Audizine Forums > Classifieds Quick-Links > Autos/Vehicles > Audi > For Sale: 2014 Audi RS7 4.0T QUATTRO TIPTRONIC - WARRANTY BANG OLUFSEN CARBON FIBER HUD LOADED!
View Full Version : For Sale: 2014 Audi RS7 4.0T QUATTRO TIPTRONIC - WARRANTY BANG OLUFSEN CARBON FIBER HUD LOADED!
I'm selling my preowned gorgeous 2014 Audi RS7 4.0T Quattro. This car is a 561hp beast! The 0-60 time is restored at 3.2 seconds by some magazines. This car has had only one owner (me). The car is in stylish and unique Nardo gray. You will get countless complements on this paint color.
The car was never tracked and driven mostly highway miles, less than 31,000 at the time of this posting. It has had all scheduled factory maintenance, without any accidents or paintwork. There is remaining 25,000 mile or over 1 year year factory warranty. The car has current Audi Care maintenance. I replaced the front and rear brake pads and rotors August 2016 ($6,000+ savings to you, be weary of other RS7s on sale that haven't had brakes yet).
The car is fully optioned with Bang Olufsen premium sound with rising tweeters, navigation, soft-close doors, head-up display, sport exhaust (with downshift pops/burbles), alcantara, deleted front license plate, iPhone interface, red brake calipers, carbon fiber package with Quattro, 21" titanium wheels. Also has adaptive cruise control, night vision assist, and lane tracking. MSRP is over $130,000!!! The Kelly Blue Book is $89,000. My loss is your gain!
The showroom photos are from the car at the dealership. The airport photos are a few months old. Since pickup, I have added custom carbon fiber mirror covers and a trunk spoiler made from genuine carbon fiber and clear coat made in Germany as a one-off piece ($3500). I have also added custom RS7 mats and four door LED "Audi logo" projection lamps which really make the car stand out. The auction includes the stock gray spoiler, gray mirror covers, and blank mats only.
There is an existing factory warranty. Clean title. Located in Central CA. No significant scratches. Minor rock chips on the hood are easily touched up. Always hand washed, never machine washed. One or two wheels with minimal curb rash (barely noticeable unless you're looking).
Where are the Airport photos?
Thanks. It is very unique. People will walk up to the car to comment.
Damned gorgeous but just a little early for my shopping time, will watch over the next month or so and see.
Did you sell it yet?


text:
Border Crossings »
What is the importance of truck crossings?
The dynamics of truck crossings through Arizona's border ports of entry reflect the composite effects of both the trade between Arizona and Mexico, and between Mexico, Canada, and other U.S. states that use Arizona's border ports for the facilitation of trade. Also reflected in truck crossing dynamics are the effects of physical and human infrastructure capacities at border ports of entry, which affects general cross border travel volumes, and wait times in particular. Among Arizona's six border ports of entry, Nogales facilitates the largest number of truck crossings. San Luis and Douglas are a distant second. In the overall picture, the Nogales border port of entry is number five behind Laredo, Hidalgo and El Paso in Texas, and Calexico East in California.
What is measured?
Trucks crossing data include commercial vehicles: the mode of transportation that carries the largest volume and value of merchandise across the border between the U.S. and Mexico. Trucks are also the primary means of transportation for Mexican fresh produce shipped through land border ports of entry to markets in the U.S. and Canada. Although data are not collected for fresh produce-carrying vs. other merchandise-carrying trucks, the concentration of fresh produce season in winter months is reflected in variability of monthly data. The selected southern border ports of entry used in this analysis are: Douglas, Lukeville, Naco, Nogales, San Luis, and Sasabe in Arizona; Calexico East in California; Santa Teresa in New Mexico; and, El Paso, Hidalgo, and Laredo in Texas.
How does Arizona compare?
To gauge Nogales' relative competitiveness in the entire U.S.-Mexico border region, data are provided for a selected number of major ports of entry in California, New Mexico, and Texas. Changes expressed in percentages allow for comparison of truck crossing dynamics at individual ports as well as for the state as a whole.
The table on this page summarizes border crossings at Arizona and major Border Ports of Entry on a monthly basis. Data are updated following release by the Bureau of Transportation Statistics. Click on any title to graph that series, and export. Or, click "download" at the base of the table to download the dataset, or customize based on a specified range.
Unavailable - Monthly Northbound Truck Crossings
The table on this page summarizes border crossings at Arizona and major Border Ports of Entry on an annual basis. Data are updated following release by the Bureau of Transportation Statistics. Click on any title to graph that series, and export. Or, click "download" at the base of the table to download the dataset, or customize based on a specified range.
Unavailable - Annual Northbound Truck Crossings
Bureau of Transportation Statistics , Office of the Assistant Secretary for Research and Technology, Border Crossing/Entry Data. Note: Zero values may indicate that no crossings took place at that particular port of entry in a given month or year. Zero values may also indicate that data cannot be recorded due to lack of facilities.
Photo of sunset with mountain panorama courtesy of Shutterstock.
Photo of delivery truck driving across desert courtesy of Shutterstock.
Important note on border crossings data:
Border crossing data presented on these pages are from the Bureau of Transportation Statistics (BTS). BTS releases border crossings as monthly data series, however, they release these data three months at a time each quarter. EBRC calculates the annual totals published here. EBRC inspects all data released from public sources for accuracy and consistency. The Center has noticed several issues of concern with the border crossing dataset. In order to improve the consistency and accuracy EBRC has made and continues incorporate the following estimates and corrections to this dataset. This means the data presented here may not always match data on the BTS website.
Missing observations in the historical monthly series are estimated when possible and sensible. These estimates are then incorporated into the annual totals published here. You may request a complete list of estimated values. EBRC believes these estimates greatly improve the accuracy and relevance of these data.
Each quarter when BTS releases three more months of data, frequently there are missing data points or incorrect data points. BTS then often corrects these errors over the course of the quarter, or the data are corrected by BTS the following quarter. After each BTS release, EBRC checks and then corrects or estimates data where possible and needed prior to publication on this website. When BTS makes corrections during the quarter EBRC incorporated these into the monthly data series and annual totals.
Because of the two issues mentioned above, EBRC does not publish new data on this website immediately upon the BTS data release. EBRC may take a week or more to check and correct the data. We will always publish a note informing our users of when the BTS data is released with an estimate for the EBRC data release on this page.
The three points above mean that data on this page may not always match data on the BTS website. It is however the opinion of EBRC that data published on this website are at any given time the most accurate and reasonable reflection of current trends in border crossings.

redpajama_set_name: RedPajamaCommonCrawl


Why should you volunteer your time with in local football?
Support the development of local football.
Play a role in the future development of the next generation of players.
Be the difference you want to see in the game.
What age can I start Volunteering?
You can volunteer at any age, as long as you are able to fulfil your allocated roles and responsibilities. For young volunteers aged 14+ the Jersey FA provide a Youth Council, called Change4Football visit the Young Volunteers page for more information.
There are a number of local clubs you can volunteer at. In the first instance you can either contact the club secretary you wish to volunteer at, or if you are not sure where you want to volunteer you can contact us here at the Jersey FA and we will be happy to signpost you to the right club. Info@JerseyFA.com or call 730433.
Most roles you will not need any Qualification however - If you are working with children you will need to pass your FA Child Safeguarding Course and Criminal Records Check.
A proof of address i.e. utility bill.
Contact Jersey FA Business Manager Robert Lawrence at Robert.Lawrence@JerseyFA.com to commence the process.
If you wish to volunteer as a coach you will need to obtain an FA level 1 Coaching Football Qualification. For more information on the next course visit the 'Get into Coaching' page.
These processes are simple and easy to complete don't let them put you off volunteering.
See the Volunteer Roles Page for more information.
Jersey FA takes Safeguarding extremely seriously. We are working to make the game as safe and enjoyable as possible. This work is delivered through Jersey FA's in conjunction with Jersey Football Combination League and Clubs.


The latest corporate sleazoid is Volkswagen. VW is charged with employing sophisticated software in 11 million cars that falsified a vehicle's true pollution output when it was being tested. The company posed as green when the cars it tampered with weren't. But don't be surprised: corporate crime happens all the time. On September 17, General Motors agreed to pay a $900 million fine for the faulty design of ignition switches that killed 124 people; the firm had known about the flaw since 2005. In banking, "too big to fail" J.P. Morgan Chase & Co and Citigroup Inc pled guilty in May 2015 to foreign exchange manipulation. They–and three foreign banks–were fined $5.7 billion. On September 21, Stewart Parnell, the owner and president of Peanut Corporation of America, and other executives, received long prison sentences (28 years for Parnell)for knowlingly shipping salmonella-infected peanut butter that sickened hundreds. In May, Duke Energy, the nation's largest untility, admitted violating environmental laws and agreed to pay a $68 million fine. Also in May, 14 people, including leading executives at FIFA, a governing body for soccer, were indicted for racketeering, money laundering and other charges extending back two decades. In an age of deregulation, corporate crime is as inevitable as boom and bust cycles.


The use of Nutri Mask plus certain specific beauty routines for each hair type will allow you to have beautiful hair full of vitality.
Since it is thinner than other types of hair it gets affected much more easily by changes in the weather, the use of hair dryers and flat irons, and hair dyes. Its lack of body makes it get tangled more easily and it is more difficult to hydrate.
After washing hair avoid rubbing it, it is ideal to wrap it with a towel allowing it to dry by itself. If hair drying is needed it is best to use at low or medium temperatures to prevent damage from artificial heat. Our cleansing and treatment line is special for sensitive hair because it is nourishing, moisturizing and strengthening.
This type of hair is identified by the diameter of each strand being greater which means it contains more protein, making it stronger. This makes it more resistant to cosmetic treatments like hair dyeing, styling, hair drying, straightening and more. Its disadvantage is that it absorbs much more humidity from the environment, causing freeze and excessive volume, this being its most frequent issue. Each strand has many layers of cuticles giving it a more coarse and unruly feel and appearance, for this reason the use of a smoothing oil is recommended in order to seal the cuticles and achieve softer, natural hair. Certain products that contain silicones relax the cuticle and initially provide this feeling of softness but its continuous use forms a non-soluble (water insoluble) layer that does not allow nutrients and moisture to penetrate each strand weakening hair and making it brittle.
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This genetic characteristic is aggravated by certain external factors like chlorine in pools, too much sun, shampoos that lather excessively, sea salt, high temperature blow drying, use of flat irons, hair dyes and environmental humidity.
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Overly active sebaceous glands give hair a shiny appearance making this type of hair get dirty more frequently. Extra oil/grease can promote the presence of bacterias related to dandruff, making it important to cleanse hair more frequently with a mild product like Oleim™ Cleansing and Conditioning Cream, which can be used daily when needed. Continuous use of Oleim™ can help your hair transition into Normal type of hair.
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This type of hair is very fragile and its curved structure has less cuticle layers making it more susceptible to heat sources. When coily hair is flat ironed too often it gets very dry with split ends. It has great volume and density. Since it has a tendency to get tangled towards the tips, deep hydration is of utmost importance as well as the use of your fingers as your main styling tool.
Nutri Mask is special for coily hair since its application before cleansing hydrates the scalp and hair, stimulating the secretion of natural oils. Using it regularly before each cleansing can substitute the use of hot oil treatments. The use of a soothing oil is recommended after straightening hair or if a natural look is desired.
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Untangling hair before cleansing it can prevent it from breaking and suffering further damage. It is also essential to use a nourishing cleanser, like Oleim™ Cleansing and Conditiong Cream, that is not alcohol based and does not contain silicones or strong detergents so natural oils can be preserved in order to provide shine and health. After cleansing, hair must not be rubbed with towel and a shirt towel is highly recommended since it is much more gentle.
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Oleim™ Nutri Mask is also designed to maintain normal hair healthy because it is not an invasive treatment and it will not cause an imbalance by drying or introducing too much oil.
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redpajama_set_name: RedPajamaGithub


As far as HSE training management is concerned, we use the ANSI standard (ANSI/ASSE Z490.1 - American National Standard Criteria for Accepted Practices in Safety, Health and Environmental Training) for the development, delivery and evaluation of HSE training programmes for clients. This is a professional standard for how to plan and conduct training programs in environmental health and safety. It establishes best management practices for how training should be formulated, presented, and assessed.
Bespoke training modules and many more….
A course for top people with an eye on valuable business benefits… Safety and health is not just a legal requirement, it's integral in many of today's successful companies. It brings huge benefits for reputation, productivity and results.
It gives anyone with leadership responsibility the practical knowledge and solutions for sustainable business advantage through good safety and health practice.
Our Leading safely course has a stand-outfeature - the diagnostictool. This sets out where you are in terms of your current safety and health practice.
The course shows where you need to get to. Making a commitment to change is how you get there.
the benefits of effective safety leadership.
access to a safety leadership community, with news from IOSH, and up-to-date discussions on themes in safety leadership, from trainers and delegates.
IOSH Managing Safely introduces managers, at all levels, to the different aspects of managing health and safety in the workplace. It provides the knowledge and understanding to manage the health and safety issues within their responsibilities and reinforces why good health and safety is essential to performing their roles successfully.
IOSH Working Safely seeks to improve the safety culture within an organisation by giving individuals a good understanding of their health and safety responsibilities, and how to keep themselves and others safe at work.It focuses on why health and safety is important and how individuals can make a real difference to the well-being of themselves and others through changing behaviour.
Based on best practice rather than legislation, it's suitable for delegates from all countries and types of organisations.
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Working safely is for people at any level, in any sector, who need a grounding in the essentials of safety and health. It gives everyone at work an understanding of why they must 'work safely' – and does that in an enjoyable way.
Successful delegates are awarded an IOSH Working safely certificate.


text:
Subjects -> PHARMACY AND PHARMACOLOGY (Total: 575 journals)
First | 1 2 3 [Sort alphabetically] [Restore default list]
Showing 401 - 253 of 253 Journals sorted by number of followers
Pharmazeutische Zeitung (Followers: 11)
Pharmazeutische Industrie (Followers: 9)
Pharmaceutical Journal (Followers: 8)
Advanced Herbal Medicine (Followers: 8)
Pharmaceutical Fronts (Followers: 7)
Molekul (Followers: 7)
Advances in Pharmacology and Pharmacy (Followers: 6)
Drug Metabolism and Pharmacokinetics (Followers: 6)
AAPS Open (Followers: 5)
Journal of Drug Research in Ayurvedic Sciences (Followers: 4)
ACS Pharmacology & Translational Science (Followers: 4)
Advances in Traditional Medicine (Followers: 3)
Canadian Journal of Pain (Followers: 3)
PharmaNutrition (Followers: 3)
Journal of Drug Delivery Science and Technology (Followers: 3)
Journal of Herbal Science (Followers: 3)
Journal of Pharmacological Sciences (Followers: 3)
Indian Journal of Drugs in Dermatology (Followers: 2)
British Journal of Pharmacy (BJPharm) (Followers: 2)
Current Research in Drug Discovery (Followers: 2)
Journal of Herbal Medicine (Followers: 2)
Asian Journal of Pharmaceutical Research and Health Care (Followers: 2)
Pharmacological Research - Modern Chinese Medicine (Followers: 2)
Archives of Pharmacy and Pharmaceutical Sciences (Followers: 2)
Current Issues in Pharmacy and Medical Sciences (Followers: 2)
International Journal of Immunopathology and Pharmacology (Followers: 2)
Advances in Pharmacoepidemiology & Drug Safety (Followers: 2)
European Journal of Medicinal Plants (Followers: 2)
Journal of Pharmaceutical Sciences and Pharmacology (Followers: 2)
Journal of Education and Science (Followers: 2)
Drug Safety - Case Reports (Followers: 2)
Current Research in Pharmacology and Drug Discovery (Followers: 1)
Advances in Medical, Pharmaceutical and Dental Research (Followers: 1)
Pediatric Pharmacology (Followers: 1)
Journal of Metabolomics & Systems Biology (Followers: 1)
Regulatory Mechanisms in Biosystems (Followers: 1)
Integrative Medicine International (Followers: 1)
Journal of Applied Pharmaceutical Research (Followers: 1)
Herbal Medicines Journal (Followers: 1)
Farmakoèkonomika : Modern Pharmacoeconomic and Pharmacoepidemiology (Followers: 1)
Separation Science plus (SSC plus) (Followers: 1)
Advanced Therapeutics (Followers: 1)
Journal of Biopharmaceutics Sciences (Followers: 1)
Expert Review of Precision Medicine and Drug Development (Followers: 1)
Psychiatry and Clinical Psychopharmacology (Followers: 1)
Farmasains : Jurnal Ilmiah Ilmu Kefarmasian (Followers: 1)
Emerging Trends in Drugs, Addictions, and Health (Followers: 1)
Journal of Medicinal Plants for Economic Development (Followers: 1)
OpenNano (Followers: 1)
Safety and Risk of Pharmacotherapy (Followers: 1)
Journal of Pharmaceutical Technology, Research and Management (Followers: 1)
Journal of the American College of Clinical Pharmacy : JACCP (Followers: 1)
Open Pharmacoeconomics & Health Economics Journal (Followers: 1)
Pharmactuel (Followers: 1)
Al-Azhar Journal of Pharmaceutical Sciences (Followers: 1)
Chinese Herbal Medicines (Followers: 1)
Drugs : Real World Outcomes (Followers: 1)
Pharmakon : Arzneimittel in Wissenschaft und Praxis (Followers: 1)
Sustainable Chemistry and Pharmacy (Followers: 1)
Medicines (Followers: 1)
Antiviral Chemistry and Chemotherapy (Followers: 1)
Future Journal of Pharmaceutical Sciences (Followers: 1)
Journal of Pharmaceutical Health Care and Sciences (Followers: 1)
East and Central African Journal of Pharmaceutical Sciences (Followers: 1)
Ethiopian Pharmaceutical Journal (Followers: 1)
Pharmacy & Pharmacology (Followers: 1)
Clinical Complementary Medicine and Pharmacology
Exploratory Research in Clinical and Social Pharmacy
Indonesian Journal of Pharmaceutical Education
Future Drug Discovery
IUPHAR/BPS Guide to Pharmacology CITE
Journal of Applied Pharmaceutical Sciences and Research
Cephalalgia Reports
Pharmacon : Jurnal Farmasi Indonesia
Research Results in Pharmacology
Journal of Toxins
Journal of Pharmaceutics & Pharmacology
Natural Product Communications
PharmaTutor
International Journal of Pharmaceutical Sciences and Developmental Research
Current Medical Science
EUREKA : Health Sciences
Iraqi Journal of Pharmacy
Revista Colombiana de Ciencias Químico-Farmacéuticas
Medicine in Drug Discovery
Frontiers in Medical Technology
International Journal of Medical and Pharmaceutical Case Reports
Asian Journal of Research in Medical and Pharmaceutical Sciences
Journal of Pharmaceutical Health Services Research
AboutOpen
Current Protocols in Pharmacology
Medical Cannabis and Cannabinoids
Ukrainian Biopharmaceutical Journal
Pharmaceutical Journal of Sri Lanka
Isan Journal of Pharmaceutical Sciences (IJPS)
Jurnal Farmasi Sains dan Komunitas (Journal of Pharmaceutical Sciences and Community)
Toxicon : X
International Journal of Pharmaceutics: X
Jurnal Farmasi dan Ilmu Kefarmasian Indonesia
Open Pharmacology Journal
ExRNA
International Journal of Pharmaceutics & Pharmacology
Antibody Therapeutics
Journal of Faculty of Pharmacy of Ankara University
Iraqi Journal of Pharmaceutical Sciences
Journal of Medicinal Botany
Journal of Medicinal Herbs and Ethnomedicine
International Journal of Pharmacokinetics
Neuropsychopharmacology Reports
Reviews on Clinical Pharmacology and Drug Therapy
Matrix Science Pharma
Contract Pharma
Journal of Cellular Neuroscience and Oxidative Stress
Istanbul Journal of Pharmacy
Acta Pharmaceutica Indonesia
Indonesian Journal of Pharmacy
EMC - Cosmetologia Medica e Medicina degli Inestetismi Cutanei
Scientia Pharmaceutica
Trends in Peptide and Protein Sciences
Acta Physiologica Hungarica
Jurnal Farmasi Sains dan Praktis
Jurnal Kefarmasian Indonesia
Pharmaceutical Historian
Planta Medica International Open
Archives of Razi Institute
Journal of Pharmaceutical Research
EJNMMI Radiopharmacy and Chemistry
FarmaJournal
Journal of Negative and No Positive Results
Pharmaciana
Folia Medica Indonesiana
Актуальні питання фармацевтичної та медичної науки та практики
International Journal of Pharmacology, Phytochemistry and Ethnomedicine
Фармацевтичний часопис
Ciência Equatorial
Iranian Journal of Pharmaceutical Research
Research & Reviews : A Journal of Drug Design & Discovery
Asian Journal of Medical and Pharmaceutical Researches
Research & Reviews : A Journal of Pharmacognosy
Ars Pharmaceutica
Pharmacognosy Communications
Egyptian Pharmaceutical Journal
Pharmacological Reports
PZ Prisma : Materialien zur Fort- und Weiterbildung
Chinese Journal of Pharmaceutical Analysis
Journal of Nanopharmaceutics and Drug Delivery
Journal of Hydrogels
Manufacturing Chemist
Pharmaceutica Analytica Acta
Journal of Pharmacovigilance
Current Pharmacology Reports
Nigerian Journal of Natural Products and Medicine
International Journal of Herbs and Pharmacological Research
Bangladesh Journal of Physiology and Pharmacology
Research Journal of Pharmacognosy
HOME > Browse by Subject
Drug Safety - Case Reports
Journal Prestige (SJR): 0.216
ISSN (Print) 2199-1162 - ISSN (Online) 2198-977X
Published by Springer-Verlag [2467 journals]
Acknowledgement to Referees
PubDate: 2019-11-16
Acute Kidney Injury Following High-Dose Methotrexate Administration in a
Day Care Hospital
Abstract: Abstract Day care is a potential alternative to inpatient care of cancer patients. Using day care reduces medical costs substantially compared to inpatient care, which is driving the transfer of many inpatient chemotherapy protocols to day care hospitals (DCHs). However, in contrast to inpatient management, day care provides limited observation time, which could increase the risk of renal toxicity when using these protocols. We present a case report of acute kidney injury following high-dose methotrexate administration in a DCH. Based on a review of the current literature on acid-base balance, we also discuss appropriate patient selection and judicious hydration and urine alkalinization so as to prevent toxicity in the day care setting.
Fournier's Gangrene in a Patient with Type 2 Diabetes Mellitus Treated
with Empagliflozin: A Case Report
Abstract: Abstract Sodium-glucose cotransporter-2 (SGLT2) inhibitors have been reported as possibly associated with Fournier's gangrene (FG). This case report describes a 34-year-old Japanese man who was diagnosed with FG after the administration of empagliflozin for type 2 diabetes mellitus (T2DM). He presented with pain and swelling in the perineum and groin 142 days after initiating empagliflozin. The clinical features, laboratory data, and computed tomographic findings were consistent with FG. Surgical drainage and debridement of necrotic tissues were performed immediately after admission to our hospital. The patient had no complications of diabetes before the onset of FG. Glycemic management was good at the time of FG onset. This case suggests a possible association between empagliflozin and FG. We report a case of FG in a patient during a period of good glycemic management following treatment with empagliflozin. We recommend further awareness of this relationship and suggest the need for additional research.
A Case of Oropharyngeal Angioedema Following Intravenous Recombinant
Tissue Plasminogen Activator (rt-PA) and Mechanical Thrombectomy
Abstract: Abstract A 72-year-old housewife presented with ischemic cerebrovascular stroke. Intravenous thrombolysis using recombinant tissue plasminogen activator (rt-PA) followed by mechanical thrombectomy under general anesthesia were attempted. The patient developed stridor and tongue swelling, in addition to hypotension and bradycardia, 60 min after completion of the rt-PA infusion. The airway was intubated, and intramuscular adrenaline, together with intravenous hydrocortisone and diphenhydramine, were administered. On the second day, the tongue edema subsided, and the cuff leak test was negative. However, extubation was not attempted due to the development of brain edema. A tracheostomy was later performed, and the patient was weaned off mechanical ventilation.
Acute Kidney Injury Following Exposure to Calcineurin Inhibitors in a
Patient with Idiopathic Membranous Nephropathy
Abstract: Abstract Membranous nephropathy (MN) is one of the most common causes of nephrotic syndrome in non-diabetic adult patients; 75% of adult patients with MN suffer from primary idiopathic membranous nephropathy (IMN). The treatment of choice is immunosuppressive therapy, with a combination of steroids and cyclophosphamide (CYF) or chlorambucil or, as second-line treatment, calcineurin inhibitors (CNIs). One of the main concerns associated with the usage of CNIs is their potential to induce nephrotoxicity. We report a case of acute kidney injury that developed on two separate occasions within days of the administration of CNIs in a 57-year-old male patient treated for MN. The patient was qualified for first-line treatment with prednisone and CYF. Due to insufficient response and bad tolerance of CYF infusions, the immunosuppressive regimen was modified and CNIs were introduced, starting with cyclosporine A (CsA). On the third day of treatment, a severe decrease in diuresis and kidney function occurred and CsA was discontinued, resulting in a return to baseline kidney function. After 2 months, the situation repeated after attempting to introduce tacrolimus.
Allopurinol-Induced Toxic Epidermal Necrolysis
Abstract: Abstract Toxic epidermal necrolysis (TEN) is an extremely rare condition characterized by separation of dermoepidermal junctions, necrosis, and subsequent detachment of the epidermis over large cutaneous areas. TEN can emerge after exposure to certain medications such as allopurinol, aromatic anticonvulsants, NSAIDs, nevirapine, and antibacterial sulfonamides. There is no standard protocol for TEN, and the therapy of choice varies from one patient to another. Some of these therapies include silver-releasing wraps/dressings, glucocorticoids, antibodies to inhibit Fas-mediated keratinocyte apoptosis, and cyclosporine A. A 35-year-old male with an allergy to antibacterial sulfonamides who was being treated for arterial hypertension and hyperuricemia with captopril and allopurinol, respectively, was admitted to hospital. The patient showed skin detachment affecting approximately 95% of his surface area, including his face, upper and lower extremities, trunk, back, oropharyngeal mucosa, anal mucosa, ocular mucosa, and genital mucosa. Intravenous methylprednisolone at a dosage of 40 mg/day for 7 days along with abrasive cures was found to be an appropriate treatment in this case.
Isoniazid-Induced Systemic Lupus Erythematosus: A Case Report
Abstract: Abstract Systemic lupus erythematosus (SLE) can be induced by various medications, such as hydralazine, procainamide, isoniazid, methyldopa, chlorpromazine, quinidine, and minocycline. A patient was admitted complaining of fever with chills and rigor. After being diagnosed with tuberculous meningitis, the patient was given antituberculosis treatment. As the patient did not improve, detailed investigations were conducted, and elevated antinuclear antibody levels were found. The consulting physician diagnosed that the patient was suffering from SLE. As isoniazid is associated with an increased risk of developing SLE, it was suspected as the culprit drug. After withdrawing isoniazid from the antituberculosis treatment regimen, the patient improved and was discharged. Based on the WHO-UMC and Naranjo's causality assessment criteria, an association between the reaction and isoniazid was deemed probable. The reaction was moderately severe (level 4b) according to the modified Hartwig and Siegel scale.
DOI: 10.1007/s40800-019-0102-y
Fatal Vitamin K-Dependent Coagulopathy Associated with
Cefoperazone/Sulbactam: A Case Report
Abstract: Abstract This case report describes a suspected and fatal adverse reaction involving vitamin K-dependent coagulopathy that might be associated with cefoperazone/sulbactam (CPZ/SAM), a combined antimicrobial formulation. We reported a patient diagnosed with acute cerebral infarction and secondary pulmonary infection who was treated with an intravenous infusion of CPZ/SAM at 3 g twice daily. After receiving treatment with CPZ/SAM, the patient developed a fatal adverse reaction of CPZ-induced hemorrhage. The Naranjo assessment score in this report was 5, suggesting that the patient's coagulation function disorder was potentially associated with the use of CPZ/SAM. To prevent vitamin K-dependent coagulopathy caused by CPZ/SAM, it is suggested to avoid cephalosporins in patients with a high risk of bleeding unless the need for cephalosporins is compelling.
Gastroschisis Following Treatment with High-Dose Methimazole in Pregnancy:
A Case Report
Abstract: Abstract Hyperthyroidism in pregnancy is associated with a increased incidence of low birth weight, preterm birth and admission to the neonatal intensive care unit. However, available treatment options are limited. In this report, we present a case of fetal gastroschisis with a history of intrauterine exposure to methimazole. A 37-year-old woman was diagnosed with Grave's disease 3 years before her pregnancy. She had a poor response to propylthiouracil and required high-dose methimazole before her pregnancy. During the first trimester, she received methimazole 120 mg/day. After her 12th week of pregnancy, she received block-and-replace therapy (levothyroxine [LT4] 50 µg/day) because of the risk of hypothyroidism, and the dose of methimazole was downtitrated to 60 mg/day. Fetal ultrasonography showed fetal growth retardation and gastroschisis at gestational week 33. The relationship between the very high doses of methimazole in the first trimester of pregnancy and the incidence of gastroschisis in this patient was not fully understood because evidence of a relationship between the use of antithyroid drugs in the first trimester and congenital abnormalities in the fetus is lacking. Furthermore block-and-replace therapy is not recommended in pregnancy because it requires a higher dose of methimazole. We recommend preconception counseling and early screening of thyroid function. The counseling should include the best timeline for pregnancy and a discussion of the risks and benefits of hyperthyroidism treatment options.
Anaphylactic Reaction to Tc-99m Macrosalb
Abstract: Abstract A 49-year-old woman developed an anaphylactic reaction to Tc-99m macrosalb used for pulmonary scintigraphy. The patient received an intravenous injection of Tc-99m macrosalb 120 MBq, containing macroaggregates of human albumin 0.14 mg. Within 1 min she developed itching all over her body, an itching throat and dyspnoea. This was followed by urticaria and facial oedema. She was diagnosed with an anaphylactic shock. The patient received clemastine and prednisone, and fully recovered after release from the hospital. According to the Naranjo assessment algorithm, the relationship between the allergic reaction and the administration of Tc-99m macrosalb should be considered as 'probable'.
Ibrutinib Caused Mediastinal Emphysema and Pneumothorax in the Treatment
of a Patient with Mantle Cell Lymphoma
Abstract: Abstract A 70-year-old Japanese man with mantle cell lymphoma underwent extensive chemotherapy and radiation because of the relapse of mantle cell lymphoma. He developed mediastinal emphysema and a pneumothorax 14 days after treatment with 560 mg of ibrutinib. The mediastinal emphysema and the right pneumothorax disappeared after the ibrutinib treatment was tapered off. The patient developed interstitial pneumonia without any infection and new lesions of mantle cell lymphoma in the lungs after restarting treatment with 560 mg of ibrutinib. In this case, the patient developed pneumonia after retreatment with ibrutinib, suggesting the small lung fibrosis that penetrated the mediastinum might have caused the emphysema and pneumothorax.
Acute Exacerbation of Fever Following Administration of Tropicamide and
Phenylephrine Ophthalmic Solution: A Case Report
Abstract: Abstract A 24-year-old woman with atopic dermatitis and persistent fever (axillary temperature of 37–38 °C for 6 months) received combination ophthalmic drops containing tropicamide and phenylephrine (Mydrin®-P), which exacerbated her fever within 15 min after instillation. Her axillary fever reached 40.1 °C but resolved the following day. No new dermatological symptoms developed. Although the patient's fever may have been caused by either tropicamide or phenylephrine, neither of which have been reported to induce fever in topical formulations, atopic dermatitis and tropicamide's inhibitory effect on perspiration under hot and humid conditions may have been the more probable cause. While drug-induced fever has been reported for other ophthalmic anticholinergic agents, this is the first reported case of possible fever exacerbation by an ophthalmic formulation of tropicamide, if the causative agent is assumed to be tropicamide.
An Interesting Case of Carbamazepine-Induced Stevens–Johnson
Abstract: Abstract A 29-year-old Black female patient was admitted to a psychiatric ward with symptoms of major depressive disorder with psychosis. The patient was started on amitriptyline 50 mg/day and haloperidol 10 mg/day. On day 4 post-admission, the preferred first-line antidepressant, fluoxetine, became available and the patient was switched from amitriptyline to fluoxetine 20 mg/day. On the same day, the dose of haloperidol was reduced to 5 mg/day. Thirteen days post-initiation of these medications the patient became talkative, associated with emotional lability, an expansive mood, irritability and restlessness. The working diagnosis was changed to bipolar affective disorder in the manic phase. Fluoxetine was discontinued and carbamazepine 600 mg/day was added to the patient's treatment regimen. Her manic symptoms started to resolve; however, 14 days post-initiation of carbamazepine, the patient had a fever; itchy, discharging eyes; respiratory distress; generalised symmetrical erythematosus rash; buccal ulceration; and conjunctival injection with difficulty opening her eyes. Carbamazepine was immediately discontinued and the patient received intravenous fluid resuscitation. The patient recovered considerably after 12 days of symptomatic and supportive management, and was transferred back to the psychiatric ward for the continuation of bipolar disorder management. Lithium therapy was instituted and the patient was subsequently discharged. Using the Algorithm of Drug causality for Epidermal Necrolysis (ALDEN) Stevens–Johnson Syndrome/toxic epidermal necrolysis (SJS/TEN) drug causality scoring system, carbamazepine and fluoxetine were evaluated as 'very probable' and 'possible' causes of SJS, respectively, in this patient. Fluoxetine-induced SJS was considered on account of previous case reports, however no evidence of causality was found in this patient. Consecutive administration with a potential increase in carbamazepine due to inhibition of cytochrome P450 (CYP) 3A4 metabolism by fluoxetine was also not ruled out. A diagnosis of carbamazepine-induced SJS was made and was considered an idiosyncratic adverse drug reaction.
Neutropenia and Thrombocytopenia Induced by Proton Pump Inhibitors: A Case
Abstract: Abstract An 85-year-old man was admitted to our hospital because of dysphagia, and was diagnosed with benign stricture of the esophagus. He was hospitalized repeatedly for balloon dilations. Pantoprazole sodium (80 mg, twice daily, intravenously) was administered each time when he was in hospital, while esomeprazole (20 mg/day, orally) was administered intermittently when he was at home. Reductions in both white blood cells and platelets were noticed about 4 months after proton pump inhibitors were introduced. Bone marrow suppression induced by proton pump inhibitors was diagnosed as proven by bone marrow biopsy. White blood cell, neutrophil, and platelet counts went back to the normal range after proton pump inhibitors were stopped. The present case shows a rare bi-cytopenia associated with proton pump inhibitors and suggests the importance of awareness of hematological adverse events during proton pump inhibitor therapy.
Curling of Hair in Two Female Patients Taking Alitretinoin
Abstract: Abstract Two female patients aged 45 and 51 years experienced curling of hair during treatment with alitretinoin (Toctino®). For one patient, the indication for use was severe chronic hand eczema, but for the second patient the indication was not reported. After 5 and 9 months, respectively, the patients developed hair texture changes and their straight hair began to curl. The dose of alitretinoin was reduced in both cases, but the patients' hair had not straightened at the time of reporting (9 months and 2 years after onset of the event). Based on the described reports received by the Netherlands Pharmacovigilance Centre Lareb, the case reports in the literature, and the possible mechanisms, we suggest a causal relationship between curling of the hair and the use of alitretinoin. Using the World Health Organization Uppsala Monitoring Centre (WHO-UMC) system for standardized case causality assessment, the association in our cases can be assessed as likely.
Acute Myocardial Infarction Following Administration of Polyethylene
Glycol Electrolyte Solution with Ascorbic Acid (MoviPrep ® ) at Home: A
Abstract: Abstract A male individual aged 82 years with hypertension who had a smoking history, but no history of cardiovascular events, developed acute myocardial infarction immediately after he took oral polyethylene glycol electrolyte solution with ascorbic acid as a pretreatment for a colonoscopy to examine anemia. He took polyethylene glycol electrolyte solution with ascorbic acid at twice (2 L/h) the rate recommended in the package insert and by the physician. The patient showed impaired consciousness 2 h after taking polyethylene glycol electrolyte solution with ascorbic acid and his family called the emergency medical service. Upon arrival of the emergency medical service, his systolic blood pressure was 60 mmHg and heart rate was 50 bpm. Systolic blood pressure and impaired consciousness were slightly improved, but compensatory shock remained, at arrival at the emergency outpatient service at our hospital. No dyspnea or rash was apparent. The patient had no subjective chest pain; however, ST-segment elevation was detected in the electrocardiogram at II, III, aVF, V3R, and V4R. He was diagnosed with ST-segment elevation myocardial infarction and underwent a coronary catheter intervention for total occlusion of the right coronary artery. His shock state was abolished by this intervention. The patient was pretreated with polyethylene glycol electrolyte solution with ascorbic acid under close watch in the coronary care unit 4 days later, with no relapse of symptoms. Advanced cancer was found in the ileocecum by colonoscopy; consequently, the patient underwent a colectomy and was discharged from our hospital and transferred to another hospital for rehabilitation on hospital day 74. A Naranjo assessment score of 4 was obtained, indicating a possible relationship of acute myocardial infarction with misuse of the suspect drug, polyethylene glycol electrolyte solution with ascorbic acid.
Metoclopramide: A Safe Alternative to Domperidone' A Case Report on
Severe Cardiac Adverse Effects in an Older Patient
Abstract: Abstract Peripheral antidopaminergic medication is frequently prescribed to treat nausea. However, domperidone is ill-famed for its severe cardiac adverse effects. Metoclopramide has been suggested as a relatively safe alternative because it has long been considered to have less significant cardiovascular adverse effects. We present an older patient who developed severe bradycardia and hypotension shortly after receiving intravenous metoclopramide. Cardiac adverse effects of metoclopramide in elderly are not frequently described in the literature, especially not in patients without a major history of cardiac disease. We recommend caution with intravenous administered metoclopramide in older patients.
Antituberculosis Drug-Induced Fixed Drug Eruption: A Case Report
Abstract: Abstract Fixed drug eruption (FDE) was caused by fixed-dose combination (FDC) of antituberculosis drugs in the form of tablet Forecox® (rifampicin [rifampin] 225 mg + isoniazid 150 mg + pyrazinamide 750 mg + ethambutol 400 mg) in a 40-year-old male patient with a history of drug allergy. The patient developed FDE after taking the third dose of tablet Forecox® for pulmonary tuberculosis. Tablet Forecox® was withdrawn and the patient recovered from the reaction after 15 days of treatment for FDE. As per World Health Organization–Uppsala Monitoring Centre (WHO-UMC) and Naranjo causality assessment criteria, the association between the reaction and tablet Forecox® was possible and probable, respectively. The reaction was moderately (Level 4b) severe according to the Modified Hartwig and Siegel scale. As there is an increased risk of allergic reaction in patients with a history of drug allergy, FDCs should not be used in order to avoid complexity in identifying the culprit drug.
Secondary Hypogammaglobulinemia After Rituximab for Neuromyelitis Optica:
Abstract: Abstract A 17-year-old male with history of neuromyelitis optica and seizures presented to the pulmonology clinic for evaluation of recurrent pneumonias. He had received rituximab for the past 6 years. Over the past 2 years, he experienced four episodes of pneumonia. In between these episodes, he would improve briefly but continued to have daily cough that was productive with yellow phlegm. He also had recurrent rhinitis and sinusitis despite multiple antibiotic courses. Review of chest X-rays revealed localized right middle lobe and right lower lobe infiltrates. An extensive workup was performed, including computed tomography (CT) of the chest and bronchoscopy to rule out congenital lesions of the right lung and foreign body aspiration. Chest CT showed right lower lobe bronchiectasis. Flexible bronchoscopy with bronchoalveolar lavage showed normal anatomy with thick mucus secretions in the right lower lobe. Immunologic evaluation was performed and revealed low levels of immunoglobulin (Ig)-G, IgM, and IgA, which had declined since initiation of rituximab. Lymphocyte subset testing was remarkable for low cluster of differentiation (CD)-19. He was referred to allergy and immunology and was initiated on immunoglobulin-replacement therapy (IGRT) for acquired hypogammaglobulinemia secondary to rituximab. There was marked clinical improvement after initiation of IGRT.


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text:
Lisa A. Porter
University of Windsor, Canada
Ashley E. Webb
Brown University, United States
Hua Bai
Iowa State University, United States
Open supplemental data
BRIEF RESEARCH REPORT article
Front. Cell Dev. Biol., 05 February 2021
Sec. Cell Growth and Division
FoxO1 Is a Novel Regulator of 20S Proteasome Subunits Expression and Activity
Marianna Kapetanou1, Tobias Nespital2, Luke S. Tain2, Andre Pahl2, Linda Partridge2* and Efstathios S. Gonos1*
1Laboratory of Molecular and Cellular Aging, Institute of Chemical Biology, National Hellenic Research Foundation, Athens, Greece
2Department of Biological Mechanisms of Ageing, Max Planck Institute for Biology of Ageing, Cologne, Germany
Proteostasis collapses during aging resulting, among other things, in the accumulation of damaged and aggregated proteins. The proteasome is the main cellular proteolytic system and plays a fundamental role in the maintenance of protein homeostasis. Our previous work has demonstrated that senescence and aging are related to a decline in proteasome content and activities, while its activation extends lifespan in vitro and in vivo in various species. However, the mechanisms underlying this age-related decline of proteasome function and the down-regulation in expression of its subunits remain largely unclear. Here, we demonstrate that the Forkhead box-O1 (FoxO1) transcription factor directly regulates the expression of a 20S proteasome catalytic subunit and, hence, proteasome activity. Specifically, we demonstrate that knockout of FoxO1, but not of FoxO3, in mice severely impairs proteasome activity in several tissues, while depletion of IRS1 enhances proteasome function. Importantly, we show that FoxO1 directly binds on the promoter region of the rate-limiting catalytic β5 proteasome subunit to regulate its expression. In summary, this study reveals the direct role of FoxO factors in the regulation of proteasome function and provides new insight into how FoxOs affect proteostasis and, in turn, longevity.
Proteostasis is a pivotal process indispensable for the majority of cellular functions, including DNA replication, the regulation of the cell cycle, metabolism, maintenance of cellular architecture, signaling pathways, development and immune responses (Powers et al., 2009; Labbadia and Morimoto, 2015). Proteostasis collapse has been documented as a key factor contributing to the progression of aging (López-Otín et al., 2013), caused by a gradual failure of the respective defense systems (Taylor and Dillin, 2011). Furthermore, several studies have demonstrated that chronic exposure to aggregated or denatured proteins contributes to the development of age-related diseases, such as Alzheimer's and Parkinson's disease (Chondrogianni et al., 2015b; Labbadia and Morimoto, 2015). The proteasome plays a pivotal role in maintaining proteostasis and as such, is involved in a multitude of biological processes (Vilchez et al., 2014). The 30S/26S proteasome is the main proteasome complex consisting of the 19S regulatory "cap" and the 20S catalytic "core" (single capped: 26S, double capped: 30S). The 20S core proteasome has barrel-like configuration and is comprised by seven different α subunits and seven distinct β subunits. Three β subunits, namely β1, β2, and β5, possess proteolytic activities with different substrate specificities (Chondrogianni et al., 2014). Our previous work has established a direct association between proteasome-mediated proteolysis and aging. Specifically, we have demonstrated that the accumulation of damaged proteins during aging is connected to an age-related downregulation of proteasome expression and activity. In addition, pharmacological or genetic induction of the proteasome improves both cellular and organismal lifespan and alleviates the pathological phenotype of protein aggregation-related diseases, such as Alzheimer's disease (Chondrogianni et al., 2015b; Mladenovic Djordjevic et al., 2021). Moreover, we have shown that human mesenchymal stem cells (hMSCs) exhibit a senescence-related decline of proteasome content and aberrations in physiological assembly of proteasome complexes during prolonged in vitro expansion, while proteasome activation via overexpression of the catalytic β5 subunit can enhance their stemness and lifespan (Kapetanou et al., 2017). Therefore, we hypothesize that the mechanisms and molecular factors that control proteasome subunit expression are crucial regulators of longevity. However, the respective underlying mechanisms remain largely obscure.
Numerous studies have identified an extensive array of genes that can alter the lifespan of several organisms. Despite this enormous volume of research, we still do not completely comprehend how these genes influence the aging process of an organism (Flatt and Partridge, 2018). A proteomic study in D. melanogaster (Tain et al., 2017) and a genetic approach in C. elegans (Vilchez et al., 2012) have suggested a potential interplay between the regulation of the proteasome and the Forkhead box-O (FoxO) transcription factors. FoxO factors control several cellular processes like autophagy and apoptosis in response to signals emanating from the environment and are important longevity determinants, downstream of insulin and insulin-like growth factor signaling (IIS). FoxO factors, under conditions of low IIS, translocate into the nucleus and bind to promoters of pro-longevity genes to regulate transcription. Nevertheless, their complex role in life-expectancy determination has not been fully elucidated yet. Here, we have dissected further the mechanism of IIS action on mammalian proteasome regulation and demonstrate for the first time that FoxO1 directly regulates the expression and activity of the 20S proteasome.
Mouse Models and Husbandry
All mice were maintained at 22°C under a 12-h light/dark cycle (lights on from 7:00 am to 7:00 pm). Mice were housed in groups of three to five same-sex littermates under specific pathogen-free conditions within individually ventilated cages (Tecniplast UK Ltd., Kettering, Northamptonshire, United Kingdom). Mice had ad libitum access to normal chow [ssniff® R/M-H phytoestrogen-poor (9% fat, 34% protein, and 57% carbohydrate) ssniff Spezialdiäten GmbH, Soest, Germany] and water. Irs1 global knockout mice were generated as described previously (Selman et al., 2008). The conditional FoxO1 (from Ron DePinho, MD Anderson Cancer Center) total knockouts were induced by tamoxifen treatment for 6 weeks using a ROSA26-CreERT2-mediated recombination (Supplementary Figure 1, mouse line from Thomas Langer, MPI for Biology of Aging) (Paik et al., 2007). Foxo1 (n = 8, 6 females and 2 males) and Irs1 (n = 7, females) KO and their littermate controls (+/+, n = 7, 5 females and 2 males and n = 7, females, respectively) were sacrificed at 16 weeks. Foxo3 total knockouts were generated by using germline Cre-mediated recombination (actin-Cre) with the conditional Foxo3 allele (from Ron DePinho, MD Anderson Cancer Center) producing the null allele. Mice were dissected and tissues were snap-frozen in liquid nitrogen.
Mouse Embryonic Fibroblast Isolation and Culture
Primary mouse embryonic fibroblasts (MEFs) were isolated from Irs1+/+ or Irs1−/− animals and cultured according to standard procedures (Qiu et al., 2016). Briefly, MEFs were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (v/v; Invitrogen), 2 mM glutamine and 1% non-essential amino-acids at 37°C, 5% CO2 and 95% humidity.
Reagents and Antibodies
LLVY-AMC, MG132 and the primary antibody against the proteasomal subunits β5 (X, MB1, ϵ; PW8895; 22.9 kDa) and α6 (C2; PW8100; 33 kDa) were purchased from Enzo Life Sciences, Inc. The ChIP Grade antibody against FoxO1 (ab39670) and HRP-conjugated anti-rabbit and anti-mouse antibodies were purchased from Abcam.
Proteasome Peptidase Assays
Liver and brain tissues were lysed in 25 mM Tris/HCl lysis buffer, pH 7.6 containing 5 mM ATP, 10% glycerol, 20 mM KCL, 1 mM EDTA, 1 mM DTT, 0.2% Nonidet P-40, 10 mM phenylmethylsulfonyl-fluoride and 10 μg/ml aprotinin (Rivett et al., 1994). The CT-L activity of the proteasome was assayed after the incubation of 10 μg of total protein for 30 min at 37°C with the fluorogenic peptide LLVY-AMC, as previously described (Georgila et al., 2014). Proteasome activity was determined as the difference between total fluoresce and fluoresce in the presence of 20 μM of the proteasomal inhibitor MG132. AMC fluorescence was measured at 360 nM excitation and 460 nM emission using a spectrofluorimeter (Tecan). A Bradford assay was used to determine protein concentration, using bovine serum albumin as standard.
Twenty μg protein of isolated protein were separated by 10% SDS-PAGE under non-reducing conditions according to standard procedures (Palmer, 2000). Following electrophoresis, protein loading was analyzed using the Stain-freeTM (Bio-Rad) imaging technology that allows the visualization of the proteins directly in the gel after a short photoactivation. Proteins were then transferred to nitrocellulose membrane (Amersham Biosciences) to be treated with the blocking buffer and were subsequently incubated with the appropriate antibodies. The primary antibodies were detected with horseradish peroxidase conjugated secondary antibodies. The detection with enhanced chemiluminescence was performed using ECL or ECL prime chemiluminescence kits (GE Healthcare) and a ChemiDoc station (Bio-Rad).
Real Time PCR Analysis
For the characterization of proteasome genes, total RNA was isolated using TRIzol (Invitrogen) and transcripted into cDNA with the cDNA iScript synthesis kit (Bio-Rad). The Real time PCR were run on the CFX Connect Real-Time PCR System (Bio-Rad). The RT-PCR primers are summarized in Supplementary Table 1. For the evaluation of FoxO1 knockout efficiency, RNA was isolated using TRIzol (Thermo-Fisher), treated with DNAse (Qiagen) and purified by isopropanol precipitation. cDNA was prepared using the SuperScript III reverse transcriptase kit (Invitrogen) as per manufacturer's instructions. TaqMan probes against Foxo1 and beta2-microglobulin were obtained from Applied Biosystems and run on a 7900HT real-time PCR system.
ChIP Analysis
Chromatin immune precipitation experiments were performed using the ChIP-IT® Express Enzymatic kit (Activemotif, cat. no 53009), as per manufacturer's instructions. Chromatin was sheared enzymatically for 5 min and precipitated with a ChIP-Grade Anti-FOXO1 antibody (ab39670, Abcam). Prior to amplification, the samples were subjected to DNA clean-up step using the Nucleospin R Gel and PCR Clean-up kit (740609.10, Macherey-Nagel). The ChIP products were then analyzed by Real-time PCR and the products were confirmed by agarose gel electrophoresis. An anti-IgG antibody was used as a negative control.
The small interfering (si)RNAs targeting murine FoxO1 and IRS1 were obtained from Thermo Fisher Scientific (s80620) and Sigma (EMU061331 MISSION® esiRNA), respectively. The siRNA targeting murine FoxO3 has been purchased from Thermo Fisher Scientific (Silencer®, 100380). Briefly, the siRNA duplexes were transfected into 70–80% confluent MEFs cultured in a in a 6-well or 96-well plate format at a final concentration of 50 nM, in presence of the TransFectinTM Lipid Reagent (1703351, Bio-Rad) at a ratio of 1:2. Transfection complexes were prepared in Opti-MEM® Reduced Serum Medium, GlutaMAXTM Supplement (51985-034, Thermo Fisher Scientific). After 24 h, the cells were harvested or transfected with the LightSwitchTM plasmids without media change.
FoxO1 Activity Assay
The isolation of nuclear proteins and the subsequent examination of FOXO1 transcriptional activity were performed with the Nuclear Extraction Kit (ab113474, Abcam) and the FOXO1 Transcription Factor Assay Kit (Colorimetric; ab207204, Abcam), respectively, according to manufacturer's instructions.
The promoter region of β5 and a mutagenized version (Supplementary Table 2) were cloned into the LightSwitchTM Promoter Reporter Vector (Active Motif). MEFs were transfected using the Transfectin reagent (1703351, Bio-Rad) and the luciferase assay was performed with the LightSwitchTM Luciferase Assay Kit (32031, Active Motif) in quadruplicates, according to manufacturer's instructions. Each well was read for 10 s and the signal of the empty vector (32021, Active Motif) was deducted from the values of sample wells.
This study was performed in strict accordance with the recommendations and guidelines of the Federation of European Laboratory Animal Science Associations (FELASA). The protocol was approved by the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen.
In silico Identification of FoxO Binding Motifs
Genomic DNA sequences were downloaded in FASTA format from www.ensembl.org/ and individual matches of known FoxO binding motifs were scanned using FIMO1 in 1 kb regions upstream of the TSS of murine 20S proteasome genes.
Statistical Analysis and Quantifications
Statistical analysis and the graphical representation of data was performed using the GraphPad Prism 5 (GraphPad Software, San Diego, CA, United States). All values were reported as mean ± SE, unless otherwise indicated. Densitometry analysis for the quantification of immunoblots was performed with Bio-Rad's Image Lab software 6.0.1. The average signal of control mice was arbitrarily set to 100% or 1.
Foxo1 Knockout Mice Exhibit Reduced Proteasome Activity and Expression in Liver and Brain
Firstly, we evaluated the proteasome status in various tissues of Foxo1 KO mice sacrificed at 16 weeks of age, in comparison to their respective control animals. We found that Foxo1 mutant mice displayed a substantial decrease in chymotrypsin like (CT-L) activity. Specifically, the data for liver and brain that are shown in Figure 1A and Supplementary Figures 2A,3A revealed a statistically significant reduction of proteasome activity by 25.8% and 22.6% in liver and brain, respectively. In support, the mRNA (Figure 1B) and protein levels (Figure 1C and Supplementary Figures 2B,3B) of representative β and α 20S subunits (catalytic β5 and α6) declined considerably in absence of FoxO1 in these tissues. However, FoxO3 depletion affected neither proteasome expression nor its activity (Supplementary Figure 6).
Figure 1. FoxO1 depletion downregulates the proteasome: (A) Mean% CT-L activities, (B) mRNA expression, and (C) Mean values immunoblot analysis of the indicated proteasome subunits in the liver and brain of the indicated control and FoxO1-depleted mice. The total protein load was used as a control for equal protein loading and GAPDH was used for RT-PCR normalization. 100% or 1 has been arbitrarily set to the average values of the control samples. Data information: *P < 0.05, **P < 0.01, ***P < 0.001. All error bars show SEM. Number of animals: Foxo1–/– n = 8, 6 females and 2 males and Foxo1+/+ n = 7, 5 females and 2 males.
Irs1 Global Knockout Enhances Proteasome Expression and Function
FoxO factors are activated when IIS activity is low to induce the expression of their target genes. To examine the effect of FoxO activation on the proteasome-mediated proteolysis, we characterized the proteasome status of the long-lived Irs1 KO mice (sacrificed at 16 weeks of age) and observed that the CT-L activity was increased (see, Figure 2A and Supplementary Figures 4A,5A) by 55.3% in the liver and by 88.2% in the brain. Additionally, there was a consistent pattern of induced mRNA (Figure 2B) and protein expression (Figure 2C and Supplementary Figures 4B,5B) of the β5 subunit in Irs1 KO mice in comparison to their relative control littermates. However, the expression of α6 was not consistently upregulated by irs1 knockdown, suggesting that IIS has a more prominent role in the regulation of β5.
Figure 2. Global Irs1 knockdown induces the proteasome: (A) Mean% CT-L activities, (B) mRNA expression and (C) Mean values immunoblot analysis of β5 and a6 proteasome subunits, in the liver and brain of the indicated control and IRS1-depleted mice. The total protein load was used as a control for equal protein loading and GAPDH was used for RT-PCR normalization. 100% or 1 has been arbitrarily set to the average values of the control samples. Data information: *P < 0.05, **P < 0.01, ***P < 0.001. All error bars show SEM. Number of animals: Irs1–/– n = 7, females and Irs1+/+, n = 7, 5 females and 2 males.
FoxO1 Directly Binds to the Murine Promoter Region of β5
Our data indicated that the expression of 20S proteasome subunits is positively regulated by FoxO1. To shed light on these observations, we performed IRS1, FoxO1, FoxO3 and double IRS1 + FoxO1 and IRS1 + FoxO3 silencing assays, using small interfering RNAs and tested the CT-L proteasome activity and the levels of the relevant β5 subunit. In support to the described in vivo data, IRS1 silencing led to doubled CT-L activity (Figure 3A) as well as to significantly increased β5 levels (Figure 3B), while FoxO1 silencing led to a downregulation of both proteasome activity and β5 content. In addition, the beneficial effects of lowered IRS1 levels on the proteasome were mediated by FoxO1 as double IRS1 + FoxO1 silencing abolished the increase in CT-L activity and β5 expression. In contrast, we observed similar effects of IRS1 and double IRS1 + FoxO3 silencing on proteasome status. Notably, we confirm that IRS1 silencing significantly enhances FoxO1 transcriptional activity (Figure 3C).
Figure 3. FoxO1 binds to the murine β5 promoter to regulate gene expression. (A) % CT-L activities, (B) Immunoblot analysis of the β5 subunit and (C) FoxO1 activity in the presence of siRNAs targeting IRS1, FoxO1, FoxO3 or control as indicated. 100% has been arbitrarily set to the average values of the control samples and the total protein load was used as a control for equal protein loading. (D) Schematic diagram of the promoter region of β5 subunit. The green area indicates the amplified chromatin immunoprecipitation products, while the tab indicates the speculatory FoxO binding site. (E) Chromatin immunoprecipitation analysis of the β5 promoter region from control or Irs1 depleted MEFs, using an anti-FoxO1 antibody. The amount of immunoprecipitated DNA was evaluated by RT-PCR. Histogram was constructed by the ratios of the immunoprecipitated DNA to the input (arbitrarily set to 100%). (F) Luciferase signal driven by the proximal wild type β5 promoter region (pβ5-lightswitch) or by a mutagenized version (mut-pβ5-lightswitch) with 8 bp substitutions on the detected FoxO binding motif in the presence of siRNAs targeting FoxO1, IRS1 or control as indicated. The signal of the empty vector has been deducted from raw values. The luminescence of wild type promoter under control conditions has been arbitrarily set to 1. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
To determine if proteasome subunit promoters are occupied by FoxO1 in vivo, we analyzed several putative binding sites of FoxO1 containing its consensus sequences 5′-TT[G/A]TTTTG-3′ (Insulin Response Element, IRE) or 5′-TT(G/A)TTTAC-3′ (Daf-16 family binding element, DBE) (Furuyama et al., 2000) that were in silico identified in the promoter regions of β1, β2, β5, and β7 subunits, using chromatin IP. Specifically, we examined putative binding motifs that were localized at −374, −577, −237, and −770 upstream of the Transcription Starting Site (TSS) of each gene, respectively. The precipitated fragments were detected using quantitative real-time PCR primers that specifically amplify DNA encompassing the putative FoxO1 binding sites (Supplementary Table 1). As demonstrated in Figure 3D a putative IRE-containing site at −237 upstream of the β5 TSS promoter was detected in FoxO1 immunoprecipitants, using a primer pair that amplified a 180 bp region starting at −281 (Other indicated proteasome subunits promoters were not amplified; data not shown). The non-specific IgG antibody failed to precipitate in vivo the proteins bound to this sequence, suggesting that mouse FoxO1 has the potential to bind to the promoter of β5. Specifically, the ChIP-to-Input ratio was 2.4% in control MEFs, suggesting that FoxO1 may be an important factor participating in the regulation of gene expression (Figure 3E). Importantly, there was a 3-fold increase in PCR detection of the FoxO1 binding site in Irs1 KO MEFs, demonstrating that low IIS activity enhances FoxO1 binding to the promoter of β5 to regulate gene expression.
To assess the functional consequences of FoxO1 binding on the putative IRE (5′-TTATTTTG-3′), the wild type β5 promoter region (−1,000 to 0 upstream the β5 TSS) or a mutagenized version with 8 bp substitutions on the detected FoxO binding motif at −237 were cloned into the pLightSwitch_Prom reporter vector, which utilizes the RenSP luciferase gene (see Supplementary Table 2). As shown in Figure 3F, the mutation on the putative IRE (mut-pβ5-lightswitch) significantly repressed luciferase activity by 26.3% compared to the wild type (pβ5-lightswitch), indicating that the respective sequence promotes transcription. Furthermore, co-transfection of siRNAs targeting FoxO1 markedly attenuated the reported activity of the pβ5-lightswitch construct by 25.2%, while IRS1 silencing yielded a 27.5% induction of the luciferase activity. Mutation of the detected IRE completely abolished the effects of FoxO1 or IRS1 silencing on promoter activity. These results indicate that the identified site is essential for the FoxO1-mediated regulation of β5 subunit expression.
FoxO transcription factors are conserved regulators of longevity downstream of insulin and insulin-like growth factor signaling. They integrate signals emanating from nutrient deprivation and stress stimuli to coordinate programs of genes involved in cellular metabolism and quality control. The evolutionary conserved function of reduced IIS in organismal lifespan extension has fueled research to understand the mechanisms underlying this pro-longevity function of FoxOs (Webb and Brunet, 2014; Fontana and Partridge, 2015; Martins et al., 2016). However, we still do not fully understand how these factors affect lifespan. In this study, we dissect the contribution of FoxO transcription factors in longevity and demonstrate for the first time using mouse models the ability of reduced IIS to promote 20S proteasome function in mammalian tissues.
The gradual age-related decline of proteostasis maintenance is considered to be an important hallmark of aging (López-Otín et al., 2013). Emerging evidence from various biological systems indicate that FoxOs orchestrate the expression of genes involved in the proteostasis network (Webb and Brunet, 2014). These findings suggest that the maintenance of proteostasis may in part underlie the ability of FoxOs to extend lifespan and to delay signs of age-related diseases. Specifically it has been found that FoxO factors promote the expression of genes involved in autophagy and the ubiquitin–proteasome system. Whereas autophagy is thought to be relatively specific to long-lived proteins and degradation under chronic starvation conditions (Mizushima and Klionsky, 2007), the proteasome system is responsible for the degradation of most short-lived and regulatory proteins (Löw, 2011). Proteasome activity declines during aging in several tissues, including brain, heart, liver, muscle and skin, accounting for the observed accumulation of damaged proteins and the inclusion bodies (Chondrogianni et al., 2015b). Conversely, an intact proteasome is correlated with extreme longevity in humans (Chondrogianni et al., 2000), while proteasome activation can increase cellular and organismal lifespan, alleviate aggregation-related pathologies and enhance stemness (Chondrogianni et al., 2000; Kapeta et al., 2010; Chondrogianni et al., 2015a; Kapetanou et al., 2017). Interestingly, studies in brain have demonstrated the link between proteasome activity and aging (Triplett et al., 2015; Kelmer Sacramento et al., 2020), while Tropea and co-workers have proposed the involvement of IIS in these processes (Wrigley et al., 2017). Other studies on skeletal muscle atrophy have addressed the role of mammalian FoxOs in the positive regulation of ubiquitin ligases. FoxO3 is a strong and direct regulator of the muscle-specific E3 ubiquitin ligases atrogin-1 and Murf-1 transcription (Sandri et al., 2004, 2006; Stitt et al., 2004). In addition to acting upstream of ubiquitination, emerging evidence suggests that FoxO factors are linked to the regulation of proteasome assembly by modulating the expression of a 19S subunit. In human embryonic stem cells (hESCs) and induced pluripotent stem cells, FoxO4 is both necessary and sufficient for expression of the 19S regulatory cap subunit PSMD11 (Rpn6) (Vilchez et al., 2012). The high proteasome activity that ESCs exhibit is considered to be critical for the prevention of senescence. Upon differentiation, PSMD11 expression declines as it is no longer under FoxO4 regulation. This is accompanied by a reduction of proteasome activity and an increase in the levels of polyubiquitinated proteins. Interestingly, FoxOs appear to have a conserved role in regulating proteasome activity. In C. elegans, FoxO/DAF-16 promotes various types of stress resistance via the activation of the PSMD11 ortholog, rpn-6 (Vilchez et al., 2012). Similarly, worms overexpressing pbs-5, the ortholog of the catalytic β5 subunit, display a daf-16 dependent increase in lifespan and resistance to proteotoxicity (Chondrogianni et al., 2015a).
However, the mechanisms underlying the exact role of FoxOs in the regulation of 20S proteasome expression and activity remained elusive. In different cell types, FoxO1 and FoxO3 factors modulate various cellular activities, while also having functional redundancies. Herein, we demonstrate that the depletion of FoxO1, but not of FoxO3, leads to a significant decline of proteasome activity in two murine tissues. Notably, the proteasome is an integral part of the cellular function and its inhibition above the observed levels is toxic to cells and tissues, while knockdown of distinct proteasome subunits is embryonic lethal (Tanaka, 2009). The detected reduction of CT-L activity is linked to the reduced mRNA and protein expression of 20S subunits, including the catalytic β5 subunit. Contrariwise, the knockdown of IRS1, which enhances FoxO1 activity and ameliorates lifespan (Taguchi et al., 2007; Kappeler et al., 2008), induces proteasome function and 20S subunits expression. In support, IIS reduction in the fly resulted in an enhanced proteasome assembly and activity in the gut accompanied by a reduction in the aberrant age-related accumulation of proteasome substrates and an increase in gut integrity with age (Tain et al., 2017). Proteasome activity was necessary for IIS-mediated longevity as treatment with low concentrations of a proteasome inhibitor abolished the beneficial effects of IIS reduction in lifespan and gut integrity. Interestingly, proteasome activation was sufficient to increase gut integrity and lifespan in D. melanogaster. Likewise, proteasomal inhibition abolished the beneficial effect of reduced IIS on the circuit function in old flies (Augustin et al., 2018).
Furthermore, we demonstrate that silencing of IRS1 leads to enhancement of FoxO1 transcriptional activity, which is in accordance with other studies in mice showing that IRS1 and IRS2 knockout prevents the repressive FoxO1 phosphorylation (Taniguchi et al., 2005; Dong et al., 2006; Cheng et al., 2009; Qi et al., 2013). Importantly, we demonstrate that FoxO1 mediates the beneficial effects of IRS1 downregulation on proteasome CT-L activity and on the protein expression of β5. Supporting the notion that FoxOs may serve discrete or tissue-specific functions (Paik et al., 2007), FoxO3 repression did not downregulate proteasome activity and was not required for the enhanced proteasome activity under conditions of diminished IRS1 expression. These results indicate that FoxO1 is a potent regulator of proteasome function downstream of IIS. FoxO transcription factors target either a conserved DNA binding sequence, 5′-TT(G/A)TTTAC-3′ (daf-16 family binding element, DBE) or the insulin response element (IRE), 5′-TT[G/A]TTTTG-3′ in the promoter regions of their target genes and subsequently regulate gene expression (Furuyama et al., 2000). Importantly, after analyzing putative FoxO1 binding sites in several 20S proteasome subunits, we show that FoxO1 directly binds to the promoter region of β5, in a region containing a candidate IRE element (5′-TTATTTTG -3′). As expected, FoxO1 binding to the β5 promoter was found increased in MEFs lacking IRS1. The functionality of the binding site detected through the ChIP-based experiment was further analyzed by cloning the wild type or a mutagenized β5 promoter into a luciferase reporter system. Supporting the notion that FoxO1 upregulates β5 expression, silencing of FoxO1 led to a significant decrease of β5 promoter activity, while IRS1 silencing led to an increase. Furthermore, the site specific mutagenesis of 8 bp of the detected IRE at position −237 downregulated gene expression and completely blunted the response to FoxO1 or to IRS1 silencing. Notably, mutation of the identified FoxO binding sequence yielded a reduction of luciferase signal similar to FoxO1 silencing. The detected variations of luciferase activity due to mutagenesis or silencing are biologically significant and fall within the typically observed range (Xiong et al., 2012; Singh et al., 2017). Moreover, as β5 is also regulated by other transcriptional factors such Nrf1 (Li et al., 2018), we did not expect that the mutation of the FoxO1 binding site would totally abolish β5 expression. Importantly, β5 in addition to being responsible for the rate-limiting chymotrypsin-like proteolytic activity of the proteasome (Kisselev et al., 1999), was shown to be sufficient to induce the expression of additional proteasome subunits and consequently to enhance 26/30S proteasome assembly and activity when overexpressed in human fibroblasts (Chondrogianni et al., 2005), in human mesenchymal stem cells (Kapetanou et al., 2017) and in C. elegans (Chondrogianni et al., 2015a). Hence, the stimulation of the β5 subunit by Foxo1 has implications on the regulation of the whole proteasome machinery. Collectively, these data demonstrate that FoxO1 binds to a functional IRE at β5 promoter to activate gene expression and enhance proteasome activity.
Taken together these data demonstrate that IIS in mice regulates proteostasis and, in turn, lifespan. Our findings provide new insights about the mechanisms regulating the activity of the proteasome and expand our knowledge of how the nutrient signaling pathways affect proteostasis and ultimately longevity. Understanding the mechanisms that mediate the beneficial effects of reduced IIS activity is of major importance for the development of effective treatments to improve health span in humans.
The animal study was reviewed and approved by the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen.
MK: conceptualization, methodology, data collection and analysis, and writing the manuscript. TN: methodology, data collection and analysis, and editing the manuscript. LT: methodology and editing the manuscript. AP: data collection and analysis. LP: conceptualization, supervision, editing the manuscript, and funding acquisition. EG: conceptualization, supervision, writing and editing the manuscript, and funding acquisition. All authors have read and approved the manuscript.
MK is a recipient of a postdoctoral scholarship, that was co-financed by Greece and the European Union (European Social Fund-ESF) through the Operational Program (Human Resources Development, Education and Lifelong Learning) in the context of the project "Reinforcement of Postdoctoral Researchers – 2nd Cycle" (MIS-5033021), implemented by the State Scholarships Foundation (IKY). This work was also supported by the EU COST Action BM1402 to LP and EG.
We kindly acknowledge Ron DePinho and Jihye Paik for providing the FoxO1 and FoxO3 conditional knockout mouse line and Thomas Langer, Carsten Merkwirth, F. Thomas Wunderlich, and Jens C. Brüning for generating and providing the ROSA26-CreERT2 mouse line.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2021.625715/full#supplementary-material
ChIP, chromatin immune precipitation; DBE, Daf-16 family binding element; FoxO, forkhead box-O; IIS, insulin/IGF-1 signaling; IRE, insulin response element; IRS1, insulin receptor substrate 1; KO, knockout; MEFs, mouse embryonic fibroblasts; RNAi, RNA inhibition; RT-PCR, real time polymerase chain reaction; TSS, transcription starting site.
^ http://meme-suite.org/tools/fimo
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Keywords: FOXO factors, insulin signaling, proteostasis, proteasome, aging, longevity
Citation: Kapetanou M, Nespital T, Tain LS, Pahl A, Partridge L and Gonos ES (2021) FoxO1 Is a Novel Regulator of 20S Proteasome Subunits Expression and Activity. Front. Cell Dev. Biol. 9:625715. doi: 10.3389/fcell.2021.625715
Received: 03 November 2020; Accepted: 18 January 2021;
Lisa Porter, University of Windsor, Canada
Hua Bai, Iowa State University, United States
Ashley Webb, Brown University, United States
Copyright © 2021 Kapetanou, Nespital, Tain, Pahl, Partridge and Gonos. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Efstathios S. Gonos, sgonos@eie.gr; Linda Partridge, l.partridge@ucl.ac.uk; Partridge@age.mpg.de
Cellular Senescence: Causes, Consequences and Therapeutic Opportunities


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Q: Altium Designer 17 DRC taking for ever Intro: My Colleague and I have been using altium Designer for Sometime now . Currently AD 17.1.6 (build 538) . The PCB we draw are 3d 4 layer maximum . the only difference between us is that the smallest grid I use is 0.1mm , while he uses the finest Grid 0.025mm.
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My thoughts: I am trying to analyze the problem and i have come up with 2 possible causes :

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*The Fine Grid is causing the program to make more calculations causing it to take more time , can anyone confirm/comment on that ??

*The 3D objects placement and clearance is causing the software to consume the extra time (unlikely) , but does anyone know  a way to prevent the DRC from looking for violations in certain rules ? so i can go one by one and eliminate until i find most time consuming rule.
If anyone is wondering about the PC : it is a core i5 processor, with 8Gb ram and GTX 660Ti VGA.

A: Start by selecting only the important rules that you want checked. The first step would be to ensure that you do not have an excessive number of rules set up in your Design -> Rules settings. Once those are cleared out you can go to Tools -> Design Rule Check and change the settings for each element under the Rules To Check heading in the left panel.
Additionally, in the Design Rule Check window change the "Stop when ___ violations found" to something manageable (for example, 100).

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