Differential Efficacy of Small Molecules Dynasore and Mdivi-1 for the Treatment of Dry Eye Epitheliopathy or as a Countermeasure for Nitrogen Mustard Exposure of the Ocular Surface.

The ocular surface comprises the wet mucosal epithelia of the cornea and conjunctiva, the associated glands, and the overlying tear film. Epitheliopathy is the common pathologic outcome when the ocular surface is subjected to oxidative stress. Whether different stresses act via the same or different mechanisms is not known. Dynasore and dyngo-4a, small molecules developed to inhibit the GTPase activity of classic dynamins DNM1, DNM2, and DNM3, but not mdivi-1, a specific inhibitor of DNM1L, protect corneal epithelial cells exposed to the oxidant tert-butyl hydroperoxide (tBHP). Here we report that, while dyngo-4a is the more potent inhibitor of endocytosis, dynasore is the better cytoprotectant. Dynasore also protects corneal epithelial cells against exposure to high salt in an in vitro model of dysfunctional tears in dry eye. We now validate this finding in vivo, demonstrating that dynasore protects against epitheliopathy in a mouse model of dry eye. Knockdown of classic dynamin DNM2 was also cytoprotective against tBHP exposure, suggesting that dynasore's effect is at least partially on target. Like tBHP and high salt, exposure of corneal epithelial cells to nitrogen mustard upregulated the unfolded protein response and inflammatory markers, but dynasore did not protect against nitrogen mustard exposure. In contrast, mdivi-1 was cytoprotective. Interestingly, mdivi-1 did not inhibit the nitrogen mustard-induced expression of inflammatory cytokines. We conclude that exposure to tBHP or nitrogen mustard, two different oxidative stress agents, cause corneal epitheliopathy via different pathologic pathways. SIGNIFICANCE STATEMENT: Results presented in this paper, for the first time, implicate the dynamin DNM2 in ocular surface epitheliopathy. The findings suggest that dynasore could serve as a new topical treatment for dry eye epitheliopathy and that mdivi-1 could serve as a medical countermeasure for epitheliopathy due to nitrogen mustard exposure, with potentially increased efficacy when combined with anti-inflammatory agents and/or UPR modulators.

Dynasore modulates store-operated calcium entry and mitochondrial calcium release in corneal epithelial cells.

Dysregulation of calcium homeostasis can precipitate a cascade of pathological events that lead to tissue damage and cell death. Dynasore is a small molecule that inhibits endocytosis by targeting classic dynamins. In a previous study, we showed that dynasore can protect human corneal epithelial cells from damage due to tert-butyl hydroperoxide (tBHP) exposure by restoring cellular calcium (Ca2+) homeostasis. Here we report results of a follow-up study aimed at identifying the source of the damaging Ca2+. Store-operated Ca2+ entry (SOCE) is a cellular mechanism to restore intracellular calcium stores from the extracellular milieu. We found that dynasore effectively blocks SOCE in cells treated with thapsigargin (TG), a small molecule that inhibits pumping of Ca2+ into the endoplasmic reticulum (ER). Unlike dynasore however, SOCE inhibitor YM-58483 did not interfere with the cytosolic Ca2+ overload caused by tBHP exposure. We also found that dynasore effectively blocks Ca2+ release from internal sources. The inefficacy of inhibitors of ER Ca2+ channels suggested that this compartment was not the source of the Ca2+ surge caused by tBHP exposure. However, using a Ca2+-measuring organelle-entrapped protein indicator (CEPIA) reporter targeted to mitochondria, we found that dynasore can block mitochondrial Ca2+ release due to tBHP exposure. Our results suggest that dynasore exerts multiple effects on cellular Ca2+ homeostasis, with inhibition of mitochondrial Ca2+ release playing a key role in protection of corneal epithelial cells against oxidative stress due to tBHP exposure.

Dynasore Protects Corneal Epithelial Cells Subjected to Hyperosmolar Stress in an In Vitro Model of Dry Eye Epitheliopathy.

Epitheliopathy at the ocular surface is a defining sign of dry eye disease, a common disorder that affects 10% to 30% of the world's population. Hyperosmolarity of the tear film is one of the main drivers of pathology, with subsequent endoplasmic reticulum (ER) stress, the resulting unfolded protein response (UPR), and caspase-3 activation implicated in the pathway to programmed cell death. Dynasore, is a small molecule inhibitor of dynamin GTPases that has shown therapeutic effects in a variety of disease models involving oxidative stress. Recently we showed that dynasore protects corneal epithelial cells exposed to the oxidant tBHP, by selective reduction in expression of CHOP, a marker of the UPR PERK branch. Here we investigated the capacity of dynasore to protect corneal epithelial cells subjected to hyperosmotic stress (HOS). Similar to dynasore's capacity to protect against tBHP exposure, dynasore inhibits the cell death pathway triggered by HOS, protecting against ER stress and maintaining a homeostatic level of UPR activity. However, unlike with tBHP exposure, UPR activation due to HOS is independent of PERK and mostly driven by the UPR IRE1 branch. Our results demonstrate the role of the UPR in HOS-driven damage, and the potential of dynasore as a treatment to prevent dry eye epitheliopathy.

Assessment of dry eye in a GVHD murine model: Approximation through tear osmolarity measurement.

Dry eye disease is one of the most frequent pathological events that take place in the course of the graft versus host disease (GVHD), and is the main cause of deterioration in quality of life for patients. Thus, demonstration of dry eye signs in murine models of oGVHD is crucial for the validation of these models for the study of the disease. Given the increasing evidence that tear osmolarity is an important player of dry eye disease, our purpose in this study was to validate the use of a reliable method to assess tear osmolarity in mice: the electrical impedance method. Then, we wanted to test its utility with an oGVHD model. Tear volume assessment was also performed, using the phenol red thread test. We found differences in tear osmolarity in mice that received a transplant with cells from bone marrow and spleen (the GVHD group) when compared with mice that only received bone marrow cells (the BM group) at day 7 (362 ± 8 mOsm/l and 345 ± 9 mOsm/l respectively; P < 0.01) and day 21 (348 ± 19 mOsm/l vs. 326 ± 15 mOsm/l; P < 0.05). We found also differences in tear volume at day 14 (2.30 ± 0.61 mm in oGVHD group and 2.89 ± 0.62 mm in BM group; P = 0.06) and at day 21 (2.10 ± 0.30 mm in oGVHD group and 2.89 ± 0.32 mm in BM group; P < 0.01). Besides this, we observed reduction in epithelial thickness between the GVHD and BM groups (37.0 ± 6.2 µm and 43.6 ± 3.3 µm respectively; P < 0.05). These data show the usefulness of the electrical impedance method to measure tear osmolarity in mice. We can also conclude that this oGVHD model mimics the tear film alterations found in human dry eye disease, what contributes to give relevance to this model for the study of GVHD.

Metabolic and Transcriptional Analysis of Durum Wheat Responses to Elevated CO2 at Low and High Nitrate Supply.

Elevated [CO2] (eCO2) can lead to photosynthetic acclimation and this is often intensified by low nitrogen (N). Despite intensive studies of plant responses to eCO2, the regulation mechanism of primary metabolism at the whole-plant level in interaction with [Formula: see text] supply remains unclear. We examined the metabolic and transcriptional responses triggered by eCO2 in association with physiological-biochemical traits in flag leaves and roots of durum wheat grown hydroponically in ambient and elevated [CO2] with low (LN) and high (HN) [Formula: see text] supply. Multivariate analysis revealed a strong interaction between eCO2 and [Formula: see text] supply. Photosynthetic acclimation induced by eCO2 in LN plants was accompanied by an increase in biomass and carbohydrates, and decreases of leaf organic N per unit area, organic acids, inorganic ions, Calvin-Benson cycle intermediates, Rubisco, nitrate reductase activity, amino acids and transcripts for N metabolism, particularly in leaves, whereas [Formula: see text] uptake was unaffected. In HN plants, eCO2 did not decrease photosynthetic capacity or leaf organic N per unit area, but induced transcripts for N metabolism, especially in roots. In conclusion, the photosynthetic acclimation in LN plants was associated with an inhibition of leaf [Formula: see text] assimilation, whereas up-regulation of N metabolism in roots could have mitigated the acclimatory effect of eCO2 in HN plants.

Quantitative RT-PCR Platform to Measure Transcript Levels of C and N Metabolism-Related Genes in Durum Wheat: Transcript Profiles in Elevated [CO2] and High Temperature at Different Levels of N Supply.

Only limited public transcriptomics resources are available for durum wheat and its responses to environmental changes. We developed a quantitative reverse transcription-PCR (qRT-PCR) platform for analysing the expression of primary C and N metabolism genes in durum wheat in leaves (125 genes) and roots (38 genes), based on available bread wheat genes and the identification of orthologs of known genes in other species. We also assessed the expression stability of seven reference genes for qRT-PCR under varying environments. We therefore present a functional qRT-PCR platform for gene expression analysis in durum wheat, and suggest using the ADP-ribosylation factor as a reference gene for qRT-PCR normalization. We investigated the effects of elevated [CO(2)] and temperature at two levels of N supply on C and N metabolism by combining gene expression analysis, using our qRT-PCR platform, with biochemical and physiological parameters in durum wheat grown in field chambers. Elevated CO(2) down-regulated the photosynthetic capacity and led to the loss of N compounds, including Rubisco; this effect was exacerbated at low N. Mechanistically, the reduction in photosynthesis and N levels could be associated with a decreased transcription of the genes involved in photosynthesis and N assimilation. High temperatures increased stomatal conductance, and thus did not inhibit photosynthesis, even though Rubisco protein and activity, soluble protein, leaf N, and gene expression for C fixation and N assimilation were down-regulated. Under a future scenario of climate change, the extent to which C fixation capacity and N assimilation are down-regulated will depend upon the N supply.

Characterization of Cell Surface Glycan Profiles in Human and Mouse Corneas Using Lectin Microarrays.

The advent of high-throughput sequencing technologies has facilitated the profiling of glycosylation genes at a single-cell level in complex biological systems, but the significance of these gene signatures to the composition of the glycocalyx remains ambiguous. Here, we used lectin microarrays to characterize the composition of cell surface glycans in human and mouse corneas and determine its relationship to single-cell transcriptomic data. Our results identify a series of cell surface glycan signatures that are unique to the different cell types of the human cornea and that correlate, to a certain extent, with the transcriptional expression of glycosylation genes. These include pathways involved in the biosynthesis of O-glycans in epithelial cells and core fucose on stromal and endothelial cell surfaces. Moreover, we show that human and mouse corneas display some structural differences in terms of cell surface glycan composition. These results could provide insights into the specialized function of individual cell types in the cornea and foster the identification of novel cornea-specific biomarkers.

Roles unveiled for membrane-associated mucins at the ocular surface using a Muc4 knockout mouse model.

Membrane-associated mucins (MAMs) are proposed to play critical roles at the ocular surface; however, in vivo evidence has been lacking. Here we investigate these roles by phenotyping of a Muc4 KO mouse. Histochemical analysis for expression of the beta-galactosidase transgene replacing Muc4 revealed a spiraling ribbon pattern across the corneal epithelium, consistent with centripetal cell migration from the limbus. Depletion of Muc4 compromised transcellular barrier function, as evidenced by an increase in rose bengal staining. In addition, the corneal surface was less smooth, consistent with disruption of tear film stability. While surface cells presented with well-developed microprojections, an increase in the number of cells with fewer microprojections was observed. Moreover, an increase in skin-type keratin K10 and a decrease in transcription factor Pax6 was observed, suggesting an incipient transdifferentiation. Despite this, no evidence of inflammatory dry eye disease was apparent. In addition, Muc4 had no effect on signaling by toll-like receptor Tlr4, unlike reports for MUC1 and MUC16. Results of this study provide the first in vivo evidence for the role of MAMs in transcellular barrier function, tear film stability, apical epithelial cell architecture, and epithelial mucosal differentiation at the ocular surface.

Galectins Differentially Regulate the Surface Glycosylation of Human Monocytes.

Monocytes are circulating blood cells that rapidly mobilize to inflamed sites where they serve diverse effector functions shaped in part by microenvironmental cues. The establishment of specific glycosylation patterns on the immune cell glycocalyx is fundamental to direct the inflammatory response, but relatively little is known about the mechanisms whereby the microenvironment controls this process. Here, we report that galectins differentially participate in remodeling the surface glycosylation of human primary CD14+CD16- monocytes under proinflammatory conditions. Using a lectin array on biotinylated protein, we found that the prototypic galectin-1 negatively influenced the expression of galactose epitopes on the surface of monocytic cells. On the other hand, the tandem-repeat galectin-8 and, to a certain extent, the chimeric galectin-3 promoted the expression of these residues. Jacalin flow cytometry and pull-down experiments further demonstrated that galectin-8 causes a profound upregulation of mucin-type O-glycosylation in cell surface proteins from primary monocytes and THP-1 cells. Overall, these results highlight the emerging role of the galectin signature on inflamed tissues and provide new insights into the contribution of extracellular galectins to the composition of the glycocalyx in human monocytes.

Does ear C sink strength contribute to overcoming photosynthetic acclimation of wheat plants exposed to elevated CO2?

Wheat plants (Triticum durum Desf., cv. Regallo) were grown in the field to study the effects of contrasting [CO(2)] conditions (700 versus 370 µmol mol(-1)) on growth, photosynthetic performance, and C management during the post-anthesis period. The aim was to test whether a restricted capacity of sink organs to utilize photosynthates drives a loss of photosynthetic capacity in elevated CO(2). The ambient (13)C/(12)C isotopic composition (δ(13)C) of air CO(2) was changed from -10.2‰ in ambient [CO(2)] to -23.6‰ under elevated [CO(2)] between the 7th and the 14th days after anthesis in order to study C assimilation and partitioning between leaves and ears. Elevated [CO(2)] had no significant effect on biomass production and grain filling, and caused an accumulation of C compounds in leaves. This was accompanied by up-regulation of phosphoglycerate mutase and ATP synthase protein content, together with down-regulation of adenosine diphosphate glucose pyrophosphatase protein. Growth in elevated [CO(2)] negatively affected Rubisco and Rubisco activase protein content and induced photosynthetic down-regulation. CO(2) enrichment caused a specific decrease in Rubisco content, together with decreases in the amino acid and total N content of leaves. The C labelling revealed that in flag leaves, part of the C fixed during grain filling was stored as starch and structural C compounds whereas the rest of the labelled C (mainly in the form of soluble sugars) was completely respired 48 h after the end of labelling. Although labelled C was not detected in the δ(13)C of ear total organic matter and respired CO(2), soluble sugar δ(13)C revealed that a small amount of labelled C reached the ear. The (12)CO(2) labelling suggests that during the beginning of post-anthesis the ear did not contribute towards overcoming flag leaf carbohydrate accumulation, and this had a consequent effect on protein expression and photosynthetic acclimation.

Membrane-associated mucins of the human ocular surface in health and disease.

Mucins are a family of high molecular weight, heavily-glycosylated proteins produced by wet epithelial tissues, including the ocular surface epithelia. Densely-packed O-linked glycan chains added post-translationally confer the biophysical properties of hydration, lubrication, anti-adhesion and repulsion. Membrane-associated mucins (MAMs) are the distinguishing components of the mucosal glycocalyx. At the ocular surface, MAMs maintain wetness, lubricate the blink, stabilize the tear film, and create a physical barrier to the outside world. In addition, it is increasingly appreciated that MAMs function as cell surface receptors that transduce information from the outside to the inside of the cell. Recently, our team published a comprehensive review/perspectives article for molecular scientists on ocular surface MAMs, including previously unpublished data and analyses on two new genes MUC21 and MUC22, as well as new MAM functions and biological roles, comparing human and mouse (PMID: 31493487). The current article is a refocus for the audience of The Ocular Surface. First, we update the gene and protein information in a more concise form, and include a new section on glycosylation. Next, we discuss biological roles, with some new sections and further updating from our previous review. Finally, we provide a new chapter on MAM involvement in ocular surface disease. We end this with discussion of an emerging mechanism responsible for damage to the epithelia and their mucosal glycocalyces: the unfolded protein response (UPR). The UPR offers a novel target for therapeutic intervention.

Screening for Higher Grain Yield and Biomass among Sixty Bread Wheat Genotypes Grown under Elevated CO2 and High-Temperature Conditions.

Global warming will inevitably affect crop development and productivity, increasing uncertainty regarding food production. The exploitation of genotypic variability can be a promising approach for selecting improved crop varieties that can counteract the adverse effects of future climate change. We investigated the natural variation in yield performance under combined elevated CO2 and high-temperature conditions in a set of 60 bread wheat genotypes (59 of the 8TH HTWSN CIMMYT collection and Gazul). Plant height, biomass production, yield components and phenological traits were assessed. Large variations in the selected traits were observed across genotypes. The CIMMYT genotypes showed higher biomass and grain yield when compared to Gazul, indicating that the former performed better than the latter under the studied environmental conditions. Principal component and hierarchical clustering analyses revealed that the 60 wheat genotypes employed different strategies to achieve final grain yield, highlighting that the genotypes that can preferentially increase grain and ear numbers per plant will display better yield responses under combined elevated levels of CO2 and temperature. This study demonstrates the success of the breeding programs under warmer temperatures and the plants' capacity to respond to the concurrence of certain environmental factors, opening new opportunities for the selection of widely adapted climate-resilient wheat genotypes.

Genotypic Variability on Grain Yield and Grain Nutritional Quality Characteristics of Wheat Grown under Elevated CO2 and High Temperature.

The progressive rise in atmospheric CO2 concentrations and temperature associated with climate change is predicted to have a major impact on the productivity and quality of food crops. Therefore, food security is highly dependent on climate change. Following a survey with 60 bread wheat genotypes, here we investigated the genetic variation in grain yield and nutritional quality among 10 of these genotypes grown under elevated CO2 and temperature. With this purpose, the biomass production, grain yield-related traits, the grain concentration of starch, total protein, phenolic compounds, and mineral nutrients, together with the total antioxidant capacity, were determined. Variation among genotypes was found for almost all the studied traits. Higher grain and ear numbers were associated with increased grain yield but decreased grain total protein concentration and minerals such as Cu, Fe, Mg, Na, P, and Zn. Mineral nutrients were mainly associated with wheat biomass, whereas protein concentration was affected by plant biomass and yield-related traits. Associations among different nutrients and promising nutrient concentrations in some wheat genotypes were also found. This study demonstrates that the exploration of genetic diversity is a powerful approach, not only for selecting genotypes with improved quality, but also for dissecting the effect of the environment on grain yield and nutritional composition.

Dynasore protects ocular surface mucosal epithelia subjected to oxidative stress by maintaining UPR and calcium homeostasis.

The mucosal epithelia of the ocular surface protect against external threats to the eye. Using a model of human stratified corneal epithelial cells with mucosal differentiation, we previously demonstrated that a small molecule inhibitor of dynamin GTPases, dynasore, prevents damage to cells and their transcellular barriers when subjected to oxidative stress. Investigating mechanisms, we now report the novel finding that dynasore acts by maintaining Ca+2 homeostasis, thereby inhibiting the PERK branch of the unfolded protein response (UPR) that promotes cell death. Dynasore was found to protect mitochondria by preventing mitochondrial permeability transition pore opening (mPTP), but, unlike reports using other systems, this was not mediated by dynamin family member DRP1. Necrostatin-1, an inhibitor of RIPK1 and lytic forms of programmed cell death, also inhibited mPTP opening and further protected the plasma membrane barrier. Significantly, necrostatin-1 did not protect the mucosal barrier. Oxidative stress increased mRNA for sXBP1, a marker of the IRE1 branch of the UPR, and CHOP, a marker of the PERK branch. It also stimulated phosphorylation of eIF2α, the upstream regulator of CHOP, as well as an increase in intracellular Ca2+. Dynasore selectively inhibited the increase in PERK branch markers, and also prevented the increase intracellular Ca2+ in response to oxidative stress. The increase in PERK branch markers were also inhibited when cells were treated with the cell permeable Ca2+ chelator, BAPTA-AM. To our knowledge, this is the first time that dynasore has been shown to have an effect on the UPR and suggests therapeutic applications.

Membrane-associated mucins of the ocular surface: New genes, new protein functions and new biological roles in human and mouse.

The mucosal glycocalyx of the ocular surface constitutes the point of interaction between the tear film and the apical epithelial cells. Membrane-associated mucins (MAMs) are the defining molecules of the glycocalyx in all mucosal epithelia. Long recognized for their biophysical properties of hydration, lubrication, anti-adhesion and repulsion, MAMs maintain the wet ocular surface, lubricate the blink, stabilize the tear film and create a physical barrier to the outside world. However, it is increasingly appreciated that MAMs also function as cell surface receptors that transduce information from the outside to the inside of the cell. A number of excellent review articles have provided perspective on the field as it has progressed since 1987, when molecular cloning of the first MAM was reported. The current article provides an update for the ocular surface, placing it into the broad context of findings made in other organ systems, and including new genes, new protein functions and new biological roles. We discuss the epithelial tissue-equivalent with mucosal differentiation, the key model system making these advances possible. In addition, we make the first systematic comparison of MAMs in human and mouse, establishing the basis for using knockout mice for investigations with the complexity of an in vivo system. Lastly, we discuss findings from human genetics/genomics, which are providing clues to new MAM roles previously unimagined. Taken together, this information allows us to generate hypotheses for the next stage of investigation to expand our knowledge of MAM function in intracellular signaling and roles unique to the ocular surface.

Growth in elevated CO2 enhances temperature response of photosynthesis in wheat.

The temperature dependence of C(3) photosynthesis may be altered by the growth environment. The effects of long-term growth in elevated CO(2) on photosynthesis temperature response have been investigated in wheat (Triticum aestivum L.) grown in controlled chambers with 370 or 700 mumol mol(-1) CO(2) from sowing through to anthesis. Gas exchange was measured in flag leaves at ear emergence, and the parameters of a biochemical photosynthesis model were determined along with their temperature responses. Elevated CO(2) slightly decreased the CO(2) compensation point and increased the rate of respiration in the light and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) V(cmax), although the latter effect was reversed at 15 degrees C. With elevated CO(2), J(max) decreased in the 15-25 degrees C temperature range and increased at 30 and 35 degrees C. The temperature response (activation energy) of V(cmax) and J(max) increased with growth in elevated CO(2). CO(2) enrichment decreased the ribulose 1,5-bisphosphate (RuBP)-limited photosynthesis rates at lower temperatures and increased Rubisco- and RuBP-limited rates at higher temperatures. The results show that the photosynthesis temperature response is enhanced by growth in elevated CO(2). We conclude that if temperature acclimation and factors such as nutrients or water availability do not modify or negate this enhancement, the effects of future increases in air CO(2) on photosynthetic electron transport and Rubisco kinetics may improve the photosynthetic response of wheat to global warming.

Acclimation to future atmospheric CO2 levels increases photochemical efficiency and mitigates photochemistry inhibition by warm temperatures in wheat under field chambers.

A study was conducted over 2 years to determine whether growth under elevated CO(2) (700 µmol mol(-1) ) and temperature (ambient + 4 °C) conditions modifies photochemical efficiency or only the use of electron transport products in spring wheat grown in field chambers. Elevated atmospheric CO(2) concentrations increased crop dry matter at maturity by 12-17%, while above-ambient temperatures did not significantly affect dry matter yield. In measurements with ambient CO(2) at ear emergence and after anthesis, growth at elevated CO(2) concentrations decreased flag leaf light-saturated carbon assimilation. The quantum yield of electron transport (Φ(PSII) ) measured at ambient CO(2) and higher irradiances increased at ear emergence and decreased after anthesis in plants grown at elevated CO(2) . At higher light intensities, but not in low light, photochemical quenching (qP) decreased after growth in elevated CO(2) conditions. Growth under CO(2) enrichment increased dark- (Fv:Fm) and light-adapted (Fv':Fm') photochemical efficiencies, and decreased the chlorophyll a:b ratio, suggesting an increase in light-harvesting complexes relative to PSII reaction centres. A relatively higher decrease in carbon assimilation than the decrease in Φ(PSII) pointed to a sink other than CO(2) assimilation for electron transport products at defined growth stages. With higher light intensities, warmer temperatures increased Φ(PSII) and Fv':Fm' at ear emergence and decreased Φ(PSII) after anthesis; in ambient-but not elevated-CO(2) , warmer temperatures also decreased qP after anthesis. CO(2) fixation increased or did not change with temperature, depending on the growth stage and year. We conclude that elevated CO(2) decreases the carbon assimilation capacity, but increases photochemistry and resource allocation to light harvesting, and that elevated levels of CO(2) can mitigate photochemistry inhibition as a result of warm temperatures.

De Novo Transcriptome Analysis of Durum Wheat Flag Leaves Provides New Insights Into the Regulatory Response to Elevated CO2 and High Temperature.

Global warming is becoming a significant problem for food security, particularly in the Mediterranean basin. The use of molecular techniques to study gene-level responses to environmental changes in non-model organisms is increasing and may help to improve the mechanistic understanding of durum wheat response to elevated CO2 and high temperature. With this purpose, we performed transcriptome RNA sequencing (RNA-Seq) analyses combined with physiological and biochemical studies in the flag leaf of plants grown in field chambers at ear emergence. Enhanced photosynthesis by elevated CO2 was accompanied by an increase in biomass and starch and fructan content, and a decrease in N compounds, as chlorophyll, soluble proteins, and Rubisco content, in association with a decline of nitrate reductase and initial and total Rubisco activities. While high temperature led to a decline of chlorophyll, Rubisco activity, and protein content, the glucose content increased and starch decreased. Furthermore, elevated CO2 induced several genes involved in mitochondrial electron transport, a few genes for photosynthesis and fructan synthesis, and most of the genes involved in secondary metabolism and gibberellin and jasmonate metabolism, whereas those related to light harvesting, N assimilation, and other hormone pathways were repressed. High temperature repressed genes for C, energy, N, lipid, secondary, and hormone metabolisms. Under the combined increases in atmospheric CO2 and temperature, the transcript profile resembled that previously reported for high temperature, although elevated CO2 partly alleviated the downregulation of primary and secondary metabolism genes. The results suggest that there was a reprogramming of primary and secondary metabolism under the future climatic scenario, leading to coordinated regulation of C-N metabolism towards C-rich metabolites at elevated CO2 and a shift away from C-rich secondary metabolites at high temperature. Several candidate genes differentially expressed were identified, including protein kinases, receptor kinases, and transcription factors.

Subconjunctival injection of mesenchymal stromal cells protects the cornea in an experimental model of GVHD.

PURPOSE: To evaluate the therapeutic effect of subconjunctival injection of human mesenchymal stromal cells (hMSCs) in the cornea of mice with graft versus host disease (GVHD). METHODS: GVHD was induced in mice after hematopoietic stem cell transplantation (HSCT) between MHC-mismatched mouse strains. Subconjunctival injection of hMSCs was applied at day 10 post-HSCT. Infiltration of CD3+ cells in the cornea and epithelial alterations were analyzed by immunofluorescence. Tear was assessed using the PRT test and TearLab Osmolarity System. qPCR was used to evaluate changes in cytokines, Pax6 and Sprr1b expression. To evaluate the effect of irradiation, we analyzed the expression of these genes in TBI mice. RESULTS: Immune cell invasion occurs in mice with GVHD, as shown by the presence of CD3+ cells in the cornea. Interestingly, eyes treated with hMSC did not present CD3+ cells. Tear osmolarity was increased in GVHD eyes, but not in treated eyes. TNFa expression was highly increased in all corneas except in Control and treated eyes. Pax6 in corneal epithelium showed a similar pattern in GVHD and Control mice, and its gene expression was enhanced in GVHD corneas. In contrast, Pax6 was reduced in GVHD + MSC corneas. We also found an increase in SPRR1B staining in GVHD eyes that was lower in GVHD + MSC mice, demonstrating that corneal keratinization is less frequent after treatment with hMSC. CONCLUSIONS: The treatment with hMSCs by subconjunctival injection is effective in reducing corneal inflammation and squamous metaplasia in ocular GVHD (oGVHD). Local treatment with hMSCs is a promising strategy for oGVHD.

Future CO2 concentrations, though not warmer temperatures, enhance wheat photosynthesis temperature responses.

The temperature dependence of C3 photosynthesis is known to vary according to the growth environment. Atmospheric CO2 concentration and temperature are predicted to increase with climate change. To test whether long-term growth in elevated CO2 and temperature modifies photosynthesis temperature response, wheat (Triticum aestivum L.) was grown in ambient CO2 (370 micromol mol(-1)) and elevated CO2 (700 micromol mol(-1)) combined with ambient temperatures and 4 degrees C warmer ones, using temperature gradient chambers in the field. Flag leaf photosynthesis was measured at temperatures ranging from 20 to 35 degrees C and varying CO2 concentrations between ear emergence and anthesis. The maximum rate of carboxylation was determined in vitro in the first year of the experiment and from the photosynthesis-intercellular CO2 response in the second year. With measurement CO2 concentrations of 330 micromol mol(-1) or lower, growth temperature had no effect on flag leaf photosynthesis in plants grown in ambient CO2, while it increased photosynthesis in elevated growth CO2. However, warmer growth temperatures did not modify the response of photosynthesis to measurement temperatures from 20 to 35 degrees C. A central finding of this study was that the increase with temperature in photosynthesis and the photosynthesis temperature optimum were significantly higher in plants grown in elevated rather than ambient CO2. In association with this, growth in elevated CO2 increased the temperature response (activation energy) of the maximum rate of carboxylation. The results provide field evidence that growth under CO2 enrichment enhances the response of Rubisco activity to temperature in wheat.