Deeply intuitive. Courageous. Real. Smart. Articulate. And… a little stubborn.
These are some of the words people have used to describe me. For as long as I can remember, I was never the early bird – you know, the first to tie my shoelaces or the first to get married. I’ve never been about following the crowd or jumping aboard the latest craze. It took me a long time to be OK with that, especially in a society that reveres “first to the top”.
“I value foundation. I’m naturally inclined to follow my own path. And to help others follow theirs.”
In a world where people and businesses are constructed by stereotypes and bound by expectations, it’s those that take the time to build their own foundation and get to know their own core and take action from that place that rise to the top. It’s the approach that’s brought me the most success and, it’s why I started Beyond Expectations Coaching + Communications – to help individuals and organizations communicate on a deeper level to effect lasting transformation.
Let’s get c l e a r.
Transformation first comes from an honest acknowledgment of the need for change within ourselves or within our organizations – a communication to ourselves from ourselves. Sometimes the message is not so clear, and that’s where I come in. Whether through my career in communications or in coaching my clients one-on-one, getting people and businesses clear on what they want and why they want it – from the inside-out – is one of my biggest strengths. By using my intuition, asking the right questions and creating an open, collaborative environment, I bring the bigger vision into focus. From there, magic happens.


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Restrictive Texas voting bill advances to floor vote
As written, HB 6 indicates that the path to ensuring “electoral integrity and security” will be through “increasing criminal sanctions” and “creating criminal offenses”. Democrats and suffrage activists said this was a voter repression tactic that would disproportionately affect color communities.
“We believe that if these provisions become law, Anglo-observer-slash-vigilantes will disrupt polling stations, especially in places where colored people are voting (from) inside the polling station, and that will make voters so uncomfortable they just leave without casting their vote, “said Nina Perales, who serves as vice president of litigation for the Mexico-US Legal Protection and Education Fund, during a press conference Wednesday.
In its current form, the bill makes it a state crime for election officials to send mail-voting requests to voters if they have not individually requested the forms.
In HB 6, even if the voter needs help for medical reasons, people helping voters to cast their votes are required to submit documents explaining why the voter needs help. The person assisting the voter must also provide their own personal contact information and indicate their relationship with the voter.
In addition, the bill extends access for election observers within polling stations, stating that an election observer can only be removed “if the election observer is engaged in activities that would constitute an electoral fraud-related offense”.
Austin, Texas residents will be lining up in record numbers on April 27, 2021 to vote early on community issues.
Voting advocates refer to this provision as an example of how the bill would encourage voter intimidation and state that the bar is very high for expelling an election observer who may otherwise behave unsavory. According to the Brennan Center for Justice, a bipartisan, independent organization that analyzes electoral rules, the bill essentially “restricts the ability of election officials to protect voters from illegal interference and harassment by” observers “”.
“If an election observer starts yelling, yelling or arguing with a voter, lying on the floor or beating pots and pans, the election officials would not be able to throw these people out. That would be.” a crime. The only recourse for the election worker would be to call the police and hope that the police would come, “Perales said.
Thursday’s vote is sure to spark another round of backlash from prominent Democrats and suffrage activists who have spent weeks deciphering the Republican urge to push double electoral bills in both the House and Senate. Former presidential candidates and locals from Texas, Julian Castro and Beto O’Rourke, and voting rights attorney, Stacey Abrams, have all spoken out against advancing the bill.
An American Airlines Boeing 737 MAX aircraft takes off on a test flight from Dallas-Fort Worth International Airport in Dallas, Texas on December 2, 2020.
In addition to high-profile political voices, corporate giants such as American Airlines, Microsoft, Dell and Unilever spoke out against the legislation that developed through the Texan legislation following the effects of the revised electoral laws in Georgia.
On Tuesday, more than 50 companies, corporate groups and industry leaders signed a letter in which they “urged lawmakers to uphold our always elusive core democratic principle: equality”.
“We stand together as a bipartisan coalition calling on all elected leaders in Texas to support reforms that make democracy more accessible and to oppose changes that would restrict voters’ access to the ballot,” the letter said.
Senator Ted Cruz speaks during a press conference outside the U.S. Capitol on April 29, 2021 in Washington, DC.
The state’s top Republicans have repeatedly expressed their opposition to corporate giants weighing on the progress of the bills as many corporations continue to grapple with their positions against the legislation. A Republican-backed proposal to financially punish companies that “publicly threatened any adverse action” against Texas in their opposition to the electoral law was even briefly discussed during the state’s budget debate in April.
Ultimately, the proposed change was not incorporated into the budget, thereby removing the comments from Texas Lt. Gov. Dan Patrick in April when asked if companies criticizing the state’s electoral laws should lose financial incentives.
“This is not a consideration, we don’t punish people for disagreeing with us,” Patrick said at the time.
“Enough is enough. Companies that are blatantly misrepresenting efforts to protect our elections must be called, singled out and cut off,” said Cruz.
Across the aisle, in one of the state’s most diverse cities, two of the region’s top Democrats – Houston Mayor Sylvester Turner, and Harris County Judge Lina Hidalgo – refused their respective “state” on Wednesday City and State to Have The county’s events are produced by the city’s chamber of commerce, the Greater Houston Partnership, via the ongoing voting rights debate.
The couple said the decision was in response to the organization’s lack of explicit opposition to advancing the state’s ballot papers, including HB 6.
“There is nothing partisan about voting suppression … if there are bills that deal with voter suppression, voter intimidation and restriction of access to polling stations – that is not partisan, it is just completely wrong,” said Turner at a press conference urge business leaders to make greater strides in opposing the law before it goes into effect.

domain: sociology


National Climate Change Information System
About the National Framework for Climate Services
The NFCS is anchored on the following Global Framework for Climate Service pillars:
Observations and Monitoring
The Observations and Monitoring pillar helps ensure that the climate observations necessary to meet the needs of end users are made, managed and disseminated, supported by relevant metadata.
User Interface Platform
User Interface Platform is the pillar of the Framework that provides a structured means for users, climate researchers and climate data and information providers to interact at all levels.
Climate Service Information System
It is the principal mechanism through which information about climate (past, present and future) is routinely collected, stored and processed to generate products and services that inform decision-making processes, often complex, across a wide range of climate-sensitive activities and enterprises.
Research, Modelling and Prediction
The Research, Modelling and Prediction pillar supports the development and improvement of tools and methods that will facilitate the transition of research to operational climate service provision and engender practical applications of climate information.
The capacity development pillar will strive to enhance existing capabilities that are needed to enable manage climate risk effectively.


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The Green Bay Packers on Monday released offensive tackle Chad Clifton. According to a report from ESPN, he did not pass a physical after off-season back surgery.
You may remember Clifton getting knocked out the game after getting blind-sided by Warren Sapp in a game against Tampa Bay. Chad needed hip surgery, but came back to play ten more seasons for the Packers. It looks like age and injuries finally caught up with him.
Chad, thank you for all you've done for the Green & Gold. You are the embodiment of a team player. Now take it easy. You deserve a little time off.

domain: sports


text:
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domain: economics


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The Harvard Graduate School of Design announces the appointment of Grace E. La (MArch ‘95) as Professor of Architecture, effective July 1, 2013.
A talented designer and experienced academic leader, La has focused her work on alternative modes of practice that connect architecture, landscape design, engineering and sustainable environments.
La is cofounder and Principal of LA DALLMAN Architects, with James Dallman (GSD M.Arch ‘92). LA DALLMAN’s built work includes the Kilbourn Tower, the Miller Brewing Meeting Center (building by Ulrich Franzen), the Hillel Student Center, the Ravine House, the Gradient House, and the Great Lakes Future and City of Freshwater permanent exhibits at Discovery World. The firm’s Crossroads Project, which transforms infrastructure for public use, includes a 700-foot-long Marsupial Bridge, a bus shelter and a media garden. LA DALLMAN is currently commissioned to design additions to the Marcus Center for the Performing Arts (original building by Harry Weese and landscape by Dan Kiley) and the Harmony Project, a 100,000-square-foot hybrid arts building for a ballet school, university dance program and medical clinic. The Harmony Project design process was awarded a 2012 grant from the National Endowment for the Arts.
The firm has received numerous professional honors and prizes in international competitions, including design awards from the American Institute of Architects. In 2011, LA DALLMAN was the first American firm awarded the Rice Design Alliance Prize. The firm was also named an Emerging Voice by the Architectural League of New York and has received a Bruner Award for Urban Excellence, Silver Medal and design engineering recognition including the national Concrete Reinforcing Steel Institute Design Award.
LA DALLMAN’s work has been featured in publications including Architect, a+t, Architectural Record, Azure, Praxis, and Topos, and in books released by Princeton Architectural Press and Routledge. Architect profiled the firm’s design culture in June 2012. La is coauthor of Skycar City (Actar, 2007), featuring the inaugural Marcus Prize Studio, which was exhibited at the 2008 Venice Biennale. She is also the cofounder and three-time editor of UWM’s Calibrations and a member of the editorial board of the Journal of Architectural Education.
La has served as a faculty member in the School of Architecture and Urban Planning at the University of Wisconsin Milwaukee (UWM) since 1999, receiving tenure in 2005. She serves as the Chair of the Planning and Coordinating Committee in the Department of Architecture, leading the school’s curriculum reform, fellowship initiatives, and strategic planning, which involves reenvisioning the school’s facilities in relation to pedagogical goals and new technologies. La received UWM’s Distinguished Undergraduate Teaching Award and has taught and lectured widely, including as a Design Critic at the GSD. She won four Faculty Design Awards from the Association of Collegiate Schools of Architecture for work that advances the reflective nature of practice and teaching; she has led collaborations with engineers, artists, scientists, craftspeople and public officials on a diverse range of civically engaged projects. Among her presentations, La has lectured at the New Museum, the Museum of Fine Arts Houston, the National Building Museum and the Carnegie Museum of Art.
La received her MArch with thesis distinction from the GSD, winning the Clifford Wong Housing Prize. She received her undergraduate degree, A.B. magna cum laude, from Harvard University in Visual and Environmental Studies.

domain: design


text:
Slice 400g chicken breast and dust with paprika. Chop and sweat 1 onion and 2 cloves of garlic in 2tbsp oil.
Add 200g peas, sweat, then add 100ml veg stock and bring to the boil. Add the chicken and simmer for 5 mins.
Add 2tbsp sesame seeds and 2tbsp grated Parmesan. Drain the pasta and mix with the chicken, season and scatter on coriander.
The world is undeniably in love with sriracha, the spicy red chilli and garlic sauce of everyone’s dreams. Although it made its way out of Asia in the 1980s, it seems to have suddenly gained popular... keep reading
Trapiche is situated in the Mendoza region of Argentina. The winery was established in 1883 and they are well known for producing quality wine at affordable prices. This is a great example of Sauvignon Blanc from the Mendoza region. Fresh aromas of grapefruit, crisp blackcurrant leaf, green pepper and grassy back notes. Buy this wine if you like: Dry whites with lively fruit and fragrant aromatics.

domain: geography


text:
COLLEGE PARK, Ga. — Two Banneker High School students were robbed on campus at gunpoint Monday evening.
Principal Dr. Duke Bradley said two former students trespassed unto the campus and approached two current Banneker students. School officials said the former students took out their weapon, forcibly took a student's cellphone and then left the building.
Officials said no one was physically harmed and images of the alleged perpetrators were caught on campus surveillance.
School officials said the case is now being handled by the Fulton County Schools Police Department.
The student who was robbed told school administrators about the incident Tuesday around 8:45 a.m.
In a press release, the school said they are deeply concerned and are revising the after school campus safety plan. They also said immediate changes will be put in place for car riding students.
"Rest assured that we take the safety of all school stakeholders very seriously and every effort will be made to protect them," Bradley said in a letter to parents.
MORE HEADLINE |

domain: law


Glen Johnson set for Liverpool exit door as part of summer cull
Glen Johnson has claimed that Liverpool must embark on an overhaul this summer in order to challenge for the Premier League title, but admits that he expects to be a high-profile victim of any Anfield cull.
Liverpool's hopes of qualifying for the Champions League next season suffered a blow with defeat at Hull City on Tuesday, with the Anfield club now seven points adrift of fourth-placed Manchester United with four games left to play.
Failure to play in the Champions League next season will lead to Liverpool missing out on at least £50 million in prize money - earnings from the competition increase from a basic £30 million next season due to the start of Uefa's broadcasting deal with BT Sport - and also leave the Merseyside club struggling to attract leading summer targets to Anfield.
However, having gone close to winning the title last season before being overtaken by the eventual champions Manchester City, Johnson believes that Brendan Rodgers, the Liverpool manager, has no option but to make wholesale changes to his squad this summer. "Definitely, there need to be changes," the England defender said.
"It's the same every year and every team is going to try and do their business in the summer to try and make themselves stronger for the following the season. The club needs to do that, too. We have some fantastic young players, but we say the same every year - we always have some good young players coming through.
"The club has got some business to do in the summer with current players and new players and it's just about trying to get the squad as strong as possible for the start."
As many as 10 players could leave Anfield this summer with Steven Gerrard, the club captain, having announced his decision to sign for LA Galaxy. Mario Balotelli, Rickie Lambert, Jose Enrique, Fabio Borini and goalkeeper Brad Jones are expected to be sold, while uncertainty surrounds the futures of Lucas Leiva, Kolo Toure and Martin Skrtel.
Liverpool are poised to resist any attempts to lure Raheem Sterling away from Anfield, despite the England winger's contractual stand-off with the club, but Johnson, an £18 million signing from Portsmouth in 2009, concedes that his own time is up at the club.
Asked if he thought he would be among those leaving, Johnson said: "Of course, I have no contract! I don't think that will change, but we have to wait and see. I have always been professional all season, always wanted to play and that's still what I doing. I've got four more games and then let's see what we do in the summer."
Liverpool's chances of finishing fourth are all but gone, but Johnson said that the players would not go through the motions in their last four games. "We are all professionals. Otherwise we would not be at a club like Liverpool. We've got four more games to concentrate on to try and get a few more points. That's what we will concentrate on doing.
"Every team wants to finish as high in the Premier League as possible. With the Europa League, you do get a lot more games, but it's a competition you would rather be in than not."
Despite the fall from runners-up position last May to the prospect of a finish as low as seventh this season, Liverpool's American owners are understood to be supportive of Rodgers as manager.
The availability of the Borussia Dortmund coach Jurgen Klopp following the end of the Bundesliga season ensures the presence of a highly respected stalking horse should Rodgers's position come under threat, but the Northern Irishman is preparing to press ahead with his rebuilding plans.
Liverpool are expected to fend off interest from Arsenal to sign James Milner on a free transfer from Manchester City, while the Burnley forward Danny Ings is known to favour a move to Anfield ahead of Manchester United, who have expressed an interest in the player.
Efforts to persuade the PSV Eindhoven winger Memphis Depay to reject similar interest from United, Paris Saint-Germain and Bayern Munich are likely to prove less successful should a failure to play in the Champions League be confirmed.
For Liverpool to overhaul United in the race for fourth, they must realistically win all of their remaining games - including a trip to champions-elect Chelsea - and hope that United lose at least two of their final four games.
However, with Gerrard poised to return against Queens Park Rangers on Saturday after missing the defeat at Hull with the after-effects of cramp, deputy captain Jordan Henderson insisted that the team would continue to fight on.
"We've just got to keep going," Henderson said.
"Obviously everybody is disappointed with the game (at Hull), but we've just got to move on as quickly as possible to the next game and hopefully win every game from now until the end of the season and see where we are." (© Daily Telegraph, London)


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Okay, okay. So this is the more Indian version of Chinese we’re talking about but we can’t do without a little of our own touch can we? Especially when it comes to Maggi!
Chinese Style Maggi Recipe
Prep the vegetables and in a bowl mix together all the sauces along with two tastemakers from maggi packets and keep aside.
Cook the maggi according to packet instructions and set aside.
Now heat the oil in a pan and add ginger and garlic, cooking them for about half a minute.
Add onions and carrots first and saute them till onions lose their colour and carrots are tender.
At this point, at the remaining vegetables and only half the amount of scallions.
Mix well and let the vegetables cook for a few minutes.
Add the sauce mix once the vegetables are cooked and mix well.
Add the cooked maggi and again mix well.
You can add a sprinkle of water if required.
Now once the maggi blends with the vegetables gravy, transfer it to a serving bowl, garnish with the remaining scallions and serve hot.

domain: food


|Partial Stability of Some Guidance Dynamic Systems with Delayed Line-of-Sight Angular Rate
Chunming Fu and Wanbiao Ma*
International Journal of Control, Automation, and Systems, vol. 12, no. 6, pp.1234-1244, 2014
Abstract : In this paper, for a class of the guidance dynamic system proposed by Moon, Kim, and Kim (Journal of Guidance, Control and Dynamics, vol. 24, no. 4, pp. 659-664, 2001) and Shafiei and Binazadeh (ISA Transactions, vol. 51, pp. 298-303, 2012), a new nonlinear missile guidance law with delayed line-of-sight (LOS) angular rate is introduced, and then partial stability is studied by using the method of Lyapunov functionals and stability theory of functional differential equations. The time delay in the LOS angular rate comes from the missile guidance dynamic system in which a low-cost sensor is used to obtain LOS rate information by image processing techniques. Furthermore, for a class of classical proportional navigation control system with delayed line-of-sight rate studied by Dhananjay, Lum, and Xu (IEEE Transactions on Control Systems Technology, vol. 21, pp. 247-253, 2013), a new stability region is obtained by using the method of Lyapunov-Razumikhin.
Functional differential equation, guidance dynamic system, LOS angular rate, partial stability.
Download PDF : Click this link


Control valves are the workhorses of process automation. They touch the process and execute the control strategy coming from the automation system. Any valve seat leakage can impact the level of control and lead to safety issues, unplanned downtime, quality issues,...
Detecting Valve Seat Leaks
The devices that touch your process such as control valves, measurement instrumentation, and analyzers have tremendous impact on the ability to control the process efficiently. Even the best control and advanced control strategies can't do what they are designed to do...
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COD WW2 update 1.16 for PS4 is now available for download. The new COD WW2 1.16 update brings bug fixes and other improvements. Apart from this, the latest COD WW2 version 1.16 also includes stability and performance improvements. Since the last patch, many players were experiencing a number of issues when trying to play the game.
Recently, a big Call of Duty WW2 update was released with Attack of the Undead Community Event and a new Community Challenge. The patch also added a new item type: Face Camos. The latest Call of Duty WW2 update 1.16 size is around 50Mb on PlayStation 4. Check out more details below.
COD WW2 Update 1.16 Changelog
- Added bug fixes and other improvements.
- COD WW2 1.16 update added performance and stability improvements.
Fixes for PC only
- Fixed Attack of the Undead Community Challenge UI issue with Zombies kill counter (All kills have still been tracking and counting properly. This was only a UI issue.)
- Fixed issue with UI displaying incorrect end date of the Attack of the Undead Community Event. The event ends on June 26 at 10AM PDT.
- Fixed issue where end of Ranked Play Season 2 Helmet rewards for the top 100 players were displaying the wrong Season number
- Distributed end of Ranked Play Season 3 Helmet rewards for the top 100 players (Check your Mail)
- Fixed issue where Camos and Paintjobs were being removed from weapons and not displaying properly
- Fixed issue with incorrect spawn site placements for S&D on Sainte Marie du Mont
- Fixed issue where party members were able to join a match via the new playlist tiles without the party leader
Check your game update and download the latest COD WW2 update 1.16.


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Most personal injury lawyers charge something known as a contingency fee. The fee benefits the client and provides the lawyer with the incentive to maximize the compensation recovered in a case.
When you hire a lawyer, make sure you understand the lawyer’s fee structure. Fee disputes can occur when lawyers and clients do not communicate their fee arrangements clearly.
How Do Lawyers Charge Legal Fees?
Private attorneys charge for legal services, and these legal fees usually take three forms:
Lawyers charge a flat fee for projects with a predictable amount of work. Through a flat fee, the lawyer quotes a fixed cost for the entire project.
Some projects that fall into this category include:
- Traffic tickets
Clients like flat fees because they provide predictability, but lawyers only offer them for specific types of legal work.
Lawyers may charge an hourly fee for projects that have an unpredictable amount of work, ensuring the lawyer gets fairly compensated for the time they spend on the project.
With an hourly fee, the lawyer quotes an hourly rate and bills the client for the amount of time spent working on a case. Clients tend to dislike hourly fees because of their inherent lack of predictability.
Lawyers can only charge a contingency fee in certain types of cases. Missouri’s Rules of Professional Conduct prevent lawyers from charging a contingency fee in criminal and divorce cases, but you will often incur a contingency fee in personal injury cases such as car accidents or slip and fall accidents.
With a contingency fee, the lawyer quotes a percentage and calculates the final bill by multiplying that percentage by the amount of compensation the lawyer has won for their client.
How Does a Contingency Fee Work?
A contingency fee can only be calculated at the end of the case once the outcome is known. When you hire a lawyer on a contingency fee basis, the lawyer works on the case without charging the client until the lawyer either wins, loses, or settles the case.
If the lawyer wins compensation for the client, the insurer or at-fault party pays the lawyer. At that point, the lawyer can calculate their contingency fee, generate a bill, collect their share of the compensation as payment, and send the rest of said compensation to the client.
Benefits of Contingency Fee
A contingency fee provides several benefits to clients, including:
No Upfront Fee
Since this particular legal fee depends on the compensation earned, a lawyer cannot charge any upfront fee. The client will sign the lawyer’s contingency fee agreement, and the lawyer will start working on the injury case.
No Fee for a Loss
A typical contingency fee falls somewhere between 30% and 40% of a client’s total compensation. If the lawyer wins or settles the case, the lawyer receives the quoted percentage of the compensation.
If the lawyer loses the case, the lawyer also receives the quoted percentage of the compensation. With that being said, though, 30% of zero is still zero, so the lawyer receives no contingency payment.
An Incentive for the Lawyer
The more the lawyer wins for the client, the larger they receive from their legal fee, which incentivizes them to receive as much as possible for the client’s injuries and damages. Thus, when hiring a contingency fee lawyer, the lawyer’s interests align with the client’s.
While the amount of the legal fee will depend on the settlement or damage award won by the lawyer, the percentage is a fixed value, which guarantees the client will receive the bulk of the compensation their lawyer wins for them.
For instance, if a lawyer quotes a 35% contingency fee, the client will receive 65% of the compensation, which will go to pay the client’s medical bills and living expenses while the client recovers from the injuries.
Questions to Ask a Contingency Fee Lawyer
When you hire a lawyer that makes use of contingency fees, make sure you ask about any additional costs, so you know exactly what you will pay for your legal representation. During your case, you will incur costs to keep the claim going.
Some examples of litigation costs include:
- Stenographer fees
- Lawsuit filing fees
- Expert witness fees
- Medical records copying fees
Most contingency fee lawyers will pay for these costs during the case, but in the end, you will need to reimburse the lawyer for any additional costs paid on your behalf.
Ask your lawyer about the process of approving litigation costs and whether you will need to reimburse the lawyer even if you lose your case.
Contact Schultz Myers Personal Injury Lawyer For Help
You will likely schedule free consultations with several lawyers before picking the one that’s best for you and your situation. Discuss legal fees with each lawyer you meet and request a copy of the lawyer’s contingency fee agreement.
Read them carefully so that if you have any questions about the agreement, you can discuss them with your lawyer before signing it.


Howard Lake-Waverly Herald, June 19, 2000
Waverly council: let the music play
By Andrea Vargo
A 3-1 vote by the Waverly City Council, Tuesday, will allow the Waverly Lions Club to have music play until 12:30 a.m. during Waverly Daze.
The decision came after several spirited exchanges between Mayor Charles Bush and disc jockey Greg Lammers.
Lammers published an ad and circulated petitions to raise support for his show to be allowed at the city celebration in July.
Bush told Lammers that police reports from the last time he played for Waverly Daze (1995, said Lammers) are the reason he is no longer allowed in Waverly at public functions.
There are no problems with Lammers' show, but there are problems with the people who attend it, Bush said.
"I promise to have the most behaved crowd you ever see," Lammers stated emotionally.
"People won't come to you if there are problems, but to me," Bush retorted.
"The council made a decision to allow music until 10 p.m. (at the May meeting) and not just limit a particular entertainer," Bush said.
He stated that Waverly Daze is a family function, and that 10 p.m. is late enough for events to finish.
Several Lions and other musicians filled the available space and spoke in support of Lammers.
The Lions Club has no official position, but individuals do, said Pat Borrell, Lions Club member.
"It is a little disconcerting that we have the largest crowd here (since I have been mayor), when we have had more important things, (like annexation and other things that impact the taxpayers)," Bush said.
Having allowed about 15 minutes for discussion, Bush attempted to move forward with the agenda.
But Councilman John Hertzog placed a motion on the floor to allow the Lions to have some music other Lammers until 12:30 a.m., and it passed three to one, with Bush voting against the measure.
Annexation of the Tom Ryan property produced the most vocal exchanges during the two public hearings Tuesday.
The final vote for both hearings was tabled until all council members assemble Friday for a special meeting. Councilman Dave Fournier was absent from this meeting.
Councilman Jerry McRaith asked how the property is to be used, who is the developer, and what is the relationship of Buzz Jonason to the project.
Jonason said it really doesn't matter, because the issue is the annexation or disapproval of it.
He said that he is an investor with developer Joe Lemmerman.
Having answered McRaith's same questions at several meetings, Ryan explained again that the council has total control of what happens to that property after it is annexed.
"It is residential property and will be developed as such," Ryan said.
McRaith said he wants to protect existing taxpayers from having to pay for city expansion.
Attorney Jennifer Ford said any city expands, stays stagnant, or decreases.
"The city has to determine its capacity (for sewer and water), fees, and such. That is why we sit here," Bush stated.
It was brought out that the developer pays for sewer and water for the proposed additions. That cost is paid by the new home owner, when the home is purchased.
The city is operating at 50 percent of capacity, planning and zoning commissioner Adrian Duske said.
If all the available space in the city were developed, the city would be beyond capacity, stated a resident. Jim Woitalla of the maintenance department agreed.
At the special Friday council meeting, the Ryan annexation was approved 4-1 with McRaith voting against the measure.
The rhetoric was heated between McRaith and Ryan, as accusations flew.
Ryan accused McRaith of improperly investigating both Ryan and Jonason, using his status as a city councilman without authorization from the council.
Ryan felt it was worth a possible lawsuit.
McRaith felt it was his right to investigate as a councilman, and said he refused to be bullied.
A conflict of interest should keep McRaith from voting on the annexation, said Ryan, since McRaith owns 40 acres in the city that are ripe for development.
McRaith voted against the annexation when the time came.
The hearing for the annexation of the LePage Addition by P&P Enterprises was uneventful, as no one had any input.
At the Friday meeting, the annexation was approved as well as a conditional use permit, allowing homes to be moved onto the lots.
The farmers market is scheduled to run June 24 to Oct. 28 from 8 a.m. to 1 p.m at Railroad Park.
Liability insurance must be supplied by the vendors (from their homeowners policy), and preregistration is required.
The registration fee for the season is $5, and a daily fee of $1 will be charged. The local Girl Scout troop will provide a supervised play area for children.Interested parties can contact Gen Johnson at 763-658-4180.
Fournier submits resignation
Waverly City Councilman Dave Fournier gave his letter of resignation to the council Friday. It becomes effective after the July 11 meeting. The council is looking for interested residents to consider for the vacancy.
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Play with 4-10 player online or via local WiFi as you attempt to prepare your spaceship for departure, but beware as one or more random players among the Crew are Impostors bent on killing everyone!
Originally created as a party game, we recommend playing with friends at a LAN party or online using voice chat. Enjoy cross-platform play between Android, iOS and PC.
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text:
If you're looking for manga similar to little heart, you might like these titles.
When a young girl nervously awaits the start of the 50th Goldfish Scoping Tournament's final round, an old lady comes up to her and gives her a poi with a ribbon tied to it. What is the story behind this special gift?
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domain: artistic


Anholt offshore wind farm is located between the headland of Djursland on the Jutland coast and the island of Anholt. The site covers an area of
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INSTALLATION OF 111 CABLES IN 4 MONTHS
As part of this project, Boskalis was contracted by Dong Energy to install 111 infield cables with a total length of around 154 km. Dong Energy subcontracted the cable manufacturer to supply three types of cable (150 mm2, 240 mm2 and 500 mm2), delivered by train on 89 drums and in 3 continuous lengths (500 mm2).
Boskalis deployed two cable installation spreads to lay cables for the Anholt Wind Farm. The first cable installation spread was the Stemat 82, assisted by anchor handling tugs, Lydia D and Nova K. The Stemat 82 is an anchored barge equipped with a large turntable which was used to install the long cables that connect the substation to the first wind turbine in each row of the wind farm.
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Safety light curtains are an essential component of automated machine guarding systems. They control the light curtain’s position in a machine by sending and receiving stop signals. Typically, these devices can be controlled by an external safety PLC or by a safety relay. But sometimes, a malfunction can cause the stop signal to be missed, requiring a manual reset. Here’s how to test safety light curtains. A simple test for automated machine guarding systems is required by OSHA.
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For the complete history of the MU, year by year, go here.

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Summary: Bridgette is Associate Professor in Health Research and teaches on the postgraduate Health Research and DClinPsychol programmes. She is also Chair of the MB ChB Undergraduate Mitigation Committee
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Bridgette Associate Professor and Chartered Psychologist. Her portfolio of research focusses around monitoring, managing, and modifying mental health and wellbeing; including the potential for e-health tools to support health and wellbeing. She is Lead for the suite of Postgraduate Programmes in Health Research. Her leadership roles include Chair of the MBChB Undergraduate Mitigation Committee.

domain: health


text:
Zinc Oxide Nanoparticles Induce Ferroptotic Neuronal Cell Death in vitro and in vivo
Received 18 February 2020
Accepted for publication 10 June 2020
Published 27 July 2020 Volume 2020:15 Pages 5299—5315
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 2
Editor who approved publication: Dr Mian Wang
Xia Qin,1,* Qianghu Tang,2,* Xuejun Jiang,3,* Jun Zhang,4 Bin Wang,4 Xuemei Liu,2 Yandan Zhang,4 Zhen Zou,4,5 Chengzhi Chen2,5
1Department of Pharmacy, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, People’s Republic of China; 2Department of Occupational and Environmental Health, School of Public Health and Management, Chongqing Medical University, Chongqing 400016, People’s Republic of China; 3Center of Experimental Teaching for Public Health, Experimental Teaching and Management Center, Chongqing Medical University, Chongqing 400016, People’s Republic of China; 4Institute of Life Sciences, Chongqing Medical University, Chongqing 400016, People’s Republic of China; 5Dongsheng Lung-Brain Disease Joint Lab, Chongqing Medical University, Chongqing 400016, People’s Republic of China
*These authors contributed equally to this work
Correspondence: Zhen Zou; Chengzhi Chen Email [email protected]; [email protected]
Purpose: Zinc oxide nanoparticles (ZnONPs) are one of the most important nanomaterials that are widely used in the food, cosmetic and medical industries. Humans are often exposed to ZnONPs via inhalation, and they may reach the brain where neurotoxic effects could occur via systemic distribution. However, the mechanisms underlying how ZnONPs produce neurotoxic effects in the brain remain unclear. In this study, we aimed to investigate the novel mechanism involved in ZnONPs-induced neurotoxicity.
Methods and Results: We demonstrated for the first time that pulmonary exposure to ZnONPs by intratracheal instillation could trigger ferroptosis, a new form of cell death, in the neuronal cells of mouse cerebral cortex. A similar phenomenon was also observed in cultured neuron-like PC-12 cell line. By using a specific inhibitor of ferroptosis ferrostatin-1 (Fer-1), our results showed that inhibition of ferroptosis by Fer-1 could significantly alleviate the ZnONPs-induced neuronal cell death both in vivo and in vitro. Mechanistic investigation revealed that ZnONPs selectively activated the JNK pathway and thus resulted in the ferroptotic phenotypes, JNK inhibitor SP600125 could reverse lipid peroxidation upregulation and ferroptotic cell death induced by ZnONPs in PC-12 cells.
Conclusion: Taken together, this study not only demonstrates that pulmonary exposure of ZnONPs can induce JNK-involved ferroptotic cell death in mouse cortex and PC-12 cells, but also provides a clue that inhibition of ferroptosis by specific agents or drugs may serve as a feasible approach for reducing the untreatable neurotoxicity induced by ZnONPs.
Keywords: zinc oxide nanoparticles, ferroptosis, neuron, cerebral cortex
Ferroptosis is a novel non-apoptotic and iron-dependent form of cell death.1 The hallmarks of ferroptosis are defined as the inactivation of phospholipid hydroperoxidase glutathione peroxidase 4 (GPX4), the enhancement of iron and the accumulation of lipid oxidation.2 Previous investigations have demonstrated that ferroptosis can be triggered by natural stimuli, synthetic agents and environmental toxicants in many types of cells.2–4 Intriguingly, the studies also reported that the engineered nanoparticles could cause ferroptotic cell death in mouse xenograft tumors, and thus suppressed the growth of tumors.5,6 These findings suggest that nanoparticles are capable of inducing ferroptosis in animals and/or cultured cells.
Zinc oxide nanoparticles (ZnONPs) are one of the most important nanomaterials that are widely used in the food, cosmetic and medical industries.7 Increasing evidence is available regarding the toxicological effects of ZnONPs in animals, particularly in the respiratory,8 cardiovascular9 and neuronal systems.10 These toxicological effects have been identified mainly through oxidative stress, inflammation and even cell death.8,11,12 ZnONPs can induce several forms of cell death in cultured cells or animals, including apoptosis,13 necrosis14 and autophagy.8,15,16 Importantly, ZnONPs recently have been found to interact with iron within the cells and significantly elevate the generation of peroxidation products (such as malondialdehyde, MDA),17,18 both of which are highly linked to the hallmarks of ferroptosis. Therefore, it is reasonable to speculate that ferroptosis may be involved in the process of cell death induced by ZnONPs. However, to date, whether ZnONPs can trigger ferroptotic cell death in vivo and/or in vitro remains unknown.
ZnONPs can distribute in various tissues of mice, such as liver, lung and kidney, etc.19 Accumulation of ZnONPs in these tissues may further contribute to the adverse effects by induction of oxidative stress, inflammatory responses and cytotoxicity.8 After systemic distribution, ZnONPs are also capable of reaching the brain via blood–brain barrier penetration or translocation along the olfactory nerve pathway.20 Consequently, ZnONPs have been demonstrated to cause spatial learning and memory deficits of animals by altering the synaptic plasticity in the hippocampus.21 In cultured cells, ZnONPs also induce the strongest cytotoxicity via induction of DNA damage and cell apoptosis in many types of neuronal cell lines, such as neural stem cells, Neuro-2a cells, SH-SY5Y cells and primary cultured astrocytes, etc.22–25
In this study, using both C57BL/6J mice and cultured PC-12 cells, we aimed to test the hypothesis that ferroptosis was involved in ZnONPs-induced neuronal cell death in vivo and in vitro. To verify the results obtained from animals and cells, a specific inhibitor of ferroptosis was used. The data showed that inhibition of ferroptosis could remarkably ameliorate the cytotoxicity caused by ZnONPs. These findings will provide a clue that inhibition of ferroptosis by specific agents or drugs may serve as a feasible approach for intervention and/or prevention of ZnONPs-induced neurotoxicity.
Materials and Methods
Chemicals and Reagents
ZnONPs, <50 nm particle size, was purchased from Sigma-Aldrich (St. Louis, MO, USA). MDA assay kit, adenosine triphosphate (ATP) assay kit and tissue iron assay kit were obtained from Nanjing Jiancheng Institute of Bioengineering (Jiangsu, China); anti-glutathione peroxidase 4 (GPX4), anti-voltage-dependent anion channel 3 (VDAC3), anti-cystine/glutamate antiporter (SLC7A11), anti-neuronal nuclei antigen (NeuN), anti-microtubule-associated protein 2 (MAP2), anti-Ki67 and anti-GAPDH antibodies were all obtained from Bioss Biological Technology Co. Ltd. (Beijing, China); β-actin antibody was obtained from ABclonal Biotechnology (Woburn, MA, USA); p-JNK (4668), p-ERK1/2 (4370) and p-p38 (4511) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Cy3 AffiniPure Goat anti-Rabbit IgG (H+L) was from EarthOx Life Sciences (San Francisco, CA, USA); 4.6-diamidino-2-phenylindole (DAPI), phenylmethanesulfonyl fluoride (PMSF) and bicinchoninic acid assay kit were all obtained from Beyotime Institute of Biotechnology (Shanghai, China); fetal bovine serum (FBS) was from Ausbian (Australia); Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Gibco Life Technologies (Grand Island, NY, USA); CCK-8 assay kit was obtained from Dojindo Laboratories (Shanghai, China); ferrostain-1 (Fer-1) was purchased from APExBIO (Houston, TX, USA); zinc-specific indicator FluoZin-3, AM was provided by Molecular Probes Inc., Invitrogen (Eugene, OR, USA); deferoxamine (DFO) was obtained from Shandong Topscience Biotech Co., Ltd. (Shandong, China). Diethylenetriaminepentaacetic acid (DTPA) was obtained from Sigma-Aldrich (St. Louis, MO, USA). SP600125 (S1460) was purchased from Selleckchem (Houston, TX, USA).
Healthy male specific pathogen-free C57BL/6J mice, aged 8 weeks and weighing 21–25 g, were purchased from Experimental Animal Center of Chongqing Medical University [Chongqing, China, license numbers: SCXK (Yu) 2012–001]. The mice were housed in propylene cages fitted with filter covers at a controlled ambient temperature (23 ± 1 °C) and relative humidity (55 ± 10%). The animals were maintained under standard laboratory conditions (12-hour light/dark cycle) and were given ad-libitum access to food and tap water. This study was approved by Institutional Animal Care and Use Committee of Chongqing Medical University. All procedures were conducted following the guidelines contained in the guide for the care and use of laboratory animals. All efforts were made to ensure minimal suffering to the animals.
ZnONPs Characterization and Preparation of Exposure Stock
The nature of particles and level of agglomeration were determined by transmission electron microscopy (TEM, Hitachi-7500, Hitachi, Ltd., Tokyo, Japan). Field emission scanning electron microscopy (Hitachi-SU8010, Hitachi, Ltd., Tokyo, Japan) with energy dispersive spectroscopy (Oxford X-MAN50, Oxford Instruments, UK) was used to detect the chemical elemental composition of ZnONPs. Size and zeta potential of ZnONPs were detected by dynamic light scattering and phase analysis light scattering (Zetasizer Nano-ZS, ModelZEN3600, Malvern Instruments Ltd., Malvern, UK). The obtained TEM image, chemical composition, Z-average, PDI and Zeta potential of ZnONPs were all shown in our previous work.8 ZnONPs were suspended in 2% heat-inactivated sibling mouse serum in MilliQ water. The solution of ZnONPs was freshly prepared as quickly as possible and sonicated with an ultrasonic cleaner (SB-5200DT, Ningbo Scientz Biotechnology Co., Ltd., China) for 20 min in an ice water bath before use.26,27
ZnONPs Intratracheal Instillation
The mice were randomly divided into four groups: control, low-dose group, medium-dose group and high-dose group. After one-week adaption, the animals were intratracheally instilled with a single dose of ZnONPs for 3 μg (low), 6 μg (medium) and 12 μg (high) per animal, and the vehicle control animals received 0.9% sterile physiological saline for instillation. The doses of ZnONPs used in the current study were calculated according to the Chinese occupational exposure limit of ZnONPs; detailed information about the process of calculation was described in our previous work.8 Moreover, these concentrations of ZnONPs were also similar to the previous studies.28,29
The animals were instilled intratracheally once as described previously by Jacobsen et al.28 In brief, after anesthesia, the mice were placed on a 40-degree slope and the trachea was intubated by an insyte catheter with a shortened needle. A total volume of 50 μL ZnONPs suspension or sterile physiological saline was instilled followed by 150 μL air by a syringe. Subsequently, the mice were held with head up and gently transferred on a 37 °C heating plate until they recovered from anesthesia. After 1, 3 and 7 days of ZnONPs administration, three mice from each group were sacrificed and the cerebral cortex tissues were quickly collected for morphological observation. At day 3 after ZnONPs exposure, eight animals from each group were used for the further designed experiments. For the rescue experiments in vivo, 100 μL Fer-1 (10 mg/kg) or vehicle was injected intraperitoneally 1 h before ZnONPs (12 μg/mouse) instillation, six mice per treatment were used. All samples were collected 3 days after ZnONPs instillation for the further indicated analysis in the rescue experiments.
The pathological changes of cerebral cortex tissues tissue were evaluated by hematoxylin–eosin (H&E) staining according to protocols described previously.4 Briefly, the tissue-containing slides were deparaffinized in xylene and dehydrated with ethanol. After staining with hematoxylin at room temperature, the sections were rinsed with distilled water and then stained with eosin. Subsequently, the sections were dehydrated by ethanol, cleared in xylene, and finally mounted with neutral balsam.
Immunofluorescence assay was conducted according to the protocols described previously.4 Briefly, the tissue-containing slides were rinsed and incubated in the blocking solution containing serum. The sections were incubated with the primary anti-NeuN (1:500), anti-MAP2 (1:500) and anti-Ki67 (1:500) antibodies at 4 °C overnight. Next day, the Cy3-conjugated secondary antibody (1:500) was incubated at room temperature followed by staining with DAPI. The sections were then observed under a fluorescent microscope (Olympus, IX53, Tokyo, Japan). The average fluorescence intensities were obtained from Image-Pro Plus software (version 6.0, Media Cybernetics, USA).
Transmission Electron Microscopy
The ultrastructure of mitochondria was observed by transmission electron microscopy as described previously.4 Briefly, the cerebral cortex tissues were rapidly collected by heart perfusion-fixed technique with 4% paraformaldehyde and 2.5% (w/v) glutaraldehyde buffer. Afterwards, the tissues were dissected and cut into 1 mm3 in size. The tissues were then fixed in glutaraldehyde and 1.0% osmic acid, and dehydrated by ethanol. After embedding, the tissue-containing slides were coated with celloidin membrane and stained with uranyl acetate and lead citrate. The slides were observed under transmission electron microscope (Hitachi-7500, Hitachi, Ltd., Tokyo, Japan).
Measurement of ATP, MDA and Iron Contents
The contents of ATP, MDA and iron were determined by commercial available kits according to the procedures of manufacturers.4 Briefly, the MDA contents were measured at a wavelength of 532 nm, the ATP contents were detected at a wavelength of 636 nm, and the levels of iron were tested at a wavelength of 520 nm by an ultraviolet spectrophotometer (UV-1750, Shimazu, Co., Ltd., Japan) and micro-plate reader (ELX808, Bio-Tek Instruments, Winooski, VT, USA), respectively.
Inductively Coupled Plasma–Mass Spectrometry (ICP-MS)
The determination of zinc ion concentration in the cerebral cortex tissue was performed as described previously.8 In brief, the tissues were rapidly dissected and homogenized after the indicated treatment. The homogenate and digestion solution were then added to a Teflon digestion tank (Sineo Microwave Chemistry Technology Co., Ltd., Shanghai, China), followed by heating on an electronic temperature control heating plate (EHC-1, Sineo Microwave Chemistry Technology Co., Ltd., Shanghai, China) until the solution was colorless and dry. After mixing, the concentration of zinc ion was detected by a PerkinElmer NexION™ 300X ICP-MS (Norwalk, CT, USA).
Western Blot Analysis
Western blot analysis was carried out according to procedures as previously described.30 Briefly, the total proteins from tissues or cells were extracted by radioimmunoprecipitation assay buffer containing PMSF and protease inhibitors. The protein concentration was determined by bicinchoninic acid method. Subsequently, the sample's protein was denatured and subjected to sodium dodecyl sulfate polyacrylamide gels. After transferring the protein to polyvinylidencedifluoride (PVDF) membrane, the membrane was blocked in 5% non-fat milk and incubated with primary antibodies (SLC7A11, 1:500; VDAC3, 1:500; GPX4, 1:500, p-JNK, 1:1000; p-p38, 1:1000; p-ERK1/2, 1:1000) overnight at 4 °C. The membrane was incubated with secondary antibodies at room temperature for 1 h. The bands were visualized by enhanced chemiluminescence reagents with a Molecular Imager Gel Doc XR System (Bio-Rad, Hercules, CA, USA) and analyzed by Image J software (NIH, Bethesda, MD, USA).
Quantitative PCR Assay
Quantitative PCR assay was performed according to the protocols described by Tang et al.4 Briefly, total RNA was extracted by TRizol reagent (KeyGen Biotech, Nanjing, China) according to the manufacturer’s instruction. cDNA was then synthesized with PrimeScript™ RT Master Mix (Perfect Real Time) obtained from TAKARA Co., Ltd. (Tokyo, Japan). Quantitative RT-PCR was conducted by SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) on CFX Connect™ Real-Time System (Bio-Rad, Hercules, CA, USA). The specific primers were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China) and are shown in Supplementary Table 1. PCR reactions were carried out under the conditions: 95 °C for 3 min, followed by amplification in 40 cycles of 95 °C for 1030 s at 55 °C for annealing, using the CFX Connect™ Real-Time System (Bio-Rad, Hercules, CA, USA).
The PC-12 Adh (ATCC CRL-1721.1TM) cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in high-glucose DMEM supplemented with 10% (v/v) FBS, 100 units/mL penicillin and 100 µg/mL streptomycin. Cells were maintained in 25-cm2 culture flask in a humidified incubator at 37 °C supplied with 5% CO2.
Measurement of Cell Viability
Cell viability was determined by using the CCK-8 assay according to protocols described previously.30 Briefly, cells were seeded into 96-well plates and then treated with designed concentration of DTPA, DFO, Fer-1, SP600125 or/and ZnONPs for 24 h. Subsequently, the cells were incubated with a CCK-8 solution at 37 °C in 5% CO2 for 4 h in the dark. The absorbance was measured at a wavelength of 490 nm by a micro-plate reader (ELX808, Bio-Tek Instruments, VT, USA). Relative cell viability was calculated and adjusted as the percentage of untreated cells.
Measurement of Intracellular Zinc Concentration
Zinc concentration in the cell was detected by FluoZin-3, AM probe using fluorescence-activated cell sorting analysis (FACS). FACS was carried out according to the protocols described previously.31 Briefly, cells were seeded into six-well plates and treated with designed concentration of DTPA and/or ZnONPs. The probe was added into the cells at the final concentration of 500 nM and incubated in the dark for 30 min. Subsequently, cells were washed by the complete medium followed by an additional incubation for 30 min in the dark. FACS analysis were performed on Becton Dickinson (BD) Influx Cell Sorter (BD Biosciences, San Jose, CA, USA) and the results were analyzed by BD FACS Software (San Jose, CA, USA).
All experimental results were reported as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by Tukey’s honestly significant different post-hoc test was applied. If the original data were in the face of heterogeneity of variance, a non-parametric Kruskal–Wallis test was used. Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA), and statistical significance was set at P< 0.05.
Pulmonary Exposure to ZnONPs Caused the Reduced Number of Neurons in Mouse Cerebral Cortex
After intratracheally administration with low- (3 μg/animal), medium- (6 μg/animal) or high- (12 μg/animal) dose ZnONPs, the cerebral cortex tissues of mice were collected on days 1, 3, and 7. H&E staining was used to observe morphological changes of cerebral cortex. The results illustrated that treatment of ZnONPs reduced the number of neuronal cells, and caused the aberrant shape, shrunken or fractured nucleus of cells in the cerebral cortex. Moreover, these pathological changes last for 7 days. These results indicated that intratracheal administration of ZnONPs led to the loss of neuronal cells in the cerebral cortex (Figure 1). In our previous work, we demonstrated that ZnONPs-induced lung damage might diminish gradually to the normal level with time on post-exposure days 7.8 Based on these findings, the time point at day 3 after ZnONPs exposure was chosen for the following designed experiments.
To verify the loss of neuronal cells induced by ZnONPs in the H&E staining, the specific neuronal protein marker, NeuN, was determined by immunofluorescence assay. The data showed that the fluorescent intensities were remarkably reduced after ZnONPs treatment as compared with the controls, suggesting that ZnONPs gradually decreased number of NeuN-positive neurons with increasing concentrations (Figure 2A). Because the reduction of NeuN labeling in the brain may be affected by depletion of the protein or loss of its antigenicity,32 expression of another neuronal marker, MAP2, was assessed. Results demonstrated that the average fluorescence intensities of MAP2-labeled cells also sharply declined in a dose-dependent manner in ZnONPs-treated mice (Figure 2B). Ki67 is a marker of cell proliferation, the initial step in the process leading to neurogenesis in the adult brain.33 To investigate the effect of ZnONPs on the adult neurogenesis, we further tested the expression of Ki67 in the cerebral cortex. Our results showed that ZnONPs caused a decrease in the number of Ki67-positive neurons (Figure 2C), indicating that the neurogenesis might be affected by ZnONPs treatment. Taken together, these findings suggest that pulmonary inhaled ZnONPs can translocate from lung to brain via systemic translocation and then cause significant reduction on the number of neurons in the cerebral cortex of mice.
Pulmonary Exposure to ZnONPs Resulted in Ferroptosis-Related Mitochondrial Oxidative Damage in Mouse Cerebral Cortex
The process of ferroptosis is typically characterized by mitochondrial dysfunction and lipid peroxidation derived from iron excess.3 Therefore, to test whether ZnONPs exposure triggers ferroptotic cell death in mouse cerebral cortex, ultra-morphological changes of mitochondria were observed by TEM. Our results showed that mitochondrial cristae were significantly reduced or vanished with severe vacuolar lesions, and the density of the mitochondrial membrane was partially condensed (Figure 3A). As shown in Figures 3B and C, the data illustrated the ATP contents were sharply reduced while the levels of iron within the tissues were significantly enhanced in response to ZnONPs exposure. Given MDA is a typical and sensitive indicator for lipid peroxidation, its contents were further detected after different doses of ZnONPs treatment. We ascertained that ZnONPs were capable of increasing the accumulation of lipid peroxidation products (Figure 3D). The protein expressions of GPX4 and SCL7A11, both of which were key regulators and used for reflecting ferroptosis, showed significant decrease of their expressions as compared with vehicle controls (Figures 3E and F). On the contrary, the opposite trends were observed in the protein expressions of VDAC3, which was found to be involved in the induction of ferroptosis (Figures 3E and F). These findings together indicate that pulmonary ZnONP exposure can result in iron accumulation and cause ferroptosis-related mitochondrial oxidative damage in the mouse cerebral cortex.
Pulmonary Exposure to ZnONPs Increased the Level of Zinc in the Mouse Cerebral Cortex
Because of their small size, inhaled ZnONPs have been found to penetrate the blood–air barrier and the blood–brain barrier, thus translocating to the brain by systemic circulation via blood.10,19,21 Indeed, in this study, by using ICP-MS analysis, our results observed that the level of zinc in the cerebral cortex was significantly increased in a dose-dependent manner, although a low concentration of ZnONPs did not cause a statistical change of the level of zinc in mouse cerebral cortex (Figure 4A). Metallothioneins (MTs) are small, cysteine-rich proteins that can bind zinc and act as a zinc reservoir. The metallothionein 2 (Mt2) isoform is predominantly found in the brain. At physiological conditions, zinc is terminally bound to the cysteine thiols of Mt2, making bead-like structures, while the expression of Mt2 can be elevated in response to an increased level of zinc.34,35 To further verify the results obtained from ICP-MS analysis, we detected the mRNA expression of Mt2 after ZnONPs treatment. Our results showed that the mRNA expression of Mt2 was significantly enhanced in ZnONPs-treated mice in a related dose-dependent manner (Figure 4B). These findings indicate that pulmonary ZnONPs exposure increased the level of zinc in the cerebral cortex.
ZnONPs Reduced Cell Viability via Increasing Concentrations of Zinc in PC-12 Cells
Because the enhancement of zinc was observed in the cerebral cortex, we further determined that the role of zinc in ZnONPs induced a reduced number of neuronal cells by using an in vitro culture system. First, we performed a CCK-8 assay to obtain the dose-related cytotoxic effects of ZnONPs on PC-12 cells. Our data showed 30 μg/mL ZnONPs treatment for 24 h significantly reduced cell viability (Figure 5A). Based on these results, this dose was chosen for the further designed experiments. Second, DTPA (200 μM), a potent metal chelator to exchange zinc for a metal of greater binding capacity, was applied to test the role of zinc in the regulation of neuron-like cell viability. Our results demonstrated that DTPA co-treatment with ZnONPs did not cause any cytotoxic effects in PC-12 cells, although DTPA itself could trigger inhibition of cell viability (Figure 5B). On the one hand, these results suggest a reduction of zinc concentration may lead to a significant decrease on cell viability; on the other hand, the obtained data also indicate that chelating with zinc by DTPA can attenuate the cytotoxicity of ZnONPs. As illustrated in Figure 5C, single ZnONPs exposure remarkably elevated the transcriptional Mt2 mRNA level compare d with untreated cells, whereas this enhancement of Mt2 mRNA expression could be relieved by co-treatment with DTPA. A similar phenomenon was observed on the intracellular zinc concentration in PC-12 cells (Figure 5D). These findings indicate that ZnONPs reduce cell viability by increasing zinc concentration within PC-12 cells.
ZnONPs Triggered Ferroptotic Cell Death in Cultured PC-12 Cells
Pulmonary exposure to ZnONPs was capable of inducing ferroptosis-related mitochondrial oxidative damage in mouse cerebral cortex. To further verify the involvement of ferroptosis in ZnONPs-triggered cell death, a selective inhibitor of ferroptosis, Fer-1 (2 μM), was used in the cultured PC-12 cells. Our data found the reduction of cell viability induced by ZnONPs was significantly relieved when co-treated with Fer-1 (Figure 6A). As the ferroptotic cell death typically occurred via the production of excess intracellular iron, the iron chelator deferoxamine (DFO, 100 μM) was used to test if ZnONPs-triggered cell death was initiated in an iron-dependent manner. Indeed, our results illustrated that, after co-treatment with DFO for 24 h, the cell viability could return to near normal levels, suggesting ZnONPs-induced cell death could be rescued by chelating iron (Figure 6B). These findings were confirmed by detection of ferroptosis-related protein expressions. As depicted in Figures 6C and D, the protein expression levels of GPX4 and SCL7A11 were markedly decreased by ZnONPs in a dose-dependent manner. Importantly, the reduced protein expressions of these two proteins could be ameliorated by co-treatment of DFO or Fer-1 (Figures 6E and F). A similar trend was also observed in the determination of MDA, a classic lipid peroxidation product, showing that Fer-1 co-treatment significantly reduced the enhanced content of MDA induced by ZnONPs (Figures 6G and H). These findings together suggest ferroptosis is involved in ZnONP-triggered cell death in PC-12 cells.
Fer-1 Inhibited ZnONPs-Induced Ferroptotic Cell Death in Mouse Cerebral Cortex
To further verify the results obtained in PC-12 cells, we pre-treated mice with Fer-1 (10 mg/kg) by intraperitoneal injection 1 h before ZnONPs administration. The method of pre-treatment of Fer-1 had been reported to exhibit better antagonistic effects against ferroptosis than either post-treatment or co-treatment.36 In this study, similar protective effects of Fer-1 against ZnONPs-induced ferroptotic cell death were also observed. Our results showed that pre-treatment of Fer-1 significantly elevated the protein expressions of GPX4 and SLC7A11 in ZnONPs-treated mice (Figure s 7A and B). Similarly, the enhanced MDA contents in ZnONPs-treated mice were significantly reduced by Fer-1 (Figure 7C). However, we did not find any changes in the mRNA expressions of Mt1, Mt2, Mt3 and Mt4 in the ZnONP-exposed mice pre-treated with Fer-1 when compared with single ZnONPs treated mice (Figure 7D). These results indicate that Fer-1 could inhibit ferroptosis in the cerebral cortex but might not influence the release of zinc ions.
Activation of c-Jun NH2-Terminal Kinases (JNK) Pathway Involved in ZnONPs-Induced Ferroptotic Cell Death
Activation of the mitogen-activated protein kinase (MAPK) pathway has been revealed to contribute to ferroptotic cell death.3 The MAPK pathway is mediated by extracellular signal-regulated kinase (ERK), JNK, and p38 protein kinases.37 After treatment with ZnONPs, we found the protein expressions of p-p38 did not show any significant changes in cerebral cortex tissues as compared with vehicle controls (Figure 8A). The protein expressions of p-ERK1/2 and p-JNK were both enhanced in ZnONPs-treated mice, but the expression of p-JNK showed greater enhancements than that of p-ERK1/2 (Figure 8A). Consistently, ZnONPs remarkably increased the level of p-JNK in a dose-dependent manner in cultured PC-12 cells (Figure 8B). To further verify the role of activation of JNK involved in ZnONPs-triggered ferroptosis, the specific inhibitor of p-JNK, SP600125 (10 μM), was used. Our results illustrated that inhibition of the JNK pathway not only significantly reduced the increased contents of MDA (Figure 8C), but also markedly reversed cell death in ZnONPs-treated PC-12 cells (Figure 8D). These findings suggest that activation of the JNK pathway may be involved in ZnONPs-induced ferroptotic cell death.
Inhalation is the main and typical route of entry to human for ZnONPs exposure in workplaces. This occurs mostly by releasing zinc oxide fumes during industrial activities, such as thermal cutting, welding of steel and melting, etc.38 Such exposure of ZnONPs via airway may result in metal fume fever in workers. The systemic inflammatory effects of ZnONPs inhalation further cause permanent pathological changes in different organs, including the brain.38–40 In the present study, to ensure consistent amounts of inhaled nanoparticles per animal, a single intratracheal instillation of ZnONPs was used. Because the bioavailability of metal oxide nanoparticles is often related to the ability of nanoparticles to deliver soluble metal ions to adjacent tissues,41 the increased levels of zinc ion and Mt2 expression observed in the mouse cerebral cortex tissues may suggest administration of ZnONPs by this way spray directly into the airway tract of animals and facilitate their absorption and distribution. Intriguingly, we also found that the pathological changes in cortex tissues at day 7 were not more serious than that at day 3 after ZnONPs treatment. Similar trends were also found in lung tissues,8 and this manifestation may be due to metabolism or excretion of nanoparticles via urine or feces with increasing time.
Growing evidence has demonstrated that ZnONPs are capable of reaching the brain because of their extremely small size.10,21,42 There are two explanations for the accumulation of inhaled ZnONPs in the brain. First, ZnONPs that are small enough can reach the lower airways and gain access to the air–blood barrier, which is the most permeable barrier in the body. ZnONPs may translocate across the air–blood barrier permitting systemic access to extra-pulmonary organs.43 They are also able to penetrate the blood–brain barrier directly by transcytosis and consequently produce neuronal injury.44 Second, inhaled ZnONPs reach the brain indirectly via an olfactory bulb–brain translocation pathway.42,45 In this study, we found administration of ZnONPs by intratracheal instillation caused the pathological changes in the cerebral cortex tissues, mainly manifested by a reduced number of neuronal cells. These results partially indicate that pulmonary exposed ZnONPs can aggregate in the brain via the above two ways to reach the central nervous system where neurotoxic effects could develop. In addition, it is now well-documented that small-sized nanoparticles (less than 50 nm) produce greater neuronal injury than larger ones.42 A previous report illustrated that nanoparticles with approximate size of 50–60 nm were able to disrupt the blood–brain barrier of an in vivo system, therefore leading to the brain pathology.46 Similarly, we observed pathological changes in the brain when mice were treated with ZnONPs under 50 nm particle size. Some studies have reported the opposite results. For instance, Deng et al. revealed no size-dependent cytotoxic effects of ZnONPs on neural stem cells after a 24 h short-term exposure.23 This discrepancy implies in vitro studies may lead to results that do not correspond to circumstances occurring in a living organism. Considering this potential discrepancy between in vivo and in vitro, in this study, we used both animals and cell models to test whether ZnONPs triggered ferroptosis in neuronal cells.
Fer-1 is a potent and selective inhibitor of ferroptosis that effectively slows the accumulation of lipid hydroperoxides and thereby inhibits cell death.47 Indeed, in this study, Fer-1 obviously inhibited the phenotype of ferroptosis induced by ZnONPs in both cultured PC-12 cells and animals. It is noteworthy that we did not find any significant alterations on the mRNA expressions of Mt1, Mt2, Mt3, Mt4, although zinc ion chelation by DTPA evidently ameliorated the cytotoxicity of ZnONPs. On the one hand, these results signify the protective effects of Fer-1 against ferroptosis are not associated with the level of zinc ion within the cells. On the other hand, it is believed that the cytotoxicity of ZnONPs may occur as a result of release of zinc ions in the biological micro-environment. Similar to other metal nanoparticles, zinc ions play a critical role in ZnONPs cytotoxicity, despite the effects of the intact nanoparticle form which cannot be excluded.48–51 Intriguingly, some epidemiological investigations have revealed that iron and zinc may interact in the body and thereby decrease the efficacy of iron supplementation.52,53 Moreover, zinc also has an inhibitory effect on iron absorption.54 These results together suggest that the elevated concentration of zinc ions in the brain may be a risk factor for the initiation of iron-dependent cell death.
Both iron metabolism and lipid peroxidation are recognized as central mediators in the initiation and execution of ferroptosis.3 However, cellular sensitivity to ferroptosis is also modulated by several other pathways, such as production of coenzyme Q10, upregulation of endoplasmic reticulum stress and activation of MAPKs pathways, etc.55,56 Among these pathways, MAPK cascades are the vital signaling pathway that regulate diverse cellular programs, including cell proliferation, differentiation, development and cell death.57 In mammals, there are more than a dozen MAPK enzymes, and the best-known conventional enzymes are ERK, p38 and JNK.57 The role of MAPKs in the regulation of ferroptosis has been revealed for several years. Blocking the ERK pathway inhibited ferroptosis in Ras-mutated cancer cells, but JNK and p38 appeared to be important in leukemia cells.55,58 Inhibition of JNK phosphorylation significantly decreased the cytotoxicity induced by ferroptosis inducers in leukemia cells.58 These results indicate that MAPKs may serve in a cell type-specific way in the process of ferroptosis. In this study, we observed a similar phenomenon in the cultured PC-12 cells, which originated from a pheochromocytoma of the rat adrenal medulla with neuronal phenotype. Our results showed ZnONPs markedly enhanced the level of p-JNK expression, but they did not activate the p-ERK and p-p38 pathway significantly. Inhibition of p-JNK by its specific agent not only relieved the overproduced lipid peroxidation product, but also reduced the cytotoxicity of ZnONPs. These findings confirm the notion that the MAPK pathway is tightly linked to the initiation of ferroptosis, but the activation of specific MAPK pathways may vary depending on the cell types, environmental stimuli or drugs.
There are several limitations regarding this study that should be considered. First, the toxicity of ZnONPs may depend on the duration of exposure time, the size of nanoparticle and the dose used, etc.10 In this study, we did not treat mice with ZnONPs for a long time based on the following two reasons. One was that the beneficial effects of a ferroptosis-specific inhibitor might not last for a long time because of drug metabolism. The other was because the toxicity of ZnONPs had been found to recover as time went by.8 Second, Fer-1 usually acts as a lipid ROS scavenger, and the inhibited effects of Fer-1 against ferroptosis may be affected by the other potential mechanisms, such as anti-oxidative effects. Third, although ferroptosis is distinct from canonical apoptosis, autophagy and necroptosis, we did not exclude whether airway exposure to ZnONPs initiated the other forms of cell death. Also, we even did not know which form of cell death dominates the cytotoxicity of ZnONPs for the neurons. Do these forms of cell death all or partially exist in response to ZnONPs exposure? Is ferroptosis independent of other known cell death pathways? These questions and related mechanisms remain unsolved and need further investigation.
In summary, in this study, we demonstrated for the first time that pulmonary exposure to ZnONPs triggered ferroptosis in the neuronal cells of mouse cerebral cortex. Similar phenomena were also observed in cultured PC-12 cells. Importantly, our results showed that inhibition of ferroptosis could alleviate ZnONPs-induced neuronal cell death both in vivo and in vitro. The molecular findings from this study indicated that ZnONPs selectively activated the JNK pathway and therefore resulted in ferroptotic phenotypes (Figure 9). These results will provide the novel evidence that inhibition of ferroptosis by specific agents or drugs may be the potential strategy for reducing untreatable neurotoxicity induced by ZnONPs airway exposure.
Figure 9 The experimental design and main results of this study are summarized.
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.
The authors declare no competing interests.
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