Legal update - Contracts – what endures beyond termination?
This is consistent with the common law position that terminating a contract only terminates the duties of the parties to perform unperformed contractual obligations. Accrued contractual obligations remain enforceable. In this sense the contract itself is not terminated – rather it is the unperformed obligations of the parties which come to an end.
So which clauses survive a termination?
In some but not all contracts, the document makes this clear with an express "survival" provision.
Because termination only brings unperformed obligations to an end, any obligations or rights which accrued prior to termination will remain. Thus in Brown v Langwoods Photo Stores Ltd the Court of Appeal affirmed that whenever a contract is cancelled under the Act, all rights based on prior breach or performance will survive.
The obligation to pay compensation/damages.
Other clauses commonly recognised as surviving termination of a contract include limitation of liability clauses, arbitration clauses and (potentially) indemnity clauses.
Limitation of liability clauses and arbitration clauses are recognised as continuing contractual obligations after termination, as affirmed by the House of Lords in Photo Production Ltd v Securicor. In this case Lord Diplock noted that cancellation will only terminate the primary contractual obligations (the parties undertakings), but not secondary obligations (clauses that are conditional on one of the parties breaching their undertakings). This is because secondary obligations arise at the time the contract is entered into and so continue unaffected after termination.
What about indemnity clauses within contracts?
Whether an indemnity clause within a contract would be treated as a primary or secondary obligation is more uncertain. An analysis of Photo Productions suggests that secondary clauses are only those that are triggered as a result of the breach of contract. Accordingly indemnity clauses would not be considered secondary obligations since their operation is not reliant on a party defaulting under the contract itself.
An indemnity contract (or clause in this context) usually gives rise to a primary liability on one party to pay the other party for loss suffered after the occurrence of a certain event. Therefore if the relevant event occurs before the contract is terminated, the indemnity clause is usually considered an enduring provision and one party is still obligated to indemnify the other even after termination of the contract. If the event occurs after the breach and termination of the contract then the indemnity clause is not enduring and any rights one party had to be indemnified for a loss would normally cease to exist along with the other primary obligations in the contract.
The extent of discharge of provisions is of course always dependent on the intention of the parties to the contract. The importance of the parties' intentions is reflected by the fact that substantive clauses (such as restraint of trade and confidentiality clauses) are often drafted in a manner that clarifies that they survive a termination of a contract.
Therefore, if a contract contains an express provision that excludes all indemnity rights upon breach and subsequent termination of the contract, the expressed intention of the parties will govern and the indemnity will be excluded. However, if no such provision was contained in the contract, based on the reasoning above an indemnity should generally remain enforceable after termination, for any events prior to the termination date.
Does the defaulting party have any rights after termination?
The fact that a party to a contract has committed a breach does not deprive them of all rights arising from the contract.
Accordingly if the defaulting party possesses an accrued right to receive performance from the other party, this is not divested upon termination. A defaulting party may also claim damages for any breach committed by the other party prior to termination of the contract.
A defaulting party can also recover sums paid, but the ability to recover monies paid will depend on the terms of the contract, and the nature of the sum paid. More particularly, whilst a deposit is not usually recoverable by a defaulting party, an instalment payment will be recoverable where it is treated as being paid conditionally, upon completion or performance of the contract.


text:
package com.google.cloud.tools.eclipse.dataflow.ui.util;

import static org.junit.Assert.assertEquals;
import static org.mockito.Matchers.any;
import static org.mockito.Matchers.anyInt;
import static org.mockito.Mockito.mock;
import static org.mockito.Mockito.never;
import static org.mockito.Mockito.times;
import static org.mockito.Mockito.verify;
import static org.mockito.Mockito.when;

import com.google.common.collect.ImmutableSortedSet;
import java.util.SortedSet;
import org.eclipse.swt.events.ModifyEvent;
import org.eclipse.swt.graphics.Point;
import org.eclipse.swt.widgets.Combo;
import org.eclipse.swt.widgets.Event;
import org.eclipse.swt.widgets.Widget;
import org.junit.Before;
import org.junit.Test;
import org.junit.runner.RunWith;
import org.mockito.Mock;
import org.mockito.Mockito;
import org.mockito.runners.MockitoJUnitRunner;

/**
 * Tests for {@link SelectFirstMatchingPrefixListener}.
 */
@RunWith(MockitoJUnitRunner.class)
public class SelectFirstMatchingPrefixListenerTest {

  @Mock
  private Combo combo;

  private ModifyEvent modifyEvent;

  private final SortedSet<String> elements =
      ImmutableSortedSet.of("collidingText1", "collidingText222", "exampleText");
  private SelectFirstMatchingPrefixListener listener;

  @Before
  public void setup() {
    Event event = new Event();
    event.widget = mock(Widget.class);
    modifyEvent = new ModifyEvent(event);

    listener = new SelectFirstMatchingPrefixListener(combo);
    listener.setContents(elements);
  }

  @Test
  public void testShortCircuitIfNewTextEqualsLastSeen() {
    when(combo.getText()).thenReturn("lastString");
    listener.lastString = "lastString";
    listener.modifyText(modifyEvent);
    assertEquals("lastString", listener.lastString);

    verify(combo, times(1)).getText();
    Mockito.verifyNoMoreInteractions(combo);
  }

  @Test
  public void testShortCircuitIfNewTextEqualsLastAutoSet() {
    when(combo.getText()).thenReturn("lastAutoSet");
    listener.lastAutoSet = "lastAutoSet";
    listener.modifyText(modifyEvent);
    assertEquals("lastAutoSet", listener.lastAutoSet);

    verify(combo, times(1)).getText();
    Mockito.verifyNoMoreInteractions(combo);
  }

  @Test
  public void testBelowPreviousNoAutofill() {
    when(combo.getText()).thenReturn("collidingText222");
    // Updates the combo to be initialized with "collidingText222" as the current value
    listener.modifyText(modifyEvent);
    when(combo.getText()).thenReturn("collidingText2");
    listener.modifyText(modifyEvent);
    verify(combo, never()).select(anyInt());
    verify(combo, never()).setSelection(any(Point.class));
  }

  @Test
  public void testBelowThresholdNoAutofill() {
    when(combo.getText()).thenReturn("s");
    // Updates the combo to be initialized with "s" as the current value
    listener.modifyText(modifyEvent);
    when(combo.getText()).thenReturn("ex");
    listener.modifyText(modifyEvent);
    verify(combo, never()).select(anyInt());
    verify(combo, never()).setSelection(any(Point.class));
  }

  @Test
  public void testIntermediateCursorNeverAutofills() {
    when(combo.getText()).thenReturn("short");
    // Updates the combo to be initialized with "short" as the current value
    listener.modifyText(modifyEvent);
    String prefix = "exampleTe";
    when(combo.getText()).thenReturn(prefix);
    when(combo.getCaretPosition()).thenReturn(prefix.length() - 2);
    listener.modifyText(modifyEvent);
    verify(combo, never()).select(anyInt());
    verify(combo, never()).setSelection(any(Point.class));
  }

  @Test
  public void testNoPrefixMatchSucceeds() {
    when(combo.getText()).thenReturn("short_text");
    // Updates the combo to be initialized with "short_text" as the current value
    listener.modifyText(modifyEvent);
    when(combo.getText()).thenReturn("also_doesnt_match");
    listener.modifyText(modifyEvent);
    verify(combo, never()).select(anyInt());
    verify(combo, never()).setSelection(any(Point.class));
  }

  @Test
  public void testPrefixMatchAutofills() {
    when(combo.getText()).thenReturn("short");
    // Updates the combo to be initialized with "short" as the current value
    listener.modifyText(modifyEvent);
    String prefix = "exampleTe";
    when(combo.getText()).thenReturn(prefix);
    when(combo.getCaretPosition()).thenReturn(prefix.length());
    listener.modifyText(modifyEvent);
    verify(combo).select(2);
    verify(combo).setSelection(new Point(prefix.length(), "exampleText".length()));
  }

  @Test
  public void testPrefixMatchMultipleMatches() {
    when(combo.getText()).thenReturn("short");
    // Updates the combo to be initialized with "short" as the current value
    listener.modifyText(modifyEvent);
    String prefix = "collidingT";
    when(combo.getText()).thenReturn(prefix);
    when(combo.getCaretPosition()).thenReturn(prefix.length());
    listener.modifyText(modifyEvent);
    verify(combo).select(0); // == "collidingText1" - it's lexicographically the earliest collision
    verify(combo).setSelection(new Point(prefix.length(), "collidingText1".length()));
  }
}

redpajama_set_name: RedPajamaGithub


Really become a crime-fighting turtle with these officially licensed Teenage Mutant Ninja Turtles 3 Finger Gloves!The light green padded gloves go up the arm and include a graphic wrist band and three fingers so you can fit right in with the others!
Wash/Care Instructions: Hand wash cold. Only non-chlorine bleach when needed. Dry flat. Do not iron.


Blockchain is like electricity--many people originally feared it, but there were the brave that understood its potential. We venture out into the depths of the cypherpunk, cryptographic world and identify the projects with the most potential to disrupt the way humans exchange value and trust. Thus, value capturing protocols that offer humanity the most freedom while providing utility for stakeholders are the projects we back.


text:
Dr. Fauci Says Stop Going Here Now
"Innocent get togethers" of small, familiar groups can put you in real danger of catching COVID-19 right now, says Dr. Anthony Fauci, the nation's top infectious disease expert. "What we're seeing throughout the country is that innocent get-togethers of family and friends, in a home where people let their guard down because they're with people they're familiar with, we're starting to see those kinds of infections," said Fauci in a CNN interview recently. For more of his warning, read on, and to ensure your health and the health of others, don't miss these Sure Signs You've Already Had Coronavirus.
Dr. Fauci Says This is Becoming an "Important Source of Infection"
Fauci detailed a typical scenario: "Eight, 10 people get together at a dinner with friends and family. One of them is infected, but with no symptoms, They put their guard down. You're sitting, you're eating, you're drinking, you take your mask off. And that's how we're starting to see infections."
"I get on the phone tonight and talk to all of my colleagues around the country—almost every one of them say that's becoming a very important source of infection, the innocent gathering," Fauci added.
Fauci advocated the development of an over-the-counter, at-home COVID-19 test that could help stem those kinds of infections. "If you had a test that you could do at home yourself, point-of-care, sensitive and specific, you can eliminate a lot of that," he said. The FDA recently approved a $50 at-home coronavirus test, but it requires a prescription.
COVID-19 cases are rising in all 50 states. In many areas, hospitals are nearing capacity. The CDC had advised Americans to avoid Thanksgiving travel and to limit gatherings to people who live in the same household, meaning those who have lived primarily in the same house for at least 14 days. Yet millions travelled over the Christmas holiday.
RELATED: 7 Tips You Must Follow to Avoid COVID, Say Doctors
Should You Attend a Group Get-Together?
Dr. Fauci had advice for Thanksgiving that you can apply to every day. "Each and every family unit should do a risk-benefit determination," Fauci advised. "You take a look at your family and you say, do I have a person there who's an elderly person, a person with an underlying medical condition that might put them at an increased risk of a severe outcome? If they get infected, do I want to take that risk right now? Or do I want to say, maybe the prudent thing to do for now is to just pull back and just keep it within the family unit that you live with."
Fauci, who is 80, added: "Everybody should at least think about the risk that you might be putting your loved ones under."
RELATED: If You Feel This, You May Have Already Had COVID, Says Dr. Fauci
More Ways to Stay Healthy
As for yourself, do everything you can to prevent getting—and spreading—COVID-19 in the first place: Wear a face mask, get tested if you think you have coronavirus, avoid crowds (and bars, and house parties), practice social distancing, only run essential errands, wash your hands regularly, disinfect frequently touched surfaces, and to get through this pandemic at your healthiest, don't miss these 35 Places You're Most Likely to Catch COVID.
Scary Mommy
Fauci Says Biden's Plan Of 100 Million Vaccinations In 100 Days Is 'Absolutely Doable'
Dr. Fauci says Joe Biden's ambitious vaccine plan is absolutely 'doable' and says two new vaccines are potentially being submitted for emergency use in a few weeks If you, like me, are counting down the days to Joe Biden's inauguration with the hope that on January 20, 2021 the U.S. becomes slightly less terrifying, you []
A Doctor Shared What It's Like to Get Both Moderna COVID Vaccine Shots
Is the second shot really that different than the first?
These Are the Top Home Organizers for Hire in the Country
Find out if they're based in your state!
To My Teen Daughter: This Is The Advice I Wish I'd Been Given When I Was Your Age
If I could teach you anything, it would be to lean into these things and embrace them with all you have.
Convert Your Pop-Fan Friends With the Best Country Love Songs of All Time
From Patsy Cline to Luke Bryan, get ready to make a mushy playlist.
Blake Shelton Says Gwen Stefani "Won't Let" Him See His Friends
With the ongoing pandemic, most of us are doing our part to stop the spread of the virus, The CDC is continuing to advise the public to practice social distancing, so that means we're currently spending a lot of our time at home without seeing our extended family or friends. For celebrities like Blake Shelton […]

redpajama_set_name: RedPajamaCommonCrawl


There are 67 Counties in Florida, but some reports show only 66 because of Duval County, which is consolidated with the City of Jacksonville. There are 379 cities in Florida (out of 411) that report regularly to the Florida Department of Revenue, but there are other incorporated municipalities that do not. The primary source of revenue for the state government is sales tax (Florida does not impose a personal income tax), but the primary revenue source for cities and counties is property tax.


The remote town of Yulara is a convenient start point for visitors setting off to the explore the sensational range of natural wonders the Red Centre has to offer.
The ancient rock formations of Kata Tjuta (also known as The Olgas) create an amazing landscape of spectacular domes and canyons.
An iconic image of outback Australia and a sacred site for the local Aboriginal people, Uluru's remarkable colour changes are a wonder to behold. Visit the lesser-explored region of the West MacDonnell Ranges and compare the ever changing landscapes of the Red Centre.
This isn't a bare bones camping experience, as you'll be treated to creature comforts such as proper beds, linen, electricity and cooling fans.
Group participation makes up a lot of this trip, so you'll be required to help with preparation of meals and cleaning up. These are the kind of tasks that bring a group closer together. Group sizes can also be pretty big, so be prepared to share this experience with a bigger group than what you might be familiar with.
Temperatures in the Red Centre can be extreme. During the day the weather can be very hot (and the Australian sun is very strong) so please bring appropriate clothing, use sun protection and drink plenty of water. In the evenings temperatures really drop, so bring base layers and warm clothing.
This trip requires a moderate level of fitness as it involves a lot of walking, allowing you to really get among nature. Please bring sturdy, comfortable walking shoes and comfortable clothing.
On most days of the trip, you'll spend several hours driving through very sparse areas of desert. Sit back to enjoy landscapes that you would miss in any other form of travel.
This afternoon, make your way to Kata Tjuta (the Olgas) and take a guided walk through the awe-inspiring Domes of Kata Tjuta. We then head to Australia's most iconic landmark - Uluru (Ayres Rock). As the sun begins to set below the horizon, enjoy some tasty snacks and watch the rich colours change against the backdrop of a blazing Red Centre sunset. The perfect end to a remarkable day is awaiting you back at camp, as you enjoy a delicious spread of food with your group. If the weather permits, sit outside under the stars.
Notes: Total driving time is approximately one hour. Total walking time is around an hour.
An early start this morning is rewarded with an Uluru sunrise. While the morning is fresh enjoy a walk around the base of Uluru, gaining insights into the land and the local Aboriginal people. After your stroll, we walk to the cave paintings near Mutitjulu Waterhole at the base of Uluru. You will be in the hands of an Anangu guide who will share the stories of this unique landscape and explain the Art, the Culture and connection to Land. This exclusive experience will give you an exclusive insight to the meaning behind Anangu art and traditional culture. The on-site Cultural Centre also houses an extensive collection of indigenous arts and crafts for you to browse.
This morning we journey through Ormiston Gorge, witnessing contrasting colours as the light reflects off the cliffs. Keep your eyes peeled for wallabies and other wildlife. Move on to Ellery Creek to enjoy panoramic views of the area, perhaps taking a dip in its still waters. After lunch, visit the world famous Simpsons Gap, an important site to the local Aboriginal communities. Watch the colour of the peaks transform under the setting sun. Head back to Alice Springs, where your trip comes to an end.
There are currently no scheduled departures on our Red Centre Adventure ex Yulara (Original) trip. If you are interested in other trips in the region visit one of the links below.
1. Trip commences in Yulara on day 1 at approximately 12pm and finishes in Alice Springs day 4 at approx 6pm.
3. This trip requires a sleeping bag.
4. This trip is available in Original and Basix styles.


text:
Q: EntityNotFoundException (cannot evaluate) I mapped model like this:
    @Entity
    public class Notification {

        @ManyToOne(fetch = FetchType.LAZY)
        @JoinColumn(name="ad")
        @Where(clause="is_deleted <> '1'")
        @JsonIgnore
        public Ad getAd() {
            return ad;
        }

    }

When Hibernate try to get this ad, it shows error:
Method threw 'javax.persistence.EntityNotFoundException' exception.
Cannot evaluate Ad_$$_javassist_1.toString()

A: As you do not have any data in your table which you are trying to retrieve that is the cause of this exception.
First insert  record in your table than try to get the data there would not be any exception
Thank you
Chandan

redpajama_set_name: RedPajamaStackExchange


Ford has announced the sportily styled Kuga ST-Line, which goes on sale in September and is priced from £24,845. It features a gloss black grille and rear skid plate, black window beltline and black alloy wheels, along with part leather sports seats.
The suspension has been lowered 10mm versus a standard Kuga, plus there are thicker anti-roll bars, stiffer bushes and revised spring and damper rates to give a more precise and responsive drive.
All of the Kuga engine range is offered with ST-Line trim. Diesel options are a 1.5-litre with 120PS and emissions of 115g/km, plus there is a 2.0-litre TDCi with either 150PS or 180PS. The 150PS engine can be specified with front- or all-wheel drive, while the 180PS engine comes with all-wheel drive only. Petrol engines are available with 120PS, 150PS or 182PS, with emissions from 143g/km to 171g/km.
The Kuga ST-Line joins the similarly-styled Fiesta, Focus and Mondeo ST-Line variants, priced from £15,645, £20,595 and £24,745, respectively. First deliveries of the Kuga ST-Line are due before the end of the year.


text:
Eric Adams will sworn in as the 110th Mayor of the Big Apple after Times Square ball drop
January 1, 2022 Lucas Carter
Out with old mayor, in with the upgraded one.
Brooklyn Borough President Eric Adams will ring the New Year at the Times Square ball drop at 12 PM Friday–then be sworn in as the 110th Mayor of the Big Apple.
Adams will quickly take over from his stand-in archetype Bill de Blasio whose last obligations as mayor will introduce 2022 at the notable New Years Eve celebration.
Adams plans to make the oath of office using a family Bible.
"Times Square has for some time been inseparable from the New Year—a place of excitement, renewal, and hope for the future," the mayor-elect said in a statement.
"These are the very topics that enlivened my mission and will illuminate my mayoralty, as I get ready to lead the city out of this challenging period," Adams said.
The incoming mayor as of late packed away an inauguration ceremony planned Jan. 1 at Kings Theater in Brooklyn because of rising Covid cases.
The COVID-19 surge additionally provoked de Blasio to cover the group size and require covers for revelers celebrating the New Year at the Crossroads of the World.


About Allison: Spanish to English interpreter and translator since 2013. Much legal interpreting experience with personal injury cases, immigration cases, and others. Also experienced with some medical interpreting for IMEs and other doctors appointments. A Language Access Volunteer with the New York Legal Assistance Group since 2014. Interpreter with AllisonToursBA in Buenos Aires, 2014. English teacher and Program Developer at Conviven Center, Buenos Aires, 2013.
Member of the ATA, NAJIT, New York Circle of Translators, ProZ, Translators Cafe, and LinkedIn.


Campsite close to La Palmyre with Water Park.
Close to La Palmyre, explore the Les Charmettes **** campsite and its water park.
Make the most of your stay at the Les Charmettes campsite and relax in our water park. Our pool complex always has expert swimmers and qualified life-guards on duty. Children can enjoy swimming in complete safety.
allowed to enter the pool area without adult supervision.
The beach has been enlarged to 700m²!


Have fun at this Phish-themed indoor cycling class hosted by Thingsyouseeatphish. This high energy ride is all about surrendering to the flow with Phish videos and glowsticks! Cycling shoes included. Please bring a water bottle.


text:
Five years after Ghost Ship: How local organizations are fighting artist displacement
In the 19th and 20th centuries, Oakland's industrial zone bustled with canneries, metal works and warehouses. As the global economy changed, industries moved out and artists moved in. The low-rent buildings, with their vaulted interiors, were suitable for live-work studios. Over the years, landlords looked the other way as tenants nested in spaces that were never coded for housing. On Dec. 2, 2016, the deadliest fire in Oakland history broke out in the Ghost Ship, a former warehouse in Fruitvale…
By Ruth Dusseault and Kristie Song on December 16, 2021
Boxes, buckets, Buicks and Chevies: Sideshows are at a crossroad
Tires screech and cars dance on the streets of Oakland as sideshow culture fills the air, along with the smell of burning rubber. Originating in Oakland in the 1980s, sideshows have gained traction in the Bay Area, exciting audiences, frustrating motorists and irritating police. Oakland recently unveiled a bold plan to crack down on sideshows, which are classified as reckless driving and punishable as a misdemeanor. Richmond City Council recently took action to deter sideshows. And a state law signed…
By Jesse Foley-Tapia on November 18, 2021
New documentary celebrates Oakland's 'last Black cowboy'
Half a dozen people sporting cowboy hats and boots stood in a queue outside of Eli's Mile High Club, chatting in hushed excitement, some squeezing together for selfies. The occasion was the Oct. 2 premiere of "Cowboy," a documentary about the life of "Oakland's last Black cowboy," 80-year-old Wilbert Freeman McAlister. He is president of the Oakland Black Cowboy Association, which is a non-profit focused on preserving the history of African Americans who were crucial to the establishment of the…
By Andrew Lopez on November 17, 2021
Fruitvale community prepares for El Día de Los Muertos festival Sunday
With marigolds, banners, altars and sugar skulls, El Día de Los Muertos observations are underway in Oakland, where a festival is planned for this weekend and altars already are up in homes and gathering places. During Sunday's downpour, in a kitchen strung with intricately patterned papel picado banners, visual artist Daniel Camacho delicately separated the marigold petals he made with orange tissue paper. It is believed that the strong scent and vibrant gold of marigolds can lead loved ones back…
By Jiyun Tsai on October 29, 2021
'Long live the legacy of Dr. Huey P. Newton': Sculpture honors Black Panthers co-founder
During an unusually stormy Sunday in West Oakland, about 150 people sang and danced in the rain to celebrate the unveiling of a sculpture honoring Huey P. Newton, who co-founded the Black Panther Party in the city. The bronze bust of Newton is the first permanent art installation to honor the activist, who started the Panthers with Bobby Seale in 1966 to provide educational, and economic support to the Black community. The party developed services to meet the everyday needs…
By Williamena Kwapo on October 24, 2021
'It's just a sweet, magical little event': Autumn Lights Festival unites artists and gardeners
This year's Autumn Lights Festival brings together over 300 artists and 70 illuminated light displays ranging from carved-out gourds to a steampunk-esque snail car fully equipped with pyrotechnics. The festival, which is celebrating its 10th year, opened to a sell-out crowd Thursday at The Gardens at Lake Merritt. Aside from art, it also gives space to local food vendors and live musicians. Director and founder of the festival, Tora Rocha, said the garden gives local artists an opportunity to show their work…
By Andrew Lopez on October 15, 2021
Pepper spray and alarms: Chinatown clinic teaches personal safety
Chinatown residents are concerned for their safety after instances of assault and robbery against seniors. The Oakland Chinatown Safety Committee hosted a personal safety and pepper spray clinic to train community members on how to be more aware of their surroundings and defend themselves.
By Kristie Song on October 11, 2021
African-American Shakespeare Co. returns to the Oakland stage with free weekend show
Oakland is the newest playground for the African-American Shakespeare Company's upcoming tour of "The Complete Works of William Shakespeare Abridged, Revised." Coming to Jack London Square this Saturday, the play challenges three actors to perform 75 characters from 37 of the bard's plays in just under an hour and a half. The Complete Works was slated to hit the stage in September 2020, but as COVID-19 devastated the theater industry, AASC joined many of the nation's theater companies in pushing…
By Corey Rose on October 6, 2021
First Fridays finally returns with food, music, fun in KONO
At his family-owned halal market Thursday morning, Temur Khwaja cut and marinated chicken and lamb for kebab at Marwa Market & Grill. "We need to prepare for First Friday tomorrow," said Khwaja, anticipation in his voice, sweat on his brow. Local businesses and restaurants in the Koreatown Northgate (KONO) neighborhood are preparing for the relaunching of the Oakland First Fridays festival tonight. First Fridays is a monthly street festival held by KONO Community Benefit District. Eighteen months after the pandemic…
By Jiyun Tsai on September 30, 2021


Dateline: Who Is Jason Blum, 'Halloween' 2018 Horror Movie Producer?
In the Dateline episode "Hollywood Heat," Josh Mankiewicz reports stories from the streets of the City of Angels. He interviews movie producer Jason Blum who's revamping the horror genre in Hollywood. Mankiewicz also interviews Scream Queen Jamie Lee Curtis (who's working on a Halloween revival with Blum), Get Out director and writer Jordan Peele, and Octavia Spencer, who's reuniting with The Help director Tate Taylor for Blum's new psychological horror film Ma.
Blum has been nominated for two Oscars: Best Motion Picture of the Year in 2017 for Get Out (written and directed by Peele) and in 2014 for Whiplash. The 49-year-old Blum has won two Emmy Awards for the 2015 HBO documentary The Jinx: The Life and Deaths of Robert Durst and the 2014 HBO movie The Normal Heart about a gay activist in the early 1980s who tries to raise awareness of HIV and AIDS (co-executive producers include Brad Pitt and Mark Ruffalo).
The original Halloween starring Jamie Lee Curtis is celebrating its 40th Anniversary in October, and Blumhouse Productions just released a photo of Michael Myers' new mask. It's available for pre-order, $59.99, at Trick or Treat Studios. The 2018 Halloween film will be in theaters on October 19.
Blum, a Vassar graduate, was born and raised in Los Angeles. His mother was an art professor, his father an art dealer. His first big time gig as a producer was for the 1995 Will Ferrell movie Kicking and Screaming. Blum is married to former Wall Street Journal writer, Harvard graduate Lauren A.E. Schuker. Dateline airs Fridays at 10pm on NBC.
Who Is Crazy Andrea In 'Homecoming Revenge' Lifetime Movie?


. Did you make her lunch too?
Pretty much. I wouldn't say stupid. I'm proven time and time again the poorer people are, the more generous and kind they are. It should be the other way around but isn't.
I once met a Japanese/Korean couple in Vietnam, chatted with them over e-mail (although a not huge amount) and when I visited Japan in 2009 I was shocked to find he'd moved out for the week (he stayed at work) and gave me his flat. They didn't "really" know me from Adam.
It's definitely an Asian thing. It seems weird on one hand to lump all Asians under the same name (Middle East, South Asian, South East Asian, Far East are hardly ethnically the same) but Asians do all seem to share the same belief for family and giving to strangers, we don't in the West. Our family beliefs are certainly vastly different. I'm not saying the Asia way is better per se but different.
bradavon wrote: . Did you make her lunch too?
I'd say the asian way is better.
Although in HK people are more distanced than maybe in other countries. And it will also depend on the individuals ofcourse.
To an extent but Asians (and I know I'm grossly generalising) have no perspective of independence. It all comes from a family perspective and that's a polar opposite to living on your own. I know I'd hate still living with my family, I like my own space too much.
I don't think it's stupid. I think it's nice that people are so generous and kind.
bradavon wrote: To an extent but Asians (and I know I'm grossly generalising) have no perspective of independence. It all comes from a family perspective and that's a polar opposite to living on your own. I know I'd hate still living with my family, I like my own space too much.
I know what you mean. In our culture you just don't move out of your parents' house unless you are getting married. Living on your own all alone is a rare thing in the East.
thelostdragon wrote: I know what you mean. In our culture you just don't move out of your parents' house unless you are getting married. Living on your own all alone is a rare thing in the East.
But you then move into another family . Being an Asian-Europe/Westerner which do you feel closer to? By that I mean do you feel the need for independence more than others in your family?
I know my Mum could never have her Mum still living with her (she's a lovely lady but can be very difficult) but she does everything for her otherwise. Orders her online shopping, collects her medicine etc... It seems like a fair compromise.


This forum is broken down by event type using thread prefixes. The prefixes make it easier for other members to search by type of event. A prefix is required for all posts. The default prefix is wedding as it's the most popular.
If you have suggestions for additional event types please reply here.
Video Jocks seem to be very quiet now. I haven't run into many people showing manly videos like I did years ago. I know Val plays mainly videos but I think that's about it. And Chuck believe plays some at events too. Nothing like the good old days.
Thanks guys. Productions and Karaoke has been added.
I'm not sure it warrants it's own category...imo most could be placed in their respective school dance or wedding or club categories.
When did you learn Photoshop.


Popping in today for a quick reminder that the Quarter 4 Link Up for all your finishes closes on Wednesday, January 9, 2019, 11 pm EST. So you still have a few more days to finish a project or two and link them up to be eligible for prizes. The link up can be found HERE or on the blog of any of our current hosts.
Please include a link to your original list in each post of your finishes (on Instagram the link is the unique tag #2018FALQ4yourname to be used in the Q4 list post, and in any Q4 finish post).
Finally, we hope you will be joining us for 2019 to do it all over again!! So get your list ready, the link up for Q1 Lists will open on January 10, 2019.


text:
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#endif

static int ok_count_;
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static int cur_running_;
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	test_func fn;
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	if (fn) {
		fn(NULL);
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		total_count_, ok_count_, fail_count_);
}

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Our Lord gave to John a name which means son of thunder, and of all the twelve apostles there was none whom He so deeply and warmly loved. It was a love of the Man in Christ for the man John. And it was shown in many ways. Peter, James, and he were the only ones Our Lord admitted to the room where he raised from death the daughter of Jairus. They alone were present at the Transfiguration and at the agony in the Garden of Gethsemane.
At the Last supper it was John who sat next to Our Lord and who rested his head on His breast. He was the only one of the Apostles present at the Crucifixion, and it was to he that Our Lord entrusted the love and protection of His greatest treasure, His mother.


Elkem Iceland, the Ferro Alloy Factory started its operation in 1979. The Icelandic government held majority ownership with the Elkem-Spiekerverket of Norway and Sumitomo in Japan until it sold its shares. Most of the workers of the factory live in the town Akranes and its surroundings.
Agricultural activities in the area have diminished considerably since the factory started. The harbour of Grundartangi was built at the same time as the factory by the communities on the Whale Bay. Nordic Aluminium (Century Aluminium) started its operation in 1998 with an average capacity of 60.000 tons per annum. In the year 2000 its capacity was increased by 30.000 tons. It also uses the harbour to receive raw materials (bauxite) and ship its products. The smelter changed ownership in the first half of 2004.
The opening of the Whale Bay Tunnel in June 1998 accelerated the development of Town Akranes and the neighbouring communities.


text:
2017 NFL draft position preview: EDGE rushers
SportsNFLLos Angeles Chargers
Texas A&M pass-rusher Myles Garrett is expected to go No. 1 overall when the Cleveland Browns kick off the NFL draft Thursday. (AP Photo/Eric Christian Smith, File)
By Rich Hammond | rhammond@scng.com | Orange County Register
PUBLISHED: April 21, 2017 at 3:33 p.m. | UPDATED: April 21, 2017 at 7:43 p.m.
The Rams and Chargers are heading in opposite directions when it comes to their defensive schemes, and that could have some impact on next week's NFL draft.
The Rams, under new defensive coordinator Wade Phillips, are transitioning from a base 4-3 front to a 3-4, one that values versatile defensive ends and linebackers, commonly known as edge rushers.
The Chargers and new coordinator Gus Bradley, are headed the other way, from 3-4 to 4-3 and might look to bulk up on the line. In total, this is a good year for teams seeking help on the edge, and it starts at the top, where Cleveland is almost certain to select Texas A&M's Myles Garrett with the No. 1 overall pick.
Garrett almost certainly will head to the Browns, who recently hired former Rams defensive coordinator Gregg Williams to lead their defense. Garrett was a first-team All-America selection in 2016 even though a knee injury limited his games and effectiveness.
It's likely that Garrett, Tennessee's Derek Barnett and Stanford's Solomon Thomas all will be selected among the first 20 picks of the draft, and UCLA's Takkarist McKinley could join that group.
Draft analyst Daniel Jeremiah called this a "good" group of edge rushers but cautioned it can be tough to grade them because of the quick-strike nature of college offenses.
"You don't get a chance to see these guys really get a good, hard rush on people at times," Jeremiah said last week. "That's a little bit of a challenge there."
There's also a challenge to determine which edge rushers can stand up in a 3-4 system, or play at the line in a 4-3 scheme, and that's something the Rams and Chargers must focus upon.
The Rams might not seek much draft help on the edge this year. They believe former defensive end Robert Quinn can translate nicely to a stand-up position, and they signed edge rusher Connor Barwin, who played under Phillips in Houston, to man the other side.
Quinn's history of injuries could give the Rams some pause, though, and if they wanted to add an edge rusher with their initial pick, at No. 37, they could have their eyes on Missouri's Charles Harris or Auburn's Carl Lawson.
The Chargers might be more likely to seek draft assistance. A year ago, they selected Joey Bosa with the No. 3 overall pick, and Bosa thrived in his rookie season but now will be asked to play more of a traditional defensive-end role under Bradley, the former Jacksonville coach.
Also, the Chargers, who obviously emphasized linebackers and interior linemen in their previous system, will need to add to their defensive-end depth, and might be able to do so in the mid-to-late rounds.
Two other edge rushers with first-round skill, Harris and Michigan's Taco Charlton, could sneak into the first round, but the top four at this position seem to have separated themselves.
MYLES GARRETT, TEXAS A&M
The scary thing is, Garrett still has room to improve some of his fundamentals. He's the clear-cut No. 1 overall pick, even though he started only nine games in 2016 because of a knee injury. Listed at 6-foot-4, 272 pounds, Garrett is tremendously explosive off the snap and a dominant pass rusher.
DEREK BARNETT, TENNESSEE
Success has followed Barnett for a while. He started as a freshman and made the SEC all-conference team for three consecutive seasons. He had some penalty problems last season, but an NFL.com analyst compared his talent to that of Hall of Famer Reggie White, and it doesn't get much better than that.
SOLOMON THOMAS, STANFORD
The product of an athletic family, Thomas is a beast at the line of scrimmage, a tough sight for any offensive lineman. Listed at 6-3, 273 pounds, Thomas actually could stand to improve his lower-body strength, but he's aggressive off the snap and should be able to thrive in a 4-3 or 3-4 defense.
TAKKARIST MCKINLEY, UCLA
Listed at 6-foot-2, 250 pounds, McKinley is beloved by scouts for his sideline-to-sideline pursuit and his speed. They're less enamored with his fundamentals and lower body, but this first-team all-conference pick should grow and develop nicely under NFL coaching tutelage and has star potential.
This is the seventh in a 10-part series previewing every position group before the 2017 NFL draft. The series previously covered quarterbacks, running backs, receivers, tight ends, offensive linemen and interior defensive linemen.
Rich Hammond | Reporter
Rich Hammond was a high school senior when the Rams left town in 1995, and now he's their beat writer for the Southern California News Group. A native of L.A., Rich broke in at the Daily Breeze as a college freshman and also has covered USC, the Kings, the Lakers and the Dodgers. He still loves sports and telling stories. Don't take the sarcastic tweets too seriously.
rhammond@scng.com
Follow Rich Hammond @Rich_Hammond
Anaheim Ducks |
An early look at the top options for Ducks in the 2023 NHL draft


WHEN IT COMES TO FOOD, QUALITY MATTERS!
WANT TO FEEL GOOD ABOUT WHAT YOU FEED YOUR BELOVED FURRY FAMILY MEMBER(S)?
QUALITY, SAFE FOODS FOR OUR BELOVED PETS!
I feed FROMM GOLD HOLISTIC PUPPY (kibble) and FROMM GOLD HOLISTIC ADULT (kibble) as a constant nutritional foundation and I rotate a number of other carefully selected, quality foods mixed in, in addition to occasional home cooked meals. CHICKEN BASED FORMULAS are at the heart of my dogs' diet, with the addition of a small amount of other meat proteins. This variety added into their consistent kibble foundation serves to keeps digestion healthy and allows the dogs to enjoy balanced nutrition while helping to eliminate the development of food allergies. I have researched both dry and canned dog food brands very carefully and came up with an "approved" list of foods that contain premium quality ingredients, no corn, no wheat, no soy, no by-products, no questionable overseas sources, high success rate, small size kibble, made in the USA, and never involved in a recall.
You can feel good about feeding your dog(s) any of the foods from the following lists.
ALS denotes a food designated as "All Life Stages." Due to their small size, Chihuahua puppies should be fed a puppy/growth formulation, as they need all the calories and nutrition they can get per bite, especially younger/smaller Chihuahua puppies. I have found that some All Life Stages formulas (such as the Victor Chicken & Rice Pate I wean puppies on) contain very similar nutritional and caloric content to puppy formulas and I include such ALS formulas in my puppies' diet, in addition to their puppy formula foundational food.
For treats, some of our favorites are Cloud Star Chewy Tricky Trainers (Liver), NutriSource Soft & Tender (Chicken or Lamb), and Earthborn EarthBites. Most importantly, check to be sure treats are wheat, corn & soy free, and made in the USA from quality USA sourced ingredients (PLEASE never feed your dog anything made in or sourced from China, they have poisoned and killed many beloved pets!).
I buy almost all of my dogs' foods, treats, toys, etc. ONLINE at one of the sites below. Some of these food/treat brands can be extremely difficult to find locally (even if you live in the city and are not out in the middle of the country like we are!), but they can all be found on one or more of the sites below, and free shipping is available!


text:
Frontiers in Cellular
and Infection Microbiology
Beyond Antimicrobials: Non-Traditional Approaches to Combating Multidrug-Resistant Bacteria View all 24 Articles
You-Hee Cho
CHA University, South Korea
Yang Zhang
University of Pennsylvania, United States
Chulhee Choi
Chungnam National University, South Korea
Open Supplemental Data
Front. Cell. Infect. Microbiol., 21 November 2018 | https://doi.org/10.3389/fcimb.2018.00410
Zinc Blockade of SOS Response Inhibits Horizontal Transfer of Antibiotic Resistance Genes in Enteric Bacteria
John K. Crane1*, Muhammad B. Cheema1, Michael A. Olyer1 and Mark D. Sutton2
1Division of Infectious Diseases, Department of Medicine, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY, United States
2Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY, United States
The SOS response is a conserved response to DNA damage that is found in Gram-negative and Gram-positive bacteria. When DNA damage is sustained and severe, activation of error-prone DNA polymerases can induce a higher mutation rate than is normally observed, which is called the SOS mutator phenotype or hypermutation. We previously showed that zinc blocked the hypermutation response induced by quinolone antibiotics and mitomycin C in Escherichia coli and Klebsiella pneumoniae. In this study, we demonstrate that zinc blocks the SOS-induced development of chloramphenicol resistance in Enterobacter cloacae. Zinc also blocked the transfer of an extended spectrum beta-lactamase (ESBL) gene from Enterobacter to a susceptible E. coli strain. A zinc ionophore, zinc pyrithione, was ~100-fold more potent than zinc salts in inhibition of ciprofloxacin-induced hypermutation in E. cloacae. Other divalent metals, such as iron and manganese, failed to inhibit these responses. Electrophoretic mobility shift assays (EMSAs) revealed that zinc, but not iron or manganese, blocked the ability of the E. coli RecA protein to bind to single-stranded DNA, an important early step in the recognition of DNA damage in enteric bacteria. This suggests a mechanism for zinc's inhibitory effects on bacterial SOS responses, including hypermutation.
The term "SOS response" as descriptor of a bacterial reactions to DNA damage was coined about 1975, the same year that the ship Edmund Fitzgerald sank in Lake Superior without sending out an SOS distress call. The SOS response triggers a constellation of changes in the bacterial cell, including cellular elongation, suspension of cell division, induction of DNA repair pathways, induction of latent bacteriophages, expression of bacteriophage-encoded toxins, and increased expression of error-prone DNA polymerases (Goodman, 2002). The latter underlies the increased mutation rate observed when the SOS response is strongly induced.
Recently, several laboratories showed that sublethal concentrations of certain SOS-inducing antibiotics induced resistance to other, unrelated antibiotics (Kohanski et al., 2010; Song et al., 2016; Bunnell et al., 2017). Their work and that of others have led to increased interest in whether SOS inhibitors could block emergence of antibiotic resistance (Nautiyal et al., 2014; Alam et al., 2016).
We initially became interested in the SOS response because of its role in inducing the expression of the Shiga toxins in Shiga-toxigenic Escherichia coli (STEC), because antibiotics induce production of the Shiga toxins Stx1 and Stx2, resulting in a worsening of the disease. Since previous work showed that zinc inhibited Stx production in STEC, we wondered if zinc's inhibitory effects were mediated by inhibition of the SOS response, and found that indeed they were (Crane et al., 2014). Indeed, zinc's inhibitory effects on the SOS response also blocked hypermutation in E. coli and Klebsiella strains (Bunnell et al., 2017).
Beaber et al. previously showed that induction of the SOS response with mitomycin C or ciprofloxacin dramatically increased the rate of horizontal antibiotic resistance transfer from E. coli to Vibrio cholerae, and from one strain of V. cholerae to another (Beaber et al., 2003). In this study, we first tested whether the inhibitory effects of zinc salts on SOS also extended to another member of the Enterobacteriaceae family, Enterobacter cloacae. We next tested whether zinc ionophores, such as A23187 or zinc pyrithione, would show increased potency against the hypermutation response. We also tested whether zinc could block the transfer of antibiotic resistance from Enterobacter to a susceptible E. coli recipient strain. Zinc salts and zinc pyrithione blocked hypermutation in E. cloacae and zinc also blocked horizontal transfer of ß-lactam resistance to another, unrelated E. coli strain. Last, we developed an electrophoretic mobility shift assay (EMSA) to determine if metals blocked binding of RecA to ssDNA. We demonstrated that zinc, but not other divalent metals, blocked the ability of RecA to bind to ssDNA in this EMSA.
Mitomycin C, ciprofloxacin (cipro), chloramphenicol (chloramph), zinc acetate, zinc pyrithione, A23187, agarose, ATP, ATP-γ-S, Sypro Orange protein stain, and copper phthalocyanine tetrasulfonate were from Sigma-Aldrich (St. Louis, MO). Gradient-impregnated antibiotic test strips were purchased from Liofilchem, Inc., (Abruzzi, Italy, and Waltham. MA). Copper pyrithione was from Combi-Blocks, San Diego, CA, while ceftazidime (ceftaz) was purchased from TCI, Tokyo, Japan. DMEM-F12 medium was from Gibco, Inc., Grand Island, NY, a subsidiary of Thermo-Fisher. DNase I was from Worthington Biochemical Corp., Lakewood, NJ. Purified RecA protein was purchased from New England Biolabs (NEB, Boston, MA). Blue Juice loading dye, SYBR-Safe DNA dye, and the 100 bp DNA ladder were from the Invitrogen division of Thermo-Fisher.
Bacterial Strains Used. Bacterial strains are described in Table 1.
Table 1. Description of bacterial strains used.
Hypermutation in E. cloacae Bacteria
As previously described, bacteria were grown overnight in LB broth at 37°C with 300 rpm shaking, then subcultured into DMEM-F12 broth medium for induction of hypermutation. The DMEM/F12 medium was supplemented with 18 mM NaHCO3 and 25 mM additional HEPES, pH 7.4, but omitted serum and antibiotics. The usual dilution from overnight was 1:100 into DMEM-F12, but this was varied if needed. After 75 min, the culture was divided into separate tubes and ciprofloxacin was added to a concentration equivalent to ½ to ¾ of the MIC for that strain. Differences in the concentration of ciprofloxacin used resulted in differences in the antibiotic resistance frequencies observed. Metals such as zinc were added at this time, and incubation was continued with 300 rpm shaking. Four hours after initiation of the subculture, the culture turbidities were measured as OD600 on a spectrophotometer, and serial dilutions were quickly plated onto LB agar to measure the total bacterial counts in each condition. In addition, 100–200 μl of undiluted culture was plated in triplicate onto plates of LB + 20 mg/L (20 μg/mL) chloramphenicol, a concentration 5 times the MIC of E_clo_Niagara. Antibiotic resistance frequencies were calculated as the (CFU of antibiotic resistant colonies/mL) ÷ (CFU of total colonies/mL). In some cases, the antibiotic resistance frequency was multiplied by 10e6 and expressed as "chloramphenicol resistance frequency per million" as previously shown, and in other cases these results were converted to a logarithmic scale. When zero colonies were observed on any of the triplicate plates, the lower limit of detection was calculated as if 1 colony had been observed; this was often the case in the control cultures, i.e., bacteria not exposed to any SOS-inducing antibiotic.
Cross-Species (Horizontal) Transfer of Antibiotic Resistance
In order to test for the transfer of genetic material from the ceftazR E. cloacae to ChloramphR fluorescent E. coli strain EC43, or vice versa, both strains were grown up in LB broth overnight, then both were subcultured into DMEM-F12 liquid medium. After 75 min, ciprofloxacin was added at the appropriate concentrations for each strain. At this time, metals such as zinc were also added. At the 4 h time point, the culture turbidities were read as OD600, and then dilutions were made in order to determine the total number of bacteria of each species in their separate cultures. Bacteria were then mixed in a 1:1 ratio, centrifuged at 1,600 g for 10 min, and then the bacteria were allowed to stand on the bench top at room temperature. After the bacteria had incubated together in the centrifuged pellets, the pellets were resuspended, and then plated on LB + 6 μg/mL ceftazidime and 20 μg/mL chloramphenicol, and the plates were inspected on the UV transilluminator box for double-resistant, green-fluorescing colonies. In pilot experiments, we tested mixing the two bacterial strains for 3, 4, and 6 h, but we never observed any transconjugant colonies at these durations of bacterial contact. Finally, we therefore allowed the centrifuged bacterial mixtures to stand on the bench top at room temperature for 20 h, and with this longer duration we did observe putative transconjugant colonies, meaning double-resistant, green-fluorescing colonies. Putative transconjugant colonies were picked and re-streaked on LB with ceftazidime + chloramphenicol. Biochemical tests were next performed to determine if the putative transconjugant colonies were E. coli that had gained resistance to ceftazidime, or E. cloacae that had developed chloramphenicol resistance and also acquired the plasmid expressing GFP. The biochemical tests done to distinguish Enterobacter from E. coli included indole production, acid production by the Methyl Red test, Voges-Proskauer test for acetoin production, and streaking on eosin methylene blue (EMB) agar. In all cases the putative transconjugants were E. coli that had become resistant to ceftazidime.
Testing for Beta-Lactamase-Encoding Genes in Enterobacter and in E. coli Transconjugants
We extracted total DNA from Enterobacter E_clo_Niagara and from the putative E. coli transconjugants that had acquired ß-lactam resistance using the AllPrep Bacterial DNA Kit from Qiagen (Hilden, Germany and Waltham, MA) according to the manufacturer's instructions. Purified DNA was sent to ID Genomics, Seattle, WA, for PCR detection of the extended spectrum ß-lactamase genes. PCR assays using broad-range primers initially identified the ß-lactamase as a member of CTX-M family, and then specific primers indicated the ß-lactamase was a CTX-M27. This "head start" from ID Genomics allowed us to then conduct our own PCR assays.
PCR Detection of CTX-M27 in E. cloacae and in Putative E. coli Transconjugants/Transformants
Plasmid DNA was prepared from the donor strain, E_clo_Niagara, recipient strain EC43, and from the putative transconjugant E. coli strains that emerged from the inter-species gene transfer experiments, using Qiagen Midi-Plasmid preparation kits (Waltham, MA). Primer pairs directed against the gene blaCTX−M27, encoding the extended spectrum beta-lactamase CTX-M27, were used for PCR and are shown in Table 2A. PCR was conducted on a Bio-Rad CFX96 real-time PCR machine using Bio-Rad iTaq Universal SYBR Green Master Mix in a final volume of 20 μL; Primer concentration was 0.5 μM, and 5 μL of the plasmid DNA prepared from each strain was used as the template for 35–39 PCR cycles using a 3-Step amplification protocol.
Table 2. Oligonucleotides used.
Electrophoretic Mobility Shift (EMSA; "Gel-Shift") Assay for recA Binding to ssDNA
Purified RecA protein from NEB was used for EMSA along with 10X Assay buffer (Tris acetate, pH 7.8, with magnesium) supplied by the manufacturer. To test for binding to ssDNA, a fluorescently labeled oligonucleotide was purchased from Integrated DNA Technologies, Inc., Coralville, IA. This 38-mer oligonucleotide was labeled at the 5′ end with 6-FAM (fluorescein) and the sequence is shown in Table 2B.
To test for RecA-DNA binding, we first did pilot experiments to determine how much fluorescent oligonucleotide was needed to produce a visible fluorescent band on agarose gels. We found that using 5 μM fluorescent oligonucleotide gave a visible band when 15 μL were loaded on an agarose gel. We next did pilot experiments to determine what molar ratio of RecA protein to oligonucleotide was needed to observe any gel shift.
The binding assay was carried out in duplicate or triplicate in 15 μL final volume in conical-bottomed 96-well plates kept on ice during reagent addition. The fluorescent oligonucleotide in assay buffer was added first, followed by zinc or other metals added to final concentrations of 0.1–1 μM. In addition, ATP or ATP-γ-S was added as noted to achieve a final concentration of 0.3 mM. Last, RecA protein was added from a concentrated stock to achieve the appropriate ratio relative to ssDNA. This ratio was varied to obtain RecA: oligonucleotide ratios of 2:1 to 5:1, as indicated in the Figure Legends. After addition of RecA, the plate was moved to the 37° incubator where it was sandwiched between pre-warmed metal blocks to speed heat transfer to the samples, and binding was allowed to proceed for 15 min. After the incubation, 2 μL of 10 X "Blue Juice" loading dye was added to the samples, and then samples were loaded into the wells of an agarose gel (1.5% agarose in 1 X TAE buffer, with SYBR-Safe DNA stain added to be able to visualize the DNA ladder).
Electrophoresis was carried out at 135 V for 35 min. After electrophoresis, the gel was visualized on the Gel-Doc E-Z fluorescent imager (Bio-Rad) using the Blue illuminator plate and the settings for SYBR Green, which also work for fluorescein. In some experiments, zinc or other metals were added to the agarose gel at the same concentration as that added to the binding assay. In this case, we used plastic rulers cut to the correct length in order to have some samples running in gel lanes with metal incorporated into the gel itself (Supplemental Figure 1). For quantitation, the image files from the Gel-Doc EZ were "inverted" to produce dark bands on a light background, then quantitated using Un-Scan-It Gel software for the MacIntosh computer (Silk Scientific, Orem, UT).
Measurement of Extracellular DNA
DNA released into culture supernatants by SOS-inducing treatments was measured in a UV spectrophotometer by A260/A280 ratio.
Data shown are means ± SD. Tests of significance were by analysis of variance (ANOVA), using Dunnett's test for multiple comparisons. Graphs were produced using GraphPad Prism (San Diego, CA).
Testing for Induction of Hypermutation in Enterobacter Cloacae Strain E_clo_Niagara
Initial experiments were performed to see if ciprofloxacin could induce hypermutation to the unrelated antibiotic, chloramphenicol, in E_clo_Niagara. For these experiments we used a chloramphenicol concentration five times the starting MIC for this strain. Figure 1A shows that a sublethal concentration of ciprofloxacin did greatly increase the chloramphenicol resistance frequency in this strain, and this increase was reversed by zinc acetate in a dose-dependent fashion. As previously observed, however, 200 μM zinc acetate was needed to observe strong inhibition of the mutator response (Figure 1A). In addition to zinc, CuSO4, and CoCl2 also were able to inhibit the hypermutation response (Figure 1B), but they were less potent than zinc, as we previously observed with E. coli and Klebsiella (Bunnell et al., 2017). In contrast to zinc, iron (as FeSO4), and manganese did not inhibit ciprofloxacin-induced hypermutation (Figure 1C; data not shown for MnCl2). Interestingly, gallium nitrate also had a mild inhibitory effect on ciprofloxacin-induced hypermutation, even though gallium is a trivalent rather than divalent element.
Figure 1. Effect of zinc and other metals on ciprofloxacin-induced hypermutation in E_clo_Niagara. (A) Effect of ciprofloxacin and zinc on the chloramphenicol resistance frequency in E_clo_Niagara. (B) Comparison of zinc with CuSO4 and CoCl2 on ciprofloxacin -induced mutation frequency. To allow comparison of separate experiments, the results were expressed as a percent of the ciprofloxacin-induced mutation frequency for each experiment, then averaged. Each line on the graph shows the mean ± SD of at least two separate experiments. (C) Lack of effect of FeSO4 on ciprofloxacin induced hypermutation. (D) Effect of gallium (III) nitrate on ciprofloxacin-induced hypermutation in E_clo_Niagara. *Significant compared to cipro alone by ANOVA; graph shown is the Mean ± SD of 3 separate experiments, expressed as % of cipro-induced.
We next tested whether metal ionophores, such as A23187, or zinc pyrithione, could improve the potency of zinc against the hypermutation response. Our results with A23187 plus zinc were unexpectedly negative, because A23187 alone, in the absence of any metal, consistently increased ciprofloxacin-induced mutation to chloramphenicol resistance (Supplemental Figure 2A, middle bar). Compared to zinc acetate alone, the addition of 100 μM A23187 unexpectedly reduced the potency of zinc (dose-response curve shifted to the right, Supplemental Figure 2B). These results may be consistent with A23187 acting more as a zinc chelator than as a zinc ionophore in E_clo_Niagara. Indeed, most of the literature on A23187 in bacterial systems has been with Gram-positive rather than Gram-negative bacteria, and it may be that A23187 is unable to conduct zinc across the double membranes of enteric Gram-negative bacteria.
Despite the unexpected results with A23187 + zinc, zinc pyrithione (ZPT) showed much more promise as an inhibitor of SOS-induced hypermutation. ZPT inhibited ciprofloxacin-induced hypermutation at concentrations near 1 μM (Figures 2A,B). While copper pyrithione (CuPT) is often viewed as more toxic to living cells than ZPT (Bao et al., 2014), we found that ZPT was ~4-fold more potent than CuPT in its ability to inhibit bacterial growth (Figure 2C). Similarly, ZPT was better than CuPT in blocking the mutator response (Figure 2D). Direct comparison of zinc acetate to ZPT showed that ZPT was ~100 fold (2 logs) more potent than zinc acetate in inhibition of SOS-induced hypermutation (Figures 2E,F). The high potency of ZPT against the SOS response might allow the effects of zinc to be exploited in situations where concentrations of zinc acetate as high as 0.2 mM cannot be achieved.
Figure 2. Effect of zinc pyrithione (ZPT) on ciprofloxacin–induced mutation response in E_clo_Niagara. ZPT is also known as 1-hydroxypyridine-2-thione zinc salt. (A) Dose-response relationship of ZPT on ciprofloxacin-induced mutation to chloramphenicol resistance, showing a single representative experiment. (B) Dose-response relationship of the ZPT on chloramphenicol resistance frequency, showing combined results from 4 separate experiments. ZPT at concentrations greater than or equal to 0.8 μM produced statistically significant reductions in chloramphenicol resistance frequency in (A,B) (< 0.05). (C) Comparison of ZPT and copper pyrithione (CuPT) on growth of E_clo_Niagara. (D) Comparison of ZPT and CuPT on inhibition of chloramphenicol resistance. Although 2 μM CuPT produced a partial inhibition of hypermutation, higher concentrations of CuPT showed a paradoxical increase back toward the ciprofloxacin-induced level; this was due to increased killing of bacteria at 4–6 μM, resulting in a drop in the denominator value (total counts). (E) Dose response relationship of zinc compounds in inhibition of mutation to chloramphenicol resistance, expressed on a log scale. ZPT curve is the mean ± SD of 4 experiments, while the zinc acetate curve is the mean ± SD of 7 separate experiments. (F) Chemical structure of zinc pyrithione.
Testing for Horizontal, Inter-species Gene Transfer in Enteric Bacteria
Having shown that the SOS response could be induced in E_clo_Niagara, we next tested whether SOS induction could trigger transfer of antibiotic resistance elements between species, as shown by Beaber et al. (2003). In contrast to the study by Beaber et al., who worked with V. cholerae, an organism that is naturally competent for uptake of extracellular DNA, E. coli and related enterics are not naturally competent, and although they can take up DNA from the environment, such DNA is often degraded and used as food rather than incorporated into their genomes (Finkel and Kolter, 2001; Chen and Dubnau, 2004; Palchevskiy and Finkel, 2006). Therefore, it was unclear whether the findings of Beaber et al. on SOS and horizontal gene transfer would apply to a different microbial system.
We tested whether the ß-lactamase of Enterobacter E_clo_Niagara could be transferred to a susceptible STEC E. coli strain, EC43. EC43 also possessed its own plasmid, encoding the green fluorescence protein (GFP) and resistance to chloramphenicol. As described in section Materials and Methods, we induced the SOS response in each bacterial strain, then mixed together the 2 strains of bacteria at a 1:1 ratio, and, after allowing the mixtures to incubate for increasing periods of time, then plated aliquots on LB agar + chloramphenicol + ceftazidime. Plates were examined on the UV transilluminator box for green-fluorescing colonies resistant to both antibiotics. In initial experiments where the E_clo_Niagara and EC43 were allowed to co-mingle for 3, 4, or 6 h before plating on antibiotic selective agar, no green fluorescing colonies were observed on the double-antibiotic plates. When the 2 species of bacteria were allowed to co-incubate for 20 h, however, green colonies resistant to both antibiotics were observed in the condition in which the SOS-response was induced with ciprofloxacin (Figure 3 and Supplemental Figure 3A). Addition of 0.2 mM zinc acetate blocked the appearance of the putative transconjugants when zinc was added to both bacteria along with ciprofloxacin (Figure 3A). Identification of the double-resistant, green colonies showed them to be E. coli that had acquired resistance to ceftazidime, rather than E_clo_Niagara that had acquired the plasmid encoding GFP and the chloramphenicol resistance marker. Control experiments with EC43 alone, not mixed with E_clo_Niagara, failed to show ciprofloxacin–induced acquisition of ceftazidime resistance (zero colonies observed in 10 separate experiments, all done in triplicate).
Figure 3. Role of the SOS response in horizontal transfer of beta-lactam resistance from E_clo_Niagara to susceptible E. coli strain EC43. (A) Induction and inhibition of inter-species gene transfer. E_clo_Niagara and E. coli EC43 were both treated separately with ciprofloxacin to induce the SOS response, then mixed and allowed to co-incubate for 20 h. The mixtures were plated on LB + ceftazidime + chloramphenicol, and examined under UV light for green fluorescing colonies resistant to both antibiotics. Since the colonies fitting the above description all tested as being E. coli, not Enterobacter, the frequency of emergence of such colonies was calculated relative to the number of EC43 bacteria placed into the original mixture. Zinc acetate concentration was 0.2 mM; *significantly increased compared to control, **significantly decreased compared to the conditions receiving ciprofloxacin. (B) Effect of ciprofloxacin applied to E_clo_Niagara alone, E. coli EC43 alone, or both strains, on the horizontal gene transfer. The concentration of zinc used in (B), far-right column, was 0.2 mM; *significantly increased compared to control, **significantly decreased compared to the conditions receiving ciprofloxacin. (C) Effect of ciprofloxacin and zinc on horizontal gene transfer, with the transconjugant frequency expressed on a log scale. 0.1 mM zinc was used in panel C; *significantly increased compared to control, †significantly decreased compared to the conditions receiving ciprofloxacin. (D) Effect of zinc and DNase I on release of double-stranded DNA into culture supernatant; *significantly decreased compared to the plus cipro condition. (E) Effect of zinc and DNase I on frequency of emergence of putative transconjugants (green fluorescing colonies resistant to both antibiotics), showing that zinc, but not DNase, blocked the transfer of beta-lactam resistance to the E. coli strain. *significantly decreased compared to the condition receiving ciprofloxacin alone. †There were no CeftazR, ChloramR E. coli colonies in the zinc-treated group, so the data points shown are calculated as if 1 colony had appeared.
We next induced the SOS response in EC43 and E_clo_Niagara separately to try to determine if SOS induction was more important in the donor strain or the recipient strain. When only the donor strain, E_clo_Niagara, was treated with ciprofloxacin, putative transconjugants remained rare (Figures 3B,C, respectively). When only E. coli EC43 was subjected to SOS induction with ciprofloxacin, putative transconjugants were regularly observed in substantial numbers (Figures 3B,C). The greatest frequency of transconjugants was observed, however, when both the donor and the recipient strain were treated with ciprofloxacin (Figures 3B,C). Zinc acetate consistently blocked the SOS-induced transfer of ß-lactam resistance phenotype. When the zinc concentration was reduced to 0.1 mM, however, the reversal of the SOS response was only partial (Figure 3C, right-hand set of points).
In the course of these experiments, we noted that induction of the SOS with ciprofloxacin, followed by a 20 h incubation, was accompanied by a release of DNA into the supernatant (Figure 3D; similar results were obtained with strain EC43 as well; data not shown). During the 20 h incubation, bacterial counts of both strains increased ~3-fold compared to the counts at the 4 h collection point, for both the antibiotic-treated and untreated conditions. Therefore, the DNA release observed is not due to massive die-off of bacteria during the 20 h incubation. Addition of zinc acetate and DNase I both significantly reduced the amount of DNA detectable in bacterial supernatants (Figure 3D). Therefore, we tested whether addition of exogenous DNase I would block the horizontal transfer of ß-lactam resistance to EC43. As shown in Figure 3E, DNase I had no effect on the frequency of appearance of the double-resistant transconjugant colonies in this assay, while zinc again showed its strong inhibitory effect. This also hinted that, although release of DNA into the supernatant occurred during our 20 h incubation, transfer of relevant DNA encoding antibiotic resistance likely does not occur by bulk release of DNA into the culture medium, but instead may require direct cell-to-cell contact.
The experiments of Figure 3 showed that the beta-lactam resistant phenotype from E_clo_Niagara, could be transferred to a susceptible E. coli strain when the SOS response was induced. We next tested whether we could detect the gene encoding the resistance had indeed been transferred to the recipient strain. The extended spectrum beta-lactamase (ESBL) gene family is large, but the ID Genomics company helped us narrow our focus to blaCTX−M27. Using this knowledge, we constructed two sets of primers directed against CTX-M27 (section Materials and Methods and Table 2). PCR with both sets of primers revealed that DNA encoding CTX-M27 was present in the donor strain E_clo_Niagara and in the E. coli transconjugants, but not present in parental E. coli strain EC43. Quantitative PCR analysis from a typical experiment showed that the number of cycles to threshold, Ct, for amplification from the plasmid preparations from the various strains were: E_clo_Niagara: 28.0 ± 0.7; EC43: NA (no amplification); Transconjugant 1: 32.2 ± 1.0; Transconjugant 2, 27.3 ± 0.5; and Transconjugant 3, 35.8 ± 0.3, using the first set of primers shown in Table 2. Similar results were observed using the second set of primers as well.
RecA-ssDNA-Zinc Interactions
Previously, we showed that zinc blocked the SOS-induced cleavage of the LexA repressor in live E. coli bacterial cells, and zinc also blocked RecA-mediated LexA cleavage in a cell-free assay using purified bacterial proteins plus cofactors (Bunnell et al., 2017). That previous work strongly suggested that zinc acted on RecA, as previously suggested by other authors (Lee and Singleton, 2004), but left open a narrow possibility that zinc might act on LexA, or on the protein-protein interface where RecA binds to LexA, possibly interfering with LexA's own proteolytic cleavage. To determine if zinc had effects on RecA alone, in the absence of LexA, we performed EMSAs using fluorescein-tagged ssDNA and purified RecA to see if zinc influenced the ability of RecA to bind to ssDNA (Svingen et al., 2001). Figure 4A shows the effect of adding increasing amounts of RecA protein in the presence of a fixed, 5 μM concentration of fluorescent 38-mer ssDNA oligonucleotide. A marked upward shift in mobility was noted in the presence of 10 μM RecA (lanes 4–6, 2:1 molar ratio of RecA to ssDNA), and the proportion of ssDNA migrating in this slower, upper band increased as the ratio of RecA to ssDNA was increased (Figure 4A, lanes 7–14). Figure 4A shows the results obtained in the absence of ATP-γ-S, but experiments in the presence of 0.3 mM ATP-γ-S were also performed, and the densitometry scans of the fluorescence images, with and without ATP-γ-S, are shown in Figure 4B. Addition of zinc markedly reduced the proportion of ssDNA migrating as the upper band, and increased the amount of ssDNA in the original, unbound position (black arrows). Gels scans of Figure 4C and a parallel experiment done in the absence of ATP-γ-S are shown in Figure 4D. As shown in Figure 4D, the inhibitory effects of zinc on RecA binding were enhanced in the presence of ATP-γ-S, consistent with the known role of ATP and ATP-γ-S in supporting the dissociation of bound RecA monomers from the ssDNA nucleofilament (Wigle et al., 2006). The inhibitory effects of zinc on RecA binding persisted even when higher concentrations of RecA were used in the assay (Figure 4E; 4:1 ratio of RecA to ssDNA). In contrast to the inhibitory effects of zinc, manganese did not inhibit ssDNA binding to RecA (Figure 4E), and neither did 1 μM FeSO4 (Figure 4F). Surprisingly, the newly discovered RecA inhibitor, copper phthalocyanine tetrasulfonate, also failed to inhibit RecA-ssDNA binding in this assay (Figure 4F). The 1 μM concentration used in Figure 4F, however, was lower than the 10–15 μM concentrations used in the report by Alam et al. however (Alam et al., 2016). Nevertheless, Figure 4 shows that RecA binding to ssDNA can be readily detected using a fluorescently labeled oligonucleotide in agarose gels, and that zinc inhibits RecA binding to ssDNA at low, 1 μM concentrations, while iron, manganese, and a phthalocyanine tetrasulfonate compound did not. In addition, although zinc acts to block RecA-induced LexA cleavage (Bunnell et al., 2017), the presence of LexA is not necessary to observe the effects of zinc in vitro. Our results suggest that zinc blocks LexA cleavage by preventing the formation of the active RecA-ATP-ssDNA nucleofilament.
Figure 4. Effect of zinc and other metals on the binding of RecA protein to a fluorescently labeled single-stranded DNA probe. Purified RecA protein and a 38-nucleotide single-stranded DNA probe labeled with fluorescein were used in an electrophoretic mobility shift assay (EMSA) in 1.5% agarose gels. (A) Effect of increasing amounts of RecA on the migration of the fluorescently labeled ssDNA. Bands shown in (A,C) are the fluorescent ssDNA, but the digital image of the gel was "inverted" to produce dark bands on a white background. Lane 1, 100 bp dsDNA molecular weight ladder, with the bands 600 and 1,500 pairs indicated. Lanes 2 and 3, 5 μM fluorescent oligonucleotide alone, with no RecA. Lanes 4–6, ssDNA + 10 μM RecA; Lanes 7–9; ssDNA + 15 μM RecA. Lanes 10–12, plus 20 μM RecA; Lanes 13–14, with 25 μM RecA. (B) Quantitative gel scan of the gel shown in (A), as well as an additional gel done in the presence of ATP-γ-S (latter gel not shown), showing that as the RecA protein is increased, the percent of the ssDNA migrating as the upper band increases. (C) Effect of zinc on the gel shift induced by RecA protein, in the presence of 0.3 mM ATP-γ-S. Lane 1, 100 bp DNA MW ladder; Lanes 2–8 all received 5 μM ssDNA probe + 10 μM RecA (2:1 ratio of RecA to ssDNA). Lanes 5 & 6, + 0.3 μM zinc acetate; Lanes 7 & 8, + 1 μM zinc acetate. Zinc was also cast into the appropriate lanes of the agarose gel as described in section Materials and Methods and Supplemental Figure 3. (D), quantitative gel scans of the gel shown in (C), plus another gel done in the absence of ATP-γ-S, are graphed as a function of the concentration of added zinc. (E) Comparison of the effect of zinc on the gel shift assay, in contrast with the lack of effect of MnCl2. With RecA added to achieve a 4:1 ratio relative to ssDNA, the inhibitory effect of 1 μM zinc was still evident, while 1 μM MnCl2 failed to inhibit the RecA-induced upward shift in the migration of the ssDNA probe; ¥, not significant compared to Oligo + RecA, by ANOVA; *significant compared to Oligo + RecA; p < 0.01 by ANOVA. (F) Lack of effect of 1 μM FeSO4 and 1 μM copper phthalocyanine tetrasulfonate (¥), reported to be a RecA inhibitor, on the gel shift induced by RecA. †Not significant compared to RecA alone by ANOVA.
Although the SOS response has been discussed in the molecular biology literature for over 40 years (Radman, 1975), appreciation of the role of the SOS pathway in generation of antibiotic resistance has been very slow to percolate into the fields of clinical microbiology and infectious diseases. Recently, however, several laboratories have shown that sublethal concentrations of antibiotics can not only induce the SOS response, but also trigger the emergence of resistance to other, unrelated antibiotics (Kohanski et al., 2010; Song et al., 2016). In addition, our laboratory and others have shown that SOS-induced hypermutation, resulting in the emergence of antibiotic resistance, can be triggered by a wider array of compounds than classical inducers such as mitomycin C, ciprofloxacin, and UV light. The SOS response is induced in E. coli by oxidant stress and by a wide variety of drugs used in human and veterinary medicine, including some antiviral drugs, cancer chemotherapy agents, antimetabolites, arsenic compounds, the herbicide paraquat, and many other antibiotics, including those used for growth promotion in farm animals (Baharoglu and Mazel, 2011; Bunnell et al., 2017). We began studying zinc because it is an inhibitor of Shiga toxin (Stx) production in STEC, but later learned that zinc was blocking the SOS response itself, not just Stx toxin release. We showed that zinc, but not most other metals tested, blocked SOS-induced hypermutation to antibiotics such as rifampin and trimethoprim, in E. coli and Klebsiella pneumoniae (Bunnell et al., 2017). As expected from the work of others (Cirz and Romesberg, 2006; Recacha et al., 2017), the hypermutation response, and zinc's inhibitory effects on SOS-induced mutation were dependent on RecA, a key sensor of DNA damage and initiator of the SOS response (Bunnell et al., 2017).
In this study, we first tested whether the inhibitory effects of zinc could be observed in another member of the Enterobacteriaceae family, E. cloacae. Zinc blocked ciprofloxacin-induced hypermutation, reducing the emergence of chloramphenicol resistance, while cobalt, copper, and gallium showed similar, but weaker activity (Figure 1). We also showed that a zinc ionophore, zinc pyrithione (ZPT), blocked SOS-induced hypermutation with an inhibitory concentration 50% (IC50) ~100-fold lower than zinc salts. This high potency compares favorably with other RecA inhibitors that have been proposed (Lee et al., 2005; Wigle et al., 2009; Alam et al., 2016). Although ZPT can have some toxicity toward mammalian cells (Priestley and Brown, 1980), it has been cleared by the United States Food and Drug Administration (FDA) for use on the skin at concentrations up to 0.25% (7.8 mM) in "leave on" products such as lotions, and 2% (63 mM) in "rinse-off" products such as shampoos (Schwartz, 2016). Older literature on ZPT tended to focus on its antifungal effects (Pierard-Franchimont et al., 2002; Reeder et al., 2011), but more recent reports emphasize the activity of ZPT against bacteria as well (Schwartz, 2016).
Inspired by the work of Beaber et al., we tested whether induction of the SOS response would trigger horizontal gene transfer from a ß-lactam resistant, ESBL-producing Enterobacter strain to a sensitive E. coli strain. We were able to demonstrate transfer of the ß-lactamase gene to the E. coli EC43 strain after a prolonged, 20 h, co-incubation. Although we have referred to the newly-resistant EC43 colonies as "putative transconjugants," we have not conclusively shown that the mechanism of transfer of the ß-lactamase is by conjugation. Jung tested a panel of 15 Enterobacter aerogenes strains for their ability to conjugate with E. coli and found only 1 of the 15 strains capable of doing so (Jung, 2014). Indeed, when we tested for transfer of the AmpC chromosomal ß-lactamase from a different E. cloacae strain (BAA-1143) to EC43, putative transconjugants were either much rarer, or, in some experiments, not observed at all (data not shown). The rare transconjugants observed with strain BAA-1143 as the donor were also unstable genetically, and gradually lost their ß-lactam resistance with repeated passage.
PCR analysis of the E. coli transconjugants that had acquired ceftazidime resistance revealed that the CTX-M27 beta-lactamase gene was present in the plasmid DNA. Therefore, it may be more accurate to describe these resistant E. coli strains as transformants (Chen and Dubnau, 2004) rather than as transconjugants. Classically, conjugation proceeds via transfer of ssDNA via a conjugation pilus to the recipient strain, whereas transformation (such as by electroporation) is usually more efficient with double-stranded plasmid DNA. McCollister et al. recently described in vivo acquisition of the CTX-M27 plasmid by a Salmonella enterica strain in a relapsed infection (McCollister et al., 2016). In that case, the patient's Salmonella strain was initially susceptible to the 3rd-generation cephalosporins and the patient was treated with ceftriaxone. After relapse, however, the Salmonella strain was resistant to ceftriaxone, and comparison of the two strains allowed the investigators to determine that the Salmonella had acquired the CTX-M27 beta-lactamase, incorporated into a plasmid. The resistance element in Salmonella could be transferred back into a susceptible E. coli strain, where it also was located on a plasmid. Although we have not conducted the detailed DNA sequencing done by McCollister et al., we believe that our results may reflect a similar process, but with gene transfer between E. cloacae and E. coli. Notably, beta-lactam antibiotics such as ceftriaxone do induce the SOS response (Drago et al., 2004; Maiques et al., 2006), and so SOS induction could have played a role in the in vivo transfer of the resistance gene in the GI tract of the patient described by McCollister et al. It is also notable that the work of McCollister et al. (2016) and Beaber et al. (2003), and the work in this study all used wild-type enteric pathogens in their studies of DNA transfer between species. Over-reliance on highly passaged laboratory strains of E. coli in the past may have prevented earlier recognition of these phenomena.
Although DNA, especially ssDNA, is often degraded upon entry into E. coli cells via the competence (Com) gene locus (Finkel and Kolter, 2001; Palchevskiy and Finkel, 2006), it is possible that when the SOS is induced, RecA becomes abundant enough that it is able to catalyze recombination of ssDNA with the recipient cell's DNA before the newly acquired DNA is degraded (Beaber et al., 2003). If so, this would explain why transfer of the ß-lactam resistance was greatest when the SOS response was induced in the recipient as well as the donor strain (Figures 3B,C). While the mechanism of the DNA transfer is not clear, what is clear is that transfer is triggered by the SOS response, and that the transfer is strongly inhibited by zinc, raising the possibility that zinc compounds could be used to block horizontal transfer of antibiotic resistance in situations where this gene transfer would be undesirable.
Other authors have suggested that a good way to exploit RecA inhibitors under development would be to reverse pre-existing antibiotic resistance, i.e., in strains already resistant to antibiotics such as the quinolones (Alam et al., 2016; Recacha et al., 2017). We suggest that a more effective strategy would be to use RecA inhibitors, including zinc compounds, to prevent the emergence of resistance in situations where sensitive microbes are still present, but induction of the SOS response would be expected or inevitable, such as when UV light, ionizing radiation, cytotoxic cancer chemotherapy, or other SOS-inducing drugs are used (Rutala et al., 2010).
Although zinc-containing RecA inhibitors show promise for preventing emergence of antibiotic resistance, a concern is whether zinc resistance could emerge as a bacterial counter-measure. Zinc resistance seems to be uncommon in bacteria, but has been reported (McHugh et al., 1975; Hau et al., 2017). Therefore, if RecA inhibitors are ever adopted for use in human or veterinary medicine, animal agriculture, or food processing, prudence and stewardship in use of these RecA inhibitors may become as necessary as prudence and stewardship in the use of antibiotics themselves.
MC did the initial hypermutation experiments with E. cloacae and began the inter-species gene transfer experiments. MO did all of the experiments with zinc ionophores. JC completed inter-species gene transfer experiments, performed the EMSA's, the PCR experiments, and wrote the manuscript. MS helped design experiments, reviewed the manuscript, and made helpful critiques.
This work was supported by funds from the Dept. of Internal Medicine, University at Buffalo, and by support from the CLIMB UP program for the summer stipend for MO.
We thank Elena Rechkina, Ph.D., ID Genomics, for help in determining the type of ß-lactamase present in E_clo_Niagara. We also thank Eric DeCourcey for assistance in ordering supplies and reagents.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb.2018.00410/full#supplementary-material
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Keywords: antibiotic resistance, RecA, electrophoretic mobility shift assay, Enterobacter cloacae, extended spectrum beta lactamase, CTX-M27, Zinc pyrithione
Citation: Crane JK, Cheema MB, Olyer MA and Sutton MD (2018) Zinc Blockade of SOS Response Inhibits Horizontal Transfer of Antibiotic Resistance Genes in Enteric Bacteria. Front. Cell. Infect. Microbiol. 8:410. doi: 10.3389/fcimb.2018.00410
Received: 01 August 2018; Accepted: 05 November 2018;
You-Hee Cho, Cha University, South Korea
Chulhee Choi, Chungnam National University, South Korea
Yang Zhang, University of Pennsylvania, United States
Copyright © 2018 Crane, Cheema, Olyer and Sutton. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: John K. Crane, jcrane@buffalo.edu


Want to see for yourself what makes us different? Click on the photos below to enjoy the virtual tour.
All My Kitties is an in-home cat boarding service set in a quiet, residential neighborhood. Our indoor cattery is directly adjacent to the outdoor "Catio", surrounded by our lush English Garden. Cats enjoy year-round views of flora and fauna overlooking Lake Washington and Mt Rainier.
See more photos of happy feline guests taking it easy over on the kitty photo blog!


If you belong to the generation which still remembers the first time ever that you've seen a cell phone, then you're already smiling with a few ideas on your mind!
Just think about it: how many years has Beckham been a part of our lives? Why is it that all generations simply adore this man?


The Gassaway Mansion at Christmas time is a beautiful site to see, especially for a wedding as beautiful as Jenn and Chad's. Getting to know them through their engagement session made us so excited for this big day! Stephen and I were thrilled to utilize all of the vintage decorations and lighting to create these images below.


A Russian oligarch has reportedly paid €5.24 million towards the salary owed to Fabio Capello, the Italian head coach of Russia's national football team.
Alisher Usmanov, the second largest shareholder in the English Premier League team Arsenal, stumped up the cash after a request from the Russian government, according to Russian media reports.
Sports Minister Vitaly Mutko asked him to give the Russian Football Union (RFS) a subsidized loan worth €5.24 million to cover part of the wages in a bid to "save Russia embarrassment", especially as it prepares to host the World Cup in 2018.
Capello and the team's general manager, Oreste Cinquini, are reportedly owed around €7.9 million and were last paid in June, the month Russia was booted out of the World Cup in the group stage.
The RFS said in November it could no longer afford to pay the wages.
Mutko said on Thursday that the salary issues would be resolved within the next couple of days.
"We spoke with Alisher Usmanov for around an hour and managed to get things sorted out," he told the news agency RBC-Sport.
"As a citizen of Russia, he would also have been embarrassed that we as a country that plans to host the next World Cup cannot even pay the head coach of Russia."
Capello's salary was estimated at €9 million when he took the job on, making him the highest-paid coach at last summer's World Cup in Brazil, according to a Forbes ranking.
But following Russia's early exit from the tournament, Capello was described as a "thief" by a far-right politician and summoned to the Russian parliament to explain himself.