62 research outputs found
Click to learn, learn to click: undergraduate synthetic organic chemistry experiments
The optimization of an undergraduate experiment for Organic Chemistry students is described to explore the concept of
click chemistry. The preparation of a terminal fluorescent alkyne and an organic azide is reported consisting of simple steps.
These are employed in the Cu(I)-catalized azide-alkyne cycloaddition to obtain a novel molecule containing a triazole ring
whose characterization allows the students to practice a variety of techniques: NMR (1H, 13C, COSY and HSQC), melting
point, thin layer chromatography, IR, fluorescence spectroscopy and mass spectrometry to confirm the structure of their
obtained product. An alternative methodology in a one-pot reaction is also explored and a full laboratory manual provided.FQM-208. GlycoChemBio: Glycochemistry and Bioconjugatio
Turning carbon dots into selenium bearing nanoplatforms with in vitro GPx-like activity and pro-oxidant activity
Selenium (Se) has been defined as the âJanus elementâ, with one face showing antioxidant activity and the other pro-oxidant
activity. The biological effect of Se depends on both dose and speciation. Se nanoparticles are attracting major interest, although
their large-scale preparation for biomedical applications is not trivial. We hypothesize that acid anhydride-coated carbon dots (AACD)
are an attractive platform for preparing nanoparticles containing chemically defined Se. The reaction of AA-CD with 3-
selenocyanatopropan-1-amine yields carbon dots bearing selenocyanate and carboxylate groups (CD-SeCN) that allow for
tuning the hydrosolubility. CD-SeCN has a Se content of 0.36 ÎŒmol per mg of nanoparticles, and they show the typical
photoluminescence of carbon dots. The selenocyanate groups (SeCN) exhibited glutathione peroxidase-like activity and
cytotoxicity. Data show that antioxidant behavior differs between normal and tumor cells, and the evaluation on HEK293 and
A549 cells reveals that the toxicity of CD-SeCN depends on dose, time, and intracellular glutathione (GSH) content. The toxicity
of CD-SeCN decreases with the time of incubation and the cell death mechanism switches from necrosis to apoptosis, indicating
that CD-SeCN is neutralized. Additionally, high levels of intracellular GSH exert a protective effect. These results support a
pharmacological potential in cancers with low levels of intracellular GSH. The use of AA-CD as nanoplatforms is a general
strategy that paves the way for the engineering of advanced nanosystemsSpanish institution
Ministerio de Ciencia, Innovacion y Universidades (No. CTQ2017-
86125-P)Unit of Excellence in Chemistry Applied to Biomedicine and the EnvironmentCentro de Instrumentacion Cientifica (Universidad de Granada)Universidad de Granada/CBUASpringer Natur
Capillary microfluidic platform for sulfite determination in wines
A microfluidic paper-based analytical device integrating a chromoreactand â a formylazo dyeâ has been fabri- cated and used for a colorimetric assay of sulfites. The chromoreactand was covalently linked to paper by vinyl sulfone chemistry. This work presents two robust capillary microfluidic devices to determine sulfite in wine without any pretreatment. One of them based on thread (”TPAD) useful to determine it in white wine and another based on paper (”PAD) to specifically determine sulfite in red wine as well as in white wine. Both are based on the selective recognition of sulfite by means of a chromoreactand that turns from orange to yellow in the presence of sulfite. The colour information acquired (H coordinate) using a digital camera readout allows for a range of appli-
cation of the ”TPAD from 7.8â
10â5 M (8.1 mg Lâ1) to 2.7â
10â3 M (279.3 mg Lâ1) with a limit of detection (LOD) of 78 ”M. The strong interference caused by the dyes present in red wine is eliminated by including a laminated paper
channel in the ”PAD structure that allows for the separation of colorants from red wine before the recognition of the sulfite. This makes it possible to adjust the ”PAD procedure to the usual sulfite concentration in wine, with an LOD of 2.2â
10â4 M (22.7 mg Lâ1) and a CV of 2.6%.This work was founded by Spanish âMinisterio de EconomĂa y Competitividadâ (Projects PID2019-103938RB-I00) and Junta de
AndalucĂa (Projects B-FQM-243-UGR18 and P18-RT-2961). The projects were partially supported by European Regional Development Funds (ERDF)
Poly(ethylene-imine)-Functionalized Magnetite Nanoparticles Derivatized with Folic Acid: Heating and Targeting Properties
Magnetite nanoparticles (MNPs) coated by branched poly (ethylene-imine) (PEI) were
synthesized in a one-pot. Three molecular weights of PEI were tested, namely, 1.8 kDa (sample
MNP-1), 10 kDa (sample MNP-2), and 25 kDa (sample MNP-3). The MNP-1 particles were further
functionalized with folic acid (FA) (sample MNP-4). The four types of particles were found to behave
magnetically as superparamagnetic, with MNP-1 showing the highest magnetization saturation.
The particles were evaluated as possible hyperthermia agents by subjecting them to magnetic fields
of 12 kA/m strength and frequencies ranging between 115 and 175 kHz. MNP-1 released the
maximum heating power, reaching 330 W/g at the highest frequency, in the high side of reported
values for spherical MNPs. In vitro cell viability assays of MNP-1 and MNP-4 against three cell
lines expressing different levels of FA receptors (FR), namely, HEK (low expression), and HeLa (high
expression), and HepG2 (high expression), demonstrated that they are not cytotoxic. When the cells
were incubated in the presence of a 175 kHz magnetic field, a significant reduction in cell viability
and clone formation was obtained for the high expressing FR cells incubated with MNP-4, suggesting
that MNP-4 particles are good candidates for magnetic field hyperthermia and active targeting.Spanish Institutions: Ministerio de Ciencia, InnovaciĂłn y
Universidades (PGC2018-098770-B-I00 and CTQ2017-86125-P)Junta de AndalucĂa (ProgramaOperativo FEDER 2014-2020, grants B-FQM-141-UGR18, A1-FQM-341-UGR-18, C-FQM-497-UGR18
Vinyl sulfone silica: application of an open preactivated support to the study of transnitrosylation of plant proteins by S-nitrosoglutathione
Background
S-nitrosylaton is implicated in the regulation of numerous signaling pathways with a diversity of regulatory roles. The high lability of the S-NO bond makes the study of proteins regulated by S-nitrosylation/denitrosylation a challenging task and most studies have focused on already S-nitrosylated proteins. We hypothesize that: i) S-nitrosoglutathione (GSNO) transnitrosylation is a feasible mechanism to account for the physiological S-nitrosylation of rather electropositive sulfur atoms from proteins, ii) affinity chromatography is a suitable approach to isolate proteins that are prone to undergo S-transnitrosylation and iii) vinyl sulfone silica is a suitable chromatographic bead.Results
The combination of vinyl sulfone silica with GSNO yielded an affinity resin that withstood high ionic strength without shrinking or deforming and that it was suitable to isolate potential GSNO transnitrosylation target candidates. Fractions eluted at 1500âmM NaCl resulted in a symmetrical peak for both, protein and S-nitrosothiols, supporting the idea of transnitrosylation by GSNO as a selective process that involves strong and specific interactions with the target protein. Proteomic analysis led to the identification of 22 physiological significant enzymes that differ with the tissue analyzed, being regulatory proteins the most abundant group in hypocotyls. The identification of chloroplastidic FBPase, proteasome, GTP-binding protein, heat shock Hsp70, syntaxin, catalase I, thioredoxin peroxidase and cytochrome P450 that have already been reported as S-nitrosylated by other techniques can be considered as internal positive controls that validate our experimental approach. An additional validation was provided by the prediction of the S-nitrosylation sites in 19 of the GSNO transnitrosylation target candidates.Conclusions
Vinyl sulfone silica is an open immobilization support that can be turned ad hoc and in a straightforward manner into an affinity resin. Its potential in omic sciences was successfully put to test in the context of the analysis of post-translational modification by S-nitrosylation with two different tissues: mature pea leaves and embryogenic sunflower hypocotyls. The identified proteins reveal an intriguing overlap among S-nitrosylation and both tyrosine nitration and thioredoxin regulation. Chloroplastidic FBPase is a paradigm of such overlap of post-translational modifications since it is reversible modified by thioredoxin and S-nitrosylation and irreversibly by tyrosine nitration. Our results suggest a complex interrelation among different modulation mechanisms mediated by NO-derived molecules.Financial Support was provided by Dirección General de Investigacion Cientıfica
y TĂ©cnica (DGICYT) (CTQ2008-01754), Junta de AndalucĂa (P07-FQM-02899),
Universidad de Jaén campus de Excelencia Internacional Agroalimentario ceiA3
and by ERDF-cofinanced grants from Ministry of Science and Innovation
(BIO2012-33904) and Junta de AndalucĂa (research groups BIO286 and BIO192).
We also acknowledge support of the publication fee by the CSIC Open Access
Publication Support Initiative through its Unit of Information Resources for
Research (URICI)
Molecular Recognition of Surface Trans-Sialidases in Extracellular Vesicles of the Parasite Trypanosoma cruzi Using Atomic Force Microscopy (AFM)
Trans-sialidases (TS) are important constitutive macromolecules of the secretome present on
the surface of Trypanosoma cruzi (T. cruzi) that play a central role as a virulence factor in Chagas disease.
These enzymes have been related to infectivity, escape from immune surveillance and pathogenesis
exhibited by this protozoan parasite. In this work, atomic force microscopy (AFM)-based single
molecule-force spectroscopy is implemented as a suitable technique for the detection and location
of functional TS on the surface of extracellular vesicles (EVs) released by tissue-culture cell-derived
trypomastigotes (Ex-TcT). For that purpose, AFM cantilevers with functionalized tips bearing the
anti-TS monoclonal antibody mAb 39 as a sense biomolecule are engineered using a covalent chemical
ligation based on vinyl sulfonate click chemistry; a reliable, simple and efficient methodology for the
molecular recognition of TS using the antibody-antigen interaction. Measurements of the breakdown
forces between anti-TS mAb 39 antibodies and EVs performed to elucidate adhesion and forces
involved in the recognition events demonstrate that EVs isolated from tissue-culture cell-derived
trypomastigotes of T. cruzi are enriched in TS. Additionally, a mapping of the TS binding sites with
submicrometer-scale resolution is provided. This work represents the first AFM-based molecular
recognition study of Ex-TcT using an antibody-tethered AFM probe.ERANet program ERANet17/HLH-0142Spanish Government PGC2018-099424-B-I0
Enhancing a de novo enzyme activity by computationally-focused ultra-low-throughput screening
Directed evolution has revolutionized protein engineering. Still, enzyme optimization by random library
screening remains sluggish, in large part due to futile probing of mutations that are catalytically neutral
and/or impair stability and folding. FuncLib is a novel approach which uses phylogenetic analysis and
Rosetta design to rank enzyme variants with multiple mutations, on the basis of predicted stability. Here,
we use it to target the active site region of a minimalist-designed, de novo Kemp eliminase. The
similarity between the Michaelis complex and transition state for the enzymatic reaction makes this
system particularly challenging to optimize. Yet, experimental screening of a small number of active-site
variants at the top of the predicted stability ranking leads to catalytic efficiencies and turnover numbers
( 2 104 M 1 s 1 and 102 s 1) for this anthropogenic reaction that compare favorably to those of
modern natural enzymes. This result illustrates the promise of FuncLib as a powerful tool with which to
speed up directed evolution, even on scaffolds that were not originally evolved for those functions, by
guiding screening to regions of the sequence space that encode stable and catalytically diverse
enzymes. Empirical valence bond calculations reproduce the experimental activation energies for the
optimized eliminases to within 2 kcal mol 1 and indicate that the enhanced activity is linked to better
geometric preorganization of the active site. This raises the possibility of further enhancing the stabilityguidance
of FuncLib by computational predictions of catalytic activity, as a generalized approach for
computational enzyme designKnut and Alice Wallenberg Foundation (Wallenberg Academy Fellowship)
2018.0140Human Frontier Science Program
RGP0041/2017FEDER Funds/Spanish Ministry of Science, Innovation and Universities
BIO2015-66426-R
RTI2018-097142-B-100FEDER/Junta de Andalucia - Consejeria de Economia y Conocimiento
E.FQM.113.UGR18Swedish National Infrastructure for computing (SNAC)
2018/2-3
2019/2-
WEARABLE DEVICE FOR REAL TIME pH MEASUREMENT IN SWEAT
Nowadays, it is more and more common to find devices that permits to everybody carry out analysis of different analytes of interest as glucose in blood or creatinine in urine by themselves, thanks to the development of the Point-of-Care (POC) devices. POCâs permit the in situ analysis of the samples, in an easy way, quickly and by the use of a small amount of sample in the sampling area of the device, obtaining result with no need of instrumentation or by the use of a very simple one. In order to match these objectives and make the device useful for everybody in any condition, the WHO has described the ASSURED guidelines for the POC devices[1].
In the recent years, and thanks to the capillary properties of different materials as paper, thread or cloth, the development of the POC devices are turning to a new strategy that implies the inclusion of the POC devices in t-shirts, bracelets or patches obtaining in this way wearables sensors. In this kind of sensor, instead of the addition of the sample in the sampling area, it moves through the device arriving to recognition/transduction area were the property of the sensor changes and can be measured and related to the concentration of the analyte.
In this work, we present a wearable POC that permits the real-time determination of the pH in sweat. For this purpose, we have developed a ÎŒCAD (Figure) that contains a pH indicator (4-[4-(2-hydroxyethanesulfonyl)-phenylazo]-2,6-dimethoxyphenol (GJM-534) [2]) covalently immobilized on cotton cloth, which color is going to change from yellow (pH around 6) to pink (pH around 9) depending on the pH. The size and shape of the ÎŒCAD (see Figure) was designed taking into account the low flow rate of sweat generated in the wrist when sweating (0.01 ÎŒL/min) including a superabsorbent material working as passive pump to avoid the saturation of sample of the ÎŒCAD.
The colorimetric device was calibrated using the H parameter from the HSV color space as analytical parameter, obtaining the calibration function and analytical parameters of the device, the reversibility of the ÎŒCAD, response time and stability.
Finally, the ÎŒCAD was integrated into a bracelet that includes a color detector and a microprocessor that registered the color of the ÎŒCAD in real-time and send the information via Bluetooth to a smartphone, obtaining and registering the pH of the sweat while doing exercise.This study was supported by project from the Spanish MINECO (CTQ2016-78754-C2-1-R)
Biological Evaluation and Docking Studies of Synthetic Oleananetype Triterpenoids
Saponins are potential wide-spectrum antitumor
drugs, and copper(I) catalyzed azideâalkyne 1,3-dipolar
cycloaddition is a suitable approach to synthesizing saponinlike
compounds by regioselective glycosylation of the C2/C3
hydroxyl and C28 carboxylic groups of triterpene aglycones
maslinic acid (MA) and oleanolic acid (OA). Biological
studies on the T-84 human colon carcinoma cell line support
the role of the hydroxyl groups at C2/C3, the influence of the
aglycone, and the bulky nature of the substituents in C28. OA
bearing a α-D-mannose moiety at C28 (compound 18)
focused our interest because the estimated inhibitory concentration 50 was similar to that reported for ginsenoside Rh2 against
colon cancer cells and it inhibits the G1âS phase transition affecting the cell viability and apoptosis. Considering that
triterpenoids from natural sources have been identified as inhibitors of nuclear factor kappa-light-chain-enhancer of activated B
cell (NF-ÎșB) signaling, docking studies were conducted to evaluate whether NF-ÎșB may be a potential target. Results are
consistent with the biological study and predict a similar binding mode of MA and compound 18 to the p52 subunit from NF-
ÎșB but not for OA. The fact that the binding site is shared by the NF-ÎșB inhibitor 6,6-dimethyl-2-(phenylimino)-6,7-
dihydrobenzo[d][1,3]oxathiol-4(5H)-one supports the result and points to NF-ÎșB as a potential target of both MA and
compound 18.This work was supported by a grant from RamoÌn Areces
Foundation (Madrid, Spain) and by grant CTQ2014-55474-
C2-1-R from the Spanish Ministerio de Economia y
Competitividad (MINECO) co-financed by FEDER funds
KRAS4A induces metastatic lung adenocarcinomas in vivo in the absence of the KRAS4B isoform.
In mammals, the KRAS locus encodes two protein isoforms, KRAS4A and KRAS4B, which differ only in their C terminus via alternative splicing of distinct fourth exons. Previous studies have shown that whereas KRAS expression is essential for mouse development, the KRAS4A isoform is expendable. Here, we have generated a mouse strain that carries a terminator codon in exon 4B that leads to the expression of an unstable KRAS4B154 truncated polypeptide, hence resulting in a bona fide Kras4B-null allele. In contrast, this terminator codon leaves expression of the KRAS4A isoform unaffected. Mice selectively lacking KRAS4B expression developed to term but died perinatally because of hypertrabeculation of the ventricular wall, a defect reminiscent of that observed in embryos lacking the Kras locus. Mouse embryonic fibroblasts (MEFs) obtained from Kras4B-/- embryos proliferated less than did wild-type MEFs, because of limited expression of KRAS4A, a defect that can be compensated for by ectopic expression of this isoform. Introduction of the same terminator codon into a Kras FSFG12V allele allowed expression of an endogenous KRAS4AG12V oncogenic isoform in the absence of KRAS4B. Exposure of Kras +/FSF4AG12V4B- mice to Adeno-FLPo particles induced lung tumors with complete penetrance, albeit with increased latencies as compared with control Kras +/FSFG12V animals. Moreover, a significant percentage of these mice developed proximal metastasis, a feature seldom observed in mice expressing both mutant isoforms. These results illustrate that expression of the KRAS4AG12V mutant isoform is sufficient to induce lung tumors, thus suggesting that selective targeting of the KRAS4BG12V oncoprotein may not have significant therapeutic consequences.We thank Marta San Roman, Raquel Villar, and Nuria Cabrera for excellent technical assistance; Mayte Lamparero and Isabel Blanco (Animal Facility) for mouse work; the Histopathology Unit for processing of mouse tissues; Lola Martinez (Flow Cytometry Unit) for her help with flow cytometry analyses; Diego Megias and Manuel Perez (Confocal Microscopy Unit) for assistance with confocal microscopy; and the Mouse Genome Editing Unit for support with the generation of the mouse strains described here. We also thank Ignacio Perez de Castro (Instituto de Salud Carlos III, Madrid, Spain) for sharing the EGFP-KRAS4B plasmid and Orlando Dominguez (Genomics Unit) and Pedro P. Lopez-Casas (Clinical Research Program) for their advice on exome sequencing. This work was supported by grants from the European Research Council (ERC-2015-AdG/695566, THERACAN), the Spanish Ministry of Science, Innovation and Universities (RTC-2017-6576-1), and the Autonomous Community of Madrid (B2017/BMD-3884 iLUNG-CM); a grant from the CRIS Cancer Foundation (to M.B.); and a grant from the Spanish Ministry of Science, Innovation and Universities (RTI2018-094664-B-I00, to M.B. and M.M.). M.B. is a recipient of an Endowed Chair from the AXA Research Fund. M.S. was supported by predoctoral contract "Severo Ochoa" (BES-2016-079096) from the SpanishMinistry of Science, Innovation and Universities. G.P. was a recipient of a "Young Ph.D." grant from the Government of the Community of Madrid. F.F.-G. was supported by a formacion de profesorado universitario (FPU) fellowship from the Spanish Ministry of Science, Innovation and Universities.S
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