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1.
J Cancer Res Ther ; 16(3): 440-444, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32719248

RESUMO

Introduction: Crystallization test is based on the principle that, when a salt crystallizes out of an aqueous solution, the crystal growth is influenced by the presence of other substances in the solution, such as blood or plant extracts. If a mixture of copper chloride solution with a small amount of whole blood is allowed to crystallize under controlled experimental conditions, an aggregate of crystals forms. Crystallization method can be used as a diagnostic aid to provide information about the systemic conditions and general health of the patient. Aim: This study aims to study the patterns of crystallization and to further determine the efficacy of crystallization test as a screening modality in premalignant lesions and oral squamous cell carcinoma (OSCC). Materials and Methods: Fifty patients of OSCC, 50 patients of premalignant lesions, and 50 healthy individuals were selected. One drop of blood was collected from the study groups to perform crystallization using cupric chloride. Statistical Analysis Used: Statistical analysis was performed using Chi-square test, Student's t-test (two-tailed), and analysis of variance. Results: The different patterns of crystals formed were studied and statistically analyzed. Conclusion: Based on the study, it was concluded that Crystallization test can be used as an effective screening modality for detection of premalignant lesions and OSCC.


Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/diagnóstico , Cobre/química , Cristalização/métodos , Leucoplasia/sangue , Neoplasias Bucais/diagnóstico , Lesões Pré-Cancerosas/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/química , Estudos de Casos e Controles , Feminino , Humanos , Leucoplasia/patologia , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Neoplasias Bucais/sangue , Neoplasias Bucais/química , Estadiamento de Neoplasias , Lesões Pré-Cancerosas/sangue , Adulto Jovem
2.
J Vis Exp ; (157)2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-32225143

RESUMO

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice of detergent is critical, because it affects both the stability and monodispersity of the complex. We recently determined the crystal structure of an active state of bovine rhodopsin coupled to an engineered G protein, mini-Go, at 3.1 Å resolution. Here, we detail the procedure for optimizing the preparation of the rhodopsin-mini-Go complex. Dark-state rhodopsin was prepared in classical and neopentyl glycol (NPG) detergents, followed by complex formation with mini-Go under light exposure. The stability of the rhodopsin was assessed by ultraviolet-visible (UV-VIS) spectroscopy, which monitors the reconstitution into rhodopsin of the light-sensitive ligand, 9-cis retinal. Automated size-exclusion chromatography (SEC) was used to characterize the monodispersity of rhodopsin and the rhodopsin-mini-Go complex. SDS-polyacrylamide electrophoresis (SDS-PAGE) confirmed the formation of the complex by identifying a 1:1 molar ratio between rhodopsin and mini-Go after staining the gel with Coomassie blue. After cross-validating all this analytical data, we eliminated unsuitable detergents and continued with the best candidate detergent for large-scale preparation and crystallization. An additional problem arose from the heterogeneity of N-glycosylation. Heterologously-expressed rhodopsin was observed on SDS-PAGE to have two different N-glycosylated populations, which would probably have hindered crystallogenesis. Therefore, different deglycosylation enzymes were tested, and endoglycosidase F1 (EndoF1) produced rhodopsin with a single species of N-glycosylation. With this strategic pipeline for characterizing protein quality, preparation of the rhodopsin-mini-Go complex was optimized to deliver the crystal structure. This was only the third crystal structure of a GPCR-G protein signaling complex. This approach can also be generalized for other membrane proteins and their complexes to facilitate sample preparation and structure determination.


Assuntos
Cristalização/métodos , Proteínas de Ligação ao GTP/metabolismo , Transdução de Sinais
3.
J Vis Exp ; (156)2020 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-32150153

RESUMO

Membrane separation has drawn attention as a novel-energy saving separation process. Zeolite membranes have great potential for hydrocarbon separation in petroleum and petrochemical fields because of their high thermal, chemical, and mechanical strength. A *BEA-type zeolite is an interesting membrane material because of its large pore size and wide Si/Al range. This manuscript presents a protocol for *BEA membrane preparation by a secondary growth method that does not use an organic structure-directing agent (OSDA). The preparation protocol consists of four steps: pretreatment of support, seed preparation, dip-coating, and membrane crystallization. First, the *BEA seed crystal is prepared by conventional hydrothermal synthesis using OSDA. The synthesized seed crystal is loaded on the outer surface of a 3 cm long tubular α-Al2O3 support by a dip-coating method. The loaded seed layer is prepared with the secondary growth method using a hydrothermal treatment at 393 K for 7 days without using OSDA. A *BEA membrane having very few defects is successfully obtained. The seed preparation and dip-coating steps strongly affect the membrane quality.


Assuntos
Cristalização/métodos , Membranas Artificiais , Zeolitas/síntese química , Óxido de Alumínio/química , Temperatura Alta , Porosidade , Zeolitas/química
4.
Nat Commun ; 11(1): 351, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31953407

RESUMO

Despite advances in directing the assembly of biomacromolecules into well-defined nanostructures, leveraging pathway complexity of molecular disorder to order transition while bridging materials fabrication from nano- to macroscale remains a challenge. Here, we present templated crystallization of structural proteins to nanofabricate hierarchically structured materials up to centimeter scale, using silk fibroin as an example. The process involves the use of ordered peptide supramolecular assemblies as templates to direct the folding and assembly of silk fibroin into nanofibrillar structures. Silk polymorphs can be engineered by varying the peptide seeds used. Modulation of the relative concentration between silk fibroin and peptide seeds, silk fibroin molecular weight and pH allows control over nanofibrils morphologies and mechanical properties. Finally, facile integration of the bottom-up templated crystallization with emerging top-down techniques enables the generation of macroscopic nanostructured materials with potential applications in information storage/encryption, surface functionalization, and printable three-dimensional constructs of customized architecture and controlled anisotropy.


Assuntos
Fibroínas/química , Nanofibras/química , Nanoestruturas/química , Peptídeos/química , Materiais Biocompatíveis/química , Cristalização/métodos , Cinética , Micelas , Impressão Tridimensional , Dobramento de Proteína , Seda/química , Engenharia Tecidual/métodos
5.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 1): 20-24, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31929182

RESUMO

Eisenia hydrolysis-enhancing protein (EHEP), which is a novel protein that has been identified in Aplysia kurodai, protects ß-glucosidases from phlorotannin inhibition to facilitate the production of glucose from the laminarin abundant in brown algae. Hence, EHEP has attracted attention for its potential applications in producing biofuel from brown algae. In this study, EHEP was purified from the natural digestive fluid of A. kurodai and was crystallized using the sitting-drop vapor-diffusion method. Native and SAD (single-wavelength anomalous diffraction) data sets were successfully collected at resolutions of 1.20 and 2.48 Šusing wavelengths of 1.0 and 2.1 Å, respectively, from crystals obtained in initial screening. The crystals belonged to space group P212121 and contained one EHEP molecule in the asymmetric unit. All 20 S-atom sites in EHEP were located and the phases were determined by the SAD method using the S atoms in the natural protein as anomalous scatterers (native-SAD). After phase improvement, interpretable electron densities were obtained and 58% of the model was automatically built.


Assuntos
Aplysia/química , Cristalização/métodos , Proteínas/química , Animais , Aplysia/enzimologia , Aplysia/genética , Aplysia/metabolismo , Cristalografia por Raios X , Hidrólise , Espectrometria de Massas , Modelos Moleculares , Conformação Proteica , Domínios Proteicos/genética , Proteínas/isolamento & purificação
6.
Int J Mol Sci ; 21(1)2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31906428

RESUMO

Although prevalent in the determination of protein structures; crystallography always has the bottleneck of obtaining high-quality protein crystals for characterizing a wide range of proteins; especially large protein complexes. Stable fragments or domains of proteins are more readily to crystallize; which prompts the use of in situ proteolysis to remove flexible or unstable structures for improving crystallization and crystal quality. In this work; we investigated the effects of in situ proteolysis by chymotrypsin on the crystallization of the XcpVWX complex from the Type II secretion system of Pseudomonas aeruginosa. Different proteolysis conditions were found to result in two distinct lattices in the same crystallization solution. With a shorter chymotrypsin digestion at a lower concentration; the crystals exhibited a P3 hexagonal lattice that accommodates three complex molecules in one asymmetric unit. By contrast; a longer digestion with chymotrypsin of a 10-fold higher concentration facilitated the formation of a compact P212121 orthorhombic lattice with only one complex molecule in each asymmetric unit. The molecules in the hexagonal lattice have shown high atomic displacement parameter values compared with the ones in the orthorhombic lattice. Taken together; our results clearly demonstrate that different proteolysis conditions can result in the generation of distinct lattices in the same crystallization solution; which can be exploited in order to obtain different crystal forms of a better quality.


Assuntos
Quimotripsina , Cristalização/métodos , Complexos Multiproteicos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Cristalografia por Raios X , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Complexos Multiproteicos/isolamento & purificação , Conformação Proteica , Proteólise , Sistemas de Secreção Tipo II/química
7.
Pharm Dev Technol ; 25(1): 40-53, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31524550

RESUMO

Dual drug nanocrystals loaded nano embedded microparticles (DNEMs) were prepared for fixed dose combination of simvastatin (SIM) and ezetimibe (EZE) using NanoCrySP technology. The purpose was to generate nanonized SIM and EZE dispersed in matrix of single crystallization inducing excipient and investigate their in vitro performance. DNEM were prepared using mannitol (MAN) as crystallization inducer (active pharmaceutical ingredients (APIs)/MAN = 3:7 w/w) using spray drying. TPGS (0.1% w/v) was used as surfactant for stabilization of nanocrystals. Crystallinity of DNEM was confirmed by solid-state characterization using DSC and PXRD. Particle size analysis was carried out using Zetasizer® and the Scherrer equation as primary techniques and SEM and TEM as orthogonal techniques. Size of both SIM and EZE in DNEM was close to 600 nm. In vitro performance was assessed using USP apparatus II in 0.025% SLS containing sodium phosphate buffer. Powder dissolution of DNEM increased 1.45 times for SIM and 1.65 times for EZE as compared to their physical mixture in discriminatory medium. MAN did not plasticize SIM or EZE by virtue of its immiscibility with the two drugs. However, MAN helped in inducing crystallization via heterogeneous nucleation. The generated DNEM were stable in terms of assay, polymorphic form and dissolution for 90 days of accelerated storage at 40 °C/75% RH.


Assuntos
Ezetimiba/química , Nanopartículas/química , Sinvastatina/química , Anticolesterolemiantes/química , Química Farmacêutica/métodos , Cristalização/métodos , Dessecação/métodos , Excipientes/química , Manitol/química , Solubilidade/efeitos dos fármacos
8.
J Pharm Biomed Anal ; 178: 112937, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31679845

RESUMO

Amorphous solid dispersions (ASDs) are single-phase amorphous systems, where drug molecules are molecularly dispersed (dissolved) in a polymer matrix. The molecular dispersion of the drug molecules is responsible for their improved dissolution properties. Unambiguously establishing the phase behavior of the ASDs is of utmost importance. In this paper, we focused on the complementary nature of (modulated) differential scanning calorimetry ((m)DSC) and X-ray powder diffraction (XRPD) to elucidate the phase behavior of ASDs as demonstrated by a critical discussion of practical real-life examples observed in our research group. The ASDs were manufactured by either applying a solvent-based technique (spray drying), a heat-based technique (hot melt extrusion) or mechanochemical activation (cryo-milling). The encountered limiting factors of XRPD were the lack of sensitivity for small traces of crystallinity, the impossibility to differentiate between distinct amorphous phases and its impossibility to detect nanocrystals in a polymer matrix. In addition, the limiting factors of (m)DSC were defined as the well-described heat-induced sample alteration upon heating, the interfering of residual solvent evaporation with other thermal events and the coinciding of enthalpy recovery with melting events. In all of these cases, the application of a single analytical technique would have led to erroneous conclusions, whilst the combination of (m)DSC and XRPD elucidated the true phases of the ASD.


Assuntos
Polímeros/química , Pós/química , Varredura Diferencial de Calorimetria/métodos , Química Farmacêutica/métodos , Cristalização/métodos , Temperatura Alta , Nanopartículas/química , Sensibilidade e Especificidade , Solubilidade/efeitos dos fármacos , Solventes/química , Tecnologia Farmacêutica/métodos , Difração de Raios X/métodos
9.
Acta Crystallogr D Struct Biol ; 75(Pt 12): 1040-1050, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31793898

RESUMO

In contrast to twinning by merohedry, the reciprocal lattices of the different domains of non-merohedral twins do not overlap exactly. This leads to three kinds of reflections: reflections with no overlap, reflections with an exact overlap and reflections with a partial overlap of a reflection from a second domain. This complicates the unit-cell determination, indexing, data integration and scaling of X-ray diffraction data. However, with hindsight it is possible to detwin the data because there are reflections that are not affected by the twinning. In this article, the successful solution and refinement of one mineral, one organometallic and two protein non-merohedral twins using a common strategy are described. The unit-cell constants and the orientation matrices were determined by the program CELL_NOW. The data were then integrated with SAINT. TWINABS was used for scaling, empirical absorption corrections and the generation of two different data files, one with detwinned data for structure solution and refinement and a second one for (usually more accurate) structure refinement against total integrated intensities. The structures were solved by experimental phasing using SHELXT for the first two structures and SHELXC/D/E for the two protein structures; all models were refined with SHELXL.


Assuntos
Aldose-Cetose Isomerases/química , Cristalização/métodos , Cristalografia por Raios X/métodos , Insulina Regular de Porco/química , Minerais/química , Modelos Moleculares
10.
Acta Crystallogr D Struct Biol ; 75(Pt 12): 1071-1083, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31793901

RESUMO

Afamin, which is a human blood plasma glycoprotein, a putative multifunctional transporter of hydrophobic molecules and a marker for metabolic syndrome, poses multiple challenges for crystallographic structure determination, both practically and in analysis of the models. Several hundred crystals were analysed, and an unusual variability in cell volume and difficulty in solving the structure despite an ∼34% sequence identity with nonglycosylated human serum albumin indicated that the molecule exhibits variable and context-sensitive packing, despite the simplified glycosylation in insect cell-expressed recombinant afamin. Controlled dehydration of the crystals was able to stabilize the orthorhombic crystal form, reducing the number of molecules in the asymmetric unit from the monoclinic form and changing the conformational state of the protein. An iterative strategy using fully automatic experiments available on MASSIF-1 was used to quickly determine the optimal protocol to achieve the phase transition, which should be readily applicable to many types of sample. The study also highlights the drawback of using a single crystallographic structure model for computational modelling purposes given that the conformational state of the binding sites and the electron density in the binding site, which is likely to result from PEGs, greatly varies between models. This also holds for the analysis of nonspecific low-affinity ligands, where often a variety of fragments with similar uncertainty can be modelled, inviting interpretative bias. As a promiscuous transporter, afamin also seems to bind gadoteridol, a magnetic resonance imaging contrast compound, in at least two sites. One pair of gadoteridol molecules is located near the human albumin Sudlow site, and a second gadoteridol molecule is located at an intermolecular site in proximity to domain IA. The data from the co-crystals support modern metrics of data quality in the context of the information that can be gleaned from data sets that would be abandoned on classical measures.


Assuntos
Proteínas de Transporte/química , Cristalização/métodos , Dessecação/métodos , Glicoproteínas/química , Albumina Sérica Humana/química , Sítios de Ligação , Gadolínio/química , Compostos Heterocíclicos/química , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Compostos Organometálicos/química , Conformação Proteica
11.
Acta Crystallogr D Struct Biol ; 75(Pt 12): 1084-1095, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31793902

RESUMO

Although often presented as taking single `snapshots' of the conformation of a protein, X-ray crystallography provides an averaged structure over time and space within the crystal. The important but difficult task of characterizing structural ensembles in crystals is typically limited to small conformational changes, such as multiple side-chain conformations. A crystallographic method was recently introduced that utilizes residual electron and anomalous density (READ) to characterize structural ensembles encompassing large-scale structural changes. Key to this method is an ability to accurately measure anomalous signals and distinguish them from noise or other anomalous scatterers. This report presents an optimized data-collection and analysis strategy for partially occupied iodine anomalous signals. Using the long-wavelength-optimized beamline I23 at Diamond Light Source, the ability to accurately distinguish the positions of anomalous scatterers with occupancies as low as ∼12% is demonstrated. The number and positions of these anomalous scatterers are consistent with previous biophysical, kinetic and structural data that suggest that the protein Im7 binds to the chaperone Spy in multiple partially occupied conformations. Finally, READ selections demonstrate that re-measured data using the new protocols are consistent with the previously characterized structural ensemble of the chaperone Spy with its client Im7. This study shows that a long-wavelength beamline results in easily validated anomalous signals that are strong enough to be used to detect and characterize highly disordered sections of crystal structures.


Assuntos
Proteínas de Transporte/química , Cristalização/métodos , Cristalografia por Raios X/métodos , Proteínas de Escherichia coli/química , Chaperonas Moleculares/química , Proteínas Periplásmicas/química , Cinética , Modelos Moleculares , Conformação Proteica
12.
AAPS PharmSciTech ; 21(1): 10, 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31802267

RESUMO

Crystal engineering approach was utilized for the development of different multicomponent solid forms of telmisartan (TEL) to improve its oral bioavailability. In this context, two cocrystals, gentisic acid (GA) and maleic acid (MA), while two eutectic mixtures, para-aminobenzoic acid (PABA) and adipic acid (AA), were successfully prepared and characterized by different analytical tools. Both the cocrystals exhibited characteristic heterosynthons, viz. OHacid⋯Narom and OHacid⋯O, to propagate new network. Structural features of coformers has been correlated with the outcomes of cocrystallization approach. Coformers having auxiliary functionality in addition to complementary functional groups have high propensity to generate cocrystals. However, multicomponent where auxiliary functionality is lacking, such combinations, is shown to form eutectic mixtures owing to strong homomeric interaction. Besides, the developed cocrystals and eutectic mixtures showed higher aqueous solubility (3-5.5-fold) and intrinsic dissolution rate (1-2.6-fold) over pure TEL. In vivo studies also revealed significant improvement in relative bioavailability (2-2.6-fold). The study also shed light on the implications of eutectic mixtures in mitigating the solubility issues of drugs which are often considered negative results of cocrystallization strategy.


Assuntos
Produtos Biológicos/síntese química , Produtos Biológicos/farmacocinética , Telmisartan/síntese química , Telmisartan/farmacocinética , Animais , Anti-Hipertensivos/síntese química , Anti-Hipertensivos/farmacocinética , Disponibilidade Biológica , Cristalização/métodos , Masculino , Ratos , Ratos Wistar , Solubilidade , Relação Estrutura-Atividade
13.
AAPS PharmSciTech ; 21(1): 16, 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31807963

RESUMO

Triclabendazole belongs to the class II/IV of the Biopharmaceuticals Classification System, and its low aqueous solubility represents a major drawback during the development of effective dosage forms. Therefore, the goal of this study was to elucidate whether polymeric solid dispersions would represent a suitable approach to overcome such disadvantage. Due to the lack of information on triclabendazole release, four different dissolution media were evaluated to analyze drug dissolution rate. The polymeric solid dispersions were characterized by X-ray diffraction and Fourier transform infrared spectroscopy. The selected final formulations were further stored for 24 months, and their physical stability was evaluated by means of X-ray diffraction and drug dissolution assays. Drug solubility studies indicated that poloxamer 407 (P407) solubilized a higher amount of drug than polyethylene glycol 6000. Drug-to-carrier ratio, nature of the selected carriers, and the type of dissolution media were important factors for increasing dissolution. By infrared spectroscopy, there were no specific interactions between the drug and polymers. The physicochemical characterization of the systems showed a detectable evidence of drug amorphization by increasing the carrier ratio. Micromeritic studies indicated that raw triclabendazole, physical mixtures, and reference formulation showed poor flow properties, in contrast to the triclabendazole:P407 solid dispersion sample. Both the crystalline properties and dissolution rate of selected samples were very similar after 24 months at room temperature. Thus, considering physical stability and dissolution studies, the development of the solid dispersion is a very suitable methodology to improve triclabendazole dissolution and, potentially, its biopharmaceutical performance.


Assuntos
Antiplatelmínticos/química , Sistemas de Liberação de Medicamentos/métodos , Triclabendazol/química , Administração Oral , Antiplatelmínticos/administração & dosagem , Varredura Diferencial de Calorimetria/métodos , Química Farmacêutica/métodos , Cristalização/métodos , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Solubilidade , Espectrofotometria Infravermelho/métodos , Triclabendazol/administração & dosagem , Difração de Raios X/métodos
14.
Pak J Pharm Sci ; 32(5): 2139-2147, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31813880

RESUMO

Different polymorphic forms can affect the performance of the drug product. In addition, isomorphic crystals show different chemical and physical properties due to the changes in the crystal habit. However, it is unclear whether the crystal habit results in different pharmacological activity. The aim of this study was to investigate whether the pharmacological effect of ibuprofen could be affected due to the variety of the crystal habit. Solvent change technique and conventional fusion method were carried out to modify the characteristics of ibuprofen. The physicochemical properties of each were investigated using powder X-ray diffraction (PXRD), Fourier transform infrared (FT-IR) spectroscopy and differential scanning calorimetry (DSC) techniques. Results of scanning electron microscopy (SEM) analysis revealed differences in the surface characteristics of the crystals obtained. Further study revealed that the samples crystallized exhibited the remarkable variation on the dissolution profiles in different dissolution medium. Moreover, in vivo antinociceptive and anti-inflammatory findings demonstrated that the crystal habit modifications resulted in the different therapeutic efficacy. Taken together, these results indicate that the modification of the crystal habit had a great influence on the in vivo pharmacological activity of ibuprofen crystals.


Assuntos
Ibuprofeno/química , Ibuprofeno/farmacologia , Analgésicos/química , Analgésicos/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Varredura Diferencial de Calorimetria/métodos , Química Farmacêutica/métodos , Cristalização/métodos , Camundongos , Camundongos Endogâmicos ICR , Microscopia Eletrônica de Varredura/métodos , Tamanho da Partícula , Pós/química , Pós/farmacologia , Ratos , Ratos Wistar , Solubilidade/efeitos dos fármacos , Solventes/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Difração de Raios X/métodos
15.
Pharm Res ; 36(12): 183, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31741058

RESUMO

Research conducted in microgravity conditions has the potential to yield new therapeutics, as advances can be achieved in the absence of phenomena such as sedimentation, hydrostatic pressure and thermally-induced convection. The outcomes of such studies can significantly contribute to many scientific and technological fields, including drug discovery. This article reviews the existing traditional microgravity platforms as well as emerging ideas for enabling microgravity research focusing on SpacePharma's innovative autonomous remote-controlled microgravity labs that can be launched to space aboard nanosatellites to perform drug research in orbit. The scientific literature is reviewed and examples of life science fields that have benefited from studies in microgravity conditions are given. These include the use of microgravity environment for chemical applications (protein crystallization, drug polymorphism, self-assembly of biomolecules), pharmaceutical studies (microencapsulation, drug delivery systems, behavior and stability of colloidal formulations, antibiotic drug resistance), and biological research, including accelerated models for aging, investigation of bacterial virulence , tissue engineering using organ-on-chips in space, enhanced stem cells proliferation and differentiation.


Assuntos
Simulação de Ausência de Peso/instrumentação , Simulação de Ausência de Peso/métodos , Ausência de Peso , Fatores Etários , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Cristalização/instrumentação , Cristalização/métodos , Dimerização , Composição de Medicamentos/instrumentação , Composição de Medicamentos/métodos , Sistemas de Liberação de Medicamentos/instrumentação , Sistemas de Liberação de Medicamentos/métodos , Descoberta de Drogas/instrumentação , Descoberta de Drogas/métodos , Resistência Microbiana a Medicamentos , Humanos , Microfluídica/instrumentação , Microfluídica/métodos , Pesquisa Farmacêutica/instrumentação , Pesquisa Farmacêutica/métodos , Fenômenos Físicos , Proteínas/química , Voo Espacial , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos
16.
Eur J Pharm Biopharm ; 145: 98-112, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31698042

RESUMO

The present study evaluates the physical stability and intermolecular interactions of Rivaroxaban (RXB) amorphous solid dispersions (ASDs) in polymeric carriers via thermodynamic modelling and molecular simulations. Specifically, the Flory-Huggins (FH) lattice solution theory was used to construct thermodynamic phase diagrams of RXB ASDs in four commonly used polymeric carriers (i.e. copovidone, coPVP, povidone, PVP, Soluplus, SOL and hypromellose acetate succinate, HPMCAS), which were stored under 0%, 60% and 75% relative humidity (RH) conditions. In order to verify the phase boundaries predicted by FH modelling (i.e. truly amorphous zone, amorphous-amorphous demixing zones and amorphous-API recrystallization zones), samples of ASDs were examined via polarized light microscopy after storage for up to six months at various RH conditions. Results showed a good agreement between the theoretical and the experimental approaches (i.e. coPVP and PVP resulted in less physically-stable ASDs compared to SOL and HPMCAS) indicating that the proposed FH-based modelling may be a useful tool in predicting long-term physical stability in high humidity conditions. In addition, molecular dynamics (MD) simulations were employed in order to interpret the observed differences in physical stability. Results, which were verified via differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR), suggested the formation of similar intermolecular interactions in all cases, indicating that the interaction with moisture water plays a more crucial role in ASD physical stability compared to the formation of intermolecular interactions between ASD components.


Assuntos
Polímeros/química , Rivaroxabana/química , Varredura Diferencial de Calorimetria/métodos , Cristalização/métodos , Portadores de Fármacos/química , Composição de Medicamentos/métodos , Estabilidade de Medicamentos , Umidade , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Termodinâmica
17.
Artigo em Inglês | MEDLINE | ID: mdl-31752321

RESUMO

The circulating pellet fluidized bed (CPFB) softening method is a highly efficient and environmentally friendly softening technology that can be used to reduce water hardness during the pretreatment process of circulating water in thermal power plants. The performance of chemical crystallization CPFB reactors was tested for increasing the concentration ratio and softening the circulating water in a thermal power plant in Dingzhou, Hebei. The results show that usage of CPFB reactors removed water hardness and Ca2+ ions with efficiencies exceeding 60% and 90%, respectively. The size of the particles discharged from the reactors was approximately 1-3 mm, and the content of CaO in these particles was found to be greater than 50%. All the discharged particles were reused in the desulfurization system in the power plant. The operational cost of the CPFB system is US$0.074 per cubic meter of water. After adopting the proposed CPFB softening method in the Dingzhou Power Plant, the concentration ratio of the circulation cooling water was increased from 4.5 to more than 9. In addition, the amount of replenished water and sewage discharge were both reduced by 150 m3/h, and the amount of scale inhibitor used in the system was reduced by more than 30%. These improvements contribute to approximately US$200,000 in annual savings in the power plant. In summary, the CPFB softening method demonstrated a high hardness removal rate, strong economic benefits, and remarkable environmental and social benefits. Therefore, this method seems ideal for softening replenished circulating cooling water, increasing the concentration ratio of the water and achieving zero liquid discharge (ZLD) in thermal power plants.


Assuntos
Cristalização/métodos , Centrais Elétricas , Esgotos/química , Águas Residuárias/química , Purificação da Água/métodos , Abrandamento da Água/métodos , Dureza
18.
Int J Pharm ; 572: 118776, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31678374

RESUMO

A comprehensive cocrystal study for the insoluble natural pharmaceutical compound xanthotoxin (XT) was conducted, in which xanthotoxin-para aminobenzoic acid (XT-PABA) and xanthotoxin-oxalic acid (XT-OA) cocrystals were obtained. The xanthotoxin cocrystals were characterized by powder X-ray diffraction, thermal analysis, and FT-IR spectra, and the crystal structures were determined by single-crystal X-ray diffraction. Crystal structures and thermal analysis showed that XT-OA was more stable than XT-PABA. Energy framework calculation indicated that H-bond and π···π interactions generated in XT-OA were stronger than that in XT-PABA and xanthotoxin. The powder dissolution experiments of xanthotoxin and its cocrystals suggested the XT-OA cocrystal might be applied as an alternative formulation of API, on account of its enhanced solubility and stability in the hydrochloric acid buffer solution (pH 1.2). The cocrystallization engineering can prolong the enhanced apparent solubility via modulating the stability.


Assuntos
Metoxaleno/química , Solubilidade/efeitos dos fármacos , Ácido 4-Aminobenzoico/química , Cristalização/métodos , Cristalografia por Raios X/métodos , Estabilidade de Medicamentos , Ligação de Hidrogênio , Ácido Oxálico/química , Difração de Pó/métodos , Pós/química , Difração de Raios X/métodos
19.
Acta Crystallogr D Struct Biol ; 75(Pt 11): 959-968, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31692470

RESUMO

A nonlinear least-squares method for refining a parametric expression describing the estimated errors of reflection intensities in serial crystallographic (SX) data is presented. This approach, which is similar to that used in the rotation method of crystallographic data collection at synchrotrons, propagates error estimates from photon-counting statistics to the merged data. Here, it is demonstrated that the application of this approach to SX data provides better SAD phasing ability, enabling the autobuilding of a protein structure that had previously failed to be built. Estimating the error in the merged reflection intensities requires the understanding and propagation of all of the sources of error arising from the measurements. One type of error, which is well understood, is the counting error introduced when the detector counts X-ray photons. Thus, if other types of random errors (such as readout noise) as well as uncertainties in systematic corrections (such as from X-ray attenuation) are completely understood, they can be propagated along with the counting error, as appropriate. In practice, most software packages propagate as much error as they know how to model and then include error-adjustment terms that scale the error estimates until they explain the variance among the measurements. If this is performed carefully, then during SAD phasing likelihood-based approaches can make optimal use of these error estimates, increasing the chance of a successful structure solution. In serial crystallography, SAD phasing has remained challenging, with the few examples of de novo protein structure solution each requiring many thousands of diffraction patterns. Here, the effects of different methods of treating the error estimates are estimated and it is shown that using a parametric approach that includes terms proportional to the known experimental uncertainty, the reflection intensity and the squared reflection intensity to improve the error estimates can allow SAD phasing even from weak zinc anomalous signal.


Assuntos
Cristalografia por Raios X/métodos , Modelos Moleculares , Termolisina/química , Cristalização/métodos , Interpretação Estatística de Dados , Conjuntos de Dados como Assunto , Funções Verossimilhança
20.
Acta Crystallogr D Struct Biol ; 75(Pt 11): 980-994, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31692472

RESUMO

When protein crystals are abruptly cooled, the unit-cell, protein and solvent-cavity volumes all contract, but the volume of bulk-like internal solvent may expand. Outflow of this solvent from the unit cell and its accumulation in defective interior crystal regions has been suggested as one cause of the large increase in crystal mosaicity on cooling. It is shown that when apoferritin crystals are abruptly cooled to temperatures between 220 and 260 K, the unit cell contracts, solvent is pushed out and the mosaicity grows. On temperature-dependent timescales of 10 to 200 s, the unit-cell and solvent-cavity volume then expand, solvent flows back in, and the mosaicity and B factor both drop. Expansion and reordering at fixed low temperature are associated with small-amplitude but large-scale changes in the conformation and packing of apoferritin. These results demonstrate that increases in mosaicity on cooling arise due to solvent flows out of or into the unit cell and to incomplete, arrested relaxation of protein conformation. They indicate a critical role for time in variable-temperature crystallographic studies, and the feasibility of probing interactions and cooperative conformational changes that underlie cold denaturation in the presence of liquid solvent at temperatures down to ∼200 K.


Assuntos
Apoferritinas/química , Cristalização/métodos , Cristalografia por Raios X/métodos , Conformação Proteica , Animais , Crioprotetores/química , Solventes , Temperatura
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