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20 Avenue de La Fosse Des Pressoirs Paris 77703 France
Location Designed in the style of a French country manor, this hotel is located at the heart of Marne-la-Vallée just 3 km from Disneyland. A shuttle bus covers the approximatelyten minutes to the world-famous leisure park. Situated around 37 km from Paris, this hotel lies within a reasonable distance of many other attractions such as golf courses, palaces and monuments. In addition, shopping facilities can be reached in around 5 minutes on foot. It is equally suitable as a stopover point for ho Facilities This hotel was built in 2003. The establishment offers travellers a total of 396 rooms. A lobby and a reception are available to guests. Individual storeys are accessible via the lift or stairs. Services such as a baggage storage service, a safe and a currency exchange service ensure a comfortable stay. Wireless internet access is available to travellers in the public areas. Various gastronomic options are available, including a restaurant, a dining area, a breakfast room, a café and a bar. Shopping facilities are also available. The grounds of the accommodation feature a playground and an attractive garden. Additional facilities at the hotel include a playroom. Those arriving in their own vehicles can leave them in the car park of the establishment. Additional services include a babysitting service, room service and a laundry service. In addition, a shuttle service is offered. Rooms Each of the rooms is appointed with air conditioning, central heating and a bathroom. Most rooms also feature a terrace for relaxation. A double bed ensures a good night's sleep. Cots and extra beds are available. A safe and a minibar are also available. Additional features include a fridge and a tea/coffee station. An ironing set is also available for guests' convenience. Little extras, including internet access, a telephone, a TV, a radio and WiFi, contribute to a great stay. The bathrooms are fitted with a shower and a bathtub. A hairdryer can also be found in each of the bathrooms. The accommodation offers family rooms and non-smoking rooms. Sports/Entertainment The hotel features various options for cooling off on hot days, including a pool, a children's pool and an indoor pool. Sun loungers and parasols are ideal for relaxation. The poolside bar serves a selection of refreshing drinks. Those wishing to enjoy sports whilst on holiday can enjoy golf on-site. The establishment offers indoor sporting activities such as a gym, or for a fee, billiards. The accommodation offers various wellness options, including a sauna and a steam bath, or for a fee, a spa, a beauty salon and massage treatments. An entertainment programme is offered for children. Meals Catering options available at the hotel include bed and breakfast and half board. Guests can choose to book options such as breakfast or dinner. Payment The following credit cards are accepted at the establishment: American Express, VISA, Diners Club and MasterCard.


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subset: commoncrawl


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Edible Birdnest farming can be considered an ideal, most exciting and a very lucrative business. This venture is suitable for those who live in parts of Cambodia, Southern Thailand, Burma, Malaysia, Vietnam, the Philippine and Indonesia. This blog is dedicated to my findings, crazy ideas, encounters with newbies, comments from friends, local news, pictures relevant to Birdnest plus my personal experiences and knowledge gained in swiftlet farming.
Sunday, May 12, 2013
A Visit To A BH In Morib, Selangor !!!
Two days ago received an SOS call from a lady who owned a BH with one solitary nest.
According to her after more than 1.5 years her house seems to attract no new birds inside.
I briefly to her what she can do and let me have a good look at it before I compile the list of things she should do.
She quickly chose the date and I complied to her request.
We met somewhere near Morib mosque and I followed her can to where ever the BH was.
After about 10 minutes we were at the location.
I took a brief look around the BH and noticed that the two story building was a bit too low sandwiched in between those palm oil trees.
Inside my heart I told myself that no matter what the owner do inside the birds will not be able to locate the BH once those palm oil trees grow taller than the BH.
Those leaves and branches will cover the whole place and it will be a very tough to lure birds into it.
This is why if you wanted to build a BH inside a palm oil plantation the design must be proper.
The most important is the height and more important is to have more open space between those palm oil trees and the BH walls.
I went inside and there was nothing that can make me surprised. Every thing that you can think of why those birds refuse to stays were shown all over the place.
1) The monkey house area was a disaster. The person who was given the task to erect this BH seems to have very little knowledge about those delicate birds.
2) The main entrance hole (window of 2' x 2') was facing an unfriendly neighbor and the owner was given no choice but to open a new hole on the rooftop of the monkey house.
3) Using the monkey house extension to open the rooftop is not a wise idea. Monkey house should be equip with side entrance and not top entrance.
4) The owner twister the direction of those tweeters at the 2'x2' window backwards. This was more like pulling the birds from the inside out. Ah ye ye ye .....
5) The windows/doors connecting the monkey house roving areas into the nesting rooms (one upstairs and one n the ground floor) were not properly thought of.
6) Both the top and ground floor nesting rooms were too bright. I keep my point that the darkness must be a 2 feet gap with no vision of your friend's face.
7) I was not very happy with the number of internal sound tweeters inside this BH. I told the owner to increase by 3.2 folds. Now only about 15 per floors and what I wanted was 50 a floor.
8) There was no external sound tweeters inside. I push my idea on the use of kite tweeters and where they should be located.
9) The number of fake nests were also very little. Since they might be tight I recommended the use of styrofoam which will not cost more than 20 sen a piece. Put about 50 pieces per floor.
10) If the can I wanted them to upgrade their external and internal sounds. Tested my marvelous cloud and during my testing there were birds inside.
11) I also wanted them to apply those pheromone. Spray a bit on those fake nests.
Wrote a brief report for them to follow my recommendations and I told them that I will be ready to drop by if they need my final advise before putting the BH back to order.
I feel grateful to have help this retired couple and I hope I can be of help to others too.
If you are facing the same situation please call 017 7551318 for any help.


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- Written by Aron
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There are just some things in life that I never get tired of. Like coffee!
Coffeehouses have been our second home the last 3 years and things have not changed after moving on the other side of the planet. Some things don't change I guess. We have been very fortunate to work for one awesome coffeehouse in Iceland called Súfistinn. Dagbjört took pictures for them as well as some graphic design and I just finished editing two videos I shot for them in August. We spent a lot of our time working in their shops and I must say when Christmas is around the corner I really miss that place. Thank God for Starbucks and their "Christsmas" butter cookie latté! It is doing a good job filling in for the Christmas drinks that Súfistinn always have this time of year.
I'm quite happy with the videos and I haven't yet decided wich one I like more. It's always hard to capture the atmosphere in places like this but I did my best when I was filming and had fun while at it. Take a look at the outcome.
Writer & co-founder of Just Us. A young videographer and web-designer that loves to travel, spend time with his wife and go on adventures around the world.


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Running a business is not easy at the best of times, but when you have a full time job as well as an online business, it can be extremely difficult. This can be made even more difficult if you also have a family. Difficult that is, if you are not well organized and make the most of every minute you have, you'll miss out on opportunities that could change your life.
This article will show you some great ways to stay on top of your commitments and become better organized in all areas of your life. [Read more...]


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In DANICO we process a wide range of metals and other materials on our hi-tech equipment and flexible manufacturing lines.
With our long experience and extensive set of equipment, we can help you with any order of simple or complex parts, at the prices, the qualities and with the delivery times you require.
We offer flexible quantities as well as the right level of sub-assemblies according to your needs.
DANICO takes care of packaging, logistics and import/export procedures.
Our special services include helping you with prototyping and technical drawings
- DanicoINTRODownload
Danico manufacturing
Our extensive set of equipment serves a wide range of purposes, from manual and CNC machining, cog-wheel production, welding, surface-treatment, heat-treatment - everything needed for completing your order.
We work with stainless steels, mild steels, cast iron, aluminum, copper alloys and plastics.
In DANICO we do simple or complex parts in small or large quantities. Our special competence is combining of welding and machining when manufacturing complex parts. One of our special services is prototyping - we help you with the first samples in very short delivery times.
At DANICO we offer any level of sub-assemblies, all the way to complete turn-key assemblies.
We help you handle packaging, logistics, import/export, installations and after marked service. We are also developing computer models, prototyping and technical drawings according to your requirements.
Cases and testimonials
A Danish manufacturer contacted us for an offer for spare-parts and sub-assemblies. Now we are working together with them supplying their casings, frames and shafts. The final assembly still take place in Denmark, but we could offer better quality and prices than their current suppliers.
In the spring of 2013 we bought the full production facilities and customer database from the Danish cog-wheel manufacture Asøma Tandhjul. Since then we have developed many new customers, including large Ukranian companies for whom we are now the preffered supplier
When needed we helped our customers with urgent deliveries directly to their customers. In one specific case, we helped to produce and deliver a new spare-part within 24-hours, so that our customer's client could get their machine started again.


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NewChurch Network www.newchurchnet.com
Assessment Centers:
In order to coordinate our Assessment & Discovery Labs we partner with Dr. George Johnson, of the Christian Evangelistic Association, in Seattle, WA. George facilitat.es the Assessments we conduct in Midland TX, Little Rock AR, and McKinney TX. firstname.lastname@example.com.
We also partner with Dr. Matthew LaGrange, of His-Story Coaching & Counseling, Grapevine TX. Matthew serves as the psychologist in our Assessment processes, having the unique advantage of having gone through the process in 2006, and having served as a campus pastor at a new site while in local ministry. email@example.com.
Regional Networks and Partners:
- Christian Evangelistic Association – Kansas.
Brad Fogo, Chairman. firstname.lastname@example.com
- Christian Church Starters – New Mexico.
Errol Stepp, Director. firstname.lastname@example.com
- Church Planters of the Rockies – Colorado.
Graydon Jessup, Director. firstname.lastname@example.com
- Arkansas Church Planting Network – Arkansas.
Lynn Cook, Chairman. email@example.com
- Love Oklahoma! – Oklahoma (a Nexus network).
Doyle Kinney, Director. email@example.com
- The 1:8 Network – Nebraska (a Nexus network).
Rowlie Hutton – Director. email@example.com
Special Partnership:
Nexus enjoys a very close relationship with Golf Course Road Church of Christ, in Midland TX. Phil Claycomb, the Nexus Director, serves on GCR's North American Missions Committee, and is the Coordinator of GCR's church-planting mission. In functional terms, Nexus and GCR collaborate so tightly that it is sometimes difficult to determine exactly where Nexus ends and GCR begins. For more information on the Golf Course Road Church of Christ church-planting ministry click here.


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Company History
The Art of Hijab, Kohl Black, and the Right Way to Pray
[March 2016]
workshop at the LaGuardia Performing Arts Center Rough Draft Festival
[March 2015]
reading at the LaGuardia Performing Arts Center Rough Draft Festival
Anna Asli Suriyah (I Come From Syria)
[August 2014]
in residence at Beit Jeddo, Bay Ridge, Brooklyn
[October 2012 and March 2013]
in residence at Brooklyn Navy Center at BLDG 92
Alice, or the Scottish Gravediggers
[October-November 2011]
in residence at The Old Stone House, Brooklyn
Workshop July 2011 The Old Stone House
Open rehearsal February 2011 The Old Stone House
Reading October 2010 A.R.T./New York
Hot Dish: Clubbed Thumb Summerworks 2010 Festival
[June 2 2010]
The Ohio Theater, New York
Piece by Avi Glickstein, starring Avi Glickstein, Lindsay Torrey and Stacey Cooper McMath
A Thousand Thousand Slimy Things
[April-May 2010]
Waterfront Museum and Barge, Red Hook, Brooklyn
shown at Martin E. Segal Theatre at the CUNY Graduate Center, April 2009.
In residence Mabou Mines, March 2009
Bushwick Starr, August 2007
[November 2009]
Access Theater Gallery
[October 2007]
Workshopped at the Bushwick Starr
Life? Or Theater? The Charlotte Salomon Project
[Summer/Fall 2006 in NY, currently available for touring]
University of Michigan, Ann Arbor, April 2007.
In residence at Brooklyn Fire Proof 2006
Mabou Mines Suite fall 2006
Voice and Vision ENVISION Retreat, Bard College, August 2005
HERE WE GO! - 365days/365plays
[January 2007]
January 2-6 in various Brooklyn locations, January 7th at The Public Theater
Mother Russia: [A Lonely Person is at Home Everywhere]
[October 2005]
Ladies' Auxiliary Telephone Bee
[Fall 2004 and ongoing]
The Tank: October 2004, February 2005, March 2005
Moral Values Festival, The Brick, June 2005
[August 2004 and ongoing]
Downstairs at the Flea, August 2004
The Brick, October 2004
Galapagos, December 2004
The Tank, February 2005
Careful of Eights
[Summer 2003]
Dixon Place, HERE American Living Room
American Theater of Actors
The Museum Play and The Telephone Call
[Winter 2002]
Present Company Theatorium
[Summer 2002]
Abingdon Theater
Desmond or Abraham and Frances
[Summer 2001]
Paradise Theater, New York International Fringe Festival


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Take a Virtual Tour
We are located at:The Gardens Rehab & Care Center
3131 Western Ave.
Kingman, Arizona 86401
(928) 718-0718
Become an Employee:
Services & Amenities
The Gardens Rehab and Care Center strives to make our residents feel comfortable in warm and friendly surroundings. We offer a full array of services, including:
We understand that each of our residents has varied, individual rehabilitation needs, and we make it a priority to help them achieve their long- and short-term goals.
We offer physical, occupational and speech therapy. The trained members of our therapy team work with each resident's physician to develop an individualized, comprehensive care plan designed to reach specific treatment goals, and we've also developed a Nursing Restorative program for our long-term care residents, to help them maintain the highest possible level of mobility.
Our licensed nursing staff provides round-the-clock care. Charge Nurses are available on every shift, to deal with any questions or concerns a resident or family member might have.
Our supportive Services include
- Responsive staff 24 hours a day
- Physical, Occupational & Speech Therapy
- Assistance with personal care and hygiene
- Laundry and linen service
- Restaurant style dining
- Housekeeping
- Salon and Barber Shop
- Secured outdoor patios
- Life enrichment activities and outings
- Semi-Private rooms with ADA approved bathrooms with showers
Beauty Salon/Barber Shop
Because we know it's important to our residents that they look and feel their best, we provide an on-site beauty salon and barber shop.


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Can you believe it? 2007 is about to be a distant memory and 2008 will be long gone before we know it. Why is that? When your younger time moves slowly and everyone tells you to enjoy it. Now that I'm older, there isn't a second taken for granted. What I would give to be 'bored' for an afternoon. 2007 will be a year our family will never forget and here's why:
Eric & I tried and got pregnant with our first child at the end of January
We went on a wonderful vacation to Florida & North Carolina in May
June, we returned home to find out that our unborn baby would be born with a birth defect, Gastroschisis
June & July we shopped, decorated, educated ourselves, met with doctors & specialists & waited
August 14th, after a routine doctors appointment we were sent to the hospital for bedrest and observation
August 15th, Eric & I welcomed our first born, Caleigh Amelia Gray at an early 28 weeks, 11 weeks to soon
August seems to be a blur.......surgeries, specialists, pumping breastmilk, trying to rationalize how unfair this all was
September came with another surgery, the meaning of patience is finally realized
November, our original due date came and went; we learned about PVL and how uncertain Caleigh's future could be, we educated ourselves
December, another surgery and now we wait.........
2007 was the most invigorating, exciting, scary, sad, emotional, physical & truly the best year of our lives, not to be forgotten.
We welcome 2008 with hope, new beginnings & eventually Caleigh's long awaited homecoming


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…but now my yard has 2″ of snow over it so all hints of green are currently hidden.
Lucky for me, I found myself able to sit down last night and make something springy and colorful without a single interruption.
No, really. This is monumental. Even the dog remained at my feet without so much as a whine.
I may have to wait for my spring garden, but at least there's this!
And yes, it's in the shop for now.


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POF Radio
I'm on air! Be sure to listen to Parade of Flesh radio show tomorrow, and every Thursday at 4PM CT on indie-verse.com or KLLI 100.3 HD3FM, or CBS Radio App on iPhone. Many ways to listen. Playlist will go up towards end of the broadcast.
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Barkev's Princess Cut Diamond Pave Tension Twist Half Bezel Set Engagement Ring
Barkev's Princess Cut Diamond Tension Twist Half Bezel Set Pave Engagement Ring Featuring 0.33 Carats Total Weight Round Cut Diamonds
- Delivery takes 3 weeks for Barkev's to create and will ship immediately after completion. Rush delivery available depending on style and upon request
- Center diamond & wedding band sold separately
- Ring can be made to accommodate any size or shape center diamond. Shown with a 1.00 carat princess cut diamond (approx 5.5mm)
- Available in 14k or 18K white gold. Also available in platinum
- Includes ring box
- Includes jewelry appraisal
- Ships fully insured to point of delivery
- All orders placed out of NY state (ordered by an out of state resident) are tax free
All rings are fully customizable by Barkev's should you prefer slight or major changes to the style you see. Alteration possibilities include alternate color gemstones, styling or metal preference
Stunning and unique this diamond engagement ring exudes confidence for the woman who wears it. Featuring a low profile cathedral shank that rises and twists to capture the channel set princess cut diamond center on point. Pave set diamonds artfully adorn the shoulders for a dramatic look of vogue


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Thru today 1/12 only, Groupon is offering 20% off, up to $50. Just go HERE and register and then enter promo code GET20 at checkout to get the savings. You can use the code up to (3) times, but you have to do different transactions.
Note: Code GET20, is valid up to $50 discount thru 1/12. Not valid on third-party-site transactions. Other exclusions may apply, see site for details. Max value $50.


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An important challenge in microbial ecology is developing methods that simultaneously examine the physiology of organisms at the molecular level and their ecosystem level interactions in complex natural systems. We integrated extensive proteomic, geochemical, and biological information from 28 microbial communities collected from an acid mine drainage environment and representing a range of biofilm development stages and geochemical conditions to evaluate how the physiologies of the dominant and less abundant organisms change along environmental gradients. The initial colonist dominates across all environments, but its proteome changes between two stable states as communities diversify, implying that interspecies interactions affect this organism's metabolism. Its overall physiology is robust to abiotic environmental factors, but strong correlations exist between these factors and certain subsets of proteins, possibly accounting for its wide environmental distribution. Lower abundance populations are patchier in their distribution, and proteomic data indicate that their environmental niches may be constrained by specific sets of abiotic environmental factors. This research establishes an effective strategy to investigate ecological relationships between microbial physiology and the environment for whole communities in situ.
Visual Overview
A fundamental question in microbial ecology addresses how organisms regulate their metabolic activities within natural communities as environmental constraints and population structures change. Recent advances in molecular biology have allowed for investigation into the physiology of organisms within natural settings, opening the door to understanding microbial metabolic responses in situ. Here, we have examined how a diverse set of organisms from microbial biofilms alters their protein complements as environmental parameters change and as ecological succession occurs. We find that, when growing in newly formed biofilms, the dominant organism within these communities exhibits a metabolism focused on rapid growth, protein synthesis, and stress defense. As community succession proceeds and secondary colonizers populate maturing biofilms, this organism's metabolism switches to one focused on synthesizing many essential cellular components, including amino acids, DNA, and carbohydrates. We also find that the metabolism of this organism is not strongly influenced by external environmental factors over the range of conditions studied. In addition, the protein complements of secondary colonizers seem to be highly responsive to changes in specific environmental parameters (e.g. pH, conductivity, temperature), which may limit their distribution across this environment. These findings provide insight into which of these environmental factors may drive community assembly in a natural microbial assemblage, and, in turn, may influence the metabolism of individual populations.
Community proteomics applied to natural microbial biofilms resolves how the physiology of different populations from a model ecosystem change with measured environmental factors in situ.
The initial colonists, Leptospirillum Group II bacteria, persist throughout ecological succession and dominate all communities, a pattern that resembles community assembly patterns in some macroecological systems.
Interspecies interactions, and not abiotic environmental factors, demonstrate the strongest correlation to physiological changes of Leptospirillum Group II.
Environmental niches of subdominant populations seem to be determined by combinations of specific sets of abiotic environmental factors.
Advances in cultivation‐independent '‐omics' techniques are beginning to allow for the study of microbial populations in natural environments at both a functional biomolecular level and a whole‐community level (proposed in Prosser et al, 2007; Little et al, 2008). The combination of environmental metagenomic and proteomic approaches opens the possibility for exploration of basic ecological rules underlying the functioning of microorganisms and the communities they form in nature (proposed by Warnecke and Hugenholtz, 2007; Raes and Bork, 2008). Recent metagenomic studies have revealed the phylogenetic diversity and functional capacity of microbial systems (Tyson et al, 2004; Tringe et al, 2005; DeLong et al, 2006; Rusch et al, 2007; Kunin et al, 2008), and community proteomics can enable the analysis of physiological characteristics in situ (Lacerda et al, 2007; Delmotte et al, 2009; VerBerkmoes et al, 2009). Although this work has greatly advanced our knowledge of natural microbial communities, comprehensive analyses of these systems are often limited by their inherent complexity. To circumvent this issue, one option is to study model communities of reduced complexity, such as biofilm communities found growing in acid mine drainage (AMD) environments (Denef et al, 2010b). Here, we integrated community proteomic and environmental data (physical, chemical, and biological) from 28 natural AMD biofilm communities to reveal relationships of the environment with the whole community, and with the molecular physiology of the individual populations.
Chemoautotrophic biofilm communities sustained by oxidation of iron and sulfur grow underground at the interface between air and sulfuric acid solutions within the Richmond Mine at Iron Mountain, California. In general, initial colonists form few microns thick films that mature into much thicker, differentiated communities with higher species richness (Wilmes et al, 2009). Although the thickness of the biofilm is correlated with maturity, other abiotic factors, such as local flow rates, seem to influence the thickness of individual films. Biofilms develop over the weeks to months and either sink and degrade or are flushed from the system by high flow rates following seasonal rainfall events. Their high biomass production rates and low species richness have enabled extensive metagenomic and proteomic characterization (Tyson et al, 2004; Lo et al, 2007; Denef et al, 2009) and make them an ideal system for exploring principles of microbial ecology at the molecular level (Denef et al, 2010b).
Sample collection and characterization
Twenty‐eight biofilm communities were collected from the air–solution interface at seven sites (Supplementary Figure S1) between January 2004 and August 2007. Temperature and solution chemical parameters (including pH, sulfate, metal, nitrate, and nitrite concentrations) were measured at each sample site (Supplementary Table S1). The average temperature and pH values were 40.9±2.8°C and 0.93±0.18 (±s.d.), respectively, and mine discharge was 196±176 l/min. ATP synthesis and all carbon fixation are driven by the high H+ and Fe2+ concentrations (182±0.07 mM) that result from the dissolution of pyrite (FeS2) (Druschel et al, 2004). The community structure (CS) (i.e. composition and population abundance) of biofilms was defined using fluorescent in situ hybridization (FISH; Supplementary Table S2) (Amann et al, 1990).
Community membership patterns
Proteomic and FISH data were used to determine community membership across the 28 biofilm communities. 2D‐LC MS/MS‐based proteomics performed on each biofilm sample identified an average of 2182±411 proteins from each community (Supplementary Table S3), and 6296 proteins across all communities (http://compbio.ornl.gov/biofilm_amd_ecological_succession). Proteins from iron‐oxidizing Leptospirillum Group II bacteria dominated all proteomic data sets (49.6±11.5% of proteins; Figure 1A). Mature biofilms with proportionally lower representation of Leptospirillum Group II had higher abundances of proteins from later biofilm colonizers. The subdominant groups included another iron‐oxidizing species, Leptospirillum Group III (13.7±5.5%), and two potentially mixotrophic archaea, G‐plasma (9.0±4.9%), and A‐plasma (4.6±3.8%) (Figure 1A). A small number of proteins also derived from Actinobacterium 1, Actinobacterium 2, and Firmicutes sp. and other archaea (E‐plasma, I‐plasma, Ferroplasma Types I and II, and ARMAN‐2). Proteins without an organismal affiliation were grouped as unassigned. This category is associated with many different low abundance organisms, and represented a relatively constant fraction of each proteome (Figure 1A; CV=0.11).
The relative abundances of community members determined by FISH were in good agreement with proteomic relative abundance data (R2=0.80; Supplementary Figure S2). Perfect concordance is not observed, though, as proteins from high abundance organisms are slightly underrepresented in proteome samples and proteins from low abundance organisms are overrepresented. The reason for this deviation from a 1:1 relationship is partly due to the dynamic exclusion filters set during the mass spectrometer run, which will bypass highly abundant peptides after they have been selected once for MS/MS analyses.
Using only FISH data (Supplementary Table S2), biofilms clustered into two distinct patterns of CS that represent different maturation stages, and are distinguished primarily by changes in the relative abundance of archaea to Leptospirillum Group II (Figure 1B). Biofilms from cluster 1 were typically thin (∼10–30 μm in thickness) (Wilmes et al, 2009), and mostly composed of Leptospirillum Group II (78±9.0% of cells). Cluster 2 biofilms were more mature, thicker (∼30–200 μm) (Wilmes et al, 2009), and had a more complex composition, with significantly higher concentrations of archaea (43±11%, t‐test—P<2e−6).
Leptospirillum Group II's wide distribution, dominance across all samples, and high proteomic activity shows its wide niche breadth within the AMD system. In contrast, Leptospirillum Group III organisms, like many other low abundance community members, seemed to have a patchier distribution as determined by FISH (Figure 1B), and their proteomes were highly variable across all communities (Figure 1A; CV=0.40). The lower abundance and more restricted distribution of these subdominant populations relative to Leptospirillum Group II indicates their comparatively smaller ecological niches within the AMD system.
Changes in organismal physiology and distribution with environmental conditions
To explore possible relationships between environmental factors and species distribution trends, we tested for significant correlations between proteomic data and biotic and abiotic environmental data. Semiquantitative measurements of protein abundance (normalized spectral abundance factor (NSAF) values) were derived from normalized spectral count values for peptides (Florens et al, 2006). Hierarchical clustering of these values allows for comparison of relative abundances of proteins across samples, enabling efficient detection of patterns within these large multivariate data sets. Communities (columns in Figure 2A) were clustered based on the abundances of all proteins within each proteome and individual proteins (rows in Figure 2A) were clustered based on their relative abundance patterns across all communities. To avoid biases due to the presence or absence of proteins in individual communities and to ensure the fidelity of the results, clustering was performed multiple times with specific subsets of the data (Supplementary Figure S3A). Twenty‐three of the communities reproducibly clustered into two groups based on their protein abundance levels (green and blue highlights in Figure 2A). Five communities could not be consistently grouped (P36, P37, P38, P40, and P43) due in part to unusual abundance patterns for some A‐plasma and plasmid proteins (Supplementary Figure S3B). A separate form of analysis, non‐metric multidimensional scaling (MDS) (Torgerson, 1952), supported the division of protein expression patterns among communities into two groups (Figure 2B).
Changes in measured environmental parameters (temperature, flow, sample collection site, pH, and CS; see Supplementary Table S1) were correlated with variations in the protein abundance patterns using the BIOENV statistical procedure (Clarke and Ainsworth, 1993; Oksanen et al, 2007). Significant correlations emerged despite the use of highly complex proteomic data sets comprised of hundreds of variables. 'Flow' was the only factor to show consistent correlation with the protein abundance patterns for all organisms (Table I).
The founding and dominant species.
For Leptospirillum Group II, CS bore the strongest correlation to protein expression patterns (Table Ia). This relationship is illustrated by an MDS created using proteomic data for this organism (Figure 3), with circles superimposed over each sample point representing the two CS clusters from Figure 1B.
Clear metabolic differences in Leptospirillum Group II that coincided with changes in the composition of the surrounding community were detected for the 23 whole‐proteome data sets classified as either high or low developmental stage. Use of the significance analysis of microarrays (SAM) technique (Tusher et al, 2001) revealed 474 proteins of Leptospirillum Group II with significant abundance level differences between stages (<4.7% false‐discovery rate (FDR); Table II). These Leptospirillum Group II proteins were usually widely distributed across all proteomes, with a majority being detected in >95% of samples (median=96.4%). In addition, these proteins generally comprised a large proportion of each individual proteome (38±2%), suggesting that the presence of surrounding community members influences the overall metabolism of the Leptospirillum Group II population.
Of the 474 Leptospirillum Group II proteins that showed a significant relationship with CS, 370 are overrepresented in high and 104 in low developmental stage biofilms. Clustering of samples using these proteins revealed two metabolic states when growing within low versus high developmental stage biofilms with high within‐cluster correlations (=0.89±0.05; Pearson correlation coefficient; Figure 4A). Leptospirillum Group II proteins associated with this metabolic reorganization were grouped into functional categories and significant biases were defined. Ribosome biosynthesis, which includes ribosome structural proteins, and transcription, which includes RNA polymerase proteins and proteins involved in transcriptional regulation, were significantly elevated in low developmental stage biofilms, as well as proteins involved in physical and chemical stress defense, unknown functions, and associated with mobile genetic elements (Figure 4B). In the high developmental stage biofilms, we detected increased investment in proteins involved in chaperone and protein turnover functions, and environmental signaling, chemotaxis, and motility (Figure 4B). There was also a shift in metabolism away from ribosome biosynthesis towards proteins involved in biosynthesis of extracellular components, carbohydrates, and amino acids.
When the effects of only physical and geochemical parameters on the Leptospirillum Group II proteome were considered, it was noted that the overall physiology of Leptospirillum Group II was largely uncorrelated with individual abiotic variables (Table II). Although some significant correlations between protein abundances and specific abiotic factors were uncovered, the total numbers of proteins were smaller than for those correlated with changes in CS, and very few proteins were uniquely correlated to a given abiotic factor (Table II). For example, only 59 of the 360 proteins with significant correlations to calcium levels were uniquely correlated with this parameter.
Of the proteins that were significantly correlated with specific physical and geochemical parameters, many perform related roles. For example, abundances of the cold shock protein from Leptospirillum Group II decreased as temperatures increased (Supplementary Table S5a) and abundances of a greater than expected number of proteins involved in fatty acid biosynthesis positively correlated with increased temperatures. Also, ∼40% of all detected ribosomal proteins showed positive correlations with nitrate (Figure 4C; Supplementary Table S5b). Additional significant and strong correlations were observed between sulfate levels and protein processing components and calcium levels and proteins involved in various biosynthesis pathways (Figure 4C; Supplementary Table S5c and S5d). Many of these proteins may ameliorate physical stresses associated with the range of abiotic conditions encountered.
Subdominant, later‐colonizing taxa.
The variable distribution of community members other than Leptospirillum Group II across samples (Figure 1) may result from constraints on their environmental niches. We found that different sets of physical and geochemical parameters were best correlated with each organism's proteome. Temperature showed the strongest correlation with protein abundance patterns of most archaea (A‐plasma, G‐plasma, and Ferroplasma Types I and II), whereas pH showed a correlation with the proteome of Leptospirillum Group III (Table Ib). We further examined these relationships by analyzing the skew in the total numbers of proteins of A‐plasma, G‐plasma, and Leptospirillum Group III that correlated positively or negatively with temperature, pH, conductivity, Fe2+ and Cu concentrations. Biases reflect differences in how individual factors may influence the abundance, activity level, or both of each organism (Figure 5A).
Source data for Figure 5A [msb201030-sup-0003-SourceData-S3.zip]
The associations of A‐plasma and G‐plasma proteins to physical factors were generally opposite, despite their close phylogenetic relationship. Temperature and copper were the exceptions, with more proteins from both organisms being positively than negatively correlated with increases in these factors. This is consistent with the BIOENV results that showed a high correlation of temperature with both organisms' proteomes (Table Ib). In contrast to both these archaea, correlations of Leptospirillum Group III proteins with abiotic factors suggest that this organism favors lower stress environments (i.e. higher pH, lower ionic strength, lower temperature, lower copper; Figure 5A).
Although more Leptospirillum Group III and A‐plasma proteins correlated positively than negatively with increasing pH (Figure 5A), their pH optima seemed to be different. Clustering of proteins with strong correlations to pH revealed that Leptospirillum Group III proteins were most abundant at pH ∼0.95 and A‐plasma proteins at pH ∼1.12 (Figure 5B).
Further support for the conclusion that Leptospirillum Group III's environmental niche is distinct from other subdominant populations was revealed when the relative abundances of this organism's proteome was considered against those of the Alphabet‐plasma group of archaea (i.e. A‐, E‐, G‐, and I‐plasma). Here, a strong negative relationship was observed between these groups, demonstrating that as one group increases in activity the other concomitantly decreases (=−0.89; Pearson correlation coefficient; Figure 5C).
Value of environmental proteomic approaches for the study of microbial communities
We have used environmental proteomic techniques to examine ecological and physiological processes in a natural model microbial community. Extensive sampling of communities across environmental and temporal gradients provided insight into relationships between environmental parameters and physiological state. Importantly, this work has examined these processes in situ, capturing processes ongoing in the natural environment. An advantage of this approach is that it is cultivation independent, enabling analysis of many coexisting difficult to culture populations. Insights were attained at a community ecology level (i.e. how population abundances within a community change as the environment changes) and at a functional molecular level (i.e. how an individual population changes its physiology as the environment changes). The approach differs from traditional CS profiling (e.g. 16S rRNA gene surveys) used in many microbial ecology studies. Although CS profiling could have shown that Leptospirillum Group II remains the dominant population throughout succession, demonstration that its metabolism changes significantly as biofilms mature relied on the application of proteomic analysis.
Community assembly patterns in AMD biofilms
The finding that Leptospirillum Group II is dominant in all biofilm developmental stages is atypical relative to most patterns of ecological succession for plant and animal communities (Connell and Slatyer, 1977). Similar to patterns of forest establishment in clearings (Glenn‐Lewin et al, 1992) and colonization of lava fields (Del Moral and Bliss, 1993), the founding colonist in the AMD system conditions the environment—in this case by fixing carbon and initiating biofilm construction (Goltsman et al, 2009)—enabling the propagation of secondary colonizers. However, in marked contrast to these macroscale examples, Leptospirillum Group II is not ultimately outcompeted and replaced by these later arrivals qas more diverse communities develop (Figure 1). Another exception to this model from macroecology are the 'fertile islands' of the African savanna established by Acacia trees (Dean et al, 1999). As in the 'fertile islands,' secondary colonists in AMD biofilms most likely depend on persistence of the initial colonist to maintain their niche. This strong and continuing dependence in these systems may be linked to environmental constraints. In both AMD communities and the 'fertile islands,' the combination of many physical challenges and the low variety of energy resources probably acts to restrict the diversity of competitors able to perform similar community‐essential roles, aiding in the initial colonist's persistence.
Another feature of plant and animal communities is that the most widely distributed species are also commonly the most abundant locally (Hanski et al, 1993). Similarly, Leptospirillum Group II was found in all communities and was also the most abundant population member (Figure 1). The ecological specialization hypothesis proposes that this is due to an ability to 'tolerate conditions and acquire sufficient resources' (Brown, 1984), an explanation that also may apply to this organism. Adaptation of this type could be accomplished either by evolution of a stable metabolism that ensures functionality over a wide range of conditions or by continuous modulation of the core metabolism as geochemical parameters change. The finding that the Leptospirillum Group II proteome is not strongly correlated with abiotic environmental perturbations indicates adaptation through a relatively stable, broadly adapted metabolism (Tables I and II). Notably, we identified two stable proteome states that are strongly correlated to microbial community composition. This finding strongly indicates that interspecies interactions directly impact the physiology of this organism.
In summary, we propose that the physiological state of Leptospirillum Group II is largely predicated on interactions with other organisms, which may include resource competition, but that its core metabolism is relatively unchanged by abiotic conditions, such as pH, temperature, and measured geochemical factors. Thus, by augmenting biotic surveys that document the spatial and temporal distribution of a bacterium with direct information about its physiological responses over a range of growth conditions, we were able to evaluate the ecological specialization hypothesis at both a physiological and species level.
Leptospirillum Group II physiological changes: ecological and evolutionary implications
The nature of the Leptospirillum Group II population proteome shift as communities diversify (Table I; Figure 4) provides insight into how biologically imposed constraints may affect physiology during ecological succession. The shift may be related to density‐dependent or complexity‐dependent effects that typify the transition from an r‐ to K‐environment (MacArthur and Wilson, 1967). In an r‐like environment where resources are abundant and predation is low (e.g. early succession where community diversity is low), it is proposed that traits such as rapid growth and dispersal are favored. Conversely, in K‐environments (e.g. late succession stages), higher biological diversity, competition for resources, and predation should favor slower growth and the ability to take up and use diminishing resources.
We propose that the functional biases comprising the metabolic shift observed for Leptospirillum Group II (Figure 4B) reflect growth in r‐ versus K‐environments. Ribosome biosynthesis, cell division, and transcription are favored in early succession stages, as expected for rapidly growing cells in thin biofilms that are not limited by resource diffusion constraints, biological competition, and/or predation. In later succession stages, processes such as the metabolism of cellular components (e.g. carbohydrates, nucleotides, amino acids) and environmental sensing are favored. Increases in the abundance of carbohydrate biosynthesis proteins may be a result of increased biomass accumulation in exopolymeric biofilm matrix. Also, deficits in the intracellular amino acid pool may be driven by the demands of general protein synthesis in earlier developmental stages. This is supported by the result that tRNA synthetases are often found in greater abundances in late developmental stage biofilms. This diversification of metabolism and enhancement of the ability to detect chemical gradients in thickening biofilms may be advantageous in a K‐like environment where density‐dependent effects are higher.
It was also observed that proteins involved in iron oxidation and the electron transport chain are overabundant in late developmental stages. These include nine subunits from the NADH dehydrogenase complex and various cytochrome‐containing enzymes (e.g. Cytochrome oxidase, Cytochrome b/b6, and Cytochrome 572, which may oxidize Fe(II) in Leptospirillum Group II; Jeans et al, 2008). Differential abundances of these enzymes may reflect increased iron oxidation by this organism in high developmental stages. In addition, some enzymes involved in the reductive TCA cycle, likely the major carbon fixation pathway in Leptospirillum Group II (Goltsman et al, 2009), are more abundant in late compared with early developmental stages. These include subunits of pyruvate synthase, aconitate hydratase, and succinyl‐CoA synthetase. However, as these constitute only a small portion of the overall pathway and given that some may have multiple roles in different metabolic pathways, it is difficult to hypothesize whether carbon fixation rates change with developmental stage.
Despite the relative stability of the Leptospirillum Group II proteome with respect to changes in abiotic factors, specific subsets of proteins correlated with abiotic perturbations and may be involved in specific environmental adaptation mechanisms (Supplementary Table S5). For example, the abundances of lipid biosynthesis proteins correlate with increasing temperature, consistent with observations of lipid composition changes in membranes of organisms under temperature stress (Allen and Bartlett, 2002). In addition, it was found that ribosomal proteins increase with nitrate levels, which are normally at nanomolar levels within the ecosystem and may be growth limiting for Leptospirillum Group II. We also observed significant and strong correlations of protein processing enzymes with sulfate concentrations and of many different metabolic pathways with calcium concentrations (Supplementary Table S5). It is not known what physiochemical interactions may be underlying these relationships, and it may be that other unmeasured abiotic factors that correlate strongly with calcium and sulfate levels may be true causal participants.
It is interesting to note that fine‐scale genetic variation occurs within Leptospirillum Group II, some of which may have functional significance in responding to changes in environmental conditions during succession (Tyson et al, 2004; Lo et al, 2007; Simmons et al, 2008). Two genotypic groups have been identified in AMD biofilms, but they do not always co‐occur (Denef et al, 2009). In most early succession stage biofilms, only the UBA genotypic group occurs. The five‐way genotypic group co‐occurs with the UBA type in most late succession stage biofilms. On the basis of strain‐resolved proteomic analysis, it was proposed that niche adaptation is associated with both differential regulation of shared genes and differences in gene content (Denef et al, 2010a). As only 4 of the 12 high developmental stage samples from our current study show greater numbers of five‐way cells than UBA cells (determined by FISH counts, Denef et al, 2010a and see Supplementary Table S2), we conclude that the proteome changes observed in here can be attributed partially to strain composition changes, but mainly to an overall shift in the metabolism of both strains found in each community. We propose that the synthesis of our results with those of Denef et al (2010a) suggests that the effects of community interactions that motivate proteomic shifts are driving genotypic divergence that will ultimately lead to speciation.
Environmental niche selection of low abundance community members
The abundances and activities of subdominant bacterial and archaeal populations that grow in late succession stages are more variable than those of Leptospirillum Group II (Figure 1). We used changes in proteomic patterns of these organisms to explore their relationships with various abiotic factors and to develop hypotheses about their possible environmental niches. The BIOENV analysis (Table Ib) revealed that the factor showing the most consistent correlation pattern for all organisms was 'flow,' which measures AMD solution discharge on the day of sampling and mirrors precipitation cycles (Supplementary Figure S4). Flow likely impacts many solution geochemical and physical parameters (Edwards et al, 1999; Druschel et al, 2004), accounting for the wide correlation with all proteomes. Temperature generally correlated strongly with the proteomes of the archaeal populations, whereas pH correlated with the proteins abundance patterns of the Leptospirillum Group III population.
To further evaluate the inferences that high temperature selects for archaea and high pH for Leptospirillum Group III in more mature biofilms, we tested for significant correlations between the abundances of individual proteins and specific abiotic factors (Figure 5A). Results indicated that the closely related archaea, A‐plasma and G‐plasma, have different environmental niches. For example, A‐plasma growth may be favored in hot, higher pH, lower ionic strength solutions, whereas G‐plasma may prefer hot, lower pH, higher ionic strength solutions (Figure 5A). In addition, the analyses demonstrated that Leptospirillum Group III activity/abundance was inversely related to the activities/abundances of various archaeal populations (Figure 5C) and that the former may prefer lower stress environments (Figure 5A). Preference for less stressful environments is consistent with this Leptospirillum Group III's patchy distribution in biofilms and its growth as small microcolonies within the biofilm interior (Wilmes et al, 2009). Other support for these niche hypotheses derives from experimentally manipulated laboratory bioreactors where biofilms were grown under low and high pH conditions (pH=0.8 and 1.4). The abundances of archaeal proteins were greater in lower pH bioreactors, whereas Leptospirillum Group III proteins were more abundant at high pH (Belnap, 2009). Thus, we conclude that specific combinations of geochemical factors restrict the environmental niches of subdominant populations within mature biofilms.
Community proteomics applied to a suite of biofilm communities enabled an unprecedented level of insight into the activities of multiple coexisting microbial members across the range of physiochemical conditions. A striking finding is that the activities of lower abundance organisms are predicated on restrictive environmental conditions. Perhaps more importantly, the dominant community member exhibits a shift from one physiological state to another that correlates with increased activity of less abundant bacteria and archaea. The strong correlation between physiological state and community membership outweighs any correlation with a single or combination of measured physical or geochemical factors. These findings support a long held, but rarely quantified, axiom in microbial ecology stating that interspecies interactions strongly shape physiological responses in microbial communities.
Materials and methods
Sample collection and metadata measurements
Collection sites for the 28 samples used in this study are shown on the map of the Richmond Mine, Iron Mountain, CA (40°40′38.42″N, 122°31′19.90″W, elevation of ∼900 m) in Supplementary Figure S1. Sample collection was performed as described earlier (Denef et al, 2009). The pH, conductivity, temperature, and Eh of all samples were determined in situ. The standards used for pH measurement were pH 1.00 and pH 1.68 (Ricca Chemical Company) together with standards prepared from standardized sulfuric acid (Fisher Scientific) for the pH range 0–2 as described (Nordstrom et al, 2000). Each in situ sample measured was bracketed by the standard with closest pH and repeated until the pH reading of the bracketing standard agreed within 0.05 pH units. The platinum electrode for the Eh measurements was checked against Zobell's solution (Ricca Chemical Company). Conductivity standards (12.9 and 111 mS/cm, Thermo Scientific Orion), corrected to in situ temperature, and used to calibrate the conductivity probe on‐site. Values of 'flow' were measured as the total outflow of AMD (l/min) from the mine on the day of sampling.
All water samples were collected in HDPE bottles and filtered as soon as possible. Samples for nitrate, nitrite and iron (II)/total iron were filtered within the mine with 0.1 μm syringe filters (Supor membrane, Pall Corporation). Samples for all other analyses were 0.1 μm filtered (Supor membrane, Pall Corporation) with either a 47 or 142 mm filtering manifold. Filtering was competed within 6 h of sample collection. Water samples were preserved for each analysis according to U.S.G.S. protocols (McCleskey et al, 2004), except for nitrate, which was stored at 4°C in a completely filled 4 ml amber glass bottle, and nitrite, which was frozen on dry ice immediately after filtering and stored at −80°C until analyzed.
Ferrous and total iron concentrations were measured spectrometrically following the ferrozine method (To et al, 1999). Nitrate and nitrite were determined at U.S.G.S., Boulder with a chemiluminescent nitric oxide detector (Sievers Analytical, Model NOA 280), using a method modified from Baeseman et al (2006). The detection limit was 10 nM for nitrate and 5 nM for nitrite. Metals were determined by inductively coupled plasma‐optical emission spectroscopy (ICP‐OES), run at either UC Berkeley with a Perkin Elmer 5300 DV or at U.S.G.S., Boulder with a Leeman Labs Direct Reading Echelle. Replicate samples run in both laboratories agreed within 5%. Sulfur was determined by ICP‐OES with the UC Berkeley instrument. Given the geochemical relationships within the AMD system, all the sulfur measured in solution is assumed to be sulfate (Druschel et al, 2004). U.S.G.S. acidic reference waters AMW 4, SCREE, and PPREE were run several times during each analytical run to monitor analytical accuracy for metals and sulfate.
Numerical representations of sampling site were calculated by using the AB drift site as the seed and assigning whole numbers to the remaining sites based on approximate distances relative to AB drift. For example, the UBA site where P35 was collected was given a value of −3 due to its outlying location relative to other sites. Conversely, samples from the C75 site (P8, P9, P10, P36, P37, P38, and P49) were given a value of +3 as this site lies on the opposite end of the mine from UBA. Values for all parameters are listed in Supplementary Table S1.
FISH of AMD biofilm samples
Characterization of the CS of each biofilm was performed using FISH (Amann et al, 1990). Protocols for biofilm disruption and oligonucleotide probe design, hybridization, microscopy, and CS estimation were followed as described earlier (Bond et al, 2000). Oligonucleotide probes and fluorescent labels used in this study for identification of individual species and groups were as follows: (1) Cy5‐LF655 (all Leptospirillum Group bacteria), (2) Cy3‐L2UBA353 (Leptospirillum Group II—UBA type bacteria), (3) FITC‐L2CG353 (Leptospirillum Group II—five‐way type bacteria), (4) Cy5‐ARC915 (all Archaea), (5) Cy3‐EUBMIX (all Eubacteria), and (6) FITC‐LF1252 (Leptospirillum Group III bacteria).
Protein extraction, mass spectrometry, and peptide identification
Whole‐cell protein fractions from each sample were extracted, prepared, and approximately equal amounts of total protein were analyzed through 24 h nano‐2D‐LC (strong cation exchange‐reversed phase)—MS/MS on a hybrid LTQ‐Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) for 23 samples using a previously described protocol (Denef et al, 2009). The remaining proteome samples (P1, P2, P26, P27, and P35) were the first proteomes collected for this project and, as such, were originally run on a stand‐alone LTQ mass spectrometer (Thermo Fisher Scientific) using similar amounts of total protein (Ram et al, 2005; Denef et al, 2009). At least three technical replicates were analyzed for each sample.
Each spectrum obtained from tandem MS scans was searched against the Biofilm_AMD_CoreDB_04232008 database containing peptide sequence information derived from the previously published UBA and five‐way community genomic data sets (Tyson et al, 2004; Lo et al, 2007) using the Sequest program (Eng et al, 1994) as outlined earlier (Tyson et al, 2004; Denef et al, 2009). Proteins contained within the search database belong to 16 genomic classifications, including: Leptospirillum Group II five‐way (2760 proteins) and UBA strains (2629 proteins), Leptospirillum Group III (2730 proteins), A‐plasma (2379 proteins), E‐plasma (1846 proteins), G‐plasma (1893 proteins), I‐plasma (1855 proteins), Ferroplasma Type I (2140 proteins) and Type II (2409 proteins), ARMAN‐2 (1006 proteins), Actinobacterium 1 (2590 proteins), Actinobacterium 2 (1770 proteins), Firmicutes sp. (1409 proteins), and plasmid (471 proteins), viral (663 proteins), and unassigned (22 045 proteins) categories. The unassigned category is highly redundant and includes many sequences for variants of proteins already included in the main assemblies, many of which occur at low abundance. Also included in this category are unresolved fragments from low abundance organisms and mobile elements. The database also included common contaminants such as trypsin, human keratins, and so on, to ensure that these would not mistakenly be classified as biofilm components.
The DTASelect program (Tabb et al, 2002) was used to parse the results using the filters described earlier (Lo et al, 2007). Proteins from Leptospirillum Group II five‐way and UBA strains were condensed into one Leptospirillum Group II genome containing unique proteins and orthologous proteins from each strain. For orthologous proteins, single spectral counts of shared peptides were summed along with unique peptides from each strain to obtain the number of total spectral counts for a given protein. Protein identification was based on the following criteria: (1) at least two peptides identified within the same run, and (2) each matched spectra must have Xcorr values >1.8 (+1), 2.5 (+2), and 3.5 (+3) and ΔCN values >0.08. Similar numbers of total spectra (∼150 000–160 000) were obtained for all runs. We have established false‐positive rates with this system at ∼1–5% with these filters in earlier studies (Ram et al, 2005; Denef et al, 2009). All databases, peptide and protein results, MS/MS spectra and Supplementary Tables are archived and made available as open access through the following link: http://compbio.ornl.gov/biofilm_amd_ecological_succession.
Proteome data preparation for statistical analyses
Spectral counts for filtered peptides with scores exceeding the above cutoffs were summed for a given protein across all three technical replicates to obtain the total number of spectra corresponding to that protein. An NSAF was calculated for each protein of the 28 different proteomes according to the method proposed by Florens et al (2006). Two values of the NSAF were calculated for each protein. The first is the result of the normalization with respect to all of the proteins in a community proteome (wcNSAF) and determines each protein's relative abundance compared with all proteins in the sample. The second normalizes individual proteins to the total proteins from the specific organism that particular protein is derived from (orgNSAF), allowing for a relative abundance within each organism's proteome to be determined. As both orgNSAF and wcNSAF values essentially represent percentages, all data were arcsine transformed (ASIN) to approximate a normal distribution. In addition, zeros were substituted for missing values across all 28 proteomes, as proteins not detected in the mass spectrometry analysis are assumed to be below the detection limit and in low abundance. Clustering of data without zeros gave similar results (data not shown). For each possible combination of paired technical replicates in each sample, a test of concordance was conducted using R2 values, which represent the proportion of variability in that is accounted for by a linear regression model for each pairwise comparison. Replicates demonstrated high reproducibility with an average R2 value of 0.974±0.010 for all pairwise comparisons across all samples (Supplementary Table S6).
Hierarchical clustering and non‐metric MDS of proteomic and FISH data
ASIN‐transformed, mean‐centered and scaled wcNSAF and orgNSAF values for each protein were clustered with the Cluster v 3.0 program (de Hoon et al, 2004). Hierarchical clustering was performed on proteins and samples using an uncentered Pearson correlation distance matrix and distances between groups were calculated using average or centroid linkage clustering methods. Cluster files were visualized using Java TreeView (Saldanha, 2004).
As biases can be introduced into clustering results based on the presence or absence of proteins across samples, multiple clustering analyses were performed on subsets of the whole‐proteome data. These subsets included proteins detected in >1% of all samples up to 100% of samples using a step size of 10% (i.e. sets containing only proteins present in >10% of samples, >20%…100%). Consistent clustering of a sample within 10 of the 11 trials allowed for confident assignment of that sample to its designated cluster (Supplementary Figure S3).
Using the vegan (Oksanen et al, 2007) and MASS packages of the R software distribution, non‐metric MDS was performed on ASIN‐transformed wcNSAF and orgNSAF values for all samples. Distance matrices were created using the Bray–Curtis dissimilarity index. This allows for a graphical representation of the relatedness between different samples based on their protein expression patterns within a two‐dimensional plane. Hierarchical clustering and MDS using ASIN‐transformed orgNSAF values gave similar results.
Hierarchical clustering of untransformed FISH data was performed similar to the protocol for proteomic data. Clustering was performed on samples using an uncentered, absolute Pearson correlation distance matrix and distances between branches were calculated using the complete linkage method. A numerical representation of the relatedness between samples was calculated by measuring the branch length between each sample and sample P4. Values of branch length are reported in Supplementary Table S1 and are used in the BIOENV analysis as the measure of CS.
Correlation of measured variables with protein expression profiles using the BIOENV analysis
Using a method developed by Clarke and Ainsworth (1993), various measured environmental (both abiotic and biotic) variables were correlated to the wcNSAF and orgNSAF values for all samples. Similar results were obtained for both and the results of the orgNSAF analyses are reported. Environmental variables included: sampling site (i.e. site), CS, total mine drainage outflow on the day of sample collection (i.e. flow), pH, and temperature (°C). Raw values of flow and temperature were log‐transformed. Values for site and CS were obtained as described above.
These correlation analyses were performed using the 'BIOENV' function within the vegan package of the R software distribution (Oksanen et al, 2007). This method involves obtaining Spearman's rank correlations between dissimilarity matrices for samples based on all environmental variables in all combinations or by themselves and a dissimilarity matrix for samples based on protein abundance data. Distance matrices using NSAF values of all samples were made using the Bray–Curtis dissimilarity index, whereas dissimilarity matrices were constructed for measured environmental variables using a Euclidean distance metric. Permutation tests were performed using an in‐house script to determine the significance of each correlation. Briefly, this script creates 1000 random permutations of the matrix of environmental variables and compares these with the real protein expression dissimilarity matrix. Top scoring random correlations are recorded and the distribution of these is compared with the real correlations to obtain an approximate P‐value for each real value.
Use of SAM for detecting significant differences in protein abundance
Although the SAM software was originally developed for detection of significant expression differences between microarray experiments (Tusher et al, 2001), it has previously been applied to proteomics data (Roxas and Li, 2008). This analysis was performed on ASIN‐transformed wcNSAF and orgNSAF values of all samples, and orgNSAF results for Leptospirillum Group II are reported. The two groups in this analysis were defined as the low and high developmental stage samples as defined in Figure 2 and Supplementary Figure S3 (blue and green highlight). The SAM software was run using the 'sam' function of the siggenes package of the R programming environment. Tests were performed using a paired experimental design and the standard F‐statistic, and at least 100 permutations were run. Differentially detected proteins identified with a <4.7% FDR were considered statistically significant.
Significant proteins from the SAM analysis were grouped into functional categories based largely on their cluster of orthologous groups classifications (Tatusov et al, 2003), and biases were evaluated. In this analysis, the total number of proteins from each functional category overrepresented in either low or high developmental stage biofilms was summed separately and divided by the total proteins for each developmental stage to give a percentage. Biases in each category were calculated by separately normalizing each percentage to the total and then subtracting the low developmental stage normalized percentage from the high developmental stage normalized percentage. Significant biases for each category were determined using a bootstrap re‐sampling technique, implemented by the perl script 'All_scrambler.pl' (Lauro et al, 2009). Re‐sampling the pool of total proteins identified in SAM created 1000 replicate subsamples (n=250 proteins). Significant differences between the numbers of proteins identified to be overrepresented in either high or low developmental stages were assessed at a 98% confidence level.
Correlations of individual protein abundances with geochemical and physical parameters
Proteins of Leptospirillum Group II with significant correlations to various environmental factors were also determined using the significance of microarrays technique (samr package of R). This analysis was performed using all ASIN‐transformed orgNSAF values from samples with corresponding metadata measurements (e.g. temperature, pH, [NO3−], [SO42−], [Cu], [As], and [Ca]). Correlations with other factors were not considered due to strong intercorrelations between them (e.g. total iron concentrations correlate with [Fe2+] levels). Tests were generally performed using a quantitative experimental design and the standard test. The exception was for tests with nitrate, where a rank test was performed due to the exponential nature of the measured nitrate values. One thousand permutations were run for tests with each parameter. Groups of proteins identified with <0.10 FDR and with ∣AB∣>0.4 were considered to have a strong and significant correlations to each individual factor. Proteins meeting these thresholds were grouped by functional categories and differences between observed and expected numbers of proteins were detected based on the distribution of the number of proteins in each functional category for the entire detected proteome of Leptospirillum Group II.
Spearman rank correlations of the abundances of proteins from subdominant populations (Leptospirillum Group III, A‐plasma, and G‐plasma) with environmental factors were determined using the 'cor' function of R. Samples were not included in this analysis if a given protein was not detected (i.e. no zero NSAF values were included in correlations). Also, proteins that were present in less than half of the samples with corresponding measures of environmental variables were not included in this analysis to prevent false correlations due to under‐sampling. Similar results were seen when wcNSAF and orgNSAF values were used (data not shown).
Strong correlations between environmental factors and abundances of proteins from low abundance organisms were defined as ∣AB∣>0.4 (Ideker et al, 2001). Biases in the number of proteins of each proteome either strongly positively or negatively correlated to individual parameters were determined. The total number of proteins with strong negative and strong positive correlations with each environmental factor was summed separately, and each sum was divided by the total number of proteins from each respective proteome ('Total' column, Supplementary Table S3). Strong biases in the total number of proteins from an organism either positively or negatively correlated with a specific factor were inferred to represent a response of that organism to that factor.
We thank Mr TW Arman, President, Iron Mountain Mines and Dr R Sugarek, EPA, for site access and Mr R Carver for on‐site assistance, P Abraham, M Lefsrud (Oak Ridge National Laboratory) for their assistance with proteomic measurements and analysis, and F Lauro for providing computational assistance. R Barnes, C Miller, and M Power are thanked for helpful reviews. This project was funded by Grant No. DE‐FG02‐05ER64134 from the US Department of Energy Genomics: GTL project (Office of Science).
Conflict of Interest
The authors declare that they have no conflict of interest.
Supplementary Information
Supplemental Figures and Tables
Figures S1–S4 and Tables S1–S6 [msb201030-sup-0001.pdf]
Source data for figure 2A [msb201030-sup-0001-SourceData-S1.zip]
Source data for figure 4A [msb201030-sup-0002-SourceData-S2.zip]
This is an open‐access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation without specific permission.
- Copyright © 2010 EMBO and Macmillan Publishers Limited


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The sheer size of the FSA's CP98 draft mortgage sourcebook gives an early taste to brokers, lenders and borrowers alike as to how life is likely to be when the rule changes are brought in.
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The industry has been set a conundrum of how to achieve regulation of the advice given without actually regulating the intermediaries who are giving the advice.
This has been a two-stage consultation and there are still many questions awaiting clarification, which the FSA says will come in the form of a published mortgage sourcebook before the end of this year. But the one definite message from CP98 is that structural change is going to happen.
The requirement for lenders to take reasonable steps to ensure that intermediaries have provided pre-application illustrations in accordance with the regulations is one that needs to be clarified.
Lenders and the CML are seeking clarification of what will actually constitute "reasonable steps". Until that has been cleared up, it will be difficult for brokers to know exactly how to prepare for that regulation.
Halifax mortgage regulation manager Celia Rowland says: "There are still a lot of ifs and buts in how the regulation will work. Intermediaries do not want 10 different lenders rooting through their files.
"But what is a reasonable step? Is it one visit to an intermediary every two years? Is it to be carried out by the lender or outsourced to a third party? We need more clarity."
Those trading through networks and mortgage clubs will have some comfort in that their parent organisations will be addressing on their behalf the problem of how information will be gathered and processed and how compliant documentation will be generated and monitored.
It is the independent brokers, often running very small operations, who face the most radical change.
The new regime describes a situation where brokers send out PAI documents from each provider discussed, so that the consumer can make clear comparative assessments between the products on offer before making any commitment, that is, before making a formal offer.
It is likely to be acceptable under the new rules for a broker to hand over the PAI documents in person and then immediately proceed to making the mortgage application. But those giving telephone-based advice may have to wait until the letters have been received before proceeding to application.
Clearly, in such a circumstance, brokers will need an internet-enabled PC as an absolute minimum to be able to access compliant documentation from lenders. But with no clear strategy from lenders as to how brokers might be able to receive all the compliance data in a consistent form yet to be formulated, it would be premature for brokers to go away and spend thousands on a software system to pro-cess the information.
Lenders do not yet know what format of documentation will be acceptable.
As fundamental a change will be the requirement for lenders to authorise every piece of advertising that brokers put out – unless that broker is itself regulated by the FSA.
This could lead to lenders producing generic packs of advertising content that brokers can adapt to their own use, which could contribute to stagnation in the sector.
Probably half of all intermediaries are not regulated and the control over their advertising will make the relationship closer to the old-style agency relationship. It could lead to a push for even greater control by lenders.
Mortgageforce managing director Robert Clifford says: "The new rules change greatly the relationship between lender and broker. It looks more like a master/servant agency relationship.
"If lenders are morally responsible for brokers' documents and advertisements, then why won't the lenders want full control and go for some type of multi-tie relationship?"
When the final version of the new rulebook is published, it will confirm that standardisation is coming and the lenders' hand will be strengthened. Independent brokers will have to scrutinise their business models as soon as it is available.
Independent Financial Services director Jim Gillespie says: "I am not frightened of change – we will have a rationalisation that will bring a cull in the same way that happened to direct salesforces. But those who survive will be more efficient mortgage broking businesses."
The road to regulation
The sheer size of the FSA's CP98 draft mortgage sourcebook gives an early taste to brokers, lenders and borrowers alike as to how life is likely to be when the rule changes are brought in.


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How fitting, a play written by playwright Martyna Majok an immigrant from Poland and based on her own mother's experiences in America, that comes to light when so much of our focus these days in our nation's capitol is around, about and disparaging immigrants.
What's lost in translation is that this country was built on the sweat and tears of immigrants. We are an immigrant nation at least we used to be. Over the years things and generations changed. In looking for a community to blame for our national woes, immigrants seem to be a good target these days.
Moxie Theatre is having a 13th birthday. By all means in the Jewish faith that's cause for celebration. So too can the Moxie's, now under the leadership of Jennifer Eve Thorn, co-founder and now executive director, celebrate its 13 year by offering "Ironbound", a deeply moving and oft times off centered comedy excellently acted, and supported by several immigrants in its midst.
It is playing through Oct. 22nd at their Rolando Theatre on El Cajon Blvd.
|Jacque Wilke
The story of Darja, a gutsy and defiant immigrant played with the same gusto as her character, Jacque Wilke shines as she jumps through a series of hoops to hang on to a life that is hell bent on bringing her down through abject poverty, an abusive second husband, an unfaithful lover and a runaway son.
Her story begins, centers and ends at a rundown bus stop not far from the factory in Elizabeth, N.J. where Darja used to work, was laid off, and is now permanently shut down.
Her story toggles back and forth over a period of 22 years. At present she is/was living as roommates with Tommy (a persuasive and convincing Eric Casalini) her errant roommate. Theirs was a rocky one and grew even more divided as she learns that he has been cheating on her with none other than the rich lady whose house she cleans as a second job to make ends meet. Fourteen times, she blasts out! She's pissed.
He wants her to get into his car and come home with him rather than wait for the bus that he knows will never come because of the late hour.
|Eric Casalini and Jacque Wilke
She stubbornly refuses unless he gives her money $3000.00 to buy a car. Her son, (not seen) a drug addict, took hers some time back and disappeared, ergo the bus stop has become her permanent encampment.
Her home away from home, the bus stop, is surrounded by broken chain link fence, an uninviting bench that she uses as her bed, and a kiosk showing bus schedules. The small surrounding area is littered with trash an old tire and whatever weeds have the guts to pop through. Credit Divya Murthy Kumar for the realistic setting; its almost another character.
Flashing back in time to a youthful Darja and her first husband at the same bus stop, musician Maks (Arusi Santi) a dreamer who won't let his immigrant status get in the way of his dreams heads off to Chicago where he wants to make it big as a blues artist. She's also pregnant.
Both immigrants he Santi's Maks is easy going and sure enough of himself as he heads to Chicaco without her. With no skills her options are more limited. She gets the factory job, a houseclening job along with an abusive second husband. She abandons him and in time the factory closes leaving her out in the cold of the bus stop.
While there she meets up a young hip-hop kid Vic (Carter Piggee) who does a second take when he sees her sleeping on the ground next to the bench. When she feels his presence and defensively jumps up, the two have to decide how they want this chance meeting to evolve.
|Carter Piggee and Jacque Wilke
With skill, Majok takes a lonely and young, almost innocent boy out of his element and a spirited, tenacious and determined hard nosed and abused forty something immigrant and faces them off in such a profound and persuasive and yes, humorous way and with such convincing dialogue that one is stunned by the outcome.
The playwright never loses sight of her characters strengths and weaknesses and with the expert direction of Jennifer Eve Thorn they spring to life bringing with them truth and defiance, frustrating bullheadedness and eventual hope that comes when Darja finally relents and goes off with Tommy.
The bus stop is empty.
|Arusi Santi with Jacque Wilke
She's antagonistic and hostile and pushes everyone in her line of fire away except when she softens around the edges as Vic tries to offer her money. In her quest to find and help her son her motherly instinct takes the place of her hostility, but that's it!
Others in her orbit mostly react. Tommy finally gives in to her blind stubbornness. Maks leaves in spite of their love and Vic, feeling sorry for her pushes cash into her hand so she can get off the streets and sleep in safety.
Life has never been easy for the immigrant then and now. With a wakening call Majok brings out the good, the bad and the ugly of it all.
Scenic designer Divya Murthy Kumar hails from India, costumer Anastasia Puatova from Russia, and actor Asuri Santi is from Mexico. Others in the creative team include Alex Crocker lighting and Haley Wolf, sound.
Imagine if half he team were not allowed here what we would be missing?
See you at the theatre.
Dates: Through Oct. 22nd
Organization: Moxie
Phone: 858.598.7620
Production Type: Drama
Where: 6663 El Cajon Blvd., San Diego, CA 92115
Ticket Prices: $30.00
Web: moxietheatre.com
Photo: Daren Scott


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This wedding cake contest is a game for the person who like challenge. You have to make the most points and you have to create the same cake that you will see. Good luck!
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Mowry lifting Bisons
Name: Lindsay Mowry
School: Bedford
Age: 14
Grade: Freshman
Sport: Soccer
Athlete of the week achievement: Mowry recorded back-to-back shutouts in goal for the Lady Bisons, shutting out Schuylkill Valley and Waynesburg Central to help Bedford reach the PIAA Class 2A semifinals.
Season update: Bedford is 23-0 and will play District 10 champion Mercyhurst Prep today in the semifinals. Mowry had seven saves against Schuylkill and 12 against Waynesburg Central.
Coach Jeff Thomas' quote: "From the start of the season until right now she keeps improving. We told her she has the right build for a goalkeeper, good height, good hands and it's showing now. She's made saves with her feet and hands, and is getting better all season. She is becoming more of a vocal leader back there, and she's just getting comfortable and building confidence."
Favorite teacher: Mr. Schrock
Favorite subject: Art
Favorite movie: "The Lone Survivor"
Favorite food: Chicken
Outside interests: Softball
Favorite professional team: No preference
Favorite college team: UCLA
Pro athlete most admired: Andrew McCutchen
Best athlete I ever competed against: Everett's Brittany Ewing
Most influential person on my athletic career: Coach Jeff Thomas
Proudest athletic achievement: Making varsity as a freshman
How I got my start in soccer: "My mom signed me up in kindergarten."
Future goal: Become a general surgeon.


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Blonde woman screwed so damn hard
Handsome young man is ready to please her mature blonde boss in the office. Horny bitch in stockings gets her sweet cunt licked and fucked with so much pleasure.
- Cast:
- Sarah Vandella
- Added:
- 01 Nov 2015
- Video Length:
- 07:00
- Download full video at
- Big Tits at Work - Brazzers Network - Join


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I heard it said that Power is one of the temptations of speakers, preachers and church leaders, I was trying to think how this would manifest itself.
Certain denominations call their leaders "Father …." or "Teacher..." when it explicitly says in the Bible "don't call anyone Father or teacher for there is only one father (God) and one Teacher (Christ)" We give these titles to men/women that we respect, that we look up to for their insight, their theology and their knowledge, but we actually have equal insight and power as all who have been "born again" have the Holy Spirit within them, and all are priests and apostles!
What I would like to know is when things go wrong, when you are having a really bad day, when you are really struggling with a situation, what do you do? What do you think?
- Do you say "O my God!" but not actually mean to ask for the help of a deity?
- Do you pray for help, recognising there is one who can and wants to help, as he has the power and resources to do so?
- Do you just give up and feel down, as you feel "Powerless"?
- Do you rely on your friends/family to "pick you up"?
There was a ruler in Capernaum who recognised Jesus' authority and knew just one word from Jesus, and his servant would be well again! So what is our excuse? Jesus said that those who follow him would be able to cast out demons, heal the sick! He gave his disciples the power and authority over demons and sickness. He had authority because he died and rose again, death, sickness nothing could hold him. If you are a follower of Christ, "He that is in you is more powerful than the spirit of the world"
So I have the Power, and if you are a born again follower of Christ so do you!
What are you waiting for? Get out there!! Either use it or get it!!


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Sounds totally legit.
Marc Elias, the attorney who infamously retained Fusion GPS to produce the largely discredited anti-Trump dossier on behalf of Hillary Clinton's 2016 presidential campaign, has been retained to represent Sen. Bill Nelson's recount efforts in Florida.
The Florida Senate race is likely headed to a recount after late reporting from Broward County lowered Gov. Rick Scott's lead to about 17,000 votes, meaning the election is within the .25% margin necessary to require a hand recount. The race has already reached the 0.5 percent threshold to require a machine recount.
In a conference call with reporters, Elias claimed that optical-scan machines may not have registered ballots that were poorly marked, and he contended that Nelson would emerge victorious from a recount, which has not yet been officially authorized.
"We believe that at the end of this process, Sen. Nelson is going to be declared the winner," Elias said.
Elias added: "We're doing this not just because it's automatic, but we're doing it to win. A significant number of ballots have not yet been counted and, because of the size of Florida, we believe the results of the election are unknown and require a recount."
Elias is widely considered one of the most powerful attorneys associated with the Democratic Party. He is chair of the Political Law Group at the Perkins Coie law firm. Elias represented Hillary Clinton's 2016 presidential campaign as well as the Democratic National Committee. He was brought in by Nelson's campaign to oversee official recount efforts. READ MORE:


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Інститут фізико-технічних та комп'ютерних наук Чернівецького Національного Університету імені Юрія Федьковича
Стаття на сайті Міжнародної комісії з оптики (ІСО), яка присвячена проведенню в Чернівцях чергової міжнародної конференції Correlation Optics-15
Correlation Optics 2015 held in Ukraine
The 12th International Conference of a series initiated in 1993 was held in Ukraine.
Despite political circumstances in Ukraine associated with Russia's occupation of Crimea and a part of Donbass, the Twelfth International Conference on Correlation Optics was held on 14–18 September at Chernivtsi National University (Ukraine). Participants from Canada, China, Denmark, Finland, France, Germany, India, Israel, Japan, Mexico, Poland, Romania, Scotland, South Korea, Spain, Sweden, Ukraine, UK, and USA, contributed about 120 research reports. The number of participants was smaller than on previous occasions, lacking the usual contributions of researchers from Russia and Crimea.
The main topics of the conference are singular optics; information content of statistical optical fields, including optical chaos, polarization optics, and coherence; optical correlation devices based on diffractive optical elements, including optical and digital holography, fractal optics, and optical sensors; optical correlation diagnostics, interferometry and microscopy of rough surfaces and random media; and new applications of correlation optics in biology and medicine.
Graphene, a material with many fascinating properties, exhibits extraordinary optical behaviour. One specific outstanding feature is the so-called surface plasmons, wave-like excitations that were predicted to exist in the sea of conduction electrons of graphene. The wavelength of graphene plasmons is 100 to 150 times smaller than the wavelength of light, enabling very strong light confinement as well as slow light, relevant for a plethora of applications such as sensors and opto-electronics.
Among invited speakers were Prof. M Berry (UK), Prof. Y Miyamoto (Japan), Prof. G Swartzlander (USA), Prof K Bliokh (Japan), Prof. S Hanson (Denmark), Prof Tim Lee (Canada); and the invitees of the OSA/SPIE Student Chapters: Prof. M Berry from the University of Bristol (UK), Prof. M Dennis from the University of Bristol (UK), and Prof. A Bekshaev from Odessa University (Ukraine).
ICO contributions helped support the participation of students from the Optical Engineering Department of the National Technical University of Ukraine "Kyiv Polytechnic Institute". The proceedings of the Conference has been published in Proc. SPIE V. 9809.


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August Wedding at The Round Barn
I never take a beautiful wedding day for granted. It was a truly lovely day for this wedding at The Round Barn in Waitsfield, VT. Featured here are some of the most beautiful anemones. I rarely rely on anemones to come in "just so perfectly", but lucky for me, they came in 2 weeks in


text: Policing St. Louis County Council calls for investigation into MetroLink policing By Jo Mannies • Jul 25, 2017 File Photo | St. Louis Public Radio Updated at 9 a.m. July 26 with more details — There will be an investigation into whether the St. Louis County officers assigned to patrol MetroLink stations and trains violated any laws, the St. Louis County Council decided Tuesday on the heels of three reports by the St. Louis Post-Dispatch. Missouri Gov. Greitens signs Blue Alert system into law as means to aid police By Jo Mannies • Jul 6, 2017 Camille Phillips | St. Louis Public Radio / St. Louis Public Radio Missouri Gov. Eric Greitens fought back tears Thursday as he explained why it was important for him to sign a measure creating a system to swiftly notify the public when an on-duty police officer is wounded. AG Sessions brings tough-on-crime message to law enforcement leaders in St. Louis By Rachel Lippmann • Mar 31, 2017 File photo | Carolina Hidalgo | St. Louis Public Radio Pledging money, research and expertise for local law enforcement, U.S. Attorney General Jeff Sessions brought a face to the Trump administration's pro-police message during a speech Friday in St. Louis. He also made general mention of the 2014 unrest in Ferguson after Michael Brown was shot and killed by a white officer, and the tensions between police and African-Americans. Missouri House moves on another labor union restriction; hair-braiding, police safety bills advance By Krissy Lane • Feb 16, 2017 File photo | Jason Rosenbaum I St. Louis Public Radio Thursday was a busy day for the Missouri House, which passed four bills, including another piece of the GOP's labor-union reform agenda. The House also sent along to the Senate two law enforcement-related bills and a measure that would deregulate the cosmetology industry. St. Louis County officials begin campaign for policing-related tax increase measure By Erica Hunzinger • Feb 8, 2017 Erica Hunzinger | St. Louis Public Radio St. Louis County voters are being tasked with another request for public funding, this time a half-cent sales tax increase for policing efforts in the April election. Officials say they're certain that the measure, which is estimated to bring in $80 million a year, is vital for public safety both in the county and in municipalities. But some communities aren't sure they'll benefit much. After the shootings in Baton Rouge, St. Paul and Dallas, a St. Louis law enforcement perspective By Kelly Moffitt • Jul 13, 2016 Kelly Moffitt | St. Louis Public Radio When citizens are shooting and killing police officers and police officers are shooting and killing citizens, something is malfunctioning in civilized society. That fact is not lost on former police officers turned UMSL criminology professors Dan Isom and David Klinger. Local researcher seeks insight into how black communities feel about neighborhood crime By Kelly Moffitt • May 26, 2016 Kelly Moffitt | St. Louis Public Radio On Thursday's St. Louis on the Air, host Don Marsh was joined by Andrea Boyles, an associate professor of criminal justice at Lindenwood University – Belleville, who has recently started a research project called "How Do Blacks Police Themselves?" Her project seeks to gain insight into how African Americans feel about neighborhood crime. On the program, we learned more about her research. Ways To Avoid Community Violence After Local Police Encounters Copyright 2018 NPR. To see more, visit http://www.npr.org/. STEVE INSKEEP, HOST: Former St. Louis police chief weighs in on police training By Evita Caldwell • May 7, 2015 Alex Heuer / St. Louis Public Radio There have been many suggestions on improving policing in our region since the unrest in Ferguson. One of the issues that has come to light is the need for changes in police training, specifically diversity training. UMSL criminology professor and former St. Louis police chief Dan Isom, and Anti-Defamation League director Karen Aroesty joined "St. Louis on the Air" host Don Marsh to talk about issues with police training including the latest recommendations from the Ferguson Commission. 'Walking the Blue Line'- addressing the racial divide with a former police officer By Evita Caldwell • May 6, 2015 Terrell Carter / Courtesy Photo Since the shooting death of 18-year-old Michael Brown and the subsequent community unrest, dialogue about racial division in the St. Louis area became a frequent topic. Additionally, many people vowed to come together and address the apparent 'invisible line' separating black and white residents in the region. Commentary: Which came first: violence or law? By M.W. Guzy • Aug 4, 2011 This article first appeared in the St. Louis Beacon, Aug. 4, 2011 - The story goes that a young man was dumped by his girlfriend. He was devastated by the rejection and adjourned to the corner bar to drown his sorrows. Sitting off to himself, he ordered a Budweiser. He drained the bottle in two draughts and ordered another. The jilted lover proceeded to pound down 12 Buds in morose silence before standing up and shouting to no one in particular, "I can't believe she left me," then ran head-first into the wall, knocking himself unconscious. Concerned patrons subsequently carried him home. He repeated this ritual every night for a week, after which he disappeared.


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This original motion picture soundtrack features an emotional mix of solo instruments, subtle string ensembles and powerful, intriguing action cues – it is composed by Gerhard Daum, produced and recorded in Berlin, Germany.
That Which I Love Destroys Me is an important documentary by Hollywood director Ric Roman Waugh. It follows two Special Operations Soldiers as they return home after fighting in the longest combat campaign in American history only to face a new battle; the effects and stigma of PTSD and reintegration as a whole.


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A video to provide an overall understanding of the offerings at Saint James School along with interviews with Msgr Cronin, students, teachers, alumni and parents.Uploaded Jan 28, 2017
A video montage of Saint James School's June activities. (Music: Sunny - Bensound.com)Uploaded Jul 11, 2017
A video montage compiled by AJ Scortino (fotobyaj.com) of the Saint James School 2016 Basketball Pep Rally.Uploaded Dec 30, 2016
A video montage of Saint James School's December Pep Rally. (Music: Bensound.com)Uploaded Jan 02, 2018