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The ability to accurately sequence long DNA molecules is important across biology. Here, Lan et al. report a droplet-based method that barcodes single DNA molecules, allowing the full-length molecules to be sequenced with multi-fold coverage using short-read next-generation sequencing.

The reaction of CAAC-CS 2 betaine ( 1 ; CAAC = cyclic(alkyl)(amino)carbene) and alkali metal reductants under ambient conditions yields carbene-stabilized carbon disulfide radical anions as crystalline alkali metal salts. The radicals 3-5 form multinuclear clusters featuring diverse metal sulfide and disulfide interactions, which promote unusual reductive coupling and cyclization of adjacent CS 2 units to C 2 S 3 heterocycles ( 6 ). The addition of crown ethers to 3-5 sequesters the alkali cations and facilitates disulfide cleavage to yield stable [CAAC-CS 2 ] ·- monomers ( 7 and 8 ). Calculated natural atomic spin populations suggest that the spin densities in the clustered and monomeric species are comparable and evenly distributed between the CAAC and CS 2 subunits. Subsequent reductions afford [CAAC-CS 2 ] 2- dianions ( 9-12 ), which can be reoxidized to radicals by comproportionation reactions with 1 . The radicals are, in turn, oxidized to betaine 1 through salt elimination reactions with transition metals. Cyclic voltammograms of 1 feature reversible 1 / 1 ·- / 1 2- couples with a small separation between the events (ΔΔ G = 11.1 kcal mol -1 ). All isolated compounds were characterized by a combination of electron paramagnetic resonance spectroscopy, heteronuclear NMR spectroscopy, infrared spectroscopy, and single-crystal X-ray diffraction. Insights into their electronic structure are supported by density functional theory calculations.

Lipoprotein X (LP-X) is an abnormal cholesterol-rich lipoprotein particle that accumulates in patients with cholestatic liver disease and familial lecithin-cholesterol acyltransferase deficiency (FLD). Because there are no high-throughput diagnostic tests for its detection, a proton nuclear magnetic resonance (NMR) spectroscopy-based method was developed for use on a clinical NMR analyzer commonly used for the quantification of lipoproteins and other cardiovascular biomarkers. The LP-X assay was linear from 89 to 1615 mg/dL (cholesterol units) and had a functional sensitivity of 44 mg/dL. The intra-assay coefficient of variation (CV) varied between 1.8 and 11.8%, depending on the value of LP-X, whereas the inter-assay CV varied between 1.5 and 15.4%. The assay showed no interference with bilirubin levels up to 317 mg/dL and was also unaffected by hemolysis for hemoglobin values up to 216 mg/dL. Samples were stable when stored for up to 6 days at 4 °C but were not stable when frozen. In a large general population cohort ( n = 277,000), LP-X was detected in only 50 subjects. The majority of LP-X positive cases had liver disease (64%), and in seven cases, had genetic FLD (14%). In summary, we describe a new NMR-based assay for LP-X, which can be readily implemented for routine clinical laboratory testing.

Purpose: To determine the role of CD49d for response to Bruton's tyrosine kinase inhibitors (BTKi) in patients with chronic lymphocytic leukemia (CLL)., Patients and Methods: In patients treated with acalabrutinib (n = 48), CD49d expression, VLA-4 integrin activation, and tumor transcriptomes of CLL cells were assessed. Clinical responses to BTKis were investigated in acalabrutinib- (n = 48; NCT02337829) and ibrutinib-treated (n = 73; NCT01500733) patients., Results: In patients treated with acalabrutinib, treatment-induced lymphocytosis was comparable for both subgroups but resolved more rapidly for CD49d+ cases. Acalabrutinib inhibited constitutive VLA-4 activation but was insufficient to block BCR and CXCR4-mediated inside-out activation. Transcriptomes of CD49d+ and CD49d- cases were compared using RNA sequencing at baseline and at 1 and 6 months on treatment. Gene set enrichment analysis revealed increased constitutive NF-κB and JAK-STAT signaling, enhanced survival, adhesion, and migratory capacity in CD49d+ over CD49d- CLL that was maintained during therapy. In the combined cohorts of 121 BTKi-treated patients, 48 (39.7%) progressed on treatment with BTK and/or PLCG2 mutations detected in 87% of CLL progressions. Consistent with a recent report, homogeneous and bimodal CD49d-positive cases (the latter having concurrent CD49d+ and CD49d- CLL subpopulations, irrespective of the traditional 30% cutoff value) had a shorter time to progression of 6.6 years, whereas 90% of cases homogenously CD49d- were estimated progression-free at 8 years (P = 0.0004)., Conclusions: CD49d/VLA-4 emerges as a microenvironmental factor that contributes to BTKi resistance in CLL. The prognostic value of CD49d is improved by considering bimodal CD49d expression. See related commentary by Tissino et al., p. 3560., (©2023 American Association for Cancer Research.)

BACKGROUNDCellular cholesterol efflux capacity (CEC) is a better predictor of cardiovascular disease (CVD) events than HDL-cholesterol (HDL-C) but is not suitable as a routine clinical assay.METHODSWe developed an HDL-specific phospholipid efflux (HDL-SPE) assay to assess HDL functionality based on whole plasma HDL apolipoprotein-mediated solubilization of fluorescent phosphatidylethanolamine from artificial lipid donor particles. We first assessed the association of HDL-SPE with prevalent coronary artery disease (CAD): study I included NIH severe-CAD (n = 50) and non-CAD (n = 50) participants, who were frequency matched for sex, BMI, type 2 diabetes mellitus, and smoking; study II included Japanese CAD (n = 70) and non-CAD (n = 154) participants. We also examined the association of HDL-SPE with incident CVD events in the Prevention of Renal and Vascular End-stage Disease (PREVEND) study comparing 340 patients with 340 controls individually matched for age, sex, smoking, and HDL-C levels.RESULTSReceiver operating characteristic curves revealed stronger associations of HDL-SPE with prevalent CAD. The AUCs in study I were as follows: HDL-SPE, 0.68; apolipoprotein A-I (apoA-I), 0.62; HDL-C, 0.63; and CEC, 0.52. The AUCs in study II were as follows: HDL-SPE, 0.83; apoA-I, 0.64; and HDL-C, 0.53. Also longitudinally, HDL-SPE was significantly associated with incident CVD events independent of traditional risk factors with ORs below 0.2 per SD increment in the PREVEND study (P < 0.001).CONCLUSIONHDL-SPE could serve as a routine clinical assay for improving CVD risk assessment and drug discovery.TRIAL REGISTRATIONClinicalTrials.gov NCT01621594.FUNDINGNHLBI Intramural Research Program, NIH (HL006095-06).

Multiple sclerosis (MS) is a complex dysimmune disorder of the central nervous system. Genome-wide association studies (GWAS) have identified 233 genetic variations associated with MS at the genome-wide significant level. Epigenetic studies have pinpointed differentially methylated CpG sites in MS patients. However, the interplay between genetic risk factors and epigenetic regulation remains elusive. Here, we employed a network model to integrate GWAS summary statistics of 14 802 MS cases and 26 703 controls with DNA methylation profiles from 140 MS cases and 139 controls and the human interactome. We identified differentially methylated genes by aggregating additive effects of differentially methylated CpG sites within promoter regions. We reconstructed a gene regulatory network (GRN) using literature-curated transcription factor knowledge. Colocalization of the MS GWAS and methylation quantitative trait loci (mQTL) was performed to assess the GRN. The resultant MS-associated GRN highlighted several single nucleotide polymorphisms with GWAS-mQTL colocalization: rs6032663, rs6065926 and rs2024568 of CD40 locus, rs9913597 of STAT3 locus, and rs887864 and rs741175 of CIITA locus. Moreover, synergistic mQTL and expression QTL signals were identified in CD40, suggesting gene expression alteration was likely induced by epigenetic changes. Web-based Cell-type Specific Enrichment Analysis of Genes (WebCSEA) indicated that the GRN was enriched in T follicular helper cells (P-value = 0.0016). Drug target enrichment analysis of annotations from the Therapeutic Target Database revealed the GRN was also enriched with drug target genes (P-value = 3.89 × 10-4), revealing repurposable candidates for MS treatment. These candidates included vorinostat (HDAC1 inhibitor) and sivelestat (ELANE inhibitor), which warrant further investigation., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: The first wave of the COVID-19 pandemic had a significant impact on the personal and professional lives of frontline nurses., Purpose: The purpose of this descriptive phenomenological study was to explore the experiences of Canadian Registered Nurses (RNs) working in Ontario or United States hospitals during the first wave of the COVID-19 pandemic., Methods: Semi-structured interviews were conducted with 36 RNs living in Ontario and employed either at an Ontario or United States hospital. Three main themes were identified across both healthcare contexts., Results: 1) The Initial Response to the pandemic included a rapid onset of chaos and confusion, with significant changes in structure and patient care, often exacerbated by hospital management. Ethical concerns arose (e.g., redeployment, allocation of resources) and participants described negative emotional reactions. 2) Nurses described Managing the Pandemic by finding new ways to nurse and enhanced teamwork/camaraderie; they reported both struggle and resiliency while trying to maintain work and home life balance. Community responses were met with both appreciation and stigma. 3) Participants said they were Looking Forward to a "new normal", taking pride in patient improvements, accomplishments, and silver linings, with tempered optimism about the future. Many expressed a reaffirmation of their identities as nurses. Differences between participants working in the US and those working in Ontario were noted in several areas (e.g., initial levels of chaos, ethical concerns, community stigma)., Conclusions: The COVID-19 pandemic has been very difficult for nursing as a profession. Close attention to post-pandemic issues is warranted.

The addition of non-benzenoid quinones, acenapthenequinone or aceanthrenequinone, to the 9-carbene-9-borafluorene monoanion (1) affords the first examples of dianionic 10-membered bora-crown ethers (2-5), which are characterized by multi-nuclear NMR spectroscopy ( 1 H, 13 C, 11 B), X-ray crystallography, elemental analysis, and UV/Vis spectroscopy. These tetraoxadiborecines have distinct absorption profiles based on the positioning of the alkali metal cations. When compound 4, which has a vacant C 4 B 2 O 4 cavity, is reacted with sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate, a color change from purple to orange serves as a visual indicator of metal binding to the central ring, whereby the Na + ion coordinates to four oxygen atoms. A detailed theoretical analysis of the calculated reaction energetics is provided to gain insight into the reaction mechanism for the formation of 2-5. These data, and the electronic structures of proposed intermediates, indicate that the reaction proceeds via a boron enolate intermediate., (© 2022 Wiley-VCH GmbH.)

The impact of the exact spatial arrangement of the alkali metal on the electronic properties of 9-carbene-9-borafluorene monoanions is assessed, and a series of [K][9-CAAC-9-borafluorene] complexes ( 1 - 4 ) have been isolated (CAAC = cyclic(alkyl)(amino) carbene, (2,6-diisopropylphenyl)-4,4-diethyl-2,2-dimethyl-pyrrolidin-5-ylidene). Compound 1 , which contains [B]-K(THF) 3 interactions, is compared to charge-separated 2 - 4 , which were prepared by capturing the potassium cations with 18-crown-6, 2.2.2-cryptand, or 1,10-phenanthroline. Notably, the 11 B NMR spectra of charge-separated borafluorene monoanions 2-4 show distinct low-field signatures compared to 1 . Theoretical calculations indicate that charge separation may be exploited to influence the nucleophilic and electron transfer properties of 9-carbene-9-borafluorene monoanions. When [K(2.2.2-cryptand)][9-CAAC-9-borafluorene] ( 3 ) is reacted with 9,10-phenanthrenequinone and 1,10-phenanthroline-5,6-dione, the carbene ligand is displaced, and new air-stable R 2 BO 2 spirocycles are formed ( 5 and 6 , respectively). Remarkably, compounds 5 and 6 display fluorescence under UV light in both the solid and solution phases with quantum yields of up to 20%. In addition, a drastic red-shift in the emission color is observed in 6 because of the presence of the nitrogen atoms on the phenanthroline moiety. Mechanistic insights into the formation of these spirocycles are also described based on density functional theory calculations.

The first structurally characterized example of a trioxaborinanone ( 2 ) is produced by the reaction of a 9-carbene-9-borafluorene monoanion and carbon dioxide. When compound 2 is heated or irradiated with UV light, carbon monoxide (CO) is released, and a luminescent dioxaborinanone ( 3 ) is formed. Notably, carbon monoxide releasing molecules (CORMs) are of interest for their ability to deliver a specific amount of CO. Due to the turn-on fluorescence observed as a result of the conversion to 3 , CORM 2 serves as a means to optically observe CO loss "by eye" under thermal or photochemical conditions.

Lipoprotein-X (LpX) are abnormal nephrotoxic lipoprotein particles enriched in free cholesterol and phospholipids. LpX with distinctive lipid compositions are formed in patients afflicted with either familial LCAT deficiency (FLD) or biliary cholestasis. LpX is difficult to detect by standard lipid stains due to the absence of a neutral lipid core and because it is unstable upon storage, particularly when frozen. We have recently reported that free cholesterol-specific filipin staining after agarose gel electrophoresis sensitively detects LpX in fresh human plasma. Herein, we describe an even more simplified qualitative method to detect LpX in both fresh and frozen-thawed human FLD or cholestatic plasma. Fluorescent cholesterol complexed to fatty-acid-free BSA was used to label LpX and was added together with trehalose in order to cryopreserve plasma LpX. The fluorescent cholesterol bound to LpX was observed with high sensitivity after separation from other lipoproteins by agarose gel electrophoresis. This methodology can be readily developed into a simple assay for the clinical diagnosis of FLD and biliary liver disease and to monitor the efficacy of treatments intended to reduce plasma LpX in these disease states.

Background: Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating low-density lipoprotein levels in plasma. While PCSK9 variants are causatively associated with familial hypercholesterolemia (FH), additional genotyping methods for FH targeting PCSK9 variants are required in a clinical setting. Loop-mediated isothermal amplification (LAMP) is a unique amplification method that amplifies a target gene under isothermal conditions (60-65 °C). However, a robust standardized method has not yet been established for LAMP-based genetic screening tests for genetic diseases, including FH. The present study aimed to develop a novel modification of the LAMP method designed to genotype single nucleotide variants (SNVs) and to apply it for the detection of PCSK9 variants., Methods: Using short quenching probes (≤ 10 nucleotides) for the loop structures of LAMP amplicons, accurate detection of SNVs was verified separately for each allele, without any additional procedures, within 3 h. The diagnostic performance of this method in detecting PCSK9 variants was validated in FH patients., Results: All PCSK9 variants tested via conventional sequencing in FH patients were successfully detected using this novel LAMP method., Conclusions: We developed a LAMP-based genotyping method to detect PCSK9 variants in FH. Compared to conventional sequencing, our genotyping method is relatively convenient and time-efficient and is suitable for the screening of FH in clinical settings. Future studies on various genes are also warranted., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Due to the unique set of stressors associated with the COVID-19 pandemic, healthcare workers in acute care settings may be facing elevated rates of mental health symptomatology. The purpose of this study was to assess levels of depression, anxiety, and stress in a sample of healthcare employees working in hospitals and their use of formal and informal mental health supports. Data was gathered over a three-week period in December 2020 as COVID cases began to rise sharply in Ontario, Canada. Results from an online survey of 650 healthcare employees suggested that overall levels of depression, anxiety, and stress were mild. However, a significant minority of participants reported severe or extremely severe levels of depression (14.4%), anxiety (21.8%), and stress (13.5%). Levels of distress were higher among women, younger participants, those who did not work directly with COVID+ patients, and those who were redeployed. Use of formal mental health supports (e.g., Employee Assistance Plans, teletherapy) was very low (<10%), with the most frequently-reported reason for not using supports being "problems not severe enough to require this service". Implications are considered for healthcare policy decisions as hospital systems attempt to address the mental health needs of their employees., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Copyright © 2022. Published by Elsevier B.V.)

A significant proportion of patients with elevated LDL and a clinical presentation of familial hypercholesterolemia do not carry known genetic mutations associated with hypercholesterolemia, such as defects in the LDL receptor. To identify new genes involved in the cellular uptake of LDL, we developed a novel whole-genome clustered regularly interspaced short palindromic repeat-Cas9 KO screen in HepG2 cells. We identified transgelin (TAGLN), an actin-binding protein, as a potentially new gene involved in LDL endocytosis. In silico validation demonstrated that genetically predicted differences in expression of TAGLN in human populations were significantly associated with elevated plasma lipids (triglycerides, total cholesterol, and LDL-C) in the Global Lipids Genetics Consortium and lipid-related phenotypes in the UK Biobank. In biochemical studies, TAGLN-KO HepG2 cells showed a reduction in cellular LDL uptake, as measured by flow cytometry. In confocal microscopy imaging, TAGLN-KO cells had disrupted actin filaments as well as an accumulation of LDL receptor on their surface because of decreased receptor internalization. Furthermore, TAGLN-KO cells exhibited a reduction in total and free cholesterol content, activation of SREBP2, and a compensatory increase in cholesterol biosynthesis. TAGLN deficiency also disrupted the uptake of VLDL and transferrin, other known cargoes for receptors that depend upon clathrin-mediated endocytosis. Our data suggest that TAGLN is a novel factor involved in the actin-dependent phase of clathrin-mediated endocytosis of LDL. The identification of novel genes involved in the endocytic uptake of LDL may improve the diagnosis of hypercholesterolemia and provide future therapeutic targets for the prevention of cardiovascular disease., Competing Interests: Conflict of interest S. V. salary and significant ownership in Global Genomics Group. M. M. M. is now an employee of Novartis Institutes of Biomedical Research, but he was not an employee when his contribution to the work was conducted. All other authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

Reactions of 9-carbene-9-borafluorene monoanion ( 1 ) with elemental selenium and selenium-containing reagents are reported. When compound 1 is reacted with grey selenium in THF, various boryl-substituted selenides and diselenides are produced ( 2-6 ), including molecules resulting from migration of the carbene ligand Dipp group (Dipp = 2,6-diisopropylphenyl). However, when a similar reaction between 1 and grey selenium is performed in toluene in the presence of 18-crown-6, boryl-substituted selenide 7 is obtained as the sole boron-containing product. As compound 7 is the monomeric variant of organoselenide 3 , 18-crown-6 promotes both product selectivity and solubility in a nonpolar solvent. Diselenide 5 , which features a trans-bent B-Se-Se-B core, was directly isolated via reaction of 1 with Se 2 Cl 2 in THF. Computational modeling suggests that the formation of 5 proceeds via a radical mechanism. This was supported by an experiment demonstrating that the CAAC-borafluorene radical also reacts with SeCl 2 to yield 5 [CAAC = (2,6-diisopropylphenyl)-4,4-diethyl-2,2-dimethyl-pyrrolidin-5-ylidene]. Energy decomposition analysis of 5 indicates a covalent borafluorene-diselenide bond (Δ E int , -168.9 kcal mol -1 ). All of the new compounds were fully characterized via single-crystal X-ray diffraction and multinuclear nuclear magnetic resonance ( 1 H, 13 C, 11 B, and 77 Se).

Multiple sclerosis (MS) is a neuroinflammatory disorder leading to chronic disability. Brain lesions in MS commonly arise in normal-appearing white matter (NAWM). Genome-wide association studies (GWAS) have identified genetic variants associated with MS. Transcriptome alterations have been observed in case-control studies of NAWM. We developed a Cross-Dataset Evaluation (CDE) function for our network-based tool, Edge-Weighted Dense Module Search of GWAS (EW&#95;dmGWAS). We applied CDE to integrate publicly available MS GWAS summary statistics of 41,505 cases and controls with collectively 38 NAWM expression samples, using the human protein interactome as the reference network, to investigate biological underpinnings of MS etiology. We validated the resulting modules with colocalization of GWAS and expression quantitative trait loci (eQTL) signals, using GTEx Consortium expression data for MS-relevant tissues: 14 brain tissues and 4 immune-related tissues. Other network assessments included a drug target query and functional gene set enrichment analysis. CDE prioritized a MS NAWM network containing 55 unique genes. The gene list was enriched (p-value = 2.34 × 10 -7 ) with GWAS-eQTL colocalized genes: CDK4, IFITM3, MAPK1, MAPK3, METTL12B and PIK3R2. The resultant network also included drug signatures of FDA-approved medications. Gene set enrichment analysis revealed the top functional term "intracellular transport of virus", among other viral pathways. We prioritize critical genes from the resultant network: CDK4, IFITM3, MAPK1, MAPK3, METTL12B and PIK3R2. Enriched drug signatures suggest potential drug targets and drug repositioning strategies for MS. Finally, we propose mechanisms of potential MS viral onset, based on prioritized gene set and functional enrichment analysis., (Copyright © 2021. Published by Elsevier Inc.)

Purpose: Nurses working during the coronavirus disease 2019 (COVID-19) pandemic have reported elevated levels of anxiety, burnout and sleep disruption. Hospital administrators are in a unique position to mitigate or exacerbate stressful working conditions. The goal of this study was to capture the recommendations of nurses providing frontline care during the pandemic., Design/methodology/approach: Semi-structured interviews were conducted during the first wave of the COVID-19 pandemic, with 36 nurses living in Canada and working in Canada or the United States., Findings: The following recommendations were identified from reflexive thematic analysis of interview transcripts: (1) The nurses emphasized the need for a leadership style that embodied visibility, availability and careful planning. (2) Information overload contributed to stress, and participants appealed for clear, consistent and transparent communication. (3) A more resilient healthcare supply chain was required to safeguard the distribution of equipment, supplies and medications. (4) Clear communication of policies related to sick leave, pay equity and workload was necessary. (5) Equity should be considered, particularly with regard to redeployment. (6) Nurses wanted psychological support offered by trusted providers, managers and peers., Practical Implications: Over-reliance on employee assistance programmes and other individualized approaches to virtual care were not well-received. An integrative systems-based approach is needed to address the multifaceted mental health outcomes and reduce the deleterious impact of the COVID-19 pandemic on the nursing workforce., Originality/value: Results of this study capture the recommendations made by nurses during in-depth interviews conducted early in the COVID-19 pandemic., (© Emerald Publishing Limited.)

Deep clustering was applied to unlabeled, automatically detected signals in a coral reef soundscape to distinguish fish pulse calls from segments of whale song. Deep embedded clustering (DEC) learned latent features and formed classification clusters using fixed-length power spectrograms of the signals. Handpicked spectral and temporal features were also extracted and clustered with Gaussian mixture models (GMM) and conventional clustering. DEC, GMM, and conventional clustering were tested on simulated datasets of fish pulse calls (fish) and whale song units (whale) with randomized bandwidth, duration, and SNR. Both GMM and DEC achieved high accuracy and identified clusters with fish, whale, and overlapping fish and whale signals. Conventional clustering methods had low accuracy in scenarios with unequal-sized clusters or overlapping signals. Fish and whale signals recorded near Hawaii in February-March 2020 were clustered with DEC, GMM, and conventional clustering. DEC features demonstrated the highest accuracy of 77.5% on a small, manually labeled dataset for classifying signals into fish and whale clusters.

Detecting acoustic transients by signal-to-noise ratio (SNR) becomes problematic in nonstationary ambient noise environments characteristic of coral reefs. An alternate approach presented here uses signal directionality to automatically detect and localize transient impulsive sounds collected on underwater vector sensors spaced tens of meters apart. The procedure, which does not require precise time synchronization, first constructs time-frequency representations of both the squared acoustic pressure (spectrogram) and dominant directionality of the active intensity (azigram) on each sensor. Within each azigram, sets of time-frequency cells associated with transient energy arriving from a consistent azimuthal sector are identified. Binary image processing techniques then link sets that share similar duration and bandwidth between different sensors, after which the algorithm triangulates the source location. Unlike most passive acoustic detectors, the threshold criterion for this algorithm is bandwidth instead of pressure magnitude. Data collected from shallow coral reef environments demonstrate the algorithm's ability to detect SCUBA bubble plumes and consistent spatial distributions of somniferous fish activity. Analytical estimates and direct evaluations both yield false transient localization rates from 3% to 6% in a coral reef environment. The SNR distribution of localized pulses off Hawaii has a median of 7.7 dB and interquartile range of 7.1 dB.

The mono- and dianions of CO 2 ( i.e. , CO 2 - and CO 2 2- ) have been studied for decades as both fundamentally important oxycarbanions (anions containing only C and O atoms) and as critical species in CO 2 reduction and fixation chemistry. However, CO 2 anions are highly unstable and difficult to study. As such, examples of stable compounds containing these ions are extremely limited; the unadulterated alkali salts of CO 2 ( i.e. , MCO 2 , M 2 CO 2 , M = alkali metal) decompose rapidly above 15 K, for example. Herein we report the chemical reduction of a cyclic (alkyl)(amino) carbene (CAAC) adduct of CO 2 at room temperature by alkali metals, which results in the formation of CAAC-stabilized alkali CO 2 - and CO 2 2- clusters. One-electron reduction of CAAC-CO 2 adduct ( 1 ) with lithium, sodium or potassium metal yields stable monoanionic radicals [M(CAAC-CO 2 )] n (M = Li, Na, K, 2-4 ) analogous to the alkali CO 2 - radical, and two-electron alkali metal reduction affords dianionic clusters of the general formula [M 2 (CAAC-CO 2 )] n ( 5-8 ) with reduced CO 2 units which are structurally analogous to the carbonite anion CO 2 2- . It is notable that crystalline clusters of these alkali-CO 2 salts may also be isolated via the "one-pot" reaction of free CO 2 with free CAAC followed by the addition of alkali metals - a process which does not occur in the absence of carbene. Each of the products 2-8 was investigated using a combination of experimental and theoretical methods., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Apolipoprotein A-I (ApoA-I) mimetic peptides are potential therapeutic agents for promoting the efflux of excess cellular cholesterol, which is dependent upon the presence of an amphipathic helix. Since α-methylated Ala enhances peptide helicity, we hypothesized that incorporating other types of α-methylated amino acids into ApoA-I mimetic peptides may also increase their helicity and cholesterol efflux potential. The last helix of apoA-I, peptide 'A' (VLESFKVSFLSALEEYTKKLNT), was used to design peptides containing a single type of α-methylated amino acid substitution (Ala/A α , Glu/D α , Lys/K α , Leu/L α ), as well as a peptide containing both α-methylated Lys and Leu (6 α ). Depending on the specific residue, the α-helical content as measured by CD-spectroscopy and calculated hydrophobic moments were sometimes higher for peptides containing other types of α-methylated amino acids than those with α-methylated Ala. In ABCA1-transfected cells, cholesterol efflux to the peptides showed the following order of potency: 6 α >K α ≈L α ≈A α ≫D α ≈A. In general, α-methylated peptides were resistant to proteolysis, but this varied depending on the type of protease and specific amino acid substitution. In summary, increased helicity and amphilicity due to α-methylated amino acid substitutions in ApoA-I mimetic peptides resulted in improved cholesterol efflux capacity and resistance to proteolysis, indicating that this modification may be useful in the future design of therapeutic ApoA-I mimetic peptides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

Background: Multiple sclerosis (MS) is a complex disease in which the immune system attacks the central nervous system. The molecular mechanisms contributing to the etiology of MS remain poorly understood. Genome-wide association studies (GWAS) of MS have identified a small number of genetic loci significant at the genome level, but they are mainly non-coding variants. Network-assisted analysis may help better interpret the functional roles of the variants with association signals and potential translational medicine application. The Dense Module Searching of GWAS tool (dmGWAS version 2.4) developed in our team is applied to 2 MS GWAS datasets (GeneMSA and IMSGC GWAS) using the human protein interactome as the reference network. A dual evaluation strategy is used to generate results with reproducibility., Results: Approximately 7500 significant network modules were identified for each independent GWAS dataset, and 20 significant modules were identified from the dual evaluation. The top modules included GRB2, HDAC1, JAK2, MAPK1, and STAT3 as central genes. Top module genes were enriched with functional terms such as "regulation of glial cell differentiation" (adjusted p-value = 2.58 × 10 - 3 ), "T-cell costimulation" (adjusted p-value = 2.11 × 10 - 6 ) and "virus receptor activity" (adjusted p-value = 1.67 × 10 - 3 ). Interestingly, top gene networks included several MS FDA approved drug target genes HDAC1, IL2RA, KEAP1, and RELA, CONCLUSIONS: Our dmGWAS network analyses highlighted several genes (GRB2, HDAC1, IL2RA, JAK2, KEAP1, MAPK1, RELA and STAT3) in top modules that are promising to interpret GWAS signals and link to MS drug targets. The genes enriched with glial cell differentiation are important for understanding neurodegenerative processes in MS and for remyelination therapy investigation. Importantly, our identified genetic signals enriched in T cell costimulation and viral receptor activity supported the viral infection onset hypothesis for MS.

Purpose of Review: To review recent lecithin:cholesterol acyltransferas (LCAT)-based therapeutic approaches for atherosclerosis, acute coronary syndrome, and LCAT deficiency disorders., Recent Findings: A wide variety of approaches to using LCAT as a novel therapeutic target have been proposed. Enzyme replacement therapy with recombinant human LCAT is the most clinically advanced therapy for atherosclerosis and familial LCAT deficiency (FLD), with Phase I and Phase 2A clinical trials recently completed. Liver-directed LCAT gene therapy and engineered cell therapies are also another promising approach. Peptide and small molecule activators have shown efficacy in early-stage preclinical studies. Finally, lifestyle modifications, such as fat-restricted diets, cessation of cigarette smoking, and a diet rich in antioxidants may potentially suppress lipoprotein abnormalities in FLD patients and help preserve LCAT activity and renal function but have not been adequately tested., Summary: Preclinical and early-stage clinical trials demonstrate the promise of novel LCAT therapies as HDL-raising agents that may be used to treat not only FLD but potentially also atherosclerosis and other disorders with low or dysfunctional HDL.

N-Heterocyclic carbene (NHC)- and cyclic (alkyl)(amino)carbene (CAAC)-stabilized borafluorene radicals have been isolated and characterized by elemental analysis, single-crystal X-ray diffraction, UV/Vis absorption, cyclic voltammetry (CV), electron paramagnetic resonance (EPR) spectroscopy, and theoretical studies. Both the CAAC-borafluorene radical (2) and the NHC-borafluorene radical (4) have a considerable amount of spin density localized on the boron atoms (0.322 for 2 and 0.369 for 4). In compound 2, the unpaired electron is also partly delocalized over the CAAC ligand carbene C and N atoms. However, the unpaired electron in compound 4 mainly resides throughout the borafluorene π-system, with significantly less delocalization over the NHC ligand. These results highlight the Lewis base dependent electrostructural tuning of materials-relevant radicals. Notably, this is the first report of crystalline borafluorene radicals, and these species exhibit remarkable solid-state and solution stability., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Objective: Highly elevated plasma levels of interleukin-10 (IL-10) are causally associated with "Disappearing HDL Syndrome" and low plasma LDL-cholesterol, but the underlying mechanism is poorly understood. Fluid-phase endocytosis, a process highly dependent on actin dynamics, enables cells to internalize relatively high amounts of extracellular fluids and solutes. We sought to investigate whether IL-10 induces lipoprotein uptake by fluid-phase endocytosis in macrophages., Methods and Results: Macrophages (RAW264.7, Kupffer and human) were incubated with vehicle (PBS) or IL-10 (20 ng/ml) for 7 days. Uptake of HDL, LDL, and/or fluid-phase endocytosis probes (albumin-Alexa680®, 70 kDa FITC-Dextran and Lucifer Yellow, LY) was evaluated by FACS. Intracellular cofilin and phosphorylated cofilin (p-cofilin) levels were determined by immunoblotting. Macrophage uptake of lipoproteins and probes was non-saturable and increased after IL-10 incubation (p < 0.0001). Furthermore, pre-incubation with fluid-phase endocytosis inhibitors (LY294002, Latrunculin A, and Amiloride) significantly reduced uptake (p < 0.05). IL-10 increased the cofilin/p-cofilin ratio (p = 0.021), signifying increased cofilin activation and hence filamentous actin. Consistently, phalloidin staining revealed increased filamentous actin in macrophages after IL-10 treatment (p = 0.0018). Finally, RNA-seq analysis demonstrated enrichment of gene sets related to actin filament dynamics, membrane ruffle formation and endocytosis in IL-10-treated macrophages (p < 0.05). IL-10 did not alter mRNA levels of Ldlr, Vldlr, Scarb1, Cd36 or Lrp1. In primary human monocyte-derived macrophages and murine Kupffer cells, IL-10 incubation also increased uptake of lipoproteins, albumin and LY (p < 0.01)., Conclusions: Interleukin-10 induces the uptake of HDL and LDL by fluid-phase endocytosis by increasing actin-filament rearrangement in macrophages, thus providing a plausible mechanism contributing to "Disappearing HDL Syndrome"., (Published by Elsevier B.V.)

Aim: To investigate palliative care nurse attitudes towards medical assistance in dying., Design: An exploratory cross-sectional study design., Methods: A mailed letter recruited participants with data collection occurring on a secure online survey platform between November 2017-February 2018. Data analyses included descriptive and bivariate statistics and stepwise linear regression., Results: Palliative care nurse attitudes towards medical assistance in dying were explained by perceived expertise in the social domain of palliative care, personal importance of religion/faith, professional importance of religion/faith, and nursing designation., Conclusion: This study reveals the perceived importance of religion, versus religious affiliation alone, as significant in influencing provider attitudes towards assisted dying. Further research is needed to understand differences in attitudes between Registered Nurses and Registered Practical Nurses and how the social domain of palliative care influences nurse attitude., Impact: Organizations must prioritize nursing input, encourage open interprofessional dialogue and provide support for ethical decision-making, practice decisions, and conscientious objection surrounding medical assistance in dying. Longitudinal nursing studies are needed to understand the impact of legislation on quality and person-centred end-of-life care and the emotional well-being/retention of palliative care nurses., (© 2019 John Wiley & Sons Ltd.)

Recent genetic studies have established that hypertriglyceridemia (HTG) is causally related to cardiovascular disease, making it an active area for drug development. We describe a strategy for lowering triglycerides (TGs) with an apolipoprotein C-II (apoC-II) mimetic peptide called D6PV that activates lipoprotein lipase (LPL), the main plasma TG-hydrolyzing enzyme, and antagonizes the TG-raising effect of apoC-III. The design of D6PV was motivated by a combination of all-atom molecular dynamics simulation of apoC-II on the Anton 2 supercomputer, structural prediction programs, and biophysical techniques. Efficacy of D6PV was assessed ex vivo in human HTG plasma and was found to be more potent than full-length apoC-II in activating LPL. D6PV markedly lowered TG by more than 80% within a few hours in both apoC-II-deficient mice and h APOC3 -transgenic (Tg) mice. In h APOC3 -Tg mice, D6PV treatment reduced plasma apoC-III by 80% and apoB by 65%. Furthermore, low-density lipoprotein (LDL) cholesterol did not accumulate but rather was decreased by 10% when h APOC3 -Tg mice lacking the LDL-receptor (h APOC3 -Tg × Ldlr -/- ) were treated with the peptide. D6PV lowered TG by 50% in whole-body inducible Lpl knockout (i Lpl -/- ) mice, confirming that it can also act independently of LPL. D6PV displayed good subcutaneous bioavailability of about 80% in nonhuman primates. Because it binds to high-density lipoproteins, which serve as a long-term reservoir, it also has an extended terminal half-life (42 to 50 hours) in nonhuman primates. In summary, D6PV decreases plasma TG by acting as a dual apoC-II mimetic and apoC-III antagonist, thereby demonstrating its potential as a treatment for HTG., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare genetic disease characterized by low HDL-C levels, low plasma cholesterol esterification, and the formation of Lipoprotein-X (Lp-X), an abnormal cholesterol-rich lipoprotein particle. LCAT deficiency causes corneal opacities, normochromic normocytic anemia, and progressive renal disease due to Lp-X deposition in the glomeruli. Recombinant LCAT is being investigated as a potential therapy for this disorder. Several hepatic disorders, namely primary biliary cirrhosis, primary sclerosing cholangitis, cholestatic liver disease, and chronic alcoholism also develop Lp-X, which may contribute to the complications of these disorders. We aimed to test the hypothesis that an increase in plasma LCAT could prevent the formation of Lp-X in other diseases besides FLD. We generated a murine model of intrahepatic cholestasis in LCAT-deficient (KO), wild type (WT), and LCAT-transgenic (Tg) mice by gavaging mice with alpha-naphthylisothiocyanate (ANIT), a drug well known to induce intrahepatic cholestasis. Three days after the treatment, all mice developed hyperbilirubinemia and elevated liver function markers (ALT, AST, Alkaline Phosphatase). The presence of high levels of LCAT in the LCAT-Tg mice, however, prevented the formation of Lp-X and other plasma lipid abnormalities in WT and LCAT-KO mice. In addition, we demonstrated that multiple injections of recombinant human LCAT can prevent significant accumulation of Lp-X after ANIT treatment in WT mice. In summary, LCAT can protect against the formation of Lp-X in a murine model of cholestasis and thus recombinant LCAT could be a potential therapy to prevent the formation of Lp-X in other diseases besides FLD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2019 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

Herein we report the synthesis and characterization of dinuclear magnesium-hydride complexes, [(IiPrMe2)Mg(μ-H)(HMDS)]2 [4, IiPrMe2 = N,N'-diisopropyl-2,3-dimethylimidazol-2-ylidine, HMDS = bis(trimethylsilyl)amide] and [(IiPrMe2)Mg(μ-H)(ASCP)]2 (5, ASCP = 2,2,5,5-tetramethyl-2,5-disila-1-azacyclopent-1-yl). Compounds 4 and 5 exhibit the lowest nuclearity of any carbene-magnesium hydride complex to date.

The first examples of N-heterocyclic carbene (NHC) and cyclic(alkyl)(amino) carbene (CAAC) stabilized borepinium and borafluorenium heterocycles are reported herein. The optical properties of the heterocyclic borenium cations were tuned by varying the Lewis base and by changing the number of atoms in the ring. More importantly, functionalizing the cationic boron ring system in the NHC-borafluorenium cation affords a temperature-sensitive molecule with reversible colorimetric "turn off/turn on" properties in solution. Notably, this is the first report of thermochromism in these cationic species. This property, which is mediated by an intermolecular boron-oxygen bond equilibrium, was examined in detail by X-ray crystallography, variable temperature-UV/Vis absorption spectroscopy (VT-UV/Vis), and density functional theory (DFT)., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the past two decades, the organometallic chemistry of the alkaline earth elements has experienced a renaissance due in part to developments in ligand stabilization strategies. In order to expand the scope of redox chemistry known for magnesium and beryllium, we have synthesized a set of reduced magnesium and beryllium complexes and compared their resulting structural and electronic properties. The carbene-coordinated alkaline earth-halides, ( Et2 CAAC)MgBr 2 ( 1 ), (SIPr)MgBr 2 ( 2 ), ( Et2 CAAC)BeCl 2 ( 3 ), and (SIPr)BeCl 2 ( 4 ) [ Et2 CAAC = diethyl cyclic(alkyl)(amino) carbene; SIPr = 1,3-bis(2,6-diisopropylphenyl)-4,5-dihydroimidazole-2-ylidene] were combined with an α-diimine [2,2-bipyridine (bpy) or bis (2,6-diisopropylphenyl)-1,4-diazabutadiene ( Dipp DAB)] and the appropriate stoichiometric amount of potassium graphite to form singly- and doubly-reduced compounds ( Et2 CAAC)MgBr( Dipp DAB) ( 5 ), ( Et2 CAAC)MgBr(bpy) ( 6 ), ( Et2 CAAC)Mg( Dipp DAB) ( 7 ), ( Et2 CAAC)Be(bpy) ( 8 ), and (SIPr)Be(bpy) ( 9 ). The doubly-reduced compounds 7 - 9 exhibit substantial π-bonding interactions across the diimine core, metal center, and π-acidic carbene. Each complex was fully characterized by UV-vis, FT-IR, X-ray crystallography, 1 H, 13 C, and 9 Be NMR, or EPR where applicable. We use these compounds to highlight the differences in the organometallic chemistry of the lightest alkaline earth metals, magnesium and beryllium, in an otherwise identical chemical environment.

Familial LCAT deficiency (FLD) patients accumulate lipoprotein-X (LP-X), an abnormal nephrotoxic lipoprotein enriched in free cholesterol (FC). The low neutral lipid content of LP-X limits the ability to detect it after separation by lipoprotein electrophoresis and staining with Sudan Black or other neutral lipid stains. A sensitive and accurate method for quantitating LP-X would be useful to examine the relationship between plasma LP-X and renal disease progression in FLD patients and could also serve as a biomarker for monitoring recombinant human LCAT (rhLCAT) therapy. Plasma lipoproteins were separated by agarose gel electrophoresis and cathodal migrating bands corresponding to LP-X were quantified after staining with filipin, which fluoresces with FC, but not with neutral lipids. rhLCAT was incubated with FLD plasma and lipoproteins and LP-X changes were analyzed by agarose gel electrophoresis. Filipin detects synthetic LP-X quantitatively (linearity 20-200 mg/dl FC; coefficient of variation <20%) and sensitively (lower limit of quantitation <1 mg/ml FC), enabling LP-X detection in FLD, cholestatic, and even fish-eye disease patients. rhLCAT incubation with FLD plasma ex vivo reduced LP-X dose dependently, generated HDL, and decreased lipoprotein FC content. Filipin staining after agarose gel electrophoresis sensitively detects LP-X in human plasma and accurately quantifies LP-X reduction after rhLCAT incubation ex vivo.

Carbenes are known to activate carbon dioxide to form zwitterionic adducts. Their inherent metal-free redox activity remains understudied. Herein, we demonstrate that zwitterionic adducts of carbon dioxide formed with cyclic(alkyl)(amino) carbenes are not only redox active, but they can mediate the stoichiometric reductive disproportionation of carbon dioxide to carbon monoxide and carbonate. Infrared spectroelectrochemical experiments show that the reaction proceeds through an intermediate radical anion formed by one-electron reduction, ultimately generating a ketene product and carbonate in the absence of additional organic or inorganic reagents., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The long-sought carbene-bismuthinidene, (CAAC)Bi(Ph), has been synthesized. Notably, this represents both the first example of a carbene-stabilized subvalent bismuth complex and the extension of the carbene-pnictinidene concept to a non-toxic metallic element (Bi). The bonding has been investigated by single-crystal X-ray diffraction studies and DFT calculations. This report also highlights the hitherto unknown reducing and ligand transfer capability of a beryllium(0) complex., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Regulation of lipid absorption by enterocytes can influence metabolic status in humans and contribute to obesity and related complications. The intracellular steps of chylomicron biogenesis and transport from the Endoplasmic Reticulum (ER) to the Golgi complex have been described, but the mechanisms for post-Golgi transport and secretion of chylomicrons have not been identified. Using a newly generated Dennd5b -/- mouse, we demonstrate an essential role for this gene in Golgi to plasma membrane transport of chylomicron secretory vesicles. In mice, loss of Dennd5b results in resistance to western diet induced obesity, changes in plasma lipids, and reduced aortic atherosclerosis. In humans, two independent exome sequencing studies reveal that a common DENND5B variant, p.(R52K), is correlated with body mass index. These studies establish an important role for DENND5B in post-Golgi chylomicron secretion and a subsequent influence on body composition and peripheral lipoprotein metabolism.

Familial LCAT deficiency (FLD) is due to mutations in lecithin:cholesterol acyltransferase (LCAT), a plasma enzyme that esterifies cholesterol on lipoproteins. FLD is associated with markedly reduced levels of plasma high-density lipoprotein and cholesteryl ester and the formation of a nephrotoxic lipoprotein called LpX. We used a mouse model in which the LCAT gene is deleted and a truncated version of the SREBP1a gene is expressed in the liver under the control of a protein-rich/carbohydrate-low (PRCL) diet-regulated PEPCK promoter. This mouse was found to form abundant amounts of LpX in the plasma and was used to determine whether treatment with recombinant human LCAT (rhLCAT) could prevent LpX formation and renal injury. After 9 days on the PRCL diet, plasma total and free cholesterol, as well as phospholipids, increased 6.1 ± 0.6-, 9.6 ± 0.9-, and 6.7 ± 0.7-fold, respectively, and liver cholesterol and triglyceride concentrations increased 1.7 ± 0.4- and 2.8 ±0.9-fold, respectively, compared with chow-fed animals. Transmission electron microscopy revealed robust accumulation of lipid droplets in hepatocytes and the appearance of multilamellar LpX particles in liver sinusoids and bile canaliculi. In the kidney, LpX was found in glomerular endothelial cells, podocytes, the glomerular basement membrane, and the mesangium. The urine albumin/creatinine ratio increased 30-fold on the PRCL diet compared with chow-fed controls. Treatment of these mice with intravenous rhLCAT restored the normal lipoprotein profile, eliminated LpX in plasma and kidneys, and markedly decreased proteinuria. The combined results suggest that rhLCAT infusion could be an effective therapy for the prevention of renal disease in patients with FLD., (U.S. Government work not protected by U.S. copyright.)

Lecithin:cholesterol acyltransferase (LCAT) and LCAT-activating compounds are being investigated as treatments for coronary heart disease (CHD) and familial LCAT deficiency (FLD). Herein we report the crystal structure of human LCAT in complex with a potent piperidinylpyrazolopyridine activator and an acyl intermediate-like inhibitor, revealing LCAT in an active conformation. Unlike other LCAT activators, the piperidinylpyrazolopyridine activator binds exclusively to the membrane-binding domain (MBD). Functional studies indicate that the compound does not modulate the affinity of LCAT for HDL, but instead stabilizes residues in the MBD and facilitates channeling of substrates into the active site. By demonstrating that these activators increase the activity of an FLD variant, we show that compounds targeting the MBD have therapeutic potential. Our data better define the substrate binding site of LCAT and pave the way for rational design of LCAT agonists and improved biotherapeutics for augmenting or restoring reverse cholesterol transport in CHD and FLD patients., Competing Interests: KM, SY, BB, LC, AG, WY, LF, DM, AS, AR, AJ, JT No competing interests declared

We have observed that marine macroalgae produce sound during photosynthesis. The resultant soundscapes correlate with benthic macroalgal cover across shallow Hawaiian coral reefs during the day, despite the presence of other biological noise. Likely ubiquitous but previously overlooked, this source of ambient biological noise in the coastal ocean is driven by local supersaturation of oxygen near the surface of macroalgal filaments, and the resultant formation and release of oxygen-containing bubbles into the water column. During release, relaxation of the bubble to a spherical shape creates a monopole sound source that 'rings' at the Minnaert frequency. Many such bubbles create a large, distributed sound source over the sea floor. Reef soundscapes contain vast quantities of biological information, making passive acoustic ecosystem evaluation a tantalizing prospect if the sources are known. Our observations introduce the possibility of a general, volumetrically integrative, noninvasive, rapid and remote technique for evaluating algal abundance and rates of primary productivity in littoral aquatic communities. Increased algal cover is one of the strongest indicators for coral reef ecosystem stress. Visually determining variations in algal abundance is a time-consuming and expensive process. This technique could therefore provide a valuable tool for ecosystem management but also for industrial monitoring of primary production, such as in algae-based biofuel synthesis., Competing Interests: The authors have declared that no competing interests exist.

Cyclic(alkyl)(amino) carbene (CAAC)-stabilized complexes of phosphorus, one of the lightest group 15 elements, are well-established and can often be obtained in high yields. In contrast, analogous CAAC compounds of bismuth, the heaviest nonradioactive member of group 15, are unknown. Indeed, reactivity increases as you descend the group, and as a result there are only a few examples of N-heterocyclic carbene (NHC)-bismuth complexes. Moreover, activated bismuth compounds often readily extrude bismuth metal, making isolation of stable complexes highly challenging. We report that CAACs react with phenylbismuth dichloride (PhBiCl 2 ) to afford Et2 CAAC-Bi(Ph)Cl 2 and Cy CAAC-Bi(Ph)Cl 2 . Significantly, these complexes represent the first structurally characterized examples of CAAC-coordination to bismuth. The CAAC-stabilized bismuth compounds can also be obtained from air-stable salts, [ Et2 CAAC-H] 2 2+ [Cl 2 (Ph)Bi(μ-Cl 2 )Bi(Ph)Cl 2 ] 2- and [ Cy CAAC-H] 2 2+ [Cl 2 (Ph)Bi(μ-Cl 2 )Bi(Ph)Cl 2 ] 2- , by deprotonation with potassium bis(trimethylsilyl)amide, K[N(SiMe 3 ) 2 ]. The electronic effects of the ligand on the bismuth center were investigated by comparing the CAAC-Bi(Ph)Cl 2 complexes to the NHC analogues, SIPr-Bi(Ph)Cl 2 (THF) and IPr-Bi(Ph)Cl 2 (SIPr = 1,3-bis(2,6-diisopropylphenyl)-4,5-dihydroimidazole-2-ylidene; IPr = 1,3-bis(2,6-diisopropylphenyl)imidazole-2-ylidene). Interestingly, the "normal" IPr-Bi(Ph)Cl 2 slowly isomerizes to the "abnormal" carbene complex, Cl 2 (Ph)Bi-IPr-H, at -37 °C. In the solid-state, the CAAC-, NHC-, and abnormal NHC-bismuth compounds exhibit Bi atomic centers in unique coordination environments. The complexes were fully characterized by NMR, elemental analysis, and single crystal X-ray diffraction studies. In addition, the bonding was probed by natural bond orbital (NBO) calculations.

Background Lecithin:cholesterol acyltransferase (LCAT) is a plasma enzyme that esterifies cholesterol. Recombinant human LCAT (rhLCAT) is now being developed as an enzyme replacement therapy for familial LCAT deficiency and as a possible treatment for acute coronary syndrome. The current 'gold standard' assay for LCAT activity involves the use of radioisotopes, thus making it difficult for routine clinical use. Methods We have developed a novel and more convenient LCAT activity assay using fluorescence-labelled cholesterol (BODIPY-cholesterol), which is incorporated into proteoliposomes as a substrate instead of radiolabelled cholesterol. Results The apparent K m and V max were 31.5  µmol/L and 55.8 nmol/h/nmoL, rhLCAT, respectively, for the 3 H-cholesterol method and 103.1  µmol/L and 13.4 nmol/h/nmol rhLCAT, respectively, for the BODIPY-cholesterol method. Although the two assays differed in their absolute units of LCAT activity, there was a good correlation between the two test assays ( r = 0.849, P < 1.6 × 10 -7 , y = 0.1378x + 1.106). The BODIPY-cholesterol assay had an intra-assay CV of 13.7%, which was superior to the intra-assay CV of 20.8% for the radioisotopic assay. The proteoliposome substrate made with BODIPY-cholesterol was stable to storage for at least 10 months. The reference range ( n = 20) for the fluorescent LCAT activity assay was 4.6-24.1 U/mL/h in healthy subjects. Conclusions In summary, a novel fluorescent LCAT activity assay that utilizes BODIPY-cholesterol as a substrate is described that yields comparable results to the radioisotopic method.

Peptides mimicking the major protein of highdensity lipoprotein (HDL), apolipoprotein A-I (apoA-I), are promising therapeutics for cardiovascular diseases. Similar to apoA-I, their atheroprotective property is attributed to their ability to form discoidal HDL-like particles by extracting cellular cholesterol and phospholipids from lipid microdomains created by the ABCA1 transporter in a process called cholesterol efflux. The structural features of peptides that enable cholesterol efflux are not well understood. Herein, four synthetic amphipathic peptides denoted ELK, which only contain Glu, Leu, Lys, and sometimes Ala, and which have a wide range of net charges and hydrophobicities, were examined for cholesterol efflux. Experiments show that ELKs with a net neutral charge and a hydrophobic face that subtends an angle of at least 140° are optimal for cholesterol efflux. All-atom molecular dynamics simulations show that peptides that are effective in promoting cholesterol efflux stabilize HDL nanodiscs formed by these peptides by the orderly covering of the hydrophobic acyl chains on the edge of the disc. In contrast to apoA-I, which forms an anti-parallel double belt around the HDL, active peptides assemble in a mostly anti-parallel "picket fence" arrangement. These results shed light on the efflux ability of apoA-I mimetics and inform the future design of such therapeutics.

This paper is a critical review of past and present languages policies in Australian schooling. We highlight the One Literacy movement that contravenes the human rights of Australia's Aboriginal students. This in turn impacts students' right to freedom of opinion and expression as stated in Article 19 of the Universal Declaration of Human Rights. The One Literacy movement operates by equating Standard Australian English literacy acquisition with Australia's global competitiveness and economic success. There is only one pathway through the Australian English curriculum with common assessments and standards. However, the Australian Curriculum provides three distinctive pathways when students from an English-speaking background learn languages other than English. We reveal this double standard, where current educational policies prioritise the languages of trade (e.g. Chinese) and accommodate speakers of these languages. Meanwhile Aboriginal-language-speaking students are not provided with the same accommodations. For educational equity, there should be a distinctive English language learner pathway that recognises that the majority of remote Aboriginal students from the Northern Territory are learning English as an additional language. We advocate for these changes because all children have a right to an appropriate education that will enable them to flourish as learners and citizens.

Apolipoprotein C-II (apoC-II) is a small exchangeable apolipoprotein found on triglyceride-rich lipoproteins (TRL), such as chylomicrons (CM) and very low-density lipoproteins (VLDL), and on high-density lipoproteins (HDL), particularly during fasting. ApoC-II plays a critical role in TRL metabolism by acting as a cofactor of lipoprotein lipase (LPL), the main enzyme that hydrolyses plasma triglycerides (TG) on TRL. Here, we present an overview of the role of apoC-II in TG metabolism, emphasizing recent novel findings regarding its transcriptional regulation and biochemistry. We also review the 24 genetic mutations in the APOC2 gene reported to date that cause hypertriglyceridemia (HTG). Finally, we describe the clinical presentation of apoC-II deficiency and assess the current therapeutic approaches, as well as potential novel emerging therapies., (Published by Elsevier B.V.)