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A set of collages explored design possibilities for the backyard. Courtesy TEN x TEN Landscape Architecture and Urbanism.
The backyard as a distinct space has not always been with us. It is, according to the cultural landscape historian Paul Groth, a relatively recent invention that was made possible by technological innovations in the 1930s and 1940s.
As Groth wrote in the journal Landscape in 1990, those innovations rid American backyards of “various sheds, laundry lines, noxious trash barrels, and ash pits.” But it was the evolution of the garage from detached to attached that signaled the moment when the backyard became “truly free, a born-again space.” Backyards are, in effect, what remained after more rugged elements were expelled.
Quarry Garden, a residential landscape designed by the Minneapolis-based firm TEN x TEN Landscape Architecture and Urbanism, distinguishes itself by reintroducing ruggedness to the backyard. It’s located in the Lowry Hill neighborhood of Southwest Minneapolis, and within the span of a year, it has pocketed both an ASLA Honor Award in Residential Design and a Landezine International Landscape Award in Private Gardens. In their recognition of the project, the Landezine awards jury described the atmosphere of the yard as “rich,” “dense,” and “mysterious.” Mystery is tough to achieve on a residential lot spanning a fifth of an acre, and that warrants a closer look.
A Study in Design Process
I met Maura Rockcastle, ASLA, and John Rasmussen at the Quarry Garden site early one Saturday morning in June. Rockcastle is a principal and cofounder, with Ross Altheimer, ASLA, of TEN x TEN. She had returned to her native Minneapolis in 2015 to found the studio after a prolific career in New York City. Rasmussen is an associate at the firm and was the lead designer and project manager on Quarry Garden. He had arrived that morning from birding with a colleague (highlights included a great horned owl and common grackle). The street was quiet, but the volume of birdsong rose as we approached the backyard.
Quarry Garden is only the third residential project by TEN x TEN, Rockcastle says. The clients, professors at the University of Minnesota, had moved from just blocks away and were looking for both architectural and landscape renovations to their new home. They hired the architects at MSR Design, who expanded the house and applied a vertical scaffolding treatment to the rear facade. For the landscape, the clients hoped to re-create a “woolly” environment that had characterized their previous backyard, Rockcastle says. They also hoped to incorporate qualities of “Japanese meditative spaces.”
Early in the design process, Rockcastle presented the clients with images of distinct Minnesotan landscapes grafted onto their lot. “We were playing with some collage techniques, trying to get a sense for how wild and rugged they might want to go.” The similarities between these rough collages and the final design are striking, and one collage in particular stands out: a snow-capped quarry wall, Mesabi gray, crowned with white birches and set against a backdrop of the clients’ vinyl-sided home. The clients’ positive response to this and other images gave Rockcastle and Rasmussen a vision toward which to work.
A diagram of the viewsheds for the design’s “outdoor rooms” concept. Courtesy TEN x TEN Landscape Architecture and Urbanism.
TEN x TEN’s office is located on the second floor of a repurposed light bulb factory in Northeast Minneapolis, about a 20-minute drive from Quarry Garden. The firm had moved into the office eight months prior but had yet to see full occupancy; the 17-person staff prefers to work from home. “I’m here all the time,” Rockcastle says. “It’s great.” The office is brightly lit by four large, north-facing windows. Sketches and mementos adorn the south wall, including a bright orange mold stamp from Mill 19, an award-winning adapted steel mill project in Pittsburgh. Several exhibition banners hang from the lofted ceilings and act as a divider between the kitchen and desks, and waist-high bookshelves frame the space between the desks and the conference room. These shelves are occupied by material samples, plaster castings, and a variety of plants in a variety of pots.
Finding Productive Tension
“We’re trying to collect a lot of materials, lots of [stuff] that inspires us,” Rasmussen says. “We do weird experiments.” He showed me some castings on the shelves: a mix of soot and soil congealed in a thin plaster batter. These were attempts to “replicate the ground plane” of a local, disused foundry. “We’re picking up charred timbers, moss growing. It connects you with the space and the place.”
Rockcastle had earlier told me that she encourages “a lot of experiments as a way of intentionally spending time [on-site]. If you’re using your hands, playing with the soil, or setting up a question or a prompt that you then photograph very specifically, it allows your sense of a place to be hyperfocused on a couple of different things, rather than just documenting randomly.”
John Rasmussen inspecting trees at a nursery. Courtesy TEN x TEN Landscape Architecture and Urbanism.
This isn’t always the case, she says. “We’re still finding our way with residential clients and what processes might be of value to them.”
For Quarry Garden, Rockcastle says she and Rasmussen decided to make “key elements, like the quarry and the deck, orthogonal and tight,” and allow the wilder counterpart to fill in, “letting the species themselves do the work of being wild.” The specific wilderness Rockcastle and Rasmussen were inspired by was the Minnesota North Woods, a boreal forest environment that Rockcastle described as “short and dense with a high proportion of pine and spruce. Birch is more in the transition zone. In the fall, it’s predominantly yellow because the birch is the primary deciduous.”
Construction of the project wrapped up in 2020. Professional photos taken of the project in September of that year show a thicket of Whitespire birch with their branches hovering just feet above a plane of Pennsylvania sedge. In the time since, the birch has grown up and a window of visibility between the lowest limbs and tallest sedge has opened. “I’m really excited to think about the succession of the birch. Like, do we get to a point where we start to bring in a couple more little guys?” Rasmussen wondered, referring to trees that would reduce visibility. He didn’t mind the limbs rising but says he missed the tight “hug” of the young birch.
The black locust decking with no-mow fescue in the foreground. Courtesy Gaffer Photography.
The ground cover was undergoing an informal review during my visit as well. TEN x TEN had specified a no-mow fescue in an area of the yard the designers refer to as “the clearing.” “Subtle characteristics” make the sedge superior to the fescue, Rasmussen says, but “from a value-added standpoint, [the no-mow] is so much cheaper.” Unfortunately, the no-mow had recently been mowed and was looking a bit defeated—a fault of the maintenance crew, who had also misidentified the garden’s sweet woodruff as a weed and uprooted it, Rasmussen told me. Landscape Renovations, the contractor that installed the landscape, would oversee maintenance in the future. And the sweet woodruff, some of which had eluded the last crew and now resembled small puffs of snow, would be worked back in.
The Big Design Move
To blur the threshold between the architecture and landscape, the designers conceived of the yard as a series of outdoor rooms. The most well-defined of these rooms is a sunken space at the back of the yard, which Rockcastle and Rasmussen consider to be the quarry proper. It’s fair to call the idea of outdoor rooms a “conventional concept,” as Landezine did, but it’s a convention because it’s an effective way of organizing space. The blurring occurs mainly in a covered outdoor lounge that faces the yard. TEN x TEN worked to ensure the yard design was visible from as much of the interior as possible, Rockcastle says. A single building and remodeling office, Terra Firma, was tasked with constructing both the home renovations and the landscape.
Out of the lounge and down the middle of the yard runs a six-foot-wide elevated walkway, surfaced with slender planks of black locust. It’s the spine of the project. “When we were modeling it, it felt powerful; it felt cool. It’s like you’re walking down a hallway…you just kind of flow,” Rasmussen says. About midway down the walkway, we stopped to inspect a Canadian hemlock. The weather had knocked out its leader branch, but Rasmussen said it would mend itself with sap and a secondary branch would take the lead, resulting in a “really cool, sculptural” hemlock.
Young birch trees cast unique shadows in both the cold and warm seasons. Courtesy Gaffer Photography.
The walkway terminates into another sculptural piece, a four-foot-wide by eight-foot-high wedge of granite. At 12,000 pounds, it’s the heaviest stone the designers specified, and it was carried over the threshold in the arms of a telehandler. Rasmussen says that “at some point, I want to drill a couple of holes through the top and on the back side. Have a water tray, so the water just sneaks through. Just a seepage.” Rockcastle wants to see light pierce the holes. “We like the idea of working with the sound of the wind in the leaves, water in the stone,” she says, creating the conditions for “those phenomenal, wild moments.”
Down three stumps of granite fashioned into steps, we descended into the quarry. Planting and grading partially obscure the quarry from view until you’re at its edge (cue the mystery). The descent is two feet and lands you on a bed of Dresser traprock and scattered forest litter. “You feel like you’re further in it” due to the top of the quarry walls sitting six inches above grade, Rasmussen says. These walls are composed of eight or so long slabs of granite procured from a local Coldspring quarry, and no material on the project exudes more character.
Roughnecks in the Garden
Known as roughbacks, each slab bears a set of evenly spaced grooves, about half an inch deep and one inch wide, along the face. The grooves are an artifact of the excavation process in which holes in the rock are drilled and packed with dynamite that, when detonated, free a 5-by-5-by-10-foot block from the quarry wall. (“Bhoosh!” Rasmussen says, mimicking the explosion sound.) Once it is freed, Coldspring shaves any defects off this block, including sections with drill holes, and it’s these sections that become the roughbacks. These specific roughbacks were tossed onto a plateau outside Coldspring’s quarry, called the graveyard, which is where they caught the eye of Rockcastle and Rasmussen.
“We didn’t know exactly what we needed,” Rockcastle says of the granite. But inventorying and deploying unused items is something of a specialty for TEN x TEN. They did it to masterful effect on Mill 19. Rockcastle says she and Rasmussen were both working on that project simultaneously with Quarry Garden. “There was a heavy influence. Giving yourself a limited set of parameters, making it a goal to reuse materials in creative ways.” The same spirit drove both projects, she says.
Rockcastle, Rasmussen, and other designers from the firm returned to the graveyard to kick the tires on the roughbacks. They brought wax pencils to tag the ones best suited for their residential quarry. “We measured and documented them in order to build a 3D model,” Rockcastle says. Rasmussen led the modeling of the stones and meticulously studied their arrangement. “It was a lovely textural study John was doing. Flipping which ones were the cut edges, which ones were the textured edges, so that when you’re sitting in the space, you have a composition of different textures.”
The best vantage on this composition is from the live-edge bench at the back of the quarry. It’s hewn from white oak and nested between the walls. “We wanted it to look like it’s floating,” Rasmussen says, so a minimalist bracketing system was used. Rasmussen considered the Japanese American woodworker George Nakashima “a huge influence,” and at one end of the bench are three butterfly joints neatly stitching a split, an incongruity typical in Nakashima’s work. Rasmussen described the joints as a “cue of care. It shows intentionality.”
The quarry is partially obscured by planting until you’re at its edge. Courtesy Gaffer Photography.
Several of the project’s minimalist elements were the result of a paring-down process. There was “a fear of overdesigning or making it more precious than it should be,” Rockcastle says. The team studied several options for a firepit in the quarry, including raised stone basins and linear structures, before taking the camouflage approach of burying a gas coil beneath the gravel and allowing the gas to seep upward. A roughback with a square void at its center sits flush with the gravel, the void framing the hidden burners. The only indication of the roughback’s purpose is an inlaid floor plate and valve that, when opened and ignited, send flames hissing out of the ground. Rasmussen demonstrated this while explaining that the gravel was igneous and, critically, solid. “It can handle being flamed. Porous rock can’t, because if you get moisture in them, you’re going to have exploding rocks.”
Rockcastle says the seating studies Rasmussen conducted used custom sculptural forms to achieve a “Noguchi-style garden, where the furniture is a really important part of the space.” These studies show wavy, primitive recliners and stools, but the clients opted for off-the-shelf furnishings. Rasmussen modeled another option for the space with an alternate wood bench, the form of which came cascading down from the elevated walkway. It’s an idea that might have seen enthusiastic implementation in a different context. But in this yard, Rasmussen believed “that would have been awful.”
Rasmussen says the decision to step down into the quarry helps to “elevate your subconscious experience of the yard,” calling it a “key moment of decision-making” between one region of the yard and the next. There aren’t many decision-making moments, however; it’s a small space. The main path pulls you from the lounge toward the back of the property, where the quarry might siphon you off. But if you instead hang a left out of the lounge, proceed past the humble vegetable garden growing tomatoes and squash, and take a right at the fence line, you’ll come upon a secluded hammock.
The yard as seen from the porch. Courtesy Gaffer Photography.
“[They] left the hammock up all summer,” Rasmussen says, pointing to the shadow it cast. That explained the bare patch of mulch beneath it. But Rasmussen used the scene to highlight that the mulch was triple-shredded. “We really want it to feel like a woodland,” he says, “kind of like you’re walking in the woods.” The mulch gave a sharp crunch as I pressed my sneaker to it, and Rasmussen nodded approvingly.
On my way out of town, I stopped at another TEN x TEN project, the Historic Fort Snelling revitalization, a recently completed landscape overlooking the Mississippi River. The fort was closed to visitors, so I paced the grounds, appreciating the nascent planting and wayfinding, which explained the significance of each species to the Dakota people. I stopped in front of a pair of limestone roughbacks, stacked one on the other but at an angle, and I imagined the arrangement studies they had undergone. I brushed the stone off with my hand and took a seat.
Aaron King is a landscape designer and writer based in Brooklyn.
Project Credits Landscape Architect TEN x TEN Landscape Architecture and Urbanism, Minneapolis. Architect MSR Design, Minneapolis. Construction Terra Firma, Saint Paul, Minnesota. Planting and Maintenance Landscape Renovations, Afton, Minnesota.
Anything rendered little might glimpse cuter, which may possibly be the primary cause for the expanding recognition of plant-based mostly micro-landscaping amid youthful people today in China. For them, micro-landscapes are not only a homey decoration, but a mode of spiritual aid from daily function or daily life.
Micro-landscapes typically take the shape of a potted micro-entire world incorporating moss, ferns, little herbs, and other crops in glass containers with rocks, themed characters, and unlimited extras. By making use of aesthetic design ideas, just one can develop a tiny-sized fairy backyard, an idyllic scene, a landscape with a compact bridge around flowing drinking water, or any green location you can think about in just just a several square inches. However, micro-landscapes did not start out out this way.
The enhancement of plant micro-landscapes owes substantially to the creation of the Wardian scenario. At the stop of the 19th century, numerous Europeans have been frenziedly gathering exotic plants from all around the environment. Having said that, because of to minimal transportation channels, unique crops could only be transported by sea, and the survival charge was exceptionally lower. In 1829, Nathaniel Bagshaw Ward, an English physician and collector residing on the outskirts of London, unintentionally found that ferns could expand very well and reproduce in closed tanks. Right after his discovery, he planted ferns in moist soil and packed them in sealed glass circumstances. The ferns had been not detrimentally influenced and grew properly for a few many years. This sort of container, just one created precisely to manage plants, would later become identified as the Wardian circumstance.
The Japanese, motivated by this creation, skillfully blended their really like for moss, bonsai, vegetation, and flowers in glass containers of several shapes, which gave these miniature gardens a perception of Zen – the predecessor of Do it yourself micro-landscaping.
With ongoing urbanization, many Chinese people today stay in significant metropolitan areas and are surrounded by metal and concrete, slowly separating them from inexperienced, all-natural landscapes. The attractiveness of Do-it-yourself micro-landscaping therefore satisfies people’s have to have to take pleasure in a touch of environmentally friendly, whether at residence or in the business office.
Micro-landscapes are effectively tiny ecosystems cultivated in glass containers. The base is usually composed of pumice substrate, sphagnum moss, and peat soil, with the pumice performing as a h2o barrier and equal to the permeable gap at the bottom of a flowerpot. The sphagnum moss is then packed as tightly as essential, acting as an isolation layer to sustain humidity and bear loading. Eventually, peat soil offers nutrition to meet the escalating requirements of plant roots.
“It can be stated that the generation approach of a micro-landscape is effortless, but to make it incredible needs expertise and aesthetics,” describes Bai Yue, the founder of Beijing NatureIn Technological know-how Co., Ltd., a micro-landscape production enterprise. “You can study how to make a person by looking at a two-minute creation movie. But you have to have to pay out unique attention to the mild due to the fact it is an significant prerequisite for the expansion of micro-landscape vegetation.”
Bai Yue began creating and marketing micro-landscape materials on-line in 2015. In advance of that, he experienced labored on the internet for much more than 10 several years. Bai usually enjoys discovering reducing-edge, superior, or wonderful items in his totally free time. Later on, he discovered himself drawn to the modest earth of plant-based micro-landscapes, creating a lot of mates as very well as an in depth buyer base thanks to his new pastime. In early 2020, Bai opened his initially offline shop when the COVID-19 epidemic strike.
“We have a great deal of relatives clients and quite a few dad and mom who invest in Do-it-yourself micro-landscapes or ecological fish tanks for their children. By means of Do-it-yourself micro-landscapes, young kinds can be nearer to nature, and grownups can acquire a break from technology. In addition to, people really like to beautify their living ecosystem with Do it yourself micro-landscapes,” mentioned Bai.
Below the ingenious layout and mindful building of micro-landscape supporters, basic resources have develop into one of a kind functions comprehensive of idyllic factors. Some fans have even been hired as micro-landscapers in Bai’s company.
Mutational landscape assessment by targeted capture sequencing in GHomas/somatotroph PitNETs
A summary of the clinical data for all 121 GHomas/somatotroph PitNETs patients is shown in Table 1. The mean basal GH level was high (15.7 ng/mL, IQR; 7.2–34.1), and the average IGF-1 SD score was 7.0 ± 2.4 (SD). The mean tumor volume was 1633.1 mm3 (IQR; 651.9–4412.4), and 23.1{6d6906d986cb38e604952ede6d65f3d49470e23f1a526661621333fa74363c48} of patients presented with a high Knosp grade (≥3). All 121 patients underwent transsphenoidal surgery (TSS), and 67 patients (55.4{6d6906d986cb38e604952ede6d65f3d49470e23f1a526661621333fa74363c48}) received preoperative SSA treatment. We evaluated biological remission by postoperative oral glucose tolerance test (OGTT) or normalization of IGF-1 SDS, resulting in 21 patients requiring additional therapy. The clinical data for all patients are provided in Supplementary Data 1. We performed targeted capture sequencing (TCS) of 36 genes (Supplementary Data 2) in all tumor tissue samples. The average sequencing depth was 1838.3 (range 29×–11973×), and 99.7{6d6906d986cb38e604952ede6d65f3d49470e23f1a526661621333fa74363c48} of the target regions were covered at least 50×. We detected a total of 83 mutations in 30 genes, with a median of 3.0 somatic coding region variants per tumor (range, 0–9). Among these mutations, 37 mutations in 18 genes were defined as recurrent (Fig. 1a).
Table 1 Clinical characteristics of GH-producing pituitary adenoma/ somatotroph pituitary neuroendocrine tumor patients and comparison of those with wild-type versus mutant GNAS.
Fig. 1: The landscape of gene mutations and CNAs in GHomas/somatotroph PitNETs.
a The landscape of gene mutations and copy number alterations (CNAs) in 121 growth hormone (GH)-producing pituitary adenomas/somatotroph PitNETs obtained by targeted capture sequencing (TCS), together with clinical and pathological annotations. b Bubble plot analysis, with age as the x-axis and the IGF-1 SD score as the y-axis. Blue circles represent GNAS wild-type, and red circles represent GNAS mutations. The circle size is relative to the Knosp grade. c Bubble plot analysis with GH change after octreotide treatment as the x-axis and tumor volume as the y-axis. Blue circles represent wild-type, and red circles represent mutations. The circle size is relative to the Knosp grade.
We observed various activating GNAS-MT (57.0{6d6906d986cb38e604952ede6d65f3d49470e23f1a526661621333fa74363c48} of patients) at known hot spots in 69 patients in our cohort; p.Arg201Cys was identified in 45 patients, p.Arg201His in 5 patients, p.Arg201Ser in 3 patients, p.Gln227Leu in 13 patients, p.Gln227Arg in 2 patients, and p.Gln227Glu in 1 patient. One patient carried both p.Arg201Cys and p.Gln870Leu mutations. We also detected 3 unreported GNAS-MT (p.Gly49Arg, p.Ser111Asn, and p.Ala249Asp). The number of patients was one. We mapped the mutations to the crystal structure of the Gsα protein (PDB: 6AU6) and found that p.Gly49 is located in proximity to the GTP binding domain (Supplementary Fig. 1a, b).
We detected several gene mutations that have previously been reported in GHomas/somatotroph PitNETs [AIP, GPR101, somatostatin receptor 5 (SSTR5), arrestin beta 1/2(ARRB1/2)] and associated with the cAMP pathway [protein kinase cAMP-activated catalytic subunit alpha (PRKACA), protein kinase cAMP-activated catalytic subunit beta (PRKACB), and protein kinase cAMP-dependent type I regulatory subunit alpha (PRKAR1A)]. AIP mutations were observed in 8 patients, and a GPR101 mutation was detected in 1 patient. SSTR5 was mutated in 3 patients. We detected 2 patients with ARRB1 mutations and 2 patients carrying ARRB2 mutations. PRKACA mutation was detected in 1 patient, and PRKAR1A and PRKACB mutations were observed in 2 patients each. Most of these mutations were not recurrent in our study, and no hotspots have previously been reported for these genes.
Copy number analysis revealed the loss of SDHx
We calculated copy numbers from the TCS data using CNVkit, as described in the Methods, and detected 21 cases with CNAs (Fig. 1a). Fourteen patients showed copy number gains, and 10 patients presented copy number losses. Seven patients harbored CNAs in more than 2 genes (range, 1–5). Notably, among the 14 patients with identified copy number gains, 7 patients harbored gains in SSTR5, 5 patients harbored gains in GPR101, and one patient presented CNAs in both genes. Among patients with identified copy number losses, 6 patients harbored losses in PRKACB, and 4 patients presented losses in succinate dehydrogenase complex (SDHx) genes, including SDHB, SDHC, and SDHD.
Clinical characteristics of patients with GNAS mutations
Although we identified 37 recurrent mutations in this study, we focused specifically on GNAS-MT. GNAS mutations (GNAS-MT) are well-known driver mutations of GHomas/somatotroph PitNETs, and more than half of the patients in our study harbored these mutations. A comparison between patients with GNAS-MT and those without mutations (GNAS-WT) is shown in Table 1 and Fig. 1b, c. The GNAS-MT group presented significantly better responsiveness to bromocriptine, consistent with a previous report that GNAS-MT adenomas are associated with higher dopamine receptor 2 mRNA expression than GNAS-WT adenomas23. A bubble plot analysis indicated that the GNAS-MT group also presented with smaller tumors and lower Knosp grades, although each dataset was associated with an extremely wide range. The GNAS-MT group tended to present with higher basal plasma GH levels and better responsiveness to octreotide loading test than the GNAS-WT group. The GNAS-MT group also showed a significantly better GH change rate following preoperative SSA treatment than the GNAS-WT group. However, the percentage of patients who required postoperative therapy was not significantly different between the two groups.
Consensus clustering-based transomics classification of pituitary adenomas/PitNETs
We performed proteomics and RNA sequencing analyses in pituitary adenomas/PitNETs (45 non-functioning pituitary adenomas/non-functioning pituitary neuroendocrine tumors [NFPAs/non-functioning PitNETs] for comparison, 60 GHomas/somatotroph PitNETs among 121 cases of the TCS cohort). The clinical data for all NFPAs/non-functioning PitNETs patients are provided in Supplementary Data 3. First, to clarify the transomics analysis, we examined the correlation between RNA and protein expression from RNA sequencing and proteomics data. Figure 2a shows a scatter plot of the correlation coefficient of a significantly positive correlation gene, with a mean Spearman’s correlation coefficient of 0.476 (Fig. 2a). This is similar to the results of the correlation analysis of RNA sequencing and proteomics in human rectal colon cancer24 and may represent tumor characteristics in pituitary tumors. Next, the whole gene was analyzed. Although 89.1{6d6906d986cb38e604952ede6d65f3d49470e23f1a526661621333fa74363c48} of the genes showed a positive mRNA-protein correlation, only 65.2{6d6906d986cb38e604952ede6d65f3d49470e23f1a526661621333fa74363c48} were significantly correlated (Fig. 2b). The average Spearman’s correlation between mRNA and protein variations was 0.330. There were uncorrelated genes and negatively correlated genes. These results suggest that there are networks that could only be identified by transomics. To test whether the concordance between protein and mRNA variation was related to the biological function of the gene product, we performed Kyoto Encyclopedia of Genes and Genomes enrichment analysis. Genes involved in several hypothalamic-pituitary-adrenal axis signaling pathways showed concordant mRNA and protein variations (Fig. 2c). These results suggest that the transomics data also accurately represented pituitary tumor characteristics. Consensus clustering subtyping was applied to RNA sequencing datasets, proteomics datasets, and the combination of these two datasets (defined as the transomics data set), exploring between 2 and 8 K-means clusters. Consensus cumulative distribution functions (CDF) and delta area (change in the CDF area) plots were generated for each dataset to determine the optimal K value (Supplementary Fig. 2). The Sankey diagram depicts the flow of cluster assignments for NFPAs/non-functioning PitNETs and GHomas/somatotroph PitNETs across data types. To clarify the significance of performing transomics analyses, we examined the classification by consensus clustering using both RNA sequencing and proteomics data. The appropriate cluster sizes were determined to be K = 5 for RNA sequencing data, K = 4 for proteomics data, and K = 4 for the transomics combination (Fig. 2d). The results showed that transomics analysis better reflects protein expression classification compared to RNA analysis.
Fig. 2: Consensus clustering-based transomics classification of pituitary adenomas/PitNETs.
a Steady-state mRNA and protein abundances were positively correlated with a mean Spearman’s correlation coefficient of 0.476. b mRNA and protein variations were positively correlated for most (89.1{6d6906d986cb38e604952ede6d65f3d49470e23f1a526661621333fa74363c48}) mRNA-protein pairs and 65.2{6d6906d986cb38e604952ede6d65f3d49470e23f1a526661621333fa74363c48} of mRNA-protein pairs showed significant correlations, with a mean Spearman’s correlation coefficient of 0.330. Negative correlations are shown in green and positive correlations in red. c mRNA and protein levels displayed dramatically different correlations for genes involved in different biological processes. Red and green indicate positive and negative correlations, respectively. d Results of unsupervised, nonnegative matrix factorization (NMF) subtyping applied to individual data types. The Sankey diagram depicts the flow of cluster assignments. e Violin plot depicting the mRNA expression of NR5A1, GATA2, and TBX19 in NFPA and GHoma. f Violin plot depicting the protein expression of nuclear receptor subfamily 5 group A member 1 (NR5A1), GATA-binding factor 2 (GATA2), and T-box transcription factor 19 (TBX19) in NFPA and GHoma. g Unsupervised multiomics subtyping via NMF identified four molecular subtypes with distinct multiomics expression patterns. NFPA: nonfunctional pituitary adenoma/non-functioning pituitary neuroendocrine tumor (PitNETs), GHoma: GH-producing pituitary adenoma/somatotroph Pituitary neuroendocrine tumor (PitNETs). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by 1-way ANOVA. n = 44 in NFPA, n = 34 in GHoma with GNAS WT, n = 28 in GHoma with GNAS MT.
RNA sequencing classification may differ from proteomics classification because the expression of RNA and protein molecules is not always correlated (Fig. 2a, b). However, similar expression patterns were identified for pituitary-specific transcription factors in the classification of NFPAs/non-functioning PitNETs and GHomas/somatotroph PitNETs (Fig. 2e, f). Although some differences were observed in the classification between the proteomics and transomics analyses, many similarities were also identified (Fig. 2d). Transomics analysis suggests that there may be molecules that exhibit variation in only protein expression that reflect the characteristics of pituitary adenomas/PitNETS to the best of our knowledge.
To explore the intrinsic cohort structure using the full complement of single-omics data, clustering was performed for mRNA and protein using non-negative matrix factorization (NMF)25, which yielded between 2 and 4 clusters for the single-omics analyses (Fig. 2g). A GO analysis of a group of molecules with significantly higher expression in Cluster 3 by proteomics revealed the terms GH synthesis, secretion and action (Supplementary Data 4). Transcriptomic data were only able to distinguish between NFPAs/non-functioning PitNETs and GHomas/somatotroph PitNETs, whereas proteomics data were able to distinguish among GHomas/somatotroph PitNETs with and without GNAS mutation. Therefore, we focused on both proteomics and genomics approaches for the characterization of GHomas/somatotroph PitNETs.
Proteogenomic characterization of nonfunctioning and GHomas/somatotroph PitNETs
The samples in which the proteins could not be identified by proteomics analysis were excluded. The protein expression of key transcription factors was compared between NFPAs/non-functioning PitNETs and GHomas/somatotroph PitNETs using uniform manifold approximation and projection (UMAP) analysis (Supplementary Fig. 3). The results showed that NFPAs/non-functioning PitNETs and GHomas/somatotroph PitNETs could be divided into 2 distinct groups, likely because NFPAs/non-functioning PitNETs and GHomas/somatotroph PitNETs have distinctly different protein expression characteristics. However, among GHomas/somatotroph PitNETs, no clear differences in protein expression characteristics were identified between cases with and without GNAS-MT. The expression of key transcription factors that contributed to pituitary differentiation visualized using a feature plot, revealed that nuclear receptor subfamily 5 Group A member 1 (NR5A1) and GATA-binding protein 3 (GATA3) are expressed at high levels in most NFPAs/non-functioning PitNETs samples, with some GHomas/somatotroph PitNETs also demonstrating high expression levels. In contrast, POU class 1 homeobox 1 (POU1F1), a key regulatory factor, was highly expressed in GHomas/somatotroph PitNETs, whereas POU1F1 and GH were not expressed in NFPAs/non-functioning PitNETs. SSTR5 was expressed heterogeneously in most GHomas/somatotroph PitNETs, with some expression observed in NFPAs/non-functioning PitNETs (Fig. 3a).
Fig. 3: Nontargeted proteomics analysis of pituitary adenomas/PitNETs.
a Uniform manifold approximation and projection (UMAP) analysis showing the clusters of NFPA and GHoma depending on tumor types: nuclear receptor subfamily 5 group A member 1 (NR5A1), GATA-binding protein 3 (GATA3), POU class 1 homeobox 1 (POU1F1), GH1, and somatostatin receptor 5 (SSTR5) using multiomics data. b Differential expression of proteins derived from nontargeted proteomics was estimated and analyzed with the Brunner–Munzel test. Venn diagram of overlapping differentially expressed molecules. c Heatmap showing differentially expressed molecules identified by proteomics analysis in NFPA and GHoma. NFPA: nonfunctional pituitary adenoma/non-functioning pituitary neuroendocrine tumor (PitNETs), GHoma: GH-producing pituitary adenoma/somatotroph Pituitary neuroendocrine tumor (PitNETs).
The Brunner–Munzel test was used to quantitatively compare protein expression levels between NFPAs/non-functioning PitNETs and GH-producing adenomas and between GNAS-WT and GNAS-MT GH-producing adenomas. A total of 7300 differentially expressed molecules were identified between NFPAs/non-functioning PitNETs and GHomas/somatotroph PitNETs, and 714 differentially expressed molecules were identified between GNAS-WT and GNAS-MT GH-producing adenomas. To evaluate the protein expression profiles of the 458 molecules that were identified as significantly differentially expressed in both comparisons (Fig. 3b, c), gene ontology (GO) analysis was performed. GO analysis (http://geneontology.org) revealed that the GNAS mutation influenced several binding functions and GPCR pathways (Supplementary Data 5). Considering these results collectively, we hypothesize that GNAS-MT induce GPCR pathways in GHomas/somatotroph PitNETs, influencing endocrinological characteristics, such as the response to drug treatments.
Correlation between the protein expression profile and clinical characteristics of Acromegaly
We first evaluated the expression of some characteristic proteins by pathological analysis. In our study, the immunohistochemistry (IHC) score for SSTR2, which is known to serve as the primary SSA receptor in GHomas/somatotroph PitNETs, did not differ between the GNAS-WT and GNAS-MT groups (Fig. 4a). Similarly, the IHC scores for GH and the CAM5.2 cytokeratin IHC pattern did not differ between the two groups (Fig. 4b, c).
Fig. 4: Protein expression levels in NFPAs/non-functioning PitNETs, GNAS-WT GHomas/somatotroph PitNETs, and GNAS-MT GHomas/somatotroph PitNETs.
Immunohistochemistry scores of (a) somatostatin receptor 2 (SSTR2) and (b) GNAS-WT and GNAS-mutant (MT) growth hormone (GH)-producing pituitary adenomas ({6d6906d986cb38e604952ede6d65f3d49470e23f1a526661621333fa74363c48}). *p < 0.05, by chi-square test. c CAM5.2 cytokeratin immunostaining pattern in the GNAS-WT and GNAS-MT groups ({6d6906d986cb38e604952ede6d65f3d49470e23f1a526661621333fa74363c48}). *p < 0.05, by chi-square test. d TBX19, (e) POU class 1 homeobox 1 (POU1F1), (f) aryl hydrocarbon receptor-interacting protein (AIP), (g) SSTR2, (h) SSTR5, (j) sigma nonopioid intracellular receptor-1 (SIGMAR1), (k) adhesion G protein–coupled receptor V1 (ADGRV1), and (l) sortilin-related VPS10 domain–containing receptor 3 (SORCS3) protein expression levels derived from nontargeted proteomics in NFPA, GNAS-WT GHoma and GNAS-MT GHoma. The protein expression values were log (base 10) transformed. i Protein expression ratio of SSTR2 to SSTR5 in NFPA, GNAS-WT GHoma, and GNAS-MT GHoma. NFPA: nonfunctional pituitary adenoma/non-functioning pituitary neuroendocrine tumor (PitNETs), GHoma: GH-producing pituitary adenoma/somatotroph Pituitary neuroendocrine tumor (PitNETs). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by 1-way ANOVA. n = 45 in NFPA, n = 21 in GHoma with GNAS WT, n = 19 in GHoma with GNAS MT.
Based on the results of the nontargeted proteomics analysis, we compared protein expression levels between three groups: NFPAs/non-functioning PitNETs, GNAS-WT GHomas/somatotroph PitNETs, and GNAS-MT GHomas/somatotroph PitNETs. The protein expression level of T-box transcription factor 19 (TBX19, also known as TPIT) did not differ among the 3 groups, whereas the expression level of POU1F1 was significantly higher in the GNAS-WT and GNAS-MT groups than in the NFPAs/non-functioning PitNETs group (Fig. 4d, e), consistent with the regulation of transcription factors during pituitary development. The protein expression levels of AIP were significantly higher in the NFPAs/non-functioning PitNETs group than in the GHomas/somatotroph PitNETs group (Fig. 4f). Conversely, SSTR2 expression was significantly higher in the GNAS-WT and GNAS-MT groups than in the NFPAs/non-functioning PitNETs group (Fig. 4g).
Next, we focused on 4 molecules: SSTR5, sigma nonopioid intracellular receptor 1 (SIGMAR1), adhesion G protein–coupled receptor V1 (ADGRV1), and sortilin-related VPS10 domain–containing receptor 3 (SORCS3). These molecules are involved in GPCR activity and were among the 458 identified differentially expressed proteins. The expression levels of these molecules were significantly different between the NFPAs/non-functioning PitNETs and GHomas/somatotroph PitNETs groups: SSTR5 expression was higher in the GHomas/somatotroph PitNETs group than in the NFPAs/non-functioning PitNETs group, and there was no difference in expression with or without GNAS-MT (Fig. 4h). The SSTR2/5 protein expression ratio was not significantly different between the NFPAs/non-functioning PitNETs and GHomas/somatotroph PitNETs groups, nor were they expressed with or without GNAS-MT in the GHomas/somatotroph PitNETs group (Fig. 4i). SIGMAR1 expression was lower in the GHomas/somatotroph PitNETs group than in the NFPAs/non-functioning PitNETs group and was lower in the GNAS-MT groups than in the GNAS-WT group (Fig. 4j). ADGRV1 expression was not significantly different between the NFPAs/non-functioning PitNETs and GHomas/somatotroph PitNETs groups, nor was it different with or without GNAS-MT in the GHomas/somatotroph PitNETs group (Fig. 4k). SORCS3 expression was higher in GHomas/somatotroph PitNETs GNAS-WT than in NFPAs/non-functioning PitNETs and lower in GNAS-MT expression than in GNAS-WT (Fig. 4l). To determine whether these GPCR-related molecules affect the clinical characteristics of acromegaly, we analyzed the correlations between the expression levels of these four proteins and clinical characteristics, including the GH change rate following the octreotide loading test and the tumor volume change rate following SSA treatment. The expression levels of ADGRV1 and SORCS3 were correlated with the GH change rate following the octreotide loading test (Supplementary Fig. 4).
The GH change rate following the octreotide loading test, which has been reported to correlate with the SSTR2 mRNA expression level, did not correlate with the SSTR2 or SSTR5 protein expression levels or with the SSTR2/5 protein expression ratio (Fig. 5a–c). We then performed correlation analyses between all of the protein expression values derived from the nontargeted proteomics analysis and the GH change rate following the octreotide loading test. We detected positive correlations for ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 (ATP2A2) and AT-rich interaction domain 5B (ARID5B) with the GH change rate following the octreotide loading test, although no correlation was observed for ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 1 (ATP2A1) (Fig. 5d–f). The protein expression levels of ATP2A2 and ATP2A1 were significantly lower in the GHomas/somatotroph PitNETs group than in the NFPAs/non-functioning PitNETs group, and ARID5B expression was significantly higher in the GHomas/somatotroph PitNETs group. These 3 molecules were expressed at significantly lower levels in the GNAS-MT group than in the GNAS-WT group (Fig. 5g–i)
Fig. 5: Correlation between protein expression levels and GH change rates in the octreotide loading test.
Correlations between (a) somatostatin receptor 2 (SSTR2) protein expression, (b) SSTR5 protein expression, (c) the SSTR2/5 ratio, (d) sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (ATP2A2) protein expression, (e) ATP2A1 protein expression, and (f) AT-rich interaction domain 5B (ARID5B) protein expression and growth hormone (GH) change ratio by octreotide loading test ({6d6906d986cb38e604952ede6d65f3d49470e23f1a526661621333fa74363c48}). Data were analyzed by Pearson’s correlation analysis. g ATP2A2, (h) ATP2A1, and (i) ARID5B protein expression levels in NFPA, GNAS-WT GHoma, and GNAS-mutant (MT) GHoma. All protein expression values were log (base 10) transformed. NFPA: nonfunctional pituitary adenoma/non-functioning pituitary neuroendocrine tumor (PitNETs), GHoma: GH-producing pituitary adenoma/somatotroph Pituitary neuroendocrine tumor (PitNETs). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by 1-way ANOVA. a–fn = 34 in GHoma with GNAS WT, n = 26 in GHoma with GNAS MT. g–in = 45 in NFPA, n = 21 in GHoma with GNAS WT, n = 19 in GHoma with GNAS MT.
To clarify the pathology of functional pituitary adenomas/PitNETS, we performed correlation analyses between the nontargeted proteomics data and the tumor volume change rate following SSA treatment because it is important to consider not only the production and secretion of hormones but also tumorigenesis. SSTR2 expression, SSTR5 expression, and the SSTR2/5 ratio did not correlate with the tumor volume change rate following SSA treatment (Fig. 6a–c). The tumor volume change rate following SSA treatment was correlated with the expression levels of WWC family member 3 (WWC3) and serine incorporator 1 (SERINC1) and tended to correlate with zinc finger AN1-type containing 3 (ZFAND3) expression (Fig. 6d–f). The expression of WWC3 was significantly lower in the GNAS-MT group than in the GNAS-WT group, whereas SERINC1 and ZFAND3 expression levels were significantly higher in the GNAS-MT group than in the GNAS-WT group (Fig. 6g–i).
Fig. 6: Correlation between protein expression levels and tumor volume change rate in the SSA test.
Correlations between (a) somatostatin receptor 2 (SSTR2) protein expression, (b) SSTR5 protein expression, (c) the SSTR2/5 ratio, (d) WWC family member 3 (WWC3) protein expression, (e) serine incorporator 1 (SERINC1) protein expression, and (f) zinc finger AN1-type containing 3 (ZFAND3) protein expression and the tumor volume change rate in the somatostatin analog (SSA) test ({6d6906d986cb38e604952ede6d65f3d49470e23f1a526661621333fa74363c48}). Data were analyzed by Pearson’s correlation analysis. g WWC3, (h) SERINC1, and (i) ZFAND3 protein expression levels in NFPA, GNAS-WT GHoma, and GNAS– MT GHoma. All protein expression values were log (base 10) transformed. NFPA: nonfunctional pituitary adenoma/non-functioning pituitary neuroendocrine tumor (PitNETs), GHoma: GH-producing pituitary adenoma/somatotroph Pituitary neuroendocrine tumor (PitNETs). *p < 0.05, **p < 0.01, ****p < 0.0001 by 1-way ANOVA. a–fn = 18 in GHoma with GNAS WT, n = 13 in GHoma with GNAS MT. g–in = 45 in NFPA, n = 21 in GHoma with GNAS WT, n = 19 in GHoma with GNAS MT.
These differentially expressed protein candidates from the nontargeted proteomics analysis were evaluated by pathological examination. Sections were stained with SIGMAR1, ATP2A2, ARID5B, WWC3, and SERINC1 (Fig. 7a–e), and IHC scoring was performed as described in the Methods. SIGMAR1 was significantly increased in the NFPAs/non-functioning PitNETs group compared with the GNAS-MT group. (Fig. 7f). ATP2A2 was significantly increased in the NFPAs/non-functioning PitNETs group compared with the GHomas/somatotroph PitNETs groups (Fig. 7g). ARID5B was significantly increased in the GNAS-WT group compared with the GNAS-MT group (Fig. 7h). There were no significant differences in WWC3 and SERINC1 among the three groups. (Fig. 7i, j). These expression patterns were similar to the trends observed using nontargeted proteomics analysis.
Fig. 7: Immunohistochemistry of differentially expressed candidates in nontargeted proteomics.
Sections were stained for (a) sigma nonopioid intracellular receptor-1 (SIGMAR1), (b) sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (ATP2A2), (c) AT-rich interaction domain 5B (ARID5B), (d) WWC family member 3 (WWC3), and (e) serine incorporator 1 (SERINC1). IHC scoring of (f) SIGMAR1, (g) ATP2A2, (h) ARID5B, (i) WWC3 and (j) SERINC1 was performed by modifying McCarty’s H-score. NFPA: nonfunctional pituitary adenoma/non-functioning pituitary neuroendocrine tumor (PitNETs), GHoma: GH-producing pituitary adenoma/somatotroph Pituitary neuroendocrine tumor (PitNETs). **p < 0.01, ***p < 0.001 by 1-way ANOVA. n = 8 in NFPA, n = 11 in GHoma with GNAS WT, n = 12 in GHoma with GNAS MT.
A space devoted to serving wine, cocktails, and beverages is a welcome addition in most homes, primarily throughout the holiday break web hosting season. The good news is, there are a lot of bar cart concepts to take into account for your possess place. There’s certain to be a little something that matches in with your personal dwelling style and design aesthetic, irrespective of whether it is an antique wooden bar cart or a midcentury stunner. And the greatest component: You really do not even need to have a correct bar cart (while including a single to your home furnishings desire list is usually an choice). An acrylic tray positioned atop a bookshelf or bar cupboard, or the best of a facet or console desk can all fill in as a makeshift but purposeful and stylish bar cart.
A glassware-and-libations pitstop for a festive soiree only wants a handful of supplies—many of which you possibly currently have on hand. Placing a bar cart alongside one another does not acquire up hours either. This is an simple Diy afternoon venture of curating and styling. “Entertaining is all about the working experience,” states designer and HomeGoods design and style specialist Beth Diana Smith, who follows a three-action formulation when prepping a bar cart: “Set the phase, provide on consume-ware, and decorate.”
How do you adorn a bar cart at dwelling?
Good designers know that at times significantly less is additional, and that adage also applies when it will come to house bar ideas and bar cart tips. “You don’t need to have to fill each inch of a bar cart,” claims Roxy Owens, founder and designer of Modern society Social. “You really don’t require to undertake a minimalist aesthetic, but if way too many items are crammed into the area, it can be overwhelming for attendees to use and it will make your room look cluttered.” Malka Helft, principal of Assume Stylish Interiors in New York, likes clustering bottles dependent on the coloration of liquor and bottle itself more than the kind of liquor.
You should also consider the bar cart design and style, or floor you are utilizing, into thing to consider. Are you styling a rattan bar cart for an out of doors bar? If so, shatterproof barware and vibrant straws and stirrers may well be in buy. But if you’re generating a vignette on a stately piece of furnishings, bar cart styling that includes glass decanters, a assertion wine rack, or a collection of amazing coupe eyeglasses could improved round out the style.
How must I inventory a bar cart?
Focus on the essentials. “Don’t sense pressured to inventory each and every final fancy-schmancy liqueur or bitter,” Owens suggests. “All you want are a couple of high-quality spirits to make the classics, a great established of bar instruments (lime squeezer, muddler, strainer, and mixing tin are important), and some appealing glassware.” In addition, purchasing a lot less suggests you can splurge on higher-excellent major-shelf spirits. If you have a surplus of wine or liquor, pick out the greatest-seeking bottles to exhibit in your residence bar and stash the relaxation out of sight in the pantry.
When it’s time to entertain, designer and luxury residence decor pro Kim Seybert says she generally has the vintage liquors on hand: gin, vodka, tequila, and bourbon. “If I know my guests favor a style of liquor, I make certain to stock it, along with contemporary-squeezed juices, soda h2o, tonic, and freshly minimize lemons,” she says. Nevertheless, Seybert urges amateur hosts to keep it easy. “Do not overextend yourself,” she continues. “Serve just one purple wine, one particular white wine, and two combined beverages. Pick a excellent wine glass for purple or white, or buy a multipurpose double aged-fashioned glass for combined beverages or wine. Use a wine bucket so the bottle can chill without the need of putting it back again in the fridge.”
Also make certain to have possibilities available for friends who do not imbibe,. For functions, continue to keep just one or two non-alcoholic beverage selections chilled. And don’t forget about extras that can seriously make all those mocktails pop. “For non-alcoholic drinks, I normally include a festive garnish like cranberries, mint sprigs, or a vibrant themed straw,” Smith carries on. “Just due to the fact it is non-alcoholic doesn’t necessarily mean it has to seem it.”
How can I spice up my bar cart?
Now that you’ve bought your basics protected, it is time for a shot of design. “Color, pattern, and texture all come into play when styling your bar cart,” Owens states. “Colorful straws and tailor made coasters insert signature design and style and functionality. Cloth cocktail napkins are a compact contact that can go a long way.”
MOUNTAIN View, Calif. – Each and every 12 months Google appears back at the tendencies of the preceding 12 months with its yearly Yr in Lookup results. So what do present day design and style, the coloration sage and landscape wallpaper have in typical? They ended up all the No. 1 look for phrase in their class this calendar year.
This 12 months, the greatest breakout research around the world according to Google was: “Is the multiverse genuine?”
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For instance, the Anchorage, Alaska and Des Moines, Iowa areas had been the only two places in the U.S. that had “quilt shops” in their top trending “near me” lookups.
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The leading 10 search phrases for several categories linked to the house are down below.
Inside Layout Styles
Contemporary
Eclectic
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Sage
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How to thoroughly clean a rusty forged iron skillet
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How to thoroughly clean an electric powered kettle
How to clean a solid iron griddle
How to clean a kitchen sink
How to clean up your hairbrush
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How to make very clear ice cubes
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Landscape wallpaper
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See also: Blog site: Piracy, cartels, cost gouging and other well timed Google queries
