Libraries of protein-encoding sequences can be generated by identification of open reading frames (ORFs) from a genome of choice that are then assembled into collections of plasmids termed ORFeome ...libraries. These represent powerful resources to facilitate functional genomic characterization of genes and their encoded products. Here, we report the generation of an ORFeome for Zymoseptoria tritici, which causes the most serious disease of wheat in temperate regions of the world. We screened the genome of strain IP0323 for high confidence gene models, identifying 4075 candidates from 10,933 predicted genes. These were amplified from genomic DNA, cloned into the Gateway® Entry Vector pDONR207, and sequenced, providing a total of 3022 quality-controlled plasmids. The ORFeome includes genes predicted to encode effectors (n = 410) and secondary metabolite biosynthetic proteins (n = 171), in addition to genes residing at dispensable chromosomes (n= 122), or those that are preferentially expressed during plant infection (n = 527). The ORFeome plasmid library is compatible with our previously developed suite of Gateway® Destination vectors, which have various combinations of promoters, selection markers, and epitope tags. The Z. tritici ORFeome constitutes a powerful resource for functional genomics, and offers unparalleled opportunities to understand the biology of Z. tritici.
Host-pathogen interactions have critical implications for the establishment of disease and for determining adaptive immune responses of the host. This study has conducted global Aspergillus fumigatus ...transcriptional analyses throughout the initiation of murine infection using a wild-type and an attenuated ΔlaeA isolate. A novel data analysis protocol was applied from which three time-series datasets were generated between 4, 8 and 14 hours post infection. This approach identified distinct temporal gene expression profiles during disease initiation whereby numerous secreted enzymes, including proteases and antigens, were upregulated between 4 and 8 hours, while a striking upregulation of genes in secondary metabolism clusters and subtelomeric loci was observed between 8 and 14 hours. In order to test the role of several upregulated secondary metabolite genes on host-pathogen interactions and virulence, two isolates mutated in non-ribosomal peptide synthetase encoding genes (ΔftmA, Δpes3), and mutants of a hybrid non-ribosomal-polyketide synthase (ΔpsoA) and a putative secondary metabolite transcription factor (ΔregA) were analysed in murine models of infection. These analyses suggest fumitremorgin C, pseurotin A and, putatively, a pigment augment A. fumigatus virulence. In contrast, the pes3 gene product favours pathogen clearance, possibly by facilitating recognition of host innate immunity. There was no evidence to support the view that defective secondary metabolism is causative of the attenuated virulence phenotype observed for the LaeA mutant. Alternative hypotheses regarding the attenuation of ΔlaeA include upregulation and downregulation of Th1 and Th2 associated antigens respectively, and deficiencies in plasma membrane transport relative to wild-type isolates. A hypothesised deficiency in maintenance of genome stability, due to LINE-1 mobilisation in the ΔlaeA isolate was tested, and subsequently discredited by a discovered lack of isogenicity among the tested strains. Work presented in this thesis also assesses the use of a mass-spectrometric method for detection of epitope tagged A. fumigatus protein in the host.
Host-pathogen interactions have critical implications for the establishment of disease and for determining adaptive immune responses of the host. This study has conducted global Aspergillus fumigatus ...transcriptional analyses throughout the initiation of murine infection using a wild-type and an attenuated ΔlaeA isolate. A novel data analysis protocol was applied from which three time-series datasets were generated between 4, 8 and 14 hours post infection. This approach identified distinct temporal gene expression profiles during disease initiation whereby numerous secreted enzymes, including proteases and antigens, were upregulated between 4 and 8 hours, while a striking upregulation of genes in secondary metabolism clusters and subtelomeric loci was observed between 8 and 14 hours. In order to test the role of several upregulated secondary metabolite genes on host-pathogen interactions and virulence, two isolates mutated in non-ribosomal peptide synthetase encoding genes (ΔftmA, Δpes3), and mutants of a hybrid non-ribosomal-polyketide synthase (ΔpsoA) and a putative secondary metabolite transcription factor (ΔregA) were analysed in murine models of infection. These analyses suggest fumitremorgin C, pseurotin A and, putatively, a pigment augment A. fumigatus virulence. In contrast, the pes3 gene product favours pathogen clearance, possibly by facilitating recognition of host innate immunity. There was no evidence to support the view that defective secondary metabolism is causative of the attenuated virulence phenotype observed for the LaeA mutant. Alternative hypotheses regarding the attenuation of ΔlaeA include upregulation and downregulation of Th1 and Th2 associated antigens respectively, and deficiencies in plasma membrane transport relative to wild-type isolates. A hypothesised deficiency in maintenance of genome stability, due to LINE-1 mobilisation in the ΔlaeA isolate was tested, and subsequently discredited by a discovered lack of isogenicity among the tested strains. Work presented in this thesis also assesses the use of a mass-spectrometric method for detection of epitope tagged A. fumigatus protein in the host.
Triage of patients is critical to patient safety, yet no clear information exists as to the utility of initial vital signs in identifying critically ill older emergency department (ED) patients. The ...objective of this study is to evaluate a set of initial vital sign thresholds as predictors of severe illness and injury among older adults presenting to the ED.
We reviewed all visits by patients aged 75 and older seen during 2007 at an academic ED serving a large community of older adults. Patients' charts were abstracted for demographic and clinical information including vital signs, via automated electronic methods. We used bivariate analysis to investigate the relationship between vital sign abnormalities and severe illness or injury, defined as intensive care unit (ICU) admission or ED death. In addition, we calculated likelihood ratios for normal and abnormal vital signs in predicting severe illness or injury.
4,873 visits by patients aged 75 and above were made to the ED during 2007, and of these 3,848 had a complete set of triage vital signs. For these elderly patients, the sensitivity and specificity of an abnormal vital sign taken at triage for predicting death or admission to an ICU were 73% (66,81) and 50% (48,52) respectively (positive likelihood ratio 1.47 (1.30,1.60); negative likelihood ratio 0.54 (0.30,0.60).
Emergency provider assessment and triage scores that rely primarily on initial vital signs are likely to miss a substantial portion of critically ill older adults.
Influenza causes substantial morbidity and mortality despite available treatments. Anecdotal reports suggest that plasma with high antibody titres to influenza might be of benefit in the treatment of ...severe influenza.
In this randomised, open-label, multicentre, phase 2 trial, 29 academic medical centres in the USA assessed the safety and efficacy of anti-influenza plasma with haemagglutination inhibition antibody titres of 1:80 or more to the infecting strain. Hospitalised children and adults (including pregnant women) with severe influenza A or B (defined as the presence of hypoxia or tachypnoea) were randomly assigned to receive either two units (or paediatric equivalent) of anti-influenza plasma plus standard care, versus standard care alone, and were followed up for 28 days. The primary endpoint was time to normalisation of patients' respiratory status (respiratory rate of ≤20 breaths per min for adults or age-defined thresholds of 20-38 breaths per min for children) and a room air oxygen saturation of 93% or more. This study is registered with ClinicalTrials.gov, number NCT01052480.
Between Jan 13, 2011, and March 2, 2015, 113 participants were screened for eligibility and 98 were randomly assigned from 20 out of 29 participating sites. Of the participants with confirmed influenza (by PCR), 28 (67%) of 42 in the plasma plus standard care group normalised their respiratory status by day 28 compared with 24 (53%) of 45 participants on standard care alone (p=0·069). The hazard ratio (HR) comparing plasma plus standard care with standard care alone was 1·71 (95% CI 0·96-3·06). Six participants died, one (2%) from the plasma plus standard care group and five (10%) from the standard care group (HR 0·19 95% CI 0·02-1·65, p=0·093). Participants in the plasma plus standard care group had non-significant reductions in days in hospital (median 6 days IQR 4-16 vs 11 days 5-25, p=0·13) and days on mechanical ventilation (median 0 days IQR 0-6 vs 3 days 0-14, p=0·14). Fewer plasma plus standard care participants had serious adverse events compared with standard care alone recipients (nine 20% of 46 vs 20 38% of 52, p=0·041), the most frequent of which were acute respiratory distress syndrome (one 2% vs two 4% patients) and stroke (one 2% vs two 4% patients).
Although there was no significant effect of plasma treatment on the primary endpoint, the treatment seemed safe and well tolerated. A phase 3 randomised trial is now underway to further assess this intervention.
National Institute of Allergy and Infectious Diseases, US National Institutes of Health.
Primary age-related tauopathy (PART) is a neurodegenerative pathology with features distinct from but also overlapping with Alzheimer disease (AD). While both exhibit Alzheimer-type temporal lobe ...neurofibrillary degeneration alongside amnestic cognitive impairment, PART develops independently of amyloid-β (Aβ) plaques. The pathogenesis of PART is not known, but evidence suggests an association with genes that promote tau pathology and others that protect from Aβ toxicity. Here, we performed a genetic association study in an autopsy cohort of individuals with PART (
n
= 647) using Braak neurofibrillary tangle stage as a quantitative trait. We found some significant associations with candidate loci associated with AD (
SLC24A4
,
MS4A6A
,
HS3ST1
) and progressive supranuclear palsy (
MAPT
and
EIF2AK3
). Genome-wide association analysis revealed a novel significant association with a single nucleotide polymorphism on chromosome 4 (rs56405341) in a locus containing three genes, including
JADE1
which was significantly upregulated in tangle-bearing neurons by single-soma RNA-seq. Immunohistochemical studies using antisera targeting JADE1 protein revealed localization within tau aggregates in autopsy brains with four microtubule-binding domain repeats (4R) isoforms and mixed 3R/4R, but not with 3R exclusively. Co-immunoprecipitation in post-mortem human PART brain tissue revealed a specific binding of JADE1 protein to four repeat tau lacking N-terminal inserts (0N4R). Finally, knockdown of the
Drosophila
JADE1 homolog
rhinoceros
(
rno
) enhanced tau-induced toxicity and apoptosis in vivo in a humanized 0N4R mutant tau knock-in model, as quantified by rough eye phenotype and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) in the fly brain. Together, these findings indicate that PART has a genetic architecture that partially overlaps with AD and other tauopathies and suggests a novel role for JADE1 as a modifier of neurofibrillary degeneration.
OBJECTIVES:To compare hemoglobin concentration (Hb), RBC use, and patient outcomes when restrictive or liberal blood transfusion strategies are used to treat anemic (Hb ≤ 90 g/L) critically ill ...patients of age ≥ 55 years requiring ≥ 4 days of mechanical ventilation in ICU.
DESIGN:Parallel-group randomized multicenter pilot trial.
SETTING:Six ICUs in the United Kingdom participated between August 2009 and December 2010.
PATIENTS:One hundred patients (51 restrictive and 49 liberal groups).
INTERVENTIONS:Patients were randomized to a restrictive (Hb trigger, 70 g/L; target, 71–90 g/L) or liberal (90 g/L; target, 91–110 g/L) transfusion strategy for 14 days or the remainder of ICU stay, whichever was longest.
MEASUREMENTS AND MAIN RESULTS:Baseline comorbidity rates and illness severity were high, notably for ischemic heart disease (32%). The Hb difference among groups was 13.8 g/L (95% CI, 11.5–16.0 g/L); p < 0.0001); mean Hb during intervention was 81.9 (SD, 5.1) versus 95.7 (6.3) g/L; 21.6% fewer patients in the restrictive group were transfused postrandomization (p < 0.001) and received a median 1 (95% CI, 1–2; p = 0.002) fewer RBC units. Protocol compliance was high. No major differences in organ dysfunction, duration of ventilation, infections, or cardiovascular complications were observed during intensive care and hospital follow-up. Mortality at 180 days postrandomization trended toward higher rates in the liberal group (55%) than in the restrictive group (37%); relative risk was 0.68 (95% CI, 0.44–1.05; p = 0.073). This trend remained in a survival model adjusted for age, gender, ischemic heart disease, Acute Physiology and Chronic Health Evaluation II score, and total non-neurologic Sequential Organ Failure Assessment score at baseline (hazard ratio, 0.54 95% CI, 0.28–1.03; p = 0.061).
CONCLUSIONS:A large trial of transfusion strategies in older mechanically ventilated patients is feasible. This pilot trial found a nonsignificant trend toward lower mortality with restrictive transfusion practice.
AbstractObjectiveTo determine if postnatal transfer or birth in a non-tertiary hospital is associated with adverse outcomes.DesignObservational cohort study with propensity score ...matching.SettingNational health service neonatal care in England; population data held in the National Neonatal Research Database.ParticipantsExtremely preterm infants born at less than 28 gestational weeks between 2008 and 2015 (n=17 577) grouped based on birth hospital and transfer within 48 hours of birth: upward transfer (non-tertiary to tertiary hospital, n=2158), non-tertiary care (born in non-tertiary hospital; not transferred, n=2668), and controls (born in tertiary hospital; not transferred, n=10 866). Infants were matched on propensity scores and predefined background variables to form subgroups with near identical distributions of confounders. Infants transferred between tertiary hospitals (horizontal transfer) were separately matched to controls in a 1:5 ratio.Main outcome measuresDeath, severe brain injury, and survival without severe brain injury.Results2181 infants, 727 from each group (upward transfer, non-tertiary care, and control) were well matched. Compared with controls, infants in the upward transfer group had no significant difference in the odds of death before discharge (odds ratio 1.22, 95% confidence interval 0.92 to 1.61) but significantly higher odds of severe brain injury (2.32, 1.78 to 3.06; number needed to treat (NNT) 8) and significantly lower odds of survival without severe brain injury (0.60, 0.47 to 0.76; NNT 9). Compared with controls, infants in the non-tertiary care group had significantly higher odds of death (1.34, 1.02 to 1.77; NNT 20) but no significant difference in the odds of severe brain injury (0.95, 0.70 to 1.30) or survival without severe brain injury (0.82, 0.64 to 1.05). Compared with infants in the upward transfer group, infants in the non-tertiary care group had no significant difference in death before discharge (1.10, 0.84 to 1.44) but significantly lower odds of severe brain injury (0.41, 0.31 to 0.53; NNT 8) and significantly higher odds of survival without severe brain injury (1.37, 1.09 to 1.73; NNT 14). No significant differences were found in outcomes between the horizontal transfer group (n=305) and controls (n=1525).ConclusionsIn extremely preterm infants, birth in a non-tertiary hospital and transfer within 48 hours are associated with poor outcomes when compared with birth in a tertiary setting. We recommend perinatal services promote pathways that facilitate delivery of extremely preterm infants in tertiary hospitals in preference to postnatal transfer.
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