Comparative annotation of viral genomes with non-conserved gene structure

MOTIVATION: Detecting genes in viral genomes is a complex task. Due to the biological necessity of them being constrained in length, RNA viruses in particular tend to code in overlapping reading frames. Since one amino acid is encoded by a triplet of nucleic acids, up to three genes may be coded for simultaneously in one direction. Conventional hidden Markov model (HMM)-based gene-finding algorithms may typically find it difficult to identify multiple coding regions, since in general their topologies do not allow for the presence of overlapping or nested genes. Comparative methods have therefore been restricted to likelihood ratio tests on potential regions as to being double or single coding, using the fact that the constrictions forced upon multiple-coding nucleotides will result in atypical sequence evolution. Exploiting these same constraints, we present an HMM based gene-finding program, which allows for coding in unidirectional nested and overlapping reading frames, to annotate two homologous aligned viral genomes. Our method does not insist on conserved gene structure between the two sequences, thus making it applicable for the pairwise comparison of more distantly related sequences. RESULTS: We apply our method to 15 pairwise alignments of six different HIV2 genomes. Given sufficient evolutionary distance between the two sequences, we achieve sensitivity of approximately 84-89% and specificity of approximately 97-99.9%. We additionally annotate three pairwise alignments of the more distantly related HIV1 and HIV2, as well as of two different hepatitis viruses, attaining results of approximately 87% sensitivity and approximately 98.5% specificity. We subsequently incorporate prior knowledge by 'knowing' the gene structure of one sequence and annotating the other conditional on it. Boosting accuracy close to perfect we demonstrate that conservation of gene structure on top of nucleotide sequence is a valuable source of information, especially in distantly related genomes. AVAILABILITY: The Java code is available from the authors.     

Habitat of the corn leafhopper (Hemiptera: Cicadellidae) during the dry (winter) season in Mexico

Although the corn leafhopper Dalbulus maidis (DeLong and Wolcott) is the most important vector of maize pathogens in Latin America, little is known about how and where it overwinters (passes the dry season), particularly in Mexico. The objectives of this study were (1) to monitor the abundance of D. maidis adults throughout the dry season in maize and maize-free habitats and (2) to determine where and how D. maidis adults, exposed or nonexposed to the maize pathogen Spiroplasma kunkelii Whitcomb, overwinter in a maize-free habitat. Work for the first objective was done during the two consecutive dry seasons of 1999-2000 and 2000-2001; the second objective was done during the dry seasons of 2003-2004 and 2005-2006. During the dry winter seasons, D. maidis was prevalent as long as maize was present in irrigated areas. The leafhopper was found in 52 of the 58 irrigated maize fields sampled in Mexico at the end of the dry seasons of 1999-2000 and 2000-2001. However, leafhopper adults were not found in nonirrigated maize-free habitats at high elevation during the dry winter season (February, March, and April), although leafhopper adults were prevalent on perennial wild grasses in January after maize harvest. Additional experiments revealed, however, that corn leafhopper adults, although few in number, survived the entire dry season in these nonirrigated maize-free fields. Also, no detectable difference in survival existed between leafhoppers exposed and those not exposed to S. kunkelli during the two dry seasons in the maize-free habitat.     

Regulation of cell diameter, For3p localization, and cell symmetry by fission yeast Rho-GAP Rga4p

Control of cellular dimensions and cell symmetry are critical for development and differentiation. Here we provide evidence that the putative Rho-GAP Rga4p of Schizosaccharomyces pombe controls cellular dimensions. rga4 Delta cells are wider in diameter and shorter in length, whereas Rga4p overexpression leads to reduced diameter of the growing cell tip. Consistent with a negative role in cell growth control, Rga4p protein localizes to the cell sides in a "corset" pattern, and to the nongrowing cell tips. Additionally, rga4 Delta cells show an altered growth pattern similar to that observed in mutants of the formin homology protein For3p. Consistent with these observations, Rga4p is required for normal localization of For3p and for normal distribution of the actin cytoskeleton. We show that different domains of the Rga4p protein mediate diverse morphological functions. The C-terminal GAP domain mediates For3p localization to the cell tips and maintains cell diameter. Conversely, overexpression of the N-terminal LIM homology domain of Rga4p promotes actin cable formation in a For3p-dependent manner. Our studies indicate that Rga4p functionally interacts with For3p and has a novel function in the control of cell diameter and cell growth.     

Persistence of high-replicative capacity in cultured fibroblasts from nonagenarians

Earlier studies on human fibroblast cultures have demonstrated an inverse relationship between the total number of population doublings (PDs) and donor age. As more recent studies were unable to replicate these findings, we set out to analyze growth characteristics of fibroblast cultures from nonagenarians who represent the extreme of human lifespan. Therefore, we obtained skin biopsies from 68 participants of the Leiden 85-plus Study, all aged 90 years. None of the 68 strains failed to proliferate and all were easily cultured under highly standardized conditions. Within a time window of 30 months, all strains displayed a high and reproducible replicative capacity that was maintained for at least 50 PDs. A decline in mitotic activity was observed between 26 and 81 PDs. Out of the 68 cell strains, 58 strains reached the post-mitotic phase with an onset between 51 and 108 PDs. The growth pattern of each senescent strain was fitted by a piecewise linear model, which allowed calculation of the transition towards the phase of decreased growth speed, as well as by a nonlinear continuous model; goodness-of-fit was high and not different between the models (both > 0.99). Growth characteristics were not associated with morbidity or mortality of the donors. We conclude that fibroblasts from nonagenarians maintain a high-replicative capacity despite a huge variability in the onset of senescence. These results cast further doubt on the association between in vitro growth characteristics of fibroblast cultures and the length of human lifespan.     

Retinal ganglion cells can rapidly change polarity from Off to On

Retinal ganglion cells are commonly classified as On-center or Off-center depending on whether they are excited predominantly by brightening or dimming within the receptive field. Here we report that many ganglion cells in the salamander retina can switch from one response type to the other, depending on stimulus events far from the receptive field. Specifically, a shift of the peripheral image--as produced by a rapid eye movement--causes a brief transition in visual sensitivity from Off-type to On-type for approximately 100 ms. We show that these ganglion cells receive inputs from both On and Off bipolar cells, and the Off inputs are normally dominant. The peripheral shift strongly modulates the strength of these two inputs in opposite directions, facilitating the On pathway and suppressing the Off pathway. Furthermore, we identify certain wide-field amacrine cells that contribute to this modulation. Depolarizing such an amacrine cell affects nearby ganglion cells in the same way as the peripheral image shift, facilitating the On inputs and suppressing the Off inputs. This study illustrates how inhibitory interneurons can rapidly gate the flow of information within a circuit, dramatically altering the behavior of the principal neurons in the course of a computation.     

Identification and characterization of ErbB-3-binding protein-1 as a target for immunotherapy

Based on immune reactivity in response to a whole-cell colon tumor vaccine and using serological identification of Ags by recombinant cDNA expression cloning, we here describe the molecular and functional identification of a novel human tumor Ag. By screening a cDNA expression library derived from the coloncarcinoma cell line HT-29 with pooled colorectal cancer patients' sera, 26 clones reactive with IgG Abs could be identified. Characterization of these cDNA clones by sequence analysis and alignment, and detailed serological analysis revealed cancer-related immunoreactivity for the ErbB-3-binding protein-1 (Ebp1). Immunohistochemical staining of colorectal tumors and neighboring normal colon tissue indicated the observed cancer-related immunogenicity of Ebp1 to be related to overexpression. Via reverse immunology, five potential HLA-A2-restricted T cell epitopes were identified, of which two (Ebp1(45-54) and Ebp1(59-67)) bound HLA-A2 with intermediate and high affinity, respectively. Analysis of their immunogenicity in vitro indicated that only the high-affinity Ebp1(59) epitope gave rise to CD8(+) T cells capable of recognizing both exogenously loaded Ebp1 peptide and endogenously expressed Ebp1 on target cells. In addition, in vivo CD8(+) T cell responsiveness against the Ebp1(59) epitope could be detected in two of nine and three of six cancer patients PBMC and tumor draining lymph nodes, respectively, but not in nine of nine healthy donors tested. These data confirm that Ebp1 is an immunogenic protein, capable of eliciting CD8-mediated responses in vivo and in vitro, providing a rationale for further exploration of Ebp1 as a possible target for anticancer immunotherapy.     

Cutting edge: conventional dendritic cells are the critical APC required for the induction of experimental cerebral malaria

Cerebral malaria (CM) is a serious complication of Plasmodium falciparum infection, causing significant morbidity and mortality among young children and nonimmune adults in the developing world. Although previous work on experimental CM has identified T cells as key mediators of pathology, the APCs and subsets therein required to initiate immunopathology remain unknown. In this study, we show that conventional dendritic cells but not plasmacytoid dendritic cells are required for the induction of malaria parasite-specific CD4+ T cell responses and subsequent experimental CM. These data have important implications for the development of malaria vaccines and the therapeutic management of CM.