Confocal Laser Scanning Microscopy Observation Research Articles

Environmental Free-Living Amoebae Isolated from Soil in Khon Kaen, Thailand, Antagonize Burkholderia pseudomallei.

Presence of Burkholderia pseudomallei in soil and water is correlated with endemicity of melioidosis in Southeast Asia and northern Australia. Several biological and physico-chemical factors have been shown to influence persistence of B. pseudomallei in the environment of endemic areas. This study was the first to evaluate the interaction of B. pseudomallei with soil amoebae isolated from B. pseudomallei-positive soil site in Khon Kaen, Thailand. Four species of amoebae, Paravahlkampfia ustiana, Acanthamoeba sp., Naegleria pagei, and isolate A-ST39-E1, were isolated, cultured and identified based on morphology, movement and 18S rRNA gene sequence. Co-cultivation combined with a kanamycin-protection assay of B. pseudomallei with these amoebae at MOI 20 at 30°C were evaluated during 0–6 h using the plate count technique on Ashdown’s agar. The fate of intracellular B. pseudomallei in these amoebae was also monitored by confocal laser scanning microscopy (CLSM) observation of the CellTracker™ Orange-B. pseudomallei stained cells. The results demonstrated the ability of P. ustiana, Acanthamoeba sp. and isolate A-ST39-E1 to graze B. pseudomallei. However, the number of internalized B. pseudomallei substantially decreased and the bacterial cells disappeared during the observation period, suggesting they had been digested. We found that B. pseudomallei promoted the growth of Acanthamoeba sp. and isolate A-ST39-E1 in co-cultures at MOI 100 at 30°C, 24 h. These findings indicated that P. ustiana, Acanthamoeba sp. and isolate A-ST39-E1 may prey upon B. pseudomallei rather than representing potential environmental reservoirs in which the bacteria can persist.

Three-Dimensional Analysis of Enamel Crack Behavior Using Optical Coherence Tomography

The aim of this study was to nondestructively analyze enamel crack behavior on different areas of teeth using 3D swept source-optical coherence tomography (SS-OCT). Ten freshly extracted human teeth of each type on each arch (n = 80 teeth) were inspected for enamel crack patterns on functional, contact and nonfunctional, or noncontact areas using 3D SS-OCT. The predominant crack pattern for each location on each specimen was noted and analyzed. The OCT observations were validated by direct observations of sectioned specimens under confocal laser scanning microscopy (CLSM). Cracks appeared as bright lines with SS-OCT, with 3 crack patterns identified: Type I – superficial horizontal cracks; Type II – vertically (occluso-gingival) oriented cracks; and Type III – hybrid or complicated cracks, a combination of a Type I and Type III cracks, which may or may not be confluent with each other. Type II cracks were predominant on noncontacting surfaces of incisors and canines and nonfunctional cusps of posterior teeth. Type I and III cracks were predominant on the contacting surfaces of incisors, cusps of canines, and functional cusps of posterior teeth. Cracks originating from the dental-enamel junction and enamel tufts, crack deflections, and the initiation of new cracks within the enamel (internal cracks) were observed as bright areas. CLSM observations corroborated the SS-OCT findings. We found that crack pattern, tooth type, and the location of the crack on the tooth exhibited a strong correlation. We show that the use of 3D SS-OCT permits for the nondestructive 3D imaging and analysis of enamel crack behavior in whole human teeth in vitro. 3D SS-OCT possesses potential for use in clinical studies for the analysis of enamel crack behavior.

Polyplex micelle installing intracellular self-processing functionalities without free catiomers for safe and efficient systemic gene therapy through tumor vasculature targeting.

Both efficiency and safety profiles are crucial for promotion of gene delivery systems towards practical applications. A promising template system was previously developed based on block catiomer of poly(ethylene glycol) (PEG)-b-poly{N'-[N-(2-aminoethyl)-2-aminoehtyl]aspartamide}-cholesteryl [PEG-PAsp(DET)-cholesteryl] with strategies of ligand conjugation at the α-terminus for specific affinity to the targeted cells and cholesteryl conjugation at the ω-terminus for structural stabilization to obtain systemic retention. Aiming for advocating this formulation towards practical applications, in the current study, the binding profile of this polymer to plasmid DNA (pDNA) was carefully studied to address an issue of toxicity origin. Quantification of free polymer composition confirmed that the toxicity mainly results from unbound polymer and polyplex micelle itself has negligible toxicity. This evaluation allowed for identifying an optimal condition to prepare safe polyplex micelles for systemic application that possess maximal polymer-binding but exclude free polymers. The identified polyplex micelles then faced a drawback of limited transfection efficiency due to the absence of free polymer, which is an acknowledged tendency found in various synthetic gene carriers. Thus, series of functional components was strategically compiled to improve the transfection efficiency such as attachment of cyclic (Arg-Gly-Asp) (cRGD) peptide as a ligand onto the polyplex micelles to facilitate cellular uptake, use of endosome membrane disruptive catiomer of PAsp(DET) for facilitating endosome escape along with use of the conjugated cholesteryl group to amplify the effect of PAsp(DET) on membrane disruption, so as to obtain efficient transfection. The mechanistic investigation respecting the appreciated pH dependent protonation behavior of PAsp(DET) permitted to depict an intriguing scenario how the block catiomers manage to escape from the endosome entrapment in response to the pH gradient. Subsequent systemic application to the pancreatic tumor demonstrated a capability of vascular targeting mediated by the cRGD ligand, which was directly confirmed based on in situ confocal laser scanning microscopy observation. Encouraging this result, the vascular targeting to transfect a secretable anti-angiogenic gene was attempted to treat the intractable pancreatic tumor with anticipation that the strategy could circumvent the intrinsic physiological barriers derived from hypovascular and fibrotic characters. The obtained therapeutic efficiency demonstrates promising utilities of the proposed formulation as a safe systemic gene delivery carrier in practical use.

In vitro degradation and cytocompatibility of g-MgO whiskers/PLLA composites

In vitro degradation properties and cytocompatibility of surface-grafted magnesia whiskers/poly(l-lactide) (g-MgO–Ws/PLLA) composite films which obtained by solution casting method were studied in this work. In vitro degradation experiments of the samples were carried out in a PBS solution at 37 °C with a pH of 7.4. Changes in weight, pH value, crystallization and melting behaviors, surface morphology and chemical composition, and tensile property of the g-MgO–Ws/PLLA composite films with the degradation time were characterized, from which the related degradation mechanism of the composite was concluded. Results showed that the weight loss of the g-MgO–Ws/PLLA composite films was slightly higher than that of the neat PLLA. The alkaline g-MgO–Ws can neutralize the acidity of degradation products of PLLA to some extent and maintain the tensile properties of the films well in the early degradation stage compared with neat PLLA. With the degradation, the crystallinity of PLLA matrix increased first and then decreased. In vitro cell culture results, based on the optical density value, alkaline phosphate (ALP) activity measurement, field emission scanning electron microscope, and confocal laser scanning microscopy observation, revealed that the g-MgO–Ws/PLLA composite films were not only in favor of cells adhesion, spreading, and proliferation, but also can significantly upregulate the ALP activity and promote the osteogenic differentiation of MC3T3-E1 cells compared with the neat PLLA film.

Dextranase from Arthrobacter oxydans KQ11-1 inhibits biofilm formation by polysaccharide hydrolysis

Dental plaque is a biofilm of water-soluble and water-insoluble polysaccharides, produced primarily by Streptococcus mutans. Dextranase can inhibit biofilm formation. Here, a dextranase gene from the marine microorganism Arthrobacter oxydans KQ11-1 is described, and cloned and expressed using E. coli DH5α competent cells. The recombinant enzyme was then purified and its properties were characterized. The optimal temperature and pH were determined to be 60°C and 6.5, respectively. High-performance liquid chromatography data show that the final hydrolysis products were glucose, maltose, maltotriose, and maltotetraose. Thus, dextranase can inhibit the adhesive ability of S. mutans. The minimum biofilm inhibition and reduction concentrations (MBIC50 and MBRC50) of dextranase were 2 U ml−1 and 5 U ml−1, respectively. Scanning electron microscopy and confocal laser scanning microscope (CLSM) observations confirmed that dextranase inhibited biofilm formation and removed previously formed biofilms.

Achievement of Release Mode under Combinational Stimuli by Acid and Reduction for Reduced Adverse Effect in Antitumor Efficacy

To reduce the adverse effect in antitumor therapy, a superparamagnetic doxorubicin (DOX)‐loaded nanogel with the release mode under the combinational stimuli present in intracellular environments of solid tumors is presented. Dextran is first modified to involve both thiol groups and carboxyl groups. The dextran derivative is then covalently coupled with previously thiolated and aminated magnetic iron oxide nanoparticles through both the linkage of disulfide bond and electrostatic interaction. Upon the optimal conditions, a nanogel is fabricated via the dual cross‐linking. The nanogel features the targeting release under combinational stimuli by acid and reduction environment with low release level in healthy tissues. In vitro release profiles reveal these release modes as expected. Cytotoxicity assay exhibits a much lower toxicity of the DOX‐loaded nanogel to COS‐7 cells than free DOX. Prussian blue staining and confocal laser scanning microscopy observation reveal the cellular uptake and subcellular release in nuclei of the nanogel. In vivo study exhibits that the nanogel presented 67.3% of inhibition rate of tumor growth and a healthy appearance. The present nanogel expresses high efficacy with low systemic adverse effect and is thus believed to offer a promising candidate for highly effective cancer therapy.image

Emerging integrated nanoclay-facilitated drug delivery system for papillary thyroid cancer therapy.

Nanoclay can be incorporated into emerging dual functional drug delivery systems (DDSs) to promote efficiency in drug delivery and reduce the toxicity of doxorubicin (DOX) used for thyroid cancer treatment. This paper reports the expansion of the basal spacing of kaolinite nanoclay was expanded from 0.72 nm to 0.85 nm, which could provide sufficiently spacious site for hosting doxorubicin molecules and controlling the diffusion rate. A targeted design for papillary thyroid cancer cells was achieved by introducing KI, which is consumed by the sodium-iodide symporter (NIS). As indicated by MTT assays, confocal laser scanning microscopy and bio-TEM observations, methoxy-intercalated kaolinite (KaolinMeOH) exhibited negligible cytotoxicity against papillary thyroid cancer cells. By contrast, DOX-KaolinMeOH showed dose-dependent therapeutic effects in vitro, and KI@DOX-KaolinMeOH was found to act as a powerful targeted therapeutic drug. Furthermore, active and passive targeting strategies played a role in the accumulation of the drug molecules, as verified by an in vivo bio-distribution analysis.

Biomimetic Approach to Stimulate Osteogenesis on Titanium Implant Surfaces Using Fibronectin Derived Oligopeptide.

Our previous studies demonstrated that a recombinant fibronectin (FN)-derived oligopeptide that we named F20 stimulated osteoblast adhesion, proliferation, and differentiation in vitro and in vivo. In the present study, we used a synthetic oligopeptide and investigated the osteogenic potential of F20 coating on titanium discs, to stimulate superior osseointegration for dental implant surface modification. Surface characteristic analysis of titanium was performed by confocal laser scanning microscopy (CLSM) observation. Synthetic F20 was coated onto the machined or SLA titanium discs by an adsorption procedure. ST2 cells were seeded on the titanium discs. We evaluated cell adhesion with SEM and CLSM observation, cell proliferation with picogreen assay, and osteoblast differentiation with real-time PCR, ALP activity assay, immunoblot assay and ALP staining. FITC-labeled F20 coating on the discs was detected by fluorescence, showing good F20 adsorption and different coating patterns according to the surface roughness. In the SEM and CLSM observations, cells were well attached on the machined surface and greater stress fiber formation was seen on discs coated with F20 than on other discs. F20 stimulated cellular proliferation, as well as osteoblast differentiation through the extracellular signalregulated kinase (Erk) signaling pathway. These cellular responses to F20 were slightly better on the machined titanium surface than the SLA surface. These results suggest that F20 promotes osteogenesis through the Erk pathway and is a suitable biomolecule for surface modification of dental implants for improved osseointegration.

Adsorption of Antibiotics on Graphene and Biochar in Aqueous Solutions Induced by π-π Interactions.

The use of carbon based materials on the removal of antibiotics with high concentrations has been well studied, however the effect of this removal method is not clear on the actual concentration of environments, such as the hospital wastewater, sewage treatment plants and aquaculture wastewater. In this study, experimental studies on the adsorption of 7 antibiotics in environmental concentration of aqueous solutions by carbon based materials have been observed. Three kinds of carbon materials have shown very fast adsorption to antibiotics by liquid chromatography–tandem mass spectrometry (LC-MS-MS) detection, and the highest removal efficiency of antibiotics could reach to 100% within the range of detection limit. Surprisedly, the adsorption rate of graphene with small specific surface area was stronger than other two biochar, and adsorption rate of the two biochar which have approximate specific surface and different carbonization degree, was significantly different. The key point to the present observation were the π-π interactions between aromatic rings on adsorbed substance and carbon based materials by confocal laser scanning microscope observation. Moreover, adsorption energy markedly increased with increasing number of the π rings by using the density functional theory (DFT), showing the particular importance of π-π interactions in the adsorption process.

Effect of hydrophobicity of polymer materials used for water purification membranes on biofilm formation dynamics

We characterized the dynamics of biofilm formation on various polymer surfaces used in the preparation of water purification membranes by using a flow cell incubation apparatus and confocal laser scanning microscopic observation. Flat films with different surface hydrophobicity were fabricated from eight kinds of polymers used for microfiltration and ultrafiltration membranes. Pseudomonas putida NBRC 100650 was used as a model strain. Firstly, the bacterial adhesion property of each polymer surface was examined by an immersion test in bacterial suspension. Bacterial adhesion increased with increasing hydrophobicity of polymer surfaces. Then, the dynamics of biofilm formation were characterized by time courses of the bacterial amounts on each polymer surface using a flow cell incubation apparatus. Uniform, flat, and thin biofilms were rapidly formed on the hydrophobic surface of polyvinylidenefluoride (PVDF). In contrast, the hydrophilic surface of polyvinyl alcohol (PVA) prevented bacterial adhesion and biofilm formation; however, localized rough and thick biofilms were formed at 200h of long-term incubation. On polyethersulfone, which had intermediate hydrophobicity between PVDF and PVA, rough and thick biofilms were observed at 68h, and their thickness and coverage increased with incubation time. The hydrophobicity of the polymer surfaces significantly affected the dynamics and structure of biofilm formation.

Genipin-crosslinked O-carboxymethyl chitosan-gum Arabic coacervate as a pH-sensitive delivery system and microstructure characterization.

The possibility of genipin-crosslinked O-carboxymethyl chitosan-gum Arabic coacervate as a pH-sensitive delivery vehicle was investigated. O-carboxymethyl chitosan-gum Arabic coacervates separated in pH 3.0, 4.5, and 6.0 were crosslinked by genipin for different durations and the crosslinked products were subjected to crosslinking degree, swelling behavior, bovine serum albumin release profile, and microstructure characterization. Genipin-crosslinking greatly improved the stability of the coacervates against the simulated gastric solution and created certain pH-sensitivity. The coacervates displayed higher swelling ratios in the simulated gastric solution than in the simulated intestine and colon solutions; meanwhile, the coacervates prepared in pH 4.5 and 6.0 swelled more severely than the complex separated in pH 3.0. Nevertheless, the bovine serum albumin release in the simulated gastric solution from the microcapsules prepared in pH 6.0 was much lower than those prepared in pH 4.5 and 3.0, whose cumulative release percentages in the three simulated solutions were 17.14%, 55.23%, and 79.79%, respectively, in crosslinking duration 2 h. X-ray diffraction, scanning electron microscopy, and transmission electron microscopy analysis revealed that genipin-crosslinking improved the regularity and compactness of coacervate structure, whereas confocal laser scanning microscopy observation indicated that O-carboxymethyl chitosan content was possibly the major reason for the different swelling and bovine serum albumin release behavior of the coacervates. It was concluded that the genipin-crosslinked O-carboxymethyl chitosan-gum Arabic coacervate was a potential intestine-targeted delivery system and its delivery performance could be tailored by varying the crosslinking degree and coacervation acidity.

Development of nanoemulsions for topical delivery of vitamin K1

Vitamin K1 (VK1) is a natural and lipophilic compound currently used in dermatological formulations. In this work, nanoemulsions containing VK1 have been proposed to overcome some issues associated to semisolid VK1-incorporating formulations. The study has been focused on the design of a lipid-free aqueous formulation, easy to prepare and with low cost of production. Thus, a simply protocol, using a low-energy method, has been used to spontaneously form the nanoemulsions. The nanoemulsion composition has been optimized to improve its physical stability during storage in different conditions. Then, the possibility to administer VK1-containing nanoemulsions by nebulization without significant alteration of the formulation was tested. Moreover, the VK1 accumulation into the skin layers have been evaluated through permeation experiments on Franz cells, ATR-FITR analysis, confocal laser scanning microscopy (CLSM) observations, and high performance liquid chromatography (HPLC) analysis. The study demonstrated that NEs represent an interesting option for the commercial development of an aqueous spray formulation for the topical delivery of VK1.

Study of Shell-Mold Thermal Resistance: Laboratory Measurements, Estimation from Compact Strip Production Plant Data, and Observation of Simulated Flux-Mold Interface

The slag film that forms between the shell and mold in steel continuous casting is key in regulating the heat transfer between them. Generally, the mechanisms proposed are related to the phenomena associated with the formation of crystals in the solid layer of the film, such as the appearance of internal pores and surface roughness, which decrease phononic conduction through the layer and interfacial gap with the mold, respectively, and the emergence of crystals themselves, which reduce the transmissivity of infrared radiation across the layer. Due to the importance of the solid layer, this study investigates experimentally the effective thermal resistance, R T, between a hot Inconel surface and a cold Cu surface separated by an initially glassy slag disk, made from powders for casting low and medium-carbon steels, denoted as A and B, respectively. In the tests, an initially mirror-polished disk is sandwiched for 10,800 seconds while the Inconel temperature, away from the disk face, is maintained steady at a value, T c, between 973 K and 1423 K (700 °C and 1150 °C)-below the liquidus temperature of the slags. The disks have a thickness, d t, between ~0.7 and 3.2 mm. Over the range of conditions studied, mold slag B shows R T 33 pct larger than slag A, and microscopic observation of disks hints that the greater resistance arises from the larger porosity developed in B. This finding is supported by high-temperature confocal laser scanning microscope observations of the evolution of the surface of slag parallelepipeds encased between Pt sheets, which reveal that during devitrification the film surface moves outward not inward, contrary with what is widely claimed. This behavior would favor contact of the slag with the mold for both kinds of powders. However, in the case of slag A, the crystalline grains growing at or near the surface pack closely together, leaving only few and small empty spaces. In slag B, crystalline grains pack loosely and many and large empty spaces arise in and below the surface. Estimation from plant data shows R T values smaller than measured ones, suggesting that the process film slag thickness must be considerably thinner than those of laboratory disks. However, the difference in thermal resistance of both powders, averaged over the mold length, is close to the dissimilarity found in laboratory.

Preparation of cellulose nanocrystals from asparagus (Asparagus officinalis L.) and their applications to palm oil/water Pickering emulsion

Nano cellulosic materials as promising emulsion stabilizers have attracted great interest in food industry. In this paper, five different sized cellulose nanocrystals (CNC) samples were prepared from stem of Asparagus officinalis L. using the same sulfuric acid hydrolysis conditions but different times (1.5, 2, 2.5, 3.0, and 3.5h). The sizes of these CNC ranged from 178.2 to 261.8nm, with their crystallinity of 72.4–77.2%. The CNC aqueous dispersions showed a typical shear thinning behavior. In a palm oil/water (30/70, v/v) model solution, stable Pickering emulsions were formed with the addition of CNC, and their sizes are in the range of 1–10μm based on the optical and confocal laser scanning microscopy (CLSM) observation. The CNC sample prepared at 3h hydrolysis time, showed a relative efficient emulsion capacity for palm oil droplets, among these CNCs. Other parameters including the CNC, salt, and casein concentrations on the emulsion stability were studied.

Dynamics of biofouling development on the conditioned membrane and its relationship with membrane performance

In this study, the impacts of a conditioning layer on the dynamics of biofouling development were investigated through batch biofouling experiments under the RO cross-flow filtration process. Fouled RO membranes extracted over the course of biofouling were prepared for measurements of extracellular polymeric substances (EPS) and total organic carbon (TOC) and confocal laser scanning microscopy (CLSM) observations. A close relationship between the biofilm dynamics and permeate flux and salt rejection were delineated. The patterns of membrane performance change demonstrated that the dominant mechanism governing the biofouling effects varied with the biofouling stage: concentration polarization dominated the early stage of biofouling, while fouling resistance was the dominant mechanism at the later stage. In addition, compared with the virgin membrane, the conditioning layer led to a severe deterioration in permeate flux and salt rejection. The results of biofilm dynamics showed the enhancement of bacterial initial attachment and biofilm maturation by the conditioning layer. Finally, there were increased biomass accumulation measured by TOC and the ratio of live to dead cells is higher on the bottom than that on the top of the biofilm on the conditioned membrane. In conclusion, conditioning layer had great influence on the counts of attached cells in the early stage of biofouling, but on the EPS concentration in the later stage of biofouling.

Sol-gel system functionalized magnetic nanocubes as remote controlled drug carriers for cooperative tumor-targeted therapy

To develop vehicles for remote controlled anticancer drug release efficiently, we report a remotely triggered drug delivery system based on magnetic nanocubes. The synthesized magnetic nanocubes with average edge length of around 30nm acted as cores, while folic acid (FA) decorated amphiphilic Pluronic® F-127 gels (F127) were employed as the coating layers. The hydrophobic anticancer drug paclitaxel was loaded during the formation of nanocarrier via hydrophobic interaction. The carrier was stable at physiological temperature and paclitaxel released with an alternating magnetic field treatment, owing to the phase change of F127 when environment temperature elevated via magnetocaloric effect. Cell viability assay and confocal laser scanning microscopy observations demonstrated that the loaded paclitaxel could be efficiently released after cellular endocytosis and induced cancer cells apoptosis with a cooperative effect of hyperthermia and chemotherapy thereby. All results suggested that FA-F127 coated magnetic nanocubes would be a promising remotely controlled drug carrier for cooperative cancer therapy.

A small molecule norspermidine in combination with silver ion enhances dispersal and disinfection of multi-species wastewater biofilms.

Detrimental biofilms have become a great concern in many areas due to their strong resistance and insensitivity to traditional antimicrobial agents. Norspermidine is a potent small molecule for biofilm dispersal. In this study, silver ion, a conventional inorganic biocide, was combined with norspermidine and used for control and removal of multi-species biofilms formed by a mixed culture from wastewater treatment systems. Results showed that silver ion (0.01-1mg/L) treatment alone failed to remove the existing wastewater biofilms. Norspermidine at the concentrations of 500-1000μM was capable to disrupt and disperse the existing biofilms with a biofilm reduction of 21-34% after 24-h exposure. The combined treatment with norspermidine (500μM) and silver ion (0.01mg/L) increased biofilm reduction to 48% (24-h exposure). The combined treatment also enhanced biofilm disinfection ratio (82%, 2-h exposure) by 2.0- and 2.6-folds compared to norspermidine (27%) or silver ion (23%) treatment alone, respectively. Confocal laser scanning microscopic (CLSM) observations found that norspermidine could disrupt biofilm matrix and promote biofilm dispersal via breaking down exopolysaccharides. The combined treatment increased the reduction in biofilm cell density and viability, possibly due to the damage of biofilm matrix, enhanced silver ion diffusion in biofilms, and increased biofilm sensitivity. These findings indicate that the combination of a small molecule norspermidine with a traditional biocide silver ion presents a novel strategy to remove and kill biofilms, which have a potential application in addressing wastewater biofilm-related issues.

Controlling effect of fractures on gas accumulation and production within the tight sandstone: A case study on the Jurassic Dibei gas reservoir in the eastern part of the Kuqa foreland basin, China

Using Dibei tight sandstone gas reservoir in the eastern part of the Kuqa foreland basin as an example, this paper discusses tight sandstone reservoir fractures characterization, its effect on storage space and gas flow capacity, and its contribution to gas accumulation, enrichment and production in tight sandstone reservoir by using laser scanning confocal microscope (LSCM) observation, mercury intrusion capillary pressure (MICP) testing, and gas-water two-phase relative permeability testing. The statistics of laser scanning confocal microscopy observation showed that the microstructural fractures width in the Dibei gas reservoir was mainly 8–25 μm, and the associated micro-fractures width was mainly 4–10 μm. Additionally, the throat radius was mainly 1–4 μm. The fractures width was significantly wider than the throat radius that served as the main channel of in gas flow. In addition, it illustrated that the samples with developed fractures became easier for gas to flow under equal porosity condition, because of lower expulsion pressure, higher mercury injection saturation, and increased gas relative permeability based on the physical simulation experiment of gas charging into core samples with saturated water, mercury injection and gas-water two-phase permeability experiments. Furthermore, it had been concluded that the fractures control tight gas in the following aspects: (1) Fractures play a significant role in reservoir property improvement. The isolated pores were linked by the fractures to form connective reservoir spaces, and dissolution is prone to occur along the fractures forming new pores. The fractures with bigger width are reservoir space as well. (2) Fractures increased fluid flow capacity because it decreased the starting pressure gradient, and it increased gas effective permeability. Thus, fractures improved the gas injection efficiency as well as gas production. (3) Fractures that developed in different time and spatial places have different effects on gas accumulation, enrichment, and production in tight sandstone reservoirs.

Potency of Massoia Bark in Combating Immunosuppressed-related Infection.

Background:As part of our search for new potential natural resources to eradicate infection, we have revealed the prominent potency of massoia bark (Massoia aromatica Becc, Lauraceae) in combating immunosuppressed-related infection.Materials and Methods:The extract was prepared by macerating the pulverized dried bark in ethanol 95%, followed by solvent evaporation. The oil was extracted from the dried bark by steam-hydrodistillation of which preparative thin-layer chromatography was performed on the oil to isolate the active constituent, C-10 massoia lactone (ML). Anti-biofilm assay against Candida albicans was conducted on polystyrene 96 wells microtiter plates, followed by a confocal laser scanning microscope observation to get three-dimensional profiles of the affected biofilms. Effects on the hyphae development inoculated on RPMI-1640 agar plates were observed for 7 days. Influences of samples on mice macrophage phagocytosis were examined by an in vitro technique. Samples concentration tested were in the range of 2.0–0.0625 mg/mL and done in triplicate.Results:Massoia bark extracts (oil and solid phase) and ML exhibited promising activities as anti-biofilm against C. albicans at IC50 0.074% v/v, 271 μg/mL and 0.026 μg/mL, respectively. The ML did not inhibit the hyphae development at the concentration tested; however, the extracts showed inhibition at 62.5 μg/mL. Macrophage phagocytosis stimulation was correlated to the ML content.Conclusion:Massoia bark is potential to be developed as anti-infective in immunosuppressed condition of which the C10 ML (C10H16O2) plays a major role in exerting activity.SUMMARY Massoia bark extracts (oily and solid phase) and C-10 Massoia lactone exhibited promising activities as antibiofilm against Candida albicans at IC50 are 0.074 %v/v, 271 μg/mL and 0.026 μg/mL respectively. The major constituent, C-10 Massoia lactone (C10H16O2) plays major role in exerting anticandida activity and potentially acts as an immunomodulator as well. However extracts showed inhibition of hyphae development of C. albicans which showed no correlation to the content of the Massoia lactone. Abbreviations used: GC/MS: Gas Chromatography/Mass Spectrometry, ML: Massoia Lactone, TLC: Thin Layer Chromatography, ATCC: American Type Culture Collection, RPMI: Roswell Park Memorial Institute, PBS: Phosphate Buffer Sterile, LSM: Laser scanning microscope, DMSO: Dimethyl sulfoxide, UV: Ultra violet, SDB: Sabouraud dextrose agar, MeOH: Methanol, LB: Luria Bertani, EtOAc: Ethyl acetate, CLSM: Confocal Laser Scanning Microscope, PI: Propidium iodide.

Effects of Fibronectin Coating on Bacterial and Osteoblast Progenitor Cells Adherence in a Co-culture Assay.

Bacterial adherence to the surface of implants functionalized with cell-adhesive biomolecules is a critical first step of infection development. This study was designed to determine how the immobilization of human plasmatic fibronectin (pFN) could impact bacterial and osteoblast cells interaction with the surface during concomitant exposition to the two cell-types. Calibrated suspensions of P. aeruginosa PAOI or S. aureus CIP4.83 bacteria and STRO-1+A osteoblast progenitor cells were mixed, co-seeded on glass coverslips coated or not with pFN and incubated at 37 °C. After 3 h of co-culture, the presence of bacteria did not modify the STRO-1+A cells adherence to glass. pFN coating significantly enhanced STRO-1+A cells, CIP4.83 and PAOI adherence to glass and bacterial interaction with STRO-1+A cells. Confocal laser scanning microscopy observations revealed that cells on the pFN-coated substrate exhibited a greater spreading, better organized network of cytoskeletal filaments, and an increased cellular FN expression than cells on the uncoated substrate. The use of fluorescently labeled pFN showed that adherent STRO-1+A cells were able to remodel and to concentrate coated pFN at the cells surface. Thus, the use of FN coating could increase the risk of bacterial adherence to the material surface, acting either directly onto the coating layer or indirectly on adherent osteoblastic cells. This may increase the infection risk in the presence of bacterial contamination.