Protocol for engineering and validating a synthetic mitochondrial intermembrane bridge in mammalian cells

Membrane contact sites are recognized as critical means of intercompartmental communication. Here, we describe a protocol for engineering and validating a synthetic bridge between the inner and outer mitochondrial membranes to support functioning of the endogenous mitochondrial contact site and cristae organizing system (MICOS). A chimeric protein,MitoT, is stably expressed in cultured mammalian cells to bridge themitochondrial membranes. This approach can be a valuable tool to study the function of the MICOS complex and associated proteins

The shape parameter of liposomes and DNA-lipid complexes determined by viscometry utilizing small sample volumes

A minicapillary viscometer utilizing \u3c0.5 ml of sample at a volume fraction of \u3c0.1% is described. The calculated a/b of DPPC/DPPG multilamellar liposome was 1.14 as prolate ellipsoids and a/b of dioleoylpropyltrimethyl ammonium methylsulfate-DNA complex at a charge ratio of 4:1 (+/-) was 3.7 as prolate ellipsoids or 4.9 as oblate ellipsoids. The deviation of shape from perfect sphere is thus expressed quantitatively in more than two significant figures. In these measurement, the necessary amount of DNA is \u3c0.5 mg

The interaction of native calf thymus DNA with FeIII-dipyrido[3,2-a:2\u2019,3\u2019-c]phenazine

The mono and bis dipyrido[3,2-a:20,30-c]phenazine (dppz) adducts of iron(III) chloride, i.e. [Fe(dppz)]Cl3 and [Fe(dppz)2]Cl3, have been synthesized and characterized. The interaction of the FeIIIdppz hydrolyzed aquo complex with native calf thymus DNA has been monitored as a function of the metal complex\u2013DNA molar ratio, by variable temperature UV absorption spectrophotometry, circular dichroism (CD) and fluorescence spectroscopy. The results obtained in solution at various ionic strength values give support for a tight intercalative binding of the FeIIIdppz cation with DNA. In particular, the appearance of induced CD bands, caused by the addition of FeIIIdppz, indicate the existence of a rigid metal complex\u2013DNA-binding leading to dominating chiral organization of FeIIIdppz species within the DNA double helix. The trend of selected CD bands with the molar concentration of FeIIIdppz emphasizes that the presence of high amounts of metal complex induces also the formation of DNA\u2013FeIIIdppz supramolecular aggregates in solution. The analysis of fluorescence measurements allowed us to calculate a value of the intercalative binding constant comparable to that obtained by UV spectrophotometric titration. Finally, the temperature dependence of the absorbance at 258 nm shows that the metal complex strongly increases the DNA melting temperature already at metal complex\u2013DNA molar ratio equal to 0.25 suggesting that metal complex intercalation effectively hinders DNA denaturation. Overall, the results of the present study point out that the FeIIIdppz aquo complex has DNA-binding properties analogous to those previously reported for the tris-chelate FeII(phen)2dppz complex (phen = 1,10-phenantroline)

A protocol to construct RNA-protein devices for photochemical translational regulation of synthetic mRNAs in mammalian cells

Here, we describe a protocol for the translational regulation of transfected messenger RNAs (mRNAs) using light in mammalian cells. We detail the steps for photocaged ligand synthesis, template DNA preparation, and mRNA synthesis. We describe steps for mRNA transfection, treatment of cells with a photocaged ligand followed by light irradiation, and analysis of the transgene expression. The protocol enables spatiotemporally regulated transgene expression without the risk of insertional mutagenesis

Dayak Onion Tuber (Eleutherine Bulbosa (Mill) (Urb) Extract Ethanol Test on The Histopatological Description of Pancreas Wistar Rat Inducted Streptozotocin

This study aimed to determine the effect of Dayak Union tuber (Eleutherine bulbosa (Mill) Urb) ethanol extract on the regeneration of pancreatic β cells of Wistar rats induced streptozotocin. This research is laboratory experimental study by using 30 rats, they are divided into 5 groups, each group consisting of 6 rats, they are group 1 (normal control), group 2 (sick control) was given Na CMC 1 % b/v, Group 3 (positive control) was given glibenclamide at a dose of 0.25 mg / KgBW, Group 4,5 and 6 were given (Eleutherine bulbosa (Mill) Urb) extract at a dose of 125 mg / KgBB, 250 mg / KgBB and 500 mg / kgBW orally for 15 consecutive days. The histopathological damage level of the pancreas was observed with HE staining using a 400x magnification Olympus Cx-21 microscope. The result of the study showed that there were secondary metabolites of alkaloids, flavonoids, saponins, steroids, and tannins in the ethanol extract of Eleutherine palmifolia (Mill) Urb); The histopathological description showed that the damage to the structure of Langerhans islands and only a dose of 500 mg / KgBB the ethanol extract of Eleutherine palmifolia (Mill) Urb) showed improvement. Keywords: Dayak Onion Tuber; histopathological; extract ethano

Electrokinetic properties of NaCl solution via molecular dynamics simulations with scaled-charge electrolytes

ACKNOWLEDGMENTS The authors thank Mrs. Flavia Silva de Castro, from the British Consulate-General in Rio de Janeiro, for her effort in promoting a closer interaction between British and Brazilian universities. This work has been supported by the São Paulo Research Foundation (FAPESP) for the project funding (Grant Nos. 2018/02713-8, 2020/13300-6, and 2021/13068-9), the “Centro Nacional de Processamento de Alto Desempenho em São Paulo” (CENAPAD-SP), and the John David Rogers Computing Center (CCJDR) at the Institute of Physics “Gleb Wataghin” of the University of Campinas for providing computational resources.Peer reviewe

Impact of copper and iron binding properties on the anticancer activity of 8-hydroxyquinoline derived Mannich bases.

The anticancer activity of 8-hydroxyquinolines relies on complex formation with redox active copper and iron ions. Here we employ UV-visible spectrophotometry and EPR spectroscopy to compare proton dissociation and complex formation processes of the reference compound 8-hydroxyquinoline (Q-1) and three related Mannich bases to reveal possible correlations with biological activity. The studied derivatives harbor a CH2-N moiety at position 7 linked to morpholine (Q-2), piperidine (Q-3), and chlorine and fluorobenzylamino (Q-4) substituents. Solid phase structures of Q-3, Q-4·HCl·H2O, [(Cu(HQ-2)2)2]·(CH3OH)2·Cl4·(H2O)2, [Cu(Q-3)2]·Cl2 and [Cu(HQ-4)2(CH3OH)]·ZnCl4·CH3OH were characterized by single-crystal X-ray diffraction analysis. In addition, the redox properties of the copper and iron complexes were studied by cyclic voltammetry, and the direct reaction with physiologically relevant reductants (glutathione and ascorbic acid) was monitored. In vitro cytotoxicity studies conducted with the human uterine sarcoma MES-SA/Dx5 cell line reveal the significant cytotoxicity of Q-2, Q-3, and Q-4 in the sub- to low micromolar range (IC50 values 0.2-3.3 μM). Correlation analysis of the anticancer activity and the metal binding properties of the compound series indicates that, at physiological pH, weaker copper(ii) and iron(iii) binding results in elevated toxicity (e.g.Q4: pCu = 13.0, pFe = 6.8, IC50 = 0.2 μM vs.Q1: pCu = 15.1, pFe = 13.0 IC50 = 2.5 μM). Although the studied 8-hydroxyquinolines preferentially bind copper(ii) over iron(iii), the cyclic voltammetry data revealed that the more cytotoxic ligands preferentially stabilize the lower oxidation state of the metal ions. A linear relationship between the pKa (OH) and IC50 values of the studied 8-hydroxyquinolines was found. In summary, we identify Q-4 as a potent and selective anticancer candidate with significant toxicity in drug resistant cells

Rings, Hexagons, Petals, and Dipolar Moment Sink-Sources: The Fanciful Behavior of Water around Cyclodextrin Complexes

The basket-like geometry of cyclodextrins (CDs), with a cavity able to host hydrophobic groups, makes these molecules well suited for a large number of fundamental and industrial applications. Most of the established CD-based applications rely on trial and error studies, often ignoring key information at the atomic level that could be employed to design new products and to optimize their use. Computational simulations are well suited to fill this gap, especially in the case of CD systems due to their low number of degrees of freedom compared with typical macromolecular systems. Thus, the design and validation of solid and efficient methods to simulate and analyze CD-based systems is key to contribute to this field. The behavior of supramolecular complexes critically depends on the media where they are embedded, so the detailed characterization of the solvent is required to fully understand these systems. In the present work, we use the inclusion complex formed by two α-CDs and one sodium dodecyl sulfate molecule to test eight different parameterizations of the GROMOS and AMBER force fields, including several methods aimed to increase the conformational sampling in computational molecular dynamics simulation trajectories. The system proved to be extremely sensitive to the employed force field, as well as to the presence of a water/air interface. In agreement with previous experiments and in contrast to the results obtained with AMBER, the analysis of the simulations using GROMOS showed a quick adsorption of the complex to the interface as well as an extremely exotic behavior of the water molecules surrounding the structure both in the bulk aqueous solution and at the water surface. The chirality of the CD molecule seems to play an important role in this behavior. All together, these results are expected to be useful to better understand the behavior of CD-based supramolecular complexes such as adsorption or aggregation driving forces, as well as to introduce new methods able to speed up general MD simulationsThis work was supported by the Spanish Agencia Estatal de Investigación (AEI) and the ERDF (RTI2018-098795-A-I00), and by the Xunta de Galicia and the ERDF (ED431C 2017/25 and Centro singular de investigación de Galicia accreditation 2016-2019, ED431G/09). P.F.G. is funded by a predoctoral research grant (BES-2016-076761) from the Spanish Ministry of Economy and Competitiveness and the European Social Fund. M.C. is funded by a predoctoral fellowship from Xunta de Galicia. R.G.-F. is a “Ramón y Cajal” fellowship (RYC-2016-20335) from the Ministerio de Ciencia, Innovación y UniversidadesS

Fluorescence studies on new potential antitumoral benzothienopyran-1-ones in solution and in liposomes

Fluorescence properties of four new potential antitumoral compounds, 3-arylbenzothieno[2,3-c]pyran-1-ones, were studied in solution and in lipid membranes of dipalmitoyl phosphatidylcholine (DPPC), egg yolk phosphatidylcholine (Egg-PC) and dioctadecyldimethylammonium bromide (DODAB). The 3-(4-methoxyphenyl)benzothieno[2,3-c]pyran-1-one (1c) exhibits the higher fluorescence quantum yields in all solvents studied. All compounds present a solvent sensitive emission, with significant red shifts in polar solvents for the methoxylated compounds. The results point to an ICT character of the excited state, more pronounced for compound 1c. Fluorescence (steady-state) anisotropy measurements of the compounds incorporated in liposomes of DPPC, DODAB and Egg-PC indicate that all compounds have two different locations, one due to a deep penetration in the lipid membrane and another corresponding to a more hydrated environment. In general, the methoxylated compounds prefer hydrated environments inside the liposomes. The 3-(4- fluorophenyl)benzothieno[2,3-c]pyran-1-one (1a) clearly prefers a hydrated environment, with some molecules located at the outer part of the liposome interface. On the contrary, the preferential location of 3-(2-fluorophenyl)benzothieno[2,3-c]pyran-1-one (1b) is in the region of lipid hydrophobic tails. Compounds with a planar geometry (1a and 1c) have higher mobility in the lipid membranes when phase transition occurs.Portugal and FEDER (Fundo Europeu de Desenvolvimento Regional), for financial support through Centro de Física (CFUM) and Centro de Química (CQ-UM) of University of Minho and through the Project PTDC/QUI/81238/2006. M.S.D. Carvalho and R.C. Calhelha acknowledge FCT for their PhD grants SFRH/BD/47052/2008 and SFRH/BD/29274/2006, respectively.Fundação para a Ciência e a Tecnologia (FCT