Effector delivery and effector characterisation in Dothistroma needle blight of pines : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Sciences) in Genetics/Molecular Plant Pathology at Massey University, Manawatū, New Zealand

The filamentous fungus Dothistroma septosporum causes a serious foliar disease, Dothistroma needle blight (DNB), on Pinus radiata in New Zealand and on many pine species worldwide. Potentially correlated to changes in climate, this disease has been on the rise for 20 to 30 years, and current countermeasures often struggle to contain the damage it causes. A molecular approach to combat DNB could be promising. Effectors are small proteins secreted by pathogens to promote host colonisation, and have been a major focus of plant pathologists in recent years. However, effector biology in pathogens of gymnosperms has received little research attention. Here, candidate effectors (CEs) were selected using a series of computational prediction tools, as well as RNAseq data from a compatible D. septosporum-pine interaction. A shortlist of 55 highly in planta expressed CEs, predicted to be secreted to the apoplast, was characterised in silico. While almost half of them lacked a predicted function, none were exclusive to D. septosporum. Seventeen effector candidates of particular interest were taken forward for functional characterisation. Specifically, these proteins were screened for induction of plant defences in the form of cell death in the model plants Nicotiana benthamiana and N. tabacum using an Agrobacterium transient expression assay. Five CEs induced cell death in these plants, suggesting recognition by the plant defence machinery. Of those five, three are similar to previously described proteins. Effector screening methods are not available for pine, thus various approaches to achieve this were trialled. A high-throughput method to collect each protein in the apoplastic wash fluid of N. benthamiana was developed. This fluid was applied to P. radiata shoots raised from tissue culture to screen for a response, with promising results. Along with an array of D. septosporum CEs, these shoots may ultimately be used to screen for resistant pine genotypes. Selection of genotypes at this early stage could speed up DNB resistance screening for pine breeding and the protocol could be transferred to related pathosystems. This research also contributes to the molecular understanding of forest diseases and effectors that may be common among pathogens of distantly related hosts

GWAS analysis reveals distinct pathogenicity profiles of Australian Parastagonospora nodorum isolates and identification of marker-trait-associations to septoria nodorum blotch

The fungus Parastagonospora nodorum is the causal agent of septoria nodorum leaf blotch (SNB) and glume blotch which are common in many wheat growing regions in the world. The disease is complex and could be explained by multiple interactions between necrotrophic effectors secreted by the pathogen and matching susceptibility genes in wheat. An Australian P. nodorum population was clustered into five groups with contrasting properties. This study was set to identify their pathogenicity profiles using a diverse wheat panel of 134 accessions which are insensitive to SnToxA and SnTox1 in both in vitro and in vivo conditions. SNB seedling resistance/susceptibility to five representative isolates from the five clusters, responses to crude culture-filtrates (CFs) of three isolates and sensitivity to SnTox3 semi-purified effector together with 11,455 SNP markers have been used for linkage disequilibrium (LD) and association analyses. While quantitative trait loci (QTL) on 1D, 2A, 2B, 4B, 5B, 6A, 6B, 7A, 7D chromosomes were consistently detected across isolates and conditions, distinct patterns and isolate specific QTL were also observed among these isolates. In this study, SnTox3–Snn3-B1 interaction for the first time in Australia and SnTox3–Snn3-D1 interaction for the first time in bread wheat were found active using wild-type isolates. These findings could be due to new SnTox3 haplotype/isoform and exotic CIMMYT/ICARDA and Vavilov germplasm used, respectively. This study could provide useful information for dissecting novel and different SNB disease components, helping to prioritise research targets and contributing valuable information on genetic loci/markers for marker-assisted selection in SNB resistance wheat breeding programme

Impairment of Cd4+ T Cell Responses during Chronic Virus Infection Prevents Neutralizing Antibody Responses against Virus Escape Mutants

We have shown previously that neutralizing antibodies (nAbs) are important contributors to the long-term immune control of lymphocytic choriomeningitis virus infection, particularly if cytotoxic T cell responses are low or absent. Nevertheless, virus escape from the nAb response due to mutations within the surface glycoprotein gene may subsequently allow the virus to persist. Here we show that most of the antibody-escape viral mutants retain their immunogenicity. We present evidence that the failure of the infected host to mount effective humoral responses against emerging neutralization-escape mutants correlates with the rapid loss of CD4+ T cell responsiveness during the establishment of viral persistence. Similar mechanisms may contribute to the persistence of some human pathogens such as hepatitis B and C viruses, and human immunodeficiency virus

Unmet Needs in Patients With Heart Failure: The Importance of Palliative Care in a Heart Failure Clinic.

Background There are increasing calls to establish heart failure (HF) clinics due to their effectiveness in the interdisciplinary management of people living with HF. However, although a recommendation exists for palliative care (PC) providers to be part of the interdisciplinary team, few of the established HF clinics include them in their teams. Therefore, in this qualitative study, we aimed to understand the unmet PC needs of patients with HF attending an already established HF clinic. Methods Secondary qualitative analysis of structured interviews undertaken within a larger study to validate the German version of the Needs Assessment Tool: Progressive Disease-Heart Failure (NAT: PD-HF). The NAT: PD-HF is a tool that aims to assess unmet needs in patients with HF. The interviews took place between January and March 2020 with patients from the ambulatory HF Clinic of a University Hospital in Switzerland. For this analysis, we transcribed and thematically analyzed the longest and most content-rich interviews until we reached data saturation at 31 participants. The interviews lasted 31 min on average (24-48 min). Results Participants (n = 31) had a median age of 64 years (IQR 56-77), the majority had reduced ejection fraction, were men, and were classified as having a New York Heart Association functional class II. Participants were in general satisfied with the treatment and information received at the HF clinic. However, they reported several unmet needs. We therefore identified three ambivalences as main themes: (I) "feeling well-informed but missing essential discussions", (II) "although feeling mostly satisfied with the care, remaining with unmet care needs", and (III) "fearing a referral to palliative care but acknowledging its importance". Conclusion Although patients who are receiving multidisciplinary management in ambulatory HF clinics are generally satisfied with the care received, they remain with unmet needs. These unmet needs, such as the need for advance care planning or the need for timely and tactful end-of-life discussions, can be fulfilled by PC providers. Including personnel trained in PC as part of the multidisciplinary team could help to address patients' needs, thus improving the quality of care and the quality of life of people living with HF

SGLT2 inhibitor therapy for transthyretin amyloid cardiomyopathy: early tolerance and clinical response to dapagliflozin.

AIMS Sodium-glucose cotransporter 2 inhibitors (SGLT2i) improve clinical outcomes in heart failure patients with reduced and preserved left ventricular ejection fraction (LVEF), but have not yet been investigated in transthyretin amyloid cardiomyopathy (ATTR-CM). This study aimed to evaluate tolerability, clinical outcomes, and changes in NT-proBNP levels and glomerular filtration rate (GFR) in ATTR-CM patients treated with dapagliflozin. METHODS AND RESULTS Patients with stable, tafamidis-treated ATTR-CM were retrospectively evaluated at the initiation of dapagliflozin and 3 months thereafter. Tafamidis-treated ATTR-CM patients without SGLT2i served as a reference cohort. Overall, SLGT2i therapy was initiated in 34 patients. Seventeen patients with stable disease on tafamidis, who were subsequently started on dapagliflozin, were included in the analysis. Patients selected for SGLT2i presented with signs of advanced disease, evidenced by higher Gillmore disease stage (stage ≥2: 53% vs. 27.5%; P = 0.041), baseline median NT-proBNP [median (IQR) 2668 pg/mL (1314-3451) vs. 1424 (810-2059); P = 0.038] and loop diuretic demand (76.5% vs. 45% of patients; P = 0.044), and lower LVEF (46.6 ± 12.9 vs. 53.7 ± 8.7%; P = 0.019) and GFR (51.8 ± 16.5 vs. 68.5 ± 18.6 mL/min; P = 0.037) compared with the reference cohort. At 3-month follow-up, a numerical decrease in NT-proBNP levels was observed in 13/17 (76.5%) patients in the dapagliflozin (-190 pg/mL, IQR: -1,028-71, P = 0.557) and 27/40 (67.5%) of patients in the control cohort (-115 pg/mL, IQR: -357-105, P = 0.551). Other disease parameters remained stable and no adverse events occurred. CONCLUSIONS In tafamidis-treated ATTR-CM patients, initiation of dapagliflozin was well tolerated. The efficacy of SGLT2i therapy in patients with ATTR-CM needs to be studied in randomized controlled trials

Impact of type 2 diabetes on life expectancy and role of kidney disease among inpatients with heart failure in Switzerland: an ambispective cohort study.

BACKGROUND Type 2 diabetes (T2D) is expected to worsen the prognosis of inpatients with heart failure (HF) but the evidence from observational studies is inconsistent. We aimed to compare mortality outcomes and life expectancy among inpatients with HF with or without T2D and explored whether chronic kidney disease (CKD) influenced these associations. METHODS We collected hospital and civil registry records of consecutive inpatients from a tertiary hospital in Switzerland with a diagnosis of HF from the year 2015 to 2019. We evaluated the association of T2D with mortality risk using Cox regression and adjusted for confounders. RESULTS Our final cohort consisted of 10,532 patients with HF of whom 27% had T2D. The median age (interquartile range [IQR]) was 75 [68 to 82] and 78 [68 to 86] for the diabetes and non-diabetes groups, respectively. Over a median follow-up [IQR] of 4.5 years [3.3 to 5.6], 5,347 (51%) of patients died. T2D patients had higher risk of all-cause mortality (hazard ratio [HR] 1.21, 95% confidence interval [CI] 1.14 to 1.29). Compared to control (i.e. no T2D nor CKD), average life expectancy (95% CI) among T2D patients, CKD, or both was shorter by 5.4 months (95% CI 1.1 to 9.7), 9.0 months (95% CI 8.4 to 9.6), or 14.8 months (95% CI 12.4 to 17.2), respectively. No difference by sex or ejection fraction category was observed. CONCLUSIONS T2D is associated with a significantly higher risk of all-cause mortality and shorter life expectancy compared to those without among middle-aged and elderly inpatients with HF; presence of CKD may further increase these risks

Interventional treatment of mitral valve regurgitation: an alternative to surgery?

Mitral regurgitation is a highly prevalent condition among elderly patients, affecting almost 10% of the general population aged 75 and older. Left untreated, severe mitral regurgitation results in high mortality and frequent hospitalisation for treatment of heart failure. Surgical treatment remains the first-line therapy for symptomatic, severe mitral regurgitation , especially for patients presenting with a primary aetiology. However, a high proportion of patients with mitral regurgitation are turned down for open-heart surgery, mainly due to advanced age, diminished left ventricular function and comorbidities. Thus, percutaneous treatment options have been recently developed as an alternative. In this article, we will review transcatheter interventional techniques at the level of the mitral valve, including implantation technique, indications and clinical results