The development of highly dense highly protected surfactant ionizable lipid RNA loaded nanoparticles

The long quest for efficient drug administration has been looking for a universal carrier that can precisely transport traditional drugs, new genomic and proteic therapeutic agents. Today, researchers have found conditions to overcome the two main drug delivery dilemmas. On the one side, the versatility of the vehicle to efficiently load, protect and transport the drug and then release it at the target place. On the other hand, the questions related to the degree of PEGylation which are needed to avoid nanoparticle (NP) aggregation and opsonization while preventing cellular uptake. The development of different kinds of lipidic drug delivery vehicles and particles has resulted in the development of ionizable lipid nanoparticles (iLNPs), which can overcome most of the typical drug delivery problems. Proof of their success is the late approval and massive administration as the prophylactic vaccine for SARS-CoV-2. These ILNPs are built by electrostatic aggregation of surfactants, the therapeutic agent, and lipids that self-segregate from an aqueous solution, forming nanoparticles stabilized with lipid polymers, such as PEG. These vehicles overcome previous limitations such as low loading and high toxicity, likely thanks to low charge at the working pH and reduced size, and their entry into the cells via endocytosis rather than membrane perforation or fusion, always associated with higher toxicity. We herein revise their primary features, synthetic methods to prepare and characterize them, pharmacokinetic (administration, distribution, metabolization and excretion) aspects, and biodistribution and fate. Owing to their advantages, iLNPs are potential drug delivery systems to improve the management of various diseases and widely available for clinical use

Exploring the mechanisms of action of human secretory RNase 3 and RNase 7 against Candida albicans

Human antimicrobial RNases, which belong to the vertebrate RNase A superfamily and are secreted upon infection, display a wide spectrum of antipathogen activities. In this work, we examined the antifungal activity of the eosinophil RNase 3 and the skin-derived RNase 7, two proteins expressed by innate cell types that are directly involved in the host defense against fungal infection. Candida albicans has been selected as a suitable working model for testing RNase activities toward a eukaryotic pathogen. We explored the distinct levels of action of both RNases on yeast by combining cell viability and membrane model assays together with protein labeling and confocal microscopy. Site-directed mutagenesis was applied to ablate either the protein active site or the key anchoring region for cell binding. This is the first integrated study that highlights the RNases' dual mechanism of action. Along with an overall membrane-destabilization process, the RNases could internalize and target cellular RNA. The data support the contribution of the enzymatic activity for the antipathogen action of both antimicrobial proteins, which can be envisaged as suitable templates for the development of novel antifungal drugs. We suggest that both human RNases work as multitasking antimicrobial proteins that provide a first line immune barrier

Unveiling the multifaceted mechanisms of antibacterial activity of buforin II and frenatin 2.3S peptides from skin micro-organs of the orinoco lime treefrog (Sphaenorhynchus lacteus)

Amphibian skin is a rich source of natural compounds with diverse antimicrobial and immune defense properties. Our previous studies showed that the frog skin secretions obtained by skin micro-organs from various species of Colombian anurans have antimicrobial activities against bacteria and viruses. We purified for the first time two antimicrobial peptides from the skin micro-organs of the Orinoco lime treefrog (Sphaenorhynchus lacteus) that correspond to Buforin II (BF2) and Frenatin 2.3S (F2.3S). Here, we have synthesized the two peptides and tested them against Gram-negative and Gram-positive bacteria, observing an effective bactericidal activity at micromolar concentrations. Evaluation of BF2 and F2.3S membrane destabilization activity on bacterial cell cultures and synthetic lipid bilayers reveals a distinct membrane interaction mechanism. BF2 agglutinates E. coli cells and synthetic vesicles, whereas F2.3S shows a high depolarization and membrane destabilization activities. Interestingly, we found that F2.3S is able to internalize within bacterial cells and can bind nucleic acids, as previously reported for BF2. Moreover, bacterial exposure to both peptides alters the expression profile of genes related to stress and resistance response. Overall, these results show the multifaceted mechanism of action of both antimicrobial peptides that can provide alternative tools in the fight against bacterial resistance

Exploiting endocytosis for transfection of mRNA for cytoplasmatic delivery using cationic gold nanoparticles

Gene therapy holds promise to cure various diseases at the fundamental level. For that, efficient carriers are needed for successful gene delivery. Synthetic 'non-viral' vectors, as cationic polymers, are quickly gaining popularity as efficient vectors for transmitting genes. However, they suffer from high toxicity associated with the permeation and poration of the cell membrane. This toxic aspect can be eliminated by nanoconjugation. Still, results suggest that optimising the oligonucleotide complexation, ultimately determined by the size and charge of the nanovector, is not the only barrier to efficient gene delivery. We herein develop a comprehensive nanovector catalogue comprising different sizes of Au NPs functionalized with two different cationic molecules and further loaded with mRNA for its delivery inside the cell. Tested nanovectors showed safe and sustained transfection efficiencies over 7 days, where 50 nm Au NPs displayed the highest transfection rates. Remarkably, protein expression was increased when nanovector transfection was performed combined with chloroquine. Cytotoxicity and risk assessment demonstrated that nanovectors are safe, ascribed to lesser cellular damage due to their internalization and delivery via endocytosis. Obtained results may pave the way to design advanced and efficient gene therapies for safely transferring oligonucleotides

Exploring the mechanism of action of human antimicrobial ribonucleases

Las ribonucleases humanas son un grupo heterogéneo de proteínas pertenecientes a la superfamilia de la Ribonucleasa A. Estas proteínas se caracterizan por su capacidad de hidrolizar ácidos ribonucleicos y por la presencia de actividad antimicrobiana frente diversos organismos patógenos como bacterias, hongos, parásitos y virus. El primer objetivo del presente estudio doctoral se centra en la caracterización de la actividad antimicrobiana y en modelos de membrana de las formas nativas de la ribonucleasa 3 purificadas a partir de eosinófilos. Las distintas formas nativas presentan modificaciones postraduccionales dadas por diversos grados de glicosilación que se correlacionan con la activación de los eosinófilos durante los procesos inflamatorios. El estudio establece la capacidad antimicrobiana de las formas nativas y su actividad en modelos de membrana. Los resultados indican que las modificaciones postrasduccionales modulan la actividad biológica de la RNasa 3, sugiriendo una contribución in vivo en su función fisiológica. Como segundo objetivo de esta tesis, se evaluó por primera vez en nuestro grupo de investigación la actividad antimicótica de las ribonucleasas 3 y 7 frente al hongo Candida albicans, el cual fue elegido como modelo patógeno eucariota. Se determinó y caracterizó la presencia de actividad frente a Candida por parte de ambas ribonucleasas humanas. Por último, el tercer objetivo de esta tesis se centra en la purificación y caracterización de la ribonucleasa 8, la más reciente ribonucleasa humana descrita, identificada inicialmente en placenta. La RNasa 8 presenta un patrón inusual de enlaces disulfuro respecto a sus proteínas homólogas. Este cambio estructural modifica la estabilidad de la proteína y expone regiones que facilitan el proceso de agregación proteica. Fue necesaria la previa optimización de un protocolo alternativo de purificación. Se analizaron sus propiedades antimicrobianas, sugiriendo su posible participación en la respuesta inmunitaria innata. Los resultados del presente estudio corroboran las propiedades antimicrobianas de diversas ribonucleasas humanas miembros de la familia de la RNasa A, sugiriendo una función ancestral en el sistema de defensa innato. El estudio contribuye a la comprensión de su mecanismo de acción y plantea su potencial uso como herramientas terapéuticas.Human ribonucleases are a heterogeneous group of proteins belonging to the superfamily of RNase A. These proteins are characterized by their ability to hydrolyse ribonucleic acids and the presence of antimicrobial activity against various pathogens including bacteria, fungi, parasites and viruses. The first objective of this doctoral study is focused on the antimicrobial characterization of native Ribonuclease 3 forms purified from eosinophils. Native forms present posttranslational modifications giving different glycosylation grades that modulate their activity during inflammatory processes. This study aims to establish the antimicrobial properties of native forms purified from eosinophils and their activity in a membrane model system. Results indicate that post-translational modifications contribute to the the protein biological activities, suggesting a related physiological role. As a second objective, we evaluated for the first time the antifungal activity of the antimicrobial RNase 3 and RNase 7 against Candida albicans, an eukaryotic pathogen selected as a simple model to test the antimicrobial mechanism of action. Both human ribonucleases displayed a high antifungal activity. Results highlighted a dual mechanism of action, where cell lysis takes place at high protein concentration, while depolarization, cell internalization and cellular RNA degradation is achieved at sublethal doses. Finally, the last objective is focused on the characterization of ribonuclease 8, also called the placental RNase, the most recent human ribonuclease described. RNase 8 has gained and lost one cysteine residue in non-conserved positions in a mechanism called "disulphide shuffling". The protein tendency to aggregate required the design of an alternative purification protocol. We analysed its antimicrobial abilities, suggesting a possible role in innate defence. The results of this study confirmed the high antimicrobial activity of several human ribonucleases from the RNase A superfamily suggesting an ancestral role in the host immune defence response. The study contributed to the understanding of their mechanism of action and set the basis for applied drug design

Exploring the mechanism of action of human antimicrobial ribonucleases

Las ribonucleases humanas son un grupo heterogéneo de proteínas pertenecientes a la superfamilia de la Ribonucleasa A. Estas proteínas se caracterizan por su capacidad de hidrolizar ácidos ribonucleicos y por la presencia de actividad antimicrobiana frente diversos organismos patógenos como bacterias, hongos, parásitos y virus. El primer objetivo del presente estudio doctoral se centra en la caracterización de la actividad antimicrobiana y en modelos de membrana de las formas nativas de la ribonucleasa 3 purificadas a partir de eosinófilos. Las distintas formas nativas presentan modificaciones postraduccionales dadas por diversos grados de glicosilación que se correlacionan con la activación de los eosinófilos durante los procesos inflamatorios. El estudio establece la capacidad antimicrobiana de las formas nativas y su actividad en modelos de membrana. Los resultados indican que las modificaciones postrasduccionales modulan la actividad biológica de la RNasa 3, sugiriendo una contribución in vivo en su función fisiológica. Como segundo objetivo de esta tesis, se evaluó por primera vez en nuestro grupo de investigación la actividad antimicótica de las ribonucleasas 3 y 7 frente al hongo Candida albicans, el cual fue elegido como modelo patógeno eucariota. Se determinó y caracterizó la presencia de actividad frente a Candida por parte de ambas ribonucleasas humanas. Por último, el tercer objetivo de esta tesis se centra en la purificación y caracterización de la ribonucleasa 8, la más reciente ribonucleasa humana descrita, identificada inicialmente en placenta. La RNasa 8 presenta un patrón inusual de enlaces disulfuro respecto a sus proteínas homólogas. Este cambio estructural modifica la estabilidad de la proteína y expone regiones que facilitan el proceso de agregación proteica. Fue necesaria la previa optimización de un protocolo alternativo de purificación. Se analizaron sus propiedades antimicrobianas, sugiriendo su posible participación en la respuesta inmunitaria innata. Los resultados del presente estudio corroboran las propiedades antimicrobianas de diversas ribonucleasas humanas miembros de la familia de la RNasa A, sugiriendo una función ancestral en el sistema de defensa innato. El estudio contribuye a la comprensión de su mecanismo de acción y plantea su potencial uso como herramientas terapéuticas.Human ribonucleases are a heterogeneous group of proteins belonging to the superfamily of RNase A. These proteins are characterized by their ability to hydrolyse ribonucleic acids and the presence of antimicrobial activity against various pathogens including bacteria, fungi, parasites and viruses. The first objective of this doctoral study is focused on the antimicrobial characterization of native Ribonuclease 3 forms purified from eosinophils. Native forms present posttranslational modifications giving different glycosylation grades that modulate their activity during inflammatory processes. This study aims to establish the antimicrobial properties of native forms purified from eosinophils and their activity in a membrane model system. Results indicate that post-translational modifications contribute to the the protein biological activities, suggesting a related physiological role. As a second objective, we evaluated for the first time the antifungal activity of the antimicrobial RNase 3 and RNase 7 against Candida albicans, an eukaryotic pathogen selected as a simple model to test the antimicrobial mechanism of action. Both human ribonucleases displayed a high antifungal activity. Results highlighted a dual mechanism of action, where cell lysis takes place at high protein concentration, while depolarization, cell internalization and cellular RNA degradation is achieved at sublethal doses. Finally, the last objective is focused on the characterization of ribonuclease 8, also called the placental RNase, the most recent human ribonuclease described. RNase 8 has gained and lost one cysteine residue in non-conserved positions in a mechanism called "disulphide shuffling". The protein tendency to aggregate required the design of an alternative purification protocol. We analysed its antimicrobial abilities, suggesting a possible role in innate defence. The results of this study confirmed the high antimicrobial activity of several human ribonucleases from the RNase A superfamily suggesting an ancestral role in the host immune defence response. The study contributed to the understanding of their mechanism of action and set the basis for applied drug design

Exploring the mechanisms of action of human secretory RNase 3 and RNase 7 against Candida albicans

Human antimicrobial RNases, which belong to the vertebrate RNase A superfamily and are secreted upon infection, display a wide spectrum of antipathogen activities. In this work, we examined the antifungal activity of the eosinophil RNase 3 and the skin-derived RNase 7, two proteins expressed by innate cell types that are directly involved in the host defense against fungal infection. Candida albicans has been selected as a suitable working model for testing RNase activities toward a eukaryotic pathogen. We explored the distinct levels of action of both RNases on yeast by combining cell viability and membrane model assays together with protein labeling and confocal microscopy. Site-directed mutagenesis was applied to ablate either the protein active site or the key anchoring region for cell binding. This is the first integrated study that highlights the RNases' dual mechanism of action. Along with an overall membrane-destabilization process, the RNases could internalize and target cellular RNA. The data support the contribution of the enzymatic activity for the antipathogen action of both antimicrobial proteins, which can be envisaged as suitable templates for the development of novel antifungal drugs. We suggest that both human RNases work as multitasking antimicrobial proteins that provide a first line immune barrier

Unveiling the multifaceted mechanisms of antibacterial activity of buforin II and frenatin 2.3S peptides from skin micro-organs of the orinoco lime treefrog (Sphaenorhynchus lacteus)

Amphibian skin is a rich source of natural compounds with diverse antimicrobial and immune defense properties. Our previous studies showed that the frog skin secretions obtained by skin micro-organs from various species of Colombian anurans have antimicrobial activities against bacteria and viruses. We purified for the first time two antimicrobial peptides from the skin micro-organs of the Orinoco lime treefrog (Sphaenorhynchus lacteus) that correspond to Buforin II (BF2) and Frenatin 2.3S (F2.3S). Here, we have synthesized the two peptides and tested them against Gram-negative and Gram-positive bacteria, observing an effective bactericidal activity at micromolar concentrations. Evaluation of BF2 and F2.3S membrane destabilization activity on bacterial cell cultures and synthetic lipid bilayers reveals a distinct membrane interaction mechanism. BF2 agglutinates E. coli cells and synthetic vesicles, whereas F2.3S shows a high depolarization and membrane destabilization activities. Interestingly, we found that F2.3S is able to internalize within bacterial cells and can bind nucleic acids, as previously reported for BF2. Moreover, bacterial exposure to both peptides alters the expression profile of genes related to stress and resistance response. Overall, these results show the multifaceted mechanism of action of both antimicrobial peptides that can provide alternative tools in the fight against bacterial resistance

Insight into the Antifungal Mechanism of Action of Human RNase N-terminus Derived Peptides

Altres ajuts: Fundació La Marató de TV3 (Marató 20180310); European Society of Clinical Microbiology and Infectious Diseases - ESCMID 2916 grantCandida albicans is a polymorphic fungus responsible for mucosal and skin infections. Candida cells establish themselves into biofilm communities resistant to most currently available antifungal agents. An increase of severe infections ensuing in fungal septic shock in elderly or immunosuppressed patients, along with the emergence of drug-resistant strains, urge the need for the development of alternative antifungal agents. In the search for novel antifungal drugs our laboratory demonstrated that two human ribonucleases from the vertebrate-specific RNaseA superfamily, hRNase3 and hRNase7, display a high anticandidal activity. In a previous work, we proved that the N-terminal region of the RNases was sufficient to reproduce most of the parental protein bactericidal activity. Next, we explored their potency against a fungal pathogen. Here, we have tested the N-terminal derived peptides that correspond to the eight human canonical RNases (RN1-8) against planktonic cells and biofilms of C. albicans. RN3 and RN7 peptides displayed the most potent inhibitory effect with a mechanism of action characterized by cell-wall binding, membrane permeabilization and biofilm eradication activities. Both peptides are able to eradicate planktonic and sessile cells, and to alter their gene expression, reinforcing its role as a lead candidate to develop novel antifungal and antibiofilm therapies

Biodiversity 2016. Status and Trends of Colombian Continental Biodiversity

This third volume of the annual report on biodiversity in Colombia continues the editorial line that begun in 2014. Using novel analytical and graphic proposals, these reports have the goal of communicating the contents to a broad public, making it available for discussion without sacrificing the quality of information. The challenge of communication continues to be a major part of the institutional project, and the new languages with which we are learning to communicate with society and other institutions are an experiment that we expect to be increasingly gratifying. The report for 2017 is already under construction and it counts on new digital technologies so the power of a colombian vital connection may be entirely expressed. The included content evidences that we are still far away from having a systematic follow-up about most of the topics related to the management of biodiversity and ecosystem services, which is the only way to evaluate the effectiveness of policies and investments made by society. In fact, a limitation that is recognized is that of identifying positive or negative changes that affect different levels of organization of life on this planet; therefore, our global navigation route of the Aichi targets is still to be verified. An additional purpose of this process includes the invitation of all Colombians to contribute in constructing and maintaining basic monitoring indicators for management since it is impossible to identify long-term trends of flora and fauna in the country without the support of institutions, researchers, and citizens. This challenge is immense in a megadiverse country such as Colombia. For this reason, the report will continue to open its pages to experts, and even indigenous peoples or local communities, for them to present their perspectives about environmental change and its effects on biodiversity in a systematic and documented manner. This has the objective of stimulating the commitment of everyone in the management of biodiversity and ecosystem services. The only way of overcoming the risk of extinction is through the active process of social learning in which all sectors assume a part of the complex responsibility in protecting the forms of life of the country, a roughly counted tenth of all creatures on Earth. I thank all the people that contributed in this Report, those who have supported us in the phases of production, and all readers and users, who are the ultimate judges of its utility.Bogotá, D. C