RESUMO
In recent years, nucleic acids have emerged as powerful biomaterials, revolutionizing the field of biomedicine. This review explores the multifaceted applications of nucleic acids, focusing on their pivotal role in various biomedical applications. Nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), possess unique properties such as molecular recognition ability, programmability, and ease of synthesis, making them versatile tools in biosensing and for gene regulation, drug delivery, and targeted therapy. Their compatibility with chemical modifications enhances their binding affinity and resistance to degradation, elevating their effectiveness in targeted applications. Additionally, nucleic acids have found utility as self-assembling building blocks, leading to the creation of nanostructures whose high order underpins their enhanced biological stability and affects the cellular uptake efficiency. Furthermore, this review delves into the significant role of oligonucleotides (ODNs) as indispensable tools for biological studies and biomarker discovery. ODNs, short sequences of nucleic acids, have been instrumental in unraveling complex biological mechanisms. They serve as probes for studying gene expression, protein interactions, and cellular pathways, providing invaluable insights into fundamental biological processes. By examining the synergistic interplay between nucleic acids as powerful biomaterials and ODNs as indispensable tools for biological studies and biomarkers, this review highlights the transformative impact of these molecules on biomedical research. Their versatile applications not only deepen our understanding of biological systems but also are the driving force for innovation in diagnostics and therapeutics, ultimately advancing the field of biomedicine.
Assuntos
Ácidos Nucleicos , Ácidos Nucleicos/uso terapêutico , Oligonucleotídeos/uso terapêutico , RNA , Materiais Biocompatíveis/uso terapêutico , Transporte BiológicoRESUMO
Thymic epithelial cells (TECs) are essential in supporting the development of mature T cells from hematopoietic progenitor cells and facilitate their lineage-commitment, proliferation, T-cell receptor repertoire selection and maturation. While animal model systems have greatly aided in elucidating the contribution of stromal cells to these intricate processes, human tissue has been more difficult to study, partly due to a lack of suitable surface markers comprehensively defining human TECs. Here, we conducted a flow cytometry based surface marker screen to reliably identify and quantify human TECs and delineate medullary from cortical subsets. These findings were validated by transcriptomic and histologic means. The combination of EpCAM, podoplanin (pdpn), CD49f and CD200 comprehensively identified human TECs and not only allowed their reliable distinction in medullary and cortical subsets but also their detailed quantitation. Transcriptomic profiling of each subset in comparison to fibroblasts and endothelial cells confirmed the identity of the different stromal cell subsets sorted according to the proposed strategy. Our dataset not only demonstrated transcriptional similarities between TEC and cells of mesenchymal origin but furthermore revealed a subset-specific distribution of a specific set of extracellular matrix-related genes in TECs. This indicates that TECs significantly contribute to the distinct compartmentalization - and thus function - of the human thymus. We applied the strategy to quantify TEC subsets in 31 immunologically healthy children, which revealed sex-specific differences of TEC composition early in life. As the distribution of mature CD4- or CD8-single-positive thymocytes was correspondingly altered, the composition of the thymic epithelial compartment may directly impact on the CD4-CD8-lineage choice of thymocytes. We prove that the plain, reliable strategy proposed here to comprehensively identify human TEC subpopulations by flow cytometry based on surface marker expression is suitable to determine their frequency and phenotype in health and disease and allows sorting of live cells for downstream analysis. Its use reaches from a reliable diagnostic tool for thymic biopsies to improved phenotypic characterization of thymic grafts intended for therapeutic use.
Assuntos
Separação Celular , Células Epiteliais/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Células Estromais/metabolismo , Timo/metabolismo , Transcriptoma , Síndrome da Deleção 22q11/genética , Síndrome da Deleção 22q11/imunologia , Síndrome da Deleção 22q11/metabolismo , Adolescente , Fatores Etários , Biomarcadores/metabolismo , Criança , Pré-Escolar , Deleção Cromossômica , Cromossomos Humanos Par 22 , Células Epiteliais/imunologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Miastenia Gravis/genética , Miastenia Gravis/imunologia , Miastenia Gravis/metabolismo , Fenótipo , Fatores Sexuais , Células Estromais/imunologia , Timo/citologia , Timo/imunologiaRESUMO
BACKGROUND: In December 2020, the United Kingdom (UK) reported a SARS-CoV-2 Variant of Concern (VoC) which is now named B.1.1.7. Based on initial data from the UK and later data from other countries, this variant was estimated to have a transmission fitness advantage of around 40-80 % (Volz et al., 2021; Leung et al., 2021; Davies et al., 2021). AIM: This study aims to estimate the transmission fitness advantage and the effective reproductive number of B.1.1.7 through time based on data from Switzerland. METHODS: We generated whole genome sequences from 11.8 % of all confirmed SARS-CoV-2 cases in Switzerland between 14 December 2020 and 11 March 2021. Based on these data, we determine the daily frequency of the B.1.1.7 variant and quantify the variant's transmission fitness advantage on a national and a regional scale. RESULTS: We estimate B.1.1.7 had a transmission fitness advantage of 43-52 % compared to the other variants circulating in Switzerland during the study period. Further, we estimate B.1.1.7 had a reproductive number above 1 from 01 January 2021 until the end of the study period, compared to below 1 for the other variants. Specifically, we estimate the reproductive number for B.1.1.7 was 1.24 [1.07-1.41] from 01 January until 17 January 2021 and 1.18 [1.06-1.30] from 18 January until 01 March 2021 based on the whole genome sequencing data. From 10 March to 16 March 2021, once B.1.1.7 was dominant, we estimate the reproductive number was 1.14 [1.00-1.26] based on all confirmed cases. For reference, Switzerland applied more non-pharmaceutical interventions to combat SARS-CoV-2 on 18 January 2021 and lifted some measures again on 01 March 2021. CONCLUSION: The observed increase in B.1.1.7 frequency in Switzerland during the study period is as expected based on observations in the UK. In absolute numbers, B.1.1.7 increased exponentially with an estimated doubling time of around 2-3.5 weeks. To monitor the ongoing spread of B.1.1.7, our plots are available online.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Suíça/epidemiologia , Reino UnidoRESUMO
Chiral phase-transfer catalysis provides high level of enantiocontrol, however no experimental data showed the interaction of catalysts and substrates. 1 Hâ NMR titration was carried out on Cinchona and Maruoka ammonium bromides vs. nitro, carbonyl, heterocycles, and N-F containing compounds. It was found that neutral organic species and quaternary ammonium salts interacted via an ensemble of catalyst + N-C-H and (sp2 )C-H, specific for each substrate studied. The correspondent BArF salts interacted with carbonyls via a diverse set of + N-C-H and (sp2 )C-H compared to bromides. This data suggests that BArF ammonium salts may display a different enantioselectivity profile. Although not providing quantitative data for the affinity constants, the data reported proofs that chiral ammonium salts coordinate with substrates, prior to transition state, through specific C-H positions in their structures, providing a new rational to rationalize the origin of enantioselectivity in their catalyses.
Assuntos
Ésteres , Cetonas , Amidas , Catálise , Compostos de Amônio Quaternário , Sais , EstereoisomerismoRESUMO
Botulinum neurotoxins (BoNTs) produced by soil bacterium Clostridium botulinum are cause of botulism and listed as biohazard agents, thus rapid screening assays are needed for taking the correct countermeasures in a timely fashion. The gold standard method relies on the mouse lethality assay with a lengthy analysis time, i.e., 2-5 days, hindering the prompt management of food safety and medical diagnosis. Herein, we propose the first paper-based antibody-free sensor for reliable and rapid detection of BoNT/A and BoNT/C, exploiting their cleavage capability toward a synthetic peptide able to mimic the natural substrate SNAP-25. The peptide is labelled with the electroactive molecule methylene blue and immobilized on the paper-based electrode modified with gold nanoparticles. Because BoNT/A and BoNT/C can cleave the peptide with the removal of methylene blue from electrode surface, the presence of these neurotoxins in the sample leads to a signal decrease proportional to BoNT amount. The biosensor developed with the selected peptide and combined with smartphone assisted potentiostat is able to detect both BoNT/A and BoNT/C with a linearity up to 1 nM and a detection limit equal to 10 pM. The applicability of this biosensor was evaluated with spiked samples of orange juice, obtaining recovery values equal to 104 ± 6% and 98 ± 9% for 1 nM and 0.5 nM of BoNT/A, respectively.
Assuntos
Técnicas Biossensoriais , Toxinas Botulínicas Tipo A , Nanopartículas Metálicas , Animais , Ouro , Limite de Detecção , Camundongos , Peptídeos , SorogrupoRESUMO
In the present work, structural features of the interaction between peptide nucleic acid (PNA)-based analogs of the tumor-suppressor microRNA-34a with both its binding sites on MYCN mRNA were investigated. In particular, the region from base 1 to 8 ("seed" region) of miR-34a was reproduced in the form of an 8-mer PNA fragment (tiny PNA), and binding to target 3'UTR MYCN mRNA, was studied by a seldom reported and detailed NMR characterization, providing evidence for the formation of anti-parallel duplexes with a well-organized structural core. The formation of PNA-3'UTR duplexes was also confirmed by Circular Dichroism, and their melting curves were measured by UV spectroscopy. Nevertheless, this study offered a valuable comparison between molecular dynamics predictions and experimental evidence, which showed great correlation. Preliminary uptake assays were carried out in Neuroblastoma Kelly cells, using short peptide conjugates as carriers and FITC fluorescent tag for subcellular localization. Moderate internalization was observed without the use of transfecting agents. The reported results corroborate the interest toward the design and development of chimeric PNA/RNA sequences as effective RNA-targeting agents.
RESUMO
Pancreatic ductal adenocarcinoma is the predominant neoplastic disease of the pancreas and it represents the fourth most frequent cause of death in cancer-related disease, with only 8% of survivors after 5-year to the diagnosis. The main issues of this type of cancer rely on fast progress (i.e. 14 months from T1 to a T4 stage), nonspecific symptoms with delay in diagnosis, and the absence of effective screening strategies. To address the lack of early diagnosis, we report a cost-effective paper-based biosensor for the detection of miRNA-492, which is recognised as a biomarker for pancreatic ductal adenocarcinoma. To design a miniaturised, sensitive, and robust paper-based platform, an electrochemical sensor was screen-printed on office paper previously wax-patterned via wax-printing technique. The paper-based sensor was then engineered with a novel and highly specific peptide nucleic acid (PNA) as the recognition element. The formation of PNA/miRNA-492 adduct was evaluated by monitoring the interaction between the positively charged ruthenium (III) hexamine with uncharged PNA and/or negatively charged PNA/miRNA-492 duplex by differential pulse voltammetry. The paper-based biosensor provided a linear range up to 100 nM, with a LOD of 6 nM. Excellent selectivity towards one- and two-base mismatches (1MM, 2MM) or scrambled (SCR) sequences was highlighted and the applicability for biomedical analyses was demonstrated, measuring miRNA-492 in undiluted serum samples.
Assuntos
Adenocarcinoma , Técnicas Biossensoriais , MicroRNAs , Ácidos Nucleicos Peptídicos , Biomarcadores , Técnicas Eletroquímicas , Humanos , MicroRNAs/genética , Ácidos Nucleicos Peptídicos/genéticaRESUMO
Biomimetic design represents an emerging field for improving knowledge of natural molecules, as well as to project novel artificial tools with specific functions for biosensing. Effective strategies have been exploited to design artificial bioreceptors, taking inspiration from complex supramolecular assemblies. Among them, size-minimization strategy sounds promising to provide bioreceptors with tuned sensitivity, stability, and selectivity, through the ad hoc manipulation of chemical species at the molecular scale. Herein, a novel biomimetic peptide enabling herbicide binding was designed bioinspired to the D1 protein of the Photosystem II of the green alga Chlamydomonas reinhardtii. The D1 protein portion corresponding to the QB plastoquinone binding niche is capable of interacting with photosynthetic herbicides. A 50-mer peptide in the region of D1 protein from the residue 211 to 280 was designed in silico, and molecular dynamic simulations were performed alone and in complex with atrazine. An equilibrated structure was obtained with a stable pocked for atrazine binding by three H-bonds with SER222, ASN247, and HIS272 residues. Computational data were confirmed by fluorescence spectroscopy and circular dichroism on the peptide obtained by automated synthesis. Atrazine binding at nanomolar concentrations was followed by fluorescence spectroscopy, highlighting peptide suitability for optical sensing of herbicides at safety limits.
Assuntos
Atrazina/farmacologia , Chlamydomonas reinhardtii/efeitos dos fármacos , Chlamydomonas reinhardtii/fisiologia , Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo , Sequência de Aminoácidos , Biomimética/métodos , Simulação de Dinâmica Molecular , Peptídeos/química , Fotossíntese/efeitos dos fármacos , Complexo de Proteína do Fotossistema II/química , Conformação Proteica , Espectrometria de Fluorescência , TermodinâmicaRESUMO
Herein we report the first organocatalysed enantioselective synthesis of gingesulfonic acids and shogasulfonic acids via a mild and convenient aminothiourea-catalysed conjugate addition of bisulfite to the olefin moiety of α,ß-unsaturated carbonyls-a technology previously reported by us. A series of optically active naturally occurring sulfonic acids are prepared in their natural and unnatural configurations, and their absolute configurations are unequivocally confirmed by single crystal X-ray diffractometry.
RESUMO
MicroRNAs are a ubiquitous class of non-coding RNAs able to regulate gene expression in diverse biological processes. Widespread miRNAs deregulation was reported in numerous diseases including cancer, with several miRNAs playing oncogenic and/or tumor suppressive role by targeting multiple mRNAs simultaneously. Based on these findings, miRNAs have emerged as promising therapeutic tools for cancer treatment. Herein, for the first time, peptide nucleic acids (PNAs) were studied to develop a new class of molecules able to target 3'UTR on MYCN mRNA without a fully complementary base pairing sequence (as miRNAs). For our proof of concept study we have selected as a model the miRNA-34a, which acts as a tumor suppressor in a number of cancers including neuroblastoma. In particular, miRNA-34a is a direct regulator of MYCN oncogene, whose overexpression is a prominent biomarker for the highly aggressive neuroblastoma phenotype. The design and synthesis of three PNA-based oligomers of different length was described, and their interaction with two binding sites on the target MYCN mRNA was investigated by molecular dynamics simulation, and spectroscopic techniques (CD, UV). Intake assay and confocal microscopy of PNA sequences were also carried out in vitro on neuroblastoma Kelly cells. Despite the presence of multiple mismatches, the PNA/RNA hetero duplexes retain very interesting features in terms of stability, affinity as well as of cellular uptake.
Assuntos
Proliferação de Células , MicroRNAs/genética , Proteína Proto-Oncogênica N-Myc/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Ácidos Nucleicos Peptídicos/farmacologia , RNA Mensageiro/antagonistas & inibidores , Química Computacional , Humanos , Microscopia Confocal , Simulação de Dinâmica Molecular , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Ácidos Nucleicos Peptídicos/síntese química , RNA Mensageiro/genética , Células Tumorais CultivadasRESUMO
Periodontal ligament stem cells (PDLSC) play an important role in periodontal tissue homeostasis/turnover and could be applied in cell-based periodontal regenerative therapy. Bacterial supernatants secreted from diverse periodontal bacteria induce the production of cytokines that contribute to local periodontal tissue destruction. However, little is known about the impact of whole bacterial toxins on the biological behavior of PDLSC. Therefore this study investigated whether proliferation, migration, inflammatory cytokines expression and transcriptional profile would be affected by exposure to endotoxins from bacterial species found in the subgingival plaque. PDLSC were cultured with the following bacterial supernatants: S. mutans, S. anginosus, P. intermedia, F. nucleatum, P. gingivalis and T. denticola. These supernatants were prepared in dilutions of 1:1000, 1:500, 1:300 and 1:50. Using quantitative RT-PCR, gene expression of selected inflammatory cytokines (IL-6, IL-8 and IL-1ß) and cell-surface receptors (TLR2, TLR4) showed upregulation of ≈2.0- to 3.0-fold, when exposed to P. intermedia, F. nucleatum, P. gingivalis and T. denticola. However, supernatants did not affect proliferation (MTT) and migration (wound scratch assays) of PDLSC. Next generation RNA sequencing confirmed modified lineage commitment of PDLSC by stimulating chondrogenesis, adipogenesis and inhibition of osteogenesis under P. gingivalis supernatant treatment compared to control. Taken together, this study shows stem cell immunomodulatory response to different periodontal bacteria supernatant and suggests that stem cell transcriptional capacity, migration/proliferation and osteogenesis may differ in the presence of those pathogens. These results bring into question stem cell contribution to periodontal tissue regeneration and onset of inflammation.
Assuntos
Bactérias/metabolismo , Diferenciação Celular , Movimento Celular , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Ligamento Periodontal/citologia , Células-Tronco/citologia , Adulto , Proliferação de Células , Sobrevivência Celular , Regulação da Expressão Gênica , Humanos , Osteogênese/genética , Células-Tronco/metabolismo , Transcrição GênicaRESUMO
Cyclopropane esters holding two quaternary centres were prepared in high yields, complete diastereoselection and up to 83% ee. The reaction described herein entailed reacting (Z)-3-substituted-2-(4-pyridyl)-acrylonitrile, a reactive class of Michael acceptor, with 2-bromomalonate esters in the presence of Cinchona derived phase-transfer catalysts. The reaction allowed multi-gram preparation of the desired products.
Assuntos
Acrilonitrila/análogos & derivados , Compostos de Amônio/química , Cinchona/química , Ciclopropanos/síntese química , Malonatos/química , Acrilonitrila/síntese química , Catálise , Ciclopropanos/química , Esterificação , Halogenação , Malonatos/síntese química , EstereoisomerismoRESUMO
(Z)-3-Substituted-2-(4-pyridyl)-acrylonitriles, a reactive class of Michael acceptors obtained exclusively as a single (Z) isomer, reacted with un-substituted isocyanoacetate esters mediated by phase-transfer catalysis to give, after base promoted cyclisation, functionalized imines in up to 94% ee and as a single diastereoisomer.
RESUMO
The reaction between 3-methyl-4-nitro-5-styrylisoxazoles and ethyl isocyanoacetate proceeded under phase transfer catalysis to give enantioenriched monoadducts in high enantiomeric excess (up to 99% ee). The resulting adducts were subsequently cyclised to give 2,3-dihydropyrroles and substituted pyrrolidines in identical high ees and as a single diastereoisomer.
Assuntos
Acetatos/química , Isoxazóis/química , Nitrocompostos/química , Pirróis/síntese química , Pirrolidinas/síntese química , Acetatos/síntese química , Catálise , Ciclização , Isoxazóis/síntese química , Metilação , Nitrilas/síntese química , Nitrilas/química , Nitrocompostos/síntese química , Pirróis/química , Pirrolidinas/química , EstereoisomerismoRESUMO
Oncogenic conversion of the RET (rearranged during transfection) tyrosine kinase is associated with several cancers. A fragment-based chemical screen led to the identification of a novel RET inhibitor, Pz-1. Modeling and kinetic analysis identified Pz-1 as a typeâ II tyrosine kinase inhibitor that is able to bind the "DFG-out" conformation of the kinase. Importantly, from a single-agent polypharmacology standpoint, Pz-1 was shown to be active on VEGFR2, which can block the blood supply required for RET-stimulated growth. In cell-based assays, 1.0â nM of Pz-1 strongly inhibited phosphorylation of all tested RET oncoproteins. At 1.0â mg kg(-1) day(-1) per os, Pz-1 abrogated the formation of tumors induced by RET-mutant fibroblasts and blocked the phosphorylation of both RET and VEGFR2 in tumor tissue. Pz-1 featured no detectable toxicity at concentrations of up to 100.0â mg kg(-1), which indicates a large therapeutic window. This study validates the effectiveness and usefulness of a medicinal chemistry/polypharmacology approach to obtain an inhibitor capable of targeting multiple oncogenic pathways.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Desenho de Fármacos , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Células NIH 3T3 , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Fosforilação/efeitos dos fármacos , Polifarmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-ret/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Herein, we describe a short synthesis of 3-methyl-4-nitro-5-alkylethenyl isoxazoles and their reactivity as Michael acceptors. The title compounds reacted with nitromethane under phase-transfer catalysis to provide highly enantioenriched adducts (up to 93% ee) which were then converted to the corresponding γ-nitroacids.
Assuntos
Isoxazóis/síntese química , Metano/análogos & derivados , Nitroparafinas/química , Catálise , Isoxazóis/química , Metano/química , Estrutura Molecular , EstereoisomerismoRESUMO
PNAs are emerging as useful synthetic devices targeting natural miRNAs. In particular 3 classes of structurally modified PNAs analogs are herein described, namely α, ß and γ, which differ by their backbone modification. Their mode and binding affinity for natural nucleic acids and their use in medicinal chemistry as potential miRNA binders is discussed.
Assuntos
MicroRNAs/química , Ácidos Nucleicos Peptídicos/química , Oligorribonucleotídeos Antissenso/químicaRESUMO
Heavily substituted cyclopropane esters were prepared in high yields, complete diastereoselection and high (up to 96%) enantioselectivity. The reaction described herein entailed reacting 4-nitro-5-styrylisoxazoles, a class of cinnamate synthetic equivalent, with 2-bromomalonate esters under the catalysis of 5 mol% of a Cincona derived phase-transfer catalyst. The reaction allowed multi-gram preparation of desired products.
Assuntos
Ciclopropanos/química , Oxazóis/química , Catálise , Ésteres , Oxazóis/síntese química , Oxirredução , EstereoisomerismoRESUMO
In this work we report the design and synthesis of kinked oligonucleotide duplexes as potential inhibitors of HMGB1, a cytokine which triggers a broad range of immunological effects. We found that the designed ligands can interact with HMGB1, as evidenced by circular dichroism spectroscopy, and are able to block some extracellular effects induced by the protein, such as cellular proliferation and migration, as we demonstrated by in vitro biological assays. After selecting the most stable and active kinked duplex, we synthesized the corresponding PNA/DNA chimeric duplex which resulted to be more resistant to enzymatic degradation, and showed a biological activity comparable to that of the natural duplex. Preliminary in vivo assays in a mouse inflammatory model, showed a significant decrease of the mortality after administration of the PNA/DNA kinked duplex to LPS-treated mice.