The ``hitchhiking Effect'''' Revisited

The number of selectively neutral polymorphic sites in a random sample of genes can be affected by ancestral selectively favored substitutions at linked loci. The degree to which this happens depends on when in the history of the sample the selected substitutions happen, the strength of selection and the amount of crossing over between the sampled locus and the loci at which the selected substitutions occur. This phenomenon is commonly called hitchhiking. Using the coalescent process for a random sample of genes from a selectively neutral locus that is linked to a locus at which selection is taking place, a stochastic, finite population model is developed that describes the steady state effect of hitchhiking on the distribution of the number of selectively neutral polymorphic sites in a random sample. A prediction of the model is that, in regions of low crossing over, strongly selected substitutions in the history of the sample can substantially reduce the number of polymorphic sites in a random sample of genes from that expected under a neutral model.     

Germ-line DNA copy number variation frequencies in a large North American population

Genomic copy number variation (CNV) is a recently identified form of global genetic variation in the human genome. The Affymetrix GeneChip 100 and 500 K SNP genotyping platforms were used to perform a large-scale population-based study of CNV frequency. We constructed a genomic map of 578 CNV regions, covering approximately 220 Mb (7.3%) of the human genome, identifying 183 previously unknown intervals. Copy number changes were observed to occur infrequently (<1%) in the majority (>93%) of these genomic regions, but encompass hundreds of genes and disease loci. This North American population-based map will be a useful resource for future genetic studies. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Constitutive protein secretion by guinea-pig seminal vesicle epithelial cells.

1. Secretion of pulse-labelled protein by the isolated epithelium of guinea-pig seminal vesicle epithelium was rapid, unaffected by cholinergic and adrenergic drugs, cyclic nucleotides or changes in the sodium, potassium and calcium concentrations of the "chase" medium. 2. Low temperature, NH4Cl, hyper- and hypo-osmolarity and membrane-stabilizing agents inhibited secretion which was also dependent on aerobic metabolism. 3. Monensin reduced secretion of the six labelled, relatively low molecular weight proteins recovered from the medium in a concentration-dependent, apparently non-specific manner.     

Conformational Changes Relevant to Channel Activity and Folding within the first Nucleotide Binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator

Deletion of Phe-508 (F508del) in the first nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to defects in folding and channel gating. NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between β-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. CFFT-001 appears to interact with β-strands S3/S9/S10, consistent with docking simulations. Decreases in T(m) from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. We hypothesize that, in full-length CFTR, shifting the conformational equilibrium to reduce H8/H9 interactions with the uniquely conserved strands S9/S10 facilitates release of the regulatory region from the NBD dimerization interface to promote dimerization and thereby increase channel open probability. These studies enabled by our NMR assignments for F508del NBD1 provide a window into the conformational fluctuations within CFTR that may regulate function and contribute to folding energetics.     

Linking predator–prey interactions with exposure to a trophically transmitted parasite using PCR‐based analyses

Parasite transmission is determined by the rate of contact between a susceptible host and an infective stage and susceptibility to infection given an exposure event. Attempts to measure levels of variation in exposure in natural populations can be especially challenging. The level of exposure to a major class of parasites, trophically transmitted parasites, can be estimated by investigating the host's feeding behaviour. Since the parasites rely on the ingestion of infective intermediate hosts for transmission, the potential for exposure to infection is inherently linked to the definitive host's feeding ecology. Here, we combined epidemiological data and molecular analyses (polymerase chain reaction) of the diet of the definitive host, the white‐footed mouse (Peromyscus leucopus), to investigate temporal and individual heterogeneities in exposure to infection. Our results show that the consumption of cricket intermediate hosts accounted for much of the variation in infection; mice that had consumed crickets were four times more likely to become infected than animals that tested negative for cricket DNA. In particular, pregnant female hosts were three times more likely to consume crickets, which corresponded to a threefold increase in infection compared with nonpregnant females. Interestingly, males in breeding condition had a higher rate of infection even though breeding males were just as likely to test positive for cricket consumption as nonbreeding males. These results suggest that while heterogeneity in host diet served as a strong predictor of exposure risk, differential susceptibility to infection may also play a key role, particularly among male hosts. By combining PCR analyses with epidemiological data, we revealed temporal variation in exposure through prey consumption and identified potentially important individual heterogeneities in parasite transmission.     

Study of model systems to test the potential function of Artemia group 1 late embryogenesis abundant (LEA) proteins

Embryos of the brine shrimp, Artemia franciscana, are genetically programmed to develop either ovoviparously or oviparously depending on environmental conditions. Shortly upon their release from the female, oviparous embryos enter diapause during which time they undergo major metabolic rate depression while simultaneously synthesize proteins that permit them to tolerate a wide range of stressful environmental events including prolonged periods of desiccation, freezing, and anoxia. Among the known stress-related proteins that accumulate in embryos entering diapause are the late embryogenesis abundant (LEA) proteins. This large group of intrinsically disordered proteins has been proposed to act as molecular shields or chaperones of macromolecules which are otherwise intolerant to harsh conditions associated with diapause. In this research, we used two model systems to study the potential function of the group 1 LEA proteins from Artemia. Expression of the Artemia group 1 gene (AfrLEA-1) in Escherichia coli inhibited growth in proportion to the number of 20-mer amino acid motifs expressed. As well, clones of E. coli, transformed with the AfrLEA-1 gene, expressed multiple bands of LEA proteins, either intrinsically or upon induction with isopropyl-β-thiogalactoside (IPTG), in a vector-specific manner. Expression of AfrLEA-1 in E. coli did not overcome the inhibitory effects of high concentrations of NaCl and KCl but modulated growth inhibition resulting from high concentrations of sorbitol in the growth medium. In contrast, expression of the AfrLEA-1 gene in Saccharomyces cerevisiae did not alter the growth kinetics or permit yeast to tolerate high concentrations of NaCl, KCl, or sorbitol. However, expression of AfrLEA-1 in yeast improved its tolerance to drying (desiccation) and freezing. Under our experimental conditions, both E. coli and S. cerevisiae appear to be potentially suitable hosts to study the function of Artemia group 1 LEA proteins under environmentally stressful conditions.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-015-0647-3) contains supplementary material, which is available to authorized users.     

Stainless steel mesh supports high density cell growth and production of recombinant Mullerian Inhibiting Substances

Summary Stainless steel mesh supported the high density growth of anchorage dependent CHO fibroblasts without the use of a special culture system. CHO cells, designated B-9, containing an amplified genomic construct of the human gene for Mullerian Inhibiting Substance (MIS), grew to a high confluent density on stainless steel meshwork while producing substantial amounts of human recombinant MIS over a long period of time. The mesh could be easily coated with various extracellular matrix proteins, such as Laminin, Fibronectin, Collagen or Matrigel, which permitted the testing of the effects of surface modifications on cell yield and recombinant protein production. Since the amount of medium per surface area required for optimal cell growth is lower than for some large volume cell culture methods, media costs can be reduced using mesh. In addition, no special cell culture equipment or complex manipulations are required. Thus, the use of meshwork for anchorage-dependent cells can increase the efficiency of growth and decrease the cost of recombinant protein production. This work is supported by NIH grant CA 17393 and American Cancer Society grant PDT 221A to P. K. D. and NIH grant EY 06535 to J. E. Editor's Statement This approach to large scale, high density cultivation of cells, one of several which are based on increasing surface area of the cultures, allows the production of large amounts of recombinant product within a research laboratory with modest bulk culture capability.     

LCR/MEL: a versatile system for high-level expression of heterologous proteins in erythroid cells.

We have used the human globin locus control region (LCR) to assemble an expression system capable of high-level, integration position-independent expression of heterologous genes and cDNAs in murine erythroleukaemia (MEL) cells. The cDNAs are inserted between the human beta-globin promoter and the second intron of the human beta-globin gene, and this expression cassette is then placed downstream of the LCR and transfected into MEL cells. The cDNAs are expressed at levels similar to those of the murine beta-globin in the induced MEL cells. Heterologous genomic sequences can also be expressed at similar levels when linked to to the LCR and beta-globin promoter. In addition we demonstrate that, after induction of differentiation, MEL cells are capable of secreting heterologous proteins over a prolonged time period, making this system suitable for use in continuous production systems such as hollow fibre bioreactors. The utility of the LCR/MEL cell system is demonstrated by the expression of growth hormone at high levels (greater than 100 mg/l) 7 days after induction. Since the expression levels seen do not depend upon gene amplification and are independent of the integration position of the expression cassette, it is possible to obtain clones with stable high-level expression within 3-4 weeks after transfection.