Journal articles on the topic 'CLC-MS/MS'

The present study summarizes the new strategies including advanced equipment and validation parameters of liquid and gas chromatography methods i.e., thin-layer chromatography (TLC), column liquid chromatography (CLC), and gas chromatography (GC) suitable for the identification and quantitative determination of different natural and synthetic bioactive compounds present in food and food products, which play an important role in human health, within the period of 2019–2021 (January). Full characteristic of some of these procedures with their validation parameters is discussed in this work. The present review confirms the vital role of HPLC methodology in combination with different detection modes i.e., HPLC-UV, HPLC-DAD, HPLC-MS, and HPLC-MS/MS for the determination of natural and synthetic bioactive molecules for different purposes i.e., to characterize the chemical composition of food as well as in the multi-residue analysis of pesticides, NSAIDs, antibiotics, steroids, and others in food and food products.

Blue orchard bees, [Osmia lignaria (Say) (Hymenoptera: Megachilidae)], have been developed as an important pollinator for orchard crops in North America over the last 40 years. The toxicity of several pesticides to O. lignaria and other Osmia species has been previously reported. However, the field-realistic toxicity of formulated premix insecticides comprised of multiple active ingredients (each with a different mode of action) to O. lignaria has not been assessed. Here, we use a customized spray tower in a laboratory setting to assess adult male and female whole-body direct contact exposure to four formulated pesticide mixtures: thiamethoxam + lambda-cyhalothrin (TLC), imidacloprid + beta-cyfluthrin (IBC), chlorantraniliprole + lambda-cyhalothrin (CLC) and methoxyfenozide + spinetoram (MS) by directly spraying anesthetized bees in Petri dishes. Separately, adult male and female whole-body direct contact exposure to formulated imidacloprid (I), beta-cyfluthrin (BC) and their 1:1 binary combination (IBC) was assessed using the same experimental method. Resulting mortality in each study was screened up to 96 h post-treatment to determine acute whole-body contact toxicity. In the first study, TLC and IBC resulted in statistically higher mortality at 24 and 48 h than the two other insecticide combinations tested. The CLC and MS combinations were slower acting and the highest mortality for O. lignaria exposed to these mixtures was recorded at 96 h. We did observe significant differences in toxicity between CLC and MS. In the second study, exposure to the 1:1 binary combination of IBC caused overall significantly higher mortality than exposure to I or BC alone. Both active ingredients alone, however, demonstrated equivalent levels of mortality to the 1:1 binary combination treatment at the 96 h observation reading, indicating increased speed of kill, but not necessarily increased toxicity. Significant differences in the onset of mortality following acute contact whole-body exposure to the formulated insecticide mixtures and individual active ingredients tested were consistently observed across all experiments in both studies.

The ClC family of chloride channels and transporters includes several members in which mutations have been associated with human disease. An understanding of the structure–function relationships of these proteins is essential for defining the molecular mechanisms underlying pathogenesis. To date, the X-ray crystal structures of prokaryotic ClC transporter proteins have been used to model the membrane domains of eukaryotic ClC channel-forming proteins. Clearly, the fidelity of these models must be evaluated empirically. In the present study, biochemical tools were used to define the membrane domain boundaries of the eukaryotic protein, ClC-2, a chloride channel mutated in cases of idiopathic epilepsy. The membrane domain boundaries of purified ClC-2 and accessible cysteine residues were determined after its functional reconstitution into proteoliposomes, labelling using a thiol reagent and proteolytic digestion. Subsequently, the lipid-embedded and soluble fragments generated by trypsin-mediated proteolysis were studied by MS and coverage of approx. 71% of the full-length protein was determined. Analysis of these results revealed that the membrane-delimited boundaries of the N- and C-termini of ClC-2 and the position of several extramembrane loops determined by these methods are largely similar to those predicted on the basis of the prokaryotic protein [ecClC (Escherichia coli ClC)] structures. These studies provide direct biochemical evidence supporting the relevance of the prokaryotic ClC protein structures towards understanding the structure of mammalian ClC channel-forming proteins.

Gating of the muscle chloride channel CLC-1 involves at least two processes evidenced by double-exponential current relaxations when stepping the voltage to negative values. However, there is little information about the gating of CLC-1 at positive voltages. Here, we analyzed macroscopic gating of CLC-1 over a large voltage range (from −160 to +200 mV). Activation was fast at positive voltages but could be easily followed using envelope protocols that employed a tail pulse to −140 mV after stepping the voltage to a certain test potential for increasing durations. Activation was biexponential, demonstrating the presence of two gating processes. Both time constants became exponentially faster at positive voltages. A similar voltage dependence was also seen for the fast gate time constant of CLC-0. The voltage dependence of the time constant of the fast process of CLC-1, τf, was steeper than that of the slow one, τs (apparent activation valences were zf ∼ −0.79 and zs ∼ −0.42) such that at +200 mV the two processes became kinetically distinct by almost two orders of magnitude (τf ∼ 16 μs, τs ∼ 1 ms). This voltage dependence is inconsistent with a previously published gating model for CLC-1 (Fahlke, C., A. Rosenbohm, N. Mitrovic, A.L. George, and R. Rüdel. 1996. Biophys. J. 71:695–706). The kinetic difference at 200 mV allowed us to separate the steady state open probabilities of the two processes assuming that they reflect two parallel (not necessarily independent) gates that have to be open simultaneously to allow ion conduction. Both open probabilities could be described by Boltzmann functions with gating valences around one and with nonzero “offsets” at negative voltages, indicating that the two “gates” never close completely. For comparison with single channel data and to correlate the two gating processes with the two gates of CLC-0, we characterized their voltage, pHint, and [Cl]ext dependence, and the dominant myotonia inducing mutation, I290M. Assuming a double-barreled structure of CLC-1, our results are consistent with the identification of the fast and slow gating processes with the single-pore and the common-pore gate, respectively.

Background: Hemophilia A is caused by deficiency of factor VIII (F.VIII), an essential blood-clotting cofactor. The current standard of care involves prophylactic or on-demand use of F.VIII. However, approximately 30% of severe hemophilia A patients develop inhibitory antibodies against the replacement factor VIII. Additionally, venous infusion of F.VIII is burdensome especially in pediatric patients. Hemlibra®, a humanized bispecific antibody (BiAb) that mimics the action of factor VIII, has been developed and approved for treating hemophilia A patients with or without inhibitors. Using Kymab's IntelliSelect® Bispecific antibody discovery platform, a fully human common light chain (CLC) bispecific antibody was developed and optimized to mimic the function of F.VIII. The biological activity of the CLC bispecific antibody was characterized using clinically relevant hemostatic assays. Methods: Kymab IntelliSelect® Transgenic mice were immunized with human factor IX (F.IX) or factor X (F.X). Isolated F.IX and F.X arms were combinatorially expressed as 2-heavy-2-light (2H2L) BiAbs. Light chain of a biologically active F.IX arm was chosen to generate CLC transgenic mice. CLC transgenic mice were immunized with F.X to identify F.X heavy chains pairing with CLC. The isolated F.X heavy chains were co-expressed with the heavy and light chains of the selected F.IX arm as CLC BiAbs and re-screened by functional assays. Biologically active BiAbs were further optimized to generate a lead BiAb, KY1049. KY1049 heterodimer was purified by a two-step method using Protein A and cation ion exchange chromatography (cIEX). The purified BiAb was analyzed by analytical HPLC and mass spectrometry (MS). The purified BiAb was also characterized using a combination of in vitro and ex vivo hemostatic assays including chromogenic FXase (FXase), activated partial thromboplastin time (aPTT) and thrombin generation assay (TGA). Binding of KY1049 to F.IX and F.X was studied using surface plasmon resonance (SPR). Results: More than 8,000 2H2L BiAbs were first screened by high-throughput chromogenic FXase assay. Functionally active 2H2L BiAbs were identified, consisting of one F.IX arm and different F.X arms. The light chain of this promiscuous F.IX arm was chosen to generate CLC transgenic mice expressing the light chain exclusively. More than 400 F.X heavy chains isolated from the CLC transgenic mice were screened to identify functionally active CLC BiAbs. Further work to iteratively and combinatorially optimize the variable domains resulted in KY1049, a lead BiAb which demonstrates robust hemostatic activity. KY1049 demonstrates a dose-dependent reduction in clotting time and a dose-dependent increase in thrombin burst, thereby functionally restoring the hemostatic activity of F.VIII-depleted plasma. Compared with a sequence identical analog (SIA) of Hemlibra®, KY1049 shows comparable efficacy in a range of concentration of IgG (0.1 to 300 nM). Importantly, KY1049 can be purified using a routine purification process. The purity and the identity of KY1049 was confirmed by mass spectrometry. KY1049 was shown to simultaneously bind F.IX and F.X using SPR in confirmation of its mode of action. Summary/conclusions: KY1049, developed using Kymab's IntelliSelect® Bispecific platform, is a potent F.VIII mimetic bispecific antibody with activities comparable to a SIA of Hemlibra®. This fully human CLC bispecific antibody can potentially provide a new treatment option for hemophilia A patients. Disclosures Wang: Kymab Ltd.: Employment, Equity Ownership, Patents & Royalties. Blackwood:Kymab Ltd.: Employment, Equity Ownership, Patents & Royalties. Magliozzi:Kymab Ltd.: Employment, Equity Ownership, Patents & Royalties. Moraes:Kymab Ltd.: Employment, Equity Ownership. Hollins:Kymab Ltd.: Employment, Equity Ownership. Sinopoli:Kymab Ltd.: Employment, Equity Ownership. Chi:Kymab Ltd.: Employment, Equity Ownership. Mitchell:Kymab Ltd.: Employment, Equity Ownership. Sellick:Kymab Ltd.: Employment, Equity Ownership. Pearce:Kymab Ltd.: Employment, Equity Ownership. Theurl:Kymab Ltd.: Consultancy, Equity Ownership; Sierra Oncoloy: Research Funding. Germaschewski:Kymab Ltd.: Employment, Equity Ownership. Galson:Kymab Ltd.: Employment, Equity Ownership. Badiali:Kymab Ltd.: Employment, Equity Ownership. Dickson:Kymab Ltd.: Employment, Equity Ownership. Bradley:Kymab Ltd.: Employment, Equity Ownership, Patents & Royalties. Lee:Kymab Ltd.: Employment, Equity Ownership, Patents & Royalties.

Cholesteric liquid crystals (CLCs) which possess a periodicity in the visible portion of the spectra, exhibit selective reflection of circularly polarized light. The ability to modulate this color through a variety of means has been explored, including work which incorporated azobenzene LCs. Two types of systems have recently been explored utilizing wavelength-specific cis-trans isomerization processes, which enable unprecedented photosensitivity. The first system exhibits large blue or red-shifted changes in reflection wavelength upon visible irradiation. The second system exploits the metastable, long-lived photoinduced isotropic state, whose return to the reflective Grandjean texture can be induced by wavelength-specific radiation. We demonstrate nonlinear transmission from both types of systems, starting with submicrowatt power levels and spanning over four orders of magnitude dynamic range. The power dependence and temporal evolution of this effect (10–100 ms) is documented here for red or green laser wavelengths. The effect for the former case is due to bandgap auto-tuning, when the laser beam is tuning the CLC Bragg reflection band to its own wavelength. For the latter case, autonomous, optical feedback due to bandgap restoration is the cause of the nonlinear transmission properties.

The development of ruthenium-based complexes for cancer treatment requires a variety of pharmacological studies, one of them being a drug’s binding kinetics to serum proteins. In this work, speciation analysis was used to study kinetics of ruthenium-based drug candidates with human serum proteins. Two ruthenium (Ru) complexes, namely [(η6-p-cymene)Ru(1-hydroxypyridine-2(1H)-thionato)Cl] (1) and [(η6-p-cymene)Ru(1-hydroxypyridine-2(1H)-thionato)pta]PF6 (2) (where pta = 1,3,5-triaza-7-phosphaadamantane), were selected. Before a kinetics study, their stability in relevant media was confirmed by nuclear magnetic resonance (NMR). Conjoint liquid chromatography (CLC) monolithic column, assembling convective interaction media (CIM) protein G and diethylamino (DEAE) disks, was used for separation of unbound Ru species from those bound to human serum transferrin (Tf), albumin (HSA) and immunoglobulins G (IgG). Eluted proteins were monitored by UV spectrometry (278 nm), while Ru species were quantified by post-column isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS). Binding kinetics of chlorido (1) and pta complex (2) to serum proteins was followed from 5 min up to 48 h after incubation with human serum. Both Ru complexes interacted mainly with HSA. Complex (1) exhibited faster and more extensive interaction with HSA than complex (2). The equilibrium concentration for complex (1) was obtained 6 h after incubation, when about 70% of compound was bound to HSA, 5% was associated with IgG, whereas 25% remained unbound. In contrast, the rate of interaction of complex (2) with HSA was much slower and less extensive and the equilibrium concentration was obtained 24 h after incubation, when about 50% of complex (2) was bound to HSA and 50% remained unbound.

Melilotus officinalis is known to contain several types of secondary metabolites. In contrast, the carotenoid composition of this medicinal plant has not been investigated, although it may also contribute to the biological activities of the drug, such as anti-inflammatory effects. Therefore, this study focuses on the isolation and identification of carotenoids from Meliloti herba and on the effect of isolated (all-E)-lutein 5,6-epoxide on primary sensory neurons and macrophages involved in nociception, as well as neurogenic and non-neurogenic inflammatory processes. The composition of the plant extracts was analyzed by high performance liquid chromatography (HPLC). The main carotenoid was isolated by column liquid chromatography (CLC) and identified by MS and NMR. The effect of water-soluble lutein 5,6-epoxide-RAMEB (randomly methylated-β-cyclodextrin) was investigated on Ca2+-influx in rat primary sensory neurons induced by the activation of the transient receptor potential ankyrin 1 receptor agonist to mustard-oil and on endotoxin-induced IL-1β release from isolated mouse peritoneal macrophages. (all-E)-Lutein 5,6-epoxide significantly decreased the percent of responsive primary sensory neurons compared to the vehicle-treated stimulated control. Furthermore, endotoxin-evoked IL-1β release from macrophages was significantly decreased by 100 µM lutein 5,6-epoxide compared to the vehicle-treated control. The water-soluble form of lutein 5,6-epoxide-RAMEB decreases the activation of primary sensory neurons and macrophages, which opens perspectives for its analgesic and anti-inflammatory applications.

Abstract Background and Aims It has been recognized that the physiological role of ClC-Ka and its mouse homolog ClC-K1 in the nephron is to confer high Cl- permeability in the thin Ascending Limb of loop of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla in the countercurrent system. Accordingly ClC-K1 KO mice display a severe polyuria resembling a Nephrogenic Diabetes Insipidus (NDI) phenotype. Thus, ClC-Ka in human nephron could be an interesting target for diuretic and anti-hypertensive drugs. Unfortunately the regulation of ClC-Ka in and its electrophysiological properties are still not completely known. Method We analyzed, by whole-cell voltage-clamp, Cl- currents in HEK293 cells co-expressing GFP-ClC-Ka and the accessory m-Cherry-tagged barttin subunit, upon intracellular Ca2+ mobilization. The patch clamp protocol consists of a holding potential from 0 mV, which is stepped to various test potentials from -195 and +125 mV for 150 ms. Pulses ended with a tail pulse to -125 mV for 20 ms. Concomitantly we monitored by live confocal microscopy, the membrane expression of either GFP-ClC-Ka or barttin-mCherry. Results In control conditions we found a linear current/voltage relationship over a broad range of membrane voltages except of hyperpolarization below -100 mV, where the channel open probability was reduced, providing the well-known hook-shaped appearance of the I-V curve for ClC-Ka channel. Interestingly, both 100 µM ATP and 400 µg/ml of dandelion root extract (DRE), both increasing intracellular Ca2+ in HEK293, significantly inhibited Cl- currents in a time dependent way. Pre-treatment of cells with either the Ca2+ chelator BAPTA-AM or the PCK inhibitor Calphostin C, reverted the inhibitory effects of both ATP and DRE. Interestingly, 1µM of phorbol meristate acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of ATP and DRE on Cl- currents. We finally demonstrated that 30 µM Heclin, an inhibitor of E3 ubiquitin ligase, did not revert the DRE- and ATP- induced Cl- currents inhibition, thus suggesting that the ClC-Ka inhibition is independent of channel ubiquitination. Accordingly, live confocal analysis showed that all the Ca2+ mobilizing compounds tested did not induced neither ClC-Ka nor Barttin internalization. We hypothesized that phosphorylation in the putative PKC consensus sites present in ClC-Ka amino acidic sequence may be involved in the inhibition of channel activity. Conclusion In conclusion, we demonstrated for the first time that the activity of ClC-Ka in renal cells could by inhibited by either receptor agonists or natural molecules able to activate an intracellular Ca2+/PKC pathway, thus opening venues for the development of new diuretic drugs.