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Reyenvas introduces its new range of reygreen products, developed to package fruits and vegetables in bulk, 100% biodegradable and compostable.
Adapted to the different needs of the farmer and agri-food marketers, our applications help to reduce the environmental impact.
Reyenvas is a manufacturer belonging to the Armando Álvarez Group. Currently we are the largest Spanish converter of polyethylene plastic films. Our long experience working with the top ten of each sector allows us to adapt and create solutions to your needs.


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What is BMI?
Body Mass Index (BMI) is a commonly used method to assess the ratio of weight to height. Calculating BMI is a quick and simple way to evaluate whether your weight is appropriate for your height.
- BMI below 18.5 – Underweight: Individuals with a BMI below 18.5 may be considered underweight and at risk of malnutrition. It is advisable to consult a doctor to establish an appropriate nutritional plan.
- BMI from 18.5 to 24.9 – Normal Weight: Individuals with a BMI in this range are considered to have a healthy body weight in proportion to their height. However, it is important to maintain healthy eating habits and engage in regular physical activity.
- BMI from 25.0 to 29.9 – Overweight: Individuals in this range may be at risk of health problems such as high blood pressure, type 2 diabetes, and heart disease. Adopting a healthier lifestyle through proper diet and regular physical activity can aid in weight loss and improve overall health.
- BMI above 30.0 – Obesity: Individuals with obesity have a significantly higher risk of serious health issues, including heart disease, stroke, type 2 diabetes, joint problems, and certain types of cancer. Seeking support from a doctor and/or a dietitian is crucial in the weight loss journey and improving overall health.
Remember that BMI is a general indicator and does not account for differences in body composition, muscle mass, or activity level. It’s always worth consulting with a doctor or nutrition specialist if you have any concerns about your weight and health.
BMI Calculator – Calculate your Body Mass Index
Enter your information below to calculate your BMI:

domain: biology


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In quarantine, we began to appreciate simple things and surround ourselves with beautiful things. It seems that online interior and flower shops have never been as popular as they are now (why growing flowers in quarantine has become a trend, we talked about here). With summer approaching, it's time to move from the rooms to the balconies, not forgetting to create comfort there. The editorial staff of BeautyHack.ru chose cool ideas for decorating a balcony on Instagram and found decor items in the photo in Moscow online stores. Believe me, repeating the interiors of popular bloggers and designers is not as difficult as it seems. The main thing is desire and inspiration. We hope that after viewing our selection you will have both.
Idea # 1: Minimalism with attention to detail
The simplest option is minimalism, which is characterized by light colors and simple shapes. However, we suggest adding bright details to this style. For example, bright pink or red flowers in a beautiful pot can be a great option.
Interior photos: @ mk.nordic
Decor items: 1) Pillow cover, Barcelona Design, 890 rubles. 2) Basket, DG Home, 1150 rubles. 3) Large-leaved hydrangea, Martin Sad, 5000 rubles. 4) Pouf-stool "Alseda", IKEA, 2799 rubles. 5) Carpet Bereber, DG Home, 18400 rubles
Idea number 2: boho style
Boho lovers should take a closer look at vintage interior stores. The main thing here is the details. Wall decorations in the form of macrame, straw pots, carpet and pillows with an unusual print - there are never too many decorative elements! It is only important to decide on the main shade of the interior.
Interior photos: @plantandwander
Decor items: 1) Decorative pillowcase, Westwing, 1450 rubles. 2) Carpet "Seoul-Benedetto", Westwing, 2664 rubles. 3) Wall decor Delya, Surdic, Westwing, 3100 rubles. 4) Succulent Sedum Nussbaumer, Enjoy Plants, 220 rubles. 5) Flower pot, H&M Home, 1699 rubles.
Idea number 3: romanticism
This option will appeal to romantic people. First, turn the balcony into a greenhouse, but choose bright flowering plants. Then add details in the style of romanticism and country: if textiles, then only with ruffles and lace, if objects, then from natural materials. Pay attention to the lighting: candles and lamps in the form of a garland will come in handy!
Interior photos: @smyk_w_kuchni_blog
Decor items: 1) Garland "Svartro", IKEA, 2799 rubles. 2) Wall lamp "Star", DG Home, 49 500 rubles. 3) Pillow with ruffles, OZON, 1170 rubles. 4) Candlestick La Forma, Dg Home, 11,990 rubles. 5) Geranium at Lechuza Balconera Cottage, Luxury Plants, 7950 rub.
Idea number 4: country
Wooden table and chairs, surrounded by plants, will turn the balcony into a real summer garden where you can relax and even have breakfast, lunch and dinner. For comfort, all that remains is to put a soft carpet on the floor, attach pots to the walls, and garlands to the ceiling. Done!
Interior photos: @my_diy_real_home
Decor items: 1) Dracaena, OBI, 2999 rubles. 2) Hanging pots H&M Home, 1699 rubles. 3) Garland "Livsor", IKEA, 1299 rubles. 4) Carpet Aftas, La Redoute, 6720 rubles. 5) Table and two chairs "Terno", IKEA, 5497 rubles.
Idea # 5: focus on the chair
An unusual but comfortable chair can become the main piece of furniture on the balcony. Next to it you can put a small table with a flower in a pot and a basket with a blanket. In this case, beauty lies in simplicity!
Interior photos: @andreanutas
Decor items: 1) Table with an oak top, TheSarai, 4270 rubles. 2) Begonia Elatior, OBI, 369 rubles. 3) Basket with handles, Furnish, 22 890 rubles. (for 2 pcs.) 4) Chair Acapulco, "First Furniture Hypermarket", 4200 rubles.
Popular by topic
The Most Popular Hardware Slimming Procedure: How Does Endosphere Therapy Work And Is It Worth The Money
What to eat to lose weight, what massage to sign up for, and how to choose the ideal volume reduction treatment? Endosphere Therapy is one of the most demanded newest hardware massage techniques in cosmetology. We learned from
Angelina Jolie Recently Gave An Unusually Many Interviews: 10 Main Thoughts From The Most Beautiful And Caring Woman On The Planet
Angelina Jolie, even during quarantine, tries to be useful. Almost every week in online conferences or interviews, she, as a philanthropist and UN Goodwill Ambassador, expresses her opinion on the topics of child abuse, home
In the last summer days, I just want to escape somewhere - away from the city, closer to nature. Have a picnic near the river, sea or at least in the park, enjoy the sun, stock up on vacation mood for future use, so that
Three new products in the ANSALIGY hair line, a collection of transferable tattoos and other events of the past week in our Friday digest. Dior Bronze Self-Tanning Water Dior introduces the first self-tanning body water
34-year-old singer Ciara will soon become a mother for the third time. She recently said that she and athlete Russell Wilson will have a son (the couple, we recall, is already raising a 3-year-old daughter Sienna and a 6-year-old son Ciara from the first

domain: artistic


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Merco resistance ropes set
The Boxing Band Set is designed for effective training of boxers and wrestlers.To buy
The cycling bag Carry 1.0 is a great accessory for everyday cycling. You can easily and quickly attach the bag to the handlebars using Velcro fasteners. When detached, you can also carry it over your shoulder thanks to the adjustable strap. This bag is made of waterproof polyester and its advantage is the inner aluminum foil, thanks to which food and drinks keep their temperature longer. The bag also offers one front zip pocket for small items, two side mesh pockets and an upper transparent pocket for your phone, map, etc.
- dimensions: 23 x 12 x 18 cm (width x depth x height)
- weight: approx. 280 g
- waterproof fabric (polyester) and waterproof zipper design
- internal insulating film to maintain temperature
- side mesh pockets
- front pocket for small items
- transparent top pocket for phone, map, etc. - 17 x 11 cm
- adjustable shoulder strap
- simple assembly without tools
Under the Merco brand, we design and have our own sports equipment manufactured. Because we like to do sports, we always try to improve the training equipment by some practical gadgets. We focus on affordable sports equipment for gyms, schools, sports clubs, leisure activities, fitness and home workout.
Merco is a popular brand on the Czech market, especially for its excellent price-performance ration of sports equipment.
|country of origin||China||Code||65297 (1)|
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Delivery to all EU countries
One of the Czech largest e-shops
Special training equipment
Time to time we send you interesting tips, instructions and discounts on equipment.
But don't worry, everything after the training. Exercise is a priority for us as well.

domain: design


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The Remote Sensing Section is a research section within the Remote Sensing Physics Branch. The section is currently engaged in the following research projects:
WindSat - Remote Sensing of Ocean Surface Winds
The wind vector affects a broad range of naval missions, including strategic ship movement and positioning, aircraft carrier operations, aircraft deployment, effective weapons use, underway replenishment, and littoral operations. Furthermore, accurate wind vector data aid in short-term weather forecasting, the issuing of timely weather warnings, and the gathering of general climatological data. WindSat is a satellite-based multifrequency polarimetric microwave radiometer developed by the Naval Research Laboratory for the U.S. Navy and the National Polar-orbiting Operational Environmental Satellite System (NPOESS) Integrated Program Office (IPO). It is designed to demonstrate the capability of polarimetric microwave radiometry to measure the ocean surface wind vector from space. The sensor provides risk reduction for the development of the Conical Microwave Imager Sounder (CMIS), which is planned to provide wind vector data operationally starting in 2010. WindSat is the primary payload on the Air Force Coriolis satellite, which was launched on 6 January 2003. It is in an 840-km circular Sun-synchronous orbit. It is currently undergoing rigorous calibration and validation to verify mission success.
Airborne Polarimetric Microwave Imaging Radiometer
The Airborne Polarimetric Microwave Imaging Radiometer (APMIR) is a passive microwave sensor being developed by the Remote Sensing Division. The instrument measures naturally occurring microwave radiation from the surface of the Earth and the atmosphere at several different frequency bands and at multiple polarizations. It will be a primary calibration tool for the Special Sensor Microwave Imager Sounder (SSMIS) and WindSat, which is currently under development at NRL. These satellite sensors will provide key data to Navy and Air Force weather forecasters. Since the geophysical parameters of interest have very small electromagnetic signatures, highly accurate sensors, such as APMIR, are needed for calibration of the satellite instruments. APMIR is carried aboard an NRL P3 Orion aircraft, and scans in azimuth and elevation. The movement of the sensor is measured and controlled to a high degree of accuracy. The first flight of the system occurred on December 19, 2001.

domain: aerospace


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A couple's dream retirement home, which became embroiled in the centre of a planning row, is being demolished by West Norfolk Council.
Rodney Wilson and his partner had planning permission to build a retirement home on land in Walsoken, near Wisbech.
But the council says the building had broken planning rules as it's 19 inches too wide and the changes it required hadn't been made.
More top news
Lifeboat crews from Hemsby in Norfolk are monitoring a number of clifftop houses that are on the brink of collapsing into the sea
Bitterly cold and windy with further snow showers.
Bitterly cold winds and some snow, especially across Essex later.

domain: news


IBNS/Every day there is one video or other which is going viral on social media but this time a video, featuring an elderly couple, has touched the hearts of millions as they wonderfully recreated the visuals of the classic Bollywood number ‘Rimjhim Gire Saawan’ from the 1979 film “Manzil”.
The original song had featured megastar Amitabh Bachchan and actress Moushumi Chatterjee.
The RD Burman classic beautifully touched the chords of Mumbai monsoon rains, making movie buffs enjoy the song for generations till now.
Now, the new video, which has gone viral, even caught the attention of Mahindra Group chairman Anand Mahindra who shared it on Twitter and wrote an emotional message that read: “This is justifiably going viral. An elderly couple re-enact the popular song ‘Rimjhim gire sawan’ at the very same locations in Mumbai as in the original film. I applaud them. They’re telling us that if you unleash your imagination, you can make life as beautiful as you want it to be…!”
In the video, the couple could be seen dressed like Bachchan and Chatterjee and recreating the scene as it is in the film.


Beginning of civil works on the major pumping station project in Valenzuela
The Department of Public Works and Highways (DPWH) said on Monday that civil works had started on a major pumping station serving a flood-prone area in the city of Valenzuela.
The project is expected to strengthen the city’s resilience to climate change, the department said in a statement.
The station is implemented by DPWH Metro Manila 3rd District Engineering Bureau and is expected to “solve the problem of permanent flooding of the city as well as nearby areas along the Meycauayan River”, he added.
Once the 234 million peso project in Barangay Veinte Reales is completed, it will complete the current network of 17 pumping stations in the city of Valenzuela.
In the DPWH’s Flood Management Master Plan for Metro Manila, measures being pursued include a stormwater catchment system that will allow some communities to store rainwater for reuse.
In February, the DPWH regional office in Metro Manila said it was assessing sites for catch basins to minimize flooding in the capital.
The World Bank estimates that the Philippines was visited by 94 destructive typhoons between 2011 and 2015, which is 9.3% more than the number of such typhoons between 2006 and 2010.
He also noted that many areas in Metro Manila are low-lying and designated as flood-prone, with insufficient protection from frequent flooding, as natural drainage is often restricted during rainfall events by high river levels and of the sea.
The World Bank provided technical and financial assistance for the department’s flood management master plan for Metro Manila. — Arjay L. Balinbin


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Some heart-soothing, heart-melting Qur’an recitation
(Begins at timestamp [1:22])
Life, Purpose, Reality
- Metaphor and Meaning:
“Please, please, not yet. I hate tidying up, it’s so boring. It’s so tiring. It’s so unfair. It’s just bad. The only good thing about tidying up is when everything is nice and clean afterwards. Is there any way to get tidy without tidying?!”
“Hardships surround Jannah and exciting temptations beautify the path to Jahannam.
The daily “tidy up time” and the corresponding anguish and protest it elicits will forever serve as a metaphor that nothing worth having comes easy, and the path there is often not the least bit enjoyable either. It is only the satisfaction of exhaling in a clear and clean space that provides you with motivation to push through. One day the ‘space’ we declutter and strive for will be clear of hardships and pain, and clean of everything the soul finds undesirable. Until then, we toil… Excruciatingly stepping on Lego one piece at a time…”
Seeing the profound realities of life’s existence does not require wild escapades and immersion into other ways of living. It requires new eyes to look deeper into what is already there. The world around us is full of the signs of Allāh, and these fill the small and seemingly inconsequential as they do the grand and mind-blowing. Whether you are sat watching an ant carry away crumbs ten times the size of its body, or trying to wrap your head around the idea of dark matter, there is metaphor and meaning in it all. Access the deeper layers beneath the surface reality of the life that we live by scrutinising things for a moment longer. Some reflection can throw up unexpected and profound insights, even – and especially – amid the so very ordinary. Sometimes, it is not new horizons we need, but new lenses through which to look at the extraordinary truths already laid bare right in front of our eyes.
“Indeed! In the creation of the heavens and the earth, and in the alternation of night and day, there are indeed signs for men of understanding. Those who remember Allāh standing, sitting, and lying down on their sides, and think deeply about the creation of the heavens and the earth, (saying): “Our Lord! You have not created (all) this without purpose; Glory is to You!”
— Qur’an (3:190-191)
Recommended YouTube Channels
- Hamza Yusuf. One of my most favourite scholars. The combined wisdom and knowledge – of literature, philosophy, politics, sociology, and more, from both Western and Islamic traditions alike – with which he speaks is astounding. Even if you do not agree with everything he says, it cannot be denied that his lectures offer some amazing insights into humanity.
- Yasmin Mogahed. An absolutely awesome scholar and speaker, Allahummabārik.
- Subhi Taha. Palestinian-Filipino, Muslim-American Subhi Taha is a YouTuber whose content I have enjoyed since 2013. Expect awkward humour, a distinguishing Texan accent, wonderfully embedded Islamic reminders, a characteristic ‘minimalistic-cultural’ aesthetic, and your extracting from his videos a unique sort of comfort from the general ‘normality’ of them. Subhi is “just an average Muslim-American”, living his truth: being Muslim, and being himself, as best as he can. Comfortably, and in a fun way, and in a way that is neither overly defensive nor reductive, when it comes to matters of Deen.
- Russell Brand. A wise one, a strange-in-a-very-good-way one, one who does not fit the ‘traditional’ mould of what ‘intelligence’ looks like. Humorous, ‘open’, idiosyncratic, and unpretentiously intelligent, Russell Brand’s ideas and short advice videos are real gems. I really believe so many of his views are perfectly aligned with Islamic ones, perhaps without his own being aware of this fact…
- ‘Smile to Jannah‘. “Conscious comedy, real issues and not reliant on immorality to make you laugh.” This is a brilliant YouTube channel whose content is ‘relatable’ and genuinely enjoyable to view, and whose messages are accessible for a range of audiences. Expect much cheesy humour (which is actually very funny, somehow!) in addition to many insights into the reality of this life, and into human psychology. Much-needed gentle reminders for us all, from someone who really ‘gets’ it.
- Lectures and infographic videos by Nouman Ali Khan. His Qur’anic explanations really do illuminate, and he conveys the Book’s messages in a way that is, I think, second to none.
Religion and ‘Spirituality’
The Test of Life
Stories / Reminders
Emotional Reassurance in Islam
- When NOT to give your Islamic Reminders
“What is a routine reminder to you can be an explosive trigger for others. Be cautious: if you are perceived as just another religious person reeling off verses like a robot, rather than a relatable empathic human being, then you risk pushing an already-distant person even further from the dīn.”
“Words are incredibly powerful. Human pain is hugely sensitive, and sometimes people’s relationship with their own faith can be complex and fragile. There is a subtle art to being able to deliver the right words of comfort a person needs and recognising when it is wiser to stay silent altogether.”
We humans are fundamentally emotional creatures. And emotional intelligence is arguably the most valuable form of intelligence that one can have; often, it is best expressed through “simple, sincere, and honest human interactions”.
- Videos by Mufti Menk, e.g:
‘Ilm and Da’wah
“Behind the ‘live your best life’ hashtags are people looking up in adulation at what is essentially a mirage.”
- Artificial Intelligence is Creating a New Madhab:
“These people are essentially living in two different worlds. They have different versions of what reality is.”
- Under Digital Threat: Muslim Children’s Spirituality
Hyper-stimulation and the ‘attention economy’. These things do have a significant impact on ‘spirituality’/religion.
“Human engagement with family, friends, and the community – together with hands-on learning, outdoor recreation, and development of ‘soft skills’ – are all part of the very broad menu of healthy development that we owe to our children.
In a world that is increasingly geared towards growth, output, productivity, and speed, we must actively take back the right to be slow, considered, and still. It is exactly there that the soul finds life.“
‘Freedom of Speech’ in France, against the postcolonial backdrop:
Uyghurs in China:
Miscellaneous (Podcasts etc.)
I am really loving the ‘Freshly Grounded’ podcast!

domain: philosophy


text: Josh has always been drawn to natural healing and alternative medicines – primarily Traditional Chinese Medicine (TCM). In 2002, while recovering at Lake Tahoe Wellness Center after a severe injury, Josh met his first teacher and learned Chen Man-Ching Yang style Taichi Quan and Taichi sword. After a year, his teacher handed the class to him, where Josh continued to teach for 2 years. Josh returned to New Hampshire and sought out Master Yang Jwing-Ming of the Yang Martial Arts Association (YMAA). He delved deep into studying Taichi and White Crane Qigong from him and, notably, started his journey into the cultivation path of Daoist Internal Alchemy. After 4 years, Josh attained the Qi Gong instructor certification from Master Yang’s school. Continuing his training over the years, he studied with many teachers and practitioners and learned aspects of Chen style Taiji and Wing Chun. In the summer of 2021, a perfect opportunity arose when Josh chanced upon Master Zhou, whose training center is not far from his home. Guided by his life experiences and inclination toward Daoist philosophy and values, Josh is now training earnestly in Wudang Taiji, Taiji Jian (straight sword), Wudang Gongfu, and Wudang Dao (saber) under Master Zhou. Josh is teaching Taiji classes in NH. Email: email@example.com.


Folding camera for 5x4 inch plates. Victor rapid rectilinear lens, US4 - 128. Bausch and Lomb shutter 1/2/5/25/100/B/T. Rack and pinion focusing, extra extension. Sliding rising and cross front. Reflecting rotating finder on baseboard. Circular ball level. Vertical swing back. Reversible back, with sprung ground glass screen. No 127548. In fitted case with two double blockform dark slides, and Premo film pack adaptor.


Big Slick - Customizable Lube Skids
Service Truck Depot lube skids allow you to turn your service truck into a lube truck with added convenience and flexibility. Lube Skids provide mobility and ease of transport. They are designed to fit a wide variety of service trucks.
Below you will find what our product offers to assist in discovering which one will best suit your needs. If you're in the market for a new service truck, see our customizable Service Trucks.
- 40/40/40 fresh product
- Radius tank corners for added strength
- 11 gauge formed shell with inserted sides
- Vented twist-lock fill opening
- Single air connection for customer provided air system
- Air operated Graco® 1” aluminum diaphragm pump
- On-load/off- load through single hose with 4-way valve
- Graco® spring rewind hose reel with ¾” suction hose, with ball valve
- 80 gallon waste oil storage tank
- Filter / regulator
- Internal product pumps for protection from damage
- Graco® 3:1 air operated reciprocating pump
- Graco® spring rewind hose reel with ½” x 35’ delivery hose, terminated with Graco® metered nozzle and holder
- Three 40 gallon product tanks
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- Service Truck Depot Lube Skids can be set on the ground, put in the bed of your truck, or mount the reel compartment remotely
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Featuring both new & used vehicles


This study investigates how the lionfish (Pterois sp.) invasion of the Western Atlantic Ocean has been socially constructed by natural scientists, the media, and stakeholders associated with various marine protected areas in the Caribbean. By examining the use of data and metaphors by these actors, I identify where invasion discourses converge and diverge. Although consensus exists regarding the non-nativeness, introduction vector, and successful establishment of lionfish throughout the region, I also identify uncertainty surrounding lionfish impact and controversies regarding lionfish management and control. The dominant discourse frames lionfish as a threat and control efforts as a war to keep the enemy at bay, and promotes lionfish hunting and consumption by humans: the “ultimate predators.” However, this view is challenged by a coalition that questions the safety, effectiveness, and morality of the practices promoted by the kill-and-eat lionfish coalition. A nascent discourse that frames lionfish as fulfilling the role of overexploited native species, primarily expressed in socioeconomic terms, is shifting lionfish impact perception from negative to positive among some stakeholder groups. Whereas the dominant discourse views humans as helping nature to regain balance through lionfish hunting, a minority coalition views lionfish as part of the ecosystem, where a new equilibrium will be reached. This study shows that scientific data and metaphors, amplified by the media, facilitated initial understanding of the lionfish phenomenon and are used to legitimize claims. In time, however, local knowledge and societal values have intermingled with scientific data, sometimes challenging scientific discourses, and contributing to a richer understanding of the invasion as a social-ecological phenomenon.
Understanding the drivers and barriers to participation in citizen science initiatives for conservation is important if long-term involvement from volunteers is expected. This study investigates the motivations of individuals from five marine protected areas (MPAs) in the Dutch Caribbean to (not) participate in different initiatives around lionfish. Following an interpretive approach, semi-structured interviews with seventy-eight informants were conducted and analyzed using thematic network analysis. Approximately 60% (n = 48) of informants indicated that they had participated in citizen science initiatives at the outset of the invasion. From this group, almost half said that they still participated in some type of data collection, but only a few did so within a citizen science context. Many informants were initially motivated to participate in lionfish detection and response initiatives due to concern for the environment. Personal meanings attached to both the data collection experiences and to the data influenced informants’ motivations to sustain or cease data collection and/or sharing. In time, the view of lionfish as a threat changed for many informants as this species’ recreational and/or commercial value increased. Enabling and constraining factors for data collection and sharing were identified at the personal, interpersonal, organizational and technical levels. Our findings have implications for the design of future citizen science initiatives focused on invasive species.


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Maybe it’s time to say goodnight? Maybe it’s easier to turn off the lights and to not fight for things anymore? Maybe I’m tired and I don’t see a lot of others out there trying to make a difference in the world or even the communities where they live. Maybe I’m tired of seeing the same people in the room trying to change things so they get better. Maybe I feel powerless in the face of all these overwhelming issues. Maybe I’m tired of quoting stats and telling stories of people who are suffering and the world not changing to help them.
But where are you? Where are you? Are you feeling powerless in the face of all this as well? Do you not Do you care?know what to do or even where to start? Did you even know that there was an issue? Did you know that there are a bunch of issues? Do you care? Because on many days I feel you don’t. On many days I feel like you’re just tuning us out. On many days I think you care more about being distracted and doing your own thing than trying to help and we really need your help. We need it so badly.
Recently and was walking to meet a friend and thinking about some of these big issues, you know the ones, and I stopped in the middle of the sidewalk and just looked and I thought do any of you care? I had this impulse from deep in my guts that started to build, that started to go through me and up to my mouth which had to forcibly keep shut to stop from screaming “Why don’t you care?”
I know you’re busy, really I do, I know you’re trying to make ends meet and that you have problems of your own and that work is hard and that you’re tired too. I know that. I really, really do. But everyone feels that way. So many of us feel that way these days.
Or maybe you feel that it’s not your problem. Maybe you feel that you gave at the office or that these people need to get a job or it’s all in their heads or that they should pull themselves up by their bootstraps or that their human debris and not worth your time.
It could be for any of these reasons or none of them. But in the end you’re not showing up, you’re not there when we need you, your nowhere in sight. Not at the ballot box when it’s time to vote, not at the meetings when we talk about poverty, not in the neighbourhood to help that person with a mental illness, not at the shelter when someone needs a place to sleep, not at city hall to demand that they start listening to everyone and not just a few, not at the plant that just got shut down. YOUR NOT THERE AD I’M TIRED AND SO ARE MT FRIENDS AND WE NEED YOUR HELP.
We need it because soon, not tomorrow or even next year, but soon it may become pointless because nothing can be done. That we’ll have no voice at the ballot box, that more people than not will be poor, that people with mental illness will all be on the street, that the environment will tank, and that it will become a long dark winter of no power and endlessly distracting ourselves so we don’t feel the loss.
Most of the people who read this first will be people who are trying to do something but maybe they’ll send it on to the people they know who aren’t doing something or don’t know how to help. It’s you I’m trying to reach here. It’s you who I’m talking to. Maybe you’re at university or maybe you work in a corporation or maybe you’re a tradespersons or maybe you’re a banker or a housewife, a painter, on welfare, or an artist but we need you to show up and give help and give input. Email me if you want to know where or how at email@example.com.
I care and do my little bit as best as I can although I’m not always sure what I can do. I did give my little donation to the “Occupy movement” I think you should write further and tell people the where and how and maybe some will listen
I agree. How about info sessions on how to get involved? I’m one of the many who care and want to help, but family and work obligations take up a lot of time.

domain: psychology


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Dry cat food in the Maxi Zoo online shop
Which dry food is right for cats?
Dry food should always be of high quality and contain all nutrients, vitamins and minerals necessary for cats. If that is the case then you can feed your cat this alone. The decisive factor is that the dry food is tailored to the needs of your cat. Several factors play a role in this – such as age, breed and life situation. House cats, for example, require less food than outdoor cats. For cats with sensitive digestion, dry food is often more digestible than wet feed. Chewing the bits is also good for cats’ dental care. It is important that your cat always has fresh water available for proper hydration. Unlike wet feed, dry food does not provide any liquid.
How much dry food does a cat need each day?
The age, weight and activity level of your cat also influence the amount of food. Outdoor cats generally need more food than indoor cats. The quality and composition of the food also play a decisive role – for example, how many calories it contains. Cat food manufacturers give general feeding recommendations on the packaging, which you can use as a guideline.
How do you accustom cats to dry food?
To get your cat used to dry food, you need to be patient. Cats are “creatures of habit”. In general, they are cautious when confronted with changes or unfamiliar things. So getting them to switch to a different kind of food can take several days or even weeks. It is best to mix a very small amount of the new food into the usual food every day. You increase this amount slightly each day – until the conversion is complete. Regular feeding times can help. It is important that your cat does not get any treats during the transition, because your well-intended snacks can spoil their appetite.

domain: pet


text:
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Making Video Dance: A Step-by-Step Guide to Creating Dance for the Screen is the first workbook to follow the entire process of video dance production: from having an idea, through to choreographing for the screen, filming and editing, and distribution. In doing so, it explores and analyses the creative, practical, technical, and aesthetic issues that arise when making screen dance. Published by Routledge on the 11th September 2018, the second edition of Making Video Dance is rigorously revised and brings the book fully up to date from a technical and aesthetic point of view.
Making Video Dance is a seminal work, the first of its kind for screendance/dance film educators. When Making Video Dance arrived on the scene, it soon became the pre-eminent resource for those interested in exploring the practice of making dance films. I whole-heartedly support the revision/additions to Making Video Dance, as its subject has only become more relevant since its first publication.
- Ellen Bromberg, Distinguished Professor, University of Utah
Katrina McPherson is an award-winning screen dance artist whose practice is informed by her background in dance and video art, as well as her wide experience as a director of arts programmes for television. Katrina’s recent directing credits include the In Motion series for BBC on-line, and collaborative screen dance works such as Uath Lochans, Coire Ruadh, and seminal works such as There is a Place, Moment, and Pace. She is an experienced lecturer and workshop leader and has taught extensively worldwide.

domain: entertainment


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domain: game


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ORIGINAL RESEARCH article
Sec. Hematologic Malignancies
Volume 11 - 2021 | https://doi.org/10.3389/fonc.2021.694331
Intratumor Heterogeneity of MIF Expression Correlates With Extramedullary Involvement of Multiple Myeloma
- 1Department of Hematology, Institute of Hematology, West China Hospital, Sichuan University, Chengdu, China
- 2Department of Anesthesiology, The Second Xiangya Hospital of Central South University, Changsha, China
- 3Hunan Provincial Key Lab of Emergency and Critical Care, Hunan Provincial People’s Hospital, Changsha, China
- 4Department of Hematology, Affiliated Hospital of North Sichuan Medical College, Nanchong, China
- 5Department of Pathology, West China Hospital, Sichuan University, Chengdu, China
- 6State Key Laboratory for Oncogenes and Related Genes, Division of Cardiology, Renji Hospital, School of Medicine, Shanghai Cancer Institute, Shanghai Jiaotong University, Shanghai, China
Macrophage migration inhibitory factor (MIF) has been shown to promote disease progression in many malignancies, including multiple myeloma (MM). We previously reported that MIF regulates MM bone marrow homing and knockdown of MIF favors the extramedullary myeloma formation in mice. Here, based on MIF immunostaining of myeloma cells in paired intramedullary and extramedullary biopsies from 17 patients, we found lower MIF intensity in extramedullary MM (EMM) versus intramedullary MM (IMM). Flow cytometry and histology analysis in xenograft models showed a portion of inoculated human MM cells lost their MIF expression (MIFLow) in vivo. Of note, IMM had dominantly MIFHigh cells, while EMM showed a significantly increased ratio of MIFLow cells. Furthermore, we harvested the extramedullary human MM cells from a mouse and generated single-cell transcriptomic data. The developmental trajectories of MM cells from the MIFHigh to MIFLow state were indicated. The MIFHigh cells featured higher proliferation. The MIFLow ones were more quiescent and harbored abundant ribosomal protein genes. Our findings identified in vivo differential regulation of MIF expression in MM and suggested a potential pathogenic role of MIF in the extramedullary spread of disease.
The proliferation of clonal plasma cells in multiple myeloma (MM) is typically confined to the bone marrow (BM). However, the extramedullary spread of MM cells, defined as extramedullary multiple myeloma (EMM), may occur at any time in the course of MM (1). Patients with EMM have inferior outcomes (2, 3). Broadly speaking, there are two main types of EMM: plasmacytomas extending from local bone lesions (para-skeletal or extramedullary bone-related, EM-B) or resulting from hematogenous dissemination (extra-skeletal or extramedullary-extraosseous, EM-E) (1, 4, 5). Previous studies have suggested that EM-E showed an even worse prognosis than EM-B (3). In addition, plasma cell leukemia (PCL) might be considered as a specific variant of EMM (5). Nevertheless, some authors have suggested that PCL should be excluded from the EMM spectrum because of PCL’s unique disease entity (4).
The pathogenesis of EMM has not been fully understood. Various genetic and microenvironment mechanisms might affect the occurrence of EMM, including the presence of 13q14 deletion, t(4;14) or TP53 mutation, frequent mutations in the RAS pathway (6–10), the impairment of C–X–C motif chemokine receptor 4 (CXCR4)/stromal-derived factor 1Alpha(SDF-1α) (11–13), differential expression of adhesion molecules, such as very-late-antigen-4 (VLA-4), CD56, CD44 and P-selection (14–17), and upregulation of angiogenic factors like CD31 and endoglin (16). We previously identified macrophage migration inhibitory factor (MIF), a soluble pro-inflammatory cytokine, as a regulator of MM BM homing (18). MIF is highly expressed in MM BM and regulated downstream adhesion molecules’ expression in MM cells; knocking down MIF in MM cells hampered MM adhesion to BM stromal cells in vitro, therefore resulting more extramedullary tumors in an SCID mice model (18). To investigate the variation of MIF in EMM versus the parental intramedullary MM (IMM), we initiated this comparative study using paired BM and extramedullary biopsies from a series of MM patients. MIF regulation and the associated single-cell trajectories in EMM were further studied using xenograft mouse models.
Materials and Methods
Formalin-fixed, paraffin-embedded (FFPE) paired tumor samples of BM and extramedullary tissues were retrospectively collected from 17 MM patients who had extramedullary involvement with the following inclusion criteria: 1) the patient was admitted to our hospital from 2014 to 2019; 2) the patient with confirmed MM had pathologically verified extramedullary involvement either at initial diagnosis (primary EMM) or during relapse/progression (secondary EMM); 3) imaging or physical examination showed measurable lesions of EMM. Both EM-B (extramedullary bone-related) and EM-E (extramedullary-extraosseous) myeloma were included in this study; 4) EMM and the corresponding IMM samples from each patient were collected at the same disease stage. Patients with solitary plasmacytoma, PCL, lymphoma with plasmacytic differentiation, or any additional malignancy were excluded. Cytogenetic examination of BM plasma cells was recommended by physicians. This study was approved by the Ethical Committee of West China Hospital, Sichuan University, China. All patients were consented for use of their medical records and samples for research.
Patient-Derived Primary MM Cells
With the approval by the Ethical Committee of West China Hospital, primary MM cells were obtained from consenting patient #16 when MM progressed with extensive extramedullary disease. Mono-nuclear cells (MNCs) were separated from his iliac BM aspirate by Ficoll-density gradient centrifugation. CD138+ myeloma cells were flow-sorted (Beckman Coulter CytoFLEX platform) using phycoerythrin (PE)-conjugated mouse anti-human CD138 antibody (BD, Biosciences, Cat.# 552026), and cryopreserved in our department tissue bank. For inoculation, the primary MM cells were resuscitated, cultured for 24 h in RPMI-1640 medium with 10% fetal bovine serum (FBS) in 5% CO2 atmosphere at 37°C, washed with phosphate buffered saline (PBS), and then resuspended in PBS at a concentration of 5 × 106/ml.
Human MM Cell Line and CRISPR Based Target Gene Knock Out
The human MM cell line ARD was a generous gift from Prof. Yiguo Hu (State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University). ARD cells were authenticated by short tandem repeat (STR) profiling (GENEWIZ, Inc. Suzhou, China) before experiment. We knocked out MIF gene in the ARD cells by the CRISPR/Cas9 system as previously described (19). The plasmid pHKO23 was also kindly gifted from Prof. Yiguo Hu. The single guided RNAs (sgRNAs) targeting human MIF gene were as follows:
CACCGAGCTCGGAGAGGAACCCGTC(fwd); AAACGACGGGTTCCTCTCCGAGCTC (rev).
Both ARD and MIF−/− ARD cells were stably infected by luciferase expressing lentivirus (HanBio Inc., Shanghai, China). All cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 g/ml streptomycin at 37°C and 5%CO2, and were mycoplasma free. Cells were resuspended in PBS at 2 × 107/ml for injection.
In Vivo Mouse Models of Human MM
All animal experiments were approved by the Animal Care and Use Committees of West China Hospital, Sichuan University and were performed in accordance with the ethical standards. To establish xenograft models, patient-derived primary MM cells or luciferase-expressing ARD cell line were implanted into six-week-old female immunocompromised B-NDG mice (NOD-Prkdcscid IL2rgtm1/Bcgen, Biocytogen Jiangsu CO., Ltd.) via tail vein injection. A total of 13 mice were used in this study, including those challenged with the primary MM cells (0.5 × 106 cells per mouse, n = 4) or wild type (w.t.) ARD cells (2 × 106 cells per mouse, n = 5), and control mice given MIF−/− ARD cells (2 × 106 cells per mouse, n = 2) or vehicle (100 μl PBS, n = 2). Mice were housed in pathogen-free conditions and inspected at least twice a week to monitor general health and disease symptoms. Mice in morbid state were sacrificed. Eyeballs were removed under anesthesia to collect blood. Subsequently, they were euthanized via cervical dislocation and dissected for further examinations.
In Vivo Bioluminescent Imaging
For tumor monitoring, cell line-derived MM bearing mice were subjected to in vivo bioluminescent imaging (BLI) using IVIS spectrum (PerkinElmer, Inc.), on days 7, 14, 21, 28 post inoculation. D-luciferin, potassium salt (Biovision, USA, Cat.# 7903), a substrate of luciferase, was injected intraperitoneally (15 mg/ml in PBS, with 150 μl per mouse). At 15 min after administration, image acquisition was performed under isoflurane anesthesia.
Immunohistochemistry Analysis and Result Quantification
Tumor specimens were fixed in 10% neutral buffered formalin, decalcified (bone tissues), paraffin-embedded and then processed for immunohistochemistry (IHC) staining. FFPE sections were deparaffinized and rehydrated, and incubated with 3% H2O2 for 10 min to block endogenous peroxidase activity. Antigens were retrieved with sodium citrate buffer (10 mM, pH 6.0, at 98°C for 10 min). Nonspecific binding was blocked with 5% normal goat serum in TBST, for 1 h at room temperature. Primary antibodies including rabbit polyclonal MIF antibody (dilution 1:400, Santa Cruz Biotechnology, Cat.# FL-115) and rabbit monoclonal CD138 antibody (dilution 1:400, Abcam, Cat.# ab128936) were applied overnight at 4°C. Then, the sections were incubated with biotinylated goat anti-rabbit IgG and streptavidin–biotin complex (SABC) following manufacturer’s instructions (Boster Biological Technology, Cat.# SA1022, China). Signal was developed by diaminobenzidine (DAB) detection kit (ZSGB-Bio, China, Cat.# ZLI-9031), followed by hematoxylin re-dying. The stained slides were imaged under an upright fluorescence microscope (AX10 imager A2/AX10 cam HRC, Zeiss, Germany). For better evaluating MIF intensity, the paired IMM and EMM samples were prepared and photographed for MIF staining synchronously. To evaluate DAB intensity of MIF staining in patients’ slides, wide-field images were captured by the TissueFAXS system (TissueGnostics) as previously described (20), and the mean MIF intensity was quantified using the StrataQuest software (TissueGnostics) followed the manufacturer’s instruction. Pathological analyses of all slides were reviewed by an expert pathologist.
Opal Multicolor Staining
We used opal multicolor staining to visualize CD138 and MIF on selected human FFPE slides with Opal 4-color manual IHC kit (PerkinElmer, Inc.) as previously described (21).
Flow Cytometry Analysis
MM cells engraftment in different murine organs was assessed by flow cytometry as previously described using Beckman Coulter CytoFLEX platform (18). Single-cell suspensions were prepared from blood, spleen, liver, lung, kidney and femurs (BM) following relevant guidelines (22). Collagenase D (Sigma-Aldrich, Cat.# 11088858001) was used (1 mg/ml, at 37°C for 30 min) for enzymatic digestion of solid organs, like lung and kidney. Myeloma cells were identified by phycoerythrin (PE)-conjugated mouse anti-human CD138 antibody (BD, Biosciences, Cat.# 552026). Since MIF was an intracellular protein, cells were fixed and permeabilized using the intracellular Fix & Perm buffer set (Invitrogen eBioscience, Cat.# 88-8824-00), followed by MIF staining with Alexa Fluor 488-conjugated mouse anti-human MIF antibody (Clone# 932606) (R&D systems, Cat.# IC2891G). 7-Aminoactinomycin D (7-AAD) (AAT Bioquest, Cat.# 17501) was applied to label non-viable cells before permeabilization. The results were analyzed by the FlowJo software.
Single Cell Sequencing and Data Analysis
Splenic cells were collected from a tumor-bearing mouse xenografted with ARD cells. CD138-positive MM cells were then sorted by PE-conjugated CD138 antibody (BD, Biosciences, Cat.# 552026) using flow cytometry technology. The construction, sequencing and processing of the single-cell transcriptome library was conducted by the Chengdu Neo-life Hope Medical Laboratory Co. Ltd.
MacoskoDropSeq pipeline was used to generate the single-cell transcriptome profile (23). The raw pair-end sequencing data were first processed using DropSeq Core computation protocol developed at McCarrll’s lab. The derived digital expression matrix was then used to generate Seurat object using Seurat R package. The downstream analyses and data visualization were performed using Seurat functions. The following Seurat functions were used in the Seurat pre-processing pipeline: NormalizeData, ScaleData were used for calculate the comparable expression values; FindVariableFeatures were used to include the variable genes that contribute to the overall similarity/variability of cellular transcriptomic profiles; RunPCA, FindNeighbors, FindClusters, RunTSNE, and RunUMAP were used to calculate the dimension-reduction coordinates for visualization and to perform unsupervised clustering. In the downstream analyses, we used Uniform Manifold Approximation and Projection (UMAP) coordinates to visualize the layout of the cells. We used Monocle2 software to explore the possible differentiation path of these xenografted plasma cells and identify the subclusters using the default software parameters (24).
We used top 10 principle components to represent the digital expression profiles of each cells, and calculate the median values of these 10 principle components of each putative clusters. Then we calculate the overall dissimilarity using the cosine distances algorithm. The abovementioned dissimilarity measure was defined in TooManyCells method (25). The hierarchical cluster was plotted based on the dissimilarity matrix of each cluster.
Statistical analyses were performed using Graphpad Prism 8 (GraphPad Softwares, USA). Comparisons of continuous variables were made by student’s t-test or analysis of variance (ANOVA). The co-expression relationship between two genes was tested by simple linear regression. Mann–Whitney U test was used to compare variables with no specific distribution from independent samples. A p <0.05 was considered statistically significant.
Differential MIF Expression in EMM Versus IMM in Patients
Previously, we showed that MIF-knockdown (MIF-KD) MM cells had decreased BM homing and were likely to form extramedullary tumors in MM mouse models (18). In this study, we retrospectively collected FFPE biopsies of IMM and EMM from 17 MM patients. The patient characteristics are shown in Table 1. Twelve males (70.6%) and five females (29.4%) were included, with a median age of 63 years (range, 44–74 years). Most of the patients had IgG myeloma (10/17, 58.8%), the others had IgA (5/17, 29.4%), IgD (1/17, 5.9%) and λ light chain (1/17, 5.9%) myeloma. Thirteen of them (76.5%) were diagnosed with primary EMM, and four with secondary EMM (23.5%). There were 10 EM-B samples (58.8%) and 7 EM-E samples (41.2%), with available imaging data for extramedullary lesions in eight cases (Supplementary Figures 1A, B). Nine patients had available cytogenetic information (9/17, 52.9%). The most frequent genetic abnormalities included deletion of chromosome 13q14, immunoglobulin heavy chain gene (IGH) translocations and 1q21 amplification.
Pathological analysis revealed higher MIF expression in IMM than in the paired EMM, as shown in the representative IHC staining in Figures 1A, B (all IHC data are available in Supplementary Figure 2). First, MM cells were positive for CD138 expression in all tumor samples (Figures 1A, B and Supplementary Figure 2). The invasive pattern in IMM varied from interstitial infiltration, focal aggregation to diffuse sheets. As for EMM, a diffuse or nest-like distribution of MM cells was more common. Second, MM cells from all tested samples generally showed cytoplasmic MIF expression. Nevertheless, differential expression of MIF was observed in EMM versus IMM (Figures 1A, B and Supplementary Figure 2). Quantification of MIF staining (Supplementary Figure 3) confirmed that MM-derived MIF expression was significantly higher in IMM than in EMM for the same patient (Figures 1C, D). In addition, we performed opal multicolor staining and immunofluorescence microscopy in selected paired FFPE samples to examine MIF expression. In agree with IHC data, lower MIF expression in EMM biopsies was observed in opal multicolor staining (Figures 2A, B). Overall, our data suggest that EMM clones tended to have down-regulated MIF expression compared with the corresponding IMM clones.
Figure 1 IHC staining showing MIF expression in EMM versus corresponding IMM. (A, B) IHC staining for CD138 and MIF in the paired samples from representative cases: the plasma cells highlighted by CD138 show higher MIF expression in IMM than in paired EMM, either for EM-B (A) or for EM-E (B; magnification 100×). (C, D) MIF intensity quantification based on wide-field IHC imaging indicates its intratumor heterogeneity; all EMM samples (n = 17) display downregulated MIF expression compared to the IMM counterparts. ****p < 0.0001, ***p < 0.001 (two-way ANOVA).
Figure 2 Immunofluorescence staining of MIF expression in EMM versus corresponding IMM. (A, B) Differential MIF expression in paired samples is also confirmed by opal multicolor staining in representative cases, including EM-B (A) and EM-E (B) (DAPI represents nuclear, red represents CD138, and green represents MIF; original magnification 40×).
Intratumor Heterogeneity of MIF Expression In Vivo in Myeloma Mouse Models
To further investigate the differential MIF expression in MM cells in vivo, immunodeficient mice were intravenously inoculated with two type of human MM cells. The primary BM MM cells were derived from a patient with advanced EM-E disease. The human MM cell line ARD was cell line-verified as single clonal PCs. MIF knockout ARD cells were used as a negative control for the animal study (Supplementary Figure 4A). All above cells showed consistent strong MIF expression in vitro (Supplementary Figure 4B). Compared with vehicle control, all the mice challenged with MM cells exhibited obvious morbidity as shown by significant weight loss, rough hair coat and limited mobility. Disease onset seemed to be earlier in ARD cell line-derived model than in the PDX model. In vivo BLI in mice revealed the formation of both IMM and EMM after inoculation with ARD or MIF−/− ARD cells, the latter resulting more evident EMM (Supplementary Figure 4C), consistent with our previous findings (18). Tumor-bearing mice became moribund approximately 4–5 weeks after inoculation and thus to be euthanized (3–4 weeks after ARD cells injection, and 4–5 weeks after primary cells injection).
Cells isolated from BM, visceral organs, and peripheral blood, were analyzed using flow cytometry for MIF expression in MM cells (CD138+ cells) in vivo. MM cells were identified in all tested samples with varied percentages, distributing mostly in BM, spleen, liver and lung (Figures 3A, B, 4A, B). In MIF−/− ARD inoculated mouse, the MM cells showed no MIF expression (Figures 3A, 4A). Both patient primary MM cells and w.t. ARD cells exhibited two populations with differential MIF expression in vivo: MIFHigh versus MIFLow. Mean fluorescence intensity (MFI) of MIF in MIFHigh cells was more than 10 folds higher than that in MIFLow cells (Figures 3C, 4C). Of note, the relative proportion of MIFHigh and MIFLow MM cells was quite different between IMM and EMM samples. In general, IMM was found to have a significantly higher ratio of MIFHigh/MIFLow MM cells compared to EMM: MM cells in BM were dominantly a MIFHigh population, while in extramedullary organs or peripheral blood, the percentages of MIFHigh cells decreased, and the MIFLow population became remarkable (Figures 3D, 4D). In the PDX scenario, MIFHigh cells still constituted the majority of EMM, except the lung lesions (Figure 3D). As to the ARD model, MIFHigh cells turned to be overwhelmed by MIFLow ones in most EMM samples (Figure 4D). In addition, IHC staining for CD138 and MIF were performed in BM and spleen samples from tumor-bearing mice. The infiltrating cells positive for CD138 were a mixture of MIFHigh and MIFLow ones, with more evident loss of MIF expression in spleen versus BM (Figures 3E, 4E).
Figure 3 Identification of MIFHigh and MIFLow MM cells in patient-derived xenografts. (A) Patient #16-derived BM CD138+ cells were intravenously injected into B-NDG mice to generate a PDX model (n = 4). They were euthanized to collect fresh tissue cells for flow cytometry within four to five weeks after inoculation. Mice received MIF−/− ARD cells were used as negative controls. Representative plots show the expression profiles of CD138 and MIF in isolated cells. Green represents CD138− cells, namely non-MM cells; light blue represents MIF− MM cells, dark blue represents MIFLow MM cells, red represents MIFHigh MM cells. (B) Percentage of infiltrating CD138+ MM cells varies considerably in collected samples (n = 4). P = 0.0017 (two-way ANOVA). (C) Compare mean fluorescence intensity (MFI) of MIF between MIFHigh and MIFLow populations in diverse samples. The mean value ± standard deviation is 362.5 ± 81.8 and 19.5 ± 5.5 respectively. ****p < 0.0001 (two-way ANOVA). (D) Ratio of MIFHigh/MIFLow MM cells is notably higher in BM compared to extramedullary samples. ****p < 0.0001 (student’s t-test). (E) Representative IHC staining images of paired FFPE samples show sheets of MIFLow CD138+ cells surrounded by MIFHigh ones, and more evident loss of MIF expression in EMM (spleen) than in IMM (the blue and red boxes indicate representative areas of MIFLow and MIFHigh MM cells, respectively).
Figure 4 Identification of MIFHigh and MIFLow MM cells in ARD cell line-derived xenografts. (A) B-NDG mice were intravenously injected with ARD cell line to establish another xenograft model (n = 5). Control mice received vehicle (PBS) or MIF−/− ARD cells. Three to four weeks after inoculation, samples were processed in the same way as described in the PDX model. Representative flow cytometry plots show MIF and CD138 expression in IMM and EMM (green, CD138- cells, namely non-MM cells; light blue, MIF- MM cells; dark blue, MIFLow MM cells; red, MIFHigh MM cells). (B) CD138+ cells (ARD cells) infiltrate all tested samples with varying degrees. P = 0.0001 (two-way ANOVA). (C) MFI of MIF differs greatly between MIFHigh and MIFLow MM cells in diverse samples, with 373.4 ± 59.3 (MIFHigh) versus 24.0 ± 8.1 (MIFLow). ****p < 0.0001 (two-way ANOVA). (D) Significantly higher ratio of MIFHigh/MIFLow MM cells in BM versus extramedullary samples. ****p < 0.0001 (student’s t-test). (E) MIFHigh and MIFLow ARD cells in vivo are also identified by IHC. Representative images show their coexistence and more evident loss of MIF expression in spleen than in paired BM (the blue and red boxes indicate representative areas of MIFLow and MIFHigh MM cells, respectively).
To summarize, based on the above animal work, we found that a portion of MM cells lost their MIF expression (MIFLow) in vivo. IMM was dominated by the MIFHigh MM cells. In EMM, by contrast, the expansion of MIFHigh cells was accompanied by a notable MIFLow population, which would attenuate the overall MIF expression. Somehow, those findings from the animal models might be consistent with our data of paired patient samples. Since a single clonal cell line was used in our model and the establishment of an MM-bearing model took only 3–4 weeks, it is not likely that the heterogeneity of MIF expression in MM cells was caused by genetic alterations.
Single-Cell Transcriptome Study Reveals the Molecular Characteristics of Heterogenous Myeloma Subpopulations
Although MIFLow MM cells in vivo were phenotypically distinct from MIFHigh ones, physically separating them from each other for molecular study was not feasible because the permeabilization procedure of MIF staining would damage the nucleic acids in cells. Based on our previous findings, we hypothesized that the MM cells in vivo might have differential levels of MIF transcripts. Therefore, total CD138+ cells sorted from splenic cells in an ARD myeloma-bearing mouse (Supplementary Figure 5A) were subjected to single-cell RNA sequencing (scRNA-seq).
We used the DropSeq technique to investigate the single-cell transcriptomic profiles of the extramedullary plasma cells harvested from an ARD myeloma-bearing mouse. Given the lineage connectivity of these xenografted cells, we plotted the putative developmental trajectory using Monocle2 software. The software color-coded seven cellular states using the default parameters (Figures 5A, B). The violin plots showed significantly higher expression of MIF and two other co-expressed genes D-Dopachrome Tautomerase (DDT) and RNA Polymerase II Subunit F (POLR2F) in cluster #5, the co-expressions were validated in BM MM cells from multiple patient datasets (GSE26760, GSE19784, GSE9782) (26–28), which suggested cluster #5 was likely the initial state of these plasma cells (Figure 5C and Supplementary Figure 5B). Using the ‘FindMarkers’ function of Seurat, we further examined the signature genes of the clusters for state #5, the putative initial points of the cell lineage, as well as states #6 and #1, two putative end states of the cell lineage (Figure 6A). We carefully examined the marker genes using functional enrichments and had some interesting hits. The initial state, #5 cluster (MIFHigh subpopulation), showed a significantly higher score of S and G2/M-phase (Figure 6B) and enriched the cell cycles, p53 signaling pathway, proteasome, etc. (Figure 6C). This finding suggests that the MIFHigh feature is likely associated with a highly proliferative cell state. For the two end states of the trajectory, compared to cluster #5 of the initial state, cluster #1(MIFLow subpopulation) gave many ribosome RNA signals, while cluster #6 gave no obvious signature expression, possibly indicating a transitional state (Figure 6A).
Figure 5 Single-cell transcriptomics exploring the trajectories of MIFHigh to MIFLow MM cells. (A) Sc-RNA seq explored the transcriptomic profiles associated with the development of MM cells in extramedullary microenvironment. MM cells labeled with the anti-human CD138 antibody were separated by flow cytometry from the spleen of an ARD MM-bearing mouse. A total of 1,424 extramedullary MM cells were plotted. The branching tree of MM cell trajectories define seven color-coded cell clusters or states: putatively developing from cluster #5 (initial state), proceeding along with several unstable intermediate states (clusters #3, #2 and #7, or cluster #4), and finally reaching cluster #1 or #6 (end state), as indicated by the red solid arrow (trajectory I) and black dotted arrow (trajectory II, probably transitional), respectively. (B) tSNE plots show seven clusters. Arrows I and II indicate the two putative cell-state transition trajectories, respectively. (C) Cluster #5 could reasonably be the initial state, since it has significantly higher expressions of MIF and two co-expressed genes DDT and POLR2F, as shown in the violin plots and scatter plots of pseudotime estimation. Likewise, the putative end state, especially cluster #1, has the least expression of MIF, DDT and POLR2F.
Figure 6 Distinct transcriptomic profiles of MM cells with differential MIF expression. (A) A heatmap of marker genes reveals distinct transcriptomic profiles of the MIFHigh initial state (cluster #5) versus the MIFLow end state (referring to cluster #1; cluster #6, lack of obvious gene expression signature, might actually be transitional). (B) Active proliferation is found in cluster #5 with a significantly higher score of S and G2/M-phase. (C) KEGG enrichment analysis indicates some important pathways, such as cell cycle, proteasome and p53 signaling pathway in cluster #5.
The presence of extramedullary myeloma, namely EMM, usually portends inferior outcomes for MM patients, even in the era of novel agents and stem cell transplantation (2, 29–31). Driven by multiple genetic and microenvironmental mechanisms, certain MM subclones are predisposed to survive outside the BM niche (5, 32). The molecular basis underlying EMM is still elusive. Although EMM typically results from contiguous or hematogenous spread of the intramedullary counterpart, EMM has its own distinctive biological features (1, 5). Here we proved the differential MIF expression between the matched IMM and EMM, both in patient samples and xenograft models.
The pleotropic biological activities of MIF have been characterized since 1960s (33–35). It is highlighted that MIF promotes progress and metastasis of various solid tumors, via activating downstream proliferation and anti-apoptotic signaling, regulating VEGF-mediated angiogenesis, inhibiting tumor suppressor P53, inducing immunosuppressive microenvironment and so on (36–43). Pathogenic role of MIF has also been identified in MM, and its expression level may serve as a surrogate for disease progression and prognosis (18, 34, 44–46). Recently, Wang et al. reported that MIF was implicated in proteasome inhibitor resistance by maintaining superoxide dismutase 1 (SOD1) activity and mitochondrial function (44). In that sense, MIFHigh MM cells seem to be associated with more aggressive tumor behavior. However, MIF itself is an important chemokine favoring MM BM homing as we previously described (18). MIFLow MM cells, generated by shRNA viral infection, would more easily egress from BM and form EMM in mice, due to impaired adhesion to the BM niche (18). Therefore, we assumed that MIFLow clones, unlike in many solid tumors, may also implicated in MM extramedullary metastasis.
This study revealed that IMM was overwhelmed by MIFHigh MM cells, while EMM showed a remarkably increased ratio of MIFLow MM cells. This finding provided more evidence to suggest the link between decreased MIF and extramedullary colonization of MM cells. The differential dominance of the MIFHigh clone in IMM versus EMM also indicated that the BM microenvironment had consistent selection pressure on MM cells to maintain the MIFHigh phenotype. Predominance of MIFLow MM cells in ARD-derived EMM was more obvious, not completely consistent with that in PDX scenario. It’s probably due to the biological difference between the two kinds of inoculated cells. Most noteworthy, ARD-derived xenograft and patient-derived xenograft, representing genetic homogeneity and heterogeneity respectively, could produce similar phenotypes in the short term after engraftment. Therefore, we inferred that an inheritable mechanism, likely involving some epigenetic changes rather than acquired genetic events, might govern the stable regulation of differential MIF expression.
Based on our ARD cell line-derived xenograft model, a further scRNA-seq was applied to explore the MIF-specific gene expression profiles in EMM. Our pseudotime analysis showed the consecutive alteration of MIF and its validated co-expressed genes along the developmental trajectory of extramedullary MM cells. Those transcriptional changes of MIF would be part of the reason for its differential expressions at the protein level. Furthermore, distinct transcriptomic profiles were revealed in the MIFHigh and MIFLow clusters, respectively. For one thing, the MIFHigh cluster featured higher proliferation, while the MIFLow cluster seemed much more quiescent. Previous in vitro studies by Joseph et al. demonstrated that mature CD138+ MM cells, being highly proliferative and chemosensitve, secreted more MIF than their quiescent, chemoresistant CD138- progenitors, and addition of MIF inhibitor or recombinant MIF factor would promote the bidirectional interconversion (47). Specifically, our data suggested the in vivo correlation between MIF gene expression and MM cells proliferation, although no significant difference was found in CD138 expression between the two clusters. For another, the MIFHigh cluster enriched the cell cycle, proteasome and P53 pathways, while the MIFLow one harbored impressively abundant ribosomal protein genes. In a sense, this part of our data was consistent with the MIF-dependent tumor-promoting phenotypes reported in literature (37, 43, 44, 47, 48).Those differentially expressed marker genes between the two clusters also suggested potential upstream regulators of MIF transcription. To our knowledge, MIF transcription is mainly affected by various transcription factors, such as specificity protein 1 (SP1), cAMP response element binding protein (CREB), inverted CCAAT box binding protein of 90 kDa (ICBP90), and Hypoxia-induced factor 1α (HIF1α), and CATT repeats or G/C polymorphism at promoter region (49), the details in MM are as yet unknown.
It should be noted that the developmental trajectory of extramedullary MM cells was putative, dynamic, and even reversible. As MIF was highly expressed in MM cells before inoculation, we artificially defined MIFHigh cluster as the initial state. In this case, adaptation to the microenvironment may account for the above findings. Another possibility is that MM cells homing to BM occasionally lose their MIF expression, and the MIFLow clone would leave BM more easily to drive the extramedullary migration. Then MIFLow cluster turned to be the alternative initial state.
In summary, our work identified the coexistence of MIFHigh and MIFLow MM cells, two distinct cell states with different proliferation ability and molecular profiles, and more obvious clonal expansion of the MIFLow ones in EMM than that in IMM. The heterogeneous MIF expression in vivo was revealed at both the protein and mRNA level. We considered the loss of MIF expression in a portion of MM cells is mainly affected by microenvironment mechanisms instead of second genetic hits. Notably, our xenograft models could not entirely mimic the clinical course of EMM. Although the causal relationship of MIFLow clone and EMM pathogenesis is still not definite, this study provides new insights into our understanding of MIF, a pathogenic effector and potential therapeutic target in MM. Nevertheless, questions remain about the exact regulation mechanisms, biological behavior and drug resistance properties of MM cells with differential MIF expression. We are conducting more studies to address those questions.
Data Availability Statement
Our scRNA-seq data presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE175660.
The studies involving human participants were reviewed and approved by the Ethical Committee of West China Hospital, Sichuan University, China. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the the Animal Care and Use Committees of West China Hospital, Sichuan University.
YHZ designed and supervised the research, and prepared the manuscript. SZ provided the patients’ samples, reviewed the results of IHC staining, assisted with data interpretation. YMZ provided the biological insight from scRNA-seq data, and prepared the manuscript. YHZ, SZ, and YMZ contributed equally to this work as co-corresponding authors. JX performed the experiments, data analysis and prepared the manuscript. NY and WLin performed the extensive scRNA-seq data analyses and prepared the manuscript. JX and NY contributed equally as co-first authors to this manuscript. PZ conducted the MIF gene knock-out experiment. FW and JH assisted with research design, and provided critical suggestions. YC, HD, YY, and YG assisted with experimental work. The other authors provided the basic clinicopathological data. All authors contributed to the article and approved the submitted version.
This work was supported by grants to YHZ from the National Natural Science Foundation of China (Nos. 81870157 and 82070219), Science and Technology Department of Sichuan Province (No. 2019YJ0028), and the Sichuan University Faculty Start Fund. Grants to JH from the National Natural Science Foundation of China (No. 81800207) and the Health Commission of Sichuan Province (No. 18PJ357). A grant to YQ from Science and Technology Department of Sichuan Province (No. 2018FZ0030).
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank the following people for their technical assistance: Prof. Yiguo Hu (State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University); Guonian Zhu and Bo Su (Core Facility, West China Hospital, Sichuan University); Li Li, Fei Chen and Chunjuan Bao (Laboratory of Pathology, West China Hospital, Sichuan Univsersity); Ping Liao (Laboratory of Anesthesia and Critical Care Medicine, Department of Anesthesiology, National-Local Joint Engineering Research Center of Translational Medicine of Anesthesiology, West China Hospital, Sichuan University).
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2021.694331/full#supplementary-material
Supplementary Figure 1 | Imagings of extramedullary myeloma lesions for biopsy in patients. The collected EMM tissue samples were bone-related (EM-B) or extraosseous (EM-E). Corresponding imaging data were available for 8 of them. Magnetic resonance imaging (MRI) or computed tomography (CT) showed the biopsied EM-B (A) and EM-E (B) lesions respectively (indicated by the red arrow).
Supplementary Figure 2 | Differential MIF expression in paired IMM and EMM. Seventeen pairs of FFPE samples were stained for CD138 and MIF. In all tested slides, the infiltrating MM cells were specifically highlighted by membranous CD138 staining, and showed cytoplasmic positivity for MIF to varying degrees. Generally, IMM had higher MIF expression in comparison to the paired EMM.
Supplementary Figure 3 | Quantification method of immunostain for MIF in paired patient samples. Quantitative analysis of MIF staining was as follows (showing a representative pair):wide-field images of BM and EM tissue slides were captured with the TissueFAXS system (TissueGnostics ), then StrataQuest software (TissueGnostics) was used to convert DAB channels of MIF staining into gray scale images and to evaluate the mean intensity of MIF in effective positive cells as dot plots show.
Supplementary Figure 4 | Supplementary data of the animal experiment. Complete knockout of the MIF gene in ARD cells was verified by western blot (A). Flow cytometry analysis showed the in vitro expression of CD138 and MIF in these three types of grafts: All of them were positive for CD138; MIF was homogenously over-expressed in both ARD cells and patient-derived primary BM MM cells, and totally absent in MIF-/-ARD cells (B). Intravenous injection of human MM cells led to both intra- and extra-medullary lesions in immunodeficient mice. More serious EMM lesions were detected in the MIF-/- ARD group. The representative in vivo bioluminescent imagings were taken at two weeks after inoculation (C).
Supplementary Figure 5 | Supplementary data of the sc-RNAseq. Parallel flow cytometry analysis confirmed the differential expression of MIF protein in CD138+ cells subjected to sc-RNAseq (A). Co-expression of DDT, POLR2F and MIF in patient MM cells from BM was validated in three microarray datasets: GSE26760, GSE19784, and GSE9782, by gene set enrichment analysis (GSEA) with Pearson correlation (R2, R-square) (B).
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Keywords: extramedullary multiple myeloma, MIF, immunohistochemistry staining, xenograft models, single-cell sequencing
Citation: Xu J, Yu N, Zhao P, Wang F, Huang J, Cui Y, Ding H, Yang Y, Gao Y, Pan L, Chang H, Wu Y, Xiang B, Gong Y, Shuai X, Hou L, Xie L, Niu T, Liu T, Zhang L, Liu W, Zhang W, Qu Y, Lin W, Zhu Y, Zhao S and Zheng Y (2021) Intratumor Heterogeneity of MIF Expression Correlates With Extramedullary Involvement of Multiple Myeloma. Front. Oncol. 11:694331. doi: 10.3389/fonc.2021.694331
Received: 13 April 2021; Accepted: 10 June 2021;
Published: 29 June 2021.
Edited by:Rakesh Verma, ARMO Biosciences, United States
Reviewed by:Tijana Martinov, Fred Hutchinson Cancer Research Center, United States
Enfan Zhang, Zhejiang University, China
Copyright © 2021 Xu, Yu, Zhao, Wang, Huang, Cui, Ding, Yang, Gao, Pan, Chang, Wu, Xiang, Gong, Shuai, Hou, Xie, Niu, Liu, Zhang, Liu, Zhang, Qu, Lin, Zhu, Zhao and Zheng. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
†These authors have contributed equally to this work

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Here’s a lovely shot from one of our weddings last December at the LA River Center and Gardens! Image via Karla Delgado Photography. Happy Friday!
This month is flying by! As usual here are a couple Instagram sneaks from our wedding last weekend at Hotel Maya. I loved their colors :: fuschia and gold. Can’t wait for the pro pics by Nathan Nowack. On Instagram, I was asked where these linens came from and these […]
This weekend we have a seriously sweet couple tying the knot at the Los Angeles River Center and Gardens. Here’s a glimpse into these lovebirds by Henry Chen Photography! If you live to be a hundred, I want to live to be a hundred minus one day so I never […]

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