Measurement of preferential solvation of some glucans in mixed solvent systems by gel-permeation chromatography

Preferential solvation of the glucans amylose, pullulan, and dextran in binary dimethyl-sulfoxide/water (DMSO/H₂O) solvent mixtures has been measured using gel-permeation chromatography. The preferential solvation behavior of the three glucans in DMSO/H₂O solvent mixtures is indistinguishable in the experiments reported. In solvent mixtures with mol ratio DMSO/H₂O less than 1:2, all three glucans are solvated preferentially by H₂O. The maximum extent of preferential solvation by H₂O is about 2.5 mol H₂O/mol of glucose residues. When the DMSO/H₂O mol ratio exceeds 1:2, DMSO solvates the glucans preferentially to a maximum extent of about 1 mol DMSO/mol of glucose residues. An interpretation of the change in preferential solvation with mixed solvent composition is suggested in terms of the known characteristics of the binary solvent system, and the relationship of preferential solvation, reported here, to the absolute solvation of the glucan chains is discussed.     

Shuttle vector plasmid propagation in human peripheral blood lymphocytes facilitated by liposome-mediated transfection

Transfection of human peripheral blood lymphocytes facilitated by a positively charged liposome preparation (Lipofectin, BRL) is 100-fold more efficient than the DEAE dextran technique for the uptake and replication of shuttle vector plasmid DNA. The yield of progeny plasmids obtained from 10 ml of blood was high enough for mutational analysis. A marked increase in the mutation frequency of the shuttle vector marker gene was noted in response to the induction of psoralen adducts in the vector. By using normal human lymphocytes, this method will permit shuttle vector analysis of DNA repair and mutagenesis in a large number of individuals. This method could also prove useful for studies of human lymphotropic viruses.     

DNA amplification fingerprinting of bacteria

Summary We have amplified short arbitrary stretches of total bacterial DNA to produce highly characteristic and complex DNA fingerprints. This DNA amplification fingerprinting (DAF) strategy involves enzymatic amplification of DNA directed by a single arbitrary oligonucleotide primer. Amplification produces a characteristic spectrum of products that is adequately resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Although DAF is simple in concept, we found that amplification parameters must be within an optimal range for reproducibility. We establish a safe window for these parameters, which include magnesium, primer and enzyme concentration as well as cycle number. The refined procedure was used to distinguish between clinical isolates of Streptococcus uberis, Klebsiella pneumoniae, and Escherichia coli. The use of template DNA concentrations higher than 1 ng·l^–1 and high MgCl₂ levels was especially important for reproductibility when amplifying small bacterial genomes. We tested a truncated Thermus aquaticus DNA polymerase, the Stoffel fragment, and found it more tolerant of reaction conditions, more efficient in the amplification of short products, and able to produce more informative fingerprints when compared to the normal thermostable polymerase from which it was derived. Because DAF produces representative fingerprints quickly and reliably from bacteria regardless of prior genetic or biochemical knowledge, we anticipate the general use of this diagnostic tool for bacterial identification and taxonomy.Correspondence to: G. Caetano-Anollés     

Measurement of surface potential and surface charge densities of sarcoplasmic reticulum membranes

Summary The binding of the anionic fluorescent probe 1-anilino-8-naphthalene-sulfonate (ANS^–) was used to estimate the surface potential of fragmented sarcoplasmic reticulum (SR) derived from rabbit skeletal muscle. The method is based on the observation that ANS^– is an obligatory anion whose equilibrium constant for binding membranes is proportional to the electrostatic function of membrane surface potential, exp(e₀/kT, where ₀ is the membrane surface potential,e is the electronic charge, andkT has its usual meaning. The potential measured is characteristic of the ANS^– bindings of phosphatidylcholine head groups and is about one-third as large as the average surface potential predicted by the Gouy-Chapman theory. At physiological ionic strength the surface potentials, measured by ANS^–, referred to as the aqueous phase bathing the surface, were in the range –10 to –15 mV. This was observed for the outside and inside surfaces of the Ca²⁺-ATPase-rich fraction of theSR and for both surfaces of theSR fraction rich in acidic Ca²⁺ binding proteins. The inside and outside surfaces were differentiated on the basis of ANS^– binding kinetics observed in stopped-flow rapid mixing experiments. A mechanism by which changes in Ca²⁺ concentration could give rise to an electrostatic potential across the membrane and possibly result in changes in Ca²⁺ permeability.The dependence of the surface potential on the monovalent ion concentration in the medium was used together with the Gouy-Chapman theory to determine the lower limits for the surface charge density for the inside and outside surfaces of the two types ofSR. Values for the Ca²⁺-ATPase richSR fraction were between 2.9×10³ and 3.8×10³ esu/cm², (0.96×10^–6 and 1.26×10^–6 C/cm²) with no appreciable transmembrane asymmetry. A small amount of asymmetry was observed in the values for the inside and outside surfaces of the fraction rich in acidic binding proteins which were ca. 6.6×10³ and ca. 2.2×10³ esu/cm² (2.2×10^–6 and 0.73×10^–6 C/cm). The values could be accounted for by the known composition of negatively-charged phospholipids in theSR. The acidic Ca²⁺ binding proteins were shown to make at most a small contribution to the surface charge, indicating that their charge must be located at least several tens of Å from the membrane surface. The experiments gave evidence for a Donnan effect on the K⁺ distribution in the fraction rich in acidic binding proteins. This could be accounted for by the known concentration of acidic binding proteins in thisSR fraction.The equilibrium constant for ANS^– was shown to be more sensitive to changes in the divalent cation concentration than to changes in the monovalent cation concentration, as predicted by the Gouy-Chapman theory. Use of these findings together with the stopped-flow rapid mixing techniques constitutes a method for rapid and continuous monitoring of changes in ion concentrations in theSR lumen.     

Rest-activity rhythms characteristics and seasonal changes in seasonal affective disorder

ABSTRACT

Identifying objectively measurable seasonal changes in 24-h activity patterns (rest-activity rhythms or RARs) that occur in seasonal affective disorder (SAD) could help guide research and practice towards new monitoring tools or prevention targets. We quantified RARs from actigraphy data using non-parametric and extended cosine based approaches, then compared RARs between people with SAD and healthy controls in the summer (n = 70) and winter seasons (n = 84). We also characterized the within-person seasonal RAR changes that occurred in the SAD (n = 19) and control (n = 26) participants who contributed repeated measures. Only controls had significant winter increases in RAR fragmentation (intra-daily variability; in controls mean winter-summer changes (log scale) = 0.05, 0.21 standard deviation, p = 0.03). In SAD participants only, estimated evening settling times (down-mesor) were an average of 30 min earlier in the winter compared with the summer (1-h standard deviation, p = 0.045). These RAR characteristics correlated with greater fatigue (Spearman r = 0.36) but not depression symptom severity. Additional research is needed to ascertain why healthy controls, but not people with SAD, appear to have increased RAR fragmentation in the winter. People with SAD lacked this increase in RAR fragmentation, and instead had earlier evening setting in the winter. Prospective and intervention studies with greater temporal resolution are warranted to ascertain how these seasonal behavioral differences relate to fatigue pathophysiology in SAD. Future research is needed to determine whether extending the winter active period, even in relatively fragmented bouts, could help reduce the fatigue symptoms common in SAD.     

AMP-activated protein kinase and ATP-citrate lyase are two distinct molecular targets for ETC-1002, a novel small molecule regulator of lipid and carbohydrate metabolism

ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid β-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca²⁺/calmodulin-dependent kinase β-independent and liver kinase β 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease.     

A Phylogenomic Perspective on the Radiation of Ray-Finned Fishes Based upon Targeted Sequencing of Ultraconserved Elements (UCEs)

Ray-finned fishes constitute the dominant radiation of vertebrates with over 32,000 species. Although molecular phylogenetics has begun to disentangle major evolutionary relationships within this vast section of the Tree of Life, there is no widely available approach for efficiently collecting phylogenomic data within fishes, leaving much of the enormous potential of massively parallel sequencing technologies for resolving major radiations in ray-finned fishes unrealized. Here, we provide a genomic perspective on longstanding questions regarding the diversification of major groups of ray-finned fishes through targeted enrichment of ultraconserved nuclear DNA elements (UCEs) and their flanking sequence. Our workflow efficiently and economically generates data sets that are orders of magnitude larger than those produced by traditional approaches and is well-suited to working with museum specimens. Analysis of the UCE data set recovers a well-supported phylogeny at both shallow and deep time-scales that supports a monophyletic relationship between Amia and Lepisosteus (Holostei) and reveals elopomorphs and then osteoglossomorphs to be the earliest diverging teleost lineages. Our approach additionally reveals that sequence capture of UCE regions and their flanking sequence offers enormous potential for resolving phylogenetic relationships within ray-finned fishes.     

Science Educational Outreach Programs That Benefit Students and Scientists

Both scientists and the public would benefit from improved communication of basic scientific research and from integrating scientists into education outreach, but opportunities to support these efforts are limited. We have developed two low-cost programs—"Present Your PhD Thesis to a 12-Year-Old" and "Shadow a Scientist”—that combine training in science communication with outreach to area middle schools. We assessed the outcomes of these programs and found a 2-fold benefit: scientists improve their communication skills by explaining basic science research to a general audience, and students'' enthusiasm for science and their scientific knowledge are increased. Here we present details about both programs, along with our assessment of them, and discuss the feasibility of exporting these programs to other universities.     

Development of a Genus-Specific Antigen Capture ELISA for Orthopoxviruses – Target Selection and Optimized Screening

Orthopoxvirus species like cowpox, vaccinia and monkeypox virus cause zoonotic infections in humans worldwide. Infections often occur in rural areas lacking proper diagnostic infrastructure as exemplified by monkeypox, which is endemic in Western and Central Africa. While PCR detection requires demanding equipment and is restricted to genome detection, the evidence of virus particles can complement or replace PCR. Therefore, an easily distributable and manageable antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection of orthopoxviruses was developed to facilitate particle detection. By comparing the virus particle binding properties of polyclonal antibodies developed against surface-exposed attachment or fusion proteins, the surface protein A27 was found to be a well-bound, highly immunogenic and exposed target for antibodies aiming at virus particle detection. Subsequently, eight monoclonal anti-A27 antibodies were generated and characterized by peptide epitope mapping and surface plasmon resonance measurements. All antibodies were found to bind with high affinity to two epitopes at the heparin binding site of A27, toward either the N- or C-terminal of the crucial KKEP-segment of A27. Two antibodies recognizing different epitopes were implemented in an antigen capture ELISA. Validation showed robust detection of virus particles from 11 different orthopoxvirus isolates pathogenic to humans, with the exception of MVA, which is apathogenic to humans. Most orthopoxviruses could be detected reliably for viral loads above 1 × 10³ PFU/mL. To our knowledge, this is the first solely monoclonal and therefore reproducible antibody-based antigen capture ELISA able to detect all human pathogenic orthopoxviruses including monkeypox virus, except variola virus which was not included. Therefore, the newly developed antibody-based assay represents important progress towards feasible particle detection of this important genus of viruses.