They arestructurally similar to yersiniabactin, which contributes to the virulenceof the human pathogen Yersinia pestis. However, the pxb-derived products feature variouschain lengths and unusual carboxamide, thiocarboxylic acid, and dithioperoxoatetermini, which are rarely found in thiotemplated biosyntheses. Here,we characterize the pxb biosynthetic logic by genedeletions, site-directed mutagenesis, and isotope labeling experiments.Notably, we propose that it involves (1) heterocyclization domainswith various catalytic efficiencies catalyzing thiazoline and amide/thioesterbond formation and (2) putative C–N and C–S bond cleavageoff-loading manners, which lead to products with different chain lengthsand usual termini. Additionally, the post-assembly-line spontaneousconversions of the biosynthetic end product contribute to productiontiters of the other products in the culture medium. This study broadensour knowledge of thiotemplated biosynthesis and how bacterial hostgenerate a chemical arsenal.Piscibactins and photoxenobactins are metallophores andvirulencefactors, whose biosynthetic gene cluster, termed  By homologous overexpression, we demonstratedthat the pxb BGC encodes the biosynthesis of an arrayof yersiniabactin-like natural products, piscibactins (1 and 2) and photoxenobactins (3–7).1 Labeling studies revealedthat salicylic acid, cysteine, and the methyl moiety of S-adenosylmethionine are the major building blocks.1 It is particularly noteworthy for their unusual termini, such ascarboxamide, thiocarboxylic acid, and dithioperoxoate  for the bacterial host, these compounds serveas virulence factors against insects1 andthereby facilitate the symbiotic nematodes killing insect prey.4Recently, we identified eroxoate 1a, which5 analysisrevealed that the pxb BGC, exemplified by the clusterin Xenorhabdusszentirmaii DSM 16338, is similar to both the ybt BGC from Yersinia pestisencoded by the pxb BGC is equivalent to the salicylatesynthase (YbtS) and the free-standing salicylate specific A domain(YbtE) encoded by the ybt BGC. Although PxbG andIrp1 are homologous to HMWP1, both PxbG and Irp1 are deficient incarbon methyltransferase (cMT1)8 specific for C-14 bismethylation of yersiniabactin and encode anadditional heterocyclization–adenylation–thiolation(Cy4–A2–T6) module and piscibactin (2) encoded by the irp BGC and hypothesized that the additional Cy4–A2–T6 module might introducethe fourth thiazoline to piscibactin (2), resulting ina full biosynthetic product.7 However,the newly identified photoxenobactins (3–7) from the pxb BGC featuring various chainlengths and termini defy such a functional prediction of the biosyntheticassembly line,1 thus motivating us to characterizethe biosynthetic logic of photoxenobactins. Here, we investigate thebiosynthesis of photoxenobactins in the overexpressing mutant X. szentirmaii PBADpxbF. Since 7 is only present in the X. szentirmaii PBADpxbF Δhfq mutant1  but isabsent in the X. szentirmaii PBADpxbF mutant, 7 is therefore not examined herein.AntiSMASH. piscida , is postulated to be identicalto that of yersiniabactin  and pxbH deletion mutants, both of which abolished production of 1–6 .The early-stage biosynthesis of photoxenobactins, until the reductionof intermediate A catalyzed by PxbH  with an alanine residue  abolished production of 2–6  led to accumulation of 1 and complete loss of 5 and 6, suggestingthat 5 and 6 are derived from the T6 domain (2 and 4 absent in the T5 inactivation mutantwere detected in the T6 inactivation mutant but with remarkablyreduced production titers (5 domain (3 possessing the same chain length as 4 is supposed to be released from the T5 domain(this is confirmed by the PxbG cMT inactivation mutant below). However, 3 was barely observed in the T6 inactivation mutant(3 is heavily dependent on the product(s) releasedfrom the T6 domain.The observed various chain lengths of piscibactinsand photoxenobactinsmight imply the existence of premature off-loading stages on PxbG,and therefore, we set out to interrupt the assembly line by inactivatingT2–4 are released from the T5 domain, 3 and 4 are terminated by a thiocarboxylic acid and carboxamide,respectively, instead of a carboxylic acid as in 2. Also, 5 and 6 derived from the T6 domainfeature a dithioperoxoate and carboxamide, respectively. At firstsight, the termini of photoxenobactins appeared to form through aminolyticand thiolytic releases by nucleophilic attacks of free ammonia andsulfide, which seemed consistent with the hydrolytic release of 2. However, if this were the case, it would have been observedthat those with identical chain lengths (1 vs 3 and 4 and 2 vs 5 and 6) would be released from the same T domain. Therefore, off-loadingsof photoxenobactins might be achieved in a different manner.It is curious that although 4) in biosynthetic assembly lines is reminiscent of the biosynthesisof myxothiazol A. The off-loading of myxothiazol A is mediated bya peptidylglycine α-hydroxylating monooxygenase domain thatis encoded in the assembly line and cleaves the glycine residue toafford a terminal carboxamide.11 Althoughno monooxygenase domains are present in the pxb BGC,it is tempting to hypothesize that (1) the T5 domain isloaded with an l-cysteine by the PxbF A1 and/orPxbG A2 domain, (2) then the Cy3 domain condensesintermediate B–S–T4 with cysteinyl–S–T5 via amide bond formation to afford the T5-boundintermediate D1, in which the amide bond formation is catalyzed bya Cy domain that has been described in in vitro reconstruction ofthe yersiniabactin biosynthesis,12 (3) and finally, C-17methine (the α-carbon of cysteinyl) is hydroxylated (intermediateD2) to trigger spontaneous C–N bond cleavage release of 4 that maintained production of 1 but gave rise to lowertiters of 2–6 , suggestingthat the terminal nitrogen is derived from the α-amino groupof cysteine and thus supporting our proposed oxidative cleavage off-loading8 is responsible for introducing a methyl group at C-17 as observedin 2, 5, and 6, we proposethat the methylation and the putative hydroxylation have direct competitionat C-17 of intermediate D1–S–T5. The cMTinactivation mutant  resulted in loss of 2, 5, and 6 without concomitant formation of their non-methylatedproducts and led to production of 1, 3,and 4, with seven- and four-fold production increaseof 1 and 4, respectively  with an alanineresidue . Consistent with previous observation of mutagenesis in the heterocyclizationdomain on the identical aspartic acid residue,17 the PxbG Cy4 D2771A mutant compromised the productionwith a twofold decrease in 5  triggers a cleavage off-loading cannot be excluded(5 is an oxidative form of a cleavage product(intermediate F3) that is prone to form a disulfide bridge via spontaneousoxidation  the PxbG Cy domains catalyzing thioesterificationis the first step in heterocyclization as the energy barrier for conversionof thioester to amide bonds is much lower than that of amide to thioesterbonds24 and (2) due to the catalytic inefficiencyof the PxbG Cy3 and Cy4 domains in heterocyclizationrelative to condensation, the thioester intermediates C1, F1, andG1 might be kinetically stable without being heterocyclized . The finding of thiocarboxylic products might serve asa snapshot of the existence of thioester intermediates during thiazolineformation on the pxb assembly line. This also indicatesthat the formation of a thiazoline catalyzed by Cy domains could bea three-step reaction involving a thioester linkage, followed by anS- to N-acyl transfer and subsequent cyclodehydration.The formation of a thiazoline unitcatalyzed by Cy domains is considered to be a two-step reaction.cyclized 5. In parpxb BGC mediates the cleavage off-loading of photoxenobactins.While the releases of 1 and 2 can be ascribedto spontaneous cyclization and hydrolysis,7 respectively (pxbI that encodes a type II thioesterase(TE) homologous to YbtT25 and PxbG TE domainsfor their possible roles in off-loading 3–7. The deletion mutant of pxbI only led toa decrease in production titers of all compounds . Thus, we assumed that in addition to removingmisprimed intermediates and nonreactive acyl residues from T domains,26 PxbI might be involved in off-loading the Tdomain-bound residue of cleavage products, and PxbG TE appears tohave an equivalent function . Taking all these data into consideration, we proposed that candidateenzyme(s) for mediating photoxenobactin off-loadings might be α-and β-hydroxylases from primary metabolic pathways, which arenot encoded by the pxb BGC. Our work of seeking theenzyme(s) is still ongoing.Next, we attempted to investigate which enzyme(s) encoded by the ectively 2, we focompounds , consistfunction 5. HoweveX. szentirmaii PBADpxbF mutant with l--cysteine, we observed that 6 showed a mass shift of +12 Da, which is made up of full preservationsof three labeled cysteine residues  in the T6 inactivation mutant  have various degrees of increases on days 3and 4. It is conceivable that production of most compounds (1–3 and 6) in the culturemedium is in part derived from the post-assembly-line conversion of 5. Moreover, the amount of 2–6 gradually decreased from day 4 until all disappeared on day 8, while 1 steadily increased, being the only pxb productsurviving during the whole course. To validate the hypothesis that 5 undergoes spontaneous transformations, we incubated 5 in various solvents and temperatures and monitored the conversionby HPLC–MS , which is predicted to be a lactamizedproduct at 40 °C and does not exist in culture media, concomitantwith the appearance of 6 , while 1 is the major thermodynamicallystable product resulting from prolonged cultivation.Interestingly, upon feedingthe residues 3b, suggewn above , we enviF mutant 6a. In co HPLC–MS 6b. We ob5) witha dithioperoxoate terminus that is the final biosynthetic productis prone to conversion into the other pxb products(1–4 and 6). Such post-assembly-linespontaneous conversions (S7) contribute to majortiters of 1–4 and 6 inthe culture medium. Indeed, homologous pxb BGCs arenot only prevalent across Xenorhabdus and Photorhabdus but also widespreadin other γ-Proteobacteria, such as Psychromonas, Salinivibrio, Shewanella, and Vibrio , which offers opportunities to study the BGC evolutionand underlying structural diversity that might result from the catalyticdiscrepancy of Cy domain(s) and enzymes mediating bond cleavage off-loadings.Again, the instability of most pxb products mightexplain why these compounds had not been described from other strainsbefore. Our discoveries thus expand the functional diversity of naturalproduct biosynthetic assembly lines and enhance our understandingof nature’s potential capability to achieve chemical innovation.In summary, we unravel a previously unknown PKS/NRPSbiosyntheticlogic that utilizes heterocyclization domains with distinct efficienciesto extend a T domain-tethered acyl/peptidyl with a cysteinyl. Thisleads to the formation of thiazoline which is a mature heterocyclicproduct, demonstrating a fully functional Cy domain and the formationof amide or thioester, which is a nascent condensation product onlyaccounting for a partly or inadequately functional Cy domain. Theresulting T domain-bound acyl/peptidyl on PxbG is off-loaded by intramolecularcyclization, hydrolysis, and putative C–N/C–S bond cleavage,which gives rise to products with different chain lengths featuringcarboxylic acid, carboxamide, thiocarboxylic acid, and dithioperoxoatetermini. It turned out that photoxenobactin C (versions 2 and S7


Stromal tumour of uncertain malignant potential (STUMP) is exceedingly rare. Diagnosis and management of STUMP present a challenge to the urologist due to the absence of specific clinical findings and its unpredictable clinical course. Thus, radical resection is often recommended. Here, we present a case of a 64-year-old male, who presented with mild obstructive voiding symptoms with a raised age-specific prostate-specific antigen (PSA) of 3.1. Magnetic resonance imaging (MRI) showed an area of suspicion, in an area thought to be the left seminal vesicle, containing a malignant lesion within it. Biopsy of this area and the prostate confirmed concurrent prostatic STUMP and Gleason 3+3=6 adenocarcinoma of the prostate, managed with robotic-assisted laparoscopic radical prostatectomy with wide local excision. Prostatic stromal tumour of uncertain malignant potential (STUMP) arises from mesenchymal tissue and is a rare entity, accounting for <1% of prostate cancer -5. SurgiHere, we present a 64-year-old patient who presented with urinary frequency and had raised age-specific prostate-specific antigen (PSA) of 3.1. This case is unique as only a few cases of concurrent prostatic adenocarcinoma and STUMP have been described in the literature, and decision-making surrounding its treatment remains poorly understood.A 64-year-old male initially presented to his general practitioner (GP) with increased urinary frequency. There was no history of urinary tract infection, haematuria, or haematospermia. He had a history of erectile dysfunction but was otherwise fit and well and did not take any regular medications. There was a history of prostate cancer with his father and breast cancer with his sister. He was a non-smoker, drank occasional alcohol, and worked in the IT sector. He had an enlarged prostate with a craggy surface on digital rectal examination. He had a raised age-specific PSA of 3.1ng/ml, which triggered a two-week wait referral from the GP to the Urologist. A subsequent MRI pelvis prostate identified no target lesion within a 34cc prostate (PSA density 0.1). Interestingly, it identified what was assumed to be a 9.6cm dilated haemorrhagic left seminal vesicle (SV) containing haemorrhage of deferring ages, with a hypercellular lesion measuring 13.8x16.0mm within  discussed this patient’s case and recommended a radical robotic-assisted laparoscopic prostatectomy (RALP) with wide local excision. The procedure was successfully performed with the da Vinci® Xi robotic platform  with a total operative time of 180 minutes, encompassing 160 minutes of console time. Intra-operatively, the SVs were found to be large and tethered but removed en bloc with the prostate. He had a successful trial without a catheter on postoperative day 12.The final histopathological report showed a pT2a R0 Gleason 3+3=6 microacinar adenocarcinoma of the prostate on the left side with negative resection margins and normal SVs  classification includes two diagnostic categories for mesenchymal tumours unique to the prostate: stromal sarcoma and STUMP . The terImmunohistochemical studies on patients with STUMP and PSS showed positivity for CD34 and progesterone receptors but no activity for oestrogen receptors. Desmin, smooth muscle actin, and HHF-35 were positive in 80%, 40%, and 50% of patients with STUMP, respectively, but this was absent in PSS patients. The expression of muscle markers in STUMP, therefore, provides a function for differentiating them from PSS. Furthermore, the Ki67 index is typically low in STUMP but high in PSS. In our case, the absence of mitosis and necrosis in the cellular stroma, low Ki67 index (<10%), and strong positivity for progesterone and desmin favour STUMP.There are no guidelines for the treatment of STUMP due to its rarity. Management of STUMP is challenging due to its diverse clinical behaviour, with the potential of progressing to stromal sarcoma and metastasis. Thus, radical resection is often recommended. Active surveillance has also been described in the literature. A case series on 14 STUMP patients who had active surveillance post biopsy or transurethral resection reported no disease progression after an average follow-up of 4.9 years . UltimatSTUMP is a rare entity, with only a few cases of concurrent STUMP and prostatic adenocarcinoma described in the literature. Diagnosis of STUMP is difficult due to the absence of specific clinical and laboratory findings. Management of STUMP presents challenge to the urologist due to its diverse and unpredictable clinical course, from being indolent to potential of progressing to stromal sarcoma and distant metastasis. There are no universal guidance on the management of STUMP due to its rarity but we recommend radical resection in the form of RALP in cases of concurrent STUMP and prostatic adenocarcinoma, with regular follow-up in the form of serial PSAs and MRI pelvis-prostate imaging.


In addition, there was a decline in the concentrations of vitamins B2 and B5. Although dietary fibers and starch are the major carbohydrates, total dietary fiber exhibited a declining trend in coarse bran, and starch exhibited a rising trend in break and reduction flour. There was a decrease in the molar ratio of phytates: zinc did not promote a significant improvement in zinc bioaccessibility. These results can be useful for generating wheat varieties rich in micronutrients as well as having better nutritional traits.Biofortification is one of the most successful approaches to enhance the level of micronutrients in wheat. In the present study, wheats with zinc biofortification (foliar fertilization and breeding strategies) were milled into five components  and their mineral content and nutritional components were evaluated. The results revealed that biofortification greatly increased the Zn concentration (by 30.58%–30.86%) and soluble Zn content (by 28.57%–42.86%) of whole flour after digestion. This improvement is mainly in break flour, reduction flour, and fine bran. Meanwhile, the contents of macronutrients including ash, lipids, and proteins and micronutrients containing iron, calcium, and vitamins (B Zinc is a component of many proteins and enzymes, playing an important role in human immunity, catalysis, and biochemical . Zinc deWheat is the most widely planted crop in the world and is consumed by humans as a staple food. It is one of the most common carriers of biofortification and is considered a feasible solution to widespread human Zn deficiency in the developing world . HoweverRecently, more attention has been directed toward improving flour extraction or recycling the wheat bran produced by milling in terms of their nutritionally valuable minerals, hormonally active compounds, vitamins, and several antioxidant compounds . Improving the proportion of bran in wheat flour and its derivative products is an effective method for increasing the content of nutrients . MeanwhiAgainst this background, in this study, zinc-fortified wheat with fertilization or breeding was selected to Bühler Laboratory Experimental Mill, and different milling fractions were obtained. The distribution of nutritional components in Zn-biofortified wheat and the effect of biofortification on Zn bioaccessibility were investigated. The resulting knowledge can be used to optimize Zn-biofortified wheat grain processing methodologies and provide more nutritional information to alleviate Zn deficiency by combining agricultural production with food processing . A field experiment was conducted in 2020–2021 at the Quzhou Experiment Station  in China. Another wheat selected from breeding program was a high-zinc variety , which was donated by the College of Agronomy and Biotechnology, China Agricultural University. No agronomic biofortified wheat  was used as the control. All grains were stored at −20 °C before use. All chemicals, solvents, and reagents were of analytical grade and purchased from Sigma–Aldrich , unless otherwise stated.The agronomic biofortified wheat variety  was donated by the College of Resources and Environment Science, China Agricultural University, which was performed by foliar fertilization with a solution containing 0.4% ZnSOThe wheat samples were hand-cleaned and tempered to 14.0% moisture content. Then, the samples were milled using the Bühler Laboratory Experimental Mill (MLU-202) to obtain break streams flour, reduction streams flour, and two types of bran (coarse and fine) . Whole fProtein content was calculated using Kjeldahl’s method according to AACC 46-10 (Nx6.25). Lipids content was calculated using Soxhlet’s extraction according to AACC 30-10. Ash content was estimated according to AACC 08-01, in which a 0.5 g sample was kept in a Muffle furnace at 550 °C for 12–18 h after carbonization. Total dietary fiber (TDF), including soluble dietary fiber (SDF) and insoluble dietary fiber (IDF), was determined using the enzyme–gravimetric method according to AACC 32-07. Starch content was determined [The calorific value was calculated using the method described in AOAC (2015). The values obtained for proteins, lipids, and carbohydrates were used to calculate the calorific value of the sample as follows: Mineral element contents were determined using an inductively coupled plasma–optical emission spectrophotometer . The samples were pretreated with wet digestion before instrument measurement . Brieflyv/v)) was added to the sample, and the sample was shaken for 60 min and centrifuged at 2500× g to extract the polyphenols. The residue was extracted using 20 mL of 70% (v/v) acetone one more time, and the supernatants were combined. Folin–Ciocalteu reagent (5 mL) and 8 mL of 15% sodium carbonate were added to the mixed solution, respectively. After incubation for 120 min, the absorbance was measured at 765 nm. The total phenolic content in cereals was calculated according to the corresponding concentration of the standard curve.First, phenol from grain was extracted, and then the content of each sample was measured using a Folin–Ciocalteu reagent . Acidic g, 0.5 mL of supernatant was removed, to which 1 mL 0.02% FeNH4(SO4)2 was added, and the solution was kept in a boiling water bath for 30 min. Then, 2 mL of 1% C10H8N2–C2H4O2S was added. After mixing, the absorbance was measured at 519 nm. The molar ratios of Phy:Fe and Phy:Zn were determined by dividing the moles of phytic acid by the moles of minerals in a 100 g sample.Phytic acid content was measured by using the colorimetric procedure . In tota1 (thiamine), B2 (riboflavin), B5 (pantothenic acid), B6 (pyridoxine), and B9 (folic acid) were quantified. Briefly, a 5 g sample was weighed and 60 mL of 0.1 mol/L HCl was added. This solution was then incubated at 121 °C for 30 min and cooled to below 40 °C. The pH was adjusted to ~4.0 with 2.0 mol/L CH3COONa, and 2.0 mL of enzyme solution was mixed in and the resulting solution was placed in an incubator at 37 °C overnight (for ~16 h). The solution was diluted in distilled water to 100 mL, and the supernatant was taken after centrifugation. The derivatized solution was filtered through a 0.45 μm Millipore filter. A reversed-phase high-performance liquid chromatography system with a C18 column  was used. The mobile phase of CH3COONa–CH3OH (65 + 35) had a flow rate of 0.8 mL/min. The injection volume was 20 μL and the excitation and emission wavelengths were 375 and 435 nm, respectively [Vitamins Bectively .n = 3). Bioaccessibility (BAC) was calculated according to the following formula:Static in vitro digestion was performed according to INFOGEST 2.0 . The actp = 0.05. All experiments were performed in three independent trials, and the results were expressed as the mean ± standard deviation.SPSS 22.0 was used for the statistical analyses. Tukey’s test was used to determine significant differences. All data were analysis of variance data determined by the least significant differences test at p < 0.05). The whole flour ash content of LZ2 (1.52 g/100 g) was higher than that of LT2 (1.30 g/100 g), but had no difference from that of JM26 (1.53 g/100 g). Ash corresponds to mineral content, and this indicates that the mineral content of wheat was improved after application of zinc fertilizer. This is in line with the conclusion that fertilization increased ash content compared with the control [p < 0.05). Previous studies reported a 25.8% increase in grain lipids content after foliar fertilization [The basic nutritional components of milling fractions of LT2, LZ2, and JM26 are presented in  control . Compare control . The liplization . The incHigh protein has a significant effect on improving the quality of wheat products, promoting excellent viscoelasticity, extensibility, and water absorption. After zinc fertilization, the protein content of LZ2 (12.75 g/100 g) was higher than that of LT2 (10.00 g/100 g) and similar to that of JM26 (12.02 g/100 g). This is consisted with the previous conclusion that suggested a significant positive correlation between zinc and protein content in wheat grains . The incp > 0.05), and the carbohydrate content of coarse bran (61.88–64.03 g/100 g) was the lowest. The variation in carbohydrates was interesting; namely, there were similar amounts of carbohydrates in different wheat types of the same milling fractions, whereas dietary fibers and starch, which are the major carbohydrates, exhibited differences in wheat grains. As presented in p > 0.05), while JM26 had a higher TDF content in fine bran (17.73 g/100 g) than that in LT2 fine bran (16.62 g/100 g) and LZ2 fine bran (15.95 g/100 g), while that of coarse bran was lower. A significant difference in SDF of whole flour was observed (p < 0.05): SDF content of JM26 (1.69 g/100 g) was the highest, followed by LZ2 (1.20 g/100 g) and LT2 (0.94 g/100 g). The fermentation of SDF in the colon contributes to colonic microbiota producing short-chain fatty acids and an acidic environment, which promotes epithelial cell proliferation, thereby increasing the absorptive surface area for utilization of minerals [TDF (including SDF and IDF), which binds with minerals to form complexes by electrostatic force or chelation, can be considered as a mineral antinutrient . The TDFminerals . The conp < 0.05). After application of zinc fertilizer, the whole flour starch content of LZ2 (66.20 g/100 g) was higher than that of LT2 (63.40 g/100 g). Studies have reported that application of Zn fertilizer markedly enhanced the amount of starch in the wheat [p > 0.05). As shown in he wheat . This ma1, VB2, VB5, VB6, and VB9. These have many positive effects on human metabolism, including fat, protein, and carbohydrate metabolism, thus benefiting human health. As shown in 1 content of whole flour ranged from 1.39 to 1.93 μg/g in the three wheat varieties. The highest amount of VB1 was found in fine bran (2.65–3.89 μg/g). Among the three wheat samples, JM26 had the highest VB1 content in whole flour and each milling fraction. Compared with LT2, LZ2 had a higher VB1 content in whole flour (1.56 μg/g) and fine bran (3.89 μg/g), representing increases of 12.2% and 6.3%, respectively. Except for VB1, the highest contents of VB2, VB5, VB6, and VB9 were detected in coarse bran, followed by fine bran and whole flour. VB5 was found to be the most abundant B vitamin in different milling fractions of selected wheat samples, with values varying from 18.17 to 38.21 μg/g for coarse bran, from 2.62 to 4.96 μg/g for whole flour, and from 0.98 to 3.29 μg/g for B flour and R flour. It was reported that the VB5 content was significantly higher in coarse bran (32.28 μg/g) than in the other fractions (3.61–20.08 μg/g) (p < 0.05) [5 in LZ2 (2.62 μg/g), mainly in R flour (0.98 μg/g) and coarse bran (18.17 μg/g), with decreases of 54.0% and 22.9%, respectively. Regarding the VB2 and VB6 contents, coarse bran had the highest amount , followed by whole flour , while R flour had the lowest amount  of the three wheat varieties. The application of zinc fertilizer reduced the content of VB2 in whole flour by 27.9%. Specifically, the contents in fine bran and coarse bran decreased by 38.3% and 7.1%, respectively. However, zinc fertilizer increased the content of VB6 in B flour and R flour, and the growth was found to be 159.5% and 83.3%, respectively. The high-zinc wheat variety (JM26) had lower VB2 and higher VB6 contents, when compared with those of LT2. The VB9 content accounted for the lowest proportion among these several B vitamins. Previous studies have also reported lower VB9 contents of wheat flour and grains  [9 were also the highest in coarse bran, with values of 112.20–225.80 μg/100 g. After biofortification, there were significant differences among LT2 (68.44 μg/100 g), LZ2 (89.75 μg/100 g), and JM26 (115.10 μg/100 g) (p < 0.05). The contents of VB9 in LZ2 and JM26 increased by 31.1% and 68.2% compared with those in LT2. The contents of VB9 in B flour and R flour increased from 20.75 and 24.11 μg/100 g to 48.57 and 42.35 μg/100 g, respectively, after zinc fertilization.Different wheat samples contain several B vitamins, such as VB < 0.05) . Comparep < 0.05). When Zn fertilizer was applied, the Zn concentration of LZ2 (37.70 mg/kg) was increased by 30.86%. Similarly, the zinc concentration of JM26 also reached 37.62 mg/kg. A positive response to biofortification has also been already reported for many other wheat varieties [42O (w/v) increased grain Zn concentration by 28%−68% depending on the variety [In terms of the distribution of zinc, the highest amount was registered for coarse bran 96.07–130.95 mg/kg), followed by fine bran (52.72–74.64 mg/kg) and whole flour (28.81–37.70 mg/kg). The other two milling fractions exhibited a very similar quantity of Zn, varying from 8.58 to 11.57 mg/kg for B flour and from 7.60 to 10.92 mg/kg for R flour. Similar observations were also reported [arieties . Foliar  variety . The tra.95 mg/kgp < 0.05). As was previously reported, the application of zinc fertilizer increased the calcium content of wheat grain [p < 0.05). Fe contents in LZ2 and JM26 increased by 35.8% and 11.8%, respectively, in B flour and by 24.2% and 18.4%, respectively, in coarse bran. This is consistent with the results of previous studies that demonstrated that Fe and Zn contents were highly positively correlated [The major quantity of Ca was found in coarse bran (982.17–1085.24 mg/kg). B flour exhibited higher Ca contents (240.83–250.87 mg/kg) compared to that in R flour, which was the milling fraction with the least amount of Ca 208.33–234.90 mg/kg). Of the three wheat samples, the higher amount was found for JM26 and LZ2 of whole flour . Compared with LT2 (343.52 mg/kg), the Ca content increased by ~6%. With zinc fertilization, LZ2 had Ca contents of 234.90 and 1085.24 mg/kg in R flour and coarse bran, respectively, which are significantly higher than those of LT2  (at grain . For theat grain . After z.90 mg/kgp > 0.05), and that in JM26 was found to be lower (12.47 mg/g) than that in LT2 and LZ2, which directly results from the genotype and plant growth conditions. Moreover, no significant difference was found in the same milling fractions among the three wheat samples (p > 0.05). It was also reported that the total amount of polyphenols in whole flour did not change significantly, nor did the antioxidant activity, after foliar Fe and Zn biofortification [As presented in fication . In difffication . Other rfication . p > 0.05). This needs to be considered in promoting whole-grain foods, which are rich in nutrients as well as antinutritional factors. As expected, the values of Phy:Zn were obviously influenced by Zn biofortification. We observed that LZ2 and JM26 had lower molar ratios in comparison to LT2 in both whole flour and each milling fraction. The molar ratio in B flour and R flour was <15, while for the control sample, it was >15, and the decline in other milling fractions reached 16.5%–37.8%. Zhao et al. also reported that foliar Zn application can decrease the value of Phy:Zn by 36% and improve the bioavailability of Zn in wheat flour and whole grain [The molar ratios of phytates and mineral are a common indicator for assessing the potential bioavailability of mineral elements. The higher the molar ratio is, the lower is bioavailability of the mineral. Molar ratios of Phy:Zn < 15 and Phy:Fe < 1 significantly improve Zn and Fe absorption, whereas molar ratios of Phy:Ca < 0.17 have been associated with Ca bioavailability. The obtained results indicated that coarse bran had higher values of Phy:Zn (35.26–49.08) and Phy:Fe (25.25–36.36) molar ratios, as well as Phy:Ca (2.39–3.04). Although coarse bran was found to be a good source of some minerals, such as Zn, Fe, and Ca, the significant content of phytates limits their bioavailability. B flour and R flour had the lowest values of Zn, Fe, and Ca . Zhang et al. found that the value of Phy:Zn in bran was higher than that in other fractions . It is nle grain . It may p > 0.05). The phytase activity in B flour (0.27–0.29 U/g) and R flour (0.19–0.25 U/g) was very similar, both being significantly lower than that in whole flour (0.41–0.48 U/g), coarse bran (0.80–0.87 U/g), and fine bran (0.54–0.59 U/g) (p < 0.05). Our results are consistent with previous studies that indicated that wheat grain phytase activity decreased along with pearling extension from the outer to inner layer [During food processing, phytates content can be degraded when phytases are activated at optimal technological conditions. Therefore, it is necessary to understand the phytase activity in wheat flour. In our study, no difference was observed among the three wheat samples . The soluble Zn in whole flour (0.28–0.40 mg/kg) exhibited lower values and the coarse bran had the lowest values . Bioaccessibility also exhibited the same pattern as the content of soluble zinc. B flour and R flour had the highest bioaccessibility (30.52%–47.62%), followed by whole flour (7.65%–8.49%), with the lowest being coarse bran (0.92%–1.40%). This is in line with previous results for whole flour ranging from 2.5% to 8.9% [According to the above analysis, Zn content in both whole flour and various milling fractions was increased by biofortification, but whether the increased Zn content can be absorbed and utilized by the human body remains an important problem. A widely used in vitro digestion model was employed to assess mineral bioaccessibility. The results related to the contents of soluble fractions and the bioaccessibility for Zn are shown in r 0.28–0. mg/kg ex to 8.9% . It was  to 8.9% . These rp > 0.05), being 7.78% and 34.50%, 8.49% and 32.59%, and 7.65% and 35.96% for LT2, LZ2, and JM26, respectively. Only in JM26 was the bioaccessibility of R flour higher, reaching 47.62%, and that of whole flour of LZ2 had a slightly higher value (8.49%). The zinc content was increased by biofortification, reducing the molar ratio of Phy:Zn and achieving higher amounts of soluble zinc after digestion. However, during the digestion process, the transport of Zn from ingestion to intestine reduces Zn content and changes its chemical form, which may not be conducive to zinc absorption. In addition, wheat flour still contains high contents of other antinutritional factors, such as fibers and phytates, and these can inhibit the release of minerals from wheat milling fractions [After zinc biofortification, regardless of fertilization or breeding methods, soluble Zn content was increased by 18.92%–21.43% in B flour, 34.48%–124.14% in R flour, and 28.57%–42.86% in whole flour, respectively. This is consistent with our previous results for the molar ratio of Phy:Zn, which exhibited lower values with treatment of biofortification in comparison to those of LT2, being conducive to zinc absorption. This demonstrates that such measures of biofortification make sense, as obtained zinc can be transported into the human gut and be ready to be absorbed after digestion. No statistical difference was observed in the Zn bioaccessibility of whole flour and B flour in the three wheat samples , but also increased contents of soluble Zn (increase by 28.57%–42.86%) after digestion, while there was no significant effect on the amount of bioaccessible zinc. Differences in macronutrients  and micronutrients  were observed after biofortification. Meanwhile, the variable responses of nutritional components in milling fractions should be taken into consideration for introducing biofortification strategies. Biofortification has led to a substantial increase in the zinc content of B flour (25.9%–34.8%), R flour (27.0%–43.7%), and fine bran (37.0%–41.6%), and the difference in dietary fiber was reflected in coarse bran and fine bran. B vitamins exhibited various changes in each milling fraction, whereas biofortification had little effect on the amounts of polyphenols, phytic acid, and carbohydrates. Combining biofortification and food processing technology is a crucial step to improving human micronutrient absorption from the perspective of the food supply chain.


Molecular simulations showed that 120 hypoCOFs that we identifiedto be promising based on these structure–performance relationsexceed the upper bound for He/CH4, He/N2, H2/CH4, and H2/N2 separations.Both real and hypothetical COFs were then studied as fillers in 25different polymers, leading to a total of 29 020 COF/polymerand hypoCOF/polymer mixed matrix membranes (MMMs), representing thelargest number of COF-based MMMs investigated to date. Permeabilitiesand selectivities of COF/polymer MMMs were computed for six differentgas separations, and results revealed that 18 of the 25 polymers canbe carried above the upper bound when COFs were used as fillers. Thecomprehensive analysis of COFs provided in this work will fully unlockthe potential of COF membranes and COF/polymer MMMs for helium separationand hydrogen purification.Hundreds of covalentorganic frameworks (COFs) have been synthesized,and thousands of them have been computationally designed. However,it is impractical to experimentally test each material as a membranefor gas separations. In this work, we focused on the membrane-basedgas separation performances of experimentally synthesized COFs andhypothetical COFs (hypoCOFs). Gas permeabilities of COFs were computedby combining the results of grand canonical Monte Carlo (GCMC) andmolecular dynamics (MD) simulations, and many COF membranes were foundto overcome the upper bound of polymeric membranes for He/H Polymershave been the most widely studied membranes for various gas separations;however, they suffer from the trade-off between selectivity and permeabilityas defined by Robeson,3 which limits their performancein commercial processes. Due to this trade-off, mixed matrix membranes(MMMs) have been developed to combine the good processabilities ofthe polymers and outstanding gas separation performances of the porousfiller materials such as zeolites,6 metal–organicframeworks (MOFs),9 and covalent organic frameworks (COFs).14Membrane-based gas separation, which utilizes the difference insolubility and diffusivity of different gases under a pressure gradient,is considered as an energy-efficient method since it does not requirea phase change and can be achieved under relatively mild conditions.18 For example, gas permeabilitiesof 13 MOFs computed using molecular simulations were utilized to predictH2/N2 separation performances of 78 MOF-basedMMMs. Several MMMs were shown to exceed the upper bound due to almost2 times higher H2 permeabilities than those of the purepolymers.17 H2/CH4 separation performances of 119 MOF-based MMMs consisting of 17 differentMOFs and 7 polymers were investigated using molecular simulations,18 and it was shown that MOF-based MMMs could increaseboth the H2 permeability and H2/CH4 selectivity of polymers.Due to the very large number and variety of MOFs, the numberofcomputational screening studies on MOF membranes escalated quickly,while several studies focused on the identification of the most promisingMOFs to be used in MMMs for gas separations.19 COFs exhibit good compatibility with the polymermatrix thanks to their organic parts. Therefore, in addition to thecommon advantages of MOFs such as structural tunability and high accessiblesurface area, the affinity and miscibility of COFs to polymers providean additional benefit to using them as fillers in MMMs.20 COF-based MMMs have recently been investigatedexperimentally for the separation of several gas pairs.29 Addition of a COF filler, NUS-2, into the polybenzimidazole (PBI)membrane significantly increased the H2/CO2 selectivityof PBI from 9.5 to 31.4 at 5 bar and 308 K and carried the polymerover the upper bound.10 Incorporation ofa Schiff base network-type COF (SNW-I) into a polyimide (PI) membraneincreased the CO2 permeability of the membrane from 6 to12.4 Barrer and the CO2/CH4 selectivity from9 to 13.4 at 4 bar and 298 K.30 COF nanosheetclusters (TpPa-1-nc) increased the selectivity of polyether-block-amide(PEBA) membranes from ∼42 to 72 and the CO2 permeabilityfrom ∼4.5 to 7.5 Barrer at 3 bar and 298 K for the separationof an equimolar CO2/N2 mixture.14 A 2D-COF-based MMM synthesized using cross-linked poly(ethyleneoxide) (XLPEO) exceeded Robeson’s upper bound with its highCO2 permeability (804 Barrer), and CO2/N2, CO2/CH4, and CO2/H2 selectivities of 61, 20, and 15, respectively, at 5 bar and308 K, which were higher than the CO2 permeability (432Barrer) and CO2 selectivities of the pure XLPEO membrane.13COFs, porous crystalline materialsconsisting of covalently bondedlight elements including boron, silicon, carbon, nitrogen, and oxygen,have been recently discovered and shown to have a high potential forgas separations due to their chemically diverse and robust structures.37 For example, Yang et al.31 demonstratedthat COF-102, -103, -105, and -108 have higher selectivities  compared to IRMOF-1 and Cu-BTC formembrane-based H2/CH4 separation at 10 bar and298 K. 295 COF membranes were investigated for CO2/N2 separation at 1 bar and 10 bar, at 298 K, and results showedthat CO2 permeabilities of COF membranes overcome thoseof MOF membranes, while MOF membranes have higher CO2/N2 selectivities than COF membranes.34 A computational investigation on the H2/CO2 separation performances of 288 COF membranes at 10 bar and 298 Khas shown that COF membranes significantly outperform polymeric membranesdue to their H2 permeabilities ranging from 599 to 1.5× 106 Barrer and H2/CO2 selectivitiesof up to 4.74.35 H2/CO2 separation performances of 794 hypoCOF membranes were shown to beeven superior, with H2 permeabilities in the range of 9× 105–5 × 106 Barrer and H2/CO2 selectivities between 2.66 and 6.14.36 A total of 572 COFs were studied for membrane-basedH2/CH4 separation at 1 bar and 298 K, and severalCOFs with large porosities showed a good combination of H2 permeability (>105 Barrer) and H2/CH4 selectivities up to 4.6.37 Incontrastto COF membranes, a very limited number of studies, only two, focusedon computational modeling of COF/polymer MMMs. Twenty-nine COFs werecomputationally evaluated as fillers in three polymers for CO2/CH4 separation, and several MMMs were found tobe above the upper bound due to the increase in their CO2 permeability up to ∼1900 Barrer and CO2/CH4 selectivity up to ∼45.38 Gulbalkan et al.39 investigated the N2/CH4 separation performances of 25 different COF/polymerMMMs and showed that MMMs with improved N2 permeabilities(∼4.5 to 104 Barrer) could be obtained upon theincorporation of COFs into polymers.Theinsight gained from the computational screening of COFs formembrane applications could be of substantial importance for decidingon the next COF filler to be used in experimental MMM studies. Recentcomputational studies predicted gas separation properties of COF membranes.40 in addition to the existenceof hundreds of polymers, experimental investigation of every possibleCOF/polymer MMM is very challenging. The number of experimental andcomputational studies on evaluating gas separation performances ofCOF-based MMMs is very limited, as demonstrated above, and these MMMshave been mostly tested for CO2 separation. In additionto these, we still do not know the potential of the hypothetical,computer-generated COFs (hypoCOFs)43 as fillers in MMMs althoughthey may have similar or even better gas separation performances thanthe experimentally available COFs. Motivated by these, we aimed toreveal the potential of all synthesized and computer-generated COFsas fillers in MMMs for a variety of gas separations. We first focusedon the most recent COF database consisting of 648 experimentally synthesizedstructures. He, H2, CH4, and N2 permeabilitiesof these COFs were predicted under realistic conditions  by performing grand canonical Monte Carlo (GCMC) and moleculardynamics (MD) simulations. Performances of 589 COF membranes and 24 100COF/polymer MMMs composed of 25 different polymers were examined forsix different gas separations, He/H2, He/CH4, He/N2, H2/CH4, H2/N2, and N2/CH4, which represent both thelargest number of COF-based membranes and the largest variety of gasseparations studied with COFs to date. Structural properties of theexperimentally synthesized COF membranes, which were identified tobe above Robeson’s upper bound, were determined, and the obtainedresults were used to select 120 representative hypoCOFs from a verylarge material database. Membrane-based gas separation performancesof these hypoCOFs were predicted by conducting GCMC and MD simulationsand compared with polymer, COF, and MOF membranes. Finally, gas permeabilitiesand selectivities of 4920 hypoCOF-incorporated MMMs were computedfor all six gas separations. Overall, we revealed the gas permeabilityand selectivity of a total of 709 COF and hypoCOF membranes and 29 020COF/polymer and hypoCOF/polymer MMMs for six gas separations. Theresults presented in this work will significantly accelerate the designof COF-based MMMs by directing the experimental efforts and sourcesto the development of the most promising COF materials.Since the number of experimentallysynthesized COFs has alreadyexceeded 600,22, He/N2, He/CH4, H2/N2, H2/CH4, and N2/CH4 separations. Weworked with 648 Clean, Uniform, and Refined with Automatic Trackingfrom Experimental Database (CURATED) COF structures.40 All of the structural properties of COFs that we used inthis work, the largest cavity diameter (LCD), the pore limiting diameter(PLD), the accessible surface area (Sacc), density (ρ), and porosity (ϕ), were computed usingZeo++ software.44 The widely applied experimentalmethod for measuring the surface area of porous materials is the Brunauer–Emmett–Teller(BET), which is based on nitrogen adsorption in porous materials.45 Therefore, we utilized a probe radius of 1.86Å (representing the radius of the nitrogen molecule) to predictthe accessible surface area of materials investigated in this work,and the number of COFs was refined as 589 to only study the COFs with Sacc > 0 m2/g as membranes.A high-throughput computational screeningapproach, as shown in 2, N2, and CH4 uptakesin COFs (NiCOF) at 1 bar and 298 K. Molecular dynamics(MD) simulations were performed to calculate the self-diffusivityof the gas i in the COF (DiCOF) at the loadings obtained from GCMCsimulations. RASPA software (version 2.0.35) was used for all molecularsimulations in this work.46 He,47 H2,48 andCH447 were modeled as nonpolarand single-site spheres, while N249 was represented with a three-site model consisting of two N atomsand a dummy atom as the center-of-mass. He and CH4 weredescribed with TraPPE models.50 The parameters forH2 were acquired from the Buch potential,48 and the parameters from the work of Makrodimitris et al.51 were used for N2. Intermolecular interactions betweenhost–gas and gas–gas were defined using the Lennard–Jones12-6 (LJ) potential. The interaction parameters for COF atoms wereobtained from the universal force field (UFF).52 The cutoff distance was set to 14 Å to truncate theLJ interactions, and Lorentz–Berthelot mixing rules were applied.The Coulomb potential was used to compute the electrostatic interactionsbetween N2 and COF atoms. Long-range electrostatic interactionswere considered using the Ewald summation.53 Density-derived electrostatic and chemical (DDEC) charges were alreadyassigned to the CURATED COFs in the database,40 and the charge equilibration method (Qeq)54 was used to assign the partial charges to hypothetical COFs thatwe studied. In GCMC simulations, 10 000 cycles were performedfor initialization, and 20 000 cycles were used for takingthe ensemble averages for gas uptakes.First, we performed grand canonical Monte Carlo (GCMC) simulationsto compute He, HDiCOF valuesat 298 K using the Nosé–Hoover thermostat.55 Self-diffusion coefficients of gases in COFswere calculated using the slope of the mean square displacements accordingto Einstein’s relation.56 Aftercarefully examining the results of MD simulations, we considered onlythe COFs with DiCOF > 10–8 cm2/sfor the accurate characterization of molecular diffusion and endedup with 588 COFs for He and H2 and 589 COFs for N2 and CH4. The gas uptakes and self-diffusivities wereused to calculate the single-component gas permeability of COFs (PiMMM) as shown with (a) in ci, Di, and fi are the uptake,self-diffusivity of gas i, and feed side pressure of the membrane,respectively, and the permeate was assumed to be at vacuum.57 Using the ratio of gas permeabilities, idealmembrane selectivities of COFs (Si/jCOF) were calculated using (b)in MD simulations were carriedout in the canonical ensemble (NVT)up to 5 ns with a time step of 1 fs to compute the 2, He/N2, He/CH4, H2/N2, H2/CH4, and N2/CH4 separations.A total of 25 polymers establishing Robeson’s upper bound foreach gas separation were chosen to generate COF/polymer MMMs. Thesepolymers are enlisted in Tables S1–S6 together with their experimentally reported gas permeabilities thatwe collected from the literature. Various theoretical permeation modelswere used in the literature59 to predict gas permeabilitiesof MMMs. The Maxwell model, the most widely used permeation modelto predict the gas separation performances of MMMs, especially atlow filler loadings (volume fraction ≤ 0.2), was used in thiswork, as shown in expression (c) in PiP represents thegas permeability of the neat polymer taken from the literature, PiCOF is the gas permeability that we calculated using molecular simulations,and PiMMM is the gas permeability of COF/polymer MMM. We used thefiller loading (φ) as 0.2 to be in the range of applicabilityof the Maxwell model.60 Selectivities ofMMMs (Si/jMMM) were calculated via expression (d) in We aimed to investigate the performances of COF-basedMMMs consistingof different types of polymers and COF fillers for He/H61 Sinceperforming molecular simulations, especially MD, for this very largematerial space is computationally challenging, we introduced an efficientapproach for the targeted selection of the most promising hypoCOFmembrane candidates. First, we focused on the physical features  of the promisingCURATED COFs, which were shown to be above Robeson’s upperbound because of their high gas permeabilities (>4 × 105 Barrer) based on the results of our molecular simulations.We definedand considered the ranges of structural properties of the promisingCOF membranes, 20 Å < LCD < 57 Å, 17 Å < PLD< 57 Å, 0.77 < ϕ < 0.95, 2000 < Sacc< 10 000 m2/g, and ρ >0.1g/cm3, to narrow down the set of hypoCOFs. We then dividedthe set of hypoCOFs into two parts: (i) 2 dimensional (2D) hypoCOFsand (ii) 3D-hypoCOFs. Sacc and ϕare the physical features having the most significant effect on thegas permeabilities of COFs, as we will discuss later, and by consideringthem, we created four mini-hypoCOF sets composed of 2D and 3D structures:hypoCOFs2D-Sacc, hypoCOFs3D-Sacc, hypoCOFs2D-ϕ, and hypoCOFs3D-ϕ. In each set, 30 hypoCOFs exist, based on the highest, mean, andthe lowest Sacc and ϕ.In the lastpart of this work, we aimed to examine the gas separationperformances of hypoCOFs selected from a very large data set of 69 840computationally designed structures.33.12, N2, and CH4 gases inCOFs and the gas permeabilities of COF membranes are shown in 2 have the lowestuptakes in COFs as a result of their low interaction energies with the COFatoms.62 The relatively higher CH4 uptakes compared to other gases is due to the stronger interactionof CH4  with the frameworks compared to N2, H2, and He .Uptakes and self-diffusivitiesfor He, H2, CH4, and N2 in COFs. Diffusion coefficients are inverselyproportional to the adsorption properties of gases since a stronglyadsorbed gas molecule within COFs cannot diffuse easily, and viceversa. CH4 and N2 molecules are heavier thanHe and H2; hence, self-diffusivities of He and H2 are relatively higher than those of CH4 and N2. This is in agreementwith the previously reported self-diffusion coefficients of the samegases in MOFs for which He and H2 diffusions were foundto be faster compared to other gas molecules (1.36 × 10–4–1.87 × 10–2 cm2/s for Heand 1.24 × 10–5–1.31 × 10–2 cm2/s for H2).63PHe, P2H, P2N, and P4CH, were computed to be in the ranges of 2.84 × 103–1.68 × 106, 5.59 × 103–2.14× 106, 4.11 × 103–8.61 ×105, and 1.58 × 104–1.39 ×106 Barrer, respectively. The lower and upper limits ofpermeability for each gas are similar due to the inverse relationshipbetween adsorbed loading and self-diffusion. The impact of diffusionproperties on determining the gas permeability seems more dominantfor He and H2, as shown in 4 are the lowest, but CH4 permeabilities of COFsare high, on the order of 104–106 Barrer,since CH4 uptakes vary in a wider range compared to theuptakes of He, H2, and N2. This indicates thatthe impact of adsorption on CH4 permeability of COFs ismore pronounced than the impact of diffusion.As shown in 2, 588 COF membranes for He/CH4, He/N2, H2/CH4, and H2/N2, and 589 COF membranes for N2/CH4 separationat 1 bar and 298 K, as shown in 63 were alsoincluded to comprehensively compare COFs and MOFs under the same conditions.We first discuss He/H2, He/CH4, and He/N2 separations as illustrated in 65 and it has to be purifiedfrom H2, CH4, and N2.We computed thepermeabilities and selectivities of 587 COF membranesfor He/H2 selectivities of587 COFs (orange data points) and 864 MOFs (black data points). Hepermeabilities of COFs were computed to be in the range of 2.84 ×103–1.68 × 106 Barrer with He/H2 selectivities in the range of 0.12–3.5. Selectivityvalues close to 1 indicate that permeabilities of He and H2 are similar due to their weak interactions with the framework atomsand lower molecular weights, causing similar uptakes and diffusivitiesas was previously shown in 3–2.09 ×106 Barrer) have the same orders as those of COFs, selectivitiesof MOFs (0.1–2.3) are lower than COFs. Therefore, it can beinterpreted that COFs could be more useful membranes than MOFs forthis separation.4 selectivitiesof 588 COFs(purple data points) and 864 MOFs (black data points) are shown in 4 separation, the selectivities ofCOFs and MOFs are similar, between 2.6 × 10–2–4.5 and 2 × 10–2–4.2, respectively.Although the selectivities for He/CH4 separation reachup to 4.5 in COFs and MOFs, there are strongly CH4-selectivematerials in both groups. Besides, in contrast to the trade-off observedin polymeric membranes, the correlation between permeability and selectivityfollows a linear trend for both COFs and MOFs. There are 499 COFsamong 588 COFs that could overcome the upper bound for He/CH4 separation, as shown in 2 selectivities of 588 COFs (pinkdata points) are shown in 2 separation, the selectivityvaries between 5.8 × 10–2–8.7 and 0.12–6.1for COFs and MOFs, respectively. COFs have a linear correlation betweenHe permeability and selectivity, and 539 COFs among 588 COFs werefound to be above the upper bound for He/N2 separation,indicating their high potential for this gas separation.He permeabilities and He/CH2/CH4 and H2/N2 separations, respectively. COFs have a strong correlationbetween permeability and selectivity for H2/CH4 and H2/N2 separations in contrast to the trade-offobserved in polymeric membranes. Calculated H2 permeabilitiesof COFs vary between 5.59 × 103 and 2.14 × 106 Barrer for H2/CH4 separation, as shownin 2 permeability range; hence, thereare MOFs with lower or higher H2 permeabilities comparedto COFs. H2/CH4 selectivities of MOFs were reportedto be up to 1 order higher (1.7 × 10–2–50.1)compared to COFs, whereas selectivities of COFs change between 4.1× 10–2 and 6.3. This can be attributed to thepresence of narrower pores in MOFs, which have stronger interactionswith CH4 molecules compared to COFs.66 A total of 416 and 535 COFs among 588 exceed the upperbound for H2/N2 and H2/CH4 separations, respectively.Hydrogenis one of the energy carriers in great demand since itcovers expectations as a clean, less-carbon-emitting alternative withhigh efficiency to overcome environmental pollution. 2 from CH4 for the purification of naturalgas. We studied N2 permeabilities and N2/CH4 selectivities of 589 COFs (yellow data points) and comparedthem with 5599 MOFs (black data points) in 2 permeabilitiesand N2/CH4 selectivities of COFs were in therange of 4.11 × 103–8.61 × 105 Barrer and 0.13–3.2, respectively. The low N2 selectivityof COFs can be explained by their large pore sizes, which let bothN2 and CH4 pass through.Membrane-based technologies arepreferred as they are less costlyand more energy-efficient compared to cryogenic distillation to separateNSacc of these materials  in Figure S1). Among the 589 COFs that we examined in this study,262, 499, 539, 535, 416, and 347 COFs were found to be above the upperbound for He/H2, He/CH4, He/N2, H2/CH4, H2/N2, and N2/CH4 separations, respectively. For all separations, PLDand LCD of COFs, which are above the upper bound (shown in red in 63 The medians of PLD (between 5 and 8.9 Å)and LCD (between 6.7 and 12.6 Å) of MOFs were found to be smallerthan those of COFs , since the gas permeability of someMOFs has a stronger dependency on the pore size compared to that ofCOFs. We provided the properties of the best-performing MOF and COFmembrane candidates, 10 materials that are located above the upperbound with the highest selectivities and permeabilities for each separation,in an Excel document as a part of the Supporting Information. Permeability is affected by adsorption and diffusionof gas molecules, which involve a complex interplay of several parametersof structures such as pore size, surface area, porosity, and chemicalenvironment. Most of the COFs naturally have larger pores than manyMOFs, and as an outcome, the PLD and LCD of COFs above the upper boundvary over a wide range compared to MOFs. Sacc of COFs and MOFs vary in a very distinct range. For COFs, the rangeof Sacc is 792.6–8533.8 m2/g, and MOFs exhibit a wider distribution with smaller surface areascompared to COFs. 4, He/N2, and He/H2 separations. Overall, After identifyingthe COFs that surpass the upper bound for eachgas separation, we examined the PLD, LCD, ϕ, and Sacc, and ϕ of COFs and their H2, CH4, He, and N2 permeabilities are demonstratedin Figure S2. A total of 142 common COFs(colored data points) out of 589 COFs (gray data points) surpassedthe upper bound for all six gas separations that we considered inthis work, with high permeability (>4 × 105 Barrerfor the strongly adsorbed gas component). These 142 COFs have PLD,LCD, Sacc, and ϕ ranges of 17–57Å, 20–57 Å, 2000–10 000 m2/g, and 0.77–0.95, respectively, and have ρ > 0.1g/cm3. When the set of hypoCOFs was refined according tothesespecified ranges, 23 341 hypoCOFs remained, and we groupedthem as 2D and 3D. Finally, based on Sacc and ϕ to achieve high gas permeability,we created four representative hypoCOF sets, each consisting of 30hypoCOFs having either 2D or 3D structures, with the highest, mean,and the lowest Sacc or ϕ, and namedas hypoCOFs2D-Sacc, hypoCOFs3D-Sacc, hypoCOFs2D-ϕ, and hypoCOFs3D-ϕ, respectively.After showing the high potential of CURATED COFs for gas separations,we studied hypoCOFs as membranes. Since it is not practical to performthe computationally demanding MD simulations for all 69 840hypoCOFs available in the database, we proposed an approach for thetargeted selection of hypoCOFs. We examined the structure–performancerelations of CURATED COFs and identified the structural propertiesof the most promising COF membranes. We then selected hypoCOFs basedon these structural properties. The relations between PLD, LCD, 2/CH4, H2/N2, He/CH4, and He/N2 separations, all hypoCOFs overcomethe upper bound with their high gas permeabilities and selectivitiesas shown in 2 and N2/CH4 separations , indicating that our computational approach for calculatingthe gas permeabilities of COF-based MMMs is valid.We have so far shownthat COFs and hypoCOFs are promising membranes due to their high gaspermeabilities and selectivities; however, problems related to robustness,scalability, and mechanical stability are known to arise during theexperimental manufacturing of membranes from crystal materials.2 separation, 2940 MMMs composed of 5 polymers and588 COFs for He/CH4 separation, 4116 MMMs composed of 7polymers and 588 COFs for He/N2 separation, 3528 MMMs composedof 6 polymers and 588 COFs for H2/CH4 separation,4116 MMMs composed of 7 polymers and 588 COFs for H2/N2 separation, and 4704 MMMs composed of 8 polymers and 588COFs for N2/CH4 separation. For He/H2 separation -TAB), He permeabilities increased up to 1.75times and most of the MMMs surpassed the upper bound. Especially MMMscomposed of 6FDA/PMDA(10/90)-TAB and Hyflon AD60X are the most promisingones since the increase in their He permeability is accompanied byhigh He/CH4 selectivities. As shown in 2 selectivity were computed to be between 38.9–1.13× 104 Barrer and 0.73–622, respectively. MMMsobtained with the incorporation of COFs into most of the polymers,such as 6FDA-TAB and TMHFBPA-I/T, outperformed the neat polymers interms of permeability. As can be seen from 2/CH4 separation,most of the COF-based MMMs are grouped around the upper bound. DAPHFDS(H)is the most promising polymer to generate MMMs since all COF-basedMMMs consisting of this polymer were above the upper bound. For H2/N2 separation, as shown in 2 permeability and selectivityvary in a wide range, 6.5–3.99 × 104 Barrerand 1.6–960, respectively. All COF-based MMMs composed of PBOI-2-Cu+, NTDA–BAPHFDS(H), PAI-3a, and a large number of COF/PIM-7and COF/PIM-1 MMMs surpass the upper bound. For N2/CH4 separation, as shown in 3 Barrer). (ii) Once polymersexhibit low permeabilities and high selectivities, COF/polymer MMMsoutperform the neat polymers generally only in terms of permeabilities.The gas permeability of MMM calculated with the Maxwell model at avolume fraction of 0.2 reduces to 1.75 times the gas permeabilityof the polymer when the gas permeability of COF is much higher thanthe gas permeability of the polymer.69 Therefore,selectivity, the ratio of gas permeabilities, does not change. (iii)Selectivities of polymers might improve, remain the same, or, in rarecases, diminish upon the incorporation of COF fillers if the polymerssuffer from low selectivities.Based on the results presented in 2, 600 MMMs (5 polymers and 120 COFs) for He/CH4, 840 MMMs(7 polymers and 120 COFs) for He/N2, 720 MMMs (6 polymersand 120 COFs) for H2/CH4, 840 MMMs (7 polymersand 120 COFs) for H2/N2, and 960 MMMs (8 polymersand 120 COFs) for N2/CH4 separation. Remarkably,hypoCOF/PTMSP, hypoCOF/PTMSP-co(95/5), and hypoCOF/Teflon AF-2400MMMs show much higher permeabilities over neat polymers compared tothe experimental COF/polymer MMMs. Incorporation of hypoCOFs intopolymers boosted the He permeabilities of polymers for He/H2, He/N2, and He/CH4 separations, as shown in 2/CH4 separation, and 111 of 120 hypoCOF/PTMSPMMMs and 113 of 120 hypoCOF/PTMSP-co(95/5) MMMs have higher H2/CH4 selectivities than the pure polymers, as shownin 2/N2 separation,as shown in 2 permeabilities of polymerswere improved with the incorporation of hypoCOF fillers although selectivitiesremain the same, as shown in We finally studied hypoCOF/polymerMMMs and showed the resultsin 42, CH4, and N2 permeabilities and He/H2, He/CH4, He/N2, H2/CH4, H2/N2, and N2/CH4 selectivities of589 COFs were computed, and the results showed that selectivitiesof COF membranes vary in the ranges of 0.12–3.5 for He/H2, 2.6 × 10–2–4.5 for He/CH4, 5.8 × 10–2–8.7 for He/N2, 4.1 × 10–2–6.3 for H2/CH4, 0.18–15.7 for H2/N2, and 0.13–3.2 for N2/CH4 separation.The surface area and porosity were identified as important structuralproperties to select the best-performing COFs exhibiting high gaspermeabilities (>4 × 105 Barrer). Based on thestructure–permeabilityrelation of the CURATED COFs that were identified to be above theupper bound for all six gas separations, 120 hypoCOFs were selectedand examined as membranes. Results revealed that all of the selectedhypoCOFs show high permeability and selectivity for each gas separationand exceed the upper bound except for He/H2 and N2/CH4 separations. Finally, 24 100 COF/polymer MMMsand 4920 hypoCOF/polymer MMMs were examined considering 25 differentpolymers, and results showed that permeability and selectivity ofseveral polymers can be improved by the incorporation of COF fillers.The highest selectivities of COF-based MMMs were calculated as 4.4for He/H2, 3041 for He/CH4, 622 for He/N2, 325 for H2/CH4, 960 for H2/N2, and 4.4 for N2/CH4 separation.In this work, we focused on a set of hypoCOFs, but there might bedifficulties in the synthesis and applications of hypothetical materials.For example, hypoCOFs, which were identified to be the best membranecandidates, may not be easily synthesized, or the synthesized structuresmight have defects. Two other important points that will shape thefuture of COF membranes other than their separation performances aretheir stability and cost. The stability of COF membranes and COF-basedMMMs needs to be tested under humid environments, in the presenceof impurity gases, and at high temperature and pressure conditions.We believe that cost-related barriers for COF membranes will be overcomewith the commercialization of COFs soon, provided that novel COF membraneprocessing methods will be discovered. We envisage that our work willinspire both experimental and computational studies for the investigationof COFs and hypoCOFs to be used in membrane-based gas separation applications.In this work, we used molecular simulations, GCMC and MD, to investigatethe membrane-based gas separation potentials of experimental and hypotheticalCOFs for six different gas separations. He, H


Then, we assess existing evidence of its dysregulation in various brain disorders, including from immunohistochemical and (phospho) proteomic data. We propose pathways by which MAP2 pathology could contribute to endophenotypes which characterize these disorders, giving rise to the concept of a “MAP2opathy”—a series of disorders characterized by alterations in MAP2 function.Microtubule-associated protein 2 (MAP2) is the predominant cytoskeletal regulator within neuronal dendrites, abundant and specific enough to serve as a robust somatodendritic marker. It influences microtubule dynamics and microtubule/actin interactions to control neurite outgrowth and synaptic functions, similarly to the closely related MAP Tau. Though pathology of Tau has been well appreciated in the context of neurodegenerative disorders, the consequences of pathologically dysregulated MAP2 have been little explored, despite alterations in its immunoreactivity, expression, splicing and/or stability being observed in a variety of neurodegenerative and neuropsychiatric disorders including Huntington’s disease, prion disease, schizophrenia, autism, major depression and bipolar disorder. Here we review the understood structure and functions of MAP2, including in neurite outgrowth, synaptic plasticity, and regulation of protein folding/transport. We also describe known and potential mechanisms by which MAP2 can be regulated  The microtubule (MT) cytoskeleton is a fundamental coordinator of neuronal structure and function. MTs, comprised of heterodimers of α- and β-tubulin subunits, are present in all cell types and provide dynamic support to enable cell migration/division, modify and maintain cellular shape, and serve as tracks for intracellular trafficking  define a set of proteins which directly bind MTs, typically serving to stabilize them , which have several features that confer unique pathogenic potential . The strBox 1IDPs are proteins which lack a stable tertiary structure. They are present in all organisms, being particularly abundant in eukaryotes, where over 30% of all proteins are estimated to have disordered regions  MAP2A and MAP2B , and low molecular weight (LMW) MAP2C and MAP2D. These four MAP2 isoforms share four of the five functional domains, with the projection domain being selective to the HMW forms. In human, MAP2A possesses a 4-residue deletion relative to MAP2B, and MAP2D contains an additional MT-binding repeat and a modified 77-residue segment in the N-terminal domain relative to the other three isoforms . Recent studies have also illuminated the importance of MT-actin crosstalk to spine morphogenesis, with actin remodeling at activated spines promoting MT entry , a component of the Ras-MAPK pathway, has been identified as the probable causal gene underlying the association signal at its locus in the largest schizophrenia genome-wide association study conducted to date  has been reported in diverse brain regions in schizophrenia and has been described as a “molecular hallmark” of the disorder , in turn suppressing tubulin polymerization  have been reported to be decreased (case:control ratio = 0.82–0.83) in DLPFC of individuals with major depression, though these changes did not remain significant after controlling for false discovery rate , the HMW and LMW forms of the protein differ by inclusion or exclusion of the projection domain. This yields differential subcellular localization patterns and MT spacing characteristics. Additionally, they appear to differ in their ability to support activity-dependent spine plasticity, with HMW forms selectively translocating into spines following chemical LTP stimulus . MAP2 has generally not been considered as a potential pathogenic agent in the tauopathies, as its presence in NFTs has been a subject of some debate , athology . This woathology , which mathology ] and limExisting data regarding MAP2 pathology is largely limited to correlational study, leaving open the possibility that changes to MAP2 immunoreactivity, expression or modification do not contribute to pathogenesis, but instead are merely incidental changes incurred by upstream processes. Despite clear evidence that disruptions to MAP2 expression and/or modification can lead to changes in dendritic and synaptic structure and function , National Institute on Aging (grant number: AG027224), and National Institutes of Health (grant number: GM97082).


Human angiotensin I‐converting enzyme (ACE) has two isoforms, somatic ACE (sACE) and testis ACE (tACE). The functions of sACE are widespread, with its involvement in blood pressure regulation most extensively studied. sACE is composed of an N‐domain (nACE) and a C‐domain (cACE), both catalytically active but have significant structural differences, resulting in different substrate specificities. Even though ACE inhibitors are used clinically, they need much improvement because of serious side effects seen in patients (~ 25–30%) with long‐term treatment due to nonselective inhibition of nACE and cACE. Investigation into the distinguishing structural features of each domain is therefore of vital importance for the development of domain‐specific inhibitors with minimal side effects. Here, we report kinetic data and high‐resolution crystal structures of both nACE (1.75 Å) and cACE (1.85 Å) in complex with fosinoprilat, a clinically used inhibitor. These structures allowed detailed analysis of the molecular features conferring domain selectivity by fosinoprilat. Particularly, altered hydrophobic interactions were observed to be a contributing factor. These experimental data contribute to improved understanding of the structural features that dictate ACE inhibitor domain selectivity, allowing further progress towards designing novel 2nd‐generation domain‐specific potent ACE inhibitors suitable for clinical administration, with a variety of potential future therapeutic benefits.7Z6Z and 7Z70, respectively, in the RCSB Protein Data Bank, www.pdb.org.The atomic coordinates and structure factors for nACE‐fosinoprilat and cACE‐fosinoprilat structures have been deposited with codes  Analysis of high‐resolution crystal structures of angiotensin I‐converting enzyme (ACE) N‐ and C‐domains in complex with fosinoprilat provide further insights into the requirements for high‐affinity domain‐specific inhibition that is required to reduce side effects of treatments that target ACE. In addition, the data highlights where modifications or redesign of fosinoprilat are needed to develop novel insecticides that act via ACE analogues. ACEangiotensin‐1‐converting enzymeAng Iangiotensin IAng IIangiotensin IIAnoACE2Anopheles gambiaeangiotensin‐1‐converting enzyme homologue from cACEangiotensin‐1‐converting enzyme C‐domainnACEangiotensin‐1‐converting enzyme N‐domainRAASrenin–angiotensin–aldosterone systemsACEsomatic angiotensin‐1‐converting enzymetACEtestis angiotensin‐1‐converting enzymeEC3.4.15.1) is a key zinc‐dependent dipeptidyl carboxypeptidase with two human isoforms: somatic ACE  and testis ACE   is the final substrate concentration of 0.5 mm Z‐FHL). IC50 were carried out in triplicate.To determine fosinoprilat binding affinity, an assay using a modified erformed . Brieflyr et al. . Initialequation , 56  and 20 mm inhibitor for 1 h . Co‐crystals were obtained with hanging drops of 1 μL of the protein‐inhibitor complex mixed with equal volume of reservoir solution . Both proteins were then incubated at 16 °C.The ACE domains were pre‐incubated with fosinoprilat in a 4 : 1 v/v ratio of protein  and i04 (cACE) at the Diamond Light Source . Crystals were kept at constant temperature 100 K) under the liquid jet during data collection. Images were collected using Eiger2 XE 16M detector . Raw data images were indexed and integrated with dials  and then[aimless  from thep4 suite . Initial [phaser . PDB codand 6F9T  were use[refmac5  followed [phenix , with coix [coot  used forlprobity  was used00 K unde [ccp4mg , and schligplot+ .The authors declare no conflict of interest.GEC performed crystallisation, collected high‐resolution X‐ray diffraction data, analysed the data and edited the manuscript. ECN performed crystallographic structure analysis and wrote the manuscript. SLUS carried out the expression and purification of nACE and cACE and the kinetic characterisation of fosinoprilat binding. REI provided fosinoprilat used in this study and edited the manuscript. EDS analysed the data and edited the manuscript. KRA supervised the study, analysed the data and edited the manuscript. All authors reviewed the manuscript.https://publons.com/publon/10.1111/febs.16543.The peer review history for this article is available at


Adapting emotion-regulation strategy use to flexibly match contextual features of a hassle is thought to aid in effective coping. We investigated if hassle domains are a pertinent feature for understanding emotion-regulation strategy selection in the everyday lives of adolescents through older adults. Participants, ranging from 14 to 88 years old (N = 325), completed an experience sampling study of approximately 9 days over a 3-week period. At each momentary assessment, participants reported on their affect, hassles, and emotion-regulation strategies. Our findings indicated that strategy use varied by domain. For example, distraction was most common in the health domain, whereas emotion expression was least likely to be used at work or school. More strategies were used when hassles were associated with multiple domains. However, greater domain differentiation was not associated with reduced hassle reactivity. Findings were similar across ages suggesting domains may similarly relate to strategy selection across the lifespan.


Architected cellular materials are a class of artificial materials with cellular architecture-dependent properties. Typically, designing cellular architectures paves the way to generate architected cellular materials with specific properties. However, most previous studies have primarily focused on a forward design strategy, wherein a geometry is generated using computer-aided design modeling, and its properties are investigated experimentally or via simulations. In this study, we developed an inverse design framework for a disordered architected cellular material (Voronoi lattices) using deep learning. This inverse design framework is a three-dimensional conditional generative adversarial network (3D-CGAN) trained based on supervised learning using a dataset consisting of voxelized Voronoi lattices and their corresponding relative densities and Young’s moduli. A well-trained 3D-CGAN adopts variational sampling to generate multiple distinct Voronoi lattices with the target relative density and Young’s modulus. Consequently, the mechanical properties of the 3D-CGAN generated Voronoi lattices are validated through uniaxial compression tests and finite element simulations. The inverse design framework demonstrates potential for use in bone implants, where scaffold implants can be automatically generated with the target relative density and Young’s modulus. An inverse design framework for 3D Voxelized geometries using a 3D conditional generative adversarial network (3D-CGAN) is proposed.The trained 3D-CGAN is capable of generating 3D Voronoi lattices with the desired target relative density and Young’s modulus.The mechanical properties of 3D-CGAN generated Voronoi lattices were experimentally and numerically validated.  TailoriOver the last few decades, extensive efforts have been dedicated to the design of new architected cellular materials to achieve characteristics such as programable mechanical responses , novel dIn recent years, deep learning algorithms have been exploited to handle these inverse design challenges ,41–50. HIn this study, we developed a deep-learning-driven inverse design framework for the direct generation of 3D voxelized architected cellular materials with user-defined relative densities  . By contIn the numerical homogenization method, numerous unit cells are considered based on a mathematical operation . However2.2.The neural network architecture yields two main outputs that are necessary for the inverse design of mechanical metamaterials: the modeling parameters, which can be used to generate geometries with additional modeling processes , and theIn this study, the inverse design of 3D architected cellular materials was implemented using a novel framework: 3D-CGAN. The 3D-CGAN was trained to generate 3D voxels of Voronoi lattices from a probabilistic space with a given label  with a photopolymer resin . The photopolymer resin is a typical plastic material with a Young’s modulus, Poisson’s ratio, and yield strength of 1.6 GPa, 0.23, and 38 MPa, respectively . The 3D- testing . These mAfter 3D-printing fabrication, the mechanical properties of these samples were examined through uniaxial compression tests using a motorized test stand . The static compression tests were performed at a vertically constant speed of 10 mm/min, following the ASTM standard D695–15. The compression strain was set to 0.15, which is adequate to achieve elastic deformation of these samples. The deformation processes were recorded using a high-speed camera placed in front of the samples. The effective Young’s moduli were calculated by linearly fitting the initial linear region of the recorded stress – strain curves.2.4.The deformation behavior was visually examined using FEM simulations. The deformation problem under a finite strain was analyzed using a nonlinear FEM simulation, where periodic boundary conditions were applied on a periodic microstructure. The detailed process of implementing the displacement and stress equations has been discussed in our previous reports ,66. FEM In the experiments, uniaxial compression deformation was imposed to 3D-printed samples composed of 3.3.1.The performance of the 3D-CGAN can be evaluated based on the similarity and stability of the training results. Here, similarity refers to the agreement between input labels , based on supervised learning. The trained 3D-CGAN was capable of rapidly generating 3D Voronoi lattices with the desired target relative density and Young’s modulus. Thus, this study demonstrates the potential application of Voronoi lattices in tissue engineering, where artificial scaffolds can be inversely generated using a given target relative density and Young’s modulus. We expect the scope of this study to be extended to the inverse design of architected cellular materials with other target properties by replacing the labels – for example, diffusivity, permeability, and conductivity – for the sake of energy storage and conservation ,72,73. F


To specially quantify the biomass of intertidal macroinvertebrates, a fundamental metric in the fields of ecology, has long remained a challenge, especially for cryptic species. This study classified intertidal macroinvertebrates by feeding mode, such as surface deposit feeders and suspension feeders, and tested whether the biomass can be estimated from their burrow opening dimension. These results indicated that the burrow opening dimension of the surface deposit feeder was available as a proxy for biomass. However, we cannot yet generalize about direct relationships between the opening dimension and biomass for suspension feeders due to a relatively low correlation between them.Biomass and abundance are fundamental parameters in ecology, conservation biology, and environmental impact assessment. Distinguishing features, such as burrow openings and feeding pellets, made by different intertidal macroinvertebrate species on the surface are used as proxies to establish the abundance of intertidal macroinvertebrates. This study investigated the feasibility of estimating biomass from the burrow opening dimensions as a proxy. We analyzed the relationship between the burrow opening dimensions and body weights of intertidal macroinvertebrates and compared surface deposit feeders with suspension feeders. Regression analysis evaluated the relationship between burrow opening diameter, body size, and biomass. The diameters of surface deposit feeder burrow openings were significantly related to biomass, but this was not the case for suspension feeders. Our results indicate that burrow opening dimensions can be used as a proxy to estimate the biomass of surface deposit feeders. However, additional studies are needed to clarify further the relationship between the burrow opening diameter and biomass of the suspension feeders. This is a preliminary study to spatially quantify the biomass of intertidal macroinvertebrates by extracting the dimension of burrow openings from drone images through object detection tools. Representing the actual spatial distribution of organisms or substances is essential to understanding the natural world and managing nature efficiently. Complex model equations are sometimes used to establish how non-uniformly spatialized items are distributed in space. However, a model is only an estimation technique; in many cases, it cannot reflect the natural state ,2. With Biomass and abundance are fundamental parameters in ecology, conservation biology, and environmental impact assessment. However, obtaining accurate and precise measurements in situ is onerous , especiaOcypode cursor [Ocypode quadrata. The OD represented the body width from 43% in Uca longisignalis [Uca annulipes [Upogebia noronhensis accounted for 89% of the carapace length [Upogebia major [Burrow structures vary greatly in size and shape. Burrow morphology and dimension are generally represented by the feeding mode, activity level, body form and size of the macroinvertebrate inhabitant ,12,13,14e cursor  to 98% ie cursor  study ofsignalis  to 97% innulipes  in the fe length , and theia major . Only a ia major ,27, but This study examined the relationship between burrow opening dimensions and body weight of intertidal macroinvertebrates between surface deposit feeders and suspension feeders. We investigated whether the biomass of intertidal macroinvertebrates could be estimated from the burrow opening dimension as a proxy. This is a preliminary study to spatially quantify the biomass of intertidal macroinvertebrates by extracting the dimensions of burrow openings from drone images through object detection tools.Macrophthalmus japonicus, Uca arcuata, Uca lactea lactea, Ocypode stimpsoni, Urechis unicinctus, and Upogebia major , which has an artificially controlled tide with a maximum range of 1.1 m [M. japonicus is an abundant species in SH, with a mean density of 10 ind. m−2. U. arcuata and U. lactea lactea samples were obtained from the DB tidal flats , which has a macro tidal range and semi-diurnal tide with a maximum tidal range of 9.8 m [−2, respectively. The O. stimpsoni and U. unicinctus samples were collected from the TA tidal flats , which has a macro tidal range and a semi-diurnal tide with a mean tidal range of 4.6 m [−2, respectively. U. major samples were obtained from the GS tidal flats , which has a semi-diurnal macro tidal regime with a mean tidal range of 6 m [−2.This study was conducted in four tidal flats along the west coast of Korea: Sihwa (SH), Daebu (DB), Taean (TA), and Geunso (GS) tidal flats . Field sia major . These sof 1.1 m . M. japoof 9.8 m . The meae of 6 m . This muOnly those showing signs of activity, such as feeding pellets, burrowing pellets, and foraging tracks of each species, were selected before sample collection to avoid including abandoned burrows. The identification of each species was determined based on the characteristics of its burrow opening and feeding or excretion trace  and the burrow opening diameters for the other five species (with a circular burrow opening). For U. unicinctus and U. major, which have two openings per burrow, we measured the diameter of both openings and took the mean value as the OD  with Tukey’s post-hoc test was used to assess differences in OD-CW (TL for U. unicinctus) ratios among six species.Differences in OD, WW, DW, and AFDW between abnormal and normal openings for M. japonicus over the entire study period at SH (p < 0.001) with R2 values of 0.84 and 0.83, respectively (p < 0.001), with an R2 ranging from 0.80 to 0.99 (We collected 124 samples of od at SH . The meaod at SH . The meaectively . The OD– to 0.99 .U. arcuata over the entire study period at DB (p < 0.001) with R2 values of 0.92 and 0.94, respectively , with an R2 ranging from 0.91 to 0.99 (We collected 176 samples of od at DB . The meaod at DB . The meaectively . The R2  to 0.99 .U. lactea lactea over the entire study period at DB (p < 0.001) with R2 values of 0.91 and 0.86, respectively (p < 0.001), with an R2 ranging from 0.71 to 0.94  with R2 values of 0.94 and 0.92, respectively (p < 0.001), with an R2 ranging from 0.88 to 0.98 , but the R2 value was low (0.11), while the OD–WW regression was the highest with a value of 0.51 (p < 0.001 and p < 0.01), with an R2 ranging from 0.10 to 0.51 (We collected 82 samples of s period . The fies period . The mea of 0.51 . The OD– to 0.51 .U. major over the entire study period at GS (p < 0.001), but the R2 values were low, with values of 0.31 and 0.14, respectively , with an R2 ranging from 0.06 to 0.69 , with R2 values of 0.11, 0.30, and 0.24, respectively. Meanwhile, the OD–WW and OD–DW regressions of the normal openings were significant (p < 0.001), but the OD–AFDW regression was not significant, with R2 values of 0.54, 0.14, and 0.05, respectively .The mean OD–CW ratios of the ectively . The mea2 value of OD–CW regression of the Ocypodidae ghost crab, Ocypode ceratophthalma, ranged between 0.82 and 0.96 [2 values of OD–CW regression for the Ocypodidae fiddler crabs U. annulipes and U. spinicarpa, with values of 0.98 and 0.91, respectively. Like the relationship between OD and CW, the CW of surface deposit feeders is highly related to their biomass. The significant relationship between body size and body weight of deposit feeders has been documented in many studies [Scopimera crabricauda, and ghost crab, O. quadrata, were significantly related to WW [This study evaluated the relationship between OD and body size and between OD and body weight of six macroinvertebrate species as a non-intrusive method to estimate the body size and weight of the burrow inhabitants. Surface deposit feeders live in isolated burrows in muddy or sandy regions and emerge at low tide to feed or engage in other surface activities nearby ,32. Sincand 0.96 ,23. Skovand 0.96  and Moutand 0.96  reported studies ,33. The ed to WW ,33. The Cardisoma guanhumi using the equation y = 32.17 + 21(x), where y = crab biomass (g) and x = burrow diameter (cm). Careel [Geolycosa xera archboldi and Geolycosa hubbelli was significantly correlated with their wet body mass, and recorded R2 values of 0.97 and 0.95, respectively. We found a significant correlation between OD and biomass of surface deposit feeders. The correlation between OD and biomass was similar (in M. japonicus and U. lactea lactea) or higher than the correlation between OD and body size (in U. arcuata and O. stimpsoni). These findings suggest that surface deposit feeders’ biomass can be accurately estimated using the OD and that OD can be used as a proxy for biomass without the need to invade or destroy their burrows.Despite the relationship between OD and body size and between body size and body weight in surface deposit feeders, attempts to estimate the biomass of marine species using their burrow opening dimensions have been limited. Only a few studies have shown the relationship between the OD and body weight of burrowing land species. Sample and Albrecht  reported. Careel  reportedU. major became abnormally narrow from August to October compared to other months. Additionally, there were no visible surface burrow openings for U. unicinctus from July to November. We presume this may be related to ecological characteristics, such as copulation, spawning, and reproduction; however, more detailed studies are needed for clarification. The mean OD of U. major differed significantly between the abnormal and normal openings. However, the mean WW, DW, and AFDW did not differ significantly; therefore, biomass could not be accurately estimated for this species using OD during this period. Our findings suggest that the ecological characteristics of species should be considered when estimating the biomass of suspension feeders through OD.The burrow openings of U. unicinctus and U. major were relatively lower than those of the surface deposit feeders due to their different feeding modes. U. unicinctus constructs a U-shaped burrow and filters suspended materials from seawater pumped through the burrow using a mucus net [U. major has a Y-shaped structure, in which two vertical passages are connected in a U-form in the upper part and a straight passage in the lower part [U. major, and another study in U. noronhensis also found a significant correlation between them [The correlations between OD and body size and OD and biomass of the suspension feeders ucus net ,35. The ucus net . The burwer part ,24,36. Twer part ,24,25, sWe evaluated the relationship between the burrow opening dimensions, body size, and body weight of six intertidal macroinvertebrate species and between surface deposit feeders and suspension feeders. The burrow ODs of the surface deposit feeders were significantly related to biomass, while those of the suspension feeders showed a relatively low relationship. These results indicate that the burrow opening dimensions can be used as proxies to estimate the biomass of surface deposit feeders. However, we cannot yet generalize a direct relationship between the burrow opening dimensions and biomass for suspension feeders based solely on our results due to their relatively low correlation. Nonetheless, this study demonstrates a non-intrusive method to estimate the biomass of cryptic species using their burrow opening dimensions extracted from drone images through object detection tools. We anticipate that any limitations to obtaining biomass data will be overcome and that a realistic biomass value will be estimated using this spatial data obtaining technology.


The epidermis in plants is the outermost cell layer and as such is crucial to mediate the plant’s interaction with its environment. In aerial tissues like leaves and stems, the epidermis protects the plant from the threats of pathogens, herbivores, and unfavorable environmental conditions. Meanwhile, the root epidermis actively increases the roots’ surface to favor water and nutrient intake, as well as to establish symbiotic relationships with beneficial fungi.Arabidopsis thaliana) trichomes act as mechanosensors, initiating Ca2+ waves that reach and alert distant cells of the presence of a threat (GLABRA2 (GL2) encodes a transcription factor (TF) and is one of the best characterized regulators of epidermal cell fate in Arabidopsis, controlling trichome differentiation, root hair-less cell file determination, as well as mucilage synthesis in seeds  and execute changes in the expression of genes, usually by recruiting additional TFs and components of the basal transcriptional machinery. TFs are modular molecules comprising distinct protein domains, each of which typically contributes to the TF activity in a different manner: the DNA binding domain determines the DNA sequence to which the TF will bind, whereas other protein domains may influence the TF’s protein interactome, intracellular transport dynamics, and protein stability. Distinguishing the contributions of individual protein domains in a TF is crucial to harness their full potential to fine-tune transcription for better yields or to efficiently design synthetic transcriptional networks .Marchantia polymorpha, the START domain binds to lipids and influences their transport (GL2 belongs to the homeodomain-leucine zipper (HD-ZIP) class IV TF family that has a plant-specific domain arrangement consisting of an HD-ZIP DNA-binding domain tandem near the N-terminus, followed by a steroidogenic acute regulatory protein-related lipid-transfer (START) domain, and a START-associated domain (STAD) near the protein C-terminus . In animransport . NeverthPlant Physiology, GL2 mutant variants that could potentially impair the HD and START domain activities. Knockout gl2 plants were transformed with the set of GL2 variants fused to a fluorescent protein, and their individual capacity to rescue the well-characterized gl2 developmental defects was scored. Confirming the predictive power of homology-based structural models, all mutant variants of GL2 failed to rescue gl2 trichome development, root hair density, and seed mucilage accumulation, indicating that the START domain, similarly to HD, is indispensable for GL2 transcriptional function. Similar phenotypic defects were quantified in domain-specific START and HD mutants, indicating that both domains are equally necessary for GL2 activity.In the current issue of HDs allow DNA binding and dictate the target site of TFs. Consequently, and based on genetic complementation assays, the START domain must govern another equally indispensable molecular mechanism for GL2 activity. The authors first analyzed the subcellular localization of the mutant GL2 variants and concluded that neither the HD nor START domains impair GL2 nuclear localization. Next, the importance of the START domain for the ability of HD-ZIP(IV) proteins to bind DNA was tested in electrophoretic mobility shift assays (EMSAs). Due to difficulties expressing recombinant GL2, a close relative, PROTODERMAL FACTOR2 (PDF2), and PDF2 mutant variants equivalent to those designed for GL2 were used in these experiments. EMSAs and western blots indicated that the START domain of PDF2 does not influence the TF DNA binding in vitro and that the START domain is necessary for homodimerization.A ChIP-seq experiment using several GL2 mutant variants revealed that the presence or functionality of the START domain does not substantially alter GL2 DNA binding specificity in vivo. Yeast-two hybrid and split-fluorescent protein assays using a comprehensive set of full-length and domain deletion GL2 variants confirmed the requirement of the START domain for effective GL2 homodimerization in yeast and in planta.Finally, Mukherjee and colleagues investigated whether the START domain could influence protein stability by applying protein synthesis inhibitors to seedlings expressing different GL2 mutant variants and quantifying the corresponding GL2 protein in a time-course experiment. The latter experiment clearly indicated that GL2 variants lacking a functional START domain were degraded more rapidly than wild-type GL2. Using a proteasome degradation inhibitor extended the lifetime of START-domain mutant GL2 variants several hours, suggesting that the proteasome degradation pathway could be controlling GL2 protein stability.M. polymorpha STAR2 protein. Pinpointing the type of lipid and the conditions under which it is bound by HD-ZIP(IV) TFs would provide us with a clearer picture of how these TFs are mechanistically regulated and perhaps reveal how these TFs may perceive and respond to readouts of cellular metabolism and/or important small signaling lipids.The authors therefore concluded the START domain is vital for HD-ZIP(IV) TF activity as it (i) enables homodimerization, therefore likely allowing a more effective recruitment of the basal transcriptional machinery; and (ii) increases protein stability, increasing the active window for each HD-ZIP(IV) molecule to conduct its cellular activity before protein turnover. Moving forward it would be very informative to address the lipid-binding capacity of the HD-ZIP(IV) START domains by means of biochemical and evo-devo approaches, that is, testing their capacity to directly bind collections of lipids, and investigating whether they can genetically complement the previously identified START domain of the Conflict of interest statement. None declared.