In Vivo Plant Bio-Electrochemical Sensor Using Redox Cycling

This work presents an in vivo stem-mounted sensor for Nicotiana tabacum plants and an in situ cell suspension sensor for Solanum lycopersicum cells. Stem-mounted sensors are mechanically stable and less sensitive to plant and air movements than the previously demonstrated leaf-mounted sensors. Interdigitated-electrode-arrays with a dual working electrode configuration were used with an auxiliary electrode and an Ag/AgCl quasi-reference electrode. Signal amplification by redox cycling is demonstrated for a plant-based sensor responding to enzyme expression induced by different cues in the plants. Functional biosensing is demonstrated, first for constitutive enzyme expression and later, for heat-shock-induced enzyme expression in plants. In the cell suspension with redox cycling, positive detection of the enzyme β-glucuronidase (GUS) was observed within a few minutes after applying the substrate (pNPG, 4-Nitrophenyl β-D-glucopyranoside), following redox reactions of the product (p-nitrophenol (pNP)). It is assumed that the initial reaction is the irreversible reduction of pNP to p-hydroxylaminophenol. Next, it can be either oxidized to p-nitrosophenol or dehydrated and oxidized to aminophenol. Both last reactions are reversible and can be used for redox cycling. The dual-electrode redox-cycling electrochemical signal was an order of magnitude larger than that of conventional single-working electrode transducers. A simple model for the gain is presented, predicting that an even larger gain is possible for sub-micron electrodes. In summary, this work demonstrates, for the first time, a redox cycling-based in vivo plant sensor, where diffusion-based amplification occurs inside a tobacco plant’s tissue. The technique can be applied to other plants as well as to medical and environmental monitoring systems

Parkin Promotes Degradation of the Mitochondrial Pro-Apoptotic ARTS Protein

<div><p>Parkinson’s disease (PD) is associated with excessive cell death causing selective loss of dopaminergic neurons. Dysfunction of the Ubiquitin Proteasome System (UPS) is associated with the pathophysiology of PD. Mutations in Parkin which impair its E3-ligase activity play a major role in the pathogenesis of inherited PD. ARTS (Sept4_i2) is a mitochondrial protein, which initiates caspase activation upstream of cytochrome c release in the mitochondrial apoptotic pathway. Here we show that Parkin serves as an E3-ubiquitin ligase to restrict the levels of ARTS through UPS-mediated degradation. Though Parkin binds equally to ARTS and Sept4_i1 (H5/PNUTL2), the non-apoptotic splice variant of <em>Sept4</em>, Parkin ubiquitinates and degrades only ARTS. Thus, the effect of Parkin on ARTS is specific and probably related to its pro-apoptotic function. High levels of ARTS are sufficient to promote apoptosis in cultured neuronal cells, and rat brains treated with 6-OHDA reveal high levels of ARTS. However, over-expression of Parkin can protect cells from ARTS-induced apoptosis. Furthermore, Parkin loss-of-function experiments reveal that reduction of Parkin causes increased levels of ARTS and apoptosis. We propose that in brain cells in which the E3-ligase activity of Parkin is compromised, ARTS levels increase and facilitate apoptosis. Thus, ARTS is a novel substrate of Parkin. These observations link Parkin directly to a pro-apoptotic protein and reveal a novel connection between Parkin, apoptosis, and PD.</p> </div

UPS mediated degradation of ARTS by Parkin protects cells from apoptosis.

<p><b>A. and B.</b> Parkin inhibits apoptosis induced by ARTS. COS-7 cells were co-transfected with 1myc-Parkin and 6myc-ARTS. Over-expression of ARTS alone was sufficient to induce apoptosis in these cells. Apoptosis is exhibited by the presence of the apoptotic markers cleaved caspase-3 (A) and H2AX (B). Thus, overexpression of Parkin can strongly inhibit ARTS-induced apoptosis. <b>C.</b> Co-transfection of 1myc-Parkin and AU5-ARTS confers a protective effect against cell death. Cell viability was quantified using XTT-based assay (see Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038837#s2" target="_blank">Methods</a>). The results are represented as a ratio between each transfection and the negative control (mean± SE, n = 6). The viability of cells transfected with empty vector was defined as the baseline viability of the assay. Cell viabilities were recorded in relation to this baseline. <b>D.</b> ARTS binds to Parkin both at the mitochondria and in the cytosol. Sub cellular fractionation was performed using 140 µg/ml digitonin. Apoptosis was induced using 1.5 µM STS for 2 h. Immunoprecipitation (IP) of ARTS from both cytosolic and mitochondrial fractions revealed basal binding of ARTS to Parkin under non apoptotic as well as under apoptotic conditions.</p

Up-regulation of ARTS levels is associated with induction of apoptosis in SH-SY5Y cells and in 6-OHDA treated rat brains.

<p>A. Western blot (WB) analysis shows a strong increase in the expression levels of ARTS upon treatment of SH-SY5Y cells with the apoptotic inducer staurosporine (STS). This elevation in levels of ARTS is associated with an increase in caspase-3 activation. GAPDH was used as a loading control. B. A significant increase in levels of ARTS is seen in response to treatment with etoposide inducing apoptosis in SH-SY5Y cells. This strong up-regulation of ARTS is associated with a corresponding increase in expression of the apoptotic marker H2AX. Actin was used as a loading control. C. Endogenous ARTS binds to XIAP in SH-SY5Y cells. Immunoprecipitation assay (IP) with monoclonal anti ARTS antibody was performed on lysates from SH-SY5Y cells. Mouse IgG served as control for co-precipitation of the specific antigen. DI. A representative picture (viewed with x40 objective) showing immunofluorescence (IF) staining using anti-ARTS antibody in <i>Substantia nigra pars compacta</i> (SNpc) of rat brain treated with 6-hydroxy dopamine (6-OHDA) as compared to control injected with saline. 6-OHDA was injected into the left medial forebrain bundle of these rats, from where it is transported to the SN. A significant increase in expression of ARTS is shown in these cells (red). DAPI staining of nuclei is shown in blue. Scale bar represents 50 µm. DII. Co-localization of ARTS (red) and cleaved caspase-3 (green) is seen in a representative rat brain section of 6-OHDA treated rat viewed using x20 objective. Scale bar represents 100 µm. DIII, DIV. Percent of ARTS/cleaved caspase-3 and ARTS/TUNEL double positive cells in SNpc of 6-OHDA treated rats compared to saline injected controls. These pictures demonstrate the levels of ARTS among apoptotic neurons. Counts were done in sections from brains after one, three and seven days following injection of 6-OHDA or saline. Figures present mean ± SEM of the ratio between the percent of double positive cells in 6-OHDA brains relative to the average percent of double positive cells in saline treated brains, at each time point. A significant increase in percentage of ARTS/caspase-3 and ARTS/TUNEL double positive SN neurons is seen 24 hours after injection of 6-OHDA as compared to controls (significance levels were calculated from data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038837#pone.0038837.s003" target="_blank">Table S1</a>; **P<0.01, ***P<0.001 for difference from same day control by ANOVA with Bonferroni post hoc test). This suggests that ARTS may play an important role in promoting susceptibility to 6-OHDA-induced apoptosis in SN neurons.</p

Although Parkin can interact with both ARTS and Sept4_i1, it selectively ubiquitinates only ARTS. AI, II

<p>. Parkin promotes degradation of ARTS through the Ubiquitin Proteasome System (UPS). COS-7 cells co-transfected with 1myc-Parkin and AU5-ARTS were treated with the proteasome inhibitor, MG132. Expression levels of ARTS were significantly decreased in the presence of Parkin, and were restored upon addition of MG132. <b>AII.</b> Densitometry analysis of WB results shown in AI. Protein levels were normalized to actin. Graph presents mean ± SEM of the densitometry values relative to actin levels. <b>B.</b> Parkin serves as an E3-ligase for ARTS. COS-7 cells co-transfected with 1myc-Parkin, AU5-ARTS and HA-8xubiquitin were subjected to in vivo ubiquitination assay followed by immunoprecipitation (IP) with either monoclonal anti-ARTS antibody or anti-Sept4_i1 antibody. Non-transfected cells and cells transfected with only 1myc-Parkin or AU5-ARTS served as controls. ARTS is ubiquitinated in the presence of Parkin. <b>C.</b> Parkin binds both ARTS and Sept4_i1. COS-7 cells were co-transfected with 1myc-Parkin together with Flag-Sept4_i1 or AU5-ARTS. Lysates were subjected to IP with anti-Parkin antibody followed by WB analysis. <b>D.</b> COS-7 cells treated with the proteasome inhibitor, MG132 were co-transfected with 1myc-Parkin or mutant Parkin T240R together with 6myc-ARTS or Flag -Sept4_i1. Immunoprecipitation was done with either anti-ARTS or anti-Sept4_i1 antibody followed by <i>In vivo</i> ubiquitination assay. Western blot analysis was performed with anti-ubiquitin antibody (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038837#s2" target="_blank">methods</a>). Parkin selectively ubiquitinates ARTS but not Sept4_i1. Mutant T240R Parkin lacking E3-ligase activity, demonstrates markedly decreased ability to ubiquitinate ARTS as compared to wt Parkin. <b>E</b>. COS-7 cells were transiently transfected with ARTS, Parkin, and the mutant Parkin T240R with compromised E3-ligase activity. The levels of cleaved caspase-3 (cCasp3) representing rate of apoptosis were visualized using western blot analyses. Co-transfection of COS-7 cells with ARTS and Parkin resulted in ARTS degradation and apoptotic suppression, as visualized by the absence of cleaved caspase-3 (cCasp3). In contrast, co-transfection with ARTS and mutant Parkin T240R restored apoptosis in these cells.</p

ARTS and Parkin exhibit co-localization in SH-SY5Y cells.

<p><b>A.</b> Immunofluorescence (IF) assay was performed on SH-SY5Y cells, which were transiently co-transfected with AU5-ARTS (red) and 1myc-Parkin (green) and treated with 1.5 µM STS. These IF results reveal co-localization of ARTS and Parkin in untreated cells, with increased co-localization as early as 30 min after apoptotic induction. <b>B.</b> Working model for the role of ARTS in healthy Parkin expressing cells, and in cells containing mutated or dysfunctional Parkin. We propose that in healthy brain cells which contain intact Parkin, the levels of ARTS are kept low through constant down regulation by Parkin. Upon initiation of a stress or apoptotic stimuli, recruitment of Parkin to dysfunctional mitochondria occurs as well as translocation of ARTS to the cytosol at the early stages of apoptosis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038837#pone.0038837-Edison2" target="_blank">[40]</a>. This may allow the binding and degradation of ARTS by Parkin and help maintain low levels of ARTS. This down-regulation of ARTS is expected to promote cell survival by preventing the inhibition of XIAP and caspase activation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038837#pone.0038837-Gottfried1" target="_blank">[33]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038837#pone.0038837-Edison2" target="_blank">[40]</a>. According to our model, in brain cells containing mutated or dysfunctional Parkin, Parkin can no longer degrade ARTS. This leads to accumulation of ARTS and initiation of apoptosis through de-repression of caspases by antagonizing XIAP. This first wave of caspases leads to Mitochondrial Outer membrane Permeabilization (MOMP) which results in release of mitochondrial factors residing in the inner membrane space (IMS) such as Cytochrome c and Smac/Diablo, causing amplified caspase activation and cell death <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038837#pone.0038837-Edison2" target="_blank">[40]</a>.</p

Parkin binds to ARTS and reduces its expression levels through the Ubiquitin-Proteasome System (UPS). A.B.

<p>Both exogenous and endogenous Parkin and ARTS bind to each other. COS-7 cells were co-transfected with AU5-ARTS (32 kD) and myc-Parkin (52 kD). Lysates were subjected to IP with anti-ARTS (I) and anti-myc (II) and then to WB analysis. These results show that exogenous ARTS and Parkin can bind to each other. <b>C</b>. Lysates of MEFs were subjected to IP with monoclonal anti-ARTS antibody. These results show that endogenous ARTS and Parkin can bind to each other. <b>D.E.</b> Parkin down-regulates exogenous and endogenous expression levels of ARTS. <b>D</b>. The expression levels of transfected AU5-ARTS and Parkin were determined in COS-7 cells, which contain low levels of endogenous ARTS. Lysates were subjected to WB analysis with monoclonal anti-ARTS and anti-Parkin antibodies. Transfection of 1myc-Parkin dramatically reduces the levels of ARTS in these cells. <b>E.</b> Endogenous levels of ARTS were determined in HeLa cells, which contain relatively high endogenous levels of ARTS. Lysates were subjected to WB analysis with monoclonal anti-ARTS and anti-Parkin antibodies. Transfection of Parkin dramatically reduces the endogenous levels of ARTS in these cells. <b>F.</b> Parkin knocked -down SH-SY5Y cells exhibit increased levels of ARTS and elevated apoptosis. A stable Parkin knockdown (Parkin KD) cell line of neuroblastoma SH-SY5Y in which Parkin expression was knocked down by short hairpin RNAs (shRNAs) was established. These Parkin KD cells were treated with 1.5 µM of STS for the indicated time periods. These Parkin KD cells exhibited elevated levels of ARTS and increased levels of apoptosis as determined by increased levels of two different markers of apoptosis, cleaved caspase-3(cCasp3), and its substrate, cleaved PARP (cPARP). <b>GI, II.</b> Parkin specifically reduces the levels of ARTS but not the non-apoptotic Sept4_i1 splice variant. COS-7 cells were co-transfected with 1myc-Parkin together with Flag-Sept4_i1 or 6myc-ARTS. The levels of ARTS but not Sept4_i1 were strongly down-regulated by Parkin. <b>GII.</b> Densitometry analysis of Western blot results shown in Fig GI. Protein levels were normalized with respect to actin. Graph presents mean ± SEM of the densitometry values relative to actin levels.</p

Newly Diagnosed Crohn’s Disease Patients in India and Israel Display Distinct Presentations and Serological Markers: Insights from Prospective Cohorts

Background: Crohn’s disease (CD) incidence is rising in India. However, features of newly diagnosed patients with CD in this population are largely unknown. The Indo-Israeli IBD GastroEnterology paRtnership (TiiiGER) aimed to investigate differences in presentation among patients with newly diagnosed CD in India and Israel, and to explore phenotype–serotype correlations. Methods: A prospective observational cohort study of consecutive adults (>18 years) conducted in two large referral centers in India and Israel (2014–2018). Clinical data, an antiglycan serological panel, and 20 CD-associated genetic variants were analyzed. Outcomes: complicated phenotype at diagnosis and early complicated course (hospitalizations/surgeries) within 2 years of diagnosis. Results: We included 260 patients (104, Indian (65.4%, male; age, 37.8); 156 Israeli (49.4%, male; 31.8, age)). Median lag time from symptoms onset to diagnosis was 10.5 (IQR 3–38) vs. 3 (IQR 1–8) months in Indian vs. Israeli patients (p p = 0.003). Complicated phenotype was associated with higher anti-Saccharomyces cerevisiae antibody (ASCA) seropositivity rate among Israeli patients (p p = 0.152). Antiglycan serology did not correlate with a complicated early course in either cohort. Conclusions: There are significant differences in patients presenting with newly diagnosed CD in India and Israel, including phenotype and distinct biomarkers at diagnosis. These differences suggest different genetic and environmental disease modifiers