The tomato Prf complex is a molecular trap for bacterial effectors based on Pto transphosphorylation.

The major virulence strategy of phytopathogenic bacteria is to secrete effector proteins into the host cell to target the immune machinery. AvrPto and AvrPtoB are two such effectors from Pseudomonas syringae, which disable an overlapping range of kinases in Arabidopsis and Tomato. Both effectors target surface-localized receptor-kinases to avoid bacterial recognition. In turn, tomato has evolved an intracellular effector-recognition complex composed of the NB-LRR protein Prf and the Pto kinase. Structural analyses have shown that the most important interaction surface for AvrPto and AvrPtoB is the Pto P+1 loop. AvrPto is an inhibitor of Pto kinase activity, but paradoxically, this kinase activity is a prerequisite for defense activation by AvrPto. Here using biochemical approaches we show that disruption of Pto P+1 loop stimulates phosphorylation in trans, which is possible because the Pto/Prf complex is oligomeric. Both P+1 loop disruption and transphosphorylation are necessary for signalling. Thus, effector perturbation of one kinase molecule in the complex activates another. Hence, the Pto/Prf complex is a sophisticated molecular trap for effectors that target protein kinases, an essential aspect of the pathogen's virulence strategy. The data presented here give a clear view of why bacterial virulence and host recognition mechanisms are so often related and how the slowly evolving host is able to keep pace with the faster-evolving pathogen.

Improved sensitivity for molecular detection of bacterial and Candida infections in blood.

The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml whole-blood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections.

Dynamic changes of the Prf/Pto tomato resistance complex following effector recognition.

In both plants and animals, nucleotide-binding leucine-rich repeat (NLR) immune receptors play critical roles in pathogen recognition and activation of innate immunity. In plants, NLRs recognise pathogen-derived effector proteins and initiate effector-triggered immunity (ETI). However, the molecular mechanisms that link NLR-mediated effector recognition and downstream signalling are not fully understood. By exploiting the well-characterised tomato Prf/Pto NLR resistance complex, we identified the 14-3-3 proteins TFT1 and TFT3 as interacting partners of both the NLR complex and the protein kinase MAPKKKα. Moreover, we identified the helper NRC proteins (NLR-required for cell death) as integral components of the Prf /Pto NLR recognition complex. Notably our studies revealed that TFTs and NRCs interact with distinct modules of the NLR complex and, following effector recognition, dissociate facilitating downstream signalling. Thus, our data provide a mechanistic link between activation of immune receptors and initiation of downstream signalling cascades.

Prf immune complexes of tomato are oligomeric and contain multiple Pto-like kinases that diversify effector recognition.

Cytoplasmic recognition of pathogen virulence effectors by plant NB-LRR proteins leads to strong induction of defence responses termed effector triggered immunity (ETI). In tomato, a protein complex containing the NB-LRR protein Prf and the protein kinase Pto confers recognition of the Pseudomonas syringae effectors AvrPto and AvrPtoB. Although structurally unrelated, AvrPto and AvrPtoB interact with similar residues in the Pto catalytic cleft to activate ETI via an unknown mechanism. Here we show that the Prf complex is oligomeric, containing at least two molecules of Prf. Within the complex, Prf can associate with Pto or one of several Pto family members including Fen, Pth2, Pth3, or Pth5. The dimerization surface for Prf is the novel N-terminal domain, which also coordinates an intramolecular interaction with the remainder of the molecule, and binds Pto kinase or a family member. Thus, association of two Prf N-terminal domains brings the associated kinases into close promixity. Tomato lines containing Prf complexed with Pth proteins but not Pto possessed greater immunity against P. syringae than tomatoes lacking Prf. This demonstrates that incorporation of non-Pto kinases into the Prf complex extends the number of effector proteins that can be recognized.

Gibberellin biosynthesis and signalling during development of the strawberry receptacle.

The enlargement of receptacle cells during strawberry (Fragaria × ananassa) fruit development is a critical factor determining fruit size, with the increase in cell expansion being one of the most important physiological processes regulated by the phytohormone gibberellin (GA). Here, we studied the role of GA during strawberry fruit development by analyzing the endogenous content of bioactive GAs and the expression of key components of GA signalling and metabolism. Bioactive GA(1) , GA(3) and GA(4) were monitored during fruit development, with the content of GA(4) being extremely high in the receptacle, peaking at the white stage of development. •Genes with high homology to genes encoding GA pathway components, including receptors (FaGID1(GIBBERELLIN-INSENSITIVE DWARF1)b and FaGID1c), DELLA (FaRGA(REPRESSOR OF GA) and FaGAI(GA-INSENSITIVE)), and enzymes involved in GA biosynthesis (FaGA3ox) and catabolism (FaGA2ox), were identified, and their expression in different tissues and developmental stages of strawberry fruit was studied in detail. The expression of all of these genes showed a stage-specific pattern during fruit development and was highest in the receptacle. FaGID1c bound GA in vitro, interacted with FaRGA in vitro and in vivo, and increased GA responses when ectopically expressed in Arabidopsis. This study thus reveals key elements of GA responses in strawberry and points to a critical role for GA in the development of the receptacle.

The IRIDICA BAC BSI Assay: Rapid, Sensitive and Culture-Independent Identification of Bacteria and Candida in Blood.

UNLABELLED: Bloodstream infection (BSI) and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. Current culture-based methods used to detect and identify agents of BSI are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. Existing methods for direct molecular detection of microbial DNA in blood are limited in either sensitivity (likely the result of small sample volumes) or in breadth of coverage, often because the PCR primers and probes used target only a few specific pathogens. There is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with BSI directly from uncultured whole blood samples. We have developed a method of extracting DNA from larger volumes of whole blood (5 ml per sample), amplifying multiple widely conserved bacterial and fungal genes using a mismatch- and background-tolerant PCR chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (PCR/ESI-MS). We describe the analytical characteristics of the IRIDICA BAC BSI Assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. The assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis), and twice the total number of positive detections. The described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours. DISCLAIMER: The IRIDICA BAC BSI Assay is not available in the United States.

Prevalence of hypertension and associated anthropometric risk factors in indigenous adults of Guatemala.

BACKGROUND: Hypertension (HT) epidemiological studies in developing regions of the world like rural Guatemala are lacking. METHODS: A sample size of 1104 subjects (552 females, all 18 years or older) was obtained through quota and geographical clustering in the entire Department of Sololá, Guatemala. Descriptive statistics and logistic regression were used. RESULTS: Average systolic, diastolic, and mean arterial pressures were significantly higher in men compared with women (116.24 vs 113.80 mm Hg, 75.24 vs 72.69 mm Hg, and 88.91 vs 86.39 mm Hg, respectively; all with P < .05). The crude prevalence of HT was 12.5% with no gender differences. Women had a significantly higher mean body mass index (BMI) than men (26.25 vs 24.71 kg/m(2), P < .001). An abnormally high waist circumference (WC) was found in 12.7% of men and in 50.7% of women. Significant associations were found between the presence of HT, age ≥55 years, and an elevated WC. The single most important isolated risk factor for HT was age in women (OR 6.76, 95% CI 3.59-12.72) and WC in men (OR 3.23, 95% CI 1.52-6.87). Increased BMIs (≥25-30 or ≥30 kg/m(2)) were not associated with HT in this study. Residing in Sololá's capital was a protective factor in women (OR 0.33, 95% CI 0.13-0.83). CONCLUSION: Hypertension and associated anthropometric risk factors are present in rural regions of Guatemala. Significant associations are found between gender, age ≥55 years, and increased WC but not with an increased BMI in this population.

Host inhibition of a bacterial virulence effector triggers immunity to infection.

Plant pathogenic bacteria secrete effector proteins that attack the host signaling machinery to suppress immunity. Effectors can be recognized by hosts leading to immunity. One such effector is AvrPtoB of Pseudomonas syringae, which degrades host protein kinases, such as tomato Fen, through an E3 ligase domain. Pto kinase, which is highly related to Fen, recognizes AvrPtoB in conjunction with the resistance protein Prf. Here we show that Pto is resistant to AvrPtoB-mediated degradation because it inactivates the E3 ligase domain. AvrPtoB ubiquitinated Fen within the catalytic cleft, leading to its breakdown and loss of the associated Prf protein. Pto avoids this by phosphorylating and inactivating the AvrPtoB E3 domain. Thus, inactivation of a pathogen virulence molecule is one mechanism by which plants resist disease.

Structural analysis of the PsbQ protein of photosystem II by Fourier transform infrared and circular dichroic spectroscopy and by bioinformatic methods.

The structure of PsbQ, one of the three main extrinsic proteins associated with the oxygen-evolving complex (OEC) of higher plants and green algae, is examined by Fourier transform infrared (FTIR) and circular dichroic (CD) spectroscopy and by computational structural prediction methods. This protein, together with two other lumenally bound extrinsic proteins, PsbO and PsbP, is essential for the stability and full activity of the OEC in plants. The FTIR spectra obtained in both H(2)O and D(2)O suggest a mainly alpha-helix structure on the basis of the relative areas of the constituents of the amide I and I' bands. The FTIR quantitative analyses indicate that PsbQ contains about 53% alpha-helix, 7% turns, 14% nonordered structure, and 24% beta-strand plus other beta-type extended structures. CD analyses indicate that PsbQ is a mainly alpha-helix protein (about 64%), presenting a small percentage assigned to beta-strand ( approximately 7%) and a larger amount assigned to turns and nonregular structures ( approximately 29%). Independent of the spectroscopic analyses, computational methods for protein structure prediction of PsbQ were utilized. First, a multiple alignment of 12 sequences of PsbQ was obtained after an extensive search in the public databases for protein and EST sequences. Based on this alignment, computational prediction of the secondary structure and the solvent accessibility suggest the presence of two different structural domains in PsbQ: a major C-terminal domain containing four alpha-helices and a minor N-terminal domain with a poorly defined secondary structure enriched in proline and glycine residues. The search for PsbQ analogues by fold recognition methods, not based on the secondary structure, also indicates that PsbQ is a four alpha-helix protein, most probably folding as an up-down bundle. The results obtained by both the spectroscopic and computational methods are in agreement, all indicating that PsbQ is mainly an alpha protein, and show the value of using both methodologies for protein structure investigation.

Caracterización epidemiológica de pacientes que presentan prueba citológica anómala persistente / Epidemiological characterization of patients with anomalous cervical citologic test

Se realizó estudios descriptivos a 50 mujeres que asistieron a la Consulta de Patología de Cuello del Policlínico Docente "Chiqui Gómez Lubián", en los que se encontró citodiagnóstico anómalo (Papanicolaou persistente), con el objetivo de determinar algunos factores epidemiológicos más relevantes que predisponen a una mayor incidencia del cáncer de cuello uterino. Mediante la revisión de historias clínicas, modelo 68-09, se obtuvieron datos en relación con sus antecedentes clínico-ginecológicos y sus resultados citohistológicos. Los factores de riesgo para la neoplasia intraepitelial cervical que han prevalecido en nuestro estudio fueron el inicio temprano de las relaciones sexuales y la presencia del papiloma virus humano. No se observó asociación de estas alteraciones con fumadoras ni con multíparas