Strong land‐use effects on the dispersal patterns of adult stream insects: implications for transfers of aquatic subsidies to terrestrial consumers

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Population-Based Incidence Rates of Diarrheal Disease Associated with Norovirus,Sapovirus, and Astrovirus in Kenya

BackgroundDiarrheal diseases remain a major cause of mortality in Africa and worldwide. While the burden of rotavirus is well described, population-based rates of disease caused by norovirus, sapovirus, and astrovirus are lacking, particularly in developing countries.MethodsData on diarrhea cases were collected through a population-based surveillance platform including healthcare encounters and household visits in Kenya. We analyzed data from June 2007 to October 2008 in Lwak, a rural site in western Kenya, and from October 2006 to February 2009 in Kibera, an urban slum. Stool specimens from diarrhea cases of all ages who visited study clinics were tested for norovirus, sapovirus, and astrovirus by RT-PCR.ResultsOf 334 stool specimens from Lwak and 524 from Kibera, 85 (25%) and 159 (30%) were positive for norovirus, 13 (4%) and 31 (6%) for sapovirus, and 28 (8%) and 18 (3%) for astrovirus, respectively. Among norovirus-positive specimens, genogroup II predominated in both sites, detected in 74 (87%) in Lwak and 140 (88%) in Kibera. The adjusted community incidence per 100,000 person-years was the highest for norovirus (Lwak: 9,635; Kibera: 4,116), followed by astrovirus (Lwak: 3,051; Kibera: 440) and sapovirus (Lwak: 1,445; Kibera: 879). For all viruses, the adjusted incidence was higher among children aged <5 years (norovirus: 22,225 in Lwak and 17,511 in Kibera; sapovirus: 5,556 in Lwak and 4,378 in Kibera; astrovirus: 11,113 in Lwak and 2,814 in Kibera) compared to cases aged ≥5 years.ConclusionAlthough limited by a lack of controls, this is the first study to estimate the outpatient and community incidence rates of norovirus, sapovirus, and astrovirus across the age spectrum in Kenya, suggesting a substantial disease burden imposed by these viruses. By applying adjusted rates, we estimate approximately 2.8–3.3 million, 0.45–0.54 million, and 0.77–0.95 million people become ill with norovirus, sapovirus, and astrovirus, respectively, every year in Kenya.     

Development of Real-Time PCR Methods for the Detection of Bacterial Meningitis Pathogens without DNA Extraction

Neisseria meningitidis (Nm), Haemophilus influenzae (Hi), and Streptococcus pneumoniae (Sp) are the lead causes of bacterial meningitis. Detection of these pathogens from clinical specimens using traditional real-time PCR (rt-PCR) requires DNA extraction to remove the PCR inhibitors prior to testing, which is time consuming and labor intensive. In this study, five species-specific (Nm-sodC and -ctrA, Hi-hpd#1 and -hpd#3 and Sp-lytA) and six serogroup-specific rt-PCR tests (A, B, C, W, X, Y) targeting Nm capsular genes were evaluated in the two direct rt-PCR methods using PerfeCTa and 5x Omni that do not require DNA extraction. The sensitivity and specify of the two direct rt-PCR methods were compared to TaqMan traditional rt-PCR, the current standard rt-PCR method for the detection of meningitis pathogens. The LLD for all 11 rt-PCR tests ranged from 6,227 to 272,229 CFU/ml for TaqMan, 1,824–135,982 for 5x Omni, and 168–6,836 CFU/ml for PerfeCTa. The diagnostic sensitivity using TaqMan ranged from 89.2%-99.6%, except for NmB-csb, which was 69.7%. For 5x Omni, the sensitivity varied from 67.1% to 99.8%, with three tests below 90%. The sensitivity of these tests using PerfeCTa varied from 89.4% to 99.8%. The specificity ranges of the 11 tests were 98.0–99.9%, 97.5–99.9%, and 92.9–99.9% for TaqMan, 5x Omni, and PerfeCTa, respectively. PerfeCTa direct rt-PCR demonstrated similar or better sensitivity compared to 5x Omni direct rt-PCR or TaqMan traditional rt-PCR. Since the direct rt-PCR method does not require DNA extraction, it reduces the time and cost for processing CSF specimens, increases testing throughput, decreases the risk of cross-contamination, and conserves precious CSF. The direct rt-PCR method will be beneficial to laboratories with high testing volume.     

Differential effects of 20-trifluoromethyl leukotriene B4 on human neutrophil functions

A leukotriene B₄ (LTB₄) analog, 20-trifluoromethyl LTB₄ (20CF₃−LTB₄), has been synthesized and evaluated with human neutrophils for effects on chemotaxis and degranulation. 20CF₃−LTB₄ was equipotent to LTB₄ as a chemoattractant (EC₅₀, 3 nM), produced 50% of maximal activity of LTB₄, and competed with [H] LTB₄ for binding to intact human neutrophil LTB₄ receptors. In contrast to chemotactic activity, 20CF₃−LTB₄ in nanomolar concentrations exhibited antagonist activity without agonist activity up to 10 μM on LTB₄-induced degranulation. The analog had no significant effect on degranulation induced by the chemoattractant peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP). Like LTB₄, 20CF₃−LTB₄ induced neutrophil desensitization to degranulation by LTB₄. The results indicate that hydrogen atoms at C-20 of LTB₄ are critical for its intrinsic chemotactic and degranulation activities. The fact that 20CF₃−LTB₄ is a partial agonist for chemotaxis and an antagonist for degranulation syggests that different LTB₄ receptor subtypes are coupled to these neutrophil functions. Desensitization of the neutrophil degranulation response to LTB₄ can result from receptor occupancy by an antagonist, and therefore, the desensitization is not specific for an agonist.     

Identification and localization of high molecular weight proteins in insect flight and leg muscle.

Thick and thin filaments in asynchronous flight muscle overlap nearly completely and thick filaments are attached to the Z-disc by connecting filaments. We have raised antibodies against a fraction of Lethocerus flight muscle myofibrils containing Z-discs and associated filaments and also against a low ionic strength extract of myofibrils. Monoclonal antibodies were obtained to proteins of 800 kd (p800), 700 kd (p700), 400 kd (p400) and alpha-actinin. The positions of the proteins in Lethocerus flight and leg myofibrils were determined by immunofluorescence and electron microscopy. p800 is in connecting filaments of flight myofibrils and in A-bands of leg myofibrils. p700 is in Z-discs of flight myofibrils and an immunologically related protein, p500, is in leg muscle Z-discs. p400 is in M-lines of both flight and leg myofibrils. Preliminary DNA sequencing shows that p800 is related to vertebrate titin and nematode twitchin. Molecules of p800 could extend from the Z-disc a short way along thick filaments, forming a mechanical link between the two structures. All three high molecular weight proteins probably stabilize the structure of the myofibril.     

Expression of rat intestinal fatty acid binding protein in E. coli and its subsequent structural analysis: a model system for studying the molecular details of fatty acid-protein interaction

A prokaryotic expression vector containing the rec A promoter and a translational enhancer element from the gene 10 leader of bacteriophage T7 was used to direct efficient synthesis of rat intestinal fatty acid binding protein (I-FABP) in E. coli. Expression of I-FABP in E. coli has no apparent, deleterious effects on the organism. High levels of expression of I-FABP mRNA in supE⁺ strains of E. coli, such as JM101, is associated with suppression of termination at its UGA stop codon. This can be eliminated by using a sup-Estrain as MG1655 and by site-directed mutagenesis of the cDNA to create an in frame UAA stop codon. E. coli-derived rat I-FABP lacks its initiator Met residues. It has been crystallized with and without bound palmitate. High resolution x-ray crystallographic studies of the 131 residue apo- and holo-proteins have revealed the following. I-FABP contains 10 anti-parallel -strands organized into two orthogonally situated -sheets. The overall conformation of the protein resembles that of a clam — hence the term -clam. The bound ligand is located in the interior of the protein. Its carboxylate group forms part of a unique five member hydrogen bonding network consisting of two ordered solvent molecules as well as the side chains of Arg¹⁰⁶ and Gln¹¹⁵. The hydrocarbon chain of the bound C16:0 fatty acid has a distinctive bent conformation with a slight left-handed helical twist. This conformation is maintained by interactions with the side chains of a number of hydrophobic and aromatic amino acids. Apo-I-FABP has a similar overall conformation to holo-I-FABP indicating that the -clam structure is stable even without bound ligand. The space occupied by bound ligand in the core of the holo-protein is occupied by additional ordered solvent molecules in the apo-protein. Differences in the side chain orientations pf several residues located over a potential opening to the cores of the apo- and holo-proteins suggest that solvent may play an important role in the binding mechanism. Comparison of the C coordinates of apo- and holo-I-FABP with those of other proteins indicates it is a member of a superfamily that currently includes (i) 10 mammalian intracellular lipid binding proteins, (ii) the photoactive yellow protein from the purple photoautotrophic bacterium Ectothiorhodospira halophila and (iii) a group of extracellular lipid binding proteins from a diverse number of phyla that have a common barrel consisting of 8 anti-parallel -strands stacked in two nearly orthogonal sheets. In summary, E. coli-derived I-FABP not only represents a useful model for assessing the atomic details of fatty acid-protein interactions and the mechanisms which regulate acquisition and release of this type of ligand, but also structure/function relationships in other superfamily members.Abbreviations I-FABP Intestinal Fatty Acid Binding Protein - r.m.s root mean square     

Comprehensive detection of single base changes in human genomic DNA using denaturing gradient gel electrophoresis and a GC clamp

We present a simple, efficient extension of denaturing gradient gel electrophoresis that allows the detection of nearly any sequence change in a defined fragment of DNA. The fragment can be obtained either by means of the polymerase chain reaction or by restriction digestion of genomic DNA. With restriction fragments of genomic DNA, sequence information is not required, and covalent modifications in genomic DNA that are lost in a PCR, such as methylation, are detectable. We describe how a GC clamp (an arbitrary, G+C-rich sequence of 30 to 60 bp) can be attached to a selected restriction fragment present in a digest of genomic DNA. The GC clamp alters the melting properties of the fragment; this change greatly increases the fraction of possible mutations that is detectable. In a 272-bp HaeIII fragment from the human beta-globin gene, we were able to detect 13 of 13 mutations tested in human genomic DNA. Four additional mutations in cloned plasmids were analyzed. The data agree with a simple theoretical model for DGGE, which predicts how two fragments, differing at a single (specified) base pair, are resolved in a gradient gel as a function of running time for the gel. The calculation assists in the design of probes and gel conditions that aid in the detection of sequence changes.