Confocal Laser Scanning Microcopy Research Articles

Staphylococcus epidermidis biofilm assembly and self-dispersion: bacteria and matrix dynamics.

Staphylococcus epidermidis, despite being a commensal of human skin and mucosa, is a major nosocomial pathogen implicated in device-associated infections. The dissemination of infection to other body sites is related to biofilm dispersal. This study focused on the dispersion stage of S. epidermidis CIP 444 biofilm, with the assessment of biofilm matrix composition in a time-dependent experiment (7days extended) with 3 independent repetitions, using confocal laser scanning microcopy (CLSM) in association with ZEN 3.4 blue edition, COMSTAT, and ImageJ software. SYTO-9, propidium iodide (PI), DID'OIL, FITC, and calcofluor white M2R (CFW) were used to stain biofilm components. The results indicated that the biomass of dead cells increased from 15.18 ± 1.81 µm3/µm2 (day 3) to 23.15 ± 6.075 µm3/µm2 (day 4), along with a decrease in alive cells' biomass from 22.75 ± 2.968 µm3/µm2 (day 3) to 18.95 ± 5.713 µm3/µm2 (day 4). When the intensities were measured after marking the biofilm components, in a 24-h-old biofilm, polysaccharide made up the majority of the investigated components (52%), followed by protein (18.9%). Lipids make up just 11.6% of the mature biofilm. Protein makes up the largest portion (48%) of a 4-day-old biofilm, followed by polysaccharides (37.8%) and lipids (7.27%). According to our findings, S. epidermidis CIP 444 dispersion occurred on day 4 of incubation, and new establishment of the biofilm occurred on day 7. Remarkable changes in biofilm composition will pave the way for a new approach to understanding bacterial strategies inside biofilms and finding solutions to their impacts in the medical field.

Topical non-aqueous nanoemulsion of Alpinia galanga extract for effective treatment in psoriasis: In vitro and in vivo evaluation

Non-aqueous nanoemulsion (NANE) of Alpinia galanga extract (AGE) was prepared using Palmester 3595 (MCT oil) as oil phase, Cremophor RH 40-Transcutol P® as surfactant-co-surfactant (Smix), and glycerin as non-aqueous polar continuous phase. The composition was optimized by applying three-level, four factor Box-Behnken design (BBD). The mean droplet size and zeta potential of the optimized AGE NANE was found to be 60.81 ± 18.88 nm and −7.99 ± 4.14 mV, respectively. The ex vivo permeation studies of AGE NANE and AGE per se on porcine skin reported flux of 125.58 ± 8.36 µg/cm2 h−1 and 12.02 ± 1.64 µg/cm2 h−1, respectively. Therefore, the enhancement ratio has shown 10-folds increase in the flux for AGE NANE when compared to extract per se. Later, confocal laser scanning microcopy confirmed that AGE NANE were able to penetrate into skin’s stratum by trans-follicular transport mechanism. The stability studies of AGE NANE confirmed its stability at 30 ± 2 °C/75 ± 5 % RH and 5 ± 3 °C. The efficacy of AGE NANE was evaluated in vivo on imiquimod (IMQ) induced mouse model. The mice treated with low and high doses of AGE NANE (groups VI and VII) showed significant (p < 0.05) amelioration of psoriasis. Results of histopathology indicated reduction in psoriasis area severity index in AGE NANE treated mice (group VI and group VII).

Probing the key foulants and membrane fouling under increasing salinity in anaerobic osmotic membrane bioreactors for low-strength wastewater treatment

Anaerobic osmotic membrane bioreactors (AnOMBRs) have attracted much attention in recent years due to their ability to produce high-quality effluent with low energy demand. Here, the performance of a thin film composite (TFC) forward osmosis (FO) membrane in an AnOMBR system is evaluated, and the key membrane foulants are examined by analyzing the characteristics of the mixed liquor and the fouling layer. As the experiment progressed, the water flux declined due to membrane fouling and salt accumulation in the bioreactor, and reached approximately 3.5 LMH by the end of each operating cycle where membrane cleaning was adopted. The conductivity increased from 3.3 mS/cm to 23.6 mS/cm over the 28-day treatment. The contaminant rejection rates of total organic carbon, total phosphorous and ammonia-nitrogen were more than 93%, 98% and 60%, respectively. Results also suggested that extracellular polymeric substances (EPS) was the major contributor to membrane fouling, and the increase of EPS in the mixed liquor with salinity was primarily composed of polysaccharides. The comprehensive characterization of the fouling layer via X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy and confocal laser scanning microcopy (CLSM) revealed the coexistence of biofouling and organic fouling. The CLSM images further demonstrated the prevalence of β polysaccharides, proteins and microorganisms on the fouled TFC FO membrane surface. In addition, the flow cytometric analysis indicated that the foulant sample was “bacterial viable”, the large number of bacteria in suspension (96.61% live cells) maybe corresponded to the high EPS content in the bulk mixed liquor.

Converting Immune Cold into Hot by Biosynthetic Functional Vesicles to Boost Systematic Antitumor Immunity.

SummaryImmune cold tumor characterized by low immunogenicity, insufficient and exhausted tumor-infiltrating lymphocytes, and immunosuppressive microenvironment is the main bottleneck responsible for low patient response rate of immune checkpoint blockade. Here, we developed biosynthetic functional vesicles (BFVs) to convert immune cold into hot through overcoming hypoxia, inducing immunogenic cell death, and immune checkpoint inhibition. The BFVs present PD1 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the surface, whereas load catalase into their inner core. The TRAIL can specifically induce immunogenic death of cancer cells to initiate immune response, which is further synergistically strengthened by blocking PD1/PDL1 checkpoint signal through ectogenic PD1 proteins on BFVs. The catalase can produce O2 to overcome tumor hypoxia, in turn to increase infiltration of effector T cells while deplete immunosuppressive cells in tumor. The BFVs elicit robust and systematic antitumor immunity, as demonstrated by significant regression of tumor growth, prevention of abscopal tumors, and excellent inhibition of lung metastasis.

Antibacterial and antibiofilm effects of α-humulene against Bacteroides fragilis

The rapid increase in antibiotic resistance has prompted the discovery of drugs that reduce antibiotic resistance or new drugs that are an alternative to antibiotics. Plant extracts have health benefits and may also exhibit antibacterial and antibiofilm activities against pathogens. This study determined the antibacterial and antibiofilm effects of α-humulene extracted from plants against enterotoxigenic Bacteroides fragilis (ETBF), which causes inflammatory bowel disease. The minimum inhibitory concentration (MIC) and biofilm inhibitory concentration (BIC) of α-humulene for B. fragilis were 2 μg/mL, and the biofilm eradication concentration (Pumbwe et al.) was in the range of 8-32 μg/mL. The XTT reduction assay confirmed that the cellular metabolic activity in biofilm rarely occurred at 8-16 μg/mL concentration. In addition, the biofilm inhibition by α-humulene was also detected via confocal laser scanning microcopy. Quantitative real-time polymerase chain reaction was also used to investigate the effect of α-humulene on the expression of resistance-nodulation-cell division (RND)-type multidrug efflux pump (bmeB1 and bmeB3) genes. As a result of qPCR, α-humulene significantly reduced the expression of bmeB1 and bmeB3 genes. This study demonstrates the potential therapeutic application of α-humulene to inhibit the growth of B. fragilis cells and biofilms, and contribute to the expansion of knowledge about biofilm medicine.

Antibiofilm and antibacterial activities of repurposing auranofin against Bacteroides fragilis.

The aim of this study is to evaluate the antibiofilm and antibacterial effects of auranofin against WT-ETBF, rETBF, WT-NTBF and clinically isolated Bacteroides fragilis strains. The minimum inhibitory concentration and biofilm inhibitory concentration of 0.25μg/ml for auranofin against B. fragilis were determined, and the biofilm eradication concentration was 1μg/ml. At an auranofin concentration of 0.5μg/ml, little cellular metabolic activity was found. Confocal laser scanning microcopy results confirmed the inhibition of biofilm by auranofin. The effects of auranofin on the outer membrane protein (ompA) gene and the RND-type efflux pump (bmeB3) gene were investigated using quantitative real-time polymerase chain reaction (qRT-PCR). The qRT-PCR results showed that treatment with auranofin significantly reduced the gene expression compared to controls without auranofin. These data indicate the applicability of auranofin as a repurposed drug due to its inhibitory effect against biofilm formation of B. fragilis. Therefore, our study demonstrates that auranofin, already approved for human use, is a promising drug that has strong antibiofilm and antibacterial activity against B. fragilis.

Mechanism of antimicrobial peptide NP-6 from Sichuan pepper seeds against E. coli and effects of different environmental factors on its activity.

A novel antimicrobial peptide named NP-6 was identified in our previous work. Here, the mechanisms of the peptide against Escherichia coli (E. coli) were further investigated, as well as the peptide's resistance to temperature, pH, salinity, and enzymes. The transmission electron microscopy (TEM), confocal laser scanning microcopy (CLSM), and flow cytometric (FCM) analysis, combined with measurement of released K+, were performed to evaluate the effect of NP-6 E. coli cell membrane. The influence of NP-6 on bacterial DNA/RNA and enzyme was also investigated. The leakage of K+ demonstrated that NP-6 could increase the permeability of E. coli cell membrane. The ATP leakage, FCM, and CLSM assays suggested that NP-6 caused the disintegration of bacterial cell membrane. The TEM observation indicated that NP-6 could cause the formation of empty cells and debris. Besides, the DNA-binding assay indicated that NP-6 could bind with bacterial genomic DNA in a way that ethidium bromide (EB) did, and suppress the migration of DNA/RNA in gel retardation. Additionally, NP-6 could also affect the activity of β-galactosidase. Finally, the effect of different surroundings such as heating, pH, ions, and protease on the antimicrobial activity of NP-6 against E. coli was also investigated. Results showed that the peptide was heat stable in the range of 60~100 °C and performed well at pH 6.0~8.0. However, the antimicrobial activity of NP-6 decreased significantly in the presence of Mg2+/Ca2+, and after incubation with trypsin/proteinase K. The results will provide a theoretical support in the further application of NP-6.

Development and characterization of an oral multispecies biofilm implant flow chamber model.

Peri-implant infections are the most common cause of implant failure in modern dental implantology. These are caused by the formation of biofilms on the implant surface and consist of oral commensal and pathogenic bacteria, which harm adjacent soft and hard tissues and may ultimately lead to implant loss. In order to improve the clinical situation, there has to be a better understanding of biofilm formation on abiotic surfaces. Therefore, we successfully developed a system to cultivate an oral multispecies biofilm model in a flow chamber system, optimized for the evaluation of biofilm formation on solid materials by direct microscopic investigation. The model contains four relevant oral bacterial species: Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis in ratios similar to the native situation. The reliability of the developed “Hanoverian Oral Multispecies Biofilm Implant Flow Chamber” (HOBIC) model was verified. Biofilm volume and live/dead distribution within biofilms were determined by fluorescence staining and confocal laser scanning microcopy (CLSM). The individual species distribution was analyzed using quantitative real time PCR with propidium monoazide pretreatment (PMA-qRT-PCR) and by urea-NaCl fluorescence in situ hybridization (urea-NaCl-FISH). This in vitro model may be used to analyze biofilm formation on dental implants in more detail and to develop future implant systems with improved material properties.

Drug-nanoencapsulated PLGA microspheres prepared by emulsion electrospray with controlled release behavior.

The development of modern therapeutics has raised the requirement for controlled drug delivery system which is able to efficiently encapsulate bioactive agents and achieve their release at a desired rate satisfying the need of the practical system. In this study, two kind of aqueous model drugs with different molecule weight, Congo red and albumin from bovine serum (BSA) were nano-encapsulated in poly (dl-lactic-co-glycolic acid) (PLGA) microspheres by emulsion electrospray. In the preparation process, the aqueous phase of drugs was added into the PLGA chloroform solution to form the emulsion solution. The emulsion was then electrosprayed to fabricate drug-nanoencapsulated PLGA microspheres. The morphology of the PLGA microspheres was affected by the volume ratio of aqueous drug phase and organic PLGA phase (Vw/Vo) and the molecule weight of model drugs. Confocal laser scanning microcopy showed the nanodroplets of drug phase were scattered in the PLGA microspheres homogenously with different distribution patterns related to Vw/Vo. With the increase of the volume ratio of aqueous drug phase, the number of nanodroplets increased forming continuous phase gradually that could accelerate drug release rate. Moreover, BSA showed a slower release rate from PLGA microspheres comparing to Congo red, which indicated the drug release rate could be affected by not only Vw/Vo but also the molecule weight of model drug. In brief, the PLGA microspheres prepared using emulsion electrospray provided an efficient and simple system to achieve controlled drug release at a desired rate satisfying the need of the practices.

In vitro biofilm development of Streptococcus pneumoniae and formation of choline-binding protein-DNA complexes.

Extracellular deoxyribonucleic acid (eDNA) is an essential component of bacterial biofilm matrices, and is required in their formation and maintenance. Extracellular DNA binds to exopolysaccharides or extracellular proteins, affording biofilms greater structural integrity. Recently, we reported evidence of intercellular eDNA-LytC complexes in pneumococcal biofilms. The LytC lysozyme is a member of the choline-binding family of proteins (CBPs) located on the pneumococcal surface. The present work shows that other CBPs, i.e. LytA, LytB, Pce, PspC and CbpF, which have a pI between 5 and 6, can bind DNA in vitro. This process requires the presence of divalent cations other than Mg(2+). This DNA binding capacity of CBPs appears to be independent of their enzymatic activity and, at least in the case of LytA, does not require the choline-binding domain characteristic of CBPs. Positively charged, surface-exposed, 25 amino acid-long peptides derived from the catalytic domain of LytB, were also found capable of DNA binding through electrostatic interactions. Confocal laser scanning microcopy revealed the existence of cell-associated LytB-eDNA complexes in Streptococcus pneumoniae biofilms. These and other findings suggest that these surface-located proteins of S. pneumoniae could play roles of varying importance in the colonization and/or invasion of human host where different environmental conditions exist.

PLGA/alginate composite microspheres for hydrophilic protein delivery.

Poly(lactic-co-glycolic acid) (PLGA) microspheres and PLGA/alginate composite microspheres were prepared by a novel double emulsion and solvent evaporation technique and loaded with bovine serum albumin (BSA) or rabbit anti-laminin antibody protein. The addition of alginate and the use of a surfactant during microsphere preparation increased the encapsulation efficiency and reduced the initial burst release of hydrophilic BSA. Confocal laser scanning microcopy (CLSM) of BSA-loaded PLGA/alginate composite microspheres showed that PLGA, alginate, and BSA were distributed throughout the depths of microspheres; no core/shell structure was observed. Scanning electron microscopy revealed that PLGA microspheres erode and degrade more quickly than PLGA/alginate composite microspheres. When loaded with anti-laminin antibody, the function of released antibody was well preserved in both PLGA and PLGA/alginate composite microspheres. The biocompatibility of PLGA and PLGA/alginate microspheres were examined using four types of cultured cell lines, representing different tissue types. Cell survival was variably affected by the inclusion of alginate in composite microspheres, possibly due to the sensitivity of different cell types to excess calcium that may be released from the calcium cross-linked alginate.

Combined use of variable pressure scanning electron microscopy and confocal laser scanning microscopy best reveal microstructure of comminuted meat gels

Observing the distribution of protein and fat phases in comminuted meat products can be helpful to understanding the mechanisms of texture development and fat/water binding. In this study variable pressure scanning electron microscopy (VP-SEM) was compared to conventional scanning electron microscopy (SEM), and contrasted with confocal laser scanning microcopy (CLSM), as tools to characterize gel morphology of cooked meat batters or non-fat pastes. Gel morphology was varied by inclusion of whey protein isolate (WPI) that gels only at high temperature, in partial substitution of myofibrillar protein (MFP). CLSM (magnification:10–1000×) revealed that, when no WPI was added, a homogeneous gel structure was produced enmeshing small, well-distributed fat particles. Substituting 30 g/100 g MFP by WPI produced a coarse gel structure with clear microphase separation of fat. VP-SEM (magnification:1000–2000×) enabled visualization of small pore structure of gel matrix whereas SEM obscured details of this, as well as of the relationship between fat globules and gel matrix that were visible by VP-SEM. Since meat gels properties can be affected by multiple morphological features, visible only at different levels of magnification, the relationships between microstructure and important properties of meat gels can be most advantageously observed when both VP-SEM and CLSM are used in tandem.

Attempt of correlative observation of morphological synaptic connectivity by combining confocal laser-scanning microscope and FIB-SEM for immunohistochemical staining technique.

Ten years have passed since a serial block-face scanning electron microscopy (SBF-SEM) method was developed [1]. In this innovative method, samples were automatically sectioned with an ultramicrotome placed inside a scanning electron microscope column, and the block surfaces were imaged one after another by SEM to capture back-scattered electrons. The contrast-inverted images obtained by the SBF-SEM were very similar to those acquired using conventional TEM. SFB-SEM has made easy to acquire image stacks of the transmission electron microscopy (TEM) in the mesoscale, which is taken with the confocal laser-scanning microcopy(CF-LSM).Furthermore, serial-section SEM has been combined with the focused ion beam (FIB) milling method [2]. FIB-incorporated SEM (FIB-SEM) has enabled the acquisition of three-dimensional images with a higher z-axis resolution com- pared to ultramicrotome-equipped SEM.We tried immunocytochemistry for FIB-SEM and correlated this immunoreactivity with that in CF-LSM. Dendrites of neurons in the rat neostriatum were visualized using a recombinant viral vector. Moreover, the thalamostriatal afferent terminals were immunolabeled with Cy5 fluorescence for vesicular glutamate transporter 2 (VGluT2). After detection of the sites of terminals apposed to the dendrites by using CF-LSM, GFP and VGluT2 immunoreactivities were further developed for EM by using immunogold/silver enhancement and immunoperoxidase/diaminobenzidine (DAB) methods, respectively.We showed that conventional immuno-cytochemical staining for TEM was applicable to FIB-SEM. Furthermore, several synaptic contacts, which were thought to exist on the basis of CF-LSM findings, were confirmed with FIB-SEM, revealing the usefulness of the combined method of CF-LSM and FIB-SEM.

Fucoidan Reduces Secretion and Expression of Vascular Endothelial Growth Factor in the Retinal Pigment Epithelium and Reduces Angiogenesis In Vitro

Fucoidan is a polysaccharide isolated from brown algae which is of current interest for anti-tumor therapy. In this study, we investigated the effect of fucoidan on the retinal pigment epithelium (RPE), looking at physiology, vascular endothelial growth factor (VEGF) secretion, and angiogenesis, thus investigating a potential use of fucoidan for the treatment of exudative age-related macular degeneration. For this study, human RPE cell line ARPE-19 and primary porcine RPE cells were used, as well as RPE/choroid perfusion organ cultures. The effect of fucoidan on RPE cells was investigated with methyl thiazolyl tetrazolium – assay, trypan blue exclusion assay, phagocytosis assay and a wound healing assay. VEGF expression was evaluated in immunocytochemistry and Western blot, VEGF secretion was evaluated in ELISA. The effect of fucoidan on angiogenesis was tested in a Matrigel assay using calcein-AM vital staining, evaluated by confocal laser scanning microcopy and quantitative image analysis. Fucoidan displays no toxicity and does not diminish proliferation or phagocytosis, but reduces wound healing in RPE cells. Fucoidan decreases VEGF secretion in RPE/choroid explants and RPE cells. Furthermore, it diminishes VEGF expression in RPE cells even when co-applied with bevacizumab. Furthermore, fucoidan reduces RPE-supernatant- and VEGF-induced angiogenesis of peripheral endothelial cells. In conclusion, fucoidan is a non-toxic agent that reduces VEGF expression and angiogenesis in vitro and may be of interest for further studies as a potential therapy against exudative age-related macular degeneration.

Development of a non-viral gene delivery vector based on the dynein light chain Rp3 and the TAT peptide

Gene therapy and DNA vaccination trials are limited by the lack of gene delivery vectors that combine efficiency and safety. Hence, the development of modular recombinant proteins able to mimic mechanisms used by viruses for intracellular trafficking and nuclear delivery is an important strategy. We designed a modular protein (named T-Rp3) composed of the recombinant human dynein light chain Rp3 fused to an N-terminal DNA-binding domain and a C-terminal membrane active peptide, TAT. The T-Rp3 protein was successfully expressed in Escherichia coli and interacted with the dynein intermediate chain in vitro. It was also proven to efficiently interact and condense plasmid DNA, forming a stable, small (∼100nm) and positively charged (+28.6mV) complex. Transfection of HeLa cells using T-Rp3 revealed that the vector is highly dependent on microtubule polarization, being 400 times more efficient than protamine, and only 13 times less efficient than Lipofectamine 2000™, but with a lower cytotoxicity. Confocal laser scanning microcopy studies revealed perinuclear accumulation of the vector, most likely as a result of transport via microtubules. This study contributes to the development of more efficient and less cytotoxic proteins for non-viral gene delivery.

Comparative study of Pluronic® F127-modified liposomes and chitosan-modified liposomes for mucus penetration and oral absorption of cyclosporine A in rats

Liposomes modified using cationic and hydrophilic nonionic polymers are 2 popular carriers for improving oral drug absorption. Cationic polymer-modified liposomes can adhere to the intestinal wall mucus (mucoadhesive type), while liposomes modified using hydrophilic nonionic polymers can penetrate across the mucus barrier (mucus-penetrating type). Chitosan-modified liposomes (CS-Lip, mucoadhesive type) and Pluronic(®) F127-modified liposomes (PF127-Lip, mucus-penetrating type) were engineered to investigate the differences between these mucoadhesive and mucus-penetrating systems in oral absorption of a poorly soluble drug, cyclosporine A (CyA). Stability of CS-Lip and PF127-Lip was studied in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). The intestinal mucus adhesion or penetration of liposomes was studied by confocal laser scanning microcopy and fluorophotometry using coumarin 6 as the fluorescent probe. The oral absorption of CyA-loaded liposomes was also studied in Sprague-Dawley rats. In vitro and in vivo studies revealed that CS-Lip tended to aggregate in SIF, to be trapped by mucus, to remain mainly in the upper portion of the intestinal tract, and to show limited penetration ability. In contrast, PF127-Lip were more stable in the SIF and SGF, were found throughout the intestinal tract, and were able to penetrate the mucus layers to reach the epithelial surface. Pharmacokinetic analysis in rats showed that the Cmax and AUC0-t of PF127-Lip were 1.73- and 1.84-fold higher than those of CS-Lip, respectively (P<0.05). In conclusion, the stability and mucus-penetrating ability of PF127-Lip in the gastrointestinal tract rendered it more suitable than the mucoadhesive CS-Lip for oral delivery CyA.

Correlative analysis of immunoreactivity in confocal laser-scanning microscopy and scanning electron microscopy with focused ion beam milling

Recently, three-dimensional reconstruction of ultrastructure of the brain has been realized with minimal effort by using scanning electron microscopy (SEM) combined with focused ion beam (FIB) milling (FIB-SEM). Application of immunohistochemical staining in electron microscopy (EM) provides a great advantage in that molecules of interest are specifically localized in ultrastructures. Thus, we applied immunocytochemistry for FIB-SEM and correlated this immunoreactivity with that in confocal laser-scanning microcopy (CF-LSM). Dendrites of medium-sized spiny neurons in the rat neostriatum were visualized using a recombinant viral vector, which labeled the infected neurons with membrane-targeted GFP in a Golgi stain-like fashion. Moreover, the thalamostriatal afferent terminals were immunolabeled with Cy5 fluorescence for vesicular glutamate transporter 2 (VGluT2). After detection of the sites of terminals apposed to the dendrites by using CF-LSM, GFP and VGluT2 immunoreactivities were further developed for EM by using immunogold/silver enhancement and immunoperoxidase/diaminobenzidine (DAB) methods, respectively. In contrast-inverted FIB-SEM images, silver precipitations and DAB deposits were observed as fine dark grains and diffuse dense profiles, respectively, indicating that these immunoreactivities were as easily recognizable as those in the transmission electron microscopy (TEM) images. Furthermore, in the sites of interest, some appositions displayed synaptic specializations of an asymmetric type. Thus, the present method was useful in the three-dimensional analysis of immunocytochemically differentiated synaptic connections in the central neural circuit.

Release kinetics of gold nanoparticles from collagen microcapsules by total reflection X-ray fluorescence

One of the main challenges in nanotechnology based drug delivery systems is the design of novel materials for patient specific therapeutic efficacy. This can be achieved taking into account changes in the expression profiles of related proteins. Here, we focus on collagen type I micro-capsules for releasing molecules using a biological stimulus elicited by over-expressed collagenolytic matrix metalloproteinase I (MMPI). We have carried out label-free real time monitoring of the release kinetics of gold nanoparticles induced by MMPI in order to define the pore opening process by synchrotron radiation based total reflection X-ray fluorescence. Scanning electron and confocal laser scanning microcopy investigations have been carried out in order to further characterize the interaction between collagen micro-capsules and MMP1. The results enable to predict and to engineer the release time for drug molecules of different sizes. Moreover, the proposed label-free approach can be adopted for the visualization of pore formation and of release kinetics for nano-engineered polymeric capsules.

The Spatial Architecture of Bacillus subtilis Biofilms Deciphered Using a Surface-Associated Model and In Situ Imaging

The formation of multicellular communities known as biofilms is the part of bacterial life cycle in which bacteria display cooperative behaviour and differentiated phenotypes leading to specific functions. Bacillus subtilis is a Gram-positive bacterium that has served for a decade as a model to study the molecular pathways that control biofilm formation. Most of the data on B. subtilis biofilms have come from studies on the formation of pellicles at the air-liquid interface, or on the complex macrocolonies that develop on semi-solid nutritive agar. Here, using confocal laser scanning microcopy, we show that B. subtilis strains of different origins are capable of forming biofilms on immersed surfaces with dramatically protruding “beanstalk-like” structures with certain strains. Indeed, these structures can reach a height of more than 300 µm with one undomesticated strain from a medical environment. Using 14 GFP-labeled mutants previously described as affecting pellicle or complex colony formation, we have identified four genes whose inactivation significantly impeded immersed biofilm development, and one mutation triggering hyperbiofilm formation. We also identified mutations causing the three-dimensional architecture of the biofilm to be altered. Taken together, our results reveal that B. subtilis is able to form specific biofilm features on immersed surfaces, and that the development of these multicellular surface-associated communities involves regulation pathways that are common to those governing the formation of pellicle and/or complex colonies, and also some specific mechanisms. Finally, we propose the submerged surface-associated biofilm as another relevant model for the study of B. subtilis multicellular communities.

Novel mucus-penetrating liposomes as a potential oral drug delivery system: preparation, in vitro characterization, and enhanced cellular uptake

BackgroundThe aim of this study was to investigate the intestinal mucus-penetrating properties and intestinal cellular uptake of two types of liposomes modified by Pluronic F127 (PF127).MethodsThe two types of liposomes, ie, PF127-inlaid liposomes and PF127-adsorbed liposomes, were prepared by a thin-film hydration method followed by extrusion, in which coumarin 6 was loaded as a fluorescence marker. A modified Franz diffusion cell mounted with the intestinal mucus of rats was used to study the diffusion characteristics of the two types of PF127 liposomes. Cell uptake studies were conducted in Caco-2 cells and analyzed using confocal laser scanning microcopy as well as flow cytometry.ResultsThe diffusion efficiency of the two types of PF127-modified liposomes through intestinal rat mucus was 5–7-fold higher than that of unmodified liposomes. Compared with unmodified liposomes, PF127-inlaid liposomes showed significantly higher cellular uptake of courmarin 6. PF127-adsorbed liposomes showed a lower cellular uptake. Moreover, and interestingly, the two types of PF127-modified liposomes showed different cellular uptake mechanisms in Caco-2 cells.ConclusionPF127-inlaid liposomes with improved intestinal mucus-penetrating ability and enhanced cellular uptake might be a potential carrier candidate for oral drug delivery.