Different Head Group Dependence on the Lipid ThermodynamicProperty of the Myelin Basic Protein in the Lipid Monolayer / 中国生物化学与分子生物学报

The 18. 5 kD myelin basic protein (MBP) isoform interacts with phospholipids and its role has been thought to maintain the stability and compactness of the myelin sheath structure. In this study, we describe the statistical thermodynamic theory of certain concentration effects on MBP in the majormyelin lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethano-lamine (POPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine(POPS)) monolayers at the air/ subphase interface via the Langmuir-Blodgett (LB) technique. A simple statistical mechanical theory is established that predicts the interaction between proteins and phosphatehead groups at low surface pressures and the second virial coefficient dependences for the PC, PE, and PS head groups are illustrated. Two-dimensional virial equation of state (2D-VES) suggested that the interaction in the monolayer structure at the MBP-myelin interface is a repulsive force, and it induces a phase change in the monolayer. This is consistent with atomic force microscope (AFM) observations of domain and aggregate structures as well as with changes in the surface morphology induced by MBP. These analyses pertaining to membrane structures will provide a theoretical and experimental basis for the establishment of the myelin membrane modeling system.

Effect of resin infiltration and bleaching on surface roughness and microhardness of human enamel with induced white spot lesions / Efeito do infiltrante resinoso e do clareamento na rugosidade e microdureza de superfície do esmalte humano com lesões de mancha branca induzidas

Objective: To comparatively evaluate the effect of resin infiltration, bleaching and bleaching followed by resin infiltration on the surface roughness and microhardness of human enamel with induced white spot lesions (WSLs) and their resistance to acidic challenge. Material and Methods: Sixty human specimens were randomly divided into three groups (n=20) according to the treatment modality applied; group I Icon® resin infiltration, group II bleaching and group III bleaching followed by Icon® resin infiltration. For each treatment modality, 10 specimens were tested for surface roughness and another 10 for microhardness. WSLs were artificially induced in all specimens and after treatment, all specimens were subjected to acidic challenge. Surface roughness was measured by the tapping mode of the atomic force microscope (AFM) and microhardness was measured by digital Vickers hardness tester at baseline, after induction of WSLs, after treatment and after acidic challenge. Results: Groups I and III showed significant reduction in surface roughness after treatment, while group II showed significant increase. Groups I and III showed significant increase in the microhardness after treatment, while group II showed insignificant increase. The three tested groups showed significant increase in surface roughness values and significant reduction in microhardness after acidic challenge. Conclusion: Resin infiltration and bleaching followed by resin infiltration reduced the surface roughness and enhanced the microhardness of the WSLs. The three treatment modalities failed to resist acidic challenge resulting in increasing surface roughness and reducing microhardness. (AU)

Objetivo: Avaliar comparativamente o efeito do infiltrante resinoso, clareamento e clareamento seguido de infiltração resinosa sobre a rugosidade e microdureza superficial do esmalte humano com lesões de manchas brancas induzidas (WSLs) e sua resistência ao desafio erosivo. Material e Métodos: Sessenta espécimes humanos foram divididos aleatoriamente em três grupos (n = 20) de acordo com a modalidade de tratamento aplicada; grupo I infiltrante resinoso Icon®, grupo II clareamento e grupo III clareamento seguido de infiltração resinosa Icon®. Para cada modalidade de tratamento, 10 corpos-de-prova foram testados para rugosidade superficial e outros 10 para microdureza. WSLs foram artificialmente induzidos em todas as amostras e, após o tratamento, todas as amostras foram submetidas ao desafio erosivo. A rugosidade de superfície foi medida por microscopia de força atômica em modo de contato intermitente (AFM) e a microdureza Vickers foi medida inicialmente, após a indução de WSLs, após o tratamento e após o desafio ácido. Resultados: Os grupos I e III apresentaram redução significativa da rugosidade superficial após o tratamento, enquanto o grupo II apresentou aumento significativo. Os grupos I e III apresentaram aumento significativo na microdureza após o tratamento, enquanto o grupo II apresentou aumento insignificante. Os três grupos testados mostraram aumento significativo nos valores de rugosidade superficial e redução significativa na microdureza após o desafio erosivo. Conclusão: O infiltrante resinoso e o clareamento seguido de infiltração resinosa reduziram a rugosidade de superfície e aumentaram a microdureza dos WSLs. As três modalidades de tratamento falharam em resistir ao desafio erosivo, resultando em aumento da rugosidade de superfície e redução da microdureza.(AU)

Microscopic morphology observation of fracture healing process promoted by panax notoginseng saponins / Observación microscópica de la morfología del proceso de curación de fracturas promovida por panax notoginseng saponins

SUMMARY: The aim of this study was to explore promoting effect of external applying Panax Notoginseng Saponins (PNS) on fractures. For this analysis 18 New Zealand male rabbits were divided into control group, splintage group and PNS group. All rabbits were performed left radius fractures and natural healing, splintage healing and splintage coated with PNS healing. 2 rabbits in each group were sacrificed on day 14, day 28 and day 42 after surgery, separately. Atomic force microscope scanning and nanoindentation tests were performed on the callus sections. The particle size and roughness in PNS group was both less than that in splintage group. The elastic modulus of callus in PNS group was consistent with normal bone tissue started from day 28 after surgery, two weeks earlier than that in splintage group. PNS could significantly reduce fracture healing time and increase strength of callus.

RESUMEN: El objetivo de este estudio fue evaluar el efecto de la aplicación externa de Panax Notoginseng Saponins (PNS) en fracturas óseas. Se usaron 18 conejos machos de raza Nueva Zelanda divididos en grupos control, entablillado y PNS. Se realizaron fracturas del radio izquierdo y cicatrización natural en todos los animales, además de la cicatrización con entablillado y entablillado recubierto con PNS. Se sacrificaron, posterior a la cirugía, dos conejos de cada grupo los día 14, 28 y 42. Se realizaron pruebas de escaneo con microscopio de fuerza atómica y nanoindentación en las secciones de callos. El tamaño de la partícula y la rugosidad en el grupo de PNS fue menor que en el grupo entablillado. El módulo elástico del callo en el grupo de PNS fue consistente con el tejido óseo normal iniciado el día 28 después de la cirugía, dos semanas antes que en el grupo de entablillado. El PNS podría redu- cir significativamente el tiempo de curación de la fractura y aumentar la fuerza del callo.

Hardness Properties of Pig Esophageal at Nanoscale Using Atomic Force Microscope / 医用生物力学

Objective To study the hardness properties of pig esophageal at the nanoscale using atomic force microscope (AFM). Methods The porcine esophagus was chosen as experimental sample to study the hardness properties of esophageal tissues at different loading rates, deflection and dwell time with AFM. Results The hardness of esophageal tissues at the nanoscale was strongly correlated with the loading rate and the deflection, which increased with the increasing loading rate and decreased with the increasing deflection of cantilever. The difference in the hardness was associated with the viscoelasticity and viscoplasticity of esophageal tissues, including contact stress, energy transition and strain plastic gradient. Conclusions The experimental results have important significance for clinical diagnosis, surgical operation and artificial material development, and reveal the changing patterns for mechanical properties of the esophageal tissues at the microscale.

In-Vitro Antimicrobial Activity Of Biological Synthesized Silver Nanoparticles Using Stenotrophomonas Maltophilia Strain Ns-24 From Non-Rhizosphere Soil

Objective: The present goals of our study were biological synthesis, characterizations of silver nanoparticles, and evaluation of its antimicrobial activity against microbial pathogens like Escherichia coli, Enterococcus faecalis, Streptococcus pneumoniae and Staphylococcus aureus. Methods: The bacterial Strain NS-24 was isolated on nutrient agar medium and was selected for the synthesis of silver nanoparticles based on its gram-negative characteristics. The characterizations of silver nanoparticles were done by UV-Visible spectroscopy, Atomic Force Microscopy (AFM), High Resolution-Transmission Electron Microscopy (HR-TEM), Scanning Electron Microscopy (SEM) with Energy Dispersive Spectroscopy (EDX), X-ray Diffraction (XRD) and Fourier Transform Infrared Spectroscopy (FTIR). Later, the molecular characterization of the Strain NS-24 was done by DNA extraction and 16S rRNA gene sequencing. Results: The UV-visible spectrophotometric observation of the Strain NS-24 supernatant and AgNO3 solution showed maximum absorbance at 423 nm. The AFM data confirmed that the particles were polydispersed and spherical in shape. Additionally, the FTIR analysis revealed the IR spectral band patterning and TEM analyzes showed the size of biological AgNPs was in the range of 12.56 nm to 27.32 nm, with an average of 18.06 nm in size. Further, the 16S rRNA gene sequencing revealed the identity of Strain NS-24 as Stenotrophomonas maltophilia. The antimicrobial activity of AgNPs was studied on different gram-negative and gram-positive bacterial strains like Escherichia coli (MTCC 40), Enterococcus faecalis (MTCC 6845), Streptococcus pneumoniae (MTCC 8874) and Staphylococcus aureus (MTCC 2825), which showed good inhibition of their growth at varying concentrations of AgNPs against all the pathogens. Conclusion: Our findings showed that the synthesized AgNPs from the isolated bacterium was small in size and had profound antibacterial activity against pathogenic micro-organisms.

Effects of High-Fat Diet on Rat Aortic Endothelial Cell Morphology and Stiffness / 医用生物力学

Objective To investigate the high-fat diet effect on morphology and stiffness of endothelial cells. Methods SD rats were randomly divided into high-fat diet group (AS group, n=3) and control group (CON group, n=3). Rat aortic endothelial cells were obtained from rat thoracic aorta by explant method. Cell morphology was observed under inverted microscopy. The mean fluorescent intensity of F-actin in two groups was calculated by immunofluorescence staining. Cell stiffness was measured by atomic force microscopy (AFM). Results The endothelial cells migrated from tissue plant on the 7th day and formed confluence after cultivation for 14 days. Endothelial cells were identified by factor Ⅷ immunofluorescence staining. Cells in AS group showed enhanced perimeter (P＜0.01), aspect ratio (P＜0.01), and area (P＞0.05), while less circularity (P＜0.01) compared with the cells in control group. The mean fluorescence intensity of F-actin in AS group was significantly higher than that in control group (P＜0.01). AFM showed that the cell stiffness of AS group was significantly higher than that of control group (P＜0.01). Conclusions High-fat diet would change the morphology and stiffness of endothelial cells, which might subsequently affect their normal function and become an important incentive to AS.

Interfacial properties and micellization of triblock poly(ethylene glycol)-poly(-caprolactone)-polyethyleneimine copolymers

This study aimed to explore the link between block copolymers' interfacial properties and nanoscale carrier formation and found out the influence of length ratio on these characters to optimize drug delivery system. A library of diblock copolymers of PEG-PCL and triblock copolymers with additional PEI (PEG-PCL-PEI) were synthesized. Subsequently, a systematic isothermal investigation was performed to explore molecular arrangements of copolymers at air/water interface. Then, structural properties and drug encapsulation in self-assembly were investigated with DLS, SLS and TEM. We found the additional hydrogen bond in the PEG-PCL-PEI contributes to film stability upon the hydrophobic interaction compared with PEG-PCL. PEG-PCL-PEI assemble into smaller micelle-like (such as PEG-PCL4006-PEI) or particle-like structure (such as PEG-PCL8636-PEI) determined by their hydrophilic and hydrophobic block ratio. The distinct structural architectures of copolymer are consistent between interface and self-assembly. Despite the disparity of constituent ratio, we discovered the arrangement of both chains guarantees balanced hydrophilic-hydrophobic ratio in self-assembly to form stable construction. Meanwhile, the structural differences were found to have significant influence on model drugs incorporation including docetaxel and siRNA. Taken together, these findings indicate the correlation between molecular arrangement and self-assembly and inspire us to tune block compositions to achieve desired nanostructure and drug loading.

Increased elastic modulus of the synovial membrane in a rat ACLT model of osteoarthritis revealed by atomic force microscopy

This study aimed to explore changes in nanoscale elastic modulus of the synovium using atomic force microscopy (AFM) in addition to investigate changes in synovial histomorphology and secretory function in osteoarthritis (OA) in a rat anterior cruciate ligament transection (ACLT) model. Sprague-Dawley rats were randomly assigned to sham control and ACLT OA groups. All right knee joints were harvested at 4, 8, or 12 weeks (W) after surgery for histological assessment of cartilage damage and synovitis in both the anterior and posterior capsules. AFM imaging and nanoscale biomechanical testing were conducted to measure the elastic modulus of the synovial collagen fibrils. Immunohistochemistry was used to visualize the expression of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and matrix metalloproteinase-3 (MMP-3) in the synovium. The OA groups exhibited progressive development of disease in the cartilage and synovium. Histopathological scores of the synovium in the OA groups increased gradually. Significant differences were observed between all OA groups except for the posterior 4W group. The synovial fibril arrangement in all OA groups was significantly disordered. The synovial fibrils in all ACLT OA groups at each time point were stiffer than those in the sham controls. OA rats displayed a significantly higher expression of IL-1β and MMP3 in the anterior capsule. In summary, synovial stiffening was closely associated with joint degeneration and might be a factor contributing to synovitis and increased production of proinflammatory mediators. Our data provided insights into the role of synovitis, particularly stiffening of the synovium, in OA pathogenesis.

Chemical and Microscopy Characterization of Trans-Endodontic Implants / Caracterización química y microscópica de implantes trans-endodónticos

Abstract Trans-endodontic implants are an artificial extension through root apex anchored in periradicular bone tissue. The aim is to improve the crown-root ratio and to provide stability to dental organ present. Zirconium oxide (ZrO2) is a material of great technological importance, having good natural color, high strength, high toughness, high chemical stability, does not suffer any corrosion, chemical and microbial resistance and excellent esthetic properties. Objective: The aim of this study was to evaluate chemical and microscopy of surface conditions of ZrO2 trans-endodontic implant. Materials and Methods: A blocks of ZrO2 were manufactured for produce trans-endodontic implants and divided in two groups: monoclinic and tetragonal phase. They were evaluated using Scanning Electroning Microscope (SEM), EnergyDispersive X-ray Spectroscopy (EDS), and Atomic Force Microscope (AFM) and Vickers Micro hardness. Results: The Monoclinic phase through AFM analysis showed roughness Ra = 0.320μm, whereas in the Tetragonal phase was 0.126μm, SEM/EDX indicated that the phases are not properly uniform and the addition of the Yttrium to favor the stabilization of the Tetragonal phase. The Vickers hardness analysis showed a value of 1500HV. Conclusion: The characterization of the surface of trans-endodontic zirconium oxide implants provides a guideline to know the surface characteristics of the material, since a greater roughness on the surface of the implant will favor the Osseo-integration capacity.

Resumen Los implantes trans-endodónticos son una extensión artificial a través del ápice radicular anclado en el tejido óseo periradicular. El objetivo es mejorar la relación corona-raíz y proporcionar estabilidad al órgano dental presente. El óxido de zirconio (ZrO2) es un material de gran importancia tecnológica, con buen color natural, alta resistencia, alta tenacidad, alta estabilidad química, no sufre corrosión, resistencia química y microbiana y excelentes propiedades estéticas. Objetivo: El objetivo de este estudio fue evaluar las condiciones superficiales de ZrO2 para su aplicación clínica a los implantes transendodónticos. Materiales y Métodos: se trituraron bloques de ZrO2 en implantes trans-endodónticos y se dividieron en: monoclínico y tetragonal. Luego se evaluaron mediante microscopía electrónica de barrido (SEM), espectroscopia de rayos X de energía dispersiva (EDS) y microscopio de fuerza atómica (AFM) y microdureza vickers. Resultados: La fase monoclínica a través del análisis AFM presenta Ra = 0.320 μm, mientras que en la fase Tetragonal es 0.126 μm, SEM / EDS muestra que las fases no son adecuadamente uniformes y la adición del Ytrio para favorecer la estabilización de la fase tetragonal. El análisis de microdureza mostro un valor de 1500HV. Conclusión: La caracterización de la superficie de los implantes trans-endodónticos de óxido de zirconio, brinda una pauta para conocer las características superficiales del material, ya que al haber una mayor rugosidad en la superficie del implante se verá favorecida la capacidad de oseointegración.

Single Molecule Method for Molecular Biology

In order to understand biological phenomena accurately, single molecule techniques using a physical research approach to molecular interactions have been developed, and are now widely being used to study complex biological processes. In this review, we discuss some of the single molecule methods which are composed of two major parts: single molecule spectroscopy and manipulation. In particular, we explain how these techniques work and introduce the current research which uses them. Finally, we present the oral biology research using the single molecule methods.

Cyclic Stretch Effects on Adipose-Derived Stem Cell Stiffness, Morphology and Smooth Muscle Cell Gene Expression

Recent investigations consider adipose-derived stemcells (ASCs) as a promising source of stemcells for clinical therapies. To obtain functional cells with enhanced cytoskeleton and aligned structure, mechanical stimuli are utilized during differentiation of stem cells to the target cells. Since function of muscle cells is associated with cytoskeleton, enhanced structure is especially essential for these cells when employed in tissue engineering. In this study by utilizing a custom-made device, effects of uniaxial tension (1Hz, 10% stretch) on cytoskeleton, cell alignment, cell elastic properties, and expression of smooth muscle cell (SMC) genes in ASCs are investigated.Due to proper availability ofASCs, results can be employed in cardiovascular engineeringwhen production of functional SMCs in arterial reconstruction is required. Results demonstrated that cells were oriented after 24 hours of cyclic stretch with aligned pseudo-podia. Staining of actin filaments confirmed enhanced polymerization and alignment of stress fibers. Such phenomenon resulted in stiffening of cell body which was quantified by atomic force microscopy (AFM). Expression of SM α-actin and SM22 α-actin as SMC associated genes were increased after cyclic stretch while GAPDH was considered as internal control gene. Finally, it was concluded that application of cyclic stretch on ASCs assists differentiation to SMC and enhances functionality of cells.

Application of atomic force microscopy (AFM) to study bacterial biofilms / 生物工程学报

Because of the nanometre resolution, piconewton force sensitivity, label-free technique and the ability to operate in liquid environments, atomic force microscopy (AFM) has emerged as a powerful tool to explore the biofilm development processes. AFM provides three-dimensional topography and structural details of biofilm surfaces under in-situ conditions. It also helps to generate key information on the mechanical properties of biofilm surfaces, such as elasticity and stickiness. Additionally, single-molecule and single-cell force spectroscopies can be applied to measure the strength of adhesion, attraction, and repulsion forces between cell-solid and cell-cell surfaces. This paper outlined the basic principle of AFM technique and introduced recent advances in the application of AFM for the investigation of ultra-morphological, mechanical and interactive properties of biofilms. Furthermore, the existing problems and future prospects were discussed.

Physicochemical and Biological Investigation of Different Structures of Carbon Coatings Deposited onto Polyurethane

The aim of this study was to examine the thrombogenic properties of polyurethane that was surface modified with carbon coatings. Physicochemical properties of manufactured coatings were investigated using transmission electron microscopy (TEM), atomic force microscopy (AFM), X-ray Photoelectron Spectroscopy (XPS), Raman spectroscopy and contact angle measurement methods. Samples were examined by the Impact-R method evaluating the level of platelets activation and adhesion of particular blood cell elements. The analysis of antimicrobial resistance against E. coli colonization and viability of endothelial cells showed that polyurethane modified with use of carbon layers constituted an interesting solution for biomedical application.

The effect of silane applied to glass ceramics on surface structure and bonding strength at different temperatures

PURPOSE: To evaluate the effect of various surface treatments on the surface structure and shear bond strength (SBS) of different ceramics. MATERIALS AND METHODS: 288 specimens (lithium-disilicate, leucite-reinforced, and glass infiltrated zirconia) were first divided into two groups according to the resin cement used, and were later divided into four groups according to the given surface treatments: G1 (hydrofluoric acid (HF)+silane), G2 (silane alone-no heat-treatment), G3 (silane alone-then dried with 60℃ heat-treatment), and G4 (silane alone-then dried with 100℃ heat-treatment). Two different adhesive luting systems were applied onto the ceramic discs in all groups. SBS (in MPa) was calculated from the failure load per bonded area (in N/mm2). Subsequently, one specimen from each group was prepared for SEM evaluation of the separated-resin–ceramic interface. RESULTS: SBS values of G1 were significantly higher than those of the other groups in the lithium disilicate ceramic and leucite reinforced ceramic, and the SBS values of G4 and G1 were significantly higher than those of G2 and G3 in glass infiltrated zirconia. The three-way ANOVA revealed that the SBS values were significantly affected by the type of resin cement (P<.001). FIN ceramics had the highest rate of cohesive failure on the ceramic surfaces than other ceramic groups. AFM images showed that the surface treatment groups exhibited similar topographies, except the group treated with HF. CONCLUSION: The heat treatment was not sufficient to achieve high SBS values as compared with HF acid etching. The surface topography of ceramics was affected by surface treatments.

Aflatoxin M1 in pasteurized, UHT milk and milk powder commercialized in Londrina, Brazil and estimation of exposure / Aflatoxina M1 em leite pasteurizado e UHT e leite em po comercializados em Londrina, Brasil e exposicao estimada.

Aflatoxin M1 (AFM1) is found in milk and other excretion products after aflatoxin B1 intake. AFM1 is carcinogenic to humans, and known levels of dairy product contamination is important to understand the risks to which the population is exposed. The occurrence of AFM1 was evaluated in 42 milk samples commercialized in Londrina, Parana State, Brazil and this rate of occurrence was used to estimate this exposure. AFM1 determina tion was ca rried out by ELISA, and was detected in 100 % samples at levels ranging from 0.01 to 0.81 µ g/kg (mean 0.13 µ g/kg). None of the samples p resente d AFM1 above the maximum permitted level by Brazilian Legislation (0.5 µ g/kg for fluid milk and 5 µ g/kg for milk powder). The estimated daily intake (EDI) of AFM1 was evaluated, and the average intake was 0.468 ng/kg body weight (b.w.) for adolescents, 0.384 ng/kg b.w. for adults and 0.559 ng/kg b.w. for the elderly. Values of EDI of AFM1 found in Londrina pose a toxicological risk to the population investigated. To the best of our knowledge, this is the first report on estimat ed AFM1 dietary exposure from Parana, Brazil.

Aflatoxina M1 (AFM1) e encontrada no leite e em outros produtos de excrecao apos o consumo de aflatoxina B1. AFM1 e carcinogenica para humanos, e avaliar os niveis de contaminacao em produtos lacteos e importante para conhecer os riscos aos quais a populacao esta exposta. A ocorrencia de AFM1 foi avaliada em 42 amostras de leite comercializadas em Londrina, Estado do Parana, Brasil, e sua ocorrencia foi utilizada para estimar sua exposicao. A determinacao de AFM1 foi avaliada por ELISA, e foi detectada em 100% das amostras, em niveis variando de 0,01 a 0,81 µ g/kg (media 0,13 µ g kg). Nenhuma das amostras apresentou niveis de AFM1 acima do maximo permitido pela Legislacao brasileira (0,5 µ g/kg para leite fluido e 5 µ g/kg para leite em po). A ingestao diaria estimada (IDE) de AFM1 foi avaliada, e a ingestao media foi de 0,468 ng/kg de peso corporal (p.c.)/dia para adolescentes, 0,384 ng/kg p.c./dia para adultos e 0,559 ng/kg p.c./dia para idosos. Valores de IDE de AFM1 encontrados em Londrina supoem um risco toxicologico para a populacao investigada. Do melhor do nosso conhecimento, este e o primeiro trabalho sobre a exposicao estimada de AFM1 do Parana, Brasil.

PURIFICACIÓN DEL ANTÍGENO 38 kDa DE Mycobacterium tuberculosis Y SU POTENCIAL USO EN DIAGNÓSTICO MEDIANTE INMUNOSENSORES PIEZOELÉCTRICOS / Mycobacterium tuberculosis 38 kDa Antigen Purification and Potential Diagnostic Use by Piezoelectric Immunosensors

Un paso crucial en el desarrollo de un inmunosensor piezoeléctrico para la detección de tuberculosis (TB), es la selección y obtención de los inmunoreactivos empleados en el inmunoensayo y la estrategia para la biofuncionalización del transductor. Diversos estudios han reportado el uso del antígeno proteico 38kDa (Ag38kDa) de Mycobacterium tuberculosis (Mtb) como un buen biomarcador de la enfermedad y el cumplimiento de las características físicas y bioquímicas para ser inmovilizado por monocapas autoensambladas (SAMs), en la superficie del electrodo de oro de cristales piezoeléctricos. Un inmunosensor piezoeléctrico desarrollado a partir de un antígeno nativo purificado de Mtb podría ser un método alternativo simple para la detección de Mtb con ventajas de rapidez y reusabilidad, contribuyendo al control y el tratamiento oportuno de la enfermedad. En este estudio se presenta el proceso de purificación del Ag38kDa a partir de proteínas de secreción filtradas de cultivo (CFP) de Mtb para ser usado como inmunoreactivo con potencial aplicación en la detección de Mtb con inmunosensores piezoeléctricos. Se obtuvieron cristales funcionalizados mediante la técnica modificada de monocapas autoensambladas (SAMs), con el antígeno nativo purificado y CFP. Las superficies biofuncionalizadas fueron caracterizadas cualitativamente con microscopía de fuerza atómica (AFM) para validar las condiciones de optimización del protocolo de inmovilización con antígenos de secreción de Mtb. Estos cristales modificados pueden ser acoplados a un sistema de caracterización de un inmunosensor piezoeléctrico para la detección de Mtb mediante un inmunoensayo competitivo directo.

The selection and procurance of the immunoreagents used in the immunoassay and biofunctionalisation transducer strategy, are a key in the piezoelectric immunosensor development for the detection of tuberculosis (TB). Many have reported the use of 38kDa protein antigen (Ag38kDa) from Mycobacterium tuberculosis (Mtb) such as good biomarker of TB disease and compliance with physical and biochemical characteristics to be immobilized by self-assembled monolayers (SAMs), in the gold electrode of piezoelectrics crystals surfaces. A piezoelectric immunosensor developed from purified native antigens of Mtb may be an alternative simple method for detection of Mtb with speed and reusable advantages, contributing to the control and early treatment of disease. In this paper, the purification process of Ag38kDa Mtb from secretory proteins filtered culture (CFP) from Mtb is presented as an immunoreactive with potential application in the detection of Mtb by piezoelectric immunosensors. Functionalized crystals were obtained by using the modified self-assembled monolayers (SAMs) technique, with purified native antigen and CFP. The functionalized surfaces were qualitatively characterized using atomic force microscopy (AFM) in order to validate the immobilization protocol optimal conditions for secretion antigens from Mtb. These modified crystals may be coupled to piezoelectric immunosensor characterization system for detecting of Mtb by a direct competition immunoassay.

Responses from mesenchymal stem cells to mechanical stimulation for variable duration / 医用生物力学

Objective To study mechanical responses from mesenchymal stem cells (MSCs) under different mechanical stimulus duration, by measuring its elastic modulus and characterizing its stress fibers. Methods High resolution images of MSCs cytoskeleton in vitro were acquired by using atomic force microscope (AFM) and laser scanning confocal microscope (LSCM). AFM cantilever with micro-bead attached probe was used to perform force-distance curve experiment on MSCs at the approaching time of 0.1，0.5, 1, 5,10 s, respectively. The elastic modulus of MSCs at 300 nm indentation depth were measured and compared. Results The rat MSCs cytoskeleton presented an intensely organized network structure. The elastic modulus of rat MSCs varied obviously for different mechanical stimulus duration. The median and quartile (QR) of MSCs elastic modulus were 10.02 (QR=9.66),1.94 (QR=7.71),3.63 (QR=19.33),17.15(QR=35.13), 23.52 kPa(QR=34.87), with probe approaching time at 0.1，0.5, 1, 5,10 s, respectively. The MSCs elastic modulus showed the tendency of increasing with stimulus duration increasing, except for the extremely short stimulus (0.1 s). Conclusions Unlike inorganic elastomer, rat MSCs possess complete and flexible mechanical load-bearing structure and can respond actively to a relatively longer mechanical stimulation, with an increase of elastic modulus. These results may provide basic data for further tissue engineering researches on mechanical regulation of MSCs behavior.

Effect of cigarette smoke extract on the interaction between thrombomodulin and thrombin by live-cell single-molecule force spectroscopy / 中国药理学通报

Aim To study the effect of CSE ( cigarette smoke extract ) on the single-molecule interactional force between thrombomodulin and thrombin by live-cell single-molecule force spectroscopy. Methods CSE was prepared by a previously reported method. The plasmid of TM-GFP was constructed and transfect-ed in COS-7 cells. The expression of TM-GFP was de-tected by fluorescence microscopy and laser scanning confocal microscopy. The transfected COS-7 cells were grouped ( 1 ) GFP -thrombin group ( 2 ) TM-thrombin group ( 3 ) CSE-TM-thrombin group ( 4 ) CSE- GFP-thrombin group. Force measurements with the thrombin modified AFM tips on the living cell surface were car-ried out on PicoSPM II with a Pico-Scan 3000 control-ler and a larger scanner. The force curves measured in living cells were recorded by PicoScan 5 software and analyzed by MATLAB R2009aMetlab. Results The single-molecule binding force of thrombomodulin and thrombin ( TM-Thr ) was determined ( 60. 90 ± 0. 82 ) pN. The binding probability for TM-Thr was about (22. 58 ± 3. 95)%. Antibody blocking binding proba-bility for TM-Thr was ( 2. 58 ± 2. 0 )%. The binding probabilities for GFP-Thr group, CSE-TM-Thr group and CSE-GFP-Thr group were significantly decreased compared with TM-Thr group ( P<0. 05 ) . The mean value of the most probable single molecular interaction force of thrombin/TM-ECD was determined as ( 45. 30 ± 1. 37 ) pN, the binding probability of thrombin and TM-ECD was ( 23. 25 ± 7. 02 )%. When the binding was blocked with the TM-MAb solution, the binding probability decreased to ( 4. 64 ± 2. 31 )%. The bind-ing probability was ( 8. 31 ± 1. 06 )% in the CSE-TM-thr-S group. When further blocked with TM-MAb, the binding probability was ( 5. 17 ± 2. 96 )%. Conclusion CSE significantly decreases the binding probability for TM-Thr to induce intravascular thrombosis.

Aflatoxin M1 preparation and identification of artificial antigens / 中国免疫学杂志

Objective:AFM1-BSA and AFM1-OVA were synthesized and then identified in this experiment.Methods: Using oximation method ,AFM1 was transformed to oxime compounds while the reaction process was monitored via TLC method aiming to identify the compounds.Coupled with carrier protein BSA and OVA respectively , we obtained AFM1-BSA and AFM1-OVA, then identified synthetic antigen via UV spectrophotometry and SDS-PAGE.Antigens were injected into experimental animals , finally obtaining the murine multi-antiserum.Eventually , the multi-antiserums were detected via indirect inhibition ELISA method to judge whether the antigens were effectively or not.Results:After oximation reaction ,the migration distance of oxime compounds in the thin layer plate was shorter.The maximum absorption peak of AFM1-BSA occurred in 274 nm,and was inconsistent with both UV absorption peaks of BSA and AFM 1.The electrophoretic velocity of AFM 1-BSA was less than that of BSA.All the titers of three immunized mice were 1×10-4 approximately;the multi-antiserum from No.3 sample had the best sensitivity ,its IC50 was 359.9 ng/ml.Conclusion:In this study,we obtained AFM1 artificial antigen and murine multi-antiserum of high sensitivity.

s size with AFM

Atomic Force Microscopy (AFM) can be used to obtain high-resolution topographical images of bacteria revealing surface details and cell integrity. During scanning however, the interactions between the AFM probe and the membrane results in distortion of the images. Such distortions or artifacts are the result of geometrical effects related to bacterial cell height, specimen curvature and the AFM probe geometry. The most common artifact in imaging is surface broadening, what can lead to errors in bacterial sizing. Several methods of correction have been proposed to compensate for these artifacts and in this study we describe a simple geometric model for the interaction between the tip (a pyramidal shaped AFM probe) and the bacterium (Escherichia coli JM-109 strain) to minimize the enlarging effect. Approaches to bacteria immobilization and examples of AFM images analysis are also described.