Deep Profiling of Plasma Proteoforms with Engineered Nanoparticles for Top-down Proteomics.

The dynamic range challenge for detection of proteins and their proteoforms in human plasma has been well documented. Here, we use the nanoparticle protein corona approach to enrich low-abundant proteins selectively and reproducibly from human plasma and use top-down proteomics to quantify differential enrichment for the 2841 detected proteoforms from 114 proteins. Furthermore, nanoparticle enrichment allowed top-down detection of proteoforms between ∼1 µg/mL and ∼10 pg/mL in absolute abundance, providing up to 10 5 -fold increase in proteome depth over neat plasma in which only proteoforms from abundant proteins (>1 µg/mL) were detected. The ability to monitor medium and some low abundant proteoforms through reproducible enrichment significantly extends the applicability of proteoform research by adding depth beyond albumin, immunoglobins and apolipoproteins to uncover many involved in immunity and cell signaling. As proteoforms carry unique information content relative to peptides, this report opens the door to deeper proteoform sequencing in clinical proteomics of disease or aging cohorts.

Chimeric mRNA-based COVID-19 vaccine induces protective immunity against Omicron and Delta variants.

The emerging SARS-CoV-2 variants of concern (VOCs) exhibit enhanced transmission and immune escape, reducing the effectiveness of currently approved mRNA vaccines. To achieve wider coverage of VOCs, we first constructed a cohort of mRNAs harboring a furin cleavage mutation in the spike (S) protein of predominant VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2). The mutation abolished the cleavage between the S1 and S2 subunits. Systematic evaluation in vaccinated mice discovered that individual VOC mRNAs elicited strong neutralizing activity in a VOC-specific manner. In particular, the neutralizing antibodies (nAb) produced by immunization with Beta-Furin and Washington (WA)-Furin mRNAs showed potent cross-reactivity with other VOCs. However, neither mRNA elicited strong neutralizing activity against the Omicron variant. Hence, we further developed an Omicron-specific mRNA vaccine that restored protection against the original Omicron variant and some sublineages. Finally, to broaden the protection spectrum of the new Omicron mRNA vaccine, we engineered an mRNA-based chimeric immunogen by introducing the receptor-binding domain of Delta variant into the entire S antigen of Omicron. The resultant chimeric mRNA induced potent and broadly nAbs against Omicron and Delta, which paves the way to developing new vaccine candidates to target emerging variants in the future.

An injectable microparticle formulation for the sustained release of the specific MEK inhibitor PD98059: in vitro evaluation and pharmacokinetics.

PD98059 is a reversible MEK inhibitor that we are investigating as a potential treatment for neurochemical changes in the brain that drive neurohumoral excitation in heart failure. In a rat model that closely resembles human heart failure, we found that central administration of PD98059 inhibits phosphorylation of ERK1/2 in the paraventricular nucleus of the hypothalamus, ultimately reducing sympathetic excitation which is a major contributor to clinical deterioration. Studies revealed that the pharmacokinetics and biodistribution of PD98059 match a two-compartment model, with drug found in brain as well as other body tissues, but with a short elimination half-life in plasma (approximately 73 min) that would severely limit its potential clinical usefulness in heart failure. To increase its availability to tissues, we prepared a sustained release PD98059-loaded PLGA microparticle formulation, using an emulsion solvent evaporation technique. The average particle size, yield percent, and encapsulation percent were found to be 16.73 µm, 76.6%, and 43%, respectively. In vitro drug release occurred over 4 weeks, with no noticeable burst release. Following subcutaneous injection of the microparticles in rats, steady plasma levels of PD98059 were detected by HPLC for up to 2 weeks. Furthermore, plasma and brain levels of PD98059 in rats with heart failure were detectable by LC/MS, despite expected erratic absorption. These findings suggest that PD98059-loaded microparticles hold promise as a novel therapeutic intervention countering sympathetic excitation in heart failure, and perhaps in other disease processes, including cancers, in which activated MAPK signaling is a significant contributing factor. Graphical abstract.

Enhanced Glucose Uptake in Human Liver Cells and Inhibition of Carbohydrate Hydrolyzing Enzymes by Nordic Berry Extracts.

A Western lifestyle with low physical activity and a diet rich in sugar, fat and processed food contribute to higher incidences of diabetes and obesity. Enhanced glucose uptake in human liver cells was observed after treatment with phenolic extracts from different Nordic berries. All berry extracts showed higher inhibition against α-amylase and α-glucosidase than the anti-diabetic agent acarbose. Total phenolic content and phenolic profiles in addition to antioxidant activities, were also investigated. The berries were extracted with 80% methanol on an accelerated solvent extraction system (ASE) and then purified by C-18 solid phase extraction (SPE). Among the ASE methanol extracts, black chokeberry, crowberry and elderberry extracts showed high stimulation of glucose uptake in HepG2 cells and also considerable inhibitory effect towards carbohydrate hydrolyzing enzymes. SPE extracts with higher concentrations of phenolics, resulted in increased glucose uptake and enhanced inhibition of α-amylase and α-glucosidase compared to the ASE extracts. Crowberry and cloudberry were the most potent 15-lipoxygenase inhibitors, while bog whortleberry and lingonberry were the most active xanthine oxidase inhibitors. These results increase the value of these berries as a component of a healthy Nordic diet and have a potential benefit against diabetes.

Elderberry and Elderflower Extracts, Phenolic Compounds, and Metabolites and Their Effect on Complement, RAW 264.7 Macrophages and Dendritic Cells.

Modulation of complement activity and inhibition of nitric oxide (NO) production by macrophages and dendritic cells may have therapeutic value in inflammatory diseases. Elderberry and elderflower extracts, constituents, and metabolites were investigated for their effects on the complement system, and on NO production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages and murine dendritic D2SC/I cells. The EtOH crude extracts from elderberry and elderflower and the isolated anthocyanins and procyanidins possessed strong complement fixating activity and strong inhibitory activity on NO production in RAW cells and dendritic cells. Phenolic compounds in the range of 0.1-100 µM showed a dose-dependent inhibition of NO production, with quercetin, rutin, and kaempferol as the most potent ones. Among the metabolites, caffeic acid and 3,4-dihydroxyphenylacetic acid showed the strongest inhibitory effects on NO production in both cell lines, without having cytotoxic effect. Only 4-methylcatechol was cytotoxic at the highest tested concentration (100 µM). Elderberry and elderflower constituents may possess inflammatory modulating activity, which increases their nutritional value.

Phenolic Elderberry Extracts, Anthocyanins, Procyanidins, and Metabolites Influence Glucose and Fatty Acid Uptake in Human Skeletal Muscle Cells.

Uptake of glucose and fatty acids in skeletal muscle is of interest for type 2 diabetes treatment. The aim was to study glucose and fatty acid uptake in skeletal muscle cells, antioxidant effects, and inhibition of carbohydrate-hydrolyzing enzymes by elderberries. Enhanced glucose and oleic acid uptake in human skeletal muscle cells were observed after treatment with phenolic elderberry extracts, anthocyanins, procyanidins, and their metabolites. The 96% EtOH and the acidified MeOH extracts were highly active. Of the isolated substances, cyanidin-3-glucoside and cyanidin-3-sambubioside showed highest stimulation of uptake. Phloroglucinol aldehyde was most active among the metabolites. Isolated anthocyanins and procyanidins are strong radical scavengers and are good inhibitors of 15-lipoxygenase and moderate inhibitors of xanthine oxidase. As α-amylase and α-glucosidase inhibitors, they are considerably better than the positive control acarbose. The antidiabetic property of elderberry phenolics increases the nutritional value of this plant and indicates potential as functional food against diabetes.

HPLC-UV method for simultaneous determination of MK-1775 and AZD-7762 in both acetonitrile-aqueous solution and mouse plasma.

A sensitive and precise method is described for the simultaneous determination of two small molecule kinase inhibitors: MK-1775 (MK) and AZD-7762 (AZD), in acetonitrile (ACN)-aqueous solution and in mouse plasma. A Nova-Pak C18 reversed phase column (3.9mm×150mm, 4µm, 60Å) was utilized in the separation using an isocratic mobile phase of 0.1% v/v triethylamine in phosphate buffer (pH=7.4): acetonitrile (ACN) (60:40, v/v), at a flow rate of 0.8mL/min. Detection wavelength was set at 310nm for both MK and AZD, and 431nm for the internal standard sunitinib (SUN). The developed method was validated following the ICH guidelines and it was shown to be accurate, precise and linear in the range of 41ng/mL to 8333ng/mL for both drugs in the ACN-aqueous solution and from 83ng/mL to 8333ng/mL for both drugs in mouse plasma samples. For the first time, the presented data suggest the suitability of this method for the simultaneous separation and quantification of MK and AZD in both ACN aqueous solution as well as in mouse plasma samples.

Effect of Phenolic Compounds from Elderflowers on Glucose- and Fatty Acid Uptake in Human Myotubes and HepG2-Cells.

Type 2 diabetes (T2D) is manifested by progressive metabolic impairments in tissues such as skeletal muscle and liver, and these tissues become less responsive to insulin, leading to hyperglycemia. In the present study, stimulation of glucose and oleic acid uptake by elderflower extracts, constituents and metabolites were tested in vitro using the HepG2 hepatocellular liver carcinoma cell line and human skeletal muscle cells. Among the crude extracts, the 96% EtOH extract showed the highest increase in glucose and oleic acid uptake in human skeletal muscle cells and HepG2-cells. The flavonoids and phenolic acids contained therein were potent stimulators of glucose and fatty acid uptake in a dose-dependent manner. Most of the phenolic constituents and several of the metabolites showed high antioxidant activity and showed considerably higher α-amylase and α-glucosidase inhibition than acarbose. Elderflower might therefore be valuable as a functional food against diabetes.

RG-I regions from elderflower pectins substituted on GalA are strong immunomodulators.

Sambuci flos, also known as elderflower, has traditionally been used and is still in use for treatment of various types of illnesses related to the immune system such as cold, flu, fever and inflammation. Pectic polysaccharides from 50% EtOH, 50°C water and 100°C water extracts from elderflowers were treated with endo-α-d-(1-4)-polygalacturonase after previous de-esterification with the intention of isolating hairy regions and relate variation in structure to immunomodulating activity. High molecular weight sub-fractions (25-29kDa) and medium molecular weight sub-fractions (6-17kDa) were isolated after enzymatic treatment in addition to oligogalacturonides. Structural elucidation indicated that RG-I regions with AG-I and AG-II sidechains were the predominant structures in the high molecular weight sub-fractions, and two of three 1,4-linked GalA units in the rhamnogalacturonan backbone were branched in either position 2 or 3. The medium molecular weight sub-fractions had monomers and linkages typical for both RG-I and RG-II. The results showed that the high molecular RG-I containing polymers exhibit the highest dose-dependent complement fixing and macrophage stimulating activities.

A new benzophenanthridine alkaloid and other bioactive constituents from the stem bark of Zanthoxylum heitzii.

Heitziquinone (7), a new benzophenanthridine alkaloid, together with five known compounds; isoarnottianamide (5), rhoifoline B (6), isobauerenol (8), 6-hydroxypellitorine (9) and sylvamide (10), were isolated as minor compounds from the hexane extract of stem bark from Zanthoxylum heitzii. Four previously reported compounds (1-4) were found, as well. Compounds 5 and 7 were both found to exist as 4:1 mixtures of two atropisomers. The structures were elucidated by 1D and 2D NMR spectroscopy and by mass spectrometry. Compounds 5-10 were identified for the first time in this species, and they are all rare natural compounds. Pellitorine (4), one of the main compounds from the hexane bark extract, was found to be responsible for the brine shrimp larvae toxicity (LC50 37 µM, 8 µg/ml) of the crude extract (LC50 24 µg/ml). Low cytotoxicity against a macrophage cell line was observed.

Structural characterization of bioactive pectic polysaccharides from elderflowers (Sambuci flos).

Elderflowers have traditionally been used and are still used for its anti-inflammatory property. Traditionally elderflowers were used as remedies against cold, flu and diuretic. The aim of this study was to relate the structure of pectic-polysaccharides from elderflowers to immunomodulating properties. Purified fractions obtained by gelfiltration and ion exchange chromatography of 50% ethanol, 50°C water and 100°C water extracts exhibited strong complement fixating activity and macrophage stimulating activity. Reduced bioactivity was observed after removal of arabinose and 1,3,6-Gal linkages by weak acid hydrolysis. Enhanced bioactivity was observed after removal of estergroups by NaOH. Relating linkage analysis to the results of the bioactivity tests, led to the assumption that the branched moieties of the arabinogalactans linked to rhamnogalacturonan region, is important for the immunomodulating activity seen in elderflowers. No cytotoxity was observed.

Structure-activity relationship of immunomodulating pectins from elderberries.

The berries of Sambucus nigra have traditionally been used and are still used to treat respiratory illnesses such as cold and flu in Europe, Asia and America. The aim of this paper was to elucidate the structures and the immunomodulating properties of the pectic polymers from elderberries. All the purified fractions obtained from 50% ethanol, 50°C water and 100°C water extracts showed potent dose-dependent complement fixating activity and macrophage stimulating activity. The isolated fractions consisted of long homogalacturonan regions, in addition to arabinogalactan-I and arabinogalactan-II probably linked to a rhamnogalacturonan backbone. Reduced bioactivity was observed after reduction of Araf residues and 1→3,6 Gal by weak acid hydrolysis. The rhamnogalacturonan region in SnBe50-I-S3-I and SnBe50-I-S3-II showed higher activity compared to the native polymer, SnBe50-S3, after enzymatic treatment with endo-α-d-(1→4)-polygalacturonase. These results indicated that elderberries contained immunomodulating polysaccharides, where the ramified regions express the activities observed.

Immunomodulating pectins from root bark, stem bark, and leaves of the Malian medicinal tree Terminalia macroptera, structure activity relations.

The root bark, stem bark, and leaves of Terminalia macroptera were sequentially extracted with ethanol, 50% ethanol-water, and 50°C water using an accelerated solvent extractor (ASE). Six bioactive purified pectic polysaccharide fractions were obtained from the 50°C crude water extracts after anion exchange chromatography and gel filtration. The root bark, stem bark, and leaves of T. macroptera were all good sources for fractions containing bioactive polysaccharides. The high molecular weight fraction 50WTRBH-I-I, being the most active fraction in the complement fixation test, has a highly ramified rhamnogalacturonan type I (RG-I) region with arabinogalactan type II (AG-II) side chains. The most abundant fractions from each plant part, 50WTRBH-II-I, 50WTSBH-II-I, and 50WTLH-II-I, were chosen for pectinase degradation. The degradation with pectinase revealed that the main features of these fractions are that of pectic polysaccharides, with hairy regions (RG-I regions) and homogalacturonan regions. The activity of the fractions obtained after pectinase degradation and separation by gel filtration showed that the highest molecular weight fractions, 50WTRBH-II-Ia, 50WTSBH-II-Ia, and 50WTLH-II-Ia, had higher complement fixation activity than their respective native fractions. These results suggest that the complement fixation activities of these pectins are expressed mainly by their ramified regions.

Effects of palladium on the optical and hydrogen sensing characteristics of Pd-doped ZnO nanoparticles.

The effect of palladium doping of zinc oxide nanoparticles on the photoluminescence (PL) properties and hydrogen sensing characteristics of gas sensors is investigated. The PL intensity shows that the carrier dynamics coincides with the buildup of the Pd-related green emission. The comparison between the deep level emission and the gas sensing response characteristics allows us to suggest that the dissociation of hydrogen takes place at PdZn-vacancies ([Pd (2+)(4d(9))]). The design of this sensor allows for a continuous monitoring in the range of 0-100% LEL H2 concentration with high sensitivity and selectivity.

Immunomodulating activity of Aronia melanocarpa polyphenols.

The immunomodulating effects of isolated proanthocyanidin-rich fractions, procyanidins C1, B5 and B2 and anthocyanins of Aronia melanocarpa were investigated. In this work, the complement-modulating activities, the inhibitory activities on nitric oxide (NO) production in LPS-induced RAW 264.7 macrophages and effects on cell viability of these polyphenols were studied. Several of the proanthocyanidin-rich fractions, the procyanidins C1, B5 and B2 and the cyanidin aglycone possessed strong complement-fixing activities. Cyanidin 3-glucoside possessed stronger activity than the other anthocyanins. Procyanidins C1, B5 and B2 and proanthocyanidin-rich fractions having an average degree of polymerization (PD) of 7 and 34 showed inhibitory activities on NO production in LPS-stimulated RAW 264.7 mouse macrophages. All, except for the fraction containing proanthocyanidins with PD 34, showed inhibitory effects without affecting cell viability. This study suggests that polyphenolic compounds of A. melanocarpa may have beneficial effects as immunomodulators and anti-inflammatory agents.

Enzyme inhibition, antioxidant and immunomodulatory activities, and brine shrimp toxicity of extracts from the root bark, stem bark and leaves of Terminalia macroptera.

ETHNOPHARMACOLOGICAL RELEVANCE: The root bark, stem bark and leaves of Terminalia macroptera have been traditionally used against a variety of ailments such as wounds, hepatitis, malaria, fever, cough, and diarrhea as well as tuberculosis and skin diseases in African folk medicine. Boiling water extracts of Terminalia macroptera, administered orally, are the most common preparations of this plant used by the traditional healers in Mali. This study aimed to investigate the inhibition of the activities of α-glucosidase, 15-lipoxygenase and xanthine oxidase, DPPH scavenging activity, complement fixation activity and brine shrimp toxicity of different extracts obtained by boiling water extraction (BWE) and by ASE (accelerated solvent extraction) with ethanol, ethanol-water and water as extractants from different plant parts of Terminalia macroptera. MATERIALS AND METHODS: 27 different crude extracts were obtained by BWE and ASE from root bark, stem bark and leaves of Terminalia macroptera. The total phenolic and carbohydrate contents, enzyme inhibition activities (α-glucosidase, 15-lipoxygenase and xanthine oxidase), DPPH scavenging activity, complement fixation activity and brine shrimp toxicity of these extracts were evaluated. Principal component analysis (PCA) was applied for total biological activities evaluation. RESULTS: Several of the extracts from root bark, stem bark and leaves of Terminalia macroptera obtained by BWE and ASE showed potent enzyme inhibition activities, radical-scavenging properties and complement fixation activities. None of the extracts are toxic against brine shrimp larvae in the test concentration. Based on the results from PCA, the ASE ethanol extracts of root bark and stem bark and the low molecular weight fraction of the 50% ethanol-water extract of leaves showed the highest total biological activities. The boiling water extracts were less active, but the bark extracts showed activity as α-glucosidase inhibitors and radical scavengers, the leaf extract being less active. CONCLUSION: The observed enzyme inhibition activities, radical scavenging properties and complement fixation activities may explain some of the traditional uses of this medicinal tree, such as in wound healing and against diabetes.

Design of interpenetrated network MWCNT/poly(1,5-DAN) on interdigital electrode: toward NO2 gas sensing.

In this paper, poly(1,5-diaminonaphthalene) was interpenetrated into the network made of multiwalled carbon nanotubes (MWCNT) on platinum interdigital electrode (IDE) by electro-polymerization of 1,5-diaminonaphthalene (1,5-DAN). The electro-polymerization process of 1,5-DAN on MWCNT was controlled by scanning the cyclic voltage at 50 mV s(-1) scan rate between -0.1 V and +0.95 V vs. saturated calomel electrode (SCE). The results of voltammetric responses and Raman spectroscopy represented that the films MWCNT/poly(1,5-DAN) were successfully created by this polymerization process. The films MWCNT/poly(1,5-DAN) were investigated for gas-sensing to NO2 at low concentration level. The gas-sensing results showed that the response-recovery times were long and strongly affected by thickness of the film MWCNT/poly(1,5-DAN). Nevertheless, these films represented auspicious results for gas sensors operating at room temperature.

Different uses of plant semiochemicals in host location strategies of the two tachinid parasitoids.

Some members of the family Tachinidae (Insecta: Diptera) deposit numerous very small eggs, termed "microtype" eggs, on the food plants of their caterpillar hosts. Parasitization is successful only when the hosts ingest these eggs. To increase the chance of hosts encountering the eggs, microtype tachinid parasitoids have to choose a suitable plant that harbors hosts and lay their eggs near the hosts. In their host location process, semiochemicals emitted by host-infested plants offer the tachinids a reliable cue. We investigated the behavioral responses of two microtype tachinid parasitoids, Pales pavida and Zenillia dolosa, to maize plants infested with their caterpillar host, Mythimna separata, in a wind tunnel. P. pavida females showed a significantly higher rate of landing on caterpillar-infested plants than on mechanically wounded or intact plants, whereas Z. dolosa landed on both the caterpillar-infested and mechanically wounded plants at significantly higher rates than on intact plants. We also examined which part of a caterpillar-infested maize leaf induces oviposition. P. pavida deposited eggs on the margin of the leaf, whereas Z. dolosa preferentially laid eggs around a caterpillar-infested area or a mechanically wounded spot. P. pavida eggs retained their parasitization ability for more than 15 days after they were deposited, whereas the eggs of Z. dolosa could not survive more than 5 days after oviposition. Our results suggest that each tachinid parasitoid employs a different host location strategy to exploit semiochemicals coming from plant-herbivore interaction as cues in order to increase their parasitization success.