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1.
Anal Chem ; 96(22): 8868-8874, 2024 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-38775341

RESUMO

Experimental methods to determine transition temperatures for individual base pair melting events in DNA duplexes are lacking despite intense interest in these thermodynamic parameters. Here, we determine the dimensions of the thymine (T) C2═O stretching vibration when it is within the DNA duplex via isotopic substitutions at other atomic positions in the structure. First, we determined that this stretching state was localized enough to specific atoms in the molecule to make submolecular scale measurements of local structure and stability in high molecular weight complexes. Next, we develop a new isotope-edited variable temperature infrared method to measure melting transitions at various locations in a DNA structure. As an initial test of this "sub-molecular scale thermometer", we applied our T13C2 difference infrared signal to measure location-dependent melting temperatures (TmL) in a DNA duplex via variable temperature attenuated total reflectance Fourier transform infrared (VT-ATR-FTIR) spectroscopy. We report that the TmL of a single Watson-Crick A-T base pair near the end of an A-T rich sequence (poly T) is ∼34.9 ± 0.7°C. This is slightly lower than the TmL of a single base pair near the middle position of the poly T sequence (TmL ∼35.6±0.2°C). In addition, we also report that the TmL of a single Watson-Crick A-T base pair near the end of a 50% G-C sequence (12-mer) is ∼52.5 ± 0.3°C, which is slightly lower than the global melting Tm of the 12-mer sequence (TmL ∼54.0±0.9°C). Our results provide direct physical evidence for end fraying in DNA sequences with our novel spectroscopic methods.


Assuntos
Pareamento de Bases , DNA , Timina , Temperatura de Transição , DNA/química , Timina/química , Espectroscopia de Infravermelho com Transformada de Fourier , Espectrofotometria Infravermelho/métodos , Conformação de Ácido Nucleico , Temperatura
2.
Spectrochim Acta A Mol Biomol Spectrosc ; 309: 123816, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38198991

RESUMO

S-adenosyl-L-methionine (SAM) is an abundant biomolecule used by methyltransferases to regulate a wide range of essential cellular processes such as gene expression, cell signaling, protein functions, and metabolism. Despite considerable effort, there remain many specificity challenges associated with designing small molecule inhibitors for methyltransferases, most of which exhibit off-target effects. Interestingly, NMR evidence suggests that SAM undergoes conformeric exchange between several states when free in solution. Infrared spectroscopy can detect different conformers of molecules if present in appreciable populations. When SAM is noncovalently bound within enzyme active sites, the nature and the number of different conformations of the molecule are likely to be altered from when it is free in solution. If there are unique structures or different numbers of conformers between different methyltransferase active sites, solution-state information may provide promising structural leads to increase inhibitor specificity for a particular methyltransferase. Toward this goal, frequencies measured in SAM's infrared spectra must be assigned to the motions of specific atoms via isotope incorporation at discrete positions. The incorporation of isotopes into SAM's structure can be accomplished via an established enzymatic synthesis using isotopically labeled precursors. However, published protocols produced an intense and highly variable IR signal which overlapped with many of the signals from SAM rendering comparison between isotopes challenging. We observed this intense absorption to be from co-purifying salts and the SAM counterion, producing a strong, broad signal at 1100 cm-1. Here, we report a revised SAM purification protocol that mitigates the contaminating salts and present the first IR spectra of isotopically labeled CD3-SAM. These results provide a foundation for isotopic labeling experiments of SAM that will define which atoms participate in individual molecular vibrations, as a means to detect specific molecular conformations.


Assuntos
Metionina , S-Adenosilmetionina , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Sais , Metiltransferases/química , Metiltransferases/metabolismo , Racemetionina , Isótopos
3.
Protein Sci ; 32(12): e4815, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37874269

RESUMO

DNA helicase activity is essential for the vital DNA metabolic processes of recombination, replication, transcription, translation, and repair. Recently, an unexpected, rapid exponential ATP-stimulated DNA unwinding rate was observed from an Archaeoglobus fulgidus helicase (AfXPB) as compared to the slower conventional helicases from Sulfolobus tokodaii, StXPB1 and StXPB2. This unusual rapid activity suggests a "molecular wrench" mechanism arising from the torque applied by AfXPB on the duplex structure in transitioning from open to closed conformations. However, much remains to be understood. Here, we investigate the concentration dependence of DNA helicase binding and ATP-stimulated kinetics of StXPB2 and AfXPB, as well as their binding and activity in Bax1 complexes, via an electrochemical assay with redox-active DNA monolayers. StXPB2 ATP-stimulated activity is concentration-independent from 8 to 200 nM. Unexpectedly, AfXPB activity is concentration-dependent in this range, with exponential rate constants varying from seconds at concentrations greater than 20 nM to thousands of seconds at lower concentrations. At 20 nM, rapid exponential signal decay ensues, linearly reverses, and resumes with a slower exponential decay. This change in AfXPB activity as a function of its concentration is rationalized as the crossover between the fast molecular wrench and slower conventional helicase modes. AfXPB-Bax1 inhibits rapid activity, whereas the StXPB2-Bax1 complex induces rapid kinetics at higher concentrations. This activity is rationalized with the crystal structures of these complexes. These findings illuminate the different physical models governing molecular wrench activity for improved biological insight into a key factor in DNA repair.


Assuntos
Reparo do DNA , DNA , DNA/química , DNA Helicases/química , Trifosfato de Adenosina/metabolismo , Cinética
4.
Biomimetics (Basel) ; 8(2)2023 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-37366830

RESUMO

Structural bioinspiration in modern material science and biomimetics represents an actual trend that was originally based on the bioarchitectural diversity of invertebrate skeletons, specifically, honeycomb constructs of natural origin, which have been in humanities focus since ancient times. We conducted a study on the principles of bioarchitecture regarding the unique biosilica-based honeycomb-like skeleton of the deep-sea glass sponge Aphrocallistes beatrix. Experimental data show, with compelling evidence, the location of actin filaments within honeycomb-formed hierarchical siliceous walls. Principles of the unique hierarchical organization of such formations are discussed. Inspired by poriferan honeycomb biosilica, we designed diverse models, including 3D printing, using PLA-, resin-, and synthetic-glass-prepared corresponding microtomography-based 3D reconstruction.

5.
J Phys Chem Lett ; 14(18): 4313-4321, 2023 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-37130045

RESUMO

The level of interest in probing the strength of noncovalent interactions in DNA duplexes is high, as these weak forces dictate the range of suprastructures the double helix adopts under different conditions, in turn directly impacting the biological functions and industrial applications of duplexes that require making and breaking them to access the genetic code. However, few experimental tools can measure these weak forces embedded within large biological suprastructures in the native solution environment. Here, we develop experimental methods for detecting the presence of a single noncovalent interaction [a hydrogen bond (H-bond)] within a large DNA duplex in solution and measure its formation enthalpy (ΔHf). We report that introduction of a H-bond into the TC2═O group from the noncanonical nucleobase 2-aminopurine produces an expected decrease ∼10 ± 0.76 cm-1 (from ∼1720 cm-1 in Watson-Crick to ∼1710 cm-1 in 2-aminopurine), which correlates with an enthalpy of ∼0.93 ± 0.066 kcal/mol for this interaction.


Assuntos
2-Aminopurina , DNA , Temperatura , Conformação de Ácido Nucleico , Ligação de Hidrogênio , Termodinâmica , DNA/química , Análise Espectral
6.
J Phys Chem B ; 127(11): 2351-2361, 2023 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-36898003

RESUMO

The carbonyl stretching modes have been widely used in linear and two-dimensional infrared (IR) spectroscopy to probe the conformation, interaction, and biological functions of nucleic acids. However, due to their universal appearance in nucleobases, the IR absorption bands of nucleic acids are often highly congested in the 1600-1800 cm-1 region. Following the fruitful applications in proteins, 13C isotope labels have been introduced to the IR measurements of oligonucleotides to reveal their site-specific structural fluctuations and hydrogen bonding conditions. In this work, we combine recently developed frequency and coupling maps to develop a theoretical strategy that models the IR spectra of oligonucleotides with 13C labels directly from molecular dynamics simulations. We apply the theoretical method to nucleoside 5'-monophosphates and DNA double helices and demonstrate how elements of the vibrational Hamiltonian determine the spectral features and their changes upon isotope labeling. Using the double helices as examples, we show that the calculated IR spectra are in good agreement with experiments and the 13C isotope labeling technique can potentially be applied to characterize the stacking configurations and secondary structures of nucleic acids.


Assuntos
Oligonucleotídeos , Proteínas , Espectrofotometria Infravermelho/métodos , Proteínas/química , DNA/química , Isótopos , Nucleotídeos
7.
Mar Drugs ; 20(11)2022 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-36354988

RESUMO

Three-dimensional chitinous scaffolds often used in regenerative medicine, tissue engineering, biomimetics and technology are mostly isolated from marine organisms, such as marine sponges (Porifera). In this work, we report the results of the electrochemical isolation of the ready to use chitinous matrices from three species of verongiid demosponges (Aplysina archeri, Ianthella basta and Suberea clavata) as a perfect example of possible morphological and chemical dimorphism in the case of the marine chitin sources. The electrolysis of concentrated Na2SO4 aqueous solution showed its superiority over the chemical chitin isolation method in terms of the treatment time reduction: only 5.5 h for A. archeri, 16.5 h for I. basta and 20 h for the S. clavata sample. Further investigation of the isolated scaffolds by digital microscopy and SEM showed that the electrolysis-supported isolation process obtains chitinous scaffolds with well-preserved spatial structure and it can be competitive to other alternative chitin isolation techniques that use external accelerating factors such as microwave irradiation or atmospheric plasma. Moreover, the infrared spectroscopy (ATR-FTIR) proved that with the applied electrochemical conditions, the transformation into chitosan does not take place.


Assuntos
Quitina , Poríferos , Animais , Quitina/química , Espectroscopia de Infravermelho com Transformada de Fourier , Poríferos/química , Eletrólise
8.
J Am Chem Soc ; 144(2): 709-722, 2022 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-34985880

RESUMO

The human copper-binding protein metallothionein-3 (MT-3) can reduce Cu(II) to Cu(I) and form a polynuclear Cu(I)4-Cys5-6 cluster concomitant with intramolecular disulfide bonds formation, but the cluster is unusually inert toward O2 and redox-cycling. We utilized a combined array of rapid-mixing spectroscopic techniques to identify and characterize the transient radical intermediates formed in the reaction between Zn7MT-3 and Cu(II) to form Cu(I)4Zn(II)4MT-3. Stopped-flow electronic absorption spectroscopy reveals the rapid formation of transient species with absorption centered at 430-450 nm and consistent with the generation of disulfide radical anions (DRAs) upon reduction of Cu(II) by MT-3 cysteine thiolates. These DRAs are oxygen-stable and unusually long-lived, with lifetimes in the seconds regime. Subsequent DRAs reduction by Cu(II) leads to the formation of a redox-inert Cu(I)4-Cys5 cluster with short Cu-Cu distances (<2.8 Å), as revealed by low-temperature (77 K) luminescence spectroscopy. Rapid freeze-quench Raman and electron paramagnetic resonance (EPR) spectroscopy characterization of the intermediates confirmed the DRA nature of the sulfur-centered radicals and their subsequent oxidation to disulfide bonds upon Cu(II) reduction, generating the final Cu(I)4-thiolate cluster. EPR simulation analysis of the radical g- and A-values indicate that the DRAs are directly coupled to Cu(I), potentially explaining the observed DRA stability in the presence of O2. We thus provide evidence that the MT-3 Cu(I)4-Cys5 cluster assembly process involves the controlled formation of novel long-lived, copper-coupled, and oxygen-stable disulfide radical anion transient intermediates.


Assuntos
Cobre/química , Dissulfetos/química , Radicais Livres/química , Metalotioneína 3/química , Oxigênio/química , Espectroscopia de Ressonância de Spin Eletrônica , Glutationa/química , Humanos , Metalotioneína 3/genética , Metalotioneína 3/metabolismo , Oxirredução , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Fluorescência , Zinco/química
9.
Int J Mol Sci ; 22(22)2021 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-34830470

RESUMO

Marine sponges were among the first multicellular organisms on our planet and have survived to this day thanks to their unique mechanisms of chemical defense and the specific design of their skeletons, which have been optimized over millions of years of evolution to effectively inhabit the aquatic environment. In this work, we carried out studies to elucidate the nature and nanostructural organization of three-dimensional skeletal microfibers of the giant marine demosponge Ianthella basta, the body of which is a micro-reticular, durable structure that determines the ideal filtration function of this organism. For the first time, using the battery of analytical tools including three-dimensional micro-X-ray Fluorescence (3D-µXRF), X-ray diffraction (XRD), infra-red (FTIR), Raman and Near Edge X-ray Fine Structure (NEXAFS) spectroscopy, we have shown that biomineral calcite is responsible for nano-tuning the skeletal fibers of this sponge species. This is the first report on the presence of a calcitic mineral phase in representatives of verongiid sponges which belong to the class Demospongiae. Our experimental data suggest a possible role for structural amino polysaccharide chitin as a template for calcification. Our study suggests further experiments to elucidate both the origin of calcium carbonate inside the skeleton of this sponge and the mechanisms of biomineralization in the surface layers of chitin microfibers saturated with bromotyrosines, which have effective antimicrobial properties and are responsible for the chemical defense of this organism. The discovery of the calcified phase in the chitinous template of I. basta skeleton is expected to broaden the knowledge in biomineralization science where the calcium carbonate is regarded as a valuable material for applications in biomedicine, environmental science, and even in civil engineering.


Assuntos
Organismos Aquáticos/química , Carbonato de Cálcio/química , Poríferos/química , Esqueleto/química , Animais , Biomineralização , Quitina/química , Espectroscopia de Infravermelho com Transformada de Fourier , Alicerces Teciduais/química , Difração de Raios X
10.
J Phys Chem B ; 125(28): 7613-7627, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34236202

RESUMO

Measuring the strength of the hydrogen bonds between DNA base pairs is of vital importance for understanding how our genetic code is physically accessed and recognized in cells, particularly during replication and transcription. Therefore, it is important to develop probes for these key hydrogen bonds (H-bonds) that dictate events critical to cellular function, such as the localized melting of DNA. The vibrations of carbonyl bonds are well-known probes of their H-bonding environment, and their signals can be observed with infrared (IR) spectroscopy. Yet, pinpointing a single bond of interest in the complex IR spectrum of DNA is challenging due to the large number of carbonyl signals that overlap with each other. Here, we develop a method using isotope editing and infrared (IR) spectroscopy to isolate IR signals from the thymine (T) C2═O carbonyl. We use solvatochromatic studies to show that the TC2═O signal's position in the IR spectrum is sensitive to the H-bonding capacity of the solvent. Our results indicate that C2═O of a single T base within DNA duplexes experiences weak H-bonding interactions. This finding is consistent with the existence of a third, noncanonical CH···O H-bond between adenine and thymine in both Watson-Crick and Hoogsteen base pairs in DNA.


Assuntos
DNA , Isótopos , Hidrogênio , Ligação de Hidrogênio , Análise Espectral
11.
Mater Sci Eng C Mater Biol Appl ; 109: 110566, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32228987

RESUMO

Marine demosponges of the Verongiida order are considered a gold-mine for bioinspired materials science and marine pharmacology. The aim of this work was to simultaneously isolate selected bromotyrosines and unique chitinous structures from A. aerophoba and to propose these molecules and biomaterials for possible application as antibacterial and antitumor compounds and as ready-to-use scaffolds for cultivation of cardiomyocytes, respectively. Among the extracted bromotyrosines, the attention has been focused on aeroplysinin-1 that showed interesting unexpected growth inhibition properties for some Gram-negative clinical multi-resistant bacterial strains, such as A. baumannii and K. pneumoniae, and on aeroplysinin-1 and on isofistularin-3 for their anti-tumorigenic activity. For both compounds, the effects are cell line dependent, with significant growth inhibition activity on the neuroblastoma cell line SH-SY5Y by aeroplysinin-1 and on breast cancer cell line MCF-7 by isofistularin-3. In this study, we also compared the cultivation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) on the A. aerophoba chitinous scaffolds, in comparison to chitin structures that were pre-coated with Geltrex™, an extracellular matrix mimetic which is used to enhance iPSC-CM adhesion. The iPSC-CMs on uncoated and pure chitin structures started contracting 24 h after seeding, with comparable behaviour observed on Geltrex-coated cell culture plates, confirming the biocompatibility of the sponge biomaterial with this cell type. The advantage of A. aerophoba is that this source organism does not need to be collected in large quantities to supply the necessary amount for further pre-clinical studies before chemical synthesis of the active compounds will be available. A preliminary analysis of marine sponge bioeconomy as a perspective direction for application of biomaterials and secondary bioactive metabolites has been finally performed for the first time.


Assuntos
Acetonitrilas , Alcaloides , Organismos Aquáticos/química , Materiais Biomiméticos , Cicloexenos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Poríferos/química , Acetonitrilas/química , Acetonitrilas/farmacocinética , Acetonitrilas/farmacologia , Alcaloides/química , Alcaloides/farmacocinética , Alcaloides/farmacologia , Animais , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacocinética , Materiais Biomiméticos/farmacologia , Linhagem Celular Tumoral , Cicloexenos/química , Cicloexenos/farmacocinética , Cicloexenos/farmacologia , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células MCF-7 , Miócitos Cardíacos/citologia
12.
Mar Drugs ; 18(2)2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-32092907

RESUMO

Structure-based tissue engineering requires large-scale 3D cell/tissue manufacture technologies, to produce biologically active scaffolds. Special attention is currently paid to naturally pre-designed scaffolds found in skeletons of marine sponges, which represent a renewable resource of biomaterials. Here, an innovative approach to the production of mineralized scaffolds of natural origin is proposed. For the first time, a method to obtain calcium carbonate deposition ex vivo, using living mollusks hemolymph and a marine-sponge-derived template, is specifically described. For this purpose, the marine sponge Aplysin aarcheri and the terrestrial snail Cornu aspersum were selected as appropriate 3D chitinous scaffold and as hemolymph donor, respectively. The formation of calcium-based phase on the surface of chitinous matrix after its immersion into hemolymph was confirmed by Alizarin Red staining. A direct role of mollusks hemocytes is proposed in the creation of fine-tuned microenvironment necessary for calcification ex vivo. The X-ray diffraction pattern of the sample showed a high CaCO3 amorphous content. Raman spectroscopy evidenced also a crystalline component, with spectra corresponding to biogenic calcite. This study resulted in the development of a new biomimetic product based on ex vivo synthetized ACC and calcite tightly bound to the surface of 3D sponge chitin structure.


Assuntos
Quitina/análogos & derivados , Quitina/química , Hemolinfa/metabolismo , Poríferos/metabolismo , Caramujos/metabolismo , Alicerces Teciduais , Animais , Biomineralização , Carbonato de Cálcio/química , Difração de Raios X
13.
Angew Chem Int Ed Engl ; 58(35): 12010-12013, 2019 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-31268220

RESUMO

Hoogsteen DNA base pairs (bps) are an alternative base pairing to canonical Watson-Crick bps and are thought to play important biochemical roles. Hoogsteen bps have been reported in a handful of X-ray structures of protein-DNA complexes. However, there are several examples of Hoogsteen bps in crystal structures that form Watson-Crick bps when examined under solution conditions. Furthermore, Hoogsteen bps can sometimes be difficult to resolve in DNA:protein complexes by X-ray crystallography due to ambiguous electron density and by solution-state NMR spectroscopy due to size limitations. Here, using infrared spectroscopy, we report the first direct solution-state observation of a Hoogsteen (G-C+ ) bp in a DNA:protein complex under solution conditions with specific application to DNA-bound TATA-box binding protein. These results support a previous assignment of a G-C+ Hoogsteen bp in the complex, and indicate that Hoogsteen bps do indeed exist under solution conditions in DNA:protein complexes.


Assuntos
Citosina/química , DNA/metabolismo , Guanina/química , Proteína de Ligação a TATA-Box/metabolismo , Pareamento de Bases , Cristalografia por Raios X , DNA/química , Conformação de Ácido Nucleico , Espectrofotometria Infravermelho , Proteína de Ligação a TATA-Box/química
14.
Biochemistry ; 58(15): 1963-1974, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30950607

RESUMO

A( syn)-T and G( syn)-C+ Hoogsteen base pairs in protein-bound DNA duplexes can be difficult to resolve by X-ray crystallography due to ambiguous electron density and by nuclear magnetic resonance (NMR) spectroscopy due to poor chemical shift dispersion and size limitations with solution-state NMR spectroscopy. Here we describe an NMR strategy for characterizing Hoogsteen base pairs in protein-DNA complexes, which relies on site-specifically incorporating 13C- and 15N-labeled nucleotides into DNA duplexes for unambiguous resonance assignment and to improve spectral resolution. The approach was used to resolve the conformation of an A-T base pair in a crystal structure of an ∼43 kDa complex between a 34 bp duplex DNA and the integration host factor (IHF) protein. In the crystal structure (Protein Data Bank entry 1IHF ), this base pair adopts an unusual Hoogsteen conformation with a distorted sugar backbone that is accommodated by a nearby nick used to aid in crystallization. The NMR chemical shifts and interproton nuclear Overhauser effects indicate that this base pair predominantly adopts a Watson-Crick conformation in the intact DNA-IHF complex under solution conditions. Consistent with these NMR findings, substitution of 7-deazaadenine at this base pair resulted in only a small (∼2-fold) decrease in the IHF-DNA binding affinity. The NMR strategy provides a new approach for resolving crystallographic ambiguity and more generally for studying the structure and dynamics of protein-DNA complexes in solution.


Assuntos
Pareamento de Bases , Proteínas de Ligação a DNA/química , DNA/química , Substâncias Macromoleculares/química , Espectroscopia de Ressonância Magnética/métodos , Conformação de Ácido Nucleico , Sequência de Bases , Isótopos de Carbono/metabolismo , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Substâncias Macromoleculares/metabolismo , Modelos Moleculares , Estrutura Molecular , Isótopos de Nitrogênio/metabolismo , Nucleotídeos/química , Nucleotídeos/genética , Nucleotídeos/metabolismo , Domínios Proteicos
15.
FEBS Lett ; 591(12): 1770-1784, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28524232

RESUMO

Noncanonical G-C+ and A-T Hoogsteen base pairs can form in duplex DNA and play roles in recognition, damage repair, and replication. Identifying Hoogsteen base pairs in DNA duplexes remains challenging due to difficulties in resolving syn versus antipurine bases with X-ray crystallography; and size limitations and line broadening can make them difficult to characterize by NMR spectroscopy. Here, we show how infrared (IR) spectroscopy can identify G-C+ and A-T Hoogsteen base pairs in duplex DNA across a range of different structural contexts. The utility of IR-based detection of Hoogsteen base pairs is demonstrated by characterizing the first example of adjacent A-T and G-C+ Hoogsteen base pairs in a DNA duplex where severe broadening complicates detection with NMR.


Assuntos
Pareamento de Bases , DNA/química , Modelos Moleculares , Adenina/análogos & derivados , Adenina/química , Adenina/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Pareamento de Bases/efeitos dos fármacos , Sítios de Ligação , Instabilidade Cromossômica/efeitos dos fármacos , Dicroísmo Circular , DNA/metabolismo , Equinomicina/química , Equinomicina/metabolismo , Equinomicina/farmacologia , Estudos de Viabilidade , Guanina/análogos & derivados , Guanina/química , Guanina/metabolismo , Ligação de Hidrogênio/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico/efeitos dos fármacos , Espectrofotometria , Espectrofotometria Infravermelho , Espectroscopia de Infravermelho com Transformada de Fourier , Estereoisomerismo
16.
Int J Biol Macromol ; 104(Pt B): 1955-1965, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28365291

RESUMO

The extraordinary biocompatibility and mechanical properties of chitinous scaffolds from marine sponges endows these structures with unique properties that render them ideal for diverse biomedical applications. In the present work, a technological route to produce "ready-to-use" tissue-engineered products based on poriferan chitin is comprehensively investigated for the first time. Three key stages included isolation of scaffolds from the marine demosponge Ianthella basta, confirmation of their biocompatibility with human mesenchymal stromal cells, and cryopreservation of the tissue-like structures grown within these scaffolds using a slow cooling protocol. Biocompatibility of the macroporous, flat chitin scaffolds has been confirmed by cell attachment, high cell viability and the ability to differentiate into the adipogenic lineage. The viability of cells cryopreserved on chitin scaffolds was reduced by about 30% as compared to cells cryopreserved in suspension. However, the surviving cells were able to retain their differentiation potential; and this is demonstrated for the adipogenic lineage. The results suggest that chitin from the marine demosponge I. basta is a promising, highly biocompatible biomaterial for stem cell-based tissue-engineering applications.


Assuntos
Materiais Biocompatíveis , Quitina , Células-Tronco Mesenquimais/citologia , Poríferos , Engenharia Tecidual , Alicerces Teciduais , Adipogenia , Animais , Materiais Biocompatíveis/química , Diferenciação Celular , Quitina/química , Criopreservação , Humanos , Teste de Materiais , Poríferos/química , Espectroscopia de Infravermelho com Transformada de Fourier , Engenharia Tecidual/métodos
17.
Int J Biol Macromol ; 104(Pt B): 1966-1974, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28347785

RESUMO

The recently discovered chitin-based scaffolds derived from poriferans have the necessary prosperities for potential use in tissue engineering. Among the various demosponges of the Verongida order, Aplysina aerophoba is an attractive target for more in-depth investigations, as it is a renewable source of unique 3D microporous chitinous scaffolds. We found these chitinous scaffolds were cytocompatible and supported attachment, growth and proliferation of human mesenchymal stromal cells (hMSCs) in vitro. Cultivation of hMSCs on the scaffolds for 7days resulted in a two-fold increase in their metabolic activity, indicating increased cell numbers. Cells cultured onto chitin scaffolds in differentiation media were able to differentiate into the chondrogenic, adipogenic and osteogenic lineages, respectively. These results indicate A. aerophoba is a novel source of chitin scaffolds to futher hMSCs-based tissue engineering strategies.


Assuntos
Quitina , Células-Tronco Mesenquimais/citologia , Poríferos , Engenharia Tecidual , Alicerces Teciduais , Adipogenia , Animais , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Quitina/química , Condrogênese , Humanos , Células-Tronco Mesenquimais/ultraestrutura , Osteogênese , Poríferos/química , Engenharia Tecidual/métodos
18.
Int J Biol Macromol ; 104(Pt B): 1626-1632, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28119189

RESUMO

Chitinous scaffolds isolated from marine demosponge Ianthella basta represent novel templates for deposition of metals such as copper and copper oxides. In contrast to traditional Extreme Biomimetics methods which are based on high temperature reactions, here, we propose an alternative way based on a well-known process - electrochemical deposition or plating. This method allows production of 3D composite materials with metallic and metal oxide structures within their surfaces. For the first time chitinous scaffolds of poriferan origin, which possess a 3D network structure, were used for the copper plating. The nanocrystallites of metallic phase obtained on chitinous fibres represents replicas of the original nanofibrous substrate.


Assuntos
Quitina/química , Cobre/química , Poríferos/química , Animais , Materiais Biomiméticos/química , Eletroquímica , Nanopartículas/química , Propriedades de Superfície
19.
Biomimetics (Basel) ; 2(2)2017 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-31105167

RESUMO

The aim of extreme biomimetics is to design a bridge between extreme biomineralization and bioinspired materials chemistry, where the basic principle is to exploit chemically and thermally stable, renewable biopolymers for the development of the next generation of biologically inspired advanced and functional composite materials. This study reports for the first time the use of proteinaceous spongin-based scaffolds isolated from marine demosponge Hippospongia communis as a three-dimensional (3D) template for the hydrothermal deposition of crystalline titanium dioxide. Scanning electron microscopy (SEM) assisted with energy dispersive X-ray spectroscopy (EDS) mapping, low temperature nitrogen sorption, thermogravimetric (TG) analysis, X-ray diffraction spectroscopy (XRD), and attenuated total reflectance⁻Fourier transform infrared (ATR⁻FTIR) spectroscopy are used as characterization techniques. It was found that, after hydrothermal treatment crystalline titania in anatase form is obtained, which forms a coating around spongin microfibers through interaction with negatively charged functional groups of the structural protein as well as via hydrogen bonding. The material was tested as a potential heterogeneous photocatalyst for removal of C.I. Basic Blue 9 dye under UV irradiation. The obtained 3D composite material shows a high efficiency of dye removal through both adsorption and photocatalysis.

20.
Int J Biol Macromol ; 78: 224-9, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25889055

RESUMO

Chitinous scaffolds isolated from the skeleton of marine sponge Aplysina cauliformis were used as a template for the deposition of polyhedral oligomeric silsesquioxanes (POSS). These chitin-POSS based composites with hydrophobic properties were prepared for the first time using solvothermal synthesis (pH 3, temp 80 °C), and were thoroughly characterized. The resulting material was studied using scanning electron microscopy, Raman spectroscopy, X-ray photoelectron spectroscopy and thermogravimetry. A mechanism for the chitin-POSS interaction after exposure to these solvothermal conditions is proposed and discussed.


Assuntos
Quitina/química , Nanocompostos/química , Compostos de Organossilício/química , Microscopia Eletrônica de Varredura , Análise Espectral Raman , Termogravimetria
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