Receptor-like kinase SOBIR1/EVR interacts with receptor-like proteins in plant immunity against fungal infection.

The plant immune system is activated by microbial patterns that are detected as nonself molecules. Such patterns are recognized by immune receptors that are cytoplasmic or localized at the plasma membrane. Cell surface receptors are represented by receptor-like kinases (RLKs) that frequently contain extracellular leucine-rich repeats and an intracellular kinase domain for activation of downstream signaling, as well as receptor-like proteins (RLPs) that lack this signaling domain. It is therefore hypothesized that RLKs are required for RLPs to activate downstream signaling. The RLPs Cf-4 and Ve1 of tomato (Solanum lycopersicum) mediate resistance to the fungal pathogens Cladosporium fulvum and Verticillium dahliae, respectively. Despite their importance, the mechanism by which these immune receptors mediate downstream signaling upon recognition of their matching ligand, Avr4 and Ave1, remained enigmatic. Here we show that the tomato ortholog of the Arabidopsis thaliana RLK Suppressor Of BIR1-1/Evershed (SOBIR1/EVR) and its close homolog S. lycopersicum (Sl)SOBIR1-like interact in planta with both Cf-4 and Ve1 and are required for the Cf-4- and Ve1-mediated hypersensitive response and immunity. Tomato SOBIR1/EVR interacts with most of the tested RLPs, but not with the RLKs FLS2, SERK1, SERK3a, BAK1, and CLV1. SOBIR1/EVR is required for stability of the Cf-4 and Ve1 receptors, supporting our observation that these RLPs are present in a complex with SOBIR1/EVR in planta. We show that SOBIR1/EVR is essential for RLP-mediated immunity and propose that the protein functions as a regulatory RLK of this type of cell-surface receptors.

Optimized agroinfiltration and virus-induced gene silencing to study Ve1-mediated Verticillium resistance in tobacco.

Recognition of pathogen effectors by plant immune receptors often leads to the activation of a hypersensitive response (HR), which is a rapid and localized cell death of plant tissue surrounding the site at which recognition occurs. Due to its particular amenability to transient assays for functional genetics, tobacco is a model for immune signaling in the Solanaceae plant family. Here, we show that coexpression of the tomato (Solanum lycopersicum) immune receptor Ve1 and the corresponding Verticillium effector protein Ave1 leads to HR only in particular tobacco species. Whereas HR is obtained in Nicotiana tabacum, no such response is obtained in N. benthamiana. Furthermore, our analysis revealed an endogenous Ve1 ortholog in Nicotiana glutinosa, as expression of Ave1 in absence of Ve1 induced a HR, and N. glutinosa was found to be resistant against race 1 Verticillium dahliae. We furthermore report the establishment of virus-induced gene silencing in N. tabacum for functional analysis of Ve1 signaling. Collectively, our data show that N. tabacum can be used as a model plant to study Ve1-mediated immune signaling.

RT-PCR detection of respiratory pathogens in newborn children admitted to a neonatal medium care unit.

BACKGROUND: This study aimed to determine the prevalence of respiratory pathogens among newborns admitted to a neonatal medium care unit (NMCU) and to identify clinical predictors. METHODS: A 1-y observational study was performed of neonates admitted to an NMCU in Amsterdam, The Netherlands. Nasopharyngeal samples were collected for the detection of respiratory viruses and bacteria by real-time PCR (RT-PCR). Cycle threshold (Ct) values were provided to estimate viral load. Predictors for the presence of study pathogens were identified. RESULTS: From October 2010 through September 2011, 334 neonates (median age 1.3 d, 53.6% male) were included. Overall, 37 respiratory pathogens were detected in 34 children (10.2%): parainfluenza-1 (n = 9), human rhinovirus (n = 7), parainfluenza-3 (n = 6), respiratory syncytial virus (RSV, n = 6), Streptococcus pneumoniae (n = 3), adenovirus (n = 2), human coronavirus (n = 2), influenza A (n = 1), and bocavirus (n = 1). Neonates with higher viral loads (Ct <35; n = 11) were more often clinically ill than those with lower viral loads (Ct ≥35; n = 23). Two variables significantly contributed to the detection of study pathogens: age (odds ratio (OR) 1.21 for each day older; 95% confidence interval 1.12-1.30) and rhinorrhea (OR 6.71; 95% confidence interval 1.54-29.21). CONCLUSION: Respiratory pathogens seem to play a role in neonates admitted to an NMCU. The influence of respiratory pathogen detection on clinical management remains to be determined.

Therapeutic drug monitoring of antiretroviral drugs at home using dried blood spots: a proof-of-concept study.

BACKGROUND: In HIV-infected patients, therapeutic drug monitoring (TDM) of antiretroviral drugs is recommended in special populations and in specific situations to optimize therapy. Currently, TDM is performed via measurement of drug plasma concentrations; however, dried blood spots (DBS) may offer a patient friendly and cost-effective alternative. Therefore, this proof-of-concept study assessed the feasibility of TDM of antiretroviral drugs using DBS with sampling at home. METHODS: Included patients were instructed to sample three DBS just before drug intake at three consecutive days at home and one DBS sample together with their routine venous sampling. All samples were analysed using liquid chromatography coupled to tandem mass spectrometry. Feasibility was investigated with a questionnaire and via determination of the calculated plasma trough concentrations. RESULTS: In total, 50 patients (48 male, mean age 50 years [range 29-69]) have been included, of whom most were virologically and immunologically well-controlled. In total, 200 DBS were collected, of which 87.5% were suitable for analysis. The questionnaire showed that most patients (68%) successfully obtained their first DBS and 51% preferred DBS over plasma sampling. Plasma trough concentrations could adequately be determined from DBS. CONCLUSIONS: This proof-of-concept study confirms the feasibility of TDM of antiretroviral drugs using DBS with sampling at home, thereby opening the possibility to obtain trough concentrations at home in populations where venous sampling is difficult.

Endoplasmic reticulum-quality control chaperones facilitate the biogenesis of Cf receptor-like proteins involved in pathogen resistance of tomato.

Cf proteins are receptor-like proteins (RLPs) that mediate resistance of tomato (Solanum lycopersicum) to the foliar pathogen Cladosporium fulvum. These transmembrane immune receptors, which carry extracellular leucine-rich repeats that are subjected to posttranslational glycosylation, perceive effectors of the pathogen and trigger a defense response that results in plant resistance. To identify proteins required for the functionality of these RLPs, we performed immunopurification of a functional Cf-4-enhanced green fluorescent protein fusion protein transiently expressed in Nicotiana benthamiana, followed by mass spectrometry. The endoplasmic reticulum (ER) heat shock protein70 binding proteins (BiPs) and lectin-type calreticulins (CRTs), which are chaperones involved in ER-quality control, were copurifying with Cf-4-enhanced green fluorescent protein. The tomato and N. benthamiana genomes encode four BiP homologs and silencing experiments revealed that these BiPs are important for overall plant viability. For the three tomato CRTs, virus-induced gene silencing targeting the plant-specific CRT3a gene resulted in a significantly compromised Cf-4-mediated defense response and loss of full resistance to C. fulvum. We show that upon knockdown of CRT3a the Cf-4 protein accumulated, but the pool of Cf-4 protein carrying complex-type N-linked glycans was largely reduced. Together, our study on proteins required for Cf function reveals an important role for the CRT ER chaperone CRT3a in the biogenesis and functionality of this type of RLP involved in plant defense.

Use of dried blood spots for the determination of plasma concentrations of nevirapine and efavirenz.

OBJECTIVES: Plasma concentrations are frequently used for therapeutic drug monitoring of antiretroviral drugs. Dried blood spot sampling offers a patient-friendly and easy alternative to plasma sampling. However, dried blood spot concentrations are not necessarily equal to plasma concentrations and therefore the objective of this work was to establish the relationship between nevirapine and efavirenz dried blood spot and plasma concentrations to facilitate clinical implementation of dried blood spot sampling. METHODS: Paired dried blood spot and plasma samples were obtained from 40 HIV-infected patients on nevirapine and 40 on efavirenz treatment. All samples were analysed using validated HPLC-tandem mass spectrometry methods for the two matrices. Theoretical plasma concentrations were calculated from dried blood spot concentrations using the formula [dried blood spot concentration/(1 - haematocrit)] × fraction bound to plasma proteins = plasma concentration. Linear regression and Bland-Altman analysis were used to compare the two methods. RESULTS: Dried blood spot and plasma concentrations of nevirapine and efavirenz correlated well (r(2) = 0.867 and 0.972, respectively), although efavirenz dried blood spot concentrations were 39.8% (SD 7.1%) lower than plasma concentrations. Theoretical plasma concentrations (using patient-specific haematocrit) of nevirapine and efavirenz were similar to measured plasma concentrations, with a mean difference between the two methods of 0.29 mg/L (SD 1.35 mg/L) and 0.08 mg/L (SD 0.31 mg/L), respectively. CONCLUSIONS: Dried blood spot concentrations of nevirapine and efavirenz were equal to plasma concentrations after correction for haematocrit and compound-specific plasma protein binding and can therefore be used in clinical practice.

Low attack rate of novel influenza A (H1N1) virus infection among healthcare workers: a prospective study in a setting with an elaborated containment plan.

PURPOSE: This study aimed to determine incidence rates of novel influenza A (H1N1) infection among healthcare personnel with different exposure risks during the 2009 H1N1 pandemic. METHODS: From August 2009 until April 2010, 66 healthcare workers from a 410 bed teaching hospital in Amsterdam were monitored. The following three different exposure groups were created: a high- (n = 26), intermediate- (n = 20), and low-risk group (n = 20). Throat swabs were collected each week and analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in order to detect the H1N1 virus. Blood was drawn at study enrollment and once monthly thereafter, and serum specimens were tested with an H1N1-specific hemagglutination-inhibition serologic assay. Influenza-like signs and symptoms were assessed weekly. RESULTS: One of 26 high-risk group participants proved H1N1 positive once by RT-PCR. This corresponds to an incidence rate in the high-risk group of 5.7/1,000 person weeks (95% CI 0-17/1,000). None of the intermediate- and low-risk group participants proved H1N1 positive by RT-PCR. Significant antibody titer rises in convalescent sera were demonstrated in three participants: one was a confirmation of the case that had proved H1N1 positive by RT-PCR; the others occurred in two asymptomatic participants belonging to the low- and high-risk groups. An influenza-like illness was assumed in four participants from the high- (n = 1), intermediate- (n = 1) and low-risk (n = 2) groups; these findings were not confirmed by positive results from either diagnostic test. CONCLUSIONS: This study demonstrates a low incidence rate of influenza A (H1N1) infection among healthcare workers during the 2009 H1N1 pandemic in a setting with high hygiene standards.

Characterization of 2009 H1N1 pandemic influenza in a population of Dutch children with influenza-like signs and symptoms.

AIM: To determine causative respiratory pathogens and describe epidemiological and clinical characteristics in a paediatric population with influenza-like illness during the 2009 H1N1-pandemic. METHODS: Observational study of 412 children visiting an outpatient clinic of a Dutch teaching hospital. RESULTS: From August to December 2009, 412 children were tested at the clinic; 32% proved H1N1-positive, confirmed by reverse-transcriptase-polymerase-chain-reaction (RT-PCR). Pathogens were detected in 65% of samples. Influenza A(H1N1) (n = 132), human rhinovirus (n = 55), respiratory syncytial virus (n = 45) and adenovirus (n = 34) were mostly identified. Co-infections were seen in 34 children (8.3%). Mean age was 6.8 and 4.2 years in H1N1-positive and H1N1-negative cases, respectively (p < 0.01). H1N1-positive outpatient children reported fever, cough and rhinorrhoea more frequently than their H1N1-negative counterparts. Of 72 hospitalized children, 31% proved H1N1-positive; all showed a relatively mild clinical illness. None of the children had been admitted to an intensive care unit or died. Oseltamivir treatment was initiated in 72 children and discontinued in 42 (63%) when RT-PCR results turned negative. CONCLUSION: The 2009 H1N1-pandemic showed a mild clinical course in a Dutch paediatric outpatient clinic population. Respiratory pathogens were detected in the majority of children with influenza-like illness and influenza A(H1N1) virus was identified in one-third. Testing symptomatic children during an influenza pandemic has effectively limited the use of oseltamivir.

The Arabidopsis thaliana DNA-binding protein AHL19 mediates verticillium wilt resistance.

Verticillium spp. are destructive soilborne fungal pathogens that cause vascular wilt diseases in a wide range of plant species. Verticillium wilts are particularly notorious, and genetic resistance in crop plants is the most favorable means of disease control. In a gain-of-function screen using an activation-tagged Arabidopsis mutant collection, we identified four mutants, A1 to A4, which displayed enhanced resistance toward the vascular wilt species Verticillium dahliae, V. albo-atrum and V. longisporum but not to Fusarium oxysporum f. sp. raphani. Further testing revealed that mutant A2 displayed enhanced Ralstonia solanacearum resistance, while mutants A1 and A3 were more susceptible toward Pseudomonas syringae pv. tomato. Identification of the activation tag insertion site in the A1 mutant revealed an insertion in close proximity to the gene encoding AHL19, which was constitutively expressed in the mutant. AHL19 knock-out alleles were found to display enhanced Verticillium susceptibility whereas overexpression of AHL19 resulted in enhanced Verticillium resistance, showing that AHL19 acts as a positive regulator of plant defense.

Adult outpatient experience of the 2009 H1N1 pandemic: clinical course, pathogens, and evaluation of case definitions.

OBJECTIVES: The aim was to describe causative agents and clinical characteristics in adult outpatients with upper airway symptoms during the 2009 H1N1 pandemic and to evaluate case definitions that are used in clinical practice. METHODS: From August through December 2009, 964 symptomatic adult outpatients were included. RT-PCR was used to detect the following pathogens: influenza A (H1N1) and B, parainfluenza 1-4, adenovirus, respiratory syncytial virus, human rhinovirus, human metapneumovirus, human coronavirus (OC43, 229E, NL63), Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella species. The Dutch GHOR, American CDC and WHO, and British HPA case definitions were evaluated. RESULTS: A respiratory pathogen was detected in 41% of tested patient samples; influenza A (H1N1) and human rhinovirus were both detected in 16%. Clinical presentation of influenza cases was significantly more serious when compared to rhinovirus or negative-tested cases. Test characteristics were almost similar for all 4 case definitions, with an average sensitivity of 66%, specificity of 70%, positive predictive value of 34% and negative predictive value of 90%. CONCLUSIONS: Influenza A (H1N1) and human rhinovirus were the major pathogens responsible for respiratory disease. The 2009 H1N1 pandemic in Amsterdam followed a mild course. Test characteristics of 4 different clinical case definitions seemed comparable but rather useless.

Economic evaluation of uterine artery embolization versus hysterectomy in the treatment of symptomatic uterine fibroids: results from the randomized EMMY trial.

PURPOSE: To investigate whether uterine artery embolization (UAE) is a cost-effective alternative to hysterectomy for patients with symptomatic uterine fibroids, the authors performed an economic evaluation alongside the multicenter randomized EMMY (EMbolization versus hysterectoMY) trial. MATERIALS AND METHODS: Between February 2002 and February 2004, 177 patients were randomized to undergo UAE (n = 88) or hysterectomy (n = 89) and followed up until 24 months after initial treatment allocation. Conditional on the equivalence of clinical outcome, a cost minimization analysis was performed according to the intention to treat principle. Costs included health care costs inside and outside the hospital as well as costs related to absence from work (societal perspective). Cumulative standardized costs were estimated as volumes multiplied with prices. The nonparametric bootstrap method was used to quantify differences in mean (95% confidence interval [CI]) costs between the strategies. RESULTS: In total, 81 patients underwent UAE and 75 underwent hysterectomy. In the UAE group, 19 patients (23%) underwent secondary hysterectomies. The mean total costs per patient in the UAE group were significantly lower than those in the hysterectomy group ($11,626 vs $18,563; mean difference, -$6,936 [-37%], 95% CI: -$9,548, $4,281). The direct medical in-hospital costs were significantly lower in the UAE group: $6,688 vs $8,313 (mean difference, -$1,624 [-20%], 95% CI: -$2,605, -$586). Direct medical out-of-hospital and direct nonmedical costs were low in both groups (mean cost difference, $156 in favor of hysterectomy). The costs related to absence from work differed significantly between the treatment strategies in favor of UAE (mean difference, -$5,453; 95% CI: -$7,718, -$3,107). The costs of absence from work accounted for 79% of the difference in total costs. CONCLUSIONS: The 24-month cumulative cost of UAE is lower than that of hysterectomy. From a societal economic perspective, UAE is the superior treatment strategy in women with symptomatic uterine fibroids.

Medicago LYK3, an entry receptor in rhizobial nodulation factor signaling.

Rhizobia secrete nodulation (Nod) factors, which set in motion the formation of nitrogen-fixing root nodules on legume host plants. Nod factors induce several cellular responses in root hair cells within minutes, but also are essential for the formation of infection threads by which rhizobia enter the root. Based on studies using bacterial mutants, a two-receptor model was proposed, a signaling receptor that induces early responses with low requirements toward Nod factor structure and an entry receptor that controls infection with more stringent demands. Recently, putative Nod factor receptors were shown to be LysM domain receptor kinases. However, mutants in these receptors, in both Lotus japonicus (nfr1 and nfr5) and Medicago truncatula (Medicago; nfp), do not support the two-receptor model because they lack all Nod factor-induced responses. LYK3, the putative Medicago ortholog of NFR1, has only been studied by RNA interference, showing a role in infection thread formation. Medicago hair curling (hcl) mutants are unable to form curled root hairs, a step preceding infection thread formation. We identified the weak hcl-4 allele that is blocked during infection thread growth. We show that HCL encodes LYK3 and, thus, that this receptor, besides infection, also controls root hair curling. By using rhizobial mutants, we also show that HCL controls infection thread formation in a Nod factor structure-dependent manner. Therefore, LYK3 functions as the proposed entry receptor, specifically controlling infection. Finally, we show that LYK3, which regulates a subset of Nod factor-induced genes, is not required for the induction of NODULE INCEPTION.

NSP1 of the GRAS protein family is essential for rhizobial Nod factor-induced transcription.

Rhizobial Nod factors induce in their legume hosts the expression of many genes and set in motion developmental processes leading to root nodule formation. Here we report the identification of the Medicago GRAS-type protein Nodulation signaling pathway 1 (NSP1), which is essential for all known Nod factor-induced changes in gene expression. NSP1 is constitutively expressed, and so it acts as a primary transcriptional regulator mediating all known Nod factor-induced transcriptional responses, and therefore, we named it a Nod factor response factor.

LysM domain receptor kinases regulating rhizobial Nod factor-induced infection.

The rhizobial infection of legumes has the most stringent demand toward Nod factor structure of all host responses, and therefore a specific Nod factor entry receptor has been proposed. The SYM2 gene identified in certain ecotypes of pea (Pisum sativum) is a good candidate for such an entry receptor. We exploited the close phylogenetic relationship of pea and the model legume Medicago truncatula to identify genes specifically involved in rhizobial infection. The SYM2 orthologous region of M. truncatula contains 15 putative receptor-like genes, of which 7 are LysM domain-containing receptor-like kinases (LYKs). Using reverse genetics in M. truncatula, we show that two LYK genes are specifically involved in infection thread formation. This, as well as the properties of the LysM domains, strongly suggests that they are Nod factor entry receptors.

Novel insight into vascular, stress, and auxin-dependent and -independent gene expression programs in strawberry, a non-climacteric fruit.

Using cDNA microarrays, a comprehensive investigation of gene expression was carried out in strawberry (Fragaria x ananassa) fruit to understand the flow of events associated with its maturation and non-climacteric ripening. We detected key processes and novel genes not previously associated with fruit development and ripening, related to vascular development, oxidative stress, and auxin response. Microarray analysis during fruit development and in receptacle and seed (achene) tissues established an interesting parallelism in gene expression between the transdifferentiation of tracheary elements in Zinnia elegans and strawberry. One of the genes, CAD, common to both systems and encoding the lignin-related protein cinnamyl alcohol dehydrogenase, was immunolocalized to immature xylem cells of the vascular bundles in the strawberry receptacle. To examine the importance of oxidative stress in ripening, gene expression was compared between fruit treated on-vine with a free radical generator and non-treated fruit. Of 46 genes induced, 20 were also ripening regulated. This might suggest that active gene expression is induced to cope with oxidative stress conditions during ripening or that the strawberry ripening transcriptional program is an oxidative stress-induced process. To gain insight into the hormonal control of non-climacteric fruit ripening, an additional microarray experiment was conducted comparing gene expression in fruit treated exogenously with auxin and control fruit. Novel auxin-dependent genes and processes were identified in addition to transcriptional programs acting independent of auxin mainly related to cell wall metabolism and stress response.