text:
She Deserved Better.... WARNING- DISTURBING PHOTOS
I met her one hundred and twenty two hours before she took her last breath.
She bolted out of the transport carrier and squatted to pee. Poor baby- it had been a long day in the car. She had
traveled from Kentucky to Pennsylvania with two transfer stops along the way.
She stood still, not moving until I encouraged her to come in. Once inside, she found a corner and huddled down,
watching the household around her. Her body still, but her eyes watching- was this a good place to be?
I left her alone, letting her adjust, hoping that she would decide to trust just one more person.
In her eight years, she had too many journeys. By the age of three, she was looking for a new home. She was adopted
again, but five years and too many accidents later, she was surrendered to the shelter. She was a good girl, her former
people said, but when she started peeing in the house she had to go.
The shelter said she was scared.
Boarded at the vet- was this better than a kennel at the shelter? Still not a home, and kind words and loving touches
came only when the staff could spare a moment for a scared little dog.
The vet said she was scared.
Off to a foster home- overburdened and overworked rescuers, trying hard to save just a few more. She hides in the
corners, and nobody notices her.
The foster home said she was scared.
"We have no homes here for an eight year old scared little dog" the rescue said. "Would you please take her? She is so
The rescue group said she was scared.
She arrives after that long day of travel, and she certainly seems scared. Not moving, not eating, just those eyes watching
all that was going on. She has to be carried outside, and her only real activity is the mad dash back inside to the corner
she has chosen as her own.
Well, of course- she must be scared. Everyone said so.
But the symptoms were there. The longstanding history of accidents, the bloody streaks and pus in her urine, the
urgency and straining when she needed to "go". The stench- that not even a nose accustomed to "doggy odors" could
There was something wrong.
I call the vet. "She just arrived, and she is sick. She needs to be seen." Three hours later, she is on the exam table. We
are sure immediately, before the tests are even run, this sweet girl has a raging urinary tract infection. She is still quiet,
allowing the vet to look at her. Her gums are pale, and blood is drawn. She never flinches, even when a second stick is
needed. She is dehydrated, and needs fluid. The vet doesn't delay, and an antibiotic injection is given right away. The
exam continues, and the vet's eyes widen. She palpates her abdomen and says "She's full of stones." We don't need an
X-ray. In the vet's words, "The signs are not subtle."
The vet checks with the surgeon, and it is decided. She should stay, and surgery will happen today. Blood results are
back, and her kidney numbers aren't good. She is anemic. Her white blood cell count is high. But all these things are
likely a result of the stones and untreated infection. Once we remove the stones, we can fix the other things. I pat her on
the head, and tell her she will feel better now that we know how to fix her problem.
She wasn't scared. She was sick and in pain. And nobody noticed.
The vet calls while she is still on the operating table. The news isn't good. Three huge bladder stones and necrotic areas
in the bladder. Her body temperature is low, and her heart rate is fluctuating. The vet is pessimistic about her chances of
recovery, but we have nothing to lose by giving her a chance.
She makes it through the surgery, and spends the next twenty four hours in a heated cage at the vet hospital. She eats a
little and pees a little- she can come home. But she is still a very sick little dog.
I prepare the corner she has chosen. Blankets, and a heating pad, and a dish of water in easy reach.
I go to get her, and when the technician brings her out she curls her head under my neck, as if to get as close as
possible, and seems to breathe a sigh of relief. "You came back" is what she seems to say.
She had been left too many times. She had been ignored too many times. Nobody realized what was really happening to
this "scared little dog."
I bring her home, and settle her on her blankets. She lays still and watches, almost like before. Now though, I get an
occasional wiggle, not even a wag, of her tail. I hope that my careful handling and gentle words have helped her begin to
believe that life will be better now.
We know that recovery will take time. She's sore from surgery, and debilitated from the infection. She rests, and I carry
her outside. I change her blankets, and get her fresh water. I am encouraged when she starts eating, and leaves the
comfort of bed for her dinner.
I scratch her chest and rub around her ears. She leans into my hand. I talk quietly to her, and tell her what a good girl
One night passes, and then another. I can see that there is less pain. She is eating well. I allow myself to hope that
maybe this brave little dog will beat the odds.
And then I hear her cry. I go in by her bed, and she is stretched out on her side, crying. As I lean down to pick her up she
stiffens, and breathes her last breath.
Her body was too damaged. The infection too strong. Her treatment too late.
I am heartbroken.
How could that be? I have only known her for five short days.
It's because SHE DESERVED BETTER.
She deserved better than her original owners who gave her up at the age of three.
She deserved better than her second owners, who didn't get her the medical care she needed to stop a simple problem
from turning into a life-threatening one.
She deserved better than to end up at a kill shelter, because her medical problems caused her accidents.
She deserved better than the numerous people who failed to see the signs of a problem, and dismissed her as a "scared
She deserved better than to die in an unfamiliar place, with unfamiliar people.
I can only hope that she felt the love and concern that surrounded her in her final days. I hope that the comfort of a warm
and soft place to sleep, and good food to eat made her last days better. I hope that those chest scratches and ear rubs
and murmured "good girls" made her feel like the amazing little dog that she was.
And I know that she deserved better.
Rest in peace, Chrissy. I will see you at the Rainbow Bridge.
© 2014 Mary G. Stoner
This article may be reprinted, reproduced, or shared without written permission only in its entirety and for non-profit use
as long as appropriate credit is given to the author/copyright holder.

domain: literature


text:
Exostemtech said it applied for a phase 1 trial of EST-P-EXO1, an osteoarthritis treatment candidate, to the Food and Drug Safety Ministry. If the company gets the nod for the study, EST-P-EXO1 will become the first exosome-based treatment that enters a clinical trial in Korea.
According to Exostemtech, EST-P-EXO1 is an experimental treatment based on exosomes extracted from human stem cells. As the drug contains stem cells’ regeneration factors and anti-inflammatory substances, it could effectively regenerate cartilage tissue damaged by osteoarthritis and suppress pain through inflammation control, the company said.
Exostemtech said it would work with Kolon Biotech, a contract manufacturing organization (CMO), to produce EST-P-EXO1 samples for the clinical trial. The manufacturing will be supported by the Korea Health Industry Development Institute’s (KHIDI) program to enhance pharmaceutical firms’ global advancement in all cycles, which picked Exostemtech as the final winner in April.
“EST-P-EXO1 can effectively improve inflammation and damaged tissues, and it can be applied to various degenerative and inflammatory diseases,” an official at Exostemtech said. “Starting with osteoarthritis, we will expand indications such as liver fibrosis.”
Exosomes are endoplasmic reticulum with 50-200 nanometers secreted by cells. They transfer information between cells. Exosome-based treatments have excellent drug delivery capability and regenerative therapeutic effects. In addition, they can be easily stored and distributed, industry officials said.
Source – Korea Biomedical Review

domain: medical


text:
PROSSER HILL COALITION, Lisa Watts–McKee, Daniel Spisak, Jacquelyn Olson, Jack Wilcox, Tom Witfield, Janice, Whitfield, Robert Heinemann, Melanie Zimmerman, Roy Wilson, Steve Bird, Randy Sunderland, Rick Olson, and Cindy Phillips, Respondents, Cross–Appellants, v. COUNTY OF SPOKANE, Silverbird LLC, and Dennis P. Reed, Dennis E. and Dawna Reed, husband and wife, Appellants.
-- August 22, 2013
Stacy A. Bjordahl, Parsons/Burnett/Bjordahl/Hume, LLP, David W. Hubert, Attorney at Law, Spokane, WA, for Appellants.Michael D. Whipple, Whipple Law Group, PLLC, Richard Kirk Eichstaedt, Center for Justice, Spokane, WA, for Respondent/Cross–Appellants.
¶ 1 After a public hearing before a county examiner, Spokane County approved a conditional use permit (CUP) for a private airstrip requested by Silverbird LLC, Dennis P. Reed, Dennis E. Reed and Dawna Reed (collectively Silverbird). But based on insufficient public notice under the Spokane County Code (SCC), the superior court, reviewing under chapter 36.70C RCW, the Land Use Petition Act (LUPA), remanded for a new hearing. Petitioners are a coalition of neighbors: Prosser Hill Coalition, Lisa Watts–McKee, Daniel Spisak, Jacquelyn Olson, Jack Wilcox, Tom Whitfield, Janice Whitefield, Robert Heinemann, Melanie Zimmerman, Roy Wilson, Steve Baird, Randy Sunderland, Rick Olson, and Cindy Phillips (collectively the Coalition). The County and Silverbird jointly appeal, contending the superior court's remand decision was error. We disagree, and additionally reject Silverbird's other contentions that we dismiss because the Coalition failed to properly name the property owners as parties and failed to issue a summons with their land use petition. We reject the Coalition's cross appeal that the superior court erred in denying its cost bill request. Accordingly, we affirm.
¶ 2 On November 24, 2009, Silverbird submitted a CUP application to the County to construct a private airstrip with a 2,500–foot long by 250–foot wide runway area. The airstrip would extend across the southern portion of two parcels owned by Dennis E. and Dawna Reed. Silverbird proposed the development of a series of high-end houses and the permanent occupancy of the site by 15 aircraft. The site and the neighboring lands are designated Rural Traditional in the County's comprehensive plan.
¶ 3 On October 12, 2010, Silverbird mailed hearing notices to property owners within 400 feet of the site. Silverbird was required to post a notice of hearing sign “on the site along the most heavily traveled street lying adjacent to the site.” Spokane County Code (SCC) 13.700.106(2)(b). The site, however, does not abut a street. The two nearest public roads are Cheney–Spokane and Jensen. The site is closest to Cheney–Spokane, which is paved and a major thoroughfare. Silverbird, however, posted its notice sign on Jensen Road, which is a dirt road that leads to Dennis E. and Dawna Reed's property. Additionally, the hearing notices erroneously state the site is located “north and west of Jensen Road.” Board Record (BR) at 205–06. The site is actually north and west of Cheney–Spokane Road. The hearing examiner denied the neighbors' request for a continuance to correct the notice deficiencies and ruled the notice requirements were met.1
¶ 4 During the CUP application hearing, several neighbors testified to their concerns over noise, elevated accident potential, threats to safety, and degraded property values. The hearing examiner granted the CUP on December 17, 2010, with conditions.
¶ 5 The Coalition then filed a LUPA petition in the superior court, challenging the hearing examiner's decision. The petition identified in its header the respondents as Spokane County, Silverbird LLC, and Dennis P. Reed. The body of the LUPA petition identified Dennis P. Reed's parents, Dennis E. and Dawna Reed, as owners of the property as well. Copies of the LUPA petition were served on Spokane County, Dennis P. Reed, Dennis E. and Dawna Reed; but, the petition was not accompanied by a summons.
¶ 6 Recognizing its error, the Coalition moved to amend the caption on January 19, 2011. Silverbird countered by filing a motion to dismiss the LUPA petition. The court granted the motion to amend the caption and denied the motion to dismiss.
¶ 7 Following a LUPA hearing, the trial court found that Silverbird erroneously placed the notice sign on Jensen Road rather than the Cheney–Spokane Road, and listed an inaccurate description of the property subject to the CUP application on the notices. The court found these errors did not amount to harmless error. The court ordered the matter remanded for a new hearing with appropriate public notice. The court declined to rule on any of the substantive issues. The court denied the Coalition's cost bill, ruling the Coalition was not a prevailing party because the court remanded versus ruling on the merits of the matter.
¶ 8 Silverbird appealed the court's orders denying the motion to dismiss, granting the motion to amend the caption, and the remand order. The Coalition cross appealed the court's cost bill denial.
A. LUPA Petition Sufficiency
¶ 9 The issue is whether the trial court erred in denying Silverbird's motion to dismiss the Coalition's LUPA petition and allowing the Coalition to amend its caption. Silverbird contends the amendment rendered the petition untimely. Silverbird contends the matter should have been dismissed because service was deficient because a summons did not accompany the petition.
¶ 10 We review an order regarding a motion to dismiss for manifest abuse of discretion. Escude v. King County Public Hosp. Dist. No. 2, 117 Wash.App. 183, 190, 69 P.3d 895 (2003). Likewise, we review the denial of a motion to amend for abuse of discretion. Karlberg v. Otten, 167 Wash.App. 522, 529, 280 P.3d 1123 (2012). Abuse occurs when the ruling is manifestly unreasonable or discretion was exercised on untenable grounds. Id.
¶ 11 A superior court hearing a LUPA petition acts in an appellate capacity with the jurisdiction conferred by law. Conom v. Snohomish County, 155 Wash.2d 154, 157, 118 P.3d 344 (2005). “[B]efore a superior court may exercise its appellate jurisdiction, statutory procedural requirements must be satisfied. A court lacking jurisdiction must enter an order of dismissal.” Id. RCW 36.70C.040(2)(b)(ii) states, “a land use petition is barred, and the court may not grant review, unless the petition is timely filed ․ and timely served on the following persons who shall be parties to the review of the land use petition ․ [e]ach person identified by name and address in the local jurisdiction's written decision as an owner of the property at issue .”
¶ 12 In Quality Rock Prods., Inc. v. Thurston County, 126 Wash.App. 250, 265–66, 108 P.3d 805 (2005), Division Two of this court held that where service is otherwise proper under the civil rules, a party's failure to include the name of a necessary party in the caption does not divest the superior court of jurisdiction absent demonstrated prejudice. In Quality Rock, other than omitting the necessary party in the petition's caption, the party complied with the procedural requirements necessary to invoke the superior court's appellate jurisdiction. Id. at 271–72, 108 P.3d 805.
¶ 13 Quality Rock is persuasive. The Coalition's petition listed in its caption the respondents as Spokane County, Silverbird LLC, and Dennis P. Reed. The body of the LUPA petition, however, identified Dennis P. Reed's parents, Dennis E. and Dawna Reed, as owners of the property in question. Copies of the LUPA petition were served on Spokane County, Dennis P. Reed, Dennis E. and Dawna Reed; however, the petition was not accompanied by a summons. Because Dennis E. Reed and Dawna Reed were notified of the LUPA petition and they participated in the County proceedings, Silverbird cannot establish prejudice resulting from the caption amendment. Following Quality Rock, the Coalition's failure to include Dennis P, Reed's parents' names in the caption does not divest the superior court of jurisdiction. The Coalition complied with the procedural requirements necessary to invoke the superior court's appellate jurisdiction. Id. at 271–72, 108 P.3d 805.
¶ 14 Silverbird contends the petition should be dismissed because the date of the amendment was over 21 days from the date of the land use decision. See RCW 36.70C.040(3) (a petition is timely if filed and served within 21 days of the land use decision). But, CR 15(c) provides that an, “amendment relates back to the date of the original pleading.” Accordingly, the Coalition's subsequent amendment did not render the petition untimely.
¶ 15 Silverbird next urges us to adopt a requirement that a summons must accompany the filing of a LUPA petition. Specifically, Silverbird contends LUPA and the civil rules require a summons be filed to grant the court jurisdiction and that the failure to file a summons is grounds for dismissal.
¶ 16 As discussed above, RCW 36.70C.040 sets forth the procedure for commencing review under LUPA, stating in part, “A land use petition is barred, and the court may not grant review, unless the petition is timely filed with the court and timely served on the [listed] persons.” Regarding service, the statute states, “Service on the local jurisdiction must be by delivery of a copy of the petition to the persons identified by or pursuant to RCW 4.28.080 to receive service of process. Service on other parties must be in accordance with the superior court civil rules or by first-class mail.” RCW 36.70C.040(5).
¶ 17 In interpreting a statute, it is our fundamental duty to ascertain and implement the legislature's intent. United States Tobacco Sales & Mktg. Co. v. Dep't of Revenue, 96 Wash.App. 932, 938, 982 P.2d 652 (1999). A court must give meaning to every word and interpret the statute as written. Enter. Leasing, Inc. v. City of Tacoma Fin. Dep't, 139 Wash.2d 546, 552, 988 P.2d 961 (1999). If two provisions conflict, courts must give preference to the most specific statute. Bowles v. Wash. Dep't of Ret. Sys., 121 Wash.2d 52, 78, 847 P.2d 440 (1993).
¶ 18 LUPA alone governs judicial review of land use decisions. Benchmark Land Co. v. City of Battle Ground, 146 Wash.2d 685, 693, 49 P.3d 860 (2002). “[T]he plain language of the ․ LUPA provisions governs.” Mellish v. Frog Mountain Pet Care, 154 Wash.App. 395, 405, 225 P.3d 439 (2010). Nothing in LUPA requires the filing of a summons. Indeed, the purpose of a summons is to give notice and provide an opportunity to answer. Sprincin King St. Partners v. Sound Conditioning Club, Inc., 84 Wash.App. 56, 60, 925 P.2d 217 (1996). As discussed in Quality Rock, “CR 4(b)'s directive that the summons provide notice and warn of potential default can be inapplicable in the LUPA context.” 126 Wash.App. at 265, 108 P.3d 805. The court further noted that although the timing and service requirements are jurisdictional, the failure to strictly comply with the procedures of the civil rules will not invalidate a LUPA proceeding. Id. at 271, 108 P.3d 805. While courts have strictly construed the timing and parties that must be served in a LUPA proceeding, the courts have not elevated other procedural requirements to a “jurisdictional threshold.” Id.
¶ 19 Here, the LUPA petition was filed with the county's auditor's office and surrounding neighbors by first class mail as prescribed by RCW 36.70C.040(5). This sufficiently invoked the court's jurisdiction. Silverbird's argument that a summons must accompany the petition is not supported by law. We are not persuaded to extend the summons requirement here.
B. Improper Notice
¶ 20 The issue is whether the trial court erred in remanding the matter to the hearing examiner based on improper notice. Silverbird contends notice was sufficient to provide the neighboring property owners an opportunity to be heard.
¶ 21 RCW 36.70C.140 outlines three possible responses for a reviewing court when considering a hearing examiner's decision: “The court may affirm or reverse the land use decision under review or remand it for modification or further proceedings.” “When reviewing a superior court's decision on a land use petition, the appellate court stands in the shoes of the superior court.” Citizens to Pres. Pioneer Park, LLC v. City of Mercer Island, 106 Wash.App. 461, 470, 24 P.3d 1079 (2001). In the context of administrative review, the court of appeals reviews the administrative agency action, not the superior court record. J.L Stomdahl & Sons, Inc. v. Cowlitz County, 125 Wash.App. 1, 6, 103 P.3d 802 (2004). This standard applies with equal force in the context of a LUPA decision. Wellington River Hollow, LLC v. King County, 121 Wash.App. 224, 230, 54 P.3d 213 (2002).
¶ 22 The SCC sets the procedure for review of project permits in Spokane County. Regarding notice, SCC 13.700.106(2)(b) requires an applicant to post a notice of hearing sign “on the site along the most heavily traveled street lying adjacent to the site.” Additionally, SCC 13.700.106(2)(a) and (c) requires neighboring property owners to receive written notice and for notice to be published in the newspaper. This notice must contain the, “project location, vicinity and address and parcel number, if applicable.” SCC 13.70.104(5).
¶ 23 We review statutory interpretation questions de novo. Estate of Haselwood v. Bremerton Ice Arena, Inc., 166 Wash.2d 489, 497, 210 P.3d 308 (2009). We discern plain meaning from the ordinary meaning of the language at issue, the context of the statute in which that provision is found, related provisions, and the statutory scheme as a “whole.” State v. Engel, 166 Wash.2d 572, 578, 210 P.3d 1007 (2009).
¶ 24 Here, the proposed airstrip is not adjacent to a road; however, the two nearest roads are Spokane–Cheney, a paved, thoroughfare in the area; and Jensen, a dirt road that leads to a long driveway that services Dennis E. and Dawna Reed's property. SCC 13.700.106(2)(b) states that notice must be posted “along the most heavily traveled street.” We give effect to a statute's plain meaning. Dep't of Ecology v. Campbell & Gwinn, LLC, 146 Wash.2d 1, 9–10, 43 P.3d 4 (2002). The plain meaning of most heavily traveled would be a paved, thoroughfare instead of a dirt road leading to a private residence. Moreover, SCC 13.700.106(2)(a) and (c) requires neighbors to receive written notice and the public to receive notice via the newspaper. Both of these methods of notice erroneously state that the site is located “north and west of Jensen Road.” BR at 205–06. The site is actually north and west of Cheney–Spokane Road.
¶ 25 While the hearing examiner ruled the notice requirements were met because nothing indicated neighboring property owners were confused or did not receive notice, it is difficult to measure the impact the faulty notice had on concerned individuals. In other words, it is difficult to prove a negative. “One purpose of specific statutory requirements for public notice of an impending land use decision is to ensure that the decision makers receive enough information from those who may be affected by the action to make an intelligent decision.” Prekeges v. King County, 98 Wash.App. 275, 281, 990 P.2d 405 (1999). The defective notice undermines the information-gathering process.
¶ 26 By comparison, in Stritzel v. Smith, 20 Wash.App. 218, 220–21, 579 P.2d 404 (1978), a landowner brought an action to reclaim land that had been sold at a tax sale. The court ordered the tax foreclosure deeds set aside because there had been only 9 days' posting of public notice of the tax sale, not 10 days as required by the tax sale statute. The court held in Stritzel that strict compliance with the posting requirement was necessary to protect the landowner's interest in having as many bidders at the sale as possible, and to guarantee him a fixed number of days to redeem the property before the sale. Stritzel, 20 Wash.App. at 221, 579 P.2d 404.
¶ 27 A neighboring landowner should be afforded a fair opportunity to be heard. See Gardner v. Pierce County Board of Commissioners, 27 Wash.App. 241, 243–44, 617 P.2d 743 (1980) (court tolled the time period to appeal a land use decision when the lack of public notice deprived a neighboring landowner of a fair opportunity to be heard). Even applying the lesser standard of substantial compliance, “[t]he key to achieving substantial compliance with a procedural statute is the satisfaction of the substance essential to the purpose of the statute.” Prekeges, 98 Wash.App. at 280, 990 P.2d 405. Posting a sign contrary to the municipalities' placement requirement and to provide notice with the wrong property description is not within the substantial compliance standard.
¶ 28 In sum, because the notice sign was not posted near the required street and notice did not contain the correct property description, as required by the clear language of SCC 13.70.104(5) and SCC 13.700.106(2)(b), the trial court did not err in remanding the matter to the hearing examiner.
C. Conditional Use Permit
¶ 29 Silverbird contends the CUP was properly granted while the Coalition contends the hearing examiner failed to consider noise, safety risks, and impacts to property value when granting the CUP. The trial court did not reach these issues, and neither do we. Based on the disposition above (affirming the court's remand for proper notice and hearing), any discussion by this court regarding the substantive issues would amount to an advisory opinion, which is disfavored by courts. State v. Norby, 122 Wash.2d 258, 269, 858 P.2d 210 (1993). Moreover, the trial court did not rule on the substantive issues. Under RCW 36.70C.040(1), the LUPA appellate process requires review first by the superior court. Thus, it would be premature for us to rule on the substantive issues.
D. Costs and Attorney Fees
¶ 30 On cross review, the Coalition contends it was the prevailing party below and should have been awarded costs under RCW 4.84.030.
¶ 31 We review a decision to award or deny costs for an abuse of discretion. Highland Sch. Dist. No. 203 v. Racy, 149 Wash.App. 307, 312, 202 P.3d 1024 (2009). “Discretion is abused when it is exercised on untenable grounds or for untenable reasons.” Id. Under RCW 4.84.030, “[i]n any action in the superior court of Washington the prevailing party shall be entitled to his or her costs and disbursements.” “[T]he determination of who is a prevailing party depends upon who is the substantially prevailing party, and this question depends upon the extent of the relief afforded the parties.” Riss v. Angel, 131 Wash.2d 612, 634, 934 P.2d 669 (1997).
¶ 32 The superior court remanded on a procedural defect. While the Coalition pointed out the deficiency in its LUPA petition, the remand did not provide its affirmative relief. By comparison, in Ennis v. Ring, 56 Wash.2d 465, 473, 341 P.2d 885 (1959), a case involving the denial of costs after an appeal court ordered a retrial, the court held, “In the event of a retrial the determination of who is the prevailing party must await the outcome of the second trial.” Because the Coalition is not the substantially prevailing party, the superior court properly denied its request for costs.
¶ 33 Both Silverbird and the Coalition request attorney fees on appeal in their reply briefs. Under RAP 18.1(b), a party requesting attorney fees, “must devote a section of its opening brief to the request.” Since neither party complied with this rule, their requests should be denied.
¶ 34 Affirmed.
WE CONCUR: SIDDOWAY, A.C.J. and KULIK, J.

domain: law


text:
What do high labor costs, succession planning and client expectations have in common? Answer: They are all huge challenges facing the accounting profession. Large numbers of corporate and firm executives quickly are approaching retirement and many will leave in the next 10 to 15 years. The challenge is clear: Firms will have to find highly skilled replacements for their senior professionals. The solution: Recruit, retain and promote top talent.
The advancement of women is critical to these goals. Yet, despite the large number of women who enter the profession every year, relatively few have advanced to the level of partner, senior executive or corporate board member. That will have to change. It’s no longer a question of addressing women’s advancement because of fairness: It’s become a business imperative.
In the last decade, a number of firms and companies began to consider ways to address this issue. Many analyzed the situation, developed plans and evaluated the outcomes. Leaders found the key to success was to show how the advancement of women benefited the organization to underscore its strategic importance to everyone. Firms with successful plans typically see the following benefits:
Reduced costs. Organizations that retain and promote a significant portion of their talent pool minimize the need to compete for experienced talent in a costly labor market.
Improved succession planning. Many firms reevaluated their selection process to identify candidates for special development programs when they realized women were receiving few invitations. They understand the need to invest in professional development, cultural change and strategic plans that prepare the next generation for leadership. They identify future leaders earlier in the process and provide them with the professional development opportunities they need to excel. Drawing from a larger, more diverse talent pool gives leaders and participants a better understanding of the issues and personal challenges they will face and results in new and creative approaches.
Enhanced relationships with clients and customers. Business owners and stakeholders in our diverse economy demand their leadership teams and outside professionals reflect the diversity of American culture and global business. Management appreciates seeing a team that represents the community, and sees the benefits of new ideas and approaches offered by teams that reflect the demographics of the community.
There is a measurable business case for women’s advancement and it’s a persuasive one. Firms that focus on the promotion and development of women and minorities not only see immediate benefits but also prepare for coming challenges. Firm leaders who recognize the value of addressing women’s advancement aren’t just doing what’s right, they’re preparing for tomorrow and doing what’s smart.
—Nancy R. Baldiga, CPA, associate professor and chair,
department of economics, College of the Holy Cross, Worcester, Mass.

domain: sociology


text:
Mozatti makes re-entrance into the music scene with the “PUNA” song featuring Iyanya. Listen, stream, buy the track.
Mozatti, A talented Singer and songwriter based in Australia joins forces with the incredible “Mr Kukere” Iyanya to release such a sexy club banger titled “PUNA” The groove of the song is irresistible, and it features some unique instrumentals and a flow that will blow your mind with its lyrical content.
This song is one that everyone who appreciates good music ought to have in their collection at all times because it’s so good.
Mozatti – PUNA (ft. Iyanya)

domain: artistic


Posted: 19 Nov 2018 15:07 EST Last activity: 20 Nov 2018 11:10 EST
Refresh Repeating Grid After Changing Pagination
Our users are wanting to know if we can make it so when they change the pagination of a repeating grid that the results change instantly, without having to click/tab away from where they input the number. Thus far I have not found anything here or elsewhere that comes close to this. Thanks in advance for any and all help!
Also onchange event currently with pagination (which updates/refresh the grid with tab/click away) would be the correct behavior since reload/refresh happens only once after that event. With the kind of requirement you have mentioned you would have to refresh for every entry in the pagination.
If i understand your question correctly then Refresh section, reload grid/list does content reload again, it will not reload specific content based on pagination number (not available from OTB). But you can customize as per required criteria.
Please elaborate your requirement more, if we misunderstood.


We’ve all got them: those shelves we try not to look at, lined with cans of old paint, older pesticides, ancient fertilizer and other stuff we can’t use anymore but can’t just toss in the trash.
The City of Decatur wants to provide an alternative to just keeping it around.
At Monday night’s meeting the city council authorized City Manager Brett Shannon to execute an interlocal agreement with the City of Fort Worth that will allow Decatur residents to dispose of hazardous household wastes at Fort Worth’s Environmental Collection Center, located at 6400 Bridge Street, just north of Interstate 30 at East Loop 820.
Decatur residents will be required to purchase a voucher at City Hall before going to the facility, for a fee of around $50. Fort Worth will charge the city $47 for each visit.
“It’s a wonderful service the citizens can use,” Public Works Director Earl Smith told the council. “If they use it, they pay. It doesn’t cost the city anything.”
Mayor Martin Woodruff said it was the kind of deal he’s been looking for.
“I’d love for our residents to have something, and I would encourage homeowners to get rid of this stuff, legally,” he said.
Shannon said the city’s waste disposal contractor, Progressive Waste Solutions, does not have a hazardous household waste program at this time.
“The county doesn’t have anything,” he said. “Progressive Waste is working on it. This would at least give our citizens a place to take it to in the meantime.”
Forty-eight entities – Tarrant, Hood, Johnson and Parker counties, the Tarrant and Upper Trinity regional water districts and 42 cities – currently utilize the service. Half require their citizens to purchase a voucher, while the other half just bear the cost with public funds.
Smith said the Fort Worth center tries to recycle as much of what they collect as possible – even mixing up all the latex paint and giving it away to participating cities and counties free of charge. They also have a “help-yourself” shelf with free chemicals, cleaners and paint in like-new condition.
The center is open 11 a.m. to 7 p.m. Thursdays and Fridays, and 9 a.m. to 3 p.m Saturdays. They also have a mobile unit – the “Crud Cruiser” – that travels to cities to collect hazardous waste from March through November.
“We could probably get them here once, maybe twice a year,” Shannon told the council. “Outside of that, citizens can just get a voucher here, go there, prove their residence and use it.”
If they take it to the landfill, they will get turned away, Shannon said.
“You can toss a dried-out paint can, but if it’s still liquid, you can’t dispose of it,” he said. “Insecticides, herbicides, car batteries – it’s all got to go down there.”
Shannon said the City of Bridgeport got a grant a few years ago and accepted hazardous materials for about a year but stopped when the grant ended.
Councilman Jason Wren suggested the city put something in its water bills to let people know the service is available. He moved to approve the agreement and Councilman Jay Davidson seconded.
It passed unanimously.
The center will accept automotive fluids, batteries, cleaners and chemicals, cooking oil, lawn, garden and pool chemicals, light bulbs, medicines, paint and painting supplies.
It will not accept ammunition and explosives, appliances and electronics, building materials, bulk trash and yard waste, butane or propane cylinders, medical waste, tires, asbestos, PCBs or radioactive items.
Business, commercial and industrial waste also cannot be accepted under Texas regulations.
Those taking waste are asked to leave the products in their original, marked containers or label them clearly. Leaking containers should be placed in a second container of like material. Items should be placed securely in the trunk of your car or the bed of your pickup, and those bringing them in are asked to remain in the vehicle and let the staff unload the items.
Participating cities include Arlington, Azle, Bedford, Benbrook, Burleson, Cedar Hill, Cleburne, Colleyville, Crowley, Dalworthington Gardens, Euless, Forest Hill, Glenn Heights, Godley, Grand Prairie, Grapevine, Haltom City, Hurst, Keller, Kennedale, Lake Worth, Lakeside, Mansfield, Midlothian, North Richland Hills, Oak Leaf, Pantego, Richland Hills, River Oaks, Roanoke, Saginaw, Sherman, Southlake, Stephenville, Trophy Club, Watauga, Waxahachie, Weatherford, Westlake, Westover Hills, Westworth Village and White Settlement.
For more information, visit fortworthtexas.gov/env/ecc.


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Next-Generation Sequencing (NGS) is increasingly being used as a molecular epidemiologic tool for discerning ancestry and traceback of the most complicated, difficult to resolve bacterial pathogens. Making a linkage between possible food sources and clinical isolates requires distinguishing the suspected pathogen from an environmental background and placing the variation observed into the wider context of variation occurring within a serovar and among other closely related foodborne pathogens. Equally important is the need to validate these high resolution molecular tools for use in molecular epidemiologic traceback. Such efforts include the examination of strain cluster stability as well as the cumulative genetic effects of sub-culturing on these clusters. Numerous isolates of S. Montevideo were shot-gun sequenced including diverse lineage representatives as well as numerous replicate clones to determine how much variability is due to bias, sequencing error, and or the culturing of isolates. All new draft genomes were compared to 34 S. Montevideo isolates previously published during an NGS-based molecular epidemiological case study.
Intraserovar lineages of S. Montevideo differ by thousands of SNPs, that are only slightly less than the number of SNPs observed between S. Montevideo and other distinct serovars. Much less variability was discovered within an individual S. Montevideo clade implicated in a recent foodborne outbreak as well as among individual NGS replicates. These findings were similar to previous reports documenting homopolymeric and deletion error rates with the Roche 454 GS Titanium technology. In no case, however, did variability associated with sequencing methods or sample preparations create inconsistencies with our current phylogenetic results or the subsequent molecular epidemiological evidence gleaned from these data.
Implementation of a validated pipeline for NGS data acquisition and analysis provides highly reproducible results that are stable and predictable for molecular epidemiological applications. When draft genomes are collected at 15×-20× coverage and passed through a quality filter as part of a data analysis pipeline, including sub-passaged replicates defined by a few SNPs, they can be accurately placed in a phylogenetic context. This reproducibility applies to all levels within and between serovars of Salmonella suggesting that investigators using these methods can have confidence in their conclusions.
Foodborne pathogens cause an estimated 9.4 million human illnesses in the U.S. each year, resulting in nearly 60,000 hospitalizations and over 1,300 deaths [1–4]. Salmonella enterica remains one of the most devastating of these foodborne pathogens with 11% of all food related deaths being attributed from exposure to this bacterium . The genus Salmonella comprises two species, S. enterica and S. bongori, both of which have been found in the food supply. Six subspecies of S. enterica have been described (I-IIIa, IIIb, IV, and VI) that can be found in a variety of mammalian and non-mammalian hosts including humans, cattle, birds, turtles, and snakes. Most non-typhoidal salmonellosis cases in mammals, including humans, come from over 1700 different Salmonella group (subspecies) I serovars. While several group I serovars such as S. Typhimurium and S. Enteritidis have been studied more widely, the genetic and phylogenetic diversity defining many of the important group I Salmonella e remains poorly understood.
One of these serovars, Salmonella enterica subsp. enterica serovar Montevideo (i.e., S. Montevideo) is one of the top ten most common serovars associated with contaminated foods. This serovar was recently associated with a Pistachio recall in 2008, and more recently, with contamination of certain pet treats http://www.fda.gov/Safety/Recalls/ucm218039.htm. Moreover, this serovar has been implicated in contamination events involving numerous meat and cheese products http://www.outbreakdatabase.com/site/search/?tag=s.+montevideo. More recently, a strain of S. Montevideo was linked to more than 240 illnesses in 38 states after being found in red and black pepper used in the production of contaminated Italian-style spiced meats [, http://www.cdc.gov/Salmonella/montevideo/montevideo_timeline2.pdf]. It is important to note that many of these highly clonal strains of S. Montevideo often confound epidemiological investigations because pulsed-field gel electrophoresis (PFGE) is unable to always distinguish outbreak-related strains from other genetically similar strains unassociated with the same outbreak. Strains of this nature often retain common PFGE patterns despite their sporadic or more historic origins.
The accurate subtyping and subsequent clustering of isolates of a bacterium associated with a foodborne outbreak event is essential for successful investigation and eventual traceback to a specific food or environmental source [6–12]. In this regard, PFGE continues to deliver useful genetic typing information by facilitating public health investigations for nearly two decades. In certain cases, however, highly clonal strains, common among some group I Salmonella e, confound epidemiological investigations because PFGE provides limited genetic differentiation of these strains. That is this approach often lacks the resolution for differentiating highly clonal bacterial isolates. In response to such events, federal public health and food safety laboratories are exploring next-generation sequencing (NGS) to define complex outbreak scenarios. NGS refers to highly parallel robotic genomic sequencers, like Roche 454 GS Titanium technology, that are being used to accomplish the whole genome sequencing (WGS) of a bacterial pathogen.
NGS is contributing long anticipated solutions to what were once viewed as insurmountable challenges, in the genetic analysis of bacterial pathogens [13–16]. Complete genome sequences from multiple bacterial strains can now be collected and analyzed in just a few days , underscoring the future potential of this technology as a molecular epidemiological tool to assist in foodborne outbreak investigations. Recent examples in the literature illustrate the ability of NGS to discern the high-resolution genetic relatedness and unrelatedness of otherwise indistinguishable isolates based on the microevolutionary genetic change that define clinical isolates, outbreak isolates found in foods, and their environmental counterparts [18–20].
These novel applications of NGS are buttressed by a massive influx of new genomic data, producing new discoveries about the critical genes that define particular pathogens, and important genomic changes associated with pathogenicity, antibiotic resistance, and unique carbon source usage [19, 20]. However, the race to sequence more bacterial pathogen genomes must be tempered by the realities and rigor of formal methods validation processes for tools deployed in epidemiological investigation http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/ucm077862.htm. This validation process is not only required for regulatory action by federal and state laboratories whose duty it is to conduct these tests, but these general procedures must be applied if the technology is to meet scientific admissibility requirements in a legal setting. Although still being developed, historical paradigms exist for the validation of sequence data. Capillary electrophoresis (CE) sequencing, for example, has been a standard technology since the early 1990s, and its accuracy has proven to be sufficient so that CE is now widely applied by a variety of federal agencies engaged in activities spanning forensic and molecular epidemiologic analyses [21, 22].
Herein, we demonstrate the value of NGS in defining the diversity of Salmonella Montevideo using a representative set of environmental, laboratory, food, and clinical strains, some of which have been associated repeatedly with contamination events in several food sources . Here, our analyses using NGS data provided far greater resolving power than previously available from other techniques. This information was essential to reconstructing both the deep phyletic relationships of this serovar and terminal relationships among highly clonal S. Montevideo isolates. Moreover, the clonally derived outbreak cluster of S. Montevideo were defined by a few single nucleotide polymorphisms (SNPs) while more geographically or temporally removed isolates showed tens to thousands of SNP differences. Additionally, NGS technology revealed considerable genomic stability and high reproducibility for SNP targets used in the clustering of closely related isolates within an important and emerging serovar of Salmonella enterica.
NGS reveals substantial intra-serovar diversity within Salmonella Montevideo
In order to explore the evolutionary genetic diversity of Salmonella Montevideo, NGS analysis was performed on 47 strains of this serovar (Table 1). This included assembling the raw reads to form contigs of overlapping sequence, annotating those contigs to determine which genes were present, and then determining homology among genes and aligning and concatenating those genetic elements for population and phylogenetic analyses. Roche-Titanium whole-genome shotgun sequencing technology [23, 24] provided 15-20× coverage for each genome reported, and downstream contig assembly and sequence alignment provided over 4.5-5 mbp of assembled contigs for each isolate. Additional data filtering yielded 72,063 variable SNP sites of which 63,987 were identified as parsimony informative (i.e., SNPs shared by two or more strains in the alignment) and subjected to phylogenetic analysis on the FDA bioinformatics, Linux based computer cluster using likelihood and parsimony methods. The resultant evolutionary tree derived from the informative SNP data yielded two important observations (Figure 1). First, S. Montevideo formed a monophyletic group of strains phylogenetically distinct from other neighboring serovars including S. Schwarzengrund, S. Pomona, and S. Javiana. Second, S. Montevideo strains partitioned into four disparate clades (designated I-IV), several of which were defined by a mixture of both natural and laboratory isolates. Clade III, for example, comprised a clinical isolate associated with tomato (206_Clinical) as well as a single strain (160_Clinical_FL) from the widely characterized subspecies I Salmonella Reference collection, SARB .
Pairwise SNP variation between these four S. Montevideo lineages is listed in Table 2. Intra-serovar SNP diversity was remarkable among the four diverged S. Montevideo genome lineages ranging from 17,600 SNPs (clade I/clade II) to 23,800 SNPs (clade II/clade IV). This latter distance was astonishing given that SNP divergence between S. Montevideo lineage I and S. Pomona, a different group I serovar, falls well within this range (i.e., 22,700 SNPs). In addition to the substantial SNP-based diversity noted among S. Montevideo lineages, genome size also fluctuated widely within this serovar (Figure 2). That is, genome length ranged from less than 4.45 million bp to about 4.75 million bp, sorting largely along intra-serovar clade divisions revealed in the phylogenetic tree (Figure 1). Most of the observed genome size differences appear to be due to the presence or absence of phage elements. The CA clinical isolate 157, for example, is bigger than the outbreak cluster in general due to phage D6. In addition, S. Montevideo strain 163 appears to be enlarged due to insertion of a plasmid pRA1, while strain 206 retains an uncharacterized phage-like sequence and elements of the SPI-7 pathogenicity island. Conversely, two smaller S. Montevideo genomes, 162 and 205, appear to be missing the putative Salmonella phage sequence relative to the outbreak cluster (i.e., clade IV, Figure 1). Akin to findings reported previously on the stress-induced acquisition and loss of phage elements in the Salmonella genome , these data signal an important role for insertions and deletions in the diversification of specific clones of S. Montevideo, and, taken together with the above SNP findings, point to a serovar of non-typhoidal Salmonella comprised of several genomically diverged and phylogenetically distinct clones [27–29].
NGS phylogenetically differentiates a clonal lineage of Salmonella Montevideo
The importance of NGS in ascertaining high-resolution phylogenetic and molecular epidemiological histories of infectious outbreak clones of bacterial pathogens has recently been noted [18, 20]. In the current study, NGS was applied for reconstructing the detailed evolutionary genetic structure of an individual clone of S. Montevideo that is largely indistinguishable using PFGE. Specifically, NGS analysis was applied to a set of S. Montevideo isolates either associated with or genetically homologous to a food contamination event of spiced Italian-style meats in the U.S. in 2009 and 2010 http://www.cdc.gov/Salmonella/montevideo/index.html. We reported previously on the success of NGS for distinguishing some of these isolates from other clonally related isolates unassociated with this spiced-meat S. Montevideo outbreak . Herein, we combined the genomes of 34 highly homogeneous S. Montevideos from food, environmental, and clinical sources from this spiced-meat outbreak with 24 newly sequenced (~15X) S. Montevideo genomes derived from clinical-food matches associated with the same spiced-meat contamination event. As an important control, historical S. Montevideos from within this clone were included that retained multiple identical PFGE patterns to the spiced-meat outbreak strains and were isolated from a variety of foods unassociated with this outbreak such as pistachios, chicken, Italian cheese, and several fishes from Indo-China. It is important to note that all of the clinical isolates included here (Figure 3) were collected in association with the spiced-meat outbreak event.
Results from the phylogenetic analysis provided several important findings relevant to the phylogenetic differentiation of clonal S. Montevideo strains (Figure 3). First, in contrast to the serovar tree presented in Figure 1, SNP diversity within this highly clonal sub-lineage of S. Montevideo was markedly lower as expected, less than 500 informative SNPs defined the entire tree. However, the resultant likelihood tree partitioned this clone into six distinct groups of isolates that were separated from neighboring groups by less than 100 parsimony informative SNPs each. Additionally, isolates associated previously with the spiced-meat outbreak clustered together in a group separate and distinct from groups of closely related S. Montevideos unassociated with this contamination event (e.g., pistachios/B, chicken/D, and fish/A). From a phylogenetic perspective, clades E and F appear to capture the scope of the outbreak. There are several reasons that support this partition. Clinical isolates (i.e., CT clinical isolates) associate closely with a drain strain from the facility forming clade E (Figure 3) and from contaminated spices collected at the facility along with a host of clinicals from several states (i.e., IA, MD, NC, and OH) nearly all of which were indistinguishable from the food isolates (clade F, Figure 3). It is also noteworthy that clade F retained a subgroup of NC isolates that were separated from the other food and clinical spiced-meat strains by just a few SNPs. However, these isolates are clear monophyletic members of clade F, one of the two outbreak clades, and may have emerged from the base of this clade through microevolutionary change. Whatever the final explanation, NGS analysis coupled with a comparative phylogenetic approach not only fully differentiated this clone of S. Montevideo, but also provided high resolution genetic information that effectively delimited the scope of the outbreak event, affirming its potential as a powerful tool for supporting molecular epidemiologic investigation of clonal outbreaks of non-typhoidal Salmonella.
SNP variation within the S. Montevideo spiced-meat clone was nearly two logs lower than what was noted for total intra-serovar diversity. Nevertheless, the signature SNPs that delineated these six subgroups (A-F) originated from various regions around the S. Montevideo genome and included a variety of genes assigned to diverse cellular functions including metabolism, DNA synthesis and repair, transport and uptake, virulence, and stress response. A list of 43 genes from which the SNPS that characterize S. Montevideo clade IV were derived is provided in Table 3. A representative SNP from each of these genes is also provided in the table along with the subgroup that it defines and its bp coordinates. Thirty of these genes were annotated previously with assigned names and functions; however, 13 additional regions that provided signature SNPs are hypothetical and, as such, are cross-referenced by locus tags only. It is notable that a partial and select set of SNPS from 25 of these 43 genes are non-synonymous, and of the 14 SNPs in Table 3 that cluster together two or more S. Montevideo subgroups in Figure 3, all but three are protein- altering in nature. These data are intriguing given an NGS report documenting positive selection among a significant subset of core genes in adapted Salmonella enterica serovars .
Although the majority of isolates composing the spiced-meat S. Montevideo clone generally exhibited a common genome length, one isolate from California (S. Montevideo 157_Clinical_CA) retained a noticeably larger genome than other members of this lineage (Figure 2). In addition to being separated from other S. Montevideos associated with the spiced-meat contamination event by nine phylogenetically informative SNPs (Figure 4A), comparative analysis revealed the presence of a 100 kb insertion with substantial homology to Enterobacterial phage D6. Since phage D6 was incomplete in GenBank (No. AY753669), a MAUVE comparison to another homologous relative, phage P1 (No. NC_005856), was helpful in suggesting that this may represent a D6-like phage insertion into contig 104 in this particular S. Montevideo genome. Based on the known length of phage D6, this particular insertion in S. Montevideo strain 157 accounts for observed variation between this genome (~ 4.75 Mb) and the other spiced-meat S. Montevideo genomes reported here (~ 4.65 Mb). Moreover, this finding underscores the utility of whole-genome scanning technologies for placing the source of size polymorphisms between otherwise homogeneous strains of Salmonella.
NGS reveals phylogenetic discordance of hyper-discriminatory PFGE enzymes in an S. Montevideo outbreak cluster
The extent of phylogenetically congruent clustering between NGS and other conventional subtyping technologies such as PFGE, MLST, or MLVA is largely unknown for most serovars of S. enterica. Congruence is important in accessing the ability of subtyping methods to accurately assign genetic relatedness among closely related strains, such as those implicated in foodborne outbreak events . Previous studies from our laboratory and elsewhere have demonstrated enhanced discrimination and accuracy for PFGE in assigning genetic relatedness of some Salmonella and E. coli O157:H7 strains by concatenating up to six different restriction enzyme patterns into single cluster analyses [6, 10, 31, 32].
The availability of whole-genome sequences of Salmonella, such as S. Montevideo, enables a comparison between the conclusions of an epidemiological investigation and the linked clusters obtained from comparative genomics of the suspect isolates. One can also examine the patterns of linkage based on other genetic tools to the epidemiological evidence such as the discriminatory power of several non-conventional PFGE enzymes in the highly homogeneous group of S. Montevideos described above. After generating PFGE patterns for the six enzymes reported previously as part of the published concatenated PFGE protocol for non-typhoidal Salmonella in a previous study of S. Enteritidis and S. Typhimurium , we overlaid individual enzyme patterns onto the S. Montevideo NGS tree presented in Figure 3 and assessed congruence (i.e. agreement) in cluster assignments between the two methods. Owing to the extreme genetic homogeneity among these strains, four of the six enzymes (i.e., Xba I, Bln I, Spe I, and Sfi I) revealed identical PFGE patterns for all 40 of the S. Montevideo isolates included in the whole-genome tree. Moreover, the predominant pac I pattern varied in only one isolate (S. Montevideo 211) from Chinese Perch. In contrast, however, Not I, an enzyme reported previously as having a high discriminatory index for S. Typhimurium and S. Enteritidis , yielded 18 distinct patterns among the 40 S. Montevideos comprising this outbreak cluster. Albeit, side-by-side comparison of Not I pattern variants with NGS subgroups delineated in the clone tree revealed evidence for homoplasy (i.e., convergent pattern evolution) for this enzyme (Figure 3). That is, NotI patterns FDA.NotI.009, -.010. and -.011 were represented by S. Montevideo isolates from different subgroups in the tree suggesting that these patterns emerged independently in distinct places during the recent evolution of these isolates. As an example, pattern FDA.NotI.009 is represented twice in group A, once in group B and D, three times in group E, and four times in group F. Thus, while the concatenation of multiple PFGE enzyme data sets may permit a more accurate clustering of closely related Salmonella e, these data sound a cautionary note when attempting to cluster outbreak strains based on any single PFGE enzyme, including highly polymorphic ones such as Not I.
Biological, laboratory, and technical replicates of Salmonella Montevideo support reproducibility of NGS applications
As the power of NGS is realized in public health settings, deployment of the technology is expected to become more commonplace. Thus, it is important to further evaluate the technology, addressing questions concerning expected variation between closely related strains, background variation, and SNP variation that may arise during sub-culturing possibly obscuring an accurate molecular epidemiological analysis of isolates associated with contamination or outbreak events. That is, does variation arise in subsequent passages of an isolate and, if so, can phylogenetic analysis overcome the potentially misleading background noise associated with this level of sensitivity? We investigated this issue by sequencing to ~15× coverage 11 S. Montevideo isolates derived from clinical-food matches associated with the 2009 spiced-Italian style meat contamination event, such that isolates were taken from the patient as well as the suspected corresponding food vehicle that sickened that particular patient. Specifically, we included seven matching S. Montevideo isolates from a single clinical/food source in Iowa and five isolates from a single clinical/food source in Connecticut. Additionally, we sequenced a single S. Montevideo food isolate (237_Lunch_Meat) for separate passages (4X) (i.e., biological variation), separate colonies from the same passage (4X) (i.e, laboratory variation), and separate sequencing reactions from a single colony (4X) (i.e, Roche technical variation). Passages were conducted as follows: the initial sample was taken from frozen stock and plated on a TSA plate. Once plated it was incubated overnight at 37 degrees C. This was followed by Day 2 were sample was taken from the Day 1 overnight plate to inoculated the day 2 TSA plate. This day 2 plate was incubated overnight at 37 degrees C. Day 3 sample was taken from the Day 2 overnight plate and inoculated a day 3 TSA plate which was then incubated overnight at 37 degrees C. Day 4 sample was from the Day 3 overnight plate to inoculated the day 4 TSA plate and incubated overnight at 37 degrees C. After each plate was grown overnight, growth was taken from that plate and grown up a broth culture for DNA extraction of each of the genomic samples. Also, all samples were not single colony isolates for any of these plates. All passages and samples are representative cultures from the full plate and not just single colony.
Whole-genome sequencing yielded an alignment of approximately 4.5 mbps for downstream analysis. A total of 639 variable SNP sites were identified of which 23 were found to be parsimony informative among the validation isolates described above. However, once the data filter was applied to the remaining SNPs (i.e., elimination of SNPs in homopolymeric tracts, adjacent to assembly breakpoints, and duplicated in other lineages), only a single informative SNP at position 3,823,524 was found remaining which was stable in the original S. Montevideo isolate (237_Lunch_Meat_IA_1) and all of its downstream genomes derived from subsequent passages, colonies, and DNA samples of this one strain (Table 4). We also searched for SNPs using the proprietary run Mapping software from Roche and found the SNP corresponding to position 3,823,524 in the WGS alignment (results not shown).
As expected, these laboratory-generated isolates were indistinguishable in a phylogenetic analysis with the single parsimony informative SNP separating the 237_Lunch_Meat_IA_1 S. Montevideo isolate series from the other Iowa matching clinical-food isolates (Figure 5). Among the replicate genomes, only two sequences, genomes from S. Montevideo isolate 237 from the second and third round passages, retained actual SNP variation that emerged on the tree. That is, save for a single nucleotide difference present in the original 237 sample and two of the four downstream passages (i.e., 237-second round and 237-third round), none of the additional biological, laboratory, or technical replicate genomes yielded nucleotide differences after alignment and quality filtration. It is important to note that these few changes did not alter relatedness or inclusivity/exclusivity among the matching food/human isolates. Rather, the only structural difference in the tree to arise from these three changes was in the form of branch length for the individual isolates affected. Additionally, it is noteworthy that, in the larger outbreak clone tree, the Connecticut and Iowa matching isolates were both phylogenetically inseparable from their sister isolates, and collectively, both strain sets sorted squarely among the spiced-meat food, environmental, and clinical isolates associated with the same contamination event (Figure 3). These findings indicate that when NGS data are quality filtered and inspected carefully using inclusivity/exclusivity criteria, the resultant stable and informative SNP data can be used effectively to phylogenetically partition closely related isolates of S. enterica (i.e., S. Montevideo). Consistent with this find, a bolus of successful applications is now accruing [17–20].
NGS provides discovery for development of novel MLVA targets
Aside from its emerging and direct role in highly homogeneous S. enterica outbreaks, it is important to recall the important function of NGS for augmenting conventional detection, identification, and subtyping methods development. Currently, several rapidly evolving regions of the Salmonella chromosome are under investigation for their utility for enhanced subtyping of highly homogeneous Salmonella strains associated with foodborne outbreaks. Specifically, select VNTRs (variable number tandem repeats) in the genomes of Salmonella and E. coli have been targeted to develop markers and probes for MLVA (multi-locus VNTR analysis), a rapid and sensitive subtyping method that fingerprints the genomes of closely related strains based on size polymorphism of VNTR sequences [33, 34].
Since MLVA protocols are developed at the serovar level for Salmonella, very few are available save for the most significant and widely studied Salmonella e (i.e., S. Typhimurium and S. Enteritidis). Here, using our NGS alignments only, we identified a polymorphic VNTR region within S. Montevideo that may serve in the development of a MLVA protocol for this important foodborne serovar as well (Table 5). The locus was identified within a cell division gene (fts N) and delineates the major S. Montevideo lineages represented in our NGS serovar tree in Figure 1. Moreover, this finding illustrates the importance of providing NGS data from multiple strains and multiple serovars in order to foster the identification of additional MLVA loci to support rapid subtyping protocols for Salmonella serovars of public health significance.
Here, we reported the use of NGS technology for describing the phylogenetic diversity of S. Montevideo, a significant serovar of S. enterica involved in numerous outbreaks and product recalls http://www.cdc.gov/ncidod/dbmd/phlisdata/Salmonella.htm#2009. Moreover, we have applied informative substitutions from these genomes to further ascertain phylogenetic relatedness among a highly homogeneous S. Montevideo clone, of which some strains were associated with a recent spiced-meat outbreak event in the U.S last year. In this instance, the investigatory utility of NGS became apparent as the unusual genetic homogeneity among both outbreak associated and non-associated S. Montevideo strains could not be resolved unambiguously with more conventional genotyping approaches. Comparative genomic molecular epidemiology produced hundreds of SNP differences across distinct lineages of S. Montevideo and even provided broad size differences among the most distantly diverged strains of this serovar. Among the S. Montevideos populating clade IV in the serovar tree, nearly all shared common pulsotypes for Xba I and Bln I as well as for several additional enzymes including Spe I, Sfi I, and Pac I. NGS combined with phylogenetic analysis, however, was able to delineate the scope of contamination by differentiating those strains associated with the spiced-meat outbreak from strains epidemiologically unrelated to this event despite the remarkable genetic identity linking these two strain sets. Given the extraordinary resolution that NGS provides--resolution best described as "nanotyping", it is not surprising that, when Salmonella isolates with divergent PFGE patterns are sequenced using NGS technology, the resultant alignments typically yield thousands of SNP differences.
For S. Montevideo, four disparate lineages of strains were observed (i.e., clades I-IV, Figure 1). One lineage, in particular (i.e., clade I in Figure 1), was characterized by a single isolate from sunflower, and it remained unclear as to whether the long branch distinguishing this isolate was due to changes that accumulated more recently from laboratory passages or whether observed variation in this strain accrued in a natural setting. Surprisingly, this sunflower isolate clustered with several S. Montevideos recently isolated from pet treats and a pet treat-manufacturing environment underscoring the potential risk associated with this and the other discrete lineages of this serovar http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm197700.htm. That is, it appears that foodborne contamination events can emerge from any of these diverged S. Montevideo lineages which are able to survive in foods and cause illness in humans. Clinical isolates were found in each of the major and separate lineages of S. Montevideo tested (Figure 1 Clades I, II, III, and IV). Moreover, such observations enforce the notion that in addition to these attributes, the risk to public health also stems from a particular Salmonella lineage simply gaining the opportunity to contaminate the human or animal food supply, rather than any one S. Montevideo lineage being more fit to persist in foods over any other.
Separation, based on SNP distances, among the four phyletic lineages of S. Montevideo reported here rivaled distances observed between S. Montevideo and other distinct Salmonella subspecies I serovars including S. Pomona, S. Javiana, and S. Schwarzengrund. Such remarkable interclade divergences suggests that the four major lineages of S. Montevideo diverged early in the evolution of this serovar, and each appears to have evolved largely independent of the others, an evolutionary pattern consistent with a hypothesis of unique host/niche adaptation for the separate lineages and sublineages that compose this serovar. This thesis is further supported by an examination of variable genes and SNPs that define various lineages of isolates within S. Montevideo clade IV. That is, 25 of 43 select informative SNPs (Table 3) defining subgroups within this clade were non-synonymous. Additionally, 78% of the representative informative SNPs clustering together two or more S. Montevideo subgroups were also found to be polymorphic. These data are reminiscent of a previous report by Soyer et al.,  which noted a potentially significant role for positive selection based on an unusual proportion of non-synonymous substitutions across the genomes of several host-adapted serovars including S. Cholerasuis, S. Typhimurium, and the agent of Typhus, serovar S. Typhi (28). Taken together, these data signal S. Montevideo as a potentially niche-adapted and evolutionarily diverse serovar among the subspecies I Salmonella e, a conclusion additionally supported by an extraordinary ecological range and natural persistence in diverse environments (e.g., S. Montevideo has been found associated with spices, produce, poultry, beef and porcine commodities to name but a few).
The results from the genome validation study reported here also merit discussion. After collecting over 50 mbp of finished bacterial sequence for multiple downstream passages, colonies, and DNA preparations of a single S. Montevideo isolate, it was clear that NGS had provided sufficiently stable data to conclude that no single potential source of variability tested (i.e., biological, laboratory or technical) was capable of altering phylogenetic conclusions uncovered during the comparative genomic investigation. That is, despite the detection of three substitutions among serially passaged genomes from a single S. Montevideo source, no re-sequenced replicate conflicted with our phylogenetic conclusions here or for strains included in a previously published letter defining an S. Montevideo spiced-meat outbreak cluster . Rather, it is clear that phylogenetic approaches are providing rational and highly reproducible analytical outcomes for high-resolution NGS data pipelines and appear to be sufficiently robust for reconstructing strain relatedness based on the hundreds and sometimes thousands of informative changes that amass from a single NGS experiment.
Global deployment of NGS technology as a direct investigatory tool has already proven to be highly successful to the public health community. In addition to the NGS application described here for one non-typhoidal Salmonella serovar, NGS has provided extraordinary insight into case studies involving: (i) traceback of tuberculosis infections in Canada ; (ii) high-resolution evolutionary linkage of global clones of Salmonella Typhi ; and (iii) identification of the origins of the Haitian Cholera outbreak  as a few examples. It is important to note, however, that NGS data can provide additional utility for development of other subtyping methods. The MLVA locus presented here is one example of how NGS can serve as a genomic "compass" in seeking out VNTR regions with sufficient rates of change to develop custom MLVA assays for other important Salmonella serovars beyond S. Typhimurium and S. Enteritidis. Additionally, as shared NGS public health databases expand, many outbreak swarms will be defined by even more rapid and efficient re-sequencing protocols that target a subset of informative SNPs relative to the differentiation of a specific outbreak clone of pathogenic bacteria.
We would like to caution that the results reported here, while extremely encouraging, do not supplant the need for independent laboratory validations to establish SOPs for their particular platforms and chemistry and kits. Such validations may include the adoption of standard practices that have worked so well with past genetic testing, including CE methods that can more easily target and validate variable sites identified by whole genome sequencing and downstream phylogenetic analyses. Clearly, given the evolutionary rates governing nucleotide change among enteric bacteria combined with the risk of intrinsic polymerase error in the sequencing process itself, each step of the pathway, from isolate collection and template preparation to the sequencing reactions, could potentially spawn artifactual variability. A careful assessment of all of these sources of variation should provide more confidence for molecular epidemiological applications including the detection and scope of disease outbreak clusters.
These results underscore the power of NGS, when coupled with phylogenetic analysis, to illuminate the genetic and evolutionary diversity of important serovars of Salmonella enterica along with the associated epidemiological pathways surrounding specific outbreak strains [17–20]. It appears that, at least in the case of Salmonella, the natural variation observed between strains is both stable and sufficient to allow for high resolution traceback of food and clinical isolates. It will be interesting to see whether ample genomic diversity can drive similar outcomes in other problematic taxa and highly clonal Salmonella serotypes. Moreover, NGS will provide the phylogenetic context on which to interpret other facile subtyping approaches that focus on more rapidly evolving genetic markers such as MLVA, rep-PCR, and CRISPRs [6–11, 35] and will provide a novel suite of SNPs that will be critical to partitioning common Salmonella outbreak strains. In public health arenas, NGS strain "nanotyping" holds the potential to revolutionize the manner in which responses to outbreaks are managed. At a minimum, we see a future where NGS methods are brought to bare on the most difficult questions involving this enteric pathogen including direct application in foodborne outbreak cases in combination with other time-tested methods of epidemiologic investigation.
Data collection and analysis pipeline methods
Roche 454 GS Titanium NGS technology was employed in this study. This platform provided longer read lengths relative to other sequencers and a relatively shorter time to raw sequence . Longer read lengths resulted in fewer contigs for draft assembly and aided in a more accurate placement of phage and plasmid sequences, both of which are commonplace among the group I salmonellae. All S. Montevideo isolates were draft shotgun sequenced using this platform and included 47 total isolates of S. Montevideo including 40 with PFGE patterns matching the spiced-meat outbreak (Figure 1, lineage IV) and 7 with unrelated PFGE patterns (Figure 1, lineages I-III). Additionally, 11 genomic replicates were sequenced for the validation experiment, including multiple colonies from the same plate (n = 4), multiple passages of an isolate (n = 4), and independent sequencing experiments (n = 4) from the same DNA source (Figure 5). Each isolate was run on a quarter of a titanium plate that produced roughly 250,000 reads per draft genome and coverage from 13× to 18×. Draft sequences for 34 of the 47 isolates were previously released as part of an outbreak case study  to test earlier hypotheses regarding the delimiting of foodborne contamination events.
De novo assemblies were created using the Roche Newbler (v 2.3) software package and the resulting contigs were annotated using NCBI's Prokaryotic Genomes Automatic Annotation Pipeline [PGAAP, ]. Phylogenetically informative SNP sites were identified using two independent alignment methods: 1) clustering of annotated open reading frames (ORFs) using reciprocal best Basic Local Alignment Search Tool [BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi] hits with a 70% sequence identity setting followed by alignment with Multiple Sequence Comparison by Log-Expectation [MUSCLE, ], and 2) multiple genome alignment of WGS contigs using Mauve . Duplicated genes were eliminated from all ORF clusters. The Mauve and ORF cluster alignments were then screened to find non-gap phylogenetically informative nucleotide positions (i.e. minor allele count ≥ 2). Informative positions from all ORF clusters and Mauve outputs were identical in the annotated protein coding regions. Informative positions for isolates in the outbreak cluster were manually checked to eliminate SNPs in homopolymers and repetitive elements. In this way, roughly 10-15 percent of the draft genome is filtered out, but the remaining SNPs are highly reproducible, providing sufficient variation for an informed molecular epidemiology interpretation .
Phylogenetic analysis of the clonal S. Montevideo data set including multiple serovars was performed on a set of 55,032 concatenated informative SNPs which encompasses the diversity within S. Montevideo. Approximately 99% of the sites in the 5 MB Salmonella genomes are phylogenetically uninformative and eliminating them dramatically reduces computation time and memory requirements. Phylogenetic analysis of the outbreak isolates was performed on the set of 43 concatenated ORFs containing informative SNPs. In all cases, phylogenetic trees were constructed using GARLI  under the maximum likelihood criterion. The phylogenetic tree in Figure 1 was constructed using GARLI under the GTR + gamma model of nucleotide evolution. The phylogenetic trees in Figure 3 and 5 were constructed using GARLI under the HKY + gamma model of nucleotide evolution.
The other related Salmonella including S. Schwarzendgrund and S. Javiana were taken from genbank (Table 1). S. Pomona was sequenced like the S. Montevideo isolates with an FDA ID number. One comparative genomics analysis suggested that S. Schwarzendgrund and S. Javiana are closely related  and our independent analyses, not shown, also would include S. Montevideo and S. Pomona in this cluster so we include all of these as outgroups.
We use the resultant phylogenetic trees to make hypotheses about the evolution of the S. Montevideo subtypes and the outbreak strains and to aid in investigation source tracking. We use these evolutionary hypotheses to identify reliable diagnostic nucleotide motifs (SNPs, rearrangements, and gene presences) for the identification of outbreak strains and for understanding the mechanisms that drive the outbreak occurrences. These methods allow both the rapid characterization of the genomes of foodborne pathogenic bacteria and can help identify the source of contamination of the food supply.
Availability of data and cultures
All NCBI S. Montevideo genomes are linked to Bioproject 61937 which lists the new accession numbers AESR00000000-AESY00000000, AHIA00000000 and AHHT00000000 - AHHW00000000. Cultures included in this study are also available upon request to anyone with valid paper work and clearances. Please direct any queries to our strain curator Dwayne Roberson, at firstname.lastname@example.org.
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We thank the NCBI rapid annotation pipeline team, Bill Klimke, Dmitry Dernovoy, Stacy Ciufo, Ruth Timme, Kathleen O'Neill, Azat Badretdin and Tatiana Tatusova, for key genome annotation services, and Charlie Wang and Guojie Cao for expert data collection. We acknowledge David Weingaertner for excellent graphical support, and would also like to thank Donald Zink, John Guzewich, Sherri McGarry, Mickey Parrish, Kathy Gombas, Roberta Wagner, Donald Kraemer, and Michael Landa from CFSAN-FDA for program support and for important epidemiological and investigatory insights as well as important discussion of our manuscript. We would also like to acknowledge our FDA-ORA regional field laboratories in Denver, Arkansas, and Atlanta for providing key outbreak and historical isolates along with the Iowa, Connecticut, North Carolina, Maryland, Ohio, California, and Rhode Island State Departments of Public Health for generous contributions of additional clinical S. Montevideo isolates. In particular, we would like to acknowledge Stacey Kinney (Connecticut Department of Health) and Mary DiMartino (Iowa Department of Health) for providing key human-food matching pairs of S. Montevideo. Mark Wilson, Peter Evans, Stephanie Defibaugh-Chavez, Kurt Lienau, Peter Gerner-Smidt, and several anonymous people also provided helpful reviews. No human subjects or animals were used in this study. All authors have read the manuscript and agree to its content, subject matter, and author line order. These data are novel and have not been previously published elsewhere. Disclosure forms provided by the authors will be available with the full text of this article.
Authors and Affiliations
Office of Regulatory Science, Center for Food Safety & Applied Nutrition, U.S. Food & Drug Administration, 5100 Paint Branch Parkway, College Park, MD, 20740, USA
Marc W Allard, Cong Li, Christine E Keys, Insook Son, Steven M Musser & Eric W Brown
Office of Food Defense, Communications, and Emergency Response, Center for Food Safety & Applied Nutrition, U.S. Food & Drug Administration, 5100 Paint Branch Parkway, College Park, MD, 20740, USA
Yan Luo & Errol Strain
Food & Environment Research Agency, Sand Hutton, York, YO41 1LZ, UK
ES and YL performed the bioinformatics analysis and analyzed the data; RS provided critical programming and bioinformatic tools; MWA, SMM, and EWB planned and conceived the experiments and co-wrote the manuscript; CL generated the sequencing libraries; and CEK and IS generated and analyzed the PFGE data. All authors read and approved the final manuscript.
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Allard, M.W., Luo, Y., Strain, E. et al. High resolution clustering of Salmonella enterica serovar Montevideo strains using a next-generation sequencing approach.
BMC Genomics13, 32 (2012). https://doi.org/10.1186/1471-2164-13-32


A strong collaboration between Nintendo and Illumination Studios; the film is a lot of fun!
The “Super Mario Brothers” are a two-brother team of plumbers in Brooklyn, hoping to take the plumbing world by storm with their skills. But when they are sucked into another dimension, they have a lot more to deal with than leaky pipes!
With three directors, Aaron Horvath and Michael Jelenic (Teen Titans Go! to the Movies) and Pierre Leduc (Illumination Studios Art Director behind Despicable Me) as well as heavy involvement from legendary Nintendo creators such as Shigeru Miyamoto, the Mario movie has been in development for a long time.
Of course, Nintendo has had an awkward past with movies: 1993 saw the infamous Super Mario Bros movie, with Bob Hoskins, and the lesser known The Wizard in 1989. Now, with this new movie, the controversy was hot with the announcement that Mario would be voiced by Hollywood go-to actor Chris Pratt. Which to many felt like a flat-footed decision made purely for marketing purposes.
But, is this movie an example of history repeating, or is it… good??
It is a good movie!
And to answer the burning questions: No, Chris Pratt isn’t the worst thing about it. Is it better than the recent Sonic the Hedgehog movies? Absolutely yes. But it isn’t without flaws.
The movie barrels along at a quick pace; a lot happens in the ninety minute runtime. The film does follow the “fish-out-of-water” storyline, with the brothers Mario and Luigi starting out in Brooklyn and working as plumbers, and then progressing to them having to adapt in the Mushroom Kingdom. The film has few breaks once this transition happens, although it isn’t as relentless as other Illumination Studio productions (looking at you, Minions)
The visuals of the movie are completely spot-on. Nintendo-aesthetics are adhered to extremely well, with dozens of references subtle and otherwise. We see nods to multiple games in the franchise, but used as means to progress story or to enable exciting sequences and fights. Most notable are references to Mario Kart, Super Mario 3D World, and the Paper Mario series.
It hosts a loaded cast as well, besides Pratt we also have Anya Taylor-Joy as Princess Peach, Jack Black as King Bowser, Seth Rogen as Donkey Kong, and Charlie Day as Luigi. They all do a good job, but especially Jack Black as the villainous tyrant, Bowser, who easily… easily steals the show. Bowser was clearly very lovingly adapted for the movie. Someone knew what they were doing!
But it isn’t all Super Shines and Rainbow Roads. With development hells comes cracks, even subtle ones, and Illumination Studios perhaps had a different vision for the project before Nintendo pumped the brakes on those plans. The first five or ten minutes are resoundingly… “Illumination > Nintendo”. There was an edge of fear going forward that this would be a spoof rather than an adaptation.
There’s also the music… Now, Nintendo has gorgeous music. Listen to the Super Mario Galaxy soundtrack, it is beautiful. This film uses orchestrated versions of Nintendo music, both subtly and bombastically, and these are wonderful. It also uses… AC/DC’s “Thunderstruck”? “Take On Me” by A-ha. Now, these are awesome tracks in their own right… but they really don’t fit here, and definitely feel like Illumination’s creative choice.
There’s also an edge of “expanding” the lore of Mario, which seemed odd. The origins of Princess Peach? Mario and Luigi’s family? It is all a little… unnecessary when you have a plumber fighting a giant turtle.
But, it is a lot of fun, and it is a great representation of Nintendo’s beloved mascot. Even though Bowser definitely owns the movie, as he rightfully should.
Additional Marshmallows: Basically all of the Mario “quotes” from the trailers… the ones people really didn’t like… were missing from the movie. Either this was done post-haste, or the trailer was designed to cause Internet drama. We might never know.