Institut Químic de Sarrià

Abstract A matching measure between two molecular structures based on density functions is described. Some examples related with an alarm pheromone activity are given; the results encourage the possible use of similarity density concepts in molecular engineering.

Articular cartilage exhibits little capacity for intrinsic repair, and thus even minor injuries or lesions may lead to progressive damage and osteoarthritic joint degeneration, resulting in significant pain and disability. While there have been numerous attempts to develop tissue-engineered grafts or patches to repair focal chondral and osteochondral defects, there remain significant challenges in the clinical application of cell-based therapies for cartilage repair. This paper reviews the current state of cartilage tissue engineering with respect to different cell sources and their potential genetic modification, biomaterial scaffolds and growth factors, as well as preclinical testing in various animal models. This is not intended as a systematic review, rather an opinion of where the field is moving in light of current literature. While significant advances have been made in recent years, the complexity of this problem suggests that a multidisciplinary approach - combining a clinical perspective with expertise in cell biology, biomechanics, biomaterials science and high-throughput analysis will likely be necessary to address the challenge of developing functional cartilage replacements. With this approach we are more likely to realise the clinical goal of treating both focal defects and even large-scale osteoarthritic degenerative changes in the joint.

Chiral polypeptides as catalysts enable stereoselective reactions. An excellent example is the reaction of chalcone with H2O2/NaOH in the three-phase system toluene-water-poly[(S)-alanine] (optical yield of (1) over 90%).

Although bone responds to its mechanical environment, the cellular and molecular mechanisms underlying the response of the skeleton to mechanical unloading are not completely understood. Osteocytes are the most abundant but least understood cells in bones and are thought to be responsible for sensing stresses and strains in bone. Sclerostin, a product of the SOST gene, is produced postnatally primarily by osteocytes and is a negative regulator of bone formation. Recent studies show that SOST is mechanically regulated at both the mRNA and protein levels. During prolonged bed rest and immobilization, circulating sclerostin increases both in humans and in animal models, and its increase is associated with a decrease in parathyroid hormone. To investigate whether SOST/sclerostin up-regulation in mechanical unloading is a cell-autonomous response or a hormonal response to decreased parathyroid hormone levels, we subjected osteocytes to an in vitro unloading environment achieved by the NASA rotating wall vessel system. To perform these studies, we generated a novel osteocytic cell line (Ocy454) that produces high levels of SOST/sclerostin at early time points and in the absence of differentiation factors. Importantly, these osteocytes recapitulated the in vivo response to mechanical unloading with increased expression of SOST (3.4 ± 1.9-fold, p < 0.001), sclerostin (4.7 ± 0.1-fold, p < 0.001), and the receptor activator of nuclear factor κΒ ligand (RANKL)/osteoprotegerin (OPG) (2.5 ± 0.7-fold, p < 0.001) ratio. These data demonstrate for the first time a cell-autonomous increase in SOST/sclerostin and RANKL/OPG ratio in the setting of unloading. Thus, targeted osteocyte therapies could hold promise as novel osteoporosis and disuse-induced bone loss treatments by directly modulating the mechanosensing cells in bone.

Abstract A sulfonated derivative of polybenzimidazole is reported, and its properties are analyzed in comparison with related polybenzimidazole proton‐conducting materials. Poly(2,5‐benzimidazole), poly( m ‐phenylenebenzobisimidazole), and poly[ m ‐(5‐sulfo)‐phenylenebenzobisimidazole] were prepared by condensation of the corresponding monomers in polyphosphoric acid. Several adducts of these polymers with phosphoric acid were prepared. The resulting materials were characterized by chemical analysis, Fourier transform infrared spectroscopy, and thermogravimetric analysis; also, the dc conductivity of doped and undoped derivatives was measured. Similar to what has been observed for the commercial polybenzimidazole polymer (also examined here for comparison), the title polymers exhibit high thermal stability. Furthermore, their doping with phosphoric acid leads to a significant increase in conductivity from less than 10 −11 Scm −1 for the undoped polymers to 10 −4 Scm −1 (both at room temperature) for their acid‐loaded derivatives. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 3703–3710, 2002

Novel boron(III) subphthalocyanines (SubPcs) soluble in organic solvents containing a variety of donor and acceptor substituent groups have been synthesized by boron trihalide-induced cyclotrimerization of adequately substituted derivatives of phthalonitrile in 1-chloronaphthalene. The choice of the substituents on the 1,2-dicyanobenzene derivatives has been made taking into account the high reactivity of the Lewis acid BCl 3 toward many functional groups. Considering this limitation, we set out to synthesize phthalodinitriles equipped with iodo, nitro, alkyl- or arylthio, alkyl- or arylsulfonyl groups that are sufficiently stable under the required reaction conditions and also provide an easily accessible set of acceptor/donor substituents. The quadratic and cubic hyperpolarizabilities of these compounds as well as their linear optical and electrochemical properties have been measured by several techniques, including EFISH (at two wavelengths), HRS, and THG, steady-state and time-resolved absorption and fluorescence, laser-induced optoacoustic calorimetry, time-resolved near-infrared emission spectroscopy, and cyclic voltammetry. β HRS has been measured at 1.46 μm, where the contamination from the multiphoton-induced fluorescence can be ruled out. β HRS reachs high values that markedly depend on substitution. It shows a clear enhancement with the acceptor character of the substituents, the highest values being obtained for the compounds bearing the strongest acceptor groups. They are comparable or even superior to many efficient second-order compounds. A main outcome of these results is that an adequate choice of the substituents offers a promising route for optimization of the quadratic response of the SubPcs. This kind of compounds is less prone to aggregation than their expanded analogues, the phthalocyanines, fluoresces with quantum yields ca. 0.25, lower than those typical for phthalocyanines, and has larger triplet quantum yields. The triplet-state lifetime is in the 100-μs time range, long enough for efficient oxygen quenching. Indeed, subphthalocyanines sensitize singlet molecular oxygen, O 2 ( 1 Δ g ), with quantum yields ranging from 0.23 to 0.75. The ground-state oxidation potentials are similar to those of phthalocyanines, while the reduction potentials are clearly more negative; i.e., they are more difficult to reduce. In contrast, electronically excited subphthalocyanines are more easily oxidized than the corresponding phthalocyanines by ca. 500 mV which results in lower photostability, especially in polar solvents.

The enzymatic hydrolysis of the glycosidic bond is catalyzed by diverse enzymes generically termed glycoside hydrolases (hereafter GHs) or glycosidases. The many sequence-based families of glycosidases have served as a rich hunting ground for enzymologists for years. Not only are these enzymes of fundamental interest, providing paradigms for enzymatic catalysis that extend beyond the bounds of carbohydrate chemistry, but the enzymes themselves play myriad essential roles in diverse biological processes. The wide utility of glycosidases, from their industrial harnessing in the hydrolysis of plant biomass to their roles in human physiology and disease, has engendered a large scientific constituency with an interest in glycosidase chemistry. A fascinating thread of this research, and one with major impact on the design of enzyme inhibitors, is the conformational analysis of reaction pathways within the diverse families. These GH families provide a large pallet of enzymes with which chemists have attempted to depict the conformational landscape of glycosidase action. In this Account, we review three-dimensional insight into the conformational changes directed by glycosidases, primarily from structural observations of the stable enzyme-ligand species adjacent to the transition state (or states) and of enzyme-inhibitor complexes. We further show how recent computational advances dovetail with structural insight to provide a quantum mechanical basis for glycosidase action. The glycosidase-mediated hydrolysis of the acetal or ketal bond in a glycoside may occur with either inversion or retention of the configuration of the anomeric carbon. Inversion involves a single step and transition state, whereas retention, often referred to as the double displacement, is a two-step process with two transition states. The single transition state for the inverting enzymes and the two transition states (those flanking the covalent intermediate) in the double displacement have been shown to have substantial oxocarbenium ion character. The dissociative nature of these transition states results in significant relative positive charge accumulation on the pyranose ring. The delocalization of lone-pair electrons from the ring oxygen that stabilizes the cationic transition state implies that at, or close to, the transition states the pyranose will be distorted away from its lowest energy conformation to one that favors orbital overlap. Over the preceding decade, research has highlighted the harnessing of noncovalent interactions to aid this distortion of the sugar substrates from their lowest energy chair conformation to a variety of different boat, skew boat, and half-chair forms, each of which favors catalysis with a given enzyme and substrate. Crystallographic observation of stable species that flank the transition state (or states), of both retaining and inverting glycosidases, has allowed a description of their conformational itineraries, illustrating how enzymes facilitate the "electrophilic migration" of the anomeric center along the reaction coordinate. The blossoming of computational approaches, such as ab initio metadynamics, has underscored the quantum mechanical basis for glycoside hydrolysis. Conformational analyses highlight not only the itineraries used by enzymes, enabling their inhibition, but are also reflected in the nonenzymatic synthesis of glycosides, wherein chemists mimic strategies found in nature.

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.

The fluorescent probe Singlet Oxygen Sensor Green is able to produce singlet oxygen under exposure to UV or visible radiation.

Preparation and characterization of membranes of poly(2,5-benzimidazole) (ABPBI) (as thin as 20 Mm) for polymer electrolyte&#13;\nmembrane fuel cells are reported. These membranes were prepared by solution casting and then impregnated in phosphoric acid&#13;\nbaths. Their characterization included thermogravimetric analyses, conductivity measurements, Fourier transform infrared spectroscopy,&#13;\nX-ray diffraction, and scanning electron microscopy. These membranes have high thermal stability and good proton&#13;\nconductivity at temperatures up to 200°C (6.2 3 1022 S cm21 at 150°C and 30% relative humidity!, similar to the well-known&#13;\ncommercial polybenzimidazole (PBI). ABPBI membrane-electrode assemblies (MEAs) using commercially available E-TEK Pt/C&#13;\nelectrodes were tested in H2 /O2 cells, obtaining power densities similar to those of PBI MEAs studied here as reference at&#13;\ntemperatures of up to 180°C, humidifying the reactants at room temperature

Background: The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC) is limited by adaptive activation of cell survival signals. We hypothesized that both signal transducer and activator of transcription 3 (STAT3) and Src-YES-associated protein 1 (YAP1) signaling are dually activated during EGFR TKI treatment to limit therapeutic response. Methods: We used MTT and clonogenic assays, immunoblotting, and quantitative polymerase chain reaction to evaluate the efficacy of EGFR TKI alone and in combination with STAT3 and Src inhibition in three EGFR-mutant NSCLC cell lines. The Chou-Talalay method was used for the quantitative determination of drug interaction. We examined tumor growth inhibition in one EGFR-mutant NSCLC xenograft model (n = 4 mice per group). STAT3 and YAP1 expression was evaluated in tumors from 119 EGFR-mutant NSCLC patients (64 in an initial cohort and 55 in a validation cohort) by quantitative polymerase chain reaction. Kaplan-Meier and Cox regression analyses were used to assess the correlation between survival and gene expression. All statistical tests were two-sided. Results: We discovered that lung cancer cells survive initial EGFR inhibitor treatment through activation of not only STAT3 but also Src-YAP1 signaling. Cotargeting EGFR, STAT3, and Src was synergistic in two EGFR-mutant NSCLC cell lines with a combination index of 0.59 (95% confidence interval [CI] = 0.54 to 0.63) for the PC-9 and 0.59 (95% CI = 0.54 to 0.63) for the H1975 cell line. High expression of STAT3 or YAP1 predicted worse progression-free survival (hazard ratio [HR] = 3.02, 95% CI = 1.54 to 5.93, P = .001, and HR = 2.57, 95% CI = 1.30 to 5.09, P = .007, respectively) in an initial cohort of 64 EGFR-mutant NSCLC patients treated with firstline EGFR TKIs. Similar results were observed in a validation cohort. Conclusions: Our study uncovers a coordinated signaling network centered on both STAT3 and Src-YAP signaling that limits targeted therapy response in lung cancer and identifies an unforeseen rational upfront polytherapy strategy to minimize residual disease and enhance clinical outcomes.

Removal of the catalytic nucleophile Glu134 of the retaining 1,3-1,4-beta-glucanase from Bacillus licheniformis by mutation to alanine yields an enzyme with no glycosidase activity. The mutant is able to catalyze the regio- and stereospecific glycosylation of alpha-laminaribiosyl fluoride with different glucoside acceptors through a single-step inverting mechanism. The main advantage of the mutant as glycosylation catalyst with respect to the kinetically controlled transglycosylation using the wild-type enzyme is that the reaction products cannot be hydrolyzed by the mutant enzyme, and glycosylation yields rise to 90%.

For a new molecular entity (NME) to become a drug, it is not only essential to have the right biological activity also be safe and efficient, but it is also required to have a favorable pharmacokinetic profile including toxicity (ADMET). Consequently, there is a need to predict, during the early stages of development, the ADMET properties to increase the success rate of compounds reaching the lead optimization process. Since Lipinski’s rule of five, the prediction of pharmacokinetic parameters has evolved towards the current in silico tools based on empirical approaches or molecular modeling. The commercial specialized software for performing such predictions, which is usually costly, is, in many cases, not among the possibilities for research laboratories in academia or at small biotech companies. Nevertheless, in recent years, many free online tools have become available, allowing, more or less accurately, for the prediction of the most relevant pharmacokinetic parameters. This paper studies 18 free web servers capable of predicting ADMET properties and analyzed their advantages and disadvantages, their model-based calculations, and their degree of accuracy by considering the experimental data reported for a set of 24 FDA-approved tyrosine kinase inhibitors (TKIs) as a model of a research project.

Porphycenes are structural isomers of porphyrins that have many unique properties and features. In the present work, the resonant two-photon absorption of 2,7,12,17-tetraphenylporphycene (TPPo) and its palladium(II) complex (PdTPPo) has been investigated. The data obtained are compared to those from the isomeric compound, meso-tetraphenylporphyrin (TPP). Detection of phosphorescence from singlet molecular oxygen, O2(a(1)Delta(g)), produced upon irradiation of these compounds, was used to obtain two-photon excitation spectra and to quantify two-photon absorption cross sections, delta. In the spectral region of 750-850 nm, the two-photon absorption cross sections at the band maxima for both TPPo and PdTPPo, delta = 2280 and 1750 GM, respectively, are significantly larger than that for TPP. This difference is attributed to the phenomenon of so-called resonance enhancement; for the porphycenes, the two-photon transition is nearly resonant with a comparatively intense one-photon Q-band transition. The results of quantum mechanical calculations using density functional quadratic response theory are in excellent agreement with the experimental data and, as such, demonstrate that comparatively high-level quantum chemical methods can be used to interpret and predict nonlinear optical properties from such large molecular systems. One important point realized through these experiments and calculations is that one must exercise caution when using qualitative molecular-symmetry-derived arguments to predict the expected spectral relationship between allowed one- and two-photon transitions. From a practical perspective, this study establishes that, in comparison to porphyrins and other tetrapyrrolic macrocyclic systems, porphycenes exhibit many desirable attributes for use as sensitizers in two-photon initiated photodynamic therapy.

Abstract Although cyclometalated Ir III complexes have emerged as promising photosensitizers for photodynamic therapy, some key drawbacks still hamper clinical translation, such as operability in the phototherapeutic window and reactive oxygen species (ROS) production efficiency and selectivity. In this work, a cyclometalated Ir III complex conjugated to a far‐red‐emitting coumarin, Ir III –COUPY, is reported with highly favourable properties for cancer phototherapy. Ir III –COUPY was efficiently taken up by HeLa cells and showed no dark cytotoxicity and impressive photocytotoxicity indexes after irradiation with green and blue light, even under hypoxia. Importantly, a clear correlation between cell death and intracellular generation of superoxide anion radicals after visible light irradiation was demonstrated. This strategy opens the door to novel fluorescent photodynamic therapy agents with promising applications in theragnosis.

Abstract Thirteen N,N ‐diazolylmethanes, including derivatives of pyrazole, imidazole, 1,2,4‐triazole, benzimidazole and indazole were prepared by reaction of azoles with methylene chloride under phase transfer catalysis conditions. The relative amounts of isomeric mixtures obtained with ‘asymmetric’ azoles or with equimolar mixtures of azoles are compared with literature results on monoalkylation of azoles. Proton and carbon‐13 nmr spectra of the N,N' ‐diazolylmethanes are discussed.

The recent Zika virus (ZIKV) outbreak demonstrates that cost-effective clinical diagnostics are urgently needed to detect and distinguish viral infections to improve patient care. Unlike dengue virus (DENV), ZIKV infections during pregnancy correlate with severe birth defects, including microcephaly and neurological disorders. Because ZIKV and DENV are related flaviviruses, their homologous proteins and nucleic acids can cause cross-reactions and false-positive results in molecular, antigenic, and serologic diagnostics. We report the characterization of monoclonal antibody pairs that have been translated into rapid immunochromatography tests to specifically detect the viral nonstructural 1 (NS1) protein antigen and distinguish the four DENV serotypes (DENV1-4) and ZIKV without cross-reaction. To complement visual test analysis and remove user subjectivity in reading test results, we used image processing and data analysis for data capture and test result quantification. Using a 30-μl serum sample, the sensitivity and specificity values of the DENV1-4 tests and the pan-DENV test, which detects all four dengue serotypes, ranged from 0.76 to 1.00. Sensitivity/specificity for the ZIKV rapid test was 0.81/0.86, respectively, using a 150-μl serum input. Serum ZIKV NS1 protein concentrations were about 10-fold lower than corresponding DENV NS1 concentrations in infected patients; moreover, ZIKV NS1 protein was not detected in polymerase chain reaction-positive patient urine samples. Our rapid immunochromatography approach and reagents have immediate application in differential clinical diagnosis of acute ZIKV and DENV cases, and the platform can be applied toward developing rapid antigen diagnostics for emerging viruses.

We report fabrication, characterization, and use of microfluidic analysis devices containing surface-immobilized cell-capturing molecules. Amino-terminated biotin ligands are immobilized onto the luminal surface of a microdevice and effectively support self-assembly of proteins, antibodies, and mammalian cells. For this purpose, chemical vapor deposition (CVD) polymerization is used to functionalize PDMS-made microfluidic devices with poly[para-xylylene carboxylic acid pentafluorophenolester-co-para-xylylene]. The resulting reactive coating shows excellent adhesion when deposited in thin films (approximately 100 nm, and the distribution of the pentafluorophenol ester groups is reasonably uniform within the microchannel inner surface, as examined by fluorescence microscopy. The utility of these devices for cell-based bioassays is demonstrated by monitoring the concentration-dependent effect of the disintegrin echistatin on cell adhesion. The described assay format could be relevant to clinical research in various fields, including angiogenesis research.

Zika and dengue are mosquito-borne diseases that present similar nonspecific symptoms but possess dramatically different outcomes. The first line of defense in epidemic outbreaks are rapid point-of-care diagnostics. Because many outbreaks occur in areas that are resource poor, assays that are easy to use, inexpensive, and require no power have become invaluable in patient treatment, quarantining, and surveillance. Paper-based sandwich immunoassays such as lateral flow assays (LFAs) are attractive as point-of-care solutions as they have the potential for wider deployability than lab-based assays such as PCR. However, their low sensitivity imposes limitations on their ability to detect low biomarker levels and early diagnosis. Here, we exploit the high sensitivity of surface-enhanced Raman spectroscopy (SERS) in a multiplexed assay that can distinguish between Zika and dengue nonstructural protein 1 (NS1) biomarkers. SERS-encoded gold nanostars were conjugated to specific antibodies for both diseases and used in a dipstick immunoassay, which exhibited 15-fold and 7-fold lower detection limits for Zika NS1 and dengue NS1, respectively. This platform combines the simplicity of a LFA with the high sensitivity of SERS and could not only improve Zika diagnosis but also detect diseases sooner after infection when biomarker levels are low.

Microrobots offer transformative solutions for non-invasive medical interventions due to their small size and untethered operation inside the human body. However, they must face the immune system as a natural protection mechanism against foreign threats. Here, non-immunogenic stealth zwitterionic microrobots that avoid recognition from immune cells are introduced. Fully zwitterionic photoresists are developed for two-photon polymerization 3D microprinting of hydrogel microrobots with ample functionalization: tunable mechanical properties, anti-biofouling and non-immunogenic properties, functionalization for magnetic actuation, encapsulation of biomolecules, and surface functionalization for drug delivery. Stealth microrobots avoid detection by macrophage cells of the innate immune system after exhaustive inspection (>90 hours), which has not been achieved in any microrobotic platform to date. These versatile zwitterionic materials eliminate a major roadblock in the development of biocompatible microrobots, and will serve as a toolbox of non-immunogenic materials for medical microrobot and other device technologies for bioengineering and biomedical applications.