Quantifying the effect of soil moisture content on segmenting root system architecture in X-ray computed tomography images

Aims

A commonly accepted challenge when visualising plant roots in X-ray micro Computed Tomography (μCT) images is the similar X-ray attenuation of plant roots and soil phases. Soil moisture content remains a recognised, yet currently uncharacterised source of segmentation error. This work sought to quantify the effect of soil moisture content on the ability to segment roots from soil in μCT images.

Methods

Rice (Oryza sativa) plants grown in contrasting soils (loamy sand and clay loam) were μCT scanned daily for nine days whilst drying from saturation. Root volumes were segmented from μCT images and compared with volumes derived by root washing.

Results

At saturation the overlapping attenuation values of root material, water-filled soil pores and soil organic matter significantly hindered segmentation. However, in dry soil (ca. six days of drying post-saturation) the air-filled pores increased image noise adjacent to roots and impeded accurate visualisation of root material. The root volume was most accurately segmented at field capacity.

Conclusions

Root volumes can be accurately segmented from μCT images of undisturbed soil without compromising the growth requirements of the plant providing soil moisture content is kept at field capacity. We propose all future studies in this area should consider the error associated with scanning at different soil moisture contents.     

Diacylglycerol Kinase δ Modulates Akt Phosphorylation through Pleckstrin Homology Domain Leucine-rich Repeat Protein Phosphatase 2 (PHLPP2)

Discovering proteins that modulate Akt signaling has become a critical task, given the oncogenic role of Akt in a wide variety of cancers. We have discovered a novel diacylglycerol signaling pathway that promotes dephosphorylation of Akt. This pathway is regulated by diacylglycerol kinase δ (DGKδ). In DGKδ-deficient cells, we found reduced Akt phosphorylation downstream of three receptor tyrosine kinases. Phosphorylation upstream of Akt was not affected. Our data indicate that PKCα, which is excessively active in DGKδ-deficient cells, promotes dephosphorylation of Akt through pleckstrin homology domain leucine-rich repeats protein phosphatase (PHLPP) 2. Depletion of either PKCα or PHLPP2 rescued Akt phosphorylation in DGKδ-deficient cells. In contrast, depletion of PHLPP1, another Akt phosphatase, failed to rescue Akt phosphorylation. Other PHLPP substrates were not affected by DGKδ deficiency, suggesting mechanisms allowing specific modulation of Akt dephosphorylation. We found that β-arrestin 1 acted as a scaffold for PHLPP2 and Akt1, providing a mechanism for specificity. Because of its ability to reduce Akt phosphorylation, we tested whether depletion of DGKδ could attenuate tumorigenic properties of cultured cells and found that DGKδ deficiency reduced cell proliferation and migration and enhanced apoptosis. We have, thus, discovered a novel pathway in which diacylglycerol signaling negatively regulates Akt activity. Our collective data indicate that DGKδ is a pertinent cancer target, and our studies could lay the groundwork for development of novel cancer therapeutics.     

Contrasting calcium localization and speciation in leaves of the Medicago truncatula mutant cod5 analyzed via synchrotron X–ray techniques

Oxalate‐producing plants accumulate calcium oxalate crystals (CaOx_(c)) in the range of 3–80% w/w of their dry weight, reducing calcium (Ca) bioavailability. The calcium oxalate deficient 5 (cod5) mutant of Medicago truncatula has been previously shown to contain similar Ca concentrations to wild‐type (WT) plants, but lower oxalate and CaOx_(c) concentrations. We imaged the Ca distribution in WT and cod5 leaflets via synchrotron X–ray fluorescence mapping (SXRF). We observed a difference in the Ca distribution between cod5 and WT leaflets, manifested as an abundance of Ca in the interveinal areas and a lack of Ca along the secondary veins in cod5, i.e. the opposite of what is observed in WT. X–ray microdiffraction (μXRD) of M. truncatula leaves confirmed that crystalline CaOx_(c) (whewellite; CaC₂O₄·H₂O) was present in the WT only, in cells sheathing the secondary veins. Together with μXRD, microbeam Ca K–edge X–ray absorption near‐edge structure spectroscopy (μXANES) indicated that, among the forms of CaOx, i.e. crystalline or amorphous, only amorphous CaOx was present in cod5. These results demonstrate that deletion of COD5 changes both Ca localization and the form of CaOx within leaflets.     

Inhibition of Dopamine Transporter Activity by G Protein βγ Subunits

Uptake through the Dopamine Transporter (DAT) is the primary mechanism of terminating dopamine signaling within the brain, thus playing an essential role in neuronal homeostasis. Deregulation of DAT function has been linked to several neurological and psychiatric disorders including ADHD, schizophrenia, Parkinson’s disease, and drug addiction. Over the last 15 years, several studies have revealed a plethora of mechanisms influencing the activity and cellular distribution of DAT; suggesting that fine-tuning of dopamine homeostasis occurs via an elaborate interplay of multiple pathways. Here, we show for the first time that the βγ subunits of G proteins regulate DAT activity. In heterologous cells and brain tissue, a physical association between Gβγ subunits and DAT was demonstrated by co-immunoprecipitation. Furthermore, in vitro pull-down assays using purified proteins established that this association occurs via a direct interaction between the intracellular carboxy-terminus of DAT and Gβγ. Functional assays performed in the presence of the non-hydrolyzable GTP analog GTP-γ-S, Gβγ subunit overexpression, or the Gβγ activator mSIRK all resulted in rapid inhibition of DAT activity in heterologous systems. Gβγ activation by mSIRK also inhibited dopamine uptake in brain synaptosomes and dopamine clearance from mouse striatum as measured by high-speed chronoamperometry in vivo. Gβγ subunits are intracellular signaling molecules that regulate a multitude of physiological processes through interactions with enzymes and ion channels. Our findings add neurotransmitter transporters to the growing list of molecules regulated by G-proteins and suggest a novel role for Gβγ signaling in the control of dopamine homeostasis.     

Suppressor of Cytokine Signaling (SOCS) 5 Utilises Distinct Domains for Regulation of JAK1 and Interaction with the Adaptor Protein Shc-1

Suppressor of Cytokine Signaling (SOCS)5 is thought to act as a tumour suppressor through negative regulation of JAK/STAT and epidermal growth factor (EGF) signaling. However, the mechanism/s by which SOCS5 acts on these two distinct pathways is unclear. We show for the first time that SOCS5 can interact directly with JAK via a unique, conserved region in its N-terminus, which we have termed the JAK interaction region (JIR). Co-expression of SOCS5 was able to specifically reduce JAK1 and JAK2 (but not JAK3 or TYK2) autophosphorylation and this function required both the conserved JIR and additional sequences within the long SOCS5 N-terminal region. We further demonstrate that SOCS5 can directly inhibit JAK1 kinase activity, although its mechanism of action appears distinct from that of SOCS1 and SOCS3. In addition, we identify phosphoTyr317 in Shc-1 as a high-affinity substrate for the SOCS5-SH2 domain and suggest that SOCS5 may negatively regulate EGF and growth factor-driven Shc-1 signaling by binding to this site. These findings suggest that different domains in SOCS5 contribute to two distinct mechanisms for regulation of cytokine and growth factor signaling.     

Outbreak of Shiga Toxin-Producing Escherichia coli (STEC) O157:H7 Associated with Romaine Lettuce Consumption, 2011

Background

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is the causal agent for more than 96,000 cases of diarrheal illness and 3,200 infection-attributable hospitalizations annually in the United States.

Materials and Methods

We defined a confirmed case as a compatible illness in a person with the outbreak strain during 10/07/2011-11/30/2011. Investigation included hypothesis generation, a case-control study utilizing geographically-matched controls, and a case series investigation. Environmental inspections and tracebacks were conducted.

Results

We identified 58 cases in 10 states; 67% were hospitalized and 6.4% developed hemolytic uremic syndrome. Any romaine consumption was significantly associated with illness (matched Odds Ratio (mOR) = 10.0, 95% Confidence Interval (CI) = 2.1–97.0). Grocery Store Chain A salad bar was significantly associated with illness (mOR = 18.9, 95% CI = 4.5–176.8). Two separate traceback investigations for romaine lettuce converged on Farm A. Case series results indicate that cases (64.9%) were more likely than the FoodNet population (47%) to eat romaine lettuce (p-value = 0.013); 61.3% of cases reported consuming romaine lettuce from the Grocery Store Chain A salad bar.

Conclusions

This multistate outbreak of STEC O157:H7 infections was associated with consumption of romaine lettuce. Traceback analysis determined that a single common lot of romaine lettuce harvested from Farm A was used to supply Grocery Store Chain A and a university campus linked to a case with the outbreak strain. An investigation at Farm A did not identify the source of contamination. Improved ability to trace produce from the growing fields to the point of consumption will allow more timely prevention and control measures to be implemented.     

Auxin-Responsive DR5 Promoter Coupled with Transport Assays Suggest Separate but Linked Routes of Auxin Transport during Woody Stem Development in Populus

Polar auxin transport (PAT) is a major determinant of plant morphology and internal anatomy with important roles in vascular patterning, tropic growth responses, apical dominance and phyllotactic arrangement. Woody plants present a highly complex system of vascular development in which isolated bundles of xylem and phloem gradually unite to form concentric rings of conductive tissue. We generated several transgenic lines of hybrid poplar (Populus tremula x alba) with the auxin-responsive DR5 promoter driving GUS expression in order to visualize an auxin response during the establishment of secondary growth. Distinct GUS expression in the cambial zone and developing xylem-side derivatives supports the current view of this tissue as a major stream of basipetal PAT. However, we also found novel sites of GUS expression in the primary xylem parenchyma lining the outer perimeter of the pith. Strands of primary xylem parenchyma depart the stem as a leaf trace, and showed GUS expression as long as the leaves to which they were connected remained attached (i.e., until just prior to leaf abscission). Tissue composed of primary xylem parenchyma strands contained measurable levels of free indole-3-acetic acid (IAA) and showed basipetal transport of radiolabeled auxin (³H-IAA) that was both significantly faster than diffusion and highly sensitive to the PAT inhibitor NPA. Radiolabeled auxin was also able to move between the primary xylem parenchyma in the interior of the stem and the basipetal stream in the cambial zone, an exchange that was likely mediated by ray parenchyma cells. Our results suggest that (a) channeling of leaf-derived IAA first delineates isolated strands of pre-procambial tissue but then later shifts to include basipetal transport through the rapidly expanding xylem elements, and (b) the transition from primary to secondary vascular development is gradual, with an auxin response preceding the appearance of a unified and radially-organized vascular cambium.     

Reduced Crowding and Poor Contour Detection in Schizophrenia Are Consistent with Weak Surround Inhibition

Background

Detection of visual contours (strings of small oriented elements) is markedly poor in schizophrenia. This has previously been attributed to an inability to group local information across space into a global percept. Here, we show that this failure actually originates from a combination of poor encoding of local orientation and abnormal processing of visual context.

Methods

We measured the ability of observers with schizophrenia to localise contours embedded in backgrounds of differently oriented elements (either randomly oriented, near-parallel or near-perpendicular to the contour). In addition, we measured patients’ ability to process local orientation information (i.e., report the orientation of an individual element) for both isolated and crowded elements (i.e., presented with nearby distractors).

Results

While patients are poor at detecting contours amongst randomly oriented elements, they are proportionally less disrupted (compared to unaffected controls) when contour and surrounding elements have similar orientations (near-parallel condition). In addition, patients are poor at reporting the orientation of an individual element but, again, are less prone to interference from nearby distractors, a phenomenon known as visual crowding.

Conclusions

We suggest that patients’ poor performance at contour perception arises not as a consequence of an “integration deficit” but from a combination of reduced sensitivity to local orientation and abnormalities in contextual processing. We propose that this is a consequence of abnormal gain control, a phenomenon that has been implicated in orientation-selectivity as well as surround suppression.