Identification of novel small RNAs in tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>)

To date, the majority of plant small RNAs (sRNA) have been identified in rice, poplar and Arabidopsis. To identify novel tomato sRNAs potentially involved in tomato specific processes such as fruit development and/or ripening, we cloned 4,018 sRNAs from tomato fruit tissue at the mature green stage. From this pool of sRNAs, we detected tomato homologues of nine known miRNAs, including miR482; a poplar miRNA not conserved in Arabidopsis or rice. We identified three novel putative miRNAs with flanking sequence that could be folded into a stem-loop precursor structure and which accumulated as 19-24nt RNA. One of these putative miRNAs (Put-miRNA3) exhibited significantly higher expression in fruit compared with leaf tissues, indicating a specific role in fruit development processes. We also identified nine sRNAs that accumulated as 19–24nt RNA species in tomato but genome sequence was not available for these loci. None of the nine sRNAs or three putative miRNAs possessed a homologue in Arabidopsis that had a precursor with a predicted stem-loop structure or that accumulated as a sRNA species, suggesting that the 12 sRNAs we have identified in tomato may have a species specific role in this model fruit species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Iron homeostasis in chronic inflammation

Inflammation induced anemia and resistance to erythropoietin are common features in patients with chronic kidney disease (CKD). Elevated levels of cytokines and enhanced oxidative stress, conditions associated with inflammatory states, are implicated in the development of anemia. Accumulating evidence suggests that activation of cytokine cascade and the associated acute-phase response, as it often occurs in patients with CKD, divert iron from erythropoiesis to storage sites within the reticuloendothelial system leading to functional iron deficiency and subsequently to anemia or resistance to erythropoietin. Other processes have also been shown to be involved in the pathogenesis of anemia provoked by the activated immune system including an inhibition of erythroid progenitor proliferation and differentiation, a suppression of erythropoietin production and a blunted response to erythropoietin. The present review concerns the underlying alterations in iron metabolism induced by chronic inflammation that result in anemia.     

Synthesis and SAR studies of potent imidazopyridine anticoccidial agents

Diaryl imidazo[1,2-a]pyridine derivatives, such as 6a and 7i, have been synthesized and found to be potent inhibitors of parasite PKG activity. The most potent compounds are the 7-isopropylaminomethyl analog 6a and 2-isopropylamino analog 7i. These compounds are also fully active in in vivo assay as anticoccidial agents at 25 ppm in feed.     

Optimization of a solid phase extraction procedure for prostaglandin E2, F2 alpha and their tissue metabolites

The primary prostaglandins PGE(2) and PGF(2 alpha) are metabolized in tissues by a series of enzymatic and non-enzymatic reactions. To measure metabolic rates and individual reaction rates it is necessary to extract the parent prostaglandins and metabolites before the separation and quantification of each compound is achieved. Here we have established and optimized a solid phase extraction (SPE) procedure to recover PGE(2), PGF(2 alpha) and their six enzymatic and non-enzymatic tissue metabolites from aqueous solutions including urine, plasma and tissue homogenate. We have used octadecyl-bonded silica gel as the stationary phase and methanol-water mixtures as binary mobile phases. The volumes and concentrations of the washing and elution solutions were optimized individually for each PG. Recoveries of all PG standards were quantitative except for PGEM, which was recovered at 80% efficiency. Biological matrix components interfered with the extraction in a PG- and matrix-specific fashion. Inclusion of 1% formic acid in the loading mixture raised recoveries from urine, plasma and tissue homogenate to >or=90%. This SPE method is the first that has been optimized by systematic elution studies for PGE(2), PGF(2 alpha) and the complement of their tissue metabolites. The procedure is simple, robust and can serve as an effective pre-purification step before downstream separation and quantification of each tissue metabolite of PGE(2) and PGF(2 alpha) from complex biological matrices.     

Anorectic estrogen mimics leptin's effect on the rewiring of melanocortin cells and Stat3 signaling in obese animals

Metabolic hormones, such as leptin, alter the input organization of hypothalamic circuits, resulting in increased pro-opiomelanocortin (POMC) tone, followed by decreased food intake and adiposity. The gonadal steroid estradiol can also reduce appetite and adiposity, and it influences synaptic plasticity. Here we report that estradiol (E2) triggers a robust increase in the number of excitatory inputs to POMC neurons in the arcuate nucleus of wild-type rats and mice. This rearrangement of synapses in the arcuate nucleus is leptin independent because it also occurred in leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice, and was paralleled by decreased food intake and body weight gain as well as increased energy expenditure. However, estrogen-induced decrease in body weight was dependent on Stat3 activation in the brain. These observations support the notion that synaptic plasticity of arcuate nucleus feeding circuits is an inherent element in body weight regulation and offer alternative approaches to reducing adiposity under conditions of failed leptin receptor signaling.     

Expression of a synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of <Emphasis Type="Italic">Escherichia coli</Emphasis> heat labile enterotoxin in rice endosperm

Epitopes often require co-delivery with adjuvant and targeting proteins to enable recognition by the immune system, and this approach may also increase the efficacy of the antigen. In this study, we assess and describe the ability of transgenic rice plants to express a fusion protein consisting of the B-subunit of the Escherichia coli heat-labile enterotoxin (LTB) and a synthetic core-neutralizing epitope (COE) of porcine epidemic diarrhea virus (PEDV), inducing an enteric disease that is seen most predominantly in piglets. Both components of the fusion proteins were detected with Western blot analysis. The fusion protein was determined to assemble into pentamers, as was evidenced by its ability to bind to GM1 gangliosides, and evidenced an average level of expression in a transgenic rice endosperm. This indicates that the expression system of the plant is capable of generating a sizable amount of antigen, possibly allowing for the successful development of an edible vaccine.     

Control of cell polarity and motility by the PtdIns(3,4,5)P3 phosphatase SHIP1

Proper neutrophil migration into inflammatory sites ensures host defense without tissue damage. Phosphoinositide 3-kinase (PI(3)K) and its lipid product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) regulate cell migration, but the role of PtdIns(3,4,5)P(3)-degrading enzymes in this process is poorly understood. Here, we show that Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1), a PtdIns(3,4,5)P(3) phosphatase, is a key regulator of neutrophil migration. Genetic inactivation of SHIP1 led to severe defects in neutrophil polarization and motility. In contrast, loss of the PtdIns(3,4,5)P(3) phosphatase PTEN had no impact on neutrophil chemotaxis. To study PtdIns(3,4,5)P(3) metabolism in living primary cells, we generated a novel transgenic mouse (AktPH-GFP Tg) expressing a bioprobe for PtdIns(3,4,5)P(3.) Time-lapse footage showed rapid, localized binding of AktPH-GFP to the leading edge membrane of chemotaxing ship1(+/+)AktPH-GFP Tg neutrophils, but only diffuse localization in ship1(-/-)AktPH-GFP Tg neutrophils. By directing where PtdIns(3,4,5)P(3) accumulates, SHIP1 governs the formation of the leading edge and polarization required for chemotaxis.     

Antibody glycosylation in COVID-19

Glycoconjugate Journal - Antibody glycosylation has received considerable attention in coronavirus disease 2019 (COVID-19) infections and recently also in vaccination. Antibody glycosylation and in...     

Upregulation of heat shock proteins and the promotion of damage-associated molecular pattern signals in a colorectal cancer model by modulated electrohyperthermia

In modulated electrohyperthermia (mEHT) the enrichment of electric field and the concomitant heat can selectively induce cell death in malignant tumors as a result of elevated glycolysis, lactate production (Warburg effect), and reduced electric impedance in cancer compared to normal tissues. Earlier, we showed in HT29 colorectal cancer xenografts that the mEHT-provoked programmed cell death was dominantly caspase independent and driven by apoptosis inducing factor activation. Using this model here, we studied the mEHT-related cell stress 0-, 1-, 4-, 8-, 14-, 24-, 48-, 72-, 120-, 168- and 216-h post-treatment by focusing on damage-associated molecular pattern (DAMP) signals. Significant cell death response upon mEHT treatment was accompanied by the early upregulation (4-h post-treatment) of heat shock protein (Hsp70 and Hsp90) mRNA levels. In situ, the treatment resulted in spatiotemporal occurrence of a DAMP protein signal sequence featured by the significant cytoplasmic to cell membrane translocation of calreticulin at 4 h, Hsp70 between 14 and 24 h and Hsp90 between 24- and 216-h post-treatment. The release of high-mobility group box1 protein (HMGB1) from tumor cell nuclei from 24-h post-treatment and its clearance from tumor cells by 48 h was also detected. Our results suggest that mEHT treatment can induce a DAMP-related signal sequence in colorectal cancer xenografts that may be relevant for promoting immunological cell death response, which need to be further tested in immune-competent animals.