A stochastic model for eye movements during fixation on a stationary target

A stochastic model describing small eye movements occurring during steady fixation on a stationary target is presented. Based on eye movement data for steady gaze, the model has a hierarchical structure; the principal level represents the random motion of the image point within a local area of fixation while the higher level mimics the jump processes involved in transitions from one local area to another. Target image motion within a local area is described by a Langevinlike stochastic differential equation taking into consideration, the microsaccadic jumps pictured as being due to point processes and the high frequency muscle tremor, represented as a white noise. The transform of the probability density function for local area motion is obtained, leading to explicit expressions for their means and moments. Evaluation of these moments based on the model are comparable with experimental results. A physiologically based criterion for the occurrence of local area changes is assumed and the renewal density of these transitions is obtained. These transitions are brought about by the occurrence of large saccades. Hence, our analysis leads us to derive expressions for the mean and moments of the occurrence of large saccades in a given time T. These predictions may be checked against experimental results.This investigation was supported by National Institutes of Health under Grants Numbers GM 16197-03, GM 16437, and RR-0712-04, by the National Science Foundation under Grant Number GK-1834X, and by the National Aeronautics and Space Administration under Grant Number NGL-05-018-022.R.Vasudevan and J.D. Smith are with the Department of Electrical Engineering, University of Southern California, Los Angeles, California. —R.Vasudevan is on leave of absence from the Institute of Mathematical Sciences, Madras, India.A.V. Phatak is now with Systems Control, Inc. in Palo Alto, California.     

Design, synthesis and pharmacological evaluation of novel tetrasubstituted thiophene analogues as anti-inflammatory agents

A new series of tetrasubstituted thiophene analogues (4a-4f, 5a-5f and 8a-8i) were designed incorporating the pharmacophoric features of COX-1 (as in fenamates), 5-LOX and the p38 MAP kinase inhibitors. The designed series was synthesized by nucleophilic addition of aryl/aroylisothiocyanate and enamine (2) yielding the addition product l-(alpha-Carbomethoxy-beta-aminothiocrotonoyl)-aryl/aroyl amines (3/7); which on reaction with substituted phenacyl bromides gave the targeted tetrasubstituted thiophene esters (4a-4f / 8a-8i). The tetrasubstituted thiophenes esters (4a-4f ) on hydrolysis with one equivalent of potassium hydroxide solution in methanol at room temperature gave corresponding acids (5a-5f ). All the targeted compounds were evaluated for their anti-inflammatory activity in carrageenin-induced rat hind paw oedema model at the doses of 10, 20 and 40 mg/kg body weight using standard drugs mefanamic acid and ibuprofen. The compounds (4c, 4e, 4f, 5f, 8a- 8i) which gave reasonable protection to the inflamed paw, eliciting good or moderate comparable anti-inflammatory activity were selected for investigating their analgesic activity using acetic acid induced writhing response test in albino mice at 10 mg/kg dose using standard drug ibuprofen and in order to arrive at possible mechanism of their anti-inflammatory activity, in vitro antioxidant nitric oxide radical scavenging assay at the concentrations of 5, 10, 15, 20, 25, 30 and 35 microg/mL were performed using standard drug ascorbic acid.     

In silico tools for predicting peptides binding to HLA-class II molecules: more confusion than conclusion

Identification of promiscuous peptides, which bind to human leukocyte antigen, is indispensable for global vaccination. However, the development of such vaccines is impaired due to the exhaustive polymorphism in human leukocyte antigens. The use of in silico tools for mining such peptides circumvents the expensive and laborious experimental screening methods. Nevertheless, the intrepid use of such tools warrants a rational assessment with respect to experimental findings. Here, we have adopted a 'bottom up' approach, where we have used experimental data to assess the reliability of existing in silico methods. We have used a data set of 179 peptides from diverse antigens and have validated six commonly used in silico methods; ProPred, MHC2PRED, RANKPEP, SVMHC, MHCPred, and MHC-BPS. We observe that the prediction efficiency of the programs is not balanced for all the HLA-DR alleles and there is extremely high level of discrepancy in the prediction efficiency apropos of the nature of the antigen. It has not escaped our notice that the in silico methods studied here are not very proficient in identifying promiscuous peptides. This puts a much constraint on the intrepid use of such programs for human leukocyte antigen class II binding peptides. We conclude from this study that the in silico methods cannot be wholly relied for selecting crucial peptides for development of vaccines.     

Characterization of resistance mechanism in transgenic Nicotiana benthamiana containing Turnip crinkle virus coat protein

Two transgenic lines, of Nicotiana benthamiana expressing Turnip crinkle virus (TCV)-coat protein (CP) gene with contrasting phenotype, the highest (#3) and the lowest (#18) CP expressers, were selected and challenged with the homologous TCV. The former, the highest expresser, showed nearly five times more CP expression than the latter. Progenies of #3 and #18 lines showed 30 and 100% infection rates, respectively. The infected progenies of #3 line showed mild and delayed symptom with TCV. This is a coat protein-mediated resistance (CP-MR), and its resistance level is directly proportional to CP transgene expression. However, CP-MR of the transgenic plants was specific only for TCV but not for heterologous viruses. Newly growing leaves of those infected progenies of #3 line did not show any visible symptoms at 4-week post-inoculation (wpi) with TCV, suggesting a reversal from infection. This was confirmed by RT-PCR analysis with the disappearance of the target at 4 wpi. This is a case of RNA-mediated resistance, and a threshold level of transgene expression may be needed to achieve the silent state. To confirm the RNA silencing, we infiltrated Agrobacterium carrying TCV-CP into leaves of progenies of #3 and performed RT-PCR analysis. The results indicate that TCV-CP’s suppressor activity against RNA silencing itself can be silenced by the homologous expression of TCV-CP in the transgenic plants. The transgenic plants containing TCV-CP seem to be a model system to study viral protection mediated by a combination of protein and RNA silencing. Ayyappan Vasudevan and Tae-Kyun Oh have contributed equally in this study.     

Tomato transcriptional changes in response to Clavibacter michiganensis subsp. michiganensis reveal a role for ethylene in disease development

Clavibacter michiganensis subsp. michiganensis (Cmm) is a gram-positive actinomycete, causing bacterial wilt and canker disease in tomato (Solanum lycopersicum). Host responses to gram-positive bacteria and molecular mechanisms associated with the development of disease symptoms caused by Cmm in tomato are largely unexplored. To investigate plant responses activated during this compatible interaction, we used microarray analysis to monitor changes in host gene expression during disease development. This analysis was performed at 4 d postinoculation, when bacteria were actively multiplying and no wilt symptoms were yet visible; and at 8 d postinoculation, when bacterial growth approached saturation and typical wilt symptoms were observed. Of the 9,254 tomato genes represented on the array, 122 were differentially expressed in Cmm-infected plants, compared with mock-inoculated plants. Functional classification of Cmm-responsive genes revealed that Cmm activated typical basal defense responses in the host, including induction of defense-related genes, production and scavenging of free oxygen radicals, enhanced protein turnover, and hormone synthesis. Cmm infection also induced a subset of host genes involved in ethylene biosynthesis and response. After inoculation with Cmm, Never ripe (Nr) mutant plants, impaired in ethylene perception, and transgenic plants with reduced ethylene synthesis showed significant delay in the appearance of wilt symptoms, compared with wild-type plants. The retarded wilting in Nr plants was a specific effect of ethylene insensitivity, and was not due to altered expression of defense-related genes, reduced bacterial populations, or decreased ethylene synthesis. Taken together, our results indicate that host-derived ethylene plays an important role in regulation of the tomato susceptible response to Cmm.     

Activation and manipulation of host responses by a Gram-positive bacterium

The interaction between tomato plants and Clavibacter michiganensis subsp. michiganensis (Cmm) represents a model pathosystem to study the interplay between the virulence determinants of a Gram-positive bacterium and the attempt of a crop plant to counteract pathogen invasion. To investigate plant responses activated during this compatible interaction, we recently analyzed gene expression profiles of tomato stems infected with Cmm. This analysis revealed activation of basal defense responses that are typically observed upon plant perception of pathogen-associated molecular patterns. In addition, Cmm infection upregulated the expression of host genes related to ethylene synthesis and response. Further analysis of tomato plants impaired in ethylene perception and production demonstrated an important role for ethylene in the development of disease symptoms. Here we discuss possible molecular strategies used by the plant to recognize Cmm infection and possible mechanisms employed by the pathogen to interfere with the activation of plant defense responses and promote disease.Key words: tomato, Clavibacter michiganensis subsp. michiganensis, ethylene, basal defense, pathogen-associated molecular patternsLittle is known on the strategies employed by Gram-positive phytopathogenic bacteria to sense the presence of the host plant, penetrate and colonize tissue, and counteract plant defense responses. Also largely unexplored are the molecular mechanisms associated with detection of Gram-positive bacteria by the host plant and with the activation of attempted defense responses.Among the most devastating Gram-positive disease agents are actinobacteria of the genus Clavibacter whose subspecies cause systemic infections of the xylem in different plant species.¹ The subspecies Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker of tomato (Solanum lycopersicum), an economically important disease causing yield losses worldwide.¹ In recent years important insight into the molecular mechanism of Cmm pathogenicity has been achieved,¹ and genome sequence of a Cmm strain has been established.² Major Cmm pathogenicity determinants are plasmid borne and include the β-1,4-endocellulase CelA,³ and the putative serine protease Pat-1.⁴ Additional genes important for virulence are located in a pathogenicity island of about 129 kb on the Cmm chromosome which has a relatively low G + C content and is required for effective Cmm colonization of tomato plants.²Tomato is an economically important crop amenable to genetic analysis and transformations. Many resources are available for this plant species, including germplasm collections, natural and induced mutants, an extensive expressed sequence tag database and an ongoing genome sequencing project.⁵ In addition, because of its experimental tractability, tomato plants have been widely used to study plant disease resistance and susceptibility. As genetic and molecular tools for both Cmm and tomato are in place, the tomato-Cmm pathosystem represents an excellent model to study the interplay between virulence determinants of a Gram-positive phytopathogenic bacterium and defense responses of a crop plant.To get insight into host responses occurring during the tomato-Cmm compatible interaction and molecular mechanisms associated with the development of wilt and canker disease symptoms, we recently analyzed gene expression profiles of tomato stems infected with Cmm.⁶ This analysis revealed a clear activation of basal defense responses, which are typically observed upon plant perception of pathogen-associated molecular patterns (PAMPs).⁷ These include production and scavenging of free oxygen radicals, induction of defense-related genes, enhanced protein turnover, and hormone biosynthesis. Interestingly, several tomato genes encoding proteins with characteristics of cell-surface receptors were differentially expressed in response to Cmm infection.⁶ These proteins can be considered as candidate receptors for Cmm PAMPs and include two receptor-like kinases, a homolog of the receptor for the fungal PAMP ethylene-inducing xylanase from Trichoderma viride,⁸ and the Ve1 resistance protein, which confers resistance in tomato to the vascular disease Verticillium wilt.⁹It remains to be elucidated what are the Cmm PAMPs perceived by tomato plants. Cold-shock protein from Gram-positive bacteria and different microbial patterns of Gram-negative bacteria, including lipopolysaccharides, flagellin, and the translational elongation factor EF-TU, were shown to act as PAMPs in plants.¹⁰ Similarly, Cmm cold shock protein or cell wall components, such as peptidoglycan, lipoteichoic acid, and lipopeptides, which function as Gram positive-derived PAMPs in animal systems¹¹, may act as PAMPs during the tomato-Cmm interaction. Additional possible Cmm PAMPs are exopolysaccharides, which are produced in large amounts by the bacterium and may interact directly with surface-exposed plant proteins.¹ The numerous extracellular cell wall degrading enzymes secreted by Cmm may also function as PAMPs, as observed for the fungal ethylene-inducing xylanase.^2,12 Alternatively, by virtue of their hydrolytic activity, these enzymes may release plant cell wall fragments that are recognized by PAMP receptors. Indeed, different β-glucan fragments released from plant cell walls were shown to elicit plant basal defense responses.^13,14How Cmm copes with the activation of basal defense responses is largely unknown. Many potential virulence determinants that might interfere with the plant defense reaction are clustered in the Cmm pathogenicity island, which is essential for effective plant colonization.² Several extracellular serine proteases are encoded in this region and inactivation of part of them by gene replacement drastically reduced Cmm colonization of tomato plants.² Although their targets are still unknown, these proteins might interfere with plant signaling pathways as it was described for certain cysteine proteases that serves in Gram-negative bacteria as suppressors of plant defenses.¹⁵ An additional candidate for interference with plant signaling may be a tomatinase, also encoded in the Cmm pathogenicity island, because hydrolysis products of α-tomatine were shown to suppress plant defense responses in a fungal system.¹⁶In addition to detecting the activation of basal defense responses, host gene expression profiling during the tomato-Cmm interaction unraveled the involvement of ethylene in disease development.⁶ In fact, Cmm infection of tomato stems was found to induce expression of host genes related to ethylene biosynthesis and response (Fig. 1).⁶ Further analysis of ethylene-insensitive Never ripe mutants and transgenic plants with reduced ethylene synthesis indicated that ethylene is required for normal development of wilting symptoms (Fig. 2), but not for the activation of defense-related genes or bacterial colonization.⁶ We hypothesize that during infection ethylene synthesis and response are manipulated by Cmm virulence determinants to promote disease. Alternatively, ethylene is released as part of the host responses activated by bacterial recognition, or as a result of tissue maceration. In line with our first hypothesis, the type III effectors AvrPto and AvrPtoB from Pseudomonas syringae pv. tomato were shown to promote enhanced disease symptoms in tomato leaves, in part, by upregulating genes involved in ethylene production.¹⁷ Interestingly, expression in tomato plants of AvrPto or AvrPtoB, and infection with Cmm resulted in the upregulation of the SlACO1 gene encoding the key enzyme of ethylene biosynthesis ACC oxidase.^6,17Open in a separate windowFigure 1Kinetics of ACC oxidase (ACO) gene expression in tomato plants inoculated with Cmm. Six-week-old tomato plants were infected with a Cmm suspension (10⁸ cfu/ml) or mock-inoculated. Total RNA was extracted from stem samples harvested at the indicated day post-inoculation (dpi) and subjected to Northern blot analysis using as probe a 550 bp fragment of the SlACO1 gene, which shares high homology with other ACO family members (upper). Ethidium bromide staining shows the amount of RNA loaded in each lane (lower).Open in a separate windowFigure 2Effect of impaired ethylene sensitivity on development of wilt symptoms in tomato plants infected with Cmm. Six-week-old plants were infected with a Cmm suspension (10⁸ cfu/ml) and examined for development of wilt symptoms during a 20-day period. The percentage of plants showing wilt symptoms was calculated in a group of at least 30 plants for the ethylene-insensitive mutant Never ripe and wild-type Pearson plants. Data are representative of two independent experiments.In conclusion, future research challenges for understanding how host responses are regulated by the plant and manipulated by a Gram-positive bacterium will be the isolation of Cmm PAMPs and their plant receptors, the identification of Cmm virulence determinants and the elucidation of their mode of action.