Despite the considerable research attention paid to block copolymer (BCP)-toughened epoxy resins, the effects of their terminal groups on their phase structure are not thoroughly understood. This study fills this gap by closely examining the effects of amino and carboxyl groups on the fracture toughness of epoxy resins at different temperatures. Through the combination of scanning electron microscopy and digital image correlation (DIC), it was found that the amino-terminated BCP was capable of forming a stress-distributing network in pure epoxy resin, resulting in better toughening effects at room temperature. In a 60 wt.% silica-filled epoxy composite system, the addition of a carboxyl-terminated BCP showed little toughening effect due to the weaker filler/matrix interface caused by the random dispersion of the microphase of BCPs and distributed silica. The fracture toughness of the epoxy system at high temperatures was not affected by the terminal groups, regardless of the addition of silica. Their dynamic mechanical properties and thermal expansion coefficients are also reported in this article. Epoxy resins and their composites are widely sought due to their excellent chemical resistance, novel mechanical and electrical insulation properties, and good adhesion to various materials. Moreover, the flexible processability of epoxy resin is not possessed by other thermosetting materials ,5,6,7,8.Epoxy-resin-based composites are often used as underfill adhesives for electronic assembly for integrated circuit (IC) chips using advanced electronic packaging technology. They fill the gap between the chip and the substrate under capillary action. After curing, the thermal stress originally concentrated on the solder joints around the periphery of the chip can be redistributed to the chip, underfill material, substrate, and all solder joints. As semiconductor silicon technology develops towards nanometer feature sizes, 3D system-level packaging with large sizes, narrow pitch, and high interconnected I/O density have almost become an inevitable trend. The high heat caused by higher power consumption, the greater warping deformation caused by large-size chips, and the flow filling problems caused by a narrow pitch and high I/O density have posed more stringent challenges to the mechanical properties of epoxy composite underfill.The toughening of epoxy systems has long been an activity of Interest ,14,15,16Poly(methyl methacrylate) (PMMA) and poly(n-butyl acrylate) (PnBA) comprise PMMA-b-PnBA-PMMA, an amphiphilic tri-BCP that is an important BCP toughener. Asada et al. studied the BCP-induced microphase separation process in epoxy in situ using small-angle X-ray scattering. They found that the separation of the microphase occurred in two steps. First, a micelle structure is formed through the self-assembly of the BCP in uncured epoxy resin. Next, the micelle grows during curing and forms miscible PMMA blocks embedded in the epoxy network . KlingleIn this study, the effects of PMMA-b-PnBA-b-PMMA tri-BCP phase structures with amino and carboxyl terminating groups on the fracture toughness of a diamine-cured epoxy resin system were compared. Additionally, the morphology and distribution of the toughening agent phase in the silica-filled epoxy system were examined. The effects of different group-terminated BCPs on the strain field around the crack tip of a single-sided notch cantilever beam (SENB) specimen were observed using digital image correlation (DIC). The toughening mechanisms of the two BCPs were compared based on microscopic fracture surface analysis and strain field characterization, and the fracture toughness of the composite system was investigated at high temperatures. The storage modulus, glass transition temperature, and thermal expansion coefficient of the material were evaluated, providing valuable guidance for the application of BCPs in electronic packaging.2 with a particle size of approximately 900 nm  was used. The chemical structures of these materials are shown in The epoxy resin used in this study was a diglycidyl ether of bisphenol F precursor purchased from Hexion  , and diethylenediamine was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China . Amino-terminated blocked tri-BCP (Amino-BCP) was purchased from Arkema , and carboxyl-terminated tri-BCP (Carboxyl-BCP) was purchased from Kuraray Co., Ltd. . Spherical SiOFirst, the BCPs were dissolved in epoxy resin at 150 °C with a ratio of 5.714 g of BCP per 100 g of resin. The mixture was stirred for 30 min until clear, and the curing agent was added to the cooled premixture and mixed using a planetary mixer  at 1500 and 2000 rpm for 2 min. For the 60 wt.% silica-filled epoxy system, silica was added with a hardener, and the same mixing procedure was used to ensure that the filler was fully dispersed. After the equipment cooled, a 6 min vacuum defoaming process was performed at 800 rpm. The mixed liquid mixture was preheated on a hot stage at 60 °C for 3 min, followed by curing in a polytetrafluoroethylene mold at 165 °C for 2 h. After cooling for 30 min, the cured epoxy resin/composite specimens were removed from the mold. 3, and the temperature range was 25–180 °C. The thermal expansion coefficients (CTEs) of the epoxy resin composites were determined using static thermomechanical analysis . A sample size of 5 × 5 × 4 mm3 and compression mode were adopted. The system was heated from 20 to 280 °C at a heating rate of 5 °C/min.The storage modulus and Tg of epoxy composites were tested using dynamic thermo-mechanical analysis  in double-cantilever mode at 1 Hz and 3 °C/min. The sample size was 60 × 13 × 3 mmQP is the peak force required to propagate a crack, B is the specimen thickness, W is the specimen width, f(W) is the geometric factor. The sample was a rectangular parallelepiped with rounded edges measuring approximately 36 × 4 × 8 mm3. Prior to the test, a sharp pre-crack was initiated ahead of the machined notch in each specimen by tapping it with a fresh razor blade. The initial crack length, a, was measured using an optical microscope and satisfied the 0.45 ≤ a/W ≤ 0.55 length requirement. The loading rate was set at 0.5 mm/min.The fracture toughness was measured using an SENB test according to the ASTM E1820 standard  in a uniThe plane strain condition requires the specimen to satisfy the following conditions to ensure its validity:The fracture surface of the cured epoxy resin composite was analyzed using a scanning electron microscope , and the phase structure of the BCP was observed. All samples were coated with a thin layer of sputtered gold to facilitate electrical conduction on the surface. The accelerating voltage was set at 10 kV.In situ observation of the strain field at the tip of the spline crack during the fracture toughness test was performed using DIC  Co., Ltd.) to obtain full-field displacement and strain distribution by comparing two images with different deformations ,50,51.The storage modulus and Tg of both the epoxy resin/BCP and epoxy resin–silica/BCP systems were tested. As shown in CTE is especially important for ensuring the suitability of epoxy composites in electronic packaging. Although the terminal groups were different, the addition of BCPs had a similar effect on the CTEs of the epoxy resins. The TMA results showed an increase in CTE1 and a decrease in CTE2 when both BCPs were introduced into the epoxy. The addition of the amino-terminated BCP led to a more significant increase of 4.8 ppm/K in CTE1 and a decrease of 11.1 ppm/K in CTE2, compared with neat epoxy. This is mainly caused by the lubricating effect of flexible BCPs in the epoxy resin matrix before Tg. However, after Tg, BCPs occupying a certain spatial position in the matrix can hinder the movement of chain segments in the matrix.2 system, the addition of both types of BCPs had little effect on CTE2, as shown in 2 was still significant, and was 3.2 ppm/K higher than that of EP/Carboxyl-BCP/SiO2. Therefore, it can be inferred that there were differences in the network structure formation of the epoxy resin between the two different terminal group BCPs, among which the structure of EP/amino-BCP/SiO2 was more conducive to the movement of chain segments.In the EP/SiO2 system, the addition of the amino-terminated BCP caused an increase of 1.5 MPa·m1/2 in its fracture toughness, whereas the addition of the carboxyl-terminated BCP reduced the facture toughness by 0.5 MPa·m1/2. At 150 °C, the effect of the BCP on both pure and silica-filled epoxy systems disappeared. This may have been caused by the significant decrease in storage modulus after the epoxy system reached Tg (around 120 °C). Note that the fracture toughness of both the resin and composite systems at high temperatures was significantly lower than that at room temperature; therefore, the difference in the BCP systems with different terminal groups was no longer obvious, suggesting that there was a significant difference in the toughening mechanism at room and high temperatures. This can be explained by the SEM observations of the fracture surfaces of the samples at different test temperatures, which are analyzed in detail in The fracture toughness results for the pure and silica-filled epoxy systems are shown in The fracture surfaces of the broken room-temperature SENB specimens were observed using SEM. As shown in Further analysis of the fracture surface of the carboxyl-terminated BCP system showed that after the crosslinking and curing reactions, a significant number of island-phase structures of different sizes were distributed in the epoxy resin matrix. These dispersed phases act as fillers. As shown in A comparison of the fracture surface results  at room 2 and EP/amino-BCP/SiO2 were not significant, and there was a large amount of residual matrix on the surface of the SiO2. However, on the fracture surface of EP/carboxyl-BCP/SiO2, most of the SiO2 surface was relatively smooth, and the dispersed phase size of the carboxyl system was significantly smaller than that prior to the introduction of SiO2. The fracture surface differences between the three groups of samples tested at high temperatures were generally small. However, by comparing the surfaces of the samples at different test temperatures, it was found that the phenomena of debonding and void growth were very common at room temperature. At high temperatures, most of the silica deboned directly, and voids caused by filler extrusion and matrix deformation could hardly be observed. Combined with the storage modulus in thermomechanical analysis, it can be seen that when the temperature of the samples reaches 150 °C, the chain segment movement is easily opened, causing the storage modulus of the epoxy resin to significantly reduce to less than 10 MPa. The matrix undergoes highly elastic deformation under shear conditions, forming the shear morphology in the cross-section. Due to the low modulus, even if the deformation of the matrix on the cross-section is significantly greater than that of the cross-section at room temperature, the energy that can be stored based on the deformation is significantly reduced, resulting in the fracture toughness of various systems at high temperatures being generally small and basically consistent.Additionally, the fracture surface of the silica-filled epoxy system was investigated . At room2 tested at room temperature, the cause of the decrease in the fracture toughness of the system after the introduction of 60 wt.% SiO2 was found. As shown in 2 was introduced, the island phase became smaller and was distributed around the SiO2, disrupting the original resin network system. This caused cracks to propagate preferentially along the deboned void edge rather than within the matrix. After further analysis of the fracture surface of EP/carboxyl-BCP/SiOUsing DIC technology, in situ observations of the crack propagation process at room temperature were performed. The strain distribution of the spline was obtained when it reached its maximum load, compared with the initial state at zero load. As shown in 2 and BCP, the network structure of the epoxy resin changed, and the molecular chains of the different systems underwent changes to their maximum strain. Generally, the wider the strain distribution, the greater the strain, the more fracture energy absorbed, and the better the fracture toughness.The strain distribution in the silica-filled epoxy system was also observed b, and th2; after 60 wt.% SiO2 was introduced, the fracture toughness at room temperature increased by 31.2%. Additionally, the fracture toughness of the epoxy resin system at high temperatures was not affected by the terminal groups of block copolymers. In terms of thermal mechanical properties, the storage modulus and Tg of EP/Amino-BCP were basically not affected, but there was a slight amplification effect on CTE1. Compared with the control group, there was an increase of 4.8 ppm/K in CTE1 without the introduction of silica; after 60 wt.% SiO2 was introduced, there was an increase of 6.1 ppm/K in CTE1. In addition, based on the characterization results of SEM and DIC, it was found that the SiO2/Epoxy de-bonding, void growth, and the stress transmission of the block copolymer are the reasons for toughening. The discussion of results in this work provide an in-depth understanding of the effect of the different toughening agents on material properties and, therefore, provide guidance for the design of epoxy-resin-based composites in underfill applications.This work proved that amino-terminated BCPs had the most obvious effect on the fracture toughness of an epoxy resin-amine curing system at room temperature. Due to the effective stress transmission of block copolymers as flexible segments in the matrix, compared with the control group, the fracture toughness at room temperature increased by 68.9% without adding SiO


Prmt1, Prmt4/Carm1 and Prmt5 represent the most prominently expressed PRMT subtypes in mouse calvarial bone and MC3T3 osteoblasts as well as human musculoskeletal tissues and mesenchymal stromal cells (MSCs). Based on effects of siRNA depletion, it appears that PRMT members have different functional effects: (i) loss of Prmt1 stimulates and (ii) loss of Prmt5 decreases calcium deposition of mouse MC3T3 osteoblasts, while (iii) loss of Carm1 is inconsequential for calcium deposition. Decreased Prmt5 suppresses expression of multiple genes involved in mineralization  consistent with a positive role in osteogenesis. Depletion of Prmt1, Carm1 and Prmt5 has intricate but modest time-dependent effects on the expression of a panel of osteoblast differentiation and proliferation markers but does not change mRNA levels for select epigenetic regulators . Treatment with the Class I PRMT inhibitor GSK715 enhances extracellular matrix mineralization of MC3T3 cells, while blocking formation of H3R17me2a but not H4R3me2a marks. In sum, Prmt1, Carm1 and Prmt5 have distinct biological roles during osteoblast differentiation, and different types histone H3 and H4 arginine methylation may contribute to the chromatin landscape during osteoblast differentiation.Osteogenic differentiation of mesenchymal cells is controlled by epigenetic enzymes that regulate post-translational modifications of histones. Compared to acetyl or methyltransferases, the physiological functions of protein arginine methyltransferases (PRMTs) in osteoblast differentiation remain minimally understood. Therefore, we surveyed the expression and function of all nine mammalian PRMT members during osteoblast differentiation. RNA-seq gene expression profiling shows that   •Protein arginine methyltransferases (PRMT) were surveyed for expression and functions during osteoblast differentiation.•Prmt1, Prmt4/Carm1 and Prmt5 are prominently expressed in osteogenic cells.•Prmt proteins have different effects on calcium deposition and/or osteoblast marker gene expression.SiRNAs for •The PRMT inhibitor GSK715 enhances calcium deposition in MC3T3 osteoblasts.•The results suggest that histone arginine methylation contributes to osteoblast differentiation. ChromatThe protein arginine methyltransferase (PRMT) family is comprised of nine evolutionarily conserved proteins that catalyze the methylation of arginine residues . These eMethylation of arginine residues is fundamental for chromatin remodeling to support different cellular processes  . MultiplBecause PRMTs have broad biological functions, a key question in bone biology is whether and how PRMT family members control osteogenic differentiation of mesenchymal stem/stromal cells (MSCs) and osteoblasts, as well as bone formation and bone homeostasis . Initial studies have suggested individual roles of PRMTs in musculoskeletal tissues, including skeletal myoblast differentiation, chondrocyte proliferation and osteoblastic cells . In this22.1RNA-seq data was retrieved from human musculoskeletal tissues including bone, cartilage, growth plate, muscle and adipose tissue, as previously reported . In addi2.210352097] that were acquired from the American Type Culture Collection. These cells were cultured at 37 °C in 5 % CO2 with complete maintenance medium, which is α-minimal essential medium (Gibco) without ascorbic acid and supplemented with 10 % fetal bovine serum , 100 units/ml penicillin (Gibco), and 100 μg/ml streptomycin (Gibco). MC3T3 cells were seeded in 6- or 12-well plates in maintenance medium . The following day (day 0), we divided the cultures into two groups. Cells were subjected to siRNA transfections with control , Prmt1 siRNA , Prmt4/Carm1 siRNA , Prmt5 siRNA . Transfections were performed using RNAiMAX as instructed by the manufacturer (Invitrogen). After 6 h, osteogenic medium consisting of complete maintenance medium supplemented with 50 μg/ml ascorbic acid (Sigma) and 4 mM β-glycerol phosphate (Sigma) was added, and the cells were cultured until harvest. Studies with the PRMT inhibitor GSK715 were performed similarly, except that instead of transfection, cells were treated with GSK715 one day after seeding  or also after three days . For both siRNA and PRMT inhibitor studies, we selected the time of harvest based on the first visual appearance of calcified extracellular matrix nodules which typically occurs between days 21 and 32 depending on the passage number, actual seeding density and type of experimental treatment.Our studies used MC3T3 E1 sc4 murine calvarial osteoblasts [PMID: 2.3−ΔΔCt method and normalized to the housekeeping gene GAPDH/Gapdh using gene specific primer sequences (Cells were lysed using TRIzol Reagent (Invitrogen) and RNA was isolated using the Direct-zol RNA MiniPrep Kit (Zymo Research) with on column DNA digestion kit (Zymo Research). Isolated RNA was reverse transcribed into cDNA using the SuperScript III First Strand Synthesis System (Invitrogen). Gene expression was quantified using real-time PCR in which each reaction was performed with 10 ng cDNA per 10 μl, QuantiTect SYBR Green PCR Kit (Qiagen), and the CFX384 Real-Time System machine (BioRad). Transcript levels were quantified using the 2equences .Table 1S2.42) were plated in 6-well plates in maintenance medium and treated with siRNA (see above) or the Class I PRMT inhibitor GSK715  as described previously  for 12 min. Upon removal of the Alizarin Red, plates were rinsed for 3 times with distilled water and imaged using a standard plate scanner. Absorption of Alizarin Red stain was quantified with ImageJ software . Because2.6t-test or two-way ANOVA for multiple comparison analyses. Histochemical assays and RT-qPCR time course experiments were performed in triplicate for each time point; each RT-qPCR measurement was performed as a technical duplicate. Experiments were replicated three times and results from a representative course are shown. Significance is denoted by asterisks in the figures .Statistical analysis and generation of figures was performed with GraphPad Prism software (version 9) using Student's unpaired 33.1PRMT or mouse Prmt genes are expressed in musculoskeletal cell types and tissues. RNA-seq data from mesenchymal versus non-mesenchymal cell types in mouse callus during fracture repair show that Prmt1, Prmt4/Carm1 and Prmt5 are preferentially expressed in mesenchymal cell types compared to non-mesenchymal cell types in callus tissue (sorted using a dsTomato fluorescent protein lineage tracer) . PRMT1, PRMT4/CARM1 and PRMT5 are the three most prominently expressed PRMT members across all tissues examined . Whereas PRMT1 is the highest expressed member in each of the tissues, PRMT4/CARM1 and PRMT5 are particularly prominent in muscle and cartilage, respectively . PRMT mRNAs were also examined in human bone marrow derived mesenchymal stem cells (BMSCs) . Similar to expression in musculoskeletal tissues, we find that PRMT1, PMRT4/CARM1 and PRMT5 are the most highly expressed PRMT members in human BMSCs, and their expression remains relatively constant during osteogenic lineage differentiation . Furthermore, Prmt1, Prmt4 and Prmt5 are also highly expressed in frontal and parietal bone of calvaria from two different mouse strains   at day 2 strains B&C. Simi strains D. Expresanalysis . Taken t3.2Prmt1, Prmt4/Carm1 and Prmt5 in mouse osteoblastogenesis, we conducted loss of function studies using siRNAs in MC3T3 cells. We examined short-term effects of siRNA depletion at both mRNA and protein levels  , while loss of Prmt4/Carm1 has no appreciable effects on calcium accumulation. In contrast, inhibition of Prmt5 dramatically decreases calcium deposition . There is no clear correlation between mineralization and Crystal Violet staining  . RT-qPCR analysis shows that siRNA depletion of each PRMT protein does not have strong numerical effects on cell cycle regulated mRNAs, including expression of the mitosis-related mRNA marker Ccnb1 or the proliferation-related Mki67 mRNA . We note that expression values at some time points exhibit statistical significance. However, the observed numerical changes cannot easily be interpreted and may not necessarily be biologically relevant. At face value, loss of CARM1 at day 7 reduces the mitosis related marker CCNB2 and increases the general proliferation marker MKI67. The latter would suggest that absence of CARM1 may decrease the G2/M population and increase the number of cells in G1 phase around day 7. Because loss of CARM1 does not alter Alizarin Red staining, these molecular changes have uncertain biological relevance. Furthermore, loss of PRMT5 results in a significant albeit quantitatively modest reduction (<2 fold) of both CCNB2 and MKI67, suggesting that PRMT5 loss may induce a quiescent cell state. The latter could potentially contribute to a reduced level of calcium deposition by preventing confluence of MC3T3 cell cultures  which are typically expressed at maximal levels around day 14 (Col1a1) and non-collagenous (Ibsp) genes and, second, of expression of mineralization specific genes (Bglap and Phospho1). Reduced Prmt1 expression significantly increases expression of Ibsp and Phospho1 at day 10 (p < 0.01), but does not appear to have statistically significant effects expression of Alpl or Bglap at any timepoint. Interestingly, Ibsp expression is reduced by Prmt1 loss at day 3  while Phosho1 expression may be accelerated at day 10 . While these changes indicate that loss of Carm1/Prmt4 has effects on osteoblast-related gene expression , and Phospho1 at day 10, while there appears to be a time-course trend for decreased expression of Bglap at days 6, 10 and 15 , with a numerical trend of decreased expression for Runx2, Osterix/Sp7 and Dlx5 during the differentiation time-course  that was not statistically validated. Aside from significant reduction of Sp7 in the siPrmt5 treatment group, expression analysis of these transcription factors upon depletion of Prmt1 or Prmt5 did not yield unambiguous results that permit a straightforward interpretation . Similarly, loss of Prmt1, Carm1 and Prmt5 mRNAs does not appear to modulate expression of epigenetic regulators known to contribute to skeletal development  . We conclude that PRMT1, PRMT4/CARM1and PRMT5 proteins have functional roles that are not directly rate-limiting for the expression of key osteoblast related transcription factors or other epigenetic regulators.Because PRMT1, PRMT4/CARM1 and PRMT5 proteins modulate distinct osteoblast markers at different stages of osteoblast differentiation, we assessed whether these epigenetic regulators may have indirect effects on osteoblastogenesis by controlling the mRNA expression of osteoblast related transcription factors  or other epigenetic regulators . The only remarkable finding of these RT-qPCR results is that 2, Brd4) , except 3.4To investigate whether the enzymatic activity of Class I PRMT enzymes  affects osteoblastogenesis, we performed studies with the pharmacological inhibitor GSK715 A. Treatmt-tests compared to controls. None of these observed changes, except for one, was statistically significant  due to the large spread in values.We tested the effects of GSK715 on MC3T3 differentiation using Alizarin Red staining . MC3T3 c2 values 0.5589 and 0.2185, respectively . Interestingly, the fitted lines for the 2× curve  shows a consistent 3 to 6 fold increase in densitometric values for cells treated with concentrations of between 1.4 and 5.9 μM GSK175; for example, comparison of staining of cells treated with vehicle  compared to a representative low dose of GSK175  reveals a 4.3 fold stimulation in Alizarin Red staining by GSK715 .The densitometric values of Alizarin Red quantification for each of the dose response curves were also fitted to a polynomial curve . The relative fit for the 2× dose response curve was better than the 1× dose response curve that have RWe note that GSK715 has biphasic effects on MC3T3 cells with apparent positive effects at low doses and negative effects of GSK715 at higher doses  & S7. NeCollectively, the findings of these pilot studies indicate that pharmacological inhibition of Class I PRMT enzymes that control nuclear H3R17me2 levels may increase calcium deposition during osteoblast differentiation. Comparison of findings obtained with GSK715 versus siRNA treatments for Prmt1, Prmt4 and Prmt5 indicates that none of these three epigenetic enzymes by themselves can account for the positive effect of the inhibitor on osteoblast mineralization. In conclusion, histone arginine methylation contributes to osteoblast differentiation and selective pharmacological inhibition of PRMTs may promote osteoblastogenesis.4PRMT1, PRMT4/CARM1 and PRMT5 mRNAs are selectively expressed in human and mouse musculoskeletal tissues. These three genes are generally most prominently expressed (i) across multiple human musculoskeletal tissues examined , (ii) in frontal and parietal bone of mouse calvaria, (iii) in human bone marrow derived MSCs and mouse MC3T3 osteoblasts, and (iv) in mesenchymal cell types present in early callus tissue during fracture repair. Loss of function studies using siRNA knockdown of Prmt1, Prmt4/Carm1 and Prmt5 in MC3T3 cells indicate that each of these Prmt members has distinct roles during osteoblast differentiation: Prmt1 loss enhances while Prmt5 loss inhibits calcium deposition, while Prmt4/Carm1 loss by itself is dispensable for calcium deposition in the extracellular matrix of differentiating osteoblasts. Gene expression analysis reveals that loss of Prmt1, Pmrt4/Carm1 and Prmt5 each modulates the expression of several but not all stage-specific mRNA biomarkers for osteoblast differentiation, although not proliferation. Finally, pharmacological inhibition with the Class I PRMT inhibitor GSK715 enhances calcium deposition in the extracellular matrix of MC3T3 cells. Collectively, these data establish that PRMT proteins have distinct effects on osteoblast differentiation.In this study, we examined the contribution of histone arginine methylation to osteoblast differentiation by surveying the expression and function of the full complement of human PRMT proteins. Our findings establish that Several technical limitations qualify our interpretations and conclusions. One major assay for the effects of PRMT loss of osteoblast differentiation is Alizarin Red staining which measures calcium deposition and not necessarily deposition of bone mineral (hydroxyapatite) in MC3T3 cells. While methods that are more definitive for hydroxyapatite  could be applied, we consider that examination of definitive effects on bone mineralization will require in vivo studies that are beyond the scope of the present work. This study does not focus on recapitulating fidelity of bone-tissue like organization, but rather we use calcium deposition as a proxy for osteoblast differentiation to understand which PRMTs modulate osteoblastogenesis. Furthermore, our study includes only a single PRMT drug (GSK715) which exhibits non-specific negative effects at higher micromolar doses. Therefore, it will be important to survey additional PRMT drugs, similar to studies we performed to screen for EZH2 inhibitors  before wOur study shows that PRMT1, PRMT4/CARM1 and PRMT5 each have different functions in osteoblast maturation and/or expression of osteoblast related markers. Results obtained for PRMT5 are most easily interpretable. Loss of PRMT5 suppresses expression of multiple genes  involved in mineralization and this finding corroborates reduced Alizarin Red staining upon siPRMT5 treatment. Our finding that PRMT1 inhibits osteoblast differentiation is in agreement with other studies indicating that PRMT1 promotes the undifferentiated state of cells . The resRecent studies suggest that PRMT proteins also have molecular effects on the activity of the H3K27me3 transferase EZH2 that normally suppresses osteoblast differentiation . One sol2 D3) dependent upregulation of CYP24A1 gene expression (2 D3). Similarly, when the CYP24A1 gene promoter remains transcriptionally silent, the CYP24A1 locus is enriched with both H3R3me2s marks and the corresponding methyl transferase PRMT5 pression . Examinase PRMT5 . Furtheritamin D . Taken titamin D  indicateIn conclusion, our study shows that PRMT1, PRMT4/CARM1 and PRMT5 proteins each have different effects on osteoblast mineralization and/or expression of osteoblast-related differentiation markers. The mechanisms by which PRMT1 and PRMT5 control osteoblast differentiation may involve effects on the histone arginine methylome, as well as direct and indirect effects on EZH2, which is a known suppressor of osteogenesis. One translationally relevant finding from our study is that the Class I PRMT inhibitor GSK715 enhances osteoblast differentiation. This finding predicts that GSK715 may potentially have bone anabolic effects in mouse models for low bone mineral density, a prediction that will be tested in future studies.10.13039/501100010659Erasmus University Medical Center (to BCJvdE), the 10.13039/100000069National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01 AR069049 to AvW & R01AR039588 to GSS), as well as the 10.13039/100009395CancerCare Manitoba Foundation (761020451 to JD).This publication was made possible through internal funding of PD performed the majority of the experiments, while EAL contributed additional supporting data, reagents and experimental ideas. PD, EAL, JARG, MAM, JPTMvL, GSS, BCJvdE, JRD & AJvW provided principal conceptual contributions pertinent to the conclusions of this work. PD prepared the first draft of this manuscript, while EAL, JARG, MAM, JPTMvL, GSS, BCJvdE, JRD & AJvW each provided critical input and suggested edits. AJvW obtained the main source of funding for this study, while EAL, GSS, BCJvdE and JRD provided auxiliary funding.Parisa Dashti: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing – original draft, Writing – review & editing. Eric A. Lewallen: Conceptualization, Funding acquisition, Investigation, Writing – review & editing. Jonathan A.R. Gordon: Conceptualization, Writing – review & editing. Martin A. Montecino: Conceptualization, Writing - review & editing. Johannes P.T.M. van Leeuwen: Conceptualization, Project administration, Supervision, Writing – review & editing. Gary S. Stein: Conceptualization, Funding acquisition, Writing – review & editing. Bram C.J. van der Eerden: Conceptualization, Project administration, Supervision, Writing – review & editing. James R. Davie: Conceptualization, Funding acquisition, Writing – review & editing. Andre J. van Wijnen: Conceptualization, Data curation, Funding acquisition, Investigation, Resources, Supervision, Validation, Writing – review & editing.The authors have no conflicts of interest to declare. All co-authors have seen and agree with the contents of the manuscript and there is no financial interest to report.


The experimental results show that with the same amount of alumina filling, the thermal conductivity and Tg (glass transition temperature) of epoxy/Al2O3 composite material decrease with the increase of alumina particle size. The maximum thermal conductivity of the composite material is 0.679 (W/mK), while the energy storage modulus of epoxy/Al2O3 composite material increases with the increase of alumina particle size, and the maximum energy storage modulus of the composite material is 160MPa. Compared with pure epoxy resin, the thermal conductivity and energy storage modulus have increased by 2.7 and 3.2 times, respectively. The epoxy/Al2O3 composite was applied to the COB (Chips On Board) type LED package, and the substrate temperature of the LED dropped to the lowest after 1.5 hours of operation using EP-A5 composite, and the temperature was stabilized at 38.2°C, indicating that the addition of 5-micron alumina composite has the best heat dissipation in the COB type LED package. These results are critical for the implementation of particulate-filled polymer composites in practical applications because relaxed material specifications and handling procedures can be incorporated in production environments to improve efficiency.Epoxy thermal conductive adhesives with high thermal conductivity and dynamic mechanical properties are important thermally conductive materials for fabricating highly integrated electronic devices. In this paper, micro-Al Due to the rapid development of microelectronics technology, the integration  and mini2O3 materials have much research in thermally conductive composites due to their high thermal conductivity, low cost and stable chemical properties, making alumina a promising filler in industrial applications [2O3 to epoxy resin to prepare 0.5 wt% b-Al2O3/EP composites, 30 wt% b-Al2O3/EP composites, and 70 wt% b-Al2O3/EP composites, and the experimental results showed that the thermal conductivity of 70% filled b-Al2O3/EP composites, the thermal conductivity of 70% b-Al2O3/EP composites is 1.13 W/(mK), which is seven times higher than that of pure epoxy resin. Zhou et al. [μm/M5μm/M0.5μm/M50nm is 2:5:1:1, the thermal conductivity of the composite rubber reaches 1.45 W/(mK), which is the highest thermal conductivity among all filled rubbers, and the highest thermal conductivity when using a single particle size Al2O3. Mai et al. [2O3 fillers and their epoxy resin composites. The experimental results showed that nano-Al2O3 coating in micro-Al2O3 showed that the modification effectively improved the infiltration between Al2O3 and epoxy resin, reduced interface defects caused by weak bonding between Al2O3 fillers and epoxy resin, and synergistically enhanced the thermal conductivity and mechanical properties of epoxy resin composites. The thermal conductivity of micro-Al2O3/EP, micro@nano-Al2O3/EP-6:1 and micro-Al2O3/EP, micro@nano-Al2O3/EP-3:1is 0.874 W/(mK),1.001 W/(mK), and 1.072 W/(mK), respectively. Compared with micro-Al2O3/EP, the modification effect is 11.45% and 12.27%, respectively. These above experimental results all indicate that nano-and micron-level alumina can increase the thermal conductivity of composites and improve thermal conductivity.However, the incorporation of metal particles usually deteriorates composites’ electrical insulation and dielectric properties, limiting their wide application in electronic packaging , 23. In ications , 27. In ications , 28. Simications . Yu et aications  added b-u et al.  added dii et al.  prepared2O3 composites with different particle sizes of micron alumina particles. It investigates the effects of different alumina particle sizes on the thermal conductivity and thermomechanical properties of epoxy/Al2O3 composites. This paper has practical significance for implementing particle-filled polymer composites in practical applications, as loose material specifications and processing procedures can be incorporated into production environments to improve efficiency.The construction of thermal conductivity networks in epoxy resins using nano alumina fillers with high specific surface area provides a very promising opportunity. However, nano fillers are difficult to disperse in epoxy resin and may exhibit agglomeration during the preparation process , 34. It 2O3 with 99.9% purity and particle sizes of 5,10,40,60, and 120 μm from Sumitomo Chemical Co., Ltd. The surface modifier was pyrolytic silica (HDK H18 type) from Wacker Chemicals (Cinha) Co., Ltd.The epoxy resin is bisphenol A epoxy resin (NPEL-128E type) produced by Nanya Electronic Materials Co., Ltd. The curing agent was 2-Aminoimidazole (GY HT110 type) manufactured by Guangzhou Guyan Electronic Materials Co., Ltd. The diluent is 4-tert-butylphenyl glycidyl ether (type 692) produced by Suzhou Senfida Chemical Co., Ltd. The thermally conductive filler was AlPreparation of epoxy resin composites filled with different particle sizes of alumina using a planetary stirring device . First, 41.5vol.% liquid bisphenol A epoxy resin and 5 vol.% diluent were added to the beaker and stirred for 5 minutes. Then add 1 vol.% H18 and blitz with a planetary blender for 10 minutes. Next, 50 vol.% alumina was added and placed in the planetary stirring device for 10 minutes, vacuuming for 10 minutes. The mixture was cooled to 0°C in a refrigerator, and finally, the curing agent was added and stirred and mixed for 15 minutes, molded on glass and cured at 100°C for 2h to prepare the cured composite. The flow chart of the sample preparation was shown in -1. Samples were mixed with KBr powder and pressed into a disc.Infrared spectra were obtained from a Nicolet 380 FTIR spectrometer  with an accumulation of 32 scans at a resolution of 0.5cmThe microscopic morphology of the composite cross-section was studied using a scanning electron microscope .Thermal conductivity was measured using a thermal conductivity meter  at room temperature using the transient planar hot plate method in the direction perpendicular to the sample plane.2O3 micro-composites was tested using the DMA242C instrument from NETZSCH. The mechanical modulus was obtained by simultaneous testing of stress and strain. The temperature range was 40°C-200°C, the heating rate was 5 K/min, the frequency was 1 Hz, the measured sample size was 10 x 10 x 2 (thickness) mm3, the applied force was 5 N, and the amplitude was 40 μm.The dynamic mechanical property of epoxy/Al2O3 composite material is bonded between the COB-type substrate and the luminaire housing, and the temperature of the selected points of the substrate and housing is tested using a TP1000 temperature data logger produced by Shenzhen Topley Electronics Co. The driving voltage of COB is 50V, the current is 1.2A, the power of COB type LED is 60W, and the working time is 1.5h.The epoxy/Al2O3, FTIR spectra of functionalized Al2O3 were recorded in 2O3, EP-A40 exhibits new absorption at 2965 and 2860 cm-1, which represent the valence stretching vibration of aliphatic C-H. The peaks at 1182,1095 and 827 cm-1 are assigned to the C-O-C stretch, Si-O-Si stretch, and epoxy group, respectively. The result of FTIR indicate that the epoxy group has been successfully introduced into the surface of Al2O3 particles through chemical grafting [2O3 granules pack together more continuously and closely to fill the space of epoxy resin structure.To demonstrate the actual occurrence of chemical reactions between epoxy resin and Algrafting , 27, 40.2O3 (30–40 W/mK) is much larger than that of pure epoxy resin (0.1–0.2 W/mK). Therefore, the addition of Al2O3 to the epoxy resin matrix will improve the thermal conductivity of the epoxy resin, and the particle size of Al2O3 will affect the thermal conductivity of the composite epoxy resin.The thermal conductivity of Alμm alumina particles filled with epoxy resin, respectively. The average interparticle distance decreases with decreasing particle size, and smaller particle size provides more linked alumina particles. Most of the alumina particles in EP-A120 are uniformly dispersed in the matrix without agglomeration, and the particles are not interconnected  are widely used in the study of viscoelastic properties of materials to measure data such as dynamic energy storage modulus (E′), loss modulus (E″) and loss tangent angle of materials under vibrational loading, as well as to test the glass transition temperature Tg value of materials. The glass transition temperature (Tg) is an important parameter affecting the material’s process and service performance . The DMAμm increases the energy storage modulus of epoxy/Al2O3 composites and has a small effect on Tg.Figs Figs 2O3 composite materials and reduce their Tg. Using a particle size of 5μm Al2O3 can effectively improve the storage modulus of epoxy resin/Al2O3 composite materials with little impact on the Tg of the composite material, which is beneficial for its application as a thermally conductive material in electronic packaging. Based on our research results, considering micron fillers may be a wise decision when designing high-performance epoxy-based electronic packaging materials with ideal comprehensive performance and balancing other functional requirements such as thermal conductivity and low cost.This article investigates the thermal conductivity and dynamic mechanical properties of five different micrometer alumina particle-doped epoxy resin composites. The thermal conductivity results indicate that many cheap small-sized spherical alumina as the main body of the thermal conductivity network can more effectively form thermal conductivity paths, significantly improving the thermal conductivity performance of epoxy composite materials. Forming an effective thermal path is a critical factor in determining thermal conductivity. In addition, DMA results revealed molecular motion at the interface between micrometer alumina particles and EP matrix. Adding alumina particles can increase the storage modulus of epoxy/AlS1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


Tribolium castaneum. However, the regulatory mechanisms of TcCTL16 proteins are still unclear. Here, spatiotemporal analyses displayed that TcCTL16 was highly expressed in late pupae and early adults. TcCTL16 RNA interference in early larvae shortened their body length and narrowed their body width, leading to the death of 98% of the larvae in the pupal stage. Further analysis found that the expression level of muscle-regulation-related genes, including cut, vestigial, erect wing, apterous, and spalt major, and muscle-composition-related genes, including Myosin heavy chain and Myosin light chain, were obviously down-regulated after TcCTL16 silencing in T. castaneum. In addition, the transcription of TcCTL16 was mainly distributed in the hemolymph. TcCTL16 was significantly upregulated after challenges with lipopolysaccharides, peptidoglycans, Escherichia coli, and Staphylococcus aureus. Recombinant CRDs of TcCTL16 bind directly to the tested bacteria (except Bacillus subtilis); they also induce extensive bacterial agglutination in the presence of Ca2+. On the contrary, after TcCTL16 silencing in the late larval stage, T. castaneum were able to develop normally. Moreover, the transcript levels of seven antimicrobial peptide genes  and one transcription factor gene (relish) were significantly increased under E. coli challenge and led to an increased survival rate of T. castaneum when infected with S. aureus or E. coli, suggesting that TcCTL16 deficiency could be compensated for by increasing AMP expression via the IMD pathways in T. castaneum. In conclusion, this study found that TcCTL16 could be involved in developmental regulation in early larvae and compensate for the loss of CTL function by regulating the expression of AMPs in late larvae, thus laying a solid foundation for further studies on T. castaneum CTLs.C-type lectins (CTLs) are a class of proteins containing carbohydrate recognition domains (CRDs), which are characteristic modules that recognize various glycoconjugates and function primarily in immunity. CTLs have been reported to affect growth and development and positively regulate innate immunity in  Each CTL has one or more carbohydrate recognition domains (CRD), known as CTL domains, which are composed of β-sheets, α-helices, and loops [Insect innate immune systems utilize soluble and membrane-bound receptors to recognize pathogen-associated molecular patterns (PAMPs) on the surface of pathogenic microorganisms . Beta-1,nd loops . Drosophila melanogaster lectin 2 (DL2) and DL3, belonging to the CTL-S type, could agglutinate E. coli and enhance melanization and encapsulation in vitro [Helicoverpa armigera CTL-3 [Ostrinia furnacalis IML-10 [D. melanogaster is required for the development of bristles and eyes [Tribolium castaneum. Animal CTLs are usually involved in innate immunity, including regulating anti-fungal immunity , enhanciin vitro ,14. Regein vitro . Helicovra CTL-3  and Ostrs IML-10 , belongis IML-10 . Studiesand eyes ,20. Simiand eyes ,22,23. HT. castaneum, a destructive insect pest of stored grain-based products, is a model organism widely used in immunological, developmental, and evolutionary biology research [T. castaneum genome using a bioinformatics approach [T. castaneum [T. castaneum [T. castaneum. In contrast, viruses are able to utilize mosquito CTLs to facilitate infection. A previous study identified an Aedes aegypti CTL, mosGCTL-1, that facilitated West Nile virus (WNV) infection in vitro and in vivo. The expression of mosGCTL-1 was upregulated after WNV infection, and it interacted with mosPTP-1, which enabled viral attachment to cells and facilitated viral entry [Aedes aegypti mosGCTL genes were first identified by Liu et al. [mosGCTL-3 could significantly enhance viral infectivity by interacting with the DENV-2 envelope protein in vitro and in vivo. However, it is not clear whether there are genes in the CTL family of T. castaneum that negatively regulate the immune response during bacterial infection and what the underlying regulatory mechanism is.research ,25. A toapproach . Earlierapproach . TcCTL5 astaneum . In addiastaneum . There ial entry . Moreoveu et al.  as key sTcCTL16 has not been studied yet. To explore the function of TcCTL16, a CTL-X type CTL, we cloned the full-length cDNA of TcCTL16 from T. castaneum and investigated its developmental and tissue-specific expression profiles, as well as its response to different elicitors. Most importantly, we investigated its role in development and pattern recognition, agglutination, and AMP regulation after bacterial infection. This research lays a solid foundation for further studies of T. castaneum CTLs.In the past 2–3 decades, many studies on the functions of insect CTLs in innate immunity have been published. Nevertheless, the function of TcCTL16 is 2511 bp in length and encodes a protein of 836 amino acids (The open reading frame (ORF) of no acids , with a no acids . As showno acids .T. castaneum formed close lineal homology groups with CTLs in Hymenoptera (H. laboriosa and A. glabripennis), Lepidoptera (M. sexta and B. mori), and Diptera (A. gambiae and D. melanogaster)  are clustered into one branch, suggesting that TcCTL3 may originate from CTL-S-type lectins  . Our res lectins .TcCTL16 was transcribed throughout all developmental stages, showing a low level at the LA stage and high levels at the LP and EA stages , the cumulative survival rate of T. castaneum was only 2% , erect wing (ewg), apterous (ap), and spalt major , were obviously down-regulated compared with the control group (Myosin heavy chain (Mhc) and Myosin light chain (Mlc), were also decreased   and Gram− bacteria (E. coli and P. aeruginosa) but had no binding ability to B. subtilis (+ bacteria (S. aureus and B. thuringiensis) and Gram− bacteria (E. coli and P. aeruginosa) but had no agglutinating ability to B. subtilis.rCTL16 was expressed in inclusion bodies from 21 (DE3) A, lane 421 (DE3) A, lane 521 (DE3) B, lane 6subtilis C. MoreovTcCTL16 RNAi reached 65% on the first day and 80% on the third and remained at 80% on the fifth day  was upregulated, while the expression of att1, att3, and def3 was not compared to the control beetles (ds-GFP + E. coli) (TcCTL16-RNAi T. castaneum infected with Gram-positive S. aureus (E. coli (TcCTL16 after RNAi could be compensated for by increasing AMP expression via the IMD pathways in T. castaneum.As shown in ed group B. FurtheE. coli) C. Moreov. aureus D and Gra from T. castaneum was functionally characterized for the first time in insects. Our results show that TcCTL16 influences growth and development in early larvae, and more interestingly, TcCTL16 silencing can be compensated for by increasing AMP expression in late larvae in T. castaneum. As a superfamily of proteins, CTLs bind to ligands with characteristic modules of one or more CRDs in a Cat manner . The funt manner ,33,34, bT. castanuem.TcCTL16, a CTL-X protein, contains one CRD domain, four CCPs, and one transmembrane domain. Interestingly, it does not contain a signal peptide but instead contains a transmembrane protein, suggesting that it may be secreted by a non-classical secretory pathway . AdditioTcCTL16 was expressed at all stages of development and was especially highly expressed in the LP and EA stages. Pupation is an important stage of metamorphosis for holometabolous insects [TcCTL16 in late pupae suggests that TcCTL16 might play an important role in the development and metamorphosis of T. castaneum. Our results showed that TcCTL16 RNAi in early larvae shortened their body length and narrowed their body width, leading to the death of 98% of the larvae in the pupal stage. This phenomenon is consistent between the uif (CTL-X) of flies and the TcCTL5 and TcCTL12 of T. castaneum, the absence of which leads to death primarily in larvae [TcCTL16 knockdown. Compared with the control group, the transcript levels of muscle regulation genes, including cut, vg, ewg, ap, salm, and Ims, were obviously down-regulated, and muscle-composition-related genes (Mhc and Mlc) were also decreased. All of the above studies indicate that TcCTL16 may be involved in the growth and development of T. castaneum by regulating muscle-related genes. We found that  insects . High exn larvae ,37. Studn larvae ,39,40. In larvae ,42. On tTcCTL16 exhibited relatively high levels in the hemolymph, which was consistent with the distribution characteristics of C-type lectins in T. castaneum [AsCTLMA15, AsCTLGA5, and AsCTL15) are mainly found in the hemolymph or hemocytes [TcCTL16 was obviously upregulated after injection with LPS, PGN, E. coli, and S. aureus. This expression pattern was consistent with the stimulation of CTLs in other invertebrates. For instance, the transcription of Eriocheir sinensis EsCTL [M. sexta immulectin-2 [Portunus trituberculatus PtCLec2 [B. mori C-type lectin 5 [TcCTL16 could be involved in the immune response to microbial infection in T. castaneum.In addition, our results showed that the mRNA of astaneum ,28,43. Semocytes ,45,46,47emocytes . Next, tis EsCTL , M. sextlectin-2 , Portunu PtCLec2 , and B. lectin 5  were ind2+-dependent ability to bind to Gram+ bacteria (S. aureus and B. thuringiensis) and Gram− bacteria (E. coli and P. aeruginosa) but had no binding activity on B. subtilis. Similar to the BmMBPs from B. mori [+ and Gram- bacteria, although with different intensities. The lack of an ability to bind rCTL16 to B. subtilis suggests that it may not be able to recognize this bacterium. We know that the binding of C-type lectins to the surface of a pathogen may be the first step in their recognition and the initiation of defense mechanisms in innate immunity [+ bacteria (S. aureus and B. thuringiensis) and Gram− bacteria (E. coli and P. aeruginosa) in a calcium-dependent manner, but it had no agglutinating ability against B. subtilis. Similar agglutination activity has also been observed in multi-CTLs from invertebrates, such as MsIML-2 from M. sexta [Litopenaeus vannamei [S. aureus, B. thuringiensis, E. coli, and P. aeruginosa and agglutinate these bacteria in a calcium-dependent manner. The function of most insect C-type lectins is to bind to bacteria by interacting with carbohydrates on the bacterial surface. Our study showed that rCTL16 displayed a Ca B. mori , they haimmunity . TherefoM. sexta  and LvPLvannamei . These rMjCC-CL from Marsupenaeus japonicus can regulate the expression of AMPs by directly activating the JAK/STAT pathway to protect against bacterial infection [TcCTL16 in regulating AMPs. Our results showed that the transcription of 7 AMPs  was obviously increased after TcCTL16 knockdown in late larvae under E. coli stimulation, suggesting that loss of TcCTL16 after RNAi could be compensated for by the increased expression of AMP genes. It has been reported that T. castaneum exhibit resistance to pathogen infection mainly through the expression of AMPs regulated by the Toll and IMD pathways [dif1 and dif2 (Toll) and rel and jnk (IMD) [rel were significantly upregulated after TcCTL16 RNAi under E. coli infection, suggesting that TcCTL16 loss could increase AMPs via the IMD pathway. Moreover, compared with the control group, the survival rate of TcCTL16-RNAi T. castaneum infected with E. coli and S. aureus increased significantly. These results directly supported the conclusion that the decreased mortality rate observed following bacterial injection of the ds-TcCTL16-treated beetles was primarily caused by enhanced larval resistibility to bacterial infection. In short, T. castaneum was able to compensate for the loss of TcCTL16 by increasing AMP expression, thus increasing the survival rate in TcCTL16 knockdown beetles.Furthermore, the recognition and agglutination activity of CTLs is the initial stage of the defense mechanism. Most CTLs can induce a range of immune responses to clear the bacteria, fungi, and viruses, such as regulating the expression of AMPs . MjCC-CLnfection . Next, apathways . The trank (IMD) . By examTcCTL16 was expressed at all stages of development, and TcCTL16 silencing in early larvae influenced the growth and development of T. castaneum. Moreover, TcCTL16 could bind to microorganisms in a Ca2+-dependent manner, and the loss of TcCTL16 in late larvae could be compensated for by the increased expression of AMP genes via the IMD immune pathway, thus decreased mortality in TcCTL16 knockdown beetles. Together, our sequence comparison and function analysis of TcCTL16 establish a solid foundation for future studies of T. castaneum CTL proteins. In summary, we functionally characterized a CTL-X protein, TcCTL16. The results showed that T. castaneum Georgia-1 (GA-1) strain was used for all experiments in this study. Beetles were reared in whole wheat flour containing brewer’s yeast (5%) at 30 °C, 40% relative humidity, and a 14:10 h light:dark photoperiod [E. coli and PGN from S. aureus were obtained from Sigma . The pET-28a (+) vectors E. coli Trans1T1 and E. coli BL21 (DE3) were purchased from TransGene . A one-step cloning kit was purchased from Vazyme .The toperiod . LPS froTcCTL16 was downloaded from Beetlebase (http://www.beetlebase.com/ accessed on 22 June 2023). TcCTL16 was amplified by RT-PCR with the primers TcCTL16-F and TcCTL16-R (http://www.ncbi.nlm.nih.gov/ accessed on 22 June 2023). The corresponding cDNA was conceptually translated, and the deduced proteins were predicted using ExPASy (http://www.expasy.org/ accessed on 22 June 2023). A pool of three 15-day-old larvae was used to isolate total RNA, and then 1 μg total RNA was converted to cDNA using a previously described method ,57. The cCTL16-R  and sequT. castaneum were obtained from Beetlebase (http://www.beetlebase.org/ accessed on 22 June 2023) and the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/ accessed on 22 June 2023). The CTL amino acid sequences of Habropoda laboriosa and Anoplophora glabripennis were obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/ accessed on 22 June 2023). The CTL sequences of Manduca sexta and Bombyx mori were obtained from the literature [Anopheles gambiae and D. melanogaster were obtained from http://cegg.unige.ch/Insecta/immunodb accessed on 22 June 2023. These sequences were aligned with ClustalW2 . The neighbor-joining trees were constructed with 1000 bootstrap replicates in MEGA7. Domain architecture prediction of the proteins was performed using SMART (http://smart.embl-heidelberg.de/ accessed on 22 June 2023). The SignalP 4.1 program was used to predict the presence and location of the signal peptide . The potential disulfide bonds and their positions were predicted using the Scanprosite program (http://www.expasy.ch/tools/scanprosite/ accessed on 22 June 2023).The CTL amino acid sequences of terature ,18. The TcCTL16 with a SYBR Green Master Mix  following the manufacturer’s instructions by the StepOnePlus Real-Time PCR System (ABI). The primer sequences (TcCTL16-qF and TcCTL16-qR) for TcCTL16 are listed in T. castaneum ribosomal protein S3 (Tcrps3) was used as the internal reference [TcCTL16 were calculated according to the 2−ΔΔCT method. The experiments were carried out in three technique repeats and three biological replicates.For the stage-specific expression study, total RNA was extracted from pools of multiple individuals in the following developmental stages: early eggs , late eggs , early larvae , late larvae , early pupae , late pupae , early adults , and late adults . For the tissue-specific expression study, total RNA was also extracted from pools of approximately 100 15-day-old larval tissues, including gut, fat body, epidermis, hemocytes, and Malpighian tubule tissues. Reverse transcription was performed using 1 μg total RNA. Quantitative real-time PCR (qRT-PCR) was employed to analyze the expression patterns of eference . The relTcCTL16 expression after LPS, PGN, S. aureus, and E. coli challenges, 12-day-old larvae were collected and separated into three groups. Approximately 60 synchronous larvae in each group were injected with LPS, PGN, S. aureus, E. coli, or physiological buffer . Pools of three randomly selected larvae were sampled for RNA isolation at 12, 24, 36, 48, 60, and 72 h post-injection to reveal the temporal expression profile of TcCTL16. TcCTL16 expression levels in the beetles challenged with polysaccharides and bacterium were determined via qRT-PCR as described in 2.4. The primer sequences of TcCTL16 (TcCTL16-qF and TcCTL16-qR) are listed in To measure E. coli BL21 (DE3) cells for protein expression. The recombinant CRD protein (designated rCTL16) was expressed in insoluble form and purified by a Ni Sepharose 6 Fast Flow column  under denatured conditions (8 M urea). The purified protein was refolded in gradient urea-TBS glycerol buffer according to Wang et al. [The partial cDNA fragment encoding the CRD domain of TcCTL16 was amplified with primers  and two Gram− bacteria , were used to test the binding spectrum of rCTL16 as previously described [g for 5 min at RT. The resulting bacterial pellets were washed three times with TBS and resuspended in TBS at 2 × 108 cells/mL. In the presence of 10 mM Ca2+, purified rCTL16 were incubated respectively with each of the five microorganisms mentioned above for 1 h at RT with gentle rotation. The microorganisms were pelleted and washed four times with 1 mL of TBS. The pellet was resuspended in 100 µL of 7% SDS solution and centrifuged at 6000× g for five min. The supernatant was collected for Western blotting as described in Five types of microorganisms, including three Gramescribed . The bac6 bacterial count in 25 µL) was incubated with rCTL16 (100 µg/mL) at 28 °C for 1 h in the presence of 10 mM Ca2+. The recombinant methyl-CpG-binding domain (rTcMBD) with a His tag from T. castaneum was used as a negative control. Agglutination reactions were observed using a compound light microscope with a 1000× objective.Five types of microorganisms, as mentioned above, were also used to detect the direct agglutination ability of rCTL16 according to a previously described method [TcCTL16 was synthesized with an in vitro transcription T7 kit for dsRNA synthesis . The DNA template for the ds-TcCTL16 preparation was generated by RT-PCR using the gene-specific primers ds-TcCTL16-F and ds-TcCTL16-R (TcCTL16 was 530 bp. The dsRNA targeting GFP (as control) was synthesized as previously described [The double-stranded RNA (dsRNA) targeting the cCTL16-R . Afterwaescribed .TcCTL16 in 200 nL was injected into the body cavity of each 12-day larva. Beetles injected with an equal volume of ds-GFP were set as control groups. Total RNA was isolated from pools of three larvae of ds-TcCTL16 and ds-GFP groups on days 1, 2, and 3 after injection. Then, a total of 1 μg RNA was converted to cDNA, and qRT-PCR was performed to quantify the TcCTL16 level using the method described in To investigate the RNAi’s efficiency, a total of 200 ng ds-TcCTL16 to test its influence on the growth and development of T. castaneum. Transcript levels of genes involved in muscle regulation and composition were measured with the primers listed in a published article [The 12-day-old larvae were injected with the ds-TcCTL16 participates in the immune response through regulating the expression of AMP and transcription factor (TF) genes in red flour beetles, we injected ds-TcCTL16 into 15-day-old larvae and then challenged them with E. coli after 3 days using the above method. When TcCTL16 was knocked down by RNAi, transcript levels of ten AMPs and four TFs were measured at 60 h post bacterial challenge with qRT-PCR using the primers listed in a published paper [To further explore whether ed paper . Three tT. castaneum were divided into four groups (30 larvae per group) to evaluate the larva survival rate for S. aureus and E. coli infection after TcCTL16 knockdown. ds-GFP was used as a control. After TcCTL16 was knocked down by ds-TcCTL16 injection, all larvae were injected with 200 nL of S. aureus (about 7.0 × 104 cells) or E. coli (about 7.8 × 104 cells) at 72 h post-dsRNA injection. The number of dead larvae was monitored every 12 h, and the survival rates were calculated. The experiments were repeated three times. t-test using the SPSS version 13.0 statistics program . All data obtained were presented as the mean ± SD from three independent experiments. Asterisks indicate statistical significance .The gene expression data and the mean values of the RNAi-treated larvae versus the mean values of the control larvae were compared by one-way analysis of variance (ANOVA) in combination with Student’s


CTNNB1 mutation and poor recurrence-free survival in EGFR-mutant LUADs. Based on these results, the entire cohort was stratified into three groups in accordance with the mutational status of Wnt/β-catenin pathway genes . The clinicopathologic characteristics and survival outcomes of these groups were then compared. Oncogenic CTNNB1 and other Wnt/β-catenin pathway gene mutations were identified in 10 (5.7%) and 14 cases (8.0%), respectively. The CTNNB1-ONC group cases displayed histopathologic features of conventional non-mucinous adenocarcinoma with no significant differences from those of the other groups. Using β-catenin immunohistochemistry, we found that the CTNNB1-ONC group displayed aberrant nuclear staining more frequently, but only in 60% of the samples. The LUADs harboring a CTNNB1-ONC exhibited significantly poorer RFS outcomes than the other groups, regardless of the β-catenin IHC status. This was a pronounced finding in the EGFR-mutant LUADs only in subgroup analysis, which was then confirmed by multivariate analysis. Nevertheless, no significant OS differences between these Wnt/β-catenin groups were evident. Hence, oncogenic CTNNB1 mutations may be found in about 6% of lung adenocarcinomas and may predict post-operative recurrence in EGFR-mutant LUADs. Aberrant nuclear β-catenin staining on IHC appears to be insufficient as a surrogate marker of an oncogenic CTNNB1 mutation.The Wnt/β-catenin pathway is known to be frequently dysregulated in various human malignancies. Alterations in the genes encoding the components of Wnt/β-catenin pathway have also been described in lung adenocarcinoma. Notably however, the clinical impacts of Wnt/β-catenin pathway alterations in lung adenocarcinoma have not been fully evaluated to date. We here investigated the prognostic implications of single gene variations in 174 cases of surgically resected lung adenocarcinoma tested using targeted next-generation sequencing. Screening of the prognostic impact of single gene alterations identified an association between  CTNNB1 gene, is the main intracellular transducer of the canonical Wnt pathway that plays significant roles in tissue homeostasis and embryonic development . Cy. Cy18]. survGroup function of R package maftools version 2.6.05 , Wnt/β-catenin gene alterations other than CTNNB1 , and wild type (WT) for Wnt/β-catenin pathway genes ), LVI was more frequent in the CTNNB1-ONC and Wnt/β-catenin-OTHER groups with statistical significance (P = 0.002). The CTNNB1-ONC group showed STAS more frequently compared to the Wnt/β-catenin-OTHER and WT groups, with marginal significance (P = 0.074). The frequencies of IASLC grade 3 and EGFR gene mutations were also found to be increased in the CTNNB1-ONC group with marginal significance (P = 0.067). None of the CTNNB1-ONC LUADs displayed KRAS or RB1 alterations. By histologic examination, oncogenic CTNNB1-altered LUADs generally displayed the features of conventional non-mucinous adenocarcinoma with predominant acinar (4/10), papillary (4/10), or solid (2/10) patterns. However, no significant histologic differences were found between CTNNB1-ONC, Wnt/β-catenin-OTHER, and Wnt/β-catenin-WT LUADs.The clinicopathologic features of the entire cohort (n = 174) are presented in CTNNB1-ONC group  showed aberrant β-catenin expression . Aberrant β-catenin expression was found to be significantly correlated with the Wnt/β-catenin pathway alteration status, which was most frequent in the pression . Additiomutation . In addimutation . These rCTNNB1-ONC group was significantly associated with a poorer RFS compared to the other groups (CTNNB1-ONC group patients did not significantly differ from the Wnt/β-catenin-WT or Wnt/β-catenin-OTHER groups (CTNNB1-ONC and CTNNB1-WT (combination of Wnt/β-catenin-WT and OTHER groups) groups  groups . RFS anaNC group .EGFR-mutant (n = 100) (EGFR-WT (n = 69) . By contrast, the Wnt/β-catenin-WT-OTHER group showed no significant survival difference (P > 0.05)  (P = 0.021)   and EGFR(n = 69)  subgroup > 0.05) . Similarnt cases . The CTN= 0.006)  and stag= 0.021)  patientssubgroup .CTNNB1 mutations were present in 5.7% of the surgically resected LUAD cases included in our current study cohort. These cases harboring an oncogenic CTNNB1 mutation were only associated with an increased frequency of LVI and showed no significant correlation with any other clinicopathologic characteristics. Cases with oncogenic CTNNB1 mutations also showed a significant association with a poorer RFS outcome, which was pronounced in the EGFR-mutant but not in the EGFR-wild type subgroup. These findings were further confirmed by multivariate survival analysis. Hence, a CTNNB1 gene mutation could be a useful indicator of a post-operative recurrence of EGFR-mutant LUAD and may require routine evaluation.Our present results demonstrated that oncogenic CTNNB1 gene mutations are known to be found in 2–3% of NSCLCs [EGFR gene mutations between populations. de Montpréville and colleagues have reported previously that CTNNB1 gene mutations are more prevalent in cases with EGFR gene mutations also [EGFR-mutant cases than previously reported Western populations, it is not surprising that we detected a higher frequency of CTNNB1 mutations. Notably, another previous study involving a Chinese LUAD cohort also found a similar CTNNB1 mutation frequency (5.3%) to that in our current series [f NSCLCs , includif NSCLCs . This isons also , which it series .CTNNB1 mutations in LUAD have not been widely investigated to date. Several studies of EGFR-mutant LUADs treated with tyrosine kinase inhibitors suggested a negative prognostic impact of CTNNB1 mutations [CTNNB1 mutations in a large cohort of surgically resected LUADs showed a trend only toward a poorer OS without statistical significance [CTNNB1 mutations are significantly associated with a poor RFS outcome in EGFR-mutant LUAD, which implicates these variations as an indicator of higher post-operative recurrence among surgically resected EGFR-mutant LUAD cases. However, we could not fully confirm a significant relationship between the presence of an oncogenic CTNNB1 mutation and OS outcomes in neither EGFR-mutant nor EGFR-wild type LUAD. Further well-controlled studies will be essential to validate the prognostic significance of CTNNB1 mutations following a surgical resection in LUAD patients.The prognostic implications of utations –28, but utations . A furthificance . We findCTNNB1 mutation and β-catenin IHC patterns in endometrial cancer and found that these mutations were present in only 15% of the cases with cytoplasmic β-catenin staining [Several studies have now indicated that the abnormal cellular localization of β-catenin, a factor thought to indicate Wnt/β-catenin signaling activation , is assostaining . FurtherCTNNB1 mutation in LUAD. Previous reports that have evaluated the diagnostic accuracy of β-catenin IHC findings in predicting a CTNNB1 mutation in endometrial cancer showed a high sensitivity and specificity [CTNNB1-mutated NSCLCs reported a heterogeneity of aberrant β-catenin staining within the same tumor, which casts doubt on the efficacy of β-catenin IHC as a predictor of a CTNNB1 mutation in NSCLC [CTNNB1 or other Wnt/β-catenin pathway genes. We speculate therefore that β-catenin IHC pattern is not a useful predictor of Wnt/β-catenin pathway gene mutations. Also, our present data suggest the possibility that the biologic mechanisms which can activate Wnt/β-catenin signaling other than a CTNNB1 mutation [EGFR-mutant LUADs.We here observed a significant level of discordance between nuclear β-catenin staining and the presence of an oncogenic cificity , 41. Nevin NSCLC . Furthermutation  may not The present study had several limitations of note. First, it was based on a retrospective case-controlled cohort which has an inherent risk of a selection bias. Even though the contributing pathologists diligently selected the most representative tumor areas for next-generation sequencing analysis, the possibility exists that these tests could not identify all subclonal mutations outside the submitted tumor area. Finally, the β-catenin immunophenotypes in our present series were investigated using TMA blocks, which may not fully encompass intratumoral heterogeneity .CTNNB1 mutations can arise in up to 5% of Asian LUAD cases and are associated with a poorer RFS in EGFR-mutant LUAD. This suggests that the CTNNB1 mutation status could serve as a prognostic marker for a higher post-operative recurrence in these cancers. Moreover, β-catenin IHC testing of tumor sections may be inadequate as a surrogate marker of CTNNB1 gene mutations in LUAD.In conclusion, oncogenic S1 Fig(A) Aberrantly diffuse and strong nuclear and cytoplasmic staining and (B) occasional weak nuclear and cytoplasmic staining (white arrows) for β-catenin was observed in the tumor cells. (C) Normal membranous staining of the tumor cells.(TIF)Click here for additional data file.S2 Fig(A) Kaplan Meier curve of recurrence-free survival (RFS) in accordance with the CTNNB1 mutation and TP53 alteration statuses within the entire cohort (n = 169). This analysis indicated an invariably poor RFS within the CTNNB1-ONC group regardless of the TP53 alteration status. (B-E) Kaplan-Meier curves of (B and D) RFS and (C and E) overall survival (OS) outcomes according to the  Wnt/β-catenin pathway alteration status and  CTNNB1 mutation status, stratified by the β-catenin IHC pattern analyzed within the EGFR-wild type subgroup (n = 69). None of these comparisons were statistically significant.(TIF)Click here for additional data file.


Background and Objectives: Checking and correctly preparing the patient for endoscopic procedures is a mandatory step for the safety and quality of the interventions. The aim of this paper is to emphasize the importance and necessity of a “team time out” as well as the implementation of a customized “checklist” before the actual procedure. Material and Methods: We developed and implemented a checklist for the safe conduct of endoscopies and for the entire team to thoroughly know about the patient’s medical history. The subjects of this study were 15 physicians and 8 endoscopy nurses who performed overall 572 consecutive GI endoscopic procedures during the study period. Results: This is a prospective pilot study performed in the endoscopy unit of two tertiary referral medical centers. We customized a safety checklist that includes the steps to be followed before, during and after the examination. It brings together the whole team participating in the procedure in order to check the key points during the following three vital phases: before the patient falls asleep, before the endoscope is inserted and before the team leaves the examination room. The perception of team communication and teamwork was improved after the introduction of the checklist. The checklist completion rates, identity verification rates of patients by the endoscopist, adequate histological labeling management and explicit recording of follow-up recommendations are some of the parameters that improved post-intervention. Conclusions: Using a checklist and adapting it to local conditions is a high-level recommendation of the Romanian Ministry of Health. In a medical world where safety and quality are essential, a checklist could prevent medical errors, and team time out can ensure high-quality endoscopy, enhance teamwork and offer patients confidence in the medical team. Endoscopic procedures represent an important part of daily practice for both gastroenterologists and nurses, enabling the timely diagnosis and precise treatment of digestive diseases. In the past 20 years, the possibilities of diagnosing and treating digestive diseases have increased considerably; GI  endoscopy procedures have become more complicated and varied, hence, the concerns about the quality of medical care and patient safety have increased . In receThe diversity of digestive pathology as well as the fast growth of the therapeutic endoscopic opportunities led to more evolved and complex procedures. The practical challenges of advanced endoscopy are approaching the complexity of a surgery. An advanced endoscopy is similar to surgery in terms of the technical complexity and invasiveness. This contains a higher risk of adverse events associated with the therapeutic interventions . The patA modern endoscopy unit needs extremely competent leaders, supported by excellent teams who can handle a high volume of procedures safely, efficiently and for the benefit of the patient. In gastroenterology, quality and safety are crucial issues that are motivated by a shared desire to advance best practices and make it easier for patients to receive evidence-based care. Patient safety should not be compromised in light of the development of endoscopic practice, and efforts to uphold and enhance GI endoscopy safety should always be made ,7. AdditThe advanced endoscopy team is part of a bigger service that provides access to therapeutic intervention, and the staff should already have experience, planned strategies and skills to deal with potential adverse events or failed procedures. If the adverse events result from inadequate planning of the intervention rather than a lack of technical skills, quality improvement initiatives may help to reduce such complications ,10.The results are determined not only by the skills of the endoscopist, but by the effectiveness of the team.-The endoscopy team;-Team leaders;-The institution where the service operates;-The political context.An effective endoscopy service depends on the following :-The endThe team itself consists of nurses, technical staff, administrative staff, anesthesiologists and endoscopic physicians. Some of the endoscopists may spend half a day or one day per week in the endoscopy department. The temporary nature of some team members can affect the team dynamics, leading to an increased disruption in the workplace. As a result, the team may not function efficiently. Providing a modern endoscopy service requires effective leadership and teamwork ,12.Each endoscopy unit has the possibility to draw up its own checklist, either in a printed or electronic version. Most checklists in digestive endoscopy do not specifically address advanced procedures; therefore, they need to be nuanced/individualized ,14. AccoIn this paper, we aim to formally evaluate the feasibility of successfully implementing a customized safety checklist in our dual center GI endoscopy units, to identify strategies and address barriers for facilitating checklist compliance and to summarize the impact on team communication and commitment to patients’ safety culture.This is a prospective pilot study performed in the endoscopy unit of two tertiary referral medical centers .This initiative was designed and approved as a quality improvement study, with the study subjects set to be the endoscopy staff comprising gastroenterology and anesthesia physicians and endoscopy nurses. The subjects from both endoscopy teams were enrolled at the same time in October 2021. The endoscopy staff members were included in the present study if they performed at least one endoscopy procedure and took part in each phase of the study. No patient information was collected during this study, hence, the exempt of approval from the Local Institutional Ethical Committee. The authors did not have access to information that could identify individual participants during or after data collection.The case diversity included elective inpatients and outpatients who underwent diagnostic and therapeutic endoscopies. All procedures were performed with the patients under propofol sedation, administered by an anesthesiologist.Real-time observation of each endoscopic procedure was performed by one designated staff member, and data were collected from October 2021 to the end of September 2022, as described in The observational studies were prospectively conducted and reported following the STROBE statement. Initially, the baseline compliance rates were inspected during the first 2 months of the study. The final data were cross-checked by the multidisciplinary team established in both institutions, and the final assessment was performed during the last 2 months. Pre- and post-intervention staff compliance rates were compared. Team satisfaction and perception of the checklist was assessed by conducting a 5-question survey, after the quality improvement interventions were applied. The staff was asked if the checklist either caused time delay, improved patients’ safety, proved to be useful if they were to have an endoscopy or if, overall, they were satisfied with the checklist implementation process.The framework for the implementation of the safety checklist is described in Development and adjustment of the endoscopy checklist;Checklist introduction seminars;Team training sessions mandatory for all endoscopy staff;Physical reminders spread around the endoscopy department;Printed paper-based checklists available in a clear designated area;Advocating the use of a safety checklist as part of the endoscopy department policy;Mandating a single individual (physician or nurse) to lead the checklist process;Actively engaging the entire endoscopy team to contribute to all phases of the checklist.Intervention strategies for the implementation of our prospectively developed checklist are as follows:Primary and secondary outcomes expected to be analyzed during the study included the following: (a) checklist completion rate, (b) identity verifications of patients, (c) validity of the endoscopy procedure appropriateness, (d) colonic preparation , (e) colonoscopy completion rate and adenoma detection rate, (f) staff satisfaction and perception about the checklist, (g) peri-procedural complications , (h) adequate histological labeling management, (i) explicit recording of follow-up recommendations .p-value of <0.05 was considered to be significant.As all collected data were categorical, comparisons between groups (nurses versus physicians) were performed using chi-square test and, alternatively, Fisher’s exact test, when not all the parameters of the chi-square test were fulfilled. A For the staff questionnaires, the frequencies and proportions of responses were computed including only the response alternatives “Yes” and “No”, and ignoring the “Hesitant” responses.Data collection was performed using Microsoft Office Excel 2019, , while the statistical assessment was carried out using MATLAB 2021a .Based on the existing checklists published in the literature and the constant feedback received during the study, we developed a personalized checklist that includes the steps to be followed before, during and after the endoscopic examination . AlthougThe “Sign in” phase starts once the patient enters the endoscopy room and comprises the following: (a) verification of patient’s identity, (b) completion of the informed consent forms, (c) verification of patient’s relevant medical documents, (d) morbidity classification according to the American Society of Anesthesiologists score, (e) assessment of relevant comorbidities , (f) any known allergies (including medication or previous difficulties with sedation/anesthesia), (g) usage of anticoagulant/antiplatelet medications, (h) a review of known infections  and (i) confirmation of proper bowel preparation.The “Time out” phase occurs before the endoscope is inserted and/or before the induction of sedation/general anesthesia. It includes the following: (a) confirmation that the patient is aware of the planned endoscopic procedure, (b) patient and procedure validation, (c) confirmation of indications, aims, potential limitations and summary of each step of the procedure, (d) confirmation that all required endoscopy equipment, endoscope(s) and accessories are available and properly functioning, (e) confirmation that the patient monitoring equipment is prepared and operational , (f) inspection of any special aspects regarding sedation or patient position and (g) assessment of whether the endoscopic procedure is difficult and whether additional preparations are required.The “Sign out” phase takes place in between the end of the endoscopic procedure and prior to the team exiting the endoscopy lab. This phase includes the following: (a) confirmation that the endoscopy report is accurate, including post-procedure instructions for patient’s recovery and eventual follow-up procedures that could be indicated, (b) confirmation that all histological samples collected during the endoscopy procedure are correctly labelled and documented, (c) documentation of patient’s post-procedure status and if any problems occurred during the procedure and (d) confirmation that all the necessary documents and information were passed on to the patient.A mixed team dedicated to quality improvement reviewed both the local and already published data in order to identify the barriers and facilitators which might influence the checklist compliance . In consThe study’s outcome rates were calculated after completing 572 consecutive endoscopic procedures, including 126 procedures during the baseline period and 446 procedures until the end of the study period. The assessment of the checklist implementation is presented in p < 0.05) was noted among the physicians, and from 42% to 91% (p < 0.05) among nurses (The subjects of this study were 15 physicians (gastroenterologists and anesthesiologists) and 8 endoscopy nurses who worked at the endoscopy units in varying degrees of frequency and performed, overall, 572 consecutive GI endoscopic procedures during the study period. When assessing the compliance rates of the physicians versus the nurses, at baseline and post-intervention, an increase from 20% to 83% . Furthermore, SMSA classification can be used to estimate the time required to perform an advanced polypectomy along with assessing the difficulty of the procedure as it is mentioned in the Team Time Out phase of the endoscopy checklist. The following would be an illustrative example of performing an endoscopic mucosal resection: Before starting, the endoscopist describes their impression, whether they expect the polypectomy to be simple, moderately challenging or difficult. This first impression may not be definitive and may change during the treatment, at which point the endoscopist should provide an update to the team. The planned technical approach is described, including where the injection needle will be used for mucosal elevation, the intended effect and positioning of the polyp after elevation, the size of the polypectomy loop and the electrical settings for cutting and/or coagulation, etc. The endoscopist continues to discuss the likelihood of adverse events , how these will be solved  and once again, asks for team confirmation that all the equipment is available and that the staff is competent and trained ,21. It iSimilar to the majority of previous published studies evaluating safety checklists in the GI endoscopy setting ,23,24, oMany factors must be taken into account when performing a safe procedure. Although each endoscopy has its own precautions and preparations, many are common and can be divided into the following three major procedural stages: pre-procedure, intra-procedure and post-procedure. While initiating our study, during the baseline phase, it became obvious that adding a checklist to the endoscopy unit’s current practices would result in low compliance, hence, intervention methods were required for reaching the goal of a minimum compliance of 75%. As the study continued, we noticed that immediate and targeted feedback seems to be a useful tool for dealing with low compliance. Team feedback in surgery was shown to improve technical performance and reduce adverse events. Post-procedural team feedback is useful, especially in the case of a significant adverse event. In our study, the feedback offered by all team members revealed that the improvement of the checklist implementation is credited to the designated individual assuming ownership for the completion and being accountable for involving the entire multidisciplinary team at each “time out” and “sign in” checklist completion. Even though the results of the staff questionnaire were not statistically significant due to the small number of participants, both physicians and nurses offered consistent and valuable responses, all supporting the usefulness of the endoscopy safety checklist.The following two main variables of interest were identified in our study: colonic preparation–colonoscopy completion rate and colonic preparation–adenoma detection rate. Both the colonoscopy completion rate and the adenoma detection rate are positively influenced by a proper colonic preparation  and, on the other hand, a poor colonic preparation  will lead to a poor adenoma detection rate and/or an incomplete colonoscopy evaluation.Due to a relatively short study period, the compact and constant number of the subjects involved in completing the safety checklist and the staff questionnaire associated with the clear protocol regarding the data collection, we did not register any missing data, which might represent one of the study strengths. A limitation of our study might be the sample size, as the number of GI endoscopy procedures were restricted by the COVID-19 pandemic. An ongoing audit is necessary, ideally with a bigger sample size, to enable subgroup analysis and reveal particular weak points or problematic practices. The baseline measurements included 126 procedures, while post-intervention, 446 procedures were assessed. To strengthen the validity of the findings, it would be advantageous to collect comparable numbers at the study’s baseline and post-intervention periods if it were to be conducted again. The “Hawthorne effect,” which occurs when team members become aware that they are being watched, is an observation bias and another limitation of the study’s scope, as the outcomes might not accurately reflect the everyday surveillance-free practice in the endoscopy lab. The installation of cameras in the endoscopy rooms and using remote video auditing, like in the study of Raphael et al. , can helAlthough the study achieved its objectives, there is still an opportunity for further development. This ought to enable more focused initiatives to lessen the standard-setting variation.In the end, we acknowledge that a customized approach and patient involvement was necessary for understanding the entire patient experience throughout the GI endoscopy department and how the endoscopy unit performed. Although there were still some items on the checklist that needed to be completed, overall, the compliance rates among the team members increased during the study period, which is very encouraging.Using a checklist and adapting it to local conditions is a high-level recommendation of the Romanian Ministry of Health. In a medical world where safety and quality are essential, a checklist could prevent medical errors, and team time out can ensure high-quality endoscopies, enhance teamwork and offer patients confidence in the medical team. The checklist implementation, in our labs, significantly strengthened the team, flattened hierarchies and improved patient approach and team communication. In a medical world where safety and quality are paramount, a checklist, implemented in all endoscopy services, could prevent procedural errors, streamline teamwork, ensure high-quality work and provide confidence to patients. Hence, standardized methods, such as checklists, promoting patient safety culture and encouraging patient involvement, are strategies for augmented patient safety.


Ecogeographical patterning supporting both principles was observed in thermally challenging environments. Finishers of hot-condition events had significantly longer legs than finishers of cold-condition events. Furthermore, hot-condition finishers had significantly longer legs than those failing to complete hot-condition events. A degree of niche-picking was evident; athletes may have tailored their event entry choices in accordance with their previous race experiences. We propose that the interaction between prolonged physical exertion and hot or cold climates may induce powerful selective pressures driving morphological adaptation. The resulting phenotypes reduce thermoregulatory energetic expenditure, allowing diversion of energy to other functional outcomes such as faster running.Both extinct and extant hominin populations display morphological features consistent with Bergmann's and Allen's Rules. However, the functional implications of the morphologies described by these ecological laws are poorly understood. We examined this through the lens of endurance running. Previous research concerning endurance running has focused on locomotor energetic economy. We considered a less-studied dimension of functionality, thermoregulation. The performance of male ultra-marathon runners ( Effective thermoregulation is a key challenge facing individuals in different environments. The apparent influence of climate on morphological traits has long been documented, and is an area of active research interest. Bergmann's and Allen's rules describe patterns of variation both within and across species in relation to the temperature of their environment. The two ecogeographic rules contend that endotherms will tend to be larger Bergmann  and dispet al. Both extinct and extant hominin species have been shown to display morphological features consistent with Bergmann's and Allen's rules , the tight 1:1 coupling of striding and breathing prevents panting  times are set at temperatures around 23°C  were recruited from the 2016 Rovaniemi150 (Finland), the 2016 Jungle Ultra (Peru), the 2016 and 2017 Al Andalus Ultimate Trail (Spain) and the 2016 Everest Trail Race . These particular races were selected on the basis of their diverse range of climates and broad similarities in terms of race distance. Two races were considered to be hot (Peru and Spain), and the other two were considered to be cold . With the exception of the Finland event, which was continuous, the remaining three events follow a similar format of five or six stages, run on consecutive days with overnight rest. An overview of the races can be seen below in Male athletes  = 24.374, p < 0.001; relative leg length hot, 49.9 ± 1.3% vs cold, 48.0 ± 1.2%, F = 55.779, p < 0.001). This suggests that there may have been a degree of self-selection  with respect to Allen's, but not Bergmann's, Rule.One-way ANCOVAs were performed comparing all race entrants (irrespective of whether or not they finished) in hot-condition races with those in cold-condition races. While there were no significant differences in either weight or body mass index , significant differences were observed with leg length and relative leg length . Consequently, age was controlled for in analyses.There is a body of literature investigating potentially important age effects on endurance running performance  = 9.862, p < 0.001; relative leg length hot 50.3 ± 0.977% vs cold 47.8 ± 1.03%, F = 20.474, p < 0.001]. These results persisted following Bonferroni corrections (both p < 0.001), and were consistent with Allen's Rule. These results are summarised in First, a one-way ANCOVA was conducted to determine statistically significant differences between the morphologies of finishers in hot (Spain and Peru) vs cold  conditions, controlling for age. Athletes who finished a hot-condition race had longer leg lengths and greater relative leg length than athletes who finished a cold-condition race .Consistent with differences between finishing athletes across conditions, athletes continued to show tentative trends in line with Allen's Rule, although not quite reaching significance. Athletes who finished the Spain race tended to, on average, exhibit larger leg length ratios (50.4 ± 1.0%) than those who did not [49.4 ± 1.8%, These results are visualised in et al. It was hypothesised that ecogeographical patterning in human morphology would be observed in ultra-marathon runners successfully completing races in hot and cold environments. Heat-adapted morphologies are considered to be characterised by smaller bodies  and substrate oxidisation. Analysis of a small number of male athletes completing 86 day and 42 day ski-trek expeditions across Greenland revealed no loss of endurance capacity, a decrease in anaerobic function (Frykman Ecogeographical patterning pertaining to Allen's Rule was observed in ultra-marathon runners competing in thermally challenging environments. Heat-adapted morphologies were evident in finishers of hot-condition events relative to non-finishers, and in finishers of hot-condition events relative to finishers of cold-condition events. Furthermore, there appeared to be a degree of niche-picking, by which athletes may have used prior knowledge to avoid exposure to environmental pressures that are detrimental to their performance.Endurance exercise in hot and cold climates imposes significant thermoregulatory demands, providing powerful selective forces that could drive the emergence of temperature-adapted morphologies. It is proposed that the interaction between environment and prolonged physical activity leads to adaptation and the evolution of environmentally appropriate morphologies. Such morphologies reduce energetic expenditure on thermoregulation, allowing energetic diversion to other processes such as faster running. This is the first study to show a correlation of functional traits with ecogeographical rules.