Optimising mechanical separation of anaerobic digestate for total solids and nutrient removal.

Mechanical separation of anaerobic digestate has been identified as a method to reduce pollution risk to waterways by partitioning phosphorus in the solid fraction and reducing its application to land. Separators have adjustable parameters which affect separation efficiency, and hence the degree of phosphorous partitioning, but information on how these parameters affect separation performance is limited in the literature. Two well known technologies were investigated, decanter centrifuge and screw press, to determine the most efficient method of separation. Counterweight load and the use of an oscillator were adjusted for the screw press, while bowl speed, auger differential speed, feed rate and polymer addition were modified for the decanter centrifuge. Separation efficiency was determined for total solids, phosphorus, nitrogen, potassium, and carbon, and the total solids content of resulting fractions was measured. The decanter centrifuge had higher separation efficiency for phosphorus in all cases, ranging from 51% to 71.5%, while the screw press had a phosphorus separation efficiency ranging from 8.5% to 10.9% for digestate of ∼5% solids (slurry/grass silage mix). Separation by decanter centrifuge partitioned up to 56% of nitrogen in the solid fraction leaving a reduced nitrogen content in the liquid fraction available for land spreading; this nitrogen would most likely need to be replaced by chemical fertiliser which would add to the cost of the system. The decanter centrifuge is better suited to cases where phosphorus recovery is the most important factor, while the screw press could be advantageous in cases where cost is a limiting factor.

The effect of biochar and acid activated biochar on ammonia emissions during manure storage.

Animal manure contains valuable plant nutrients which need to be stored until field application. A significant proportion of slurry nitrogen is volatilized in the form of ammonia (NH3) during storage. This impacts human health, biodiversity, air and water quality and thus urgent action is needed to reduce NH3 emissions. In this experiment, we evaluated the NH3 emission mitigation potential of biochars derived from miscanthus (MB) and solid separated anaerobic digestate (DB), and orthophosphoric acid activated MB (AMB) and DB (ADB) as well as lightweight expanded clay aggregate (LECA) during four months of liquid manure storage. A slurry without amendment was included as a control (Ctrl). Acid activated and non-activated biochars were applied on top of the slurry maintaining a 7 mm thick surface layer, while LECA was applied in a 2 cm thick layer. NH3 emissions were measured by photoacoustic analyzer. In comparison to Ctrl, acid activated biochar decreased (p < 0.05) NH3 emissions during the slurry storage. Activated biochar reduced the emissions by 37-51% within the first month of slurry storage and achieved a 25-28% emissions reduction efficiency throughout the four month period due to the reduction in emission mitigation efficiency as the storage period progressed. LECA reduced NH3 emissions by 21% during storage. Losses of NH3 as a percentage of total ammoniacal N were 29-31% for activated biochars, 35-39% for non-activated biochars and 33% for LECA. In conclusion, acid activated biochars and LECA could be good floating-covers to mitigate NH3 emissions during manure storage, but activated biochars may have better mitigation potential than LECA.

Prediction of Lignin Content in Ruminant Diets and Fecal Samples Using Rapid Analytical Techniques.

The measurement of lignin content in ruminant diet and fecal samples is important for digestibility studies, but it is typically time-consuming and costly. The work reported involves correlation of traditional wet chemistry data with those from three rapid instrumental techniques, Fourier transform infrared spectroscopy (FTIR), conventional thermogravimteric analysis (TGA), and high-resolution TGA (MaxRes TGA) to predict the lignin content of diets and feces from digestibility trials. Calibration and performance data indicate that the FTIR model is acceptable for screening, while the conventional and MaxRes TGA predictions are high accuracy for quantitative analysis. Cross validation and model performance data reveal that MaxRes TGA provides the best-performing predictive model. This work shows that MaxRes TGA can accurately predict lignin content in ruminant diet and fecal samples with distinct advantages over traditional wet chemistry: namely, the requirement of small sample size, ease of sample preparation, speed of analysis, and high sample throughput at considerably lower cost.

Irx4 Marks a Multipotent, Ventricular-Specific Progenitor Cell.

While much progress has been made in the resolution of the cellular hierarchy underlying cardiogenesis, our understanding of chamber-specific myocardium differentiation remains incomplete. To better understand ventricular myocardium differentiation, we targeted the ventricle-specific gene, Irx4, in mouse embryonic stem cells to generate a reporter cell line. Using an antibiotic-selection approach, we purified Irx4+ cells in vitro from differentiating embryoid bodies. The isolated Irx4+ cells proved to be highly proliferative and presented Cxcr4, Pdgfr-alpha, Flk1, and Flt1 on the cell surface. Single Irx4+ ventricular progenitor cells (VPCs) exhibited cardiovascular potency, generating endothelial cells, smooth muscle cells, and ventricular myocytes in vitro. The ventricular specificity of the Irx4+ population was further demonstrated in vivo as VPCs injected into the cardiac crescent subsequently produced Mlc2v+ myocytes that exclusively contributed to the nascent ventricle at E9.5. These findings support the existence of a newly identified ventricular myocardial progenitor. This is the first report of a multipotent cardiac progenitor that contributes progeny specific to the ventricular myocardium. Stem Cells 2016;34:2875-2888.

Lineage Reprogramming of Fibroblasts into Proliferative Induced Cardiac Progenitor Cells by Defined Factors.

Several studies have reported reprogramming of fibroblasts into induced cardiomyocytes; however, reprogramming into proliferative induced cardiac progenitor cells (iCPCs) remains to be accomplished. Here we report that a combination of 11 or 5 cardiac factors along with canonical Wnt and JAK/STAT signaling reprogrammed adult mouse cardiac, lung, and tail tip fibroblasts into iCPCs. The iCPCs were cardiac mesoderm-restricted progenitors that could be expanded extensively while maintaining multipotency to differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells in vitro. Moreover, iCPCs injected into the cardiac crescent of mouse embryos differentiated into cardiomyocytes. iCPCs transplanted into the post-myocardial infarction mouse heart improved survival and differentiated into cardiomyocytes, smooth muscle cells, and endothelial cells. Lineage reprogramming of adult somatic cells into iCPCs provides a scalable cell source for drug discovery, disease modeling, and cardiac regenerative therapy.

Irx4 identifies a chamber-specific cell population that contributes to ventricular myocardium development.

BACKGROUND: The ventricular myocardium is the most prominent layer of the heart, and the most important for mediating cardiac physiology. Although the ventricular myocardium is critical for heart function, the cellular hierarchy responsible for ventricle-specific myocardium development remains unresolved. RESULTS: To determine the pattern and time course of ventricular myocardium development, we investigated IRX4 protein expression, which has not been previously reported. We identified IRX4+ cells in the cardiac crescent, and these cells were positive for markers of the first or second heart fields. From the onset of chamber formation, IRX4+ cells were restricted to the ventricular myocardium. This expression pattern persisted into adulthood. Of interest, we observed that IRX4 exhibits developmentally regulated dynamic intracellular localization. Throughout prenatal cardiogenesis, and up to postnatal day 4, IRX4 was detected in the cytoplasm of ventricular myocytes. However, between postnatal days 5­6, IRX4 translocated to the nucleus of ventricular myocytes. CONCLUSIONS: Given the ventricle-specific expression of Irx4 in later stages of heart development, we hypothesize that IRX4+ cells in the cardiac crescent represent the earliest cell population in the cellular hierarchy underlying ventricular myocardium development.

Image-inspired 3D multiphoton excited fabrication of extracellular matrix structures by modulated raster scanning.

Multiphoton excited photochemistry is a powerful 3D fabrication tool that produces sub-micron feature sizes. Here we exploit the freeform nature of the process to create models of the extracellular matrix (ECM) of several tissues, where the design blueprint is derived directly from high resolution optical microscopy images (e.g. fluorescence and Second Harmonic Generation). To achieve this goal, we implemented a new form of instrument control, termed modulated raster scanning, where rapid laser shuttering (10 MHz) is used to directly map the greyscale image data to the resulting protein concentration in the fabricated scaffold. Fidelity in terms of area coverage and relative concentration relative to the image data is ~95%. We compare the results to an STL approach, and find the new scheme provides significantly improved performance. We suggest the method will enable a variety of cell-matrix studies in cancer biology and also provide insight into generating scaffolds for tissue engineering.

Rapid analysis of purified cellulose extracted from perennial ryegrass (Lolium perenne) by instrumental analysis.

Dried, milled perennial ryegrass samples were processed using chemical and physical treatments and the extracted cellulose products were analysed for yield, crystallinity by X-ray Diffraction (XRD) and for purity using Thermogravimetric Analysis (TGA), Pyrolysis-Gas Chromatography/Mass Spectrometry (Py-GC/MS) and Fourier Transform Infrared (FTIR) spectroscopy. Extraction protocols examined the use of chemical chelation, acid and alkaline hydrolysis, along with physical degradation methods. Highest product yields were obtained using single step chemical protocols followed by physical processing, however, these products had low crystallinity and higher amorphous fraction content. Multistep chemical processing to completely remove hemicellulose and lignin with an alkali refluxing step, delivered lower yielding cellulose products of greater crystallinity and purity. In combination, the four instrumental techniques highlighted removal of amorphous fractions, providing rapid, accurate compositional data on the extracted cellulose products.

Spatial and temporal analysis of extracellular matrix proteins in the developing murine heart: a blueprint for regeneration.

The extracellular matrix (ECM) of the embryonic heart guides assembly and maturation of cardiac cell types and, thus, may serve as a useful template, or blueprint, for fabrication of scaffolds for cardiac tissue engineering. Surprisingly, characterization of the ECM with cardiac development is scattered and fails to comprehensively reflect the spatiotemporal dynamics making it difficult to apply to tissue engineering efforts. The objective of this work was to define a blueprint of the spatiotemporal organization, localization, and relative amount of the four essential ECM proteins, collagen types I and IV (COLI, COLIV), elastin (ELN), and fibronectin (FN) in the left ventricle of the murine heart at embryonic stages E12.5, E14.5, and E16.5 and 2 days postnatal (P2). Second harmonic generation (SHG) imaging identified fibrillar collagens at E14.5, with an increasing density over time. Subsequently, immunohistochemistry (IHC) was used to compare the spatial distribution, organization, and relative amounts of each ECM protein. COLIV was found throughout the developing heart, progressing in amount and organization from E12.5 to P2. The amount of COLI was greatest at E12.5 particularly within the epicardium. For all stages, FN was present in the epicardium, with highest levels at E12.5 and present in the myocardium and the endocardium at relatively constant levels at all time points. ELN remained relatively constant in appearance and amount throughout the developmental stages except for a transient increase at E16.5. Expression of ECM mRNA was determined using quantitative polymerase chain reaction and allowed for comparison of amounts of ECM molecules at each time point. Generally, COLI and COLIII mRNA expression levels were comparatively high, while COLIV, laminin, and FN were expressed at intermediate levels throughout the time period studied. Interestingly, levels of ELN mRNA were relatively low at early time points (E12.5), but increased significantly by P2. Thus, we identified changes in the spatial and temporal localization of the primary ECM of the developing ventricle. This characterization can serve as a blueprint for fabrication techniques, which we illustrate by using multiphoton excitation photochemistry to create a synthetic scaffold based on COLIV organization at P2. Similarly, fabricated scaffolds generated using ECM components, could be utilized for ventricular repair.

Endogenous fluorescence signatures in living pluripotent stem cells change with loss of potency.

The therapeutic potential of stem cells is limited by the non-uniformity of their phenotypic state. Thus it would be advantageous to noninvasively monitor stem cell status. Driven by this challenge, we employed multidimensional multiphoton microscopy to quantify changes in endogenous fluorescence occurring with pluripotent stem cell differentiation. We found that global and cellular-scale fluorescence lifetime of human embryonic stem cells (hESC) and murine embryonic stem cells (mESC) consistently decreased with differentiation. Less consistent were trends in endogenous fluorescence intensity with differentiation, suggesting intensity is more readily impacted by nuances of species and scale of analysis. What emerges is a practical and accessible approach to evaluate, and ultimately enrich, living stem cell populations based on changes in metabolism that could be exploited for both research and clinical applications.

Extracellular matrix promotes highly efficient cardiac differentiation of human pluripotent stem cells: the matrix sandwich method.

RATIONALE: Cardiomyocytes (CMs) differentiated from human pluripotent stem cells (PSCs) are increasingly being used for cardiovascular research, including disease modeling, and hold promise for clinical applications. Current cardiac differentiation protocols exhibit variable success across different PSC lines and are primarily based on the application of growth factors. However, extracellular matrix is also fundamentally involved in cardiac development from the earliest morphogenetic events, such as gastrulation. OBJECTIVE: We sought to develop a more effective protocol for cardiac differentiation of human PSCs by using extracellular matrix in combination with growth factors known to promote cardiogenesis. METHODS AND RESULTS: PSCs were cultured as monolayers on Matrigel, an extracellular matrix preparation, and subsequently overlayed with Matrigel. The matrix sandwich promoted an epithelial-to-mesenchymal transition as in gastrulation with the generation of N-cadherin-positive mesenchymal cells. Combining the matrix sandwich with sequential application of growth factors (Activin A, bone morphogenetic protein 4, and basic fibroblast growth factor) generated CMs with high purity (up to 98%) and yield (up to 11 CMs/input PSC) from multiple PSC lines. The resulting CMs progressively matured over 30 days in culture based on myofilament expression pattern and mitotic activity. Action potentials typical of embryonic nodal, atrial, and ventricular CMs were observed, and monolayers of electrically coupled CMs modeled cardiac tissue and basic arrhythmia mechanisms. CONCLUSIONS: Dynamic extracellular matrix application promoted epithelial-mesenchymal transition of human PSCs and complemented growth factor signaling to enable robust cardiac differentiation.

Imaging cardiac extracellular matrices: a blueprint for regeneration.

Once damaged, cardiac tissue does not readily repair and is therefore a primary target of regenerative therapies. One regenerative approach is the development of scaffolds that functionally mimic the cardiac extracellular matrix (ECM) to deliver stem cells or cardiac precursor populations to the heart. Technological advances in micro/nanotechnology, stem cell biology, biomaterials and tissue decellularization have propelled this promising approach forward. Surprisingly, technological advances in optical imaging methods have not been fully utilized in the field of cardiac regeneration. Here, we describe and provide examples to demonstrate how advanced imaging techniques could revolutionize how ECM-mimicking cardiac tissues are informed and evaluated.

Pitx2c modulates cardiac-specific transcription factors networks in differentiating cardiomyocytes from murine embryonic stem cells.

AIM: The knowledge of the molecular signals that control cell differentiation into cardiomyocytes is critical to apply cell-based therapies and repair an injured heart. The transcription factor Pitx2 has essential roles in the development of different organs including the heart. Although a direct role of Pitx2 in the developing myocardium has recently been reported, the molecular pathways driven by Pitx2 as well as its cardiac target genes remain largely unexplored. The aim of this study was to unravel the molecular mechanisms driven by Pitx2 during the process of cardiomyocyte differentiation in vitro in mouse embryonic stem cell-derived cardiomyocytes. METHODS AND RESULTS: Pitx2c was overexpressed in the R1-embryonic stem cell line. mRNA levels and protein distribution of several specific cardiac genes were analyzed by real-time PCR and immunohistochemistry experiments in R1-embryonic stem cell-derived beating areas at different stages of in vitro differentiation. Our results show that overexpression of Pitx2c in embryonic stem cell-derived cardiomyocytes is able to dynamically upregulate several cardiac-enriched transcription factors such as Isl1, Mef2c and Gata4. Additionally, Pitx2c induces the expression of chamber-specific cardiac genes such as Tbx5, Nppa and Cx40. These data were validated in an in vivo model of Pitx2 loss of function. CONCLUSION: Taken together, these results demonstrate that Pitx2 plays a major role reinforcing the transcriptional program of cardiac differentiation.

Multiphoton flow cytometry to assess intrinsic and extrinsic fluorescence in cellular aggregates: applications to stem cells.

Detection and tracking of stem cell state are difficult due to insufficient means for rapidly screening cell state in a noninvasive manner. This challenge is compounded when stem cells are cultured in aggregates or three-dimensional (3D) constructs because living cells in this form are difficult to analyze without disrupting cellular contacts. Multiphoton laser scanning microscopy is uniquely suited to analyze 3D structures due to the broad tunability of excitation sources, deep sectioning capacity, and minimal phototoxicity but is throughput limited. A novel multiphoton fluorescence excitation flow cytometry (MPFC) instrument could be used to accurately probe cells in the interior of multicell aggregates or tissue constructs in an enhanced-throughput manner and measure corresponding fluorescent properties. By exciting endogenous fluorophores as intrinsic biomarkers or exciting extrinsic reporter molecules, the properties of cells in aggregates can be understood while the viable cellular aggregates are maintained. Here we introduce a first generation MPFC system and show appropriate speed and accuracy of image capture and measured fluorescence intensity, including intrinsic fluorescence intensity. Thus, this novel instrument enables rapid characterization of stem cells and corresponding aggregates in a noninvasive manner and could dramatically transform how stem cells are studied in the laboratory and utilized in the clinic.

Identification of RING finger protein 4 (RNF4) as a modulator of DNA demethylation through a functional genomics screen.

DNA methylation is an important epigenetic modification involved in transcriptional regulation, nuclear organization, development, aging, and disease. Although DNA methyltransferases have been characterized, the mechanisms for DNA demethylation remain poorly understood. Using a cell-based reporter assay, we performed a functional genomics screen to identify genes involved in DNA demethylation. Here we show that RNF4 (RING finger protein 4), a SUMO-dependent ubiquitin E3-ligase previously implicated in maintaining genome stability, plays a key role in active DNA demethylation. RNF4 reactivates methylation-silenced reporters and promotes global DNA demethylation. Rnf4 deficiency is embryonic lethal with higher levels of methylation in genomic DNA. Mechanistic studies show that RNF4 interacts with and requires the base excision repair enzymes TDG and APE1 for active demethylation. This activity appears to occur by enhancing the enzymatic activities that repair DNA G:T mismatches generated from methylcytosine deamination. Collectively, our study reveals a unique function for RNF4, which may serve as a direct link between epigenetic DNA demethylation and DNA repair in mammalian cells.

The microwell control of embryoid body size in order to regulate cardiac differentiation of human embryonic stem cells.

The differentiation of human embryonic stem cells (hESCs) into cardiomyocytes (CMs) using embryoid bodies (EBs) is relatively inefficient and highly variable. Formation of EBs using standard enzymatic disaggregation techniques results in a wide range of sizes and geometries of EBs. Use of a 3-D cuboidal microwell system to culture hESCs in colonies of defined dimensions, 100-500 microm in lateral dimensions and 120 microm in depth, enabled formation of more uniform-sized EBs. The 300 microm microwells produced highest percentage of contracting EBs, but flow cytometry for myosin light chain 2A (MLC2a) expressing cells revealed a similar percentage (approximately 3%) of cardiomyocytes formed in EBs from 100 microm to 300 microm microwells. These data, and immunolabeling with anti-MF20 and MLC2a, suggest that the smaller EBs are less likely to form contracting EBs, but those contracting EBs are relatively enriched in cardiomyocytes compared to larger EB sizes where CMs make up a proportionately smaller fraction of the total cells. We conclude that microwell-engineered EB size regulates cardiogenesis and can be used for more efficient and reproducible formation of hESC-CMs needed for research and therapeutic applications.

Characterization of recycled mushroom compost leachate by chemical analysis and thermogravimetry-mass spectrometry.

Recycled compost leachate (RCL or euphemistically named "goody water") can be a potent source of foul odor on mushroom substrate production sites and contributes to composting smells. A complex mixture of sulfur compounds, fatty acids, and nitrogen containing compounds is responsible for odor production. Fifty samples, collected from 14 compost production sites in Ireland and the U.K. over a 2 year period, were analyzed for chemical properties and by thermogravimetry-mass spectrometry (TG-MS) for compositional differences. Results indicated that aerated samples had lower values of electrical conductivity, redox potential, and dry matter content than nonaerated samples and that the higher thermal stability of aerated samples measured by TGA could be attributed to greater mineralization of the substrate due to aerobic processes. The lower temperatures noted for peak evolution of methane, water, and carbon dioxide from TG-MS analysis suggested that a more energetic process had occurred in aerated RCL storage facilities, producing greater decomposition of macromolecules that volatilized at lower temperatures. Chemical composition, thermal stability of the freeze-dried leachate, pyrolysis profiles, and relative amounts of pyrolysis products were all markers of as to how effective control measures could influence RCL quality.

An adaptable hydrogel array format for 3-dimensional cell culture and analysis.

Hydrogels have been commonly used as model systems for 3-dimensional (3-D) cell biology, as they have material properties that resemble natural extracellular matrices (ECMs), and their cell-interactive properties can be readily adapted in order to address a particular hypothesis. Natural and synthetic hydrogels have been used to gain fundamental insights into virtually all aspects of cell behavior, including cell adhesion, migration, and differentiated function. However, cell responses to complex 3-D environments are difficult to adequately explore due to the large number of variables that must be controlled simultaneously. Here we describe an adaptable, automated approach for 3-D cell culture within hydrogel arrays. Our initial results demonstrate that the hydrogel network chemistry (both natural and synthetic), cell type, cell density, cell adhesion ligand density, and degradability within each array spot can be systematically varied to screen for environments that promote cell viability in a 3-D context. In a test-bed application we then demonstrate that a hydrogel array format can be used to identify environments that promote viability of HL-1 cardiomyocytes, a cell line that has not been cultured previously in 3-D hydrogel matrices. Results demonstrate that the fibronectin-derived cell adhesion ligand RGDSP improves HL-1 viability in a dose-dependent manner, and that the effect of RGDSP is particularly pronounced in degrading hydrogel arrays. Importantly, in the presence of 70mum RGDSP, HL-1 cardiomyocyte viability does not decrease even after 7 days of culture in PEG hydrogels. Taken together, our results indicate that the adaptable, array-based format developed in this study may be useful as an enhanced throughput platform for 3-D culture of a variety of cell types.

Dynamic expression patterns of leucine-rich repeat containing protein 10 in the heart.

Leucine-rich repeat containing protein 10 (LRRC10) is a heart-specific factor whose function remains unknown. Examination of the intracellular location of the gene products is a critical step in determining the biological functions of the protein. Our expression analyses in mice indicate that LRRC10 is exclusively expressed from the precardiac region in early embryos to the adult heart. LRRC10 expression is markedly elevated upon birth, suggesting its role in the embryonic as well as adult hearts. Of interest, LRRC10 exhibits dynamic intracellular expression patterns in cardiomyocytes. Cardiomyocytes from embryos and newborns show diffuse cytoplasmic and nuclear staining of LRRC10. In contrast, striking striations are observed in adult cardiomyocytes, which are colocalized with the markers for the Z-line, sarcoplasmic reticulum (SR), and transverse (T)-tubule by double immunostaining. Further investigation by electron micrographs places LRRC10 in a diad region where the SR interacts with the T-tubule that locates along the Z-line.