My name is David. You can find me on the internet as David, Twitter, GitHub. I work for a company called Michelara, like the drink. If you've never had one, Michelara is a great drink that us Mexicans drink all the time. It's made with ice, clam juice, magical Mexican sauce, chili powder, salt, lime, and, of course, beer. And it's delicious. You should get one if you've never had one. But anyway, the name of my talk is Tips and Tricks for New Developers. I'm going to tell you, I'm going to give you a little bit of context why this talk, what I decided to give this talk. So, long time ago, Galaxy Far Far Away 2009, Las Vegas, my first RailsConf. And it was a great RailsConf. Back then, I was building, I just moved on to Rails. Had my first job. I was being paid to write my first Rails app. And I was building a huge e-commerce platform but knew nothing about, you know, the real stuff. Like, what, once you're past, like, the tutorial phase of Rails, like, what happens next, right? And I remember that there were several talks at this Rails, at this particular RailsConf that sort of changed my view and helped me a lot. Things like solving the riddle of search using Sphinx with Rails. Like, it made me figure out that it might not be a good idea to filter my products with SQL or do searches with, like, you know, like searches. So, I learned that there was this thing that you could use to search. We ended up using Solr, by the way, not Sphinx, but anyway, I sort of learned something new. I learned about WebRat. You probably don't remember WebRat, but it was what we used to test applications before Capybara and all this fancy new stuff that we have. But I learned about it. And then the most important one, or one of the most important ones, is I learned about Git. I was using SVN before that and VisualSourceSafe before that. So I learned about Git. I think it's one of the best docs I've ever heard. It started with Git in 60 seconds, I remember, clearly. And the speaker sat there and just basically read that in less than 60 seconds. It was amazing. And then he proceeded to do the proper doc, right? And another important thing that I learned at that RailsConf is how to put, like, what was I to do once I had my application. I learned that there was this thing called mongrel that it was our web server. I don't remember about mongrel, of course. I learned that I had to, you know, set it up in different ports. You had a process, a mongrel process, that started on each of the ports. And then you had NGINX on top or Apache on top of it that sort of, like, did the processing around. And then if you wanted to scale, well, you scaled like that. Just added more servers and HEProxy to do the processing and you would be fine. You needed more power, you just added another one of those. And that was back then in 2009 before Puma and all this new fancy stuff that we have. So I remember Las Vegas, right? So I think that we still need talks for beginners at RailsConf. We do need to know how to do, like, microservices and how to handle a gazillion of requests per second and all of that, but we need talks for beginners so people can come here and learn stuff that they don't even know they needed to know, right? So this talk is for beginners. Most of the things that you learn here, maybe you already know it, maybe not, but I do want you that this talk is for beginners. And it's just based on my experience, what I do when I start a new Rails application, like the things that I always use, the things that I usually do and all that. So hope it helps for you.  So let's begin. First of all, let's talk about configuration files. When you start a new project, right, you use your Rails new command. The first thing that you want to do, well, it's gonna do your bundle stuff, it's gonna copy files and all that. The first thing that you want to do, I consider this a courtesy to our developers, is please copy your database file into an example file and then ignore the original one so we can always have on hand a configuration file that we can base on. It's awful to go into a new project and then start your Rails server, your database jammer file and then you realize there's nothing, you have to go Google it, paste it and all that. It's just time consuming. So please keep an example file of your database file. It's gonna pay out in the future. And do so for any other configuration file that you think that it's needed for the project to run. Please create sample files and copy them and just ignore the real ones from Git. And while we're talking about configuration files, did you know that you can have as many as you want and you can separate those in different files, like, for example, you can have a payments jammer file and then you can just call it from the application like this, use config for and just send the name of the file as a sim and then you will get that particular configuration. So you can use it to just organize your configuration files better and everyone will be happier when it's cleaner and you know exactly what kind of configuration is on each of the files. And then remember that wherever there's a string or an integer on the code that's comparing to something else, you probably want to use a constant but a setting on one of those config files. So remember that if you see code like this in your Rails application, try to change it to something like this. Use a constant for that 30. And if you want to go, like, if you think it's going to change constantly, just move it all the way over to a configuration file. You're going to be very happy that you did this when your boss comes and say, hey, I need to change that to 10 minutes and then 15 minutes later he's going to say, like, hey, change it to 20 minutes. This way you can, you know where to change something. Just restart your server and, you know, you're done instead of, like, looking for that setting in the code base. Let's talk about the database.  I've seen it a lot. Do not yet ignore the schema file. Please. There are two teams where, or I've joined applications where there's no schema file and then I ask, like, what happened to it? And there were too many conflicts with it, so we just got rid of it. Like, that's the point, you know? So please do not ignore your schema file. It's very important that you're always able to restore your database state with either DB setup or a DB reset, especially if you're new to the project.  So please, please do not ignore that file. It's very important that you find those conflicts and then talk to your fellow developers and say, hey, I added this column. Hey, yeah, me too, so let's figure it out, right? Instead of just ignoring it. This is a tricky one. Don't change your data in your database migrations. This is hard to achieve, so I'm going to change a little bit the tip to be careful when you change data in your database migrations. It is pretty common that you do something like this. You add a column to one of the tables that you already have, and then you decide that you want to fill it with something, maybe based on the current data that you already have, like here, right? We're splitting the code column into two columns. So you do something like this, right? You add the new columns, you go through all the companies, you split it, and then get rid of the old column. The problem with this kind of code, first of all, is that it's not future-proof. It's not future-proof because, for example, what happens in the future, your company model is now the organization model, for example. This migration won't run anymore. And while migrations are not supposed to last that long, they're just, you know, they help you to keep track of history, of how the database evolved. Your schema RB file is the one that's supposed to have, like, the real data structure, database structure. It's still nice to sort of, you know, try to keep it as coherent as possible. The other problem that you might find with this kind of code is that you can, you know, have longer deploy times.  But you forgot that there's, like, three million companies in your database. So now it's going through all of them in production, and, you know, it's doing all of that, and you can't, like, your database is in a weird state. Maybe there's more migrations that are coming, and the site is, can't run without those migrations, so you're doing maintenance mode or whatever, and your boss is angry because it's taking long, and you forgot about the three million rows. So try not to do that.  And the other one, so this code, for example, this is going to happen in production, I guarantee it. You forgot that maybe code is nil, so it blows up, you know, you're stuck with a migration weird state on the database, and everyone's yelling at you because you have to log into the server, change that code real quick, run the migrations again. I've been there, so it's a really bad idea to do this. So one way to fix it, prefer SQL over Ruby. If you're gonna do it, you're gonna be careful, but if you're gonna do it, you might be better off like this. Do your thing, update, use regular SQL to do your updates, and move on, right? This is faster, of course, because it's not iterating through all of the objects, it's not instantating anything, so it's just updating whatever needs to be done. If there's nil, it's not gonna blow up, so this is way faster. So try to think your changes in data as SQL and prefer this. Obviously, it's gonna be safer to do this, but the best thing to do, in my opinion, is to use a gem like this, Rails data migrations. There's a lot of gems that do the same thing, but I think this is the best one because it works with Rails 5, by the way, and this gem is gonna give you a different set of break tasks for your data migrations, right? So, for example, if you had this regular migration, once you have the gem, you're adding a column and then doing an update on all the records on the table, what you would do, in this case, is create a data migration, you know, set authors active. It will create a special migration in a different folder, and then in that migration, you will add your data changes, right? So you don't need them anymore on the regular migration. So now, when you deploy, you will run first your database migrations that will change the structure, and then with a different break task, you will run your data migrations. So it's a separate process, and it makes everything cleaner. And it also keeps track of how data is changing using the same, like, the timestamp that the database migration uses. So this is way better. At least, maybe, first, make all the migrations that change the structure, and then add migrations later to change the data, right? Let's talk about background jobs. It's pretty useful, to say, to use background jobs. You have something in place like SadKick, Rescue, DelayJob. I still used DelayJob a couple of months ago. And what I noticed is that it's regularly a bad idea to pass objects to your methods that go in the background. There's a few things that got me wrong, but what I mean is this. Let's say you have a job class that has this perform method, and you probably want to do this, right? You have an order and a user, and you're going to assign it to it. What I mean is that it's better if you do this. Just pass the IDs, and then find again your objects on the job, and just do whatever needs to be done. The problem with using objects is that they're usually converted into Jaml, and they end up being, like, huge strings that, if you're not careful, and there are special characters in the description field or something like that, it just makes everything blow up, and you will not notice until you see, like, all the alerts coming up. It's better if you just use the ID. It's smaller to just, you know, pass an integer around instead of a whole object as Jaml, and it's just going to be better. It doesn't matter in terms of performance, because it's already on the background, so just let it do it, right? And speaking of background jobs, always, when you start a new application, always, always deliver your email later.  Always, when you start, you start thinking about delivering your email later. Always. And the same rule applies for mailers. Instead of passing objects, try to pass IDs and just pull them over from the code on the mailer, right? This way, even if you don't have anything in place right now to sort of delay the sending of the mailers, in the end, you're just going to need to add that delay method if you're using sidekick or delay job or if you're using active job, just use deliver later, and that will be it. But use the integers. Don't use the objects. For the same reasons as the background jobs, okay? Let's talk about REST. This is my favorite lesson for all new developers. You have to be RESTful all the time. REST is like the core of Rails and MBC and everything that you're doing, and it's simpler. It's very simple to sort of explain, but it's hard to, like, actually implement it. Let me tell you why. REST, to me, it means that in your controllers, you only have these actions. Nothing more. Actions should always be index, show, new, create, edit, update, or delete. No other action, but it's pretty common to find in code bases things like this, right? You have the products controller and then you have an action to deactivate the product and you create your route and use get and member, whatever, right? This is wrong. This is not RESTful. When you think about RESTful, you should think that for every request, there's a something, a resource that's changing. So in this case, when you're deactivating the product, what's changing, it's the product state. So the correct thing to do here is to create a product state controller and you're updating it, right? This is more RESTful, much more RESTful. For example, if you have a shopping cart, right? Instead of having an action to apply a coupon, what's happening, you are creating a discount or a shopping cart discount. So create a controller for the discount and you are creating it. This is RESTful, right? This is very common. If you have contacts, you have a search, so you create a search action wrong. That's totally wrong. You create the contacts search controller, search as controller, and then you have an action for that search. This is RESTful, right? You have something that is changing because that's what RESTful is supposed to be. So the way to make sure that you're complying with this rule, it's very easy. You don't have controller actions that are not on that list. It's always index, show, edit, new, create, update or delete. That's it. If you have an action that's named differently, wrong, right? Okay. Now, a few Ruby gems that I think must be on all projects. I find them useful. You can add them, so I hope this helps for you too. This one you probably heard of a lot, Rubocop. Rubocop, it's going to analyze your code. It's going to check how it's written, and it's going to tell you where you're doing things right and when you're doing things wrong. And it's automated, so it's pretty cool.   If you add it right now, there's going to be like 500 errors and you don't have the time to fix them. That's okay. What you can do is you can add this, run it with this option, auto gen config, and it's going to create a Rubocop todo.jammel file that's going to include an exception for everything that it found. So that way, you just add it to your, as a sort of an include file to your main Rubocop file, and that way you have like a sort of a clean slate at that point in time, and then you are supposed to come back later. That's why it's called a todo file, and remove all those exceptions and fix them. That's what you're supposed to do. You probably won't, but it's fine, right? At least from that moment, like for example, right there, it's green, at least from that moment on, if there's something, like if you write bad Ruby code, it's going to tell you starting from there, instead of having to fix all other like 500 or whatever errors you find. Auto Gen Config also helps for a new project, so you can like pull configurations that you might use, like change the rules depending on your style. For example, there's this one where it configures Rubocop to how many columns, how many characters you see per line. I usually have it at 80, 90, you can set it to 130. I know there's people that say, we have large monitors, like why do we need this? The thing is that, you know, your vision is only, like it's really narrow, so it's better if you have less characters to read at once. You just have to like sort of nest code around, so it looks better if you try to remove the characters on your lines. Try that one. This one, style and documentation, I usually move it to the main file because I'm not going to write comments on all my classes or methods or anything, so I just get rid of it, because no one has time for that, right? And this one, I think it's important, this is the one I value the most about Rubocop, the metrics method length. It forces you to make methods that are no longer the 10 lines. It's hard to, you know, comply with this one, but if you do, your code will be so much cleaner and so much readable, so trust me, try this one. I'll allow you, if you go to 12 or 13 lines, that's fine, but don't go over that.  It's gonna, you know, you're supposed to try to abstract all the, like the code into smaller methods and then test those, unit testing, that's why it's called unit testing. So keeping this one, it's like, really, it's gonna make your code so much readable and so much better. And then if you want to run this before you push code to your, to git, you can use a hook. If you add that file, .git, slash hooks, slash pre-push, it's gonna run RuboCop before it actually pushes the code to the repo and if it fails, then the push will be canceled.  It's very useful, for example, us, we have a CI server that actually runs RuboCop and our repos are filled with comments that, you know, really like fix that RuboCop thing. That RuboCop, because the build fails when RuboCop fails. So it's usual to push, wait for the CI server, fail, why, because, you know, maybe you went over with the characters or whatnot, so you have to make a commit that only fixes that. F RuboCop, dammit RuboCop, there's a lot of commits like this on our repos. So what I tell the guys is just to use a hook, so, you know, you run RuboCop before actually pushing and if it doesn't pass, then you won't push and you can fix it and not curse at it. Annotate, this is in our gem, that I don't see often and I don't know why, but I love it. Annotate, what it does is when you say that you have a model user with username, password, active, whatever, when you run Annotate on a Rails project, well, after you do your migrations, what it's gonna do, it's gonna, well, annotate your models with information about the database, automatically, basically. So if you add columns, you just run Annotate again and it's gonna add that information at the top of your classes. And it's gonna do so for models, test files, unit test files, and factories if you use. So it works as a reference, a quick reference if you are working on a model about the columns that it has. Instead of, you know, going to the database or checking schema or whatnot, you just have it right there at the top of the model and you can see it real quick. So it's pretty useful to have these annotations handy. And this one is pretty useful, too. If you use routes as an option, then it will annotate your routes file. So you don't have to rake routes every time you need to figure out where my controller should go. You will already have it.  So it basically autocompletes only on what's open.  So it's pretty cool. Very useful. But I don't know why people don't use it. So now you know. Please use it, annotate. Very useful. Bullet. Bullet detects the N plus one problem as it's happening. As you are developing. If you don't know what the N plus one problem is, it's very common in Rails. But it's something like this. So let's say you have a book method that belongs to author, has many comments. Then you have the author that has, you know, name, whatever. And then you have the comment that belongs to user, belongs to book. And your index file looks like, sorry, your index page looks like this, right? So when you go to this page, the controller usually looks like this probably. And then when you look at that page, your log file will look like this.  Like three queries, four queries per row. And, you know, it's just getting all the objects one by one because that's what you told Rails to do. It doesn't know what else to do. So this is called the N plus one problem. It's very common when you're developing Rails application. So if you use bullet and you configure it, let's say, for example, what I'm doing here is, well, enabling it. I wanted to show on the console. I wanted to show on the Rails logs. And to add the footer, what happens is as you load that page, you will notice that there is a footer that says, hey, you know, you're probably better off if you include the author on the query or if you include the comments on the query. And it's gonna gel at you from the console too, from the Rails logger, right?    And then when you load your page, you will now have, you know, the queries for each of the classes.  So this is way better at M-Bullet. You know, we just tell you, as soon as you're developing, that you're doing something not wrong, but maybe weird. And another similar gem, oink. This detect memory leaks before it's too late. I don't know if you've worked with Rails applications that have memory leaks. You have a memory leak when your applications start growing, like, I don't know, 1.2 gigs in RAM, and then you need to restart. Actually, that's the best way to fix memory leaks on Rails application, just set up Monet. And if it goes past the threshold, just restart the server, done, right? Because it's so hard to find memory leaks on the code on Rails. It's very, very hard. Once it's there, it's not gone ever. Like, you're stuck with it. Deal with it, basically. That's a solution. So oink, when you have it on your code base, and you, it's some sort of middleware, so you initialize it, and it's gonna add at the bottom of your logs, like when you're developing disinformation, which is basically how much memory it's using, and the number of objects that you are instantiating in that request. So if you're developing something like this, and you see that you have, like, 500 comment objects instantiated, something must be wrong, right? There's something wrong going on. And it's actually kind of safe to use in production. I wouldn't enable it all the time, but if you're sort of trying to figure out where the problem is, I wouldn't enable it, and then just let it log a few requests and try to find where I'm instantiating a lot of objects. So it's very useful for them. And, wow, right on the 30 minutes. That's it. That's all I have for you right now. Like I said, I had to remove a bunch of them of, like, advice, but this is, like, what I thought was most important. And that's it. Thank you. Thank you.


Migration is one of the most complex matters of our time. It can be an opportunity for all. But too often, it's an unpredictable and dangerous road for many. The road to safe migration lies in understanding its complexity. So, where do we start? Let's begin by understanding that migrants, like all human beings, belong to families, circles of friends, communities, national and global societies. All these levels affect decision-making. We need to address each one of them if we want to find a sustainable way forward. That's why we created Compass, a global initiative to protect the most vulnerable while guiding safe migration better, together. We make sure that people on the move are safe and protected, and if they wish to go home, we help them return and face the challenges of reintegration. We also support governments and partners through training and coordination. Collect data to learn from our experience to better understand migration and inform new policy. Together, we have the power to make the cycle of migration safer. Join us.


You're watching News Made Easy and I'm on India Chakravarthy. Friends, there's going to be another economic boom that's going to come very soon. You're going to hear about it, but it's only going to be for the top 1-2% of Indians. The rest of India is going to continue to face the economic hardships that they've been facing for a very, very long time. Now, who are these 1-2% of people? Many of you who are watching this video are likely to be amongst them. Because if you're household income, and household I mean that it includes probably 2 people, on an average 1.25 people per household when we talk about working, earning members in every family. And if your household income is about 10 lakh rupees or more, then you are considered to be an affluent person in India. That's what most economists take as the threshold. That's about 83,000 rupees. Most of these such people who earn that kind of money stay in big cities in India. And they stay in the better of localities in these cities itself. Not necessarily the poshest or the tiniest places, because after all with about 80,000, 80, 85,000 rupees a month, it's very unlikely that you're going to be living in the richest part of your city, given the EMI is required for that or the rents required for that. But if you have 2 kids, you're probably sending them to the good private schools of the city, paying 6-7,000 rupees per month on their education. You go for occasional lunches and dinners to mid-rump restaurants. And if you earn more than that, obviously to top-rump restaurants, you probably have got that car and you're probably going to be replacing it every 6-7 years. You have smartphones, more than one in the family. You have laptops and desktops in the family. You subscribe to English newspapers. You probably have Netflix and Prime Video that you watch, even Hotstar. And you generally also spend on household gadgets, because these are the people, those who earn more than 10 lakhs a year. These are the buying classes of India. These are the people who do almost all of big ticket purchases in India. Alright, let's try and gauge how many people there are who earn more than 10 lakh rupees a year. A year, remind you, not month. If I do a bit of number crunching on the income tax data for 2017-18, which is un-money earned in 2017-18 and tax paid, which is also called the assessment year of 2018-19, then you will see that there were about 5 million odd families, households. And this requires a bit of number crunching and assumptions. But I'd say roughly about 5 million households who earned more than 10 lakh rupees a year. These are tax-paying households and because of various kind of stringent actions, one really can't expect that tax is not being paid by the visibly affluent. So this would more or less be the correct number. Mahesh Vyas of CMI wrote recently that now in 2021, there are just about 3 million such households left who earn more than 10 lakh rupees a month. And he says that before that, before the pandemic hit us, which is March 2021, in 2020, there were about 4 million. So during COVID, about 1 million or 10 lakh affluent families slipped below the threshold. And between 2018 and 2020, before that, because of the slowdown, pre-COVID slowdown, another million had slipped through as well. So effectively, currently, there are about 3 million odd families who can buy, who can really buy goods. And they are the people who drive it. So that 5 to 3 million slide, that is what has shown up as despondency. That is what has shown up as the slowdown that you see, cars not selling, gadgets not selling, houses not selling, even hospitality businesses in trouble. So that is what had caused the affluent had slipped. A significant section of the affluent had slipped below that threshold and won't be spending that much anymore. But how much does it really take for that recovery to take place? Very little. All you need is about broadly 400 to 500,000 jobs. 400 to 500,000 job openings where people have a sense that, we can go to another job and earn more. As soon as that happens, there will be a sense of recovery that takes place. Amongst this top 1%, remember, 3 million households is just 1%, less than 1% of India's population, 5 million households is less than 2% of India's population. So that is all that the recovery is going to affect, that top 2%. And it doesn't make take much. And already there are surveys that tell us that nearly 3 lakh new jobs, open positions exist in white collar jobs. Right now in September, they're better paying. We're hearing of how IT salaries, you know, tech salaries have shot up sharply. And that is going to show up in increased consumption. And you're going to say how much can that happen? But it has happened. All the booms that we've seen since liberalization has actually been driven just by that top 2% to 3% of people, not more than that. That is why in India, we sell about 2.7 million cars a year at peak, 2.7, 2.8 million cars a year. When it's at its peak, it tells you that other than the replacement demand, there is very little new demand for goods in India, except for replacement and upgrade. That is all, that top 1% to 2%. Yes, there is a demonstration effect. There is a multiplier effect that probably trickles down to another 8-9% of people. It's just that top 10% who are consuming in this country. 90% of people do not consume. When that top 2% drops a little bit, they lose out. We see a sense of panic. We feel bad effects set into the economy. Let's talk about slowdown. When that top 2% again starts buying, we will hear a talk of boom. It's going to come. Be ready. But remember, this is a fake boom like previous booms, and it is going to go back to slowdown sooner than later because it only can be sustained by those top 1-2%. Remaining people in India will continue to face the economic hardships that they've faced for the last 30 years. That's the show today. Keep watching NewsClick and do subscribe to us as well.


Ukrainian troops near the pulverized eastern city of Bakhmut on May 3 said they were repelling Russian attacks. Soldiers dug into a trench complex, manned a heavy gun and fired repeatedly at Russian positions. Brigade commander Oleksandr told Reuters, they storm our positions almost daily, but we repelled the attacks. There are two area here where the fighting is intense. Otherwise, it is more or less fine. Russian military vowed on May 2 not to give up the city as it prepares to launch a long-promised spring counter-offensive against Russian forces. The battle has symbolic importance for both sides, with Ukraine still holding on to some parts of the city after months of fierce fighting against regular Russian troops and the Wagner mercenary force. Russia sees Bakhmut, once home to 70,000 people, as a stepping stone to attacking other Ukrainian cities.


OK.  And I start by saying that this story may challenge how we think about the genetics of Mendelian disorders and also how we think about the genetics of complex disease. Because not too long ago, we saw genetic disorders as divided into two major categories, those that were simple single gene disorders, things like Gauchy disease, cystic fibrosis, PKU, and disorders that we knew were genetic, but we considered to be complex multi gene disorders like asthma diabetes, ADHD like you heard last month, and Parkinson's disease. The Mendelian disorders were considered simple because they fit into this single gene disorder category. But we started to note as we realized we started to see this boundary between these two discrete categories becoming blurred when things like environment and epigenetics, there we go, modifier genes and risk factors started to blur the boundaries that distinguished them. In fact, I think today we see these disorders more as a continuum, where on one side you have disorders where there's a single primary gene that has a large effect and then perhaps multiple modifier genes or risk factors that contribute. And on the other end of the spectrum, you have disorders where there's multiple primary genes with small effects, and you might also still have modifying aspects. So today I'm going to I want to introduce the two disorders that I'm going to speak about. The first is Gauchet disease, which is a rare autosomal recessive enzyme deficiency with a variable age of onset and multi organ involvement. On the other hand, I'm also going to speak about Parkinson's disease, which in contrast is a common disorder affecting about 1.5% of the population over 65. It's a complex multi gene disorder that's more late onset. Symptoms we'll discuss. And it is distinguished by the loss of dopaminergic neurons and the formation of Lewy bodies. These are these aggregates that you see on autopsy in the brain. And it mainly affects the substantia nigra and brainstem. I just wanted to review the anatomy where this was. So how are these two seemingly unrelated disorders connected? I think throughout this talk, I'm going to show you how this is being approached by clinical studies, pathologic studies, imaging, genetic studies, cell biology, protein work. And I also hope that you will learn that Mendelian disorders can at times provide a unique window into complex diseases like Parkinson's disease. So to begin a little bit more about Parkinson's disease, start with some definitions. I'm going to use the word Parkinsonism. And that's a term that describes the motor features of Parkinson's disease. Parkinson's disease is clinically and pathologically defined. There's actually rigid criteria put out by the UK Parkinson's Disease Society Brain Bank criteria. It includes bradykinesia, the slowing of initiation of voluntary movements and the reduction of speed or amplitude of repetitive actions, and at least one of the following, muscular rigidity, arrest tremor, and postural instability. Actually, it's more than one entity. We can call them the Parkinson's diseases. First of all is the classic idiopathic Parkinson's disease, which is thought to be largely sporadic and not necessarily running through families. Then there's environmental causes of Parkinson's disease, the classic being the MPTP story or the frozen addicts. There are the Lewy body dementias that overlap some with Parkinson's disease, but pathologically are really distinct entities. And these include dementia with Lewy bodies and the Lewy body variant of Alzheimer's disease. And then there's this category that's lumped as Parkinson's plus, including things like multiple system atrophy and progressive super nuclear palsy, which I'm not going to really go into very much today. The list of genetic forms of Parkinson's disease is growing. They're kind of numbered now as gene park 1, park 2, et cetera. I think they're up to at least 15 park genes, some, but not all, are associated with Lewy body pathology. The pattern of an inheritance of these park genes is variable. There's some that are autosomal dominant. Perhaps the most famous of these is with the first, park 1, first described by Bob Nussbaum across the street at NIH. And this is a very rare cause of hereditary Parkinson's disease. And it's caused by point mutations and multiplications of alpha-synuclein. But its discovery is an example of why it's important to find a gene because it really taught us a lot about the central importance of alpha-synuclein and the pathogenesis of this disorder. Another more common autosomal dominant form is park 8 or lark 2. This is fairly frequent, especially in some populations like Ashkenazi Jews and North Africans. And it's inherited with incomplete penetrates. In a pedigree, you can have the mutation and not have Parkinson's disease. And it has variable pathologic findings. In addition, there's some variants. There's some park genes that are inherited in an autosomal recessive fashion. Park 2, the first one identified, is associated with an early onset, slowly progressive form. And it's more common in early onset patients, where in that group, it accounts for about 10% to 20% to these patients have mutations in this gene. And the other two listed here are also more rare and associated with an early onset. So why is it important to discover these genes? This is a slide that I borrowed from John Hardy. And it's very, very current. Because what he tried to do was he took some of these park genes by their other names and tried to see how they might be interrelated. And it seems that many of them fall into two pathways, either the lysosomal pathway or the mitochondrial pathway. Actually, both are probably critical in PD pathogenesis. And as we're discovering these genes, we're learning more and more about these pathways. In fact, where I'm going to focus is on GBA. So GBA is the gene mutated in Gaussier disease. Going back full circle. Gaussier disease is the most common of the lysosomal storage disorders. It's also the most common inherited disorder in the Ashkenazi Jewish population. It's caused by the deficiency of glucoserebracyase, which is the enzyme that cleaves this glucose moiety off of the lipid glucoserebracy. It's a disorder primarily of the reticular endothelial system, where lysosomes within macrophages become engorged with the storage product. And most of you from medical school probably might only remember this one image of Gaussier disease, because it frequently appeared on board exams. This is the classic appearing Gaussier macrophage. And it's defined as having sort of a wrinkled tissue paper appearing cytoplasm. And it's so chuck-full that the nuclei often get displaced laterally. When you look at these cells by EM, it's quite remarkable also.  But in these patients, these become so engorged with lipid that they totally get distorted. And this is a Gaussier lysosome. So the history, I think, is kind of unique. And I always show the slide when I speak to audiences with students, because it was described by Philippe Gaussier while he was a medical school student. In fact, it was his medical school thesis. And this was in 1882. It took almost half a century before they realized the nature of the stored material, that it was glucose-reverside. And much of the progress in research, this may be my personal perspective having spent a career in the clinical center, but I do think that this is sort of a poster child for research that happened at NIH. Because in the last 40-some years, 50 years, many of the major advances came out of the clinical center. Beginning with identification of the enzymatic defect by Roscoe Brady in the 60s, the gene was first cloned by Ed Ginz in 1981. Enzyme replacement, the first effective therapy was developed and went through clinical trials at NIH. First animal models were created at NIH. And this association with Parkinsonism also. Well, what kept me interested in Gaussier disease now over two decades has been that there's really vast clinical heterogeneity in this presumably single gene disorder. It's been grouped into three types, type one, type two, and type three. They're very generalized groupings and they're basically based on, type one has absence and type two and three, the presence and rate of progression of neurologic manifestations. So by far, type one Gaussier disease is the most common form. It is very heterogeneous in itself. It can present at any age with variable progression. Common signs and symptoms are hepatosplenomegaly, bone involvement, anemia and thrombocytopenia, though there can be other organs involved. And while some of our patients are fairly incapacitated even in childhood, on the other end of the spectrum, we know there are many asymptomatic individuals. In fact, just calculating back by the gene frequency, we suspect that more patients have Gaussier disease that are not recognized than those that reach medical attention. Like all forms, it's autosomal recessive and panethnic, but it is more common among Ashkenazi Jews where the carrier frequency is about one in 14 to one in 16. The most common finding is splenomegaly, most often painless splenomegaly, and these spleens can be enormous, sometimes brimming over the pelvic rim. The most, one of the more common causes of morbidity is skeletal disease, and there can be fractures, osteopenia, a whole gamut, but the classic finding is this Erlenmeyer flask deformity of the distal femur, which radiologists consider almost bathenomonic. In contrast, type II Gaussier disease fortunately is a little more stereotypic and far more rare. It usually presents at a few months of life, though we also determined that it could also be a disorder of the neonate, and almost uniformly it results in death by two or three years of age. The common signs and symptoms include a pattern of splenomegaly, but babies also develop seizures, neurologic deterioration that's progressive and devastating, and this is pan-ethnic and fairly rare. Usually I imagine there's a couple dozen cases each year in the United States. Type III, again, is heterogeneous because it's sort of a waste basket of anybody who has any form of neurologic manifestations that didn't die in early infancy. So the neurologic involvement is pretty diverse, but pretty uniformly, most patients with type III Gaussier disease have slowing and looping of the horizontal saccades, which is very characteristic. They also can develop a taxia dementia and myoclonic epilepsy. The degree of visceral and bone involvement is variable, but it can be vast, as you can see from this young man here. And again, this is pan-ethnic, though there's thought to be a geographic isolate in the northern most regions of Sweden where it's seen a little bit more frequently. So having worked on this disorder for some time now, I've actually begun to think of it much more as a continuum of phenotypes, ranging from, again, asymptomatic octogenarians that are just diagnosed by accident when they come in for something else late in life or at autopsy even, to fetuses that succumb in utero and a whole gamut of presenting phenotypes that come out in between. In fact, perhaps the major distinction is that some patients have brain involvement and some don't. And what we're going to focus on today is this part of the spectrum, those that develop Parkinsonian manifestations. So the gene I mentioned was cloned in early 1980s. It was pretty quickly realized that there was a very homologous sequence known as a pseudogene, which is a copy of the gene that was located only 16 kilobases downstream on chromosome 1q21. The pseudogene sequence shares 96% of the sequence of glucose ribosidase. And this is important for two reasons. One, when you're doing the molecular diagnosis, you need to make sure that you're looking at the gene and not the pseudogene. And also, because having this sequence nearby has been the source of different mutant alleles that we encounter in patients. To date, more than 300 mutations have been identified in patients. They include point mutations, frameships, deletions, and insertions. I tried a few years ago to graph the distribution of these mutations according to, it's a fairly small gene, so it spans 11 exons and the mutations encounter almost throughout the gene. Some are more common and some are private. Some of the more common ones I've shown here. The ones that are denoted in pink are ones that are actually sequence that you would see in the pseudogene that somehow has been incorporated as a mutation into the gene itself. And two that you'll hear of more frequently are M370S, which is the most common Ashkenazi Jewish mutation and probably the most common Type I mutation worldwide. And L44P, that's often associated with some neurologic manifestations. So how can you explain such heterogeneity in a single gene disorder? It's long been known that neither the amount of lipid that's stored in these patients nor the amount of residual enzyme activity and all patients that live have at least some residual enzyme activity, but the amount doesn't correlate well with the patient's phenotype. So when I came to NIH in the late 80s, I saw that some gene had just been cloned and sequenced and what I would do is learn how to find mutations and then I'd be able to explain that the severe mutations cause the severe disease and the mild mutations where there is some dramatic patients and it would all be wrapped up and I'd finish my fellowship and move on. But it turned out that it became a lot more complicated. There were some correlations, but we found that clinically different patients can have the same genotype. Conversely, clinically similar patients can have many different genotypes. In fact, siblings with the same genotype can have different clinical manifestations and responses to therapy, even identical twins. So it kept me in business for many more years to come. One of the examples is a paper that we published in 2005 where we evaluated at the Clinical Center 32 patients that were all homozygous for this mutation, L44P. And among the 32, they all had that slowed horizontal, saccotic eye movement so they would all be considered type three. But the degree of systemic and neurologic manifestations was highly variable. We had patients with severe psychomotor delay, even autism, and yet on the other end of the spectrum we had successful college students. In fact, one of the patients actually played a jeopardy for several seasons on television. So the residual enzyme activity also was quite variable among these patients that all shared the same genotype. These are just four of the 32, just showing the spectrum of what we encountered. So I think that by focusing on cohorts where they have the same genotype and also siblings where there's a difference between genotypes, it may give us some insight into some of these factors that modify a single gene disorder. So just to rehash, the distinguished, the two different groups that were once considered simple Mendelian single gene disorders and complex disorders are blurred by many different things that could include molecular chaperones, post-translational processing of protein, modifier genes, et cetera. So now I'm going to go and begin my story of the relationship of Gauchier disease and Parkinson disease. And I was asked to begin a little bit with a case report. So this was actually the first patient that I saw back in 1996 that began the whole story for me. She was diagnosed with Gauchier disease in her late teens, was mild, she did have a splenectomy but had few problems with it and pretty much was not treated and had not really thought about her diagnosis. And when she was 42, she developed a tremor with progressive rigidity, mast facies, difficulty initiating movements and rapid deterioration of her gait. She was followed in New Hampshire. They actually tried a paladotomy with no improvement and they also tried the enzyme replacement therapy for Gauchier disease, again with no improvement of her Parkinsonism. We saw her first at NIH, I believe when she was about 49, I'm gonna see if I can get the, you have to go back to, there you go. This is an old video when we first started doing this but you can appreciate her mast facies, her rigidity, her difficulty initiating movements, rapid tremor. So we followed her for another five or six years. She developed progressive dementia and she died at age 54. So we first thought that this could very well be a coincidence. After all, having a rare disorder does not make you immune for having another more common disorder. But we started, when I went through the literature and talked to other colleagues around the world, it seemed that all the Gauchier centers seemed to have more cases of Parkinson than you would have imagined. So we started to try to collect those cases and in 2003, we published a series of 17 such patients. They included Ashkenazi Jewish patients and non-Jewish patients from countries around the world, males and females. And they had many different genotypes. That in 370S was common but it was not necessarily shared by everyone. Clinically, when we looked at these patients that had Gauchier and Parkinsonism, when we looked at their Gauchier manifestations, most had relatively mild Gauchier manifestations but their Parkinson manifestations were more classic, always including tremor, bradykinesis, rigidity, mask, facies, and some actually had cognitive decline or dementia. When we looked at the age of diagnosis of Gauchier disease, it was relatively late at 35 as the mean. When we looked at the age of onset of Parkinsonian symptoms that was relatively early, 48 years of age, the dopest response was mostly favorable with somewhat variable. None who received the enzyme replacement therapy had any improvement. And interestingly, some of these probands had siblings or parents that had Parkinson disease. Well, that observation made us think that we should explore what's going on in these families. We asked whether carriers could also be more susceptible to Parkinsonism. So for a period of about 18 months, we did a prospective study closely interviewing all Gauchier probands that came to the clinical center. There were about 45 of them. And interestingly, about 12 had relatives with Parkinsonism. Often this was a parent or a grandparent who was either an obligator known to be a Gauchier carrier. And then this was explored in other centers, especially the Gauchier clinic in Jerusalem, where they found about the same finding, about 25% of Gauchier probands had a first degree relative with Parkinsonism. So this led us to propose that heterozygos might also be at an increased risk for Parkinsonism. One of my favorite stories was when we interviewed the parents of this child who was a child with about seven with type three Gauchier disease. As I was taking the history from the mother, I started to ask about the father and the mom said, oh, he's here at NIH, but while we were here, he decided to go enroll in a familial Parkinson disease protocol that Bob Nussbaum was conducting. And so Bob enrolled him and actually went out to visit his family. And there were several generations of Parkinsonism. And when he went to visit, he collected DNA on the people that have circles around their symbol. And those that had the Parkinsonian manifestations were all L4-4P carriers, whereas those without clinical symptoms did not have any mutations in gluca cerebrosidase. So this encouraged us to continue in this direction. So then we decided that the next chapter sort of went on in patients with Parkinson disease that had no known Gauchier disease. And again, this was sort of a serendipitous finding. Several years ago, a student who had been a Howard Hughes medical student in our laboratory back in the, I believe early 90s, called me and said that she was now chief neuropathology fellow at Mass General and she was doing an autopsy of a patient with Parkinson disease. She read on the chart that the patient also had Gauchier disease. She googled them, discovered that that was something I was interested in and was calling me to ask if there was any tissue or anything that she'd like me to say. And I was thrilled and I said, but meanwhile, could you get me a couple age-matched Parkinson subjects so that if I do any studies I'll have a comparison? So the three tissue samples arrived in the laboratory and we just decided that we should confirm just to make sure that nothing got mixed up. And when we measured the enzyme activity, one of them was very, very low, but the other two were lower than we expected. So I said, what the heck, go ahead and let's go get the brain samples and extract a little DNA and we'll sequence it just to confirm which is which. Well, it turned out that the patient with very low enzyme activity had Gauchier disease. It had two copies of N370S, which is a common mutation. But the two that were supposed to just be plain Parkinson disease both also had one copy of a mutation in glucosyriversidase. So we got very excited and we went back to this brain collection and then to several other brain banks that are collected around the country and in a few months had assembled 57 brain samples for patients with the pathologic diagnosis of Parkinson disease, extracted DNA and sequenced them. And eight of the 57 carried mutations in glucosyriversidase and we went back and said, you know, is this a coincidence? Could this disorder be more frequent? So we actually asked for controls from the same brain banks, didn't find any mutations among the controls. Now I have to say that this was not an easy finding to publish. Everybody had objections to it. One of the problems was we didn't know ethnicity of the brain donors and they thought there might be a bias towards Ashkenazi Jews or I can't even remember all of the complaints that I had but I think I sent the manuscript out about to spy for six different journals. But eventually the finding has come accepted and it really has propelled the community studying the genetics of Parkinson disease in a new research direction. And there have been many different replication studies published around the world. Part of the skepticism of the initial studies I think was because when they said there wasn't enough power, people that study common disorders want to have large, large, large cohorts and also because large genome-wide association studies that were being conducted around the country on Parkinson disease had never picked up this gene so they thought it must be some kind of mistake. But as I said, replication studies kept coming on. These are some that just were in a few years, some of them quite large studies and in all of them you can appreciate that the percentage of Parkinson patients that had mutations in Glucose rebrisidase was much higher than in their control populations. In fact, I was really gratified one day to be Googling Parkinson genetics and I got to the Michael J. Fox website which apparently kept a list of the top 10 Parkinson genes of the time and Glucose rebrisidase was now on the number one place and this is still before many people believe the whole story. In fact, one of our initial skeptics, John Hardy, who's very well known in the Parkinson genetics field, wrote an editorial where he conceded that the Glucose rebrisidase example was an illustration of how an important genetic risk factor for a complex disease can actually evade detection by systematic analysis. It only came into the radar because of clinical observation and for us clinicians, those kind of statements are incredibly rewarding. If you just go through Medline right now, PubMed and look at the number put in Glucose rebrisidase in Parkinson, when we started this endeavor, there were one or two case reports, but right now there's over 200 papers that's almost rising exponentially. Part of what propelled this and got it defining to be so widely accepted was a study that we put together a few years ago where because everybody was complaining that we didn't have power in any of these individual studies, we assembled a collaboration of 16 centers around the world that had been looking at Glucose rebrisidase mutations in Parkinson disease and putting the data together and this included centers from four continents, marked some of them on the map, some of them had a few cases, some of them had large numbers, some of them sequence DNA, some of them screened for a few mutations, but when we put them all together, we had over 5,600 cases and we had about 5,000 controls. And the bottom line was that around the globe, subjects with Parkinson disease are at least five times more likely to have a mutation in this gene, giving an odds ratio of 5.43. Now, I mentioned that the way they identified mutations varied from place to place. There was a subset of almost 2,000 subjects where the entire Glucose rebrisidase gene was sequenced and in that group, actually the mutation frequency was higher, it was almost 7%. And interestingly, when we looked at those mutations, we got different distribution of mutations depending on ethnicity, but importantly, a lot of groups that only screened for N370SNL44P because it was easier to just screen for two than to have to do whole gene sequencing on large numbers, but if you only focused on those two, you would fail to detect at least 42% of mutations in non-Ashkenazi Jewish cohort and this is important because I think that the results of this study were probably an underestimate. We also looked at the clinical characteristics where we were provided the clinical information and that was a subset of about 3,000 patients. When we looked at different aspects of Parkinson disease, including whether it was a symmetric onset, whether there was Bradykinesia tremor, all kinds of findings, those in blue are those that had the GBA mutations, those in red are those without, and the profile was really not that different. Where we did see some differences was, we heard that there was a family history of Parkinson disease more frequently among subjects that had mutations. I guess that could be expected. Response to Aldopa was slightly less good, but statistically significant and there was a tendency to see more cognitive changes among those that had mutations and in fact, I think that many subjects with cognitive decline might have been excluded from this initial study because the diagnostic criteria for Parkinson disease might have, they might have been excluded and had been considered to have other forms of dementia. The other interesting finding was that the patients that had mutations had an earlier onset of Parkinsonism mean of about 4.3 years. So the conclusions of the study was that subjects with Parkinson disease have at least five times more likely to have mutation in this gene, that the screening techniques were crucial, that you can't just get away with screening for two common mutations. The age of onset was earlier, but in general, the Parkinsonian symptoms varied a lot and didn't differ greatly from subjects that didn't have mutations. And I think that by the end, everybody was willing to conclude that mutations in this gene are the most significant genetic risk factor for Parkinson disease identified to date. Well, it did come out of the New England Journal, but I didn't impress my at the time 19 year old son who was a computer guy. And my findings only reached his attention when it was discussed in Wired Magazine, which had an article about 23andMe. This is a direct to consumer website where they were collecting subjects with Parkinson disease and doing screens and they looked at Lucas Reversides and found that in a matter of minutes they could replicate our findings and actually had very close findings to what we had initially seen. So I always like to pause at this point and say yes, but the majority of patients that we see with Gauchier disease and the majority of Gauchier carriers do not develop Parkinson disease. So somehow this mutation in this gene must be a risk factor, but not predictive of developing Parkinsonism. And this for patients, especially our patients that are taught about recessive and dominant inheritance, it becomes very complicated to explain. And what I try to say is that when we look at the patient and that's examining the phenotype and we usually think of a model and say in Gauchier disease, mutations in a single gene result in the phenotype, but it's got to be a lot more complicated than that. There's got to be modifier genes, sometimes multiple genes, health factors, lifestyle and environmental exposure that all compose the kaleidoscope of what makes up what we see clinically in our patients. So I mentioned how this has become a finding that's difficult to talk to the Gauchier community about because it's still a little bit hard to know what it means for them. So what the question I'm frequently asked is what is the frequency of Parkinson disease in patients with Gauchier disease and Gauchier carriers? And it was kind of hard to assess. There is an international Gauchier registry run by the company that treats patients and they tried to look at their patient group. I think they have about 5,000 patients registered. They found Parkinson disease and reported in 68 of 1130 patients over age 60, deciding that the probability was about 5.7% by age 70, nine to 12% by age 80, but that still says that you can get to age 80 and 80 some percent or plus may not never develop Parkinson disease. And there was no predictive Gauchier profile. There's been other studies from individual clinics and I think that we still need better and bigger studies, but right now we can sort of assume that the lifetime risk is about five to eight times higher than the general population. So in our group at the NIH for over a decade now we've been following clinically patients with Gauchier disease and Parkinsonism. We find that these patients can have classic Parkinson disease, a more aggressive early onset form or familial form and we kind of have seen it all. These are three patients that I picked out. One professor who was only diagnosed in his late 60s. Oops, we've followed him now for over a decade. He hasn't exhibited any neurocognitive changes. He's a patient with an early onset and then one who onsetted in his 50s who pretty rapidly started to develop cognitive changes. We see multiple genotypes, so N370S is somewhat common. Most of them are responsive to Aldopa. None of them have improved when they were treated for Gauchier disease. Smell, olfactory testing and cognitive testing indicate that impairment is relatively common in this group. Among the first 12 that we published, the mean age of diagnosis of Parkinson disease was about 49. The mean UDPRS score was about 26. So now we are prospectively following these patients both studying, looking at their clinical features and doing some PET imaging. The goals of the study and the physician and coordinator who are working on the study are here in the audience today if people have more specific questions. It's being conducted in collaboration with Karen Berman and NIMH. And our goals are to better characterize the Parkinson phenotype associated with glucosuribrosidase to look at fluorodopa uptake and evaluate PET as a surrogate marker in these subjects and to see if we can establish the earliest signs of Parkinson disease and at-risk individuals. So with that in mind, we recruit patients that have both Gauchier disease and Parkinson disease. We have some controls that have Parkinson disease alone. And then we're looking at patients with Gauchier disease who don't have Parkinson disease but have a family history either in a parent or sibling and also some carriers that also have the strong family history. When patients come to us, they undergo a pretty uniform physical and neurologic examination. They undergo neurocognitive assessment by a single neuropsychologist. They do the scratch test for all factory testing and they're given multiple surveys looking for different non-motor symptoms of Parkinsonism. They also have imaging studies including MRI for structural abnormalities. They're all given fluorodopa PET scanning and water studies looking for cerebral blood flow patterns and also transcranial ultrasonography looking at the midbrain structures. We've recently published our first set of PET studies. This was done on about 44 patients and we looked at both fluorodopa PET, which this is a summary of what we do but they're not individual studies here, they're composite studies where you put the entire cohort together. So this is a group that just has Parkinson disease and this is a group that has Gauchier disease and Parkinsonism. And if you don't see differences that's the correct observation. It turns out that the pattern of dopamine loss in these two groups seems to be similar. Where we did see some differences was in the flow studies. These are rendered templates of the brain where we show areas where there's a difference in the two different groups. And it turns out that there's less activity in areas that are affected in neurodegenerative disorders like Alzheimer's, which may in part explain some of the cognitive impairment that we're seeing in the patients. Interestingly in 14 patients with Gauchier disease and seven carriers who had no Parkinson disease themselves but of positive family history only two showed any evidence of dopamine loss. So that might be somewhat reassuring to some of our patients and it definitely needs longitudinal follow up, excuse me. We've also been able to perform some neuropathologic assessments. In fact, that first patient where I showed you the video was the first autopsy that we were able to get done at the NIH. These patients do have Lewy bodies. They stay in positive for Alpha-Synuclein as you'd expect. And they also have Gliosis of hippocampal layers CA2 to CA4 which are regions that are also affected in some of the Lewy body disorders. When we used some of these brain samples to explore mechanism, we did this immunochemistry studies where we stained the tissue both with antibody to Glucose Reversidase which would appear as red and Alpha-Synuclein which is green. So when we looked at brain samples from patients with Parkinsonism that had mutations in Glucose Reversidase, we saw staining in the Lewy bodies both for Alpha-Synuclein and Glucose Reversidase. When we looked at Lewy bodies and subjects that had Parkinsonism that didn't have mutations, we didn't see the staining or saw very little of it staining for Glucose Reversidase. So we think that may be an important clue as we explore pathogenesis. Now another, clinically we were observing, as I mentioned a few times, that there seemed to be some more cognitive changes in the group that had mutations. This prompted us to go back and do a second multi-center analysis this time of patients that had the diagnosis of dementia with Lewy bodies because this disorder is more associated with neurocognitive changes. This is much rarer. We went back and collected data from 11 centers. There were 720 cases with dementia with Lewy bodies and a large number of age-matched controls. Of the 721, 450 were autopsied and had autopsy confirmation of the finding. And 80% had full Glucose Reversidase sequencing. And the association was really quite significant. The odds ratio this time was over eight. When we looked at this cohort again, the age of diagnosis of mutation carriers was about five years earlier than those without mutations. And the mutations were associated with higher PD scores. So it seems that mutations in this gene may even play a greater role in dementia with Lewy bodies than straightforward Parkinson disease. There have been pilots that he's published in multiple system atrophy and essential tremor but they have not shown the similar association. So why would being a carrier for one genetic disorder put you at risk for an unrelated illness, the million dollar question? Well, at first I thought that this was an extreme situation but thinking about it and talking to people we sort of came up with a list of other situations where having a mutation in one gene that was known to cause a disorder could put you at risk for another complex disease. And I think that, thinking about this, it may not be such a rare phenomena. Glucose Reversidase now fits in that list and those of you who follow the New England Journal just last month there was a report of TREM2 which is a gene that is associated with a neurodegenerative disorder and bone cyst disorder being a risk factor for Alzheimer's disease. Mutations in this gene are seen four times more commonly than in the general population. So how might this be happening? Well, most of our mutations lead to a misfolded protein and this conformational change in protein can have important consequences. First of all, it can lead to an unstable protein which is degraded leading to the functional defect which is what we see in Gauchat disease. You have degraded protein, you don't have the enzyme, you have the phenotype. But it's also possible that you could have a change in the protein and come up with another stable protein with a new function. This is known as the dominant negative effect or having this mutation could lead to alterations in transport or protein-protein interactions leading to organelle dysfunction, traffic jamming as the newly formed protein is processed through the cell or it could cause aggregation of a protein, all three of these leading to what we consider a gain of a toxic function. So in this example of glucosireberosidase, we know that the formation of insoluble alpha-synucreate aggregates leads to the neuronal cell death that's seen in patients. So perhaps having this mutant protein around could increase aggregate formation or lead to organelle dysfunction, particularly in the lysosome, leading to decreased clearance of aggregates. And this is what I'd consider a gain of function hypothesis supported by the fact that mutations are seen in heterozygotes as well as homozygotes. And also by what I showed you where we saw that there was glucosireberosidase present in Lewy bodies in these patients. But the alternate hypothesis is that having mutation can lead to the unstable or deficient protein, which is degraded. You don't have enough enzyme. You have lipid accumulation. And then it's this accumulation that provokes neuronal cell death. And this is more a loss of function hypothesis. And it's supported by the fact that some of our patients actually have no alleles. They actually have mutations that don't make protein. So it's hard to describe a misfolded protein when one's not made it at all. And also when you use inhibitor to glucosireberosidase called CBE, you see differences in alpha-synuclein. So the jury is still out on how this plays to be. There was an important paper that came out about a year ago from Boston showing that in cell that we actually collaborated with where they showed using all kinds of things, silenced RNA, neuronal cells, IPS cells, the elegance, human and mouse brain studies, that they came up with a hypothesis that there's a bi-directional feedback group where having this lipid glucosal ceramide promotes the formation of soluble alpha-synuclein oligomers. Having increased number of these oligomers blocks the trafficking from the ER to the Golgi, which would in turn lead to less enzyme and more lipid causing a self-propelling cycle. I actually think that this is interesting but it still may be more complicated. One of the findings that we published in collaboration with Jennifer Lee's group in NHLBI was that at least in the test tube there seems to be a molecular link between alpha-synuclein and glucosireberosidase. And they showed this by a lot of fancy techniques including fluorescence measurements where you see that when you put the two proteins together there's a shift, NMR, where you see the shift actually indicating that the interaction is at the C terminus of alpha-synuclein. And then we actually did some pull-down experiments and brain extracts showing that glucosireberosidase can be immunoprecipitated together with alpha-synuclein. Now all three of these findings were only seen at pH 5.5, which is closer to the lysosomal pH, and not at pH 7, not with mutant glucosireberosidase and not with other lysosomal enzymes. So we sort of put this together as a model with this cartoon where we postulate that perhaps the C terminal of alpha-synuclein interacts with glucosireberosidase, that's this glowing region, whereas the N terminal helix could bind to the glycolipid rich vesicles in the lysosome. Jennifer's gone on to establish that membrane-bound alpha-synuclein interacts with glucosireberosidase and actually inhibits its activity. So we speculate that maybe this binding at lysosomal pH could facilitate alpha-synuclein degradation, have a beneficial effect, or prevent aggregation. What we found is that's really needed to propel these studies or some better models. One of the problems is that the mouse models that we have there don't really mimic the human phenotype. Skin fibroblasts, which we have over the years collected on our patients, are useful for some things, like extracting DNA and RNA and protein studies, but they don't show the lipid storage that you would need in a model. So that prompted us to explore using patient-derived induced pluripotent stem cells or IPS cells. IPS cells are reprogrammed skin fibroblasts that can grow for extensive periods and be used to form any type of cell. And this was our first IPS cell colonies from a patient with type two Gauchier disease. We've worked to, working to develop this, and it's right now in progress. We've managed to make our first IPS cell lines and differentiate them into first Gauchier macrophages. And to our delight, we were able to show a storage phenotype. This is a control IPS cell. This is a Gauchier IPS cell. When you feed them ghost cells that are labeled, you can see the lipid accumulate only in the Gauchier macrophages and not in the controls. We've now begun to generate neurons and we're in the process of establishing cell lines from patients that have both Parkinson disease and Gauchier disease. And these show that markers for neurons are positive in these IPS derived patient neurons. So how do I fit this all together? I really still don't know for sure. We know that alpha-synuclein is degraded by the proteasome or mitochondrial pathways and also by lysosomal pathways. And I think since this is a lysosomal enzyme, our part of the story has to do with the lysosome. So let's speculate that when the lysosome is functioning normally, you somehow need to have wild-type glucosaribrosidase and alpha-synuclein and somehow they come together and the net result is that the alpha-synuclein is degraded like it needs to be. However, when you have mutant glucosaribrosidase around, speculate that you have, of course, diminished glucosaribrosidase activity and less protein around. And somehow the net result here is that less alpha-synuclein is degraded and it accumulates in aggregates contributing to neuronal cell death. Now if this is the model, why doesn't every patient that have Gauchier disease go on to develop Parkinson disease? Well, it's gotta be a lot more complicated than that. But we know that Parkinson disease is a disorder that occurs as we begin to age. And with aging, the number of lysosomes that cells have just go down, they function less well. And also the amount of alpha-synuclein we produce goes up. So perhaps there's this homeostasis necessary and having mutations around the stirubs that I think that will still have a lot of research ahead. And I just want to, for a few minutes, turn to implications for therapy of these findings. So I mentioned a few times therapy for Gauchier disease. There is an enzyme replacement. It has been very successful in improving quality of life, especially in patients with type one Gauchier disease because it improves hemoglobin and platelets and it shrinks spleen and liver size. But it's incredibly costly, ranging from about 100 to 300 or $400,000 per year per patient. It's a treatment, it's not a cure, so you have to stay on it. The response is highly variable. It's an IV preparation, so it's inconvenient. And importantly, it doesn't cross the blood-brain barrier, so it doesn't help any of the neurologic manifestations associated with the disease. So we've been working with the new, not new now, but the then new NIH Center for, the NCGC, the NIH blocking on the name, Center for, somebody help me, what is it called? NCGC, chemical genomics, gotcha. Okay, so when we've been working with Chris Austin and Wazing several years ago, we developed our first screen and had a nice paper in PNAS. What we were looking for are what's known as chemical chaperone, thinking that chemical chaperone therapy may be very advantageous, first for Gauchier disease. Because in Gauchier disease, we know that glucosuribrosidase is synthesized in the ER. It's glycosylated and folded, but it doesn't attain its functional tertiary status structure until it gets into the lysosome. However, mutin glucosuribrosidase, when you have a mutation, can be, as I mentioned, just misfolded and degraded. So that if you can find a small chemical that binds to the active site and enhances folding, you may be able to deliver it to the lysosome where it can do its work. And we think that this might correct the enzyme deficiency in patients with Gauchier disease. So we approached it with high throughput screening where our goals were to identify activators or inhibitors of the enzyme and that would work as pharmacological chaperones. When you work with the NCGC, what you have to do is first have an assay that's miniaturizable. They all have to be done in a 1536 wealth format, which takes some time. Fortunately, we could miniaturize the assay because we had the commercially available enzyme and we had a fluorogenic readout. So once we had the assay, we provided the assay and the enzyme to the NCGC. And they, with robotics, can screen seven concentrations of these libraries. When you've started with 60,000, I think we're up to half a million compounds. And robots screen plates of compounds that have different concentrations. And it's really amazing. This is something that a postdoc would probably not be able to do in a decade. And we can get results in a matter of hours to days. We then came up with a new screening approach that was published in PLOS this year where instead of using the commercially available enzyme, we thought we should screen mutant enzyme, but we didn't have that available. So what we did is we took a frozen patient spleen sample and used that as the source of mutant enzyme and performed the screen of about a quarter of a million compounds and this time we actually found some really interesting compounds, both activators and inhibitors. Our lead compound is considered a non-inhibitory chaperone. And I'll show you, we believe that it can reverse lipid storage and enhance enzyme activity. So we hope to be able to develop this further. And I suspect that the small molecule therapy may stabilize or activate glucosyriversidase to treat Gauchatia disease, but it could also have implications for glucosyriversidase-associated Parkinson's disease. And actually some of the companies are now looking to this. Now the screens are just one step of the process. First then they have to, the compounds have to be confirmed and then you have to optimize the chemical structure and the medicinal chemists become involved and then you provide functional studies to determine cell toxicity from the compounds and then you need to have a phenotype that's corrected by the compounds and we've been working our way along this process. So with our lead non-inhibitory chaperone we show with four different assays that we increase the enzymatic activity. When we go to patient fibroblasts, these are patient fibroblasts treated with DMSO and these are patient fibroblasts treated with this compound. Green is glucosyriversidase and red is LAMP, which is a lysosomal marker. So in our patients we don't see very much glucosyriversidase going to the lysosome but when we add the compound we see this yellow which shows that we are seeing transfer to the lysosome. The compound appears to be selected for glucosyriversidase and it didn't activate other hydrolysis that we tested and to our delight we were able to make use of our new models, both macrophages collected from patients and IPS derived macrophages. So these are non-treated. This is, oh, sorry. This is a control line. This is patient macrophages and this is IPS derived macrophages. So we're looking at storage of the lipid and you don't see it in controls. We add these fluorescently tagged ghost cells to increase the amount of storage that we see. We still see very little in controls but we see a lot of it in the patient macrophages and then when we add our compound we're seeing that the storage is cleared which is nice and reassuring and hopefully that will help us get to the next stage of development. So I wanna go back and say that understanding the links between these two disorders is very rewarding and frustrating but I think that it will ultimately teach us about the pathogenesis of both disorders leading to improved genetic counseling and better therapeutic strategies. So I hope that you can appreciate now that the complexity that we're seeing in Mendelian disorders like Gauchier disease may ultimately give us a nice window into other complex disorders. So I wanna acknowledge a lot of different people. First of all, my group who's done all the work. Many of whom are shown here are listed. Many close collaborators both at NIH and around the world and I always like to give special thanks to the patient's family members and referring physicians who have contributed so much to these studies. Thank you. Thank you. Thank you. We're gonna have a number of questions about one and that is environmental exposures as a possible destination in Parkinson's disease. So that's a difficult question in Parkinson's disease and it's being challenged in major studies. If I could come up with DNA from a cohort that was known to have a strong environmental exposure, I would really like to be able to sequence glucose-reversidase to see if there's some association but at this point it hasn't been done. Other comments or questions? You can compare and raise the question. Oh, okay. And individuals have a strong family history and yet have no manifestations of either the Gauchies or the Parkinson's. And is it not only because they don't have the abnormalities or have you studied them, perhaps they have a resistant gene that could protect them from this and use that observation? So the question is patients that have the mutation but don't have either disorder, is there a protective phenomena? I won't say that patients that have two mutations in the glucose-reversidase gene don't have Gauchier disease, they just might have a very mild form because by definition if they have two mutations, they have the disease. But the other question is really important, is there some kind of protective gene? And one way we're hoping to look at that is we've been trying to collect Sib pairs where both patients have Gauchier disease but only one developed Parkinson's disease to see if we can tease out what's protecting the one that didn't get it. But at this point, we don't know. Can you tell us what chemical class or compounds that you've identified? Well, I'm not the chemist, I don't even remember the name of it, but it has been published. I can give you the reference. Yeah. Other comments or questions? If not, please join me in thanking Dr. Walsh.


How's it going everyone and welcome back to another video. So one of my favorite companies in the world has reached out to me to test out the new Rode Vlogger Kit with the Rode Wireless GO 2. I've been using their products ever since I started making videos and always had a great experience with their products. So I'm quite excited to share with you guys this filmmaking kit setup for mobile phones. Now smartphones have come a long way and most of the smartphones out there are capable of shooting high quality videos. So if you're someone that is considering getting into mobile vlogging or generally wants to capture great content with their smartphone then make sure to stick around as this could level up your mobile filmmaking skills. So Rode has sent me the Vlogger Kit iOS edition since I'm using the iPhone 12 Pro Max but if you're using an Android they also have a USB type C edition and for devices that have a 3.5 millimeter jack you also have the option to choose the universal Vlogger Kit. To give you an idea I will first talk about what's in the box of the Rode Vlogger Kit and Rode Wireless GO 2. Show you how I set it up with the iPhone 12 Pro Max, some test footage, pricing and my final thoughts on it. Keep in mind that all the products mentioned will be in the video description below as well as a timestamp if you want to skip or go back to a particular part of the video. With that said, let's jump into the video. I would first like to start off by showing you what's in the box of the Rode Vlogger Kit. So you will get the Rode VideoMic Mi L which is a directional microphone that helps capture better audio by reducing the noise in the background. You will also receive a windshield that helps block wind noise and the built-in microphone on smartphones produce decent audio quality but when facing windy conditions outside you won't have the option to add a windshield to the built-in microphone and therefore will have unwanted noise in your audio. For me, good audio is very important as it's half the viewing experience even if the video quality isn't that good. You will also receive a rubber clip to better support the microphone, a smart grip made out of metal to mount your smartphone and a tripod for handheld or static shots. The tripod also has a ball head for positioning your camera in different angles. You also get a micro LED on camera light to better light your shots as well as a diffuser for a more softer lighting. You will also get eight colored filters for creative use and a USB-C cable to charge the LED light. Rode has also provided me with the Rode Wireless Go 2 for a hands-free setup which is a compact dual-channel wireless microphone system. The Wireless Go 2 is not included in the Vlogger Kit and has to be bought separately but what I really like about it is not only the great audio quality but also the small size and how extremely versatile it is to use. But I will talk about that more later. In it, you will receive two transmitter, one receiver, three furry windshields, a USB-C and a USB-A cable, a TRS to TRS cable for analog outputs and a carry pouch. So as you can see, I've already set up the iPhone 12 Pro Max with the Rode Vlogger Kit and to show you how I've set this up, basically I have the Rode VideoMic Me L plugged into the side of the iPhone 12 Pro Max with the windshield.  Now I like to keep things simple and use the standard camera app as I can quickly access this on the home screen. I prefer using the front-facing camera as I can monitor myself and easily set and lock focus and exposure before filming. The resolution and frame rate are set to 4K 25 frames per second because I live in Switzerland, which is in a PAL region. You can enable the video format in the camera settings on your iPhone. So if you live in an NTSC region, I recommend using 24 frames per second. I also keep grid enabled to better set my composition. Now, once the microphone is plugged in, you don't have to do any further adjustments since the iOS has an automatic gain control that regulates the input signals to make sure the audio doesn't distort. You have the option to monitor your audio by plugging your headphones on the back of the Rode VideoMic Me L. So one thing that bothers me is that with the windshield attached, some of the screen is covered over here and usually the record button is right over here. But what you can do is you can easily press the volume key to trigger recording. As for the LED light, there are three output levels. I like to keep it at the first level and I only use the LED light if there is not sufficient light, for example, if I'm filming indoors. Now I always keep the diffuser attached as this produces a more pleasing look on my face. I'm gonna use the built-in LED light so that you guys can see how it looks like. This is level one, level two, level three. This is how it will look like with the LED light on. The LED light lasts around four hours, which is more than enough for my usage. Let me now show you how it looks like with the Rode Vlogger kit. Hey, what's up guys? So I'm in the middle of Zurich. I'm here with George. He's currently setting up his time-lapse and it's quite cloudy out here. I'm in the middle of the road. There are cars passing by me in the background and there's also a construction site going on down there. And that's great because I would like to do a comparison between using the Rode VideoMic Miele, which is plugged in right now, versus the built-in mic so that you guys can see the difference. Future Bennett here. I apologize. The audio recording on the built-in microphone on my iPhone 12 Pro Max didn't go well because I didn't restart the camera app. I actually just plugged out the external mic and then started recording. And that caused the audio to distort. There are a lot of wind noise because it's also quite windy up here. So anyways, this is how it sounds like using the Rode VideoMic Miele plugged in. It's a little bit windy outside. Probably also hear birds singing. But yeah, with the shotgun mic it's more directional. So my voice will probably be more present. So I'm now gonna switch over to the built-in microphone on the iPhone 12 Pro Max. So this is how it sounds like with the built-in microphone on my iPhone 12 Pro Max. It shouldn't sound that bad because there's actually not a lot of wind noise going on. You might hear a little bit more ambient noise. When there's a lot of wind, you should get an external mic with a windshield because that will help improve the audio a lot. So let's now head back. For the hands-free setup, I've set up the Rode Wireless Go 2 on top of the cold shoe mount and connected with my iPhone using the Rode SC15, which has to be purchased separately. I then add the furry windshield, clip the transmitter onto my shirt, turn on both devices, and I'm basically ready to shoot. A second transmitter is also included, which is perfect for recording two audio sources at the same time. If you're traveling with a companion, this could be quite useful for filming yourself and the second person from different distances and maintaining a good audio quality. Okay guys, so we're now using two transmitters. I have the second channel hooked onto George and we should have two audios running right now. So George, what are you doing? I'm trying to shoot a lot of time lapse here, but the clouds are not moving very well. Yeah, that's true. And as I walk away, you guys can still hear George pretty clearly. Hey Bennett, where are you going? See? The Wireless Go 2 can operate up to seven hours and the transmission range is 200 meters, which again is more than enough for my usage. If you additionally want to mount the LED light, you will have to get the Rode DCS1, which is a dual cold shoe mount. When using two transmitter for vlogging, I like to keep the audio split instead of merged, which allows the audio to be recorded separately. I can adjust the settings in the Rode Central app, which is a great feature Rode added for more control over the Rode Wireless Go 2. Also when recording in audio split tracks, I can selectively mute or lower one person's audio. So if you're out of vlogging with someone, both having a mic on, you can decide how you want to hear the audio in your final edit, which is amazing. Another great feature I have activated is the backup recording, which records the audio on the internal storage of the transmitters. Each transmitter can record up to 24 hours of audio. Once turned on, it will automatically start the backup recording, and if the memory is filled up, it will begin deleting older files, which is great. This way I don't have to worry about it. The recordings can be accessed through the Rode Central app, where it can playback and export the files. So using the Rode Vlogger Kit has been a great experience. You have everything you need to film a great vlog. The simple setup makes it very easy to vlog. This way I can focus more on storytelling. I also like the versatility of the setup as I can use the Rode Wireless Go 2 for a hands-free setup and can also include a second person in my vlog and film from different distances while maintaining good audio quality. The idea of vlogging with a smartphone is great because the video quality is good, the setup is fast and doesn't feel heavy and looks more discreet since everyone carries a smartphone nowadays. The Rode Vlogger Kit is available for around $145. The Wireless Go for $299. You will definitely get a lot of value for its price and it doesn't have to be just vlogging. You can use the Vlogger Kit and the Rode Wireless Go 2 for live streams or for your YouTube videos. This package is designed for a high quality video and audio recording to a smartphone. But of course, you can also use the Rode Wireless Go 2 with a larger camera as well. But for me, this setup feels complete. Let me know in the comments if this video was helpful and make sure to leave a like and subscribe so that you don't miss out on the newest filmmaking tutorials. Follow me on Instagram at benergrazer and for those that are new to smartphone filmmaking, make sure to download my free smartphone filmmaking guide where I will show you the tools you need to get started. The link will be in the video description below. Before I end this video, I just wanted to thank you all for being part of this big community. We're soon reaching 40,000 subscribers and this journey has been amazing so far and I get a lot of questions but also messages from you guys saying that my videos helped you a lot and I'm super grateful for that and happy to see that I can somehow help you guys get better at filmmaking. So keep it up. Thank you so much for watching. Stay creative and have a good one.