Antibacterial Hydrogels Derived from Poly(γ-glutamic acid) Nanofibers.

Biocompatible hydrogels with antibacterial properties derived from γ-polyglutamic acid (γ-PGA) were prepared from bulk and electrospun nanofibers. The antibacterial drugs loaded in these hydrogels were triclosan (TCS), chlorhexidine (CHX) and polyhexamethylene biguanide (PHMB); furthermore, bacteriophages were loaded as an alternative antibacterial agent. Continuous and regular γ-PGA nanofibers were successfully obtained by the electrospinning of trifluoroacetic acid solutions in a narrow polymer concentration range and restricted parameter values of flow rate, voltage and needle-collector distance. Hydrogels were successfully obtained by using cystamine as a crosslinking agent following previous published procedures. A closed pore structure was characteristic of bulk hydrogels, whereas an open but structurally consistent structure was found in the electrospun hydrogels. In this case, the morphology of the electrospun nanofibers was drastically modified after the crosslinking reaction, increasing their diameter and surface roughness according to the amount of the added crosslinker. The release of TCS, CHX, PHMB and bacteriophages was evaluated for the different samples, being results dependent on the hydrophobicity of the selected medium and the percentage of the added cystamine. A high efficiency of hydrogels to load bacteriophages and preserve their bactericide activity was demonstrated too.

Conducting polymer nanoparticles for a voltage-controlled release of pharmacological chaperones.

Pharmacological chaperones (PCs) are low-molecular weight chemical molecules used in patients for the treatment of some rare diseases caused primarily by protein instability. A controlled and on-demand release of PCs via nanoparticles is an alternative for cases in which long treatments are needed and prolonged oral administration could have adverse effects. In this work, pyrimethamine (PYR), which is a potent PC consisting of pyrimidine-2,4-diamine substituted at position 5 by a p-chlorophenyl group and at position 6 by an ethyl group, was successfully loaded in electroresponsive poly(3,4-ethylenedioxythiophene) nanoparticles (PEDOT NPs). The PYR-loading capacity was 11.4 ± 1.5%, with both loaded and unloaded PEDOT NPs exhibiting similar sizes (215 ± 3 and 203 ± 1 nm, respectively) and net surface charges (-26 ± 7 and -29 ± 6 mV, respectively). In the absence of electrical stimulus, the release of PC from loaded NPs is very low (1.6% in 24 h and 18% in 80 days) in aqueous environments. Instead, electrical stimuli that sustained for 30 min enhanced the release of PYR, which was ∼50% when the voltage was scanned from -0.5 V to 0.5 V (cyclic voltammetry) and ∼35% when a constant voltage of 1.0 V was applied (chronoamperometry).

Nanotheranostic Interface Based on Antibiotic-Loaded Conducting Polymer Nanoparticles for Real-Time Monitoring of Bacterial Growth Inhibition.

Conducting polymers have been increasingly used as biologically interfacing electrodes for biomedical applications due to their excellent and fast electrochemical response, reversible doping-dedoping characteristics, high stability, easy processability, and biocompatibility. These advantageous properties can be used for the rapid detection and eradication of infections associated to bacterial growth since these are a tremendous burden for individual patients as well as the global healthcare system. Herein, a smart nanotheranostic electroresponsive platform, which consists of chloramphenicol (CAM)-loaded in poly(3,4-ethylendioxythiophene) nanoparticles (PEDOT/CAM NPs) for concurrent release of the antibiotic and real-time monitoring of bacterial growth is presented. PEDOT/CAM NPs, with an antibiotic loading content of 11.9 ± 1.3% w/w, are proved to inhibit the growth of Escherichia coli and Streptococcus sanguinis due to the antibiotic release by cyclic voltammetry. Furthermore, in situ monitoring of bacterial activity is achieved through the electrochemical detection of ß-nicotinamide adenine dinucleotide, a redox active specie produced by the microbial metabolism that diffuse to the extracellular medium. According to these results, the proposed nanotheranostic platform has great potential for real-time monitoring of the response of bacteria to the released antibiotic, contributing to the evolution of the personalized medicine.

The influence of Ni2+ and other ions on the trigonal structure of DNA.

We present a new structure of a DNA dodecamer obtained in the presence of Ni2+ ions. The DNA forms Ni-guanine cross-links between neighboring molecules. Our results show that an adequate dosage of Ni2+ may help to form well-defined DNA nanostructures. We also compare our structure with other dodecamers which present unique features and also crystallize in trigonal unit cells, strongly influenced by the counterions associated with DNA. In all cases, the DNA duplexes form parallel pseudo-helical columns in the crystal, similar to DNA-protamine and native DNA fibers.

Scaffolds for Sustained Release of Ambroxol Hydrochloride, a Pharmacological Chaperone That Increases the Activity of Misfolded ß-Glucocerebrosidase.

Ambroxol is a pharmacological chaperone (PC) for Gaucher disease that increases lysosomal activity of misfolded ß-glucocerebrosidase (GCase) while displaying a safe toxicological profile. In this work, different poly(ε-caprolactone) (PCL)-based systems are developed to regulate the sustained release of small polar drugs in physiological environments. For this purpose, ambroxol is selected as test case since the encapsulation and release of PCs using polymeric scaffolds have not been explored yet. More specifically, ambroxol is successfully loaded in electrospun PCL microfibers, which are subsequently coated with additional PCL layers using dip-coating or spin-coating. The time needed to achieve 80% release of loaded ambroxol increases from ≈15 min for uncoated fibrous scaffolds to 3 days and 1 week for dip-coated and spin-coated systems, respectively. Furthermore, it is proven that the released drug maintains its bioactivity, protecting GCase against induced thermal denaturation.

Functional and structural analysis of AT-specific minor groove binders that disrupt DNA-protein interactions and cause disintegration of the Trypanosoma brucei kinetoplast.

Trypanosoma brucei, the causative agent of sleeping sickness (Human African Trypanosomiasis, HAT), contains a kinetoplast with the mitochondrial DNA (kDNA), comprising of >70% AT base pairs. This has prompted studies of drugs interacting with AT-rich DNA, such as the N-phenylbenzamide bis(2-aminoimidazoline) derivatives 1 [4-((4,5-dihydro-1H-imidazol-2-yl)amino)-N-(4-((4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)benzamide dihydrochloride] and 2 [N-(3-chloro-4-((4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)-4-((4,5-dihydro-1H-imidazol-2-yl)amino)benzamide] as potential drugs for HAT. Both compounds show in vitro effects against T. brucei and in vivo curative activity in a mouse model of HAT. The main objective was to identify their cellular target inside the parasite. We were able to demonstrate that the compounds have a clear effect on the S-phase of T. brucei cell cycle by inflicting specific damage on the kinetoplast. Surface plasmon resonance (SPR)-biosensor experiments show that the drug can displace HMG box-containing proteins essential for kDNA function from their kDNA binding sites. The crystal structure of the complex of the oligonucleotide d[AAATTT]2 with compound 1 solved at 1.25 Å (PDB-ID: 5LIT) shows that the drug covers the minor groove of DNA, displaces bound water and interacts with neighbouring DNA molecules as a cross-linking agent. We conclude that 1 and 2 are powerful trypanocides that act directly on the kinetoplast, a structure unique to the order Kinetoplastida.

Sperm nuclear basic proteins of tunicates and the origin of protamines.

Sperm nuclear basic proteins (SNBPs) are the chromosomal proteins that are found associated with DNA in sperm nuclei at the end of spermiogenesis. These highly specialized proteins can be classified into three major types: histone type (H-type), protamine-like type (PL-type), and protamine type (P-type). A hypothesis from early studies on the characterization of SNBPs proposed a mechanism for the vertical evolution of these proteins that involved an H1 → PL → P transition. However, the processes and mechanisms involved in such a transition were not understood. In particular, it was not clear how a molecular transition from a lysine-rich protein precursor (H1 histone) to the arginine-rich protamines might have taken place. In deuterostomes, the presence of SNBPs of the H-type in echinoderms and of protamines in the higher phylogenetic groups of vertebrates had long been known. The initial work on the characterization of tunicate SNBPs attempted to define the types and range of SNBPs that characterize this phylogenetically intermediate group. It was found that tunicate SNBPs belong to the PL-type. In this work we discuss how the study of SNBPs in the tunicates has been key to providing support to the H1 → PL → P transition. Most significantly, it was in tunicates that a potential molecular mechanism to explain the lysine-to-arginine transition was first reported.

Crystal structure of a complex of DNA with one AT-hook of HMGA1.

We present here for the first time the crystal structure of an AT-hook domain. We show the structure of an AT-hook of the ubiquitous nuclear protein HMGA1, combined with the oligonucleotide d(CGAATTAATTCG)(2), which has two potential AATT interacting groups. Interaction with only one of them is found. The structure presents analogies and significant differences with previous NMR studies: the AT-hook forms hydrogen bonds between main-chain NH groups and thymines in the minor groove, DNA is bent and the minor groove is widened.

Spermiogenic nuclear protein transitions and chromatin condensation. Proposal for an ancestral model of nuclear spermiogenesis.

We have chosen three species (Sparus aurata, Dicentrarchus labrax, and Monodonta turbinata) that represent different transition patterns in the composition and structure of spermiogenic nuclei. The transition patterns of these species are representative of spermiogenesis in a large number of animal species. We analyze: (a) nuclear protein exchange; (b) chromatin condensation pattern; and (c) histone acetylation during spermiogenic development. In the simplest spermiogenesis histones and nucleosomes remain in mature sperm. Chromatin of spermatids is organized into 20 nm granules, simultaneous with a nuclear volume reduction. The granules coalesce in the final stage of spermiogenesis. Granular chromatin is correlated with acetylation of histones H3 and H4, whereas final coalescence is associated with histone deacetylation. We also studied two other spermiogenesis where a basic protein substitutes histones. Each species has a very different substituting protein. One has a typical protamine of 34 amino acids; the other has a sperm nuclear basic proteins (SNBP) of 106 amino acids. In both, the structural transitions and histone acetylation pattern are similar: in early spermiogenesis chromatin is organized into 20 nm granules, and histones are significantly acetylated, while the nuclear volume decreases. Subsequently, acetylated histones are displaced by the protamine or SNBP. Histone substitution causes chromatin remodelling and additional reduction in nuclear volume. We analyze these three cases together with earlier works and propose that the formation of 20 nm granules containing acetylated H3 and H4 accomplishes the minimum functional requirement to be considered the most evolutionarily ancestral chromatin conformation preceding condensation in animal spermiogenesis.

The sperm proteins from amphioxus mirror its basal position among chordates and redefine the origin of vertebrate protamines.

The sperm nuclear basic proteins (SNBPs) that participate in chromatin condensation in spermatozoa belong to 3 groups: histone (H), protamine-like (PL), and protamine (P) type. They share a common origin with histone H1 resulting from the segregation of PL components, corresponding to different regions of an H1 precursor molecule (N-terminal, winged-helix, C-terminal domains), becoming independent and following a subsequent process of parallel vertical evolution (H <--> PL <--> P). In the present work, we describe the sequence and primary structure of the main SNBP component in the sperm of the cephalochordate Branchiostoma floridae (amphioxus), revealing that it represents the deuterostome counterpart of the PL-III SNBP component from molluscs corresponding to the H1 N-terminal region. Until now, this has been a missing piece needed to complete the evolutionary history of SNBPs in metazoan genomes. The discovery of this PL lineage in deuterostomes definitively validates the parallel vertical evolution of SNBPs across metazoans, giving further support to the "basal" position of amphioxus among chordates, with respect to tunicates. Sequence analyses suggest that later on in evolution, the appearance of positively selected arginine-rich protamines, derived from the H1 C-terminal region, led to the extinction of this PL lineage in the genomes of early protostomes and deuterostomes. Given that tunicates are now viewed as a sister group of vertebrates, the lysine to arginine transition responsible for the origin of vertebrate protamines must be set a step back from tunicates.

A unique vertebrate histone H1-related protamine-like protein results in an unusual sperm chromatin organization.

Protamine-like proteins constitute a group of sperm nuclear basic proteins that have been shown to be related to somatic linker histones (histone H1 family). Like protamines, they usually replace the chromatin somatic histone complement during spermiogenesis; hence their name. Several of these proteins have been characterized to date in invertebrate organisms, but information about their occurrence and characterization in vertebrates is still lacking. In this sense, the genus Mullus is unique, as it is the only known vertebrate that has its sperm chromatin organized by virtually only protamine-like proteins. We show that the sperm chromatin of this organism is organized by two type I protamine-like proteins (PL-I), and we characterize the major protamine-like component of the fish Mullus surmuletus (striped red mullet). The native chromatin structure resulting from the association of these proteins with DNA was studied by micrococcal nuclease digestion as well as electron microscopy and X-ray diffraction. It is shown that the PL-I proteins organize chromatin in parallel DNA bundles of different thickness in a quite distinct arrangement that is reminiscent of the chromatin organization of those organisms that contain protamines (but not histones) in their sperm.

Analysis of the stability and function of nucleoplasmin through cysteine mutants.

Xenopus laevis nucleoplasmin is a pentameric nuclear chaperone. The relation between the structure and the multifunctional aspects of the molecule has not yet been clearly established. In the course of analysing a C-terminally His-tagged recombinant version of the region equivalent to the trypsin resistant core (r-NP142) of the molecule, we found that this domain exhibited a substantially decreased oligomerization potential. To better understand the role of the three cysteines of nucleoplasmin on its pentameric functional structure, we have selectively mutated these residues to serine and generated three mutants (C15S, C35S, and C45S) both for the complete recombinant nucleoplasmin (r-NP) and the truncated r-NP142 non-tagged forms. We demonstrate that there are no disulphide bridges stabilizing either the monomer or the pentamer. Neither C15S nor C35S has any structural effects, while the mutation C45S abolishes the ability of r-NP142 to pentamerize. This structural impairment suggests that hydrophobic interactions of Cys 45 are critical for the stability of the protein. Our studies allow to analyse for the first time the structural and functional properties of nucleoplasmin in its monomeric form.

Mutation of the small acidic tract A1 drastically reduces nucleoplasmin activity.

Xenopus laevis nucleoplasmin is a molecular chaperone that mediates sperm decondensation and nucleosome assembly. Nucleoplasmin has three acidic tracts (A1, A2 and A3) and until recent years the long polyglutamic tract A2 was thought to be the binding site for basic proteins. However, the latest publications in this field show that neither A2 nor A3 is indispensable for histone and sperm-specific protein binding. In this work, we show that the mutation of only four acidic amino acid residues of the small A1 tract drastically reduces nucleoplasmin decondensing activity, pointing out this region as the potential binding site for sperm proteins.

Chromatin organization during spermiogenesis in Octopus vulgaris. I: Morphological structures.

In the process of the chromatin remodeling that occurs during spermiogenesis in some animal species, it is possible to distinguish between two separate aspects: the chromatin condensation pattern itself (granular, fibrillar, or lamellar), and the architecture of this pattern, that is to say, its arrangement within the nucleus. In the cephalopod Octopus vulgaris these two aspects are clearly differentiated. The condensation pattern develops from 25 nm fibers to fibers with a tubular aspect and with a progressively increasing diameter (40-60 nm and then to 80 nm), to end finally in the form of very thin fibers (3-5 nm) product of the coalescence and dissolution of the major fibers. The main directive force that governs this process lies in the global change that occurs in the proteins that interact with all (or the major part) of the genomic DNA. The condensation pattern by itself in this species does not present a fixed order: most of the fibers appear without any predominant spatial direction in the spermiogenic nuclei. However, as the nuclei elongate, the chromatin fibers arrange in parallel following the elongation axis. This parallel disposition of the chromatin fibers appears to be mediated by two specific areas, each of which we call a "polar nuclear matrix" (PNM). These matrices differentiate in the basal and apical nuclear poles adjacent to the centriolar implantation fosse and the acrosome, respectively. The areas that constitute the PNM have the following characteristics: (a) they are the only areas where DNA is found anchored to the nuclear membrane; (b) they are the zones from which the chromatin condensation pattern (fibers/tubules) begins; and (c) they are most probably the points through which the mechanical forces originating from nuclear elongation are transmitted to chromatin, causing the chromatin fibers/tubules to adopt an almost perfectly parallel disposition. Finally, we discuss the importance of the architecture of the chromatin condensation pattern, as it is one of the determining factors of the spatial organization of the mature sperm genome and chromosome positioning.

Histone H1 and the origin of protamines.

We present evidence that chordate protamines have evolved from histone H1. During the final stages of spermatogenesis, the compaction of DNA in many organisms is accomplished by the replacement of histones with a class of arginine-rich proteins called protamines. In other organisms, however, condensation of sperm DNA can occur with comparable efficiency in the presence of somatic-type histones or, alternatively, an intermediate class of proteins called protamine-like proteins. The idea that the highly specialized sperm chromosomal proteins (protamines) and somatic chromosomal proteins (histones) could be related dates back almost to the discovery of these proteins. Although this notion has frequently been revisited since that time, there has been a complete lack of supporting experimental evidence. Here we show that the emergence of protamines in chordates occurred very quickly, as a result of the conversion of a lysine-rich histone H1 to an arginine-rich protamine. We have characterized the sperm nuclear basic proteins of the tunicate Styela montereyensis, which we show consists of both a protamine and a sperm-specific histone H1 with a protamine tail. Comparison of the genes encoding these proteins to that of a sister protochordate, Ciona intestinalis, has indicated this rapid and dramatic change is most likely the result of frameshift mutations in the tail of the sperm-specific histone H1. By establishing an evolutionary link between the chromatin-condensing histone H1s of somatic tissues and the chromatin-condensing proteins of the sperm, these results provide unequivocal support to the notion that vertebrate protamines evolved from histones.

Interaction of nucleoplasmin with core histones.

Nucleoplasmin is one of the most abundant proteins in Xenopus laevis oocytes, and it has been involved in the chromatin remodeling that takes place immediately after fertilization. This molecule has been shown to be responsible for the removal of the sperm-specific proteins and deposition of somatic histones onto the male pronuclear chromatin. To better understand the latter process, we have used sedimentation velocity, sedimentation equilibrium, and sucrose gradient fractionation analysis to show that the pentameric form of nucleoplasmin binds to a histone octamer equivalent consisting of equal amounts of the four core histones, H2A, H2B, H3, and H4, without any noticeable preference for any of these proteins. Removal of the histone N-terminal "tail" domains or the major C-terminal polyglutamic tracts of nucleoplasmin did not alter these binding properties. These results indicate that interactions other than those electrostatic in nature (likely hydrophobic) also play a critical role in the formation of the complex between the negatively charged nucleoplasmin and positively charged histones. Although the association of histones with nucleoplasmin may involve some ionic interactions, the interaction process is not electrostatically driven.

Nucleoplasmin interaction with protamines. Involvement of the polyglutamic tract.

Different recombinant forms of nucleoplasmin including truncations at the carboxyl-terminal end of the molecule (r-NP121, r-NP142) have been expressed and purified. All of them were found to oligomerize, forming pentameric complexes which, according to their hydrodynamic properties, can be modeled by oblate ellipsoids of constant width. In this model, the highly charged carboxyl ends appear to be arranged around a pentameric core along the plane defined by the major axes of the ellipsoid. Importantly, all the recombinant forms appear to be able to decondense protamine-containing sperm nuclei. However, although the stoichiometry with which protamines bind to these forms appears to be constant (2.5 mol of protamine/mol of nucleoplasmin pentamer), the efficiency with which they remove protamines from the sperm DNA decreases in the following order: o-NP > r-NP142 > or = r-NP >> r-NP121. Therefore, the main polyglutamic tract of nucleoplasmin (which is absent in r-NP121), while enhancing the efficiency of protamine removal, is not indispensable for sperm chromatin decondensation in the biological model we have used.