<Emphasis Type="Italic">Scoredist</Emphasis>: A simple and robust protein sequence distance estimator

Background  

Distance-based methods are popular for reconstructing evolutionary trees thanks to their speed and generality. A number of methods exist for estimating distances from sequence alignments, which often involves some sort of correction for multiple substitutions. The problem is to accurately estimate the number of true substitutions given an observed alignment. So far, the most accurate protein distance estimators have looked for the optimal matrix in a series of transition probability matrices, e.g. the Dayhoff series. The evolutionary distance between two aligned sequences is here estimated as the evolutionary distance of the optimal matrix. The optimal matrix can be found either by an iterative search for the Maximum Likelihood matrix, or by integration to find the Expected Distance. As a consequence, these methods are more complex to implement and computationally heavier than correction-based methods. Another problem is that the result may vary substantially depending on the evolutionary model used for the matrices. An ideal distance estimator should produce consistent and accurate distances independent of the evolutionary model used.     

Improved profile HMM performance by assessment of critical algorithmic features in SAM and HMMER

Background  

Profile hidden Markov model (HMM) techniques are among the most powerful methods for protein homology detection. Yet, the critical features for successful modelling are not fully known. In the present work we approached this by using two of the most popular HMM packages: SAM and HMMER. The programs' abilities to build models and score sequences were compared on a SCOP/Pfam based test set. The comparison was done separately for local and global HMM scoring.     

Identification of clustered microRNAs using an <Emphasis Type="Italic">ab initio</Emphasis> prediction method

Background  

MicroRNAs (miRNAs) are endogenous 21 to 23-nucleotide RNA molecules that regulate protein-coding gene expression in plants and animals via the RNA interference pathway. Hundreds of them have been identified in the last five years and very recent works indicate that their total number is still larger. Therefore miRNAs gene discovery remains an important aspect of understanding this new and still widely unknown regulation mechanism. Bioinformatics approaches have proved to be very useful toward this goal by guiding the experimental investigations.     

Simultaneous imaging of GFP,CFP and collagen in tumors <Emphasis Type="Italic">in vivo</Emphasis>using multiphoton microscopy

Background  

The development of multiphoton laser scanning microscopy has greatly facilitated the imaging of living tissues. However, the use of genetically encoded fluorescent proteins to distinguish different cell types in living animals has not been described at single cell resolution using multiphoton microscopy.     

Sarcopenia is not due to lack of regenerative drive in senescent skeletal muscle

Sarcopenia, loss of skeletal muscle mass, is a hallmark of aging commonly attributed to a decreased capacity to maintain muscle tissue in senescence, yet the mechanism behind the muscle wasting remains unresolved. To address these issues we have explored a rodent model of sarcopenia and age-related sensorimotor impairment, allowing us to discriminate between successfully and unsuccessfully aged cohort members. Immunohistochemistry and staining of cell nuclei revealed that senescent muscle has an increased density of cell nuclei, occurrence of aberrant fibers and fibers expressing embryonic myosin. Using real-time PCR we extend the findings of increased myogenic regulatory factor mRNA to show that very high levels are found in unsuccessfully aged cohort members. This pattern is also reflected in the number of embryonic myosin-positive fibers, which increase with the degree of sarcopenia. In addition, we confirm that there is no local down-regulation of IGF-I and IGF-IR mRNA in aged muscle tissue; on the contrary, the most sarcopenic individuals showed significantly higher local expression of IGF-I mRNA. Combined, our results show that the initial drive to regenerate myofibers is most marked in cases with the most advanced loss of muscle mass, a pattern that may have its origin in differences in the rate of tissue deterioration and/or that regenerating myofibers in these cases fail to mature into functional fibers. Importantly, the genetic background is a determinant of the pace of progression of sarcopenia.     

Eric Kandel: the future of memory

Watching ice floes glide by on the Hudson River from Eric Kandel's office, one gets a sense of placid reflection tempered by constant action-an apt analogy for Kandel's ability to calmly manage several ongoing projects and commitments at once. In addition to his well-lauded, ongoing research at Columbia University Medical Center's New York State Psychiatric Institute, Kandel has written several books on neurobiology, behavior, and memory. In addition to being a Nobel Laureate Scientist, he is well-known as an editor of the seminal textbook Principles of Neural Science. He and his colleagues are in the midst of working on a new edition of Principles, and he is working on a scientific autobiography. MI sat down with Dr. Kandel and discussed with him a range of topics including childhood and early career influences, intramural research at the NIH, the HHMI, ethical considerations of altering memory and, of course, Aplysia.     

Compact and ordered collapse of randomly generated RNA sequences

As the raw material for evolution, arbitrary RNA sequences represent the baseline for RNA structure formation and a standard to which evolved structures can be compared. Here, we set out to probe, using physical and chemical methods, the structural properties of RNAs having randomly generated oligonucleotide sequences that were of sufficient length and information content to encode complex, functional folds, yet were unbiased by either genealogical or functional constraints. Typically, these unevolved, nonfunctional RNAs had sequence-specific secondary structure configurations and compact magnesium-dependent conformational states comparable to those of evolved RNA isolates. But unlike evolved sequences, arbitrary sequences were prone to having multiple competing conformations. Thus, for RNAs the size of small ribozymes, natural selection seems necessary to achieve uniquely folding sequences, but not to account for the well-ordered secondary structures and overall compactness observed in nature.     

Swarming of mosquitoes

Experiments have been made on the swarming of a number of mosquitoes, especially Culex fatigans, under laboratory conditions and the influence of light intensity, temperature, different color swarm markers; and time factors, both in regard to duration of the change of light and the time lapse between experiments, have been studied.

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Résumé Des expériences sur l'essaimage des moustiques mâles (surtout Culex fatigans) ont été faites au laboratoire. La cage dans laquelle se formaient les essaims était placée dans une chambre dont l'éclairage, la température et l'humidité pouvaient être réglés.Les essaims qui comprenaient le plus grand nombre d'individus et qui duraient le plus longtemps, étaient obtenus quand le changement d'éclairement (crépuscule artificiel) durait plus de 20 minutes et quand la température était de 24–25° après une durée de plus de cinq heures depuis le dernier changement.Dans ces conditions artificielles les moustiques faisaient des essaims tous les matins et tous les soirs. Le commencement de l'essaimage le soir, et sa fin le matin avaient lieu à un même éclairement (1,6 log lux). Si l'éclairement était tenu inférieur à 1,6 log lux, les essaims duraient toute la nuit jusqu'à ce que la lumière soit relevée.Quelques observations sur Culex nigripalpus et Anopheles quadrimaculatus indiquent que les déliminations de l'éclairement permettent l'essaimage. Chez. C. nigripalpus ils étaient presque les mêmes que celles de Culex fatigans. Psorophora howardii et Aëdes taeniorhynchus ne font pas d'essaimage dans la petite cage mais la dernière espèce fait un essaim dans une cage plus grande après un éclairement subit.

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Contribution No. 113, Florida State Board of Health, Entomological Research Center, Vero Beach, Florida. This investigation was supported by Public Health Research Grant E-1492, from the National Institute of Allergy and Infectious Diseases, Public Health Service.     

Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling

A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.     

Microarrays based on affinity-tagged single-chain Fv antibodies: sensitive detection of analyte in complex proteomes

Protein-based microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform global proteome analysis. However, the process of designing adequate protein microarrays is a major inherent problem. In this study, we have evaluated a protein microarray platform based on nonpurified affinity-tagged single-chain (sc) Fv antibody fragments to generate proof-of-principle and to demonstrate the specificity and sensitivity of the array design. To this end, we used our human recombinant scFv antibody library genetically constructed around one framework, the n-CoDeR library containing 2 x 10(10) clones, as a source for our probes. The probes were immobilized via engineered C-terminal affinity tags, his- or myc-tags, to either Ni(2+)-coated slides or anti-tag antibody coated substrates. The results showed that highly functional microarrays were generated and that nonpurified scFvs readily could be applied as probes. Specific and sensitive microarrays were obtained, providing a limit of detection in the pM to fM range, using fluorescence as the mode of detection. Further, the results showed that spotting the analyte on top of the arrayed probes, instead of incubating the array with large sample volumes (333 pL vs. 40 microL), could reduce the amount of analyte required 4000 times, from 1200 attomole to 300 zeptomole. Finally, we showed that a highly complex proteome, such as human sera containing several thousand different proteins, could be directly fluorescently labeled and successfully analyzed without compromising the specificity and sensitivity of the antibody microarrays. This is a prerequisite for the design of high-density antibody arrays applied in high-throughput proteomics.