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As we step into the year 2023, the automotive industry is buzzing with excitement and anticipation for the next big thing in car design. Manufacturers and designers are pushing boundaries, exploring new technologies, and reimagining the concept of transportation. This article takes you on a journey into the future, discussing the trends, innovations, and challenges involved in designing the next car.
The Rise of Electric Vehicles
One of the most significant trends in car design is the increasing dominance of electric vehicles (EVs). With a focus on sustainability and reducing carbon emissions, EVs have gained immense popularity. Designers are now faced with the challenge of creating sleek, aerodynamic designs that maximize range and efficiency while maintaining a distinctive visual appeal.
Integration of Autonomous Driving
Another major aspect of future car design is the integration of autonomous driving technology. As self-driving cars become more prevalent, designers are rethinking the interior layout to create comfortable and functional spaces. The focus is on creating an environment that promotes relaxation, productivity, and entertainment, rather than traditional driver-centric designs.
Efficiency and Lightweight Materials
In an effort to improve fuel efficiency and reduce environmental impact, car designers are exploring lightweight materials and advanced manufacturing techniques. Materials like carbon fiber, aluminum, and composites are being utilized to reduce weight without compromising safety or performance. These innovative materials also offer greater design flexibility, enabling designers to push the boundaries of creativity.
Challenges in Designing the Next Car
While the future of car design is exciting, it comes with its fair share of challenges. One of the primary concerns is striking the right balance between aesthetics and functionality. Designers must create visually appealing cars that also meet the demands of practicality, safety, and performance.
Designing cars that comply with ever-evolving regulations is another challenge. Safety standards, emission norms, and other regulatory requirements can significantly impact the design process. Designers must stay abreast of these regulations and find innovative solutions to meet them without compromising on design principles.
Changing Consumer Preferences
Understanding and predicting consumer preferences is crucial for successful car design. With evolving lifestyles and increasing emphasis on sustainability, designers must anticipate the needs and desires of future car buyers. Balancing individuality, connectivity, and eco-friendliness is the key to designing cars that resonate with the target audience.
The Future of Car Interiors
With the advent of autonomous driving technology, car interiors are undergoing a revolution. The focus is shifting from traditional driver-centric designs to passenger-centric layouts. Interior spaces are being reimagined as versatile environments that cater to various activities such as work, entertainment, relaxation, and socializing.
Connected and Interactive Experiences
Seamless connectivity and interactive experiences are becoming essential in car interiors. Touchscreens, voice commands, gesture controls, and augmented reality are just a few of the technologies that will shape the future of car interiors. Designers are working to integrate these features seamlessly, making the driving experience more intuitive and enjoyable.
Sustainable Materials and Ergonomic Designs
Car interiors are also embracing sustainable materials and ergonomic designs. Natural and recycled materials, eco-friendly fabrics, and low-VOC (volatile organic compound) components are gaining popularity. Designers are prioritizing comfort, health, and sustainability to create interiors that align with the evolving needs and values of consumers.
1. How will electric vehicles impact car design?
Electric vehicles will impact car design by influencing the shape, weight, and ergonomics of cars. The absence of an internal combustion engine allows for more creative freedom in design, resulting in sleeker and more aerodynamic cars. Additionally, the need for efficient battery placement and cooling systems will influence the overall layout and packaging of the vehicle.
2. What role will autonomous driving play in future car design?
Autonomous driving will revolutionize car design by shifting the focus from driver-centric to passenger-centric interiors. With self-driving cars, the interior layout can be optimized for comfort, productivity, and entertainment. The integration of advanced technologies like AI, voice recognition, and augmented reality will also transform the driving experience.
3. How are lightweight materials changing car design?
Lightweight materials like carbon fiber and aluminum are revolutionizing car design by reducing weight and improving fuel efficiency. These materials offer greater design flexibility and allow for the creation of sleeker and more aerodynamic shapes. However, they also come with challenges in terms of cost, manufacturing processes, and ensuring safety standards.
4. How are car interiors evolving?
Car interiors are evolving to become more connected, interactive, and versatile. With autonomous driving technology, the focus is shifting towards creating comfortable and functional spaces that cater to various activities. Seamless connectivity, advanced infotainment systems, and personalized experiences are some of the key features shaping the future of car interiors.
5. How are designers addressing sustainability in car design?
Designers are addressing sustainability in car design by incorporating eco-friendly materials, reducing waste in manufacturing processes, and optimizing energy efficiency. The use of recycled and renewable materials, eco-conscious manufacturing techniques, and the adoption of circular economy principles are some of the ways designers are working towards creating more sustainable cars.


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Weekly email list
- Orioles Agree To Deal With Ariel Miranda
- Right-Hander Norge Ruiz Leaves Cuba, Will Seek Deal With MLB Club
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- Mariners Acquire Welington Castillo From Cubs For Yoervis Medina
- Bruce Chen Announces Retirement
- Red Sox Outright Allen Craig
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Some links for your Tuesday morning…
- Jon Paul Morosi of FOX Sports says there's no guarantee the Dodgers will spend the money they saved in the Juan Pierre deal.
- Newsday's David Lennon says Omar Minaya had plans to speak with John Lackey today and concludes that the pitcher wasn't interested in joining the Mets.
- ESPN.com's Jerry Crasnick says there's speculation that the Jays will deal Lyle Overbay now that they're set to obtain Brett Wallace.
- ESPN.com's Buster Olney hears that the Red Sox are looking at righty outfielders such as Xavier Nady to contribute in a utility role.
- Newsday's Ken Davidoff doesn't expect the Yankees to commit to another nine-figure salary and sign Matt Holliday, especially since they need pitching.
- Rob Neyer of ESPN.com says the $130MM or so the Cardinals have apparently offered Holliday is "too much risk for too many years."
- The Marlins signed Clay Hensley to a minor league deal, according to MLB.com's Joe Frisaro. The 30-year-old righty didn't pitch in the majors last year.
- Juan C. Rodriguez of the South Florida Sun Sentinel says the Marlins, who will watch Aroldis Chapman throw, have "legit" interest in the 21-year-old lefty.
- Gordon Edes of ESPNBoston points out that the Red Sox have tons of cash, even if they like to downplay their wealth.
- Dan Shaughnessy of the Boston Globe likes the Lackey signing for the Red Sox.
- Ken Rosenthal of FOX Sports reports that Kelvim Escobar will pitch in front of teams this week in Venezuela. We know the Mets recently offered him a minor league deal.
- Lots of people have said the Brewers are a threat to sign Mark Mulder. GM Doug Melvin confirmed on 1250 WSSP in Milwaukee that he thinks the Brewers are the "frontrunners" to sign the lefty (Hat Tip: MLB.com's Adam McCalvy).
- Dave Cameron of U.S.S. Mariner says the Mariners could use a player like Luke Scott.
- Jeff Zrebiec of the Baltimore Sun wonders how seroiusly the Orioles are interested in Japanese pitcher Hisanori Takahashi. The O's met with his agents at the Winter Meetings.
- Mark Feinsand of the New York Daily News says the Yankees are glad to see Roy Halladay leave the AL East. I'm sure Yankees fans agree.
- Joel Sherman of the New York Post says the Yanks would be comfortable playing Melky Cabrera in left and using Johnny Damon as the primary DH.
- The A's sent three people to watch Aroldis Chapman throw in Houston, according to Susan Slusser of the San Francisco Chronicle.
Full Story | Comments | Categories: Aroldis Chapman | Baltimore Orioles | Boston Red Sox | Hisanori Takahashi | John Lackey | Johnny Damon | Kelvim Escobar | Los Angeles Dodgers | Luke Scott | Lyle Overbay | Mark Mulder | Matt Holliday | Melky Cabrera | Miami Marlins | Milwaukee Brewers | New York Mets | New York Yankees | Oakland Athletics | Seattle Mariners | St. Louis Cardinals | Toronto Blue Jays | Xavier Nady
Links for Monday…
- MLB.com's T.R. Sullivan wonders if Bobby Crosby could be a match for the Rangers as a utility infielder.
- MLB.com's Mychael Urban has an update on free agent lefty Mark Mulder.
- Maury Brown of The Biz of Baseball analyzes Tim Lincecum's possible 2010 salary, and predicts the pitcher might submit $16.8MM against the Giants' $8MM figure.
- ESPN's Buster Olney tweets that Hideki Matsui's marketability is not a factor for the Yankees.
- Alex Speier of WEEI examines the chances of Roy Halladay remaining a workhorse over the next several seasons.
- Derrick Goold of the St. Louis Post-Dispatch doesn't see geography as a big factor for Matt Holliday. Meanwhile, Bernie Miklasz of the P-D doesn't understand Albert Pujols' reasoning as to why he needs to wait to discuss an extension.
- MLB.com's Brian McTaggart asks readers to predict the Astros' arbitration decisions for free agents. Jose Valverde and LaTroy Hawkins are tough calls in that it makes sense but we're not sure if the Astros will. We'll have predictions on all Type A/B free agents later today.
- Tom Haudricourt of the Milwaukee Journal-Sentinel explains why he feels only Felipe Lopez will get an arbitration offer from the Brewers.
- MLB.com's Joe Frisaro says the Marlins are leaning against offering arbitration to Type B free agents Kiko Calero and Nick Johnson. Calero might be worth it.
- Jon Weisman of Dodgers Thoughts says the Dodgers' arbitration decisions won't necessarily be influenced by the McCourts' divorce.
- Staying with this theme, FanGraphs' Dave Cameron looks at the factors that should influence a team's arbitration-offer decision.
- Ken Rosenthal and Jon Paul Morosi of FOX Sports look at the market for Miguel Tejada.
- Morosi suggests ten teams that should talk to the Tigers about Miguel Cabrera, Curtis Granderson, and Edwin Jackson.
- Jacob Jackson of Athletics Nation has a detailed offseason plan for the A's.
- Righty Miguel Batista told Geoff Baker of the Seattle Times that he has a few contenders interested in his services.
- Notes from NPB Tracker's Patrick Newman: the Orioles are eyeing pitcher Hisanori Takahashi, and Hideki Okajima would like a contract extension (he's under team control through 2012 as an arbitration-eligible player).
- Dejan Kovacevic of the Pittsburgh Post-Gazette says the fates of Jeff Karstens, Robinzon Diaz, and Justin Thomas will be decided today.
- Elmer Dessens doesn't have a contract with the Mets quite yet, writes Adam Rubin of the New York Daily News.
Full Story | Comments | Categories: Albert Pujols | Baltimore Orioles | Bobby Crosby | Boston Red Sox | Curtis Granderson | Detroit Tigers | Edwin Jackson | Elmer Dessens | Felipe Lopez | Hideki Matsui | Hideki Okajima | Hisanori Takahashi | Houston Astros | Jeff Karstens | Jose Valverde | Kiko Calero | LaTroy Hawkins | Los Angeles Dodgers | Mark Mulder | Matt Holliday | Miami Marlins | Miguel Batista | Miguel Cabrera | Miguel Tejada | Milwaukee Brewers | New York Mets | New York Yankees | Nick Johnson | Oakland Athletics | Pittsburgh Pirates | Robinzon Diaz | Roy Halladay | San Francisco Giants | St. Louis Cardinals | Texas Rangers | Tim Lincecum | Toronto Blue Jays
Some Sunday night links:
- In his latest mailbag, Paul Hoynes of the Cleveland Plain Dealer writes that the Indians are in the hunt for a first baseman that hits right-handed, in the event that Matt LaPorta isn't healthy by Opening Day.
- Patrick Newman of NPB Tracker passes along the news that the San Francisco Giants are "showing mild interest" in Hisanori Takahashi, via Twitter. Earlier this week, the 34-year-old reliever made it known that he wants to make the jump to MLB.
- Padres closer Heath Bell says that former GM Kevin Towers was never able to spend the way he wanted to and was "kind of a puppet", according to Kevin Tomase of the Boston Herald. Bell also said that he hopes new GM Jed Hoyer has full rein over the club's baseball decisions.
- According to a report from MLB.com's Bill Ladson, the odds of Livan Hernandez returning to the club are "less than 50-50." Over the last three years, the 34-year-old pitcher has recorded an ERA of 5.45 whilst eating 568 innings for five different clubs.
A few more items for Wednesday evening…
- Chad Jennings of The Journal News thinks the Yankees ought to at least call Toronto about right-handed ace Roy Halladay. "I have no idea what the asking price might be," writes Jennings, "but Halladay is better than [John] Lackey and he’ll surely cost fewer prospects than Felix Hernandez."
- As noted by the Boston Globe's Nick Carfado, MLB executive vice president Rob Manfred said Wednesday the revenue sharing figures thrown out by agent Scott Boras during last week's GM meetings "have no basis in reality" and that Boras is living in a "fantasy land."
- According to Ryo Shinkawa of NPB Tracker, right-hander Ryota Igarashi and left-hander Hisanori Takahashi filed for international free agency on Wednesday.
- Sam Mellinger of the Kansas City Star expects Zack Greinke to move on once his contract with the Royals is through. "The good news is that the team has Greinke under contract three more years," writes Mellinger. "The bad news is they have a lot of improving to do in that time."
- Joe Strauss and Bernie Miklasz of the St. Louis Post-Dispatch took to the radio Wednesday afternoon with a few opinions on the Cardinals' offseason plans. Both feel Matt Holliday is only an average defender and that the Cardinals could be looking to add starting pitching via trade this offseason.
According to Bobbie Dittmeier of MLB.com, Japanese lefthander Hisanori Takahashi declared free agency on Tuesday, and said he wants to try his luck in the Major Leagues.
"I decided I wanted to try out for the Majors three days ago," Takahashi said, according to sponichi.co.jp. "If I didn't have confidence in myself, I wouldn't have made the decision. I want to go to a team that needs me. I'd be extremely happy if I'm used as a starter."
"It's a dream I have," Takahashi said. "And I'd like to attain that dream. Seeing (Koji) Uehara in the Majors, that made me want to go to the Majors even more. I want to return to the days of being a rookie and take on this new challenge."
A former teammate of Uehara and Hideki Matsui, the 34-year-old Takahashi had a 2.94 ERA in 25 games last year, striking out 126 in 144 innings. He has spent his entire career with the Yomiuri Giants, and owns a career 3.70 ERA with 7.25 K/9.
Reliever Ryota Igarashi has already made it known that he plans to pursue a career in the big leagues next year.

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Binding, WBW 30/30/30, ABS
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Save 15% on 4 or more and 25% on 12 or more!
Plastic makes a good binding/purfling due to its resiliency and ease in bending. You can rip the laminated bindings to use as purfling. Although our plastic binding is cut as cleanly as possible, it might be necessary to run the binding strips over a file (in a vice) prior to installation to further smooth out the edge.
Caution: Never rout for your bindings, backstrips, or purflings before you receive them as dimensions can vary slightly.
The chemical names for plastic acronyms are as follows:
- ABS - Acrylonitrile butadiene styrene
- PVC - Polyvinyl chloride
- CAB - Cellulose Acetate Butyrate
- Boltaron™ - an extruded ABS/PVC Alloy.
NOTE: We offer the 4 different plastic compositions because not all dimensions are available in one particular type of plastic. All 4 compositions behave similarly and can be glued up using our binding adhesives.
- Shipping Restrictions - Location
- No Restrictions
- 49" or 124.4cm
- .220" or 5.5mm
- .090" or 2.2mm
No customer reviews for the moment.

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Priyanka Chopra and Nick Jonas Attend Same Mumbai Cultural Opening as Tom and Zendaya
Our boy Nick was spotted hanging out in India this week, keeping his bae company while she hit up a ton of events. What a guy! The 30-year-old crooner tagged along with Priyanka, who's a ripe old 40, to the...
Zendaya and Tom Holland Look Amazing at Cultural Center Opening in Mumbai
Tom Holland and Zendaya attended the Nita Mukesh Ambani Cultural Centre's inauguration, and let me tell you, they looked chic as heck! They even hung out with Gigi Hadid, NBD. Tom kept it classic with a black tux and bowtie,...
Will Smith Spotted in Mumbai, India, For the First Time Since Being Banned From The Academy Awards for TEN YEARS!
53-year-old 'Gemini Man' actor Will Smith has been spotted for the very first time in the city of Mumbai, India, since being banned from The Academy Awards for ten years, after slapping actor Chris Rock...Read about it here. Smith...
MONSOON SZN: Priyanka Chopra and Nick Jonas Enjoy Inappropriate Date in Mumbai!
35-year-old 'Quantico' actress Priyanka Chopra and singer Nick Jonas, 25, continued their inappropriate romance across the ocean, as they were spotted on a romantic date in Mumbai, India! Priyanka wore a very racy dress that she paired with pink lipstick...
Justin Bieber Surprises Bus in India Full of Kids!
23-year-old 'Sorry' singer Justin Bieber surprised a bus full of kids in Mumbai yesterday! Bieber hopped on the bus and greeted all of the excited kids who shook his hand and greeted him. Bieber is currently in India for his Purpose...
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Jennifer Garner Takes Jennifer Lopez’s Daughter Emme to Disneyland
It turns out that Jennifer Garner and Jennifer Lopez's kids are not only step-siblings, but they're also one big...

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Metabolome analysis is an emerging method that provides insight into intracellular and physiologic responses. Methotrexate (MTX) is an antifolate that suppresses DNA syntheses by inhibiting dihydrofolate reductase. High-dose methotrexate treatment with deferred radiotherapy is a standard protocol in primary central nervous system lymphoma (PCNSL) treatments. However, most cases come to relapse-acquired resistance, in which the role of metabolic pathways is largely unknown.
Metabolome analysis in methotrexate-resistant PCNSL-derived cells (designated as TK-MTX and HKBML-MTX) was performed to detect alternative metabolites and pathways.
The metabolomic analyses using capillary electrophoresis-time-of-flight mass spectrometry detected 188 and 169 peaks in TK- and HKBML-derived cells, respectively, including suppression of central carbon metabolism, lipid metabolism, nucleic acid metabolism, urea cycle, branched chain and aromatic amino acids, and coenzyme metabolism. Particularly, whole suppressive metabolic pathways were demonstrated in TK-MTX, whereas HKBML-MTX indicated partially enhanced pathways of the urea cycle, amino acid metabolism, and coenzyme metabolism. Reciprocally detected metabolites for glycolysis, including induced glucose and reduced glycogen, and induced lactate and reduced pyruvate, in addition to increased lactate dehydrogenase activity, which is involved in Warburg effect. Thereby, ATP was increased in both methotrexate-resistant PCNSL-derived cells. Furthermore, we specifically found that PI3K/AKT/mTOR and RAS/MAPK signaling pathways were activated in TK-MTX but not in HKBML-MTX by growth rate with inhibitors and gene expression analysis, suggestive of cell type–specific methotrexate-resistant metabolic pathways.
These results can help us understand targeted therapies with selective anticancer drugs in recurrent CNS lymphoma–acquired resistance against methotrexate.
Methotrexate (MTX) is an antifolate that suppresses DNA syntheses by inhibiting dihydrofolate reductase. High-dose methotrexate treatment with deferred radiotherapy is a first-line treatment in primary central nervous system lymphoma (PCNSL) treatments, whereas the median overall survival of patients with PCNSL shows a poorer prognosis and most cases come to relapse-acquired resistance to methotrexate. Here, we generated the two independent methotrexate-resistant PCNSL-derived cells, TK-MTX and HKBML-MTX, and demonstrated that TK-MTX and HKBML-MTX indicated whole suppressive metabolic pathways and partially enhanced pathways, respectively. Furthermore, we clarified enhanced glycolysis pathways including altered metabolites involved in the Warburg effect in both methotrexate-resistant PCNSL-derived cells with different manners. PI3K/AKT/mTOR and RAS/MAPK pathways were activated in TK-MTX but not in HKBML-MTX by growth rate with inhibitors and gene expression analysis, suggestive of cell type–specific methotrexate-resistant metabolic pathways. Hence, the results would help us understand targeted therapies with selective anticancer drugs in recurrent CNS lymphoma–acquired resistance against methotrexate.
Primary central nervous system (CNS) lymphoma (PCNSL) is an aggressive CNS tumor that is a rare type of extranodal non-Hodgkin lymphoma (NHL) including diffuse large B-cell lymphoma (DLBCL) localized into the brain, eye, spinal cord, and meninges, which is distinct from systemic DLBCL (1, 2). Most PCNSLs are immune privileged site–associated DLBCLs according to the WHO diagnostic criteria (1). PCNSLs account for 3% of all primary CNS tumors and 1% of NHLs in adults (3). Methotrexate is an antifolate that inhibits dihydrofolate reductase (DHFR) activity in purine and thymidine syntheses (4, 5), which controls the expression of glucocorticoid receptors in blood cells (6). High-dose methotrexate (HD-MTX) is used as a first-line treatment in PCNSL (7). Despite intensive treatments with the combinatorial use of HD-MTX–containing polychemotherapy regimens, including cyclophosphamide, pirarubicin, etoposide, vincristine, procarbazine with or without rituximab (8), and deferred whole brain radiotherapy, the median overall survival (OS) of patients with PCNSL associated with poorer prognosis is approximately 4 years (9, 10). Moreover, second-line treatments are also required for 10%–35% of patients with refractory diseases and for another 35%–60% or more who have relapse-acquired resistances (11).
The metabolism of cancer cells differs from that of most normal cells (12, 13). Cancer cells require altered metabolomic pathways to incorporate nutrients efficiently for immortal proliferation (14, 15) and for survival in different metabolic environments outside of normal tissues (16, 17). There are a few studies on metabolome analyses in NHL and DLBCL as revealed in searches of the Human Metabolome Database (HMDB). For example, electrospray ionization-liquid chromatography-tandem mass spectrometer (ESI-MS/MS) analyses in urine from 30 patients with NHL identified a low-mass ion pattern and decreased hypoxanthine (18). A recent metabolomic profiling demonstrated that panobinostat, a modified histone deacetylase inhibitor, alters lipid metabolism and the downstream survival signaling in DLBCL (19). In particular, panobinostat induces metabolic adaptations resulting in de novo pathways for choline and PI3K activation (19). However, to date, few studies have reported on the metabolomic analyses in CNS lymphoma with relapse-acquired resistance against methotrexate.
Here, we generated two independent methotrexate-resistant PCNSL-derived cell lines to conduct metabolome analyses and evaluate the differences between their metabolomic pathways and those of both original cell lines. Especially, we focused on glycolysis with lactate dehydrogenase (LDH) activity, ATP synthesis, glutamate metabolism, and mitochondrial activity. We also examined the gene expression for PI3K/AKT/mTOR signaling upstream of the glycolysis pathway, and defined cell type–specific characteristics of the two PCNSL-derived cells.
Materials and Methods
Primary central nervous system lymphoma (PCNSL) cell lines TK and HKBML were purchased from JCRB Cell Bank [National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka, Japan] on November 10, 2015, and RIKEN Cell Bank (RIKEN BRC, RIKEN BioResource Center, Tsukuba, Japan) on March 10, 2015, respectively, and were grown in RPMI1640 medium with 10% FBS and Ham's F-12 medium (Nacalai Tesque Inc.) with 15% FBS (Thermo Fisher Scientific), respectively, in 5% CO2 at 37°C. methotrexate-resistant cells were generated as described previously (20, 21). Briefly, TK cells (passage 23) were cultured with 1.0 × 10−6 mol/L methotrexate for 6 weeks following preculture with lower concentrations of methotrexate for 9 weeks. HKBML cells (passage 24) were cultured with 1.0 × 10−7 mol/L methotrexate for 6 weeks following preculture with lower concentrations of methotrexate for 4 weeks. TK and HKBML cells used in all experiments were cultured with 10% and 15% serum, respectively, in each medium. Mycoplasma contamination test was performed using e-Myco Mycoplasma PCR Detection Kit (iNtRON Biotechnology, Inc.) on February 20, 2020, which detected no contamination in the cells used.
Metabolic extracts were prepared from 4–5 × 106 cells of TK-derived cells and 3–5 × 106 cells of HKBML-derived cells with methanol containing an internal standard solution (Human Metabolome Technologies, Inc.) and analyzed using a capillary electrophoresis (CE)-connected electrospray ionization-time-of-flight mass spectrometry (ESI-TOFMS) and CE-MS/MS system, according to the manufacturer's instruction (Human Metabolome Technologies, Inc.). Briefly, after removal of culture medium, cells were washed and then collected, according to the protocol E-140014 (Human Metabolome Technologies, Inc.). Cationic and anionic compounds were analyzed with the cation and anion mode of CE-TOFMS system with fused capillary inner diameter (i.d.) 50 μm × 80 cm (Agilent Technologies), respectively. Peaks detected by CE-TOFMS were extracted to obtain peak information, including mass-to-charge ratio (m/z), migration time (MT), and peak area (S/N > 3) using automatic integration software MasterHands (Keio University, Tsuruoka, Japan). Condition of measurements was as follows: (cation mode) sample injection; pressure injection 50 mbar, 10 seconds, CE voltage; positive, 27 kV, MS ionization; ESI Positive, MS capillary voltage; 4,000 V, MS scan range; m/z 50–1,000, sheath liquid; HMT Sheath Liquid (p/n: H3301-1020), and (Anion mode) sample injection; pressure injection 50 mbar, 25 seconds, CE voltage; positive, 30 kV, MS ionization; ESI Negative, MS capillary voltage; 3,500 V, MS scan range; m/z 50—1,000, sheath liquid; HMT Sheath Liquid (p/n: H3301-1020). The peaks were annotated by the HMT metabolite database (Human Metabolome Technologies, Inc.), based on their MTs in CE and m/z values determined by TOFMS. The tolerance annotation range of peaks was referred as ±0.5 min for MT and ±10 ppm for m/z. Concentrations of metabolites were estimated with normalization of the peak area of each metabolite (100 μmol/L) with respect to the area of the internal standard (200 μmol/L) and standard curves, which were detected by a one-point calibration. Relative area was calculated with a following formula: (target peak area)/(internal control area)/(sample volume). A metabolic pathway map was drawn with a Visualization and Analysis of Networks containing Experimental Data (http://vanted.ipk-gatersleben.de/). Triplicate samples were examined and relative areas of peaks were compared among groups with Welch t test (P < 0.05).
Cell proliferation assay
Cell proliferation assay was performed as described previously (22). Briefly, cells were seeded at 1 × 104 cells per well in 96-well plates. Cell proliferation was measured using a Cell Count Reagent SF, according to the manufacturer's instruction (Nacalai Tesque Inc.). Briefly, 10 μL of the Reagent SF solution was added to each well and incubated for 30 minutes in a 5% CO2 incubator at 37°C. The absorbance at 450 nm was measured with SpectraMax M2e (Molecular Devices, Tokyo, Japan).
Glycolysis, ATP synthesis, and glutamate metabolism
For glycolysis analysis, the amounts of glucose, glycogen, pyruvate, and lactate, and LDH activity were measured by colorimetric and fluorometric detection using Glucose Assay Kit (Ex/Em = 535/587 nm; Abcam), Glycogen Assay Kit (Ex/Em = 535/587 nm; Abcam), Pyruvate Assay Kit (Ex/Em = 535/587 nm; BioVision, Inc.), Lactate Assay Kit-WST (λ = 450 nm; Dojindo), and Cytotoxicity LDH Assay Kit-WST (λ = 490 nm; Dojindo), respectively, in accordance with the manufacturer's instructions. ATP synthesis was measured by fluorometric detection using the ATP Assay Kit (Ex/Em = 535/587 nm) (Abcam), according to the manufacturer's instruction. For glutamate metabolism, the amounts of glutamate and α-ketoglutamate, and glutamate dehydrogenase (GDH) activity were measured by colorimetric and fluorometric detection using Glutamate Assay Kit (Ex/Em = 540/590 nm) (Abcam), Alpha Ketoglutarate (alpha KG) Assay Kit (Ex/Em = 535/587 nm; Abcam), and Glutamate Dehydrogenase Activity Assay Kit (λ = 450 nm) (Abcam), respectively, according to the manufacturer's instructions. The absorbance and fluorescence signals were measured with SpectraMax M2e (Molecular Devices).
Mitochondrial activity assay
MitoBright Red (Ex/Em = 561/617 nm; Dojindo) and Mito-FerroGreen (Ex/Em = 488/550 nm; Dojindo) were used to evaluate activities of the mitochondrial membrane potential and the amount of Fe(II) oxide at Fe/S clusters in the mitochondria, respectively, according to the manufacturer's instructions. The fluorescence signals were measured with SpectraMax M2e (Molecular Devices).
Altered amounts of metabolites in the PCNSL-derived cells were clustered with the hierarchical method using the JMP built-in modules (SAS Institute, Inc.), as described previously (23).
Principal component analysis
Principal component analysis (PCA) was used to classify the metabolites detected by metabolomic analyses into the subgroups. PCA was performed using normalized values of metabolites using JMP built-in modules (SAS Institute Inc.), as described previously (8, 24).
In vivo tumorigenicity assay
The methotrexate-resistant cells and the parent cells were transplanted into right and left subcutaneous tissues in the back of nude mice (BALB/cSlc-nu/nu; Japan SLC, Inc.), respectively (25). Xenografts were grown for 11–23 weeks and resected, followed by measure for weights (g) and volumes (cm3). Volumes were calculated with the formula as follows: 0.5 × width (cm) × width (cm) × length (cm). Rapamycin (5 mg/kg body weight), PD0325901 (5 mg/kg), and Chrysin (1 mg/kg) were administered into the mice harboring xenografts with intraperitoneal (i.p.) injection. Mice were sacrificed at day 7 to day 9 using isoflurane anesthesia. All of the experimental protocols were approved by the Animal Care and Use Committee at Kyoto Prefectural University of Medicine (M30-57454).
The data were presented as means ± SD of multiple samples. Statistical analyses were performed using JMP built-in-modules (SAS Institute Inc.) and Microsoft Excel (Microsoft Japan Co., Ltd.; ref. 26). P < 0.05 and false discovery rate (FDR) q < 0.01 were considered statistically significant.
Generation of methotrexate-resistant PCNSL-derived cells
To know how differences of metabolomic profiles of methotrexate-resistant PCNSL cells compared with nonresistant cells, we first generated two independent methotrexate-resistant PCNSL-derived cells, including methotrexate-resistant TK and HKBML cells (Fig. 1A). For cell viability calculations, the values of inhibitory concentration 50 (IC50) in control TK cells and methotrexate-resistant TK (TK-MTX) cells were 3.73 × 10−8 mol/L and 8.76 × 10−6 mol/L, respectively, showing 234-fold resistance against methotrexate in TK cells. While in HKBML-derived cells, the values of IC50 in the control HKBML cells and methotrexate-resistant HKBML (HKBML-MTX) cells were 1.17 × 10−10 mol/L and 2.14 × 10−8 mol/L, respectively, showing 183-fold resistance against methotrexate in HKBML cells. These results indicate that degrees of methotrexate resistances in both PCNSL cells are similar, whereas the basal resistance of TK is 319-fold compared with that of HKBML.
Metabolome analysis in the methotrexate-resistant PCNSL-derived cells
We considered the difference between methotrexate-resistant and nonresistant PCNSL cells and then conducted metabolome analyses among them including methotrexate-resistant and nonresistant TK and HKBML cells (n = 3; Supplementary Figs. S1 and S2). Hierarchical clustering analysis showed the two distinct subgroups between the methotrexate-resistant and nonresistant cells in TK and HKBML-derived cells (Fig. 1B). PCA also showed compact component sets of metabolites detected in TK-derived cells (86.3% of 188 peaks including 102 cations and 86 anions) and HKBML-derived cells (65.2% of 169 peaks including 98 cations and 71 anions; Fig. 1C). As summarized in the Venn diagrams (Fig. 1D–I), most metabolites detected in TK-MTX cells decreased in the urea cycle (Fig. 1E), lipid metabolism (Fig. 1F), branched chain and aromatic amino acids (Fig. 1G), nucleic acids metabolism (Fig. 1H), and coenzyme metabolism (Fig. 1I). Some metabolites detected in HKBML-MTX cells increased in central carbon metabolism (Fig. 1D), urea cycle (Fig. 1E), lipid metabolism (Fig. 1F), and coenzyme metabolism (Fig. 1I), in addition to decreases of most of the metabolites detected in central carbon metabolism (Fig. 1D) and branched chain and aromatic amino acids (Fig. 1G). These findings clearly indicated that most of the metabolites detected in TK-MTX cells decreased, whereas some metabolites detected in HKBML-MTX cells increased compared with the corresponding control cells, suggesting differences in cell type–specific metabolic pathways between the two PCNSL-derived cells.
Cell type–specific metabolic pathways in the methotrexate-resistant PCNSL-derived cells
On the basis of data of discrete metabolites detected, we found that suppression in TK-MTX cells (0.167- to 0.241-fold) and activation in HKBML-MTX cells (1.014-3.273-fold) altered the following pathways: protein metabolism, lipid metabolism, cell function, apoptosis, osmolytes, sugar metabolism, transmethylation, liver disease, and renal disease, compared with the corresponding control cells (Supplementary Table S1). Meanwhile, both TK-MTX (0.248-0.489-fold) and HKBML-MTX cells (0.925-0.619-fold) suppressed electron carrier, purine bases, collagen, arthritis, imidazole compounds, nucleotide sugars, essential amino acid, and methylglyoxal, compared with the corresponding control cells (Supplementary Table S1). Furthermore, based on the data from Human Metabolome Database (HMDB) category, as a representative data, TK-MTX cells were specifically suppressed in arthritis (0.314-fold, P = 0.000411), guanidino compounds (0.186-fold, P = 0.005687), electron carrier (0.329-fold, P = 0.007003), but not in HKBML-MTX cells, compared to the corresponding control cells (Supplementary Table S2). HKBML-MTX cells were specifically activated in renal disease (1.564-fold, P = 0.047601), lipid metabolism (2.07-fold, P = 0.003866), pyrimidine bases (1.158-fold, P = 0.020303), and neuropsychiatric disorder (2.449-fold, P = 0.022399), but these were suppressed in TK-MTX cells, compared with the corresponding control cells (Supplementary Table S2). Summarized cell type–specific metabolites detected in TK-MTX and HKBML-MTX cells, glucose 1-phosphate and fructose 1,6-diphosphate, lactic acid, and acetyl coenzyme A (divalent) decreased specifically in TK-MTX cells (<0.11-fold, P < 0.049), compared with the control TK cells (Supplementary Table S3), indicating that TK-MTX cells are dysregulated in glycolysis. While in HKBML-MTX cells, amino acids including Ala, Trp, His, Phe, and Tyr decreased (<0.5-fold, P < 0.035), but Gln and Asp increased (>1.6-fold, P < 0.047), compared with the control HKBML cells (Supplementary Table S3), indicating that HKBML-MTX cells are dysregulated in amino acid metabolisms.
Glycolysis, ATP synthesis, glutamate metabolism, and mitochondria activity in the methotrexate-resistant PCNSL-derived cells
Considering the abovementioned metabolic profiles, we decided to further investigate glycolysis, ATP synthesis, glutamate metabolism, and mitochondria activity. We reevaluated the amounts of metabolites in the glycolysis pathway using biochemical analysis techniques (Fig. 2A–C). The amounts of glucose and ATP significantly increased in both TK-MTX (1.276-fold and 1.613-fold, respectively) and HKBML-MTX (1.102-fold and 1.421-fold, respectively) cells, compared with the corresponding control cells (Fig. 2A). However, glycogen, pyruvate and lactate significantly decreased in both TK-MTX and HKBML-MTX cells, compared with the corresponding control cells (Fig. 2A). LDH activities were upregulated in both TK-MTX (1.465-fold) and HKBML-MTX (1.83-fold) cells with statistical significances, compared with the corresponding control cells (Fig. 2B and C). These results suggest that cellular glycogen storages were released, and concentrated glucose was processed into pyruvate and also to lactate by LDH, and then the ATP level increased. The amount of glutamate increased in TK-MTX cells (1.303-fold, P < 0.01), but not in HKBML-MTX. The α-keto-glutamate and the glutamate dehydrogenase (GDH) activities significantly decreased in both TK-MTX (0.585-fold and 0.969-fold, respectively) and HKBML-MTX (0.72-fold and 0.603-fold, respectively) cells, compared with the corresponding control cells (Fig. 2D). Furthermore, the activities of mitochondrial membrane potentials and the amount of Fe(II) oxides at the Fe/S cluster into mitochondrial lumen significantly decreased in both TK-MTX (0.67-fold and 0.95-fold, respectively) and HKBML-MTX (0.53-fold and 0.9-fold, respectively) cells, compared with the corresponding control cells (Fig. 2E). These results suggest that glutamate metabolism that provides glutamate as fuels into tricarboxylic acid (TCA) cycle was downregulated in both TK-MTX and HKBML-MTX with decreased GDH activities and suppressed mitochondrial activities. Moreover, glycolysis is more dominant than aerobic metabolism including TCA cycle in both methotrexate-resistant PCNSL cells.
Candidate pathways regulating glycolysis in the methotrexate-resistant PCNSL-derived cells
We next examined the upstream signaling regulating glycolysis in the methotrexate-resistant PCNSL-derived cells. First, we investigated cellular signaling pathways in proliferation (Fig. 3, Supplementary Fig. S3, see Supplementary text). Growth suppression of TK cells with 1.6 μmol/L LY294002 as a PI3K inhibitor (0.45-fold, P < 0.05) was recovered by acquired methotrexate resistance, whereas both control HKBML and HKBML-MTX cells were suppressed by LY294002 (0.8-fold and 0.76-fold, P < 0.05, respectively; Fig. 3A and B). Growth suppression of TK cells with 0.4 μmol/L PD0325901 (0.66-fold, P < 0.05), a MAPK inhibitor, was also recovered by acquired methotrexate resistance, whereas HKBML-MTX cells were sensitive to PD0325901 compared with the control HKBML cells (0.73-fold, P < 0.05; Fig. 3C and D). Besides, growth suppression of TK cells with 2.1 μmol/L rapamycin as a mTOR inhibitor (0.74-fold, P < 0.01) was also recovered by acquired methotrexate resistance, whereas HKBML-MTX cells showed a sensitivity for rapamycin, compared with the control HKBML cells (0.78-fold, P < 0.05; Fig. 3E and F). These results suggest that PI3K, MAPK, and mTOR signaling pathways are activated in the TK-MTX cells, but not in HKBML-MTX cells, thus the inhibitors are hard to suppress the growth of TK-MTX cells (1.2-8.57-fold, P < 0.01), but function in the growth suppression of HKBML-MTX cells (0.06-0.84-fold, P < 0.01), compared with the corresponding control cells (Supplementary Fig. S4). However, such malformed growth suppression was not associated with apoptosis and/or cellular senescence because of low signals for caspase-3/7 activity (Supplementary Fig. S5A and S5B) and senescence-associated (SA)-β-galactosidase activity (Supplementary Fig. S5C and S5D), compared with the appropriate positive controls.
Subsequently, we examined the amounts of glucose, pyruvate, lactate, and ATP, and the LDH activity in the TK-MTX and HKBML-MTX cells with inhibitors (Fig. 4; Supplementary Fig. S6, see Supplementary text). TK-MTX cells treated with PD0325901 showed decreased glucose (0.74-fold, P < 0.01), compared with the control TK cells (Fig. 4A). HKBML-MTX cells showed increased glucose (1.36-fold, P < 0.01), and furthermore, LY29402 and PD0325901 also increased glucose concentration (1.24-fold, P < 0.05 and 1.07-fold, P < 0.01, respectively), whereas rapamycin decreased that in HKBML-MTX cells (0.83-fold, P < 0.01), compared with the control HKBML cells (Fig. 4B). TK-MTX cells showed decreased pyruvate (0.44-fold, P < 0.01), whereas LY294002, PD0325901, and rapamycin increased pyruvate concentration in TK-MTX cells (8.94-fold, P < 0.01; 1.75-fold, P < 0.05; and 16.41-fold, P < 0.01, respectively), compared with the control TK cells (Fig. 4C). HKBML-MTX cells showed decreased pyruvate (0.44-fold, P < 0.01), whereas rapamycin increased pyruvate concentration (4.16-fold, P < 0.01) in HKBML-MTX cells, compared with the control HKBML cells (Fig. 4D). TK-MTX cells showed decreased lactate (0.34-fold, P < 0.01), and furthermore, LY294002, PD0325901, and rapamycin also decreased lactate concentration in TK-MTX cells (0.13-fold, P < 0.01; 0.29-fold, P < 0.01; and 0.03-fold, P < 0.01, respectively) compared with the control TK cells (Fig. 4E). HKBML-MTX cells showed decreased lactate (0.51-fold, P < 0.05), and LY294002 also decreased lactate concentration in HKBML-MTX cells (0.66-fold, P < 0.05), compared with the control HKBML cells (Fig. 4F). LDH activities were also tested with the inhibitors (Fig. 4G and H). The LDH activity in TK-MTX cells was upregulated (2.83-fold, P < 0.01), whereas LY294002, PD0325901, and rapamycin showed the reduced activities of LDH in TK-MTX cells (0.14-fold, P < 0.01; 0.42-fold, P < 0.01; and 0.23-fold, P < 0.01, respectively), compared with the control TK cells (Fig. 4G). Similarly, The LDH activity in HKBML-MTX cells was upregulated (1.66-fold, P < 0.01), whereas LY294002, PD0325901, and rapamycin showed the reduced activities of LDH in HKBML-MTX cells (0.52-fold, P < 0.01; 0.45-fold, P < 0.05; and 0.67-fold, P < 0.01, respectively), compared with the control HKBML cells (Fig. 4H). We also examined ATP synthesis with the inhibitors (Fig. 4I and J). The ATP increased in TK-MTX cells (3.22-fold, P < 0.01), and LY294002 also increased ATP concentration in TK-MTX cells (2.57-fold, P < 0.01), compared with the control TK cells (Fig. 4I). HKBML-MTX cells showed increased ATP (1.53-fold, P < 0.01), and LY294002 also increased ATP concentration in HKBML-MTX cells (1.65-fold, P < 0.05), whereas PD0325901 and rapamycin decreased ATP concentration (0.48-fold, P < 0.01, and 0.01-fold, P < 0.01, respectively), compared with the control HKBML cells (Fig. 4J). Considering the abovementioned results, these data indicate that both TK-MTX and HKBML-MTX cells tend to upregulate glycolysis with LDH activity and ATP synthesis, whereas the inhibition of PI3K, MAPK, and mTOR signaling pathways especially compete the effects of methotrexate resistance in such metabolic pathways in the TK-MTX cells, suggesting that TK cells have a potential methotrexate resistance than HKBML cells, which is consistent with the results in cell proliferation, as already shown in Fig. 1A.
Differential gene expression for PI3K, MAPK, and mTOR signaling pathways in the methotrexate-resistant PCNSL-derived cells
We furthermore investigated changes in gene expression related to PI3K signaling (PIK3R1, PIK3CA, PDK1, PTEN, AKT1, and GSK3B), cell proliferation (MYC, PCNA, and MKI67), mTOR complex formation (MTOR, RPTOR, RICTOR, and DEPTOR), glucose metabolism and transport (G6PT1, G6PC, and GLUT2), and transcription factors related to such signaling pathways (BCL2/6, FOXO1, GATA1/2/3, HIF1A, NFKB1/2, and TP53) in the methotrexate-resistant PCNSL-derived cells (Fig. 5A and B; Supplementary Table S4). The expression levels of the following genes were significantly upregulated in the TK-MTX cells compared with the control TK cells: MYC (3.96-fold), PIK3R1 (3.76-fold), MTOR (2.69-fold), BCL6 (2.5-fold), MKI67 (2.02-fold), RPTOR (1.81-fold), GATA2 (1.77-fold), NFKB2 (1.71-fold), PIK3CA (1.66-fold), TP53 (1.49-fold), PTEN (1.47-fold), and G6PT1 (1.42-fold). However, the expression levels of the following genes were significantly downregulated in the TK-MTX cells as compared with the control TK cells: GLUT2 (0.32-fold), NFKB1 (0.42-fold), PCNA (0.65-fold), and FOXO1 (0.66-fold; Fig. 5A). As compared with the control HKBML cells, the following proteins were significantly upregulated in the HKBML-MTX cells: MTOR (5.41-fold), BCL6 (3.08-fold), HIF1A (2.29-fold), FOXO1 (2.13-fold), GLUT2 (1.99-fold), MYC (1.62-fold), G6PC (1.47-fold), BCL2 (1.36-fold), MKI67 (1.13-fold), G6PT1 (1.13-fold), and NFKB2 (1.02-fold). However, the expression levels of the following genes were significantly downregulated in the HKBML-MTX cells compared with in the control HKBML cells: PIK3R1 (0.14-fold), DEPTOR (0.65-fold), AKT1 (0.69-fold), RPTOR (0.76-fold), and GATA3 (0.78-fold) (Fig. 5B). Summarizing these expression data, the upregulation of MAPK signaling, cell proliferation (MYC, BCL6, and MKI67), and B-cell differentiation (NFKB2) was considered to contribute to the growth of TK and HKBML cells reprogrammed by methotrexate (Fig. 5C and D). The upregulation of PI3K signaling (PIK3R1 and PIK3CA), mTOR signaling (mTOR and RPTOR), and multipotent transcription factor GATA2, and the downregulation of FOXO1 for proapoptotic signaling likely contributed to the growth of TK-MTX cells (Fig. 5C). The upregulation of glucose transport (GLUT2, G6PC, and G6PT1) and hypoxia transcription factor HIF1A contributed specifically to the growth of HKBML-MTX cells (Fig. 5D).
Phosphorylation of the PI3K–AKT–mTOR and MAPK were confirmed with protein analyses, suggesting that these signaling pathways are activated in the methotrexate-resistant PCNSL cells (Supplementary Figs. S7 and S8, see Supplementary text). Furthermore, global expression analyses demonstrated that downregulated function of membrane activity, glucose, and central carbon metabolism and upregulated stem cell property exist in HKBML-MTX, and downregulated function of negative regulation of MAPK, ECM disassembly, B-cell receptor signaling, and NK-cell–mediated cytotoxicity and upregulated function of glycosylation of glycoproteins and EGF-like signaling pathway are served in TK-MTX (Supplementary Fig. S9; Supplementary Table S5, see Supplementary text). Besides, the GO analyses denoted that tissue stem cell function and immune system process are activated in both HKMBL-MTX and TK-MTX and showed that downregulated signaling of endogenous galectin-1 exists in HKBML-MTX. In the GSEA, TK-MTX also showed the downregulation of cell projection, neural development, and blood cell differentiation (Supplementary Table S6, see Supplementary text).
Tumorigenicity of PCNSL xenografts with blockades for mTOR and HIF-1α pathways in vivo
To investigate tumorigenicity of PCNSL cells in vivo, we transplanted PCNSL-derived cells into subcutaneous tissues in the back of nude mice. Survival rates of the xenografts of the cells were 11.62% (n = 5/43) in TK and 25% (n = 13/52) in HKBML, suggestive of low viabilities and effects of tumor microenvironments. The xenografts of PCNSL-derived cells were grown in animals and examined drug sensitivities using rapamycin (mTOR inhibitor), PD0325901 (MEK inhibitor), and Chrysin (HIF1A inhibitor; Fig. 6; Supplementary Fig. S10). The measurements in tumor weight and volume clarified that TK-MTX was engrafted than TK (1.47-fold; **, P = 0.0078 in weight, 1.62-fold; *, P = 0.0167 in volume), but the both TK and TK-MTX were suppressed with rapamycin (Fig. 6A–C). While, HKBML could be suppressed by rapamycin (0.08-fold; P = 0.1381 in weight, 0.31-fold; *, P = 0.0366 in volume), but HKBML-MTX sustained prominent tumors (Fig. 6D). In addition, HKMBL-MTX evaded tumor suppression by rapamycin (8.42-fold; ***, P = 5.17 × 10−5 in weight, 8.5-fold; *, P = 0.0201 in volume) (Fig. 6E and F). Besides, HKBML-MTX showed a susceptibility to PD0325901 in tumor weight (0.2-fold; **, P = 0.0028). Furthermore, HKBML showed a Chrysin resistance in tumor volume (5.61-fold; **, P = 0.0039), whereas HKBML-MTX acquired a Chrysin sensitivity (0.09-fold; P = 0.0635 in weight, 0.43-fold; *, P = 0.0204 in volume; Fig. 6D–F). These results indicated that the mTOR pathway inhibition was effective for suppression of TK, TK-MTX, and HKBML in vivo, but not in HKBML-MTX, and that the in vivo growth suppression of HKBML-MTX could be caused by the HIF-1α pathway inhibition.
Comparing the exo-metabolomics of differentiating murine B lymphoma cells and primary B cells with one-dimensional proton nuclear magnetic resonance (NMR) spectroscopy and LC/MS, lactate production increased with reduced essential amino acids. Moreover, immunoglobulin secretion was paralleled by Ala, Glu, and Gln as carbon and energy sources (27). With antigen-binding small B lymphocytes, a proteomic metamorphosis is activated to generate antibody-secreting cells (28). Methotrexate is an antifolate that is a DHFR inhibitor in purine and thymidine syntheses (5). Meanwhile, a serine hydroxymethyltransferase (SHMT) converts Ser into Gly and a tetrahydrofolate-bound one-carbon unit for purine and thymidine synthesis and for cell growth (29). In Burkitt lymphoma and DLBCL, gas chromatography-mass spectrometry (GC-MS) and nanoLC-sequential window acquisition of all theoretical (SWATH)-MS clarify decreased pyruvic acid content, and downregulated glycolysis but upregulated one-carbon metabolism in Burkitt lymphoma, compared with DLBCL (30). In anaplastic large cell lymphoma, integrated phosphoproteomic and metabolomic analyses reveal that constitutively active tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) induces a metabolic shift toward aerobic glycolysis, lactate production, and biomass production. The metabolic shift is mediated by ALK phosphorylation at Y105 of the tumor-specific isoform of pyruvate kinase 2 (PYK2) protein, resulting in decreased enzymatic activity (31).
Altered metabolites and metabolomic pathways are undoubtedly biomarkers for cancer development and progression (32). In serum lipid analysis on a subset of individuals from a population-based NHL case–control study from 100 patients with DLBCL, significantly elevated levels of endocannabinoid and 2-arachidonoylglycerol (2-AG) were detected in the serum of DLBCL patients (33). NMR-based serum metabolomics identifies the following metabolites as high-risk signatures for patients with DLBCL: Lys and Arg; the degradation product cadaverine and a compound in oxidative stress (2-hydroxybutyrate) in the refractory/early relapse group (n = 27), and Asp, Val and ornithine, and a metabolite in the glutathione cycle, pyroglutamate in long-term progression-free patients (n = 60) (34). In Burkitt lymphoma mice, NMR-based metabolomics demonstrate that the serum metabolites are altered during the metabolism of energy, amino acids, fatty acids, and choline phospholipids (35). The diagnostic potential of differential metabolites also clarifies potential biomarkers including Glu, glycerol, and choline with a high diagnostic accuracy; in contrast, Ile, Leu, pyruvate, Lys, α-ketoglutarate, betaine, Gly, creatine, Ser, lactate, Tyr, Phe, His and formate, discriminating Burkitt lymphoma mice from normal mice (35). In cutaneous T-cell lymphoma (CTCL), a class of NHL, and a heterogeneous group of skin-homing T-cell neoplasms, ultrahigh performance liquid chromatography–quadrupole (UHPLC-Q) TOF/MS and further LC/MS-MS determined the 36 potential biomarkers associated with CTCL, which are derived from CTCL plasma metabolic perturbations (36). In skin and plasma of CTCL mice, UHPLC-QTOF/MS also demonstrate that increased l-glutamate and decreased adenosine monophosphate are the most essential metabolic pathways of CTCL tumors and tumor adjacent skins (37). Data from GC-MS and UHPLC-QTOF/MS show that glycerophospholipid metabolism, tryptophan metabolism, and purine metabolism are altered pathways in serum samples from 31 patients with CTCL (38).
Metabolomics is a comprehensive method to identify and quantify metabolites from biological samples including cells (19), sera (35), and urines (18). However, few studies discuss glycolysis in CNS lymphoma. Here we generated two independent methotrexate-resistant PCNSL-derived cells as models of refractory and relapse-acquired methotrexate resistance, designated as TK-MTX and HKBML-MTX, and then conducted metabolomic analyses to know how changes in endogenous metabolites in methotrexate-resistant PCNSL-derived cells affect their viability. The metabolomic data from a capillary electrophoresis (CE)-connected electrospray ionization-time-of-flight mass spectrometry (ESI-TOFMS) and CE-MS/MS, and the annotation by HMDB demonstrated cell type–specific metabolic pathways with changes of glycolysis and amino acid metabolism in TK-MTX and HKBML-MTX cells, respectively, although noncharacterized metabolites should be addressed. Furthermore, we verified the differentially concentrated metabolites using biochemical methods in vitro, which were focused on glycolysis (with LDH), ATP synthesis, glutamate metabolism (with GDH), and mitochondrial activity, based on the metabolomic data. Consequently, TK cells were more dominant than HKBML cells in proliferation following methotrexate exposure, whereas the reprogrammed TK-MTX cells were governed by PI3K/AKT/mTOR and RAS/MAP-Kinase signaling pathways. Conversely, the HKBML-MTX cells acquired sensitivities at mTOR and MAPK signaling pathways without proapoptotic and cellular senescence activities. Besides, the genes related to the signaling pathways including PI3K/AKT/mTOR, RAS/MAP-Kinase and the transcription factors involved were also dysregulated in the methotrexate-resistant PCNSL-derived cells (e.g., increases of MYC, PIK3R1, PIK3CA, MTOR, and RPTOR in TK-MTX cells and increases of MYC, G6PT1, G6PC, GLUT2, HIF1A, and BCL6 in HKBML-MTX cells). Considering suppressive electron carriers in both methotrexate-resistant PCNSL-derived cells (Supplementary Table S1), these observations suggest that PCNSL cells are reprogramed by methotrexate into the cells harboring the metabolism via aerobic glycolysis than oxidative phosphorylation pathway, such as the Warburg effect (39). TK cells were sensitive to oxidative stress but HKBML cells were resistant (Supplementary Figs. S11–S13, see Supplementary text). Furthermore, HKBML had a potential activity of reactive oxygen species (ROS) and enhanced HIF1α activity under oxidative stress (Supplementary Fig. S11). Thus, there would be cell type–specific and oxidative stress–dependent signaling pathways via HIF1α and c-MYC for glycolysis in differential manners (Supplementary Fig. S12). On the other hand, of great interest was that HKBML-MTX cells showed sensitivity for mTOR inhibitors and MEK inhibitors. This suggests a possibility that, after diagnosing HKBML cell types with metabolomic biomarkers, mTOR and/or MAPK signaling pathways could be promising therapeutic targets for growth suppression of CNS lymphomas with refractory and relapse-acquired methotrexate resistances. While, a recent study clarified that TK-MTX was sensitive to bortezomib than parent TK, but HKBML was not affected (20). The qPCR results from PCNSL tissues clarified that the subgroups with higher expression of MTOR and PIK3R1 showed poor prognoses, also suggesting that PI3K and mTOR pathways are crucial for biology in the methotrexate-resistant PCNSL with poor survival (Supplementary Table S7; Supplementary Figs. S14–S16, see Supplementary text).
We also tested in vivo tumorigenicity of the PCNSL-derived cells and their drug resistance/sensitivity using nude mice. The results demonstrated that the mTOR inhibition caused the growth suppression of the xenografts of the PCNSL-derived cells except for HKBML-MTX, although TK-MTX and HKBML-MTX were resistant and sensitive to rapamycin, respectively, in vitro. Moreover, PD0325901 MEK inhibitor slightly failed growth suppression of HKBML and HKBML-MTX in vivo. These results are not consistent with the results from in vitro experiments in the current study. However, such discrepancies might be explained by the differences of tumor microenvironments in vitro and in vivo, which should await future studies. The microenvironments might also be associated with a low survival rate of xenografts in nude mice. On the other hand, the HIF-1α inhibition by Chrysin clearly induced growth suppression of the HKBML-MTX xenografts, but not in the HKBML. This is consistent with the results from in vitro experiments, such as the antioxidative stress cell growth, ROS activity, HIF1A expression, high GSH/GSSG ratio, and glutathione reductase activity. These in vivo results also suggested cell type–specific manners for cell survival and growth and signaling pathways in PCNSL. Thus, we must diagnose such cell-types before treatments in future. This study would be a hint for understanding the signaling at the methotrexate and folate metabolisms, drug discovery, and development for de novo biomarkers and pathways in PCNSL.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conception and design: Y. Takashima, R. Yamanaka
Development of methodology: R. Yamanaka
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Takashima, A. Hayano, R. Yamanaka
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y. Takashima, R. Yamanaka
Writing, review, and/or revision of the manuscript: Y. Takashima, R. Yamanaka
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y. Takashima, A. Hayano, R. Yamanaka
Study supervision: R. Yamanaka
This work was supported by JSPS KAKENHI (grant numbers 16H05441, to R. Yamanaka; 18K09001, to Y. Takashima). The authors thank Dr. Junya Fukai (Wakayama Medical University), Dr. Yasuo Iwadate (Chiba University), Dr. Koji Kajiwara (Yamaguchi University), and Dr. Hiroaki Hondoh (Toyama Prefectural Central Hospital) for providing PCNSL samples and clinical data.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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It is the most popular song in America. references to country and cowboy imagery it does not embrace enough elements of.
That spirit was clearly in evidence Wednesday evening at St. James Plantation’s SeaSide Club in Southport, where more than.
Music Player Lenovo 64 Bits Simply put, it is able to wirelessly play video music from mobile to Mac/Windows 10 and computer to Sony/LG/Samsung smart TV with 4K UHD/HDR display. In case needed, anyone could use this 64-bit 4K. May 26, 2017 · Windows media DVD 64bit player FOR WINDOWS 10 64 BIT. It seems I may be wasting my time
It was a summer of music festivals, with the Atlanta pop festival and Woodstock both occurring before. And no one cared.".
Today in Music History for Sept. In 1991, Garth Brooks released his album "Ropin’ the Wind." It made music history by becoming the first album to enter both the Billboard country and pop charts at.
SuperM, the "Avengers of K-pop", as CEO of SM Entertainment. are already on the way. Just today, Taemin, Mark and Taeyong.
New York – Australian pop group Human Nature sat down and. When asked about the title of the current chapter of their.
from bank accounts and election campaigns to the creation of pop music – the show’s focus. It’s a peculiar anime in just how.
If we’re to judge an album by its cover, “Lover,” Taylor Swift’s latest which was released today. of “Lover” and the most.

domain: artistic


«RS Motor» has a fully equipped workshop in Porriño (Pontevedra, Spain) with the necessary experience, equipment and supplies to perform maintenance works on the gas engines we have under our maintenance or not. We also carry out reconditioning works for the most important elements of the engine.
An engine overhaul is a major maintenance intervention, where the engine is removed and disassembled part by part, following approved assembly processes. Worn elements are replaced and the reconditioned engine is returned to its initial state of use.
Due to the complexity of the procedure, we generally take the overhauled engine to our workshop. Here we have all the necessary means and equipment to carry out these works with all the guarantees on engines up to 4.50 MW.
Sometimes, and where downtime makes it unfeasible to remove the engine to our workshop, we can perform the overhaul on an exchange block, minimizing downtime-machine and resuming production in minimal time.
Reconditioning an engine involves disassembling its mechanical elements, inspecting and verifying the state of the engine, cleaning the mechanical parts with ultrasounds and carrying out controls and/or hydraulic tests. Worn elements are replaced and the reconditioned engine is restored to zero hours.
Engine elements usually reconditioned in the «RS Motor» workshop are:
- Cylinder heads.
- Water pumps.
- Plate heat exchangers.
- Oil pumps.
- Connecting rods (dimensional control & renew of bushings).
- Zero pressure valves.
- Water jackets.
- Throttle valves.
- Gas mixers.
- Fuel pumps.
In case you need to repair, reconditioning, or carry out a preventive or corrective maintenance, «RS Motor» is specialist on the following brands and types:
- DEUTZ / MWM TBG616 / TCG2016;
- DEUTZ / MWM TBG620 / 2020;
- DEUTZ / MWM TBG632 / 2032;
- and many more.
- Jenbacher type 2;
- Jenbacher type 3;
- Jenbacher type 4 and,
- Jenbacher type 6 (it does not matter the number of cylinders).
- Guascor / Siemens FG / FGLD / SFGLD 180;
- FG / FGLD / SFGLD / HGM 240;
- FGLD / SFGLD 360;
- FGLD / SFGLD 480 and,
- SFGLD / SFGM / HGM 560.
Please, do not hesitate to contact us.


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Less Than Jake / The Expendables Denver 2009
Original concert poster for Less Than Jake and The Expednables at the Ogden Theatre in Denver, Colorado. 11x17 card stock. Art by Maria DiChiappari.
Genre: Maria DiChiappari
Dimensions: 11x17 inches
Condition: MINTDate: 2009
Posters > Music Posters
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Please call us at 303-443-3102 with the SKU numbers of any items you may wish to purchase at the store prior to arrival.


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The Foo Fighters emerged from the morass of suicide and potent musical legacy that was Nirvana to establish themselves - against all odds - as one of the most popular rock bands in the world. Deflecting early critical disdain, Dave Grohl has single-handedly reinvented himself and cemented his place in the rock pantheon. This is his story, from his pre-Nirvana days in hardcore band Scream to his current festival-conquering status as a Grammy-winning, platinum-selling grunge legend reborn. Across the seven Foo Fighters albums, the whole sordid yet legendary Nirvana tale, the pre-history in the nascent Seattle scene and right up to date via Grohl's flirtations with bands such as Queens of the Stone Age, this is an utterly comprehensive and insightful chronicle of Dave Grohl's remarkable life.Drawing on new interviews with key figures in the Grohl story, this definitive biography of one of modern rock's most influential figures is now updated to include the world conquering double album "In Your Honour", the mellow acoustic brilliance of their "Skin and Bones" film and album, and their recent multi-platinum opus "Echoes, Silence, Patience and Grace".
"synopsis" may belong to another edition of this title.
Martin James once found himself watching The Prodigy backstage with Grohl, both clambering up a lighting rig to share a better view. James is a music and popular culture journalist who has enjoyed editorial positions at Muzik, Melody Maker, as well as contributing to The Independent, The Guardian, Mixmag and Urb magazine. Martin has also written books on Fatboy Slim, The Prodigy and Moby.
"About this title" may belong to another edition of this title.
Book Description Music Press Books, 2008. Paperback. Condition: New. Never used!. Seller Inventory # P111906191050
Book Description Music Press Books, 2008. Condition: New. book. Seller Inventory # M1906191050

domain: entertainment


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Michael R Gold, MD is a Cardiologist (Heart Specialist) - General, Cardiologist (Heart Specialist) - Clinical Cardiac Electrophysiology practicing in Charleston, SC
He has not yet shared a personalized… Read more ›
|University Of Colorado School Of Medicine||Medical Degree||1985|
|University of Co School of Medicine, Denver Co||Other Degree||1985|
|Residency - Mass Gen Hospital, Cardiovascular Diseases||Not Specified|
|Residency - Massachusetts General Hospital Program||Not Specified|
Sub-Specialty: Cardiovascular Disease
|Internal Medicine||Not Specified|
Michael R Gold, MD has not yet indicated the hospitals that he is affiliated with.
Michael R Gold, MD has not yet listed the medications that he commonly prescribes.
Michael R Gold, MD has not yet added any information about his practice's billing policies and payment options.

domain: medical


Transport and Infrastructure Minister Ian Borg explained that there has been a 10% increase in superyacht registration in Malta, as there are currently over 800 registered vessels over 24 metres in length. This was during an official visit to the Monaco Yacht Show earlier this week.
The Minister gave his support to the industry, adding that there was a lot of potential for further investment in the equivalent industry in Malta. The resulting increase in registration was deemed by the Minister as a clear sign of the government’s work to provide the right type of infrastructure for the industry to flourish.
“This is the direction in which the government shall continue to progress, all the while working with stakeholders within the sector so that an already good relationship will continue to improve,” added Dr Borg.
The Minister also announced that commercial yachts can now benefit from a dead-weight tonnage tax scheme which was just approved by the EU. This was done to encourage further yacht registration in Malta, thus solidifying the island’s name in the sector.
Dr Borg was also joined by high officials within Transport Malta, who have been a part of the Monaco Yacht Show for the last nine years. TM has also published guidelines to facilitate regulations regarding non-commercial yachts who can carry over 12 passengers, and for the renting of commercial yachts.


AWE Tee Shirt [Size Options: Small]
In Store Brisbane - Ships within 24 Hours
This Limited Edition AWE - Condor Maxfort Tee Shirt is a treated polyester fabric with moisture wicking, anti-microbial, and anti-odor finishes that provides maximum performance and comfort.
AWE (Australian Warrior Expo), a successful event of the year "For Those Who Serve" from Military, Law Enforcement, and Emergency Services.
Features and Specifications of Limited Edition AWE - Condor Maxfort Tee Shirt
- Highly breathable polyester
- Moisture wicking
- Odor resistant
- Tagless label
- Colour: Black Only
Great buyBy: Kevin on 17 May 2019Very nice touch sending the shirts in the shoulder bag, thankyou, appreciate the professional touch, shirts are great and look good.


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POP HEALTH FORUM: A HIMSS EVENT
Chicago, IL - October 2 - 3, 2017
Smita Patel, D.O., is a neurology, integrative medicine and sleep disorders specialist at NorthShore University HealthSystem. She believes in empowering patients to learn about health and wellness, nutrition and integrative approaches in treating disease.
As a Senior Clinician Educator at the University of Chicago Pritzker School of Medicine, she plays an active role at the NorthShore Neurological Institute and its Center for Brain Health, which focuses on primary prevention to address the unmet public health challenges of Alzheimer’s and related neurodegenerative diseases.
She is board certified in neurology and sleep medicine, and is a member of the American Board of Psychiatry and Neurology. She earned her doctorate in osteopathy from Midwestern University's Chicago College of Osteopathic Medicine. She interned and fulfilled her residency at the Medical College of Wisconsin, and served her fellowship at both University of Chicago and University of Arizona.
A Chicago-based neurologist shares best practices in using the electronic medical record (EMR) in delaying – even preventing – the onset of Alzheimer’s disease, now reaching epidemic proportions in America. She'll detail data and analytics, care coordination and patient engagement with regards to brain health and look at how specialists identify patients at high risk for Alzheimer’s disease and related disorders years before disease onset. She'll also describe how these patients can be integrated into long-term brain health care and surveillance to delay or prevent disease.
The only certainty in healthcare is uncertainty but what does this uncertainty mean for population health management? How does it affect strategy, purchasing, and implementation? Do you stay the course? Put the brakes on? Or some of both?
Our panel of experts will address this head on. Likewise, in keeping with the panel’s state-of-the-industry format, they’ll also address topics key to pop health success, such as clinical workflow improvement, decreasing utilization, data visualization, choosing vendors, measuring results, and more.
This session will provide attendees with a great overview and set the stage for the sessions to follow.

domain: health


10 Best Programming Language To Learn In 2022 programing language
Why Ruby Is Easy To Learn
Compared to Python, which focuses on providing a single, simple solution for every problem, Ruby aims to allow multiple approaches that achieve the same end. He has 25+ years experience as a programmer and QA leader, and holds several Microsoft certifications. The average Matlab developer takes home an average salary of $100,760 each year.
- It’s also a highly influential language, making it a useful one to know the basics of.
- It is a highly secured programming language because in java, there is no concept of explicit pointers.
- It helps marketers and data scientists to easily analyze, present, and visualize data.
- Larger companies tend to develop their internal software applications using C# or Java and their Web applications using PHP.
- It’s an interpreted language and can easily be embedded with languages like HTML.
The piece of code given below performs a basic task of printing “hello world! We must know that keyboard, scanner, mouse, microphone, etc are various examples of input devices, and monitor, printer, speaker, etc are examples of output devices. Python evolved from the language to write automation scripts or swiftly prototype apps to the first-class language for web and mobile development. It powers all kinds of websites and web applications as a server-side scripting language. Of Kotlin programmers use the language to build Android apps, 31% for back-end applications, 30% for SDK/libraries.
It is very popular in the field of high-performance computing. It is a structured and compiled programming language that is a subset of Fortran95. Fortran 2003, a revised version of Fortran supports object-oriented programming. Algorithmic Language, as it is called, is actually a family of imperative programming languages that was developed in the middle 1950s. It proved instrumental in the creation of programming languages like BCPL, B and C.
Again, the TIOBE index isn’t about the best programming language. As with many coding languages, the best way to learn is to get as much practice as you can with the language. There are many resources available that will take you through the basics, such as how the language works and what the basic syntax looks like. It’s a high-level procedural language that has become one of the most widely-used ones out there. Despite its age, it’s still a relatively complex language, yet its influence can be seen in many others. C#, C++, Java, Python, and more all take elements of inspiration from C.


The husband and I yearned to get away from the humdrum of life and decided on an adventurous holiday on the Big Island with a pit stop in Pearl Harbor.
We flew into Hilo, drove South to Volcano National Park and then flew out of the Kailua Kona Airport.
Kailua Kona is all about the ocean. We spent most days in the water followed by romantic evenings snuggled up on our lanai watching the sunset.
Day 1, 6 PM
After a 2.5 hour drive from Volcano National Park along the southern coast of the Island, we reached our rental at the Hale Kona Kai and were immediately blown away by the breathtaking views from the lanai!
The husband brewed some hot chai and we spent the rest of the evening watching an incredible sunset. From our perch, we could see shoals of Yellow Tang and a sea turtle lazily munching away.
Day 2, 7 AM : Snorkel in Kahalu’u
We grabbed our snorkels and headed to Kahalu’u Beach Park.
The sheltered bay with shallow water makes it an easy snorkeling ground. At 7 AM, the tide was still in and there were just a handful of other snorkelers.
We spotted sea urchins, tangs, surgeon fish, parrot fish and a whole host of other marine life whose names still elude us.
We spent a couple of hours in the water and came back to a group of ukulele singers at their weekly meeting serenading everyone in the park.
Day 2, 11 AM : Kona Coffee
After a quick breakfast, we headed to the historic Greenwell Coffee Farm to learn more about Kona’s famous (and expensive) coffee!
The farm has guided tours every 30 minutes – the farm has been operational since the late 1800’s, they purchase red coffee berries from hundreds of farmers all across Kona and that coffee is becoming a tough business with the wave of invasive pests and beetles destroying crops in Hawaii. For coffee aficionados, the tour also provides an opportunity to try different roasts and flavors of coffee.
Day 2, 5 PM : Night Snorkel with Manta Rays
Getting ready for our night snorkeling to view giant manta rays up close!
At 5 PM, we turned up at the Big Island Divers shop in Kona. They fitted us out with dive suits and fins. ( These guys definitely know what they are doing and I would highly recommend them!)
We then drove to the boat launch area. At 6 pm, a guide from the dive group gave us a short introduction to the trip – what to expect, the marine life and the Manta Rays. Bright lights put in the water attract tons of plankton. The Manta Rays emerge from the depths of the ocean to feed on the plankton.
We had great weather when we set out to the dive/snorkel spot. We cruised for an hour to the snorkel spot. By the time we got there, there were a couple of other boats and people already out in the water.
We got on our gear and dove straight in. We floated around a buoy which emitted light into the water. 2 minutes in and we saw a huuuuuuuuge manta ray coming straight at us. It did a barrel role as it fed on the plankton.
It was a mesmerizing sight to see these huge, gentle giants hover and somersault underneath us. One came up close and crashed into my fellow snorkeler.
We were in for a splendid night and witnessed a mating trail. There was one large female Manta Ray followed by a train of 6 hopeful males. The husband got some amazing shots of that night.
This was one of our most phenomenal nights on the island and the Manta rays hovering over those blue lights reminded me of something out of science fiction.
We got back to shore around 9PM and were greeted by a light drizzle. We went to bed dreaming of those Manta Rays…
Day 3, 6AM : Swim with Dolphins
After the close encounters with the Mantas, we woke up early and drove down to Keauhou Bay, the meeting point for our snorkeling trip to Kealakekua Bay.
After cruising in the water for about 20 minutes we spotted a pod of spinner dolphins. These dolphins were resting after a nighttime of feeding and hunting in the ocean. We swam in the water making sure to keep a respectful distance from these playful creatures.
We were thrilled when one of the dolphins leaped out of water and did a few spins before diving back in.
The husband got some beautiful videos as the pod swum gracefully by. Watch the videos with the SOUND ON to listen to the dolphins talking to each other!
After an hour of swimming with the dolphins, we got back onto the boat and headed to Kealakekua Bay.
Kealakekua Bay is a historic site where Captain Cook encountered Hawaiian natives. The Captain Cook monument is constructed at the site where he was killed by the natives of the island. This bay has some of the best snorkeling in Hawaii – the waters are deep and crystal clear with beautiful coral reefs and shoals of colorful fish. Because access is restricted there is very less coral bleaching taking place here. After swimming around for a couple of hours, we got back to the boat and headed back to shore.
We reached back around 12:30 PM and were greeted by the news that a 6.9 magnitude earthquake had just hit Kona!!! Because we were in the water, we hadn’t felt it!!!
The earthquakes were from the volcanic eruption in Kilueaua. We were lucky to have been so close to the volcano only the day before with lava flowing right by us!
Day 3, 4 PM : Farmer’s Market
We strolled to the nearby Farmers Market and bought some papayas, litchis, rambutans and longans……
The papayas and the longans are the sweetest that I’ve tasted.
For dinner, we headed to a local favourite – Broke Da Mouth Grindz – a hole in the wall away from touristy Ali’i Drive. I apologize for not taking any pics here but I was too busy stuffing my face with the Furikake chicken! It was so good that we came back here the next day!
Day 4, 10 AM: Pu’uhonua O Honaunau
Our last day in Hawaii, we decided to visit the place of refuge.
Years ago in Hawaii, you were punished by death if you broke one of the sacred laws (women were forbidden from eating bananas!!! ). The only way to save your life was to make your way to the place of refuge. Once you arrived here, a priest absolved you of your sins and you were free to go back to your old life.
We picked up one of the audio tours and strolled around the place. It’s quiet, serene and hard to imagine that Hawai’i had some brutal repercussions from breaking the law.
On our way back to Kona, we stopped at the famous South Kona Fruit Stand and splurged on some smoothies.
We headed back to Hale Kona Kai, said our byes to the resident turtle and packed our bags with a heavy heart……sad at having to leave behind this beautiful, exotic island.


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Artist Robert Kaindl is
inspired by nature and the classic Murano (Italy) Glass blowing
techniques, while infusing his own elegant yet organic style.
The Ostrea Series emulates this ancient mollusk re-imagined by
utilizing a vibrant modern color pallet. This petite
collection ranges in scale from 7" to 13" OD.
HOME GALLERY ARTIST CONTACT
Kaindl Glass Art, LLC | Seattle Washington USA


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Summary of SRI Activities in Guinea
In 2003, Dr. Peng Jimeng, deputy director of the China National Hybrid Rice Research and Development Center introduced hybrid varieties and SRI methods in Guinea. In 2012, a Peace Corps Volunteer and her colleague from the National School of Agriculture and Livestock (ENAE) in Lower Guinea attended a three-day SRI workshop that was held in early September 2012 in Ouémé, Benin. Following the workshop, SRI training was held at the Kanakan’s National Agriculture school, which trained 60 students and professors with the intention of incorporating SRI into field trainings.
Also in 2012, Dr. Mamadou Billo Barry, scientific director at the Institut de Recherche Agronomique de Guinée (IRAG), made a presentation and gave an interview on the potential for adoption of the SRI in Guinea at the Regional Workshop on the System of Rice Intensification (SRI), which was held in Ouagadougou, Burkina Faso and organized by the West and Central African Council for Agricultural Research and Development (CORAF/WECARD), the National Center of Specialization for Rice (NCOS-Rice Mali) and SRI-Rice (Cornell University) within the framework of the West Africa Agricultural Productivity Program (WAAPP). Due to growing interest in SR in Guinea following the workshop in Ouagadougou, Burkina Faso, the project Improving and Scaling up the System of Rice Intensification in West Africa, which resulted from the workshop, was formally launched in January 2014. The first phase of the project is running from January 2014 – June 2016. For more information about the SRI-WAAPP project view the project website and the project brochure. The SRI-WAAPP National Facilitator for Guinea is Mamadou Billo Barry, based at L’Institut de Recherche Agronomique de Guinée (IRAG). A 2016 map (right) shows SRI-WAAPP sites in Guinea.
- Peace Corps SRI Training in Yorokogueyah[January 30, 2017] Peace Corps Guinea’s thirteen Agroforestry Extension trainees arrived in Guinea in December 2016, and participated in two training sessions on the System of Rice Intensification in January 2017. At the training site in Yorokogueyah (Dubreka), Hillary Mara, Guinea RPCV ‘14 and SRI-Rice team member, led an introductory session on the basic principles of SRI as well as background on the importance of rice cultivation in Guinea. Trainees engaged on technical, biological, and socio-economic aspects of SRI. Trainees then practiced improved seed sorting methods. A nursery was prepared and about 12 days later, trainees had the hands-on experience of transplanting young seedlings following SRI management techniques. Trainees experimented with using both rakes and ropes as markers. The trainees leave for their respective communities in early February and will soon begin to identify possible partners and sites for testing SRI with community members in the coming rainy season.
- Farmers Impressed by Peace Corps SRI Plots Near Dubreka[November 30, 2016 ] Rainfed lowland rice planted by Peace Corps volunteers during an SRI training in Dubreka, Guinea in early August have reached maturity, demonstrating impressive growth and tillering (photo at right). Peace Corps Agroforestry Program Manager, Kalifala Fofana, notes: “The farmer near the field is appreciating it at every occasion. If I look at each rice plant coming only from a single seedling, it is amazing!” Hillary Mara, SRI team member, will be leading another SRI training for Peace Corps volunteers in Guinea this December.
- SRI Training for Extension Agents, Farmers, Community Leaders, and Peace Corps Volunteers[August 24, 2016] Hillary Mara, graduate student in the Cornell Institute of Public Affairs, spent 6 weeks in Guinea during mid-2016 training field agents and staff at the NGO NARSEME (Nature ressources pour les services écosystémique et les moyens d’existence), farmers, rural community leaders, and Agroforestry Peace Corps volunteers in the SRI methodology.
In the small rural community of Madina Oula, Kindia region, Martin Kourouma, Head of Project Development with NARSEME, and Mara shared the SRI information with NARSEME field agents, key farmers and resource people in the community. Following sessions on SRI theory and seed sorting, they worked with farmers to build demonstration and comparison plots, and a nursery for SRI seedlings. This work, which was replicated in the nearby town of Badet Kanty, was followed later with transplanting and weeding sessions, including the use of a rope-marker for plant spacing and a upland rice weeder, appreciated for its remarkable efficiency. Mara remarked, “Farmers were especially intrigued by the 95% reduction of seed: as the only purchased input for small holder rice farming, this represents a significant reduction in expenses for farmers and is very appealing to subsistence farmers.” During August, Mara also traveled to the Peace Corps training site in Dubreka and led a training for over 20 Agroforestry volunteers. (See article and photo album.)
- SRI-WAAPP Sites Expanded[May 2016] SRI continues to expand the areas of Guinea where rice is cultivated, as can be seen in the SRI-WAAPP project site map. The map, which is current as of 2016, shows 20 sites where farmers have received training in SRI since the SRI-WAAPP formally began in 2014. For more information, visit the SRI-WAAPP Guinea web page.
- Regional Project to Scale Up SRI Formally Launched[January 1, 2014] As one of 13 participating countries in the World Bank-financed regional project “Improving and Scaling up the System of Rice Intensification in West Africa” (SRI-WAAPP) that formally began in January 2014, Guinea participates in the project in regional workshops, trainings and meetings is undertaking nationally funded SRI activities through the WAAPP. Part of the larger and on-going West Africa Agricultural Productivity Program (WAAPP), SRI-WAAPP grew out of demands for technical and training assistance in SRI from most of the 13 countries, which resulted in a commissioned project development with an initial regional workshop to design the project in Ouagadougou, Burkina Faso in July 2012. The first phase of the project is running from January 2014 – June 2016. For more information about the SRI-WAAPP project view the project website and the project brochure. The project’s Regional Coordination Unit is a partnership between Mali’s National Center of Specialization in Rice (CNS-Riz) who houses the regional coordinator and SRI-Rice as the technical and strategic partner for this project, The SRI-WAAPP National Facilitator for Guinea is Mamadou Billo Barry, based at L’Institut de Recherche Agronomique de Guinée (IRAG). For more about SRI activities in Guinea through the SRI-WAAPP project, visit the project’s Guinea page. Since the project’s initial planning workshop in 2012, Guinea has participated in the numerous regional workshop (see reports for the various national and regional SRI-WAAPP activities).
- SRI Trainings and Trials Undertaken Following West Africa SRI Training in BeninA Guinea Peace Corps Volunteer and her colleague from the National School of Agriculture and Livestock (ENAE) in Lower Guinea attended a three-day SRI workshop that was held in early September 2012 in Ouémé, Benin. The event was a collaboration between the West Africa Food Security Partnership, SRI-Rice at Cornell University, and the Conseil de Concertation des Riziculteurs du Benin (CCR-B), and was hosted at the Solidarité Agricole Integrée (SAIN) teaching farmer and research center in Kakanitchoé, Ouémé.
Following the training, the Guinea participants to the workshop from ENAE conducted SRI trainings at the Kankan’s National Agriculture School. Over 60 agricultural students and professors attended. ENAE plans to integrate SRI into its trainings, conduct SRI trials and install demonstration fields. During their internships, students at ENAE will then be able to transfer their knowledge and skills on SRI to the NGOs, development projects, agriculture centers and other organizations that host their internships.
- Scientific director at the Institut de Recherche Agronomique de Guinée (IRAG) Attends SRI Workshop in Burkina Faso
Dr. Mamadou Billo Barry, scientific director at the Institut de Recherche Agronomique de Guinée (IRAG), made a presentation and gave an interview (at right) on the potential for adoption of the SRI in Guinea at the Regional Workshop on the System of Rice Intensification (SRI), which was held in Ouagadougou, Burkina Faso. The workshop, which was held July 26 and 27, 2012, was organized by the West and Central African Council for Agricultural Research and Development (CORAF/WECARD), the National Center of Specialization for Rice (NCOS-Rice Mali) and SRI-Rice (Cornell University) within the framework of the West Africa Agricultural Productivity Program (WAAPP). Based on information Dr. Barry learned at the workshop, he plans to set up trials in several plots in lowland periurban areas and also to engage with other governmental and non-governmental groups in Guinea.
- SRI Introduced into Guinea along with Hybrid Varieties from China
In 2003, Dr. Peng Jiming, deputy director of the China National Hybrid Rice Research and Development Center, while introducing hybrid varieties in this country also introduced SRI methods. The combination produced yields as high as 9 t/ha in a country where the current average yield is below 2 t/ha. The China National Hybrid Rice Center is introducing SRI methods in many areas that it is promoting its hybrid rice in Africa.
- Diallo, Mamadou Alimou. 2018. Comment produire le riz de manière intensive: Système de Riziculture Intensive (SRI). Agrofast Guinee website. December 1. [French langauge article from Guinea that describer the steps to planting SRI.]
- Mara, Hillary. 2016. SRI in Guinea: Training extension agents, farmers, community leaders, and Peace Corps Volunteers. August 24 feature article, SRI-Rice website. [Article on Hillary Mara’s 2016 SRI trainings with NGOs and the Peace Corps in Guinea, West Africa.]
- 2014. Le Système de Riziculture Intensive (SRI) en Guinée: Des rendements du riz jusqu’à plus de10 tonnes par ha confirm és par la recherche! L’Institut de Recherche Agronomique de Guinée (IRAG). SRI West Africa website.
- 2014. Note sur l’introduction et l’adaptation du Système de Riziculture Intensive (SRI) en Guinée. L’Institut de Recherche Agronomique de Guinée (IRAG). Octobre 2014.
- Jiming, Peng. Studies on the Use of Chinese Hybrid Rice with Modified SRI in Guinea. China National Hybrid Rice Research and Development Centre, Hunan, China. System of Rice Intensification website. (3p., 377KB pdf)
- Mara, Hillary. 2017. Rice Production in Guinea and SRI; Peace Corps Guinea Agroforestry PST. 54 slides. Presented at SRI Peace Corps Guinea Agroforestry PST, Dubreka, Guinea, January 9, 2017. SRI-Rice channel, Slideshare.net. [uploaded January 26, 2017]
- 2016 (October 12). SRI Line Spacing, Madina Oula, Guinea. 1:03 min. SRI-Rice channel, YouTube. [Description of SRI field preparations by the NGO Narseme in Guinea.]
- 2015: (April 6). Video SRI Guinée. 18.26 min. SRI-Rice channel, YouTube. [French language TV clip with farmer SRI interviews.]
- 2012 (August 23). First West Africa SRI Workshop, Ouagadougou, 8/26-27/12: Mamadou Billo Barry (Guinea) (French). 2:24 min. Produced by SRI-Rice. SRI Rice channel, YouTube. [Comments (in French) by Dr. Mamadou Billo Barry, scientific director at the Institut de Recherche Agronomique de Guinée (IRAG), Conakry, while attending the First West Africa System of Rice Intensification (SRI) Workshop, Ouagadougou, Burkina Faso, July 26-27, 2012.]
- Potentialites pour le SRI en Guinée
Powerpoint presented at the West Africa Regional SRI Workshop, Ouagadougou, Burkina Faso, July 26-27, 2012. 6 slides.
- The SRI-Rice Guinea Photo Collection contains photographs of SRI fields taken in Guinea

domain: agronomy