Dimer Fraction Research Articles

The infrared spectrum of YI3 in the vapour phase

The vibrational spectrum of the vapour phase above YI3 was measured by infrared spectroscopy. The spectrum revealed four strong bands, three of which could be assigned to the fundamantal infrared active vibrations of the YI3 monomer molecule. The observed frequencies agree very well with quantum-chemical calculations reported in literature. The presence of dimer molecules, the fraction of which is significant in the vapour according to literature, is also discussed in the context of assigning the fourth strong band in the experimental spectrum. Although its position matches well with a strong fundamental of the dimer, its intensity suggest a dimer fraction that is far outside the range for the other rare-earth triiodides.

Interplay between protease and reverse transcriptase dimerization in a model HIV-1 polyprotein.

The Gag-Pol polyprotein in human immunodeficiency virus type I (HIV-1) encodes enzymes that are essential for virus replication: protease (PR), reverse transcriptase (RT), and integrase (IN). The mature forms of PR, RT and IN are homodimer, heterodimer and tetramer, respectively. The precise mechanism underlying the formation of dimer or tetramer is not yet understood. Here, to gain insight into the dimerization of PR and RT in the precursor, we prepared a model precursor, PR-RT, incorporating an inactivating mutation at the PR active site, D25A, and including two residues in the p6* region, fused to a SUMO-tag, at the N-terminus of the PR region. We also prepared two mutants of PR-RT containing a dimer dissociation mutation either in the PR region, PR(T26A)-RT, or in the RT region, PR-RT(W401A). Size exclusion chromatography showed both monomer and dimer fractions in PR-RT and PR(T26A)-RT, but only monomer in PR-RT(W401A). SEC experiments of PR-RT in the presence of protease inhibitor, darunavir, significantly enhanced the dimerization. Additionally, SEC results suggest an estimated PR-RT dimer dissociation constant that is higher than that of the mature RT heterodimer, p66/p51, but slightly lower than the premature RT homodimer, p66/p66. Reverse transcriptase assays and RT maturation assays were performed as tools to assess the effects of the PR dimer-interface on these functions. Our results consistently indicate that the RT dimer-interface plays a crucial role in the dimerization in PR-RT, whereas the PR dimer-interface has a lesser role.

Innovative Microemulsion Loaded with Unusual Dimeric Flavonoids from Fridericia platyphylla (Cham.) L.G. Lohmann Roots.

Fridericia platyphylla (Cham.) L.G. Lohmann is a species native to the Brazilian cerrado, with promising bioactivity. The organic fraction of the roots is rich in unusual dimeric flavonoids, reported as potential candidates for cancer treatment. The exploration of these flavonoids is very important, considering their diverse biological activities and the need for innovative therapeutic options. This work aimed to develop and characterize a microemulsion loaded with a non-polar fraction (DCM). The constituents were chosen, and the pseudo-ternary diagram was constructed to determine the region of microemulsion formation. The microemulsions blank (ME), with 3% (ME3) and 5% (ME5) of fraction DCM, were characterized in terms of droplet size, zeta potential, and polydispersity index. Both MEs showed particle sizes <100 nm; only ME3 exhibited better values for polydispersity index and zeta potential and was therefore selected for further study. The organoleptic and physicochemical characteristics were evaluated, revealing limpidity and transparency typical of these microstructures, physiologically acceptable pH, refractive index of 1.42±0.01, and density of 1.017 g/cm3±0.01. The stability tests showed good stability profiles even after exposure to extreme thermal conditions, with minimal changes in pH and the content of the incorporated fraction. The in vitro release study demonstrated that ME3 enabled the controlled release of the fraction, with a cumulative amount released over 60% within 6 h. Furthermore, fraction DCM and ME3 exhibited no toxicity in Tenebrio molitor larvae. The developed microemulsion exhibited excellent properties, so this study represents the first successful attempt to develop a formulation that incorporates the dimeric flavonoid fraction.

Speciation analysis of manganese against the background of its different content in the blood serum of dairy cows.

Studies in the field of microelement speciation in the body of farm animals, in particular dairy cattle, are almost completely absent. The average concentration of Mn in the blood serum of all the studied animals (n = 80) was 2.5 μg/L, which corresponds to normal values. Of the total number of animals, 21% were the cows with the low normal values (serum Mn concentration ≤ 2 µg/L, i.e. less than Q25 of the total sample) and 25% were the animals with the high normal values (serum Mn concentration ≥ 2.72 µg/L, i.e. more than Q75 of the total sample). The data obtained in the course of the study indicate that the change in the Mn level, even in the range of normal values, is accompanied by the redistribution of this element over various protein fractions. The six found Mn blood serum forms are presumably represented by α2-macroglobulin (tetramer, dimer, and monomer), transferrin/albumine, manganese citrates, and "free" metal ions. The analyzed fractions of Mn found in the blood serum of cows had the following hierarchy of concentrations: in the group with low-normal values of Mn ("free" Mn >> tetrameric form of α2-macroglobulin >> transferrin/albumine >> dimeric form of α2-macroglobulin >> monomeric form of α2-macroglobulin >> citrate), in the group with high normal manganese values ("free" Mn >> monomeric form of α2-macroglobulin >> transferring/albumine >> citrate >> tetrameric form of α2-macroglobulin >> dimeric form of α2-macroglobulin). In the group with high normal Mn values relative to the group with low normal values, there was a percentage decrease in the tetrameric fraction of a2-macroglobulin from 17.2 to 4.4%, dimeric fraction of a2-macroglobulin from 6.9 to 2.2%, "free" Mn from 54.3 to 44.4% and an increase in monomeric fraction of a2-macroglobulin from 6.7 to 23.1%, transferrin/albumine from 10.1 to 17.7%, citrate from 4.8 to 8.2%. Our data demonstrate the features of Mn redistribution of dairy cows, which can be used for an extended assessment of the microelement status of animals.

Galloylated proanthocyanidins in dentin matrix exhibit biocompatibility and induce differentiation in dental stem cells.

Grape seed extract contains a complex mixture of proanthocyanidins (PACs), a plant biopolymer used as a biomaterial to improve reparative and preventive dental therapies. Co-polymerization of PACs with type I collagen mechanically reinforces the dentin extracellular matrix. This study assessed the biocompatibility of PACs from grape seed extract on dental pulp stem cells (DPSCs) in a model simulating leaching through dentin to the pulp cavity. The aim was to determine the type of PACs (galloylated vs. non-galloylated) within grape seed extract that are most compatible with dental pulp tissue. Human demineralized dentin was treated with selectively-enriched dimeric PACs prepared from grape seed extract using liquid-liquid chromatography. DPSCs were cultured within a 2D matrix and exposed to PAC-treated dentin extracellular matrix. Cell proliferation was measured using the MTS assay and expression of odontoblastic genes was analyzed by qRT-PCR. Categorization of PACs leaching from dentin was performed using HPLC-MS. Enriched dimeric fractions containing galloylated PACs increased the expression of certain odontoblastic genes in DPSCs, including Runt-related transcription factor 2 (RUNX2), vascular endothelial growth factor (VEGF), bone morphogenetic protein 2 (BMP2), basic fibroblast growth factor (FGF2), dentin sialophosphoprotein (DSPP) and collagen, type I, alpha 1 (COLI). Galloylated dimeric PACs also exhibited minor effects on DPSC proliferation, resulting in a decrease compared to control after five days of treatment. The non-galloylated dimer fraction had no effect on these genes or on DPSC proliferation. Galloylated PACs are biocompatible with DPSCs and may exert a beneficial effect on cells within dental pulp tissue. The observed increase in odontoblastic genes induced by galloylated PACs together with a decrease in DPSC proliferation is suggestive of a shift toward cell differentiation. This data supports the use of dimeric PACs as a safe biomaterial, with galloylated dimeric PACs exhibiting potential benefits to odontoblasts supporting dentin regeneration.

Designing highly multiplex PCR primer sets with Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE)

One major challenge in the design of highly multiplexed PCR primer sets is the large number of potential primer dimer species that grows quadratically with the number of primers to be designed. Simultaneously, there are exponentially many choices for multiplex primer sequence selection, resulting in systematic evaluation approaches being computationally intractable. Here, we present and experimentally validate Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE), a stochastic algorithm for design of multiplex PCR primer sets that minimize primer dimer formation. In a 96-plex PCR primer set (192 primers), the fraction of primer dimers decreases from 90.7% in a naively designed primer set to 4.9% in our optimized primer set. Even when scaling to 384-plex (768 primers), the optimized primer set maintains low dimer fraction. In addition to NGS, SADDLE-designed primer sets can also be used in qPCR settings to allow highly multiplexed detection of gene fusions in cDNA, with a single-tube assay comprising 60 primers detecting 56 distinct gene fusions recurrently observed in lung cancer.

Перспективы применения синтетических углеводородных основ — дисперсионных сред буровых промывочных жидкостей на неводной основе

The possibility of using a light (by-product) fraction of the process of producing synthetic high viscosity index, low pour point polyalphaolefin base oils based on octene-1 as part of environmentally safe non-aqueous process fluids used in drilling wells in the most difficult mining and geological conditions, including offshore drilling,is investigated. It is shown that the dimeric fraction of octene-1 meets the requirements for the dispersion media of drilling fluids.

Перспективы применения синтетических углеводородных основ — дисперсионных сред буровых промывочных жидкостей на неводной основе

The possibility of using a light (by-product) fraction of the process of producing synthetic high viscosity index, low pour point polyalphaolefin base oils based on octene-1 as part of environmentally safe non-aqueous process fluids used in drilling wells in the most difficult mining and geological conditions, including offshore drilling, is investigated. It is shown that the dimeric fraction of octene-1 meets the requirements for the dispersion media of drilling fluids.

Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System.

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 kDa. While the cellular function of FL HTT is not entirely understood, a mutant expansion of the polyQ tract above ~36 repeats is associated with Huntington's disease (HD), with the polyQ length correlating roughly with the age of onset. To better understand the effect of structure on the function of mutant HTT (mHTT), large quantities of the protein are required. Submilligram production of FL HTT in mammalian cells was achieved using doxycycline-inducible stable cell line expression. However, protein production from stable cell lines has limitations that can be overcome with transient transfection methods. This paper presents a robust method for low-milligram quantity production of FL HTT and its variants from codon-optimized plasmids by transient transfection using polyethylenimine (PEI). The method is scalable (>10 mg) and consistently yields 1-2 mg/L of cell culture of highly purified FL HTT. Consistent with previous reports, the purified solution state of FL HTT was found to be highly dynamic; the protein has a propensity to form dimers and high-order oligomers. A key to slowing oligomer formation is working quickly to isolate the monomeric fractions from the dimeric and high-order oligomeric fractions during size exclusion chromatography. Size exclusion chromatography with multiangle light scattering (SEC-MALS) was used to analyze the dimer and higher-order oligomeric content of purified HTT. No correlation was observed between FL HTT polyQ length (Q23, Q48, and Q73) and oligomer content. The exon1-deleted construct (aa 91-3,144) showed comparable oligomerization propensity to FL HTT (aa 1-3,144). Production, purification, and characterization methods by SEC/MALS-refractive index (RI), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, Native PAGE, and Blue Native PAGE are described herein.

Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 kDa. While the cellular function of FL HTT is not entirely understood, a mutant expansion of the polyQ tract above ~36 repeats is associated with Huntington's disease (HD), with the polyQ length correlating roughly with the age of onset. To better understand the effect of structure on the function of mutant HTT (mHTT), large quantities of the protein are required. Submilligram production of FL HTT in mammalian cells was achieved using doxycycline-inducible stable cell line expression. However, protein production from stable cell lines has limitations that can be overcome with transient transfection methods. This paper presents a robust method for low-milligram quantity production of FL HTT and its variants from codon-optimized plasmids by transient transfection using polyethylenimine (PEI). The method is scalable (>10 mg) and consistently yields 1-2 mg/L of cell culture of highly purified FL HTT. Consistent with previous reports, the purified solution state of FL HTT was found to be highly dynamic; the protein has a propensity to form dimers and high-order oligomers. A key to slowing oligomer formation is working quickly to isolate the monomeric fractions from the dimeric and high-order oligomeric fractions during size exclusion chromatography. Size exclusion chromatography with multiangle light scattering (SEC-MALS) was used to analyze the dimer and higher-order oligomeric content of purified HTT. No correlation was observed between FL HTT polyQ length (Q23, Q48, and Q73) and oligomer content. The exon1-deleted construct (aa 91-3,144) showed comparable oligomerization propensity to FL HTT (aa 1-3,144). Production, purification, and characterization methods by SEC/MALS-refractive index (RI), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, Native PAGE, and Blue Native PAGE are described herein.

Porphyrin Binding and Irradiation Promote G-Quadruplex DNA Dimeric Structure.

Nucleic acid sequences rich in guanines can organize into noncanonical DNA G-quadruplexes (G4s) of variable size. The design of small molecules stabilizing the structure of G4s is a rapidly growing area for the development of novel anticancer therapeutic strategies and bottom-up nanotechnologies. Among a multitude of binders, porphyrins are very attractive due to their light activation that can make them valuable conformational regulators of G4s. Here, a structure-based strategy, integrating complementary probes, is employed to study the interaction between TMPyP4 porphyrin and a 22-base human telomeric sequence (Tel22) before and after irradiation with blue light. Porphyrin binding is discovered to promote Tel22 dimerization, while light irradiation of the Tel22-TMPyP4 complex controls dimer fraction. Such a change in quaternary structure is found to be strictly correlated with modifications at the secondary structure level, thus providing an unprecedented link between the degree of dimerization and the underlying conformational changes in G4s.

Identification of a Catalytic Active but Non-Aggregating MDM2 RING Domain Variant

As a key regulator of the tumour suppressor protein p53, MDM2 is involved in various types of cancer and has thus been an attractive drug target. So far, small molecule design has primarily focussed on the N-terminal p53-binding domain although on-target toxicity effects have been reported. Targeting the catalytic RING domain of MDM2 resembles an alternative approach to drug MDM2 with the idea to prevent MDM2-mediated ubiquitination of p53 while retaining MDM2′s ability to bind p53. The design of RING inhibitors has been limited by the extensive aggregation tendency of the RING domain, making it challenging to undertake co-crystallization attempts with potential inhibitors. Here we compare the purification profiles of the MDM2 RING domain from several species and show that the MDM2 RING domain of other species than human is much less prone to aggregate although the overall structure of the RING domain is conserved. Through sequence comparison and mutagenesis analyses, we identify a single point mutation, G443T, which greatly enhances the dimeric fraction of human MDM2 RING domain during purification. Neither does the mutation alter the structure of the RING domain, nor does it affect E2(UbcH5B)–Ub binding and activity. Hence, MDM2-G443T facilitates studies involving binding partners that would be hampered by the low solubility of the wild-type RING domain. Furthermore, it will be valuable for the development of MDM2 RING inhibitors.

CRISPR/Cas12a-mediated labeling of MET receptor enables quantitative single-molecule imaging of endogenous protein organization and dynamics.

SummarySingle-molecule localization microscopy (SMLM) reports on protein organization in cells with near-molecular resolution and in combination with stoichiometric labeling enables protein counting. Fluorescent proteins allow stoichiometric labeling of cellular proteins; however, most methods either lead to overexpression or are complex and time demanding. We introduce CRISPR/Cas12a for simple and efficient tagging of endogenous proteins with a photoactivatable protein for quantitative SMLM and single-particle tracking. We constructed a HEK293T cell line with the receptor tyrosine kinase MET tagged with mEos4b and demonstrate full functionality. We determine the oligomeric state of MET with quantitative SMLM and find a reorganization from monomeric to dimeric MET upon ligand stimulation. In addition, we measured the mobility of single MET receptors in vivo in resting and ligand-treated cells. The combination of CRISPR/Cas12a-assisted endogenous protein labeling and super-resolution microscopy represents a powerful tool for cell biological research with molecular resolution.

Structural basis for allosteric regulation of pyruvate kinase M2 by phosphorylation and acetylation

Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme and transcriptional coactivator and is critical for tumor metabolism. In cancer cells, native tetrameric PKM2 is phosphorylated or acetylated, which initiates a switch to a dimeric/monomeric form that translocates into the nucleus, causing oncogene transcription. However, it is not known how these post-translational modifications (PTMs) disrupt the oligomeric state of PKM2. We explored this question via crystallographic and biophysical analyses of PKM2 mutants containing residues that mimic phosphorylation and acetylation. We find that the PTMs elicit major structural reorganization of the fructose 1,6-bisphosphate (FBP), an allosteric activator, binding site, impacting the interaction with FBP and causing a disruption in oligomerization. To gain insight into how these modifications might cause unique outcomes in cancer cells, we examined the impact of increasing the intracellular pH (pHi) from ∼7.1 (in normal cells) to ∼7.5 (in cancer cells). Biochemical studies of WT PKM2 (wtPKM2) and the two mimetic variants demonstrated that the activity decreases as the pH is increased from 7.0 to 8.0, and wtPKM2 is optimally active and amenable to FBP-mediated allosteric regulation at pHi 7.5. However, the PTM mimetics exist as a mixture of tetramer and dimer, indicating that physiologically dimeric fraction is important and might be necessary for the modified PKM2 to translocate into the nucleus. Thus, our findings provide insight into how PTMs and pH regulate PKM2 and offer a broader understanding of its intricate allosteric regulation mechanism by phosphorylation or acetylation.

Molecular Mechanism of Light-Induced Conformational Switching of the LOV Domain in Aureochrome-1.

Light oxygen voltage-sensing (LOV) domains are widely found in photoreceptor proteins of plants, algae, fungi, and bacteria. Structural studies of LOV domains suggest that Phe and Gln residues located in the proximity of the chromophore undergo conformational changes upon illumination; however, the molecular mechanism associated with activation of the effector domain remains to be elucidated. Photozipper (PZ) protein is an N-terminally truncated aureochrome-1 comprising a LOV domain and a basic leucine zipper domain. Blue light (BL) induces PZ dimerization and subsequently increases its affinity for target DNA. In this study, we prepared PZ mutants with substitutions of F298 and Q317 and performed quantitative analyses in dark and light states. Substitutions of Q317 significantly reduced the light-induced changes in PZ affinity for the target DNA, especially in the case of the high affinities observed in the dark state. Upon illumination, all PZ mutants showed increased affinity for the target sequence, which demonstrated a clear correlation with the dimer fraction of each PZ mutant. These results suggest the existence of a conformational equilibrium and that its shift by a synergistic interaction between the chromophore and protein moiety probably enables BL-regulated switching of aureochrome-1.

Mechanistic Insights into Cytochrome P450 Inactivation by Azole Drugs in Acanthamoeba castellanii

Cytochrome P450 (CYPs) are heme containing enzymes that oxidize substances using iron and are capable to metabolize wide variety of xenobiotics and play key role in the activation or inactivation of various therapeutic agents. Studies have described more than 50,000 CYPs in most forms of life i.e. archaea, viruses, protists, bacteria, animals, plants and fungi.One of the major advancements in pharmacology is the discovery of antifungal activity of azole compounds. These compounds showed a very significant advancement in the management of fungal infections. Major enzyme inhibited by these azoles is 14 alpha‐demethylase, a cytochrome P450 enzyme, which is involved in the conversion of lanosterol to ergosterol.As these CYPs descend from a common ancestor and share structural features, we tried to repurpose these antifungal drugs against Acanthamoeba castellanii, an opportunistic pathogen and an emerging cause of granulomatous amebic encephalitis and amebic keratitis and have been associated with cutaneous lesions and sinusitis.In the present study, we structurally characterized recombinant CYP51 from Acanthamoeba castellanii (AcCYP51), which is also an important enzyme in the biosynthesis of ergosterol. Interestingly, structure‐based analysis of ligand free and ligand bound forms of AcCYP51 made very remarkable observations. Here, we observed that ligand free AcCYP51 tends to dimerize both in solution (as observed in size exclusion chromatography,SEC) and in the crystals (1.8 Å) via N‐terminal swapping forming a large homo‐dimerization interface exceeding 2000 Å2. Spectral analysis of dimeric and small monomeric fractions of AcCYP51, which we obtained after SEC, have shown that only dimeric form of AcCYP51 has spectral characteristics typical of functionally competent CYP enzymes. This is for the first time that a strong and functionally competent CYP dimer was defined structurally. Further structural analysis of inhibitor (azole) bound AcCYP51 have shown that binding of inhibitors promoted dimer dissociation and we obtained AcCYP51‐inhibitor complexes in a monomeric form (2.9 Å). We have used here different azole inhibitors (according to their potency against Acanthamoeba ) and observed that according to their in‐vivo potency, these inhibitors elucidated differences in their mode of action. With a less potent inhibitor AcCYP51 was capable to restore the monomer structure whereas with the most potent inhibitor AcCYP51failed to fully restore the monomer structure. This failure of CYP51 to fully refold upon dimer dissociation, in vivo may cause an irreversible loss of a structurally aberrant target enzyme through proteolytic degradation. This proposed inactivation mechanism is consistent with the superior potency of this molecule against A. castellaniicells.Repurposing of the antifungal azole drugs (known as conazoles) emerges as a promising strategy to combat Acanthamoeba infections. The drug‐target crystal structures determined in this work and the mechanistic insights thus gained may be further exploited for the medicinal chemistry efforts aimed at improving potency and bioavailability of these drugs for applications in human diseases caused by infection with Acanthamoeba.

Meso-1,4-phenylene bridged nickel norcorrole dimer

Nickel norcorrole dimer 3 linked with a phenylene bridge was prepared by a nickel-mediated homolytic cross-coupling of meso-phenylene bisdipyrrin nickel(II) dimer 2b. The structures of 2b and 3 were elucidated by X-ray diffraction analysis. Each nickel norcorrole fraction of the nickel norcorrole dimer 3 exposed a bowl-like structure, which conserved anti-conformation. Structural distinctions between the monomeric and dimeric nickel norcorroles were established by vibrational spectroscopic analysis and assessed with density functional theory calculations. Enhanced flexibility of the norcorrole planes in solution states of monomeric nickel norcorrole was compromised to that of bowl-shaped norcorroles in the solid states of nickel norcorrole dimer 3, where C[Formula: see text] was the formulated conformation. The deformation of the planarity derived from the objection of laser pulses effectuated vibration shifts of specific Raman-active motions toward a higher-frequency region, as associating with a paratropic nature of the [Formula: see text]-electron delocalization circuit of norcorrole. Computational simulation exposed a reliable drift of the Raman frequencies.

Phase Behavior of Amorphous Solid Dispersions of Felodipine: Homogeneity and Drug-Polymer Interactions.

In the current investigation, the role of drug-polymer hydrogen bonding (H-bonding) with respect to the phase behavior of amorphous solid dispersions (ASDs) is studied in depth on a nanometer level. Melt-quenched dispersions of felodipine (FEL) with poly(vinylpyrrolidone), or PVP, poly(vinylpyrrolidone-co-vinylacetate), or PVP/VA, and poly(vinylacetate), or PVAc, were prepared at drug loadings of 50-90% w/w. Modulated differential scanning calorimetry (MDSC) was used to detect microscopic homogeneity for each set of ASDs. A single composition dependent glass transition temperature (Tg) was observed over the entire composition range in MDSC data for each set of ASDs; however some samples within each set of ASDs showed a crystallization exotherm and corresponding melting endotherm in the first heating scan. Solid-state nuclear magnetic resonance spectroscopy (SSNMR) was further employed to understand phase homogeneity in these systems. The proton spin-lattice relaxation times in the laboratory and rotating frame (1H T1 and T1ρ) for the drug and individual polymer for each set of ASDs were measured to evaluate phase homogeneity. On the basis of proton relaxation measurements, it was revealed that FEL:PVP and FEL:PVP/VA ASDs exhibited better compositional homogeneity than FEL:PVAc ASDs. The strength and the extent of H-bonding were studied by using 13C SSNMR spectra. In addition, deconvolution of the carbonyl region of amorphous FEL revealed that 40% of amorphous FEL molecules were hydrogen bonded (H-bonded) through dimers and the remaining 60% were free/non H-bonded. The dimer fraction decreased as the polymer content increased for each set of ASDs, while the free fraction increased. This indicated that the polymers containing hydrogen bond acceptor groups disrupted dimers and formed intermolecular H-bonding interactions with FEL. The strength and extent of FEL:polymer H-bonding was rank ordered as PVP > PVP/VA > PVAc. These findings were also confirmed through DFT calculations on these systems. Our results suggest that drug-polymer H-bonding interaction may impact the phase homogeneity in ASDs formulated by a specific method. The data from the current study further demonstrate that SSNMR is a powerful tool for characterizing phase homogeneity in ASDs with sub-50 nm resolution. In addition, SSNMR can provide insights into drug-polymer interactions and speciation in ASDs.

Primary myogenesis in the sand lizard (Lacerta agilis) limb bud

Our studies conducted on reptilian limb muscle development revealed, for the first time, early forelimb muscle differentiation at the morphological and molecular level. Sand lizard skeletal muscle differentiation in the early forelimb bud was investigated by light, confocal, and transmission electron microscopy as well as western blot. The early forelimb bud, filled with mesenchymal cells, is surrounded by monolayer epithelium cells. The immunocytochemical analysis revealed the presence of Pax3- and Lbx-positive cells in the vicinity of the ventro-lateral lip (VLL) of the dermomyotome, suggesting that VLL is the source of limb muscle progenitor cells. Furthermore, Pax3- and Lbx-positive cells were observed in the dorsal and ventral myogenic pools of the forelimb bud. Skeletal muscle development in the early limb bud is asynchronous, which is manifested by the presence of myogenic cells in different stages of differentiation: multinucleated myotubes with well-developed contractile apparatus, myoblasts, and mitotically active premyoblasts. The western blot analysis revealed the presence of MyoD and Myf5 proteins in all investigated developmental stages. The MyoD western blot analysis showed two bands corresponding to monomeric (mMyoD) and dimeric (dMyoD) fractions. Two separate bands were also detected in the case of Myf5. The observed bands were related to non-phosphorylated (Myf5) and phosphorylated (pMyf5) fractions of Myf5. Our investigations on sand lizard forelimb myogenesis showed that the pattern of muscle differentiation in the early forelimb bud shares many features with rodents and chicks.

The domain swapping of human cystatin C induced by synchrotron radiation

Domain swapping is observed for many proteins with flexible conformations. This phenomenon is often associated with the development of conformational diseases. Importantly, domain swapping has been observed for human cystatin C (HCC), a protein capable of forming amyloid deposits in brain arteries. In this study, the ability of short exposure to high-intensity X-ray radiation to induce domain swapping in solutions of several HCC variants (wild-type HCC and V57G, V57D, V57N, V57P, and L68V mutants) was determined. The study was conducted using time-resolved small-angle X-ray scattering (TR-SAXS) synchrotron radiation. The protein samples were also analysed using small-angle neutron scattering and NMR diffusometry. Exposing HCC to synchrotron radiation (over 50 ms) led to a gradual increase in the dimeric fraction, and for exposures longer than 150 ms, the oligomer fraction was dominant. In contrast, the non-irradiated protein solutions, apart from the V57P variant, were predominantly monomeric (e.g., V57G) or in monomer/dimer equilibrium. This work might represent the first observation of domain swapping induced by high-intensity X-rays.