Deuterated Clopidogrel Analogues as a New Generation of Antiplatelet Agents

Clopidogrel (CPG) is an antithrombotic prodrug that needs hepatic cytochrome P450 (CYP) enzymes for its bioactivation. The clinical effects of CPG have been associated with high intersubject variability and a certain level of resistance. Recently, comprehensive biotransformation studies of CPG support that the observed clinical uncertainty stems from the low bioactivation efficiency, which is attributed to extensive attritional metabolism (e.g., hydrolysis of the methyl ester functionality and oxidation of the piperidine moiety). With the goal of potentiating the desired thiophene 2-oxidation through minimal structural modification, we have adopted the strategy of <i>targeted metabolism shift</i> and have designed and synthesized deuterated piperidine analogues of CPG. In vitro studies showed that the prodrug activation percentages have been significantly increased for the deuterated analogues as a result of stability enhancement of the piperidine moiety. In a pharmacological study with a rat model, oral administration of the deuterated analogues also demonstrated higher inhibitory activity than that of CPG against adenosine diphosphate (ADP) induced platelet aggregation. These deuterated analogues represent a new generation of antiplatelet agents with the potential to overcome the major clinical drawbacks of CPG

sj-pdf-1-imr-10.1177_03000605221123882 - Supplemental material for Refractory postherpetic neuralgia in a multiple myeloma patient with lenalidomide maintenance therapy: a case report

Supplemental material, sj-pdf-1-imr-10.1177_03000605221123882 for Refractory postherpetic neuralgia in a multiple myeloma patient with lenalidomide maintenance therapy: a case report by Xueqin Cao, Gang Wu, Bo Jiao and Xianwei Zhang in Journal of International Medical Research</p

Additional file 1 of Evaluation of aphid resistance on different rose cultivars and transcriptome analysis in response to aphid infestation

Supplementary Material 1

Expression of SA-related genes in transgenic <i>Arabidopsis</i> overexpressed <i>PtrWRKY89</i> after inoculation of <i>Pst</i>DC3000.

<p><i>ICS1</i>: <i>ISOCHORISMATE SYNTHASE 1</i>, <i>PAD4</i>: <i>PHYTOALEXIN DEFICIENT 4</i>, <i>EDS1</i>: <i>ENHANCED DISEASE SUSCEPTIBILITY 1</i>, <i>MYB44</i>: <i>ARABIDOPSIS THALIANA MYB DOMAIN PROTEIN 44</i>, <i>NPR1</i>: <i>NONEXPRESSER OF PR GENES 1</i>, <i>CPR5</i>: <i>CONSTITUTIVE EXPRESSION OF PR GENES 5</i>, <i>PR1/PR2/PR5</i>: <i>PATHOGENESIS RELATED GENES 1/2/5</i>. <i>Arabidopsis UBC</i> gene was used as an internal control.</p

Constitutive expressing <i>PtrWRKY89</i> in <i>Arabidopsis</i> plants showing susceptibility to <i>Botrytis cinerea</i>.

<p>(A) Disease response of inoculated plants at 7 days after <i>B</i>. <i>cinerea</i> infection. (B) Transcript accumulation of <i>B</i>. <i>cinerea Actin</i> gene in these inoculated plants. <i>Arabidopsis UBC</i> gene was used as an internal control. Values represent means of three replicates and error bars indicated standard deviation. Two asterisks indicated a statistically significant difference between WT and transgenic plants (**, <i>P</i><0.01 by Student’s <i>t</i> test).</p

Overexpression of Poplar <i>PtrWRKY89</i> in Transgenic <i>Arabidopsis </i>Leads to a Reduction of Disease Resistance by Regulating Defense-Related Genes in Salicylate- and Jasmonate-Dependent Signaling

<div><p>The plant hormones jasmonic acid (JA) and salicylic acid (SA) play key roles in plant defenses against pathogens and several WRKY transcription factors have been shown to have a role in SA/JA crosstalk. In a previous study, overexpression of the poplar WRKY gene <i>PtrWRKY89</i> enhanced resistance to pathogens in transgenic poplars. In this study, the promoter of <i>PtrWRKY89</i> (<i>ProPtrWRKY89</i>) was isolated and used to drive <i>GUS</i> reporter gene. High GUS activity was observed in old leaves of transgenic <i>Arabidopsis</i> containing <i>ProPtrWRKY89-GUS</i> construct and <i>GUS</i> expression was extremely induced by SA solution and SA+MeJA mixture but not by MeJA treatment. Subcellular localization and transactivation assays showed that PtrWRKY89 acted as a transcription activator in the nucleus. Constitutive expression of <i>PtrWRKY89</i> in <i>Arabidopsis</i> resulted in more susceptible to <i>Pseudomonas syringae</i> and <i>Botrytis cinerea</i> compared to wild-type plants. Quantitative real-time PCR (qRT-PCR) analysis confirmed that marker genes of SA and JA pathways were down-regulated in transgenic <i>Arabidopsis</i> after pathogen inoculations. Overall, our results indicated that PtrWRKY89 modulates a cross talk in resistance to <i>P</i>. <i>syringe</i> and <i>B</i>. <i>cinerea</i> by negatively regulating both SA and JA pathways in <i>Arabidopsis</i>.</p></div

<i>PtrWRKY89</i> overexpressing <i>Arabidopsis</i> plants showed more susceptible to <i>Pst</i>DC3000 than wild-type plants.

<p>(A) Disease symptoms of wild-type (WT) and transgenic plants after 3 days of <i>Pst</i>DC3000 infection. (B) The quantification of total chlorophyll content in transgenic and WT plants at 3 days post infection. (C) Growth of <i>Pst</i>DC3000 in <i>planta</i> 3 days after inoculation. Values represent means of three replicates. Error bars indicate standard deviation. Asterisks indicate a statistically significant difference between WT and transgenic plants (*, <i>P</i><0.05 by Student’s <i>t</i> test).</p

Expression analysis of the <i>PtrWRKY89</i> promoter.

<p>(A) The promoter of <i>PtrWRKY89</i> was cloned and ligated into vector pCXGUS-P to drive <i>GUS</i> expression and the resulting construct was introduced into <i>A</i>. <i>thaliana</i>. Transgenic seedlings were grown on MS media and then transplanted in soil for GUS staining. <i>GUS</i> expression was observed in various tissues of transgenic plants, including (B) flowers, (C) siliques, (D) 5-week-old seedlings, (E) old leaves, (F) young leaves and (G) roots.</p

Expression patterns of the <i>PtrWRKY89</i> promoter driven <i>GUS</i> gene in response to salicylic acid (SA), methyl jasmonate (MeJA) with low concentration and their mixed solution, respectively.

<p>SA solution (100 μΜ), MeJA solution (100 μΜ) and SA+MeJA mixture (100 μΜ + 100 μΜ) were sprayed onto the surface of two-week-old transgenic <i>Arabidopsis</i> seedling containing the <i>ProPtrWRKY89-GUS</i> construct. The control plants (CK) were treated by H₂O. After treatments of 24 h, the seedlings were collected from MS medium. (A) The GUS staining of the seedlings. (B) The transcript profiles of <i>PtrWRKY89</i> were analyzed by quantitative RT-PCR (qRT-PCR). Error bars were obtained from three biological replicates. <i>Arabidopsis UBC</i> (AT5G25760) expression was used as a control and gene-specific primers were used for qRT-PCR analysis were described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149137#pone.0149137.s005" target="_blank">S1 Table</a>. Two asterisks indicate a statistically significant difference by Student's <i>t</i>-test (**, <i>P</i> < 0.01).</p

Subcellular localization and transactivation assay of PtrWRKY89.

<p>(A) <i>PtrWRKY89</i> was ligated into pCX-DG vector to generate <i>GFP</i>:<i>PtrWRKY89</i> construct. The resulting construct and empty vectors were transformed into epidermal cells of onion (<i>Allium cepa</i>) and stained with DAPI, respectively. The fusion protein displayed its localization to the cell nucleus as manifested by GFP (lower column) and <i>GFP</i> driven by the <i>CaMV 35</i> promoter was localized to both the cytoplasm and the cell nucleus (upper column). Overlay and bright field images of the epidermal cells were also shown. (B) <i>PtrWRKY89</i> was cloned into pGBKT7 vector with DNA binding domain of GAL4 and introduced into Gold2 yeast cells. The transformants were grown on SD medium lacking tryptophan (Try) for the sake of positive clone selection and then on SD medium without Try, histidine (His) and adenine (Ade) for the transactivation assay. The clones grown on SD (-Try/His/Ade) were stained by X-α-gal. GAL4-BD (empty vector) was a negative control.</p