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Mycoplasma hyorhinis and M. hyosynoviae are agents associated with arthritis in pigs. This study investigated the tonsillar detection patterns of M. hyorhinis and M. hyosynoviae in a swine population with a history of lameness. The plausibility of dual PCR detection of these agents in dams at one and three weeks post-farrowing and their offspring at the same time was determined. The association between M. hyorhinis and M. hyosynoviae detection in piglets and potential development of lameness in wean-to-finish stages was evaluated by correlating individual piglet lameness scores and PCR detection in tonsils. Approximately 40% of dams were detected positive for M. hyorhinis and M. hyosynoviae at both one and three weeks post-farrowing. In first parity dams, M. hyorhinis was detected in higher proportions (57.1% and 73.7%) at both weeks of sampling compared to multi-parity dams. A lower proportion of first parity dams (37.5%) were detected positive at week one with M. hyosynoviae and an increase in this proportion to 50% was identified in week three. Only 8.3% of piglets were detected positive for M. hyorhinis in week one compared to week three (50%; p<0.05). The detection of M. hyosynoviae was minimal in piglets at both weeks of sampling (0% and 0.9%). Lameness was scored in pigs 5–22 weeks of age, with the highest score observed at week 5. The correlation between PCR detection and lameness scores revealed that the relative risk of developing lameness post-weaning was significantly associated with detection of M. hyorhinis in piglets at three weeks of age (r = 0.44; p<0.05).The detection pattern of M. hyorhinis and M. hyosynoviae in dams did not reflect the detection pattern in piglets. Results of this study suggest that positive detection of M. hyorhinis in piglets pre-weaning could act as a predictor for lameness development at later production stages.
Citation: Roos LR, Surendran Nair M, Rendahl AK, Pieters M (2019) Mycoplasma hyorhinis and Mycoplasma hyosynoviae dual detection patterns in dams and piglets. PLoS ONE 14(1): e0209975. https://doi.org/10.1371/journal.pone.0209975
Editor: Xiuchun Tian, University of Connecticut, UNITED STATES
Received: April 9, 2018; Accepted: December 15, 2018; Published: January 3, 2019
Copyright: © 2019 Roos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: An Excel file containing all data has been added as supplemental information.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Mycoplasma hyorhinis and M. hyosynoviae are considered commensal microorganisms of the upper respiratory tract and tonsils of pigs [1,2]. These bacterial agents are “facultative” pathogens of swine known to cause arthritis and/or polyserositis . Often, M. hyorhinis associated arthritis and polyserositis are reported in nursery pigs, especially between six and ten weeks of age, [4,5] and infection in pigs older than three months of age usually results in mild arthritis . Mycoplasma hyosynoviae is mostly identified as a causative agent of arthritis  in finishing pigs [7,8] and the lesions are known to be restricted to the joints together with the involved synovial membranes. Therefore, infectious arthritis, particularly Mycoplasma-associated clinical arthritis is a concern to swine producers, and can impact both animal well-being, and performance during all stages of swine production.
It has been suggested that M. hyorhinis can persist chronically in joints [9,10], however, bacterial isolation of the microorganism from clinically affected pigs might not be definitive for diagnosis of disease . Similarly, M. hyosynoviae might also be detected in joints with no apparent clinical presentation or macroscopic joint lesions observed [8,12]. The prevalence of M. hyorhinis and M. hyosynoviae prior to weaning has been described using several sample types and tests, such as bacterial culture from tonsil tissue and synovial fluids [2,13,14], serum tested by ELISA and complement fixation [12,13,15], as well as oral fluids and nasal swabs tested by real-time PCR [3,16]. Furthermore, M. hyorhinis has also been reported to be detected from pneumonic lungs in fattening pigs by real time PCR. Nevertheless, the mechanisms of systemic spread and disease development of these two microorganisms are still unknown. Additionally, other microorganisms including, but not limited to, Haemophilus parasuis, Erysipelothrix rhusiopathiae, and Streptococcus suis can produce a similar type of arthritis in pigs .
Mycoplasma hyorhinis is assumed to be transmitted from dams to piglets shortly after birth , with decreased transmission between piglets after 8 weeks of age . In the case of M. hyosynoviae, the likelihood of transmission is mostly limited to pigs older than 4–8 weeks of age , with various investigations showing detection of M. hyosynoviae in the tonsils of suckling pigs [11,14,21,22]. However, limited studies have investigated whether these two microorganisms are associated at any point in colonizing newborn pigs, or whether one bacterial species limits colonization with the other. Therefore, it was hypothesized that M. hyorhinis and M. hyosynoviae can be detected simultaneously in dams and their piglets using PCR from tonsillar swabs prior to weaning. Thus, the objectives of this study were to determine the dual detection patterns of M. hyorhinis and M. hyosynoviae in dams and piglets prior to weaning, and to evaluate the association between the detection of the two bacterial species and their correlation with pig lameness in later production stages. To the best of our knowledge, this is one of the first studies looking at the detection pattern of both microorganisms in tonsils of dams and piglets in the pre-weaning phase.
Materials and methods
1. Ethics statement
Conventional crossbred lines of sows and piglets were included in this study. Animals were sampled following protocols approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC approval number: 1404-31453A) and handled according to the sow farm standard procedures.
2. Animals and housing
A 2,000-sow farm located in the Midwest, USA, with unknown M. hyosynoviae prevalence at enrollment, but with a history of clinical disease (based on attending Veterinarian’s clinical evaluation and PCR positive diagnosis results) in the production system was selected for this investigation. All animals in this study were housed under commercial conditions and management practices standard in the swine industry. Gilts were purchased from an external multiplier. During the study period, the sow farm was negative for M. hyopneumoniae and endemically infected for porcine reproductive and respiratory syndrome virus.
According to sow-farm management practices, dams were vaccinated against porcine parvovirus, E. rhusiopathiae, and Leptospira spp. three weeks prior to farrowing. In general, piglets were treated with a combination of lincomycin, dexamethasone, and flunixin as a broad spectrum prophylactic measure to decrease lameness signs caused by common arthritogenic pathogens like S. suis. Nevertheless, it is important to mention that the dams and piglets enrolled in this study did not receive antimicrobial treatments, one month prior to, and throughout the course of the study. However, all piglets were administered farm immunizations. Piglets were processed (castrated and dock-tailed) within three days of age, cross-fostered if needed, and vaccinated at processing and one day prior to weaning against porcine circovirus type 2. The duration of the lactation period was between 18–23 days of age, with an average of 21 days.
For this study, one hundred- twenty piglets, individually ear-tagged, with even distribution by gender, were randomly selected at three days of age from 30 dams of various parities. Dams were randomly selected after stratification by parity. The sample size was estimated based on a 10% expected prevalence and a 95% confidence. The estimates for sensitivity and specificity of PCR in oral fluids were derived from previous studies conducted [3, 16]. However, one of the dams was culled due to agalactia, leading to a final dam sample size of 29. At the end of the study, all animals were retained in the farm and completed their production cycle. A client consent form was approved and signed by the swine producer prior to the start of the investigation.
3. Experimental design
A graphical representation of the experimental design is shown in Fig 1. Tonsillar swabs were collected in the farrowing rooms from dams and piglets at one and three weeks post-farrowing. The overall goal of this study was to detect the pathogens in dams and piglets just after farrowing and just before weaning. Therefore, sampling times were chosen in order to avoid disruption of routine farm management practices. Sampling on week one post-farrowing occurred after processing and cross-fostering of piglets, whereas sampling on week three occurred prior to weaning. Further, selected weaned piglets (n = 60) were observed throughout 22 weeks of age and the lameness scores were recorded at weeks 5, 7, 10, 13, 16, 19 and 22. Approximately two piglets per dam were followed for lameness scoring.
4. Sample collection
The tonsillar swab collection process involved the use of a mouth gag and a headlamp to aid in the location of the tonsils in the mouth cavity. Tonsillar swabs were collected by rubbing a sterile swab (BBL CultureSwab, Sparks, MD, USA) on the tonsils surface. The process was performed in a similar way regardless of the age, and pigs were manually restrained during collection.
After weaning and at the wean-to-finish barn, lameness scores were determined individually by evaluating the selected pigs. Lameness score evaluation was performed by the same investigator throughout the study, using a 0 to 4 scale as previously proposed . Briefly, a score of 0 described a scenario where the pig gets up immediately from a lying position and moves freely in the pen with balanced weight on all four limbs. For score 1, pig rises immediately but a reluctant movement is observed, with short steps and uneven distribution of body weight. For score 2, pig moves slowly in the pen with short steps and reduced weight in the sore limb, or pig rises slowly and the affected limb was not weight bearing. Score 3, the pig is reluctant to rise, with muscle shivering when standing and lifts the sore limb from the floor, or pig refuses to walk or walks on three limbs only. Finally, for score 4, pig only rises when forced and when standing has marked signs of pain (e.g. reluctance to move, limping and vocalization).
5. Sample processing and testing
Tonsillar swabs were transported to the laboratory and stored at -20°C until use. DNA was extracted using MagMAX-96 Viral RNA isolation kit and MagMAX Express-96 Magnetic Particle Processor (Life Technologies, Grand Island, NY, USA). For M. hyorhinis genetic material detection, a real-time PCR was performed with QuantiFast Probe PCR kit (Qiagen Inc., Germantown, MD, USA), according to manufacturer’s protocol, employing custom primers and probe . Similarly, a real-time PCR assay was performed to detect M. hyosynoviae with Path-ID qPCR Master Mix Kit (Life Technologies, Grand Island, NY, USA), according to manufacturer’s protocol. The primers and probes were synthetized based on the Standard Operating Procedures (SOP) routinely followed at the University of Minnesota, Veterinary Diagnostic Laboratory for M. hyosynoviae detection (UMN-VDL SOP .0078). Samples were considered positive by real-time PCR for M. hyorhinis and M. hyosynoviae when Ct ≤ 37.
6. Statistical analysis
The proportion of dams and piglets detected with M. hyorhinis and M. hyosynoviae genetic material in tonsillar swabs were compared separately within the week of sampling using a two-sample test for equality of proportions with 95% confidence interval. For the proportions with zero detection, the Clopper-Pearson method was used to determine the confidence interval. The association between M. hyorhinis and M. hyosynoviae detection was evaluated by Pearson’s Chi-squared test as a non-parametric 2-sample test for equality of proportions, with Yates continuity correction for small sample size. Throughout the study, dam and piglets were tested separately, with separate results for each population, as both the independent variables were of interest for hypothesis testing. In addition, the number of dams with dual M. hyorhinis and M. hyosynoviae detection, M. hyorhinis or M. hyosynoviae detection only, and no detection at each week of sampling were also evaluated descriptively. Proportions were calculated as the number of M. hyorhinis or M. hyosynoviae detections divided by the total number of pigs tested in the group in each week of sampling.
A comparison of two population proportions was also performed in order to evaluate dam parity association with detection of M. hyorhinis and M. hyosynoviae in tonsillar swabs at each week of sampling. Moreover, the relative risk of positive M. hyorhinis detection at week three post-farrowing was determined considering previous positive M. hyorhinis detection at week one post-farrowing as exposure. A similar analysis was also performed for M. hyosynoviae detection. Furthermore, proportions of positive detection in piglet tonsillar swabs prior to weaning were compared with positive results of individual lameness scores (score of 1 or greater) on each week of sampling after weaning by Chi-square test. Selected piglets were individually scored for lameness (0–4) and the weekly mean scores were individual based, instead of pen based. Statistical significance was considered when p-values were lower than 0.05. Statistical analysis and graphical representation were performed in R v3.2 (R Core Team) .
The detection pattern of M. hyorhinis and M. hyosynoviae in tonsillar swabs collected from dams and piglets tested by real-time PCR is shown in Fig 2. In week one post-farrowing, M. hyorhinis was detected in tonsillar swabs of 72% (95% CI: 55.7–88.3%) of dams and 8.3% (95% CI: 3.4–13.2%) of piglets. In the case of M. hyosynoviae, 55% (95% CI: 36.9–73.1%) of tonsillar swabs from dams were detected positive, one-week post-farrowing, while no piglets were detected positive the same week. At week three post-farrowing, M. hyorhinis was detected in tonsillar swabs of 65% (95% CI: 47.6–82.4%) of dams and 50% of piglets (95% CI: 41–58.9%) whereas M. hyosynoviae was detected in tonsillar swabs of 48.3% (95% CI: 30.1–66.5%) of dams and 0.9% (95% CI: 0–2.6%) of piglets.
Proportion of dams and piglets detected positive with M. hyorhinis are represented using black squares and dams and piglets positive for M. hyosynoviae are represented with grey squares. Error bars depict the 95% confidence interval. Detection of M. hyorhinis was significantly higher than M. hyosynoviae detection in tonsillar swabs collected from piglets at weeks one (M. hyorhinis: 8.3%; 95% CI: 3.4–13.2% % and M. hyosynoviae: 0%; 95% CI: 0–0.034) and three (M. hyorhinis: 50%; 95% CI: 41–58.9% and M. hyosynoviae: 0.9%; 95% CI: 0–2.6%) of sampling (p<0.05). M. hyorhinis detection in piglets was significantly higher at week 3 (50%; 95% CI: 41–58.9%) compared to week 1 (8.3%; 95% CI: 3.4–13.2%) (p<0.05). * p<0.05 between pathogens or ages at collection.
The detection of M. hyorhinis was significantly higher in tonsillar swabs of piglets at week three compared to week one (p<0.05). Detection of M. hyorhinis was significantly higher than M. hyosynoviae detection in piglet tonsillar swabs at both weeks of sampling (p<0.05). The proportion of dams detected with M. hyorhinis or M. hyosynoviae at either week of sampling was not statistically significant (p>0.05).
Individual M. hyorhinis and M. hyosynoviae detection in tonsillar swabs collected from dams at each week post-farrowing are shown in Fig 3. Among 29 dams, in both weeks of sampling, 51.7% (15/29) remained positive for M. hyorhinis in tonsillar swabs, whereas 34.5% (10/29) were detected positive for M. hyosynoviae. Twenty-one percent dams (6/29) were detected M. hyorhinis positive only at week one, 13.8% (4/29) were positive at week three alone, and 13.8% (4/29) remained negative for M. hyorhinis detection throughout the study. Similarly, in the case of M. hyosynoviae, 20.6% (6/29) of dams were positive only at week one, 13.8% (4/29) positive at week three alone, and 31% (9/29) of dams were negative at both weeks of sampling. In week one, 41.3% (12/29) of dams were detected positive for both M. hyorhinis and M. hyosynoviae, whereas in week three only 37.9% (11/29) were dual detected positive.
Each circle denotes individual dam and fill pattern indicates the detection pattern.
Considering M. hyorhinis detection results at both weeks in tonsillar swabs collected from dams, the estimated relative risk of positive detection in week three post-farrowing was 1.4286 (95% CI: 0.6789- 3.0059) if dams were positive at week one (p<0.05). In other words, dams with positive detection at week one were 1.43 times at risk of a positive detection at week three compared to dams with no detection at week one. In the case of M. hyosynoviae detection, the estimated relative risk of positive detection in week three post farrowing was 1.78 (95% CI: 0.88–3.6), if positive at week one although these differences were not statistically significant.
The detection pattern of M. hyorhinis and M. hyosynoviae in tonsillar swabs collected from dams with respect to parity is shown in Fig 4. Among the 29 dams sampled, 14 were first-parity dams (P0) and 15 were multiple parity dams (P1 or older). In week one post-farrowing, among the total M. hyorhinis positive dams (n = 21), first parity dams (P0) corresponded to 57.1% (12/21) and multiple parity dams (P1 or older) corresponded to 42.9% (9/21) of M. hyorhinis detection in tonsillar swabs. Among the total M. hyosynoviae positive dams (n = 16), 37.5% (6/16) was detected from the first parity dams whereas multiple parity dams corresponded to 62.5% (10/16) of M. hyosynoviae detection in tonsillar swabs.
Proportion of dams detected positive with M. hyorhinis are represented using black squares and dams positive for M. hyosynoviae are represented with grey squares. Error bars depict the 95% confidence interval limit. *Detection of M. hyorhinis in first parity dams was significantly higher at week three post-farrowing compared to week one (p<0.05).
In week three post-farrowing, first parity dams contributed to 73.7% (14/19) of M. hyorhinis detection in tonsillar swabs whereas multiple parity dams contributed 26.3% (5/19) of the total M. hyorhinis positive dams (n = 19). Similarly, first parity dams corresponded to 50% (7/14) of M. hyosynoviae detection in tonsillar swabs and multiple parity dams corresponded to 50% (7/14) of the total M. hyosynoviae positive dams (n = 14). M. hyorhinis detection was significantly higher in tonsillar swabs collected from first parity dams when compared to multiple parity dams (p<0.05). Nevertheless, no association was observed between M. hyorhinis and/or M. hyosynoviae detection and parity at either week of sampling.
Mean individual lameness scores measured in the wean-to-finish phase are shown in Table 1. The estimated relative risk of developing lameness at least once during post-weaning (from week 5 to week 22) was 1.862 (95% CI: 1.508–2.463) if the piglets were detected positive for M. hyorhinis at week three. There was a significant association between positive detection of M. hyorhinis by real-time PCR in tonsillar swabs at week three and a positive lameness score during its post-weaning age (r = 0.44; p<0.05). However, the association between positive detection of M. hyosynoviae and lameness score in post-weaning was not established due to fewer numbers of positive cases in week three. Therefore, from the estimated relative risk of lameness reported in piglets in the M. hyorhinis positive group, the proportion of events are statistically different after controlling for the week of detection (p<0.05) compared to that of the M. hyosynoviae positive group.
This investigation was aimed to evaluate the detection of M. hyorhinis and M. hyosynoviae in dams and piglets sampled in farrowing rooms using tonsillar swabs real-time PCR testing. Dams appeared to be consistently positive for both M. hyorhinis and M. hyosynoviae. On the other hand, M. hyorhinis and M. hyosynoviae were detected in a small proportion of piglets in week one, except for M. hyorhinis immediately prior to weaning, which was detected in half of the sampled piglet population.
M. hyorhinis was detected in a higher proportion of first parity dams than in multiple parity dams in both weeks of sampling, although this difference was only significant on week three of sampling. Detection of M. hyosynoviae, however, was higher in multiple parity dams in the first week of sampling, yet an increase in PCR detection was observed in first parity dams in week three. The pattern of increasing detection between weeks one and three post-farrowing observed for both microorganisms in first parity dams may reflect a more recent transmission event and consequent colonization. Nevertheless, the role of first parity dams in the M. hyorhinis and M. hyosynoviae detection dynamics has not been previously reported. Furthermore, dams were detected with both microorganisms simultaneously at a high proportion in weeks one (41.3%) and three (37.9%) post-farrowing. Yet the association between M. hyorhinis and M. hyosynoviae, and the possible competitive mechanisms of dual colonization has to be further elucidated. To our knowledge, this is one of the first investigations to evaluate M. hyorhinis and M. hyosynoviae dual detection in dams and their offspring.
M. hyorhinis was detected in piglets at both weeks of sampling prior to weaning, although in a significantly higher proportion at week three post-farrowing. In contrast, M. hyosynoviae was only detected in piglets at week three post-farrowing, and in a very low proportion. Hence, these results could support the hypothesis that the colonization status of piglets in lactation might influence the timing of disease development, which has been reported in the nursery stage for M. hyorhinis [9,25], and in the finishing stage for M. hyosynoviae [2,7,8]. This was in line with the observed high association of M. hyorhinis detection and lameness after weaning in the study. However, the actual cause of the lameness was not determined.
It is important to take into account that maternally derived antibodies may play a protective role against M. hyosynoviae colonization prior to weaning [13,26], delaying the transmission between piglets post-weaning [14,26], with few colonized piglets acting as carriers. It has been shown that M. hyorhinis maternally derived antibodies decrease at approximately seven weeks of age, whereas antibodies agains M. hyosynoviae seem to persist until 11 weeks of age , which may contribute to the earlier colonization by M. hyorhinis. Nonetheless, detection of M. hyorhinis and M. hyosynoviae specific and maternally-derived antibodies was not performed in this study.
In commercial settings, various sample types have been employed for detection of M. hyorhinis and M. hyosynoviae using real-time PCR. Neto et al.  suggested that pen-based oral fluids and tonsillar scrapings collected from post-weaning pigs were capable of detecting both pathogens; while, nasal swabs were identified as a sensitive sample for M. hyorhinis detection only. Colonization with M. hyorhinis in dams and piglets prior to weaning has been reported to be lower than 10% in nasal swabs tested by real-time PCR [16, 23]. However, our results revealed a higher proportion of M. hyorhinis detection using the same PCR technique, on tonsillar swabs. Interestingly, when evaluated for another swine mycoplasma (M. hyopneumoniae), nasal swabs were reported as a more sensitive sample when compared with tonsillar swabs . Nevertheless, it is important to note that M. hyopneumoniae is not considered a commensal microorganism of the nasal cavities and is mainly detected in the lower respiratory tract of pigs.
Despite the diagnostic sensitivity of tonsillar swabs for detection of M. hyorhinis and M. hyosynoviae not being yet reported, a potential hypothesis is that the sensitivity would fall between that of nasal swabs and tonsillar scrapings, with the latter likely identified as the most sensitive sample at the individual pig level . Due to the collection technique, tonsillar swabs may be considered a less invasive in vivo sample than tonsil scrapings, yet, less tonsil tissue may be harvested in the process of tonsillar swabs collection. In addition, the association between tonsillar detection and disease development needs further investigation, considering that the tonsils are a selective tissue for M. hyorhinis and M. hyosynoviae natural colonization and may not imply disease association. Variation from the observations in this study is also expected in different farms and systems, as sample collection can carry intrinsic result diversity, although detection of M. hyorhinis or M. hyosynoviae is extremely common in most swine farms. Moreover, it is important to note that this study was performed in a herd with previous history of lameness.
Control of M. hyorhinis and M. hyosynoviae in the field can be challenging due to the commensal nature of these microorganisms , and their potential multi-factorial aspect of disease presentation [2,19]. Mycoplasma hyorhinis and M. hyosynoviae transmission would likely continue among piglets in later production stages. Information on M. hyorhinis and M. hyosynoviae epidemiology are sparse and efforts must be made to improve the understanding of the colonization dynamics and disease processes of these microorganisms in order to aid decision-making by practitioners and swine producers in the application of control strategies.
This investigation aimed to assess for the first time M. hyorhinis and M. hyosynoviae dual detection in dams and their offspring prior to weaning. Under the conditions of this investigation, dams appeared to be consistently positive for both M. hyorhinis and M. hyosynoviae prior to weaning. In contrast, higher detection was observed in piglets at week three, in comparison to week one post-farrowing, with M. hyorhinis, while detection of M. hyosynoviae was remarkably minimal. The relative risk of developing lameness in postweaning piglets was highly associated with the detection of M. hyorhinis at three weeks of age. In addition, a high detection of M. hyorhinis in dams and piglets was obtained in this study with the use of tonsillar swabs as a sample type.
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- 24. R Core Team, R: A language and environment for statistical computing [Computer software]. 2015; R Foundation for Statistical Computing, Vienna, Austria. Available from http://www.r-project.org/.
- 25. Goiš M, Kuksa F, Skišák F. Experimental infection of gnotobiotic piglets with Mycoplasma hyorhinis and Bordetella bronchiseptica. Zentralblatt für Veterinärmedizin R B. Blackwell Publishing Ltd; 2010;24: 89–96.
- 26. Lauritsen KT, Hagedorn-Olsen T, Jungersen G, Riber U, Stryhn H, Friis NF, et al. Transfer of maternal immunity to piglets is involved in early protection against Mycoplasma hyosynoviae infection. Vet Immunol Immunopathol. Elsevier; 2017;183: 22–30. pmid:28063473
- 27. Sibila M, Calsamiglia M, Segales J, Rosell C. Association between Mycoplasma hyopneumoniae at different respiratory sites and presence of histopathological lung lesions. Vet Rec. British Medical Journal Publishing Group; 2004;155: 57–58. pmid:15285285

domain: biology


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Samajwadi Party chief Akhilesh Yadav on Saturday demanded that all future elections be conducted through ballot papers as ‘electronic voting... ...http://www.rediff.com/news/report/cant-rely-on-evms-faith-on-ballot-papers-akhilesh/20170415.htm
BS Reporter Hyderabad, 8 AprilThe National Company Law Tribunal (NCLT) at Hyderabad has issued an interim order to stop demolition of erstwhile... ...http://www.business-standard.com/article/current-affairs/nclt-stays-demolition-of-erstwhile-hyderabad-stock-exchange-property-117040800470_1.html
Former Commonwealth Games gold-medallist Manoj Kumar (69kg) advanced to the quarters but World Championships medallists Vikas Krishan (75kg) and... ...http://www.dnaindia.com/sport/report-manoj-advances-shiva-vikas-bow-out-of-thailand-international-2381476
The announcement of Mogul - the Gulshan Kumar story, the biopic on the emperor of music, which sees Akshay Kumar play the title role, has already... ...http://www.santabanta.com/bollywood/120702/akshay-and-bhushan-kumar-sign-mogul-in-front-of-lord-shiva/
Adarsh's all-round effort went in vain as LRDE Sports Club went down to Hutti Gold Mines Cricket Club by two wickets in the KSCA Group II, Division... ...http://www.deccanherald.com/content/603624/hutti-notch-nervy-win.html
Mukesh is an epithet for Hindu God...

domain: news


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About this Vendor
World's Most Beautiful Photo Booth Photos!
Photo Booths are fun for all occasions. We offer a beautiful custom booth great for indoors and outdoors, you will always have a attendant to man the booth, we have a variety of props and can customize them to your event, sit back and enjoy all the laughter from your guests. Setup and breakdown is on our time giving you the full "Facetime". All packages come with props and the option to purchase a scrapbook keepsake. 10% Discount on events Monday thru Thursday.
$ – Inexpensive
A big THANK YOU to Facetime Photo’s (Snow and Associates) for the photo booth services provided for Derrick and Brittany Larkins’ wedding reception on Saturday, July 9, 2016 at the University of Maryland, College Park Samuel Riggs Alumni Center. Facetime Photo’s delivered on all of their features. I so loved the props and the spacious photo booth. I also found the printouts to be the most elegant and fun to look at compared other photo booths! Every time I look at the photos I just smile. Thank you for being a part of making Derrick & Brittany’s the ‘best wedding ever’. The role you & your team played in making that happen did not go unnoticed. Mother-of-the-Bride Sherry
We were extremely happy with the photobooth! She had a huge assortment of props, and incorporated the backdrop we wanted to use in the photobooth. The pictures came out quickly and the option to have them texted/emailed as well was awesome. She was very attentive and adjusted the booth and camera to fit the number of guests in the booth. The pictures were high quality and printed fast. I also was given a pen drive at the end of the night with all of the pictures on them which was great.
You might say that Facetime Photo was the diamond in the rough at our wedding....basically they were a happy surprise. Getting a photo booth was a bit of a last minute decision on our part and we couldn't really find exactly what we were picturing in our mind. Not sure how we decided to go with them but we are SO HAPPY that we did. Honestly this was such a hit with our guests, my only regret is not having them there longer. They came and set up....must have been quick and quiet because I didn't see them until it was photo time, they had amazing and a plethora of props and they were there to help people have fun taking pics. Then somehow at the end of the night they handed us a mockup of the best photos from the night, like when did they have time to do that?! And they handed us a flash drive with all the photos, which were so fun to go through later. We HIGHLY recommend them for any wedding, party or event. You will not be disappointed!!
Meet The Events Director
We are a family business run by myself and my mom or one of my daughters. I am the character of the group I was definitely cut out to entertain. We are very people friendly and easy to talk to. We will also party with you while keeping your guest amused. We go the extra mile for our clients because we love referrals. We are very knowledgeable in arts and design, photography and computers.

domain: artistic


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“I wondered why somebody didn’t do something. Then I realized, I am somebody.”
— Author Unknown
To many people, giving time is simply volunteering. You do it because it’s the “right thing to do.” Or, you do it because you were asked.
Yet, giving time is much more fundamental to our well-being than just volunteering or doing good things. It’s a foundation on which we can build a better life for ourselves while we build better neighborhoods and communities. It’s a safe and solid way to become more integrated into our communities, to build networks and friendships, and learn to act with intention. It’s a foundation to build meaningful purpose into our lives.
Plus, it’s free.
Giving times works in these important ways:
- Perception. Giving time changes our perception of time so we become more open to new opportunities. That gives us more time. Read this study to see how some Ivy League professors put that to the test.
- Intent. The more intent you have to help someone in our community, the more giving time will come back to you. Serving ourselves is limiting. Serving others is empowering.
- Connection. Isolation is one of the under-reported scourges of humanity. When we’re connected within our communities, we’re happier and more productive. Giving time helps connect you with like-minded people.
- Immediacy. Helping others doesn’t have to wait until you can quit your job, take a life-changing trip, or wait until you’re retired. It can start now by helping someone in your family in a small way – do the dishes, pick up after them, give them a ride, be extra kind.
- Happiness. Being open to doing more, having the intent to help others, being connected, and doing it now gives more than just a sense of satisfaction. It makes a real difference in every aspect of our lives.
If you start giving your time with intention, everyday, you’ll find that you will start to make a real difference in your life. Slowly at first, but it will change. People will start to treat you differently because you treat them differently. You’ll start to ask “how” can I do this, rather than “why.” You’ll start to see opportunities rather than problems.
Eventually, you will lay your head down on your pillow some night and you’ll realize that “Yes, I made a difference.”
We *do* have time to make a difference.
Start by giving time today.
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I totally agree that giving back is so important. Giving allows us to make such a big and positive difference in the world. We’ve recently made a video on just this topic and you’re welcome to check it out 🙂
Wonderful information I like to read about tech and news I read at Techy Light. But you are awesome, Thanks.
Pingback: Gifts you Can Give that Don't Cost the Earth · Articles

domain: sociology


text: The KR-3’s wings are removable and the aircraft is trailerable. The new fuselage is 9” longer than the original, with the C of G moved forward 8” and the airfoil changed from GA(W)-1 to basically a Clark Y 4418 tapering to 4412. Following the trend-setting construction techniques of the KR-1 and KR-2, the KR-3 amphibian is quick and easy to build from wood, foam, Dynel and epoxy resin. The engine is a 2100cc Rev-master VW conversion with a three bladed Rand-Robinson propeller. The tricycle gear is hydraulically actuated. Retraction and extension are a matter of flipping a micro switch to the appropriate position and pumping a light handle located between pilot and passenger. Turbocharging permits the KR-3 to operate efficiently at altitudes up to 15,000 feet.

domain: design


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GLOUCESTERSHIRES’S police and crime commissioner is supporting a new initiative aimed at tackling rural crime.
Martin Surl is one of 18 police and crime commissioners to sign up to a new National Rural Crime Network (NRCN).
Once established the NRCN will provide an online resource for police and other community safety practitioners to work together, share information, develop training and link into other mechanisms for reporting crime and suspicious behaviour in rural communities.
“Recent events within our county have shown just how vulnerable some individuals may feel at times and I hope this will show how seriously I regard the issue of rural crime”, said Mr. Surl.
“Our rural crime prevention officer Jon Palfrey worked tirelessly to raise the profile of rural crime and, in the process, created a system of rural crime liaison officers which is the envy of many other forces.
“Establishing a National Rural Crime Network will make it easier for the police and others who protect the countryside to share best practice and enable us to challenge rural crime in Gloucestershire even more successfully”. The NRCN was the brainchild of the Rural Services Network, a ‘not for profit’ organisation which represents a diverse range of rural service providers in the public, private and voluntary sectors.
Rural Services Network spokesman Nick Payne said “The impact of rural crime is just as serious as it is elsewhere and it’s becoming an increasing problem as austerity bites and police resources are stretched thinner. “There is good collaborative work already occurring in some localities but it is widely acknowledged that sharing of best practice is patchy and urgently needs to be improved. The Network will ensure that is effectively coordinated and sustained,”
Another eight police and crime commissioners are considering joining the scheme.


Take a look at today’s photograph, you are looking at a brand-new basement floor that was poured yesterday morning, that really is new. And this was accomplished in the middle of winter.
In this basement scenario there are several things to understand and consider:
- Radiant Heat – look at the far end of the basement and if you look carefully you can see the Red Pipes going down into the cement. Those pipes will be connected to a condensing boiler for radiant heat.
- Under the floor 3” of rigid foam was put down to conserve the heat in the basement. The radiant heating tubes are laid on the top of the foam inside the concrete where the warm water’s heat is radiated out into the concrete.
- On both the right and left walls there are built in buttresses to support the stress against the 56’ of concrete wall. These buttresses were poured as one part of the wall and then incorporated within the floor. Since everything is tied together is creates a very strong structure
- Look carefully and you will see what looks like white tubes protruding up out of the floor. Those pipes are for a basement lavatory that will be installed.
- One of the white pipes protruding up through the floor is part of The Radon Gas Extraction system that has been installed.
- Great care and precautions have been taken to have the moisture from this basement floor vented to the outside.
When the family room down in the basement is finished this is going to be an exciting addition to this beautiful modular home here in Chittenden County Vermont.
We already have another home on order that will be going to Orleans County here in Vermont. That is one of our most northern counties. A portion of the county is the International Border with Canada.
Would you like a finished Family Room in your basement?


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Sagard NewGen has acquired a minority stake in Asobo Studio
Founded in 2002, Asobo Studio is a leading personal computer and console video game development studio. Internationally renowned, the company has worked together with major industry players, including Microsoft, Disney/Pixar, Ubisoft and Focus Home Interactive, and has developed or co-developed 24 games, including key entertainment franchises as well as original creations.
Released in August 2020, the latest entry in the Microsoft Flight Simulator series sets a milestone in Asobo Studio’s journey. It strengthens the established partnership with Microsoft and marks the beginning of the studio’s long-term commitment with the franchise.
François Carlot, Managing Director at Lincoln, commented, “We are proud to have advised Sagard NewGen on their investment in Asobo Studio, one of the most technologically advanced and creative studios globally.”
This marks Lincoln’s fifth transaction closed in this sector over the past 36 months.
About Sagard NewGen
Sagard NewGen fund was launched in 2020 to support leading companies in the healthcare and technology sectors. The fund was established to make majority and minority investments, financing the expansion strategy of European profitable companies (revenues of up to €150 million). Sponsored by Sagard Holdings and supported by Sienna Capital, Sagard NewGen extends the platform’s European base alongside Sagard MidCap. Sagard NewGen provides management teams with bespoke support, a high value-added ecosystem with truly international reach via its offices in Paris, France; Montréal and Toronto, Canada; and New York, U.S.
About Asobo Studio
Asobo Studio is a French video game development company based in Bordeaux, France, founded in 2002. The studio has developed 24 video games so far, which include the critically acclaimed Microsoft Flight Simulator (released in August 2020 – 91% Metacritic score, two million players) and multi-award winning A Plague Tale: Innocence (May 2019 – more than one million copies sold worldwide to date). Asobo Studio employs approximately 220 full-time developers.
Sagard NewGen is very pleased with this collaboration. The professionalism of Lincoln International's teams and their expertise in the video game industry were major assets in helping Sagard NewGen stand out in this highly competitive transaction.
Meet our Senior Team
View More Transactions
Lincoln International is pleased to announce that SiteLock, a portfolio company of Abry Partners, has been sold to Sectigo, a portfolio company of GI Partners
Lincoln International is pleased to announce that it has advised CAPZA on the financing of its acquisition of Eidosmedia
Lincoln International is pleased to announce that it has advised Chequers Capital on the sale of Wörner Medizinprodukte Holding GmbH to DCC plc
Lincoln International is pleased to announce the sale of Medix Specialty Vehicles, a portfolio company of LFM Capital, to Demers Braun Crestline, a portfolio company of Clearspring Capital Partners
Lincoln International is pleased to announce that it has advised Brembo S.p.A. on its acquisition of J. Juan Group
Lincoln International advised Alphalink founders and BNP Paribas Développement, a minority shareholder, on securing senior and mezzanine facilities
Lincoln International is pleased to announce that it has advised Stirling Square Capital Partners on the acquisition of Consensus Support Services, a leading specialist adult care provider in the UK, from Caring Homes Group
Lincoln International is pleased to announce the completion of a $112 million Series C financing by Wasabi, a leading pure-play provider of cloud object storage

domain: finance


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Satoshis per bitcoin news
Please eh your inbox for our sole email. Sign up to Day Magnates I already have an satoshis per bitcoin news. Already emerging the email we saw you and price on the top to purchase your account. Bitcoin Metre recently found in two; which excitation should be bad Bitcoin Cash..


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The Sculpture and Art Foundation CIC sees the launch of the new annual arts fayre ‘We Are Creative’ at Lichfield Cathedral. The exciting new event is open to the public with free entry on the 29th September – 10am-4pm
Organisers, The Sculpture and Art Foundation CIC are inviting creatives to take a space within the event, as a wonderful opportunity to show off their talents within the stunning interior of the Cathedral.
Open to artists and craftspeople and those with creative visual art skills We Are Creative will become an annual event in Lichfield. “We want to provide a platform for creative people to show what they can do, what they can make and to help show off the vast number of talented individuals who live, work and create in the region”, Kathryn Walker, Director of The Sculpture and Art Foundation.
The event promises to be the start of a new and inspiring annual project, providing opportunity for artists, designers and craftspeople to come together in one place to sell, discuss and share ideas with visitors and other creatives.
Visitors can expect an exciting new showcase of Artists and their artwork with artist-led workshops, talks, demonstrations and children’s activities. There will be food and drink stalls and handmade gifts for sale as well as original art works and unique crafts.
“We Are Creative has space for up to 60 stalls in the Cathedral and several spaces outside and we are keen that artists and craft-makers from all styles and creative disciplines apply for a space in the event. Visitors can take advantage of the chance to come and experience new art forms, speak with artists and get involved with our free activities.’ Natasha Hettihewa-Young, project co-manager.
In an area rich in artistic talent this is a fantastic opportunity to show off artists work to a diverse audience and be part of this festival of Artists and Creatives.
Anyone who would like to have a space in the event should contact the Sculpture and Art Foundation via K.email@example.com for booking form and further information.
For more information check out the organisers website www.sculptureandartfoundation.com, facebook : @sculptureandartfoundation.com and twitter @SculptureandArt
‘Composition’ Exhibition 30th June – 15th July 2018
Composition is the first of a new annual 2 week exhibition bringing diverse work within the fabric of the 800 year old Cathedral. The second week of the exhibition coincides with the Lichfield Festival, and the exhibition will attract an estimated 20,000 visitors.
The exhibition is open to all professional and practising artists, or recent graduates.
The exhibition is open to all fine art media – inclusive of painting, sculpture, ceramics and photography, however textiles, conceptual and installation artworks cannot be displayed.
Sculptural works must be easily transportable and movable by the installation team.
Composition is key to all art forms, and the basis of many artworks. The exhibition is looking to reflect the theme through the selected works. It is therefore open to style, media and expression. The organisers are looking for a wide selection of works from different styles and artistic perspectives.
Artists can submit up to 2 artworks for consideration.
Artist should submit digital images of work. Digital images must be clear and accurate depictions of the artwork.
Artwork should be produced within the last 2 years.
It is free to submit work and no charge applied to exhibition.
For more information and submission please email the following details to :
Submission should include:
Name and contact details
Title, media and size (+ approx weight in terms of sculpture) of submitted artwork
Cv or Link to website.
Images provided as JPEG or TIFF up to 1MB is size (for sculptural works additional images may be requested and Indication as to whether a plinth is provided as part of the work)
Cost of the artwork inclusive of 40% commission must be indicated.
Acknowledgement of insurance to cover the cost of damage or replacement of the artwork.
Artworks will be selected by
Kathryn Walker, Director the Sculpture and Art Foundation CIC
Sally Ann Newstead, Lichfield Cathedral, and formerly of Sotheby’s
Peter Walker, Lichfield Cathedral Artist in Residence and Cathedral Artistic Director.
Deadline for submission April 30th – Notification will be made before the end of May 2018
Terms and Conditions:
Artists must bring selected works to the handing in day, and collect unsold work from on specified dates (information to be provided upon selection) at no cost to the organisers. The artist is responsible for the condition of the artwork during transport, and packing and removal from site.
Any artwork deemed unfit for display on delivery will be rejected by the organisers and must be removed.
40% Commission is taken on any works sold. Any sale made after the close of the exhibition and directly as a result of direct display during the exhibition is liable for commission to the organisers.
Artist must own copyright of the work submitted and the work must be original.
Participation in the exhibition permits agreement for the artwork to be photographed and utilised for press and social media. Agreement also allows visitors to take photos for personal or non commercial.
The organisers have no liability to the artist for loss or damage, to the artwork, frame or base, or to the artists reputation during the installation. The artwork must be insurance by the artist during the exhibition.
Private opening on the evening of the 29th June
Any works not removed on the dates specified will be subject to a fixed fine.
The Sculpture and Art Foundation CIC, Lichfield Cathedral and Lichfield Festival have no liability associated with display of the artworks.
Number of works selected will be based on space and relationship to the exhibition space.
Additional terms and conditions may apply


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The purposes of this study were to 1) examine the immune and oxidative stress responses following high-intensity interval training (HIIT); 2) determine changes in antioxidant enzyme gene expression and enzyme activity in lymphocytes following HIIT; and 3) assess pre-HIIT, 3-h post-HIIT, and 24-h post-HIIT lymphocyte cell viability following hydrogen peroxide exposure in vitro. Eight recreationally active males completed three identical HIIT protocols. Blood samples were obtained at preexercise, immediately postexercise, 3 h postexercise, and 24 h postexercise. Total number of circulating leukocytes, lymphocytes, and neutrophils, as well as lymphocyte antioxidant enzyme activities, gene expression, cell viability (CV), and plasma thiobarbituric acid-reactive substance (TBARS) levels, were measured. Analytes were compared using a three (day) × four (time) ANOVA with repeated measures on both day and time. The a priori significance level for all analyses was P < 0.05. Significant increases in superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities were observed in lymphocytes following HIIT. No significant increases in lymphocyte SOD, CAT, or GPX gene expression were found. A significant increase in TBARS was found immediately post-HIIT on days 1 and 2. Lymphocyte CV in vitro significantly increased on days 2 and 3 compared with day 1. Additionally, there was a significant decrease in CV at 3 h compared with pre- and 24 h postexercise. These findings indicate lymphocytes respond to oxidative stress by increasing antioxidant enzyme activity. Additionally, HIIT causes oxidative stress but did not induce a significant postexercise lymphocytopenia. Analyses in vitro suggest that lymphocytes may become more resistant to subsequent episodes of oxidative stress. Furthermore, the analysis in vitro confirms that lymphocytes are more vulnerable to cytotoxic molecules during recovery from exercise.
- oxidative stress
- antioxidant enzymes
- high-intensity interval training
routine moderately intense exercise stimulates the immune system and enhances resistance to infectious diseases (27, 28). However, during prolonged/strenuous exercise or a heavy schedule of training and competition, there is often immunosuppression during the recovery period (23). The postexercise immune response is similar to that seen in both infection and inflammation, consisting of both neutrophilia and lymphocytopenia (35, 37). The mechanisms associated with these exercise-induced immune changes include oxidative stress, as well as neuroendocrine factors such as catecholamines, growth hormone, and cortisol (28). While there is substantial evidence linking prolonged/strenuous exercise to oxidative stress and immunosuppression, comprehensive investigation of postexercise inflammation endogenous antioxidant defense interactions is lacking.
Oxidative stress occurs when reactive oxygen species (ROS)/reactive nitrogen species (RNS) production and antioxidant defenses become imbalanced. Redox regulation via ROS/RNS and the antioxidant defenses represents a tightly controlled system that can have both deleterious and beneficial effects within the cellular environment. For instance, low concentrations of ROS upregulate the expression of superoxide dismutase (SOD) and glutathione peroxidase (GPX) in skeletal muscle (15). Catalase (CAT), SOD, and GPX present in mononuclear cells scavenge both exogenous and endogenous ROS. Additionally, lymphocyte SOD and GPX activities increase in response to acute oxidant exposure in vitro and following exhaustive endurance exercise, while neutrophils exhibit decreased antioxidant enzyme activities following intense exercise (3, 35–37). Considering that acute exercise increases ROS production (7, 18) and immune cells express antioxidants, it is important to understand the integrated dynamics between oxidative stress and immune cell function. Increases in oxidative stress during exercise and the subsequent immune response during recovery from exercise provide a unique opportunity to examine the relationship between stress and immunity. Therefore, using exercise as a model to induce stress that yields both an increase in ROS and an acute immune response may facilitate a better understanding of relationships between these two systems.
Traditional endurance training (ET) [55%-65% maximal oxygen consumption (V̇o2max), 60–120 min] induces a variety of beneficial adaptations, while large doses impose deleterious oxidative stress and immunosuppressive responses (34, 41). While ET yields long-term adaptations to improve health and longevity, it has also been shown to be a double-edged sword: when performed for a prolonged duration or at a strenuous intensity, it can cause oxidative stress and suppress the immune system (18, 23). High-intensity interval training (HIIT), by contrast, involves brief intermittent bouts of exercise (usually ≥90% of V̇o2max) separated by a few minutes of rest or low-intensity exercise during the recovery period (14). Recent investigations demonstrate that HIIT stimulates muscular and vascular adaptations previously associated with ET (4, 5, 13, 32). Specifically, HIIT has been shown to stimulate mitochondrial biogenesis and oxidative enzyme activity (13, 14, 19). HIIT also improves peripheral arterial stiffness, GLUT4 expression, and insulin sensitivity (19, 32). Perhaps the most unique aspect of these HIIT findings is the fact that they transpire in response to lower weekly training time and volume compared with ET (12). Very little information exists regarding the postexercise inflammation response to HIIT. Therefore, the purposes of this study were to investigate the acute immune and oxidative stress response during 1 wk of HIIT. A further objective was to ascertain whether changes in lymphocyte antioxidant gene expression and enzyme activity occurred following HIIT, and if these changes augment lymphocyte cell viability in vitro.
METHODS AND PROCEDURES
Male volunteers aged 19–35 yr meeting the following inclusion criteria were invited to enroll in the study: 1) peak oxygen consumption (V̇o2peak) between 35 and 55 ml·kg−1·min−1; 2) healthy, as determined by Medical Par Q Screening; 3) currently engaging in no more than 3 days/wk of moderate strength and/or endurance training (self-reported); and 4) currently not taking any antioxidant supplementation or nonsteroidal anti-inflammatory drugs (NSAIDS).
Preliminary Procedures and Assessments
Qualifying volunteers were scheduled for an initial visit to the Auburn University Thermal Lab for further screening. On arrival, volunteers were provided with and asked to sign an institutionally approved informed consent document before any screening processes. Volunteers meeting all of the inclusion criteria then continued with anthropometric measurements and preliminary physiological assessments.
Anthropometric measurements including height and weight were obtained. Height was determined to the nearest 0.25 in. with a stadiometer, and weight was measured to the nearest 0.25 kg using a calibrated scale (Michelli Scales, Harahan, LA). Three-site skinfold assessment was obtained using skinfold calipers (Lange Skinfold Caliper, Beta Technology, Cambridge, MD), and body density was calculated. Participants performed a 30-s Wingate maximal anaerobic power test on a cycle ergometer with resistance determined by body weight (0.075 kp/kg body wt) during the first visit and then performed an incremental V̇o2peak test on an electronically braked cycle ergometer (Quinton Excalibur, Quinton Instrument, Bothell, WA) during the second visit (separated by at least 48 h from the first visit). Each participant pedaled at 100 W for 5 min, 150 W for 3 min, 200 W for 3 min, and then at wattage increases of 25 W/min until volitional exhaustion. Oxygen uptake was measured with an automated metabolic testing system (True Max 2400 Metabolic Testing System, Parvo Medics, Salt Lake City, UT). The highest observed oxygen uptake averaged for 45 s was considered the V̇o2peak. V̇o2 values were plotted against work rate for the first three workloads, and a linear regression line was used to calculate %V̇o2peak at a given work rate (Table 1).
Participants performed a HIIT protocol, consisting of three exercise sessions separated by 48 h, over the course of 1 wk. Participants were asked to refrain from other physical activity during the course of the study and to continue similar dietary practices. Participants performed each exercise session at the same time of day. Participants arrived to the laboratory for exercise following an overnight fast. They refrained from food and beverage consumption (with the exception of water) until the 3-h postexercise blood sampling period.
On arrival to the lab, participants provided a urine sample for determination of hydration status. Samples were analyzed for specific gravity using a refractometer (American Optical, Keene, NH), and the participant was cleared to exercise if urine specific gravity was <1.020 g/ml. Venous blood samples were taken from the antecubital vein before exercise, immediately postexercise, 3 h postexercise, and 24 h postexercise each day.
Participants performed a HIIT protocol on an electrically braked cycle ergometer (Quinton Excalibur, Quinton Instrument). Participants performed a 20-min protocol, consisting of 4 min of cycling at 15% of maximum anaerobic power (Max-AP) followed by 30 s at 90% of Max-AP. These workloads were based on pretrial Wingate tests. This cycle was repeated four times within each protocol, ending with 2 min at 15% of Max-AP. It should be noted that the cycle ergometer did not transition wattages in a square-wave fashion. Wattage increased/decreased at each power transition at a rate of 150 W/s.
Blood Sampling Procedures
Participants reported to the lab on testing days after an overnight fast. A venous butterfly needle (23 ga × 3/4; Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ) was inserted into a superficial vein in the antecubital region of the arm and then capped with an intermittent injection port (Kawasumi Laboratories, Tampa, FL). Venous blood samples were collected with suitable vacutainers with EDTA as the anticoagulant. Immediately following blood draw, fresh whole blood was used to quantify leukocytes, lymphocytes, neutrophils, hemoglobin, and hematocrit using an automated analyzer system (Coulter LH750, Hematology Analyzer). Hemoglobin and hematocrit concentrations from whole blood samples were used to estimate plasma volume shifts resulting from the exercise sessions via the method of Dill and Costill (8).
Blood (8 ml) was collected in EDTA tubes mixed with an equal volume of phosphate-buffered saline and carefully layered onto a lymphocyte separation solution (8 ml) and centrifuged at 400 g at 22°C for 30 min. The lymphocyte layer was removed and the slurry was washed twice with phosphate-buffered saline and centrifuged for 10 min at 260 g at 22°C. It is important to note this isolation procedure yields 20–25% of monocytes and 75–80% of lymphocytes (we will use the general term lymphocytes throughout the manuscript). The isolated lymphocytes were 1) used for gene expression studies, 2) used for antioxidant enzyme activity assays, or 3) cultured in RPMI 1640 medium/10% fetal bovine serum and 2% phytohemagglutinin (PHA-GIBCO) for cell viability studies.
Gene Expression in Lymphocytes
Real-time PCR analyses of CuZn-SOD, Mn-SOD, GPX, and CAT were performed in lymphocyte RNA samples. Total lymphocyte RNA was extracted using the RNeasy Plus Kit according to the manufacturer's instructions (Qiagen, Valencia, CA). Complementary cDNA was synthesized using iScript first-stand synthesis kit (Bio-Rad) containing 200 ng total RNA. Real-time PCR was performed on a myIQ real-time PCR detection instrument using SYBR green. The gene-specific primers used for real-time PCR were as follows: superoxide dismutase 1 Cu/Zn-SOD (accession no. AY450286), forward 5′-AGGCCTCGCACTTCTCGAA, reverse 5′-CTACAGGTACTTTAAAGCAACTCT; superoxide dismutase 2 Mn-SOD (accession no. BC035422.1), forward 5′-GGCCTGATTATCTAAAAGCTATTTGG, reverse 5′-CGATCGTGGTTTACTTTTTGCA; GPX (accession no. NM_002084), forward 5′-TTCCCGTGCAACCAGTTTG, reverse 5′-TTCACCTCGCACTTCTCGAA; and catalase (accession no. NM_001752), forward 5′-TTTTCCCAGGAAGATCCTGAC, reverse 5′-ACCTTGGTGAGATCGAATGG. The thermocycler was programmed to an initial denaturation at 94°C for 10 min, followed by 35 cycles of 94°C for 15 s, 60°C for 30 s and 68°C for 1 min. Melt curve analysis showed the presence of 1 amplicon, and no primer dimerization. PCR efficiency was determined by 10-fold cDNA dilutions followed by real-time PCR. Data were normalized to two housekeeping genes: glyceraldehyde 3-phosphate dehydrogenase, forward 5′-GCAAATTCCATGGCACCGT, reverse 5′-GCCCCACTTGATTTTGGAGG; and acidic ribosomal phosphoprotein Po (Arbp) (accession no. X03205), forward 5′-CGGCTACCACATCCAAGGAA, reverse 5′-GCTGGAATTACCGCGGCTGC. Data were analyzed using a modification of the ΔΔCt method as described by Vandesompele et al. (39), and gene expression was expressed as a fraction of that occurring preexercise.
Lymphocyte Antioxidant Enzyme Activities
The activities of SOD, CAT, and GPX were measured in lymphocytes. All activities were determined with a SmartSpec plus spectrophotometer (Bio-Rad, Hercules, CA). Cell numbers were calculated for each sample. Cell samples were ruptured via the addition of 500 μl of deionized H2O per 2 × 106 cells. All assays were performed in duplicate.
Total SOD activity was measured using the method of McCord and Fridovich (21). This assay utilizes the xanthine/xanthine oxidase method to generate O2·−. Cytochrome c in the solution is reduced in the presence of O2·− and was monitored at an absorbance of 550 nm. CAT activity was measured by the spectrophotometric method of Aebie (1) based on the decomposition of hydrogen peroxide (H2O2). This reaction depends on the concentration of H+ donor and the steady-state concentration or rate of production of H2O2 in the solution. The active catalase-H2O2 complex I is formed first. The decomposition of H2O2, in which a second molecule of H2O2 serves as the H+ donor for complex I, proceeds rapidly. The decomposition of H2O2 was read at an absorbance of 240 nm. GPX activity was measured using the spectrophotometric method of Flohe and Gunzler (10). This assay utilizes a coupled reaction of GPX with glutathione reductase (GR). GPX catalyzes the oxidation of reduced glutathione (GSH) to oxidized glutathione (GSSG), while GR utilizes NADPH as the substrate to reduce GSSG back to GSH. Absorbance of NADPH per minute was read at 340 nm.
Thiobarbituric acid reactive substances (TBARS) assay was performed in plasma as a marker of plasma oxidative stress levels and determined using a TBARS Kit (Cayman Chemical, Ann Arbor, MI, catalog no. 1009055). Plasma was obtained following centrifugation of whole blood (400 g, 30 min). Equal volumes of plasma and sodium dodecyl sulfate solution were added to a 5-ml tube (100 μl of each solution). A color reagent, consisting of thiobarbituric acid (TBA), acetic acid, and sodium hydroxide, was added to the 5-ml tube (4 ml of color reagent). Samples were boiled in a water bath for 1 h and centrifuged for 10 min at 1,600 g at 4°C. Samples were loaded into 96-well plates, and absorbance was measured spectrophotometrically at 540 nm.
Lymphocyte Cell Viability Studies
Lymphocyte samples from preexercise, 3 h postexercise, and 24 h postexercise for each exercise session were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 2% phytohemagglutinin. Lymphocytes (1 × 106 cells/ml) were cultured and exposed to H2O2 (200 μM–1 mM) or its vehicle for 4 h. Cell viability was determined by the Trypan blue exclusion assay. Following the 4-h incubation period, the culture medium was removed from the culture plate and placed into 6-ml centrifuge tubes. Cells were centrifuged at 500 g for 10 min at 22°C. The supernatant was aspirated and the cell pellet was resuspended in 1 ml PBS. Ten microliters of cell suspension was mixed with 10 μl of Trypan blue dye, and the numbers of live and dead cells were assessed using a hemocytometer (on average 200 cells were counted). Cell viability was calculated using the following equation: viable cell % = (total number of viable cells per ml of aliquot)/(total number of cells per ml of aliquot) × 100.
A three (day) × four (time) ANOVA with repeated measures on both day and time was used to compare superoxide dismutase, catalase, glutathione peroxidase (enzyme activity), TBARS, total leukocyte number, neutrophil number, lymphocyte number, and cell viability for each day. Gene expression of antioxidant enzymes was compared with day 1 preexercise expression. Gene expression was also assessed within days using preexercise expression as control. Significant differences were further explored utilizing a Tukey post hoc analysis. The significance level for this study was set at P < 0.05. Data were analyzed with the Statistical Package for the Social Sciences (SPSS, version 16.0, SPSS, Chicago, IL).
In total, nine male volunteers met the study inclusion criteria and agreed to participate in the study. One participant withdrew from the study because of time commitment; therefore eight participants completed the entire study protocol. Participant baseline physiological characteristics are presented in Table 1.
All participants were able to complete the three exercise trials. Each exercise session lasted ∼20 min and was identical in power settings and cycle ergometer settings for each participant. Participants completed the exercise protocol following an overnight fast. Exercise was performed during the same time of day and was separated by 48 h. Percentage of V̇o2peak was based on each participant's power output at V̇o2peak.
Effect of Exercise on Leukocyte, Neutrophil, and Lymphocyte Counts
Leukocyte, neutrophil, and lymphocyte cell numbers for pre-, post-, 3 h post-, and 24 h postexercise for each of the three exercise days are depicted in Fig. 1. There was no significant difference in cell number between exercise days for any of the cell types. Circulating leukocytes increased significantly immediately post- and 3 h postexercise compared with preexercise and returned to baseline by 24 h postexercise on days 1 and 2, while day 3 only showed a significant increase immediately postexercise. Neutrophil number increased significantly immediately post- and 3 h postexercise compared with preexercise on days 1 and 2, while day 3 was only significantly elevated 3 h postexercise (P < 0.05). Lymphocyte number increased significantly immediately postexercise compared with preexercise and returned to preexercise values by 3 h postexercise on days 1, 2, and 3. (P < 0.05). A significant decrease in lymphocytes from preexercise values was observed 24 h postexercise on day 2 (P < 0.05).
Plasma Oxidative Stress Determinant Following Exercise
Oxidative stress was measured indirectly using a TBARS assay kit. Plasma TBARS levels were determined preexercise, postexercise, and 3 h postexercise for each day of exercise (Fig. 2). TBARS levels were significantly elevated immediately postexercise compared with preexercise on days 1 and 2, and 3 h postexercise on day 2 (P < 0.05). There were no significant differences in TBARS levels on day 3 of exercise. These data show that oxidative stress occurred following HIIT on both day 1 and day 2 but did not significantly increase on day 3 of HIIT.
Lymphocyte Antioxidant Gene Expression Following Exercise
Antioxidant gene expression for CuZn-SOD, Mn-SOD, CAT, and GPX were assessed in lymphocytes following HIIT. Gene expression was assessed between days and within days. All changes in gene expression between days were compared with preexercise on day 1 (control value). To determine gene expression changes within days, all 3-h postexercise values were compared with preexercise expression. No significant differences in antioxidant gene expression were observed at any of the sampling times. Antioxidant gene expression comparing each day and time point with preexercise day 1 is shown in Fig. 3.
Changes in Lymphocyte Antioxidant Enzyme Activities Following Exercise
SOD, GPX, and CAT enzyme activities were determined in lymphocytes following three HIIT sessions (Table 2). Enzyme activities were determined preexercise, postexercise, 3 h postexercise, and 24 h postexercise.
CAT activity was significantly elevated from pre- to postexercise on day 1 (95.4 ± 89%) and day 2 (66 ± 76%) (P < 0.05). No significant differences in CAT activity were found at any other sampling times.
There were no significant differences in activity between days 1, 2, or 3 at any sample time for SOD. SOD activity was significantly elevated postexercise on day 1 (45.6 ± 23%), day 2 (41 ± 83%), and day 3 (50 ± 34%), and 3 h postexercise day 1 (44 ± 11%), day 2 (33 ± 88%), and day 3 (36 ± 22%) compared with preexercise levels (P < 0.05). SOD activity returned to preexercise levels by 24 h.
GPX activity was significantly elevated from pre- to postexercise on day 1 (67 ± 18%) and day 3 (36 ± 24%) (P < 0.05). No significant differences were found pre- to postexercise on day 2. Additionally, postexercise GPX activity was significantly elevated from 3 h and 24 h postexercise on day 1, day 2, and day 3 (P < 0.05). GPX activity returned to preexercise levels by 3 h postexercise.
Effect of Exercise on Lymphocyte Viability Following H2O2 Treatment In Vitro
Each concentration of H2O2 treatment was analyzed independently to determine if cell viability improved between days 1, 2, and 3 following treatment. A significant increase in cell viability was observed at day 2 and day 3 compared with day 1 during the 3-h postexercise period for the 1 mM H2O2 challenge (P < 0.05). Cell viability data 3 h postexercise are presented in Fig. 4. There were no significant differences observed between the other three concentrations.
To determine if cells were less resistant to an oxidative insult at a particular time period pre- and postexercise, we used the mean cell viability following the 1 mM H2O2 challenge for all 3 days at preexercise, 3 h postexercise, and 24 h postexercise. Cell viability was significantly reduced at 3 h postexercise compared with preexercise and 24 h postexercise (P < 0.05). Cell viability at each time period is presented in Fig. 5.
This is the first study to examine postexercise oxidative stress to high-intensity cycle exercise and subsequent antioxidant fortifications within the immune system. Specifically, the purposes of this study were to examine the acute immune and oxidative stress responses following 1 wk of HIIT. The key finding of this study is that within the context of acute post-HITT exercise inflammation and oxidative stress, changes in antioxidant enzyme activity and cell viability were observed in lymphocytes. Specific study findings indicate 1) HIIT did not induce a postexercise lymphocytopenia as seen following traditional ET; 2) HIIT did elicit oxidative stress; 3) lymphocyte adaptations to HIIT include increases in SOD, GPX, and CAT activity, but not increased gene expression; and 4) analysis of cell viability in vitro revealed lymphocytes were more resistant to H2O2 on days 2 and 3 of exercise compared with day 1, and lymphocytes were least resistant to the H2O2 challenge during the postexercise recovery period. A cornerstone of the current experimental design was to examine the oxidative stress and immune system responses to a serial regimen of HITT exercise trials. Given the many beneficial adaptations observed with HITT exercise, we sought to understand whether this approach would also avoid the well-established immunosuppressive effects of ET (23, 24, 27, 36, 37). By extension, the current post-HIIT findings are consistent with previous investigations and demonstrated a 44% increase in circulating leukocytes immediately postexercise, a 79% increase in circulating neutrophils 3 h postexercise, and a 72% increase in circulating lymphocytes immediately postexercise. However, the lymphocytopenia was not observed during the recovery period, as lymphocytes returned to preexercise values at 3 h post-HIIT. This significant finding, as recently shown, demonstrates that HIIT induces short-term increases in both aerobic and anaerobic conditioning (4, 5, 13, 14). Thus HIIT may be an ideal mode of training for improving health, with less immunosuppression compared with ET. It is also worth noting in the current exercise protocol that the 48-h recovery between HIIT trials provided an avenue to achieve a HIIT stimulus without overtraining effects. However, the authors acknowledge that the acute immune responses following 1 wk of HIIT may not represent the immune response following chronic HIIT. Therefore, further work is needed to determine the long-term effects of HIIT on immunity.
The postexercise oxidative stress response is less well defined compared with postexercise immune function. However, recent well-controlled studies have demonstrated that high-intensity exercise is associated with a marked rise in oxidative stress biomarkers (2, 31). While we did not measure rate of perceived exertion (RPE) directly in the current study, our protocol elicited maximal efforts during all three HITT trials. In response significant increases in plasma TBARS levels were observed immediately postexercise on days 1 and 2 and at the 3-h time point postexercise on day 2 (Fig. 2). TBARS were not elevated in response to day 3 HITT exercise. This is among the first investigations to examine the acute blood oxidative stress response to serial exercise sessions. Current findings indicate a transient oxidative stress response to HIIT; thus future oxidative stress research should incorporate protocols that use multiple exercise sessions and additional sampling periods to confirm findings observed following a single session.
The fundamental link between the immune and oxidative stress responses to prolonged endurance or high-intensity exercise are undeniable (31, 35). Elevated ROS production following exercise permeates the nucleus and induces DNA cell damage in lymphocytes, implicating exercise-induced oxidative stress may contribute to previous observations of postexercise lymphocyte apoptosis (20, 40). We sought to determine if the exercise-associated oxidative stress altered lymphocyte antioxidant enzyme gene expression and/or activity. No changes in CuZn-SOD, Mn-SOD, CAT, or GPX mRNA expression occurred following HIIT (Fig. 3). However, even within a relatively small sample size, CAT, GPX, and most consistently, SOD enzyme activity did increase following 3 days of HITT exercise (Table 2). Lymphocyte enzymatic antioxidant defenses have shown the ability to adapt to exercise-induced oxidative stress. Previous investigations have reported increases in CuZn-SOD, Mn-SOD, and heme oxygenase-1 (HO-1) mRNA expression in lymphocytes following various forms of exercise (6, 38). A significant downregulation in CuZn-SOD, Mn-SOD, and GPX mRNA levels have previously been reported in skeletal muscle of untrained rats, yet to our knowledge, this has not been observed in lymphocytes (17, 26). These data suggest that changes in antioxidant enzyme activity following HIIT are products of posttranscriptional modifications, increased mRNA stability, or decreased activity of regulatory modulators, opposed to pretranscriptional regulation.
Prior investigations (6, 37) of lymphocyte antioxidant enzyme activities have yielded equivocal results. Tauler et al. (35) examined antioxidant enzyme activities following both maximal and submaximal cycle ergometer tests. They found no significant changes in antioxidant enzyme activities following the maximal test, while GPX activity increased following submaximal exercise (35, 36). A later study by the same group examined the immune and oxidative stress responses following a mountain cycling stage and reported both an increase in oxidative stress and lymphocytopenia (37). They observed significant increases in lymphocyte GPX and SOD activities, yet this increased antioxidant activity was unable to prevent lymphocyte oxidative damage. Our data showed significant increase in each antioxidant enzyme measured; differences in CAT and GPX activity were observed between HIIT sessions, whereas SOD activity responded similarly to each HIIT session (Table 2). It is difficult to compare results between studies, as different modes of exercise were used and the aforementioned studies mentioned used only a single sampling period. Lymphocytes appear to respond to oxidative stress challenges by increasing antioxidant defenses, although the activation of these antioxidant enzymes may be unique to exercise mode and duration.
The immunosuppression observed following exercise of a strenuous and prolonged nature has been linked to a decrease in circulating lymphocytes and a blunted natural killer cell activity (29). Oxidative stress, catecholamines, growth hormone, and cortisol have all been coupled to lymphocyte function. While cortisol has often been linked to postexercise immunosuppression (23, 25, 27), Peters et al. (30) assessed lymphocyte DNA damage and apoptosis in well-trained athletes and found that concentrations of apoptotic lymphocytes were not related to changes in serum cortisol concentrations (30). We did not measure hormonal markers in this study since our emphasis was the link between oxidative stress and immune function. Prior work has shown increases in lymphocyte antioxidant enzyme activities, yet this increase was not sufficient to prevent postexercise lymphocytopenia (37). The HIIT protocol used in the current study is quite different from traditional prolonged ET utilized in previous investigations. Although we did not measure growth hormone in this study, prior work in our lab (consisting of similar participants and the same HIIT protocol) revealed 60-fold increases in serum growth hormone concentrations from pre- to postexercise (Foster EB, Fisher G, Sartin JL, Elsasser TD, Wu. G, Pascoe DD, unpublished observations). We find this particularly interesting as human growth hormone has been shown to diminish various forms of lymphocyte cell death in vitro by upregulating an essential antiapoptotic protein, Bcl-2 (16, 22). Therefore, it is possible that the increased growth hormone in conjunction with the increase in antioxidant enzyme activity following HIIT may be responsible for maintaining lymphocyte function. Future studies should be performed to determine if the increased growth hormone following HIIT is responsible for the preservation of circulating lymphocytes.
The lymphocyte concentration increase seen during exercise is thought to be due to recruitment of all lymphocyte subpopulations into circulation, specifically from tissue pools such as the spleen, lymph nodes, and the gastrointestinal tract (28). Additionally, animal models have shown a redistribution of lymphocytes from the circulation back into the tissue pools following exercise (33). Thus, examining changes in lymphocyte cell viability following an oxidative stress challenge in vitro may better illustrate lymphocyte ability to resist exercise-induced oxidative stress. In this study, lymphocytes were cultured from preexercise, 3 h postexercise, and 24 h post-HIIT blood samples and exposed to H2O2 for 4 h. Prior investigations found that H2O2 concentrations up to 200 μM induced changes in cytokines without affecting cell viability (9). Therefore, 200 μM, 500 μM, and 1 mM concentrations of H2O2 were used for the oxidative stress challenge. No significant changes in lymphocyte cell viability for control, 200 μM, and 500 μM H2O2 exposure were observed; thus cell viability does not appear to be affected at these concentrations. A unique finding from this study was the observation of a significant increase in lymphocyte cell viability at 3 h postexercise on day 2 (12%) and day 3 (13%) compared with day 1 of HIIT (Fig. 4). These data suggest that lymphocytes may adjust following exercise and become more resistant to subsequent episodes of oxidative stress. Additionally, a significant decrease in lymphocyte cell viability was observed at 3 h postexercise compared with both pre- and 24 h postexercise (Fig. 5). These data support the postulate that lymphocytes may be more vulnerable to cytotoxic agents during recovery from exercise.
Strengths of this study included obtaining multiple samples over several exercise sessions, which enabled us to assess the transient immune, antioxidant, and oxidative stress responses to HIIT. Additional strengths include closely matching the age, body fat%, and fitness levels of the participants, and incorporating an in vitro experimental design with the in vivo experiments. A key limitation in this study is the measurement of only a single oxidative stress biomarker. Additionally, measuring oxidative stress within the lymphocytes would have strengthened our conclusions.
In conclusion, this is the first study to examine the acute oxidative stress and immune cell viability to high-intensity serial exercise sessions. Postexercise oxidative stress was associated with elevations in lymphocyte SOD, GPX, and CAT activity in a manner appearing consistent with posttranscriptional regulation. These findings were further associated with improved lymphocyte viability to an H2O2 challenge in vitro. In light of the fact that HIIT did not impair the immune system in vivo, this HIIT model may be an ideal form of exercise to improve health and performance without overstressing the immune system. Future research assessing these same variables, in addition to hormonal regulation, over longer training cycles should be conducted. Carefully designed experiments measuring both the immune and oxidative stress response over extended periods of time are needed. These types of studies would provide data necessary to better understand the link between oxidative stress and the immune system.
No conflicts of interest, financial or otherwise, are declared by the author(s).
- Copyright © 2011 the American Physiological Society


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Callum McManaman, star of Wigan Athletic's stunning FA Cup triumph over Manchester City, is likely to be out for three months with an ankle injury, manager Roberto Martinez revealed on Friday.
The 22-year-old damaged ankle ligaments in Tuesday's 4-1 loss at Arsenal, which condemned Wigan to relegation from the Premier League, and will be unable to represent England at the Under-21 European Championship in Israel in June.
McManaman now faces a challenge to return to fitness in time for the start of next season, but Martinez took solace from the fact he will not require an operation.
"It's good news, because we expected the worst, but good news means he's going to be out for the best part of three months, but he doesn't need surgery and that's really positive," Martinez said.
"But probably that puts him out of the under-21s, which is a real shame because he really deserved to finish the season with that experience."
Martinez's future at Wigan is in doubt after he was installed as one of the favourites to succeed David Moyes at Everton, but the Spaniard urged McManaman to stay put at the DW Stadium despite the club's relegation to the Championship.
"If one player needs to stay at the club and become an important part of this football club, it's Callum," he said.
"He's been a phenomenal player for us, but he's someone who, at 22, needs to work on certain aspects to become the player he can be. He will be a top, top player, and probably the next 10 months are vital for him to be perfect in the programme that he has to fulfil.
"The Championship, for him to play week in, week out and have an important role, it will be the perfect challenge for him."


Design of the Ufag C.i was conventional for the time. The biplane wings were of equal span and staggered with the upper assembly ahead of the lower. The wings featured single bays and parallel struts. The engine was fitted to the forward compartment of the slab-sided fuselage, powering a two-blade wooden propeller. The powerplant was a single Hiero 6-cylinder liquid-cooled in-line engine developing 230 horsepower. The pilot sat immediately behind the engine compartment and made use of one or two forward-facing 8mm synchronized machine guns. His observer was seated directly aft and manned a trainable 8mm machine gun of his own. The fuselage tapered into a conventional empennage with large-area horizontal planes and a small-area vertical tail. The undercarriage was fixed and made up of two main landing gear wheels and a tail skid. The landing wheels were situated under the engine compartment, just ahead of the lower wings, and braced with struts.
Performance specifications indicated a top speed of 118 miles per hour as well as a ceiling of 16,075 feet. Engine endurance was limited to 3 hours.
Phonix lent its production talents to the C.I, constructing at least 40 examples of the type.
Text ©2003-2016 www.MilitaryFactory.com. All Rights Reserved. No Reproduction Permitted. Email corrections/comments to MilitaryFactory at Gmail dot com. Material presented throughout this website is for historical and entertainment value and should not to be construed as usable for hardware restoration, maintenance or general operation. Please consult original manufacturers for such information.


text:
Kevins Longleaf Pine & Quail Placemats
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Kevin's exclusive Longleaf Pine and Bobwhite Quail Fabric was designed from original paintings of a local Georgia artist. The beautiful and rare Longleaf Pine forests found in the Red Hills and the Bobwhite quail who live in this habitat are the inspiration. We manufactured this fabric in a wonderfully soft, brushed-finished cotton canvas for durability.


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text: Welcome to Audiko, where it’s possible to download ringtones for free for your cell. Today we have a ringtone of “Pas le temps” by Faf Larage as well as 45 other ringtones by Faf Larage. “Pas le temps” is a top-rated song from the “Rap Stories” album that was composed by Faf Larage at their label together with other 5 tracks. You can listen to all 4 Faf Larage – “Pas le temps” ringtone variations available here and download it for free directly to your mobile. You can also take a glance at other 1 albums produced by Faf Larage for all time. “Pas le temps” is referred to french, and you can download a huge amount of french artists absolutely for free. And finally, we have collected lyrics for this track and you can view it here as well. So feel free to browse, listen, download ringtones at Audiko.net. Make your phone sound cool!


IN BRIEF: Tourist shot in face by youth
A DUBLIN youth has been sentenced to 12 months' supervised probation after he admitted firing a pellet gun at a tourist.
The 17-year-old shot a Brazilian man in the face causing a superficial cut, Dublin Children's Court heard.
The youth, now aged 18, pleaded guilty to assault causing harm and the attempted robbery of the tourist, at Railway Street, Dublin, last June. The teen, who is in a FAS training course, also admitted driving without insurance receiving a six-year road ban.
Last month, the youth, who has 14 prior convictions, spent a week in custody on remand.
McCourt sells LA Dodgers
Los Angeles Dodgers owner, Frank McCourt, has agreed to sell the bankrupt baseball team for $2bn (€1.5 bn) to a group that includes former LA Lakers basketball star Magic Johnson.
The agreement will lead to a transfer of the team by the end of April. It is subject to approval in the federal bankruptcy court.
As part of the agreement McCourt and partners would acquire the land sur-rounding Dodger Stadium for $150m dollars (€112m).
Romney iPads stolen from car
Laptops and iPads containing sensitive information about Republican presidential front-runner Mitt Romney were stolen from one of his campaign cars in San Diego.
The items belonging to two Romney campaign staffers were taken as well as two radios.
The thefts last Friday were first reported by KGTV, a local San Diego TV station.
A Romney spokeswoman said the campaign didn't suspect the items were stolen for political reasons.
Actress faces fraud charge
Actress and model Donna Air is to stand trial accused of fraud.
The 32-year-old former Byker Grove star is charged with fraudulently obtaining a parking permit by false representation from London's Royal Borough of Kensington and Chelsea.
In December, Air appeared at Isleworth Crown Court and denied the charge.
She will return to the court for trial today.


by JL Yastine
The Sovereign Investor
I hear it all the time from Sovereign Society members — in person, in phone calls and in emails: “The financial world doesn’t make sense to me.”
How can the stock market gain so much ground (notwithstanding the weakness of the past few days), or the Federal Reserve continue to talk about restraining the U.S. economy by raising interest rates … despite headlines that say:
- The United States’ economy is barely growing at all.
- U.S. corporate profits are on the decline.
- U.S. exports of goods and services have fallen for 19 consecutive months.
No wonder one of the big brokerages recently said: “We have never had so many clients start meetings with statements like ‘We are totally lost.’”


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Hey, Vietnamese foodies, did you know there is a good place in Rockwall? We tried the steak pho and the iced coffee this week, and both were very good with good prices. It's Pho Garden, located in the strip mall in the SW quadrant of the I30-Horizon Road intersection. It's near the old Kroger's location, if you're old enough to remember that.
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domain: geography


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April 8, 2013
7 years old
He tends to be a little clingy to me in public (but not so much at home).
Bernese Mountain Dog
Hi. My name is Aileen and I have a Bernese Mountain Dog named Django. We usually go to the dog beach but I would also like him to meet other dogs in different settings.

domain: pet


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Pretty Little Liars Season 3 Episode 7 Crazy : HANNA IS IN THE HOT SPOT,IN AN ALL NEW EPISODE OF “PRETTY LITTLE LIARS,”TUESDAY, JULY 24TH AT 8:00 PM ET/PT ON ABC FAMILY
Burbank, CA (July 6, 2012) – When things look dire for Hanna, the Liars must turn to Mona to help their friend out, in “Crazy,” an all new episode of ABC Family’s hit original series, “Pretty Little Liars,” airing Tuesday, July 24th (8:00 – 9:00 PM ET/PT).
With new key evidence in Alison’s murder case, Hanna fears that “A” may be framing her after a visit from Det. Wilden. With paranoia setting in, Aria decides that she has to turn to Mona for answers if she is going to help Hanna. But is Aria ready to face her former tormentor, and more to the point, is Mona ready to give Aria the answers the PLLs are desperately looking for? Meanwhile, someone from Ali and Jason’s past arrives in Rosewood and piques the girls’ interest.
“Pretty Little Liars” is produced by Alloy Entertainment in association with Warner Horizon Television and is executive produced by I. Marlene King, Oliver Goldstick and Leslie Morgenstein. The series stars Troian Bellisario, Ashley Benson, Tyler Blackburn, Holly Marie Combs, Lucy Hale, Ian Harding, Laura Leighton, Chad Lowe, Janel Parrish, Shay Mitchell and Sasha Pieterse.

domain: entertainment