TP53 variants are associated with multiple neoplastic diseases, and thus have come to serve as a paradigm for genetic analyses in this setting. We searched 14 independent, globally distributed datasets and recovered TP53 SNPs from 202,767 cancer-free individuals. In our analyses, 19 new missense TP53 SNPs, including five novel variants specific to the Asian population, were recurrently identified in multiple datasets. Using a combination of in silico, functional, structural, and genetic approaches, we showed that none of these variants displayed loss of function compared to the normal TP53 gene. In addition, classification using ACMG criteria suggested that they are all benign. Considered together, our data reveal that the TP53 coding region shows far more polymorphism than previously thought and present high ethnic diversity. They furthermore underline the importance of correctly assessing novel variants in all variant-calling pipelines associated with genetic diagnoses for cancer.Infrequent and rare genetic variants in the human population vastly outnumber common ones. Although they may contribute significantly to the genetic basis of a disease, these seldom-encountered variants may also be miss-identified as pathogenic if no correct references are available. Somatic and germline  TP53 status is becoming mandatory in a number of clinical situations. In patients with Li–Fraumeni syndrome or families with hereditary breast and ovarian cancer syndrome, the early identification of germline TP53 mutations has been shown to be highly beneficial for disease surveillance , 33TP53 al Table . This deA1 Table . One of a Tables ,  S11. Aa Tables ,  S11. Ba Tables ,  S11. FUntil recently, human genetic variation studies were largely restricted to constitutional variants occurring at frequencies above roughly 1–5%. That threshold was due to not only technological and cost limitations but also our own lack of knowledge of human genome diversity. Consequently, most medical genetic analyses were developed based on information derived from these common variants. Today, high-throughput DNA sequencing technologies enable the analysis of thousands of samples and the identification of infrequent SNPs. Recent explosive population growth has led to a great number of rare SNPs occurring at frequencies below 0.5% , 35. TheTP53 gene is a paradigm for such analyses. Indeed, the accurate diagnosis of somatic mutations in it is mandatory in multiple clinical settings, for example in chronic lymphocytic leukemia, where it is used to improve patient stratification and optimize therapeutic decisions [TP53 mutations for genetic counseling is proving pertinent in such pathologies as hereditary breast and ovarian cancer syndrome and pediatric cancer [The ecisions . Furtherc cancer , 36.TP53 variants but their results were heterogeneous and thus a source of discord.Recently, the ACMG—Association for Molecular Pathology provided a systematic method for the interpretation of sequence variants for genetic disorders . UnfortuTP53 SNPs have been identified and defined as benign constitutional variants [TP53 gene mutations have been described in more than 100,000 patients. However, in most of these cases, matched normal DNA was not available and, thus, the somatic origin of the mutation was determined by consulting reference databases. It is therefore highly likely that some of those variants were infrequent constitutional variants mistakenly identified as somatic mutations.Historically, two exonic missense variants . SomaticTP53 SNPs in the human population and (ii) what is the potential pathogenicity of these variants?In the present study, using a combination of in silico, in vitro, structural, and genetic approaches, we addressed a two-tiered question: (i) is it possible to identify novel and infrequent The use of multiple independent population databases has proven very helpful for the selection of novel potential SNPs. Although gnomAD is an outstanding resource for population analyses, the addition of novel datasets of individuals from different ethnic groups has led to a more accurate selection of potential SNPs .TP53 [TP53 protein domain [In silico predictive software enables the assessment of the effects of amino acid substitutions on protein structure or function with, in principle, no imperative need for functional studies. However, in silico and functional approaches each have their own strengths and weaknesses. Depending on the algorithm, the performance of in silico approaches will vary based on the specific gene and protein being considered. As all algorithms focus on protein function based on the homology of sequences and/or the physical properties of amino acids, confusion has arisen between deleteriousness and pathogenicity, which should be considered as two different features. Great caution has been advised for the use of these algorithms, including for TP53 –41. Concn domain .TP53 is the availability of three unbiased large-scale functional studies for all potent TP53 missense variants [TP53 variants. p.N235S has been defined as pathogenic, likely pathogenic or VUS in different studies [TP53, we strongly advocate for the use of functional studies to assess TP53 variant deleteriousness.One major advantage for variants –18. A covariants . Althoug studies , 44–46. TP53 mutation on protein function and their repercussions on the various pathways regulated by TP53 are highly heterogeneous. Although a few variants can be considered as amorph with a total loss of function, the complex structure of the TP53 protein is known to give rise to variants with multiple consequences with hypomorph, neomorph or antimorph properties [TP53 regulates transcription through specific binding to highly degenerated response elements in promoters or introns of target genes [TP53 for these various biological sites is variable, and several TP53 variants can display only partial loss of their DNA-binding activity, allowing the mutant protein to bind only to a subset of TP53 response elements. This feature is linked to differential transactivation activities that may depend on p53 protein levels, as well as target sequences [TP53 is reflected by our transcriptional analysis performed on endogenous genes . Upon p53 induction, p21WAF1/CIP1 activates the transcriptional repressor DREAM, leading to the shutdown of more than 200 cell-cycle genes [TP53 mutations [TP53 variants toward the WAF1 promoter and the DREAM pathway is essential for promoting tumorigenicity remains to be elucidated. As of this writing, there is no “gold standard” assay to define a TP53, suppressive effect, but the observation of defective transcription in the large majority of TP53 variants found in human cancer indicates that this activity is a key target. Although none of the variants in the present study displayed any obvious functional defects, we cannot exclude that any one of them may be partially defective for a particular function. Nonetheless, their high frequency in the human population and low frequency in cancer mutation databases suggest that any deleterious clinical impacts will be very low.The consequences of operties –49. TP53et genes . The affequences , 52. Thines Fig. . Genes ale genes . Derepreutations . WhetherTP53 variants found recurrently in multiple population datasets from different ethnic groups was indistinguishable from wild-type TP53. Through the analysis of multiple national datasets, we were able to definitively validate nine of the 21 variants as BA1, i.e., the ACMG stand-alone criterion for benignity. Seven other variants met the ACMG criterion BS1 + BS3, also indicating benignity. Thus, we identified 16 variants that may only be defined as natural polymorphisms. Among those variants, we identified for the first time five variants that were specific to the Asian population (present only at very low frequency in other populations). Our analysis of the UMD_TP53 database confirmed the Asian origin of the five variants and furthermore showed that they were mistakenly defined as somatic pathogenic mutations. We also identified another TP53 variant specific to the Indian population. Our analysis emphasizes the need to exercise great caution when using population datasets to check patient cohorts. Matching the ethnicity of the patient or the cohort to the reference datasets appears essential for increasing the accuracy of the analysis [Using different functional assays, we showed that a subset of 21 germline analysis . Also, sanalysis . Such “fTP53 activity but whether it is associated with an increased risk of cancer remains highly controversial [We cannot exclude that a number of the variants described in the present study may be associated with specific hereditary traits associated or not with cancer, or with other genetic characteristics. To illustrate this point, variant rs1042522 (p.P72R) does not impair oversial . Similaroversial .TP53 mutation diagnostics, they should be updated with the present data. The 2020 release of the UMD_TP53 database as well as the web application Seshat have been updated with these novel data.Most of the variants described in set 21 are described as pathogenic or likely pathogenic in multiple databases and pipelines for the assessment of pathogenicity. To improve the accuracy of TP53. Unfortunately, the quantity of research for other cancer genes is much more limited. Nevertheless, the availability of large, independent datasets representing ethnic populations should be sufficient to identify novel benign variants and emphasize genetic heterogeneity. For an accurate assessment of somatic and germline variants, human genome reference databases used in analytical pipelines must include accurate ethnicity information to ensure that genetic analyses provide the most pertinent information possible for patients.This analysis was made possible by the numerous functional and structural analyses available for TP53 variants identified in this study are provided in the Supplementary Information.Details on the cell lines, western blot, luciferase assay, plasmid construction, and flow cytometry analysis for the experimental validation of TP53 variants from a range of population databases were downloaded from their respective portals . This latter handles all types of TP53 gene variations, such as substitutions, insertions, duplications, deletions, and more complex insertion/deletions [TP53. This is an essential issue for avoiding problems due to the use of multiple genome references  by the various NGS pipelines as well as noncompliant nomenclature. To minimize database contamination by potential carriers of germline TP53 variants, the non-cancer version of gnomAD was used in the present analysis. Taken together, exonic TP53 variants issued from gnomAD correspond to 56% of the variants issued from the 14 cohorts.ls Table . The orieletions . The curTP53 variants were downloaded from the UMD_TP53 database (Released 2017), which includes 80,402 mutations (6870 different TP53 variants) identified in tumors, in cell lines (somatic mutations) or in patients with hereditary cancers (germline mutations) [Cancer-associated tations) . This reTP53 variants in this work is in accordance with the Human Genome Variation Society’s guidelines using the NM_000546.5 transcript sequence and the full-length protein NP_000537.3 [The sequence nomenclature used for 000537.3 .TP53 variants called the “CSD” [TP53 variants were extracted from four different nonoverlapping datasets on various types of cancer. The Sanger dataset includes 4299 variants extracted from the latest version of the UMD_TP53 mutation database excluding all NGS studies  of TP53. We created the CSD by combining these four datasets to define a core of 471 recurrent TP53 variants found at least once in each database [We previously defined and validated a specific set of pathogenic he “CSD” . Brieflyy et al. ). The otts: TCGA , ICGC [6ts: TCGA , and MSKts: TCGA . Each ofdatabase . The foudatabase .TP53 database includes three sets of functional data for TP53 variants. The first set was described in detail in a previous report [TP53 variants and a green fluorescent protein reporter plasmid were constructed. TP53 activity was tested by measuring the fluorescent intensity of green fluorescent protein that is controlled by eight different promoter sequences regulated by the p53 protein after 3 days of growth at 37 °C. The second set of functional data integrated in the UMD_TP53 database corresponds to the analysis of 5300 TP53 variants performed by Kotler et al. [TP53 was assessed in H1299 cells. The third set corresponds to the study by Giacomelli et al. [TP53 variants  were chosen for in silico analyses based on their occurrence in multiple datasets (more than 2). We also added the five Asian-specific variants and eight of the ten outlier germline variants found in the UMD_TP53 database. The variant p.T125R, found at high frequency as a germline variant in the database, was excluded because missense and synonymous mutations in this codon close to a splice site are known to impair TP53 splicing [A subset of 27 splicing , 65. Thesplicing .TP53 variants as well as rs1042522 (p.P72R) and rs1800371 (p.P47S).Set 21, used for in vitro analyses, is derived from set 27 using more stringent criteria. It includes variants found in multiple datasets (more than 4) at frequencies higher than 0.0005. Asian and outlier germline variants were retained but variants p.R248Q, p.R283H, and p.R156H found, respectively, in five, four, and four datasets were not included as they are well-known pathogenic variants included in the CSD. Set 21 thus included 19 novel Models of p53 DBD mutants were generated using the SWISS-MODEL server , with PDhttps://sites.google.com/site/jpopgen/dbNSFP). This database compiles prediction or conservation scores from multiple prediction algorithms [dbNSFP v3.5 was downloaded from the Jpopgen website (gorithms . PredictFive other predictors not included in dbNSFP were also used: Revel , PON-P2 TP53 database includes data from 4600 publications. A manual survey of the database was performed for the five TP53 variants suspected to be constitutional variants specific to the Asian population. Publications describing any one of these variants were retrieved for analysis. In most cases, the hospitals and/or institutions used for patient recruitment were available.The 2017 release of the UMD_Supplementary informationSupplementary Figures and TablesTable S2Table S3Table S5


Magnesium silicate is an inorganic compound used as an ingredient in product formulations for many different purposes. Since its compatibility with other components is critical for product quality and stability, it is essential to characterize the integrity of magnesium silicate in different solutions used for formulations. In this paper, we have determined the magnitude of dissociation of synthetic magnesium silicate in solution with positively charged, neutral, and negatively charged compounds using Scanning Electron Microscopy (SEM), Energy Dispersive X-ray Spectroscopy (EDS), and Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS). The EDS results were verified through Monte Carlo simulations of electron-sample interactions. The compounds chosen for this study were positively charged cetylpyridinium chloride (CPC), neutral lauryl glucoside, and negatively charged sodium cocoyl glutamate and sodium cocoyl glycinate since these are common compounds used in personal care and oral care formulations. Negatively charged compounds significantly impacted magnesium silicate dissociation, resulting in physio-chemical separation between magnesium and silicate ions. In contrast, the positively charged compound had a minor effect on dissociation due to ion competition, and the neutral compound did not have such an impact on magnesium silicate dissociation. Further, when the magnesium ions are dissociated from the synthetic magnesium silicate, the morphology is changed accordingly, and the structural integrity of the synthetic magnesium silicate is damaged. The results provide scientific confidence and guidance for product development using synthetic magnesium silicate. In these fields, synthetic magnesium silicates also have potential in applications where natural magnesium silicates are used, including active ingredients or excipients for oral and topical pharmaceuticals and cosmetics11. The synthetic magnesium silicate, Magnesol, is used broadly in the biodiesel industry due to its adsorptive properties12. The active sites formed from the free silonal groups on the surface of magnesium silicate make it an excellent adsorbent13 resulting in extensive studies on its use as an adsorbent16, especially for used oils21 and wastewater treatment28. The adsorption properties are also exploited in chromatography, where synthetic magnesium silicate, sold as Florisil, is used widely in analytical and preparative chromatography applications33. Additional applications and studies of synthetic magnesium silicate include its use as a paint additive34, cement additive35, anticorrosion coating36, drug carrier in organic–inorganic hybrid materials37, and a polymer filler49. Due to the significant role of magnesium silicates in various industries, characterizing its stability in conjunction with other compounds and at different conditions for formulations is vital.Synthetic magnesium silicates have broad applications in industry and are used extensively in pharmaceuticals, cosmetics, biodiesel purification, and chromatography. Laponite, a commercially available55. Methods to control the physicochemical properties and morphology of magnesium silicates have also been examined, including the effects of surface modification62, the effects of additives during synthesis, including nonionic surfactants16, and sodium hydroxide13, as well as the effects of pH on synthesis49 and adsorption capacity63. Compared with the polycrystalline structure of natural magnesium silicate minerals, synthetic magnesium silicates commercially available are often amorphous and porous, potentially resulting in more significant dissociation in a liquid dispersion system. For example, in acidic solutions, Mg2+ ions can be leached out, i.e., dissociate from the silicate ion pairs, known as “acid leaching” of silicates64. Reaction of the free Mg2+ ions with other species in the solution can generate magnesium complexes that may alter product integrity and physical properties upon storage. Several studies have shown changes in magnesium silicate properties and morphology upon the addition of surfactants and ionic compounds41 or as an effect of pH63. However, to the best of our knowledge, studies on synthetic magnesium silicate stability under these conditions are lacking. Consequently, there is a need to determine the stability of synthetic magnesium silicate in various solutions and monitor possible changes in morphology.Synthetic and natural magnesium silicates have similar application potentials due to similarities in their structures. In the literature, there have been many studies on the synthesis of magnesium silicates65 and self-repairing lubricant technology66. The compounds chosen for the SEM/EDS study were positively charged cetylpyridinium chloride (CPC), neutral lauryl glucoside, and negatively charged sodium cocoyl glutamate since these compounds are often used in formulations for personal care or oral care products. The EDS results were verified through Monte Carlo simulations of electron-sample interactions using CASINO software67. Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) was used to verify and confirm the observed disassociation of Mg2+ ions from magnesium silicate. The results provide scientific confidence and guidance for product development using synthetic magnesium silicate and facilitate the use of SEM/EDS to characterize magnesium silicate integrity.Here, Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDS) were used to determine the dissociation of synthetic magnesium silicate as a function of pH in positively charged, neutral, and negatively charged compounds. EDS has previously proven helpful for the study of magnesium silicate in the literature on scale inhibition in oil production. Representative 1000× magnification SEM images of the raw synthetic magnesium silicate particles (D) and the particles collected from each compound solution at each pH are shown in Fig. To better understand the difference between the effect of positively charged, neutral, and negatively charged compounds on magnesium silicate dissociation, SEM/EDS analysis was conducted. The 3D structure of each compound is shown in Fig. From the SEM images in Fig. 2+ ion dissociation.Point EDS spectra were measured for at least six particles for each of the nine samples. Only silicon (Si), magnesium (Mg), and oxygen (O) were included as elements of interest, and a ratio of the resultant percent Si over the percent Mg was calculated. A plot of the average ratios is shown in Fig. 67 was used to simulate 200,000 electrons targeting MgSiO at 10 keV, with a beam radius of 125 nm. A MgSiO density (ρ) of 1.71896 g/cm3 and weight fractions of 0.1 for Mg, 0.39 for Si, and 0.51 for O determined from the typical Mg, Si, and O ratio measured in EDS point spectra of the neutral compound were used for the simulation. The software was used to generate characteristic X-rays for each element. The classical Φ(ρz) curves and radial distributions are shown in Fig. ρz) is a relative generated intensity that varies with depth, first suggested by Castaing68 where the mass depth, ρz, is the product of the density of the sample, ρ, and the depth, z69. The Φ(ρz) curves give information on the X-ray generation depth of each element in the sample, while the radial distributions give lateral information. For each element, we can determine that the X-rays are only measured at a depth less than 700 nm into the sample and less than 1500 nm radially from the center of the beam. Since the typical particle size used for EDS point spectra was 6–10 μm, we can be positive that the results are obtained from the particle with no interference from the filter paper or substrate.To verify point EDS spectra were an accurate measurement of the solid particles, simulations of Monte Carlo electron-sample interactions were performed. CASINO softwareFrom the EDS mapping results in Fig. 2+ ions with charged compounds, simple solutions were prepared of magnesium chloride with two negatively charged compounds, sodium cocoyl glycinate and sodium cocoyl glutamate, as well as neutral lauryl glucoside separately. For both negatively charged compound solutions, a significant amount of precipitate was formed. No precipitate was formed in the lauryl glucoside solution. The differences are related to the charges. Lauryl glucoside is neutral and does not react with the positive Mg2+ ions. Therefore, no precipitate can be formed. In contrast, sodium cocoyl glycinate and sodium cocoyl glutamate are negatively charged compounds, and the interaction between the negative heads on the compounds and positive Mg2+ ions is the root cause for the precipitate formation. To confirm this hypothesis, the precipitates formed were analyzed by LC-HRMS.To further understand the interaction from dissociated Mg2+ ion chelated with two moles of negative ions from glycinate or glutamate functional groups at the compound head. The schematics of the formation of magnesium complexes formed with the negatively charged compound are shown in Fig. The precipitate was collected by filtration, dried, and redissolved into methanol. The methanol solution was analyzed by LC-HRMS. Figure 2), silicon dioxide (SiO2), and water according to Eq. , lauryl glucoside , and sodium cocoyl glutamate  were prepared by dissolving 1 mol of each compound in 30 mL of deionized water. After the synthetic magnesium silicate  was filtered (Whatman Grade 2 filter paper) and dried in a vacuum oven at 100 °C, 1 mol (based on anhydrous molar mass) was dispersed in each of the solutions with a stirring rate of 400 rpm for a minimum of 20 min at room temperature. Solid samples were collected from these solutions at the natural pH of each solution (approximately 10). The solid samples were collected after five series of centrifugation at 8000 rpm for 10 min. The supernatant was discarded, and the sample was redispersed in DI water between each centrifugation step. The pH of each solution was then adjusted with hydrochloric acid  to approximately 7 and 5, with solid collected at each pH to obtain a total of nine solid samples. The unique labels for each of the nine samples are shown in Table Further, to determine the formation of magnesium complexes, a simple magnesium salt, magnesium chloride, was combined in solution with the charged compounds. First, 10% magnesium chloride  and 10% lauryl glucoside were prepared individually in deionized water. The two solutions were randomly mixed with variable ratios, and no precipitation was observed. The pH ranged from 6 to 10. For the control experiment, solutions of 10% sodium cocoyl glycinate  and 10% sodium cocoyl glutamate were prepared in deionized water. Precipitation was observed when magnesium chloride solution was mixed with sodium cocoyl glycinate solution and sodium cocoyl glutamate solution separately. The precipitate was collected and redissolved in methanol solution and analyzed using LC-HRMS.A workstation  was used to analyze the solid samples obtained at each pH. SEM imaging was optimized at an accelerating voltage of 2 kilovolts and a beam current of 200 picoamperes. EDS point spectra and mapping images were obtained at an accelerating voltage of 10 kilovolts and a beam current of 2 nanoamperes. SEM samples were prepared by dispersing each sample in 1.5 mL of 2-propanol and allowing 10 μL of the dispersion to dry on a membrane filter used as a support substrate for imaging purposes. The resultant solid samples on membrane filters were mounted and coated with gold/palladium through a sputtering process to reduce charging during SEM/EDS analysis.The precipitate made from magnesium chloride or synthetic magnesium silicate with negatively charged compounds was filtered and washed with water. The precipitate was dried and redissolved into methanol for LC-HRMS analysis with direct injection technology. The analysis was carried out with a Q-Exactive™ Orbitrap™ mass spectrometer  equipped with a heated electrospray ionization source (HESI-II). Samples were analyzed in full-scan MS mode under positive polarity conditions using electrospray ionization. The compound and source parameters were optimized. The samples were introduced by direct injection and delivered by an HPLC mobile phase of 90% methanol–water. The optimized parameter settings were: sheath and auxiliary gas flow rate at 30 and 10 respectively, spray voltage 3.2 kV, capillary temperature 320 C, S-lens RF level 50, auxiliary gas heater temperature 400 C. Software used for operating the LC-HRMS instrument was Xcalibur™ (version4.1).Supplementary Information 1.Supplementary Information 2.


Poly (PPTA) is a high-performance polymer that has been utilized in a range of applications. Although PPTA fibers are widely used in various composite materials, laminar structures consisting of PPTA and ultra-high-molecular-weight polyethylene (UHMWPE), are less reported. The difficulty in making such composite structures is in part due to the weakness of the interface formed between these two polymers. In this study, a layered structure was produced from PPTA fabrics and UHMWPE films via hot pressing. To improve the interlayer adhesion, oxygen plasma was used to treat the PPTA and the UHMWPE surfaces prior to lamination. It has been found that while plasma treatment on the UHMWPE surface brought about a moderate increase in interlayer adhesion (up to 14%), significant enhancement was achieved on the samples fabricated with plasma treated PPTA (up to 91%). It has been assumed that both surface roughening and the introduction of functional groups contributed to this improvement.  Poly (PPTA) is a high-performance polymer with high toughness, high tensile strength, and chemical stability at high temperatures. PPTA fibers and fabrics have been widely utilized as reinforcement in composite materials ,2,3,4. TUltra-high molecular weight polyethylene (UHMWPE) is another high-performance polymer. Due to its good mechanical, chemical, and biocompatible properties, it has gained great popularity in many applications including medical devices, body armors, marine infrastructure, and automobiles ,11,12. V® composite with pure UHMWPE samples. There have been some efforts to produce PPTA/UHMWPE composites. For example, Hofsté et al. prepared composites from chopped PPTA fibers and UHMWPE powder . The autIn addition to infusing PPTA fibers into UHMWPE powder-based matrix, knitting PPTA and UHMWPE yarns is another method to fabricate PPTA/UHMWPE composites ,23. AdheAs above-mentioned, the weak interfacial bonding was found to adversely affect the performance of the composites ,28,29. F® fibers and Kevlar® fiber/bismaleimide composites. They found that the oxygen–plasma treatment could change the chemistry and the morphology of the surface, which in turn improved the water resistance, dielectric properties, and interlaminar shear strength (40% higher than that of the untreated) [® fibers/epoxy composites using micromechanical techniques and surface wettability test. They attributed the increased interfacial shear strength (14.8% higher for PBO composite and 16% higher for Kevlar® composite in comparison to those without treatment) and work of adhesion (13.5% higher for PBO composite and 11.7% higher for Kevlar® composite in comparison to those without treatment) to induced hydrogen bonding and covalent bonding as a result of plasma treatment [® fabric/phenolic composites could be improved with plasma treatment by introducing nitrogenous and oxygenic groups as well as roughening of the surface [Among these methods, plasma treatment is of particular interest because of: (i) its effectiveness in surface modification without changing the bulk properties of the processed materials; (ii) ease of implementation; and (iii) minimal environmental impact (no acids or organic solvents are needed) . There atreated) . Park anreatment . Guo et  surface . As previously discussed, the majority of the efforts to make PPTA/UHMWPE composites were focused on PPTA fibers and UHMWPE powders. There is limited information in the open literature on PPTA/UHMWPE laminates made with continuous UHMWPE film material. In this study, we report the research on producing a laminar structure consisting of PPTA fabrics and UHMWPE films using a facile hot-pressing protocol. Oxygen plasma treatment was applied to both materials and their surface characteristics, both topological and chemical, were altered. Our results showed that the interlaminar adhesion could be enhanced by treating either the PPTA or the UHMWPE surfaces. However, significant improvement was achieved when the plasma treatment was conducted on the PPTA fabrics. With the controllable interlaminar adhesion, future work will focus on PPTA/UHMWPE composite panels, targeting body armor applications. 2, thickness: 0.37 mm, weave: plain) woven from Kevlar® 49 fiber and UHMWPE film (thickness: 0.2 mm) were purchased from Goodfellow Corp. . The epoxy glue (JB weld epoxy) was purchased from JB Weld Company .The PPTA fabrics (area density: 218 g/mThe UHMWPE film cut into pieces (1” × 1.5”) was sonicated with acetone, rinsed with deionized water, and dried with nitrogen. Oxygen plasma treatment of UHMWPE and PPTA fabrics (1.5” × 2”) was performed using a Harrick Plasma cleaner PDC-32G (3” diameter × 6.5” length Pyrex chamber)  for different times. Surface free energies of UHMWPE with different plasma power and treatment time were calculated from contact angle measurements . A plasmHeat press machine used in preparation of laminate was purchased from Across International, Inc. . Two pieces of PPTA fabrics and one piece of UHMWPE film were stacked to a laminate structure with the UHMWPE film in between, as shown in Differential scanning calorimetry (DSC) was used to study the thermal behavior of the UHMWPE film , whose mThe peel test was conducted according to ASTM D903-98 using a universal testing machine . Sample configuration is shown in The surface morphologies of PPTA fabric and UHMWPE film after plasma treatment were characterized using scanning electron microscopy , atomic force microscopy . The surface chemical group were analyzed by Fourier transform infrared spectroscopy  and X-ray photoelectron spectroscopy . For XPS, a 100 μm monochromatic Al Kα (1486.6 eV) X-ray beam was used to irradiate the sample surface. The C1s peak was calibrated to 285.0 eV and CasaXPS was utilized to carry out peak fitting procedure. Failure analysis was conducted by examining the surface morphologies after peeling test with SEM.The morphology of the PPTA fabric is shown in AFM analysis was further carried out to examine the surface morphologies of both PPTA fabric and UHMWPE film as a result of the plasma treatment . The untThe AFM results of UHMWPE with different treatment times are shown in −1 and 1537 cm−1 correspond to the stretching vibration and the bending vibration of the N–H bonds, respectively. The peaks at 1394 cm−1 and 1111 cm−1 correspond to the stretching vibration of C–N. Peaks at 1610 cm−1 and 1510 cm−1 correspond to the stretching vibration of C=C. The stretching vibration of C=O in amide carbonyls and the bending vibration of C–H result in two peaks at 1640 cm−1 and 822 cm−1, respectively [−1 and 1300–1255 cm−1 can be ascribed to C–N stretching and the symmetrical deformation of the –NO2 group [−1. This peak corresponds to the stretching mode of oxygen containing group C–O, which may be introduced by the plasma treatment. FTIR was performed to investigate possible chemical changes induced by the plasma treatment. ectively . The peaO2 group . On clos−1, 2850 cm−1, 1473 cm−1 and 719 cm−1, which correspond to the non-symmetric stretching vibration, the symmetric stretching, the bending vibration, and the in-plane vibration of –CH2– bond, respectively [−1 and 1198 cm−1, which correspond to the C=O stretching and C–O stretching, respectively. The peak at 1078 cm−1 related to the C–O stretching mode became stronger after plasma treatment. The emergence of these oxygen containing bonds is believed to be a result of the plasma treatment. ectively . Two smaSince plasma treatment only modifies the topmost surface  while the FTIR detection depth is usually on the level of micrometers , the relIn addition to FTIR, the X-ray photoelectron spectroscopy (XPS) technique was also used to examine the surface chemistry of both the PPTA fabric and the UHMWPE film samples. The content of carbon, however, showed an opposite trend, with a decrease from 80.05% (untreated) to 63.71% (1 min plasma treatment). The deconvoluted C1s peaks of untreated PPTA and those treated for 1 min, 2 min, 5 min and 10 min are compared in The XPS results of UHMWPE films with different plasma treatment times are shown in The average peeling forces for all samples included in this study were summarized in The treatment time of the PPTA fabrics had a significant effect on the improvement of adhesion. As shown in The morphology of both PPTA and UHMWPE surfaces after the peel-off test is shown in Laminate samples were fabricated from PPTA fabrics and UHMWPE films by hot pressing method. Oxygen plasma treatment was utilized to enhance the interlamellar adhesion between PPTA fabrics and UHMWPE film. SEM and AFM results showed that plasma treatment could modify the surface texture, while FTIR and XPS analyses demonstrated the surface functionalization induced by the plasma treatment. The 180-degree peel test result showed that the adhesion of the PPTA/UHMWPE laminate could be significantly enhanced when the PPTA fabrics were treated with the oxygen plasma. It was likely that the process of oxygen plasma treatment introduced active functional groups and cleaned and roughened the fiber surface, thereby improving the bond between UHMWPE and modified PPTA fabrics. PPTA/UHMWPE composites with controllable interlaminar adhesion for body armor applications will be studied in future work.


In general, there were no effects of 24 h pre-exercise lower leg compression, including those treated with menthol and camphor applications on running kinematics, skin temperature, heart rate, or recovery perception in athletes undertaking consecutive running exercises.Compression and temperature manipulation are discussed as strategies to improve performance markers and recovery in sports. Here, we investigate the effects of compression stockings made with fabric, either combined or not with heating and cooling substances, on variables related to running performance and recovery. Ten trained runners  with no experience of using compression garments performed an intense running session of 10 km, then wore a stocking for 24 h , and were evaluated on the following day, after running 5 km. The different types of compression stockings used 24 h before exercise did not affect running kinematics ( Athletes are interested in compression garments to improve athletic capacity and exercise recovery, but there is still a lack of evidence regarding their biomechanics and comforting effects. While compression can affect muscle soreness, muscle damage and inflammatory markers, performance, fatigue, and thermoregulation markers are usually unaltered . BiomechPrevious studies investigating the lower extremity biomechanics in participants subject to compression showed controversial results. Hip and thigh compression may influence the performance of jump landing tasks due to decreased dynamic valgus in the landing  and reduThe effects of compression on running performance parameters have attracted the interest of scientists, but knowledge about their effects on running kinematics, for example, is limited. Moreover, evidence to support the claim of below-knee compression garments in improving recovery in runners is also limited . The mecFor two consecutive 5 km running sessions with a 1 h recovery period, wearing compression stockings in between did not influence performance time and rate of fatigue. When the same participants were grouped according to their perception of the efficacy of compression stockings (being classified as believers or not), the runners with a stronger belief in the effects of compression showed improved running performance after 1 h compression compared to those with a neutral or negative perception of compression effects . When 4 Considering that different studies report different responses to compressive garments, outcomes are often discussed as dependent on blood flow and oxygenation, and therefore body temperature could play a role. For example, manipulating temperature is a physiological strategy for improving muscle contraction, which helps to explain why a warming-up period before exercise can benefit performance . The useIt is challenging to combine temperature manipulations while delivering compression. One option is to use chemical compounds combined with the textile material. A compression fabric with menthol application promotes the perception of freshness and reduces heating discomfort . On the The research was advertised in local running clubs, and experienced runners were invited to join the experiment. They were invited to visit the laboratory eight times within 12 weeks when no competition was scheduled. The eight visits always included a block of two consecutive days of indoor running, one for each of the control or compression garment conditions. We were able to include ten experienced male competitive runners in the study. They had mean ± standard deviation age 45 ± 9 years old, body mass 69 ± 7 kg, height 166 ± 4 cm, and have frequently been training over the past 14 ± 13 years, with a 5 km personal record of 20:13 ± 3:54 min . All participants were members of running clubs from the local community, had been training and running for at least one year uninterruptedly, had no history of injury or pain, had participated in amateur competitive events with distances from 5 to 42 km, and did not have any previous experience of using compression. Participants not completing all the evaluation sessions in the expected period of 12 weeks were excluded. The participants had never worn leg compression garments before and did not classify themselves as believers in compression effects during an interview, which included questions such as “do you know what compression garments are?” and “do you believe they can improve performance during running?” The local institution ethics committee approved this research, and all participants signed a consent term. All procedures complied with the Helsinki declaration.Compression and control garment conditions were randomized. On day 1, participants ran 10 km at their competitive pace. On day 2, they ran 5 km at the same speed as day 1, and the data were collected. Garments were provided after the end of exercise on day 1 and worn continuously for the following 24 h. Each garment condition was evaluated at least one week apart, and participants always used the same shoes. Running was performed without the garments. Four different garments were considered. They were all long socks from the knee popliteal line to the foot. The compression garments were stockings made of 10% polyamide, 75% polyester, and 15% elastomer. Three compression models were used as the standard and two others that included camphor and menthol to induce the perception of hot and cold. The garments’ compression was gradual, with an average of 21–24 mmHg pressure reported by the manufacturer. A finishing textile process in a padding machine applied camphor and menthol; the concentration of the solution was: 7% of the respective substance (camphor or menthol), 3% of polyvinyl pyrrolidone (PVP), 35% of ethanol, and 55% of water. After impregnation, the garments were dried for polymerization. The control stocking was a model of the same color and size but made of 100% cotton. Cotton socks are standard for a runner, and we therefore considered it as a control. The purpose of compression garments is to promote greater compression than cotton socks. All the stockings were brand new, from the same manufacturer, and had identical designs and appearance. Sizes were adjusted for each participant considering leg circumference and according to the manufacturer’s sizing chart.Running was performed on a motorized treadmill with control for speed and inclination set at 1% . Running was performed at a competitive race pace corresponding to 90% of the best 10 km personal record [https://kinovea.org, accessed on 6 July 2021). Spherical reflexive markers of 14 mm diameter were placed on the shoe at the fifth metatarsal, lateral malleolus, lateral knee epicondyle, great trochanter, and acromion on the right side of the body. Joint angles were defined in the sagittal at the foot strike as illustrated in Figure 3 . Ten complete strides were analyzed for each kilometer considered [Trunk, hip, knee, and ankle sagittal plane angles determined at the foot strike instant, step frequency, and step length were determined by 2D video analysis . Measurensidered . All parOn day 2, participants were questioned about their perception of recovery regarding the exercise performed the day before. The perception of recovery was assessed considering a visual scale ranging from 0 (very poorly recovered/extremely tired) to 10 (very well recovered/highly energetic) . For botSkin temperature was determined before and immediately after each running session using a thermal infrared camera  with Noise Equivalent Temperature Difference (NETD) < 0.05 °C, and measurement uncertainty of ±2 °C or 2%. To record the images, the camera was positioned 1 m away from the participant and with the lens aligned perpendicular to the body region of interest (ROI) and turned on 10 min before taking the images to ensure its stabilization. The images were recorded while the participant was standing up wearing underpants after a room thermal adaptation of 10 min. An anti-reflective panel was placed behind the participant to minimize the effects of infrared radiation reflected from the wall. All recommended procedures regarding environmental conditions  and participant preparation regarding drinking alcohol or caffeine, smoking, using cosmetics, having large meals, using ointments, sunbathing, engaging in physical activity, and undergoing physiotherapy treatments were followed to minimize influence factors. The TISEM checklist was completed to certify that all the important protocol and thermographical analysis aspects were attended to . Four ROEach ROI was defined with a similar area for all participants. Temperature data) were analyzed using commercial software , taking an emissivity factor of 0.98  into accp > 0.05). Repeated-measures ANOVA with two factors (measurement moment and garment condition) was applied for kinematic  and comfort parameters. Resting heart rate, maximum heart rate, and recovery rate were assessed with repeated-measures ANOVA with one factor (garment condition). For leg perimeter and skin temperature parameters, repeated-measures ANOVA with three factors  was applied. When significance was found, the Bonferroni post hoc test was applied to identify the differences. The significance level was set as 0.05.Data from the different conditions are reported as mean ± standard deviation. The normality of data distribution was checked using the Shapiro–Wilk test  of 6.46%.p = 0.15; main effect of moment p = 0.24, stocking * moment p = 0.37; stride rate: stocking condition p = 0.16, moment p = 0.26, and stocking condition * moment p = 0.37). The mean stride length and stride rate were 0.79 ± 0.06 m and 4.3 ± 0.2 stride/s, respectively. No differences in angular kinematics were observed between compression and control conditions .The stride length and stride rate did not differ between the different garment conditions (stride length: main effect of stocking conditions p > 0.05). Although the garment conditions main effect was significant for anterior thigh, when considering the measurement of mean skin temperature and maximum skin temperature and its interaction with measurement moment for the anterior leg of minimum temperature, the post-hoc analysis did not reveal differences between garment conditions (p > 0.05). The main effect of each factor and its interactions with the other factors of the repeated-measures ANOVAS models are shown in p = 0.81) and maximal heart rate (p = 0.13) between the stocking conditions (p = 0.13). Perception of recovery evaluated on day 2 did not differ between the garment conditions (p = 0.86), presenting an average value of 7.4 (0.4) points  points .Our study aimed to determine the effects of 24 h lower leg compression using different compression garments, including those inducing perceptions of hot and cold, on aspects of running performance by considering the kinematic assessment of technique, intensity, comfort, and recovery following consecutive running exercises. Our main findings were that the different types of compression, including chemical compounds, such as menthol and camphor, or not on the lower leg did not affect the parameters evaluated. Importantly, rest and maximal heart rate did not differ between experimental conditions, suggesting that a similar intensity was performed for all tests. Under the test conditions performed here, compression did not present any perceived advantage for the runners.One could argue that the magnitude of compression provided by the garment can influence the results. However, a previous study showed that compression effects were not associated with the magnitude of pressure applied . The preIn a previous study, compression administrated to the hip and thigh elicited changes in frontal plane kinematics towards a movement pattern considered protective against knee injuries during jump landing . Here, wThere are controversial results in the literature about the effects of compression garments on thermal stress. If compression garments increase skin temperature, this could be negative for performance due to the reduced thermal gradient between the core and the skin . We did The use of fabrics that promote thermal interactions between the stocking and the skin did not influence recovery perception and exercise intensity. Positive effects were described for menthol in thermal perception, ventilatory responses, analgesia , and incOur study has limitations. The compression garment may influence arterial blood pressure both locally and in the body, as well as the percentage of the total body water. We did not measure these parameters that influence the vein return and final cardiac output. The maximal or the resting heart rate is not the best physiological variable to assess the impact of compression on cardiovascular function. We used a 2D video for kinematics analysis, as it has been reported that 2D video analysis is accurate enough to evaluate the variables that we assessed during treadmill running . The resIn this study, we investigate the effects of compression stockings made with fabric, either combined or not with heating and cooling substances, on variables related to running performance and recovery. Overall, no effect of 24 h lower leg compression including menthol and camphor applications was found on characteristics of running performance considering kinematics, heart rate, and perceived recovery of runners performing consecutive running sessions at a competitive pace.


The blaCTX-M locus was only detected during spring and was more prevalent downstream of intensive dairy farms. Loci associated with STEC were detected in both the autumn and spring, again predominantly downstream of intensive dairying. This cross-sectional study suggests that targeted testing of environmental DNA is a useful tool for monitoring waterways. Further studies are now needed to extend our observations across seasons and to examine the relationship between the presence of these genetic elements and the incidence of disease in humans.The emergence of clinically significant antimicrobial resistance (AMR) in bacteria is frequently attributed to the use of antimicrobials in humans and livestock and is often found concurrently with human and animal pathogens. However, the incidence and natural drivers of antimicrobial resistance and pathogenic virulence in the environment, including waterways and ground water, are poorly understood. Freshwater monitoring for microbial pollution relies on culturing bacterial species indicative of faecal pollution, but detection of genes linked to antimicrobial resistance and/or those linked to virulence is a potentially superior alternative. We collected water and sediment samples in the autumn and spring from three rivers in Canterbury, New Zealand; sites were above and below reaches draining intensive dairy farming. Samples were tested for loci associated with the AMR-related group 1 CTX-M enzyme production ( There ianimals) . Environanimals) . TherefoAntimicrobial resistant infectious diseases are emerging as one of the most pressing global public health issues . AntimicEscherichia coli—are recognized as an important group of bacteria that carry AMR in clinical settings  enzymes provide resistance to beta-lactam antibiotics, including first and third generation cephalosporins, carbapenems and penicillins . The proplasmids . Some ESoli–STEC–, a groupoli–STEC–). STEC aoli–STEC– and USA oli–STEC– and incroli–STEC–. STEC haal route .Molecular characterization is routinely used to classify cultured bacterial isolates, as culturing alone is unable to identify all the genetic components imparting AMR or virulence . Pre-isoE. coli (uidA), with the remaining six frequently associated with group 1 CTX-M β-lactamase production (blaCTX-M) and human-pathogenic STEC virulence  and STEC serotype (O157 rfbE and O26 wzy).In this case study we tested eDNA collected from sites on three Canterbury, New Zealand rivers during autumn (May) and spring (September), which coincides with seasonal peaks in human STEC cases . We evalTwo substrates, water and sediment, were sampled, collected from the Ashley, Rangitata, and Selwyn rivers once in austral autumn and spring, 2018. Collections were made at two sites along each river; these sites were 10–15 km apart with one above and the other below reaches draining intensive dairy farms . At each2O to a final volume of 50 ml. At sites where the level of suspended sediments was high, an additional 1:50 dilution was also prepared to ensure ease of enumeration. For each sample we prepared three technical replicates at each dilution .Water column sample aliquots were diluted 1:10, 1:5, and 1:2.5 with sterile MilliQ H2O added, and the mixture then agitated vigorously for 30 s. Aliquots of the resulting supernatant were diluted 1:500, 1:200, 1:100, and 1:50 with MilliQ H2O to a final volume of 50 ml. For each sample we prepared three technical replicates at each dilution .For sediment samples, 2 g of wet sediment was first transferred to a 5 ml microtube, 3 ml MilliQ HFor each dilution the total 50 ml volume was vacuum filtered through a single sterile 0.45 µm cellulose ester membrane filter .E. coli (red/magenta colonies) were counted.Bacterial culturing followed United States Environmental Protection Agency method 1603 . Each fiThree 500 ml aliquots of water from each site were vacuum filtered through separate 0.45 µm cellulose ester membrane filters. Environmental DNA was extracted from half of each filter using the NucleoSpin® soil kit  following the manufacturer’s instructions. For each sediment sample eDNA was extracted from three 0.5 g aliquots of wet sediment, using the NucleoSpin® soil kit.bla gene associated with group 1 CTX-M β-lactamases (blaCTX-M) using the primers of rfbE for O157 and wzy for O26, the stx1 and stx2 toxin genes, and the intimate attachment and effacing gene, eae, using the primers reported by 3 CFU/ml   targeting the 3 CFU/ml  with a sclusive) . As an a E. coli  , 1 pM of each primer, and 2.5 μl of DNA template. Thermocycling was performed in a T1 thermocycler  using standard cycling conditions including an initial denaturation at 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min, with a final extension at 72 °C for 5 min. Amplification products were visualized using SYBR Safe  following electrophoresis in 2% Tris-acetate-ethylenediamine tetraacetic acid agarose gels.E. coli were consistently higher from sediment than water column samples , colonies indicative of  samples . MoreoveuidA locus was successfully amplified from every sample (blaCTX-M) and the three gene loci associated with pathogenic STEC  varied with location, time and substrate. The antibiotic resistance gene was detected in both substrates, with equal frequency, in samples from the Selwyn and Rangitata rivers. The stx1, stx2 and eae genes were more frequently detected in water samples in the autumn but in sediment samples in the spring. In both the autumn and spring, serotype genes (O26 wzy and O157 rfbE) were amplified from the same sample as one or more toxin genes (stx1 or stx2) and the effacement gene (eae). The O26 marker was more frequently recovered from water, while that for O157 was more common in sediment.The y sample . In conti.e., stx1, stx2, eae and rfbE) were present in 25% of the samples. All three virulence and both serogroup genes were detected in the autumn, both substrates from the Ashley and the water from the Rangitata had both serotype markers in the ‘below intensive dairy’ samples. Additionally, the stx1, eae, and rfbE genes were present in water from the site ‘below intensive dairy’ on the Ashley River in spring. The blaCTX-M gene associated with AMR was not detected in any autumn samples.In autumn, the genes associated with pathogenic STEC O157  was detected 4 times more often than that associated with the O26 serogroup (wzy). In three samples stx1, stx2 and eae were all detected  genes were detected in all three of the rivers sampled. Every sample tested positive for the uidA gene, consistent with E. coli being isolated at all six sites, from both substrates, and at both sampling times using traditional plating.In our analyses the genetic markers indicative of stx1, stx2 and eae) and O serotype (O26 wzy and O157 rfbE) were present in every river, their presence varied by location, substrate, and season. Most importantly, all the genes necessary for human pathogenic STEC  were detected within a single sample 11 times and AMR genes were only detected from samples that contained all the genes for human pathogenic STEC. These results suggest that explanations for the distribution of these genes across the landscape are likely to be complex and involve a range of factors.Using relatively small sample volumes we were able to detect genes associated with both AMR and human pathogenic STEC. Although all the genes associated with human pathogenic STEC virulence  in the spring and in three of 12 samples (25%) in the autumn whereas the AMR gene was only detected in the spring from two rivers. Detection of the AMR gene during the spring may reflect patterns of antibiotic use on the surrounding farms. Antibiotics are often administered to drying-off cows during winter as well as during spring calving and early milking , potentiE. coli were consistently higher in sediments than the water column. This is a common finding frequently attributed to substrate stability; sediments are less mobile than flowing water, therefore a longer-term and more stable habitat for bacteria   , disturbe.g., counting colonies visually assessed to be E. coli) and the presence of genes associated with virulence and/or AMR. Moreover, the virulence or AMR status of isolated bacteria are not routinely assessed leading to delays in risk management. Understanding the relationship between pathogenic bacteria and AMR in recreational waterways would require more intensive sampling over a broader geographic range and the inclusion of multiple substrates in sampling protocols. In most cases monitoring is conducted on samples retrieved from the water column. Such samples may not accurately reflect the microbial community that recreational users of the waterway are potentially exposed to. Our results suggest sediments may act as an important reservoir of bacteria and resuspension of these sediments by waterway users could potentially increase exposure to pathogenic strains.Monitoring of recreational waterways for human pathogens is a complex but important task. In part this can be attributed to the metabolic diversity of the bacteria themselves and the apparent lack of a relationship between faecal indicator bacteria (There is a growing appreciation of new technologies that enable the presence and persistence of zoonoses to be monitored without the need for microbial culturing . This re


Hermetia illucens (Hi) larvae included in a low-FM diet on gut microbial communities of rainbow trout (Oncorhynchus mykiss), in terms of both composition and function of microbiome. A feeding trial was conducted using 192 trout of about 100-g mean initial weight. Fish were fed in quadruplicate (4 tanks/diet) for 131 days with two diets: the control (Ctrl) contained 20% of FM as well as other protein sources, whereas the Hi diet contained 15% of Hi larvae meal to replace 50% of the FM contained in the Ctrl diet. High-throughput sequencing of 16S rRNA gene was used to identify the major feed and gut bacterial taxa, whereas Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis was performed on gut bacterial genomes to identify the major active biological pathways. The inclusion of IM led to an increase in Firmicutes, mainly represented by Bacilli class and to a drastic reduction of Proteobacteria. Beneficial genera, such as Lactobacillus and Bacillus, were enriched in the gut of fish fed with the Hi diet, whereas the number of bacteria assigned to the pathogenic Aeromonas genus was drastically reduced in the same fish group. The metagenome functional data provided evidence that dietary IM inclusion can shape the metabolic activity of trout gut microbiota. In particular, intestinal microbiome of fish fed with IM may have the capacity to improve dietary carbohydrate utilization. Therefore, H. illucens meal is a promising protein source for trout nutrition, able to modulate gut microbial community by increasing the abundance of some bacteria taxa that are likely to play a key role in fish health.With demands and reliance on aquaculture still growing, there are various challenges to allow sustainable growth and the shift from fishmeal (FM) to other protein sources in aquafeed formulations is one of the most important. In this regard, interest in the use of insect meal (IM) in aquafeeds has grown rapidly. Accordingly, the aim of the present study was to assess the effects of dietary IM from The online version contains supplementary material available at 10.1007/s10695-020-00918-1. Aquaculture is growing rapidly and becoming integral in global food resources, supplying around half of the world’s seafood supply. One of the most important challenges that aquaculture sector is currently facing is the shift from fishmeal (FM) to other protein sources in aquafeed formulations and considerable efforts have been made so far to achieve this , essential amino acids, lipids, minerals, and vitamins, having a nutritional profile similar to FM , common housefly (Musca domestica), yellow mealworm (Tenebrio molitor), lesser mealworm (Alphitobius diaperinus), house cricket (Acheta domesticus), banded cricket (Gryllodes sigillatus) and field cricket (Gryllus assimilis).The EU Regulation No. 2017/893 (Annexe II of 24th May 2017) authorises the use in fish feeds of insect-derived proteins originating from seven species, namely, black soldier fly (Hermetia illucens) is considered one of the most promising species to be used in feeds for salmonids, i.e. rainbow trout (Oncorhynchus mykiss) and Atlantic salmon   larvae satisfy these requirements as they contain a very high percentage of protein (36–48% DM) and fat (31–33% DM) and an essential amino acid profile similar to FM . All procedures involving fish comply with the guidelines of the European Union Council (2010/63/EU) for the use and care of experimental animals. The Ethical Committee of the University of Turin (protocol no. 143811) approved the experimental protocol.Hermetia illucens (Hi) larva meal to replace 50% of the FM contained in the Ctrl diet. Hermetia illucens larva meal was provided by MUTATEC . Due to differences in chemical composition between Hi and FM and to ensure isonitrogenous, isolipidic, and isoenergetic diets, the level of inclusion of porcine haemoglobin and wheat starch slightly changed.Two diets were formulated to be isonitrogenous, isolipidic, and isoenergetic Table . The firAll feeds were prepared through cold pelleting at the experimental facility of the Department of Agricultural, Forest and Food Science (DISAFA) of the University of Turin . Briefly, all grounded ingredients were mixed with oil and desired consistency for pelleting was gained by adding water to the mixture. Each diet was cold pelleted using a 2.5-mm die meat grinder and the obtained pellet was dried at 50 °C for 48 h. Diets were stored in dark bags at a controlled temperature and humidity conditions.3 connected to a flow through an open system supplied with artesian well water . Fish were manually fed with two experimental diets in quadruplicate (four tanks/diet). The feeding rate was restricted to 1.4% of biomass for all the duration of the trial (131 days). Fish mortality was checked and recorded every day. At the end of the feeding trial, eight fish/dietary groups (2 fish/tank) were sacrificed by over anaesthesia with MS-222 . The intestine was aseptically isolated from each fish, and the faecal matter was obtained by squeezing out and scrapping the intestinal mucosa with a sterile spatula, in order to collect both the digesta- and the mucosa-associated microbiota (transit and resident microbiota). The microbiota samples were then transferred into a sterile 2-mL tube containing 800 μL of Xpedition™ Lysis/Stabilization Solution  and then stored at room temperature, until DNA extraction (within 48 h).A total of 192 rainbow trout with an initial mean body weight of about 100 g were randomly distributed in 8 outdoor fibre glass tanks of 0.4 mThe amplification of the V4 region of the bacterial 16S rRNA gene and amplicon library construction were conducted as previously reported by our group , and merged. Filtered reads were dereplicated; singletons and chimeric sequences were removed using QIIME DADA2 denoise-paired command. All sequences were then clustered into operational taxonomic units (OTUs) at a 97% similarity cut-off. OTUs were classified using the reference Greengenes v. 13.8 as reference database (http://greengenes.lbl.gov/) down to genus level. Chloroplasts as well as sequences that were eukaryotic were removed. Sequences that had a frequency lower than 0.005% were removed from the dataset. Alpha rarefaction curves were plotted to determine the adequacy of sequencing depth. Alpha diversity indexes  were calculated to explain the species richness and diversity in each sample. Good’s coverage estimator was used to assess the percentage of the total species that are represented in a sample. Principal coordinates analysis (PCoA) was conducted to visualize similarities or dissimilarities of data based on unweighted UniFrac and weighted UniFrac distance metric  at the default setting  at 99% of similarity. The corresponding biom table was generated using the tools export function and used as input for the PICRUSt pipeline. In brief, PICRUSt was first used to correct biom tables for 16S rRNA copy numbers and subsequently used to predict KEGG (Kyoto Encyclopedia of Genes and Genomes) orthologues (KO). The maximum allowed Nearest Sequenced Taxon Index (NSTI) value was set to 2 to control for the overall accuracy of the metagenomic predictions. The output data generated with PICRUSt were subsequently uploaded to the Statistical Analysis of Metagenomic Profiles (STAMP) software package  , Student’s t test or nonparametric Mann-Whitney U test was applied depending on normality and homoscedasticity of the data. All analyses were performed using Past3 software , mainly represented by Bacilli class than the Ctrl feed (40%). Conversely, microbiota associated with Ctrl showed a higher relative abundance of Proteobacteria (58%), principally belonging to Alpha- and Gammaproteobacteria classes  or entropy (Shannon and evenness) Table .Table 3AR = 0.46, pseudo-F = 3.32, q < 0.05) in the composition of the microbiota between Ctrl and Hi15 feeding groups only in the unweight UniFrac analysis  Fig. , but notp < 0.05) by dietary insect meal   Table , Table 5ols Fig. , Table 5PICRUSt was applied to predict the functional potential of the intestinal microbiome of rainbow trout. Level 3 KEGG orthologue function prediction was used. Our analysis revealed 217 predicted metabolic pathways  was significantly increased by dietary supply of 15% of insect meal in our study. Previously, we found an increase of species richness only in the digesta-associated , but not in mucosa-associated (autochthonous), gut microbiota of rainbow trout fed with increasing levels of Hi meal (10–30%)   may be due to the prebiotic effect of chitin, and, as proposed by Bruni et al.  from prokaryote cells acts as intercellular signal, principally for leukocytes. Chaperones and foldase have been defined “moonlighting” proteins since they may act as homeostatic immune regulators and, under certain circumstances, contribute to tissue pathology as well ESM 2(PDF 37 kb)ESM 3(PDF 37 kb)ESM 4(PDF 156 kb)ESM 5(PDF 232 kb)


Biliary atresia (BA) is a severe cholangiopathy of early infancy that destroys cholangiocytes, obstructs ductular pathways and if left untreated, culminates to liver cirrhosis. Mechanisms underlying the etiological heterogeneity remain elusive and few studies have attempted phenotyping BA. We applied machine learning to identify distinct subtypes of BA which correlate with the underlying pathogenesis.The BA microarray dataset GSE46995 was downloaded from the Gene Expression Omnibus (GEO) database. Unsupervised hierarchical cluster analysis was performed to identify BA subtypes. Then, functional enrichment analysis was applied and hub genes identified to explore molecular mechanisms associated with each subtype. An independent dataset GSE15235 was used for validation process.FCGR2A, suggesting an autoimmune response targeting bile ducts. Subtype 2 was associated with immune receptor activity, cytokine receptor, signaling by interleukins, viral protein interaction, suggesting BA is associated with viral infection. Subtype 3 was associated with signaling and regulation of expression of Robo receptors and hub gene ITGB2, corresponding to embryonic BA. Moreover, Reactome pathway analysis showed Neutrophil degranulation pathway enrichment in all subtypes, suggesting it may result from an early insult that leads to biliary stasis.Based on unsupervised cluster analysis, BA patients can be classified into three distinct subtypes: Autoimmune, Viral and Embryonic subtypes. Functional analysis of Subtype 1 correlated with Fc Gamma Receptor (FCGR) activation and hub gene The classification of BA into different subtypes improves our current understanding of the underlying pathogenesis of BA and provides new insights for future studies. Biliary atresia (BA) is a severe neonatal disease that often ends with cholestasis and progressive hepatic failure. Although BA is a rare disease, with variable incidence ranging from 1 in 5,000 to 1 in 19,000 live births per year, it is the leading cause of end-stage liver disease in children and the leading indication for pediatric liver transplantation worldwide , 3. ElucPathological jaundice and acholic stools in the first few months of life are the clinical hallmarks of BA. Depending on time of disease onset, BA patients were initially divided into “embryonic/developmental” BA <20%) and “perinatal/acquired” BA (>80%) . The pre0% and “pde novo classification for BA. Then, validation was carried out using an independent BA dataset. Finally, we explored the mechanisms associated with each BA subtype, by identifying related immune cells and the hub genes.A growing body of evidence suggests the presence of activated immune cells in liver tissue of BA patients at the time of diagnosis, and an association with autoimmunity, amplification of epithelial injury, and bile duct obstruction –8. With Two datasets [GSE46995  and GSE1R package “CancerSubtypes”  was usedt-test and p<0.01 was used as the threshold value to test whether expression of DEGs was statistically significant. The representative DEGs were identified by a Venn plot method. For example, to identify the DEGs of subtype 1, a Venn plot of (subtype 1 + subtype 2) v/s (subtype 1 +subtype 3) was drawn to obtain the overlap of DEGs. p<0.05 represented statistically significant difference. Screening of immune and stroma cells for each subtype was performed by “tableone” and “t-test” and p<0.05 was used as the selection criteria. The screening method used to identify each BA subtype’s representative immune and stroma cells was the same as screening DEGs.R package “tableone” was used to describe baseline characteristics. Comparison of genes between subtypes was performed using https://string-db.org/). The hub genes were analyzed, and the PPI network was visualized by cytoscape plug-in Molecular Complex Detection (MCODE) and CytoHubba of Cytoscape software version 3.7.2. MCODE was used to screen the modules of the PPI network identified by degree cutoff =2, node score cutoff =0.2, K core =2 and a maximum depth=100. Then, the importance of these hub genes was calculated by cytoscape plug-in CytoHubba, using the topological algorithm “degree”. In the PPI network, genes shown in red indicated more frequent interactions with other proteins.We performed enrichment analysis and Reactome pathway analysis using R packages “clusterProfiler” and “ReactomePA” to provide a functional interpretation of the representative genes. Then, the protein-protein interaction (PPI) network analysis was constructed by STRING (A flowchart of our study is illustrated in A Venn plot was used to identify representative genes for each subtype (See method section 2.4 for more details). Subtypes 1, 2 and 3 consisted of 839, 1173 and 1447 representative differentially expressed genes, respectively  enrichment and Reactome pathway analysis. MF analysis of genes from Subtype 1 showed correlation with GDP binding and fatty acid binding, while reactome analysis showed enrichment in neutrophil degranulation, rRNA processing and Fc Gamma Receptor (FCGR or FcγR) activation pathways  pathways. FCGR2A, formerly known as CD32, plays an important role in the clearance of immune complexes by phagocytic cells such as macrophages and neutrophils. Prior studies have demonstrated strong evidence that FCGR2A is not only involved in immune system activities but also with autoimmunity  in subtype 3 was not significantly different from the healthy group . Clinical studies have consistently shown poorer prognosis of CMV positive patients after HPE , 31. Intvia other methods. Accordingly, our attempt to classify BA by integrating multi-omics data requires further validation via in vivo and in vitro studies.Nonetheless, hospitals performing more than five cases per year had better surgical outcomes compared with hospitals performing a smaller number of HPE . At fourThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ZC, JG, and XL designed this study and took responsibility for the integrity of the data and the accuracy of the data analysis. XP and JC analyzed the data and wrote the article, SC, ZG, GL and YC contributed to revise the article. All authors contributed to data interpretation, and reviewed and approved the final version.This work was supported by a grant from the National Key R&D Program of China (2018YFC1315400), Guangdong Key Field R&D Plan (2019B020228001) and 5010 Project of Sun Yat-sen University (No. 2018024).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Antioxidant addition altered the mechanical and barrier properties of the films. At 34% relative humidity (RH), increasing the concentration of quercetin increased the tensile strength and decreased the elongation at break of the composite films. Increasing quercetin and TBHQ contents increased the film water solubility and water vapor transmission rate. Intermolecular interactions between the antioxidants and films, as found in Fourier transform infrared (FT-IR) spectra and XRD micrographs, were related to the changed film functionalities. In food application studies, the cassava starch/gelatin films containing quercetin and TBHQ retarded the oxidation of lard (more than 35 days) and delayed the redness discoloration of pork. Cassava starch/gelatin composite films integrated with quercetin and TBHQ can be utilized as active packaging that delays oxidation in foods.Edible and active packaging are attractive for use in food packaging applications due to their functionality and sustainability. This research developed new antioxidant active food packaging materials from cassava starch/gelatin (7:3  The improvement of active biodegradable films has been increasing because of their potential substitution for some petrochemicals in the food packaging industry and their perceived sustainable choice. Biodegradable films are normally produced from agricultural wastes that contain abundant biopolymeric materials such as polysaccharides, proteins, lipids, or the combination of these components. In efforts to improve functionality, films containing combinations of polymers have been investigated, including those based on polysaccharide and protein blends such as methylcellulose–wheat gluten , soluble2. Owing to their chemical stability, low cost, and availability, synthetic antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tertiary butylhydroquinone (TBHQ) are widely used in the food industry [As food additives, antioxidants are commonly used to improve the oxidation stability of lipids and to extend the shelf life, especially for fatty products and foods sensitive to Oindustry . In the industry . TBHQ isindustry  because industry . Howeverindustry . Many naindustry .Punica granatum L.) peel powder has great potential as an active film with antioxidant and antimicrobial properties, and thus, it can help maintain the quality and prolong the shelf life of food products. Shao et al. [Staphylococcus aureus, Bacillus subtilis, and Escherichia coli. Fang et al. [Recently, antioxidants have been incorporated into polymer films to advantageously utilize their antioxidative properties in both the films and products to which they are applied . Hargenso et al.  added teg et al.  producedg et al.  added epg et al.  found thg et al.  developeThe cassava starch/gelatin films used as a starting point in this study were effectively prepared and characterized in a previous study . Other rThe films were made by cassava starch . Gelatin and 2,2-diphenyl-1-picryl hydrazyl (DPPH) were acquired from Sigma  and Fluka , respectively. Antioxidants, quercetin, and tertiary butylhydroquinone (TBHQ) were purchased from Alfa Aesar  and Fluka , respectively. Magnesium chloride and magnesium nitrate from Ajax Finechem  were used for controlling environmental RH is storage chambers to 34% and 54% RH, respectively. Folin–Ciocalteu reagent, glacial acetic acid, methanol, chloroform, and glycerol were bought from Merck . w/solid w cassava starch/gelatin) was added as a plasticizer. To achieve starch gelatinization, film solutions were heated to 80 °C with continuous agitation. Then, the film-forming solutions were cast on Teflon plates and dried for 24 h at room temperature (25 °C).The process of casting the cassava starch/gelatin film was adapted from methods published by Tongdeesoontorn et al. ,27. The Films were cut into 100 × 25.4 mm strips and preconditioned in the environmental chambers at 34 and 54% RH at 25 °C for 48 h . Tensile−1 using a Nicolet 6700 Fourier transform infrared (FT-IR) spectrometer  following the method of Tongdeesoontorn et al. [Transmission infrared spectra of the films were documented at 25 °C in the range of 4000–400 cmn et al. . Differential scanning calorimetry analyses were done as described by Tongdeesoontorn et al.  by usingThe X-ray diffraction patterns of cassava starch/gelatin films with and without antioxidants were collected using an X’Pert MPD X-ray diffractometer  at 40 kV and 35 mA in the 2θ range of 5–50°. Film samples were preconditioned at 25 °C, 54% RH for 7 days prior to analysis. The surface and cross-section microstructure of the films were observed by a Scanning Electron Microscope  with gold sputtering. iw) of films (about 0.3 g) were checked, and then films were added into beakers with 50 mL of distilled water at 25 °C and agitated intermittently for 24 h. The solutions comprising film residues were filtered with dried Whatman filtered paper No.1. The residues on filtered papers were dried at 80 °C for 24 h and then weighed to determine the final dry weight (fw). Tests were achieved in triplicate, and the water solubility (%) was calculated using Equation (1):Film solubility was investigated as a percentage of solubilized film in water following a method of Tongdeesoontorn et al. . Films wWVTR) of films was determined at 25 ± 1 °C using the ASTM E96-80 method [2) of the cup was sealed over with each film sample. Cups were stored in desiccators containing saturated salt solution (MgCl2 and Mg(NO3)2 for 34% and 54% RH, respectively). The WVTR was calculated using the following Equation (2):W/∆t is the amount of water gain (g/day) and A is the test area (m2).The water vapor transmission rate  was used to compare the short-term antioxidant potencies of antioxidants in the cassava starch/gelatin films ,34. Film2 and kept at 30 °C, 40%RH for 35 days. The control was put in beaker without any package. The peroxide value of lard was analyzed at 1-week intervals.The effects of antioxidant (quercetin and TBHQ) addition to films on the quality changes of lard during storage were studied using methods adapted from previous publications ,35. Lardv/v). Then, saturated potassium iodide (1 mL) was added, and the solution was retained in the dark for 10 min. After stabilization, 30 mL of distilled water was added into the solution, which was followed with 1 mL of starch solution (1% w/v), and solutions were then titrated with 0.01 N Na2S2O3 until colorless. PVs were calculated as follows (Equation (5)):S, B, N (mol equiv/L), and W denote the titration quantity of sample, the titration quantity of blank, the normality of Na2S2O3, and the sample weight , respectively [The peroxide value (PV) measures primary products of lipid oxidation and is used to determine the oxidative state of lipid-containing foods . Lard waAntioxidant film applications on fresh pork were studied ,17,36. FAll results were analyzed statistically by ANOVA analysis and Duncan’s multiple range tests to determine the significant differences between group samples using a statistical program SPSS v. 10.0 at a confidence interval of 95%.Cassava starch/gelatin (7:3) films are reported to have good mechanical properties . To deteIncreasing storage RH decreased the TS of all films, as expected, since water plasticizes films. Adding antioxidants to the films had type and concentration-dependent effects on TS. The cassava starch/gelatin films with lower amounts of quercetin showed higher TS than the control at 34%RH, which is attributed to the intermolecular hydrogen bonding interactions formed between hydroxyl groups of quercetin (hydrogen bond donors) and the starch/gelatin molecules. Intermolecular hydrogen bond networks can increase the strength of films . IncreasThe elongation at break (EAB) traits of the films are shown in By comparing the mechanical properties of quercetin and TBHQ films, it is evident that cassava starch/gelatin composite films containing quercetin had a higher tensile strength than TBHQ-containing films. Additionally, cassava starch/gelatin films with quercetin were less flexible than films with TBHQ. The composite films with the highest TS were films made from 50 mg quercetin/200 mL film solutions and stored at 34%RH, while the highest EAB films were made from 100 mg TBHQ/200 mL film solutions and stored at 54%RH. These findings were not consistent with a report by Rachtanapun et al. , who stu−1  stretching within the carboxylic group (amide I), and the peak at 1550 cm−1 is correlated to the N–H bond (amide II) stretching of gelatin [2 stretching is causing the band at 1076 cm−1 [−1 and 2929–2926 cm−1, respectively. With antioxidant addition, the band of C–OH bending of the cassava starch/gelatin film that appeared at 1322 cm−1 was displaced to 1326–1323 cm−1. With the addition of quercetin and TBHQ, minor differences in the absorption band strength at 1650 and 1550 cm−1 were observed in the composite films. These findings were consistent with the FT-IR spectra of fish gelatin films containing BHT and α-tocopherol [FT-IR spectroscopy was used as a tool to investigate the structure and possible interactions between cassava starch/gelatin films and antioxidants (quercetin and TBHQ). Special note should be made of the peaks between 3272 and 2930 cm−1 , referri−1 . In addi bending , is disc gelatin . CH–O–CH076 cm−1 . Adding copherol . Increascopherol .−1 correlated with a decrease in peaks at 3273–2930 cm−1. This discovery confirmed the theory that because of the intermolecular interactions between antioxidant polyphenol compounds with hydroxyl and amino groups of the gelatin matrix, there may be a clear structural change in the films [A rise in the absorption bands in the FT-IR range at 1650 and 1550 cmhe films ,43. Morehe films  and the he films , who fouhe films . It is apparent from the FT-IR analyses that the incorporation of quercetin and TBHQ could form hydrogen bonding and thus occupy the cassava starch/gelatin matrix functional group, consequently reducing the free hydrogen group that can form hydrophilic bonding with water . These sDSC thermograms of the films are provided in The X-ray diffraction patterns of the control films and antioxidants, along with those of the antioxidant-containing films, are shown in The decreased peak intensity in the films at 28° is related to the re-arrangement and possible blockage of starch/gelatin crystallization ,50. A coThe scanning electron micrographs of the cross-sections of composite films at different concentration of quercetin and TBHQ are presented in All composite films showed similar surface images to the microstructure of cassava starch-gum bio-based films , which aThe SEM image of cassava starch/gelatin film showed a dense structure with a few areas of roughness a, which Water solubility is an important trait of many composite films. The water solubility can be essential for some applications in the food industry, such as food or additives encapsulation , while fWater vapor transmission rates (WVTRs) of cassava starch/gelatin films with and without TBHQ and quercetin are shown in As the concentration of antioxidants increased, the total phenolic content of cassava starch/gelatin films also increased significantly . This rep ≥ 0.05) on the DPPH scavenging activity of cassava starch/gelatin films (data not shown), which represented the similar antioxidant activity of all cassava starch/gelatin films with different antioxidant contents. The DPPH radical scavenging activity of the cassava starch/gelatin films were not affected by the storage time.The results displayed that increasing quercetin and TBHQ concentration had no effect  was determined for lard samples stored in cassava starch/gelatin films containing quercetin or TBHQ during storage to document the film effects on lipid oxidation . After aMeat surface discoloration largely depends on the oxidation rate of the red oxymyoglobin into metmyoglobin, which gives the meat less attractive brown color . The effp < 0.05) redness than the control (uncovered pork). The redness of uncovered pork reduced rapidly and decreased by more than 50% after 8 days of storage, while the redness of pork covered by quercetin and TBHQ films showed a lower drop of redness than control . Pork covered with TBHQ films showed a lower redness reduction than quercetin films. After 12 days, the redness of pork concealed with TBHQ films decreased slightly (less than 20% reduction in redness). Increasing the content of quercetin decreased the rate of color change, but variations in TBHQ concentration did not have a major difference in reducing pork redness. These findings suggested that the addition of quercetin and TBHQ in the cassava starch/gelatin film provided good results in the prevention of overall discoloration. Moreover, these findings also corresponded with the inhibition of pork oxidation by anti-oxidative plastic films coated with horseradish extract [In this research, pork samples were coated with cassava starch/gelatin films containing different concentrations of quercetin and TBHQ, and color of pork was then monitored for 12 days. The percent reduction in redness was measured, as shown in  extract , using t extract  and porkThe oxidation of myoglobin in pork can be inhibited by the cassava starch/gelatin antioxidant composite films . This reThe addition of quercetin and TBHQ as well as environmental relative humidity influenced the mechanical properties of cassava starch/gelatin films. Cassava starch/gelatin films containing quercetin displayed better tensile strength but lesser break elongation than control films. Conversely, films containing TBHQ showed lower tensile strength but higher elongation at break than control films. Changes in polymer matrix structures in the presence of the different antioxidants were documented by FT-IR and XRD analyses. These structural changes corresponded to differences in mechanical and barrier properties of the films. Increasing the content of quercetin and TBHQ increased the film solubility in water and the WVTR of composite films. Throughout the storage period (30 days), both total phenolic content and antioxidant activity stayed in films. For a potential application, cassava starch/gelatin film comprising quercetin and TBHQ were found to slow down the oxidation of lard (more than 35 days) and postpone the redness discoloration of pork. The developed quercetin-containing and TBHQ-containing cassava starch/gelatin films have the ability to be used as active biodegradable films for food products with the potential to delay oxidation reactions in packaged products thereby enhancing product shelf-life and quality.