Willow

Substrates consisted of glucurono-xylans or glucurono-xylooligosaccharides. The glucurono-xylooligosaccharides UX and UXXX were purchased from Megazyme International (County Wicklow, Ireland), whereas UXX and XUXX were made as follows: 5 g of birch wood xylan (Sigma) was digested to completion with either CjXyn10A (19 (link)) or NpXyn11A (20 (link)) xylanases in 50 mm sodium phosphate, 12 mm citrate buffer, pH 6.5, containing 1 mg/ml of BSA (PC buffer) to completion. The glucurono-xylooligosaccharides generated by CjXyn10A, UXX, or NpXyn11A, XUXX, were purified from neutral xylooligosaccharides using Dowex chromatography (9 (link)). The structure of the products were confirmed by incubation with CjGlcA67A, which generated xylotriose from the CjXyn10A product but no undecorated xylooligosaccharide from the NpXyn11A product, whereas BoAgu115A released xylotriose and xylotetraose from glucurono-xylooligosaccharides generated by the GH10 and GH11 xylanases, respectively. The deduced structure of the two glucurono-xylooligosaccharides is entirely consistent with the glucuronoxylan binding mode of the two xylanases (21 (link), 22 (link)). Enzyme assays, in which polysaccharides or glucurono-xylooligosaccharides were the substrates, were carried out in PC buffer at 37 °C using enzyme purified to electrophoretic homogeneity by immobilized metal ion affinity chromatography. The concentration of enzyme varied from 10 nm for the wild type glucuronidase to 10 μm for the least active variants of BoAgu115A. For kinetic assays, glucuronic acid was detected using the α-d-glucuronidase assay kit in which the uronic acid released is oxidized to glucarate with the concomitant reduction of NAD⁺ to NADH, which was monitored continuously at 340 nm and quantified using a molar extinction coefficient of 6220 m⁻¹ cm⁻¹. The molar concentration of the GlcA/MeGlcA in the glucuronoxylans was quantified by digesting 100 μg of the polysaccharides to completion with BoAgu115A. To measure the activity of the BoAgu115A mutants the xylotetraose reaction product released from XUXX was monitored by high performance anion-exchange chromatography (HPAEC) as described previously (18 (link)). The reaction was carried out in 20 mm sodium phosphate buffer, pH 7.0, at a substrate concentration (1 mm) that was well below the K_M. Thus, the initial rate of hydrolysis of the glucurono-xylooligosaccharides gives a direct readout of k_cat/K_M (19 (link)). The glucuronidase-catalyzed reactions were also subjected to Polysaccharide Analysis using Carbohydrate gel Electrophoresis (PACE)⁴ as follows: a alcohol-insoluble residue was prepared from mature Arabidopsis thaliana wild type and gux1gux2 stems as well as wild type willow, barley, sugar cane, and Miscanthus stems, as previously described (23 (link)). Alcohol-insoluble residue (500 μg) was pre-treated with 20 μl of 4 m NaOH for 1 h, neutralized with HCl, and ammonium acetate buffer, pH 6.0, added to a final concentration of 0.1 m and a final volume of 500 μl. The Arabidopsis alcohol-insoluble residue was digested to completion with xylanases (CjXyn10A and NpXyn11A), a glucuronoxylanase (BoGH30; Bacova_03432), and GH67 and GH115 α-glucuronidases (CjGlcA67A and BoAgu115A) as stated in the text and then dried in vacuum, whereas the other xylans were just digested with BoAgu115A. Released mono- and oligosaccharides were labeled with 8-aminonaphthalene-1,3,6-trisulfonic acid (Invitrogen) and separated by gel electrophoresis as previously described (24 (link)). Xylooligosaccharides (Xyl_1–6, Megazyme Int.) were derivatized alongside each set of samples and run as standards within each gel.

The clinic is run by three board-certified plastic surgeons and one orthotist. It is open to patients with head deformity with a referral letter from primary care pediatricians.
Head deformity was evaluated according to the modified Argenta classification ^{(14) (link)} and anthropometric measurements (Figure 1a and b). The anthropometric measurements of the cranial vault were obtained using a craniometer and tape measure. The reliability of these evaluation methods is reported in past studies ^{(15) (link), (16) (link), (17) (link)}. According to the method proposed by Loveday et al. ^{(18) (link)}, head circumference, cranial asymmetry (CA), and cephalic index (CI) were used. CA and cranial vault asymmetry index (CVAI) describe the severity of asymmetry. We defined CA of less than 5 mm as normal skull, 5-9 mm as mild, 10-14 mm as moderate, 15-19 mm as severe, and ≥20 mm as very severe deformity. To enable a comparison with past reports, we also described CVAI, and CVAI of less than 3.5% was defined as normal, 5%-6% as mild, 7%-9% as moderate, 10%-13% as severe, and ≥14% as very severe ^{(14) (link)}. CI describes the brachycephalic or dolichocephalic tendency. CI of less than 79 was defined as dolichocephaly and greater than 94 as brachycephaly based on the Japanese standard ^{(19) (link)}.
The patients were evaluated and treated according to the algorithm (Figure 2). The criteria for helmet therapy were Argenta ≥ II. When the parents consented with the treatment, helmet therapy was initiated.
For helmet production, infant’s head was scanned using a surface scanner (OMEGA Scanner, Ohio Willow Wood Company, Ohio, USA). The helmet was designed by a single plastic surgeon (T.A.) using specialized software (OMEGA Tracer, Ohio Willow Wood Company, Ohio, USA) supervised by a senior plastic surgeon (T.K.). The helmet (Michigan Cranial Reshaping Orthosis, Danmer Products, Michigan, USA) was fabricated in the US and sent to us (Figure 1c and d) ⁽²⁰⁾ . The family was instructed to wear the helmet for 23 h a day after setting a break-in period of 7-14 days, during which the wearing time is gradually increased, and to visit the clinic after 3 to 4 weeks for adjustment. The helmet was continually used until the helmet was tight or until the parents were satisfied with the infant’s head shape. At the completion of the treatment, the patient was re-evaluated using the Argenta classification, anthropometric measurements, and surface scanner. For the time period of this study, the cost of the helmet was partially covered by our research fund, and patients paid 100,000 Japanese yens.

We conducted our research at the RNUP in Toronto, Ontario, Canada (43.8188° N, 79.1728° W). The RNUP is the first urban national park in Canada and is part of a pilot project carried out by Parks Canada to conserve urban biodiversity, Indigenous cultural landscapes, and agricultural heritage of the area [27 ]. It is an ecologically protected zone established in 2015 under the Rouge National Urban Park Act [28 ] that encompasses 80 km² of forests, meadows, rivers, wetlands, and fragments of rare habitats such as oak savannah and Carolinian woodlands [27 ]. Situated at the center of the Canada’s largest metropolitan area (Fig 1), the park is surrounded by major highways, freight and passenger railways, residential, commercial, and industrial developments, and agricultural lands [27 ].
Our study site is situated in the southern portion of the RNUP. In the early 1990s, the area was restored to a wetland complex of vernal pools, and more permanent ponds of various sizes with littoral vegetation including alders (Alnus spp.), cattails (Typha spp.), sedges (Carex spp.), and willows (Salix spp.) [21 ]. More recently, invasive species, such as European common reed (Phragmites australis), garlic mustard (Alliaria petiolate), purple loosestrife (Lythrum salicaria), and reed canary grass (Phalaris arundinacea) have become ubiquitous. Once restoration efforts were completed, the Toronto Zoo’s Adopt-A-Pond Wetland Conservation Program began wetland surveys to evaluate species occurrence in the area. The surveys found three at-risk turtle species: Painted Turtle (Chrysemys picta), Snapping Turtle (Chelydra serpentina), and the globally endangered [33 ] Blanding’s Turtle. In Canada, Painted and Snapping turtles are designated as ‘Special Concern’ by the Committee on the Status of Endangered Wildlife in Canada (COSEWIC) [34 , 35 ], and Blanding’s Turtle is designated as ‘Endangered’ [36 ].
In 2005, the Blanding’s Turtle population within the park boundary was known to be comprised of three adult turtles (two males and one female) and a juvenile. Two additional adult turtles (one male and one female) were discovered in 2006 in an adjacent creek approximately 4 km from the RNUP (Toronto Zoo [Unpublished]). Given that the Blanding’s Turtle population in the RNUP was presumed functionally extinct, the Toronto Zoo initiated a headstarting program in 2012 to supplement the wild population [21 ]. A preliminary population viability analysis (PVA) showed that 40 headstarted turtles with 1:1.5 male:female sex ratio would need to be released each year for 20 years to reach a self-sustaining population of 150 adult Blanding’s Turtles (Toronto Zoo [Unpublished]). The first release occurred in 2014 with 10 juveniles, followed by 21 in 2015, 36 in 2016, 49 in 2017, 49 in 2018, 48 in 2019, 57 in 2020 for a total of 270 headstarted turtles released to date (Toronto Zoo [Unpublished]). An additional 184 hatchlings were released without headstarting because the number of eggs that hatched exceeded the capacity of the Toronto Zoo rearing facility.

10 mg of Salix mucronata dried leaves or Tritticum spelta were minced with (100 mL) 50% or 100% ethanol separately for one day, and then filtered off. Each filtrate was collected, evaporated under reduced pressure using rotary evaporator (Telstar) separately, then lyophilized and weighed. The obtained ethanolic extract (powder), that containing active ingredient, was dissolved at concentration of 1 mg/mL distilled water.