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The content of introductory chemistry courses in the Australian universities is described from an analysis of notebooks provided by students who had taken the courses. Variations in content and treatment in physical, inorganic, and organic chemistry are described. (AL)

Cryopreservation has facilitated considerable advances in both medical technology and scientific research. However, further developments have been limited by the relatively low number of effective cryoprotective agents. Even after fifty years of research, most protocols rely on the same two toxic agents, i.e. dimethylsulfoxide or glycerol. Ionic liquids are a class of promising solvents which are known glass formers and may offer a less-toxic alternative. The research presented here investigates ten protic ionic liquids as potential cryoprotective agents. The liquids are screened for key properties including cellular toxicity, permeability and thermal behaviour. The most promising, ethylammonium acetate, was then tested as a cryoprotective agent on a model cell line and was found to be as effective as the common cryoprotectant, dimethylsulfoxide. This work reports the first use of a protic ionic liquid as an effective cryoprotective agent for a mammalian cell line. This will inform the development of a suite of potential new ionic liquid-based cryoprotectants that could potentially allow the cryopreservation of new cell types.

Cross-linking mass spectrometry (XL-MS) is a powerful tool for studying protein-protein interactions and elucidating architectures of protein complexes. However, one of the inherent challenges in MS analysis of cross-linked peptides is their unambiguous identification. To facilitate this process, we have previously developed a series of amine-reactive sulfoxide-containing MS-cleavable cross-linkers. These MS-cleavable reagents have allowed us to establish a common robust XL-MS workflow that enables fast and accurate identification of cross-linked peptides using multistage tandem mass spectrometry (MSn). Although amine reactive reagents targeting lysine residues have been successful, it remains difficult to characterize protein interaction interfaces with little or no lysine residues. To expand the coverage of protein interaction regions, we present here the development of a series novel sulfoxide-containing cross-linker that target acidic residues (dihydrazide sulfoxide (DHSO)), cystine residues (bismaleimidesulfoxide (BMSO)), and finally a heterobifunctional cross-linker that targets lysine on one end and a non-specifically targets residues (Succinimidyl diazirine sulfoxide (SDASO)) on the other. We demonstrate that cross-linkers create cross-linked peptides that display the same predictable and characteristic fragmentation pattern during collision induced dissociation as amine-reactive sulfoxide-containing MS-cleavable cross-linked peptides, thus permitting their simplified analysis and unambiguous identification by MSn. Furthermore, we applied these linkers to either characterize the yeast 26S proteasome (SDASO) or the Cop9 signalosome (DSSO, DHSO and BMSO) demonstrating both the feasibility of SDASO’s photocross-linking of large protein complexes for the first time and the ability of multi-chemistry data for the integrative structure modeling of protein complexes to determine the interaction and structural dynamics of CSN, respectively. Moreover, our platform targeting various chemistries with cross-linking is robust and captures interactions complementary to residue-specific reagents, providing the foundation for future applications of multi-chemistry targeting approach to studying protein complexes.

Deep eutectic solvents (DESs) are tuneable solvents with attractive properties for numerous applications. Their structure–property relationships are still under investigation, especially at the solid–liquid interface. Moreover, the influence of water on interfacial nanostructure must be understood for process optimization. Here, we employ a combination of atomic force microscopy and molecular dynamics simulations to determine the lateral and surface-normal nanostructure of the DES choline chloride:glycerol at the mica interface with different concentrations of water. For the neat DES system, the lateral nanostructure is driven by polar interactions. The surface adsorbed layer forms a distinct rhomboidal symmetry, with a repeat spacing of ~0.9 nm, comprising all DES species. The adsorbed nanostructure remains largely unchanged in 75 mol-% DES compared with pure DES, but at 50 mol-%, the structure is broken and there is a compromise between the native DES and pure water structure. By 25 mol-% DES, the water species dominates the adsorbed liquid layer, leaving very few DES species aggregates at the interface. In contrast, the near-surface surface-normal nanostructure, over a depth of ~3 nm from the surface, remains relatively unchanged down to 25 mol-% DES where the liquid arrangement changed. These results demonstrate not only the significant influence that water has on liquid nanostructure, but also show that there is an asymmetric effect whereby water disrupts the nanostructure to a greater degree closer to the surface. This work provides insight into the complex interactions between DES and water and may enhance their optimization for surface-based applications.

Hypothesis Deep eutectic solvents (DESs) are an attractive class of tunable solvents. However, their uptake for relevant applications has been limited due to a lack of detailed information on their structure-property relationships, both in the bulk and at interfaces. The lateral nanostructure of the DES-solid interfaces is likely to be more complex than previously reported and requires detailed, high-resolution investigation. Experiments We employ a combination of high-resolution amplitude-modulated atomic force microscopy and molecular dynamics simulations to elucidate the lateral nanostructure of a DES at the solid-liquid interface. Specifically, the lateral and near-surface nanostructure of the DES choline chloride:glycerol is probed at the mica and highly-ordered pyrolytic graphite interfaces. Findings The lateral nanostructure of the DES-solid interface is heterogeneous and well-ordered in both systems. At the mica interface, the DES is strongly ordered via polar interactions. The adsorbed layer has a distinct rhomboidal symmetry with a repeat spacing of ~0.9 nm comprising all DES species. At the highly ordered pyrolytic graphite interface, the adsorbed layer appears distinctly different, forming an apolor-driven row-like structure with a repeat spacing of ~0.6 nm, which largely excludes the chloride ion. The interfacial nanostructure results from a delicate balance of substrate templating, liquid-liquid interactions, species surface affinity, and packing constraints of cations, anions, and molecular components within the DES. For both systems, distinct near-surface nanostructural layering is observed, which becomes more pronounced close to the substrate. The surface nanostructures elucidated here significantly expand our understanding of DES interfacial behavior and will enhance the optimization of DES systems for surface-based applications.

Protein arginine methyltransferase 5 (PRMT5) overexpression in hematologic and solid tumors methylates arginine residues on cellular proteins involved in important cancer functions including cell-cycle regulation, mRNA splicing, cell differentiation, cell signaling, and apoptosis. PRMT5 methyltransferase function has been linked with high rates of tumor cell proliferation and decreased overall survival, and PRMT5 inhibitors are currently being explored as an approach for targeting cancer-specific dependencies due to PRMT5 catalytic function. Here, we describe the discovery of potent and selective S-adenosylmethionine (SAM) competitive PRMT5 inhibitors, with in vitro and in vivo characterization of clinical candidate PF-06939999. Acquired resistance mechanisms were explored through the development of drug resistant cell lines. Our data highlight compound-specific resistance mutations in the PRMT5 enzyme that demonstrate structural constraints in the cofactor binding site that prevent emergence of complete resistance to SAM site inhibitors. PRMT5 inhibition by PF-06939999 treatment reduced proliferation of non–small cell lung cancer (NSCLC) cells, with dose-dependent decreases in symmetric dimethyl arginine (SDMA) levels and changes in alternative splicing of numerous pre-mRNAs. Drug sensitivity to PF-06939999 in NSCLC cells associates with cancer pathways including MYC, cell cycle and spliceosome, and with mutations in splicing factors such as RBM10. Translation of efficacy in mouse tumor xenograft models with splicing mutations provides rationale for therapeutic use of PF-06939999 in the treatment of splicing dysregulated NSCLC.

Traditional manufacturing approaches to embed active materials, such as piezoelectrics, into metals can be problematic due to their high process temperatures or long curing times of the adhesives utilized to bond the active component to the metal. This paper presents the development and characterization of aluminum embedded with a piezoelectric polyvinylidene fluoride (PVDF) sensor using a low-temperature, solid-state metal joining process called ultrasonic additive manufacturing (UAM). The embedded sensor is subjected to a pre-compression that is empirically optimized by varying the depth of the pocket and tape foils welded above the sensor, thus eliminating reliance on a bonding medium to mechanically couple the metal and the sensor. The technology presented in this study could enable rapid fabrication of functionalized metal structures that can be used in non-destructive testing, energy harvesting, and load monitoring applications.

A copper-containing antibiotic Bacteria require transition metal ions for biological processes and must also protect themselves against excess metal, which is toxic. Patteson et al . explored how the environmental bacterium Pseudomonas aeruginosa uses a five-enzyme pathway to synthesize a small-molecule complex, fluopsin C, which is built from cysteine and contains a copper ion. The biosynthesis involves unusual enzymatic transformations that convert cysteine to a thiohydroximate, two of which chelate a copper ion in the final natural product. Fluopsin C protects P. aeruginosa from excess copper and also acts as a broad-spectrum antibiotic against other bacteria. —VV

BackgroundDirect biomarkers such as phosphatidylethanol (PEth) have the capability to detect heavy alcohol use, but it is unclear how strongly self-reported reduction in alcohol use correlates with reduction in PEth. We sought to explore the strength of correlation between reductions in self-reported alcohol use and change in PEth among a sample of women living with HIV (WLWH) who participated in a clinical trial to reduce heavy alcohol use. We also sought to determine whether this correlation was stronger in women with lower body mass index (BMI) and women without an alcohol use disorder (AUD).Methods81 WLWH (mean age=48.7, 80% Black) engaging in a randomized trial of naltrexone versus placebo with a positive baseline PEth (≥8ng/ml), and alcohol use data at baseline, 2, and 7months were included in this analysis. Spearman correlation coefficients were compared to measure the correlation between baseline PEth and number of drinks per week by demographic, biological, and alcohol use factors. Mini-International Neuropsychiatric Interview was used to screen for AUD. Further analyses were stratified by BMI and AUD. Spearman correlation coefficients were calculated for the change in PEth and the change in number of drinks per week over 7months, including 3 time-points: baseline, 2, and 7months.ResultsAt baseline, the correlation between baseline PEth and the number of drinks per week was significantly stronger for those with a BMI ≤25 compared to those with a BMI>25 (r=0.66; r=0.26, respectively). Similarly, the correlation between baseline PEth and number of drinks was stronger for those who did not screen positive for AUD compared with those who did (r=0.66; r=0.25, respectively). When stratifying by BMI, a low-to-moderate correlation (r=0.32, p=0.02) was present for persons with a BMI>25; when stratifying by AUD, a moderate correlation (r=0.50, p&nbsp

Leucine-rich repeat kinase 2 (LRRK2) encodes a complex protein that includes kinase and GTPase domains. Genome-wide association studies have identified dominant LRRK2 alleles that predispose their carriers to late-onset idiotypic Parkinson's disease (PD) and also to autoimmune disorders such as Crohn's disease. Considerable evidence indicates that PD initiation and progression involve activation of innate immune functions in microglia, which are brain-resident macrophages. Here we asked whether LRRK2 modifies inflammatory signaling and how this modification might contribute to PD and Crohn's disease. We used RNA-Seq-based high-resolution transcriptomics to compare gene expression in activated primary macrophages derived from WT and Lrrk2 knockout mice. Remarkably, expression of a single gene, Rap guanine nucleotide exchange factor 3 (Rapgef3), was strongly up-regulated in the absence of LRRK2 and down-regulated in its presence. We observed similar regulation of Rapgef3 expression in cells treated with a highly specific inhibitor of LRRK2 protein kinase activity. Rapgef3 encodes an exchange protein, activated by cAMP 1 (EPAC-1), a guanine nucleotide exchange factor that activates the small GTPase Rap-1. Rap-1 mediates cell adhesion, polarization, and directional motility, and our results indicate that LRRK2 modulates chemotaxis of microglia and macrophages. Dominant PD-associated LRRK2 alleles may suppress EPAC-1 activity, further restricting motility and preventing efficient migration of microglia to sites of neuronal damage. Functional analysis in vivo in a subclinical infection model also indicated that Lrrk2 subtly modifies the inflammatory response. These results indicate that LRRK2 modulates the expression of genes involved in murine immune cell chemotaxis.

Enabling extreme fast charging (XFC, ≤10–15 min charging) requires a comprehensive understanding of its implications. While lithium plating is a key bottleneck for the anode, the full extent of limitations for the cathode are not well-understood, particularly in extended-cycle settings with well-defined battery designs and conditions. This article presents cycle-life implications of XFC on cathodes at multiple length scales, combining electrochemical analyses, degradation modeling, and post-test characterizations. The comprehensive test matrix includes 41 well-defined gr/NMC pouch cells under varied fast-charge rates (1–9C) and state-of-charges cycled up to 1000 times. Cathode issues remain minimal in early cycling, but begin to accelerate in later life, when distinct cracking is found and identified as a fatigue mechanism. The bulk structure of cathodes remains intact, but distinct particle surface reconstruction is observed; however, this shows less pronounced effect on cathode aging than does cracking.

Betaine (BET) has shown to be effective in improving body composition and performance, although research in women is lacking. This study investigated the effects of BET supplementation on markers of metabolic flexibility, body composition, and anaerobic performance in college females. Twenty-three active subjects with 21.8 ± 3.0 years of age, 66.6 ± 8.8 kg body mass, 1.6 ± 0.1 m height, and 23.2 ± 5.3% body fat performed a graded exercise test on a cycle ergometer consisting of 4 incremental, 3 min stages for collection of fat and carbohydrate oxidation rates. Three 10 s sprint tests were then completed against a resistance of 7.5% of body mass, separated by 2.5 min of recovery. The study comprised 3 phases: (a) pre-supplementation, (b) randomization to supplement for 2-weeks with either 2.4 g/day BET or placebo (parallel design), and (c) post-supplementation. Repeated-measures analysis of variance were conducted to determine interactions or main effects. There were no group differences for substrate oxidation rates (

Inflammation driven by the NLRP3 inflammasome in macrophages is an important contributor to chronic metabolic diseases that affect growing numbers of individuals. Many of these diseases involve the pathologic accumulation of endogenous lipids or their oxidation products, which can activate NLRP3. Other endogenous lipids, however, can inhibit the activation of NLRP3. The intracellular mechanisms by which these lipids modulate NLRP3 activity are now being identified. This review discusses emerging evidence suggesting that organelle stress, particularly involving mitochondria, lysosomes, and the endoplasmic reticulum, may be key in lipid-induced modification of NLRP3 inflammasome activity.

In human milk fat, palmitic acid (16:0) is esterified to the middle (sn-2 or β) position on the glycerol backbone and oleic acid (18:1) predominantly to the outer positions, giving the triacylglycerol (TG) a distinctive stereoisomeric structure that is believed to assist nutrient absorption in the infant gut. However, the fat used in most infant formulas is derived from plants, which preferentially esterify 16:0 to the outer positions. We have previously showed that the metabolism of the model oilseed Arabidopsis thaliana can be engineered to incorporate 16:0 into the middle position of TG. However, the fatty acyl composition of Arabidopsis seed TG does not mimic human milk, which is rich in both 16:0 and 18:1 and is defined by the high abundance of the TG molecular species 1,3-olein-2-palmitin (OPO). Here we have constructed an Arabidopsis fatty acid biosynthesis 1-1 fatty acid desaturase 2 fatty acid elongase 1 mutant with around 20% 16:0 and 70% 18:1 in its seeds and we have engineered it to esterify more than 80% of the 16:0 to the middle position of TG, using heterologous expression of the human lysophosphatidic acid acyltransferase isoform AGPAT1, combined with suppression of LYSOPHOSPHATIDIC ACID ACYLTRANSFERASE 2 and PHOSPHATIDYLCHOLINE:DIACYLGLYCEROL CHOLINEPHOSPHOTRANSFERASE. Our data show that oilseeds can be engineered to produce TG that is rich in OPO, which is a structured fat ingredient used in infant formulas.

High postprandial triglycerides are a strong cardiovascular disease risk factor. However, the extent triglycerides rise during daily living due to multiple meals remains poorly defined, especially in at-risk individuals. The aim of this case study was to quantify 24-h triglycerides in an individual with normal-weight obesity (NWO), metabolic syndrome (MetS), and type 2 diabetes (T2D) consuming a Western-style diet. In the morning, an indwelling catheter was inserted into a forearm vein and metabolic markers were measured hourly for 18-h and every 3-h for the last 6-h. The participants with NWO and T2D both experienced peak triglycerides ~280 mg/dL, which is considered an adverse response. The participant MetS had a similar relative change in triglycerides, but only reached 214 mg/dL. Interestingly, The participant with NWO displayed the greatest triglyceride area under the curve. Overall, we report that all three participants’ triglycerides nearly tripled with Western diet consumption, despite considerably different risk-profiles.

Nonselective antagonists of muscarinic acetylcholine receptors (mAChRs) that broadly inhibit all five mAChR subtypes provide an efficacious treatment for some movement disorders, including Parkinson's disease and dystonia. Despite their efficacy in these and other central nervous system disorders, antimuscarinic therapy has limited utility due to severe adverse effects that often limit their tolerability by patients. Recent advances in understanding the roles that each mAChR subtype plays in disease pathology suggest that highly selective ligands for individual subtypes may underlie the antiparkinsonian and antidystonic efficacy observed with the use of nonselective antimuscarinic therapeutics. Our recent work has indicated that the M4 muscarinic acetylcholine receptor has several important roles in opposing aberrant neurotransmitter release, intracellular signaling pathways, and brain circuits associated with movement disorders. This raises the possibility that selective antagonists of M4 may recapitulate the efficacy of nonselective antimuscarinic therapeutics and may decrease or eliminate the adverse effects associated with these drugs. However, this has not been directly tested due to lack of selective antagonists of M4. Here, we utilize genetic mAChR knockout animals in combination with nonselective mAChR antagonists to confirm that the M4 receptor activation is required for the locomotor-stimulating and antiparkinsonian efficacy in rodent models. We also report the synthesis, discovery, and characterization of the first-in-class selective M4 antagonists VU6013720, VU6021302, and VU6021625 and confirm that these optimized compounds have antiparkinsonian and antidystonic efficacy in pharmacological and genetic models of movement disorders.

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. However, repeated inhalation toxicity data on NNK, which is more directly relevant to cigarette smoking, are currently limited. In the present study, the subacute inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9–10 weeks age; 16 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.8, 3.2, 12.5, or 50 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.03, 0.11, 0.41, or 1.65 mg/L air) for 1 h/day for 14 consecutive days. Toxicity was evaluated by assessing body and organ weights; food consumption; clinical pathology; histopathology observations; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); O6-methylguanine DNA adduct formation; and blood and bone marrow micronucleus frequency. Whether the subacute inhalation toxicity of NNK followed Haber’s Rule was also determined using additional animals exposed 4 h/day. The results showed that NNK exposure caused multiple significant adverse effects, with the most sensitive endpoint being non-neoplastic histopathological lesions in the nose. The lowest-observed-adverse-effect level (LOAEL) was 0.8 mg/kg BW/day or 0.03 mg/L air for 1 h/day for both sexes. An assessment of Haber’s Rule indicated that 14-day inhalation exposure to the same dose at a lower concentration of NNK aerosol for a longer time (4 h daily) resulted in greater adverse effects than exposure to a higher concentration of NNK aerosol for a shorter time (1 h daily).

The organocatalytic transformation of resorcinols is extremely rare. In this article, we report a highly enantioselective, organocatalytic intramolecular cyclization of these systems by a Friedel-Crafts-type 1,4-addition using a Jorgensen-Hayashi-like organocatalyst with a large silyl protecting group, and show that heat improves reaction yield with virtually no detriment to enantioselectivity. A variety of bicyclic resorcinols were obtained with excellent enantioselectivities (up to 94%). To show the utility of these constructs, and as part of a wider project involving the synthesis of cannabinoid-like compounds, the resorcinol formed was used to generate both 'normal' and 'abnormal' cannabidiol (CBD) derivatives which were shown to have anticonvulsant activity.

Fodder beet (FB) is a source of readily fermentable carbohydrate that can mitigate early spring herbage deficits and correct the negative energy balance experienced during early lactation in pastoral dairy systems of New Zealand. However, the low-fiber and high-soluble carbohydrate content of both FB bulb and spring herbage are factors that promote subacute ruminal acidosis, impairing rumen function and limiting the marginal milk production response to supplement. In a crossover experiment, 8 Holstein Friesian × Jersey early-lactation dairy cows were used to test the effect of supplementing 16 kg of dry matter (DM) of a grazed perennial ryegrass herbage with 6 kg of DM/d of FB bulb (FBH) versus herbage only (HO) on changes in rumen function and grazing behavior. Following 20 d of adaptation to diets, DM disappearance (%) of FB bulb (FBH cows only) and herbage were measured in sacco, separately. Cows were fasted overnight, and the ruminal contents were bailed the following morning (~0930 h) again to determine the pool size of volatile fatty acids, ammonia, and particle size of digesta, as well as to estimate the rate of ruminal outflow and degradation of neutral detergent fiber. The FBH diet did not alter DM intake, milk yield, or milk solid (fat + protein) production compared with HO. Supplementation of herbage with FB reduced ruminal pH compared with HO between ~0800 h and 1300 h each day. During each period, 1 cow experienced severe subacute ruminal acidosis (pH 180 min/d) during final adaptation to the target FB allocation. The FBH diet reduced the ruminal pool of acetate and ammonia, but increased the ruminal pool of butyrate and lactate compared with HO. When fed FB, rumination and grazing time increased and grazing intensity declined compared with cows fed HO. Despite increased rumination, the comminution of large particles declined 28% between the first and second rumen bailing when cows were fed FB, and in sacco DM disappearance of perennial ryegrass declined 18% compared with cows fed HO. These results indicate that grazing dairy cows supplemented with FB (40% of daily intake) increase rumination and mastication intensity to counteract reduced ruminal degradation of ryegrass herbage due to low ruminal fluid pH.

Tailoring the pore environments of metal-organic frameworks (MOFs) is key to improving their performance and expanding their applicability. Postsynthetic methods, wherein an already synthesized MOF undergoes further chemical reactions, present many advantages for such tailoring and lead to much interesting new chemistry. However, this method has seldom been pushed farther than two reaction steps on the organic component. Here we report a three-step sequence starting from an alkenyl group on the biphenyl backbone of an IRMOF-9 analogue. The alkene is converted to an oxirane group and subsequently to a 1,2-azidoalcohol. The ultimate product is a framework functionalized with an aziridine ring. The reaction efficiency of each step is high, which suppresses the formation of undesired functional groups and the buildup of unintended multivariate frameworks. The synthesis of each framework was attempted via a direct synthetic method employing the appropriately functionalized biphenyldicarboxylate ligand. In general, this met with failure, which demonstrates the power and utility of postsynthetic methods for preparing new materials.

INTRODUCTION: The promise of the natural immunoregulator, Galectin-1 (Gal1), as an immunomodulatory therapeutic is challenged by its unstable homodimeric conformation. Previously, a Gal1 homodimer stabilized via covalent poly(ethylene glycol) diacrylate (PEGDA) cross-linking demonstrated higher activity relative to the non-covalent homodimer. METHODS: Here, we report Gal1 homodimers formed using an alternative thiol-Michael addition linker chemistry. RESULTS: Poly(ethylene glycol) bismaleimide (PEGbisMal) reacted with Gal1 at multiple sites with greater efficiency than PEGDA. However, multiple PEGbisMal molecules were conjugated to Gal1 C130, a Gal1 mutant with one surface cysteine (cys-130) and two cysteines thought to be buried in the solvent-inaccessible protein core (cys-42 and cys-60). Site-directed mutagenesis demonstrated that cys-60 was the site at which additional PEGbisMal molecules were conjugated onto Gal1 C130. Compared to WT-Gal1, Gal1 C130 had low activity for inducing Jurkat T cell death, characterized by phosphatidylserine exposure and membrane permeability. PEG cross-linking could restore the function of Gal1 C130, such that at high concentrations Gal1 C130 cross-linked by PEGbisMal had higher activity than both WT-Gal1 and Gal1 C130 cross-linked by PEGDA. Mutating cys-42 and cys-60 to serines in Gal1 C130 did not affect the cell death signaling activity of the Gal1 C130 homodimer cross-linked by PEGbisMal. PEGylated Gal1 C130 variants also eliminated the need for a reducing agent, such as dithiothreitol, which is required to maintain WT-Gal1 signaling activity. CONCLUSION: Collectively, these data demonstrate that thiol-Michael addition bioconjugation leads to a PEG-cross-linked Gal1 homodimer with improved extracellular signaling activity that does not require a reducing environment to be functional.

Luteolin (5,7,3′,4′-tetrahydroxyflavone) belongs to the flavone subclass of flavonoids. Luteolin and its glycosides are present in many botanical families, including edible plants, fruits, and vegetables. While the beneficial properties of luteolin have been widely studied, fewer studies have investigated its toxicity. In the present study, using human lymphoblastoid TK6 cells and our newly developed TK6-derived cell lines that each stably express a single human cytochrome P450 (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C18, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7), we systematically evaluated luteolin-induced cytotoxicity and genotoxicity, and the role of specific CYPs in the bioactivation of luteolin. Treatments with luteolin for 4–24 h induced cytotoxicity, apoptosis, DNA damage, and chromosome damage in a concentration-dependent manner. Subsequently, we observed that luteolin-induced cytotoxicity and genotoxicity, measured by the high-throughput micronucleus assay, were significantly increased in TK6 cells transduced with CYP1A1 and 1A2. In addition, key apoptosis and DNA damage biomarkers, including cleaved PARP-1, cleaved caspase-3, and phosphorylated histone 2AX (γH2A.X), were all significantly increased in the CYP1A1- and 1A2-expressing cells compared with the empty vector controls. Analysis by LC-MS/MS revealed that TK6 cells biotransformed the majority of luteolin into diosmetin, a less toxic O-methylated flavone, after 24 h; the presence of CYP1A1 and 1A2 partially reversed this process. Altogether, these results indicate that metabolism by CYP1A1 and 1A2 enhanced the toxicity of luteolin in vitro. Our results further support the utility of our TK6 cell system for identification of the specific CYPs responsible for chemical bioactivation and toxicity potential.

Author(s): Hahn, Judith A; Murnane, Pamela M; Vittinghoff, Eric; Muyindike, Winnie R; Emenyonu, Nneka I; Fatch, Robin; Chamie, Gabriel; Haberer, Jessica E; Francis, Joel M; Kapiga, Saidi; Jacobson, Karen; Myers, Bronwyn; Couture, Marie Claude; DiClemente, Ralph J; Brown, Jennifer L; So-Armah, Kaku; Sulkowski, Mark; Marcus, Gregory M; Woolf-King, Sarah; Cook, Robert L; Richards, Veronica L; Molina, Patricia; Ferguson, Tekeda; Welsh, David; Piano, Mariann R; Phillips, Shane A; Stewart, Scott; Afshar, Majid; Page, Kimberly; McGinnis, Kathleen; Fiellin, David A; Justice, Amy C; Bryant, Kendall; Saitz, Richard | Abstract: BackgroundObjective measurement of alcohol consumption is important for clinical care and research. Adjusting for self-reported alcohol use, we conducted an individual participant data (IPD) meta-analysis to examine factors associated with the sensitivity of phosphatidylethanol (PEth), an alcohol metabolite, among persons self-reporting unhealthy alcohol consumption.MethodsWe identified 21 eligible studies and obtained 4073 observations from 3085 participants with Alcohol Use Disorders Identification Test-Consumption (AUDIT-C) positive scores (≥3 for women and ≥4 for men) and PEth measurements. We conducted 1-step IPD meta-analysis using mixed effects models with random intercepts for study site. We examined the associations between demographic (sex, race/ethnicity, and age) and biologic (body mass index-BMI, hemoglobin, HIV status, liver fibrosis, and venous versus finger-prick blood collection) variables with PEth sensitivity (PEth≥8nng/ml), adjusting for the level of self-reported alcohol use using the AUDIT-C score.ResultsOne third (31%) of participants were women, 32% were African, 28% African American, 28% White, and 12% other race/ethnicity. PEth sensitivity (i.e., ≥8nng/ml) was 81.8%. After adjusting for AUDIT-C, we found no associations of sex, age, race/ethnicity, or method of blood collection with PEth sensitivity. In models that additionally included biologic variables, those with higher hemoglobin and indeterminate and advanced liver fibrosis had significantly higher odds of PEth sensitivity; those with higher BMI and those living with HIV had significantly lower odds of PEth sensitivity. African Americans and Africans had higher odds of PEth sensitivity than whites in models that included biologic variables.ConclusionsAmong people reporting unhealthy alcohol use, several biological factors (hemoglobin, BMI, liver fibrosis, and HIV status) were associated with PEth sensitivity. Race/ethnicity was associated with PEth sensitivity in some models but age, sex, and method of blood collection were not. Clinicians should be aware of these factors, and researchers should consider adjusting analyses for these characteristics where possible.

Deployable space structures are being built from thin-walled fiber-reinforced polymer composite materials due to their high specific strength, high specific stiffness, and designed bistability. However, the inherent viscoelastic behavior of the resin matrix can cause dimensional instability when the composite is stored under strain. The extended time of stowage between assembly and deployment in space can result in performance degradation and in the worst case, mission failure. In this study, the viscoelastic properties of candidate commercial polymers consisting of difunctional and tetrafunctional epoxies and thermoplastic and thermosetting polyimides were evaluated for deployable boom structures of solar sails. Stress relaxation master curves of the candidate polymers were used to predict the relaxation that would occur in 1 year at room temperature under relatively low strains of about 0.1%. A bismaleimide (BMI) showed less stress relaxation (about 20%) than the baseline novolac epoxy (about 50%). Carbon fiber composites fabricated with the BMI resin showed a 44% improvement in resistance to relaxation compared to the baseline epoxy composite.

The development of a polyarginine cell-penetrating peptide (CPP) could enable the treatment of age-related macular degeneration, with drugs like bevacizumab, to be administered using eye drops instead of intravitreal injections. Topical formulations have a vast potential impact on healthcare by increasing patient compliance while reducing the financial burden. However, as the ocular preparations may contain several doses, it is essential to understand the stability of the bevacizumab+CPP conjugate produced.In this work, we examine the stability of a bevacizumab solution with and without cell-penetrating peptide using dynamic light scattering and circular dichroism to assess the physical stability. We use HPLC to assess the chemical stability and ELISA to assess its biological activity. We also examine the potential of the CPP to be used as an antimicrobial agent in place of preservatives in the eye drop.The structural stability of bevacizumab with and without the CPP was found not to be affected by temperature: samples stored at either 20°C or 4°C were identical in behavior. However, physical instability was observed after five weeks, leading to aggregation and precipitation. Further investigation revealed that the addition of the polypeptide led to increased aggregation, as revealed through dynamic light scattering and concentration analysis of the peptide through HPLC. Complexing the bevacizumab with CPP had no effect on biological stability or degradation.Our findings suggest that the shelf life of CPP+bevacizumab complexes is at least 38 days from its initial formulation. Currently, the mechanism for aggregation is not fully understood but does not appear to occur through chemical degradation.

The ability to replicate the load-bearing properties of articular cartilage is attractive for many engineering applications, particularly bearings where low friction, low wear, and high durability are required. Hydrogels are widely used materials spanning many diverse applications owing to their lubricity and unique mechanical/chemical properties. The poor mechanical characteristics of conventional hydrogels, especially their compressive behavior, limit their application in load-bearing applications despite their favorable properties such as poro/viscoelasticity and lubricity. This paper demonstrates a cartilage-inspired approach to produce a structure that benefits from water-swelling resistant and ultrafast recovery behavior of elastomers as well as the stress-relaxation and energy dissipation properties of hydrogels. A method is presented in this work to fabricate interconnected macro-porous elastomers based on sintering poly(methyl methacrylate) beads. The porous elastomer imparted structural support and resilience to its composite with an infused-grafted hydrogel. At 30% strain and depending upon the strain rate, the composite exhibited a load-bearing behavior that was 14–19 times greater than that of pristine hydrogel and approximately 3 times greater than that of the porous elastomer. The equilibrium elastic modulus of the composite was 452 kPa at a strain range of 10%–30%, which was close to the values reported for the modulus of cartilage tested with similar experimental parameters defined in this study. The dissipated energy for the composite at strain rates of 1 and 10–3 s–1 was enhanced by 25-, 25-, 5-, and 15-fold as compared to that for the pristine hydrogel and the porous elastomer, respectively. The cyclic loading tests at two strain rates showed that the composite immediately recovers its load-bearing properties with the maximum load recovery staying above 95% of its initial values throughout the testing. The permeability of the structures was measured experimentally, and the results showed a decrease of permeability by 3 orders of magnitude following hydrogel grafting.

The leukocyte immunoglobulin-like receptor A3 (LILRA3) is a soluble protein primarily expressed by peripheral blood monocytes and is abundant in sera of healthy donors. Extracellular LILRA3 is anti-inflammatory and displays neuro-regenerative functions in vitro. However, its intracellular expression, distribution, and function(s) remain unknown. Using a combination of high-resolution confocal and super-resolution microscopy, we identified intracellular expression of native LILRA3 in the nucleus of peripheral blood monocytes and in vitro-derived macrophages. This unexpected nuclear localization of LILRA3 was confirmed in LILRA3-GFP-transfected HEK293T cells. Western blot of proteins fractionated from primary macrophages and the transfected HEK293T cells confirmed nuclear localization of the native and expressed LILRA3 proteins. Interestingly, most of the LILRA3 in the nucleus was in a monomeric form like the biologically active secreted protein, while that in the other cellular compartments was in mixed monomeric, dimeric, and oligomeric forms. The predominant presence of monomeric LILRA3 in the nucleus was independently corroborated in transfected live HEK293T cells using the number and molecular brightness (N&B) analysis method. Immunoprecipitation and mass spectrometric peptide sequencing studies revealed that nuclear LILRA3 co-immunoprecipitated with several nuclear proteins involved in host protein synthesis machinery via direct interactions to a key multifunctional RNA-binding protein, the Ewing sarcoma breakpoint region 1 protein (EWS) (data are available via ProteomeXchange with identifier PXD024602). The biological significance of the nuclear expression of LILRA3 and its interaction with these key proteins remain to be elucidated.

At the basis of cell shape and behavior, the organization of actomyosin and its ability to generate forces are widely studied. However, the precise regulation of this contractile network in space and time is unclear. Here, we study the role of the epithelial-specific protein EpCAM, a contractility modulator, in cell shape and motility. We show that EpCAM is required for stress fiber generation and front-rear polarity acquisition at the single cell level. In fact, EpCAM participates in the remodeling of a transient zone of active RhoA at the cortex of spreading epithelial cells. EpCAM and RhoA route together through the Rab35/EHD1 fast recycling pathway. This endosomal pathway spatially organizes GTP-RhoA to fine tune the activity of actomyosin resulting in polarized cell shape and development of intracellular stiffness and traction forces. Impairment of GTP-RhoA endosomal trafficking either by silencing EpCAM or by expressing Rab35/EHD1 mutants prevents proper myosin-II activity, stress fiber formation and ultimately cell polarization. Collectively, this work shows that the coupling between co-trafficking of EpCAM and RhoA, and actomyosin rearrangement is pivotal for cell spreading, and advances our understanding of how biochemical and mechanical properties promote cell plasticity., The organization and force-generating property of actomyosin dictate the plasticity and behaviour of cells but the spatio-temporal regulation of this network is unclear. Here, the authors show that coupling between EpCAM/RhoA co-trafficking and actomyosin rearrangement is pivotal during cell spreading and polarization.