BackgroundThere has been a notable increase in interest in the transcriptional regulator Kaiso, which has been linked to the regulation of clonal hematopoiesis, myelodysplastic syndrome, and tumorigenesis. Nevertheless, there are no consistent data on the binding sites of Kaiso in vivo in the genome. Previous ChIP-seq analyses for Kaiso contradicted the accumulated data of Kaiso binding sites obtained in vitro. Here, we studied this discrepancy by characterizing the distribution profile of Kaiso binding sites in Caki-1 cells using Kaiso-deficient cells as a negative control, and compared its pattern on chromatin with that in lymphoblastoid cell lines.ResultsWe employed Caki-1 kidney carcinoma cells and their derivative, which lacks the Kaiso gene, as a model system to identify the genomic targets of Kaiso. The principal binding motifs for Kaiso are CGCG and CTGCNAT, with 60% of all binding sites containing both sequences. The significance of methyl-DNA binding activity was confirmed through examination of the genomic distribution of the E535A mutant variant of Kaiso, which cannot bind methylated DNA in vitro but is able to interact with CTGCNA sequences. Our findings indicate that Kaiso is present at CpG islands with a preference for methylated ones. We identified Kaiso target genes whose methylation and transcription are dependent on its expression. Furthermore, Kaiso binding sites are enriched at CpG islands, with partial methylation at the 5' and/or 3' boundaries. We discovered CpG islands exhibiting wave-like methylation patterns, with Kaiso detected in the majority of these areas. Similar data were obtained in other cell lines.ConclusionThe present study delineates the genomic distribution of Kaiso in cancer cells, confirming its role as a factor with a complex mode of DNA binding and a strong association with CpG islands, particularly with methylated and eroded CpG islands, revealing a new potential Kaiso target gene—SQSTM1, involved in differentiation of acute myeloid leukemia cells. Furthermore, we discovered the existence of a new class of CpG islands characterized by wave-like DNA methylation.

Abstract A modular synthesis of novel series of 1,7‐difluorinated BODIPYs has been elaborated. First, the acid‐catalyzed condensation of ethyl 3‐aryl‐4‐fluoro‐1H‐pyrrole‐2‐carboxylates with aromatic aldehydes gives the corresponding dipyrromethane‐1,9‐dicarboxylates. The latter are subjected to the exhaustive reduction with lithium aluminum hydride to transform the ester moieties into methyl groups. The subsequent oxidation of the resulting 1,9‐dimethylated dipyrromethanes followed by the boron difluoride complexation afforded a family of novel core‐fluorinated BODIPYs in up to 74 % yield. Photophysical properties of the resulting BODIPYs were tuned by varying of the starting fluoropyrroles and aromatic aldehydes and were studied by UV‐visible and fluorescence spectroscopy. As a result, the fluorescence quantum yields of the obtained compounds reached up to 99 %. In addition, their ability to generate singlet oxygen and electrochemical properties were also evaluated. As a result, a new promising family of fluorophores with a good combination of the fluorescence and photosensitizing properties was obtained. It was found that conversion of ester groups into methyl ones at the 3,5‐positions of the BODIPY core is a crucial step toward fluorescence enhancement. In addition, DFT calculations were performed to elucidate a relationship between electronic structure, geometry and photophysical properties of these BODIPYs.

Transient Receptor Potential Vanilloid 1 (TRPV1) and Ankyrin 1 (TRPA1) are nonselective cation channels expressed in primary sensory neurons and several other non-neuronal structures such as immune cells, keratinocytes, and vascular smooth muscle cells. They play important roles in nociception, pain processing and their chanellopathies are associated with the development of several pathological conditions. They are located in cholesterol- and sphingolipid-rich membrane lipid raft regions serving as platforms to modulate their activations. We demonstrated earlier that disruption of these lipid rafts leads to decreased TRP channel activation and exerts analgesic effects. Cyclodextrins are macrocyclic molecules able to form host-guest complexes with cholesterol and deplete it from the membrane lipid rafts. The aim of this study was to investigate 8 structurally different (methylated and non-methylated) CD derivatives on cell viability, mitochondrial membrane potential, membrane composition and activation abilities of the TRPV1 and TRPA1 channels. We showed that non-methylated derivatives have preferable safety profiles compared to methylated ones. Furthermore, methylated derivatives reduced mitochondrial membrane potential. However, all investigated derivatives influence the ordered cell membrane structure depleting membrane cholesterol and inhibit the TRPV1 agonist capsaicin- and the TRPA1 agonist allyl isothiocyanate-induced Ca2+-influx. This mechanism of action might provide novel perspectives for the development of peripherally acting analgesics via indirectly decreasing the generation and transmission of nociceptive signals.

Temozolomide (TMZ) is considered a first line chemotherapy drug for glioblastoma (GBM). Unfortunately, the GBM without methylation of O6-methylguanine-DNA methyltransferase (MGMT), accounting for about 70% of all GBM, shows an inherent resistance to TMZ treatment. Aberrant accumulation of neutral lipids, primarily triglycerides (TGs) and cholesteryl esters (CEs), in lipid droplets (LDs) has been recognized as metabolic vulnerability for GBM therapy. However, it is not known whether MGMT methylation affects lipid accumulation in GBM. Herein, we employed label-free Raman spectromicroscopy, which integrated stimulated Raman scattering (SRS) microscopy and confocal Raman spectroscopy, to quantitatively analyze both the amount and composition of intracellular LDs in intact GBM tissues obtained from patients who had undergone resection surgery. Our results showed significant reductions in both the LD amount and the CE percentage in MGMT unmethylated GBMs (MGMT methylation < 15%) compared to MGMT methylated ones (MGMT methylation ≥ 15%). Due to a big variation of lipid accumulation in the MGMT methylated GBMs, these patients were further divided into hypermethylated group (MGMT methylation ≥ 50%) and intermediate-methylated group (MGMT methylation 15∼50%), according to the significantly different median survival rates of these two groups. Remarkable differences in LD amount, CE percentage, and also lipid saturation degree were found between the hypermethylated group and the other two groups, but not between the unmethylated and intermediate-methylated groups. To elucidate the possible underlying mechanism, we analyzed the differential expression of lipid metabolism-related genes in GBM with different levels of MGMT methylation using The Cancer Genome Atlas Program (TCGA) dataset. It was shown that the genes related to lipid oxidation and lipid efflux were upregulated, and the genes related to lipid synthesis were downregulated in unmethylated group. These findings unravel the relationship between MGMT methylation and lipid accumulation in GBM, which may offer new opportunities for the diagnosis and treatment of TMZ-resistant GBM.

The conserved H/ACA RNPs consist of one H/ACA RNA and 4 core proteins: dyskerin, NHP2, NOP10, and GAR1. Its assembly requires several assembly factors. A pre-particle containing the nascent RNAs, dyskerin, NOP10, NHP2 and NAF1 is assembled co-transcriptionally. NAF1 is later replaced by GAR1 to form mature RNPs. In this study, we explore the mechanism leading to the assembly of H/ACA RNPs. We performed the analysis of GAR1, NHP2, SHQ1 and NAF1 proteomes by quantitative SILAC proteomic, and analyzed purified complexes containing these proteins by sedimentation on glycerol gradient. We propose the formation of several distinct intermediate complexes during H/ACA RNP assembly, notably the formation of early protein-only complexes containing at least the core proteins dyskerin, NOP10, and NHP2, and the assembly factors SHQ1 and NAF1. We also identified new proteins associated with GAR1, NHP2, SHQ1 and NAF1, which can be important for box H/ACA assembly or function. Moreover, even though GAR1 is regulated by methylations, the nature, localization, and functions of these methylations are not well known. Our MS analysis of purified GAR1 revealed new sites of arginine methylations. Additionally, we showed that unmethylated GAR1 is correctly incorporated in H/ACA RNPs, even though with less efficiency than methylated ones.

Kikuchi-Fujimoto Disease: A Case Report

Simultaneous quantitative analysis of catecholamines (CAs) and their respective acid metabolites in biological fluids can become a powerful tool for diagnosis and treatment of various diseases. However, selective sample preparation procedure is necessary to isolate the aforementioned analytes from the matrix. Therefore, we propose a synthetic strategy for the preparation of a molecularly imprinted sorbent with binding sites for both CAs and their respective acid metabolites. The synthesis combines noncovalent and semicovalent imprinting methods. Methylenebisacrylamide, 4-vynilbenzyl-N,N,N-trimethylammonium (3,4-dimethoxyphenyl)acetate, and 3-phenylpropylacrylate were used as the cross-linker, functional monomer 1/“dummy” template 1, and functional monomer 2/“dummy” template 2 under precipitation polymerization conditions, respectively. The polymer was characterized using Fourier Transform Infrared Spectroscopy and Scanning Electron Microscopy. The binding properties were studied by constructing adsorption isotherms and conducting competitive binding tests. The performance of the molecularly imprinted polymers (MIPs) toward CAs and the acids was described by good imprinting factors (3.1–5.6), fast-binding equilibrium, and the ability to differentiate nonmethylated CAs from methylated ones. The MIP was used to simultaneously isolate the analytes from human plasma under dispersive solid-phase extraction conditions with subsequent detection using optimized UHPLC/MS/MS procedure. As a result, no major interferences were detected which suggests that the impurities were well separated from the analytes.

Intraocular lesions have been infrequently reported in patients with Gaucher disease type 3 (GD3). We previously reported siblings with GD3 who responded well to the combination of enzyme replacement therapy (ERT) and substrate reduction therapy (SRT). Here we report progressive bilateral vitreous and preretinal deposits with declining visual acuity requiring bilateral vitrectomies in one of these siblings. These ocular manifestations had progressed despite combined ERT and SRT with improvement in visual acuity after vitrectomies. Vitrectomy fluid analysis performed for the first time by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) identified a high concentration of glucosylceramide (GluCer) in the patient (262.842 nM) compared to a sample (0.428 nM from a patient without a lysosomal storage or known hereditary metabolic disorder). The GluCer detected in our patient was resolved into 12 different isoforms including two methylated ones. No evidence of galactosylceramide (GalCer) was detected. The development of these intraocular manifestations and their characterization by UPLC-MS/MS indicate a need for ongoing ophthalmologic evaluation of all GD patients and for new therapies that can cross the blood–retinal and blood–brain barriers for patients with GD and other neuropathic lysosomal storage disorders.

Coatings for high temperatures (HT &gt; 400 °C) are obtained from interpenetrating polymer network (IPN) binders formed by simultaneous polymerization of silicone and epoxide pre-polymers. A ceramic layer; mainly composed of silica and fillers; remains on the metal surface after a thermal treatment at 450 °C. The layer adhesion and the inorganic filler’s distribution have been investigated by, firstly, exchanging the organic substituents (methyl and phenyl) of the silicone chains and, secondly, by adding conductive graphene nanoplatelets with the aim to assure a uniform distribution of heat during the thermal treatment. The results are evidence that different substituent ratios affect the polymer initial layout. The adhesion tests of paint formulations are analysed and were related to instrumental analyses performed using glow discharge optical emission spectroscopy (GDOES); thermal analyses (TG/DTA and DSC); electron microscopy with energy dispersive X-ray analysis (SEM-EDX). A greater resistance to powdering using phenyl groups instead of methyl ones; and an improved distribution of fillers due to graphene nanoplatelet addition; is evidenced.

Absolute quantification of DNA methylation using microfluidic chip-based digital PCR

DFT calculations have been performed for a series of push-pull nitriles [(R2N)n(X═Y)iC≡N, where i = 0, 1, or 2, n = 1, 2, or 3, R2N = H2N, Me2N, or C4H8N, X = CH, N, or P, Y = CH or N]. The possible protonation N-sites (N-cyano, N-imino, and N-amino) have been examined and their proton affinities (PA) estimated. For all compounds in the series, even for those containing the guanidino, phosphazeno, and diphosphazeno pushing groups, the N-cyano atom is the favored site of protonation. The n-π conjugation strongly decreases the PA value of the pushing amino group in favor of the pulling cyano one. Nitriles with the phosphazeno groups [(R2N)3P═N-P(R2N)2═N and (R2N)3P═N] exhibit the strongest basicity in the series. Some of them (with PA > 1000 kJ mol(-1)) are stronger bases than DMAN, the so-called "proton sponge". Nitriles bearing the guanidino group [(R2N)2C═N] are less basic than those with the phosphazeno group [(R2N)3P═N] but more basic than those with the formamidino group (R2N-CH═N) containing the same substituent R. The N-imino atoms, present in the transmitter group (X═N, X = CH, N, or P), display PA values lower than those of the N-cyano site by more than 30 kJ mol(-1). When proceeding from the unsubstituted derivatives (R = H) to the methylated ones (R = Me), the Me groups at the N-amino atom increase the PA value of the N-cyano site for Me2N-X═Y-C≡N (X, Y = CH or N) by ca. 30-60 kJ mol(-1). For the guanidino and phosphazeno derivatives containing two and three amino groups, respectively, this effect is not additive. The four Me groups for (Me2N)2C═N-C≡N and the six Me groups for (Me2N)3P═N-C≡N increase the PA(N-cyano) values by only 30-50 kJ mol(-1). The C≡N bond lengths of the neutral forms are well correlated with the PA(N-cyano) values.

Glioblastoma multiforme (GBM) is the most common brain tumor in adults. The role of high in normal-1 (HIN-1) as a potential biomarker in combating this disease is being described for the first time in this study. A combination of O6-methylguanine DNA methyltransferase (MGMT) and HIN-1 methylation could be a possible biomarker in therapy choice. Interestingly, survival data shows a similar trend for the methylation of MGMT and for unmethylation of HIN-1 and vice versa. Eighty-eight paraffin-embedded brain tumors were analyzed to screen methylation rates of different genes and evaluate the association between genes methylation and clinicopathologic variables. Our study is the first of its kind to indicate that MGMT and HIN-1 methylation status are inverted (97.7% of methylated ones) and could be new markers in the study of GBM prognosis, especially in the therapy selection.

Methylone, 3,4-methylenedioxypyrovalerone (MDPV), and mephedrone are psychoactive ingredients of 'bath salts' and their abuse represents a growing public health care concern. These drugs are cathinone derivatives and are classified chemically as β-ketoamphetamines. Because of their close structural similarity to the amphetamines, methylone, MDPV, and mephedrone share most of their pharmacological, neurochemical, and behavioral properties. One point of divergence in their actions is the ability to cause damage to the CNS. Unlike methamphetamine, the β-ketoamphetamines do not damage dopamine (DA) nerve endings. However, mephedrone has been shown to significantly accentuate methamphetamine neurotoxicity. Bath salt formulations contain numerous different psychoactive ingredients, and individuals who abuse bath salts also coabuse other illicit drugs. Therefore, we have evaluated the effects of methylone, MDPV, mephedrone, and methamphetamine on DA nerve endings. The β-ketoamphetamines alone or in all possible two-drug combinations do not result in damage to DA nerve endings but do cause hyperthermia. MDPV completely protects against the neurotoxic effects of methamphetamine while methylone accentuates it. Neither MDPV nor methylone attenuates the hyperthermic effects of methamphetamine. The potent neuroprotective effects of MDPV extend to amphetamine-, 3,4-methylenedioxymethamphetamine-, and MPTP-induced neurotoxicity. These results indicate that β-ketoamphetamine drugs that are non-substrate blockers of the DA transporter (i.e., MDPV) protect against methamphetamine neurotoxicity, whereas those that are substrates for uptake by the DA transporter and which cause DA release (i.e., methylone, mephedrone) accentuate neurotoxicity. METH (a) enters DA nerve endings via the DAT, causes leakage of DA into the cytoplasm and then into the synapse via DAT-mediated reverse transport. Methylone (METHY) and mephedrone (MEPH; b), like METH, are substrates for the DAT but release DA from cytoplasmic pools selectively. When METH is combined with METHY or MEPH (c), DA efflux and neurotoxicity are enhanced. MDPV (d), which is a non-substrate blocker of the DAT, prevents METH uptake and efflux of DA. Therefore, bath salts that are substrates for the DAT and release DA (METHY, MEPH) accentuate METH neurotoxicity, whereas those that are non-substrate blockers of the DAT (MDPV) are neuroprotective.

Spectroscopy and dynamics of topotecan anti-cancer drug comprised within cyclodextrins

Abstract Low‐k dielectric films were deposited by PECVD from mixtures of hexamethyldisiloxane (HMDSO), ethylene, and oxygen. After deposition the films were thermally annealed in order to reduce their permittivity. Different deposition conditions were investigated in order to accomplish the best trade‐off between low permittivity and low thickness loss upon annealing. Soft plasma conditions were required in order to allow organic species incorporation in the film and achieve dielectric constant as low as 2.2 with a thickness loss of 14% upon thermal annealing treatment. Results were also compared to those obtained with divinyltetramethyldisiloxane, which is similar in structure to HMDSO but with two vinyl groups replacing methyl ones. The morphological analysis showed that feeding the plasma with a large amount of ethylene, CHx‐enriched nanodomains were obtained over a siloxane matrix. Finally, the plasma phase composition was also investigated by infrared spectroscopy and correlated with the film chemistry. magnified image

Evaluation of the cytotoxicity of β-cyclodextrin derivatives: Evidence for the role of cholesterol extraction

On the bioreactivity of triorganotin aminobenzoates. Investigation of trialkyl and triarylyltin(IV) esters of 3-amino and 4-aminobenzoic acids

DNA methyltransferase (cytosine-5) 1 (Dnmt1) is the principal enzyme responsible for maintenance of CpG methylation and is essential for the regulation of gene expression, silencing of parasitic DNA elements, genomic imprinting and embryogenesis. Dnmt1 is needed in S phase to methylate newly replicated CpGs occurring opposite methylated ones on the mother strand of the DNA, which is essential for the epigenetic inheritance of methylation patterns in the genome. Despite an intrinsic affinity of Dnmt1 for such hemi-methylated DNA, the molecular mechanisms that ensure the correct loading of Dnmt1 onto newly replicated DNA in vivo are not understood. The Np95 (also known as Uhrf1 and ICBP90) protein binds methylated CpG through its SET and RING finger-associated (SRA) domain. Here we show that localization of mouse Np95 to replicating heterochromatin is dependent on the presence of hemi-methylated DNA. Np95 forms complexes with Dnmt1 and mediates the loading of Dnmt1 to replicating heterochromatic regions. By using Np95-deficient embryonic stem cells and embryos, we show that Np95 is essential in vivo to maintain global and local DNA methylation and to repress transcription of retrotransposons and imprinted genes. The link between hemi-methylated DNA, Np95 and Dnmt1 thus establishes key steps of the mechanism for epigenetic inheritance of DNA methylation.

Field‐theoretic simulations of polyelectrolyte complexation

Study of different parameters affecting the derivatization of acidic herbicides with trimethylsulfonium hydroxide to make them suitable for gas chromatography analysis