text:
Lost Idol: New Wave of Myanmar Youth Activists Look Beyond Daw Aung San Suu Kyi
TIMELINE: China-Myanmar Relations
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Myanmar State Counselor Daw Aung San Suu Kyi waits for the arrival of her delegation before a Japan Myanmar Summit meeting at the Palace State Guest House in Tokyo, Japan. on Oct. 9. / Reuters
By Reuters 3 December 2018
YANGON — Myanmar youth activist and television host Thinzar Shun Lei Yi would once have called herself one of Daw Aung San Suu Kyi's greatest fans. Now, she is one of her most vocal critics.
The 27-year-old belongs to a small but high-profile group of liberal activists, many former die-hard Daw Aung San Suu Kyi supporters, who are growing increasingly disillusioned with the administration they voted into power with sky-high hopes three years ago.
"I lost my idol. I'm confused, frustrated and lost," said Thinzar Shun Lei Yi, who hosts an "Under 30" talk show on a popular local website.
"Most of the activists and youths are now thinking: 'What is next,' 'What will happen?' 'What can we do?' At this stage, Daw Aung San Suu Kyi is going her own way and nobody can intervene, and she won't listen to civil society organizations," she said.
While Daw Aung San Suu Kyi continues to inspire devotion among many ordinary Burmese, the emergence of a dissenting youth movement — driven by anger over her handling of ethnic minorities, including the Muslim Rohingya, as well as curbs on the media and civil society — presents a new challenge for her administration.
At stake is the future of Myanmar's transition towards democracy after years of military rule. With a general election looming in 2020, the country's first civilian government in decades is confronted by growing divisions among activists who once coalesced around her National League for Democracy (NLD) party.
NLD spokesman U Myo Nyunt said the party was trying to win over young people, increasing the budget for education and supporting vocational training programs.
"The youth and the people expected a lot from our government," he said. "We couldn't live up to their expectations, we admit. But we are doing our best."
Daw Aung San Suu Kyi took power in 2016 after a landslide election win, vowing to continue democratic reforms and end the country's long-running civil wars.
Since then, the administration has come under pressure over its response to a military crackdown against the Rohingya minority that the United Nations has described as "ethnic cleansing" with "genocidal intent," as well as faltering peace talks with ethnic armed groups and a stagnating economy.
Activists say the civilian government has also become increasingly authoritarian, failing to use its overwhelming parliamentary majority to scrap colonial-era laws used to stifle dissent, while tightening restrictions on civil society.
In recent months, they have staged several protests, including an anti-war march in the commercial capital of Yangon in May that ended in scuffles. A total of 17 people were charged with unlawful protest, including Thinzar Shun Lei Yi. Their trial is ongoing.
"Sensitive issues are banned, and protesters arrested and beaten," she said. "The National League of Democracy, the party using the name of democracy, must respect democracy and human rights."
According to free speech organization Athan, which means "Voice" in Burmese, 44 journalists and 142 activists have faced trial since Daw Aung San Suu Kyi's government took power.
They include Reuters reporters Ko Wa Lone, 32, and Ko Kyaw Soe Oo, 28, who were sentenced to seven years in prison in September after being convicted of breaking the colonial era Official Secrets Act.
The journalists are appealing their conviction to the country's High Court, citing evidence of a police setup and lack of proof of a crime. Daw Aung San Suu Kyi said in September their jailing had nothing to do with freedom of expression. The government says Myanmar's courts are independent.
Athan's founder, poet and activist Maung Saung Kha, was among the protesters charged alongside Thinzar Shun Lei Yi in May. Four months later, in September, they both helped organize another demonstration, this time for free speech.
Facing the crowd, Maung Saung Kha — who is still an NLD member — donned the orange shirt traditionally worn by his party's lawmakers and draped a green jacket resembling military garb over it. Armed with a copy of the state-run daily newspaper The Mirror, he began beating journalists gathered nearby.
"The government has failed to use its power to protect people's rights," he told Reuters.
Myo Nyunt, the party spokesman, said the government was cooperating with non-governmental organizations, but their activities needed to be examined case-by-case.
"If it is not related to security or not a divisive issue among ethnics, we accept them," he said. "We are going forward to democracy, so we acknowledge the role of NGOs. But we have concerns that NGOs are being influenced by sponsors instead of being independent."
"Acknowledge Rohingya"
While she has no control over the military, Daw Aung San Suu Kyi has faced international criticism for failing to defend the Rohingya, more than 730,000 of whom fled a sweeping army crackdown in western Rakhine State in 2017, according to UN agencies. The crackdown was launched in response to insurgent Rohingya attacks on security forces.
Myanmar denies almost all the allegations of atrocities made by refugees, saying the army was carrying out a legitimate campaign against terrorists.
While many among Myanmar's Buddhist majority revile the Rohingya, the young activists offer a rare sympathetic voice.
"We acknowledge Rohingya. We totally denounce the fact that they are referred to as 'Bengali,'" said Maung Saung Kha, referring to a term commonly used in Myanmar to imply the Rohingya are interlopers from Bangladesh, despite a long history in the country.
"We haven't seen any acknowledgement or punishment for the things that happened," he said. "The refugees will not come back as long as these people think of them as less than humans, and that it is not a crime to kill them."
Khin Sandar, another young activist facing unlawful protest charges, spent months campaigning for the NLD ahead of the 2015 election but lost faith in Daw Aung San Suu Kyi over her handling of the Rakhine crisis.
Her family was affected in a wave of communal violence in 2012, when not only Rohingya but members of the Kaman Muslim minority, who also face discrimination but unlike the Rohingya are considered Myanmar citizens, were driven from their homes. They live in crowded internal displacement camps outside the Rakhine State capital Sittwe and are subjected to severe restrictions on movement.
In a speech after last year's violence, Daw Aung San Suu Kyi said all residents of Rakhine "have access to education and healthcare services without discrimination."
"My own nephew and nieces are still living in the Sittwe camps and they don't have those rights," said Khin Sandar. "I was shocked. How can she say that in her speech?" Afterwards, she said, she quit her job as researcher for an NLD lawmaker.
While the youth activists represent only a small segment of Myanmar society, they are increasingly influential in the grassroots activism scene, while their protests and public comments have attracted significant attention from media and from their vast social media followings.
Mostly in their 20s and 30s, they highlight the gulf between Myanmar's young population — the median age is 27 — and its ageing leadership, comprised of mostly men in their 60s and 70s.
"Myanmar is a very conservative country. These young people, especially from Yangon, are now challenging that," said Myat Thu, a political analyst from the Yangon School of Political Science.
"In order to have a revolution of ideas, not many people need to know. They will spread it gradually."
Topics: Activist, NLD, Rohingya, Youth
Reuters ...


As technologists … we are often called upon to figure out HOW things work in a system.
First, and possibly foremost, understanding WHY something works the way it does gives a far better understanding of HOW it works. Understanding HOW it works, just means you understand the mechanics of how you interact with something. Understanding WHY it works means you understand the underlying mechanism (even if it's just at a high level).
Additionally, if all you understand is HOW something works, when it works in slightly different ways you might not be prepared for them. Understanding WHY something works the way it does will give you more insight into different behaviors that might be encountered.
Knowing that, if you change a physical file, you must recompile the programs that use it. Understanding what format level identifiers and how they are bound into program objects.
When you create a new message file, it should have a specific CCSID assigned to it. Understanding what CCSID's mean and how the system handles the various values.
In the end, understanding WHY something works will make you a much more skilled technologist than simply understanding HOW it works.
And, for those of you who already know WHY something works … if someone asks you HOW it works … don't stop and just explaining the HOW, explain the WHY also. The person who's asking the question might be interested, and probably should know.


text:
package org.wisdom.framework.csrf.controller;

import org.wisdom.api.DefaultController;
import org.wisdom.api.annotations.*;
import org.wisdom.api.http.HttpMethod;
import org.wisdom.api.http.Result;
import org.wisdom.api.templates.Template;
import org.wisdom.framework.csrf.api.CSRF;
import org.wisdom.framework.csrf.api.AddCSRFToken;

@Controller
public class CSRFController extends DefaultController {

    @View("csrf")
    Template template;

    @View("dialect")
    Template templateWithDialect;

    public CSRFController() {
        logger().info("Starting controller");
    }

    @Route(method = HttpMethod.GET, uri = "/csrf")
    @AddCSRFToken
    public Result getPage(@HttpParameter(AddCSRFToken.CSRF_TOKEN) String token) {
        if (token == null) {
            return internalServerError("Token expected " + context().request().data());
        }
        return ok(render(template, "token", token));
    }

    @Route(method = HttpMethod.GET, uri = "/csrf/dialect")
    @AddCSRFToken
    public Result getPageUsingDialect() {
        return ok(render(templateWithDialect));
    }

    @Route(method = HttpMethod.POST, uri = "/csrf")
    @CSRF
    public Result submitted(@FormParameter("key") String key) {
        if (key == null) {
            return internalServerError("Key expected");
        }
        return ok(key);
    }
}

redpajama_set_name: RedPajamaGithub


text:
Главная Новости и события Новости SW Business Aviation debuts with a new G650
SW Business Aviation debuts with a new G650
Azeri operator Silk Way (SW) Business Aviation, a subsidiary of the holding company Silk Way, is debuting at Jet Expo this year with its Gulfstream G650 ultralong-range business jet at the static park. This is the second example of the type received by the company. The first aircraft was delivered in March 2015 and has been operated in the interest of the Azeri government. The second airframe is available for charter bookings. SW Business Aviation says this G650 has an exclusive interior design and offers everything necessary to keep passengers comfortable during long-haul flights. "Our company is very much interested in the Russian bizav market; while we may be of no immediate medium-term interest to Russian customers due to the abundance of offerings on the market, the G650 is a fundamentally new product," says an SW Business Aviation representative. "There are fairly few G650 charters in Europe, and we hope that Russian clients will appreciate the benefits of choosing this newest aircraft on transcontinental routes." The first half of 2015 was very busy for the operator. The second G650 was delivered in June, adding to the company's capacity and expanding its route network. SW Business Aviation bore the brunt of bizav services associated to the European Games hosted by Baku on June 12-28. "Our traffic grew by 30% in 2014 because we started operating our two new Gulfstream G280 super-midsize jets," the company says. "The most popular destinations out of Baky are Moscow, London, Dubai, and Istanbul." Based at Baku's Heydar Aliyev International Airport, SW Business Aviation is a major bizav operator in the Caspian region. The backbone of its fleet is represented by nearly the entire Gulfstream product line, including the G550, G450, G200, and G280. There are also a dedicated Boeing 727-200 for small delegations and a VIP-configured 20-seat ATR 42-500.

redpajama_set_name: RedPajamaCommonCrawl


text:
Biological and Structural Characterization of a Host-Adapting Amino Acid in Influenza Virus
Shinya Yamada,
Affiliation Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan
Masato Hatta,
Affiliation Influenza Research Institute, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, United States of America
Bart L. Staker,
Affiliations Emerald BioStructures, Inc., Bainbridge Island, Washington, United States of America, Seattle Structural Genomics Center for Infectious Disease, Washington, United States of America
Shinji Watanabe,
Masaki Imai,
Kyoko Shinya,
Affiliation Department of Microbiology and Infectious Diseases, Kobe University, Hyogo, Japan
Yuko Sakai-Tagawa,
Mutsumi Ito,
Makoto Ozawa,
Affiliations Influenza Research Institute, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, United States of America, Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo, Japan
Tokiko Watanabe,
Saori Sakabe,
Affiliations Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan, ERATO Infection-Induced Host Responses Project, Saitama, Japan
Chengjun Li,
Jin Hyun Kim,
Peter J. Myler,
Affiliations Seattle Structural Genomics Center for Infectious Disease, Washington, United States of America, Seattle Biomedical Research Institute, Seattle, Washington, United States of America, Departments of Global Health and Medical Education & Biomedical Informatics, University of Washington, Seattle, Washington, United States of America
Isabelle Phan,
Affiliations Seattle Structural Genomics Center for Infectious Disease, Washington, United States of America, Seattle Biomedical Research Institute, Seattle, Washington, United States of America
Amy Raymond,
Eric Smith,
Robin Stacy,
Chairul A. Nidom,
Affiliations Faculty of Veterinary Medicine, Tropical Disease Centre, Airlangga University, Surabaya, Indonesia, Collaborating Research Center-Emerging and Reemerging Infectious Diseases, Tropical Disease Centre, Airlangga University, Surabaya, Indonesia
Simon M. Lank,
Affiliation Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America
Roger W. Wiseman,
Benjamin N. Bimber,
David H. O'Connor,
Affiliations Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America, Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, Wisconsin, United States of America
Gabriele Neumann,
Lance J. Stewart ,
* E-mail: kawaokay@svm.vetmed.wisc.edu (YK, for virological research aspects); lstewart@embios.com (LJS, for structural research aspects)
[ ... ],
Yoshihiro Kawaoka
Affiliations Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan, Influenza Research Institute, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, United States of America, Department of Microbiology and Infectious Diseases, Kobe University, Hyogo, Japan, Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo, Japan, ERATO Infection-Induced Host Responses Project, Saitama, Japan, Creative Research Initiative, Sousei, Hokkaido University, Sapporo, Japan
[ view less ]
Tokiko Watanabe
Shinya Yamada Masato Hatta ... Yoshihiro Kawaoka
Two amino acids (lysine at position 627 or asparagine at position 701) in the polymerase subunit PB2 protein are considered critical for the adaptation of avian influenza A viruses to mammals. However, the recently emerged pandemic H1N1 viruses lack these amino acids. Here, we report that a basic amino acid at position 591 of PB2 can compensate for the lack of lysine at position 627 and confers efficient viral replication to pandemic H1N1 viruses in mammals. Moreover, a basic amino acid at position 591 of PB2 substantially increased the lethality of an avian H5N1 virus in mice. We also present the X-ray crystallographic structure of the C-terminus of a pandemic H1N1 virus PB2 protein. Arginine at position 591 fills the cleft found in H5N1 PB2 proteins in this area, resulting in differences in surface shape and charge for H1N1 PB2 proteins. These differences may affect the protein's interaction with viral and/or cellular factors, and hence its ability to support virus replication in mammals.
Influenza viruses that originate from avian species likely have to acquire adapting amino acid changes to replicate efficiently in mammals. Two amino acid changes in the polymerase PB2 protein—a glutamic acid to lysine change at position 627 or an aspartic acid to asparagine change at position 701—are known to allow influenza viruses of avian origin to replicate efficiently in mammals. Interestingly, the pandemic H1N1 viruses (which possess an avian-like PB2 gene) do not encode the 'human-type' amino acids PB2-627K and PB2-701N. Here, we report that a basic amino acid at position 591 of PB2 can compensate for the lack of PB2-627K and allows efficient replication of highly pathogenic H5N1 and pandemic H1N1 viruses in mammalian species. We also present the X-ray crystal structure of the C-terminal portion of a pandemic H1N1 PB2 protein. The basic amino acid at position 591 fills a distinctive cleft found in the PB2 proteins of H5N1 viruses. We also speculate on the biological significance of the altered surface of the H1N1 PB2 protein.
Citation: Yamada S, Hatta M, Staker BL, Watanabe S, Imai M, Shinya K, et al. (2010) Biological and Structural Characterization of a Host-Adapting Amino Acid in Influenza Virus. PLoS Pathog 6(8): e1001034. https://doi.org/10.1371/journal.ppat.1001034
Editor: Daniel R. Perez, University of Maryland, United States of America
Received: March 9, 2010; Accepted: July 12, 2010; Published: August 5, 2010
Copyright: © 2010 Yamada et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a National Institute of Allergy and Infectious Diseases Public Health Service research grants (R01 AI069274), by an NIAID-funded Center for Research on Influenza Pathogenesis (CRIP, HHSN266200700010C), by Grant-in-Aid for Specially Promoted Research, by a contract research fund for the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases from the Ministry of Education, Culture, Sports, Science and Technology, and by grants-in-aid from the Ministry of Health and by ERATO (Japan Science and Technology Agency). This research was also funded by NIAID under Federal Contract No. HHSN272200700057C which supports the Seattle Structural Genomics Center for Infectious Disease (www.SSGCID.org). Support for deep sequence analysis was provided by US National Center for Research Resources grant P51 RR000167 to the Wisconsin National Primate Research Center. Dr. Shinya was supported by Precursory Research for Embryonic Science and Technology (PRESTO). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Influenza viruses pose an ongoing threat to human health, as underscored by the current H1N1 influenza pandemic and the sporadic transmission of highly pathogenic avian H5N1 influenza viruses to humans [1], [2]. Viral determinants of virulence and transmissibility are still poorly understood, although lysine at position 627 of the polymerase subunit PB2 (PB2-627K) is now known to be important for avian influenza virus adaptation to mammals [3], [4]. Most avian influenza viruses (with the exception of the Qinghai Lake-lineage of H5N1 viruses) possess glutamic acid at this position (PB2-627E), whereas most human influenza viruses (with the notable exception of the current pandemic H1N1 viruses) have lysine (PB2-627K). In addition, replacement of the aspartic acid at position 701 of PB2 (PB2-701D) found in most avian influenza viruses with asparagine (PB2-701N) conferred high pathogenicity to an H5N1 influenza virus in mice [5]. These mammalian-type amino acids (i.e., PB2-627K and PB2-701N) are found in some H5N1 (http://www.flu.lanl.gov) or H7N7 [6] influenza viruses isolated from humans, are selected during replication of H5N1 viruses in humans [7], and facilitate virus transmission in ferret [8] and guinea pig models [9]. Collectively, these results have led to the concept that PB2-627K or PB2-701N are critical for efficient influenza virus replication in mammalian species. Nevertheless, the pandemic H1N1 viruses and some H5N1 influenza viruses isolated from humans do not possess these amino acids. Here, we sought to identify additional amino acid changes that facilitate virus adaptation in mammalian species.
Results/Discussion
Lysine at position 591 of the PB2 protein confers efficient replication to an H5N1 influenza virus in mammals
An H5N1 influenza virus isolated from an infected person in Indonesia in 2005 (A/Indonesia/UT3006/05, UT3006) replicated more efficiently than an avian H5N1 virus (A/chicken/Indonesia/UT3091/05 virus; CkUT3091) in normal human bronchioepithelial (NHBE) cells (Fig. 1), despite having avian-type amino acids at PB2-627 and PB2-701. In addition, the mouse lethal dose 50 (MLD50) of UT3006 is 180 plaque-forming units (PFU), indicating appreciable virulence in mice. Reverse genetics approaches demonstrated a critical role of the UT3006 PB2 segment in facilitating more efficient growth of UT3006-CkUT3091 reassortants in NHBE cells (Fig. 1A). Although the UT3006 and CkUT3091 PB2 proteins both encode the avian-type amino acids at positions 627 and 701, they differ by nine other amino acids (62-R/K, 117-T/I, 288-R/Q, 344-M/V, 524-T/I, 526-K/R, 591-Q/K, 676-K/T, 756-T/M; where the first amino acid indicates the residue found in CkUT3091 and the second indicates that found in UT3006). Five of these changes (at positions 62, 117, 524, 526, and 591) are not commonly found in avian influenza viruses, suggesting that they may play a role in mammalian adaptation. Introduction of the UT3006 sequence at these five sites appreciably increased the replicative ability of CkUT3091 in NHBE cells (CkUT3091-5aa, Fig. 1B), although virus titers did not reach the level of UT3006 virus. The respective changes were tested individually for their ability to facilitate avian influenza virus replication in human cells and introduction of the PB2-Q591K change appreciably enhanced the growth properties of CkUT3091 in NHBE cells (Fig. 1B). In contrast, the CkUT3091 variant possessing the PB2-T117I mutant was comparable in its growth to the parental CkUT3091 virus (Fig. 1B). The mutants containing PB2-R62K, PB2-T524I, or PB2-K526R changes were not viable, suggesting that these residues require compensatory changes at other sites. These findings suggest that, in the absence of PB2-627K or 701N, PB2-591K is a key amino acid for efficient influenza virus replication in human cells. Hence, replacement of the highly conserved glutamine at position 591 with lysine may facilitate mammalian adaptation in the context of an avian-type PB2 protein.
Figure 1. Amino acid PB2-591 is important for H5N1 virus replication in mammalian cells.
A. We inoculated the human UT3006 virus, the avian CkUT3091 virus, or CkUT3091-based reassortant viruses possessing single genes of UT3006 (CkUT3091-UT3006PB2, -UT3006PB1, -UT3006PA, -UT3006HA, -UT3006NP, -UT3006NA, -UT3006M, and -UT3006NS) into normal human bronchioepithelial (NHBE) cells at a multiplicity of infection of 0.0005 and cultured infected cells at 37°C. The culture supernatants were harvested at the indicated times and subjected to plaque assays in NHBE cells to determine virus titers. B. Growth curves in NHBE cells of UT3006, CkUT3091, and CkUT3091 variants possessing UT3006-derived PB2 substitutions at position 117 (PB2-T117I), 591 (PB2-Q591K), or at all five positions indicated in the text (PB2-5aa). The CkUT3091 virus possessing the UT3006-PB2 gene (CkUT3091-UT3006PB2, see Fig. 1A) is shown again for comparison. Experiments to determine viral growth kinetics were carried out twice, with three explicates each. Shown are data from one experiment.
Next, we tested CkUT3091 and its mutant possessing the PB2-Q591K substitution (CkUT3091-PB2-Q591K) for their virulence in mice. All mice infected intranasally with high doses of virus [106 or 105 PFU of virus] lost more than 25% of their pre-infection weight and had to be euthanized on day 4 or 5 post-infection (Fig. 2A and B). At lower infection doses (102–104 PFU), most animals infected with CkUT3091 virus survived (Figs. 2C–E), while all mice infected with CkUT3091-PB2-Q591K had to be euthanized. At the lowest doses tested (101 PFU), infected animals did not experience significant weight loss and survived (Fig. 2F). The MLD50 was 104.3 PFU for CkUT3091 and 50 PFU for the mutant possessing PB2-Q591K (CkUT3091-PB2-Q591K). These findings demonstrated that the glutamine-to-lysine mutation at position 591 of PB2 increased the virulence of an avian H5N1 influenza virus in mice.
Figure 2. Amino acid PB2-591K increases the virulence of an avian H5N1 virus in mice.
Mice (4 per group) were inoculated with the indicated amounts of CkUT3091 virus (blue squares) or CkUT3091 virus possessing the PB2-Q591K substitution (red circles) and observed daily for changes in body weight. Animals that lost more than 25% of their pre-infection weight were euthanized. The numbers in the graphs indicate the numbers of surviving mice per group. A.–F. Infection of mice with 106 pfu (A), 105 pfu (B), 104 pfu (C), 103 pfu (D), 102 pfu (E), or 101 pfu (F) of virus.
Lysine at position 591 of the PB2 protein is selected during H5N1 virus replication in ostrich cells
We previously reported that the human-adapting amino acids PB2-627K and PB2-701N are not only selected during replication of avian influenza viruses in mammalian cells, but also during replication of avian H5N1 viruses in cultured primary ostrich cells or ostriches [10]. Although not fully understood, the selection processes in ostriches thus appear to mimic those in mammalian cells. Since the significance of PB2-591 had not been recognized at the time, we focused our previous analysis on PB2-627 and PB2-701. Here, we therefore reexamined all amino acid changes acquired during replication of A/duck/Vietnam/5001/04 (H5N1) or A/duck/Vietnam/NCVD-18/03 (H5N1) in ostrich cells in vitro or in vivo. PB2-591K was not detected after replication of A/duck/Vietnam/NCVD-18/03 in an infected ostrich, but 12 of 73 PB2 molecular clones derived from viruses isolated from the trachea of an ostrich infected with A/duck/Vietnam/5001/04 (originally possessing PB2-591Q) had acquired the PB2-591K substitution (Table 1). Interestingly, five passages of A/duck/Vietnam/5001/04 in ostrich embryonic cells also yielded one clone (out of 16) with a PB2-Q591R mutation, as found in pandemic H1N1 viruses (see below). Comparable to mammalian cells, the replication of avian H5N1 influenza viruses in ostriches thus resulted in the selection of a basic amino acid at position 591; it is interesting to note that both PB2-591K (as found in some mammalian-adapted H5N1 viruses) and PB2-591R (as found in pandemic H1N1 viruses) were detected. The observed differences in adaptation to ostriches between the two duck viruses may result from differences in their genetic composition. The selection of a basic amino acid at PB2-591 in ostrich cells that seem to mimic the selective pressure in mammals provided further support for the role of PB2-591 in avian influenza virus adaptation to mammalian species.
Table 1. Mammalian-type amino acids in the PB2 protein of ostrich isolates.
All 12 ostrich PB2 clones possessing the PB2-591K mutation encoded PB2-627E (i.e., the wild-type, avian-type amino acid; Table 1). Conversely, the remaining 61 clones acquired the PB2-627K (mammalian-type) mutation but encoded the wild-type amino acid at position 591 (PB2-591Q) (Table 1). These findings from our in vivo selection study are consistent with three published ostrich sequences (Supplementary Figure S1) that show the PB2-591Q/627K or PB2-591K/627E combinations, but not mutations at both positions (i.e., PB2-591K/627K). Hence, PB2-591K appears to be selected in combination with PB2-627E, but not in combination with PB2-627K, suggesting that PB2-591K may functionally compensate for the lack of a positive charge at position 627.
Role of PB2 amino acids 591, 627, and 701 in pandemic H1N1 viruses
Our data indicated that a basic amino acid at PB2-591 can compensate for PB2-627K in mammalian adaptation; interestingly, these two residues are positioned near each other on the surface of PB2 [11], [12]. These findings offered an explanation for the efficient replication of the newly emerged pandemic H1N1 viruses in humans: although these viruses lack mammalian-type amino acids at position 627 and 701, they encode a basic amino acid (i.e., arginine) at position 591 (PB2-591R). In fact, a recent study demonstrated that PB2-590S and PB2-591R are important for efficient polymerase activity in an in vitro assay, and for efficient replication of a mixed human/avian influenza virus (with the polymerase and nucleoproteins of avian virus origin) in human cells [13]. Importantly, the additional introduction of PB2-627K did not increase the replicative ability of the mutant virus further [13], consistent with our finding that the combination of basic amino acids at positions 591 and 627 is not selected for in vivo. However, these studies were not carried out with authentic pandemic H1N1 virus and therefore leave in question the biological significance of PB2-591R for the efficient replication of pandemic H1N1 viruses in humans.
To test the significance of specific PB2 amino acid changes in the background of authentic pandemic H1N1 viruses in animal models, we first generated the following PB2 protein variants that were all based on A/California/04/09 (Cal04), an early pandemic H1N1 virus that we previously characterized in vitro and in several animal models [14]: (i) wild-type PB2, (ii) PB2-627K (mammalian-type) instead of PB2-627E (avian-type), (iii) PB2-701N (mammalian-type) instead of PB2-701D (avian-type), or (iv) PB2-591Q (consensus amino acid at this position) instead of PB2-591R (found in pandemic H1N1 viruses). These variants were assessed for their polymerase activity in minireplicon assays, essentially as described [15]. Replacement of PB2-591R with PB2-591Q reduced the polymerase activity (Supplementary Figure S2), further indicating a critical role of PB2-591R in efficient pandemic H1N1 virus replication. Replacement of PB2-627E with PB2-627K, or of PB2-701D with PB2-701N, had mild or no effects. Herfst et al. [16] recently reported increased polymerase activity in minireplicon assays for pandemic H1N1 variants possessing the PB2-627K or PB2-701N mutations, but used different pandemic H1N1 virus isolates.
Next, the PB2 mutations described above were introduced into the genetic background of Cal04 virus, using reverse genetics. Growth curves in Madin-Darby canine kidney (MDCK) cells revealed no significant differences (Supplementary Figure S3), as has been reported by others for the PB2-627K and PB2-701N variants [16], [17]. Intranasal inoculation of BALB/c mice with 104 or 105 PFU of virus resulted in increased weight loss for the PB2-627K and PB2-701N variants (Supplementary Figure S4); however, virus titers in the lungs and nasal turbinates on days 3 and 6 post-infection were comparable for all variants tested (Supplementary Table S1). Similar findings (i.e., weight loss in mice, but no increase in virus titers) were recently reported by others for pandemic H1N1 viruses possessing PB2-627K or PB2-701N [16], [17]. The effects of mutations in PB2 on pandemic H1N1 viruses were further tested in competitive transmission studies in ferrets, an established animal model in influenza virus research; in addition, pandemic H1N1 viruses are known to efficiently transmit in ferrets [14], [18], [19]. Wild-type Cal04 (PB2-591R/627E/701D) and its PB2 variants (PB2-591R/627K/701D, PB2-591R/627E/701N, or PB2-591Q/627E/701D) were mixed at a 1∶1 ratio based on plaque-forming units; however, deep sequencing revealed a slight excess of wild-type Cal04 PB2 vRNA over mutant PB2 vRNAs (Fig. 3B–D). Three ferrets (one per cage) were infected intranasally with virus mix. One day later, one ferret each was placed in a cage adjacent to an infected ferret ('contact' ferret) to monitor virus transmission by aerosols. At the indicated time points, nasal wash samples were assessed for the relative ratios of the two viruses (Figs. 3B–D; and Supplementary Table S2). In both infected and contact ferrets, mutations at position PB2-627 (PB2-591R/627K/701D) or PB2-701 (PB2-591R/627E/701N) did not provide a replicative advantage to the respective viruses when compared to wild-type Cal04 virus (PB2-591R/627E/701D). This is consistent with recent findings that E627K or D701N mutations did not affect the virulence or transmissibility of pandemic H1N1 viruses in ferrets [16]. However, in competition studies between wild-type Cal04 virus and a mutant virus possessing PB2-591Q (PB2-591Q/627E/701D), the relative amounts of Cal04 virus increased over time in infected animals (Fig. 3D), indicating that in the background of PB2-627E/701D, PB2-591R provides a replicative advantage over PB2-591Q (the amino acid commonly found at this position). Interestingly, two out of three contact animals showed enrichment of the PB2-591Q mutant virus on day 3 after co-housing; thus, although further studies with larger numbers of animals are needed, we speculate that PB2-591Q might enhance transmission, but not replication in the new host. Collectively, our findings supported the hypothesis that PB2 possessing 591R supports efficient viral replication in mammals, so that there is no strong selective pressure to acquire the mammalian-type amino acids at position 627 and 701.
Figure 3. Competitive transmission studies in ferrets for wild-type and mutant pandemic H1N1 viruses.
A. Overview of PB2 amino acids at positions 591, 627, and 701. Shown are A/California/04/09 (H1N1; Cal04) and its mutants tested in competitive transmission studies. B.–D. The indicated pairs of viruses were mixed at a 1∶1 ratio and three ferrets (one per cage) were infected intranasally with 106 plaque-forming units of virus mix. One day later, one ferret each was placed in a cage adjacent to an infected ferret ('contact' ferret); that is, for each set of viruses, three pairs of infected/contact ferrets were tested. Virus populations in the nasal washes of infected and contact ferrets were assessed at the indicated time points. Grey bars; percent wild-type PB2 molecules. Colored bars; percent mutant PB2 molecules. NA, not applicable; ND, not determined (nasal wash samples with virus titers of less than 102 plaque-forming units per ml were not processed for sequence analysis). Competition studies were carried out for Cal04 (PB2-591R/627E/701D) and Cal04PB2-627K (PB2-591R/627K/701D) (B), Cal04 (PB2-591R/627E/701D) and Cal04PB2-701N (PB2-591R/627E/701N) (C), and for Cal04 (PB2-591R/627E/701D) and Cal04-PB2-591Q (PB2-591Q/627E/701D) (D).
Based on our experimental data and the evaluation of published influenza virus sequences, we suggest the following scenario: efficient replication in mammalian species can be conferred by PB2-591Q/627K [found in some avian H5N1 viruses that acquired the ability to replicate in mammalian cells [7] (http://www.flu.lanl.gov), and found in human influenza viruses], PB2-591K/627E [found in some avian H5N1 viruses that acquired the ability to replicate in mammalian cells, (http://www.flu.lanl.gov), and identified in this study], or PB2-591R/627E (found in pandemic H1N1 viruses). However, the combination of PB2-591R/K and PB2-627K does not seem to further enhance replication in mammalian cells (Figs. 3B and C). Currently, it is not clear why PB2-591R is found in pandemic H1N1 viruses, while PB2-591K is selected in avian H5N1 viruses. In addition, PB2-701N also confers to H5N1 viruses the ability to replicate in mammalian cells in the absence of PB2-627E [5]. Moreover, other amino acids changes in PB2 may confer efficient replication in mammalian species as well (for example, [20], [21]).
Crystal structure of the C-terminal portion of PB2 from a 2009 H1N1 pandemic virus
Previous X-ray crystallographic studies have demonstrated the close proximity of residues 591 and 627 on the surface of PB2 [11], [12]. This has lead to the suggestion (based on modeling) that PB2-591R neutralizes the PB2-627E in pandemic H1N1 viruses, and thereby partially restores the positively charged surface of the 627 domain of PB2 [13]. To address this critical question, we present here the high-resolution crystal structure of the C-terminal domain of PB2 from a 2009 H1N1 pandemic virus (A/Mexico/InDRE4487/2009) (Fig. 4A); the PB2 proteins of A/Mexico/InDRE4487/2009 and Cal04 are identical in the region crystallized; i.e., amino acid residues 538–741. For comparison, we also present the crystal structure of the C-terminal portion of an H5N1 PB2 protein encoding PB2-627K (A/Vietnam/1203/04; Fig. 4B).
Figure 4. X-ray crystal structures of pandemic H1N1 and avian H5N1 PB2 proteins.
Shown are the X-ray crystal structures of a pandemic H1N1 (PB2-591R/627E) and an H5N1 PB2 protein encoding PB2-627K (PB2-591Q/627K). A. The surface of H1N1-PB2 in the region of PB2-591R and PB2-627E (PDBID:3KHW). Color of surface is mapped by qualitative vacuum electrostatics generated by PYMOL (27). Blue = relative positive charge, Red = relative negative charge. Residue S590, R591, and E627 are shown as stick representations underneath the surface contour and are identified with black arrows. The side chain of R591 intrudes into the cleft shown in Fig. 4B. B. The surface of H5N1-PB2 in the region of PB2-591Q and PB2-627K (PDBID:3KC6) shows a distinct cleft in comparison to the surface of H1N1 PB2-591R and PB2-627E (PDBID:3KHW). C. Close-up view of the interactions of H1N1 PB2-591R with neighbouring residues. H1N1-PB2 residues A587, S590, R591, and E627 are shown in sticks model, green = carbon, red = oxygen, blue = nitrogen. The conformation of H1N1 PB2-591R (PDBID:3KHW) is shown oriented away from PB2-627E making hydrogen bonding and water-mediated hydrogen bonding interactions to the carbonyl oxygen of residue PB2-587A. PB2-591R fills the cleft shown in Figure 4B. Residue S590 sits underneath E627 and blocks the conformational freedom of E627.
The PB2 protein of the pandemic H1N1 virus shows an altered polar surface when compared to the avian H5N1 PB2-627K protein. Interestingly, our structure of the pandemic H1N1 PB2 protein reveals that PB2-591R fills the cleft found in H5N1 PB2 proteins in this area (compare Fig. 4A and 4B). In particular, the side chains of R591 and S590 in the structure of H1N1 PB2 occupy what would otherwise be the positively charged cleft in the H5N1 PB2. PB2-591R is located 4 Ångstrom from PB2-627E, with the side chain of R591 rotated away from PB2-627E and participating in hydrogen-bonded and water-mediated hydrogen bond networks with the main chain carbonyl backbone between residues A587 and T588 (Fig. 4C), even though the two residues have space to assume rotomers that could form a neutralizing salt bridge (see Fig. 4C). The R591 residue in H1N1 PB2 thus affects both the shape and charges on the surface of the protein, which may affect its interaction with other viral and/or host factors.
A previous study [22] suggested that an inhibitory host factor may suppress the activity of avian-type polymerases (possessing PB2-627E), but not that of human-type polymerases (possessing PB2-627K). In this scenario, the positively charged residue (PB2-591R) that rotates away from PB2-627E may prevent the inhibitory host factor from interacting with PB2 by either charged repulsion or steric hindrance by filling the distinct cleft on the surface of the H5N1 PB2. Alternatively, the positive charge associated with PB2-627K or PB2-591R may facilitate interaction with a stimulatory host factor. Thus, efficient replication in mammalian cells may require a PB2 structure that prevents the action of the inhibitory host factor, or facilitates the action of the stimulatory host factor. Our finding that PB2-591Q/627K (found in human viruses) and PB2-591R/627E (found in pandemic H1N1 viruses) adopt different conformations (i.e. a positively charged, 'cleft' vs. a positively-charged residue that fills the cleft and is located next to a negatively-charged patch) probably favors the former possibility, since it is less likely that they would both enhance interaction with the same host protein, but the positive charge introduced by either change could easily disrupt interaction with an inhibitory host protein.
A serine at position 590 of PB2 is found in pandemic H1N1 viruses (but not consistently in older human H1N1 isolates), and this residue (PB2-590S) has been suggested to play a role in the enhanced replicative ability of pandemic H1N1 viruses in mammals [13]. Our results that a basic amino acid at PB2-591 provided a replicative advantage in mammals to avian H5N1 viruses (which possess a glycine at PB2-590), argue against a critical role of PB2-590S in the adaptation of H5N1 influenza viruses to mammals. Our H1N1 structure shows that while the side chain of PB2-590S is in close proximity (<4 Ångstroms) to 627E, the hydroxyl moiety points away from the glutamatic acid and does not participate in hydrogen bonds or polar interactions with the side chains of 591R or 627E. Furthermore, the side chain atoms of PB2-590S also protrude into the positively charged cleft and may provide a steric platform that reduces the conformational flexibility of neighboring residue 627E. These findings suggest that PB2-590S constrains any side chain at position 627. This constraint may be critical for PB2-627K which may need conformational freedom to reach its binding partner. The constraint by PB2-590S on PB2-627E may be less critical, and may in fact help to shield the negative charge.
Our study identifies a new marker (PB2-591R or K) for influenza virus adaptation in mammals that compensates for the lack of PB2-627K. This finding provides an explanation for the efficient replication of pandemic H1N1 viruses (which possess PB2-591R) in mammals. Our X-ray crystal structure of the C-terminal portion of a pandemic H1N1 PB2 protein reveals changes in surface shape and charge created by PB2-591R which may prevent an inhibitory host factor as suggested by Mehle & Doudna [22] from binding to PB2, hence allowing efficient influenza virus replication. Although a few human H1N1 isolates have now been found to possess PB2-627K (http://www.promedmail.org/pls/otn/f?p=2400:1001:19224::NO::F2400_P1001_BACK_PAGE,F2400_P1001_PUB_MAIL_ID:1010,79432), this mutation did not appear to increase the severity of disease, and does not seem to spread in human populations, consistent with our conclusion that the PB2-627K mutation does not provide a significant replicative advantage to pandemic H1N1 viruses. Based on findings with H5N1 influenza viruses, it was feared that the introduction of PB2-627K into pandemic H1N1 viruses could increase the pathogenicity of the pandemic viruses. However, our data and recent findings by others [13] indicate that PB2-627K does not provide a replicative advantage in the background of a PB2 protein possessing a basic amino acid at PB2-591. From a public health perspective, the notion that the introduction of PB2-627K into pandemic H1N1 viruses is rare and unlikely to create a more pathogenic variant is thus reassuring.
Viruses and cells
Human embryonic kidney (293 or 293T) cells were maintained in Dulbecco's modified essential medium (DMEM) containing 10% fetal calf serum and antibiotics. Madin-Darby canine kidney (MDCK) cells were maintained in Eagle's minimal essential medium (MEM) containing 5% newborn calf serum. Normal human bronchioepithelial cells (NHBE) were obtained from Lonza (Walkersville, MD) and maintained in serum-free and hormone-supplemented growth medium according to the manufacturer's instructions. All cells were incubated at 37°C with 5% CO2.
All influenza viruses used in this study were amplified in MDCK cells. All viruses were stored at −80°C until their use in experiments. The titers of stock viruses were determined by plaque assays in MDCK cells (for H1N1 viruses) or NHBE cells (for H5N1 viruses). All experiments with H5N1 viruses were performed in enhanced biosafety level 3 (BSL3) containment laboratories at the University of Wisconsin-Madison, which are USDA approved for such use by the CDC and the U.S. Department of Agriculture, or in BSL3 containment laboratories at the University of Tokyo (Tokyo, Japan), which are approved for such use by the Ministry of Agriculture, Forestry and Fisheries, Japan.
Construction of plasmids and generation of wild-type and mutant influenza viruses
The cDNAs of the A/chicken/Indonesia/UT3091/05 (H5N1; CkUT3091), A/Indonesia/UT3006/05 (H5N1; UT3006), and A/California/04/09 (H1N1; Cal04) viruses were synthesized by reverse transcription as previously described [23], [24]. Viruses were amplified and titrated as described above. All viruses were sequenced to ensure the absence of unwanted mutations.
Mutations in the PB2 genes of CkUT3091 or Cal04 viruses were generated by PCR amplification of the respective RNA polymerase I PB2 construct with primers possessing the desired mutations (primer sequences will be provided upon request). All constructs were sequenced to ensure the absence of unwanted mutations.
Minireplicon assays
To assess the viral polymerase activity, we performed luciferase activity-based minireplicon assay as described previously [15] using the following plasmids; expression plasmids for Cal04-PB2, -PB1, -PA, and -NP (0.2 µg each), pPolI-WNA-Fluc (0.02 µg), which express NA gene encoding firefly luciferase gene, and pGL4.74[hRluc/TK] (Promega, 0.02 µg). The luciferase activity in the plasmid-transfected 293 cells was measured by Dual-Luciferase Reporter Assay System (Promega) at 24 h post-transfection.
Growth curves in NHBE or MDCK cells
NHBE cells (Lonza) were infected with the indicated viruses at a multiplicity of infection (m.o.i) of 0.0005. One hour later, the viral inoculum was removed and replaced with bronchial epithelial growth medium (SAGM; Cambrex) containing bovine pituitary extract (BPE; 30 µg/ml), hydrocortisone (0.5 µg/ml), human epidermal growth factor (hEGF; 0.5 ng/ml), epinephrine (0.5 µg/ml), transferrin (10 µg/ml), insulin (5 µg/ml), triiodothyronine (6.5 ng/ml), bovine serum albumin – fatty-acid free (BSA-FAF; 50 µg/ml), retinoic acid (RA; 0.1 ng/ml), gentamycin (30 µg/ml) and amphotericin B (15 ng/ml). Infected cells were incubated at 37°C and 5% CO2. At the indicated times post infection, the virus titers in the cell culture supernatant were determined by plaque assays in NHBE cells. For the determination of viral growth kinetics in MDCK cells, cells were infected at an m.o.i. of 0.001; at the indicated time points, virus titers were determined by plaque assays.
Virulence in mice
Six-week-old female BALB/c mice (Japan SLC Inc., Shizuoka, Japan; The Jackson Laboratory, Bar Harbor, Maine, USA) were used in this study. Baseline body weights were measured prior to infection. Four mice per group were anesthetized with isoflurane and intranasally inoculated with 101–106 PFU (50 µl) of CkUT3001 or CkUT3091-PB2-Q591K, or with 103 or 106 PFU of Cal04 possessing wild-type or mutant PB2 protein. Body weight and survival were monitored daily for 14 days; mice with body weight loss of more than 25% of pre-infection values were euthanized. For virus titration in organs, mice were infected intranasally with 105 PFU of virus and euthanized on days 3 and 6 postinfection.
Ferret competition-transmission study
We used six-to-seven-month-old female ferrets (Triple F Farms, Sayre, PA), which were serologically negative by hemagglutination inhibition (HI) assay for currently circulating human influenza viruses (including pandemic H1N1 viruses). The animals were housed in adjacent transmission cages that prevent direct and indirect contact between animals but allow spread of influenza virus through the air. Three ferrets were intranasally inoculated with 106 plaque-forming units (pfu; in 500 µl) of the mixture of Cal04 and mutant virus at a 1∶1 ratio (0.5×106 pfu of Cal04 plus 0.5×106 pfu of mutant virus). One day after infection, three naive ferrets were each placed in a cage adjacent to an inoculated ferret (contact ferrets). Hence, we tested three pairs of infected/contact ferrets for each virus. Nasal washes from ferrets were collected on days 1, 3, 5, and 7 post-infection or co-housing and titrated for virus titers in MDCK cells. Samples with greater than 1×102 pfu/ml of virus were subjected to sequence analysis as described below. All animal experiments were carried out in accordance with institutional guidelines.
Sequence analysis of viruses obtained from infected or contact ferrets
Viral RNA was extracted from nasal wash samples and twice from the inoculum (to verify the 1∶1 ratio of the two test viruses). The cDNAs were synthesized as previously described [23]. A panel of PCR primer pairs was used to generate 410 base pair amplicons of the PB2 region covering 591Q, 627K, and 701N of A/California/04/2009. All primers contained Roche/454 Titanium Amplicon adapter sequences and distinctive multiplier identifier (MID) tags engineered at their 5′ termini. After gel purification and normalization to 108 molecules/µl, emulsion PCR was performed on four pools containing 14 MID-tagged amplicons each.
The resulting pools were pyrosequenced in individual regions of a 70×75 PicoTiterPlate with a 16-well gasket on a Roche/454 GS FLX instrument following protocols provided by the manufacturer (www.454.com). An average of 2221 sequence reads was obtained for each sample (range: 705–5102). All sequences from a single time point were aligned to the PB2 sequence of A/California/04/09 (Roche/454 AVA software). The frequencies of wild-type and mutant amino acids at residues 591, 627, and 701 were tabulated from the aligned sequences. To confirm the pyrosequencing results, independent PB2 sequences from 37 of the 42 samples were generated using alternative sets of PCR primers and pyrosequenced using the Roche/454 FLX chemistry.
X-ray crystal structure determination of PB2 C-terminal domains
Codon engineered synthetic genes for the PB2 gene of influenza A virus strains A/Vietnam/1203/04 (H5N1) (AAT73550) and A/Mexico/InDRE4487/09 (H1N1) (ACQ73384) were designed in Gene Composer (Emerald BioSystems, Bainbridge Island, WA) using a combined E. coli and baculovirus codon usage table with a threshold setting of 0.2% and synthesized (DNA 2.0, Menlo Park, CA) [25], [26]. The C-terminal amino acid residues 538–741 from both strains were cloned into an engineered pET vector using the Polymerase Incomplete Primer Extension (PIPE) cloning technology [27]. Cloning, expression, and purification followed reported protocols from the Seattle Structural Genomics Center for Infectious Disease [28]. Constructs were expressed in vectors containing an N-terminal 6×His Smt fusion protein [29]. Site specific removal of the 11kDA 6×His Smt fusion partner was accomplished with 0.2 µg Ulp1 protease/mg of recombinant target [29], [30]. Final protein buffer composition following size exclusion chromatography (Superdex 75, GE Healthcare, Piscataway, NJ) was 25 mM Hepes/NaOH pH 7.2, 500 mM NaCl, 2 mM DTT, 5% glycerol. Protein crystallization trials were conducted by sitting drop vapor diffusion at 16°C in 96-well crystallization plates. Crystallization conditions for H5N1-PB2(538–741) were 5% octyl β-D-1-thioglucopyranoside (SBOG), 100 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) pH 10.5, 1.2 M sodium phosphate, 0.2 M lithium phosphate, 0.8 M potassium phosphate at protein concentration of 8 mg/ml. Crystallization conditions for H1N1-PB2(538–741) were 20% w/v PEG 3350, 200 mM ammonium citrate at protein concentration of 24 mg/ml. All crystal drop sizes used were 0.4 µl crystallant plus 0.4 µl protein. Crystals were harvested by transfer into drops containing crystallant plus either 25% glycerol or ethylene diol as cryoprotectant and flash frozen in liquid nitrogen. X-ray data were collected on a Rigaku (Rigaku Americas, The Woodlands, Texas) FR-E+ X-ray generator equipped with a Saturn-944 CCD detector. One degree images were collected for 30 seconds and scaled with HKL2000 [31]. Structures were determined by molecular replacement with the coordinates from PDBID 3CW4 using CCP4/PHASER [32]. Iterative refinement and modeling of coordinates was conducted using REFMAC [33] and COOT [34]. Final crystallographic refinement statistics are presented in Table 2. Structure figures were generated using PYMOL [35].
Table 2. X-ray crystallographic data collection and refinement statistics.
Our experiments in mice, ostriches, and ferrets followed the University of Tokyo's Regulations for Animal Care and Use and the University of Wisconsin-Madison's Animal Care and Use Protocol. They were approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo (approval number: 19–29) and the Animal Care and Use Committee of the University of Wisconsin-Madison (protocol number V00806), which acknowledged and accepted both the legal and ethical responsibility for the animals, as specified in the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, 2006 (Japan) and in the Animal Welfare Act and associated Animal Welfare Regulations and Public Health Service Policy (USA).
Alignment of PB2 protein sequences of influenza A viruses isolated from ostriches, emus, and rheas. Shown is the region covering amino acid positions 591, 627, and 701.
Polymerase activity of Cal04 PB2 variants in vitro. 293 cells were transfected with plasmids for the expression of a virus-like RNA encoding luciferase, and with plasmids encoding the Cal04 PB1, PA, NP, and wild-type or mutant PB2 proteins. Forty-eight hours later, luciferase activity was assessed.
Growth curves of wild-type and mutant Cal04 viruses in MDCK cells. Cells were infected with wild-type or mutant Cal04 viruses at an m.o.i. of 0.001. At the indicated times post-infection, the virus titers in the cell culture supernatant were assessed by plaque assays. Experiments were carried out in triplicate.
Virulence of wild-type and mutant Cal04 viruses in mice. BALB/c mice were infected with 105 PFU (A) or 104 PFU (B) of the indicated viruses and assessed daily for weight losses. For mice infected with the PB2-627K or PB2-701N variants, one mouse each had to be euthanized on day 5 or 6, respectively, due to body weight loss of more than 25% of the respective pre-infection body weight.
Biological features of PB2 variants in mice
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Competitive transmission studies in ferrets for wild-type and mutant pandemic H1N1 viruses
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We thank M. McGregor, R. Moritz, A. Hanson, S. Basnet, C. Doyle, L. Burley, and R. Nelson for technical assistance. We also wish to thank Chris Wright and Evette Vlach at the University of Illinois at Urbana-Champaign High-Throughput Sequencing Center and Steve Wolinsky, Kevin Kuntsman, Kristin Mars and Jennifer Kuntsman at Northwestern University for outstanding support with the Roche/454 pyrosequencing studies. Special thanks to Don Lorimer, Taryn Haffner, Michelle Dietrich, and Becky Poplawski at Emerald BioStructures for their support in molecular biology, protein crystallization and X-ray data collection.
Conceived and designed the experiments: M. Hatta, B. Staker, S. Watanabe, T. Watanabe, P. Myler, I. Phan, A. Raymond, E. Smith, R. Stacy, S. Lank, R. Wiseman, B. Bimber, D. O'Connor, L. Stewart, Y. Kawaoka. Performed the experiments: S. Yamada, M. Hatta, B. Staker, M. Imai, K. Shinya, Y. Sakai-Tagawa, M. Ito, M. Ozawa, T. Watanabe, S. Sakabe, C. Li, J. Kim, P. Myler, I. Phan, A. Raymond, E. Smith, R. Stacy, S. Lank, R. Wiseman, B. Bimber, D. O'Connor, L. Stewart. Analyzed the data: B. Staker, P. Myler, I. Phan, A. Raymond, E. Smith, R. Stacy, S. Lank, R. Wiseman, B. Bimber, D. O'Connor, L. Stewart, Y. Kawaoka. Contributed reagents/materials/analysis tools: C. Nidom. Wrote the paper: G. Neumann, Y. Kawaoka.
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Is the Subject Area "H1N1" applicable to this article?
Influenza viruses
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One of every three bites of our food originates from bees pollinating the flowers that produce our fruits, nuts, vegetables and even coffee–coffee production has been found to double when purposely pollinated by honey bees. Each one of us benefits from a healthy honey bee population.
Honeybee populations have been declining for decades, (due to colony collapse disorder and other stressors) and they continue to struggle for survival. Honey bees are biological indicators, meaning that honey bee health reflects the general health of the environment. Bee losses are possibly a symptom of a much greater environmental problem.
Dandelions and clover could not be any easier to grow–all you have to do is nothing.
Let dandelions, clover, and other bee-friendly plants grow in your garden and yard.
The benefits of a community garden go way beyond helping to Save the Bee. They help improve air and soil quality, increase plant and animal biodiversity, and when you plant fruit, vegetable and grain crops, you will have a source of fresh food that does not need to be "trucked in" to your community.
Bees do not stand a chance against toxic chemicals designed to kill weeds and pests.
You can actually see the effects of spraying toxic chemicals on a bee or a beehive–the bees die.
What you don't see right away are the effects on you, your family and your pets. Weed killers and pesticides are marketed as being safe as long as you follow the instructions and allow the chemicals to dry before permitting children or pets to enter the area where the chemical has been applied. But, there is plenty of evidence that these chemicals are not safe for any living things, and some have been linked to certain cancers. Organic weed and pest control is a safer and healthier choice for you, your family and the bees.
When bees nest inside your home you might be inclined to grab a can of pesticide and get them out. Or, you might call an exterminator who will use toxic chemicals and wipe out the entire colony. Consider instead a bee-friendly option that will save the bee–humane bee removal. Many pest removal services are educated about the plight of the honey bee and now offer humane bee removal. They'll save the bee without using toxic chemicals and they'll remove the honeycomb, which if left intact, will create a mess and attract other animals and insects. Contact your local beekeeping association or search online for "humane bee removal" or "live bee removal" to find a local service.
CCorn-basedsweeteners are spectacularly energy intensive in growth and production, and cause a plethora of ethical problems.
Honey is a great alternative but cheap, highly processed honey is very likely to be manufactured by companies that are not concerned about the treatment of their bees and the quality of the honey. The honey you can get from small local beekeepers is typically raw honey in its sweet, pure, most natural state. It's unfiltered, minimally processed and contains protein-packed bee pollen and enzymes.


Are you looking for aviation part 251A243026? ILSmart can help.
ILSmart is the world's most active aviation parts marketplace. You can find aviation spare parts such as 251A243026 and many others through a simple search. Go to www.ILSmart.com to find out more or Contact your local ILS representative.


text:
E.Palau killed in car wreck
Two killed in tragic accident
originally published 12/22/06 on KOFantasyBoxing.com
The boxer-trainer relationship is most similar to that of father and son; both work hard to bring the best out of each other at all times and when one member of the partnership hurts both men feel it. When tragedy strikes it's like losing a part of one's self. Such is the case for former fighter and current trainer Sean "Fitzy" Fitzgerald whose junior middleweight charge, Enrique Palau lost his life earlier this week in a fatal car accident just a few days after notching his seventh straight win as a professional. Fitzgerald spoke with KOFantasyBoxing about the senseless and tragic death.
"It's terrible," said Fitzgerald. "His mother is devastated."
Palau, 27, and Amanda Snaras , 25, a "childhood friend" of Enrique's, were both killed early Wednesday morning on their way home from a Christmas Party when the car Palau was driving struck a rock wall at the end of Grafton Street in Worcester, MA. Palau leaves behind two children, ages four and one.
"The woman he was with she had two children too so it's a double tragedy." Fitzgerald, still very distraught over the accident, described what a blow this has been to so many people close to both victims. "I was his trainer but I've also been a father figure to him."
Palau, who had spent five years in prison for armed robbery, turned his life around once paired with Fitzgerald and exposed to the disciplined life of a prize-fighter. Palau had won amateur titles and was on his way to becoming a regional star after seven straight victories his first year as a professional. On December fifteenth he fought at The Castle in Boston, a fight to be aired on January second on NESN.
"Anytime you go from getting in trouble like that and then having the gang unit of the police department behind you and pulling for you, being involved actively in your life and helping you out, it always takes you higherナThis kid made a hundred percent turn around. And now he's snuffed out and I don't understand it. If he died when he was not boxing and being a punk it's different. I'm just happy that he turned his life around and now he'll go out like a hero."
"I've got kids in my gym, high school kids, sophomores and juniors that are really upset. They looked up to Enrique," Fitzgerald explained.
"He had a big heart. He believed in his ability. He had really big balls and good power. But unfortunately things happen for a reason. I don't know why God did it though."
NESN will have details about a trust fund for Palau's family on the January second broadcast of their Battle-Zone Boxing Show which will feature Palau's final bout with Hollister Elliott. Once available KOFantasyBoxing will post details about where donations or well-wishes can be sent.
This writer spoke to Enrique Palau just a few days before his most recent bout and after the interview I was left with such a positive vibe from the young man. His motivation and enthusiasm were infectious and it was a shock to hear of his untimely death.
KOFantasyBoxing extends their condolences to the Palau and Snaras families, Sean Fitzgerald and all those affected by this tragic accident.
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I had a thought while I was at Starbucks today. I was reading a book for Doctrine of Holiness, and this thought that has nothing at all to do with the book came into my head. Do we still need evangelism?
So it seems like the original idea of evangelism worked. I don't know to much about Christianity oversees, but it seems like everyone in America knows about Christianity, about Jesus and the Church. I would bet that a high percentage of everyday Americans could give a pretty basic theology of Christianity and be fairly accurate. So if the definition of evangelism is "telling the world about Jesus", then why do we still need evangelism? It sure seems like people who stand on street corners with a bullhorn or handing out tracts or going door to door aren't telling anybody anything new.
I believe we either have to redefine evangelism or do away with it all together. Because the problem is, the people that are still evangelizing are really annoying and not doing so great for our image. Evangelism as we know it must fundamentally change, because everybody already knows who Jesus is, or at least they know about the Jesus that has been presented to them. Whether or not this portrayal is correct is incredibly important, but I don't want to address it at length. here…another time, perhaps.
But what I will say is this: as cliche as it sounds, the Church in America no longer needs to tell people about Jesus, the Church needs to show people Jesus. And I'm not talking about just the little things like helping people move or sitting by the awkward guy at lunch. I mean corporally, the Church needs to show the world who Jesus is by feeding the hungry, eradicating poverty, clothing the naked, caring for creation, etc. You know, the kind of world change things we could accomplish if we cared enough to try. The way I see it, the Church is an enormous body of people that are supposed to be united around one Person, who taught compassion, love, and peace. If any group in the world has the potential to fix the world's problems, it's the Church.
To quote a favorite author of mine, or rather to paraphrase him, people who "evangelize" typically ask two questions: "If you died tonight, do you know where you would go?" and "If the world ended today, what would happen to you?" When really, the better questions are, "If you lived for 50 more years, what kind of person would you want to become?" and "If the earth was around for another 500 years, what kind of world do we want to create?" That's the kind of evangelism that the world needs, if you can call it that.
Good points, Kevin! For me, it is helpful to look at where we got our word for "evangelism". This word comes from the Greek word euangelion, which means "gospel". By adding the '-ism' we've given action to this word and turned it into a verb. So, how we practice and live is essential to evangelism. I would say that in evangelism we are attempting to participate in a culture (a very specific, holy, and Christ-oriented culture), and invite others to join us.
I enjoy your thoughts and look forward to future posts!
I agree that in America there is really no need for evangelism. What about in the countries in the world that don't know about Christ or Christianity. I may not be very far in my studies but I have learned that there are some unreached people groups in the world. So towards that effort I would say that we should continue evangelical training. It is important that the whole world knows of Christ. At least that's what I think the Great Commission meant.
So then, what is your new system?
i completely agree, but i also think we make a mistake before that point. i think we're in way too big of a rush to get people to the altar to have a crisis experience that we don't let people "count the cost" like Jesus talked about… we tell them one aspect of Christianity that sounds good and then emotionally deceive people into a decision without telling them the rest of it. Not to day that we do this intentionally or with evil intentions, but this it seems to pan out. and then we don't disciple them. seems like a flawed system to me.


text:
A Mixed-Method Study of Same-Sex Kissing Among College-Attending Heterosexual Men in the U.S.
A Mixed-Method Study of Same-Sex Kissing Among College-Attending Heterosexual Men in the U.S. Eric Anderson, Matthew Ripley, Mark McCormack. Sexuality & Culture, https://link.springer.com/article/10.1007/s12119-018-9560-0
Abstract: This is the first research to assess the prevalence of same-sex kissing among college-attending, heterosexual men in the United States. We utilized a mixed-method study of 442 quantitative surveys and 75 in-depth interviews with participants from 11 universities in order to understand the frequency, context and meanings of same-sex kissing. We found that the prevalence of kissing on the cheek among these participants was 40%, and kissing on the lips 10%. Both types of kisses were predicted by positive attitudes toward gay men and both types of kissing were generally described as non-sexual expressions of affection. We situate these empirical results within contemporary theoretical debates about masculinities and contend that the meanings associated with heterosexual masculinity are undergoing a profound shift in U.S. culture. This trend of same-sex kissing needs further attention to fully understand these shifts and the emerging homosocial and tactile experiences of young American men.
The more incentives people have to behave self-servingly, the more they perceive the victims of that behavior as dissimilar, i.e., the more they purposely & proactively distance themselves psychologically
Motivated dissimilarity construal and self-serving behavior: How we distance ourselves from those we harm. Laura J. Noval, Andrew Molinsky, Günter K. Stahl. Organizational Behavior and Human Decision Processes, Volume 148, September 2018, Pages 145-158. https://doi.org/10.1016/j.obhdp.2018.08.003
• We introduce an incentivized form of psychological distance called "Motivated Dissimilarity Construal" (MDC).
• The more incentives people have to behave self-servingly, the more they perceive the victims of that behavior as dissimilar.
• Anticipated discomfort about self-serving behavior increases MDC.
• MDC is possible when people consider an acquaintance but less likely when they consider a friend.
• MDC increases self-serving behavior by reducing discomfort about that behavior.
Abstract: It is well established that people are more likely to act in a self-serving manner towards those dissimilar to themselves. Less well understood is how people actively shape perceptions of dissimilarity towards victims in order to minimize their own discomfort. In this paper, we introduce the concept of Motivated Dissimilarity Construal (MDC) – the act of purposely and proactively distancing oneself psychologically from the victim of one's own self-serving behavior. In doing so, we challenge the notion that potential victims of self-serving acts are perceived objectively and independently of a decision maker's motivation, as traditional rationalist models of decision making might suggest. Across three experiments, we demonstrate how, why and when MDC is likely to occur, and discuss implications of these findings for theory and research on behavioral ethics and interpersonal similarity.
In addition to previously reported risk factors, patient's subjective impressions of penile size negatively impacts sexual life in about 10% of men considered healthy, while objective penile length does not play significant role in erectile function
Does underestimated penile size impact erectile function in healthy men? Brunno CF Sanches et al. International Journal of Impotence Research, volume 30, pages158–162 (2018). https://www.nature.com/articles/s41443-018-0039-1
Abstract: The aim of this study is to assess the impact of objective (stretched) and subjective penile size in the erectile function in a urological check-up program on a cross-sectional study including 689 men aged 35–70 years. IIEF-5 questionnaire, physical examination (penile length, prostate volume, blood pressure, body mass index-BMI), metabolic syndrome (MS), comorbidities, habits (sexual intercourse frequency, physical activity, alcohol, and tobacco use), level of education, serum glucose, total testosterone, estradiol, PSA, lipid profile, and self-perceptions (ejaculation time and subjective penile size) were examined in multivariate models using logistic and linear regressions. Penile objective mean length was 13.08 cm ± 2.32 and 67 (9.72%) patients referred small penis self-perception. Seventy-six (11.03%) participants had severe erectile dysfunction (ED), 75 (10.88%) had mild to moderate and moderate ED, 112 (16.25%) had mild ED and 426 (61.83%) had no ED. Risk factors for ED that held statistical significance were self-perceived small penis (OR = 2.23, 95% CI 1.35–3.69, p = .0017), sexual intercourse frequency (per week) (OR = 0.45, 95% CI 0.38–0.52, p < .0001), satisfactory ejaculation time (no vs. yes, OR = 2.06, 95% CI 1.46–2.92, p < .0001), comorbidity (yes vs. no, OR = 2.01, 95% CI 1.46–2.76, p < .0001), age >65 years (OR = 2.93, 95% CI 1.53–5.61, p < .0001), tobacco use (yes vs. no, OR = 1.41, 95% CI 1.02–1.96, p < .0375), regular physical activity (no vs. yes, OR = 1.59, 95% CI 1.13–2.23, p < .0083), serum total testosterone < 200 ng/dl (OR = 3.48, 95% CI 1.69–7.16, p = 0.0009), serum glucose > 100 mg/dl (OR = 1.69, 95% CI 1.18–2.43, p = 0.0044) and systolic blood pressure > 130 mmHg (OR = 1.60, 95% CI 1.16–2.19, p = 0.0037). Results suggest that in addition to previously reported risk factors, patient's subjective impressions of penile size negatively impacts sexual life in about 10% of men considered healthy, while objective penile length does not play significant role in erectile function.
A Mixed-Method Study of Same-Sex Kissing Among Col...
The more incentives people have to behave self-ser...
In addition to previously reported risk factors, p...


text:
La Familia: Una Proclamación para el Mundo es una declaración emitida por La Iglesia de Jesucristo de los Santos de los Últimos Días en 1995, que define la posición oficial de la Iglesia respecto a la sexualidad humana y la familia. Fue primeramente anunciada por el Presidente de la Iglesia Gordon B. Hinckley durante la Reunión Mundial de la Sociedad de Socorro el 23 de septiembre de 1995 y lleva el sello oficial de la Primera Presidencia y el Quórum de los Doce Apóstoles de la iglesia.

Historia
La Proclamación fue emitida poco después de que la iglesia se viera involucrada en medio de importantes debates legales y acciones legislativas en relación con el matrimonio entre personas del mismo sexo en Hawái y otros estados de los Estados Unidos. En marzo de 1995, el estado de Utah aprobó una ley estatal haciendo ilegal el matrimonio entre homosexuales y luego en abril de ese mismo año, Hawái revaluó sus estatutos en relación con prohibiciones sobre el matrimonio entre personas del mismo género resolviendo el asunto en sus cortes.

El documento se leyó en público por primera vez durante una conferencia de mujeres de la iglesia, el 23 de septiembre de 1995, después de lo cual, en octubre de ese año se discutió extensamente en la conferencia general de la igleisia. Numerosas copias han sido impresas y enmarcadas para ser mostradas en las casas de los hogares de los miembros de la iglesia, una tradición que continúa, más de dos décadas después.

Contenido
La Proclamación no contiene nuevas declaraciones sobre doctrina o política de la Iglesia de Jesucristo de los Santos de los Últimos Días. Es, sin duda, la declaración oficial más completa emitida por la Iglesia de Jesucristo de los Santos de los Último Días sobre la posición de la iglesia en relación con los géneros y el sexo. La proclamación puede ser dividida en tres secciones, cada una de ellas con una lista de puntos claves: afirmaciones, directivas y admoniciones.

Afirmaciones
 Todos los seres humanos, hombres y mujeres, son creados a la imagen de Dios.
 El ser hombre o mujer es una característica esencial de la identidad y el propósito eternos de los seres humanos en la vida premortal, mortal, y eterna.
 En la vida premortal, los hijos y las hijas espirituales de Dios lo conocieron y lo adoraron como su Padre Eterno, y aceptaron Su plan.
 Las ordenanzas y los convenios sagrados disponibles en los templos de la iglesia permiten que las personas regresen a la presencia de Dios y que las familias sean unidas eternamente.

Directivas
 Los sagrados poderes de la procreación se deben utilizar sólo entre un hombre y una mujer legítimamente casados, como esposo y esposa.
 Los padres tienen la responsabilidad sagrada de educar a sus hijos dentro del amor y la rectitud, de proveer para sus necesidades físicas y espirituales, de enseñarles a amar y a servirse el uno al otro.
 Hay más posibilidades de lograr la felicidad en la vida familiar cuando se basa en las enseñanzas de Jesucristo y los principios de la fe, la oración, el arrepentimiento, el perdón, el respeto, el amor, la compasión, el trabajo y las actividades recreativas edificantes.
 El padre debe presidir sobre la familia con amor y rectitud y tiene la responsabilidad de protegerla y de proveerle las cosas necesarias de la vida.
 La responsabilidad primordial de la madre es criar a los hijos. En estas responsabilidades sagradas, el padre y la madre, como iguales, están obligados a ayudarse mutuamente.

Advertencias

 Las personas que violan los convenios de castidad, que abusan de su cónyuge o de sus hijos, o que no cumplen con sus responsabilidades familiares, un día deberán responder ante Dios.
 La desintegración de la familia traerá sobre el individuo, las comunidades y las naciones las calamidades predichas por los profetas antiguos y modernos.

Estado

El presidente Gordon B. Hinckley leyó esta proclamación como parte de su mensaje en
la Reunión General de la Sociedad de Socorro, el 23 de septiembre de 1995, en Salt Lake City, Utah, E.U.A.

La proclamación fue firmada por los tres miembros de la Primera Presidencia y los doce miembros del Consejo de los Doce Apóstoles, y las copias producidas por la Iglesia incluyen esas quince firmas en la parte inferior, enfatizando su naturaleza autorizada. La Iglesia enseña que cada uno de los quince firmantes es un apóstol, como así también un profeta, un vidente y un revelador; que la Primera Presidencia y el Quórum de los Doce actuando en conjunto con la Primera presidencia cuando está activa, están capacitados para recibir revelación de Dios para guiar a la Iglesia y enseñar al mundo entero; y que sus pronunciamientos, cuando son impulsados por el Espíritu Santo, poseen la autoridad de una escritura.

Enlaces externos
 Texto de la proclamación

Referencias

Familia
Doctrinas, creencias y prácticas mormonas
Textos de La Iglesia de Jesucristo de los Santos de los Últimos Días

redpajama_set_name: RedPajamaWikipedia


text:
Beer-lovin' softball team raises spirits and funds for Kansas community
by: Emily Younger
Posted: Nov 9, 2021 / 07:41 PM CST / Updated: Nov 10, 2021 / 06:45 AM CST
GARDEN PLAIN, Kan. (KSNW) – What started as a beer-drinking softball league more than a decade ago has turned into a fundraising machine.
"They go hand in hand. There is nothing wrong with drinking beer and raising money at the same time," said Michael Scheer.
"We decided, hey, why don't we hang out more, and since we are hanging out, having fun, why not raise money?" said Brent Randolph.
That 'why not' attitude turned into an annual softball tournament known as the One Pitch Classic. Since 2009, the group behind the tourney, now called the Pearlsnaps, has raised thousands of dollars and put on numerous other events for people and places in their hometown.
"The mission now really is to hang out and have fun, but at the same time, promote the town of Garden Plain, and you know if somebody in the community needs something, we can raise money for it, why not do it while we are having fun," explained Randolph.
Wichita's Miracle League knocks inclusion out of the park
Latest fundraiser helps girl battling cancer
All of the proceeds from the most recent One Pitch Classic, held in October, benefited Madison Francis. Madison, 5, was diagnosed with a form of spindle cell sarcoma in August.
"On August 1, my husband and I were getting ready for bed, and we noticed a really large lump in her stomach. It was not there the night before, so we were a bit concerned, so we took her to a local hospital. They did some scans. They told us it was a mass. They didn't really know what it was, so they sent us to the oncologist in Kansas City, and on August 2, she got a biopsy, and a week later, we were told it is stage four," said Jerika Francis, Madison's mother.
Jerika is a Garden Plain native. She went to school with several of the guys on the Pearlsnaps.
"We have that tournament every year anyways. We obviously have fees to play in it. Why not give those fees to her. We don't need the money, and it's just great to be able to help people," said Randolph.
Randolph said the tournament raised nearly $8,000 for the Francis family. It is a big relief for Jerika as she recently had to quit her job to care for Madison as they travel back and forth from Kansas City and their home in Norwich.
"I can't even express how grateful my family is. Just from the bottom of our hearts, like, it's amazing, and I'm very proud to call Garden Plain my hometown," Jerika sobbed as she explained her deep appreciation for the Pearlsnaps to KSN.
Walking Patriot: Kansas soldier uses TikTok fame to inspire kindness
Other Pearlsnaps' 'fun'draisers
From softball tournaments to Christmas caroling, the Pearlsnaps are not short on creative ways to raise money.
"Most of us have kids that have to do some type of fundraising. No one likes going door to door and selling a candy bar or popcorn," Randolph said. "His town likes to socialize, so we do social events that people like to attend.
The group has even tried its hand at the annual Wagonmasters Chili Cookoff in Wichita. While they have not secured a win yet, they intend on continuing to try.
They also create t-shirts for many of their events with the sales going back to the community or a community cause. In the last decade, the Pearlsnaps estimates the team has raised about $20,000.
"It's community-wide. It's not just us. When we started, when we promoted this most recent softball tournament, we had people as soon as we put a banner up, we had people blowing up our inboxes saying how can we contribute? What can we do and that's just how this community operates. Everyone wants to give time, talent, or treasure in some sort of fashion to help someone in need. It just kind of snowballs from us," explained Scheer.
11 year old's jewelry-making skills send Kansas veterans to D.C.
How did they get their name?
The name Pearlsnaps comes from the men's authentic Western-wear attire.
"My dad, he used to always wear these around the house when he was working on Saturdays. He had this old pearl snap with the sleeves cut off, and then, I inherited some over the years, so I started wearing them," explained Scheer.
Scheer's style then took off. He gifted some of his teammates some pearl snaps.
"We said, 'you know we should wear these on game days. This should be our formal jersey,' and that's how we became the Pearlsnaps," he said.
Harvey County sheriff's deputy struggling with COVID
Dealers are spiking drugs with fentanyl, and killing even more Kansans during the pandemic


Arm in arm with hubby donald himself in a three piece tuxedo melania trump made her way to marine one in a sweeping pale yellow chiffon gown that draped off her shoulders calling to mind. Melania trump stunned in a yellow gucci dress meeting putin in helsinki. First lady melania trump dressed for the state dinner with the prime minister of england in a jmendel gown.
Melania trump stuns in sweeping grecian goddess j.
Melania trump yellow gown. The first lady and president trump are on a state visit to the uk. Mendel dress melania trump wore during the first familys first official visit to london last week. Mendel goddess gown to wear against the dramatic. First lady melania trump chose a dramatic j.
Mendel gown for dinner at churchills palace. On this weeks episode of who wore it best social media has decided to take on the yellow j. For a formal dinner hosted by the british prime minister teresa may first lady melania wowed in a gorgeous yellow gown with a dramatic cape train. Heres everything you need to know about the showstopping yellow ball gown melania trump wore to blenheim palace last.
Melania trump wore a yellow gown for a gala dinner at blenheim palace on thursday. First lady melania trump wowed many with her latest fashion pick on thursday chosen for a black tie dinner hosted by englands prime minister theresa may at blenheim palace in oxfordshire england. Melania trump wore a dramatic yellow gown for an evening with officials at blenheim palace last night and comparisons to disney princesses were immediately being drawn on social media. The first lady of the united states has started the uk state visit in style.


text:
/*
 * Open Source Software published under the Apache Licence, Version 2.0.
 */

package io.github.vocabhunter.analysis.grid;

import io.github.vocabhunter.analysis.core.CoreConstants;
import io.github.vocabhunter.analysis.core.VocabHunterException;
import org.apache.poi.ss.usermodel.*;
import org.apache.poi.ss.usermodel.Row.MissingCellPolicy;

import java.io.IOException;
import java.nio.file.Path;
import java.util.List;
import java.util.function.Predicate;
import java.util.stream.IntStream;
import java.util.stream.StreamSupport;
import javax.inject.Singleton;

@Singleton
public class ExcelGridReaderImpl implements ExcelGridReader {
    @Override
    public List<GridLine> readGrid(final Path file, final Predicate<String> filter) {
        try (Workbook workbook = WorkbookFactory.create(file.toFile())) {
            Sheet sheet = workbook.getSheetAt(0);
            DataFormatter dataFormatter = new DataFormatter(CoreConstants.LOCALE);

            return StreamSupport.stream(sheet.spliterator(), false)
                .map(r -> row(r, filter, dataFormatter))
                .toList();
        } catch (final IOException e) {
            throw new VocabHunterException("Unable to read spreadsheet " + file, e);
        }
    }

    private GridLine row(final Row row, final Predicate<String> filter, final DataFormatter dataFormatter) {
        List<GridCell> cells = IntStream.range(0, row.getLastCellNum())
            .mapToObj(i -> row.getCell(i, MissingCellPolicy.RETURN_BLANK_AS_NULL))
            .map(c -> cell(c, filter, dataFormatter))
            .toList();

        return new GridLine(cells);
    }

    private GridCell cell(final Cell original, final Predicate<String> filter, final DataFormatter dataFormatter) {
        String text;

        if (original == null) {
            text = "";
        } else {
            text = dataFormatter.formatCellValue(original).trim();
        }

        return new GridCell(text, filter.test(text));
    }
}


To keep pace with population growth and increasing urban density, VINCI Construction helps meet the need for housing, office and hotel infrastructure. VINCI Construction combines innovation capacity and ability to adapt to a wide variety of socioeconomic environments to help cities and governments address major societal issues.
The hotel complex in the heart of Paris's La Défense business district opened in February 2015. VINCI Construction came up with ingenious solutions to implement its design.
Inaugurated at the end of 2015, the 170-metre Odeon Tower overlooks the beaches in Monaco.
Freyssinet worked on the renovation of the MLC tower, a standout feature of the Sydney, Australia skyline.
The conversion of the Macdonald warehouse in Paris's 19th arrondissement next to the ring road is a major part of the Paris North East urban renewal programme.
In London's King's Cross neighbourhood, VINCI Construction UK has built a complex for BNP Paribas Real Estate Property Development UK comprising 40,000 m² of office space with shops on the ground floor.