase in prescription of both oxygen and as required Novorapid® (for patients prescribed dexamethasone at risk of corticosteroid-induced hyperglycaemia).•Furthermore, a qualitative survey of prescribers showed a high level of awareness of the protocol, and an improvement in confidence in adherence to guidance following implementation of the protocol.•E-prescribing protocols can be a useful tool to simplify prescribing and improve adherence to guidance, particularly when created with input from end-users and the wider multidisciplinary team.
The evidence around COVID-19 management is continuously evolving. Ensuring awareness of, and adherence to current guidance is challenging. As the second wave of COVID-19 emerged, we recognised the urgent need for better standardisation of patient care in the context of increasing patient load and acuity and the resulting redeployment of staff.
COVID-19 patients admitted to adult medical wards were identified via their positive swab results. An e-prescribing protocol which included five drugs was introduced and adherence to prescribing guidelines assessed via the electronic noting and prescribing system. Doctors’ views of the prescribing protocol were assessed.
Following introduction of the protocol, adherence to guidelines improved. The proportion of patients either prescribed dexamethasone or with a valid contraindication documented increased from 85% to 97% and for remdesivir this increased from 60% to 79%. There was also significant improvement in the prescription of ‘as required’ insulin for patients on steroids (26% to 48%) and oxygen (43% to 79%).
93% of doctors surveyed were aware of the e-prescribing protocol and 81% had used it. Confidence in adhering to the protocols increased from an average of 3.3 to 4.5 out of 5 and 93% of respondents agreed that the protocol was easy to use.
Overall, this demonstrates that electronic prescribing protocols can be effective in increasing adherence to guidelines and doctors felt this was a useful tool. This is especially important in a pandemic situation in which many doctors were redeployed outside of their usual specialties.
Abstract
Introduction
Increased mortality has been demonstrated in older adults with coronavirus disease 2019 (COVID-19), but the effect of frailty has been unclear.
Methods
This multi-centre cohort ...study involved patients aged 18 years and older hospitalised with COVID-19, using routinely collected data. We used Cox regression analysis to assess the impact of age, frailty and delirium on the risk of inpatient mortality, adjusting for sex, illness severity, inflammation and co-morbidities. We used ordinal logistic regression analysis to assess the impact of age, Clinical Frailty Scale (CFS) and delirium on risk of increased care requirements on discharge, adjusting for the same variables.
Results
Data from 5,711 patients from 55 hospitals in 12 countries were included (median age 74, interquartile range IQR 54–83; 55.2% male). The risk of death increased independently with increasing age (>80 versus 18–49: hazard ratio HR 3.57, confidence interval CI 2.54–5.02), frailty (CFS 8 versus 1–3: HR 3.03, CI 2.29–4.00) inflammation, renal disease, cardiovascular disease and cancer, but not delirium. Age, frailty (CFS 7 versus 1–3: odds ratio 7.00, CI 5.27–9.32), delirium, dementia and mental health diagnoses were all associated with increased risk of higher care needs on discharge. The likelihood of adverse outcomes increased across all grades of CFS from 4 to 9.
Conclusion
Age and frailty are independently associated with adverse outcomes in COVID-19. Risk of increased care needs was also increased in survivors of COVID-19 with frailty or older age.
Data on vaccine immunogenicity against SARS-CoV-2 are needed for the 40 million people globally living with HIV who might have less functional immunity and more associated comorbidities than the ...general population. We aimed to explore safety and immunogenicity of the ChAdOx1 nCoV-19 (AZD1222) vaccine in people with HIV.
In this single-arm open-label vaccination substudy within the protocol of the larger phase 2/3 trial COV002, adults aged 18–55 years with HIV were enrolled at two HIV clinics in London, UK. Eligible participants were required to be on antiretroviral therapy (ART), with undetectable plasma HIV viral loads (<50 copies per mL), and CD4 counts of more than 350 cells per μL. A prime-boost regimen of ChAdOx1 nCoV-19, with two doses was given 4–6 weeks apart. The primary outcomes for this substudy were safety and reactogenicity of the vaccine, as determined by serious adverse events and solicited local and systemic reactions. Humoral responses were measured by anti-spike IgG ELISA and antibody-mediated live virus neutralisation. Cell-mediated immune responses were measured by ex-vivo IFN-γ enzyme-linked immunospot assay (ELISpot) and T-cell proliferation. All outcomes were compared with an HIV-uninfected group from the main COV002 study within the same age group and dosing strategy and are reported until day 56 after prime vaccination. Outcomes were analysed in all participants who received both doses and with available samples. The COV002 study is registered with ClinicalTrials.gov, NCT04400838, and is ongoing.
Between Nov 5 and Nov 24, 2020, 54 participants with HIV (all male, median age 42·5 years IQR 37·2–49·8) were enrolled and received two doses of ChAdOx1 nCoV-19. Median CD4 count at enrolment was 694·0 cells per μL (IQR 573·5–859·5). No serious adverse events occurred. Local and systemic reactions occurring during the first 7 days after prime vaccination included pain at the injection site (26 49% of 53 participants with available data), fatigue (25 47%), headache (25 47%), malaise (18 34%), chills (12 23%), muscle ache (19 36%), joint pain (five 9%), and nausea (four 8%), the frequencies of which were similar to the HIV-negative participants. Anti-spike IgG responses by ELISA peaked at day 42 (median 1440 ELISA units EUs; IQR 704–2728; n=50) and were sustained until day 56 (median 941 EUs 531–1445; n=49). We found no correlation between the magnitude of the anti-spike IgG response at day 56 and CD4 cell count (p=0·93) or age (p=0·48). ELISpot and T-cell proliferative responses peaked at day 14 and 28 after prime dose and were sustained to day 56. Compared with participants without HIV, we found no difference in magnitude or persistence of SARS-CoV-2 spike-specific humoral or cellular responses (p>0·05 for all analyses).
In this study of people with HIV, ChAdOx1 nCoV-19 was safe and immunogenic, supporting vaccination for those well controlled on ART.
UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca.
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