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By SJMA Forest Ambassador, Jojo Matson
Alpine tundra ecosystem services
May you be mindful that the alpine tundra is an incredibly special place for our entire ecosystem near and far. Everything underneath your feet is alive. The tundra ecosystem acts as a giant and delicate sponge created by a network of roots reaching up to two feet deep. This sponge holds our snowpack through the winter and reduces avalanche potential. It holds our soils together allowing moisture from snowmelt and spring monsoon storms to absorb into the ground instead of running off or triggering a mudslide. What most of you might notice are the abundant flower varieties that feed large populations of pollinators and herbaceous animals.
Every impact to that delicate sponge compresses the network of roots. The more compressed it becomes, the less effective it is at holding the ecosystem together. Look along the trail on the edges of the footpath and see how far it has compressed from the surrounding soil from feet and tires. Notice areas of increased water flow in the trail and the rapid erosion that takes place within a short amount of time. Once the erosion begins, it is very hard to stop on its own and is amplified and widened when we try to avoid the impacted areas. At some point, the impact might be irreversible, and the trail will become a large gully for water runoff.
May you be mindful where your feet, your furry friend’s feet, or your tires go today.
Alpine tundra, visitors in an ecosystem homestead
May you be mindful that the alpine tundra is a unique and diverse ecosystem that supports lifeforms near and far in an incredible balanced network of traveling and permanent dwellers. Some of these neighborhoods are confined to the tops of our mountains and are bound by the environmental boundaries of elevation, temperature, and water. This neighborhood is a collection of specialized flowers and plants and the specialized creatures that eat them. One you may be familiar with is the pika, a small and adorable furry creature that greets you as you enter the high country with loud chirps. The pika is very special because it can only survive in our high cool mountaintops. They cannot withstand temperature above 77 degrees F. They also do not sleep the winter away but survive awake underneath the snowpack. It spends all summer collecting, drying, and storing plants in stashed piles underneath rocks all over the tundra. Their knowledge of each plant’s necessary drying and storage time is miraculous. All the while, they spread seeds and propagate the environment they need to survive in the next year. They are as brilliant as they are adorable.
When you visit the tundra, you will notice there are not many places to hide. A few boulders, scree rock slopes, and valleys of flowers. The pikas have storage stacks that are in safe regions for them to use later. When our feet or out furry friend’s feet go near or invade the storage areas, it can lead to the pika getting hurt or stopping collection of food at that location.
May you be mindful while you visit the tundra where your feet and your furry friend’s feet go. We are there for a short time, and the pika calls it home.
The heartstrings for the trees
May you be mindful that the San Juan Forest is a uniquely biodiverse ecosystem ranging from a dry desert range to the high alpine mountaintops. Everything under your feet is alive including the streams, soils, and the network of mycorrhizal and root systems holding the soils together. Every change in elevation and moisture creates a specialized ecosystem composition that is as unique as a fingerprint to anywhere else.
Notice the changes to the areas that have seen impacts from repeated use to the areas that have been left untouched. Notice what life does or does not survive underfoot. See that the giant roots that grow across the trail are the heart strings for the beautiful trees that stretch above you. Their roots are not deep, they spread wide as they grip to the side of a mountain. These trees are already taxed from impacts of smoky air, insect infestations, and rising temperatures all while they do the important job of stabilizing our mountains and cleaning our air.
May you be mindful of those heart strings while you are out underneath their canopy.


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This category is for primary resources relating to the laws of Georgia, including the state Constitution, case law, and statutory law.
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Georgia -- American Law Sources Online
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Guide to Law Online: U.S. Georgia | Law Library of Congress
The Guide to Law Online contains a selection of Georgia legal, judicial, and governmental sources accessible through the Internet | Links provide access to primary documents, legal commentary, and general government information about specific jurisdictions and topics.
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domain: library


Hamdi Mani of Arizona State University explains what radio frequency interference is, what causes it, and the challenges that radio astronomers face.
Created by Margaret Blumm of the ASU Low-Frequency Cosmology Lab (LoCo).
The LoCo Lab's Danny Jacobs explains how we use an octocopter at our telescope in Australia. Be sure to catch the footage from a test flight at the end of the video!
Videos about radio astronomy created by the Arizona State University Low-Frequency Cosmology Lab. Go LoCo!
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text:
Personal bankruptcy rose 12.2 per cent over the last 12 months, according to a study released on Tuesday which also found that the situation is particularly problematic across the middle class.
The study, undertaken by the University of Melbourne's Centre for Corporate Law and Securities Regulation, found that the last 18 years has seen a significant hike in the rate of personal insolvency, including bankruptcy. The 261 per cent rise is attributed to credit factors, gambling and the cost of caring for dependants.
Given that economic times were considered good during most of this period, said the study's author, Professor Ian Ramsay, the growing rates of personal insolvency were increasingly concerning. "Now that we are experiencing very difficult economic times, the rate of personal insolvency is increasing and we can expect to see it grow across more parts of Australian society," Ramsay said.
Personal insolvency was found to be a growing phenomenon across the middle class, with bankrupt individuals increasingly coming from higher status occupations, as well as higher levels of personal and housing incomes.
Ramsay said these finding should challenge some common stereotypes regarding bankrupts - especially the misconception that they tend to be mainly students or the unemployed.
The problem is also growing around older Australians. In 1997, 26.25 per cent of bankrupts were aged 45 or older but in 2008, 45 per cent fitted that age group.
- Angela Priestley

domain: economics


Buffalo Loses Cultural Gift After Just 6 Years
How long can a gifted fountain last? Apparently not that long. Was it the Buffalo weather? A poor design? The details are limited but the fact remains, the fountain that stood for just under 7 years in Lafayette Square, is gone.
Channel 2 News followed up on a story that we had on Tuesday morning. Liz and I noticed crews removing a large pile of debris that was the fountain that stood just outside the Buffalo Public Library in Lafayette Sq.
The fountain was a gift from Buffalo's sister city in Turkey. It was November of 2010 when the mayor or Bursa, Turkey was in Buffalo to unveil the fountain which met it's demise this week.
According to the story from Channel 2,
We checked with the City of Buffalo, and a spokesperson said the library removed it. So, we asked the library, and they told us the city took it down.
A city spokesman called us back to clarify that the city removed the fountain early Tuesday morning saying it was deteriorating and crumbling to the point that it was a safety hazard.


text:
FASDEP targets about 240,000 farmer beneficiaries, -says coordinator
Nov 26, 2013, 9:48 AM
The Minister of Forestry and the Environment, Hon. Fatou Ndey-Gaye, last week officially commissioned four brand new vehicles for the department of forestry.
Speaking at the official commissioning held at the Ministry of Forestry, Fatou Ndey Gaye expressed delight at the development.
“These four brand new double-cabin
She added that the development would address their mobility constraints.
She said that it’s another important milestone in the history of the department of forestry in The Gambia in its endeavour to enhance the delivery of services.
The minister further stated that the department of forestry was very fortunate to have access to the National Forestry Fund in place and was able to obtain annual approvals from the Ministry of Finance and Economic Affairs which supports the department to supplement the National Development estimate budget to cater for logistic, infrastructuraldevelopment and operational costs in order to promote the protection, management, development, and sustainableuse of forest resources and to promote community forestry program in the country.
“This year, the department of forestry was able to purchase four brand Toyota pickups in addition to two pickup that were bought last year from the same fund, thanks to the support of the president of The Gambia for his timely intervention in this crucial moment,” she remarked.
According to her, this year marks the first year the department of forestry under the auspices of the Ministry of Forestry and the Environment has led The Gambia to the winning of the silver medal in the 2011 Future Awards by the World Future Council and International Policy Research Organisation in New York as one of the countries with the world most inspiring and innovative forest policies.
In receiving the keys of the vehicles, the director of forestry, Abdoulie Sanneh, thanked the president of The Gambia for ensuring that his department is enhanced with the necessary mobility to ensure effective service delivery.
He further called for the collective role of all Gambians in the protection and safeguarding of the country’s natural resources.
He also used the occasion to thank the Ministry of Finance and Economic Affairs for approving 2.9 million dalasi for the purchase of the vehicles.
Director Sanneh further commended Gambia Public Procurement Authority GPPA for processing the vehicles on time.
The beneficiaries would put the vehicles into good use, he assured.

domain: politics


Samsung has started the global rollout of the latest software update for its Galaxy Note10 lineup with Germany and the update includes important refinements of the face unlock and the gesture-based navigation system.
The firmware versions for the Note10 and Note10+ phones are N975FOXM2BTB3, N975FXXU2BTB5 and N975FXXU2BTB3 respectively, and are now being distributed to units in Germany. The update requires a download of around 260MB big and improves the facial recognition algorithm along with the gestures used for navigation.
And if you haven't received the update yet, wait patiently and it would come in the following days.
my Note 10+ finger print sensor sucks big time... why can you improve the fingerprint sensor?
iPhone Face ID not fooled in fake head test as Android rivals fail. A 3D-printed head has shown that while Apple's Face ID is a secure biometric authentication system, other facial recognition systems used by Android-based smartphones are able to be ...
Best 3D face unlock ever, but truth hurts cause android can’t get 3D face unlock to date even pixel 4 3D face just a garbage 3D face unlock that works even if your eyes closed lol.


text: Jinny Tavee, MD Make an Appointment Ask a Question Refer Patient Jinny Tavee, MD, is a neurologist at National Jewish Health. Dr. Tavee serves as Chief of the Division of Neurology & Behavioral Health. Chief, Division of Neurology & Behavioral Health Professor Department of Medicine Castle Connolly & 5280 Top Doctors 2023 Previously a 5280 Top Doctor America’s Top Doctors 2015 — Castle Connolly, Medical, Ltd. Recognized in America’s Top Doctors — Castle Connolly, Inc. Best Doctors in America® 2019-2020 — Best Doctors, Inc. Recognized in Best Doctors in America® — Best Doctors, Inc. America’s Top Doctors 2020 — Castle Connolly Medical, Ltd. Email Profile Print Profile Overview Contact Info & Locations Patient Ratings & Comments Programs & Services ALS Clinic Department of Medicine Division of Neurology & Behavioral Health Neurology Program Sarcoidosis Program Specialties & Conditions Amyotrophic Lateral Sclerosis (ALS) Brachial and Lumbosacral Plexopathy Chronic Inflammatory Demyelinating Polyneuropathy (CIDP) Guillain-Barre Syndrome Motor Neuron Disease Myasthenia Gravis Myositis/Myopathy Neurosarcoidosis Peripheral Neuropathy Radiculopathy Research Areas Mind-Body Treatments for Neurologic Disorders Neurosarcoidosis Peripheral Neuropathy Radiation Plexopathy Special Interests Dr.Tavee sees adult patients with neuromuscular disorders and has a special interest in neurosarcoidosis as well as peripheral neuropathy. She is currently looking at how the immune system may cause small fiber neuropathy in sarcoidosis and other autoimmune disorders, and is investigating potential treatment options to help patients with this condition. She is also involved in research studies evaluating the effects of integrative holistic therapies on chronic neurological diseases and has led meditation retreats for patients, physicians and U.S. Marines returning from the Gulf War through the Wounded Warriors program. Education Education 1998 University of Missouri Kansas City School of Medicine, Bachelor of Arts and Doctor of Medicine Internships 1999 Cleveland Clinic Foundation, Cleveland, OH, Internship Residency 2001 Cleveland Clinic Foundation, Cleveland, OH, Neurology Residency 2002 Cleveland Clinic Foundation, Cleveland, OH, Chief Resident Fellowship 2003 Cleveland Clinic Foundation, Cleveland, OH, Neuromuscular Fellowship Awards & Recognition 2019-2020: John A. Kessler Neurology Teacher of the Year, Northwestern University 2007-2008: Neurology Staff Teacher of the Year, Cleveland Clinic 2006: ACP Research Competition – 2nd place, U.S. Navy May 2001: Neuroscience Research Day 2nd Place, Cleveland Clinic Foundation May 2002: Neuroscience Research Day 2nd Place, Cleveland Clinic Foundation 1998: Graduation with Honors: University of Missouri School of Medicine 1998: Missouri Outstanding Scholar in Medicine Award 1996: Alpha Omega Alpha Honor Society 1994: U.S. Naval Health Sciences Scholarship Board Certification American Board of Electrodiagnostic Medicine American Board Of Psychiatry and Neurology: Clinical Neurophysiology American Board Of Psychiatry and Neurology: Neurology American Board of Psychiatry and Neurology: Neuromuscular Medicine American Board Integrative Holistic Medicine Professional Memberships 2015-Present: American Board of Psychiatry and Neurology Clinical Neurophysiology Committee 2013-2017 and 2020-Present: American Neurological Association, Member 2013-Present: American Association of Sarcoidosis and Other Granulomatous Disorders, Member 2006-Present: American Academy of Neurology, Member 2003-Present: American Association of Neuromuscular and Electrodiagnostic Medicine Publications Muppidi S, Guptill J, Jacob S, Li Y, Farrugia M, Guidon A, Tavee J, Kaminski H, Howard J, Cutter G, Wiendl H, Maas M, Illa I, Mantegazza R, Murai H, Utsugisawa K, Nowak R. COVID-19 Associated risks and effects in myasthenia gravis (CARE-MG): Development of an International Physician Registry and Initial Data. Lancet Neurology 2020;12:970-971. Tavee J, Karwa K, Ahmed Z, Thompson N, Parambil J, Culver DA. Sarcoidosis-associated small fiber neuropathy in a large cohort: Clinical aspects and response to IVIG and anti-TNF alpha treatment. Respiratory Medicine. 2017 May 31;126:135-8. Tavee J, Stern BA. Neurosarcoidosis. Chest Clinics N Am 2015;36:643-56. Tavee J, Rensel M, Pope-Planchard S, Stone L. Effects of Meditation on Pain and Quality of Life in Multiple Sclerosis and Polyneuropathy: A controlled study. Int J MS Care 2011;113:163-169. Tavee J, Zhou L. Small Fiber Neuropathy: a burning problem. Cleveland Clinic Journal of Medicine 2009;76:297-305. Teaching & Professional Positions 2021-present: Chief, Neurology Division National Jewish Health, Department of Medicine Denver, CO 2021-present: Director, EMG laboratory National Jewish Health, Department of Medicine Denver, CO 2021-present: Associate Professor National Jewish Health, Department of Medicine Denver, CO 2018- 2020: Director, EMG Neurodiagnostic Testing Center Northwestern University Feinberg School of Medicine, Davee Department of Neurology Chicago, IL 2018-2020: Co-Director, Peripheral Neuropathy Multidisciplinary Care Center Northwestern University Feinberg School of Medicine, Davee Department of Neurology, Chicago, IL 2017-2020: Medical Director, Neuromuscular Division Northwestern University Feinberg School of Medicine, Davee Department of Neurology Chicago, IL 2016-2017: Associate Professor of Medicine Northwestern University Feinberg School of Medicine, Davee Department of Neurology Chicago, IL 2016-2017: Associate Professor of Medicine Cleveland Clinic Lerner College of Medicine of Case Western Reserve University 2014-2017: Neuromuscular Fellowship Program Director Cleveland Clinic Foundation, Neurologic Institute, Neuromuscular Center Cleveland, OH 2007-2017: Cleveland Clinic Foundation, Neurologic Institute, Neuromuscular Center Cleveland, OH 2003-2007: Chief, EMG/Neuromuscular Section U.S. Navy, Naval Medical Center Portsmouth Portsmouth, VA Ask a Question through MyChart Sign in to your MyChart account to communicate with your care team, manage appointments, and more. Create an Account Contact Information Office: 877.225.5654 Patient Ratings The Patient Rating score is an average of all responses to care provider related questions on our independent rating system, the Press Ganey Patient Satisfaction Survey. This survey is about the patient care experience and does not address crucial characteristics like medical decision-making, prescribing the best therapy, and patient outcomes. Responses are measured on a scale of 1 to 5, with 5 being the best score. Learn more about our patient satisfaction survey. Comments Comments are collected in our Press Ganey Patient Satisfaction Surveys. Patients are de-identified to protect confidentiality and patient privacy. Learn more about our patient satisfaction survey.

domain: health


Immediately after the Pulse nightclub massacre, Sen. Marco Rubio (R-FL) made a point of telling conservative Christians that they needed to be more tolerant. Now he’s invoking Sodom and Gomorrah as his idea of tolerance.
Six years ago, Rubio told a gathering of the Florida Renewal Project, a notably anti-LGBTQ conference, that American history “has been marred by discrimination against and rejection of gays and lesbians.” What Christians need to do was show more love and less condemnation, Rubio told the gathering.
“Abandoning judgment and loving our LGBT neighbors is not a betrayal of what the Bible teaches, it is a fulfillment of it,” Rubio said. “Jesus showed us how to do this. Jesus showed us that we do not have to endorse what people do in order to accept them for who they are: children of a loving and merciful God.”
That’s the kind of play-acting as a statesman that gets you fawning coverage in The New York Times. It looked like a bold move–go straight (so to speak) to the source to challenge the prevailing approach, risking political capital, showing some compassion immediately after a national tragedy. It also appealed to the pundit class, who remembered the GOP’s 2012 post-election analysis, which said that the party needed to ratchet back the anti-LGBTQ rhetoric.
Of course, none of Rubio’s rhetoric was anything other than posturing. Now that Florida’s Don’t Say Gay legislation is barreling toward becoming law, Rubio is using the very kind of language that would thrill the hearts of the very same Christians he was lecturing in 2016.
Last Monday, U.S. Surgeon General Dr. Vivek Murthy accurately noted that Don’t Say Gay “sends a signal to LGBTQ+ youth that they are not fully accepted.” Senator Tolerance didn’t like that.
First, Rubio tweeted that Murthy was arguing that schools “should be having discussions with children from 5 to 8 years old about sexual orientation & gender identity.” Apparently dissatisfied with relying upon the standard defense, Rubio decided to up the ante. Just fourteen minutes after that tweet, he came out with the fire and brimstone.
Hear the word of the LORD, princes of Sodom! Listen to the instruction of our God,people of Gomorrah!
— Marco Rubio (@marcorubio) March 15, 2022
As a combination of pandering, hypocrisy, and sheer offensiveness, that tweet is hard to beat. Of course, that pretty much sums up Rubio’s career, along with a large dollop of clownishness. (Remember his obsessive water-swilling when he responded to the State of the Union address in 2013?)
The truth is that talk is just that–talk. When it comes to actually putting faith into action, Rubio has always fallen short. His voting record on LGBTQ issues in Congress started out as dismal and quickly plummeted, earning him a zero from HRC in the most recent session.
Now that he is up for re-election this year, Rubio is going to go all-in on the DeSantis/Trump approach to politics: all snarl, bullying, and sharp elbows. The problem is that, as Rubio proved in his failed 2016 presidential run, he’s not really good at playing that role.
But he is good at sucking up. He’s managed to get into Trump’s good graces, and that’s enough to make him the favorite in the race. Unfortunately, that means we’ll get to see more of the man Trump called Lil Marco trying to pander to the worst impulses of his party instead of trying to correct them.


THE Sony announced this week the WF-1000XM3, which are your new portable wireless earbud headsets noise cancellation. Also known by their English name "ear buds", the headphones promise more battery life and improved noise cancellation (NC) over the previous version of the product, the WF-1000X.
The WF-1000XM3 earphones have a capacitive surface that can be used for different controls by gestures and touches, including holding the finger or hand over the headset to disable noise cancellation in case anyone is talking. with the user or person wanting to pay more attention to the surround sound for any reason. But even without you turning off cancellation, the handset has smart security measures called Adaptive Sound Control which, according to Sony, are able to detect "important noises". It is the case, for example, of a car honking in the street, that the headphones would automatically detect and let the sound pass to alert the user.
And the update released by Samsung s weighs 1.36 MB
In the technical settings part, each handset weighs just under 9g and comes with a case that can be used to recharge gadgets. Along with the case, the portable set weighs a total of 77g. The headphones are compatible with Bluetooth 5.0 and promise a range of up to 10 meters from the connected device.
The promise of battery life of up to 6 hours playing music continuously. With noise cancellation turned off, the range goes up to 8 hours, according to Sony. The full charge of the battery filled in just 1.5 hours recharging.
Sony's new WF-1000XM3 headphones will hit stores in August at the suggested price of $ 230, approximately $ 876 in direct conversion at the current price. They will be released in black and silver color options. We really are a long way from Sony's first Walkman.
Picture credits: Sony
Source: GSMArena. (TagsToTranslate) Headset (t) In-Ear Headphones (t) Sony (t) wf-1000xm3 (t) Sony wf-1000xm3 (t) Noise Canceling (t) bluetooth


text:
Oil Spray for Citrus Trees
Citrus trees include grapefruit, oranges, lemons, kumquats and limes which -- depending on the species -- grow in U.S. Department of Agriculture plant hardiness zones 9 through 12. These fruit trees can come under attack from damaging insects and diseases, which can affect the fruit production of your citrus. To kill, treat or prevent problems, oil sprays are one option.
Neem, jojoba and horticultural oil are some of the oil sprays available. Plant-based oils -- such as neem and jojoba -- are made using oil extracted from various parts of certain plants. Made from highly refined petroleum, horticultural oil has been used for centuries to keep pests off plants. When applied properly, these oil sprays can control various insect or disease problems on citrus. Oil sprays degrade quickly with little to no residual effect on the environment.
Leafrollers, aphids, mealybugs, thrips, whiteflies, cutworms and scale insects are some of the insects that infest citrus plants. These insects cause varying degrees of damage but generally will leave the citrus leaves wilted, curled, discolored and distorted. The damaged leaves may fall from the citrus tree prematurely. Aphids, mealybugs, thrips, whiteflies and scale insects feed on the juices inside the leaves by inserting a straw-like mouthpart to suck the liquid out of the plant. Leafrollers eat leaves and mature fruit and roll leaves up, securing them with a silky thread. For the oil spray to effectively control the insects, the pest must be entirely covered with the oil. To increase the effectiveness of the oil, thoroughly cover the citrus plant -- including the tops and undersides of the leaves -- with the spray.
Horticultural and neem oil control insects -- such aphids and whiteflies -- that carry viruses that infect plants. In addition, they protect the citrus plant from various fungal diseases, such as powdery mildew, rusts, black spot and scab. Neem oil works by preventing the fungal spores from adhering to the plant while horticultural oil disrupts the fungal pathogen's cell wall and prevents it from attaching itself to the citrus plant. These oil sprays have a low toxicity to humans, birds and bees but may cause skin irritation. They are toxic to fish, so don't use them around ponds and make sure there's no runoff that could enter a body of water.
No matter which oil spray you use, follow the manufacturer’s directions printed on the label. Most of these sprays cannot be applied when temperatures are higher than 90 degrees Fahrenheit. Higher temperatures may cause the oil to burn the citrus plant. Wait for a calm day to apply the oil spray to prevent spray drifts and choose a day when rain isn’t in the forecast for 24 to 48 hours. A second application seven to 14 days after you applied the first treatment may be necessary. The oil spray label will have specific instructions for the type and brand of oil spray you are using.
- University of California Integrated Pest Management Program: Citrus
- Missouri Botanical Garden: Gardening Help FAQs
- University of California Integrated Pest Management Program: Leafrollers
- University of California Integrated Pest Management Program: Thrips
- University of California Integrated Pest Management Program: Whiteflies
- Missouri Botanical Garden: Oil Sprays
- Oregon State University Extension Service: Least Toxic and Organic Pesticides for Gardeners
- Purdue University Extenison: Using Organic Fungicides
Amanda Flanigan began writing professionally in 2007. Flanigan has written for various publications, including WV Living and American Craft Council, and has published several eBooks on craft and garden-related subjects. Flanigan completed two writing courses at Pierpont Community and Technical College.

domain: agronomy


text:
It was indeed Chu Zhaoyi who was holding him…
He found it somewhat unbelievable, watching Chu Zhaoyi’s anxious expression, seeing the way he rushed into a nearby pavilion.
Within the broad arms, he could hardly feel the jostling of the run.
In his previous life, Fu Nian had also yearned for such treatment, but he had never experienced it even at the point of death.
Now that he was experiencing it… Fu Nian didn’t think too much about it. After mustering some strength, he simply flipped over and rolled out of Chu Zhaoyi’s arms.
Upon landing, he inadvertently received a kick because Chu Zhaoyi was still in motion. The blow landed on his shoulder wound, making Fu Nian suppress the urge to grimace in pain.
After landing, the situation seemed even more awkward.
While lying prone on the ground, Fu Nian saw Chu Zhaoyi’s steps coming to a halt. Judging by his shadow, he seemed to be looking down at him, although he didn’t say anything.
However, just lying there, Fu Nian felt that it was inappropriate. After contemplating for a while, he tremulously spoke up, “Subordinate… pays respects to Your Majesty.”
After speaking, another silence ensued.
Fu Nian began to regret not remaining obedient and letting Chu Zhaoyi hold him. Things wouldn’t have been so awkward if he had.
After a prolonged standoff, Fu Nian finally remembered that he seemed to be covered by a dragon robe. He hastily removed his clothes, as carefully as he could manage due to the pain, and extended both hands with clothes in hand, “Your, Your—”
Before he could finish, Fu Nian felt the clothes in his hands being taken away. Immediately, his body tightened. Turning his head, he saw the man in front of him with a stern expression, using the dragon robe to conceal the torn parts of his clothing and then fastening the buttons.
“Wearing torn clothes in public is uncouth.”
After a brief silence, Fu Nian finally heard this person speak.
Indeed, appearing disheveled before the Emperor was rather impolite.
“Since you can walk, stand up and walk by yourself.”
Hearing the command, Fu Nian didn’t have a chance to retort. After getting up from the ground, he took slow steps forward.
Passing by a rockery, as expected, Fu Nian saw the imperial physician in the pavilion, setting up a mat and preparing medicine on the side.
“Why are you so severely injured? Sit down quickly.”
Upon hearing the physician’s voice, Fu Nian didn’t waste time and hurriedly knelt down on the mat.
“With such severe injuries, you shouldn’t have walked. There were clearly stretchers sent, but those clever fellows must have slacked off again.”
“He’s already taken the hemostatic powder.” Chu Zhaoyi stood by, watching the physicians scurrying about, and suddenly spoke in a cold tone.
“May Your Majesty be in good health.” The physician finally noticed Chu Zhaoyi was also present, “My eyesight isn’t good. I, I didn’t see that you were here, Your Majesty, your—”
“It’s all right. The dragon robe is on him. His clothes are all torn, which is uncouth.” After Chu Zhaoyi finished speaking, he glanced at the physician, whose face was drenched in sweat, “And Chu Yao?”
“Reporting to Your Majesty, Chu Yao just left.” The physician answered while hesitating, scissors in hand. Logically, with such injuries, they should have completely cut away the clothes, but who would have thought that Your Majesty would just drape the dragon robe like that? He was momentarily unsure whether he should continue cutting.
Seeing the physician in a dilemma, Chu Zhaoyi said, “Go ahead and cut, it’s just clothes.” After that, Chu Zhaoyi still had his hands behind his back, observing the physician’s actions in treating the wound.
Fu Nian had refrained from speaking and moving.
The clothes on his right shoulder had been completely cut away, including parts of the dragon robe stained with blood. It wasn’t cold with his arm exposed in the summer air, but Fu Nian was curious.
Chu Zhaoyi had mentioned being uncouth, but he had been scrutinizing the clothes associated with that.
Of course, Fu Nian naturally didn’t dare to ask aloud.
After cleaning the wound and applying medicine, Fu Nian watched as the physician immobilized his right arm. Besides the gauze, many wooden boards were placed on his shoulder.
Fu Nian attempted to move his arm a bit, but apart from excruciating pain, his arm could hardly budge.
“You’ve injured your bone; you must refrain from moving for a while. If the bone heals crooked, it will need to be broken again.” Seeing his intention to move, the physician timely warned, “The wooden boards are to prevent you from moving.”
Indeed, with his shoulder pierced, it would be strange if the bone wasn’t injured.
“Is there any other injury?”
“No. Thank you.” Fu Nian finished speaking and attempted to get up from the mat.
However, his right shoulder was entirely immobile, severely restricting his movement.
When standing up, Fu Nian clenched his teeth, using his left hand to support himself against a pillar to barely stand upright.
As he turned around, Fu Nian suddenly realized.
Why was Chu Zhaoyi still here…
Fu Nian hadn’t even taken a step when he heard Chu Zhaoyi command.
Fu Nian could only turn around, facing away from Chu Zhaoyi.
Senior Brother had also mentioned that his back looked almost the same as in the previous life. Of course, it was only the back that resembled it. The appearance and voice had no connection to the past life.
Before he could finish thinking, Fu Nian suddenly felt his clothes being slashed from behind.
Immediately after, a strong force tore off the upper half of his clothes.
If he wasn’t wearing a waistband, this situation might have been even more embarrassing.
Without looking back, Fu Nian knew who it was.
“I didn’t permit you to speak.”
As the words fell, Fu Nian felt his belt being forcefully removed as well.
Right after, Fu Nian heard a sigh.
“Help him put on his clothes.” Chu Zhaoyi ordered without a ripple in his expression.
Fu Nian still didn’t move.
Hearing the physicians’ salutations, he dared to turn around, feeling a bit more at ease as he leaned against the wall.
“Actually, this old man doesn’t know why Your Majesty suddenly tore your clothes… Miss, would you please help the imperial guard put on his clothes.”
Fu Nian quietly allowed the palace maid to help him change his upper garments.
He knew what Chu Zhaoyi was sighing about.
In the previous life, there had been a brand below the small of his back, and all the Shadow Guards bore such marks. The designs were uniform, differing only in position. They served to identify bodies, just in case.
His brand was an exception.
It served as evidence of a past intimate connection with that man. The design was also distinct from the other Shadow Guards’. It was placed in a more private spot due to its excessively suggestive pattern.
After leaving the palace, Fu Nian hadn’t paid much attention to the hidden brand, and alone, he couldn’t see his back anyway.
However, this time when he had his body checked upon entering the palace, he found that the brand had disappeared. In its place were scars that hadn’t faded, and not a trace of the brand could be seen.
Undoubtedly, when his body was brought back to the palace, this brand was used for identification.
So, that’s why Chu Zhaoyi had acted that way just now and left disappointed.
After dressing, Fu Nian finally dared to speak, “Has Your Majesty already left?”
“Yes, Your servant is a maid by Your Majesty’s side. His Majesty entrusted me with the message that if you have something to say, I can pass it on.”
Fu Nian heard the gentle voice of the maid who had helped him dress.
Though her appearance seemed unfamiliar, Fu Nian glanced at her before turning away.
“If you have a request, please speak, and Your servant will definitely deliver it.”
After thinking for a while, Fu Nian spoke up, “Could you please remind His Majesty about the reward for winning the competition?”
After saying this, Fu Nian heard the girl’s voice choke.
After a long pause, she replied, “Your, Your servant understands.”
Only then did Fu Nian relent.
He had thought that since Chu Zhaoyi had been standing here for so long, he might give him the reward all at once. Fu Nian even dared to hope that Chu Zhaoyi might be in a good mood and give him a bit more.
Now, it seemed that all his hopes were indeed far-fetched, just as much a pipedream as hoping for Chu Zhaoyi to show the slightest hint of affection in the previous life.
After his right shoulder became immobile, Fu Nian’s walking speed slowed considerably.
Finally returning to his residence, before he even reached the doorstep of the courtyard, Fu Nian realized that it seemed like everyone was looking at him.
Fu Nian knew he had lost face, so he could only press against the wall and walk with his head down.
Back in the courtyard, he saw Rendong waiting for him. Fu Nian thought to himself that he finally saw someone he recognized, so he hurriedly supported himself against Rendong and walked faster back inside.
Shutting the door, Fu Nian let out a sigh of relief as he sat on the bed, gesturing for Rendong to find a stool as well.
Though the room wasn’t spacious, it was much more private than the shared quarters of the new guards. Rendong looked around but hesitated to sit, so he leaned against the wall, standing. “Congratulations, Big Brother! They say you’re practically guaranteed to serve in front of the Emperor after this competition! They said there was a palace maid before who the Emperor personally saved, and later the Emperor arranged a marriage for her with a powerful official, giving her a surname and a full name. According to the standards of a princess, she was married out of Ze Wang’s residence and recognized Ze Wang as her godfather.”
Fu Nian was aware of this matter. Originally, Chu Zhaoyi had planted an informant among the palace maids, and she was carrying some important documents on her person. Unexpectedly, she “accidentally” fell into the water, and Chu Zhaoyi even jumped in to save her for the sake of those documents.
“…I’m different,” Fu Nian didn’t explain anything.
One reason was that there was no need to explain to outsiders. Another reason was that Fu Nian was still pondering the events from just now.
Chu Zhaoyi had intentionally checked the brand on his back and sighed when he found it empty.
—But even before confirming, Chu Zhaoyi had unhesitatingly embraced him and treated his wounds, even using his own dragon robe to cover him, soothing and medicating him with gentle words.
Fu Nian wasn’t quite sure of Chu Zhaoyi’s mindset at the time.
Was he confusing him with his previous self, the lapdog?
…Or was it that even though he was kind to people, cared for them, and had various emotions, he simply wasn’t deserving of it in his previous life?
“Why are you different? Well, you don’t look happy at all. Is the wound too painful?”
“Probably.” He had just drunk the pain-relieving soup, and as long as he didn’t move his right shoulder, it wouldn’t be particularly painful. “I’ll rest for a while.”
“Okay, then I’ll leave—”
Before Rendong could finish, Fu Nian heard the loud voice of the eunuch from inside the courtyard.
“By the command of the Emperor, the imperial decree states, ‘Imperial Guard, Lian Nian, listen to the order—'”
Hearing that Li Gonggong had arrived, Fu Nian hastily stumbled to his feet, opened the door, and went outside. He knelt down on the ground. “Today, due to the outstanding performance of Imperial Guard Lian Nian in the martial arts competition, you are rewarded with 200 taels of gold—”
Fu Nian felt a little happy; he finally got the money he was owed.
He was about to accept the decree when he realized that Li Gonggong wasn’t finished.
“Furthermore, you are granted a jade waist pendant representing your service at the front hall. We hope that Imperial Guard Lian Nian will continue to achieve success in the front hall in the future—”
Fu Nian’s heart sank.
As expected, this kind of high-profile attention meant trouble, not fortune.
“Imperial Guard Lian Nian, accept the decree,” Li Gonggong saw that he didn’t react and reminded him.
Fu Nian didn’t respond and didn’t accept the decree.
A decree with a derogatory meaning had to be accepted, but if it was a reward or promotion, refusing not only wouldn’t result in harm, but could even strengthen the bond between monarch and subject. At least, that’s what was written in the history books. He hadn’t seen anyone who refused a reward being executed.
“Lian Nian, accept the decree,” Rendong couldn’t hold back and softly reminded him.
Fu Nian remained silent, quietly organizing his words in his mind.
After a long pause, he spoke with a calm and composed tone, “This subordinate is naturally dull-witted and incapable of fulfilling tasks at the front hall. I am unable to bear Your Majesty’s kindness, and therefore, I cannot accept the decree. I hope Your Majesty will understand.”
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domain: literature


US-based private equity firms Advent International, Carlyle Group and General Atlantic have been shortlisted to buy a controlling stake in the Indian arm of Spanish fashion brand Pepe Jeans, Mint reported, citing two people aware of the development.
Pepe Jeans is seeking a valuation of Rs 2,000 crore for its India unit, the report said.
Citing a person in the know, the report said Advent joined the fray two weeks ago.
Earlier, Pepe Jeans had held talks with US buyout firm KKR & Co. and home-grown private equity firm Everstone Capital Advisors, said the second person in the report.
The Netherlands-based Pepe Jeans Europe BV is the holding company of Pepe Jeans India.
Kotak Investment Banking was given the mandate by Pepe Jeans to give advice on the sale process, according to the report.
Pepe Jeans India’s net sales stood at Rs 427.6 crore and profit after tax at Rs 49.2 crore for the financial year ended March 2017.
Founded in 1973 in London, Pepe Jeans Group operates through two main brands -- Pepe Jeans and Hackett.
In 2015, L Catterton Asia and Lebanese investment firm M1 Group acquired a majority stake in Pepe Jeans Group.
In another development, a consortium of Indian private equity firm WestBridge Capital and share investor Rakesh Jhunjhunwala is aiming to match the Rs 6,500 crore offer of ICICI Lombard General Insurance Co. for Star Health Insurance Co., The Economic Times reported, citing two people aware of the development.
The consortium is in talks with shareholders of Star Health Insurance to match the offer of the highest bidder, ICICI Lombard, said a person in the know. WestBridge-Jhunjhunwala had offered Rs 6,100 crore for the insurer.
PremjiInvest, the family office of Wipro chairman Azim Premji, and UK-based financial services company Prudential Plc. had made a joint bid of Rs 5,900 crore for the company, the report said.
Star Health Insurance’s board will meet on May 9 to decide on the bids, according to the report.
The standalone health insurance firm’s promoter, Star Health Investments Pvt. Ltd, along with its investors, had started the process for the company’s sale in early 2017.
Its investors include ICICI Venture, Tata Capital Growth Fund, Sequoia Capital, Oman Insurance Company and Apis Partners.
Star Health started its operations in 2006. It offers health insurance, overseas medi-claim policies and personal accident cover. The company’s gross direct written premium was Rs 2,488.17 crore for the nine months ended December 2017.


It takes a very special director to make scenes of sky-diving, free climbing, big-wave surfing and BASE jumping something to yawn at. Yet Ericson Core must be that kind of miracle worker, because “Point Break,” his update of the 1991 cult classic, is basically a cavalcade of extreme sports, but with less drama than a highlight reel.
Written by Kurt Wimmer, the remake has the same basic architecture of the original. There’s still an FBI agent named Johnny Utah (Luke Bracey, taking over for Keanu Reeves), who goes undercover to investigate a string of crimes. And Johnny still gets starry-eyed after meeting a brawny, mystical anarchist named Bodhi (Edgar Ramirez, standing in for Patrick Swayze).
Yet this is a completely different movie, because Wimmer and Core seem to believe that what people loved about the first “Point Break” wasn’t the cheesy dialogue or the bromance-induced moral conundrum, but all of the adrenaline.
A rush, it turns out, only goes so far. A little more character development, for example, wouldn’t hurt.
In the updated version, Johnny used to be an extreme poly-athlete, specializing in dirt biking and winter sports. But an accident years ago shifted his priorities, and he wound up in the FBI. Still, his tattoos are evidence that he can only put his past so far behind him.
In his new career, Johnny has been monitoring a group of Robin Hood-esque criminals who carry out over-the-top heists, such as robbing a bank on the top floor of a skyscraper, then breaking through the windows on motorcycles and parachuting to safety.
Johnny has it all figured out. These criminals are less interested in the money than in completing the “Ozaki Eight,” a series of death-defying trials that honor the forces of nature. It’s up to Johnny to infiltrate the group and try to stop them before their next job.
That is, if he doesn’t fall for Bodhi first – in a purely platonic way.
Some of the visuals are truly spectacular, most notably a sequence in which people wearing wingsuits sail through the air around the Alps after throwing themselves off the Jungfrau. But where the earlier film was punctuated by action, this too self-important version is the opposite. The story is an afterthought.
Cast: Luke Bracey, Edgar Ramirez
Director: Ericson Core


text:
Full body harness, Y-front, made of thick black leather with a strap width of 40mm. Very masculine look due to the breast-emphasizing design and the strap thickness of 3mm. The leather harness can be optimally adjusted using stable roller buckles and snaps. All adjustment holes reinforced with metal eyelets. Large welded round and D rings. In addition, the leather harness is divisible and can therefore only be worn as an upper body harness.
Including stainless steel cock ring, diameter 50mm, interchangeable with snap button loops.
Made by Slingking™.
Strap width: 40mm
Size: chest circumference from 90cm to 110cm
|Shipping weight:||2,00 Kg|
|Product weight:||1,75 Kg|

domain: design


text:
Ian Kelly (‘23) was Luther’s individual top performer at last weekend’s American Rivers Conference Cross Country Championships, finishing 10th overall and earning All-Conference honors. Wartburg College, nationally ranked in NCAA DIII men’s and women’s cross country, won the team title in both races. The championships were held at Luther’s cross country course for the first time since 2011.
Kelly, with his time of 25:49.7, led the Luther men as the team finished fourth overall, while the Luther women finished sixth. This race was the third time that Kelly has achieved All-Conference honors, becoming the 23rd Luther runner to achieve that feat.
“Being named all-conference means a lot to me,” Kelly said. “It shows off all the hard work I have been putting into running this season and throughout the summer. I wouldn’t be in the conference without my teammates and coaches pushing me to do my best.”
Kelly was followed in the race by Sam Schillinger (‘22), who placed 22nd with a time of 26:12.6. Tom Altier (‘22) in 32nd with a time of 26:30, and Alex Heidorn (‘25) placed 36th with a time of 26:43. The Norse women were lead by Lauren Berg (‘22) in 32nd place with a time of 24:37, while Heather Hostager (‘22) finished in 38th place at 24:44, and Anneke Knauss (‘22) finished in 39th place with a time of 24:48.
One advantage that Luther had going into the championship races was competing on their home course . The course is seen by some runners as difficult, so many Norse competitors knew their familiarity would serve them well.
“Racing at home is always exciting, and one of the main challenges is controlling that energy,” Ryan Rogers (‘22) said. “Because this was such a major race, it’s easy to get wrapped up in the excitement and start the race too fast, ruining your chances of finishing strong. However, if you can take the first half of the race smart, home meets can be the perfect place to run personal bests.”
According to runners like women’s team member Berit Skogen (‘23), this season has been tough for cross country team members who have been dealing with injuries and other challenges. She felt there was a big payoff in getting to run a major race in front of a home crowd.
“A lot of us took this opportunity to race and pulled through in some awesome ways,” Skogen said. “We executed our race plan of working up through the field in the second half of the race, and our results showed that.”
This race was the second to last that all Luther runners will run, with the top performers traveling next to Saukie Golf Course in Rock Island, IL. At this upcoming race on November 13, runners will compete in the NCAA DIII Midwest Regional Championships, with a goal of qualifying for the National Championships. The remaining runners will be racing again at Luther on November 5 in the annual Saga Cup.

domain: news


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Cabled observations are used in order to relay compromised data to scientists, particularly when located in remote parts of the ocean. They are ocean graphic research platforms which record still images or videos under water and are connected to the surface by undersea cables. By using these cables in comparison to sonar or radio frequency communications, cabled observations allow a persistent study of underwater. A single cable can support multiple observation sites and can then provide data in 2D or 3D at the surface. However, there are some major drawbacks to this method. Large quantities of information (images and video content) have to be stored and analysed in order to find the information scientists require.
To help overcome this, a group of researchers in DCU’s Water Institute in collaboration with Insight, Centre for data Analytics and other universities in Ireland are carrying out a project on how to filter the information which is required in different scenarios. The project is titled: Understanding underwater video content, investigating object detection and recognition for the Smart Bay camera platform. The project is being carried out on the Smart Bay cabled observatory in Galway Bay.
The long term goal of this project is to enable a higher-level knowledge exploitation of underwater imagery by semantic labelling and indexing of images and video data to improve data mining and retrieval. Object detection algorithms will be applied to the content from the underwater cameras. The result of object detection can be used to automatically identify potentially interesting video segments thereby reducing the amount of video content which has to be analysed.
When applying the network to the still image dataset, the results provided an estimation of the density of the fish. However, when this method was applied to video content, the performance decreased considerably. The encouraging results obtained from the still imagery, lead the researchers to optimize the model and introduce regularization techniques and make it more generalizable in order to improve the quality on the video content.
At present, the method to capture video content is done in a way that frames from the video are captured and it allows the researchers to train a model to analyse the content. However, the only drawback of this method is that the model needs to be re-trained each time new data is available.

domain: geography


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Endoplasmic reticulum stress from unfolded proteins is associated with the proliferation of pancreatic tumor cells, making the many regulatory molecules of this pathway appealing targets for therapy. The objective of our study was to assess potential therapeutic efficacy of inhibitors of unfolded protein response (UPR) in pancreatic cancers focusing on IRE1α inhibitors. IRE1α-mediated XBP-1 mRNA splicing encodes a transcription factor that enhances transcription of chaperone proteins in order to reverse UPR. Proliferation assays using a panel of 14 pancreatic cancer cell lines showed a dose- and time-dependent growth inhibition by IRE1α-specific inhibitors (STF-083010, 2-Hydroxy-1-naphthaldehyde, 3-Ethoxy-5,6-dibromosalicylaldehyde, toyocamycin). Growth inhibition was also noted using a clonogenic growth assay in soft agar, as well as a xenograft in vivo model of pancreatic cancer. Cell cycle analysis showed that these IRE1α inhibitors caused growth arrest at either the G1 or G2/M phases (SU8686, MiaPaCa2) and induced apoptosis (Panc0327, Panc0403). Western blot analysis showed cleavage of caspase 3 and PARP, and prominent induction of the apoptotic molecule BIM. In addition, synergistic effects were found between either STF-083010, 2-Hydroxy-1-naphthaldehyde, 3-Ethoxy-5,6-dibromosalicylaldehyde, or toyocamycin and either gemcitabine or bortezomib. Our data suggest that use of an IRE1α inhibitor is a novel therapeutic approach for treatment of pancreatic cancers.
UPR; pancreatic cancer; IRE1α
X-box binding protein-1 (XBP-1) is an important regulator of a subset of genes during endoplasmic reticulum (ER) stress. In the current study, we analyzed endogenous XBP-1 expression and localization, with a view to determining the effects of ER stress on the developmental competency of preimplantation embryos in mice. Fluorescence staining revealed that functional XBP-1 is localized on mature oocyte spindles and abundant in the nucleus at the germinal vesicle (GV) stage. However, in preimplantation embryos, XBP-1 was solely detected in the cytoplasm at the one-cell stage. The density of XBP-1 was higher in the nucleus than the cytoplasm at the two-cell, four-cell, eight-cell, morula, and blastocyst stages. Furthermore, RT-PCR analysis confirmed active XBP-1 mRNA splicing at all preimplantation embryo stages, except the one-cell stage. Tunicamycin (TM), an ER stress inducer used as a positive control, promoted an increase in the density of nuclear XBP-1 at the one-cell and two-cell stages. Similarly, culture medium supplemented with 25 mM sorbitol displayed a remarkable increase active XBP-1 expression in the nuclei of 1-cell and 2-cell embryos. Conversely, high concentrations of TM or sorbitol led to reduced nuclear XBP-1 density and significant ER stress-induced apoptosis. Tauroursodeoxycholic acid (TUDCA), a known inhibitor of ER stress, improved the rate of two-cell embryo development to blastocysts by attenuating the expression of active XBP-1 protein in the nucleus at the two-cell stage. Our data collectively suggest that endogenous XBP-1 plays a role in normal preimplantation embryonic development. Moreover, XBP-1 splicing is activated to generate a functional form in mouse preimplantation embryos during culture stress. TUDCA inhibits hyperosmolar-induced ER stress as well as ER stress-induced apoptosis during mouse preimplantation embryo development.
Resveratrol, trans-3, 4’, 5,-trihydroxystilbene, suppresses multiple myeloma (MM). The endoplasmic reticulum (ER) stress response component IRE1α/XBP1 axis is essential for MM pathogenesis. We investigated the molecular action of resveratrol on IRE1α/XBP1 axis in human MM cells.
Human MM cell lines ANBL-6, OPM2, and MM.1S were utilized to determine the molecular signaling events following the treatment with resveratrol. The stimulation of IRE1α/XBP1 axis was analyzed by Western blot and reverse transcription polymerase chain reaction. The effect of resveratrol on the transcriptional activity of spliced XBP1 was assessed by luciferase assays. Chromatin immunoprecipitation (ChIP) was performed to determine the effects of resveratrol on the DNA binding activity of XBP1 in MM cells.
Resveratrol activated IRE1α as evidenced by XBP1 mRNA splicing and the phosphorylation of both IRE1α and its downstream kinase JNK in MM cells. These responses were associated with resveratrol-induced cytotoxicity of MM cells. Resveratrol selectively suppressed the transcriptional activity of XBP1s while it stimulated gene expression of the molecules that are regulated by non-IRE1/XBP1 axis of the ER stress response. Luciferase assays indicated that resveratrol suppressed the transcriptional activity of XBP1s through sirtuin 1 (SIRT1), a downstream molecular target of resveratrol. ChIP studies revealed that resveratrol decreased the DNA binding capacity of XBP1 and increased the enrichment of SIRT1 at the XBP1 binding region in the XBP1 promoter.
Resveratrol exerts its chemotherapeutic effect on human MM cells through mechanisms involving the impairment of the pro-survival XBP1 signaling and the activation of pro-apoptotic ER stress response.
Endoplasmic Reticulum Stress Response; IRE1α; Multiple Myeloma; Resveratrol; XBP1
The unfolded protein response (UPR) is an adaptive signaling pathway utilized to sense and alleviate the stress of protein folding in the endoplasmic reticulum (ER). In mammals, the UPR is mediated through three proximal sensors PERK/PEK, IRE1, and ATF6. PERK/PEK is a protein kinase that phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 to inhibit protein synthesis. Activation of IRE1 induces splicing of XBP1 mRNA to produce a potent transcription factor. ATF6 is a transmembrane transcription factor that is activated by cleavage upon ER stress. We show that in Caenorhabditis elegans, deletion of either ire-1 or xbp-1 is synthetically lethal with deletion of either atf-6 or pek-1, both producing a developmental arrest at larval stage 2. Therefore, in C. elegans, atf-6 acts synergistically with pek-1 to complement the developmental requirement for ire-1 and xbp-1. Microarray analysis identified inducible UPR (i-UPR) genes, as well as numerous constitutive UPR (c-UPR) genes that require the ER stress transducers during normal development. Although ire-1 and xbp-1 together regulate transcription of most i-UPR genes, they are each required for expression of nonoverlapping sets of c-UPR genes, suggesting that they have distinct functions. Intriguingly, C. elegans atf-6 regulates few i-UPR genes following ER stress, but is required for the expression of many c-UPR genes, indicating its importance during development and homeostasis. In contrast, pek-1 is required for induction of approximately 23% of i-UPR genes but is dispensable for the c-UPR. As pek-1 and atf-6 mainly act through sets of nonoverlapping targets that are different from ire-1 and xbp-1 targets, at least two coordinated responses are required to alleviate ER stress by distinct mechanisms. Finally, our array study identified the liver-specific transcription factor CREBh as a novel UPR gene conserved during metazoan evolution.
The endoplasmic reticulum (ER) is an intracellular organelle where proteins fold and assemble prior to transport to the cell surface. The ER contains a finely tuned quality control apparatus to ensure that improperly folded proteins are retained in the ER lumen. A variety of physiological demands, environmental perturbations, and pathological conditions compromise protein folding in the ER and lead to the accumulation of unfolded proteins. The unfolded protein response (UPR) is an evolutionarily conserved intracellular adaptive signaling pathway that alleviates protein-folding defects in the ER. The unfolded protein signal is transmitted from the ER to the nucleus by three pathways involving the proteins ATF-6, PEK-1, and IRE-1/XBP-1. However, it is not known how these three pathways coordinate downstream transcriptional activation to mediate either cell adaptation or cell death. The authors have studied the nematode Caenorhabditis elegans to present a comprehensive genetic and gene expression analysis of the three UPR pathways. The findings demonstrate that the UPR regulates the expression of hundreds of genes in the presence, as well as the absence, of ER stress in a manner that is more complex and diverse than previously known.
Recent evidences suggest that endoplasmic reticulum (ER) stress was involved in multi pathological conditions, including diabetic nephropathy (DN). X-box binding protein 1(XBP1), as a key mediator of ER stress, has been proved having the capability of preventing oxidative stress. In this study, we investigated the effects of spliced XBP1 (XBP1S), the dominant active form of XBP1, on high glucose (HG)-induced reactive oxygen species (ROS) production and extracellular matrix (ECM) synthesis in cultured renal mesangial cells (MCs) and renal cortex of STZ-induced diabetic rats. Real time PCR and Western blot were used to evaluate the mRNA and protein levels respectively. Transfection of recombinant adenovirus vector carrying XBP1S gene (Ad-XBP1S) was used to upregulate XBP1S expression. XBP1S siRNA was used to knockdown XBP1S expression. ROS level was detected by dihydroethidium (DHE) fluorescent probe assay. The results showed that HG treatment significantly reduced XBP1S protein and mRNA level in the cultured MCs while no obvious change was observed in unspliced XBP1 (XBP1U). In the mean time, the ROS production, collagen IV and fibronectin expressions were increased. Diphenylene-chloride iodonium (DPI), a NADPH oxidase inhibtor, prevented HG-induced increases in ROS as well as collagen IV and fibronectin expressions. Transfection of Ad-XBP1S reversed HG-induced ROS production and ECM expressions. Knockdown intrinsic XBP1S expression induced increases in ROS production and ECM expressions. Supplementation of supreoxide reversed the inhibitory effect of Ad-XBP1S transfection on ECM synthesis. P47phox was increased in HG-treated MCs. Ad-XBP1S transfection reversed HG-induced p47phox increase while XBP1S knockdown upregulated p47phox expression. In the renal cortex of diabetic rats, the expression of XBP1S was reduced while p47phox, collagen IV and fibronectin expression were elevated. These results suggested that XBP1S pathway of ER stress was involved in HG-induced oxidative stress and ECM synthesis. A downstream target of XBP1S in regulating ROS formation might be NADPH oxidase.
Upon endoplasmic reticulum (ER) stress, mammalian cells induce the synthesis of a transcriptional activator XBP1s to alleviate the stress. Under unstressed conditions, the messenger RNA (mRNA) for XBP1s exists in the cytosol as an unspliced precursor form, XBP1u mRNA. Thus, its intron must be removed for the synthesis of XBP1s. Upon ER stress, a stress sensor IRE1α cleaves XBP1u mRNA to initiate the unconventional splicing of XBP1u mRNA on the ER membrane. The liberated two exons are ligated to form the mature XBP1s mRNA. However, the mechanism of this splicing is still obscure mainly because the enzyme that joins XBP1s mRNA halves is unknown. Here, we reconstituted the whole splicing reaction of XBP1u mRNA in vitro. Using this assay, we showed that, consistent with the in vivo studies, mammalian cytosol indeed had RNA ligase that could join XBP1s mRNA halves. Further, the cleavage of XBP1u mRNA with IRE1α generated 2′, 3′-cyclic phosphate structure at the cleavage site. Interestingly, this phosphate was incorporated into XBP1s mRNA by the enzyme in the cytosol to ligate the two exons. These studies reveal the utility of the assay system and the unique properties of the mammalian cytosolic enzyme that can promote the splicing of XBP1u mRNA.
Rapid growth of a tumor can overwhelm the vasculature that supplies it with nutrients and oxygen. Inside such tumors, cells undergo endoplasmic reticulum (EnR) stress but can survive such adverse microenvironments by an adaptive mechanism called the unfolded protein response (UPR). X-box binding protein-1 (XBP-1) is a critical transcriptional activator of the UPR and is responsible for regulating the function of genes in cell survival. An unconventional splicing of the XBP-1(U) mRNA results in two proteins: XBP-1(S) that is often increased in a variety of human cancers, and any translated proteins from the unspliced XBP-1(U) mRNA that acts as a dominant negative of endogenous XBP-1(S) action. In cancer cells, over-expression of XBP-1 can confer drug resistance by preventing drug-induced cell cycle arrest and mitochondrial permeability and apoptosis while down-regulation of XBP-1 increases the sensitivity to killing by hypoxia. XBP-1 is also implicated in cellular de-differentiation, oncovirus infection and the epithelial-to-mesenchymal transition (EMT). Given that XBP-1 mediates a wide range of responses in tumorigenesis, it is logical to focus on XBP-1 as an anti-cancer therapeutic target. Furthermore, combining inhibitors of XBP-1 with other anti-UPR drugs may enhance the activity of some anti-neoplastic therapies.
Cardiomyocyte apoptosis is a hallmark of coxsackievirus B3 (CVB3)-induced myocarditis. We used cardiomyocytes and HeLa cells to explore the cellular response to CVB3 infection, with a focus on pathways leading to apoptosis. CVB3 infection triggered endoplasmic reticulum (ER) stress and differentially regulated the three arms of the unfolded protein response (UPR) initiated by the proximal ER stress sensors ATF6a (activating transcription factor 6a), IRE1-XBP1 (X box binding protein 1), and PERK (PKR-like ER protein kinase). Upon CVB3 infection, glucose-regulated protein 78 expression was upregulated, and in turn ATF6a and XBP1 were activated via protein cleavage and mRNA splicing, respectively. UPR activity was further confirmed by the enhanced expression of UPR target genes ERdj4 and EDEM1. Surprisingly, another UPR-associated gene, p58IPK, which often is upregulated during infections with other types of viruses, was downregulated at both mRNA and protein levels after CVB3 infection. These findings were observed similarly for uninfected Tet-On HeLa cells induced to overexpress ATF6a or XBP1. In exploring potential connections between the three UPR pathways, we found that the ATF6a-induced downregulation of p58IPK was associated with the activation of PKR (PERK) and the phosphorylation of eIF2α, suggesting that p58IPK, a negative regulator of PERK and PKR, mediates cross-talk between the ATF6a/IRE1-XBP1 and PERK arms. Finally, we found that CVB3 infection eventually produced the induction of the proapoptoic transcription factor CHOP and the activation of SREBP1 and caspase-12. Taken together, these data suggest that CVB3 infection activates UPR pathways and induces ER stress-mediated apoptosis through the suppression of P58IPK and induction/activation of CHOP, SREBP1, and caspase-12.
The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers transcriptional and translational reprogramming. This unfolded protein response (UPR) protects cells during transient stress and can lead to apoptosis during prolonged stress. Two key mediators of the UPR are PKR-like ER kinase (PERK), which phosphorylates the α subunit of eukaryotic translation initiation factor 2 (eIF2α), resulting in decreased protein synthesis, and the α subunit of inositol-requiring enzyme 1 (IRE1α), which initiates cytoplasmic splicing of the mRNA encoding the transcription factor X-box binding protein 1 (XBP1). XBP1 induces transcription of genes involved in protein quality control. This report describes cross talk between these two pathways: phosphorylation of eIF2α was required for maximal induction of spliced XBP1 (XBP1s) protein levels via a mechanism that involved stabilization of XBP1s mRNA. By using mouse embryo fibroblasts deficient in UPR signaling pathways, we demonstrate that stress-induced stabilization of XBP1s mRNA requires cytoplasmic splicing of the mRNA and inhibition of its translation. Because the XBP1s protein promotes transcription of its own gene, the UPR-induced mRNA stabilization is part of a positive feedback loop that induces XBP1s protein accumulation and transcription of target genes during stress. We propose a model in which eIF2α phosphorylation-mediated control of mRNA turnover is a molecular switch that regulates the stress response transcription program and the ER's capacity for protein folding during stress.
Inositol-requiring enzyme 1 (IRE1) is the most highly conserved signaling node of the unfolded protein response (UPR) and represents a potential therapeutic target for a number of diseases associated with endoplasmic reticulum stress. IRE1 activates the XBP-1 transcription factor by site-specific cleavage of two hairpin loops within its mRNA to facilitate its nonconventional splicing and alternative translation. We screened for inhibitors using a construct containing the unique cytosolic kinase and endoribonuclease domains of human IRE1α (hIRE1α-cyto) and a mini-XBP-1 stem-loop RNA as the substrate. One class compounds was salicylaldehyde analogs from the hydrolyzed product of salicylaldimines in the library. Salicylaldehyde analogs were active in inhibiting the site-specific cleavage of several mini-XBP-1 stem-loop RNAs in a dose-dependent manner. Salicyaldehyde analogs were also active in inhibiting yeast Ire1 but had little activity inhibiting RNase L or the unrelated RNases A and T1. Kinetic analysis revealed that one potent salicylaldehyde analog, 3-ethoxy-5,6-dibromosalicylaldehyde, is a non-competitive inhibitor with respect to the XBP-1 RNA substrate. Surface plasmon resonance studies confirmed this compound bound to IRE1 in a specific, reversible and dose-dependent manner. Salicylaldehydes inhibited XBP-1 splicing induced pharmacologically in human cells. These compounds also blocked transcriptional up-regulation of known XBP-1 targets as well as mRNAs targeted for degradation by IRE1. Finally, the salicylaldehyde analog 3-methoxy-6-bromosalicylaldehyde strongly inhibited XBP-1 splicing in an in vivo model of acute endoplasmic reticulum stress. To our knowledge, salicylaldehyde analogs are the first reported specific IRE1 endoribonuclease inhibitors.
Enzyme Inhibitors; ER Stress; Protein Folding; RNA Processing; Transcription Factors; IRE1; RNase; XBP-1
Rio1 kinase is an essential ribosome-processing factor required for proper maturation of 40 S ribosomal subunit. Although its structure is known, several questions regarding its functional remain to be addressed. We report that both Archaeoglobus fulgidus and human Rio1 bind more tightly to an adenosine analog, toyocamycin, than to ATP. Toyocamycin has antibiotic, antiviral and cytotoxic properties, and is known to inhibit ribosome biogenesis, specifically the maturation of 40 S. We determined the X-ray crystal structure of toyocamycin bound to Rio1 at 2.0 Å and demonstrated that toyocamycin binds in the ATP binding pocket of the protein. Despite this, measured steady state kinetics were inconsistent with strict competitive inhibition by toyocamycin. In analyzing this interaction, we discovered that Rio1 is capable of accessing multiple distinct oligomeric states and that toyocamycin may inhibit Rio1 by stabilizing a less catalytically active oligomer. We also present evidence of substrate inhibition by high concentrations of ATP for both archaeal and human Rio1. Oligomeric state studies show both proteins access a higher order oligomeric state in the presence of ATP. The study revealed that autophosphorylation by Rio1 reduces oligomer formation and promotes monomerization, resulting in the most active species. Taken together, these results suggest the activity of Rio1 may be modulated by regulating its oligomerization properties in a conserved mechanism, identifies the first ribosome processing target of toyocamycin and presents the first small molecule inhibitor of Rio1 kinase activity.
The unfolded protein response (UPR) is an evolutionary conserved adaptive reaction for increasing cell survival under endoplasmic reticulum (ER) stress conditions. X-box-binding protein-1 (Xbp1) is a key transcription factor of UPR that activates genes involved in protein folding, secretion, and degradation to restore ER function. The UPR induced by ER stress was extensively studied in diseases linked to protein misfolding and aggregations. However, in the porcine system, genes in the UPR pathway were not investigated. In this study, we isolated and characterized the porcine Xbp1 (pXbp1) gene in ER stress using porcine embryonic fibroblast (PEF) cells and porcine organs. ER stress was induced by the treatment of tunicamycin and cell viability was investigated by the MTT assay. For cloning and analyzing the expression pattern of pXbp1, RT-PCR analysis and Western blot were used. Knock-down of pXbp1 was performed by the siRNA-mediated gene silencing.
We found that the pXbp1 mRNA was the subject of the IRE1α-mediated unconventional splicing by ER stress. Knock-down of pXbp1 enhanced ER stress-mediated cell death in PEF cells. In adult organs, pXbp1 mRNA and protein were expressed and the spliced forms were detected.
It was first found that the UPR mechanisms and the function of pXbp1 in the porcine system. These results indicate that pXbp1 plays an important role during the ER stress response like other animal systems and open a new opportunity for examining the UPR pathway in the porcine model system.
IRE1-alpha is an integral membrane protein of the endoplasmic reticulum (ER) that is a key sensor in the cellular transcriptional response to stress in the ER. Upon induction of ER stress, IRE1-alpha is activated, resulting in the synthesis of the active form of the transcription factor XBP1 via IRE1-mediated splicing of its mRNA. In this report, we have examined the role of IRE1-alpha and XBP1 in activation of the hepatitis B virus S promoter by ER stress. Cotransfection experiments revealed that overexpression of either IRE1-alpha or XBP1 activated this promoter. Conversely, cotransfected dominant-negative IRE1-alpha or small interfering RNA directed against XBP1 decreased the activation of the S promoter by ER stress, confirming an important role for the IRE1-alpha/XBP1 signaling pathway in activation of the S promoter. However, XBP1 does not bind directly to the S promoter; rather, a novel S promoter-binding complex that does not contain XBP1 is induced in cells undergoing ER stress in an XBP1-dependent manner. This complex, as well as transcriptional activation of the S promoter, is induced by ER stress in hepatocytes but not in fibroblasts, despite the presence of active XBP1 in the latter. Thus, the hepatitis B virus S promoter responds to a novel, cell type-restricted transcriptional pathway downstream of IRE1-alpha and XBP1.
The cytosolic chaperone Hsp72 directly modulates stress sensing in response to the accumulation of unfolded proteins in the endoplasmic reticulum and promotes cell survival.
Endoplasmic reticulum (ER) stress is a feature of secretory cells and of many diseases including cancer, neurodegeneration, and diabetes. Adaptation to ER stress depends on the activation of a signal transduction pathway known as the unfolded protein response (UPR). Enhanced expression of Hsp72 has been shown to reduce tissue injury in response to stress stimuli and improve cell survival in experimental models of stroke, sepsis, renal failure, and myocardial ischemia. Hsp72 inhibits several features of the intrinsic apoptotic pathway. However, the molecular mechanisms by which Hsp72 expression inhibits ER stress-induced apoptosis are not clearly understood. Here we show that Hsp72 enhances cell survival under ER stress conditions. The UPR signals through the sensor IRE1α, which controls the splicing of the mRNA encoding the transcription factor XBP1. We show that Hsp72 enhances XBP1 mRNA splicing and expression of its target genes, associated with attenuated apoptosis under ER stress conditions. Inhibition of XBP1 mRNA splicing either by dominant negative IRE1α or by knocking down XBP1 specifically abrogated the inhibition of ER stress-induced apoptosis by Hsp72. Regulation of the UPR was associated with the formation of a stable protein complex between Hsp72 and the cytosolic domain of IRE1α. Finally, Hsp72 enhanced the RNase activity of recombinant IRE1α in vitro, suggesting a direct regulation. Our data show that binding of Hsp72 to IRE1α enhances IRE1α/XBP1 signaling at the ER and inhibits ER stress-induced apoptosis. These results provide a physical connection between cytosolic chaperones and the ER stress response.
The endoplasmic reticulum (ER) is responsible for production and folding of secreted proteins. When the protein folding machinery cannot keep up with demand, misfolded proteins accumulate, leading to a state of ER stress that contributes to diseases such as cancer, neurodegeneration, diabetes, and myocardial infarct. The unfolded protein response (UPR) is an intracellular signaling network activated in response to ER stress. It initially tries to restore normal ER homeostasis, but if the damage is too severe cell death pathways mediated by cytosolic and mitochondrial proteins are activated. The molecular mechanisms involved in the transition of the UPR from a protective to an apoptotic phase are unclear. IRE1α is an ER membrane protein that acts as a sensor of ER stress. A number of proteins can interact with IRE1α to regulate its function, which includes an RNase activity responsible for inducing the unconventional splicing of the transcript for a downstream signaling protein called XBP-1. Here, we report that Hsp72, a stress-inducible cytosolic molecular chaperone, can bind to and enhance the RNase activity of IRE1α, providing an important molecular link between the heat shock response and the ER stress response. Importantly, increased production of active XBP-1 was necessary for Hsp72 to exert its prosurvival effect under conditions of ER stress. Our results suggest a mechanism whereby Hsp72 overexpression helps cells adapt to long-term ER stress in vivo by enhancing the pro-survival effects of the IRE1α/XBP1 branch of the UPR.
Eukaryotic cells respond to stress caused by the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum by activating the intracellular signaling pathways referred to as the unfolded protein response (UPR). In metazoans, UPR consists of three parallel branches, each characterized by its stress sensor protein, IRE1, ATF6, and PERK, respectively. In Drosophila, IRE1/XBP1 pathway is considered to function as a major branch of UPR; however, its physiological roles during the normal development and homeostasis remain poorly understood. To visualize IRE1/XBP1 activity in fly tissues under normal physiological conditions, we modified previously reported XBP1 stress sensing systems (Souid et al., Dev Genes Evol 217: 159–167, 2007; Ryoo et al., EMBO J 26: 242-252, 2007), based on the recent reports regarding the unconventional splicing of XBP1/HAC1 mRNA (Aragon et al., Nature 457: 736–740, 2009; Yanagitani et al., Mol Cell 34: 191–200, 2009; Science 331: 586–589, 2011). The improved XBP1 stress sensing system allowed us to detect new IRE1/XBP1 activities in the brain, gut, Malpighian tubules, and trachea of third instar larvae and in the adult male reproductive organ. Specifically, in the larval brain, IRE1/XBP1 activity was detected exclusively in glia, although previous reports have largely focused on IRE1/XBP1 activity in neurons. Unexpected glial IRE1/XBP1 activity may provide us with novel insights into the brain homeostasis regulated by the UPR.
UPR; ER stress; XBP1; Drosophila; Glia
X-box-binding protein 1 (XBP1) is a key modulator of unfolded protein response (UPR), which is involved in a wide range of pathological and physiological processes. The active/spliced form of XBP1 (XBP1s) messenger RNA is generated from unspliced form by IRE1 during UPR. However, post-translational modulation of XBP1s remains largely unknown. Here, we demonstrate that XBP1s is a target of acetylation and deacetylation mediated by p300 and SIRT1 respectively. p300 increases acetylation and protein stability of XBP1s, and enhances the transcriptional activity of XBP1s. SIRT1 deacetylates XBP1s and inhibits the transcriptional activity of XBP1s. Deficiency of SIRT1 enhances the XBP1s-mediated luciferase reporter activity in HEK293 cells and the upregulation of XBP1s target gene expression under ER stress in mouse embryonic fibroblasts (MEFs). Consistent with XBP1s favoring cell survival under ER stress, Sirt1−/− MEFs display a greater resistance to the ER stress-induced apoptotic cell death compared with Sirt1+/+ MEFs. Taken together, these results suggest that acetylation/deacetylation constitutes an important post-translational mechanism in controlling protein levels as well as transcriptional activity of XBP1s. This study provides a novel insight into molecular mechanisms by which SIRT1 regulates UPR signaling.
acetylation; SIRT1; p300; unfolded protein response; X-box-binding protein 1; cell death
To investigate the anti-apoptotic mechanism of leptin in non-small cell lung cancer.
Materials and Methods
The influences of leptin on apoptosis were investigated, analyzing the mechanism that triggers growth of A549 cells. The effects of leptin on cell proliferation were examined by XTT analysis. Leptin, C/EBP homologous protein (CHOP), phosphorylated-PKR-like ER kinase (p-Perk), inositol requiring proteins-1, spliced X-box transcription factor-1 (XBP1), cleaved activating transcription factor-6 (ATF6), eukaryotic translation initiation factor-2α, caspase-12 and CHOP protein were detected in four groups by western blot, and endoplasmic reticulum (ER) stress related mRNA were detected by reverse transcription PCR.
The expression of leptin in A549 and leptin transfected cells inhibited cisplatin activated ER stress-associated mRNA transcription and protein activation. Two ER stress unfolded protein response pathways, PERK and ATF6, were involved, and XBP1 and tumor necrosis factor receptor-associated factor 2 (TRAF2) were increased significantly when treated with cisplatin in A549-siRNA against leptin cells. Furthermore, CHOP expression was inhibited upon leptin expression in A549, LPT-PeP and LPT-EX cells.
Leptin serves as an important factor that promotes the growth of A549 cells through blocking ER stress-mediated pathways. This blocking is triggered by p-Perk and ATF6 via inhibition of CHOP expression.
Apoptosis; ER stress; cell growth; leptin; TRAF2; XPB1
PURPOSE: Tumors encounter endoplasmic reticulum stress during tumor growth and activate an adaptive pathway known as the unfolded protein response (UPR). Because this pathway is induced by the tumor microenvironment, it is a promising target for cancer therapy. We have previously demonstrated that X-box binding protein 1 (XBP-1), a key regulator of the UPR, was required for survival under hypoxia and critical for tumor growth in tumor xenografts. In this study, we investigated the role of XBP-1 in regulating tumor angiogenesis. METHODS: We used an intradermal angiogenesis model to quantify the effect of XBP-1 on angiogenesis. We also used a human tumor xenograft model to assay for tumor growth delay. We determined vascular endothelial growth factor (VEGF) expression by quantitative polymerase chain reaction and ELISA. Finally, we stained human pancreatic adenocarcinoma specimens for XBP-1 expression and correlated the expression pattern of XBP-1 with CD31 (endothelial cell marker) expression. RESULTS: We demonstrated that XBP-1 is essential for angiogenesis during early tumor growth. Inhibiting XBP-1 expression by short-hairpin RNA sequence specific for XBP-1 reduced blood vessel formation in tumors from mouse embryonic fibroblast cells and human fibrosarcoma tumor cells (HT1080). Expressing a dominant-negative form of IRE1α also reduced blood vessel formation in tumors. Moreover, expression of spliced XBP-1 (XBP-1s) restored angiogenesis in IRE1α dominant-negative expressing cells. We further demonstrated that XBP-1-mediated angiogenesis does not depend on VEGF. CONCLUSIONS: We propose that the IRE1α-XBP-1 branch of the UPR modulates a complex proangiogenic, VEGF-independent response that depends on signals received from the tumor microenvironment.
Persistent paraprotein production in plasma cells necessitates a highly developed rough endoplasmic reticulum (ER) that is unusually susceptible to perturbations in protein synthesis. This biology is believed to account for the exquisite sensitivity of multiple myeloma (MM) to the proteasomal inhibitor bortezomib (BTZ). Despite remarkable response rates to BTZ in MM, BTZ carries the potential for serious side-effects and development of resistance. We, therefore, sought to identify therapeutic combinations that effectively disrupt proteostasis in order to provide new potential treatments for MM. We found that sulforaphane, a dietary isothiocyanate found in cruciferous vegetables, inhibits TNFα-induced Iκβ proteasomal degradation in a manner similar to BTZ. Like BTZ, sulforaphane synergistically enhances the cytotoxicity of arsenic trioxide (ATO), an agent with clinical activity in MM. ATO and sulforaphane co-treatment augmented apoptotic induction as demonstrated by cleavage of caspase-3, -4 and PARP. The enhanced apoptotic response was dependent upon production of reactive oxygen species (ROS) as demonstrated by glutathione depletion and partial inhibition of the apoptotic cascade after pretreatment with the radical scavenger N-acetyl-cysteine (NAC). Combination treatment resulted in enhanced ER stress signaling and activation of the unfolded protein response (UPR), indicative of perturbation of proteostasis. Specifically, combination treatment caused elevated expression of the molecular chaperone HSP90 (heat shock protein 90) along with increased PERK (protein kinase RNA-like endoplasmic reticulum kinase) and eIF2α phosphorylation and XBP1 (X-box binding protein 1) splicing, key indicators of UPR activation. Moreover, increased splicing of XBP1 was apparent upon combination treatment compared to treatment with either agent alone. Sulforaphane in combination with ATO effectively disrupts protein homeostasis through ROS generation and induction of ER stress to culminate in inhibition of protein secretion and apoptotic induction in MM. Our results suggest that sulforaphane deserves further investigation in combination with ATO in the treatment of MM.
sulforaphane; arsenic trioxide; unfolded protein response; Iκβ; endoplasmic reticulum stress; reactive oxygen species
The transcription factor X-box-binding protein-1 (XBP-1) plays an essential role in activating the unfolded protein response in the endoplasmic reticulum (ER). Transcribed XBP-1 mRNA is converted to its active form by unconventional cytoplasmic splicing mediated by inositol-requiring enzyme-1 (IRE-1) upon ER stress. We report activation of the IRE-1/XBP-1 pathway in effector CD8+ T cells during the response to acute infection. Transcription of unspliced XBP-1 mRNA is up-regulated by IL-2 signals, while its splicing is induced after TCR ligation. Splicing of XBP-1 mRNA was evident during the expansion of Ag-specific CD8+ T cells in response to viral or bacterial infection. An XBP-1 splicing reporter revealed that splicing activity was enriched in terminal effector cells expressing high levels of killer cell lectin-like receptor G1 (KLRG1). Overexpression of the spliced form of XBP-1 in CD8+ T cells enhanced KLRG1 expression during infection, whereas XBP-1−/− CD8+ T cells or cells expressing a dominant-negative form of XBP-1 showed a decreased proportion of KLRG1high effector cells. These results suggest that, in the response to pathogen, activation of ER stress sensors and XBP-1 splicing contribute to the differentiation of end-stage effector CD8+ T cells.
The trichothecene mycotoxin deoxynivalenol (DON) induces systemic expression of the interleukin-6 (IL-6) and other proinflammatory cytokines in the mouse. The purpose of this study was to test the hypothesis that DON triggers an endoplasmic reticulum (ER) stress response in murine macrophages capable of driving IL-6 gene expression. DON at concentrations up 5000 ng/ml. was not cytotoxic to peritoneal cells. However, DON markedly decreased protein levels but not the mRNA levels of glucose-regulated protein (GRP) 78 (BiP), a chaperone known to mediate ER stress. Inhibitor studies suggested that DON-induced GRP78 degradation was cathepsin and calpain dependent but was proteosome-independent. RNAi-mediated knockdown of GRP78 resulted in increased IL-6 gene expression indicating a potential downregulatory role for this chaperone. GRP78 is critical to the regulation of the two transcription factors, X-box binding protein 1 (XBP1) and activating transcription factor 6 (ATF6), which bind to cAMP-response element (CRE) and drive expression of CRE-dependent genes such as IL-6. DON exposure was found to increase IRE1α protein, its modified products spliced XBP1 mRNA and XBP1 protein as well as ATF6. Knockdown of ATF6 but not XBP1 partially inhibited DON-induced IL-6 expression in the macrophages. Three other trichothecenes (satratoxin G, roridin, T-2 toxin) and the ribosome inhibitory protein ricin were also found to induce GRP78 degradation suggesting that other translation inhibitors might evoke ER stress. Taken together, these data suggest that in the macrophage DON induces GRP78 degradation and evokes an ER stress response that could contribute, in part, to DON-induced IL-6 gene expression.
deoxynivalenol (DON); interleukin-6; ER stress, translation inhibition
Past studies have shown that melanoma cells have largely adapted to endoplasmic reticulum (ER) stress. In this study, we report that melanoma cells under ER stress are more resistant to apoptosis induced by the microtubule-targeting chemotherapeutic drugs, docetaxel and vincristine, and this is, at least in part, due to activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway mediated by the X-box-binding protein 1 (XBP-1) axis of the unfolded protein response. Treatment with the ER stress-inducer tunicamycin (TM) or thapsigargin before the addition of docetaxel or vincristine reduced the levels of apoptosis induced by the drugs. This was associated with inhibition of mitochondrial release of apoptogenic proteins and activation of Bax and Bak. Induction of ER stress resulted in the rapid activation of the PI3K/Akt pathway that seemed to be important in antagonizing docetaxel and vincristine, in that inhibition of Akt blocked the effect of pretreatment with TM on apoptosis induced by the drugs. Neither docetaxel nor vincristine triggered ER stress in melanoma cells, but the basal activity of XBP-1 signaling seemed to play a role in the protection against the drugs because small interfering RNA knockdown of XBP-1 enhanced docetaxel- and vincristine-induced apoptosis. In addition, inhibition of XBP-1 decreased the constitutive levels of activation of Akt and blocked the activation of Akt induced by TM. Taken together, these results identify activation of the PI3K/Akt pathway by XBP-1-mediated signaling of the unfolded protein response as a resistance mechanism against docetaxel and vincristine in melanoma cells under ER stress.
X-box binding protein 1 (XBP-1), a basic leucine zipper transcription factor, plays a key role in the cellular unfolded protein response (UPR). There are two XBP-1 isoforms in cells, spliced XBP-1S and unspliced XBP-1U. XBP-1U has been shown to bind to the 21-bp Tax-responsive element of the human T-lymphotropic virus type 1 (HTLV-1) long terminal repeat (LTR) in vitro and transactivate HTLV-1 transcription. Here we identify XBP-1S as a transcription activator of HTLV-1. Compared to XBP-1U, XBP-1S demonstrates stronger activating effects on both basal and Tax-activated HTLV-1 transcription in cells. Our results show that both XBP-1S and XBP-1U interact with Tax and bind to the HTLV-1 LTR in vivo. In addition, elevated mRNA levels of the gene for XBP-1 and several UPR genes were detected in the HTLV-1-infected C10/MJ and MT2 T-cell lines, suggesting that HTLV-1 infection may trigger the UPR in host cells. We also identify Tax as a positive regulator of the expression of the gene for XBP-1. Activation of the UPR by tunicamycin showed no effect on the HTLV-1 LTR, suggesting that HTLV-1 transcription is specifically regulated by XBP-1. Collectively, our study demonstrates a novel host-virus interaction between a cellular factor XBP-1 and transcriptional regulation of HTLV-1.
Expression of the Cat-1 gene (cationic amino acid transporter-1) is induced in proliferating cells and in response to a variety of stress conditions. The expression of the gene is mediated via a TATA-less promoter. In the present study we show that an Sp1 (specificity protein 1)-binding site within a GC-rich region of the Cat-1 gene controls its basal expression and is important for induction of the gene during the UPR (unfolded protein response). We have shown previously that induction of Cat-1 gene expression during the UPR requires phosphorylation of the translation initiation factor eIF2α (eukaryotic initiation factor 2α) by PERK (protein-kinase-receptor-like endoplasmic reticulum kinase), one of the signalling pathways activated during the UPR. This leads to increased translation of the transcription factor ATF4 (activating transcription factor 4). We also show that a second signalling pathway is required for sustained transcriptional induction of the Cat-1 gene during the UPR, namely activation of IRE1 (inositol-requiring enzyme 1) leading to alternative splicing of the mRNA for the transcription factor XBP1 (X-box-binding protein 1). The resulting XBP1s (spliced XBP1) can bind to an ERSE (endoplasmic-reticulum-stress-response-element), ERSE-II-like, that was identified within the Cat-1 promoter. Surprisingly, eIF2α phosphorylation is required for accumulation of XBP1s. We propose that the signalling via phosphorylated eIF2α is required for maximum induction of Cat-1 transcription during the UPR by inducing the accumulation of both ATF4 and XBP1s.
activating transcription factor 4 (ATF4); cationic amino acid transporter-1 (Cat-1); endoplasmic reticulum stress; specificity protein 1 (Sp1); unfolded protein response; X-box-binding protein 1 (XBP1)
Viral infection causes stress to the endoplasmic reticulum. The response to endoplasmic reticulum stress, known as the unfolded protein response (UPR), is designed to eliminate misfolded proteins and allow the cell to recover by attenuating translation and upregulating the expression of chaperones, degradation factors, and factors that regulate the cell's metabolic and redox environment. Some consequences of the UPR (e.g., expression of chaperones and regulation of the metabolism and redox environment) may be advantageous to the viral infection; however, translational attenuation would not. Thus, viruses may induce mechanisms which modulate the UPR, maintaining beneficial aspects and suppressing deleterious aspects. We demonstrate that human cytomegalovirus (HCMV) infection induces the UPR but specifically regulates the three branches of UPR signaling, PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE-1), to favor viral replication. HCMV infection activated the eIF2α kinase PERK; however, the amount of phosphorylated eIF2α was limited and translation attenuation did not occur. Interestingly, translation of select mRNAs, which is dependent on eIF2α phosphorylation, did occur, including the transcription factor ATF4, which activates genes which may benefit the infection. The endoplasmic reticulum stress-induced activation of the transcription factor ATF6 was suppressed in HCMV-infected cells; however, specific chaperone genes, normally activated by ATF6, were activated by a virus-induced, ATF6-independent mechanism. Lastly, HCMV infection activated the IRE-1 pathway, as indicated by splicing of Xbp-1 mRNA. However, transcriptional activation of the XBP-1 target gene EDEM (ER degradation-enhancing α-mannosidase-like protein, a protein degradation factor) was inhibited. These results suggest that, although HCMV infection induces the unfolded protein response, it modifies the outcome to benefit viral replication.

domain: medical


text:
In my previous article, I talked about why gathering photo reference matters. Now, I'd like to share some practical tips to help you jumpstart your own process.
1. Rent, Buy, or Borrow Costumes
For my astronaut illustration, I visited a local costume shop and rented an astronaut suit. The folds and buckles in the suit served as great reference for the final product. If I had simply worn pants, a shirt, and tried to imagine an astronaut design after-the-fact, the results wouldn't have been as convincing.
Invest in the details, especially if you don’t know enough from experience to render from imagination. Amazon.com is a great internet resource to buy props from if you're looking to build a wardrobe.
You could also borrow from a family member or friend. It's free (usually), and you (typically) wouldn't be pressured to return it by the end of the week!
2. Hire a Model, Enlist the Help of a Willing Friend, or Do It Yourself!
If you can afford it, hiring a model is an ideal way to gather good reference. You have complete control of the setup and can focus on capturing the best pose and look for your work.
If you don’t feel comfortable working with a complete stranger, try asking friends or family for their cooperation. Share your vision for the finished project and, if they’re willing to help, offer to pay them for their time.
For most of my work, I model for the reference. To do this, I usually put a timer on my camera and rush to get into position, tweaking and altering as deemed necessary. This is the cheapest way to get your reference, however, it costs time and limits your range of experimentation.
3. Choose Lighting that Best Captures the Mood
Research different types of lighting and decide which one fits the mood you’re wanting to convey. Butterfly, Rembrandt, rim, and loop lighting are just a few to look up. I used a ceiling window to get a butterfly light for my astronaut piece. Invest in a clamp lamp and experiment with moving the light around your subject for new and interesting looks.
4. Experiment, Experiment, Experiment!
Take pictures based on the thumbnails you created, but don’t be afraid to experiment and try different angles. You may find an image that better conveys your narrative than your initial concept.
5. What Camera Should You Use?
Regarding what camera to use, I can only speak from experience. Before smartphones had developed such fantastic lenses, I used a simple Nikon camera. Now, I use either my iPhone SE or iPad Pro 12.9, depending on the need.
1. Get a costume!
2. Do all the poses!
3. Research all the lighting!
5. Use what you got!
You're doing better than you think. Keep going!

domain: hobby