The alloys were developed in a vacuum electric arc furnace and then studied from a structural, mechanical and in vivo assessment (on rabbits) perspective. The effect of the silicon addition was clearly seen in both the structural and the mechanical characteristics, standing out as beta alloys with a dendritic structure and lowering the mechanical properties as a result of the silicon addition. In experimental rabbits, the proliferation of mesenchymal stem cells was observed in the periosteum and peri-implant area, differentiating into osteoblasts and then into osteocytes. Osteoclasts were discovered within the cartilaginous islands that provide structural support to newly formed bone, playing a primary role in bone remodeling. The newly formed spongy tissue adhered to the fibrous capsule that surrounds the alloy, ensuring good osseointegration of metallic implants. The overexpression of Osteopontin, Metalloproteinase-2 , and Metallopeptidase-9  underscores the processes of osteogenesis, bone mineralization, and normal bone remodeling.Biomaterials are currently a unique class of materials that are essential to improving the standard of human life and extending it. In the assent of the appearance of biomaterials that contain non-toxic elements, in this study, we examine a system of Ti25Mo7Zr15Ta From the perspective of protecting health, biomaterials can be defined as “materials that possess new properties that make them suitable to come into immediate contact with living tissue without causing an immune rejection or an adverse reaction” ,2,3.Scientists can now model the properties of materials at high levels thanks to the knowledge they have accumulated over the previous 70 years. In order to meet the numerous service requirements of today’s complex society, tens of thousands of different materials have been developed with specific properties ,5.In reparative medicine, a broad diversity of biomaterials are used. Currently, commercially pure titanium (C.P.-Ti), Ti6Al4V, Ti6Al7Nb, CoCr and stainless-steel alloys  are usedxZr alloys may enhance the adhesion, proliferation, and differentiation of osteogenic cells. In in vivo studies, alloys with a higher percentage of Ta have shown increased osteogenic activity, with titanium alloys containing Ta-30Zr emerging as a promising alternative [The identification and testing of the effectiveness of new types of implants are the essential stages for the design and development of the medical device industry. The most commonly utilized materials in medical applications are still titanium and its alloys because of their exceptional characteristics. They are mostly utilized in the domains of orthopedics, dentistry and cardiovascular medicine for the replacement of hard tissues because of their high tensile strength, satisfactory biocompatibility and good resistance to corrosion or wear ,8,9,10. ernative . Howeverernative ,14,15,16Tantalum is considered to be non-toxic and of medium biocompatibility . In vitrSilicon (Si), which can be found as silicide and solid solution, is a crucial element in titanium (Ti) alloys. Some studies have demonstrated that the addition of silicon to titanium alloys can improve strength, creep resistance and oxidation resistance, as well as reduce fluidity, particularly at ambient temperature ,24,25,26The biocompatibility of implant materials is the determining characteristic for the osseointegration process and implicitly for implant stability, reliability and durability. The concept of intrinsic biocompatibility is defined as “the ability of a material to induce an appropriate host response, in the case of a specific application”. In implantology, the concept of biocompatibility of materials must be combined with that of the bifunctionality of the implant, which implies the in vivo functional performance of the implant ,28,29,30It is crucial to design materials with superior biocompatibility and high durability. Although a variety of materials are now used as biomaterials, titanium alloys are quickly replacing them as the most in-demand material for most applications ,37,38,39xSi  system developed for medical applications. This study includes microstructure analyses, mechanical testing, and in vivo investigation of the chemical and biological characteristics of the alloys to evaluate their performance. This scientific study explores the microstructural and biological properties of a new Ti25Mo7Zr15TaThe production of titanium alloys is a complex process that poses several challenges, including the need for protective conditions and the high cost of metallurgical equipment. Titanium has a strong tendency to react with oxygen, hydrogen and nitrogen, which can cause gas dissolution in the melted metal during alloy production. To address these issues, specialized metallurgical equipment is required to enable processing under vacuum or a controlled argon atmosphere, which provides the necessary protection against gas contamination .xSi = 0.5; 0.75; 1%).To ensure biocompatibility, high-purity chemical elements  were used to produce Ti-25Mo-7Zr-15Ta-xSi alloys. Upon analyzing the Ti-Mo binary diagram, it was observed that there is a broad range of potential chemical compositions that can provide excellent chemical stability for the alloy. The alloys obtained were Ti25Mo7Zr15Ta, Ti25Mo7Zr15Ta0.5Si, Ti25Mo7Zr15Ta0.75Si and Ti25Mo7Zr15Ta1Si. The effect of silicon on the alloys was analyzed by keeping the content of other alloying elements constant and by increasing the silicon content .3 and 85 mL H2O, for a duration of 30 s.The metal samples obtained from the Ti-Mo-Zr-Ta-Si system were examined using metallographic analysis. For the purpose of analyzing optical microstructures, it is recommended to utilize a Zeiss Axio Imager A1 upright microscope, which is equipped with multiple objectives. This microscope is manufactured by Carl-Zeiss-Strasse located in Oberkochen, Germany. In order to examine the metallographic structure of the samples, they were first polished and then subjected to etching with a reactive solution composed of 10 mL HF, 5 mL HNOThe identification of phases was conducted using Panalytical X’Pert Pro MPD equipment, manufactured by Malvern Panalytical Ltd. in the Malvern, UK. The data obtained from the equipment were processed using the Highscore Plus program.In mechanical engineering, indentation hardness tests are employed to determine a material’s resistance to deformation. Various tests are available where the material is indented until an impression is formed, either on a macroscopic or microscopic level.The properties of the TiMoZrTaSi alloys obtained were evaluated through indentation using a Tribometer UMT 2 apparatus equipped with a Rockwell diamond penetrator . Instrumented indentation involves applying a sharp tip to the material’s surface and recording a force–displacement curve. These results provide valuable insights into the material’s mechanical behavior, such as hardness, elastic moduli, and plastic deformation. An essential aspect of instrumented indentation testing is the precise control of the tip’s force or displacement, which can be measured continuously throughout the indentation process. Advanced technology now allows accurate force control within a broad range.Oryctolagus cuniculus) aged 8–10 months. A control rabbit received a stainless-steel orthopedic rod. Experimental protocol followed the regulations of the European and national legislation on the matter of experimentation on animals and ISO 10993-1:2018 procedures [Interaction between the alloys and bone tissues was observed after surgical implantation into the tibial compacta of mature rabbits .Anti-osteopontin , anti-MMP2 and anti-MMP9 :SC-6840) were used for immunohistochemical staining. After dewaxing and microwaving in a 10 mmol citrate acid buffer (pH 6) at 95 °C for 10 minutes, the sections were cooled and then washed twice in PBS for 5 minutes. They were subsequently incubated overnight with primary antibodies (diluted 1:100) at 4 °C in a humid chamber. Slides were washed 3 times in PBS for 5 min and incubated with the secondary antibodies.goat anti-rabbit IgG secondary antibody for OPN, MMP2, and MMP9. Slides were developed with 3,3’-diaminobenzidine (DAB) and counter-stained with hematoxylin.We used The chemical characterization is based on the quantitative and qualitative chemical analysis of the samples taken from the ingots obtained. The chemical composition of the alloys obtained in the vacuum arc remelting furnace is presented in The chemical composition was measured for 10 different points of the samples. By analyzing the chemical composition, the constituent elements of the alloys were determined: titanium, molybdenum, tantalum, zirconium and silicon. The alloys were homogeneous, without metal inclusions. The energy-dispersive spectroscopy (EDS) elemental mapping results of the elements found in the alloys can be found in We conducted optical microscopy analysis on metallographically prepared samples, which were sanded with SiC metallographic paper  in an aqueous medium and polished with a 3 µm diamond suspension, until a mirror surface was obtained for samples with dimensions of 15.00 × 10.00 × 5.00 mm. To visualize the structure under an optical microscope, the surface of the samples was attacked with a special reagent. From The mechanical characteristics of titanium alloys, particularly their resistance to fatigue, can be influenced by their dendritic structure. In order to improve fatigue resistance and overall mechanical strength, titanium alloys should generally have a fine and homogenous dendritic structure ,46.However, additional characteristics, like the cooling rate and alloy composition, may also have an impact on the dendritic structure. A dendritic structure that is finer can be produced by rapid cooling, even though a dendritic structure that is coarser can be produced by faster cooling. Moreover, the dendritic structure and its resulting properties can be impacted via specific alloying elements ,48.When creating and choosing titanium alloys for use in medical implants and other applications where mechanical strength and durability are crucial, it is vital to take into account the dendritic structure in general.The addition of beta-stabilizing elements can significantly enhance the properties of titanium alloys, making them more suitable for a wide range of applications. In our case, the beta-stabilizing elements of the introduced titanium alloys, such as Mo, Ta and Si, influenced the structure toward a dendritic structure.xSi  alloys. XRD may be used to determine the various crystal phases that are present in titanium alloys. The alpha phase and the beta phase, the two main crystal phases of titanium alloys, each have a unique crystal structure. The structure of the studied alloys shows a majority beta-type structure, which was also confirmed via optical microscopy.3): 4.49, the volume of the cell (106 pm3): 35.38, Z: 2.00 and RIR: 9.61.The XRD diffractograms highlight that the alloys exhibit one compound: β-Ti (reference code: 01-089-4913). The main characteristics for the Ti alloys obtained  are as fTo assess the mechanical qualities of the materials, particularly titanium alloys, indentation tests are frequently performed. In these tests, a controlled load is applied, while a hard indenter, such as a diamond tip, is driven into the material’s surface. Measurements of hardness, elastic modulus and plastic deformation behavior can all be made using the resulting indentation.Indentation testing on titanium alloys can reveal important details about the material’s mechanical properties at micro- and nanoscales. For instance, nanoindentation studies can be used to gauge the elastic modulus and hardness of distinct alloy phases, like the alpha and beta phases. These measurements can shed light on how alloying components and manufacturing parameters affect the material’s mechanical properties.Indentation tests can be performed to examine the deformation behavior of titanium alloys at micro- and nano-scales in addition to analyzing their bulk mechanical properties. Researchers can learn details about the material’s plastic deformation behavior, including the size and distribution of deformation zones and the level of strain hardening, by examining the shape and size of the indentation. For some applications, this knowledge can be used to enhance the design and processing of titanium alloys.Indentation tests are generally a good method for assessing the mechanical qualities of titanium alloys. At micro- and nanoscales, they can offer insightful data about the hardness, elastic modulus and plastic deformation behavior of a material, which can be used to improve the alloy’s characteristics and functionality for a variety of applications.In recent years, the continuous search for titanium alloys with improved mechanical and biological properties has yielded a variety of novel compositions. A crucial aspect of these newly developed alloys is the optimization of their Young’s modulus to closely resemble that of human cortical bone (15–30 GPa), which is crucial for orthopedic applications to minimize the stress shielding effect.For our Ti25Mo7Zr15Ta1Si alloy, we observed that Young’s modulus is higher than that of cortical bone (71.72 GPa). However, a remarkable trend was noted: with an increase in Si content, there was a noticeable decrease in Young’s modulus. This offers the possibility of tuning the mechanical properties of the alloy by varying the Si content, a significant advantage for specific biomedical applications.Compared with a classic titanium alloy commonly used in orthopedic applications, Ti-6Al-7Nb has the highest Young’s Modulus (105 GPa). Its high modulus, however, can result in stress shielding effects when used as an implant material.Another alloy, Ti-19Nb-14Zr stands out as the alloy with the lowest modulus, coming in at just 14 GPa. This makes it considerably more elastic and closer to the modulus of human bone, potentially reducing stress shielding effects.Our Ti25Mo7Zr15TaxSi alloy, depending on the amount of Ta, has an average modulus of 79.22 GPa. This places it as mid-range in comparison to the other alloys. Notably, it matches the modulus of Ti-12Mo-5Zr, suggesting similarities in their mechanical properties (64 GPa). What is important, however, is that by varying the Si content in the system, there is potential to tune its Young’s modulus, a unique capability not necessarily present in these other compositions.The addition of silicon is seen in alloys like Ti-15Mo-1Si (43 GPa) and Ti-20Mo-1Si (26 GPa) generally seems to reduce the modulus. The lowest is Ti-15Mo-0.5Si at 20 GPa, which is quite close to the modulus of bone.The primary objective of the X-ray analysis was to determine the position of the alloys in both the control and experimental rabbits, as well as to detect any relevant reactions in the surrounding tissues to the alloy surface. Throughout the 60-day experimentation period, no abnormal radiological events were observed .CT Analysis: We utilized a high-resolution CT system  with a 20 µm/slice scanning resolution to assess peri-implant tissue density. The resulting images were processed into both 3D and 2D formats using Image Software (Version 2.21 Synego viewer). Density measurements were made using Hounsfield Units (HU), revealing that the alloy structure exhibited higher density than surrounding bones and tissues, leading to the creation of artifacts.Regarding the radiodensity of the surrounding tissues of the peri-implant ,50, the In the control rabbits, mesenchymal stem cells were located in the inner layer of the periosteum and differentiated into osteoblasts, responsible for synthesizing preossein and collagen fibers see . PreosseA passive layer, ranging from 3 to 10 nm, covers the titanium alloy and has the capacity to attract calcium and phosphate ions, leading to the binding of specific proteins such as collagen type I and osteocalcin to osteoblasts. This aspect was also observed in our experiment ,54,55,56For all implants, stem cells are present in the cambium area, which differentiate into osteoprogenitor cells that later become osteoblasts involved in osteoid synthesis . AdjacenIn the case of the Ti25Mo7Zr15Ta alloy, the proliferation of osteoprogenitor cells and osteoid formation can be observed in cartilage islands. The newly formed bone tissue consists mainly of fibro-cartilaginous tissue, including rare islands of mineralization. The inner layer of the periosteum continues with islands of mineralization of the newly formed bone tissue . In the In the case of the Ti25Mo7Zr17Ta0.75Si alloy, the peri-implant fibrous capsule is thinner and denser, and the newly formed bone tissue consists of an alternation of cartilage islands with mineralized areas and the formation of bone lamellae . The perOPN is produced via embryonic stroma, macrophages, fibroblast cells or bone cells, such as osteoblasts, in different stages of differentiation, and osteocytes are involved in the wound-healing process of bones, contributing to osteoclast genesis and activity ,62,63,64In this study, the overexpression of OPN in the bone cells is an indicator of progressive osteogenesis. In the control rabbit, OPN was observed, and it exhibited overexpression in the bone interface area in the experimental rabbits see  16,23,5,523,59In the current study, high MMP-9 activity was highlighted in osteoprogenitor cells and osteoblasts. Our results suggest that MMP-9 is the downstream molecular signal of OPN, thus contributing to cell migration ,66. MMPsIn recent years, the medical field has seen a surge in the use of metallic biomaterials, especially for orthopedic and dental implants. However, their use is not just limited to these fields; new applications are continually being discovered. Each of these materials is carefully assessed and undergoes stringent testing to ensure they meet the required safety and efficacy standards for medical applications.While traditional alloys like C.P.-Ti, Ti6Al4V and Ti6Al7Nb have played a significant role in medical implantology, the research world has constantly been looking for improved materials with better mechanical and biocompatible properties. Newly developed Ti-alloys, like the one presented in this work, offer potential enhancements over their predecessors.x = 0, 0.5, 0.75, 1 wt.%) alloy, which was synthesized in a vacuum electric arc furnace. This alloy was rigorously characterized in terms of its structure, mechanical properties and biocompatibility.Our study focuses on the development of a new Ti25Mo7Zr15TaxSi , the development of peri-implant bone regeneration was followed. Thus, both in the control and experimental rabbits, at the implantation gap, intense proliferative activity was observed in the internal layer of the periosteum and on the periphery of the peri-implant fibrous capsule. The presence of cells positive for OPN, MMP-2, and MMP-9 indicates the proliferation and migration of cells that enhance osseointegration and extracellular matrix production. These molecules facilitate cell adhesion, leading to callus remodeling and the redirection of trabeculae in newly formed bone along the force lines of the osteon system, ultimately promoting the integration of the alloy into the bone. Due to their biocompatibility and strength, titanium alloys have generally shown to be a dependable and successful material for medical implants, making them the perfect option for surgical implants that must stay in the body for extended periods of time.The Ti25Mo7Zr15TaxSi alloy system exhibits a lower modulus of elasticity compared to classic alloys, such as C.P.-Ti, Ti6Al4V and Ti6Al7Nb, and offers superior biocompatibility. Titanium alloys, owing to their biocompatibility and strength, have stood the test of time as dependable materials for medical implants. Their reliability becomes even more crucial for surgical implants that are designed to remain within the body for prolonged durations. The newly developed Ti-Mo-Zr-Ta-Si system, based on our findings, possesses the potential for medical applications, most notably within the orthopedic sphere, without any risk of bodily rejection.


Abdominal ultrasound (US) is a widely used, first-level technique for the diagnosis of hepatocellular carcinoma (HCC). However, the prognostic meaning of different US patterns of HCC has not been investigated yet. In this study, we present a novel four-type US classification based on HCC morphology; we aimed to assess the impact of US patterns on overall survival (OS) and recurrence-free survival (RFS) in a cohort of HCC patients treated with radiofrequency thermal ablation and with a long-term follow-up. We demonstrated that specific US patterns are independent predictors of both OS and RFS and, therefore, could help identify patients at a higher risk of worse prognosis in clinical practice.p = 0.048) and tumor dimension (p = 0.034) compared to pattern 1A. Conclusions: Our findings demonstrate that different US patterns correlate with different survival outcomes and tumor behavior in patients with HCC. Prospective studies are needed to confirm these results.Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. Abdominal ultrasound (US) is by far the most widely used first-level exam for the diagnosis of HCC. We aimed to assess whether different ultrasound patterns were related to tumor prognosis. Methods: We retrospectively reviewed all patients with a new diagnosis of HCC (single nodule) and undergoing radiofrequency thermal ablation (RFTA) at our clinic between January 2009 and December 2021. Patients were classified according to four HCC ultrasound patterns: 1A, single capsulated nodule; 1B, well capsulated intra-node nodule; 1C, cluster consisting of capsulated nodules; and 2, non-capsulated nodule. Results: 149 patients were analysed; median follow-up time was 43 months. US patterns 1A (32.9%) and 1B (61.1%) were the most commonly seen. Median overall survival (OS) and recurrence-free survival (RFS) from RFTA were 54 months  and 22 months , respectively. Pattern 1A showed the best OS. Compared to pattern 1A, 1B was independently associated with worse OS  vs. 46 months ) and RFS  vs. 18 months ). Patterns 1C and 2 were associated with worse RFS compared to 1A, while no difference was seen for OS. Among baseline clinical variables, pattern 1B exhibited higher histological grade ( Hepatocellular carcinoma (HCC) accounts for more than 80% of primary liver tumors worldwide ,2, makinDespite improvements in therapeutic approaches, the five-year and ten-year survival rates are about 20% and 10%, respectively . To defiTransabdominal ultrasound (US) is by far the most common imaging technique used to screen and diagnose HCC, thanks to the absence of ionizing radiation and its widespread distribution . An annuHowever, to our knowledge, few studies have investigated the predictive role of US imaging features for HCC prognosis; rather, studies have mainly focused on the dynamic and quantitative parameters assessed by CEUS ,19,20. TThus, the aim of this study was to assess the correlation between the US morphological patterns and survival outcomes in a cohort of patients with HCC.We performed a single-center, retrospective, observational study. From January 2009 to December 2021, all patients diagnosed with a single nodule of HCC that were treated with radiofrequency thermal ablation (RFTA) were consecutively enrolled. At enrolment, the following data were collected: date of birth, gender, date of diagnosis, etiology of liver disease, Child-Pugh score at diagnosis, degree of HCC on histological examination, size of the nodule, date and cause of death if the patient was deceased, and date of the last follow-up for surviving patients, evaluated up to 1 August 2022 through telephone calls and the informatic system of the Lombardy Region. For final inclusion in the study analyses, patients with at least one of the following criteria were excluded: previous diagnosis of HCC, multifocal HCC, neoplastic thrombosis, metastatic lymph nodes, ASA score > 3, or mixed HCC-cholangiocarcinoma (CCC) histotypes according to a biopsy examination. Patients lost to follow-up, whose survival could therefore not be calculated, were also excluded. We included only patients treated with the same therapeutic approach  in order to obtain a homogeneous cohort, thereby avoiding biases related to different therapies. The follow-up time was calculated between the date of RFTA and the date of death or last follow-up. A minimum follow-up time of 6 months was considered.The study was approved by the local Ethics Committee and was performed according to the Helsinki declaration. All patients gave their written informed consent for inclusion in the study, clinical data collection, and for the conservation of biologic materials.The US morphological subtypes of HCC nodules were developed before starting the enrollment, based on the following experience. Over the years, all the ultrasound-detected HCC nodules that were candidates for surgical treatment were evaluated by intraoperative ultrasound before their surgical removal. We tried to correlate the ultrasound appearance of the nodule with its macroscopic appearance when removed and dissected. Through this experience, four ultrasound patterns were individuated. We defined as 1 the category of capsulated nodules, divided as follows: 1A—single, capsulated nodule; 1B—intra-node node, well capsulated; and 1C—cluster formation consisting of capsulated nodules. All nodules without a capsule, i.e., with ill-defined margins, were included in category 2. Example images of the four US nodule subtypes with the relevant macroscopic appearance after surgical resection are shown in The primary aim of the study was to investigate a correlation between ultrasound patterns and overall survival. The secondary aims were to correlate the ultrasound pattern with recurrence-free survival and with histological grade and other baseline clinical variables.The categorical variables were absolute frequency and percentage. The continuous variables with normal distribution were mean ± SD, whereas the continuous variables without normal distribution were median and range. Time-to-event data, namely, overall survival (OS) and recurrence-free survival (RFS), were calculated from the first procedure until the event  or the last follow-up visit and were estimated by means of Kaplan-Meier analysis. The correlation between US patterns and survival outcomes was assessed by means of Cox regression analysis; the results were expressed as hazard ratio (HR) and 95% CI. Specifically, survival analyses were performed by calculating the hazard ratio of death; the US pattern exhibiting better survival in the one-by-one Kaplan-Meier analysis was taken as the reference for comparisons with other patterns . Variables found to be significant in the univariate analysis were entered in the multivariate model. Correlations between the US patterns and the baseline clinical variables were assessed using the Mann-Whitney analysis and the Chi-Square or Fisher’s exact tests for continuous and categorical variables, respectively. Analyses were performed using R Statistical Software “Survival” package , and significance was established at the 0.05 level (two-sided).Between January 2009 and December 2021, 149 patients received a diagnosis of HCC and were treated with RFTA . The predominant HCC etiology was HCV (76.5%). The most represented ultrasound pattern was 1B (61.1%). Only 3.3% and 2.7% of patients demonstrated patterns 1C and 2, respectively. All of the demographic and baseline clinical features of the patients are reported in During follow-up, calculated between the date of RFTA and the date of death or last follow-up, 102 patients (68.5%) died, of whom 64.7% died due to liver-related disease . The 1- and 5-year cumulative probabilities of OS from RFTA were 91%  and 48% , respectively, with a median OS of 54 months  .One hundred and two patients (68.5%) experienced an HCC recurrence during follow-up, with a median RFS of 22 months  and 1-year and 5-year cumulative probabilities of RFS of 66%  and 27% , respectively.p = 0.008) and C , nodule size , and ultrasound pattern 1B  were found to be correlated with increased mortality. In our multivariate regression analysis  for pattern 1A, 51  for pattern 1B, 46  for pattern 1C, and 40  for pattern 2 a. The 1Aanalysis , all thop = 0.001 and HR 4.11; 95%CI, 2.69–10.87; p < 0.001, respectively), nodule size , and all ultrasound patterns  were independently associated with increased mortality.The median RFS for US pattern 1A was 34 months , 18 months for 1B , 9 months for 1C , and 11 months for 2  b. The unp = 0.034)  . All othThis is the first study to investigate the prognostic value of HCC morphology as assessed by transabdominal US. Additionally, we present a novel US classification of HCC nodules based on the two main groups of capsulated and non-capsulated nodules, with the capsulated ones being further divided into three subgroups. These patterns had been defined in the years before this study and correspond not only to the US image but also to the true macroscopic morphology of the tumor when dissected after surgical resection; this may therefore make our US classification and our results more reproducible. It is also worth noting that the four ultrasound patterns were homogeneous for all the baseline parameters recorded and that we considered only patients treated with the same therapeutic approach , thereby reducing the risk of biases. We evaluated relevant outcomes, including the overall survival and the recurrence-free survival.Patterns 1A  and 1B  were by far the most commonly found, covering roughly 95% of the cases. In our survival analysis, pattern 1A showed the highest survival probabilities over time and was used as a reference to compare with other patterns. As the main finding, our study showed that patterns 1B, 1C (nodule cluster appearance), and 2 (non-capsulated nodule) were independently correlated with worse prognosis. Particularly, pattern 1B was associated with increased overall mortality, while all three patterns correlated with increased risk of recurrence over time. Patterns 1C and 2 failed to demonstrate a correlation with OS, possibly because of the small sample size of these groups. The only analogy we could find in the literature was with a previous study assessing the prognostic role of preoperative US features in a cohort of HCC patient undergoing liver resection, in which the authors found that a mixed echogenicity pattern was associated with shorter survival . This siOther studies have evaluated the prognostic impact of CEUS quantitative parameters on survival outcomes, showing that some variables, such as rise time and time to peak, were associated with worse OS and RFS ,19. HoweIn agreement with existing data in the literature ,23, our Regarding histological grade, although the guidelines recommend biopsy only when a nodule > 10 mm has a doubtful contrast enhancement behavior in two imaging methods in cirrhotic patients , in our Finally, our results confirm the prognostic role of tumor size, which was proved to be independently correlated with both OS and RFS. This finding agrees with several previous reports ,29,30,31This study had some limitations: its retrospective nature, which makes it necessary to confirm our results in larger, prospective cohorts; the small sample size of US patterns 1C and 2, which, although sufficient to demonstrate a correlation with RFS, limited our ability to analyze such a correlation with OS. The strengths of the study were the homogeneity of the study cohort, consisting of patients all treated with the same HCC therapeutic approach and regularly followed up thereafter, the relevance of the analyzed outcomes, and the long-term median follow-up that made these outcomes more robust.In conclusion, our study demonstrates, for the first time, that the US pattern of HCC nodules at diagnosis may be interpreted as an indicator of OS, RFS, and histological grade. Further studies are needed to confirm these data prospectively in order to assess if the US pattern can help in identifying a subgroup of patients at increased risk of worse outcome, thus aiding the selection of a suitable therapeutic approach and follow-up.


Aspergillus and Penicillium families. OTA commonly contaminates food and beverages, resulting in animal and human health issues. The toxicity of OTA is known to cause liver damage and is still being researched. However, current findings do not provide clear insights into the toxin mechanism of action. The current studies focusing on the use of potentially protective compounds against the effects of the toxin are insufficient as they are mainly conducted on animals. Further research is required to fill the existing gaps in both fields (namely the exact OTA molecular mechanism and the prevention of its toxicity in the human liver). This review article is a summary of the so far obtained results of studies focusing on the OTA hepatotoxicity, its mode of action, and the known approaches of liver cells protection, which may be the base for expanding other research in near future.Ochratoxin A (OTA) is considered as the most toxic of the other ochratoxins synthesized by various fungal species belonging to the  Aspergillus and Penicillium fungi. Among them, it can be distinguished into ochratoxin A (OTA), its methyl ester, ochratoxin B (OTB) and its methyl and ethyl esters, ochratoxin C (OTC), 4-hydroxyochratoxin A (4-OH OTA), and ochratoxin α (OTα). OTA is known as the most toxic . Th. Th42]. OTA biosynthesis requires multiple genes as it is a complex process. Studies conducted on various OTA-producing strains show similarities in toxin biosynthesis.An15g07920 gene OTA was used to identify hepatocellular uptake in isolated rat hepatocytes. Obtained results showed, that OTA transport was saturation, temperature, and energy dependent. Furthermore, the OTA uptake was reduced by different bile acids, sulfobromophthalein, and the thrombin inhibitor CRC 220m which are known to be a substrate for organic anion-transporting polypeptides (OATPs) . The impThe liver microsomes are involved in OTA metabolism. Studies have shown that, cytochrome P450 (CYP450) is responsible for the formation of hydroxylated forms of OTA such as (4S)-4-OH OTA and (4R)-4-OH OTA in human, pig, rat and rabbit ,66, and The results of the studies indicate the formation of similar OTA metabolites. Researchers agree on several types that occur in both animals and humans, suggesting resemblances in livers OTA metabolism in various organisms. However, these studies do not show the formation of acyl hexose and acyl pentose conjugates in the human but focusing on rat model, which may indicate the existing difference among animals and humans. Moreover, in the case of 10-hydroxyochratoxin A its formation is characteristic for rabbit. (4R)-4-OH OTA and (4S)-4-OH OTA are present in all mentioned studies, suggesting that, these are major metabolites generated in the livers.A broad spectrum of OTA’s activity was shown in many studies. Most of them focused on multiorgan toxicity, with an emphasis on kidneys . HoweverGayathri et al. demonstrated that OTA induced apoptosis of the HepG2 liver cell line, chromatin fragmentation, generate reactive oxygen species (ROS) production, and causes DNA damage. Furthermore, based on the results from the mitochondrial trans-membrane potential and the concentration of caspases and apoptotic-associated proteins (Bax and Poly(ADP-ribose) Polymerase (PARP)), the authors suggested that, the apoptotic cell death was mediated mainly by mitochondria . SimilarIn study conducted by Qi et al., the miRNA expression profile in the livers of rats treated with OTA was examined. Results showed that, in the high-dose group, 47 miRNAs were upregulated, and 26 were downregulated in comparison to control livers cells. In the medial-dose group, 59 miRNAs were upregulated, and 47 were downregulated. Overlapping results shown 29 upregulated and 14 downregulated miRNAs. The differences in the expression were also studied in the mRNA profile in order to identify the mechanisms and pathways influenced by OTA treatment. In the high-dose group, OTA treatment was associated with circulatory, endocrine, digestive, and excretory systems, signal transduction, and lipid metabolism, which are solely linked with different body systems. On the other hand, in the medial-dose group, the differences were linked with amino acid, carbohydrates, cofactor and vitamin metabolism, and signaling molecules and overall interaction, which were mainly related to energy metabolism. The obtained results showed that, the doses of OTA may induce different toxicity mechanisms in the liver. Furthermore, to verify the identified abnormalities in miRNA and mRNA expression profile, the authors performed two-dimensional electrophoresis, to evaluate the proteomic alterations. Obtained results showed that, the expressions of 61 proteins were significantly different in the high-dose and control group. Bioinformatic analysis indicated various proteins expressions associated with cysteine and methionine metabolism, peroxisome proliferator-activated receptor (PPAR) signaling, arginine, and proline metabolism, primary bile acid biosynthesis, and metabolism of xenobiotics by CYP450 .Wang et al. performed a study, in which ducklings were treated with oral gavage of OTA for 2 weeks. Results showed that, OTA administration induced changes in the intestinal microbiota composition, and caused the accumulation of lipopolysaccharides (LPS) and liver inflammation, which was confirmed by molecular techniques and histological analysis. Ducks livers had augmented expression of TLR-4, Myd88, and p-p65 (at the mRNA and protein level), which are the known factors that stimulate NF-ĸB pathway ,77.As mentioned in the pck gene, which encodes the PEPCK, died within three days of birth. Furthermore, one-day-old pck-null mice had strongly elevated hypoglycemic. Results of this study showed, that PEPCK is essential for life and its disorders may lead to impaired gluconeogenesis, thus emphasizing its crucial role in this metabolic pathway [Gluconeogenesis is a metabolic pathway, mainly occurring in the liver and kidneys, in which noncarbohydrate metabolic substrates are transformed into glucose. Gluconeogenesis is performed serially in eleven enzyme-catalyzed reactions and its regulation is dependent on the amount of pyruvate carboxylase, phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase, and glucose-6-phosphatase ,79. The  pathway . A study pathway . This ph pathway . Thus, Opck2 gene, pfkl and pgk1 were also downregulated. Phosphofructokinase, which is encoded by the PFKL gene, is crucial liver-type enzyme that catalyzes the conversion of D-fructose 6-phosphate to D-fructose 1,6-bisphosphate, which is a main step in glycolysis. Phosphoglycerate kinase 1, encoded by the PFGK1 gene, is an enzyme that catalyzes the conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate in glycolysis [In the study performed by Hundhausen et al., OTA-treated Hep2G cells were used in the screening analysis of the mRNA profile by microarrays. Results showed that besides the ycolysis . At the PPAR belongs to the nuclear receptor superfamily of ligand-inducible transcription factors. The three main isoforms are presented in mammals: PPARα , which was first identified in 1990, PPARβ/δ (NR1C2), and PPARγ (NR1C3). PPARs are able to control the expression of genes associated with inflammation, lipid metabolism, adipogenesis, and metabolic homeostasis. PPARα is mainly expressed in liver, brown adipose tissue, and heart, and is linked with the oxidation pathways of fatty acid. PPARβ/δ shows parallel functions as PPARα is also expressed in the liver and heart ,85. PPARFabp2, Cd36 (associated with lipid uptake), Me1, Lpin2, Fads2 (associated with lipogenesis), Plin2 and Fsp27 (associated with lipid droplet assembly) genes. Similar results were obtained for the HepG2 cell line, however, the expression of FADS2 was not statistically significant. Furthermore, the concentration of PPARγ was also increased. To verify the connection between PPARγ activity and elevated steatosis, authors used PPARγ antagonist – GW9662. Results showed that suppressed PPARγ prevented lipid accumulation in hepatocytes. The possible explanation of this phenomenon was linked with post-translational ubiquitination, which was experimentally confirmed. It is also worth mentioning that both Cluster of Differentiation 36 (CD36) and Fatty Acid Binding Protein 2 (FABP2) had a crucial role in the lipid accumulation, which was shown after the gene silencing experiment [In the study conducted by Zhen et al., OTA administration was shown to impact PPARγ. Firstly, the authors showed that OTA caused the accumulation of lipid droplets in Hep2G cells and isolated primary hepatocytes in a dose-dependent manner. Secondly, the effect of OTA was also confirmed in vivo. After 12 weeks of OTA treatments, mice showed a sign of early steatosis, which confirms the lipid accumulation in hepatocytes. Further performed bioinformatic analysis showed predominantly altered mRNA genes expression associated with the PPAR signaling pathway. Results obtained from next-generation sequencing and qPCR of primary hepatocytes showed upregulation of periment . Based oUpregulation of genes associated with the PPAR signaling pathway (according to KEGG pathways) were also observed in the chicken hepatocellular carcinoma cell - LMH  after exposure to OTA .Deregulation in the PPAR signaling pathway was also demonstrated in the Qi et al.  study, i3+ ions, facilitating their easier reduction to Fe2+ ions, which in the presence of oxygen initiate the lipid peroxidation process. This mechanism has been proposed in reconstituted system comprising phospholipid vesicles, Fe3+ ions, and the flavoprotein NADPH-cytochrome P450 [2●−, HOO●, and OH●) was also observed in the Guerra et al. study [p < 0.05) in the mRNA expression and 9% in the protein expression (p < 0.05) of Nrf2 in rabbit’s liver [OTA has been implicated in wide range of harmful, toxicological effects, including DNA damage. Currently, the exact mechanisms of action are not fully understood, but it is hypothesized that oxidative and nitrative stress may play a significant role in genotoxicity and liver disorders. Several distinct mechanisms involved in the oxidation and reduction processes induced by OTA have been reported. One of these mechanisms involves the capacity to chelate Feome P450 . Other rome P450 ,93. In tome P450 . The dimome P450  and Grosome P450 . The augl. study . What isl. study . The repl. study . Howeverl. study . On the l. study . Similar’s liver . These r’s liver . Accordi’s liver  and DAVI’s liver ,102. The’s liver . In the ’s liver . Further’s liver . Thus inThe discrepancy among studies may result from the presence of various differentiating factors, such as cell line type, origin, exposure time, applied dosage, as well as the form of OTA administration. Therefore, a greater number of studies focusing on determining the activity of the Nrf2-ARE pathway and GSH metabolism in liver cells are necessary.2•− will result in the formation of Fe2+ and H2O2, which subsequently, through the Fenton reaction, will produce HO•, leading to oxidative DNA damage [The metabolism of OTA presents similarities and analogies with the chlorophenol toxin—pentachlorophenol (PCP). The use of PCP in research on OTA is relatively common, particularly due to the fact that OTA is referred as natural chlorophenol mycotoxin ,107. In A damage ,110. In A damage . OTA in A damage . FurtherA damage , thus enA damage .The information gathered in this subsection requires however more detailed investigations. The majority of conducted studies were performed on human kidney cell lines or the kidneys of various animals. However, it should not be forgotten that, the uncertainties indicated in our hypotheses may also involve the liver. Given its significant role in the metabolism of various chemical compounds, the presence of the same molecular pathways, as well as the highest concentration of CYP450, which plays a crucial role in OTA metabolism. Conducting more detailed studies on liver cells could provide immense implications in determining the molecular mechanism of hepatotoxicity, and consequently, identifying hepatoprotective agents that could potentially save the health and lives of many individuals.p < 0.01) [tp53, bad, apafl, bcl2a1, mcl-1, bnip2, bnip3, birc5, caspase 7). It was suggested that OTA apoptosis might be associated with DNA damage and subsequent activation of p53-target genes, especially bad and bax, which are p53-target genes also generated on the protein level. Furthermore, obtained results showed that OTA-treated hepatocytes had a significantly elevated rate of nuclear apoptotic events, based on the DAPI staining. Moreover, the activity of caspase-3, -7, -8, and -9 was provoked by OTA treatment. Thus, the authors suggested that observed disturbances (increased activity of caspase-3 and -9) may be related to oxidative stress and damaged mitochondria in the intrinsic pathway [Liver damage may also be caused due to induced apoptosis, the programmed cell death. Atroshi et al. showed that, the mice livers after two weeks of OTA administration had an 8.3-fold augmented amount of apoptotic bodies compared to the not-treated, control livers ( < 0.01) . In the  pathway . Similar pathway ,115. Bes pathway . In the In the studies focused on the role of OTA on hepatocyte apoptosis, it was also shown that OTA stimulates secretion of TNFα, which is crucial ligand for extrinsic pathway . HoweverApart from studies that highlight the OTA hepatotoxicity, there are also reports that emphasize the protective properties of certain substances which can reduce the toxin harmful effects on the liver. Unfortunately, due to the fact that the experiments were conducted on animals models, only a few of them focus on the use of human cells, thus leaving a gap in this field. Animals are used in biological research and medicine due to their anatomical and physiological similarities to humans, especially in the case of mammals. However, the results cannot be directly translated to humans . Pound eA. Gayathri et al. reported that, vitamin E can reduce ROS levels and increases the viability of the HepG2 cell line as vitamin co-administration ensures protection against OTA-induced cytotoxicity . The supIn the study performed by Shalaby Nile tilapia were fed with OTA contaminated food in two concentrations—400 and 600 µg. The results showed a significant decrease in the levels of liver total protein and aspartate aminotransferase (AST) activity. In the case of alanine aminotransferase (ALT), its activity was significantly higher in comparison to control. The administration of 500 µg of vitamin C improved these parameter by increasing both total protein levels and AST activity while decreasing the ALT action . In the Quercetin has an anti-inflammatory effect in HepG2 cells as it weakens the ROS generation and NO accumulation, induces Nrf-2 nuclear translocation and expression and leads to suppression of NF-ĸB expression and retention in the cytoplasm. Pretreatment of cells with quercetin followed by OTA leads to lower expression of cyclooxygenase-2 (COX-2) . It was Coadministration of honey or quercetin with OTA for 15 days significantly reduced DNA damage in mice’s livers. Both reduced the length of tails visible in the comet assay .MEL, produced by the pineal gland, participates in various physiological functions. This compound was used by Sutken et al. in the study performed on rats. After four weeks, the OTA-treated rats expressed significantly higher levels of MDA and hydroxyproline (Hyp), while glutathione peroxidase (GPx) level was lower. The addition of MEL (OTA+MEL) markedly decreased levels of MDA and Hyp and increased GPx activity in the liver. It was observed that MEL at dosage of 10 mg/kg can reduce parenchymal and stromal degeneration in the liver . MEL pro10 worked similarly to MEL, since it significantly decreased MDA and Hyp levels and increased GPx activity. In comparison to the control, OTA+MEL results were closer, while OTA+CoQ10 were significantly different—higher for MDA and Hyp and lower for GPx. Moreover, CoQ10 treated group had decreased mononuclear cell inflammation and congestion in sinusoids [The addition of CoQinusoids .As reported, the zinc supplementation significantly reduced ROS production and decreased the activity of SOD. Zinc also helped to maintain the DNA integrity. The presence of OTA increased the mRNA expression of metallothionein1-A (MT1A), metallothionein2-A (MT2A), and superoxide dismutase 1 (SOD1), while zinc supplement additionally increased the expression of MT1A and MT2A but did not affect the expression of SOD1. Based on the findings, it is suggested that supplementing zinc can protect human liver cells from the negative effects of OTA, including oxidative stress, 8-hydroxy-2′-deoxyguanosine formation, DNA strand breaks, and DNA hypomethylation .In vivo study showed protective effect of selenomethionine (SeMet) on rabbit liver. SeMet in concentration 0.4 mg/kg keeps positive impact as it regulates Nrf2/HO-1 expression by activating the Nrf2 signaling pathway and increasing HO-1 expression. Since this compound weakens the impact of OTA, it increases the antioxidant effect and protects the liver from oxidative damage .Emblica officinalis known as amla has been used in the acient Indian Ayurveda as a potent rasayana. In the study from 2010 concerning protective influence of amla on OTA mouse liver cells, it is reported that after oral administration its aqueous extract led to significant improvement in lipid peroxidation caused by OTA in mouse liver as it decreased the levels of MDA [s of MDA .Hippophae rhamnoides L., commonly known as SBT, is naturally distributed across Asia and Europe. Various parts of this plant have been used in traditional medicine [medicine . In vivomedicine .Nigella sativa is widely used as medicinal plant  [d Unani) . Its oild Unani) .In vivo study, where rats were treated with olive oil with OTA, showed reduced ability against oxidative stress. Whereas, the addition of curcumin increases the activity of antioxidant enzymes such as SOD, catalase (CAT), and GPx. Moreover, curcumin weakens the effect of the toxin on liver inflammation, steatosis, and necrosis . CurcumiO-β-glucopyranoside (C3G), an anthocyanin family member which naturally occurs in pigmented oranges, blackberries, strawberries, and cranberries, is known for its antioxidant properties. In the study conducted by Guerra et al. [Cyanidin-3-a et al.  effectiva et al. . This coa et al. . After fa et al. .Yemeni green coffee is a source of caffeoyl-quinic acids one of the intense antioxidative substance. According to report by Nogaim et al. , Yemeni Green tea-mediated zinc oxide nanoparticles (ZnONPs) appear to be hepatoprotective in albino rats by reducing the lethal effect of OTA. The presence of OTA damages liver cells and leads to necrosis. As a result, the levels of biomarkers associated with liver function, such as ALT and AST, tend to rise. The use of ZnONPs has significantly improved the ALT and AST parameters by decreasing their levels. Furthermore, it was observed the improvement during histopathological examination. The animals that received a combination of OTA and ZnONPs exhibited moderate degeneration in their livers, whereas those who consumed only the toxin experienced severe alterations .Palabiyik et al. reported a significant decrease in selenium, zinc, and copper levels in OTA-treated rats. The addition of lycopene did not affect the liver zinc level, whereas it markedly increased selenium and copper compared to the group treated with OTA only . While OSilybum marianum (milk thistle) is a plant that is native to the Mediterranean region and is now grown and cultivated worldwide [orldwide . Sil is orldwide  and primorldwide . OTA cauorldwide . The in orldwide .Silibinin possess a protective effect on rats’ isolated perfused livers and Kupffer cells against OTA toxicity. It had the inhibitory effect on OTA-mediated TNF-α release. Moreover, silibinin completely restored glutamate dehydrogenase (GLDH) and lactate dehydrogenase (LDH) levels . AccordiThe results obtained after coadministration with OTA three different compounds: ammonium glycyrrhizin (CAG), L-arginine (L-Arg), and glucurolactone (GA) were similar to results with Sil. The apoptotic rates were decreased after their pretreatment to 14.90 ± 1.50, 18.76 ± 1.55 and 27.50 ± 2.16%, respectively. SOD activity and GSH levels were significantly higher comparing to group with OTA only. Moreover, the MDA levels and Caspase-3 mRNA expression were markedly decreased while Bcl-2 mRNA expression was significantly increased after the pretreatment with CAG, L-Arg and GA .These coThe results indicate the similarities in the action of individual compounds. However, what should be taken into account, all of these compounds are able to mitigate the effects of ochratoxin A, although they are not drugs that will completely eliminate its toxic effect.Many studies indicate that OTA contamination in food products is still a prevalent and inevitable problem. This poses a serious threat. Exposing people to the toxin ingestion will result in the occurrence of health problems related to the hepatotoxic effect of OTA. Based on the available experimental studies, it can be suggested that the basic mechanism responsible for hepatocytes damage  is related to the induction of oxidative stress. The disturbed balance between free radicals and antioxidants leads to significant hepatocytes damage and significantly affects the metabolism of these cells. Unfortunately, there are many unresolved issues and unanswered questions in the field of the effects of OTA on hepatocytes. The following questions were created due to the discrepancy among results in the above-mentioned studies: Does OTA/OTA-HSA affect PEPCK activity in liver? Does PFKL and PFGK1 expression changes affect glycolysis in liver? What are the molecular differences among described pathways between kidney and liver? What is the reason of different functioning processes after OTA administration between liver and kidneys? To answer these questions, the detailed analysis of gluconeogenesis, glycolysis, PPAR signaling pathway and overall multi-omics should be performed on different cell types.The results of the conducted experiments clearly indicate that OTA has a detrimental effect on hepatocytes. However, existing analyzes are often performed on different cell lines or organisms, with different incubation times, concentrations, and routes of OTA administration. These discrepancies significantly affect the proposed assumptions, as not all findings can be extrapolated to the human model. One of the main issues is a lack of studies on the human organism, which obstruct the comparison of obtained results concerning whether the impact of OTA in an animal model will have the same molecular background in the case of humans. An example is the ability of OTA to perfuse organs by binding to HSA. In rats, plasma albumin was shown to have a structural and quantitative differences in the binding sites and in various stereoselective behavior than in HSA . MoreoveA very promising tool for future studies on human liver is the liver-on-a-chip (LOC) device. This technology provides the application of various cell types co-culturing that build human organs. Furthermore, used microfluidic device enable control of fluid flow. Thus, the microenvironment of the cultured cells can be strictly monitored and easily changed . ResultsIn addition, the results also show that there are compounds that can positively influence by minimization of the toxic effects of OTA and thus protect the hepatocytes. However, most of the available data as it was mentioned are based on animals, so they do not provide clear answers as they would not be equally effective in humans. For this reason, there is still a need for broader research that could explain the molecular mechanisms of OTA-induced hepatotoxicity, and further research on hepatocytes protective compounds. The published results indicate the relationship between the protective effect on the liver against the toxic effects of OTA and antioxidants, which should be taken into account when taking further research steps.


Thus, this study aims to verify the association of food consumption according to the degree of processing with body image (dis)satisfaction in adolescents from the 1997/1998 birth cohort of São Luís, Maranhão. (2) Methods: A cross-sectional study was conducted with 2515 adolescents aged between 18 and 19. Food consumption was stratified based on the NOVA classification in culinary preparations, processed foods, and ultra-processed foods, categorized into tertiles. Body image (dis)satisfaction was evaluated based on the Stunkard Body Scale and was classified as satisfaction, dissatisfaction with thinness, and dissatisfaction with excess weight. Multinomial logistic regression was used for associations. (3) Results: Among the adolescents, 77% considered themselves dissatisfied with their body image, with 42.8% dissatisfied with being thin and 34.2% dissatisfied with excess weight. However, food consumption, according to the degree of processing, was not associated with body image (dis)satisfaction. (4) Conclusion: This work highlighted the prevalence of body image dissatisfaction among adolescents but found no association between body (dis)satisfaction and food consumption according to the degree of processing. Adolescence involves several physical changes, biological, psychological, cognitive, emotional, and behavioral characteristics of the period known as puberty . Among oBody image comprises the mental conception of the real body . In BrazFood processing can be considered one of the main determinants of food quality and health ,19. In tUltra-processed foods  are markers of an unhealthy diet due to their nutritional imbalance and the damage they can cause to health . The conOn the other hand, the inverse association is also possible, in which the perception of body image is a determining factor in eating behavior ,31. FromTo date, few studies ,35 have This cross-sectional study originated from a birth cohort conducted with adolescents born in São Luís, state of Maranhão, Brazil, in 1997/1998. This study uses data from the third phase of the 1997/1998 São Luís Birth Cohort ,37. The first phase of this cohort used one in seven deliveries performed between March 1997 and February 1998 as a sample basis, in ten public and private hospitals in the city, with proportional sharing according to the number of births in each unit, totaling a sample of 2493 live births. The cohort was followed up in 2005/2006, with a participation rate of 72.7%, totaling 673 children aged 7 to 9 years. Another follow-up was carried out in 2016 with 684 adolescents aged between 18 and 19 years. However, the decision to open the cohort was made to increase the size of this sample due to the small number of individuals who agreed to participate in the study in previous phases, in addition to preventing future losses and increasing the sampling power. Thus, new participants born in São Luís in 1997, who were not initially part of the cohort, were drawn. These individuals were identified using the Brazilian Live Birth Information System, school or university registration, and social network, recruiting 1828 new adolescents, which were included in the study along with the initial sample, totaling 2515 participants in the final sample . The newData collection was carried out between January and November 2016 and consisted of individual interviews and body composition assessments.The assessment of (dis)satisfaction with body image was performed using the Body Scale by Stunkard, et al., . This scThus, body (dis)satisfaction was evaluated based on the subtraction of the IBI number from the RBI number, with the result ranging from minus eight to eight. Participants whose subtraction result was equal to zero were considered satisfied, i.e., those whose RBI was equal to the IBI. Negative results (from minus eight to minus one) were considered dissatisfied due to thinness, and positive results (from one to eight) were considered dissatisfied due to excess weight . This clFood consumption was assessed using the validated food frequency questionnaire (FFQ) . The 24 The reported consumption frequency was converted to daily frequency and then associated with serving size to estimate each food item’s total energy and daily energy consumed. The nutritional composition of the diet was obtained based on the nutritional composition table of foods consumed in Brazil, which is based on food composition tables and includes information from food labels and regional recipes ,44,45.Although the NOVA classification divides foods into four groups, this study categorized the FFQ items into three groups: 1. culinary preparations  , 2. proc2 = body mass/height2). Weight and height were measured using a high-precision scale coupled to the BOD POD and an Altura Exata stadiometer, respectively . Nutritional status was classified based on BMI by age, according to the reference values of the growth curves of the World Health Organization (WHO) [2 at 18 years old or BMI ≤ 25.4 kg/m2 at 19 years old; female, BMI ≤ 24.8 kg/m2 at 18 years old or BMI ≤ 25.0 kg/m2 at 19 years old), and overweight or obesity .Based on the literature ,24,34, ton (WHO) , categorp ≤ 0.05. The prevalence of body (dis)satisfaction categories for each food group according to the degree of processing was assessed using analysis of variance (ANOVA). The consumption of culinary preparations, processed, and ultra-processed foods related to (dis)satisfaction with body image was evaluated using multinomial logistic regression analysis (crude and adjusted models), calculating the Odds Ratio (OR) with their respective 95% confidence intervals (95%CI). The satisfaction with the body image category was considered a reference in the multinomial regression models. The models were adjusted for age, skin color, years of schooling, economic class, leisure time physical activity, screen time, alcohol consumption, smoking, and BMI. The analyses were performed by using the statistical program Stata version 14.Descriptive analyses were performed for (dis)satisfaction with body image according to exposure, and the prevalence and respective intervals were estimated with 95% confidence (95%CI). The chi-square test was used to examine the differences between the prevalence of body (dis)satisfaction categories and the characteristics of the sample. The level of statistical significance was set at The study was approved by the Research Ethics Committee of the University Hospital of the Federal University of Maranhão . Written informed consent was obtained from all subjects by signing the informed consent form.Of the 2515 study participants, 52.5% were female, 69.3% were 18 years old, 63.6% had brown skin color, 88.0% had 9 to 11 years of schooling, 50.1% were from economic class C, and 70.1% had a family income ≤3 minimum wages. Regarding lifestyle, 53.0% did not practice leisure time physical activity, 63.4% had more than 5 h of screen time per day, 41.5% consumed alcoholic beverages, and 3.5% were smokers .p < 0.001), with the prevalence of dissatisfaction with thinness being higher among boys (54.7%) than among girls (32.7%), and dissatisfaction with excess weight being more prevalent among girls (44.8%) than among boys (22.9%). In the sample, 77% of adolescents considered themselves dissatisfied with their body image, with 42.8% dissatisfied with thinness and 34.2% dissatisfied with excess weight. The differences by gender were statistically significant (p < 0.001), among those dissatisfied with excess weight and higher consumption of rice (p < 0.001), processed foods (p < 0.001), and industrialized bread (p < 0.001) among adolescents dissatisfied with thinness satisfaction with body image, demonstrating greater consumption of culinary preparations, such as fruits, chicken, fish, and seafood satisfaction.Regarding the high prevalence of dissatisfaction with body image (77%), the findings corroborate other studies conducted in Brazil with adolescents aged between 10 and 16 years ,54,55. FThis study showed a higher percentage of dissatisfaction with thinness (42.8%) than with excess weight (34.2%), corroborating findings among adolescents aged between 12 and 18 years in Tunisia , with a In this study, rice and industrialized bread consumption were higher among adolescents dissatisfied with thinness. Similarly, a study  conducteRegarding the consumption of culinary preparations and processed food groups, no significant association with any category of body (dis)satisfaction was observed. However, Hernández Camacho, et al., , who invAdolescence is marked by greater consumption of ultra-processed foods , which, Note that body image can be represented by two different dimensions: perceptual and attitudinal. The perceptual dimension encompasses the individual’s perception of body size and shape. The attitudinal dimension comprises the subject’s emotions and attitudes towards their body ,69,70. TFew studies have investigated the association of food consumption according to the degree of food processing with body (dis)satisfaction in adolescents  and younThe main strengths of this study are the large sample size, the use of validated instruments, the use of highly valid methods for measuring body composition, and the use of the NOVA food classification. Additionally, the silhouette scale allows the analysis of issues that require the individual’s opinion since it is a subjective measure .One limitation of this study was the sample loss due to difficulties locating the adolescents, which required incorporating other individuals not in the original cohort. Additionally, memory bias in response to food consumption questions and overestimation of consumption by the FFQ can also be considered limitations of this study. These limitations occur because the same food can fit into more than one group due to the difficulty of fitting it into a single category according to the degree of processing. To minimize these limitations, the interviewers were trained to minimize recall bias, ensuring proper questionnaire application. Furthermore, to minimize the overestimation of consumption caused by the difficulty of separating foods between groups, each food was assigned the NOVA classification category in which it is most used satisfaction in adolescents, most of the young people in this study were dissatisfied with their body image, mainly with thinness. Note the seriousness of these results since body dissatisfaction is linked to several determinants related to health, which can lead to anxiety , depressThis study provided relevant information about the high prevalence of body image dissatisfaction among adolescents but showed no association between body (dis)satisfaction and food consumption according to the degree of food processing.


Arbuscular mycorrhizal fungi (AMF) are widely distributed microorganisms in the soil, playing an important role in vegetation succession, plant community diversity, and improving soil physicochemical properties. In this study, morphological identification and high-throughput sequencing technology were used to comprehensively analyze the AMF community composition and diversity at different succession stages of Songnen saline-alkali grassland. To determine the root colonization status of plants collected in the field, a colonization system was established using late-succession plants as host plants to verify the existence of mycorrhizal symbiosis and the matching phenomenon of AMF in Songnen saline-alkali grassland. The results indicated that both morphological methods and high-throughput sequencing technology showed that glomus was the dominant genus of AMF in Songnen saline grassland. Redundancy analysis (RDA) and linear regression analysis showed that electrical conductivity (EC) and pH were the main environmental factors affecting AMF species diversity and community structure in the succession sequence of Songnen saline grassland. In addition, the results of root colonization identification and the colonization system test in the field showed that AMF successfully colonized vegetation at different succession stages and had mycorrhizal symbiosis. The results of this study could help to understand the AMF community of Songnen saline-alkali grassland as well as provide a reference and basis for optimizing the AMF community structure of Songnen saline-alkali grassland through human intervention in the future and using mycorrhizal technology to restore and rebuild the degraded ecosystem of Songnen saline-alkali grassland. On thrassland . At the me place .The degree of soil salinization is an important factor affecting plant growth, and high concentrations of salt or alkali will cause plant yield reduction or death ,10. PlanIn the current research, the diversity of AMF in the soil is often analyzed by spore morphological methods, and classical morphological identification has become the basis for the application of single species . HoweverL. chinensis was often grown as a mixture of late-successional plants with associated species such as Chloris virgata, Medicago sativa, and Lepidium apetalum, and the mycorrhizal network system had been shown to work between host plants and associated species [Recently, the analysis of previous studies on mycorrhizal function found that the mycorrhizal effect of mixed cultures was better than that of single species , and the species ,27. HoweL. chinensis, a plant in the late succession stage of Songnen saline-alkali grassland, was used as the host plant to further clarify whether there is an ecological matching effect of AMF communities in different vegetation community succession stages, which provides a more reliable scientific basis for optimizing the AMF community structure and rational use of mycorrhizal effect in Songnen saline-alkali grassland by human intervention in the future.Both plants in the saline grassland of Songnen and AMF exist and function in a community manner. So, are there differences in the community structure and diversity of AMF at different succession stages? What factors drive the succession of AMF communities? Is there a local adaptation or ecological matching phenomenon in AMF-plant symbiosis? In order to better solve these problems, we used high-throughput sequencing technology supplemented by morphological identification to analyze the structure characteristics and diversity of AMF communities in the root enclosure and plant roots at different succession stages in the Songnen saline-alkali grassland. The relationship between AMF community structure and soil physicochemical properties at different succession stages in Songnen saline-alkali grassland was clarified, and on this basis, S. glauca of the Amaranthaceae family and P. tenuiflora and L. chinensis of the Poaceae family are the main plant communities of the saline grassland of Songnen. Among them, S. glauca is the early succession (I.), P. tenuiflora is the middle succession (II.), and L. chinensis is the late succession (III.), which constitutes the succession series of the Songnen saline grassland community.The study site is located in the southwest of Songnen Plain, Heilongjiang Province, China . It belongs to the middle temperate continental monsoon climate and has a hot and rainy summer, cold and dry winter, and large temperature difference; the highest temperature in summer is 30 °C, the lowest temperature in winter is −25 °C, and the annual average temperature is 2.8 °C; and it has an annual frost-free period of approximately 140 days, annual evaporation of 1073.3–1368.4 mm, and annual precipitation of 350–550 mm mainly concentrated in July–September. In August 2022, the roots and root enclosures of Alkali Plant, Star Grass, and Lamb Grass were collected in the saline-alkali grassland of Songnen City, Zhaodong City, Heilongjiang Province. We selected three repeat plots (10 m × 10 m), each of which had a minimum aggregation length of 100 m. Each site adopts the “multi-point mixed sampling method”  and the −1), diluted it into a solution of different concentrations, added 3,5-dinitrophenol color development, and drew a standard curve colorimetric at a wavelength of 508 nm on a spectrophotometer. Urease activity was determined by sodium phenol colorimetry. After the N standard solution (1000 mg·L−1) was diluted at different times and sodium phenol and sodium hypochlorite were added for color development, the standard curve was developed on the spectrophotometer at the wavelength of 578 nm. Catalase activity was determined by potassium permanganate titration and, finally, easily extractable soil protein associated with saccomycin was determined by 20 mM citrate (pH = 7.0), and the difficult-to-extract soil protein associated with saccomycin was determined by 50 mM citrate (pH = 8.0). Bovine serum protein was configured into a standard protein solution, different concentrations of standard protein solution were added to Coomassie Brilliant Blue Reagent, and two minutes later, the standard curve was drawn on a spectrophotometer at a wavelength of 595 nm.The chemical properties of the soil were determined by the methods of Boshidan , the enz® soil DNA kit , the instructions were followed for the total DNA extraction of microbial communities. DNA extraction quality was detected using 1% agarose gel electrophoresis, and DNA concentration and purity were determined using NanoDrop2000; PCR amplification of the 18S rRNA gene fragment using AMV4-5NF (5′-AAGCTCGTAGTTGAATTTCG-3′) and AMDGR (5′-CCCAACTATCCCTATTAATCAT-3′) was as follows: Predenaturation at 95 °C for 3 min, 27 cycles , then stable extension at 72 °C for 10 min. Finally, it was stored at 4 °C (PCR instrument: ABI GeneAmp® 9700). The PCR reaction system was as follows: 5× TransStart FastPfu buffer 4 μL, 2.5 mM dNTPs 2 μL, upstream primer (5 uM) 0.8 μL, downstream primer (5 uM) 0.8 μL, TransStart FastPfu DNA polymerase 0.4 μL, template DNA 10 ng, ddH2O supplement to 20 μL. Each sample was repeated three times.According to the E.Z.N.A.PCR products from the same sample were mixed and recovered using 2% agarose gel, purified using the AxyPrep DNA Gel Extraction Kit , electrophoresis assayed by 2% agarose gel, and quantified with Quantus™ Fluorometer . We used the NEXTflexTM Rapid DNA-Seq Kit  for library construction: (1) Connector link; (2) use of magnetic bead screening to remove the connector self-connecting fragment; (3) enrichment of library templates by PCR amplification; and (4) magnetic beads recovered PCR products to obtain the final library.AMF spores in plant rhizosphere soil were separated by wet sieve decantation-sucrose centrifugation, and the Petri dish containing the sieve was placed under a double-tube solid anatomical microscope and AMF spores with the same morphological characteristics were transferred one by one to a 1.5 mL centrifuge tube and classified and numbered. After spores were prepared, their size, color, spore wall hierarchy, and thickness were observed under a light microscope, and the reaction after staining Melzer’s reagent was recorded. Referring to the Mycorrhizal Identification Manual  and the Distribution frequency (F) = number of occurrences of a genus or species of AMF/number of soil samples × 100%Relative abundance (RA) = number of spores of a genus or species of AM fungus/total number of AMF at the sampling point × 100%Average of Importance Value (IV) = (F + RA)/2 × 100%.The roots collected in the wild were cleaned, the lateral roots were cut into 1 cm root segments, and then the root segments were submerged in FAA fixative solution  and fixed for 12 h. Observing the colonization of AMF in the root system with reference to the research method of Philips et al. , the rooS. glauca, P. tenuiflora, and L. chinensis were used as in situ soil agents, and the soil-soaking solution filtered by the 38μm filter membrane was used as a bacterial filtrate to exclude bacterial interference. The seeds were collected from the saline grassland of Songnen, soaked in clean water, disinfected by 0.1% HgCl2, and sown at a low temperature of 48 h. A total of 5 groups were set up, two groups of controls (in situ soil and peaty soil) and three groups of experimental groups , and each group was repeated 5 times, for a total of 25 pots of seedlings, and all the sown pots were placed in the plant light culture chamber for cultivation. After 60 days of incubation, the fresh weight, dry weight, and root colonization status of each treated L. chinensis were tested, and the fresh weight (Wf) and dry weight (Wd) of the plants were accurately weighed using an electronic balance according to the method of Liu Ping and Li Mingjun [The saline soil of Songnen was used as the cultivation substrate to establish a potting test colonization system. The soil samples and plant roots collected from the rhizosphere of the community of  Mingjun . The pla Mingjun .https://github.com/Open Gene/fastp, version 0.20.0, 19 May 2023) software, and splicing using FLASH [http://www.cbcb.umd.edu/software/flash, version 1.2.7, 19 May 2023) software: (1) Filter the bases below the mass value of the tail of reads, set a window of 50 bp, and if the average mass value in the window is lower than 20, cut off the back-end base from the window, filter the reads below 50 bp after quality control, and remove reads containing N bases. (2) According to the overlapping relationship between PE reads, the paired reads are merged into a sequence, and the minimum overlap length is 10 bp. (3) The maximum allowable mismatch ratio of the overlap region of the splicing sequence is 0.2, and the screening does not conform to the sequence. (4) The sample is distinguished according to the barcode and primers at both ends of the sequence, and the sequence direction is adjusted, the number of mismatches allowed by the barcode is 0, and the maximum number of primer mismatches is 2. Using UPARSE [http://drive5.com/uparse/, version 7.1, 20 May 2023), OTU clustering of sequences based on similarity of 97% [http://rdp.cme.msu.edu/,version 2.2, 20 May 2023) are used to annotate the species taxonomy of each sequence.The original sequencing sequence was quality controlled using fastp  . Dry weight and fresh weight results were presented in histograms using SigmaPlot 14.0.A one-way analysis of variance (ANOVA) was conducted to analyze all results using the SPSS 25.0 statistical analysis package. All data were presented as the mean ± standard deviation (SD) of the three independent replications, and Duncan’s multiple range test was applied with the level of significance fixed at L. chinensis stage was the largest, with 43 species, and 34 and 33 species were isolated and identified in the root enclosure soil of the P. tenuiflora stage and the S. glauca stage, respectively. Glomus and Acaulospora contain the largest number of species with 20 and 14 species. Ambispora, Paraglomus, Sclerocystis, Racocetra, Rhizophagus, Dominikia, and Archaeospora each identified 1 species. After calculation, the separation frequency, relative abundance, and important values were obtained, respectively. Furthermore, 92.59%, 8.90%, and 50.75% of the dominant species sp1 (IV ≥ 50%) were obtained in the succession process of Songnen saline-alkali grassland, but the existing morphological information could not classify it into specific species. In addition, the six most common strains (30% < IV < 50%) were obtained from G. reticulatum, G. clarum, Ac. laevis, Cl. walkeri, Cl. claroideum, and Rh. intraradices during the succession of pine saline grasslands. There were 18 common strains (10% < IV < 30%) and 36 rare strains (0% < IV < 10%). The specific spore image is supplemented with A total of 62 species of AMF in 17 genera were isolated in three succession stages . Among t−1. pH and EC values decreased significantly with successional sequences, and the pH and EC values in the S. glauca stage were 0.09 and 2.22 times higher than those in the L. chinensis stage, respectively. In contrast, the organic matter, organic carbon, whole N, and total extracted GRSP were the highest in the L. chinensis stage, followed by the P. tenuiflora and S. glauca stages. Similarly, bicarbonate and total Mg remain stable in successional phases. The pH, EC, carbonate, whole K, all Ca, all Na, and urease stages of L. chinensis were lower than those in the stage of star grass and the S. glauca stage, while the soil proteins associated with organic matter, organic carbon, all N, all P, C/N, catalase, sucrase, and E-GRSP were higher than those in the stage of P. tenuiflora and S. glauca stage, which also indicated that the degree of soil salinization in the late succession stage was low and the nutrient content was high.We found differences in soil physicochemical properties, enzyme activity, and GRSP at different plant succession stages . All ploL. chinensis community, 113,549 sequences from the P. tenuiflora community, and 122,145 sequences from the S. glauca community. Based on 97% species similarity, a total of 171 operational taxa (OTUs) were detected in the entire succession sequence community, of which the maximum number of OUT was 136 in the root enclosure and 124 in the plant root , and the lowest values of the Shannon and Sobs diversity indices occurred in the S. glauca stage. The maximum value of the Simpson index occurred in the S. glauca stage, which was significantly higher than that of the other two stages (p < 0.05), and the lowest value of the Simpson index occurred in the L. chinensis stage.The dilution curve indicates that our sequencing depth is sufficient to cover most species in all samples . The ShaS. glauca stage and the P. tenuiflora and L. chinensis stages, while the difference in AMF community structure in the P. tenuiflora and L. chinensis stages was not significant.Based on the UniFrac phylogenetic distance algorithm, all samples were clustered by NMDS analysis to detect the differences between the community structures of AMF at different succession stages. The results showed that there were significant differences in AMF community composition between different plant succession stages . There wAccording to the results of the Mantel Test, some environmental factors significantly related to the AMF community structure were selected for RDA analysis . Analysip < 0.05) for Shannon, Sobs, and Simpson, and EC was significant for Shannon and Simpson (p < 0.05), but not for the Sobs index. Peroxidase and calcium ion content showed a certain correlation but not a significant effect on AMFα diversity.According to the results of RDA analysis, the four factors with the greatest influence on AMF community structure were obtained, and linear regression analysis was used for them and AMFα diversity . The resS. glauca community , and the succession of vegetation, the colonization rate (%), colonization intensity (%), vesicle abundance, and arbuscular abundance (%) increased gradually.Studies have shown that AMF successfully colonize the ommunity A–C, the ommunity D–F, and ommunity G–I that  samples , the chaL. chinensis with AMF communities of different successional stages, the colonization structures, such as hyphae, vesicles, and arbuscular, of AMF could be observed in plant roots (p < 0.05). Similarly, as can be seen from p < 0.05).After 60 days of the establishment of the colonization system of the late-successional plant nt roots A–F. OnlyDi. eburnea and Do. aurea were isolated for the first time in the saline grassland of Songnen, indicating that there are abundant AMF resources in the saline habitat. Glomus identified the largest number of species, followed by Acaulospora, Claroideoglomus, and Gigaspora [Glomus and Acaulospora were absolutely dominant in different succession stages, which was in line with the view of broad-spectrum symbiotic fungi [Glomeraceae sp1. However, it had a certain degree of inhibition on the germination and bud tube elongation of dominant strains isolated from other plant root enclosures such as G. mosseae, Cl. etunicatum, and Rh. intraradices [Glomeraceae sp1 image information had not been published in other habitat studies and its ecological function had not been explored, this strain had a strong survival ability in a saline-alkali environment, and the discussion of this strain should be strengthened in future studies.Sixty-two species of AMF17 genera were isolated from the root enclosure soil of the three vegetation succession stages, and multiple species not reported in similar studies were found in this experiment, among which igaspora ,42. Amonic fungi . Joinperic fungi  found tharadices ,46. SincS. glauca community had the highest EC value, pH value, and carbonate and bicarbonate content, which also confirmed that the salinization degree of the S. glauca community was the highest. Total phosphorus content was lower in the three succession stages, which may be related to the presence of more carbonates and bicarbonates in the soil [S. glauca and P. tenuiflora, thus ensuring the supply of the N element. The activity of sucrase showed a trend of change according to the succession series, which may be related to the degree of salinization.Soil EC and pH are the main factors controlling the evolution of saline-alkali ecosystems . As the the soil , and thethe soil . The conthe soil . These ithe soil , sucrasethe soil , and catthe soil . Urease L. chinensis had a more developed root system than S. glauca and P. tenuiflora and could form a good co-effect with AMF so that AMF could absorb and use 20% of plant photosynthetic fixed carbon and transmit it to the soil through hyphae, spores, mycorrhizal residues, etc., and finally fix it in the soil in the form of balloonmycin-related soil protein to increase the carbon source in the soil [L. chinensis stage could adsorb or bind fine soil particles through physical action, form stable soil aggregates, improve the soil structure [GRSP was a kind of soil glycoprotein produced during the decay of AMF mycelium, spores, and other propagators and was also a key component of soil organic matter and organic carbon in Songnen saline-alkali grassland . In the the soil . The abutructure , and makGlomus. It may be that the spores of the genus Glomus are relatively small, able to produce a large number of spores in a short period of time, and are easily transmissible [L. chinensis stage were significantly different from those of the previous two stages, which may be due to the influence of soil environmental conditions, the salinization degree of the L. chinensis stage being the lowest, and the fact that it had a more suitable environment for AMF than the S. glauca and P. tenuiflora stages.In this study, the abundance of AMF in soil and plant roots in Songnen saline grassland was unexpectedly higher than that of other ecosystems, such as agricultural ecosystems and forest ecosystems ,57, indimissible , which iThe EC value and pH showed a significant negative correlation with AMF diversity, which directly verified the claim that the salinization degree affects AMF growth and population distribution . With thL. chinensis was inoculated with fungi at different succession stages, it was found that L. chinensis had a more significant effect on the biomass increase in host plants than other agents. This may be because L. chinensis stage fungi and L. chinensis host plants can form a better mycorrhizal symbiosis network system, expand the host plant root system, increase the effective use of N, P, and other elements in the soil by plant roots [L. chinensis stage fungal agent increase the biomass of host plants more significantly. The colonization rate and intensity of L. chinensis on host plants were higher than those in other treatment groups, which further indicated that AMF plants in Songnen saline-alkali grassland had mycorrhizal symbiosis matching phenomena .It is widely believed that there is a mycorrhizal symbiotic match  between the host plant and AMF ,64. Prevnt roots , and theL. chinensis was more significant, indicating that there was a phenomenon of mycorrhizal symbiosis matching in Songnen saline-alkali grassland. Songnen saline grassland is an unique ecosystem, affected by a variety of factors, which provides a theoretical basis and data support to better understand the impact of environmental change on the structure and function of AMF in the Songnen saline-alkali grassland ecosystem.In summary, the diversity and community structure of AMF at different succession stages in the Saline Songnen grassland were fully understood by morphology and high-throughput sequencing. The results showed that Glomus was the dominant genus of AMF in Songnen salin-alkali grassland. RDA analysis showed that the soil EC value, pH, calcium content, and hydroperoxide activity had the greatest influence on AMF community structure, and linear regression analysis showed that the EC value and pH had the greatest influence on AMF species diversity. In addition, in order to verify whether there is a mycorrhizal symbiotic match between AMF and host plants in the Songnen saline grassland, a colonization system was established. The results showed that the promotion effect of