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When poor drainage, bad attitude and poor sanitation intertwine - Graphic Online
When poor drainage, bad attitude and poor sanitation intertwine
BY: Kwabena Agyei-Boahene
Ghana seems to be no stranger to floods, but this year's flooding in June, which claimed nearly 20 lives in the country, was the most painful of all in recent times.
It was sad to hear that people died so easily as a result of a normal rainfall.
Frankly, no excuse can be offered to ease the pain and devastation that the loved ones of those who lost their lives are still going through.
Ghana has witnessed flooding on so many occasions and on each occasion, innocent lives are lost and property worth millions of cedis destroyed.
Gradually, flooding is leaving a permanent mark on this country.
This is very terrible and distressing because it seems pointless to remain trapped in this condition annually.
This problem can, however, be linked to three major reasons: poor drainage, poor sanitation and bad attitude.
Poor drainage
Undeniably, the way drains are constructed in this country is not only poor but an eyesore. Our drainage systems are so bad that they cause pavement distresses and premature failure of our roads.
Major drains in the big cities, for instance, are not well constructed to connect water flow to water courses, creeks, rivers and the sea to ensure a neat drainage discharge.
Again, there seems to be no proper coordination between drainage designers and road engineers to ensure that there is always a proper flow and control of stormy waters.
There are no substantive plans which constantly ensure routine maintenances and the dredging of drains that are constructed.
Human attitude is also an important factor.
Shoddy work has become deeply rooted in the fabric of the Ghanaian society and why we allow this to happen can best be explained by leadership.
Shoddily constructed drains do not only destroy roads but bring hardship on people.
Most individuals and companies neglect their duties by failing to do the jobs they have been paid to do.
This harrowing situation makes it difficult for us to pursue a clean environment.
Certain people deliberately litter the environment by dumping refuse on streets and in gutters, forgetting that it will eventually block the drains and result in flooding when it rains.
They pollute the environment without paying attention to health implications such as malaria, cholera and, in certain cases, death.
Although human behaviour is ethical, there is little to show for it.
Influential people and organisations in the country have turned a blind eye to this problem. District chief executives and religious bodies have ignored their roles in this regard.
For instance, the Catholic and Presbyterian churches have policies on the environment but they are not well utilised.
It is very important to highlight how problematic, irritating, disturbing and damaging our environment has become as a result of poor sanitation.
As a country, we do not pay proper attention to waste and how it should be managed.
Refuse is dumped haphazardly in places such as streets, highways, gutters, communication and water hydrants. Although most of the refuse we generate in Ghana, particularly in the cities, come from packaging, we lack the expertise to handle, collect and recycle them.
Sadly, the country's record in tackling sanitation is not encouraging.
By setting up more recycling factories in Ghana, the government would be providing jobs for a section of the jobless group, reduce unemployment and promote the use of reusable and recycled materials.
The factories can also recover organic materials for energy and fertiliser production.
But all these cannot be achieved without the necessary financial capital to set the factories up.
This is why the government should develop a unique national waste management strategy for the country.
Luckily, sanitation has been pushed to the top of the political agenda with the establishment of the Ministry of Sanitation.
President Nana Addo Dankwa Akufo-Addo has also set himself a target to ensure a clean environment.
Therefore, fresh strategic programmes should be developed for this country with a special focus on children and religious organisations.
They should be put on the and made to understand their responsibilities as change agents.
This will help lock in them a culture change that could help save this country.
Henceforth, coconut sellers, street traders, shop owners, big supermarkets, sachet and water producers, polythene bag producers, restaurant and nightclub operators should all be roped in and made aware that they do have a stake in making this country clean.
They must support any government environmental clean-up programme aimed at curbing floods and beautifying the environment.


Mark it down, ya'll… April 21st we celebrate the 11th anniversary of Record Store Day!
To commemorate this historic day, and YOU, the people who keep the wax business alive, we are throwing our annual RSD weekend banger with CASKET LOTTERY on Saturday and Bad Dreamer, Instant Karma, and Hi Lux on SUNDAY. As per usual, we will also be ordering every gosh darn title on the RSD 2018 list which you can oggle at right here.
We highly recommend subscribing to our email list and/or checking the blog daily for the latest updates on which Special Releases/Indie Exclusives that will be shipped and available at our location.
RSVP to our 2018 RSD Party here.
MARK THEM CALENDARS FOR APRIL 21ST/22ND.


It is becoming more common these days in Gym foyers to have a Meet & Greet type area rather than the traditional reception desk. If you have turnstiles, it is behind the reception desk that things like the push buttons to manually open the turnstiles are located. Also, leisure centres are expanding their offering and often have a softplay area with security gates. These areas can be some distance away from the point of sale for tickets. So where do you locate the push buttons? The answer is very simple: wireless remote push buttons. Take a look at our video below.
These buttons can usually be retrofitted to existing installations as well. Please contact us or call 01295 660 566 for further information.


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U.S. manufacturing output surges in June, but production still below pre-pandemic level
WASHINGTON (Reuters) - U.S. factory output increased for a second straight month in June as motor vehicle production accelerated amid the reopening of businesses, but the nascent improvement in manufacturing activity was overshadowed by surging new COVID-19 infections.
The Federal Reserve said on Wednesday manufacturing production increased 7.2% last month after climbing 3.8% in May.
Still, factory output was 11.1% below its level in February.
Economists polled by Reuters had forecast manufacturing output rising 5.6% in June.
Businesses have resumed operations after being shuttered in mid-March in an effort to slow the spread of the coronavirus.
But there has been a resurgence in new infections of the respiratory illness, especially in the highly populated South and West, prompting some authorities in these regions to either shut down businesses again or pause reopenings.
Production at factories contracted at a 47.0% annualized rate in the second quarter after falling at a 5.5% pace in the January-March period. Manufacturing, which accounts for 11% of the U.S. economy, is also being constrained by the fallout from the Trump administration's trade war with China.
Global supply chains remain fragile and demand for crude oil is weak, leading to lower prices that are undercutting spending by oil producers on drilling and shaft exploration equipment. Economists expect business spending to have contracted in the second quarter, the fifth straight quarterly decline.
Motor vehicles and parts production vaulted 105.0% in June after rising 120% in May. Still auto production remained nearly 25% below its pre-pandemic level.
The surge in manufacturing output combined with a 4.2% increase in utilities to offset a 2.9% slump in mining, leading to a 5.4% rise in industrial production in June. That followed a 1.4% gain in May. Industrial production contracted at a 42.6% rate in the second quarter, the largest decline since World War II, after decreasing at a 6.8% pace in the first quarter.
Capacity utilization for the manufacturing sector, a measure of how fully firms are using their resources, rose 4.6 percentage points to 66.9% in June. Overall capacity use for the industrial sector rose 3.5 percentage points to 68.6%. It is 11.2 percentage points below its 1972-2019 average.
Officials at the Fed tend to look at capacity use measures for signals of how much "slack" remains in the economy — how far growth has room to run before it becomes inflationary.
Reporting by Lucia Mutikani; Editing by Chizu Nomiyama and Andrea Ricci

redpajama_set_name: RedPajamaCommonCrawl


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Sour el Ghozlane Weather ForecastSour el Ghozlane Weather Forecast
Sour el Ghozlane (Bouira, Algeria) forecast issued: 12 am Mon 30 Jan 2023 local time
Issued: 12 am Mon 30 Jan 2023 local time
Sour el Ghozlane
Sour el Ghozlane Weather Forecast. Providing a local hourly Sour el Ghozlane weather forecast of rain, sun, wind, humidity and temperature.
The Long-range 12 day forecast also includes detail for Sour el Ghozlane weather today. Live weather reports from Sour el Ghozlane weather stations and weather warnings that include risk of thunder, high UV index and forecast gales. See the links below the 12-day Sour el Ghozlane weather forecast table for other cities and towns nearby along with weather conditions for local outdoor activities.
Sour el Ghozlane is 852 m above sea level and located at 36.15° N 3.69° E. Sour el Ghozlane has a population of 50204. Local time in Sour el Ghozlane is CET.
Sour el Ghozlane Weather Today
Mostly dry. Mild temperatures (max 9°C on Wed afternoon, min 2°C on Sun night). Wind will be generally light.
Sour el Ghozlane Weather (4–7 days)
Mostly dry. Very mild (max 12°C on Sat afternoon, min 4°C on Thu night). Wind will be generally light.
10 Day Sour el Ghozlane Weather
Mostly dry. Very mild (max 13°C on Mon afternoon, min 9°C on Tue afternoon). Wind will be generally light.
Sour el Ghozlane Weather Next Week
Mostly dry. Very mild (max 12°C on Thu afternoon, min 8°C on Tue night). Wind will be generally light.
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Sour el Ghozlane Weather
Live weather stations near Sour el Ghozlane
Sour el Ghozlane Live Weather Today: Recent reports from weather stations near Sour el Ghozlane
Bejaia/Soummam – Abane Ramdane Airport
Jijel - Achouat
State of sky unchanged.
Light winds from the SE
(7 km/h at SE°).
Chlef Airport
212 km WSW
234 km ESE
274 km S
* NOTE: not all weather stations near Sour el Ghozlane update at the same time and we only show reports from the nearest stations that are deemed current. Weather reports submitted by any ship (SYNOP) that is close to Sour el Ghozlane within an acceptable time window are also included.Read More
Sour el Ghozlane Location map
Algeria Weather Map
Sour el Ghozlane Weather Photos
See weather photos from Algeria.
Upload your own Sour el Ghozlane weather image:Upload
Weather forecast for places near Sour el Ghozlane:
'Ain Bessem
16 km Mostly dry. Mostly dry. Mostly dry. Mostly dry.
Sidi Aissa
Draa el Mizan
45 km Some drizzle, heaviest during Mon afternoon. Mostly dry. Mostly dry. Mostly dry.
Lakhdaria
Melouza
48 km Mostly dry. Mostly dry. Some drizzle, heaviest during Tue afternoon. Mostly dry.


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Biography – SMITH, RICHARD – Volume IX (1861-1870) – Dictionary of Canadian Biography
SMITH, RICHARD, mining engineer and administrator, industrialist, and politician; b. 30 Jan. 1783 at Tipton, Staffordshire, England, son of Thomas Smith and Mary Morris; d. 21 July 1868 near Lichfield, Staffordshire.
Son of a south Staffordshire coal operator, Richard Smith studied at the Royal School of Mines and was knowledgeable about coal mines "from my youth upwards." On 12 June 1811 he married Elizabeth Fereday, daughter of Samuel Fereday, an important Black Country coal and iron master. Smith's future dimmed, however, when he and Fereday suffered financial disaster in the collapse of the coal and iron boom at the end of the Napoleonic wars. An experienced practical expert on coal mining by this time, Smith re-established himself in London, and also managed coal operations in Wales and Portugal during this period.
In 1826 George IV by royal prerogative gave his brother, the Duke of York, a 60-year lease on all the unworked mineral resources in Nova Scotia and the duke sublet the rights to his creditors, the London jewellers Rundell, Bridge, and Rundell. On the advice of the British cabinet the General Mining Association, the jewellery firm's mining arm, hired Richard Smith to establish their coal mining operations in Nova Scotia.
On his arrival at Pictou in the early summer of 1827 Smith settled his large colony of men and machinery at a site seven miles up the East River which he named Albion Mines (now Stellarton) where small-scale coal operations had existed for about 20 years. The nearby leaseholders agreed to sell to the GMA, and under Smith's supervision brickworks, buildings, wharves, coke ovens, a sawmill, and a foundry were built, and track was laid for a horse-drawn railway. Though Smith found the gas in the ground "abundant, almost beyond precedent, and the water exceedingly troublesome," by September the GMA's first coal was raised from a newly opened, 212-ft pit. On 7 December a 20-horsepower steam engine, probably the first in Canada, started to pump water and hoist coal at the mine; its 75-ft stack became a local landmark. Equally ostentatious was Smith's impressive brick mansion, Mount Rundell, on a landscaped estate overlooking the industrial operations. Soon a steady stream of visitors viewed the curiosities of the coal mines and enjoyed the gay social life led by the GMA envoy and his wife at Mount Rundell.
In 1830 Smith turned to the important coal resources of Cape Breton Island, where GMA activities had been supervised since 1826 by the young mining engineer Richard Brown, later a chief agent for the GMA. Here too, after some confusion over the terms of the company's grant, the GMA had taken over the existing lease from small-scale operators, completing its monopoly of the colony's coal resources. A growing community of men, machinery, and buildings was established at Sydney Mines, where coal had been mined since 1785. Under the supervision of Smith and Brown a new shaft was sunk on the main seam there and steam-powered engines installed, but progress was delayed for two years by problems with the pumping machinery and heavy water flow in the mine. Smaller mines were also opened at Bridgeport and Little Bras d'Or. By 1833 more than 900 men were employed by the GMA in Nova Scotia and coal production had tripled to more than 50,000 tons.
Smith had also plunged energetically into the political life of the colony. On behalf of the GMA he lobbied the assembly for a special clause to specify the GMA's mineral rights in all land grants, resisted the idea of an export tax on coal favoured by some assemblymen, secured stricter patrol of coal smuggling on the Cape Breton coast, demanded stiff punishment of alleged arsonists when a fire erupted at Albion Mines in 1832, and generally safeguarded the company's interests. The GMA's liberal outlay of capital won local acclaim, as did Smith's personal skill and energy in developing the coal operations. However, Smith also met growing criticism of the monopoly control and generous lease provisions the GMA enjoyed. Indeed Smith's insistence on strict enforcement of these tarnished his image as a benefactor of the colony. The assembly frowned most on the low rents and royalties, for the coal mines promised to be a major source of colonial revenue. By 1837 Reformers like Joseph Howe* had become staunch opponents of the GMA, but only in 1857 were mineral rights finally repatriated and the GMA's monopoly restricted.
Bitterness peaked in 1832 when Smith contested a new assembly seat in Cape Breton County against William Young*, an ally of the nascent Reform group [see Doyle]. On the hustings the contest between "monopolist" and "patriot" was violent: at Sydney, Smith's supporters armed themselves with bludgeons, and in the rural sectors Young's backers seized the polls. Young was declared elected, but on Smith's request the assembly investigated his charges of intimidation and irregularities, unseated Young, and installed Smith. The latter served part of the 1833 and 1834 sessions, but returned to England in May 1834.
On his departure Smith probably had few regrets. He had disliked the rigours of life in Nova Scotia. Aggressive and overbearing, Smith left behind "some enemies as well as some friends," according to the Novascotian, and even Joseph Smith, who succeeded his uncle at the mines, thought it wise to rename one of the company's steamboats, then known as the Richard Smith. Yet Smith also took with him many tributes. The Halifax Mechanics' Institute voted him an honorary member; and Howe noted that Smith had "in very trying and difficult circumstances displayed intelligence, activity and a copiousness of scientific resource, very rarely combined in the same individual – and that have seldom, if ever, been witnessed in Nova Scotia."
In England Smith resumed his career as one of the "professional gentlemen" who supervised domestic industrial expansion. From 1836 to 1864 he managed the Earl of Dudley's coal, iron, and limestone holdings near Birmingham, developing an extensive network of mines, canals, railways, and ironworks. In 1857 he opened the Round Oak Ironworks, a model enterprise which earned an international reputation. He also served for a time as justice of the peace and as deputy lord lieutenant for Staffordshire. He retired at about the age of 81.
On the whole the record of Smith's stint in Nova Scotia was a laudable one. He left the coal industry with a production capacity far ahead of demand: despite Smith's efforts and to the disappointment of the GMA, Nova Scotia coal made slow headway in American markets. Smith claimed to have started coal mining on an "enlarged and scientific footing," and in this way inaugurated the industrial revolution in Nova Scotia. For this achievement, though, Smith must share the credit with his able deputies, mining engineers Joseph Smith at Albion Mines and Richard Brown at Sydney Mines, and with the hundreds of nameless "Miners, Colliers, Engineers and Mechanics" also dispatched by the GMA. Without the Duke of York and his jewellers, Nova Scotia might have supplied the capital and entrepreneurship to develop the coal mines, a project Samuel Cunard had in mind before the arrival of the GMA. But the colony could not supply men with the practical experience and knowledge of Smith and his subordinates.
David Frank
PANS, MG 1, 89 (S. G. W. Archibald papers, correspondence, 1813–35); 151–59 (Richard Brown papers, documents, 1859–1914); RG 1, 194–96; 458–64; RG 5, R, 18, 1833; RG 21, A, 2–3; M, 17–20. PRO, CO 217/146–57. Richard Brown, The coal fields and coal trade of the island of Cape Breton (London, 1871; repr. Stellarton, N.S., 1899). G.B., Parl., House of Commons paper, 1835, V, 603, pp.1–360, Report from the select committee on accidents in mines, together with the minutes of evidence, and index, pp.223–36, 249–52. N.S., House of Assembly, Journal and proc., 1824–36, 1857–58. Acadian Recorder, 1827–34. Colonial Patriot (Pictou, N.S.), 1827–34. Mechanic and Farmer (Pictou, N.S.), 1839. Novascotian, 1827–34. Pictou Observer (Pictou, N.S.), 1831–34. Times (London), 1825–34, 1868. Directory of N.S. MLAs. J. M. Cameron, The Pictonian colliers (Halifax, 1974). C. O. Macdonald, The coal and iron industries of Nova Scotia (Halifax, 1909). George Patterson, A history of the county of Pictou, Nova Scotia (Montreal, 1877). R. P. Fereday, "The career of Richard Smith," Acorn [house magazine of Round Oak Steel Works Limited, Brierley Hill, West Midlands, Eng.], 1966–67. H. B. Jefferson, "Mount Rundell, Stellarton, and the Albion Railway of 1839," N.S. Hist. Soc., Coll., XXXIV (1963), 79–120. J. S. Martell, "Early coal mining in Nova Scotia," Dal. Rev., XXV (1945–46), 156–72.
CUNARD, Sir SAMUEL (Vol. 9)YOUNG, Sir WILLIAM (Vol. 11)DOYLE, LAURENCE O'CONNOR (Vol. 9)HOWE, JOSEPH (Vol. 10)ARCHIBALD, THOMAS DICKSON (Vol. 11)HUNTINGTON, HERBERT (Vol. 8)KEMPT, Sir JAMES (Vol. 8)
HOWE, JOSEPH
DOYLE, LAURENCE O'CONNOR
CUNARD, Sir SAMUEL
HUNTINGTON, HERBERT
KEMPT, Sir JAMES
ARCHIBALD, THOMAS DICKSON
David Frank, "SMITH, RICHARD," in Dictionary of Canadian Biography, vol. 9, University of Toronto/Université Laval, 2003–, accessed January 31, 2023, http://www.biographi.ca/en/bio/smith_richard_9E.html.
Permalink: http://www.biographi.ca/en/bio/smith_richard_9E.html
Author of Article: David Frank
Title of Article: SMITH, RICHARD


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Lake Waccamaw, NC
Best Libraries around Lake Waccamaw, NC
Andrews Branch Library
105 North Morgan street
Andrews, SC
Baker Memorial Public Library
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Monday-Friday, 9 a.m. – 6 p.m. Saturday 9 a.m. – 1 p.m. Closed Sunday
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Hours: Mon: 10:00 am - 6:00 pm Tue: 10:00 am - 7:00 pm Wed: &nbs...
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The Bordeaux Branch Library serves over 30,000 people who live in western Fayetteville. The 10,000 square foot facility opened in December 1985 adj...
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Odessa International Airport LTD – a joint venture established in November 2011 by Odessa City Council and the private investor "Odessa Airport Development", financed construction of a new airport terminal and infrastructure worth 45 million dollars.
Odessa International Airport belongs to the group of the largest airports of Ukraine and by the airlines it is connecting Odessa with many cities of Ukraine, CIS, and also Western Europe, Asia, and Africa countries.
The aerodrome has one artificial runway (2800x56m), 57 parking places, taxiways network and one main taxiway. The Airport is equipped by air traffic control aids, the artificial runway is equipped by radio landing systems, light-signaling equipment, meteorological equipment providing its round-the-clock operation by ICAO 1st Category meteorological minimum from two headings of runway.
The passenger terminal is designed for international and domestic flights handling with capacity of 400 passengers per hour.
The Airport is located in the southwest part of Odessa. The distance from the city center to the airport is 7,5 km. It occupies the area of 570.3 ha.
Odessa International Airport LTD – a joint venture established in November 2011 by Odessa City Council and the private investor "Odessa Airport Development", financed construction of a new airport terminal and infrastructure worth 45 million dollars.


text:
AID Vol.2 No.3 , September 2012
Serum Procalcitonin and Neutrophil Toxic Granules Guided Management of Post-Operative K. pneumoniae Septic-Shock in Laminectomy—A Case Report
Indira Chivukula, Paul K. Marx, Kamaraju S. Ratnakar, G. Subbaiah, Venkataraman Sritharan*
Molecular Diagnostics and Biomarkers Laboratory, Department of Laboratory Medicine, Global Hospitals, Hyderabad, India.
Academic and Research, Global Hospitals, Hyderabad, India.
Institute of Orthopedics and Spine Surgery, Hyderabad, India.
Abstract: Introduction: We still rely on clinical diagnosis for initiating empirical antibiotic therapy and await blood culture for confirmation of infection, species identification and drug sensitivity. Differential blood cell count (WBC and neutrophils) and recording of toxic granules in the neutrophils are established methods for indirect diagnosis of infection though they are not used in the diagnosis of sepsis per se. Serum Procalcitonin is considered as a good biomarker in the management of sepsis. Materials and Methods: Whole blood and serum were used for laboratory analysis. We have combined the detection of toxic granules in the peripheral blood smear and serum PCT levels for diagnosis and monitoring the recovery of a patient with septic shock. A 63 year old laminectomy patient was transferred 2 days after the surgery to our hospital with several co-morbidities and complications. He was in septic shock and was put on Continuous Renal Replacement Therapy, with ionotropic support and IV fluids via nasogastric feeding and administration of Activated Protein C. Blood culture and daily measurements of serum Procalcitonin, differential blood cells count, and observation of toxic granules in neutrophils were done. Results: The blood culture showed infection due to K. pneumoniae resistant to carbapenems. WBC and Neutrophil counts were quite variable and showed incoherent response to treatment. Serum PCT was 24.57 ng/mL on the next day of admission and it peaked at 30.2 ng/mL on day 3. Its level started decreasing from the 4th day. Toxic granules disappeared when serum PCT levels reached < 1 ng/mL. The patient responded well to treatment and he was discharged on the 16th day upon request. Conclusion: This case is presented as an example of managing sepsis with a combination of a conventional hematology marker and a modern biomarker. Resource poor hospitals with inadequate microbiology services, may evaluate and use these two biomarkers not only for the total management of sepsis but also to reduce the cost of critical care.
Keywords: Sepsis; Procalcitonin; Toxic Granules
Cite this paper: I. Chivukula, P. K. Marx, K. S. Ratnakar, G. Subbaiah and V. Sritharan, "Serum Procalcitonin and Neutrophil Toxic Granules Guided Management of Post-Operative K. pneumoniae Septic-Shock in Laminectomy—A Case Report," Advances in Infectious Diseases, Vol. 2 No. 3, 2012, pp. 72-75. doi: 10.4236/aid.2012.23011.
[1] J.-L. Vincent and E. Abraham, "The Last 100 Years of Sepsis," American Journal of Respiratory and Critical Care Medicine, Vol. 173, No. 3, 2006, pp. 256-263. doi:10.1164/rccm.200510-1604OE
[2] S. Chatterjee, S. Todi, S. Sahu and M. Bhattacharyya, "Epidemiology of Severe Sepsis in India," Critical Care, Vol. 13, Suppl. 1, 2009, p. 345.
[3] L. A. Al-Gwaiz and H. H. Babay. The Diagnostic Value of Absolute Neutrophil Count, Band Count and Morphologic Changes of Neutrophils in Predicting Bacterial Infections," Medical Principles and Practice, Vol. 16, No. 5, 2007, pp. 344-347. doi:10.1159/000104806
[4] A. Nakamura, H. Wada, M. Ikejiri, T. Hatada, H. Sakurai, Y. Matsushima, J. Nishioka, K. Maruyama, S. Isaji, T. Takeda and T. Nobori, "Efficacy of Procalcitonin in the Early Diagnosis of Bacterial Infections in a Critical Care Unit," Shock, Vol. 31, No. 6, 2009, pp. 586-591. doi:10.1097/SHK.0b013e31819716fa
[5] L. Simon, F. Gauvin, D. K. Amre, P. Saint-Louis and J. Lacroix. "Serum Procalcitonin and C-Reactive Protein Levels as Markers of Bacterial Infection: A Systematic Review and Meta-Analysis," Clinical Infectious Diseases, Vol. 39, No. 2, 2004, pp. 206-217. doi:10.1097/SHK.0b013e31819716fa
[6] A. van de Vyver, E. F. Delport, M. Esterhuizen and R. Pool, "The Correlation between C-Reactive Protein and Toxic Granulation of Neutrophils in the Peripheral Blood," South African Medical Journal, Vol. 100, No. 7, 2010, pp. 442-444.
[7] P. Schuetz, W. Albrich, M. Christ-Crain, J. Chastre and B. Mueller, "Procalcitonin for Guidance of Antibiotic Therapy," Expert Reviews in Anti Infectives Therapy, Vol. 8, No. 5, 2010, pp. 575-587. doi:10.1586/eri.10.25
[8] P. Emmanuel Charles, E. Kus, S. Aho, S. Prin, J.-M. Doise, N.-O. Olsson, B. Blettery and J.-P. Quenot, "Serum Procalcitonin for the Early Recognition of Nosocomial Infection in the Critically Ill Patients: A Preliminary Report," BMC Infectious Diseases, Vol. 9, No. 1, 2009, pp. 49-58.
[9] J. U. Jensen, L. Heslet, T. H. Jensen, K. Espersen, P. Steffensen and M. Tvede, "Procalcitonin Increase in Early Identification of Critically Ill Patients at High Risk of Mortality," Critical Care Medicine, Vol. 34, No. 10, 2006, pp. 2596-2602. doi:10.1097/01.CCM.0000239116.01855.61
[10] C. Pierrakos and J. L. Vincent, "Sepsis Biomarkers: A Review," Critical Care, Vol. 14, No. 1, 2010, p. R15. doi:10.1186/cc8872
[11] J. C. Singh and N. S. Kekre, "Procalcitonin: A Marker for Renal Parenchymal Infection in Children?" Indian Journal of Urology, Vol. 22, No. 2, 2006, pp. 162-63.
[12] S. K. Todi, "Top Stories of 2009," Indian Journal of Critical Care Medicine, Vol. 14, No. 1, 2010, pp. 3-7. doi:10.4103/0972-5229.63027
[13] U. Sudhir, R. K. Venkatachaliah, T. Anilkumar, M. Yogeshwar Rao and P. Kempegowda, "Significance of Serum Procalcitonin in Sepsis," Indian Journal of Critical Care Medicine, Vol. 15, No. 1, 2011, pp. 1-5. doi:10.4103/0972-5229.78214
[14] M. Sinha, S. Desai, S. Mantri and A. Kulkarni, "Procalcitonin as an Adjunct Biomarker in Sepsis," Indian Journal of Anesthesia, Vol. 55, No. 3, 2011, pp. 266-270. doi:10.4103/0019-5049.82676
[15] M. Meisner, "PCT in the Differential Diagnosis of Viral and Bacterial Infections," In: M. Meisner, Ed., Procalcitonin—A New Innovative Infection Parameter Biochemical and Clinical Aspects, 3rd Edition, Thieme Verlag, Stuttgart, 2000, pp 136-37.


text:
All About New Year's Resolutions
This year, we resolve to write more blog posts about New Year's resolutions. And look at that, we did it!
• Ancient Babylonians started the idea of New Year's resolutions about 4,000 years ago, although the new year in that society started in the middle of March and lined up with crop-planting season. As part of Akitu, a 12-day religious and social festival, Babylonians promised the gods that they would pay debts and return borrowed items. In exchange, such was the deal, the gods would provide and ensure a healthy harvest.
• About 2,000 years ago, in Ancient Rome, the calendar as we know it today took shape, with the new year officially beginning on the first day of the month of January. The namesake of the month: the two-faced god Janus. Romans believed that each of Janus's faces looked in opposite directions, one to the past and one to the future. In recognition of the latter, Romans offered up sacrifices and also promised to behave correctly in the new year.
• New Year's resolutions also have roots in Christianity, specifically the Methodist faith. Sect founder John Wesley devised a special New Year's Eve religious service in 1740, which included biblical readings, singing hymns, and congregants assuring themselves, each other, and their higher power that they'd act in a morally upstanding way in the year to come.
• No, not everybody makes New Year's resolutions. According to researchers, slightly less than half of all American adults make a specific resolution. Of that number, about half were successful in fully meeting their stated goals.
Uncle John's Greatest Know on Earth Bathroom Reader
• What are the most common New Year's resolutions? They mostly involve health. The most popular are exercising more, losing weight, and eating more healthily. Other frequently stated resolutions: getting organized, learning a new hobby or skill, traveling more, and saving money.
• It's tough to turn some resolutions into realities, but it's not impossible. According to life coaches, psychologists, and other experts, there are ways to make these life changes easier to accomplish. They say it's important to be specific with a resolution, and to break it down into smaller ones with built-in benchmarks to measure and appreciate progress. Experts also advise to only take on one or two resolutions and to seek out friends and family in your mission — not only to provide support but also accountability.
It's a brand new year! Learn new trivia day after day with the 2020 Uncle John's Greatest Know on Earth Bathroom Reader. It's available now.
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In the wake of post-polls violence in Khulna, a journalist has been reported brutally wounded with sharp weapons.
On Friday at 9pm, Md Shahjahan Siraj, treasurer of Koyra Press Club and Koyra correspondent for The Daily Khulnanchal and Ajker Sangbad, was attacked and brutally wounded when he was returning home.
He was rescued by locals and taken to a health complex. Later, he was transferred to Khulna Medical College and Hospital.
Locals also caught one of the attackers red-handed with a chopper, Shahin, and handed him over to the police.
According to the locals, the attack on Journalist Siraj was due to his news reporting concerning recent upazila elections.
However, this is not the only incident of post-polls violence in Khulna. There has been an increase in violence in different districts of Khulna after the upazila parishad elections.
Several attacks, lawsuits, and vandalism have been reported in Tekheda, Rupsha, and Koyra upazilas. Political leaders and activists of these areas have fled.
In the upazila elections, Upazila Awami League President, GM Mohsin Reza, contested against and lost to rebel contestant SM Shafiqul Islam, former convenor of the upazila Jubo League. Once the election was over, fights broke out in different places.
On April 4, the Khulna Press Club organized a press conference where Md Ayub Ali Sana, office secretary of Khulna Bangabandhu Legal Assistant Association, said: "On the night of voting, supporting-activists of SM Shafiqul Islam attacked the house of upazila Awami League's Sports Secretary, Abu Bakar Siddique.


Agonist stimulation of Group I metabotropic glutamate receptors (mGluRs) initiates their coupling to the heterotrimeric G protein, Gαq/11, resulting in the activation of phospholipase C, the release of Ca2+ from intracellular stores and the subsequent activation of protein kinase C. However, it is now recognized that mGluR5a also functions as a receptor for cellular prion protein (PrPC) and β-amyloid peptide (Aβ42) oligomers to facilitate intracellular signaling via the resulting protein complex. Intracellular mGluR5a signaling is also regulated by its association with a wide variety of intracellular regulation proteins.
In the present study, we utilized mass spectroscopy to identify calmodulin kinase IIα (CaMKIIα) as a protein that interacts with the second intracellular loop domain of mGluR5. We show that CaMKIIα interacts with both mGluR1a and mGluR5a in an agonist-independent manner and is co-immunoprecipitated with mGluR5a from hippocampal mouse brain. CaMKIIα positively regulates both mGluR1a and mGluR5a endocytosis, but selectively attenuates mGluR5a but not mGluR1a-stimulated ERK1/2 phosphorylation in a kinase activity-dependent manner. We also find that Aβ42 oligomers stimulate the association of CaMKIIα with mGluR5a and activate ERK1/2 in an mGluR5a-dependent manner. However, Aβ42 oligomer-stimulated ERK1/2 phosphorylation is not regulated by mGluR5a/CaMKIIα interactions suggesting that agonist and Aβ42 oligomers stabilize distinct mGluR5a activation states that are differentially regulated by CaMKIIα. The expression of both mGluR5a and PrPC together, but not alone resulted in the agonist-stimulated subcellular distribution of CaMKIIα into cytoplasmic puncta.
Taken together these results indicate that CaMKIIα selectively regulates mGluR1a and mGluR5a ERK1/2 signaling. As mGluR5 and CaMKIIα are involved in learning and memory and Aβ and mGluR5 are implicated in Alzheimer's disease, results of these studies could provide insight into potential pharmacological targets for treatment of Alzheimer's disease.
G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors and function to transduce extracellular signals into intracellular responses [1,2]. GPCRs are composed of seven transmembrane domains that can be activated by a variety of stimuli including photons, odorants, hormones, amino acids, peptides and neurotransmitters . Glutamate, the major excitatory neurotransmitter in the central nervous system, signals via the activation of both metabotropic GPCRs and ionotropic receptors [3-6]. There are eight different GPCRs that respond to glutamate and mediate the metabotropic glutamate receptor signaling in the brain [5-7]. These receptors are classified into three subgroups based on sequence similarity and G protein coupling specificity. Group I metabotropic glutamate receptors, (mGluR1 and mGluR5), regulate excitatory synaptic signaling response via their coupling to the heterotrimeric G protein, Gαq/11, which upon activation stimulates the activity of the enzyme phospholipase C to generate the production of two second messengers, diacylglycerol and inositol 1, 4, 5 trisphosphate. Inositol 1, 4, 5 trisphosphate acts as a ligand that binds to inositol phosphate receptors localized to the endoplasmic reticular membrane to release intracellular Ca2+ stores, in turn Ca2+ functions in combination with diacylglycerol to activate protein kinase C .
Group I mGluRs serve as molecular scaffold proteins to regulate both extracellular and intracellular signaling complexes. Specifically, mGluR5 functions as a receptor to mediate intracellular Ca2+ signaling and extracellular-regulated protein kinase (ERK1/2) activation by cellular prion protein (PrPc) and β42-amyloid (Aβ42) oligomers [8,9]. PrPc can physically associate with mGluR5, and binding of Aβ to PrPC generates mGluR5-mediated increases in intracellular Ca2+ concentrations in neurons . Negative allosteric modulators of mGluR5 weaken the interaction of PrPc with mGluR5, while positive allosteric modulators increase this interaction. Silent allosteric modulators of mGluR5 disrupt Aβ induced interactions of mGluR5 with PrPc , confirming the physical interaction of these proteins and suggesting a mechanism by which PrPc and Aβ can regulate mGluR5 signaling. It is now recognized that mGluR5 activation by both PrPc and Aβ42 oligomers also plays an important role in the pathophysiology associated with Alzheimer's disease (AD) [9,11]. Consistent with this concept, mGluR5 selective antagonists improve the cognitive deficits observed in AD mouse models and the genetic deletion of mGluR5 results in a reduction in both soluble Aβ42 oligomers and β-amyloid plaques and results in the amelioration of cognitive dysfunction observed in APPswePSIΔE9 AD transgenic mice .
Group I mGluRs, in addition to their coupling to Gαq/11 heterotrimeric G proteins, also interact with and scaffold a variety of intracellular signaling proteins that play a pivotal role in regulating mGluR1/5 signaling, endocytosis, subcellular localization and synaptic activity [3,12]. These proteins include, but are not limited to, Homer, calmodulin, SIAH-1a, β-arrestin1, casein kinase 1, protein phosphatase 2a, phospholipase D2, RalA, Pyk2, and Fyn [9,13-22]. β-Arrestin1 has been shown to be involved in mGluR1-mediated ERK1/2 phosphorylation, as have Pyk2, PKC and Src [22,23]. Phospholipase D2 and RalA regulate the constitutive endocytosis of Group I mGluRs, as well as a number of other GPCRs [17,24,25]. In addition, Homer interactions with Group I mGluRs regulates their G protein signaling, coupling to the activation of ERK1/2 and plays a role in Group I mGluR-mediated regulation of synaptic activity [26-28]. Both mGluR1a and mGluR5a encode a protein phosphatase 1γ binding motif and protein phosphatase 2A has been implicated in N-methyl-D-aspartate (NMDA) receptor-stimulated increases in mGluR5 activity by promoting the dephosphorylation of desensitized receptors [21,29].
Emerging studies reveal that Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) can also regulate several GPCRs. CaMKIIα has been shown to regulate behavioral responses to cocaine by regulating the D3 dopamine receptor by binding to third intracellular loop of the receptor to mediate its desensitization . CaMKIIα-dependent desensitization has also been reported for the D1/D2 heterodimer  and the Histamine H1 receptor . In contrast, CaMKIIα binding to the second intracellular loops of D1/D2 receptor heteromers increases signaling . Moreover, antagonism of CaMKIIα activity has been shown to reduce mGluR1 internalization [34,35]. CaMKIIα has been shown to play a direct role in regulating the desensitization of mGluR1a inositol 1, 4, 5 trisphosphate signaling by physically interacting with the C-terminal tail of the receptor . Recently, CaMKIIα has also been shown to interact with the proximal C-terminal tail of mGluR5 in vitro . In rat striatal neurons, inactive CaMKIIα binds constitutively to mGluR5 and mGluR5- mediated Ca2+ release results in the dissociation of C-tail bound CaMKIIα and recruitment to the NMDA receptor where it can phosphorylate the GluN2B subunit .
In the present study, we utilized a cell permeant Tat peptide from HIV  coupled to a peptide encoding the second intracellular loop (IL2) of mGluR1/5 followed by a FLAG epitope tag to perform a proteomic screen to identify neuronal proteins that interact with Group I mGluRs. As a consequence, we have identified a series of known Group I-interacting proteins including: protein phosphatase 1γ, protein phosphatase 2A and Gαq11 in the proteomic screen, as well as CaMKIIα, β, δ, and γ as novel proteins that interact with IL2 of mGluR1/5. We find that CaMKIIα positively regulates the endocytosis of both mGluR1a and mGluR5a, but has differential effects on mGluR1/5-stimulated ERK1/2 phosphorylation. Furthermore, Aβ42 oligomers stimulate mGluR5-mediated ERK1/2 phosphorylation and CaMKIIα association with mGluR5, but CaMKIIα does not regulate Aβ42 oligomer-stimulated ERK1/2 phosphorylation. In addition, mGluR5a and PrPC expression results in the subcellular redistribution of CaMKIIα. Taken together our observations indicate that CaMKII regulates agonist-stimulated Group I mGluR signaling, but not Aβ42 oligomer-mediated signaling via mGluR5a.
In order to identify potentially novel mGluR1/5 IL2 interacting proteins, we performed a proteomic screen using a Tat-tagged mGluR1/5 IL2 peptide conjugated to a FL-epitope tag to screen by mass spectroscopy proteins in neurons that may bind to mGluR1/5. To do this, a mixed culture of 107 cortical and striatal neurons was incubated with the mGluR1/5 Tat peptide (70 μM final concentration) for 2 hours. We found that a number of novel and known interacting proteins were identified (Table 1). The known mGluR1/5 interacting proteins include Gαq/11, protein phosphatase 1γ catalytic subunit, and the regulatory subunit of protein phosphatase 2A [19,21]. Amongst the novel mGluR1/5 IL2-interacting proteins identified were CaMKIIα, β, δ, and γ. As CaMKII antagonism was previously reported to antagonize mGluR1a internalization , we examined whether CaMKIIα interacted with mGluR1a and mGluR5a to regulate either their signaling or endocytosis.
To validate the potential interaction of CaMKIIα with Group I mGluRs, HEK 293 cells were transfected with either FL-epitope-tagged mGluR1a or mGluR5a along with either GFP or GFP-CaMKIIα. We found that GFP-CaMKIIα was co-immunoprecipitated with both FL-mGluR1a and FL-mGluR5a in an agonist-independent manner (Figures 1A and B). The co-immunoprecipitation of GFP-CaMKIIα with FL-mGluR1a was not dependent upon CaMKIIα catalytic activity, as the pretreatment of HEK 293 cells with 5 μM KN-93 did not affect the extent of GFP-CaMKIIα co-immunoprecipitated with the receptor (Figure 1C). Endogenous CaMKIIα could be selectively co-immunoprecipitated with endogenous mGluR5a using a rabbit polyclonal mGluR5a antibody, but not Rab11 antibody, from hippocampal mouse brain lysates (Figure 1D).
Co-immunoprecipitation of GFP-CaMKIIα with FLAG-mGluR1a and FLAG-mGluR5a. Representative immunoblot showing GFP-CaMKIIα co-immunoprecipitation with either A) FL-mGluR1a or B) FL-mGluR5a from HEK 293 cells transiently transfected as labeled with 3 μg of pcDNA3.1 of either FL-mGluR1a or FL-mGluR5a along with either 0.5 μg of plasmid cDNA encoding either pEGFP or GFP-CaMKIIα and treated with 50 μM quisqualate for the times indicated in the Figure. Bar graphs show the quantitative densitometric analysis of GFP-CaMKIIα co-immunoprecipitated with either FL-mGluR1a or FL-mGluR5a. The data represents the mean ± SD of 6 independent experiments. C) Representative immunoblot showing GFP-CaMKIIα co-immunoprecipitation with FLAG-mGluR5a in HEK 293 cells treated with or without 5 μM KN-93 that were transiently transfected with 2 μg of FL-mGluR5 along with 0.5 μg of GFP-CaMKIIα. Bar graphs show the quantitative densitometric analysis of GFP-CaMKIIα co-immunoprecipitated with FL-mGluR5a in the absence and following pretreatment with 5 μM KN-93 for 1 h. The data represents the mean ± SD of 6 independent experiments. D) Shown is a representative immunoblot of endogenous CaMKII co-immunoprecipitated with endogenous mGluR5. 1 mg of adult CD-1 mouse hippocampal tissue lysate was incubated with protein G sepharose beads and either polyclonal rabbit anti-Rab11 or anti-mGluR5 antibody. Shown is a representative immunoblot from 4 independent experiments.
CaMKIIα interactions with the mGluR1/5 IL2 were identified by mass spectroscopy following the incubation of neurons with a cell permeant mGluR1/5 IL2 peptide. To confirm this interaction we purified GST and GST-IL2 from bacteria and assessed the ability of these constructs to co-precipitate either GFP-CaMKIIα or GRK2 (positive control) from HEK 293 cell lysates . We found that GST-IL2 effectively co-precipitated both GFP-CaMKIIα and GRK2 (Figure 2A). We then utilized a series of GST-mGluR1/5 IL2 alanine scanning mutants that we previously employed to identify IL2 residues required for GRK2 binding in an attempt to further delineate specific mGluR1/5 IL2 residues required for GFP-CaMKIIα (Figure 2B) . However, we were unable to discern discrete IL2 residues required for GFP-CaMKIIα binding (Figure 2B). Moreover, FL-mGluR1b, that lacks an extended carboxyl-terminal tail and two mGluR1b mutants defective in GRK2 binding (K691A and K692A), was still effectively able to co-immunoprecipitate GFP-CaMKIIα (Figure 2C). Thus, although CaMKIIα interacts with the IL2 of mGluR1/5, there appeared to be no specific IL2 residues that are essential for CaMKIIα binding.
Purified GST-fusion proteins encoding the mGluR1/5 IL2 loop domain interact with GFP-CaMKIIα. A) Shown is a representative immunoblot showing the co-immunoprecipitation of G protein-coupled receptor kinase 2 and GFP-CaMKIIα with a GST mGluR1/5 IL2 domain fusion protein. 1 μg of GST protein was incubated with 500 μg of HEK293 cell lysates over-expressing either GRK2 or GFP-CaMKIIα. The immunoblots are representative of 3 independent experiments. B) Upper panel, shows the schematic representation of GST-IL2 fusion protein alanine scanning mutants previously used to identify the IL2 residues required for GRK2 binding to Group I mGluRs . Middle panel shows a representative immunoblot showing the co-immunoprecipitation of GFP-CaMKIIα with the GST-IL2 fusion protein alanine scanning mutants and the expression of the GST fusions. Lower panel shows the densitometric analysis of the relative co-immunoprecipitation of GFP-CaMKIIα with the GST-IL2 fusion protein alanine scanning mutants compared to the wild-type IL2 GST fusion protein. The data represents the mean ± SD of 3 independent experiments. C) Representative immunoblots showing GFP-CaMKIIα co-immunoprecipitated FL-mGluR1b and FL-mGluR1b mutants (K691A and K692A) that do not bind GRK2. Also shown are FL-mGluR1a immunoprecipitates and GFP-CaMKIIα expression in cell lysates. Bar graph shows the relative co-immunoprecipitation of GFP-CaMKIIα with the FL-mGluR1b and FL-mGluR1b mutants (K691A and K692A). Data shown represents the means ± SD of three independent experiments.
It has previously been reported that the antagonism of CaMKII activity can negatively regulate the internalization of mGluR1a and its alternatively spliced variants . Therefore, we examined whether the overexpression of CaMKIIα would increase the agonist-stimulated endocytosis of either FL-mGluR1a or FL-mGluR5a. We found that the over-expression of GFP-CaMKIIα promoted the agonist-stimulated endocytosis of both FL-mGluR1a and FL-mGluR5a (Figures 3A and B). In agreement with the previous work of Mundell et al., 2002, the CaMKII antagonist KN-93 blocked the CaMKIIα-dependent increase of FL-mGluR1a endocytosis in GFP-CaMKIIα over-expressing cells  (Figure 3A). Thus, it appears that CaMKIIα contributes to the endocytosis of mGluR1a in a kinase activity-dependent manner.
CaMKIIα overexpression increases agonist-stimulated mGluR1a and mGluR5 endocytosis. A) Upper blot shows a representative immunoblot for cell surface biotin-labeled mGluR1a in HEK 293 cells transfected with 3 μg of pcDNA3.1 encoding FL-mGluR1a along with 0.5 μg of plasmid cDNA encoding either GFP or GFP-CaMKIIα following 50 μM quisqualate treatment following 1 h pre-treatment in the presence and absence of KN-93. Lower blot shows the total cell lysates (50 μg) for mGluR1a. The bar graph shows the densitometric analysis of biotin-labeled cell surface mGluR1a immunoblots normalized to total mGluR1a biotinylation. Data represents the mean ± SD of 7 independent experiments. *P < 0.05 versus GFP transfected cells. B) Upper blot shows a representative immunoblot for cell surface biotin-labeled mGluR5a in HEK 293 cells transfected with 3 μg of pcDNA3.1 encoding FL-mGluR5a along with 0.5 μg of plasmid cDNA encoding either GFP or GFP-CaMKIIα following 50 μM quisqualate treatment. Lower blot shows the total cell lysates (50 μg) for mGluR5a. The bar graph shows the densitometric analysis of biotin-labeled cell surface mGluR1a immunoblots normalized to total mGluR5a biotinylation. Data represents the mean ± S.E.M. of 4 independent experiments completed in duplicate. *P < 0.05 versus GFP transfected cells, #P < 0.05 versus GFP CaMKIIα control.
Group I mGluRs couple to the activation of ERK1/2 phosphorylation via a number of different mechanisms, that include Gαq/11-mediated activation of Ca2+ signaling leading to the activation of both protein kinase C and Src, as well as their interaction with both Pyk2 and Homer [22,40]. Therefore, we examined whether over-expression of either CaMKIIα or an autophosphorylation incompetent CaMKIIα-T286A mutant altered either FL-mGluR1a- or FL-mGluR5a-stimulated ERK1/2 phosphorylation. We found that neither GFP-CaMKIIα nor CaMKIIα-T286A over-expression affected FL-mGluR1a-stimulated ERK1/2 phosphorylation in response to 50 μM quisqualate stimulation for either 5 or 10 min (Figure 4A). In contrast, CaMKIIα over-expression significantly attenuated FL-mGluR5a stimulated ERK1/2 phosphorylation in response to 5 and 10 min treatment with 50 μM quisqualate, whereas the expression of CaMKIIα-T286A mutant had no effect on quisqualate stimulated ERK1/2 phosphorylation in FL-mGluR5a expressing cells (Figure 4B).
Effect of CaMKIIα overexpression on mGluR1a- and mGluR5a- -stimulated ERK1/2 phosphorylation. A) Shown are representative immunoblots for FL-mGluR1a expression, p-ERK1/2 activity and total-ERK1/2 expression in HEK 293 cells transiently transfected with 3 μg of pcDNA3.1 encoding FLAG-mGluR1a along with 0.5 μg of plasmid cDNA encoding either GFP, GFP-CaMKIIα or GFP-CaMKIIα-T286A in response to 50 μM quisqualate treatment for 0, 5 and 10 min. Bar graph shows the densitometric analysis of ERK1/2 phosphorylation normalized to both basal and total ERK1/2 protein expression. Data represents the mean ± SD of five independent experiments. *P < 0.05 versus GFP transfected control cells. B) Shown are representative immunoblots for, FL-mGluR5a expression, p-ERK1/2 activity and total-ERK1/2 expression in HEK 293 cells transiently transfected with 3 μg of pcDNA3.1 encoding, FL-mGluR5a along with 0.5 μg of plasmid cDNA encoding either GFP, GFP-CaMKIIα or GFP-CaMKIIα-T286A in response to 50 μM quisqualate treatment for 0, 5 and 10 min. Bar graph shows the densitometric analysis of ERK1/2 phosphorylation normalized to both basal total ERK1/2 protein expression. Data represents the mean ± SD of five independent experiments. *P < 0.05 versus GFP transfected control cells.
Aβ42 oligomers were recently shown to signal via mGluR5a . Therefore, we examined whether Aβ42 oligomer-stimulated activation of mGluR5a would alter the agonist-stimulated association of GFP-CaMKIIα with FL-mGluR5a. We found that 200 nM Aβ42 oligomer treatment of HEK 293 cells transfected with FL-mGluR5a and GFP-CaMKIIα resulted in a significant time-dependent increase in GFP-CaMKIIα co-immunoprecipitated with FL-mGluR5a (Figure 5), a response we did not observe in response to receptor agonist stimulation (Figures 1A and B). Given that CaMKIIα negatively regulated agonist-stimulated mGluR5a-dependent ERK1/2 phosphorylation, we examined whether Aβ42 oligomer treatment of mGluR5a resulted in ERK1/2 phosphorylation and whether this activation was regulated by CaMKIIα interactions. We found that treatment of FL-mGluR5a expressing cells with 200 nM Aβ42 oligomers stimulated a significant increase in ERK1/2 phosphorylation, but that Aβ42 oligomer-stimulated ERK1/2 phosphorylation via the activation of FL-mGluR5a was not affected by CaMKIIα overexpression (Figure 6A). Similar to quisqualate-stimulated mGluR5a activation of ERK1/2 phosphorylation, Aβ42 oligomer-stimulated ERK1/2 phosphorylation could be antagonized by the pretreatment of HEK 293 cells with 1 mM of the PKC inhibitor Bisindolymaleimide I (Bis I) (Figure 6B). Thus, Aβ42 oligomers could stimulate ERK1/2 phosphorylation in an mGluR5a- and PKC-dependent manner that was not regulated by CaMKIIα, despite the fact that Aβ42 oligomers increased CaMKIIα association with mGluR5a.
Aβ42 oligomers increase CaMKIIα co-immunoprecipitation with mGluR5a. A) HEK 293 cells were transiently transfected with 2 μg of pcDNA3.1 encoding FLAG-mGluR5a and 0.5 μg of plasmid cDNA encoding GFP-CaMKIIα. HEK 293 cells were treated with 200 nM of Aβ42 oligomer for 0, 2, 5, 15 and 30 min. B) The bar graph shows the densitometric analysis of the relative co-immunoprecipitation of GFP-CaMKIIα with FL-mGluR5a following Aβ42 oligomer treatment normalized to total mGluR5a protein expression. Data represents the mean ± SD of 5 independent experiments. *p < 0.05 versus untreated cells.
Aβ42 oligomers stimulate ERK1/2 phosphorylation in an mGluR5a and PKC dependent manner that is independent of CaMKIIα overexpression. A) Shown are representative immunoblots of p-ERK1/2 activity and total ERK1/2 expression in HEK 293 cells transfected with 2 μg of pcDNA3.1 encoding FLAG-mGluR5a along with either 0.5 μg of plasmid cDNA encoding either GFP or CaMKIIα and treated with 100 nM Aβ42 oligomer for 5 and 10 min. Bar graph shows the densitometric analysis of ERK1/2 phosphorylation normalized to both basal and total ERK1/2 protein expression. Data represents the mean ± SD of 4 independent experiments. *P < 0.05 versus non-transfected (NT) cells. B) Shown are representative immunoblots of p-ERK1/2 activity and total ERK1/2 expression in HEK 293 cells transfected with 2 μg of pcDNA3.1 encoding FLAG-mGluR5a and treated with either 50 μM quisqualate or 100 nM Aβ42 oligomer in either the presence or absence of 1 μM Bis-1. Bar graph shows the densitometric analysis of ERK1/2 phosphorylation normalized to both basal and total ERK1/2 protein expression. Data represents the mean ± SD of 7 independent experiments. *P < 0.05 versus non-transfected (NT) cells.
Aβ42 oligomers were previously shown to interact with PrPC with high-affinity and this complex was recently shown to utilize mGluR5a as a scaffold to facilitate Aβ42 oligomer-dependent intracellular signaling [9,41,42]. Moreover, Aβ42 oligomers were shown to stimulate the subcellular redistribution of CaMKII resulting in impaired α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking . Therefore, we assessed whether the co-expression of mGluR5a and PrPC would alter the subcellular localization of GFP-CaMKIIα in HEK 293 cells. The expression of mGluR5a and PrPC did not alter the subcellular distribution of GFP in HEK 293 cells (Figure 7A). In addition, 30 μM quisqualate treatment did not affect the diffuse cytosolic distribution of GFP-CaMKIIα when it was expressed alone in HEK 293 cells (Figure 7B). Co-expression of either mGluR5a or PrPC alone with GFP-CaMKIIα did not alter the subcellular localization of GFP-CaMKIIα in response to 30 μM quisqualate treatment (Figures 7C and D). However, co-expression of both mGluR5a and PrPC along with GFP-CaMKIIα resulted in the subcellular redistribution of GFP-CaMKIIα in intracellular cytoplasmic puncta in 13% of transfected HEK 293 cells following 30 μM quisqualate treatment (Figures 7E and F). Thus, mGluR5a and PrPC were both required for agonist-stimulated alterations in the subcellular localization of GFP-CaMKIIα.
Effect of mGluR5a and PrP C expression of GFP-CaMKIIα subcellular localization following agonist stimulation. HEK 293 cells were transiently transfected with different combinations of GFP-CaMKIIα (0.5 μg), FLAG-mGluR5a (2 μg), PrPC (2 μg) or empty pEGFP (2 μg) pcDNA3.1 plasmid cDNA. Shown are representative confocal microscopic images of HEK 293 cells transfected with A) pEGFP (control) along with both FL-mGluR5a and PrPC, B) GFP-CaMKIIα alone (green), C) GFP-CaMKIIα along with FL-mGluR5a (red), D) GFP-CaMKIIα along with PrPC (blue), and E) GFP-CaMKIIα along with both FL-mGluR5a and PrPC. All transfections were treated with 30 μM quisqualate for 20 min. F) Quantification of the number of cells exhibiting GFP-CaMKIIα puncta, number of cells imaged is shown in brackets. Data is representative 4 different experiments.
In the present study, we screened for novel proteins that may interact with the IL2 domain of mGluR1 and mGluR5. In doing so, we identified a number of functionally interesting putative Group I mGluR-interacting substrates, several of which were previously described as Group I mGluR-interacting proteins [19,21]. Specifically, we found that Gαq/11, the catalytic subunit of protein phosphatase 1γ and the regulatory subunit of protein phosphatase 2A, known mGluR5-interacting proteins, associate with the mGluR1/5 IL2 domain. Here, we show that CaMKIIα also interacted with the mGluR1/5 IL2 domain, in addition to its reported interaction with the mGluR1a and mGluR5a carboxyl-terminal tail [36,37].
CaMKIIα is known to both interact with and contribute to the desensitization of ionotropic glutamate receptors, as well as Gαq/11-coupled dopamine D1 and D3 receptors [30,31,44]. CaMKIIα is reported to interact with mGluR1a via its association with the mGluR1a carboxyl-terminal tail in a Ca2+-dependent manner and contributes to the subsequent attenuation of endogenous mGluR1a signaling in striatal neurons . CaMKIIα has also been shown to interact with the carboxyl-terminal tail of mGluR5a in rat striatal neurons in a calcium dependent manner . These two proteins dissociate upon activation of mGluR5 calcium and CaMKII subsequently binds to and phosphorylates the adjacent GluN2B subunit of the NMDA receptor, suggesting a mechanism by which mGluR5/CaMKIIα interactions could potentially mediate long-term potentiation . We find here that CaMKIIα can be co-immunoprecipitated with both mGluR1a and mGluR5a in an agonist-independent manner and that it interacts with a GST-fusion protein encoding IL2 of mGluR1/5. This indicates that both the IL2 and carboxyl-terminal tail domains play a role in regulating CaMKIIα interactions with Group I mGluRs. The interaction of CaMKIIα with mGluR1a is reported to phosphorylate and desensitize agonist-stimulated mGluR1a signaling in striatal neurons and CaMKII activity is suggested to regulate G protein-coupled receptor kinase 2 (GRK2)-mediated desensitization of mGluR1a [35,36]. However, we previously found that DHPG-stimulated inositol phosphate signaling in mouse striatal neurons is mediated predominantly by mGluR5a . Thus, either rat striatal neurons express mGluR1a at different levels than those found in mice or the interaction of CaMKII with mGluR5a in rat striatal neurons may also contribute the regulation of mGluR5a in these cultures. This desensitization may also involve CaMKII-mediated regulation of GRK2-dependent Group I mGluR desensitization, as endogenous GRK2 expression is essential for regulating mGluR5 signaling in mouse striatal neurons and both CaMKII and GRK2 bind to the mGluR1/5 IL2 domain [35,39,45].
Previous studies have demonstrated that many regulatory proteins interact with both the IL2 and carboxyl-terminal tail domains of mGluR1/5, including heterotrimeric G protein, GRK2, phospholipase D1, RalA, Arf6 and Pyk2 [3,17,22,39]. Thus, it is not unprecedented that binding to the intracellular face of an integral membrane protein would involve multiple intracellular receptor domains. However, although desirable, it is difficult, if not impossible to distinguish the relative importance of CaMKIIα interactions with the IL2 or carboxyl-terminal tail domains of mGluR1/5. Furthermore, previous studies identified interactions between the IL3 of the D3 dopamine receptor and CaMKII and the carboxyl-terminal tail of mGluR1a and CaMKII, indicating that both loop and tail domains of GPCRs may contribute to CaMKII interactions [27,30]. However, both of these studies failed to examine whether other intracellular domains of the receptor could also interact with CaMKII and rather only examined in vitro interactions with GST fusion proteins, which may have led to the potential misconception that CaMKII binds to a single discrete domain in both receptors.
We show that CaMKIIα overexpression increases the agonist-stimulated internalization of both mGluR1a and mGluR5a and that this is dependent upon the catalytic activity of the enzyme as KN-93 treatment blocked CaMKIIα-dependent enhancement of mGluR1a endocytosis. These results are similar to those previously reported for the GABAB receptor, another Class C receptor . The antagonism of CaMKIIα also reduces both the heterologous and agonist-stimulated internalization of mGluR1a and its alternative splice variants [34,35]. Interestingly, the agonist-stimulated internalization of mGluR1a appears to be impaired by its extended carboxyl-terminal tail, as the mGluR1b and mGluR1c splice variants exhibit enhanced internalization, which is effectively blocked by KN-93 . In contrast, both we and Mundell and colleagues (2002) find that internalization of mGluR1a in the absence of CaMKIIα overexpression in HEK 293 cells is not effectively blocked by KN-93 treatment. Thus, although the C-terminus of mGluR1a contains the consensus site for CaMKIIα phosphorylation, the extended C-terminus may impede interactions with IL2, which we find is also involved in the interaction of CaMKIIα with the receptor. This impediment may be overcome in neurons where CaMKIIα is an abundantly expressed protein.
We find that the overexpression of CaMKIIα differentially effects the regulation of ERK1/2 phosphorylation in response to the activation of both mGluR1a and mGluR5a. Specifically, mGluR1a-stimulated ERK1/2 phosphorylation is unaffected by the overexpression of wild-type CaMKIIα. In contrast, over-expression of wild-type CaMKIIα results in the attenuation of ERK1/2 phosphorylation in response to agonist stimulation of mGluR5a. Consistent with these observations, CaMKII is known to affect DHPG-mediated ERK1/2 phosphorylation in striatal neurons . Specifically, the treatment of striatal neurons with the CAMKII inhibitor, KN-62, resulted in attenuation of DHPG-induced ERK1/2 phosphorylation in the rat striatum . We also find that the attenuation of mGluR5a-mediated ERK1/2 phosphorylation by CaMKIIα is dependent upon CaMKIIα activity, as the overexpression of the autophosphorylation-incompetent CaMKIIα-T286A mutant did not alter mGluR5a-stimulated ERK1/2 phosphorylation. CaMKIIα has been previously shown to phosphorylate the carboxyl-terminal tail at a CaMKII consensus sequence R/K-x-x-S/T (Thr871) localized within the carboxyl-terminal tail of mGluR1a . This consensus site is also conserved within the carboxyl-terminal tail of mGluR5a (Ser860). mGluR1a also contains three additional putative consensus sites for CaMKII phosphorylation and is phosphorylated at a secondary site by CaMKII at Thr945, which is not conserved in mGluR5a . The mGluR5a carboxyl-terminal tail encodes an additional seven putative CaMKII phosphorylation consensus sequences that are not conserved in mGluR1a (Ser853, Ser879, Ser871, Ser984, Ser1016, Thr1156 and Thr1165). Thus, the differential phosphorylation-dependent regulation of mGluR1a- and mGluR5a-mediated ERK1/2 phosphorylation may be the consequence of mGluR5a phosphorylation at sites not present in the mGluR1a C-terminal tail.
We and others show that CaMKII can be co-immunoprecipitated with full length mGluR1a/5a and GST fusion proteins encoding the IL2 and carboxyl-terminal tail domains of mGluR1a/5a. However, this does not necessarily indicate that CaMKII forms a stable complex with the receptors required to phosphorylate and desensitize the receptors or regulate their endocytosis and activation of ERK1/2 phosphorylation . It is entirely possible that CaMKII is activated in response to Group I mGluR-stimulated Ca2+ release downstream of G protein activation as opposed to forming a constitutive component of the mGluR1a/5a signaling complex. Nevertheless, regardless of whether the regulation of Group I mGluR desensitization, endocytosis and ERK1/2 phosphorylation requires a stable physical interaction with the receptor, transient receptor interactions and/or the phosphorylation of downstream regulatory proteins, CaMKII clearly plays an important role in regulating Group I mGluR activity.
Recently, it has been reported that mGluR5a functions as the co-receptor for PrPC and functions as a scaffold/co-receptor for both PrPC- and Aβ42 oligomer-mediated intracellular signaling [42,48]. Aβ42 oligomer binding to mGluR5 stimulates the clustering of mGluR5 and increases Ca2+ signaling . We find here that Aβ42 oligomer treatment of HEK 293 cells increases the association of CaMKIIα with mGluR5a and activates mGluR5a-dependent ERK1/2 signaling to an extent that is similar to agonist stimulation. Despite the fact that Aβ42 oligomer treatment leads to increased CaMKIIα association with mGluR5a, unlike what we observe for agonist-stimulated ERK1/2 phosphorylation, CaMKIIα interactions do not antagonize Aβ42-stimulated ERK1/2 phosphorylation. Nevertheless, both agonist- and Aβ42- stimulated ERK1/2 phosphorylation are protein kinase C-dependent suggesting a role for Ca2+ signaling. Thus, although both mGluR5a agonists activate ERK1/2 phosphorylation via a convergent downstream mediator, quisqualate and Aβ42 stabilize mGluR5a activation states that appear to be differentially regulated by CaMKIIα.
Aβ42 oligomers have previously been demonstrated to activate both p38 MAPK and ERK1/2 via their association with the α7 nicotinic acetylcholine receptor . Furthermore, Aβ42 oligomer-mediated activation of the β2-adrenergic receptor results in the β-arrestin-dependent activation of ERK1/2 and genetic deletion of the β2-adrenergic receptor ameliorates the pathophysiological deficits associated with the APPswe/PS1ΔE9 mouse model of AD . In addition, Aβ42 oligomers induce alterations in the subcellular localization of CaMKIIα leading to altered AMPA receptor trafficking . We find that the co-expression of both PrPC, an Aβ42 oligomer binding partner and mGluR5a, the co-receptor for both Aβ42 oligomers and PrPC , results in the redistribution of GFP-CaMKIIα into intracellular cytoplasmic puncta. The mechanisms regulating CaMKII clustering in HEK 293 cells and neurons is mediated by similar mechanisms . Thus, this observed change in CaMKIIα distribution in neurons leading to altered AMPA receptor localization in response to Aβ42 oligomer treatment of neurons may involve the association of Aβ42 oligomers and PrPC with mGluR5a.
In summary, we confirm that mGluR5a acts as a binding partner for CaMKIIα and demonstrate that in addition to associating with the mGluR5a C-terminal tail, CaMKIIα interacts with the IL2 domain of mGluR5a and mGluR1a . Moreover, we find that CaMKIIα overexpression facilitates the endocytosis of both mGluR1a and mGluR5a in a manner that is dependent upon the catalytic activity of the enzyme. In contrast, CaMKIIα selectively regulates agonist-stimulated mGluR5a-mediated ERK1/2 phosphorylation, but not Aβ42 oligomer-mediated ERK1/2 phosphorylation. Moreover, CaMKIIα subcellular localization is altered by mGluR5a and PrPC expression. Taken together these data suggest that not only does CaMKIIα regulate mGluR5a activity, but that Aβ42 oligomer signaling via its association with mGluR5a and PrPC may contribute to pathophysiological alterations in mGluR5a cell signaling associated CaMKIIα.
Adult CD-1 mice were from Charles River (Wilmington, MA). Human Embryonic Kidney (HEK293) Cells were from American Type Culture Collection (Manassas, VA). Cell culture reagents were from Invitrogen (Burlington, ON): Minimal Essential Media (MEM), Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS) and 0.25% Trypsin-EDTA. OmniPur Bovine Serum Albumin (BSA) was from VWR (Mississauga, ON). L-quisqualic acid (quisqualate) and KN-93 were from Tocris Bioscience (Minneapolis, MN). Biotinylation reagents EZ-Link Sulfo-NHS-SS-Biotin and NeutrAvidin Agarose Resin, as well as Pierce ECL Western Blotting Substrate and SuperSignal West Dura Chemiluminescent Substrate were purchased from Thermo Scientific (Rockford, IL). Myo-[3H] Inositol was purchased from Perkin Elmer (Waltham, MA). Protein G Sepharose beads were from GE Healthcare (Oakville, ON). ANTI-FLAG M2 Affinity Gel was purchased from Sigma-Aldrich (St. Louis, MO). A DC Protein Assay Kit was purchased from BioRad Laboratories (Mississauga, ON). Kodak X-Omat Blue Film was from Fisher Scientific (Ottawa, ON). Aβ42 peptide was purchased from American Peptide (Vista, CA, USA). Bisindolymaleimide I (Bis-1) hydrochloride was purchased from Calbiochem (San Diego CA, USA). CaMKII (pan), CaMKIIα, Phospho-p44/42 MAPK, p44/42 MAPK were purchased from Cell Signaling Technology (Danvers, MA). Rabbit mGluR1a and mGluR5a antibodies were purchased from Millipore (Billerica, MA), GFP antibody was purchased from Invitrogen (Mississauga, ON) and secondary mouse and rabbit antibodies were purchased from GE Healthcare (Oakville, ON) and BioRad (Mississauga, ON), respectively. GFP-CaMKIIα construct was from Dr. Paul De Koninck (Laval University, PQ).
Human embryonic kidney (HEK) 293 cells were cultured in MEM with 8% FBS. Cells were plated on 100 mm dishes and transfected using a modified calcium phosphate method  with cDNA amounts indicated in the Figure Legends. For transfection, cDNA was diluted to 450 μL in sterile distilled water, 50 μL 2.5 M CaCl2 added, 500 μL 2X HEPES-buffered saline (0.38 M NaCl, 0.05 M HEPES, 1.5 mM Na2HPO4, pH 7.05) added drop-wise and mixed gently before transfection mixture was added to cells. Cells were washed 16–20 hours post transfection and then allowed to recover in new media before experimentation. For co-immunoprecipitation, cells recovered for 24 hours. For all other experiments, cells recovered for 6–8 hours and then were reseeded into 6-well dishes and allowed to recover for 18 hours.
Neuronal cultures were prepared from cortical and striatal brain tissue from CD-1 E14.5 mouse embryo brains. Animal procedures were approved by The University of Western Ontario Animal Care Committee. After dissection, the tissue was submitted to trypsin digestion followed by cell dissociation using a fire-polished pasteur pipette. Cells were plated on poly-L-ornithine coated dishes in neurobasal media supplemented with N2 and B27 supplements, 2 mM of glutamax, 50 μg/ml penicillin, and 50 μg/ml streptomycin. Cells were incubated at 37°C and 5% CO2 in a humidified incubator and cultured for 12 days in vitro (DIV) with media replenishment every 4 days as previously described . Neurons (1 x 107 cells) seeded in each of three 100 mm dishes were incubated for 60 min with a 20 μM final concentration of a Tat-mGluR1/5-FLAG peptide (Ac-YGRKKRRQRRRIARILAGSKKKICTRKPRFMSDYKDDDDK-NH2) that encoded a cell permeant HIV Tat peptide at its amino-terminal end, followed by the amino acid sequence corresponding to the conserved second intracellular loop domains of mGluR1/5 and followed by a FLAG epitope tag. Subsequently, neurons were washed 3 times with PBS and solubilized in 1% Triton-X lysis buffer (25 mM HEPES, pH 7.5, 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA and 1% Triton X-100) containing protease inhibitors (1 mM AEBSF and 20 μg/mL of both leupeptin and aprotinin). Cellular debris was precipitated by centrifugation at 13,000 g for 30 min at 4°C. Cellular lysates were pre-cleared by incubation with a 20 μL volume of Protein A agarose beads for 6 h. Lysates were then incubated with a 40 μL volume of FLAG M2 resin for 4 h and washed 3X with Lysis buffer and 3 times with 50 mM NH4HCO3 pH 7.8. Subsequently, co-immunoprecipitates were eluted with 500 mM NH4OH at pH 11 in three 100 μL volumes and lyophilized to remove NH4OH. The samples were then resuspended in a 100 μL volume of H2O and subjected to a second round of lyophilization. Subsequently, samples were resuspended in a 50 μL volume of NH4HCO3 pH 8.0 and directly digested with sequencing-grade trypsin (Promega). The resulting peptide mixture was then analyzed by liquid chromatography-tandem mass spectrometry using a LTQ-XL Linear Ion Trap Mass Spectrometer (Thermo Scientific). The acquired tandem mass spectra were searched against a FASTA file containing the human NCBI sequences using a normalized implementation of SEQUEST running on the Sorcerer platform (Sage-N Research). The resulting peptide identifications returned by SEQUEST were filtered and assembled into protein identifications using peptide and protein prophets (Institute of Systems Biology, Seattle) as described previously .
HEK 293 cells were transiently transfected with various cDNA constructs as described in the Figure Legends. One day after transfection, cells were starved for 1 hour in HBSS (1.2 mM KH2PO4, 5 mM NaHCO3, 20 mM HEPES, 11 mM Glucose, 116 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 2.5 mM CaCl2, pH 7.4) and subsequently stimulated as indicated in the Figure Legends. Cells were washed on ice with cold Phosphate-Buffered Saline (PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.2) and then lysed on a rocking platform for 15 minutes at 4°C for using 0.1% Triton-X 100 lysis buffer (0.025 M HEPES, 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton-X) with added protease inhibitors: 1 mM AEBSF, 10 μg/ml leupeptin, and 5 μg/ml aprotinin. Lysate was collected and centrifuged at 15,000 RPM for 15 minutes at 4°C. 250 μg of each lysate was incubated with FLAG (FL)-agarose beads (50 μl bead slurry) for 1–2 hours. Beads were washed three times with cold PBS. Samples were eluted using 3x SDS sample buffer with 2-mercaptoethanol and separated by SDS-PAGE and co-immunoprecipitated proteins were detected by Western Blot.
The hippocampus was removed from CD-1 Adult Mice and placed in 0.5% Triton-X 100 lysis buffer with protease inhibitors. It was homogenized using a polytron and solubilized for 2 hours at 4°C. Lysate was then centrifuged at 15,000 RPM for 15 minutes at 4°C and 1 mg of protein was incubated with protein G-sepharose with or without mGluR5 antibody (Millipore, 1.5 μg) to immunoprecipitate mGluR5. Samples were eluted using 3x SDS sample buffer with 2-mercaptoethanol and separated by SDS-PAGE. Membranes were immunoblotted for immunoprecipitated mGluR5 (Millipore, 1:1000) and co-immunoprecipitated CaMKIIα (Santa Cruz, 1:250).
GST-mGluR1a-IL2 and mutants were cloned into a pGEX4T1 vector and transformed into E.coli recombinant bacteria . E. coli bacteria were grown at 37°C with shaking until OD600 was 0.6-1.0. Cultures were then induced with 1 mM isopropyl 1-thio-β-D-galactopyranoside for 3 hours at 23°C. Cells were pelleted and lysed in lysis buffer (500 mM NaCl, 0.5% NP-40, 50 mM Tris pH7.6, 5 mM EDTA, 5 mM EGTA) containing protease inhibitors (2 mM AEBSF, 50 mg/ml aprotinin, 20 mg/ml leupeptin) and sonicated (3 times for 10 seconds) at 4°C. Insoluble material was pelleted at 15000 g for 15 minutes at 4°C. 50 μl of Glutathione-Sepharose bead slurry was incubated overnight with 1 ml of solubilized protein to purify GST-fusion constructs. Glutathione-sepharose beads were then washed 3 times in PBS and 500 μg of HEK 293 cell lysates overexpressing GFP-CaMKIIα was added to the GST-fusion peptide bound to matrix and rotated for 1 hour at 4°C. Glutathione-sepharose beads were then washed 6 times in PBS and eluted with 3X SDS loading buffer containing β-mercaptoethanol. Samples were subjected to SDS-PAGE and membranes were immunoblotted with GFP to determine if GFP-CaMKIIα was pulled down with the GST-mGluR1a-IL2 peptides as described previously .
Twenty-four hours after transfection, cells were reseeded into 6-well dishes. The following day, cells were starved in DMEM overnight. On the day of experiment, cells were starved for an additional hour in HBSS. Cells were then stimulated at 37°C with either 50 μM quisqualate or 100 nM Aβ42 oligomers as indicated in the Figure Legends. For experiments using the PKC inhibitor, 1 μM Bis-1 was added 30 minutes prior to stimulating cells. Cells were subsequently lysed in lysis buffer (25 mM HEPES, 300 mM NaCl, 1.5 mM MgCl2, 200 μM EDTA, 0.1% Triton-X) containing protease and phosphatase inhibitors (1 mM AEBSF, 25 mg/ml aprotinin, 10 mg/ml leupeptin, 10 mM NaF, 100 μM Protein concentration was determined using a Bradford protein assay. The lysates were mixed with SDS loading buffer containing β-mercaptoethanol prior to gel loading. ERK1/2 phosphorylation was determined by immunoblotting for phospho-ERK1/2 and total ERK1/2 and the ratio was normalized to basal levels.
HEK293 cells were transiently transfected with receptor (FL-mGluR1a and FL-mGluR5a) and either pEGFP (control) or GFP-CaMKIIα (3 μg of receptor and 0.5 μg of GFP constructs). Cells were serum starved for 1 hour in HBSS for at 37°C on the morning of the experiment. Cells were washed and incubated on ice for 20 minutes in HBSS. Plasma membrane proteins were biotinylated at 4°C with EZ-Link Sulfo-NHS-SS-Biotin in HBSS and then incubated at 4°C in 100 mM glycine in HBSS for 30 minutes to quench biotinylation. Cells were then stimulated with 50 μM quisqualate for 0, 5 and 15 minutes, which allowed the receptor to internalize. Remaining cell surface biotin was stripped using 100 mM sodium 2-mercaptoethanesulfonate (MesNa) in TE Buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8.6) with 0.2% BSA. A control without stimulation or stripping was kept on ice and used to assess amount of total cell surface receptor. Cells were lysed, biotinylated protein pulled down with NeutrAvidin agarose resin (50 μL bead slurry), eluted with 3x SDS sample buffer containing 2-Mercaptoethanol, separated by SDS-PAGE and immunoblotted for mGluR1a and mGluR5a (Millipore, 1:1000). Internalization of the receptor at various time points with and without GFP-CaMKIIα was compared to GFP transfected control cells. Results are expressed as percent cell surface internalization. Protocol was modified slightly for mGluR1a experiments. Following serum starving, cells were pretreated for 1 hour with or without 1.0 μM KN-93 in HBSS. For stimulation, cells were stimulated with 50 μM quisqualate for 0 and 15 minutes.
Lyophilized Aβ42 peptides stored at -80°C were allowed to equilibrate to room temperature prior to dilution to 1 mM with 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). HFIP was evaporated in a vacuum centrifuge in order to form Aβ42 peptide films and films were then stored at -80°C. Prior to use, Aβ42 peptide films were diluted in dimethylsulphoxide (DMSO) to 1 mM and sonicated for 10 minutes in a Branson sonicator. Aβ42 peptides were then subsequently diluted to 100 μM in ice-cold F-12 cell culture media (phenol free red), vortexed immediately for 30 seconds, and incubated at 4°C for 24 hours in order to form Aβ42 oligomers.
Confocal microscopy was performed using a Zeiss LSM-510 laser scanning microscope equipped with a Zeiss 63X 1.4 numerical aperture oil immersion lens. Live cell imaging was performed on HEK293 cells in 35 mm glass-bottomed plates. mGluR5a was labeled with rabbit anti-FLAG conjugated Zenon Alexa Fluor 647 antibody and PrPC was labeled with mouse anti-PrPC conjugated Zenon Alexa Fluor 555 IgG2B antibody. Visualization of labelled proteins with GFP-CaMKIIα was performed by triple excitation (488/543/647 nm), emission band pass from 505–530 (GFP), long pass at 560 (Alexa Fluor 555) and 660 (Alexa Fluor 647) filter sets. For internalization experiments, mGluR5a was labelled with rabbit anti-FLAG-conjugated Zenon Alexa Fluor 555 antibody. Visualization of antibody-labelled receptor with GFP-CaMKIIα was performed by dual excitation (488/543 nm) and emission band pass from 505–530 (GFP) and long pass at 560 (Alexa Fluor 555) filter sets. Receptor was stimulated with the addition of 30 μM quisqualate (final concentration) for 20 minutes.
Immunoblots were quantified using Image Lab software. GraphPad Prism software was used to analyze data for statistical significance as well as to analyze and fit dose–response curves. Statistical significance was determined by either an unpaired two-tailed t-test or by one-way ANOVA followed by Tukey's post hoc multiple comparison's test.
Fitore Raka and Andrea R Di Sebastiano contributed equally to this work.
S. S. G. F. holds a Tier I Canada Research Chair in Molecular Neurobiology and is a Career Investigator of the Heart and Stroke Foundation of Ontario.
This work was supported by the Canadian Institutes of Health Research [grant number MOP-119437] to S. S. G. F.
FR and ARD performed experiments, data analysis and writing of the manuscript. SCK performed experiments and data analysis. FMR and CMG assisted with many of the experiments and provided intellectual input. FAC assisted with the confocal microscopy experiments and provided intellectual input. SA: Assisted in the discovery of CaMKIIα as a novel second intracellular loop interacting protein of Group I mGluRs. SSGF: Principle investigator, contributed to the experimental design, data analysis and writing of the manuscript. All authors read and approved the final manuscript.


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Finding Sophie Scholl in the ICPL databases
by Jennifer Eilers on March 30th, 2017
This morning a crowd of very excited middle schoolers from a local school bounded up to the second floor of the library to do research. Having other excited researchers flooding up the quiet, second floor stairs buoys the heart of a librarian like nothing else. I spoke with their teacher about the project they were working on. The students wanted to know how media played a role in the resistance movements against the Nazi party in WWII.
The group of girls I talked to were going to put on a play about the White Rose Movement. I had never heard about this movement before or Hans and Sophie Scholl. Before even coming up to the desk, the girls had basically cleaned the library out of all obvious available books and DVDs we had on the subject, so, my challenge was to see what else I could find about the group to point them to.
I did some basic research using Wikipedia to kind of get me started. The White Rose movement was a leaflet and graffiti campaign that was headed by Munich University students Hans Scholl and Sophie Scholl, Alexander Schmorell, Willi Graf, and professor, Christoph Probst. Hans and Sophie were the first of the group to be tried by the People's Court for distributing propaganda materials decryin g the Nazi regime. The two were beheaded on February 22, 1943 for treason. One of the pamphlets written by the group was smuggled out of the country, printed by the Allied forces, and dropped over German cities. Before their deaths the group had distributed close to 15,000 leaflets.
Within our collection's access, I was able to find a burial record in Ancestry.com for Sophie Scholl which shows the memorial and the headstones marking her burial plot. I found a newspaper article in the New York Times written in 1947 about how German schools were using Sophie Scholl's heroism to revive the broken spirits of school children. There was a thorough article in the History Review about the White Rose movement and how it compared with other more affluent and organized political groups opposing the Nazis. And finally there was a small article publishing the song lyrics that were believed to be the inspiration for the movement's name.
I'm always surprised by the breadth of the materials we have access to through our databases. There were more full text articles beyond those I've listed above. If you're doing some research either for your own knowledge or for a school project see what you can find or ask one of us at the Info Desk to help you! Needless to say after helping these girls research this topic, I became fascinated with Sophie Scholl myself. I've already put holds on some of the books the girls had checked out in our collection. You can check them out as well:
Sophie Scholl the Final Days
We will not be silent : the White Rose student resistance movement that defied Adolf Hitler
Women Heroes of World War II : 26 stories of Espionage, Sabotage, Resistance, and Rescue
Rad Women Worldwide : Artists and Athletes, Pirates and Punks, and Other Revolutionaries Who Shaped History
Published in From the Information Desk
One Response to "Finding Sophie Scholl in the ICPL databases"
Michelle Morey says:
Thanks for this truly awesome piece! As a teacher librarian I loved reading it and hearing about some rad local kids.
Why is my Christmas Cactus blooming in March?
National Bookmobile Day Celebration
Jennifer Eilers at the Library