text:
Q: onclick is not fired in a scrollable div I'm not able to capture the click event on a div which is inside the scrollable div in some devices like Tab 3 and Galaxy grand. But it is working absolutely fine in other android devices.
My parent div has style
#parentDiv { overflow:true}

I have tried all methods like
$(document).on("click","#id"+i,function(){
    alert("tapped");
});
$(document).on("tap","#id"+i,function(){
    alert("tapped");
});
$(document).on("swipeleft","#id"+i,function(){
    alert("tapped");
});

where "id"+i are the dynamically generated divs inside parentDiv
Even I tried replacing the divs with "button" and "a" tag, still does not work

A: I am not getting your problem, but I can give you an idea to debug the problem. I don't think, it is because of the scrollable HTML. What you can do is, while iterating the elements add an onlick attribute to with some alert statement. If it works the problem is somewhere in your script.
For example,
JS
var ele = '';
for(var i=0; i<arr.length; i++){
  ele += 'element +' i '+'
}

A: I have face similar problem and it is simple to solve, as you did not detail in case you are adding or midifying the divs dinamically.
Be sure the div exist in the dom at the moment the ready page triggers, if you are adding the div or parent dinamically the trick is to attach the click event inside the parent and filter by children, that way the click event is attached to an existent element.
The parent should exist since the beginning, if you are targetting the same event to all child divs use a class instead, if no your ids are ok.
Something like
$('#parentDiv').on('click', '.myClassDiv', function (){…});

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Knowledge Unlatched Pilot is given Department for Employment and Learning backing
Knowledge Unlatched (KU) is pleased to announce that the Department for Employment and Learning (DEL) has agreed to make a grant of up to £550 to universities in Northern Ireland who participate in the KU Pilot.
Read PDF version
This grant contribution will be used to reduce the participation fee paid by university libraries in Northern Ireland by 50%.
Employment and Learning Minister Dr Stephen Farry said: "We are pleased to support this new and timely model. It is essential that the Department supports innovation in scholarly communication as part of our wider aim to maximise the dissemination of publicly funded research. This project is a good example of how we might move forward to sustainable academic publishing and open access."
The financial backing from DEL will enable university libraries in Northern Ireland to secure the benefits of participating in the KU Pilot. Full details of how to pledge can be viewed on the Jisc Collections website where universities are invited to sign up.
Frances Pinter, Executive Director of KU, added: "We're delighted to see this support coming from Northern Ireland with DELNI enabling university libraries to participate in facilitating open access for books."
This grant will be available to higher education institutions in Northern Ireland who sign up to the KU Pilot Collection. The deadline for signing up is 28 February 2014.
1. The Department for Employment and Learning aims to promote learning and skills, to prepare people for work and to support the economy. Further information on Department and its programmes can be found at: http://www.nidirect.gov.uk/skills-to-succeed or http://www.delni.gov.uk/
2. Jisc Collections supports the procurement of digital content for education and research in the UK. Jisc is managing the pledging process of the KU Pilot for UK participants.
3. Knowledge Unlatched is committed to changing the current business model for publishing to one with libraries sharing the costs of scholarly books. Knowledge Unlatched believes that by working together libraries and publishers can create a sustainable route to Open Access for scholarly books and secure long-term cost savings for their own institutions by sharing the costs of making HSS monographs available on a Creative Commons licence.

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An after-school program for students in Lincoln, Mass.
All full-time LEAP staff members are certified in CPR and first aid, and they must undergo at least 20 hours a year of continuing education as required by the state Department of Early Education and Care.
I have a B.A. in early childhood education from the University of Massachusetts at Amherst and an A.S. in art and have been certified in outdoor education through the Outdoor Leadership Program at Greenfield Community College. I've been working at LEAP for 13 years and love the new role I've been so honored to take on. I look forward to working with staff, parents and the community in making LEAP continue to excel as the amazing after-school program that it is and can be!
I was lucky enough to begin working at LEAP in August of 2015. I attended Bently College (now a university) and majored in accounting. My job combines working with the "numbers" in the office and interacting with the children during the day. The best of both worlds! I keep pretty busy outside of LEAP with swimming, tennis, and golf. I also am involved with a couple of bands. The best time of the day is when the kids arrive with their eager faces looking forward to being there. That always makes me smile!
I have been involved with LEAP in some way since 2004. This year I am excited to return to LEAP as the Site Coordinator. The past three years I have been working in international sales to try out the corporate world, but my love for kids has brought me back to this wonderful program. I have worked with children of all ages and I'm eager to share with them my passion for arts and sports. I'm excited to assist Katie and work with the staff on new methods of teaching so that we can make LEAP the best it can be!
I started working at LEAP in the spring of 2016 and became the Kindergarten Coordinator at the beginning of the 2016-2017 school year. I have a BA in Sociology from UMASS, Lowell, and I love working with children of all age groups. In previous years I have worked at the YMCA in Newtown. I also have a passion for all things Halloween. I work as the Creative Director at Fright Kingdom in Nashua, New Hampshire, and I own my own business making props and costumes for various Haunted Attractions. I love learning new things from the students and spending time doing all sorts of different activities. LEAP is such a wonderful place to work and I'm so happy to be here!
I am so excited to be returning to LEAP part time this year! Although I work full time now as a custodian at the schools, I couldn't give up the fun times here at LEAP. Every Wednesday I have some time to join the crew before work. Get ready for lots of LEGOs, Fort Building, and hiking around Lincoln—just a few of my specialties. In my free time, I'm taking classes at MCC towards my BS in business, and I love music and the great outdoors.
This is my first year here at LEAP but I've been in Lincoln a while now. I graduated LS in 2014 and have worked at the Commons, Donelans, and I help coach soccer and basketball for the Lincoln middle school. I love sports and I am psyched to run some rec activities and get to know the kids. In the future I am considering returning to school for sports management, but maybe I'll find a new passion here at LEAP. Can't wait for an awesome school year!
I am so excited to be joining LEAP this year for the first time! I worked at Lincoln Day Camp over the summer and enjoyed the kids and the campus so much I just had to come back! This year, I will be a sophomore at Middlesex Community College, where I am considering majoring, or adding a minor in early childhood education. I am a super outgoing and enthusiastic person and I love sports and nerdy things like Dragonball Z and Batman!
I'm so happy I get to be a part of LEAP this school year! After graduating from UMass Boston in 2016 with a double major in English and Anthropology, I started working at the Lincoln Smith and Brooks schools as a Special Education tutor. I was also the Arts and Crafts teacher this year at the Lincoln Summer Camp. All of the kids who attend LEAP are familiar faces to me, many of who I get to see everyday at school! I really love being a part of the Lincoln community and educating children is my biggest passion in life so I am very excited to work at LEAP this year!
I am elated to be joining Leap this year for the first time! I grew up in Lincoln and attended Lincoln Sudbury Regional High School before transferring to Middlesex School in Concord, MA in order to pursue soccer and a rigorous education system that included school on Saturdays. I graduated Middlesex in 2015 and went on to play soccer at Union College up in Schenectady NY for 1 year before returning to Boston in order to act in a couple of movies: The Burning Woman and Pretty Woman (2018). My education presses onward via coding and other nightly/morning classes. However the main reason I returned to Boston was to join Boston Cares, a non-profit that's apart of the helping hands network. I've tutored adults and kids in math,science, English, and coached Lincoln youth soccer for two years. Get ready for homework to become fun-work and tons of creative recreational games! I am extremely grateful to be apart of such a wonderful team here at leap!!
I am very excited to be the newest member of the LEAP team! I have been working with kids for over three years now ranging from kindergarten to 8th graders. I've worked at the Umana school in East Boston, ran basketball and baseball camps in Acton, and worked at the summer camp in Boxbrough. I just spent a year at URI and am happy to be back home, continuing my education in the fall at Middlesex Community College. I am currently pursuing my degree in Early Education and child development and I am so happy to be working at LEAP to gain experience towards my future career goals. I have to thank my childhood friend Griffin introduced me to this awesome program, and Katie for allowing me the opportunity to work with these amazing kids!


text:
Q: Import cv2 error but opencv-python is already installed I've been trying to make a project with opencv, so I followed the YouTube video (It's in python), the video told me to go to cmd (win10) and use the command "pip install opencv-python" so that I can use the command import cv2. But the problem is after I did everything it still gave me an error.
I've done some research on it and nothing seem to work. I tried restarting my laptop or reinstall opencv, but nothing works for me. I'm thinking maybe it's because the PATH was wrong so it couldn't access it? But I've never worked with these before so I don't know what I'm supposed to do. I've seen some people with the same problem but the solution doesn't seem to work.
I'm using:
python 3.10.8
windows 10
IDLE Shell 3.10.1

A: Welcome to Stack Overflow!
Try installing with pip3
pip3 install opencv-python


Thanks to CMGer Don Thuotte.
Happiness is always a big topic in the world of science, psychology, and spirituality. Google happiness and you will many things from "Positive Psychology," to Certification in the Science of Happiness, and a variety of inspired TED talks.
After pouring through endless blog posts and websites, I decided I couldn't read one more set of "how to be happy" guidelines from the experts. What I wanted was down to earth, real-people experiences.
I informally surveyed ten of my more successful pals about their personal happiness. Some had everything going well for them in all areas, others were in rough spots in their marriages, some had health issues, and a few were struggling financially. All agreed however, that no matter what their challenges were, they had the ability to create a general state of happiness. Also, these people didn't seem to view challenges as problems, but rather, as opportunities to shift into better possibilities. Here is the collective response to the question, "What is happiness for you?
1. Get in alignment with your heart and head in regard to what you are doing. Or stop doing it. Or start doing what brings you into alignment. There's a feeling of power and peace when one is operating from a place of inner integrity.
2. Consider an attitude adjustment. There are times in life when we don't seem to get what we intend. Or people don't behave the way we would like. Years ago I found a book about a successful businessman who had had a spiritual awakening as a result of changing how he did business. He chose to see everyone and everything through a filter of generosity and trust. Those were the qualities he eventually experienced over and over again. The Universe seems to mirror back to us exactly what we convey in our actions, emotions and viewpoints.
4. Acknowledge yourself, acknowledge others. Look for all the ways you contribute, create, explore, and love in life. Feeling good is what happiness really is. When you take clear inventory of just how much you really accomplish in life, you will feel good. Acknowledging others goes a long way toward creating good will by telling people in words, writing, or actions that you recognize and appreciate them.
5. Let go of trying to control outcomes. Letting go puts us in a place of feeling trust in life. We can rarely control how things turn out, but we can control our interpretations and responses to the results. Letting go also opens up our ability to perceive new solutions, let more inner energy flow, and relax and de-stress.
6. Do things that raise your self-esteem and don't do things that lower it. Stay in integrity as much as possible and forgive yourself when you don't.


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Why does my image not always display on my PDF?
Why is my Credit Note PDF showing negative currency amounts when the other currency columns are positive?
Why does my PDF generate with an error sometimes and not others?
How I can add our T&C's to the bottom of our quotation PDF?
How can I view and edit a downloaded XSL template file? Which application should I use?


Includes AT120Eb Cartridge and AT-HS10BK Headshell.
The AT120Eb cartridge features a vector-aligned dual magnet design and A-T's exclusive Paratoroidal signal generator. .3 x .7 mil elliptical.
This product has been discontinued. Please see similar model VM530EN/H.
Will this work on my sl-d2 turntable? I can not find a needle that fits my current cartridge... any suggestions would be great!!
Yes, it appears your listed turntable will accommodate a standard 1/2 inch mount cartridge. You may confirm with the turntable manufacturer.
The new Audio Technica turntable I just purchased comes with an ATN 95E stylis. (1) Is the 120EB/HSB combo compatible with my AT turntable LP120Pro and (2) if it is will this significantly improve the sound quality?
Yes, the AT120EB cartridge is an upgrade from the AT95E cartridge may be used on the AT-LP120-USB turntable.
What will the counter weight setting be on my AT LP120 using this cartridge? Is there a link I can go to that has this information?
If I buy this combo will I need to buy a needle as well?
Thank you for your inquiry. No the needle is the orange plastic piece. It is included with the cartridge. However, If you would like to purchase an extra needle you will need the ATN120EB.
Hi. I recently purchased a brand new Roland TT99 with an AT95ECD cartridge with on a Roland headshell. Does this combo kit fit the Roland TT99 tonearm? Thank you.
Thank you for your inquiry. Your headshell does take a half inch mount, which is what this cartridge is. Please note this is a low compliance cartridge which is best suited for tone arm with high tone arm mass. I cannot find any information about the effective tone arm mass for the turntable. You may need to ask Roland.
Hello, what is the average time of life of this cartridge/needle? I already got one and I'm about to use it but I thought it would be convenient to know that. Thanks!
Thank you for your inquiry. The cartridge should last years. For the stylus it depends on several factors, especially the tracking force. You should get between 500 and 1000 hours on the stylus.
Does the cartridge come pre-aligned in the headshell or does it need to be adjusted once installed?
The cartridge should be pre-aligned. However, checking the alignment is an excellent approach before use. If you need assistance with aligning your cartridge, please visit the following link: http://blog.audio-technica.com/audio-solutions-question-of-the-week-how-do-i-align-the-cartridge-on-the-at-lp120-usb-and-at-lp1240-usb-turntables/ If you have any further questions, please contact the Audio-Technica United States (ATUS) Audio Solutions Team at: audio-solutions@atus.com Thanks!


text:
Secondary Effects Doctrine
By David L. Hudson Jr.
Other articles in Legal Terms and Concepts Related to Speech, Press, Assembly, or Petition
The secondary effects doctrine is used when content-based laws, many times regulations of adult-oriented businesses, are aimed at the secondary effects of protected expression. The laws can more easily pass First Amendment scrutiny. Before the secondary effect doctrine was introduced, Boston created an "adult entertainment district," as shown in this 1974 photo, in hopes it would keep the sex business from spreading to other parts of the city. (AP Photo/J. Walter Green, used with permission from the Associated Press)
The secondary effects doctrine permits normally unconstitutional content-based regulation to be treated as if it were content-neutral.
The doctrine took shape in the context of regulation of adult entertainment businesses, which allegedly impose adverse side effects — for example, increased crime and decreased property values — on the communities in which they are situated. In effect, the doctrine allows for a reduced degree of judicial scrutiny in the instance of regulation of adult-oriented expression. This contrasts with the strict scrutiny requirement normally accorded regulation of First Amendment–protected expression.
Critics claim that the secondary effects doctrine has proven fertile ground for abuse, enabling government officials and the communities they represent to conceal their distaste for adult entertainment behind claims of harmful effects. Justice William J. Brennan Jr. warned in his dissent in Boos v. Barry (1988) that the doctrine "could set the court on a road that will lead to the evisceration of First Amendment freedoms."
Regulations on adult entertainment businesses led to secondary effects doctrine
Adult entertainment establishments are subject to an array of zoning and licensing requirements. A typical regulation provides that adult businesses cannot be within 500 feet of a church, school, playground, or another adult-oriented business. Others dictate the distance between patrons and performers, limit the hours of operation, or prohibit totally nude dancing. Strict licensing requirements must be observed when opening adult businesses.
The Supreme Court introduced the secondary effects doctrine in its decision in Young v. American Mini Theatres (1976). The city of Detroit had adopted an anti-skid row ordinance preventing adult businesses from locating within 1,000 feet of any two existing adult businesses or within 500 feet of any residential area. The theater that challenged the law contended that the zoning ordinance was a content-based law that targeted businesses because officials did not like the expressive messages conveyed by the adult material displayed.
The Court expanded the secondary effects doctrine in City of Renton v. Playtime Theatres, Inc. (1986). The city of Renton, Washington, passed an adult-business zoning law in 1981 that prevented adult businesses, like the Renton theater pictured here, from locating within 1,000 feet of any residential area, school, park, or church. The Court upheld the ordinance based on the secondary effects rationale. (AP Photo/Barry Sweet, used with permission from the Associated Press)
Court cited neighborhood deterioration as 'secondary effect' of adult business concentration
The Court reasoned that the law was not passed to silence offensive expression but to prevent the deterioration of neighborhoods. In a footnote, Justice John Paul Stevens characterized such neighborhood deterioration as a "secondary effect," writing that it is "this secondary effect which these zoning ordinances attempt to avoid, not the dissemination of 'offensive' speech."
The Court expanded the doctrine a decade later in City of Renton v. Playtime Theatres, Inc. (1986). The city of Renton, Washington, passed an adult-business zoning law in 1981 that prevented adult businesses from locating within 1,000 feet of any residential area, school, park, or church. Two adult businesses challenged the law on First Amendment grounds, pointing out that Renton officials had offered no evidence of harmful secondary effects to the city itself.
The Court upheld the ordinance based on the secondary effects rationale. In delivering the opinion of the Court, Chief Justice William H. Rehnquist wrote that "our result is largely dictated by our decision in Young."
Law targeting secondary effects of protected expression could be considered neutral
He noted that the zoning law resembled a content-based law but ruled that it could be considered content-neutral because the law targeted the secondary effects of the businesses, not their expressive content. The Court also ruled that a city does not have to conduct its own study to justify its reliance on the secondary effects argument. Instead, the city could rely on studies conducted in other cities.
The Court extended the secondary effects doctrine from land-use regulations to the content of adult expression in Barnes v. Glen Theatre, Inc. (1991).
In Barnes, a narrow majority of the Court upheld an Indiana public-nudity law applied to totally nude dancing at strip clubs.
In his concurring opinion, Justice David H. Souter relied on the secondary effects doctrine even though the restriction seemingly imposed a direct regulation on the expressive content of nude dancing.
In City of Erie v. Pap's A.M. (2000), the Court applied the secondary effects rationale to uphold a city law banning totally nude dancing. The case involved his club, the only club in Erie, Pennsylvania, featuring all-nude dancers. (AP Photo/Gene J. Puskar, used with permission from the Associated Press)
Court applied secondary effects doctrine to uphold city's ban on nude dancing
In City of Erie v. Pap's A.M. (2000), the Court applied the secondary effects rationale to uphold a city law banning totally nude dancing.
Ironically, both Justice Stevens, who first used the term "secondary effects" in Young, and Justice Souter, who extended the secondary effects doctrine beyond zoning cases in Barnes, dissented in Pap's A.M. Souter admitted he had made a mistake in Barnes: "I may not be less ignorant of nude dancing than I was nine years ago, but after many subsequent occasions to think further about the needs of the First Amendment, I have come to believe that a government must toe the mark more carefully than I first insisted."
In City of Los Angeles v. Alameda Books (2002), the Court upheld a Los Angeles ordinance prohibiting a single adult establishment from functioning as both an adult bookstore and an adult arcade showing adult films. Justice Sandra Day O'Connor wrote that "it is rational for the city to infer that reducing the concentration of adult businesses in a neighborhood, whether within separate establishments or in one large establishment, will reduce crimes."
Secondary effects doctrine also used for laws involving noise, harm to children
The secondary effects doctrine has been applied in cases far removed from the land-use regulation of adult businesses.
Some of the secondary effects cited by government officials include noise, security problems, reduced privacy, appearances of impropriety, employment discrimination, negative effects of gambling, competition in the video-programming market, sexual arousal of readers, and harm to children.
A federal judge in Kentucky used the secondary effects rationale to uphold the constitutionality of a public high school dress code, determining that the code was really aimed at the "secondary effects of student dress," such as gang activity, violence, and inability to identify campus visitors.
David L. Hudson, Jr. is a law professor at Belmont who publishes widely on First Amendment topics. He is the author of a 12-lecture audio course on the First Amendment entitled Freedom of Speech: Understanding the First Amendment (Now You Know Media, 2018). He also is the author of many First Amendment books, including The First Amendment: Freedom of Speech (Thomson Reuters, 2012) and Freedom of Speech: Documents Decoded (ABC-CLIO, 2017). This article was originally published in 2009.
Barnes v. Glen Theatre, Inc. (1991)
Boos v. Barry (1988)
City of Erie v. Pap's A.M. (2000)
City of Los Angeles v. Alameda Books (2002)
City of Renton v. Playtime Theatres, Inc. (1986)
Content Based
Content Neutral
David Souter
Expressive Conduct
Harmful to Minors Laws
Licensing and Permit Laws
Nude Dancing
Young v. American Mini Theatres (1976)
Zoning Laws
Paul, Bryant, Daniel Linz, and Bradley Shafer. "Government Regulation of 'Adult' Businesses Through Zoning and Anti-Nudity Ordinances." Communications Law and Policy 6 (2001): 355-391.
Hudson, David L., Jr. "The Secondary Effects Doctrine: The 'Evisceration of First Amendment Freedoms.' " Washburn Law Journal 37 (1997): 55–99
Hudson, David L. Jr. Adult Entertainment and the Secondary Effects Doctrine. Nashville, Tenn.: First Amendment Center, 2002.
Hudson, David L. Jr. "The Secondary-Effects Doctrine: Stripping Away First Amendment Freedoms." Stanford Law and Policy Review 23 (2012): 19-30.
Hudson, David L. Jr. "Secondary-effects doctrine." Freedom Forum Institute, Sept. 13, 2002.
David L. Hudson Jr.. 2009. Secondary Effects Doctrine [electronic resource]. The First Amendment Encyclopedia, Middle Tennessee State University (accessed Jan 29, 2023). http://www.mtsu.edu:8443/first-amendment/article/26/secondary-effects-doctrine


I've never been the sort of believer who talks freely about hearing God's voice. It always seemed so out there to me; like something people who are a bit "off" did – or worse, something people who are putting on a Christian front do to disarm or deceive others or make excuses for their behavior.
I've never been ready to classify myself into either category.
But the truth is, I've heard God's voice. Time and again, over the course of my life.
I felt that in a real way. I knew.
Over the course of your 9 years with us Izzy, you have shown us how to be better people, how to have real Faith, and how what Grace looks like. We've always known that you hear God's voice. And because of that I've always known that God was real; with us in every moment. Even those hard times when your mommy was sick, when Max and Kate were sick, in cold sterile hospital rooms, in dark nights when I felt lost in my journey as the head of our household and as a father. I spent too many years trying to wrestle the answers out of Him.
I heard God's voice many times over the last decade. Usually through you, my 3 children. He also spoke to me one day at the bottom of the ocean, as I fought for my senses after hitting my head on the ocean floor, spraining my shoulder, stumbling back onto the beach in a heap. Broken.
And trust Me, He said.
So I did. But it took me 2 years. I quit a job that was no longer making me happy or feel like I was following God's will in my life.
It was scary. But things are great now. So much better than I could have imagined when fear was telling me to rely on myself rather than trusting in Him. I reached a point where I didn't have the strength anymore. So I surrendered to His command in my life.
That's all he wanted, I guess.
Today, less than 2 months later, I start a brand new job that I believe God had in mind all along.
Last night you showed us again, Izzy, what Grace and trust and Faith look like. I cried tears of real joy as you made a public declaration of the Faith you've always had.
You were baptized last night at the closing of VBS. You wore a small necklace around your neck that reads: "Believe". Your Nana, Papaw, Grandma, Grandfather, Aunt, Uncle, cousins, friends, brother, sister, and Mommy and Daddy all shared the moment with you. Your past and future sins washed white as snow, you chose this dress to wear afterwards.
We came back to the house afterwards and had cake and icecream. I could feel the change in our home. It's never been bad. But I can feel a difference. We're listening now. Not doubting. Trusting. And following.
I'm so proud of you, Isabella Hope.
ps. You did great at the County Championship Swim Meet on Saturday too. Here are a few pics we took!
100 IM – your event!
Awesome. This one blew me away. Congratulations.
Erik, Ellie, and Dr. G, thank you all for the kind words! I'm always happy when my letters to my kids are relevant to others as well.
If only we all had the Faith of a child. So true that our children are often the face of Jesus to us parents.
Congrats to Izzy! And to you.
I hope you don't mind me sharing this. reading your letter, the words from psalm 23 leapt out at me.
Children are the light to our world and the inspiration for us to love as God our father loved us.


EASY & LOW MAINTENANCE LARGE FAMILY HOME WITH AIR CON & FANS!
PLEASE CALL THE OFFICE ON 3818 1822 ONE HOUR PRIOR TO ANY INSPECTION TIMES TO CONFIRM. UNFORTUNATELY IF YOU DO NOT CALL WE CANNOT GUARANTEE THE VIEWING WILL STILL BE PROCEEDING.
This lovely 4 bedroom, 2 bathroom low set brick home is centrally located and ready for your family to enjoy! With air conditioning to the living areas and ceiling fans to all bedrooms it is an ideal home!
26 Elkhorn Street, Bellbird Park QLD 4300 was last sold in 2004 and 65 other 4 bedroom house in Bellbird Park have recently been sold.
In this street, the most recent rental of another house with 4 beds, 2 baths, 1 parking space was at number 29, which leased for $310 4 months ago.


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Mortal Kombat 11 - How to Link to Mobile Game And What Are The Rewards
By Aidan O'Brien on Tue, 04/23/2019 - 06:15
Mortal Kombat 11 is releasing today, and if you link it to the Mortal Kombat Mobile game, you can get a new announcer voice. By linking the two, you will get access to the Kronika announcer voice for Mortal Kombat 11, and you will receive a variety of other rewards including konsumables, hearts, koins, and souls each day when you play Mortal Kombat 11 if you have logged in to Mortal Kombat Mobile on the same day.
For the mobile game you will receive 40 Skulls, and a special pack that includes a Mortal Kombat 11 character for your Mortal Kombat Mobile game, and free Souls each day you log into the game if you are also logged into Mortal Kombat 11 on the same day. For the first 30 days, you will receive 40 free Souls each day. After 30 days you will receive five free Souls each day indefinitely!
How To Link To Mobile
Linking the two is quite simple. On the mobile game, click on Settings, and then click Console Link. The app will then give you a six digit code to use.
After that, just open up Mortal Kombat 11 on either your PS4, Xbox One, or PC, and select the Link To Mobile option in the Settings menu. Put in the six-digit code, and you are all linked up!
And an important piece of information that you should be aware of, from the Warner Bros. FAQ, is that your Mortal Kombat 11 Console/PC Account and your Mortal Kombat Mobile Account can only be linked to ONE other account. The link is permanent and cannot be changed at this time. This means you will want to make sure that you are logged into the Mortal Kombat 11 Console/PC Account and Mortal Kombat Mobile account that you plan to use to play each game regularly if you want to receive all of your rewards.
The Gamepur crew is pretty hyped for the release of Mortal Kombat 11, and have already been hard at work to provide people with some quality content. If you want to know how to get Shao Khan if you didn't pre-order the game, how to complete the "Enough Already" achievement, the Krypt Chest locations at the Palace Entrance, or how microtransactions work in the game, then we already have some guides that you should find very helpful. We will bring you all sorts of new guides and content over the coming days as well, so stay tuned for more.
link to mobile
Mortal Kombat Mobile
Marvel Ultimate Alliance 3 Review: A Frantic Fun Time
By Adam Newell on Thu, 07/18/2019 - 19:00
Colossus and Cyclops are coming to Marvel Ultimate Alliance 3 as free DLC on Aug. 30
By Misael Duran on Thu, 07/18/2019 - 17:52
Pokemon Go: What are Shadow Pokemon?
By Adelaide Weiss on Thu, 07/18/2019 - 17:42
Gamepur.com © 2010-2019


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Savor the joys of Japanese cuisine with a "Shokado bento" box filled with fresh ingredients from the Setouchi.
LOCAL FLAVORS SOMETHING SPECIAL ON YOUR PALATE SPRING SUMMER AUTUMN WINTER
OKAYAMA SETOUCHI
The "Hiru-Gozen", featuring the bounty of the Seto Inland Sea, is a beauty to behold.
A square box of some sort arrives. Opening it, a cry of wonderment escapes. Before me sits a beautiful assortment of items reflecting the four seasons of Japan. This is the 3,390 yen "Shokado bento". Its division into square compartments ensures that each part of the lunch remains separate from the others in taste and flavor, a way of serving food that is uniquely Japanese, allowing enjoyment of each of the different items in the box. First invented by a traditional Japanese restaurant around 1930 for use in a tea ceremony, this type of bento box eventually became a national phenomenon. This former inn, which boasts expansive views of the Seto Inland Sea, offers traditional Japanese foods like tempura and sashimi that are a luxurious tribute to the waters below. Bingoya is located up a hill and affords views of a small harbor of the Seto Inland Sea. Another reason for its popularity is that diners can enjoy the atmosphere of a traditional Japanese inn built in the Sukiya-zukuri architectural style.(This information is as of December 2017)
Bingoya
956 Tamashima Agasaki, Kurashiki City, Okayama [Map]
11:30-14:30 (OS 14:00) / 17:00-22:00 (OS 21:30) * Hours negotiable
Acsess
10 minutes from JR Shin-Kurashiki Station by Ikasa Bus Corporation (take bus for Yorishima Sogoshisho-mae), alighting at Showabashi Stop. One minute on foot from bus stop.
Enjoy night views of the Mizushima Coastal Industrial Zone
A café in a traditional house evoking Japan's four seasons and nature


It's my pleasure to welcome you to the College of Arts and Sciences' sixth annual Community Semester. The Community Semester began with former Dean John Murray, after conversations with CAS faculty, and I am honored to continue the tradition again this year. Over the years of the Community Semester, one of our most important goals has been to share some of the amazing research and creative work of our faculty and students with the residents of Terre Haute and the greater Wabash Valley. As the largest and most diverse college at Indiana State University, we produce some fascinating and nationally recognized scholarship and creative work. This year our theme is "1968," and we have a wide range of events that promise to hold your interest. Our theme reflects the fact that we are marking 50 years since that momentous time in the history of the United States and much of the world. Events and discoveries in (and around) that year continue to reverberate in the United States today, helping to shape the modern world in which we live. I hope you can join us for some terrific events that will prompt us to think about the important changes initiated during that period that continue to affect us today.
All of our events are free and open to the public. We have worked hard to make sure there is something for all ages. We also have events that represent the arts, the sciences, the social sciences, and the humanities. We hope that you attend as many events as you can, and we are truly honored to share the College of Arts and Sciences with you.
Please click the link below for the full program of events.


The calculation of partial derivatives is a fundamental need in scientific computing. Automatic differentiation (AD) can be applied straightforwardly to obtain all necessary partial derivatives (usually first and, possibly, second derivatives) regardless of a code's complexity. However, the space and time efficiency of AD can be dramatically improved—sometimes transforming a problem from intractable to highly feasible—if inherent problem structure is used to apply AD in a judicious manner.
provide many easy to understand applications, examples, and MATLAB templates.
This book will prove useful to financial engineers, quantitative analysts, and researchers working with inverse problems, as well as to engineers and applied scientists in other fields.
Thomas F. Coleman is Professor, Department of Combinatorics and Optimization and Ophelia Lazaridis University Research Chair, University of Waterloo. He is also the director of WatRISQ, an institute composed of finance researchers that spans several faculties at the university. From 2005 to 2010, Dr. Coleman was Dean of the Faculty of Mathematics, University of Waterloo. Prior to this, he was Professor of Computer Science, Cornell University. He was also the director of the Cornell Theory Center (CTC), a supercomputer applications center, and founded and directed CTC-Manhattan, a computational finance venture. Dr. Coleman has authored three books on computational mathematics, edited six conference proceedings, and published over 80 journal articles in the areas of optimization, automatic differentiation, parallel computing, computational finance, and optimization applications.
Wei Xu is Research Manager at Global Risk Institute (GRI), Toronto. Before joining GRI, Dr. Xu was Visiting Professor at the University of Waterloo. Previously, he was Associate Professor at Tongji University, Shanghai. He cofounded Shanghai Raiyun Information Technology Ltd., a risk management services and solutions provider, and currently serves as its Director of R&D. His research is featured in over 30 publications and he has coauthored a book on risk management.
Click the button below to add the Automatic Differentiation in MATLAB Using ADMAT with Applications to your wish list.


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In line with latest Government's guidelines on home moving, the housing market remains open, and all our offices in England, Scotland and Wales continue to operate. Health and safety remains our main priority, and we continue to follow a number of strict measures to protect our customers and staff. More information
Winchester, Canterbury, York? Who tops the value table?
Comparing the cathedral cities of Winchester, Canterbury and York using Zoopla property values data, has Winchester very much at the top of the values tree.
Zoopla's index shows house prices in Winchester at the close of 2013 are on average £435,044. This is substantially higher than Canterbury at £267,084 and York at £237,961.
As you would expect, the average rents for these cathedral cities follow the same ranking, with Winchester top, commanding £1,454 per month average, followed by Canterbury at £1,225. York's average rent stands at £978 per month.
The available stock of rental properties in these towns is a different story. Winchester desperately needs more good quality rented accomodation to meet demand. Zoopla data shows just 191 properties available to rent in Winchester in December, versus 826 in York and 789 in Canterbury.
Come and talk to us today, for free, impartial guidance on investing in buy-to-let property in the Winchester area, whether first time or portfolio expansion.
Copyright © 2021 Martin & Co (UK) Ltd
Martin & Co (UK) Ltd


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Cloning, expression, and characterization of a peptidoglycan hydrolase from the Burkholderia pseudomallei phage ST79
Nittaya Khakhum1,
Umaporn Yordpratum2,
Atcha Boonmee3,
Unchalee Tattawasart2,
Jorge L. M. Rodrigues4,5 &
Rasana W. Sermswan1
The lytic phage ST79 of Burkholderia pseudomallei can lyse a broad range of its host including antibiotic resistant isolates from within using a set of proteins, holin, lysB, lysC and endolysin, a peptidoglycan (PG) hydrolase enzyme. The phage ST79 endolysin gene identified as peptidase M15A was cloned, expressed and purified to evaluate its potential to lyse pathogenic bacteria. The molecular size of the purified enzyme is approximately 18 kDa and the in silico study cited here indicated the presence of a zinc-binding domain predicted to be a member of the subfamily A of a metallopeptidase. Its activity, however, was reduced by the presence of Zn2+. When Escherichia coli PG was used as a substrate and subjected to digestion for 5 min with 3 μg/ml of enzyme, the peptidase M15A showed 2 times higher in lysis efficiency when compared to the commercial lysozyme. The enzyme works in a broad alkaligenic pH range of 7.5–9.0 and temperatures from 25 to 42 °C. The enzyme was able to lyse 18 Gram-negative bacteria in which the outer membrane was permeabilized by chloroform treatment. Interestingly, it also lysed Enterococcus sp., but not other Gram-positive bacteria. In general, endolysin cannot lyse Gram-negative bacteria from outside, however, the cationic amphipathic C-terminal in some endolysins showed permeability to Gram-negative outer membranes. Genetically engineered ST79 peptidase M15A that showed a broad spectrum against Gram-negative bacterial PG or, in combination with an antibiotic the same way as combined drug methodology, could facilitate an effective treatment of severe or antibiotic resistant cases.
A bacteriophage or phage is the virus of bacteria that is very specific to its host. It can replicate, multiply after transfection and either lyse the host or become integrated into its genome (Golkar et al. 2014). Eastern Europe such as Poland and Georgia reported the phage as an alternative therapy for infectious diseases (Abedon et al. 2011). Phage therapy has also been evaluated in the United States (Ho 2001). The discovery of antibiotics changed the paradigm of curing infectious diseases and helped protect enormous numbers of people who suffered from bacterial infections in the twentieth century, however, the numbers of antibiotic-resistant bacteria have also increased (Golkar et al. 2014). Even though the post-antibiotic era is not at hand, the use of broad-spectrum antibiotics has proven to disturb the beneficial bacterial community in humans and animals, especially gut microbiota, that affect immunity (O'Hara and Shanahan 2006; Round and Mazmanian 2009). Therefore, the use of phages as an alternative treatment came under the spotlight again because the phage is highly specific, can overcome antibiotic resistant bacteria and are available in large quantities in nature.
The enzymes related to the lysis mechanism of the phages that are used to lyse the bacterial host during the release of their progeny have been studied and revealed the classical holin-endolysin lysis system found in phages of both Gram-positive and Gram-negative bacteria (Young et al. 2000). Holin is used to make pores in the cytoplasmic membrane that assists endolysin to access and cleave peptidoglycan to lyse the bacteria from the inside out. In addition, some phages of Gram-negative bacteria also have Rz/Rz1 or lysB/lysC accessory proteins that help in the lysis of the process (Berry et al. 2008). Endolysins have been considered as a new class of antibiotics as they can destroy the peptidoglycan (PG) of Gram-positive bacterial cell walls. Endolysin has five specific activities on PG which are muramidase (lysozyme), transglycosylase, glucosaminidase that digests N-acetylmuramic acids (NAM) and N-acetylglucosamine (NAG), amidase that digests NAM and peptides and endopeptidases digested within the peptide chain of PG (Borysowski et al. 2006). The enzymes or the phages themselves are extensively applied in several fields, for example, the food industry and biological control of unwanted bacteria (Ruyter et al. 1997) including pathogenic bacteria in medicine as it shows neither toxicity nor stimulates hyperimmune sera in the mouse model (Jado et al. 2003).
Burkholderia pseudomallei is a Gram-negative soil bacterium that causes severe septic infectious disease called melioidosis. The disease can be found in both humans and animals in endemic areas (Leelarasamee and Bovornkitti 1989). This pathogenic bacterium is intrinsically resistant to several antibiotics and it can produce high levels of biofilms that protect the bacterium from the killing by either antibiotics or the host immune response (Sawasdidoln et al. 2010; Pibalpakdee et al. 2012; Mongkolrob et al. 2015). The drug of choice is a third generation cephalosporin such as ceftazidime that needs long-term treatment to prevent relapse. Currently, there is no commercial vaccine available (Limmathurotsakul et al. 2015). Phages that have shown some specificity in lysing B. pseudomallei have been reported (Sariya et al. 2006; Yordpratum et al. 2011; Gatedee et al. 2011; Kvitko et al. 2012; Guang-Han et al. 2016). The genome of ST79, a novel lytic phage that lyses B. pseudomallei was sequenced and submitted to GenBank (GI:509141608) (manuscript in preparation). The lysis cassette of ST79 was also characterized (Khakhum et al. 2016) and its modified phages were shown to lyse a wide range of B. pseudomallei isolates and could significantly reduce biofilm formation of the bacteria especially at the early stage of attachment (Kulsuwan et al. 2015).
In this study, the peptidase M15A, known as endolysin or peptidoglycan hydrolase from the ST79 lytic phage that could lyse a broad spectrum of B. pseudomallei and other Gram-negative bacteria from within was cloned, expressed and characterized. More information on the enzymes and phages themselves could facilitate the application of them as adjunct standard antibiotic therapy for B. pseudomallei.
Bacterial strains and ST79 phage
Burkholderia pseudomallei strain P37 was isolated from a blood sample from a patient admitted to Srinagarind Hospital, Faculty of Medicine, Khon Kaen University, Thailand. The B. pseudomallei lytic phage ST79, isolated from soil in the northeast of Thailand, was used as a source of the peptidase M15A for cloning (Yordpratum et al. 2011). Escherichia coli BL21 (DE3) was used as the host for cloning and protein expression processes (Thermo Fisher Scientific, Waltham, MA, USA). Eighteen Gram-negative bacteria, five of which were E. coli host strains; Top10, LMG194 (Invitrogen, CA, USA), DH5α, BL21 (DE3) and XL1-Blue (Thermo Fisher Scientific, Waltham, MA, USA), two B. pseudomallei isolates, P37 and G1; two Burkholderia mallei isolates, EY2233 and EY2237; Burkholderia thailandensis UE5 (kindly provided by MORU, Mahidol University, Thailand), Klebsiella pneumoniae, Vibrio parahaemolyticus, Pseudomonas vasculitis, P. aeruginosa, Acinetobacter baumannii, Salmonella gr. D, Shigella gr. D and Citrobacter freundii and seven Gram-positive bacteria included Enterococcus sp., Streptococcus epidermidis, Staphylococcus aureus, Bacillus sp., Micrococcus sp., β-streptococcus gr. B and Corynebacterium diphtheria were obtained from the Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand and used in this study. All of bacterial strains and ST79 phage were deposited in culture collection belonging to World Data Centre For Microorganism (WDCM) as MRCKKU (registration number 1130). ST79 phage is available for research collaborators.
Bioinformatic analysis
The peptidase M15A amino acid sequence (YP_008060500.1) from the ST79 phage genome (NC_02134.1) was submitted to BLASTP homology search (Altschul et al. 1997) in the NCBI database (http://www.ncbi.nlm.nih.gov/). The peptidase information resource and sequence analysis were performed by the MEROPS batch Blast tool (Rawlings and Morton 2008). The Interproscan 4 software v.4.8 (Zdobnov and Apweiler 2001) was used to analyze protein functional domains. The protein conserved domain and structure was predicted using Pfam (Finn et al. 2016) and SWISS-MODEL (Biasini et al. 2014).
ST79 phage propagation and DNA extraction
The ST79 lytic phage was propagated in liquid culture using B. pseudomallei strain P37 as the propagating strain (Yordpratum et al. 2011). The phage DNA was extracted with a phenol–chloroform extraction method as described elsewhere (Sambrook and Russell 2001).
Cloning and expression of peptidase M15A and Western blot analysis
The peptidase M15A gene was amplified from the ST79 genomic DNA by the polymerase chain reaction (PCR) using in house designed peptidase M15A forward primer (5′-TATAAAGAGCTCTATGCAGTTGACGGACCATTTC-3′) with SacI restriction site (underlined sequence) and a peptidase M15A reverse primer (5′-ATAATAGGTACCTCATGCGCCCACCGTGTA-3′) with the KpnI restriction site (underlined sequence). The PCR product was cloned into the pQE31 vector (Qiagen, Hilden, Germany) containing the N-terminal 6xhistidine tag and transformed into E. coli BL21 (DE3). E. coli cells containing the recombinant plasmid with peptidase M15A gene were propagated in Luria and Bertani (LB) medium containing 100 µg/ml ampicillin until mid-exponential phase (OD600 nm = 0.6) and induced with 1 mM final concentration of isopropyl-β-d-thiogalactopyranoside (IPTG) (Sigma, St. Louis, Missouri, USA). Cells were further cultured for 4 h and collected as a pellet followed by sonication (10 cycles of 30 s pulses and 30 s rest at 200 Watts in an ice bath, MSE Soniprep 150, MSE, London, UK) in 5 ml lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole pH 8.0). The protein supernatant and pellet fractions were separated by 15 % SDS-PAGE (Sambrook and Russell 2001) using the Novex Sharp Pre-Stained Protein Standard (Thermo Fisher Scientific, Waltham, MA, USA) as the protein molecular weight marker, then blotted onto nitrocellulose by a semi-dry blotting system (BioRad, CA, USA). The proteins on the membrane were detected by anti-6X His tag® antibody (HRP) (Abcam, Cambridge, UK) and chemiluminescent SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA).
The recombinant E. coli BL21 (DE3) containing the peptidase M15A gene-plasmid was induced by IPTG and the His-tagged proteins inside E. coli cells were purified using Ni–NTA agarose (Qiagen, Hilden, Germany) by a gravity-flow chromatography method. SDS-PAGE was used to analyze the eluted fractions for the 18 kDa of the peptidase M15A protein. The fraction containing the protein was refolded by buffer exchanging with 25 mM sodium acetate buffer, pH 6.5 and filtered through the Amicon® Ultra 10 K centrifugal filter device (Millipore, Darmstadt, Germany) according to the manufacturer's instructions. The purified protein was quantified by the bicinchoninic acid assay (BCA assay) (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions using a Spectronic 20D+ spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
Zymogram analysis
The peptidase M15A enzyme activity was observed using zymogram or renaturing SDS-PAGE (Piuri and Hatfull 2006) with some modifications as follows: An overnight culture of E. coli XL1-Blue (0.2 % w/v) was autoclaved and added to the SDS-PAGE solution (15 % w/v) before polymerization. The purified peptidase M15A enzyme was mixed with 2× sample-refolding buffer (0.5 mM Tris–HCl pH 6.8, 20 % glycerol, 0.2 % bromophenol blue), then loaded into the SDS-PAGE containing E. coli. After electrophoresis, the gel was incubated at 37 °C for 16 h in 1 % triton X-100, 25 mM Tris–HCl pH 8.5 and washed once with water and stained for 3 h with 0.5 % methylene blue in 0.01 % KOH. The peptidoglycan hydrolase activity of peptidase M15A on E. coli lysate was observed as a clear zone.
The outer membrane of E. coli XL1-Blue was permeabilized by chloroform to expose the peptidoglycan for digestion as described by Lavigne et al. (2004) with some modifications and used as the substrate for peptidase M15A. In brief, bacteria were grown until the mid-exponential phase (OD600 nm = 0.6), then centrifuged at 4000g at 4 °C, for 15 min to collect the cell pellet. The outer membranes were permeabilized by chloroform-saturated 0.05 M Tris–HCl buffer, pH 7.5 and gently shaken at room temperature for 45 min. The permeabilized E. coli were then washed, resuspended in 10 mM phosphate buffer, pH 8.0 and adjusted to OD600 nm = 0.6–1.0. The E. coli suspension of 270 µl was added into 96-well BD Falcon microplates (BD Bioscience, San Jose, CA, USA), then the purified peptidase M15A enzyme was added at the amount of 0.1 (0.3 μg/ml), 0.5 (1.6 μg/ml), 1 (3 μg/ml) and 5 μg (16 μg/ml) (30 µl) and the turbidity reduction at OD600 nm was measured by a Gen5 microplate reader (Biotek, Vermont, USA) as the kinetic assay for 15 min (1 min time intervals). The chicken egg white lysozyme (Sigma, St. Louis, Missouri, USA) (3 μg/ml) was used as a positive control and 10 mM phosphate buffer, pH 8.0, was used as a negative control (Briers et al. 2007a).
The effect of pH, temperature and divalent metal ions
To evaluate the effect of pH on enzymatic activity, permeabilized E. coli XL1-Blue cells were resuspended in 900 μl of 10 mM citrate buffer for pH 3.0–5.5, 10 mM phosphate buffer for pH 6.0–7.0 or 10 mM Tris–HCl buffer for pH 7.5–9.0 and then 100 μl of enzyme (5 μg) was added. The percentage of OD600 nm reduction was determined after incubation at 30 °C for 15 min. The effect of temperatures at 25, 30, 37 and 42 °C on enzyme activity were tested in the same manner at pH = 8.0.
The effect of metal ion, particularly Zinc, on enzyme activity was observed using 100 μl (5 μg) of purified peptidase M15A mixed with 900 μl of permeabilized E. coli XL1-Blue cells (OD600 nm ~ 1.0) in various concentrations of ZnCl2 (5, 10, 50 and 100 μM), MgCl2, MnCl2 or CaCl2 (100 and 1000 μM). The relative lytic activity was calculated as follows: {ΔOD600 nm sample (endolysin added) − ΔOD600 nm (buffer only)}/initial OD600 nm (Plotka et al. 2015) and compared with the control without metal ions for 100 % of relative activity (Son et al. 2012).
Spectrum of antibacterial lytic activity
A total of 18 Gram-negative and seven Gram-positive bacteria were tested for the peptidoglycan hydrolase spectrums. Gram-negative bacteria were permeabilized by chloroform and their peptidoglycans were used as substrates for enzyme digestion as previously described (Briers et al. 2007a). For Gram-positives, each strain was grown to mid-exponential phase then centrifuged at 3000g to collect cell pellets. The pellets were washed and resuspended in 10 mM phosphate buffer pH 8.0, the OD600 nm to 0.6–1.0 was adjusted. To test the spectrum of antimicrobial lytic activity, 5 and 20 μg in 100 μl of purified peptidase M15A was added into 900 μl permeabilized cell suspensions of Gram-negative or cell suspension of Gram-positive. The score was estimated from % relative lytic activity after incubation at 30 °C for 15 min. The % relative activity was defined as—(no lytic activity), + (1–30 %), ++ (31–60 %) and +++ (61–100 %) (Son et al. 2012).
The data of OD600 nm in the turbidity reduction test at different pHs and temperatures were analyzed by a Student's t test and with a p value of <0.05 considered as significant.
Peptidase M15A sequence analysis
The protein sequence of peptidase M15A from the lytic phage ST79 showed a conserved domain as Peptidase_M15_3 (PF08291) when analyzed by BLASTP and Pfam (Fig. 1a). The Interproscan 4 (version 4.8) protein functional domain indicated a Hedgehog signaling/DD-peptidase zinc-binding domain (SSF55166). The SWISS-MODEL protein structure prediction was matched with the 1lbu.1.A template, which was muramoyl-pentapeptide carboxypeptidase (MEROPS data). When the MEROPS batch Blast tool was used to detect peptidases and their non-peptidase homologues sequences in ST79 genome, it showed common amino acids among peptidases found in other bacterial genome, such as: B. glumae, B. thailandensis, B. cenocepacia, Burkholderia sp. CCGE1002, Asticcacaulis excentricus and Pseudomonas putida but not B. pseudomallei and B. mallei (Fig. 1b). This study's analysis also identified a homologue catalytic domain from amino acid positions 3 to 134 (total 132 residues) with peptidase from P. putida (MER087996). The active site residues were located at the tryptophan (W117H) position and metal ligands at histidine (H77), aspartic acid (D84) and histidine (H119) (Fig. 1c). The analysis also indicated the presence of motifs HXXXXXXD and WXH, which were typical for peptidase M15 subfamily A.
The bioinformatics analysis of ST79 peptidase M15A protein sequence. a The Peptidase_M15_3 conserved domain analyzed using Pfam; #HMM: consensus of the Hidden Markov Models (HMMs), Capital letters indicated the most conserved positions, #MATCH: the match between the query sequence and the HMM, ' + ' indicates a positive score which can be interpreted as a conservative substitution, #PP: posterior probability, which is the degree of confidence in each individual aligned residue, 0 means 0–5 %, 1 means 5–15 % and so on and 9 means 85–95 %, '*' means 95–100 % posterior probability, #SEQ: query sequence, '-' indicated deletions in the query sequence with respect to the HMM, b Multiple alignment of peptidase protein sequences from the MEROPS database showed identity of peptidase M15A sequence from ST79 phage with peptidase enzymes from B. glumae, B. thailandensis, B. cenocepacia, Burkholderia spp. CCGE1002, A. excentricus and P. putida using the CLUSTAL X program. Conserved amino acids of all sequences were marked with an asterisk. c The full amino acids sequence analysis of ST79 peptidase M15A using MEROPS batch Blast. The peptidase unit was labeled in green, active site in red, metal ion ligand in orange and the conserved domain in underlined letters
Expression, purification and zymogram analysis of the Peptidase M15A
The peptidase M15A gene was successfully cloned and expressed in E. coli BL21 (DE3). The estimated molecular weight of purified peptidase M15A on SDS-PAGE was approximately 18 kDa (with 6xhis tag) (Fig. 2). For zymogram analysis, the peptidase M15A enzyme in the gel lysed the peptidoglycan substrate from E. coli in which it appeared as a transparent band (Fig. 2).
The SDS-PAGE analysis of proteins and zymogram analysis of the lytic activity of Peptidase M15A against E. coli peptidoglycan. The Coomassie Brilliant blue stained SDS-PAGE gel shows protein molecular weight marker (M), induced E. coli cells containing pQE31 vector (lane 1), induced cells containing peptidase M15A/pQE31 plasmid (lane 2), 5 μg of purified peptidase M15A protein (lane 3) and the zymogram that is stained with 0.1 % methylene blue in 0.01 % KOH of SDS-PAGE refolding gel (lane 4). The clear lysis of PG substrate by the enzyme on zymogram located at the same position of the over-expressed protein band in lane 2 and purified enzyme in lane 3. The arrow indicates the 18 kDa lytic band of peptidase M15A
Lytic activity test
At the concentration of 1.6 μg/ml onward at 5 min of digestion, the enzyme reduced the turbidity of permeabilized E. coli XL1-Blue more than the lysozyme. When longer times of 10 and 15 min were observed, the turbidities from each concentration including lysozyme were similar. When the same concentrations of lysozyme and purified peptidase M15A (3 μg/ml) were compared at 5 min of digestion, the lysozyme gave a 22 % relative lytic activity while the peptidase M15A resulted in 52 %. The purified peptidase M15A digested the substrate approximate 2 times more than the lysozyme at this point (Fig. 3).
The turbidity reduction curve of peptidase activity against chloroform-treated E. coli. The peptidase M15A activity of various amounts was used to lyse chloroform E. coli XL1-Blue cell suspensions. The graph of turbidity reduction using peptidase M15A of 0.3 μg/ml (filled triangle), 1.6 μg/ml (filled circle), 3 μg/ml (open triangle) and 16 μg/ml (open circle) were evaluated. Phosphate buffer (filled square) was used as a negative control and 3 μg/ml of lysozyme (filled diamond) was used as positive control. Each point represents the mean of triplicate experiments and error bars indicate the standard deviation (SD)
Effect of pH, temperature and divalent metal ions on the enzyme activity
The peptidase M15A showed highest the activity at pH 7.5–9.0 with relative activity above 60 % (Fig. 4a). The enzyme could work in broad temperature ranges of 25, 30, 37 and 42 °C (Fig. 4b). The enzyme activity was decreased when Zn2+ concentrations were increased (Fig. 5). On the other hand, the 100 μM of Mg2+ and Mn2+ could only increase approximately 10 % of relative activity. When the 1000 μM concentration of Mg2+ and Mn2+ was used, the enzyme activity was reduced to 50.3 and 65.6 %. The Ca2+ ion showed little effect on the enzyme activity as seen by an approximately 6 % increase with 100 μM of the ion and a 5 % decrease when 1000 μM was used (Table 1).
The effect of pH and temperature on the peptidase M15A lytic activity against E. coli XL1-Blue peptidoglycan. The optical density at 600 nm of cell suspensions was used to observe the effects of pH a and temperature b on the peptidase M15A lytic activity against E. coli XL1-Blue peptidoglycan. The grey bars show negative controls without enzyme and black bars indicate the reaction with enzyme. p value <0.05 (*) and 0.01 (**) indicate the significance. Each column represents the mean of triplicate experiments and error bars indicate SD
The percentage of E. coli XL1-Blue peptidoglycan reduction when treated with peptidase M15A in the presence of Zn2+. The effect of Zn2+ on peptidase M15A activity was calculated from the difference between optical densities at 600 nm of enzyme treated cells and untreated cells. Each column represents the mean of triplicate experiments and error bars indicate SD
Table 1 The effect of divalent metal ions on lytic activity of peptidase M15A enzyme
Eighteen chloroform permeabilized Gram-negative and seven Gram-positive bacteria were used for susceptibility tests against 5 and 20 μg of the peptidase M15A (Table 2). The enzyme lysed the peptidoglycan from all Gram-negative bacteria investigated. The enzyme effectively lysed E. coli, K. pneumoniae, Shigella gr. D and C. freundii and moderately lysed Burkholderia spp., V. parahaemolyticus, P. vasculitis, P. aeruginosa, A. baumannii and Salmonella gr. D (36–82 %). For Gram-positive, it only lyzed Enterococcus sp. (49 %).
Table 2 Spectrum of antibacterial lytic activity against Gram-negative and Gram-positive bacteria
The increase in antibiotic-resistant bacteria makes the use of possible phage therapy as an alternative treatment for bacterial infections as one of multiple options for treatment. Similar to the concept of a mixed viral vaccine, phages also could be used as a portion of a cocktail for broad host range lysis and more phage could be added into suit the resistance situation (Chan et al. 2013). PG is the major component of Gram-positive bacteria and also the lining under the outer membrane of Gram-negative bacteria. Endolysins are a group of PG hydrolyzing enzymes well characterized in phages for their function on the release of the progeny out of the bacterial host during the lytic cycle. The information on the specificity of the phage and its enzymes against bacteria could facilitate their use safely.
A novel lytic phage ST79 and its modified phage that lyses B. pseudomallei has been reported to effectively lyse a broad range of the bacterium (Yordpratum et al. 2011; Kulsuwan et al. 2015). ST79 endolysin-like protein, peptidase M15A, was identified from the phage genome sequence and predicted to contain 149 amino acids (approximately 16 kDa) with catalytic but not the binding domain (Khakhum et al. 2016). This was similar to other lysins from phages that infect Gram-negative bacterial hosts, which contain single catalytic domains with a molecular mass of 15–20 kDa (Nelson et al. 2012). Endolysins KZ144 and EL188 from a Pseudomonas phage, however, were shown to contain both lytic and N-terminal binding domains (Briers et al. 2007b).
The peptidase M15A amino acid sequence analyzed by MEROPS showed a high identity with conserved amino acid sequences of the peptidase M15 subfamily A that is typical for metallopeptidases with a metal binding part (Rawlings and Morton 2008). A similar prediction was observed with BLASTP and Pfam results, detecting a conserved domain of peptidase M15_3_superfamily. The peptidase M15A from ST79 phage has 132 amino acid residues with active sites containing a Zn2+ ion-binding site at the following amino acids: His77, Asp84 and His199. The peptidase enzyme of a Streptomyces phage also contains His154, Asp161 and His197 of the D-Ala-D-Ala carboxypeptidase zinc specific cleavage site. When the Zn2+ ion was present, the activity of peptidase M15A from ST79 phage was inhibited while that of Streptomyces works more effectively (Courvalin 2006). Likewise, the Zn2+ inhibition effect is also found in the phage T5 endolysin, in which a 10 mM concentration completely inactivated the enzyme immediately after addition. It was also observed that this specific endolysin requires Ca2+ instead of Zn2+ or Mn2+ at the stage of the phage developmental cycle (Mikoulinskaia et al. 2009). Activity of the peptidase M15A was inhibited with addition of 100 μM Zn2+ (10 % of relative activity remain) while 100 μM of Mg2+, Ca2+ and Mn2+ caused a slightly increased enzyme activity. When 1000 μM of Mg2+, Mn2+ and Ca2+ were used, the activity was decreased. On the contrary, Zn2+ and Mn2+ are required for full enzymatic activity of LysB4 endolysin from the B. cereus-infecting phage B4 (Son et al. 2012) and this requirement is also seen in Ply500 endolysin from the Listeria monocytogenes phages (Loessner et al. 1995).
Characterization of some biochemical properties of the peptidase M15A showed a broad range of optimal pHs varying from 7.5 to 9.0 (% relative lytic activity >60 %), which is in alkalophilic range similar to other previously reported peptidases as observed for phage T5 endolysin (optimum pH 8.5) (Mikoulinskaia et al. 2009), lysB4 (pH 8.0–10.0) (Son et al. 2012), transglycosylase endolysin of the phage SPN1S (pH 7.0–10.5), and the highly thermostable Ts2631 amidase endolysin from the Thermus scotoductus phage vB_tsc2631 (7.0–11.0) (Plotka et al. 2015). The Peptidase M15A worked at 25–37 °C and also 42 °C which is the optimum temperature for cultivation of B. pseudomallei in the laboratory (Chen et al. 2003; Palasatien et al. 2008). The enzyme from phages mostly works in an alkalophilic pH.
In this study, all the chloroform permeabilized Gram-negative bacteria were prepared and used as a substrate to test the specificity of endolysin against PG (Briers et al. 2007a). The PG from 18 strains of Gram-negative bacteria including drug resistant strains B. pseudomallei G1, B. mallei EY2233 can be lysed by ST79 peptidase M15A. The enzyme may be more specific to peptidoglycans of Gram-negatives as the enzyme can lyse only Enterococcus sp. among eight Gram-positive bacteria tested. The amino acid composition and sequence of PG in Gram-negative bacteria is known to have a low variation and the PG type belong to A1γ. Even though PG from C. diphtheriae contains A1γ as in Gram-negative bacteria (Schleifer and Kandler 1972), its cell wall is distinct from others with a predominance of meso-diaminopimelic acid in the murein wall and multiple repetitions of arabinogalactan (Besserer et al. 2006). The PG is A1γ type, similar to Bacillus sp., but has modification like deacetylation and resists lysozyme digestion (Davis and Weiser 2011). Both of them were resistant to the ST79 peptidase M15A. For other Gram-positive bacteria, S. epidermidis, S. aureus, Micrococcus spp. and β- streptococcus group B, their PG belongs to the A3α type (Schleifer and Kandler 1972), preventing lysis by the peptidase M15A. Interestingly, Enterococcus sp., which is a Healthcare–Associated Infections (HAI) bacterium, was effectively lysed by ST79 peptidase M15A. The enzyme could act on the D-Ala-D-Ala termini of Enterococcus cell wall peptidoglycan (Arthur et al. 1996). Therefore, the action of the ST79 peptidase M15A may be specific to the peptidoglycan type A1γ of Gram-negative bacteria and to the Gram-positive Enterococcus sp.
Even though, in general, endolysin cannot attack the PG which is located under the outer membrane in Gram-negative bacteria, but permeabilized the outer membrane for example with 10 mM EDTA in combination with 50 mg/ml of the Pseudomonas endolysin EL188 can decrease the viable P. aeruginosa cells by 3 or 4 orders of magnitude in 30 min (Briers et al. 2011). Interestingly, LysAB2, the endolysin from A. baumannii phage ϕAB2 was reported to have an antibacterial effect against both Gram-negative (A. baumannii, E. coli, Salmonella enterica) and Gram-positive (Streptococcus sanguis, S. aureus, B. subtilis) strains (Lai et al. 2011). LysAB2 contains a C-terminal amphipathic region that is necessary for the antibacterial activity as also reported in the Lys1521 endolysin from a B. amyloliquefaciens phage that contains two cationic C-terminal regions. The cationic region was demonstrated to permeabilize the outer membrane of P. aeruginosa (Muyombwe et al. 1999). It is therefore possible to genetically engineer endolysin to have the cationic C-terminal regions to lyse Gram-negative bacteria from outside the same way as suggested by Nelson et al. (2012).
Lysozyme, also known as muramidase, is a well-known endolysin enzyme that is generally used as a standard for comparisons of endolysin activity. For example, A. baumannii phage phiAB2 endolysin activity was only 30 % activity of chicken egg white lysozyme (Lai et al. 2011). When the ST79 peptidase M15A activity was compared with chicken egg white lysozyme, it could reduce PG substrate approximately 2 times higher in lysis efficiency than lysozyme. Nevertheless, the transglycosylase SPN1S endolysin from Salmonella typhimurium-infecting phage can reduce 50 % OD of a 1 ml EDTA pretreated cell suspension when only 50 nanograms was used. This phage enzyme, therefore, has approximately 30 times higher activity than lysozyme (Lim et al. 2012) and also more than ST79 peptidase M15A.
In conclusion, ST79 peptidase M15A is specific to A1γ PG and cleaves PG at the peptide chains. The enzyme can work in a broad alkaligenic range and temperature, has higher activity when compared to lysozyme and is also active against a broad range of Gram-negative bacterial PG and also Enterococcus sp. that make the enzyme an outstanding one for further development. The cationic amphipathic C-terminal in some endolysins that showed permeability to Gram-negative bacteria may be genetically engineered into ST79 peptidase M15A and used as an adjunct to standard antibiotic therapy for B. pseudomallei infection. The combination of a compound that permeabilizes B. pseudomallei's outer membrane with the enzyme that attacks PG may provide a more effective treatment in severe or drug resistant cases. Intensive investigation is, however, definitely required.
PG:
peptidoglycan
NAM:
N-acetylmuramic acids
NAG:
N-acetylglucosamine
Luria and Bertani medium
IPTG:
isopropyl-β-d-thiogalactopyranoside
Healthcare–Associated Infections
EDTA:
Ethylenediaminetetraacetic acid
optical density
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NK, UY, AB, UT, JLR and RWS designed the experiments. NK performed experiments. NK and RWS drafted the manuscript. All authors read and approved the final manuscript.
We would like to thank Prof. James A. Will for editing the manuscript via Publication Clinic, Khon Kaen University, Thailand.
Not applicable since this article does not contain any studies with human participants or animals performed by any of the authors.
This work was supported by The Royal Golden Jubilee Ph.D. program (Grant No. 4.O.KK/52/I.1.N.XX) for NK and RWS and the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the heath cluster, project "Specific Health Problem in Greater Mekong Sub-region (SHeP-GMS)" of Khon Kaen University.
Department of Biochemistry and Melioidosis Research Center, Faculty of Medicine, Khon Kaen University, 123 Mitraparb Road, Khon Kaen, 40002, Thailand
Nittaya Khakhum
& Rasana W. Sermswan
Department of Microbiology and Melioidosis Research Center, Faculty of Medicine, Khon Kaen University, 123 Mitraparb Road, Khon Kaen, 40002, Thailand
Umaporn Yordpratum
& Unchalee Tattawasart
Department of Microbiology, Faculty of Science, Khon Kaen University, 123 Mitraparb Road, Khon Kaen, 40002, Thailand
Atcha Boonmee
Department of Land, Air and Water Resources, University of California, Davis, 3308 Plant and Environmental Sci. Bld., Davis, CA, 95616, USA
Jorge L. M. Rodrigues
Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA
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Correspondence to Rasana W. Sermswan.
Khakhum, N., Yordpratum, U., Boonmee, A. et al. Cloning, expression, and characterization of a peptidoglycan hydrolase from the Burkholderia pseudomallei phage ST79. AMB Expr 6, 77 (2016) doi:10.1186/s13568-016-0251-7
Endolysins


text:
Former S&T college dean Russell Buhite dies
Historian and former minor league first baseman Russell Buhite in 2014. Photo by Sam O'Keefe
Dr. Russell D. Buhite, a professor emeritus of history and political science and a former dean at S&T, died May 27. He was 79. Per his wishes, no memorial service is planned.
A noted scholar in U.S. diplomatic history, he was the author of 12 books, one of which was nominated for the Pulitzer Prize in Letters.
Before he became an author and a history professor, Buhite was a minor league first baseman and outfielder for the New York Giants, the Baltimore Orioles and the Washington Senators. His experiences informed his book, The Continental League.
Buhite joined Missouri S&T as dean of the College of Arts and Science in 1997 and held that position until 2002. He taught for 25 years at the University of Oklahoma, serving 10 years as chair of the history department, and was head of the history department at the University of Tennessee.
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