Estrogen receptor β‐mediated nuclear interaction between IRS‐1 and Rad51 inhibits homologous recombination directed DNA repair in medulloblastoma

In medulloblastomas, which are highly malignant cerebellar tumors of the childhood genotoxic treatments such as cisplatin or γ‐irradiation are frequently associated with DNA damage, which often associates with unfaithful DNA repair, selection of new adaptations and possibly tumor recurrences. Therefore, better understanding of molecular mechanisms which control DNA repair fidelity upon DNA damage is a critical task. Here we demonstrate for the first time that estrogen receptor beta (ERβ) can contribute to the development of genomic instability in medulloblastomas. Specifically, ERβ was found highly expressed and active in mouse and human medulloblastoma cell lines. Nuclear ERβ was also present in human medulloblastoma clinical samples. Expression of ERβ coincided with nuclear translocation of insulin receptor substrate 1 (IRS‐1), which was previously reported to interfere with the faithful component of DNA repair when translocated to the nucleus. We demonstrated that ERβ and IRS‐1 bind each other, and the interaction involves C‐terminal domain of IRS‐1 (aa 931–1233). Following cisplatin‐induced DNA damage, nuclear IRS‐1 localized at the sites of damaged DNA, and interacted with Rad51—an enzymatic component of homologous recombination directed DNA repair (HRR). In medulloblastoma cells, engineered to express HRR‐DNA reporter plasmid, ER antagonist, ICI 182,780, or IRS mutant (931–1233) significantly increased DNA repair fidelity. These data strongly suggest that both molecular and pharmacological interventions are capable of preventing ERβ‐mediated IRS‐1 nuclear translocation, which in turn improves DNA repair fidelity and possibly counteracts accumulation of malignant mutations in actively growing medulloblastomas. J. Cell. Physiol. 219: 392–401, 2009. © 2008 Wiley‐Liss, Inc.     

High‐Performance and Stable Perovskite Solar Cells Based on Dopant‐Free Arylamine‐Substituted Copper(II) Phthalocyanine Hole‐Transporting Materials

A power conversion efficiency (PCE) as high as 19.7% is achieved using a novel, low‐cost, dopant‐free hole transport material (HTM) in mixed‐ion solution‐processed perovskite solar cells (PSCs). Following a rational molecular design strategy, arylamine‐substituted copper(II) phthalocyanine (CuPc) derivatives are selected as HTMs, reaching the highest PCE ever reported for PSCs employing dopant‐free HTMs. The intrinsic thermal and chemical properties of dopant‐free CuPcs result in PSCs with a long‐term stability outperforming that of the benchmark doped 2,2′,7,7′‐Tetrakis‐(N,N‐di‐p‐methoxyphenylamine)‐9,9′‐Spirobifluorene (Spiro‐OMeTAD)‐based devices. The combination of molecular modeling, synthesis, and full experimental characterization sheds light on the nanostructure and molecular aggregation of arylamine‐substituted CuPc compounds, providing a link between molecular structure and device properties. These results reveal the potential of engineering CuPc derivatives as dopant‐free HTMs to fabricate cost‐effective and highly efficient PSCs with long‐term stability, and pave the way to their commercial‐scale manufacturing. More generally, this case demonstrates how an integrated approach based on rational design and computational modeling can guide and anticipate the synthesis of new classes of materials to achieve specific functions in complex device structures.     

Sequence specificity of psoralen photobinding to DNA: a quantitative approach.

The effects of different DNA sequences on the photoreaction of various furocoumarin derivatives was investigated from a quantitative point of view using a number of self-complementary oligonucleotides. These contained 5'-TA and 5'-AT residues, having various flanking sequences. The furocoumarins included classical bifunctional derivatives, such as 8-methoxy- and 5-methoxypsoralen, as well as monofunctional compounds, such as angelicin and benzopsoralen. Taking into an account the thermodynamic constant for noncovalent binding of each psoralen to each DNA sequence, the rate constants for the photobinding process to each fragment were evaluated. The extent of photoreaction is greatly affected by the DNA sequence examined. While sequences of the type 5'-(GTAC)n are quite reactive towards all furocoumarins, 5'-TATA exhibited a reduced rate of photobinding using monofunctional psoralens. In addition terminal 5'-TA groups were the least reactive with 5- and 8-methoxypsoralen, but not with angelicin or benzopsoralen. Also 5'-AT-containing fragments exhibited remarkably variable responses toward monofunctional or bifunctional psoralen derivatives. As a general trend the photoreactivity rate of the former is less sequence-sensitive, the ratio between maximum and minimum being less than 2 for the examined fragments. The same ratio is about 3.4 for 8-methoxypsoralen and 6.2 for 5-methoxypsoralen. This approach, in combination with footprinting studies, appears to be quite useful for a quantitative investigation of the process of covalent binding of psoralens to specific sites in DNA.     

Investigation of the dark interaction between furocoumarins and DNA

The complexes between some furocoumarins and DNA have been studied using various physicochemical techniques. Flow-dichroism measurements data strongly support the intercalation of the planar furocoumarin molecules between two base pairs of duplex DNA. The equilibrium dialysis and spectrophotometric data show relatively low values of the association constants of the complexes and a small number of molecules able to intercalate in DNA, thus indicating that furocoumarins have a relatively low affinity for DNA in the complex formation. The biological and photobiological consequences connected with these results are discussed.The binding curves obtained using some polynucleotides and various DNA samples having different composition with regard to base pairs, have shown that the regions of the macromolecule having alternate sequences of purine and pyrimidine represent sites useful for intercalation. No preference has been observed for A-T or G-C.     

Finding flies in the mushroom soup: Host specificity of fungus‐associated communities revisited with a novel molecular method

Fruiting bodies of fungi constitute an important resource for thousands of other taxa. The structure of these diverse assemblages has traditionally been studied with labour‐intensive methods involving cultivation and morphology‐based species identification, to which molecular information might offer convenient complements. To overcome challenges in DNA extraction and PCR associated with the complex chemical properties of fruiting bodies, we developed a pipeline applicable for extracting amplifiable total DNA from soft fungal samples of any size. Our protocol purifies DNA in two sequential steps: (a) initial salt–isopropanol extraction of all nucleic acids in the sample is followed by (b) an extra clean‐up step using solid‐phase reversible immobilization (SPRI) magnetic beads. The protocol proved highly efficient, with practically all of our samples—regardless of biomass or other properties—being successfully PCR‐amplified using metabarcoding primers and subsequently sequenced. As a proof of concept, we apply our methods to address a topical ecological question: is host specificity a major characteristic of fungus‐associated communities, that is, do different fungus species harbour different communities of associated organisms? Based on an analysis of 312 fungal fruiting bodies representing 10 species in five genera from three orders, we show that molecular methods are suitable for studying this rich natural microcosm. Comparing to previous knowledge based on rearing and morphology‐based identifications, we find a species‐rich assemblage characterized by a low degree of host specialization. Our method opens up new horizons for molecular analyses of fungus‐associated interaction webs and communities. Fruiting bodies of fungi constitute an important resource for thousands of other taxa. The structure of these diverse assemblages has traditionally been studied with labour‐intensive methods involving cultivation and morphology‐based species identification, to which molecular information might offer convenient complements. To overcome challenges in DNA extraction and PCR associated with the complex chemical properties of fruiting bodies, we developed a pipeline applicable for extracting amplifiable total DNA from soft fungal samples of any size. Our protocol purifies DNA in two sequential steps: (a) initial salt–isopropanol extraction of all nucleic acids in the sample is followed by (b) an extra clean‐up step using solid‐phase reversible immobilization (SPRI) magnetic beads. The protocol proved highly efficient, with practically all of our samples—regardless of biomass or other properties—being successfully PCR‐amplified using metabarcoding primers and subsequently sequenced. As a proof of concept, we apply our methods to address a topical ecological question: is host specificity a major characteristic of fungus‐associated communities, that is, do different fungus species harbour different communities of associated organisms? Based on an analysis of 312 fungal fruiting bodies representing 10 species in five genera from three orders, we show that molecular methods are suitable for studying this rich natural microcosm. Comparing to previous knowledge based on rearing and morphology‐based identifications, we find a species‐rich assemblage characterized by a low degree of host specialization. Our method opens up new horizons for molecular analyses of fungus‐associated interaction webs and communities.     

Mutagenic compounds in an extract from Rutae Herba (Ruta graveolens L.). II. UV-A mediated mutagenicity in the green alga Chlamydomonas reinhardtii by furoquinoline alkaloids and furocoumarins present in a commercial tincture from Rutae Herba

A commercial tincture prepared from Rutae Herba (Ruta graveolens L.) exhibited a moderate photomutagenicity in an arginine-requiring mutant strain of Chlamydomonas reinhardtii. In the tincture some furocoumarins, e.g., bergapten, psoralen, imperatorin, and 3 furoquinoline alkaloids (dictamnine, gamma-fagarine, skimmianine) were detected. All compounds revealed photomutagenic properties but their activities were quite different. Bergapten was the most potent furocoumarin. Dictamnine, the furoquinoline with the strongest effect, reached only about 10% of the activity of bergapten. Based on the amount of these compounds in the tincture and their activities we conclude that bergapten is mainly responsible for the photomutagenicity of the tincture. The lower phototoxicity and photomutagenicity of the furoquinoline alkaloids may be due to the fact that furoquinolines form only monoadducts with DNA in the presence of UV-A in contrast to furocoumarins which also form biadducts.     

Synthesis and fluorescence properties of 1,2,4‐triazolo[3,4‐b]‐1,3,4‐thiadiazol derivatives and their terbium complexes

Eight novel 1,2,4‐triazolo[3,4‐b]‐1,3,4‐thiadiazol derivatives have been designed and synthesized, and their corresponding Tb³⁺ complexes were also prepared successfully. The fluorescence properties and fluorescence quantum yields of the target complexes were investigated, the results showed that the ligands were an efficient sensitizer for Tb³⁺ luminescence, and the target complexes exhibited characteristic fluorescence emissions of Tb³⁺ ion. The fluorescence intensity of the complex substituted by chlorine was stronger than that of other complexes. The substituents' nature has a great effect upon the electrochemical properties of the target complexes. The results showed that the introduction of the electron‐withdrawing groups tended to decrease the oxidation potential and highest occupied molecular orbital energy levels of the target Tb³⁺ complexes; however, introduction of the electron‐donating groups can increase the corresponding complexes' oxidation potential and highest occupied molecular orbital energy levels. Copyright © 2014 John Wiley & Sons, Ltd.     

Phylogenetic analysis of HpnP reveals the origin of 2‐methylhopanoid production in Alphaproteobacteria

Hopanoids are bacterial steroid‐like lipids that can be preserved in the rock record on billion‐year timescales. 2‐Methylhopanoids are of particular interest to geobiologists because methylation is one of the few chemical modifications that remain after diagenesis and catagenesis. 2‐Methylhopanes, the molecular fossils of 2‐methylhopanoids, are episodically enriched in the rock record, but we do not have a robust interpretation for their abundance patterns. Here, we exploit the evolutionary record found in molecular sequences from extant organisms to reconstruct the biosynthetic history of 2‐methylhopanoids using the C‐2 hopanoid methylase, HpnP. Based on HpnP phylogenetic analysis, we find that 2‐methylhopanoids originated in a subset of the Alphaproteobacteria. This conclusion is statistically robust and reproducible in multiple trials varying the outgroup, trimming stringency, and ingroup dataset used to infer the evolution of this protein family. The capacity for 2‐methylhopanoid production was likely horizontally transferred from the Alphaproteobacteria into the Cyanobacteria after the Cyanobacteria's major divergences. Together, these results suggest that the ancestral function of 2‐methylhopanoids was not related to oxygenic photosynthesis but instead to a trait already present in the Alphaproteobacteria. Moreover, given that early 2‐methylhopane deposits could have been made solely by Alphaproteobacteria before the acquisition of hpnP by Cyanobacteria, and that the Alphaproteobacteria are thought to be ancestrally aerobic, we infer that 2‐methylhopanoids likely arose after the oxygenation of the atmosphere. This finding is consistent with the geologic record—the oldest syngenetic 2‐methylhopanes occur after the rise of oxygen, in middle Proterozoic strata of the Barney Creek Formation.     

Design,Synthesis, and Biological Evaluation of 2‐(2‐Bromo‐3‐nitrophenyl)‐5‐phenyl‐1,3,4‐oxadiazole Derivatives as Possible Anti‐Breast Cancer Agents

Breast Cancer (BCa) is the most often diagnosed cancer among women who were in the late 1940’s. Breast cancer growth is largely dependent on the expression of estrogen and progesterone receptor. Breast cancer cells may have one, both, or none of these receptors. The treatment for breast cancer may involve surgery, hormonal therapy (Tamoxifen, an aromatase inhibitor, etc.) and oral chemotherapeutic drugs. The molecular docking technique reported the findings on the potential binding modes of the 2‐(2‐bromo‐3‐nitrophenyl)‐5‐phenyl‐1,3,4‐oxadiazole derivatives with the estrogen receptor (PDB ID: 3ERT). The 1,3,4‐oxadiazole derivatives 4a – 4j have been synthesized and described by spectroscopic method. 2‐(2‐Bromo‐6‐nitrophenyl)‐5‐(4‐bromophenyl)‐1,3,4‐oxadiazole ( 4c ) was reconfirmed by single‐crystal XRD. All the compounds have been tested in combination with generic Imatinib pharmaceutical drug against breast cancer cell lines isolated from Caucasian woman MCF‐7, MDA‐MB‐453 and MCF‐10A non‐cancer cell lines. The compounds with the methoxy (in 4c ) and methyl (in 4j ) substitution were shown to have significant cytotoxicity, with 4c showing dose‐dependent activation and decreased cell viability. The mechanism of action was reported by induced apoptosis and tested by a DNA enzyme inhibitor experiment (ELISA) for Methyl Transferase. Molecular dynamics simulations were made for hit molecule 4c to study the stability and interaction of the protein?ligand complex. The toxicity properties of ADME were calculated for all the compounds. All these results provide essential information for further clinical trials.     

Deficient repair of chemical adducts in α DNA of monkey cells

We have examined excision repair of DNA damage in the highly repeated α DNA sequence of cultured African green monkey cells. Irradiation of cells with 254 nm ultraviolet light resulted in the same frequency of pyrimidine dimers in α DNA and the bulk of the DNA. The rate and extent of pyrimidine dimer removal, as judged by measurement of repair synthesis, was also similar for α DNA and bulk DNA. In cells treated with furocoumarins and long-wavelength ultraviolet light, however, repair synthesis in α DNA was only 30% of that in bulk DNA, although it followed the same time course. We found that this reduced repair was not caused by different initial amounts of furocoumarin damage or by different sizes of repair patches, as we found these to be similar in the two DNA species. Direct quantification demonstrated that fewer furocoumarin adducts were removed from α DNA than from bulk DNA. In cells treated with another chemical DNA-damaging agent, N-acetoxy-2-acetylaminofluorene, repair synthesis in α DNA was 60% of that in bulk DNA. These results show that the repair of different kinds of DNA damage can be affected to different extents by some property of this tandemly repeated heterochromatic DNA. To our knowledge, this is the first demonstration in primate cells of differential repair of cellular DNA sequences.     

Photobiophysics of furanocoumarins

Furocoumarins (psoralens) are photosensitizers of plant origin, which increase the sensitivity of biological objects to near ultraviolet (UV-A, 320-400 nm). In combination with UV-A, they are successfully used for treating many dermal and autoimmune diseases (PUVA therapy and photophoresis). Along with therapeutic effects, the furocoumarin photochemotherapy induces a number of side-effects (erythema, edema, hyperpigmentation, and premature aging of skin). All photobiological effects of furocoumarins result from their photochemical reactions. Therefore, in order to advance the therapy, it is necessary to know the photochemical mechanisms of induction of both side- and therapeutic effects. The types of photoreactions of furocoumarins classified with respect to reactive photoproducts interacting with substrate were considered. Primary emphasis was placed on reactions proceeding with the participation of photooxidation products of furocoumarins. Among these photoproducts, at least two types can be distinguished. Some of them possess membranotoxic properties, others produce the immunosuppressory action in vivo. The photochemical mechanisms of the formation of the photoproducts of furocoumarins are different. It was found that, by varying the illumination conditions (intensity of UV-A radiation or the concentration of the photosensitizer), it is possible to obtain the photoproducts of furocoumarins that have either membranotoxic or immunosuppressory properties. It was found that the mechanisms of the immunosuppressive action of the photooxidation products of furocoumarins have some features in common with those underlying the PUVA therapy and photophoresis. It is assumed that the photochemical basis of the therapeutic action of furocoumarins is the reactions with the involvement of the products of their photooxidation.     

Post-irradiation dark recovery of photodamage to DNA induced by furocoumarins

Summary Photosensitization processes provoked by furocoumarins on various biological systems seem to be in connection with the photoreactions that these substances give bothin vitro andin vivo with pyrimidine bases of nucleic acids; in particular linear furocoumarins (psoralen) photoreact with native DNA giving both monofunctional and bifunctional additions (forming in this last case inter-strand cross-linkings) while angular furocoumarin (angelicin) can give only monofunctional additions. Previous studies on the possible recovery of this damage to DNA provoked both by linear and angular furocoumarins demonstrated that no repair underwent either by means of photosplitting experiments or through photoreactivation processes.In this paper are reported direct results indicating that the photodamage to DNA is repairable through post-irradiation dark recovery both operating on microbial cultures and on guinea-pig skin. In both biological systems monofunctional additions appear much more easily repairable than bifunctional additions; in any case bifunctional additions (which produce inter-strand cross-linkings) clearly appear to be repairable.     

A cytotoxicity,optical spectroscopy and computational binding analysis of 4‐[3‐acetyl‐5‐(acetylamino)‐2‐methyl‐2,3‐dihydro‐1,3,4‐thiadiazole‐2‐yl]phenyl benzoate in calf thymus DNA

In this study the interaction mechanism between newly synthesized 4‐(3‐acetyl‐5‐(acetylamino)‐2‐methyl‐2, 3‐dihydro‐1,3,4‐thiadiazole‐2‐yl) phenyl benzoate (thiadiazole derivative) anticancer active drug with calf thymus DNA was investigated by using various optical spectroscopy techniques along with computational technique. The absorption spectrum shows a clear shift in the lower wavelength region, which may be due to strong hypochromic effect in the ctDNA and the drug. The results of steady state fluorescence spectroscopy show that there is static quenching occurring while increasing the thiadiazole drug concentration in the ethidium bromide‐ctDNA system. Also the binding constant (K), thermo dynamical parameters of enthalpy change (ΔH°), entropy change (ΔS°) Gibbs free energy change (ΔG°) were calculated at different temperature (293 K, 298 K) and the results are in good agreement with theoretically calculated MMGBSA binding analysis. Time resolved emission spectroscopy analysis clearly explains the thiadiazole derivative competitive intercalation in the ethidium bromide‐ctDNA system. Further, molecular docking studies was carried out to understand the hydrogen bonding and hydrophobic interaction between ctDNA and thiadiazole derivative molecule. In addition the docking and molecular dynamics charge distribution analysis was done to understand the internal stability of thiadiazole derivative drug binding sites of ctDNA. The global reactivity of thiadiazole derivative such as electronegativity, electrophilicity and chemical hardness has been calculated.     

Isothiocyanate groups of 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonate (DIDS) inhibit cell penetration of octa‐arginine (R8)–fused peptides

Delivering biomolecules, such as antibodies, proteins, and peptides, to the cytosol is an important and challenging aspect of drug development and chemical biology. Polyarginine—a well‐known cell‐penetrating peptide (CPP)—is capable of exploiting its positive charge and guanidium groups to carry a fused cargo into the cytosol. However, the precise mechanism by which this occurs remains ambiguous. In the present study, we established a new method of quantitatively assessing cell penetration. The method involves inducing cell death by using a polyarginine (R8) to deliver a peptide—ie, mitochondrial targeting domain (MTD)—to the cytosol. We found that 4,4′‐diisothiocyanatostilbene‐2,2′‐di‐sulfonate (DIDS)—an anion channel blocker—inhibited the ability of octa‐arginine (R8)–fused MTD to penetrate cells. Other anion channel blockers did not inhibit the penetration of peptides fused with R8. Comparison of DIDS with other structurally similar chemicals revealed that the isothiocyanate group of DIDS may be primarily responsible for the inhibitory effect than its stilbene di‐sulfonate backbone. These results imply that the inhibitory effect of DIDS may not be derived from the interaction between stilbene di‐sulfonate and the anion channels, but from the interaction between the isothiocyanate groups and the cell membrane. Our new MTD method enables the quantitative assessment of cell penetration. Moreover, further studies on the inhibition of CPPs by DIDS may help clarify the mechanism by which penetration occurs and facilitate the design of new penetrative biomolecules.     

Histone deacetylases: salesmen and customers in the post‐translational modification market

HDACs (histone deacetylases) are enzymes that remove the acetyl moiety from N‐?‐acetylated lysine residues in histones and non‐histone proteins. In recent years, it has turned out that HDACs themselves are also subject to post‐translational modification. Such structural alterations can determine the stability, localization, activity and protein—protein interactions of HDACs. This subsequently affects the modification of their substrates and the co‐ordination of cellular signalling networks. Intriguingly, physiologically relevant non‐histone proteins are increasingly found to be deacetylated by HDACs, and aberrant deacetylase activity contributes to several severe human diseases. Targeting the catalytic activity of these enzymes and their post‐translational modifications are therefore attractive targets for therapeutical intervention strategies. To achieve this ambitious goal, details on the molecular mechanisms regulating post‐translational modifications of HDACs are required. This review summarizes aspects of the current knowledge on the biological role and enzymology of the phosphorylation, acetylation, ubiquitylation and sumoylation of HDACs.     

DNA‐binding activity and cytotoxic and cell‐cycle arrest properties of some new coumarin derivatives: a multispectral and computational investigation

Coumarins are the most important class of natural compounds found widely in various plants. Many coumarin derivatives with different biological and pharmacological activities have been synthesized. In this study, the antiapoptotic and cytotoxic effects and DNA‐binding properties of some synthetic coumarin derivatives (4b, 4d, 4f, 4 g (DBP‐g), 4 h and 4j) against K562 cell lines were investigated using different techniques. MTT assay indicated that the DBP‐g compound was more active than other derivatives, with a IC₅₀ value of 55 μM, and therefore this compound was chosen for further investigation. Apoptosis induction was assessed using acridine orange/ethidium bromide double‐staining and cell‐cycle analysis. In addition, in vitro DNA‐binding studies were carried out using ultraviolet–visible light absorption and fluorescence spectroscopy, as well as viscosity measurement and molecular modelling studies. In vitro results indicated that DBP‐g interacted with DNA through a groove‐binding mode with a binding constant (K_b) of 1.17 × 10⁴ M^?1. In agreement with other experimental data, molecular docking studies showed that DBP‐g is a minor groove binder. Overall, it can be concluded that DBP‐g could be used as an effective and novel chemotherapeutic agent.     

Synthesis of the N‐methyl Derivatives of 2‐Aminothiazol‐4(5H)‐one and Their Interactions with 11βHSD1‐Molecular Modeling and in Vitro Studies

11β‐Hydroxysteroid dehydrogenase type 1 (11β‐HSD1) is an enzyme that affects the body's cortisol levels. The inhibition of its activity can be used in the treatment of Cushing's syndrome, metabolic syndrome and type 2 diabetes. In this study, we synthesized new derivatives of 2‐(methylamino)thiazol‐4(5H)‐one and tested their activity towards inhibition of 11β‐HSD1 and its isoform – 11β‐HSD2. The results were compared with the previously tested allyl derivatives. We found out that methyl derivatives are weaker inhibitors of 11β‐HSD1 in comparison to their allyl analogs. Due to significant differences in the activity of the compounds, molecular modeling was performed, which was aimed at comparing the interactions between 11β‐HSD1 and ligands differing by substituent at the amine group (allyl vs. methyl). Modeling showed that the absence of the allyl group can lead to the rotation of whole ligand molecule which affects its interaction with the enzyme.