Rocks in the auxin stream : wound-induced auxin accumulation and ERF115 expression synergistically drive stem cell regeneration

Plants are known for their outstanding capacity to recover from various wounds and injuries. However, it remains largely unknown how plants sense diverse forms of injury and canalize existing developmental processes into the execution of a correct regenerative response. Auxin, a cardinal plant hormone with morphogen-like properties, has been previously implicated in the recovery from diverse types of wounding and organ loss. Here, through a combination of cellular imaging and in silico modeling, we demonstrate that vascular stem cell death obstructs the polar auxin flux, much alike rocks in a stream, and causes it to accumulate in the endodermis. This in turn grants the endodermal cells the capacity to undergo periclinal cell division to repopulate the vascular stem cell pool. Replenishment of the vasculature by the endodermis depends on the transcription factor ERF115, a wound-inducible regulator of stem cell division. Although not the primary inducer, auxin is required to maintain ERF115 expression. Conversely, ERF115 sensitizes cells to auxin by activating ARF5/MONOPTEROS, an auxin-responsive transcription factor involved in the global auxin response, tissue patterning, and organ formation. Together, the wound-induced auxin accumulation and ERF115 expression grant the endodermal cells stem cell activity. Our work provides a mechanistic model for wound-induced stem cell regeneration in which ERF115 acts as a wound-inducible stem cell organizer that interprets wound-induced auxin maxima

DE NOVO ORGANOGENESISI AND TISSUE REPAIR IN THE ARABIDOPSIS ROOT

Ph.DDOCTOR OF PHILOSOPHY (FOS

Erythropoietin Protects Cardiomyocytes from Cell Death during Hypoxia/Reperfusion Injury through Activation of Survival Signaling Pathways

<div><p>Hypoxia/Reoxygenation (H/R) cardiac injury is of great importance in understanding Myocardial Infarctions, which affect a major part of the working population causing debilitating side effects and often-premature mortality. H/R injury primarily consists of apoptotic and necrotic death of cardiomyocytes due to a compromise in the integrity of the mitochondrial membrane. Major factors associated in the deregulation of the membrane include fluctuating reactive oxygen species (ROS), deregulation of mitochondrial permeability transport pore (MPTP), uncontrolled calcium (Ca²⁺) fluxes, and abnormal caspase-3 activity. Erythropoietin (EPO) is strongly inferred to be cardioprotective and acts by inhibiting the above-mentioned processes. Surprisingly, the underlying mechanism of EPO's action and H/R injury is yet to be fully investigated and elucidated. This study examined whether EPO maintains Ca²⁺ homeostasis and the mitochondrial membrane potential (ΔΨ_m) in cardiomyocytes when subjected to H/R injury and further explored the underlying mechanisms involved. H9C2 cells were exposed to different concentrations of EPO post-H/R, and 20 U/ml EPO was found to significantly increase cell viability by inhibiting the intracellular production of ROS and caspase-3 activity. The protective effect of EPO was abolished when H/R-induced H9C2 cells were treated with Wortmannin, an inhibitor of Akt, suggesting the mechanism of action through the activation Akt, a major survival pathway.</p></div

Evolution of the RNase P RNA structural domain in Leptospira spp

We have employed the RNase P RNA (RPR) gene, which is present as single copy in chromosome I of Leptospira spp. to investigate the phylogeny of structural domains present in the RNA subunit of the tRNA processing enzyme, RNase P. RPR gene sequences of 150 strains derived from NCBI database along with sequences determined from 8 reference strains were examined to fathom strain specific structural differences present in leptospiral RPR. Sequence variations in the RPR gene impacted on the configuration of loops, stems and bulges found in the RPR highlighting species and strain specific structural motifs. In vitro transcribed leptospiral RPR ribozymes are demonstrated to process pre-tRNA into mature tRNA in consonance with the positioning of Leptospira in the taxonomic domain of bacteria. RPR sequence datasets used to construct a phylogenetic tree exemplified the segregation of strains into their respective lineages with a (re)speciation of strain SH 9 to Leptospira borgpetersenii, strains Fiocruz LV 3954 and Fiocruz LV 4135 to Leptospira santarosai, strain CBC 613 to Leptospira kirschneri and strain HAI 1536 to Leptospira noguchii. Furthermore, it allowed characterization of an isolate P2653, presumptively characterized as either serovar Hebdomadis, Kremastos or Longnan to Leptospira weilii, serovar Longna

Pre-treatment of EPO maintains mitochondrial membrane potential in H/R induced H9C2 cells.

<p>The effect of EPO on Membrane potential was determined by colocalization studies using Rhodamine-123 and DCFH-DA. H9C2 cells were subjected to H/R with or without pre-treatment with 20 U/ml EPO for 24 hrs. H9C2 cells pretreated with EPO and control cells showed Rhodamine-123 fluorescence only in the perinuclear region about 58% and 52% colocalzation respectively whereas H9C2 cells subjected to H/R showed Rhodamine-123 fluorescence both in perinuclear region and cytosol about 94.7% co localization in Figure 5 A. Data are presented as means ± SEM of the ratios from three independent experiments * denotes p<0.001 for analyses compared to H/R.</p

Pre-treatment of EPO in H/R induced H9C2 cells had uniformly green nuclei with intact plasma and nuclear membranes.

<p>The effect of EPO on apoptosis and necrosis was determined using Acridine/Orange (Ao/EtBr) double staining assay. H9C2 cells were subjected to H/R with or without pre-treatment with 20 U/ml EPO for 24 hrs. (A) EPO stabilizes plasma membrane and nuclear membrane showed byuniform green nuclei. In H/R induced cells, early apoptotic cells were indicated by intact membrane but nuclei was condensed and green. Late-stage apoptotic cells were indicated by bright orange-stained nuclei. Necrotic cells were indicated by red stained nuclei. (B) Represents the quantification of the above image. Data are presented as means ± SEM of the ratios from three independent ratios *p<0.001 for analyses compared to H/R.</p

Pre-treatment of EPO increases intracellular p-Akt in H/R induced H9C2 cells.

<p>The effect of EPO on p-Akt was determined. H9C2 cells (A) were subjected to 8 hrs hypoxia and 30 mins of reperfusion with or without pre-treatment with 20 U/ml EPO and 1 µM Wortmannin before 30 mins for 24 hrs and stained with p-AKT antibody. EPO increase p-Akt. (B) Represents the quantification green florescence, which indicates the increase in levels of p-Akt. Data are presented as means ± SEM of the ratios from three independent experiments * denotes p<0.001 for analyses compared to H/R.</p

Characterization of H9C2 cells by Anti α – Sarcomeric actin.

<p>Panel A shows DAPI, Panel B shows α-sarcomeric actinin and Panel C shows merged image of DAPI and α-sarcomeric actinin.</p

Pre-treatment of EPO decreases caspase-3 activity in H/R induced H9C2 cells.

<p>The effect of EPO on caspase-3 activity was determined using caspase-3 colorimetric assay. H9C2 cells were subjected to H/R with or without pre-treatment with 20 U/ml EPO for 24 hrs and incubated with 1 µM of Wortmannin 30 mins before EPO treatment. EPO decrease caspase-3 activity after H/R. H9C2 cells pretreated with wortmannin showed increase in caspase-3 activity. Data are presented as means ± SEM of the ratios from three independent experiments * denotes p<0.001 for analyses compared to H/R.</p