Persistencia de malezas gramíneas en cultivos de trigo del sudeste bonaerense

En la presente tesis se estudió la persistencia de especies poáceas en cultivos de trigo del sudeste de Buenos Aires. En dicha región, Avena fatua L. y Lolium multiflorum Lam. son las malezas poáceas más importantes, tanto por la dificultad de control como por sus efectos competitivos sobre el cultivo. A los efectos de cuantificar la persistencia de dichas especies, se estudió la composición de la comunidad de malezas en dos momentos del ciclo: preaplicación de herbicidas y precosecha. Individuos de ambas malezas fueron registrados en ambos momentos como consecuencia de “escapes” al control realizado con herbicidas, siendo A. fatua más constante que L. mutiflorum. Posteriormente, se estudiaron los procesos que definen la persistencia de ambas malezas. Los resultados obtenidos indican que el ajuste del momento de emergencia es jerárquicamente el factor más importante para explicar la persistencia de A. fatua. Se demostró que los modelos de germinación son diferentes según las semillas provengan de un lote agrícola o de una condición de no cultivo, siendo estas diferencias de naturaleza genética. Por otro lado, la variabilidad en la supervivencia a los herbicidas es el factor que mejor explica la persistencia de L. multiflorum, habiéndose documentado resistencia cruzada a los herbicidas inhibidores de la ALS, pyroxsulam, imazamox y flucarbazone, sin antecedentes previos en la región. Los índices de resistencia encontrados presentan variación con la temperatura ambiente en post-aplicación del herbicida, habiéndose registrado mayor resistencia con mayor temperatura. Además, se comprobó que los individuos resistentes presentan menor tiempo a floración que los susceptibles. Tal atributo puede significar una ventaja demográfica para dichas poblaciones. Queda así demostrada la persistencia de A. fatua y L. multiflorum durante el ciclo del cultivo más allá de las prácticas de control realizadas y la participación de dos procesos demográficos distintos (establecimiento y supervivencia) en dicha persistencia

Homeotic Gene <i>teashirt</i> (<i>tsh</i>) Has a Neuroprotective Function in Amyloid-Beta 42 Mediated Neurodegeneration

<div><p>Background</p><p>Alzheimer's disease (AD) is a debilitating age related progressive neurodegenerative disorder characterized by the loss of cognition, and eventual death of the affected individual. One of the major causes of AD is the accumulation of Amyloid-beta 42 (Aβ42) polypeptides formed by the improper cleavage of amyloid precursor protein (APP) in the brain. These plaques disrupt normal cellular processes through oxidative stress and aberrant signaling resulting in the loss of synaptic activity and death of the neurons. However, the detailed genetic mechanism(s) responsible for this neurodegeneration still remain elusive.</p> <p>Methodology/ Principle Findings</p><p>We have generated a transgenic <i>Drosophila</i> eye model where high levels of human Aβ42 is misexpressed in the differentiating photoreceptor neurons of the developing eye, which phenocopy Alzheimer's like neuropathology in the neural retina. We have utilized this model for a gain of function screen using members of various signaling pathways involved in the development of the fly eye to identify downstream targets or modifiers of Aβ42 mediated neurodegeneration. We have identified the homeotic gene <i>teashirt</i> (<i>tsh</i>) as a suppressor of the Aβ42 mediated neurodegenerative phenotype. Targeted misexpression of <i>tsh</i> with Aβ42 in the differentiating retina can significantly rescue neurodegeneration by blocking cell death. We found that Tsh protein is absent/ downregulated in the neural retina at this stage. The structure function analysis revealed that the PLDLS domain of Tsh acts as an inhibitor of the neuroprotective function of <i>tsh</i> in the <i>Drosophila</i> eye model. Lastly, we found that the <i>tsh</i> paralog, <i>tiptop</i> (<i>tio</i>) can also rescue Aβ42 mediated neurodegeneration.</p> <p>Conclusions/Significance</p><p>We have identified <i>tsh</i> and <i>tio</i> as new genetic modifiers of Aβ42 mediated neurodegeneration. Our studies demonstrate a novel neuroprotective function of <i>tsh</i> and its paralog <i>tio</i> in Aβ42 mediated neurodegeneration. The neuroprotective function of <i>tsh</i> is independent of its role in retinal determination.</p> </div

A paralog of <i>tsh</i>, <i>tio</i>, also exhibits a neuroprotective function.

<p>(A) A cartoon depicting full length type Tio protein and various truncated constructs used in this study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080829#B49" target="_blank">49</a>]. The full length Tio protein consists of a PLDLS domain and four DNA binding Zinc finger domains [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080829#B49" target="_blank">49</a>]. A membrane specific marker Disc large (Dlg, white channel) was used to mark the outline of cells and pan neural marker Elav (red channel) marks nuclei of the photoreceptor neurons. Targeted misexpression of Aβ42 and deletion constructs of the (B, C) first zinc finger domain (GMR>Aβ42+<i>t</i><i>i</i><i>o</i>∆Zn1), (D, E) second zinc finger domain (GMR>Aβ42+<i>t</i><i>i</i><i>o</i>∆Zn2), (F, G) third zinc finger domain (GMR>Aβ42+<i>t</i><i>i</i><i>o</i>∆Zn3) resulted in the significant rescue of Aβ42 mediated neurodegeneration in the adult eye, which is comparable to the full length Tio protein. However, targeted misexpression of (J, K) the fourth zinc finger domain (GMR>Aβ42+<i>t</i><i>i</i><i>o</i>∆Zn4), and (L, M) deletion constructs of the PLDLS domain (GMR>Aβ42+<i>t</i><i>i</i><i>o</i>∆PLDLS) resulted in a significantly stronger rescue. Note that this rescue is stronger than the one seen with (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080829#pone-0080829-g002" target="_blank">Figure 2C</a>) full length Tio protein. The magnification of (B, D, F, H, J, L) the adult eye SEM micrographs is 180X and (C, E, G, I, K, M) the eye-antennal imaginal disc confocal images is 20X. </p

PLDLS domain of Tsh acts as a suppressor of its neuroprotective function.

<p>(A) A cartoon depicting full length Tsh protein and various truncated constructs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080829#B49" target="_blank">49</a>] used in this study. The full length Tsh protein consists of a PLDLS domain and three DNA binding Zinc finger domains. (B-E) Scanning electron micrographs (SEM) of the adult eyes generated by targeted misexpression of various deletion constructs of <i>tsh</i> in the GMR>Aβ42 background to test their requirement in its neuroprotective function. A membrane specific marker Disc large (Dlg, white channel), was used to mark the outline of cells and pan neural marker Elav (red channel) marks nuclei of the photoreceptor neurons. Targeted misexpression of Aβ42 and deletion constructs of <i>tsh</i> lacking the (B, C) first zinc finger domain (GMR>Aβ42+<i>t</i><i>s</i><i>h</i>∆Zn1), (D, E) second zinc finger domain (GMR>Aβ42+<i>t</i><i>s</i><i>h</i>∆Zn2), and (F, G) third zinc finger domain (GMR>Aβ42+<i>t</i><i>s</i><i>h</i>∆Zn3) resulted in a significant rescue of the Aβ42 mediated neurodegeneration in the imaginal disc and the adult eye, respectively. Note that the extent of rescue by deletion constructs lacking various Zn finger domains is comparable to the full length Tsh protein. (H, I) Targeted misexpression of Aβ42 and deletion construct of the PLDLS domain (GMR>Aβ42+<i>t</i><i>s</i><i>h</i>∆PLDLS) results in a significantly stronger rescue both in the (H) eye imaginal disc and the (H) adult eye. Note that this rescue is stronger than the one seen with (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080829#pone-0080829-g002" target="_blank">Figure 2C</a>) full length Tsh protein. The magnification of (B, D, F, H) the adult eye SEM image is 180X, and (C, E, G, I) the eye-antennal imaginal disc is 20X.</p

Genetic modifiers of amyloid-beta 42 (Aβ42) mediated neurodegeneration in the <i>Drosophila</i> eye.

<p>In a gain-of-function forward genetic screen, the candidate genes of interest were misexpressed along with Aβ42 in the differentiating neurons of the developing eye. The effect of upregulation of the gene of interest on the Aβ42 mediated neurodegenerative phenotype is assayed in the adult eye. In comparison to the (a) wild-type compound eye, (B) misexpression of Aβ42 (GMR>Aβ42) results in strong neurodegeneration in the adult eye, as evident from the highly reduced size, glazed appearance and fusion of ommatidia. However, targeted misexpression of (C) <i>teashirt</i> (tsh) with Aβ42 (GMR>Aβ42+<i>t</i><i>s</i><i>h</i>) results in a significant rescue of the neurodegenerative phenotype. (D-J) Targeted misexpression of Aβ42 along with the retinal determination genes (D) eyeless (ey), (GMR>Aβ42+<i>e</i><i>y</i>), (E) eyes absent (eya), (GMR>Aβ42+<i>e</i><i>y</i><i>a</i>), (F) sine <i>oculis</i> (so), (GMR>Aβ42+<i>s</i><i>o</i>), (G) <i>dacshund</i> (dac), (GMR>Aβ42+<i>d</i><i>a</i><i>c</i>), and (H) <i>eya</i> and so (GMR>Aβ42+<i>e</i><i>y</i><i>a</i>+<i>s</i><i>o</i>) did not show any significant rescue of the Aβ42 (GMR>Aβ42) mediated neurodegenerative phenotype. Even though (D) GMR>Aβ42+<i>e</i><i>y</i> and (E) GMR>Aβ42+<i>e</i><i>y</i><i>a</i> cause a subtle increase in the eye field but the neurodegenerative phenotype is not rescued. Furthermore, black necrotic spots are also seen suggesting that members of the core retinal determination genes machinery cannot rescue Aβ42 (GMR> Aβ42) mediated neurodegeneration. Targeted misexpression of negative regulator of eye development (D) <i>homothorax</i> (hth), (GMR>Aβ42+<i>h</i><i>t</i><i>h</i>), (E) dominant negative <i>hth</i> (hth^ENR), (GMR>Aβ42+<i>h</i><i>t</i><i>h</i>^<i>ENR</i>) also did not affect Aβ42 mediated neurodegeneration. Other signaling pathways tested in the gain-of-function screen were (K, L) Wingless (Wg), (M) Decapentaplegic (Dpp), (N) Notch (N) and (P) Hedgehog (Hh). (K) Upregulation of Wg (GMR>Aβ42+<i>w</i><i>g</i>) enhances whereas (L) downregulating Wg signaling by using an antagonist of Wg signaling shaggy (GMR>Aβ42+<i>s</i><i>g</i><i>g</i>) can significantly rescue Aβ42 mediated neurodegeneration. (M) Activation of Dpp signaling (GMR>Aβ42+<i>d</i><i>p</i><i>p</i>) can significantly rescue Aβ42 mediated neurodegeneration. However upregulation of (N) <i>N</i> (GMR>Aβ42+<i>N</i>), (O) optomotor blind (omb), (GMR>Aβ42+<i>o</i><i>m</i><i>b</i>) and (P) patched (GMR>Aβ42+<i>p</i><i>t</i><i>c</i>) did not rescue the neurodegenerative phenotype. The magnification of all brightfield images of the adult is 10X.</p

Ectopic induction of Tsh can rescue Aβ42 mediated neurodegeneration by blocking cell death.

<p>(A-C) Third instar eye-imaginal discs stained for 22C10 (marks the axonal sheath, in green channel), pan neural marker Elav (blue channel) and TUNEL that marks the nuclei of dying cells (red and split channels). (A, A') Wild-type eye imaginal disc showing random cell death in a few cells in the developing eye field, however, (B, B') the number of TUNEL positive dying cells nuclei increases dramatically in the GMR>Aβ42 background. (C, C') Targeted misexpression of <i>tsh</i> (GMR>Aβ42+<i>t</i><i>s</i><i>h</i>) significantly reduces the number of dying cell nuclei in the developing eye imaginal disc. (D) Quantitatively, the number of TUNEL cells have been counted and recorded with all five constructs shown. These phenotypes of enhancement of the neurodegenerative phenotype and rescue, based on the number of TUNEL positive cells, are significant as seen by the calculation of P-values based on the one-tailed t-test using Microsoft Excel 2010. Note that the number of dying cells increased more than three folds in the GMR>Aβ42 background as compared to the wild-type eye imaginal disc. The number of dying cells in GMR>Aβ42+<i>t</i><i>s</i><i>h</i> background is reduced to half as compared to GMR>Aβ42. Although the number of dying cells in GMR>Aβ42+<i>t</i><i>s</i><i>h</i> background it is still more than the wild-type eye disc. (E-G) Photoreceptor cells projections in third instar larva visualized using Chaoptin (MAb24B10, green channel) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080829#B65" target="_blank">65</a>] staining. MAb24B10 marks the retinal axons from the neural retina to the brain, and proneural marker Elav (red channel). (E') In the wild type larva, retinal axons projection pattern from the photoreceptors in the retina to the optic lobes in the brain. Note that ommatidial axonal bundle from eye field contacts the brain at two locations in a highly organized fashion in the wild type. (E', F') Misexpression of (F') Aβ42 (GMR>Aβ42) results in aberrant retinal axon targeting from the neural retina to the brain (E") as compared to the wild type. (G') Targeted misexpression of <i>tsh</i> in the Aβ42 background (GMR>Aβ42+ <i>tsh</i>) results in a strong restoration of retinal axonal targeting. The magnification of confocal images of (A-C) the Eye-antennal imaginal disc is 20X, and (E-G) the retinal axon is 60X.</p

Ectopic induction of Tsh expression can rescue Aβ42 mediated neurodegeneration.

<p>(A-C) Scanning Electron Micrographs (SEM) of the adult <i>Drosophila</i> eye, (A) Wild type, (B) Misexpression of Aβ42 (GMR>Aβ42) in the differentiating photoreceptors of the developing eye results in a highly reduced eye due to lack of ommatidia and glazed surface due to extensive neurodegeneration. (C) Targeted misexpression of <i>tsh</i> and Aβ42 (GMR>Aβ42+<i>t</i><i>s</i><i>h</i>) in the differentiating photoreceptors of the eye leads to a significant rescue of the Aβ42 mediated neurodegenerative phenotype. (D-F) The <i>tsh</i> transcriptional status [marked by β-galactosidase reporter (<i>lacZ</i>; green channel)] and Tsh protein (Rabit Anti-Tsh antibody, blue channel) levels were tested in different genetic backgrounds in the developing eye-imaginal disc. Pan neural marker Elav (red channel) marks the neuronal fate. (D, D') <i>tsh</i> reporter is localized in bands both posterior and anterior to the morphogenic furrow (MF) in disc proper, (D") whereas Tsh protein is mainly restricted anterior to the MF in disc proper. (E, E') In the GMR>Aβ42 background similar compartmental patterns of <i>tsh</i> transcription (E, E") were seen. However, (E") Tsh expression is reduced as compared to (D") its expression in the wild type eye. (F- F") Targeted misexpression of <i>tsh</i> and Aβ42 (GMR>Aβ42+<i>t</i><i>s</i><i>h</i>) in the GMR domain of the eye-imaginal disc (F') show no deviation in <i>tsh</i> transcription from wild type, however (F") strong induction of Tsh expression is seen in the GMR domain. (G-I) In developing pupal retina Tsh protein and pan neural marker Elav were seen. The developing photoreceptors (marked by Elav in the red channel) in the pupal retina are arranged in a highly organized fashion (G, G') which is severely disrupted in the GMR>Aβ42 construct with fusion of ommatidia (marked by white arrow heads) and gaping holes (as marked by the yellow outline). (I, I') In the GMR>Aβ42+<i>t</i><i>s</i><i>h</i> pupal retina, ommatidial organization is restored as distinct ommatidial clusters are seen and no ommatidial fusion was seen (H') as compared to GMR>Aβ42. (G, G") Tsh is present in the developing ommatidia but did not show strong nuclear localization in the GMR>Aβ42 retina. (I, I") Strong induction of Tsh is present in both primary and secondary cells of the retina in the GMR>Aβ42+<i>t</i><i>s</i><i>h</i> construct. The magnification of (A-F) SEM micrographs of the adult eye is 180X, and confocal images of (D-F) the eye imaginal disc is 20X and (G-I) the pupal retina is 40X.</p

A Novel Use of Gentamicin in the ROS-Mediated Sensitization of NCI-H460 Lung Cancer Cells to Various Anticancer Agents

Aminoglycosides are broad-spectrum antibiotics that are used for the treatment of severe Gram-negative and Gram-positive bacterial infections. While bactericidal effects of aminoglycosides are due to binding to the 30S subunit of the bacterial ribosome, aminoglycosides can affect protein synthesis, intracellular calcium levels, and levels of reactive oxygen species (ROS) in eukaryotic cells. While aminoglycosides can be cytotoxic at high concentrations, our results show that at much lower doses, gentamicin can be implemented as a sensitizing agent for the NSCLC cell line NCI-H460, increasing the efficacy of camptothecin, digitoxin, and vinblastine <i>in vitro</i>. We have also established that this sensitization is reliant on the ROS response generated by gentamicin

Characterization of a morphogenetic furrow specific Gal4 driver in the developing <i>Drosophila</i> eye

<div><p>The ability to express a gene of interest in a spatio-temporal manner using Gal4-UAS system has allowed the use of <i>Drosophila</i> model to study various biological phenomenon. During <i>Drosophila</i> eye development, a synchronous wave of differentiation called Morphogenetic furrow (MF) initiates at the posterior margin resulting in differentiation of retinal neurons. This synchronous differentiation is also observed in the differentiating retina of vertebrates. Since MF is highly dynamic, it can serve as an excellent model to study patterning and differentiation. However, there are not any Gal4 drivers available to observe the gain- of- function or loss- of- function of a gene specifically along the dynamic MF. The <i>decapentaplegic (dpp)</i> gene encodes a secreted protein of the transforming growth factor-beta (TGF-beta) superfamily that expresses at the posterior margin and then moves with the MF. However, unlike the MF associated pattern of <i>dpp</i> gene expression, the targeted <i>dpp</i>-Gal4 driver expression is restricted to the posterior margin of the developing eye disc. We screened GMR lines harboring regulatory regions of <i>dpp</i> fused with Gal4 coding region to identify MF specific enhancer of <i>dpp</i> using a GFP reporter gene. We employed immuno-histochemical approaches to detect gene expression. The rationale was that GFP reporter expression will correspond to the <i>dpp</i> expression domain in the developing eye. We identified two new <i>dpp-</i>Gal4 lines, <i>viz</i>., <i>GMR17E04</i>-Gal4 and <i>GMR18D08</i>-Gal4 that carry sequences from first intron region of <i>dpp</i> gene. <i>GMR17E04</i>-Gal4 drives expression along the MF during development and later in the entire pupal retina whereas <i>GMR18D08</i>-Gal4 drives expression of GFP transgene in the entire developing eye disc, which later drives expression only in the ventral half of the pupal retina. Thus, <i>GMR18D08</i>-Gal4 will serve as a new reagent for targeting gene expression in the ventral half of the pupal retina. We compared misexpression phenotypes of Wg, a negative regulator of eye development, using <i>GMR17E04</i>-Gal4, <i>GMR18D08</i>-Gal4 with existing <i>dpp</i>-Gal4 driver. The eye phenotypes generated by using our newly identified MF specific driver are not similar to the ones generated by existing <i>dpp</i>-Gal4 driver. It suggests that misexpression studies along MF needs revisiting using the new Gal4 drivers generated in our studies.</p></div

Schematic presentation of <i>dpp</i> enhancer and CRM lines using GMR collections.

<p>The two newly identified <i>dpp</i> CRM lines are <i>GMR18D08</i>, <i>GMR17E04</i>[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196365#pone.0196365.ref056" target="_blank">56</a>] are in one big intron region in <i>dpp</i> gene sequence of <i>Drosophila melanogaster’s</i> 2L chromosome. Both of these CRM drive expression in the developing eye. These CRMs do not overlap with each other and are present upstream of known <i>dpp</i> CRM element(s) shown in BS 3.0 lacZ (GenBank U63852.1) in 3’ region of disk <i>dpp</i>.</p