Structured Mobilization for Critically Ill Patients: A Pragmatic Cluster-randomized Trial.

Rationale: Small trials and professional recommendations support mobilization interventions to improve recovery among critically ill patients, but their real-world effectiveness is unknown. Objective: To evaluate a low-cost, multifaceted mobilization intervention. Methods: We conducted a stepped-wedge cluster-randomized trial across 12 ICUs with diverse case mixes. The primary and secondary samples included patients mechanically ventilated for ⩾48 hours who were ambulatory before admission, and all patients with ICU stays ⩾48 hours, respectively. The mobilization intervention included 1) designation and posting of daily mobilization goals; 2) interprofessional closed-loop communication coordinated by each ICU's facilitator; and 3) performance feedback. Measurements and Main Results: From March 4, 2019 through March 15, 2020, 848 and 1,069 patients were enrolled in the usual care and intervention phases in the primary sample, respectively. The intervention did not increase the primary outcome, patient's maximal Intensive Care Mobility Scale (range, 0-10) score within 48 hours before ICU discharge (estimated mean difference, 0.16; 95% confidence interval, -0.31 to 0.63; P = 0.51). More patients in the intervention (37.2%) than usual care (30.7%) groups achieved the prespecified secondary outcome of ability to stand before ICU discharge (odds ratio, 1.48; 95% confidence interval, 1.02 to 2.15; P = 0.04). Similar results were observed among the 7,115 patients in the secondary sample. The percentage of days on which patients received physical therapy mediated 90.1% of the intervention effect on standing. ICU mortality (31.5% vs. 29.0%), falls (0.7% vs. 0.4%), and unplanned extubations (2.0% vs. 1.8%) were similar between groups (all P > 0.3). Conclusions: A low-cost, multifaceted mobilization intervention did not improve overall mobility but improved patients' odds of standing and was safe. Clinical trial registered with www.clinicaltrials.gov (NCT03863470).

Negatively charged phospholipids accelerate the membrane fusion activity of the plant-specific insert domain of an aspartic protease.

Various plants use antimicrobial proteins/peptides to resist phytopathogens. In the potato, Solanum tuberosum, the plant-specific insert (PSI) domain of an aspartic protease performs this role by disrupting phytopathogen plasma membranes. However, the mechanism by which PSI selects target membranes has not been elucidated. Here, we studied PSI-induced membrane fusion, focusing on the effects of lipid composition on fusion efficiency. Membrane fusion by the PSI involves an intermediate state whereby adjacent liposomes share their bilayers. We found that increasing the concentration of negatively charged phosphatidylserine (PS) phospholipids substantially accelerated PSI-mediated membrane fusion. NMR data demonstrated that PS did not affect the binding between the PSI and liposomes but had seminal effects on the dynamics of PSI interaction with liposomes. In PS-free liposomes, the PSI underwent significant motion, which was suppressed on PS-contained liposomes. Molecular dynamics simulations showed that the PSI binds to PS-containing membranes with a dominant angle ranging from -31° to 30°, with respect to the bilayer, and is closer to the membrane surfaces. In contrast, PSI is mobile and exhibits multiple topological states on the surface of PS-free membranes. Taken together, our data suggested that PS lipids limit the motion of the anchored PSI, bringing it closer to the membrane surface and efficiently bridging different liposomes to accelerate fusion. As most phytopathogens have a higher content of negatively charged lipids as compared with host cells, these results indicate that the PSI selectively targets negatively charged lipids, which likely represents a way of distinguishing the pathogen from the host.

Insights into the mechanism of membrane fusion induced by the plant defense element, plant-specific insert.

In plants, many natural defense mechanisms include cellular membrane fusion as a way to resist infection by external pathogens. Several plant proteins mediate membrane fusion, but the detailed mechanism by which they promote fusion is less clear. Understanding this process could provide valuable insights into these proteins' physiological functions and guide bioengineering applications (i.e. the design of antimicrobial proteins). The plant-specific insert (PSI) from Solanum tuberosum can help reduce certain pathogen attack via membrane fusion. To gain new insights into the process of PSI-induced membrane fusion, a combined approach of NMR, FRET, and in silico studies was used. Our results indicate that (i) under acidic conditions, the PSI experiences a monomer-dimer equilibrium, and the dimeric PSI induces membrane fusion below a certain critical pH; (ii) after fusion, the PSI resides in a highly dehydrated environment with limited solvent accessibility, suggesting its capability in reducing repulsive dehydration forces between liposomes to facilitate fusion; and (iii) as shown by molecular dynamics simulations, the PSI dimer can bind stably to membrane surfaces and can bridge liposomes in close proximity, a critical step for the membrane fusion. In summary, this study provides new and unique insights into the mechanisms by which the PSI and similar proteins induce membrane fusion.

Toxocara polymerase chain reaction on ocular fluids in bilateral granulomatous chorioretinitis.

To report a rare case of bilateral granulomatous chorioretinitis complicated by bilateral peripapillary choroidal neovascular membranes. This is the first reported case in Australia where intravitreal injections of anti-vascular endothelial growth factor ranibizumab were used to successfully treat choroidal neovascular membrane caused by granulomatous chorioretinitis. This is also the first reported case in Australia of Toxocara polymerase chain reaction being performed on intraocular fluids.

Glycated hemoglobin vs fasting plasma glucose as a predictor of left ventricular dysfunction after ST-elevation myocardial infarction.

BACKGROUND: The World Health Organization and the American Diabetes Association recommend a level of glycated hemoglobin (HbA1c) ≥ 6.5% as diagnostic for diabetes. However, concordance between fasting plasma glucose (FPG) and HbA1c levels in acutely unwell patients is unknown. Furthermore, the prognostic value of HbA1c for left ventricular (LV) dysfunction is unclear. This study aimed to evaluate the concordance between HbA1c levels and FPG in consecutive patients with acute ST-elevation MI (STEMI) and compare their prognostic value in predicting LV dysfunction and elevated filling pressures on echocardiography. METHODS: A total of 142 patients with a first incidence of STEMI were prospectively recruited. LV diastolic function was defined as mean septal and lateral early diastolic velocities (average e'); filling pressure was the ratio of transmitral E velocity to average e' (average E/e'). RESULTS: Mean FPG and HbA1c levels were 7.7 ± 2.8 mmol/L and 6.5% ± 1.6%, respectively. Of 109 patients without previous diabetes, HbA1c levels identified an additional 18 patients (16.5%) as having diabetes, and the concordance with FPG was poor. Between diabetic and nondiabetic patients, there were no differences in LV end-diastolic volume (116 ± 37 vs 118 ± 43 mL; P = 0.78), end-systolic volume (69 ± 33 vs 68 ± 35 mL; P = 0.93), and ejection fraction (42 ± 12 vs 44 ± 11%; P = 0.49). On multivariable analyses, average e' was independently associated with HbA1c (ß = -0.161; P = 0.045) but not FPG (P = 0.82). Similarly, average E/e' was independently associated with HbA1c (ß = 0.168; P = 0.04) but not FPG (P = 0.32). Receiver operating characteristic curve analysis showed that an HbA1c cutoff of 6.4% (area under the curve, 0.68; P = 0.002) was associated with an elevated LV filling pressure. CONCLUSIONS: Only HbA1c was independently associated with impaired LV diastolic function and increased filling pressures after STEMI.

Structural characterization of a novel glucan from Achatina fulica and its antioxidant activity.

A novel glucan designated AFPS-IB was purified from Achatina fulica (China white jade snail) by anion-exchange and gel-permeation chromatography. Chemical composition analysis indicated AFPS-IB was composed of glucose, fucose, rhamnose, mannose, and galactose in a molar ratio of 189:2:1:1:2 and with an average molecular weight of 128 kDa. Its structural characteristics were investigated by Fourier transform infrared spectroscopy (FTIR), high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GC-MS), methylation analysis, nuclear magnetic resonance (NMR) spectroscopy ((1)H,( 13)C, H-H COSY, HSQC, TOCSY, and NOESY), and atomic force microscopy (AFM). The glucan mainly consisted of a backbone of repeating (1→4)-α-d-glucose residues with (1→6)-ß-d glucosyl branches at random points on the backbone glucose. Antioxidant studies revealed AFPS-IB showed significant DPPH (2,2-diphenyl-1-picrylhydrazyl) radical, superoxide anion (O2(-)) scavenging activities and high reduction potential. This study suggested that AFPS-IB could be a new source of dietary antioxidants.

siRNA-induced liver ApoB knockdown lowers serum LDL-cholesterol in a mouse model with human-like serum lipids.

Increased serum apolipoprotein (apo)B and associated LDL levels are well-correlated with an increased risk of coronary disease. ApoE⁻/⁻ and low density lipoprotein receptor (LDLr)⁻/⁻ mice have been extensively used for studies of coronary atherosclerosis. These animals show atherosclerotic lesions similar to those in humans, but their serum lipids are low in apoB-containing LDL particles. We describe the development of a new mouse model with a human-like lipid profile. Ldlr CETP⁺/⁻ hemizygous mice carry a single copy of the human CETP transgene and a single copy of a LDL receptor mutation. To evaluate the apoB pathways in this mouse model, we used novel short-interfering RNAs (siRNA) formulated in lipid nanoparticles (LNP). ApoB siRNAs induced up to 95% reduction of liver ApoB mRNA and serum apoB protein, and a significant lowering of serum LDL in Ldlr CETP⁺/⁻ mice. ApoB targeting is specific and dose-dependent, and it shows lipid-lowering effects for over three weeks. Although specific triglycerides (TG) were affected by ApoB mRNA knockdown (KD) and the total plasma lipid levels were decreased by 70%, the overall lipid distribution did not change. Results presented here demonstrate a new mouse model for investigating additional targets within the ApoB pathways using the siRNA modality.

Integrating experimental and analytic approaches to improve data quality in genome-wide RNAi screens.

RNA interference (RNAi) not only plays an important role in drug discovery but can also be developed directly into drugs. RNAi high-throughput screening (HTS) biotechnology allows us to conduct genome-wide RNAi research. A central challenge in genome-wide RNAi research is to integrate both experimental and computational approaches to obtain high quality RNAi HTS assays. Based on our daily practice in RNAi HTS experiments, we propose the implementation of 3 experimental and analytic processes to improve the quality of data from RNAi HTS biotechnology: (1) select effective biological controls; (2) adopt appropriate plate designs to display and/or adjust for systematic errors of measurement; and (3) use effective analytic metrics to assess data quality. The applications in 5 real RNAi HTS experiments demonstrate the effectiveness of integrating these processes to improve data quality. Due to the effectiveness in improving data quality in RNAi HTS experiments, the methods and guidelines contained in the 3 experimental and analytic processes are likely to have broad utility in genome-wide RNAi research.

Fluorogenic substrates for high-throughput measurements of endothelial lipase activity.

Endothelial lipase (EL) has been shown to be a critical determinant for high density lipoprotein cholesterol levels in vivo; therefore, assays that measure EL activity have become important for the discovery of small molecule inhibitors that specifically target EL. Here, we describe fluorescent Bodipy-labeled substrates that can be used in homogeneous, ultra-high-throughput kinetic assays that measure EL phospholipase or triglyceride lipase activities. Triton X-100 detergent micelles and synthetic HDL particles containing Bodipy-labeled phospholipid or Bodipy-labeled triglyceride substrates were shown to be catalytic substrates for EL, LPL, and HL. More importantly, only synthetic HDL particles containing Bodipy-labeled triglyceride were ideal substrates for EL, LPL, and HL in the presence of high concentrations of human or mouse serum. These data suggest that substrate presentation is a critical factor when determining EL activity in the presence of serum.

SAR studies: designing potent and selective LXR agonists.

Counterscreening compounds from a Merck PPAR program discovered lead 1, as a nanomolar LXR/PPAR dual agonist. SAR optimization developed a series of heterocyclic LXR agonists having excellent selectivity over all PPAR isoforms and possessing high LXR affinity and strong in vivo potency.

Different roles of liver X receptor alpha and beta in lipid metabolism: effects of an alpha-selective and a dual agonist in mice deficient in each subtype.

Liver X receptor (LXR) alpha and LXRbeta are closely related nuclear receptors that respond to elevated levels of intracellular cholesterol by enhancing transcription of genes that control cholesterol efflux and fatty acid biosynthesis. The consequences of inactivation of either LXR isoform have been thoroughly studied, as have the effects of simultaneous activation of both LXRalpha and LXRbeta by synthetic compounds. We here describe the effects of selective activation of LXRalpha or LXRbeta on lipid metabolism. This was accomplished by treating mice genetically deficient in either LXRalpha or LXRbeta with an agonist with equal potency for both isoforms (Compound B) or a synthetic agonist selective for LXRalpha (Compound A). We also determined the effect of these agonists on gene expression and cholesterol efflux in peritoneal macrophages derived from wild-type and knockout mice. Both compounds raised HDL-cholesterol and increased liver triglycerides in wild-type mice; in contrast, in mice deficient in LXRalpha, Compound B increased HDL-cholesterol but did not cause hepatic steatosis. Compound B induced ATP-binding cassette transporter (ABC) A1 expression and stimulated cholesterol efflux in macrophages from both LXRalpha and LXRbeta-deficient mice. Our data lend further experimental support to the hypothesis that LXRbeta-selective agonists may raise HDL-cholesterol and stimulate macrophage cholesterol efflux without causing liver triglyceride accumulation.