Welcome to MOOC course on applications of Interactomics using genomics and proteomics technologies. In today's lecture MS Nikita Gehoi a final year PhD student in my proteomics laboratory at IT Bombay will talk to you about one of the applications of protein microarrays in cancer research. As you are aware that we can have different type of protein microarray platforms of course if you need the purified proteins that is much more laborious and big task you can also have the you know genes printed on the chip, you can have the cdn clones, you can have the tissue or the cell lysates printed or you can have antibodies, you can have many ways of making arrays however you know if you have the purified protein based protein microarray that can have lot more impact because it can have you know tremendous value for many applications. So, for this project we collaborated with a collaborator from Johns Hopkins Dr. Heng Yu and his lab and they have made a human proteome arrays hue proterase where all the possible proteins of you know human so far around 19,000 human proteins have been printed on the chip and these proteins are printed in duplicate and along with these proteins there are many spots which are control features. So, by using this kind of array platform Nikita has tried to use for various type of you know brain tumor patients who are suffering from glioma and meningiomas how to screen these patient serum sample on hue proterase and try to identify the potential auto antibodies and biomarkers obtained from this experiment. So, in this lecture she is going to talk to you and provide you an overview of how an experiment using hue proterase can be performed and also various you know details about performing such experiment your experimental requirement the protocol details and finally above data analysis. So, the basic workflow of different type of protein microarrays are very similar as we have been discussing in different lectures. However, there are several small difference which you will notice when you are using an upper base platform versus you are using a purified protein platform or you are going to use the reverse phase protein arrays base platform. So, the number of proteins on these hue prot ships are much larger than the various other arrays platform which we talked including the parasite protein based arrays for malaria research which we have discussed each slide which we are using here for the from the hue prot ships those are used only for one patient sample. So, it means if you have an array on which there are thousands of protein printed almost 40,000 spots are there. So, then only one patient sample can be probed on the whole arrays whereas if you have a smaller number of genes printed on the chip then you can have multiple wells and you can use many sample screening on the same platform. So, these are the small details which I am sure as we go along in the lectures you will understand that how to do different type of field experiment also multiplexing capabilities these things only will be clearer when we reach towards the end of the course when we can discuss about comparison of different platforms. So, in this case when we are talking about the clinical sample usage on the entire ship the sample handling because very critical even the entire assay reproducibility become very crucial. So, these things are going to be discussed in much more detail. So, Rikita is going to demonstrate the entire experiment in the context of auto antibody signatures of brain tumors. So, let us have this lecture and try to understand the workflow involved using purified protein from hue prot ships and its applications in brain tumor research. Hello all today we will be talking about one of the applications of protein microarray in cancer research which is to detect the presence of auto antibody in cancer patients. I am Nikita Gahoy and I am currently a senior PhD student working on detection of auto antibodies in cancer patients. So, let us start with basics of protein microarrays. So, if you can see in the slide these are this is just a basic image of protein microarray slide and these are spots you see are actually the proteins that are printed on the slide. Majorly protein microarray contains array of a lot of proteins which are immobilized on the on a glass surface and then what are auto antibodies. Cancers are a cause of lot of mutations because of which aberrant molecules are produced either they are produced in a higher level or because of altered protein structures or because the cell death mechanism cells release a lot of differentially produced molecules which acts as antigen and then body produces antibody against it. So, this can be produced because of a large number of reasons like neo epitopes like the proteins are modified because of different slice variants and which body does not recognize as cell and then antibodies are produced against it. Then sometimes there are proteins which should be present at certain concentration but then they are produced at a larger concentration and then body reacts against it and produces antibody against it. See post-translationally modified proteins or sometimes it is the expression of proteins are supposed to be present in the in some different organ but then they are expressed at other location of the body which leads to production of antibody. So, these antibodies are produced actually against your cell proteins which now acts as foreign and body the immune surveillance they take them as something foreign and creates antibody against it. So, in the study design of this experiment will be we first take a patient cohort we take C-RA or CSF from the patient and then these are the chips which contains around 19,000 proteins watch and duplicate. So, we incubate the slide with the C-RA and then wherever the antibodies are produced again the against the proteins this antibody will go and bind to the slide to the protein and then we can detect the signal using Psi 3 or Psi 5 dyes. So, let us talk about the experimental procedure. So, the experimental procedure is very similar to that of western blotting where the slide contains a lot of protein molecules and then there are a lot of spaces where the area is empty and then first we do a blocking experiment where we where we use BSA and super block to block the extra spaces where protein is not available. Once that is done we incubate the slide with patient C-RA and anti GST antibody. Anti GST antibody we put because each and every protein in this chip has a GST tag. So, just for the quality control of the slide these every protein is for should bind with the anti GST antibody and we see a response against it and against whichever protein the auto antibody is produced the auto antibody from the serum will go and bind to it. So, we use a cocktail of serum which contains the primary antibody from the human and anti GST antibody cocktail to incubate it with the slide. After incubation for 2 hours we wash the slide and then we incubate it with the secondary antibody which contains Psi 3 and Psi 5 anti antibodies and then we scan it at dual channel. Now, let us move on to the hands on experiment of how auto antibodies are detected using protein microarray. So, this is how the slide looks like and this is just a normal glass slide which contains 19000 protein spots. This is the area where the spots are printed in duplicates. This is the area where the barcode is present and you cannot touch the slide where the spots are present. You have to immediately touch the slide here only you have this is one of the precautions otherwise fingerprints can result in erroneous results. So, this is one of the slide which I am taking and I am putting it in this box. This is the another slide this is the experiment where I am showing 2 slides that can be screened together for screening 2 different patients. So, this is another slide the first step is blocking of the slide. So, for that we use BSA and super block solution and then this is the slide in the box we will just put the BSA and super block solution on to the slide. Make sure that you put it on the barcode region and then the slide is kept for incubation for 2 hours. So, that all the extra spaces except for proteins are blocked properly. So, this is done using a rocker and we started and the rocker keeps on moving for 2 hours. The blocking is done speed is kept as such the slide is covered properly with the with the blocking solution. So, once this blocking step is completed we take this blocking buffer out we decant it carefully decant the buffer in the discard. So, make sure that the slide does not fall and remove the blocking buffer properly. Once that is done take the slide carefully without touching the protein spots. So, for that you can just touch the slide at the barcode region and then you have we place the slide carefully into the cassette. So, this is the cassette where we place the slide for washing we will do the same for the another patient. So, this is how the cassette looks like and we keep the slide keep the protein area facing each other. Now, we take the cassette and place it into the buffer for washing this buffer is one XTBST and the PH is set at 7.4 so that the protein structure remains intact. Now, we just start so here you see there is a magnetic bead which is used for the circulation of the buffer. So, we start the magnetic bead and the circulation of the buffer make sure that the slides are washed properly so that the extra blocking buffer which is present over the protein spots can get removed properly. This is this washing step is performed thrice for 10 minutes each. So, once the first step of the washing is complete for the 10 minutes what we do is we flip the slide we stop these magnetic bead and then we flip the slide so that the washing is uniform from both the ends make sure that you touch the slide at the corner or at the barcode region and then we flip the slide and place it again into the washing buffer same is repeated for the another slide touch the slide at the corners and place it back into the buffer and now we start the washing again. So, this is done thrice so that all the extra blocking the agent is removed and the proteins are now available for the primary antibody incubation. So, once again make sure that you close the bead before taking the cassette out and then now we remove this cassette and just to remove the extra buffer what we do is we rinse it with water. So, this cassette contains miliki water we take out the slide and then we rinse it in the in the water to remove the extra solution. The slide is then spin down to dry and to remove all the extra buffer. So, we remove extra we remove the water by placing the cassette on the tissue and then we spin it at 900 at 900 rpm for 2 minutes once the slide is dried we take we place the slide back into the boxes and put the cocktail of primary antibody. So, this cocktail contains serum and anti-GST antibody in BSA and TBST. So, now this is done for two patients so we will place the primary antibody into different boxes. So, for patient 2 we will place it onto the sample onto the slide. Now again this slide is incubated for with this human serum and anti-GST antibody for 2 hours on the rocket. Once the incubation is performed for 2 hours we decan the serum sample make sure that the slide is kept properly and we do not disturb it. Now we take the slide out and put it back in the cassette the same procedure is repeated for the second sample make sure that the magnetic bead is placed before you place the cassette into the into the TBST otherwise the magnetic bead might hit the slide and we can see we can get a blotch in the slide. So, the here you can see the magnetic bead is already kept with the TBST there and then we start the washing again. So, once this washing is complete 3 times 10 minutes we will again remove the cassette rinse it with water to remove the extra buffer and spin it for 2 minutes at 900 RPMs. The slide once dried is then again placed in the box for the secondary antibody incubation.  So, here we will put the solution on to the strip. So, now we will do the same thing for the second sample second patient sample and then since this is a life life sensitive reaction make sure that the slides are covered with aluminum foil or with some black box so that the light does not interfere with the reaction. So, then we will start the incubation for 2 hours in dark after 2 hours we will stop the reaction we will we will do the same procedure but then everything will be performed in dark. Once the slide is dried we take a we go ahead for the scanning of the slides. So, for scanning make sure that the scanner is on for at least 20 minutes so that the lasers are stabilized. Now, so this is one of the process slide which I am going to place on inside the scanner. So, in this scanner we keep the slides in the inverted position that is the surface that contains all the proteins should be facing downwards. So, this is the this is the area where the slide is kept this is the slide holding area place the slide opposite. So, that the proteins spots are facing the uh lasers. Now, we will start the software we will start the scanning. So, this is GenePix Pro software which comes along this scanner. So, this is how the interface looks like. So, now because it's it was a dual channel experiment where we have used both the antibodies together we will scan it at 635 for the size 5 labels and at 532 for the size 3 labels. Once we have set the parameters now we can also adjust the PMT gain. So, PMT gain tells you the saturation or the spot intensity of the slides. So, here I will show you I will give you a demo how the PMT gain can change the contrast of the slide. I will demonstrate how the PMT gain can affect the slide intensities. So, for now if we can see it is getting scanned at 350 uh 350 PMT gain. See suppose if I increase it to 650 you can see how the scanning has increased. Now, if I reduce it to say 200 the intensity of these spots will be reduced. So, for the PMT gain settings we have to make sure that not all the spots are getting saturated. Also we have to make a balance that we are not losing a lot of intense spots. So, for that there is an option called as auto PMT. We have to fill in the details here. Once you press the apply button it will scan at three different PMT settings and it will tell what is the best PMT settings for the slides to be scanned. So, this is how the scanning parameters are defined. Once the scanning parameters are defined we go to the scan area and then we scan the slide. So, this is how the scan slides looks like and here this is a scanning of dual channel. This is how the one of the blocks look like. So, you can see in the red channel how the spots are visible. Also in the green channel how the spots are looking. Now we will lay the grid onto the slide to know how where what is the location of which protein is spot onto the slide. So, this is how the grid looks like. Each grid is unique for each version of the slides. So, now we take the grid and we try to place it to map where the proteins are. So, this is how the grid is laid and then we try to resize the grid for each and every block. So, now you can see the difference between the two blocks. The software has tried to align all the spots with the protein location and this is how the grid is laid this is repeated for each and every block. So, once this grid is laid we then for each and every block we then process the data and then we get the data in form of .gpr file. Hope you have got a basic feel of how the micro experiment is performed to detect the presence of auto antibody in the cancer patients. So, this is one of the applications of protein microarray which can be used to screen multiple number of proteins using a very small amount of patient sample and to detect the abnormalities in the patients. Thank you. Okay. So, I hope you you got a good understanding of how different type of microarray platforms can be used for different applications although the underlying principle remains the same. And many of these steps if you have done the basic you know biochemistry experiments of SDS page and followed by western blots they are very similar you know the various steps are very similar different washing steps are involved and it depends on your application what you want to get out of these arrays you have to provide the either purified protein for the protein interactions or look at the you know serum samples for looking at the auto antibody signature or you can think about any other analyte which you can use as a part of the primary antibody or the first step and then it will be secondary antibody followed by detection of signal. So, depending on your application you are looking at the ptms biomarkers interactors you can select the workflows but remaining steps remain very similar in different type of platforms. So, this experiment you must have noticed that the slight handling becomes very tricky especially during the washing steps and particularly when you have multiple slides you know coming from different patient samples they are all hybridized at the same time. So, handling multiple slides becomes crucial however it is really important to have multiple slides you know parallel processed otherwise you are going to if you process you know only two slides on one day and another two slides on tomorrow then you are going to have much more variability as compared to if you screen 10 slides on the same day then all the 10 patients get the similar kind of treatment and your chances of getting higher reproducibility and better normalization can be achieved. So, we will continue this discussion about using the purified protein based arrays in the next lecture as well and you will learn about how the micro data could be analyzed especially coming from such you know large data sets where many type of control spots become very crucial and your considerations about how to ensure that what we are identifying is the right spot it is not an artifact it is not a false positive and these considerations can only come when we talk in detail about data analysis aspect. So, we will talk to you about micro experiment and same application and data analysis in next class. Thank you.


Iwo Jima and Phoebe's ready group return to his home port of Naval Station Norfolk, December 20th, following a regularly scheduled deployment to the 5th and 6th Fleet areas of responsibility. 1100 sailors and Marines, they've gone for 9 months, doing a fantastic job, a flawless job, called on again and again with changes and new tasks that come along, a risen occasion every time they've been, I couldn't ask for any more, I'm so proud of these kids I could bust and I'm ready to get them turned over to their families here as soon as possible. Daddy, we're so excited, we've been waiting a long, long, night, Monday, waiting to go run up to them and hug them and give them big gloves to keep them. Sailors will welcome home by families and friends just in time for the holidays. In engineering, so usually I have to wait, he's usually one of the last people off the ship, so we wanted to make sure that that wouldn't happen this time, it's going to be awesome to have him Along with the Iwo Jima was a USS New York which completed its first deployment. Reporting for N-Pace East, I am Petty Officer Tamikia Perdue.


Yeah, I mean, obviously, disappointing to lose. And especially when it happens like that, D-Led, our guys have always, it's a resilient group, and you've got to give credit to Washington. They made a play at the end, thought we had the look we wanted, and they got tipped the ball up, and they caught it. So fortunately, that's the way it went. But our guys understand it's always going to sting, but you get back tomorrow, we'll get up, and we'll get back to work and try to correct things and come out swinging, because we've got a big one next weekend in Pittsburgh. I'm not going to go on a scheme, but there's a lot of options in that play, Mike. And the way the Washington defender played it, thankfully, Mark is able to get out of it. And so you come back, and we got plenty of time. We still had our timeouts, and those are decisions you make, and you've got to live with. And so like the look, but unfortunately, the result, not what you're looking for. And hats off to their D-line. I mean, it wasn't like they tipped a lot of balls today and really didn't get a lot of pressure the way our line played, but they made one more play right at the end. And so, yeah, it's natural. You live with that, but that's part of coaching in the NFL. So you've got to be ready to make the calls and give Washington credit they made the play. I'm not going to get in the scheme. We had a wide open look, and we liked Mike. They made a play. Ball tipped, and then it bounced right there, and I got up under it.  Well, I mean, we knew they were going to limit possessions. The way they came out, they tried to throw it. They were aggressive early.  And we threw us a couple. We caught one, and AJ got close on the other. They tried to transition to a run.  They had the ball. They had a pretty long drive. They backed us up. We didn't get out of there. And then they held the ball for most of the third quarter. That's the way it goes. It was probably going to be a low possession game. And then we only had a few more drives after that. We had four total drives in the second half. We had the backed up. We had the drive that came back. They were able to get the field goal. And then the one drive that they actually, they got the sack. And we punted them. We had the long drive at the end. But this one ended the inside of the final play call there. Is that something that you will stew over tonight? Or do you have to let that get? I mean, how do you personally handle it? Yeah, you know, OK. This is an emotional game. And the easy thing to do is to look for some kind of excuse and whatever at the end of the day. Josh, I mean, that's what you live with as a play caller. I like the, you like the look. But again, they had a say and they got tipped the ball. So yeah, sure. You go back and you replay a lot of things that lead up to it. This, that, whatever. I mean, ultimately, when you can take the emotion out, then you've got to be as objective as you can be. And that's what we try to pride ourselves on. And like I said, unfortunately, we came up short. So as you live with those decisions. What was the strategy of letting it run all the way down to take over first? Because I got the ball after half. Wait, challenging to see if they banged the timeout. I thought they would try to do it. They didn't. So I felt it was advantageous with our timeouts and the situation. So that's the game you're playing. They know they're getting the ball after half. So you're trying to, in a situation, not let them double up. And then wanted to go for it. But it was also kind of a stare off to see if they'd use their timeout and they didn't. So I was like, great, let it run down. Use our, we still had plenty of timeouts left. Again, they made a play up front. We were short. They threw it right back to us. We got it down, FERC got in there and had a shot to try to end the half with points. So that's what went into it.  You know, it's a good defense. It really wasn't that different. I mean, that's a credit to them. I mean, a lot of times when you're playing teams at that stato front, they usually don't do a whole lot. And that's not a shot. That's just the reality of it. That's smart. Play to your strengths. So not really. Kind of been seeing if they're going to call it a timeout. Is there a point where you have to maybe? So we've been in a pretty good situation. You got an example of what you're trying to recalibrate? Last year. I'm just saying like, like. I just mean like when you're playing. I'm just trying to. You always look at things that things it's, you get there in a low possession game, when they get the ball, they defer. They get the ball. That's how you steal possession. It's pretty common. The double up turn we use. And that's what they didn't try to do it. And so in that small possession, if you punt it there, you take a timeout or they took one early, they're going to have plenty of time to come down and score. Get in the field, go raise, milk the clock. They get the ball to half, try to bleed it. And so you always look at everything. But our guys have been pretty good at the end of half. Look, it's just you're dealing with what's reality. I mean, there's only excuses, Mike. I mean, you'd love to have everybody. I mean, they're missing guys too. I mean, it's just reality around the national football league. So I don't, of course, you'd love to have all your guys. But that's not, that's not what we lost. As a tip, can I give Mike his play throughout the game? Pretty solid. I mean, they ran the ball pretty well, made some big time plays. Again, we came up short. There's a lot of things that dissect D-Led when you lose. But our guys were, they played pretty physical up front against good front. Obviously, we didn't get it done. Thank you. Coach, did you lack the look in anything about that? Yeah, I thought it was a great play call. CPU was open. Unfortunately, he got his hand up, made a play on it, and bounced their way. And they're being recorded, right? No, I think that's tough, right?  You know, sometimes you can kind of feel defenders in your face, but it's hard to say that you're really kind of thinking about that in that moment. Oh, it was my decision. I thought I had a one-on-one opportunity with the defensive end, and I thought he was crashing down on CP, and maybe I had a chance to get around. And you all are trying to battle and stay in a playoff place. Yeah, it is tough. You know, one of these games, as you're progressing through the season, get bigger and bigger. And, you know, unfortunately, we didn't pull this one out, but at the same time, we got to bounce back and get ready for Pittsburgh. No, we're nowhere near that. Where we stand today, we have an opportunity, you know, however that finishes out in Tampa, you know, obviously we're game back, or maybe however it is, whatever it is. But no, I'm not looking at that right now. You know, when the season's done, maybe you can go back and look at it, and you can pinpoint certain things. But at this moment, no. It's tough. It's tough without Kyle, right? He's just an unbelievable player. And I thought multiple guys kind of stepped up into that role, and you have to, when you're replacing a guy like Kyle, whether it was OZ, whether it was Peru, you know, and FERC. So I thought our guys came out and filled that role nicely. You know, unfortunately, we just didn't make enough place. I think he just came down to the moment, right? Like, you just got to go out there and make a play. I thought our guys had a great job of understanding that this is it. You got to go make a play. And OZ did an unbelievable job in that play. It kind of came out that way.  And the way that they were playing the zone read just gave me opportunities to make plays. Yeah, well, it's nice, right? But like at the same time, like I've been in this long enough, you don't want others to control your destiny. You want to take care of business. You know, fortunately, we left one out there today, but we have opportunities coming up to write the ship and get going. Yeah, just trying to get up as fast as I can.  Worked out. OZ found a window. And you know, at that point in time, just kind of it's kind of chaotic. But you just find find opportunity to get your guard of all. All right. Thank you, guys. Safe travels. Yeah. Thank you.


Lecture 9 As-Salaam-Alaikum. Welcome to virtual university's course on business and technical communication. In today's lesson, we will continue looking at the seven C's of effective communication. In the previous lesson, we looked at completeness and conciseness. And in today's lesson, we will look at consideration and concreteness. In this lecture, you will learn to see your material from your reader's point of view. This means that you will learn to see if your material that you are using, if that is acceptable and comprehensible to your reader or not. A lot of the times what we write is comprehensible to us, but it might not be comprehensible to our reader. So we have to look at it from the reader's point of view as well. You will also learn that you is more desirable than I or we in most instances. A lot of the time when we are writing for technical communication, we use the pronouns I and we. However, it is more desirable to use the pronoun you because you are addressing the reader and it is important to keep the reader in mind and to let the reader know that he or she is important. Also, we will learn that readers like to see these benefits. They like to be addressed directly and they like you to read the material from their point of view. So you need to show that these benefits are a prominent part of your message. You will also learn to consciously use positive words rather than negative words in your communication. You will also learn if you were precise in using facts and figures whenever possible. A lot of the times you are using facts and figures in your communication, in reports, in presentations, etc. and you need to check if you were precise in the facts and the figures that you were using. This is very important so that you do not give the wrong message across. We will also see if you were using active voice more than the passive voice. We've talked about this previously, very briefly, that it is better to use active voice rather than passive voice in a lot of cases because the passive voice makes the message a lot more complicated. Also, we will look at action in verbs and see if the action is in verbs rather than in nouns and also check if you try occasionally using vivid image-building words. However, you need to be careful when you are using these vivid image-building words in business communications. You cannot use them too frequently because then it makes the message too colorful and you might not always want that. One main C of business communication is consideration. Consideration means preparing every message with the message receiver in mind. We talked about this earlier where we said you need to keep the reader in mind and this is really what consideration is. You need to keep not only yourself in mind because you are the one who is writing the message but you need to keep more importantly, you need to keep the reader in mind because it is the reader who needs to understand your message. You need to be sure that you don't lose your temper in your message, you don't accuse and you don't charge your reader without facts. A lot of the times when we are writing a negative message or when we are replying to a message which might not be very pleasant we need to be careful that we do not resort to these things. There are many such messages which are not very positive and we need to be very careful not to say anything which is related to rudeness and in which we feel that the reader is particularly angry or you are being rude to them. Consideration also means that you are foremost aware of the desires of the reader their problems, their circumstances, emotions and probable reaction to your thoughts. Now probable reactions means that you need to be aware of how they will react when they read your message. When you are writing a message, you need to keep in mind that when the reader will read this message what will be his opinion on this message and how he will react after reading this message. This is very important and this is the most important thing of consideration that you have understood the other person. Consideration is the same. You also need to consider that you need to handle the matter from the reader's point of view and this thoughtful consideration is also called the you attitude the human touch or understanding human nature. The you attitude means that the writer is thinking of the reader and he is telling the reader that you the reader are more important than I the writer. There are three specific ways to indicate consideration. The first, as we said earlier, you need to focus on you instead of I or we. You need to show audience benefit or interest in the receiver and you need to emphasize positive pleasant facts. Emphasizing positive pleasant facts means that if there is something negative in the message we should not say it clearly or negatively but we should first emphasize the positive things. Now, looking at the first, focusing on you instead of I or we. Using you does help project a you attitude. You need to use the word you and target the reader more consciously, more directly so that the reader feels more important and the reader knows that you have kept them in mind when you have been writing the message. However, overuse of you can lead to negative reaction especially if the message that you are giving is negative. If you are using you too much to portray a negative message it can seem accusing so you need to be careful. We will say it in everything and we will put it on the reader so it can be possible that the effect of it is not so favorable. That is why we need to use you but it does not mean that we should use you at all times. We need to mix I or we but we need to emphasize you more because the reader will read it and it is more important to us. Let's move on. Focusing on you instead of I or we. You need to create considerate audience-oriented messages and focus on how message receivers will benefit, what they will receive and what they want or need to know. This again is an important consideration. You need to know and you need to show the reader that you are aware of their needs and their perceptions. You need to know what they want to hear and what they want to read and according to your communication you have written. In some cases this focusing on the benefits of the message receiver and what they will receive can be accomplished by emphasis. You may downplay your own feelings to make a point. This basically means that you may emphasize on certain points which will be favorable to the reader and downplay other points. For example, the we attitude would be I am delighted to announce that we have extended our office hours to make shopping more convenient. Now in this sentence there is emphasis on I. I am delighted. I am delighted. However, if we take this from your attitude it will become you will be able to shop evenings with the extended office hours. So now instead of saying we have decided you are saying you will be able to do this. Our decision has been reduced but we have emphasized the benefit of the reader. Another example, the we attitude we are sure you must be frustrated by the length of time to ship your documents to Australia and the you attitude for the same thing would be the documents will be reaching Australia within a week. Now in this example we have taken away all pronouns there is no I, there is no you and it is merely stating that the documents will be reaching Australia at a certain time. This lays more emphasis on the action of what is going to happen other than on the writer or the reader. Now in this example we have taken away all pronouns there is no I, there is no we, there is no you. It is merely stating that the documents will reach Australia at a specific time. The emphasis here is more on the action rather than the feelings of the people because they are not important, it is not important whether we are aware that the person will be frustrated or not. What is more important is what will actually happen because we are aware that the reader is more concerned with having the documents reach Australia in time. So many times it happens that we will remove the I and the you and we will emphasize the action on it because the reader is asking about the action or asking for the result of the action. So we do not need to put the I and the you. Showing consideration for the audience involves more than just using you instead of I or we. Messages that use we can be receiver oriented if we includes the receiver of the message. Many times it happens that we will include the receiver in the we. When we are saying we, we do not necessarily mean just we who are on the side of writing but we can mean you and I. When we say we, it is possible that we are on one side or it is possible that we are on the other side. So we can use we in that sense to include the receiver instead of using you all the time. Sometimes avoiding you can reduce potential defensiveness or allow the recipient to save face. This basically means that if we are using you too much then it might seem accusing to the reader that we might put too much responsibility on the reader. So if we reduce the use of you then the responsibility will be removed from the reader. These decisions that we have to use I or you depend on the content of the message. What we are trying to give in that message and what kind of message. An extreme example of a negative situation is a collection letter with you or your in almost every sentence. If the sentences are insulting, sarcastic, tactless or untrue accusations. Now for example if we need to, if you are a collection agency for example you are sending a bill to somebody and they haven't paid the bill and now you are going to be sending a letter following that bill saying that you haven't paid. If you are emphasizing too much on you then it will seem very negative and it will seem very accusing and tactless that you haven't paid the bill and you haven't paid the bill. So in this type of cases it is better to reduce the use of you. The you in negative situations can be avoided by employing passive voice and making the receiver part of the group. Like we said earlier that what we are talking about is not about the action but about inverting it and instead of saying you have not paid the bill you can say the bill has not been paid by you. So the emphasis in that comes from you and comes from the bill. Or you can say we have not received the bill. So the emphasis on the bill was not on that but it comes from you that you have not received the bill. But it seems a little bit of an accusation. It does not seem like you are blaming the next person. Even though it seems like you are blaming the next person. For example an intensive way of saying something might be you failed to enclose your check in the envelope. Now this seems very intense it seems very accusatory that you did not put the check it is your fault. You have blamed the person but if you say it in another way then you can say in a considerate way you can say the check was not enclosed. This is passive voice. Another way would be the envelope did not have a check in it. This is a depersonalized way of saying it. There is no indication on any person. You are saying there was no check in the envelope. It can be anyone's fault but there is no accusation on anyone. Another example would be an intense way of saying saying it would be your contract tells you plainly that for example you need to give one month's notice before you leave the job. And a more considerate way would be I am always happy to discuss the contract terms with new employees. Now in this the receiver becomes part of a group when you are saying I am happy to discuss terms with new employees then the receiver is part of the group of new employees and therefore he is not he does not seem to be standing apart as a single person who is on one side with the organization on the other side. So it is often that instead of saying you you share a group so that they feel alone with you. In another example you are completely off base in your proposal. Off base means that you are completely moving away from the point or what you had to tell is completely wrong. And the considerate way of saying we have different interpretations of the utility of the proposal. This is sender oriented with acceptance of responsibility. Now this basically means that the sender the person who is writing the message is going to find what you have said we might not agree with it but it's not necessarily because what you have said is wrong it can also be because we are interpreting it in a different way. It's not necessary to say what you said and it's not necessary to say what you said or if you do not like it then it is wrong. It also means that and its interpretation is different. We understand it in some other way. You understood it in some other way. Basically, the seawater is given a face-saving opportunity by doing something like this. It is exactly their fault, but instead of saying it, if you say it like this, then they feel a little less ashamed. Another way would be to say that the proposal plan has three aspects that need clarification. Now in this, even if you have found the proposal plan to be off base, you are not saying that directly. You are basically asking for clarification. This way the reader gets to give some clarification. The reader gets to explain their viewpoint without feeling that they have been accused of being wrong. Another way of showing consideration is that you show audience benefit or interest in the receiver. The first way of showing consideration we said was to use you more than I or V and the second way is to show audience benefit. Readers may react positively when benefits are shown to them. Whenever possible and true, show how your receivers will benefit from whatever the message asks or announces. Now obviously if you are asking for something in a message or if you are announcing something, maybe a new product, you need to show your readers how they will benefit from buying that product or how they will benefit from coming to your company or dealing with your company instead of showing them how you will benefit. For example, if you read an ad in a newspaper advertising computers, you are more attracted to the fact that those computers will benefit you rather than being attracted to the fact that the sale of those computers is going to benefit the company. Whenever you are going to buy something, as a reader, when you are reading an ad or reading a letter you need to have your own benefit. You don't have to look at it as a reader. You don't have to care about how much the company will benefit from it. You just want it to benefit you. That is why when you are writing, you should also consider that the reader should keep you in his place and you should think about how he will benefit from it so that he is interested in buying your product and dealing with your company. Whatever your situation is, your communication is better for yourself. The benefits must meet recipient's needs, address their concerns or offer them rewards. Now in whatever way, depending on who your audience is, the benefits that you are going to be showing in the letter should match the type of audience that you are catering for. You need to keep their needs in mind. You need to keep their concerns in mind. If your letter is going to an audience that is very old, then for them, depending on their concerns, their needs, the benefits will be written in that letter. Obviously, they will not write the benefits that will attract a 6 year old child. That is why you need to keep your audience in mind and you also need to think about what their needs and concerns are. Depending on what letter you are going to write or what correspondence you are going to do or if there will be an advertisement or a report or a proposal, depending on that, keep in mind what will attract that audience. Now, also you must keep in mind that what you offer must be perceived as benefits by the receiver. A lot of the times, what you are offering, you might think, oh this is a very good benefit for my reader, but the reader might not be looking at that as a benefit. You must identify legitimate benefits of your position, policy or product and then put yourself in the place of the receivers to assess their perspective. As I said before, see if the benefits are legitimate, see if the benefits are attractive enough and then put yourself in the position of the reader. The receivers will be more likely to react favorably and do what you suggest. If you show that the benefits are worth the effort and cost you ask them. Now obviously, if you are going to be announcing, for example, a new product, then the readers or the audience will only be interested in that new product if they show that it is worth it or if they know that it is worth it, your product then has to be attractive to the person who is buying that product and therefore the communication that you are sending out with that product or in relation to that product should announce those benefits and the reader should be able to see clearly that the product is worth it and that the product is worth it. So, if you are going to be announcing for example, a new product, then the reader should be able to see clearly that those benefits do exist. If you are giving that benefit to the reader, you have made a new policy, you work in an insurance company and you have a new policy about it or you are selling cars, receivers will be more likely to react favorably and do what you suggest. If you show that the benefits are worth the effort and cost you ask them. Now obviously, if you are writing a proposal for your employer, then and the employer is the audience and the receiver in this case, then they are more likely to react favorably if whatever you have requested in your proposal, whatever benefits you have asked for or whatever changes you want made in the company and you feel that there will be benefits for the company if they seem to be favorable and if they seem to be cost effective, if you want to bring any change in your company and you have written a proposal for your boss, then until your employer and your boss do not see that it is beneficial, they will not be willing to act on it. That is why it is important that they also see the benefits in their favor. In situations where actual direct benefit is impossible or irrelevant to the subject matter, the message should at least show concern for the recipient. There are many situations where you will not have direct benefit, but at least then you can show that you have concern for the reader. It is not necessary that you are benefiting from it, but at least if they have this idea that you are communicating with them, you are also concerned for them then they are also favorable for you as a writer. Also, mainly inserting the word you does not ensure the you attitude as shown in this sentence. You will be glad to know that we now have a walk-up window open 7 to 9 a.m. and 3 to 5 p.m. every weekday. Now, you might be announcing something, you might say, we have a window or we have a walk-in window or a counter open from this time to this time, but a lot of readers might say, so what? The fact that you are using you does not mean that you are actually, you have actually highlighted the benefit that the reader will get out of what you are announcing. Another way to show consideration is to emphasize pleasant and positive facts. This means stressing what can be done instead of what can not be done. If we will tell negative things for everything, that this cannot be done in this way, then obviously it is not so pleasant. However, if you will say that this is the problem but this is the solution or this is the problem then we can tackle it in this way. If you write it in your communication or if you will tell it, then it is obviously more positive. The other one was negative. So it is better that you stress what is positive and not stress what is negative. Then stress the solution which is positive. Also, you must focus on words your recipient considers favorable. A lot of the words, you might be considering favorable so please focus on words that you feel that the recipient will find favorable. An example of emphasizing positive pleasant points it would be negative to say it is impossible to open an account for you today. A positive way of saying it would be as soon as your signature card reaches us we will gladly open an account for you. The first thing in which you said it will be impossible to open an account, your client will feel that you should not do this work. Or it is very difficult and it cannot be done. Maybe they will go to some other bank to open their account. But if you will tell them the solution and basically you will tell them why they cannot open an account today then it is more effective communication. Then they can act on it and they will feel that you are a person for them and you are giving them a solution on which they can act. Also, another example we do not refund if the returned item is sold and unsealable. Basically you are saying that we do not take any sold item back if that item is opened or destroyed or cannot be sold. So in this, your client is making a direct refusal that we cannot take this item. What happens is we refund when the returned item is clean and resealable. Rather than giving the negative things soiled and unsealable you will say we will refund we will happily refund what you want to refund but only when it is clean and we can sell it again. That is the same thing. Both of you are making a refusal that this is not clean and cannot be sold. But the other one you are saying we will definitely take it but we will take the clean item instead of saying it is dirty. Another case of saying something negatively would be when you travel on the company expense you will not receive first class fare. This might be something that is obvious to a lot of people but if you say it this way it sounds very negative. A positive way of saying this would be your approved fare is not for tourist class. The positive way of saying this would be when you travel on company expense your approved fare is for economy class. Now in this by saying the approved fare is for economy class basically you are telling the reader what benefit he has. You are saying the benefit you are getting is that you are getting fare for economy class. Whereas in the other way of saying it you are saying that you are not getting the benefit of travelling first class. If you are getting the benefit then it is better that the benefit is less it is not the first class it is economy class but the benefit you are giving you should emphasize it. Do not emphasize that you are not giving it. Amongst the positive words to which the reader reacts favorably some of them are benefit, cordial, happy, generous, loyal, pleasure, thanks, thoughtful etc. Keep using cordial words in your communication that makes the reader feel better rather than using negative words. Amongst the negative words the reader might react favorably to may be due to past experience with some words the words are blame, complaint, failed, fault, regret, reject, trouble, unfair and many others. All these are negative blame, complaint etc. All these have negative connotations and the reader will not have a good reaction if it is written in a letter or a proposal or correspondence. So it is better that the positive words which the reader reacts favorably should be used more and these negative words should be used less. Obviously there will be many situations where you will have to use negative words in different ways and the rest of the text will be a little tactful. So these were some points that we need to keep in mind when we want to write with consideration. Let's briefly have a look at a checklist so that you remember all the points and then we will move on to concreteness. For consideration, for considerate writing you need to make sure that your material is from the reader's point of view. Also that you is more desirable than I or we in most instances you need to keep this in mind. You also need to remember that readers like to see these benefits and you need to be sure that benefits are a prominent part of your message and also consciously use positive words. If you do all these things then you are being considerate as a writer. Now coming to the fourth C amongst the seven C's is concreteness. Communicating concretely means being specific, definite and vivid rather than being vague and general. Now concreteness as we just read on the slide if you are concrete, if you are vivid if you are very clear in your message then your message is concrete. If you are vague and very general in what you are saying then your message is not concrete. If you use such vocabulary and such words in your message which do not have a very close meaning then your message will not have a close meaning. It will feel very vague that maybe you do not know what you are trying to say and you are talking about what you are saying here and there. It is better to use the vocabulary and the message you want to give A lot of the times misunderstanding of words have produced tragedies both in war and in peace and in business and non-business situations. So if you are vague then it leads to misunderstandings and these misunderstandings can lead to large problems or small problems but whatever the case, vagueness does lead to misunderstandings. So it is better to be concrete and more precise in what you are saying so that there are no misunderstandings. Often concreteness means using denoted, direct, explicit often dictionary based words rather than connotative words. Denotative words will be the direct meaning which the reader will get to know. If you search for the word dictionary then it will have the same meaning which you are trying to give in the message. Connotative words will be whose meaning in the dictionary will not be the meaning in your message. You are saying something and you are using some other asl words whose direct meaning cannot reach the audience. So it is better to use your message in a concrete way using denoted words whose direct meaning is the actual meaning. The benefits to business professionals of using words and figures are that your receiver knows exactly what is desired from them and also that when you supply specific surreal, you increase the likelihood that your message will be interpreted the way you intended. Obviously when you say something clearly or in a toast way or in a specific way the chance of misunderstanding will be reduced and the chance of understanding and understanding will increase. So it is better to use concrete words and keep the vagueness in your communication The following guidelines should be followed to compose concrete convincing messages. You need to use specific facts and figures. You need to put actions to words and use vivid image building words. Now using specific facts and figures. It is desirable to be precise and concrete both in written and oral technical communications. When we talk about facts and figures they are used in technical communication more often. In common communication we use very few figures. Obviously when we are doing casual non-business communication we don't need numbers and figures. It is very important to be precise and precise and use as many facts and figures as possible to understand our knowledge but more important is that our message is more concrete and the reader knows what is the situation actually. A vague sentence would be the students GMAT scores are higher a more concrete way of saying this would be in 1996 the GMAT scores averaged 600 by 1997 they had risen to 60. Now in the first instance the vague situation the students GMAT scores are higher higher than what? Higher than whose GMAT scores? If you are telling something high or low you will have to tell some figures to the reader to know what is higher or what is lower. It is better to give more concrete information to the reader in a concise way. To give facts and figures it is not about extending your sentences you have to keep the conciseness that we talked about in the last lecture but you have to another example would be Eastern Europe is making progress in obtaining investment. Now this is a very correct statement and if you say that this company or this country is getting more investment but it is not very concrete it does not give any countries within in this example it does not give any countries within Eastern Europe what is the amount of investment in Eastern Europe? What is the amount of investment in any sector? These things are not communicated in this message a better way would be in 1990 the investment in Eastern Europe was 30 million dollars today that figure has increased by 12 percent so here again you are comparing you are contrasting with what was before and what is now and you are also giving figures another vague example would be she is a brain now this sounds very casual it is obviously very vague it sounds very casual and it also sounds a bit slangy that is why it is called a concrete precise way her grade point average in 1996 was 3.9 on a 4 point scale this shows obviously if someone has a 3.9 GPA out of 4 they will be very intelligent their mind will be very good but she is a brain or she is very brain instead of saying it like this by looking at the facts and figures it clearly shows how intelligent they are there are however some exceptions to the facts and figures rule when it is not possible to be specific you may not have precise facts and figures so it might not be possible to be specific you may also not wish to use facts and figures when you wish to be diplomatic instead of saying something harsh like you have missed 3 invitations to my office you may be more tactful by saying I have sent you several reminders to see me in my office now basically instead of using the figure of 3 you are just saying several that is really the only difference but it is because you might not want to pinpoint exactly how many times you invited that person or you summoned that person to your office and exactly how many times they refused to come so instead of saying something harsh you just wrap it in a more tactful word and it is the same message that goes across also you might not want to use figures when exact figures are unimportant a lot of the times it is not very important to give exactly what the figure is if you are saying something like in this example more than half the committee was present it might be enough because the reader knows what the full number of people on the committee was and also it is not important if the committee was 10 people and 5 were present or if it was 20 people and 10 were present what is important is that there was a 50% attendance or that half the people were there and half the people were not there so in that sense then it might not be very important to use a direct figure here you need to to show concreteness you need to also put action in your verbs you need to use active rather than passive verbs and put action in your verbs rather than in your nouns and infinitives when you speak in an active voice then it is more direct and it looks more concrete when you put action in your verbs then it is a clear message instead of putting action in nouns for example using the active voice a passive verb has 3 characteristics the subject does not do the acting in the sentence also the verb consists of 2 or more words for example 2b and number 3 the word by is expressed or implied by whom etc let's have a look for example in passive voice sentence where the subject receives the action the tests were administered by the professors here the action is it is the subject who is receiving the action the test was administered by whom by the professors the active way of saying the same thing would be the professors administered the test here it is the subject who is performing the action it is not an action which is being received by the subject but an action which is being taken by the subject it is more direct it is more active it is the professor who is in control of the situation so another example where the subject receives the action would be the grades of the students will be sent to you by the school the active way of saying this would be much more direct where the subject is performing the action the school will send students their grades the sentence is much more direct because it is the active voice and it is much clearer the use of active verbs makes the sentences more specific for example the dean decided is more specific than a decision has been made it makes the sentences more personal you will note is both personal and specific rather than it will be noted active verbs also make sentences more concise the figures show as compared to it is shown by the figures and they make sentences more emphatic they give more emphasis to the subject it makes the sentences more concise figures show as compared to it is shown by the figures also they make the sentences more emphatic it gives more emphasis to the sentences the students held a contest more emphasis to the subject as compared to a contest was held by the students so all these things obviously make your communication clearer more effective more concrete rather than being vague by using passive voice also when you want to avoid personal blunt accusations you use passive voice earlier we looked at active voice and how to make your communication more direct but there are times when you don't want to be so direct especially when it might being direct might seem that you are being blunt and that you are making an accusation there and also when it might seem that you are giving a negative message there it might be better to use passive voice for example the October check was not included is more tactful than saying you failed to include the October check because again in this first by the use of you you are giving emphasis to the subject to the receiver thereby you are accusing the receiver of not including the check so it is better to use passive voice in this type of case so that the receiver or the reader doesn't get blamed also attendance at the meeting is required is less hard than you must attend the meeting by saying at the meeting is required by all employees you are basically one you are including the reader as a group instead of pointing out to the reader and also you are using the passive voice giving a notice but you are not giving a direct order also when you want to stress the object of the action you would use passive voice instead of active voice you are invited is much better than we invite you because here you want to give the emphasis on you rather than we your savings account is insured up to something up to 100,000 rupees in this you have intentionally stressed your savings account rather than we have insured your account you will also put actions in verbs not in nouns to make your message more concrete a lot of the times the action can be hiding in a quiet noun for example, professor will give consideration to the report here the action of giving consideration is not directly stated because consideration the verb has been included in the noun or the verb of consider has been included in the noun of consideration it would be better to say professor will consider the report this is more direct and the action is very obvious for example you can have an action hiding in an infinitive the duty of the secretary is to check all the incoming mail and to record it the action in verb would be the secretary checks all the incoming mail and records it you will also use vivid image building words to make your message more complete and you can make your message forceful by using comparisons sensory appeals, figurative language concrete nouns and well chosen adjectives comparisons can be for example a bland image would be this is a long letter here there is no real comparison and it is a very bland statement a more vivid image would be this letter is three times as long as you said it would be this actually gives you an idea of exactly how long a letter it is from what was what the intention was that is why you made it more concrete you clarified and compared the image would be better in the reader's mind using figurative language literally would be her work in groups was exemplary and figurative language would be she could be called the spark plug of the group by saying this you are making an image and you are making the image more concrete and you are making your message more concrete now at the end a quick checklist let's see if you have got all the points of concreteness and when you finish writing your messages it would be good to have a look at this checklist as well you need to check if you were precise in using facts and figures whenever possible no mistake was made in your figures no mistake was made let me tell you did you use active voice more than passive is there action in verbs rather than in nouns and also did you try occasionally to use vivid image building words but in business writing remember use them sparingly you do not want to use image building words and vivid vocabulary too much in business writing in this lesson we have looked at two more seas of the seven seas of effective communication we have looked at concreteness and consideration and we will complete this in the following lectures that's all for this lesson for today Allah Hafiz


The 31st District State Senate race is contentious, and it's also very expensive. According to the Kentucky Registry of Election Finance, as of the 15-day reporting deadline for campaign contributions and disbursements, Democratic challenger Glenn Martin Hammond and incumbent Democrat Ray Jones have collectively received nearly $600,000 in the campaign and have made disbursements totaling nearly $485,000. Jones has out-collected Hammond by more than two and a half times, and he's outspent Hammond, despite Hammond's critical campaign against Jones by more than five times. Hammond's political spokesperson, Kim Javidin, said an expensive campaign was expected. You can't be the 16-year-entrenched career politician like Ray Jones on the cheek. Jones has spent years climbing the political ladder in Frankford, developing relationships with wealthy liberals from Louisville, doling out cold severance money to his political allies. All of those favors add up in the form of political contributions, and to compete with that little money, you know, it takes money. Jones said his large amount of spending has largely been to defend his character and his record against attacks by Hammond's campaign. I wish that this race had been focused on issues instead of, you know, mud-slinging and just downright dirty politics. You know, when you take commercials and distort what I say, take a joke and distort what I say to try to make your campaign look better, there's something wrong with that. And I'm proud of what we've done, and I'm proud to have the support of so many people throughout the 31st district. As of the 32-day campaign finance filing deadline, the race was the most expensive of any political race for state or local office, and with just a few days remaining until the May 17th primary election, the spending is not done. We'll have a strong presence, and, you know, on the ground, Glenn has been working relentlessly, campaigning on the ground throughout the district in all five counties. We'll have a major presence on radio and television, and we really feel good about going into these last few days. I would think that most of the advertising and that type of thing has already been done. So now it's getting your votes out, talking to your friends, talking to your supporters, making sure you're getting people out to vote. None of us can predict what the turnout's going to be in this democratic primary, but I think there's a lot of enthusiasm for our campaign. The primary election is next Tuesday, May 17th, reporting in Pike County for EKB News. I'm Chris Anderson.


Good evening everyone. We are back again with the case of the month. A 25-year-old female presented with vague history of neck pain. MRI with contrast of brain including the base of skull was asked for. The MR showed heterogeneous altered signal intensity lesions involving the basis venoid, left pterygoid plates including the occipital condyles with associated mild soft tissue component. These lesions showed diffusion restriction, heterogeneous enhancement on post-contrast study with multiple areas of peripheral ring enhancement and central necrosis. In addition, localizer sequences of thorax were obtained which showed an expansile lesion involving the anterior aspect of right third rib. CT cuts of the base of the skull showed permeative osteolytic lesions in the mentioned bones with no evidence of matrix mineralization. In view of the lesion involving right third rib, CT screening of chest and abdomen was done. CT showed expansile leitic lesion involving the rib. A permeative leitic lesion was seen in spinous process of D11 vertebra. Leitic lesions showing a radiolucent rim and central dense sclerotic bone were seen in left pubic bone and right iliac bone consistent with button sequestrum. Erosions and irregularity were seen along the articular aspects of right sacroiliac joint consistent with sacroiliatus. The cranial vault was normal. Lung fields were clear with no evidence of medastinal or hyalurrimphidinopathy. Domen and Pelvis showed no obvious lesion on the plain CT. Permeative leitic lesions in base of skull can be seen in osteomyelitis. Similar lesions can also be seen in metastasis. Although not common, similar lesions have been described in eosinophilic granuloma or Langerhensel histiocytosis as well. Button sequestrum has typically been described in tuberculosis and eosinophilic granuloma or Langerhensel histiocytosis. Unilateral sacroiliatus involving the right SI joint in this case would be typically seen in infective sacroiliatus. Expansile leitic lesion involving the anterior aspect of right third rib can be seen in multiple etiologies such as osteomyelitis, eosinophilic granuloma, metis, fibrous dysplasia among other etiologies. On the basis of all these findings, multiple lesions in multiple locations, differential diagnosis in decreasing order of their likelihood were multifocal osteomyelitis, histiocytosis and metastasis. Myeloma was excluded as no lesions were seen in the skull vault or in the mandible. The patient was sent back to us for FNAC which revealed pus. No acid-fast bacilli were seen on microscopic examination. The sample was sent for CB NAT which is a PCR-based examination for tuberculosis and the report came back positive for tuberculosis. The take-home message from this case would be not all multifocal leitic lesions are metastasis or myeloma. Localizer sequences on MRI and CT screening of other parts can yield a great deal of information and help in narrowing down to probable differential diagnosis. Thank you.