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History of Busting Out and dragon boating for breast cancer survivorship
In January 1998 the concept of a dragon boat team for breast cancer survivors in Ottawa began. By June 1998 22 paddlers entered the Ottawa Dragon Boat Festival for the first time as team Busting Out. Since then, the program has grown immensely. A program of Breast Cancer Action Ottawa, Busting Out dragon boat team is comprised of only breast cancer survivors.
In 1996, Dr Don MacKenzie conducted a study to determine whether women who were treated for the disease should avoid rigorous upper body exercise. At the time, it was thought that exercise could cause lymphedema, a debilitating and chronic side effect of treatment and surgery. The sports medicine professor trained 24 survivors to paddle in a dragon boat, and at the end of the three month season none of the volunteers had developed lymphedema. After the study was over, the women discovered additional benefits of dragon boating, including improved fitness and health, and the camaraderie and support of the other women. They decided to continue training together, and the next year the first dragon boat races were organized. What started as a study is now an international success. Women and men who have survived breast cancer all over the world paddle in dragon boats. International festivals are held every four years at various locations in the world.
Busting Out is always looking to share the journey to better health and ongoing support for breast cancer survivors. If you are a survivor, join our team. If you know a survivor, share our story.


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Caribou Herds of Northwest Alaska, 1850-2000 by Ernest S. Burch Jr.
By Ernest S. Burch Jr.
In his ultimate, significant ebook Ernest S. "Tiger" Burch Jr. reconstructs the distribution of caribou herds in northwest Alaska utilizing facts and data from examine performed over the last a number of a long time in addition to resources that predate western technology by means of multiple hundred years. also, he explores human and common components that contributed to the dying and restoration of caribou and reindeer populations in this time. Burch presents an exhaustive checklist of released and unpublished literature and interviews that might intrigue laymen and specialists alike. The unflinching review of the jobs that people and wolves performed within the dynamics of caribou and reindeer herds will unquestionably strike a nerve. Supplemental essays ahead of and after the incomplete paintings upload context in regards to the writer, the undertaking of the ebook, and the significance of either.
Read or Download Caribou Herds of Northwest Alaska, 1850-2000 PDF
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Additional resources for Caribou Herds of Northwest Alaska, 1850-2000
Example text
And, like their wild cousins, double their weight by the time they are two weeks old. They are just as precocious as caribou calves but, at least in a close herding regime, do not travel nearly as far or as fast. Reindeer calves also usually have less to fear from non-human predators, being saved by their owners for their own predatory purposes. The more open the herding regime, the more closely the first few weeks of a reindeer's life resemble that of a caribou calf. Adult Alaska reindeer cows weigh approximately 165–230 lbs.
The observations of trained biologists are considered "scientific," especially if they are expressed statistically. "Anecdotal data," on the other hand, consist of the observations or recollections of Natives, explorers, travelers, anthropologists, or others who were not trained as biologists and were not involved in formally organized research projects focused on R. tarandus. Most biologists seem to consider anecdotal data to be worthless. My own attitude toward the use of anecdotal data was well expressed by Henry Dobyns: "one either uses such data as may be available and learns something, however inadequate, or abjures such data and learns nothing" (Dobyns 1976:7; see also Krech 1978b:89).
Documenting any but the most extreme changes in the caribou population more precisely at this distance in time is impossible, and there is no sense in deluding ourselves on the point. 14 The narrower time range here is possible because the amount of information available on it is greater by several orders of magnitude than that existing for the initial period. The reason for a five-year instead of a oneyear "window" is to provide information on annual variation in herd sizes and movements. Changes in the Landscape Over the 150-year period covered by this study15 there have been many changes in the landscape of Northwest Alaska, but most of them had no known major impact on its R.


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A new app, Farlight, brings a captivating drawing experience to your Windows Phone
by Nisha
6 years
The makers of Intellicam bring you Farlight. Farlight is the app if you are looking for a mesmerizing and relaxing drawing experience. The app lets users apply touch to paint and with just a few strokes create stunning animated ab...


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Slavic Bible Church
Of Colorado Springs, CO
|Main | Pictures | Address | Bible for One Year
|Reading in September
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Ecclesiastes 4; 5; 6:1-12; 2 Corinthians 6:14-18; 7:1-7; Psalm 47:1-9; Proverbs 22:16;
|September 4
Ecclesiastes 4; 5;
Oppression, Toil, Friendlessness
1 Again I looked and saw all the oppression that was taking place under the sun: I saw the tears of the oppressed— and they have no comforter; power was on the side of their oppressors— and they have no comforter. 2 And I declared that the dead, who had already died, are happier than the living, who are still alive. 3 But better than both is he who has not yet been, who has not seen the evil that is done under the sun. 4 And I saw that all labor and all achievement spring from man's envy of his neighbor. This too is meaningless, a chasing after the wind. 5 The fool folds his hands and ruins himself. 6 Better one handful with tranquillity than two handfuls with toil and chasing after the wind. 7 Again I saw something meaningless under the sun: 8 There was a man all alone; he had neither son nor brother. There was no end to his toil, yet his eyes were not content with his wealth. "For whom am I toiling," he asked, "and why am I depriving myself of enjoyment?" This too is meaningless— a miserable business! 9 Two are better than one, because they have a good return for their work: 10 If one falls down, his friend can help him up. But pity the man who falls and has no one to help him up! 11 Also, if two lie down together, they will keep warm. But how can one keep warm alone? 12 Though one may be overpowered, two can defend themselves. A cord of three strands is not quickly broken.
Advancement Is Meaningless
13 Better a poor but wise youth than an old but foolish king who no longer knows how to take warning. 14 The youth may have come from prison to the kingship, or he may have been born in poverty within his kingdom. 15 I saw that all who lived and walked under the sun followed the youth, the king's successor. 16 There was no end to all the people who were before them. But those who came later were not pleased with the successor. This too is meaningless, a chasing after the wind.
Ecclesiastes 5
Stand in Awe of God
1 Guard your steps when you go to the house of God. Go near to listen rather than to offer the sacrifice of fools, who do not know that they do wrong.
2 Do not be quick with your mouth, do not be hasty in your heart to utter anything before God. God is in heaven and you are on earth, so let your words be few.
3 As a dream comes when there are many cares, so the speech of a fool when there are many words.
4 When you make a vow to God, do not delay in fulfilling it. He has no pleasure in fools; fulfill your vow. 5 It is better not to vow than to make a vow and not fulfill it. 6 Do not let your mouth lead you into sin. And do not protest to the temple messenger, "My vow was a mistake." Why should God be angry at what you say and destroy the work of your hands? 7 Much dreaming and many words are meaningless. Therefore stand in awe of God.
Riches Are Meaningless
8 If you see the poor oppressed in a district, and justice and rights denied, do not be surprised at such things; for one official is eyed by a higher one, and over them both are others higher still. 9 The increase from the land is taken by all; the king himself profits from the fields.
10 Whoever loves money never has money enough; whoever loves wealth is never satisfied with his income. This too is meaningless.
11 As goods increase, so do those who consume them. And what benefit are they to the owner except to feast his eyes on them? 12 The sleep of a laborer is sweet, whether he eats little or much, but the abundance of a rich man permits him no sleep. 13 I have seen a grievous evil under the sun: wealth hoarded to the harm of its owner, 14 or wealth lost through some misfortune, so that when he has a son there is nothing left for him. 15 Naked a man comes from his mother's womb, and as he comes, so he departs. He takes nothing from his labor that he can carry in his hand. 16 This too is a grievous evil: As a man comes, so he departs, and what does he gain, since he toils for the wind? 17 All his days he eats in darkness, with great frustration, affliction and anger.
18 Then I realized that it is good and proper for a man to eat and drink, and to find satisfaction in his toilsome labor under the sun during the few days of life God has given him—for this is his lot. 19 Moreover, when God gives any man wealth and possessions, and enables him to enjoy them, to accept his lot and be happy in his work—this is a gift of God. 20 He seldom reflects on the days of his life, because God keeps him occupied with gladness of heart.
1 I have seen another evil under the sun, and it weighs heavily on men:
2 God gives a man wealth, possessions and honor, so that he lacks nothing his heart desires, but God does not enable him to enjoy them, and a stranger enjoys them instead. This is meaningless, a grievous evil.
3 A man may have a hundred children and live many years; yet no matter how long he lives, if he cannot enjoy his prosperity and does not receive proper burial, I say that a stillborn child is better off than he. 4 It comes without meaning, it departs in darkness, and in darkness its name is shrouded. 5 Though it never saw the sun or knew anything, it has more rest than does that man- 6 even if he lives a thousand years twice over but fails to enjoy his prosperity. Do not all go to the same place?
7 All man's efforts are for his mouth, yet his appetite is never satisfied. 8 What advantage has a wise man over a fool? What does a poor man gain by knowing how to conduct himself before others? 9 Better what the eye sees than the roving of the appetite. This too is meaningless, a chasing after the wind. 10 Whatever exists has already been named, and what man is has been known; no man can contend with one who is stronger than he. 11 The more the words, the less the meaning, and how does that profit anyone? 12 For who knows what is good for a man in life, during the few and meaningless days he passes through like a shadow? Who can tell him what will happen under the sun after he is gone?
2 Corinthians 6:14-18; 7:1-7;
Psalm 47:1-9;
For the director of music. Of the Sons of Korah. A psalm.
1 Clap your hands, all you nations; shout to God with cries of joy.
2 How awesome is the LORD Most High, the great King over all the earth!
3 He subdued nations under us, peoples under our feet. 4 He chose our inheritance for us, the pride of Jacob, whom he loved. Selah 5 God has ascended amid shouts of joy, the LORD amid the sounding of trumpets. 6 Sing praises to God, sing praises; sing praises to our King, sing praises. 7 For God is the King of all the earth; sing to him a psalm [a] of praise. 8 God reigns over the nations; God is seated on his holy throne.
9 The nobles of the nations assemble as the people of the God of Abraham, for the kings [b] of the earth belong to God; he is greatly exalted.
Psalm 47:7 Or a maskil (probably a literary or musical term)
Psalm 47:9 Or shields
Proverbs 22:16;
16 He who oppresses the poor to increase his wealth and he who gives gifts to the rich—both come to poverty.


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Somerset County Council Adopted Minerals Plan
by GFFM • March 19, 2015 • News
Somerset County Council resolved to adopt the Somerset Minerals Plan: Development Plan document up to 2030 on 18 February 2015.
The part of the plan that we are most interested in is Energy Minerals…
Most governments across the globe are looking for greater energy security. This has a local impact, as industry seek to discover and exploit accessible reserves of energy minerals (such as oil, gas and coal) without causing unacceptable environmental impacts.
Somerset has extracted coal in the past; however, further coal mining in Somerset is unlikely. The county potentially has oil and gas reserves; for example within coal beds or possibly within its shale resource. Minerals policy needs to ensure there is a robust local policy on this issue, providing clarity to industry and also to local communities who might be concerned about what any proposals for exploration, appraisal and/or production might mean.
For more on this go to Chapter 9 of the Minerals Plan.


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Area : Calabasas, California
Description : Our client is one of the largest tool and equipment retailers in the country. They are seeking a Director of Retail Controls to lead a number of retail analytics strategy initiatives. This role also oversees workforce management analytics and helps drive labor budget strategy for the business.
- Coach and develop a team of high performers and provide clear goals and expectations to enable success
- Work across a variety of functions to own retail analytics data - including human capital, finance, inventory, marketing, and operations
- Oversee the creation, application, and management of annual and monthly labor forecasts using a comprehensive workforce management strategy
- Partner with Finance team to manage and maintain the alignment of labor expense to budgets and forecasts
- Lead the design and development of retail reporting platforms, collaborating with stakeholders to understand the needs of the business, as well as with key partners including Finance, Business Strategy, IT, LP and others
- Formulate, propose, and garner support and approval for new continuous improvement, reporting, and business intelligence initiatives
- Proactively identify areas of opportunity related to sales, workload, payroll hours and costs and develop and implement strategic plans for improvement
- Bachelor's Degree in Finance, Business, Accounting, Economics, Information Technology, or related field
- Master's degree preferred
- 7+ years of experience with multi-unit retail in Finance, Retail Operations, Analytics, or similar business area
- Field retail experience, a plus
- Advanced knowledge of Excel, PowerPoint, and data modeling
- Experience with Kronos Workforce Management, Oracle, Cognos, Netezza, and/or Tableau a plus
Published : Jul 16, 2018
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Original Research ARTICLE
Effect of a Ropy Exopolysaccharide-Producing Bifidobacterium animalis subsp. lactis Strain Orally Administered on DSS-Induced Colitis Mice Model
- 1Department of Microbiology and Biochemistry of Dairy Products, Instituto de Productos Lácteos de Asturias – Consejo Superior de Investigaciones Científicas, Villaviciosa, Spain
- 2CIBER-EHD, Department of Pharmacology, ibs.GRANADA, Center for Biomedical Research, University of Granada, Granada, Spain
- 3Geisel School of Medicine at Dartmouth, Hanover, NH, USA
- 4Universidad Autónoma de Chile, Santiago, Chile
Exopolysaccharide (EPS)-producing bifidobacteria, particularly Bifidobacterium animalis subsp. lactis strains, are used in the functional food industry as promising probiotics with purported beneficial effects. We used three isogenic strains of B. animalis subsp. lactis, with different EPS producing phenotypes (mucoid-ropy and non-ropy), in order to determine their capability to survive the murine gastrointestinal tract transit, as well as to evaluate their role in improving clinical outcomes in a chemically-induced colitis model. The three strains were able to survive in the intestinal tract of C57BL/6J mice during the course of the intervention study. Furthermore, the disease activity index (DAI) of the animal group treated with the ropy strain was significantly lower than of the DAI of the placebo group at the end of the treatment. However, no significant differences were found among the three strains. The analysis of several immune parameters, such as TNFα and IL-10 quantified in blood plasma and lymphocyte populations enumerated in mesenteric nodes, showed some significant variations among the four experimental animal groups. Remarkably, a higher capability of the ropy strain to increase regulatory T-cells in mesenteric lymphoid nodes was demonstrated, suggesting a higher ability of this strain to regulate inflammatory responses at mucosal level. Our data indicate that strains of B. animalis subsp. lactis producing EPS that confer a mucoid-ropy phenotype could represent promising candidates to perform further studies targeting intestinal inflammatory processes.
The human gut is one of the most densely populated ecosystems inhabited by a community of microorganisms that actively contributes to the health of the host. Microbiota establishes a symbiotic relationship with the host, but also their members must co-exist in a balanced state. Indeed, disturbances in microbiota composition (dysbiosis) and function, as well as in the microbiota-host cross-talk, are the basis of prevalent gastrointestinal diseases, and also of some extra-intestinal pathology (Tojo et al., 2014). Among others, microbiota shifts have been related to chronic inflammatory disorders, such as inflammatory bowel disease (IBD) (Huttenhower et al., 2014). Up to now, dietary interventions toward restoring the unbalanced microbiota, such as the oral administration of probiotics, have been a realistic approach for human applications (Reid et al., 2011; Collins, 2014). Hill et al. (2014) have recently reviewed the probiotic concept and they have adopted, with a minor grammatical correction, the definition "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host," previously proposed by the FAO and WHO organizations (FAO/WHO. Food and Agricultural Organization of the United Nations and World Health Organization, 2001). Research on probiotics has generated a vast amount of literature, mainly related to the characterisation of the probiotic potential of Lactobacillus and Bifidobacterium, the genera most commonly used for human consumption. However, only a small number of strains have been evaluated for the prevention or treatment of gastrointestinal disorders, such as IBD, diverse diarrheas, irritable bowel syndrome (IBS) or necrotising enterocolitis (NEC) (WGO. World Gastroenterology Organisation, 2011; Sanders et al., 2013). In relation to bifidobacteria, specific strains belonging to the species Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium bifidum, and Bifidobacterium animalis subsp. lactis, have been tested in human intervention studies resulting in positive effects on gastrointestinal dysfunctions (Tojo et al., 2014).
B. animalis subsp. lactis is the species that has been most successfully introduced in food formulations, since it is able to deal with technological challenges during food manufacture (Prasanna et al., 2014). However, analysis of the available genomes shows a very low genetic variability among members of this subspecies (Milani et al., 2013). One of the features shared in all of their genomes is the presence of a cluster involved in the synthesis of exopolysaccharides (EPS) which are carbohydrate polymers surrounding the cell wall (Hidalgo-Cantabrana et al., 2014b). These EPS play a relevant role for the producing strain because they are involved in protection and niche colonization, but EPS also act as intermediaries in the dialog established between bacteria and host (Hidalgo-Cantabrana et al., 2014b). It has been proposed that EPS are able to interact with receptors located in the gut epithelium (Lebeer et al., 2010b) and they could act as effector molecules eliciting different immune responses (Hidalgo-Cantabrana et al., 2012).
In recent years our group has been working on the study of biological properties of EPS synthesized by bifidobacteria. One of the B. animalis subsp. lactis strains from our collection displayed a mucoid-ropy phenotype, denoted by the formation of a long filament from the colony, and it was able to synthesize a high molecular weight (HMW)-EPS. Recently, working with three recombinant strains, we demonstrated the involvement of a mutation (C> 266 >T) in wzz gene in the occurrence of this phenotype and in the synthesis of the HMW-EPS fraction. This gene encodes a hypothetical membrane anchored protein, with a predicted large soluble domain, which is theoretically involved in the chain length determination of the polymer (Hidalgo-Cantabrana et al., 2015). The acquisition of the ropy phenotype favored the in vitro survival and adhesion of the recombinant strain to the gut environment. In turn, this strain also elicited an in vitro and ex vivo anti-inflammatory cytokine profile (Hidalgo-Cantabrana et al., 2015). Thus, in a step forward the current work aims to in vivo evaluate the probiotic potential of the ropy B. animalis subsp. lactis Balat_1410S89L checking its capability to counteract an inflamed state of colitis chemically induced by dextran sodium sulfate (DSS) administration.
Materials and Methods
Bifidobacterial Strains and Preparation of Lyophilized Bacteria in Milk
Three isogenic EPS-producing B. animalis subsp. lactis strains from the IPLA-CSIC collection were used in this study to test their probiotic capabilities in an in vivo model. Based on the type strain DSM10140, recombinant strains were obtained from a previous study (Hidalgo-Cantabrana et al., 2015): strain ΔBalat_1410 (DSM10140 lacking the gene Balat_1410 complemented with pAM1, non ropy), strain Balat_1410 (strain ΔBalat_1410 complemented with pAM1 + Balat_1410, non ropy), and Balat_1410S89L (strain ΔBalat_1410 complemented with pAM1 + Balat_1410S89L, ropy). Glycerol stocks kept at −80°C were plated on agar MRSC [MRS Difco (BD, Biosciences, San Diego, CA) containing 0.25% L-cysteine-HCl (Sigma-Chemical Co., St. Louis, MO)] supplemented with erythromycin (2.5 μg/ml) (MRSCE). Plates were incubated at 37°C under anaerobic conditions (80% N2, 10% CO2, 10% H2) in an MG500 chamber (Don Whitley Scientific, West Yorkshire, UK). A single colony per strain was used to inoculate 50 ml MRSCE broth and after 20 h this culture was used to inoculate 2% (v/v) 1 L fresh medium. After 20 h incubation, cultures were centrifuged, washed once with PBS and resuspended in 100 ml tindalized 11% skimmed milk (Difco). Then, bacterial suspensions in milk were lyophilized for 48 h in the Virtis Freezemobile 12EL equipment (SP Scientific, NY). Each lyophilized bifidobacterial stock was tested for bacterial viability and enumeration by counting in agar-MRSCE and incubated under standard conditions. For oral administration, each lyophilized bifidobacteria was resuspended daily in water (at 5 × 109 cfu/ml) and 100 μl was administered per mouse.
Animals, Experimental Designs and Sample Collection
All studies were carried out following the Directive 2010/63/EU of the European Parliament and the Council on the protection of animals used for scientific purposes (Directive 2010/63/EU, 2010). The experimental protocol was approved by the Ethical Committee of Laboratory Animals of the University of Granada (Spain) (Permit Number CEEA-2010-286). Male C57BL/6J mice (7–9 weeks old, approximately 20 g) were obtained from Janvier Labs (St Berthevin Cedex, France) and kept under conventional conditions with a standard pelleted diet and sterilized water for 1 week before beginning the experiments.
Bifidobacteria Survival Study
Animals (32 mice) were randomly assigned to four experimental groups with 8 mice per group: three bifidobacterial groups, each orally receiving 100 μl/day (5 × 108 cfu) of the corresponding milk-bifidobacterial suspension in water, and one placebo (control) group which received 100 μl/day of skimmed milk. Placebo and bifidobacteria suspensions were orally administered for 9 days by means of an intra-gastric cannula. Every day, the drinking water and food intake was measured for each group of mice, whereas the body weight was measured individually for each animal. Stool samples were taken, in duplicate, at group level every 2 days, including the first and last day. At the end of the intervention period (day 10), each mouse was anesthetized with isoflurane and then, the animals were sacrificed by cervical dislocation by expert and qualified persons, according to the Federation of European Laboratory Animal Science Associations (FELASA). The colon was excised, its content was collected and its length and weight was measured after washing with PBS. Stool and colon-content samples were immediately processed as described in the next section.
Dextran Sodium Sulfate (DSS) Induced Colitis Study
Mice (40 animals) were randomly assigned to five groups, each with 8 animals, as described in Figure 1. The reference (non-colitic, non-placebo/bifidobacteria fed) group received daily, through an intra-gastric cannula, 100 μl water during the 15-day experimental period. The other four groups were fed with bifidobacteria, or a placebo, and treated with DSS. During the 15 days of the experimental period these four groups received a placebo (100 μl/day milk, group 1), or different bifidobacterial strains (100 μl/day 5 × 108 cfu milk-bifidobacterial suspensions in water, for groups 2, 3, and 4). After 9 days of bifidobacteria/placebo feeding, colitis was induced in the four groups by adding DSS (3%, w/v, 36–50 kDa, MP Biomedicals, CA) in the drinking water (Mähler et al., 1998). This DSS-treatment was kept for a period of 6 days unless the application of humane end-point, as defined below, was needed. At the end of the experimental period (day 15) animals were anesthetized and a blood sample was directly extracted from the heart using heparinized tubes. The blood plasma was obtained by centrifugation (9,300xg, 4°C, 20 min). The animals were then euthanatized in order to collect colon and colon-content, as described above, as well as mesenteric lymphoid nodes. Samples were immediately processed or stored at −20°C until their analysis.
Figure 1. Experimental design to check the capability of EPS-producing B. animalis subsp. lactis strains to counteract the dextran sodium sulfate (DSS)-induced colitis. Group 1 (placebo) daily receiving 100 μl skimmed milk and Groups 2–4 daily receiving 100 μl milk with 5 × 108 cfu bifidobacteria: group 2, strain ΔBalat_1410 (DSM10140 lacking the gene Balat_1410 complemented with pAM1, non ropy); group 3, strain Balat_1410 (strain ΔBalat_1410 complemented with pAM1 + Balat_1410, producing a non-ropy EPS); group 4, strain Balat_1410S89L (strain ΔBalat_1410 complemented with pAM1 + Balat_1410S89L, producing a ropy EPS). Reference group (non DSS-induced colitis, non-placebo/bifidobacteria fed group) daily receiving 100 μl water.
Drinking water and food intake was measured at group level each day, whereas body weight, stool consistency and the presence of gross blood in feces were evaluated daily and scored (Table 1) for each mouse during the experimental period. The disease activity index (DAI) was calculated as previously reported by Cooper et al. (1993): total score [body weight decrease + stool consistency + rectal bleeding] / 3. The ulcerative colitis (UC) disease is considered for DAI ≥ 1.5 and the humane endpoint was fixed at DAI = 3. Stool samples were taken at group level every 2 days to check viability of bifidobacteria.
Table 1. Disease activity index (DAI) score used to evaluate the DSS-induced colitis. DAI index was calculated as total score (body weight decrease + stool consistency + rectal bleeding) divided by 3.
Microbial Quantification in Fecal Samples
Bifidobacterial Quantification by Counting
Stool samples, collected in both studies as previously indicated, were homogenized in PBS (0.1 g/ml) using a Heidolph (Heidolph Instruments GmbH, Schwabach, Germany) stirrer for 1 min. Then, 100 μl was used to obtain serial dilutions in PBS which were plated onto the surface of TOS propionate agar medium (Merck, Darmstadt, Germany), supplemented with lithium-mupirocin as recommended by the manufacturer, to selectively enumerate total bifidobacteria. Additionally, agar lithium-mupirocin-TOS was supplemented with 2.5 μg/ml erythromycin (TOS+Ery) which is the selective marker of the plasmid present in the three B. animalis subsp. lactis strains under study. All plates were incubated at 37°C in an anaerobic jar using Oxoid-Anaerogen™ (Thermo Fisher Scientific Inc., Waltham, MA) sachets to generate anaerobic conditions for 72 h.
Bifidobacterial Identification by 16S rDNA Amplification
Several colonies were picked up from TOS+Ery plates of each of the four experimental groups belonging to the bifidobacteria survival study. The DNA from a total of 67 colonies was isolated using the GenElute Bacterial Genomic DNA kit (Sigma) according to the manufacturer's instructions, but adding a prior step of bacterial lysis with mutanolysin and lysozyme. Primer uses were: pA/pH (Edwards et al., 1989). The PCR mixture (final volume 50 μl) was: 1 μl chromosomal DNA, 0.20 μM of each primer, 200 μM dNTPs (Amersham Bioscience, Upsala, Sweden) and 2.5 U Taq DNA-polymerase (Eppendorf, Hamburg, Germany). After an initial cycle at 95°C, the PCR amplification consisted of 30 cycles: 95°C for 1 min, 55°C for 50 s and 68°C for 2 min, and ended with a final extension step of 68°C for 10 min. Reactions were carried out in the thermal cycler UnoCycler (VWR International Eurolab S.L., Barcelona, Spain). Purification and sequencing of the amplicons were performed in Macrogen (Seoul, Korea). The free Chromas 1.45 software (Technelysium Pty Ltd., Australia) was used to process sequences which were compared to those held in the GenBank database (http://www.ncbi.nlm.nih.gov/genbank) using the BLASTn tool.
Gene Expression Analyses in Colonic Samples by Reverse Transcriptase qPCR
After colon excision, PBS-washed colonic tissue was sectioned in small fragments which were placed in RNAlater stabilization reagent (Qiagen GmbH, Hilden, Germany) and then stored at −80°C until RNA extraction. Total RNA was isolated using RNeasy Mini Kit (Qiagen) according to the manufacturer's recommendations. Purity and RNA concentration were determined with the NanoDropTM 2000 spectrophotometer. Then, 3 μg RNA was reverse transcribed to obtain cDNA using oligo(dT) primers and reagents from Promega (Promega, Southampton, UK) and the TProfessional basic thermocycler (Biometra GmbH, Goettingen Germany).
The amplification was performed in an Eco™ Real-Time PCR System (Illumina, San Diego, CA USA) using 20 ng of cDNA and specific primers (Table S1). The 2−ΔΔCt method was used to normalize expression results (Livak and Schmittgen, 2001). The values of the house-keeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were first used to normalize the values obtained for each of the genes under study. These values were additionally normalized to those obtained in the reference group, thus this group had a normalized value of 0. Finally the relative expression of each gene was calculated as 2−ΔΔCt, thus the reference group had the relative expression value of 1.
Cellular Population in Mesenteric Lymphoid Nodes
The mesenteric lymphoid nodes were carefully mashed with wet slides to decrease friction and the solutions were filtered through a 70 μM cell strainer. Cells were isolated, counted and plated on anti-CD3 (clone17A2, eBioscience, San Diego, CA, USA) and anti-CD28 (clone 37.51, eBioscience)—coated plates for FcγR blocking. Then, the cells were transferred to polystyrene tubes for surface staining with anti-CD4 (PerCP-Cy™5.5, clone RM4-5 BD Pharmigen™, Franklin Lake, NU, USA), anti-CD45 (APC-eFluor®780, clone 30-F11, eBioscience) and Viability Dye (eFluor®660, eBioscience) for 15 min at 4°C in the dark. The cells were then fixed, permeabilized with the Fixation/Permeabilization kit (eBioscience) and intracellular staining was done with anti-Foxp3 (PE, clone FJK-16s, eBioscience) for 30 min at 4°C in the dark. Data collection was performed using a flow cytometer CANTO II (BD Biosciences San Diego, CA, USA).
Cytokine Levels of Blood Samples
The levels of tumor necrosis factor (TNF)-α and interleukin (IL)-10 cytokines in blood plasma were quantified using the mouse TNFα Quantikine and mouse IL-10 Quantikine ELISA kits (R&D Systems, Abingdon, UK) following the manufacturer's instructions.
Statistical Analyses
All considered variables were distributed normally (Kolmogorov-Smirnov test was used to check normality); thus, they are described as mean ± standard deviation. Robust Student-Welch or ANOVA tests were used in order to check the equality among groups for all variables, and post-hoc T3 de Dunnet or LSD tests were used to compare each two groups. Standard linear regression models were employed to compute the size of the effect (95% confidence interval) of the different treatments on the DAI; to do this, crude data were adjusted by the initial conditions (DAI score the first day of DSS-induced colitis) and the placebo group was used as reference. Differences below 0.05 were considered statistically significant. All analyses were made with the free statistical software R (www.r-project.org).
Results and Discussion
B. animalis subsp. lactis Strains Are Able to Colonize the Murine Gut
Orally administered probiotics should survive the upper gastrointestinal tract (GIT) transit and reach the colon alive, where they should persist for a certain time in order to be able to exert their beneficial effect. Therefore, an animal experimentation procedure was done to assess the viability of the three B. animalis subsp. lactis strains under study aiming to check whether the presence of the ropy HMW-EPS surrounding Balat_1410S89L strain could exert a protective role. Additionally, although B. animalis has QPS (qualified presumption of safety) status (EFSA BIOHAZ Panel, 2013), we evaluated the lack of toxicity of our strains, at this oral-dose administered to the animals, by measuring animal behavior and wellbeing parameters. In this regard, no differences (p > 0.05) were found in water and food intake, body weight, stool consistency or colon anatomy between the placebo (skimmed milk-fed) and each of the bifidobacteria-fed groups (Table 2). These results support the fact that the three bifidobacterial strains used in this study had no side effects at the concentration level administered to this murine model.
Table 2. Mean and standard deviation of general parameters measured in four experimental groups of mice to evaluate the absence of toxicity of the bifidobacterial strains administered for 10 days at dose of 5 × 108 cfu per mouse and day.
The bifidobacterial population in the fecal samples was enumerated using agar-TOS (Figure 2A) which is a selective medium for bifidobacteria growing in milk products. After 2 days of administration, the bifidobacterial levels increased in all groups about 1 log unit, including the placebo receiving skimmed milk. However, during prolonged treatment the population of bifidobacteria in the placebo group tended to decline to reach the initial values, whereas counts in the three bifidobacteria-fed groups remained stable. Similarly, counts obtained in TOS+Ery, which is the medium containing the selective marker erythromycin of the plasmid present in the three strains under study, were significantly higher (p < 0.05) in the three bifidobacterial-fed groups with respect to the placebo (Figure 2B). Levels of erythromycin-resistant bacteria in the microbiota of placebo-fed mice were around 4 log units and remained unaltered for the duration of the experimentation time. After 2 days of administration, levels of erythromycin-resistant bifidobacteria increased (on average) 0.7 log cfu/g in the mice receiving bifidobacteria; the highest count increase (1.7 log cfu/g) was reached after 4 days, remaining at similar values until the end of the experimentation period. In general, no statistical differences among the three bifidobacteria-fed groups were found and, additionally, the colonies chosen to partially sequence the gene 16S rDNA were identified as members of Bifidobacterium genus; those isolated from Balat_1410S89L fed animals displayed a ropy phenotype as well (see Table S2). Altogether, these results show that our bifidobacterial strains were able to survive as members of the intestinal microbiota of mice for at least 9 days, following a daily intake of 5 × 108 cfu. The increase in total bifidobacteria observed in this study could be related to the ability of EPS-producing B. animalis subsp. lactis to raise the number of other bifidobacterial species in the intestinal microbiota of rodents as previously described (Salazar et al., 2011). This effect could be related to the ability of members from this microbial community to use EPS as fermentable substrates (Salazar et al., 2009). However, the increase of erythromycin resistant members in feces is indicative of a higher survival rate in the gut of the specific strains used in this work. The in vivo performance of the three strains is rather similar, since there was no statistical difference in the number of bifidobacteria present in the fecal samples among the three groups of mice. Thus, it seems that the presence of HMW-EPS on the surface of the ropy Balat_1410S89L strain does not confer an additional protection of survival in mice gut. Fanning et al. (2012) found that fecal counts (made in Reinforced Clostridium agar with 50 mg/L mupirocin) of mice fed with EPS+ B. breve UCC2003 were significantly higher than those obtained with two non-EPS producing mutant strains. Thus, these authors conclude that the presence of an EPS on the surface of the wild type B. breve was involved in its higher survival rate in the mice gut. On the contrary, in vivo studies carried out with different EPS-producing Lactobacillus johnsonii strains showed that mutants with deletion of genes from the eps cluster had an increased residence time in the animal's gut (Denou et al., 2008; Horn et al., 2013). It has also been shown that the loss of EPS production in Lactobacillus rhamnosus GG improves the in vitro adhesion of the strain to Caco2 cells (Lebeer et al., 2009); however, the presence of the EPS surrounding the wild type GG strain improves its in vivo performance allowing the strain to persist for longer periods because the EPS protects against the antimicrobial peptide LL-37 (Lebeer et al., 2010a). The intrinsic characteristics of EPS, such as composition and size, seem to promote different effects on bacterial survival under gut challenges. In some cases, polymers could prevent the exposure of molecules from the bacterial envelope involved in adhesion, but EPS can also act as a shield against host immune defense (Hidalgo-Cantabrana et al., 2012).
Figure 2. Counts in agar-TOS to enumerate total bifidobacterial population (A) and agar-TOS+Ery (supplemented with 2.5 μg/ml erythromycin, the antibiotic marker of strains under study) to enumerate erythromycin-resistant bacteria (B) in fecal homogenates obtained from four experimental groups of mice along the experimental period. Placebo group (), feed B. animalis subsp. lactis ΔBalat_1410 (), feed B. animalis subsp. lactis Balat_1410 (), and feed B. animalis subsp. lactis Balat_1410S89L (). Within each treatment day: statistical differences with respect to the control group are marked with an asterisk (p < 0.05) and differences among the three bifidobacterial groups are denoted with different letters (p < 0.05).
The Ropy B. animalis subsp. lactis Balat_1410S89L Reduces DSS-Induced Damage
As mentioned above, in vitro and ex vivo tests (using human tissues) showed different immune modulation capabilities of the three isogenic bifidobacteria, the ropy strain Balat_1410S89L having an anti-inflammatory profile (Hidalgo-Cantabrana et al., 2015). To expand on these observations, we wanted to address whether this predicted anti-inflammatory ability is effective in vivo, modifying the immune response in a model of gut inflammation such as mice with DSS-induced colitis. This is a well-established animal model of acute mucosal inflammation that has been used for several decades (Wirtz et al., 2007) and it has been validated as a model for the translation of mice data to humans (Melgar et al., 2008).
In our experimental procedure, four groups of mice were pre-treated with an oral administration of bifidobacteria, or placebo, for 9 days before the administration of DSS in drinking water, which was continued for an additional 6 days (Figure 1). As was expected, in accordance with results shown in the previous section, the pre-feeding for 9 days in the four groups did not modify the wellbeing and behavior parameters of the mice. This is also consistent with the absence of significant variations in the disease activity index (DAI) score of these groups (values lower than 0.5, data not shown). The DAI score was kept close to 0 in the non-colitic, non-placebo/ bifidobacteria-fed (reference) group during all experimental procedures. In the four colitic groups, after the second day (day 11) of DSS treatment, the DAI values increased continuously (Figure 3A). The maximum score was obtained at the end of the experimental procedure, after 6 days of DSS colitic induction. In general, no statistical differences were detected on each day of treatment among the placebo and the three bifidobacteria-fed groups during DSS-treatment; the exception was the group fed with the strain Balat_1410S89L, which showed a significantly (p < 0.05) lower DAI score than the placebo group at day 15. These results were confirmed when DAI data were adjusted by means of linear regression models taking into consideration the DAI of placebo group each day of DSS-induction (Figure 3B). This model allowed us to determine the effect of the treatments on the DAI with respect to the placebo (value = 0). Thus, negative values of the adjusted DAI indicate a reduction in the severity of the disease in comparison with the placebo group. The highest reduction in adjusted DAI obtained for B. animalis subsp. lactis Balat_1410S89L fed animals at the end of the treatment was mainly associated with a reduction in diarrhea and rectal bleeding. This led to a 44% reduction in the severity of the disease with respect to the placebo (p < 0.007), whereas, in the other two bifidobacteria-fed animals this reduction was not statistically significant, although the reduction of the illness was 23.2% for the strain ΔBalat_1410 and 16.7 % for the strain Balat_1410. In spite of the remarkable differences among the three strain-fed groups (disease severity in Balat_1410S89L was reduced by almost two and three-fold compared to ΔBalat_1410 and Balat_1410, respectively), these differences were not deemed statistically significant. Therefore, we cannot ascertain that the protective effect against DSS-induced colitis exerted by strain B. animalis subsp. lactis Balat_1410S89L is attributed to the HMW-EPS surrounding the bacteria. Some authors have reported the capability of probiotic strains to reduce the DAI index of animals under DSS-induced colitis. However, a mixture of several bacteria, or combinations of bacteria-bioactive compounds, are often tested, thus the positive effect cannot be assigned to a single bacterium (Dai et al., 2013). Only a few reports have shown the efficacy of single bifidobacterial strains (Hayes et al., 2014; Zheng et al., 2016).
Figure 3. Mean values of the disease activity index (DAI, Table 1) calculated for each experimental group. The coefficient of variation (SD/mean) percentage of these values varied between 9 and 70%. Each bifidobacteria-fed group was compared with the placebo throughout the DSS-treatment and statistical differences (p < 0.05) are marked with an asterisk (A). The DAI score of each bifidobacterial group was adjusted, as indicated in material and methods, to values of placebo at the first day of DSS-treatment (value = 0). Negative values indicate a higher reduction in the disease severity with respect to the placebo. These adjusted data were compared with placebo during the 5 days of DSS-treatment and statistical differences (p < 0.05) are marked with an asterisk (B). Reference group (); DSS-induced colitis groups: placebo (), feed B. animalis subsp. lactis ΔBalat_1410 (), feed B. animalis subsp. lactis Balat_1410 (), feed B. animalis subsp. lactis Balat_1410S89L ().
Different B. animalis subsp. lactis Strains Elicit Specific Host Responses
In order to understand the mechanism(s) involved in the attenuation of the DSS-colitis by the strain Balat_1410S89L several immune parameters, targeting adaptive as well as innate responses, were measured in different tissues. In our study, the expression of several genes related to the reinforcement of the intestinal epithelium barrier, such as those coding for chemokines, proteins of tight junctions and mucins, showed no statistical variations (p > 0.05) among the four groups of animals (Table 3). Only the non-ropy EPS-producing strain Balat_1410 was able to slightly induce (p < 0.05) the expression of Mcp1, a chemokine able to attract monocytes, as well as the secretory protein TFF3, which is synthetized by goblet cells and acts to stabilize the mucus layer. Thus, it seems that, based on these results of gene expression, our strains were not able to modify the epithelial barrier in a remarkable way. However, it was reported that probiotics are able to reinforce the intestinal barrier function by multiple mechanisms (Lebeer et al., 2010a; Rao and Samak, 2013). The efficacy of Bifidobacterium infantis BB-02 to reduce intestinal injury in NEC was due to its ability to keep claudin-4 and occludin located in the tight-junction of bifidobacteria-fed mice (Bergmann et al., 2013). It was also demonstrated that bioactive factors released by B. infantis (one of the strains present in the VSL#3 mixture) were also able to reduce gut permeability by acting upon tight junction proteins (Ewaschuk et al., 2008). Similarly B. bifidum OLB6378 also favored the location of tight-junction and adherent-junction proteins, also being able to induce the synthesis of mucin-3, TFF3, and IL-6 (Khailova et al., 2009). These works, among others, show that bifidobacteria are able to improve the gut barrier function in a strain dependent manner. Regarding expression of cytokine genes in colonic tissue, statistical differences (p < 0.05) were detected only for pro-inflammatory IL-1β and IFNγ genes and the anti-inflammatory IL-10 (Table 3); in general, the two first genes were expressed at a lower rate in the placebo group with respect to the three bifidobacteria-fed groups, whereas IL-10 gene had significantly lower expression rate only for the ropy strain Balat_1410S89L. Thus, it seems that this strain induced the expression of genes that could lead to a mild pro-inflammatory response, since the ratio TNFα/IL-10 was significantly higher than that obtained for the other three groups (data not shown). A similar pro-Th1 profile was obtained with wild-type EPS-producing B. animalis subsp. lactis strains (one of them also producing a ropy HMW-EPS) co-cultivated in vitro with PBMC and GALT (gut associated lymphoid tissue) cells isolated from naïve rats (Hidalgo-Cantabrana et al., 2014a). Indeed, in the current study the concentration of these cytokines measured in blood plasma (Figure 4A) gives TNFα/ IL-10 ratio values of 1.69 ± 0.97 for placebo, 4.32 ± 2.15 for strain ΔBalat_1410, 2.77 ± 1.85 for strain Balat_1410, and 10.28 ± 9.15 for strain Balat_1410S89L. The ratio obtained for the animals fed with the ropy strain was significantly (p < 0.05) higher than that obtained for the other three groups of mice. However, it is worth noting that none of the three strains, or the placebo, was able to induce a strong immune systemic response since the values of both cytokines were near (or lower) than value 1, which is the value of the reference animal group. Thus, this suggests that our strains, or the skimmed milk (placebo) used as a protectant for bifidobacteria delivery, did not contribute to increase the inflammatory process induced by DSS.
Figure 4. Mean values of relative TNFα and IL-10 cytokines measured in blood samples (A) and relative number of CD4+CD25+ (T helper cells) and FOXP3 (T regulatory cells) quantified in mesenteric lymphoid nodes (B). Samples were collected after 6-days of DSS-treatment. Values of each DSS-colitic animal group were referred to as the average of those obtained in the reference group (value = 1). Placebo group (), feed B. animalis subsp. lactis ΔBalat_1410 (), feed B. animalis subsp. lactis Balat_1410 (), feed B. animalis subsp. lactis Balat_1410S89L (). Within each parameter: statistical differences among the four groups are denoted with different letters (p < 0.05).
Immune cells isolated from the lymphoid nodes associated with the gastrointestinal tract were quantified by flow cytrometry and significant (p < 0.05) variations among the four experimental groups were detected (Figure 4B). Mice orally receiving the ropy Balat_1410S89L or the non-ropy Balat_1410 strains presented a significant reduction in the population of CD4+/CD45+ cells, which are the markers for helper T-lymphocytes. The number of this population was even slightly lower than that of the reference group (value 1). In addition, the group receiving the strain Balat_1410S89L was the only one significantly different from the placebo regarding the number of cells expressing Foxp3+, which acts as a regulatory factor for differentiation and function of regulatory T-lymphocytes. Besides, values of Foxp3+ cells in this group of animals, which also showed a reduced severity in DAI score, were slightly higher than the value of the animal reference group. Thus we suggest that the oral administration of the ropy strain could favor the recruitment of Treg cells to the intestinal mucosa, which could lead to a decrease in the severity of the damage caused by DSS. Jeon et al. (2012) showed in vivo that B. breve Yakult strain was able to ameliorate T cell-dependent colitis in mice through the induction of Treg cells toward producing IL-10. Similar results were obtained with a mixture of five probiotic strains and a mice model mimicking human Crohn's disease (Kwon et al., 2010). These authors also conclude that the efficacy of probiotics was due to an enrichment of CD4+ Foxp3+ Treg in the inflamed area. In recent years our group has been working with two models of isogenic, ropy and non-ropy EPS-producing B. animalis subsp. lactis strains: wild-types and the recombinant strains used in the current work. The results regarding the immune response that both types of strains could elicit are consistent when immune cells from the same origin are used. That is, all types of strains are able to induce a mild pro-inflammatory (Th1) response when a murine (rats or mice) model is used, either by in vitro (Hidalgo-Cantabrana et al., 2014a) or in vivo (the current work) approaches. However, the same EPS-producing strains tested in vitro or ex vivo, with immune cells and colonic tissue from human origin, had a reduced capability to elicit an immune response, or displayed an anti-inflammatory cytokine profile (López et al., 2012; Hidalgo-Cantabrana et al., 2015). These results underline the strong influence of the biological model used to test immune capability of strains with probiotic potential. Variations obtained with both models could be related to the differences in composition and function of the microbiota inhabiting the human and mouse intestines (Wos-Oxley et al., 2012).
Finally, due to the absence of statistical differences among the three strains we cannot conclude without doubt that the protective effect of DAI score promoted by strain Balat_1410S89L was due to the synthesis of the HWM-EPS. However, we can hypothesize that the ropy HMW-EPS (produced in higher amounts by this strain) could act as a physical barrier, like a biofilm, protecting from chemical insults and, consequently, reducing the severity of the mucosa damage. In this regard, we have previously shown that the EPS purified from the wild ropy strain B. animalis subsp. lactis A1dOxR was able to in vitro counteract the cytotoxic effect of bacterial toxins upon humans (Caco2 monolayers) or animals (rabbit erythrocyte) cells (Ruas-Madiedo et al., 2010). Synthesis of EPS by bifidobacteria in intestinal environmental conditions has not been proved to date; however, production of EPS by commensal Bacteroides fragilis in the gut of mice has recently been proved (Geva-Zatorsky et al., 2015). Thus, this fact would support our hypothesis that the HMW-EPS from strain B. animalis subsp. lactis Balat_1410S89L forms an EPS-biofilm protecting, to a certain degree, the intestinal epithelium from DSS-induced damage.
In this work we have shown that the ropy strain B. animalis subsp. lactis Balat_1410S89L was able to protect mice from injury caused by DSS. To date, we have not found modifications in the expression of genes related to the reinforcement of the intestinal barrier that could explain the attenuation of chemically-induced damage; the most relevant immune parameter that could be related to this fact is the high capability of strain Balat_1410S89L to induce Treg cells in mesenteric lymphoid nodes which, in turn, could lead to a localized reduction of the inflammation at mucosal level. In spite of the only difference among the three isogenic recombinant strains tested in this study being the production of a ropy HMW-EPS, we cannot without doubt attribute the capability to reduce the DSS-induced damage to the polymer produced by Balat_1410S89L.
There is a lack of consistency in the immune modulation capability of the ropy HMW-EPS-producing B. animalis subsp. lactis strains due to the high influence of the biological model used. However, the ability of the strain Balat_1410S89L to survive in the mice gut and the absence of adverse effects on the animals, together with previous in vitro and ex vivo evidence upon human cells, makes it feasible that the ropy (wild-type) B. animalis subsp. lactis could be a good candidate to check its anti-inflammatory ability in patients suffering from intestinal inflammation.
Author Contributions
JG, AM, and PR contributed with the conception, experimental design and results interpretation of this study. CH carried out all experiments, some of them performed with the collaboration of FA, AR, and TV. PM was in charge of the statistical analyses. PR was in charge of writing the drafted manuscript. All authors performed a critical revision of the manuscript and approved the final version.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was partially financed by FEDER European Union funds through the projects AGL2012-33278 and SAF2011-29648 of the Spanish Ministry of Economy and Competitiveness (MINECO), as well as to the projects AGR-6826 and CTS-164 of the "Junta de Andalucía." CH acknowledges his FPI fellowship to MINECO and FA acknowledges her fellowship to Junta de Andalucía. The CIBEREHD is funded by the "Instituto de Salud Carlos III."
Supplementary Material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/article/10.3389/fmicb.2016.00868
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López, P., Monteserín, D. C., Gueimonde, M., de los Reyes-Gavilán, C. G., Margolles, A., Suárez, A., et al. (2012). Exopolysaccharide-producing Bifidobacterium strains elicit different in vitro responses upon interaction with human cells. Food Res. Int. 46, 99–107. doi: 10.1016/j.foodres.2011.11.020
Mähler, M., Bristol, I. J., Leiter, E. H., Workman, A. E., Birkenmeier, E. H., Elson, C. O., et al. (1998). Differential susceptibility of inbred mouse strains to dextran sulfate sodium induced colitis. Am. J. Physiol. Gastrointest. Liver Physiol. 274, G544–G551.
Melgar, S., Karlsson, L., Rehnström, E., Karlsson, A., Utkovic, H., Jansson, L., et al. (2008). Validation of murine dextran sulfate sodium-induced colitis using four therapeutic agents for human inflammatory bowel disease. Int. Immunopharmacol. 8, 836–844. doi: 10.1016/j.intimp.2008.01.036
Milani, C., Duranti, S., Lugli, G. A., Bottacini, F., Strati, F., Arioli, S., et al. (2013). Comparative genomics of Bifidobacterium animalis subsp. lactis reveals a strict monophyletic bifidobacterial taxon. Appl. Environ. Microbiol. 79, 4304–4315. doi: 10.1128/AEM.00984-13
Prasanna, P. H. P., Grandison, A. S., and Charalampopoulos, D. (2014). Bifidobacteria in milk products: an overview of physiological and biochemical properties, exopolysaccharide production, selection criteria of milk products and health benefits. Food Res. Int. 55, 247–262. doi: 10.1016/j.foodres.2013.11.013
Rao, R. K., and Samak, G. (2013). Protection and restitution of gut barrier by probiotics: nutritional and clinical implications. Cur. Nutr. Food Sci. 9, 99–107. doi: 10.2174/1573401311309020004
Reid, G., Younes, J. A., Van der Mei, H. C., Gloor, G. B., Knight, R., and Busscher, H. J. (2011). Microbiota restoration: natural and supplemented recovery of human microbial communities. Nat. Rev. Microbiol. 9, 27–38. doi: 10.1038/nrmicro2473
Ruas-Madiedo, P., Medrano, M., Salazar, N., de los Reyes-Gavilán, C. G., Pérez, P., and Abraham, A. G. (2010). Exopolysaccharides produced by Lactobacillus and Bifidobacterium strains abrogate in vitro the cytotoxic effect of bacterial toxins on eukaryotic cells. J. Appl. Microbiol. 109, 2079–2086. doi: 10.1111/j.1365-2672.2010.04839.x
Salazar, N., Binetti, A., Gueimonde, M., Alonso, A., Garrido, P., González del Rey, C., et al. (2011). Safety and intestinal microbiota modulation by the exopolysaccharide-producing strains Bifidobacterium animalis IPLA R1 and Bifidobacterium longum IPLA E44 orally administered to Wistar rats. Int. J. Food Microbiol. 144, 342–351. doi: 10.1016/j.ijfoodmicro.2010.10.016
Salazar, N., Ruas-Madiedo, P., Kolida, S., Collins, M., Rastall, R., Gibson, G., et al. (2009). Exopolysaccharides produced by Bifidobacterium longum IPLA E44 and Bifidobacterium animalis subsp. lactis IPLA R1 modify the composition and metabolic activity of human faecal microbiota in pH-controlled batch cultures. Int. J. Food Microbiol. 135, 260–267. doi: 10.1016/j.ijfoodmicro.2009.08.017
Sanders, M. E., Guarner, F., Guerrant, R., Holt, P. R., Quigley, E. M. M., Sartor, R. B., et al. (2013). An update on the use and investigation of probiotics in health and disease. Gut 62, 787–796. doi: 10.1136/gutjnl-2012-302504
Tojo, R., Suárez, A., Clemente, M. G., de los Reyes-Gavilán, C. G., Margolles, A., Gueimonde, M., et al. (2014). Intestinal microbiota in health and disease: role of bifidobacteria in gut homeostasis. World J. Gastroenterol. 20, 15163–15176. doi: 10.3748/wjg.v20.i,41.15163
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Wos-Oxley, M. L., Bleich, A., Oxley, A. P. A., Kahl, S., Janus, L. M., Smoczek, A., et al. (2012). Comparative evaluation of establishing a human gut microbial community within rodent models. Gut Microb. 3, 1–16. doi: 10.4161/gmic.19934
Zheng, B., van Bergenhenegouwen, J., van de Kant, H. J. G., Folkerts, G., Garssen, J., Vos, A. P., et al. (2016). Specific probiotic dietary supplementation leads to different effects during remission and relapse in murine chronic colitis. Benef. Microbes 7, 205–213. doi: 10.3920/bm2015.0037
Keywords: Bifidobacterium, exopolysaccharide, mucoid, ropy, immune modulation, DSS-colitis, mouse model
Citation: Hidalgo-Cantabrana C, Algieri F, Rodriguez-Nogales A, Vezza T, Martínez-Camblor P, Margolles A, Ruas-Madiedo P and Gálvez J (2016) Effect of a Ropy Exopolysaccharide-Producing Bifidobacterium animalis subsp. lactis Strain Orally Administered on DSS-Induced Colitis Mice Model. Front. Microbiol. 7:868. doi: 10.3389/fmicb.2016.00868
Received: 24 April 2016; Accepted: 23 May 2016;
Published: 09 June 2016.
Edited by:Marco Ventura, University of Parma, Italy
Reviewed by:Douwe Van Sinderen, University College Cork, Ireland
Chiara Ferrario, University of Parma, Italy
Copyright © 2016 Hidalgo-Cantabrana, Algieri, Rodriguez-Nogales, Vezza, Martínez-Camblor, Margolles, Ruas-Madiedo and Gálvez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Patricia Ruas-Madiedo, email@example.com


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This was taken in Norway at 7am on a May morning while sailing up Midfjorden bound for Adalsnes.
In the clear, calm and sunny conditions my early rise was rewarded by two hours of stunning scenery and views.
Canon 60D with 17-40 f4 lens and polarising filter, 1/1000 at f8, ISO 400.
Pic of the Week is in association with Eastwood Photographic Society — photgraphy for all.


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Romantic Getaway in Yosemite
October 19, 2012
You have to be there to feel the magnificence of Yosemite National Park! There is no better feeling than holding hands with the person you love while watching the angles of the mountains shine with a bronze-golden light from the setting sun. The deep valleys, massive rock structures, and the lakes, are breathtaking. Mother Nature's beauty can be felt at its fullest here.
There is something for everyone at the park! We spent 6 days there, last summer, and enjoyed every moment of it! There are amazing hikes around the East Entrance. One of my favorites is an easy 5-6 mile trek to the Devils Postpile and Rainbow falls. The Devils Postpile is hexagonal columns of cooled and shrunk volcanic lava and it is amazing!
The end of that trail took us to a pretty waterfall. The rainbow was nice but did not come up in my pictures because of the way the sun reflected :-( We were not super impressed by the falls, having been to the Niagara Fall region a couple of times! But it was pretty nonetheless!
Everything is so beautiful, that pictures cannot compare to actually being there to experience it. You can camp, hike, canoe and ski, depending on the weather! There is a beautiful scenic gondola ride up the mammoth mountain. The peak is at 11,053 feet and the view is gorgeous!
Out of everything, if I had to pick one favorite, serene, romantic landscape, it would be the lakes. We visited Lake Mono and Lake Tenaya. The still water and almost beach-like sandy areas next to the lakes make the place very picnic friendly. We had sandwiches, orange juice and grapes while throwing stones in water, trying to see whose stones went in furthest.
So if you love nature and a vast range of diverse rugged landscape and you and your sweetheart want to do something active, fun and romantic, this is the place to be in. Yosemite National Park will not disappoint you :-)
If you have your own stories and tips to share about travelling there, please feel free to comment below.
Thank you and Keep Smiling!
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Semmes Middle School has a strong belief in parental involvement. You are invited to participate in the many activities we have each year. We encourage you to volunteer and see what a wonderful school, faculty and staff we have. We have a Parent Resource Center with computers available for parent use during the school day. If you need assistance on the computer or do not have a computer at home, you are welcome to come in any time.
Parent involvement is one of the key factors for student success. Your participation will build a strong and productive partnership between family and school.
We also have brochures on important topics as well as videos you may check out or view here. Our Parent Program has an "Open Door" policy. If we can help you in any way, just let us know.
The Mobile County Public School System is committed to ensuring that the content on this website is accessible to everyone, including those with disabilities and users of assistive technology. For more information, see our Section 504 Grievance Procedures.
Copyright © 2019 Semmes Middle


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Romantic Hotel Puglia
Romantic Hotel in Puglia – Looking to surprise your loved one with a romantic hotel get-away or an unforgettable romantic experience in Puglia? We have the perfect honeymoon hotels and romantic places lined up for newly weds. Puglia's best romantic resorts that are ideal for celebrating anniversaries or just simply enjoying intimate moments with your partner.
WorldTop7 has recommended romantic small hotels in Puglia with the most romantic settings. Have memorable candle-light dinners and sultry intimate moments with rose petal sprinkled pillows at any of our wide selection of choices of the Puglia's most romantic hotels.
Choose among one of our many suggestions for Puglia's best and most romantic hotels.
Romantic Hotels Puglia
Masseria Torre Coccaro Puglia Italy
Masseria Torre Coccaro Masseria Torre Coccaro is a luxurious authentic regional and local property which is a favourite golfers' choice,
Masseria San Domenico Hotel Fasano Puglia Italy
Masseria San Domenico Hotel Fasano Masseria San Domenico is the 15th century watch tower restored and turned into a luxury
Masseria Salinola Puglia Italy
Masseria Salinola Masseria Salinola is a charming and romantic getaway set in an 16th century historic limestone structure that used
Masseria Lamacoppa Puglia Italy
Masseria Lamacoppa Masseria Lamacoppa is an elegant countryside Manor surrounded by 70 hectares of lush lemon and olive trees. The
La Sommita Relais & SPA Puglia Italy
La Sommita Relais & SPA La Sommita Relais & SPA is predominantly booked and visited by romantic couples, which perfectly
La Chiusa delle More Peschici Puglia Italy
La Chiusa delle More Peschici La Chiusa delle More Peschici is located in the region of Peschici; giving both the
Masseria Torricella Puglia Italy
Masseria Torricella Masseria Torricella is a wonderful countryside escape, especially for families with toddlers since guests three years of age
Il Convento di Santa Maria Puglia Italy
Il Convento di Santa Maria A crumbling local convent until its restoration in 2005 by Alastair McAlpine and his wife
Barsento Masseria Fortificata Puglia Italy
Barsento Masseria Fortificata Barsento Masseria Fortificata in the quaint town of Putignano in the Puglia Region is a beautiful and


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TECHNO FOAM MFX 5.1 "Aluminum"
Foam maker cannon./cannon for foam production
Our Techno line assures high-engineering products with high plug-and-play performances, groundbreaking design and "handicraft" Manufacturing.
This connotation gives uniqueness to your working equipment, making them rough-looking but extremely strong.
The entire products range spirit is enclosed by this acronym.
We was able to make a single machine that will allow you to get more effects simultaneously
Those who decide to invest in Tech Foam MFX 5.1 will buy a machine that is worth almost 2!
In fact with the substitution of the "mfx head" with an "accessory" the machine will automatically turn into a "shoots foam" or "shoots confetti" cannon with a saving of almost 50%.
Our goal was to build an important volume machine that gives excellent results without compromising the weight. Our choice to use an aluminum structure was successful!
The finish ennobled the super light alloy, forming a single body between metal, bottom and poxy coating previously fixed in the oven making it indestructible and "unscratchable" in the unmistakable Starlights original color.
Foam maker cannon foam parties, indestructible, design for large environments ( capable to shoot foam over 25 meters/ 82 ft.), futuristic design, exellent ratio between production and range.
Adjustable inclination with knobs and tightening eased by a lever safety device.
The axis rotation is comfortable and smooth thanks to the use of 6 bearings located at the base ( supplied with the support of 4 removable feet).
For professional use only.
We highly recommend this accessory because it's not only a simple LED lighting system is a truly scenography which enriches your machine making it "monstrously" eye-catching, attractive and catalyst for the public. Your customers will be pleased to host these special effect machines, they will make the surroundings even more modern and hitech just with their presence. (Often supporting scenography and animation too).
The function can be disabled if wanted.
Tech Foam MFX 5.1 can be customized on demand:
– customized colors or airbrushed.
– with the client logo and personalized colors.
* these version may require more time for the delivery than the standard versions and a possible additional charge.
(Please make your purchase carefully).
Very high quality components and Productions rigorously made in Italy.
Cannon, pump and tubes.
Removable rocking support on 4 aluminum feet.
Ready to use solution tank.
For an Optimal Performance and for users safety (since the "total body" contact with the public) we highly recommend to only use guaranteed and certified products made in Italy like our Fluids Foam
Power AC 240V 50-60 Hz
Consumption …
Direct Control….
IP Protection IP56
Shooting Distance up to 25 m
Inox Connection
Fluid Consumption
Size Base..


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Breaking down BaliPure's win over PetroGazz
by rick olivares
The young BaliPure National University Water Defenders outplayed the PetroGazz Angles in a five-set win (22-25, 25-23, 28-26, 22-25, 15-12) at the Filoil Flying V Centre.
It was obvious that outside BaliPure NU's imports in Janisa Johnson and Alexis Mathews (and veterans Rose Doria and Audrey Paran) that high-level experience was still lacking for some players especially in the first set that resulted in a loss.
And it was on display for much of the first set before the team settled down.
What was noteworthy?
A one-two combination to watch in Cagande-Johnson
Watching the Joyme Cagande and Janisa Johnson connection was like watching Gary Payton throw up the ball to former Seattle Supersonics teammate Shawn Kemp. I remember how one time, Kemp complained at the level of difficulty of some of Payton's lobs. Ever the loquacious one, Payton replied that he's catching the lobs anyway for a dunk.
It's the same with Cagande and Johnson as the former, who is only an incoming freshman at NU, tries to find consistency in her game. But what was most impressive was how Johnson adjusts to the set in mid-air for a variety of shots that were truly impressive. She reads the blocks well and is able to either break the wall, get a check ball with a softer hit, smash one down the line, or even drop on behind the defense.
Johnson top scored with 31 points while Cagande finished with 32 excellent sets; 14 more than veteran setter Chie Saet who won a championship with La Salle during her college days.
In the first set, Cagande had difficulty finding her teammates not only because of first-ball problems but also in mixing up her sets. Come the second set, she finally utilized Alexis Mathews better providing a wing and middle attack. When Roselyn Doria came in for a struggling Ivy Lacsina, the middle attack and defense continued to be solid even when Mathews rotated out.
But Johnson…. Despite her standing at about 5'8" what an athlete! I can only imagine the damage she and former Pocari Sweat import Crystal Rivers inflicted on foes on their way to the recent French Cup that they won.
Robles is a rookie?
Speaking of size… another who impressed was open spiker Princess Robles who is a little shorter than Johnson. For those who have followed the NU high school team, you know what you're getting with her high-flying game. Some might say, beginner's luck in the opening day loss to Philippine Navy-Iriga, when Robles scored 15 points. Against PetroGazz, she once more displayed no nerves (except for a poor third set where she struggled with her shots and placement) en route to 17 points.
Said the Iloilo native, Robles at the post-game dinner at Mann Hann: "Kung ano ibigay ni coach (Babes Castillo), ibibigay ko lahat. Basta yun ang mindset ko – give it my all every time. Natural lang sa akin ibigay yung best kasi yun ang natutunan ko."
That superb floor defense by Jennifer Nierva
While Johnson, Mathews, and Robles were huge for BaliPure NU, the Purest Water Defenders got things going when libero Jennifer Nierva began to play better in the late first set. Nierva had 19 excellent digs and 25 excellent receptions (to lead all players from both sides) was inspirational.
This young BaliPure NU team, even at 1-1, has greatly impressed. Volleyball fans should watch for this team.


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GMP News No. 800
GMP News
26 September 2006
Initiative by Members of EU
Parliament Calls for
Therefore, the declaration calls for inspections of all manufacturers of active pharmaceutical ingredients by EU authorities in order to obtain a GMP certificate - no matter if the manufacturing sites are located within or outside the Community. Besides, the group proposes the introduction of traceability of the active ingredients. This should be reached by discouraging the relabelling of active ingredients.
Till 4 December 2006, comments are compiled and evaluated. If the
requirements proposed in the declaration are implemented, the consequences
will be far-reaching. The present regulation requiring the manufacturer
of the medicinal product to verify the GMP compliance of an active ingredient (usually
by means of an audit) will be enhanced by supervision on the part of the
The GMP-compliant manufacture of active pharmaceutical ingredients is a
prerequisite for their use in medicinal products in any case (even if this
initiative will not be passed in the European Parliament). For this
reason, ECA supports and/or co-sponsors qualification seminars and
APIC/CEFIC is organising:
APIC/CEFIC is organising (co-sponsored by ECA):


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By Anonymous, Trending Writer
As it has become the norm for business to bow to political correctness and societal agendas that demand unquestioning inclusion, it is refreshing to know there are companies who are still willing to stand by their conservative beliefs.
The 1st Amendment gives citizens freedom of speech, press, religion and assembly, but it commonly gets thrown up as a shield from words and ideas that a person does not agree with or simply not like to hear. The people who demonize companies with certain conservative values and push to have them change their belief systems are perpetrating a hypocrisy in itself. These individuals push modern agendas onto companies and are infuriated when met with refusal. However if an outside force tells them how to act, they would be just as stubborn.
Chick-Fil-A is one of the most beloved fast food chains, however they are not open on Sunday due to CEO Dan Cathy's religion. In addition, in 2012, remarks surfaced about Mr. Cathy being "very much" against gay marriage, and subsequent protests and boycotting occurred. He went on to explain, "We know that it might not be popular with everyone, but thank the Lord, we live in a country where we can share our values and operate on biblical principles". They have no issue hiring or serving LGBT customers, but it remains hypocritical for one side to try and force another to change their religions views, while taking no accountability for the seriousness of their request.
John H. Tyson, the current chairman of Tyson Food Corporation and a born-again Christian, also believes his values should not be pushed aside when he walks through the front door. If he were to quell his beliefs, it would set a precedent for his workers that they should repress their religion at work as well. By standing by his beliefs, Mr. Tyson advocates for an atmosphere that is described as "giving people permission to live their faith seven days a week." They are not a company who is bigoted towards a certain faith, rather just led by a boss who believe his religious values should not be stifled.
If any company failed to accommodate a lower level employee's religious choices, they would be brought to court under Title VII of the Civil Rights Act, so why should the CEO should be held to different standards?
A version of this article appeared in the Tuesday, March 21st print edition.
Contact the writer through the editor at


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Raelynn Alker
Carrick High Class of 2014
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Why ILS?
We're different than other 3PL providers.
In fact, Cardinal Health Integrated Logistics Services is solely dedicated to providing supply chain solutions to the healthcare industry.
Our expertise in healthcare can help you:
• Drive supply chain efficiency
• Reduce your risk and complexity
• Deliver quality
• Improve your bottom line
Your products, our promise: This is the network built just for you.
We are the complete 3PL solution for healthcare, combining a nationwide network of distribution centers, transportation logistics and regulatory expertise. We are a complete source for medical devices and supplies to every point of care—including hospitals, surgery centers, offices and even patients' homes. We collaborate closely with every client to customize the right solution, from enhancing transportation to offering a fully outsourced, turnkey supply chain. And through it all, our proprietary quality and regulatory systems help you remain compliant. So you can keep your products moving—no matter what. Because we are your healthcare 3PL.
Case Study
Healthcare has never been more challenging or essential than it is today. There's greater pressure on margins, new regulations and more complexity than ever before


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The NWIRC is a strategic partner for innovators – entrepreneurs and inventors – to help bring their ideas to life. We assist by building a customized strategy for success through an expert business toolkit, access to an exclusive Just-In-Time Mentorship™ program and an expansive community of local resources.
Check out the new Innovation Lab!
Whether you are an inventor or entrepreneur, NWIRC helps you evaluate the idea, identify your needs and create a customized road map for success.
Every business needs a roadmap to help navigate the pitfalls. Using years of entrepreneurial expertise, we build customized strategies to meet the individual needs of your business.
Every business needs a toolkit to keep the business moving forward. We have an experienced toolkit full of business expertise and know-how to keep you on track.
Diane and the NWIRC network helped me focus my business plan and financials into a format that I could present to potential investors. With renewed focus and a thought out presentation, I held the confidence and information that lead me to financial investors. While my business grew, NWIRC continued to provide advice on how to find an accountant or how to start another business! It has been a privilege to be part of NWIRC's enterprise program and I plan to continue utilizing the resources NWIRC provides as my business demands.
~Leah Paisano
We are here to help!
Contact us today to find out more about our programs or to volunteer as part of our Entrepreneurial Innovation Community.
Recent Posts
Got Ideas? Prototyping with everyday objects.
Over the last few months I have been writing articles for this column after talking to experts about issues that are important to "inventors". However, this series is not just about getting a sneak peekinto the invention process; I really hope it will spark the...read more
Artisan dog products
By: Lara Merriam-Smith Name of inventor/product developer: Kellie and Katya Szirom Product: Artisan dog products. Hometown: Bellingham How to buy: Bellingham Whole Foods Market, Hohl Feed & Seed, Whatcom Farmers Co-ops, our website....read more
This monthly series is designed to introduce you to people right here in Whatcom County that invented products that solve everyday problems. Invention is a process accessible to anyone with an idea and we need people with great ideas to solve big and small problems....read more


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The Opioid Epidemic in 6 Charts Designed To Deceive You
I do not know Dr. Andrew Kolodny, personally, and, aside from one brief phone call last year, I have had no contact with him. Therefore I cannot know his motivation for becoming a driving force behind "opioid reform"— a concept which would border on hysterically funny if not for the tragedy that it is causing in this country.
I'm always skeptical about these "Everyone on the bandwagon" crusades and I know people who were denied the pain medication they needed due to it. My guess is that Dr. Kolodny qualifies as a Social Justice Warrior. I HATE SJW's!


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IT Salary Survey - Be-IT's fourth annual survey of IT and digital pay
Posted on 6th February 2018
This is the fourth year we have produced this survey and we know, from the huge spike in our website traffic, that it's avidly read by IT professionals in Scotland and beyond. Carried out by an independent third party, one of the key features of Be-IT's salary survey is that we look at rates of pay at different levels of experience. As always with studies of this kind, it's important to remember that the market does vary and skills appreciate according to supply and demand. Other factors affecting salaries are location, company and individual performance and experience, technological change and people's ability to keep up with new trends.
You can download the salary survey here.
Posted in Recruitment News, Salary Survey
.. Back to Blog


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Caotica is internet marketing and communication providing firm, responsible for design, web development, internet marketing and communication strategies to deliver effective, creative and user-friendly solutions across a wide range of world wide web.
A story of Caotica began in the early days of year 2004. In the beginning there was one person (founder Martin Palmet) doing things and sharing love for internet on a blog. The more people learned about what he could do, the more people asked him to make things for them.
Over the years it was fun and educating (still is), but mostly done as a part job, and there is only 24 hours in one day. It was only a matter of time before Caotica was born – in the end of 2008.
Web development and marketing
Today, Caotica's core business is strictly internet marketing and web development. Customers are mostly smaller and medium size companies. Caotica's mission is to help it's clients to be aware of all the wonders in the world of internet and help them to achieve it's goals through this medium.
It is absolutely correct to say that our work is our passion and we pride ourselves in our ability to deliver professional projects on time and beyond all expectations of quality and customer service.
Approachable, professional and dedicated
Caotica doesn't do mediocre and believe the level of service and care you receive should be as exceptional as the work we produce. We're down-to-earth, approachable, professional and dedicated to exceeding your expectations.
Achieving your business goals is our most important goal. With our flexible approach, creative talent and contagious passion for what we do, you'll love working with us. We will achieve great things with your project.
Martin Palmet – founder and project manager
Meet Martin Palmet, the founder and project manager for your next inspiring goal. Since founding the small web agency, Martin has been part of smaller and bigger web projects for local and international companies. He has also been hired to work in international companies as Development Manager as well as Web Manager.


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Eko by Pedini
Modern kitchen design is what European cabinets are known for. Pedini developed Eko to allow more people to be able to own an advanced, modern design, eco-friendly contemporary European kitchen, at an affordable price.
About Pedini
Pedini Have been manufacturing kitchens for over fifty years. Their journey has taken them from handcrafted kitchens to the state of the art in technology and design and now one of the leading kitchen manufacturers in the world.


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Know more about this business than we do? Cool! Please submit any corrections or missing details you may have.Help us make it right
Be the first NOLA.com user to add photos or videos of Ino Therapeutics
Since 2009, Ino Therapeutics has been providing Industrial Gases from Port Allen. Ino Therapeutics has estimated annual revenues of $240,000.00 and also employs an estimated 3 employees.
Ino Therapeutics provides services in the Port Allen, LA area. Call us today for our latest specials. Powered by Blabble Networks.


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FACT Report: October 7, 2015
Squeezing Conservatives Out of the Republican Party (October 7, 2015)
Recently the Tennessee Education Association sent a letter to some of Tennessee's Republican legislators touting how it had encouraged its members to vote in Tennessee's open Republican primaries, noting that member turnout in "key races" had "increased by more than 300%."
No doubt the TEA wants its members voting in Republican primaries, because there are so few Democrats even left in the legislature to vote for. But Republicans need to remember that the TEA is an affiliate of the National Education Association that routinely pushes for very liberal social policies.
So, rather than seeing this as a welcome sign, the Tennessee Republican Party better realize that leaving the primaries open is an invitation for liberals to take over the Party. And if the Party won't close the primaries, then conservatives may find themselves without influence in either party.
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