In control animals, the bone cavities were filled with their own blood clots after bone trepanation and bleeding. The periods for monitoring bone regeneration were 3, 5, and 7 days and 2, 3, 4, 6, 8, and 10 weeks. The control of bone regeneration was carried out using multiple morphological and histological analyses. Results showed that the following process is an obligatory process and is accompanied by the binding and release of angiogenic implantation: the chitosan construct actively replaced early-stage defects with the formation of full-fledged new bone tissue compared to the control group. By the 7th day, morphological analysis showed that the formation of spongy bone tissue could be seen. After 2 weeks, there was a pronounced increase in bone volume (p < 0.01), and at 6 weeks after surgical intervention, the closure of the defect was 70–80%; after 8 weeks, it was 100% without violation of bone morphology with a high degree of mineralization. Thus, the use of modified chitosan after filling eliminates bone defects of critical size in the maxillofacial region, revealing early signs of bone regeneration, and serves as a promising material in reconstructive dentistry.A biomaterial is proposed for closing extensive bone defects in the maxillofacial region. The composition of the biomaterial includes high-molecular chitosan, chondroitin sulfate, hyaluronate, heparin, alginate, and inorganic nanostructured hydroxyapatite. The purpose of this study is to demonstrate morphological and histological early signs of reconstruction of a bone cavity of critical size. The studies were carried out on 84 white female rats weighing 200–250 g. The study group consisted of 84 animals in total, 40 in the experimental group and 44 in the control group. In all animals, three-walled bone defects measuring 0.5 × 0.4 × 0.5 cm The successive stages of bone regeneration are often a long-term, chronic process. It is known that with a large area of bone damage, physiological repair is not able to eliminate the defect on its own, and the use of many replacement polymer structures does not provide the expected regeneration effect ,2,3. The2) in the chitosan molecule, for example, with weak ascorbic acid, makes it possible to increase the efficiency of controlling angiogenic reactions in the periodontium ). During the choosing statistical tests for assessing the type I error (α) and the sensitivity of the criterion  distribution was carried out on the basis of the Shapiro–Wilk test, as well as on the basis of the graphical method (Quantile–Quantile plot). Most of the variables obtained obeyed the normal distribution law, and the cases of deviation of the variables from the normal distribution were not pronounced. For variables deviating from the normal distribution, the following methods of data transformation were used to achieve compliance with the normal distribution: with right-sided (positive) skewness, square roots were taken from the obtained values, and in the case of left-sided (negative) skewness, the formula Plain microscopy of histological preparations in the area of the postoperative defect of the lower jaws of healthy rats removed after 3 days of this experiment revealed extensive foci of hemorrhagic impregnation B. Single2, which corresponded to the area of the field of view displayed on the monitor screen at × 400 magnification. The presented histomorphometric criterion was not studied after the experiment period of 3 weeks due to the complete elimination of pathomorphological signs of inflammation. As shown in p < 0.01), while in the group of animals with implanted biopolymer “CH–SA–HA”, the objective indicator of cell infiltration from the 3rd to the 7th day of this experiment increased by 57% (p < 0.01), while in the control group, the same range of changes was only 36% (p < 0.01)  . Thus, the processes occurring at week 1 correspond to the first stage of bone tissue regeneration (the formation of an osteoid island) and are characterized by the appearance of inflammatory infiltrate cells, the number of which increases every day, and osteoblastic differon cells that trigger the process of osteogenesis. When comparing the ongoing process of regeneration in the animals of the control and experimental groups, it follows that in the animals of the experimental group, the inflammation process is less pronounced; osteoblasts appear in greater numbers earlier, and osteoid islands are formed as early as in the first week.p < 0.01). In pairwise comparisons, the numerical density of the inflammatory infiltrate in the experimental group was significantly lower (p < 0.01) at all estimated time intervals of the experiment, except for the period of 3 weeks. In pairwise comparisons, the digital values of the numerical density of the inflammatory infiltrate in the peripheral zone of the bone defect (experiment) were more than two times lower (p < 0.01) compared with the values in the zone of the bone defect proper . The application of a large bone injury in the lower jaw triggers the mechanism of early bone resorption in both groups with an active osteoclastic reaction, especially at the periphery of the defect , followeOn the 2nd and 3rd weeks of the experiment, formed bone beams were visualized in the area of the inflicted defect, on the surfaces of which numerous osteoblasts were detected, mostly of a cubic shape A,B. The p < 0.01). Filling the surface of the bony trabeculae with cell mass leads to the thickening of the trabeculae and contraction of the intertrabecular spaces and the inactive free bone surface. In the first 2 weeks, the increase in the thickness of bone trabeculae in this experiment was 100%, and in the control, 83%. The walls of the bone cavity in control after 3 weeks are in the form of thin bone beams with pronounced intertrabecular spaces and poor cell mass , during which spongy bone is formed, represented by bone trabeculae and wide inter-trabecular spaces filled with developing bone marrow. Reticular fibrous bone tissue is involved in the construction of this bone. The processes occurring during these periods are characterized by a decrease in the number of inflammatory infiltrates and high osteoblastic activity. Animals of the experimental group in the indicated time periods are characterized by a high volumetric density of the newly formed bone tissue.After 6 weeks , the actIt should be pointed out that the activity in the dynamics of osteogenesis in the control group is significantly lower than the results of this experiment. The inactive zone of the bone cavity walls (FS%) occupies a significant area during the first 6 weeks of the regeneration process .During the 6th and 8th weeks, spongy bone tissue predominates at the defect site with oriented bone trabeculae, flattened osteoblasts, and single osteoclasts A. Later,Thus, 6–10 weeks of this experiment correspond to the last stage of osteogenesis (formation of a compact bone or differentiation of reticufibrous bone tissue into lamellar bone tissue). At 6–8 weeks, high osteoblastic activity is still observed; the volume of the resulting bone increases, and osteoclast cells are active, participating in the processes of tissue reorganization. All of these processes are most active in the experimental group of animals. By the 10th week of the experiment, lamellar bone tissue is formed, which is characterized by the presence of osteons in the bone trabeculae and the formation of Haversian canals. Animals of the experimental group are characterized by a high volumetric density of the emerging bone in earlier periods of regeneration.p < 0.01) of the influence of the experimental period factor on the variable under consideration. Even though the growth rate for the entire period of this experiment (70 days) was 0.94% per day in the group with implanted CH–SA–HA biopolymer and 0.96% per day in the control group, a high rate of bone tissue growth in the focus regeneration was observed in the time interval of 6–14 days. In the group with implanted biopolymer, the growth rate was 28.9% per day, and in the control group, this figure was 27.2% per day. Statistical analysis based on Welch’s t-test showed that this difference was significant (p < 0.01). Analysis over a randomly selected period of 1–28 days also revealed significant differences (p < 0.01) in favor of the group of animals implanted with CH–SA–HA biopolymer  established the significance 6·5H2O), found in human dentin, is due to a higher activity of inclusion in the early process of bone regeneration [Analysis of the results of regeneration of the spongy and compact parts of the bone in the works of other authors shows that the use of the product of the apatite base of the bone octacalcium phosphate  [p < 0.0001), as determined by one-way ANOVA. At the same time, a comparative analysis of the formation of vessels in the regeneration process did not reveal the advantage of either chitosan or bone allograft . A comparison of data from the experimental groups yielded no statistically significant differences in terms of osteoclast numbers .Numerous publications on bone regeneration after the formation of a critical-size bone cavity show that self-implantation of a multilayer microfiber of chitosan with a molecular weight of 200–300 kDa and a degree of deacetylation of 90% into a 5 mm femoral defect using mesenchymal stem cells differentiated in osteoblasts within 12 weeks does not lead to the final filling of the defect. Compared with the use of a bone allograft, the results of the regeneration volume were 1.5 times lower ( < 0.01) . A blindp = 0.0077). The difficulty of closing large bone defects is confirmed in detailed studies by Casillas-Santana, M.A. et al., 2023. Using colloidal chitosan in combination with nano-hydroxyapatite or silver nanoparticles in this study, the authors did not achieve complete regeneration of a bone defect of a critical size after 6–8 weeks of observation and achieved only 75% of healing [165, BMP2) and a program of simultaneous elution into the bone cavity will allow for closing bone defects of critical size of any localization [Counting the number of osteoblasts in the defect zone revealed a clear advantage of the bone allograft (one-way ANOVA;  healing . This relization . Currentlization .Obtaining a morphological picture of early bone formation due to the active formation of the osteoid surface, starting from 5 to 7 days after surgery, and confirmation of this process during the first month after injury indicates the role of the polymer structure in the advanced regeneration of the cancellous part of the bone. The authors believe that the creation of specialized technologies and technical means for obtaining osteogenic matrices for direct transplantation into bone tissue defects in animals and humans is very important for subsequent widespread use in medical practice. Successfully constructed osteogenic matrices, when in contact with the wall of the bone cavity, are able to reduce or even exclude certain stages of osteogenesis, for example, the cartilaginous stage of development, and trigger the mechanisms of early bone formation. The absence of a well-perfused substrate in the affected area in the early stages after extensive trauma to the bone tissue, the lack of the ability of synthetic materials that do not contain growth factors and osteogenic cells to cover large bone defects of a critical size  is one o− alginate and NH3+ of chitosan. In addition, NH3+ groups can interact with PO43− hydroxyapatite groups, linking the framework together and forming a more compact structure. In this case, porosity and degradation rate decrease, and mechanical stability increases [It is known that the use of a chitosan membrane as a single polymer leads to the formation of new bone and cementum in single-wall intraosseous defects in large experimental animals . Physicancreases . If we ancreases , leads tncreases . The higncreases . The higncreases . Long-tencreases ,74. Howencreases  and rapi


The synovial plica of the elbow is a fold of synovial tissue, which is said to be a remnant of the embryonic septa of normal articular development and is located around the radiocapitellar joint. The objective of the present study was to provide morphometric properties of the synovial plica of the elbow and its relation to surrounding structures in asymptomatic patients.A retrospective study was conducted to establish the morphometric characteristics of the synovial plica of the elbow. The results of 216 consecutive patients, who for different reasons during the five year period of time underwent magnetic resonance imaging (MRI) of an elbow, were analyzed.Plica was found in a total of 161 of 216 elbows (74.5%). The mean width of the plica was set to be 3.00 mm (SD: 1.39). The mean length of the plica was established at 2.91 mm (SD: 1.13). An analysis of sexual dimorphism was also included. Potential correlations were analyzed for each of the categories and age.The synovial plica of the elbow is a clinically relevant anatomical structure. Analyzing the morphometric parameters of the synovial plica is necessary to properly evaluate synovial plica syndrome, which can commonly be confused with other sources of lateral elbow pain such as tennis elbow, oppression of the radial and/or posterior interosseous nerve, or snapping of the triceps tendon. The authors suggest that the thickness of the plica may not be the golden diagnostic feature as there are no statistically significant differences in this parameter between symptomatic and asymptomatic patients. A precise and accurate diagnosis of synovial fold syndrome and/or differentiation from other sources of lateral elbow pain must be performed, as the surgical treatment, even if performed properly, will be unsuccessful because of a misdiagnosed source of pain. The synovial plica of the elbow is a fold of synovial tissue, which is said to be a remnant of the embryonic septa of normal articular development and is located around the radiocapitellar joint . HistoloPathologies of the plicae become symptomatic in people with chronic inflammation caused by repetitive athletic activities, such as throwing, or direct trauma . ChronicDespite the significant clinical relevance , 5, precA retrospective study was conducted to establish the morphometric characteristics of the synovial plica of the elbow. The results of 216 consecutive patients, who underwent magnetic resonance imaging (MRI) of an elbow, were analyzed in the Department of Radiology of Jagiellonian University Medical College, Cracow, Poland, in October 2022. The condition for the inclusion of the MRI result into the present study was the certainty that the patient did not have any symptoms of lateral elbow pain at the time of the examination. For this purpose, patients’ medical histories were also analyzed and patients who had MRI examination performed due to lateral elbow pain were excluded. Additional exclusion criteria were established as follows: (1) previous elbow trauma affecting the plica and/or its close anatomical area, (2) significant artifacts that prevented accurate and precise imaging and/or measurement of the plica and/or its close anatomical area, (3) low quality and illegible images, and (4) absence of the plica in the studied material after the initial evaluation. Eventually, a total of 161 elbows met the required criteria.All examinations were performed using 3.0 Tesla MRI scanner . To the further analysis, only T1-weighted and STIR (short tau inversion recovery) sequences were included. Detailed scan parameters were the following: (1) for T1-weighted turbo spin echo (TSE) sequence—TR 500 ms, TE 14 ms, flip angle 90, NEX 1, slice thickness 3 mm, matrix 560 × 560, FOV 240 × 240 × 71, (2) for STIR TSE sequence—TR 5239 ms, TE 30 ms, inversion time 190 ms, flip angle 90, NEX 2, slice thickness 3 mm, matrix 400 × 400, FOV 240 × 240 × 71.The MRIs were analyzed on a dedicated workstation in the Anatomical Department of Jagiellonian University Medical College, Cracow, Poland. To ensure the highest possible quality of the visualizations and measurements and minimize potential bias, Materialise Mimics Medical version 22.0 software  was used. 3-dimensional (3D) reconstructions of each scan were developed, employing a set of settings adjusted to each scan.Each plica and its close anatomical area were fully visualized at the beginning of each evaluation. Subsequently, a set of measurements was executed on each elbow as the following: (1) width of the plica, measured at its widest point, (2) length of the plica, measured at its longest diamension, (3) width of the radiohumeral joint gap, measured at the widest point of the plica, (4) width of the radiohumeral joint gap, measured at the middle of the radius articular surface, (5) width of the radiohumeral joint gap, measured at the medial end of the joint, (6) the distance from the edge of the radiohumeral joint to the plicas’ apex, (7) the length of the radius articular surface in the radiohumeral joint, (8) the length of the ulnar articular surface in the ulnohumeral joint, (9) the length of the common radio-ulnar articular surface in the proximal radio-ulnar joint, (10) the distance from the plicas’ edge to the apex of the proximal radio-ulnar articular surface, (11) the length of the radius head, (12) the thickness of the humerus, measured at its widest point, (13) the distance from the most lateral point of the radius head to the plicas’ edge, (14) the distance from the most lateral point of the lateral epicondyle of the humerus to the plicas’ edge, (15) plicas’ contact area with the humerus, (16) plicas’ contact area with the radius, (17) the length of the lateral edge of the radiohumeral joint, (18) the shortest distance from the plicas’ edge to the radial nerve bifurcation point, (19) the shortest distance from the plicas’ edge to the extensor carpi radialis longus (insertion), (20) the shortest distance from the plicas’ edge to the extensor carpi radialis brevis (insertion), (21) the shortest distance from the plicas’ edge to the extensor digitorum (insertion). In order to maintain objectivity and repeatability of the measurements, the same pattern was followed while establishing distances between structures. Each measurement was obtained twice by two independent researchers with radiological experience (surgeon and radiologist), and a mean value was established afterward. For radiological measurements, the interobserver and intraobserver reliability were calculated based on ten randomly selected patients using the reliability statistics of the interclass correlation (ICC). Both ICCs coefficients presented excellent values (ICC>0.95).The measurement methods used in this study are presented in Fig. p < 0.05. Mann-Whitney and Wilcoxon signed rank tests were used to establish potential differences between the groups. Spearman’s rank correlation coefficient was used to determine possible correlations between the parameters.Statistical analysis was performed with STATISTICA v13.1 . The frequencies and percentages presented qualitative features. The Shapiro-Wilk test was used to assess the normal distribution. Quantitative characteristics were presented by medians and upper and lower confidence intervals , as well as means and standard deviation (SD), depending on verified normality of the data. Statistical significance was defined as Plica was found in a total of 161 of 216 elbows (74.5%). MRI results were obtained from patients between 18 and 67 years of age, with a mean age of 37.44 years (SD: 12.34). Out of those, 53 (32.9%) were from women and 108 (67.1%) were from men.The mean width of the plica was set to be 3.00 mm SD: 1.39). The mean length of the plica was established at 2.91 mm (SD: 1.13). The mean width of the radiohumeral joint gap, measured at the widest point of the plica was set to be 4.63 mm (SD: 1.19). The mean width of the radiohumeral joint gap, measured at the middle of the radial articular surface was established at 2.56 mm (SD: 0.76). The mean width of the radiohumeral joint gap, measured at the medial end of the joint was set to be 3.89 mm (SD: 0.92). The mean distance from the edge of the radiohumeral joint to the plicas’ apex was established at 5.02 mm (SD: 1.55). The mean length of the radius articular surface in the radiohumeral joint was set to be 20.67 mm (SD: 2.32). The mean length of the ulnar articular surface in the ulnohumeral joint was established at 22.44 mm (SD: 2.90). The mean length of the common radio-ulnar articular surface in the proximal radio-ulnar joint was set to be 10.26 mm (SD: 1.77). All of the results mentioned above and more detailed ones can be found in Table .39. The p < 0.05). All the results and more detailed descriptions of sex differences are presented in Table An analysis of sexual dimorphism was also included. Regarding all categories mentioned in Table R = −0.16 ; p = 0.01). However, some categories correlate with each other. The associations between the categories are summarized in Table Potential correlations were analyzed for each of the categories and age. The only parameter that was statistically significantly correlated with age was the length of the edge of the radiohumeral joint . Similar conclusions appear after a comparison of our results with the results of the study by Luzuriaga et al. [p = 0.50). We did not compare the results of the current study with the results of Celikyay et al. [Our analysis of plicae in an asymptomatic group of individuals shows that the mean width  of the synovial fold is 3.00 mm (SD: 1.39). This would contradict the results of the study by Awaya et al. , where ta et al. . In theiy et al.  researchThe present study is the first to provide detailed measurements of the relations of the synovial plica with other anatomical structures of the elbow. We measured the distances from the lateral edge of the plicae to numerous structures, such as the point of bifurcation of the radial nerve and the extensors, mainly the extensor carpi radialis longus and extensor digitorum, among others. Furthermore, the contact area between the plica and the humerus and radius was also measured. In future research, these parameters should be measured in symptomatic patients because they could potentially provide physicians with more parameters for the diagnosis of symptomatic synovial plica syndrome. Physicians should also take into account synovial fold syndrome when diagnosing other lateral elbow conditions such as lateral epicondylitis (most commonly known as tennis elbow), oppression of the radial and/or posterior interosseous nerve, or snapping of the triceps tendon , 14. DifThe present study is the first to examine the potential differences between women and men in plica parameters and to establish potential correlations between these measurements and the age of the patients. It may be concluded that age should not necessarily be taken into account when examining the synovial fold syndrome, as none of the parameters, besides the length of the edge of the joint, statistically significantly correlated with age. However, the statistically significant difference between women and men was established in most of the categories. Therefore, when physicians compare their measurements with the results presented in our study, it is crucial to make the comparison with the same gender group as the patients’, rather than with the overall results. Furthermore, a comparison of symptomatic and asymptomatic plicae in future studies should also be performed with respect to the sex of the patients.This study is not without limitations. The authors did not compare the results obtained using magnetic resonance imaging with other imaging methods, such as ultrasound, which is used more frequently in daily clinical practice. Additionally, more measurements should be evaluated in further studies, in other positions of the elbow, as the plica may change some of its measurements while moving. The authors believe that a meta-analysis and/or more primary studies should be performed in the future to provide a homogeneous conclusion on plica thickness in the diagnosis of synovial fold syndrome.The synovial plica of the elbow is a clinically relevant anatomical structure. Analyzing the morphometric parameters of the synovial plica is necessary to properly evaluate synovial plica syndrome, which can commonly be confused with other sources of lateral elbow pain such as tennis elbow, oppression of the radial and/or posterior interosseous nerve, or snapping of the triceps tendon. The authors suggest that the thickness of the plica may not be the golden diagnostic feature as there are no statistically significant differences in this parameter between symptomatic and asymptomatic patients. The most detailed evaluation appears to be based on the measurements of plicas’ contact areas with the humerus and radius, width of the joint gaps, length of the edge of the joint and plicas length and width. Therefore, when physicians compare their measurements with the results presented in our study, it is crucial to make the comparison with the same gender group as the patients’, rather than with the overall results. A precise and accurate diagnosis of synovial fold syndrome and/or differentiation from other sources of lateral elbow pain must be performed, as the surgical treatment, even if performed properly, will be unsuccessful because of a misdiagnosed source of pain.


The increasing number of food frauds, mainly targeting high quality products, is a rising concern among producers and authorities appointed to food controls. Therefore, the development or implementation of methods to reveal frauds is desired. The genetic traceability of traditional or high-quality dairy products  represents a challenging issue due to the technical problems that arise. The aim of the study was to set up a genetic tool for the origin traceability of dairy products. We investigated the use of Short Tandem Repeats (STRs) to assign milk and cheese to the corresponding producer. Two farms were included in the study, and the blood of the cows, bulk milk, and derived cheese were sampled monthly for one year. Twenty STRs were selected and Polymerase Chain Reactions for each locus were carried out. The results showed that bulk milk and derived cheese express an STR profile composed of a subset of STRs of the lactating animals. A bioinformatics tool was used for the exclusion analysis. The study allowed the identification of a panel of 20 markers useful for the traceability of milk and cheeses, and its effectiveness in the traceability of dairy products obtained from small producers was demonstrated. The free movement of safe food is an essential aspect of the European Union’s (EU) internal market and trade and contributes significantly to the health and well-being of citizens and to their social and economic interests. According to EU law, food fraud is an intentional action for the purpose of deceiving consumers and obtaining a financial gain. Estimates on the economic impact of food fraud vary significantly among authors, from a cost of $10–15 billion to $40 billion, and others suggesting a value of at least $65 billion globally ,3. In thIn this context, an effective control system represents the best guarantee to protect the safety and the quality of production and contributes to safeguarding the economic value of products on the markets .Dairy products, particularly milk, rank among the top 10 food categories with the most reported instances of food fraud ,7,8. AmoDairy products also represent a high source of food-borne disease; therefore, dairy product fraud can have not only an economic impact but also implications on the health of consumers ,9. The tGiven the general interest and importance of the topic, new methods for the authentication of these products are needed ,10,11. AMolecular traceability methods are currently applied for the identification of fraud and traceability and authentication of food products ,9,10,11.While several molecular methods have already been developed for species/breed detection in dairy products ,20,21,22The genetic traceability of dairy products poses some technical challenges as cheese comes from bulk milk (BM), which contains DNA from different cows of the farm and from different lactation periods and undergoes consistent changes during manufacturing processes, such as ripening, that may compromise the quality and quantity of the extracted DNA, making it unsuitable for subsequent analyses .Currently, no studies in the literature have focused on the development of biomolecular methods which would be useful for attributing dairy products to the farm of production. The aim of this study was to set up a genetic tool for tracing the farm of origin. In particular, we investigated the use of microsatellites (STRs) to identify in the BM and in the derived cheese (CHE) a “DNA fingerprint” of the farm of origin which would be useful to assign dairy products to the corresponding producers.Although DNA pooling is a technique already proposed as a method to reduce analysis costs in quantitative and population genetic studies ,25,26,27Given the absence of similar studies and the difficulties related to the matrix, method, and interpretation of the results, we decided to conduct this study under controlled conditions, developing the protocol and evaluating its effectiveness in small farms.Results showed that BM and derived CHE express an STR profile composed of a subset of STRs of the lactating animals. A bioinformatics tool was used for the exclusion analysis. The study enabled the identification of a panel of 20 markers which would be useful for the traceability of milk and cheeses.Two farms were included in the study. The first farm, located in the province of Biella (BI) , was composed of 12 cows belonging to the Pezzata Rossa d’Oropa breed, and the second farm, located in the province of Imperia (IM) , hosted 14 Bruna Alpina breed cows. The two farms were chosen considering: (i) a low number of animals (between 10 and 20); (ii) the farmer’s openness to produce cheese with the BM of the sampling day, to store it separately from the rest of the production for 60 days, and finally to sell it for research purposes; (iii) localization in traditional cheese production areas where cheese traceability could be an issue.Samples were collected by official veterinarians of the Local Health Authority. Blood samples were collected once during the annual health testing of the animals, and milk samples of each cow (M) were collected once at the beginning of the study. BM and derived CHE were sampled monthly for one year. The producers made cheese with the BM produced on the sampling day and the cheese ripening period was 60 days.Blood samples were collected using Vacutainer tubes containing Ethylenediaminetetraacetic acid (EDTA) and stored at −20 °C until further analysis.g for 10 min (min) at 4 °C. The cell pellet was washed twice with PBS and centrifugation at 473× g for 2 min was performed after each washing. Then, the pellet was placed in a 1.5 mL Eppendorf and centrifugated at 21,250× g for 3 min. PBS was removed after the centrifugation, and the dry pellet was stored at −20 °C until DNA extraction.M and BM samples were processed for isolation of somatic cells immediately after they arrived in the lab (40 mL for each M and 50 mL for BM). Samples were centrifuged at 841× In collaboration with the Department of Veterinary Sciences and Technologies for Food Safety (VSA), University of Milan, a group of 62 STRs was selected from a panel of 280 already tested on Brown cattle  26]. Th. Th26]. TM Genomic DNA Mini Kit  was used for blood and somatic cells samples. For DNA extraction from CHE, the QIAamp Fast DNA Stool Mini Kit  was used with some modification: 200 mg of each CHE sample was aliquoted and homogenized by beads in lysis buffer using tissue grinding tubes and incubated at 70 °C for 5 min. The producer protocol of extraction was then used. The concentration of DNA extracted from all three matrices was checked using NanoDrop ND-1000 spectrophotometer  and samples were stored at −20 °C until use.Commercial DNA purification kits were used to isolate DNA from the different samples based on matrix type, according to the manufacturer’s instructions. Pure Link2, 0.4 mM dNTPs, 1 UI of Taq DNA polymerase , and 0.5 μM of each primer (Thermo Fisher Scientific). From 20 to 100 ng of DNA extracted from blood  was analyzed. A negative control containing no template was added at each run to ensure the absence of contaminations. The thermal cycling conditions were initial Taq activation step at 95 °C for 15 min followed by 45 cycles of denaturation at 95 °C for 30 s (sec), primer-specific annealing temperature for 45 s, extension at 72 °C for 1 min, followed by a final extension at 72 °C for 30 min. PCR products were displayed by electrophoresis on 2% (w/v) agarose gel. In order to verify their specificity, amplified DNA products with unexpected length were purified from agarose gel using IllustraTM, AutoSeqTM G-50 Dye Terminator removal kit , and sequenced by Big-Dye Terminator kit v.3.1 (Thermo Fisher Scientific), according to the manufacturer’s instructions. Capillary electrophoresis was performed using 3130 Genetic Analyzer (Thermo Fisher Scientific). The sequences obtained were compared with the sequences deposited in Genebank through BLAST software (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 18 August 2023)) using the blastn algorithm. A reduced panel of STR was selected according to the following parameters: (1) amplification and presence of polymorphism in the analyzed samples; (2) amplification length between 90 and 300 bp.For each STR, an end-point PCR was performed to evaluate the correct amplification of each locus. Each PCR was performed in a total volume of 25 μL containing 1X Q solution, 1X PCR buffer, 1.5 mM MgCl2, 0.4 mM dNTPs, 1 UI of Taq DNA polymerase , and 0.5 μM of each primer (Thermo Fisher Scientific). From 20 to 100 ng of template were analyzed. A negative control containing no template was added at each reaction to ensure the absence of contaminations. The amplification profile was carried out as follows: initial denaturation at 95 °C for 15 min, followed by 45 cycles at 95 °C for 30 s, primer-specific annealing temperature for 45 s, and extension at 72 °C for 1 min, followed by final extension at 72 °C for 30 min. PCR-amplified products were displayed by electrophoresis on 2% (w/v) agarose gel. Amplified DNA products with unexpected length were sequenced and analyzed as described in the previous paragraph. The sequences obtained were compared with the sequences deposited in Genebank through BLAST software using the blastn algorithm.The panel of 50 STRs selected in the first step was tested by end-point PCR on DNA samples extracted from six cheeses from both BI and IM farms. Each PCR reaction was performed in a total volume of 25 μL containing 1X Q solution, 1X PCR buffer, 1.5 mM MgClThe STRs successfully amplified in the two different matrix samples showing the variable length of amplification products among subjects were selected. The forward primers were labeled with the FAM or HEX fluorophore  in order2, 0.4 mM dNTPs, 0.5 μM of each primer , and 1 UI of Taq DNA polymerase . A control not containing DNA was amplified in each run to assess the absence of contaminations. The thermal cycling conditions were initial inactivation at 95 °C for 15 min, followed by 45 cycles at 95 °C for 30 s, annealing at 54 °C for 45 s and extension at 72 °C for 1 min, followed by final extension at 72 °C for 30 min. The annealing temperature was set at 56 °C for the reactions containing the STRs MB064_HBB and BMS4044. Each sample was tested three times.The selected STRs  were ampTM (Thermo Fisher Scientific) were used to assign the allele size (AS).The analysis of the amplification products was performed by capillary electrophoresis using ABI PRISM 3130xl Genetic Analyzer (Thermo Fisher Scientific) and POP-7TM separation polymer. GeneMapperTM v. 5 software and size standard ROX 500In order to guarantee the absence of conditioning in the assignment of AS, no information was given to the reader operator regarding the composition  of the GO (group of origin from each farm) during the evaluation of the BM and CHE samples. Some samples were performed several times to verify the attribution agreement of the AS. The AS was attributed only to alleles amplified with a high degree of uncertainty.Genotyping data were analyzed with FSTAT version 2.9.4 software to estimate and test the genetics parameters of the two GOs .To demonstrate the ability of the selected panel to trace the origin of BM and CHE, we decided to implement an algorithm able to verify the correlation between the fingerprinting of the product (BM or CHE) and the genetic characteristics of the GO. The algorithm was developed in collaboration with the Polytechnic of Turin; details can be found in Rossi et al. (2018) .vice versa). The threshold value according to which the correspondence between LM or FOR and the GO can be rejected may be low (restrictive approach) or high (more tolerant approach). The latter case can be applied, for example, for supposed errors in reporting the number and identity of the cows included in the GO.The developed algorithm verified how much the “fingerprinting” of the product (BM or CHE) is correlated with the genetic characteristics of the cows included in the group of production. Based on the profiles of each animal of the farm, the software created an ideal linear combination corresponding to the “fingerprinting” of the farm. It then compared this combination with the result of the BM or CHE analysis and attributed a correlation value, indicative of the actual origin of the sample from the candidate group of animals. In particular, the analysis was divided into two separate and complementary parts: the test step and the simulation step. In the first step, the presence of all BM and CHE alleles is checked in the GO alleles set and a “penalty score” is produced that represents an increasing value proportional to the number of unmatching between BM and/or CHE and the GO. In the simulation step, the evolutionary algorithm generates a linear combination of cows in the farm, which correlates as much as possible with the signature of BM and/or CHE. The more the penalty score tends to zero, the more an appropriate match between the dairy products and the cows’ group exists, and the simulation step could be considered reliable , it is possible to obtain a linear combination of cows to recreate the “signature” of the BM or CHE, according to a group of cows which have produced BM or CHE. The value obtained by the simulation process ranges from 0 to 1  and can be used as a further index to be added to the penalty score when attributing the origin of the product to the GO. The hypothetical score equal to zero and correlation equal to one is the best case of attribution.The genotyping data of each farm and the respective BM and CHE obtained using our STR panel were analyzed with the developed algorithm in order to verify the possible attribution and/or exclusion of the origin of the products. Then, a cross-analysis was performed by testing the correlation between the data of the dairy products of one farm and those of the GO signature of the other producer.Twenty-six EDTA blood and individual milk from the BI (n = 12) and IM (n = 14) farms and twenty-three samples of bulk milk (BI n = 12 IM = 11) were collected. All samples were uniquely identified and processed as described above and then stored at −20 °C until further analyses.Twenty-three cheese samples were collected: 11 from IM and 12 from BI farm. All cheese samples were aliquoted in a 2 mL tube and stored at −20 °C.The number of animals involved in the production of BM and CHE in each sampling varied from a minimum of four to a maximum of seven for the BI farm and from a minimum of nine to a maximum of fourteen for the IM ones.DNA was extracted from all collected samples. The quantity of extracted DNA from the milk samples resulted in quantities between 6.5 and 25.5 ng/µL with quality values, calculated on the basis of the 260/280 and 260/230 ratio, between 1 and 1.6 in the first case and 0.8 and 1.9, respectively. The DNA extracted from the cheese samples resulted in quantities between 3.5 and 19.5 ng/µL with quality values between 1 and 1.6 (260/280) and 0.2 and 0.5 (260/230).In the first phase, an efficient method for the genotyping of the individuals included in the study starting from DNA obtained by blood  was set up. Fifty STRs were initially selected according to the cited parameters. Markers showing no amplification or a single fragment in all samples analyzed were excluded. Detailed information on microsatellites  is shown in The STRs were analyzed by end-point PCR using BM and CHE samples (six different samples each) and the developed protocol. Markers showing no amplification in one or two different matrices were excluded in subsequent analyses.Among the analyzed STRs, BM0143 and INRA133 markers produced fragments with greater and shorter lengths than expected, respectively, in all samples. Sequencing analysis confirmed the markers’ specificity and the presence of microsatellites. The INRA133 marker produced an unspecific fragment amplification, shorter than 200 bp, that was not considered when genotyping the samples.Within the group of 50 STRs, 20 markers were selected . The forThe selected markers showed: (1) high peak definition and characteristic shadow bands in the electrophoretic trace; (2) absence of null alleles; (3) robustness of the amplification reaction; (4) size of the amplification product shorter than 300 bp; (5) high polymorphism.All blood samples from the two farms were genotyped and each analysis was replicated three times. IM and BI samples showed variable number of alleles, with some alleles shared and others specific to each farm .In order to verify the feasibility of the individual milk instead of the blood sample for the genotyping, seven samples from BI and nine samples from IM were randomly chosen and tested by six markers  with the developed protocol. Individual milk genotyping showed the same results obtained by blood analysis.All BM and CHE samples were tested with the 20 STR markers, and three replicates for each sample were set up. An allelic profile was not obtained in 24 out of 480 cases (5%) for BM and in 29 out of 460 cases (6.3%) for CHE samples.The markers with the highest number of non-genotyped samples (five each) were BM3507, INRA133, and BMS0607. The genotyping of all samples  detected a total of 109 and 119 alleles in the LM of the BI and IM farm, respectively. In the BI CHE and IM CHE samples, 119 and 122 alleles were detected, respectively . For BM The alleles assignment of the BM and CHE samples was carried out several times in separate sessions in order to verify its reproducibility, and consistent results were achieved in the different sessions. However, the loss of one of the longer alleles occasionally occurred.Not all alleles present in each GO were always detected in the corresponding dairy products, showing a loss of alleles between the genotyping of subjects included in GOs and that of BMs and CHEs. For five markers , the number detected in BM end/or CHE exceeds the number of alleles detected in the corresponding GO, which explains some “penalty score” values.The BM and CHE samples showed an STR profile represented by a subset of markers belonging to animals on lactation at the time of sampling (GO). Furthermore, the BM and CHE produced by distinct groups of animals in different months showed different STR profiles when all 20 markers analyzed were considered.In order to verify the effectiveness of the selected panel and the implemented algorithm in tracing the GO of dairy products, STR profiles of six BMs (three from BI and three from IM) and six CHEs (three from BI and three from IM) were submitted to the algorithm evaluation.The penalty score values were similar for both farms and ranged from 0.06 to 0.20 for BM and CHE of BI and from 0.00 to 0.29 for BM and CHE of IM. Correlation values range from 0.44 to 0.80 for the BI LM and FOR and from 0.44 to 0.75 for the IM samples.vice versa are reported in The values of correlation and penalty score obtained by comparing the BM and CHE produced by BI farm with the GO of IM and Improving food traceability is crucial in order to hinder food counterfeiting, to guarantee the quality and the safety of food products, and to safeguard risk categories .Chromatographic and molecular methods are the major approaches applied in the detection of food fraud . MoleculProtein-based, spectroscopy, and chromatographic techniques are also available for the detection of adulteration of dairy products ,14,15,30The traceability of dairy products with PDO or PGI labels has become an important issue for authentication because they have high commercial and nutritional value and the breeds and geographical origins from which these products are derived have been used as market symbols and have encouraged rural development and breed conservation . DNA-basDespite the several molecular methods available, fraudulent techniques continue to diversify and evolve, challenging current techniques for determining food authenticity; therefore, the development of more efficient and reliable molecular tools is necessary .Although primarily for financial gain, in many cases food fraud can have direct and/or indirect consequences on food safety . In thisProteome and metabolome have been used for finding an unequivocal and reliable fingerprint to correctly identify species/breed or the geographical origin of dairy products ,15.In the present study, we established for the first time a biomolecular method based on the analysis of a panel of microsatellites to identify in bulk milk and in derived cheeses a “DNA fingerprint” of small dairy farms and to assign dairy products to the corresponding producers. Two small Italian farms were involved and a blood and milk sample from each cow, bulk milk, and the deriving cheeses were collected during a period of 12 months. Due to the different organizations in the production method, sampling did not take place simultaneously for the two farms, but at the end of the sample collection period, the expected number was reached.DNA quality is one of the main factors influencing the dairy products’ authentication by molecular methods, being affected by the manufacturing process, extraction method employed, and chemical composition of the matrix . Among oThe extraction protocols adopted in our study were effective and suitable, showing that it is possible to obtain DNA of good quality and quantity for the analysis of microsatellites, even from difficult matrices such as high-fat containing milk and ripened cheeses. The difficulty in extracting DNA of good quality from ripened dairy products is known, and indeed, the extraction yield was higher for milk than for cheese. Ripening can reduce the integrity of DNA, thereby affecting DNA amplification detection ,22. RentTests performed on single milk samples to verify reproducibility and the possibility of replacing blood for individual typing gave positive results, although in three cases only one of the two alleles was amplified at the first run, probably due to the allelic dropout. Therefore, it is advisable to repeat the analysis at least three times (or more in case of doubtful results) when individual milk is used for the typing of subjects to limit the loss of genetic information.The difference in DNA quantity and purity could explain the slight differences in the number of alleles detected among the GO and the respective BM and CHE ,22,32. TAs predictable, not all the alleles present in each GO were detected in the corresponding dairy products, showing a loss of alleles between the GO and BM or CHE samples genotyping. This finding may depend both on the intrinsic characteristics of the genotyping by microsatellites  and on the features of BM and CHE matrices which influence the extraction efficiency and purity of the obtained DNA ,22,32. FNo qualitative differences in terms of reduction in the method efficiency and of genotyping results were observed in relation to the number of animals included in the GO on the basis of the lactation period of each cow (from a minimum of 4 to a maximum of 14). This allows us to state that the method of analysis developed in this study is not affected by the different numbers of animals involved, at least up to a maximum of 14 cows.Among the DNA markers studied for traceability purposes in livestock to differentiate breeds, STRs are widely studied but none have been implemented as a tool for breed differentiation or dairy product authentication . SardinaDespite their limited use for the traceability and authentication of foods of animal origin, STRs are widely used for agri-food traceability including varietal identification and adulteration detection due to their high reproducibility, polymorphism degree, possible standardization, and straightforward detection systems .Our results showed that the STRs profile detected in BM and CHE is composed of a subset of markers among those existing in lactating animals at the time of the collection. Furthermore, BM and CHE produced in different months showed different STRs profiles. In some cases, a difference in a few alleles among the GO, BM and CHE was observed; indeed, alleles not included in the corresponding GO have been occasionally detected in CHE and BM. Nevertheless, those alleles could be of cows hosted in the farm of origin (but not producing milk in the sampling period); therefore, we supposed a low level of contamination by equipment or instruments. Overall, the farm of IM and BI showed variable allelic lengths, with some shared alleles and others characteristic of each farm, which explain the Fst value . These dConsidering the two farms separately and applying the algorithm implemented by Rossi et al. (2018) , satisfaThe correlation values from 0.44 to 0.75 highlight the existence of a correlation between the GO signature and the fingerprinting of the corresponding BM and CHE products, although the data are also influenced by the presence of contaminated alleles.vice versa, highlights higher penalty score values (0.54–0.86), indicating little compatibility of the products with the GO. In this simulation, the correlation values are instead low (0.10–0.42), pointing out the absence of linear correlation between GO and dairy products and supporting an exclusion of the GOs as producers of the dairy products. In Rossi et al. (2018) [p < 0.05).The test performed by crossing the data obtained by BM and CHE of the IM GO with those of the BI farm, and . (2018) , we repoTherefore, our study developed a protocol of analysis with a selected panel of highly informative STRs and demonstrated the effectiveness of a tool combining the molecular analysis of those STRs markers and the informatic evaluation based on a heuristic algorithm for the genetic traceability of dairy products.The proposed protocol shows many advantages of DNA-based approaches for dairy products traceability: sensitivity, repeatability, and reliability ,15,16,22One of the problems related to DNA-based approaches for food authentication is the lack of procedure standardization and universality ,16. The Although the implemented method can be improved by entering as input data the relative quantities of each detected allele, it provides numerical values, namely penalty scores and correlation values, which are comparable and evaluable from a statistical point of view and useful for the traceability of the dairy products. In fact, these values could be useful mainly in assessing the probability that a traditional dairy product does not come from a production farm (exclusion approach) and therefore in providing a measure of the level of potential counterfeiting.Given the need to genotype all the cows in the GO, the method we present is not suitable for use in routine test, but it could be a helpful tool to verify suspected fraud, particularly for traditional dairy products produced by small farmers. Based on the results obtained, an applied study should be carried out on a larger scale, i.e., on a greater number of farms and/or farms with a greater number of lactating cows than that of the farms recruited in this project; the ultimate aim should be the availability of a tool which is useful to producers and control authorities both for product certification and for fighting against fraud.Food fraud has become a global problem, and prevention is increasingly the focus of producers and government authorities responsible for ensuring food quality and safety. Among the most adulterated foods are milk and dairy products, mainly due to the nutritional value of milk, the global demand, the reduced shelf life, and the lack of new methods for authenticating these products.Although there are several molecular methods for the detection of adulterant species and the authentication of the breed from which the foods are produced, there is still an unmet demand for techniques to trace dairy products back to the farm of origin.Here, we developed for the first time a molecular method, based on the analysis of a panel of microsatellites, to assign milk and cheese to the corresponding producers in the context of food traceability and counter-fraud measures.Our study allowed the development of a DNA extraction and amplification protocol that can be applied to high-fat and processed food. We identified a panel of 20 highly informative markers and demonstrated that dairy products expressed an STRs profile composed of a subgroup of STRs belonging to the animals from which the dairy product originated.The panel of STRs and the implemented algorithm proved their usefulness in the traceability of dairy products obtained from small producers by the assessment of the correlation between the genetic fingerprinting of the product (BM or CHE) and the genetic characteristics of the GO.Considering the constant demand for the development and improvement of analytical methods, our study represents an innovative approach, although improvable, which becomes part of the range of techniques that can be used to prevent food fraud.


The risk transmission process between international construction projects largely contributes to the dilemma of risk management of international construction projects. Firstly, this paper adopts methods such as literature review and brainstorming to identify the risks in international construction projects from all aspects and all stages. Connections between risks is built by the Delphi method and further construct the international construction project risk network. Combined with “ucinet”, a network visualization analysis tool, overall feature parameters and local feature parameters are presented for analysis as the focus. Starting from this, the risk transmission in complex construction projects is analyzed to identify key risks and transmission relationships and reveal inherent laws of risk transmission. Accordingly, when formulating risk prevention strategies for international engineering projects, it is proposed that measures to curb risk transmission should be effectively adopted from both key risks and their transmission relationships. International construction projects are complex and highly integrated projects with long cycles and high inputs. They are closely linked to people’s livelihoods and are of great significance to the national economy and social development . Risks The risk transmission in international construction projects is essentially the transmission path and process between risk nodes. Complex network analysis has been widely used in risk management . InternComplexity is one of the most important and basic features of international construction projects. The complexity of international construction projects is determined by the differences and dependencies of many interacting components . The coProject risks are uncertain events or conditions that may arise during the implementation of a project, and will exert a positive or negative impact on the project’s original objectives when occurring , includHowever, most of the views are that international construction project risks will have a negative impact on the project . As intRisk assessment is a basic method of risk management for international construction projects. Risk assessment for international construction projects includes expert experience and qualitative risk analysis based on subjective judgment , trade In addition, many scholars have adopted the complex network theory to address problems in the field of project management. Based on analyses of industrial project construction, the chain of construction, agent relationships and agent networks, Han.et al. (2021)  establisThe international construction project is a strong national tool to enhance a country’s international competitiveness, and the most powerful “material” used to tell Chinese stories in a more appealing way and shape China’s international image. Meanwhile, international construction projects usually have a span of time and space, a large investment scale, and a far-reaching impact on the host country’s society and economy. While once the risks of international construction projects occur, negative effects of varying degrees can be brought about, and the ensuing governance pain points can affect the social, economic, and environmental development of the host and home countries over a long period of time and space. Therefore, the correlation between risks needs to be analyzed on the basis of the identification of risk elements in international construction projects, and the risk transmission mechanism needs to be clarified through the network nature of the international construction project organization. Therefore, this study combines the complex network theory with the risk transmission in international construction projects to further investigate the nature of nodes and edges in complex networks and their interactions and reveal the risk transmission mechanism in the whole life cycle stages of international construction projects.The international construction project is essentially an open, dynamic, and adaptive system where various components influence each other and with a high degree of uncertainty. It is often faced with a more complex risk system than domestic construction projects. Existing studies have attempted to isolate individual risks from the project risk system and identify the categories and risk sources to which each risk belongs but have failed to adequately consider the impact of the interaction between international construction project risks on risk management. International construction project risks are interrelated and influence each other, which is the root cause of the formation of international construction project risks. Due to the dynamic nature of international construction risks, it is difficult to find a precise boundary to distinguish the impact of individual risks. According to the analysis of literature review, complex network analysis can break through the traditional risk transmission relationship analysis of international construction projects. Therefore, this study uses complex networks, combined with the whole life cycle theory to describe the net-like interaction relationship among risks of international construction projects and analyze the risk transmission mechanism of international construction projects and othThe informed consent of all experts participating in the questionnaire was obtained by telephone prior to the publication of this study.Ri represents the risk element number, and the one-way arrow line indicates there is a causal relationship between the risk elements.Visualization of the international construction project risk network matrix can visually reveal the relationship between stakeholders and risk factors in the international construction project risk network. At present, the relatively common complex network visualization software includes Ucinet, Pajek, NetMiner, etc. Therefore, this paper uses Ucinet to analyze the risk network in different life cycle stages of international construction projects, where There are 26 nodes and 103 relationships in the risk factor impact network of international construction projects ; block 2 and block 8 are located in the sending position (only having sending relationships); and block 7 is located in the receiving position (only having receiving relationships). The blocks at the core of the block model theory should have both a receiving and a sending relationship; therefore, block 1, block 3, block 4, block 5 and block 6 in the transmission position have the prerequisites to be at the core of the block. According to further analysis, block 3 mainly assumes the role of receiving which influences the relationship, and there is only one sending relationship being passed to block 7, which only takes on a receiving role and does not play a sending role and causes the relationship not to be transmitted. And therefore, it can be judged that block 3 is not in a core position. Block 1, block 4, block 5, and block 6 are therefore at the core position of the risk network and are the core blocks of the whole life cycle risk network for international construction projects.In complex networks, local parameters mainly include betweenness centrality analysis, intermediary analysis, and closeness centrality. Among them, the betweenness centrality analysis and intermediary analysis can judge the key risks in the complex network of international construction projects. While the betweenness centrality analysis of the line can identify the relationship between key risks. This study describes the individual network characteristics of the whole life cycle risks of international construction projects through these two parameters.(1) Intermediary analysisA influences node C through node B. When three nodes are in different groups, node B will play a different role. Node B assumes the coordinator role when nodes A, B, and C are in the same group; and node B takes on the gatekeeper role when node A is in one group and nodes B and C are in another; when nodes A and B are in one group and node C is in another, node B takes on the role of the agent; when nodes A and C are in one group and node B is in another, node B takes on the role of advisor; when nodes A, B, and C are in separate groups, node B takes on the role of liaison. The intermediary roles in the risk network for international construction projects are derived from the “Ucinet” analysis, which can be seen in R3 , R19 , R13 (management risk), R14 (contract risk), R15 , R24 (market risk), and R21  are the core risks of the whole life cycle risk network of international construction projects, and these 7 categories of risks together bear 55% of the intermediaries.In the social network analysis, intermediaries are generally classified according to Gould and Fernandez’s theory, which includes five roles: coordinator, gatekeeper, agent, advisor, and liaison. Suppose that in a social network, node (2) Betweenness centrality analysis of the nodeR3 , R19 , R15 , R14 (contract risk), R13 (management risk), R24 (market risk), and R18 (economic risk) are the core risks of the whole life cycle risk network of China’s overseas water conservancy and hydropower projects. And there is one difference between these 7 core risks and the results of the intermediary analysis.The betweenness centrality of the node refers to the probability of the node falling between other nodes, and the probability indicates the degree of the node playing a bridging role in the network. The higher the betweenness centrality of the node, the greater the probability, and the greater the bridging role, indicating that the node is at the core position of a complex network. In the whole life cycle risk network of international engineering projects, if a node is on more than one transmission path and controlling this node can help control multiple paths so as to curb the risk transmission, then the risk will be considered to be at the core position. The betweenness centrality of nodes of the whole life cycle risk network of international construction projects is calculated by “Ucinet”, which can be seen in (3) Betweenness centrality analysis of the lineThe betweenness centrality of the line refers to the probability of the line falling between other lines, and the probability indicates how much control the relationship line has over the whole network. The higher the betweenness centrality of the line, the greater the probability and the stronger the controlling role, indicating that the relationship line is at the core position of the social network. In the whole life cycle risk network of China’s overseas water conservancy and hydropower projects, if the betweenness centrality of a relationship line is higher, the more the relationship line transmission should be controlled to curb the risk spreading. The betweenness centrality of lines of the whole life cycle risk network of international construction projects is calculated by using “Ucinet”. Due to the large number of risk nodes, the complexity of the relationship lines, and the length of the calculated results, they are not shown here and can be asked from the author if any reader needs them.By combining the previous analyses of overall network characteristics and individual network characteristics, key risk factors for the whole life cycle of international construction projects can be identified as follows:R3 , R19 , R13 (management risk), R14 (contract risk), R15 , R24 (market risk), and R21  together bear 55% of the intermediaries, representing the core risks of the whole life cycle risk network of international construction projects. The result of the betweenness centrality analysis of the nodes shows that the top 7 risks in the betweenness centrality among 26 risks are R3 , R19 , R15 , R14 (contract risk), R13 (management risk), R24 (market risk), and R18 (economic risk), indicating that these 7 types of risks have a greater probability of being in between other nodes and play a greater bridging role. And they are the core risks of the whole life cycle risk network of China’s overseas water conservancy and hydropower projects.Firstly, the result of the intermediary analysis describing the individual network characteristics is that the top 7 risks in the total number of intermediaries among 26 risks are the core individual risks. R3 , R19 , R13 (management risk), R14 (contract risk), R15 , R24 (market risk), R21 , and R18 (economic risk).Therefore, according to the individual network characteristics, the core risks of the whole life cycle risk network of China’s overseas water conservancy and hydropower projects include R15, R3, R19), block 4 , block 5 , and block 6  are in the core position of the network risks and are core blocks of the whole life cycle risk network of international construction projects. Among the core risk factors, R3 , R19 , and R15  are located in core block 1; R24 (market risk), R21 , and R18 (economic risk) are located in core block 4; and R13 (management risk) and R14 (contract risk) are located in core block 6.Secondly, the overall network characteristics show that block 1 , R19 , R13 (management risk), R14 (contract risk), R15 , R24 (market risk), R21 , and R18 (economic risk) are the key risk factors of the whole life cycle risk network of international construction projects.In summary, 8 categories of the key risk factors for the whole life cycle risk network of international construction projects are obtained through the identification of key risk factors above. But this only shows that these 8 categories of risks should be paid high attention to and does not reveal how the key risks of the whole life cycle risk of international construction projects are transmitted. In addition to the key factors of risk network nodes, the relationships of the overall risk network (the connections between nodes) also play an important role in risk transmission. The calculation of the betweenness centrality of the lines in a risk network can assess objectively the extent to which a relationship is located in other relationships and identify the relationships that are highly communicative in the network so as to improve the objectivity and comprehensiveness of the identification of risk network characteristics. This can further improve the efficiency of risk management and block the transmission of some risks throughout the network powerfully.R3→R19, R3→R22, R3→R2, R19→R15, R19→R24, R13→R14, R13→R19, R21→R18, R3→R1, and R13→R22 are the 10 relationships with the greatest betweenness centrality of the lines, i.e., they are located with a high probability in other relationship lines and with 5strong control over the overall risk network transmission, and they are the key relationships for the whole life cycle risk transmission of international construction projects. Besides, the nodes corresponding to the relationship lines are closely related to the key risk factors R3 , R19 , R13 (management risk), R14 (contract risk), R15 , R24 (market risk), R21  and R18 (economic risk) identified previously. Namely, if the above key risk factors can be effectively managed, their closely related key relationships may not be considered separately.Through the analysis of the calculated results of the betweenness centrality of the lines of the whole life cycle risk network of international construction projects, it can be found that there are 103 lines with betweenness centrality larger than 0. Due to the limited space, only the top 10 relationships are taken and included in This study builds a risk-related network model for the whole life cycle of international construction projects from the perspective of the network and accurately captures the key risks throughout the construction process of international construction projects through methods such as parametric analysis and visual model. After a combination of the above results, it can be found that governmental institutions, owners, and contractors are the major sources of risks in international construction projects in terms of the subject of risk generation. In terms of the stages of risk generation, the key risks in the international construction project risk network are mainly found in the construction and implementation stage and the operation and maintenance stage throughout the project life cycle. These two stages are the most critical stages in the project life cycle, with the longest time span, the largest number of participants and the strongest interactions among different parties. Therefore, the number of risk factors increases exponentially during these stages and interactions between various nodes enhance obviously, with the emergence of risk factors. Through the analyses above, it can be found that the management risk, contract risk, market risk, and financial and tax risk are all important risks emerging in the construction stage, and there is an interaction between these network nodes and many other neighboring risk nodes. Therefore, it plays a significant role in risk transmission. Besides, social risk in the investment and decision stage investment decision stage is also important transmission node that cannot be neglected, indicating that the risks in other stages of the whole life cycle of international construction projects will lead to the occurrence of this risk, and it will also react to other risks. And if such important node is removed, the connectivity and tightness of the whole risk network will be greatly reduced. Therefore, it is an important part of the risk control work of international construction projects to find ways and means to prevent risks from the key nodes of the risk network.There are certain shortcomings in the research of this paper: this paper uses Delphi method to determine the correlation between risk factors in the whole life cycle of international engineering projects, but Delphi method may lead to unreliability of the research results due to the defects such as incomplete expertise of experts, and we will further optimize the research method in the subsequent research to compensate for the defects of this paper.S1 File(XLSX)Click here for additional data file.


Generally, the dynamic modulus and bending stiffness modulus are used to evaluate the mechanical properties of asphalt mixture, and they are also used as basic parameters for asphalt mixture design. Therefore, a study was conducted on changes in the dynamic modulus and bending stiffness modulus of hot-recycled asphalt mixture with high levels of reclaimed asphalt pavement (RAP) under the influence of different factors: dosage of regenerant, curing temperature, and curing time. The performance of reclaimed asphalt pavement (RAP) was first evaluated. Then, the hot-recycled asphalt mixture was adjusted and designed in order to conduct modulus experiments, composed of the dynamic modulus test and three-point bending test. Finally, the influencing factors were not only qualitatively but also quantitatively analyzed to clarify the change laws of the mechanical parameters of hot-recycled asphalt mixture. The results showed that the modulus of the recycled asphalt mixture first decreased, then increased, and then decreased with increasing dosage of regenerant. As the curing time or temperature increased, the modulus first decreased and then increased. In terms of the dynamic modulus of the hot-recycled asphalt mixture, the curing time had the greatest impact, followed by dosage of the regenerant and curing temperature. For bending stiffness modulus, the influence of dosage of the regenerant was the greatest, followed by curing time and curing temperature. For the bending stiffness modulus of hot-recycled asphalt mixture, the curing conditions had a greater influence compared with the dynamic modulus. Asphalt pavement is widely used in high-grade highways due to its significant advantages such as comfortable driving, easy maintenance, and good smoothness. However, under cyclic vehicle loads and long-term exposure to harsh natural environments, asphalt pavement deteriorates and develops distress such as rutting, cracks, and potholes. During the maintenance and repair of asphalt pavement, large quantities of reclaimed asphalt pavement are produced. RAP has great recycling value because it contains non-renewable resources such as aged asphalt and aggregates. Its application in recycled asphalt mixture  has obvious economic and social benefits, and the performance of asphalt mixture is guaranteed ,2,3,4. RWhen the proportion of RAP in the recycled mixture is high, it is necessary to add a regenerant to reactivate aged asphalt and restore its performance . The degAsphalt mixtures are road construction materials with viscoelastic properties, and evaluation of their mechanical performance has always been a hot topic in the field of road engineering research ,26,27. TThe regenerant employed in this study was self-made. The related properties are recorded in The RAP came from the Changchun–Siping section of the Changyu highway, which had been in service for nearly 15 years. The pavement structure of this section of road consisted of upper, middle, and lower layers. The upper layer was 4 cm modified SMA-16, the middle layer was 5 cm AC-20, and the lower layer was 6 cm AC-25. JTG/T F41-2017 requires that RAP used for recycled mixture has appropriate gradation and strength. Therefore, it was necessary to undertake performance testing to determine whether the milled recycled old material met the corresponding requirements.Normally, the grading design of hot-recycled asphalt mixture is determined based on RAP gradation. However, for the original pavement asphalt mixture, after a long service time, part of the coarse aggregate was refined under the effects of the environment and vehicle load. Therefore, it was necessary to screen the recycled material, determine the gradation of RAP, and then perform the grading design of the hot-recycled asphalt mixture.An asphalt mixture extractor was used to extract the recycled material with trichloroethylene as the solvent. The extracted liquid was distilled using a rotary evaporator for asphalt recovery, and the asphalt content in the RAP was calculated to be 4.2%. The passing rates of each grade aggregate before and after extraction were used to prepare a grading curve, as shown in The penetration, 60 °C dynamic viscosity, softening point, and 15 °C ductility of the recycled asphalt were measured in accordance with JTG/T F41-2017, and the results are shown in According to the indicators of RAP performance, hot-recycled asphalt mixture of AC-16 is designed to contain 80% RAP, 14% fine aggregates with particle sizes of 0–3 mm, and 6% coarse aggregates with particle sizes greater than 13.2. The grading curve was prepared based on this grading to meet the required upper and lower limits, from JTG F40-2004, as shown in The optimum asphalt content of the recycled asphalt mixture was determined to be 4.9%. The Marshall results conducted on the basis of JTG E20-2011 are displayed in Based on the principle of a single variable, 15 groups of samples were set with the dosage of regenerant, curing time, and curing temperature as shown in To eliminate the influence of boundary effects on the experiment, a Superpave gyratory compactor (SGC) was employed to prepare the samples for the dynamic modulus test, and different regenerants were added according to the designed grading. The molded specimens were φ 150 mm × 170 mm (high) cylinders, and then the specimens were cured in the environment described in The uniaxial compression dynamic modulus testing of the asphalt mixture was conducted using a universal testing machine (UTM-100) under unconfined conditions according to JTG E20-2011. An offset sine wave axial compressive stress was applied to the specimen at a certain loading frequency, in a selected temperature environment. The recoverable axial strain of the specimen was obtained, the dynamic modulus and phase angle of the asphalt mixture were calculated, and then the mechanical properties of the asphalt mixture were evaluated. The temperatures selected for the experiment were −10 °C, 5 °C, 20 °C, 35 °C, and 50 °C. The loading frequencies of the compressive stress were 0.1 Hz, 0.5 Hz, 1 Hz, 5 Hz, 10 Hz, and 25 Hz.Based on the time–temperature equivalence principle, the generalized modified sigmoidal model and the Christensen–Anderson–Marasteanu (CAM) model, the master dynamic modulus curve and the phase angle master curve of the hot-recycled asphalt mixture were fitted. The trend and relative position of each master curve were observed, and the effects of the regenerant dosage and curing conditions on the dynamic modulus of the hot-recycled asphalt mixture were summarized.At a specific temperature and loading rate, the three-point bending test can be used to determine the maximum load and mid-span deflection of a small beam when it is damaged and then to obtain the bending stiffness modulus of the beam. Based on JTG E20-2011, this test was used to evaluate the low-temperature tensile performance of the asphalt mixture, which is a mechanical parameter used in mixture design. At −20 °C, a small beam prepared from hot-recycled asphalt mixture was loaded at a rate of 50 mm/min by UTM-100 until the small beam was damaged.The master curves of the uniaxial compression dynamic modulus parameters of the hot-recycled asphalt mixture with 0%, 5%, and 10% regenerant dosages are shown in (1)The dynamic moduli of hot-recycled asphalt mixture with three dosages of regenerant followed the same trend, and the trend of the values was 0%, 10%, and 5% successively. Thus, increasing the amount of regenerant increased the viscosity of the recycled asphalt, but the dynamic modulus of the hot-recycled asphalt mixture first decreased and then increased with increasing proportions in the mixture.(2)The regenerant had little effect on the phase angle of the hot-recycled asphalt mixture, and there was some scatter in the curves of the phase angle. However, within the frequency range of the test (0.1–25 Hz), the phase angle of the hot-recycled asphalt mixture was slightly less for 5% than 10% regenerant, and the trend of the dynamic modulus was similar.The following conclusions were obtained by analyzing the relative positions of the curves in the diagram:In a scatter diagram of data from the three-point bending tests of small beams prepared from hot-recycled asphalt mixture with different dosages of regenerant, the stiffness moduli changed with curing conditions, as shown in Comparing the relative positions of each point for different curing conditions showed that the bending stiffness modulus followed the same trend for hot-recycled asphalt mixture with 5% and 10% regenerant dosages, and the values of the bending stiffness moduli of the temperature test group were not significantly different. If the minimum values only of the bending stiffness modulus were compared, then the bending stiffness modulus was slightly greater for hot-recycled asphalt mixture with 5% than 10% regenerant dosage.The curves of the uniaxial compression dynamic modulus of the hot-recycled asphalt mixture with regenerant dosages of 10% and 5% and cured at 60 °C, 100 °C, and 135 °C for 4 h are shown in (1)With a regeneration time of four hours, the dynamic modulus of hot-recycled asphalt mixtures with 5% and 10% rejuvenator dosage was similar under different regeneration temperatures. The dynamic modulus was lower at 60 °C compared with 100 °C. At a regeneration temperature of 135 °C, the dynamic modulus of hot-recycled asphalt mixtures was the highest, approaching the value of asphalt mixture with no added rejuvenator. For hot-recycled asphalt mixtures with 10% rejuvenator dosage, the dynamic modulus at a regeneration temperature of 100 °C was slightly higher than at 135 °C.(2)When the dosage of the rejuvenator was 5%, the dynamic modulus value of the hot-recycled asphalt mixture at 135 °C for three hours was almost the same as the dynamic modulus value of the hot-recycled asphalt mixture at 100 °C for four hours. When the dosage of the rejuvenator was 10%, the dynamic modulus value of the hot-recycled asphalt mixture at 135 °C for three hours was similar to the dynamic modulus value of the hot-recycled asphalt mixture at 60 °C for four hours. Therefore, when the regeneration temperature varies, adjusting the regeneration time can achieve a similar modulus for the hot-recycled asphalt mixture.(3)The results of the dynamic modulus under the two rejuvenator dosages indicate that the larger the dosage of the rejuvenator, the more significant was the effect of the regeneration temperature on the dynamic modulus of the hot-recycled asphalt mixture.After analyzing the relative positions of the primary dynamic modulus curves in the figure, the following conclusions can be drawn:The scatter plot of three-point bending test data for small beams in Comparing the relative values of the bending stiffness modulus of the hot-recycled asphalt mixture under different curing temperatures in the figure shows that when the curing time was 4 h, the trends of the bending stiffness modulus for 5% and 8% regenerant were consistent and gradually increased with increasing curing temperature. At the three curing temperatures selected in the experiment, as the curing temperature increased, the bending stiffness modulus of the hot-recycled asphalt mixture continuously increased. That is, within this curing time and temperature range, as the curing temperature increased, the bending stiffness modulus of the hot-recycled asphalt mixture first decreased and then increased.Graphs of the uniaxial compression dynamic modulus for hot-recycled asphalt mixture cured at 135 °C for 1 h, 2 h, 3 h, and 4 h for regenerant dosages of 10% and 5% are shown in (1)When the curing temperature was 135 °C, the dynamic modulus of the hot-recycled asphalt mixture with a 10% regenerant dosage decreased with curing for 4 h, 1 h, 3 h, and 2 h.(2)The recovery of the dynamic modulus recovery was generally consistent for hot-recycled asphalt mixture with 5% and 10% regenerant, but the dynamic modulus was higher for specimens cured for 2 h than other lengths of time.(3)According to the values of the dynamic modulus for two types of regenerant dosages, the larger the regenerant dosage, the more significant was the effect of curing time on the dynamic modulus of the hot-recycled asphalt mixture.By analyzing the relative positions on each graph, the following conclusions were obtained:According to the three-point bending test data for the small beams, the scatter plot in Overall, with increasing regenerant dosage, the modulus of the hot-recycled asphalt mixture first decreased, then increased, and then decreased. As the curing time or temperature increased, the modulus first decreased and then increased, as shown in (1)The dynamic modulus and bending stiffness modulus data of the hot-recycled asphalt mixture showed that the dynamic modulus was indeed slightly lower for the 10% than the 5% dosage. Although there was no peak, it satisfied the trend of the modulus curve.(2)The dynamic modulus of the hot-recycled asphalt mixture with 10% regenerant was slightly larger than that of the control group without the addition of recycled agent, for a curing temperature of 100 °C. However, the dynamic modulus of the hot-recycled asphalt mixture with both types of regenerant dosages reached its minimum at a curing temperature of 60 °C and then increased with increasing curing temperature, which was basically consistent with the overall trend.(3)Although the dynamic modulus of the hot-recycled asphalt mixture with the 5% regenerant dosage reached its maximum value after 2 h of curing, its overall trend of change followed the basic trend. The dynamic modulus of the hot-recycled asphalt mixture with 10% regenerant first decreased and then increased, with a minimum value after 2 h of curing, which was consistent with the basic trend.(4)Comparing the minimum bending stiffness modulus of hot-recycled asphalt mixture with different regenerant dosages showed that for the recovery degree of low-temperature performance, the performance was slightly better for hot-recycled asphalt mixture with 5% than 10% regenerant, which was consistent with the changes in basic behavior.(5)When the curing time was 4 h, as the curing temperature increased, the bending stiffness modulus of the hot-recycled asphalt mixture showed an upwards trend. Although it deviated from the basic trend of first decreasing and then increasing, it also satisfied the latter half of the trend.(6)When the curing temperature was 135 °C for the hot-recycled asphalt mixture with 10% regenerant, the bending stiffness modulus was lower for the asphalt cured for 4 h than 3 h. However, when the curing time was within 3 h, the bending stiffness modulus of the hot-recycled asphalt mixture with two types of regenerant dosages first decreased and then increased, and the curing temperature was the lowest at 2 h. Therefore, the trend in the bending stiffness modulus within 3 h of curing was consistent with the basic trend, which first decreased and then increased.To more intuitively observe the changes in the dynamic modulus of the hot-recycled asphalt mixture under different curing conditions, the average change ratio of the dynamic modulus was summarized. This ratio compared the hot-recycled asphalt mixture under different curing conditions to that without curing, as shown in When the curing time was 4 h, the dynamic moduli of the hot-recycled asphalt mixture with two types of regenerant dosages were basically the same as those under the curing environments of 100 °C and 135 °C. Therefore, the dosage of regenerant mainly affected the dynamic modulus of the recycled mixture cured at low temperature. The scatter plot of the change rate of dynamic modulus values with curing time when the curing temperature was 135 °C is shown in In Based on the data in the table, a scatter plot of the change rate of the dynamic modulus values with changes in curing conditions is shown in The scatter plot of the change ratio of the dynamic modulus with 5% recycled agent is shown in To further evaluate mechanical parameters of hot-recycled asphalt mixture with RAP, the grey correlation analysis for the correlation degree of influential factors was calculated. This method is always applied to reveal the change among data sequences ,31,32. TThe matrix for influencing factors can be described as follows:Then, a non-dimension matrix can be derived from the normalization:ρ is the recognition coefficient, defined as 0.637. Finally, the grey correlation coefficient is calculated as:The correlation coefficient can then be calculated:The results of the grey correlation analysis are depicted in Based on the uniaxial compression dynamic modulus test and the three-point bending test on the small beams, the results of this study summarize the influence of the regenerant dosage, curing temperature, and curing time on the dynamic modulus and bending stiffness modulus of the hot-recycled asphalt mixture with high RAP.(1)In the design of hot-recycled asphalt mixture, mixtures with similar responses can be obtained by changing the dosage of regenerant, curing time, curing temperature, and other parameters.(2)For the dynamic modulus of the hot-recycled asphalt mixture, the influence of the curing time was the largest, followed by the regenerant dosage, and finally the curing temperature. For the bending stiffness modulus, the regenerant dosage had the greatest influence, followed by the curing time, and then the curing temperature.(3)The influence of each factor was greater for the bending stiffness modulus than the dynamic modulus of the hot-recycled asphalt mixture, and the modulus change rate of the former was approximately 1.5 times that of the latter.