Chiral Magneto-Electrochemistry

Magneto-electrochemistry (MEC) is a unique paradigm in science, where electrochemical experiments are carried out as a function of an applied magnetic field, creating a new horizon of potential scientific interest and technological applications. Over time, detailed understanding of this research domain was developed to identify and rationalize the possible effects exerted by a magnetic field on the various microscopic processes occurring in an electrochemical system. Notably, until a few years ago, the role of spin was not taken into account in the field of magneto-electrochemistry. Remarkably, recent experimental studies reveal that electron transmission through chiral molecules is spin selective and this effect has been referred to as the chiral-induced spin selectivity (CISS) effect. Spin-dependent electrochemistry originates from the implementation of the CISS effect in electrochemistry, where the magnetic field is used to obtain spin-polarized currents (using ferromagnetic electrodes) or, conversely, a magnetic field is obtained as the result of spin accumulation

Modulatable endosomalytic, intracellularly biodegradable vectors for gene delivery

In order to achieve efficient gene delivery, we have designed p$K_a$ modulatable oligopeptides (2COPs) by combining with lysine, histidine and cysteine residues which will bind DNA extracellularly, internalize via endocytosis, provide a tunable endosomal release mechanism, and provide a degradable backbone in order that the DNA can be released once in the cytoplasm. The reducible polycations (RPCs) were synthesized from 2COPs. The sizes, surface charges and the stability of RPC polyplexes under the simulated physiological conditions extra- and intracellularly suggested that these RPCs are promising vectors. In addition, the transfections revealed that the RPCs can facilitate endosomal buffering and intracellular reduction and are non-toxic to cells. The nuclear targeting signal (TAT) was incorporated into these vectors. The reducible copolycations (RcPCs) were synthesized via oxidative polymerisation between 2COPs and TAT. The RcPC polyplexes are ~100 nm, and are positively charged. Gel shift assay revealed that RcPCs have less potential than RPCs to be used as vectors as they are less stable extracellularly than the RPCs. In addition, chloroquine was required to enhance the transfection of RcPCs. Furthermore, there is no improvement in transfection of RcPC compared to RPCs. Therefore, this suggests that the incorporation of TAT does not improve the transfection

Enzymatic synthesis and characterization of oligopeptides, co-oligopeptides and hydroxy acid capped polypeptides in mixed aqueous organic media

Proteolytic enzymes have been used for the synthesis of homo-oligomers and hydroxy acid capped peptides with amino acids and hydroxy acid esters as substrates in aqueous and aqueous organic biphasic and triphasic systems. However, successful incorporation of hydroxy acids in oligopeptide substrates has not been demonstrated. Use of monophasic media comprising of acetonitrile/water in which oligopeptide substrates were soluble was examined for the synthesis of oligomers of neutral and polar amino acids like Met, Tyr, Lys, Gly, Asp and Glu and tailored co-oligomers of Lys-Met and Arg-Met. Effect of reaction parameters such as starting substrate concentration, system composition, reaction time, temperature, and time of addition of second substrate on the peptide synthesis process was evaluated. The HPLC and ESI-MS characterization revealed the formation of oligomers and co-oligomers in yields of~ 80%. Results also show that systematic change of reaction media composition and sequential addition of amino acid substrates would serve as the best approach for the synthesis of cooligopeptides of Lys-Met and Arg-Met in acetonitrile/water system. Use of anti-oxidants during oligomerization is essential. Traditionally mercaptoethanol has been used. However, because of their toxicity, such anti-oxidants pose a problem for utilization. To over come this, the effectiveness of L-Cysteine as an anti-oxidant for protease catalysis was also evaluated. Results show that L-Cysteine can be used as an effective replacement for mercaptoethanol in peptide synthesis. Finally, hydroxy acid capping of peptides was carried out in acetonitrile/water system. Results show that oligopeptides of Met, Phe and Insulin could be readily capped with a hydroxy acid in a monophasic system comprising of acetonitrile/water --Abstract, page iv

Programi i baze podataka o peptidima i proteolitičkim enzimima na internetu – kratak osvrt na 2007. i 2008. godinu

Bioinformatics methods have become one of the most important tools in peptide science. The number of available peptide databases is growing rapidly. The number of online programs able to process peptide sequences to extract information concerning their structure, physicochemical and biological properties is also increasing. Many of such programs were designed to manipulate protein sequences, but they have no built-in restrictions disabling their application to process oligopeptides containing less than 20 amino acid residues. Publications addressing these programs cannot be found in literature databases using the keyword \u27peptide\u27 or \u27peptides\u27, in connection with the term \u27bioinformatics\u27 or related terms, thus a reference source summarizing data from such publications seems necessary. This paper provides a brief review of bioactive peptide databases and sequence alignment programs enabling the search for peptide motifs, determination of physicochemical properties of amino acid residues, prediction of peptide structure, the occurrence of posttranslational glycosylation and immunogenicity, as well as the support of peptide design process. The review also includes databases and programs providing information about proteolytic enzymes. The databases and programs discussed in this paper were developed or updated between September 2007 and December 2008.Bioinformatičke metode postale su jedan od najvažnijih alata u području istraživanja peptida. Sve je veći broj dostupnih baza podataka o peptidima, a i „online” programa koji obradom aminokiselinskih sljedova daju informacije o strukturi peptida te njihovim fizikalno-kemijskim i biološkim svojstvima. Mnogi od tih programa dizajnirani su za obradu aminokiselinskih sljedova proteina, ali nemaju ugrađenu restrikciju njihove primjene na oligopeptide koji sadrže manje od 20 aminokiselinskih ostataka. Radovi o takvim programima ne mogu se pronaći u bazama publikacija uporabom ključnih riječi „peptid” ili „peptidi”, u kombinaciji s pojmom „bioinformatika” ili sličnim terminima. Stoga je važno sažeti rezultate objavljenih radova u jednom izvoru. U ovom je radu dan kratak pregled baza podataka o bioaktivnim peptidima i programima za analizu aminokiselinskoga slijeda koji omogućuju: pronalazak peptidnih motiva; određivanje fizikalno-kemijskih svojstava aminokiselinskih ostataka; predviđanje strukture peptida, pojave posttranslacijske glikozilacije i imunogenosti; te programsku podršku za dizajn peptida. Također su prikazani programi i baze podataka o proteolitičkim enzimima. Svi navedeni programi i baze razvijeni su i ažurirani od rujna 2007. do prosinca 2008

Development of nanobody-based surfaces for on-demand smart switchable biosensing

The biosensing field has been evolving with the advances of nanotechnology, medicine and with the need for diagnostics and health monitoring. Biosensors are facing high demands related to sensitivity, long term stability and on demand sensing. Control over detection provides the opportunity for on-demand biosensing and smart materials such as stimuli-responsive surfaces that can control a wide range of bio-interactions. However, there are few smart surfaces for on demand biosensing and these are limited to control over simple molecular structures. To address these challenges, this research used single domain antibodies, known as nanobodies (Nbs), with great potential as biorecognition elements for sensors due to their small size, specificity and robustness. Different strategies for Nbs immobilisation on gold sensors were optimised to retain their maximum functionality. The experimental data and detailed computational simulations confirmed the formation of stable, well-oriented nanobody monolayers. Furthermore, this work explored challenges related with Nbs’ orientation and antigen dimensions, emphasizing crucial factors to consider when designing nanobody-based biosensors. Envisioning new smart electrically-responsive surfaces, preliminary studies showed the stability and high response of Nbs under applied potentials. Additionally, electrically-responsive oligopeptides, potential switching units for nanobody-antigen binding control, were designed and investigated. These oligopeptides are required to conceal the Nbs binding site, only exposing it to bind the antigen upon applied potential. In conclusion, this work provides a step forward with the vision of combining stimulus-responsive surfaces with nanobodies for on-demand biosensing, contributing for the design and fabrication of stable, reliable, and robust biosensing platforms for a wide range of medical, biotechnological, environmental, and food applications

Self-consistent field theory for the interactions between keratin intermediate filaments

Background: Keratins are important structural proteins found in skin, hair and nails. Keratin Intermediate Filaments are major components of corneocytes, nonviable horny cells of the Stratum Corneum, the outermost layer of skin. It is considered that interactions between unstructured domains of Keratin Intermediate Filaments are the key factor in maintaining the elasticity of the skin. Results: We have developed a model for the interactions between keratin intermediate filaments based on self-consistent field theory. The intermediate filaments are represented by charged surfaces, and the disordered terminal domains of the keratins are represented by charged heteropolymers grafted to these surfaces. We estimate the system is close to a charge compensation point where the heteropolymer grafting density is matched to the surface charge density. Using a protein model with amino acid resolution for the terminal domains, we find that the terminal chains can mediate a weak attraction between the keratin surfaces. The origin of the attraction is a combination of bridging and electrostatics. The attraction disappears when the system moves away from the charge compensation point, or when excess small ions and/or NMF-representing free amino acids are added. Conclusions: These results are in concordance with experimental observations, and support the idea that the interaction between keratin filaments, and ultimately in part the elastic properties of the keratin-containing tissue, is controlled by a combination of the physico-chemical properties of the disordered terminal domains and the composition of the medium in the inter-filament region. Keywords: Stratum corneum, Skin keratins, Intermediate filaments, Unstructured terminal domains, Bridging attractio

Composition, thermodynamics, and morphology: A multi-scale computational approach for the design of self-assembling peptides

Peptide self-assembly has generated significant interest as a means for the bottom-up fabrication of highly tunable biocompatible nanoaggregates. Individual peptides can be synthesized to include non-natural π-conjugated subunits, endowing assembled aggregates with a range of optical and electronic properties that render them useful in applications as biocompatible organic electronics. The immense number of possible peptides, however, causes the exhaustive traversal of sequence space to be intractable. This massive composition space lends itself toward the use of computer simulation and data science tools to understand molecular aggregation and guide experimental synthesis and design. In this dissertation, I present work employing a hierarchy of molecular modeling techniques to identify self-assembling peptides with specific photophysical properties by probing thermodynamic and structural characteristics of peptide aggregation. We employ classical molecular dynamics simulation to probe the key molecular forces governing the morphology and free energy of oligomerization, time dependent density functional theory to predict photophysical properties as a function of aggregate morphology, and data-driven quantitative structure property models to perform high-throughput virtual screening of chemical space to identify promising peptide chemistries. This work establishes a multi-scale framework for the principled computational design of self-assembling π-conjugated peptides with engineered photophysical properties

Subpoblaciones quimiotípicas en cianobacterias planctónicas. Ecología y aplicación en herramientas avanzadas de monitorización

Tesis doctoral inédita, leída en Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 08-11-201

The Chlamydia effector TarP mimics the mammalian leucine-aspartic acid motif of paxillin to subvert the focal adhesion kinase during invasion

Host cell signal transduction pathways are often targets of bacterial pathogens, especially during the process of invasion when robust actin remodeling is required. We demonstrate that the host cell focal adhesion kinase (FAK) was necessary for the invasion by the obligate intracellular pathogen Chlamydia caviae. Bacterial adhesion triggered the transient recruitment of FAK to the plasma membrane to mediate a Cdc42- and Arp2/3-dependent actin assembly. FAK recruitment was via binding to a domain within the virulence factor TarP that mimicked the LD2 motif of the FAK binding partner paxillin. Importantly, bacterial two-hybrid and quantitative imaging assays revealed a similar level of interaction between paxillin-LD2 and TarP-LD. The conserved leucine residues within the L(D/E)XLLXXL motif were essential to the recruitment of FAK, Cdc42, p34Arc, and actin to the plasma membrane. In the absence of FAK, TarP-LD-mediated F-actin assembly was reduced, highlighting the functional relevance of this interaction. Together, the data indicate that a prokaryotic version of the paxillin LD2 domain targets the FAK signaling pathway, with TarP representing the first example of an LD-containing Type III virulence effector