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Let's Talk About It: Do our fathers affect how we look for love and respect?!
A couple of weekends back, I was watching a Gangland marathon on the History channel with my mom. One episode that stood out was "From Girl to Gangsters", which featured female members of the Bloods and Crips in California. You can catch the first 10 minutes of the episode below:
There are many things happening and suggestions as to why they may happening even in the first 10 minutes of the episode. One message that was prevalent is that girls join gangs for love and/or respect. The clip cites broken homes as one of the causes of this; resulting in women seeking "love" and "respect" in destructive ways. And though we always talk about the effects of fatherless homes in the way women look at love and respect, we never look at the effects of fathers being present in their daughter's lives. So let's really talk about it, "Do our fathers affect how we look for love and respect?!"
We often don't talk about the father/daughter relationship in our community. This may be due to the fact that many fathers are not present in their child's lives to even talk about it. But for those with their fathers in their lives, the story is usually different. One person in the Gangland episode that caught my attention was Sylvia aka Rambo. She was from a middle class background and had both her mother and father in her life. She became a gang member after her brother was murdered by members of an opposing gang and her father was imprisoned. Unlike many of the other women on the show, Sylvia wasn't solely seeking love, but also respect and power, which she acquired by putting in her work. Later in the episode, Sylvia went on to say, "My dad told me, I'm going to teach you everything a woman need to know. You ain't gon' need a man, but for one thing: the bedroom". For many of us with our fathers in our lives or a constant male presence, we can attest to a similar reality check about life and love.
Biggie said it best with, "Get my daughter this college plan, so she don't need no man." Dads are traditionally the disciplinary and are infamous for bringing out harsh realities in many situations, lol. I think of dads as giving us the tools we need to navigate life, while moms nurture us as we take life on. With our dads around, we see that not only do we require nurturing in love but respect as well. In that expectation of respect is our education and character. And while Sylvia's dad taught her how to steal cars and physically fight, even for those of us who are formally educated, we can attest to not being as dependent on men for anything outside of love and respect. Unfortunately until more men in our community recognize and value what we bring to the table, the way our fathers and the male figures in our lives teach us, many of us "educated" women find ourselves very lonely. A plan devised by dads of daughters many years ago, I'm sure, lol. The key as always is a honest conversation with your partner about your expectation for love and respect. xx
PS Shoutout to the dads that do what they are supposed to do when choosing (or forced ) to raise their bundles of joy, especially us girls!! And big shoutout to the all the single moms, that play both nurturer and disciplinary. True superheroes xx
Related posts:
- Let's Talk About It: The Cool Homegirl v. The Crazy Girlfriend, what separates us?!
- "To Submit or Not To Submit…": A Reflection on Love and Independence
- Let's Talk About It: A Real Look at Sisterhood and Friendship amongst black women
- Let's Talk About It: "Representing" and What It Means to You?!
- Let's Talk About It: Is Waiting for Adam a bigger problem than we think?!


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Robyn's third and newest CD "Waterproof," produced by John Jennings (Mary-Chapin Carpenter) and featuring Christine Lavin, brings to life a fresh set of original songs for thinkers and dreamers. Reflecting at once a joyful and sober perspective on the world, it's an expansive chronicle of life's journeys...uplifting, activist, even spiritual, yet deeply personal. It's a compassionate embrace of the fragile and a paean to hope and resilience.
Robyn's second CD (and first solo effort). The eagerly anticipated Many Moons, released May 2009, has beautiful and tasty guest appearances by Michael Lille, Mark Graham, Kat Eggleston, Larry Murante, Joe and Karena Prater (Cat Loves Crow), and Hans York; and features Paul Elliott on fiddle and Cary Black on bass. One of the most-played CDs on folk radio in 2009, reaching #16 on the Folk-DJ airplay chart in 2009 and #11 on the Washington Roots Radio airplay chart. With no special promotion or publicist, Many Moons caught the ear of more than 70 DJs, many of whom cited it as a beloved favorite.
"Robyn Landis radiates excellence with every word, every note and every song. Many Moons is clearly one of the most exciting projects released by a singer/songwriter in North America this year--maybe even in the last few years and possibly for several more yet to come. This girl has what it takes--and then some!"
~ Dennis Brunnenmeyer, Nevada City Limits, KVMR Radio, CA
"I really love Many Moons...truly one of my favorite new releases I've received in the last many, many months. I have this system of marking songs on new CDs for future airplay and nearly the entire cd is marked as 'high priority'! Really, just a lovely record...wow! And I really love the flow of the record, an absolute treat to listen to from beginning to end."
~ Mark Michaelis, "Acoustic Harmony," WGDR Radio, VT
"It is good. It is very good. My gosh, it is really, really very good. I listened, I listened again. I really enjoy your singing, the songs, the arrangements. The first cut on Many Moons drew me in, and I was pretty much compelled to not skip around, and that does not often happen to me these days. Great work!
~ Rik James, "Americana Backroads," KGLT-FM, Bozeman, MT
"It is always a joy to find artists, emerging ones or otherwise, that have a special gift that I was not previously aware of...you are one of those."
~ Craig Huegel, "Our Kind of Folk," WSLR, Sarasota, FL
"Each time I hear a Robyn Landis song I feel a delicious surprise and I think, "Gosh, that's a beautiful song. Gosh, what a beautiful voice." ...Robyn was voted by listeners into two spots on the Top 20 this month, no. 5 and 11. "
~ Kelley Martin, Acoustic Pie Radio, April 2010
The first CD by acoustic duo Short and Sweet. Contains five songs by Robyn, five songs of Steve Amsden's, and two co-writes. Also features stellar Northwest musicians Paul Elliott (fiddle), Cary Black (bass) and Dan Tyack (steel).
© 2016 Robyn Landis

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Tam Coc - Van Long - Kenh Ga Hot Spring Tour
Duration: 2 Days 1Night
Style Tour: Private tour
Tour Route: Hanoi - Hoa Lu - Tam Coc - Van Long Nature Reserver - Kenh Ga Hot Spring - Hanoi.
Bai Dinh - Trang An - Tam Coc - Bich Dong - Hoa Lu Tour
Duration: 2Days 1Night.
Style tour: Private tour.
Trang An, the eco tourism attraction located in the East of Ninh Binh, with area of 2000 ha. The area caves Trang An eco belonging Truong Yen Commune, Xuan Ninh, Ninh Hai (Hoa Lu district),
Read more: Bai Dinh - Trang An - Tam Coc - Bich Dong - Hoa Lu Tour
1 Day Hanoi - Phat Diem - Tam Coc Tour
Brief description : Private tour, discovering the unique church that were built by green stone, sightseeing the peaceful countryside of Vietnam. Discovering dry halong bay Vietnam with rowing boat a long the rice paddies and through many caves and visiting one of ancient citadel in Vietnam.


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Title: Salt-Lake-City die Mormonen-Stadt
Map Maker:
Bibliographische Institut
Place / Date: Hildburghausen / 1858
Coloring: Uncolored
Size: 6 x 4 inches
Condition: VG+
Price: $125.00
Inventory ID: 37704
One of the earliest obtainable views of Salt Lake City, Utah.
This early view of Salt Lake City, shows an idealized image of the City on a hill in the foreground, with the lake in the distance, wrapping around the Wasatch Mountains in the style of an Alpine Lake.
Related Categories:
Maps of the Rocky Mountains (Colorado, Wyoming, Montana, Utah, New Mexico)
Maps of Southwest America (Nevada, Arizona, Colorado, Utah, New Mexico, Texas)
Western US


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Is this attraction free to the public?
Is this attraction a good picnic spot?
Does this attraction have good sunset views?
Does this attraction have good sunrise views?
Is this attraction good for couples?
Is this attraction good for big groups (greater than 5)?
Is this attraction good for small groups (less than four)?
Would this be a good sunny day activity?
Is gratuity suggested at this attraction?
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Thanks for helping!
We've camped on Bumpkin several times and visited by kayak many times more. Each of the harbor islands are spectacular places to visit. Near the heart of the city but it feels so...
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Beach was nothing but rocks - no sand at all. Facilities were ok - clean at least. Not sure I would go back but had a good time with family.
We've camped on Bumpkin several times and visited by kayak many times more. Each of the harbor islands are spectacular places to visit. Near the heart of the city but it feels so far away!
Via kayak or boat - or when the tide is super low you can walk - a visit to this harbor island is just plan fun!
Overseen by the State of Massachusetts, u can reserve campgrounds on several,of the Boston Harbor Islands. There are decent sanitary facilities provided and some nice trails, from which you might see deer, turkeys, pheasants and even coyotes, so keep your foodstuffs safely tucked away. You can set up tents on the campsites and have magnificent views of Boston as well...
The boat will take you to this picturesque
island where you can spend to day exploring, picnic or even camp out! It's a great family place.
An interesting place to visit, fish from and walk out on the sand spit at low tide. There are even prehistoric horseshoe crabs on the spit sometimes--don't touch them!!!!
My husband and I just returned from a 2 night camping trip on Bumpkin Island; we had such a great time! We took the ferry from Hingham Shipyard ($14 round trip/person plus $1/day for parking) and arrived on the island early afternoon.
The two rangers, Max and Kimm, were on the dock to meet us. They gave us a map...
I toured the islands on the ferry last summer and it was great. This Memorial Day, we rented sea kayaks instead and setting off from Hingham, we paddled over to Slate Island, ate lunch (Slate is not too user-friendly). After lunch, we paddled accross the channel, avoiding some bigger boats. The weather was cool, overcast, and cloudy, so the seas...
Is it safe and pleasant to swim on this island? Where do we get in the fairy to get there? Is there any shade on the island? Thank you.
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Booking Information
How to Book
Online (Recommended)
Booking online is the easiest and quickest way to secure your tickets and is available 24 hours a day, seven days a week.
You can book online with this mobile friendly website for all events, choosing where you want to sit from the interactive seating plan.
By Phone
By calling 020 3285 6000 between 10.00am and 6pm Monday to Saturday.
Lines are closed on Sundays and Bank Holidays
In Person
The Box Office is situated in the main foyer of the theatre.
Box Office Opening Times
|Monday to Saturday
|Sunday and Bank Holidays
|Show Days
|10am until 15 minutes after the last performance begins
|Open 2 hours prior to the show and close 15 mins after the last performance begins
|Non-Show Days
|10am until 6pm
Friday 14th April 10am – 8.45pm
Saturday 15th April 10am – 7.45pm
Sunday 16th April CLOSED
Monday 17th April CLOSED
Tuesday 18th April 10am – 7.45pm
What's On
Booking Fees
There is no transaction fee. The standard booking fee for the majority of shows and events is £2.50 per ticket.
There are no booking fees for Groups of 10 or more (call the Box Office to book fee free), or Churchill Friends. Some events may carry a different booking fee, which is clearly shown on the information page.
Print at home: Free
Postage fee: £2.
Where offered, concession ticket prices apply to those:
- Over 60
- Under 16
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One free personal assistant ticket is available for those who are registered disabled and require a personal assistant for their visit.
Proof of eligibility will be required for all concessions either when purchased or on entry to an event.
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The Churchill Theatre does not accept American Express or ATG Gift Vouchers.
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If you are booking 10+ tickets for the same show, please call our box office on 020 3285 6000
- No bookings fees
- Seats can be reserved without payment
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Children under 18 months may be admitted to family shows when sat on parents' or guardians' lap. Please mention when booking.
All children over the age of 18 months must have their own seat.
Customer Care Charter
View our Customer Care Charter here
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Join our email or postal mailing list to keep up-to-date with all shows and events at the Churchill Theatre as they are put on sale.
Snow White And The Seven Dwarfs
Sat 2 - Sun 31 Dec 2017
The Fairest Pantomime Of Them All!
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Never Miss a Show!
Join our email or postal mailing list to keep up-to-date with all shows and events at the Churchill Theatre as they are put on sale.
Snow White And The Seven Dwarfs
Sat 2 - Sun 31 Dec 2017
The Fairest Pantomime Of Them All!
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Conceived and designed the experiments: VK HJ MBS MS SM. Performed the experiments: VK HJ MBS. Analyzed the data: VK HJ MBS MS SM. Contributed reagents/materials/analysis tools: MBS AMB MS SM. Wrote the paper: VK HJ MBS MS SM.
Many pathogens are equipped with factors providing resistance against the bactericidal action of complement. Yersinia enterocolitica, a Gram-negative enteric pathogen with invasive properties, efficiently resists the deleterious action of human complement. The major Y. enterocolitica serum resistance determinants include outer membrane proteins YadA and Ail. Lipopolysaccharide (LPS) O-antigen (O-ag) and outer core (OC) do not contribute directly to complement resistance. The aim of this study was to analyze a possible mechanism whereby Y. enterocolitica could inhibit the antibody-mediated classical pathway of complement activation. We show that Y. enterocolitica serotypes O:3, O:8, and O:9 bind C4b-binding protein (C4bp), an inhibitor of both the classical and lectin pathways of complement. To identify the C4bp receptors on Y. enterocolitica serotype O:3 surface, a set of mutants expressing YadA, Ail, O-ag, and OC in different combinations was tested for the ability to bind C4bp. The studies showed that both YadA and Ail acted as C4bp receptors. Ail-mediated C4bp binding, however, was blocked by the O-ag and OC, and could be observed only with mutants lacking these LPS structures. C4bp bound to Y. enterocolitica was functionally active and participated in the factor I-mediated degradation of C4b. These findings show that Y. enterocolitica uses two proteins, YadA and Ail, to bind C4bp. Binding of C4bp could help Y. enterocolitica to evade complement-mediated clearance in the human host.
To cause disease in humans, pathogenic bacteria have to evade the versatile immune system of the host. An important part of innate immunity is the complement system that is composed of over 30 proteins on host cells and in blood able to detect and destroy foreign material. To survive, bacteria can bind complement regulator proteins onto their surfaces and thus inhibit the activation of complement. Previously, it has been shown that food-borne diarrhoea-causing Yersinia enterocolitica can survive in human serum because of two bacterial surface proteins, YadA and Ail. These proteins have been shown to bind a complement alternative pathway regulator, factor H. Here, we show that both proteins also bind the classical and lectin pathway inhibitor, C4b-binding protein. These results together explain the serum resistance of Y. enterocolitica. The ability to evade complement attack is apparently important for the pathogenicity of Yersinia enterocolitica.
Yersinia enterocolitica is a food-borne enteric pathogen of humans and animals. The invasive strains predominantly belong to serotypes O:3, O:9, O:5,27 and O:8. Successful colonization of the intestinal tract is the prerequisite for Y. enterocolitica infection. Bacteria pass through the acidic content of the stomach and reach the small intestine. Soon after invading the M cells, bacteria enter the lamina propria and encounter innate and adaptive immune responses [1]. Y. enterocolitica can cause enterocolitis, mesenteric lymphadenitis and, as a post-infectious complication, reactive arthritis [2]. Typically, infections lead to specific antibody responses. Mechanisms whereby Y. enterocolitica can evade the immune system killing and why it causes reactive arthritis are not fully understood.
The complement system is a crucial constituent of the innate immunity. Its activation via the classical (CP), lectin (LP) or alternative pathway (AP) may lead to the killing of microbes by direct lysis or by complement opsonin-enhanced phagocytosis [3]. Many pathogens, however, can resist complement attack [4]. Under microbe-free circumstances activation of the complement system must be effectively controlled as excessive activity could cause complement consumption, host cell damage or inflammation. C4b-binding protein (C4bp) down-regulates complement activity by acting as a fluid-phase inhibitor of the CP or LP [5]. The predominant form of this large (570 kDa) octopus-shaped glycoprotein [6] consists of seven identical α-chains and one β-chain [7],[8]. The α- and β-chains are composed of eight and three complement control protein (CCP) domains each, respectively. The chains are bundled together by disulphide bonds at their most C-terminal parts [9]. C4bp inhibits CP and LP activation at steps that involve C4b. The N-terminal domains of the C4bp α-chains bind C4b to prevent the assembly of the CP C3-convertase (C4b2a), accelerate its natural decay and render C4b susceptible for factor I (FI) -mediated cleavage and inactivation [10].
Distinct pathogenic microorganisms have been demonstrated to be able to bind host complement regulators, such as factor H (FH) or C4bp, to exploit their protective properties and prevent complement activation on the microbial surfaces. These pathogens include e.g. Streptococcus pyogenes, S. pneumoniae, Neisseria gonorrhoeae, N. meningitidis, Borrelia burgdorferi, Escherichia coli K1, Moraxella catarrhalis, Bordetella pertussis, Haemophilus influenzae and Candida albicans [11]–[25].
Y. enterocolitica resists efficiently complement-mediated killing [26]–[29]. This resistance depends mainly on two outer membrane proteins YadA and Ail [29]–[33], both expressed exclusively at 37°C. YadA, encoded by the virulence plasmid (pYV), is a trimeric (monomer 43–45 kDa), lollipop-shaped protein composed of the head, neck, coiled-coil stalk and membrane anchor domains. The trimer projects 30 nm out from the outer membrane to form a fibrillar matrix covering the bacterial surface [34],[35].
Ail is a 17 kDa protein encoded chromosomally. It is predicted to comprise eight membrane spanning β-strands and four extracellular loops located close to the cell membrane [36],[37]. Ail seems to be masked to some extent by the distal parts, O-antigen (O-ag) and the outer core (OC), of lipopolysaccharide (LPS) [30]. Unlike in many other Gram-negative bacteria, in Y. enterocolitica serotype O:3, the O-ag homopolymer and the OC hexasaccharide are linked to the inner core forming a branched structure. Though both O-ag and OC are needed for colonization of the gut [38],[39] their role in serum resistance appears to be indirect [30].
Similar to other pathogens also Y. enterocolitica binds the AP inhibitor FH [40] and we recently demonstrated that Y. enterocolitica serotype O:3 bacteria in fact use both YadA and Ail to bind the AP regulator FH (Biedzka-Sarek et al., submitted for publication) but no studies on possible regulation of the CP or C4bp binding to the bacterium have been reported. Since Y. enterocolitica efficiently escapes all the complement activation pathways we examined in this study whether Y. enterocolitica also interacts with the major CP and LP regulator, C4bp. We show that C4bp binding to Y. enterocolitica is also mediated by YadA and Ail. Since the proteins are located at different layers on the bacterial surface, Ail is masked by O-ag and OC while YadA is well surface-exposed. In consequence, Ail binds C4bp when not blocked by O-ag and OC, while YadA-mediated C4bp-binding occurs regardless of the LPS expression status. As an end result the Y. enterocolitica –bound C4bp retains its function thereby modulating CP and LP activation on the bacterial surface.
Human serum samples devoid of anti-Yersinia antibodies were collected from healthy human donors and stored at −70°C. Serum was heat-inactivated (HIS) by incubation for 30 min at 56°C. C4bp with protein S was purified from pooled human plasma as described previously [41]. C4b, factor I and factor H were supplied by Calbiochem. C4bp was labeled with 125I (NEN, Boston, MA) using the Iodogen method [42]. Triton X-114 (Tx-114) and heparin sodium salt were purchased from Sigma Chemicals. Phosphate-buffered saline (PBS), Veronal-buffered saline (VBS, 1.8 mM Na-barbital, 3.3 mM barbituric acid, 147 mM NaCl, pH 7.5) or Tris-based solutions were used as assay buffers. 0.1% gelatin – VBS (GVBS) or hypotonic 1/3 GVBS was used in 125I-C4bp binding assays.
The bacterial strains and plasmids used in this study are listed in Table 1. For the C4bp-binding and inhibition assays as well as for the serum adsorption assay, bacteria were grown in the RPMI 1640 medium at 37°C. This medium increases YadA expression. Prior to use, exponential-phase bacteria were washed with VBS or PBS. When appropriate, antibiotics were added to the growth medium at the following concentrations: kanamycin (Km), 100 µg/ml in agar plates and 20 µg/ml in broth, chloramphenicol (Clm), 20 µg/ml, and ampicillin, 50 µg/ml.
To generate a YadA-negative strain of Y. enterocolitica O:8 the yadA -gene of pYV8081 was cloned in a 4.4 kb XbaI-PvuI fragment (nt 45398–49838 of pYV8081, accession number NC_008791) between the EcoRV and XbaI sites of pTM100 to generate plasmid pYMS3221x. The kanamycin resistance GenBlock (KmGB) removed by AccI digestion from pUC4K was cloned into the ClaI site within the yadA gene of pYMS3221x to obtain pYMS3223. The expression of YadA was abolished by the KmGB-insertion (data not shown). pYMS3223 was transformed into E. coli S17-1 and mobilized into the wild type Y. enterocolitica O:8 strain 8081 and recombinants, which had lost the vector plasmid due to double crossing-over between pYV8081 and pYMS3223, were screened for by looking for KmR ClmS strains and one such strain was named as YeO8-116. YeO8-116 did not express YadA as verified by SDS-PAGE and autoagglutination tests (data not shown). Restriction digestions and Southern hybridizations of the isolated virulence plasmid of YeO8-116 showed that it carried the KmGB and an inactivated yadA -gene. In addition, YeO8-116 was calcium dependent, and produced the Yop proteins as released proteins identical to the wild type strain (data not shown). The strain Ye08-116 has been used also in earlier studies [43]–[45]. Plasmid pTM100 was electroporated into the E. coli JM109 strain. Triparental conjugation was used to mobilize the plasmids pTM100 and pTM100-ail (from JM109/pTM100 and JM109/pTM100-ail) with a help of E. coli HB101/pRK2013 to YeO3-c-Ail-OCR. The matings were performed as described elsewhere [30].
Bacteria grown to mid-logarithmic phase were suspended in hypotonic 1/3 GVBS. A 50 µl (3×108) aliquot of the bacterial suspension was incubated with 50 µl of radiolabeled C4bp (5,000–20,000 cpm) for 30 minutes at 37°C. The assays using BSA (0–300 nM/assay), unlabeled C4bp (0–300 nM/assay) or factor H (0–300 nM/assay) as competitors were performed with 40 µl of bacteria (3×107) in reaction mixtures containing 125I-C4bp, or 125I-BSA as a control. The effects of heparin (0–1000 µg/ml) and NaCl (50–650 mM) on binding were assayed in reaction mixtures containing 125I-C4bp and 40 µl of bacteria (3×108). After incubation the mixtures were centrifuged through 20% (w/v) sucrose in 1/3 GVBS to pellet the bacteria with the bound radiolabeled protein. Tubes were frozen, the bottoms of the tubes were cut out and radioactivities in the pellets and supernatants were measured with a γ-counter (Wallac, Finland). The ratios of bound to total activities were calculated.
Bacteria (3×108) were incubated in 5% heat-inactivated serum at 37°C for 30 min. Thereafter, the bacteria were washed 5–6 times with 400 µl of PBS or 1/3 PBS and bound proteins were eluted with 0.1 M glycine-HCl (pH 2.7) or PBS, respectively. Supernatants were collected and those acidified due to elution with glycine were additionally neutralized with 1 M Tris-HCl (pH 7.5). Samples of the last wash and elution fractions were subjected to a non-reducing 8% SDS-PAGE gel electrophoresis and subsequently transferred onto nitrocellulose membranes. The membranes were blocked and incubated with 17,500 diluted sheep anti-human C4bp antiserum (The Binding Site, Birmingham, UK) and further with 110,000 diluted peroxidase-conjugated donkey anti-sheep antiserum (Jackson Immunoresearch). The proteins were detected by enhanced chemiluminescence.
The cofactor assay was performed to analyze the effect of C4bp on FI-mediated cleavage of C4b. Bacteria (4×109) were incubated with C4bp (final concentration 50 µg/ml) in 40 µl of PBS for 30 min at 37°C with shaking. After washing for four times with PBS, bacteria (109) were pelleted and resuspended in 30 µl of PBS containing FI (final concentration 50 µg/ml) and C4b (final concentration 35 µg/ml). The reactions were incubated for 45 min at 37°C with shaking. A reaction where C4b and FI were incubated for 45 min at 37°C with 50 µg/ml of C4bp was used a positive control. In addition, a negative control comprising of C4b and FI incubated without C4bp, was included. After incubation the samples were centrifuged, supernatants were collected, mixed with Laemmli buffer and subjected to 12.5% SDS-PAGE and immunoblotting using rabbit anti-human C4c antiserum (DAKO, 15,000). The bound anti-human C4c antibodies were detected using HRP-conjugated swine anti-rabbit IgG (DAKO; 15,000). Supernatants were also analyzed by immunoblotting with sheep anti-human C4bp antiserum to exclude unbound C4bp.
The extracts Tx-YadA (from E. coli JM103/pYMS4450), Tx-Ail (from E. coli JM109/pTM100-ail) and vector control extracts from E. coli strains JM103/pL2.1 and JM109/pTM100 were prepared as described previously [46] with slight modifications. Briefly, bacteria were grown overnight at 37°C in 400 ml of Luria broth supplemented with appropriate antibiotics. Bacteria were centrifuged (3,000×g, 15 min) and incubated on ice for 1 h in 20 ml of lysis buffer (10 mM EDTA, 50 mM glucose, 25 mM Tris-HCl [pH 8.0], 5 mg/ml of lysozyme). Triton X-114, prepared as described previously [47], was then added to the lysate to a final concentration of 5%. The extraction was carried out by incubating the mixture at 4°C for 24 h with slow rocking. Subsequently, the mixture was incubated overnight at 37°C to separate the water and Tx-114 phases followed by centrifugation (4,000×g, 10 min) to clear the phases. The Tx-114 phase was recovered and stored at 4°C.
The OG-Ail and vector control extracts from E. coli JM109/pTM100-ail and JM109/pTM100 were prepared using OG as described elsewhere [37].
Samples from Tx-YadA, Tx-Ail and OG-Ail as well as from the three control extracts (see above) were run into a 7.5–17.5% SDS-PAGE gel. Proteins were electrotransferred onto a nitrocellulose membrane. Membranes were blocked with 5% skimmed milk and incubated with the radioactively labeled C4bp (106 cpm/assay) or 5% NHS. Binding of the protein was detected by autoradiography or immunoblotting with rabbit anti-human C4bp (5 µg/ml) as described above. YadA on the membrane was detected by immunoblotting with the monoclonal antibody 3G12 [48].
The GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) accession number for the gene sequence of Ye O:8 YadA is X13881. For the protein sequences the accession numbers are CAE53849 for Ail (Ye serotype O:3) and CAA32086 for YadA (Ye 6471/76 serotype O:3).
In order to study whether Y. enterocolitica binds the classical and lectin pathway complement inhibitor C4bp serotype O:3, O:8, O:9 and biotype 1A strains were incubated with 125I-C4bp and phase-separated from unbound 125I-C4bp by centrifugation through 20% sucrose. As shown in Fig. 1, all strains belonging to pathogenic pYV-positive serotypes O:3, O:8 and O:9 bound 125I-C4bp with a binding capacity of ~20–40%. The less virulent pYV-negative biotype 1A Y. enterocolitica strain (27675) bound 125I-C4bp significantly less or not at all. The binding percentages by strains that did not bind C4bp (including E. coli K12 C600) ranged between 1–8% under the conditions used (not shown).
To examine cofactor activity of the Y. enterocolitica–bound C4bp, serotype O:3 wild type bacteria and Ail-expressing YeO3-028-OCR strain were incubated with purified C4bp as described in Experimental procedures. A clinical isolate, less virulent Ye biotype 1A strain was included as a control because the strain bound C4bp clearly less than the other two strains used. Unbound C4bp was removed by extensive washing with PBS. The bacteria with surface-bound C4bp were subsequently incubated with FI and C4b. Following the incubation, the C4bp-cofactor activity was verified by immunodetection of C4b-cleavage products in the supernatants using anti-C4c antibodies. As a polyclonal antibody the anti-C4c antibody detects the α′-chain, the β-chain and cleavage fragments of the α′-chain. As shown in Fig. 2, Y. enterocolitica-bound C4bp displayed cofactor activity for FI-mediated cleavage of C4b with both strains YeO3 and Ye-028-OCR. This is indicated by the appearance of the C4b α′-chain cleavage fragments (Fig. 2). The molecular weight of the cleavage fragment generated by Y. enterocolitica-bound C4bp corresponds to the fragment generated in the absence of bacteria by FI incubated with C4bp and C4b (Fig. 2, positive control lane). Clearly less cleavage products were seen with the biotype 1A strain, as expected based on the low binding of radiolabeled C4bp. No cleavage was observed when bacteria were incubated with FI and C4b suggesting that C4bp bound to bacterial surface is essential for the observed C4b cleavage (data not shown).
Serum resistance of Y. enterocolitica depends greatly on the expression of the pYV-encoded YadA protein [29]–[31],[49],[50]. Thus, we first examined the role of YadA in the acquisition of serum C4bp. To this end we incubated the wild type O:3 and O:8 strains (YeO3 and 8081, Table 1) and their YadA-negative pYV-positive derivatives (YeO3-028 and YeO8-116, respectively) in heat-inactivated serum (HIS). After extensive washings, the bacteria-bound serum proteins were eluted and subjected to immunoblotting using anti-C4bp antiserum. Material eluted from the wild type O:3 and O:8 strains contained C4bp while strains lacking YadA displayed significantly less C4bp in the eluted fractions (Fig. 3). This suggested that YadA is involved in C4bp binding. Residual C4bp-binding by YadA-negative strains, however, suggested a role for other Y. enterocolitica factor(s) in C4bp-binding.
To identify C4bp receptors on Y. enterocolitica O:3 surface a set of 12 strains expressing YadA, Ail, LPS O-ag and OC in different combinations was tested for the ability to bind 125I-C4bp in 1/3 GVBS buffer containing 50 mM NaCl. YadA was indispensable for the maximal C4bp binding. Both pYV- and YadA-negative strains (YeO3-c and YeO3-028, respectively) displayed equally low levels of bound C4bp suggesting that the main factor responsible for the binding is pYV-encoded YadA (Fig. 4). Almost all YadA-negative strains bound much less C4bp than the wild type strain. The two sole exceptions were strains expressing Ail in the absence of both O-ag and OC (Fig. 4, YeO3-c-OCR and YeO3-028-OCR). Both Ail-expressing strains were found to bind 125I-C4bp. The fact that the removal of either O-ag (Fig. 4, YeO3-028-R, YeO3-R1) or OC (Fig. 5, YeO3-028-OC, YeO3-c-OC) was not sufficient to promote Ail-C4bp interaction suggests that either of them can block Ail-mediated C4bp-binding to the bacterial surface. To confirm Ail-mediated C4bp binding, we complemented in trans the YeO3-c-Ail-OCR strain (YadA−, Ail−, O-ag−, OC−) with pTM100-ail carrying the cloned ail gene. The resulting strain YeO3-c-Ail-OCR/pTM100-ail restored C4bp-binding ability, while the vector control YeO3-c-Ail-OCR/pTM100 strain was unable to bind the CP-regulator (Fig. 5). C4bp binding was shown to be specific since no binding of 125I-labeled control protein, BSA, to any of the tested strains was detected (Fig. 5). In addition, the strain with trans-complemented ail (YeO3-c-Ail-OCR/pTM100-ail) displayed about four-fold higher C4bp binding capacity (80%) than the YeO3-c-OCR strain expressing Ail from the chromosomally-located ail gene (Figs. 4 and and5).5). This difference, however, can be explained by the overexpression of Ail by pTM100-ail due to a copy-number effect (data not shown).
Neither O-ag nor OC contributed to C4bp binding since YadA- Ail- negative strains expressing full LPS (YeO3-c-Ail) or its rough (YeO3-c-Ail-R) or OC-less (YeO3-c-Ail-OC) derivatives bound only negligible amounts of C4bp (Fig. 4).
To see a direct interaction of YadA and Ail with C4bp we tested the binding of serum C4bp to TritonX-114 extracted YadA and Ail, Tx-YadA and Tx-Ail, respectively, in affinity blotting. C4bp binding to YadA trimer was observed (Fig. 6). Binding to Tx-Ail, however, could not be detected (data not shown). Also β-octylglucoside-extracted Ail (OG-Ail) failed to bind C4bp when tested in a ligand blotting assay (data not shown). This suggests that Ail loses its appropriate conformational structure upon extraction and/or processing for SDS-PAGE.
Electrostatic forces and ion pairings have been suggested to be essential for C4bp binding to C4b [51]. The nature of C4bp binding to YadA and Ail was examined using the wild type strain YeO3 that expresses both YadA and Ail (but the latter is blocked by O-ag and OC as demonstrated above), mutant strain YeO3-028-OCR that expresses unblocked Ail, and YeO3-c-Ail-OCR/pTM100-ail expressing Ail in trans. Bacteria were incubated with 125I-C4bp in 1/3 GVBS alone or supplemented with NaCl to create a salt-concentration gradient ranging from 50 to 650 mM. After centrifugation through 20% sucrose bound 125I-C4bp was measured using a gamma-counter. In general, at low salt concentrations both YadA- and Ail-mediated C4bp-binding was the highest and showed a tendency to decrease with increasing salt concentrations (Fig. 7A). The C4bp binding to YeO3-c-Ail-OCR/pTM100-ail was not affected by an increase in salt concentration. YadA-mediated C4bp-binding, however, was more salt sensitive than Ail-mediated binding. A two-fold decrease in C4bp binding to YadA (strain YeO3) was observed already at NaCl concentration of 100 mM while Ail-mediated C4bp-binding at this salt concentration was not affected. Thus, C4bp binding to YadA depends on ionic interactions between the proteins. The fact that C4bp-binding to YadA, similarly as that to C4b [51], was almost completely abolished in the presence of 250 mM salt (Fig. 7A), suggests that YadA and C4b have affinity for C4bp at site(s) with similar properties.
Heparin binds to the N-termini of C4bp α-chains, i.e. to CCP1-3 [52]–[54]. We tested whether heparin inhibits the binding of C4bp to YadA- or Ail-expressing strains (Fig. 7B). Bacteria were incubated with 125I-C4bp in the presence of heparin (0–1000 µg/ml). As shown in Fig. 7B heparin efficiently and dose-dependently inhibited 125I-C4bp binding to Ail, while significant reduction of 125I-C4bp binding to YadA could only be observed at the highest heparin concentration of 1000 µg/ml. Delayed response to heparin was observed with the strain over-expressing Ail (YeO3-c-Ail-OCR/pTM100-ail).
The relative binding of 125I-C4bp to Ail-expressing strain Ye-028-OCR could be dose-dependently reduced to 55% by adding 0–300 nM factor H. C4bp binding to YeO8 wt strain was not affected by the addition of 0–300 nM factor H (data not shown).
To verify whether YadA- and Ail-mediated 125I-C4bp-binding was specific, wild type and YeO3-028-OCR strains were incubated with 125I-C4bp in the presence of unlabeled C4bp or BSA (0–300 nM). In this assay, ten-fold less bacteria (3×107) were used when compared to the heparin and salt inhibition experiments. Under these conditions 125I-C4bp binding to both, YadA (wild type) and Ail (YeO3-028-OCR and YeO3-c-Ail-OCR/pTM100-ail), was significantly inhibited in the presence of unlabeled C4bp (Fig. 7C). YadA-mediated 125I-C4bp-binding was inhibited to a greater extent than that of Ail. The presence of BSA did not affect the 125I-C4bp binding neither to YadA nor to Ail (Fig. 7D). With ten-fold higher numbers of bacteria, the inhibition with unlabeled C4bp could not be observed possibly because of excessive amounts of C4bp-binding surface proteins, YadA and Ail (data not shown).
The complement system is an essential part of host defense against many microorganisms. A number of pathogens, however, have evolved mechanisms to subvert complement activation at different steps of the cascade. To survive and establish infection in the gut and surrounding tissues, Y. enterocolitica must resist complement-mediated opsonization and lysis. It is thus equipped with surface factors that confer resistance to serum, such as the outer membrane proteins, YadA and Ail [29]–[33],[55]. Although it has been shown that Ail promotes resistance to complement killing, the mechanism of Ail-mediated serum resistance has remained unknown. YadA, in turn, has been shown to be the major serum resistance determinant of Y. enterocolitica [29],[30],[49]. Thus, not surprisingly, mechanisms underlying YadA-mediated resistance have for long been of interest. It has been speculated that the formation of YadA-composed velvet-like coat on the bacterial surface could by itself act as a shield protecting against complement [56],[57]. There is evidence, however, for YadA-mediated binding of the alternative pathway regulator FH and inhibition of the complement cascade at both C3 and C9 levels [40],[58]. This manifests as a reduced binding of C3b and as a failure of the membrane attack complex to incorporate into the outer membrane of Y. enterocolitica [49]. Lipopolysaccharide O-ag and OC are involved in complement-resistance indirectly [30]. They block outer membrane proteins, such as small-sized Ail, thereby having a negative influence on bacterial resistance to serum [30].
This study demonstrated a novel immune evasion mechanism of Y. enterocolitica, C4bp binding by YadA and Ail proteins. All serotypes tested, O:3, O:8 and O:9, were shown to bind the host complement regulator C4bp to avoid opsonophagocytosis and bactericidal action of serum (Fig. 1). The bacteria also acquired this CP-inhibitor from serum, as demonstrated by serum adsorption assays (Fig. 3). In addition, binding of purified radiolabeled C4bp to Y. enterocolitica could be observed (Figs. 1 and and4).4). This shows that the binding is direct and does not involve other serum proteins. Importantly, FI cofactor assay showed that C4bp bound to Y. enterocolitica surface was functionally active (Fig. 2). By binding C4bp Y. enterocolitica can thus inhibit antibody-mediated CP, and the LP.
C4bp receptors on Y. enterocolitica surface were identified using a set of serotype O:3 mutants expressing YadA, Ail, O-ag and OC in different combinations (Fig. 4). Analyses of 125I-labeled C4bp binding to Y. enterocolitica O:3 strains showed that YadA was crucial for capturing this CP regulator. Therefore, YadA- or pYV-negative mutants bound only marginal amounts of C4bp, exceptions being the strains expressing Ail in the absence of O-ag and OC (Fig. 4). Thus, Ail could bind C4bp solely when accessible on the outer membrane. This was additionally confirmed by trans-complementing ail in a strain missing all four factors (YeO3-c-Ail-OCR). Since this strain lacks O-ag and OC, C4bp binding to Ail was strongly favored (Fig. 5). The demonstration of C4bp receptors as YadA and Ail correlated with the previously published serum resistance results of these Ye O:3 strains [30]. These results revealed that the major serum resistance determinant of Y. enterocolitica was YadA and that the removal of LPS O-ag and OC potentiated Ail-mediated complement resistance of YadA-negative strains [30]. It is possible that during infection the production of LPS, O-ag, and OC is suppressed. Similar phenomenon has been observed for Salmonella [59],[60]. In addition, LPS was shown not to contribute to serum resistance directly. Accordingly, in the present work we observed binding of C4bp neither to O-ag nor to OC (Fig. 4).
YadA is a member of a large family of surface proteins of Gram-negative bacteria. These trimeric autotransporter proteins exert many functions and are required for full virulence of pathogenic species. Some of these proteins, such as Actinobacillus actinomyctemcomitans Omp100, E. coli EibD, Haemophilus ducreyi DsrA and Moraxella catarrhalis UspA1 and UspA2, confer resistance to serum [14], [61]–[66]. Interestingly, DsrA, UspA1 and UspA2 have been shown to capture C4bp [14],[63]. Apparently, C4bp-binding is a mechanism shared by multiple members of this family of autotransporters. The interaction between YadA and C4bp appeared to be ionic strength-dependent (Fig. 7A). Interestingly, salt inhibited YadA-C4bp interaction similarly to that between C4bp and C4b [51]. The fact that the positively charged cluster of amino acids between CCP1 and CCP2 is involved in C4b binding [54] would suggest that these CCPs are needed also for the YadA-C4bp interaction. Heparin inhibition assay, however, showed discordant results (Fig. 7B). The electronegative polysaccharide, heparin, alike C4b, binds to CCP1-2, and thus partially competes with C4b for C4bp binding [52]–[54]. Heparin inhibition data showed an initial increase in C4bp binding to YadA at low heparin concentrations (Fig. 7B). This could be theoretically explained by the binding of C4bp oligomers, formed in the presence of heparin, to YadA. Higher doses of heparin inhibited rather weakly C4bp binding to YadA, and 50% inhibition of the binding could only be observed at the highest heparin concentration of 1 mg/ml (Fig. 7B). Thus, the C4bp binding sites for C4b and YadA do not seem to be identical, but most likely are overlapping. Electrostatic forces thus mediate the YadA-C4bp complex formation.
Ail belongs to a family of β-barrel outer membrane proteins that include Salmonella enterica serovar Typhimurium PagC and Rck, and Enterobacter cloacae OmpX [67]–[71]. These proteins, though highly similar in structure, do not appear to share many of their functions. The only protein sharing serum resistance phenotype with Ail is Rck [72]. Here we provided evidence for Ail-mediated C4bp binding. The mechanism of C4bp binding to Ail appeared to be different from that of C4b and YadA, as heparin efficiently and dose-dependently inhibited the binding of C4bp to Ail (Fig. 7B). This observation suggested that Ail-binding involved the CCP1-3 domains of C4bp α-chain. The Ail-C4bp interaction was also less sensitive to salt when compared to that of YadA-C4bp (Fig. 7A) or C4b-C4bp interactions [51]. Thus, other than electrostatic forces, e.g. hydrophobicity, could also be involved in this interaction. Ail binding sites on C4bp, however, are most likely not fully equivalent, though possibly overlapping, with those involved in C4b-C4bp or YadA-C4bp interactions.
Y. pestis, the causative agent of plague, has been shown to be resistant against complement-mediated killing and to bind C4bp [25]. In contrast to Y. enterocolitica, the surface protein Ail (also called OmpX) seems to be solely responsible for the serum resistance property in Y. pestis [73],[74]. Y. pestis Ail protein shares about 70% sequence identity with Y. enterocolitica Ail. As a result of several gene mutations, however, Y. pestis does not express YadA or O-ag [75]. Here we have shown that in YadA-negative Y. enterocolitica strain, removing the Ail-masking O-ag and OC greatly enhances the binding of C4bp (Fig. 4). Based on these previous findings and our current results about Ail binding C4bp, it is probable that constitutively expressed Ail binds C4bp also on the surface of Y. pestis and is, at least partly, responsible for the high complement resistance.
In summary, this study provides the first evidence that Y. enterocolitica acquires the CP regulator C4bp in a functionally active form able to promote degradation of C4b. The binding depends on the two outer membrane proteins YadA and Ail, the latter binding C4bp only when well surface-exposed, i.e., not blocked by O-ag or OC (Fig. 8). Y. enterocolitica is thus able to take advantage of the captured C4bp and is likely to be able to prevent both C4b-mediated opsonization and formation of the CP C3-convertase (C4bC2a). Consequently, the bacteria can avoid complement-mediated lysis and increase their chances to survive in the human host. It is also remarkable that Y. enterocolitica uses both YadA and Ail to recruit both the AP and CP regulators FH and C4bp, respectively. This way the pathogen can ascertain that it will be protected from complement activation during different phases of infection.
The authors thank Reija Venho for technical help in the construction of Y. enterocolitica O:8 yadA mutant and Marjatta Ahonen for technical assistance.
The authors have declared that no competing interests exist.
This work has been supported by funding from the Academy of Finland, European Union Network of Excellence EuroPathogenomics (MB-S and MS), the Sigrid Jusélius Foundation, and the Helsinki University Hospital (EVO) Funds.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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text: Ayun Halliday is the Chief Primatologist of The East Village Inky zine and author of the self-mocking autobiographies No Touch Monkey! And Other Travel Lessons Learned Too Late, The Big Rumpus Dirty Sugar Cookies: Culinary Observations, Questionable Taste, and Job Hopper. Little children know Ayun as the author of Always Lots of Heinies at the Zoo, illustrated by Dan Santat. Teens know her as the author of Peanut, a graphic novel illustrated by Paul Hoppe. And Luddite vagabonds may recognize her as the author of the analog guidebook, The Zinester's Guide to NYC. Her dream is to play the Nurse in Romeo and Juliet.


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""It takes tremendous patience to be a wildlife photographer". Do u agree with the statement?" in Wildlife Photography Classes
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This property is Real Estate Owned, or REO. The property was foreclosed and repossessed by a lender when the owner failed to make payments. A bank is now the owner. Since banks are not in business to own homes, they are usually interested in selling foreclosed homes quickly at attractive low prices. A list price is published but reasonable offers can be submitted for consideration. Offers can be submitted through the official Listing Agent but in some cases, no agent is listed and offers can be submitted the bank owner directly.
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Lathyrus pratensis
Observed: 9th August 2011 By: synan
Added: 24th August 2011
Other observations of Meadow Vetchling (Lathyrus pratensis)
Species with which Meadow Vetchling (Lathyrus pratensis) interacts
24 August 2011 - 5:34pm synan
Thank you allrounder. I may have photographed the wrong plant, unless this also has hollow stems.
24 August 2011 - 6:44pm allrounder
I based my id mostly on the leaf shape. Also, Greater Birdsfoot trefoil has 9-12 flowers in the head and has a 'tighter' look to it. Meadow Vetchling has a 'looser' look. Also, habitat can plays a role, MV is mostly found in dry grassy habitats whereas GBFT is most often found in damp grassy places such as marshes and fens. Hope this helps. Gary
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Beijing (XFN-ASIA) - Air China (SHA 601111; HK 0753) said it
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Chinese-Style Broccoli Salad Recipe2016-07-28
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- 2 heads fresh broccoli
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Cut broccoli into bite-sized florets and peel the tough skin off stem and quarter it into 2 inch pieces cut slantwise.
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The New-England Weekly Journal, 1727-1741.
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We know everyone is busy and often our RSS readers get filled with too many interesting articles to keep up with. Here are summaries of a few of our posts and some by other authors that we particularly enjoyed.
Why A Technology Leader Should Code
The military teaches that a leader should be "technically and tactically" proficient. Military leaders owe it to their subordinates to understand the equipment that the unit employed and the basic combat tactics that would be followed. This concept is transferable to technology companies; the CTO owes it to their subordinates to understand the technology. They also owe it to the business to understand the economic aspects of the business and be able to straddle these two worlds. Additionally, periodically having to code a feature and deploy it will provide the engineering manager a better understanding and appreciation for what her engineers go through on a daily basis. Read more…
What Is That Delay Costing?
Most technologists know that the slower the page the more likely the user will flee the page or the transaction flow and not make the purchase. Research is teaching us that it may be less important to reduce actual delay rather than create a system where users will be less likely to attribute the delay to the site. An example that we sometimes see is to give the user the option of selecting a low or high graphic site in order to provide the users with the control. Users will likely perceive this as an active effort on the part of the SaaS provider to minimize download time and thus attribute delays to themselves, their computer, their ISP, etc but not the site. Read more…
DevOps is an umbrella concept that refers to anything that smoothes out the interaction between development and operations and is a response to the growing awareness of the disconnect between development and operations. There is an emerging understanding of the interdependence of development and operations in meeting a business' goals. While not a new concept, we've been living and suggesting ARB and JAD as cornerstones of this coordination for years, DevOps has recently grown into a discipline of its own. Read more…
Google Megastore
Google provided a paper detailing their design and development of "Megastore." This is a storage system developed to meet the requirements of today's interactive online services and according to the paper it blends the scalability of a NoSQL datastore with the convenience of a traditional RDBMS in a novel way, providing strong consistency and high availability. The system's underlying datastore is Google's Bigtable but it additionally provides for serializable ACID semantics and fine-grained partitions of data. Read more…
Scalability at the Cost of Availability
One subtle concept that is sometimes misunderstood is that if not careful an increase in scalability can actually decrease your availability. The reason for this is the multiplicative affect of failure with items in series. If two pieces of hardware independently have 99.9% uptime, when combined into a single system that relies on both to respond to requests, the availability of the system to go down to 99.9% x 99.9% = 99.8%. Read more…
8 Lessons We Can Learn From The MySpace Incident
Robert Scoble wrote a case study, MySpace's death spiral: insiders say it's due to bets on Los Angeles and Microsoft, in which he reports that MySpace insiders blame the Microsoft stack on why they lost to Facebook. Some lessons can be gleaned from this including All computer companies are technology companies first and Enterprise Programming != Web Programming and Intranet != Intranet. Read more…
Aztec Empire Strategy: Use Dual Pipes For High Availability
The Aztecs built the great aqueduct 600 years ago but even then thought about uninterrupted supply. This post states that the purpose of the twin pipes was to keep water flowing during maintenance. When one pipe got dirty, the water was diverted to the other pipe while the dirty pipe was cleaned. Read more…
Research Update and Request for Help
Marty and Mike will both be presenting their research at the 2011 Academy of Management Conference. Marty's research deals with tenure based conflct and Mike's research is focused on social contagion (a.k.a. viral growth). You can read the abstracts and full text for both papers here.
We are continuing our research and could use your help. Please consider completing one or both surveys.
If you are an executive team member at a startup, please take this survey and pass it along to your colleagues within your company.
If you participate in any of the following social networks (Facebook, Friendster, LinkedIn, Twitter, MySpace, Ning, Orkut, or Yahoo!360), please take this survey and pass it along to your friends or colleagues.
Thanks for your support!


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Amazon offers the ASUS ROG STRIX Radeon Rx 470 4GB OC Edition AMD Graphics Card (STRIX-RX470-O4G-GAMING) for a discounted price of $149.99 after $20 rebate plus free shipping. Limited time offer. You save $50 off the price! That's the lowest price we could find. This has a 4.3 out of 5 star rating on Amazon. You should have no issues with running the vast majority of games out there on high settings with this card.
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PCMag Digital Group


text: 03 Dec 2016 Secret London: 14 odd attractions you never knew were there Previous slide Next slide 16 of 16 View All Skip Ad Best of the rest Wilton's Music Hall (wiltons.org.uk) near Tower Hill, for its live shows, quirky events and the very fine Mahogany Bar; Dennis Severs' House (dennissevershouse.co.uk) in Spitalfields, "the closest you will ever get to living in grubby 18th-century London"; the gruesome Old Operating Theatre (thegarret.org.uk) in Southwark; The London Wall (examples at Tower Hill and Barbican); London's smallest police station (the southeast corner of Trafalgar Square); and Hyde Park's pet cemetery (which can only be seen by appointment with local police). -- Taken from National Geographic London Book of Lists: The City's Best, Worst, Oldest, Greatest, and Quirkiest (National Geographic Books; ISBN 978-1-4262-1382-3) by Tim Jepson and Larry Porges. GETTY Back to image


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OTOBI Limited get jobs opportunity
Today OTOBI Limited get jobs circular 2016.OTOBI Limited is company type job.They take young people with high salary.Its a great offer for unemployed people.OTOBI Limited related all current information has been found my website www bd cakri.com.People who interested this job they can apply until 30 November 2016.
OTOBI Limited get jobs opportunity
OTOBI Limited
Job Description / Responsibility
- Daily production report generation
- Prepare requisition of raw materials & internal components based on production schedule & forecast (weekly/ monthly)
- Prepare purchase requisition of local item of ETO product
- Follow up materials stock & receiving status against requirement
- Materials (stock) transfer from others plant
- Master data correction (BOM & Routing in SAP)
Job Nature
Educational Requirements
- Bachelor's Degree in any discipline or Diploma in computer science/ computer engineering
Experience Requirements
- 0 to 2 year(s)
- The applicants should have experience in the following area(s):
- The applicants should have experience in the following business area(s):
Manufacturing (FMCG)
Job Requirements
- Age 20 to 30 year(s)
- Hands on experience with MS-Excel
- Knowledge of entry procedure
- Willingness to work in shift duty is essential
- Values: Analytical, Hard working, team spirited etc.
Job Location
Salary Range
- Negotiable
Other Benefits
- As per company rules
Applicant must enclose his/her Photograph with CV.
Application Deadline : Nov 30, 2016
Company Information
OTOBI Limited
Get more details connect with our Facebook Fan Page and keep on your eye with my website http://bdcakri.com/
Address : Otobi Centre, Plot – 12, Block CWS(C), Gulshan South Avenue, Gulshan – 1, Dhaka – 1212
company : Furniture manufacturing & marketing.


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Bachelor degrees from accredited colleges and universities can be important stepping-stones toward a successful career.The most common type of undergraduate program is a bachelor's degree, usually awarded after four years of successful study
A landlocked country found in the Central Europe region with Hungarian as their official language. The country's mathematics and science approach are outstanding. It homes the world's first technology institute. It is here that the first steam engine in Europe was built back in 1722.
Budapest is the capital city of Hungary and the ninth largest in the European Union. As the largest in the country, it serves as a center for commercial and industrial activities among others. It has close to twenty institutions of higher learning within its borders.
Request Information Bachelor Degrees in Photography in Budapest in Hungary 2017
Photography is able to simultaneously recreate the world of facts, imagination, fiction and dreams. We encourage our students to express their ideas through the means offered by this creative outlet; we teach them how to use the visual design language of photography with analog- and digital technical tools. [+]


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Katerina KalchenkoKaterina Kalchenko is a Energy EFT Master Practitioner Trainer in Kiev, Ukraine, Ukraine
Katerina Kalchenko is a professional member of the Guild of Energists.
Katerina Kalchenko is listed in: Energy EFT Master Practitioners in Ukraine Trainers in Ukraine
These websites are provided to all professional members of the Guild of Energists (GoE). The GoE is the worlds largest modern energy organisation and supports techniques such as Energy EFT and EMO. To find out more about the GoE and how to join see:


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ARLINGTON, Texas -- For the record, Roy Williams finished with five catches for 83 yards and no touchdowns.
But one reception, a 47-yarder, will be remembered as what turned things around in the Cowboys' 30-27 loss to the New Orleans Saints on Thursday.
As the Cowboys were trying to run out the clock or increase their lead to two scores, Williams caught a nice pass from Jon Kitna and appeared headed toward the end zone. As he was running down the field, Williams switched the ball from his right to his left hand to avoid a defender.
Williams didn't see Malcolm Jenkins, who came from behind and stripped him of the ball with 3:03 to play in the fourth quarter. The turnover gave the Saints new life, and they would eventually score the go-ahead touchdown when Drew Brees found Lance Moore on a 12-yard slant with 1:55 to play.
"I had two hands on it, but the guy made a great play," said Williams, who sat at his locker in full uniform as reporters surrounded him. "I'm accountable for it. I lost the ballgame. I will say that. I let my teammates down."
Jenkins said he made a mistake in coverage on the play and tried to do something after Williams ran past him.
Williams has endured heavy criticism from the media and fans since the Cowboys made a trade for him in 2007. He hasn't had a 1,000-yard season or become the playmaker Jerry Jones thought he was getting from the Lions.
A variety of reasons led to this, including the emergence of Miles Austin and Dez Bryant, and Williams himself.
"It's not on him; it's 45 of us," Austin said. "It's on everybody. We all have to be in a position that even if something happens. You know we'll still come away with it."
Said Kitna of Williams: "He made great plays all day. Even after that he made a great play to get us a first down on that last drive. He was trying to to do the right thing. He had two hands on the football. Trying to go down, it just kind of got you off balanced where you're almost running backwards. It's kind of one of those scary feelings."
Still, Williams believes he cost his team the game and said he could have almost jumped from the Saints' 11, where he was stripped, to the end zone.
"It's late in the game," Williams said of the fumble. "That's the nail in the coffin right there. The nail in the coffin. We had the momentum swung our way. We were there headed to a W. That's a W if I just get tackled or just long jump my way into the end zone. Somehow, some way, we win the ballgame. Just fall down, we win."


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Train Sim World: CSX Heavy Haul Review (PC)
Train Sim World: CSX Heavy Haul is the first release in a new franchise for Dovetail Games. If freight is your thing, this first person simulation is for you
Train Sim World: CSX Heavy Haul is the first release in a new franchise for Dovetail Games. If freight is your thing, this first person simulation is for you
Whether it's as a pure character, devoting their life to god, or as a schemer making their deals with the devil, there is a lot of fun in Monks & Mystics!
Toy Odyssey highlights the best and worst in roguelites. When it gets going it can be exhilarating. But it can take you an awful long time to get there.
Aside from the lack of license there is very little to complain about. Playsport Games and SEGA have delivered a gem of a sport sim in Motorsport Manager.
Let us hope that the robot haulers of the future are even half as persistent as the pearly white 18-wheelers in Clustertruck.
Batman: The Telltale Series is an essential Batman story. It's a pretty good Telltale game too, almost up to the standards of their greatest titles.
Its difficulty may prove to be a bit much for some, but fans of challenging platformers like Super Meat Boy should add Flywrench to their collection.
If you have an Oculus with Touch controllers, then there really is no reason that you should not be checking out SUPERHOT VR.
While some may struggle to look past the dated visuals of Mound & Blade: Warband, those who can may find an intricate game that rewards perseverance.
Rive, means to tear or spilt apart violently. It is a good title for a game that was announced as the final product from developer Two Tribes.


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Exceptional multi family home (2 grand main residences), Completely rebuilt in 2007. Very high quality materials. 6 bdrms (could be 7),9 baths(7 full 2 half) 5 fireplaces, an elevator, 3 laundry rooms, outdoor kitchen overlooking the lake. 2 kitchens (1 is at main home 2nd is a full canning kitchen). lots of great parking (4 cars inside garage & 2 additional behind wall). personal bthrms for every bdrm, radiant heat in bthrm floors. 3 mstr bdrms have private balconies. besides all this there is a separate cabana which is a perfect office or in-law/guest suite. Perfectly level parcel with a boat dock includes boat lift. Radiant heated floors in bthrms.Last 3 photos are LOP generic.
上市日期: 2016年4月26日


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After bronze in Los Angeles, the ultimate prize in Seoul - and the attention that it brought to a sport which had been unsung for generations - was supposed to herald a new era of success.How could anything go wrong? Britain had struck gold despite minimal financial assistance, and now sponsors were queueing up to throw money at the sport.
That 1988 triumph did indeed yield an unprecedented legacy - sponsored leagues, full-time coaching positions for many leading players and a new pounds 9m national hockey stadium in Milton Keynes which was baptised in March with an international between England and India.
The failure to win any medal at the 1992 Olympics in Barcelona did not unduly hinder the sport's growth in popularity and participants, but a far-from-satisfactory build-up to Atlanta, which came to a head a couple of months ago when the players controversially ousted their coach, David Whitaker, suggests that all is not set fair.
Steve Batchelor, a member of both the 1984 and 1988 British teams, feels that playing standards may have declined since he retired despite the fact that many more players are effectively full-time. "I don't have much contact with the sport at international level these days," Batchelor said, "but I went to England's 2-1 defeat by Germany at Milton Keynes a few weeks ago and I was a bit disappointed with what I saw. Having said that, the squad's trip to the West Indies is just what they need. They will do some light training out there, but most important they will be getting away from the spotlight and relaxing. Also, in a tournament of just 12 sides, you've always got a chance."
Roger Self, whose 10-year career as Great Britain team manager was crowned by the Seoul gold medal, is in a better position than most to assess what is needed in Atlanta. He acknowledges that a successful Olympiad is essential but believes the present side can confound their critics.
"We do have a lot of natural ability in this country," Self said. "Over the last 12 weeks of preparing for the Olympics, the design side of things seems to have been completely fouled up. But I remember the Denmark footballers hauling themselves off their holiday beaches at a moment's notice to win the European Championship in 1992. It could be that the team spirit created because things have not gone well will work in our favour.
"Our own preparations in 1988 were not much better than this time, especially as we went to Pakistan just before the Games and got rolled over five times. The key thing then, as now, was that there was optimism about our prospects."
In reality, it is not possible to feel too optimistic about those prospects, especially when the team's captain, Jason Laslett, says "our draw in Atlanta is the toughest I've ever known". The opening game on 21 July is against South Korea, a team expected to thrive in Georgia's summer heat. The other members of Britain's six-strong group are Holland, Australia, South Africa (all touted as potential medal winners) and Malaysia. Only the leading two sides after the round-robin section will progress to the semi-finals.
Including the Olympic pre-qualifying tournament in Barcelona, the players have turned out (either as England or GB) in nearly 40 internationals over the past seven months, producing mainly moderate performances. They have also visited Belgium, Egypt, Sardinia, Malaysia, Atlanta and Amsterdam - a punishing schedule by anyone's standards, and one which ultimately cost its architect, Whitaker, his job.
The players reportedly resented being asked to put their careers on hold to devote themselves full-time to the Olympic cause while their coach, who was reportedly paid more than pounds 25,000 for fewer than 100 days a year by the Great Britain Hockey Board, was quite often absent pursuing his own profession as a motivational consultant.
Rumours abound that the players have been exhausted physically and mentally by their schedule and are depressed about their preparations for Atlanta, but Laslett claims that morale is high. "It's been a long haul - the last few months have been chaos but I think overall we have prepared as well as we could. The two coaches who took over David Whitaker's responsibilities, John Copp and James Duthie, were with us anyway so there hasn't been much disruption."
Things looked up last Thursday when GB thrashed Italy 7-1 at Bisham Abbey in their farewell game with the penalty corner specialist Calum Giles netting a hat-trick. Self believes that result could be just the boost the team needs. "From what I've seen, they're useful, young and holding their corner," he said. "Now let's hope they can get their act together like we did in 1988." For one person, at least, Seoul is anything but consigned to oblivion.
- More about:
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Grow your business by managing supplier relationships: e-book on supplier relationship management
Failing to strengthen your relationships with business partners may damage your growth prospects
Partners support your business strategy, supply goods and services and deliver your products to end customers. If your partner relationships are not as strong as they could be, you may be missing out on opportunities to grow your business. This is when supplier relationship management, managing supplier relationships with technology and e-invoicing network can play a key role.
Download this supplier relationship management ebook to discover how technology and e-invoicing can help you in strengthening your customer and supplier relationships to provide visibility, productivity, compliance and growth.
"Consider moving to a vendor that offers a more comprehensive solution that includes a procurement network to support supplier collaboration"
Source: Technology Insights for Procurement Insights, Gartner 2016


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Not many websites let you see the inside of the bag, so let me show you.
On one side, we have a zippered pocket (where you can store stuff you don't want other people to see). On the other side, there's a cell phone pocket and a flat pocket you can store your keys/ipod. I'm sorry it's kind of hard to see. But it's there! I purposely used my cell phone to model it for you.
There's a story that came with this bag:
I signed up to be a Karmaloop representative last summer around the same time I started this blog. I didn't heavily advertise it or anything because that wasn't my goal for this blog. I did, however, post my rep code to coupon websites to help generate some exposure as well as let my readers know about deals I find on Karmaloop, such as this HK bag. Either way, in December, someone used my rep code and bought a generous amount of stuff, so I got quite a number of points from that. I didn't even know because Karmaloop doesn't notify their reps when they make a sale. It was just a fluke that I even saw it.
Anyway, as a Karmaloop rep, once I earn a certain amount of points through sales, I can submit a number of banner designs to earn additional points within a time frame after the sale was made. So from the points I made through sales and submitting in creative banners, I managed to trade in the points for a gift card and used it towards this handbag!
I feel really lucky and am so grateful for whoever used my rep code back in December... So, how do you like my new handbag?


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Maximum current/power transfer for minimal lags
100 ft. of 18 Gauge OFC Speaker Wire
100ft. of 18 AWG Oxygen Free Copper Speaker Wire Cable • Translucent Jackets
Only 1 Left!
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60-Day Money-Back Guarantee
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Availability: Out Of Stock
The adventures of Peter Parker unfold in his college years after a radioactive spider bite transforms him into the caring crime fighter, Spider-Man. His fearsome battles against the forces of evil sit uncomfortably alongside a job as freelance photographer at the Daily Bugle, romantic encounters with the editor's secretary Betty Brant and caring for his elderly Aunt May Parker.
The 1980s syndicated sequel to the original animated series elevates Marvel's® most famous character, Spider-Man, from comic books to the television screen. Adapted from the newspaper sketches of John Romita Sr., this DVD release features the first seven spine-tingling episodes of the series.
A tidal wave of crime engulfs New York as the webbed wonder Spider-Man swings in to protect the city from an amazing ensemble of classic villains including Doctor Octopus, the Lizard, Sandman, Magneto and Mysterio. Meanwhile a six-part story arc kicks off with his battles against Doctor Doom.
Episode #1 Bubble, Bubble, Oil and Trouble
The mechanical mastermind Doctor Octopus launches a spate of diamond thefts in a slick plot to steal the world's oil supplies. Can Spider-Man put the skids on his priceless plan?
Episode #2 Doctor Doom, Master of the World
World stability is undermined as would-be-ruler Doctor Doom brainwashes the President and the entire United Nations assembly. Only Spider-Man can prevent a robot invasion leading to diplomatic destruction.
Episode #3 Lizards, Lizards, Everywhere
Jungle madness descends on the city as reptiles from Central Park Zoo go on the rampage. With the Lizard planning to turn New York into a swamp land, Spider-Man has to hunt down the amphibian aggressor.
Episode #4 Curiosity Killed the Spider-Man
New Year arrives with a bang when the jewel thief Black Cat creates bedlam at the Bugle. Spider-Man gets caught up in a game of cat and mouse as she sets her beady eyes on a famous artifact.
Episode #5 The Sandman is Coming
Sandman steals radioactive soil samples from space to unleash a wave of bank robberies. Hopes of a happy ending are sinking fast as the villain slips Spider-Man's grasp and Peter Parker faces expulsion from college.
Episode #6 When Magneto Speaks....People Listen
The Earth is held to ransom when Magneto hijacks power supplies by satellite. Spider-Man engineers a hi-tech showdown with the manipulative mutant in a bid to shield the nation from a communications disaster.
Episode #7 The Pied Piper of New York Town
The youth of New York are entranced by disco music at the hands of nightclub owner and master hypnotist Mysterio. Spider-Man will have to record his greatest hit to escape an explosive ending.
Due to the fact that this is archive footage there may be some imperfections in the picture quality.


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The boys love playing with this cardboard box! They played construction site for hours.
Wednesday, March 15, 2017
Tuesday, March 14, 2017
GOBBLE IT UP REVIEWS : Rose Art
GOBBLE IT UP REVIEWS : Rose Art: Rose Art is a brand we include in our Easter baskets every year. From art products to outdoor activities, they have whatever we need. I...
Friday, October 21, 2016
My Lil pumpkins!
I decided to do this post to share some really adorable play spaces besides a few pictures of my sweet grand babies...!
Is this not the sweetest...such a fun day picking out pumpkins!
and don't you just love making indoor forts on rainy days
Here is Thomas making pumpkins out of pumpkin scented clay
Below is another fun day... Thomas and Luke at the farm market!
Take a look around it is so darn cute!
after that they spent time at this adorable pet clinic
And a little time...under the sea! This is such a cute idea!
Picking our own string beans was such a treat!
I hope you all enjoyed my pictures ......I will be around...not as often as I'd like...but I hope you still enjoy stopping by once in awhile.
Wednesday, October 12, 2016
Happy Fall!
This is a wooden pumpkin that I had my hubby cut it out for me.
I then painted it to my liking.
Tuesday, May 5, 2015
Vintage Bettys Sign!
My sister, Connie and I are opening up a vintage thrift shop in the very near future! Our shop is also going to be named Vintage Bettys after my mom. This is a picture before the final editing of the sign...that is a picture of my mom when she was on her honeymoon in the poconos. Tell me what you think about the sign...Does it need something more?...Opinions are welcome.
Monday, February 16, 2015
Paper Clay snowman!
Thursday, January 1, 2015
Merry Christmas and wishing everyone a Happy New Year!
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Welcome to the end of the world4.39 / 5.00 2,740 Views
Living Dolls: No Evil 024
Wouldn'tyou just like to... leave conflict.4.29 / 5.00 680 Views
Cortex's Airship
What happened to Cortex's airship?4.24 / 5.00 2,559 Views
i liked the part with the cars
For the 4th attempt you should have made him like" i can't do it i can not jump" Then have the other guy push him out of the plane. hahahaha
Pretty damn good
Lol. That was funny when he got ran over by all those cars. And at the end when you put that part about the guy getting his nuts blown off! Very funny!
i loved it
since a love blood and gore i think this is the bomb
if you could make another one with more attempts it would have been even better.
even if u dont like it stay until the end loool!!!
not too gd but its all worth it at the end looooooooooooooooooooool
Yay, this is going into my favs. now, nice one man, i love yer stuff.....it rules!!!


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Northern Soul & Motown Night - Tranmoor Lane
Armthorpe Coronation club Tranmoor Lane DN3 3BS GB
Soul Nights
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Northern Soul & Motown Night with DJ Mark Taylor & guests playing the great sounds of the twisted wheel, mojo, casino, ect, all in one room, club prices on drinks, free entry, free parking
Venue Details
Event AddressArmthorpe Coronation club, Tranmoor Lane, Armthorpe Doncaster, DN3 3BS, United Kingdom
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Cambridge have showed alarming switches of form, allowing their reserves a sniff in the first pair of two-minute rows and showing real authority in the second.
There was more excitement in the veterans' race, in which the oarsmen have to average more than 40 years and to have rowed for the university. Curiously both colours rely on reserve crewmen from Isis and Goldie. Perhaps they try harder and carry on rowing longer to work off unfulfilled ambitions.
The Cambridge veterans led from the start but allowed Oxford to take a small lead as they came towards Barn Elms. However, after that the race was wrecked with a series of clashes in which, among other damage, the Oxford No 6, Tom Cadoux Hudson, completely sheared his stretcher and was obliged to complete the race pressing on air. Cambridge needed no second chance and cruised on to win by a length.Reuse content