Single-cell multi-omics in the medicinal plant Catharanthus roseus.

Advances in omics technologies now permit the generation of highly contiguous genome assemblies, detection of transcripts and metabolites at the level of single cells and high-resolution determination of gene regulatory features. Here, using a complementary, multi-omics approach, we interrogated the monoterpene indole alkaloid (MIA) biosynthetic pathway in Catharanthus roseus, a source of leading anticancer drugs. We identified clusters of genes involved in MIA biosynthesis on the eight C. roseus chromosomes and extensive gene duplication of MIA pathway genes. Clustering was not limited to the linear genome, and through chromatin interaction data, MIA pathway genes were present within the same topologically associated domain, permitting the identification of a secologanin transporter. Single-cell RNA-sequencing revealed sequential cell-type-specific partitioning of the leaf MIA biosynthetic pathway that, when coupled with a single-cell metabolomics approach, permitted the identification of a reductase that yields the bis-indole alkaloid anhydrovinblastine. We also revealed cell-type-specific expression in the root MIA pathway.

Genome-wide association identifies a missing hydrolase for tocopherol synthesis in plants.

The tocopherol biosynthetic pathway, encoded by VTE genes 1 through 6, is highly conserved in plants but most large effect quantitative trait loci for seed total tocopherols (totalT) lack VTE genes, indicating other activities are involved. A genome-wide association study of Arabidopsis seed tocopherols showed five of seven significant intervals lacked VTE genes, including the most significant, which mapped to an uncharacterized, seed-specific, envelope-localized, alpha/beta hydrolase with esterase activity, designated AtVTE7. Atvte7 null mutants decreased seed totalT 55% while a leaky allele of the maize ortholog, ZmVTE7, decreased kernel and leaf totalT 38% and 49%, respectively. Overexpressing AtVTE7 or ZmVTE7 partially or fully complemented the Atvte7 seed phenotype and increased leaf totalT by 3.6- and 6.9-fold, respectively. VTE7 has the characteristics of an esterase postulated to provide phytol from chlorophyll degradation for tocopherol synthesis, but bulk chlorophyll levels were unaffected in vte7 mutants and overexpressing lines. Instead, levels of specific chlorophyll biosynthetic intermediates containing partially reduced side chains were impacted and strongly correlated with totalT. These intermediates are generated by a membrane-associated biosynthetic complex containing protochlorophyllide reductase, chlorophyll synthase, geranylgeranyl reductase (GGR) and light harvesting-like 3 protein, all of which are required for both chlorophyll and tocopherol biosynthesis. We propose a model where VTE7 releases prenyl alcohols from chlorophyll biosynthetic intermediates, which are then converted to the corresponding diphosphates for tocopherol biosynthesis.

Quantitative Single-Cell Mass Spectrometry Provides a Highly Resolved Analysis of Natural Product Biosynthesis Partitioning in Plants.

Plants produce an extraordinary array of natural products (specialized metabolites). Notably, these structurally complex molecules are not evenly distributed throughout plant tissues but are instead synthesized and stored in specific cell types. Elucidating both the biosynthesis and function of natural products would be greatly facilitated by tracking the location of these metabolites at the cell-level resolution. However, detection, identification, and quantification of metabolites in single cells, particularly from plants, have remained challenging. Here, we show that we can definitively identify and quantify the concentrations of 16 molecules from four classes of natural products in individual cells of leaf, root, and petal of the medicinal plant Catharanthus roseus using a plate-based single-cell mass spectrometry method. We show that identical natural products show substantially different patterns of cell-type localization in different tissues. Moreover, we show that natural products are often found in a wide range of concentrations across a population of cells, with some natural products at concentrations of over 100 mM per cell. This single-cell mass spectrometry method provides a highly resolved picture of plant natural product biosynthesis partitioning at a cell-specific resolution.

The biosynthesis of thymol, carvacrol, and thymohydroquinone in Lamiaceae proceeds via cytochrome P450s and a short-chain dehydrogenase.

Thymol and carvacrol are phenolic monoterpenes found in thyme, oregano, and several other species of the Lamiaceae. Long valued for their smell and taste, these substances also have antibacterial and anti-spasmolytic properties. They are also suggested to be precursors of thymohydroquinone and thymoquinone, monoterpenes with anti-inflammatory, antioxidant, and antitumor activities. Thymol and carvacrol biosynthesis has been proposed to proceed by the cyclization of geranyl diphosphate to γ-terpinene, followed by a series of oxidations via p-cymene. Here, we show that γ-terpinene is oxidized by cytochrome P450 monooxygenases (P450s) of the CYP71D subfamily to produce unstable cyclohexadienol intermediates, which are then dehydrogenated by a short-chain dehydrogenase/reductase (SDR) to the corresponding ketones. The subsequent formation of the aromatic compounds occurs via keto-enol tautomerisms. Combining these enzymes with γ-terpinene in in vitro assays or in vivo in Nicotiana benthamiana yielded thymol and carvacrol as products. In the absence of the SDRs, only p-cymene was formed by rearrangement of the cyclohexadienol intermediates. The nature of these unstable intermediates was inferred from reactions with the γ-terpinene isomer limonene and by analogy to reactions catalyzed by related enzymes. We also identified and characterized two P450s of the CYP76S and CYP736A subfamilies that catalyze the hydroxylation of thymol and carvacrol to thymohydroquinone when heterologously expressed in yeast and N. benthamiana Our findings alter previous views of thymol and carvacrol formation, identify the enzymes involved in the biosynthesis of these phenolic monoterpenes and thymohydroquinone in the Lamiaceae, and provide targets for metabolic engineering of high-value terpenes in plants.

Less Evaluative Measures of Personality in Job Applicant Contexts: The Effect on Socially Desirable Responding and Criterion Validity.

Researchers have long sought to mitigate the detrimental effects of socially desirable responding on personality assessments in high-stakes contexts. This study investigated the effect of reducing the social desirability of personality items on response distortion and criterion validity in a job applicant context. Using a 2 × 2 repeated measures design, participants (n = 584) completed standard (International Personality Item Pool) and less evaluative (Less Evaluative Five Factor Inventory) measures of Big Five personality in a low-stakes context and then several weeks later in a simulated job applicant context. Self-report criteria with objective answers, including university grades, were also obtained. In general, the less evaluative measure showed less response distortion than the standard measure on some metrics, but not on others. Declines in criterion validity in the applicant context were smaller for the less evaluative measure. In the applicant context, however, validities were similar across the two measures. Correlations across contexts for corresponding traits (e.g., low-stakes extraversion with high-stakes extraversion) were also similar for both measures. In summary, reducing socially desirable item content might slightly reduce the substantive content required to predict criteria in low-stakes contexts, but this effect appears to be partly offset by reduced response distortion for less evaluative measures in applicant contexts.

Responses in randomised groups of healthy, adult Labrador retrievers fed grain-free diets with high legume inclusion for 30 days display commonalities with dogs with suspected dilated cardiomyopathy.

Early responses in healthy adult dogs fed grain-free diets with high inclusion of split peas (20%) and lentils (40%) that may lead to canine diet-induced dilated cardiomyopathy (DCM) were investigated. To help understand the clinical relevance of the findings, a survey of electronic health records (EHR) was conducted of dogs with and without suspected DCM for comparison. Control and Test diets were fed to Labrador retriever dogs for 30 days (n = 5 and 6, respectively). Blood and urine samples collected at baseline and days 3, 14 and 28/30 were analyzed for hematology, clinical biochemistry and taurine concentrations. The EHRs of dogs at Banfield® Pet Hospitals in the 2-year period 2018-2019 were surveyed, revealing 420 dogs diagnosed with DCM, which were compared with 420 breed, gender and age-matched healthy control dogs. Compared to baseline values, feeding the Test diet for 28 days caused progressive, significant (p < 0.001) decreases in red blood cell counts (RBC), hematocrit and total hemoglobin by 7.7, 8.3 and 6.3%, respectively, and a 41.8% increase in plasma inorganic phosphate. Commonalities in these parameters were observed in clinical DCM cases. Regarding taurine status, Test dogs transiently increased whole-blood (23.4%) and plasma (47.7%) concentrations on day 14, while taurine:creatinine ratio in fresh urine and taurine in pooled urine were reduced by 77 and 78%, respectively, on day 28/30. Thus grain-free, legume-rich Test diets caused reduced RBC and hyperphosphatemia, findings also indicated in dogs with suspected DCM. Changes in taurine metabolism were indicated. The data will aid in generating hypotheses for future studies.

An updated gene atlas for maize reveals organ-specific and stress-induced genes.

Maize (Zea mays L.), a model species for genetic studies, is one of the two most important crop species worldwide. The genome sequence of the reference genotype, B73, representative of the stiff stalk heterotic group was recently updated (AGPv4) using long-read sequencing and optical mapping technology. To facilitate the use of AGPv4 and to enable functional genomic studies and association of genotype with phenotype, we determined expression abundances for replicated mRNA-sequencing datasets from 79 tissues and five abiotic/biotic stress treatments revealing 36 207 expressed genes. Characterization of the B73 transcriptome across six organs revealed 4154 organ-specific and 7704 differentially expressed (DE) genes following stress treatment. Gene co-expression network analyses revealed 12 modules associated with distinct biological processes containing 13 590 genes providing a resource for further association of gene function based on co-expression patterns. Presence-absence variants (PAVs) previously identified using whole genome resequencing data from 61 additional inbred lines were enriched in organ-specific and stress-induced DE genes suggesting that PAVs may function in phenological variation and adaptation to environment. Relative to core genes conserved across the 62 profiled inbreds, PAVs have lower expression abundances which are correlated with their frequency of dispersion across inbreds and on average have significantly fewer co-expression network connections suggesting that a subset of PAVs may be on an evolutionary path to pseudogenization. To facilitate use by the community, we developed the Maize Genomics Resource website (maize.plantbiology.msu.edu) for viewing and data-mining these resources and deployed two new views on the maize electronic Fluorescent Pictograph Browser (bar.utoronto.ca/efp_maize).

Quantitative trait loci and differential gene expression analyses reveal the genetic basis for negatively associated ß-carotene and starch content in hexaploid sweetpotato [Ipomoea batatas (L.) Lam.].

KEY MESSAGE: ß-Carotene content in sweetpotato is associated with the Orange and phytoene synthase genes; due to physical linkage of phytoene synthase with sucrose synthase, ß-carotene and starch content are negatively correlated. In populations depending on sweetpotato for food security, starch is an important source of calories, while ß-carotene is an important source of provitamin A. The negative association between the two traits contributes to the low nutritional quality of sweetpotato consumed, especially in sub-Saharan Africa. Using a biparental mapping population of 315 F1 progeny generated from a cross between an orange-fleshed and a non-orange-fleshed sweetpotato variety, we identified two major quantitative trait loci (QTL) on linkage group (LG) three (LG3) and twelve (LG12) affecting starch, ß-carotene, and their correlated traits, dry matter and flesh color. Analysis of parental haplotypes indicated that these two regions acted pleiotropically to reduce starch content and increase ß-carotene in genotypes carrying the orange-fleshed parental haplotype at the LG3 locus. Phytoene synthase and sucrose synthase, the rate-limiting and linked genes located within the QTL on LG3 involved in the carotenoid and starch biosynthesis, respectively, were differentially expressed in Beauregard versus Tanzania storage roots. The Orange gene, the molecular switch for chromoplast biogenesis, located within the QTL on LG12 while not differentially expressed was expressed in developing roots of the parental genotypes. We conclude that these two QTL regions act together in a cis and trans manner to inhibit starch biosynthesis in amyloplasts and enhance chromoplast biogenesis, carotenoid biosynthesis, and accumulation in orange-fleshed sweetpotato. Understanding the genetic basis of this negative association between starch and ß-carotene will inform future sweetpotato breeding strategies targeting sweetpotato for food and nutritional security.

Genome diversity of tuber-bearing Solanum uncovers complex evolutionary history and targets of domestication in the cultivated potato.

Cultivated potatoes (Solanum tuberosum L.), domesticated from wild Solanum species native to the Andes of southern Peru, possess a diverse gene pool representing more than 100 tuber-bearing relatives (Solanum section Petota). A diversity panel of wild species, landraces, and cultivars was sequenced to assess genetic variation within tuber-bearing Solanum and the impact of domestication on genome diversity and identify key loci selected for cultivation in North and South America. Sequence diversity of diploid and tetraploid Stuberosum exceeded any crop resequencing study to date, in part due to expanded wild introgressions following polyploidy that captured alleles outside of their geographic origin. We identified 2,622 genes as under selection, with only 14-16% shared by North American and Andean cultivars, showing that a limited gene set drove early improvement of cultivated potato, while adaptation of upland (Stuberosum group Andigena) and lowland (S. tuberosum groups Chilotanum and Tuberosum) populations targeted distinct loci. Signatures of selection were uncovered in genes controlling carbohydrate metabolism, glycoalkaloid biosynthesis, the shikimate pathway, the cell cycle, and circadian rhythm. Reduced sexual fertility that accompanied the shift to asexual reproduction in cultivars was reflected by signatures of selection in genes regulating pollen development/gametogenesis. Exploration of haplotype diversity at potato's maturity locus (StCDF1) revealed introgression of truncated alleles from wild species, particularly Smicrodontum in long-day-adapted cultivars. This study uncovers a historic role of wild Solanum species in the diversification of long-day-adapted tetraploid potatoes, showing that extant natural populations represent an essential source of untapped adaptive potential.

Stable aqueous dispersions of optically and electronically active phosphorene.

Understanding and exploiting the remarkable optical and electronic properties of phosphorene require mass production methods that avoid chemical degradation. Although solution-based strategies have been developed for scalable exfoliation of black phosphorus, these techniques have thus far used anhydrous organic solvents in an effort to minimize exposure to known oxidants, but at the cost of limited exfoliation yield and flake size distribution. Here, we present an alternative phosphorene production method based on surfactant-assisted exfoliation and postprocessing of black phosphorus in deoxygenated water. From comprehensive microscopic and spectroscopic analysis, this approach is shown to yield phosphorene dispersions that are stable, highly concentrated, and comparable to micromechanically exfoliated phosphorene in structure and chemistry. Due to the high exfoliation efficiency of this process, the resulting phosphorene flakes are thinner than anhydrous organic solvent dispersions, thus allowing the observation of layer-dependent photoluminescence down to the monolayer limit. Furthermore, to demonstrate preservation of electronic properties following solution processing, the aqueous-exfoliated phosphorene flakes are used in field-effect transistors with high drive currents and current modulation ratios. Overall, this method enables the isolation and mass production of few-layer phosphorene, which will accelerate ongoing efforts to realize a diverse range of phosphorene-based applications.

Silicon-Phosphorene Nanocavity-Enhanced Optical Emission at Telecommunications Wavelengths.

Generating and amplifying light in silicon (Si) continues to attract significant attention due to the possibility of integrating optical and electronic components in a single material platform. Unfortunately, silicon is an indirect band gap material and therefore an inefficient emitter of light. With the rise of integrated photonics, the search for silicon-based light sources has evolved from a scientific quest to a major technological bottleneck for scalable, CMOS-compatible, light sources. Recently, emerging two-dimensional materials have opened the prospect of tailoring material properties based on atomic layers. Few-layer phosphorene, which is isolated through exfoliation from black phosphorus (BP), is a great candidate to partner with silicon due to its layer-tunable direct band gap in the near-infrared where silicon is transparent. Here we demonstrate a hybrid silicon optical emitter composed of few-layer phosphorene nanomaterial flakes coupled to silicon photonic crystal resonators. We show single-mode emission in the telecommunications band of 1.55 µm ( Eg = 0.8 eV) under continuous wave optical excitation at room temperature. The solution-processed few-layer BP flakes enable tunable emission across a broad range of wavelengths and the simultaneous creation of multiple devices. Our work highlights the versatility of the Si-BP material platform for creating optically active devices in integrated silicon chips.

Solution-Based Processing of Monodisperse Two-Dimensional Nanomaterials.

Exfoliation of single-layer graphene from bulk graphite and the subsequent discovery of exotic physics and emergent phenomena in the atomically thin limit has motivated the isolation of other two-dimensional (2D) layered nanomaterials. Early work on isolated 2D nanomaterial flakes has revealed a broad range of unique physical and chemical properties with potential utility in diverse applications. For example, the electronic and optical properties of 2D nanomaterials depend strongly on atomic-scale variations in thickness, enabling enhanced performance in optoelectronic technologies such as light emitters, photodetectors, and photovoltaics. Much of the initial research on 2D nanomaterials has relied on micromechanical exfoliation, which yields high-quality 2D nanomaterial flakes that are suitable for fundamental studies but possesses limited scalability for real-world applications. In an effort to overcome this limitation, solution-processing methods for isolating large quantities of 2D nanomaterials have emerged. Importantly, solution processing results in 2D nanomaterial dispersions that are amenable to roll-to-roll fabrication methods that underlie lost-cost manufacturing of thin-film transistors, transparent conductors, energy storage devices, and solar cells. Despite these advantages, solution-based exfoliation methods typically lack control over the lateral size and thickness of the resulting 2D nanomaterial flakes, resulting in polydisperse dispersions with heterogeneous properties. Therefore, post-exfoliation separation techniques are needed to achieve 2D nanomaterial dispersions with monodispersity in lateral size, thickness, and properties. In this Account, we survey the latest developments in solution-based separation methods that aim to produce monodisperse dispersions and thin films of emerging 2D nanomaterials such as graphene, boron nitride, transition metal dichalcogenides, and black phosphorus. First, we motivate the need for precise thickness control in 2D nanomaterials by reviewing thickness-dependent physical properties. Then we present a succinct survey of solution-based exfoliation methods that yield 2D nanomaterial dispersions in organic solvents and aqueous media. The Account subsequently focuses on separation methods, including a critical analysis of their relative strengths and weaknesses for 2D nanomaterials with different buoyant densities, van der Waals interactions, and chemical reactivities. Specifically, we evaluate sedimentation-based density gradient ultracentrifugation (sDGU) and isopycnic DGU (iDGU) for post-exfoliation 2D nanomaterial dispersion separation. The comparative advantages of sedimentation and isopycnic methods are presented in both aqueous and nonaqueous media for 2D nanomaterials with varying degrees of chemical reactivity. Finally, we survey methods for forming homogeneous thin films from 2D nanomaterial dispersions and emerging technologies that are likely to benefit from these structures. Overall, this Account provides not only an overview of the present state-of-the-art but also a forward-looking vision for the field of solution-processed monodisperse 2D nanomaterials.

Relationship between duration of preoperative symptoms and postoperative ileus for small bowel obstruction.

BACKGROUND: Factors associated with postoperative ileus and increased resource utilization for patients who undergo operative intervention for small-bowel obstruction are not extensively studied. We evaluated the association between total duration of preoperative symptoms and postoperative outcomes in this population. MATERIALS AND METHODS: We performed a retrospective review of patients who underwent surgery for small-bowel obstruction (2013-2016). Clinical data were recorded. Total duration of preoperative symptoms included all symptoms before operation, including those before presentation. Primary endpoint was time to tolerance of diet. Secondary endpoints included length of stay, total parenteral nutrition use, and intensive care unit admission. Association between variables and outcomes was analyzed using univariable analysis, multivariable Poisson modeling, and t-test to compare groups. RESULTS: Sixty-seven patients were included. On presentation, the median duration of symptoms before hospitalization was 2 d (range 0-18 d). Total duration of preoperative symptoms was associated with time to tolerance of diet on univariable analysis (Pearson's moment correlation: 0.28, 95% confidence interval: 0.028-0.5, P = 0.03). On multivariable analysis, ascites was correlated with time to tolerance of diet (P < 0.01), but total duration of preoperative symptoms (P = 0.07) was not. Length of stay (Pearson's correlation: 0.24, 95% confidence interval: -0.02 to 0.47, P = 0.07) was not statistically different in patients with longer preoperative symptoms. Symptom duration was not statistically associated with intensive care unit (P = 0.18) or total parenteral nutrition (P = 0.3) utilization. CONCLUSIONS: Our findings demonstrate that preoperative ascites correlated with increased time to tolerance of diet, and duration of preoperative symptoms may be related to postoperative ileus.

Impact of choice of future climate change projection on growth chamber experimental outcomes: a preliminary study in potato.

Understanding the impacts of climate change on agriculture is essential to ensure adequate future food production. Controlled growth experiments provide an effective tool for assessing the complex effects of climate change. However, a review of the use of climate projections in 57 previously published controlled growth studies found that none considered within-season variations in projected future temperature change, and few considered regional differences in future warming. A fixed, often arbitrary, temperature perturbation typically was applied for the entire growing season. This study investigates the utility of employing more complex climate change scenarios in growth chamber experiments. A case study in potato was performed using three dynamically downscaled climate change projections for the mid-twenty-first century that differ in terms of the timing during the growing season of the largest projected temperature changes. The climate projections were used in growth chamber experiments for four elite potato cultivars commonly planted in Michigan's major potato growing region. The choice of climate projection had a significant influence on the sign and magnitude of the projected changes in aboveground biomass and total tuber count, whereas all projections suggested an increase in total tuber weight and a decrease in specific gravity, a key market quality trait for potato, by mid-century. These results demonstrate that the use of more complex climate projections that extend beyond a simple incremental change can provide additional insights into the future impacts of climate change on crop production and the accompanying uncertainty.

Layer-by-Layer Sorting of Rhenium Disulfide via High-Density Isopycnic Density Gradient Ultracentrifugation.

Isopycnic density gradient ultracentrifugation (iDGU) has been widely applied to sort nanomaterials by their physical and electronic structure. However, the commonly used density-gradient medium iodixanol has a finite maximum buoyant density that prevents the use of iDGU for high-density nanomaterials. Here, we overcome this limit by adding cesium chloride (CsCl) to iodixanol, thus increasing its maximum buoyant density to the point where the high-density two-dimensional nanomaterial rhenium disulfide (ReS2) can be sorted in a layer-by-layer manner with iDGU. The resulting aqueous ReS2 dispersions show photoluminescence at ∼1.5 eV, which is consistent with its direct bandgap semiconductor electronic structure. Furthermore, photocurrent measurements on thin films formed from solution-processed ReS2 show a spectral response that is consistent with optical absorbance and photoluminescence data. In addition to providing a pathway for effective solution processing of ReS2, this work establishes a general methodology for sorting high-density nanomaterials via iDGU.

Solution-Processed Dielectrics Based on Thickness-Sorted Two-Dimensional Hexagonal Boron Nitride Nanosheets.

Gate dielectrics directly affect the mobility, hysteresis, power consumption, and other critical device metrics in high-performance nanoelectronics. With atomically flat and dangling bond-free surfaces, hexagonal boron nitride (h-BN) has emerged as an ideal dielectric for graphene and related two-dimensional semiconductors. While high-quality, atomically thin h-BN has been realized via micromechanical cleavage and chemical vapor deposition, existing liquid exfoliation methods lack sufficient control over h-BN thickness and large-area film quality, thus limiting its use in solution-processed electronics. Here, we employ isopycnic density gradient ultracentrifugation for the preparation of monodisperse, thickness-sorted h-BN inks, which are subsequently layer-by-layer assembled into ultrathin dielectrics with low leakage currents of 3 × 10(-9) A/cm(2) at 2 MV/cm and high capacitances of 245 nF/cm(2). The resulting solution-processed h-BN dielectric films enable the fabrication of graphene field-effect transistors with negligible hysteresis and high mobilities up to 7100 cm(2) V(-1) s(-1) at room temperature. These h-BN inks can also be used as coatings on conventional dielectrics to minimize the effects of underlying traps, resulting in improvements in overall device performance. Overall, this approach for producing and assembling h-BN dielectric inks holds significant promise for translating the superlative performance of two-dimensional heterostructure devices to large-area, solution-processed nanoelectronics.

Thermally labile components of aqueous humor potently induce osteogenic potential in adipose-derived mesenchymal stem cells.

Adipose-derived mesenchymal stem cells (ASCs) hold promise for use in cell-based therapies. Their intrinsic anti-inflammatory properties are potentially useful for treatments of inflammatory conditions such as uveitis, while their ability to differentiate along multiple cell lineages suggests use in regenerating damaged or degenerated tissue. However, how ASCs will respond to the intraocular environment is poorly studied. We have recently reported that aqueous humor (AH), the fluid that nourishes the anterior segment of the eye, potently increases alkaline phosphatase (ALP) activity of ASCs, indicating osteogenic differentiation. Here, we expand on our previous findings to better define the nature of this response. To this end, we cultured ASCs in the presence of 0, 5, 10, and 20% AH and assayed them for ALP activity. We found ALP activity correlates with increasing AH concentrations from 5 to 20%, and that longer treatments result in increased ALP activity. By using serum free media and pretreating AH with dextran-coated charcoal, we found that serum and charcoal-adsorbable AH components augment but are not required for this response. Further, by heat-treating the AH, we established that thermally labile components are required for the osteogenic response. Finally, we showed myocilin, a protein present in AH, could induce ALP activity in ASCs. However, this was to a lesser extent than untreated 5% AH, and myocilin could only partially rescue the effect after heat treatment, documenting there were additional thermally labile constituents of AH involved in the osteogenic response. Our work adds to the understanding of the induction of ALP in ASCs following exposure to AH, providing important insight in how ASCs will be influenced by the ocular environment. In conclusion, increased osteogenic potential upon exposure to AH represents a potential challenge to developing ASC cell-based therapies directed at the eye.

Annealing free, clean graphene transfer using alternative polymer scaffolds.

We examine the transfer of graphene grown by chemical vapor deposition (CVD) with polymer scaffolds of poly(methyl methacrylate) (PMMA), poly(lactic acid) (PLA), poly(phthalaldehyde) (PPA), and poly(bisphenol A carbonate) (PC). We find that optimally reactive PC scaffolds provide the cleanest graphene transfers without any annealing, after extensive comparison with optical microscopy, x-ray photoelectron spectroscopy, atomic force microscopy, and scanning tunneling microscopy. Comparatively, films transferred with PLA, PPA, PMMA/PC, and PMMA have a two-fold higher roughness and a five-fold higher chemical doping. Using PC scaffolds, we demonstrate the clean transfer of CVD multilayer graphene, fluorinated graphene, and hexagonal boron nitride. Our annealing free, PC transfers enable the use of atomically-clean nanomaterials in biomolecule encapsulation and flexible electronic applications.

The formation of cortical actin arrays in human trabecular meshwork cells in response to cytoskeletal disruption.

The cytoskeleton of human trabecular meshwork (HTM) cells is known to be altered in glaucoma and has been hypothesized to reduce outflow facility through contracting the HTM tissue. Latrunculin B (Lat-B) and Rho-associated protein kinase (ROCK) inhibitors disrupt the actin cytoskeleton and are in clinical trials as glaucoma therapeutics. We have previously reported a transient increase in HTM cell stiffness peaking at 90 min after Lat-B treatment with a return to pretreatment values after 270 min. We hypothesize that changes in actin morphology correlate with alterations in cell stiffness induced by Lat-B but this is not a general consequence of other cytoskeletal disrupting agents such as Rho kinase inhibitors. We treated HTM cells with 2 µM Lat-B or 100 µM Y-27632 and allowed the cells to recover for 30-270 min. While examining actin morphology in Lat-B treated cells, we observed striking cortical actin arrays (CAAs). The percentage of CAA positive cells (CPCs) was time dependent and exceeded 30% at 90 min and decreased after 270 min. Y-27632 treated cells exhibited few CAAs and no changes in cell stiffness. Together, these data suggest that the increase in cell stiffness after Lat-B treatment is correlated with CAAs.