Deep proteogenomics; high throughput gene validation by multidimensional liquid chromatography and mass spectrometry of proteins from the fungal wheat pathogen Stagonospora nodorum

BACKGROUND: Stagonospora nodorum, a fungal ascomycete in the class dothideomycetes, is a damaging pathogen of wheat. It is a model for necrotrophic fungi that cause necrotic symptoms via the interaction of multiple effector proteins with cultivar-specific receptors. A draft genome sequence and annotation was published in 2007. A second-pass gene prediction using a training set of 795 fully EST-supported genes predicted a total of 10762 version 2 nuclear-encoded genes, with an additional 5354 less reliable version 1 genes also retained. RESULTS: In this study, we subjected soluble mycelial proteins to proteolysis followed by 2D LC MALDI-MS/MS. Comparison of the detected peptides with the gene models validated 2134 genes. 62% of these genes (1324) were not supported by prior EST evidence. Of the 2134 validated genes, all but 188 were version 2 annotations. Statistical analysis of the validated gene models revealed a preponderance of cytoplasmic and nuclear localised proteins, and proteins with intracellularassociated GO terms. These statistical associations are consistent with the source of the peptides used in the study. Comparison with a 6-frame translation of the S. nodorum genome assembly confirmed 905 existing gene annotations (including 119 not previously confirmed) and provided evidence supporting 144 genes with coding exon frameshift modifications, 604 genes with extensions of coding exons into annotated introns or untranslated regions (UTRs), 3 new gene annotations which were supported by tblastn to NR, and 44 potential new genes residing within un-assembled regions of the genome. CONCLUSION: We conclude that 2D LC MALDI-MS/MS is a powerful, rapid and economical tool to aid in the annotation of fungal genomic assemblies

Single-trial event-related potential extraction through one-unit ICA-with-reference.

Objective: In recent years, ICA has been one of the more popular methods for extracting event-related potential (ERP) at the single-trial level. It is a blind source separation technique that allows the extraction of an ERP without making strong assumptions on the temporal and spatial characteristics of an ERP. However, the problem with traditional ICA is that the extraction is not direct and is time-consuming due to the need for source selection processing. In this paper, the application of an one-unit ICA-with-Reference (ICA-R), a constrained ICA method, is proposed. Approach: In cases where the time-region of the desired ERP is known a priori, this time information is utilized to generate a reference signal, which is then used for guiding the one-unit ICA-R to extract the source signal of the desired ERP directly. Main results: Our results showed that, as compared to traditional ICA, ICA-R is a more effective method for analysing ERP because it avoids manual source selection and it requires less computation thus resulting in faster ERP extraction. Significance: In addition to that, since the method is automated, it reduces the risks of any subjective bias in the ERP analysis. It is also a potential tool for extracting the ERP in online application

Floor vibration serviceability in a multistory factory building

ArticleExperimental and analytical modal analysis and in-operation vibration measurements were performed on the massive concrete structural floors of several structurally connected ‘units’ of a six-level, multitenant industrial complex with total floor usable area exceeding 0.1 km2. The aim of the systematic study was to characterize vibration sources and factors that affect vibration serviceability, which is a major concern when changing usage patterns lead to conflicting requirements for vibration generation and tolerance for different types of industrial/ commercial user. This was a rare investigation aiming to provide information on specific performance and relevant technologies for occupancy decisions by tenants and building management of similar structures. Floors evaluated were within different types of industrial singleoccupant unit stacked up to six levels and having multibay floors with spans up to 12 m with first vibration mode frequencies greater than 8 Hz. These “high-frequency floors” display typical transient response behavior to footfalls, with response levels controlled by modal mass. Units were studied in typical operational conditions including warehousing, instrument assembly and testing, light electronic/mechanical manufacturing, and machining. Vibration sources included internal and external vehicles, human footfalls, and machinery. The study showed the most onerous form of loading to be forklift trucks and that higher level floors of the same type were least serviceable. Experimental modal analysis showed a surprising range of modal properties for nominally identical floors of the same type and the relevance to performance of modal mass

Aqueous Angiography with Fluorescein and Indocyanine Green in Bovine Eyes.

PurposeWe characterize aqueous angiography as a real-time aqueous humor outflow imaging (AHO) modality in cow eyes with two tracers of different molecular characteristics.MethodsCow enucleated eyes (n = 31) were obtained and perfused with balanced salt solution via a Lewicky AC maintainer through a 1-mm side-port. Fluorescein (2.5%) or indocyanine green (ICG; 0.4%) were introduced intracamerally at 10 mm Hg individually or sequentially. With an angiographer, infrared and fluorescent images were acquired. Concurrent anterior segment optical coherence tomography (OCT) was performed, and fixable fluorescent dextrans were introduced into the eye for histologic analysis of angiographically positive and negative areas.ResultsAqueous angiography in cow eyes with fluorescein and ICG yielded high-quality images with segmental patterns. Over time, ICG maintained a better intraluminal presence. Angiographically positive, but not negative, areas demonstrated intrascleral lumens with anterior segment OCT. Aqueous angiography with fluorescent dextrans led to their trapping in AHO pathways. Sequential aqueous angiography with ICG followed by fluorescein in cow eyes demonstrated similar patterns.ConclusionsAqueous angiography in model cow eyes demonstrated segmental angiographic outflow patterns with either fluorescein or ICG as a tracer.Translational relevanceFurther characterization of segmental AHO with aqueous angiography may allow for intelligent placement of trabecular bypass minimally invasive glaucoma surgeries for improved surgical results

SUMO-1 targets RanGAP1 to kinetochores and mitotic spindles

RanGAP1 was the first documented substrate for conjugation with the ubiquitin-like protein SUMO-1. However, the functional significance of this conjugation has not been fully clarified. We sought to examine RanGAP1 behavior during mitosis. We found that RanGAP1 associates with mitotic spindles and that it is particularly concentrated at foci near kinetochores. Association with kinetochores appeared soon after nuclear envelope breakdown and persisted until late anaphase, but it was lost coincident with nuclear envelope assembly in telophase. A mutant RanGAP1 protein lacking the capacity to be conjugated to SUMO-1 no longer associated with spindles, indicating that conjugation was essential for RanGAP1's mitotic localization. RanBP2, a nuclear pore protein that binds SUMO-1–conjugated RanGAP1 during interphase, colocalized with RanGAP1 on spindles, suggesting that a complex between these two proteins may be involved in mitotic targeting of RanGAP1. This report shows for the first time that SUMO-1 conjugation is required for mitotic localization of RanGAP1, and suggests that a major role of SUMO-1 conjugation to RanGAP1 may be the spatial regulation of the Ran pathway during mitosis

Long-range forces between two excited mercury atoms and associative ionization

The long-range quadrupole-quadrupole ($\sim R^{-5}$) and leading dispersion ($\sim R^{-6}$) interactions between all pairs of excited Hg($6s6p$) $^3P_0$, $^3P_1$, $^3P_2$, and $^1P_1$ atoms are determined. The quadrupole moments are calculated using the {\it ab initio} relativistic configuration-interaction method coupled with many-body perturbation theory. The van der Waals coefficients are approximated using previously calculated static polarizabilities and expressions for the dispersion energy that are validated with similar systems. The long-range interactions are critical for associative ionization in thermal and cold collisions, and are found to be quite different for different pairs of interacting states. Based on this knowledge and the short-range parts of previously calculated potential curves, improved estimates of the chemi-ionization cross sections are obtained.Comment: accepted in Phys Rev

In vivo and ex vivo regulation of visfatin production by leptin in human and murine adipose tissue : role of mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways

Visfatin is an adipogenic adipokine with increased levels in obesity, properties common to leptin. Thus, leptin may modulate visfatin production in adipose tissue (AT). Therefore, we investigated the effects of leptin on visfatin levels in 3T3-L1 adipocytes and human/murine AT, with or without a leptin antagonist. The potential signaling pathways and mechanisms regulating visfatin production in AT was also studied. Real-time RT-PCR and Western blotting were used to assess the relative mRNA and protein expression of visfatin. ELISA was performed to measure visfatin levels in conditioned media of AT explants, and small interfering RNA technology was used to reduce leptin receptor expression. Leptin significantly (P < 0.01) increased visfatin levels in human and murine AT with a maximal response at leptin 10–9 M, returning to baseline at leptin 10–7 M. Importantly, ip leptin administration to C57BL/6 ob/ob mice further supported leptin-induced visfatin protein production in omental AT (P < 0.05). Additionally, soluble leptin receptor levels rose with concentration dependency to a maximal response at leptin 10–7 M (P < 0.01). The use of a leptin antagonist negated the induction of visfatin and soluble leptin receptor by leptin. Furthermore, leptin-induced visfatin production was significantly decreased in the presence of MAPK and phosphatidylinositol 3-kinase inhibitors. Also, when the leptin receptor gene was knocked down using small interfering RNA, leptin-induced visfatin expression was significantly decreased. Thus, leptin increases visfatin production in AT in vivo and ex vivo via pathways involving MAPK and phosphatidylinositol 3-kinase signaling. The pleiotropic effects of leptin may be partially mediated by visfatin

Genetic diversity of Asian water buffalo (Bubalus bubalis): mitochondrial DNA D-loop and cytochrome b sequence variation

Swamp and river buffalo mitochondrial DNA (mtDNA) was sequenced for 303 bp of the cytochrome b gene for 54 animals from 14 populations, and for 158 bp of the D-loop region for 80 animals from 11 populations. Only one cytochrome b haplotype was found in river buffalo. Of the four haplotypes identified in swamp buffalo, one found in all populations is apparently ancestral both to the other swamp haplotypes and to the river haplotype. The phylogenetic relationships among the 33 D-loop haplotypes, with a cluster of 11 found in swamp buffalo only, also support the evolution of domesticated swamp and river buffalo from an ancestral swamp-like animal, most likely represented today by the wild Asian buffalo (Bubalus arnee). The time of divergence of the swamp and river types, estimated from the D-loop data, is 28 000 to 87 000 years ago. We hypothesise that the species originated in mainland south-east Asia, and that it spread north to China and west to the Indian subcontinent, where the rive type evolved and was domesticated. Following domestication in China, the domesticated swamp buffalo spread through two separate routes, through Taiwan and the Philippines to the eastern islands of Borneo and Sulawesi, and south through mainland southeast Asia and then to the western islands of Indonesia