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Milton council to hold hearing on Granary master plan
Friday, November 4, 2022
Milton planners grant preliminary approval for Granary master plan
Tuesday, October 4, 2022
Milton planners begin review of Granary master plan
Saturday, September 10, 2022
DEStorage proposal begins review process in Milton
Tuesday, August 16, 2022
The Granary asks for special development district designation
Tuesday, August 9, 2022
Milton council gives final approval to Granary annexation
Friday, May 6, 2022
Granary annexation a smart move for Milton
Friday, March 11, 2022
Milton council approves Granary annexation
Thursday, March 10, 2022
Milton council to vote on Granary annexation
Friday, March 4, 2022
Milton council tables Granary vote
Thursday, February 10, 2022
Milton Town Council unanimously approved the master plan for the proposed Granary at Draper Farm development, paving the way for the project to go to the planning and zoning commission to begin the site-plan review process.
Council held a public hearing Nov. 7 on the master plan, which serves as a road map for how the development will be planned. While details are still to be worked out in the site-plan process, the master plan allows city officials to look at street design and how the pieces of the proposed 1,350-unit development will fit together.
The Granary is planned for a 450-acre site off Sand Hill Road to be built out over a 20-year period. Besides a mix of single-family homes and townhouses, the development also includes 110 acres of dedicated open space, a small-batch brewery, a walking/biking trail, an amphitheater and 60,000 square feet of potential commercial space. The parcel is zoned R-2 residential with a large-parcel development overlay; the development is slated to take place over 10 phases. 
The public hearing began with comments by David Hutt and Zac Crouch, the attorney and engineer for developer Convergence Communities. Crouch said issues raised by the planning and zoning commission regarding street design have been addressed in the latest plan. Street design is a major issue because the streets will be dedicated and maintained by the town after the development is complete. 
During the planning and zoning review, alleyways were viewed as a concern, in part due to their effect on lot sizes. Concerns also included ownership and width of the alleyway. The commission and engineers from Convergence Communities agreed on the minimum width of 16 feet for alleys that only serve single-family homes and have two points of access to a public street. When a lot fronts open space, the alley must be a minimum of 24 feet wide. Either way, the commission agreed the alleys are wide enough for large trucks such as trash trucks. 
Councilwoman Lee Revis-Plank raised concerns about how the alleyways are located in the densest areas of the development. 
“I don’t think it’s safe to have that density of population without having wide enough streets,” she said.
Crouch said the alleyways do not include any parking and are not intended to be main access roads. He said the alleyways are intended to service garages. Councilwoman Randi Meredith defended the use of the alleyways, saying a wider road would encourage more speeding. 
Several comments came from residents of neighboring Bangor Lane, which is not part of Milton, but would be directly affected by the development. Their questions centered around buffers to nearby Diamond Pond, traffic, and how the development will fit in with Milton. Mayor John Collier said many of those issues will be settled in the site-plan review process, but plans for the project have included buffers around Diamond Pond. 
 
The price of liberty is eternal vigilance.

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Potter Noah Hughey-Commers has been creating new wood-fired stoneware items for this year’s Artisans Studio Tour, which takes place Saturday and Sunday and features 39 artisans in 21 different studios.
Johannah Willsey of Phoenix Handcraft, who will display furniture and wall art in forged steel, hand-cut glass mosaic and wood with Kyle Lucia, will be on hand at the Charlottesville studio of quilt artist Mary Beth Bellah. Look for Studio 13 on the Artisans Studio Tour.
Twice a year, potter Noah Hughey-Commers fires up his kiln at Muddy Creek Pottery in Lovingston and spends seven days tending flames. What can be a solitary pursuit the rest of the year becomes an event that unifies fellow artisans in fellowship, community and mutual support.
“I’ve just finished a busy show season,” said Hughey-Commers said. “I got back this morning and lit up my kiln, which I only do about twice a year. It’s kind of a community thing. I host about nine other potters, and it takes about ten of us to fire the kiln.
“I do a four-day firing with three days of low heat before that. It’s a wood firing trying to melt the wood ash.”
Keeping the kiln at a consistent 2,400 degrees means the fire must be fed around the clock to ensure the finest results for Hughey-Commers’ 300 or so pots and another 500 created by the colleagues who help stoke the flames. By representing both individual artistic achievements and community teamwork that lifts small businesses and amplifies a shared love of making objects by hand for daily use and decoration, the resulting creations can symbolize the Artisans Studio Tour.
The 28th annual Artisans Studio Tour, scheduled for 10 a.m. to 5 p.m. Saturday and Sunday, will take visitors on self-guided journeys to 21 different Central Virginia studios to meet 39 artists and artisans in Charlottesville and Albemarle, Greene, Madison, Nelson and Amherst counties.
Visitors who drop by Hughey-Commers’ studio can get a closer look at how a favorite coffee cup or vase has made the transformation from clay to class. Adam McNeil also will be there with his own collection of functional ceramics.
“I’m lucky in that my space is in the same building as my kiln,” Hughey-Commers said. “They’re in the place where it is made to connect the dots.”
The tour will give visitors a chance to glimpse the processes behind ceramics, fiber arts, jewelry and wood items that reflect a wide variety of aesthetic approaches and tastes.
“I specialize in functional dinnerware, and I make a lot of ornamental vases,” Hughey-Commers said. “I make a lot of teaware and coffeeware.”
There’s a lot to take in on the tour, and he recommends checking out the website at www.artisansstudiotour.com and picking up the maps provided at studios along the way so you can plan your own individual tour. Hoping to sample local ciders or tuck in a short mountain hike amid the fall colors? Just add your own tour stops along the way for local color and flavor. It’s also possible to check out one group of studios on Saturday and a totally different combination on Sunday.
“We send out a really helpful map,” Hughey-Commers said. “I would just pick a zone. There are so many great B&Bs, if you want to make a weekend of it. There are so many great wineries in Nelson County, where I am, and breweries.”
And if you’re a fan of nature’s own palette, Hughey-Commers said the beauty of Central Virginia you’ll encounter along the tour will enhance the experience.
“You’ll get to see the mountains in a whole new way,” he said.
Head to artisansstudiotour.com to map out your own personal tour and learn more about the following stops:
Studio 1: Hean Cabinetry at 3226 Lonesome Mountain Road in Charlottesville. (434) 962-3918. Fred Hean’s furniture, accessories and custom cabinetry.
Studio 2: Phineas Rose Studios at 1112 Graves Mill Road in Madison. (540) 948-4248. Alec and Richard Gordon’s workshop and furniture showroom and Ninika Gordon’s jewelry showroom. Ninika Gordon will demonstrate Viking knitting with silver wire. Shari Jacobs will be there with wheel-thrown porcelain and stoneware.
Studio 3: Noon Whistle Pottery at 328 Main St. in Stanardsville. (434) 985-6500. Holly Horan’s mugs and John Pluta’s sculptures.
Studio 4: Mud Dauber Pottery at 4225 Earlysville Road in Earlysville. (434) 973-7943. Suzanne Crane’s wheel-thrown stoneware and Becky Garrity’s pottery for daily use.
Studio 5: Blaise Gaston at 686 Fairhope Ave. in Earlysville. (434) 973-1801. Blaise Gaston’s fine furniture; Jan Elmore’s wall-mounted sculptures, mirrors, painted paper collages and jewelry; and Nancy Ross with primarily wheel-thrown pottery finished by hand.
Studio 6: Heller and Heller Custom Furniture at 1611 Keith Valley Road in Charlottesville. (703) 716-2939. Dave Heller’s decorated furniture and craft items and Steve Palmer’s hand-built and wheel-thrown pottery.
Studio 7: Nan Rothwell Pottery Studio at 700 Wilder Drive in Charlottesville. (434) 263-4023. Nan Rothwell’s functional stoneware pottery.
Studio 8: Inleaf Studio at 721 Park St. in Charlottesville. (434) 882-1569. Lotta Helleberg’s nature-inspired wall hangings, artist books, scarves and other fiber creations.
Studio 9: Gabriel Ofiesh at 908 E. High St. in Charlottesville. Gabriel Ofiesh’s gold and silver jewelry with cabochon gems and pearls, plus his patented Orbit collection.
Studio 10: Tom Clarkson Pottery at 1213 Meriwether St. in Charlottesville. (434) 963-2650. Tom Clarkson’s ash-glazed stoneware pottery.
Studio 11: taviametal and Rani Morris at 1304 E. Market St. in Charlottesville. (434) 466-4042. Tavia Brown’s industrial yet delicate jewelry; goldsmith Rani Morris’ geometric, historic and South Asian designs; Susan Haas’s blown-glass pieces; and Stuart Howe’s functional pottery.
Studio 12: Short 18th Studio at 1510 Short 18th St. in Charlottesville. (434) 242-5813. Mary Cassell’s artistically designed pots.
Studio 13: Mary Beth Bellah, Quilt Artist, at 1855 Arrowhead Valley Road in Charlottesville. (434) 409-9213. Mary Beth Bellah’s three-dimensional and wall-hung art quilts; Elizabeth Krome’s functional and decorative clay work; furniture and wall art in forged steel, hand-cut glass mosaic and wood by Kyle Lucia and Johannah Willsey; and Katherine Maloney’s collectible and functional animal-inspired ceramics.
Studio 14: Wood Art by Kirk McCauley at 4264 Edge Valley Road in North Garden. ( 434) 293-7525 or (434) 953-6309. Kirk McCauley’s artistic and functional wood bowls, lamps, cowboy hats and whisky barrel furniture.
Studio 15: Pettigrew Woodworks at 2435 Rock Branch Lane in North Garden. (804) 363-6189. Alex Pettigrew’s functional and decorative home and office accessories crafted from local woods; Jessie Rublee’s functional stoneware pottery.
Studio 16: Muddy Creek Pottery at 470 Ponton Lane in Lovingston. (434) 760-3280. Noah Hughey-Commers’ functional wood-fired dinnerware; Adam McNeil’s functional ceramics finished in a high-fire wood kiln.
Studio 17: Tye River Pottery at 1289 Falling Rock Drive in Amherst. (434) 263-4065. Kevin Crowe’s functional pots. The kiln will be firing during the tour.
Studio 18: CM Glass Art at 160 Greenwood Farm in Greenwood. (804) 986-0853. Cherie Mehler’s functional and decorative fused-glass art.
Studio 19: The Barn Swallow at 796 Gillums Ridge Road in Charlottesville. (434) 979-4884. Janice Arone’s stoneware and porcelain clay vessels; Mary Ann Burk’s functional porcelain and stoneware; and Liz Hanson Metalsmith’s contemporary jewelry in sterling silver and 18-karat gold.
Studio 20: Sunset Farm Studio at 5026 Jones Mill Road in Crozet. (434) 242-0113. Fiber artist Sarah Tremaine’s natural garments, accessories, gifts and home décor; Cathy Vaughn will demonstrate coppersmithing techniques and tools.
Studio 21: Frederick Williamson Bowls at 5623 Sugar Ridge Road in Crozet. (434) 823-1882. Fred Williamson’s fine wood pieces made from local trees, including globes, open forms and salad bowls.
28th annual Artisans Studio Tour
10 a.m. to 5 p.m. Saturday and Sunday
21 studios in Charlottesville, Crozet, Lovingston, Stanardsville, Madison, Greenwood, North Garden, Earlysville and Amherst
www.artisansstudiotour.com
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Editor’s Note: Exhibits shares information about exhibitions presented on display or online in Charlottesville and Albemarle, Buckingham, Fluv…

“Blackity Black Black” embraces and celebrates diverse expressions of Black heritage and will be a statement of people, presence and pride. Self-identified Black artists residing in Virginia are eligible to apply. There is no application fee. For details, contact the curation team at [email protected].

A two hour ceremony was held in Massachusetts on Saturday to mark the symbolic return of about 150 items considered sacred by the Sioux peoples that had been stored at a small Massachusetts museum for more than a century. The ceremony included representatives of the Oglala Sioux and Cheyenne River Sioux Tribes. The artifacts will be officially handed over during a private ceremony. The items include weapons, pipes, moccasins and clothing. Several of the items are thought to have a direct link to the 1890 Wounded Knee Massacre in South Dakota. The items had been held by the Founders Museum in Barre, Massachusetts.

A new exhibit celebrating King Tut is about to land in several cities with a very modern twist, taking a digital look at the boy king for the Instagram age. Not a single golden treasure or artifact is on view at “Beyond King Tut: The Immersive Experience.” Instead, high-resolution digital projections of Tutankhamun’s world are splashed along walls and floors as visitors walk through nine galleries that chart his life, death and world. The exhibit, which opens Friday in New York City, coincides with the 100th anniversary of the tomb’s discovery. “Beyond King Tut” is open in Washington, D.C., and will be in Los Angeles and Vancouver, British Columbia, on Nov. 4.
Second Street Gallery will present “Artists in Conversation: Megan Marlatt + Akemi Ohira” at 11 a.m. Saturday. Exhibiting artist Marlatt and f…
Editor’s Note: Exhibits shares information about exhibitions presented on display or online in Charlottesville and Albemarle, Buckingham, Flu…

The Columbus Museum of Art has opened a comprehensive exhibit on artist and illustrator Maurice Sendak. “Wild Things are Happening” is the first major retrospective of Sendak’s work since his 2012 death and the largest and most complete to date. Sendak today is best known as the author and illustrator of such children’s book classics as “Where the Wild Things Are” and “In the Night Kitchen.” But Sendak also gained fame as a designer for opera, theater, film, and television. Lynn Caponera is the executive director of the Maurice Sendak Foundation in Ridgefield, Connecticut. She says one of the exhibit’s goals is to help people understand that Sendak was a serious artist in many mediums.
Potter Noah Hughey-Commers has been creating new wood-fired stoneware items for this year’s Artisans Studio Tour, which takes place Saturday and Sunday and features 39 artisans in 21 different studios.
Johannah Willsey of Phoenix Handcraft, who will display furniture and wall art in forged steel, hand-cut glass mosaic and wood with Kyle Lucia, will be on hand at the Charlottesville studio of quilt artist Mary Beth Bellah. Look for Studio 13 on the Artisans Studio Tour.
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The Mercedes-AMG One is now the fastest road-legal vehicle on the legendary Nürburgring track.
The near 13-mile circuit is the most famous — and most dangerous — in Germany.
Just 275 Mercedes-AMG One cars will be made and are already sold out despite costing $2.6 million.
The most powerful car ever produced by Mercedes-Benz has set a new record for street-legal vehicles on the legendary Nürburgring-Nordschleife racetrack in Germany.
A Mercedes-AMG One driven by Maro Engel zoomed round the track in just 6 minutes and 35.183 seconds, beating the previous record by a full eight seconds despite “less-than-ideal” track conditions, per Mercedes.
The 1,049bhp hypercar topped speeds of 210 mph as it set the new record on October 28.
The Mercedes-AMG One is legal to drive on public roads, but just 275 are due to be built. The run is already sold out despite the cars costing $2.6 million apiece.
The car is a plug-in hybrid and features Formula One technology. Each has a turbocharged 1.6-litre V6 engine, four electric motors and an 8.4kWh battery, per the Daily Mail.
The previous record by a road-legal production car around Nürburgring was set by the Porsche 911 GT2 RS Manthey-Racing in June 2021, which clocked the circuit in 6 minutes and 43.300 seconds.
 
Engel described his record-breaking lap in the Mercedes ATG-One as an “unforgettable experience.”
The German driver added: “I didn’t expect that we would be able to set such a lap time with these track conditions. In some crucial areas of the track, it hadn’t dried completely yet and was therefore tricky. That was a special challenge.”
The Nürburgring is the world’s most dangerous racetrack. It is just under 13 miles long and has more than 150 corners.
Read the original article on Business Insider
Chief medical adviser to the president Dr. Anthony Fauci, who's served under seven administrations, said he's stepping down from his position in December. But the immunologist won't be retiring completely. He discusses what's next for his career with "CBS Saturday Morning" co-host Michelle Miller.
With odds of a split U.S. Congress rising, Treasury Secretary Janet Yellen warned that lawmakers' failure to raise the statutory limit on U.S. debt posed a "huge threat" to America's credit rating and functioning of U.S. financial markets. Yellen told Reuters in an interview in New Delhi on Friday that cooperation is still possible with Republicans on some issues, but lifting the debt ceiling is a non-negotiable item. Some Republicans have threatened to use the next hike in the $31.4 trillion debt ceiling as leverage to force concessions from U.S. President Joe Biden, a Democrat.
The Giants got wide receiver Kenny Golladay back in the lineup on Sunday after he missed four games with a knee injury, but he spent most of the day on the bench. Golladay dropped a pass just before halftime of the 24-16 Giants win and he spent the second half watching from the bench. After [more]
U.S. President Joe Biden on Sunday met Australian Prime Minister Anthony Albanese and discussed their security pact and issues surrounding the Taiwan Strait, the White House said. The AUKUS security pact between the United States, the United Kingdom and Australia is seen as an effort by the Western allies to push back against China's growing power and influence, particularly its military buildup, pressure on Taiwan and deployments in the contested South China Sea.
Stocks could gain this year but the path will be volatile and absent a signal by the Fed it will start cutting rates soon, analysts say.
The Hall of Fame running back catches up with his old friend and center postgame after the Colts defeat the Raiders.
Six people were killed, and dozens wounded in Turkey after a bomb exploded on Istiklal Avenue, a popular tourist destination in Istanbul on Sunday.
Federal prosecutors are asking a judge to sentence disgraced Theranos CEO Elizabeth Holmes to 15 years in prison for lying to investors and putting patients' safety at risk.
Elon Musk has just put himself in an unenviable position. The Tesla CEO has been busy revamping Twitter for a few weeks now, after taking over the social network on October 27 for $44 billion. The billionaire, who has incurred a debt of $13 billion to pay for the acquisition, wants to find sources of income as soon as possible to make the platform profitable.
Republican election deniers are projected to constitute more than a third of the new House, which could give Trump a leg up.
The Pininfarina Battista can hit 62 mph in under two seconds, making it one of the fastest-accelerating cars in the world. Only 150 will ever be made.
Singapore-based crypto exchange said its chief executive will go live on YouTube on Monday to answer questions around some transactions on the platform that had sparked speculation and fund withdrawals. Chief executive Kris Marszalek said on Twitter on Sunday that the exchange had mistakenly sent 320,000 ether to another exchange called Gate.io on Oct. 21. Marszalek said the ether was recovered and returned to the exchange, but that failed to calm a jittery market.
Terry Barker was a retired American Airlines pilot. Another former airline employee from Tarrant County, Len Root, also died in the crash.
Another worker rescued one of the woman’s dogs days later.
A Russian Iskander-K missile carrying an unexploded cluster-bomb warhead that hit a residential area of the city of Zaporizhzhya on Nov. 12 has been successfully disposed of, Zaporizhzhya Oblast military administration head Oleksandr Starukh said on Telegram on Nov. 13.
The 2022 midterms saw several candidates who questioned the results of the 2020 presidential election lose their bids for office.
The Outer Space Treaty says space should be free for use by all nations and that celestial bodies should be used exclusively for peaceful purposes.
A bomb rocked a bustling pedestrian avenue in the heart of Istanbul on Sunday, killing six people, wounding several dozen and leaving panicked people to flee the fiery blast or huddle in cafes and shops. Turkish President Recep Tayyip Erdogan called the blast a “treacherous attack” and said its perpetrators would be punished. Sunday’s explosion was a shocking reminder of the anxiety and safety concerns that stalked the Turkish population during years when such attacks were common.
"Perhaps it is because your real account sounds like a parody?" Musk tweeted when asked by the senator how Twitter will prevent impersonations.
“The idea that you can inoculate yourself from criminal charges by just declaring yourself a candidate for some high office is ludicrous,” said Barbara McQuade.

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Communications Biology volume 5, Article number: 1232 (2022) Cite this article
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Here we use a combination of two-photon Fluorescence Lifetime Imaging Microscopy (FLIM) of NAD(P)H free/bound ratio in living HIs with post-fixation, immunofluorescence-based, cell-type identification. FLIM allowed to measure variations in the NAD(P)H free/bound ratio induced by glucose; immunofluorescence data allowed to identify single α and β cells; finally, matching of the two datasets allowed to assign metabolic shifts to cell identity. 312 α and 654 β cells from a cohort of 4 healthy donors, 15 total islets, were measured. Both α and β cells display a wide spectrum of responses, towards either an increase or a decrease in NAD(P)H free/bound ratio. Yet, if single-cell data are averaged according to the respective donor and correlated to donor insulin secretion power, a non-random distribution of metabolic shifts emerges: robust average responses of both α and β cells towards an increase of enzyme-bound NAD(P)H belong to the donor with the lowest insulin-secretion power; by contrast, discordant responses, with α cells shifting towards an increase of free NAD(P)H and β cells towards an increase of enzyme-bound NAD(P)H, correspond to the donor with the highest insulin-secretion power. Overall, data reveal neat anti-correlation of tissue metabolic responses with respect to tissue insulin secretion power.
Blood glucose levels are finely tuned in a narrow range by the regulated secretion of the pancreatic islet hormones insulin and glucagon in response to fluctuations in glucose and other metabolites concentration^1,2,3,4. A cascade of highly regulated biochemical processes connects glucose influx to hormone secretion in specialized cells within the islets of Langerhans of the mammalian pancreas, i.e., the α and β cells^2,3,4,5,6. Such regulation is progressively lost in diabetes, leading to onset and worsening of the disease^7,8. Given the importance of these processes for systemic glucose homeostasis and for our comprehension of their mis-regulation in pathology, non-invasive and fast strategies capable of monitoring quantitatively α and β cells’ metabolic responses in living islets are highly desirable. Typically, the efficiency of the overall process can be described by monitoring the amount (and timing) of secreted hormones with respect to the amount of the administered stimulus by quantitative ELISA-based assays. These assays provide a useful experimental platform to monitor the general responsiveness of α and β cells to stimulation and the possible alteration of their behavior in the pathological condition⁹, although they average out the biochemical processes that occur in between stimulation and secretion. On the other hand, the possibility to monitor directly molecular processes within living α and β cells by using suitably labeled target molecules (e.g., by introducing genetically encoded fluorescent proteins) and optical microscopy is challenged by the need of not to alter the chemical identity and endogenous stoichiometry of the biochemical components involved in the metabolic response of these cells¹⁰. In this regard, in the pioneering works by the Piston’s Group, two-photon excitation microscopy was used to image and quantify the intrinsic NAD(P)H auto-fluorescence from both dispersed mouse-derived α/β cells and intact pancreatic islets under glucose stimulation^11,12,13. The authors were able to measure the change in whole-cell NAD(P)H levels in response to glucose stimulation and spatially resolve NAD(P)H signals from the cytoplasm and mitochondria of cells³. Using this intensity-based approach, additional studies were conducted to evaluate islet metabolic response to several stimuli, both in mouse¹⁴ and human¹⁵ systems. However, NAD(P)H intensity-based measurements may contain artifacts due to the heterogeneity of fluorophore concentration and to the differing quantum yields of NAD(P)H species¹⁶. To bypass this limitation, Fluorescence Lifetime Imaging Microscopy (FLIM) emerged as a potential useful tool as it is minimally affected by cell absorption/scattering and/or fluctuation in excitation intensity and, in addition, it can selectively discriminate the free and protein-bound forms of NAD(P)H molecules^17,18. So far, FLIM on NAD(P)H, especially in combination with the phasor-based graphical and fit-free data-analysis approach^19,20,21,22, found widespread use in the study of cell metabolism and its possible alteration during processes such as disease progression, differentiation, cell fate, cell division^21,22,23,24. For what concerns islet metabolism, phasor-FLIM analysis of NAD(P)H species was used to monitor the metabolic status of both mouse- and human-derived intact islets under different stimuli, including pulsed glucose stimulation. For instance, in the pivotal works by Gregg and collaborators on aging and obesity, phasor-FLIM of NAD(P)H signal was used in both mouse- and human-derived intact islets under different stimuli^25,26. The authors, however, interpreted the observed metabolic responses as deriving exclusively from β-cells because of their dominance in the islet, effectively forgoing to identify α cells and quantify their fractional contribution to overall islet response. Similarly, Haythorne and collaborators measured islets from both healthy and diabetic mouse models using phasor-FLIM of NAD(P)H signal but attributed the obtained metabolic response again to the (dominant) contribution of β cells²⁷. Of note, Wang and collaborators recently set out to discriminate the specific responses of α and β cells to glucose stimulation²⁸. The authors started from dispersed primary mouse α and β cells and used a combination of phasor-FLIM of NAD(P)H signals to study metabolism and immunofluorescence to identify cell types²⁸. Interestingly, a neat discordant response to glucose from the two types of cells was observed, with α cells shifting towards a decrease in the bound/free NAD(P)H ratio and β cells towards a concomitant increase of the same ratio²⁸. The authors extended similar conclusions to α and β cells measured within the intact mouse islet, where cell-cell interactions and possible paracrine effects persist, but for cell-type identification they relied in this case mainly on the assumption that α cells are located in the periphery and β cells in the core of the mouse islet²⁸. Finally, the authors measured human-derived islets from donors with and without T2D but, due to the more dispersed distribution of α and β cells in the human islets compared to mouse islets, they were unable to reliably identify cell types and thus characterize their specific response to glucose²⁸. Here, after validation of phasor-FLIM by using an immortalized cellular model of β cells (i.e., Insulinoma 1E cells, hereafter INS-1E cells, Fig. S1), we addressed the specific metabolic responses of α and β cells from living human-derived Langerhans islets by using an experimental protocol consisting of three main phases, namely: (i) NAD(P)H auto-fluorescence lifetime imaging in live islets at both low (2.2 mM) and high glucose (16.7 mM), with subtraction of the lipofuscin intrinsic signal, typical of human pancreatic islets (ii) islet fixation and immunostaining using antibodies against glucagon and insulin to identify single α and β cells, respectively, (iii) matching of immunofluorescence and live-islet acquisitions to extract single-cell information from phasor-FLIM data. By this approach, 312 α cells and 654 β cells were identified from 15 total islets (HI) of 4 non-diabetic human organ donors, and analyzed. While demonstrating the effectiveness of an optical-microscopy-based protocol to measure the specific responses of α and β cells in a living human Langerhans islet, present results suggest an emergent relationship between the optical signature of HIs and their insulin secretion power of potential biomedical/clinical interest.
The experimental approach is schematically represented in Fig. 1. It consists of three main phases, namely: (i) NAD(P)H auto-fluorescence lifetime imaging at a focal plane in live islets at both low (2.2 mM) and high glucose (16.7 mM), with subtraction of the lipofuscin intrinsic signal, to produce whole-islet metabolic data (Fig. 1a–d), (ii) islet fixation and immunostaining using antibodies against glucagon and insulin to identify single α and β cells and achieve single-cell and cell-type resolution (Fig. 1e, f), (iii) spatial matching of immunofluorescence and live-islet acquisitions to extract single-cell information from phasor-FLIM metabolic data (Fig. 1g). To start, we calibrated our system using an immortalized cell line that is a recognized model of β cells: Insulinoma 1E (INS-1E) cells (Fig. S1; experimental details can be found in the “Methods” section). Results on INS-1E cells are reported in Fig. S1: in brief, glucose stimulation induces a neat shift of INS-1E cells towards an increase in bound/total NAD(P)H ratio (hereafter referred to as “shift towards bound NAD(P)H”; conversely, a shift towards a decrease in bound/total NAD(P)H ratio will be referred to as “shift towards free NAD(P)H”), in keeping with previous results by some of us²⁹ and others²⁸. The bound/total NAD(P)H ratio is calculated from the barycenter of the phasor using Eq. 3 in “Methods” and Fig. S2. At this point, we started experiments on HIs. Before imaging, stabilization of HIs was achieved by using agarose at 1% in SAB medium, which allowed reducing islet mobility throughout the imaging/stimulation experiments while preserving islet viability (see “Methods” for further details). Islet auto-fluorescence was elicited by 2-photon excitation at 740 nm and collected in the 420-460-nm emission range. The optical-sectioning depth was chosen in the 10–15 µm range from the glass and kept constant for all the acquisitions: this choice was dictated by the observation that light penetration (and consequently imaging) deteriorates with depth, due to absorption/scattering by the tissue. As control, the efficiency of light penetration in a live islet stained with Hoechst was compared with the same experiment conducted after islet fixation and clearing with the TDE/PBS solution^30,31 (Fig. S3, see also “Methods” for further details). At the selected optimal depth, FLIM measurements were performed and analyzed using the phasor approach, allowing the lifetimes measured at each pixel in the image to appear in the corresponding region of the phasor plot. (Fig. 2a, b). As can be appreciated from the phasor plot reported in Fig. 2b, a rather broad distribution of lifetimes is measured in the live-islet optical section, positioned along the expected “metabolic segment”. Contrary to INS-1E cells, in this case a main correction needs to be made before assigning the measured lifetimes univocally to NAD(P)H. As suggested in previous works, in fact, the auto-fluorescence signal coming from lipofuscin-containing aggregates, which are typical of α and β cells in HIs, can potentially interfere with NAD(P)H imaging and lifetime analysis²⁹. Indeed, at the imaging conditions selected here, intense punctuate signal was detected in the majority of cells (Fig. 2c). Interestingly, considering the 4-donors cohort used in this study, the overall amount of lipofuscin detected by imaging was found to correlate with donor age and Body Mass Index (BMI) (Fig. S4a, b; refer also to Tabs. S1 and S2 for donor-related parameters), a result in keeping with previous systematic studies conducted on human islets³⁰. Lipofuscin was subtracted by introducing a filter based on the intensity (see “Methods” for further details). This correction eliminates a lifetime component with a peculiar location in the phasor plot, i.e., close to free NAD(P)H on the metabolic segment (Fig. 2d), that would affect the estimate of the phasor barycenter and the calculation of the bound/total NAD(P)H ratio (Fig. 2e). Yet it must be noted that, since the amount of lipofuscin does not change after stimulation, its subtraction has a negligible effect on the calculation of the metabolic shift (Fig. S4c, d). At this point, the signal left after lipofuscin subtraction was attributed to NAD(P)H auto-fluorescence, and the basic experiment of glucose stimulation was performed by acquiring two consecutive FLIM images for each islet: the first at low glucose concentration (2.2 mM, after incubation in the SAB solution for 45 min), the second after exposure of the islet to high glucose concentration (16.7 mM). All stimulated islets were measured within 15 min from glucose administration. The corrected bound/total NAD(P)H ratios from all measured islets (N = 15), before and after stimulation, are reported in Fig. 2f. Overall, glucose stimulation induced a metabolic shift towards bound NAD(P)H in the majority of the islets. This average result is in keeping with previous observations on human islets^26,27 but, as discussed above, it does not contain any information on the specific contribution from α and β cell populations in the islet.
a Human pancreatic islet imaging requires immobilization (using agarose gel, see Methods) and correct focal plane positioning, due to the high scattering properties and absorption process of the tissue by chromophores and pigments (i.e., lipofuscin), which prevent deep light penetration. At the selected plane (b), tissue auto-fluorescence lifetime was measured at 740 nm and collected in the 420–460-nm range. The first measurement was performed at 2.2 mM glucose concentration and subtraction of lipofuscin auto-fluorescence was performed to univocally assign the remaining signal to NAD(P)H (see methods for a detailed description regarding the correction). c Stimulation was performed by increasing glucose concentration from 2.2 to 16.7 mM: 3 minutes after the stimulation a second FLIM image was acquired and corrected for lipofuscin signal as well. At this level, the phasor barycenter from the two FLIM images can be calculated and their coordinates along the metabolic axis define the so-called metabolic shift and its magnitude. d To obtain single-cell metabolic data, the islet was fixed and then incubated with anti-glucagon and anti-insulin antibodies to recognize α and β cells, respectively. Within this step, the islet was imaged again at the same focal plane used for metabolic imaging, but in a standard 2-channel immunofluorescence experiment (α-cells in “red” and β-cells in “green”). e FLIM images at 2.2 and 16.7 mM are compared with the immunofluorescence one with subsequent cell type recognition and single-cell ROI creation in both FLIM images (f). At this point, each recognized cell has an associated phasor plot. g Single-cell metabolic shifts are calculated in the same way as whole-islet ones, namely by determining the barycenter coordinate shift along the metabolic axis from 2.2 to 16.7 mM.
a, b Likewise immortalized cells, FLIM images of living human islets depict the distribution of fluorescence intensity with each pixel having a corresponding point in the phasor plot. The islet metabolic status can be modeled using the phasor barycenter and its movement along the metabolic axis upon increasing glucose concentration from 2.2 and 16.7 mM. Before assigning signals to NAD(P)H, the contribution of lipofuscin bodies (c) needs to be subtracted. Lipofuscin signal (d) potentially interferes with barycenter calculus, causing barycenter displacement towards free NAD(P)H condition (n = 15 islets) (e), yielding lipofuscin correction a necessary task for the correct identification of islet metabolic state. Considering the islet as a whole, metabolic response for glucose stimulation (n = 15 islets) displays mild to moderate bound-shift (f), yielding a statistically significant bound-shift on average (n = 15 cells) (g), presumably due to the higher amount of β cells in the islet which thus have higher contribution in barycenter calculus. At this point, no cell type recognition has been performed yet. Statistical difference obtained using Paired Samples Wilcoxon Test, with significance α = 0.05 (**P < 0.01). Scale bar: 20 μm.
To retrieve information on cell identity, islets were fixed and stained using anti-glucagon and anti-insulin antibodies readily after the FLIM acquisition at 16.7 mM glucose concentration (Fig. 3a). After the fixation/staining step, particular attention was paid to finding the same islet optical section used for metabolic imaging. As an exemplary case, the same islet of Fig. 2 is reported also in Fig. 3a, b to highlight that antibody-based identification of α and β cells was conducted in the same optical section used for NAD(P)H auto-fluorescence imaging. Each FLIM image was compared with the immunostained one in order to identify cell type (i.e., α or β) and cell shape; this latter was exploited to draw single-cell ROIs (highlighted by green/red contour lines in Fig. 3b). With this information gained, single-cell metabolic shifts could be finally extracted from the comparison of FLIM acquisitions at 2.2 and 16.7 mM glucose concentration. In the exemplary case shown in Fig. 3c, single-cell metabolic shifts are reported and classified according to cell type (α cells by red bars, β cells by blue bars) and magnitude (bar height; according to Eq. 4 metabolic shift towards bound NAD(P)H are “positive,” those towards free are “negative”). In this particular case, 10 α cells and 49 β cells were identified in the selected optical section of the islet: a markedly heterogeneous spectrum of responses to glucose can be appreciated in both cell types, with metabolic shifts either towards bound or free NAD(P)H. Obviously, information on cell identity and metabolism can be combined together to produce maps which contain the spatial distribution, the magnitude and direction of the metabolic response to glucose of each cell (reported in Fig. S5). The workflow described so far was completed for all the islets included in this study (an additional example is reported in Fig. S6). Worthy of mention, FLIM measurements of metabolic shifts can be used to estimate the number of NAD(P)H molecules involved in the metabolic change, by means of Eq. 5 (see “Methods”). For the islet of Fig. 3, for instance, the highest shifts measured in the two directions are of about 0.06 (cells “36” and “43,” Fig. 3c). Assuming an average concentration of NAD(P)H in the cell of approximately 100 μM³¹, and an average volume of the cell of 1280 μm³³², Eq. 5 (see “Methods”) leads to an estimate of approximately 4.6·10⁶ NAD(P)H molecules which are shifting towards their bound form in cell 36 or towards their free form in cell 43. Interestingly, these FLIM-based estimates are in keeping with previous ones obtained by others using NAD(P)H as test molecule¹¹. In addition, it should be kept in mind that what is measured by FLIM are the so-called “fractional intensities” of the two NAD(P)H forms, which are related to their actual molar fractions by means of the quantum yields (QYs) of the pure bound and free species³³. Taking into account that the average QY for the bound NAD(P)H form is ~8.5 times larger than that for the free NAD(P)H form (estimated by the difference in lifetimes^20,34), the measured fractional intensities can be corrected and used to extract the molar fractions of free and bound NAD(P)H from FLIM data (Eq. 6). From these calculations, the stationary-state metabolic status appears with a predominance of NAD(P)H in the free form (80% for both α cells and β bells) as compared with NAD(P)H in the bound form (20% for both α cells and β bells). Overall, from the N = 15 islets analyzed a total of 312 α cells and 654 β cells were identified, which yielded a relative abundance of the two types of cells of 32% and 68% for α and β cells, respectively (Fig. 3d) in keeping with expectations for human islets^3,35 (α/β cell proportions for each measured islet are reported in Tab. S3). Interestingly, having at our disposal the identity (i.e., α or β) of each single cell measured allowed us to correlate the amount of lipofuscin detected per single cell to the cell type. As reported in Fig. S4c, it was found that β cells contain substantially more lipofuscin (approximately twofold) as compared to α cells, again in keeping with previous estimates³⁶. Most importantly, cell identity was used to extract the average metabolic response of all the α and β cells collected in our experiments: results are reported in Fig. 3e and show an average response of both cell populations (α cells in red, β cells in green) towards bound NAD(P)H. This result apparently contradicts the markedly discordant metabolic trajectories of α cells and β cells observed by Wang and co-workers in disaggregated and intact mouse islets²⁸, as discussed extensively above. Yet, the population-averaged data reported in Fig. 3e do not exclude the possibility of single-cell metabolic shifts towards free NAD(P)H. Of note, in fact, if metabolic shifts are not averaged together but just classified according to their magnitude and sign (i.e., positive if towards bound NAD(P)H, negative if towards the free form), both α and β cells display metabolic responses in both directions, with a prevalence of cells engaged in a shift towards bound NAD(P)H (i.e., approximately 60% in both cell types) (Fig. S7).
a Immediately after 16.7 mM glucose FLIM imaging, cells are fixed to preserve their shape and structure and then incubated with anti-glucagon and anti-insulin antibodies which in turn recognize α (red) and β -cells (green). b Comparison of 2.2 and 16.7 mM FLIM images with immunofluorescence image allow us to recognize cell borders and identity. At this point, single-cell segmentation occurs by tracing ROIs on the two FLIM images (here reported only the 2.2 mM as representative image). Each segmented cell has a unique and equal ROI number in both 2.2 and 16.7 mM FLIM images. Then, single-cell barycenter are calculated, converted in Bound/total NAD(P)H ratio and their difference between 2.2 and 16.7 mM state determines the single-cell metabolic shifts (c). Each cell ROI represents a single cell with red bars indicating bound-shift and blue ones free-shift. d Considering all the segmented cells, the relative abundance of α (~30%) and β cells (~70%) is in keeping with literature³ and their average response is bound-shifted for both cell types (e), in apparent contrast with literature. f If single-cell metabolic shifts are averaged at islet level, α cells (n = 312 cells from 15 islets obtained by 4 different donors) show shift towards free NAD(P)H in 60% of islets with the remaining 40% shifting towards bound NAD(P)H; by contrast, for what concerns β cells (n = 654 cells from 15 islets obtained by 4 different donors), approximately 80% of the islets display a metabolic shift towards bound NAD(P)H and only the remaining 20% towards free NAD(P)H. Statistical difference obtained using Paired Samples Wilcoxon Test, with significance α = 0.05 (****P < 0.0001). Scale bar: 20 μm.
The similarity in α- and β-cell responses described so far at the population level does not necessarily mean that the two cell types cannot show discordant metabolic trajectories if grouped and analyzed at the islet- or even at the donor-level. To check whether this is the case, α and β cells were first grouped according to the islet from which they originated (Fig. 3f). Each point in the plot now represents the average response of either α (red) or β (green) cells which belong to the same islet. Interestingly, if grouped at this level, the majority of islets (approximately 80% over a total of N = 15 islets from N = 4 donors, compared to 60% from single-cell data) show an average β-cell-specific metabolic response towards bound NAD(P)H (Fig. 3f, green points). By contrast, only 40% of the islets show α-cell-specific metabolic response in the same direction (Fig. 4a, red points), compared to the 60% calculated from single-cell data. Thus, single-cell data grouped at the islet level suggest a non-random distribution of α- and β-cell metabolic responses among different islets. To further investigate this point, single-islet data were further traced back to the respective donor and correlated to its insulin secretion power, for both α and β cells (red and green data points and linear interpolations in Fig. 4): noteworthy, robust and concordant average responses of both α and β cells towards bound NAD(P)H correspond to the donor with the lowest insulin secretion power (i.e., Donor-1 data points); then, such responses differentially decrease in magnitude (i.e., α-cell responses decrease faster than β-cell ones) in donors with intermediate insulin-secretion powers (i.e., Donor-2 and Donor-3 data points); finally α- and β-cell responses end up to be discordant in the donor with the highest insulin-secretion power (i.e., Donor-4 data points), with α cells shifting towards free NAD(P)H and β cells in the opposite direction, a result in line with what previously observed by Wang and collaborators²⁸. Further investigations will be needed to clarify whether these results reflect a regulatory action imparted on α cells by paracrine factors associated to glucose-induced secretion by β cells (e.g., insulin, zinc, GABA, etc.), in addition to glucose itself. Worthy of mention, if α- and β-cell responses from each donor are summed together (i.e., reconstituting the whole-islet metabolic response), marked linear anti-correlation with respect to the donor secretion power emerges (Fig. 4, black data points; Pearson’s correlation coefficient: −0.997). Interestingly, the 4 donors included in this study are representative of the overall distribution of insulin-secretion indexes typically measured by the ELISA assay^37,38: the reference gray curve in Fig. 4 is calculated on a total of N = 17 donors (insulin secretion indexes reported in Tab. S1). Again, further studies will be needed to clarify whether this result, even if devoid of information on the two cell types, is potentially revelatory of a marked general relationship between the optical measurement of islet auto-fluorescence lifetime (in the form of a metabolic shift) and the secretion power of the respective donor (and vice versa).
Donor-averaged metabolic shift of β cells (green points, n = 654 cells from 15 islets obtained by 4 different donors) always display a metabolic shift towards bound NAD(P)H, in accordance with present literature; by contrast, α cells averaged at donor level (red points, n = 312 cells from 15 islets obtained by 4 different donors) show a decreasing trend, progressively shifting from “positive” ∆bound/total NAD(P)H values to “negative” ones. In addition, the sum of α- and β-cell responses per each donor (black points) shows a markedly linear trend (fitting equation and R² coefficient reported in the graph legend). The gray curve represents the Gaussian fitting of independently measured (thorough ELISA assays) insulin Stimulatory Index (normalized for insulin content) of n = 17 donors, whose detailed description in reported in Tab. S1. Data are presented as mean ± SE.
Here we measured the metabolic response to glucose in 312 α and 654 β cells in 15 human islets from a cohort of 4 healthy donors exploiting fit-free phasor-FLIM analysis of NAD(P)H signals. The metabolic status of a cell was defined by the average position of FLIM data along the so-called “metabolic segment” in the phasor plot, a segment connecting the characteristic lifetimes of the two main forms of NAD(P)H: free and enzyme-bound. Thus, technically, collected data afford a quantitative estimate of the balance of free and bound NAD(P)H in the cell (in this report, analogously to previous ones²⁸, such balance is expressed by the bound/total NAD(P)H ratio). In our experimental protocol, two consecutive measurements of bound/total ratio are performed, the first at 2.2 mM glucose, the second upon shifting cells to 16.7 mM glucose.
A thorough understanding of cellular responses must inevitably take into account the high complexity of cell biochemistry, in terms of enzyme identity and enzyme expression level, and the differences among cell types, in this case those regarding α and β cells. In fact, variations in the identity and/or expression of enzymes involved in NAD(P)H processing may reflect into variations of the free/bound NAD(P)H balance (and of its response to stimuli). For instance, some of us recently demonstrated that non-secretory cells show indeed a different response to glucose stimulation as compared to INS-1E cells³⁹. By the same reasoning, it should be kept in mind that a similar variation in the balance of free and bound NAD(P)H may have different biochemical explanations in different cell types, depending on the peculiarities of cell biochemistry. Worthy of mention, cell biochemistry is not supposed to change during the whole FLIM assay (which lasts few minutes in total); we are prompted to speculate, instead, that the increase in glucose concentration makes cell biochemistry settle down to a different value of the balance of free and bound NAD(P)H. In other words: upon shifting the culturing conditions from 2.2 to 16.7 mM glucose, the same cell biochemistry rearranges NAD(P)H bound/free ratio to a new value, towards an increase in either bound or free NAD(P)H.
Such reasoning naturally applies to α and β cells, which show specific differences, among others, in glucose handling⁴⁰, expression of key metabolic enzymes⁴¹ and enzymatic activity⁴². For instance, the high levels of glycerol 3-phosphate dehydrogenase expression⁴² as well as the tight coupling between glycolysis and Krebs cycle⁴⁰ in β cells are in line with the well-known commitment of these cells to rapidly increase ATP levels in response to glucose and supports the idea that the measured increase in bound NAD(P)H may reflect activation of the oxidative phosphorylation pathway, as already proposed in the literature^25,28,39. By contrast, for instance, α cells are expected to use much more the lactate dehydrogenase enzyme (which uses NAD(P)H as cofactor) as compared to β cells⁴⁰, and this may reflect into a substantial contribution of this pathway in generating bound NAD(P)H upon glucose stimulation.
In conclusion, it is worth mentioning that reported results have potential implications from both the methodological and biomedical/clinical point of view. Concerning the methodological approach, we demonstrated the effectiveness of an optical-microscopy-based protocol to measure the specific responses of α and β cells in a living human-derived Langerhans islet, thus opening to similar investigations under different conditions of biomedical/clinical interest. Yet, a few lines of future development on this side can be envisioned. In any biophysical measurement, in fact, a compromise has to be found for the resolution achievable in the two main dimensions of interest, the spatial and the temporal one. Concerning the spatial dimension, for instance, it was shown here that light penetration in the tissue is quite inefficient due to the presence of hundreds of cells tightly packed in 3D in the islet. Although using multiphoton infrared microscopy the effective penetration-depth did not allow imaging of the entire islet and, as consequence, a complete single-cell identification and analysis. Preliminary results obtained using tissue-clearing procedures, as reported here in Fig. S3, are encouraging: along with an increase in imaging performances, these procedures are also compatible with metabolic imaging. In fact, Chacko and co-workers recently demonstrated that the lifetime-based NAD(P)H metabolic contrast in a sample is preserved even after chemical fixation, thus opening to the opportunity to generate useful auto-fluorescence signatures from unstained tissues exposed to different stimuli⁴³. Of course, an approach based on fixed samples would lose the privileged access to temporal information preventing, for instance, to measure the metabolic response of the same islet in time. On the other hand, concerning the temporal dimension, the present approach exploits live samples to monitor the metabolic shift but then requires fixation for cell identification. To avoid the fixation step, lentivirus-based transduction systems may represent a valuable option. In fact, by allowing the delivery of genetically-encoded fluorescent proteins (FPs)⁴⁴, these systems could be used to identify α and β cells by means of specific FP-tagged protein markers. From a biomedical/clinical perspective, we uncovered the optical signature of the metabolic response to glucose of α and β cells in the human islet. These two populations of cells show a markedly heterogeneous spectrum of responses, encompassing metabolic shifts towards either an increase or a decrease of the bound/free NAD(P)H ratio, in apparent contradiction with results obtained in mouse models²⁸. However, it was interesting to note that, if single-cell responses are traced back to the respective donor and correlated to its insulin secretion power, emergent collective properties can be highlighted: in detail, robust and concordant average responses of both α and β cells towards bound NAD(P)H correspond to low insulin-secretion power while robust but discordant responses (i.e., α cells shifting towards free and β cells towards bound NAD(P)H) correspond to high insulin-secretion power. This in turn speaks in favor of a coordinated response of α and β cells in the islet and of a possible role of paracrine factors associated to glucose-induced insulin secretion in modulating the response of the two populations of cells. Then, if α- and β-cell responses are summed together, marked linear anti-correlation with respect to the amount of insulin secreted by each donor emerges highlighting a possible relationship between the FLIM signature of the islet and its insulin-secretion power. Additional investigations, however, will be needed to increase the statistics at the donor level and thus understand how general such relationship is. If confirmed it would have the merit of being independent from cell-type recognition (and thus from tissue fixation) and thus potentially useful to estimate the donor insulin-secretion power from a rather simple optical measurement on a subset of tissue-derived islets.
The pancreases of 4 non-diabetic donors were used for the isolation and study of islets. The characteristics of islet donors are listed in Tabs. S1 and S2. The procedures were approved by the Ethics Committee of the University of Pisa (21 November, 2013, #2615). Islets were isolated by collagenase digestion followed by density gradient purification, as reported in other works³⁹, before 1 November, 2022. Maintenance culture was at 37 °C and 5% CO₂ atmosphere, in M199 culture medium complemented with 10% bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, 750 ng/mL amphotericin B, and 50 µg/mL gentamicin, at 5.5 glucose concentration. A complete list of reagents is reported in Tab. S4.
INS-1E cells were kindly provided by Prof. C. Wollheim, University of Geneva, Medical Center and cultured in RPMI 1640 medium with 11 mM glucose supplemented with 10% heat-inactivated fetal bovine serum, 100 Units/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate, and 50 μM β-mercaptoethanol at 37 °C in a humidified 5% CO₂ atmosphere.
Insulin secretion was measured as described in other works³⁹. Briefly, batches of 15 handpicked human islets were pre-incubated for 45 min with a Krebs’ solution containing 3.3 mM glucose, then challenged acutely (45 min) with 3.3 and 16.7 mM glucose. The supernatant was collected and stored at −20 °C. Total insulin content was extracted with an acid ethanol solution and insulin levels were quantified by the Insulin ELISA kit following the manufacturer’s protocol (Mercodia AB, Uppsala, Sweden). Insulin stimulation index was calculated as ratio of insulin release at 16.7 mM glucose over release at 3.3 mM glucose, each expressed as percentage of the total insulin content.
Whole islets (250-500 μM diameter) were plated on an 8-well coverglass (Sarstedt; 94.6170.802, see Fig. S8). Next, islets were embedded in a solution consisting of 1% agar (%wt/vol) dissolved in SAB (114 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 2.5 mM CaCl₂, 1.16 mM MgSO₄, 20 mM HEPES at pH = 7.4, and 2.2 mM glucose). After agar gelation, islets were equilibrated in a low-glucose buffered solution (SAB) at 37 °C for 45–90 min. After imaging at the low glucose concentration (2.2 mM), human islets were treated with glucose (stock solution 0.5 M) to reach a final concentration of 16.7 mM and imaged after 3–5 min.
For the clearing process, live human islets were soaked in 1% agarose and then stained with Hoechst 33342 (dilution 1:1000 in M199 culture medium complemented with 10% bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, 750 ng/mL amphotericin B, and 50 µg/mL gentamicin.) for 1 h at 37 °C and 5% CO₂ atmosphere. After imaging, the samples were fixed in 4% PFA following three washes in PBS, 5 min each. For the clearing procedure, the fixed specimens were equilibrated with 30% TDE/PBS (v/v) for 30 min and 68% TDE/PBS (v/v) for 1 h and imaged by two-photon microscopy. For the penetration depth analysis, cellular nuclei of two independent islet samples labeled with Hoechst were detected by Fiji using Region Of Interests (ROIs) of 4 × 4 μm (depth 0 μm: 12 nuclei; depth 10 μm: 30 nuclei; depth 20 μm: 36 nuclei; depth 30 μm: 40 nuclei; depth 40 μm: 40 nuclei; depth 50 μm: 40 nuclei). Mean values with associated standard deviation were calculated by Fiji and plotted using Origin (OriginLab Corporation, Origin 2019b).
Live islet imaging by an Olympus FVMPE-RS microscope coupled with a two-photon Ti:sapphire laser with 80-MHz repetition rate (MaiTai HP, SpectraPhysics) and FLIMbox system for lifetime acquisition (ISS, Urbana Champaign). NADH has been excited at 740 nm and the emission collected by using a ×30 planApo silicon immersion objective (NA = 1.0) in the 420–460 nm range. Calibration of the ISS Flimbox system has been performed by measuring the known mono-exponential lifetime decay of Fluorescein at pH = 11.0 (i.e., 4.0 ns upon excitation at 740 nm, collection range: 400–570 nm). To prepare the calibration sample, a stock of 100 mmol/L Fluorescein solution in EtOH has been prepared and diluted in NaOH at pH 11.0 for each calibration measurement. For each measurement, a 512 × 512 pixels image of FLIM data was collected until 30 frames were acquired. The acquisition time was typically in the order of 1-2 minutes.
Human islets fluorescent maps present bright spots due to lipofuscin, which is commonly removed by means of an arbitrary intensity threshold because more brilliant than other fluorescent components. In order to obtain a faster and less user-dependent analysis, an automatic signal subtraction algorithm has been developed. First, it calculates the intensity distribution in the FLIM image and, since the lipofuscin bodies pixels can be seen as outliers, it is possible to discriminate lipofuscin by applying the formula:
where Q₃ is the third quartile and IQR = Q₃–Q₁ is the interquartile range. The already obtained threshold has been used to create a binary mask comprising all pixels below the already calculated threshold and then, by applying the mask on FLIM images, the lipofuscin-containing pixels are excluded from subsequent analyses. The effect of correction is shown in Fig. 2. Though, the algorithm also presents limitations. For example, it is sensitive to illumination gradients caused by tissue scattering, but this is generally prevented by properly choosing the measurement focal plane, otherwise a pre-processing stage can be performed using Fiji or writing customized code using image processing libraries (e.g., scikit-image or OpenCV). Another limitation could be represented by abnormal cells which are almost entirely constituted by lipofuscin, but this was not the case in our experiments.
The metabolic state measured by FLIM is presented as Bound/total NAD(P)H between 0 and 1. This Bound/total NAD(P)H is calculated from the barycenter ((underline{g},underline{s})) of the phasor:
where g_i and s_i are the coordinates of ith point and N the number of pixels (e.g., in a 512 × 512 image, N = 262144). Conversion of barycenter coordinates ((overline{g},overline{overline{s}})) into Bound/total NAD(P)H occurs by means of the following equation:
where f₁ and f₂ are the fractional intensities of the free and bound NAD(P)H or, namely, the barycenter distances from the two extremes of the metabolic-axis on the universal circle (τ₁ = 0.4 ns and τ₂ = 3.4, see Fig. S2), which in turn represent the characteristic lifetimes of the free and bound forms of NAD(P)H according to present literature²⁰. The FLIM-based measurement of metabolic shifts can be defined as the difference between the two relevant conditions measured, i.e., 16.7 and 2.2 mM:
and allows to estimate the number of NAD(P)H molecules involved in the metabolic change (Delta {N}_{{{{{rm{NAD}}}}}left({{{rm{P}}}}right){{{rm{H}}}}}) by the equation:
where [NAD(P)H] is the average NAD(P)H concentration (mol/m³) in the cell, V_cell (μm³) the average cellular volume, and N_A is the Avogadro constant (mol⁻¹).
To obtain biologically relevant data, however, correction of the measured fractional intensities needs to be performed taking into account the difference in brightness between the two forms, i.e., bound NAD(P)H is approximately 8.5 times brighter as compared to free NAD(P)H^20,36. Based on this, Eq. 1 can be rearranged into:
where ({bar{f}}_{i}) are the brilliance-corrected values present in Eq. 2, namely:
where ({epsilon }_{i}) is the molar extinction coefficient and ({phi }_{i}) the quantum yield of the fluorescent specie.
After metabolic imaging, human islets were fixed in 4% paraformaldehyde (PFA; stock solution 32% paraformaldehyde, 10× PBS) at room temperature (RT) for 1 h in gentle shaking, followed by 3 washes with PBS, 5 min each at RT. Then, the fixed samples were permeabilized in 0.1% TritonX-100 in PBS (PBST) for 30–45 min at 37 °C in gentle shaking. After permeabilization, the primary antibodies (mouse anti-Insulin, rabbit anti-Glucagon both diluted 1:500; see Tab. S3) were incubated for 3 days at 37 °C in gentle shaking. After 3 washes for 10 min each at 37 °C with PBST, the human islets were incubated with the secondary antibodies donkey anti-mouse IgG AF 488 and donkey anti-rabbit IgG AF 594 for 24 h at 37 °C in gentle shaking and then, washed 3 times for 10 h each at 37 °C. The stained human islets were then acquired using a two-photon Ti:sapphire laser (Olympus FVMPE-RS) at the excitation light of 800 nm. The insulin-secondary antibody fluorescence signal was collected at 380–570 nm, while the glucagon-secondary antibody signal was collected at 570–680 nm. Microscope parameters (zoom and focus) were maintained equal to the previous acquisitions (auto-fluorescence images), to perform an efficient islet matching. For single-cell identification, auto-fluorescence and immunostained images were paired and α and β cells were manually segmented using Fiji (Tools; ROI Manager). A custom script finally merges ROIs with raw FLIM.R64 files and automatically performing all the already described tasks, i.e., lipofuscin subtraction and metabolic shift calculation.
Data are presented as mean ± SE. Statistical difference has been assessed using Paired Samples Wilcoxon Test, with significance α = 0.05 on Origin software (OriginLab Corporation, Origin 2019b). In total, 321 α cells and 654 β cells have been analyzed which have been obtained from 15 human Langerhans Islets from 4 different donors (additional information are reported on Tabs. S1–S3).
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
The data underlying the findings of this study are available upon requests to the authors.
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This work has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No 866127, project CAPTUR3D). P.M. and M.T. receive support from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreements No 115,797 (INNODIA) and 945,268 (INNODIA HARVEST). These Joint Undertakings receive support from the Union’s Horizon 2020 research and innovation program and “EFPIA,” “JDRF,” and “The Leona M. and Harry B. Helmsley Charitable Trust.” The authors want to acknowledge Professor Antonio Lucacchini for contributing to the interpretation of the biochemical data.
These authors contributed equally: Fabio Azzarello, Luca Pesce.
NEST Laboratory – Scuola Normale Superiore, Piazza San Silvestro 12, Pisa, Italy
Fabio Azzarello, Luca Pesce, Valentina De Lorenzi, Gianmarco Ferri & Francesco Cardarelli
Department of Clinical and Experimental Medicine, Islet Cell Laboratory, University of Pisa, Pisa, Italy
Marta Tesi, Silvia Del Guerra & Piero Marchetti
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F.A. and L.P. performed experiments, analyzed data, and wrote a manuscript draft; V.D.L. performed immunofluorescence experiments and analyzed data; G.F. analyzed data; M.T. and S.D.G. isolated human islets and performed insulin secretion assays; P.M. designed research and wrote the manuscript; F.C. designed and supervised research, analyzed data, and wrote the manuscript.
Correspondence to Francesco Cardarelli.
The authors declare no competing interests.
Communications Biology thanks Suman Ranjit and Guy Rutter for their contribution to the peer review of this work. Primary handling editors: Chao Zhou and Manuel Breuer. Peer reviewer reports are available.
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Azzarello, F., Pesce, L., De Lorenzi, V. et al. Single-cell imaging of α and β cell metabolic response to glucose in living human Langerhans islets. Commun Biol 5, 1232 (2022). https://doi.org/10.1038/s42003-022-04215-w
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Isaac Hale, Daily Herald file photo

Traffic flows Wednesday, June 27, 2018, at the intersection of State and Center Streets in Orem.
On Wednesday, the Orem City Council received an updated presentation on the city’s transportation plan.
Sam Kelly, city engineer, introduced the plan study with the help of John Dorny, of Horrocks Engineering, who was brought on to help.
The original plan was developed in 2015. The 2022 update takes the travel level on roads through the next 30 years.
“This is an update from the 2015 Master Plan, so it wasn’t a complete redo,” Dorny said. “We looked at the counts from 2015 and realized they were outdated so we did provide new data and gathered (traffic) counts.”
Dorny noted that Orem has a unique level of trip generation due to the number of students living and commuting in the city.
Harrison Epstein, Daily Herald

Cars drive through the intersection of State Street and Center Street in Orem on Wednesday, April 13, 2022.
“It’s a higher level of trips per house and that is showing up in the master plan for the transportation study,” he said.
Among the information presented is how traffic is expected to grow, and just what the road capacity will look like in years to come.
“We also consult the General Plan. We want to make sure that the master plan doesn’t contradict the General Plan because then you get developers picking which plan they follow,” Dorny said.
Dorny indicated that there was not a full allowance for public input because there were open houses before the 2015 Transportation Master Plan and this is just an update. Still, residents will be able to make their voices heard.
“The public will be notified to come in and comment about it,” he said.
Courtesy Orem city

Orem State Street, a major arterial road in the city, is shown in this undated photo.
The city has been divided into the following classifications to provide optimal traffic flow throughout Orem. The variety of roads include:
The study showed the various levels of service roads would undertake through 2050. Through a variety of images mapping the city, the council will know what areas will need to be addressed first. Not all streets will need to be enlarged over the years, but it is a fact they will be busier.
The Transportation Master Plan update will have the additional document items and maps available online for residents who are interested.
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The new Google Pixel Buds Pro BT (Bluetooth), ANC (Noise Cancelling) earphones are not only great value at $299 but are among the sweetest sounding we have heard, putting many more expensive buds to shame. Colours Charcoal, Fog and Lemongrass.
We cannot objectively test the Frequency response of in-ear earphones. We use a series of test tracks that exercise bass, mid and treble. Our opinion – it has a neutral sound signature (good) that can handle heaps of bass and bright vocals well.
We use Fail (below expectations), Pass (meets expectations) and Exceed (surpasses expectations or is the class leader) against many of the items below. We occasionally give a Pass(able) rating that is not as good as it should be and a Pass ‘+’ rating to show it is good but does not quite make it to Exceed.
You can click on most images for an enlargement.
It is Google Style – a white ceramic-looking USB-C/Qi charge case  (with a reset button) and mid-height-profile buds. It comes with S/M/L ear tips (11/12/13mm), and the App has a seal test to help you select the best fit.
It is a closed-back design yet has vents to help widen the soundstage.
The buds fit well and stay in your ear despite having no wing tips. Google has sensors that actively measure the ear canal pressure and can vent that if necessary. We tested with eight hours of continuous use – and got to 7.6 hours before it needed a charge. These are among the more comfortable we have tested, although at 6.2g each, they are a fraction heavier than the competitors.
They are IPX4 sweat-resistant, but the lack of stability fins means it is best not to use them in physical sports.
Battery life is over 7/20-hours (buds/case) ANC On and 11/31 ANC off. USB-C charge time is less than 80 minutes (5V/1A/5W) and about 120 minutes on Qi. Five minutes gives about an hour of listening time.
It has large 11mm drivers that produce lots of well-controlled bass, flat mids and treble at relatively low distortion rates. The neutral sound signature can be controlled by a full EQ that downloads the settings to the buds.
Out of the box, we tested with several genres, and while it is pretty good, the EQ makes a huge difference. You can select from Default, Heavy Bass, Light Bass, Balanced (mid-recessed), vocal boost, and Clarity (1-4kHz boost). Or adjust the EQ’s low bass (<40Hz), Mid, Treble and Upper Treble. Excellent, and the EQ is the secret sauce that makes these better performers than most competitors.
Like all closed-back buds, the sound stage is just within your head, but Dolby Atmos content expands that with 3D spatial and 2D horizontal sound object placement.
It has volume EQ that adjusts the sound depending on sound levels – it works well at lower volumes.
These are fine for video lip sync at <200ms but forget gaming.
It supports multi-point pairing and fast pairing, and mono listening.
But it only supports SBC and AAC (no aptX), which may be a deal breaker to an audiophile. That is logical, as the Pixel 6 and 7 don’t have a Qualcomm SoC and aptX support.
It has a BT Find my Buds feature.
The ANC mixes passive (correct fit) and active noise cancelling. The fit right (ear seal check) will block deep rumble and general chatter.  There is little noise leakage.
It picks up the surroundings quite well using its external mics and allows you to converse without removing them.
It has average call quality and limited noise cancellation, but overall, it is acceptable for most and-free use.
If you use one bud, you lose the phone controls.
Naturally, it has Google Assistant and is always in listening mode.
Transcribe mode lets you hear 80+ spoken translated languages in the buds and a transcript on the phone. 
Right in-ear detection for single-bud use.
Google has finally got the formula right – decent ANC, great music quality, long battery life, excellent comfort and style. It now has competitive $299 earphones.
My daily drive is the $399 Sennheiser Momentum 3 Sennheiser True Wireless Momentum 3 BT/ANC earphones (review and update) (mainly for music quality). While the Google Pixel Buds Pro is tempting, the Momentums is worth the extra $100 investment.
BTW the Sony WF-1000XM4 are remarkable BT, ANC earphones (long-term review) at $389 is very good but lacks Qualcomm aptX codecs.
If you compare them to the following, it meets or exceeds these excellent buds.
I would insist on Qualcomm aptX codecs (Sennheiser and Jabra).
Features: 85 – has it all except Qualcomm, aptX
Value: 95 – in the zone and presents excellent value
Performance: 90 – good battery life, fast and Qi charge, and a good sound signature
Ease of Use: 85 – needs some setup, and the App can be a little daunting
Design: 85 – typical Google style
 

 
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CyberShack offers consumers a one-stop-shop for the most current information in the tech space, including news, lifestyle and products, as well as reviews on the latest gadgets and games. It also runs competitions and has links to the latest episodes of Cybershack TV.

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The Wanderer

            On November 2, Mattapoisett’s 2022 update of the town’s Master Plan was released by the Southeastern Regional Planning and Economic Development District (SRPEDD), which has been working with a volunteer group of residents on the project since August 2020. Due to the restrictions imposed on public gatherings during the height of the coronavirus pandemic, the volunteers pushed on using virtual technology, thusly giving voice to their opinions, concerns and hopes for a town they were willing to help.
            The committee was comprised of Robert Burgman, Carole Clifford, Kate Connelly, Paul Criscuolo, Mary Dermody, Carlos DeSousa, Yasin Flefleh-Vincent, Shirley Haley, Kate Haley, David Horowitz, Nate Ketchel, Robin LePore, Janice Robbins, Mike Rosa, Colby Rottler, Donna Shea, Aaron Smith, Tom Tucker and Chrystal Walsh.
            Throughout the process, the team was facilitated by SRPEDD Principal Comprehensive Planner Lizeth Gonzalez, who guided discussions in a format intended to ferret out the most pressing needs of the community with an eye towards a variety of strategic goals.
            The Master Plan model is regulated by Massachusetts State Law Chapter 41, Section 81D. The plan addresses all planning elements: Land Use, Housing, Economic Development, Open Space and Recreation, Natural and Cultural Resources, Services and Facilities, Transportation and Circulation and, due to Mattapoisett’s waterfront location, Resilience.
            The Master Plan draft is now on view at the Mattapoisett Library in hardcopy until the end of November and via the SRPEDD weblink srpedd.s3.amazonaws.com/wp-content/uploads/2022/11/01163008/Draft-MattapoisettMP-11022022.pdf.
            During the Planning Board’s November 7 meeting, Nate Ketchel, acting chair for the board and the Master Plan Committee, said that the public is invited from now until the end of November to review the draft and offer comments. Comments may be submitted to Gonzalez at [email protected]. Ketchel said that next steps after the review process has ended is to make final edits and then present the completed plan to the Select Board.
            Detailed in the draft plan are population and school-enrollment numbers critical for one of the biggest topics currently under review by the Edward J. Collins Jr. Center for Public Management at UMass Boston, school consolidation. The Master Plan Committee discussed Mattapoisett Public Schools in several categories. The following text in the draft demonstrates their efforts. “Residents are proud of the quality of education and the relationships their children have with the town itself, especially the Village.” However, the committee also recognized the need for planning that would address decreases in enrollment and the future of a town supporting two elementary schools.
            Of the Town Hall, the operations of which have been said to need a completely new location given the grave condition of the structure, the Master Plan Committee thought it might be redeployed as a cultural center despite its location in a flood zone.
            The committee also looked at the needs of a growing senior population. In their discussion regarding Housing, one of the main chapters in the plan, both families with school-age children and senior citizens were viewed as needing more affordable housing.
            Under the Open Space and Recreation header, the committee wondered about building affordable housing units on the former Holy Ghost grounds. SRPEDD wrote, as informed by the committee, “Mattapoisett’s inventory of town-owned land appropriate for development is limited; however, the town should pursue state funding to prepare key sites such as the Holy Ghost Grounds on Park Street for redevelopment.”
            The 2022 Master Plan draft lays out potential economic-growth opportunities along the Village Business District on Route 6. The committee believes there is untapped potential for multiuse structures that can be constructed in a visually appealing manner.
            Again, the 2022 Master Plan draft is available now for review at the Mattapoisett Library in hardcopy and via SRPEDD’s weblink.
            The balance of business handled by the Planning Board on November 7 was the continuation of a Site Plan Review for the application submitted by Sun Partners Solar for a 198.8KW array on property located at 156 Acushnet Road. The hearing was continued to November 21.
            The board completed review of a Form-C Definitive Plan submitted by JBL Fairhaven Road LLC, for a proposed subdivision. Although the board completed review, including hearing from engineer David Davignon of Schneider, Davignon & Leone, Inc., including his responses to comments from peer-review consultant Ken Motta of Field Engineering, rather than close the hearing the board elected to give Motta an opportunity to rebut Davignon’s responses to the peer review. The hearing was continued to November 21.
            The next meeting of the Mattapoisett Planning Board is scheduled for Monday, November 21, at 7:00 pm.
Mattapoisett Planning Board
By Marilou Newell

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Bloomberg Markets European Open kick starts the trading day, breaking down what’s moving markets and why. Francine Lacqua and Tom Mackenzie live from London bring you an action-packed hour of news no investor in Europe can afford to miss.
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US stocks gained surer footing after a choppy morning session, extending a rally sparked by a slowdown in inflation. The dollar fell, declining for a fourth week.
The S&P 500 closed near session highs in the biggest weekly gain since June. The tech-heavy Nasdaq 100 climbed more than 1.5%, notching its best week in two years. Cash Treasury trading is closed for Veterans Day.

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Short interest in VictoryShares US EQ Income Enhanced Volatility Wtd ETF (NASDAQ: CDC) fell by a sizeable amount throughout October. On October 31st, the total number of shares borrowed to cover short positions was 24,500, representing a drop of 51.4% from the previous day’s 50,400 shares. Considering the typical daily volume of trade, which is 188,100 shares, the short-interest ratio is currently at 0.1 days.
In addition to that, the corporation announced a monthly dividend, which was distributed on November 10th. As of November 9th, stockholders were each given dividends amounting to $0.118 per share. This dividend became payable on November 8th, which was a Tuesday. This equates to a dividend payment of $1.42 annually and a dividend yield of 2.22%.
On Friday, the share price of CDC was $63.66 when the market opened. The price of the VictoryShares US EQ Income Enhanced Volatility Wtd ETF has ranged from a low of $59.18 to a high of $74.46 over the past 52 weeks. The stock’s moving average over the past fifty days is $62.79, and its moving average over the past 200 days is $66.36.
Several institutional investors have recently boosted the amount of stock they hold in their portfolios. EverSource Wealth Advisors LLC invested 5.4% more money in the VictoryShares US EQ Income Enhanced Volatility ETF during the third quarter than in the previous quarter. EverSource Wealth Advisors LLC now has 39,501 shares of the company’s stock, valued at $2,345,000 following the acquisition of an additional 2,009 shares during the time in question. In addition, Calton & Associates Inc. raised its holdings in the VictoryShares US EQ Income Enhanced Volatility Wtd ETF by 59.7% during the third quarter. After purchasing an additional 24,569 shares during the period, Calton & Associates Inc. now has 65,739 shares of the company’s stock, valued at $3,902,000. During the period, these shares were acquired. During the third quarter, Vantage Financial Partners LLC and Silver Oak Securities Incorporated increased their holdings in the VictoryShares US EQ Income Enhanced Volatility Wtd ETF. Vantage Financial Partners LLC invested the amount of $7,197,00, while Silver Oak Securities Incorporated increased its holdings by 2.2%. Silver Oak Securities Incorporated now owns 582,491 shares of the company’s stock after purchasing an additional 12,645 shares. The value of the company’s stock held by Silver Oak Securities Incorporated is $34,577. Last but not least, during the third quarter, Cambridge Investment Research Advisors Inc. increased the percentage of VictoryShares US EQ Income Enhanced Volatility Wtd ETF that it held by 196.8%. After making further purchases during the time frame, Cambridge Investment Research Advisors Inc. now holds a total of 454,956 shares of the company’s stock, which are worth a combined total of $27,006,000.

Ronald Kaufman is a veteran analyst and researcher with an expertise in the fields of Pharma, Cyber, FoodTech and Blockchain. He has been published on entrepreneur.com, GuruFocus, Finextra Research and others. He is currently a researcher at the Future Markets Research Tank (FMRT), where he does deep-dive market analysis and research in a number of industries.

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By Owen Tonks For Mailonline
Published: 14:50 EST, 12 November 2022 | Updated: 14:50 EST, 12 November 2022

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Keith Levene, a guitarist with The Clash and Public Image Ltd, has died at the age of 65.
The news was confirmed by his friend, author Adam Hammond, who wrote on Twitter: ‘It is with great sadness I report that my close friend and legendary Public Image Limited guitarist Keith Levene passed away on Friday 11th November. 
‘There is no doubt that Keith was one of the most innovative, audacious and influential guitarists of all time.
Sad news: Keith Levene, a guitarist with The Clash and Public Image Ltd, has died at the age of 65 (pictured in 2012)
‘Keith sought to create a new paradigm in music and with willing collaborators John Lydon and Jah Wobble succeeded in doing just that. 
‘His guitar work over the nine minutes of Theme, the first track on the first PiL album, defined what alternative music should be.
‘As well as helping to make PiL the most important band of the age, Keith also founded The Clash with Mick Jones and had a major influence on their early sound. 
Making music: Keith joined Public Image Ltd in 1978 with John Lydon after the split of the Sex Pistols (pictured in 1982)

Speaking out: The news was confirmed by his friend, author Adam Hammond, who shared some heartfelt words on Twitter
‘So much of what we listen to today owes much to Keith’s work, some of it acknowledged, most of it not.
‘Our thoughts and love go out to his partner Kate, sister Jill and all of Keith’s family and friends. 
‘The world is a darker place without his genius. Mine will be darker without my mate.’
A cause of death was not confirmed by Adam. 
On stage: Keith was a member of The Clash but left before they recorded music and he went on to perform with Public Image Ltd (Keith pictured with PiL members John Lydon, Martin Atkins and Jah Wobble in 1980)
Keith first started working in the music industry when he was employed as a roadie for Yes in the 1970s.
He went on to found The Clash with Mick Jones in 1976 and convinced Joe Strummer to leave The 101ers and join their band.
Keith left The Clash before they started recording music and he went on to join Public Image Ltd in 1978 with John Lydon after the split of the Sex Pistols.
He left Public Image Ltd in 1983 to record solo material and work in music production. 
Moving on: Keith left Public Image Ltd in 1983 to record solo material and work in music production (Keith (left) seen with John Lydon in 1981)
Published by Associated Newspapers Ltd
Part of the Daily Mail, The Mail on Sunday & Metro Media Group

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Over 50 species previously thought to be mute make sounds, according to a new study  CNN
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Thunderstorms. Gusty winds and small hail are possible. High 69F. E winds shifting to W at 10 to 20 mph. Chance of rain 90%..
Mainly clear skies. Low 41F. Winds N at 10 to 20 mph.
Updated: November 14, 2022 @ 1:19 am
Curly-leaf parsley is an attractive plant and can be used in your landscape as a border.
Flat-leaf parsley is used more in food and is full of vitamins.
Curly-leaf parsley is an attractive plant and can be used in your landscape as a border.
Flat-leaf parsley is used more in food and is full of vitamins.
Aah, herbs. Not only do they smell good, but they are pretty, add flavor to foods, attract pollinators and contain vitamins and minerals. I love cooking with herbs.
Herbs are easy to grow, providing they are in a sunny area with well-drained soil. They need about 1 inch of water a week. They can be grown in containers, gardens and even in your flower bed. Herbs should be grown close to your house and a water source.
Cool-weather herbs that you can plant now include cilantro/coriander, chives, dill, mint, oregano, sage, thyme, lavender and parsley. I read where arugula was thought to be an herb. My favorites to cook with are cilantro, rosemary, dill and oregano.
Cilantro/coriander (Coriandrum sativum) is a two-herb in one. You get the plant, but you can let it go to seed. Cilantro is known for its use in Mexican food dishes, salsa, salads, pinto beans, rice, pasta and marinades. It is best to use the young leaves since the older leaves can be strong flavored.
Coriander seeds can be used in pastries, cooked fruit and sausage. In the Bible, the coriander seeds were compared to the taste of manna.
Cilantro is one of the most used herbs. Cilantro grows about 2 feet tall and will get pinkish white flowers in early summer. I let mine go to seed because they attract pollinators. The plants prefer to grow in sun to partial shade. When I harvest large bundles of cilantro, I blend leaves of cilantro with olive oil in a blender and freeze in silicone ice trays. When frozen, I take them out and place in plastic bag and use as needed when cooking.
Dill (Anethum graveolens) was prescribed 5,000 years ago by ancient Egyptian doctors. In the Bible, it is mentioned as a tax payment. Dill is an annual and a member of the carrot family. It has delicate looking fern-like leaves with 6-inch clusters at the top and yellow flowers in the summer.
They should be planted 10 inches apart in full sun. Plants can grow 5 feet tall and should be harvested in the morning. Seeds and the leaves can be harvested and used in soups, salads, cheese, egg, fish, pork, lamb, poultry, apples, vinegar, vegetables, pickles, sauces, bread and salt-free types of herb seasonings. My favorite is dill leaves on grilled fish.
Dill is a favorite of the swallowtail butterfly. Plant extra for them. Dill tastes best fresh. I have grown “Bouquet” and “Mammoth” variety and had good luck with both of them.
Parsley (Petroselinum sativum) is known as the most used but least eaten herb. It is usually used as a garnish and then thrown away. It is loaded with vitamins and minerals.
Two types of parsley are used. Flat leaf or Italian parsley is stronger in flavor, larger and hardier than curly leaf or French parsley. Curly leaf parsley makes an attractive border plant.
Many people like flat leaf for its flavor but it can go to seed more quickly than curly leaf. Parsley can be used in salads, salad dressings or with chicken or pork.
One article suggested that parsley could be dipped in batter and deep fried. If you decide you want to use parsley as a garnish, it can be chewed to help freshen our breath. Parsley is good for attracting beneficial insects as well as the swallowtail butterfly.
It is said that herbs should be considered in every garden. I agree and have my favorites. Bon appetit and happy herb gardening.
The Gardeners’ Dirt is written by members of the Victoria County Master Gardener Association, an educational outreach of Texas A&M AgriLife Extension – Victoria County. Mail your questions in care of the Advocate, P.O. Box 1518, Victoria, TX 77901; or [email protected], or comment on this column at VictoriaAdvocate.com.
REFERENCES:
BOOKS
Masabni, Joseph G., Easy Gardening for Texas, Texas A&M AgriLife Extension Service, 2016, p. 209.
Adams, William D. and LeRoy, Thomas R., The Southern Kitchen Garden, Taylor Trade Publishing, 2007, p. 58-60.
Barrett, Judy, What Can I Do With My Herbs? How to Grow, Use & Enjoy These Versatile Plants, Everbest Printing Co., 2010, 41-43, 47-49, 102-104.
Oster, Maggie, Ortho’s All About Herbs, Meredith Publishing Company, 1999, p. 66, 69, 83.
PERIODICALS
Blachman, Vicki, Texas Gardener Magazine, Suntex Communications, Inc., September/October 2012, p. 20-23.
Leander, Patty G., Texas Gardener Magazine, Suntex Communications, Inc., November/December 2020, p. 12-15.
Blachman, Vicki, Texas Gardener Magazine, Suntex Communications, Inc., January/February 2009, p. 28-32.
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Gloria Wallace said:
Offering condolences 🙏🏽 to the Family, from the Wallace Family!
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Mary Helen Pena said:
My deepest condolences to Karen and family for your loss. My prayers and thoughts are with you. May you find comfort in each other.
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Isaac Jefferson said:
To the family and friend ,you have my sincere condolences rest in peace my
brother.
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NASHVILLE, Tenn. (WSMV) – Metro Parks and the Metro Historical Commission have finalized the latest Fort Negley Master Plan after a year of research, planning and public engagement, the groups announced Thursday.
The vision of the plan is to create an open-air museum of natural significance that speaks through the cultural landscape to reveal history from the Civil War to the civil rights era.
Community engagement and feedback played a critical role in the development of the plan. Throughout a series of five events beginning in the fall of 2021, community members were asked to participate in interactive activities that ultimately laid the groundwork for the design process. Activities included an expert panel session, open houses, opportunities for reflection and multiple physical scale models to facilitate feedback. The consultant team was available to provide expert guidance on best and innovative practices for the treatment of historic and cultural sites.
More than 175 participants and more than 1,175 comments were received during these meetings.
Numerous stakeholder groups were also engaged periodically throughout the design process to share knowledge and to provide updates regarding the master plan. Over the course of the entire master planning process, more than 20 meetings were held with various groups.
“The master plan represents a dedicated effort by a multitude of diverse individuals and organizations. The public input, stakeholder discussions, and Metro Nashville’s dedication to supporting an inclusive and thorough planning process have yielded what we believe to be a plan for the successful and sustainable transformation of the Fort Negley site,” Metro Parks Director Monique Horton Odom said in a news release.
“The recommendations laid out in this master plan represent an opportunity to provide the site its proper treatment; to create a lasting landscape of memory, hope and resilience for Nashville and for the nation. The new plan establishes a long-term vision for the preservation, interpretation and development of the park,” Richie Jones, partner at HDLA Landscape Architecture, who served as the prime contractor for the master plan, said in a news release. “Our entire team of planners, designers, historians and neighborhood advocates participated in crafting the final product.”
Fort Negley Park is a 64-acre park in the heart of Nashville. Recognized as one of the nation’s most important historical sites, it is home to distinct natural and cultural assets and was designated a “Site of Memory” by the UNESCO Slave Route Project in 2019 and is listed in the National Register of Historic Places.
Click for information about the Fort Negley master plan.
Copyright 2022 WSMV. All rights reserved.

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It’s the tale of the two Pagans.
NYPD cops grabbed a man held on Rikers Island named Andrew Pagan and brought him to federal court Wednesday, thinking they had their suspect in a gun and meth ring operating in the city.
But it turns out they had the wrong Andrew Pagan, and it all took a day and some red faces to sort it out.
The mistake was set in motion when a veteran detective with the NYPD’s Gun Offender Monitoring Unit named Debra Lawson concluded that the Andrew Pagan they were looking for was being held on Rikers Island, sources said.
Rikers Island (Seth Wenig/AP)
Lawson and other law enforcement officers went to the Anna M. Kross Center, where this Pagan, 26, has been held a Bronx gun case since March. They took him to Brooklyn federal court to be seen before U.S. Magistrate Judge Vera Scanlon, court and Department of Correction records show.
Scanlon presided over his arraignment Wednesday and he was taken to the Metropolitan Detention Center in Brooklyn.
Everything seemed fine, until the cops realized they had the wrong Andrew Pagan.
In fact, the actual suspect, Andrew A. Pagan, wasn’t even in Rikers. Authorities finally figured out where he works, arrested him there on Thursday morning and hauled him into court.
Later that day, Assistant U.S. Attorney John Enright had to explain the mess to Judge Scanlon.
He told Scanlon that another NYPD detective with Lawson “pieced together, post-arraignment, that Andrew Pagan was not in fact Andrew A. Pagan.”
”The NYSID [New York state identification] numbers didn’t match. And then they immediately began to take corrective steps,” Enright said. “He was, in fact, identified as not, in fact, being in Rikers Island or otherwise incarcerated.”
“The government apologizes greatly for this error,” Enright added.
The prosecutor later said that the reason the government didn’t notice it was the wrong guy was because another assistant US attorney , was subbing for him at Wednesday’s arraignment.
”There was no way really for me to independently have identified that this was not the right person,” said lawyer Karume James, the Federal Defender who represented the wrong Pagan on Wednesday. He added that it’s “obviously the purview of law enforcement and the government to ensure that they actually have the right person. The only documents that I was printed yesterday were just an indictment with the person’s name and charges.”
James said Thursday he was able to obtain an NYPD report with the correct Pagan’s picture on it and though their birthdays were similar, it was clear the authorities had the wrong man.
The correct Pagan’s lawyer, Federal Defender Benjamin Yasser, called the lapse “just a lack of any due diligence.”
”I just want to mention one small fact that makes this error particularly inexcusable,” he said, noting the correct Andrew A. Pagan, who is charged in federal indictment was charged with the same offense in Brooklyn state court nearly a year ago, and that he’s out on bail in that case.
”In that case, if you reviewed the most basic criminal history records, you would see he was released on bail. There is no way that the Andrew A. Pagan wanted in this indictment could have been in Rikers,” Yasser said.
Scanlon ordered the charges vacated. The wrong Pagan was held overnight in the Metropolitan Detention Center and then sent back to Rikers.
The right Pagan was released on a $100,000 bond. He hasn’t missed a state court date since his arrest in December 2021. He owns two businesses, a smoke shop, and a music production company.
“Due to an administrative error, the wrong individual was taken from Rikers Island, where he was already in state custody,” the Correction Department said in a statement.
“When the error was discovered, the individual was returned to Rikers Island and the appropriate person was apprehended and charged federally with distribution of a controlled substance and firearm possession.”
The NYPD did not immediately respond to a request for comment.
Copyright © 2022, New York Daily News
Copyright © 2022, New York Daily News

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The Ministry of National Defense on Saturday declined to comment on the authenticity of an audio recording of an apparent confrontation between a Taiwanese navy vessel and a Chinese navy destroyer off eastern Taiwan.
In the recording, a person, allegedly an officer on the Chinese destroyer Xiamen, can be heard saying that there is no border of Taiwan’s contiguous zone during a radio exchange with the guided-missile destroyer ROCS Ma Kong.
The recording, posted on social media, was allegedly made by the Marshall Islands-flagged bulk carrier Lady Neeti, which was sailing nearby when the exchange took place.
Photo: Huang Liang-chieh, Taipei Times
In the recording, a man’s voice can be heard identifying his vessel as the Ma Kong and warning another ship that it is approaching the outer edge of the nation’s contiguous zone, 44.5km from the baseline.
Another man’s voice replies: “Xiamen to Ma Kong, the so-called ‘24 nautical-mile [44.5km] baseline’ does not exist. Please check your terminology. Our ship has the right to sail here. Please maintain a safe distance of at least 3 nautical miles.”
“This is Ma Kong. We are currently sailing on a course of 230°. Please adjust your course as appropriate,” the first voice is heard as saying, to which the second voice replies: “Xiamen copies.”
The ministry declined to go into details about its military deployments, saying only that it had taken appropriate response measures to all incursions by Chinese military vessels.
Institute for National Defense and Security Research analyst Su Tzu-yun (蘇紫雲) said the best way to prepare Taiwanese mentally for Chinese military incursions is to make information about the incursions more transparent and accessible.
Since US House of Representatives Speaker Nancy Pelosi’s visit to Taiwan in early August, Chinese warships have routinely carried out incursions past the median line of the Taiwan Strait and sailed in the waters off Taiwan’s east coast in an attempt to create a new “status quo,” Su said.
Taiwan could learn from Japan’s model of issuing alerts whenever North Korea fires missiles toward it, he added.
Separately, the ministry said 10 Chinese fighter jets crossed the median line on Saturday.
They were among 36 Chinese aircraft detected near Taiwan that day, the ministry said, adding that three Chinese naval vessels had also been spotted in the area.
The ministry said that six Shenyang J-11 and four J-16 planes flew across the median line.
The military also spotted four Chengdu J-10 fighter jets, a Y-8 anti-submarine warfare plane, and three H-6 strategic bombers in Taiwan’s southwestern air defense identification zone, the ministry said.
In addition, three Chinese uncrewed aerial vehicles — a Harbin BZK-005 high-altitude, long-range drone, a Guizhou BZK-007 and a CASC Rainbow CH-4 — were detected in the same area, it said.
The ministry said it scrambled combat air and naval patrols, and deployed defense missile systems to track the Chinese aircraft.
More than 90 percent of Taiwanese consider learning Mandarin helpful for work and interpersonal relationships, a survey released by National Taiwan Normal University’s (NTNU) Chinese Language and Technology Center showed. The survey, conducted on Tuesday and Wednesday, collected 1,092 valid samples from Taiwanese aged 21 to 60, the center said on Thursday. The study also used news reports on classical Chinese education posted on Professional Technology Temple (PTT), the nation’s largest online bulletin board system, it added. The survey was publicized after an announcement by the University Admission Committee last month that subjects of the General Scholastic Ability Test are to be included
INTERNATIONAL LIST: National Taiwan University retained 19th spot for the third consecutive year, while China’s Peking University topped the list Nine Taiwanese universities entered the top 100 in the Quacquarelli Symonds (QS) Asia University Rankings released on Tuesday. National Taiwan University (NTU) retained 19th spot for the third consecutive year, the ranking showed. NTU was the only higher education institution from Taiwan that made the top 20 in the ranking, which lists 760 Asian universities. The eight other Taiwanese universities in the top 100 are National Cheng Kung University (36th), National Tsing Hua University (38th), National Yang Ming Chiao Tung University (43rd), National Taiwan University of Science and Technology (59th), National Taiwan Normal University (70th), National Taipei University of Technology (73rd), National Sun
T1 League officials yesterday formally introduced US NBA star Dwight Howard to basketball fans in Taipei, ahead of his first game for the Taoyuan Leopards. Howard is the biggest former NBA name to play in Taiwan, as the eight-time NBA All-Star and five-time rebound king promised to dominate play and bring a championship title to the Taoyuan club. “This is going to be an awesome time, I can’t wait to get out there and play,” Howard told a press conference, adding that he takes the sport seriously, and aims to help the team win the league’s championship title. “This is not me coming
BEWARE: While reporting on the successes police have had in cracking down on human trafficking, officials warned that people are still being conned by job scams The kidnapping and torture of more than 60 people involved Taiwan’s major gangs, police officials said as authorities warned that jobseekers are still being conned by human traffickers. At a news conference in Taipei yesterday, National Police Agency Director-General Huang Ming-chao (黃明昭) praised judicial agencies and local police for their diligence, when announcing that nine suspects had been arrested in a case of alleged kidnapping and human trafficking by a gang based in the Hsinchu area. “Our government is taking strong action, with law enforcement agencies undertaking operations across the nation. We are clamping down on snakehead operations involving human trafficking to

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Belgrade, Serbia – Seprember 19th: Keith Flint from English band The Prodigy performing at Warrior’s Dance festival on Seprember 19th, 2012 in Belgrade, Serbia  ·  Source: Zamrznuti tonovi/Shutterstock/Gearnews
Without a doubt one of the most impactful electronic acts of the 1990s, The Prodigy concocted their own brand of industrial breakbeat EDM and swiftly took it to a global audience.
Not only was this due to Liam Howlett’s unique approach to music production, but the hard-hitting nature of the records and live shows meant they could play festivals such as Rock Am Ring and Wacken Open Air, which weren’t ever geared toward electronic music.
For a band named after a synth, it’s easy to get fooled into thinking synthesizers were the focal point of The Prodigy’s sound. In actual fact, the true genius of the music comes from Liam’s record collection and his appreciation for Hip-Hop sampling.
The anthemic synth hooks are certainly a feature, but classics like Voodoo People, Breathe, and Mindfields are all built around the innovative reconstitution of existing drum breaks and melodic elements from other records.
The use of digital sampling didn’t stop there either, because often individual drum hits and even synths were resequenced with old-school samplers simply to attain the character of the sound they produce.
Let’s take a look at some of the key gear choices that became the method in the madness:
When it was launched in 1989, the Roland W-30 was a groundbreaking workstation that combined sampling, sequencing, and synthesis into a single keyboard. As he was looking for a sequencer, Liam got hold of one right away and it soon became his primary songwriting tool.
The gritty 12-bit digital sampling engine is responsible for the characteristically industrial sound of the early Prodigy releases. The workflow clearly resonated with Liam, as he continued to use the W-30 in live shows, even after he found other solutions for sequencing.
Roland W-30
The W-30 was in use until The Fat Of The Land in 1997 when the post-production was done on Cubase for the first time.
Steinberg Cubase Pro 12
Another not-so-secret weapon in The Prodigy’s arsenal was the Boss SE-70 multi-effects unit, introduced in 1993. Although it wasn’t particularly innovative or affordable, it did have an outstanding selection of effects algorithms, including a vocoder, ring modulator, and pitch tracker.
Although designed for guitarists, there was a huge wealth of effects that could be used on any instrument, with up to 16 effects per algorithm. The SE-70 also had some hidden tweaks for getting interesting distortion effects when you set the reverb mix on full, and this made it essential to The Prodigy’s sound, especially on The Fat Of The Land.
BOSS SE-70
You won’t easily find such an extensive multi-effects unit in an accessible price range these days, but the Roland VT-4 certainly produces some exciting results.
Roland VT-4
The noxious-sounding Prophecy was one of Korg’s early forays into virtual analogue synthesis in 1995. It might be monophonic, but this monster is equipped with 13 DSP-based oscillator algorithms – which included VPM(similar to FM) and physical modeling – and a workstation-level synthesis engine.
Programming this beast took some patience, but once you got the hang of the rather tedious interface you could create some absolutely ridiculous sounds. It was used extensively by Howlett on The Fat Of The Land, where you can hear it distinctively on Firestarter and Smack My *itch Up.
Korg Prophecy
The Prophecy takes a unique approach to synthesis, which you can still do to a certain extent on the Korg Modwave.
Korg Modwave
The SP-12 and SP-1200 samplers are easily some of the most influential music-making tools ever built. The uniquely chunky sound instantly appealed to Howlett, who was a big fan of Hip-Hop sampling and used the SP-1200, particularly for one-shots and individual drum hits.
You can hear it on the drum break intro to Firestarter, or the retriggered drumbeat of Spitfire where the Lo-Fi character still manages to get pushed to the front of the mix. From Daft Punk and the French House movement to RZA and the Wu-Tang Clan, the sound of SP-1200 has become a timeless part of music production history.
E-mu SP-1200
Although SP-1200 was reissued, you can get great results using the workflow of the Elektron Digitakt. You can read more about getting the old-school sampler sound here.
Elektron Digitakt
If you’re not familiar with the Nord sound, it’s an incredibly raw and lively virtual analogue synth that rose to prominence in the 1990s. It was used for leads and effects on Funky Sh*#, and also became a regular fixture in The Prodigy’s live rig.
Nord synthesizers provide a relatively straightforward interface for creating sounds and, being digital, you don’t get some of the inconsistencies and maintenance headaches you would normally associate with vintage analogue synths.
Nord synthesizers were also used by Nine Inch Nails during The Fragile era, so you can be sure they have the ability to cut right through the mix.
Clavia Nord Lead
The original Nord Lead was discontinued back in 1997, but you can get similar results with the modern Nord Lead A1.
Clavia Nord Lead A1
What are some of your favourite instruments used by Liam Howlett and The Prodigy?
Please let us know in the comments below!
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How do you like this post?
I heard that Liam did a lot of the Prodigy recordings with Propellerhead‘s Reason DAW. But even if you have all the studio tools, you will never sound like this band because they had a very special culture, taste, background, and the ability to create a very individual artistic result!
Ahh, love the Prodigy so thank you for putting this together 🙂
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13 Nov · Thanks for the suggestion, but at close to $500, the Slate VSX is far from being a budget set of headphones.
13 Nov · Gotta say it’s weird as heck not having Slate VSX on this list.
13 Nov · Ahh, love the Prodigy so thank you for putting this together 🙂

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Updated At: Nov 12, 2022 09:22 PM (IST)

Teamology is one of the best PR agency in Chandigarh specializing in promoting  brands for their diversified clients all across India and Dubai. After creating a benchmark for themselves in PR in all metros of the country, Teamology has expanded their footprint in Chandigarh to promote the businesses of the city.
 
Public relations is nothing but editorial coverage for business promotion which are published in offline and online media. So it is more authentic than advertising and adds more value than paid media advertisements. 
 
The city of Chandigarh is witnessing a progressive growth in its business environment in the recent past which in turn led to an increased need for PR activities as industries these days are in a race to establish and promote their brands for sustainability. 
 
Although Teamology as a whole caters their optimum services to a wide range of industries in Chandigarh, one key area which the company has been seeing outstanding success is the entertainment sector.  
 
PR in entertainment includes promotions of movies, music, TV, web series etc. Punjabi movies and music industry have always been very rich and now the Punjab government is taking up initiative to set up the first film city of North India in Chandigarh which will further boost the area’s entertainment sector. Punjabi songs have a history of creating sensations all over the country and with most shooting of Punjabi music videos of recent times having taken place in Chandigarh, the music industry of Chandigarh is also booming. 
 
Teamology is actively working with a lot of Chandigarh based movie and music production houses, actors and singers. Some of the PR practices exercised by Teamology are –

●       Releasing teaser promos, trailers, and other content related to press releases for the movies. 
●       Organizing promotional campaigns to build positive images of the movies and arouse deep interest in public, media and record companies about the music or singer.
●       Announcing fresh releases of films by holding press conferences. 
●        Organizing talk shows for the cast members to appear. 
 
Teamology, the best PR agency in India is dominating the PR market of Chandigarh with their acclaimed PR campaigns helping Punjabi movie and music production houses, singers and actors to jump on the branding bandwagon. 
 

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The Tribune, now published from Chandigarh, started publication on February 2, 1881, in Lahore (now in Pakistan). It was started by Sardar Dyal Singh Majithia, a public-spirited philanthropist, and is run by a trust comprising four eminent persons as trustees.
The Tribune, the largest selling English daily in North India, publishes news and views without any bias or prejudice of any kind. Restraint and moderation, rather than agitational language and partisanship, are the hallmarks of the paper. It is an independent newspaper in the real sense of the term.
The Tribune has two sister publications, Punjabi Tribune (in Punjabi) and Dainik Tribune (in Hindi).
Remembering Sardar Dyal Singh Majithia
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New Jersey, United States – Market Research Intellect has recently published a research report titled, “Global Music Publishing Administration Software Insight, Forecast To 2028” assessing various factors impacting its trajectory. The Global Music Publishing Administration Software report offers a high-quality, accurate, and comprehensive research study to equip players with valuable insights for making strategic business choices. The research analysts have provided deep segmental analysis of the Global Music Publishing Administration Software on the basis of type, application, and geography. The vendor landscape is also shed light upon to inform readers about future changes in the market competition. As part of competitive analysis, the report includes detailed company profiling of top players of the Global Music Publishing Administration Software . Players can also use the value chain analysis and Porter’s Five Forces analysis offered in the report for strengthening their position in the Global Music Publishing Administration Software .
Leading players of the Global Music Publishing Administration Software are analyzed taking into account their market share, recent developments, new product launches, partnerships, mergers or acquisitions, and markets served. We also provide an exhaustive analysis of their product portfolios to explore the products and applications they concentrate on when operating in the Global Music Publishing Administration Software . Furthermore, the report offers two separate market forecasts – one for the production side and another for the consumption side of the Global Music Publishing Administration Software . It also provides useful recommendations for new as well as established players of the Global Music Publishing Administration Software .
Get Sample PDF (Including Tables and Figures, Charts & Graphs) of Music Publishing Administration Software Market Research Report@ https://www.marketresearchintellect.com/download-sample/?rid=198501 
The report endeavors to offer a 360-degree analysis of the global Music Publishing Administration Software Market on the back of an insightful study of the prevailing demand and supply trends, important fiscal statistics of major players sustaining in the market, and the influence of latest economic advancements on the market. Developments in each geographical region is charted using authentic historical data with a view to help gauge the future trajectory of the market across the globe. SWOT analysis is conducted to identify the strengths, weaknesses, opportunities, and threats that these companies forecast to witness during the forecast period.
Key Companies:
Segment by Types:
Segment by Applications:
Get | Discount On The Purchase Of This Report @ https://www.marketresearchintellect.com/ask-for-discount/?rid=198501 
Impact of COVID-19 on Music Publishing Administration Software Market Industry: The coronavirus recession is an economic recession happening across the world economy in 2020 due to the COVID-19 pandemic. The pandemic could affect three main aspects of the global economy: production, supply chain, and firms and financial markets. The report offers complete version of the Music Publishing Administration Software Market will include the impact of the COVID-19 and anticipated change on the future outlook of the industry, by taking into the account the political, economic, social and technological parameters.
Music Publishing Administration Software Market: Regional Analysis Includes:
⇨ Asia-Pacific (Vietnam, China, Malaysia, Japan, Philippines, Korea, Thailand, India, Indonesia, and Australia)
⇨ Europe (Turkey, Germany, Russia UK, Italy, France, etc.)
⇨ North America (the United States, Mexico, and Canada)
⇨ South America (Brazil etc.)
⇨ The Middle East and Africa (GCC Countries and Egypt)
Table of Contents
Global Music Publishing Administration Software Market 2015-2026, With Breakdown Data of Capacity, Sales, Production, Export, Import, Revenue, Price, Cost and Gross Margin
Chapter 1. Report Overview
Chapter 2. Market Snapshot
2.1 Major Companies Overview
2.2 Music Publishing Administration Software Market Concentration
2.3 Six-Year Compound Annual Growth Rate (CAGR)
Chapter 3.Value Chain of Music Publishing Administration Software Market
3.1 Upstream
3.2 Downstream
3.3 Porter’s & Five Forces Analysis and SWOT Analysis
Chapter 4. Players Profiles
4.1 Company Profiles
4.2 Product Introduction
4.3 Production, Revenue (2015-2020)
4.4 SWOT Analysis
Chapter 5. Global Music Publishing Administration Software Market Analysis by Regions
5.1 Music Publishing Administration Software Market Status and Prospect (2016-2026)
5.2 Music Publishing Administration Software Market Size and Growth Rate (2016-2026)
5.3 Music Publishing Administration Software Market Local Capacity, Import, Export, Local Consumption Analysis (2015-2026)
Chapter 6. North America Music Publishing Administration Software Market Analysis by Countries
Chapter 7. China Music Publishing Administration Software Market Analysis by Countries
Chapter 8. Europe Music Publishing Administration Software Market Analysis by Countries
Chapter 9. Asia-Pacific Music Publishing Administration Software Market Analysis by Countries
Chapter 10. India Music Publishing Administration Software Market Analysis by Countries
Chapter 11. Middle East and Africa Music Publishing Administration Software Market Analysis by Countries
Chapter 12. South America Music Publishing Administration Software Market Analysis by Countries
Chapter 13. Global Music Publishing Administration Software Market Segment by Types
Chapter 14. Global Music Publishing Administration Software Market Segment by Applications
Chapter 15. Music Publishing Administration Software Market Forecast by Regions (2020-2026)
Chapter 16. Appendix
Browse the Full Report or TOC of Music Publishing Administration Software Market @ https://www.marketresearchintellect.com/product/global-music-publishing-administration-software-market-size-and-forecast/
Report Includes Following Questions:
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About Us: Market Research Intellect
Market Research Intellect provides syndicated and customized research reports to clients from various industries and organizations with the aim of delivering functional expertise. We provide reports for all industries including Energy, Technology, Manufacturing and Construction, Chemicals and Materials, Food and Beverage, and more. These reports deliver an in-depth study of the market with industry analysis, the market value for regions and countries, and trends that are pertinent to the industry.  
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Market Research Intellect
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