Extensive de novo mutation rate variation between individuals and across the genome of Chlamydomonas reinhardtii.

Describing the process of spontaneous mutation is fundamental for understanding the genetic basis of disease, the threat posed by declining population size in conservation biology, and much of evolutionary biology. Directly studying spontaneous mutation has been difficult, however, because new mutations are rare. Mutation accumulation (MA) experiments overcome this by allowing mutations to build up over many generations in the near absence of natural selection. Here, we sequenced the genomes of 85 MA lines derived from six genetically diverse strains of the green alga Chlamydomonas reinhardtii. We identified 6843 new mutations, more than any other study of spontaneous mutation. We observed sevenfold variation in the mutation rate among strains and that mutator genotypes arose, increasing the mutation rate approximately eightfold in some replicates. We also found evidence for fine-scale heterogeneity in the mutation rate, with certain sequence motifs mutating at much higher rates, and clusters of multiple mutations occurring at closely linked sites. There was little evidence, however, for mutation rate heterogeneity between chromosomes or over large genomic regions of 200 kbp. We generated a predictive model of the mutability of sites based on their genomic properties, including local GC content, gene expression level, and local sequence context. Our model accurately predicted the average mutation rate and natural levels of genetic diversity of sites across the genome. Notably, trinucleotides vary 17-fold in rate between the most and least mutable sites. Our results uncover a rich heterogeneity in the process of spontaneous mutation both among individuals and across the genome.

The route of infection determines Wolbachia antibacterial protection in Drosophila.

Bacterial symbionts are widespread among metazoans and provide a range of beneficial functions. Wolbachia-mediated protection against viral infection has been extensively demonstrated in Drosophila. In mosquitoes that are artificially transinfected with Drosophila melanogaster Wolbachia (wMel), protection from both viral and bacterial infections has been demonstrated. However, no evidence for Wolbachia-mediated antibacterial protection has been demonstrated in Drosophila to date. Here, we show that the route of infection is key for Wolbachia-mediated antibacterial protection. Drosophila melanogaster carrying Wolbachia showed reduced mortality during enteric-but not systemic-infection with the opportunist pathogen Pseudomonas aeruginosaWolbachia-mediated protection was more pronounced in male flies and is associated with increased early expression of the antimicrobial peptide Attacin A, and also increased expression of a reactive oxygen species detoxification gene (Gst D8). These results highlight that the route of infection is important for symbiont-mediated protection from infection, that Wolbachia can protect hosts by eliciting a combination of resistance and disease tolerance mechanisms, and that these effects are sexually dimorphic. We discuss the importance of using ecologically relevant routes of infection to gain a better understanding of symbiont-mediated protection.

PrfA regulation offsets the cost of Listeria virulence outside the host.

Virulence traits are essential for pathogen fitness, but whether they affect microbial performance in the environment, where they are not needed, remains experimentally unconfirmed. We investigated this question with the facultative pathogen Listeria monocytogenes and its PrfA virulence regulon. PrfA-regulated genes are activated intracellularly (PrfA 'ON') but shut down outside the host (PrfA 'OFF'). Using a mutant PrfA regulator locked ON (PrfA*) and thus causing PrfA-controlled genes to be constitutively activated, we show that virulence gene expression significantly impairs the listerial growth rate (µ) and maximum growth (A) in rich medium. Deletion analysis of the PrfA regulon and complementation of a L. monocytogenes mutant lacking all PrfA-regulated genes with PrfA* indicated that the growth reduction was specifically due to the unneeded virulence determinants and not to pleiotropic regulatory effects of PrfA ON. No PrfA*-associated fitness disadvantage was observed in infected eukaryotic cells, where PrfA-regulated virulence gene expression is critical for survival. Microcosm experiments demonstrated that the constitutively virulent state strongly impaired L. monocytogenes performance in soil, the natural habitat of these bacteria. Our findings provide empirical proof that virulence carries a significant cost to the pathogen. They also experimentally substantiate the assumed, although not proven, key role of virulence gene regulation systems in suppressing the cost of bacterial virulence outside the host.

Studies over the existence of a certain impulse-based fuzzy integrodifferential equations of the Sobolev type.

The present investigation employs impulses and a non-local constraint to prove the existence are some various types of abstract differential and integrodifferential equations related to the Sobolev type. Semigroup theory, specifically variants of constant formula, is utilized to get the analytical results for those equations. Furthermore, findings using the Banach fixed point approach were examined using fuzzy numbers with values spanning the [Formula: see text] range, which includes the normal, convex, upper semi-continuous, and compactly supported interval. A description is given for each situation to illustrate the principle.

Importance of non-technical skills in anaesthesia education.

Rising concern about patient safety has resulted in growing interest in non-technical skills (NTS) among anaesthesiologists. Growing evidence suggesting the use of good NTS training in patient safety in simulated as well as real-world environment made them important in medical education. Both technical skills (TS) and NTS are interdependent. Successful task performance depends on effective integration of both TS and NTS for any given situation. Development of tools for assessing the NTS of an anaesthesiologist in improving health care outcomes is challenging. Teaching, understanding and evaluating NTS among anaesthesiologists in improving health care outcomes is a domain which is supposed to be a rich seam for future studies.

Kinetic investigation of para-nitrophenol reduction with photodeposited platinum nanoparticles onto tunicate cellulose.

Photodeposition is a specific method for depositing metallic co-catalysts onto photocatalysts and was applied for immobilizing platinum nanoparticles onto cellulose, a photocatalytically inactive biopolymer. The obtained Pt@cellulose catalysts show narrow and well-dispersed nanoparticles with average sizes between 2 and 5 nm, whereby loading, size and distribution depend on the preparation conditions. The catalysts were investigated for the hydrogenation of para-nitrophenol via transfer hydrogenation using sodium borohydride as the hydrogen source, and the reaction rate constant was determined using the pseudo-first-order reaction rate law. The Pt@cellulose catalysts are catalytically active with rate constant values k from 0.09 × 10-3 to 0.43 × 10-3 min-1, which were higher than the rate constant of a commercial Pt@Al2O3 catalyst (k = 0.09 × 10-3 min-1). Additionally, the Pt@cellulose catalyst can be used for electrochemical hydrogenation of para-nitrophenol where the hydrogen is electrocatalytically formed. The electrochemical hydrogenation is faster compared to the transfer hydrogenation (k = 0.11 min-1).

Large discrete resistance jump at grain boundary in copper nanowire.

Copper is the current interconnect metal of choice in integrated circuits. As interconnect dimensions decrease, the resistivity of copper increases dramatically because of electron scattering from surfaces, impurities, and grain boundaries (GBs) and threatens to stymie continued device scaling. Lacking direct measurements of individual scattering sources, understanding of the relative importance of these scattering mechanisms has largely relied on semiempirical modeling. Here we present the first ever attempt to measure and calculate individual GB resistances in copper nanowires with a one-to-one correspondence to the GB structure. Large resistance jumps are directly measured at the random GBs with a value far greater than at coincidence GBs and first-principles calculations. The high resistivity of the random GB appears to be intrinsic, arising from the scaling of electron mean free path with the size of the lattice relaxation region. The striking impact of random GB scattering adds vital information for understanding nanoscale conductors.

Oral Bacterial Infection and Shedding in Drosophila melanogaster.

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Characterization of epidermal growth factor in mouse testis.

Considerable evidence exists to suggest that epidermal growth factor (EGF) influences spermatogenesis directly. The tissue source of this EGF, however, is not yet clear. In this study we examine whether the testis itself can serve as a source of EGF. Gel filtration fractions of acid extracted testes exhibited the ability to displace 125I-EGF from testis membranes. The testicular fractions containing the 125I-EGF displacement activity coeluted within the same range as those of submandibular gland (SG) fractions containing mature EGF and prepared in an identical fashion. Next, we employed specific antisera probes to investigate first, whether the testis synthesizes this EGF displacement activity and second, to determine the cell distribution of the testicular EGF. Two types of antisera probes were employed: 1) commercially available antisera to mature EGF (EGFm), i.e. the 6,000 M(r) peptide, and 2) polypeptide specific antisera to the C-terminus of the EGF precursor (EGFp), i.e. the 140,000 M(r) integral membrane molecule which exhibits seven EGF-like repeats in addition to the EGFm. Metabolic labeling of testis with 35S-methionine was performed, followed by immunoprecipitation with the anti-EGFm antisera. Parallel studies using kidney and SG were used as positive controls. Fluorograms exhibited a prominent band at M(r) 140,000 for testis and kidney, corresponding to the EGFp. There was, in addition, a M(r) 50,000 band present for the testis. In SG, a band at M(r) 6,000, corresponding to EGFm, in addition to bands at M(r) 21,000 and 46,000 were observed also. Immunoblotting of testis, kidney, and SG membrane preparations with the specific antisera to either the EGFm or EGFp also resulted in identifying the EGFp at M(r) 140,000, as well as other lower mol wt bands. Preadsorption of anti-EGFm antisera with excess EGFm eliminated all of the specific bands that were immunoblotted. Peroxidase immunocytochemistry of testis, kidney, and SG was also performed using the specific antisera to either EGFm or EGFp. EGFp and EGFm staining in SG and kidney was identical to previously published results in which the distribution of EGFm in these tissues was established. In testis, EGFm immunostaining showed positive results in Sertoli cells, pachytene spermatocytes and round spermatids. In contrast, EGFp immunostaining was limited to pachytene spermatocytes and round spermatids. These results suggest that the testis must now be included in the list of tissues capable of synthesizing EGFp. Specifically, EGFp synthesis appears limited to the post meiotic germ cells.

Relationship between submandibular gland epidermal growth factor and spermatogenesis in C3H mice.

Epidermal growth factor (EGF), a potent mitogen produced primarily in the submandibular gland of adult male mice, has been implicated in modulating processes known to be of vital importance in the regulation of spermatogenesis. In the present investigation we demonstrate that submandibular gland EGF from adult male mice is indeed capable of displacing radiolabeled EGF from testicular membranes. Scatchard analysis of this binding site reveals that it is of high affinity (Kd = 0.77 nM) and low capacity (Bmax = 8.15 fmol/mg protein). Cross-linking of 125I-EGF to the identical membrane preparation resulted in the SDS-PAGE/autoradiography identification of a single band at approximately 170 kDa. Next, we examined the cellular distribution of the EGF receptor in the testis using biotin-streptavidin immunoperoxidase and employing different antisera probes generated to a conserved sequence of the EGF receptor. The Scatchard and cross-linking data described above, along with the immunocytochemistry results, suggest strongly that there is only one functional binding site for EGF in the adult testis and that this receptor is present in Sertoli and Leydig cells.

Characterization of epidermal growth factor receptor in testis, epididymis and vas deferens of non-human primates.

The presence of the epidermal growth factor receptor (EGFR) in testis, epididymis and vas deferens of monkeys was demonstrated using a polyclonal antibody (RK2) raised against a peptide-specific sequence of the intracellular domain of the human EGFR. Immunoblotting of membrane preparations revealed a specific band at approximately 170 kDa corresponding to those of controls, A431 and monkey liver cells. Cryostat sections were stained by biotin-streptavidin peroxidase immunocytochemistry. The liver showed positive staining along the basolateral membranes of the hepatocytes lining the sinusoids. The testis showed positive staining indicating the presence of EGFR in Leydig cells, Sertoli cells and peritubular cells. In the epididymis, immunostaining of the EGFR was observed on both the basolateral and the luminal borders of the epididymal epithelium. Immunofluorescence studies revealed a similar pattern of EGFR distribution in the epididymis and indicated that the luminal immunostaining was vesicular. In the vas deferens, positive immunostaining was detected in a pattern very similar to that observed in the epididymis. There was no positive staining in the interstitium of the epididymis or in the smooth muscle cell layers of the vas deferens. The sections of all tissues treated with pre-immune serum were negative. These results suggest that EGF in the primate testis may act at the level of somatic cells. In addition, the basolateral and luminal EGFR staining in the epididymis and vas deferens suggest that these cells respond to an EGF, or EGF-like, source both at the basal, luminal or at both sides of the cells, or that these tissues serve as sites of EGF transcytosis across the epithelium.

An improved method of encapsulation of doxorubicin in liposomes: pharmacological, toxicological and therapeutic evaluation.

We describe here an improved method of encapsulating doxorubicin in liposomes using phosphatidylcholine, cholesterol and synthetic tetramyristoyl cardiolipin. With this new composition of lipids the entrapment of doxorubicin was found to be > 90%. Cytotoxicity studies using vincristine-resistant HL-60/VCR leukaemia cells showed that liposome-encapsulated doxorubicin reverses multidrug resistance 5-fold compared with conventional doxorubicin and at levels equivalent to that obtained using liposomes with natural cardiolipin. In normal mice, liposome-encapsulated doxorubicin was much less toxic than the conventional drug. A dose of 25 mg kg-1 i.v. of conventional doxorubicin produced 100% mortality in mice by day 14, whereas liposomal doxorubicin exhibited only 10% mortality by day 60. Liposomal doxorubicin demonstrated enhanced anti-tumour activity against murine ascitic L1210 leukaemia compared with conventional doxorubicin. At a dose of 15 mg kg-1, liposomal doxorubicin increased the median life span with 12 of 18 long-term (60 days) survivors compared with only 3 of 18 with conventional drug. Mice injected i.v. with liposomal doxorubicin had plasma levels 44-fold higher than conventional doxorubicin, producing significantly higher (P < 0.02) area under the plasma concentration curve. An altered tissue distribution was also observed with liposomal doxorubicin; cardiac tissue demonstrating at least 2-fold lower levels with liposomal doxorubicin probably accounting for its lower toxicity. This altered pharmacokinetics of liposome-encapsulated doxorubicin, providing enhanced therapeutic advantage and the ability to modulate multidrug resistance, could be useful in a clinical setting.