League of Fat Bastards Cigars has appointed Eddie Tarazona as the company’s new director of sales for the USA and Americas
For more than 20 years, Tarazona ran his own cigar company, Tarazona Cigars. In 2022, he campaigned for a U.S. Congressional seat, representing Florida&#8217;s 18th Congressional District, though <a href="https://halfwheel.com/eddie-tarazonas-bid-for-u-s-house-of-representatives-falls-short/413253/">fell short</a> in the race for the seat
“I am honored that Adam and the team have entrusted me with his role
not just to share our incredible cigars but to be part of the critical cause to tackle men’s mental health,&#8221; said Tarazona in a press release
&#8220;I’m excited for the opportunity to help grow its impact across the US and the Americas
It’s a real pleasure to be joining something bigger than myself.”
The company said that Tarazona&#8217;s first task will be to build a sales team for the U.S
the brand’s vp of business development for the Asia-Pacific
Middle East and North Africa regions to further develop the brand in other areas of the world
“In building his own brand for the last twenty years
he has been there and done it,&#8221; said Adam J
the founder and ceo of League of Fat Bastards Cigars
&#8220;I love his work ethic and his aim to always find a win for everybody
‘do it with passion’ and that fits so well with our mission-focussed brand.”
League of Fat Bastards Cigars currently has distribution in the USA
while distributors in several European countries
will be receiving their first shipment in the near future
I strive to capture the essence of a cigar and the people behind them in my work – every cigar you light up is the culmination of the work of countless people and often represents generations of struggle and stories
it’s about so much more than the cigar – it’s about the story behind it
the experience of enjoying the work of artisans and the way that a good cigar can bring people together
I’m the public address announcer for the Colorado Rockies and Arizona Diamondbacks during spring training
as well as for the Salt River Rafters of the Arizona Fall League
and previously the Arizona Rattlers of the Indoor Football League
I also work in a number of roles for Major League Baseball
I covered the Phoenix and national cigar scene for Examiner.com
Utah — A Utah DoorDash driver has now been arrested and faces sexual battery charges after he allegedly touched a Taco Bell employee inappropriately multiple times
Angel Tineo Tarazona was arrested on Monday
According to court documents obtained by FOX 13 News
Saratoga Springs police were called to the Taco Bell at 1300 E
The victim called the police to the restaurant to report the incident
The victim explained that she gave Tarazona the order he needed when he pointed to the menu behind her
she says Tarazona put his hand on her butt and pulled her towards him
who denied any inappropriate touching occurred
Tarazona explained to police that he had simply been placing his hand on her hip to teach her to dance
This story was supported by Tarazona's wife who claimed to have witnessed the interaction and stated that nothing inappropriate occurred
officers trespassed Tarazona from the location as they continued their investigation
officers received video footage of the incident from a lobby camera inside the Taco Bell
Police say the video shows Tarazona pointing to the menu and then placing his hands on the worker and pulling her towards him
Angel Tarazona then allegedly went to the bathroom for 2 minutes before calling the victim over again
Detectives say he again pointed to the menu and
placed his hands on her and pulled her in towards him
Tarazona was arrested at his home in Orem Monday and faces a sexual battery charge
Utah (KUTV) — A DoorDash driver was arrested after he allegedly touched a Taco Bell employee inappropriately several times as he was picking up an order
was arrested on suspicion of sexual battery
officers with the Saratoga Springs Police Department responded to Taco Bell on 1300 East State Street
Tarazona was still at the scene when officers arrived
The caller told oficers that after she gave the order to Tarazona he pointed at the menu and asked a question
When she turned around to look at the menu
who denied anything happened but said he &quot;put his hand on her hip to teach her to dance,&quot; the affidavit states
Tarazona&#x27;s wife claimed she was a witness and that nothing inappropriate had happened
officers obtained security footage that showed Tarazona touching the victim inappropriately several times
Tarazona was arrested from his home and booked into the Utah County Jail
WELLINGTON — <a href=https://www.palmbeachpost.com/story/sports/polo/2024/02/24/buena-vibra-wins-rain-delayed-us-open-womens-polo-championship-in-wellington/72725435007/>Buena Vibra</a> rallied in the second half to defend its title in the U.S. Open Women’s Polo Championship Sunday at <a href=https://www.palmbeachpost.com/story/sports/2024/12/27/palm-beach-county-winter-polo-season-wellington/77239927007/>National Polo Center</a>'s U.S
In front of the largest crowd to witness a women's tournament
to win its second consecutive women's title
La Fe Eastern Hay (Naomi Tachibana Marlough
Women's Open awarded $25,000 in prize money from U.S
divided between first- ($17,000) and second-place ($8,000) teams
Each team also received $2,500 to donate to the polo charity of their choice
More: <a href=https://www.palmbeachpost.com/story/sports/polo/2024/12/31/polo-concord-equity-group-port-mayaca-george-miller-memorial/77321516007/ rel=noreferrer target=_blank>Concord Equity Group rallies to win George Miller Memorial in polo opener</a>
La Fe Eastern Hay, led by 10-goaler Hope Arellano, dominated the first half jumping out to leads of 4-1, 6-3 and 7-5 at the half. Arellano scored six goals in the first three chukkers, finishing with eight goals.
The momentum shifted to Buena Vibra, which translates to "Good Vibes," in the second half. Buena Vibra scored three consecutive goals in the fourth quarter with Hine tying the game at 8 with 54 seconds left.
Hine gave Buena Vibra its first lead of the game early in the fifth chukker when she converted a 30-yard penalty for a 9-8 advantage. Hine finished with eight goals, including four penalty conversions.
"I'm so happy, it still hasn't sunk in yet," said Hine, the top scorer with 10 goals. "I'm so happy to be a part of this team. I think we were all a little nervous to play on this incredible field. There was the pressure of being in the finals. In the second half, we went back to playing basics."
Buena Vibra outscored La Fe Eastern Hay 3-1 in the final chukker. La Fe Eastern Hay had its share of scoring opportunities but couldn't capitalize.
"I don't even know how I am feeling," said 14-year-old Valentina Tarazona, who grew up playing in the Wellington-based Polo Training Foundation. "I can't believe I won. It feels surreal to win again." Her sister Giuliana is 15.
"I was a little bit nervous when I got here," said Giuliana, 15. "Definitely in the first chukker I was frozen. It pushed me to get over the nerves when they were leading. We were definitely off in the first half but we came back. It's always been a dream to win with my sister."
Cande Fernández Araujo, a 9-goaler, and her horse, Machitos Felpa, swept Most Valuable Player and Best Playing Pony honors. She is rated 10 goals in Argentina.
"It's amazing, I've never won the U.S. Open before," Araujo said. "Playing on this field in front of all these people is incredible. It's really a big achievement for me. We just did the little things with force and intelligence to win."
The U.S. Open Women's Polo Championship was presented by Brad and Kathy Coors Foundation. The next major women's tournament is the WCT Finals in April at Grand Champions Polo Club. The event honors the legacy of Hall of Famer Sunny Hale, who created the WCT and increased interest in women's polo to what it is today. Women were only allowed to join the USPA in 1972.
In the semifinals, Buena Vibra advanced with an 8-6 win over Mint Eco Car Wash and La Fe Eastern Hay advanced with an 11-9 victory over North Star.
06 Apr 2025 10:00:00 GMT?.css-1txiau5-AnswerContainer{color:var(--GlobalColorScheme-Text-secondaryText2);}FC Andorra won 1–0 over SD Tarazona on Sun
This is 31 of the Primera Federacion - Group 1
Predicted lineups are available for the match a few days in advance while the actual lineup will be available about an hour ahead of the match
The current head to head record for the teams are FC Andorra 1 win(s)
Have scored 6 goals in their last 5 matches
Who won between FC Andorra and SD Tarazona on Sun
06 Apr 2025 10:00:00 GMT?FC Andorra won 1–0 over SD Tarazona on Sun
06 Apr 2025 10:00:00 GMT.InsightsHave scored 5 goals in their last 5 matches
FC Andorra is playing home against SD Tarazona on Sun
It wasn’t to be. Despite dominating from start to finish, Barça Atlètic drew 1-1 with SD Tarazona at the Estadi Johan Cruyff this Saturday. Although the visitors went ahead through Llácer, Toni Fernández equalised before the break. The blaugranes couldn’t find a winner in the second half and remain in the relegation zone, now 11 games without a victory with 10 games to go.
As expected, Barça looked to control possession, while Tarazona set up defensively and eyed chances on the counter. Breaking their five-man backline proved difficult early on. The first real chance came in the 26th minute when Cédric hit the crossbar after a Dacosta cross.
Barça’s misfortune grew when Llácer capitalized on a defensive lapse to make it 1-0 to the visitors. But Toni Fernández responded quickly with a fine left-footed strike from the edge of the box to level the score on 36 minutes, with no further change before half-time.
Barça stayed calm and controlled the second half, with Toni and Dacosta providing width and threat. Cédric remained the main danger, having a goal ruled out for a tight offside and narrowly missing another chance.
Sergi Milà made several changes—bringing on Trilli, Guille, Jan Virgili, Darvich, and Jofre Torrents—but Barça lacked precision in the final third. Guille had two late chances, and Trilli tested Fuoli with a powerful shot in stoppage time, but the score stayed as it was and it all ended 1-1.
Barça Atlètic: Kochen, Farré (Torrents, 79'), Cortés, Sergi Domínguez (Trilli, 66'), Mbacke, Garrido, Soma (Darvich, 79'), Rubén López (Guille Fernández, 66'), Toni Fernández, Raúl Dacosta, Cédric (Virgili, 70'). 
SD Tarazona: Fuoli, López (Álvarez, 75'), Trilles, Camus, Vadik, Llácer (Chechu Martínez, 40'), Fuentes, Cedeño, Mena, Agüero (Buyla, 75'), Pradera (Areso, 83').
Goals: 0-1, Llácer (min 30); 1-1, Toni Fernández (min 36).
Referee: Juncal Moreira. Yellows for Dacosta &amp; Toni Fernández (Barça) and Chechu Martínez, Vadik &amp; Trilles (Tarazona).
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<a href="https://ksltv.com/crime-public-safety/">CRIME</a>
6:01 PM                    | Updated: Apr 2
FILE - A sign hangs at a Taco Bell on May 23
Declaring a mission to liberate "Taco Tuesday" for all
to force Wyoming-based Taco John's to abandon its longstanding claim to the trademark
BY <a href="https://ksltv.com/author/ksltv/" style="font-weight:500;">KSL TV</a>
LEHI — A DoorDash driver is being accused of inappropriately grabbing a Taco Bell employee while asking her a question
the incident happened Friday at a Taco Bell in Lehi
Saratoga Springs police officers said they received reports from the victim who was touched inappropriately by a DoorDash driver
later identified as 59-year-old Angel Tarazona
police said that Tarazona and the victim were there and both were separated immediately
&#8220;The complainant explained after she gave the driver the order
(Tarazona) pointed at the menu and asked a question about an item
he put his hand on her buttocks and pulled her towards him,&#8221; the affidavit stated
Tarazona denied inappropriately touching her but admitted to placing his hands on her hips to teach her how to dance
Tarazona&#8217;s wife claimed to be a witness and said she didn&#8217;t see anything inappropriate
Police trespassed Tarazona from the Taco Bell and began to further investigate
police obtained security footage of when the alleged incident happened
&#8220;The video shows (Tarazona) points to the menu as if to ask a question
he places his hand on her lower back and pulls her body towards him,&#8221; the affidavit stated
The footage showed Tarazona going into the bathroom for about two minutes and calling for the victim again
Tarazona grabbed her again and pulled her toward him
Tarazona was located by another police agency at his him
Tarazona was booked into the Utah County Jail on suspicion of sexual battery
Editor&#8217;s Note: The story originally said that Tarazona was arrested on Friday
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15 Feb 2025 16:00:00 GMT?.css-1txiau5-AnswerContainer{color:var(--GlobalColorScheme-Text-secondaryText2);}CD Arenteiro won 2–1 over SD Tarazona on Sat
This is 24 of the Primera Federacion - Group 1
The current head to head record for the teams are CD Arenteiro 1 win(s)
Haven&#x27;t scored in their last 2 matches
Have scored 5 goals in their last 5 matches
Have kept the most clean sheets in the competition (11)
Who won between CD Arenteiro and SD Tarazona on Sat
15 Feb 2025 16:00:00 GMT?CD Arenteiro won 2–1 over SD Tarazona on Sat
15 Feb 2025 16:00:00 GMT.InsightsHave scored 5 goals in their last 5 matches
CD Arenteiro is playing home against SD Tarazona on Sat
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Barça Atlètic picked up their first point away from home this season thanks to a goalless draw at Tarazona in a game that could have gone either way with chances for both sides. 
The home side were happy to delegate possession to the blaugranes and look to cause problems on the counter attack. A stop start first half saw debut keeper Yaakobishvili make an excellent save for Barça as the home side threatened. The contest was an even one with both teams cancelling each other out. 
After the break the game opened up. Unai Hernández almost scored one of the goals of the season but keeper Fuoili saved well. Tarazona had a goal disallowed in a frenetic final period in which Barça also went close to taking the win. Barça Atlètic are next in action in a game against Sestao away from home on Sunday. 
Tarazona: Fuoli; Camus, Trilles, Murria, Llacer; Marc Álvarez (Iker Gil 82'), Cedeño (Romero 77'), Buyla, Javi Martín (Pradera 66'); Fuentes (Manu Rico 82') and Cubillas (Areso 66').
Barça Atlètic: Yaakobishvili; Cuenca (Trilli 61'), Olmedo, Cortés, Edu Sánchez; Pau Prim (Pedro Soma 76'), Guillermo (Rubén López 61'), Unai; Toni Fernández (Ureña 61'), Brberá and Iván Cédric (Juan Hernández 87').
19 Jan 2025 18:30:00 GMT?.css-1txiau5-AnswerContainer{color:var(--GlobalColorScheme-Text-secondaryText2);}SD Amorebieta vs SD Tarazona on Sun
This is 20 of the Primera Federacion - Group 1
The current head to head record for the teams are SD Amorebieta 0 win(s)
Who won between SD Amorebieta and SD Tarazona on Sun
19 Jan 2025 18:30:00 GMT?SD Amorebieta vs SD Tarazona on Sun
19 Jan 2025 18:30:00 GMT ended in a 2–2 tie.InsightsHave scored 4 goals in their last 5 matches
SD Amorebieta is playing home against SD Tarazona on Sun
01 Mar 2025 18:00:00 GMT?.css-1txiau5-AnswerContainer{color:var(--GlobalColorScheme-Text-secondaryText2);}SD Tarazona won 2–1 over Lugo on Sat
This is 26 of the Primera Federacion - Group 1
The current head to head record for the teams are Lugo 0 win(s)
Have scored 4 goals in their last 5 matches
Who won between Lugo and SD Tarazona on Sat
01 Mar 2025 18:00:00 GMT?SD Tarazona won 2–1 over Lugo on Sat
01 Mar 2025 18:00:00 GMT.InsightsHave scored 8 goals in their last 5 matches
Lugo is playing home against SD Tarazona on Sat
15 Mar 2025 17:00:00 GMT?.css-1txiau5-AnswerContainer{color:var(--GlobalColorScheme-Text-secondaryText2);}Barca Atletic vs SD Tarazona on Sat
This is 28 of the Primera Federacion - Group 1
The current head to head record for the teams are Barca Atletic 2 win(s)
Who won between Barca Atletic and SD Tarazona on Sat
15 Mar 2025 17:00:00 GMT?Barca Atletic vs SD Tarazona on Sat
15 Mar 2025 17:00:00 GMT ended in a 1–1 tie.InsightsHave scored 6 goals in their last 5 matches
Barca Atletic is playing home against SD Tarazona on Sat
26 Jan 2025 14:30:00 GMT?.css-1txiau5-AnswerContainer{color:var(--GlobalColorScheme-Text-secondaryText2);}SD Tarazona won 1–0 over Zamora on Sun
This is 21 of the Primera Federacion - Group 1
The current head to head record for the teams are SD Tarazona 1 win(s)
Have scored 7 goals in their last 5 matches
Who won between SD Tarazona and Zamora on Sun
26 Jan 2025 14:30:00 GMT?SD Tarazona won 1–0 over Zamora on Sun
26 Jan 2025 14:30:00 GMT.InsightsHave scored 4 goals in their last 5 matches
SD Tarazona is playing home against Zamora on Sun
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08 Feb 2025 17:00:00 GMT?.css-1txiau5-AnswerContainer{color:var(--GlobalColorScheme-Text-secondaryText2);}Real Sociedad B won 1–0 over SD Tarazona on Sat
This is 23 of the Primera Federacion - Group 1
The current head to head record for the teams are SD Tarazona 0 win(s)
Have kept the most clean sheets in the competition (10)
Have scored 8 goals in their last 5 matches
Real Sociedad B have won the previous 3 matches against SD Tarazona
Who won between SD Tarazona and Real Sociedad B on Sat
08 Feb 2025 17:00:00 GMT?Real Sociedad B won 1–0 over SD Tarazona on Sat
08 Feb 2025 17:00:00 GMT.InsightsHave scored 5 goals in their last 5 matches
SD Tarazona is playing home against Real Sociedad B on Sat
ha compartido su trayectoria y vivencias en el programa &#8216;Eméritos&#8217; de TRECE
Con una vida marcada por su labor en Roma tras el Concilio Vaticano II y su episcopado en una de las diócesis más pequeñas de España
retiros espirituales y la escritura de su último libro
mantiene su compromiso con la oración y el servicio
Tras media vida en Roma poniendo en pie los documentos que surgieron del <a href="https://www.cope.es/religion/vivir-la-fe/cultura-y-fe/libros-ecclesia/noticias/comentario-teologico-del-concilio-vaticano-20230614_2762596" data-mrf-link="https://www.cope.es/religion/vivir-la-fe/cultura-y-fe/libros-ecclesia/noticias/comentario-teologico-del-concilio-vaticano-20230614_2762596">Concilio Vaticano II</a>
en el año 2011 pasaría a ser obispo de una de las diócesis más pequeñas de España
Pero lejos de una vida retirada se mantiene muy activo
 “El Papa quería que entrásemos toda la Iglesia en una sinfonía de oración poniendo el Padrenuestro en el centro de la oración”
También encuentra tiempo para visitar su pueblo natal
Un pueblo agrícola que destaca por el “espárrago
que en esos años fue una fuente de riqueza con tres fábricas
Su padre sin embargo era transportista y su madre ama de casa. El mayor de una familia con cuatro hermanos, Eusebio Hernández abandona Cárcar a los doce años para ingresar en el seminario con los <a href="https://agustinosrecoletos.org/">Agustinos Recoletos.</a>
El obispo emérito de Tarazona vio nacer su vocación a comienzo de los años cincuenta gracias al agustino Jesús Pardo Ojer
en el Amazonas y nacido en el mismo pueblo navarro que Eusebio Hernández
Jesús Pardo Ojer estaba un día con los niños cerca el río
y él estaría rezando y los niños brasileños jugando al fútbol
se lanzaron a por el balón y la barca dio la vuelta enseguida y comenzaban a ahogarse los cuatro niños
un infarto y murió en la orilla del río Purús
La ordenación sacerdotal llegó en un día y año icónico: San Fermín de 1968
Las revueltas de París habían llegado a los seminarios españoles
donde incluso los futuros sacerdotes organizaban pequeñas huelgas: “Hicimos una pequeña huelga a un profesor en 1967
Éramos un curso un poco revoltoso y nos dijeron que como pena todos a Filipinas
porque entonces se consideraba una penitencia ir a Filipinas”
donde los ecos procedentes de París eran más intensos en las universidades: “Viví intensamente estos años en la residencia y compartí la vida con estudiantes
me tocó hasta correr delante de los caballos de los grises y meterme debajo de los pupitres
porque cuando entraban no había contemplaciones
Fueron años de grata experiencia en el que pasamos un poco de miedo
los estudios se hacían difíciles porque no era fácil compaginar los estudios con el ambiente que había”
En 1975, el obispo emérito de Tarazona fue trasladado a Roma, donde se incorporó a la Congregación para la Vida Consagrada, en el campo de las conferencias que agrupan los institutos religiosos, que en el caso de España es la <a href="https://confer.es/">CONFER</a>
donde Eusebio Hernández contribuyó a crear las conferencias episcopales en diferentes países
“Cuando llegué habría unas ochenta conferencias episcopales y cuando me fui había 150
Pensar que cuando llegué estaba el Muro de Berlín y no había posibilidad de crear conferencias en los países del Este
y me tocó ir a Rusia a crear esta conferencia
Y eso de las conferencias me permitió conocer países de Asia
la India&#8230; Esto nos hacía sensibles a la problemática que iban surgiendo como la Teología de la Liberación
la opción por los pobres&#8230; estos problemas los vi en primera mano”
Eusebio Hernández se ha confesado amante del Concilio Vaticano II: “De Gaulle decía que fue el acontecimiento más importante de la historia de la humanidad
salir para ver qué sucede en el mundo para poder responderles”
El obispo lo recuerda como años apasionantes pero también de tensiones
“al haber cantidad de salidas” de sacerdotes que se secularizaron: “Me tocó cerrar algún Instituto de Teología Intercongregacional en México donde había una Teología de la Liberación fuerte
los obispos estaban en desacuerdo y tuve que poner orden en el conflicto”
Tras 35 años en Roma, <a href="https://www.cope.es/podcasts/benedicto-xvi-el-papa-de-la-tormenta">el Papa Benedicto XVI </a>le nombra obispo de Tarazona en 2011
recuerda esta etapa “con un cariño especial”
destacando la sencillez y humildad de sus 60.000 feligreses
Uno de los recuerdos del obispo fue al final de su ministerio en la diócesis aragonesa, el 2022, cuando estalló la <a href="https://www.cope.es/programas/la-linterna/el-video-blog-de-exposito/monologos/te-fiarias-dos-personalidades-trump-putin-mano-paz-ucrania-20250213_3095582.html">guerra de Ucrania. </a>El Obispado se volcó en la ayuda: “Surgió gente que quería ir a Ucrania a llevarles comestibles
ropa&#8230; Salieron ocho furgonetas hasta la frontera y se trajeron las furgonetas de vuelta llena de gente”
Todos ellos fueron acogidos en el seminario de Tarazona: “Estuvieron en las mejores habitaciones
buenas camas&#8230; Al principio a alguno le molestó pero les dije que eran los más necesitados
Llegamos a tener hasta cien ucranianos en el seminario
Era la urgencia más necesaria y había que ayudarles”
<a class=link-es href="./?lang=es"></a>   <a class=link-pt href="./?lang=pt-pt"></a>   <a class=link-en href="./?lang=en"></a>
A Utah DoorDash driver has been arrested and facing sexual battery charges
Allegedly he touched a Taco Bell employee inappropriately on multiple accastions
In Saratoga Springs the victim had been called the police to the Taco Bell
When the police got there both of the parties were in the restaurant
The victim states that she was giving Tarazona the order when the victim turned
thats when he put his hand on her butt and pulled her towards him
he denied it by saying he had been trying to teach her a dance and thats why he placed his hand on her hip
Tarazona&#8217;s wife claimed she witnessed the interaction and nothing inappropriate occurred
officers recived video footage of the incident and it shows that everything the victim said was true
Tarazona was arrested at his home in Orem and now faces sexual battery charges
<a href="https://www.fox13now.com/news/crime/utah-doordash-driver-accused-of-touching-taco-bell-worker-inappropriately">Read More</a>
Donald Trump has implemented tariffs on several uninhabited islands as a part of an confused effort to establish equal trade
The White House published a list of all the countries that have been slapped with &#8220;reciprocal tariffs&#8221;
Social media users were the ones to point out
that the list includes uninhabited islands and territory where the only significant settlement was U.S
One of the uninhabited islads include Heard and McDonald Islands one of the most remote places on Earth
was slapped with 10% &#8220;reciprocal tariff.&#8221; Along with the British Indian Ocean Terriroty was also slapped with a 10% reciprocal tariff
despite the fact the he only inhabitants of the islands are U.S.-U.K
<a href="https://www.mediaite.com/politics/whoops-trump-slaps-tariffs-on-uninhabited-islands/">Read More</a>
a 35 year old man has been arrested after police said he accidentally shot his friend in the groin with an AK-47
has been booked into Salt Lake County Jail as an investigation of aggravated assult resulting in serious injury
illegal discharge of a firearm causing injury
two counts of domestic violence in the presence off a child and providing alcohol to a minor
The 19 year old victim had been at Angell&#8217;s house Tuesday night to &#8220;hang out&#8221; the two &#8220;began to drink and smoke weed&#8221; thats when &#8220;Jason then informed (the victim) that he wanted to show him his new gun.&#8221; That is when Angell pulled out the AK-47
The victim staes &#8220;he told Jason multiple times to make sure the gun was clear
but he did not appear to know how to use the gun
in manner that (the victim) was not able to describe
pointed the gun at the victim and shot him.&#8221; Angell told the investigators that &#8220;the gun just went off&#8221; and that when she saw the victim with his arms in the air and saying &#8220;You just shot me.&#8221;
<a href="https://www.ksl.com/article/51287668/magna-man-accused-of-accidentally-shooting-friend-with-ak-47">Read More </a>
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Over 60 people perished in Paiporta when a wave of water rushed down the Poyo canal 
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The mangled car in which Jorge Tarazona’s three-year-old niece and sister-in-law perished in last week’s catastrophic flooding in <a href="/topic/spain">Spain</a> now hangs halfway off the ragged edge of a highway
His brother managed to survive, clinging to a fence. He and his family had been caught in traffic driving home to Paiporta on <a href="/topic/valencia">Valencia</a>’s southern outskirts, Tarazona said. They had no chance to escape when the tsunami-like wave quickly overflowed the nearby drainage canal and swept away everything in its path.
“They did not have time to do anything,” Mr Tarazona told The Associated Press, a week after the 29 October flash floods. “My brother was dragged away and ended up clinging to a fence.” His sister-in-law “could not get out and died with her little girl”.
Mr Tarazona had ridden a bike back to the site and taped a note on the car asking for whoever eventually removed the wreck off the side of the highway, to call him.
“It all happened so fast,” he said, tears coming to his eyes. “In half an hour the current had carried away the car. There was no time, no time. She managed to send me the location of their car hoping for a rescue.
“The next day she was found dead inside,” he said.
It’s unclear if the two are included in the official toll of the 217 confirmed dead as fatalities tick up, eight days after the deadliest floods in Spain this century.
Paiporta has been labelled by Spanish media as the “ground zero” of the natural disaster that has also left 89 people still missing, while officials say the real figure could be higher. 
Over 60 people perished in Paiporta when a wave of water rushed down the Poyo canal that cuts through its centre. Frustration over the survivors’ sense of abandonment exploded in Paiporta on Sunday when a crowd greeted Spain’s royals and officials with a barrage of mud and other objects. 
Prime minister Pedro Sanchez was rushed away and the royal couple had to eventually cancel the visit after speaking to several distraught neighbours amid a chaotic scene.
The Civil Guard said they rescued two people who had been trapped in their Paiporta home, almost a miraculous exception among the tragedy.
The mayor of Paiporta, a middle-class community of 30,000, pleaded on Tuesday for a “higher authority” to step in and take control of her municipality because the floods had made it impossible to go on. Mayor Maribel Albalat said all the municipal buildings, from the town hall to the local police, had been severely damaged and that many of the local civil servants “are in a state of shock”.
“Paiporta is a strong village, but this overwhelms our capacities as a local administration,” she said.
The air-throbbing “thup, thup, thup” of the huge, two-propeller Chinook helicopters that have flown overhead with the arrival of the army has added to the post-apocalyptic atmosphere.
The destruction, however, went far beyond Paiporta and covered a huge swath of municipalities, above all on the southern flank of Valencia city on the Mediterranean coast. Seventy-eight localities had at least one person die from the floods. Police have expanded their search to the nearby marshes and coastline, where the waters carried some away.
The residents, businesses and town councils of the affected localities can apply for financial help from a €10.6bn (£8.8bn) relief package from Spain’s government. The regional Valencia government, which is being slammed for not alerting the populace of the danger in time, has asked the central government in Madrid for €31bn to ensure the recovery.
Over a week later, the clean-up goes on to get rid of tons of mud and debris that clog street after street, filling thousands of ground floors, and destroying living rooms and kitchens. Neighbourhoods were left without shops and supermarkets after all their products were ruined. Many houses still don´t have drinking water.
An impromptu army of volunteers were the first helpers on the ground, shovelling and sweeping away the sticky brown mire covering everything, and helping to start removing pile after pile of debris that made access to cars impossible in many areas. 
Authorities eventually mobilised 15,000 soldiers and police reinforcements to help firefighters search for bodies and start extracting thousands of wrecked cars strewn over streets and sunk in canal beds. 
At every corner, cars are piled on top of one another or smashed into buildings, light poles, trees and bridge overpasses.
“There is still so much to do,” said volunteer Juanma Baztan Lopez, who is helping churn through the muck in Catarroja, which borders on Paiporta, in his four-wheel drive. He has helped transport doctors to people in need, deliver essential products, and tow away wrecked cars.
 “It will take a year to get this back to normal,” he said.
Jorge Tarazona attaches a poster to a car in Paiporta, Valencia, Spain
22 Mar 2025 15:00:00 GMT?.css-1txiau5-AnswerContainer{color:var(--GlobalColorScheme-Text-secondaryText2);}SD Tarazona won 2–0 over Sestao on Sat
This is 29 of the Primera Federacion - Group 1
The current head to head record for the teams are Sestao 1 win(s)
Who won between Sestao and SD Tarazona on Sat
22 Mar 2025 15:00:00 GMT?SD Tarazona won 2–0 over Sestao on Sat
22 Mar 2025 15:00:00 GMT.InsightsHave scored 5 goals in their last 5 matches
Sestao is playing home against SD Tarazona on Sat
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hooded individuals arbitrarily detained Carlos Correa
a renowned human rights defender and executive director of Venezuelan NGO Espacio Público
His detention is solely based on his human rights work
similarly to four other defenders currently arbitrarily detained for their activism in Venezuela: Javier Tarazona
We demand Nicolás Maduro ensures their immediate and unconditional release and
Source: <a href="https://ghanasoccernet.com/" rel="nofollow noopener" target="_blank">ghanasoccernet.com</a>
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<a rel="nofollow" href="Ghana-forward-Jerry-Afriyie-marks-impressive-CD-Lugo-debut-in-SD-Tarazona-clash-1973729?audio=1">Listen to Article</a>
<a href="Ghana-forward-Jerry-Afriyie-marks-impressive-CD-Lugo-debut-in-SD-Tarazona-clash-1973729?gallery=1"> </a>Afriyie joined CD Lugo in February on loan
<a href="Watch-highlights-of-Jerry-Afriyie-s-CD-Lugo-debut-against-SD-Tarazona-1973687?gallery=1"> </a>Jerry Afriyie during his first game for CD Lugo
<a target='_blank' href='/GhanaHomePage/people/person.php?ID=3622'><a target='_blank' href='/GhanaHomePage/people/person.php?ID=3622'>Black Stars</a></a> forward Jerry Afriyie made his first appearance for Spanish club CD Lugo in their league game against SD Tarazona
The 18-year-old climbed off the bench to star for Lugo despite their 2-1 defeat to Tarazona
Afriyie replaced Jon Cabo after the break and immediately made an impact
winning a penalty which was converted by Brian Mendoza
The visitors raced into a two-goal lead before the hour mark through a brace from Adrián Fuentes
who netted his first with a strong header before he added his second from the spot
Afriyie joined CD Lugo in February on loan after sealing a deal to Al-Qadsiah from Ghanaian lower-tier side Thoughts FC
The Ghana U20 star will spend the rest of the season at Lugo as part of his development before moving to Saudi to connect with his parent club
The versatile forward is expected to make the <a target='_blank' href='/GhanaHomePage/people/person.php?ID=3622'><a target='_blank' href='/GhanaHomePage/people/person.php?ID=3622'>Black Stars</a></a> team for the upcoming World Cup qualifiers against Chad and Madagascar later in March
Watch highlights of Afriyie's debut below:
𝗥𝗘𝗦𝗨𝗠𝗘𝗡 | <a href="https://twitter.com/hashtag/LugoTarazona?src=hash&ref_src=twsrc%5Etfw">#LugoTarazona</a>
1-2 | <a href="https://twitter.com/CDeportivoLugo?ref_src=twsrc%5Etfw">@CDeportivoLugo</a> - <a href="https://twitter.com/SDTarazona?ref_src=twsrc%5Etfw">@SDTarazona</a>
⚽ <a href="https://twitter.com/adryyfuentes?ref_src=twsrc%5Etfw">@adryyfuentes</a> (0-1
⚽ <a href="https://twitter.com/adryyfuentes?ref_src=twsrc%5Etfw">@adryyfuentes</a> (0-2
📺 Partido completo: <a href="https://t.co/aMfSPND3Ug">https://t.co/aMfSPND3Ug</a><a href="https://twitter.com/hashtag/PrimeraFederaci%C3%B3n?src=hash&ref_src=twsrc%5Etfw">#PrimeraFederación</a> | <a href="https://twitter.com/hashtag/VersusELearning?src=hash&ref_src=twsrc%5Etfw">#VersusELearning</a> <a href="https://t.co/E7ldxn7WqP">pic.twitter.com/E7ldxn7WqP</a>
<a class="MkLink" aria-label="Go to home page" href="/en"></a>WomenAndrea Tarazona and Levante UD Extend Contract Until 2026There are no reactions yet
Be the first!Levante UD and goalkeeper Andrea Tarazona (born in Riba-roja
2004) have agreed to extend their contract until June 30
This renewal adds an additional season to the previously signed agreement and officially includes the young goalkeeper as a first-team player
This formal change recognizes Tarazona's vital role in achieving the goals of Levante UD's women's first team since her debut
The U-20 international for Spain made her first appearance for Levante in the 2022-23 season opener against Alhama de Murcia
securing a 3-2 victory for Sánchez Vera's team
Tarazona has played 34 official matches across all competitions
she recorded the second-best goal-conceding record in the top national league
Tarazona joined Levante UD in the summer of 2020 to play for their B team
becoming a key figure in the team's project
Her development includes regular first-team training
marking her as one of the most promising talents to emerge from Levante's academy in recent years
SUMMER 2015Won the bronze medal at the Phillips 66 National Championships in the 400 Free Relay with a 3:45.54 ...&nbsp;She finished fourth in the 200 Fly at the Arena Pro Swim Series ..
Also made the B Final in the 100 Fly (15th) and the D race in the 50 Fly (28th) ..
Had a pair of top-10 finishes at the Los Angeles Invitational
placing sixth in the 200 IM championship final (2:18.93) and 10th in the 100 Fly consol race (1:00.90)
2014-2015Swam career-bests in the 100 Fly (52.82)
and the 200 IM (1:59.10) at the NCAA Championships ..
Set new school records in the 400 Medley Relay (3:34.25) at NCAAs ...&nbsp;Qualified for NCAAs in the 200 Medley Relay while setting a new UCLA record (1:37.17) ..
Swam a season-best 2:00.47 in the 200 IM at Pac-12s and won the B Final of the 100 Fly (season-best 52.94) ..
Set a season-best 1:55.15 in the 200 Fly A Final to place second ..
Earned First-Team CSCAA Scholar All-America honors and Second-Team Pac-12 All-Academic honors ..
SUMMER 2014Tarazona swam at the Phillips 66 U.S
placing second in the B Final (10th overall) in the 100 Butterfly with a personal-best 59.71 &hellip; also had a 10th-place finish and personal-best swim of 2:10.49 in the 200 Fly &hellip; part of the fifth-place 400 Freestyle Relay team (3:47.81) and 21st in the 50 Fly (27.40) &hellip; also swam at the Los Angeles Invitational
finishing seventh in both the 200 Individual Medley (2:19.19) and 100 Fly (1:00.68)
2013-2014Was a CSCAA Honorable Mention All-America after finishing 11th in the 200 Butterfly (1:54.88) at the NCAA Championships &hellip; was also a part of the 12th-place 800 Freestyle Relay team (7:06.49)
26th in the 400 Individual Medley (season-best 4:11.74) and 27th in the 100 Fly (personal-best 52.89
4th in UCLA history) &hellip; placed second in the 200 Fly at the Pac-12 Championships with a personal-best 1:54.80
which is second in school history &hellip; also had three other Top 10 finishes
going fifth as part of the 400 Free Relay (3:18.07) and seventh in both the 100 Fly (52.71) and 400 IM (4:14.62) &hellip; won six individual events in dual meets
including the 200 Fly three times against USC (1:56.50)
Arizona State and Arizona &hellip; also claimed the 400 IM against ASU and the 100 Fly and 200 Free at San Diego &hellip; was a part of the 200 Medley and 400 Medley winning teams against Utah and Washington State &hellip; second in the 200 Fly against California
Utah and Oregon State/UCSB/FGCU and third versus Stanford &hellip; second in the 200 IM against WSU and Arizona and second in the 100 Fly versus WSU and OSU/UCSB/FGCU &hellip; finished second in the 200 Fly at the Texas Invitational (1:56.37)
also placing 10th in the 400 IM and 100 Fly and 13th in the 200 IM &hellip; won the 200 Fly at the UNLV Fall Invitational
12th in the 100 Fly and 17th in the 400 IM &hellip; was named Second Team Pac-12 All-Academic and made the Athletic Director&rsquo;s Honor Roll all three quarters
making the finals in the 200 Butterfly (2:12.53
13th) and the 400 Individual Medley (4:52.62
won the Championship final in the 200 IM at the Los Angeles Invitational and was a part of the winning 400 Free Relay squad ..
was also second in the 200 Fly and the 400 Medley Relay (4:26.85) and third in the 400 IM and the 100 Fly ..
seventh in the 100 Fly (1:02.25) and eighth in the 200 IM (2:19.32) and the 400 IM at the Speedo Grand Challenge in Irvine
2012-2013Earned CSCAA Honorable Mention All-America accolades thanks to a 13th-place finish and school-record breaking swim in the 800 Freestyle Relay (7:06.23) at the NCAA Championships ..
also placed 17th in the 200 Butterfly (1:56.61)
22nd in the 400 Individual Medley (4:11.72
third all-time at UCLA) and 38th in the 200 IM (career-best 1:59.31) at the NCAA&rsquo;s ..
11th in the 400 IM (4:12.50) and 20th in the 200 IM (2:00.76) at the Pac-12 Championships
also swimming on the sixth-place 200 Medley Relay (1:39.44) and the 800 Free Relay (7:07.61) teams and setting a personal best in the 100 Fly (53.39) ..
out-touching 2012 Olympic bronze medalist Caitlin Leverenz for the second time this season ..
San Diego State/Kansas and Oregon State/UC Santa Barbara/Utah and finished second in that event at USC ..
finished third in the 200 Fly at the AT&amp;T Winter National Championships (Austin
won the 200 Fly at the Arena Invitational in Long Beach
also placing third in the 800 Free Relay and fourth in the 200 Medley Relay and the 400 Medley Relay ..
won the 400 IM against UC Davis/Washington State ..
named to the Athletic Director&rsquo;s Honor Roll for the Spring and Winter quarters ..
also earned Pac-12 All-Academic Honorable Mention accolades
SUMMER 2012Competed in the 100/200 Butterfly and 200/400 IM at the U.S
2011-2012Placed 18th in the 200 Fly and 32nd in the 400 IM at the NCAA Championships
11th in the 400 IM (freshman school record
4:12.94) and 12th in the 200 IM at the Pac-12 Championships ..
placed second in the 200 Fly and fourth in the 400 IM vs
finished second in both the 200 Fly and 100 Fly vs
was second in the 400 IM and third in the 200 IM against Stanford ..
placed first in the 400 IM and second in both the 200 Fly and 100 Fly against Oregon State/Utah ..
finished first in the 200 Breast and was second in the 200 Fly vs
placed second in the 400 IM and third in the 200 Fly vs
CLAREMONT HIGH SCHOOLCLUB: CLAREMONTMember of the National Junior PanPac team in 2010 where she won the 200m Fly title &hellip; All-American in the 100 Fly and 200 IM all four years of high school &hellip; Scholastic All-American &hellip; First Team all-league &hellip; league MVP her senior year &hellip; two-time CIF champion in the 100 Fly (&lsquo;10/&rsquo;11) ..
also won the 2009 CIF title in the 200 &hellip; four-time National Youth Team member &hellip; competed on the National Junior World Cup team in 2009 &hellip; holds age group records in the 200m and 200-yd fly events (15-16) and IM &hellip; competed for the Claremont Club under coach John Ries
where she was inducted into the Hall of Fame
&hellip; daughter of Jennifer and John Tarazona &hellip; has two brothers
DJ and Dylan &hellip; majoring in psychology
WELLINGTON — Buena Vibra had to wait a week but it was worth it after winning the rain-delayed U.S. Open Women's Polo Championship Friday at the <a href=https://www.palmbeachpost.com/story/sports/polo/2023/12/29/world-polo-league-gauntlet-of-polo-series-set-for-2024-season/72049782007/>National Polo Center</a>
The youngest team in the tournament rallied in the sixth chukker to defeat 90210
in front of a good crowd to win its Open debut on U.S
The final was rained out last Sunday and rescheduled a week later
The foursome of 1-goaler Valentina Tarazona
4-goaler Cory Williams and 8-goalers Clara Cassino and Milly Hine was the only team to finish the eight-team tournament
presented by Brad and Kathy Coors Foundation
Tarazona is the youngest player to win the Women's U.S
breaking Hope Arellano's six-year record at age 14
Last year at 12 the Colombian was one of the youngest to compete in the Open as a sub
"It means a lot to me to be the youngest player
Her being 14 and playing was incredible to me
I dreamed about playing the Open since I was a little girl
the leading scorer with a team-high eight goals
oldest daughter of polo great Adolfo Cambiaso
I almost forgot I had to play," Hine said about the rescheduled game
"I kept thinking it's a week and then it was like
"We met last week for what we thought was going to be the final
We met last night again like we do before every game
We went out there and showed them what we were capable of and enjoyed it
were greeted with pink smoke near the players' tent as they rode off the field after the game
Cassino scored back-to-back goals tying the game following up a missed tail shot by Williams and scoring again to give Buena Vibra a 12-11 lead with 1:11 left
Hine then scored an insurance goal with a 30-yard penalty conversion
It was the first time all four players won a U.S
"To win this kind of final is a dream," Cassino said
"This is what I have been dreaming my whole life playing this kind of tournament with these teams
Winning is the best thing that can happen to a polo player
"I think the most important thing are the horses
It's not easy to play against that organization
We needed a good strategy and to know what we were going to do
We needed to be very well-prepared and what we needed to do for this final."
7-goaler Catalina Lavinia and 8-goaler Mia Cambiaso finished the tournament with a 3-2 record and won $8,000
which means "good vibes," knocked off defending champion La Fe (Naomi Tachibana Marlough
More: <a href=https://www.palmbeachpost.com/story/sports/polo/2024/02/02/first-black-woman-to-play-in-us-open-womens-polo-championship-making-history/72448808007/ target=_blank>Shariah Harris makes history as first Black woman to play in US Open Women's Polo Championship</a>
sponsored by Grand Champions Polo Club President Melissa Ganzi
ending Shariah Harris' history-making journey
The Work To Ride alum was the first Black woman to compete in the Women's U.S
we didn't even know if all of us were going to be playing together
"For us to come together so last minute and make it to the semis and to have a real shot at making it to the final I think is definitely something we should be proud of
The amount of growth we had from our very first game together to now is crazy
NEW: The Effects of the US Foreign Aid Freeze on Freedom House
and use of the justice system as a tool for political persecution may amount to crimes against humanity
The undersigned organizations wholly reject and express profound concern about the July 2 detention and incarceration of the three human rights defenders (HRDs) from the NGO Fundacion Redes (Fundaredes)
as well as activists Rafael Tarazona and Omar García
Their detention is the latest event reflecting rapidly escalating political persecution and criminalization of the HRDs in Venezuela who independently monitor
and report on the country’s critical human rights situation
the Bolivarian Intelligence Service (SEBIN
by its Spanish acronym) detained the three activists
outside the office of the Prosecutor General of the state of Falcon
The activists had been denouncing harassment by SEBIN the previous day
the three Fundaredes’ activists were transferred to Caracas and presented before a terrorism tribunal
where they were charged with inciting hatred
and terrorism—charges frequently used in Venezuela to criminalize HRDs and journalists seeking to defend fundamental freedoms
All three activists remained in prison after being denied access to their lawyers
their lawyers were denied access to the case filings
hampering any possibility of providing an adequate defense
we remind Venezuelan authorities that arbitrary detentions and incarceration
imposing false charges for political reasons
as well as the consistent use of the justice system as a tool for political persecution
all amount to possible crimes against humanity
as recognized by the United Nations’ Independent Fact-Finding Mission for Venezuela
the detention of the Fundaredes’ activists only one day after the publication of the UN Office of the High Commissioner for Human Rights’ (UNOHCHR) report on Venezuela’s feeble compliance with UN recommendations
reinforces the Maduro government’s consistent lack of commitment to international cooperation or respect for international standards.&nbsp; 
Venezuela should guarantee the basic conditions for HRDs to perform their work freely and safely
We demand that Venezuelan authorities immediately release Javier Tarazona
and cease to criminalize the critical work of HRDs to document abuses and defend the rights of Venezuelans.&nbsp; 
Fundaredes is one of the few organizations that monitor
and report on violence and human rights abuses committed by armed groups in Venezuela’s border regions—dangerous and difficult-to-access areas where access to information is scarce
Fundaredes has been&nbsp; documenting the armed conflict between Venezuela’s armed forces and a faction of former FARC dissidents that began in March 2021 in the state of Apure
Fundaredes’ activists have consistently faced harassment by Maduro government officials for their work
such that the Inter-American Commission on Human Rights (IACHR) issued precautionary measures for Tarazona
The conflict in Apure displaced more than 5,000 Venezuelans across the border to the Colombian department of Arauca
Fundaredes has faced increasing surveillance and harassment by state security forces for documenting and reporting on grave human rights violations
Fundaredes’ documentation on the links between Maduro government officials and the Colombian guerilla groups ELN and FARC dissidents have also fed recent accusations
Venezuela’s Prosecutor General Tarek William Saab further justified the activists’ detention
arguing that Fundaredes’ reporting is baseless and undermines Venezuela’s security
Freedom in the World has been widely used by policymakers
Donate today to help us ensure the future of this vital resource.&nbsp;
Eddie Tarazona of Tarazona Cigars has come up short in his bid to be the Republican candidate for Florida&#8217;s 18th Congressional District
an area that covers a large amount of land to the west and northwest of Lake Okeechobee in central Florida
Tarazona billed himself as a conservative leader that is “pro-Constitution
pro-freedom and pro-you,” who promised to “take the fight to the establishment and stand for Florida and #AmericaFirst.”
Tarazona issued the following statement about the outcome of the primary election to his campaign&#8217;s Twitter account:
— Eddie Tarazona for Congress (FL-18) (@EddieTarazonaFL) <a href="https://twitter.com/EddieTarazonaFL/status/1562252701798473728?ref_src=twsrc%5Etfw">August 24, 2022</a>
was arbitrarily detained on 2 July 2021 after attempting to report harassment from security officers at the Attorney General’s Office in the city of Coro (Western Venezuela)
His pre-trial hearing took place on December 16
Javier Tarazona is a prisoner of conscience
having been arbitrarily detained for his human rights work
Tarazona’s health has seriously deteriorated due to lack of medical treatment
We urge the authorities to release him immediately and unconditionally
FundaREDES is a Venezuelan human rights organization that promotes and defends human rights in the bordering states of Táchira
Its work includes documentation and reporting of human rights violations and human rights abuses by non-state actors in these regions
they have focused on documenting the violence in Apure state
in what has been claimed to be a conflict with FARC non-demobilized groups
Tarazona Cigars next release draws inspiration from the Cigar City
The Tarazona La Tampeña will debut in a 6 x 52 toro size that uses a habano criollo wrapper from Jalapa
Nicaragua over a Nicaraguan binder and Nicaraguan fillers
&#8220;pays homage to the women that worked the cigar factories of Tampa during its golden years especially the women in our family that came over from Cuba to Tampa in the late 1800’s due to the cigar trade.&#8221;
It has an MSRP of $16 per cigar and will be limited to 1,000 boxes of 10 cigars
Tarazona says it will begin shipping on June 8
Tarazona says this is the first cigar in its new Discovery Series
I am an editor and co-founder of halfwheel.com/Rueda Media
I previously co-founded and published TheCigarFeed
I have written about the cigar industry for more than a decade
covering everything from product launches to regulation to M&amp;A
I handle a lot of the behind-the-scenes stuff here at halfwheel
wearing sweatshirts year-round and eating gyros
Eddie Tarazona of Tarazona Cigars has <a href="https://twitter.com/EddieTarazonaFL/status/1522994783601541121">announced</a> that he is launching a candidacy for a seat in the U.S
Tarazona has filed paperwork with the Federal Election Commission and is running as a Republican for Florida&#8217;s newly-created 18th Congressional District, an area that covers a large amount of land to the west and northwest of Lake Okeechobee in central Florida. In a <a href="https://twitter.com/EddieTarazonaFL/status/1522994783601541121">Tweet</a> announcing his candidacy
Tarazona called himself a conservative leader that is &#8220;pro-Constitution
pro-freedom and pro-you,&#8221; who promised to &#8220;take the fight to the establishment and stand for Florida and #AmericaFirst.&#8221;
He said he plans to launch his <a href="http://www.tarazonafl.com">website</a> on Monday
which will contain more details about his candidacy
Tarazona told halfwheel that he will continue to run it during his campaign
he will likely be handing off a chunk of the day-to-day duties
He said that he feels this is an opportunity to serve his country
much like he did during his time in the U.S
He added that he thinks having a person from the premium cigar industry in Congress will help the chances of pushing back against FDA regulation and other issues facing the industry
as well as provide a perspective as to how a healthy and thriving cigar industry would help the conditions in countries where cigars are produced
which in turn could help with international stability and immigration issues
Tarazona said that he is the only candidate to have entered the race for the District 18 seat
though both federal and state records still show multiple candidates for the seat
That appears to be related to a recent redistricting of Florida&#8217;s congressional districts and a lag in getting the websites of both the Federal Elections Commission and the Florida Department of State caught up with the changes
several of the candidates listed for the District 18 seat have already updated their own websites to show that they are running for other districts following that redrawing of the map
It is that redrawing that might also present another challenge for Tarazona
as legal challenges have arisen after the state&#8217;s legislature recently approved the revised map of its districts
a plan that was pushed for and signed into law by Gov
Ron DeSantis amidst a significant amount of controversy
The governor and Republican legislators have been accused of gerrymandering the districts to reduce the influence of Black voters throughout the state while making the vast majority of districts into Republican strongholds
there has been speculation that DeSantis pushed for the changes to help ensure he would win Florida&#8217;s electoral votes should he run for president in 2024
as well as moved three districts considered to be highly competitive into ones with a significant partisan lean
The net effect has been forecast at a gain of four seats in the House for the GOP
including the League of Women Voters of Florida
the Black Voters Matter Capacity Building Institute
have already filed a lawsuit in state court over the redistricting
They say the new map violates the state&#8217;s constitution as it dilutes the voting power of minorities while also being drawn to directly benefit one political party
A federal lawsuit is also expected to be filed
though the generally slow speed at which the court system moves may prevent a decision from being rendered prior to the upcoming elections
The groups have also filed a request for a temporary injunction
though that is centered around another district in northern Florida
The Florida primary election is scheduled for Aug
with the winning candidates advancing to the general election on Nov
The calendar may have just turned to September
but the newest release from Tarazona heading to stores is focused on the end of October
The cigar is the Tarazona La Bruja Broomstick
The cigar features three Ecuadorian-grown wrappers
Underneath those is a Nicaraguan binder and fillers
the latter of which come from the country&#8217;s Condega and Jalapa regions
The wrappers are cut in such a way so as to resemble a broom handle
while the exposed fillers are designed to evoke an image of a broom&#8217;s bristles
The cigar is offered in a 7 1/8 x 57 figurado vitola and comes with an MSRP of $17 cigar or $170 for a box of 10 cigars
Tarazona has a follow-up slated for the Christmas season, a 5 x 50 vitola called <a href="https://halfwheel.com/pca-2021-tarazona/396528/">Misfit Toys</a> that comes in gift-wrapped ten packs featuring art by David Angelo Roman
an artist who has done work for the Rick and Morty franchise
Tarazona released two brand new limited edition cigars that were named after Eddie Tarazona’s business partner
There have been two different sizes announced so far for the Caraballo828 Edición Limitada line so far:
Here is what I wrote in my <a href="https://halfwheel.com/caraballo828-silverback-edicion-limitada/370838/">original review</a> a little more than one year ago:
then the Tarazona Caraballo828 Silverback Edición Limitada was seemingly custom-built for your enjoyment
that aforementioned combination that this blend features so prominently comes at a cost: specifically having negative impacts on the complexity
nuance and—albeit to a lesser extent—the balance that is present in the cigars
while it shows flashes in the second third
the full strength basically waits to hit you virtually all at once in the final third
which when combined with the overwhelming earth flavors leads to a very linear profile
earth-dominant blends should seek these out immediately
but those looking for almost any other flavors will find them in short supply
As with the cigars I smoked for my first review
this Tarazona Caraballo828 Silverback Edición Limitada is covered in a dark espresso brown wrapper that is sandpaper rough to the touch and features a copious amount of oil
While there are just as many very prominent veins running up and down the length as I remember from the samples in my first review
this cigar is quite a bit more firm when squeezed than those were
The aroma from the wrapper is a combination of bitter dark chocolate
while the cold draw brings flavors of coca nibs
the Tarazona Caraballo828 Silverback Edición Limitada starts off with a very obvious combination of gritty earth and dark chocolate
along with secondary flavors of freshly brewed espresso
There is plenty of black pepper to be had on the retrohale
albeit not nearly was much as I remember from the first time around nor enough to drown out the distinct pomegranate sweetness that makes itself known early on and never lets up
The second half of the Tarazona features a touch less black pepper and spice on both the retrohale and on my tongue
Although the main flavors continue to be the same gritty earth and dark chocolate that dominated the first half
the flavors somehow seem a bit more rich on the palate
and leather flit in and out in various amounts as the cigar burns down
while the pomegranate sweetness on the retrohale increases a tad and remains at that constant level until the end of the cigar
the Silverback Edición Limitada features an excellent draw throughout after a Dickman cut
but the burn needs touching up twice: once in the first third and again at the end of the second third
Smoke production was both dense and copious for the entire cigar and the overall strength increased evenly from a very strong mild at the beginning to end a just under the full mark by the end of the cigar
When it comes to the imperfect process that makes up the art of aging cigars
blends like the Tarazona Caraballo828 Silverback Edición Limitada fascinate me
I wondered whether the earth note that dominated the profile melt together with the other flavors enough to provide more balance to the profile
While there is still quite a bit of both earth and black pepper in the profile
there is also significantly more sweetness on the retrohale and the overall profile is both more balanced and more enjoyable
I do think that some more time will probably continue the trend of increasing both the balance and complexity
the Silverback Edición Limitada is a much better cigar now than it was when it was released
I have worn many hats in my life up to this point: I started out as a photojournalist for the Dallas Morning News and the Fort Worth Star-Telegram
then transitioned to photographing weddings—both internationally and in the U.S.—for more than a decade
After realizing that there was a need for a cigar website containing better photographs and more in-depth information about each release
SmokingStogie quickly became one of the more influential cigar blogs on the internet
extremely hard-to-find and expensive cigars
and it was one of the predecessors to halfwheel
Saint Patrick&#8217;s Day is the cue for many cigar companies to release limited edition candela-wrapped cigars and this year Tarazona will join the tradition
Its newest release is the Tarazona Corned Beef &amp; Cabbage
a 6 x 52 barberpole that uses three different wrappers: Ecuadorian Candela
Ecuadorian Connecticut and Mexican San Andrés maduro
Underneath is a Nicaraguan binder and Nicaraguan filler
There will be just 600 bundles of 15 cigars made and the MSRP is set at $9 per cigar
The Tarazona Corned Beef &amp; Cabbage will be shown off at next week&#8217;s TPE 2022 trade show and begin shipping to retailers in February
will soft launch the Corned Beef &amp; Cabbage before it begins shipping to other stores
<a href="/articles/s41467-020-16937-8/metrics" data-track="click" data-track-action="view metrics" data-track-label="link" rel="nofollow">Metrics details</a>
Multi-omic studies combine measurements at different molecular levels to build comprehensive models of cellular systems
The success of a multi-omic data analysis strategy depends largely on the adoption of adequate experimental designs
and on the quality of the measurements provided by the different omic platforms
the field lacks a comparative description of performance parameters across omic technologies and a formulation for experimental design in multi-omic data scenarios
we propose a set of harmonized Figures of Merit (FoM) as quality descriptors applicable to different omic data types
we formulate the MultiPower method to estimate and assess the optimal sample size in a multi-omics experiment
MultiPower supports different experimental settings
and includes graphical for experimental design decision-making
an algorithm to estimate sample size for machine learning classification problems based on multi-omic data
the so-called multi-platform or multi-omics studies are becoming popular
the success of a multi-omic project in revealing complex molecular interconnections strongly depends on the quality of the omic measurement and on the synergy between a carefully designed experimental setup and a suitable data integration strategy
multi-omic measurements should derive from the same samples
and variance distributions similar if the planned approach for data integration relies on correlation networks
and analysis expectations are frustrated by underpowered experimental design
and the lack of a realistic integration method
these tools do not apply to non-sequencing omics (i.e.
metabolomics) and are not conceived to compare platform performance nor support multi-omic experimental design choices
Figures of Merit (FoM) are performance metrics typically used in analytical chemistry to describe devices and methods
and dynamic range; descriptors also applied to omic technologies
the definition of each FoM acquires a slightly different specification depending on the omic technology considered
and each omic platform possesses different critical FoM
comprehensive coverage of the targeted space (i.e.
while this is not the case for shotgun proteomics
which is strongly biased toward abundant proteins
we currently lack both a systematic description of FoM discrepancies across omic assays and a definition of a common performance language to support discussions on the multi-omic experimental design
the multi-omics field currently lacks a comparative description of performance metrics across omic technologies and methods to estimate the number of samples required for their multiple applications
we propose a formal definition of FoM applicable across several omics and provide a common language to describe the performance of high-throughput methods frequently combined in multi-omic studies
We leverage this harmonized quality control vocabulary to develop MultiPower
an approach for power calculations in multi-omic experiments applicable to across omics platforms and types of data
an R method to obtain the optimal sample size required by ML approaches to achieve a target classification ER
together with the MultiPower and MultiML calculations proposed here constitute a framework for quality control and precision in the design of multi-omic experiments
</a>a Calibration line for omics measuring the levels of the target features (MS platforms and gene-based sequencing platforms). b Calibration line for omics not measuring concentrations but finding genomic regions, where a biological event occurs (region-based sequencing platforms).
</a>The table summarizes critical aspects of each FoM and omic
while MS platforms are able to measure hundreds to thousands of metabolites in a single sample
Sensitivity in sequencing platforms depends on the number of reads associated with the feature
a parameter influenced by sequencing depth
Features with an elevated number of reads are more accurately measured
and hence smaller relative changes can be detected
where the goal is to identify genomic regions where a certain event occurs
the above definition is difficult to apply
and sensitivity is described in terms of true positive rate or recall
the proportion of true sites or regions identified as such
given the number of reads in the seq output
reproducibility for DNA variant calling is associated with the balance between read coverage at each genome position and the technology sequencing errors
or exclude features when repeatedly falling under the LOD
LOD is assumed to be zero and data do not contain missing values
features with few counts in many samples risk exclusion from downstream analyses
The dynamic range of an omic feature indicates the interval of true signal levels that can be measured by the platform, while the linear range represents the interval of true signal levels with a linear relationship between the measured signal value and the true signal value (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41467-020-16937-8#Fig1">1</a>)
These FoM influence the reliability of the quantification value and
as detection of the true effect size depends on the width of these ranges
linear ranges usually span 3–4 orders of magnitude
while dynamic ranges increase to 4–5 orders and can be extended using the isotopic peak of the analytes
A combination of analytical methods can increase the dynamic range
as different instruments may better capture either high or low concentration metabolites
NMR has a high dynamic range and can measure highly abundant metabolites with precision
although it is constrained by a high detection limit
the dynamic range strongly depends on sequencing depth
and values can range from zero counts to up to hundreds of thousands
linear range boundaries are difficult to establish and may require the use of calibration RNAs (spike-ins)
linear ranges may be feature dependent and affected by sequence GC content
which then requires specific normalization
while in proteomics selectivity strongly depends on sample complexity
which determines whether a given feature is detected or not
selectivity relates to competition of fragments to undergo sequencing
a highly expressed transcript or a highly accessible chromatin region) may outcompete low-abundant elements
this problem is difficult to address by means other than increasing the sequencing depth
the application of normalized libraries can partially alleviate selectivity problems; however
at the cost of compromising expression level estimations
variant detection is compromised at repetitive regions and can be alleviated with strategies that increase read length
The coverage of a platform is defined here as the proportion of detected features in the space defined by the type of biomolecule (aka feature space)
Targeted MS platforms measure a small subset of compounds with high accuracy; hence the coverage is restricted and lower than for untargeted approaches
As sample complexity is frequently much larger than the sampling capacity of current instruments
identification is limited to compounds with the highest abundances
Repeating sample measurement excluding the features identified in the first run is an efficient strategy to improve coverage
although this requires increased instrument runtime and sample amounts
Coverage in seq-based methods strongly depends on sequencing depth and can potentially reach the complete feature space associated with each library preparation protocol
capture of antisense transcripts imposes strand-specific protocols
and microRNAs require specific small RNA protocols
coverage also relates to the applied protocol
Whole-genome bisulfite sequencing has greater genome-wide coverage of CpGs when compared to Reduced Representation by Bisulfite Sequencing (RRBS)
while RRBS and MeDIP provide greater coverage at CpG islands
region-based sequencing approaches can cover the whole reference genome
with coverage depending on the efficiency of the protocol employed to enrich the targeted regions
</a>a Relationship between omic platforms (MS in blue and sequencing in red)
MS- and seq-based omics have different key properties that determine their FoM
which in turn affect differently to power parameters used as input by MultiPower to compute sample size in multi-omics experiments
MultiPower works either with no prior data and with pilot data (red boxes)
The blue box contains the parameters to be set by users
and purple boxes represent the data types accepted by MultiPower and the rest of power parameters estimated by MultiPower from prior data when available or given by the user
MultiPower solves an optimization problem to estimate the optimal sample size and to provide a power study of the experiment (gray boxes)
Each color represents a different omic data type
b Expected percentage of differentially expressed (DE) features for each omic
c Pooled standard deviation (PSD) per gene and per omic for the estimated DE features (pseudo-DE features
Boxplots represent the median and interquartile range (IQR)
Whiskers depict the minimum and maximum of data without outliers
which are the values outside the interval (Q1 − 1.5 × IQR
d–g MultiPower results with parameters same sample size in all the omics
d Statistical power curves for each omic for sample sizes between 2 and 35
Squared dots indicate power at the optimal sample size (n = 16)
e Statistical power curves for each omic when considering different percentiles of PSD
Squared dots indicate power for the dispersion used in power calculations for each omic (75th percentile)
f Curve relating the initial Cohen’s d to the optimal sample size needed to detect each magnitude of change
the red arrows and text highlight the magnitude of change to be detected (Cohen’s d = 1.98 in this case)
g Statistical power per omic using the optimal sample size (n = 16) with Cohen’s d = 0.8
and the maximum sample size allowed by the user (n = 4) with Cohen’s d = 1.98
</a>a Given a multi-omic dataset with O different omic types and NO samples per omic and a classification vector Y
MultiML obtains the maximum number of common observations Nmax (Nmax ≤ NO) for the combination of omic types specified by the user
MultiML then creates subdatasets (ticks) having different number of observations (nt) from the available Nmax and obtains the variables that best explain each tick through LASSO regression
Taking provided machine learning algorithm ML and cross-validation method CV
the selected variables at each tick are used to predict the classification error rate (ER) on a different random subdatasets of size nt
This variable selection-ER prediction process is repeated through m iterations to obtain an average ER and confidence interval for each tick
a first-order-smoothed penalized P-spline regression is adjusted to estimate the learning curve that will allow the prediction of classification ERs for sample sizes larger than Nmax
b–d MultiML results on the TCGA Glioblastoma data
b Predicted sample size for different omics combinations using a target ER of 0.01
Margin of error is given as number of samples
c Example of MultiML graphical output with the predictive P-spline for a combination of four omics types
An increasing number of observations and omic types were run as input data in MultiML having as ERtarget
the ER achieved with complete data for four omics (69 observations)
their ER are recovered from the actual data and their deviation from ERtarget is calculated as ΔError
The ΔError is plotted against the size of the input data
Accuracy in ER predictions increases with the size of input data and the number of omics
where ERs rapidly decrease as the number of samples increases to reach a stable classification performance
This graph can be used to calculate the number of samples required at different ER levels
We concluded that the sample size could be accurately predicted when input data represents ~40–60% of the required sample size
the field has yet to address aspects that are essential to understand the complexity of multi-omic analysis
such as the definition of performance parameters across omic technologies and the formulation of an experimental design strategy in multi-omic data scenarios
we addressed both issues by proposing FoM as a language to compare omic platforms
and by providing algorithms to estimate sample sizes in multi-omic experiments aiming at differential features analysis (MultiPower) or at sample classification using ML (MultiML)
FoM have traditionally been used in analytical chemistry to describe the performance of instruments and methods
These terms can also be intuitively applied to sequencing platforms
but we noticed that the meaning and relevance of FoM slightly differ for both types of technologies
we explain FoM definitions across omic platforms and discuss which of these metrics are critical to each data type
and identification are FoM with critical influence on the number of features comprising the omics dataset
which in turn affects the power of the technology to identify features with true signal changes
Power diminishes as the number of features increases due to the application of multiple testing corrections to control false positives
Reproducibility and dynamic range may also be very different across platforms
and these have a direct impact on the within-condition variability and across-condition differences of the study
while sequencing depth critically affects many of the described FoM of sequencing platforms
the choice of a targeted or untargeted method strongly influences FoM values
The number of features detected by the omic platform together with the different measurement variabilities across features and the magnitude of change to be detected
represent major components of power calculations for omics data
The highly heterogeneous nature of these factors across omics platforms calls for specific methods for power calculations in multi-omic experiments
MultiPower solves the optimization problem of obtaining the sample size that minimizes the cost of the multi-omic experiment
while ensuring both a required power per omic and a global power
MultiPower indicates the sample size required to detect a targeted effect size given a significance threshold
MultiPower graphically represents the relationship between sample size
and facilitates exploration of alternative experimental design choices
Estimates for MultiPower parameters are optimally calculated from pilot data
although they can also be manually provided
count and binary data to facilitate the integration of omic technologies of different analytical nature
Data should have been properly preprocessed and eventual batch effects
we showcase MultiPower functionalities using sequencing
although the method could be applied to other technologies
the optimal sample size can be computed under two different requirements: an equal sample size for all platforms ensuring a common minimal power
or different sample size per omic to achieve the same power
This is relevant for the choice of downstream statistical analysis
Methods that rely on co-variance analysis typically require uniform sample sizes and MultiPower will provide this while revealing the differences in power across data modalities
Methods that combine data based on effect estimates allow sample size differences and can benefit from the equally powered effect estimation
We illustrate the MultiPower method in three scenarios
where different parameterizations are assessed and include both controlled laboratory experiments and cohort data to highlight the general applicability of the method
By discussing interpretations of power plots and the factors that contribute to sample size results
we provide a means to make informed decisions on experimental design and to control the quality of their integrative analysis
The MultiPower approach is not directly applicable to ML methods used for sample classification
as in this case the basic parameters of the power calculation—significance threshold and effect size—are not applicable
sample size estimation is relevant as multi-omic approaches are frequently used to build classifiers of biological samples
feature relationships within the multivariate space are instrumental in ML algorithms
The MultiML strategy calculates samples size for multi-omic applications
where the classification ER can be used as a measure of performance
MultiML is itself a learning algorithm that learns the relationship between sample size and classification error
and uses this to estimate the number of observations required to achieve the desired classification performance
MultiML has been designed to be flexible for the ML algorithm and to evaluate multiple combinations of omics types in order to identify optimized multi-omic predictors
a uniform description of performance parameters across omic technologies
and offers computational tools to calculate power and sample size for the diversity of multi-omics applications
We anticipate MultiPower and MultiML will be useful resources to boost powered multi-omic studies by the genomics community
which we broadly classified into two groups: quantitative or analytical FoM include sensitivity
To describe how they apply to omic technologies
we distinguish between MS and seq-based platforms
we also consider two subgroups: “feature-based” and “region-based”
a genome annotation file defines the target features to be quantified
the definition of the target feature to be measured (usually genomics regions) is part of the data analysis process
Applications of RNA-seq to annotate genomes could be considered a region-based assay
We consider here omic assays with a dynamic component regarding the genotype
while analytical FoM are defined at the feature level
omic platforms by nature measure many features simultaneously and consequently
the FoM may not be uniform for all of them
we consider FoM globally and discuss how technological or experimental factors affect the FoM of different ranges of features within the same platform
The MultiPower R method (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41467-020-16937-8#Fig3">3b</a>) performs a joint power study that minimizes the cost of a multi-omics experiment
while requiring both a minimum power for each omic and an average power for all omics
MultiPower calculations are defined for a two groups contrast and implemented in the R package to support the application of the method to single and multiple pairwise comparisons
The parameters required to compute power can be estimated from multi-omic available data (pilot data or data from previous studies) or
The method considers multiple testing corrections by adjusting the significance level to achieve the indicated FDR
MultiPower accepts normally distributed data
and optimal sample size (number of replicates or observations per condition) can be computed either requiring the same or allowing different sample sizes for each omic
the monetary cost is considered as an additional parameter in the power maximization problem
MultiPower can be used to both design a new multi-omic experiment and to assess if an already generated multi-omic dataset provides enough power for statistical analysis
MultiPower minimizes the total cost of the multi-omics experiment while ensuring a minimum power per omic (Pi) and a minimum average power for the whole experiment (A). Equation (<a data-track="click" data-track-label="link" data-track-action="equation anchor" href="/articles/s41467-020-16937-8#Equ1">1</a>) describes the optimization problem to be solved to estimate the optimal number of biological replicates for each omic (xi):
ci is the cost of generating a replicate for omic i
The effect size is calculated with the fold change (ωi) between both groups as well as the average counts (μi)
For binary data with 0/1 or TRUE/FALSE values indicating
or if a transcription factor is bound or not
the goal is comparing the percentages of 1 or TRUE values between two populations
the parameters needed to estimate power are related to the difference between proportions to be detected (the effect size) and the sample size
where m is the number of features in a particular omic
r1 is the expected number of true detections
an assorted range of experimental designs can be found
All omic assays may be obtained on the same biological samples or individuals
which would result in identical replicates number for all data types
this is not always possible due to restrictions in cost or biological material
sample size differs among omic types and yet the data are to be analyzed in an integrative fashion
MultiPower contemplates these two scenarios
Under the first scenario, adding the constraint of equal sample size for all omics (xi = x for all I = 1, …, I) to the optimization problem in Eq. (<a data-track="click" data-track-label="link" data-track-action="equation anchor" href="/articles/s41467-020-16937-8#Equ1">1</a>) results in a straightforward solution
the minimum sample size required to meet the constraint on the minimum power per omic (xi) is calculated and the initial estimation of x is set to x = maxI{xi}
the second constraint on the average power is evaluated for x
x is increased until the constraint is met
the cost per replicate does not influence the optimal solution xopt
Under the second scenario, allowing different sample sizes for each omic, the optimization problem in Eq. (<a data-track="click" data-track-label="link" data-track-action="equation anchor" href="/articles/s41467-020-16937-8#Equ1">1</a>) becomes a nonlinear integer programming problem
as the statistical power is a nonlinear function of xi
The optimization problem can be transformed into a 0–1 linear integer programming problem by defining the auxiliary variables \(z_n^i\) for each omic i and each possible sample size n from 2 to a fixed maximum value nimax
where \(z_n^i = 1\) when the sample size for omic i is n
The new linear integer programming problem can be formulated as follows:
where fi(n) is the power for sample size n
The MultiPower R package implements the described method together with several functionalities to support both the selection of input parameters required by the method and the subsequent interpretation of the results. The R package and user’s manual are freely available at <a href="https://github.com/ConesaLab/MultiPower">https://github.com/ConesaLab/MultiPower</a>
The MultiPower package requires different parameters to compute the optimal sample size
As the choice of parameters can be challenging
MultiPower can estimate them from pilot or similar existing data
The multi-omic pilot data must have two groups and at least two replicates per group
The algorithm assumes data are already preprocessed
Both normally distributed and count data are accepted
We recommend raw count data to be provided for sequencing technologies as MultiPower deals with the sequencing depth bias
when count data contains other sources of technical noise
we recommend previous transformations to meet normality (e.g.
with log or voom transformation) and indicating MultiPower that data are normally distributed
We also recommend removing low count features from sequencing data
these must be removed or imputed before running MultiPower
the power.prop.test() function from stats R package is used
MultiPower transforms parameters provided by the user to fit specific arguments required by these functions
in such a way that magnitudes are maintained roughly comparable for all data types
MultiPower computes the Cohen’s d value for all omic features and applies this value to estimate the set M1 of DE features (those with d &gt; d0)
We recommend setting the same Cohen’s d initial value (d0) for all the omics although MultiPower allows different values for each one of them
The equivalent to Cohen’s d when comparing proportions in groups A and B is Cohen’s h, which can be defined as h = |φA − φB|, where φi = 2 arcsin √pi, and pi is the proportion of 1 or TRUE values in group i. Classification in Supplementary Table <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41467-020-16937-8#MOESM1">7</a> is also valid for Cohen’s h
the fold change of pseudo-DE features (ω) is estimated as the percentile P100−k of the fold changes corresponding to pseudo-DE features with a PSD between percentiles Pk−5 and Pk+5
the proportions chosen to estimate power correspond to the P100−k of pseudo-DE features for Cohen’s d
and are stored in the delta output parameter of MultiPower
As variability is not considered for this data type
dispersion power plots are not generated in this case
Once the power parameters are obtained for each omic and the user sets the minimum power per omic and the average power for the experiment, the optimization problem in Eq. (<a data-track="click" data-track-label="link" data-track-action="equation anchor" href="/articles/s41467-020-16937-8#Equ4">4</a>) is completely defined and MultiPower makes use of lpmodeler and Rsymphony R packages to solve it
Note that the application of these packages to solve the problem is only needed when the number of replicates for each omic differs
MultiPower returns a summary table with the provided and/or estimated power parameters
While parameter estimation from previous data is recommended for MultiPower analysis
MultiPower requires parameters to be provided by users
we provide recommendations for critical MultiPower parameters
The average value for the standard deviation per omic feature and condition partially depends on the reproducibility of omics technology
Overestimating this parameter guarantees that the sample size fits the power needed but may lead to too large sample sizes
a good value for the standard deviation is 1
Value for the expected proportion of DE features per omic should be set according to results seen in similar studies
A high percentage is expected for cell differentiation processes or diseases like cancer
while small perturbations or other types of diseases may induce fewer changes
Typical values for the minimum fold change between conditions and the mean of counts for the DE features when using count data are 2 and 30
but again these values depend on the sequencing depth of the experiment and the magnitude of the expected molecular changes
After obtaining the optimal sample size with MultiPower
especially when this sample size exceeds the available budget for the experiment:
How much reduction of the sample size can we afford without losing too much power
If power cannot be decreased but the sample size has to be reduced
how will this reduction influence the effect size to be detected
Can we remove any omic platform with negligible changes between conditions
since this platform imposes a too large optimal sample size
To provide answers to these and similar questions, MultiPower returns several diagnostic plots (see Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41467-020-16937-8#Fig4">4d–g</a> for instance)
sample size plot shows variations in power as a function of the sample size
dispersion plot also displays the power curves but for different dispersion values (PSD in normal data or ϕ parameter in count data)
the power for the estimated optimal sample size or the fixed dispersion value is represented by a square dot
If the optimal sample size estimated by MultiPower exceeds the available budget
researchers may opt for increasing the effect size (given by the Cohen’s d) to be detected and allowing a smaller sample size without modifying the required power
the postMultiPower() function can be applied
which computes the optimal sample size for different values of Cohen’s d
from the initial value set by the user to dmax = mini in I{P90i(d)}
where P90i(d) is the 90th percentile of the Cohen’s d values for all the features in omic i
This choice ensures sufficient pseudo-DE features to estimate the rest of the parameters needed to compute power
Although MultiPower algorithm is essentially defined for a two groups comparison
the MultiPower R package supports experimental designs with multiple groups
Assuming that a pilot dataset is available
the MultiGroupPower() function automatically performs all the possible pairwise comparisons (or those comparisons indicated by the user) and returns both a summary of the power and optimal sample size for each comparison
and a numerical and graphical global summary for all the comparisons
the global optimal sample size is computed as the maximum of optimal sizes obtained for the individual comparisons
the solution given by MultiPower method does not allow a different sample size for each group
Users must be aware that different optimal sample size could be obtained in this case
each omics data matrix is reduced to its significant variables and analysis is performed maintaining a three-way N × P × O structure
and O are the different omics in the study
returning either an overall classification ER or a balanced ER calculated on the left-out samples
only ER is available as both methods give similar results
MultiML also provides different prediction distances for PLS-DA and RF used to assign a category to samples
These prediction distances can be defined as a model with H components
Given NnewInds new individuals and their corresponding omic data matrix Xnewinds
the predicted response variable Ŷnewinds can be computed as follows:
where W is a p (variables) × H matrix containing the loading vectors associated with X; D is a p × H matrix containing the regression coefficients of X on its H latent components; and B is an H × n (individuals) matrix containing the regression coefficients of Y on the H latent components associated to X
The predicted scores (Tpred) are computed as:
The degrees of freedom of this model are the number of observation subsets evaluated (ticks) minus 1
The model is used to predict the sample size required to obtain a given classification error
MultiML is a computationally intensive algorithm
The function RequiredTimeTest() allows users to estimate the time required to run the full predictive model at the local installation
For users who can benefit from parallelization options
we have implemented the slurm_creator() R function that creates a
sh script to run MultiML calculations in a SLURM cluster
The function Previous_CER() allows the utilization of a previous MultiML result with N samples in a new MultiML calculation that expands the size of the prior dataset by M samples
thereby significantly accelerating the calculation of the new model
The plot also includes the fitted penalized smooth spline model to graphically obtain the sample size for an ERtarget not achievable with the pilot dataset
The user can also create a comparative plot of all omic combinations to determine the best contributing data modality to an accurate classification by using Comparative_ERPlot() function
Further information on research design is available in the <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41467-020-16937-8#MOESM2">Nature Research Reporting Summary</a> linked to this article
The MultiPower and MultiML methods are available at GitHub repository <a href="https://github.com/ConesaLab/MultiPower">https://github.com/ConesaLab/MultiPower</a>
Strategies for integrated analysis of genetic
and gene expression variation in cancer: addressing the challenges
DNA accessibility and gene expression data to build regulatory maps in an organism
A multivariate analysis approach to the integration of proteomic and gene expression data
A multiway approach to data integration in systems biology based on Tucker3 and N-PLS
A survey of best practices for RNA-seq data analysis
ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia
esATAC: an easy-to-use systematic pipeline for ATAC-seq data analysis
SAAP-RRBS: streamlined analysis and annotation pipeline for reduced representation bisulfite sequencing
Using MetaboAnalyst 3.0 for comprehensive metabolomics data analysis
Galaxy-M: a Galaxy workflow for processing and analyzing direct infusion and liquid chromatography mass spectrometry-based metabolomics data
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This work has been funded by FP7 STATegra project agreement 306000 and Spanish MINECO grant BIO2012–40244
work in the Imhof lab has been funded by the (DFG; CIPSM and SFB1064)
has been funded by the University of Florida Startup funds
Institute for Food and Agricultural Research
Munich Center of Integrated Protein Science LMU Munich
Biological and Environmental Sciences and Engineering Division
Electrical and Mathematical Sciences and Engineering Division
King Abdullah University of Science and Technology
North-West University (Potchefstroom Campus)
and drafted the manuscript; L.B.-N.: implemented and tested the MultiML method
and drafted the manuscript; D.G.-C: contributed to FOM analysis
and MultiML methods; A.S.: contributed to FOM analysis of proteomics data; A.I.: contributed to FOM analysis of proteomics data; T.H.: discussed FOM analysis of metabolomics data; J.T.: supervised parts of the work; J.A.W.: contributed to FOM definition and analysis; and A.C.: supervised
All authors contributed to and approved the final version of the manuscript
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Volume 7 - 2020 | <a class="ArticleLayoutHeader__info__doi" href="https://doi.org/10.3389/fmars.2020.00569">https://doi.org/10.3389/fmars.2020.00569</a>
This article is part of the Research TopicChemically Mediated Interactions Between Marine Macrophytes and Microbes<a class="Link Link--linkType Link--maincolor Link--medium Link--icon Link--chevronRight Link--right" aria-label="View all 9 articles" href="https://www.frontiersin.org/research-topics/9468/chemically-mediated-interactions-between-marine-macrophytes-and-microbes/magazine" target="_self" data-event="customLink-linkType-a_viewAll9Articles">View all 9 articles</a>
The dinoflagellate genus Prorocentrum is globally represented by a wide variety of species found upon benthic and/or epiphytic substrates
Many epibenthic Prorocentrum species produce lipophilic polyether toxins
some of which act as potent protein phosphatase inhibitors and tumor-promoters associated with Diarrheic Shellfish Poisoning (DSP)
Most members of the Prorocentrum lima species complex (PLSC) commonly found in the tropics and sub-tropics are toxigenic
Epiphytic and planktonic bacteria co-occur with toxigenic Prorocentrum but reciprocal allelochemical interactions are under-investigated
The aim of the present study was to identify the culturable bacteria collected together with isolates of the PLSC from seagrass (Thalassia testudinum) and macroalgae along tropical Atlantic coasts of Mexico
and to explore potential species interactions with selected isolates
Twenty-one bacterial genera belonging to Proteobacteria
and Bacteroidetes were identified by amplification of the 16S rRNA gene marker from nine clonal Prorocentrum cultures
with &#x03B3;-proteobacteria comprising the dominant class
A positive correlation was found between the bacterial genera associated with two Prorocentrum clones and the esterified toxin analog DTX1a-D8
but there was no apparent correlation between the other PLSC clones and their associated bacteria with the other five DSP toxins detected
No bacteriostatic or allelochemical response was found for cell- and culture medium extracts of five Prorocentrum isolates assayed for bioactivity against Staphylococcus sp
was only effective in reducing bacterial load in the initial growth stages
but did not yield axenic cultures or lower bacterial cell densities throughout the culture cycle
Antibiotic treatment did not impair growth or survival of the dinoflagellate
There was no significant correlation between Prorocentrum cell volume
or cellular toxin concentration over the entire time-series culture cycle
Benthic Prorocentrum and associated bacterial communities comprise highly diverse and characteristic microbiomes upon substrates
but this study provides little evidence that allelochemical interactions among Prorocentrum cells and associated bacteria originating from epibenthic substrates play a definable role in growth and toxigenicity
The extent to which associated bacteria affect growth and polyketide toxin production by epibenthic Prorocentrum in natural microbiomes or in monoclonal cultures remains a controversial issue
the current study aimed to determine the composition and diversity of the bacterial flora associated with unialgal cultures of the PLSC from natural populations and different substrates
from seagrass (Thalassia testudinum) and macroalgae from tropical Mexican reef systems
The second objective was to identify putative effects of associated bacteria on growth and cellular toxin content and composition throughout a culture cycle under controlled environmental conditions
and to evaluate potential allelochemical interactions between Prorocentrum and bacteria in culture
was prepared from heat-sterilized seawater stock at salinity 36
Clonal isolates were cultured by incubation at 25 &#x00B1; 1&#x00B0;C on a 12:12 h light:dark cycle and illumination of 50 &#x03BC;mol photons m&#x2013;2 s&#x2013;1
Geographical origin of the monoclonal isolates of the Prorocentrum lima species complex (PLSC) obtained for this study
was initially brought into culture and subjected to preliminary analysis
including of the associated bacterial community
the culture soon exhibited heavy contamination by cyanobacteria
and hence was excluded from further analysis of cell morphology
Cultures were maintained at 24 &#x00B1; 1&#x00B0;C on a light:dark cycle of 14:10 h and illumination of 86 &#x03BC;mol photons m&#x2013;2 s&#x2013;1 measured with a LI-1000 light sensor (DataLogger LI-COR
Specimens of Prorocentrum isolates from stable cultures were analyzed in detail by light and scanning electronic microscopy (SEM) for morphological characteristics. Cells stained with 0.2% Calcofluor White M2R in aqueous solution (<a href="#B20">Fritz and Triemer, 1985</a>) were studied under epifluorescence microscopy (Axio Scope.A1
Germany) with filter set 18 shift free EX BP 390-420 (excitation)
Light micrographs were taken with an Axiocam 506 color digital camera (Zeiss
Cultured dinoflagellate cells for SEM were fixed with 2% glutaraldehyde for 90 min
Specimens were washed in 1.5 mL distilled water (5&#x00B0;C) and centrifuged at 1200 &#x00D7; g at 5&#x00B0;C for 6 min
The wash procedure was repeated four times
Samples (1.5 mL each) underwent a graded ethanol dehydration series (10
with centrifugation as for the sample wash at each dehydration step
a drop (250 &#x03BC;L) of hexamethyldisilazane (HDMS) air-drying agent was placed upon the sample on aluminum SEM stubs
and specimens were gold sputter-coated for 5 min
Bacteria for SEM analysis were inoculated into marine broth (Difco
United States) and incubated for 6 days under the previously referred conditions
Each sample (1.5 mL) was fixed with 100 &#x03BC;L of 25% glutaraldehyde for 45 min
Specimens then were washed and dehydrated as described for Prorocentrum cells
1 mL of HDMS:ethanol 1:1 was added to the microtube for 3 min
1 mL of pure HDMS was added for 3 min; 10 &#x03BC;L of sample was pipetted onto round coverslips and gold sputter-coated as above
Specimens of dinoflagellates and bacteria were observed with a JEOL JSM6360LV electron microscope equipped with a backscattered electron detector under 8 kV voltage acceleration and at 15 mm working distance
Sequence reactions were prepared with big dye (v3.1) (Applied Biosystems
United States) following manufacturer&#x2019;s instructions
and processed in a 3730xl DNA Analyzer (Applied Biosystems-Hitachi
Sequences were deposited in GenBank with the accession numbers MN853604&#x2013;MN853648
The software R (<a href="#B59">R Core Team, 2018</a>) was used to obtain the rarefaction curves to identify the sampling effort of bacterial genera among the isolates
A non-metric multidimensional scaling (NMDS) analysis
an indirect gradient approach for producing an ordination plot based upon a distance matrix
a Bray-Curtis similarity matrix was plotted to detect differences in the distribution of bacterial genera associated with the Prorocentrum isolates
excluding Pseudomonas and Microbacterium due to their cosmopolitan character
The identified toxins from each isolate were transformed and then fitted onto the NMDS ordination plot
Permutation tests (n = 1000) were used to test the significance of vector fits
and only significant vectors were depicted (p &#x003C; 0.1)
Dense actively growing Prorocentrum cultures
cultivated in plastic Petri plates under the conditions detailed in Section &#x201C;Sample Collection and Initial Dinoflagellate Culture,&#x201D; were harvested with a 1 mL micropipette after sub-sampling 2 mL of stirred culture for cell counts in a Sedgewick Rafter cell-counting chamber under a light microscope (Olympus BH2
The remaining cells were transferred into 2 mL cryotubes and centrifuged at 5000 &#x00D7; g at 4&#x00B0;C for 5 min
The cell pellets were resuspended in 0.2 &#x03BC;m-filter-sterilized seawater (5&#x00B0;C) and centrifuged again under the same conditions
The supernatant was again decanted and the cryotubes were placed in a thermomixer at 100&#x00B0;C for 5 min to inactivate esterase enzymes that could modify the toxin profile
Cell pellets were stored frozen (&#x2212;20&#x00B0;C) for later extraction
Cell pellets were resuspended in 500 &#x03BC;L 50% methanol in FastPrep tubes
After adding 0.9 g FastPrep lysing matrix D (Thermo Savant
cells were homogenized by reciprocal shaking in a FastPrep FP120 (Bio 101
Homogenized samples were centrifuged at 16,000 &#x00D7; g for 15 min at 11&#x00B0;C
The supernatant from each sample was transferred into a 0.45 &#x03BC;m pore-size spin-filter (Millipore Ultrafree
Germany) and centrifuged at 11,000 &#x00D7; g for 1.5 min at 11&#x00B0;C
filtrates were transferred into 2 mL LC-autosampler vials (Agilent
Germany) for analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)
The analytical system consisted of an SCIEX- 4000 Q-Trap (Sciex
triple quadrupole mass spetrometer equipped with a TurboSpray&#x00AE; interface coupled to an 1100 liquid chromatograph (LC) (Agilent
The LC equipment included a solvent reservoir
and a temperature-controlled column oven (G1316A)
Separation of toxins was achieved following previous protocols (<a href="#B30">Krock et al., 2008</a>; <a href="#B49">Nielsen et al., 2013</a>) after injection of 5 &#x03BC;L extract onto a C8 analytical column packed with 3 mm HypercloneTM 3 &#x03BC;m BDS C8 130 &#x00C5; 50 &#x00D7; 2 mm (Phenomenex
The flow rate was 0.2 mL min&#x2013;1 and gradient elution was performed by eluents A (water
Initial conditions were 12 min column equilibration with 5% B
followed by a linear gradient to 100% B in 10 min and isocratic elution until 16 min with 100% B
The program was then returned to initial conditions until 19 min (total run time: 31 min)
Analytical standards of OA (1 ng &#x03BC;L&#x2013;1)
and DTX 2 (DTX2) (500 pg &#x03BC;L&#x2013;1) from the Institute for Marine Biosciences
were used to identify and quantify DSP toxins in extracts of Prorocentrum cells
Due to lack of standards for derivatives such as OA diol-ester (OA-D8) and DTX 1-diol ester (DTX1-D8)
cell quotas were expressed as OA and DTX1 equivalents
considering the following detection limits for OA (47 pg &#x03BC;L&#x2013;1)
Data acquisition and processing were performed with the Analyst Software (Version 1.5
were selected for larger-volume batch cultures based on relative toxin composition
These isolates were scaled-up into 2.8 L glass Fernbach flasks and harvested (total volume = 1 L) in active growth phase by centrifugation in 45 mL aliquots at 1600 &#x00D7; g at 10&#x00B0;C for 10 min (Eppendorf 5810R
Cell pellets were combined for each isolate into a single tube and freeze-dried
The dried pellets were sequentially extracted with hexane
and ethanol:water (1:1) after ultrasonication (Sonorex Digitec
After the pellet had precipitated at each step
the supernatant was transferred into a new scintillation vial and 2 mL of the next solvent was added
The supernatant was rotary-evaporated (Laborota 4002
Crude residues were dissolved in their respective solvent (&#x223C;500 &#x03BC;L) and stored at &#x2212;20&#x00B0;C prior to disk application
The pooled supernatants for each isolate were passed through a solid phase extraction (SPE) cartridge (Bond Elut PPL
The column was first conditioned with methanol
the column was washed with Milli Q deionized water to remove unbound compounds and then eluted with 1 mL methanol to release target molecules
Cell pellet extracts and SPE-concentrated supernatants were tested by disk diffusion assay (<a href="#B4">Bauer et al., 1966</a>) for their effect against bacterial growth
and methanol] was evaporated on sterile 8 mm paper (two replicates per extract) for &#x223C;30 min at room temperature
Cell equivalents were calculated based on the number of harvested cells and the extract volume applied in the assay (PA1: 382.5
Aliquots (60 &#x03BC;L) of overnight-grown (&#x223C;108 cells mL&#x2013;1) bacterial target strains Staphylococcus sp
HEL66 were spread over the surface of marine agar plates (Difco
United States) with a sterile glass spreader
The dried filters containing the extracts were placed symmetrically upon the agar surface (four filters per plate)
Paper disks with each solvent served as controls
The agar plates were incubated at 20&#x00B0;C for 24 h and the inhibition zones recorded
The final 2 L culture (cell concentration: 4.84 &#x00D7; 105 cells L&#x2013;1) was retained untreated by either cell washing or antibiotics and served as a growth control of the initial conditions from the bulk culture
The treated and control cultures were incubated in 5 L glass bottles for acclimation under the same conditions detailed in Section &#x201C;Sample Collection and Initial Dinoflagellate Culture.&#x201D; After 4 days
the content from each bottle was distributed into 150 &#x00D7; 12 mm plastic Petri dishes after stirring for 20 min for homogenization to yield approximately equal cell numbers among the plates
Replicate (n = 5) cultures from each treatment and control were harvested every 3&#x2013;5 days from Day 0 to 35
The final harvest was extended to the beginning of senescence phase
but only two treated replicates were evaluated at Day 47 and no control sample was considered at this point
An aliquot of 1.5 mL was pipetted from a well-mixed sample for Prorocentrum cell counts at each time-point for each replicate. Cells were immediately fixed with acidic Lugol&#x2019;s iodine solution and counted in a Sedgewick-Rafter chamber with an inverted microscope (AxioVert.A1, Carl Zeiss, Oberkochen, Germany) at 100X magnification (according to <a href="#B60">Reguera et al., 2016</a>)
Prorocentrum cell size was measured by the software Capture Express with an HDMI 16MDPX camera (DeltaPix
Denmark) and volume calculated by the following equation:
Associated bacteria in the well-mixed culture aliquots were fixed with 0.25 mL 20% phosphate-buffered saline (PBS)-formaldehyde and prepared for counting following the fluorescence method of <a href="#B57">Porter and Feig (1980)</a>
The bacterial cells were stained by adding 0.5 mL of preserved sample to 5 mL sterilized water and 25 &#x03BC;L of 4,6-diamidino-2-phenylindole (DAPI
with incubation (20 min) at 4&#x00B0;C in the dark
0.2 &#x03BC;m pore size black polycarbonate Nucleopore Track-Etch filter (Sigma&#x2013;Aldrich
Germany) mounted over a 25 mm Whatman GF/C glass microfiber filter (Sigma&#x2013;Aldrich
Filters were placed on a glass slide and the bacteria counted by epifluorescence microscopy (Axioskop 2 plus
Statistical analysis was performed with the software Infostat V.2018 (<a href="#B14">Di Rienzo et al., 2018</a>) with inclusion of mean values and standard deviations
An analysis of variance (two-way ANOVA) was followed by a post hoc Tukey&#x2019;s HSD test for mutually significant differences in interactions among multiple variables; namely
and toxin content among the treated (washed cells plus antibiotics and washed cells) and non-treated cultures
all these variables were analyzed and compared at each timepoint throughout the 47 day growth experiment
All data complied with the assumptions of normality and homoscedasticity
Pearson&#x2019;s correlation coefficient (r) was determined to establish a linear correlation among the same variables
Morphometric characteristics (n = 20) of cultured cells of the Prorocentrum lima species complex (PLSC) from the Gulf of Mexico and Caribbean coasts of Mexico observed by epifluorescence microscopy after staining with Calcofluor White M2R
Scanning electron microscopy of cultured Prorocentrum showed some dinoflagellate cells with attached bacteria, but most bacteria appeared as free-living coccoid or rod-like forms (sometimes filaments) often clumped in mucilaginous aggregates in the culture medium (<a href="#F1">Figure 1</a>)
Bacteria attached to Prorocentrum cells: (a) PA3; (b) PA19
and isolated from cell aggregates versus culture medium: (c) Thalassospira sp
isolated exclusively from cells belonging to PA11; (d) Kocuria sp
isolated exclusively from the culture medium of PA11
The phylogenetic tree of bacteria isolated from nine Prorocentrum cell cultures shows the phylogenetic association pattern among four dominant classes: Actinobacteria, Flavobacteriia, &#x03B1;-Proteobacteria, and &#x03B3;-Proteobacteria (<a href="#F2">Figure 2</a>)
Class &#x03B3;-Proteobacteria was the most abundant
with 11 genera within the orders Alteromonadales
Class &#x03B1;-Proteobacteria comprised five genera
whereas Actinobacteria presented four genera
Genus Euzebyella was the only representative of the class Flavobacteriia
Prorocentrum isolate PA17 showed the highest number of bacterial genera (13 of 21 identified genera)
whereas PA1 exhibited the lowest abundance
Neighbor-joining phylogenetic tree of bacterial isolates from Prorocentrum cell cultures from the Veracruz Reef System and the Mexican Caribbean coast
The tree is constructed from 16S rRNA sequences with classification according to the Ribosomal Database Project
and with 10,000 bootstrap repetitions (blue dots)
The tree scale indicates the evolutionary distance among the isolates
The branch color denotes the corresponding bacterial class: Actinobacteria (blue); Flavobacteriia (orange); &#x03B1;-Proteobacteria (red); &#x03B3;-Proteobacteria (green)
the pie charts show the proportion of colonies of each isolate associated with Prorocentrum cell aggregates (D) versus the surrounding culture medium (CM)
whereas the colored stacked histogram bars (non-cumulative) indicate the number of colonies of each bacterial species (isolate) found in the nine Prorocentrum cultures
Abundance of cultured bacterial genera/species (A) and classes (B) from dinoflagellate cell-aggregates (D) and culture medium (CM)
Prorocentrum isolates PA19 and PA26 were clustered mainly by the abundance of Halomonas and Marinobacter
while PA2 and PA12 were clustered by Marispirillum and Sphingobium
Isolates PA11 and PA17 were clustered primarily by the high abundance of Alteromonas
which showed a positive correlation with DTX1a-D8
Other Prorocentrum clones and their associated bacterial isolates were not significantly correlated with the identified DSP toxins
Bacterial genera isolated exclusively from specific Prorocentrum isolates and culture compartments: Prorocentrum cell aggregates versus surrounding medium
NMDS plot based upon a Bray&#x2013;Curtis matrix of the distribution of clones of the Prorocentrum lima species complex (PLSC) in relation to the abundance of their associated isolated bacterial genera and DSP toxin composition in culture
The scale of the axes is arbitrary as is the orientation of the plot
with closeness of the clustered elements indicating the degree of similarity and association
The symbol shape denotes the bacterial class; color indicates genera; and size denotes abundance
Vector length indicates magnitude and correlation of the toxin with the Prorocentrum clones and their isolated associated bacteria
Relative toxin composition (%total concentration) of DSP toxins (OA and DTX analogs) from cultured benthic Prorocentrum isolates from the Veracruz Reef System and the Mexican Caribbean coast
The agar disk diffusion assay showed no inhibition zones nor apparent changes in growth of bacteria near the disk area for either Prorocentrum cell pellet extracts or supernatants
Solvent controls were similarly negative for growth effects
The first assay series did show an apparent reduction in the number of colonies of Staphylococcus sp
around the disks for the 50% ethanolic extract of Prorocentrum PA1 cells
but subsequent application of extracts in a dilution series (1:10)
Since there was no apparent effect on bacterial growth
Epifluorescence microscopy after DAPI staining of bacteria in Prorocentrum PA1 cultures showing apparent increase in bacterial cell density over time in all treatments: AB
there were no significant differences among treatments (two-way ANOVA
p &#x003C; 0.05 and Tukey&#x2019;s HSD test
Time-series of bacterial cell density from Prorocentrum PA1 cultures under various treatments
Error bars indicate mean &#x00B1; standard deviation (SD) of replicate cell counts
Prorocentrum cell volume remained constant through the growth cycle and among treatments
with only minor non-significant fluctuations on a daily basis among treatments (two-way ANOVA
Time-series of bacterial cell density along the growth curve of Prorocentrum PA1 subjected to alternative treatments
but the toxin quota varied significantly during the first 16 days of the experiment
Toxin cell quotas in antibiotic-treated (AB) and washed cultures without antibiotics (WT) were substantially lower at Day 4 compared to initial values (Day 0)
where the AB culture reached minimum quota (1.58 &#x00D7; 102 fmol cell&#x2013;1)
WT and NT control reached minimum values: 1.36 &#x00D7; 102 and 1.43 &#x00D7; 102 fmol cell&#x2013;1
During late exponential to stationary growth phase from Day 16 to 47
although AB-cultures reached the highest value (3.36 &#x00D7; 102 fmol cell&#x2013;1) on Day 25
Variation of total DSP toxin cell quota over time-series growth subjected to alternative treatments
Total cell quota (fmol cell&#x2013; 1) calculated as sum of all detectable DSP toxin analogs: OA
Error bars indicate mean &#x00B1; standard deviation (SD) of replicate measurements
These generalities have been defined with respect to dense pelagic blooms in the water column
but it is not clear that such growth dynamics apply to consortia of bacteria&#x2013;dinoflagellates in benthic systems
where the &#x201C;blooms&#x201D; of dinoflagellates and associated bacteria are more spatially constrained upon and adjacent to the substrates
the high differential selection inherent in culture experiments means that the results from co-culture of bacterial assemblages and dinoflagellates isolates cannot be simply extrapolated back to natural benthic systems as representative of in situ bacterial composition and diversity
Previous studies on cultured isolates of toxigenic benthic Prorocentrum also noted, as found herein for tropical Atlantic populations, that the microbiota from the bulk culture medium and the phycosphere could be considerably different (<a href="#B58">Prokic et al., 1998</a>; <a href="#B56">Perez et al., 2008</a>)
Even though five bacterial genera were exclusively isolated from Prorocentrum cell aggregates among dinoflagellate clones from tropical coasts of Mexico
the distribution of the identified cultivable bacteria between cell-aggregates and surrounding medium was very similar
This is likely attributable to the unique and nutrient-rich microhabitat provided by Prorocentrum cells
but genus Roseobacter was not found among bacterial isolates in the current study
Isolate PA14 was heavily colonized by cyanobacteria shortly after bacterial isolation
which may indicate the association of CFB-member Euzebyella with the senescence of this Prorocentrum in high cell density culture
Further experiments are required to determine the algicidal potency of specific bacterial isolates against benthic Prorocentrum clones
it is now doubtful that bacteria alone can produce OA analogs
they do not possess the complete genetic machinery
None of the other Prorocentrum strains and their isolated bacteria showed any correlation with the identified toxins in this study
implying that these bacteria do not interfere directly with toxin production or composition in the dinoflagellates
This provides further evidence that toxins are produced by Prorocentrum alone and not by their associated extracellular bacteria
Other OA-producing Prorocentrum species were also tested under the same conditions
but no antimicrobial activity was found; these authors suggested the presence of specific antimicrobial compounds (presumably not DSP toxins) for P
Prorocentrum species can either inhibit or stimulate, or have no effect, on bacterial growth, depending on the target bacteria (<a href="#B13">De Vera et al., 2018</a>)
The bioactivity screening in the experiments reported here did not reveal any apparent allelochemical effect on the bacterial growth of Staphylococcus sp
HEL66 from either cell-extracts or supernatants from Prorocentrum PA1
Initial results showed a slightly positive growth inhibition
but this was not sustained through the dilution series
it is possible that higher biomass equivalents in the cell extracts could yield bioactivity against bacteria from Prorocentrum metabolites
but not at environmentally realistic exposure concentrations
The bacterial isolates for the bioassays originated from the North Sea and were deliberately selected
rather than bacterial isolates from the Prorocentrum cultures from Mexico
to eliminate any conceivable possibility of resistance or co-evolution from prior exposure
Negative evidence of allelochemical interactions of Prorocentrum metabolites
antibiotic effect against these two na&#x00EF;ve strains
does not preclude the possibility that allelochemicals and/or polyketide toxins could be effective growth inhibitors against other potentially competing bacteria in their own native benthic environment
and even against other strains of Staphylococcus and Vibrio
Further experiments considering extracts prepared from axenic versus non-axenic Prorocentrum cultures would help to resolve the issue of bioactivity against its naturally occurring associated bacteria (i.e.
isolated from Prorocentrum cultures or directly from natural assemblages)
Disk diffusion assays with purified DSP toxins would demonstrate the specific potency against bacteria
in the absence of potentially interfering components in crude extracts
This corresponds with the exponential growth phases from PA1 of about 20&#x2013;25 days under all treatments
and this likely interferes with the exposure of these bacteria to the antibiotics
The selected antibiotic cocktail and final concentration were chosen with respect to conventional methods to achieve axenic cultures of phytoplankton but were ineffective for dense assemblages of benthic dinoflagellates in culture
it is appropriate to be somewhat skeptical of previous references to &#x201C;axenic&#x201D; cultures of benthic dinoflagellates
where convincing evidence of bacterial absence is not provided
The cell toxin quota in strain PA1 decreased at the beginning of the culture cycle, i.e., during acclimation and lag growth phase, but then remained stable through the growth curve, while toxin composition remained stable. Similarly, in a previous study, no significant differences in toxin composition between axenic and non-axenic cultures were found for the closely related toxigenic benthic species P. hoffmannianum (<a href="#B42">Morton et al., 1994</a>)
Further experiments considering N and/or P deprivation and micronutrient and trace element availability would provide a closer estimation of the potential interactions between Prorocentrum and associated bacteria in the natural benthic environment
Association of specific bacterial assemblages with the evolution of HABs in the pelagic zone
in particular with the high abundance of &#x03B3;-Proteobacteria
may also be represented in the benthic realm for dense aggregations of benthic dinoflagellates
it is premature to conclude that the culturable bacterial community yields an appropriate insight into the bacteria&#x2013;dinoflagellate interactions modulated in natural blooms
A metagenomic analysis of the bacterial and microeukaryotic assemblages (including the toxigenic dinoflagellates) is required as the first step for such a comparison
High inorganic nutrient loads in culture medium promote optimal growth of cultured benthic dinoflagellates
which in turn yield enhanced organic substrates for associated bacterial growth
The high component of &#x03B3;-Proteobacteria in the Prorocentrum cultures may indicate that this microcosm reflects a mature high cell density &#x201C;bloom&#x201D; with concomitant selection features
The different taxonomic distribution of cultivable bacteria among culture compartments
Prorocentrum cell aggregates versus the surrounding medium
does suggest that the phycosphere constitutes a unique microenvironment with high selective capacity for bacteria
The lack of bioactivity of cell extracts and culture supernatants against selected model bacteria does not rule out the possible production of allelochemical metabolites
but does not support arguments for high general potency against bacteria
related to phosphatase inhibition by DSP toxins
Production of large volume axenic dinoflagellate cultures is essential to unraveling the potential role of bacterial interactions in growth and toxin production
This approach would allow to assay the effect of taxonomically identified specific consortia of naturally co-occurring bacteria on axenic cultures of P
lima to determine effects on growth and toxin production
current studies provide little evidence that extracellular bacteria play a critical role in regulation of DSP toxin production in Prorocentrum or in the modulation of growth under non-nutrient limited conditions in culture
The raw data supporting the conclusions of this article will be made available by the authors
All authors contributed actively to the preparation of data content and writing of this manuscript
and YO conducted the dinoflagellate field sampling
LD-R and AC prepared preliminary isolates for culture
LD-R and YO performed the morphotaxonomic analysis of Prorocentrum isolates
and AC maintained the clonal strains at UNAM
MP-Z and SP were responsible for bacterial identification and phylogenetic analysis
UT-J and JT conducted the bioactivity assays against bacteria
UT-J performed the toxin extractions and data analysis for quantification of DSP toxins under supervision of BK and JT
UT-J and AC conceived and designed the time series experiment
The coordination of the drafting of the manuscript was led by UT-J
Financial contribution for the preparation and publication of this manuscript and associated research activities was provided to AC
and JT via the PACES II Research Program (Topic II Coast: WP3) of the Alfred-Wegener-Institut
Helmholtz-Zentrum f&#x00FC;r Polar- und Meeresforschung
The Deutscher Akademischer Austauschdienst (DAAD) made possible the research stay of LD-R at the AWI in order to complete this project on benthic dinoflagellates from Mexico
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest
The authors thank Manuel Victoria-Muguira of Dorado Buceo for boat access and logistical support with field sampling
and Manuel Rodr&#x00ED;guez-G&#x00F3;mez from the Acuario de Veracruz for donating filtered seawater
Laura Elena G&#x00F3;mez-Liz&#x00E1;rraga from the Scanning Electron Microscopy Laboratory at Instituto de Ciencias del Mar y Limnolog&#x00ED;a
provided micrographs and Hugo P&#x00E9;rez-L&#x00F3;pez assisted with SEM sample preparation
The help of Annegret M&#x00FC;ller for technical assistance in the laboratory and Thomas Max for LC-MS/MS measurements of the DSP toxins at AWI is much appreciated
Jennifer Bergemann assisted with the antimicrobial assays within laboratory facilities graciously provided by Tilmann Harder
The Supplementary Material for this article can be found online at: <a href="https://www.frontiersin.org/articles/10.3389/fmars.2020.00569/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fmars.2020.00569/full#supplementary-material</a>
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Mauricio Tarazona is the head of Treasury and Trade Solutions (TTS) for Panama
with the responsibility to lead the areas of international trade
he managed the TTS business for Costa Rica from 2012 to 2015
Tarazona Joined Citi Peru in 2005 as a product manager for trade finance and services covering Peru and Bolivia and since then has taken on new positions with expanded roles
In 2010 he became head of the trade and corporate lending business for Peru
Tarazona lead the identification and disbursement of major structured trade/supply chain/EAF and lending deals in excess of $1.4 billion
financial institutions and public-sector entities
Tarazona has over 15 years of banking experience and worked in regional and global financial institutions mainly in the areas of international trade
He is currently a member of the board of the ACH Clearing System in Panama (Telered) and throughout the years has participated in business audits around Latin America
and business assessments in countries such as Brazil
Tarazona holds a BA in economics and an MBA from Universidad de Lima
in addition to post-graduate studies in finance and international business
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