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Q: STXXL equivalent in Java I'm searching a collection framework designed for huge datasets in Java that behaves transparently, like STXXL does for C++.
It should transparently swap to disk, but in a much more efficient manner than plain OS-based VM swapping. A StringBuffer/String drop-in replacement would be a big plus.

A: These fill needs only partially:
Oracle Berkeley DB Java Edition database backed collections:
http://www.oracle.com/technology/documentation/berkeley-db/je/java/index.html
Joafip persistent collections:
http://joafip.sourceforge.net/javadoc/net/sf/joafip/java/util/package-summary.html

A: Terracotta


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HomeFACTOR AFFECTING TEACHING AND LEARNING OF BUSINESS STUDIES IN JUNIOR SECONDARY SCHOOL IN IKERE LOCAL GOVERNMENT AREA OF EKITI STATE .
FACTOR AFFECTING TEACHING AND LEARNING OF BUSINESS STUDIES IN JUNIOR SECONDARY SCHOOL IN IKERE LOCAL GOVERNMENT AREA OF EKITI STATE .
This study investigates factors affecting the teaching and learning of Business studies in junior secondary schools in Ikere Local Government Area of Ekiti State. This study was a descriptive research design which comprises of all students from four selected secondary schools in Ikere Ekiti. The sample for the study was made up of One hundred (100) students. Simple random Sampling was employed to select the respondnets.Questionnaires were used as instrument for data collected. The data were analyzed using percentage method . The result shows that lack of fund, in availability of instructional materials, government policy, non-availability of related textbooks and lack of upgrading knowledge by so-called teachers of business studies are the factors affecting the teaching and learning of business studies.Based on the results, the researcher therefore recommended that the schools should ensure that their environment were favorable to teach the subject by ensuring that there are adequate class rooms this will encourage the students to learn. That teachers should ensure that they handle business studies as a subject in such a way that will encourage students to take the subject and Government should employ more teachers and provide resources for teaching and learning business studies in schools
1INTRODUCTION
Background of the study
Business Education are integrals part of general education that is concerned with education for business and education about business.
Business Education can be traced from the time when commercial secondary modern school were established in some part of the country and commercial subject such as typewriting , shorthand, commerce, principle of account and the use of English were being offered at the moment in the secondary schools in the country.
Some years back government policy on education was indebted to train more business personnel, so that business subject can be introduced the secondary school curriculum, with the aim of providing capable and competent hands to handle the finance and administration of the country. The national policy on education (2004) revised education stipulated that Business Education should be taught in tertiary institution such as College of Education, College of Technology, Polytechnics and in Universities.
Education is an important aspect of economic development. When citizens of a country are educated that is, a key to social economic and political stability (Ojiambo, 2009). The Nigeria government has considerably invested in education which is evidenced by the free primary education and the free day secondary education (Ojiambo, 2009). The main purpose of education is to transmit wisdom and knowledge from one generation to another, this prepares young people to be members of the society, maintain it and develop it.
Education also has positive influence on population, health nutrition and also increases value and efficiency for labour (Kising U, 2012). The unemployment problems facing the country can best be addressed through equipping the young people with skills that can lead to self-employment, the curriculum therefore should equip the learners with skills and more so entrepreneurial skills and this therefore indicates the importance of business education in the school curriculum.
The Business Education which was earlier referred to as Business education was introduced into public high schools in the U.SA. by tax payers demand during last part of nineteeth century. Parents insisted that public schools should provide Business education, otherwise they would take their children to private business colleges. Many district school introduced Business education courses, shorthand, typewriting and book keeping. Since then Business education has been undergoing scrutiny and transformation (Popham et al, 2005). In Nigeria Business Education in junior secondary school curriculum came about as a means of laying the foundation for national technological and economic advancement as articulated in the National Policy on Education (Federal Ministry of Education, 2011). The designers of the Business Education curriculum apparently considered that, in line with the current Nigerian Vocational curriculum philosophy (National Board for Technical Education, 1984), the obvious way to develop a curriculum that would give a broad introduction to the field of Business was to do away with the traditionally compartmentalized single subjects. Consequently, five such subjects (book-keeping, commerce, office practice, shorthand and typewriting) were brought together as units of a new subejct to be called Business Education . (Ekpenyoung 2006). The curriculum aimed at giving students knowledge and skills that should enable them to adapt to changes in office technology and develop a broad understanding of Business activities, the structure and functions of Business institutions and their inter-relationships.
In Nigeria a number of education commissions and education committees have been put in place to review the education system in Nigeria. the commissions have seen the Business Education evolve to its current status in the Nigerian education system. Fraser Report (2009) on Education recommended introduction of industrial education in schools as a means of providing skills to native labourers and artisans in large numbers for various purposes. During the colonial period, the colonial government opened a number of industrial schools in areas not effectively served by missionaries, some of the courses offered included Carpentry, agriculture, typing and blacksmithing (Kising 4, 2012), typing and office practice is a constituent of Business Education currently.
Onywoki (2011), the introduction of secondary Business education programme was based on the results of the manpower survey and its aim was to produce highly skilled manpower required for the growing commercial enterpises in the country. Specifically, the project was aimed at introducing Business education subjects in selected general secondary schools in nigeria, for example accounting, shorthand and typing with office practice and commerce. Because of the success of the project, there were about 120 secondary schools teaching Business education in Nigeria by 2000s (Oluoch, 2012).
According to Shiundu and Omulando (2012) integration of subjects emphasizes the relationship between various curriculum areas in an attempt to interrelate content or learning experiences in order to enable students perceived a unity of knowledge. Anuda (2000) asserts that by integrating the aspects of these disciplines, overlaps and cost have greatly been reduced. Business Education aims to provide the leaner with opportunities to acquire basic Business knowledge, skills and positive attitude for development of self and nation, being a dynamic subject it takes into account the need to address contemporary issues, trends in Business and current economic issues in society.
Onywoki (2011), Accounting, Commerce, Economics, Entrepreneurship and Office Practice are distinct subjects of study and have intrinsic values for the learners and teachers. Therefore, the distinctiveness of each discipline of study has to be preserved.
Statement of Problem
Business education in Nigeria is at its developmental stage at the initial introduction of this course into our education system there was no interest nor motivation from the government. According to Nsofor (1979) "The government was not only uninterested in including commercial subjects in the educational curriculum it made a deliberate and calculated attempt to frustrate the efforts of come of the philantiopicts". Due to lack of attention which business education received at the introduction stage, efforts by the private proprietors no attention to crumple down. The proprietors gave no attention to the training of business teachers. This may be due to professional complications and financial burden which were not within their reach.
It is discouraging to note that despite all the problem business education faces at the earlier stage and the opportunities at had offended to the country, much has not been done to improve the situation. Schools, students still today lack qualified business teachers which may be as a result of the period of former inadequate when compared with the complexities and dynamic nature of knowledge required. A major problems that face business education today is lack of equipment which are very necessary for proper mastery of the subject. As a result of these problems, there has been less interest and motivation on the part of the students who offer business subjects in school.
Another problems which will inhibit a smooth teaching of business studies in secondary school is lack of libraries. The important of which cannot be over emphasized nor will not occupy lengthy discussion in this work.
In every teaching and learning process, there is bound to be some factors which may not allow for smooth running of the process such as inadequate time table, inadequate instruction materials these factors will definitely affect the achievement or attainment of the set objectives which may not thereafter have negative effect on the skills to be imparted on the learners. This situation will no doubt, affect the management and administration of today's business.
Thus, this study is to examine those problems which can face learning and teaching of business studies and seek for ways of solving such problem.
Objective of the Study
This study is on the survey of the factors influencing the teaching and learning of business studies in junior secondary schools in Ikere local government area of Ekiti state
This study was guided by the following specific research purpose:
To find out the factor affecting teaching and learning of business studies
To identify social -based factors in teaching and learning of business study
To determine the non-institutional factors in teaching and learning of business studies
To suggest solution to the identified problems
Specifically, this study investigated the following questions:
What are the factors affecting the teaching and learning of business studies in Ikere local government area?
What are the social based factors affecting teaching and learning of business studies in Ikere Local Government Area of Ekiti State?
What are the non-institutional based factors affecting teaching and learning of business studies in Ikere Local Government Area of Ekiti State?
what are the suggested solution to the problem identified?
The research work is limited to junior secondary school students . The research is carried out to determine the factors affecting teaching and learning of business students in five selected secondary schools in Ikere local government area
Limitation of the Study
The following are the problems countered during the course of the research
Time: The time allocated for the research work was merged with other academic works, thereby not giving enough time for a wider coverage.
Human attitude: Respondents were not easily pinned down. Also the researcher needs to be on ground to explain the question the questionnaire to them to ensure that it was correctly filled.
This study will enable the teachers of Business studies to identify those factors affecting student's performance, thereby helping them to proffer solution to them.
The government too will benefit from this study in that, it will beam it's search high on provision of teaching material and other teaching facilities that should be made available for the use in the classroom
The post FACTOR AFFECTING TEACHING AND LEARNING OF BUSINESS STUDIES IN JUNIOR SECONDARY SCHOOL IN IKERE LOCAL GOVERNMENT AREA OF EKITI STATE . appeared first on TY Computer Institute.

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Euroluce Aydinlatma Mumessillik
4 out of 4 search engines are rated 'best engineering college'
June 17, 2021June 17, 2021| adminadmin| 0 Comment | 9:25 am
Search engines have come under fire recently for their ranking of engineering colleges.
While the ranking is based on the quality of their engineers, it is based largely on their graduates, rather than their students, who tend to be more competitive and less likely to leave school.
A recent article from Business Insider, however, revealed a new trend: the number of engineering schools ranked by search engines like Google, Yahoo, and Baidu has dropped dramatically in the last two years, while the number that are rated "best engineering schools" has stayed the same.
This could be due to the fact that search engines have begun to move away from their former reputation as the top engines for business and engineering.
The article, titled "Engineering School Rankings Drop Amid A Massive Tech Bubble," claims that the number ranking engineering schools dropped by 11% over the last three years, from 11.8 in 2016 to 10.4 in 2017.
The number ranking business schools fell by 7%.
Baidus ranking dropped by 6%.
Google ranked first by a 5% margin, while Yahoo ranked third by a 3% margin.
Yahoo is the first search engine to drop from the top of the list in the same period.
Google and Biz, for their part, remain in the top 10, while Bing and Yahoo are both at the top.
While this may be due in part to the impact of Google and Bing on the economy, the article suggests that Google and Yahoo have also benefited from the "bubble" in the tech industry that began with the rise of artificial intelligence.
"While the bubble may be a recent phenomenon, it's a trend that's been evident in the U.S. economy since the dot-com bust of 2001," the article states.
"This phenomenon has created a shortage of qualified, skilled engineers and created a bottleneck for hiring them, leading to a decline in the number and quality of jobs."
This article is based off of a previous article that Business Insider published, titled The 10 Best Engineering Schools in America, which ranked engineering schools based on their number of graduates, their graduates' average SAT scores, and their overall average GPA.
Business Insider also compiled a list of the top engineering schools in the world.
Google, Baidhu, and Yahoo also make the list.
These four companies, and others, have all been ranked among the best engineering schools by Google and the other top search engines.
These companies are likely the result of the success of Google, which was ranked the fourth best search engine by Google last year.
The company also has some of the best search engines on the web, and has been ranked in the #1 position in the US since 2009.
While Baiduan, Google, and Bing have all improved their rankings since the end of 2016, there are other search engines that have been ranked higher in the past, and have been around longer.
The top five Google searches in 2017, according to the search engine's own data, are "best computer," "best laptop," "largest, most expensive home," and "best TV."
These are the top five most searched searches on Google, according.
Google has consistently ranked at or near the top for the past decade.
In 2018, it became the third-largest search engine in the United States.
The Google News ranking, which measures the search traffic and engagement of Google+ members, also showed that Google is still a top search engine.
The search engine has a massive audience and has an impressive amount of traffic.
According to Google, Google+ has over 300 million monthly active users.
It also has a sizable following, as its top 100,000 members in 2018 have over 3 million monthly searches.
It has been gaining popularity with students, and as a result, there has been a growing number of companies making the switch from their main search engine, Bing, to their own search engine of choice, Google.
In the last few years, Google has been steadily increasing its ranking, and many are moving away from Bing in favor of Google.
This is particularly true for Microsoft, which has dropped out of the "top 10" list, down to #13, in 2018.
Other search engines, such as Bing, Yahoo and Bancor, have also fallen down the rankings.
Microsoft has been at the bottom of the rankings for several years, but this year, it finally started making some headway.
The Bing search engine was at the end, ranking #14 in 2018, while Google's search engine dropped from #9 to #10 in 2018 and now is ranked #5.
Bancors search engine ranked #9 in 2018 before being ranked #6 in 2018 after Microsoft dropped out.
Google's Bing has been in the rankings of search engines for years, and it was the number one search engine for the first three months of 2017.
Bing is also the most widely used search engine globally.
Microsoft, Google and Facebook have all increased their rankings in
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'We are ready to die': Israelis say they'll sacrifice if necessary'We are ready to die': Israelis say they'll sacrifice if necessary
Israel's army chief said Wednesday that the country will go to war if the Palestinians don't give up their right to return to the West Bank."We are prepared to die,"
Hellcat Engine is the #1 paid engineering job on Google, but you can make more by doing something elseHellcat Engine is the #1 paid engineering job on Google, but you can make more by doing something else
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Q: Laravel firstOrNew overwriting all entries I'm having problems with the firstOrNew() method. Please see my method below.
public function updateUnique($attributeId, $data)
{
    $attribute = \App\Clients\Attribute::firstOrNew(
        [
        'mem_id' => $data->lead_id,
        'attribute_id' => $attributeId,
        'type' => $this->updateType($data->pk)
        ] );

    $attribute->mem_id = $data->lead_id;
    $attribute->type = $this->updateType($data->pk);
    $attribute->attribute_id = $attributeId;
    $attribute->attribute = $data->value;

    $attribute->save();
}

This worked absolutely fine until I added the property public $primaryKey = "mem_id"; on the model Attribute.
All entries in the database are updated if "first". The 'new' aspect works ok.
As soon as I remove that it works. I'd like to try and understand what's going on?

redpajama_set_name: RedPajamaStackExchange


This well decorated unit is nestled among the trees for your own private enjoyment. Peak-a-boo views of the Pacific Ocean abound throughout. Abutting natural wetlands you are ensured peace and quiet to enjoy the plush decor. The large dining room table provides the perfect setting for family meals or games. The large kitchen is well stocked and functions well for multiple chefs. It overlooks the living room and dining room so you are always part of the conversation. The master bedroom has its own private bathroom including jetted tub and separate vanity. Beach access is just a few steps away.
Only problem we had was the lamp on the right side of the bed would not work. I changed the bulb, which was definitely bad, tried other outlet, still wouldn't work.
We had an amazing time in Westport. The condo was absolutely perfect and had everything we needed. Jet tub and sleep number bed were amazing bonuses. The sunsets on the beach were the highlight of trip. Thank you so much!
I really enjoyed my stay at the condo. Everything about it made me feel comfortable, appreciate my stay, and enjoy my vacation time. Excellent choice for a vacation spot!
Great facility and great location!
We rented this unit for my parents for the 4th of July weekend. The rest of our family have a beach house in North Cove so this was a great family time for all of us. A special thank you to Trish for arranging an early access to the unit for my parents.
Beautiful condo, perfect weekend getaway. Will definitely stay again. It was clean, spacious, and in a great location.
I LOVE staying at the Condo's! As tempting as it is to keep this location a secret I have to be honest and give a fabulous review! It's Peaceful, beautiful, clean, well kept, well stocked, so close to the shore and a beautiful beachwalk. Perfect for long walks, exploring the town, watersports, clam digging, fishing, writing, you name it. Well done Vacations by the Sea...I'll be back!
I enjoyed very much my stay at this lovely condo which was very clean, very comfortable and most cheerful with beautiful views of the trees and part of the ocean. I couldn't have had a better vacation place.
Wonderful place to stay. We will come back for sure. Could put some toys and more games for kids.
Had all the essentials for two quiet days away from the kids and work. Comfortable, very clean and homey. Only suggestion would be room darkening shades in the bedroom as it faces the front with bright security lights. I'm one of those people who needs a dark bedroom to sleep well.
This is a great little condo! Everything is like new. We loved the little children's table and stools. The condo is set away from the other main buildings in the complex, so it was fairly quiet - minus the groups celebrating Saturday evening. We also loved the little playground, beach access, and pool. We would stay again.
We stayed just one night but it was great. The condo was spotless, the bed comfortable, the deck perfect for relaxing while listening to the waves. It is a 1 minute walk to the beach and a gorgeous sunset. Highly recommend it.
This place was the best. New, clean, great location. I would stay here again.
Love the location and amenities. The condo is lovely; very quiet and peaceful. I had trouble getting the internet to work, which was a problem, since I was in the area on business. The condo was overly warm, and the refrigerator smelled like crab. Otherwise, I loved my stay and will definitely come back.
Thanks for staying and we're glad to hear you enjoyed the peace and quiet. Please let us know of any difficulties with the internet during your stay and we'll do our best to resolve them. Often the equipment just needs to be reset. Fresh seafood is both blessing and a curse here on the coast, while we clean each condos thoroughly between guests, occasionally an odor can linger for a little while, especially if guests depart and arrive on the same date. See you next time!
I took my husband here to celebrate his birthday. The unit was spotless and very tastefully decorated. We were very pleasantly surprised to discover a sleep number bed! We have one at home and have become very accustomed to that comfort so this was a real plus for us.
A very nice touch was the spa robes that greeted us on the bed. It really made us feel like we were at an upscale resort.
It was a very stormy weekend and I especially loved my peek a boo view of the ocean.
We were very impressed with the unit and the entire complex. We will be back, for sure!
The only bad part about our stay was having to leave. We could not have asked for more.
The whole experience was absolutely wonderful. The office staff was very friendly and helpful. The condo was very clean and comfortable. My daughter refers to it as "our home away from home" because it was so inviting.
Glad to hear your family had a wonderful stay and think of this condo as your home away from home! See you again soon.
This was our second visit, and we love staying here. All the comforts of home at a great location for a family vacation. Thanks!
This was our first condo rental and it exceeded our expectations in a variety of ways. There were all the comforts of home and more. Easy access to the beach for long walks, minutes from the heart of Westport, and important for us privacy and well maintain units.
I'm very happy to hear your first condo rental was with us and you had such a great time. We try to maintain the condos in our care to the highest standards and value our guests comfort and privacy. Hope to see you again soon in the future.
So short, only over night...not enough time to evaluate adequately.
Sorry you could only stay with us just for a night, next time we hope you can stay longer and take advantage of our outdoor heated swimming pool and hottub. Both are open until 10pm during hte summer season!
Rental exceeded my expectations and beach was great.
last minute overnight trip--this spot made everything that much more perfect, thanks!
Thanks for staying and we hope the waves were great for your last minute surf trip. Aloha!
4th or 5th time back so we must like it!
See you for number 6! Thanks for being such a frequent, loyal customer.
We thoroughly enjoyed our stay at the resort. The setting is great from the miles of beach to the walk to the park. The unit was clean and well appointed. The only tiny negative thing was that we thought there should be a second lamp in the living room between the chair and the couch but this didn't take away from the overall great experience. We will visit again!
Our condo had everything we needed for a relaxing vacation. There is not a whole lot to do in town, so we spent most of our time around the condo. The beach is very close. We had lovely picnics, collected pretty rocks and whole sand dollars, and played in the sand. We borrowed puzzles and rented movies from the clubhouse every night of our stay. The pool was the perfect size and temperature for the kids to enjoy. We will definitely return.
Near the end of season. Very quiet and relaxing. Just what we needed. I'll be back!
I didn't realize that the condos were so close to the beach, it was great. My daughter loved the pool so much, though, it was hard to get her out of it long enough to go to the beach!
As owner of another unit, I was interested to see VBTS from a renter's perspective, was very pleased with the experience.
Vacations by the Sea is my "Happy Place"
You may laugh, but seriously, whenever I do something dumb like stub my toe or go through something traumatic like my son being airlifted to the hospital after an accident, the happy place my mind goes to to keep my sanity is the ocean at Westport and the condos at Vacation by the Sea. It's a visualization that never leaves my mind and the relaxing non-touristy atmosphere is just what my inner-doctor ordered. We look forward to returning in the future again.
Thanks for the wonderful compliment and we are very honored you consider our condos a happy place to retreat to relax. See you in the future!
This was a great trip! Our first time to the WA coast and it was such a great experience. Loved the condo and the close location to the beach. Also loved the long Fisherman's memorial trail. We took a very long walk and it was so quiet and peaceful. We are definitely going to return!
Beautiful place on the beach!
We had a great time! And everyone was so hospitable!
Thanks so much for staying with us at Vacations by the Sea! We enjoyed having you hope you'll come back with the family soon!
Very enjoyed our staying and all of our family wanna be there again.
we had a great time. My only disappointment was the fact that we were not allowed to steam seafood on the back deck. I understand if we were on a level other than the 1st floor, but our room was on the 1st floor and the back deck was concrete. We enjoy cooking and without the opportunity to do so put a damper on our trip.
We're very happy you had a wonderfuil time and gave us such high marks for your stay. Unfortunately it is the Home Owners Associations rule that no BBQ's or fire sources be used on the decks and patios of the condos. Thanks for staying with us and we hope to see you again soon!
Wish we could have stayed longer. But very comfortable and nice.


The PC Music track features Clairo on vocals.
Danny L Harle has teamed up with Superorganism to drop a new remix of his track 'Blue Angel' (feat. Clairo).
The PC Music released track comes after Superorganism remixed Gorillaz' 'Humility' last month. Superorganism's self-titled debut album was released back in March.
Danny is fresh from releasing his recent remix of Superorganism's 'Nightime' via Domino and working with Nile Rodgers on the first new Chic album in almost 30 years.
Listen to 'Blue Angel' below.
Superorganism are one of the acts involved in the European Talent Exchange Programme. For more information on ETEP, and the artists and festivals involved, head to their website.


text:
Touching Gravitas
Feb 5 - Mar 19, 2022
10572 115 st, Edmonton, AB T5H 3K6
Wed-Fri | 12pm – 6pm
Sat | 12pm – 5pm
NOTE: The Gallery is closed for installation until our next show opens on January 21. Staff is available during office hours.
See current gallery hours
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SNAP is pleased to present Touching Gravitas, an exhibition by Liz Ingram and Bernd Hildebrandt, in the gallery from February 5 – March 19, 2022.
This exhibition results from years of collaboration and intimate contact between Bernd Hildebrandt, Liz Ingram and a special site in the boreal forest of Alberta. Consisting of printed works on paper, fabric print installations, video works, objects, and an artist bookwork, this exhibition strives to give the viewer an experience that re-establishes understanding, sense of place and responsibility within nature. Images of body contact with trees, moss, rich muskeg peat, and water speak of 'otherly' connections and love within nature. Other works allude to precariousness – to climate changes that are causing fire, floods and other devastating occurrences. A fragile and rather melancholic artist bookwork evokes a sense of touch, memory and the passing of time, utilizing images and text gathered during a prolonged stay in Buenos Aires, the city of Ingram's birth.
The gallery will be open for drop ins on Thursdays and Fridays, and by appointment on Saturdays. Please click here to book an appointment.
Online Artist's Talk on Saturday, February 12th at 4pm MST: Register here
This exhibition pays tribute to the forty-year anniversary of the Society of Northern Alberta Print-Artists. It also marks forty years of our marriage – a time of living, loving, raising two sons, playing, and working together. Our collaborative art production is focused on transitional states between material presence and the ephemeral, investigating what it means to be human within nature. Our prime inspiration for image and word source material has been and continues to be a particular place/lake/stream in the boreal forest of Alberta. We have recorded and presented our personal interactions within its 'nature', through evocative photographic and printed images, poetic text, tactile materials and various interpretive media. These have been intimate and personal as in the prints and artist bookworks, and spatial and sensual as in our tent/image/text installations.
Our most recent collaborative works utilized site-specific installations of images and words which attempt to re-awaken an awareness of our inextricable and fundamental oneness with nature, and our need to connect through our senses. This process has become more poignant and essential than ever in today's world of the transient, virtual, digitized realities contrasted with the rapid extinction of species and transformation of land, sea and atmosphere. Using printed images, paper, ink, light, and fabric, we are striving to give the viewer an experience that affirms their understanding, sense of place and responsibility within nature.
Liz Ingram was born in Argentina and grew up in New Delhi, Mumbai and Toronto. For over forty years she taught at the University of Alberta in Edmonton, Canada, and is currently Distinguished University Professor Emerita. She works in various media including etching, lithography, digital print, and installation. Her artworks have been exhibited in numerous solo and duo exhibitions, and over 300 group exhibitions in North and South America, Europe, the Middle East and the Far East. She has received awards for her prints at juried exhibitions in Canada, Slovenia, Poland, Korea, Brazil, Estonia, India and Finland. Other awards include: Royal Canadian Academy of Arts membership, 1998; the J. Gordin Kaplan Award for Research Excellence at the University of Alberta, 2008; the City of Edmonton Hall of Fame, 2008; the University Cup, 2011; the Royal Society of Canada membership, 2011; the Order of Canada membership, 2017; and the University Distinguished Alumni Award, 2021. Recent solo/duo exhibitions include: Touching Gravity, 'ace AIR Gallery, Buenos Aires, Argentina, 2018; Water/Bodies, FAB Gallery, University of Alberta, Edmonton, Canada, 2017; Site Collaborations, Anna Leonowens Gallery, Halifax, NS, 2017; Liz Ingram: Print Encounters, Prince Takamado Gallery, Embassy of Canada, Tokyo, Japan, 2015. www.lizingram.com
With a background in industrial and graphic design, and sculpture Bernd Hildebrandt plans exhibit spaces, designs display furniture and installs exhibitions that integrate objects, images and words. Working as an independent exhibit designer he has designed a number of extensive object based exhibits including: Edgar Degas: the Figure in Motion, Art Gallery of Alberta, Discovering Dinosaurs, for University of Alberta Museums and various exhibits (2007 – 2017) showcasing Chinese robes and scroll paintings from The Mactaggart Art Collection, University of Alberta. An interest in writing poetry has sparked numerous collaborations with Liz Ingram in which his poems are integrated into the fabric of her often large-scale images and prints. Working together has been very productive and has resulted in: temporary installations (2011-2018) in Poland, Germany, Argentina, Edmonton; permanent installations at the Southern Alberta Jubilee Auditorium (April 2011); and gallery exhibitions at the Glenbow Museum (2011), the Chelsea Art Museum in New York (2012), FAB Gallery, Edmonton (2017)and 'ace AIR Gallery, Buenos Aires, Argentina, (2018). www.hilxing.com
SNAP is happy to provide this programming at no cost to participants.
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Emerging Artists in Residence – Winter Window Showcase


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Next-generation sequencing of small RNAs from inner ear sensory epithelium identifies microRNAs and defines regulatory pathways
Anya Rudnicki1,
Ofer Isakov2,
Kathy Ushakov1,
Shaked Shivatzki1,
Inbal Weiss1 nAff3,
Lilach M Friedman1,
Noam Shomron2 &
Karen B Avraham1
The mammalian inner ear contains sensory organs, the organ of Corti in the cochlea and cristae and maculae in the vestibule, with each comprised of patterned sensory epithelia that are responsible for hearing and balance. The development, cell fate, patterning, and innervation of both the sensory and nonsensory regions of the inner ear are governed by tight regulation involving, among others, transcription factors and microRNAs (miRNAs). In humans, mutations in specific miRNA genes are associated with hearing loss. In mice, experimental reduction or mutations of miRNAs in the inner ear leads to severe developmental and structural abnormalities. A comprehensive identification of miRNAs in the sensory epithelia and their gene targets will enable pathways of auditory and vestibular function to be defined.
In this study, we used Next-Generation Sequencing (NGS) to identify the most prominent miRNAs in the inner ear and to define miRNA-target pairs that form pathways crucial for the function of the sensory epithelial cells. NGS of RNA from inner ear sensory epithelial cells led to the identification of 455 miRNAs in both cochlear and vestibular sensory epithelium, with 30 and 44 miRNAs found in only cochlea or vestibule, respectively. miR-6715-3p and miR-6715-5p were defined for the first time in the inner ear. Gene targets were identified for each of these miRNAs, including Arhgap12, a GTPase activating protein, for miR-6715-3p, implicating this miRNA in sensory hair cell bundle development, actin reorganization, cell adhesion and inner ear morphogenesis.
This study provides a comprehensive atlas of miRNAs in the inner ear sensory epithelia. The results provide further support of the essential regulatory role of miRNAs in inner ear sensory epithelia and in regulating pathways that define development and growth of these cells.
miRNAs play an essential role in inner ear development [1]. miRNAs are small non-coding RNAs that regulate gene expression post-transcriptionally. By binding to sequences in the 3' untranslated region (UTR) of mRNAs, a miRNA can inhibit target mRNAs by translational suppression and mRNA destabilization [2]. miRNAs have been implicated in hearing loss in humans, since mutations in the seed region of miR-96 or its gene are associated with hearing loss in three extended families [3, 4]. miRNAs were also reported in other human ear pathologies, including an elevation of miR-21 in cholesteatomas [5] and in vestibular schwannomas [6]. miRNAs were found to regulate the otitis media inflammatory response [7]. Their study in humans, however, has been hampered by the unavailability of inner ear RNA from human subjects, making the mouse an invaluable model for studying miRNA development and regulation in the inner ear [8]. In an ENU-induced mutant mouse exhibiting deafness and vestibular dysfunction, a mutation in the miR-96 seed region was found to be responsible for hearing loss, presumably due to the alteration in expression and hence function of both direct and indirect gene targets [9]. A number of mouse mutants affecting miRNA regulation through Dicer have been created, leading to developmental inner ear defects [10, 11]. These conditional knock-out mouse mutants have been instrumental in demonstrating that miRNAs are vital for inner ear morphogenesis and development of the sensory epithelia and sensory neurons.
Expression of over 300 miRNAs have been reported in mouse and rat inner ears thus far, identified by microarray analysis [8, 10, 12, 13], with the majority of work performed on the triad mir-96, −182, and −183 [14]. In order to further identify and characterize miRNAs in the mammalian inner ear, we used NGS for the first time to identify miRNAs in cochlear and vestibular sensory epithelia. Using this method, we identified over 500 inner ear miRNAs, both in common between and unique to each tissue. We further validated and characterized the expression of two miRNAs, miR-6715-3p and miR-6715-5p, in the mouse inner ear. Vezatin, an adherens junctions transmembrane protein previously implicated in deafness [15] and Arhgap12, a Rho GTPase activating protein [16], were verified to be gene targets of miR-6715-3p. Protocadherin 19, a member of the δ2 subclass of nonclustered protocadherins and associated with epilepsy and mental retardation [17], is a gene target of miR-6715-5p. Arhgap12, newly described in the inner ear, is expressed in the epithelial cell-cell junctions of the hair and supporting cells. The miR-6715-3p–Arhgap12 miRNA target pair defines a new regulatory pathway. Understanding these interactions may shed light on known and novel miRNAs, their effect on the development of normal and impaired hearing, and the mechanisms leading towards deafness.
RNA-seq derived miRNA transcription profile of the mouse inner ear sensory epithelium
In order to identify miRNAs in the auditory and vestibular sensory epithelium, we sequenced short RNA molecules from cochlear and vestibular sensory epithelia by high–throughput RNA sequencing (RNA-seq). For the cochlea, the sensory epithelium consisted of the organ of Corti, including hair cells, supporting cells, and cells of the greater and lesser epithelial ridges, as well as mesenchymal and neuronal cells. For the vestibule, the sensory epithelium was derived from the saccule, utricle and the lateral and anterior cristae and included hair cells and supporting cells. The advantage of using high–throughput sequencing is that this technique allows for assessment of transcript expression in a given sample at a large dynamic range, from minute quantities to highly abundant ones, and does not require a-priori knowledge for the identification of modified and novel RNA molecules in addition to known ones. cDNA libraries from small RNAs were prepared from dissected sensory epithelium of post-natal day (P)0 mice. Subsequent analysis of sequence reads was done using miRNAkey, a tool for miRNA differential expression analysis [18]. Processed reads were aligned to the mature Mus musculus miRNA database (http://www.mirbase.org), allowing one mismatch to occur between a read and the reference, due to isomiRs [19]. Sixty-six and 61.4% of the total reads from cochlear and vestibular sensory epithelia, respectively, were aligned to known mouse miRNAs, indicating a successful isolation of miRNAs. The majority of mapped reads in cochlear and vestibular sensory epithelia, 96.9% in the cochlea and 97.6% in the vestibule, were uniquely mapped to a single miRNA. Overall, 485 and 499 known miRNAs had an RPM (reads per million) mapping higher than 1 in the cochlear and vestibular sensory epithelium, respectively (Additional file 1: Table S1). These lists include all miRNAs that have been studied thus far in the mouse inner ear sensory epithelia, as well as miRNAs that have not been previously detected. Nine miRNAs were most highly expressed in the cochlea and the vestibule, with a similar order in both, except for miR-204-5p, which was the fourth most prevalent in the cochlea and the lowest in the vestibule (Table 1).
Table 1 Most highly expressed miRNAs in cochlear and vestibular sensory epithelium
Differential expression among the known miRNAs was inferred by miRNAkey, based on the widely-accepted assumption that the number of reads mapped to each miRNA is relative to its level of expression [20, 21]. The reads numbers in each sample were normalized to RPM values, and the relative expression levels of the different miRNAs in the two samples were calculated. In general, miRNA expression was similar in the cochlear and vestibular sensory epithelia. The majority of miRNAs with RPM > 1 were found in both tissues (455, or 86%), with 30 found only in the cochlea and 44 found only in the vestibule (Figure 1A, Additional file 1: Table S1). The rates of the highest expressing miRNAs in each tissue were also highly similar between tissues (Pearson coefficient = 0.99; P < 10−8) (Figure 1B). We defined a miRNA as differentially expressed if the fold change between samples was higher than 4 and P < 10−4 (Bonferroni corrected). Eleven miRNAs were differentially expressed in cochlear and vestibular sensory epithelia (Table 2).
miRNA expression similarity in the cochlear and vestibular sensory epithelia. A. Tissue similarity and common expression of inner ear miRNAs. High similarity of miRNA expression in the cochlear and vestibular sensory epithelium can be observed with the majority (455) of the 529 miRNAs, expressed with an RPM > 1, in both tissues. B. The relative proportion of each miRNA among the most highly expressed miRNAs in each tissue. The total amount (100%) represents the sum of RPK values of each miRNA. The Y-axis represents the proportion of each miRNA out of this total. Each miRNA is color-coded and indicated in the legend.
Table 2 Differentially expressed miRNAs in cochlear and vestibular sensory epithelium
Targets for most highly expressed miRNAs
miRNAs and targets exert their function through regulation of biological processes. As each miRNA can have multiple targets, we performed an analysis to discover additional mRNA genes that are computationally predicted to be targets of the three most highly expressed miRNAs, miR-182, miR-181a and miR-26a. Among numerous predictions, a few were of particular interest due to their involvement in auditory function. The targets were defined according to their presence in the list of genes that are associated with deafness in the Mouse Genome Informatics (MGI) (http://www.informatics.jax.org/diseasePortal/) database and predicted by two prediction algorithms, TargetScanMouse and miRanda (Table 3). All miRNA targets presented are involved in auditory function and hearing loss. As a few of the predicted targets overlap, we postulated that there might be underlying regulatory networks prompted by these miRNAs. This was further illustrated using gene ontology (GO) of biological-based processes, performed by geneMANIA (http://www.genemania.org), where 6/16 genes fell in the categories 'sensory perception of sound' and 'sensory perception of mechanical stimulus,' while 5/16 genes fell in the categories 'inner ear development' and 'ear development' (Additional file 2: Table S2). These predictions may set the basis for signaling pathways in the inner ear, subject to experimental validation.
Table 3 Predicted mRNA targets for the most abundant miRNAs in the sensory epithelia of the inner ear
Identification of inner ear miRNAs
In order to identify inner ear miRNAs, the genes associated with "impaired hearing" (MP:0006325) and "deafness" (MP:0001967) were downloaded from MGI (Additional file 3: Table S3). Since these genes are crucial for inner ear development and function, we predicted that miRNAs that reside in their introns may have a role in the inner ear. Out of the hearing associated genes, ten had pri-miRNAs in their introns (Additional file 4: Table S4). One pri-miRNA, expressed in both cochlear and vestibular sensory epithelia with an RPM > 1, was found in the intron of deafness-related gene β-tectorin (Tectb) (Figure 2A), which is expressed in the mouse inner ear and associated with altered hearing [22]. This pri-miRNA produces two mature miRNAs, miR-6715-3p and miR-6715-5p (Figure 2B). These miRs were supported by 2.08 and 5.06 reads per million library reads, respectively (Figure 2C). These miRs were also found in an analysis of sequences from mouse data sets derived from different tissues [23]. Provisional ID chr19_45771, available on miRBase (miR-6715; MI0025026), was detected by an extremely low number of reads (0.0021-0.0126 per million library reads) in oocytes, embryonic day (E)7.5 embryo, E15.5 brain, hippocampus, heart, skin, muscle cells, lung and liver by RNA-seq.
The genomic location, predicted structure and relative level of expression of miR-6715-3p and miR-6715-5p. A. pre-miR6715 is located in the second intron of the Tectb gene. B. miRDeep2 prediction of the RNA secondary hairpin structure of the unprocessed miR-6715-3p, miR-6715-5p, and loop. The seed region of miR-6715-3p is indicated in blue and of miR-6715-5p in green. C. Middle density plot shows the distribution of reads in the predicted precursor sequence, as produced by miRDeep2.
Expression of new inner ear miRNAs
The expression of miR-6715a-3p and miR-6715a-5p, detected by RNA-seq, was validated by qRT-PCR analysis (Figure 3A). We found both miRNAs to be expressed in the sensory epithelium of the mouse cochlea, at E16, P0 and P8. The expression of both miRNAs was dynamic and increased with age, showing higher expression at P8 compared to P0 and E16, and at P0 compared to E16 (P < 0.005). The two miRNAs were undetected in brain, liver, kidney and heart. This is consistent with the previous RNA-seq detection [23]. The inner ear expression, and the absence or very low expression of the miRNAs in other tested tissues, suggests that they are inner ear-specific miRNAs. The expression prior to the onset of hearing suggests a developmental role for these miRNAs.
miR-6715-3p and miR-6715-5p expression in the inner ear. A. qRT-PCR analysis and validation of novel miRNAs in the mouse cochlea sensory epithelium. miR-6715-3p and miR-6715-5p exhibited increased expression with age between E16 and P8 (*) P < 0.05, (**) P < 0.005. n = 5. B. In situ hybridization of P0 mouse cochlear and vestibular sections. miR-6715-3p and miR-6715-5p expression was found in the hair and supporting cells of both cochlea and vestibule, spiral and vestibular ganglia, stria vascularis, basilar membrane and in Reissner's membrane. sa, saccule; ut, utricle; sg, spiral ganglia; sm, scala media; st, scala tympani; sv, scala vestibule; ohc, outer hair cells; ihc, inner hair cells; hc, hair cells. Bar: 100 μm.
In order to explore the spatial expression pattern of the two inner ear miRNAs, in situ hybridization analysis was performed on inner ears derived from P0 C57/BL6 mice, at the same age the RNA-seq was performed. Consistent with the qRT-PCR results, expression of the two miRNAs was observed in P0 mice (Figure 3B). A clear staining of both miRNAs was observed in many parts of the inner ear, in spiral and vestibular ganglia, basilar membrane and Reissner's membrane, the stria vascularis and spiral ligament. In a higher magnification, in the cochlea, expression of both miRNAs was observed in the organ of Corti, in the spiral limbus and in particular in the hair and supporting cells. In the vestibular system, staining was observed in sensory epithelia of the saccule and the utricle, in hair and supporting cells.
Target pathways in auditory and vestibular function
In order to determine what pathway might be impacted by miR-6715-3p and miR-6715-5p regulation, we searched for potential targets. Targets were predicted by TargetScan Custom, which searches for a complementary 3'UTR given a putative seed sequence. miR-6715-3p and miR-6715-5p were each predicted to target 201 and 206 targets, respectively, with ten overlapping targets (Additional file 5: Table S5). We examined which potential targets were inner ear-expressing genes, genes associated with hearing loss in human or mice, or genes encoding proteins involved in pathways relevant for inner ear function. These included protocadherin 19 (Pcdh19) [24] as a candidate target for miR-6715-5p, and vezatin (Vezt) [15], Scn8a [25] and Arhgap12 [16] as candidate targets for miR-6715-3p.To test whether these potential targets have a direct interaction with miR-6715-3p and miR-6715-5p, we examined luciferase activity by inserting the 3'UTR of the potential targets downstream of a luciferase gene. Transfections of these constructs, together with a pre miR-6715 expressing vector into HEK293T cells, and measurement of the relative luciferase activity compared to a mutant 3'UTR construct, were indicative of targeting. Pcdh19 was found to be a direct target of miR-6715a-5p (Figure 4A), with a reduction of luciferase activity by approximately 35%. Vezt and Arhgap12 were found to be direct targets of miR-6715-3p, with a reduction of luciferase activity by 55% and 15%, respectively (Figure 4B, C). Snc8a is not a direct target of miR-6715a-3p, as luciferase activity was not reduced (Figure 4D).
miRNA-target prediction validation and expression. Using TargetScan, potential targets of miR- 6715-3p and miR-6715-5p were identified, based on the complimentarily of the seed region and 6-7 nt sequence of the target mRNA. To test the possibility of miR-target regulation, luciferase assays were performed. A. Protocadherin19 as a potential target of miR-6715-5p. Wild-type 3'UTR had approximately 65% of luciferase activity as compared to the mutant. (*) P < 0.05. B. Vezatin as a potential target of miR-6715-3p. Wild-type 3'UTR had approximately 45% of luciferase activity as compared to the mutant (*) P < 0.05. C. Activator for the Rho-type GTPases Arhgap12 as a potential target of miR- 6715-3p. Wild-type 3'UTR had approximately 85% of luciferase activity as compared to the mutant. (*) P < 0.05. D. Scn8a is not a direct target for miR-6715-3p, as the luciferase activity was not reduced.
Arhgap12 was chosen for further analysis, due to its putative role as a GTPase activating protein (GAPs) for small G proteins and expression in epithelial cell–cell junctions [16]. Temporal expression of Arhgap12 mRNA was evaluated by qRT–PCR in the cochlear sensory epithelium. In the cochlea, Arhgap12 was expressed at E16, P0 and P8, with a small, though not significant, increase in age (Figure 5). This protein was not previously characterized in the inner ear. We found Arhgap12 expression, using a commercial antibody examined for specificity by peptide competition (Additional file 6) in the organ of Corti, in hair and supporting cells, the stria vascularis, spiral ligament and Reissner's membrane (Figure 6A, B). The strongest expression was observed in nerve cells, in the spiral ganglia (Figure 6C). In the vestibule, expression was found in the hair and supporting cells (Figure 6D). In whole mount preparations, Arhgap12 was found to be present in hair and supporting cells (Figure 7A-C). Its expression was observed in the same hair cell and cuticular plate planes as ZO-1, but did not overlap (Figure 7A-C). The outer hair cell and Deiter cell junctions expresses ZO-1, while Arhgap12 appears absent in these junctions. Arhgap12 appears in the kinocilium (Figure 6B).
qRT-PCR analysis of Arhgap12 expression in the mouse cochlea sensory epithelia. Arhgap12 exhibited a similar expression pattern at E16, P0 and at P8 in cochlear sensory epithelia, with no significant differences between ages. n = 5.
Arhgap12 protein expression in mouse inner ear sections. Immunohistochemistry of Arhgap12 (green) in P0 inner ear sections, with hair cells stained by myosin VI (red) and nuclei stained by Draq5 (blue). A. Arhgap12 was detected in the organ of Corti, in hair and supporting cells, stria vascularis, spiral ligament and Reissner's membrane. B. Magnification of the organ of Corti, with localization of Arhgap12 in epithelial cells. C. Arhgap12 is localized to the spiral ganglia. D. In the vestibular system, Arhgap12 is present in hair and supporting cells in the crista. Bar: A, 100 μm; B-D, 50 μm.
Arhgap12 protein expression in mouse inner ear whole-mount preparations. Immunohistochemistry of Arhgap12 (green) in whole mount preparations of P0 mouse cochleas, with cell junctions stained by ZO-1 (red) and actin stained by phalloidin (blue). A. Arhgap12 is localized to the hair and supporting cells of the sensory epithelium. B. In a higher magnification of A, Arhgap12 is localized to the hair bundle and the kinocilium. C. Additional plane showing mutual exclusion of localization of Arhagp12 and ZO-1. hc, hair cells. Bar: A, 10 μm; B, 5 μm.
The function of miRNAs and their targets in the mammalian inner ear are now being discovered, as the link between miRNAs and hearing and deafness has been established. The inner ear is a complex tissue with multiple cell types, with miRNAs involved in fine-tuning the multitude pathways required for functional hearing. While there are a relatively small number of miRNAs in mammals, their role in regulation far surpasses their number, since each miRNA can exert its effect on hundreds of downstream targets [26]. Hundreds of miRNAs have been discovered in the inner ear; only a subset of these miRNAs have validated targets (reviewed in Table 1[27]).
Our study was designed to profile the general expression of miRNAs in the inner ear and a comparison of the auditory and vestibular epithelia. We chose the time point of p0 since work by our group [10, 13] and others have demonstrated that miRNAs are expressed and functional at this stage, including miRNA-96, which is essential for hearing [3, 9]. RNA-seq analysis led to the identification of over 500 miRNAs, with miR-182 as the most highly expressed miRNA in both sensory epithelia (Table 1) and accounting for more than 50% of the 20 most highly expressed miRNAs. A few targets have been confirmed for miR-182, including Sox2, Clic5 and Tbx1 [28–30]. The second most abundant miRNA in the cochlear and vestibular sensory epithelia, miR-181a-5p, has no known validated targets in the inner ear. This miRNA has been studied in other systems and found to have a pro-proliferative role in cultured human myeloid leukemia cells [31] and regulates thymic selection in mouse T-cells [32]. The role of this miRNA has been studied in the avian inner ear, where it has been shown to promote proliferation [33].
We validated a number of targets for miR-6715-3p and miR-6715-5p. Predicted targets for the miRNAs described in this study have direct or potential implications for hearing loss. Pcdh19 is expressed in the zebrafish inner ear [24]. Mutations in protocadherin 19 (PCDH19) have been associated with X chromosome-linked epilepsy and mental retardation and it is a member of the protocadherin family of proteins [17]. This family includes protocadherin 15 (Pcdh15), with a known function in the tip links of the stereocilia of the inner ear in conjunction with cadherin 23 [34] and has been associated with multiple mutations in both non-syndromic and syndromic hearing loss in humans [35, 36].
Vezatin (Vezt) is an adherens junctions transmembrane protein, and while it is ubiquitously expressed in these junctions, it is especially prevalent in in hair and supporting cells of the mouse inner ear [15]. Vezt mutant mice suffer from late onset progressive hearing loss, due to hair cell apoptosis. Most compelling, the resistance of the organ of Corti to mechanical stress was reduced when vezatin was absent in the inner ears of conditional mutants. These studies led to the prediction that vezation is required to maintain junction integrity and is crucial to protect the junctions from mechanical stress due to sound trauma.
Arhgap12, a Rho GTPase activating protein (GAP) family member, is a component of cell–cell junctions in kidney epithelial cells [16]. While not previously known to be expressed in the inner ear, its target, Rac1, localizes to hair and supporting cells, specifically in the stereocilia and kinocilium of the hair cells. It was found to be involved in hair cell morphogenesis, specifically in hair bundle development, epithelia actin assembly and cell adhesion. Inactivation of Rac1 specifically in the otic epithelium leads to defects in cochlear morphogenesis, hair bundle orientation and cellular organization of the organ of Corti [37]. Arhgap12 was found to target Rac1 and inactivates it by increasing GTP hydrolysis [38]. Here we suggest a key role for miR-6715-3p in targeting Arhagap12 and in this fashion, influencing Rac1 in the inner ear (Figure 8).
A newly identified miRNA-target regulatory pathway in the inner ear. A suggested pathway for miR-6715-3p and Arhgap12, whereby miR-6715-3p down-regulates expression of Arhgap12, which in turn blocks the hydrolysis of Rac1 (Ras-Related C3 Botulinum Toxin Substrate 1) GTPase. This may result in increased cell adhesion, actin reorganization and inner ear morphogenesis.
Arhgap12, a GAP protein, is involved in controlling the time period in which Rac1 will remain active. In MDCK cells, Arhgap12 expression overlaps with Rac1 expression [38, 39], probably due to the regulation of Arhgap12 on Rac1 at this time point. In the inner ear, besides the actin-rich areas, Rac1 is localized to hair cells and in particular, the kinocilium [37]. In accordance with our data, Arhgap12, at P0, with expression in the hair cells and kinocilium, might be regulating by Rac1 at this time, when the hair bundle is still developing and needs to be under close regulation. The kinocilium is necessary for proper development [40] and may have a structural role in guiding the architecture of the hair bundle and/or be involved in G-protein signaling in the kinocilium. If the latter is true, then Arhgap12 may regulate its GTPase activity. In our model of the miR-6715-3p circuit in the inner ear, we propose that the miRNA indirectly enhances the action of Rac1 and therefore may promote hair bundle development, actin reorganization, cell adhesion and inner ear morphogenesis.
The identification of inner ear-related miRNAs by RNA-seq analysis demonstrates that the dataset is reliable not only for characterizing expression profiles of known miRNAs, but also for discovery of novel miRNAs in the inner ear. Further investigation of these miRNAs may shed light on their regulatory roles in various molecular pathways underlying the development of the embryonic inner ear.
RNA isolation
All procedures involving animals were approved and met the guidelines described in the National Institutes of Health Guide for the Use of Laboratory Animals and approved by the Animal Care and Use Committee of Tel Aviv University (M-10-087). Cochlear and vestibular sensory epithelia were dissected from 12 inner ears of 6 P0 C57Bl/6 J mice. The cochlear sensory epithelia included the organ of Corti with some attached membranes, while the vestibular sensory epithelia included the saccule, utricle, lateral crista and anterior crista. The dissected sensory epithelia were placed immediately in Qiazol lysis buffer (Qiagen) on ice and stored at −80°C for several hours. The sensory epithelia was then thawed and total RNA was isolated, using the miRNeasy Mini Kit (Qiagen) without DNAse, which enables purification of total RNA of >18 nt. The isolated RNAs from the samples were precipitated in sodium acetate and glycogen in 80% ethanol (Ambion protocol), in order to further clean and concentrate the RNA samples. The precipitated RNA samples were dissolved in nuclease-free water and their quality was assessed by 1.2% agarose gel (90 V).
Twelve cochlear epithelia were pooled and twelve vestibular epithelia were pooled, and each pool was used to create a library for MPS and sequenced. No biological repeats were performed. miRNA libraries for MPS were created using the TruSeq SmallRNA SamplePrep Kit (Illumina), with one modification. Instead of running the samples on acrylamide gel in order to isolate the miRNA band, the samples were loaded on a 4% agarose E-Gel and the purification was performed with the Qiagen Kit. The libraries were multiplexed and 2.5 pM of each library was sequenced at SR × 36 bp using the Illumina Genome Analyzer IIx in the same lane at the Functional Genomics Laboratory at Tel Aviv University.
miRNA sequence analysis
A total of 8,763,589 and 10,048,818 raw sequences of small RNAs were found in the cochlear and vestibular samples, respectively. Small RNA adapter sequences were clipped using fastq-mcf (ea-utils: http://code.google.com/p/ea-utils/), discarding sequences shorter than 16 nucleotides and adapter dimers. Following adapter clipping, 8,340,741 and 9,441,679 reads remained. Differential expression of the resulting sequences was performed using miRNAkey. Specifically, sequence alignment was performed using Burrows-Wheeler Aligner (BWA) [41] against mouse mature miRNA sequences (downloaded from miRBase (Release 20: June 2013; http://www.mirbase.org/), while allowing one mismatch between read and reference. The read count for each read was normalized using the RPM method. Differential expression was tested using chi-squared proportion testing and corrected using the Bonferroni correction for multiple comparisons. Novel miRNA prediction was performed using miRDeep2 [42].
Cochlear sensory epithelia of E16, P0 and P8 C57BL/6 mice was dissected. Small RNAs were extracted using the miRNeasy Mini Kit (QIAGEN). Custom-made probes, designed by Applied Biosystems, were used to detect miRNAs. miRNAs and U6B RNA (endogenous control) were reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The qRT-PCR reaction was conducted using the FastStart Universal Probe Master (Roche) in the StepOne Plus qRT-PCR machine (Applied Biosystems). All miRNAs expression was normalized to the expression of U6B. At each age, sensory epithelia from 4–5 mice were pooled and five RT-PCR experiments were performed.
Inner ears of P0 C57BL/6 mice were dissected and fixed with 4% paraformaldehyde. Whole mount in situ hybridization analysis was performed using the Exiqon protocol. miRCURY LNA™ microRNA detection custom-made probes, labeled with digoxygenin (DIG) (Exiqon), were used for the detection of novel miRNAs. Probes were hybridized with the tissue, 20-22°C below Tm of the probe. Probes were detected with the anti-DIG-AP (alkaline phosphatase conjugated) antibody (Roche), and the color reaction was developed using the NTB/BCIP (Sigma). The ears were frozen and cryosectioned to 10-18 μm sections and mounted. Images were taken using the Zeiss Aviovert200 M microscope. Three experiments were performed, and 3–5 ears were included in each experiment.
miRNA target prediction
Target prediction for all miRNAs was predicted by TargetScan Custom (Release 5.2; http://www.targetscan.org/vert_50/seedmatch.html), TargetScanMouse (Release 5.2, http://www.targetscan.org/mmu_50/) and/or miRanda (http://www.microrna.org/microrna/home.do). Chosen potential targets were compared to gene expression databases (SHIELD; https://shield.hms.harvard.edu/) and known hearing loss genes and loci (Hereditary Hearing Loss Homepage, http://hereditaryhearingloss.org).
Luciferase assay
3' UTRs of chosen potential targets were cloned into the pGL3 luciferase reporter vector, downstream to the luciferase reporter gene, creating the 3'UTR-wt-pGL3 vector. The first three nucleotides of the miRNA binding site in the 3'UTR were mutated by site directed mutagenesis, creating the 3'UTR-mut-pGL3 vector. The miR-6715 miRNA expression vector was created by cloning the genomic polycistronic miR-6715-3p and miR-6715-5p into the miRvec expression vector (obtained as a gift from Reuven Agami) [43]. HEK293T cells were transiently transfected with either 3'UTR-wt-pGL3 or with 3'UTR-mut-pGL3, for each of the tested targets, together with the corresponding miRNA expression vector and Renilla expressing vector. The luciferase reporter assay was performed 48 hours following transfection using the Dual-Luciferase® Reporter assay system (Promega). Three experiments were conducted, with duplicates.
Whole mount and paraffin sections of P0 C67/BL6 mouse inner ear were prepared for staining as previously described [44]. Myosin VI was used to stain hair cells, phalloidin to stain actin and Draq5 to stain nuclei. The following antibodies were used: goat-anti-Arhgap12 antibody (Santa Cruz), 1:100; mouse-anti-ZO-1 (Zymed), 1:100; rabbit-anti-myosin VI (Proteus BioSciences) 1:200; and Draq5 1:600 (Abcam). Antibody specificity of Arhgap12 was assessed by a competition assay in HCT116 cells, with blocking by an Arhagap12 peptide (Santa Cruz) (Additional file 6).
For qRT-PCR and luciferase assays, the Student's two-tailed t test P values of less than 0.05 were considered to be statistically significant and those of less than 0.005 were considered to be highly statistically significant. The data in the figures are presented by mean + SEM. Differential expression of miRNAs between paired samples was measured using a chi-squared statistic. P-values are calculated for the null hypothesis of no differential expression between the two samples. Final P-values were corrected using the Bonferroni correction for multiple hypotheses testing [18].
The RNA-seq data from this study is available in the NCBI Sequence Read Archive (SRA) (http://www.ncbi.nlm.nih.gov/sra), under accession number SRP043019.
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This study was supported by the Israel Science Foundation (grant no. 1320/11), National Institutes of Health (NIDCD) R01DC011835, I-CORE Gene Regulation in Complex Human Disease Center No. 41/11 and a fellowship from the Edmond J. Safra Center for Bioinformatics at Tel Aviv University. We thank Varda Oron-Karni for her aid with RNA-seq.
Inbal Weiss
Present address: Clalit Health Services, Migdal HaEmek Clinic, Tel Aviv, Israel
Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, 69978, Israel
Anya Rudnicki, Kathy Ushakov, Shaked Shivatzki, Inbal Weiss, Lilach M Friedman & Karen B Avraham
Department of Cell and Developmental Biology, Sackler Faculty of Medicine and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, 69978, Israel
Ofer Isakov & Noam Shomron
Anya Rudnicki
Ofer Isakov
Kathy Ushakov
Shaked Shivatzki
Lilach M Friedman
Noam Shomron
Karen B Avraham
Correspondence to Karen B Avraham.
AR, LMF, NS and KBA designed and coordinated the study. AR and SS collected the inner ear samples. AR, KU, SS, IW and LMF generated the experimental data. OI and LMF performed the computational analysis of the data. AR, OI, KU, NS and KBA contributed to the writing of the manuscript. All authors read and approved the final manuscript.
Anya Rudnicki, Ofer Isakov contributed equally to this work.
Additional file 1: Table S1: miRNAkey output table for cochlear vs. vestibular miRNA differential expression. miRNA differential expression analysis includes the read count, RPM, fold change and comparison statistics for each miRNA transcript. (XLS 137 KB)
Additional file 2: Table S2: Predicted targets categorized by gene ontology (GO) of biological-based processes. (DOCX 63 KB)
Additional file 3: Table S3: Genes associated with "impaired hearing" and "deafness" impairment in MGI. (XLS 33 KB)
Additional file 4: Table S4: Expression of pri-miRNAs found inside introns of "impaired hearing" and "deafness" genes, as defined in MGI. (XLS 27 KB)
Additional file 5: Table S5: Predicted targets for miR-6715-3p and miR-6715-5p. Source: TargetScanHuman Custom Release 5.2: June 2011. (XLSX 15 KB)
Additional file 6: Specificity of the Arhgap12 antibody was tested by a peptide competition assay. HCT116 cells were stained with an Arhgap12 antibody and an Arhgap12 peptide. No staining of Arhgap12 was observed in the blocked cells. Bar: 25 μm. (TIFF 2 MB)
Rudnicki, A., Isakov, O., Ushakov, K. et al. Next-generation sequencing of small RNAs from inner ear sensory epithelium identifies microRNAs and defines regulatory pathways. BMC Genomics 15, 484 (2014). https://doi.org/10.1186/1471-2164-15-484
Sensory epithelia


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Kenneth Oppel Books In Order
Publication Order of Barnes and The Brains Books
A Bad Case Of Ghosts (1993) Hardcover Paperback Kindle
A Crazy Case Of Robots / A Bad Case Of Robots (1993) Hardcover Paperback Kindle
A Strange Case Of Magic / A Bad Case of Magic (1994) Hardcover Paperback Kindle
An Incredible Case Of Dinosaurs (1994) Hardcover Paperback Kindle
A Weird Case Of Super-Goo / A Bad Case Of Super Goo (1996) Hardcover Paperback Kindle
A Creepy Case of Vampires (2010) Hardcover Paperback Kindle
Publication Order of Silverwing Books
Silverwing (1997) Hardcover Paperback Kindle
Sunwing (1999) Hardcover Paperback Kindle
Firewing (2000) Hardcover Paperback Kindle
Darkwing / Dusk (2007) Hardcover Paperback Kindle
Publication Order of Matt Cruse Books
Airborn (2004) Hardcover Paperback Kindle
Skybreaker (2005) Hardcover Paperback Kindle
Starclimber (2008) Hardcover Paperback Kindle
Publication Order of The Apprenticeship of Victor Frankenstein Books
This Dark Endeavor (2011) Hardcover Paperback Kindle
Such Wicked Intent (2012) Hardcover Paperback Kindle
Publication Order of The Overthrow Books
Bloom (2020) Hardcover Paperback Kindle
Hatch (2020) Hardcover Paperback Kindle
Thrive (2021) Hardcover Paperback Kindle
Publication Order of Standalone Novels
Colin's fantastic video adventure. (1985) Hardcover Paperback Kindle
The Live Forever Machine (1990) Hardcover Paperback Kindle
Cosimo Cat (1991) Hardcover Paperback Kindle
Dead water zone (1992) Hardcover Paperback Kindle
The Devil's Cure (2000) Hardcover Paperback Kindle
Kings Taster (2007) Hardcover Paperback Kindle
Half Brother (2010) Hardcover Paperback Kindle
The Boundless (2014) Hardcover Paperback Kindle
The Nest (2015) Hardcover Paperback Kindle
Every Hidden Thing (2016) Hardcover Paperback Kindle
Inkling (2018) Hardcover Paperback Kindle
Ghostlight (2022) Hardcover Paperback Kindle
Publication Order of Short Stories/Novellas
The Klack Bros. Museum (2013) Hardcover Paperback Kindle
Publication Order of Picture Books
Cosmic Snapshots (1993) Hardcover Paperback Kindle
Follow That Star (1994) Hardcover Paperback Kindle
Emma's Emu (1999) Hardcover Paperback Kindle
Peg and the Whale (2000) Hardcover Paperback Kindle
Peg And The Yeti (2004) Hardcover Paperback Kindle
Publication Order of Chapbooks
Galactic Snapshots (1996) Hardcover Paperback Kindle
Publication Order of Anthologies
Other Worlds (2013) Hardcover Paperback Kindle
Kenneth Oppel was born in the year 1967, in the mill town of Port Alberni located on Vancouver Island, BC. However, despite the fact that Opel was born in Port Alberni, he spent a bigger part of his childhood days in Victoria, BC, and Halifax, Nova Scotia. When Kenneth Oppel turned 12 years, he made up his mind that all he wanted was to become an author. Oppel began by first penning down science fiction novels and then eventually moved to sorcery and sword tales. During the summer holidays, when Kenneth Oppel was fourteen, Oppel began to pen down a humorous narrative about a boy who was addicted to video games. The narrative eventually turned out to be a long narrative, and Kenneth Oppel was forced to rewrite the narrative for the next summer. As luck would have it, one of the family friends knew renowned author Roald Dahl in person.
The family friend presented the narrative to Roald Dahl, and after reading the script, Roald Dahl decided to pass down the narrative to his agent. A few months later, Oppel received a letter informing him that the agency wanted to publish Oppel's story and try to sell it.
Airborn is the first installment in the Matt Cruise book series, and it follows the story of one Matt Cruise, a cabin boy who works on the Aurora spaceship. Aurora is a luxury cruise liner in a different world, where Jules's dreams become a reality. Unlike a majority of the alternate history narratives, author Kenneth Oppel does not give so much when it comes to world development. The author does not specify the year that the story takes place and the extremely strange mix of technology makes things extremely hard to place the narrative. Matt Cruise is a boy who has always felt at home flying aboard Aurora, a luxury spaceship. Before, Aurora belonged to his father; however, it now belongs to Matt. Matt is hopeful that one day he is going to become a junior sailor. One evening, while Matt was on watch duty, he notices a balloon in distress. With the assistance of his team, Matt can rescue the balloonist, who before dying asks Matt whether he saw them.
The encounter with the old man sticks in Matts' head as he tries to establish what the old man had seen. It does not take long before Kate de Vries arrives and ultimately turns Matt's world inside out. Apart from being highly intelligent, Kate is a fast-talking woman who many at times might be a little bit headstrong. Kate is the granddaughter of the man who Matt had rescued from the balloon. Kate has traveled all the way to prove that her grandfather was not a crazy man. Furthermore, she is there to prove that her grandfather had seen some extremely marvelous creatures, while on his normal trips. Readers are going to fall in love with Kate from the word go. Matt, on the other hand, was extremely smart, dedicated and loyal as well. Matt's opinion of his captain was quite evident, and author Kenneth Oppel did tell and show with Matt's character development.
Apart from mishaps, Airborn was also filled with adventures and pirates as well. Author, Kenneth Oppel did a fantastic job of creating real characters and in the process getting the readers to care about these characters.
Silverwing is a narrative that focuses on Shade, an extremely young bat, and his day-to-day adventures. Shade was always a rebel, and as expected went against one of the main rules of the bat kingdom, never look at the sun. In the process, Shade's home was burnt to the ground. As a result, the entire colony had no option but to relocate to their winter home, down south. In the midst of a storm, Shade gets separated and eventually meets with a banned bat, Marina. Marina and Shade embark on a series of adventures, flying across the cities and meeting with two gigantic bats, Throbb and Goth. In the beginning, Throbb and Goth appeared to be nice bats, however, along the way, Shade discovers that these two bats were cannibals. Throbb and Goth, then forced Marina and Shade to take them to where their colony had settled. Along the way, the two giant bats were stopped, and both Marina and Shade eventually made it to safety.
With that said, the main theme in Silverwing is friendship. If Shade would have not met with Marina, then he would have died. Due to the fact that Marina and Shade trusted each other, they survived. The Ending of Silverwing was extremely sweet, though as expected it was a set up for the future books in the series. All the characters in Silverwing, were quite realistic and readers are going to feel as if they are watching real bats with actions and descriptions.
Starclimber
Starclimber is the third installment in the Matt Cruise book series. In Starclimber, we meet once again with the protagonist, Matt Cruise, and Kate De Vries. Starclimber begins as Matt Cruise and Kate are about to embark on an ultimate quest, which is filled with spaceships, romance, adventure, and space monkeys as well. After going through some extremely intense space training, Matt Cruise hopes that he is going to be the one who will be selected for the outer space mission. As luck would have it, Matt Cruise is shortlisted for the mission. Kate and Matt clamor onto the spaceship, the Starclimber. Initially, Matt felt extremely miserable when Kate announced she was engaged. What Matt does not know is that this will not be the only time when he feels this type of sadness.
It does not take long before a huge disaster strike, and Matt's survival skills are ultimately put to the test. With that said, the Starclimber is an excellent conclusion to the Matt Cruise book series. Readers can easily devour Starclimber in less than three days. Matt Cruise was very likable and did not disappoint at all. Tobias was also well developed and quite likable as well. The choice of words that author Kenneth Oppel used in his descriptions was well selected and rightfully placed as well.
Book Series In Order » Authors » Kenneth Oppel


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Norvell Theater
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Details Events
The Norvell Theater seats 251 people and hosts five youth productions per season, seasonal educational workshops and summer theater camps.
Piedmont Players Youth Theatre presents Stuart Little
Dates vary between January 31, 2020 - February 8, 2020
Piedmont Players Youth Theatre presents Stuart Little January 31 - February 1 and February 7-8 at 7:30 p.m.
Stuart Little presented by Piedmont Players Theatre
This is the endearing classic about a mouse named Stuart Little who is born into an ordinary New York family. The many adventures—both big and small—of Stuart...
Piedmont Players Youth Theatre presents Romeo and Juliet
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Piedmont Players Youth Theatre presents Romeo and Juliet at the Norvell Theatre February 21-22, 28-29 at 7:30 p.m.
Romeo & Juliet present by Piedmont Players Theatre
An age-old vendetta between two powerful families erupts into bloodshed. A group of masked Montagues risk further conflict by gatecrashing a Capulet party. A...
Piedmont Players Youth Theatre presents Madagascar
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Piedmont Players Youth Theatre presents Madagascar at the Norvell Theatre April 23-25, April 29 - May 2 at 7:30 p.m. April 26 & May 2 at 2:30 p.m.
Piedmont Players Youth Theatre presents Frozen, Jr.
Dates vary between July 16, 2020 - July 25, 2020
Piedmont Players Youth Theatre presents Frozen, Jr. at the Norvell Theatre July 16-18, 22-25 at 7:30 p.m. July 19 & 25 at 2:30 p.m.