Multimodal characterization of murine gastruloid development.

Gastruloids are 3D structures generated from pluripotent stem cells recapitulating fundamental principles of embryonic pattern formation. Using single-cell genomic analysis, we provide a resource mapping cell states and types during gastruloid development and compare them with the in vivo embryo. We developed a high-throughput handling and imaging pipeline to spatially monitor symmetry breaking during gastruloid development and report an early spatial variability in pluripotency determining a binary response to Wnt activation. Although cells in the gastruloid-core revert to pluripotency, peripheral cells become primitive streak-like. These two populations subsequently break radial symmetry and initiate axial elongation. By performing a compound screen, perturbing thousands of gastruloids, we derive a phenotypic landscape and infer networks of genetic interactions. Finally, using a dual Wnt modulation, we improve the formation of anterior structures in the existing gastruloid model. This work provides a resource to understand how gastruloids develop and generate complex patterns in vitro.

HP1 proteins regulate nucleolar structure and function by secluding pericentromeric constitutive heterochromatin.

Nucleoli are nuclear compartments regulating ribosome biogenesis and cell growth. In embryonic stem cells (ESCs), nucleoli containing transcriptionally active ribosomal genes are spatially separated from pericentromeric satellite repeat sequences packaged in largely repressed constitutive heterochromatin (PCH). To date, mechanisms underlying such nuclear partitioning and the physiological relevance thereof are unknown. Here we show that repressive chromatin at PCH ensures structural integrity and function of nucleoli during cell cycle progression. Loss of heterochromatin proteins HP1α and HP1ß causes deformation of PCH, with reduced H3K9 trimethylation (H3K9me3) and HP1γ levels, absence of H4K20me3 and upregulated major satellites expression. Spatially, derepressed PCH aberrantly associates with nucleoli accumulating severe morphological defects during S/G2 cell cycle progression. Hp1α/ß deficiency reduces cell proliferation, ribosomal RNA biosynthesis and mobility of Nucleophosmin, a major nucleolar component. Nucleolar integrity and function require HP1α/ß proteins to be recruited to H3K9me3-marked PCH and their ability to dimerize. Correspondingly, ESCs deficient for both Suv39h1/2 H3K9 HMTs display similar nucleolar defects. In contrast, Suv4-20h1/2 mutant ESCs lacking H4K20me3 at PCH do not. Suv39h1/2 and Hp1α/ß deficiency-induced nucleolar defects are reminiscent of those defining human ribosomopathy disorders. Our results reveal a novel role for SUV39H/HP1-marked repressive constitutive heterochromatin in regulating integrity, function and physiology of nucleoli.

Multiscale light-sheet organoid imaging framework.

Organoids provide an accessible in vitro system to mimic the dynamics of tissue regeneration and development. However, long-term live-imaging of organoids remains challenging. Here we present an experimental and image-processing framework capable of turning long-term light-sheet imaging of intestinal organoids into digital organoids. The framework combines specific imaging optimization combined with data processing via deep learning techniques to segment single organoids, their lumen, cells and nuclei in 3D over long periods of time. By linking lineage trees with corresponding 3D segmentation meshes for each organoid, the extracted information is visualized using a web-based "Digital Organoid Viewer" tool allowing combined understanding of the multivariate and multiscale data. We also show backtracking of cells of interest, providing detailed information about their history within entire organoid contexts. Furthermore, we show cytokinesis failure of regenerative cells and that these cells never reside in the intestinal crypt, hinting at a tissue scale control on cellular fidelity.

Poly(methyl methacrylate) Surface Modification for Surfactant-Free Real-Time Toxicity Assay on Droplet Microfluidic Platform.

Microfluidic droplet reactors have many potential uses, from analytical to synthesis. Stable operation requires preferential wetting of the channel surface by the continuous phase which is often not fulfilled by materials commonly used for lab-on-chip devices. Here we show that a silica nanoparticle (SiNP) layer coated onto a Poly(methyl methacrylate) (PMMA) and other thermoplastics surface enhances its wetting properties by creating nanoroughness, and allows simple grafting of hydrocarbon chains through silane chemistry. Using the unusual stability of silica sols at their isoelectric point, a dense SiNP layer is adsorbed onto PMMA and renders the surface superhydrophilic. Subsequently, a self-assembled dodecyltrichlorosilane (DTS) monolayer yields a superhydrophobic surface that allows the repeatable generation of aqueous droplets in a hexadecane continuous phase without surfactant addition. A SiNP-DTS modified chip has been used to monitor bacterial viability with a resazurin assay. The whole process involving sequential reagents injection, and multiplexed droplet fluorescence intensity monitoring is carried out on chip. Metabolic inhibition of the anaerobe Enterococcus faecalis by 30 mg L-1 of NiCl2 was detected in 5 min.

Self-assembled photoadditives in polyester films allow stop and go chemical release.

Near-infrared (NIR) triggered chemical delivery allows on-demand release with the advantage of external tissue stimulation. Bioresorbable polyester poly-l-lactic acid (PLLA) was compounded with photoadditives of neat zinc oxide (ZnO) nanoparticles and 980→365nm LiYF4:Tm3+, Yb3+ upconverting nanoparticles (UCNP). Subsequently, neat ZnO and UCNP blended PLLA films of sub-50µm thickness were knife casted with a hydrophobic small molecule drug mimic, fluorescein diacetate. The PLLA films displayed a 500 times increase in fluorescein diacetate release from the 50mW NIR irradiated PLLA/photoadditive film compared to non-irradiated PLLA control films. Larger ratios of UCNP/neat ZnO increased photocatalysis efficiency at low NIR duty cycles. The synergistic increase results from the self-assembled photoadditives of neat zinc oxide and upconverting nanoparticles (UCNPs), as seen in transmission electron microscopy. Colloidal ZnO, which does not self-assemble with UCNPs, had less than half the release kinetics of the self-assembled PLLA films under similar conditions, advocating Förster resonance energy transfer as the mechanism responsible for the synergistic increase. Alternative to intensity modulation, pulse width modulation (duty cycles from 0.1 to 1) of the low intensity 50mW NIR laser diode allowed tailorable release rates from 0.01 to 1.4% per day. With the low intensity NIR activation, tailorable release rates, and favorable biocompatibility of the constituents, implanted PLLA photoadditive thin films could allow feedback mediated chemical delivery. STATEMENT OF SIGNIFICANCE: Upconverting nanoparticles and zinc oxide nanorods were found to spontaneously self-assemble into submicron particles in organic solvents. Exposure of the submicron particles to near-infrared light allows stop and go chemical release from biocompatible polymers. Sample preparation of thin films is done with ease through physical mixing of the photoadditives followed by air-dried knife casting. A colloidal ZnO variant that does not self-assemble with upconverting nanoparticles had slower chemical release, suggesting that synergistic chemical release is brought upon by highly efficient energy transfer mechanisms when the nanoparticles are less than 10nm apart. Never before seen composite particles of UCNP/ZnO are displayed, which shows the close interaction of the photoadditives within the polymer matrix.

Novel Sensor-Enabled Ex Vivo Bioreactor: A New Approach towards Physiological Parameters and Porcine Artery Viability.

The aim of the present work is to design and construct an ex vivo bioreactor system to assess the real time viability of vascular tissue. Porcine carotid artery as a model tissue was used in the ex vivo bioreactor setup to monitor its viability under physiological conditions such as oxygen, pressure, temperature, and flow. The real time tissue viability was evaluated by monitoring tissue metabolism through a fluorescent indicator "resorufin." Our ex vivo bioreactor allows real time monitoring of tissue responses along with physiological conditions. These ex vivo parameters were vital in determining the tissue viability in sensor-enabled bioreactor and our initial investigations suggest that, porcine tissue viability is considerably affected by high shear forces and low oxygen levels. Histological evaluations with hematoxylin and eosin and Masson's trichrome staining show intact endothelium with fresh porcine tissue whereas tissues after incubation in ex vivo bioreactor studies indicate denuded endothelium supporting the viability results from real time measurements. Hence, this novel viability sensor-enabled ex vivo bioreactor acts as model to mimic in vivo system and record vascular responses to biopharmaceutical molecules and biomedical devices.

Rapid fluorescence-based measurement of toxicity in anaerobic digestion.

A rapid fluorescence measurement based on resazurin reduction was developed and applied for the detection of toxicants/inhibitors to anaerobic digestion metabolism. By initially using a pure facultative anaerobic strain, Enterococcus faecalis as a model organism, this technique proved to be fast and sensitive when detecting the model toxicant, pentachlorophenol (PCP). The technique revealed significant metabolic changes in Enterococcus faecalis with a PCP spike ranging from 0.05 to 100 mg/L, and could detect PCP's toxicity to E. faecalis at a concentration of only 0.05 mg/L in 8 min. Furthermore, by extending this technique to a mixed anaerobic sludge, not only could the effect of 0.05-100 mg/L PCP be determined on anaerobic digestion metabolism within 10 min, but also its rate of biogas production. These results suggest that a resazurin-based fluorescence measurement can potentially be incorporated into a microfluidic system to develop a biosensor for the real-time monitoring, control and early warning of toxicant/inhibitor loads in the influent to an anaerobic digestion system.

Tunable chemical release from polyester thin film by photocatalytic zinc oxide and doped LiYF4 upconverting nanoparticles.

Once manufactured or implanted, polyester release kinetics tend to be fixed with little modulation possible for optimal local chemical concentrations. Here, a typical implantable polyester was fabricated into thin films (∼50 µm thick) with additives of photocatalytic ZnO nanoparticles, lanthanide-doped LiYF4 nanoparticle upconverting nanoparticles, or a combination thereof and irradiated with either 6 mW ultraviolet (365 nm) light emitting diodes or 50 mW near-infrared (980 nm) laser diodes to induce polymer photooxidation. Irradiated polyester films with the aforementioned photoadditives had enhanced release kinetics up to 30 times more than nonirradiated, neat films with extended release times of 28 days. Near-infrared, ZnO-mediated photocatalysis had the highest light on/light off ratio release kinetics of 15.4, while doped LiYF4 upconversion nanoparticles paired with ZnO nanoparticles had the highest linear R(2) correlation of 0.98 with respect to duty cycle and release kinetics. Future applications of the technology will aim toward modulation of previously developed polymeric reagents/drugs for real-time, feedback-optimized release.

Toxicants inhibiting anaerobic digestion: a review.

Anaerobic digestion is increasingly being used to treat wastes from many sources because of its manifold advantages over aerobic treatment, e.g. low sludge production and low energy requirements. However, anaerobic digestion is sensitive to toxicants, and a wide range of compounds can inhibit the process and cause upset or failure. Substantial research has been carried out over the years to identify specific inhibitors/toxicants, and their mechanism of toxicity in anaerobic digestion. In this review we present a detailed and critical summary of research on the inhibition of anaerobic processes by specific organic toxicants (e.g., chlorophenols, halogenated aliphatics and long chain fatty acids), inorganic toxicants (e.g., ammonia, sulfide and heavy metals) and in particular, nanomaterials, focusing on the mechanism of their inhibition/toxicity. A better understanding of the fundamental mechanisms behind inhibition/toxicity will enhance the wider application of anaerobic digestion.