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Abstract The SP/KLF family of transcription factors harbour three C-terminal C2H2 zinc fingers interspersed by two linkers which confers DNA-binding to a 9-10 bp motif. Mutations in KLF1, the founding member of the family, are common. Missense mutations in linker two result in a mild phenotype. However, when co-inherited with loss-of-function mutations, they result in severe non-spherocytic hemolytic anemia. We generate a mouse model of this disease by crossing Klf1 +/− mice with Klf1 H350R/+ mice that harbour a missense mutation in linker-2. Klf1 H350R/− mice exhibit severe hemolysis without thalassemia. RNA-seq demonstrate loss of expression of genes encoding transmembrane and cytoskeletal proteins, but not globins. ChIP-seq show no change in DNA-binding specificity, but a global reduction in affinity, which is confirmed using recombinant proteins and in vitro binding assays. This study provides new insights into how linker mutations in zinc finger transcription factors result in different phenotypes to those caused by loss-of-function mutations.

Impaired respiratory function secondary to acute or chronic respiratory disease poses a significant clinical and healthcare burden. Intrapulmonary percussive ventilation (IPV) is used in various clinical settings to treat excessive airway secretions, pulmonary atelectasis, and impaired gas exchange. Despite IPV’s wide use, there is a lack of clinical guidance on IPV application which may lead to inconsistency in clinical practice. This scoping review aimed to summarise the clinical application methods and dosage of IPV used by clinicians and researchers to provide guidance. A two-staged systematic search was conducted to retrieve studies that used IPV in inpatient and outpatient settings. MEDLINE, EMBASE, CINAHL, Scopus, and Google scholar were searched from January 1979 till 2022. Studies with patients aged ≥16 years and published in any language were included. Two reviewers independently screened the title and abstract, reviewed full text articles, and extracted data. Search yielded 514 studies. After removing duplicates and irrelevant studies, 25 studies with 905 participants met the inclusion criteria. This is the first scoping review to summarise IPV application methods and dosages from the available studies in intensive care unit (ICU), acute inpatient (non-ICU), and outpatient settings. Some variations in clinical applications and prescribed dosages of IPV were noted. Despite variations, common trends in clinical application and prescription of IPV dosages were observed and summarised to assist clinicians with IPV intervention. Although an evidence-based clinical guideline could not be provided, this review provides detailed information on IPV application and dosages in order to provide clinical guidance and lays a foundation towards developing a clinical practice guideline in the future.

Abstract Background Right ventricular (RV) function is tightly coupled to afterload, yet echocardiographic indices of RV function are frequently assessed in isolation. Normalizing RV function for afterload (RV-PA coupling) using a simplified ratio of tricuspid annular plane systolic excursion (TAPSE)/ tricuspid regurgitant velocity (TRV) could help to identify RV decompensation and improve risk stratification in critically ill patients. This is the first study to explore the distribution of TAPSE/TRV ratio and its prognostic relevance in a large general critical care cohort. Methods We undertook retrospective analysis of echocardiographic, clinical, and mortality data of intensive care unit (ICU) patients between January 2012 and May 2017. A total of 1077 patients were included and stratified into tertile groups based on TAPSE/TRV ratio: low (

Key messages Increased pulmonary vascular resistance and RA-PA uncoupling is present in a significant subset of patients with sepsis, and measurement with TTE is feasible. Measurement of PAAT and RV-PA uncoupling could aid in improving definitions of right ventricular dysfunction. The therapeutic and prognostic role of these parameters in sepsis remains unknown, and further prospective studies are warranted.

This project addresses the issue of detecting intruders from hiding behind privacy-protecting anonymity networks. The freely available Tor and the SOCKS proxy services have been popular tools that provide circuit-based anonymous connections to network users. However, recent security breaches reveal that SSH and HTTPS have been used to launch attacks by malicious users by taking advantage of these services to hide their identities. This paper investigates strategies to detect SSH and HTTPS connections via the circuit-based anonymity networks, to help servers and websites defend against anonymous intruders. We evaluate our approaches with SSH and HTTPS connections and show that they achieve high performance for both applications. Our detection algorithms are based on the extra latency delays introduced by the presence of the anonymity networks. Since the latency disparity is sensitive to the location of the anonymity network, our algorithms must be evaluated in the most challenging scenarios. The detection rates for all four combinations of SSH/HTTPS applications via Tor/SOCKS networks were very high, with a low false-positive rate. To demonstrate the robustness of our approach in the Tor case, we tested our method in multiple Tor circuit node selection strategies. The approach can be applied to other applications meeting the same criteria.

Identification of host factors associated with severe influenza infection could provide insights into treatment options. Here, the authors provide transcriptomic analyses of blood from >100 influenza infected patients and show that changes in circulating neutrophils are associated with severe influenza infection.

Abstract Background Diagnosis of significant coronary artery disease (CAD) and acute coronary artery occlusion in ICU can be difficult, and an inappropriate intervention is potentially harmful. Myocardial contrast perfusion echo (MCPE) examines ultrasound contrast intensity replenishment curves in individual myocardial segments measuring peak contrast intensity and slope of return as an index of myocardial blood flow (units = intensity of ultrasound per second [dB/s]). MCPE could possibly serve as a triage tool to invasive angiography by estimating blood flow in the myocardium. We sought to assess feasibility in the critically ill and if MCPE could add incremental value to the clinical acumen in predicting significant CAD. Methods This is a single-centre, prospective, observational study. Inclusion criteria were as follows: adult ICU patients with troponin I > 50 ng/L and cardiology referral being made for consideration of inpatient angiography. Exclusion criteria were as follows: poor echo windows (2 patients), known ischaemic heart disease, and contrast contraindications. Seven cardiologists and 6 intensivists blinded to outcome assessed medical history, ECG, troponin, and 2D echo images to estimate likelihood of significant CAD needing intervention (clinical acumen). Clinical acumen, quantitative MCPE, and subjective (visual) MCPE were assessed to predict significant CAD. Results Forty patients underwent MCPE analysis, 6 (15%) had significant CAD, and median 11 of 16 segments (IQR 8–13) could be imaged (68.8% [IQR 50–81]). No adverse events occurred. A significant difference was found in overall MCPE blood flow estimation between those diagnosed with significant CAD and those without (3.3 vs 2.4 dB/s, p = 0.050). A MCPE value of 2.8 dB/s had 67% sensitivity and 88% specificity in detecting significant CAD. Clinical acumen showed no association in prediction of CAD (OR 0.6, p = 0.09); however, if quantitative or visual MCPE analysis was included, a significant association occurred (OR 17.1, p = 0.01; OR 23.0, p = 0.01 respectively). Conclusions MCPE is feasible in the critically ill and shows better association with predicting significant CAD vs clinical acumen alone. MCPE adds incremental value to initial assessment of the presence of significant CAD which may help guide those who require urgent angiography.

Abstract Background Mutations in the transcription factor, KLF1, are common within certain populations of the world. Heterozygous missense mutations in KLF1 mostly lead to benign phenotypes, but a heterozygous mutation in a DNA-binding residue (E325K in human) results in severe Congenital Dyserythropoietic Anemia type IV (CDA IV); i.e. an autosomal-dominant disorder characterized by neonatal hemolysis. Results To investigate the biochemical and genetic mechanism of CDA IV, we generated murine erythroid cell lines that harbor tamoxifen-inducible (ER™) versions of wild type and mutant KLF1 on a Klf1 −/− genetic background. Nuclear translocation of wild type KLF1 results in terminal erythroid differentiation, whereas mutant KLF1 results in hemolysis without differentiation. The E to K variant binds poorly to the canonical 9 bp recognition motif (NGG-GYG-KGG) genome-wide but binds at high affinity to a corrupted motif (NGG-GRG-KGG). We confirmed altered DNA-binding specificity by quantitative in vitro binding assays of recombinant zinc-finger domains. Our results are consistent with previously reported structural data of KLF-DNA interactions. We employed 4sU-RNA-seq to show that a corrupted transcriptome is a direct consequence of aberrant DNA binding. Conclusions Since all KLF/SP family proteins bind DNA in an identical fashion, these results are likely to be generally applicable to mutations in all family members. Importantly, they explain how certain mutations in the DNA-binding domain of transcription factors can generate neomorphic functions that result in autosomal dominant disease.

Abstract Background Right ventricle (RV) size and function assessment by echocardiography (echo) is a standard tool in the ICU. Frequently subjective assessment is performed, and guidelines suggest its utility in adequately trained clinicians. We aimed to compare subjective (visual) assessment of RV size and function by ICU physicians, with advanced qualifications in echocardiography, vs objective measurements. Methods ICU specialists with a qualification in advanced echocardiography reviewed 2D echo clips from critically ill patients on mechanical ventilation with PaO2:FiO2

BackgroundPulmonary complications such as pneumonia, pulmonary atelectasis, and subsequent respiratory failure leading to ventilatory support are a common occurrence in critically ill patients. Intrapulmonary percussive ventilation (IPV) is used to improve gas exchange and promote airway clearance in these patients. The current evidence regarding the effectiveness of intrapulmonary percussive ventilation in critical care settings remains unclear. This systematic review aims to summarise the evidence of the effectiveness of intrapulmonary percussive ventilation on intensive care unit length of stay (ICU-LOS) and respiratory outcomes in critically ill patients.Research questionIn critically ill patients, is intrapulmonary percussive ventilation effective in improving respiratory outcomes and reducing intensive care unit length of stay.MethodsA systematic search of intrapulmonary percussive ventilation in intensive care unit (ICU) was performed on five databases from 1979 to 2021. Studies were considered for inclusion if they evaluated the effectiveness of IPV in patients aged ≥16 years receiving invasive or non-invasive ventilation or breathing spontaneously in critical care or high dependency units. Study titles and abstracts were screened, followed by data extraction by a full-text review. Due to a small number of studies and observed heterogeneities in the study methodology and patient population, a meta-analysis could not be included in this review. Outcomes of interest were summarised narratively.ResultsOut of 306 identified abstracts, seven studies (630 patients) met the eligibility criteria. Results of the included studies provide weak evidence to support the effectiveness of intrapulmonary percussive ventilation in reducing ICU-LOS, improving gas exchange, and reducing respiratory rate.InterpretationBased on the findings of this review, the evidence to support the role of IPV in reducing ICU-LOS, improving gas exchange, and reducing respiratory rate is weak. The therapeutic value of IPV in airway clearance, preventing pneumonia, and treating pulmonary atelectasis requires further investigation.

Abstract Background Transthoracic 3D cardiac analysis is enticing in its potential simplicity and wealth of data available. It has been suggested to be accurate vs magnetic resonance imaging in relatively stable patients, but feasibility and agreement with conventional echocardiographic assessment of stroke volume (SV) have not been thoroughly assessed in critically ill patients, who are traditionally harder to image. The objectives of this study were to compare 3D transthoracic volumetric analysis vs Doppler assessment of SV (which is suggested to be accurate in the critically ill) and Simpson’s biplane assessment in a cohort typical of the intensive care unit (ICU), where accurate assessment is important: mechanically ventilated patients with a significant ventilation/perfusion (V/Q) mismatch. We hypothesised that it would be feasible but might lack agreement. Methods Patients were imaged within 24 hours of admission. Inclusion criteria were adult patients, V/Q mismatch present (defined as a ratio of arterial oxygen partial pressure to fractional inspired oxygen

Gemtuzumab ozogamicin (GO) is an anti-CD33 antibody-drug conjugate for the treatment of acute myeloid leukemia (AML). Although GO shows a narrow therapeutic window in early clinical studies, recent reports detailing a modified dosing regimen of GO can be safely combined with induction chemotherapy, and the combination provides significant survival benefits in AML patients. Here we tested whether the survival benefits seen with the combination arise from the enhanced reduction of chemoresidual disease and leukemic initiating cells (LICs). Herein, we use cell line and patient-derived xenograft (PDX) AML models to evaluate the combination of GO with daunorubicin and cytarabine (DA) induction chemotherapy on AML blast growth and animal survival. DA chemotherapy and GO as separate treatments reduced AML burden but left significant chemoresidual disease in multiple AML models. The combination of GO and DA chemotherapy eliminated nearly all AML burden and extended overall survival. In two small subsets of AML models, chemoresidual disease following DA chemotherapy displayed hallmark markers of leukemic LICs (CLL1 and CD34). In vivo, the two chemoresistant subpopulations (CLL1+/CD117− and CD34+/CD38+) showed higher ability to self-renewal than their counterpart subpopulations, respectively. CD33 was coexpressed in these functional LIC subpopulations. We demonstrate that the GO and DA induction chemotherapy combination more effectively eliminates LICs in AML PDX models than either single agent alone. These data suggest that the survival benefit seen by the combination of GO and induction chemotherapy, nonclinically and clinically, may be attributed to the enhanced reduction of LICs.

Abstract Background Left ventricular diastolic dysfunction is suggested to be associated with higher mortality in severe sepsis and septic shock, yet the methods of diagnosis described in the literature are often inconsistent. The recently published 2016 American Society of Echocardiography and European Association of Cardiovascular Imaging (ASE/EACVI) guidelines offer the opportunity to apply a simple pragmatic diagnostic algorithm for the detection of diastolic dysfunction; however, it has not been tested in this cohort. Aims We sought to assess the applicability in septic patients of recently published 2016 ASE/EACVI guidelines on diastolic dysfunction compared with the 2009 ASE guidelines. Our hypothesis was that there would be poor agreement in classifying patients. Methods Prospective observational study includes patients identified as having severe sepsis and septic shock. Patients underwent transthoracic echocardiography on day 1 and day 3 of their ICU admission. Patients with normal and abnormal (ejection fraction

Abstract Background Left ventricular diastolic dysfunction is associated with mortality outcomes in severe sepsis and septic shock. There are ongoing issues with diagnosing diastolic dysfunction in this cohort, partly owing to the poor applicability of traditional parameters in the hyperdynamic circulation. In this feasibility study, we sought to assess the utility of a novel parameter (septal e′/s′) to identify diastolic dysfunction in patients with severe sepsis and septic shock who had normal systolic function against the 2016 American Society Echocardiography and European Association of Cardiovascular Imaging (ASE/EACI) guidelines on diastolic dysfunction. Methods In this prospective observational pilot study, patients identified as having severe sepsis and septic shock underwent transthoracic echocardiography on day 1 and day 3 of their intensive care unit admission. In patients with normal systolic function, septal e′/s′ was calculated using the peak modal velocity of the s′ compared with the e′ from the septal annulus tissue Doppler imaging and compared with their diastolic grade according to the 2016 ASE/EACI guidelines on diastolic dysfunction. Results On day 1 of admission, 44 of 62 patients with severe sepsis and septic shock had normal systolic function. There was a strong association of those with diastolic dysfunction having a reduced septal e′/s′ compared with patients with normal diastolic function (AUC 0.91). A similar relationship was seen with patients who had indeterminate diastolic dysfunction. On day 3, 37 patients had normal systolic function. Again, there was a strong association of those with diastolic dysfunction and a reduced septal e′/s′ (AUC 0.95). Conclusions A reduction in septal e′/s′ may indicate diastolic dysfunction in patients with severe sepsis and septic shock who have normal systolic function. As opposed to limited traditional measures of diastolic dysfunction, it is applicable in those with hyperdynamic systolic function.

BackgroundIncreased postoperative cardiac troponin (cTn) independently predicts short-term mortality. Previous studies suggest that preoperative cTn also predicts major adverse cardiovascular events (MACE) and mortality after noncardiac surgery. The value of preoperative and perioperative changes in cTn as a prognostic tool for adverse outcomes has been sparsely investigated.Methods and findingsA systematic review and meta-analysis of the prognostic value of cTns for adverse outcome was conducted. Adverse outcome was defined as short-term (in-hospital or 30 days) MACE and/or all-cause mortality, in adult patients undergoing noncardiac surgery. The study protocol (CRD42018094773) was registered with an international prospective register of systematic reviews (PROSPERO). Preoperative cTn was a predictor of short- (OR 4.3, 95% CI 2.9-6.5, pConclusionOur study indicates that although preoperative cTn and perioperative change in cTn might be valuable predictors of MACE and/or all-cause mortality in adult noncardiac surgical patients, its overall prognostic performance remains uncertain. Future large, representative, high-quality studies are needed to establish the potential role of cTns in perioperative cardiac risk stratification.