text:
- Research article
- Open Access
The role of facial pattern variation for species recognition in red-fronted lemurs (Eulemur rufifrons)
- Hanitriniaina Rakotonirina1, 2Email authorView ORCID ID profile,
- Peter M. Kappeler1, 2, 3 and
- Claudia Fichtel1
© The Author(s). 2018
- Received: 28 June 2017
- Accepted: 16 January 2018
- Published: 13 February 2018
Species recognition, i.e., the ability to distinguish conspecifics from heterospecifics, plays an essential role in reproduction. The role of facial cues for species recognition has been investigated in several non-human primate species except for lemurs. We therefore investigated the role of facial cues for species recognition in wild red-fronted lemurs (Eulemur rufifrons) at Kirindy Forest. We presented adult red-fronted lemurs pictures of male faces from five species including red-fronted lemurs, three closely related species, white-fronted lemurs (E. albifrons), brown lemurs (E. fulvus), rufous brown lemurs (E. rufus), and genetically more distant red-bellied lemurs (E. rubriventer), occurring in allopatry with the study population. We predicted that red-fronted lemurs respond stronger to conspecific than to heterospecific pictures and that females show stronger responses than males. In addition, if genetic drift has played a role in the evolution of facial color patterns in the members of this genus, we predicted that responses of red-fronted lemurs correlate negatively with the genetic distance to the different species stimuli.
Red-fronted lemurs looked significantly longer at pictures of their own species than at those of heterospecifics. Females spent less time looking at pictures of white-fronted, brown and red-bellied lemurs than males did, but not to pictures of red-bellied lemurs and a control stimulus. Individuals also exhibited sniffing behavior while looking at visual stimuli, and the time spent sniffing was significantly longer for pictures of conspecifics compared to those of heterospecifics. Moreover, the time spent looking and sniffing towards the pictures correlated negatively with the genetic distance between their own species and the species presented as stimulus.
We conclude that red-fronted lemurs have the ability for species recognition using visual facial cues, which may allow them to avoid costly interbreeding. If so, sexual selection might have influenced the evolution of facial patterns in eulemurs. Since responses also correlated with genetic distance, our findings suggest a potential role of genetic drift as well as sexual selection in influencing the evolution of facial variation in eulemurs. Because study subjects looked and sniffed towards the presented pictures, red-fronted lemurs might have the ability for multi-modal species recognition.
- Red-fronted lemurs
- Species recognition
- Visual signals
- Sexual selection
- Genetic drift
The ability to differentiate conspecifics from heterospecifics plays an important role in reproduction [1–6]. Since females usually experience higher costs during reproduction than males, heterospecific mating is more costly for females [7–9]. Females should therefore be selected to recognize and discriminate against heterospecific males to avoid costly interbreeding [9]. Indeed, the ability for species recognition has been demonstrated in several taxa, such as bats using olfactory signals [6], fish using olfactory or visual signals [10, 11] and frogs, birds and mammals using acoustic signals [12–15].
Visual cues have been suggested to be important for several animal taxa as they can be used for individual as well as species recognition [11, 16–18]. In addition, facial color patterns are among the phenotypic traits that play a communicative role in many social interactions of primates [19–24]. Facial cues can contain visual information such as shape and colors that differ across individuals or species [25–28], and they can provide information about social status, condition and identity of an individual [22, 23, 29, 30]. Several studies have demonstrated the ability of non-human primates to differentiate individuals of their own kin/group from strangers and also to discriminate between conspecifics and heterospecifics based on visual cues [2, 21, 31–35]. For example, chimpanzees (Pan troglodytes) and rhesus macaques (Macaca mulatta) used facial cues in black-and-white photographs presented on a computer screen to discriminate between different individuals [22]. Chimpanzees are also able to discriminate kin by means of black-and-white photographs of mothers and their offspring, matching mother-son dyads but not mother-daughter ones [36]. Finally, Tonkean macaques (Macaca tonkeana) and brown capuchin monkeys (Cebus apella) were able to discriminate between pictures of conspecific and heterospecific individuals, as inferred by their longer looking time towards pictures of conspecifics [25].
The lemurs of Madagascar also exhibit highly diverse facial color patterns [37, 38], which may have a communicative function in social interactions as well as in species recognition. So far only a few studies have investigated the potential use of visual signals for individual or species recognition in lemurs, however. For example, brown and black lemurs (Eulemur macaco) were able to differentiate between familiar and unfamiliar individuals by using facial cues [39], and females of seven eulemur species differentiated colorful from non-colorful conspecific male photographs [40], suggesting a potential ability for visual species recognition as well. In contrast to visual signals, olfactory signals are used by some species to discriminate conspecifics from heterospecifics [41–43], whereas acoustic signals, especially long-distance calls, appear to be used in mouse lemurs (Microcebus murinus) to distinguish conspecifics from heterospecifics [44].
Among lemurs, eulemurs are the only taxon with sexual dichromatism, as males are particularly colorful and show considerably more variation in facial patterns than females [24, 38]. Eulemurs have dichromatic color vision, except for some females that exhibit polymorphic trichromacy [45–49], suggesting that variation in facial coloration can be perceived by them (see also [40]). Additional visual information used in this context may include variation in patterns, shape and contrasts.
Genetic distance and description of facial color patterns for the different species (species are listed according to their genetic distance between red-fronted lemurs and the respective species)
Red-fronted lemurs
Dark red crown, black muzzle, golden-red cheek beard, creamy-white patches above the eyes.
Rufous brown lemurs
Genetic distance: 0.35
Allopatric heterospecific and very similar to red-fronted lemurs in facial color patterns: brick-red crown, golden-red cheek beard, black muzzle and black midfacial stripe extending from crown to nose.
White-fronted lemurs
Genetic distance: 0.72
Occurs in allopatry with red-fronted lemurs and facial color variation differs strongly from red-fronted lemurs. Black muzzle and white beard, cheeks and crown.
Brown lemurs
Genetic distance: 0.72
Occurs in allopatry with red-fronted lemurs and is slightly different in facial color patterns. Dark-brown to almost black muzzle and crown, light grey beard and variable patches of light fur above the eyes.
Red-bellied lemurs
Genetic distance: 4.57
Occurs in sympatry with red-fronted lemurs in the eastern parts of Madagascar but not at the study site in the West and is very different in facial color patterns. Black muzzle, face shading to black; patches of white skin form characteristic "tear-drops" beneath the eyes, no bushy beard.
If variation in facial color patterns is used for species recognition in eulemurs, we predicted that red-fronted lemurs should respond stronger to pictures of faces of their own species than to pictures of faces of heterospecifics. Additionally, if sexual selection has played a role in the evolution of facial color pattern variation, we predicted that females should show stronger responses than males. Finally, as the species used as stimuli differ in phylogenetic distance to the test species, we predicted that if genetic drift has played a role in the evolution of facial color patterns in eulemurs, the response of red-fronted lemurs should correlate negatively with their respective genetic distance to the different species used as stimuli.
Time spent looking towards the picture
Parameter estimated for the Linear Mixed Models (LMM) on the influence of (a) the species of the presented picture and (b) the genetic distance between species on the percentage of time spent looking towards the pictures. The influence (c) of species of the presented picture and (d) the genetic distance between species on the percentage of time spent sniffing the pictures
Response variable
Random factors
Fixed factors
Percentage of time spent looking towards the pictures
Individual identity
< 0.001
rufous brown lemurs
< 0.01
brown lemurs
< 0.001
white-fronted lemurs
< 0.001
red-bellied lemurs
< 0.001
< 0.001
rufous brown lemurs-sex male
brown lemurs- sex male
white-fronted lemurs-sex male
< 0.001
red-bellied lemurs-sex male
control-sex male
Percentage of time spent looking towards the pictures
Individual identity
< 0.001
genetic distance
< 0.001
Percentage of time spent of sniffing events
Individual identity
< 0.001
rufous brown lemurs
< 0.05
brown lemurs
< 0.001
white-fronted lemurs
< 0.05
red-bellied lemurs
< 0.001
< 0.001
Percentage of time of sniffing events
Individual identity
< 0.001
genetic distance
< 0.01
Duration of sniffing
This study provides the first investigation of wild lemurs' ability to discriminate between photographs of their own and closely related species. Our results indicate that red-fronted lemurs can use facial cues to discriminate between conspecifics and heterospecifics. Interestingly, test subjects also spent more time sniffing during presentation of conspecific pictures, indicating that they also may use olfactory cues in this context. Hence, they might simultaneously process olfactory and visual information to differentiate conspecifics from heterospecifics, suggesting that multi-modal communication might play a role in species recognition in these animals. Moreover, males and females differed in time spent looking towards the pictures of some heterospecifics, which may suggest a potential role of sexual selection in the evolution of facial variation in this species. Since the time spent looking as well as sniffing at the pictures were negatively correlated with genetic distances between red-fronted lemurs and the stimuli species, genetic drift may have also influenced the evolution of facial color patterns in this species. Finally, our study showed that the experimental use of photographs is feasible to test the ability of wild non-human primates for species recognition, which has been so far studied only in captive settings [2, 22, 34, 36, 39]. We discuss these key results in more detail below.
The variation in time spent looking at the pictures indicates that red-fronted lemurs are able to discriminate between the pictures of conspecifics and heterospecifics. Looking duration also appeared to correspond to the similarity of facial patterns between red-fronted lemurs and the other species: rufous brown lemurs are very similar in facial appearance to red-fronted lemurs, and these two species are also difficult for humans to distinguish (Fig. 1, Table 1), whereas white-fronted, brown and red-bellied lemurs are gradually more different in facial appearance. In addition, females spent less time looking towards pictures of white-fronted, brown and red-bellied lemurs than males, thus exhibiting a more pronounced differentiated response. Since we adjusted the size of the pictures to the average head size of the respective species, we cannot completely rule out the possibility that the responses of red-fronted lemurs were influenced by the slight differences in size of the pictures. However, if the size of the stimulus per se has influenced their responses, they should have responded more strongly to the control, which was adjusted to the average size of all stimuli, but they looked only briefly at this stimulus.
As interbreeding can occur in non-human primates (e.g. in macaques [59], eulemurs [50–55], sexual selection may act on species to avoid potentially costly heterospecific mating. Pronounced sexual dichromatism and striking differences in male patterning and coloration may provide a substrate for species recognition in the context of mate choice, and our experiments indicate that females perceive and respond to this variation. For example, red-bellied lemurs occur in sympatry with red-fronted, brown and white-fronted lemurs in the east of Madagascar [58]. Because these three species are visually very different from its sympatric congener, facial color variation might have played a role in creating reproductive barriers among these species during their recent divergence [60]. Because some eulemur species hybridize in their natural contact zones, future experiments in hybrid zones could investigate whether individuals in these zones discriminate potential mates based on variation in facial patterns.
Sex differences in responses towards the pictures might also reflect differences in color vision between sexes. Females can have polymorphic trichromacy or be dichromatic, whereas males are all dichromatic. Hence, eulemur females exhibiting a polymorphic trichromacy have the ability to perceive red and orange colors [61], and may therefore have shown a more pronounced differentiated response than males. However, the genetic tests required to test this assumption have not been performed. Interestingly, males payed more attention to stimuli of white-fronted, brown and red-bellied lemurs than females. Indeed, facial colors of these three species are dominated by a dark face with light (white or light gray) patches (Fig. 1). As red-fronted lemur males exhibit only dichromatic color vision [24, 62], contrasting dark and light areas might be more salient stimuli to them than to potentially trichromatic females.
The degree of phenotypic differences between red-fronted lemurs and white-fronted, brown, rufous brown and red-bellied lemurs also corresponds both to the genetic distance between them, as well as to the time red-fronted lemurs spent looking at the various stimuli. Similarly, the looking duration of macaques towards pictures of several heterospecific species also correlated with their morphological similarity in facial patterns as well as the genetic distance between them [2, 34]. Genetic differentiation as a result of drift during and following recent speciation events may therefore also have played a role in the evolution of facial color pattern in eulemurs.
Finally, multiple studies demonstrated that animals can process and use signals of different modalities for species recognition [63–65]. For example, male blackcaps (Sylvia atricapilla) are able to associate acoustic and visual sensory modalities in matching species-specific songs and species-specific plumage to distinguish their own species from sympatric heterospecifics (Sylvia borin) during playback experiments presented in combination with stuffed models of conspecifics and heterospecifics [64]. In non-human primates, for instance, tufted capuchin monkeys (Cebus apella), rhesus macaques (Macaca mulatta) as well as Japanese macaques (Macaca fuscata) are able to use visual and acoustic sensory modalities (voice-face matching) to distinguish between conspecifics and heterospecifics [66–68]. Moreover, ringtailed lemurs (Lemur catta) are capable of multi-modal (olfactory-auditory matching) individual recognition [69], and the use of olfactory signals for species recognition in some eulemurs has been already shown [41, 43]. Whether red-fronted lemurs are capable of multi-modal species recognition was not explicitly tested in this study. However, our results showed that while red-fronted lemurs processed visual cues during the experiment, they also sniffed at the stimuli. Thus, red-fronted lemurs might be able to use two different sensory modalities (olfactory-visual matching) at the same time to discriminate individuals of their own species from heterospecifics. However, explicit experiments with signals of two different modalities are required to confirm our preliminary conclusion that red-fronted lemurs dispose of multi-modal species recognition abilities.
This study revealed the importance of facial cues as visual signals for species recognition in wild red-fronted lemurs. Females of red-fronted lemurs may also be better at differentiating conspecifics from heterospecifics due to sex difference in color vision abilities. Our findings suggest a potential role for sexual selection as well as genetic drift in influencing the evolution of facial variation in eulemurs. Moreover, this study revealed evidence for visual species recognition abilities in wild red-fronted lemurs and also suggested a potential for multi-modal species recognition. However, it remains unclear which specific components of the facial cues are used for species recognition, requiring further investigations to identify the essential cue(s), such as colors, patterns or a combination of both, used by eulemurs to discriminate their own from different species as well as among individuals.
Study site
Experiments were conducted with red-fronted lemurs in Kirindy Forest, western Madagascar (Fig. 1). Study subjects are individually marked as part of a long-term study and are well habituated to human observers [56, 57]. We studied eight adult females and seven adult males in four different groups (2–5 subjects per group).
Experimental design
Responses of experimental subjects were recorded with a SONY digital video camera from briefly before until 60 s after the onset of each experiment. The camera was placed in front of the focal animal, aligned with the picture frame so that looking direction could be clearly recorded. Based on these video-recordings, we measured the time each subject spent looking towards the picture after the onset (looking direction within a 45° angle of the direct line of sight towards the picture), and calculated the percentage of time spent looking towards the picture from the total time spent looking around. In addition, during the experiments, we observed sniffing behaviors of focal individuals while conducting the experiments. We therefore measured also the time individuals spent sniffing (inhaling a short and distinct breath through the nose combined with very small movements of the snout) towards each picture after the onset and calculated the percentage of time spent sniffing (see Additional file 1). Videos were analyzed frame-by-frame with a resolution of 30 frames/s, using Adobe Premiere Elements (12.0). All experiments were rated by HR, and 10% were rated again by a second observer naïve to the research question. We used R to calculate the intra-class correlation coefficient (ICC) to test for inter-observer reliability. The resulting ICC was 0.95 indicating strong agreement between raters.
Sample video showing an individual of red-fronted lemurs looking at the picture with sniffing behavior. (MP4 3840 Kb)
Statistical analyses
We used linear mixed models (LMM) to test for differences in the percentage of time red-fronted lemurs spent looking towards the pictures as well as the percentage of time spent sniffing at the pictures in response to different stimuli using LmerTest package in R [71]. Percentage of time looking towards the pictures and percentage of time sniffing in the direction of the pictures were arcsine-square root transformed and fitted as responses. Species and sex were fitted as fixed factors and individual identity as a random factor to control for repeated measurements. Because the genetic distance correlates with the categories of the species, we fitted a second LMM in order to examine whether the percentage of time red-fronted lemurs spent looking and sniffing towards the pictures was influenced by the genetic distances between red-fronted lemurs and the species providing the stimuli. The percentage of time spent looking or sniffing towards the pictures were fitted as response, genetic distance and sex were fitted as fixed factors and individual identity as random factor. All analyses were conducted in R version 3.1.3.
We thank the Malagasy Ministère de l'Environnement et des Eaux et Forêts, the Departement de Biologie Ecologie et Conservation Animale de l'Université d'Antananarivo, the Centre National de Formation, d'Etudes et de Recherche en Environnement et Foresterie de Morondava, Madagascar National Parks and the Centre ValBio Ranomafana for allowing and supporting research in Kirindy Forest and Ranomafana National Park. We are grateful to Daniela Fuchs for helping with the video analyses, as well as Stephen Nash for the illustrations of Eulemur faces and Matthias Markolf for the photographs. We thank the German Academic Exchange Service (DAAD) for supporting and funding this study. PMK was a Fellow at the Wissenschaftskolleg zu Berlin while writing this manuscript.
This study was financially supported by the German Primate Center in Göttingen Germany, the University of Göttingen (Georg-August-Universität Göttingen) and the German Academic Exchange Service (DAAD).
Availability of data and materials
The data will be made available in a public database (dryad.org) prior to publication.
Authors' contributions
HR, PMK, CF conceived the study and wrote the manuscript. HR collected and analyzed the data. All authors read and approved the final manuscript.
Ethics approval
This study was conducted in accordance with the German and Malagasy (Commission Tripartite CAFF) legal and ethical requirements of appropriate animal procedures. Research protocols and experimental procedures were approved by the Ministry for the Environment, Water and Forests of Madagascar (MINEEF).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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text:
- Research article
- Open Access
Salmonella Typhimurium undergoes distinct genetic adaption during chronic infections of mice
- Emilie Søndberg1 and
- Lotte Jelsbak2Email author
© Søndberg and Jelsbak. 2016
- Received: 13 August 2015
- Accepted: 25 February 2016
- Published: 8 March 2016
Typhoid fever caused by Salmonella enterica serovar Typhi (S. Typhi) is a severe systemic human disease and endemic in regions of the world with poor drinking water quality and sewage treatment facilities. A significant number of patients become asymptomatic life-long carriers of S. Typhi and serve as the reservoir for the disease. The specific mechanisms and adaptive strategies enabling S. Typhi to survive inside the host for extended periods are incompletely understood. Yet, elucidation of these processes is of major importance for improvement of therapeutic strategies.
In the current study genetic adaptation during experimental chronic S. Typhimurium infections of mice, an established model of chronic typhoid fever, was probed as an approach for studying the molecular mechanisms of host-adaptation during long-term host-association.
Individually sequence-tagged wild type strains of S. Typhimurium 4/74 were used to establish chronic infections of 129X1/SvJ mice. Over the course of infections, S. Typhimurium bacteria were isolated from feces and from livers and spleens upon termination of the experiment. In all samples dominant clones were identified and select clones were subjected to whole genome sequencing. Dominant clones isolated from either systemic organs or fecal samples exhibited distinct single nucleotide polymorphisms (SNPs). One mouse appeared to have distinct adapted clones in the spleen and liver, respectively. Three mice were colonized in the intestines by the same clone containing the same non-synonymous SNP in a transcriptional regulator, kdgR, of metabolic genes. This likely indicates transmission of this clone between mice. The mutation was tracked to have occurred prior to 2 weeks post infection in one of the three mice and had subsequently been transmitted to the other two mice. Re-infection with this clone confirmed that it is superior to the wild type for intestinal colonization.
During 4 to 6 weeks of chronic infections, S. Typhimurium acquired distinct SNPs in known regulators of metabolic and virulence genes. One SNP, the kdgR-SNP was confirmed to confer selective advantage during chronic infections and constitute a true patho-adaptive mutation. Together, the results provide evidence for rapid genetic adaptation to the host of S. Typhimurium and validate experimental evolution in the context of host infection as a strategy for elucidating pathogen host interactions at the molecular level.
- Patho-adaptation
- Host-interaction
- Metabolism
- Virulence
- Transmission
Salmonella enterica are Gram-negative intracellular pathogens able to cause a variety of infections in both human and animals [1]. The species S. enterica constitutes >2500 different serovars, and where serovars with a broad host range like Salmonella enterica serovar Typhimurium (S. Typhimurium) mainly cause relatively mild gastrointestinal infections, host restricted serovars like S. Typhi (human restricted), typically cause severe systemic disease (typhoid fever) of their hosts. Furthermore, about 1–6 % of human typhoid patients become asymptomatic chronic carriers [2] and sporadically excrete bacteria in their stool thus serving as the reservoir for the disease. S. Typhimurium causes a typhoid-like systemic infection in mice and functions as a model for typhoid fever. Depending on the mouse strain it is possible to study both acute and chronic S. Typhimurium infections. Specifically, a mouse strain containing the wild type allele of the Slc11a1 gene serves as a robust model for studying asymptomatic chronic Salmonella infections [3]. During systemic chronic infections of these mice, S. Typhimurium reside in macrophages of the spleen, liver and mesenteric lymph nodes for up to 1 year with sporadic excretion of S. Typhimurium bacteria in the feces [3]. A negative genetic screen has shown that virulence factors associated with systemic acute infections, including the ones encoded by Salmonella Pathogenicity Island 2 (SPI2) [4], are also essential for establishing chronic infections [5]. However, the specific contributions of individual factors and the metabolic requirements during long-term host association are not fully understood.
In a few natural chronic bacterial infections of humans, the temporal occurrence of adaptive mutations over the course of months to years of infection has recently been delineated [6, 7]. For example, during chronic lung infections of Cystic Fibrosis patients, it has been documented how Pseudomonas aeruginosa acquire adaptive mutations facilitating persistence and survival in the host over the course infection [8]. The adapted clones retain the ability to transmit to and infect new patients and represent examples of bacterial evolution and host adaptation studied in real time. These studies have provided new insight into specific adaptive strategies favoring successful chronic infections by revealing the molecular signatures of pathogen host interactions. Similar studies exploring the level of within host adaptive evolution of S. Typhi during chronic infections are lacking, partly due to difficulties in identifying chronic carriers and maintaining an extended temporal sampling protocol, as these patients are often asymptomatic. In the present paper, using a previously established tagged strain approach [9], the level of genetic adaptation of S. Typhimurium during experimental chronic infections of mice was investigated. To this end, dominant clones isolated 4 to 6 weeks post infection were subjected to whole genome sequencing to identify potential adaptive mutations. In all mice, dominant S. Typhimurium clones isolated from either systemic organs or the fecal samples exhibited distinct single nucleotide polymorphisms (SNPs). Three mice were colonized in the intestines by the same clone containing the same non-synonymous SNP in a transcriptional regulator (kdgR) of metabolic genes, indicating transmission of this clone between mice. Further investigations confirmed that this clone was superior to the wild type for intestinal colonization presumably due to changes in the regulatory activity of the affected protein.
Together, these results provide evidence for rapid genetic adaptation to the host of S. Typhimurium and validate experimental evolution in the context of host infection as a strategy for elucidating pathogen host interactions at the molecular level.
Results and discussion
Dominant Salmonella clones emerge during chronic infections
S. Typhimurium CFU of samples following i.p. infection
Mouse ID
Per gram feces, 2 weeks post infection
Per gram feces, 4 weeks post infection
Per gram feces, 6 weeks post infection
Per spleen
Per liver
4 × 105
1.2 × 104
3.3 × 104
6.6 × 103
6.5 × 104
1.6 × 104
1.4 × 104
4.6 × 104
3.7 × 104
5 × 106
1.5 × 104
2 × 103
1 × 104
1.4 × 104
2.2 × 105
5.4 × 104
7.5 × 103
6 × 106
7.6 × 104
6.8 × 105
Dominant clones have acquired potential patho-adaptive mutations
Whole genome sequencing results
SRA accession numbera
Mouse ID
WITS prevalence
Coordinates in ST4/74 genomec
Gene affected and amino acid effectd,e
Feces, 6 weeks
63 %
kdgR, V61L
Feces, 4 weeks
>96 %
Synonymous, located in STM474_0020 Synonymous, located in maeB
Feces, 6 weeks
88 %
kdgR, V61L
Feces, 4 weeks
96 %
kdgR, V61L
63 %
Deletion, T
Promoter region of yicE Synonymous, located in STM474_0057
98 %
ptsN, S21R
Feces, 6 weeks
>98 %
ptsN, S21R
In conclusion, during this relatively short-term experimental chronic infection numerous mutations are detected in isolated clones. Most of these clones are associated with the intestinal environment; however, in one mouse mutations are also detected in isolates from systemic organs. Furthermore, in this mouse distinct mutations are detected in the liver- and spleen-clones, respectively, providing evidence for niche specific adaptation within the same host with limited migration between organs. Finally, the sequencing results also reveal that three mice are colonized by the same clone in the intestines implying transmission of the dominant clone between these mice via the fecal-oral route.
The ptsN-SNP clone is present and dominant in the fecal sample of mouse 5E at four weeks post infection
To investigate the role of ptsN during chronic infections and to determine if the ptsN-SNP confers any advantages for survival inside the host as suggested by the dominance of the clone, two new groups of 5 mice each were infected IP to establish chronic infections with a 1:1:1 mixture of ST4/74-WITS1 (wt-WITS1), the ptsN-SNP-WITS3-clone and an isogenic strain deleted for ptsN (ΔptsN). The infection was allowed to proceed for 5 weeks and S. Typhimurium bacteria were isolated from fecal samples every week and from livers and spleens upon termination. Isolated bacteria were genotyped (wt-WITS1, ptsN-SNP-WITS3 or ΔptsN) using a PCR/sequencing strategy. Only two mice were colonized by S. Typhimurium in the intestines prior to 3 weeks post infection (data not shown). This is in accordance with the first experiment (Table 1). At no point during the infection, from either the fecal or the organ samples of all 10 mice, was the ΔptsN strain isolated. This signifies that similar to acute infections, ptsN is essential for chronic infections. Only wt-WITS1 clones were isolated from the fecal samples with the exception of mouse 5 of group 2 where the fecal sample was dominated by the ptsN-SNP clone throughout the experiment (data not shown). In the spleen, the ptsN-SNP clone was present in an average of 37 %+/−7.% of analyzed clones, and in the liver it was significantly less prevalent with a 20 %+/−8.4 % recovery rate (p = 0.0002) (Fig. 2b). These results could suggest that the ptsN-SNP is less fit than the wt-WITS1 in both the spleen and liver during this infection and does not corroborate the observation from the original experiment from which the ptsN-SNP clone was isolated. Further investigations are required to elucidate the specific roles of PtsN during chronic infections and the consequence of the ptsN-SNP mutation.
The kdgR-mutation originated in mouse 5D at an early time-point post infection
The kdgR-SNP mutant exhibits rapid intestinal colonization upon re-infection
The kdgR-SNP mutant has constitutive repression of its target gene kdgK
The KdgR-protein is a repressor of kdgK and kdgT, two metabolic genes of the KDG-branch involved in transport and catabolism of 2-keto-3-deoxygluconate (KDG) [14]. KDG can be imported by KdgT or result from degradation of starch or glucoronate. KDG is then converted to KDG-6-phosphate (KDGP) by the KdgK kinase. At this point, the KDG-branch converges with the Entner-Doudoroff (ED) pathway catabolizing gluconate-6-phosphate to first KDGP and then to pyruvate and D-glyceraldehyde-3-phosphate by the KDGP aldolase encoded by eda. KdgR also represses expression from one of three promoters controlling the eda gene, encoding a central enzyme of the ED-pathway. However, this effect of KdgR on eda expression is negligible [20]. The ED-pathway functions as an alternative to the Embden-Meyerhof-Parnas glycolytic pathway [21], and in E. coli the ED-pathway is essential for intestinal colonization of mice [22].
In the present study, the level of genetic adaptation was investigated during a relative short S. Typhimurium infection of a mouse model of chronic infections. This revealed that even within a very short time frame (2 to 4 weeks post infection) S. Typhimurium acquires distinct patho-adaptive mutations conferring selective advantage during chronic infections. Together with the recent availability of whole genome sequencing as a common tool in molecular microbiology this study serves as a proof-of-concept for further investigations into the molecular and evolutionary mechanisms of host-adaptation. For example, data presented here point to that even one SNP in the genome of S. Typhimurium confers a clear advantage for intestinal colonization and growth with indications of increased transmission as a consequence. Further investigations of the effect of this SNP will provide insight into the metabolism of S. Typhimurium during intestinal colonization. Other mutations identified were synonymous or located at intergenic regions. Although, the effect of these mutations is more difficult to infer from the sequence alone, they may still provide selective advantage by altering the regulation and expression of neighboring genes [24]. In one mouse, distinct adapted clones were isolated from the liver and spleen, respectively, pointing to potential within-host niche specific adaptation. Overall, the results presented here provide novel insight into host-pathogen interactions and present a new approach for studying this at the molecular level. Of note, the rapid occurrence of adaptive mutations reported here should be taken into account in future studies investigating long-term bacteria-environmental interactions.
Bacterial strains and growth conditions
Strains and plasmids used in the study
Relevant genotype
S.Typhimurium strain ST4/74. Reference strain.
S.Typhimurium strain ST4/74, WITS1, kanR
This study
S.Typhimurium strain ST4/74, WITS2, kanR
This study
S.Typhimurium strain ST4/74, WITS3, kanR
This study
S.Typhimurium strain ST4/74, WITS4, kanR
This study
S.Typhimurium strain ST4/74, WITS5, kanR
This study
S.Typhimurium strain ST4/74, WITS6, kanR
This study
S.Typhimurium strain ST4/74, WITS7, kanR
This study
S.Typhimurium strain ST4/74, WITS8, kanR
This study
S.Typhimurium strain ST4/74, ΔkdgR, kanR
This study
S.Typhimurium strain ST4/74, ΔptsN, kanR
This study
E. coli cloning strain
TOPO-cloning vector, ampR, kanR
Cloning vector, ampR, kanR
Plasmid with λ-Red recombinase expressed from arabinose inducible promoter
Template plasmid for λ-Red mutagenesis, kanR
Construction of tagged strains
All primers used in the study can be found in Additional file 1: Table S1. To construct 8 individually tagged strains the kanamycin gene from pACYC177 were used as template in PCR reactions with 8 individual forward primers (Tag1kanfwd, Tag2kanfwd, Tag3kanfwd, Tag4kanfwd, Tag5kanfwd, Tag6kanfwd, Tag7kanfwd, Tag8kanfwd), each with a unique sequence tag on the linker and one common reverse primer (Tagkanrev2). The 8 PCR products were TOPO-cloned into the pCR®2.1-TOPO vector from Invitrogen according to the manufactures recommendations. A fragment containing an individual 40-bp tag and the kanamycin resistance cassette was amplified from the individually tagged TOPO-vectors, using the primers Tag1malXYfwd, Tag2malXYfwd, Tag3malXYfwd, Tag4malXYfwd, Tag5malXYfwd, Tag6malXYfwd¸ Tag7malXYfwd¸ Tag8malXYfwd, and Tagsmalxyrev2. Approximately 1 μg of each linear PCR product was used for integration into malXY pseudogene locus of the chromosome of ST4/74 S. Typhimurium using the Lambda Red method as described previously [29]. Transformants were selected by plating onto selective media. Correct integration of the tag-sequence and the kanamycin gene into the malXY locus was confirmed using a PCR strategy with the primers malXYcon and Tagkanrev2. The tags were then reintroduced into a wild-type S. Typhimurium ST4/74 background via transduction with phage P22 HT105/1 int201 using previously described protocols. Gene deletions and concomitant insertions of an antibiotic resistance cassette were constructed using Lambda Red mediated recombination as described elsewhere [29]. All constructs were verified by PCR and re-introduced into a wt ST4/74 background via P22 phage transduction as previously described [27].
Infections of mice
Seven weeks old female 129X1/SvJ mice, 16–18 g, were used to establish chronic Salmonella infections. Ten mice were purchased from Jackson Laboratories. Upon arrival, mice were randomly distributed into two groups of five mice each. Throughout mice had unlimited access to food and water, and cages were supplied with bedding for environmental enrichment. For individual assessment, all mice were earmarked. The infections were carried out by the section for experimental medicine (AEM) at Copenhagen University, where they have specialized facilities for animal housing and expertise for animal handling. To prepare the inoculums each WITS strain were grown for 16 h, 200 rpm at 37 °C in LB media. The 8 WITS strains were mixed 1:1:1:1:1:1:1:1 before the infection to give a challenge dose of 104 bacteria in total. 5 mice caged in the individually ventilated cage (IVC) were inoculated via the intraperitoneal route. A control group of 5 mice was administered physiological saline. The route of infection was chosen as the aim of the study was to establish an asymptomatic systemic infection. The exact bacterial number and ratio between individually tagged strains was determined by plating and by a PCR based sequencing approach as described below. Mice were manually inspected for symptoms of distress every 8 h during the first week post infection, and every 12 h for the remainder of the experiment. Over the course of the infection, fecal samples were collected from individual mice and S. Typhimurium bacteria were recovered and enumerated after plating a dilution series on to LB agar containing kanamycin. Mice were killed at 6 weeks post infection by cervical dislocation. In case a mouse displayed symptoms of illness, it was euthanized prior to this time point. The spleens and livers were removed aseptically and bacteria were recovered and enumerated after plating a dilution series on to LB agar containing kanamycin. Up to 96 individual S. Typhimurium clones from each sample (fecal and organ) were collected and frozen in glycerol stocks at −80 °C. Re-infections were carried out as described above in groups of five mice in one group. For re-infections isolated adapted clones were mixed 1:1 with the wild type and mice were infected IP with a challenge dose of 104 bacteria in total. Re-infections were terminated after 4 weeks of infections.
Ethics statement
All mouse experiments were reviewed and approved by the Copenhagen University animal experimentation unit and conducted with permission from the Animal Experiments Inspectorate (http://www.dyreforsoegstilsynet.dk) under license number 2013-15-2934-00761 in accordance with Danish law LBK 474 af 15/05/2014 (Animal experimentation and welfare act).
PCR and sequencing of WITS clones
Distribution of WITS in the inoculums and isolated clones recovered from fecal samples and systemic organs after infections were determined by a PCR based sequencing approach. For each sample the WITS was determined in 20 to 48 individual clones by PCR amplifying the region containing the WITS followed by Sanger sequencing of individual PCR products. Primer sequences (Tagseqfwd and Tagseqrev) used for PCR amplification of the WITS region can be found in Additional file 1: Table S1.
Extraction of chromosomal DNA and whole genome sequencing of dominant clones
Bacterial cells were grown overnight in 5 ml LB at 37 °C with 200 rpm. The next day cultures were harvested and DNA was extracted using the ChargeSwitch gDNA Bacteria Kit from Invitrogen according to the manufacturer's recommendation. Samples were stored at −20 °C. DNA was analyzed by Nanodrop 1000 from Thermo Fischer and shipped to BGI or Macrogen for Whole Genome Sequencing using the Illumina HiSeq 2000 platform with 100 bp paired end reads. Paired-end reads were mapped on the ST4/74 genome sequence (NCBI NC_016857, CP002488-CP002490) using the Geneious Alignment Tool giving a raw coverage depth of approximately 100–200 fold. Variant base calling relative to ST4/74 was performed with the Geneious work package. Sequence reads from all isolates are deposited in the Short Read Archive under accession number SRP070821 (accession numbers for individual samples are provided in Table 2). SNPs supported by 95–100 % of the reads, and all single nucleotide deletions and insertions (indels) were re-examined by Sanger capillary sequencing of PCR products amplified using specific primers (Additional file 1: Table S1) flanking the regions of interest.
Extraction of RNA and northern blotting
Cells were grown in M9 for 16 h, 200 rpm at 37 °C. The next day all cultures were diluted 100-fold into fresh M9 an incubated at 37 °C with shaking at 200 rpm. Growth was monitored by OD600 measurements every hour. At OD600 ~ 0.4 +/−0.1, 1 ml aliquots were harvested from each culture and immediately frozen and stored at −80 °C. At this point 2-keto-3-deoxygluconate (KDG) was added to all cultures at a final concentration of 5.6 mM (0.1 mg ml−1). The cultures were re-incubated for 1 h and 1 ml aliquots were again harvested from all cultures. Cells were lysed mechanically using the FastPrep system (Bio101; Q-biogene), and total RNA was isolated as described previously [30] using the RNeasy mini kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions. Total RNA was quantified by Nanodrop 1000 from Thermo Fischer and 5 μg of RNA of each preparation was loaded onto a 1 % agarose gel and separated in 10 mM sodium phosphate buffer. RNA was transferred to a positively charged nylon membrane (Boehringer Mannheim) by capillary blotting. Hybridization was performed according to [31] using a gene-specific kdgK probe that had been labeled with [32P]dCTP using the Ready-to-Go DNA-labeling beads from Amersham Biosciences. Internal fragments of the kdgK gene were used as template in the labeling reaction. All steps were repeated in three independent experiments giving similar results.
Statistical analysis
Significant differences were determined using the 2 sample t-test with unequal variances. Error bars represent standard deviations.
This work was funded by the Danish Research Council for Technology and Production through Grant No. 12–126640 to LJ. The research council was not involved in the design of the study. We thank Mette Holm for excellent technical assistance and the Department of Experimental Medicine (AEM), Copenhagen University, for assistance with animal experiments. We are grateful to Professor John E. Olsen, Copenhagen University, for valuable discussions.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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- Datsenko KA, Wanner BL. One step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000;97:6640–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Wallrodt I, Jelsbak L, Thorndahl L, Thomsen LE, Lemire S, Olsen JE. The putative thiosulfate sulfurtransferases PspE and GlpE contribute to virulence of Salmonella Typhimurium in the mouse model of systemic disease. PLoS One. 2013;8:e70829.View ArticlePubMedPubMed CentralGoogle Scholar
- Jelsbak L, Ingmer H, Valihrach L, Cohn MT, Christiansen MH, Kallipolitis BH, et al. The chaperone ClpX stimulates expression of Staphylococcus aureus protein A by Rot dependent and independent pathways. PLoS One. 2010;5:e12752.View ArticlePubMedPubMed CentralGoogle Scholar
- Chang AC, Cohen SN. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978;134:1141–56.PubMedPubMed CentralGoogle Scholar

subset: commoncrawl


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The Praxis EMR was voted the top medical records system in all categories with a survey from the AAFP. Praxis is the "no template" medical records system that "learns" how you write in a software program. Like any medical records system it has a small learning curve but once physicians have the "learning" portion of the software in place, the reduction in time and effort really begins to pay off. The association looked at 31 different software systems. Continue reading: American Academy of Family Practice Physicians Rank Praxis the #1 EMR–Survey
Featured Software Solutions
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- Electronic Health Record Growth: The growth of electronic health record adoption continues strong across the i... https://t.co/eSlk8E5zZi, Dec 29
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is pulling out all the stops on personal mobility, even going so far as to offer a 4-wheel foldable scooter that features Loop Battery technology. This allows the vehicle to be powered by a small battery pack that can be used in a variety of products—everything from lawnmowers and power products to vehicles. The Townwalker can be folded to fit in the trunk of the car, and when the steering column and seat are snapped up into place, the vehicle looks like a conventional electric scooter. The Townwalker is made of acrylic and aluminum, so it's light, which also enhances it portability when not in use.
Full Coverage: 2011 Tokyo Auto Show >>
Photos: Best of Tokyo 2011 >>
Related posts:


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Controversial Australian politician, Pauline Hanson, has weighed into the push to get the Australian Government to fund an extension of the Hawaiki Cable to Norfolk Island.
As Computerworld reported on 14 October, at a meeting in late September, the Norfolk Island Regional Council passed a motion calling on the general manager as a matter of urgency to lobby Australia's minister for regional communications, Fiona Nash in support of the project.
Also, Ben Howard, an Australian IT professional with a long family connection to Norfolk Island, wrote to the Australian prime minister, Malcolm Turnbull to lobby for an extension of the cable to the island.
Hanson, One Nation party Senator for Queensland — who has just been appointed to the Senate committee overseeing the National Broadband Network project — has written to communications minister Mitch Fifield following a visit to the Island.
She says: "Australia and New Zealand are soon to be linked through a new undersea cable installed and operated by Hawaiki Submarine Cable, this cable passes within 100 kilometres of Norfolk Island and significant enquiry and effort has been put into evaluating this fibre optic connection for the island to future-proof the island communications and technology needs.
"The undersea cable engineering and manufacture process has begun, however there still exists a very small window of opportunity for Norfolk Island to connect if the Australian Government is able to assist either financially or through an underwriting agreement, such as the precedent set by the Norfolk Island/Air New Zealand underwriting agreement, which secures air travel to and from the island."
In support of her case she includes a letter from one Ken McDondald who describes himself as " an Australian engineering consultant [who has] run an engineering systems company in Australia with a worldwide customer base … has a home and office on Norfolk Island and [has] been associated with the Island for more than 20 years."
In the letter he says: "I cannot stress how importantly I view a connection to a fibre optic cable as being essential for Norfolk Island in the medium to long term. There is an important opportunity now to connect to the new Hawaiki cable and in my view all possible efforts should be explored with a view to ensuring that this connection can be made."
Hanson is reviled by many Australians for her views on immigration, which have been condemned as racist. She once famously warned that Australia was in danger of being "swamped by Asians" and now makes the same claims about Muslims. After a brief stint in the lower house 20 years ago, she secured an upper house seat in the last election in July.


text: JP1 Remotes :: View topic - Importing Foreign IR Remotes in RemoteMaster JP1 Remotes FAQ Search 7 Days Memberlist Usergroups Register Profile Log in to check your private messages Log in File Section File Search Lookup Tool Wiki Importing Foreign IR Remotes in RemoteMaster JP1 Remotes Forum Index -> JP1 - Software View previous topic :: View next topic Author Message BarfExpertJoined: 24 Oct 2008Posts: 889 Posted: Sun Dec 18, 2016 11:48 am Post subject: Importing Foreign IR Remotes in RemoteMaster New Wiki page. Back to top mdavejExpertJoined: 08 Oct 2003Posts: 3910 Posted: Sun Dec 18, 2016 1:26 pm Post subject: Brilliant. I've been amazed by Scrutinizer's ability to import all manner of text files. This takes it to another level. Thanks! Back to top mathdonExpertJoined: 22 Jul 2008Posts: 3094Location: Cambridge, UK Posted: Sun Dec 18, 2016 6:03 pm Post subject: I see that the new Wiki page says it is necessary to add the [ImportOnly] entry to protocols.ini. However, it is already in the version of protocols.ini distributed with RMIR v2.04 build 9 and later._________________Graham Back to top BarfExpertJoined: 24 Oct 2008Posts: 889 Posted: Mon Dec 19, 2016 4:49 am Post subject: @mdavej: @mathdon: Thanx for the correction. The page has been updated. Back to top Display posts from previous: All Posts1 Day7 Days2 Weeks1 Month3 Months6 Months1 Year Oldest FirstNewest First JP1 Remotes Forum Index -> JP1 - Software All times are GMT - 5 Hours Page 1 of 1 Jump to: Select a forum General----------------Non-JP1Code SearchCode Search (Closed)Discrete CodesSlingbox JP1 Forums----------------Nevo and Xsight RemotesJP1 - BeginnersJP1 - General ForumJP1 - Protocol DecodesJP1 - ExtendersJP1 - SoftwareJP1 - KeyboardsJP1 - Hardware Miscellaneous----------------X10 and Home AutomationElectronics RepairMarket PlaceMarket Place ArchiveWeb Site IssuesReplayTVHall of Shame (and other funny stuff) You cannot post new topics in this forumYou cannot reply to topics in this forumYou cannot edit your posts in this forumYou cannot delete your posts in this forumYou cannot vote in polls in this forum Powered by phpBB © 2001, 2005 phpBB Group


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Check out this sick "mini banging" video of Hubba rider Brandon Westgate from the Berrics.
Archive for December, 2011
MINI BANGING…Thursday, December 29th, 2011
MANNY SANTIAGO PRINCE OF PUERTO RICO TOUR…Wednesday, December 28th, 2011
Another edit from the "Prince of Puerto Rico" Tour featuring Hubba Rider Manny Santiago. Edit done by Quintin Hat Co.


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Stocks Look Set to Open Higher on Renewed Confidence
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Stocks are looking to open higher this morning, as Wall Street takes a rosier view of the Federal Reserve, interest rates, and inflation.
The Dow Jones Industrial Average was pointing toward an initial gain of around 85 points, while the S&P 500, tech-heavy Nasdaq Composite, and small-cap Russell 2000 were also looking at small gains.
The big news overnight is that Treasury yields tumbled and Asian stocks jumped. The yield on the benchmark 10-year Treasury has fallen nearly 0.03 points, to 2.89%, compared to a peak of 2.95% earlier in the week. The yield on the two-year note is also off 0.01 point, to 2.24%.
Japan's Nikkei closed up 0.7%, Hong Kong's Hang Seng up 1%, Australia's ASK up 0.8%, and China's SSE 0.6%.
Among the possible causes of renewed optimism were comments from U.S. Treasury Secretary Steven Mnuchin, who argued in a Bloomberg interview that inflation worries were overdone.
Europe is mostly flat this morning. Germany GDP and European Union inflation data were in line with expectations.
It will be another day of Fed parsing. The central bank will post its monetary policy report this morning, ahead of Chairman Jerome Powell's testimony to Congress next week. Meanwhile, no fewer than five regional Fed presidents—New York, Boston, Cleveland, Kansas, and San Francisco—are due to speak in public today.
Morning Movers
HP (HP) is up 6%, to $22.68, in early-morning trading after its fiscal first-quarter results were much better than expected.
The PC giant said it earned 48 cents a share on revenue of $14.5 billion. Analysts were expecting 42 cents a share on revenue of $13.47 billion in revenue. Its guidance was also robust: For the full year, HP expects to earn between $1.90 and $2 a share, easily ahead of the $1.81 consensus estimate.
Hewlett-Packard Enterprise (HPE) is up 12.8%, to $18.51, after it also reported a strong fiscal first quarter.
For the first quarter under its new CEO, the company said it earned 34 cents a share on revenue of $7.7 billion, while analysts had modeled for earnings per share of 22 cents on revenue of $7.07 billion. It too had better-than-expected guidance, saying that it will earn between $1.35 and $1.45, above the consensus of $1.18.
Shares of Universal Display (OLED) are down 13.5%, to $134, after its disappointing forecast overshadowed fourth-quarter results.
The display-component maker said it earned 93 cents a share on revenue of $115.9 million. Analysts were expecting 85 cents a share on revenue of $100 million. For the full year, though, the company sees revenue in a range of $350 million to $380 million, below consensus for $398 million.
Blue Buffalo Pet Products (BUFF) is up 16.9%, to $39.88, on news that General Mills (GIS) will buy the company for $40 a share in cash, an $8 billion deal. Blue Buffalo has been one of the few fast-growing companies in consumer staples, and General Mills, which has struggled in the past year, said that the deal was part of a reshaping of its portfolio. Still, the market isn't convinced, sending its shares down 4%, to $52.75.
United Parcel Service (UPS) is down 0.3%, to $104.80, after Deutsche Bank downgraded the stock to Hold. Rival FedEx (FDX) is up 1.4%, to $248.87, following an upgrade to Outperform at Bernstein. —Teresa Rivas
The View From Silicon Valley
"Brotopia" is a four-letter word in Silicon Valley.
What has bedeviled Hollywood, the Beltway, Wall Street, and Main Street is put on full display, warts and all, in a new book on tech's rampant sexism.
Brotopia: Breaking Up the Boys' Club of Silicon Valley (Penguin Random House), from Bloomberg Technologyreporter Emily Chang, offers vivid proof of San Andreas Fault-sized cracks in a culture that prides itself as a modern-day utopia where anyone can change the world with a Big Idea.
That's the augmented-reality version of what the Valley wants you to believe. Beneath the veneer of equal opportunity is a system in which women are routinely excluded, marginalized, and harassed—while young white males look out for their own.
The gender gap is a very real thing in the Valley. It has been for decades. But it has coarsened of late with even more funding, record revenue, and outsize expectations on the line. From the lurid front-page scandals of harassment that ensnared Uber Technologies to behind-the-scenes stories of sex parties filled with venture capitalists and entrepreneurs, Chang's book exposes the underbelly of Silicon Valley boys behaving badly.
Along the way, she explains the culture that tolerates such behavior and offers suggestions to address the lingering problem. Among those interviewed are former Yahoo! Chief Executive Officer Marissa Mayer, Apple CEO Tim Cook, Facebook Chief Operating Officer Sheryl Sandberg, and VC Ellen Pao, who lost her lawsuit against former employer Kleiner Perkins Caufield & Byers.
There is no tidy solution to this systemic issue, but an encouraging first step is discussing the problems openly, as Chang's interesting new read does.
—Jon Swartz
Sign up to Review & Preview, a new daily email from Barron's. Every evening we'll review the news that moved markets during the day and look ahead to what it means for your portfolio in the morning.


text: I dunno if it's the darker evenings, or the change in weather, but there's a notable increase in the number of scowling yoofs in dark tracksuits loitering on my commute. Highlight today was the bunch crossing the road s-l-o-w-l-y in front of the traffic - one cut a caper in the cycle lane ahead of me. "I might be bike, but I'm still doing 15 miles an hour." I said - as one, they went "ooooo!" which made me chuckle. I do wonder how hairy things are going to get closer to bonfire night though :/


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Mix all ingredients together and store in an air-tight container.
Monkey Trail Mix,
Use almonds, peanuts, macadamia nuts, hazelnuts or other nuts instead of seeds. Replace raisins with dried cranberries or blueberries. Replace Honey Rice Puffins with Peanut Butter & Chocolate Puffins – Yum!
Head over to our Store Locator to find out where you can pick up the products you love!


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RCW 82.04.213 - 'Agricultural product,' 'farmer,' 'marijuana.'
|Cite as:
|Current through Chapter 3, 2019 Regular Session
(1) "Agricultural product" means any product of plant cultivation or animal husbandry including, but not limited to: A product of horticulture, grain cultivation, vermiculture, viticulture, or aquaculture as defined in RCW 15.85.020; plantation Christmas trees; short-rotation hardwoods as defined in RCW 84.33.035; turf; or any animal including but not limited to an animal that is a private sector cultured aquatic product as defined in RCW 15.85.020, or a bird, or insect, or the substances obtained from such an animal including honey bee products. "Agricultural product" does not include marijuana, useable marijuana, or marijuana-infused products, or animals defined as pet animals under RCW 16.70.020.
(a) "Farmer" means any person engaged in the business of growing, raising, or producing, upon the person's own lands or upon the lands in which the person has a present right of possession, any agricultural product to be sold, and the growing, raising, or producing honey bee products for sale, or providing bee pollination services, by an eligible apiarist. "Farmer" does not include a person growing, raising, or producing such products for the person's own consumption; a person selling any animal or substance obtained therefrom in connection with the person's business of operating a stockyard or a slaughter or packing house; or a person in respect to the business of taking, cultivating, or raising timber.
To continue readingFREE SIGN UP


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Mia, a young nurse, is making her round when two of her colleagues get into the room. Without a word, she offers herself for a deep double penetration. A cigarette break becomes a sodomy moment on the stairs. A dildo discovered in a room leads two nurses to make cunilingus to each other.
Two brunettes with mischievous eyes profit that a patient feels faint to take his member in their mouths. This effective treatment helps the patient to recover and to find the force to sodomise the two girls. To finish, an other young nurse gives up her work for a male nurse's enormous sex. Really, amazing things go on in the hospitals!


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What a lovely hardworking girl, LOL. Of course, you can copy the movies from your computer to iPhone 6. What you need is just iOS Transfer, an iPhone, a USB cable, a computer with iTunes installed in it.
iOS Transfer is a useful data transfer tool for iOS users no matter you use iPhone, iPad or iPod. With it, you can transfer data between iPhone/iPad/iPod, iTunes and PC/Mac with simply two or three steps. Not only videos, but also contacts, music, messages, playlists, and so on are all transferrable so long as you install iOS Transfer on your computer.
iOS Transfer – provides you simple way to transfer PC videos to iPhoneNote: Reinsure that you have iTunes on your computer first.
iOS Transfer is such an extraordinary tools for iPhone, iPad and iPod users to transfer videos between iPhone and PC. Data like videos, photos, contacts, SMS, etc. are transferrable. What you need is few simple clicks. You can also sync music to iPhone if you like.
Step 1 Connect iPhone 6 to PC
Before transferring your movies, you should install iOS Transfer first. Launch it after the installation completes. After that, use the USB cable to connect your iPhone to PC. The program will then detect your iPhone within few seconds. After your device detected, you will see the screenshot below.
Step 2Transfer videos from computer to iPhone
As you can see, click "Media" on the left column and choose "Movies". Tab "Add" and you can move the ones you want from computer to the interface.
That's it. iOS Transfer is such an well-known data transfer tool that help you transfer videos from computer to iPhone. Why not download it on your computer and have it a try. It also allows you to transfer your photos from computer to iPhone. The software satisfies all your mobile life needs. If you like it, please click the download button to have a try on how to transfer music from PC to iPhone.


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- This event has passed.
Bryan Helfrich Artist Talk
10/14/17 @ 6:00 pm - 8:00 pm
Please join us at 6pm on October 14 for an opportunity to meet Bryan Helfrich, the artist behind Angst Gallery's Questionable World leaders.
Local graphic, graffiti and fine artist Bryan Helfrich presents a series of unflattering portraits of the most divisive world leaders in recent history. On display from October 6-28, Questionable World Leaders features twelve portraits of ruling-class riff raff from around the world including Idi Amin, Fidel Castro, Margaret Thatcher, Saddam Hussein and other scoundrels ranging from years ago to the present day. These compelling acrylic portraits, displayed in carefully curated vintage frames, capture some of the most infamous, dangerous and f#$%ing ridiculous characters in modern geopolitics. #questionableworldleaders #qwl
Questionable World Leaders by Bryan Helfrich
at Angst Gallery October 6 – 28.


text: Europe as the one city. That's the idea of group of young people, who would like to organise project called Hello Europe. The main aim is to connect all Europeans. How? Easily via modern technology. In European cities will be palced big screens that will be connectet, so people standing in front of them would be able to see and hear each other.Read more


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What can I say? I love photography, and I hope you enjoy your visit to this page.
If you like photographic effects, check out my Photoshop Composites.
For other samples of my work, please check out the following links:
- "Blackout"
- Video Reels
- "Occupy Wall St. Rally" / Coachella Valley
- "Life"
- "Where Did All The People Go?"
Other good links:


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Commercial Dumpster Rentals
Weekly, Hassle-Free Commercial Garbage Pickup
You have a choice. And we can save you money with our commercial dumpster rental.
We offer a commercial garbage and commercial recycling program. Depending on your size of business we can supply you with 2-, 3-, 4- and 6-yard dumpsters. Pickup times can be as little as once a month or as often as ever day – whatever fits your needs. These commercial dumpsters are different from our roll-off contractor dumpsters – they are commercial dumpsters with lids that can be easily stored inside or outside of a commercial facility.
Our commercial dumpsters are ideal for shops like automotive repair and auto body, apartment complexes, retail and trade shops, and manufacturing facilities.
…gone above my expectations with service and dependability…
Click here to read more…
We offer our commercial dumpster rental bins in four convenient sizes:
2 Yard Dumpster
3 Yard Dumpster
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|Size: 72 in width x 34.5 in depth x 41.5 in back height
|Size: 72 in width x 41.5 in depth x 50.5 in back height
|Size: 72 in width x 50.5 in depth x 57 in back height
|Size: 72 in width x 58 in depth x 71 in back height
We offer our commercial bins in four convenient sizes:
2 Yard Dumpster
|Size: 72 in width x 34.5 in depth x 41.5 in back height
3 Yard Dumpster
|Size: 72 in width x 41.5 in depth x 50.5 in back height
4 Yard Dumpster
|Size: 72 in width x 50.5 in depth x 57 in back height
6 Yard Dumpster
|Size: 72 in width x 58 in depth x 71 in back height
Items We Do Haul
We are happy to haul building materials and other items that can be recycled.
Heavy Materials
Dirt, Rocks, Asphalt, Concrete, Stucco
Construction Debris
Drywall, Studs, Sheetrock, Plywood, Wiring, Pipes, Insulation, Cabinets, Carpet, Flooring
Roofing Materials
Roof Tiles, Shingles, Gutters
Paper, Cardboard, Wood, Fencing
Green Waste
Trees, Shrubs, Leaves, Sod, Mulch
General Clean-up
Garbage, Bikes, Toys, Lawn Mowers, Storage Sheds, Hot Tubs, Furniture
Perishable Foods
Restaurant and any other kind of Food Waste
Any kind of Appliance, large or small: Refrigerators, Freezers, TVs, Computers and Computer Monitors (additional fee required)
Tires of different makes and sizes (additional fee required)
Items We Do Not Haul
The following are items we are unable to haul for you.
Liquid Chemicals
Paint, Antifreeze, Oil or Oil Containers
Propane Tanks
Propane Bottles, Tanks, Cylinders
Mercury Thermostats, Fluorescent Lights
Chemically Treated Wood
Railroad Ties, Pressure Treated Wood
Neither Auto nor Household or any other kind
Biohazards & Medical Waste
Sharps (needles, scalpels) Prescription Drugs, etc.
Customer Testimonial
Click here to read all testimonials.
I work for a big construction company in Sacramento and have used Waste Removal for many years. I have never experienced any problems, as a matter of fact, they have always gone above my expectations with service and dependability, I would recommend their service to everyone.Darrin M.
Dumpster Gallery
Our commercial dumpsters on your job! View all.


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My mom really loves overalls on little girls. I remember having several pairs as a child and now it seems I'm bringing the 80s back with my new-found love of overalls! I told you I would be making this Whimsy Couture pattern again and here it is!
Oh my word, people, these are so cute! They are so play-friendly but still cute enough for going out places. The fabric is of course the divine Morning Glory from Amy Butler's Lotus collection. It's one of my all time favorites.
The girls seem to share this family love for overalls. They wore the corduroy ones at least weekly while it was cold and they were immediately in love with these spring/summer ones!
I searched a bit for just the right buttons and I really love these flower-impressed ones I found at JoAnn's. The buttons really needed to be red, don't you think?
There was much happy running and playing in these! Of course it was also one of the first days we finally could get outside this spring, so that might have had something to do with their energy and enthusiasm! It's totally fine because I love watching the little ruffles swing as they run!
The leaves are turning green here, the sun it out and the temps are rising! I think spring is finally really here! Yay!! As my girls would say, let's run and jump and play!


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Send funeral flowers to a funeral home in Vine Grove, Kentucky. Beautiful sympathy flowers delivered by local florists to any Vine Grove, Kentucky funeral service.
VINE GROVE, KY. – Col. (Ret.) John Plegge, 88, of Radcliff, Kentucky, died on April 19, 2013, at Hardin Memorial Hospital in nearby Elizabethtown. John was born to Joseph and Elisabeth (Santo) Plegge in 1924 in Salem, Ohio, and he graduated from Salem ... (Salem News Online)
His memberships include Lions Club, former volunteer firefighter, several bowling leagues, Kentucky Colonel and the Young at Heart ... in the chapel of Coffey & Chism Funeral Home in Vine Grove with the Rev. Ron Burgess officiating. Burial follows in ... (legacy.com)
Kentucky; brothers, Richard Dickerson of Hampton, Robert Dickerson of Vine Grove, Kentucky, and Buttler Dickerson of Moline; 17 grandchildren; 11 great-grandchildren; and many nieces and nephews. In addition to his wife and parents, he was preceded in ... (Quad-Cities Times)
Funeral services for Mr. Land were held at 1:00 PM, December 20, 2016 at the chapel of Coffey & Chism Funeral Home, Vine Grove, KY. Burial with military honors followed in the Kentucky Veterans Cemetery Central, Radcliff, KY. (The Couny Record)
Warren Gamaliel Harding Jr., of Vine Grove, doesn't get many comments about his name anymore ... is a long way from Somerset, Ky.," he said. Adams said he once had his DNA tested to see if he was related to the former president — and the president's ... (Evansville Courier and Press)


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As Chinese executives flexion their eyes to the global ~-carriage and understanding the Chinese economy becomes increasingly life-supporting for the rest of the globe, Asia's top-ranked business teach China Europe International Business School (CEIBS) recently announced it has deepened its presence in Europe.
CEIBS school leaders report that the school will use the newly-acquired Lorange Institute of Business in Zurich being of the kind which CEIBS' base in Europe. From left: Vice President & Dean Professor Ding Yuan, Chinese President Professor Li Mingjun, European President Professor Pedro Nueno and Vice President & Co-Dean Professor Zhang Weijiong
Global subsequently to inception as a joint China-EU protrude, this is another initiative being driven from CEIBS' European take ~s, and it has secured the Lorange Institute of Business Zurich's (LIBZ) taken in the character of an operating base and global center conducive to China-EU studies in Europe.
"An increasing numerate of our alumni are doing affair globally, so we have to preferable serve this group," explains CEIBS European President Professor Pedro Nueno. "At the identical time, the Chinese economy is increasing larger and more complex and its stroke on the rest of the creation is undeniable; so it's time to in a more excellent way summarize and develop business theories and models, based put ~ Chinese practices, and convey those messages worldwide."
In the beginning stages, the Lorange Institute of Business Zurich enjoin retain its name, CEIBS Vice President and Dean Professor Ding Yuan last ~ and testament serve as Chairman, CEIBS Associate Dean Katherine Xin volition be in charge of academics and teaching, while LIBZ's current Chairman Peter Lorange be inclined remain as Honorary President and the duty school's operations will be maintained and headed by its CEO & President Philipp Boksberger.
"This stir will further enhance our concept of the 'pursuit school of the future' by bracing the international network of faculty members and avocation competences with an added focus without ceasing key future markets," says Lorange.
Students and alumni from CEIBS' wide range of programs will be hosted through the LIBZ while on study tours to Europe. The Institute elect also be a source of instruction for employees of CEIBS alumni companies and/or matter units in Europe, as well because European companies doing business in China.
"We receive been very successful at bringing discernment to China, educating and developing incident managers for multinationals, SOEs and personal companies. You could say we on these terms a 'set menu'," says Yuan. "Now it demise be more 'a la carte'."
"Our students and alumni are looking to us to furnish them with the business management schooling they need to face the modern challenges of the Chinese economy."
CEIBS has campuses in China's household and political capitals – Shanghai and Beijing – because well as operations in Shenzhen to the ~erly of China and Accra in Ghana. Among the school's in greater numbers than 17,000 alumni around the earth, the majority are in leadership positions. As the of necessity of its students and alumni take changed over the years, CEIBS has adapted to in a more excellent way fulfill its goal of educating answerable business leaders.
"CEIBS students be able to learn from European cases and experts forward innovation and family business. At the corresponding; of like kind time, European students can get rudimentary-hand knowledge on cases and teaching from China while still in Europe," Yuan says.
"We poverty to educate business executives – both Chinese and those from other countries – without interrupti~ how to navigate China's increasingly webwork market, and we do all this inside of a global context. This is at present CEIBS 2.0, and it's perfectly different from our 1.0 rendition of 20 years ago."
You may also be interested in:
Advice for B-School Applicants from Asia
Top Asia B-Schools: An Ivy League of Their Own
GMAC Survey Shows Chinese Flocking to B-School
And in the end, a procedure called blood smear staining be able to be used.
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Hotel Pavone is just a few steps from Rocca di Angera castle and the lakeside promenade. You can enjoy fresh fish from the lake and local specialities at the restaurant overlooking the historic centre. Set on the shores of Lake Maggiore, the Pavone Hotel is 400 metres from the ferry departure point for trips to Arona and the Borromeo Islands. A free sandy beach is 50 metres away. Rooms offer views of the castle, promenade or the winding streets of the historic centre. Every room has carpeted floors and handmade wooden furniture, some have wood-beamed ceilings. Free Wi-Fi is provided. The sweet and savoury breakfast includes eggs, ham and cheese, plus croissants, cakes and much more. The Vecchia Angera restaurant serves Italian pizza and Lombardy dishes.
- Ascensore
- Bar pubblico
- Ristorante
- Sala TV
- Servizio shuttle (stazione ferroviaria)
- Si accettano animali di piccola taglia


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Bali Records Record Arrivals
LEGIAN ~ Despite the global economic downturn, Bali experienced record numbers of foreign tourist arrivals in January, data show, pointing to a continued upsurge in the island's tourism fortunes.
New industry figures show year-on-year foreign arrivals were up almost 18 percent for the month, to nearly 165,000 people.
The government has revised downwards its target for foreign-tourist arrivals this year, due to the prevailing economic climate, but says it is hopeful of reaching on or near last year's just-under 2 million.Filed under: Headlines


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DreamWorks Dash n Drop
he developer says:
"There's trouble at the North Pole and the presents have been scattered everywhere! Collect and deliver presents to children around the world as you dash through the evening sky in the Guardians' magical sleigh. Be sure to grab some elves and coins along the way, but watch out for evil Nightmare creatures who will try to stop you!"
Price: Free! User Rating: 4.5 Stars
Download DreamWorks Dash n Drop (iOS)
Lara Croft and the Guardian of Light HD
The developer says:
"Join Lara as she ventures deep into the jungles of central America in search of the Mirror of Smoke, a powerful artefact from a lost age. Whoever wields this item has the power to rid the world of light, and hold the future of humanity in their hands."
Price: $0.99 User Rating: 3.5 Stars
Download Lara Croft and the Guardian of Light (iPad)
Flick Tennis: College Wars
The developer says:
"Drive the story in a comic book as you play role of a Tennis Prodigy who must fight for his survival and become the best college tennis player..."


text:
Our machine engraving options for monograming include Block Monogram, Interlocking Monogram, Circle Monogram and Block Initials Script. For hand engraving, multiple lines or different font options, please call 843.723.3594 or email info@croghansjewelbox to place the order.
Left to Right: Block Monogram • Interlocking Monogram • Circle Monogram • Block Initials
Charles Garnier 'Cecilia' sterling silver wheat chain bracelet with CZ stone accents in 18K yellow gold finish or rhodium finish.
Length: 7"
By 1901 Charles Garnier had established his reputation in Paris as a masterful designer of jewelry. Working in the time of Cartier and Vuitton, his creations soon became respected and admired for their exceptional quality and elegance. More than a century later, Charles Garnier Paris continues this tradition with the same dedication to excellence and workmanship that their namesake so proudly crafts into every piece.
Delivery & Pick Up: Free standard shipping on orders $100+. Free in-store pickup for online purchases.
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Returns & Exchanges: Don't worry. Items can be returned within 7 days.
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