ステーションを始めますアミューキー、ヒタッチ、ダイキー、シェクトリック今日はここで感謝します今日はここで幸せです今日、私たちのコントロビューションについて話します日本のハーブレッジファーリックのコミュニテーションについてお話しますとにかく、ハーブレッジファーリックは世界中で使用されています私たちがブロックシャンのテクノロジーを使用している人もいます私たちは英語の話をしていますそれから、英語の話を読んでいる英語の読み方があります英語の話をしている英語の話をしていますこのヴァリアーのハーブレッジファーリックのコミュニテーションは地域によって翻訳するお話をしています日本のファーリックのコミュニテーションはこれでもあります今日は、私たちのコミュニテーションについてお話します私たちの絶対についてお話をしていますOKはいおすすめということをお願いしますOK.So after this session, I hope more people will start the translation effort.So the first topic is motivation.Why we are here?Fabric is a great blockchain platform, so many people use it.However, English speakers have had time to keep up withfrequent updated hybrid document.There are two problem cases.The first case is translation tools do not provide enough quality.There are famous translation tools like DeepAir or Google Translate.Those are great tools, but they cannot understand the technical context.So automatically, translation documents do not provide good quality.In other case, some local engineers write blog posts in their local language,but those articles are updated soon because Fabric is updated frequently.For example, some guys write how to deploy Fabric V1.4,but when Fabric V2.2 is released, those documents are outdated.In short, no native speakers have language barriers.To overcome those language barriers,Hyperfabric Documentation Working Group has started initiativeto translate official documents into local languages.The goal is to enable more known English speakers to learn how to use Fabric in their local languages.If the document is available in the local language,more people will adapt hybrid technologies.Under the Working Group, there are 11 language teams.Each local community has their own contributors and they are working on translation.Japanese Working Group is the fourth group.The Documentation Working Group helps start new language translation.They provide good guidelines and communication tools.So, why we translate?I think we have three good reasons.The first one is learning blockchain technology.To translate the document, we need to understand the technology in detail.And the second reason is learning open source contribution.Contributing code is little difficult.So, translation is a good starting point to understand the contribution process and tools.Lastly, we can connect with local community.We can get feedbacks from local guides and we can connect with client companies in local community.These are the motivations.So, the next topic is community development.This graph shows the overview of Japanese Documentation Working Group.Contributors pick up English document and then they translate it into Japanese.And they submit the doc to the JTAR repository and mention the jacket and the document is erased.Contributors follow contribution process and they use communication tools.These contribution process and communication tools are supported byHabitat staffs and the Fabric Documentation Working Group.This graph is the latest version.So, when we start in the team and there is no concrete process.So, we faced a lot of problems.So, we solve those problems and we build community management like this one by Troy and Ella.So, let me share some problems and solutions today.This is the history of our community building.Launching translation working group.Develop contribution process.Standize translation rule and recruit contributors.So, first topic is studying a translation team.Studying a translation team is building a new open source community.However, we do not have enough knowledge and experience for organizing a new community.We didn't know how to build a translation team.How to call for contributors.So, our parent group, that is Hybrid Fabric Documentation Working Group helped us to organize a community.They provided us communication tools and contribution guides.Next, Japanese also helped us.They called for contributors in Japan because they have connections with local engineers.I am one of them.After studying the team, we had a communication program because original members used emails to connect with others.So, communication was closed.So, new members cannot access to communication logs.The other program is English is required to ask questions at FabricDog.ItemChannel.This is also a communication barrier.So, to solve the question, we asked hybrid staffs to create a new channel for JavaScript working group.Thanks to their effort, now we have Weki, Disco, and GitHub Zoom for JavaScript working group.So, we communicate in Japanese and everyone can see the communication log.This is how we solve the communication program.Next topic is setting the milestone.You know, Fabric has various active versions.Which version has to do with it?So, which version is best for translation?To answer this question, we said criteria.The criteria includes LTS version, number of users at blockchain services,and it's for a long time.When we started the project, Fabric 2.2 was the latest LTS.However, most blockchains provided Fabric V104 for their cloud service.So, readers read articles in V104.Concerning the situation, V104 may be a good candidate.But it may not be used for a long time.For the future, we decided to translate V2.2.It was a good decision because now most cloud providers provide Fabric in 2.4.So, it was a good decision.After setting the milestone, we decided the priority of translation because there aremany documents.So, where do we start the translation?To answer this question, Fabric <IP_ADDRESS>.1 group provided as priority guidelines.There are three levels.First topics documents for document contributors.They include contribution guide and grocery.Next topic, next priority.It has high priority documents for developers.For example, key concepts, getting started, tutorials are equaled.The topics for developers.For example, operation guides and architecture reference are equaled.So, we follow this guideline.So, we first translate first topics and satisfy next topics.Now, we are working on others.So, the next step is developing contribution process.Contribution process is important to collaborate efficiently.The first solution is contributor role.Japanese digression working group consists of engineers,fabric code contributors, and professional translators.They had different level of experience about hybrid fabric.So, we need to find a way to collaborate efficiently as one team.So, we introduced three ways for contribution.Translator is a role for traffic document.They don't need to have any requirement.Reviewer checks the translated documents.They need to have previous experience about hybrid fabric.For example, fabric code contributor, fabric application developer,and the certification holder of the explanation.Tweakup support is a role for support using tools.So, each contributor can pick up a role as they like.Translator flow is also important for collaboration.So, we decided a translation flow.The requirement is simple contribution processbecause our community has been about open source contributions.So, we looked for translation process as an open source community.The most famous one was Sphinx.But we gave up using following Sphinxbecause the contribution process is very complicated.Fabric has modified formats like .me and .rstbut Sphinx only supported .rst.So, we gave up using Sphinx.Instead, we introduced more simple translation flow.We just followed Fabric's GitHub contribution process.So, each contributor picks up a file.They translate.And they just keep the document forward.So, it's simple, but we think it's effectively working.And next topic is task management.So, there are many pages waiting translation.So, we need to collaborate as one team.So, we use GitHub cover board.Yeah, I know you are familiar with cover board.So, this event shows the cover board.And the left side is to the tasks.And the next lane is a progress task.And the right lane is task completed.So, each contributor can pick up an issue.And we can share who is working on what tasks and what is left.So, the next step is standardized translation rule.The purpose is to keep the quality and productivity of translation.So, we introduced translation rule book and terminology.So, good documentation has common styles throughout all pages.But we didn't have common styles.So, when a translator submits a request,reviewers had to compare with the previous documents.So, contributors and reviewers had no time to check the styles.But we wanted to focus on the content itself.So, to solve the problem,we introduced a translation rule book.Translator checked the documents before submitting.Reviewers used the checklist as a checking point.The rule book itself is updated anytimebecause we may find some new problems and tips.This use shows the examples of translation rule book.It includes terminology, Japanese style,no broker format, PR template.For example, no broker format is usedto check if the documentation format is not broken.For example, the link to other documentsmay be broken after translation.So, we need to check it.Once example is PR template.We have a rule.One comment has only one pagebecause we want to avoid a large conflict.So, we introduce this rule.And we also have a rule that as a tag.jgpbecause all language groups share only one gt repository.So, adding the tag is usefulto pick up only Japanese document issues.And let me explain the terminology in detail.This is very important topics.We studied the translation.We used fabric grocery to standardize terminology.It contains 47 pairs of English and Japanese words.However, it did not work enough.So, 47 words are not enough to cover all words.So, we initiated a special subgroupfor creating new terminology for Japanese.So, we collected terminologiesfrom previously translated documents.So, we found 463 terms.And we categorized those words into four levels.Level one is the highest priority.Those words are fabric words, like order or endorsement.Level two is production words, like cryptocurrency.Level three is additional terms.And level four is common IQ words.This terminology is still in progress.So, we managed those articles on github.This is an example of our terminology.The left lane is English and the right lane is Japanese.So, continue to check if they use correct terminologybefore submitting a pro request.Create a terminology to introduce another problembecause we have over 400 words here.So, checking all words is heavy work.So, how much to translate those reviewsfor the terminology?To answer this question, we introduced a practical policybased on the levels.So, contributors must follow level one and two.It means that we need to followfabric words and blockchain words.And checking level three and four is additional.So, this is how we balance the qualityand productivity of translation.And the last topic is recruiting new contributors.A year after the launch of the team,the number of active contributors decreased.It often happens at open source quality.So, we need to do how more new contributors.So, we had meetups with Black Spanish Japan.At the meetup, we introduced our translation activityand we shared the very little contributionand explained translation flow.After the meetup, remember to join the meetup.Thank you.He is also the new contributor.So, we are very happy to have you with us.This is the current status of our team.We have completed translating 90% of the document.We hope we will complete all tasks soon,probably by the end of this year.As the translated document is available here.So, if you have time, please check it.And there are many languages here.So, please check it.We have French, Chinese, Spanish, and so on.Okay.So, next topic is translation tips.So, I pass my mic to Daiki.Go ahead.Thank you, Kondo-san.So, from this section,I will explain three translation tips.So, at first, it's the point of translation rule.As we mentioned before, we made a translation rule.So, I introduced the point of a translation tipsand a solution for a language-specific manner.In Japanese, there are two points of view.End of the sentence and punctuation mark.So, both of them, there are many types in Japaneselike punctuation mark, 10.And sometimes Japanese use dot and comma depend on documentslike thesis.It's actually up to people.So, we put a solution for a language-specific mannerto a translation rule.And another thing is a permutationto working members.And, you know, that's the important thingto use translation rule by working members continually.So, we tried many things like share by Wikiand put to review process like that.So, next is a sentence not to be translated.Sometimes it's better to not be translated to local languagebecause sometimes you have a technical problem or contents.And, for example, we have a technical problemwhen we translate link sentence.We couldn't go to jump to your pages.So, we guess it's a language-specific problembecause Japanese is not Latin language.So, we decided that link sentence is not to translate itinto Japanese.And, after that, we put them to your translation rule.You know, it's so important thing.So, last, it's for reviewer to understand easily.You know, Japanese is so complicated sentencebecause conclusion sentence is last.For example, in terms of grammar,Japanese and English is subject and verb and object,while Japanese is subject, object and verb.So, that's why Japanese is so complicated.So, for example, you translate this example sentenceby machine translation, you get left side sentence.So, that line is conclusion sentence.So, by machine translation,it's conclusion sentence is separated.So, we endeavor to try to make sentence shorterand conclusion first.Maybe it's very easy to understand.So, next, challenges for our work better.First, call for more contributors.So, actually, we have only seven active members.So, to activate our working group,we have to recruit more contributors.So, we try to do something like these things.Second, catch up to next LTS version.We know that the next LTS version will be released soon.So, we have to think about how to get new release versioninto documentation.Third, evaluation for Japanese documents.So, we want to know how useful Japanese documentation is.So, we think that we use metrics for page viewers too,any kinds of tool.But, you know, we have some ideas.But, we do not have a specific plan.So, actually, we would like to discuss with you.So, last conclusion.We showed four things, motivation.We want to remove the barrier for no English speakerto understand hyperlature fabric technology.And, through documentation activity,we can understand hyperlature fabric deeply.Community development.So, we tried many, many things to build, develop,and improve constantly for our work better.Translation tips.We showed three translation tips for solution specific problems.So, last challenge.We will try to three things for our work better.You know, our work activity is still developing.So, thank you so much for listening.And, special thanks for relaxed foundation staffs,fabric documentation working group.And, thank you for everything Japanese documentation working group members.Tsujita-san, Shimosa-san, Sato-san, Uchiyama-san, Maekawa-san.Thank you so much.So, thank you for listening.So, if you have any queries, please let me know.Regarding another language, actually, do you know it?The quality?Yes, so I think contribution process is very important for collaboration.So, because if we work on translation by one person, it's very hard work.So, collaborating with other people are very good ways to translation.So, to work efficiently with other guys, contribution is very important.So, Fabric documentation work group provides good baseline,but each language has different situation.So, I think it's a good idea to improve those basic guidelines to fit their own language.For example, Japanese team introduced terminology for Japanese,because English and Japanese don't fit one-to-one.So, we need to decide the appropriate words for Japanese.So, yeah, I think creating the contribution process is important for the language community, too.Welcome.Go ahead.You mentioned that you have tried using Google Translate.Have you tried other translating tools like DPL, for instance?Yeah, we also tried Google Translate and DPL.Yeah, of course, they provide translation documents,but they cannot understand the technical context for blockchain or high-performance fabric.So, sometimes they display weird translation.So, yeah, we need to translate by ourselves to provide good quality documentation for leaders.Property, I understand that Japanese are very particular on details of documentation.So, is it okay to ask you to express your personal opinion about quality of the public documentation?I understand this is a tricky question, but you don't need to answer it.You don't have to answer anything.Yeah, I think it's a good question.So, I think fabric contributors provide good documentationbecause it's very well organized.So, they provide the getting started tutorials,the quality concept, and the reference architecture.So, we can check the token at best of our levels.So, Bias can use tutorials and getting started.And if an operator who are working on production leveltake care of these reference architecture or operational guides,so, I think the documentation is very organized.So, yeah, of course sometimes I cannot understand some of them,but yeah, overall I think fabric provides good quality of the documentation.Does it make sense?City, does it fit your expectations as a Japanese industry?I'm sorry?I understand it's a good documentation,but does it fit your expectations as a Japanese engineer?I'm not satisfied with the documentation.You would like to have more details.Oh, and I know, yeah, in Japanese culture,editors, customers want to build quality of the documentation.But this is the open source community.So, if you want to improve the quality of the documentation,we can contribute.So, yeah, I think it's a challenge,but our next step is to contribute to fabric documentation itself.So, after completing the translation,yeah, we can propose some improvementsfor fabric documentation itself.So, yeah, I think it's a good contribution from our side.Yeah, thank you.Any other comments or questions?Generic question, I'm sorry.I have questions because we work in Japan a lotover 20 years, kind of from the standard of culture.So, what is the main issue in introducing morelike a hyper-logic technology of working in Japan?So, it's more generic question.Oh, thank you.You have an idea?Okay, let me go first.So, I think it's similar as a language.You know, we have many political concepts,but we have only a full production level services.So, you know, what is the root cause of its issues,but it's helpful as to go to the production level.So, it's a combination in Japanese production world.You're welcome.Thank you.You have any comments?Actually, in terms of technological documentation in English,I think it's not enough to build like prototype hyper-logic system.So, actually, it's not technological problem,but, you know, English documentation is also important, I think.So, I want to community to improve documentation contentsmore deeply.Thank you so much.Standards that all the documentations would followso that it's easy to translate it to different languages.Just like we talked about some standards,shall we have those standards when we make documentation in English?So, it's much easier for you guys to translate it tothe language also.Thank you.Yeah, I think it's a good suggestion.So, I think we can share some levels.So, yeah, we can standardize at the sub-levels becauseconditional process and terminology are common problemsin other languages.So, I think it's a good idea to share knowledge betweendifferent language groups.And they can also customize some points to improvetheir all-language specific problem.Yeah.Thank you for your advice.So, digitizing the documents, like I've been workingwith some organizations who have like 40, 50, or even 70old documents, which are still relevant for them.And they wanted to digitize them.So, as part of the deal, which is many challenges.But one thing that we understood was, if we weretelling on the same point, if the documentation, when itwas done, it followed certain principles.Like, this is the format, this is the form.This is how a paragraph should start.If it followed those things, then the job was veryeasier.The same job that would take someone a year could bereduced very well to a month.12 times or 15 times, you can reduce that if the documentation was done in a particular object.So that you can build tools and reusable components,which can actually stand through it, which can actually read through and find out, these are the documentation marks.These are the sum of the headings.So all those things it could figure out.So that was my experience of NEO.And we did about 4 million pages.We converted 4 million pages of different books and different formats into a very specialized XML format.OK.Yeah.Thank you for sharing your experience.OK.So thank you for coming here.We got a very good feedback from you.You appreciate it.Thank you so much.Thank you so much.


Till now we have mostly discussed about certain fundamental aspects of microfluidics and of course with reference to some interesting applications but in the last few lectures we will be mainly concentrating on some of the applications. Now when we discuss about applications of microfluidics if you recall one of our introductory lectures we mentioned a lot of applications which focused on biological issues. So many times if somebody asks us that what are the applications of microfluidics it is very tempting to say bio and non-bio applications. What it essentially indicates is that bio applications are very very common and very very significant. So bio applications in microfluidics are concerned are of concern from the viewpoint of several practical applications and the entire paradigm of bio applications of microfluidics that entire paradigm is often termed as bio microfluidics. Now when we discuss about bio microfluidics we have to keep in mind that there are whole lots of issues of bio microfluidics and I do not have any intention of touching upon all the issues of bio microfluidics in this particular course rather my objective will be to give you some glimpses of ideas relating to biological applications of microfluidics. So in the first part of the lecture we will be discussing about bio microfluidics of cells and DNA. So in bio microfluidics of cells and DNA we will be mainly concerned about the interfacing of microfluidics with cells or with DNA or in general with cellular biology or with genomics for instance. So as we know that microfluidics deals with miniaturized devices and is mainly concerned with laminar flow it requires lower reagent volume and reagent quantities one can use it for high throughput screening, microfluidic devices tend to consume less power and one of the very big and important issues of concern why we intend to study cellular biology using microfluidics is that microfluidics can selectively address cellular length scales. Not only that one can have precise volumetric and thermal control of samples and you know in human body there is some natural microfluidics, bio microfluidics like for example in human body you have a hierarchy of blood vessels like you have large arteries, large veins, small arteries, small veins, arterioles, venules and then micro capillaries. The micro capillaries are like microfluidic channels. So blood flow through micro capillaries is a natural bio microfluidics taking place in human bodies. So as I mentioned that bio microfluidics is an agglomeration of biology with microfluidics and there are whole lots of applications that we talk about. Now as I mentioned in the first part of my lecture today we will be concerning about cellular bio microfluidics and we will also give you some idea of multicellular bio microfluidics and but to begin with like we have to clearly understand that why are we going for cellular microfluidics. What are the outstanding biological problems that we can solve using the ideas of cellular microfluidics. Now there are several issues let me talk about one outstanding problem say one particular problem in cancer biology that is cancer metastasis. So you know that one of the most lethal stages of cancer comes into the picture when the cancer cell from its origin moves to a distant location within the same human body by flowing across the blood stream. So when the cancer cell migrates in this process it has to pass through the hierarchy of blood vessels that I talked about and it also has to be passing through the micro capillaries which are like microfluidic channels. Now the condition in the microfluidic channel is highly stressful so that it may be very difficult for a normal cell to survive under such a stressful condition but the cancer cell has excellent adaptability so that it can get easily transported through the micro capillaries and that is how metastasis can be sustained. So a big outstanding question is what are the physical attributes of a cancer cell which gives them the capability of migrating successfully through the stressful condition of the microfluidic capillaries in human bodies. So understanding cancer progression is a very important and outstanding topic that can be discussed that can be I should not say that is yet satisfactorily resolved but that can be probed to some extent successfully by using microfluidics. Now not only cancer biology one has to understand that cells are typically of the length scales of let us say you talk about human bodies typical biological cells are of length scales of around 10 microns or so. So the length scale of a cell is very much compatible to the length scale of a microfluidic system or a microfluidic channel therefore you can capture cellular length scales you can capture the dynamics over cellular length scales in a microfluidic platform. Not only that you can control the fluid flow in micrometers in precisely defined geometries of microfluidic cell culture that can mimic the in vitro cellular environment and also can facilitate simultaneous manipulation and analysis starting from a single cell level to tissues cultured on fully integrated and automated chips. In diseased conditions different properties of cells like morphology membrane malleability etcetera significantly affected so the alterations in the cellular properties affect the dynamics of cell in the physiological environment. So cellular dynamics in a microfluidic channel can give rise to the detection or indication of the possible existence of a disease. So because the conditions are different for diseased condition and normal condition not only that microfluidics offers the possibility of delivering not only the chemical but also the mechanical signals providing an extra degree of control. Now I can understand that this particular audience is not a biology specialist neither me is a biology specialist, I am an engineer by training. So I think that it is appropriate that we touch upon some of the basic ideas or some of the basic concepts before we move on to our discussion like for example what is a cell. So the cell is the basic structural functional and biological unit of all known living organisms. They are the smallest units of life that can replicate independently and are often called as the building blocks of life. Now cells within the human body contain thousands of genes proteins and other chemicals enclosed within the cellular membranes and each cell responds to the chemical signals from the body or the environment and modifies its behavior in response to the signals. Cellular diseases like sickle cell anemia, Alzheimer's disease and cancer occur when cells dysfunction. This may include the development of too many cells, deficiencies in existing cells or dysfunction or loss of essential cells. So as I discussed about the cells relating to abnormalities in the diseased condition. So for example you can see in this view graph the biconcave shape, biconcave disc shape of a red blood cell. Now this can be critically affected by genetic or acquired pathological conditions. So various diseases I will not go into the details will alter the morphology of the red blood cell and when the morphology of the red blood cell is altered if one can identify that properly then that can give rise to the detection of several possible diseased conditions. Now cells in channels underlying motivation. So importance of single cell analysis because of natural cell to cell viability, variability in biochemical processes such as mRNA and protein expression levels, dynamical signaling processes and the inevitable stochastic molecular responses, population average bulk assays are often misleading compared to single cell analysis. So one usually goes for cell culture platforms I mean there are traditional cell culture platforms but in microfluidics one can go for alternative cell culture platforms using microfluidics principles and there are several advantages of microfluidics based cell culture platforms vis-a-vis conventional cell culture platforms. Why cell culture platforms are important because without cell culture, without cell culture it is not possible to study the behavior of cells. So one of the basic premises of studying cellular biology is cell culture and to culture cells in a microfluidic system the paradigm is significantly different as compared to the classical system. So here we compare the conventional versus microfluidic cell culture system. In the conventional system there are large fluid volumes that prevent rapid changes. In a microfluidic system there are small volumes that allow dynamic control. So regarding addition of nutrients and removal of metabolites in conventional system it is infrequent and manual exchange of large volumes. In a microfluidic system it is precisely measured continuous or transient but it is fully controlled. Stimulation with drugs, proteins and simultaneous imaging this is a very very important consideration because many of the diseased conditions of cells or many physiological or biophysical conditions of the cells can be obtained by imaging. Now in a conventional system it is mostly not feasible that you simultaneously give drugs and proteins and take the imaging but in a microfluidic system it is feasible. Parallelization of cellular assays in conventional system it is not feasible in a microfluidic system it is feasible. Automation of cell culture tasks in a conventional system it is possible but bulky expensive fluid handling robots must be used. In a microfluidic system it is possible with a high capability of automation in compact inexpensive format you do not need to use bulky robots. Single cell manipulation and analysis in conventional system it is manually involved inaccurate and low throughput system and most importantly in microfluidic system you can do single cell manipulation and analysis very accurately with high throughput. So there are several salient features of microfluidic cell culture precise control of cell numbers and density in a given area or volume and placement of cells in complex geometries, accuracy and throughput of biological assays by orders of magnitude, spatio temporal control over the concentration of biomolecules allowing for instance the generation of diffusion based gradients of the given molecules, low consumption of costly reagents, automation of microfluidic cell culture systems allows culturing of cells for several weeks under precisely defined conditions without any manual intervention. And it provides the possibility to deliver not only chemical but also mechanical signals providing an extra degree of control over the culture cells. So there are several steps involved in a microfluidic cell culture system like basically you prepare microfluidic channels through soft lithography which is very common in bio microfluidic applications we have discussed about this earlier then for cell culture you prepare the cells and then see the cells inside the microfluidic channel and then you do an analysis which is mostly microscopy based. Now many times you may not be satisfied with a simple microfluidic channel but you can make a complex network of biomimetic microfluidic devices and you can study the cellular behaviour. The whole issue is that like earlier we have discussed one example where we are having a targeted drug delivery through a complex network of microfluidic channels and the drug is targeted towards cancer cells. So what it will do to the cancer cells basically is first it will create a blockage of supply of oxygen to the cancer cells or nutrients to the cancer cells. So it will cause ischemia to the cancer cells not only that what it will do is it will supply the necessary drug or the necessary chemical to the cancer cells. So in order to do so basically the drug loaded molecule the drug loaded beads so to say I have shown you an example video that how these beads are designed to pass through the microfluidic networks and in this way you can create an artificial animal model. So instead of making a whole lot of trials on a animal patient or a human patient and then waiting for the results of trials you can design your drug treatment or drug based treatment or chemotherapy say in a cancer treatment. You can design the treatment and a priori assess the success by making an artificial network of microfluidic channels and then targeting the drug loaded beads to the desired cells which are having cancerous traits. So in this way one can to some extent replace the traditional animal based studies with a in vitro microfluidic system. Now what are the outstanding issues in a cell culture based system? So in a cell culture system the micro system is important and interesting the small scale system because we have already discussed that it can resolve the dynamics over cellular length scales. So to that extent the micro system is fine but the question is why do we require a fluid flow? Well we may require a microfluidic conduit but why do we require fluid flow? So let us look into this. So if there is no fluid flow in a cell culture system then dissolved oxygen concentration increases and carbon decreases and carbon dioxide concentration increases in the vicinity of the cell. So this is because of lack of oxygen supply, toxic metabolic products accumulate, pH, osmotic balance is disrupted leading to cell death. So if you are trying to culture cells without giving rise to cell death then obviously it is a matter of concern. So why do you need to culture cells? Because you intend to study some biological aspects of the cell. Now I mean you may say that well we may not have explicit supply of oxygen but the cancer cell or the normal cell whatever is being cultured it is cultured in a microfluidic platform and it let us say the platform has PDMS substrate that is the microchannel is made of PDMS for example or PMMA. Now these are porous materials right. So through the pores of the PDMS wall oxygen can be supplied well to some extent yes but it is not good enough to supply the necessary oxygen uptake rate. It is there but it is not sufficient. So that means you must somehow supply nutrients to the cell by virtue of something else and that something else is fluid flow. So that brings fluid mechanics into the picture in a cellular microfluidic environment. Not only that you think of a biological scenario you think of an outstanding problem in biology that to understand fully the dynamics of a cell in a condition when there is incipient blood flow under a dynamic environment. Now if you want to mimic that in a in vitro system that is in a artificial system then in a in a in vitro system if you want to implement that then what you what you require you require a fluid flow past a cell. I mean the fluid ideally should be a blood analog but even if it is not a blood analog at least some like aqueous solution which should pass around the cell to mimic the biophysical environment in a real in vivo system. So we can understand the motivation of fluid flow in a cell culture based bio microfluidic system. So one is a biophysical motivation that the motivation to represent the biophysical scenario in the human body and the other is like to keep the cell alive you require to supply nutrients and to supply nutrients fluid flow is one of the common means. Now here comes an interesting point when you supply nutrients to a cell by virtue of fluid flow then that nutrient will try to keep the cell alive but the fluid flow itself creates a stress in the microfluidic environment and the cell may die because of stress. So on one side the fluid flow is trying to keep the cell alive by supplying the necessary nutrients on the other side it is exerting stresses on a cell which if go beyond the critical limit can give rise to cell damage or even cell death. So the moral of the story is you require an optimal flow rate not only that you require an optimal static incubation time before you start the fluid flow. Now one has to understand as a passing remark that when we are talking about cellular assays in a microfluidic system one also needs to discuss a little bit about subcellular assay that is measuring the chemical or physical function of property within a length scale that is less than typical cellular dimensions that is also possible in the microfluidic based cell culture arrangement. So I can give you one or two examples like intracellular transport from one side to the other has been studied by exposing different parts of the cell with different diet act macromolecules. If the size of the macromolecule is large then diffusive mixing can be negligible. So you can study intracellular transport also not just transport outside the cell but what happens as a transport phenomenon within the cell that is a matter of great interest because within the cell there is an aqueous environment there is a more solid type environment there is so there is a combination of aqueous and solid type environment there is transfer of mass and momentum within the cell. So the cell itself is a nice and interesting system to study and that can be done. Another example I can talk about you can in a reproductive system when you have sperms you can selectively isolate motile and non-motile sperms only motile sperms are able to move cross stream and non-motile ones are dragged with the laminar flow. Now we will move on to some issues of microfluidic cell culture based studies and with certain preliminaries I will essentially give you some glimpse of some research that we have done in our group in this particular area typically related to studying the adaptability of mammalian cells in physiological confinements to understand the mechanisms of cancer progression. So to begin with like see as I told you that in microfluidics you have a significant contribution coming from the engineering issues and one has to understand that you are basically interfacing the engineering on the fluid mechanics with the biological scenario and traditionally I mean long times back people from these two disciplines did not use to talk with each other very much but with the advent of microfluidics and with the like emerging growth of biophysics and biomechanics in general it is now customary that I mean researchers from these two I mean apparently or seemingly disparate disciplines they combine together to solve outstanding problems in biology and that is how that is where the future of biological research is directing to it is mostly interdisciplinary and not the traditional biological research that I mean some people perceive about. So let us say that you have a setup like this so I will point out what are the relevant things you have a pump like for example a syringe pump or a peristaltic pump that drives the flow through the microfluidic system the microfluidic channel has height h and width w. So you have let us say adhering cell on a substrate I will talk about the specific nature of the microfluidic wall and you have a microscope based imaging system of the cell and the image of the cell studied under certain conditions will give us indication of the stress distribution on the cell question is how that is possible we will come into that. So it is possible through a particular type of technique called as traction force microscopy. So in the traction force microscopy what you basically do is you embed some particles these particles are embedded inside the microfluidic substrate. So these are fluorescent beads or fluorescent particles and when the cell is sitting on a substrate and the cell gets deformed these particles are displaced and once these particles are displaced you can take the image of these particles and based on the image of these particles you can get by some mathematical analysis the stress distribution on the cell that could be eventually responsible for this displacement. So fluorescent level beads are embedded into an easily deformable substrate and it is easier said than done because the substrate has to be significantly deformable for this method to work. Now not only that the other point is that the displacement field of the bead embedded substrate is calculated by comparing the fluorescent images taken at identical microscopic location in presence and in absence of cells adhering to the substrate. Then images are compared by dividing both of these images in small view ports is spanning over 10 pixel by 10 pixel and locating the coordinates of the cross correlation function maximum through a 2D fast Fourier transform algorithm. So this entirely falls within the paradigm of image processing. So you can see that on one side you have micro channel fluid flow which is like a sort of an activity of a mechanical or a chemical engineer or a physicist and on another side you have image processing which is commonly dealt with by people from electrical sciences and then based on these images we will be trying to solve some outstanding problems in biology which is typically the work of a biologist. So how these activities are coordinated and how these activities are in interfaced that is a very interesting and nice aspect of not only interdisciplinary research in biology in general but biomicro fluidics in particular. Now from the experimentally obtained displacement field the traction force is determined by the unconstrained Fourier transform traction cytometry or FTTC method. Now as I told you that what are the unique features of this system? See this traction force microscopy has been known for quite some time but only a few years back maybe around 5 years back our group has been successful in implementing this in a microfluidic environment which was not possible before. So how is it possible? You use a unique ultra soft substrate PDMS ultra soft PDMS. So normal PDMS will have base is to cross linker around 10 is to 1 instead of that you make ultra soft PDMS it is better easier said than done but like it is quite difficult to make this kind of ultra soft PDMS with base is to cross linker ratio of 65 is to 1. And then not only that you make the substrate more cytocompatible that is compatible to cells by modification of the PDMS substrate using APTMS and polydialycin.  Then smaller diameter of marker fluorescent beads like 0.046 plus minus 0.006 micron can be placed very close to each other. So typically 8 number of beads in 1 micron square area allowing enhanced spatial resolution of the TFM. So in this way you can basically probe the dynamics of a cell over length scales of microns. So as I told you that what are the problems that you can study. So for example the traction force that is exerted on the cell as a function of time you can study with different fluid flow rates. So you can see that like after some time you can see that the media has to be replaced for the cell to survive. So optimum static incubation period static incubation period is the period over which you do not supply fluid to the fluid flow to the cellular environment. This increases with increasing volume per cell. So the traction force decreases after the optimum time. So after the critical time if you do not supply fluid flow if you do not supply nutrient the cell is progressively going towards death. And you can program a cell death in this way and so you stop the nutrient supply to the cell that means you do not have any supply of nutrient to the cell. So after a critical time the traction force will decrease and the cell that signals that the cell is naturally going towards death. And in fact if the static incubation is controlled in this way it can give rise to a controlled cell death or apoptosis. So like as I told you that there are several image processing issues involved here. So you can see that these are the forms of these are the various facets of the experiment. So these are the fluorescent beads and like this is the experimental setup. So you can see this is the microfluidic pump and you can see the microscopes and the microfluidic setup here and then you have a computer where you study the images. So the typical images so you can see that you can see the contour of the cell adhering to the substrate. The red regions are the regions of high stress concentration and the blue regions are the regions of low stress concentration and intermediate colors represent intermediate effects. So you can see that as a result of this study you can guess get a nice color map of distribution of stress on the cell. So with different flow rates you can see that the stress distribution on the cell is changing and this is purely fluid mechanics. So fluid mechanics as applied to a deformable body. So if you have a cell a big attribute to the cell that it is deformable. So in terms of fluid mechanics it is the effect of fluid flow on a deformable substrate but a substrate with not only deformability but a natural adaptability which is not possessed by an inanimate object. So as I tell in some of my lectures that in terms of fluid mechanics a cell is very similar to something like a vesicle but the outstanding difference is that a cell has life whereas a vesicle has no life. So by virtue of the aspects of this living system the cell will respond to the incipient fluid flow in a different manner as compared to what is done by a vesicle or any other deformable object. So you can relate the traction force and deformation and like so you can see the centroid of bit displacement can be plotted as a function of time and this displacement is eventually correlated with the traction force that is distributed on the cell. Now so this is fine but this is a purely engineering platform but what kind of biological information we can extract from this engineering platform. Effect of micro confinement. Now what are the effects of micro confinement that we are interested to study in a cellular bio microfluidic environment. One important aspect is tissue matrix confinement. So mechanical and chemical effects of interstitial flows that can be carefully studied. Shear stress near one Pascal is capable of affecting the cell physiology and proliferation. Not only that there are chemical effects also there are chemicals which are continuously secreted from cells. So there are certain chemicals and we will study this very carefully. These are these may be autocrine morphogens growth factors like EGF and all these. These molecules bind to cell surface receptors. So if you look into this cartoon these are the receptors and the red dots are the molecules which are called as ligands which will bind to the receptors and this can help the cell to assess the micro environment. So cells are continuously releasing these chemicals of these molecules and then these molecules are binding to the receptors located on the surface and in once this binding is there then the cell can understand its micro environment. But it is very important to understand that where does fluid flow come into the picture. The convective flux alters the special distribution of the growth factors like for example the EGF or the epidermal growth factor. Now there are several other examples where the effect of micro confinement of the cell can be important like blood flows through capillaries. Cancer cells preferably adhere and survive within small capillaries during dissemination. Size of blood vessels influences blood clotting dynamics and mechanical signal transduction through EGFR is that is the epidermal growth factor receptor is one of the critical issues by which mechanotransduction or transduction of mechanical signals in a biophysical system may be possible. So how do we study this problem? So we make a simple schematic so that we can also study this problem from a mathematical perspective that is we study the fluid flow and the species transport. So let us say that you have H as the micro channel height and H cell is a typical length scale of the microfluidic system. So the confinement factor is denoted by H by H cell. So that means if the smaller the value of H by H cell that means that the stronger is the confinement and in this particular cartoon the epidermal growth factor is shown by the green colored circles and the corresponding receptors by the Y shaped symbols. So it is pretty clear that the EGFs are secreted from the cell and they are captured by the Y shaped ligands which are located on the cell surface. So as a problem of microfluidics how do we theoretically solve it? We solve the species transport equation with a fluid flow which is typically a pressure driven fluid flow and the boundary conditions are a reactive boundary condition at the substrate depending on the reactions that we are interested to study. So if in the absence of any reaction the right hand side of the equation which involves the reaction kinetic features will be equal to 0. So although in many cases we are not interested to study reactions I have purposefully put the reactive surface boundary condition to make the model look more generic. So then we study the EGF concentration as a function of the Peclet number. If you recall the Peclet number is the ratio of the advection to diffusion strength or the diffusion time scale by advection time scale and you can see that EGF concentration near the cell surface increases with increasing confinement. So confinement is a very important problem. See in microfluidics there are 2 important issues that we need to address while modeling a real biophysical system in a human body. One is confinement another is flexibility. The confinement is something the confinement effect is something which we want to capture through the results that I am going to present here. The flexibility is a more critical problem because we can make microfluidic channels we can make microfluidic channels with a good level of flexibility but that flexibility may not essentially mimic the flexibility of the flexibility of a blood vessel in a human body. So as I tell you and I often make this remark as teachers we always tell you that what are the problems either we as a as our own group or other research groups have been able to solve. Hardly we discuss in the class that what are the outstanding problems and it is not a bad idea that we bring out an outstanding problem here. So if you want to make an artificial system which mimics the microcapillaries in human bodies one aspect is confinement which people have been able to successfully make. But the other aspect is a flexibility and the flexibility real human body system the flexibility of a capillary in a human real human body system is very very complex in nature and that engineers have not yet been successful in manufacturing or fabricating microfluidic systems which mimic the flexibility of the blood vessels in human bodies. I mean there are several research reports which make flexible microchannels and these are reported well those are flexible those are those have control flexibility but that does not essentially mimic the complicated nature of the distensibility of the blood vessel as a function of the local blood pressure as the blood is flowing through the capillary. So in this particular results we do not bring out the effect of flexibility but we bring out the effect of confinement and we have clearly shown that EGF concentration increases with increasing confinement. Then with this understanding now what are the issues that we need to resolve with this little bit of background on like creating attraction force microscopy based environment for studying the stress distribution. What are the questions that you would like to answer? Does the size of the surrounding confinement play a controlling role in dictating the effective survival capability of a biological cell? Where from this question is coming? We have seen that the EGF concentration increases with stronger confinement that means the size of the confinement might play a controlling role in the biophysical response of a cell and then it can give rise to a distinct survival capability of the cell depending upon the confinement. The second question is a more critical question. Well the effect of confinement has to be there on a biological cell but how is it different in cancer cells and normal cells? So does the cancer cell possess better stress responsive ability within a micro confinement or does it possess a worse stress responsive ability within a micro confinement? We need to not only get a qualitative answer to this question but also a quantitative answer that means that we know that confinement has its role on the survival ability of the cell. How is this role different between cancer cells and normal cells? So this is a cartoon by which I want to demonstrate the whole idea. So you know there are different types of cells. So in this particular cartoon we have focused on adhering cells. Now when you look into the adhering cells you need to carefully understand that as human beings we stand on a surface with the help of our legs. In a similar way a cell sits on a substrate with the help of its own legs which are called as focal addition points. Now focal addition points on the basal surface are these ones and then what happens when there is a stress on the cell by the incipient blood flow then these focal addition points are first disrupted. Just like if I am walking and somebody is kicking at my back then first my legs will be destabilized and I would be mechanically destabilized by virtue of the destabilization of my feet. Similarly the focal addition points will be disrupted. Now by a unique mechanism this disturbance this mechanical disturbance will be propagated through the cell in terms of propagation of biological signal and then what will happen? The most distant part of the cell membrane which is called as the apical cell membrane will understand that there is a stress. So when the apical cell membrane understands the stress then what it will do? To cut the story short there are different types of entities in the cell membrane. In cell membrane there is a fluid type of entity which is called as lipid and there is a solid type of entity which is called as lipid raft. Now with the stressful condition two possible things can happen. One is the lipid raft can move from cell membrane to inside the cell just to escape from the stressful condition in the cell membrane and otherwise if it is still residing on the cell membrane then the lipid raft may break all together. So I give an analogy like this. Let us say that a decoy has attacked my house. Now if a decoy has attacked my house see I am not a very brave person. So I will not be keen to face the decoys with my own fighting ability but I will initially try to hide myself in the bathroom instead of facing the decoy. So the lipid rafts instead of facing the stressful condition they want to escape to go from cell membrane to inside the cell. Now in this condition what will happen? The cell membrane which had lipid rafts now will get devoid of lipid rafts under the stress. So the cell membrane will become more fluidic or more malleable because it has now less lipid rafts than lipids and in this process because it is malleable it can adjust its shape in a microfluidic confinement to survive the stressful condition and this is the short version of the long story that we try to unveil through our experiments. So I will try to go through the experimental results on shear adaptation of cancer cells in a microfluidic confinement and the confinement effect. So what we initially wanted to study is that like what is the mobile fraction of in the cell membrane. So that means basically you can also talk it in terms of the diffusion coefficient. So what you can do is that you can make a study which is called a SPRAP, Fluorescent Recovery after photo bleaching. So you tag the cell membrane with fluorescent dyes and then what you do is that you photo bleach a part of the cell membrane and then after photo bleaching the fluorescent dye molecules will diffuse from the surrounding because of the concentration gradient and the faster is the fluorescent recovery after photo bleaching that means higher is the diffusion coefficient and higher is the membrane fluidity. Why is membrane fluidity important? Because membrane fluidity essentially is related to the depletion of lipid rafts from the cell membrane. So there are two important phenomena which are taking place. One is the stressed induced focal addition disassembly that is the disruption of the focal addition points that is the legs of the cell which are the connections between the legs of the cell and the microfluidic substrate and lipid raft internalization. So there are two events. One is focal addition disassembly another is lipid raft internalization and there is a time lag between these two. This time lag is the response time. This is the time that the cell needs to respond to its micro environment. So understanding or getting a feel of the micro environment is focal addition disassembly and responding to that is lipid raft internalization. So shorter the response time that means the cell is responding quickly to the stress pool condition. So we have studied the variation in the response time in the parametric space of h star that is h by h cell and shear stress and we have seen from this map you can understand that the response time decreases if the shear stress is increased beyond the critical value or so the first is the response time decreases if the shear stress is increased beyond the critical value or when the h star falls below critical limit that is h by h cell falls below 70 micron and the stress is greater than 10 dynes per centimeter square. So this is called as confinement effect. The confinement and the stressful condition combined together can reduce the response time of the cell for adjusting it to its micro environment. Now the second question that we addressed that we tried to address the confinement effect is more exclusive for cancer cells. So we have studied several cancer cell lines and normal cell lines and our study has revealed that cancer cells are essentially more malleable than normal cells. So under a stressful condition the lipid draft dynamics is more prolific in cancer cells the lipid draft internalization is more prolific in cancer cells that makes the cancer cell possibly better shape adaptive or shape adjusting or malleable to survive the stress adaptive capability to survive in a stressful condition in a microfluidic confinement. So the cancer cells adapt themselves better that is how they survive in a metastatic condition and if we can understand the biophysics of this mechanism of cancer cell adaptation in a microfluidic confinement then this can give rise to a critical understanding of the metastasis of cancer cells from a pure biophysical viewpoint. Now molecular origin of confinement effect. So we have discussed about the EGF and EGFR and we will try to understand that how the EGFR activated EGFR concentration is related to the confinement effect and to relate it to the molecular origin we have plotted a ratio eta which is activated EGFR by EGFR and we have found that a decrease in the response time is equivalent to an increase in the activated EGFR. So that means that in the confinement in a highly stressful condition EGFR is more prolifically activated. So that is not that is one thing not only that beyond the critical stress the EGFRs form clusters. So you can see in this view graphs clusters of EGFR that we have been we have picturized. So now the EGFR can be activated in two ways one is ligand mediated another is ligand independent and flow induced the ligand is the EGF. So it is basically called as a ligand receptor model ligand is the EGF and what binds with the ligand or the ligand itself binds with its receptor that is the EGF receptor or EGFR. So you can use some anti EGF which blocks only the ligand dependent activation and you can use a chemical which can block both ligand dependent and ligand independent and flow induced activation. Now a very interesting point that we have observed is that below a critical shear stress the activation is purely ligand dependent fluid flow does not play a big role beyond the critical shear stress the activation is predominantly ligand independent and primarily fluid flow induced. So that is where you can see that the molecular biology of the cell or the molecular behaviour of the cell is influenced by fluid flow. So this is a very interesting point where fluid mechanics is interfacing with molecular biology. So you can see that like normally the EGF distribution or EGFR activation that should not be ideally dependent on the fluid mechanics but by the traditional thoughts those are dependent on the biophysical or the biochemical features within the cell. Now we have shown that how these can be tuned by altering the stress condition as well as the confinement effect. So with the confinement effect you have both the ligand dependent and ligand independent activation. So you have the ligand dependent activation which is dependent on the EGF concentration and ligand independent activation of EGFR which is dependent on the fluid flow. So EGFR activation depends on EGF which is ligand dependent and also on fluid flow which depends on the confinement effect as well as the stress due to the fluid flow. So just to conclude on our discussions on cellular dynamics I want to highlight on a few other applications not just the model for understanding the biophysics of cancer progression like for example how can we use this for medical diagnostics. I can give an example of cell solder. So you can see here I will explain this figure the cell solution is focused into smaller confinement so that the whole fluid comes under a beam of laser okay and only one cell is included in a single cross section. So why is it important is very is because very narrow channel fabrication is costly. So you can instead focus the cellular solution to pass through a very narrow passage not a artificial channel but a focused narrow area by which it will pass. Then a laser beam of specified wavelength illuminates the fluid. If the cell passing through the illuminated volume emits a fluorescent light a voltage operated valve is activated and the fluid is further switched towards a particular location where the cells will be collected finally. So you can sort specific targeted cells from a cellular solution by using this. So for example in a blood sample of a specific volume the number of leukemia cells can be determined in this process. So cellular bio microfluidics you can also use for medical diagnostic applications. So we have discussed about some aspects of cellular bio microfluidics and in the next lecture we will be mainly focusing on microfluidics with DNA and some aspects of microfluidics based medical diagnostics which is one of the very important areas of bio microfluidics. Thank you very much.


Okay guys It is 454 a.m. Coming into the second morning of our camping trip. I don't know if you can hear it kind of slacked up right now, but It's pouring down rain So I went out and made some checks and made sure everything was all buttoned down And so far no leaks right mama Huh So I just I think she going back to sleep going back to sleep I went outside and checked everything. I had to close this I Had this side open here To get a breeze in so went outside to do that and You know zip that up so so far Seems to be pretty decent no leaks. I did not waterproof this tent because You know you got to have at least 24 hours For it to dry when she do that and I knew I wasn't going to have that so I didn't want it just washing off It would have been a waste of time So Hopefully I won't leak But if it does it'll be because I didn't want to prove it but So far I'm not seeing any leaks so Good morning, YouTube 454 you said yep 454 So I think the mic's picking it up So as I didn't smoke the cigarette while I was out there, so Speaking of cigarettes, I don't know where I put loads of packs Put them somewhere with something Inside something I think I don't know because I only got one left in here Crap All right, I'll let you know what's happening Okay guys as you can tell from the intro It rain I'll tell you what it actually poured harder than that. I mean it got bad. I think our leak stopped but still I'm going to It was coming in somewhere right in here So what I'm gonna do It was just a little drip and when it dripped onto the screen, you know how it kind of splatters she could feel it Every so often so it wasn't like getting wet in there or anything like that You could just see it on the screen. So I'll take some I'll unzip this and then I'll take my fingers and I'll go down along each each side of that with some Vaseline petroleum jelly And then I'll zip it up and then I'll take my finger and I'll run it Down through here and across the seam here just to make sure but it will look like it's coming in about here But I'll run it down through and seems to so it seemed though. We didn't get what though didn't we mama? Not really Yeah, but you can't count that wouldn't really No, we didn't get wet So I'm making her coffee and her eggs and Bruno's got a couple eggs there and I'm making I made some bacon half a pound of bacon there Because I'm gonna save the rest for the pork chops tonight.  Uh-huh, and she goes, oh, I guess so I Thought it was put a hole in it. Yeah, I did too hard. I did too. It's like great But I don't know if you can see or not, but the clouds are moving fast because we're only like Five minutes that way is the Gulf Coast. Yeah the bay So and we might be able to get there too with our boat. Yeah, maybe I don't know. We'll see but we're gonna take the boat out today as long as it stays like this I'm on my And I'll kick you back on When we do other stuff What we did was we went out on the boat for about five hours It rained 85 percent of the time we was out there. It was nice Then we pulled back into a cove did a little fishing kind of windy for that had a couple hits, but that's it but uh And bring my camera because it was underneath the front seat of the truck And when we got down there Guy said well, they're telling me to charge you guys to park here Or you can just take your trailer back to your campsite And I said it doesn't say that in the website. He said yes, it does.  I said it says it's free. No overnight parking No, it doesn't I've never seen that and I've worked here I said well, you better start studying your job a little bit more. So she brought the truck back. It's not that far And uh Then walked back and we've done some boating Um, so my camera got left in the truck, but monkey's got some footage So maybe she'll let me throw some of that in so I'm starting to fire here Everything's pretty wet, but it seems to be taking hold pretty good. I moved the firing Just until it gets started so it can have more air I'm going to make some pork chops. So Bruno had a good time He was a good puppy. He only tried to jump out of the boat once. I don't know what he seen but Never seen him do that before but then after that he never did it again. So I don't know uh crazy dog, but uh So yeah, I think we're going to go back down and do some night fishing off the dock So that should be interesting.  It was uh 89 degrees At 10 57 10 47 this morning With a real feel like 92 or something It's hot, but at least on the boat, you know, you can get some air. You can take off and get some air So I got a story for you about that old boat I had it up to I think the fastest I ever had it was 21 so when she's with me, you know, we've had it up to 19 Uh, when we first got it it would run 16 Then I've had it up to 19 with her in it So I was out boating around while she came back to get the truck so um So I was boating around this guy on a bass tracker just come out And he pulls up beside me Because I was, you know, tweaking the motor it'll be some man that thing runs pretty good How fast can you get all that little thing? I said I've had it up to 21. He said well, you were doing faster than 21 there. I guarantee. I see you think so He said you want to check I said, yeah, he says whenever you're ready go So what I did was raise the motor up half an inch So it doesn't get on plane as fast, but it's got more top in now So, you know, it kind of does this when I'm by myself it does this Does this for a minute, and then it'll start leveling out So I got on it and I'm doing this wheelie and he's beside me And all once this thing just started to sit down and it was like a missile And I was running hard boy giving it all she had and I heard him because I seen him falling back And I heard him Throttle his up to catch me and he caught up to me and he kept giving me a thumbs up and then when we leveled off he went like this So we slowed down we stopped out there and we was talking He said how fast you think he's going now? I said, I don't know. That felt pretty good 22-23.  He said oh, by the way, my name is tim man so we talked for a minute and He was heading out towards the gulf to go fishing But while we was out in the gulf today And it was rough that thing that boat was airborne several times. I said, it's too choppy I'm turning around. She's like why that's Not in this old boat Once we got turned around coming back You know with the waves where it was okay, but it was really choppy. I said, you know, these rivets are almost 52 years old When this boat coming apart and nobody was around so we come back in and boated around We used about two and a half gallons of gas out of six gallons and we boated for five hours And when we sat for about 30 minutes the rest of the time we were We were under power. Sometimes we used a trolling motor. So about 85 percent of the time We were under power and wide open. So that's pretty good.  Okay guys, here's the pork chops. There's monkeys Okay, we're heating up the stew And there's some bacon I had on top of the chops To give it some more flavor also We're cooking the bacon. So we're going to bacon too bacon a little bit of stew and some uh Potato salad right mama But she already tried that. How was it? Very good. Yeah, I marinated them all you used was Was kikaman Yeah Now our season salt Uh, just some paprika. I wish we had smoked paprika, but Don't really need it because it's on the fire Uh cayenne pepper And some lemon pepper All right guys, we're gonna have a bite to eat And wait for dark, huh? Maybe we'll take a napto dark Hey guys, there's a little hack for you Check this out. I'm gonna turn you off of night vision All right, I'm gonna turn this flashlight on Okay It really does nothing but it shoots straight up in there, right? But you take a gallon of water And you have a table lamp Cool beans Cool beans got the boat lit up because it's cool But Check it out. Nice little table lamp.   Hang on stop stop It's all right. Okay.  Yeah, come this way Listen to me here. You do it. No You do it honey. Listen to me come here I got Bruno. Okay Let's see your line Okay, bring him up here and put him this way Bring him up here. That's better than mine.  What is it? Yeah Look at that Nice Second fish in your life She gave one. Oh Good job. I have caught what three? Yeah, three and two crab.  There you go Hey, honey. Yeah, look at that right down there. What is it? Look see it. Uh-huh. Where my light is. Yeah. What is that? Isn't that um the stingray? No, that's an eel. See it? It's an eel. See it? I don't know if the camera's picking it up, but there's your second one. Yay All right. Yeah, you just hold that light. Oh, I see him. Yeah, I seen. Yeah, but your light's better Let me get both of these lights Wow. No, go down put your light down a little bit more. There you go. Up just a bit right there Yeah, I see him really good. Where's your light there? He's jumping I think it's an eel. Look at this snake No, I think it's an eel All right, hold your light there Good job monk number two Number two. Yay I just unhooked her and rebaited her Bruno, stay Oh, he's trying to leave me They're getting bigger Okay guys, I don't know if you can see us I've got a fish on here This is my fourth fish. I've caught five crabs and an eel I'm gonna have to set you down here for a second The monkey's caught two fish One was about a pounder and her second one was about a two pounder Trying to keep in the Light here. I don't know if night vision will be any better. Let's try it Okay, I got your night vision now Maybe that'll be a little better All right, that's a nice one. Let me take off the night vision for a second And I'll shine a light on him Hang tight This is the biggest one yet. He's every bit as white as my boot. Here you go He'd probably go four pound All right Four fish five crabs and an eel cool All right, I gotta get him off my hook so Okay guys, so I'm back from fishing Monkey, what'd you catch? Where you at? Two fishes Huh, I caught two fishies two fishies. Uh-huh some kind of fishies cat cat fish. Yeah One was bigger than the other, huh? Yeah. Yeah Yeah and I caught four Four cat fish You saw the last one I didn't film mine. I should have been but I had to film that one because I knew it was pretty good size Wasn't a monster, but he would have been good eating. I caught five crabs And an eel Yeah I wasn't sure if it was a snake, but I thought man I never seen a snake go down there on the bottom before I got him up out of the water He was like two and a half feet long and he was just Yeah shaking around finally he got off of there, but I did catch him. Yeah So had a good night, huh? Yeah, it's after it's after three o'clock right now. Yeah Yeah, so Yeah Bruno's in the tent. Yeah, but I can't pick it up because of the screen. He's in there on the suitcase I only got the boat all lit up Got our light there Turn that off again. I'll show you this again That's amazing How much light this thing gives off? Yeah so Back on night vision All right guys So that's day two. We're out of here tomorrow Man, we was down there before nine o'clock Worry. Yeah, because when we was just getting ready to leave I heard somebody asked what tunnel was over there and they said 830 So he's down there before nine.  I wanted to go talk to him. She wouldn't let me because He was netting fish. Yeah, okay fine No big deal right, but it was doing it right in front of where we're fishing Shining his blights all over us.  I wanted to go talk to him.  You know, have some respect.  You know, it's just one thing after another all right guys, so Day two, hopefully I got enough footage for you guys. So We'll be heading out tomorrow. So I'm gonna say good night Monkey did good. I'm proud of her. Good job, monk. Yay. I got two fishies. Yeah That second one was about a two pounder. Yeah That last one when I filmed it. I I put my boot beside him. He was every bit as white as my boot Yeah, so he's about four pound made maybe five. Yeah So, all right guys, I'm gonna say say shea berry the myth the man alleged I'm gone for now We're gonna hang out for a few minutes and we're gonna get some sleep We can sleep in a little bit tomorrow and then we got to go so All right. Bye guys


Aloha, I'm Kayli Iakina, and although I'm a trustee in the Office of Hawaiian Affairs, the views I express today are purely my own and do not represent any government organization. Transparency is a difficult area for politicians and policymakers. Typically, they profess to understand the value of open government, but it's astounding how quickly they can carve out exceptions to withhold information from the public. House Bill 1768, a bill currently working its way through the state legislature. If enacted, it would disclose salary information about government workers in the form of broad ranges, as opposed to exact compensation. Supporters of the bill, like the Hawaii Government Employees Association, cite the need for some level of privacy for state employees, which many people can sympathize with. On the other hand, early versions of the bill didn't specify the ranges that should be provided, making any information it would provide virtually useless for the purpose of studying public employee compensation. Salary information was crucial to the Grassroot Institute of Hawaii's efforts to expose excessive overtime pay on Kauai, and it has been at the heart of other overtime scandals. To reduce this to a question of privacy overlooks the strong public interest in knowing how taxpayer money is being spent, especially when corruption and self-dealing might be hidden in those salaries. In its latest incarnation, HB 1768 caps the range at $15,000 in improvement on its previous versions. However, in the effort to balance privacy and the public interest, we must remember that government employees are in a different category than the typical private citizen. They hold a position of public trust, and with that comes a higher standard of openness about the details of their employment, including their compensation. Ehana Kako, let's all work together. I'm Keli Akinah, aloha.


My name is Dr. Muthu Sayez from AVMC, my topic for paper presentation is a comparative study of CT findings of colorectal carcinoma with their operative and historical findings. Introduction. Colorectal cancer is one of the most common GIT diagnosis that results in significant more detailed mortality. CT is being used for preoperative assessment of the growth and the involvement of adjacent structures, including the fat and pelvic muscles. At CT, colorectal cancer typically appears as a discrete soft tissue mass that narrows the colonic lumen. Colorectal cancer can also manifest as vocal colonic wall ticketing and lumen laryngeal. And complications of primary colonic malignancies such as obstruction, perforation and pistula can be readily visualized with CT. The local extension of tumor appears as an extra colic mass or simply as thickening and implantation of pericolic fat. Extra colic spread is also suggested by laws of flat planes between the colon and adjacent organs. Considering these points, the present study was planned with the Indian officials to assess the extent and the spread of colorectal malignancy on CT scan and to correlate CT findings with pre-op and histopathological diagnosis. The aim of the study is to assess the accuracy of CT scan findings and the spread of colorectal malignancy and to correlate these findings with the operative and the histological findings. Materials and method, this is a retrospective and prospective study which was carried out in AVMC and hospital to the cherry in the department of radio diagnosis for the period of 2 years. A total of 31 biopsy 21 patients showing a variable bowel wall ticketing involved in the colon rectum on the CECT were included in the study. The tumors were staged based on the CT scan findings and were compared with the operative and histopathological findings. The staging of colorectal carcinoma using pre-modalities like the operative, histopathology and CT scans has been subject to the interest of accurately defining the extent of disease. So this is an axial contrast-enhanced CECT image showing a heterogeneously enhancing and symmetrical wall ticketing involving the rectum. Here, this is a CECT sagittal, heterogeneously sagittal CT image showing heterogeneously enhancing wall ticketing involving the rectum with loss of patterns between the rectum and prostate that suggest to work in involvement. Now just the axial contrast-enhanced CT image showing a certain potential wall ticketing at the hepatic flexure. So now we will see the PNM classification of post staging of colorectal cancer. The primary tumor, Tx primary tumor cannot be accessed. T0 is no evidence of any primary tumor. Tis is a colorectal carcinoma. T1, the tumor invokes a sublimposa. T2, it invokes a muscularis propria. And T3, here the tumor invokes the muscularis propria into the subcerosa then into the pererectal tissues. T4, tumor directly invokes the other organ or structure and perpetrates the visceral petronium. The regional lymph nodes Nx, the regional lymph nodes cannot be accessed. N0, no regional lymph nodes, catastrophes. N1, catastrophes in 1, 2, 3 pericolic or perirectal lymph nodes. N2 is metastasis in more than 4 pericolic or perirectal lymph nodes. Distance metastasis, Mx, the distant metastasis cannot be accessed. N0, no distant metastasis. M1, distant metastasis. This is the discussion. Rectum was one of the most common site of involvement followed by the recto-signal involvement. Metastasis was observed in 5 cases out of 31 melignan cases. 5 of the 7 cases were correctly staged as T1, T2 lesions on safety. And having a sensitivity of 83.3% and specificity of 92% and positive predictive value of 71.4% and negative predictive value of 95.8% in the diagnosis of T1 and T2 lesions. 15 of the 16 cases were correctly staged as T3 lesions. And CT had a sensitivity of 82.2% and specificity of 93.8% and positive predictive value of 93.8% and negative predictive value of 86.7% in the diagnosis of T3 lesions. All 8 cases were correctly staged as T4 lesions and CT had a sensitivity of 100% specificity of 100% and positive predictive value of 100% and negative predictive value of 100% in the diagnosis of T4 lesions. Conclusion. Hereby I conclude that CT is an excellent modality in diagnosis of melignan lesion of colon and rectum. These are my reference. Thank you.