This work focuses on developing light environments for the effective regulation of morphogenesis and ex vitro conditions adaptation in micropropagated raspberry plants on the basis of photomorphogenetic control of physiological processes using light-emitting diodes (LEDs). In experiments with cloned plants growing ex vitro in stressful conditions during acclimation, the effects of optical radiation of various spectral combinations from different photosynthetically active radiation (PAR) spectral regions were studied. The data on the plant development and state of the photosynthetic apparatus, features of photosynthetic gas exchange and transpiration, accumulation of photosynthetic pigments, light curves of photosynthesis, and data on growth processes in light modes using combined quasimonochromatic radiation  with various ratio of the distinct spectral regions were obtained. Photosynthetic apparatus functional activity under different light conditions was studied with chlorophyll fluorescence determination, and plant stress responses to growing under artificial spectral light conditions were characterized. The experiments were accompanied by detailed plant phenotyping at the structural and functional levels. Plant acclimation and photosynthetic improvements in response to added far-red and green light wavelengths to the main red-blue spectrum have been elucidated. Our photobiological studies are aimed at developing effective methods for regulating morphogenesis and obtaining high-quality planting material of woody garden plants propagated vegetatively using in vitro technologies, based on photomorphogenetic regulation of physiological processes using light-emitting diodes (LEDs). The use of blue and red LEDs with spectral power distributions optimal for photosynthesis affords considerable energy savings. On the other hand, the recent availability of high-brightness diodes emitting light of other spectral regions opens new perspectives for precise horticultural lighting, including plant clonal micropropagation in vitro and ex vitro. The studies on the physiological mechanisms of light action in plants, taking into account the new experimental possibilities opened with the use of LEDs, allow us to approach the development of light modes at a fundamentally new level , ensurinLight spectral composition role in the control of plant morphogenesis (including in vitro growth) is well recognized. Changes in light spectral environment influence plant shoot and root formation, growth rate, and metabolic processes direction ,3. VarioBesides light effects on plant photosynthetic responses, light spectral quality determines numerous photomorphogenetic responses in plants, providing better plant adaptation to the light environment of coenosis, seasonal changes, etc. First of all, plant photomorphogenesis depends on the ratio of blue, red, and far-red photon fluxes. At the same time, green light addition often enhances created artificial light environment effects on the morphophysiological processes in plants both in vitro and ex vitro ,19.Numerous studies have focused on the role of red, blue, and their combinations in the control of plant growth and development ,21,22,23The specific microenvironment conditions in vitro with limited gas exchange, low light, and carbohydrates content in the nutrient medium often promote the production of reactive oxygen species and trigger oxidative stress in plants  that canThe studies on the physiological mechanisms of light action on plants, taking into account the new experimental possibilities opened with the use of LEDs, allow us to approach the development of light modes at a fundamentally new level, ensuring the success of plant adaptation in vitro, as well as their ability to overcome the stress of transfer to ex vitro conditions.There are many reports on the successful LEDs applications to promote in vitro growth and morphogenesis in various plant species due to the possibility to optimize light environment spectral quality ,40. BettRubus idaeus L., micropropagation in vitro. Red LEDs increased multiplication and rooting of shoots of two raspberry cultivars in comparison to the fluorescent lamps [At the same time, there are few publications on the effects of LED lighting on raspberry, nt lamps , while int lamps .Our objective was to characterize micropropagated raspberry plants acclimation ex vitro in various light environments after transplanting from in vitro conditions. We estimated their photosynthetic and growth adaptation to diverse spectra containing combinations of red, far-red, and blue light  and combinations of red, green, and blue light (the second series) to determine how light signals in complex spectra interact to influence growth, photosynthesis, and acclimation.Two experiments with different light treatments were carried out. Both experiments were conducted simultaneously, and the plant material from the same stock was used.Rubus idaeus L., cv. Orange Miracle, following in vitro phase, were transplanted in the pots with a peat-based substrate and grown ex vitro at different ratios of red (R)–far-red (FR) light; blue light PPFD was at the same level in all the spectral treatments  possible decrease in accordance with experimental set-up; combined R632 + R660 PPFD had been adjusted to the same level in all treatments.In this experiment, raspberry plants, eatments . Taking orptance , we haverP and frP, respectively, affected raspberry plants acclimation ex vitro. In response to treatment 1 conditions, 20% of plants did not survive after transplanting into a new stressful environment. In the other treatments 2–4, 100% of plants survived. Differences in the ratio of red–far-red light influenced plant growth and morphology (660 (treatment with 100% FR) inhibited leaf growth significantly by the end of the first month of vegetation . Below, in One commonly reported plant morphological response is specific leaf weight (SLW). SLW represents an investment by the plant per unit of leaf area developed, so that plants with the same plant-level net photosynthesis could have very different leaf areas due to differences in SLW and different net photosynthesis rates per unit leaf area. SLW was not affected by various spectral treatments. It is important to note that SLW decreased significantly in all the treatments after the second month of vegetation. This response could be attributed to the plant adaptation to the low irradiation level in the experiment . Chlorophyll a non-photosynthetic quenching (NPQ) characterizes absorbed light energy heat dissipation; it was decreasing significantly in response to the decrease in the FR light proportion in the experimental optical radiation spectra from 1 to 4.The maximum quantum efficiency of photosystem II (PSII) photochemistry (Fv/Fm) was the highest in treatments 1 and 2 with a high ratio of the FR radiation . It was Application of light sources with triple peak spectral outputs at 659 nm, 518 nm, and 450 nm with various ratios of red, green, and blue light, respectively , Table 2Transition to different light regimes influenced plant growth and morphogenesis .According to the experimental layout, PPFD in treatments 1–3 consisted almost of the same amount of G light (maximum variation in PPFD level within 12%), and the ratio R–B was changing—2:1, 1:1, and 1:2, respectively. During the first 30 days after transplanting, enhanced stem growth and rapid internode elongation were observed in treatment 3 with an increased portion of B. Since a great part of assimilates was invested in the stem growth, this retarded leaf setting (decreased leaf number) and leaf area development. In other treatments, there were no significant differences between these phenotype parameters. Increased R light portion in treatments 1, 4, and 5 resulted in rapid leaf growth and dry biomass accumulation, and 30 days later (60 days after transplanting), these characters were smoothed in all five treatments. The initial delay in leaf growth in treatments 2 and 3 with increased blue light portion resulted in their slow leaf and stem biomass accumulation. SLW was the same in all the treatments at both samplings. Interestingly, there was no reduction of SLW in older plants, as observed in Decreasing R–B ratio from light treatment 1 to treatment 3 resulted in the increase of net photosynthesis rate in plants  and a de2 m−2 s−1, was observed in treatment 3 with R–B = 1:2. The highest photosynthesis quantum effectiveness was also registered in treatment 3, as well as the lowest light compensation point.The lowest photosynthesis intensity at the saturating PPFD was observed in treatment 4 (control), with the highest portion of R light in the spectrum , Table 3a and b accumulation was observed in the control plants  was decreasing significantly in response to the decrease of the R–B light ratio . The maximum quantum efficiency of PSII photochemistry (Fv/Fm) was comparable in all the spectral treatments . It was The consequences of plant raising in vitro influence their growth after transplanting ex vitro. First, they affect plant water regime, photosynthetic capacity, and growth. All these physiological functions are interdependent, and limitations in any of them influence the whole physiological machinery increasing various disorders. At the same time, all these functions are light-sensitive, and plants effectively use light signaling to cope with environmental changes. From this viewpoint, the idea to use light manipulations with LEDs for fine-tuning plant physiological processes looks very promising.The study of the physiological mechanisms of light action on plants, taking into account the new experimental possibilities opened with the use of LEDs, allows us to approach the development of light modes at a fundamentally new level, ensuring the success of plant adaptation in vitro, as well as their ability to overcome the stress of transfer to ex vitro conditions. Plant photosynthesis action spectrum matches with blue and red regions of photosynthetically active radiation in the natural environment ,5,6. In Disrupted water balance control due to the low stomata functional activity after the in vitro environment  can seriIncreased plant growth is usually associated with a higher fraction of far-red light ,61. AccoRelatively low R–FR ratio in our experiment can be perceived by plants as a signal triggering shade-avoidance response, and in spite of the minimum direct contribution of FR to photosynthesis, its addition can enhance photoassimilation . NeverthInterestingly, a high portion of FR in treatments 1 and 2 resulted in a rapid growth of roots which is favorable for the production of planting material. Above-ground plant habit was more attractive in treatment 4, excluding FR radiation. Absorbed photons energy can be used in the light reactions of photosynthesis to drive photochemistry (electron transport), while some amount of light energy is dissipated as heat  or re-emitted as fluorescence. The ratio of the photon excitation energy stream division between the photochemical reactions on the one hand and NPQ and chlorophyll fluorescence, on the other hand, determines the effectiveness of photosynthetic apparatus (primary photosynthesis reactions) .PSII) is usually lower than Fv–Fm [The maximum quantum efficiency of PSII photochemistry (Fv–Fm) was relatively low in Treatments 3 and 4. It indicates how effectively PSII uses absorbed light energy to reduce the primary quinone acceptor of PSII (QA); in practice, this indicator can be used to assess stress in plants ,53. Valuan Fv–Fm . A low Fan Fv–Fm ,13,56. LHigher chlorophyll content in leaf blades in the treatments with decreased FR PPFD provides better conditions for the increased light absorption, as it was demonstrated earlier for woody plants . The lowThus, in our experiments, higher effectiveness of photosynthetic apparatus (primary light reactions) was observed in response to the irradiance with a decreased ratio of FR light. However, plants in these treatments had a higher transpiration rate that can be regarded as a disadvantage at the initial phase of adaptation.Speaking about light environment optimization for plant growing, one must keep in mind that the effects of the blue and red lights are not equal: red light is perceived in addition to photosynthetic apparatus by phytochromes only, whereas blue light is absorbed by both phytochromes and blue light receptors (cryptochromes and phototropins). In this respect, blue light exciting a larger set of photoreceptors is functionally more versatile . Of courIn our experiment, blue light significantly enhanced photosynthetic capacity relative to treatments lacking blue light, which is consistent with other findings, e.g., analyses of Rubisco activity and potential rate of photosynthetic electron transport . In leafmax with increasing blue light percentage could be associated with an increase in specific leaf weight, chlorophyll content per area, and stomatal conductance, as it was observed before [max growth in our case (in treatments with blue light percentage increase from 25% to 48%) was not so significant as in [An increase in Ad before . Howevernt as in , where b2 fixation than targeted red–blue light treatment like it was shown for tomato and poinsettia but not in cucumber at ambient CO2 concentrations [A broader spectrum can result in higher COtrations . The leaf higher photosynthetic capacity was connected with an increased portion of blue light in the spectrum. A higher blue–red light ratio in our experiments resulted in plant photomorphogenetic responses, e.g., they produced leaves with properties typical for the plants grown at high PPFD levels. This could be advantageous for the production of “high-light acclimated” plants at the nursery with a poor irradiance, as it was considered earlier .a non-photosynthetic quenching (NPQ) characterizes absorbed light energy heat dissipation. It has decreased significantly only in response to red light proportion fall from 50% to 37% . Both high ΦPSII and high chlorophyll content may increase the ETR, CO2 assimilation rate, and potentially the growth rate of plants [A higher level of blue light in LED treatments increased Fv–Fm; there are data that blue light enhances PSII photochemistry relative to red light . Chloropf plants , as it wThe green spectral region portion in the artificial PPFD provides better light penetration into the canopy and optimize plant growth adaptations in the coenosis ,76. AddiOur studies have shown that in triple spectral combinations increased portion of green light or blue light enhanced red light effects on the net leaf photosynthesis . Red andHigh-light crop species could respond to the fraction of photons in the green region with either shade tolerance (leaf expansion) or shade avoidance (stem elongation). . In addiPlant responses discussed above are concerning biomass production first. However, depending on the object and technology applied, other goals could be important. In micropropagation, these are stress-resistance and ex vitro survival, as it was mentioned in this article title. Thus, calculated assimilation/transpiration ratio (WUE) was increasing from regime 1 to regime 3 . Stomatal conductance decreased significantly in the treatments with high blue light portion (R–B = 1:1 and 1:2). If we compare stomatal conductance, net photosynthesis, and respiration–photosynthesis ratio  in treatments 1 and 5 , the favorable effect of green light on the assimilation also becomes evident. WUE in treatment 5 was 1.55 (65% higher than in treatment 1). The experimental work consisted of 2 experiments with different experimental layouts conducted simultaneously.Rubus idaeus L., plants of the evergreen cultivar Orange miracle were used in our studies. Plants produce vigorous shrubs and are characterized with a high shoot producing capacity. Shoots are covered with thorns. Berries of medium size with orange color and good taste and aroma. Medium draft resistance.Raspberry, Microplants of raspberry Orange miracle grown in vitro were used for planting ex vitro and further research. Aseptically cultured microplants were used for cutting preparation. We used a node with two axillary buds as an ex-plant.−1, respectively. During the rooting stage, we used QL medium with half salt concentration, containing 1.0 mg L−1 of indole-3-butyric acid (IBA). The pH level of all nutrient media was adjusted to 5.9–6.2.During the micropropagation stage, microplants were clonally propagated by cuttings. To induce the formation of adventitious buds and axillary shoots, ex-plants were cultured on Quoirin and Lepoivre medium (QL), containing 6-benzylaminopurine (6-BAP) and indole-3-acetic acid (IAA) at concentrations of 1.0 and 0.5 mg LThe sterility of the medium was obtained by autoclaving at 120 °C, 1.3 atm. for 25 minutes. Procedures of cutting preparation were performed in a sterile laminar box. The tools were fired in the burner’s flame immediately before the operation. Ex-plant disinfection was not performed due to the source material asepsis. −2 s−1, photoperiod 16 h.Microplants were cultured in a light room with the temperature of 22–24 °C; lighting was provided with OSRAM L36/25 fluorescent white lamps with at photosynthetic photon flux density 150–180 µmol mca. 15 days), they were used for further ex vitro adaptation in growth chambers  with various light treatments according to experiments’ layout. When microplants formed a well-developed root system  with pH 6.0-6.5 and complete macro- and micronutrient supply including 150 mg LPlant chambers were illuminated with lamps consisting of various light-emitting diode (LED) bars specifically designed to provide a custom spectrum in each chamber. Fixtures consisted of light modules with tunable light-emitting diodes varying in the wavelength and spectral composition of the emitted light over wide ranges. ∆λ0.5 = 623 ÷ 641 nm, λmax= 632 nm), long-wave red , far-red , and blue . In all the light treatments, photosynthetic photon flux density (PPFD) 75 µmol m−2 s−1 was maintained, photoperiod 18 h. Spectra of the resulting lamp systems were measured with a spectrometer UPRtek PG100N (Taiwan). To measure the PPFD in the PAR region, an LI-191R quantum sensor with an LI-250A data logger  was used. In the experiment on the effects of R–FR light ratio, four types of high-performance narrow-band 3 w LEDs  were used: short-wave red , green , and blue . Warm “white” COB with Tcolor = 2500K , also containing FR in the spectrum, was used as a reference treatment in this series  were used: red (∆λs series .Four plants per each treatment were destructively harvested twice: 30 and 60 days after transplanting. The number of leaves (>1 cm) per plant was counted and total leaf area was measured using a leaf area meter LI-3000A . Shoot fresh weight was measured using an electronic balance. Subsequently, shoots were oven-dried to a constant weight at 70 °C for dry weight determination. Specific leaf weight (SLW) was calculated by dividing leaf weight by leaf area (dry weight per unit leaf area).2 concentration was maintained at 400 ± 12.0 µmol mol−1, air temperature 21–23 ° C, air humidity 60 ± 4.0%. Light curves were approximated using model [Plant leaf photosynthetic rate and transpiration analyses were carried out using a LI-6400XT Portable Photosynthesis System . During the measurements, COng model  with theng model .Chla = 9.784 × D662 − 0.990 × D644; CChlb = 21.426 × D644 − 4.650 × D662; CChla+Chlb = 5.134 × D662 + 20.436 × D644; Ccar = 4.695 × D440.5 − 0.268 × CChla+Chlb. Here, CChla, CChlb, and Ccar are the concentrations of chlorophylls a and b and carotenoids in acetone. Dλ is the optical density of the extract at the corresponding wavelength λ. Pigment content was calculated per dry weight.The quantitative analysis of pigments (chlorophylls and carotenoids) included their extraction from the plant tissues using acetone, separation of the mixture into individual components, and spectrophotometry. The concentrations of pigments in 100% acetone leaf tissue extracts were calculated according to Holm–Wettstein as follows: Ca fluorescence in PSII was measured using Junior-PAM fluorimeter . Minimum (Fo) and maximum (Fm) fluorescence rates were determined after 15 min leaf exposition in darkness. Maximum quantum efficiency of PSII Fv–Fm = (Fm–Fo)/Fm was calculated after [PSII)·PPFD·0.5. Fluorescence parameters were determined in 4–6 biological replicates. Chlorophyll ed after . Relativp ≤ 0.05.For each light treatment, four replications were tested during plant phenotyping. Statistical analysis of physiological parameters was performed using analysis of variance (ANOVA) with MS Excel software and AGROS software . In the graphs and tables, means ± standard error (SE) are presented; means followed by the same letter were not different at Plant acclimation and photosynthetic improvements in response to added far-red and green light wavelengths to the main red–blue spectrum have been studied along with the changing red–blue light ratio.Water stress and light stress  follow in vitro plants transition ex vitro. Acclimation to different growth-light spectra assumes photosystem excitation balance maintenance and plant water status stabilization due to the stomata apparatus improvement and other morphological and functional adaptations. Plants can tune their stoichiometry to retain high photosynthetic quantum yield in the artificial non-typical light environment . They haSharp plant responses and significant differences between the light treatments in our experiments were observed during the first period after transplanting, at the critical phase of acclimation. It is clear that at this stage, increasing the fractions of green and blue photons in photosynthetic photon flux could benefit plant survival. After this water-saving treatment, tuning of growth and photosynthesis by manipulations with the R–FR ratio and R–B ratio becomes more critical. Thus, in development, we suggest a dynamic (changing in time) light regime combining the advantages of distinct spectra studied above at the critical periods of their action during the initial plant growth. Or course, this dynamic light environment optimization requires further research. There are data on the LED spectra effects on the production of the reactive oxygen species (ROS) in the electron transport system and activation of various plant defense mechanisms . Studies


In RMVs a total of 1559 DEGs were found, of which 1004 genes were higher expressed in RMVs than in RT. Moreover, 4244 DEGs between BMVs and BT were identified, of which 1956 genes were ≥ twofold enriched in BMVs. Using these DEGs, we comprehensively analyzed the actual expression levels and highlighted their involvement in critical functional structures in RMVs and BMVs, such as junctional complex, transporters and signaling pathways. Our work provides for the first time the transcriptional profiles of rat RMVs and BMVs. These results may help to understand why retina and brain microvasculature show different susceptibilities to stressors, and they might even provide new insight for pharmacological interventions.Although retinal microvessels (RMVs) and brain microvessels (BMVs) are closely related in their developmental and share similar blood-neural barriers, studies have reported markedly different responses to stressors such as diabetes. Therefore, we hypothesized that RMVs and BMVs will display substantial differences in gene expression levels even though they are of the same embryological origin. In this study, both RMVs and BMVs were mechanically isolated from rats. Full retinal and brain tissue samples  were collected for comparisons. Total RNA extracted from these four groups were processed on Affymetrix rat 2.0 microarray Chips. The transcriptional profiles of these tissues were then analyzed. In the present paper we looked at differentially expressed genes (DEGs) in RMVs (against RT) and BMVs (against BT) using a rather conservative threshold value of ≥  ± twofold change and a false discovery rate corrected for multiple comparisons ( Hence, the pathways of transendothelial solute exchange largely characterize the microcirculation in a given organ. The most complex design of microvessels (MVs) is present in the brain and in the retina, the latter derived from midbrain structures at an early stage of ontogenetic development. In these organs, highly specialized barriers termed blood–brain barrier (BBB) and inner blood-retina barrier (iBRB) separate the intra- and extravascular spaces. The most intriguing features of these barriers include (1) tight intercellular junctions to effectively prevent paracellular exchange of hydrophilic compounds, (2) a paucity of vesicular activity to minimize unselective transport, and (3) expression of a wide bunch of ion channels and transport systems including ATPases, symporters and antiporters, and carriers to enable selective transendothelial movement of solutes , a member of the ATP-binding cassette (ABC) efflux transporter family6 and of high and low affinity neurotransmitter / organic acid transporters7. Furthermore, indirect evidence comes from studies addressing the level of expression/activity in isolated MVs or purified endothelial cells compared with the respective full tissue13.Despite the close ontogenetic relationship between brain and retina and the common vascular supply there are remarkable differences between the retina MV (RMV) and brain MV (BMV) compartments, for instance in the response to systemic challenges such as chronic hyperglycemia in diabetes, arterial hypertension, and in the retinopathy of prematurity model with early postnatal hypoxia-hyperoxia challenges14. Pericytes surround the abluminal side of the capillary wall and contribute to microvascular development, network stabilization and remodeling, and blood flow regulation17. In fact, the ratio of endothelial cells to pericytes is suspiciously high in the retina and brain13, pointing to their prominent role in RMVs and BMVs physiology within the neurovascular unit14. Therefore, to fully characterize the blood-neuronal barrier properties, isolated RMVs and BMVs consisting of endothelial cells and pericytes are in many respects superior to the use of individual cell types such as isolated endothelial cells or pericytes. Therefore, we have previously developed a method, which allows high yield high purity isolation of the microvascular compartment from rat brain and retina. We characterized these isolates histologically and by semi-quantitative real time polymerase chain reaction (qRT-PCR) methodology which showed significant enrichment of markers of microvascular-specific cells and a distinct depletion of parenchymal cell markers  compared to full tissues13. Based on these results we have now performed a transcriptome-wide analysis to systematically compare RMVs and BMVs obtained from the same group of rats. We hypothesize that these two types of MVs will display substantial differences in gene expression levels even though they are of the same embryological origin. This heterogeneity at the transcriptome level may translate into differences in physiological responses, and it may also play a role in the differential susceptibility of the iBRB and BBB to stressors like high blood glucose.Although the BBB and iBRB are prominent features of the RMV and BMV endothelial cells, the microvascular structure and function in the retina and brain require close interaction with the second type of mural cells, the pericytesA total of 20,743 genes were measured using Affymetrix rat 2.0 microarrays for transcriptome analysis of RMVs and BMVs and full retina tissue (RT) and brain tissue (BT). The gene expression data were at first analyzed by principal components analysis (PCA) which revealed 4 clusters with wide separation of RT and BT samples and somewhat less but still clear distinction of the RMVs and BMVs samples Fig. . The priIn the transcriptional comparison between RMVs and RT we identified 1,559 differentially expressed genes , of which 1,004 genes were higher expressed, while 555 genes were lower expressed in RMVs than in RT  displayed a significant agreement of the data obtained by microarray and qRT-PCR  . These observations strongly suggest good suitability and reliability of our microarray data for comparative gene expression analysis in the microvascular compartments.For validation of the microarray data qRT-PCR measurements were performed for 6 target genes and 4 reference genes   junctional complex formation, 2) endothelial transporters, and (3) endothelial / pericyte signaling pathways. Nine genes encoding for junctional complex proteins displayed a ≥ twofold enrichment in at least one MV compartment over the respective tissue (Tab.le  endothelWe identified 355 SLC and 47 ATP-binding cassette (ABC) transporter genes in the arrays. Among the SLCs, 56 genes showed a more than twofold enrichment in at least one MV compartment  pathway, transforming growth factor beta (TGF-β) signaling pathway and Notch signaling pathway Table . CompareTo our knowledge, this is the first study in which a detailed gene expression analysis was performed in isolated rat RMVs. Using a threshold of ≥ twofold enrichment in comparative transcriptome analysis we identified 1,004 genes enriched in RMVs over corresponding RT. Furthermore, we also comprehensively analyzed the whole transcriptional profile of BMVs and the respective BT. Significantly enriched genes were mainly related to vascular biological processes in both, RMV and BMV compartments. Our study also enables in-depth comparison of RMVs and BMVs, which is of major interest since embryologically the microvascular bed of the retina is deemed a derivative of the brain circulation. Despite this close ontogenetic relationship, the overall gene expression profiles of RMVs and BMVs displayed two distinct albeit closely neighbouring, clusters in the PCA analysis. Since both, RMVs and BMVs are characterized by a tight endothelial barrier and a suspiciously high endowment with pericytes, we scrutinized the classes of junctional complex, membrane transporter, and endothelial / pericyte signaling pathway genes.13. In this paper, we provided an in-depth characterization of the microvascular compartments showing that both, RMVs and BMVs were histologically devoid of any contamination by tissue fragments. Moreover, on the gene expression level, endothelial cell and pericyte marker genes displayed a distinct enrichment and a distinct depletion of astroglial, neuronal, and photoreceptor marker genes suggesting high purity of the isolated RMV and BMV compartments. This conclusion receives further support by the present study using microarray methodology. Again, each total RNA isolate underwent a strict quality control procedure using a chip-based electrophoretic method. Extracts were only used for further analysis if the RNA integrity number was at least 8 as recommended previously18. For bioinformatics analysis, a threshold value of an at least twofold enrichment was applied to identify DEGs. This value was chosen to limit the number of hits, although a somewhat lower enrichment factor, ≥ 1.5 fold has been suggested suitable for identifying significant DEGs when complex tissues are studied19.Isolation of RMVs and BMVs was performed in exactly the same manner as described previously2 fold changes in RMVs and BMVs displayed a good correlation with the respective values obtained by microarray analysis. Secondly, 8 target genes measured by microfluidic card methodology as reported in our previous paper13 also revealed a good agreement of the enrichment factors (again expressed as log2 fold changes in RMVs and BMVs) with the respective values obtained in the present microarray study. The panel of target genes selected in both approaches contains endothelial and pericyte marker genes with some overlap (Slc2A1 and PDGFRb). The consistency of results derived from a wide range of genes and different starting materials provides strong support to the reliability of our microarray data.In order to validate the enrichment factors obtained by the microarray measurements we used results from qRT-PCR. Firstly, in samples of total RNA from the tissue isolates produced in the present study the expression levels of 6 target genes and 4 reference genes were measured. The degree of enrichment, expressed as log20. Functionally, iBRB and BBB are similar, since the permeability surface product for the small molecular tracers mannitol and sucrose has been found largely comparable21. In our study, both RMVs and BMVs expressed high levels (≥ twofold enrichment in at least one MV compartment) of a number of junction-related genes. These results clearly emphasize the functional importance of the tight intercellular junctions, yet also suggest some differences in their make-ups in RMVs and BMVs. The overall enrichment of junctional complex genes in RMVs and BMVs is not necessarily conflicted by a markedly lower expression level of Cx43 in BMVs than in BT. In fact, there is a high level in BT of Cx43 which has previously been shown to be the predominant Cx subtype in adult brain tissue, abundantly expressed in astrocytes22.The RMVs and BMVs are characterized by the expression of tight barriers, termed iBRB and BBB. These are characterized by the formation of intercellular macromolecular complexes consisting of tight junctions, adherens junctions, and gap junctions between the endothelial cells23. The tightness of the iBRB and BBB, which has been related to the expression level of junctional genes, also determines the susceptibility to endogenous or exogenous insults. For example, Tien et al. found that the downregulation of Cx43 in the retina induced by Cx43 siRNA or diabetes following streptozotocin injection, contributed to compromised retinal vascular homeostasis24. More recently, using immunohistochemistry and vasomotor response assessment, Ivanova and coworkers reported that Cx43 gene expression level in rat retinal capillaries was significantly decreased by hyperglycemia and that this decrease contributed to the vasomotor decline of the inner retinal capillaries25. Furthermore, it has been demonstrated that in vitro downregulation of the Cx43/ZO-1 complex in RMV or BMV endothelial cells contributes to the breakdown of the iBRB and BBB27. In the present study, we demonstrate that in physiological conditions the expression levels of Cx43, Cx40, and Ocln were significantly lower in RMVs compared to BMVs while the difference for ZO-1 did not reach the predetermined DEG level. Based on these observations, we speculate that the lower baseline expression of many tight junction-associated genes in RMVs might contribute to a higher susceptibility to e.g. hyperglycemia-induced damage of the iBRB compared to the BBB.In agreement with the present study, distinctly high expression levels of junctional complex genes have previously been found in rat cortical MVs, despite major differences in the methods used for vessel isolation and gene quantification9 studying 19 in- and efflux transporter mRNAs identified 5 of these  with a suspiciously high expression level. Other studies have shown that Slc7a1 , Slc7a5 , Slc16a1 (monocarboxylate transporter 1 [MCT-1]) and Slc22a5 (organic transport transporter 2 [OCTN2]) mRNA were expressed in the TR-iBRB2 cell line31. In our study employing whole transcriptome analysis, we identified 355 SLC transporter genes of which 56 genes were ≥ twofold enriched in at least one MV compartment. Moreover, we identified 8 out of 47 ABC transporter genes to display a ≥ twofold enrichment in at least one MV compartment. Our findings expand the database of transporter genes expressed in the iBRB and also increase our knowledge of similarities and differences between iBRB and BBB. For instance, we found that the expression level of Slc2A1, which is a major transporter of glucose across the barriers, was expressed at comparable levels in both RMVs and BMVs. In contrast, a previous study described a markedly higher protein expression level in rat RMVs than BMVs with hyperglycemia-induced downregulation to occur only in RMVs12. In this previous study, RMVs and BMVs were isolated by different methods which may well have affected the analysis outcome. In our study, both RMVs and BMVs were isolated in an identical manner, thus allowing direct comparison of the results from both MV compartments. Our data show that in addition to Slc2A1, the insulin-sensitive glucose transporter Slc2A4 (Glut-4) also displayed a significant enrichment in RMVs and BMVs, although at much lower levels than Slc2A1. Using immunofluorescence microscopy, Slc2A4 has previously been localized in individual neurons of different brain areas32 and in endothelial cells of MVs isolated from the rat hypothalamus33. However, our data suggest a more widespread expression in the microvasculature of the brain and similarly in the retina, at a markedly lower level in RMVs than in BMVs. The presence of Slc2A4 is considered of functional importance in regulating glucose transport into neuronal tissues during hypoglycemia34. Based on our results we therefore speculate that RMVs may be less responsible compared to BMVs to changes in blood glucose concentrations, which subsequently may lead to impaired glucose control in the retina.The tight interendothelial junctions of the iBRB and BBB effectively block the paracellular passage of hydrophilic solutes. Therefore, these compounds depend on a transcellular route to enter (or leave) the parenchymal tissue. However, this cannot be simple diffusion . In the light of the paucity of transport vesicles, the movement of bulk amounts of many solutes requires the presence of endothelial transporters, which, therefore, take center stage in the barrier function. Systematic studies of the transporter gene expression patterns in the rodent iBRB have been limited until recently due to the difficulty in isolating MVs of sufficient quality and purity. Instead of RMVs, retinal vascular endothelial cells and a conditionally immortalized retinal capillary endothelial cell line (TR-iBRB2) have been used as iBRB models. Using these models, Tachikawa et al.35. This protein effects transport of lactate and pyruvate, and the high expression level found in the present study may be taken to indicate a prominent role in RMVs although the functional importance of this is not yet clear.Quite uniquely, another member of the Slc family, Slc16a1 showed a considerably higher expression level in RMVs than in BMVs. Previously, Slc16a1 has been localized to brain endothelial cells, mainly on the luminal surface16. The communication between endothelial cells and pericytes in BMVs has been shown to contribute to the formation and maintenance of the BBB integrity36, and of the iBRB alike37. Moreover, endothelial cell—pericyte interaction is of outmost importance in the regulation of (micro)vascular development, stabilization, maturation, and remodeling as reviewed elsewhere38. In accord, we found many genes of these signaling pathways in the pool of common genes highly enriched in the BMVs and RMVs yielding significant enrichment scores for vasculature-related pathways. By far the highest enrichment score was for the NOTCH signaling pathway, which has been suggested to play a pivotal role in the regulation of arteriogenesis and angiogenesis39. Despite the agreement of BMV and RMV gene expression within the common gene pool, slight differences between these MV compartments were noted. These differences are reflected in the higher number of individual genes enriched over tissue in BMVs compared to RMVs, and they are also reflected in the distinct clustering of RMVs and BMVs seen in the PCA.In recent years, several critical signaling pathways of communication between endothelial cells and pericytes such as PDGFb / PDGFRb signaling, Tie2 / Ang signaling, TGF-β signaling and Notch signaling have been described41, and similar measurements should also be applicable to the RMV and BMV compartments isolated by our novel protocol. Nevertheless, our study by increasing the knowledge of gene expression patterns in retina and brain microvascular systems might well be instrumental to understand why retina and brain show different susceptibilities to stressors and even provide new targets for specific pharmacological interventions.In summary, we provide for the first time comparative transcriptional profiles of rat retinal and cerebral microvasculature from the same rats. In spite of largely comparable results in the groups of genes in our focus some remarkable differences were encountered as well, first of all higher expression levels of solute carrier genes in BMVs. These differences may provide further insight into the mechanism(s) underlying the higher susceptibility of retina tissue to systemic stressors such as diabetes and arterial hypertension, and they may hopefully generate new protective approaches such as stimulation of carrier(s) in RMVs. Most resasonably,the results of the current study call for a follow-up on the protein expression level, and the lack of such data on protein is a limitation of our study. In fact, a shotgun type of proteome survey would probably best reflect our transcriptional approach. First studies in this direction have already been presented for full retina and brain tissue samples42. Male Wistar rats purchased from Janvier  were used in this study. They were housed following a standard 12-h light/dark cycle in a temperature-controlled environment, and with free access to food and tap water. Six rats (aged 12 weeks with a body weight of 546 ± 17 g) were anesthetized deeply with CO2 inhalation and sacrificed. Brains were dissected by removing meninges, superficial vessels, choroid plexus and white matter. Eyes and brain hemispheres were snap-frozen in liquid nitrogen and stored at -80 °C until use.This study was approved by the Animal Ethics Committee at the regional council in Karlsruhe (Germany). Application, approval, and animal care complied with the ethical regulations of the Directive 2010/63/EU, and all experimental procedures as well as data analysis and reporting met the ARRIVE guidelines as introduced by Kilkenny and coworkers13. Briefly, frozen eyes from individual rats were cryosectioned . A section of 50 µm was collected for RNA extraction from total retina tissue. The other sections were transferred into a glass tube containing 3 ml phosphate-buffered saline ; PBS: NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10 mM, KH2PO4, 1.8 mM, pH 7.4). This retinal preparation was homogenized using a motor-driven homogenizer . For BMVs isolation, serial sections were prepared from an individual hemisphere, of which three sections were taken for total RNA extraction from full tissue. The remaining sections were homogenized in the same way as for RMVs isolation. Thereafter, the brain and retinal suspension were individually transferred onto a density gradient column (3 ml PBS / 31% dextran in the lower phase and 3 ml PBS / 18% dextran in the upper phase) and centrifuged for 15 min (1300 g). Finally, both RMVs and BMVs were captured after filtration over a 60 µm nylon mesh. All the procedures were performed at ≤ 4o C.The retinal and brain microvessels were isolated as described previouslyThe samples were immersed in 350 µl lysis buffer  plus 3.5 µl mercaptoethanol (Sigma), and then pulled ten times through a 22-gauge needle. Total RNA was obtained from individual MV samples or RT and BT with RNeasy® Plus Micro kit  according to the manufacturer's protocol. The concentration and quantity were determined using the Agilent 6000 Pico kit (RMVs and BMVs), or Agilent Nano kit  on an Agilent 2100 bioanalyzer . Only samples with an integrity number (RIN) > 8.0 were used for microarray processing.http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE110675.For each sample, 1 ng of total RNA was amplified using the GeneChip® WT Pico Reagent Kit (Affymetrix) according to the manufacturer's protocol. 20 μg of cRNA was used as input for the second cycle of cDNA reaction. 5.5 μg of single-stranded cDNA was used as input for the fragmentation reaction. The Affymetrix Genechip WT Terminal labeling kit was used for fragmentation and biotin labeling. Finally, the samples were hybridized to the whole-transcriptome Rat Gene 2.0 ST microarrays (Affymetrix) on the Genechip Fluidics Station 450 (Affymetrix), scanned using the Genechip Scanner 7G (Affymetrix) and the raw intensity values stored in CEL files by the GeneChip® Operating Software (Affymetrix). These raw CEL files were normalized using the Affymetrix® Expression Console Software  and the adjusted intensity values were transformed to log2 format. The complete microarray dataset is available at Gene Expression Omnibus (GEO) database . To assess the microvascular gene expression profiles, we identified DEGs between MVs and the respective full tissue using a threshold of ≥ twofold enrichment and a FDR corrected for multiple comparisons (p < 0.05). In a second pairwise comparison, we identified DEGs between RMVs and BMVs with the same parameters applied as described above.http://geneontology.org/). Pathways analysis was performed using the PANTHER Classification System43. Enrichment scores were obtained using a Bonferroni adjusted p < 0.05 level of significance.To investigate the functional classifications of these DEGs, biological process categories were analyzed using the Gene Ontology (GO) Consortium database  using the assay-on-demand primer and probe sets (from Applied Biosystems) listed in Table -ΔCt methodology we calculated the ratio of the relative expression level for each of the 6 selected target genes to the expression level of 4 reference genes . The enrichment factors in RMVs and BMVs for each of the selected target genes were calculated for the microarray and qRT-PCR measurements (applying the 2-ΔΔCt approach) and the mean values obtained from each experimental group used for correlation analysis.Aliquots of total RNA were taken from all samples and cDNA synthesis was performed as previously described


In summary, this study has identified the regulatory regions of genes in three tissues that play key roles in defining health and economically important traits and has set the precedent for the characterization of regulatory elements in ovine tissues using the Rambouillet reference genome.The Ovine Functional Annotation of Animal Genomes (FAANG) project, part of the broader livestock species FAANG initiative, aims to identify and characterize gene regulatory elements in domestic sheep. Regulatory element annotation is essential for identifying genetic variants that affect health and production traits in this important agricultural species, as greater than 90% of variants underlying genetic effects are estimated to lie outside of transcribed regions. Histone modifications that distinguish active or repressed chromatin states, CTCF binding, and DNA methylation were used to characterize regulatory elements in liver, spleen, and cerebellum tissues from four yearling sheep. Chromatin immunoprecipitation with sequencing (ChIP-seq) was performed for H3K4me3, H3K27ac, H3K4me1, H3K27me3, and CTCF. Nine chromatin states including active promoters, active enhancers, poised enhancers, repressed enhancers, and insulators were characterized in each tissue using ChromHMM. Whole-genome bisulfite sequencing (WGBS) was performed to determine the complement of whole-genome DNA methylation with the ChIP-seq data. Hypermethylated and hypomethylated regions were identified across tissues, and these locations were compared with chromatin states to better distinguish and validate regulatory elements in these tissues. Interestingly, chromatin states with the poised enhancer mark H3K4me1 in the spleen and cerebellum and CTCF in the liver displayed the greatest number of hypermethylated sites. Not surprisingly, active enhancers in the liver and spleen, and promoters in the cerebellum, displayed the greatest number of hypomethylated sites. Overall, chromatin states defined by histone marks and CTCF occupied approximately 22% of the genome in all three tissues. Furthermore, the liver and spleen displayed in common the greatest percent of active promoter (65%) and active enhancer (81%) states, and the liver and cerebellum displayed in common the greatest percent of poised enhancer (53%), repressed enhancer (68%), hypermethylated sites (75%), and hypomethylated sites (73%). In addition, both known and  Regulatory element characterization and chromatin state determination in relevant tissues was identified as a critical need for implementing precision breeding within the livestock industry by the Agricultural Animal Genomics Community . To thisIn vivo analysis of regulatory elements will allow researchers to test hypotheses of biological function of putative causal mutations in relevant production tissues. Understanding the phenotypic influences of genetic variance that lie in promoter and enhancer regions is important for trait prediction and the improvement of sheep production.Sheep production for meat, milk, and wool is an important agricultural industry across the globe with more than one billion sheep suited to a diverse range of climates . This diFunctional variants that are causally implicated in phenotypic variation are increasingly found to lie outside of transcribed regions within DNA regulatory elements . These rN = 2 for each tissue by design , the data collected here also provide a resource for evaluating the larger effort by permitting estimation of interindividual variation in the appearance and tissue distribution of regulatory elements. Three tissues were selected for this study based on their prominence in defining production traits and to span tissues of endodermal, mesodermal, and ectodermal origin and because each presents unique procedural challenges for performing chromatin immunoprecipitation with sequencing (ChIP-seq) assays. The liver is an endodermal-derived tissue that is a key metabolic component of the alimentary system  which denotes promoters and acetylation of histone 3 lysine 27 (H3K27ac) which denotes active enhancers . The monDNA methylation data derived from whole-genome bisulfite sequencing (WGBS) were incorporated to validate regulatory regions and chromatin states. In mammals, several groups have identified CpG islands that lack methylation are located at gene promoters . RepressOur objective for this study was to identify the locations of gene regulatory elements in sheep by characterizing histone modifications, CTCF binding, and DNA methylation for the cerebellum, liver, and spleen. Defining regulatory elements in the sheep genome will provide the basis for a greater understanding of the mechanisms that underpin phenotypic variation in important health and production traits in sheep.Tissue was collected postmortem from two pairs of healthy half siblings (one ewe and one wether per pair) totaling four yearling crossbred sheep  as approved by the Washington State University Institutional Animal Care and Use Committee. Small pieces of liver, spleen, and cerebellum tissues were collected within 30 min postmortem, briefly rinsed with ice cold 1 × PBS, and promptly snap frozen in liquid nitrogen. Samples were transferred from liquid nitrogen directly into a −80°C freezer for storage.g for 1 min at 4°C. Chromatin was incubated with 1 μg of antibody overnight at 4°C in a Hula mixer for 16 h. The following morning, protein G-coated magnetic beads were added and incubated 2 h at 4°C in the Hula mixer. The sample was washed twice with a low salt and once with a high salt buffer. Cross-links were reversed by incubating the sample at 65°C for 30 min at 400 rpm in a thermomixer. Purification was performed with the DNA Purification Buffers and Spin Columns Kit following the manufacturer’s instructions .Chromatin immunoprecipitation (ChIP) was performed using commercial antibodies for the histone modifications H3K4me3 , H3K4me1 , H3K27ac , H3K27me3 , and CTCF  with SimpleChIP Plus Enzymatic Chromatin IP Kit according to the manufacturer’s instructions  . BrieflyPurified DNA samples were quantified using the Qubit dsDNA HS Assay Kit . The DNA size and integrity were verified using a Fragment Analyzer . Libraries were prepared with the TruSeq ChIP Library Preparation Kit  for 75 base pair paired-end reads following the manufacturer’s instructions and sequenced to at least 20 million mapped reads for “narrow” histone marks H3K4me3, H3K27ac, and CTCF libraries and at least 40 million mappable reads each for “broad” histone marks H3K4me1 and H3K27me3 libraries.Whole-genome bisulfite sequencing was performed as a service by Novogene  on the liver, spleen, and cerebellum in all four animals. Briefly, DNA extracted from these tissues was subjected to agarose gel electrophoresis to test for DNA degradation and potential RNA contamination. The DNA was then quantified using a Nanodrop spectrophotometer  and a Qubit2.0 fluorometer . Lambda phage DNA was spiked in as a negative control for DNA methylation. Since lambda phage DNA lacks DNA methylation, all the cytosines in its DNA should be converted to uracil during bisulfite conversion. Any unchanged cytosine in the lambda phage DNA can thus be used to determine the efficiency of bisulfite conversion. For library construction, DNA samples were fragmented into 200–400 bp using sonication . Next, end repair, A-ligation, and methylation sequencing adapter ligation was performed. The adapter sequences were 5′ adapter (5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTA CACGACGCTCTTCCGATCT-3′) and 3′ adapter (5′-GATC GGAAGAGCACACGTCTGAACTCCAGTCACATCACGATC TCGTATGCCGTCTTCTGCTTG-3′). Following this, the DNA library was subjected to bisulfite treatment . Library concentration was first quantified by Qubit2.0, diluted to 1 ng/μl before checking insert size on Agilent 2100 , and quantified with more accuracy by quantitative PCR (effective concentration of library > 2 nM). Libraries were then pooled per sample and sequenced paired-end.Oar_rambouillet_v1.0 with Bowtie2  (+2 to −2 kb), exonic, intronic, near a transcription termination site (TTS) (+1 to −1 kb), and intergenic using the Homer annotatePeaks.pl function  were trimmed using Trimgalore v0.4.5 with default parameters. Cleaned data for each sample was aligned to the sheep reference genome rameters . The X-crameters . The mets v0.0.6 . Regionss v0.0.6 .LearnModel function in ChromHMM was implemented with given chromatin states of two through 20 for each animal, and the model with the optimal number of chromatin states was examined using the CompareModels function in ChromHMM  at an average depth of 21 × (range 18× to 26×). Bisulfite conversion rate was ∼99.9% for all the samples. Mapping statistics for each tissue sample per sheep are displayed in Whole-genome bisulfite sequencing of cerebellum, liver, and spleen samples from the four sheep generated a total of 986, 1,070, and 904 million paired end reads, respectively, with a read length of 2 × 150 bp. The number of reads uniquely mapped to the reference genome was 84.24, 78.86, and 82.48% for the cerebellum, liver and spleen, respectively. The uniquely mapped bases covered the reference genome , which is consistent with several tissues in cattle . ChromosOar_rambouillet_v1.0 genome annotation file and these classifications are displayed in The proximity of H3K4me3 peaks to TSS was investigated by comparing consensus H3K4me3 peaks and CAGE data generated by ALB), a gene that encodes a plasma protein synthesized in hepatocytes and expected to be active in the liver, has one promoter and two enhancers annotated in humans that are within 2 kb upstream from the start of the gene , a gene expected to be active in the spleen and encodes a membrane protein involved with macrophage development, displayed sequence pileup for active marks H3K4me3 and H3K27ac and low levels of DNA methylation directly upstream (PAX6) is known to be involved in the development of neural tissues and maturation of granule neurons in the cerebellum and is known to have a promoter and multiple enhancers both upstream and downstream of the gene  is a gene that encodes a meiosis-specific protein involved in the synapsis of sister chromatids that is not expected to be active in the liver, spleen, or cerebellum  and cerebellum , but the correlation in the spleen was markedly lower , and overall, the correlations between the spleen samples were lower than the liver and cerebellum for all four histone marks. This is evident in H3K27me3 in the spleen  than in the liver  and cerebellum . The correlations of DNA methylation signal between samples ranged from 0.70 to 0.76, with the liver and cerebellum displaying the greatest correlation between the two males (0.76). However, sex differences in correlations were not observed, as each female has a moderate to high correlation with both the other female (0.54–0.84) and both males (0.44–0.92) for each mark within all three tissues.A principal component analysis was performed with the DNA methylation data to investigate similarity and differences between samples and tissues. Eigenvalues were calculated based on the position of CG methylation signal in all animals for all three tissues, and the first two eigenvalues (PC1 and PC2) were plotted . SamplesAverage methylation levels were calculated and compared in each of the three tissues in both the CG and non-CG sites . Non-CG The relative positions of the combination of specific histone marks provide a more complete definition of the overall regulatory chromatin state than individual peak calling. Regulatory elements were defined for two animals (M1 and M2) using a hidden Markov model employed by ChromHMM which assigns 200 bp bins across the genome to a given number of chromatin states based on the combined histone modification signal profiles , 2017. TThe signal of all the histone marks and the nine chromatin states for each tissue is displayed as heatmaps in The correlation of DNA methylation status with predicted chromatin state was examined by estimating the number of hyper- and hypomethylated regions per Mb within the boundaries of the regulatory elements in the nine defined chromatin states. The greatest number of hypomethylated regions was observed in active enhancer regions in the liver and spleen and in active promoter regions in the cerebellum, as expected if our process was correctly identifying regulatory elements and classifying them as actively transcribed genes. The greatest number of hypermethylated regions was observed in poised enhancers and CTCF in the liver, weak poised and poised enhancer regions in the spleen, and poised enhancer regions in the cerebellum, also consistent with the process correctly classifying regulatory elements.The chromosomal segments spanned by regulatory elements, as defined by the histone mark peaks, were combined and summarized to estimate the overall extent and percent of the genome representing regulatory elements and their chromatin state among the three tissues examined. Chromatin states from the ChromHMM analyses were categorized and combined into promoter, active enhancer, poised enhancer including weak poised enhancers, repressed enhancer including weak repressed enhancers, and quiescent or low signal categories and averaged for each tissue . PromoteThe locations of assigned promoter chromatin states were compared with TSS generated from CAGE data for the liver, spleen, and cerebellum. Both the signal distribution and heatmap plots display a strong signal before and after the TSS in all three tissues . This siSimilarities and differences of promoters, enhancers, and methylated regions within and between tissues were examined and percentages of overlap are displayed in P < 0.00001) CTCF-binding motifs both known from previous research and de novo was identified across the genome in the liver, spleen, and cerebellum , compartments of chromatin interactions, across the genome . The locrebellum . Of thesrebellum . Three mThe goal of this study was to characterize regulatory elements in ovine liver, spleen, and cerebellum using ChIP-seq and WGBS. The three selected tissues, the liver, spleen, and cerebellum, each represent a different developmental origin and are important to metabolism, immune response, and motor control, respectively. We have demonstrated the successful application of the micrococcal nuclease ChIP protocol across these tissues and the bioinformatic pipeline for the analysis of ChIP-seq in sheep. Furthermore, this study has incorporated the value of coupled histone modification and DNA methylation data toward a better understanding of regulatory regions in the sheep genome.Micrococcal nuclease was used to shear the chromatin because it provided a consistent and reproducible shearing across samples and tissue types. A limitation of the micrococcal nuclease may be increased likelihood of the appearance of duplicated reads due to similarity of cut sites in the chromatin; however, several studies have not found substantial bias when duplicates were removed . FurtherSequence read pileups were examined in IGV near genes known to be active and inactive in humans and expected to be conserved across species. This provided a means of examining genes with known promoters and expression patterns as positive and negative controls for both ChIP-seq experiments and WGBS and provided insight into the potential similarity of regulatory elements across species. Several genes known to be active across different mammalian species in the liver, spleen, and cerebellum showed a sequence pileup of active histone marks which likely indicated the presence of active regulatory elements. Inversely, genes known to be active during meiotic processes and quiescent during adult stages in several mammalian species showed no sequence pileup of histone marks and presence of DNA methylation, which likely indicates inactivity of regulatory elements.Consistency of regulatory element identification by ChIP-seq and DNA methylation for each tissue between the four individual animals was evaluated by calculating Spearman and Pearson correlations, respectively. Correlations between samples for both ChIP-seq and DNA methylation were within the ranges previously reported with sequence data . FurtherThe CG methylation signal for all four samples clustered distinctly by tissue in a principal component analysis, indicating clear differences in DNA methylation between tissues . This fiThe enrichment of individual histone marks was examined by identifying peaks in each sample. The number of peaks identified in these sheep liver, spleen, and cerebellum samples was consistent with other studies in sheep adipose, cattle liver, cattle muscle, cattle rumen epithelium, human liver, and mouse liver  Villar . Many chOar_rambouillet_v1.0 genome. In the liver, spleen, and cerebellum, the most TSS were identified near (within 2 kb of distance on either side) to H3K4me3 peaks, which is not surprising. Many H3K27ac and H3K4me1 peaks, which indicate the presence of active or poised enhancers, were located in intronic regions. Repressed enhancers marked by H3K27me3 were located mostly in intergenic regions, along with CTCF, which may be indicative of insulated TAD boundaries not in close proximity of genes. Further work with additional animals in combination with RNA expression and TSS analyses is needed to examine regulatory element activity outside of previously annotated regions of the sheep genome.Trimethylation of histone 3 lysine 4 peaks were enriched annotated TSS in all three tissues. The peaks and heatmap signature signals are similar to several other ChIP-seq experiments in human PBMCs and CD14+ cells, as well as mouse liver . Peaks fThe genomic segments identified by histone mark peaks were evaluated for overlap between marks and CTCF binding. This broader view of the regulatory landscape lends a better understanding of gene regulation at each location than individual marks . Active Hypermethylated and hypomethylated regions of the sheep genome were defined across liver, spleen, and cerebellum tissues. The number of hypermethylated and hypomethylated regions per Mb in each of the nine chromatin states was quantified. The data presented in this study demonstrate an enrichment of hypermethylated regions in chromatin states with prominent H3K4me1 (primarily poised enhancers) and hypomethylated regions in active enhancers and promoters enriched with H3K27ac and H3K4me3. These results agree with previous research in humans and mice which indicate that active enhancer activity is inversely correlated with DNA methylation . InteresApproximately 20% of the sheep genome was assigned to a chromatin state category including promoters; active, poised, and repressed enhancers; and CTCF in the liver, spleen, and cerebellum. In cattle, a previous study similarly assigned approximately 30% of the genome to either a chromatin state or areas with open chromatin in rumen epithelium . The locThe CCCTC-binding factor (CTCF) along with cohesins was shown to be present at the boundaries of TADs in humans and mice . Dependide novo motifs identified in more than one tissue. A large number of CTCF-binding motifs are similar in sequence across mammalian species including cattle (Drosophila melanogaster), and yeast (Saccharomyces cerevisiae) within the HOMER motif database Kimberly Davenport (University of Idaho)Stephen White Michelle Mousel Alisha Massa (Washington State University)Kim Worley (Baylor College of Medicine)Alan Archibald Emily Clark Brian Dalrymple James Kijas (CSIRO)Shannon Clarke (AgResearch)Rudiger Brauning (AgResearch)Timothy Smith Tracey Hadfield (Utah State University)Noelle Cockett (Utah State University)PRJEB41457.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI BioProject, accession no: Sheep were housed and cared for under Animal Subject Approval Form (ASAF) 04618 titled “Maintenance of MCF and OPPV free sheep flocks” approved by the Washington State University Institutional Animal Care and Use Committee (IACUC) and euthanized for tissue collection under ASAF 6003.MM, MH, SW, SM, TS, and BM designed the study. KD, AM, MM, MH, and BM collected the samples. BM, KD, MM, SW, AM, SM, and SB facilitated the ChIP-Seq and methylation experiments, data analyses, and drafted the manuscript. TS facilitated ChIP-seq library preparation and sequencing. KD, AM, SB, SM, MM, MH, SW, TS, and BM discussed and interpreted results. SM, MM, SW, NC, TS, and BM acquired funding. All authors contributed to the article and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Deactivation based on sintering phenomena is one of the most costly issues for the industrial application of metal nanoparticle catalysts. To address this drawback, mesoporous silica encapsulation is reported as a promising strategy to stabilize metallic nanoparticles towards use in high temperature catalytic applications. These protective shells provide significant structural support to the nanoparticles, while the mesoporosity allows for efficient transport of the reactants to the catalytically active surface of the metallic nanoparticle in the core. Here, we extend the use of gold nanorods with mesoporous silica shells by investigating their stability in the CO oxidation reaction as an example of high temperature gas phase catalysis. Gold nanorods were chosen as the model system due to the availability of a simple, high yield synthesis method for both the metallic nanorods and the mesoporous silica shells. We demonstrate the catalytic activity of gold nanorods with mesoporous silica shells at temperatures up to 350 °C over several cycles, as well as the thermal stability up to 500 °C, and compare these results to surfactant-stabilized gold nanorods of similar size, which degrade, and lose most of their catalytic activity, before reaching 150 °C. These results show that the gold nanorods protected by the mesoporous silica shells have a significantly higher thermal stability than surfactant-stabilized gold nanorods and that the mesoporous silica shell allows for stable catalytic activity with little degradation at high temperatures. Gold nanorods stabilized with mesoporous silica shells are characterized and used as the catalyst for a model CO oxidation reaction to counter the costly sintering phenomena found in many industrial nanoparticle catalysts. Recent experiments and computational simulations conclude that for a variety of structurally-sensitive catalytic reactions, the activity and selectivity can be improved by manipulating the arrangement of atoms on the surface.7 Therefore, different catalytic activity and selectivity may be achieved by engineering NPs with different crystal facets, as elegantly enabled by colloidal synthesis.8Metallic nanoparticles (NPs), particularly noble metals, like platinum (Pt), palladium (Pd) and gold (Au), are of great importance in the chemical industry, and in the fields of biomedical engineering, sensing and catalysis.9–11 where the organic capping agents used as stabilizers for the NPs will decompose. In addition, due to their small size and high surface area, NPs are prone to sinter and coalesce when subjected to high temperatures.12 Therefore, it is anticipated that the highly active facets of shape-selected nanocrystals will gradually be destroyed at elevated temperature. Specifically, first the capping agents used during synthesis will thermally decompose, followed by recrystallization into the thermodynamically stable Wulff-shape structures and/or sintering into larger superstructures. Currently, in industrial applications, most heterogeneous catalytic processes take place on small spherical NPs, or NPs of undetermined shapes and non-uniform distributions because of their large surface area.13 Moreover, at elevated temperatures, catalytic processes on bare NPs usually take place on NPs adsorbed or grown on support materials, such as alumina,14 titanium oxide,15 and porous carbon,16 to maximize NP dispersion and improve their structural stability at elevated temperatures. As a consequence of the described intrinsic thermal instability of NPs with a well-defined shape, when used in catalysis, their application is usually restricted to mild thermal conditions and/or limited reproducibility. For instance, in Bai's work,17 bare Au nanorods (NRs) were tested for the reduction of nitro compounds, performed at ambient temperature, while Park, et al. reported a catalytic test of bare Pt NPs for CO oxidation up to 240 °C.18 Simultaneously, recent applications of unsupported catalysts indicate that the traditional need for a support to enhance the catalytic activity of NPs may be unnecessary when the NPs are optimized to the reaction.19,20When applying NPs as heterogeneous catalysts, it is important to consider the ultimate goal of providing efficient catalysis on an industrial scale. However, many industrial catalytic applications are normally performed above 300 °C,21,22 The first method used to synthesize such structures, also called the surface permeable etching method, begins with the addition of silica monomers to a colloidal solution of pre-synthesized, facetted NPs and obtains porosity through the subsequent mesoporogen of these metal–silica core–shell structures.23 A second method also begins with a colloidal NP solution, but the porous structure is achieved by mixing the silica precursor and suitable amount of surfactants simultaneously.24 The porous coating provided by both of these methods allows for NPs with both shape stability and reactant penetration through the shell, allowing the reactants to interact with the active surface on the NPs during reactions. Recently, Zhang, et al. demonstrated that ultrasmall Au nanospheres encapsulated by porous silica exhibited stable activity for CO oxidation at high temperatures without compromising the original shape and size.25 Additionally, TTAB-stabilized Pt nanocubes with mesoporous silica shells produced inspiring, thermally stable catalysis for CO oxidation, with a stable catalytic performance recorded at 750 °C.26 In fact, in many cases, CO oxidation serves as a prototypical reaction for heterogeneous catalysts, including Au. While the mechanism of the catalysis varies depending on the particle orientation, particle size, particle shape and presence of a support oxide material,15,16,19,22,27 typically the heterogeneous catalysts serve to convert CO to CO2, since CO is a toxic gas, while CO2 is identified as an energy carrier for the eventual formation of renewable energy.28,29 Thus, the CO oxidation reaction is both industrially relevant and vitally important to environmental cleanup.In a quest to overcome the thermal instability of NPs with well defined shapes, several recent publications have demonstrated that the use of the metal/mesoporous silica core–shell NPs, which can be achieved through several synthetic methods, are of great promise.5,30 Therefore, the surface of Au NRs needs to be tailored for thermal stability as well as increased catalytic activity through the restructuring of the surface atoms and removal of CTAB.31 Recent examples include the coating of Au NRs with silica for the reduction of 4-nitrophenol30 and the use CTAB-capped Au nanorods functionalized by ionic liquid termination for the catalysis of nitro-compound reductions.17 However, as these reactions can occur at relatively low temperatures (<60 °C), the surface reconstructions were primarily highlighted for the increased catalytic activity, rather the high-temperature stability that is needed for a variety of other reactions, including CO oxidation.Au NRs are less frequently reported in catalytic applications than spherical Au NPs due to low temperature sintering and aggregation, as well as the presence of the surfactant, hexadecyltrimethylammonium bromide (CTAB), that isolates the active Au surface from external reactants.2 for gas phase CO oxidation, which requires higher temperatures than the aforementioned solution based nitro-compound reductions. This reaction allowed us to highlight the effect of the mSiO2 coating, resulting in an enhanced thermal stability and improved catalytic performance of CTAB-stabilized Au NRs. Specifically, we synthesize Au NRs with high shape purity, and the corresponding Au NRs@mSiO2, using the surface permeable etching method. Subsequently, we compare the thermal stability, CO oxidation performance and durability of Au NRs and Au NRs@mSiO2 when catalyzing a high temperature oxidative/reductive reaction. As the main results, we find that the Au NRs@mSiO2 demonstrate steady activity and only minor degradation after several heating and catalytic treatments, as a consequence of their high thermal stability confirmed using electron microscopy.Herein, we extend the use of Au NRs@mSiO3), followed by rinsing with copious amounts of Milli-Q water.All chemicals were obtained from the commercial suppliers as mentioned below and used without further purification. Milli-Q water  was used for all solution preparation. All glassware and magnetic bars were cleaned using freshly prepared aqua regia (6 : 1 v/v HCl : HNO4·3H2O), l-ascorbic acid, silver nitrate (AgNO3), sodium borohydride (NaBH4), hydrochloric acid , 3-mercaptopropyltrimethoxysilane (MPTMS), sodium silicate solution, tetraethyl orthosilicate (TEOS), sodium hydroxide (NaOH), and potassium iodide (KI). Other chemicals obtained from additional suppliers were sodium oleate , nitric acid , ethanol  and iodine .Chemicals were obtained from Sigma Aldrich, including hexadecyltrimethylammonium bromide , hydrogen tetrachloroaurate trihydrate  images were obtained with a FEI Tecnai T20 operated at 80 and 200 kV. TEM samples were prepared by drop-casting NP suspensions on copper TEM grids with carbon membranes . Samples for TEM analysis before and after the catalytic tests were drop-cast on specially designed TEM windows composed of a silicon frame covered by a thin layer silicon nitride, which can survive the high temperatures needed for the catalytic tests. Zeta-potential was measured with a Malvern Panalytical Zetasizer Nano ZS. UV-Vis absorption spectra were measured using a temperature-controlled (27 °C) Agilent Cary-60 spectrophotometer with a xenon ash lamp (80 Hz) as a light source. Catalytic tests were performed using a Insplorion X1  connected with a PrismaPlus QME 220 mass spectrometer to measure the reaction output.et al.'s synthesis method32 was followed for high yield synthesis of Au NRs. First, a Au seed solution is prepared by reducing a mixture of HAuCl4  and CTAB  with NaBH4  while stirring vigorously (1200 rpm) for 2 minutes. The brown seed solution was then aged for 30 minutes prior to growth. The growth solution consisted of a mixture of CTAB (7 g) and NaOL (1.234 g) dissolved in warm water , which was cooled to 30 °C after the surfactant powders were completely dissolved. AgNO3  was added to this solution and aged for 15 minutes, followed by the addition of HAuCl4 , which was stirred (700 rpm) for 90 minutes, until the solution became colorless. The pH of the solution was adjusted with HCl  and stirred (400 rpm) for a further 15 minutes, followed by the addition of ascorbic acid  under vigorous stirring for 30 seconds. Finally, the seed solution (0.8 mL) was injected and stirred for 30 seconds before the solution was aged (without stirring) for 17.5 hours, resulting in an opaque, red-brown solution. The final products were isolated via centrifugation  and washed further, using both ultra centrifugal filters  at 5100 rpm for 15 minutes and normal centrifugation . After washing, the highly concentrated NRs solution was carefully re-dispersed in Milli-Q water. The concentration of the NRs colloidal suspension was adjusted to 1 mM Au after the washing, to prepare for the silica coating. We have assumed that the amount of Au NRs remains invariant during the mesoporous silica coating.Ye 33 with two modifications. First, the strictly washed Au NRs suspension from the previous sections is coated instead of applying the Au NPs from Zhou's protocol. Additionally, the centrifugation speed was changed to 3200 rpm to fit the size requirements of our Au NRs. Briefly, this method is a surface permeable etching method that begins with the modification of the Au NR  surface achieved by stirring with MPTMS  followed by the growth of a thin silica shell with sodium silicate . Subsequently, a thick silica shell is formed through the addition of TEOS  and NaOH  and mesopores are obtained through an etching step using NaOH  and CTAB . A detailed synthetic diagram of this process is included in the ESI S1.1.†The coating of a mesoporous silica shell is achieved by following Zhou's worket al.34 1.5 mL I2/KI solution  was introduced into the Au NRs@mSiO2 suspension  at room temperature, while stirring at 500 rpm. After 3 minutes, the suspension was recollected by centrifugation . The sedimented NPs at the bottom of the centrifugal tube were dispersed into 0.3 mL Milli-Q water for TEM imaging. A parallel spectroscopic test was performed to show the full etching procedure, with the suspension transferred to a quartz cuvette after 5 minutes of reaction time.The core-etching protocol was adapted from the work of Chul Cho, 2 was examined by monitoring the CO-oxidation catalytic reaction. In this work, a plug-flow type reactor has been utilized and a ‘glass pocket’ was integrated inside the externally heated quartz tube as previously reported by our recent work20 and Fredriksson et al.35,36 This unique reactor design with minimum dead volume enhances the quadrupole mass spectrometer (QMS) ion current for reaction products from our unsupported nanoparticle catalysts. Total gas flow during the reaction was set to 100 mL min−1 through the quartz tube and only 1 mL min−1 portion was directed to pocket reactor. Au NRs and Au NRs@mSiO2 samples  were drop-cast on separate, pre-diced 2 cm2 silica wafers and these wafers were placed in a flow reactor  solution over two days. The thicker silica shell was grown onto these modified Au NRs through the addition of TEOS to the Au NRs solution, followed by 24 hours of reaction, stirred at room temperature. TEM images of these Au NRs@SiO2, in 41,42 The longitudinal peak also features a shoulder at approximately 950 nm, resulting from the nanorods that did not get efficiently coated.Normally, due to the lack of an –OH group, the surface of metallic NPs, stabilized only by the surfactant used in synthesis, does not have good affinity to SiO2 were treated with NaOH, a typical mesoporogen, and additional CTAB in order to form the porous structure necessary for effective catalytic function. TEM images of these Au NRs@mSiO2 are shown in 43 but contaminates the porous structure, resulting in pores with inadequate resolution for imaging .via calcination. This demonstrates that the mesoporous silica shell improves the thermal stability of these Au NRs, but does not block the movement of reactants to the inside, metallic core. Accessibility of Au NR in the core was further confirmed through reactivity towards gas phase CO oxidation reaction at high temperature, as will be discussed in next section in detail.In addition to the thermal stability, the accessibility of the Au surface through the mesoporous shell before calcination  was inved 850 nm  in the o2 was evaluated by using high temperature CO oxidation as a model reaction.44The catalytic performance of bare Au NRs and the corresponding Au NRs@mSiO5,30,45 As indicated by the aforementioned TEM images, the CTAB present on the surface decomposes and the ‘bare’ Au NRs lose their rod-like shape beyond 150 °C. Further catalytic treatments, performed up to 350 °C, exhibit expectedly low CO oxidation activity on these ‘bare’ Au NRs  is governed by the total number of exposed accessible active sites of the Au NRs@mSiO2, which may be partially hindered by the dimensions of the porous structure of the coating. However, there is only a small degradation after several heating cycles, with the Au NRs@mSiO2 maintaining 95% of their initial activity after three cycles. The CO ion current calibration, as well as the CO2 production of bare Au NRs and Au NRs@mSiO2 in units of CO2 ion current are reported in the ESI S5.3 and S5.4,Surfactant-stabilized Au NRs were initially tested following a low temperature (150 °C) pre-treatment, and show no discernible CO conversion activity, which can be attributed to the inaccessibility of the Au surface due to the surfactant, CTAB, as well as the formation of larger Au domains corresponding to aggregation .5,30,45 ’ Au NRs . Howevero 350 °C . This cao 350 °C  and the 2, we imaged the sample before and after the catalytic treatments. As demonstrated in In order to confirm the thermal and catalytic stability of the Au NRs@mSiO2 compared to the surfactant-stabilized Au NRs. We have demonstrated and compared the catalytic activity, as well as the durability of both surfactant-stabilized Au NRs and Au NRs@mSiO2, respectively, using a CO oxidation model reaction with multiple heating cycles. Combined with the TEM results before and after these catalytic tests, we show that the increased catalytic activity and low degradation rate of the Au NRs@mSiO2 are attributed their excellent thermal stability and the removal of the surfactants at these high temperatures.In summary, we have synthesized Au NRs with an aspect ratio of 5.8 ± 1.8 and successfully coated these colloidal NRs with a ∼50 nm thick mesoporous silica shell. We characterized the thermal stability and surface accessibility of these Au NRs@mSiOYC synthesized and characterized the samples and contributed to the written manuscript. SL contributed to the characterization of the samples and wrote the manuscript. ZS and CT performed the catalytic experiments and contributed to the written manuscript. CL supervised the catalysis experiments. KMP supervised the nanoparticle synthesis and characterization. All authors have given approval to the final version of the manuscript.The authors declare no competing financial interests.RA-011-D1RA01577J-s001