Cell- and Tissue-Specific Transcriptome Analyses of Medicago truncatula Root Nodules

Legumes have the unique ability to host nitrogen-fixing Rhizobium bacteria as symbiosomes inside root nodule cells. To get insight into this key process, which forms the heart of the endosymbiosis, we isolated specific cells/tissues at different stages of symbiosome formation from nodules of the model legume Medicago truncatula using laser-capture microdissection. Next, we determined their associated expression profiles using Affymetrix Medicago GeneChips. Cells were collected from the nodule infection zone divided into a distal (where symbiosome formation and division occur) and proximal region (where symbiosomes are mainly differentiating), as well as infected cells from the fixation zone containing mature nitrogen fixing symbiosomes. As non-infected cells/tissue we included nodule meristem cells and uninfected cells from the fixation zone. Here, we present a comprehensive gene expression map of an indeterminate Medicago nodule and selected genes that show specific enriched expression in the different cells or tissues. Validation of the obtained expression profiles, by comparison to published gene expression profiles and experimental verification, indicates that the data can be used as digital “in situ”. This digital “in situ” offers a genome-wide insight into genes specifically associated with subsequent stages of symbiosome and nodule cell development, and can serve to guide future functional studies

Paracetamol in therapeutic dosages and acute liver injury: causality assessment in a prospective case series

Background: Acute liver injury (ALI) induced by paracetamol overdose is a well known cause of emergency hospital admission and death. However, there is debate regarding the risk of ALI after therapeutic dosages of the drug. The aim is to describe the characteristics of patients admitted to hospital with jaundice who had previous exposure to therapeutic doses of paracetamol. An assessment of the causality role of paracetamol was performed in each case. Methods: Based on the evaluation of prospectively gathered cases of ALI with detailed clinical information, thirty-two cases of ALI in non-alcoholic patients exposed to therapeutic doses of paracetamol were identified. Two authors assessed all drug exposures by using the CIOMS/RUCAM scale. Each case was classified into one of five categories based on the causality score for paracetamol. Results: In four cases the role of paracetamol was judged to be unrelated, in two unlikely, and these were excluded from evaluation. In seven of the remaining 26 cases, the RUCAM score associated with paracetamol was higher than that associated with other concomitant medications. The estimated incidence of ALI related to the use of paracetamol in therapeutic dosages was 0.4 per million inhabitants older than 15 years of age and per year (99%CI, 0.2-0.8) and of 10 per million paracetamol users-year (95% CI 4.3-19.4). Conclusions:Our results indicate that paracetamol in therapeutic dosages may be considered in the causality assessment in non-alcoholic patients with liver injury, even if the estimated incidence of ALI related to paracetamol appears to be low

The Cytosolic Nucleoprotein of the Plant-Infecting Bunyavirus Tomato Spotted Wilt Recruits Endoplasmic Reticulum–Resident Proteins to Endoplasmic Reticulum Export Sites

In contrast with animal-infecting viruses, few known plant viruses contain a lipid envelope, and the processes leading to their membrane envelopment remain largely unknown. Plant viruses with lipid envelopes include viruses of the Bunyaviridae, which obtain their envelope from the Golgi complex. The envelopment process is predominantly dictated by two viral glycoproteins (Gn and Gc) and the viral nucleoprotein (N). During maturation of the plant-infecting bunyavirus Tomato spotted wilt, Gc localizes at endoplasmic reticulum (ER) membranes and becomes ER export competent only upon coexpression with Gn. In the presence of cytosolic N, Gc remains arrested in the ER but changes its distribution from reticular into punctate spots. Here, we show that these areas correspond to ER export sites (ERESs), distinct ER domains where glycoprotein cargo concentrates prior to coat protein II vesicle–mediated transport to the Golgi. Gc concentration at ERES is mediated by an interaction between its cytoplasmic tail (CT) and N. Interestingly, an ER-resident calnexin provided with Gc-CT was similarly recruited to ERES when coexpressed with N. Furthermore, disruption of actin filaments caused the appearance of a larger amount of smaller ERES loaded with N-Gc complexes, suggesting that glycoprotein cargo concentration acts as a trigger for de novo synthesis of ERE

Fate map of Medicago truncatula root nodules

Legume root nodules are induced by N-fixing rhizobium bacteria that are hosted in an intracellular manner. These nodules are formed by reprogramming differentiated root cells. The model legume Medicago truncatula forms indeterminate nodules with a meristem at their apex. This organ grows by the activity of the meristem that adds cells to the different nodule tissues. In Medicago sativa it has been shown that the nodule meristem is derived from the root middle cortex. During nodule initiation, inner cortical cells and pericycle cells are also mitotically activated. However, whether and how these cells contribute to the mature nodule has not been studied. Here, we produce a nodule fate map that precisely describes the origin of the different nodule tissues based on sequential longitudinal sections and on the use of marker genes that allow the distinction of cells originating from different root tissues. We show that nodule meristem originates from the third cortical layer, while several cell layers of the base of the nodule are directly formed from cells of the inner cortical layers, root endodermis and pericycle. The latter two differentiate into the uninfected tissues that are located at the base of the mature nodule, whereas the cells derived from the inner cortical cell layers form about eight cell layers of infected cells. This nodule fate map has then been used to re-analyse several mutant nodule phenotypes. This showed, among other things, that intracellular release of rhizobia in primordium cells and meristem daughter cells are regulated in a different manner

Nod factor receptors form heteromeric complexes and are essential for intracellular infection in Medicago nodules

Rhizobial Nod factors are the key signaling molecules in the legume-rhizobium nodule symbiosis. In this study, the role of the Nod factor receptors NOD FACTOR PERCEPTION (NFP) and LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) in establishing the symbiotic interface in root nodules was investigated. It was found that inside Medicago truncatula nodules, NFP and LYK3 localize at the cell periphery in a narrow zone of about two cell layers at the nodule apex. This restricted accumulation is narrower than the region of promoter activity/mRNA accumulation and might serve to prevent the induction of defense-like responses and/or to restrict the rhizobium release to precise cell layers. The distal cell layer where the receptors accumulate at the cell periphery is part of the meristem, and the proximal layer is part of the infection zone. In these layers, the receptors can most likely perceive the bacterial Nod factors to regulate the formation of symbiotic interface. Furthermore, our Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicates that NFP and LYK3 form heteromeric complexes at the cell periphery in M. truncatula nodules

Long-term therapy with nevirapine and tropism

Objective: A more potent effect on the residual viraemia was ascribed to nevirapine (NVP) with respect to other antiretroviral drugs; moreover a selection of X4 strains was described in patients (pts) with undetectable viraemia; our aim was to study viro-immunological parameters and tropism for co-receptor in pts on a long term successful therapy with NVP. Methods: 14 pts on HAART from 130 months (GL, median value, range 118–156 months) without occurrence of blips, as assessable with the available methods at that time, were retrospectively selected from a single center cohort (Bolzano). Tropism for V3 was determined by population sequencing on blood, and using geno2pheno algorithm; cellular HIV DNA load was analysed by in-house Real-Time. A further eighteen months (mo) follow up was then observed. Data were compared with those obtained from a control group of 50 naïve pts (GS), evaluated after a 36-mo successful therapy (median, range 12–84) with various drug combinations, with median baseline (BL) CD4 of 50/μl, comparable value with the GL cohort. Results: In 7 pts a R5-tropic (GLR5, FPR median 84.8%) and in 7 an X4-tropic strain (GLX4, FPR median 1.1%) was demonstrated. BL data of GLR5 were 46 y old, CD4 54/l, HIV-RNA 104,000 cps/ml; HAART from 142 mo, with NVP from 125 (one after 70 mo on NVP switched to protease from 57); at follow up CD4 were 679/l, HIV-DNA 60 cps/106 PBMCs (range<5–252). GLX4 were 46 y, at BL 38 CD4/l, HIV-RNA 250,000 cps/ml; in HAART from 121 mo, with NVP from 97; at follow up CD4 902/l, HIV-DNA 60 cps/106 PBMCs (range<5–225). Six out of seven pts of the two groups were on treatment with abacavir+lamivudine (ABC+3TC) and one with tenofovir+emtricitabine. In the subsequent 18 mo four blips were observed (21–71 cps/ml); the backbone was changed to raltegravir in two GLR5 and one GLX4 for convenience. In the 50 GS pts at follow up an X4 strain was found in 50% of 14 efavirenz-treated, in 16% of 6 NVP, and 63% of 30 protease. Discussion: In a group of very long-term treated pts with NVP plus two NRTI (ABC+3TC in 12 out of 14), a tropism for CXCR4 was demonstrated in 50%, without significant differences in the CD4 gain and in the HIV-DNA load archived in the peripheral blood. With respect to pts on various therapies from a median of 36 mo, the type of archived virus does not seem to have a role on the outcome of a very long therapy, 130 mo, with NVP+ABC+3TC; this therapy does not seem able to select a special tropism in pts