Lydia-marie Joubert - Academia.edu (original) (raw)

Papers by Lydia-marie Joubert

Stem Cells and Development, 2014

In mammals, the permanence of many forms of hearing loss is the result of the inner ear's inabili... more In mammals, the permanence of many forms of hearing loss is the result of the inner ear's inability to replace lost sensory hair cells. Here, we apply a differentiation strategy to guide human embryonic stem cells (hESCs) into cells of the otic lineage using chemically defined attached-substrate conditions. The generation of human otic progenitor cells was dependent on fibroblast growth factor (FGF) signaling, and protracted culture led to the upregulation of markers indicative of differentiated inner ear sensory epithelia. Using a transgenic ESC reporter line based on a murine Atoh1 enhancer, we show that differentiated hair cell-like cells express multiple hair cell markers simultaneously. Hair cell-like cells displayed protrusions reminiscent of stereociliary bundles, but failed to fully mature into cells with typical hair cell cytoarchitecture. We conclude that optimized defined conditions can be used in vitro to attain otic progenitor specification and sensory cell differentiation.

Introduction: The initial steps in the process of high-resolution scanning electron microscopy (S... more Introduction: The initial steps in the process of high-resolution scanning electron microscopy (SEM) of biological samples are well described [1, 2] and involve various steps of aldehyde fixation and dehydration, followed by critical point drying with liquid CO 2 , or chemical drying with hexamethyldisilazane, or cryo-fixation and lyophilization. In all cases the samples need to be mounted onto an instrument-compatible platform (i.e. the specimen stub) with a conductive adhesive layer, which may consist of carbon or copper tape, colloidal graphite or silver paint. Finally the specimen is coated with a thin conductive film of metal, typically gold and/or palladium, iridium, or platinum. In cases where heavy metals may interfere with the secondary electron (SE) or backscattered electron (BSE) signals needed for the detection of valuable information, carbon evaporation can be used to provide a conductive coating on the sample. "Cleanliness is next to godliness" Precision meas...

Eukaryotic cell, Jan 10, 2015

Aspergillus fumigatus is commonly responsible for lethal fungal infections among immunosuppressed... more Aspergillus fumigatus is commonly responsible for lethal fungal infections among immunosuppressed individuals. A. fumigatus forms biofilm communities that are of increasing biomedical interest due to the association of biofilms with chronic infections and their increased resistance to antifungal agents and host immune factors. Understanding the composition of microbial biofilms and the extracellular matrix is important to understanding function and, ultimately, to developing strategies to inhibit biofilm formation. We implemented a solid-state NMR approach to define compositional parameters of the A. fumigatus extracellular matrix (ECM) when biofilms are formed in RPMI 1640 nutrient medium. Whole biofilm and isolated matrix networks were also characterized by electron microscopy, and matrix proteins were identified through protein gel analysis. The (13)C NMR results defined and quantified the carbon contributions in the insoluble ECM, including carbonyls, aromatic carbons, polysacch...

Acid Mine Drainage needs to be treated, before it is recharged in rivers and dams. The biological... more Acid Mine Drainage needs to be treated, before it is recharged in rivers and dams. The biological treatment of AMD can be applied using degradation products of cellulose, e. g. Volatile Fatty Acids as carbon sources. The aim of this study was to demonstrate that microbes originating from compost and rumen are able to ferment Grass Cuttings to produce Volatile

The complex character of distillery wastewater comprises high concentrations of sugars, lignins, ... more The complex character of distillery wastewater comprises high concentrations of sugars, lignins, hemicelluloses, dextrans, resins, polyphenols and organic acids which are recalcitrant to biodegradation. Microorganisms play a key role in the production and degradation of organic matter, environmental pollutants, and cycling of nutrients and metals. Due to their short life cycles microbes respond rapidly to external nutrient loading, with major

PLoS Pathogens, 2012

Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and h... more Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.

Water SA, 2009

ABSTRACT Copyright: 2007 Water Research Commission A series of lignocellulosic fungi, capable of ... more ABSTRACT Copyright: 2007 Water Research Commission A series of lignocellulosic fungi, capable of cellulase and/or xylanase production, were isolated from soil to be used for cellulose degradation and nitrate removal from nitrate-rich wastewater in simple one-stage anaerobic bioreactors containing grass cuttings as source of cellulose. The fungal consortium, consisting of six hyphomycetous isolates, some of which belong to the genera Fusarium, Mucor and Penicillium, was able to remove a significant portion of the nitrate from the treated water. The results were obtained for three bioreactors, i.e. FR, FRp and AFRp, differing in volume and mode of grass addition. Bioreactor AFRp received autoclaved grass, instead of non-autoclaved grass containing natural microbial consortia, as supplied to FR and FRp. Nitrate removal in FR amounted to 89% removal efficiency, while this was 65% and 67% in FRp and AFRp, respectively. The residual chemical oxygen demand (COD) concentration in FR was higher than 600 mg/ℓ, while it was 355 and 379 mg/ℓ in FRp and AFRp, respectively. The similar nitrate removal results for AFRp and FRp indicated that the micro-organisms attached to grass cuttings did not seem to affect the nitrate removal in the reactor. This observation has led to the conclusion that the fungal consortium was, except for being able to degrade cellulose within the grass cuttings, also responsible for nitrate removal from the synthetic nitrate-rich wastewater

Tissue Engineering Part A, 2014

A biocompatible hydrogel consisting of extracellular matrix (ECM) from human tendons is described... more A biocompatible hydrogel consisting of extracellular matrix (ECM) from human tendons is described as a potential scaffold for guided tissue regeneration and tissue engineering purposes. Lyophilized decellularized tendons were milled and enzymatically digested to form an ECM solution. The ECM solution properties are assessed by proteome analysis with mass spectrometry, and the material's rheological properties are determined as a function of frequency, temperature, and time. In vivo application of the gel in a rat model is assessed for remodeling and host cell repopulation. Histology for macrophage invasion, fibroblast repopulation, and nanoscale properties of the gel is assessed. Gel interaction with multipotent adipoderived stem cells (ASCs) is also addressed in vitro to assess possible cytotoxicity and its ability to act as a delivery vehicle for cells. Proteome analysis of the ECM-solution and gel mass spectroscopy identified the most abundant 150 proteins, of which two isoforms of collagen I represented more than 55% of the sample. Rheology showed that storage (G') and loss (G″) of the ECM solution were stable at room temperature but displayed sigmoidal increases after ∼15 min at 37°C, matching macroscopic observations of its thermo responsiveness. G' and G″ of the gel at 1 rad/s were 213.1±19.9 and 27.1±2.4 Pa, respectively. Electron microscopy revealed fiber alignment and good structural porosity in the gel, as well as invasion of cells in vivo. Histology also showed early CD68(+) macrophage invasion throughout the gel, followed by increasing numbers of fibroblast cells. ASCs mixed with the gel in vitro proliferated, indicating good biocompatibility. This ECM solution can be delivered percutaneously into a zone of tendon injury. After injection, the thermoresponsive behavior of the ECM solution allows it to polymerize and form a porous gel at body temperature. A supportive nanostructure of collagen fibers is established that conforms to the three-dimensional space of the defect. This hydrogel holds the distinctive composition specific for tendon ECM, where tissue-specific cues facilitate host cell infiltration and remodeling. The results presented indicate that injectable ECM materials from tendon may offer a promising alternative in the treatment of tendinopathies and acute tendon injuries.

Review of Scientific Instruments, 2011

This paper describes the design and operation of a simple airtight container devised to facilitat... more This paper describes the design and operation of a simple airtight container devised to facilitate the transfer of atmosphere-sensitive samples from a glovebox to the vacuum chamber of an analytical instrument such as a scanning electron microscope. The use of this device for characterizing the microstructure of highly hygroscopic strontium iodide ceramics by scanning electron microscopy is illustrated as an application example.

Nature Cell Biology, 2012

Many of the more than 20 mammalian proteins with N-BAR domains control cell architecture and endo... more Many of the more than 20 mammalian proteins with N-BAR domains control cell architecture and endocytosis by associating with curved sections of the plasma membrane. It is not well understood whether N-BAR proteins are recruited directly by processes that mechanically curve the plasma membrane or indirectly by plasma-membrane-associated adaptor proteins that recruit proteins with N-BAR domains that then induce membrane curvature. Here, we show that externally induced inward deformation of the plasma membrane by cone-shaped nanostructures (nanocones) and internally induced inward deformation by contracting actin cables both trigger recruitment of isolated N-BAR domains to the curved plasma membrane. Markedly, live-cell imaging in adherent cells showed selective recruitment of full-length N-BAR proteins and isolated N-BAR domains to plasma membrane sub-regions above nanocone stripes. Electron microscopy confirmed that N-BAR domains are recruited to local membrane sites curved by nanocones. We further showed that N-BAR domains are periodically recruited to curved plasma membrane sites during local lamellipodia retraction in the front of migrating cells. Recruitment required myosin-II-generated force applied to plasma-membrane-connected actin cables. Together, our results show that N-BAR domains can be directly recruited to the plasma membrane by external push or internal pull forces that locally curve the plasma membrane.

Microscopy and Microanalysis, 2010

ABSTRACT Stem cell research is no longer in its infancy. It has matured into a discipline with fa... more ABSTRACT Stem cell research is no longer in its infancy. It has matured into a discipline with far-reaching applications. When little is known about a subject, the questions people raise are almost invariably ethical. As knowledge grows, however, people become amoral and more concerned with information i.e. more narrowly intellectual [1]. The words of Charles Spurgeon (1834-1992) that 'every generation needs regeneration' takes on new meaning in the era of stem cell biology [2] and there is a sense of urgency to move new developments quickly into the clinical setting. Visualization of stem cells is one of the core set of techniques that are applied to unlock the potential of stem cell biology. Various differentiation protocols revolve around fluorescence microscopy, applying different molecular markers. However, the limited resolution of light microscopy, and an inability to resolve surface features, has put Scanning EM in great demand to fill this gap. It is often also the preferred technique to apply before TEM investigation is started. The additional benefit of Variable pressure (VP) SEM that enabled visualization of hydrated samples, eg biomaterials onto which stem cells are grafted before clinical application, has proved to be an indispensible tool in our recent research. The ethical debate that ensued from Human Embryonic Stem cells (HESC) prompted large efforts to find alternative approaches to use adult stem cells or progenitor stem cells instead [3]. The concept that certain stem cells can trans-differentiate into cell types outside their normal lineage has been raised recently. We currently study adipose stem cells from humans, dogs and mice, and turn these into bone and other stem cells. HESC cells generate cardiomyocytes, cells that contract rhythmically and will be engineered to restore contractile function of the heart. Light microscopic detection of 'beating' cells on glass coverslips are followed SEM visualization, after marking the location of the (contracting) cells of interest. Otic progenitor cells, i.e. cochlear stem cells, form floating colonies, so-called neurospheres that develop through different stages, from compact solid spheres into irregular partially hollow spheres and ultimately large hollow spheres, when cultured in non-adherent conditions [4]. Surface properties, cell junctions and inner and outer surface features are resolved by SEM, applying both secondary (SE) and backscatter electron (BSE) detection. For transplantation or grafting of stem cells, biomaterials, hydrogels and various protein or polysaccharide scaffolds are developed. The structure and porosity of these scaffolds in their natural hydrated state are visualized by VP-SEM, with coolstage control of hydration [5]. Control of porosity is imperative for diffusion of growth media and proliferation of cells. Stem cells grown on such scaffolds are aldehyde-fixed and post-fixed in OsO4 to increase contrast, thereby enabling detection of fully hydrated cells on the hydrogel surface. Low voltage SEM is in great demand for visualization of surface features not resolved by light or fluorescence microscopy, and not enabled by TEM and other technologies. Low magnification low voltage SEM, as well as VP-SEM, enable stem cell biologists from all branches of Medical Sciences to bridge the gap in understanding cell shape, cell surface and attachment features, and is imperative before embarking on higher resolution technologies and application.

Microscopy and Microanalysis, 2010

ABSTRACT Instrumentation and Techniques SymposiaTo Infinity and Beyond: SEM to X-Ray Fluorescence... more ABSTRACT Instrumentation and Techniques SymposiaTo Infinity and Beyond: SEM to X-Ray Fluorescence Imaging to Determine Micronutrients in Helicobacter PyloriArticle author queryjoubert lm [PubMed] [Google Scholar]amieva m [PubMed] [Google Scholar]tan s [PubMed] [Google Scholar]webb s [PubMed] [Google Scholar]lai b [PubMed] [Google Scholar]L-M Jouberta1, M Amievaa1, S Tana1, S Webba1 and B Laia2a1 Stanford University

Microscopy and Microanalysis, 2009

Hydrogels are an important class of biomaterials with exceptional promise in biomedical applicati... more Hydrogels are an important class of biomaterials with exceptional promise in biomedical applications [1,2]. Their use in reconstructive surgery, bioadhesives and as controlled drug release devices additionally do not incur the typical immunological reactions that occur with living organ ...

Microscopy and Microanalysis, 2012

Journal of Applied Microbiology, 2005

Determining the response of different microbial parameters to copper oxychloride in acidic sandy ... more Determining the response of different microbial parameters to copper oxychloride in acidic sandy loam soil samples using cultivation-dependent and direct microscopic techniques. Culturable microbial populations were monitored for 245 days in a series of soil microcosms spiked with different copper oxychloride concentrations. Microbial populations responded differently to additional Cu. Protistan numbers and soil metabolic potential decreased. Experiments with more soil samples revealed that metabolic potential was not significantly affected by < or =100 mg kg(-1) additional Cu. However, a negative impact on protista was noted in soil containing only 15 mg kg(-1) EDTA-extractable Cu. The negative impact on protistan numbers was less severe in soils with a higher phosphorous and zinc content. Bacterial populations responded differently, and protista were most sensitive to elevated Cu levels. Protistan numbers in soil from uncultivated land were higher and seemed to be more sensitive to additional Cu than the numbers of these organisms in soil originating from cultivated land. Protistan sensitivity to small increases in Cu levels demonstrates the vulnerability of the soil ecosystem to Cu perturbations, especially when the importance of protista as link in the flow of energy between trophic levels is considered.

Zeitschrift für Physikalische Chemie, 2011

ABSTRACT Experiments were conducted in a gas-driven shock tube to investigate shock wave-induced ... more ABSTRACT Experiments were conducted in a gas-driven shock tube to investigate shock wave-induced damage to Bacillus thuringiensis Al Hakam endospores over a wide range of post-shock temperatures in non-oxidative gas environments. The results were compared with previous studies on B. atrophaeus and B. subtilis and demonstrate that B. thuringiensis Al Hakam exhibited a qualitatively similar response to rapid shock heating, even though this strain has a significantly different endospore structure. B. thuringiensis Al Hakam endospores were nebulized into an aqueous aerosol, which was loaded into the Stanford aerosol shock tube, and subjected to shock waves of controlled strength. Endospores experienced uniform test temperatures between 500 and 1000 K and pressures ranging from 2 atm to 7 atm for approximately 2.5 ms. During this process the bio-aerosol was monitored using in situ time-resolved laser absorption and scattering diagnostics. Additionally, shock-treated bio-aerosol samples were extracted for ex situ analysis including viability plating, flow cytometry and scanning electron microscopy (SEM) imaging. B. thuringiensis Al Hakam endospores lost the ability to form colonies at post-shock temperatures above 500 K while significant breakdown in morphology was observed only for post-shock temperatures above 700 K. While viability loss and endospore morphological deterioration adhere to a similar framework across all endospore species studied, phenomena unique to B. thuringiensis Al Hakam were noted in the SEM images and optical extinction data. This initial characterization of the response of B. thuringiensis Al Hakam spores treated with shock/blast waves shows that these methods have potential for spore inactivation and detection.

Cellular Microbiology, 2011

inflammasome, which leads to the activation of caspase-1 and the processing and secretion of acti... more inflammasome, which leads to the activation of caspase-1 and the processing and secretion of active IL-1b, IL-18 and pyroptosis. Previous studies have reported that F. novicida and LVS mutants in specific genes (e.g. FTT0584, mviN and ripA) induce elevated inflammasome activation and hypercytotoxicity in host cells, leading to the proposal that F. novicida and LVS may have proteins that actively modulate inflammasome activation. However, there has been no direct evidence of such inflammasome evasion mechanisms. Here, we demonstrate for the first time that the above mutants, along with a wide range of F. novicida hypercytotoxic mutants that are deficient for membrane-associated proteins (DFTT0584, DmviN, DripA, DfopA and DFTN1217) or deficient for genes involved in O-antigen or LPS biosynthesis (DwbtA and DlpxH) lyse more intracellularly, thus activating increased levels of AIM2dependent pyroptosis and other innate immune signalling pathways. This suggests that an inflammasome-specific evasion mechanism may not be present in F. novicida and LVS. Furthermore, future studies may need to consider increased bacterial lysis as a possible cause of elevated stimulation of multiple innate immune pathways when the protein composition or surface carbohydrates of the bacterial membrane is altered.c mi_1643 1586..1600

Stem Cells and Development, 2014

In mammals, the permanence of many forms of hearing loss is the result of the inner ear's inabili... more In mammals, the permanence of many forms of hearing loss is the result of the inner ear's inability to replace lost sensory hair cells. Here, we apply a differentiation strategy to guide human embryonic stem cells (hESCs) into cells of the otic lineage using chemically defined attached-substrate conditions. The generation of human otic progenitor cells was dependent on fibroblast growth factor (FGF) signaling, and protracted culture led to the upregulation of markers indicative of differentiated inner ear sensory epithelia. Using a transgenic ESC reporter line based on a murine Atoh1 enhancer, we show that differentiated hair cell-like cells express multiple hair cell markers simultaneously. Hair cell-like cells displayed protrusions reminiscent of stereociliary bundles, but failed to fully mature into cells with typical hair cell cytoarchitecture. We conclude that optimized defined conditions can be used in vitro to attain otic progenitor specification and sensory cell differentiation.

Introduction: The initial steps in the process of high-resolution scanning electron microscopy (S... more Introduction: The initial steps in the process of high-resolution scanning electron microscopy (SEM) of biological samples are well described [1, 2] and involve various steps of aldehyde fixation and dehydration, followed by critical point drying with liquid CO 2 , or chemical drying with hexamethyldisilazane, or cryo-fixation and lyophilization. In all cases the samples need to be mounted onto an instrument-compatible platform (i.e. the specimen stub) with a conductive adhesive layer, which may consist of carbon or copper tape, colloidal graphite or silver paint. Finally the specimen is coated with a thin conductive film of metal, typically gold and/or palladium, iridium, or platinum. In cases where heavy metals may interfere with the secondary electron (SE) or backscattered electron (BSE) signals needed for the detection of valuable information, carbon evaporation can be used to provide a conductive coating on the sample. "Cleanliness is next to godliness" Precision meas...

Eukaryotic cell, Jan 10, 2015

Aspergillus fumigatus is commonly responsible for lethal fungal infections among immunosuppressed... more Aspergillus fumigatus is commonly responsible for lethal fungal infections among immunosuppressed individuals. A. fumigatus forms biofilm communities that are of increasing biomedical interest due to the association of biofilms with chronic infections and their increased resistance to antifungal agents and host immune factors. Understanding the composition of microbial biofilms and the extracellular matrix is important to understanding function and, ultimately, to developing strategies to inhibit biofilm formation. We implemented a solid-state NMR approach to define compositional parameters of the A. fumigatus extracellular matrix (ECM) when biofilms are formed in RPMI 1640 nutrient medium. Whole biofilm and isolated matrix networks were also characterized by electron microscopy, and matrix proteins were identified through protein gel analysis. The (13)C NMR results defined and quantified the carbon contributions in the insoluble ECM, including carbonyls, aromatic carbons, polysacch...

Acid Mine Drainage needs to be treated, before it is recharged in rivers and dams. The biological... more Acid Mine Drainage needs to be treated, before it is recharged in rivers and dams. The biological treatment of AMD can be applied using degradation products of cellulose, e. g. Volatile Fatty Acids as carbon sources. The aim of this study was to demonstrate that microbes originating from compost and rumen are able to ferment Grass Cuttings to produce Volatile

The complex character of distillery wastewater comprises high concentrations of sugars, lignins, ... more The complex character of distillery wastewater comprises high concentrations of sugars, lignins, hemicelluloses, dextrans, resins, polyphenols and organic acids which are recalcitrant to biodegradation. Microorganisms play a key role in the production and degradation of organic matter, environmental pollutants, and cycling of nutrients and metals. Due to their short life cycles microbes respond rapidly to external nutrient loading, with major

PLoS Pathogens, 2012

Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and h... more Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.

Water SA, 2009

ABSTRACT Copyright: 2007 Water Research Commission A series of lignocellulosic fungi, capable of ... more ABSTRACT Copyright: 2007 Water Research Commission A series of lignocellulosic fungi, capable of cellulase and/or xylanase production, were isolated from soil to be used for cellulose degradation and nitrate removal from nitrate-rich wastewater in simple one-stage anaerobic bioreactors containing grass cuttings as source of cellulose. The fungal consortium, consisting of six hyphomycetous isolates, some of which belong to the genera Fusarium, Mucor and Penicillium, was able to remove a significant portion of the nitrate from the treated water. The results were obtained for three bioreactors, i.e. FR, FRp and AFRp, differing in volume and mode of grass addition. Bioreactor AFRp received autoclaved grass, instead of non-autoclaved grass containing natural microbial consortia, as supplied to FR and FRp. Nitrate removal in FR amounted to 89% removal efficiency, while this was 65% and 67% in FRp and AFRp, respectively. The residual chemical oxygen demand (COD) concentration in FR was higher than 600 mg/ℓ, while it was 355 and 379 mg/ℓ in FRp and AFRp, respectively. The similar nitrate removal results for AFRp and FRp indicated that the micro-organisms attached to grass cuttings did not seem to affect the nitrate removal in the reactor. This observation has led to the conclusion that the fungal consortium was, except for being able to degrade cellulose within the grass cuttings, also responsible for nitrate removal from the synthetic nitrate-rich wastewater

Tissue Engineering Part A, 2014

A biocompatible hydrogel consisting of extracellular matrix (ECM) from human tendons is described... more A biocompatible hydrogel consisting of extracellular matrix (ECM) from human tendons is described as a potential scaffold for guided tissue regeneration and tissue engineering purposes. Lyophilized decellularized tendons were milled and enzymatically digested to form an ECM solution. The ECM solution properties are assessed by proteome analysis with mass spectrometry, and the material's rheological properties are determined as a function of frequency, temperature, and time. In vivo application of the gel in a rat model is assessed for remodeling and host cell repopulation. Histology for macrophage invasion, fibroblast repopulation, and nanoscale properties of the gel is assessed. Gel interaction with multipotent adipoderived stem cells (ASCs) is also addressed in vitro to assess possible cytotoxicity and its ability to act as a delivery vehicle for cells. Proteome analysis of the ECM-solution and gel mass spectroscopy identified the most abundant 150 proteins, of which two isoforms of collagen I represented more than 55% of the sample. Rheology showed that storage (G') and loss (G″) of the ECM solution were stable at room temperature but displayed sigmoidal increases after ∼15 min at 37°C, matching macroscopic observations of its thermo responsiveness. G' and G″ of the gel at 1 rad/s were 213.1±19.9 and 27.1±2.4 Pa, respectively. Electron microscopy revealed fiber alignment and good structural porosity in the gel, as well as invasion of cells in vivo. Histology also showed early CD68(+) macrophage invasion throughout the gel, followed by increasing numbers of fibroblast cells. ASCs mixed with the gel in vitro proliferated, indicating good biocompatibility. This ECM solution can be delivered percutaneously into a zone of tendon injury. After injection, the thermoresponsive behavior of the ECM solution allows it to polymerize and form a porous gel at body temperature. A supportive nanostructure of collagen fibers is established that conforms to the three-dimensional space of the defect. This hydrogel holds the distinctive composition specific for tendon ECM, where tissue-specific cues facilitate host cell infiltration and remodeling. The results presented indicate that injectable ECM materials from tendon may offer a promising alternative in the treatment of tendinopathies and acute tendon injuries.

Review of Scientific Instruments, 2011

This paper describes the design and operation of a simple airtight container devised to facilitat... more This paper describes the design and operation of a simple airtight container devised to facilitate the transfer of atmosphere-sensitive samples from a glovebox to the vacuum chamber of an analytical instrument such as a scanning electron microscope. The use of this device for characterizing the microstructure of highly hygroscopic strontium iodide ceramics by scanning electron microscopy is illustrated as an application example.

Nature Cell Biology, 2012

Many of the more than 20 mammalian proteins with N-BAR domains control cell architecture and endo... more Many of the more than 20 mammalian proteins with N-BAR domains control cell architecture and endocytosis by associating with curved sections of the plasma membrane. It is not well understood whether N-BAR proteins are recruited directly by processes that mechanically curve the plasma membrane or indirectly by plasma-membrane-associated adaptor proteins that recruit proteins with N-BAR domains that then induce membrane curvature. Here, we show that externally induced inward deformation of the plasma membrane by cone-shaped nanostructures (nanocones) and internally induced inward deformation by contracting actin cables both trigger recruitment of isolated N-BAR domains to the curved plasma membrane. Markedly, live-cell imaging in adherent cells showed selective recruitment of full-length N-BAR proteins and isolated N-BAR domains to plasma membrane sub-regions above nanocone stripes. Electron microscopy confirmed that N-BAR domains are recruited to local membrane sites curved by nanocones. We further showed that N-BAR domains are periodically recruited to curved plasma membrane sites during local lamellipodia retraction in the front of migrating cells. Recruitment required myosin-II-generated force applied to plasma-membrane-connected actin cables. Together, our results show that N-BAR domains can be directly recruited to the plasma membrane by external push or internal pull forces that locally curve the plasma membrane.

Microscopy and Microanalysis, 2010

ABSTRACT Stem cell research is no longer in its infancy. It has matured into a discipline with fa... more ABSTRACT Stem cell research is no longer in its infancy. It has matured into a discipline with far-reaching applications. When little is known about a subject, the questions people raise are almost invariably ethical. As knowledge grows, however, people become amoral and more concerned with information i.e. more narrowly intellectual [1]. The words of Charles Spurgeon (1834-1992) that 'every generation needs regeneration' takes on new meaning in the era of stem cell biology [2] and there is a sense of urgency to move new developments quickly into the clinical setting. Visualization of stem cells is one of the core set of techniques that are applied to unlock the potential of stem cell biology. Various differentiation protocols revolve around fluorescence microscopy, applying different molecular markers. However, the limited resolution of light microscopy, and an inability to resolve surface features, has put Scanning EM in great demand to fill this gap. It is often also the preferred technique to apply before TEM investigation is started. The additional benefit of Variable pressure (VP) SEM that enabled visualization of hydrated samples, eg biomaterials onto which stem cells are grafted before clinical application, has proved to be an indispensible tool in our recent research. The ethical debate that ensued from Human Embryonic Stem cells (HESC) prompted large efforts to find alternative approaches to use adult stem cells or progenitor stem cells instead [3]. The concept that certain stem cells can trans-differentiate into cell types outside their normal lineage has been raised recently. We currently study adipose stem cells from humans, dogs and mice, and turn these into bone and other stem cells. HESC cells generate cardiomyocytes, cells that contract rhythmically and will be engineered to restore contractile function of the heart. Light microscopic detection of 'beating' cells on glass coverslips are followed SEM visualization, after marking the location of the (contracting) cells of interest. Otic progenitor cells, i.e. cochlear stem cells, form floating colonies, so-called neurospheres that develop through different stages, from compact solid spheres into irregular partially hollow spheres and ultimately large hollow spheres, when cultured in non-adherent conditions [4]. Surface properties, cell junctions and inner and outer surface features are resolved by SEM, applying both secondary (SE) and backscatter electron (BSE) detection. For transplantation or grafting of stem cells, biomaterials, hydrogels and various protein or polysaccharide scaffolds are developed. The structure and porosity of these scaffolds in their natural hydrated state are visualized by VP-SEM, with coolstage control of hydration [5]. Control of porosity is imperative for diffusion of growth media and proliferation of cells. Stem cells grown on such scaffolds are aldehyde-fixed and post-fixed in OsO4 to increase contrast, thereby enabling detection of fully hydrated cells on the hydrogel surface. Low voltage SEM is in great demand for visualization of surface features not resolved by light or fluorescence microscopy, and not enabled by TEM and other technologies. Low magnification low voltage SEM, as well as VP-SEM, enable stem cell biologists from all branches of Medical Sciences to bridge the gap in understanding cell shape, cell surface and attachment features, and is imperative before embarking on higher resolution technologies and application.

Microscopy and Microanalysis, 2010

ABSTRACT Instrumentation and Techniques SymposiaTo Infinity and Beyond: SEM to X-Ray Fluorescence... more ABSTRACT Instrumentation and Techniques SymposiaTo Infinity and Beyond: SEM to X-Ray Fluorescence Imaging to Determine Micronutrients in Helicobacter PyloriArticle author queryjoubert lm [PubMed] [Google Scholar]amieva m [PubMed] [Google Scholar]tan s [PubMed] [Google Scholar]webb s [PubMed] [Google Scholar]lai b [PubMed] [Google Scholar]L-M Jouberta1, M Amievaa1, S Tana1, S Webba1 and B Laia2a1 Stanford University

Microscopy and Microanalysis, 2009

Hydrogels are an important class of biomaterials with exceptional promise in biomedical applicati... more Hydrogels are an important class of biomaterials with exceptional promise in biomedical applications [1,2]. Their use in reconstructive surgery, bioadhesives and as controlled drug release devices additionally do not incur the typical immunological reactions that occur with living organ ...

Microscopy and Microanalysis, 2012

Journal of Applied Microbiology, 2005

Determining the response of different microbial parameters to copper oxychloride in acidic sandy ... more Determining the response of different microbial parameters to copper oxychloride in acidic sandy loam soil samples using cultivation-dependent and direct microscopic techniques. Culturable microbial populations were monitored for 245 days in a series of soil microcosms spiked with different copper oxychloride concentrations. Microbial populations responded differently to additional Cu. Protistan numbers and soil metabolic potential decreased. Experiments with more soil samples revealed that metabolic potential was not significantly affected by < or =100 mg kg(-1) additional Cu. However, a negative impact on protista was noted in soil containing only 15 mg kg(-1) EDTA-extractable Cu. The negative impact on protistan numbers was less severe in soils with a higher phosphorous and zinc content. Bacterial populations responded differently, and protista were most sensitive to elevated Cu levels. Protistan numbers in soil from uncultivated land were higher and seemed to be more sensitive to additional Cu than the numbers of these organisms in soil originating from cultivated land. Protistan sensitivity to small increases in Cu levels demonstrates the vulnerability of the soil ecosystem to Cu perturbations, especially when the importance of protista as link in the flow of energy between trophic levels is considered.

Zeitschrift für Physikalische Chemie, 2011

ABSTRACT Experiments were conducted in a gas-driven shock tube to investigate shock wave-induced ... more ABSTRACT Experiments were conducted in a gas-driven shock tube to investigate shock wave-induced damage to Bacillus thuringiensis Al Hakam endospores over a wide range of post-shock temperatures in non-oxidative gas environments. The results were compared with previous studies on B. atrophaeus and B. subtilis and demonstrate that B. thuringiensis Al Hakam exhibited a qualitatively similar response to rapid shock heating, even though this strain has a significantly different endospore structure. B. thuringiensis Al Hakam endospores were nebulized into an aqueous aerosol, which was loaded into the Stanford aerosol shock tube, and subjected to shock waves of controlled strength. Endospores experienced uniform test temperatures between 500 and 1000 K and pressures ranging from 2 atm to 7 atm for approximately 2.5 ms. During this process the bio-aerosol was monitored using in situ time-resolved laser absorption and scattering diagnostics. Additionally, shock-treated bio-aerosol samples were extracted for ex situ analysis including viability plating, flow cytometry and scanning electron microscopy (SEM) imaging. B. thuringiensis Al Hakam endospores lost the ability to form colonies at post-shock temperatures above 500 K while significant breakdown in morphology was observed only for post-shock temperatures above 700 K. While viability loss and endospore morphological deterioration adhere to a similar framework across all endospore species studied, phenomena unique to B. thuringiensis Al Hakam were noted in the SEM images and optical extinction data. This initial characterization of the response of B. thuringiensis Al Hakam spores treated with shock/blast waves shows that these methods have potential for spore inactivation and detection.

Cellular Microbiology, 2011

inflammasome, which leads to the activation of caspase-1 and the processing and secretion of acti... more inflammasome, which leads to the activation of caspase-1 and the processing and secretion of active IL-1b, IL-18 and pyroptosis. Previous studies have reported that F. novicida and LVS mutants in specific genes (e.g. FTT0584, mviN and ripA) induce elevated inflammasome activation and hypercytotoxicity in host cells, leading to the proposal that F. novicida and LVS may have proteins that actively modulate inflammasome activation. However, there has been no direct evidence of such inflammasome evasion mechanisms. Here, we demonstrate for the first time that the above mutants, along with a wide range of F. novicida hypercytotoxic mutants that are deficient for membrane-associated proteins (DFTT0584, DmviN, DripA, DfopA and DFTN1217) or deficient for genes involved in O-antigen or LPS biosynthesis (DwbtA and DlpxH) lyse more intracellularly, thus activating increased levels of AIM2dependent pyroptosis and other innate immune signalling pathways. This suggests that an inflammasome-specific evasion mechanism may not be present in F. novicida and LVS. Furthermore, future studies may need to consider increased bacterial lysis as a possible cause of elevated stimulation of multiple innate immune pathways when the protein composition or surface carbohydrates of the bacterial membrane is altered.c mi_1643 1586..1600