OxG Palm hybrid yields oil known as “palm oil with a higher content of oleic acid” (HOPO), with <35% SFA and >50% oleic acid. Characterizing novel high oleic oils is the starting point to find processes that can functionalize them such as oleogelation. This study compared the thermophysical properties of HOPO to SPO using Differential Scanning Calorimetry, shear rheology, polarized light microscopy, and texture analysis to characterize the differences between these oils. HOPO had a lower onset crystallization temperature (Δ7 °C) and its rheological behavior followed similar trends to SPO; however, large viscosity offsets were observed and were correlated to differences in crystallization temperatures. The maximum peak force of SPO was an order of magnitude higher than that of HOPO. Overall similar trends between the oils were observed, but differences in firmness, crystal morphology, and viscosity were not linearly correlated with the offset in crystallization temperature. This study quantified differences between these oils that will better enable industry to use HOPO in specific applications.Standard Palm Oil (SPO) is widely used as a food ingredient partially due to its unique thermophysical properties. However, the American Heart Association recommends a saturated fat consumption of <5% of the caloric intake per day. The  Elaeis guineensis, largely due to its versatility as a food ingredient [Edible fats and oils play a key role in the human diet, providing energy, essential fatty acids for cellular development, and delivery of liposoluble phytonutrients . As an igredient  and highgredient . The typgredient . The fatgredient ,4. Howevgredient . The AHAgredient ,6,7. Palgredient . HoweverElaeis oleifera X Elaeis guineensis (OxG) palm [In 2019, the Codex Alimentarius included a new definition for a “palm oil with a higher content of oleic acid” in the standard for named vegetable oils. Grown primarily in Colombia, Ecuador, and Brazil, this oil is obtained from the interspecific hybrid xG) palm . A highexG) palm . FurtherxG) palm . HOPO alxG) palm  and caroxG) palm  when comxG) palm . As the xG) palm , it is nThe crystallization process of fats and oils is dependent on both intrinsic and extrinsic factors. Intrinsic factors include the fatty acid profile, triglyceride composition, and the presence of naturally occurring minor components. Minor components include free fatty acids (FFA), monoglycerides, diglycerides, and impurities. Major extrinsic factors include the cooling rate, degree of undercooling, annealing time, and shear rate during the formation of the crystalline structure. Differential Scanning Calorimetry (DSC) is commonly used to study exothermic and endothermic reactions caused by temperature changes under constant cooling and heating regimes. Phase transitions are identified by deviations from the baseline heat requirement to change temperature. Once crystallized, the firmness of the oil is an additional parameter that varies with temperature and solid fat content , which dNumerous researchers have correlated the physical properties of fats to their crystalline structure ,18. MicrCurrently, there is limited knowledge on the crystallization kinetics and physical properties of HOPO and its products . Publicaw/w were not reported. The literature values for SPO are shown in The fatty acid profile obtained in this study for HOPO was 27.8% palmitic acid (C16:0), 2.8% stearic acid (C18:0), 55.5% oleic acid (C18:1), and 11.9% linoleic acid (C18:1). These results matched previously published data for HOPO shown in It was confirmed that HOPO had a lower concentration of palmitic and stearic fatty acids when compared to SPO ; this was expected, and it was also observed by Mozzon et al. . ConsequThe results from this analysis showed SPO’s thermogram consists of two-phase transition zones for the crystallization and melting stages. This was consistent with the thermograms reported in previous studies . The cryWhile most of the work in this study focused on temperatures above 0 °C (in the common range of food industry applications), the first large melting peak for HOPO had an onset of −5.45 °C which ends at 3.5 °C, while for SPO, the peak extends up to 10 °C. This indicates that the lower-melting fraction of the HOPO will be almost completely melted at temperatures below 0 °C. The melting profile from the thermogram showed complete melting at 30.03 °C for HOPO and 37.99 °C for SPO see . The fulIn the cooling diagram, the reduced energy required to transition at E and F likely corresponds to the lower concentration of di-saturated and fully saturated TAGs (~65% vs. ~53%) in HOPO when compared to SPO. These results were consistent with the findings of Mozzon et al. , who alsAs shown in As shown in Even when similar phase transitions occur at 7 °C apart in the thermograms , the stiThe characterization of the crystalline microstructure of fats can help better assess the mechanical properties of fats. In polarized microscopy, fat crystals appear as white and the liquid oil in the background as black. The samples were crystallized at 5, 10, 15, and 20 °C for 24 h and images were taken in duplicate .Spherulites were the most common structures identified in both oils. For HOPO, structures ranged from 18.1 ± 1.1 µm at 20 °C to 8.6 ± 1.3 µm at 5 °C and between 12.9 ± 1.1 µm at 20 °C and 8.8 ± 1.2 at 5 °C for SPO. The ideal spreadable fat consists of small (<30 µm) and largely dispersed crystals . The β pShear rheology has long been utilized to describe the first and second crystallization temperatures of palm oil under shear by notable jumps in viscosity at constant cooling rates . The sam−1) are shown in −1 and 1350 s for higher shear rates; SPO crystallization started at 1150 s at 0.1 s−1 and 1200 s for higher shear rates. The viscosity of HOPO was higher than SPO at lower shear rates (0.1 and 1 s−1) at the offset of −10 °C. In [−1 plateaus at two orders of magnitude higher than that of shears of 10 s−1 and 100 s−1.The dynamic viscosities vs. temperature and shear rates , and saturated fatty acids, represented as palmitic acid (29.92% vs. 43.52%).The effect of the FA profile on the melting/cooling behavior during DSC analysis was observed for both oils. The thermograms had similar shapes; however, a ~7 °C offset in the crystallization and melting profiles was identified. The onset crystallization temperature started at 7.90 °C in HOPO, whereas it occurred at 16.45 °C in SPO. HOPO completely melted at 29 °C and SPO at 37 °C. These findings are important to establish the optimal crystallization temperatures for HOPO and how these will affect its mechanical and rheological characteristics. Major crystallization events occurred at 16.45 °C, 14.05 °C, 4.64 °C, and −1.18 °C in SPO and were consistent with the literature. The corresponding values for HOPO were 7.9 °C, 5.81 °C, −1.27 °C, and −8.11 °C, which offset the maximum usable temperature of HOPO.A texture analysis for the oils near major crystallization temperatures found the highest stiffness value to be 2.495 ± 0.182 kg at 5 °C for HOPO and 5.064 ± 0.546 at 5 °C for SPO, with the difference that SPO deformed as force was applied, while HOPO was more brittle and broke at the maximum stiffness.The microstructure of HOPO was temperature-sensitive with well-defined spherulites at 20 °C, but irregular and ambiguous structures at 15 °C and 10 °C. The microstructure of SPO did not experience major structural changes with the decrease in temperature. Under different cooling regimes, the viscosity profiles were similar between the oils at higher shears; however, there was a shift in the induction time of nearly 4 min for crystallization, occurring at around 6–8 °C in HOPO at high shears, 10 °C less than for SPO.This study expands the understanding of the physical properties of HOPO, suggesting that low-temperature applications can harness the soft but viscous texture of HOPO while taking advantage of it being a nutritious alternative to oils with higher saturated fatty acid concentrations. Additionally, it was shown how the processing conditions, shear, and cooling temperatures play a key role in the applications of HOPO as an ingredient. Given its low viscosity at room and refrigerator temperatures, HOPO can be used as a fluid fat component in the making of functional fats such as shortenings, oleogels, soft margarines, or spreads, which require a soft texture at refrigerated storage conditions.E. Guineensis and E. Oleifera OxG was supplied by Thin Oil Products . The specifications of HOPO are summarized as an Iodine Value (IV) of >67, a melting point of 27 °C, and moisture of <0.05%. Refined, bleached, and deodorized standard palm oil (SPO) from E. Guineensis was supplied by Catania Oils . The specifications of SPO are summarized as an Iodine Value (IV) of >52.7 and a melting point of 39.7 °C.Refined, bleached, and deodorized (RBD) high oleic palm oil (HOPO) from the interspecific hybrid Some variability in the fatty acid profile for palm oil in the literature is expected as regional variability has been identified ,37. Due To examine the crystallization and melting events under static (non-sheared) conditions, a Differential Scanning Calorimeter (DSC) Q1000 (TA Instruments) was used. Triplicate samples of each oil weighing 8–9.8 mg were weighed into aluminum pans to the nearest 0.1 mg and tightly sealed. An empty, hermetically sealed aluminum pan was used as a baseline. The experiment was conducted in a controlled helium atmosphere chamber. Samples were equilibrated at 80 °C for 10 min, crystallized at 10 °C/min at −60 °C (held for 10 min), and melted at 5 °C/min at 80 °C (held for 10 min) as described by the AOCS Official Method Cj 1-94 . TA Univfinal bath1 − Tfinal bath2 = 25 °C) and therefore simulate similar cooling rates for each of the final temperatures. The 24 h conditioning facilitated the formation and stabilization of the crystals. The tests were performed immediately after removing the samples from each water bath. The puncture test was performed on a TA-XTplus texture analyzer  equipped with a 6 mm diameter cylindrical probe (probe at room temperature). The probe was inserted into the sample at 2 mm/s to a depth of 10 mm, and removed at 2 mm/s. The maximum force encountered during the insertion was recorded as the peak force, while the area under the force vs. time curve was recorded as the work of penetration. As per the method, the force area is correlated to the firmness of the sample. The test was replicated in n = 4 vials under identical conditions.A standard puncture test from Texture Technologies Corp  was condTo examine the morphology and size of fat crystals at various crystallization temperatures, the samples were observed using a Nikon Eclipse Ci  microscope equipped with polarized filters, similar to the methods described by Tang and Marangoni . To prep−1. Different final crystallization temperatures were chosen due to the sensitivity of the instrument ; therefore, the offset temperature of the first crystallization for each oil was chosen.Shear rheology was used to assess crystallization under sheared conditions, similar to the methods described in . The dynt-test. The level of significance for all tests was α = 0.05. Statistical analyses were performed with GraphPad Prism 8.0 .One-way ANOVA was used to identify differences between means with temperature as the independent variable in the texture analysis, and Tukey’s HSD test was used to determine the statistical significance. DSC onset, peak, and offset temperatures were compared using an unpaired


MicroRNA (miRNA) 107 expression is downregulated but Wnt3a protein and β-catenin are upregulated in degenerated intervertebral disc (IVD). We investigated mir-107/Wnt3a-β-catenin signaling in vitro and in vivo following hyperbaric oxygen (HBO) intervention. Our results showed 96 miRNAs were upregulated and 66 downregulated in degenerated nucleus pulposus cells (NPCs) following HBO treatment. The 3′ untranslated region (UTR) of the Wnt3a mRNA contained the “seed-matched-sequence” for miR-107. MiR-107 was upregulated and a marked suppression of Wnt3a was observed simultaneously in degenerated NPCs following HBO intervention. Knockdown of miR-107 upregulated Wnt3a expression in hyperoxic cells. HBO downregulated the protein expression of Wnt3a, phosphorylated LRP6, and cyclin D1. There was decreased TOP flash activity following HBO intervention, whereas the FOP flash activity was not affected. HBO decreased the nuclear translocation of β-catenin and decreased the secretion of MMP-3 and -9 in degenerated NPCs. Moreover, rabbit serum KS levels and the stained area for Wnt3a and β-catenin in repaired cartilage tended to be lower in the HBO group. We observed that HBO inhibits Wnt3a/β-catenin signaling-related pathways by upregulating miR-107 expression in degenerated NPCs. HBO may play a protective role against IVD degeneration and could be used as a future therapeutic treatment. Low back pain (LBP) is directly associated with reduced quality and expectancy life . InterveWnt (wingless and int-1) signaling is activated upon binding of several members of the Wnt protein family to the Frizzled/low-density lipoprotein receptor-related protein 5 or 6 (Fz-LRP5/6) receptor complex. This causes β-catenin stabilization and translocation to the nucleus, where it binds to the lymphoid enhancer factor (LEF) and T-cell factor (TCF) transcription factors to activate Wnt target gene expression . Wnt proβ-catenin signaling  plasmid (Promega) was used to transfect the cells in each well. The pGL4.74[hRluc/TK] plasmid containing the Renilla luciferase gene was used as an internal control for normalizing the transfections. Transient transfections using Lipofectamine LTX and PLUS reagent (Invitrogen) were performed according to the manufacturer’s instructions. Eight hours after transfection, the transfection medium was changed to DMEM/F-12 containing 5% FBS, and the cells were exposed to HBO. On days 3 and 5, the cells were re-transfected once and exposed to HBO as described above. After an additional 24 h of culturing, the cells were washed with PBS and harvested using 100 μL/well of passive lysis buffer (Promega). The cell lysates (20 μL) were evaluated for luciferase activity using a Dual-Luciferase Reporter Assay Kit (Promega). Luciferase activity was measured according to the manufacturer’s instructions.The activity of the Wnt signaling pathway was detected by a Wnt signal reporter assay using the TOP/FOP TCF reporter kit . Cells were seeded in 24-well tissue culture plates at 5 × 10g. NE-PER nuclear and cytoplasmic extraction reagents  were used to isolate cytoplasmic and nuclear extracts from the cells. The protein content was quantitated using a protein assay kit , and separated by 7.5% SDS-PAGE to detect β-catenin  and TATA binding protein .At 24 h after 3 HBO treatments, the cells were rinsed with PBS, treated with 0.05% trypsin, and collected by centrifugation at 800× −ΔΔCt method to calculate the relative mRNA levels of each target gene.At 24 h after 3 HBO treatments, cellular RNA was isolated using an RNeasy Mini Kit  and reverse-transcribed into cDNA with the ImProm-II Reverse Transcription System (Promega). For real-time PCR detection of Wnt3a, MMP-3, and -9 RNA transcripts, cDNA was analyzed on an ABI PRISM 7900 Sequence Detection System using TaqMan PCR Master Mix (Applied Biosystems). The cycle threshold (Ct) values were obtained, and the data were normalized to GAPDH expression using the 2The levels of MMP-3 and -9 in condition media (CM) after hyperbaric or normobaric treatments were determined using a commercial immunoassay kit . At 24 h after each treatment, 200 μL of CM was sampled and analyzed according to the manufacturer’s instructions. All measurements were performed in duplicate. The results were normalized to μg DNA. The DNA content was determined using DNAzol Reagent  according to the manufacturer’s instructions.We used the external compression device that was developed by Kroeber et al.  to creatSix IVD degenerated rabbits were used and divided into two groups: (I) HBO group, which included 3 rabbits exposed to HBO with 100% oxygen at 2.5 ATA for 1.5 h daily, 5 days a week after surgery in a HBO animal chamber , and (II) control group, housed in cages containing normal air. After 4 weeks of mechanical loading, the custom-made external loading devices were removed and Safranin-O staining was used to identify disc degeneration.At intervals of 1, 2, and 4 weeks after operation, venous blood was obtained from the rabbit ears in the control and HBO groups. Blood serum was obtained after centrifugation and analyzed for agKS  content by competitive indirect ELISA ,37. BrieThe degenerated IVD was sent for immunohistochemically analysis. Tissue blocks were fixed in 10% formalin, decalcified with 20% EDTA, and embedded in paraffin. Five-micron sections were cut and stained with Safranin-O. Tissue sections were deparaffinized and incubated with proteinase K  for 60 min at 37 °C. Endogenous peroxidase activity was blocked with 1% hydrogen peroxide. The presence and distribution of Wnt3a and β-catenin was determined using antibodies to detect Wnt3a  and β-catenin  with exposure at 4 °C overnight. Subsequently, a HRP linking 2° Ab was used for 30 min. Bound immunoglobulin was detected using a Dako REAL Envision Detection System  and 0.1% methyl green (Dako) was used for counterstaining.t-test to analyze the control/HBO ratio in this study. Data were represented as mean ± standard division (SD). The p-values for the paired Student’s t-test were performed using the SPSS software package . A p-value of <0.05 was considered statistically significant.Each human disc yielded one sample. The control and HBO samples separated from the same (Control group: without HBO treatment) so we used a paired n = 5). MiR-107 was chosen for further investigation  Target Scan 7.2 and (b) miRnalyze software. We found that Wnt3a, an important stimulator of canonical pathway, was likely a direct target of miR-107, as the 3′ UTR of Wnt3a contained a potential binding element for miR-107 with a 7-nt match to the miR-107 seed region a,b. Furtp < 0.01, n = 4), whereas mutation of the miR-107 binding sites abolished this inhibitory effect of miR-107  in the degenerated NPCs  or a mutant variant (Mut) of the Wnt3a 3′ UTR containing the target sequence was cloned into a dual-luciferase reporter vector a. Overexted NPCs b. These p < 0.001, n = 4) whereas transfection with anti-miR-107 upregulated the mRNA level of Wnt3a in NPCs following HBO intervention , whereas knockdown of miR-107 partly reversed Wnt3a protein expression in NPCs following HBO intervention . These data indicate that Wnt3a was negatively mediated by miR-107 at the post-transcriptional level in NPCs following HBO treatment, as overexpression of miR-107 following HBO intervention significantly inhibited the mRNA  and phosphorylated LRP6  were downregulated after culturing for 5 d with HBO intervention , whereas the FOP flash activity  was not affected. HBO downregulated the activity of the Wnt/β-catenin signaling.To evaluate the effects of HBO on the activity of the Wnt/β-catenin signaling, we analyzed the activity of both TOP flash (containing the wild-type TCF binding sites) and FOP flash (mutant TOP flash) in NPCs following HBO treatment. p < 0.001, n = 4) and MMP-9 . After mechanical loading, serum KS levels rose sharply in week 1, then dropped in week 2 and week 4 in the control group. Serum KS levels were also increased in the HBO group in week 1, but were significantly lower than that of the control group 2  and 4 weeks  after loading. Serum KS level was significantly increased after mechanical loading and gradually returned to near baseline after HBO treatment (The serum keratan sulfate (KS) levels in both groups are shown in reatment .p < 0.001, n = 3). In addition, stronger expression was observed in the β-catenin positive staining intensity ratio in the control group . IHC demonstrated that HBO treatment markedly suppressed Wnt3a and β-catenin expression in repaired IVD tissue.In the repaired IVD tissue, a reduced staining intensity ratio of Wnt3a (as a percent of the positive stained area) was observed in the cytoplasm and cartilage matrix of the HBO group  family, which consists of 10 members. Upon binding to FZD/LRP receptor complexes, components of the steady degradation complex for β-catenin—including Dvl, Axin, Gsk-3β, and others—are recruited to the receptors, and β-catenin degradation is blocked, causing its accumulation and translocation to the nucleus where it activates Tcf/Lef-dependent transcription of target genes [Wnt pathways are usually segregated into three divisions. The canonical pathway is the first and most intensely studied pathway involving the ligands Wnt1, Wnt2, Wnt3, and Wnt8. These ligands bind to LRP5/6 co-receptors along with receptors of the Frizzled  is an enzyme that belongs to the zinc metalloproteinases family involved in the degradation of the extracellular matrix. MMPs can cleave or degrade clotting factors, chemotactic molecules, cell–cell adhesion molecules, latent growth factors, and cell surface receptors. Out of 24 MMPs, MMP-9 is the only one that is both undetectable in healthy tissues and highly expressed in inflammation and in several diseases, including cancer . Pulsed Activation of Wnt/β-catenin has been reported to inhibit NPCs proliferation and trigger cell senescence, thereby promoting IDD aggravation . Li et aOur data suggest that HBO inhibiting the Wnt3a/β-catenin signaling pathway by upregulating miR-107 expression in human degenerated disc may be feasible as a therapeutic for degenerative disc disease. However, there are some restrictions in this study. Because other miRNAs may also be relevant for the regulation of Wnt signaling in NPCs following HBO intervention, transfection with anti-miR-107 only partly suppressed the effects of HBO treatment . In the HBO inhibits Wnt3a/β-catenin signaling-related pathways by upregulating miR-107 expression in degenerated NPCs. HBO may play a protective role against IVD degeneration and could be used as a future therapeutic treatment.


Musa spp.) holds significant global economic importance, but faces numerous challenges, which may include diverse abiotic and biotic factors such as drought and various diseases caused by fungi, viruses, and bacteria. The genetic and asexual nature of cultivated banana cultivars makes them unattractive for improvement via traditional breeding. To overcome these constraints, modern biotechnological approaches like genetic modification and genome editing have become essential for banana improvement. However, these techniques rely on somatic embryogenesis, which has only been successfully achieved in a limited number of banana cultivars. Therefore, developing new strategies for improving somatic embryogenesis in banana is crucial. This review article focuses on advancements in banana somatic embryogenesis, highlighting the progress, the various stages of regeneration, cryopreservation techniques, and the molecular mechanisms underlying the process. Furthermore, this article discusses the factors that could influence somatic embryogenesis and explores the prospects for improving the process, especially in recalcitrant banana cultivars. By addressing these challenges and exploring potential solutions, researchers aim to unlock the full potential of somatic embryogenesis as a tool for banana improvement, ultimately benefiting the global banana industry.The cultivation of bananas and plantains ( Musa spp.) are important staple food crops grown in over 136 countries in the tropics, with global production of 170 million tons annually (FAOSTAT2021). It is a valuable food security and cash crop as it can be cultivated in diverse environments and produces fruits throughout the year in these favorable weather conditions. Smallholder farmers mainly cultivate banana for domestic consumption and local or regional markets; only about 15% of production enters international markets. Hundreds of cultivars of bananas are grown and consumed worldwide, but large-scale farmers mainly grow the Cavendish type of dessert bananas for commercialization in local and international markets. Other dessert banana cultivars, such as ‘Sukali Ndiizi’, ‘Gros Michel’, ‘Rasthali’, and ‘Lakatan’, are also produced in several countries worldwide, where they are sold for economic and social benefits. Plantain is grown largely in Central and West Africa, and East African Highland banana (EAHB) is cultivated in East Africa, especially in the Great Lakes region of Africa, and is a source of livelihood for millions of people . On. On23]. Embryogenic cells from cell suspension cultures need to develop into somatic embryos e and theAcclimatization is the final step before the plants can be introduced to the farm or greenhouses for further growth and development. Ex-vitro banana plants are easier to acclimatize than those from other plant species and do not require special substrates such as peat moss or vermiculite. Well-rooted plants i can be Cryopreservation is a crucial technique in plant biotechnology that ensures long term and safe storage of important germplasm . It takeGenerating ECS of most elite banana cultivars remains a challenge because most of these cultivars are irresponsive to the available protocols. In the past, most somatic embryogenesis protocols for plants were developed based on a trial-and-error approach; however, increasing reports on somatic embryogenesis and recent advances in molecular biology uncover molecular mechanisms underpinning the process, providing novel research directions. Here, we summarize the factors to consider when optimizing somatic embryogenesis in banana.Most banana cultivars have not responded to somatic embryogenesis using the most responsive explants like scalps and immature male flowers. Thus, the focus should shift to explant selection and manipulation before callus initiation. Factors such as age and lushness affect the response of an explant to somatic embryogenesis. For instance, the response of immature male flowers to somatic embryo induction is better when the male buds are collected within three weeks after emergence, which also varies with the genotype ,41. AlsoSomatic embryogenesis is induced by supplementing the medium with different combinations of exogenous auxins and cytokinins. Typically, higher auxin concentration relative to cytokinin is needed for the induction and proliferation of somatic embryos in banana . DespitePlant tissue culture medium additives such as amino acids and antioxidants also play a significant role in somatic embryo induction and germination. Amino acids provide organic nitrogen and serve as precursors for endogenous synthesis of proteins and PGRs that facilitate somatic embryogenesis. Proline and glutamine are associated with embryo maturation, which improves embryo germination . TryptopConditions under which plant tissue cultures are maintained significantly affect the outcome of an experiment and, therefore, should be considered when optimizing somatic embryogenesis in banana. These conditions mainly include temperature, light, and humidity but can be extended to include medium conditions such as gelling strength, pH, and frequency of sub-culture/medium change. Temperature regulates many physiological processes in plants and thus plays a crucial role in somatic embryogenesis. The optimum temperature for somatic embryogenesis can vary with genotype and the embryogenesis stage. For instance, enhanced somatic embryo induction was observed at a lower temperature of 17 °C than at 28 °C in radiata pine . In contAgave tequilana [Araujia sericifera [Light requirement during somatic embryogenesis also varies with genotype and stage of embryogenesis ,53. In bequilana . Moreovericifera , suggestOther factors such as humidity, gelling strength, pH, and frequency of sub-culture also affect somatic embryogenesis. For example, Strosse et al.  reportedMolecular studies have revealed specific genes and signaling pathways that regulate plant somatic embryogenesis. These genes comprise transcriptional factors, cell cycle proteins, and stress/defense-related proteins that modulate endogenous hormone synthesis and other signaling pathways . TranscrA. thaliana [Medicago truncatula [Elaeis guineensis [Betula platyphylla [GhWUS from Gossypium hirsutum in A. thaliana promoted somatic embryogenesis [WUS (MaWUS2) is upregulated during the late stage of ECS, revealing that it potentially plays a crucial role in ECS proliferation in banana [WUS is a homeodomain TF modulating the formation of apical shoot meristem, which is crucial in somatic embryogenesis . WUS expthaliana . Upreguluncatula , Elaeis ineensis , and Bettyphylla . Recentlogenesis . A banann banana .BBM gene encodes AP2/ERF domain transcription factor implicated in multiple plant morphogenic responses [A. thaliana [Capsicum annuum [Glycine max [Zea mays [BBM (MaBBM2) is highly expressed at all stages of ECS development relative to NECS, suggesting that it could play an essential role in somatic embryo induction and proliferation in banana [esponses ,65. It aesponses . BBM hasthaliana , Capsicum annuum , Glycinecine max , and ZeaZea mays . Shivanin banana .Daucus carota, SERK genes are upregulated during somatic embryo formation up to the globular stage, indicating their essential role in the initiation and early stages of somatic embryogenesis [MaSERK1 expression in banana showed that it is upregulated in the embryogenic callus of immature male flowers compared with the non-embryogenic callus. Its expression is also higher during ECS proliferation, suggesting that it may be vital in the formation and proliferation of embryogenic callus in banana [The SERK family of TFs can serve as a marker of somatic embryogenesis in several plant species ,71,72. In banana .MaLEC2) in banana is higher in NECS, and it remains unclear whether MaLEC2 plays a negative regulatory role in banana somatic embryogenesis [The LEC family of TFs plays significant roles in somatic embryogenesis and has been demonstrated to induce somatic embryo formation in heterologous systems ,75,76,77ogenesis .MaPIN1 as a positive regulator and MaCRE2 and MaCRE3 as either negative regulators or insignificant in banana somatic embryogenesis [Besides TFs, other genes that regulate somatic embryogenesis have been identified in banana. Kumaravel et al. identified many genes involved in somatic embryo induction, development, and germination. Specifically, calcium-binding mitochondrial carrier protein and calcium-dependent protein kinase (CDPK) were upregulated 34.2 folds in embryogenic callus relative to non-embryogenic callus of banana cv. Rasthali . CDPK faogenesis . Furtherogenesis . Defenseogenesis . Cysteinogenesis ,82. Notaogenesis . In contogenesis .Musa spp., insights from studies involving other plant species could inform future related studies in banana. Studies have shown that DNA methylation influences somatic embryogenesis [A. thaliana demonstrated that de novo DNA methylation and its maintenance can affect somatic embryogenesis [A. thaliana and other plant species [Apart from DNA sequence variations, epigenetic factors have recently emerged as significant contributors to somatic embryogenesis . This efogenesis . Specifiogenesis ,86,87. Aogenesis . Notablyogenesis . Many ex species ,90,91. P species . Histone species . Also, t species .MusaDMR6 in banana using CRISPR/Cas9-mediated gene editing resulted in enhanced resistance to BXW disease [Genome editing via CRISPR/Cas9 has recently gained significance as the method of choice for genome engineering of crops. CRISPR/Cas9 tools have been recently optimized for banana and plantain crops using embryogenic cell suspension in several laboratories. A CRISPR/Cas9-based genome editing system for bananas has been established, targeting the knockout of the phytoene desaturase (PDS) as a visual marker gene . The end disease .In addition, the CRISPR/Cas9 was used to generate β-carotene-enriched Cavendish banana cultivar “Grand Naine” . FurtherLEC, WUS, and BBM. Although not yet reported in Musa spp., the overexpression of these TFs can significantly promote somatic embryogenesis and transformation efficiencies in diverse plant species, including dicotyledonous and monocotyledonous plants.The main obstacle hindering banana improvement via modern biotechnology is the recalcitrance of elite cultivars to somatic embryogenesis. Although manipulation of explants, medium components, and culture conditions can improve the frequency of somatic embryogenesis, it is improbable that such approaches will work for the most recalcitrant cultivars like the EAHBs. In addition, optimizing somatic embryogenesis via medium variation is time-consuming and less precise since it relies on trial-and-error approaches. Studies have also revealed that although many genes that mediate somatic embryogenesis are shared across diverse plant species, some are limited to certain plant species . Thus, fLEC 2 overexpression induced somatic embryogenesis in the seedlings of A. thaliana, confirming its potential role in auxin signaling [WUS induced callus formation and somatic embryogenesis by 400 folds [AtWUS from A. thaliana in cotton enhanced callus differentiation and positively modulated other TFs, including FUS3, LEC1, and LEC2 [A. thaliana revealed that it is mainly upregulated in plant tissues undergoing differentiation and morphogenesis [BBM from Brassica napus and A. thaliana in Nicotiana tabacum promoted cell differentiation. Still, they exhibited different responses depending on the plant species, suggesting that components of its signaling pathway may vary between species [A. thaliana and Theobroma cacao overexpressing the TcBBM gene from T. cacao. However, subsequent suppression of embryo development was observed in the T. cacao transgenic lines, implying that overexpression of BBM genes can adversely affect some genotypes’ later stages of somatic embryogenesis [BBM and WUS genes enhanced somatic embryogenesis and transformation of recalcitrant maize, sorghum, and rice cultivars [MaWUS2 and MaBBM2 could play a crucial role in somatic embryogenesis, suggesting that their overexpression has a great potential in revolutionizing somatic embryogenesis and transformation of recalcitrant banana cultivars [For example, ignaling . In coff00 folds . The hetand LEC2 . In addiogenesis . Heterol species . Notablyogenesis . Recent ultivars ,107,108.Somatic embryogenesis and the establishment of ECS in banana are highly dependent on genotype, which has limited banana genetic engineering for decades. Moreover, the process is time-consuming, and the maintenance of ECS is expensive and laborious. As a result, only a few banana cultivars have seen significant advancements through this method, while elite cultivars like the EAHBs have been left behind. Somatic embryogenesis in banana can be optimized by manipulating explants, culture medium components, and growth room conditions. However, overcoming the challenges posed by highly recalcitrant cultivars, particularly EAHB requires additional approaches. Fortunately, ongoing molecular studies provide new insights into the process and will likely contribute to future breakthroughs. Notably, recent investigations on the heterologous expression of morphogenic genes in monocots offer promising insights. This approach is anticipated to play a critical role in overcoming the genotype barrier associated with somatic embryogenesis and ultimately enabling the genome engineering of banana.By leveraging these molecular advancements, researchers can work towards improving the efficiency and effectiveness of somatic embryogenesis in bananas. It could lead to the successful somatic embryogenesis of elite banana cultivars like the EAHBs, which were previously challenging to incorporate into biotechnology programs. The enhanced somatic embryogenesis has the potential to revolutionize banana improvement efforts through modern biotechnology and open new possibilities for improving the traits and characteristics of this globally important crop.


The analysis of target protein co-expression, observed in the renal vesicles/immature glomeruli, ampullae/collecting ducts, convoluted tubules, metanephric mesenchyme of developing kidneys, but proximal convoluted tubules, distal convoluted tubules and glomeruli of postnatal kidneys, was performed using double immunofluorescence and semi-quantitative methods. The overall expression of acetylated α-tubulin and inversin during normal kidney development increases with higher expression in yotari mice as the kidney acquires mature morphology. An increase in β-catenin and cytosolic DVL-1 levels, indicating a switch from non-canonical to canonical Wnt signaling, is found in the postnatal kidney of yotari mice. In contrast, healthy mouse kidney expresses inversin and Wnt5a/b in the postnatal period, thus activating non-canonical Wnt signaling. Target protein expression patterns in kidney development and the early postnatal period observed in this study could indicate that switching between canonical and non-canonical Wnt signaling is crucial for normal nephrogenesis, while the defective Dab1 gene product in yotari mice may promote CAKUT due to interfering with this process.This study aims to determine the protein expression patterns of acetylated α-tubulin, inversin, dishevelled-1, Wnt5a/b, and  The development of the metanephric kidney starts with two types of tubulogenesis. Initially, the ureteric bud (UB) branches off the mesonephric duct, invades the overlying metanephric mesenchyme (MM), and undergoes branching morphogenesis ,2. This −/−Dab1 mutation causes CAKUT to be displayed as kidney hypoplasia [Disabled 1 (DAB1), an intracellular adapter protein found in mice, is essential for a signal transduction pathway that coordinates the formation of neural networks and neuronal positioning in the developing brain. During kidney development, the canonical reelin/DAB1 pathway is crucial for cell fate decisions and differentiation . A homozpoplasia . Reelin poplasia .β-catenin in the absence of Wnt ligands. The destruction complex is bound to the cell membrane and disabled by DVL when one of the nineteen Wnt ligands binds to the frizzled receptor and the LDL receptor-related protein 5/6 coreceptors, enabling β-catenin to avoid degradation and accumulate in the nucleus, thus activating canonical Wnt signaling [β-catenin, which is bound to the T cell factor/lymphoid enhancer factor (TCF/LEF) in the nucleus. This leads to complex impacts on cellular responses such as proliferation, survival, and differentiation [2+ pathway is one of the non-canonical Wnt pathways which causes intracellular calcium to be released and triggers calcium-dependent processes. Another non-canonical pathway is the Wnt/planar cell polarity (PCP) pathway, which is responsible for creating cell polarity and altering the arrangement of the cytoskeleton, leading to cell migration. In addition, the activation of several other transcription factors has also been observed [Glycogen synthase kinase-3 (GSK-3), a component of the protein-degrading complex, includes Axin, adenomatous polyposis coli (APC), and casein kinase 1, which typically targets cytosolic ignaling . Genes tntiation . The nonntiation . The Wntobserved . β-catenin depended on Wnt pathway by targeting cytoplasmic dishevelled (Dsh or DVL-1) for degradation, lead to nephronophthisis type II, an autosomal recessive cystic kidney disease characterized by extensive renal cysts, situs inversus, and renal failure [Ift20, which encodes intraflagellar transport protein-20, results in cilia loss and causes misorientation of cell division and cystic kidney disease in mice [In recent years, primary cilia have been shown to have key roles in kidney pathophysiology . For exa failure . Inversi failure ,22. Ther failure ,24. Cros in mice . Wnt5a deletions display a range of CAKUT phenotypes, from duplex kidneys and hydronephrosis to unilateral or bilateral renal agenesis [Wnt5a has a role in PCP regulation in mice, and disruption in the PCP pathway has been shown to result in kidney cyst formation [β-catenin-mediated canonical signaling acts transiently to induce the mesenchyme, as the downregulation of β-catenin activity is essential for the transition to the fully epithelial state of the renal vesicle [β-catenin signaling acts on tubular epithelial cells by upregulating certain of its target genes, some of which are important regulators of the tubular EMT process in the context of CAKUT, including fibroblast-specific protein 1 (FSP-1), matrix metalloproteinase 7 (MMP7), Snail, and Twist [β-catenin signaling consistently reduces renal fibrotic lesions and ameliorates kidney injury in a variety of chronic kidney disease models, indicating that targeting this signaling pathway may be an effective therapeutic intervention [β was discovered to enhance the EMT process by upregulating β-catenin and Wnt signaling. When compared to control cells, Arl13b and Ift20-knock down cells had higher levels of EMT triggered by TGF-β, which resulted in decreased cilia elongation and higher expression of EMT markers like fibronectin, α-SMA, and collagen III [Mice with agenesis . Wnt5a hormation . β-caten vesicle . Wnt/β-cnd Twist . Inhibitrvention . Throughagen III ,31. disabled 1 (−/−Dab1) mutation results in renal hypoplasia (CAKUT spectrum) accompanied with the foot process effacement in the glomeruli and the loss of functional structures [β-catenin in the developing and early postnatal −/−yotari Dab1 and wild-type mouse kidneys to determine whether alterations in Wnt signaling are present and their possible relation with the CAKUT spectrum.We previously showed a slight increase in the expression of α-tubulin and inversin and a decreased expression of DVL-1 in healthy postnatal human kidneys . Additioructures ,32,33. TThe Shiga University of Medical Science’s Guidelines for the Care and Use of Laboratory Animals permitted the use of animals in this research. The University of Split School of Medicine’s Ethical Committee granted its approval for the study, which was conducted according to the Declaration of Helsinki’s criteria .−/− yotariDab1 mutant mice characterized by an autosomal recessive mutation of the Dab1 gene, displaying phenotypes such as tremors, an unsteady gait, and early death around the time of weaning [Yotari (yot) and C57BL/6N wild-type mice were kept apart and housed in groups in typical polycarbonate cages with unrestricted food and water access in a temperature-controlled environment. (23 ± 2 °C). The photoperiod consisted of 12 h of artificial light and 12 h of dark. The following PCR primers were used for genotyping: yotari—GCCCTTCAGCATCACCATGCT and CAGTGAGTACATATTGTGTGAGTTCC; wild type—GCCCTTCAGCATCACCATGCT and CCTTGTTTCTTTGCTTTAAGGCTGT [yotari and wt) were analyzed. The mice were anesthetized by 1.5% sodium pentobarbital and transcardially perfused using phosphate buffer saline  and 4% paraformaldehyde (PFA) in 0.1 M PBS [This experiment used homozygous  weaning ,33,34. YAAGGCTGT . To acqu.1 M PBS . Their kThe tissue was first fixed and dehydrated using graded ethanol solutions  before being serially sliced into five µm-thick sections, embedded in paraffin blocks, and mounted on slides. The hematoxylin-eosin (HE) staining of every 10th section confirmed proper tissue preservation. The mounted tissue samples were heated in a sodium citrate buffer  for 20 min at 95 °C in a water steamer and then progressively cooled down to room temperature after deparaffinization in xylol and rehydration in graded ethanol and distilled water. After washing in 0.1 M PBS, protein-blocking buffer  was applied for 20 min to prevent non-specific staining. Sections were consequently incubated in a humidity chamber overnight with primary antibodies . The folβ-catenin were estimated by dividing the area of overlap using Adobe Photoshop. The quantity of positive cells in each group was averaged. Any level of nuclear, cytoplasmic, or membrane staining was considered positive. Different kidney structures’ staining intensity was semi-quantitatively assessed at four levels: the absence of any reactivity (−), mild reactivity (+), moderate reactivity (++), and strong reactivity (+++). Negative cells were categorized as cells with the absence of any immunoreactivity. Three researchers independently assessed the images while being blinded to the mouse strain and the time points. Interclass correlation analysis was used to examine the interrater agreement and produced a coefficient >0.75, suggesting excellent agreement [An immunofluorescent microscope  equipped with a Nikon DS-Ri2 camera  was used to analyze the sections. Non-overlapping visual fields were recorded at a magnification of x40 and constant exposure periods in order to determine the immunoexpression of proteins of interest. At least ten images of the embryonic kidney structures, namely, renal vesicles/immature glomeruli (Rv/G), ampullae/collecting ducts (A/Cd), convoluted tubules (Ct), and metanephric mesenchyme (MM), were taken at E13.5 and E15.5, but twenty images of the postnatal kidney structures, namely, glomeruli (G), proximal convoluted tubules (pct), and distal convoluted tubules (dct), were captured at P4 and P14. All analyzed images were processed with ImageJ software  and Adobe Photoshop  . The co-greement .yotari mice. The percentage of positive cells was represented as mean value standard deviation (SD), with p < 0.05 being the threshold for statistical significance.The statistical analysis was conducted using GraphPad Prism 9.0.0 software . In order to identify significant variations in the percentage of positive cells, immunoexpression was compared using a two-way ANOVA test and Tukey’s multiple comparison test between Rv/G, A/Cd, Ct, and MM at E13.5 and E15.5 and G, pct, and dct at P4 and P14 of wild-type and Yotari (yot) −/−Dab1 and wild-type (wt) mouse kidneys were used to assess the protein expression patterns of acetylated α-tubulin, inversin, DVL-1, Wnt5a/b, and β-catenin markers in the glomeruli (G), proximal (pct), and distal convoluted tubules (dct) at postnatal days P4 and P14. Embryonic structures including renal vesicles/immature glomeruli (Rv/G), ampullae/collecting ducts (A/Cd), convoluted tubules (Ct), and metanephric mesenchyme (MM) were examined using mice at gestation days E13.5 and E15.5. Focus was particularly placed on the co-expression of inversin with DVL-1 and Wnt5a/b with β-catenin. The obtained markers showed positive expression patterns but with differences in intensity  during E13.5, with a statistically significant increased expression in yot compared to wt mice .Acetylated α-tubulin was strongly expressed in the perinuclear cytoplasm and apical membrane of postnatal kidney structures, specifically the visceral epithelial cells (podocytes) of Bowman’s capsule and the dct; however, the pct showed weak protein expression in both mouse types e–h. The yot dct j. Postnayotari mice during the postnatal period . Ct showed greater expression in wild-type compared to yotari mice at E15.5 . Wild-type mice at E15.5 showed higher protein expression of DVL-1 compared to yotari in renal vesicles, convoluted tubules and metanephric mesenchyme . During P14, inversin immunoreactivity was present in the cytoplasm of the parietal layer of Bowman’s capsule in wt mice. Inversin reactivity drops in dct as the kidney acquires mature morphology (yotari mice (p < 0.0001). DVL-1 expression in G and dct was elevated significantly from P4 to P14 in yotari mice (p < 0.001). Co-expression of inversin and DVL-1 characterized cellular compartments of tubular cells in the pct and dct  . β-catenin immunofluorescence staining was significantly increased in the ampullae but decreased in the convoluted tubules of E13.5 wt compared to yotari (p < 0.001). During E15.5 β-catenin immunoreactivity was increased in wt collecting ducts and convoluted tubules when compared to yotari mice. The co-expression of Wnt5a/b and β-catenin was observed in the metanephric mesenchyme during the development of wild-type and yotari kidneys  . Yotari yotari at day E13.5 and E15.5 . There was no statistically significant difference in Wnt5a/b expression in dct and G of wt and yot mice at P4 or P14, and the same was true for β-catenin expression in the G and pct of wt and yot mice (β-catenin protein in the dct compared to P4 (p < 0.0001). β-catenin of yotari P14 was also decreased in relation to P4 but remains statistically higher when compared to wild-type β-catenin at day P14  mouse model to wild-type mice, was to determine the differences in spatio-temporal distribution patterns of acetylated α-tubulin, inversin, DVL-1, Wnt5a/b, and β-catenin in both embryonic and postnatal developmental stages and their correlation with the switch between canonical and non-canonical Wnt signaling, resulting with CAKUT when interrupted. Nephrogenesis, extending even into the early postnatal period of mice, is composed of complex processes precisely coordinated by the interaction of a large number of genes, whereas CAKUT has been associated with mutations in more than 20 genes so far ,39. Due yotari mice. Primary cilium-mediated Wnt pathway activation during kidney development enables cell proliferation, differentiation, and tissue morphogenesis. Many children with impaired primary cilia function and disrupted Wnt signaling develop chronic kidney disease (CKD) . Primaryβ-catenin signaling pathway regulates cell fate, proliferation, and survival during the early stages of kidney morphogenesis. Using β-catenin responsive TCF/β-gal reporter mic, canonical Wnt signaling activation was found in the epithelia of branching UB and induction during the progression of the MM to tubular differentiation [β-catenin is activated in animal models of both acute kidney injury and chronic kidney disease, which might function as a protective or harmful mechanism for the kidneys [yotari mice, which could lead to CAKUT. The canonical Wnt/ntiation ,43. In cntiation . The conntiation ,28. Wnt/ kidneys . Our stuyotari mice as kidneys acquired mature morphology, except for acetylated α-tubulin in the MM where overall expression is decreased and inversin in the Ct where higher expression is obtained in wild-type mice. On the contrary, a study by Solic et al. showed decreasing overall expression of these markers as kidney structures mature [yotari mice, compared to the decreasing expression in our previous study. There was an occasional co-expression of the inversin and DVL-1 proteins, mostly in the MM during nephrogenesis, pct, and dct during the postnatal period. Similar to the study of Solic I et al., our results showed a slight increase in the expression of inversin but a decrease of DVL-1 in healthy postnatal kidneys that corroborates the conclusion that the non-canonical Wnt pathway remains active after birth, but the canonical Wnt pathway is silenced [Results of this study determine that overall protein expression of acetylated α-tubulin and inversin during normal kidney development is increased, with higher expression in s mature . DVL-1 psilenced . These fsilenced .The localization of inversin (cystoprotein) at the base of the primary cilium in renal tubular cells is considered to be obligatory in canonical Wnt signaling inhibition through cytosolic, but not membrane-bound DVL degradation, in the maintenance of normal tubular elongation and positioning, and its mutation subsequently provoked abnormal proliferation in tubular cells, resulting in CAKUT spectrum disease through the cystogenesis process ,47. Membyotari mutant mice group. This investigation revealed a considerably lower percentage of β-catenin positive cells in the convoluted ducts and convoluted tubules of yotari mice at all observed embryogenic time points, indicating that β-catenin is needed for normal kidney morphogenesis and development. Our results demonstrated greater expression of β-catenin protein in the distal convoluted tubules of postnatal yotari kidneys compared to the control group, implying the switch from non-canonical to canonical Wnt/β-catenin signaling. Regular Wnt signaling pathway activation has been demonstrated to occur during tubulogenesis, but excessive canonical Wnt pathway activity in transplanted kidneys has been discovered to have a positive predictive value toward renal fibrosis [β-catenin has a renoprotective effect, whereas tubule-specific ablation of β-catenin exacerbates damage [β-catenin could be a potential therapeutic target against kidney fibrosis by reducing the hyperactivity of Wnt/β-catenin by natural compounds, particular inhibitors, or gene modification [Therefore, we explored whether these markers’ expression and the staining pattern are altered in different mice kidney phases when comparing the healthy control group to the fibrosis ,49. Studs damage . Howevers damage . Taking fication . Wnt5a in mice resulted in pleiotropic but severe kidney phenotypes, including agenesis, fused kidney, hydronephrosis, and duplex kidney/ureter [yotari, which confirms that non-canonical Wnt/PCP signaling remains active after birth in a healthy kidney.Wnt5a/b, which binds to both the canonical and non-canonical Wnt pathways, is a ligand for the seven-transmembrane receptor frizzled-5 and the tyrosine kinase orphan receptor 2. It is crucial for controlling developmental pathways throughout embryogenesis and the processes of oncogenesis. Global knockout of y/ureter . Wnt5a/by/ureter ,52. The y/ureter . Our resyotari mice exhibit higher expression compared to wild-type mice. In the postnatal period, a significant increase in Wnt5a/b expression is observed in proximal convoluted tubules of wild-type kidneys compared to yotari, which confirms its role in nephrogenesis. The co-expression of Wnt5a/b and β-catenin was observed during nephrogenesis in metanephric mesenchyme of wild-type and yotari mice, but only occasionally in dct and pct during the postnatal development.Wnt5a/b immunoexpression was observed in all structures during gestation, except the Ct at E13.5 in both mouse types, where β-catenin throughout different kidney development phases. Their balance and expression in all investigated kidney structures imply their important role in normal kidney development. We suggest that their interchange determines transcription in the canonical Wnt pathway or order of cell migration and polarization during kidney development in a non-canonical signaling pathway. The critical finding of this study is that β-catenin and cytosolic DVL-1 levels are decreased in the MM of embryonic kidneys of yotari mice, compared to wild-type mice, while the levels of inversin and Wnt5a/b are increased. This implies that the switch from canonical to non-canonical Wnt signaling occurs earlier in yotari mice, possibly causing a premature end to nephrogenesis, which could be a contributing factor to their renal hypoplasia phenotype. Furthermore, β-catenin and cytosolic DVL-1 are upregulated in the postnatal kidneys of yotari mice, implying a switch from non-canonical to overactivated canonical Wnt signaling, which could be another contributing factor to renal hypoplasia in yotari mice or a possible compensatory mechanism. In contrast, healthy kidneys expressed inversin and Wnt5a/b in the postnatal period, thus maintaining non-canonical Wnt signaling. Therefore, disturbances of α-tubulin, inversin, DVL-1, Wnt5a/b, and β-catenin found in diseased yotari kidneys might be the underlying pathological mechanism and a result of the switch from non-canonical to canonical Wnt pathway in the developing and postnatal kidneys, resulting in CAKUT and impairment of kidney function, such as chronic kidney failure. During normal kidney morphogenesis, the switch between the canonical and non-canonical Wnt-signaling pathway might be indicated by the protein immunoexpression pattern dynamics of acetylated α-tubulin, inversin, DVL-1, Wnt5a/b, and β-catenin in developing and early postnatal kidneys differs between −/−yotari Dab1 and wild-type mice, indicating alterations in Wnt signaling. Wild-type kidneys display increased levels of β-catenin and cytosolic DVL-1 during the embryonic period, while increased inversin and Wnt5a/b levels characterize the postnatal period, indicating that a switch from canonical to non-canonical Wnt signaling is crucial for normal nephrogenesis. On the contrary, embryonic kidneys of yotari mice show decreased levels of β-catenin and cytosolic DVL-1 with increased levels of inversin and Wnt5a/b, implying an early switch from canonical to non-canonical Wnt signaling. The postnatal kidneys of yotari mice demonstrate a decreased inversin and Wnt5a/b levels and increased β-catenin and cytosolic DVL-1 levels, indicating a switch from non-canonical to overactivated canonical Wnt signaling, possibly resulting in a CAKUT spectrum disease.The protein expression of acetylated α-tubulin, inversin, DVL-1, Wnt5a/b, and


In malignant cerebral infarction decompressive hemicraniectomy has demonstrated beneficial effects, but the optimum size of hemicraniectomy is still a matter of debate. Some surgeons prefer a large-sized hemicraniectomy with a diameter of more than 14 cm (HC > 14). We investigated whether this approach is associated with reduced mortality and an improved long-term functional outcome compared to a standard hemicraniectomy with a diameter of less than 14 cm (HC ≤ 14).Patients from the DESTINY  registry who received hemicraniectomy were dichotomized according to the hemicraniectomy diameter (HC ≤ 14 cm vs. HC > 14 cm). The primary outcome was modified Rankin scale (mRS) score ≤ 4 after 12 months. Secondary outcomes were in-hospital mortality, mRS ≤ 3 and mortality after 12 months, and the rate of hemicraniectomy-related complications. The diameter of the hemicraniectomy was examined as an independent predictor of functional outcome in multivariable analyses.Among 130 patients  age 55 (11) years), the mean hemicraniectomy diameter was 13.6 cm. 42 patients (32.3%) had HC > 14. There were no significant differences in the primary outcome and mortality by size of hemicraniectomy. Rate of complications did not differ . Age and infarct volume but not hemicraniectomy diameter were associated with outcome in multivariable analyses.In this post-hoc analysis, large hemicraniectomy was not associated with an improved outcome or lower mortality in unselected patients with malignant middle cerebral artery infarction. Randomized trials should further examine whether individual patients could benefit from a large-sized hemicraniectomy.https://www.drks.de; Unique Identifier: DRKS00000624).German Clinical Trials Register (URL: The online version contains supplementary material available at 10.1007/s00415-023-11766-3. In malignant cerebral infarction space-occupying brain edema is associated with a mortality of up to 80% despite intensive care treatment . Early hThe volume gained by hemicraniectomy directly correlates with the diameter of the bone flap. Current recommendations are based on in vitro models which calculated a minimum diameter of 12 cm to create an additional volume of at least 80 ml . While hBetween January 2010 and July 2016 patients with large ischemic infarction were prospectively included into the DEcompressive Surgery for the Treatment of malignant INfarction of the middle cerebral arterY—Registry (DESTINY-R) study in 30 neurological and neurosurgical departments in Germany and Austria.A detailed description of the study design has been published previously . The triFor this analysis patients who fulfilled the following inclusion criteria were selected from the complete study cohort: (1) ischemic infarction of at least 50% of the middle cerebral artery territory confirmed by computed tomography (CT) or magnetic resonance imaging (MRI) with corresponding clinical signs of a severe hemispheric syndrome, with or without additional infarction of the ipsilateral anterior or posterior cerebral artery territory, (2) hemicraniectomy performed according to the decision of the treating physician independent of study participation, (3) available postoperative imaging data measuring hemicraniectomy diameter, and (4) completed follow-up assessment of functional outcome conducted after 12 months. Patients with concomitant acute contralateral and/or infratentorial infarction or additional acute traumatic brain injury were excluded. The timing of hemicraniectomy and choice of the surgical technique  were left to the discretion of the treating neurosurgeon with a reference to the current guidelines .After study inclusion, sociodemographic factors, clinical parameters , pre-morbid functional status, vascular risk factors as well as surgical and medical treatment data were documented in a case report form . AdditioThe primary outcome was defined as the functional status according to the modified Rankin Scale score (mRS) after 12 months. Clinical outcome was dichotomized as mRS 0–4 versus 5 and 6 according to the definition of the pooled analysis of the three European hemicraniectomy RCTs . SecondaTIFT (Tension Imaging and Fiber Tracking) software [Neuroradiological parameters were processed centrally and blinded to functional outcome on the basis of CT or MRI scans on admission and after neurosurgery using software , 17, 19.software . In contTo simplify the measurement of the hemicraniectomy diameter on postoperative imaging data, the bone flap was considered as an ellipse oriented in sagittal axis see Fig. : First, All patients were dichotomized in two groups based on the diameter of the bone flap according to the larger major axis defining diameter ≤ 14 cm as “standard hemicraniectomy” (HC ≤ 14) and > 14 cm as “large hemicraniectomy” (HC > 14) , 14, 21.2 test were used as appropriate to identify differences between the two treatment-groups (HC ≤ 14 vs. HC > 14). To prevent confounding by indication, i.e., performing larger diameter of hemicraniectomy for larger infarctions, linear regression analysis between infarct volume at study inclusion and diameter of hemicraniectomy was performed. Using receiver-operating characteristic (ROC) analysis, threshold levels of hemicraniectomy diameter were calculated to evaluate the cut-off suggested by current guidelines of 14 cm. Diameter of bone flap and parameters of neurosurgery and conservative therapy were entered in a binary logistic regression model to evaluate their impact on functional outcome adjusted for age and infarct volume on admission. Results were expressed as odds ratios with 95%-Confidence Interval . The level of significance was defined as p < 0.05.Statistics were performed using the SPSS 26 software package . Descriptive analyses were calculated for all variables. Student´s t test, Mann–Whitney U test, or χ2 vs. HC > 14: 119.4cm2, p < 0.001) were larger than in the HC ≤ 14 group  were female, mean age (SD) was 54.5 (11.0) years, and 94 (72.3%) patients were younger than 60 years at study inclusion. One year after hemicraniectomy 27 (20.8%) patients had a mRS of 0–3 and 79 (60.8%) patients had a mRS of 0–4. 29 (22.3%) patients had died before the 12 months follow-up. Causes of death during hospital stay  were transtentorial herniation (N = 9), cardiac arrest (N = 2), pneumonia (N = 1), pulmonary embolism (N = 1), and unknown (N = 2). Causes of death during the follow-up period after discharge were not assessed. Depending on the decision of the operating neurosurgeon 88 (67.7%) patients underwent standard hemicraniectomy (≤ 14 cm) and 42 (32.3%) patients were treated by large hemicraniectomy (> 14 cm). Large hemicraniectomy was performed at 10 out of 30 study centers and 30 out of 42 (72%) cases were performed at 3 study centers. No significant differences in baseline demographic or clinical characteristics were found between the two hemicraniectomy groups  = 0.216, p = 0.643), suggesting an absence of confounding by indication and the unselected use of large over standard hemicraniectomy. In addition, ROC analyses did not determine a certain hemicraniectomy diameter other than 14 cm that could predict outcome , in-hospital mortality , or mortality after 12 months  with a sensitivity or specificity higher than chance.Neurosurgery-related parameters and complications regarding hemicraniectomy or intensive care are shown in Table In multivariable binary regression analyses with outcome as dependent variable, adjusted for age, volume of infarction, number of complications, frequency of wound healing disorders, frequency of subdural hematomas, differences in conservative therapy, and type of duraplasty performed, the diameter of hemicraniectomy was no significant predictor of outcome Table . The addDespite early prophylactic hemicraniectomy almost 20% of patients who suffer a malignant middle cerebral artery infarction still die. Most deaths occur early and are due to herniation which raises the question if some patients might benefit from a larger hemicraniectomy or if a larger hemicraniectomy in general could be beneficial . None ofMortality in this study population was similar compared to patients treated with hemicraniectomy in the pooled analysis of RCTs in younger patients with malignant infarction . FunctioSince infarct volume can predict outcome after decompressive hemicraniectomy , the assIn RCTs, the rates of hemicraniectomy-related complications were comparatively lower  , than inIn our cohort osmotherapy and muscle relaxation were applied more frequently in the standard hemicraniectomy group. Although both treatments had no impact on functional outcome in multivariable analyses, the increased use of osmotherapy and muscle relaxation in patients with smaller hemicraniectomies may be indicative of a poorer control of intracranial pressure compared to patients with larger bone flaps.Despite the strengths of a prospective multicenter registry, our study is not without limitations: it is a non-randomized study and the decision of timing, diameter, and technique of the hemicraniectomy was left at the discretion of the treating neurosurgeon. Hemicraniectomy with duraplasty as a procedure was not standardized , neitherIn conclusion, our results do not provide evidence that large hemicraniectomy is beneficial regarding functional outcome or mortality as compared to standard hemicraniectomy in unselected patients with malignant middle cerebral artery infarction treated by hemicraniectomy. Although our results indicate that standard hemicraniectomy may be associated with less complications and is sufficient in most cases, selected patients may still benefit from a larger diameter hemicraniectomy to achieve the best possible outcome. This relevant question should be addressed in a randomized controlled trial.Supplementary file1 (DOCX 32 KB)Below is the link to the electronic supplementary material.


We report on atomic level of detail analyses of polymer composite models featuring epoxy resin interfaces to silica, iron oxide, and cellulose layers. Using “reactive” molecular dynamics simulations to explore epoxy network formation, resin hardening is investigated in an unprejudiced manner. This allows the detailed characterization of salt-bridges and hydrogen bonds at the interfaces. Moreover, our sandwich-type composite systems are subjected to tensile testing along the interface normal. To elucidate the role of relaxation processes, we contrast (i) direct dissociation of the epoxy-metal oxide/cellulose contact layer, (ii) constant strain-rate molecular dynamics studies featuring (visco-)elastic deformation and bond rupture of the epoxy resin, and (iii) extrapolated relaxation dynamics mimicking quasi-static conditions. While the fracture mechanism is clearly identified as interface dissociation of the composite constituents, we still find damaging of the nearby polymer phase. The observed plastic deformation and local cavitation are rationalized from the comparably large stress required for the dissociation of salt-bridges, hydrogen bonds, and van der Waals contacts. Indeed, the delamination of the contact layers of epoxy resins with slabs of silica, magnetite, and cellulose call for a maximum stress of 33, 26, and 21 MPa, respectively, as compared to 84 MPa required for bulk epoxy yielding.Macromolecules 53(22): 9698–9705). The underlying interaction models are split into conventional Generalized Amber Force Fields (GAFF) for non-reacting moieties and a recently developed reactive molecular mechanics potential enabling epoxy bond formation and cleavage (ACS Polymers Au 1(3): 165–174).Molecular dynamics simulations using the Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) code were augmented by a Monte Carlo–type procedure to probe epoxy bond formation ( Industrial needs for resilient materials of light-weight and low-cost are often addressed by composites based on epoxy resins . This thIn the presence of additional particles, fibers or substrate surfaces, the epoxy-linker curing reactions need to adapt to the corresponding boundaries—and  polymer network formation is altered by hydrogen bonding, salt-bridges, etc. at the interface. The latter may be sufficiently strong to enable the use of epoxy resins as adhesives and surface coatings . Upon fuWhile a large body of experimental research and industrial development efforts is dedicated to the empirical engineering of constituents and processing parameters, in-depth understanding is increasingly collected from atomic force and electron microscopy –7. LikewFor the interpretation of polymer toughening from fiber and/or particle incorporation, the commonly discussed aspects range from frontal wakes and bridging effects in cracks to larger scale crack pattern formation . These cHowever, in lack of detailed information of the molecular scale processes at polymer-fiber/particle interfaces, these approaches often rely on ad hoc assumptions and empirical parameterization. To this end, much  experimental information such as stress-strain diagrams is needed for model development, whereas rigorous benchmarking is often limited to checks of self-consistency. An appealing alternative to such semi-empirical materials modelling could be given from atomic simulations. The latter are typically based on molecular mechanics that coarsen atomic interaction forces as observed from first principles calculations. Thanks to quantum chemistry, we may hence employ atomistic simulation data as a non-empiric route to assessing the inputs needed for meso-scale particle or continuum models –15.In what follows, we exemplify this strategy by characterizing the interaction forces governing interfaces of epoxy resins to commonly used composite constituents and substrate materials. Along this line, we account for silica as prominent species for particle incorporation, whereas cellulose reflects an important compound for polymer reinforcement from fibers, respectively. In turn, iron oxide, here magnetite, is addressed because of its importance for steel surfaces (mimicking corrosion passivation from bluing). Apart from atomic-scale analyses, a key focus is dedicated to the critical stress and mechanical work needed for interface separation—which is suggested as direct inputs for coarse-grained particle concepts and continuum models.2 sized slab of 1.8-nm thickness and comprises 3024 Si, 5799 O2-, and 498 OH- ions, respectively; details on model preparation from melt quenching are provided in refs. [2) [2 surface charge density at pH=7 [The silica-epoxy (i), magnetite-epoxy (ii), and cellulose-epoxy (iii) interfaces are described as sandwich models featuring 2D periodic slabs of the two constituents that are staggered using 3D periodic boundary conditions. As a starting point to model system, (i) we employ an amorphous silica model developed in an earlier study . It reflin refs. , 15. Theefs. [2) . While e at pH=7 –20, in o2- ions of the magnetite slab were protonated  to mimic the effect of air humidity. While the explicit surface structure of humid magnetite surfaces is still under debate [2 slab features a thickness of 1.3 nm and comprises 700 Fe2+, 1300 Fe3+, 2500 O2-, and 300 OH- ions, respectively.In turn, (ii) the iron oxide slab was constructed from the unit cell replicated 7 × 7 × 1.7 to a super cell of magnetite with (100) surface. In analogy to the silica surface, also the outermost Or debate , our modβ(1-4) linked, D-glucose molecules. Upon geometry optimization from energy minimization, fiber-fiber hydrogen bonding leads to a well-defined 6.0×6.0 nm2 slab of 1.8-nm thickness.The third model (iii) mimics a cellulose slab formed by 5 stacks of cellulose sheets—each of which featuring 7 parallel cellulose fibers. The nanometer scale fiber models were described by oligomer chains of 11 covalently connected, i.e., 2, 5.7×5.7 nm2, and 6.0×6.0 nm2 sized layer segments of models (i), (ii), and (iii), respectively, whereas the polymer precursors are added along the remaining dimension. For each system, 1024 EPON (bisphenol-F-diglycidyl ether) and 512 DETDA  molecules were introduced and 3D-periodic boundary conditions are applied to create sandwich-style bulk systems.All three slab models were first relaxed in vacuum to achieve well-defined 2D-periodic layers. Next, the simulation cells were fixed in two dimensions according to the 7.2×7.6 nmBenefitting from our earlier development of a reactive molecular mechanics model for describing the polymerization reaction , we explThe atomic interactions of the EPON, DETDA, and hardened epoxy networks are described by the reactive molecular mechanics model reported in ref. . Therein−1—as suggested by Fennel [The van der Waals pair potentials were cut off at a distance delimiter of 1.2 nm. In turn, the more long-rage Coulomb interactions were described by the damped-shifted force approach . For thiy Fennel . The epoy Fennel . The cony Fennel . Using tz (upper epoxy block) and −Δz for the lower epoxy block of the sandwich models. Consequently, the cell vector along interface normal (z direction) of the 3D-periodic simulation models are elongated by 2∙Δz. For this, cell vector elongation is implemented at a 0.015, 0.15, and 1.5 m/s using three independent molecular dynamics runs, respectively. The temperature is kept at 300 K—which allows relaxation as governed by the crossing of energy barriers to epoxy reorganization [z-direction, the thermostat is only applied to velocities along x and y directions, respectively.The separation of the composite interfaces is explored by two rather dissimilar strategies. We firstly explored (A) block-wise separation of the two entities without structural relaxation. This block-wise separation mimics the idealized case of brittle fracture by separation of the contact layers of the sandwich models. Thus, we argue that this “brute-force” approach refers to an upper estimate of composite fragmentation at extremely high speed. In turn, tensile testing is also explored (B) by an iterative procedure of gradual displacement steps. In each step, we move the two epoxy blocks embedding the metal oxide or cellulose slabs by shifting all atoms collectively by ΔOn this basis, run (A) is confined to block-wise separation, whereas only run (B) enables visco-elastic relaxation and gradual bond rupture in the epoxy resin. Subsequently, we used our slowest deformation runs (B) for σ along the pulling direction z—which as directly calculated as the virial σzz, which only reflects atomic interaction forces directed along z. Moreover, the work of separation is calculated from the integral of the stress profiles σzz(s) as functions of the sandwich separation by s = Σ Δz.To compare the resulting fracture pathways, we monitor the stress The realistic account of epoxy composite interfaces calls for the explicit consideration of the formation process of the material. Akin to the experimental preparation of such composites, also simulation models should not be based on simply attaching blocks of cured polymer to other constituents. Instead, the guest constituents are typically introduced to the epoxy/hardener liquid mixture before or at the very beginning of the polymerization reactions. Thus, polymer network formation occurs in the direct presence of filler particles or fibers.To ensure the unbiased investigation of the interplay of polymer-polymer interactions and the forces of the polymer-metal oxide/cellulose interfaces all sandwich models were subjected to a combined Monte Carlo/molecular dynamics simulation procedure of gradual network formation and relaxation . To thisIn brief, relaxation of the interfaces and the unreacted EPON/DETDA precursor liquid is allowed from 2.5-ns scale molecular dynamics simulation runs at 1 atm and 460 K using the conventional, non-reactive force-fields. Next, we switched to our reactive force field  within a2, whereas other binding motifs appear negligible. The underlying O-H distances were found in the range of 0.2–0.25 nm thus indicating rather weak hydrogen bonds. For comparison, the hydrogen bonds within the uppermost cellulose layer show O-H distances of 0.12–0.15 nm and the overall density of cellulose-cellulose hydrogen bonds amounts to 1.3 per nm2, respectively.Illustrations of the fully relaxed sandwich models are shown in Fig. 3O4 slab is observed. The underlying Fe-O and Fe-N distances of 0.18–0.32 nm however indicate strong electrostatic interactions. Finally, the silica-epoxy interface features Si-O (but no Si-N) salt-bridges at an occurrence of 0.22 contacts per square nanometer and distances of 0.23–0.32 nm, in parallel to hydrogen bonding donated by the silanol groups to O (0.24 per nm2) and N (0.05 per nm2) acceptors of the polymer, respectively. While representative structural motifs are highlighted in Fig. In turn, the magnetite-epoxy interactions are clearly dominated by Fe-O and Fe-N salt-bridges at occurrences of 0.86 and 0.02 contacts per square nanometer, respectively. Practically no hydrogen bonding between the polymer and the (surface-hydroxylated) Fes were instead taken from a series of single point calculations. In turn, the derivatives of these energy profiles indicate the adhesive forces of the interfaces. While this procedure offers very rapid assessment of overall interface energies, the mechanisms of epoxy-cellulose and epoxy-metal oxide dissociation are surely more complex. Indeed, run A reflects the limiting case of concerted salt-bridge and hydrogen bond cleavage that gives rise to rather unrealistic estimates of the maximum stress σmax.Two types of molecular dynamics runs were performed to elucidate interface separation. In run A, we scanned the separation energy by block-wise displacement of the polymer domains and the cellulose/metal oxide slabs. To provide an upper estimate of rapid interface dissociation, in run A no structural relaxation was performed and energy profiles as functions of the displacement proper relaxation of the sandwich models during tensile testing, we performed nanosecond scale molecular dynamics simulation in run B. For this, we implemented epoxy and cellulose/metal oxide separation at constant deformation rate To go beyond concerted bond dissociation, and thus enable  in ref. . In Fig.entclass1pt{minimas(σmax) = 0.16–0.26 nm, full interface dissociation is observed at separation by more than 1 nm.Before the dissociation of hydrogen bonds and salt-bridges at the interfaces, the epoxy resin features elastic and visco-elastic deformation. Upon gradual release of interfacial bonds, also the polymer network experiences significant plastic deformation. This particularly holds for the magnetite-epoxy sandwich model. As a consequence, we do not observe abrupt decline of the adhesion at the interface, but a more complex interplay of polymer network rearrangement and step-wise dissociation of hydrogen bonds and salt-bridges. The latter process extends over nanometer scale displacements of the composite constituents. While our sandwich models show maximal stress at σzz(s) of tensile testing the silica, cellulose, and magnetite interfaces to epoxy resin are shown in Fig. s = s(σmax), 1 and 2 nm, respectively), additional runs at constant s (pull-and-hold) were performed. On the basis of exponential relaxation, we describe the time-dependent profiles asThe underlying stress profiles s(σmax) lead to A = 3.6, 3.6, and 4.5 MPa and τ = 3, 1.5, and 7 ns for epoxy interfaces to silica, cellulose, and magnetite, respectively.where σmax to be reduced by 60–75%. On the other hand, also the maximum elongation before bond dissociation is drastically reduced.To one side, we derived the maximum stress extrapolated for mentclass2pt{minimσmax as well as the underlying displacement s(σmax) to describe the linear increase in stress within 0 ≤ s ≤ s(σmax). In turn, (ii) yielding is modelled for s(σmax) < s ≤ ssep by assuming a linear decline of the stress until σ(ssep) = 0. While σmax and s(σmax) are well-defined properties assessable from the stress profiles  and centimeter scale fibers , respectively . For the3O4 rather than pure iron or rusted surfaces, our simulations address machine parts that were pre-treated via bluing processes [The current sandwich-type model systems arguably reflect only the first step within a series of studies needed for the comprehensive understanding of particle/fiber-enforced epoxy composites. However, for the somewhat simpler system of steel-epoxy resin interfaces, we suggest the layer-wise contact as a quite realistic model for understanding adhesion. Focusing on magnetite Ferocesses .