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Huawei may be hurting by the US sanctions, but that's not stopping the company from setting new records. The Chinese tech giant has already shipped 200 million smartphones in 2019, 64 days faster than it did last year. The numbers were largely driven by the success of products like the P30 and Mate 30 series, the company said through a press release on Wednesday.
Huawei, however, didn't reveal as to how many of those 200 million smartphones were shipped outside of China. With the company's smartphone business outside China largely looming uncertain, it is highly likely that most of them were sold within its home country. Nevertheless, it's still a big achievement considering Huawei had just surpassed the 100 million mark in October 2016.
US trade ban hurting
From three million units in the whole year of 2010 to 200 million units in less than ten months in 2019, Huawei's emergence as the world's second-largest smartphone vendor has been phenomenal. However, recent developments, unfortunately, pose a serious threat to the company's future in the global market.
In August 2019, the US government added Huawei to the entity list, effectively blocking the company from launching new smartphones with Google services. This isn't really a big deal in China, but it's inimical to the company's global smartphone business.
Google services, which include Play Store, Youtube, Search, and more, are integral parts of Android smartphones outside of China. Without them, Huawei will find it difficult to sell smartphones in the global market. Of course, the company is already working on an Android alternative, named HarmonyOS. But it'll admittedly take years in the making. So unless something changes really quickly, Huawei's smartphone sales are highly likely to go down from here on.
Special edition Mate 30 Pro 5G announced
To celebrate the 200 million milestone, Huawei has announced that it will be launching a "Commemorative Edition" of the Huawei Mate 30 Pro 5G soon. This limited edition handset will be available in the Chinese market from November 1, 2019. The smartphone will come with 8GB of RAM and 512GB of internal storage space. It can be purchased in Forest Green and Orange colors from the Huawei Vmall. The company didn't share the pricing details, though.
As for the regular Mate 30 Pro 5G, prices start at CNY 6,899 (roughly $975) for the base variant with 8GB RAM and 256GB storage capacity. The 8GB+512GB variant costs CNY 7,899 ($1115). The smartphone packs a quad-camera setup at the back and features a 6.53-inch Full HD+ OLED display. Under the hood is a HiSilicon Kirin 990 5G chipset. The smartphone goes on sale in China on November 1.


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subset: commoncrawl


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Buck Tilton, M.S., has worked in outdoor education for more than twenty-five years. He is an EMT instructor/coordinator and the executive director of the Wilderness Medicine Institute, Inc., based in Pitkin, Colorado. Frank Hubbell, D.O., has worked in medical education for more than twenty years. He is an EMT instructor/coordinator, has a private practice in family medicine, and is active in mountain search and rescue.Tilton, Buck is the author of 'Medicine for the Backcountry' with ISBN 9781570340024 and ISBN 1570340021.[read more]
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What To Expect
The flower arranging can take anywhere from one to two hours, depending on the complexity and format of the arrangement. Alice's Table provides aprons for you to wear for the duration of the event. Working with flowers can be messy, so make sure to wear something you won't mind getting a little dirty.
Purchased tickets but something came up? You can transfer your ticket to another class as long as you contact us at least 48 hours before the event. Please note we do not offer refunds per our refund policy.
What Should I Bring?
You don't need to bring anything out of the ordinary with you - we provide the flowers, vases, and tools you will need to create your arrangement.
About the Venue
Unique among Salem, MA restaurants, The Roof offers Salem's only rooftop bar and restaurant serving the necessities—strong drinks and simple food. Located at the top of The Hotel Salem, this seasonal dining space features an outdoor kitchen and expansive views of Salem's cobblestone streets and bustling harbor. Expect to find some of the best-crafted cocktails in the area, as well as tacos, rice bowls, and oysters.


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Employees are still having difficulty navigating and processing simple payroll tasks and requests, resulting in uncertainty about their following paycheck. This design case study will attempt to develop a solution to this problem.
This was created with Lucidchart, Balsamiq, and Figma.
How do current programs differentiate their payroll process between salary and hourly employees?
What struggles do employees face when requesting for PTO, tracking their hours, etc.?
How long does HR track certain special requests & for how long does it take for them to get back to that employee?
Systems need to be simple enough to navigate through, but they need to still be able to provide access to all of the necessary information possible.
A ticketing service needs to be integrated that sends both group notifications and emails on a weekly basis.


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- © 2013 American Society of Plant Biologists. All rights reserved.
In plants, mounting an effective innate immune strategy against microbial pathogens involves triggering local cell death within infected cells as well as boosting the immunity of the uninfected neighboring and systemically located cells. Although not much is known about this, it is evident that well-coordinated cell–cell signaling is critical in this process to confine infection to local tissue while allowing for the spread of systemic immune signals throughout the whole plant. In support of this notion, direct cell-to-cell communication was recently found to play a crucial role in plant defense. Here, we provide experimental evidence that salicylic acid (SA) is a critical hormonal signal that regulates cell-to-cell permeability during innate immune responses elicited by virulent bacterial infection in Arabidopsis thaliana. We show that direct exogenous application of SA or bacterial infection suppresses cell–cell coupling and that SA pathway mutants are impaired in this response. The SA- or infection-induced suppression of cell–cell coupling requires an ENHANCED DESEASE RESISTANCE1– and NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1–dependent SA pathway in conjunction with the regulator of plasmodesmal gating PLASMODESMATA-LOCATED PROTEIN5. We discuss a model wherein the SA signaling pathway and plasmodesmata-mediated cell-to-cell communication converge under an intricate regulatory loop.
The basal immune response to microbial pathogens requires accumulation of the defense hormone salicylic acid (SA) (Vlot et al., 2009; Rivas-San Vicente and Plasencia, 2011; Fu and Dong, 2013). Plant protein receptors that recognize pathogen-associated molecular patterns or effectors trigger a mitogen-activated protein kinase cascade and a burst of reactive oxygen species that together activate multiple downstream responses (Wiermer et al., 2005; Spoel and Dong, 2012). The core genetic components known to regulate upstream events of SA biosynthesis include ENHANCED DESEASE RESISTANCE1 (EDS1), PHYTOALEXIN DEFICIENT4, and SENESCENCE-ASSOCIATED GENE101 (Wiermer et al., 2005; Dempsey et al., 2011; Rietz et al., 2011). Lipase-like proteins that are encoded by these regulatory genes function together in specific combinations to enhance defense gene expression. Although the underlying molecular mechanisms are not fully known, EDS1 facilitates the expression of ISOCHORISMATE SYNTHASE1 (ICS1), which encodes an SA biosynthetic enzyme that plays a key role in immunity against bacterial infection and other stresses (Wildermuth et al., 2001; Vlot et al., 2009; Dempsey et al., 2011). Following ICS1-based hyperaccumulation of SA, one of the master regulators of SA signal transduction, NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), is activated by changes in redox conditions within the cell (Tada et al., 2008). As a transcriptional coactivator, NPR1 migrates into the nucleus and brings about major shifts in gene expression patterns, such as induction of pathogenesis-related genes and secretion pathways (Wang et al., 2005; Fu and Dong, 2013).
The critical role of SA as an immune signal has been well documented (Vlot et al., 2009; Rivas-San Vicente and Plasencia, 2011; Fu and Dong, 2013). Direct application of SA activates various pathogenesis-related gene expressions, induces resistance to virulent microbial pathogens, elicits hypersensitive response cell death, and establishes systemic acquired resistance (Malamy et al., 1990; Ryals et al., 1996; Mur et al., 2008; Shah, 2009; Coll et al., 2011). An elegant recent study predicts that a gradient of SA may form such that cells local to the infection site begin hyperaccumulating SA, priming them for cell death, while their neighboring uninfected cells acquire a boost of immunity owing to a lower concentration of SA building up within them (Fu et al., 2012). The molecular mechanisms determining the cellular boundaries between the dying and healthy neighboring cells have yet to be discovered. However, it is conceivable that well-orchestrated local cell communication would be essential in order to confine the hypersensitive response within infected cells so that the detrimental spread of cell death–triggering signals into neighboring healthy cells is prevented (Rustérucci et al., 2001; Rinne and van der Schoot, 2003; Lee and Lu, 2011). In this view, plants would also require a mechanism that coordinates SA-based defense reactions with intercellular connectivity for the full and safe execution of basal immune responses.
Plasmodesmata (PD) allow for direct cytoplasmic connections in the plant and facilitate local molecular exchange among neighboring cells (Robards and Lucas, 1990; Blackman and Overall, 2001; Oparka and Roberts, 2001; Cilia and Jackson, 2004; Maule, 2008; Lucas et al., 2009; Burch-Smith et al., 2011; Sevilem et al., 2013). As PDs are initially formed during cell division, virtually all cells are born with plasmodesmal connections with their sister cells by default. However, these primary PD connections are not permanently set for the rest of a cell's life. Rather, they undergo various types of structural modifications and degeneration/regeneration processes to meet the specific needs of cells that may set out rapid expansion, different developmental phases or differentiation, or adaption processes in response to the changes in various physiological and environmental conditions (Ehlers and Kollmann, 2001; Roberts and Oparka, 2003; Lucas and Lee, 2004; Burch-Smith et al., 2011; Burch-Smith and Zambryski, 2012). For instance, PD frequency and density change as cells grow and develop (Gunning, 1978; Seagull, 1983; Ehlers and Kollmann, 1996; Burch-Smith and Zambryski, 2010; Ehlers and van Bel, 2010) or during shifts from vegetative to reproductive phases (Ormenese et al., 2000; Ormenese et al., 2002; Ormenese et al., 2006); PDs differentiate from simple to complex forms (Faulkner et al., 2008; Fitzgibbon et al., 2013); PDs are completely disintegrated during guard cell maturation (Wille and Lucas, 1984); PD permeability undergoes temporal regulation by environmental conditions, such as daylength and temperature (Ormenese et al., 2006; Bilska and Sowinski, 2010; Rinne et al., 2011), etc.
Permeability, dilation, or structure of PDs can be also altered during infection by microbial pathogens (Heinlein, 2002; Benitez-Alfonso et al., 2010; Schoelz et al., 2011; Ueki and Citovsky, 2011). For example, plant viruses spread their infectious materials cell to cell (Benitez-Alfonso et al., 2010) through either regulating PD dilation (Waigmann et al., 1994) or modifying PD structure (van Lent et al., 1991; Pouwels et al., 2003,; 2004). Unlike viruses, bacterial pathogens are mostly epiphytic in their lifestyle, and their mode of infection does not require them to move cell to cell (Hou et al., 2009). However, bacterial infection induces the closure of PD, and a loss of PD regulation or a constitutive closure of PD in Arabidopsis thaliana confers either susceptibility or resistance, respectively, to virulent strains of Pseudomonas syringae (Lee et al., 2011). We have previously proposed that PLASMODESMATA-LOCATED PROTEIN5 (PDLP5) of Arabidopsis acts as a molecular link between the regulation of PD-mediated cell-to-cell coupling and innate immunity (Lee et al., 2011). PDLP5 is a type I transmembrane protein sharing structural and a minimal sequence homology to seven other PD-located proteins (PDLPs) (Thomas et al., 2008). Using immunolocalization in combination with correlative electron microscopy, we mapped the central cavity region as the subdomain of PD that PDLP5 associates with Lee et al. (2011). Interestingly, a recent review reported an immunogold labeling of another PDLP family member, PDLP1, along the length of the PD channel (Maule et al., 2011), which suggests the subdomain of PD that PDLP members are targeted to may not be the same.
More studies are necessary before assigning either specific or redundant functions to each PDLP isoform. However, PDLP5 seems to perform a unique function integral to immune responses among the PDLP isoforms. For example, lack of PDLP5 results in a loss of the regulation of basal PD permeability and an increased susceptibility to bacterial infection, whereas single knockouts of PDLP1, PDLP2, or PDLP3 had no effect on PD permeability (Thomas et al., 2008). PDLP5 alone is transcriptionally and translationally induced by infection with the virulent bacterial pathogen P. syringae pv maculicola (Pma) or by direct application of exogenous SA. The PDLP5 gene is also called HOPW1-1-INDUCED GENE1 , which was named based on its transcriptional induction upon infection by P. syringae secreting HOPW1-1 effector (Lee et al., 2008).
Consistent with the finding that PDLP5 gene induction is regulated by SA, both endogenous and SA-induced expression of PDLP5 transcript were significantly compromised in eds1, npr1, and ics1 mutants (Lee et al., 2011). Moreover, an ectopic overexpression of PDLP5 induces PD closure, SA accumulation, and basal immunity against virulent bacterial pathogens, whereas the loss-of-function mutant pdlp5-1 allows for an abnormally extensive PD permeability and enhanced disease susceptibility (Lee et al., 2011). Finally, an introduction of the bacterial gene encoding SA hydroxylase, NahG, suppressed the outward plant morphological phenotypes associated with PDLP5 overexpression, namely, growth retardation and spontaneous lesion development. Taken together, these data indicate that SA accumulation through a positive feedback regulation plays a crucial role in PDLP5 function.
Here, we report that SA signaling components are required to regulate cell-to-cell connectivity as well as the epistatic relationship between the SA pathway and PDLP5. We show that direct application of SA to Arabidopsis Columbia-0 (Col-0) induces PD callose deposition and closure, while in the absence of PDLP5, application of exogenous SA was not sufficient to induce these responses. Furthermore, SA mutants defective in SA accumulation or signal transduction are compromised in PD closure upon infection by virulent bacterial pathogens, and the capacity of PDLP5 to activate PD callose deposition and closure is largely dependent on the intact SA biosynthesis and signaling pathway. These results together firmly establish that crosstalk between PDLP5 and the SA pathway is essential for regulating PD permeability during a pathogen defense response.
Direct Application of SA Induces PD Closure
To gain insight into the potential role of SA in modulating cell–cell coupling, we first investigated whether exogenous application of SA has an effect on PD permeability by employing the Drop-and-See (DANS) assay that we developed in a previous study (Lee et al., 2011). The DANS assay uses membrane-permeable, nonfluorescent carboxyfluorescein diacetate as a probe, which acquires fluorescence once released into the cytoplasm through cleavage by cellular esterases and becomes membrane impermeable. The DANS assay has proven to be an effective, noninvasive approach for a real-time, in situ assessment for the extent of molecular diffusion through PD. To test the effect of SA on PD permeability, 3-week-old wild-type Col-0 plants were sprayed with buffer in the absence or presence of SA at 1, 10, or 100 µM, followed by DANS assays on the fourth and fifth rosette leaves (see Supplemental Figure 1 online). Compared with the buffer control, treating wild-type Col-0 plants with 100 µM SA for 24 h resulted in a significant reduction in PD permeability (Figure 1A). Treating the plants with 100 µM SA for 24 or even for 48 h did not cause any obvious stress or yellowing of the plants. Also, there was no cell death detectable by microscopy (see Supplemental Figure 2 online). Based on these results, we chose to use 100 µM SA and a 24-h time point as an optimal treatment condition for our study to assess the effect of SA on PD permeability.
SA Accumulation Is Required for PDLP5-Mediated PD Closure
Having found that SA treatment induces PD closure, we then tested whether SA accumulation is required for the reduced PD permeability phenotype, which is manifested by transgenic plants overexpressing PDLP5 under the control of the 35S promoter (hereafter called PDLP5 plants). To this end, comparative PD permeability assays were performed on 3-week-old wild-type Col-0, PDLP5, NahG, and homozygous F3 progenies of the cross between PDLP5 and NahG plants (hereafter called PDLP5NahG) grown under the same environmental conditions. NahG encodes a bacterial SA hydroxylase (Delaney et al., 1994) and was employed as a genetic means to negate SA hyperaccumulation in the PDLP5 plants. As previously shown in many SA hyperaccumulating mutants (Lorrain et al., 2003; Brodersen et al., 2005), introduction of NahG blocked the spontaneous lesion formation phenotype of PDLP5 plants. Compared with the wild-type Col-0 control, the PD permeability based on the DANS assay is substantially reduced in PDLP5 (Figures 1B and 1C). However, this PD inhibition phenotype also was completely suppressed by the introduction of NahG, indicating that PDLP5 requires SA accumulation to induce the closure of PD.
We predicted that NahG would restore the PD permeability in PDLP5 to a similar level as wild-type Col-0 if NahG simply eliminates the PDLP5-induced SA hyperaccumulation. Surprisingly, PDLP5NahG exhibited greatly enhanced PD permeability, allowing for ∼20% higher diffusion of the fluorescent reporter than the wild type (Figures 1B and 1C). Notably, this level of extensive PD permeability was previously observed in pdlp5-1 (Lee et al., 2011). The level of PDLP5 transcript in PDLP5NahG was comparable to that in the parental line PDLP5 (Lee et al., 2011), eliminating the possibility that the restored PD permeability in PDLP5NahG may reflect a potential fluctuation or instability in PDLP5 expression. Thus, the enhanced PD permeability in PDLP5NahG led us to ask whether the overexpression of NahG alone could enhance PD permeability. Indeed, subsequent DANS assays on NahG plants revealed that an overexpression of NahG alone can significantly enhance the dye diffusion through PD (Figures 1B and 1C). Fluorescent dye absorption on the adaxial surface in transgenic or mutant lines was comparable to that in wild-type plants, as we had shown previously for the PDLP5 and pdlp5-1 (Lee et al., 2011; see Supplemental Figure 3A online). In addition, overall epidermal cell sizes on both adaxial and abaxial sides of PDLP5, NahG, and PDLP5NahG were comparable to those of wild-type Col-0 plants (see Supplemental Figures 3B and 3C online). Thus, the aberrant PD permeability shown in NahG or PDLP5NahG was apparently not due to an alteration in epidermal surface property or cell size.
The ability of NahG to suppress the phenotypes associated with SA hyperaccumulation has been largely attributed to its SA hydroxylase activity, which disables SA accumulation (Lawton et al., 1995). However, there was a report that a certain phenotype manifested by the introduction of NahG is linked to an inappropriate production of an SA degradation by-product, catechol (van Wees and Glazebrook, 2003). We therefore tested the possibility that the enhancing effect NahG has on PD permeability is linked not necessarily to the lack of SA accumulation, but rather to an increased catechol production. The DANS assays performed 24 h after spraying wild-type Col-0 with 100 µM catechol (van Wees and Glazebrook, 2003) demonstrated that this catechol treatment has no effect on the PD permeability (Figure 1D). These results indicate that the inability to accumulate SA, rather than catechol accumulation, attributes to extensive opening of PD in NahG plants.
In contrast with the repressive effect of SA on symplastic dye diffusion in wild-type Col-0 (Figure 1A), the same SA treatment failed to induce PD closure in NahG (Figure 1E), as expected. However, application of an SA agonist, benzo(1,2,3) thiadiazole-7-carbothioic acid (BTH), which is not degradable by NahG, could induce PD closure in NahG to a similar extent shown in BTH-treated wild-type plants (Figure 1F). Moreover, BTH treatment could restore the reduced PD permeability phenotype in PDLP5NahG, whereas such treatment did not have any additional effect in PDLP5 plants. Collectively, our data provide strong experimental evidence that SA both acts as a signal to restrict cell-to-cell coupling and is required for PDLP5-mediated PD closure.
Inability to Accumulate SA Leads to the Loss of the Regulation of PD during Defense
In response to virulent Pma infection, cell-to-cell diffusion through PD becomes highly restricted in Arabidopsis Col-0 (Lee et al., 2011). Having established that exogenous application of SA promotes PD closure, we speculated that the Pma-induced PD closure might also be attributed to cellular activation of SA biosynthesis and accumulation resulting from innate immune responses. To test this hypothesis, the PD permeability of NahG upon Pma infection was examined in comparison to Col-0 wild-type plants; we reasoned that the inability to accumulate SA would make NahG insensitive to Pma infection in terms of the PD response. Indeed, we found that bacterial infection failed to induce PD closure in NahG as well as in PDLP5NahG, while infected wild-type Col-0 exhibited a substantial reduction in PD permeability compared with the mock-treated plants (Figure 1G). The SA deficiency in PDLP5NahG also suppressed the PDLP5 pathogen-resistant phenotype, indicating that SA buildup is in fact required for both the regulation of PD and basal immunity in PDLP5 plants. These results underscore the dual role of SA as a hormonal signal that not only activates defense but also regulate symplastic cell-to-cell connectivity during the plant response to microbial pathogens.
SA Induces PD Callose Deposition
Pma infection was previously found to stimulate callose deposition at PD (Lee et al., 2011), but at that time, it was unknown which signaling molecules or pathway regulated that response. However, in light of the results described above, it seemed prudent to test the possibility that callose deposition restricting PD during the defense response to Pma was also regulated by SA. To this end, we examined the effect of SA on PD callose deposition by treating 3-week-old Col-0 plants with 100 µM SA spray for 24 h, followed by aniline blue staining for callose detection in the rosette leaves and quantification of the fluorescence foci intensity per square micron. Compared with the mock control, the leaves treated with SA accumulated over twofold higher PD callose (Figure 2A). This result together with the finding that PD permeability is highly enhanced in NahG suggested that a lack of SA accumulation may avert deposition of PD callose in NahG. Indeed, the basal PD callose deposition in NahG was substantially lower, comprising only 30% of the wild-type level (Figure 2B). Moreover, consistent with the suppressive effect of NahG on the reduced PD permeability phenotype of PDLP5, NahG also eliminated the phenotype of PDLP5 associated with hyperaccumulation of PD callose (Figure 2C). These data support the idea that SA is a crucial hormonal factor regulating cell-to-cell permeability via recruiting callose to PD.
SA Does Not Rescue the Enhanced PD Permeability Phenotype of pdlp5-1
Since a high PDLP5 expression level within cells stimulates SA accumulation, one could argue that the restricted PD phenotype of PDLP5 is due simply to SA hyperaccumulation triggering closure through another, PDLP5-independent mechanism. If this were indeed true, then supplying pdlp5-1 with exogenous SA would induce a near PDLP5-level of PD closure because the loss of PDLP5 would not stop SA from exerting its PD-closing function. To test this possibility, we performed DANS assays on Col-0 and pdlp5-1 that were either mock treated or treated with 100 µM SA. In stark contrast with the wild-type control, the SA-induced PD closure response was fully impaired in pdlp5-1, which retained a higher PD permeability even in the presence of exogenously supplied SA (Figures 3A and 3B). We next addressed whether PDLP5 was essential for closing PD during a response to bacterial pathogen by performing DANS assays on pdlp5-1 following Pma infection. This experiment showed that Pma-induced PD closure is also fully impaired in pdlp5-1 (Figure 3C). This result, together with the loss of SA sensitivity demonstrated in pdlp5-1 (Figures 3A and 3B), indicates that PDLP5 is indeed a key molecular player for translating SA signaling to PD closure during defense responses. Collectively, our data provide strong experimental evidence that PDLP5 and SA exert their effect on PD in an interdependent manner such that restriction of PD requires both components.
PDLP5 Is an Essential Molecular Link between the SA-Based Defense Response and PD Callose Deposition and Closure
Numerous reports have presented a correlation between PD closure and callose deposition (Radford et al., 1998; Simpson et al., 2009; Lee et al., 2011; Vatén et al., 2011; Zavaliev et al., 2011; Koh et al., 2012), supporting the general consensus that enhanced PD callose deposition is indicative of PD closure and, thus, inhibition of cell-to-cell coupling. Our study has shown that the relative amount of PD callose deposition is inversely correlated with the extent of PD permeability but directly correlated with the level of PDLP5 expression (Lee et al., 2011). However, it was unclear to us whether the increase in PD callose level during infection was also dependent on PDLP5 or any other factors (such as SA hyperaccumulation). We addressed this question by examining PD callose levels in pdlp5-1 following application with exogenous SA. If pdlp5-1 responds normally to the SA treatment in terms of elevated PD callose level, it would mean that SA, not PDLP5, is responsible for augmenting PD callose. However, while wild-type Col-0 responded to exogenous SA by inducing a significant amount of PD callose deposition, this response was also fully impaired in pdlp5-1 (Figure 3D). This result confirms that the induced PD callose response upon SA accumulation is indeed dependent on PDLP5, further supporting that PDLP5-mediated PD closure is most likely mediated through callose deposition at PD.
SA Defense Pathway Mutations Are Epistatic to PDLP5
Based upon the data described above, it is clear that SA regulates PD permeability in conjunction with PDLP5. To gain further insight into the functional relationship between PDLP5 and SA, we determined epistatic relationships between the genetic components of SA pathway and PDLP5 using PDLP5 and SA mutants. We chose the SA mutants eds1-2, ics1-1, and npr1-1, which lack the key upstream regulator of SA signaling pathway EDS1, SA biosynthetic enzyme ICS1, and SA downstream regulator NPR1, respectively (Cao et al., 1994; Aarts et al., 1998; Wildermuth et al., 2001). We isolated homozygous F3 progenies of PDLP5eds1, PDLP5ics1, and PDLP5npr1 and then examined which genetic mutations can suppress the PD permeability phenotype in PDLP5. The homozygosity of the SA pathway mutations and the homogeneous PDLP5 expression level in those double mutants were confirmed by transcript analysis using RT-PCR (see Supplemental Figure 4A online). Morphological comparisons of PDLP5eds1, PDLP5 ics1, and PDLP5 npr1 to the PDLP5 parental line showed that all three mutations suppressed the stunted growth phenotype of PDLP5 to a certain extent (Figure 4A). Furthermore, the highly elevated SA level found in the PDLP5 parental line was also diminished (Figure 4B), with a subsequent reduction or abolition of the SA hyperaccumulation marker PR1 (see Supplemental Figure 4B online), confirming that abnormal induction of PR1 in PDLP5 depends on these genetic components. These results indicate that PDLP5-regulated positive SA feedback must work through the basal SA defense pathway components EDS1, ICS1, and NPR1.
We next examined PD permeability phenotype in PDLP5eds1, PDLP5ics1, and PDLP5npr1. Fluorescent dye absorption on the adaxial surface and overall epidermal cell sizes on both adaxial and abaxial sides in SA mutants and crosses were comparable to those in wild-type plants (see Supplemental Figure 3 online). Subsequent DANS assays on these plants demonstrated that all three mutations were able to suppress the restricted PD permeability in PDLP5 (Figure 4C). However, whereas both eds1 and npr1 were able to fully suppress the reduction in PD permeability, ics1 was only partially effective. This result suggests that PDLP5-mediated PD closure requires an EDS1- and NPR1-dependent SA pathway.
Basal PD Permeability Is Normal in SA Mutants
Considering a potential mechanism by which SA acts on PD, we propose two simple possibilities. One is that SA is a chemical agonist that directly affects PDLP5 or other PD components. The other is that SA acts indirectly as a hormonal signal that activates a downstream signaling pathway(s) on which PDLP5 activity/function relies. To gain insight into the mechanistic relationship between SA and PD permeability, we decided to investigate the latter possibility by examining PD permeability phenotypes of SA mutants. To this end, basal PD permeability was first measured by performing DANS assays on the SA pathway mutants eds1, ics1, and npr1. This test showed that the basal level of PD opening in these mutants under normal growth conditions was comparable to that of wild-type controls (Figures 5A and 5B). Notably, eds1 showed a slightly lower PD permeability than wild-type Col-0, a result that was perplexing considering the endogenous SA level in this mutant was lower than Col-0 (Figure 4B). Since eds1 is derived from the Landsberg erecta (Ler) ecotype, we subsequently tested whether the low PD permeability detected in eds1 reflects a difference between Ler and Col-0 ecotypes. Indeed, the DANS assay showed that wild-type Ler plants exhibited slightly lower PD permeability compared with wild-type Col-0, and the PD permeability of eds1 was comparable to wild-type Ler (Figure 5C).
Induced PD Closure Response Is Compromised in SA Mutants
Since NPR1 is the most downstream regulator, responsible for regulating many of the critical genetic responses to SA accumulation during defense (Wang et al., 2005), we decided to determine if NPR1 was also essential for regulating the pathogen-induced reduction in PD permeability. Indeed, the PD closure response normally induced by Pma infection was fully impaired in the absence of functional NPR1 either in wild-type or PDLP5 backgrounds (Figure 6A). Furthermore, quantitative analyses of aniline blue–stained PD callose revealed that basal PD callose levels in the wild type and npr1-1 were comparable to each other. However, the loss of NPR1 could fully suppress the hyper PD callose accumulation phenotype in PDLP5npr1 (Figure 6B). These data establish the dependence of PDLP5 on NPR1 in modulating PD callose accumulation and, hence, PD closure during immune responses.
Although signaling downstream of SA is largely dependent on NPR1, some SA responses are NPR1 independent (Ferrari et al., 2003; Blanco et al., 2005). To determine whether SA-induced PD closure relies solely on an NPR1-dependent pathway, we performed additional DANS assays using npr1 and PDLP5npr1 following SA application. This experiment showed that exogenous SA application was not able to complement the loss of NPR1 (Figure 6C), thus validating that SA-induced PD closure requires NPR1 or its downstream factor(s). By contrast, SA application restored normal PD closure response in PDLP5eds1 and PDLP5ics1 (see Supplemental Figure 5 online).
In conclusion, we demonstrated that SA is a critical signaling molecule for regulating cell-to-cell connectivity and that SA-directed PD closure requires PDLP5 as the molecular link between the SA pathway and PD modulation. PDLP5 functions to close PD during immune responses by working simultaneously with an NPR1-dependent pathway to trigger a high level of callose deposition at PD during infection. And without either PDLP5 or NPR1, Arabidopsis cannot close PD in response to pathogen infection.
We had shown in a recent study that the SA pathway plays a role in regulating PD by upregulating a PD inhibitor, PDLP5, during immune responses against bacterial pathogens (Lee et al., 2011). In this article, we provided experimental evidence supporting that an accumulation of SA is critical to block PD in Arabidopsis and established an epistatic relationship between SA biosynthetic/signaling components and PDLP5. First, exogenous application of SA or BTH induces PD callose deposition, which results in PD closure. Second, defects in SA accumulation or signaling in NahG and npr1, respectively, compromise the ability to close PD in response to either SA treatment or bacterial infection. Third, in the absence of PDLP5, plants were also not able to stimulate PD callose deposition; thus, there was no PD closure after treatment with exogenous SA or during bacterial infection. Finally, dissection of epistatic relationships revealed that the physiological and cellular phenotypes associated with overexpression of PDLP5, such as SA hyperaccumulation, increased PD callose deposition, and severe restriction of PD permeability, are suppressed in eds1, ics1, npr1, and NahG. Taken together, these data provide insight into how PD closure is regulated during defense. Namely, bacterial pathogen infection triggers the basal SA defense pathway, and SA accumulation activates both NPR1 and PDLP5, which must work in tandem to produce a complete PD closure response, via increasing PD callose deposition. This response is reinforced by a positive feedback loop through the SA defense pathway, which requires the components EDS1, ICS1, and NPR1 (Figure 7).
SA Accumulation via ICS1 Is Required but Not Sufficient for PDLP5-Mediated PD Closure
In Arabidopsis, SA is synthesized primarily via the isochorismate-using pathway in the chloroplast and secondarily via the Phe ammonia-lyase pathway. ICS1, a biosynthetic enzyme in the isochorismate pathway, is not required for the maintenance of the basal level of SA but is mainly responsible for SA accumulation during bacterial infection (Wildermuth et al., 2001). Interestingly, the SA levels in PDLP5eds1 and PDLP5npr1 are elevated but are not as high as in the PDLP5 parental line. By contrast, PDLP5ics1 was found to have a similar SA level to that of ics1 alone (Figure 4B), which indicates that SA accumulation in PDLP5 is fully dependent on ICS1 function. However, while the basal PD permeability of eds1, ics1, and npr1 was no different from that in each of their wild-type ecotypes (either Col-0 or Ler; Figure 5), both the eds1 and npr1 mutations fully suppressed PDLP5-induced PD closure, whereas ics1 could only partially suppress the PDLP5-restricted PD phenotype (Figure 4C). These data suggest the presence of an additional factor(s) independent of ICS1-amplified SA that is responsible for PD closure via PDLP5 through EDS1- or NPR1-triggered changes in defense signaling. We speculate that factors from both ICS1-dependent and -independent pathways may work together to produce the maximum activity/function of PDLP5.
Do SA and PDLP5 Impede Dye Movement through Induction of Cell Death or PD Modification?
We have shown in our previous study that overexpression of PDLP5 results in hyperaccumulation of SA and spontaneous lesions (Lee et al., 2011). It is possible that cell death might have impeded dye movement in PDLP5 plants to some extent apart from or in addition to PD callose deposition. The PDLP5 line used in this study was an intermediate line that has a mild cell death phenotype. Moreover, cell death in PDLP5 plants progresses with aging, becoming visible at 4 to 5 weeks after germination starting from the edges of the oldest leaves. All our DANS assays and callose staining were done on the central regions of the fourth and fifth rosette leaves of 3-week-old plants, in which there are few to no lesions. Also, treatment with 100 µM SA for 24 h or longer did not cause lesions to form but still resulted in a reduction in PD permeability. Based on these pieces of circumstantial evidence, it appears that cell death might be a later response than or eventual outcome of PD closure.
What might be the direct role that SA and SA pathways play in regulating PD? Could SA affect the subcellular localization of PDLP5? We tested this possibility, but PDLP5 localization at PD remained the same 24 or even 48 h after SA treatment (see Supplemental Figure 6 online). It is possible that SA or SA pathways may induce a component that PDLP5 requires for its function; our data indicate that PDLP5 cannot function to close PD in the absence of this SA-dependent component, factor X (Figure 7). SA was shown to facilitate secondary PD formation in Arabidopsis seedlings grown on SA-containing medium for several days (Fitzgibbon et al., 2013). It would be interesting to determine whether there is a close link between structural modification of PD and the SA pathway during immune responses and whether SA together with PDLP5 induces PD closure through such modification.
Basal PD Callose/Permeability Requires SA in Conjunction with PDLP5 but Not npr1
In contrast with ics1, NahG fully suppressed the restricted-PD phenotype in PDLP5. This result is not too surprising, as several previous reports have documented that NahG and ics1 do not always suppress phenotypes associated with SA hyperaccumulation in a similar manner (Nawrath and Métraux, 1999; Heck et al., 2003; Brodersen et al., 2005; Jagadeeswaran et al., 2007; Vogelmann et al., 2012). One obvious possibility is that NahG is more efficient at eliminating SA in the PDLP5 background than ics1, considering their efficacies in suppressing the PDLP5 phenotype. Along this line, we could argue that SA is the major factor PDLP5 requires to close PD and that SA synthesized independently of ICS1 might play a role in both maintaining basal PD permeability and responding to pathogen infection. Thus, NahG is more potent than ics1 in suppressing PDLP5 because it degrades SA from all sources within the plant, while ics1 only prevents ICS1-dependent SA accumulation. Consistent with this idea, treating NahG plants with nondegradable SA analog BTH induced PD closure, as was also the case for BTH-treated wild-type plants.
While the highly enhanced basal PD permeability in the NahG background was not seen in ics1, eds1, or npr1, the permeability observed in the NahG background was quite similar to that in pdlp5-1 (Figure 3). An intriguing question here is whether there is a common factor that NahG and pdlp5-1 share to open PD beyond the basal level. One may logically suggest that the enhanced PD permeability in pdlp5-1 has to do with greatly reduced or eliminated SA, similar to NahG. However, our previous study has already shown that the SA content in pdlp5-1 was not different from that in the wild type (Lee et al., 2011), underscoring that a loss of PDLP5 results in an enhancement in basal PD permeability regardless of having a normal SA level. Another possibility is that there is not sufficient SA in the NahG plants to maintain normal levels of PDLP5. Indeed, RT-PCR data show that the PDLP5 expression level in NahG is lower than that in ics1 and the wild type (see Supplemental Figure 7 online), supporting the pdlp5-1 phenotype in NahG (i.e., enhanced PD permeability). However, given that PDLP5 alone does not close PD in the absence of SA accumulation, we conclude that PDLP5 or SA cannot work alone but rather both are required to close PD. In the case of npr1, the level of SA and/or PDLP5 must still be above the threshold required for maintaining basal PD callose accumulation/permeability. The fact that basal PD callose/permeability is abolished in both NahG and pdlp5-1 (Figures 2C and 3D) but not in npr1 is consistent with our conclusion that basal PD regulation requires PDLP5 only in conjunction with SA but not NPR1.
SA-Induced PD Callose Accumulation via Specific Callose Synthases?
Callose accumulation in planta is induced in response to fungal infection at the penetration site, upon elicitor treatment and bacterial infection, and by other developmental or mechanical factors (Jacobs et al., 2003; Nishimura et al., 2003; DebRoy et al., 2004; Xie et al., 2011). How might SA pathways and PDLP5 specifically affect PD callose accumulation? SA has been implicated in upregulating a subset of Arabidopsis callose synthase (CALS) transcripts in an NPR1-dependent manner (Dong et al., 2008). It is tempting to speculate that the loss of PD callose in PDLP5npr1 is due to the lack of expression of the same NPR1-dependent CALS genes, CALS1/GSL6 and CALS12/GSL5/PMR4, described by Dong et al. (2008). However, knocking down those CALS genes by RNA interference was reported earlier not to affect callose accumulation either at the cell plate or PD (Jacobs et al., 2003; Nishimura et al., 2003), eliminating the possibility that these isoforms are involved in PD callose deposition. Increases in CALS transcript levels may not necessarily correlate with upregulation of callose synthase activity because callose synthase requires additional protein components to form an active complex and other factors for catalytic activation (Brownfield et al., 2009). Also, callose deposition is regulated by the balance between callose synthase and hydrolase activities (Beffa et al., 1996; Levy et al., 2007; Zavaliev et al., 2011); thus, the role of β-1,3-glucanses would need to be taken into consideration.
Recently, two Arabidopsis CALS genes, CALS10/GSL8 and CALS3/GSL12, were shown to affect PD callose accumulation and play important roles during plant development (Guseman et al., 2010; Vatén et al., 2011). The loss-of-function mutant of CALS10/GSL8, chor, is compromised for callose deposition at the cell plate, cell wall, and PD and is seedling lethal. Knocking out CALS3/GSL12 did not alter the level of PD callose deposition, but gain-of-function mutations caused hyperaccumulation of PD callose in root cells. Another isoform, CALS7/GSL7, was shown to be specifically expressed in phloem and required for both basal and wound-induced callose deposition at the sieve plates (Xie et al., 2011). Collectively, these data suggest that specific CALS genes may be responsible for PD callose deposition in certain tissues in specific developmental stages and/or in response to physiological cues or environmental challenges. In this study, we showed that direct application of SA to Arabidopsis leaves induces a substantial amount of PD callose, which suggests that the PD callose induction by bacterial infection that we previously reported (Lee et al., 2011) is likely mediated through elevated SA concentration. In addition, the hyperaccumulation of PD callose induced by PDLP5 overexpression was suppressed in npr1, underscoring the critical role of this component (Figure 6). Currently, we are investigating which CALS/hydrolases might be responsible for SA-induced PD callose accumulation during immune responses and the mechanisms by which they may regulate both basal and induced PD permeability in plants.
Crosstalk between Defense Signaling and PD Regulation via PDLP5
Based upon the results presented in this study, we propose a model illustrating how PDLP5 is integrated into the defense signaling cascade to regulate cell-to-cell connectivity in Arabidopsis (Figure 7). Bacterial pathogen infection activates the basal immune pathway through EDS1 and ICS1, stimulating SA biosynthesis, which leads to the upregulation of PDLP5 gene expression, which is partially dependent upon NPR1. However, the high expression of PDLP5 also triggers a feedback loop through the same pathway, soon causing a hyperaccumulation of SA in the tissue, which reinforces the defense response. What happens next will require further exploration to gather essential details, but from the experimental evidence that we have at hand so far, PD closure during an SA-based defense response absolutely requires both NPR1 and PDLP5. Since it is highly unlikely that NPR1 and PDLP5 interact directly, there is probably an NPR1-dependent "factor X" that must work together with PDLP5 for proper PD callose accumulation and closure during SA-dependent defense. Further biochemical characterization of PDLP5 and identification of the genes that are responsible for the PD callose deposition during immune responses should provide insight into the mechanism by which the restriction of PD is achieved during basal immunity.
Plant Materials and Growth Conditions
Seeds of the Arabidopsis thaliana mutants eds1-2, ics1, and npr1-1 were provided by the H. Bais lab (University of Delaware) and transgenic NahG seeds from the X. Dong lab (Duke University). Plants were grown in soil in a 22°C, 60% humidity, 16/8-h-light/dark growth chamber. Crosses were made by removing the sepals, petals, and immature stamens from an unopened bud of an SA pathway mutant and then coating the stigma with pollen from either PDLP5 or pdlp5-1 plants. Segregating F2 seeds from the offspring of the crosses were screened with specific primers for each mutant (see Supplemental Table 1 online) using RT-PCR (see section below), and F3 seeds used in this study were collected from double homozygous mutants.
Genomic and RT-PCR
Total genomic DNA was isolated using DNA extraction buffer containing 200 mM Tris-HCl, pH 8.0, 250 mM NaCl, 25 mM EDTA, pH 8.0, and 0.5% SDS, followed by isopropanol precipitation. Genomic DNA resuspended with nano-water was treated with 10 µg/mL RNase A solution (Sigma-Aldrich) at 65°C for 15 min, and 100 to 300 ng DNA was used as template per PCR. Total RNAs were isolated using the Trizol (Invitrogen) method for RT: RNAs were first treated with 2 units of DNaseI (New England Biolabs) for 20 min at 37°C, and 1 µg of each RNA sample was used in a 20 μL total RT reaction containing 0.5 mM deoxynucleotide triphosphate, 0.5 µg oligo(dT), 20 units of Murine RNase inhibitor (NEB), and 20 units of M-MuLV reverse transcriptase (NEB) at 42°C for 1 h. One-twentieth of the cDNAs from RT was used per PCR reaction as follows. Both genomic and RT-PCRs were performed in a 25-μL reaction volume using gene-specific primers (see Supplemental Table 1 online) and Taq DNA polymerase (GenScript). All PCRs were performed using a Bio-Rad DNA Engine Peltier thermal cycler. The genomic PCR amplification profile was three cycles of 94°C for 1 min, 52°C for 2 min, and 72°C for 6 min followed by 25 cycles of 94°C for 1 min, 60°C for 2 min, and 72°C for 6 min. The RT-PCR profile was 28 cycles of 94°C for 30 s, 55°C for 45 s, and 72°C for 35 s. Samples were resolved in ethidium bromide–stained 1% agarose gels and visualized and imaged with an Alpha-Imager HP, followed by densitometric quantification using the Image J-NIH program. At least three biological and three technical repeats were performed per sample for quantification.
Confocal Microscopy Imaging, DANS Assay, and Callose Quantification
Plant samples were imaged on a Zeiss AxioObserver inverted light microscope using an LSM 510 META scanhead on a Zeiss LSM 5 LIVE DUO confocal microscope as described before (Lee et al., 2011). Dye loading assays and callose staining were performed on fourth and fifth rosette leaves of 3-week-old plants as described previously (Lee et al., 2011) in the presence or absence of chemical treatments. Briefly, a droplet of 1 mM 5(6)-carboxyfluorescein diacetate (CFDA) was loaded on the adaxial leaf surface of intact Arabidopsis plants for 5 min, followed by removal of dye by pipetting and imaging the abaxial leaf surface under a Fluar ×5/0.25 objective lens, using 488-nm laser excitation with a 505- to 550-nm band-pass emission filter. Aniline blue stains were detected using a C-Apochromat ×40/1.20-W Korr UV-VIS-IR objective and 405-nm laser excitation with a 420- to 480-nm band-pass emission filter. For the effect of bacterial infection, dye loading assays and callose staining were performed on systemic rosette leaves (fourth and fifth) 24 h after infection by bacterial infiltration on lower leaves.
Chemical Treatments, Bacterial Infection, and SA Measurement
For SA and catechol treatments, 3-week-old plants grown in soil were sprayed with 100 μM SA, BTH, or catechol dissolved in double-distilled water containing 0.01% Silwet-77 or 10 mM MES, pH 5.8, and samples were collected 24 h after treatment. Pma ES4326 infection was performed as described before (Lee et al., 2011). Briefly, 3- to 4-week-old Arabidopsis plants were infected by infiltration (OD600 = 0.001) and incubated for 2 d before DANS and callose quantification assays. Statistical difference between samples was analyzed using a t test at P < 0.001 or analysis of variance (ANOVA) in conjunction with Fisher's LSD test.
Total SA content was measured by HPLC analysis using 25-d-old plant extracts according to Wang et al. (2011). Briefly, 100 to 200 mg of whole plant tissues was extracted with methanol, dried, and resuspended in 500 μL of 100 mM sodium acetate, pH 5.5. For total SA measurement, 40 units of β-glucosidase (Sigma-Aldrich G-0395) were added to a set of duplicated tubes to digest glucosyl-conjugated SA for 1.5 h at 37°C. All samples were precipitated with an equal volume of 10% TCA and centrifuged at 10,000g for 10 min. The supernatant was further extracted twice with 1 mL of extraction solvent (ethylacetate:cyclopentane:2-propanol 100:99:1, v/v). The top phase was collected and dried in a fume hood overnight. The residual fraction was resuspended in 0.5 mL of 55% methanol by vortex, filtrated through a 0.2-mm nylon spin-prep membrane (Fisher 07-200-389), and subjected to reverse-phase HPLC analysis using an RF2000 fluorescence detector.
Accession Numbers
DNA sequence data from this article can be found in the GenBank/EMBL database under accession numbers AT1G70690, AT1G74710, AT3G48090, AT1G64280, and X83926.1.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. DANS Assays on Mock- or SA-Treated Wild-Type Col-0 Plants.
Supplemental Figure 2. SA Treatment for 24 or 48 h Does Not Induce Microlesion Formation.
Supplemental Figure 3. Confocal Images Showing Adaxial Leaf Surfaces 5 min after Loading with Fluorescent Dye in DANS Assays.
Supplemental Figure 4. Genotyping and Marker Gene Phenotyping to Confirm Mutations in Desired Genes and Homozygosity of Crosses.
Supplemental Figure 5. PD Closure Response in PDLP5ics1 and PDLP5eds1 Plants upon 100 μM SA Treatment.
Supplemental Figure 6. SA Treatment Has No Effect on PDLP5 Subcellular Localization.
Supplemental Figure 7. RT-PCR Showing the Relative Expression Level of PDLP5.
Supplemental Table 1. PCR Primers.
This research was supported by grants provided by the National Science Foundation (IOB 0954931) and partially by the National Center for Research Resources (5P30RR031160-03) and the National Institute of General Medical Sciences (8 P30 GM103519-03) from the National Institutes of Health to J.-Y.L. Publically available Arabidopsis mutant lines were obtained from the ABRC.
X.W., R.S., W.C., and C.Z. performed research. X.W., R.S., H.L., and J.-Y.L. analyzed the data. J.-Y.L. directed the research and wrote the article.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Jung-Youn Lee (lee{at}dbi.udel.edu).
↵[C] Some figures in this article are displayed in color online but in black and white in the print edition.
↵[W] Online version contains Web-only data.
- salicylic acid
- plasmodesmata
- PD-located protein
- Pma
- P. syringae pv maculicola
- Col-0
- Columbia-0
- benzo(1,2,3) thiadiazole-7-carbothioic acid
- Ler
- Landsberg erecta
- 5(6)-carboxyfluorescein diacetate
- analysis of variance
- Received February 13, 2013.
- Revised May 2, 2013.
- Accepted May 23, 2013.
- Published June 7, 2013.


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|Cerebral venous thrombosis in a Mexican multicenter registry of acute cerebrovascular disease: The RENAMEVASC study
|Ruiz-Sandoval, J.L.
Chiquete, E.
Banuelos-Becerra, L.J.
Torres-Anguiano, C.
Gonzalez-Padilla, C.
Arauz, A.
Leon-Jimenez, C.
Murillo-Bonilla, L.M.
Villarreal-Careaga, J.
Barinagarrementeria, F.
Cantu-Brito, C.
Cantu-Brito, C.
Arauz-Gongora, A.
Ruiz-Sandoval, J.L.
Villarreal-Careaga, J.
Murillo-Bonilla, L.
Rangel-Guerra, R.
Barinagarrementeria, F.
|Issue Date:
|Background: Cerebral venous thrombosis (CVT) is a rare form of cerebrovascular disease that is usually not mentioned in multicenter registries on all-type acute stroke. We aimed to describe the experience on hospitalized patients with CVT in a Mexican multicenter registry on acute cerebrovascular disease. Methods: CVT patients were selected from the RENAMEVASC registry, which was conducted between 2002 and 2004 in 25 Mexican hospitals. Risk factors, neuroimaging, and 30-day outcome as assessed by the modified Rankin scale (mRS) were analyzed. Results: Among 2000 all-type acute stroke patients, 59 (3%; 95% CI, 2.3-3.8%) had CVT (50 women; female:male ratio, 5:1; median age, 31 years). Puerperium (42%), contraceptive use (18%), and pregnancy (12%) were the main risk factors in women. In 67% of men, CVT was registered as idiopathic, but thrombophilia assessment was suboptimal. Longitudinal superior sinus was the most frequent thrombosis location (78%). Extensive (>5 cm) venous infarction occurred in 36% of patients. Only 81% of patients received anticoagulation since the acute phase, and 3% needed decompressive craniectomy. Mechanical ventilation (13.6%), pneumonia (10.2%) and systemic thromboembolism (8.5%) were the main in-hospital complications. The 30-day case fatality rate was 3% (2 patients; 95% CI, 0.23-12.2%). In a Cox proportional hazards model, only age <40 years was associated with a mRS score of 0 to 2 (functional independence; rate ratio, 3.46; 95% CI, 1.34-8.92). Conclusions: The relative frequency of CVT and the associated in-hospital complications were higher than in other registries. Thrombophilia assessment and acute treatment was suboptimal. Young age is the main determinant of a good short-term outcome. © 2012 by National Stroke Association.
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|Producción científica UdeG (prueba)
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|120 × 120 × 6 cm
Unframed, Unframed with 4 L-pins, Unframed with Hanging Brace, Framed – White, Framed – Black, Framed – Natural, Framed – Teak


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Dota 2 Matchmaking System Noobs
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Beverly Johnson's Diet Once Consisted of Cocaine, Champagne & Coffee: The skinnier you were, the more fabulous you were.
In her new memoir — The Face That Changed It All — Beverly Johnson is revealing some of the more darker moments of her life.
The retired 63-year-old super model has had a legendary career and was first African-American face on the cover of American Vogue back in 1974.
I've talked about bulimia, anorexia, depression and losing my daughter in a custody battle but never about drugs.
When she was just getting her start in the modeling world, in the early seventies, she was rejected and told that she was too fat. At the time, she 5'9″ and 125 pounds.
To become a size zero, she admits to starving herself until she weighed just 103 pounds. She says:
I was eating nothing, zero. I drank black coffee, a sip of broth if things got tough, and in the evening, a glass of champagne as a pick me up. We didn't even drink water. We thought it was was fattening.


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76 Bayview Street
Runaway Bay, QLD, 4216
- 4bed
- 3bath
- 2car
Stunning Standalone Home in 'Runaway Cove' EstateThis executive style home is accessed through the secure gated estate of 'Runaway Cove' giving you access to all the facilities with the benefit of living in a standalone house. The owners have just put in new carpet throughout, new tiles, all new internal paint and are even throwing in regular garden and lawn maintenance. Pets are considered on application and the owners will consider a 6 or 12 month lease.
Offering 4 Large Bedrooms,3 Living areas,3 Bathrooms, Formal dining room/ Theater room, Air-conditioning, Study, 2 car garage with drive through & outside storage facility/ wine room .
The house comes unfurnished and is available from the 8th of April or possibly sooner.
Features include:
* Master bedroom with parents retreat, walk in robe, ensuite with spa & double bow sink.
* Chef style kitchen with electric cooking, dishwasher, stone bench tops and water views
* Formal lounge, dining and casual dining area next to the kitchen
* Two large family rooms plus study or possible 5th bedroom
* Air-conditioned
* Purpose built insulated storage room or wine cellar
* Shared Pontoon
* Double garage with drive through storage area for the caravan or trailer
* Manicured lawns and large back yard for the children or pets- regular lawn and garden maintenance included
* 'Runaway Cove' facilities include: Tennis court, Lagoon style pool, gymnasium & boat ramp
* Walking distance to a variety of local amenities such as: Sports super centre, Cycling and walking tracks, Runaway Bay leagues, cricket, soccer & netball clubs, Library, school, daycare centre, a variety of restaurants, Runaway Bay shopping village.
* A short drive to Harbour Town, Gold Coast hospital, Griffith university, Gold Coast great beaches and easy access to the M1 motorway onto Brisbane / Coolangatta.
Tennis court, Lagoon style pool,gymnasium,boat ramp


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Wage Funding and Skills Training leads to Success in Immigrant Employment
ECO Canada is managing a pilot project entitled Environmental Immigrant Bridging Program. It is funded by the Foreign Credential Recognition Program of the Canadian Government.
The outlay
Moreover, it is distributing $300,000, for wage fund support, towards employers, to hire newcomers, in their first work experience, in Canada as a part of the environmental role. So far, 100% newcomers who reached the end of this program gained the full-time employment.
This program gives equal to 50% of the newcomer's salary, to a maximum of $10,000, for six months, in a full-time, new, and environmentally relevant position, to the eligible employers. It provides employability training for extending valuable support to the newcomers in job search. This also helps in growing skills preferred by their employers. The joint approach of funding and training helps these newcomers to break entry barriers. They are in a position to establish themselves in a fine career that influences their skill set and knowledge in a positive manner.
The future scenario
22% of the environmental sector will retire in the subsequent 8 years. The result will be a noteworthy job gap in the middle and senior level positions. The Newcomers will be in a position to provide the necessary skills and also fill up these gaps. Besides, there will be a diverse perspective in the industry.
This program permits the employers to explore fresh recruitment opportunities and build a strong capacity in their organization. The employers claim of loving this program to find a candidate with better credentials and a desire to contribute. The teams which struggle to find a right balance between present resources and growth aim are relieved of the financial risk.
Many job seekers in the environment domain arrive in Canada and look for a fresh start, but struggle to find proper work in their desired field in which they have huge experience.
The challenges
Their journey is challenging but the support they receive through ECO Canada boosts their morale. The support in work creates a positive impact and shapes the lives of fresh arrivals.
This aim of this employment program is to improve opportunities for fresh Canadians who face a struggle in finding a meaningful work commensurate with their talent, skill and background.
It also allows employers to work with a lesser risk. The pilot is a success; and has paved the way for fresh arrivals to get great jobs. Moreover, the employers receive skilled talent who make an immediate contribution to the place of employment in the environmental sector.
Such programs must be continued by the government so that newcomers can shift into the workplace quickly and benefit the Canadian society.
Finally, this program helps to create an inclusive online training program for Canadians who show an interest to enter the environmental sector in Canada.


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Blue Buffalo Blue Wilderness Large Breed Adult Salmon Recipe Dry Dog Food
Blue Buffalo Blue Wilderness Large Breed Adult Salmon Recipe Dry Dog Food
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- Weight24 LBS
BLUE Wilderness Grain Free Large Breed Adult Salmon dog food is a high protein low carb dog food with all natural and healthy ingredients. Wilderness salmon is the best dog food for your active pet.


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HomeReady® Mortgage Program is a Hot Topic with Buyers!
This July, the HomeReady program will broaden its income requirements even more, making the idea of homeownership possible for low to moderate income buyers. What is HomeReady? HomeReady® offers low down payments, and flexible sources of funds for buyers, making it a good option for first time home buyers, or anyone who needs expanded options […] …read more
4 Great Reasons to Consider Buying a Home Today Instead of Waiting
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Home Affordability-What is the cost of waiting?
Home Affordability Always the biggest question when someone is looking to buy a home. "How much do I qualify for"? The true question you are asking is "what is my home affordability?". Per dictionary.com, Affordable: Adjective 1. That can be afforded; believed to be within one's financial means: noun 2. Usually affordable. Items expenses,etc., […] …read more
Purchasing a home in Colorado's competive market
Purchasing a home in Colorado's competitive market takes strategy. Many of you think that the only way to purchase in this market is that you have more money to put down and great credit. This is not necessarily the case. Let's go over some facts and how to put yourself into a better position […] …read more
Denver Real Estate- Rising Mortgage Rates – Have You Concerned?
Here we are, the days are getting longer! You would think the Denver real estate market would be about to wake up from its winter slumber. Wait this is not 2012. So, unless you have been under a rock, that is not the case in Denver. The news to start 2018 in Denver real estate has been […] …read more