I've discussed before the merits of learning from your children: especially in the world of social networking. I recently observed at close quarters another excellent application.
My daughter is shortly off to University. Putting aside my own feelings at seeing her fly the nest, I remember my own university experience. I attended a sixth form college with very little contact with the outside world. I attended a couple of formal university open days, applied to a few and was given an offer based on achieving target grades. I waited until my exam results emerged and, if in doubt, rang the university for confirmation. I filled in some more forms for accommodation and then turned up on day one excited, bewildered and a little nervous.
The road to university is very different these days and much better for it. Along the way to acceptance my daughter has attended summer schools, sat entrance examinations, visited the universities through open days had interview days and corresponded with the academic departments. At each stage she has added Facebook friends and used the online student forums. This meant that as each was waiting to hear results or offers they were able to consult and console with their own support network. It's much better not hearing anything if you know that no-one else has heard anything either! It also supplies a safety net for asking questions and setting expectations. For the universities this must undoubtedly provide valuable insight into the student experience which can only improve their recruitment in future.
Once she was accepted she was contacted by two students from her university who have been allocated as her college accommodation "parents". They have introduced themselves and made her feel welcome. She will have two people to meet immediately on arrival who can show her the ropes, ensuring she gets fed and watered but also engaged with the social side from the start – University can be a lonely place for anyone not naturally outgoing. This has taken away a lot of the trepidation and given her something to look forward to and us a lot of peace of mind. She was then contacted by a subject "brother" – someone studying her course who will help her out with the subject – where the lectures are and someone she can ask about the workload. She has also been in touch with a group of other people who have now been accepted onto her course or her accommodation who are sharing the same experiences.
Although I am delighted for my daughter and relieved as a parent I am interested with the parallel with the workplace. The obvious links are with the recruitment process. How much better to welcome in a new employee through the interview preparation and then through their notice period by social networking. Could you get a quicker return on the salary investment and a happier new recruit who is more engaged with the company culture?
In 1929 Frigyes Karinthy summarised the future potential for friendship networks in a world where communications and travel were easier and cheaper. Six Degrees Of Separation was the model which suggested that two people anywhere in the world could be connected with each other by a chain of introductions. In the 1960s Stanley Milgram conducted experiments to prove the theory. Earlier this year Facebook announced that Facebook users were 4.74 degrees apart on average.
This is a file from the Wikimedia Commons by Dannie_walker.
It figures that if you want to communicate a message to as many people as possible you need to understand a little more about these interconnections. Firstly a tweet which is retweeted can rapidly go a long way and secondly you may be interested in curtailing the connections in some way.
Firstly, the bigger the numbers, the harder it is to even monitor the conversation. It would make sense that you would want a mutual social relationship with your community and so you would "see" all their comments, statuses and posts. The sheer volume of noise this generates means it is difficult, even with the best monitoring tools, to make sense of what is happening – even harder to engage in a meaningful way with your community. This is the equivalent of the doors to the trade show opening and a wall of people making their way directly to your stand. The people manning the stand would be completely overwhelmed and be unable to identify the real prospects amongst the timewasters.
The objective is not just to acquire and keep the community captive. The challenge is to nurture and develop relationships with the community on as personalised basis as possible. A monitoring and listening approach will look out for references to you, your company, your products, your competitors and market issues. The hierarchy would suggest that with limited resources you want to make sure that the defensive position is protected first – that the mentions of you and your products are picked up and responded to. This could still leave a large part of the community untapped and feeling unloved. This is similar to having a stand and just handing out promotional literature. It's a fairly blunt marketing strategy. The best use of trade shows involves taking the right people to one side and discussing their detailed requirements over a coffee.
The final reason why numbers are unimportant is that you will be judged by the company you keep. The Social Influence scorekeepers look not only at numbers but the strength of the community you interact with. As your social community is often visible, if it is largely made up of pornbots and people from different continents it doesn't reflect well on your housekeeping. It may mean that potential followers decide not to. Again, taking the trade show analogy, we all scout the whole venue first and make value judgements over which stands are worth visiting. Of course there will be those you have heard of in advance but there will also be those that look popular or have interesting content worth exploring. Similarly you avoid the ones where the staff look uninterested or uninteresting and the stands which are full of students. As a supplier you need to create a positive first impression just to have a chance of developing the relationship further.
With those caveats, how should you build your Twitter community?
To start with – really think carefully about your Twitter name and profile. The name is important for making it easy for people to refer to you and contact you. The hard work is done by Twitter when you reply to someone but if you want to DM someone or refer to them in a normal tweet, underscores and numbers can mean you never get the message. Keep it as simple as possible. If all of the remotely basic versions of your company name are taken choose a different path. For example if you are a joiner called John Smith it is far better to go for @HappyJoiner than @john__smith377. Think in advance also about how other people at your company could get involved. If this is likely @JoinerJohn could easily be supplemented in future by @JoinerRichard but @HappyJoiner would only suggest Richard is in a bad mood or unrelated. Research shows that the photograph plays a really important part in whether people look at your tweets or not. As a result, never leave the egg image, and pick a photograph which is of a person or an image large enough that it can be seen clearly as a thumbnail. Twitter profiles are one of those areas where you don't want to be too arty. You then have a limit of 160 characters. In this space you must say clearly what you do, try and differentiate yourself, explain where you are (a bugbear of mine) and who you want to follow you. The Twitter search will be used by people so think of the words you would want to be searchable in your profile. You can't say everything you want to in 160 characters but you do also have space to add a url so have a clear strategy for where you are going to send people for more information.
I find that using lists and targets is the best way to stay on top of things. In Twitter there are some real constraints which you need to manage your way around. The most obvious of these is are the 2000 barrier on follows and the dangers of being suspended if you unfollow too rapidly. It would be much easier if you could spend hours searching for "your kind of Twitterer", follow them all and then wait for them to follow back. Unfortunately Twitter puts a set of hurdles in place to force true engagement. The first of these is that you can't follow more than 2000 accounts unless you also have 2000 followers (approximately). There are similar hurdles as the number increases but the problem is the same. As you get nearer to the limit you find yourself carefully unfollowing to give yourself headroom.
I find it easiest to stay focussed from the start and not automatically follow everyone back. When I follow people I really try and concentrate on people that I want to engage with – potential customers, influencers, influences, relevant suppliers, customers and competitors. If the Twitter account doesn't meet the profile: don't follow! Of course this can appear rude if you ignore a follower by not following back. The simple way to address this is to publish a charter of what your account is about and who you want to follow you. When choosing who to follow always look at their most recent tweets – what's the point of following someone who last tweeted three months ago? Similarly – what's their balance of follows and followers. If you follow someone who has 13,000 followers but only follows 100 is their really any point? If you want to learn from their pronouncement then maybe but the chances are they won't ever see one of your tweets. Interaction and referencing is the best approach with these accounts.
I also find it useful to maintain lists from the start. Lists are a really good way of grouping Twitter followers and followers according to whatever criteria you want. Make sure you make the lists secure if you don't want to offend them by seeing how you categorise them (people will be notified they have been added to a list unless you do so). This will also come in handly when you want to be proactive – you can quickly see how many followers of a particular type you have (a quick check is to fill lists right up until you reach the 500 limit and then create a second list called Customer2 for example). These can be exported into spreadsheets and databases for further analysis. This is a very tedious job to do later so its best to stay on top of it as part of your operating procedures from the start.
One of these lists should be targets. These are people who initially you would follow and who you would love to follow you back. These may be journalists, executives, or key customers. They will often have thousands of followers and be extremely selective about who they follow. Importantly they are also likely to be very busy. If you don't know who you are aiming for you are never going to engage with them. Tweetdeck is a good tool for monitoring their conversation in different lists – this target list may only have one or two tweets a day and most of them will not give you an opportunity. However, if you monitor in real time you can also react at a time when you know they are catching up on their social media and may well see your tweet. To give yourself a chance of getting a follow you need to be seen first and foremost and you then need to give them a reason to! Examples of reasons may be that you retweet their comments (everyone likes retweets), that you provide more information on a subject they are tweeting about currently, or simply that they entertain you and they retweet your own creation. It's a fine balance between stalker and someone who is genuinely interested in them so make sure you control frequency and content carefully.
Finally, regularly have a purge of follows. There are some good tools available which analyse people you follow who don't follow you back. The same tools can manage removal of the oldest first and also give some indication of the likelihood you should expect a follow back based on the relative numbers of follows/followers. I regularly have a look at unfollowing up to 50 at a time. This gives headroom for following more people who may be interested in participating in your community without causing concern with Twitter. This is a good practice also because some of these may not be following back only because they overlooked you. Unfollowing and following back may be all it takes to recruit!
I really believe that at the end of every working day it is important to know whether you are winning or losing. It's a viewpoint that ensures you stop doing pointless things and push harder on the things that work. When times are tough it puts it in perspective if you can look back at previous victories and can see that the current challenges are only on the journey to something good – showing the light at the end of the tunnel. Fundamental to this are very clear objectives and targets.
Social media is no different . We're putting in the effort for a reason and it is important to understand, and more specifically for our bosses to understand, what the return looks like. I've blogged previously to indicate that the Social Media metric is Return on Influence. The emphasis on monetising social media is spawning the term social commerce.
In my mind the challenge is to think of social media as a channel. With my contact centre manager hat on I am used to understanding the return on investment of my activities. My clients understand their customers and they believe that the right balance of cost and service for their customers is to target me on answering 80% of their calls within 20 seconds for example. I know that to achieve this Erlang calculations tell me the number of people I need to have answering telephone calls at each part of the day. I can calculate my total costs by considering the amount of shrinkage I need to allow for holidays, sickness, lieu days, training, toilet breaks etc. Looking at it from the client's side I can consider the value of each sale they make and the conversion rate against inbound calls. By factoring in the margin they make on each item I can work out a return on their investment in my contact centre. The important thing about inbound activities, whether they are telephone, white mail, web chat or email is that they are demand driven. In a purely inbound world the handle on the sausage machine turns and 1000 contacts produces x number of orders and so x number of products need to be manufactured to meet the demand. The emphasis in call centre operations these days is to minimise cost through first time resolution and to focus on customer satisfaction (which may require a few more touch points).
Social media turns the model on its head because, used well, it seeks out the conversation. I will discuss the merits of cars many times more than I will contact an automotive company's help line. The challenge for automotive manufacturers is to build a relationship with me so that when the time comes to monetise my interest I understand the brand. It is much, much more complex though because I have a part to play in the influence of others too. Everyone likes to be seen as a clever consumer and so we tend to think that the car we drive/the holiday we chose/the clothes we wear were the best choices and we rate them accordingly. Bazaarvoice research as indicated by the infographic shows the difference between the Millennials (those born between 1981 and 2000 also known as Generation Y) and the Baby Boomers (1946-1964) in the importance of User Generated Content to their purchasing decisions. The bottom line is that the smart social commerce players want the potential purchasers of the car I drive to talk to me about my experiences, not send them a brochure.
So coming back to targets, the objective of the social commerce practitioner is to create and grow a carefully focussed community and to groom it with a light touch. Whereas my contact centre with its traditional channels is governed by demand, the perfect community would be the place where THE conversations happen. Only the Community Manager fully understands that the Community exists, its scale and its influence because it is built on layers of blogs, websites, Twitter feeds, Pinterest pages, and Facebook pages. Original content is created to feed the interest of participants but also the movers and shakers will participate. Taking the automotive example, it's a big day when Jeremy Clarkson comments on your blogpost. And here is a further challenge; while it may be a great day if he likes what you have to say, woebetide it if he decides that today is the day to vent his spleen. Of course Mr Clarkson is subject to social pressure too – he wants to be seen building brand Clarkson by being Clarkson-esque but carrying some serious weight too. If your offer great cars and great service he is going to face a backlash if he decides to buck the trend.
I like to think of social media like water. It is a very precious resource that needs protecting and keeping pure but it is also an unpredictable natural resource – you will never master it. At best you can protect against the extreme damage and channel it to provide benefits in a wide range of ways but knowing that your best laid plans at some point have to change due to either drought or flood.
Initially your community of customers will be hugely grateful for the supply of fresh water you have provided for them to bathe, drink, create water features, enjoy as a natural spectacle, fish, swim, sail etc The moment you become complacent however they will start to scrutinise what you are doing more closely and challenge the price and service you provide!
So, at the end of today, how well are you managing the water? Market research is a key measure which indicates how well you are doing. You will never get the precision of knowing that a particular advertisement played well but the brand perception should move in accordance with your activities. There will be instances where the influence of social media can be captured precisely. Within the contact centre we regularly capture which specific advertising treatment triggered the purchase and the same crude mechanism could identify those customers who are following on Twitter for example. The customer database will grow with today's name address, telephone number and email address being appended with Twitter name. Permission to contact by mail with carefully chosen offers could be altered to requesting permission to "friend" on Facebook.
Your customer satisfaction scores should also improve if you are proactively listening and engaging rather than relying on them getting in touch for help. Associated with this will be the day-to-day little wins – the new customer who takes the time to explain that the reason they are purchasing was a positive review they saw on Amazon or recommendations from Twitter followers.
As a call centre manager it's easy to slip into the lazy habit of imagining your customer holding a telephone to their ear in their hallway. This wouldn't matter if it wasn't that we tend to design all our products and services around a particular image and the image in our head can often be from times gone by. We have been programmed to design projects with a payback over a number of years but our world and our customers are changing.
30 years ago the typical customer communication was via post. In my first working office I remember the telex machine stood as a silent reminder of a channel dying out. The fax machine was used regularly as a way of sending images. I wrote letters which went through the typing pool before being sent, hopefully to receive a response within a week. We prospected in those days by sending an introductory letter followed up by a telephone call a few days later.
Then came email, a wonderful invention geared towards improving communication within our business, and shortly afterwards came the web with access to some interesting bulletin boards of information. Around the same time the first handheld devices I remember – the Psion organiser – emerged as an alternative to the Filofax. It incorporated a diary, contacts database and clock. The smartest sales reps around would flip one open at a moment's notice. The original model in 1984 cost £99 – around £285 in today's money and I remember being extremely envious of them but being unable to justify such enormous expense. At the same time the PC was developing and I remember selling a proprietary standalone computer, second-hand, for the equivalent of £2,500. It could just about run a spreadsheet and a word processing package. The first mobile phones were enormous and they were rationed like gold dust – expensive to buy and expensive to use. This was at the back end of a generation who were grateful for telephones, never mind mobile phones, and were brought up to ration their use. I worked with Americans at this time who were astonished at the British cultural aversion to using the phone.
Over the years the handsets got smaller but still did pretty much the same thing. Their usage became more widespread but I still remember the shock when teenagers started to get mobile phones. What would they use them for – who would they call? They were clear luxury items.
The IBM PC launch started a revolution too. The difference with the IBM PC was actually the Microsoft operating system and the access to software it enabled – at that stage no-one knew about Microsoft and everyone knew about IBM. The thing that impressed me about the first IBM PC I saw was the graphics rather than the machine itself. The disks, the chips and the graphics capabilities improved rapidly. The first laptops I remember were actually "luggable" devices used by auditors on the move. Very heavy and with very poor monitors. Compaq launched some comparatively tiny machines and a new market was created.
Around the mid 1990s I began to really dabble with gadgetry and I had a string of pretty good but very expensive HTC and HP devices which involved using a stylus and could access mobile websites in a text only form using WAP. HP had an online shop of apps – there must have been a couple of hundred to choose from at one point. The growth of the mobile networks in coverage and bandwidth and the development of broadband with wireless internet have changed the face of technology in the 2000s.
The launch of the iPhone happened in 2007. Since then there has been the meteoric rise of the Blackberry, now tailing off. The seeming dominance of Apple based on vastly more apps and ease of use is now being eroded first by Android and potentially by Windows 7. The phones at first got smaller and are now getting bigger. Tablets have been around for at least 20 years but the iPad transformed their market and now tablets are getting smaller.
1. At the time, each of these stages seemed revolutionary. You couldn't imagine wanting or needing anything more. The first Psion Organiser was every bit as groundbreaking and revolutionary as the iPhone.
2. The pace of change is phenomenal and it is impossible to pick long-term winners. The days of a dominant supplier are numbered.
3. While much of the change is driven by technology making things possible, a significant part of the change is driven by consumers.
While we need to spend a significant amount of time thinking about a social media strategy based on applications we also need to think about where and how the applications are being used. We need to build strategies and platforms to take advantage of opportunities with customer behaviour across all platforms and not try to pick winners. Anyone who doesn't adapt their website to serve customers using a mobile device is missing a trick. Service strategies reliant on customers quoting a long account number from memory or a recent bill, making a phone call, or writing an email are completely outdated.


92.1 & 93.5 Wild West Country » Is a Carrie Underwood Workout Really a Prize?
Planking. Push ups. Jump squats. Russian twists. In any other universe, that would be more of a punishment than a prize.
But for one San Diego woman, it was the win of a lifetime, because she was doing it all with Carrie Underwood.
Through her local radio station , Leslie was the chosen one to go to San Antonio and work out with Underwood.
And even though Leslie says she never felt intimidated and that Underwood never pressured her or judged her, it does sound like she couldn't help but compare herself to the star.
"I could not keep up. She's a beast. I was doing 5 to 8-pound weights, while she was doing 20. She did half-hour planks, I swear to God. I could do a minute and a half plank, and then I was on the treadmill, and she was still planking," she said.
Oh, and Underwood was singing Van Halen songs the whole time.
"She's just tiny and she's beautiful and she's perfect," Leslie confirmed.


text:
Comparison of benign peritoneal fluid- and ovarian cancer ascites-derived extracellular vesicle RNA biomarkers
Cindy M. Yamamoto1Email authorView ORCID ID profile,
Melanie L. Oakes1,
Taku Murakami1,
Michael G. Muto2,
Ross S. Berkowitz2 and
Shu-Wing Ng2, 3
Journal of Ovarian Research201811:20
Received: 2 November 2017
Extracellular vesicles (EVs) are considered as a new class of resources for potential biomarkers. We analyzed expression of specific mRNA and miRNA in EVs derived from ovarian cancer ascites and the ideal controls, peritoneal fluids from benign patients for potential early detection and prognostic biomarkers.
Fluids were collected from subjects with benign cysts or endometrioma (n = 10), or low/high grade serous ovarian carcinoma (n = 8). EV particles were captured using primarily ExoComplete filterplate or ultracentrifugation and analyzed by nanoparticle tracking analysis, ELISA, and scanning electron microscopy. EV RNAs extracted from two ascites and three peritoneal fluids were submitted for next-generation sequencing. The expression of 34 mRNA and 18 miRNAs in the EVs isolated from patient fluids and cell line media was determined using qPCR.
EVs isolated from patient samples had concentrations greater than 1010 EV particles/mL and 30% were EpCAM-positive based on ELISA. EV particle sizes averaged 113 ± 11.5 nm. The qPCR studies identified five mRNA (CA11, MEDAG, LAMA4, SPINT2, NANOG) and six miRNA (let-7b, miR23b, miR29a, miR30d, miR205, miR720) that were significantly differentially expressed between cancer ascites and peritoneal fluids. In addition, CA11 mRNA was decreased to 0.5-fold and SPINT2 and NANOG mRNA were significantly increased up to 100-fold in conditioned media of cancer cells compared to immortalized ovarian surface and fallopian tube epithelial cell lines, the hypothesized cells of origin for ovarian cancer development.
This study indicates that EV mRNA profiles can reflect the disease stage and may provide a potentially novel source for discovery of biomarkers in ovarian cancer.
Extracellular vesicles
Peritoneal fluids
Ovarian cancer is the fifth-leading cause of cancer deaths in women [1]. With a lack of early obvious symptoms, women are frequently diagnosed with advanced stage disease. Approximately 60% of women are diagnosed at stage 3 or higher where the 5-year survival rate is below 30% [2]. In contrast, only 15% of cases are diagnosed at stage 1, when the tumor is localized to the primary site and patients have a 5-year survival rate of about 92%. In addition, patients with metastatic ovarian cancer frequently experience high recurrence rates within 16–22 months after conventional platinum-based combination chemotherapy. Identification of novel, specific and sensitive biomarkers for screening, monitoring or prediction may improve clinical outcomes and survival.
Screening tests currently used for ovarian cancer detection include pelvic examination, transvaginal ultrasound, and cancer antigen 125 (CA125). If an adnexal mass is detected by physical examination and/or ultrasound, surgery is ultimately needed for the confirmed ovarian cancer diagnosis and staging. CA125 is more routinely used as a marker for disease recurrence and treatment response [3]. It has been shown to be elevated in 80% of epithelial ovarian carcinomas, but its increase in other conditions such as endometrial, pancreatic and breast cancer and certain benign conditions have limited its use as an early screening marker [4, 5]. In addition, annual screening with both CA125 and transvaginal ultrasound has not reduced ovarian cancer mortality compared with usual care [6]. Further research is necessary to discover and identify biomarkers that would be effective in early ovarian cancer screening.
Recently, extracellular vesicles (EVs) have been analyzed for their potential as ovarian cancer disease biomarkers [7]. EVs comprise of exosomes (30–100 nm) and microvesicles (100–1000 nm) which are either actively released from cells by fusion of multivesicular bodies to plasma membrane or formed by direct budding of the cell membrane into the extracellular space, respectively. Exosomes and microvesicles contain proteins, lipids and nucleic acids such as mRNA and miRNA from their cell of origin. The increasing evidence of their roles in cell-to-cell communication [8, 9], their high abundance in plasma (1012 per mL), and highly stable nature, are several of the reasons for the increased interest to identify EV-based biomarkers. In ovarian cancer, EVs have been explored in ascites and urine for miRNA and protein surface markers [10, 11]. Because of the demonstrated roles of EVs in communication and tumor progression, we initiated a study to examine the expression of EV mRNA and miRNA in ovarian cancer ascites and compared the expression with an ideal but difficult to obtain control source, peritoneal fluids from females inflicted with benign gynecologic diseases. These studies indicate that malignant ascites EVs package quantifiable mRNA and miRNA that can potentially provide insights into diagnostic biomarkers and therapeutic targets.
The experimental design is separated into two parts: 1) preliminary characterization of clinical samples and 2) evaluations of the biomarkers in cultured cell line. For the characterization of clinical samples, four main studies were performed: 1) size and concentration of EVs, 2) preliminary qPCR screening of mRNA biomarkers identified through previous literature, 3) pilot RNA-sequencing of EV mRNA, and 4) RT-qPCR validation of mRNA and screening of miRNA. The mRNA biomarkers identified through the RNA-seq qPCR validation were evaluated in several appropriate cell lines.
Biofluid collection
Ascites from advanced stage ovarian cancer patients were collected from the peritoneal cavity using Yankauer suction connected to a drainage bag, or bulb suction from volumes smaller than 500 mL. Bulb suction was also used to collect small volumes (1–5 mL) of peritoneal fluids from patients with non-malignant conditions. Samples were collected at the Brigham and Women's hospital under informed consent and Internal Review Board approval. Fluids were transferred into 50 cm3 tubes and centrifuged at 2000 x g for 15 min at 4 °C to remove cell debris. The supernatant was then stored at − 80 °C until further use.
Immortalized human fallopian tube secretory epithelial cell line (FTSEC) was kindly provided by Dr. Ronny Drapkin [12]. Ovarian surface epithelial (OSE7, HOSE1–15) and high-grade serous ovarian cancer (SKOV3, OVCA3) cell lines have been previously described [13]. FTSEC cells were cultured in DMEM/Ham's F-12 1:1 (Cellgro, Mediatech, Inc. Manassas, VA) supplemented with 2% Ultroser G serum substitute (Pall Corp., Port Washington, NY). Ovarian epithelial cell lines were cultured in a mixture of medium 199 and MCDB105 medium (1:1) (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA).
Standard media was replaced with exosome-free fetal bovine serum (System Biosciences, SBI, Palo Alto, CA) containing media 24 h prior to conditioned media collection. Cell counts were determined at the time of conditioned media collection and ranged from 2 to 9 × 105 cells/mL. Conditioned media were transferred to 50 cm3 tubes and centrifuged at 2000×g for 15 min at 4 °C to remove cells and cell debris. The supernatant was then stored at − 80 °C until further use.
Differential ultracentrifugation and EV characterization
Ascites and peritoneal fluid samples were initially centrifuged at 2000 x g for 10 min to remove large debris. The supernatant was collected and further centrifuged at 10,000 x g for 30 min. EVs in the supernatant were then collected by ultracentrifugation at 100,000 x g for 1 h, washed with PBS, then collected again at 100,000 x g for 1 h in a Ti90 rotor. EV pellet was resuspended in PBS and stored at − 80 °C until further use. Nanoparticle tracking analysis of the EVs was conducted by Nanosight LM10 (Particle Characterization Laboratories, Inc., Novato, CA). Samples were diluted from 1:5 to 1:50 and applied to ELISA assay for EpCAM detection (Thermo Scientific, Frederick, MD) at 450 nm.
Ascites samples were pre-centrifuged at 3000 x g for 15 min at 4 °C before applying to the ExoComplete filterplate (Hitachi Chemical Diagnostics, Inc., (HCD), Mountain View, CA). EVs were captured on the filter membrane after centrifugation at 2000 x g for 5 min. Filters were fixed with 4% paraformaldehyde, and blocked with casein before incubation with 1:200 anti-human CD63 (Clone H5C6, BioLegend, Dedham, MA) for 1 h with gentle shaking. Samples were washed 3 times with casein PBS followed by incubation with 1:40 goat anti-mouse IgG gold colloid 9.0–11.0 nm (Sigma-Aldrich, St. Louis, MO) for 2 h. EVs were washed 3 times with casein PBS and PBS. Samples were fixed again with 4% paraformaldehyde for 5 min. Samples were washed once with PBS and 2 times with distilled water before incubation with 100 μL Silver Enhancement solution (BBI Solutions) for 10 min. Filters were washed with distilled water and air-dried overnight before analysis with SEM using Hitachi S-4800.
Next generation RNA sequencing
Ascites (n = 2) and peritoneal fluid (n = 3) samples were centrifuged at 2000 x g for 10 min at 4 °C after thawing as described above. The ExoComplete filterplate (HCD) captured EVs from 400 μL of centrifuged ascites and peritoneal fluid supernatant. Total RNA from EVs attached to the filter membrane was isolated using miRNeasy kit (Qiagen, Valencia, CA). Total RNA was monitored for quality control using the Agilent Bioanalyzer Nano RNA chip and Nanodrop absorbance ratios for 260/280 nm and 260/230 nm. Library construction was performed according to the Illumina TruSeq mRNA stranded protocol. The input quantity for total RNA was within the recommended range and mRNAs and noncoding RNAs with poly(A) tails was enriched using oligo dT magnetic beads. The enriched poly(A)+ RNA was chemically fragmented. First strand synthesis used random primers and reverse transcriptase to make cDNA. After second strand synthesis, the ds cDNA was cleaned using AMPure XP beads, cDNA was end repaired and then the 3′ ends were adenylated. Illumina barcoded adapters were ligated on the ends and the adapter ligated fragments were enriched by nine cycles of PCR. The resulting libraries were validated by qPCR and sized by Agilent Bioanalyzer DNA high sensitivity chip. Concentrations for the libraries were normalized and then multiplexed together. Multiplexed libraries were sequenced using paired end 100 cycles chemistry for the HiSeq 2500. The version of HiSeq control software was HCS 2.2.58 with real time analysis software, RTA 1.18.64. FASTQ files were input into Maverix Biomics platform mRNA-seq for differential expression in eukaryotes version 2.5. Additionally, Ingenuity Pathway Analysis (IPA) (Qiagen) was employed to identify biological pathway modulation.
Extracellular vesicle mRNA analysis
Ascites (n = 8), peritoneal fluid (n = 10), and cell culture conditioned media (2 mL) were processed by first thawing for 10 min at 37 °C and then placed on ice. For preliminary qPCR screening of mRNA in clinical samples, ascites (n = 8) and peritoneal fluid (n = 2) were used. Thawed samples were centrifuged at 2000 x g for 10 min at 4 °C. Three hundred fifty μL of supernatant was applied to ExoComplete filterplate (HCD) and centrifuged. For cell culture conditioned media, samples were centrifuged as above and supernatants were applied to EV collection tubes (HCD). EVs were then captured onto the filter membrane after repeated centrifugation. From this step, procedures for mRNA analysis for ascites, peritoneal fluid, and cell culture conditioned media are identical. Exocomplete lysis buffer is applied to the EVs captured on the filter and incubated at 37 °C for 10 min. Centrifugation of the filterplate or filter tips from the collection tube was performed at 2000 x g for 5 min at 4 °C to transfer lysate to the mRNA Capture Plate for hybridization of mRNA to the oligo(dT)-covalently linked wells. After wash steps, on-plate random-primed cDNA synthesis using MMLV was performed at 37 °C for 2 h. For qPCR analysis, 2 μL of cDNA was used with Sso Advanced SYBR mix (Bio-Rad, Hercules, CA) and gene-specific primers (Additional file 1). Real-time PCR was performed on a ViiA7 (Thermo Fisher Scientific, Inc., Waltham, MA) instrument using the following profile: initial denaturation at 95 °C for 10 min, 40 cycles of 95 °C for 30 s and 65 °C for 1 min, melting curve analysis. Ct values greater than 36 were set to 36 cycles for data analysis. Real-time PCR data was processed by Data Assist v3.01 (Thermo Fisher Scientific, Inc.) and analyzed by Excel.
Extracellular vesicle miRNA analysis
Ascites (n = 8) and peritoneal fluid (n = 10) were thawed for 10 min at 37 °C and then placed on ice. Four hundred μL ascites and peritoneal fluid were centrifuged at 2000 x g for 10 min at 4 °C. Supernatant was applied to ExoComplete filterplate and centrifuged at 2000 x g for 5 min at 4 °C. Lysis buffer from miRNeasy (Qiagen), was applied to the wells in the filterplate. Total RNA was isolated per manufacturer's protocol. Synthesis of cDNA was performed using miScript RT kit (Qiagen). The cDNA was diluted 1:4 and 1 μL was used with the miScript PCR assay (Qiagen) for qPCR screening. For miRNA analysis, Excel and DataAssist v3.01 was used with the following parameters: cut-off value of Ct = 36, SNORD61 selected as endogenous reference RNA.
The statistical analysis was performed using Microsoft Excel software. The statistical significance of the differences was determined by applying the Student's t-test.
Patient characteristics and samples
The patient characteristics and clinical information were obtained for ovarian cancer ascites samples (Additional file 2). All samples were collected at the time of diagnosis. Eight patients were diagnosed with serous type ovarian cancer: seven with high grade, and a single patient was diagnosed with low grade serous type. The ages ranged from 48 to 80 years with an average age at diagnosis of 64 ± 12 years old. Using the available data, CA125 levels were elevated with average values at 1289 ± 541 U/mL at diagnosis and average progression-free and overall survival were 19 ± 8 and 33 ± 22 months, respectively. For peritoneal fluid samples, no malignant cells were identified in peritoneal washings or from ovary, fallopian tube, uterus and cervix pathology report (Additional file 3).
Ascites and peritoneal fluid EV characterization
Ascites and peritoneal fluid extracellular vesicles were isolated using differential ultracentrifugation and characterized for size and concentration using nanoparticle tracking analysis (Table 1). This preliminary characterization indicated average sizes of ascites and peritoneal fluid EVs were not statistically different and averaged 113 ± 11.5 nm. CD63-positive particles < 200 nm in diameter were observed in scanning electron micrographs of ovarian cancer ascites samples applied to the ExoComplete EV capture filterplate and correlate to size estimates from nanoparticle tracking analysis (Fig. 1). EV concentrations ranged from 1010 to 1012particles/mL and were also not statistically significant between the two sample types. EVs from both ascites and peritoneal fluids were also evaluated for EpCAM surface marker expression by ELISA. EpCAM is a proposed surface marker of ovarian cancer-derived exosomes. Only one of each sample type was found to be positive for EpCAM. The single ascites and peritoneal fluid-derived EV samples had an EpCAM concentration of 670 pg/mL and 1.44 ng/mL, respectively. The other samples were below the limit of detection (50 pg/mL) for the ELISA assay.
Ascites (n = 3) and peritoneal fluid (n = 3) EV characteristics
EV Sample Sourcea
EpCAM ELISAa
Concentration (particles/mL)
Size (nm)
2.17E + 10
Peritoneal Fluid
aEV isolated by differential centrifugation
b10E9 – 10E11 EVs applied
a,b Scanning electron microscopy of ovarian cancer ascites EVs captured on the membrane of the ExoComplete filterplate. Ovarian cancer ascites was applied to the filterplate, fixed, labeled with anti-CD63 primary monoclonal antibody and anti-mouse IgG colloidal gold with silver enhancement. Black arrows indicate selected extracellular vesicles with diameters < 200 nm captured on the membrane. Images were obtained with accelerating voltages of 2.0 kV in detection mode
Next generation RNA sequencing and qPCR validation
A literature search for potential ovarian cancer biomarkers identified 50 mRNA candidates (Additional file 4) which were then evaluated in a preliminary qPCR screening of ovarian cancer ascites (n = 8) and peritoneal fluid EVs (n = 2). Three mRNAs, NANOG, SPINT2, ZEB2, were found to be significantly elevated (> 2-fold, p-value < 0.05) in ascites in the preliminary screening (Additional file 5). To further identify differentially expressed genes and additional mRNA markers, next generation poly(A)+ RNA sequencing was performed on selected samples of ascites (n = 2) and peritoneal fluid (n = 3). RNA sequencing mapping percentages to hg19 assembly ranged from 50% to 87%, and greater than 70% of the reads were kept after quality assessment (Additional file 6). The read distributions indicate that both samples have a higher percentage of reads mapping to exons and 3' UTR compared to introns and 5' UTR as expected based on sequencing preparation methodology (Additional file 7). Intergenic regions ranged from 2.5–15.9% for peritoneal fluid EVs and 24–47% for ovarian cancer ascites EVs.
Pathway analysis of RNA-seq differential gene expression data from ovarian cancer ascites and peritoneal fluid EVs identified organismal injury, cancer and reproductive system diseases as the main categories of diseases and disorders (Additional file 8). In terms of function, results were consistent with the biological environment of an advanced stage disease. Signaling pathways within growth, malignant tumor and advanced malignancy categories were predicted to be up-regulated whereas tumor cell death pathways were predicted to decline.
RNA-seq differential gene expression analysis identified 114 genes with statistical significance (p < 0.05) (Additional file 9). From this list, 30 genes selected for qPCR validation based on fold change, p-value, abundance and function were measured in eight ovarian cancer ascites and ten peritoneal fluid samples (Additional file 10). SPINT2, one of the three mRNA found to be differentially expressed in the initial screen based on literature-identified biomarkers was identified in the RNA-seq analysis. The remaining two genes, NANOG and ZEB2, were added to the list of 30 genes in the final RNA-seq qPCR validation assays. ACTB was selected as a reference gene for mRNA normalization. Of the selected mRNA for qPCR validation, five were found to be significantly (p < 0.05) differentially expressed in ovarian cancer ascites and peritoneal fluid (Fig. 2). Three mRNAs, CA11, LAMA4, MEDAG, were .01–.28-fold lower expressed and two mRNA, SPINT2 and NANOG, were 3.2–5.8-fold higher expressed in ovarian cancer ascites versus peritoneal fluid EVs.
Relative gene expression in peritoneal fluids (n = 10) and ovarian cancer ascites (n = 8) EV samples. a-e CA11, MEDAG, LAMA4, SPINT2, NANOG normalized to ACTB are shown as 2^-ΔCT gene expression levels with average and SD indicated by horizontal lines. Maximum gene expression was set at a cut-off of 10. Gene expression values for each individual subject are represented as solid circle and square symbols for ascites and peritoneal fluids, respectively. Statistical significance for (a-d) is p < 0.05 and (e) is p < 0.005 using Student's t-test
Ascites and peritoneal fluid EV miRNA analysis
Small RNAs are abundant within extracellular vesicles. Here, we quantitate specific miRNA previously identified to be involved in ovarian cancer progression or invasiveness [14–18]. There were six miRNAs, let-7b, miR23b, miR29a, miR30d, miR205, miR720, which were found to be significantly (p < 0.05) decreased .01–.21-fold in ovarian cancer ascites compared to benign peritoneal fluid when normalized to SNORD61 (Fig. 3).
Relative miRNA expression levels in peritoneal fluids (n = 10) and ovarian cancer ascites (n = 8) EV samples. a-f Let7b, miR205, miR23b, miR29a, miR30d, miR720 normalized to SNORD61 are shown as 2^-ΔCT expression levels with average and SD indicated by horizontal lines. Gene expression values for each individual subject are represented as solid circle and square symbols for ascites and peritoneal fluids, respectively. Statistical significance for (a-d) is p < 0.05 and (e) is p < 0.005 using Student's t-test
Multivariate discriminate analysis
The combined mRNA and miRNA raw qPCR data were used in multivariate discriminate analysis (Additional file 11). The predictors, LAMA4, CA11, MEDAG, NANOG, SPINT2, let-7b, miR23b, and miR29a were found to be sufficient to classify 87.5% and 100% of ovarian cancer (n = 8) and disease control (n = 10) groups, respectively.
Specific EV mRNA from normal and cancer cell lines
The EVs released from normal human fallopian tube epithelial (FTSEC194), ovarian surface epithelial (OSE7, HOSE1–15), and high grade serous ovarian cancer (SKOV3, OVCA3) cell lines were analyzed for specific mRNA that were differentially expressed in ovarian cancer ascites and peritoneal fluids (Fig. 4). The immortalized normal fallopian and ovarian surface epithelial cells were used as controls. Using 3 mL of conditioned media from each cell line, we confirm that CA11 mRNA is 0.1–0.5-fold lower abundance and NANOG is 50-fold higher abundance in EVs released from high-grade serous ovarian cancer cells, OVCA3. Although CA11 expression appeared to be elevated in SKOV3 cells, mRNA levels for CA11 as well as NANOG were not statistically significant compared to normal cells. SPINT2, however, was found to be significantly elevated in both SKOV3 and OVCA 3 cells compared to normal cells. LAMA4 was expressed in 0.1-fold lower abundance in OVCA3, but up to 2-fold higher abundance in EVs originating from SKOV3 cells compared to ovarian surface epithelial cell-derived EVs (HOSE1–15). The two high grade serous ovarian cancer cell lines, SKOV3 and OVCA3, demonstrated similar expression patterns for SPINT2 and NANOG, but distinct relative expression for CA11 and LAMA4. MEDAG mRNA was present in very low levels and was not reproducibly detected in all cell lines.
Specific mRNA expression normalized to ACTB from immortalized fallopian (FTSEC194), ovarian surface epithelial (OSE7, HOSE1–15) and ovarian cancer cell lines (SKOV3, OVCA3) EVs released in conditioned media. a SPINT2, (b) NANOG, (c) CA11, (d) LAMA4 mRNA quantitation (2^-ΔCT) is shown as column graph with average and SD (n = 2) and statistical significance indicated by a bar representing p < 0.05 by Student's t-test
Extracellular vesicles (EVs), including exosomes and microvesicles, are small membranous particles released from all cells and found in many biofluids. These vesicles are thought to provide a mode of cellular communication and deliver their cargo of protein, DNA and RNA, to target cells [10, 11]. As a result, EVs have been a novel source of biomarkers in a wide range of diseases. In this study, we analyzed EV mRNA and miRNA from ovarian cancer ascites and benign peritoneal fluids to determine if they can provide biological insight into metastasis and be a potential source of novel diagnostic biomarkers.
The average size and concentrations of EVs isolated from ovarian cancer ascites and peritoneal fluids were < 120 nm and at least 1010 particles/mL and are within range of EVs isolated from other biofluids such as plasma and urine. The variability in particle concentration observed between samples is consistent with what has been observed in previous literature [11]. In addition, EpCAM has been proposed as a marker for ovarian cancer-derived exosomes [11], but only 1 out of 3 of the ovarian cancer and peritoneal fluid EV samples were positive for EpCAM. This may be reflective of the typically low presence of adenocarcinoma cells (< 0.1%) within ovarian cancer ascites and the presence of endometrial epithelial cells in peritoneal fluids [19, 20]. The ovarian cancer ascites EV sample did not demonstrate a higher EpCAM concentration compared to the peritoneal fluid EV sample.
The ovarian cancer ascites and peritoneal fluid EVs were analyzed for differential mRNA expression. The mRNAs in this study were selected based on a combination of next generation sequencing (NGS) results and previous literature. The EV characterization and RNA sequencing were pilot analyses of the EVs and identification of new targets. IPA results confirmed RNA-seq is a methodology which may identify relevant biomarkers for diagnosis. The qPCR validation of the RNA-seq results employed more samples to confirm the gene expression pattern. Based on these qPCR analyses, the mRNAs confirmed to be decreased in ovarian cancer ascites compared to peritoneal fluid EVs included LAMA4, CA11, and MEDAG. EVs from a high grade serous ovarian adenocarcinoma cell line OVCA3 also contained less abundance of CA11 compared to controls from immortalized epithelial fallopian and ovarian cells. Recently, fallopian tube secretory epithelial cells have been proposed to be the precursor tissue for high grade serous ovarian cancer, and immortalized human fallopian tube secretory epithelial cells are being used for studying early-stage development of high grade serous ovarian cancer [21]. Based on the publicly available genotype-tissue expression (GTEx) database, CA11 mRNA has high baseline expression in brain and medium expression in tissues such as ovary. Although no previous studies were found to link CA11 with gynecological cancers, this mRNA was down-regulated in human gastric cancer [22]. CA11 is a member of carbonic anhydrase family known to participate in biological processes such as formation of aqueous humor, CSF and saliva. Similar to CA11, MEDAG was in lower abundance in ovarian cancer ascites EVs. MEDAG expression, however, was either at the limit of detection or undetectable in the fallopian and ovarian cell lines used in this study suggesting low baseline expression of MEDAG in these tissues. The GTEx database confirms this observation and shows MEDAG expressed at higher levels in visceral adipose and arterial tissues. MEDAG is a gene involved in processes that promote adipocyte differentiation, lipid accumulation, and glucose uptake in mature adipocytes. Because ovarian cancer cells bind preferentially to omental fat and use human omental adipocytes as an energy source, the decrease observed in ovarian cancer ascites MEDAG EV mRNA may reflect changes in the ascites microenvironment [23]. A previous study using microarray has also shown a lower MEDAG and LAMA4 expression in ovarian cancer compared to normal epithelial cells [24]. LAMA4 is a member of the major family of non-collagenous constituents of basement membranes. The decrease in LAMA4 observed in malignant ascites EV and OVCA3 EV, in contrast to the increase in SKOV3 EV, are interesting and warrants further experimental studies to evaluate the relationship. Ascites EVs could be providing cell-to-cell communication through unique molecular profiles promoting cancer cell survival within the ascites milieu.
In contrast to LAMA4, CA11, and MEDAG, the 2 mRNAs, SPINT2 and NANOG, were found to be increased in ovarian cancer ascites compared to peritoneal fluid EVs. SPINT2 was also in higher abundance in OVCA3 and SKOV3 EVs compared to controls. Interestingly, although SPINT2 is a putative suppressor, we demonstrate an increase in cancer ascites [25]. Müller-Pillasch et al. also reports that SPINT2 expression was elevated in pancreatic cancer [26]. NANOG, on the other hand, is a DNA binding homeobox transcription factor involved in embryonic stem cell proliferation, renewal and pluripotency. NANOG is increased in expression from normal tissue, benign, borderline, and malignant tumors of ovarian serous cystadenocarcinomas and protein is found selectively associated with high-grade ovarian serous carcinoma [27, 28]. NANOG mRNA was found to be significantly increased in OVCA3 EVs compared to OSE7 control. Based on previous studies relating to these mRNA, CA11 and MEDAG may be involved in maintaining malignant ascites microenvironment, while LAMA4, NANOG and SPINT2 activities could regulate ovarian cancer progression and metastasis.
There were six miRNA biomarkers, let7b, miR205, miR23b, miR29a, miR30d, and miR720, significantly down-regulated in ovarian cancer ascites EVs compared to peritoneal fluids. These miRNAs have previously been shown to be involved in ovarian cancer progression, invasion or metastasis. The let-7 family regulates downstream gene targets involved in self-renewal of mesenchymal stem cells derived from human embryonic stem cells. Let-7b is often dysregulated in ovarian cancer and is associated with poor prognosis [29]. Recently, miR205 was shown to be elevated in ovarian cancer tissue and associated with tumor growth and metastasis in ovarian cancer [30]. In pre-surgical plasma, miR720 was elevated in women who had short overall survival (< 2 years) when compared to women with long overall survival (> 4 years) after their diagnosis [31]. In contrast, miR23b expression is lower in epithelial ovarian carcinoma and borderline tumors than in normal ovarian tissues and benign tumors consistent with the lower abundance observed in ovarian cancer ascites in this study. MiR23b was shown to target cyclin G1 and suppress ovarian cancer tumorigenesis and progression [14]. MiR29a was also shown to have tumor suppressive effects and may contribute to cisplatin resistance of ovarian cancer cells [32, 33]. MiR30d also functions as a suppressor of ovarian cancer progression notably by decreasing Snail expression and blocking TGF-b1-induced EMT process [34].
Malignant ascites presents in approximately 30% of women with ovarian cancer and is frequently tapped to relieve symptoms. This fluid is composed of lymphocytes, epithelial cells, and EVs, and provides clues into ascites formation and metastatic progression. Functional analysis of each specific mRNA and miRNA will be needed to determine the biological significance of their differential expression in ovarian cancer.
Here, we demonstrate that EVs from ovarian cancer ascites contain distinct RNA expression signatures from benign peritoneal fluids, control samples that are rarely available to research. The two mRNA markers SPINT2 and NANOG that are upregulated in cancer ascites relative to peritoneal fluids may have potential as diagnostic biomarkers. Through continued liquid biopsy investigations, an understanding of the mechanisms involved in advanced stage disease and development of chemo-resistant disease may lead to alternative therapeutic targets and improved palliative care.
CA125:
Cancer antigen 125
EOC:
EV:
FTSEC:
Fallopian tube secretory epithelial cell
No external source of funding was used for this study.
CMY and SWN designed the study and contributed to writing the manuscript. CMY, MO, and TM collected, analyzed and/or interpreted data. MM and RB collected peritoneal and ascites samples. All authors read and approved final manuscript.
Human samples were collected at Brigham and Women's hospital under informed consent and Internal Review Board approval.
CMY, MO, and TM are employees of Hitachi Chemical Co. America, Ltd. R&D Center and the two authors, CMY and SWN have submitted a provisional patent (Appl. No. 62/507,091) entitled, "Methods for detecting ovarian cancer using extracellular vesicles for molecular analysis".
Additional File 1: Primer sequences (5′ to 3′) for qPCR validation. (DOCX 13 kb)
Additional File 2: Clinical information from patient ascites samples. (DOCX 13 kb)
Additional File 3: Clinical information from benign peritoneal fluid (PF) sample pathology reports. (DOCX 12 kb)
Additional File 4: Preliminary set of genes with primer sequences for screening based on literature search. Additional genes (MET, EGFR, EPCAM, CLDN3) were quantitated using Qiagen Quantitect Primer Assays. (DOCX 13 kb)
Additional File 5: Volcano plot displays p-values vs. fold change of ovarian cancer ascites (n = 8) and peritoneal (n = 2) EVs. Three mRNA (NANOG, SPINT2, ZEB2) show values above the fold change boundary of 2 (2-fold change) and a p-value of 0.05. Plot generated using. Data Assist v3.01 software. (PPTX 58 kb)
Additional File 6: mRNA sequencing reads and percentage aligned to hg19 assembly. (DOCX 12 kb)
Additional File 7: The distribution of EV mRNA sequencing reads mapping to human genome annotations. The percentage of reads (ave. ± SD) overlapping genomic features including exons, introns, UTR, and intergenic regions are shown for peritoneal fluids (n = 3) and ovarian cancer ascites samples (n = 2). (PPTX 115 kb)
Additional File 8: Ingenuity Pathway Analysis summary of top diseases and disorders, top canonical pathways, and top molecular and cellular functions. P-values are calculated using the right-tailed Fisher Exact Test and number of molecules are based on Ingenuity Knowledge Base with information contained in Canonical Pathways coming from specific journal articles, review articles, textbooks and HumanCyc. (DOCX 22 kb)
Additional File 9: Differentially expressed genes from RNA sequencing analysis. Significantly increased RNA (p < 0.05) from ovarian cancer ascites (n = 2) compared to benign peritoneal fluid (n = 3) are listed below as either up-regulated or down-regulated in ascites compared to peritoneal fluids. (DOCX 22 kb)
Additional File10: Next generation RNA sequencing data were plotted for each sample of ovarian cancer ascites (OC) and benign peritoneal fluids (DC). RPKM values for each corresponding gene are indicated in blue and the 30 selected genes for qPCR validation are labeled in red. Solid blue lines indicate linear regression. Both over- and under-expressed genes were selected for validation. (PPTX 318 kb)
Additional File 11: Multivariate discriminant analysis of mRNA and miRNA qPCR data. Linear discriminant functions are listed for ovarian cancer and disease control groups. (DOCX 12 kb)
Hitachi Chemical Co. America, Ltd. R and D Center, 1003 Health Sciences Rd, Irvine, CA 92617, USA
Department of Obstetrics, Gynecology and Reproductive Biology, Gynecologic Oncology Division, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA
Department of Obstetrics and Gynecology, Tuft Medical Center, 800 Washington Street, Boston, MA 02111, USA
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redpajama_set_name: RedPajamaCommonCrawl


text:
Tennis: Lleyton Hewitt's 2013 Wimbledon Run
Our guest tennis writer is back! Melinda Samson gives us a bit of a wrap on Lleyton Hewitt's potential Wimbledon draw…
Will Lleyton Hewitt get the luck of the draw at Wimbledon 2013?
Although Lleyton Hewitt has a current ATP world ranking of 82, on his day, which it has been recently, he is still one of the best players around on grass.
Hewitt won Wimbledon in 2002 and is four times champion at the Queen's Club (2000,01,02 and 06), a major grass court tournament. Since Hewitt's Wimbledon win only three other players have won the tournament being Roger Federer, Rafael Nadal and Novak Djokovic.
Hewitt's Form on Grass Courts in 2013
Semi-finalist at the Aegon Championships (Queen's Club), London
1st round defeated Michael Russell 4-6, 6-1, 6-3
2nd round defeated Grigor Dimitrov (seeded 10) 6-4, 6-3
3rd round defeated Sam Querrey (seeded 6) 7-6, 5-7, 7-6
Quarterfinals defeated Juan Martin Del Potro (seeded 3) 6-2, 2-6, 6-2
Semi-finals defeated by Marin Cilic (seeded 5) 6-4, 4-6, 6-2
Hewitt's Grand Slam Form 2011 to Present
At Grand Slam tournaments in recent years, it has to be said that the draw hasn't often been kind to Hewitt, matching him against top players in the early rounds. Even so, Hewitt has always made his fans proud, running down every ball and never giving up a point lightly.
Here's a review of Hewitt's grand slam form over the past few years
French Open – 1st round defeat by Gilles Simon (ranked 18) 3-6, 1-6, 6-4, 6-1, 7-5
Australian Open – 1st round defeat by Janko Tipsarevic (ranked 9) 7-6, 7-5, 6-3
US Open – 3rd round defeat by David Ferrer (ranked 5) 7-6, 4-6, 6-3, 6-0
Wimbledon – 1st round defeat by Jo-Wilfried Tsonga (ranked 6) 6-3, 6-4, 6-4
French Open – 1st round defeat by Blaz Kavcic (ranked 99) 7-6, 6-3, 6-7, 6-3
Australian Open – 4th round defeat by Novak Djokovic (ranked 1) 6-1, 6-3, 4-6, 6-3
Wimbledon – 2nd round defeat by Robin Soderling (ranked 5) 6-7, 3-6, 7-5, 6-4, 6-4
Australian Open – 1st round defeat by David Nalbandian (ranked 21) 3-6, 6-4, 3-6, 7-6, 9-7
Other than the 2012 French Open Hewitt has only been beaten in Grand Slams, since 2010, by players ranked 21 or higher at the time of the match. During this time only two of these defeats were in straight sets.
If this continues it will likely take a top 20 player to defeat Hewitt at 2013 Wimbledon and in more than three sets.
Let's just hope he gets the luck of the draw at Wimbledon in 2013 and faces at least one or two low ranked players on the way to the second week.
This is a guest article by Melinda Samson aka GrandSlamGal. In 2012 Melinda achieved one of her life goals by attending all four tennis grand slam tournaments in the same year.
If you're going to Wimbledon this year, or planning to in future, get your Free copy of the Wimbledon e-Companion, full of tips, info and website links to maximise your fan performance.
Great stuff Melinda! Thank you 🙂 ~ theaussieword
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here, right now, at this present moment.
as the loving, liberating, light of Truth.
Superior Energy in whom the many are One Complete Perfection.
light, Our Word is Satsang.
clear and logical manner, this ancient, yet very modern, spiritual knowledge.
back to our Ancient, yet very modern, Self.
humble offerings of devotion and love.
have always been, albeit, unconsciously.
liberating us from Maya. Thank You for helping us to remember You.
true self knowledge, dignity, and self respect at all levels of consciousness.
Sobriety and dignity at all levels of consciousness demand that we regain our sanity.
and transcending the body, mind, intellect, and ego.
way we look at the world the world we look at, and our behavior toward it, changes.
due to association with You, Supreme Spirit, the Highest, Greatest, Purest, Power of all.
happens, one sees and experiences this Self as ones natural condition, ones Original Being.
of the seemingly vast and variegated energy generally known as the Cosmic Manifestations.


Punta Mita is ready to offer exciting experiences for residents and visitors over the summer. The residential and tourism complex located north of Puerto Vallarta will host a series of culinary, music and sporting events where the fun is guaranteed.
The ultimate event for the most discerning foodies. This year's edition will feature France and Japan as guests countries. Cuisines of both nations have been appointed Intangible Heritage of Humanity, making this a not-to-be-missed event. From July 5 – 7, Yasuo Asai (Asai Kaiseki Cuisine executive chef) will delight the palates of attendees at the Punta Mita Residents Beach Club. Finally, Frédéric Lobjois (Au Pied de Cochon executive chef) will offer an haute cuisine tasting menu from July 14 -19.
This summer camp is aimed at children aged 5 to 12 and will have two editions taking place from July 23 – 27 and from August 6 -10. Punta Mita Ocean Sports SUP Kids Camp intends to raise environmental awareness while people enjoy the wonders of the ocean in a safe and responsible manner. The program features activities such as Stand Up Paddle competitions, water sports and science lectures at the Punta Mita Ocean Sports education center.
The 6th edition of the Punta Mita Beach Festival will take place August 4 & 5, featuring the best gastronomy, mixology, sports and music in a charming natural setting. This is definitely a must for this season.
This will be the first Punta Mita Surf Camp, a four-day event dedicated to this water sport originated in the Polynesian Islands. Attendees will have the chance to meet special guests such as Coco Nogales, Diego Cadena and Fernando Stalla.
"Punta Mita Surf Camp is the perfect occasion to enjoy multiple water sports: surf, foilboarding, outrigger and underwater fishing, everything overseen by Fernando, Coco and Diego to ensure everyone's security," said Adam Finer, Punta Mita Water Sports director.
Along with these activities, hotels Four Seasons Punta Mita Resort and The St. Regis Punta Mita Resort have scheduled other gastronomic and entertainment experiences for their guests. We encourage you to check out their Facebook pages by clicking the links.
For more information about Punta Mita and each of these events, visit www.puntamita.com.


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Patriarche
Supporting the transition from local agency to the international group.
We designed and produced the Patriarche group's new digital showcase: an emblematic project for a fast-growing group whose history is based on four pillars that we were keen to highlight in this redesign.
Patriarche.
www.patriarche.fr
Web responsive development
Make the agency's website the showcase of augmented architecture
"We are augmented architects."
The Patriarche Group wanted to coincide the redesign of its website with this change of direction which is crucial if it is to play a role in this changing world.
As Patriarche. adjusts its thinking, it is also adjusting its discourse and the way it presents it.
The old version of the site had qualities of animation and highlighting of projects but lacked narrative, diagrams and the international angle was not sufficiently emphasised. These key points were then reworked in this new version of the site.


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When it comes to beverages, frothy can be delicious.
In what may be the least inspiring description of fizzy drinks ever written, a group of food engineers explained, "Aeration in beverages, which is manifested as foam or bubbles, increases the sensory preference among consumers."1
Stock markets can fizz up, too. Share prices bubble, enthusiastic investors invest, and prices go even higher. In a frothy market, share prices often rise above estimates of underlying value. The terms that describe this financial market phenomenon include irrational exuberance, animal spirits, and overconfidence.2, 3, 4
Last week, there was speculation about whether some parts of the U.S. stock market have gotten frothy. Eric Platt, David Carnevali, and Michael Mackenzie of Financial Times wrote about an initial public offering (IPO) of stock by a hospitality company. They reported:5
"…[the share price] more than doubled in value on Thursday in a return to the kind of mammoth pops that came to define the dotcom boom of the late 1990s…It's a sign of frothiness, a sign of incredible demand, a sign of a retail investor that…just wants to get in."
That observation about investor enthusiasm for stocks was supported by the AAII Sentiment Survey. Last week, 48.1 percent of participants were bullish. The long-term average is 38 percent. (Some believe the survey is a contrarian indicator. When bullishness is stronger than usual, contrarian investors would adopt a bearish stance, and vice versa.)6, 7, 8
Despite IPO fervor, major U.S. stock indices finished lower last week. The decline has been attributed to a lack of new stimulus from Congress rather than concerns about high share prices. Inflation worries may have played a role, too. Last week, headlines in both The Economist and The Wall Street Journal suggested the post-pandemic world may include higher inflation.9, 10, 11
Wall Street's fear gauge, the CBOE Volatility Index edged higher last week, reported Ben Levisohn of Barron's. It's at about 24.6, which is above its long-term average of 19. That's a sign markets may be volatile during the next few weeks.12
A HOLIDAY SEASON LIKE NO OTHER. The coronavirus, which The Economist estimates has infected one-in-five Americans, is reshaping holiday traditions this year.13 "COVID-19 is playing on shoppers' psyches as they weigh its impact on their health and finances. But, as we've seen with previous periods of recession, as well as those of growth, consumers are resilient and will adjust their habits to adapt," reported Deloitte's 2020 holiday retail sales consumer survey report:14
33 percent of survey participants were in a worse financial position than last year, especially those in lower income groups.
40 percent planned to spend less on the holidays than they did last year because they are concerned about the economy.
51 percent were anxious about shopping in stores because of COVID-19. Deloitte estimated shoppers would spend about $390 of their holiday budgets in stores and $892 online.
The Deloitte survey identified four types of holiday shoppers:14
The Festive Shopper: "These shoppers are all about buying gifts for others, keeping up with conventional shopping routines. They also outspend other types of shoppers."
The Conscious Shopper: "These shoppers care about society and are willing to 'put their money where their mouth is' by paying more for socially responsible products."
The Deal-Seeker: These shoppers "…enjoy the search for the right gift at the right price and will spend weeks shopping. [They're] up for browsing and sifting through digital platforms, including social media, for attractive options."
The Efficient Shopper: These shoppers "…aren't in the mood to browse or get caught up in time-sucking events, like Black Friday. Shopping is a task, and they'll seek out the easiest way to get it done."
Do you recognize yourself in any of these descriptions?
"Beware the investment activity that produces applause; the great moves are usually greeted by yawns."
--Warren Buffett, Investor15
1 https://link.springer.com/article/10.1007/s12393-020-09213-4
2 https://www.investopedia.com/terms/i/irrationalexuberance.asp
3 https://www.investopedia.com/terms/a/animal-spirits.asp
4 https://www.investopedia.com/terms/f/froth.asp
5 https://www.ft.com/content/cfdab1d0-ee5a-4e4a-a37b-20acfc0628e3 (or go to https://peakcontent.s3-us-west-2.amazonaws.com/+Peak+Commentary/12-14-20_FinancialTimes-Wall_Street_IPO_Bonanza_Stirs_Uneasy_Memories_of_90s_Dotcom_Mania-Footnote_5.pdf)
6 https://www.aaii.com/sentimentsurvey?
7 https://www.aaii.com/journal/sentimentsurveyarticle
8 https://www.investopedia.com/terms/c/contrarian.asp
9 https://www.marketwatch.com/story/dow-poised-to-skid-200-point-amid-setbacks-on-u-s-fiscal-relief-package-and-brexit-11607691156
10 https://www.economist.com/leaders/2020/12/12/after-the-pandemic-will-inflation-return (or go to https://peakcontent.s3-us-west-2.amazonaws.com/+Peak+Commentary/12-14-20_TheEconomist-After_the_Pandemic_Will_Inflation_Return-Footnote_10.pdf)
11 https://www.wsj.com/articles/goodbye-covid-hello-inflation-11607618398 (or go to https://peakcontent.s3-us-west-2.amazonaws.com/+Peak+Commentary/12-14-20_WSJ-Goodbye_COVID_Hello_Inflation-Footnote_11.pdf)
12 https://www.barrons.com/articles/airbnb-and-other-soaring-ipos-cant-lift-the-stock-market-why-thats-good-news-51607735116?refsec=the-trader (or go to https://peakcontent.s3-us-west-2.amazonaws.com/+Peak+Commentary/12-14-20_Barrons-The_Dow_Snapped_Its_Two-Week_Winning_Streak-Why_this_Weeks_Pause_was_Good-Footnote_12.pdf)
13 https://www.economist.com/graphic-detail/2020/12/10/almost-one-in-five-americans-may-have-been-infected-with-covid-19 (or go to https://peakcontent.s3-us-west-2.amazonaws.com/+Peak+Commentary/12-14-20_TheEconomist-Almost_One_in_Five_Americans_may_have_been_Infected_with_COVID-19-Footnote_13.pdf)
14 https://www2.deloitte.com/us/en/insights/industry/retail-distribution/holiday-retail-sales-consumer-survey.html (or go to https://peakcontent.s3-us-west-2.amazonaws.com/+Peak+Commentary/12-14-20_Deloitte-2020_Holiday_Retail_Sales_Consumer_Survey-Footnote_14.pdf)
15 https://wiseowlquotes.com/warren-buffett/