An application of the standardised reference extract quantification strategy in the quality control of ginseng infusions by liquid chromatography with mass spectrometric detection.

INTRODUCTION: Limited availability of individual standards is a bottleneck for quality control of functional foods and natural medicines. The use of standard mixtures or secondary standards is a possible alternative in this case. Earlier, an approach known as standardised reference extract (RE) strategy was introduced for HPLC-UV analysis of different plant materials; however, its application in HPLC-MS analysis has not been investigated. OBJECTIVE: To establish an HPLC-MS-based RE method for determination of ginsenoside content in ginseng infusions using commercially available extract reference material of Panax quinquefolius L. RESULTS: The developed HPLC-MS method was validated as precise (1.1%-9.4% intra-day variation; 1.6%-12.8% inter-day variation) and highly sensitive [limit of detection (LOD): 1-40 ng/mL; limit of quantification (LOQ): 4-120 ng/mL]. The stability of samples was satisfactory (5.7%-16.3%). The RE quantification method was compared with the external standard method, and the obtained difference was not significant, mostly in the range of 5%-10%. Matrix effects for the diluted samples of RE and ginseng infusions, determined via the standard addition method, were in the range of 85%-115% and 80%-126%, respectively, and were also positively correlated with the ginsenoside concentration. Eleven batches of ginseng infusions from different manufacturers were analysed using the established method. CONCLUSION: The method for HPLC-MS-based ginsenoside quantification using RE as a secondary standard was established for the first time. The results of this study demonstrate that the application of the standardised RE strategy in HPLC-MS can minimise the matrix effect-related error in addition to the cost-effective quality control of herbal products, foods, and traditional medicines.

Application of digital holographic tomography in antitumor effect of cantharides complex on 4T1 breast cancer cells.

The study focuses on a methodology providing noninvasive monitoring and evaluation of the antitumor effect of traditional Chinese medicine, cantharides complex (canth), on 4T1 breast tumor cells. Digital holographic tomography (DHT) and developed data post-processing algorithms were used for quantitative estimation of changes in optical and morphological parameters of cells. We calculated and compared data on the refractive index, thickness, and projected area of 4T1 breast tumor cells in control untreated specimens and those treated with doxorubicin hydrochloride (DOX), canth, and their combinations. Post-treatment changes in cellular morphology recorded by DHT demonstrated that the two drugs led to noticeably different morphological changes in cells that can be presumably associated with different pathways of their death, apoptosis, or necrosis. The effect of combined treatment with these two drugs strongly depended on their relative concentrations and could lead to changes characteristic either for DOX or for canth; however, being more profound than those obtained when using each drug solely. The results obtained by DHT are in a good correspondence with commonly used cell viability analysis and immunofluorescent analysis of changes in cellular cytoskeleton.

Engineered Tug-of-War Between Kinesin and Dynein Controls Direction of Microtubule Based Transport In Vivo.

Bidirectional transport of membrane organelles along microtubules (MTs) is driven by plus-end directed kinesins and minus-end directed dynein bound to the same cargo. Activities of opposing MT motors produce bidirectional movement of membrane organelles and cytoplasmic particles along MT transport tracks. Directionality of MT-based transport might be controlled by a protein complex that determines which motor type is active at any given moment of time, or determined by the outcome of a tug-of-war between MT motors dragging cargo organelles in opposite directions. However, evidence in support of each mechanisms of regulation is based mostly on the results of theoretical analyses or indirect experimental data. Here, we test whether the direction of movement of membrane organelles in vivo can be controlled by the tug-of-war between opposing MT motors alone, by attaching a large number of kinesin-1 motors to organelles transported by dynein to minus-ends of MTs. We find that recruitment of kinesin significantly reduces the length and velocity of minus-end-directed dynein-dependent MT runs, leading to a reversal of the overall direction of dynein-driven organelles in vivo. Therefore, in the absence of external regulators tug-of-war between opposing MT motors alone is sufficient to determine the directionality of MT transport in vivo.

Superplastic Behavior and Microstructural Features of the VT6 Titanium Alloy with an Ultrafine-Grained Structure during Upsetting.

In this paper, the superplastic behavior of the two-phase titanium alloy VT6 with an ultrafine-grained (UFG) structure produced by equal-channel angular pressing is examined. The deformation of specimens with a UFG structure was performed by upsetting in a temperature range of 650-750 °C and strain rate range of 1 × 10-4-5 × 10-1 s-1. Under these conditions, an increased strain-rate sensitivity coefficient m was observed. The calculation of apparent activation energy showed values in a range of 160-200 kJ/mol while the superplastic flow of the VT6 alloy was occurring. When superplastic behavior (SPB) was impeded, the energy Q grew considerably, indicating a change in mechanism from grain-boundary sliding (GBS) to bulk diffusion. A change in temperature and strain rate influenced the development of superplastic flow and the balance of relaxation processes. Microstructural analysis shows that the UFG state is preserved at upsetting temperatures of 650 and 700 °C. A decrease in strain rate and/or an increase in upsetting temperature promoted a more active development of recrystallization and grain growth, as well as α2-phase formation. In a certain temperature and strain-rate range of the UFG VT6 alloy, α2-phase plates were found, the formation of which was controlled by diffusion. The effect of the α2-phase on the alloy's mechanical behavior is discussed.

Effect of the Texture of the Ultrafine-Grained Ti-6Al-4V Titanium Alloy on Impact Toughness.

In this work, the strength properties and impact toughness of the ultrafine-grained (UFG) Ti-6Al-4V titanium alloy produced by severe plastic deformation (SPD) in combination with upsetting were studied, depending on the direction of crack propagation. In the billets processed by equal-channel angular pressing (ECAP), the presence of anisotropy of ultimate tensile strength (UTS) and ductility was observed, conditioned by the formation of a metallographic and crystallographic texture. At the same time, the ECAP-processed UFG alloy exhibited satisfactory values of impact toughness, ~0.42 MJ/m2. An additional upsetting of the ECAP-processed billet simulated the processes of shape forming/die forging and was accompanied by the development of recovery and recrystallization. This provided the "blurring" of texture and a reduction in the anisotropy of UTS and ductility, but a difference in impact toughness in several directions of fracture was still observed. It is shown that texture evolution during upsetting provided a significant increase in the crack propagation energy. The relationship between microstructure, texture and mechanical properties in different sections of the material under study is discussed.

Microstructural Features and Microhardness of the Ti-6Al-4V Alloy Synthesized by Additive Plasma Wire Deposition Welding.

Wire arc additive manufacturing (AM) is able to replace the traditional manufacturing processes of Ti alloys. At the same time, the common drawback of Ti workpieces produced by AM via wire deposition welding is the formation of a coarse-grained dendritic structure, its strong anisotropy and, consequently, lower strength as compared to a monolithic alloy. In this work, a new method is proposed for the enhancement of the strength properties of the Ti-6Al-4V alloy synthesized by AM via wire deposition welding, which involves the use of a wire with an initial ultrafine-grained (UFG) structure. The UFG wire is characterized by a large number of defects of the crystalline lattice and grain boundaries, which will enable increasing the number of "crystallization centers" of the α-phase, leading to its refinement. The macro- and microstructure, phase composition and microhardness of the Ti-6Al-4V alloy samples were investigated. The microhardness of the alloy produced by layer-by-layer deposition welding using a UFG wire was shown to be on average 20% higher than that of the samples produced by a deposition welding using a conventional wire. The nature of this phenomenon is discussed, as well as the prospects of increasing the mechanical characteristics of Ti alloys produced by additive manufacturing.

Conformational exchange at a C2H2 zinc-binding site facilitates redox sensing by the PML protein.

The promyelocytic leukemia protein, PML, plays a vital role in the cellular response to oxidative stress; however, the molecular mechanism of its action remains poorly understood. Here, we identify redox-sensitive sites of PML. A molecule of PML is cysteine-rich and contains three zinc-binding domains including RING, B-box1, and B-box2. Using in vitro assays, we have compared the sensitivity of the isolated RING and B-box1 domains and shown that B-box1 is more sensitive to oxidation. NMR studies of PML dynamics showed that one of the Zn-coordination sites within the B-box1 undergoes significant conformational exchange, revealing a hotspot for exposure of reactive cysteines. In agreement with the in vitro data, enhancement of the B-box1 Zn-coordination dynamics led to more efficient recruitment of PML into PML nuclear bodies in cells. Overall, our results suggest that the increased sensitivity of B-box1 to oxidative stress makes this domain an important redox-sensing component of PML.

Microstructure of the Advanced Titanium Alloy VT8M-1 Subjected to Rotary Swaging.

In this study, the microstructural behavior of the advanced Ti-5.7Al-3.8Mo-1.2Zr-1.3Sn-0.15Si (VT8M-1) alloy during rotary swaging (RS) was investigated. VT8M-1 has increased heat resistance and is considered a replacement for the Ti-6Al-4V alloy. It was shown that, during RS, the evolution of the primary a phase is characterized by the formation of predominantly low-angle boundaries according to the mechanism of continuous dynamic recrystallization. The density of low-angle boundaries increases three times: from 0.38 µm-1 to 1.21 µm-1 after RS. The process of spheroidization of the lamellar (a + b) component is incomplete. The average size of globular a and b particles was 0.3 µm (TEM). It is shown that the microstructures after RS (ε = 1.56) and equal-channel angular pressing (ECAP) (ε = 1.4) are significantly different. The temperature-velocity regime and the predominance of shear deformations during ECAP contributed to a noticeable refinement of the primary a-phase and a more complete development of globularization of the lamellar (a+b) component. EBSD studies have shown that RS leads to the formation of a structure with a higher density of low- and high-angle boundaries compared to the structure after ECAP. The results are useful for predicting alloy microstructure in the production of long rods that are further used in forging operations.

A Novel Interaction Between RAD23A/B and Y-family DNA Polymerases.

The Y-family DNA polymerases - Pol ι, Pol η, Pol κ and Rev1 - are most well-known for their roles in the DNA damage tolerance pathway of translesion synthesis (TLS). They function to overcome replication barriers by bypassing DNA damage lesions that cannot be normally replicated, allowing replication forks to continue without stalling. In this work, we demonstrate a novel interaction between each Y-family polymerase and the nucleotide excision repair (NER) proteins, RAD23A and RAD23B. We initially focus on the interaction between RAD23A and Pol ι, and through a series of biochemical, cell-based, and structural assays, find that the RAD23A ubiquitin-binding domains (UBA1 and UBA2) interact with separate sites within the Pol ι catalytic domain. While this interaction involves the ubiquitin-binding cleft of UBA2, Pol ι interacts with a distinct surface on UBA1. We further find that mutating or deleting either UBA domain disrupts the RAD23A-Pol ι interaction, demonstrating that both interactions are necessary for stable binding. We also provide evidence that both RAD23 proteins interact with Pol ι in a similar manner, as well as with each of the Y-family polymerases. These results shed light on the interplay between the different functions of the RAD23 proteins and reveal novel binding partners for the Y-family TLS polymerases.

The B-box1 domain of PML mediates SUMO E2-E3 complex formation through an atypical interaction with UBC9.

The small, ubiquitin-like modifier SUMO is covalently attached to substrates by the enzyme UBC9. SUMO conjugation of substrates often requires an E3 ligase, which ensures substrate specificity by simultaneously binding UBC9 and the substrate. E3 SUMO ligases commonly use a RING domain to engage UBC9. The Promyelocytic Leukemia protein (PML) has been implicated as a probable SUMO ligase. Although PML does contain a RING domain, which is expected to recruit UBC9, we demonstrate that PML RING does not bind UBC9 in vitro. Instead, we show that isolated PML B-box1 possesses UBC9-binding activity and map the B-box1 binding site on UBC9. This site also binds the upstream E1 enzyme that transfers SUMO to UBC9. The overlap of these two binding sites suggests that UBC9 cannot interact with its E1 and E3 partners simultaneously. Furthermore, we present a model of the PML dimer that supports the accessibility of B-box1 for UBC9 binding in the context of the full-length PML.

Microstructure and Mechanical Properties of ß-Titanium Ti-15Mo Alloy Produced by Combined Processing including ECAP-Conform and Drawing.

At present, researchers pay great attention to the development of metastable ß-titanium alloys. A task of current importance is the enhancement of their strength and fatigue properties. An efficient method for increasing the strength of such alloys could be severe plastic deformation. The object of this study was a medical metastable ß-titanium alloy Ti-15Mo (ASTM F2066). The alloy in the (α + ß) state was for the first time deformed by combined processing, including equal channel angular pressing-conform and drawing. Such processing enabled the production of long-length rods with a length of 1500 mm. The aim of the work was to study the effect of the combined processing on the alloy's microstructure and mechanical properties. An ultrafine-grained structure with an average size of structural elements less than 100 nm was obtained. At the same time, high strength and ductility (σuts = 1590 MPa, δ = 10%) were achieved, which led to a record increase in the endurance limit (σ-1 = 710 MPa) under tension-compression terms.

Linear and Nonlinear Elastic Properties of Polystyrene-Based Nanocomposites with Allotropic Carbon Fillers and Binary Mixtures.

We report measurements of linear and nonlinear elastic properties of polystyrene-based nanocomposites with six types of nanofillers, including single and binary mixtures of allotropic carbon nanoparticles. Composite samples were fabricated by the same technology and contained the same filler concentration (5% wt.), which allowed for a direct comparison of their properties. It was shown that the most significant variations of linear and nonlinear elastic properties occur in different nanocomposites. In particular, the most pronounced enhancements of linear elastic moduli (in about 50%) obtained in tensile and flexural tests and in dynamic mechanical analysis were recorded in the sample filled with spherical fullerene nanoparticles. While the most profound rise of absolute values of nonlinear elastic moduli (tens of times) was obtained in the sample filled with the mixture of carbon nanotubes and graphene. The observed tendencies demonstrated the synergistic effect of fillers of different dimensionality on the elastic properties of nanocomposites.

The protein kinase A-anchoring protein moesin is bound to pigment granules in melanophores.

Major signaling cascades have been shown to play a role in the regulation of intracellular transport of organelles. In Xenopus melanophores, aggregation and dispersion of pigment granules are regulated by the second messenger cyclic AMP through the protein kinase A (PKA) signaling pathway. PKA is bound to pigment granules where it forms complexes with molecular motors involved in pigment transport. Association of PKA with pigment granules occurs through binding to A-kinase-anchoring proteins (AKAPs), whose identity remains largely unknown. In this study, we used mass spectrometry to examine an 80 kDa AKAP detected in preparations of purified pigment granules. We found that tryptic digests of granule protein fractions enriched in the 80 kDa AKAP contained peptides that corresponded to the actin-binding protein moesin, which has been shown to function as an AKAP in mammalian cells. We also found that recombinant Xenopus moesin interacted with PKA in vitro, copurified with pigment granules and bound to pigment granules in cells. Overexpression in melanophores of a mutant moesin lacking conserved PKA-binding domain did not affect aggregation of pigment granules but partially inhibited their dispersion. We conclude that Xenopus moesin is an AKAP whose PKA-scaffolding activity plays a role in the regulation of pigment dispersion in Xenopus melanophores.

Actin dynamics is essential for myosin-based transport of membrane organelles.

Actin filaments that serve as "rails" for the myosin-based transport of membrane organelles [1-4] continuously turn over by concurrent growth and shortening at the opposite ends [5]. Although it is known that dynamics of actin filaments is essential for many of the actin cytoskeleton functions, the role of such dynamics in myosin-mediated organelle transport was never studied before. Here, we addressed the role of turnover of actin filaments in the myosin-based transport of membrane organelles by treating cells with the drugs that suppress actin-filament dynamics and found that such a suppression significantly inhibited organelle transport along the actin filaments without inhibiting their intracellular distribution or the activity of the myosin motors. We conclude that dynamics of actin filaments is essential for myosin-based transport of membrane organelles and suggest a previously unknown role of actin-filament dynamics in providing the "rails" for continuous organelle movement resulting in the increased distances traveled by membrane organelles along the actin filaments.

Persistent growth of microtubules at low density.

Microtubules (MTs) often form a polarized array with minus ends anchored at the centrosome and plus ends extended toward the cell margins. Plus ends display behavior known as dynamic instability-transitions between rapid shortening and slow growth. It is known that dynamic instability is regulated locally to ensure entry of MTs into nascent areas of the cytoplasm, but details of this regulation remain largely unknown. Here, we test an alternative hypothesis for the local regulation of MT behavior. We used microsurgery to isolate a portion of peripheral cytoplasm from MTs growing from the centrosome, creating cytoplasmic areas locally depleted of MTs. We found that in sparsely populated areas MT plus ends persistently grew or paused but never shortened. In contrast, plus ends that entered regions of cytoplasm densely populated with MTs frequently transitioned to shortening. Persistent growth of MTs in sparsely populated areas could not be explained by a local increase in concentration of free tubulin subunits or elevation of Rac1 activity proposed to enhance MT growth at the cell leading edge during locomotion. These observations suggest the existence of a MT density-dependent mechanism regulating MT dynamics that determines dynamic instability of MTs in densely populated areas of the cytoplasm and persistent growth in sparsely populated areas.

Machine Learning Assisted Classification of Cell Lines and Cell States on Quantitative Phase Images.

In this report, we present implementation and validation of machine-learning classifiers for distinguishing between cell types (HeLa, A549, 3T3 cell lines) and states (live, necrosis, apoptosis) based on the analysis of optical parameters derived from cell phase images. Validation of the developed classifier shows the accuracy for distinguishing between the three cell types of about 93% and between different cell states of the same cell line of about 89%. In the field test of the developed algorithm, we demonstrate successful evaluation of the temporal dynamics of relative amounts of live, apoptotic and necrotic cells after photodynamic treatment at different doses.

Relaxation dynamics of alkyl derivatives of fluorescein MitoFluo and C8-Fl in solutions with liposomes.

We present results of experimental and theoretical studies of excited state dynamics in two alkyl derivatives of fluorescein, MitoFluo and C8-Fl in solutions with liposomes. The liposomes DOPC and soybeanPC + 20% Cardiolipin (Azo-Cl), modelling cellular and inner mitochondrial membranes, respectively, were used in experiments. Both types of liposomes were shown to reduce significantly the fluorescence quantum yield as compared to that of pure fluorescein derivatives in solutions, while DOPC liposomes also caused a noticeable (ca 10 nm) red shift of fluorescence maximum. The study of fluorescence polarization decay has been carried out where important fluorescence parameters: polarization anisotropy, fluorescence lifetimes, and rotational diffusion times have been determined. It was shown that the isotropic fluorescence decay of C8-Fl in liposome containing solutions was single-exponential and the anisotropic decay was double-exponential for both types of lyposomes. In the case of MitoFluo both isotropic and anisotropic fluorescence decays were fitted satisfactory only with double-exponential functions. The interpretation of the experimental data obtained was supported by ab initio calculations of the structure and excitation properties of MitoFluo and C8-Fl in aqueous solution. The analysis of anisotropic fluorescence decay allowed for isolation of the contributions of fluorescein derivatives free in solution from those embedded in liposomes. Also, the experimental data suggest that MitoFluo interacts with liposomes more effectively than C8-Fl. Basing on the experimental and theoretical results obtained we conclude that free C8-Fl and MitoFluo molecules in solution were mostly in their dimer forms.

Laser cladding of bioactive glass coating on pure titanium substrate with highly refined grain structure.

Free from toxic elements biomaterial potentially applicable for load bearing biomedical implants was obtained for the first time by laser cladding of S520 bioactive glass onto ultrafine-grained commercially pure titanium. The cladding process affected the refined structure of the substrate inducing martensitic transformation near its surface. The α' acicular martensite gradually passes into relatively large grains with increasing distance from the substrate surface, which subsequently are transformed into smaller grains of about 2 µm in diameter. Both the melted zone, where the martensite crystalline structure was found, and the HAZ are characterised by relatively lower hardness in comparison with that of the substrate core indicating increased ductility. Such a combination of zones with different properties may have a synergistic effect and is beneficial for the obtained biomaterial. A characteristic region in the form of about 3 µm width band was formed in the melted zone at about 10 µm below the titanium surface. The results of EDS analysis indicate that several glass elements moved into the region while the titanium content in the same area was decreased. High bioactivity of the coated S520 glass was revealed by in vitro testing with SBF solution and almost complete reduction of P concentration occurred after 14 days.

Cytoplasmic dynein is involved in the retention of microtubules at the centrosome in interphase cells.

Cytoplasmic dynein is known to be involved in the establishment of radial microtubule (MT) arrays. During mitosis, dynein activity is required for tethering of the MTs at the spindle poles. In interphase cells, dynein inhibitors induce loss of radial MT organization; however, the exact role of dynein in the maintenance of MT arrays is unclear. Here, we examined the effect of dynein inhibitors on MT distribution and the centrosome protein composition in cultured fibroblasts. We found that while these inhibitors induced rapid (t(1/2) approximately 20 min) loss of radial MT organization, the levels of key centrosomal proteins or the rates of MT nucleation did not change significantly in dynein-inhibited cells, suggesting that the loss of dynein activity does not affect the structural integrity of the centrosome or its capacity to nucleate MTs. Live observations of the centrosomal activity showed that dynein inhibition enhanced the detachment of MTs from the centrosome. We conclude that the primary role of dynein in the maintenance of a radial MT array in interphase cells consists of retention of MTs at the centrosome and hypothesize that dynein has a role in the MT retention, separate from the delivery to the centrosome of MT-anchoring proteins.

Eco-friendly synthesis of fused pyrano[2,3-b]pyrans via ammonium acetate-mediated formal oxa-[3 + 3]cycloaddition of 4H-chromene-3-carbaldehydes and cyclic 1,3-dicarbonyl compounds.

Various substituted polycyclic pyrano[2,3-b]pyrans were synthesized via the condensation of 4H-chromene-3-carbaldehydes and their areno-condensed analogues with hetero- and carbocyclic 1,3-dicarbonyl compounds in acetic acid. Ammonium acetate was used as a green catalyst for the reaction. The process also involves the subsequent Knoevenagel condensation and 6π-electrocyclization of the 1-oxatriene intermediates formed. Fused pyridines were isolated as the products of the conjugated addition of ammonia to 1-oxatriene intermediates while using carbocyclic 1,3-dicarbonyl compounds and increasing the reaction time, indicating the reversibility of the electrocyclization stage. The calculated values of the Gibbs free energies and reaction rate constants for the 1-oxatriene - 2H-pyran equilibrium also testified to the irreversibility of pyrano[2,3-b]pyran formation in the case of using of heterocyclic 1,3-dicarbonyl compounds.