A Hierarchical Approach to Protein Molecular Evolution

Biological diversity has evolved despite the essentially infinite complexity of protein sequence space. We present a hierarchical approach to the efficient searching of this space and quantify the evolutionary potential of our approach with Monte Carlo simulations. These simulations demonstrate that non-homologous juxtaposition of encoded structure is the rate-limiting step in the production of new tertiary protein folds. Non-homologous ``swapping'' of low energy secondary structures increased the binding constant of a simulated protein by $\approx10^7$ relative to base substitution alone. Applications of our approach include the generation of new protein folds and modeling the molecular evolution of disease.Comment: 15 pages. 2 figures. LaTeX styl

Eye movements are coordinated with pectoral fin beats during locomotion in a marine teleost fish

Animals must simultaneously engage multiple functional systems in order to navigate, feed, and survive in complex environments. Nearly all vertebrates perform rapid gaze-shifting eye movements called saccades, but we know little about the behaviour of saccades during rhythmic locomotion. This study examines how saccades are coordinated with locomotor movements in a pectoral-fin-propelled teleost fish, Cymatogaster aggregata, the shiner surfperch. Twelve individuals were filmed swimming in a flow tank at 10 cm s−1, and timing data were analyzed using circular statistics. Results reveal that Cymatogaster generates saccades non-uniformly throughout the pectoral fin cycle. Saccades primarily occur during fin abduction, when a large amount of thrust is produced, and rarely occur during the thrust-free refractory phase. Because vision is known to be impaired during saccades, we hypothesize that Cymatogaster synchronizes saccades with periods of high acceleration in order to stabilize retinal images during low-acceleration phases, which are nearly saccade-free

Znaczenie kliniczne migotania przedsionków prowokowanego podczas stymulacji przezprzełykowej

Wstęp: U części pacjentów bez udokumentowanych spontanicznych zaburzeń rytmu w trakcie wykonywania przezprzełykowej stymulacji przedsionków (TAS, transesophageal atrial stimulation) można sprowokować migotanie przedsionków (AF, atrial fibrillation). Celem badania było ustalenie, czy pacjenci ci różnią się pod względem wybranych parametrów od grupy, w której nie sprowokowano AF. Materiał i metody: Badanie objęło 68 chorych, których podzielono na 2 grupy: I - ze sprowokowanym AF i II - w której arytmia nie wystąpiła. U pacjentów wykonano TAS, standardowe zapisy EKG, rejestrację EKG metodą Holtera oraz badanie echokardiograficzne. Wyniki: Wyniki wskazują na znamienne różnice automatyzmu węzła zatokowego, przewodzenia zatokowo-przedsionkowego i śródprzedsionkowego, czasu refrakcji lewego przedsionka, maksymalnej i średniej częstości rytmu zatokowego, częstości ekstrasystolii nadkomorowych oraz wymiaru i wskaźnika lewego przedsionka. Pozostałe parametry nie wykazały różnic istotnych statystycznie. Wnioski: Migotanie przedsionków prowokowane podczas TAS identyfikuje pacjentów z zaburzeniami automatyzmu węzła zatokowego, upośledzonym przewodzeniem zatokowo-przedsionkowym i przewodzeniem śródprzedsionkowym, a także charakteryzuje chorych z długim okresem refrakcji lewego przedsionka. Pacjenci, u których sprowokowano AF mają znamiennie wyższą maksymalną i średnią częstość rytmu zatokowego, a także częściej występuje u nich ekstrasystolia nadkomorowa. Chorzy ci różnią się od grupy kontrolnej większym wymiarem lewego przedsionka i częstszym występowaniem w EKG zaburzeń depolaryzacji przedsionków, więc należy ich systematycznie kontrolować pod kątem występowania napadów AF w przyszłości

Emergency healthcare providers perception of workplace dangers in the polish Emergency Medical Service: a multi-centre survey study

INTRODUCTION: There are many risk factors that account for hazards in paramedics’ and ambulance nurses’ profession. Driving a vehicle, having contact with patients, making difficult medical decisions, doing night shifts and working in a stressful environment, all of those features negatively affect their health. The aim of the study was to evaluate paramedics’ and ambulance nurses attitude towards personal safety, to assess their subjective feeling of danger, as well as identify types of hazards they experience. MATERIAL AND METHODS: The study was carried out via a diagnostic survey method, an anonymous questionnaire. Among 572 responders there were nurses and paramedics, who work in non-physician medical rescue teams in Poland. RESULTS: Most of the surveyed medics (40.5%) have rated the level of danger of their occupation to 4 on a scale from 1 to 5, with the greatest hazard being posed by patients under the influence of designer drugs. As many as 43% of medics have had back-related problems and 41% have suffered injuries at work. Notwithstanding, a majority of respondents have admitted that if they could plan their career again, they would choose the same profession. CONCLUSIONS: Prehospital healthcare providers have generally rated their work as dangerous. More attention should be paid to teach first responders how to deal with aggression and how to handle stress. Efforts should be made to increase paramedics’ and nurses’ awareness about health problems related to shift work

Biodiversity inventories and conservation of the marine fishes of Bootless Bay, Papua New Guinea

Background: The effective management and conservation of biodiversity is predicated on clearly defined conservation targets. Species number is frequently used as a metric for conservation prioritization and monitoring changes in ecosystem health. We conducted a series of synoptic surveys focusing on the fishes of the Bootless Bay region of Papua New Guinea to generate a checklist of fishes of the region. Bootless Bay lies directly south of Port Moresby, the capital of Papua New Guinea, and experiences the highest human population density of any marine area in the country. Our checklist will set a baseline against which future environmental changes can be tracked. Results: We generated a checklist of 488 fish species in 72 families found in Bootless Bay during a two-week sampling effort. Using incident-based methods of species estimation, we extrapolate there to be approximately 940 fish species in Bootless Bay, one of the lowest reported numbers in Papua New Guinea. Conclusions: Our data suggest that the Bootless Bay ecosystem of Papua New Guinea, while diverse in absolute terms, has lower fish biodiversity compared to other shallow marine areas within the country. These differences in faunal diversity are most likely a combination of unequal sampling effort as well as biophysical factors within Bootless Bay compounded by historical and/or contemporary anthropogenic disturbances.</p

Rapid Assembly of Multiple-Exon cDNA Directly from Genomic DNA

Backgrouud. Polymerase chain reaction (PCR) is extensively applied in gene cloning. But due to the existence of introns, low copy number of particular genes and high complexity of the eukaryotic genome, it is usually impossible to amplify and clone a gene as a full-length sequence directly from the genome by ordinary PCR based techniques. Cloning of cDNA instead of genomic DNA involves multiple steps: harvest of tissues that express the gene of interest, RNA isolation, cDNA synthesis (reverse transcription), and PCR amplification. To simplify the cloning procedures and avoid the problems caused by ubiquitously distributed durable RNases, we have developed a novel strategy allowing the cloning of any cDNA or open reading frame (ORF) with wild type sequence in any spliced form from a single genomic DNA preparation. Methodology. Our Genomic DNA Splicing technique contains the following steps: first, all exons of the gene are amplified from a genomic DNA preparation, using software-optimized, highly efficient primers residing in flanking introns. Next, the tissue-specific exon sequences are assembled into one full-length sequence by overlapping PCR with deliberately designed primers located at the splicing sites. Finally, software-optimized outmost primers are exploited for efficient amplification of the assembled full-length products. Conclusions. The Genomic DNA Splicing protocol avoids RNA preparation and reverse transcription steps, and the entire assembly process can be finished within hours, Since genamic DNA is more stable than RNA, it may be a more practical cloning strategy for many genes, especially the ones that are very large and difficult to generate a full length cDNA using oligo-dT primed reverse transcription. With this technique, we successfully doned the full-length wild type coding sequence of human polymeric immunoglobulin receptor, which is 2295 bp in length and composed of 10 exons. © 2007 An et al.published_or_final_versio

Pairwise selection assembly for sequence-independent construction of long-length DNA

The engineering of biological components has been facilitated by de novo synthesis of gene-length DNA. Biological engineering at the level of pathways and genomes, however, requires a scalable and cost-effective assembly of DNA molecules that are longer than ∼10 kb, and this remains a challenge. Here we present the development of pairwise selection assembly (PSA), a process that involves hierarchical construction of long-length DNA through the use of a standard set of components and operations. In PSA, activation tags at the termini of assembly sub-fragments are reused throughout the assembly process to activate vector-encoded selectable markers. Marker activation enables stringent selection for a correctly assembled product in vivo, often obviating the need for clonal isolation. Importantly, construction via PSA is sequence-independent, and does not require primary sequence modification (e.g. the addition or removal of restriction sites). The utility of PSA is demonstrated in the construction of a completely synthetic 91-kb chromosome arm from Saccharomyces cerevisiae

The core-independent promoter-specific interaction of primary sigma factor

Previous studies have led to a model in which the promoter-specific recognition of prokaryotic transcription initiation factor, sigma (σ), is core dependent. Most σ functions were studied on the basis of this tenet. Here, we provide in vitro evidence demonstrating that the intact Bacillus subtilis primary sigma, σA, by itself, is able to interact specifically with promoter deoxyribonucleic acid (DNA), albeit with low sequence selectivity. The core-independent promoter-specific interaction of the σA is −10 specific. However, the promoter −10 specific interaction is unable to allow the σA to discern the optimal promoter spacing. To fulfill this goal, the σA requires assistance from core RNA polymerase (RNAP). The ability of σ, by itself, to interact specifically with promoter might introduce a critical new dimension of study in prokaryotic σ function

Anatomy of Escherichia coli σ(70) promoters

Information theory was used to build a promoter model that accounts for the −10, the −35 and the uncertainty of the gap between them on a common scale. Helical face assignment indicated that base −7, rather than −11, of the −10 may be flipping to initiate transcription. We found that the sequence conservation of σ(70) binding sites is 6.5 ± 0.1 bits. Some promoters lack a −35 region, but have a 6.7 ± 0.2 bit extended −10, almost the same information as the bipartite promoter. These results and similarities between the contacts in the extended −10 binding and the −35 suggest that the flexible bipartite σ factor evolved from a simpler polymerase. Binding predicted by the bipartite model is enriched around 35 bases upstream of the translational start. This distance is the smallest 5′ mRNA leader necessary for ribosome binding, suggesting that selective pressure minimizes transcript length. The promoter model was combined with models of the transcription factors Fur and Lrp to locate new promoters, to quantify promoter strengths, and to predict activation and repression. Finally, the DNA-bending proteins Fis, H-NS and IHF frequently have sites within one DNA persistence length from the −35, so bending allows distal activators to reach the polymerase

Antibody Phage Display Libraries: Contributions to Oncology

Since the advent of phage display technology, dating back to 1985, antibody libraries displayed on filamentous phage surfaces have been used to identify specific binders for many different purposes, including the recognition of tumors. Phage display represents a high-throughput technique for screening billions of random fusion antibodies against virtually any target on the surface or inside cancer cells, or even soluble markers found in patient serum. Many phage display derived binders targeting important tumor markers have been identified. Selection directed to tumoral cells’ surfaces lead to the identification of unknown tumoral markers. Also the improvement of methods that require smaller amounts of cells has opened the possibility to use this approach on patient samples. Robust techniques combining an antibody library displayed on the phage surface and protein microarray allowed the identification of auto antibodies recognized by patient sera. Many Ab molecules directly or indirectly targeting angiogenesis have been identified, and one of them, ramucirumab, has been tested in 27 phase I–III clinical trials in a broad array of cancers. Examples of such antibodies will be discussed here with emphasis on those used as probes for molecular imaging and other clinical trials