Although glucocorticoids are a profoundly important class of anti-inflammatory and immunosuppressive agents, their actions in dendritic cells (DCs) are not well understood. We found that ...dexamethasone, a potent glucocorticoid, selectively induced apoptosis in mature, but not in immature, DCs in healthy mice, in mice with experimental airway inflammation, and in vitro in bone marrow–derived DCs. Distinct glucocorticoid receptor (GR) translational isoforms expressed in immature and mature DCs probably contribute to the DC maturational stage-specific glucocorticoid sensitivity. The GR-D isoforms were the predominant isoforms in immature DCs, whereas the proapoptotic GR-A isoform was the main isoform in mature DCs. Ectopic expression of the GR-A isoform in immature DCs increased glucocorticoid sensitivity and RU486, a selective GR antagonist, inhibited the glucocorticoid sensitivity of mature DCs. Furthermore, the distinct expression pattern of GR isoforms in immature and mature murine DCs was also observed in human monocyte–derived DCs. These studies suggest that glucocorticoids may spare immature DCs and suppress mature DCs and inflammation via differential expression of GR translational isoforms.
•Mature, but not immature, dendritic cells are sensitive to glucocorticoid-induced apoptosis.•Mature, but not immature, dendritic cells express proapoptotic glucocorticoid receptor translational isoforms.
Translational isoforms of the glucocorticoid receptor α (GR-A, -B, -C1, -C2, -C3, -D1, -D2, and -D3) have distinct tissue distribution patterns and unique gene targets. The GR-C3 isoform-expressing ...cells are more sensitive to glucocorticoid killing than cells expressing other GRα isoforms and the GR-D isoform–expressing cells are resistant to glucocorticoid killing. Whereas a lack of activation function 1 (AF1) may underlie the reduced activity of the GR-D isoforms, it is not clear how the GR-C3 isoform has heightened activity. Mutation analyses and N-terminal tagging demonstrated that steric hindrance is probably the mechanism for the GR-A, -B, -C1, and -C2 isoforms to have lower activity than the GR-C3 isoform. In addition, truncation scanning analyses revealed that residues 98 to 115 are critical in the hyperactivity of the human GR-C3 isoform. Chimera constructs linking this critical fragment with the GAL4 DNA-binding domain showed that GR residues 98 to 115 do not contain any independent transactivation activity. Mutations at residues Asp101 or Gln106 and Gln107 all reduced the activity of the GR-C3 isoform. In addition, functional studies indicated that Asp101 is crucial for the GR-C3 isoform to recruit coregulators and to mediate glucocorticoid-induced apoptosis. Thus, charged and polar residues are essential components of an N-terminal motif that enhances the activity of AF1 and the GR-C3 isoform. These studies, together with the observations that GR isoforms have cell-specific expression patterns, provide a molecular basis for the tissue-specific functions of GR translational isoforms.
Organic production is one of the fastest growing food sectors globally. However, average yield in organic vegetable production is up to 33% lower than in conventional production. This difference ...could be due to higher fertilization rates in conventional, compared to organic, farming. We aimed to compare yield and quality characteristics of carrots produced under equal nitrogen fertilization rates over four years in organic and conventional conditions. We found a 14.5% higher marketable, and 10.0% lower discarded, yield in the organic compared to the average conventional treatments. In addition, carrots managed organically had 14.1% lower nitrate and 10.0% higher vitamin C content than carrots managed conventionally. There were no convincing effects of cultivation system on the nitrogen, total sugar, or dry matter content of carrots. Organically managed carrots were free of pesticide residues, while several residues were found in carrots managed conventionally. Our study reveals that organic management of carrots may exceed that of conventional methods in yield and several quality characteristics, while being free of pesticide residues. Organic fertilizer gave an advantage over mineral fertilizer, when equal rates of nitrogen were used in both production systems.
Polyphenols of fruits and vegetables form an important part of human dietary compounds. Relatively little is known about accumulation of phenolics during fruits ripening process. The goal of this ...work was to study the changes in antioxidant activity and in content of 30 polyphenols during ripening of tomato fruits. Five organically and conventionally grown tomato cultivars were investigated at three different ripening stages. Phenolic compounds were extracted with methanol and extracts were analyzed by HPLC-DAD-MS/MS. During ripening, four different changing patterns were observed: (1) high level in green fruits with minimal changes; (2) continuous increase with maximum level in red-ripe fruits; (3) decrease; (4) increase and achieving maximum level at half-ripe stage. Similar change patterns were found for organic and conventional fruits. The accumulation patterns of phenolic compounds were similar in standard-type tomatoes but differed in several cases in cherry-type cultivar. Although contents of some polyphenols decreased during ripening, total phenolics and free radical scavenging activity increased in all studied cultivars and in case of both cultivation modes. The changes in content of phenolic compounds during ripening were greatly influenced by cultivars, but cultivation mode had only minor impact on dynamics in polyphenols contents in tomato fruits.
Abstract only
RNA splicing and processing provide important means of genetic control to diversify gene function, and fine‐tune protein production. RNA regulation is a critical step in the expression ...of most genes, and its misregulation often results in human disease. Rbfox1 is a RNA binding protein that regulates alternative splicing and RNA stability, and plays an important role in regulating alternative splicing (AS) in neurons, skeletal muscle, and the heart. Recently, Rbfox1 was identified as a critical regulator of Mef2 splicing to protect the heart from failure. However, its role in protecting the heart from failing is likely not limited to Mef2 splicing. To begin to study the role of Rbfox1 in heart failure, we generated cardiomyocyte specific Rbfox1 gene‐deletion mice (cRbfox1−/−). Cardiomyocyte specific deletion of Rbfox1 was confirmed by Western blotting and immunohistochemistry. cRbfox1−/− mice showed mild hypertrophy and depressed cardiac function under homeostatic conditions, which did not deteriorate with age. Pressure overload by trans‐aortic constriction (TAC) caused exaggerated cardiac hypertrophy and accelerated heart failure in cRbfox1−/− compared to wildtype mice. Since Rbfox1 is a known RNA binding protein, we performed RNA sequencing and found significant changes in calcium regulated genes. We measured calcium dynamics and noted a significantly delayed Calcium reuptake into the Sarcoplasmic Reticulum, which is likely caused by decreased SERCA2a expression on Western blot. In conclusion, Rbfox1 is important to maintain cardiac function by regulating calcium signaling. We are currently unraveling the mechanisms via which Rbfox1 regulates Serca2a function.
Support or Funding Information
Acknowledgement:
This work was supported by National Institutes of Health grants and Lillehei Scholar Award (to JVB).
•Limited nitrogen availability after roller-crimping reduced cabbage yields.•Late termination of cover crops reduced nitrogen availability for cabbage.•Sufficient fertilisation maintained cabbage ...yield under roller-crimping.•Termination time and method of cover crops did not affect nitrogen leaching risk.•Roller-crimping changed resource allocation between shoot and root growth of cabbage.
Agro-ecological service crops (ASCs), also known as cover crops, green manures or catch crops, can improve organic vegetable production in terms of weed suppression, nitrogen (N) recycling, or addition of N through symbiotic N2 fixation by legumes. Traditionally, ASCs are terminated through full incorporation into the soil (FI), but alternative termination without tillage is available by roller-crimping (RC). The applicability of RC to Northern and Western European climates has only been studied to a limited extend. The objective of this study was therefore to investigate the N dynamics involved in FI and RC of legume and cereal ASCs in organic cabbage production across Northern and Western European climates. Field experiments were conducted at one location in Estonia and in Denmark, and at three locations in Belgium during two cropping cycles (2015/2016 and 2016/2017) to assess the effect of ASC termination method in spring (FI and RC) and ASC species (pea, pea/cereal mixtures and cereals), compared with a bare soil control (BS), on soil mineral N content, N accumulation, yield, and root growth of a following crop of cabbage. Agro-ecological service crops, and in particular cereals, reduced N availability for succeeding cabbage compared with BS by 37–73%. Similar marketable cabbage yield was obtained under FI and BS in two out of four cases in Belgium, due to higher availability of soil mineral N when terminated earlier (April/May) than RC (May/June). However, FI reduced marketable yields by 36–98 % in the other cases in Belgium and in two out of four cases in Estonia and Denmark, where FI was conducted in May/June. Overall, cabbage marketable yields were reduced by 68–100% under RC compared to BS and FI in seven out of nine cases mainly due to reduced soil mineral N availability. Soil mineral N availability was lower under RC likely because of a slower mineralisation of ASCs and soil organic matter. Besides, the termination of ASCs close to the time of cabbage planting under RC increased pre-emptive competition on N. Cabbage marketable yield could be maintained under RC at fertilisation by 236 kg N ha−1 following pea in Denmark. Cabbage root growth was generally increased with higher above ground plant biomass across treatments in Estonia and Denmark, while the relationship was reverse under RC in East Flanders, indicating changes in resource allocation. Soil mineral N at harvest did not differ among treatments in deeper depth, showing no higher risk of N leaching from FI or RC. In conclusion, RC showed major yield reductions in the first and second year after conversion from spring tillage. Therefore, further investigations should verify improved yields under longer-term RC practice, e.g. due to build-up of soil organic matter, before implementation can be recommended in organic vegetable production in Northern and Western Europe.
Although cardiac c-kit
cells are being tested in clinical trials, the circumstances that determine lineage differentiation of c-kit
cells in vivo are unknown. Recent findings suggest that endogenous ...cardiac c-kit
cells rarely contribute cardiomyocytes to the adult heart. We assessed whether various pathological stimuli differentially affect the eventual cell fates of c-kit
cells.
We used single-cell sequencing and genetic lineage tracing of c-kit
cells to determine whether various pathological stimuli would result in different fates of c-kit
cells.
Single-cell sequencing of cardiac CD45
c-kit
cells showed innate heterogeneity, indicative of the existence of vascular and mesenchymal c-kit
cells in normal hearts. Cardiac pressure overload resulted in a modest increase in c-kit-derived cardiomyocytes, with significant increases in the numbers of endothelial cells and fibroblasts. Doxorubicin-induced acute cardiotoxicity did not increase c-kit-derived endothelial cell fates but instead induced cardiomyocyte differentiation. Mechanistically, doxorubicin-induced DNA damage in c-kit
cells resulted in expression of p53. Inhibition of p53 blocked cardiomyocyte differentiation in response to doxorubicin, whereas stabilization of p53 was sufficient to increase c-kit-derived cardiomyocyte differentiation.
These results demonstrate that different pathological stimuli induce different cell fates of c-kit
cells in vivo. Although the overall rate of cardiomyocyte formation from c-kit
cells is still below clinically relevant levels, we show that p53 is central to the ability of c-kit
cells to adopt cardiomyocyte fates, which could lead to the development of strategies to preferentially generate cardiomyocytes from c-kit
cells.
Abstract only
Cardiac regeneration is partly regulated by c‐kit
+
cardiac progenitor cells (CPC). Doxorubicin (DOX) is an effective anticancer chemotherapeutic which induces cardiotoxicity. To what ...extent c‐kit
+
CPCs are activated in response to DOX treatment and their fate remains unknown. Here, we used the following genetic mouse models to characterize the contribution of c‐kit
+
CPCs to cardiac repair mechanisms after DOX; c‐kit Cre‐IRES‐GFP mice to mark all cells that currently express the kit locus, c‐kit MerCreMer(MCM) cross‐bred to R26‐GFP reporter mice to mark CPC derived cells using inducible genetic lineage tracing, and c‐kit MCM cross‐bred to R26‐mT/mG double fluorescent reporter mice to differentiate de novo cardiomyocyte from cell fusion. Two‐month‐old mice were treated with DOX (20 mg/kg cumulative dose). Echocardiography one week after DOX injection showed significantly depressed cardiac function accompanied by cardiomyocyte apoptosis using immunohistochemistry for cleaved caspase 3. Furthermore, DOX activates expression of GATA4 specifically in CPCs. Presumably as a consequence of increased Gata4 expression, genetic lineage tracing of CPCs showed a significant increase (25‐fold) in cardiomyocyte formation, mostly newly formed. Interestingly, this was not at the expense of endothelial fate commitment. In conclusion, DOX enhances c‐kit
+
CPC cardiomyocyte commitment and differentiation. We are currently unraveling the underlying molecular pathways to enhance the myogenic potential of c‐kit
+
CPCs to repair injured hearts.
Abstract only
Alternative splicing increases the ability to respond to stress. One of the most important alternative splicing regulators is the family of Rbfox proteins. Rbfox1 is specifically ...expressed in brain, heart and skeletal muscle. Here, we generated cardiac specific Rbfox1 deletion mice using Nkx2.5‐cre (cRbfox1‐/‐), which showed depressed baseline cardiac function measured by echocardiography. We confirmed cardiomyocyte specific deletion of Rbfox1 by Western blotting and immunohistochemistry. Morphometry analysis identified a small but significant increase in cardiac hypertrophy at baseline. We next isolated adult cardiomyocytes, which showed slower calcium reuptake kinetics. To assess the role of Rbfox1 mediated alternative splicing in the cardiac stress response, we induced pressure overload by trans‐aortic constriction (TAC) in two‐month‐old WT and cRbfox1‐/‐ mice. cRbfox1‐/‐ mice showed an increased propensity to develop heart failure with depressed cardiac function. The slight increase in cardiac hypertrophy we noted at baseline was further enhanced. We further showed increased cardiomyocyte apoptosis by cleaved caspase 3 staining and cardiac fibrosis by fast green and sirius red staining. In conclusion, Rbfox1 is important to maintain cardiac function and loss of Rbfox1 function sensitizes cardiomyocytes to pressure overload induced cardiac dysfunction and cardiomyocyte apoptosis. We are currently unraveling which Rbfox1 mediated alternatively spliced genes drive these events.
PDF
