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TransLoc's New On-Demand Microtransit Model Sees Broad Adoption by Transit Agencies Nationwide
TransLoc
Public transit agencies and cities take the lead on innovative approach to delivering flexible demand response transit options for all
Sacramento Regional Transit (SacRT) SmaRT Ride shuttle (Photo: Business Wire)
DURHAM, N.C.–(BUSINESS WIRE)–Microtransit solutions provider TransLoc is experiencing unprecedented adoption and expansion of what the company calls agency-owned microtransit. Acquired by Ford Smart Mobility in early 2018, TransLoc's accelerating growth is being fueled by a reimagining of public mobility within transit agencies as cities embrace economic growth, equitable mobility for every citizen and the creation of smarter, more sustainable cities now and into the future.
Microtransit solutions provider @TransLoc is experiencing unprecedented adoption and expansion of what the company calls "agency-owned microtransit" with more than 40 cities—by far the largest roster of any microtransit company in the industry today.
"While ride-hailing services, such as Uber and Lyft, have demonstrated to transit agencies that on-demand services can be successful, other attempts to help transit agencies bridge the gap have missed the mark," said Doug Kaufman, CEO of TransLoc. "We are taking a fundamentally different approach, and clearly the market is responding. This next generation of microtransit is designed to empower transit agencies to be central to new on-demand services, with improved convenience and reliability that have begun to bring new riders to public transit."
TransLoc's approach, which enables transit agencies to be central to new flexible and on-demand microtransit services, is attracting cities from across the U.S. at an accelerating pace. TransLoc is currently partnered with more than 40 cities—by far the largest roster of any microtransit company in the industry today.
One of the earliest adopters of TransLoc's agency-owned approach to microtransit, Sacramento Regional Transit District (SacRT), recently announced an expansion of its microtransit pilot service called SmaRT Ride. Within six weeks of the service's launch, SmaRT Ride expanded from its original service area to include several additional suburban towns and points of interest to meet rider demands. SmaRT Ride ridership has increased by almost ten times more than previous service levels. As a result, SacRT launched the next phase of SmaRT Ride in mid-summer, further expanding services into other diverse communities around Sacramento.
"The transition to this app-driven on-demand service has completely changed our transit system and made it viable for all of our residents," said Steve Miller, Mayor of Citrus Heights, California in the Sacramento metro area. "The feedback's been fantastic, SmaRT Ride has really been received well. We've seen our ridership numbers grow. I like bragging that Citrus Heights was the first ever city-wide microtransit system, not only in the nation, but the world. We have to find ways to provide mobility in creative ways to our residents."
As a result of SmaRT Ride's success, SacRT was recently awarded a $12 million grant by the Sacramento Transportation Authority (STA) to promote microtransit service in residential and commercial areas that are underserved by public transit.
"One of the benefits of microtransit is that you're not following our schedule. We're, more or less, following yours," said Mark Lonergan, Deputy General Manager and Chief Operating Officer at SacRT. "We all know how to run demand-response service. You need to be open to try and run it in a better way. And that's what microtransit does. That's what companies like TransLoc have to offer us: a better way to reach the public, to schedule their trips in a way that is very customer-friendly."
About TransLoc
Hailed by Fast Company as one of the world's most innovative companies in transportation, TransLoc, a Ford Smart Mobility company, is the technology provider of flexible agency-owned microtransit solutions for public transit to deliver the ultimate rider experience. By offering the expertise to plan and deliver flexible, future-proof transportation solutions with the company's cloud-based OnDemand microtransit system built for rapid deployment and hands-free operations, TransLoc is enabling transit agencies nationwide to remain central to the future of modern transportation. TransLoc has partnered with hundreds of transit agencies to reach a higher level of service and win the confidence of the riding public for more than a decade. TransLoc is also proud to be a Google Partner. To learn more, visit TransLoc.com or follow us on Twitter at @TransLoc.
SHIFT Communications for TransLoc
Meg Brenner
TransLoc@shiftcomm.com


Organization of the Islamic Conference Secretary General Ekmeleddin Ihsanoglu has strongly condemned the Israeli troops' assault on the Al-Aqsa Mosque and warned of dangerous consequences.
Ihsanoglu said the illegal entry of Israeli police into the Al-Aqsa Mosque compound on Sunday violated international law protecting houses of worship.
He associated the move with Tel Aviv's decision to designate two shrines on Palestinian territory as Israeli national heritage sites, saying the decision would impede efforts to revive the Middle East peace process.
"Such a breach, which comes days after the Israeli government's decision to illegally add the Ibrahimi Mosque and the Mosque of Bilal bin Rabah to the list of Israeli heritage sites, is a dangerous development in the Israeli scheme to stifle Islamic sanctities," Ihsanoglu said in a statement issued on Sunday.
The statement added, "Any damage to the Al-Aqsa Mosque and other holy places will have serious consequences with unpredictable danger to international peace and security."
Clashes erupted on Sunday morning when Israeli troops using stun grenades, tear gas, and rubber bullets broke into the courtyard of the Al-Aqsa Mosque compound in occupied Jerusalem Al-Quds, beating Palestinian worshippers with the butts of guns and clubs.
At least 17 Palestinians were wounded and seven others were detained during the fierce confrontations between Palestinian activists using stones, shoes, and bare fists and armed Israeli troops in and around the Al-Aqsa Mosque.
Muslims believe the frequent Israeli attacks on the Al-Aqsa Mosque compound are part of the Judaization campaign that targets the holy city of Jerusalem Al-Quds and are meant to provoke the sentiments of Muslims.


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Rubin 'Hurricane' Carter's Final Wish02:29
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Carter's dying wish is to get another innocent man released from incarceration. (Hamish Blair/Getty Images)
I have a soft spot for "where are they now" stories, though I've heard enough of them to understand that sometimes they are not in a good place.
Almost 15 years ago, in connection with a fine biography by Jim Hirsch titled Hurricane: The Miraculous Journey of Rubin Carter, I met Mr. Carter, who had been a fearsome and successful middleweight boxer, though by the end of his career he was losing as often as he won. After I'd interviewed him, we sat and talked for some time. He spoke softly and smiled often. My older daughter, who was 12 at the time, had joined me at work that day. She still remembers Rubin Carter with enthusiasm.
[sidebar title="50 Years After Clay vs. Liston, Where Does Boxing Stand?" align="right"]It's been 50 years since the heavyweight boxing match between Muhammad Ali — then Cassius Clay — and Sonny Liston. Bill Littlefield reports on the significance of that bout and the state of boxing today.[/sidebar]In 1966, Carter had been convicted of murder and sent to prison. 19 years later, he was released by a judge who ruled that his conviction had been a miscarriage of justice, a judgment with which Bob Dylan, among others, concurred.
What does a man who has been imprisoned for almost two decades for a crime he didn't commit do when he gets out?
Carter became involved with an organization in Toronto called the Association in Defense of the Wrongly Convicted. For a time he served as its director. He has been credited with helping to free a score of people who should never have been incarcerated. He was awarded honorary doctorates of law by Griffith University in Australia and York University in Canada.
I only know where Carter is now because the New York Daily News recently published an op-ed that he'd written. Early in that piece, Carter acknowledges that he has terminal cancer and is, as he puts it, "quite literally on my death bed." In the next sentence he writes that his single regret is that he has not yet been able to insure that a man named David McCallum, who was convicted of murder when he was 16 years old, will be released from prison. Carter calls for District Attorney Ken Thompson to grant David McCallum a full hearing by the Brooklyn Conviction Integrity Unit.
Rubin Carter is no saint. He would be the first to acknowledge that. He had a criminal record when he joined the army, which is where he learned to box. In the op-ed piece he writes, "If I find a heaven after this life, I'll be quite surprised." But what an exceptional thing that a man who was imprisoned for nearly 20 years before a judge ruled that he should never have been there could step away from the bitterness to which he would seem to be entitled and say that his one regret is that he's been unable to be of sufficient service to a fellow human being.
That's where Rubin Carter is now.

redpajama_set_name: RedPajamaCommonCrawl


The Aquobot T2 was a great machine for the first year. Since then I have sent it back for repairs at least twice per year at a cost on average of $350.00 per repair.
I am very disappointed with the durability. When I spend $1200 for a pool cleaner I expect it to last for several years.
I have sent it for repairs and gotten it back and it lasted only 3 days before it needed another $350 repair. I am switching to something I can count on.
I am totally disgusted with this product!!!!


Cabins USA offers a wide selection of Pigeon Forge cabin rentals that are perfect for your Smoky Mountains vacation. With all of the exciting things you can plan on a Pigeon Forge getaway, our rentals rest in prime locations so you can experience it all.
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Cabins USA's Pigeon Forge cabins offer the ideal location close to all the exciting attractions of the Smokies. Renting a cabin gives Smoky Mountain visitors the advantage of enjoying fun-filled days near the Pigeon Forge Parkway as well as the chance to relax at one of our peaceful, spacious cabin retreats.
With amenities such as hot tubs, jacuzzis, game rooms, and theater rooms, you'll have everything you need to relax and unwind in one of our terrific rentals. We also boast vacation properties for every occasion. Whether you need a cabin for a romantic getaway for two, a family reunion, or a large group retreat, you can find a comfortable, affordable, and convenient space for you and your group of any size.
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For your stay in Pigeon Forge, you can enjoy convenience, privacy, and luxurious amenities in a beautiful log cabin. Whether you're in need of a rental with mountain views or a cabin that's close to great attractions like Dollywood, The Titanic Museum, and The Island - we've got you covered!
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Cabins USA strives to provide quality, affordable accommodations in Pigeon Forge and the surrounding areas for visitors staying in the Smoky Mountains. The beautiful mountains around Pigeon Forge offer a haven for visitors looking for rest, renewal, and relaxation. Better yet, there are tons of great shops, attractions, and restaurants that make Pigeon Forge a thrilling vacation destination with something for everyone.
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Many eye diseases, including diabetic retinopathy and macular degeneration, exhibit increased permeability of blood vessels in the retina.
These leaky vessels in the central part of the eye can lead to abnormal fluid accumulation and vision loss. Eye injections targeting a specific cytokine, vascular endothelial growth factor (VEGF), have transformed care for these diseases; however, not all patients respond well.
Research by University of Michigan Kellogg Eye Center shows that inhibiting a specific molecule, atypical protein kinase C (aPKC), either genetically or pharmacologically, reduces increased vessel permeability and blocks inflammation.
Blocking a PKC may help protect vision in patients with potentially blinding eye diseases.
"Our data reveal aPKC as an interesting target both for vascular permeability and inflammation and developing aPKC inhibitors may provide a new therapeutic option for blinding eye diseases," says David A. Antonetti, Ph.D., the Roger W. Kittendorf Research Professor of Ophthalmology and Visual Sciences.
"Our research may help patients with diabetic retinopathy, the leading cause of blindness in working age adults in the United States, and may also lead to new treatments for uveitis, a spectrum of diseases that leads to inflammation of the eye, as well as for retinal vein and artery occlusions," Antonetti says in a Journal announcement.
In a recent study in the American Journal of Pathology, he and colleagues explore the protective effect of an experimental small-molecule aPKC inhibitor. The pre-clinical effects are promising.
Good vision requires retinal neurons to send signals to the brain, and retinal neurons must be protected and kept in a healthy microenvironment within the eye.
The microenvironment is maintained, in part, by the blood-retinal barrier.
However, injury or chronic disease can weaken the blood-retinal barrier and increase vessel permeability.
Although VEGF and TNF, a protein that causes inflammation, possess distinct signaling mechanisms, both eventually activate a common pathway, aPKC signaling, to change vessel permeability. Further, aPKC promotes inflammation.
Using genetic mouse models, Antonetti and colleagues showed small molecule inhibitors to aPKC reduced vascular permeability and inflammation, according to the Pathology study findings.
Antonetti, Kellogg's scientific director, is one of the world's experts in the blood-retinal barrier that is essential to sight. He has spent two decades studying how this barrier develops and how diseases such as diabetic retinopathy damage it and lead to vision loss.
He is part of a team that is advancing approaches to protect and restore the barrier so that vision can be saved and restored.
The team, along with other University of Michigan colleagues, also is investigating the formation and loss of the blood-brain and blood-retinal barrier in cancer and stroke.
The work may not only benefit the millions of people with diabetes, but has implications for those who might have a stroke and other brain diseases.


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redpajama_set_name: RedPajamaGithub


PeakFreak is a plugin that converts audio input that falls into a certain frequency and amplitude range into midi notes of a certain length.
This can for example be useful if you want to trigger the bass drum from your sound expander via MIDI automatically by the bass drum from an audio track.
Good to see at least 1 developer grappling with the most needful thing (imho) in this category (audio to midi).


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Home Tags Cuba
Tag: Cuba
Paquito D'Rivera: Letters to Yeyito
If there is anyone who communicates through music and life it could be said that few people do it better than Paquito D'Rivera. If you have ever been to one of his concerts, you will know that he loves to introduce his songs with anecdotes...
Jazz at Lincoln Center Orchestra with Wynton Marsalis: Live in Cuba
We hear so much big band music these days that we may be at risk of forgetting where the priceless modern day markers really...
Artist Profile: Iván 'Melón' Lewis
Introducing Cuban Pianist, Composer, Bandleader Iván 'Melón' Lewis Born in Cuba, Iván 'Melón' Lewis received extensive training in classical music and graduated from the prestigious...
Tiempo Libre: Panamericano
The musical and theatrical finery of Panamericano is going to be quite the sensation in the weeks and months to come, and several years...
Talking In Cuban: Palacio de la Rumba
Palacio de la Rumba Big Band is an exceptional ensemble formed by Australian composer and saxophonist Gai Bryant On a trip to Cuba to research...
Pianist, Composer Alfredo Rodríguez
Alfredo Rodríguez (born October 7, 1985) is a Grammy-nominated Cuban composer and Cuban Jazz Pianist. He was trained at Manuel Saumell Conservatory, then at...
Ignacio Berroa has been recognized by many as one of the greatest drummers of our times. His numerous contributions to the American music scene have earned him a place among a selected group of artists known to have set new musical trends...
Estrellas de Areito: Los Héroes
Although much of the world was welcoming of Cuba after the Revolution and although many artists did flee the country for a new world of freedom and...
Buena Vista Social Club Featuring Eliades Ochoa and Omara Portuondo
http://youtu.be/Vt1E1zv0hiA http://youtu.be/jIuHqoAu1gc As Cuban revolutions go, it was an entirely peaceable uprising - but its impact could not have been more profound. On the release of...
Singer-Songwriter Xiomara Laugart
Originally from the Guantanamo province of Cuba, Xiomara Laugart began her career at the age of 15, performing several different expressions of traditional and contemporary Cuban music. During the 1980s, she entered the Adolfo Guzman Contest...
Mario Adnet, Elio Villafranca at JALC
Sounds of Brazil: Mario Adnet February 20-22, 2015 Dizzy's Club Coca-Cola - Jazz at Lincoln Center, NYC - Broadway and 60th Street, 5th Floor Set Times: 7:30...
Frank Emilio, Guillermo Barreto y su Cuban-Jazz Combo: Drume Negrita
The clave that is pronounced in Afro-Cuban music is almost stealthily present in Jazz and has been lurking secretly in that idiom ever since...
Adonis Puentes, Pancho Amat release "Veinte Años"
Cuban-Canadian Grammy nominee teams up with Cuban legend for a tribute to a Trova icon. Adonis Puentes and Pancho Amat release Veinte Años: Homenaje...
Julián Cabrera: Musician, Rumbero, Gentleman and Scholar
Julián Cabrera's style on the conga drum was rock-solid, never flashy, he preferred to keep the rhythm simple but lively, and, because he was...
Chucho Valdés At Flato Markham Theatre
On Friday, September 26, 2014, legendary Cuban Piano Master, Dionisio Jesús Valdés Rodríguez, better known as Chucho Valdés, offered a magnificent solo performance at...
Chucho Valdés Solo Concert in Markham
Jazz.FM91 Presents Chucho Valdés: With more than a half-century of innovation behind him, Cuban pianist Chucho Valdés is at the peak of his performing...
Cuba: The History that Anacaona Made
They were called the "Queens of Havana" in the 30's when it was anathema for young women to be seen at nightclubs, let alone...
Jane Bunnett: Finalist Premier's Award for Excellence in the Arts
Four-time JUNO Award winner, two-time Grammy nominee, and Officer of the Order of Canada, soprano saxophonist/flautist Jane Bunnett has been chosen as a finalist...
The Incredible Renesito Avich
René Avich Wanton, new exponent of the Tres guitar in Cuba. Winner of several national competitions: Festival Contest "Ignacio Piñeiro", "Cubadisco Award 2011", "Competition...
Jane Bunnett and Maqueque
The celebrated Canadian soprano saxophonist is embracing voices again. But these voices are of a slightly different sort. The Creole Choir...


A high waistband gives these Puma leggings classic comfort and support, while dryCELL wicking technology and sleek stretch fabric provide performance and a flattering look.
Hits at calf; approx. inseam: 28"


There are all kinds of opportunities in real estate: To invest, to build or rebuild. I have learned that there are major classification of types which go into these provinces of real estate.
In 1969, Peter Drucker wrote: "measured by the yardsticks of the economist, the last half-century has been the Age of Continuity." To me that means that there was a predictability -- rules you could learn and live by. The first half of this new century will be known as the Age of Discontinuity, which is the title of his great book written back then -- rules that were no longer valid. Even Drucker would not have expected volatility and mistrust to become the watchwords of the new millennium.
Continuity, like success, breeds arrogance. So much of success is pure luck and timing -- being at the right place at the right time. But the right place has no arrow above it, pointing it out as "the place to make money." Timing is mostly cumulative experience that becomes intuition -- the gut-feeling that this is the right place and time.
I have known many people gifted with that sense and even more who grew an arrogance that smelled up their organization. There was no room for personal growth; people hung around because they were well-paid and got used to the tyranny of the master -- though it ate them up inside and sacrificed their very souls.
1. Platinum Players: Already notably experienced, they have discretionary money to invest without any one deal crushing them. They have earned self-confidence with their success and accumulated knowledge. These are calculated risk-takers who gather as much feeling and evidence that they can feed into their intuition until it sparks the conclusion.
2. God-Complex Players: Decision makers to whom others look to save their lives and heal them. Notably, they are doctors who are used to making judgements and building a defense mechanism that protects them from feelings of failure and guilt. They are innocent of how this mechanism can make them terrible investors in something about which they know nothing. They need professional assistance in deciding risk and abstinence.
3. Innocent Players: Long for success and have role models they believe are successful in the field in which they will invest. Their innocence calls for professional guidance to illuminate the way, for they are on a severe learning curve and there is no exception to this.
Knowledge: Real knowledge rather than just information or the compilation of statistics. I believe in knowledge that is gained through personal experience and investigation of subjects like timing, location, demand and satisfaction of specific demand. If it is important enough not to lose money, then personalized investigation is wise and even necessary.
Field investigation: Visiting types of real estate to get a sense of differentiation between success and failure; when one touches it and speaks to the person who built, designed or financed it -- whatever "it" may be -- one gains insight into specific land use and real estate types.
Borrowing the experience of others: Making contacts with lenders who have lent financing for projects; finding why and what they would do differently now. This I call "original research into case studies," and it is invaluable. It takes time and patience but is worthwhile.
Timeliness and perspective: There are windows of opportunity that open and close to form "timing" itself, which I believe is the most important ingredient to success. Taking the time to investigate lessons learned from case studies is so important. Impatience is probably the single most common reason for failure. It's like money burning a hole in your pocket; it attempts to accelerate the investigation and decision process, thereby penalizing the entire process. Fast Company magazine built an immediate reputation on its analysis of the new economy, and concluded that the old economy had a different rhythm. The new economy needed to be faster, more nimble and accelerate its entire management velocity. The magazine was wrong and it was a dangerous illusion.
Being busy: Busy people may make faster decisions, but they make larger mistakes in judgement. I have also concluded that most successful people are very busy ones who keep cutting down on the time they take to gather information and make decisions. They become more impatient, and less mature in their judgements.
In conclusion, take your time to find who you are, what you want to accomplish and on whom to rely as your sounding board. Since you will experience discontinuity between today and the last time you went through the process, your learning curve must be cultivated out of patience and astute observation.


Well now......look who has made a new milestone. Thought you could hit #500 and have nobody notice? Uh-uh.....congrats!
Congratulations my friend. For those of you who don't know Henry, he is a first class gentleman, 70 something years young but you wouldn't know it. I've cached with him many times and you won't have to wait for him. He piled up a bunch of caches this spring while recovering from shoulder surgery which has kept him off the links but not from the hunt for caches.
And he has hidden some tough caches as well!
So kudos to a first class gentleman. I'm sure 1000 will be in your future soon!
Congratulations Henry! It was nice to finally meet you at Gobblers event. Thanks for the FTF runs against Dubord and I look forward to more hides from you!
Congrats and on to many more.
Hat's off to you for 500. And now for that hard question I always ask.....which one was your favorite?
Congrats Henry You Are Nipping At My Heels Keep On Caching. Let Me Know If You Want A Ride To Do The Yorktown Caches.
About a year ago, my nephew lexmano invited me to go geocaching with him and his wife Barb. I didn't have any idea what it was all about so I went along for an introduction. The first find was in Augusta at the old Cony High School. The cache was named Gargoyle. I reached in a hole and there was the nano. I'd have to say that one of my favorites has to be a mail box named RFD in the Unity area. It has a false back that had me fooled.
I'd have to say that one of my favorites has to be a mail box named RFD in the Unity area. It has a false back that had me fooled.
My uncle Henry, or Bub as I have always known him, runs rings around Barb & I. We need to use technology to keep ahead of him. He has picked up this hobby with great enthusiasm. My mother is jealous because when I call her now, I am usually asking if she knows where Bub is!!
His numbers are slowing down because he has reached that place many of us have reached wherein there are no more local caches we have not found. I will try to get him out on a road trip soon to help his numbers.
congratulations my friend. We'll have to go cachin g togather again soon. Yoda can get us places you can't in a car. I know there are a few still in the area. Real nice to see your first and five hundreth finds were caches placed by me!
I'd have to say that one of my favorites has to be a mail box named RFD in the Unity area. It has a (spoiler deleted by WhereRWe?) that had me fooled.
I gotta agree that this was a very good cache. Had RULost2? totally flustrated! LOL! I usually stay in the car at these quick-grab-by-the-side-of-the-road caches, but I had to talk her through this one.


text:
Transcription profiles of the responses of chicken bursae of Fabricius to IBDV in different timing phases
Changbo Ou1,2,3,
Qiuxia Wang2,3,
Yanhong Zhang3,
Weili Kong4,
Shouping Zhang3,
Yan Yu3,
Jinyou Ma3,
Xingyou Liu2,3 &
Xianghui Kong1
Virology Journal volume 14, Article number: 93 (2017) Cite this article
Infectious bursal disease virus (IBDV) infection causes immunosuppression in chickens and increases their susceptibility to secondary infections. To explore the interaction between host and IBDV, RNA-Seq was applied to analyse the transcriptional profiles of the responses of chickens' bursas of Fabricius in the early stage of IBDV infection.
The results displayed that a total of 15546 genes were identified in the chicken bursa libraries. Among the annotated genes, there were 2006 and 4668 differentially expressed genes in the infection group compared with the mock group on day 1 and day 3 post inoculation (1 and 3 dpi), respectively. Moreover, there were 676 common up-regulated and 83 common down-regulated genes in the bursae taken from the chickens infected with IBDV on both 1 and 3 dpi. Meanwhile, there were also some characteristic differentially expressed genes on 1 and 3 dpi. On day 1 after inoculation with IBDV, host responses mainly displayed immune response processes, while metabolic pathways played an important role on day three post infection. Six genes were confirmed by quantitative reverse transcription-PCR.
In conclusion, the differential gene expression profile demonstrated with RNA-Seq might offer a better understanding of the molecular interactions between host and IBDV during the early stage of infection.
Infectious bursal disease virus (IBDV) is an important small RNA virus in the family of Birnaviridae, which may lead to severe immunosuppressive effects and pathological damage in young chickens. It mainly targets early B cells, especially those in the gut-associated lymphoid organ, the bursa of Fabricius [1]. After infection with IBDV, the bursa collected from chickens displays oedema and haemorrhage, or even necrosis, accompanied by a large volume of T cells infiltration and a cytokine storm [2]. Especially when chickens mix infection with IBDV and other pathogens, such as E. coil and Newcastle disease virus, farms can suffer large economic losses. Therefore, it is urgent to learn the pathways of IBDV invasion and replication in host cells and the interactions between IBDV and host, in order to determine effective strategies for the prevention and control of IBDV infection.
Transcriptional profiling analysis has displayed a great deal of information about host-pathogen interactions, including many important biological processes, cellular components and molecular functions. Thomas Ruby et al. have analysed gene expression levels in the bursae of young chickens from the resistant and susceptible lines by using a novel cDNA microarray and shown that the changes in gene expression in the target tissue during the early stages of infection of young chickens with IBDV [1]. Similarly, Wong RT et al. have screened differentially expressed genes in IBDV-induced apoptotic chicken embryonic fibroblasts by using cDNA microarrays and unravelled the candidate physiological pathways involved in host-virus interactions on a molecular level in vitro [3]. However, cDNA microarray is a closed system which only detects those specific genes. It can not offer comprehensive transcriptional landscape described in the cells and bursae post IBDV infection. We still do not entirely understand how the host defends against IBDV and how the infection influences the biological metabolism of the host at different stages of infection. With the development of high throughput sequencing, it is feasible to obtain transcript splice-variants, isoformas and new genes and compare the biological metabolism and immune responses of hosts at different infection points [4], which will provide new insights for understanding the pathogenesis of IBDV and the antiviral immunity of the host.
Birds and viruses
A classical IBDV strain CJ801 was kindly provided by Prof. Jue Liu of the Beijing Academy of Agriculture and Forestry, Beijing, China. All the studies were performed on 3-week-old specific-pathogen-free (SPF) chickens (Vital Bridge Co. Ltd., Beijing, China). The experimental procedures and animal management were approved by the Institutional Animal Care and Use Committee at Henan Institute of Science and Technology.
Experiment design
Eighteen SPF white leghorn chickens were randomly divided into two groups with 9 chickens for each group: the mock group (the healthy group) and the IBDV-inoculated group (the infection group). Chickens from the infection group were inoculated with IBDV CJ801 stock at a dosage of 103 EID50/0.1 mL through eye-nose drops [5, 6]. The chickens of the mock group were mock challenged with PBS. All chickens were kept in two separate isolator and they were free to drink and eat with 12 h light and 12 h dark. On days 1, 3 and 7 after infection, 3 chickens of each group were randomly selected and killed under anesthesia for bursa collection [7]. Each bursa was immediately put into liquid nitrogen and then stored in 80 °C refrigerator. RNA sequencing was performed with total RNA from bursae from each group at the first two time points and completed by a commercial company (Shanghai Biotechnology Corporation, Shanghai, China).
RNA extraction and purification
Total RNA was separately extracted from each bursa using RNAiso Plus Total RNA extraction reagent (TAKARA, China) following the manufacturer's instructions and checked for RNA integrity number (RIN) to inspect RNA integrity by an Agilent 2100 Bioanalyzer (Agilent technologies, Santa Clara, CA, US). Qualified total RNA was further purified by RNeasy Micro Kit (QIAGEN, GmBH, Germany) and RNase-Free DNase Set (QIAGEN, GmBH, Germany). The qualified RNA meets the following requirements: the RIN value was not less than 7.0, while the ratio of 28 s/18 s should be between 1.8 and 2.2.
cDNA library construction and sequencing
We used at least 3 μg of high-quality mRNA for each library construction. The mRNA was fragmented by incubating at 94 °C in fragmentation buffer to yield a size range of 400–500 bp, verified by an Agilent 2100 Bioanalyzer (Agilent). We used SuperScript II Reverse Transcriptase (Invitrogen) for the library construction. According to the corresponding process shown in the cBot User Guide, we completed the Cluster generation and the first sequencing primer hybridisation on cBot in Illumina HiSeq 2500 sequencer. Then, sequencing was performed using the Illumina HiSeq 2500 according to its standard protocols. The data for each sample was not less than 4.0 G while the percentage of Q20 (bases of Q > =20/all bases of sequencing) was not less than 90%.
After the sequencing was completed, the obtained image data was transformed into raw reads and stored in a FASTQ format. The raw reads were cleaned by a short-reads pre-processing tool (FASTX-Toolkit, version 0.0.13). The low-quality reads, including adapter, ribosome RNA and sequences shorter than 25 nt with Q < 20 at 3′ end, were removed. The resultant clean reads from each sample library were used for further analyses. The clean reads were mapped to the chicken genome (galGal4, Ensembl release 85) by a mapping tool TopHat2 (version: 2.0.9). The differentially expressed genes with fold changes ≥ 2 or ≤ 0.5 and False Discovery Rate (FDR) < 0.05 were analyzed using the web-based tools in DAVID to identify enriched gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, group functionally related genes and cluster the annotation terms. The raw sequencing data have been deposited in the Gene Expression Omnibus (GEO) at the NCBI under accession number GSE94500.
Confirmation of RNA-Seq data by quantitative real-time PCR
To verify the correctness of the RNA-Seq results, some genes were randomly selected for quantitative real-time PCR (qRT-PCR) experiments. CCR2 and CCR4 were used for their chemotaxis effects on monocyte, macrophage and dendritic cells. IL-6 is an important inflammatory factor. IFN-γ is over-expressed during IBDV infection and plays an important role in host antiviral responses. MHC-I molecule is to display intracellular protein to cytotoxic T cells. Like IL-2, IL-15 exerts a huge role in the process of virus infection. So, CCR2, CCR4, IL-6, IFN-γ, MHC-I, IL-15 were chosen in the study while β-actin was used as housekeeping gene [8]. The primers used in this study are listed in Table 1. Total RNA was extracted from chicken bursae by total RNA Kit (Omega Bio-Tek, Guangzhou, China) and then reverse transcribed into cDNA using M-MLV Reverse Transcriptase (Promega, CA, USA) following the manufacturer's instructions. The qRT-PCR was performed using a 7300 Real-Time PCR System (ABI, Britain) with SYBR® Select Master Mix (ThermoFisher Scientific, China). The reaction conditions were 30 s at 95 °C, 40 cycles of 5 s at 95 °C, 25 s at 58 °C and 30 s at 72 °C, followed by a final step of melting curve analysis. After amplification, the relative fold change of the differentially expressed genes was calculated through the 2−ΔΔCT method [9]. All samples were performed in 3 replicates as technical repeats to ensure the reproducibility of the amplification.
Table 1 Genes and primers for real-time PCR used in the text
VP2 gene expression during IBDV infection in chickens
After being challenged with IBDV, chickens from the infected group displayed some clinical symptoms on 1 dpi, such as decreased feed intake, fluffed feathers and diarrhoea. Autopsy results displayed haemorrhaging and necrosis on the bursae of Fabricius. However, chickens from the mock group displayed very healthy. To further confirm the success of viral infection, IBDV VP2 genes in the bursa from the infected group were quantified by real-time PCR on 1 and 3 dpi. The results showed that VP2 gene expression significantly increased in the infected group and was about 21 and 79 times higher than those of the mock group on 1 and 3 dpi, respectively (Fig. 1).
Quantification of the infectious bursal disease virus (IBDV) VP2 gene levels in the bursae of Fabricius in the mock group and the infection group by real-time PCR. The data were expressed as the average ratio of IBDV/β-actin. **Indicates P < 0.01 when samples taken from day 1 postinfection in the infection group were compared with those from day 3 in the infection group
Identification of differentially expressed genes between the mock group and the infection group on day 1 and day 3
To analyse the gene expression of IBDV-infected chickens, the total RNA was prepared from the bursae of Fabricius of IBDV or mock-infected chickens on days 1 or 3 after infection. In this study, we identified a total of 15546 genes in the chicken bursa libraries. As shown in Fig. 2a, among the annotated genes, we identified 2006 and 4668 differentially expressed genes in the infection group compared with the mock group on 1 and 3 dpi, respectively (FDR ≤ 0.05). Out of these, 1618 genes were the common differentially expressed genes with 388 and 3050 specific differentially expressed genes, respectively, on 1 and 3 dpi. Among the differentially expressed genes, there were 946 up-regulated and 179 down-regulated significantly differentially expressed (SDE) genes on 1 dpi while 2142 up-regulated and 1597 down-regulated SDE genes were identified on 3 dpi in the infection group with a fold-change ≥ 2 or ≤ 0.5 (Fig. 2b-c). Among these SDE genes, there were 676 common up-regulated and 83 common down-regulated genes in the bursae taken from the chickens infected with IBDV on both 1 and 3 dpi (Fig. 2b-c).
Identification and expression of the bursae collected from chickens inoculated with IBDV on day 1 and day 3. a The Venn diagram showing unique and common differentially expressed genes of the bursae collected from the infection group on day 1 and day 3 postinoculation with IBDV compared with the mock group. b The Venn diagram showing unique and common upregulated expressed genes, of which the fold changes were ≥ 2. c The Venn diagram showing unique and common downregulated expressed genes, of which the fold changes were ≤ 0.5
The host mainly displayed strong immune responses in the early stage of virus infection
The functions and pathways of the 799 differentially expressed transcripts were analysed based on the Gene Ontology (GO) project and KEGG using the SBC Analysis system. Top gene ontology clusters of the common SDE genes between the infection group and the mock group were mainly grouped into molecular function and biological processes, such as binding, response to stress, external stimulus, biotic stimulus, external biotic stimulus, virus, lipopolysaccharides and molecules of bacterial origin, immune responses and cytokine activity (Table 2).
Table 2 Top gene ontology clusters of SDE genes between the mock group and the infection group
KEGG pathway enrichment analysis showed that the differentially expressed genes were mainly clustered into the following pathways: cytokine-cytokine receptor interaction, influenza A, Herpes simplex infection, cell adhesion molecules and some signaling pathways (such as JAK-STAT, Toll-like receptor and RIG-I-like receptor) (Table 3).
Table 3 Top KEGG pathways associated with SDE genes between the mock group and the infection group
There were still some characteristic differentially expressed genes in different stages of virus infection
To compare the differentially expressed genes of bursae from different infection phase, we analysed the gene expressions of chickens from days 1 and 3 post inoculation. The heat-map has displayed that there were several differentially expressed genes between the day 1 samples and the day 3 samples (Fig. 3).
Expression profiles (heat maps) of SDE genes between days 1 and 3 post infection with IBDV (day 1: sample 1–3; day 3: 4–6). The color in the heat-map represents the normalised isotig number. The color in the heat-map represents gene expression changes. Red indicates high gene expression activity; green indicates low gene expression and black indicates no activity. SDE gene clusters were labeled in the left of the heat-map line
Compared with samples taken from day 1 postinfection with IBDV, KEGG analysis has shown that the highly expressed genes of samples collected from day 3 mainly focus on glutathione metabolism (ANPEP, GSTA, HPGDS and GSTA3), drug metabolism-cytochrome P450 (GSTA, HPGDS and GSTA3), adherens junction (EGFR1, SRC and YES1), ECM-receptor interaction (ITGA6 and LAMB1), tight junction (e.g CTTN, MYH11 and OCLN), focal adhesion (such as ITGA6, LAMB1 and MYL9), amino sugar and nucleotide sugar metabolism (e.g GNPNAT1, PGM1 and TSTA3). The decreased expression genes were mainly clustered into DNA replication (e.g. DNA2, FEN1 and PCNA), cell cycle (e.g CCNA2, CDK1 and HDAC2), intestinal immune network for IgA production (IL10 and IL6), ribosome (MRPS6, RPL13 and so on), Toll-like receptor signalling pathway (CCL4, IL8L2 and so on), Fanconi anaemia pathway (e.g. RAD51 and RMI2), JAK-STAT signaling pathway (CSF3, IFNA3, PIK3R5 and so on), AGE-RAGE signalling pathway in diabetic complications (e.g. NFKB1, TGFBR2 and VCAM1) and cytokine-cytokine receptor interaction (e.g. CCL4, IFNA3 and IL18) (detailed information is shown in Additional file 1: Table S1).
Validation of the RNA-Seq data by qRT-PCR
To validate the results of the RNA sequencing, qRT-PCR was carried out to determine the differentially expressed genes. Six (CCR2, CCR4, IL-6, IFN-γ, MHC-I, IL-15) genes were chosen for the qRT-PCR tests. The qRT-PCR results showed a similar expression pattern to that observed in the RNA sequencing analysis, even though the fold changes measured by the two methods were not entirely the same (data not shown, see Additional file 1: Table S2). The results revealed that the RNA sequencing results could represent all the gene expression variations.
IBDV can cause immune suppression of poultry and bring great economic losses for the poultry industry. The host generally displays a series of antiviral responses to IBDV infection, including natural and acquired immunity. Moreover, the host's responses have different characteristics in different stages of infection. Therefore, studying the hosts' immune responses in different stages of infection is very important for better understanding of the pathogenesis of pathogenic microbes. It is both reasonable and feasible to obtain these differentially expressed genes by the RNA sequencing along with the progress of transcriptome sequencing technology. In this manuscript, we report for the first time the transcriptome changes of chicken bursae during the early stage of IBDV infection by RNA-seq. The results displayed that, even if all samples were taken from the infection group, gene changes were not exactly the same for the days 1 and 3 post inoculation. Meanwhile, there were differentially expressed genes between the infection group and the mock group post-infection.
Following infection and replication of IBDV, lots of T cells infiltrate the bursae of infected chickens [10], while IBDV causes injury to B cells and macrophages [11], thus inducing a so-called "cytokine storm", such as proinflammatory cytokines, antiinflammatory cytokines, chemokines, interleukins, nitric oxide (NO) and so on. Obviously, it was no doubt that the primarily observing immune responses by host were displayed by the primary or secondary infected cells on 1 dpi. On 3 dpi, these immune signals might still be present, but they were being diluted and confounded by the responses of different (myeloid & lymphoid) cell types, some of which will be displaying their own activated phenotype. Therefore, it was difficult to work out the signals coming from the resident stromal or immigrating immune cells, especially with regard to the activation of those cells. In this study, though similar results were shown in IBDV-infected DF1 cells at the early stage of infection [12], significant elevations of some important genes expression levels were displayed in the infection group and inflammatory response genes, antiviral related genes and these characteristic differentially expressed genes on 1 and 3 dpi were mainly observed.
Inflammatory cytokine storm during the stage of IBDV infection
Inflammatory response genes, such as NOS2, IL6 and TNFRSF1B, were up-regulated while some positive regulation of inflammatory response genes, such as TLR3, STAT5 and LRRC32, were also elevated post-infection with IBDV. The chemokines (CCL4, CCL19, CXCL12) and their receptors (CCR5, CCR7 and CXCR4) were involved in inflammatory responses. Among these genes, LRRC32 (leucine rich repeat containing 32, also known as GARP) is critical for tethering TGF-β to the cell surface, and rearrangements of LRRC32 or a neighbouring gene may be important for the pathogenesis of hibernomas [13, 14]. The main sources of these chemokines are dendritic cells, T cells or white cells and their effector cells are T cells or other immune cells [15, 16]. Thus, these genes might be involved in virus antigen presentation and virus killing during IBDV infection.
Tumour necrosis factor α (TNF-α) is a pleiotropic cytokine and could respond to a wide range of stimuli [17]. Along the TNF-mediated signalling pathway, several gene expressions were largely influenced during IBDV infection, such as TNFSF10 and TNFAIP2. In the present study, TNF receptor-associated factor 2 (TRAF2) was differentially up-regulated by two times compared to the mock group and has been shown to be essential for TNF-α-mediated activation of JNK. This could contribute to the activation of NF-kappaB and anti-apoptotic signals [18]. Moreover, tyrosine phosphorylation of signal transducer and activator of transcription 2 (STAT2) have been reported to be induced by activation of JAK kinases because of type I IFN binding to cell surface receptors, leading to activating the expression of interferon-stimulated genes and driving the cell into an antiviral state [19]; it was also increased during IBDV infection. Tumour necrosis factor receptor superfamily member 4 (TNFRSF4), a receptor for TNFSF4/OX40L/GP34, is a costimulatory molecule implicated in long-term T cell immunity and could act as a receptor for human herpesvirus 6B [20, 21]. Tumor necrosis factor alpha-induced protein 2 (TNFAIP2) functions as an important pro-inflammatory gene and plays vital role in the process of inflammatory response [22].
Strong host antiviral immune responses were activated by IBDV infection
After IBDV infection, some anti-infection genes were significantly changed without regard to the first day or third day post inoculation, including defense response to virus, positive regulation of T cell-mediated cytotoxicity and interferon-γ production and innate immune response. For response to virus, a total of 29 and 39 genes in the samples of chickens infected with IBDV on days 1 and 3 post inoculation, respectively, showed severe changes. Among the genes associated with responses to virus, TLR3, IRF1, GATA3, SAMHD1 and RSAD2 were all significantly up-regulated on both day 1 and day 3 post infection. TLR3, mainly expressed in the surface of dendritic cells (DCs), macrophages and fibroblasts, recognizes viral dsRNA and is critically involved in innate antiviral responses [23]. The up-regulated expression of TLR3 has been shown in chickens and chicken embryo fibroblasts (CEFs) infected with IBDV, of which similar results were displayed in our study. Moreover, as one promoter regulator of the IFN-α/β genes and MHC-I antigens in chicken fibroblast cell line C32 [24, 25], IRF-1 was up-regulated to 12- and 7-fold changes in the infection group on days 1 and 3, respectively, while the fold change of the IRF-1 gene expression in CEFs was 23-fold in a previous study on day 3 following infection with IBDV [26]. GATA binding protein 3 (GATA3) belongs to the GATA family of transcription factors and plays an important role in T cell development. It has been displayed to improve the secretion of IL-4, IL-5 and IL-13 from Th2 cells and induce Th0 cells differentiation into a T cell subtype [27]. Therefore, over-expression of GATA3 might be involved with virus inhibition of infiltrated T cells in this study. SAM domain and HD domain-containing protein 1 (SAMHD1), is a cellular enzyme in dendritic cells, macrophages and monocytes, responsible for blocking the replication of HIV by depleting the intracellular pool of deoxynucleoside triphosphates [28,29,30,31]. Radical S-adenosyl methionine domain-containing protein 2 (RSAD2, also known as CIG5, viperin or CIG33) with 361 amino acids is usually upregulated and displays antiviral defence responses against some pathogens, such as bovine respiratory syncytial virus (BRSV) and hepatitis C virus [32, 33].
Host responses displayed significant differences on days 1 and 3 post inoculation
In our study, on day 1 post-infection, the cytokine-cytokine receptor interaction pathway, JAK-STAT signaling pathway, AGE-RAGE signaling pathway in diabetic complications, Toll-like receptor signaling pathway and intestinal immune network for IgA production were activated and related genes were largely up-regulated in the infection group (Fig. 3). However, on day 3 post-infection, many metabolism pathways were activated and participated in the process of viral infection. Wong has described that transcription profiles of IBDV-infected cells were also different at 24, 48, 72 and 96 h post-infection, which was in accordance with our results [3]. Hui et al. also reported that different isoforms were involved in IBDV infection at different time-points post-infection, such as the IFIT5-IRF1/3-RSAD5 pathway in the DF1 cells that was triggered in the early infection stage [12].
Early host responses were vital for prevention of viral infection and replication. Our study showed that CSF3 (Myelomonocytic growth factor) increased in the bursae of IBDV-infected chickens at 1 day post infection, which could induce leukotrienes and exert anti-HIV-1 effects [34]. FOS (FBJ murine osteosarcoma viral oncogene homolog), as a cellular immediate-early gene was markedly induced before expressions of the Epstein-Barr Virus (EBV) transactivator genes were activated [35], which was similar in the process of IBDV infection. Likewise, host immune-related genes, such as VCAM1, NFKB1 and TLR2/3, were also elevated at the early stage in the infection group, which were consistent with many previous reports [36,37,38].
In the current study, several metabolic pathways-associated genes were involved on day 3 post-infection with IBDV, such as MYL9, GSTA and GMPPA, and these corresponding genes changed significantly compared with those on day 1. Among them, many metabolic pathways played vital roles in tissue injury and cytoskeleton repair, which might meet higher metabolic demands for virus replication and bursal injury repair during the middle stage of IBDV infection. It has been suggested that glycosaminoglycan could participate in a variety of biological processes, including cell-matrix interactions and activation of chemokines, enzymes and growth factors, and be used as a candidate to enhance chronic wound repair [39, 40]. Sun et al. have displayed that an array of factors involved in glycerophospholipid metabolism, arachidonic acid metabolism and tryptophan metabolism pathways were activated during human hepatitis C virus infection and arachidonic acid metabolism was also an up-regulated marker of neuro-inflammation in HIV-1 transgenic rats [41, 42]. Moreover, glycosphingolipid biosynthesis played an important role in nephritis development and CD1d-mediated lipid antigen presentation [43, 44]. Glutamine is used in the tricarboxylic acid cycle and human cytomegalovirus infection could activate the mechanisms that switch the anaplerotic substrate from glucose to glutamine to meet the biosynthetic and energetic needs of the viral infection [45].
Host (including fibroblast, DF1 and bursal cells) immune responses against IBDV have been widely reported, and many genes involved in viral infection have been studied. However, there were few reports on bursae-virus interactions in the early stage of IBDV infection by RNA-Seq. The results presented in this study have shown that lots of T cell infiltrated into bursae post IBDV infection accompanied with host immune responses and inflammatory responses. Overall, the host mainly displayed antiviral responses in the early stage of viral infection while many metabolic pathways were involved in the middle stage of viral infection. Therefore, finding an effective method of promoting IBDV-infected cells apoptosis and improving antiviral responses might be the best way to delay virus replication in the early stage. Moreover, inhibition of metabolic pathways may be an effective method of inhibiting the spread of the virus and the formation of a large amount of connective tissue in the middle or latent stages of viral infection.
EBV:
IBDV:
Infectious bursal disease virus
qRT-PCR:
Quantitative real-time PCR
SDE:
Significantly differentially expressed
SPF:
Specific-pathogen-free
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Ye J, Gradoville L, Miller G. Cellular immediate-early gene expression occurs kinetically upstream of Epstein-Barr virus bzlf1 and brlf1 following cross-linking of the B cell antigen receptor in the Akata Burkitt lymphoma cell line. J Virol. 2010;84(23):12405–18.
Zhang Q, Huang C, Yang Q, Gao L, Liu HC, Tang J, Feng WH. MicroRNA-30c Modulates Type I IFN Responses To Facilitate Porcine Reproductive and Respiratory Syndrome Virus Infection by Targeting JAK1. J Immunol. 2016;196(5):2272–82.
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Lee CC, Wu CC, Lin TL. Chicken melanoma differentiation-associated gene 5 (MDA5) recognizes infectious bursal disease virus infection and triggers MDA5-related innate immunity. Arch Virol. 2014;159(7):1671–86.
Peplow PV. Glycosaminoglycan: a candidate to stimulate the repair of chronic wounds. Thromb Haemost. 2005;94(1):4–16.
Taylor KR, Gallo RL. Glycosaminoglycans and their proteoglycans: host-associated molecular patterns for initiation and modulation of inflammation. FASEB J. 2006;20(1):9–22.
Sun H, Zhang A, Yan G, Piao C, Li W, Sun C, Wu X, Li X, Chen Y, Wang X. Metabolomic analysis of key regulatory metabolites in hepatitis C virus-infected tree shrews. Molecular & Cellular Proteomics. 2013;12(3):710–9.
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Sundararaj KP, Thiyagarajan T, Molano I, Basher F, Powers TW, Drake RR, Nowling TK. FLI1 Levels Impact CXCR3 Expression and Renal Infiltration of T Cells and Renal Glycosphingolipid Metabolism in the MRL/lpr Lupus Mouse Strain. J Immunol. 2015;195(12):5551–60.
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This work was supported by the National Natural Science Foundation of China (No.31402169), China Postdoctoral Science Foundation funded project (No.2015 M570630), Key Scientific Research Project Plan of Henan Province (No. 15A230002) and postdoctoral fund of Henan Institute of Science and Technology.
CO and QW designed the experiment and drafted the manuscript. YZ and SZ participated in the experiment and bioinformatics analysis of the sequencing data. YY and JM performed the molecular-related experiments. WK, XL and XK participated in the design of the experiment, the discussion and helped to draft the manuscript. All authors read and approved the final manuscript.
College of Life Science, Henan Normal University, Xinxiang, 453007, Henan, China
Changbo Ou
& Xianghui Kong
Postdoctoral Research and Development Base, Henan Institute of Science and Technology, Xinxiang, 453003, Henan, China
, Qiuxia Wang
& Xingyou Liu
College of Animal Science and veterinary medicine, Henan Institute of Science and Technology, Xinxiang, 453003, Henan, China
, Yanhong Zhang
, Shouping Zhang
, Yan Yu
, Jinyou Ma
Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, 14642, USA
Weili Kong
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Correspondence to Xingyou Liu or Xianghui Kong.
Supplementary Tables 1–6. (DOC 194 kb)
Ou, C., Wang, Q., Zhang, Y. et al. Transcription profiles of the responses of chicken bursae of Fabricius to IBDV in different timing phases. Virol J 14, 93 (2017). https://doi.org/10.1186/s12985-017-0757-x
Accepted: 26 April 2017
Transcription profiles
Bursa of Fabricius
Veterinary DNA viruses


text:
Q: Capturar preço de uma div em C# através do HTML Agility Pack Amigos, saudações! Sou iniciante em C# e estou tentando capturar o produto com o menor valor através da plataforma 'enjoei' e posteriormente, o mesmo aparecer em uma textbox, por exemplo. O código feito até o momento foi esse:
    HtmlAgilityPack.HtmlDocument doc;
    string pesquisarprecoenjoei;

    private async void descobrirprecoenjoei()
    {

        HtmlWeb web = new HtmlWeb();
        var doc = await Task.Factory.StartNew(() => web.Load("https://www.enjoei.com.br/camisa-do-flamengo-adidas/s?ref=products_search&sid=9a784ff9-da43-44f4-817c-bf2dedef19bd-1637161303630&q=camisa+do+flamengo+adidas&sr=near_regions&o=low_price"));
        var preco = doc.DocumentNode.SelectNodes("//div[@class='c-product-feed__list']//html//body//main//section//div//div//div//div//div//div//div//div//div//div//div//div//span//span");
        var inner = preco.Select(node => node.InnerText);

    }

Porém, ocorre o seguinte erro: "System.ArgumentNullException: 'Valor não pode ser nulo.
Arg_ParamName_Name'"
O caminho em XPath é: /html/body/main/section/div/div/div/div/div[3]/div[1]/div/div/div[1]/div/div[2]/div[1]/span[1]/span[1]
Como proceder? Muito obrigado desde já!

A: User conforme abaixo para retornar todos os preços da página de pesquisa:
var precos = doc.DocumentNode.SelectNodes("//span[@class='c-product-card__price']");

Como SelectNodes retorna uma coleção, você precisará iterar pelos resultados usando foreach (por exemplo):
foreach (var preco in precos)
{
    var inner = preco.InnerText;
}

redpajama_set_name: RedPajamaStackExchange


text:
Scutula Tul. (skutula) – rodzaj grzybów z rodziny Byssolomataceae.

Systematyka i nazewnictwo
Pozycja w klasyfikacji według Index Fungorum: Byssolomataceae, Lecanorales, Lecanoromycetidae, Lecanoromycetes, Pezizomycotina, Ascomycota, Fungi.

Synonimy nazwy naukowej: Hollosia Gyeln.

Nazwa polska według W. Fałtynowicza.

Niektóre gatunki
 Scutula aggregata Bagl. & Carestia 1889
 Scutula dedicata Triebel, Wedin & Rambold 1997
 Scutula epiblastematica (Wallr.) Rehm 1890 – skutula zewnętrzna
 Scutula epicladonia (Nyl.) Zopf 1906
 Scutula heeri (Hepp ex A. Massal.) P. Karst. 1885 – skutula Heera
 Scutula krempelhuberi Körb. 1865 – skutula Krempelhubera
 Scutula miliaris (Wallr.) P. Karst. 1853 – skutula zaniedbana
 Scutula solorinaria (Nyl.) P. Karst. 1885
 Scutula stereocaulorum (Anzi) Körb. 1865
 Scutula tuberculosa (Th. Fr.) Rehm 1906
Nazwy naukowe na podstawie Index Fungorum. Uwzględniono tylko taksony zweryfikowane. Nazwy polskie według W. Fałtynowicza.

Przypisy

Byssolomataceae

redpajama_set_name: RedPajamaWikipedia


text:
Social Networks Influential, Not Always Trusted
Nearly one in five US Internet users (18%) say they have purchased a product because of something they have seen on a social networking website—such as Facebook or Twitter—yet social sites continue to receive low trust and privacy ratings from consumers of all ages, according to a survey from Vision Critical.
Even so, 28% of Internet users age 18-34 say they have purchased a product because of something they have seen on a social networking site.
Below, other findings from Vision Critical's three-country survey on social media.
Trustworthiness of People, Media
Though online social networking sites bring consumers closer to the most-trusted parts of their lives—their family and friends—consumers place little trust in the sites themselves: just 12% say they fully trust social networking sites, while only 8% trust online forums, blogs, and reviews.
Trust is greater toward traditional media channels, such as TV (38%), radio (45%), and print newspapers (33%), but face-to-face encounters with family (80%) and friends (70%) are the most trustworthy.
Concerns About Privacy, Sharing of Personal Info
Internet users are concerned about privacy and the unauthorized sharing of their personal information with social networking sites: 63% of US consumers say they are strongly or moderately concerned about their privacy on social networking sites, and 55% are strongly or moderately concerned about social sites' selling their personal information to advertisers.
However, despite lingering trust and privacy issues, 48% say online social networks are a good place for brands and products to advertise to consumers, and 33% enjoy learning about brands and products via social sites.
Looking for real, hard data that can help you match social media tools and tactics to your marketing goals? The State of Social Media Marketing, a 240-page original research report from MarketingProfs, gives you the inside scoop on how 5,140 marketing pros are using social media to create winning campaigns, measure ROI, and reach audiences in new and exciting ways.
Trustworthiness of Brand Placements
Nearly one-half (47%) of US consumers consider a brand message on a social network most trustworthy when it's in the form of a discussion or recommendation from friends, family, or contacts within a social networking site.
Coupons or special offers are deemed the second most-reliable method of brand and product placement (33%), followed by product photos and videos (22%), sponsorships (19%), and pages dedicated to a brand or product (19%). Traditional banner ads (15%) are the least-trusted type of brand placement among those tested.
"This research suggests that while consumers may have very real concerns about the trustworthiness of online social networks, this emerging medium can still be a fertile ground for marketers and advertisers in terms of brand building, product marketing, and advertising," said Matt Kleinschmit, Vision Critical's SVP of Media.
"The key for advertisers is to successfully navigate the 'public' and 'private' characteristics of social networks, and harness those elements that will facilitate organic brand advocacy."
About the data: Findings are based on a survey conducted by Vision Critical of some 4,000 randomly selected adults—1,007 from the US, 1,022 from Canada, and 2,003 from the UK—from March 3 to 7, 2010.
Continue reading "Social Networks Influential, Not Always Trusted" ... Read the full article
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The Romney campaign, Fox news pundit, and campaign surrogate excuses for why Mitt Romney's name still appeared on SEC filings after he says he left Bain Capital just plays into the disconnect the average American has with Mitt Romney. We hear these excuses and we are reminded of all the wealthy elites gaming the system in the run-up to the financial disaster which resulted in the Great Recession.
No, I'm not saying Romney is directly responsible for the bad economy, but he is friends with the people who are responsible, and he advocates the policies which helped these friends game the system and crash the economy.


text:
Elia Andrioletti (* 9. Juni 1952 in Vertova, Region Lombardei) ist ein italienischer Endurosportler. Während seiner sportlichen Karriere wurde er mehrmals Europa- und italienischer Meister.

Karriere
Andrioletti begann seine sportliche Karriere 1970. 1973 wurde er in der Klasse bis 100 cm³ mit einer KTM italienischer Enduro-Meister. Fortan fuhr er ausschließlich auf Motorrädern der österreichischen Marke. Im folgenden Jahr verteidigte er seinen nationalen Titel. 1976 gewann er in der Klasse bis 175 cm³ die Enduro-Europameisterschaft und wurde erneut italienischer Meister. Außerdem wurde er in diesem Jahr Klassensieger beim italienischen Endurorennen Valli Bergamasche. Im folgenden Jahr wiederholte er seine Siege bei der Europameisterschaft und bei der Valli Bergamasche.

1978 wechselte er in die Klasse der Motorräder bis 350 cm³. Erneut gelang es ihm, die Europameisterschaft und den Klassensieg bei der Valli Bergamasche zu erringen. Außerdem gewann er die Roof of Africa in Südafrika. 1979 war er Mitglied der siegreichen italienischen Trophy-Nationalmannschaft bei der Internationalen Sechstagefahrt in Deutschland. 1980 war er wiederum Mitglied der Siegermannschaft bei der Sechstagefahrt in Brioude in Frankreich. Bei der Valli Bergamasche gewann er in der Klasse bis 125 cm³. 1981 gewann er erneut die Roof of Africa. Im gleichen Jahr nahm er mit dem KTM-Werksteam an der Rallye Paris-Dakar teil die er jedoch nicht beendete.

Bei seinen zwölf Teilnahmen bei der Internationalen Sechstagefahrt gewann er neun Goldmedaillen und drei Mal in seiner Klasse.

Wichtigste Erfolge
 Italienischer Enduro-Meister 1973, 1974, 1976
 Enduro-Europameister 1976, 1977, 1978
Internationale Sechstagefahrt 1979, 1980

Literatur

Weblinks
 Infos auf www.motowinners.it (ital.)

Endurosportler
Motorradweltmeister
Motorrad-Rennfahrer (Italien)
Italiener
Geboren 1952
Mann
Italienischer Meister (Motorradsport)


text:
Police 'not interested' in Wyman's affair with 13-year-old Mandy Smith, who claims she slept with him when she was 14
Guitarist Bill Wyman says he contacted police to talk about their relationship
He describes involvement with teenager as 'emotional and special'
Wyman and Smith married when she was 18 but separated soon afterwards
Former Rolling Stone admits he does not talk about relationship with Smith as it upsets wife and three daughters aged 14 to 18
By Neil Sears for the Daily Mail and Jaya Narain for MailOnline
Former Rolling Stone Bill Wyman has said that he approached police over widely reported claims he had sex with a minor – and was told that they were not interested.
The ageing rocker, now 76, famously began courting Mandy Smith when she was 13 and he was 48.
They married, disastrously, when she was 18, but her mother joined them on honeymoon and they spent less than a week of married life together.
Newlyweds: Bill Wyman, then 49, and 18-year-old Mandy Smith on their wedding day in 1989
Wyman and Ms Smith: She claims they slept together when she was just 14
The way they were: Wyman says it was 'emotional and special ' when he became involved with Mandy Smith
Rock king: Bill Wyman with Ronnie Wood picking up their Ivor Novello awards in 1991
She claimed they had sex when she was 14, and her sister called for the ex-Stone to be prosecuted.
Wyman insists he has nothing to be ashamed of – but amid a swirl of celebrity paedophile allegations sparked by the unmasking of the late Jimmy Savile, the star has finally revealed he took the initiative to contact detectives himself.
Revealed: Suicide-row DJ Liz Kershaw was mystery broadcaster... I was cursed by Freddie's fortune: Queen star's lover got...
He and Miss Smith had married in 1989, parting and divorcing soon afterwards.
He later left the Rolling Stones under a cloud, and despite playing with the now super-rich band last year, it appears he is yet to be fully reconciled with them.
But Wyman is still forthright about his relationship with the girl more than three decades his junior who became his second bride.
Now married to third wife Suzanne Accosta, by whom he has daughters Katherine, 18, Jessica, 17, and Matilda, 14, Wyman said of his love affair with a schoolgirl: 'It was very emotional and special at the time. It wasn't how it was reported to be and it was the only time it ever happened in my life.
'A lot of people understood, but a lot didn't, and the media certainly didn't. They treated me like c**p.'
Child bride: Mandy Smith today. She is no longer in contact with the former Rolling Stone
Ageing rock star: Bill Wyman was reunited with the Stones to mark 50 years of the band
Admitting he avoided talking about Miss Smith because it upset his wife and teenage girls – 'who are the age she was' – Wyman said: 'We all have a skeleton in the cupboard, it's just if you're a taxi driver in Halifax no one ever hears about it.
'But if you are a celebrity everyone does. In my case it was publicised to the world and that wasn't really fair, I don't think.'
He said: 'I went to the police and I went to the public prosecutor and said, "Do you want to talk to me? Do you want to meet up with me, or anything like that?" and I got a message back, "No".
'I was totally open about it.'
The Metropolitan Police refused to comment on Wyman's claims.
The star, who married first wife Diane in 1959, went through his first divorce in 1969 after joining the Rolling Stones plunged him into a world of groupies, from whom he reportedly picked favourites while playing bass on stage.
He has boasted of bedding a thousand young women, and first met 13-year-old Mandy Smith when she was dancing at London's Lyceum ballroom with her elder sister.
He has said she made him feel as if he had been 'whacked over the head with a hammer', and he promptly began helping her find work as a model.
He has claimed she was 'a woman at 13' and pointed out her mother did not object to their relationship, despite his age and reputation.
For two-and-a-half years their affair was a closely guarded secret, but when it became known they wed in a civil ceremony, then went on Terry Wogan's TV show.
Their divorce led to a £580,000 payout to Miss Smith, and he married for a third time just five months later.
But Wyman had not quite escaped his past, and events later took a still more bizarre turn when his son Stephen, 30, became engaged to Miss Smith's mother Patsy.
Together: Rocker Bill Wyman and his wife Suzanne Accosta who have three children
Bill Wyman: Police 'not interested' in former Rolling Stone's affair with 13-year-old Mandy Smith, who claims she slept with him when she was 14