Ladies and gentlemen, on behalf of the Institute of International and European Affairs, I'd like to welcome you all to today's event. My name is David Donohue. Our guest today is Svetlana Tikhanovskaya, who will speak to us on the topic of Belarus on the brink of now. Svetlana will look at the current situation in Belarus and at possible ways forward for the opposition there, of which she is a very prominent member in the aftermath of the election last year and also of the further crisis caused by the Lukashenko regime's diversion of a Ryanair jet, as we know in May, and the arrest of an activist who was on board. Svetlana is perhaps Belarus's best known human rights activist and opposition leader. As we all know, she stood as she contested the presidential election last August as the main opposition candidate following the detention of her husband, Sergei Tikhanovskaya. Since the election and its heavily disputed results, she's been based in Lithuania as the official representative of Belarus's democratic opposition in exile. She heads the Coordination Council to facilitate a peaceful democratic transfer of power in Belarus. Before running for the presidency, she was an English teacher and interpreter. And as those of us living in Ireland know, she spent a number of summers here as a teenager near Rosquay. And in fact, Svetlana, it has been very touching, I think, for all of us to see the coverage of your reunion with your host family this week. You've had a very busy trip to Ireland this week, and we're all a little more grateful to you for taking time out from that schedule to speak to us at the Institute. A few housekeeping points, Svetlana will be keeping her opening remarks fairly brief to about five minutes, and both her remarks and the Q&A session will be on the record. Please feel free to send in questions or comments as they occur to you during the session, and you would use the Q&A function for that, which you'll see at the bottom of your screens. And we'll try to get to as many as possible. And Svetlana will be joined by her colleague, Vranek Via Chorka, who is a senior advisor to her for the Q&A session. Finally for those using Twitter, the handle is at IIEA. So with that Svetlana, once again, you're very, very welcome, and please take the floor. Thank you.  As you know, Ireland is an exceptional country for me. I was 14 when I came here for the first time as a so-called Chernobyl kid. I came here three times and had a terrific summer with my host family. I saw them yesterday in Roskri and I can't express how grateful I am for their love and kindness. You know, apparently Belarus political crisis began a long time ago. In 1996, a year when I first came to Ireland, Aleksandr Lukashenko conducted a political coup d'etat. He endowed himself with unlimited powers. Before that he dismissed parliament and destroyed opponents. And many alternative parties, media outlets, civil society groups, got destroyed. And thousands of people had to flee the country and look for a better future abroad. Most prominent political leaders disappeared and got killed by the regime's cronies a few years later. All elections since then were falsified. And it was hard to imagine that something might change in Belarus. And as an absolute majority of Belarusians, I was never involved in politics before 2020. Of course, I was following the news and I saw this horrible injustice ordinary Belarusians face daily. But I didn't see a way to change it. But it changed last year when Belarusians woke up. And after the election was announced, my husband decided to run. Never soon he got jailed, so I decided to come and register instead of him. This is how I became a presidential candidate, advocating for democratic reforms, but first of all for the release of political prisoners. And it was already evident that the regime will not give up its power. And it was also apparent that people will not give up either. And on August 9th, millions of Belarusians expressed their wish for a peaceful and democratic transition of power. And predictably, elections were stolen. And moreover, those who joined the peaceful protest faced rubber bullets, gas and truncheons. The protests continued despite police violence and torture. And since last August, more than 35,000 Belarusians were arrested. Thousands were tortured, thousands were prosecuted and 555 political prisoners and at least 10 died in relation to protest. Not a single case was initiated against the government officials for all these abuse. My husband, Sergei Tikhanovsky, is one of the hundreds of political prisoners. He didn't see his children for more than one year already. My only contact to him is through lawyer. And of course, he can't tell everything he feels there behind the bars. But I know for sure that he's strong and he will get through this. Unfortunately, very little information gets to us from those behind the bars. Prisoners are deprived of medical assistance. There are no windows, so they can't see the sun. Prisoners are not delivered or delivered with a mob delay. Women are stripped searched.  Journalist Igor Loisik protested against prison conditions. He almost died in 40 days hunger strike, twice committed suicide attempts. And he says that he can't handle it. In one of his latest letters to his wife, he said that he feels like a prisoner in Auschwitz. In the last two weeks, regimes thugs raided more than 30 media outlets and human rights organizations.  The regime is literally destroying civil society. The human rights catastrophe in Belarus may seem to prove the strength of the repressive machine and the weakness of the unarmed resistance. But in reality, it demonstrates the weakness of the dictatorship that has no other means left to keep in power but violence. And the regime tries to stop time and to return to the worst practices of the Soviet era. But Belarusians have already chosen the future as democratic and will not change their minds. You know, the crisis has outgrown Belarus borders already. The regime hijacked the Iran airplane, organized the flow of migrants to the neighboring countries and openly threatened to flood Europe with trucks and nuclear waste. For some, it might seem as many separate problems. However, the source of problems is the same, uncountable, unpredictable and non-transparent regime which stole elections to raise Belarusians and impose security threats to entire Europe. So I urge the international community to focus on the problem, not only the consequences. Until Belarusians have a possibility to freely choose their leadership, the type of accidents, attacks, provocations and repressions against Belarusian citizens are going to continue. New free and fair elections this year will help to solve the crisis. Violence must end and all political prisoners must be unconditionally released and rehabilitated. And we ask the world to support these demands. And you may ask, what will make the regime to release people and conduct free elections? My answer is pressure, from inside and outside. People in the regime, and this is not about only ex-president, must feel that there is no other solution to the crisis to step down and conduct new elections. And the country and multiple initiatives bravely continue. They work despite terror and repressions and then it helps. So I call on the international community to provide assistance and emergency funding for media, human rights defenders, legal defense efforts, local civic initiatives and activists, striking committees and emerging professional associations such as athletes, cultural workers, doctors and teachers and of course students that are expelled for their political views. I would love to draw special attention to supporting media initiatives. Vibrant and sustainable media are the basements for a resilient and robust society. I also ask to support families of the repressed. As one of the Chernobyl kids, I can say that we face now the new Chernobyl, Chernobyl of human rights. And so many Belarusians, especially children are traumatized and in need of rehabilitation. And it would be great to develop a special program to let children or political prisoners spend time in Ireland. I also ask to support Belarusian relicans and diaspora. Thousands of people were forced to leave the country fleeing prosecution last year and this year. But just as the Irish abroad became a great force that helped to restore independence, the Belarusian diaspora is rapidly developing networks of solidarity all over the globe. So I welcome the support of these initiatives, especially in the sphere of culture that we celebrated last week. Pressure from outside must include sanctions, international legal mechanisms and legitimization of the regime. And I really welcome the principal position of Ireland. And I thank your country for condemning the regime and supporting the European sanctions against it. The crackdown on media and human rights defenders must get a strong response from the international community. So now I ask to support the fifth European Union sanctions package that should include a broad list of individuals and companies and close the loopholes in the sanctions imposed recently against potash and oil sector. I also call Ireland along with other like-minded countries to promote the topic of Belarus at the UN Security Council. Organizing an area formal hearing on the human rights abuses and torture in Belarus can help to draw attention of the world to Belarus. And it's essential to use all available international mechanisms to bring perpetrators to justice. The case of Freynair plane hijacking should be filed to the International Court of Justice. And Ireland as an affected country can help with that. We also ask the UN Human Rights Council and the Office of the High Commissioner to put the Belarus crisis on the agenda. Even though Belarus is not a member of the International Criminal Court, preliminary examination of the regime's crimes can be still launched. You know, I look in the future with optimism. One year ago, Belarusians chose to live in free and democratic Belarus. Right now we have to protect this choice. No one will bring freedom to us. And it's our task, of course. But with international support and your solidarity, the path to freedom will be faster and less painful. And I dream that everyone who is behind bars or in forced exile will be able to return home. So please help us pave the way for new democratic Belarus. Thank you. And Guru Mahagat. Thank you very much, Svetlana. And especially for the last words in Irish, you obviously put your time and lost weight to good use. I thought maybe you might have picked up a temporary accent, but I'm sure you have very, very good memories of your time there. We have a number of questions coming in, Svetlana, and some comments, all extremely supportive of what you're doing. Let me just first mention a comment from David Joyce, who is with the Irish trade union movement. He just wanted to underline the solidarity of Irish trade unions to you and all who are struggling for democracy and human rights in Belarus. He witnessed the level of oppression at a meeting of the ILO in June where the Belarus authorities openly threatened brothers and sisters in the independent trade union BKDP. So basically David Joyce wants to underline the readiness of the Irish trade union movement to do everything possible in solidarity with you. I have a question from Tom Ferris, which let me just read it out. Yeah, is the European Union doing enough to support the Belarusian people's struggle towards a peaceful democracy? You touched on that a little bit, Svetlana, but it would be useful just to talk a bit more about the EU's response as such. What has happened up to now and what could potentially still be done? Could I hand that to you? Thank you. I really appreciate all the efforts of the European Union and other countries like the USA, UK and the Ukraine, Canada for their strong position. And after August event, all the democratic countries declared that our elections were fraudulent and that Lukashenka's regime is not legitimate. And it was a very supportive message to all Belarusians that the whole world with us and be continued our fight being assured that democratic countries will be standing with us. And I have to say that through packages of sanctions that were imposed right after their elections, they were more moral, I'd say. They were not strong enough and it was like a message to regime that we see you and we are going to stand with Belarusians. And after this, especially after this picture of beautiful demonstrations, it disappeared because Lukashenka with the help of violence and guns and tortures was succeeded to suppress those huge demonstrations. Attention to Belarus was decreasing step by step. And after December, we didn't have big conferences or huge attention to Belarusian question and Lukashenka felt impunity. And it ends with hijacking of the plane. And after this, after this hijacking, the attention of democratic countries, of course, raised and new adequate sanctions, rather strong sanctions for package and sectoral sanctions were imposed. And, you know, of course, it threatened regime because they maybe didn't expect such a strong answer and especially it's about closing airspace above Belarus. And but even then, you know, regime couldn't think strategically. They started to take revenge on the countries that are supporting Belarusian society and started to send illegal immigrants to the borders of Lithuania. And, you know, I'm sure that European countries want to help. But so as it is our pain, and this is our beloved people and relatives are in jail suffering from humiliation and ill treatment. It's never enough for us. And I'm sure that European Union can do more. But if there are, you know, some doubts about sanctions, I'm sure we'll discuss it a little bit more later. But as for assistance to civil society. You know, the answer, the response can be really much stronger because maybe not all the countries understand the scale of the repressions and the scale of needs of Belarusian civil society at the moment. So many prisoners inside country, their relatives need help. But what is more prominent that Lukashenko's regime destroyed mass media inside their country. He's destroying human rights defenders in the country. And a lot of journalists had to flee the country. A lot of people had to flee the country. And all those relatives, all those businesses that were lost in Belarus and also had to be relocated, they need assistance. And, you know, European Union want to help. But sometimes it helps because of, you know, some level of bureaucratization very slow. But we really need this help to be emergency help. You know, all those money that were launched to help Belarusians, for example, in November, they came to organizations maybe in March or April. But people are suffering now. And sometimes it's necessary maybe to launch new program, emergency program that will be able to bring this assistance rather fast to to to relic and to organizations that had to flee the country to help them to restore. And for example, if we take journalists, you know, we have traditional mass media like to buy different local newspapers that are being closed now. But also we have emergent journalists like YouTube channels, telegram channels, because we have, you know, Belarusians have become so creative now. And while closing traditional mass media, we have to open new one to show the truth, to search information inside the country to show the world. And this emergency journalists, journalism need assistance like, you know, technical support to buy equipment to relocate people. And it's only one part journalist, but there are, as I already said, businesses, people who flee the country, you know, without nothing, just taking one clothes and leaving in rush. So if it's possible, you know, to reorganize this, reorganize this help, you know, to to react more effectively and more rapidly, it would be of much appreciate, you know, and, you know, Lithuania asked for five fifth package of sanctions, and I hope that European Union will impose those sanctions rather fast, not just delaying this, because, you know, more democratic countries are delaying their response. It's more easily for regime to get used to this and find ways to bypass those restrictions of sanctions. And of course, it's necessary to close loopholes that were in the sectoral sanctions and in the fourth sanction list. So we just, we have to look at the problem from two sides, pressure on the regime and assistance to civil society and European Union can be really helpful.  And we are trying to use technologies to provide this assistance to the inside country. And now we see that only cryptocurrency is available in Belarus and European Union is rather, you know, is hesitating about using this method of sending money. But, you know, we have very unconventional situations, so unconventional ways of assistance also have to be launched, you know, just, we are always, you know, trying to assist in this question to European Union representatives to help them to, you know, to transfer this money to people who are in need to give to provide all the information how it's better to do this. But of course, there are problems in getting this assistance, but, you know, we together with European Union countries do our best to improve this. Thank you very much, Fadana. You make very powerful points there about supporting media freedom, about supporting civil society and de-bureaucratising the packages of sanctions. I think those points are all very, very well taken. A question from Stephen Frayn, who is with the Institute. What is your vision for Belarus sort of eventually taking its place as a free and democratic country within Europe? Do you see it seeking membership of the EU and or the Council of Europe, looking further afield to a further head to when there is a peaceful transfer of power? What kind of relations do you see Belarus aspiring to at that point with other European countries? You know, I see new Belarus as safe and prosperous country, and now I don't have the right to say what the future we will have according to in relation to European Union. It's up to Belarusian people to decide. My task and task of Belarusian people now is to bring our country to new elections where they will be able to choose the president they really want and after this to solve all their other questions. We are not, now our strategy is not pro-European or pro-Russian or anti-Australian. You know, we have our aim and after gaining this aim, we will decide where to move to the West, East, North or South.  That's our task now and only then we, you know, we will decide our movements. Thanks for that. We have quite a number of questions coming in about the relationship with Russia. No surprise, one from Alan Dukes, who's a former Irish government minister and director general of the Institute. Alan notes that support from Putin is essential for Lukashenko. What do you think the EU can do to get Putin to change his position? I mean, the relationship with Russia is very, very complicated. What's your own assessment of that relationship? You know, putting pressure on Lukashenko's regime and isolating him politically and economically. You are European countries sending clear message to Kremlin that Lukashenko is toxic. You know, and it's, you should like increase the price for Kremlin to support and Lukashenko. You know, you, at this very moment, you know, we are not Russia, and we always send this message, we should be, our crisis is not like crisis of two countries, we are a separate country, we are not appendix of Russia. And, but of course we all understand that Kremlin influences Lukashenko, first of all. But at this very moment, it's important to clarify your position that it's the subject of Belarusian people to fight against the regime. No, it's not, you know, like Russian issue.  And on the other hand, you should send clear message that all the deals that are signed between two countries, they are not illegitimate, they are not legal, and will be, of course, they can be recalled or, you know, in the future. You know, because our country now can be the threat of losing independence and independence is the main value for Belarusian people. And we don't know what Lukashenko is doing now behind the curtains, maybe he's selling enterprises or maybe he's selling our independence. And we want European Union countries to be guarantors that all the deals, you know, will be revised in the future. You know, that's it. Fernando, one further question that I made just about Putin, do you think that Putin is already looking to the post Lukashenko situation? I mean, it's no secret that there has been no love loss between the two of them over the years. But do you think Russia, in view of the events of the last year, is beginning to shift its own thinking towards who would come after him? And is there much speculation about that among Belarus opposition groups? What kind of successor could emerge? You know, Kremlin supported Lukashenko after fraudulent elections because Kremlin wasn't prepared for such huge uprising also. They didn't prepare their own pro-Russian candidate, for example. And now it's not comfortable for Russia to have Lukashenko the power because it means not stable situation in Belarus. But they can't afford that democracy will come to Belarus through revolution, through uprising. And we don't see that they have really clear strategy about Belarus now. They are continuing to support Lukashenko on diplomatic level, they accept him, they make pictures with him, but obviously this support is not very evident now. They would like to solve this crisis as well, but they don't know how. You know, they understand that Lukashenko's toxic is not possible to build normal relationship with person who isn't accepted, not inside the country, not on the international arena. But they have to follow the so-called support of him not to allow democratic forces to win. But again, it's not about Russia. On all our meetings, I had to persuade, not persuade, but to underline that we are not part of the Russia. Look, we are separate country. Of course, we have bilateral interest, but we want to be, Russia is our neighbors, and we always will be in trade relationship, political relationship with them, but we want to continue this. But we want this relationship more transparent and clear in future, and we always send messages to Russia that we want to talk. We want to see Russia as the part of the mediation table, the dialogue table. You know, the same as Lithuania as our other neighbors. We welcome them if they want to be there, you know, they want to be resolution of the crisis, not to deep this crisis, not to freeze this crisis. And but we, you know, we have to deal with what we have. We continue our efforts to communicate to representatives of Kremlin the same as with the representatives of the regime. We are open for dialogue and we always like, you know, showing this. We are not closed. We want to be civilized, we want to find civilized way to resolve the crisis, to think about people, first of all, not about power. Thank you very much for that. And a lot of questions coming in about Roman Potasevich and his partner Sophia. Can you tell us anything about the latest situation in relation to Roman and what prospects there are of having him released from custody? You know, Roman is under home arrest now, but home arrest doesn't mean freedom and rehabilitation. He's still like on the hook, if I may say so, in English again. Yeah, he's, he's living, I suppose, in a separate house, but he's living for sure with KGB people inside and he's, you know, being monitored by them and, you know, he's hostage. And maybe they will use him somewhere once again, like as you saw his interviews praising, you know, Lukashenko, but he could be tortured, he could be threatened, I don't know, with what they have methods to influence and threaten people. So his parent doesn't have any contact with him. So we know that relatives maybe of Sophia or Romans relative can bring food to, you know, to him. And I hope that he's well, but he don't know for sure. Yeah. Just still on the subject of Lithuania, the migration crisis or to be more precise Lukashenko's exploitation of migrants in order to put more pressure on Lithuania. How do you see that impacting on the current situation? For sure Lukashenko and his regime wants to take revenge. You know, they want to show that look we are so strong, we can do everything we want, we can threaten European society as well. They are threatening not only Belarusian side country but also Europeans, European countries and you know, European Union is a real power and countries have to show their teeth if I may say so now and to be consistent in their policy. Lukashenko regime has only violence and such disgusting methods of so-called foreign policy, you know, and it's not, it's unacceptable and European Union has to show the strong answer for these actions and not to return to business as usual.  You know, this time should be different and Belarusians are sure that this time we can be success story and they're ready to fight as long as possible because people are being jailed and new people appear who replace volunteers, who replace activists and we have very small space of acting inside country but we use every like point of this. Our workers are being organized in striking communities, they are preparing a national white strike one day and our volunteers, we have a rather big net of volunteers inside country that widespread. We're trying to reach Svetlana again just a momentary interruption. Yeah, I'm here, sorry. They were great. Yeah, Svetlana, yeah. So I wanted to say that people are continuing to fight as much as they can. It's not, it's very difficult, extremely difficult under such pressure, but they understand their responsibility, responsibility for those who are already in jail. You know, those people sacrificed with their freedom and some with lives with the health for to give us opportunity to continue and we can't betray those people and the democratic society, international society also can't betray, betray, you know, prisoners and all the Belarusians who understand that we are responsible for the changes, but with the help of international community, this will be much easier and faster. So something like this. Yeah. Thanks Svetlana. You've spoken very clearly about what the European Union should do, or I'm sure will do. What about the Council of Europe and the OSCE as other organizations where the kind of the human rights were met, covering all parts of Europe? Are there particular things you'd like to see the Council of Europe and maybe the OSCE do in support of your activities? You know, we are grateful to all international structures that are trying to help us and Council of Europe can be really very vocal and supportive to our fight, but I'll give the word to Franek now just for him to explain the situation. Thank you. Okay. Hi Franek. Yes, hi. Hi, good afternoon. We are trying to mobilize and use leverage OSCE, United Nations, Council of Europe. Of course it's very limited, especially considering the fact that Belarus is not very active member of OSCE and Belarus is not member of Council of Europe because of death penalty. And this makes us in the situation when we want to get Belarus being discussed, but it is blocked in OSCE by Russia and by Lukashenko as represented on one hand. And in Council of Europe, all the decisions and resolutions, they're not implemented because Belarus is not a member of this. But what we see right now, Parliamentary Assembly of OSCE, issuing resolutions perhaps doesn't have a direct impact, but it has accumulative effect because it's like analyzing and giving assessments to the current state of affairs. We see Council of Europe presenting reports almost every month on torture, on human rights, on media. And this also helps to show both to Belarusians, to elites, and to the international community that things are not normal in Belarus. So it's also helping a lot. What can be done more? Some like international high level events. We were trying to hold such events under OSCE auspices, but unfortunately it did not happen because of COVID, but also because of some member states were not interested in doing this. But instead of this, we saw impressive Moscow Mechanism developed by OSCE member states, which didn't need consensus because Moscow Mechanism allowed to impose, you know, mechanism without consensus of all OSCE member states. And this Moscow Mechanism became the basis for many other policies and policies implemented by governments, parliaments, but also different stakeholders when they went public, they were referring Moscow Mechanism. On the United Nations, that's a separate story, United Nations have very different layers that can be involved. Today, Svetlana has met the Special Rapporteur on human rights defenders. And yesterday we saw a crackdown on human rights defenders. And this is exactly the case where UN Special Rapporteur can raise awareness to speak out and to help Belarusian human rights defenders to get the help and attention. UN have Human Rights Council as well. Human Rights Council adopted the resolution which gave special mandate to high commissioner on human rights. But what we have to do, we have to help this experts group created by Michel Bachelet, you know, to be more efficient, you know, to support it. It's not enough just to launch the framework. It's very important to make this framework working to make it more practical, so to give it political power, only then it will be working. And sometimes, you know, very negative thing and we're disappointed that many things are discussed regarding Belarus when there is media attention. And then when media attention is going down, you know, there is no more political will. This political will is disappearing. And it's very difficult to keep this attention span on a high level. Thank you very much, Vanik. Can I come to a question from Shona Murray of Euronews? Shona asks, what would it take for new and fair elections to materialize? I mean, this obviously is one of your major objectives, Svetlana. We've read about supposed efforts by our supposed intentions on Lukashenko's part to bring in new constitutional reforms and referendums. I mean, to be honest, we've been there before. I'm sure there's a lot of skepticism about that. But what do you think it will actually take to bring about the fair elections that you have demanded? You know, we in Belarus think that only consistency will bring us to new elections. We have to follow our strategy, dialogue and new elections. And we are preparing for this. You know, we are in process of revolution, of course, but we are thinking about transition period and about future as well. We had a wonderful conference on new elections and we estimated that new elections can be held in 40 days after some changes will happen. Just we will be prepared for transition period. We will be prepared for new elections and the period after elections. I have to repeat this. Pressure, assistance to civil society for them to survive and to be sustainable at the moment to continue their fight. And you know, regime is making mistakes, you know, and it's also, it can happen very quickly. It seems maybe that nothing is happening in Belarus, but this situation is changing constantly. You know, who knows what could influence the situation. Nobody expected to run a plane hijacking. Nobody expected this flow of migrants to Lithuania. Nobody knows what new Lukashenka will invent in the country that will raise people again.  You know, there can be a lot of scenarios of this, but we will, our task is to be prepared for the moment when, when quick changes will, you know, have to be done. Okay, sorry for my English. And like we are doing everything.  It's not, it's not done behind the curtains as the regime always did. And now they are pretending they are also working on a new constitutional reform, but it's off. It's all, can I say bluff? You know, they are not going to, yeah, they are not going to do anything is they are just like, want to keep more time to keep the regime in the power, and only our strong actions, joint actions inside the country and joint actions in all the democratic countries that you're saying the European Union countries, you know, you European Union and the you're saying looking at each other. You know, what will be the next step of European Union who will be braver so we will join their sanctions, you know, and you know that inside Belarus all the processes are going on, because all those businesses who now are supporting the regime, they understand that, you know, regime is toxic now. And all those sanctions can impose businesses of this businessman and they have to choose. So should they continue supporting this strange person who is acting, you know, such, you know, in a moral way, or should they pay attention to new Belarus and join a civil society, you know, at least not, they can't, they may not support Lukashenko at the moment but they can change the attitude to him. And they know that they, you know, these businesses have already experienced that in past, even if you are friend of Lukashenko he easily can put you in jail in couple of months. Everybody is under threat, supporting the regime not supporting one mistake and you are in jail, even from from those who are loyal to this regime. So we have to show, you know, now we are talking about sanctions, about all those atrocities and violence, it's so painful but we have to look into the future as well. We have to show new Belarus, and this comprehensive plan that was launched by European Union was accepted with gratitude inside Belarus, because for 27 years, you know, the regime told us nobody will join. Okay, we can hear you. Yes, yes, yes. Okay. Let me just post one, which has come from Philip Beck of the IAA. Is there a role for Irish professionals who would adopt similar Belarus professionals who are political prisoners through organizations like Liberico, Amnesty International and so on, such as teachers, teacher to teacher, engineer to engineer, doctor to doctor. So the background is that there have been recent reports about Irish politicians doing this, and this is very encouraging so the question really is, it wants to see whether that could be done on a broader scale. The political prisoners became the super action which united many parliaments around the world on both sides of the Atlantic and it's helped to raise awareness not only about the prominent figures not only about presidential candidates but also about regular man and women, doctors, teachers, people of different professions and ages, who were kidnapped and jailed by Lukashenko regime. And there are some other initiatives that also allow foreigners to help Belarus and civil society and political prisoners. For example, it's possible to buy food packages for political prisoners online. So for example, you want to help the family you can go online you can pay the for the package and volunteers on the ground will form this package and will give to the prisoner you want to, you want to support and help. Encourage different civic groups, especially universities, schools, high schools, professional associations to help their peers colleagues in Belarus. Sometimes with teachers doctors workers it's much easier because there are formed institutions, but in some like in you with some specific professions, it could be difficult to find peers. So, in case if you are represented like popular profession like doctor, we recommend you to find them, or to support or to get in touch with the fund medical solidarity fund, which was formed and under after events last August, and which allows which which help doctors who are repressed by Lukashenko regime. You can find this initiatives online, but also we will be happy on the website of St. Lanets, you can you can check it out or but we will be also happy to share, you know the contacts for different groups and whom to help with whom to be in touch. But again, I think youth, especially high schools and universities, universities could be could be mutually beneficial for Belarusians to feel the support, but for foreigners also to learn from Belarusians what they feel what they're getting through. And especially for high school students in Ireland, I think it will be important experience to get to hear directly from and people in Belarus what they're what they suffer what they think about what they feel. Thank you very much. So then we're coming towards the end of this of this session. And I just wanted really to come back to what you would like Ireland to do. You've you said it at the beginning, you've sketched out a number of things on which we can be helpful. And I'm sure that colleagues at the Irish mission to the UN will have noted what you said about an area formula meeting. I think that that makes a lot of sense. And we are on the Security Council at the moment, as you know, and I'm sure that thought will be given to ways in which we can support the moves towards peaceful political change in Belarus. Is there anything you'd like to say, finally, just directed specifically at Ireland? We are obviously part of the consideration of sanctions within the EU, and you've had meetings with the T-Shark and the the the Foreign Minister this week. But is there anything that you are there any messages for the Irish people that you would like to close off with? All the political messages I declared in my speech, you know, about putting children of political prisoners in your Chernobyl program, you know, for children could visit Ireland and have rest about about area formula about assistance to Belarusians. But you asked about Irish people, ordinary people, you know, really a lot can be done. And, for example, when yesterday I was at my host family, I asked Henry Dean, you know, he visit church every day. And I told him to print the addresses of prisons where political prisoners are situated and give it to every person who is in church and say them, right, come home and try to relate it to political prisoners. And it will take 10 minutes of your life, but it will make the whole day for those who are behind the bars. They are so happy to receive letters from people, especially abroad. You know, it can be done in universities, in high schools, you know, you can even organize such lectures where you talk about democracy, that you are got used to it. You stop to value this, but there are countries that are where people losing their lives and freedom just, you know, fighting for these values. You can support those people and leave 10 minutes of this lecture for writing letters. It's very, very important step. Usually people can retweet our messages, can be active on Instagram, for example, to promote the idea of Belarusian people and never be afraid to make a small step. Because when we are millions, there are already millions of steps. So I ask Irish people to be with us and it's better to do something than not to do anything at all. So I really grateful for those who are already doing something and I'm grateful for those who will find a strength and do something else or something, you know, open something new in themselves. And because, you know, I had to repeat that we believe that Belarus can be success story, and I ask you to be the part of this, you know, success to democracy. Svetlana, thank you so much for a very powerful and disturbing, I have to say, account of what's happening in Belarus at the moment and of the challenges which you and your colleagues as Democrats are facing. We on this call have benefited greatly from your insights, but also your inspiration and from the generosity with which you have answered the questions. Thanks also to Vranek. We've covered a lot of ground, we've taken a lot of your time, but I'm sure I speak on behalf of everybody and thank you warmly for what you've told us, and especially with those parting messages, which I'm sure strike home for all of us. Lovely to see you in Ireland again, and we look forward to seeing you hopefully in person on a future occasion. In the meantime, warmest thanks and solidarity with everything that you and your colleagues are doing. Thank you for being with us. Thank you gentlemen and ladies for your attention. Goodbye.


This problem is not on your note sheet, but it is important to know how to do for your assignment. So I'm going to show you, it's kind of like a coordinate proof, but what it asks you is to identify if A, B, C, D is a trapezoid. After you've done that, it then asks to identify whether or not it's an isosceles trapezoid. So what we know about a trapezoid is that one pair of opposite sides has to be parallel. Well, in order to prove segments are parallel, we need to show that they have the same slope. So the way that you're going to do this problem, or the way that you're going to start it, is you're going to find the slope for every single combination of A, B, C, C, D, and A, D. So you're actually going to do the slope formula four times. So what we're going to do is we're going to find the slope of A, B. And again, we're just using the slope formula. So 8 minus a negative is going to be plus 4, and 0 minus a negative is going to be plus 8. And I'm just using the points A and B. So that gives me 12 over 8, and that reduces, 4 goes into 12 three times, 4 goes into 8 twice. So the slope of A, B is 3 over 2. Next I'm going to do the slope of B, C. So if you want to pause for a minute and go ahead and calculate the slope of B, C, and then come back and check to see if you get the answer right. I'm going to pause the video and fill in the rest of the slope formula. Okay, so the slope of B, C you should have gotten 0. Now I'm going to do the same thing for C, D and for A, D. Again, I'm going to pause the video and do the work of finding the slope for C, D and A, D. You can go ahead and do the same on your paper. Okay, so now what we're looking at is these four slopes. I have a slope of 3 over 2, a slope of 0, a slope of 3 over 2, and a slope of negative 3. Remember, all we need to have a trapezoid is one pair of opposite sides being parallel. And sure enough, we see right here we have 3 over 2 and right here we have 3 over 2. So it is a trapezoid because A, B is parallel to C, D. Now by the way, I didn't draw this on a graph. That might help you. It's really nice to be able to visualize these things. I didn't have a graph to draw on for this video, but of course if you were doing your assignment or a quiz or a test, definitely I would suggest that you plot these points so that you're actually looking at the picture because that's going to hopefully help you. Okay, so this is a trapezoid because A, B is parallel to C, D. So we have one pair of opposite sides parallel. Now let's move to this question or this part of the problem where it says identify if it's isosceles or not. So remember that in order to have an isosceles trapezoid, the legs have to be congruent. Now remember that the legs are the non-parallel sides of the trapezoid. So that means I have to show that B, C is congruent to A, D because those are the legs. They're the non-parallel sides. Again, if you draw a picture that might help you see that. But again, if you can remember that the legs are the non-parallel sides, you can figure it out that way. So how do I show that two segments are congruent? Well, I have to show that they're the same length. So now I'm going to switch gears and I'm going to move my screen down here a little bit and what we're going to do is we're going to use the distance formula to show whether or not B, C is congruent to A, D. So I'm just going to go to the next screen to finish this proof to see if this is isosceles or not. So here we have our points B, C and A, D and what we want to figure out is B, C congruent to A, D because if it is, then we know we have an isosceles trapezoid. So we're just going to go ahead and do the distance formula. So using B and C, go ahead, plug the numbers in to the distance formula and see what you get. I'm going to pause the video and fill in the values. So hopefully you got an answer of 6 for B, C. 6 minus 0 squared is 36. 8 minus 8 squared is 0. So you get the square root of 36, which is 6. Now go ahead and do the same thing for A, D. So when you put in the values for A, D, you can see what happens here is we get negative 6 plus 8 squared and negative 10 plus 4 squared. When we add that together, 4 plus 36, we get 40, the square root of 40. Well clearly, 6 and square root of 40 are not equal. So that means that B, C does not equal A, D. So therefore, and these little dots when they're in the form of a triangle, I just messed it up, I mean therefore. So therefore, this figure A, B, C, D is a trapezoid because we saw that we had one pair of parallel sides, but it's not isosceles.


hello and welcome back to the craft box my name is brain I'm joined with Finn and today we are on for another rune to 100 yes hi hi bro silverfield because we need to unlock the bridge so we need to get some wood some wooden planks get wood minecraft 101 get wood first achieve episode one get wood episode yeah get that that wood minecraft let's play coming in the near future definitely not basically getting pulled in northernland is like the easiest thing to do like you can get pulled so quick I'm worried about that I'm not gonna lie yeah use them use the teleporters as much as you can there's only one but use it if you need to just because you are probably gonna be the one who gets pulled I mean I could but you might probably I mean let's look at who's been pulled so far this episode and we'd have to disagree this is targeting this is targeting I don't approve of this targeting okay yeah it is very easy to be pulled out to me right now I'm eating across you're eating across okay good job I left a mark look at the other side I've got the prostrates of leaving the mark I've done this a bit too many times a bit too many good job I've been pulled told you Grindelweeds Grindelweeds yep please if we ever hit episode 420 oh that could be so plausible that's probably possible I mean we're looking at the the amount of episodes that we've done and how far through the game we actually are 420 is a viable outcome yeah that amount that road to 100 episode 100 yeah I can't wait for that one it's gonna be a fun episode don't even know what it is no do I because we probably won't even know it's episode 100 yeah we'll just be like together videos and out of what we record and all of a sudden it'll be episode 100 and we're like whoa what we're gonna come episode 100 geez didn't even do anything special when we get to level 100 we're gonna have a great dark more stream three hour dark more stream do you think that'll be fun I think that'll be fun I think no I think yes I think well we have to do dark more at some time anyway we have to do dark more at some time yes but also save me it'll be fun it'll be fun with viewers you know it won't just be us eating my way and eating your way downtown walking fast and zoom walking fast that's what it that's what it says right walking fast yeah zooming along making my way down walking fast zooming along that's that's the song and I'm down exactly our shields are going out I don't think that's part of the song though the tower show but yeah I think we should have another one let's play that will last like three episodes I think those ones are my favorite oh mine as well yeah favorite only only good I think they're the best minecraft series on the intimate oh they are definitely I personally love the factions one that lasted three episodes oh yeah yeah 100% and we I don't even think we built a base did we build a base we did but it got rated within like three episodes that's why we're gonna do it stopped oh gosh you know oh they want me to piano don't really for the piano I'm gonna hit him with the home with that again hit him with the kaboom who would win one piano or one towery boy I'm gonna guess the towery boy oh my god tower shields go luckily I have that one piece that comes in clutch sometimes you know so much damage this wasn't even enchanted like look so much damage look at this boom so much damage oh it's got a weakness I was only doing it for the weakness you know


Good evening, wherever you may be. My name is Jeff Lejeune. I'm a food safety officer here at FAO in the Division of Food Systems and Food Safety. And behalf of the FAO AMR Working Group, I'd like to walk you in today's webinar. We have the fortunate opportunity today to hear from Dr. Lawrence Goodrich. We'll introduce in a moment, talking about antifungal resistance. What's all the hype about it? So I have a few introductory comments that I'd just like to go through. And we'll start at all of you. Thank you already for keeping your microphones on mute. So not to disturb the others as we go through. If you could, it would be helpful to rename yourself on Zoom with your organization and your name. So when we refer to you, if you're asking questions, we can get back to you specifically. Caveat here, the views presented today are those of Dr. Lawrence Goodrich and not those necessarily. In terms of the chat, we have the chat there is available to post questions and we'll aim to answer those questions as we go through. Just keep in mind that we'd ask you to refrain from posting any kind of advertisements or commercial services or branding in that chat box there. The last thing I'll mention that this meeting is being recorded, which we will be able to share after the meeting. I would ask you if you do not agree to be recorded, either to not say anything or just leave the meeting and then you will not be recorded. The same goes for the chat discussion. We'll be recorded and shared over your time. So this is for every convention that's at the end. We will, this is a monthly webinar series. We will have another seminar next month on the 8th of September and being enrolled in this, this serve we can inform you of the topic in the speaker. So without any further ado, I would like to introduce the speaker, Dr. Lawrence Goodrich, who is a Leone Family Professor of Food Safety and the Director of the Canadian Research Institute for Food Safety at the University of Guelph, located in Guelph, Ontario, Canada. His primary research is in the development of approaches for risk assessments for food-borne microorganisms, achieving this goal by combining both phenotypic and genotypic analysis and methods to characterize both virulence and antimicrobial resistance in food-borne microorganisms. The specific research studies support, I'm just trying to figure out how to quick share on my screen here. There we go, sorry. This is specific to research areas and identifying mechanisms of resistance in bacteria and in fungi and the role of mobile genetic elements. He also identifies new anti-microbial compounds that may be used to control the growth of pathogenic bacteria. And I know in the last two years, as many people have leveraged this knowledge in terms of addressing the COVID pandemic. With no further ado, I think I'll pass it over to Dr. Goodrich to begin. The format here is about a 30-minute presentation followed by adequate times for questioning and answer. Larry, please, welcome. Great, thank you, Jeff. And I will just share my screen here. Can you see my screen? Looks great, please proceed. Great, thank you. So good afternoon, everyone. Or good morning or good evening, depending on where you're joining us. And I'd like to thank you, Jeff and FAO, for inviting me to share my research today, where we'll be talking about antifungal resistance. So to begin, we know that antimicrobial resistance is an emerging threat to global public health. Currently, there are approximately 700,000 deaths caused by AMR every year. And it's estimated that by 2050, there'll be 10 million deaths caused, which will exceed the deaths caused by various other illnesses or reasons for death, as you can see on the right side of the screen, including cancer. So we really need to begin to study AMR with the goal of trying to understand the mechanisms by which resistance is developed and also with the goal of trying to develop alternative approaches for the treatment of pathogenic microorganisms. So one aspect of AMR is antifungal resistance, which has not received the same attention that antibiotic or I mean antibacterial resistance has received. And so today we will focus on antifungal resistance. And so this is related to the use of fungicides or fungicides that would either kill or inhibit the growth of pathogenic fungi and pathogenic meaning pathogenic to humans. And the issue here is that there's only three types of antifungal drugs. And so because of that, unlike antibiotics that are used to treat bacteria, we really are limited to how we can treat these pathogens. And the concern is that bloodstream infections that can develop in humans, if they are resistant to these, if they should become resistant to these three types of antifungal drugs that can lead to serious health outcomes and even death. And there are several, well, there are actually many different fungi that are resistant to antifungal drugs, but the two main genera of fungi that are of primary concern are Aspergillus and Candida. And within Candida, Candida aureus, which is a new species that is particularly resistant to antifungal drugs and can spread in healthcare settings is of major concern. So when we talk about fungicides or also different types of fungicides, but really it's the asyl compounds that are of concern here. And they've been used in agricultural practices since the mid 1960s to control the growth of fungi on food crops and to stop spoilage. And you can see on the slide that the crops that asyl compounds have been used on and are currently used. So the situation with the asyl compounds are somewhat analogous to, if we think of bacteria, the use of antibiotics in food production. In that case, the use in food animals. And we've certainly seen the rise of antimicrobial resistance, the rapid rise of antimicrobial resistance and not just antimicrobial resistance, but resistance to multiple drugs in bacteria because of the use in agriculture. And that has led to now global approaches to try to reduce the use of those compounds. And so it's the same with fungi because of the use of fungicides in food production, there is concern that this is driving the emergence and rapid emergence of antifungal resistance. So this slide here just shows the mechanisms by which resistance can develop to asyls in fungi. And so I should also point out that the concern here, again, like the use of antibiotics in bacteria is the fact that once resistance develops to compounds used as fungicides or fungic stats in agriculture, this could also lead to resistance to asyl or fungi. To compounds to antifungals used in human health and even veterinary medicine. So if we look at, if we start here at section A, we see that this is showing the use of fungicides in agriculture. And as I've said, this may lead to development of resistance in fungi, primarily through the development of mutations that allow the fungi to essentially inhibit the compound, the asyl compound. And we'll talk about that in a little bit more. Then if we go down to section B here, we see humans are exposed to the either resistant or sensitive spores of in this case aspergillus fumigatus, which is one of the main species of concern, and they become ill. And then if the patients are then treated in section C with the medical triazole compounds, then this further leads to resistance or selection of resistance strains. Patients can also become infected by inhaling sensitive aspergillus spores. And then if the patients are treated in section C, the aspergillus spores, and then if they were, so in certain people, these spores can cause an opportunistic infection. And so if they receive long-term asyl therapy, then this could drive resistance. And that is section E here. So they've inhaled it, and some of the patients will develop a resistant infection through the long-term exposure of the fungi to the medical triazole compounds. Or in E, the patient could recover. So we're really worried here about section F, and this is the whole basis for this work. So our objective in this project was to determine the prevalence of asyl resistant aspergillus in food crops and their environments, and then also to characterize the resistant islets to identify resistance mechanisms. And really the focus here is on the potential environmental and clinical resistance linkage. So by that, what I mean is it's, you know, the role that agriculture plays in leading to development of fungal resistance to triazole compounds, I would say remains unproven. And by that, what I mean is, while there's been a lot of studies showing a potential linkage, there really hasn't been a definitive concrete proof, at least that the entire scientific community agrees with. And so one way to try to develop that proof is to look for resistance mechanisms that appear to be specific or develop specifically in agricultural islets of aspergillus that are resistant. And then to see if those same resistance mechanisms can then be found in clinical islets of the fungi. And so that was one aspect of our study here. So we looked at several food crops, including tomatoes, wheat and barley, and we sampled them to collect the fungal islets. These foods were produced in North America in various countries in North America. And so we purchased the tomatoes from grocery stores or other retail settings and the barley and wheat came directly from farmers. And we sampled a number of aliquots to isolate fungi. And because there's a diversity of resistance mechanisms and so forth, we collected a large number of islets per plate from per food crop and we did our work that way. So this just shows the methods once we had collected the islets, this just shows two approaches that we used to determine susceptibility to azo compounds. And so we used an azo compound called the phenoconazole which is a broad spectrum azo compound used as a fungicide in agriculture. And so on the panel on the left, we added that to an agar called potato dextrose agar which is typically used to grow fungi. And then we mixed that and put that into a petri plate and once it solidified, then we added, we actually inoculated or we streaked individual fungal islets that we'd isolated from the tomato wheat and barley onto the petri plate. So we used 30 micrograms per mil with the phenoconazole compound on the plate because that's really according to scientific literature what delineates resistance, full resistance from sensitivity. And then in the second approach on the right, we used a micro plate assay. So we took the fungal islets at the top and we inoculated them into broth. And then we had broth with the phenoconazole compound and we added that to the micro tire plate. And then once the fungal islets had independently grown in their individual broth tubes, we then added that to the micro plate as well. Again, the cutoff was 30 micrograms per mil that we use. And so here we incubated the plates for up to 48 hours at 37 degrees Celsius. And we looked for growth, which was determined by optical density. And each micro plate had a control strain or two control strains, one that was sensitive to the compound and one that was resistant. And so resistance was determined by OD as well as comparing to the control. So before we go further, it's in order for you to understand some of the results that I will show today is important to understand the mechanisms of resistance to asal drugs that are currently the current mechanisms that are understood to cause resistance to asal drugs. So there's three major mechanisms in panel A here. And two of them really, I should say, are based around the CYP51A gene. And so in panel A, this is showing the presence of mutations that are responsible for a substitution effect, which is based on causing structural modifications of the CYP51A enzyme. And so what this does, it leads to reduced asal affinity intake. And so there are several amino acid changes that have been reported in the literature. And so these designations here, G54, P216, et cetera, have been identified and associated with that. In panel B, we have overexpression of the CYP51A gene due to various insertions in its promoter region. So here you see the designation TR34, for example, there are other insertions that have been recognized, TR46, TR53, for example. And these insertions in the promoter region are combined with a substitution at codon 98 of the gene, which leads to a substitution of leucine to histidine. So leucine to histidine, hence the designation L98H. So this combination leads to overexpression of the CYP51A gene, which also again leads to reduced or complete elimination of asal uptake. And then finally, panel C, which is a overexpression of efflux transporter. So this is basically a pump that pumps out toxic compounds out of the cell, much like in bacteria, we see efflux transporters playing a major role in antibacterial resistance. And so this basically just pumps out the toxic compound, in this case, the asal compound out of the cell. So A, section A and section B are the main roots of resistance that have received the most attention in the literature. And so we really focused our attention on these two mechanisms of resistance in our work. So this slide shows that those two mechanisms in a bit more detail. So here we see in some more detail, the CYP51A gene and its promoter, showing the locations of the modification. So in panel A, this is a wild type strain, a wild type strain, which is sensitive to asal compounds. And so you can see in the promoter region, we don't have a tandem repeat or inclusion of additional amino acids. And you see the L98 amino acid downstream of the promoter. In panel B, which is a asal resistance strain, we see this TR, the tandem repeat, which could be 34 amino acids long, 46 amino acids long or 53. And we also see this substitution of the leucine to a histidine, and it's both of these compound, both of these changes that lead to the over expression of the CYP51A gene and resistance. So the first thing we did was, what we want to look at these resistance mechanisms and try to understand which, what were the most common ones, so that we could then look for that in our isolates. So we took genome sequences of triase or resistance strains of aspergillus shumigatus from the NCBI database, and we identified the six most common mutations of the CYP51A gene. So we've already talked about the tandem repeats, the L98H mutation, and we looked at two other mutations, M220 mutation and G54. So those latter two would be in panel A of the diagram I showed you, the amino acid substitutions. And then we identified sequences, the sequences as either environmental. So what I mean by that is we identified in the genomes that had these mutations, we identified those genomes that's coming from environmental isolates of aspergillus shumigatus, clinical isolates of aspergillus shumigatus, or paired clinical or environmental isolates. And this identification came from their original publications. So in other words, what were their sources, what were the sources of the isosidium mutations? And again, this was done in an attempt to see whether we could identify any specific mutations that seem to be only associated with environmental isolates and then to check for those in clinical isosidium as a way to try to understand the role that the use of fungicides in agriculture plays in terms of developing resistance in clinical isosidium. So once we had those sequences, then we checked for their presence in our fungal isosidium using polymerase chain reaction. So we designed several primary sets in our lab to amplify a single 744 base pair amplicon that was part of the CYP-51A gene. And the amplicon includes the promoter region and all of the common areas for the mutations and tandem repeats of the six most common mutations that we had studied. And we just use a standard protocol with a 40 cycle amplification of the DNA and then a kneeling temperature of 56 degrees Celsius. So let's get to some results. So we identified 85 isolates or we chose 85 isolates from three different countries in North America from which we obtained the food crops and isolated the fungi. So you can see in this chart that the male is barley and wheat and the number of isolates coming from each country. So of the 85 isolates approximately 31% were observed to be resistant to the 30 micrograms per male of the phenoconazole and we identified resistant isolates regardless of whether we streaked them out on the plate containing the phenoconazole or whether we use the microplate assay. And so in the pictures on the right, you're seeing the growing fungi. So in picture A1 here and D1, but these are two different isolates grown on the pateodectros agar without the phenoconazole and plates A2 and B2 are the same isolates grown on the pateodectros agar with the 30 micrograms of the phenoconazole. So you can see A1 and A2 are the two isolates B1 and B2 are the same isolates. So you can see A1 in particular had no problem growing in the presence of the azole compound whereas B1, there was a few resistant colonies but nothing as prominent as A2. So this shows our bioinformatic analysis which is assessment of the gene mutations in the NCBI database. So you can see in the row labeled mutation or sorry, in the column labeled mutation, you can see the six most common mutations that we focused on the tandem repeats either 34, 46 or 53 amino acids long and then the amino acid substitutions including the L98H which is a very common one. And so in the clinical assets, we saw the majority of, and I should say that the tandem repeats and this L98H mutation are linked if you can remember that diagram that showed a few slides ago. So we can see that the majority of the clinical mutations tend to have this TR34 and L98H. If we look at environmental isolates, we see that there are a number of them that have the TR34 mutation and the L98H mutation but we see for this TR46, this is 46 amino acid substitution, we do see a much higher number in environmental isolates than in clinical isolates. And then this represents papers that looked at both environmental and clinical isolates that were resistant. So it appears that this TR46 could be used as a marker for environmental resistance as compared to clinical resistance. So what this slide shows here is the PCR analysis or PCR analysis of the CYP51A mutations. So again, we amplified the primers that we designed were initially designed to amplify a 744 base pair region which includes the promoter region and all the region of the CYP51A mutation that could contain the mutations and tandem repeats. So the way this works is that, so here if we look on the left side of the screen, this is a, and I should add that we were looking for either the absence of a band or a band that had increased size above the 744 predicted size. So 744 somewhere around here. And what I wanna point out is that, and these are different isolates. The ones in the red box are sensitive isolates that we included. And so if we saw, for example, an increase in the molecular weight above 744 base pairs, that could mean that that was due to the presence of tandem repeats in the promoter region which would lead to an increased molecular weight. If we saw the absence of a band, there are faint bands here, for example, which could mean that the amplicon did not amplify, this might be a nonspecific band because of the presence of mutations. These amino acid substitution or substitutions in the codon leading to differential amino acids, which would cause the PCR reaction to fail because the primers would not be recognized by the DNA strands. So those were the two mechins we were looking for here. On the right side, this just shows why we use capillary electrophysicists. It's a great approach to see small changes in the molecular weight of an amplicon that one could not see with conventional gel electrophysicists, for example. So this is showing isolate A12, M28, so which looks to be, here you see the size of the molecular weight. So that looked to be around 744, but actually when we look at it, it's 787, as you can see by the peak over here. So that would seem to indicate that there's a tandem repeat there. So the original PCR that amplifies the 744 base spray region worked well, but we want to really try to drill down and see if we could really tease out the different resistance mechanisms. So again, remember that the main one seems to be a tandem repeat in the promoter region, which could either be 34, 46, or 53 amino acids in size, coupled with a mutation downstream leading to a transfer of leucine to histidine. And so we designed, well, we obtained primers, which had been previously designed in the literature, in the scientific literature, and we use those to tease out these differences. So this is what you're seeing here. So at the top is the L98H PCR assay, where we're expecting a 143 base per band. So this is what you're seeing here at this size. And if there was a substitution in the given fungal respective isolate, then we would expect to see a failure here. Because the primers wouldn't find. So in lane seven, that's what you're seeing here. We do see increased banding here, increased molecular weight banding. So that was as unclear why we're seeing that. And we'll get to that in more detail later. And then here is a TR that stands for tandem repeat. So in this case, the band is supposed to be 100 base pairs in length or in size, the amplicon. And so we're seeing that here. So if there's a tandem repeat, we'd expect to see an increase in the molecular weight. So we're seeing that in lane five and also in lane seven. So again, these could indicate the presence of tandem repeats in the promoter region. So, we thought that we had evidence of mutations in these isolates. So we wanted to take a closer look at that. So what we did was we sequenced three isolates. We actually sequenced four isolates, but I'm presenting the results of three because one of them was actually, once we analyzed the sequences, we found that it was contaminated. It was a mixture of two fungal species. So we sequenced the genomes of three isolates. So to do that, we isolated DNA using the kaigen DNA power soil pro kit. And we subjected the genomes to long read DNA sequencing using the Oxford nanopore grid ion platform. And you can see the flow cells that we used. And so this generated anywhere from 150 to 300,000 reads, sequencing reads with a median length of 20 kb. So following sequencing, we assembled the sequence reads using a pipeline called fly version 2.9.1. And long read sequencing can be prone to error correction. So we used a program called Medaka to correct for those errors. And then we wanted to then extract two targets we focused on in the genomes, the CYP 51A gene that we've talked about, extensively here, and another gene ERG 11, which I'll talk about later. Excuse me. And then once we'd extracted those, we aligned the different sequences with a program called muscle. So here we see the results of our sequencing. So we picked a nice set from tomato, one from wheat and one from barley. Now the first thing I wanna point out is if we go to the far right column, you'll see that the goal here was to work with aspergillus fumigatus. However, when we sequenced these fungal acids, they did not turn out to be aspergillus, but rather penicillium and to be more precise, penicillium lumens. So penicillium and aspergillus are actually very closely related. And so, and as we'll see, the fact that these acids are penicillium that did not really subtract from our analysis. What we're also seeing is some statistics here that really show the quality or can be used to determine the quality of the sequencing. So the overall size of the genomes range from 28.6 to 28.7 gigabases, which is good because they're all identified using a program called Kraken II as the same, not only the same species, but really the same islet. The number of contigs range from seven for the tomato islet to 10 for barley, which again shows that the sequencing was high quality. And then we see other statistics that can be used like the end count and gaps, which are really indications of missing data and completeness. So there's no missing data here. So again, it really shows that the sequencing was very high quality and the end 50, this column here really denotes the median content length, which is an indicator of the completeness and overall quality of the assembly, which is about 4.5 gigabase for all of them. So again, overall just a very good sequencing approach and good sequencing data, which gives us confidence that when we extracted the various gene homologs, we could be confident in what we're seeing. So I just put this slide up here again to highlight the fact that we had aimed to isolate Aspergill's fumigas, but we got Penicillium Rubens. This is a recent paper from 2021 that came out in which the authors report two new ASL resistance mechanisms in the CYP51A gene in Penicillium and they show that one of the mutations could be transferred to ASL sensitive Aspergill's fumigatus isolates, which subsequently became ASL resistance. So we see that these again, these two species are closely related and resistance mechanisms in the Penicillium can cause resistance in the Aspergillus. So even though we identified Penicillium and not Aspergillus, interrogating their genomes for their resistance mechanisms can certainly be applied to Aspergillus as well. So what this shows here, this is a genomic tree and of Aspergillus as well as 16 other different fungal species. So as we've talked about point mutations in the CYP51A gene, it is really a predominant mechanism of resistance to ASL drugs in Aspergillus. So the CYP51 proteins belong to what is called the CYP super family and are conserved across different fungal genera. So this is what we've done here is assess the diversity of the CYP51A gene by constructing a phylogenetic tree of all protein sequences from fungi and the NCBI database. And what we did was we can destructed the tree from multiple sequence alignments of amino acid sequences of approximately 1,100 gene sequences from the fungi. And so in the tree sequences can originate from the same genus have the same color. And we can see the Aspergillus in the gray here formed most of the sequences in the NCBI database which isn't really surprising because this is the genus that has been the most studied for ASL resistance. Although, and then we see that in different genomes though the CYP51A gene is conserved across different fungal genera. So we see the different ones here, the different colors and you can see we've labeled them here but you can also see what they are here. And even though they come from different genera they cluster together in the tree. And so since this tree was constructed based on point mutations this could suggest a similar mutation or similar mutation patterns in the gene sequences from different fungal genera. So again, in terms of our work with penicillium this shows that our analysis would be is still useful in terms of trying to delineate these mutations that are related to fungal resistance. Larry, I can jump in here if we can try to wrap things up quickly but we're getting a lot of questions and we'd like to make sure we have time for to answer those questions. Yeah, okay, I only have, I think it's two or three more slides. So this is a phylogenetic tree that was constructed from the multiple sequence alignments of amino acid sequences of six CYP51A sequences in the NCBI database. This is aspergillus. What you're seeing in the pink with the star are three islets that we sequenced the penicillium rubens and their CYP51A genes. And we can see that they cluster nicely with the aspergillus although they do form a separate clade within the tree. So what this is showing is the alignment of our three islets with another islet. And this is where we did this alignment to try to identify known CYP51A mutations that have been associated with azoresistence. So what you're seeing are the alignments at the top. This is a sequence from NCBI that came from our resistant islet of aspergillus from McGillus and then here the remaining lines are or in the rows are islets. And so this is a partial sequence of CYP51A genes. So what I want to highlight is the mutations in red. We found a number of mutations in red particularly in this islet, BM32, which comes from Barley. And you can see amino acid substitutions. These have been previously associated or reported, I should say in azoresistence fungal islets. So we are seeing some mutations in these islets, these resistant islets that have been previously reported. So the last thing I want to talk about is a gene called ERG11, which is also mutations of which lead to azoresistence in Candida albicans. So you might remember at the start of the talk, I said that aspergillus and Candida are two main genera of concern with respect to azoresistence and development of human infection. So we want to see, there are homologs of ERG11 in our penicillin mysid. So we want to see if we could identify any resistance mutations in that gene. And so we did the alignments again. Here you see Candida albicans or I mean Candida auris islet from NCBI and our three islets that we aligned and we looked for mutations associated with this gene in the penicillin, but we did not see any there. So to summarize, azoresistence continues to emerge, which we know as a threat to human health. And as I said at the start of the talk, the link between environmental fungicide use and clinical azoresistence while likely remains unproven definitively. We did identify azoresistence and penicillin rubens based on known CYP51 mutations. And in future work, since we have the entire genome sequences will be aimed at identifying novel azoresistence mechanisms in food and environmental fungal islets that have not previously been reported. And this will include sequencing additional islets from our collection. So with that, I'd like to acknowledge the funding for this project from FAO, as well as the two people who did primary, the majority of the work and the primary work on this, Dr. Opiemi Lawal, who's a postdoctoral fellow in my lab, as well as Jesse Huffman, who's a PhD candidate. And with that, I thank you for your time and we'll take some questions. All right, thanks, sir. If you could stop sharing your screen, that'd be helpful. We don't need to look at it, a question mark. And I'll share mine. A number of questions in the chat, and maybe I'll start with this first one, kind of moderators privilege a little bit too. You talked about the CYP51 gene. Do you know what that does in a normal, what is it's role in a normal fungi? And the question is, mutations in that gene, does that add to any fitness cost to the fungi and maybe even specifically from the chat, is there relations between mitochondrial dysfunction? And obviously someone knows a little bit more about this than I do and the mutations in that CYP51. Okay, so CYP51A is a gene coding for cytochrome P450, 14 alpha sterile demethylase. So it's a complex gene. So it's really there to demethylate compounds. And so this is how it breaks down. Well, so it will do that at a low level in azole compounds. However, if you have these mutations and what that leads to is, as I said, overexpression of the gene, which therefore increases its demethylase activity and therefore it leads to breakdown of the azole compound. And then sorry, you asked the second part. Well, is there a fitness cost or an advantage to survival and the environment or potentially survival and clinical hosts? Well, if you remember the slide that I showed of the plate with the isolates where we showed, we streaked them out in the presence and absence of the azole compound and you could see one of them grew just as well. So it depends on the isolate, it depends on the type of mutation like the tandem repeats in the primordial region depending on how overexpressed that gene is. So I would say depending on the isolate, you can see that there could be a fitness cost or there may not be. And given the fact that these are the most common mutations that have been shown and there are a number of clinical isolates that have them, I would say that there really isn't a fitness cost in the most successful isolates. And of course, I think the advantage, the obvious advantage of this is that it can survive in the environment with these compounds. One thing I didn't say about azole compounds is because they've been widely used in agriculture, the compounds are really everywhere in that environment. So not just the soil, but even the air. And so these fungi really have the ability to interact with the compounds and then become resistant. So I think these mutations are a direct result of that and it does improve their ability to survive in that given environment. Great, thank you. Another question is, and unlike bacteria, well, as you said, like bacteria, these mutations that you're talking about are not located on mobile genetic elements. And so the question is, how important are they in terms of, is it total expansion due to selective pressure that's the main issue as compared to transmission? Do you anticipate a continued expansion or if we were to reduce the use of azoles in agriculture, do you think the prevalence of these organisms would decrease in the environment over time? Yeah, so that's a great question. So the first part was, you know, unlike bacteria, these mutations, at least the ones I presented are not on mobile genetic elements, which is true. I would, however, remind the audience that in bacteria, there are mutations, particularly in efflux pumps that are not located on mobile genetic elements. And I did show briefly that that is one mechanism, albeit not the main one that is present also in these fungi. So with respect to what we're seeing here, yeah, I think what we're seeing are clones from resistant islets that developed, and then they've just been disseminated. You know, one thing that would be interesting to see, which we haven't looked at and we may try, it depends on what information we can glean from the scientific literature, but it'd be very interesting to see, and I see there's a question here about information regarding farmers, and I was just about to discuss this. So it'd be very interesting to see if farmers tend to have a higher, if the number of, if the resistant fungal infections tend to be higher in farmers than in the general population, because they're there and potentially breathing in the spores, the fungal spores and so forth, which we've just said, tend to become resistant in that environment because of their interaction with the compounds. But yes, I think what we're seeing are clones of these. So if we remove the azo compounds, I think that we may not, I don't think we'd see necessarily a decrease. Again, it looks like these isos once they become resistant, there's not really a fitness trade-off. So it'd be interesting to see, but I think the goal of removing the compounds is to stop the emergence of new clones and new mechanisms. In the scientific literature, there have been some groups that are now starting to look at the entire genomes like we plan to. And some groups have found, in preliminary evidence, some new mechanisms for resistance. So I think that's what we would try to do. All right, so you have several different things talking about the problem for human health. One is the transmission problem from the potential, from the environment and humans, and what is the attributable fraction that causes the resistant infections. And then the other one, and what is the impact of using azols to the prevalence of these and magnitude in the environment. We'd like to stay on time here. We're one minute over, I need to close up. I think we appreciate your presentation, Larry. And when we attack antimicrobial resistance, we need to go from farm to table. We need to go from the molecular to the global scale. And I think your presentation here really go into some of the molecular sequence-based data and how that can inform the questions for developing more practical on farm or agriculture or in clinic solutions that address this problem. And so I'd like to thank you for your time. I'd like to remind the audience that there will be another webinar related to antimicrobial resistance on the same time. There will be a 12.30 from our headquarters time in Rome on September 8th. And we look forward to having you join us again. The recordings should be available of this presentation future on our YouTube channel. Thank you very much for everyone, for your time and your participation. Have a good day. Goodbye.


Shartru cat vs. Bombay cat. What are the differences between these two cat breeds? With his cloak of gray fur, sober as amongst robes, it's no wonder that the Shartru was associated with the Carthusian order in France. The quiet mousers would have been perfect companions for members of the silent, solitary order. It's a pretty legend, but there is no real evidence that the cats were kept by the Carthusians, although perhaps they were simply not considered important enough to mention. The Shartru might well be compared to a mime, silent but communicative and sometimes silly. Cat breeders are an experimental lot, creating distinctive new breeds either by building on natural genetic mutations or by crossing breeds to achieve a new look, color or pattern. The Bombay, named for the exotic port city of India, has no connection with the subcontinent but was created from crosses between sablebremese and black American shorthairs to resemble a black panther in miniature. The Bombay is a smart cat who loves to play and will thrive with a family who is willing to teach tricks, play games, and provide plenty of interactive toys. The Shartru has the sturdy, powerful body of a working cat wrapped in a short, thick, water-repellent coat. Besides his beautiful fur, his most distinctive feature is his deep orange eyes, set in a rounded, broad head. He has full cheeks and a sweet, smiling expression. Medium-sized ears sit high on the head. His body type is sometimes described as primitive, being husky and robust with broad shoulders and a deep chest. His relatively short and fine-boned legs rest on round, medium-sized paws that look almost dainty. A lively, flexible tail is heavy at the base, tapering to an oval tip. Don't let his ample body and solid muscle mass fool you. This is a supple, agile cat who typically weighs 7 to 16 pounds at maturity. Pick him up and you may be surprised by his heft.  The medium-short double coat has a slightly woolly texture, which varies depending on the cat's age and gender, as well as the climate in which he lives. For instance, mature males have the heaviest coats, while females or young cats may have a thinner coat or one that feels silky. The coat can be any shade of blue-gray. The tips of the fur look as if they have been lightly brushed with silver. Kittens may have the faint imprint of tabby markings, cold ghost barring, or tail rings, but by the time they mature the coat should have a bright, even tone. Completing the look is slate-gray nose leather, blue lips, and rose-toed pawpads. Except for his dramatic black coat, the Bombay looks much like the Burmese, but with a few physical differences such as a larger, longer body and longer legs. He has a rounded head with medium-size ears set wide apart, eyes that range in color from gold to copper, and a straight, medium-length tail. The short, fine coat feels satiny to the touch and shines like patent leather. Although the gene for the black coat is dominant, a sable-colored kitten is sometimes born in a Bombay litter. Some associations permit these kittens to be registered as Burmese. The Bombay is a medium-size cat. When lifted, he feels heftier than he looks. The breed develops slowly and males may not reach full size and musculature until they are two years old. Hello! This video is sponsored by Beemix Pets. Are you looking for high-quality cat collars at an affordable cost? Check out BeemixPets.com. Use coupon code KittenLife, to get 20% off. The chartreux might well be compared to a mime, silent but communicative and sometimes silly. Short playtimes interspersed with naps and meals are his idea of the perfect day. When he is not displaying his superb timing and pouncing ability, killing toys with abandon, or performing acrobatic flips as he chases a lower toy, he is an attentive and gentle companion who likes to stay near his people and observe their doings. He appreciates any attention he receives, especially if it involves scratching him beneath the chin or between the ears, but he's not demanding, content to follow you around devotedly, sleep on your bed and snuggle with you if you're not feeling well. He rarely uses his voice, preferring to direct your actions with a glance from his pumpkin-colored eyes. If need be, however, he may communicate with a small meow or chirp. Make a habit of observing him carefully and being familiar with his actions since it is unlikely that he will vocalize any discomfort or make any sound to let you know where he is. This is an adaptable cat with a middle-of-the-road personality. He's not a social butterfly, but he's not a shy wallflower, either. Expect him to watch and wait before deciding whether to greet a guest or otherwise involve himself in a situation. His calm nature makes him suited to staying home alone while you are out earning the money for his treats and toys, but he won't object to keeping company with another cat or dog. That same good nature makes him a good travel companion for an RV enthusiast or long-distance truck driver. As long as you keep his routine similar each day, he will be a happy camper. The chartreuse has a sunny, polite disposition that makes him a pleasure to live with. This is one cat who does his best to obey the rules. Always treat him kindly and patiently, and you will be rewarded with a friend for life. The lively and affectionate Bombay loves people and is adaptable to many different environments and lifestyles. His calm nature makes him a good apartment dweller, and he is amenable to life with other pets, although he definitely wants to be top cat. Expect to find the Bombay hogging the warmest spot in the house. That includes under the covers at bedtime. Most will converse with their people in a distinctive but not loud voice. Bombays are often good at playing fetch, and some have learned to walk on leash. This is a smart cat who loves to play and will thrive with a family who is willing to teach him tricks, play games with him and provide him with plenty of interactive toys. The chartreuse short, FitCode is easy to care for with weekly brushing. The coat sheds in the spring and may require extra brushing during that time. A bath is rarely necessary. If your chartreuse does require a bath, remember that it can take time to get the water repellent coat wet enough for bathing. The Bombay short, SleepCode is easily cared for with a few strokes of the hand or at most weekly brushing or rubdown with a chamois to remove dead hair, distribute skin oil and polish the coat to its gleaming best. A bath is rarely necessary. Brush the teeth to prevent periodontal disease. Daily dental hygiene is best, but weekly brushing is better than nothing. Trim the nails every couple of weeks. Wipe the corners of the eyes with a soft, damp cloth to remove any discharge. Use a separate area of the cloth for each eye so you don't run the risk of spreading any infection. Check the ears weekly. If they look dirty, wipe them out with a cotton ball or soft damp cloth moistened with a 50 to 50 mixture of cider vinegar and warm water. Avoid using cotton swabs, which can damage the interior of the ear. Keep their litter box spotlessly clean. Cats are very particular about bathroom hygiene. It's a good idea to keep them as an indoor only cat to protect them from diseases spread by other cats, attacks by dogs or coyotes, and the other dangers that face cats who go outdoors, such as being hit by a car. Cats who go outdoors also run the risk of being stolen by someone who would like to have such a beautiful cat without paying for it. The tolerant and gentle chartreuse fits nicely in a home with children. He is more likely to walk away than to scratch if he doesn't like the way he is being handled. Parents with young children should always supervise interactions to make sure the cat isn't mistreated. He is happy to live with cat-friendly dogs, too, thanks to his amiable disposition. The outgoing Bombay is a perfect choice for families with children and cat-friendly dogs. He loves the attention he receives from children who treat him politely and with respect. He lives peacefully with cats and dogs who respect his authority. Always introduce pets slowly and in controlled circumstances to ensure that they learn to get along together. If you enjoyed this video, kindly press the like button. Also don't forget to subscribe with notifications on so that you don't miss out on videos like this. Thank you for watching.


Green synthesis is the watchword for the combination of nanoparticles, NPs, by plants or their metabolites. This innovation is particularly compensating as far as decreasing the poisonous quality caused by the conventionally integrated NPs. In the green strategies, plant metabolites and natural substances are utilized to orchestrate the NPs for the pharmaceutical and other applications. This article was authored by Amangore and Narendra Kumar Jain.


This paper presents a new time domain diffuse correlation spectroscopy, TDDCS, system based on a superconducting nanowire single photon detector, SNSPD. The SNSPD has a high quantum efficiency, a narrow timing response, and a negligible dark count noise, making it ideal for use in TDDCS systems. This system was compared to a typical single photon avalanche diode, SPAD, detector and was found to be faster and more efficient than the SPAD. Additionally, the SNSPD enabled the authors to perform in vivo measurements of pulsatile blood flow index, BFI, on the adult human forehead. This article was authored by Veronica Parfencheva, Lorenzo Colombo, Pranav Lankar, and others.


What about the extra symptoms from autistic burnout? Sensory sensitivity increases, you may find that things that you could usually deal with, perhaps walking outside, perhaps going to the gym, they're a lot harder to deal with than usual. The people that are around you, the noises, the lights even, they can become a lot more intense. Your ability to manage that and the amount of time that you can spend in those highly sensory environments is lower. And so it can really have an impact on a lot of different areas of your life.