Coral larval settlement induction using tissue-associated and exuded coralline algae metabolites and the identification of putative chemical cues.

Reef-building crustose coralline algae (CCA) are known to facilitate the settlement and metamorphosis of scleractinian coral larvae. In recent decades, CCA coverage has fallen globally and degrading environmental conditions continue to reduce coral survivorship, spurring new restoration interventions to rebuild coral reef health. In this study, naturally produced chemical compounds (metabolites) were collected from two pantropical CCA genera to isolate and classify those that induce coral settlement. In experiments using four ecologically important Caribbean coral species, we demonstrate the applicability of extracted, CCA-derived metabolites to improve larval settlement success in coral breeding and restoration efforts. Tissue-associated CCA metabolites induced settlement of one coral species, Orbicella faveolata, while metabolites exuded by CCA (exometabolites) induced settlement of three species: Acropora palmata, Colpophyllia natans and Orbicella faveolata. In a follow-up experiment, CCA exometabolites fractionated and preserved using two different extraction resins induced the same level of larval settlement as the unfractionated positive control exometabolites. The fractionated CCA exometabolite pools were characterized using liquid chromatography tandem mass spectrometry, yielding 145 distinct molecular subnetworks that were statistically defined as CCA-derived and could be classified into 10 broad chemical classes. Identifying these compounds can reveal their natural prevalence in coral reef habitats and facilitate the development of new applications to enhance larval settlement and the survival of coral juveniles.

Serial small- and wide-angle X-ray scattering with laboratory sources.

Recent advances in X-ray instrumentation and sample injection systems have enabled serial crystallography of protein nanocrystals and the rapid structural analysis of dynamic processes. However, this progress has been restricted to large-scale X-ray free-electron laser (XFEL) and synchrotron facilities, which are often oversubscribed and have long waiting times. Here, we explore the potential of state-of-the-art laboratory X-ray systems to perform comparable analyses when coupled to micro- and millifluidic sample environments. Our results demonstrate that commercial small- and wide-angle X-ray scattering (SAXS/WAXS) instruments and X-ray diffractometers are ready to access samples and timescales (≳5 ms) relevant to many processes in materials science including the preparation of pharmaceuticals, nanoparticles and functional crystalline materials. Tests of different X-ray instruments highlighted the importance of the optical configuration and revealed that serial WAXS/XRD analysis of the investigated samples was only possible with the higher flux of a microfocus setup. We expect that these results will also stimulate similar developments for structural biology.

Millimeter-scale topography facilitates coral larval settlement in wave-driven oscillatory flow.

Larval settlement in wave-dominated, nearshore environments is the most critical life stage for a vast array of marine invertebrates, yet it is poorly understood and virtually impossible to observe in situ. Using a custom-built flume tank that mimics the oscillatory fluid flow over a shallow coral reef, we isolated the effect of millimeter-scale benthic topography and showed that it increases the settlement of slow-swimming coral larvae by an order of magnitude relative to flat substrates. Particle tracking velocimetry of flow fields revealed that millimeter-scale ridges introduced regions of flow recirculation that redirected larvae toward the substrate surface and decreased the local fluid speed, effectively increasing the window of time for larvae to settle. Regions of recirculation were quantified using the Q-criterion method of vortex identification and correlated with the settlement locations of larvae for the first time. In agreement with experiments, computational fluid dynamics modeling and agent-based larval simulations also showed significantly higher settlement onto ridged substrates. Additionally, in contrast to previous reports on the effect of micro-scale substrate topography, we found that these topographies did not produce key hydrodynamic features linked to increased settlement. These findings highlight how physics-based substrate design can create new opportunities to increase larval recruitment for ecosystem restoration.

Characterising a PDMS based 3D cell culturing microfluidic platform for screening chemotherapeutic drug cytotoxic activity.

Three-dimensional (3D) spheroidal cell cultures are now recognised as better models of cancers as compared to traditional cell cultures. However, established 3D cell culturing protocols and techniques are time-consuming, manually laborious and often expensive due to the excessive consumption of reagents. Microfluidics allows for traditional laboratory-based biological experiments to be scaled down into miniature custom fabricated devices, where cost-effective experiments can be performed through the manipulation and flow of small volumes of fluid. In this study, we characterise a 3D cell culturing microfluidic device fabricated from a 3D printed master. HT29 cells were seeded into the device and 3D spheroids were generated and cultured through the perfusion of cell media. Spheroids were treated with 5-Fluorouracil for five days through continuous perfusion and cell viability was analysed on-chip at different time points using fluorescence microscopy and Lactate dehydrogenase (LDH) assay on the supernatant. Increasing cell death was observed in the HT29 spheroids over the five-day period. The 3D cell culturing microfluidic device described in this study, permits on-chip anti-cancer treatment and viability analysis, and forms the basis of an effective platform for the high-throughput screening of anti-cancer drugs in 3D tumour spheroids.

Ptychographic X-ray tomography reveals additive zoning in nanocomposite single crystals.

Single crystals containing nanoparticles represent a unique class of nanocomposites whose properties are defined by both their compositions and the structural organization of the dispersed phase in the crystalline host. Yet, there is still a poor understanding of the relationship between the synthesis conditions and the structures of these materials. Here ptychographic X-ray computed tomography is used to visualize the three-dimensional structures of two nanocomposite crystals - single crystals of calcite occluding diblock copolymer worms and vesicles. This provides unique information about the distribution of the copolymer nano-objects within entire, micron-sized crystals with nanometer spatial resolution and reveals how occlusion is governed by factors including the supersaturation and calcium concentration. Both nanocomposite crystals are seen to exhibit zoning effects that are governed by the solution composition and interactions of the additives with specific steps on the crystal surface. Additionally, the size and shape of the occluded vesicles varies according to their location within the crystal, and therefore the solution composition at the time of occlusion. This work contributes to our understanding of the factors that govern nanoparticle occlusion within crystalline materials, where this will ultimately inform the design of next generation nanocomposite materials with specific structure/property relationships.

Evaluation of microflow configurations for scale inhibition and serial X-ray diffraction analysis of crystallization processes.

The clean and reproducible conditions provided by microfluidic devices are ideal sample environments for in situ analyses of chemical and biochemical reactions and assembly processes. However, the small size of microchannels makes investigating the crystallization of poorly soluble materials on-chip challenging due to crystal nucleation and growth that result in channel fouling and blockage. Here, we demonstrate a reusable insert-based microfluidic platform for serial X-ray diffraction analysis and examine scale formation in response to continuous and segmented flow configurations across a range of temperatures. Under continuous flow, scale formation on the reactor walls begins almost immediately on mixing of the crystallizing species, which over time results in occlusion of the channel. Depletion of ions at the start of the channel results in reduced crystallization towards the end of the channel. Conversely, segmented flow can control crystallization, so it occurs entirely within the droplet. Consequently, the spatial location within the channel represents a temporal point in the crystallization process. Whilst each method can provide useful crystallographic information, time-resolved information is lost when reactor fouling occurs and changes the solution conditions with time. The flow within a single device can be manipulated to give a broad range of information addressing surface interaction or solution crystallization.

Dynamic Crystallization Pathways of Polymorphic Pharmaceuticals Revealed in Segmented Flow with Inline Powder X-ray Diffraction.

Understanding the transitions between polymorphs is essential in the development of strategies for manufacturing and maximizing the efficiency of pharmaceuticals. However, this can be extremely challenging: crystallization can be influenced by subtle changes in environment, such as temperature and mixing intensity or even imperfections in the crystallizer walls. Here, we highlight the importance of in situ measurements in understanding crystallization mechanisms, where a segmented flow crystallizer was used to study the crystallization of the pharmaceuticals urea: barbituric acid (UBA) and carbamazepine (CBZ). The reactor provides highly reproducible reaction conditions, while in situ synchrotron powder X-ray diffraction (PXRD) enables us to monitor the evolution of this system. UBA has two polymorphs of almost equivalent free-energy and so is typically obtained as a polymorphic mixture. In situ PXRD analysis uncovered a progression of polymorphs from UBA III to the thermodynamic polymorph UBA I, where different positions along the length of the tubular flow crystallizer correspond to different reaction times. Addition of UBA I seed crystals modified this pathway such that only UBA I was observed throughout, while transformation from UBA III into UBA I still occurred in the presence of UBA III seeds. Information regarding the mixing-dependent kinetics of the CBZ form II to III transformation was also uncovered in a series of seeded and unseeded flow crystallization runs, despite atypical habit expression. These results illustrate the importance of coupling controlled reaction environments with in situ XRD to study the phase relationships in polymorphic materials.

Controlling the fluorescence and room-temperature phosphorescence behaviour of carbon nanodots with inorganic crystalline nanocomposites.

There is a significant drive to identify alternative materials that exhibit room temperature phosphorescence for technologies including bio-imaging, photodynamic therapy and organic light-emitting diodes. Ideally, these materials should be non-toxic and cheap, and it will be possible to control their photoluminescent properties. This was achieved here by embedding carbon nanodots within crystalline particles of alkaline earth carbonates, sulphates and oxalates. The resultant nanocomposites are luminescent and exhibit a bright, sub-second lifetime afterglow. Importantly, the excited state lifetimes, and steady-state and afterglow colours can all be systematically controlled by varying the cations and anions in the host inorganic phase, due to the influence of the cation size and material density on emissive and non-emissive electronic transitions. This simple strategy provides a flexible route for generating materials with specific, phosphorescent properties and is an exciting alternative to approaches relying on the synthesis of custom-made luminescent organic molecules.

Rapid Mapping of Protein Interactions Using Tag-Transfer Photocrosslinkers.

Analysing protein complexes by chemical crosslinking-mass spectrometry (XL-MS) is limited by the side-chain reactivities and sizes of available crosslinkers, their slow reaction rates, and difficulties in crosslink enrichment, especially for rare, transient or dynamic complexes. Here we describe two new XL reagents that incorporate a methanethiosulfonate (MTS) group to label a reactive cysteine introduced into the bait protein, and a residue-unbiased diazirine-based photoactivatable XL group to trap its interacting partner(s). Reductive removal of the bait transfers a thiol-containing fragment of the crosslinking reagent onto the target that can be alkylated and located by MS sequencing and exploited for enrichment, enabling the detection of low abundance crosslinks. Using these reagents and a bespoke UV LED irradiation platform, we show that maximum crosslinking yield is achieved within 10 seconds. The utility of this "tag and transfer" approach is demonstrated using a well-defined peptide/protein regulatory interaction (BID80-102 /MCL-1), and the dynamic interaction interface of a chaperone/substrate complex (Skp/OmpA).

The Effect of Additives on the Early Stages of Growth of Calcite Single Crystals.

As crystallization processes are often rapid, it can be difficult to monitor their growth mechanisms. In this study, we made use of the fact that crystallization proceeds more slowly in small volumes than in bulk solution to investigate the effects of the soluble additives Mg2+ and poly(styrene sulfonate) (PSS) on the early stages of growth of calcite crystals. Using a "Crystal Hotel" microfluidic device to provide well-defined, nanoliter volumes, we observed that calcite crystals form via an amorphous precursor phase. Surprisingly, the first calcite crystals formed are perfect rhombohedra, and the soluble additives have no influence on the morphology until the crystals reach sizes of 0.1-0.5 µm for Mg2+ and 1-2 µm for PSS. The crystals then continue to grow to develop morphologies characteristic of these additives. These results can be rationalized by considering additive binding to kink sites, which is consistent with crystal growth by a classical mechanism.

3D visualization of additive occlusion and tunable full-spectrum fluorescence in calcite.

From biomineralization to synthesis, organic additives provide an effective means of controlling crystallization processes. There is growing evidence that these additives are often occluded within the crystal lattice. This promises an elegant means of creating nanocomposites and tuning physical properties. Here we use the incorporation of sulfonated fluorescent dyes to gain new understanding of additive occlusion in calcite (CaCO3), and to link morphological changes to occlusion mechanisms. We demonstrate that these additives are incorporated within specific zones, as defined by the growth conditions, and show how occlusion can govern changes in crystal shape. Fluorescence spectroscopy and lifetime imaging microscopy also show that the dyes experience unique local environments within different zones. Our strategy is then extended to simultaneously incorporate mixtures of dyes, whose fluorescence cascade creates calcite nanoparticles that fluoresce white. This offers a simple strategy for generating biocompatible and stable fluorescent nanoparticles whose output can be tuned as required.

HyCCAPP as a tool to characterize promoter DNA-protein interactions in Saccharomyces cerevisiae.

Currently available methods for interrogating DNA-protein interactions at individual genomic loci have significant limitations, and make it difficult to work with unmodified cells or examine single-copy regions without specific antibodies. In this study, we describe a physiological application of the Hybridization Capture of Chromatin-Associated Proteins for Proteomics (HyCCAPP) methodology we have developed. Both novel and known locus-specific DNA-protein interactions were identified at the ENO2 and GAL1 promoter regions of Saccharomyces cerevisiae, and revealed subgroups of proteins present in significantly different levels at the loci in cells grown on glucose versus galactose as the carbon source. Results were validated using chromatin immunoprecipitation. Overall, our analysis demonstrates that HyCCAPP is an effective and flexible technology that does not require specific antibodies nor prior knowledge of locally occurring DNA-protein interactions and can now be used to identify changes in protein interactions at target regions in the genome in response to physiological challenges.

Methylation of yeast ribosomal protein S2 is elevated during stationary phase growth conditions.

Ribosomes, as the center of protein translation in the cell, require careful regulation via multiple pathways. While regulation of ribosomal synthesis and function has been widely studied on the transcriptional and translational "levels," the biological roles of ribosomal post-translational modifications (PTMs) are largely not understood. Here, we explore this matter by using quantitative mass spectrometry to compare the prevalence of ribosomal methylation and acetylation for yeast in the log phase and the stationary phase of growth. We find that of the 27 modified peptides identified, two peptides experience statistically significant changes in abundance: a 1.9-fold decrease in methylation for k(Me)VSGFKDEVLETV of ribosomal protein S1B (RPS1B), and a 10-fold increase in dimethylation for r(DiMe)GGFGGR of ribosomal protein S2 (RPS2). While the biological role of RPS1B methylation has largely been unexplored, RPS2 methylation is a modification known to have a role in processing and export of ribosomal RNA. This suggests that yeast in the stationary phase increase methylation of RPS2 in order to regulate ribosomal synthesis. These results demonstrate the utility of mass spectrometry for quantifying dynamic changes in ribosomal PTMs.

Secreted proteoglycans directly mediate human embryonic stem cell-basic fibroblast growth factor 2 interactions critical for proliferation.

Human embryonic stem (ES) cells can be maintained in an undifferentiated state if the culture medium is first conditioned on a layer of mouse embryonic fibroblast (MEF) feeder cells. Here we show that human ES cell proliferation is coordinated by MEF-secreted heparan sulfate proteoglycans (HSPG) in conditioned medium (CM). These HSPG and other heparinoids can stabilize basic fibroblast growth factor (FGF2) in unconditioned medium at levels comparable to those observed in CM. They also directly mediate binding of FGF2 to the human ES cell surface, and their removal from CM impairs proliferation. Finally, we have developed a purification scheme for MEF-secreted HSPG in CM. Using column chromatography, immunoblotting, and mass spectrometry-based proteomic analysis, we have identified multiple HSPG species in CM. The results demonstrate that HSPG are key signaling cofactors in CM-based human ES cell culture.

Mass spectrometry identifies and quantifies 74 unique histone H4 isoforms in differentiating human embryonic stem cells.

Epigenetic regulation through chromatin is thought to play a critical role in the establishment and maintenance of pluripotency. Traditionally, antibody-based technologies were used to probe for specific posttranslational modifications (PTMs) present on histone tails, but these methods do not generally reveal the presence of multiple modifications on a single-histone tail (combinatorial codes). Here, we describe technology for the discovery and quantification of histone combinatorial codes that is based on chromatography and mass spectrometry. We applied this methodology to decipher 74 discrete combinatorial codes on the tail of histone H4 from human embryonic stem (ES) cells. Finally, we quantified the abundances of these codes as human ES cells undergo differentiation to reveal striking changes in methylation and acetylation patterns. For example, H4R3 methylation was observed only in the presence of H4K20 dimethylation; such context-specific patterning exemplifies the power of this technique.

Derivation of human embryonic stem cells in defined conditions.

We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells, but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions.

Basic fibroblast growth factor support of human embryonic stem cell self-renewal.

Human embryonic stem (ES) cells have most commonly been cultured in the presence of basic fibroblast growth factor (FGF2) either on fibroblast feeder layers or in fibroblast-conditioned medium. It has recently been reported that elevated concentrations of FGF2 permit the culture of human ES cells in the absence of fibroblasts or fibroblast-conditioned medium. Herein we compare the ability of unconditioned medium (UM) supplemented with 4, 24, 40, 80, 100, and 250 ng/ml FGF2 to sustain low-density human ES cell cultures through multiple passages. In these stringent culture conditions, 4, 24, and 40 ng/ml FGF2 failed to sustain human ES cells through three passages, but 100 ng/ml sustained human ES cells with an effectiveness comparable to conditioned medium (CM). Two human ES cell lines (H1 and H9) were maintained for up to 164 population doublings (7 and 4 months) in UM supplemented with 100 ng/ml FGF2. After prolonged culture, the cells formed teratomas when injected into severe combined immunodeficient beige mice and expressed markers characteristic of undifferentiated human ES cells. We also demonstrate that FGF2 is degraded more rapidly in UM than in CM, partly explaining the need for higher concentrations of FGF2 in UM. These results further facilitate the large-scale, routine culture of human ES cells and suggest that fibroblasts and fibro-blast-conditioned medium sustain human ES cells in part by stabilizing FGF signaling above a critical threshold.

Biochemical analysis of chromatin containing recombinant Drosophila core histones.

To investigate the effects of histone modifications upon chromatin structure and function, we studied the assembly and properties of chromatin that contains unmodified recombinant core histones. To this end, we synthesized the Drosophila core histones in Escherichia coli. The purified histones were lacking covalent modifications as well as their N-terminal initiating methionine residues. The recombinant histones were efficiently assembled into periodic nucleosome arrays in a completely purified recombinant system with Drosophila ATP-utilizing chromatin assembly and remodeling factor (ACF), Drosophila nucleosome assembly protein-1, plasmid DNA, and ATP. With the Gal4-VP16 activator and a crude transcription extract, we found that the transcriptional properties of ACF-assembled chromatin containing unmodified histones were similar to those of chromatin containing native histones. We then examined ACF-catalyzed chromatin remodeling with completely purified factors and chromatin consisting of unmodified histones. In these experiments, we observed promoter-specific disruption of the regularity of nucleosome arrays upon binding of Gal4-VP16 as well as nucleosome positioning by R3 Lac repressor and subsequent nucleosome remobilization upon isopropyl-beta-D-thiogalactopyranoside-induced dissociation of R3 from the template. Thus, chromatin assembly and remodeling by ACF can occur in the absence of histone modifications.

Chromatin assembly using Drosophila systems.

To successfully study chromatin structure and activity in vitro, it is essential to have a chromatin assembly system that will prepare extended nucleosome arrays with highly defined protein content that resemble bulk chromatin isolated from living cell nuclei in terms of periodicity and nucleosome positioning. The Drosophila ATP-dependent chromatin assembly system described in this unit meets these requirements. The end product of the reaction described here has highly periodic extended arrays with physiologic spacing and positioning of the nucleosomes.