End Of Primer Research Articles

Searching in microbial genomes for encoded small proteins

As the price of genome sequencing has been rapidly decreasing and can be expected to keep on doing so in the next 10 years, the speed at which new microbial genome sequences become available will increase accordingly. In most genome projects, the first step after acquiring a genome sequence is predicting protein‐encoding open reading frames (pORFs). Small proteins or peptides, loosely defined as less than 50 amino acids, encoded in microbial genomes have been largely underestimated. Recent focused functional genomics efforts have led to the identification of a number of new small proteins encoded in genomes of both Gram‐negative and Gram‐positive bacteria, and fungi (Kastenmayer et al., 2006; Li et al., 2008; Hemm et al., 2010; Hobbs et al., 2010; Bitton et al., 2011). Increasing evidence demonstrates that small proteins participate in a wide array of cellular processes and exhibit great diversity in their mechanisms of action. A recent review (Hobbs et al., 2011) highlights examples of small proteins that, in addition to the well‐conserved small ribosomal proteins, participate in cell signalling or regulation, act as antibiotics and toxins/anti‐toxins, alter membrane features, act as chaperones, stabilize protein complexes or serve as structural proteins (Table 1) (Fig. 1). Table 1 Types and functions of small proteins. Figure 1 Structure of small proteins. Small secreted proteins exhibit diversity in their three‐dimensional structures and can contain unique intramolecular linkages or modified amino acids. For example, the mature form of (A) subtilosin (PDB: 1PXQ) is ... Failure to recognize a pORF encoding a small protein means that these important cell constituents will be missed. Here, we give a brief summary of which problems arise in searching for such encoded small proteins, and what we could do to improve the search process.

The sugar ring conformation of 4'-ethynyl-2-fluoro-2'-deoxyadenosine and its recognition by the polymerase active site of HIV reverse transcriptase.

4' Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is the most potent inhibitor of HIV reverse transcriptase (RT). We have recently named EFdA a Translocation Defective RT Inhibitor (TDRTI) because after its incorporation in the nucleic acid it blocks DNA polymerization, primarily by preventing translocation of RT on the template/primer that has EFdA at the 3'-primer end (T/PEFdA). The sugar ring conformation of EFdA may also influence RT inhibition by a) affecting the binding of EFdA triphosphate (EFdATP) at the RT active site and/or b) by preventing proper positioning of the 3'-OH of EFdA in T/PEFdA that is required for efficient DNA synthesis. Specifically, the North (C2'-exo/C3'-endo), but not the South (C2'-endo/C3'-exo) nucleotide sugar ring conformation is required for efficient binding at the primer-binding and polymerase active sites of RT. In this study we use nuclear magnetic resonance (NMR) spectroscopy experiments to determine the sugar ring conformation of EFdA. We find that unlike adenosine nucleosides unsubstituted at the 4'-position, the sugar ring of EFdA is primarily in the North conformation. This difference in sugar ring puckering likely contributes to the more efficient incorporation of EFdATP by RT than dATP. In addition, it suggests that the 3'-OH of EFdA in T/PEFdA is not likely to prevent incorporation of additional nucleotides and thus it does not contribute to the mechanism of RT inhibition. This study provides the first insights into how structural attributes of EFdA affect its antiviral potency through interactions with its RT target.

Erratum to: Transitivity of generic semigroups of area-preserving surface diffeomorphisms

Theorem 12 in [2] claims that for a Moser generic diffeomorphism f and an invariant residual domain U , there are no periodic points in the boundary of U . This is claimed to be a direct consequence of [3]. However, Mather’s theorem is weaker than that; specifically, it says that the rotation number of the prime ends compactification on any end of U is irrational. On the sphere, Mather’s result implies what is claimed in Theorem 12, and the proof (which can be found in [1]) uses the existence of homoclinic intersections for periodic points of f guaranteed by a theorem of Pixton. This can also be done in T2 using [4]. However, for higher genus, the claim remains open. We will prove a weaker version of Theorem 12 of [2]. To do this, we define for each f a Cr -residual set G f , and we prove that the statement of Theorem 12 of [2] holds if we additionally require that the residual domain U be g-invariant for some g ∈ G f . This is enough for our proofs. The modifications needed in the statements and proofs of [2] are as follows:

Efficacy of Transcatheter Arterial Infusion Alone or Combined with Transcatheter Arterial Chemoembolization on Advanced Hepatocellular Carcinoma

Objective: Interventional therapy for advanced hepatocellular carcinoma (HCC) is still controversial. This retrospective study was to evaluate the efficacy of transcatheter arterial infusion (TAI) alone or combined with transcatheter arterial chemoembolization (TACE) on advanced HCC. Methods: The study population consisted of 132 advanced HCC patients with Child-pugh A/B. Tumor in all patients was involved with main trunk of portal vein and/or inferior vena cava, or local lymph node metastasis, or distant metastasis. TAI alone or combined with TACE were performed in 65 patients with advanced HCC (interventional treatment group), 67 patients were treated with traditional Chinese herbal drug (Chinese herb group). The prime end point was overall survival (OS), and prognostic factors were analysed. Results: The median OS was 205 days [95% confidence interval (CI), 155-255 days] in interventional treatment group and 127 days (95% CI, 70-184 days) in Chinese herb group (P <0.05). The 6-month, 1-year, and 2-year OS rates were 58.9%, 29.1%, 7.7% in interventional treatment group, and 33.3%, 12.3%, 1.8% in Chinese herb group. The portal vein thrombosis, ECOG performance status were independent prognostic factors for OS. Conclusion: Interventional treatment could greatly prolong the OS of advanced HCC patients.

Update on cystic fibrosis-related diabetes

The aim is to provide a detailed review of recent publications on cystic fibrosis-related diabetes (CFRD) with a particular focus on the interplay between cystic fibrosis (CF) lung disease and diabetes. CFRD is a form of diabetes that is distinct from type 1 or type 2 diabetes. CFRD remains very common and increases in prevalence with increasing age so that one in two middle-aged CF persons have CFRD. People with CFRD have lower lung function, worse nutrition, more frequent hospitalization, and worse mortality than CF people without diabetes. The excess mortality previously noted in women with CFRD compared with CF women without diabetes or CF men is much less apparent. CFRD is due to insulin deficiency and peripheral insulin resistance is much less a factor. Genetic susceptibility and oxidant stress are key risk factors for developing CFRD. The lung is the prime end organ target in CFRD and mortality is due to respiratory failure, not vascular complications. Insulin is the mainstay of therapy and early recognition and institution of therapy appear to improve health outcomes. CFRD remains one of the most important co-morbidities in CF. Early recognition of the disease and therapeutic intervention may diminish the negative impact that diabetes has on lung health in CF. Although a clearer understanding of the role of oxidant stress and genetics in the pathogenesis of CFRD is being elucidated, much needs to be learned before more targeted, specific therapies can be developed for this distinct form of diabetes.

Invariant subspaces for operators whose spectra are Carathéodory regions

In this paper it is shown that if an operator T satisfies ‖ p ( T ) ‖ ⩽ ‖ p ‖ σ ( T ) for every polynomial p and the polynomially convex hull of σ ( T ) is a Carathéodory region whose accessible boundary points lie in rectifiable Jordan arcs on its boundary, then T has a nontrivial invariant subspace. As a corollary, it is also shown that if T is a hyponormal operator and the outer boundary of σ ( T ) has at most finitely many prime ends corresponding to singular points on ∂ D and has a tangent at almost every point on each Jordan arc, then T has a nontrivial invariant subspace.

Structure and Properties of 9′-cis Neoxanthin Carotenoid Radicals by Electron Paramagnetic Resonance Measurements and Density Functional Theory Calculations: Present in LHC II?

The radical intermediates formed upon catalytic or photooxidation of the carotenoid 9'-cis neoxanthin inside MCM-41 molecular sieves were detected by pulsed Mims and Davies electron nuclear double resonance (ENDOR) spectroscopies and characterized by density functional theory (DFT) calculations. Mims ENDOR spectra (20 K) were simulated using the hyperfine coupling constants predicted by DFT, which showed that a mixture of carotenoid radical cations (Car(+)) and neutral radicals (#Car) is formed. The DFT relative energies of the neutral radicals formed by proton loss from the C5, C5', C9, C9', C13, and C13'-methyl groups of Car(+) showed that #Car(9') is energetically most favorable, while #Car(9), #Car(13), #Car(13'), #Car(5'), and #Car(5) are less favorable for formation by 2.6, 5.0, 5.1, 22.5, and 25.6 kcal/mol. No evidence for formation of #Car(5') and #Car(5) was observed in the EPR spectra, consistent with DFT calculations. The epoxy group at the prime end and the allene bond at the unprime end prevent protons loss at the C5 and C5'-methyl groups by reducing the conjugation so crucial for the neutral radical stability. Previous CV measurements for allene-substituted carotenoids show that once the radical cations are formed, proton loss is rapid. These examined properties and the known crystal structure of the light harvesting complex II (LHC II) suggest the absence of the neutral radicals of 9'-cis neoxanthin available for quenching the excited states of Chl, consistent with its observed nonquenching properties.

A fixed point theorem for branched covering maps of the plane

It is known that every homeomorphism of the plane which admits an invariant non-separating continuum has a fixed point in the continuum. In this paper we show that any branched covering map of the plane of degree d, |d| ≤ 2, which has an invariant, non-separating continuum Y , either has a fixed point in Y , or is such that Y contains a minimal (by inclusion among invariant continua), fully invariant, non-separating subcontinuum X. In the latter case, f has to be of degree −2 and X has exactly three fixed prime ends, one corresponding to an outchannel and the other two to inchannels.

Metabolic Engineering of ω3-Very Long Chain Polyunsaturated Fatty Acid Production by an Exclusively Acyl-CoA-dependent Pathway

omega3-Very long chain polyunsaturated fatty acids (VLCPUFA) are essential for human development and brain function and, thus, are indispensable components of the human diet. The current main source of VLCPUFAs is represented by ocean fish stocks, which are in severe decline, and the development of alternative, sustainable sources of VLCPUFAs is urgently required. Our research aims at exploiting the powerful infrastructure available for the large scale culture of oilseed crops, such as rapeseed, to produce VLCPUFAs such as eicosapentaenoic acid in transgenic plants. VLCPUFA biosynthesis requires repeated desaturation and repeated elongation of long chain fatty acid substrates. In previous experiments the production of eicosapentaenoic acid in transgenic plants was found to be limited by an unexpected bottleneck represented by the acyl exchange between the site of desaturation, endoplasmic reticulum-associated phospholipids, and the site of elongation, the cytosolic acyl-CoA pool. Here we report on the establishment of a coordinated, exclusively acyl-CoA-dependent pathway, which avoids the rate-limiting transesterification steps between the acyl lipids and the acyl-CoA pool during VLCPUFA biosynthesis. The pathway is defined by previously uncharacterized enzymes, encoded by cDNAs isolated from the microalga Mantoniella squamata. The conceptual enzymatic pathway was established and characterized first in yeast to provide proof-of-concept data for its feasibility and subsequently in seeds of Arabidopsis thaliana. The comparison of the acyl-CoA-dependent pathway with the known lipid-linked pathway for VLCPUFA biosynthesis showed that the acyl-CoA-dependent pathway circumvents the bottleneck of switching the Delta6-desaturated fatty acids between lipids and acyl-CoA in Arabidopsis seeds.

On prime ends and local connectivity

Let U be a simply connected domain on the Riemann sphere whose complement K contains more than one point. We establish a characterization of local connectivity of K at a point in terms of the prime ends whose impressions contain this point. Invoking a result of Ursell and Young, we obtain an alternative proof of a theorem of Torhorst, which states that the impression of a prime end of $U$ contains at most two points at which $K$ is locally connected.

P3-270: An association study between progranulin gene polymorphisms and Alzheimer's disease

Mutations in the progranulin gene (PGRN) leading to haploinsufficiency of the progranulin protein have been shown to cause tau-negative frontotemporal dementia linked to chromosome 17q21. Although the precise biological function of progranulin in neurons has not yet been determined, the initial findings suggest that progranulin is crucial for the neuronal survival and that the down regulation of this protein leads to neurodegeneration. Here we wanted to investigate whether genetic variability in the PGRN gene influences also the risk of developing the more common neurodegenerative brain disease, Alzheimer's disease (AD). To study the genetic association between PGRN and AD, we genotyped four single nucleotide polymorphisms (SNPs) (rs3785817, rs4792939, rs850713, and rs5848) in clinically well-defined AD patient and control cohort originating from Eastern Finland. Over 500 late onset AD patients were compared to 650 non-demented, age-matched control subjects using single locus and haplotype approaches. After performing allele, genotype and haplotype analyses of SNPs, no significant associations were found. However, upon stratifying the population based on APOE status, a significant association was found in APOE ϵ4 negative subgroup with SNPs rs850713 (p<0.05) and rs5848 (p=0.01), which are located at the 3'-prime end of PGRN gene. Furthermore, the same SNPs showed a significant association among men and the assessment of combined effect of APOE status and gender revealed strong single locus and haplotype association with SNPs rs850713 and rs5848 in men not carrying the APOE ϵ4 allele (p<0.01). Although we did not find any of the studied PGRN SNPs to be associated with AD in the whole case-control cohort, the fact that SNPs rs850713 and rs5848 were associated in APOE and gender stratified subgroups suggests that genetic variability in the PGRN gene may also influence the risk of AD in certain conditions. Further studies are required to corroborate our findings.

Significant Decrease in Dialysate Albumin Concentration during Molecular Adsorbent Recirculating System (M.A.R.S.) Therapy

The molecular adsorbent recirculating system (M.A.R.S.) is widely used as liver support therapy in patients with hepatic dysfunction. The goal of this study was to measure changes in dialysate albumin and bilirubin concentrations during clinical MARS treatments. Eight patients with acute liver dysfunction and hyperbilirubinemia were enrolled in this study. Five of them received a total of 10 treatments with MARS, in which 600 mL of 20% human albumin was used as dialysate, continuously regenerated by two adsorbent columns in the circuit. Three patients received 4 treatments of a modified MARS, in which the two adsorbent columns were bypassed in the first course for 4 h, and then connected to the circuit in the second course for another 4 h. The total, conjugated and unconjugated bilirubin (TB, CB, UCB) and albumin concentrations in serum and albumin dialysate were dynamically measured, and the adsorbent column inlet pressures were recorded during each session. In one session, dialysate albumin levels were measured during the priming process, at the time points prior to the priming process, immediately after priming, and at the end of the treatment. During MARS therapies, the reduction ratio of serum TB, CB and UCB was 26.6+/-9.0%, 29.5+/-9.6% and 14.8+/-12.3%, respectively. The molar ratio of TB/albumin in serum was approximately 20-fold higher than dialysate at all time points. A significant albumin concentration decrease from baseline in the dialysate was found (mean+/-SD, 34.6+/-16.6%). For the first four hours of modified treatments, in which only albumin dialysis without albumin regeneration by adsorbent columns was performed, the dialysate albumin decrease was substantially smaller (mean, 8.3+/-1.5%). After switching to standard MARS, there was a further decrease in the dialysate albumin concentration of 35.1+/-14.5%. In one session, dialysate albumin concentrations were measured during the priming process, and levels decreased from 196.9 g/L to 144.4 g/L. Adsorber inlet pressure increased from 40+/-10 mmHg at the start of priming to 150+/-50 mmHg at the end of priming, and further increased to 340+/-100 mmHg at the end of treatment. There is a significant reduction in dialysate albumin concentration during MARS therapy. Binding of albumin to the adsorbent columns used for albumin regeneration is largely responsible for this decrease.

The scaling limits of planar LERW in finitely connected domains

We define a family of stochastic Loewner evolution-type processes in finitely connected domains, which are called continuous LERW (loop-erased random walk). A continuous LERW describes a random curve in a finitely connected domain that starts from a prime end and ends at a certain target set, which could be an interior point, or a prime end, or a side arc. It is defined using the usual chordal Loewner equation with the driving function being $\sqrt{2}B(t)$ plus a drift term. The distributions of continuous LERW are conformally invariant. A continuous LERW preserves a family of local martingales, which are composed of generalized Poisson kernels, normalized by their behaviors near the target set. These local martingales resemble the discrete martingales preserved by the corresponding LERW on the discrete approximation of the domain. For all kinds of targets, if the domain satisfies certain boundary conditions, we use these martingales to prove that when the mesh of the discrete approximation is small enough, the continuous LERW and the corresponding discrete LERW can be coupled together, such that after suitable reparametrization, with probability close to 1, the two curves are uniformly close to each other.

ALIS‐FLP: Amplified ligation selected fragment‐length polymorphism method for microbial genotyping

A DNA fingerprinting method known as ALIS‐FLP (amplified ligation selected fragment‐length polymorphism) has been developed for selective and specific amplification of restriction fragments from TspRI restriction endonuclease digested genomic DNA. The method is similar to AFLP, but differs in that only one specific restriction enzyme (TspRI) is used. The cohesive ends of the DNA fragments are ligated with two types of oligonucleotide. A long oligonucleotide containing the primer site and the specific 9 nt 3 prime end, which is complementary to specific 9 nt, cohesive 3 prime end of the TspRI genomic DNA fragment, and a short, degenerated, oligonucleotide covering the remaining TspRI cohesive ends. Other cohesive ends are covered by a short degenerated oligonucleotide lacking the primer site. The ligation mixture is used as a template for amplification using a single primer corresponding to the 5 prime end of the long, specific oligonucleotide. The selection of TspRI digested genomic DNA fragments for amplification is achieved by sequence selective ligation of the specific long oligonucleotide carrying the primer site to both ends of the specific target fragment. This technique allows for differentiation of the organisms without previous knowledge of their DNA sequence. The usefulness of the method is confirmed by genotyping of 70 previously characterized clinical E. coli isolates. The grouping obtained was identical to the results of REA‐PFGE. Versatility of the method is highlighted, i.e. its combining the advantages of the AFLP technique with a simple, rapid and cheap polymerase chain reaction product detection method.

Finding exonic islands in a sea of non-coding sequence: splicing related constraints on protein composition and evolution are common in intron-rich genomes.

Biased usage of amino acids near exon-intron boundaries is phylogenetically widespread and characteristic of species for which there are expected to be problems defining exons.

Multiple Hybridization-extension Sequencing (MHES) on Microarray

Sequencing-by-synthesis (SBS) by fluorescein-labelled nucleotide incorporating into a target DNA template has been greatly concerned on microarray. The extended fluorophore-base must be required to be quenched prior to sequencing the next one. However, the low quenching efficiency has been an obstacle in length-read. Here, we present a new sequencing strategy, multiple hybridization-extension sequencing (MHES), to resolve the above problem. First, the sequencing primers hybridize to the ssDNA template immobilized on microarray. The first 3-5 bases next to the primer's end are sequenced by SBS of Cy5-dNTP. The extended primers are rapidly removed by lambda DNA exonuclease. Then, the same primers hybridize to the same ssDNA templates again. The sequenced bases are polished by natural dNTP. The other 3-5 bases next to the polished primer's end are sequenced. According to this principle, the unknown sequences of a target DNA could be sequenced after primers' hybridization-extension multiple times. Although the fluorescein-labelled nucleotides are also needed, it is unnecessary to quench the fluorophore-bases in the process of sequencing. It has been successfully demonstrated that 10 bp fragment from synthetic template and 10 bp fragment from DTBNP1 gene were accurately sequenced. The new method has a great potential in read-length and high-throughput sequencing on microarray.

Bifurcations of basins of attraction from the view point of prime ends

In dynamical systems examples are common in which two or more attractors coexist, and in such cases the basin boundary is nonempty and the basins often have fractal basin boundaries. The purpose of this paper is to describe the structure and properties of unbounded basins and their boundaries for two-dimensional diffeomorphisms. Frequently, if not always, there is a periodic saddle on the boundary that is accessible from the basin. Carathéodory and many others developed an approach in which an open set (in our case a basin) is compactified using so-called prime end theory. Under the prime end compactification of the basin, boundary points of the basin (prime ends) can be characterized as either type 1, 2, 3, or 4. In all well-known examples, most points are of type 1. Many two-dimensional basins have a basin cell, that is, a trapping region whose boundary consists of pieces of the stable and unstable manifolds of a well chosen periodic orbit. Then the basin consists of a central body (the basin cell) and a finite number of channels attached to it, and the basin boundary is fractal. We present a result that says {a basin has a basin cell} if and only if {every prime end that is defined by a chain of unbounded regions (in the basin) is a prime end of type 3 and furthermore all other prime ends are of type 1}. We also prove as a parameter is varied, the basin cell for a basin B is created (or destroyed) if and only if either there is a saddle node bifurcation or the basin B has a prime end that is defined by a chain of unbounded regions and is a prime end of either type 2 or type 4.

Area-Preserving Surface Diffeomorphisms

We prove some generic properties for $C^r$, $r=1, 2, ..., \infty$, area-preserving diffeomorphism on compact surfaces. The main result is that the union of the stable (or unstable) manifolds of hyperbolic periodic points are dense in the surface. This extends the result of Franks and Le Calvez \cite{FL03} on $S^2$ to general surfaces. The proof uses the theory of prime ends and Lefschetz fixed point theorem.

Fractional iteration in the disk algebra: prime ends and composition operators

In this paper we characterize the semigroups of analytic functions in the unit disk which lead to semigroups of operators in the disk algebra. These characterizations involve analytic as well as geometric aspects of the iterates and they are strongly related to the classical theorem of Carath\'eodory about local connection and boundary behaviour of univalent functions.

Indecomposable continua and the Julia sets of polynomials, II

We find necessary and sufficient conditions for the connected Julia set of a polynomial of degree d ⩾ 2 to be an indecomposable continuum. One necessary and sufficient condition is that the impression of some prime end (external ray) of the unbounded complementary domain of the Julia set J has nonempty interior in J. Another is that every prime end has as its impression the entire Julia set. The latter answers a question posed in 1993 by the second two authors. We show by example that, contrary to the case for a polynomial Julia set, the image of an indecomposable subcontinuum of the Julia set of a rational function need not be indecomposable.