Galaxea fascicularis holobiont from nearshore and offshore reefs. These nearshore reefs were more turbid, eutrophic, and warm than offshore reefs. However, coral physiological parameters did not differ significantly. Corals under stressful nearshore environments had low symbiont diversity and selected more tolerant Symbiodiniaceae. The bacterial diversity of offshore corals was significantly higher, and their community composition varied obviously. Diffusion limitations and environmental heterogeneity were essential in structuring microbial communities. Functional annotation analysis demonstrated significant differences between nearshore and offshore corals in bacterial functional groups. Environmental stress significantly reduced the complexity and connectivity of bacterial networks, and the abundances of keystone taxa altered considerably. These results indicated that corals could thrive nearshore through holobiont plasticity to cope with multiple environmental stresses.Global change and local stressors are simultaneously affecting the nearshore corals, and microbiome flexibility may assist corals in thriving under such multiple stressors. Here, we investigated the effects of various environmental variables on  Reef-building stony corals are the basis for the formation of coral reef ecosystems, often referred to as “holobionts” consisting of animal hosts, Symbiodiniaceae, as well as other microbial partners, including bacteria, archaea, fungi, and viruses . CollaboHowever, multiple stressors associated with climate change, overfishing, and overexploitation may lower the resilience of corals to environmental changes in coastal ecosystems . RecentlGalaxea fascicularis is widely distributed in the Indo-Pacific region, and it is an essential ecological and dominant species in the SCS  sites to investigate microbial communities (Symbiodiniaceae and bacteria) and explore their relationship with environmental factors.Coral reefs in the South China Sea (SCS) are distributed in areas ranging from nearshore coast to uninhabited, remote regions . Therefo the SCS . Due to icularis . Meanwhicompared . To bettHainan Island is located on the tropical northern edge of the Indo-Pacific Ocean in the SCS  and biomass (mg/cm2) per unit of coral surface area.The coral surface was rinsed with a Water-Pik containing seawater (filtered through a 0.45-μm filter) until only the white coral skeleton remained. The volume of the rinsed solution was measured in a measuring cylinder, then 10 ml of rinsed solution was measured, and the number of zooxanthellae was counted and recorded under a microscope using a blood cell counter  and ITS2 reverse (5′-GGGATCCATATGCTAAGTTCAGCGGGT-3′) were used to for amplifying the ITS variable region of Symbiodiniaceae rRNA gene . The bacThe high-quality reads were clustered into operational taxonomic units (OTUs) at 97% identification. BLASTn was used to select the most abundant OTU sequences as representative sequences for comparison with the ITS2 database , and nont-test to determine the spatial differences between the two regions. The Shannon index of Symbiodiniaceae and bacteria was calculated in the “vegan” package. After testing the data normality and homoscedasticity of variances, the difference in diversity index between the two groups was compared by t-test (p < 0.05). Non-metric multidimensional scaling (NMDS) and analysis of similarity (ANOSIM) based on the Bray–Curtis dissimilarity index were performed to investigate the patterns of microbial community structure. Community similarity (1- dissimilarity of the Bray–Curtis distance) and log-transformed data of geographic distance were used to study the distance attenuation relationship (DDR). Based on the results of the detrended correspondence analysis (DCA), Redundancy analysis (RDA) was used to determine the association between the communities and environmental factors . Finally, the “igraph” package and Gephi  were used to build and visualize co-occurrence networks . Among them, the mean temperature of seawater at the nearshore station (HN) was 31.47 ± 0.12°C, while the mean temperature at the offshore station (XS) was 29.73 ± 0.15°C (in situ of HN (32.66 ± 0.06‰) was significantly lower compared with XS . The mean values of pH, turbidity, ammonium, and nitrite at HN were significantly higher compared with XS . For dissolved oxygen, nitrate, and phosphate contents, there was no significant difference between the two sea areas. In general, compared with the offshore coral reef sites in the Xisha Islands, the temperature and water quality of the offshore coral reefs in northwest Hainan were higher. It was worth noting that the symbiont density (2.27 ± 0.23 ·106 cells/cm2) in the healthy state collected at the HN station was slightly higher compared with the XS station  was slightly lower compared with XS (8.11 ± 0.76 mg/cm2). This was interesting because there was no significant difference between the two in different sea areas.The PCA results and permutational multivariate analysis of variance (PERMANOVA) showed that the environmental conditions of the northwest coast of Hainan Island and the Xisha Islands were best differentiated . Using the RDA of Symbiodiniaceae community composition and the screened important influencing factors , the two axis pairs could explain 41.79% of the overall variation in the Symbiodiniaceae composition . After quality control, 465,889 sequences belonging to the Symbiodiniaceae family were identified, and these high-quality sequences were clustered to form 25 Symbiodiniaceae OTUs. The Shannon index of Symbiodiniaceae was lower in HN (0.36 ± 0.04) compared with XS (0.55 ± 0.15), while there was no significant difference . NMDS anposition . The resAt the family level , the bacBased on FAPROTAX functional database, bacterial functions of different stations were identified. PCA results showed significant differences in microbial functional characteristics between HN and XS samples . In genet = 6.35, p < 0.001; 2 = 0.35, p < 0.001; p = 0.001). The bacterial Shannon diversity index was significantly decreased with increasing temperature, turbidity, and ammonium content but increased with increasing salinity . The RDA results showed that explanatory variables accounted for 58.2% of the total variation, among which salinity (11.66%), temperature (9.15%), ammonium (8.69%), turbidity (7.25%), nitrite (3.68%), and pH (3.24%) could significantly affect the coral bacterial community . Moreover, these parameters were significantly positively correlated with bacterial diversity  and salinity but significantly negatively correlated with temperature, turbidity, and ammonium , and the abundant inorganic nitrogen content stimulated the symbiotic algae to utilize nutrients for their growth and reproduction  can be found at: WZ: conceptualization, investigation, methodology, and writing-original draft. MZ, XL, JX, HW, and RC: investigation. XL: conceptualization, writing-review and editing, and supervision. All authors contributed to the article and approved the submitted version.This work was financially supported by the National Natural Science Foundation of China , the Natural Science Foundation of Hainan Province (2021RC169), the Hainan Provincial Key Research and Development Program (ZDYF2020177), and the Foundation of Hainan University [KYQD(ZR)1805].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To assess the reporting quality of published economic evaluations of the negotiated oncology drugs listed for China’s 2020 National Reimbursement Drug List (NRDL).A comprehensive search was conducted to identify economic evaluation studies of negotiated oncology drugs listed in China’s 2020 NRDL using the PubMed/MEDLINE, Embase, Web of Science, CNKI, SinoMed, and WanFang Database up to March 31, 2021. The Consolidated Health Economic Evaluation Reporting Standards (CHEERS) checklist scored the reporting quality between 0 and 100. A linear regression analysis was employed to examine the influence of various characteristics on the reporting quality scores.Eighty papers were included in the study, with the majority published during the past decade. Furthermore, more than half of the articles  were written in English. The average CHEERS score was 74.63 ± 12.75 and ranged from 43.48 to 93.75. The most inadequately reported items included choice of model, characterization of heterogeneity, and discussion, as well as currency, price date and conversion. Higher scores were associated with articles published from 2019 to 2021 and English publications.The economic evaluation studies of negotiated oncology drugs listed in 2020 NRDL had moderate reporting quality. The Chinese economic evaluation publications could improve the reporting quality if the CHEERS checklist is consistently implemented. Also, the Chinese journals maybe explore introducing a reporting standard for economic evaluations.The online version contains supplementary material available at 10.1186/s12913-022-08858-7. With the growing demand for medical care, health spending and pharmaceutical expenditure has increased rapidly over the past few decades , 2. DrugCancer is one of China’s leading causes of death . In 2019In May 2016, the results of the first pilot drug price negotiation in China were announced, and this negotiation successfully negotiated three drugs, two of which are oncology drugs. The outcome of the NRDL price negotiations from 2017 to 2020 shows that eighteen negotiated oncology drugs were included in the list in 2017 , accountAs an indispensable part of the NRDL price negotiation, economic evaluations are designed to inform decisions that estimate the cost and effectiveness trade-off of two or more drugs, programs, or interventions, which play a significant role in decision-making on insurance coverage and reimbursement prices . TherefoA systematic review of the literature was conducted on March 31, 2021, to identify economic evaluation studies relating to negotiated oncology drugs in China’s 2020 NRDL. We used the keywords “pharmacoeconomic”, “cost-effectiveness analysis”, “cost-benefit analysis”, “cost-utility analysis”, “cost minimization analysis”, “cost analysis” and the generic name for negotiated oncology drugs on the list to search in the three English-language  and three Chinese-language databases (China Knowledge Resource Integrated Database (CNKI), WanFang Database, and Chinese Biomedical Literature Database (SinoMed)). The specific search formula was combined with keywords and adjusted appropriately to different databases. In addition to database searches, the reference lists of included review studies were screened to identify additional studies that could have been missed during the systematic search.Inclusion criteria were: (1) original research on the economic evaluations of negotiated oncology drugs displayed in 2020 NRDL; (2) conducted in China; (3) manuscript published in English or Chinese.Exclusion criteria were: (1) repeatedly published and reported studies; (2) meeting abstracts, letters to the editor, expert opinion, reviews, and introduction to the pharmacoeconomic methodology; (3) studies unrelated to negotiated oncology drugs on the list or non-pharmacoeconomic; (4) studies conducted in other regions or countries.Two researchers independently carried out the literature search using the strategy determined in advance, then screened the studies by reading the title, abstract and full texts to identify whether the study meets inclusion and exclusion criteria. This process resolved discrepancies through consensus discussion or a third reviewer’s adjudication. Two reviewers also extracted data from full articles for methodological details, study designs, and analysis and interpretation of results into summary tables. If there were differences, they discussed them first. In the case of failure to achieve consensus, the third reviewer resolved them.The quality of the included studies was evaluated using the 24-item Consolidated Health Economic Evaluation Reporting Standards (CHEERS) checklist . the IntThe CHEERS is a checklist composed of twenty-four items to evaluate six main categories: title and abstract, introduction, methods, results, discussion, and other  . Each chTwo reviewers independently appraised the studies in the quality assessment process. When results differed, reviewers resolved discrepancies by discussion. If they cannot reach an agreement, the third reviewer resolves conflicts. Ethical approval was not necessary because the present study did not involve patients.Descriptive summary statistics were estimated for basic information and economic evaluation characteristics of included studies. The reporting quality scores’ means and standard deviation (SD) were calculated. The Shapiro–Wilk W test rejected the null hypothesis of normal distribution of CHEERS scores. We applied the non-parametric test to examine the difference in quality scores among the various characteristics. It has been shown that linear regression models are fairly robust to violations of the normality assumption . TherefoFigure Of these studies, most of them (83.75%) conducted the cost-utility analysis, and only one article conducted the cost-minimization analysis. Most (82.5%) of studies used modeling for their analyses, and 23.75 and 47.5% were health insurance and healthcare system perspective, respectively. In addition, two-fifth (38.75%) of studies used the 6-10-year time horizon for analysis, 20% used the 1 ~ 5 year, and 18.75% used a more than 10-year time horizon. Most studies were funded by the government (40%). Non-small cell lung cancer (46.25%), colorectal cancer (16.25%), and hepatocellular carcinoma (10%) were the most common tumor type for these studies.Figure Overall, the average CHEERS score of all articles was 17.68 ± 3.41 and ranged from 9.5 to 22.5 (Supplement Table P < 0.001). There was a significantly rising time trend in reporting quality scores: 68.81 (± 12.12) between 2009 and 2015), 73.69 (± 14.15) between 2016 and 2018, and 78.06 (± 10.78) after 2018 . Regarding the author numbers, the articles with fewer authors assessed lower scores than those with more than six authors (P = 0.013). Regarding the type of economic evaluation, the mean score of articles reporting a CUA was 78.43 (± 9.27), which was significantly higher than those articles reporting a CEA and CMA (P < 0.001). The mean score of articles that used modeling design was 78.47 (± 9.12), significantly higher than the articles using prospective study design 49.40 (± 3.00), or the articles using retrospective study design 59.39 (± 13.14) (p < 0.001). The studies that used a longer time horizon for analysis had higher scores than those articles with a time horizon of less than one year (P < 0.001). In terms of source of funding, studies funded by the pharmaceutical industry had the highest mean scores (85.16 ± 6.34), followed by the government (79.58 ± 10.11), and studies with not mention funding sources (63.72 ± 10.55) had the lowest scores (P < 0.001). There are significant differences in the mean scores among articles that used different study perspectives, and the study did not mention that the study perspective had the lowest scores (P < 0.001).Table P < 0.05) and English publications (P < 0.01). Studies without the disclosed source of funding and study perspective (P < 0.05) were statistically significant factors of lower scores.Table To the best of our knowledge, this is the first systematic review to examine the reporting quality of economic evaluation studies focusing on the negotiated oncology drugs included in China’s NRDL for 2020. A drug price negotiation package should include an economic evaluation study. Transparent clear reporting and high-quality studies are essential for supporting decision-making in the process . FurtherThe overall mean score of reporting quality of economic evaluations in the present study was 17.68, and the scores ranged between 9.5 and 22.5, which showed less than 75% adherence to the CHEERS 2013 checklist. The CHEERS score was nearly the same as the reporting quality score of health economic evaluation research in India, Myanmar, Cambodia, and Laos, ranging between 17 to 17.8 . Before The CHEERS scores can be divided into three categories: high quality for scores over 75, medium quality for scores between 50 and 75, and low quality below 50 , 112. IfOur study showed the mean score of studies from 2019 to 2021 was higher than those of studies between 2009 and 2015. The possible reason is that China Guidelines for Pharmacoeconomic Evaluations and Manual (2015 version) were published, which further contributes to guide the implementation and reporting of economics evaluation studies. In addition, the implementation of the NRDL price negotiations in recent years in China is similar to the reimbursement of drugs in many countries . It willThere was a significant difference between the average quality scores of the studies published in Chinese and those published in English. The findings also were consistent with Jiehua Cheng et al . This diThere were some limitations in this study. Firstly, the CHEERS was intended to qualitatively evaluate the report quality of studies without specific rules for quantitative assessment , 119. WeThis study reveals moderate reporting quality of economic evaluations of negotiated oncology drugs listed in the 2020 NRDL. The number and reporting quality of economic evaluations of negotiated oncology drugs in mainland China have improved. However, most studies, especially those published in Chinese, do not fully report CHEERS items, significantly decreasing the studies’ transparency. Therefore, the reporting quality of economic evaluations conducted in mainland China should continue to improve. Also, the Chinese journals maybe explore introducing a reporting standard for economics evaluations, not only based on the CHEERS checklist.Additional file 1: Supplementary Table 1. The 24-item CHEERS evaluation applied on the 80 included economic evaluation studies.


Osteogenesis imperfecta (OI) is a genetic disorder of connective tissue characterized by low bone mass and spontaneous fractures, as well as extra-skeletal manifestations, such as dental abnormalities, blue sclera, hearing loss and joint hypermobility. Tendon ruptures have been reported in OI patients. Here, we characterized the biomechanical, structural and tissue material properties of bone and tendon in 5-week-old female osteogenesis imperfecta mice (oim), a validated model of severe type III OI, and compared these data with age- and sex-matched WT littermates. Oim tendons were less rigid and less resistant than those of WT mice. They also presented a significantly higher rate of pentosidine, without significant modification of enzymatic crosslinking. The oim bones were less resistant and avulsion fractures were evident at high tendinous stress areas. Alterations of trabecular and cortical bone microarchitectures were noticed in young female oim. Bone tissue material properties were also modified, with a less mature and more mineralized matrix in association with lower collagen maturity. Our data suggest that the tendon-to-bone unit is affected in young oim mice, which could explain tendon ruptures and bone fragility observed in OI patients. COL1A1 or COL1A2 genes. Consequently, defects in collagen molecular structure, and possibly mineral alterations, lead to decreased bone strength, resulting in low-trauma fractures, skeletal deformities and growth deficiency [Osteogenesis imperfecta (OI) is a disorder of connective tissue generally associated with dominantly inherited pathogenic variants in ficiency .Aside from skeletal alterations and symptoms, teeth, which are also mineralized tissues rich in collagen type I, are commonly implicated in OI . They exBone and tendon are made up of dense, regular connective tissue deposited by specialized cells, osteoblasts and tenocytes, with organic extracellular matrices composed mainly of collagen. While type I is the major form of collagen in bone and tendon, other types are also present, including type III. Bone and tendon organic extracellular matrices also contain non-collagenous proteins, including proteoglycans and glycoproteins, whose functions are to promote cell attachment and control collagen fibrillogenesis and hydration of matrices. Among all the glycoproteins found in tendon, tenomodulin is known as a marker of tendon maturation and plays a role in proliferation .However, the main difference between the two tissues is the mineralization of bone extracellular matrix by tablets of poorly crystalline substituted hydroxyapatite.Bone and tendon mechanical properties are highly dependent on collagen structure and organisation and, for bone only, on the structure of the mineral tablets and the degree of mineralization. Some elements play a cardinal role; namely secondary conformation (triple helix and beta sheet), hydroxyproline or bound-water content, porosity and cross linking, whether enzymatic (including mature pyridinoline and immature dehydrodihydroxylysinonorleucine) or non-enzymatic, such as advanced glycation end-products (pentosidine) .In a preliminary histological study, we highlighted a different proportion of thick collagen fibres, as well as fibrocartilage islets, in tendons of oim mice . The ostThe main objective of the present study was to characterize the biomechanical, structural and tissue material properties in 5-week-old female oim and to compare these data with age- and sex-matched WT littermatesp < 0.0001), CT imaging highlighted frailer but also less radiopaque bones in oim than in WT mice  B.Histological analysis at the elbow highlighted very thin cortical bone, as well as thin and rare bone trabeculae, in oim ulna epiphyses C. The bo2 = 0.21, p = 0.07). This suggests a veritable osteotendinous unit, likely with an adaptation of tendon development according to mechanical constraints.Moreover, there seemed to be a relationship between the triceps brachii tendon thickness and the BV/TV in the proximal epiphysis . As illustrated by individual stress–strain and stress evolution curves, oim tendons were less rigid and less resistant than WT ones (p = 0.17) (p = 0.14). When the tendon was deformed to 15% of its initial length, the stress values were lower for oim tendons, indicating a lower rigidity (The mass (0.54 ± 0.04 mg) and the length (4.6 ± 0.2 mm) of oim flexor digitorum longus tendons were significantly lower than WT ones  C,D. No srigidity B. Indeedrigidity F.p = 0.0002; p = 0.0003; p = 0.0021; p = 0.0001, respectively). As all parameters were decreased, the work to fracture (p = 0.0001).The load–deformation curves show that the oim bones had a lower deformation and fractured with a lower load than the WT bones A. The ulfracture F was alsp < 0.0001), trabecular number  and trabecular thickness . At the cortical level (p = 0.022), Tt.Ar , Ma.Ar  and cross-sectional moment of inertia in the medio-lateral axis  were observed. On the other hand, cortical porosity was significantly increased by 63% (p = 0.004) in oim as compared with WT controls.Assessment of bone microstructure was performed after three-point bending. Unfortunately, three oim bones broke close to the distal metaphysis and hampered the microCT analysis. al level E–I, signp < 0.01) and β-sheet content  and a significant reduction in collagen NH stretch-bound water  in oim as compared with WT animals. None of the other studied tendon material parameters were significantly different between the two groups of animals. The expression of collagen III in oim tendons was significantly lower than in WT mice, but other components and regulator did not seem to be impacted. Indeed, expression of decorin and tenomodulin looked identical in both groups  and HPO4 content  and a significant reduction in the mineral crystallinity  as compared with WT animals. None of the other mineral parameters were significantly different between the two groups of animals. At the organic matrix level (p = 0.041). None of the other organic parameters showed a significant difference between the two groups of animals.We performed the analysis of bone tissue material properties at the cortical bone . In ordeix level E, we fou4 content.Our study provides an in-depth investigation of bone and tendon biomechanical, structural and compositional properties in young oim. The oim tendons were slighter, less rigid and less resistant than WT ones. We also found differences in the collagen matrix, since oim presented significantly higher rates of pentosidine and beta sheets. The rates of enzymatic crosslinking were not significantly different but oim tendons were more porous and had lower bound water than WT tendons. In bone tissue, we found a significant reduction in biomechanical properties associated, with alterations of bone microstructure  and bone tissue material properties, especially collagen maturity, the phosphate/amide ratio, mineral crystallinity and HPOAs expected, and as described in another study , OI-relaThe histological results suggested that the oim tendons were slighter than the WT ones. However, due to the mutation, oim have a short stature phenotype and the difference in tendon size could be only explained by this smaller size of the mice.The tensile tests gave very interesting information on the mechanical properties of oim tendons. Tendon ruptures described in the literature do not seem to be due to a lack of flexibility, since the oim tendons looked more stretchable than WT ones. Based on our mechanical data, we hypothesized that collagen reticulation would be lower in oim than WT mice. Surprisingly, we did not observe differences in the rates of enzymatic cross-links between groups. In the literature, some data show that pyridinoline may be identical or higher in oim . For thePreviously, reductions in the mean crystal thickness and degree of alignment of mineral crystals, as well as an elevated degree of mineralization, were reported in oim . Our resThe homotrimeric collagen of oim is known to form larger kinks and to freely rotate with a much larger angle than heterotrimers. In bone, it has been shown that collagen packing is affected, since there is a larger lateral spacing in fibrils and a larger gap/overlap ratio . HoweverRates of NH groups in collagen besides bounded water were significantly higher in oim than in WT triceps brachii tendons. The ratio 3325/2949 is a spectroscopic biomarker of collagen bound-water correlated with mechanical resistance , which iThe biomolecular analysis showed that the non-collagenic matrices of tendons did not appear to be impacted since the amounts of tenomodulin and decorin were identical. It could be hypothesized that the decrease in mechanical properties in the tendon was almost exclusively explained by collagen structure damage. Unfortunately, it was not possible to quantify the elastin with Miller staining since there was no marking on the tendons. We observed a lower expression of collagen III in oim than in WT tendons. As this type is known to be present in cases of cicatrization and lesions, this led us to consider that the repair process occurs differently in oim.Current treatments for OI aim to increase bone strength, prevent fracture and improve quality of life. They utilize a multidisciplinary approach include drug treatment, orthopedic surgery, physiotherapy and preventive dental care . IntraveTo conclude, our data suggest that the tendon-to-bone unit is affected in young oim, which could explain the tendon ruptures and bone fragility observed in OI patients. It would be interesting to evaluate the effects of age and treatments on this complex and whether such alterations are found in other models of OI with grading severity. Further studies conducted in humans are definitely required to evaluate whether such modifications of bone and tendon material properties are also present in human patients.oim/J, JAX stock #001815) were obtained from the Jackson laboratory . Heterozygous mice were crossed, and 5-week-old female homozygous oim (n = 10) and wild-type littermates (n = 10) were used for this study. The genotype was monitored with PCR to amplify the purified genomic DNA from cut tail samples, using the following primers: forward—(5′-3′) GGCTTTCCTAGACCCCGATGCTTAG; reverse for WT—(5′-3′) GTCTTGCCCCATTCATTTGTC; and reverse primer for OI—GTCTTGCCCCATTCATTTGTT. All procedures were undertaken in accordance with the Belgian federal law and protocols for the care of animals, and handling and care of mice were approved by the ethics committee for animal research of the Université Catholique de Louvain (2020/UCL/MD/02). The mice were housed at 24 °C with a 12–12 h light–dark cycle and received standard pellet chow  and water ad libitum. Mice received subcutaneous calcein (30 mg/kg) 10 days and 2 days before euthanasia. On the day of euthanasia, at 5 weeks, mice were weighed and scanned with an in vivo computed tomograph  under sevoflurane overdose inhalation. Elbows, bone samples, tail and flexor digitorum longus tendons were dissected within 10 min after death.Heterozygous mice  was assessed in three transverse slices of each bone with the XCT540 software of the pQCT. After dissection, the elbow was fixed in 4% formalin, decalcified, embedded in paraffin and sliced into 5 µm thick sections, which were stained with Sirius Red and then observed under an optical microscope. The bone volume/total volume (BV/TV) ratio of the olecranon epiphysis and the triceps brachii tendon thickness were measured in the same sections with ImageJ software (version 1.52A).The ulna epiphysis was scanned with a pQCT Research SA+ . Eight transverse slices were obtained with thickness of 150 µm; the voxel size was 0.07 mm and the threshold was 280 mg/cmFlexor digitorum longus tendons were used for the tensile test with a mechanical device developed in a laboratory of our institution  and equiThree-point bending was performed on the right femur as previously reported . Bones wMicroCT analyses were performed on the right femur after three-point bending with a Skyscan 1272 microtomograph  operated at 70 kV and 140 μA, with 1000 ms integration time. The isotropic pixel size was fixed at 4 μm and the rotation step at 0.25°, and exposure was performed with a 0.5 mm aluminium filter. The trabecular volume of interest was located 0.5 mm above the growth plate at the distal end and extended 2 mm up. The cortical volume of interest was located 3 mm above the distal growth plate and extended by 0.5 mm. All histomorphometry parameters were measured according to guidelines and nomenclature proposed by the American Society for Bone and Mineral Research .−1, with an integration time of 5 s and 15 accumulations. Enzymatic collagen crosslinking (intensity: ratio: 1670/1690 cm−1), non-enzymatic collagen crosslinking (intensity ratio 1345/920 cm−1), hydroxyproline content (intensity ratio: 872/920 cm−1), nanoporosity (intensity ratio: 1296/920 cm−1), glycosaminoglycan content (intensity ratio: 1380/920 cm−1) and β-sheet content  were computed. In order to estimate organic matrix-related water, we also recorded three spectra per tendon in the spectral range from 2700 to 3800 cm−1, following Unal et al. [−1. Collagen-bound water (intensity ratio: 3243/2940), collagen NH stretch (intensity ratio: 3330/2940), and collagen OH stretch (intensity ratio: 3457/2940) were computed.After dissection, the elbow was fixed in 4% formalin, decalcified and embedded in paraffin. Paraffin blocks were trimmed and the triceps brachii tendon composition was analysed by confocal Raman microspectroscopy using a Renishaw InVia Qontor microscope equipped with a 785 nm laser diode . Spectral acquisitions were made with a laser power set at 10 mW using a 1200 lines/mm holographic grating and a 20× objective . Prior to acquisition, the Raman system was calibrated with a silicium standard at the 520 nm peak. In order to estimate tendon tissue material properties, three spectra were recorded for each tendon in the range from 800 to 1800 cm−1 was computed and a nanoporosity ratio  was calculated. After pMMA subtraction, the spectrum was vector-normalized for the v1, v3 phosphate band and analysed with a routine script in Matlab R2021b , as previously reported [4 peak at 960 cm−1), crystal size index (area ratio: ~1076 cm−1/~1058 cm−1), acid phosphate content (area ratio: ~1127 cm−1/~1096 cm−1), carbonate/phosphate ratio  and collagen maturity (area ratio: ~1660 cm−1/1690 cm−1) were computed. The secondary structure of collagen matrices was also computed following Belbachir et al. [Right femurs were embedded undecalcified in pMMA after dehydration and infiltration, as previously reported . One micreported . Phosphar et al. .® films  using ECLTM Western Blotting Detection Reagents . Protein levels were normalized to beta-actin . Films were scanned and quantified by densitometry using ImageJ software (Version 1.52A).The tail tendons were homogenized in radioimmunoprecipitation assay buffer containing a protease inhibitor cocktail and PhoSTOP . Protein concentration was determined using a bicinchoninic assay protein assay kit . A total of 20 µg of proteins were loaded per well on 8% SDS-PAGE gel and migration was performed in migration buffer  under 120 V for 90 min. Proteins were transferred onto nitrocellulose blotting membranes  with a constant amperage of 350 mA for 90 min in transfer buffer . Membranes were blocked for 1 h at room temperature with Tris-buffered saline (TBS) containing 0.1% Tween and 5% BSA. Primary antibodies—decorin , tenomodulin  and collagen III —were then incubated on membranes at 4 °C overnight. Membranes were washed in TBS-Tween and incubated with the peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, membranes were developed on CL-X Posuret-test or the non-parametric Mann–Whitney test. Differences were considered significant at p < 0.05 .Data are presented as means ± SEM. Statistical analysis was performed using an unpaired Student’s


Textbook outcome (TO) is a multidimensional measure reflecting the ideal outcome after surgery. As a benchmarking tool, it provides an objective overview of quality of care. Uniform definitions of TO in hepato-pancreato-biliary (HPB) surgery are missing. This study aimed to provide a definition of TO in HPB surgery and identify obstacles and predictors for achieving it.A systematic literature search was conducted using PubMed, Embase, and Cochrane Database according to PRISMA guidelines. Studies published between 1993 and 2021 were retrieved. After selection, two independent reviewers extracted descriptive statistics and derived summary estimates of the occurrence of TO criteria and obstacles for achieving TO using co-occurrence maps.Overall, 30 studies were included. TO rates ranged between 16–69 per cent. Commonly chosen co-occurring criteria to define TO included ‘no prolonged length of stay (LOS)’, ‘no complications’, ‘no readmission’, and ‘no deaths’. Major obstacles for achieving TO in HPB surgery were prolonged LOS, complications, and readmission. On multivariable analysis, TO predicted better overall and disease-free survival in patients with cancer. Achievement of TO was more likely in dedicated centres and associated with procedural and structural indicators, including high case-mix index and surgical volume.TO is a useful quality measure to benchmark surgical outcome. Future definitions of TO in HPB surgery should include ‘no prolonged LOS’, ‘no complications’, ‘no readmission’, and ‘no deaths’. Textbook outcome (TO) reflects the ideal outcome after surgery and is a useful quality measure to benchmark surgical outcome. We are the first to propose a uniform definition of TO in hepato-pancreato-biliary surgery, including ‘no prolonged length of stay’, ‘no complications’, ‘no readmission’, and ‘no mortality’. In light of these changes, reliable measures that assess patient-centred outcome and overall quality of surgical care are indispensable. Ideally, these measures will help guide treatment decisions and ensure that treatment meets appropriate standards.Hepato-pancreato-biliary (HPB) surgery frequently involves complex procedures, notably those operations undertaken for malignant disease. Many procedures are prone to complications that influence postoperative course and can have a detrimental effect on outcome. Advances in surgical technique and perioperative care have made surgery safer, leading to increased proclivity to consider patients with more advanced disease or co-morbidities as candidates for surgery3. TO is achieved when all prespecified parameters are fulfilled according to an all-or-none principle. In this regard, TO can provide a global picture and an overall reflection of surgical quality and care. In turn, identification of parameters that have the greatest influence on TO achievement can help to target single issues and make specific changes accordingly to improve healthcare quality. In this respect, TO has been suggested as a new benchmarking tool to measure internal surgical quality and provide quality-of-care information to patients. Indeed, patients prefer summary measures regarding quality-of-care information5.Textbook outcome (TO) is a composite measure, originally described in colorectal cancer surgery, that aims to reflects the ideal surgical outcome in a single indicator6–9. Ideally, included parameters should be disease and surgery specific.A uniform definition of TO in HPB surgery is presently missingversus major), survival, hospital performance ), type of hospital, hospital designation (including Magnet status), and socioeconomic factors.This study aimed to systematically review literature to investigate existing definitions of TO in HPB surgery and provide the basis for a uniform definition of this composite measure. The primary objective was to assess parameters used for defining TO. Secondary objectives were TO achievement rate; identifications of factors influencing TO achievement; relation between TO and minimally invasive surgery, type of resection  OR (textbook AND outcome*). The reference list of each article was searched for further relevant literature. Duplicate articles, editorials, and conference abstracts were excluded. The articles were screened and filtered by title and abstract. A full-text assessment and review of the remaining studies was conducted. Data from the included articles were extracted independently by the two reviewers using double-data extraction. Inconsistencies were resolved by consensus. In the case of disagreement, a third reviewer was consulted so that consensus was reached .A systematic literature search was conducted using PubMed, Embase, and Cochrane Database according to the PRISMA guidelinesAll studies reporting TO in patients 18 years or older who underwent either hepatic, biliary, or pancreatic surgery or a combination of these for any condition  were considered eligible for inclusion. Multivisceral resections including other compartments  were excluded.11, teaching status, and Magnet designation12), expenditure, social vulnerability, and racial diversity.The following data were collected: author details, year of publication, country, recruitment interval, study population, sample size, diagnosis, procedure, definition of TO with all included components (including the percentage of the respective components reached), occurrence of TO, independent predictors of TO, any data with regard to hospital status and performance  indicating the amount of co-occurrence. Co-occurrence maps were generated first including all studies on hepatobiliary surgery for both benign and malignant disease  and second, focusing on only subgroup analyses of pancreatic cancer  and only hepatobiliary malignancies . Summary graphs were also provided . Summary variables included ‘no prolonged length of stay (LOS)’ in either the 50th or 75th percentile, ‘no readmission’, and ‘no deaths’ at 30 days, 90 days, in-hospital, and any time, ‘no complications’ either overall or three or more, and ‘no bile leak’ grade B/C, or any as defined by the International Study Group for Liver Surgery14.Co-occurrence maps were computed to visualize how many times each TO criterion was considered. Co-occurrence is defined as an above-chance frequency of occurrence of two terms/criteriaRates of patients that achieved each TO criterion were derived from the original publication or, where not available, computed by the authors of this study. In the presence of subgroups, averages were weighted by the number of participants within each subgroup.To help readers visualize the contribution of each parameter to TO achievement, negative (indicated in blue) and positive (indicated in red) contributors were displayed using word clouds. The size of each word/term reflects the strength of its contribution to TO. Where identical terms were present across multiple studies, their OR values were averaged. Studies with missing values were excluded. To indicate the relevance of each contributor to achieving TO in the context of a particular diagnosis, diagnostic labels in parentheses following terms (where applicable) were added.Fig. 1). The eight studies excluded did not reported the relevant outcomes.A total of 650 studies were identified after de-duplication. Of these, 14 conference abstracts were excluded. Screening by title led to the exclusion of 576 studies, 22 studies were excluded after abstract review, leaving 38 studies that underwent full-text assessment, with 30 meeting eligibility criteria , six were international, and five were from France. The remaining studies were from UK (one), The Netherlands (one), Spain, and France . There were 28 multi-institutional studies. The sample size of patients per study ranged from 78 to 42 551. Ten studies had a sample size lower than 1000, six between 1000–10 000, and 14 more than 10 000.The characteristics of the included studies are shown in n = 18). Among those, six included only patients with pancreatic ductal adenocarcinoma (PDAC), eight focused on liver cancer (three hepatocellular carcinoma (HCC), three intrahepatic cholangiocarcinoma (ICC), one HCC and ICC, and one primary and secondary malignancy), and four looked at HPB malignancies as a whole.Most studies were focused on patients receiving HPB surgery for a malignant indication . Multiple definition existed for these parameters. ‘No prolonged LOS higher or equal to the 75th percentile’, ‘no readmission within 90 days’, ‘no deaths within 90 days’, and ‘no complications’ were the most used . Figure 2c,d shows the results for subgroup analysis with regard to PDAC  and hepatobiliary malignancies .Parameters used to define TO are shown in Table 1).The rate of patients achieving TO ranged between 15.8–69.1 per cent, 16.8–60.3 per cent, and 15.8–69.1 per cent in all candidates, pancreatic surgery candidates , and liver surgery candidates  respectively. Median TO rate was 38 per cent. Patients with malignant disease presented lower TO rates than the median and patients with benign disease. Patients with benign disease had a TO rate higher than the median.  and 5.6 (95 per cent c.i. 1.7 to 18.2)33. One study investigating patients who underwent HPB surgery for any indication reported that a laparoscopic approach was an independent predictor of TO achievement with an OR of 1.52 (95 per cent c.i. 1.34 to 1.73)15. One study investigating TO with respect to operating approach among patients with PDAC failed to identify a difference in TO rates between minimally invasive and open pancreaticoduodenectomy30.Seven studies investigated the TO rate with regard to the operating approach. All studies found better TO rates in patients undergoing a laparoscopic procedure compared with an open procedure. Two of these studies included only patients with ICC26. PDAC  and a dilated pancreatic duct (more than 3 mm)  were identified as independent prognostic factors of TO achievement after pancreatoduodenectomy (OR 1 or higher) in one study8.TO ranged between 32.5–58.3 per cent, and 55.8–67.4 per cent after pancreaticoduodenectomy and distal pancreatectomy respectively39.Among hepatobiliary procedures, previous liver resection, ICC, gallbladder carcinoma, increasing tumour size (more than 3 cm), minor resection of posterior/superior segments, non-anatomical resection,  major resection, biliary reconstruction, and major vascular invasion were all associated with decreased likelihood of achieving TO (OR 1 or less)11.Hospital CMI was strongly associated with the probability of achieving TO in one study. TO rate was 45.7 per cent and 53 per cent in facilities with low and high CMI respectively. A low as well as an average CMI were significantly associated with lower odds of achieving TO  when compared with high CMI. Even after adjusting for HPB surgical volume at the hospital level, CMI had a beneficial effect on the odds of achieving TOTable 2).Twelve studies evaluated the relationship between TO and surgical volume. Seven studies reported a significant positive correlation, whereas five did not  had higher odds of TO  and lower odds of complications, prolonged LOS, and deaths22.One study suggested that dedicated cancer centres provide higher-value surgical care for patients with HPB malignancies. Patients treated in dedicated cancer centres, even when presenting with a higher co-morbidity burden, had higher odds of achieving TO  compared with those treated in other hospitals17. Socioeconomic factors independently associated with achievement of TO were lower patient education, government insurance, low county-level racial diversity, and Black race27.Patients with a high social-vulnerability index (SVI) were less likely to achieve TO after HPB surgery  in one study. A high SVI was an independent predictor of complications and 90-day deaths after both pancreatic and liver surgery as well as prolonged LOS in liver surgery23.Four studies investigated the relationship between expenditure (insurance payments) and TO. All reported that payments for patients achieving TO were markedly lower (about $10 000 less) than payments for patients who did not achieve TO35, the remaining nine studies did 39 (Table S1).Eleven studies assessed the impact of TO on survival. While two studies did not report better survival rates when TO was achievedet al. reported that among patients with HCC who achieved TO, 5-year overall survival (OS) was higher (69.6 per cent versus 56.9 per cent) compared with patients who did not. TO achievement was associated with 40 per cent decreased risk of death with respect to all-cause deaths 38. Another study reported that TO was independently associated with 26 per cent and 37 per cent decreased hazards of death among ICC  and patients with HCC  respectively39. Similarly, in a study by Sweigert et al. on patients with PDAC, TO achievement was associated with better OS (median 27 versus 19.9 months) and lower risk of long-term deaths 6. Three studies on PDAC concluded that TO was associated with better OS 30, a 4.8-month increase in median survival at any facility27, and that all components of TO  were independently associated with the 5-year OS24. Ultimately, two studies investigating the impact of TO on disease-free survival (DFS) in patients with HCC and pancreatic neuroendocrine tumours reported that TO was independently associated with DFS 33.Tsilimigras Fig. 4).Several studies investigated patient characteristics and perioperative factors and their association to TO achievement , the Model for End-Stage Liver Disease score, and an albumin-bilirubin grade 2/3 were independent predictors of TO in patients with hepatic malignancies39. Metabolic syndrome, ASA score of 3 or higher, and previous hepatic surgery were negatively associated with TO in patients undergoing liver surgery for any liver disease23.Parameters associated with decreased odds of achieving TO in multivariate analysis included Charlson–Deyo co-morbidity index 3 or higher, older age (age more than 70 years), and neoadjuvant chemotherapy15.More than one previous hospital admission was associated with decreased odds of TO (odds decreasing by the increasing number of previous stays) in patients undergoing HPB surgery for any indication28. Omission of neoadjuvant chemotherapy in patients undergoing distal pancreatectomy, and a dilated pancreatic duct (more than 3 mm) as well as diagnosis of PDAC in patients receiving pancreatoduodenectomy were associated with higher likelihood of achieving TO8. A Barcelona clinic liver cancer stage 0 (versus B/C) and an albumin-bilirubin grade 1 (versus 2/3) were independent predictors of TO in patients with HCC38. In patients with ICC, age 60 years or younger, no preoperative jaundice, and omission of neoadjuvant chemotherapy increased the likelihood of achieving TO36.Female sex was an independent predictor of TO in patients undergoing HPB surgery both for cancer or any indication41.In this systematic review the median rate of TO achievement was 38 per cent, ranging widely from 15 to 70 per cent. Major obstacles in achieving TO were ‘complications’, ‘prolonged LOS’, and ‘readmission’. While identification of these parameters might be of great benefit to guide improvement initiatives, it is important to note that they are themselves influenced by patient and procedural factors. LOS and readmission not only depend on surgical quality but also on cultural norms and healthcare policies as payment schemes and access to long-term rehabilitation facilitiesversus pancreaticoduodenectomy and minor versus major liver resection), high CMI and surgical volume, and higher socioeconomic status all influenced TO positively. Age, co-morbidity status, and neoadjuvant chemotherapy were also reported as independent predictors of TO after multivariable analysis in some studies39. Indeed, the retrospective fashion of all these studies severely limits the reliability of this data.Many factors influenced TO achievement. Laparoscopic approach, less-extended surgery . Aiming to reach high TO rate should not lead to operations being offered only to the ‘best’ candidates and avoiding those at higher risk. To avoid these biases, TO should be used in addition to other quality metrics such as CMI29. TO does not include a patient perspective. As patient-reported and clinically defined outcomes may well differ, future studies should further investigate this relationship and incorporate patient-reported outcomes in TO definitions7.TO definition TO is a promising multidimensional measure reflecting the ideal outcome after surgery but is still heterogeneously defined. A current definition of TO in HPB surgery should include the terms ‘no prolonged LOS’, ‘no complications’, ‘no readmission’, and ‘no deaths’. Under proper risk adjustment, TO in addition to other quality metrics, might become a useful tool to aid clinical decision-making, provide information on surgical quality for the patient, and assist preoperative patient selection.zrac149_Supplementary_DataClick here for additional data file.


Circulating tumor-derived endothelial cell (CTEC) is a new potential tumor biomarker to be associated with cancer development and treatment efficacy. However, few evidences are available for breast cancer.  Eighty-nine breast cancer patients were recruited, and preoperative and postoperative blood samples were collected. Besides, 20 noncancer persons were enrolled as controls. An improved subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) method was adopted to codetect CD31+ aneuploid CTEC and CD31− aneuploid circulating tumor cell (CTC). Then, the clinical significance of CTCs and CTECs on breast cancer screening and prognosis prediction was evaluated and compared. P=0.043), while there was no significant difference in CTC. CTEC  had better performance than CTC (AUC = 0.795) to distinguish breast cancer patients from controls by receiver operator characteristic curve analysis. Preoperative CTEC count ≥ 2 was a significant risk factor for reducing PFS of breast cancer patients.  The positive rate of CTCs and CTECs in newly diagnosed breast cancer patients was 68.75% and 71.88%. Among detected aneuploid circulating rare cells, CTEC accounts for a greater proportion than CTC in breast cancer patients. CTEC-positive rate and level were significantly higher in breast cancer patients with lymph node metastasis (LNM) than those without LNM ( CTECs may function as a reliable supplementary biomarker in breast cancer screening and prognosis prediction. Breast cancer is the most common cancer in women worldwide . About 2As a liquid biopsy method, circulating tumor cell (CTC) is a commonly used biomarker for breast cancer diagnosis and prognosis prediction. Previous studies have shown that CTCs numbers in peripheral blood were directly associated with the prognosis of breast cancer patients –8. PersiThe technical approaches of enrichment and identification were multifarious, mainly based on polyploidy , 17, CK In this study, we applied the SE-iFISH method developed by Cytelligen  to codetect aneuploid CTC and CTEC . A totalA total of 89 female breast cancer patients and 20 noncancer controls were recruited from 3201 Hospital Affiliated to Xi'an Jiaotong University School of Medicine and Quanzhou First Hospital Affiliated to Fujian Medical University between April 2018 and April 2020. The clinical characteristics of 89 breast cancer patients are given in In order to detect CTCs and CTECs, 7.5 mL of peripheral blood was collected by acid citrate dextrose (ACD) anticoagulant tube  from 20 noncancer controls and 32 breast cancer patients. There are 72 patients after surgery who received CTCs and CTECs tests, while 23 of them received twice, after surgery and postoperative adjuvant chemotherapy. The design of this study is shown in This study involves human participants and was approved by the Ethical Committee of Department of Oncological Surgery, 3201 Hospital Affiliated to Xi'an Jiaotong University and Quanzhou First Hospital Affiliated to Fujian Medical University. The experiments were conducted after collecting the informed consent of each subject, and the study conforms with The Code of Ethics of the World Medical Association (Declaration of Helsinki) in the British Medical Journal (18 July 1964).g for 8 min at room temperature to remove plasma. Then, the lower layer of cells was transferred into centrifuge tubes containing 3 mL of hCTC separation matrix, and red blood cells were discarded by centrifugation at 450 × g for 8 min at room temperature. Buffy coat cells were collected in new tubes and then incubated with an immunomagnetic particle-conjugatedanti-CD45 antibody for 20 min with gentle shaking. WBCs bound to immunobeads were depleted using a magnetic separator. The solution without beads was centrifuged at 450 × g for 8 min following rinsed twice by hCTC buffer at room temperature. Finally, the cell pellet was completely mixed with cell fixative, and the mixture was coated on glass slides and dried overnight at 30°C, which would be identified by immunostaining-fluorescence in situ hybridization (iFISH).CTCs and CTECs were enriched with a Human Circulating Rare Cell Subtraction Enrichment kit  according to the manufacturer's instructions and previous studies' suggestions . In brieFor CTCs and CTECs identification, the dried monolayer cells on the coated and formatted CTC slides were rinsed and incubated with saline-sodium citrate buffer for 10 min following dehydration in ethanol for 2 min. Centromere probe 8 (CEP8) spectrum orange  was added to the CTC slides, which were denatured at 76°C for 10 min and hybridized for 4 h at 37°C. Then, the hybridization slides were subsequently darkly incubated with AlexaFluor® 594-conjugatedanti-CD45 IgG (spectrum red) and AlexaFluor® 488-conjugatedanti-CD31 IgG (spectrum green) for 2 h at room temperature. Finally, DAPI (spectrum blue) was added to the CTC slides and was subjected to fluorescence microscope scanning and analyses.The CTC were confirmed by CD31−/CD45−/DAPI+/CEP8 > 2 , and thet-test, while nonparametric tests  were used if they are not consistent with normal distribution. Receiver-operating characteristic (ROC) curve was used to assess the diagnostic efficacy of variables between controls and patients. Kaplan–Meier survival curves and log-rank tests were used to compare the differences in progression-free survival (PFS) rate between the two groups. Cox proportional hazards regression analyses were carried out to identify risk factors of poor prognosis. P < 0.05 was considered to indicate a statistically significant difference.Statistical analysis and graphical plots were performed using SPSS 22 (IBM Corp.), GraphPad Prism 6 , and R project. The differences of categorical variables in distribution among groups were analyzed using the chi-square test or the Fisher exact test. Differences of continuous variables with normal distribution among groups were compared by the P=0.033) and level (P=0.043) were significantly higher in breast cancer patients with LNM than those without LNM, while there was no significant difference in CTC-positive rate (P=0.325) and level (P=0.6005). CTC was detected in all triple-negative breast cancer (TNBC) patients , and its positive rate was much higher than that in HER2+  and luminal A/B  classification. P=0.0188). However, there was no significant difference in the level of CTC and CTEC between stage 0–II and III-IV patients. A novelty of the results was that elder patients (>50 years) had a significantly higher CTEC-positive rate than younger patients (P=0.050) and overweight patients (BM ≥ 24.0) had a higher CTC-positive rate (P=0.024) than other patients (BMI < 24.0).nd level , P=0.043ive rate , P=0.325r = 0.28, P=0.01). Meanwhile, CEA had markedly positive correlations with CA19-9  and CA15-3 , and CA19-9 was positively correlated with CA15-3 . No significant correlation was found between novel biomarkers (CTC and CTEC) and traditional biomarkers . These results indicated CTC and CTEC were independent of traditional cancer biomarkers.To comprehensively understand the relationship and difference between novel and traditional cancer biomarkers, we calculated Spearman's rank correlation coefficient of CTC, CTEC, CEA, CA12-5, CA19-9, and CA15-3 . CTC wasP < 0.0001). Results of ROC curve analysis showed that CTEC (AUC = 0.859) had better performance than CTC (AUC = 0.795) for breast cancer screening. After summing the aneuploid cells (CTC plus CTEC), the AUC was widely promoted to 0.914. This result suggested aneuploid cells from peripheral blood were an ideal biomarker for breast cancer screening.To evaluate the clinical application value of CTC and CTEC for breast cancer screening, 20 noncancer participants were recruited to detect CTC and CTEC as controls. CTC was not detected in 18 of 20 (90%) controls, and all the 20 controls' CTEC (100%) was negative. For breast cancer patients, the positive rate of CTC and CTEC was 68.75% (22/32) and 71.88% (23/32), respectively. The mean value of CTC and CTEC was significantly higher in breast cancer patients than those in controls  and postoperative quantity of CTEC also had no significant influence on PFS  as the end of follow-up. The median follow-up time was 37 months (3–62 months). During the follow-up period, 14 patients had recurrence, 1 patient was lost of follow-up, and 74 patients were progression-free. We separately plotted the progression-free survival curves according to the CTC and CTEC values before and after the surgical operation. Preoperative CTEC count ≥2 was a significant risk factor for reducing the PFS of breast cancer patients (e on PFS . Surprise on PFS .The tumor microenvironment (TME) is a complex system comprised of cancer cells and their surrounding cells such as endothelial cells, cancer-associated fibroblasts, immune cells, and so on . CTCs weIn this study, we used an improved SE-iFISH method, which could codetect and distinguish aneuploid CTEC by adding an anti-CD31 antibody from aneuploid CTC. Then, 89 breast cancer patients and 20 noncancer controls were enrolled to receive CTC and CTEC tests of peripheral blood. First, we exhibited the cytogenetic characteristics of CTCs and CTECs during the course of breast cancer. We found that higher CTECs quantity and positive rate were significantly correlated with LNM of breast cancer, but no significant difference was found in CTC level. Since endothelial cells make up the lining of the tumor vasculature and lymphogenous cancer metastases are mainly impacted by lymphatic vessel-related lymphangiogenesis , an actiIn fact, many studies have shown that CTECs are associated with the development of cancers. CTECs counts were found to be increased in nonsmall cell lung cancer (NSCLC) patients compared with controls and were considered as the high-risk factor of recurrence . CTECs hIn this study, we found surgery had no obvious effect on the heteroploid distribution of CTCs and CTECs. However, preoperative and postoperative CTC/CTEC count had different influences on PFS of breast cancer. Preoperative CTEC count equal to or greater than 2 cells/7.5 mL was the risk factor of shorter PFS of breast cancer, while no similar phenomena appeared on postoperative CTC or CTEC number. Previous studies have proved that the level of CTC or CTEC will rise within a short period when patients were receiving treatment such as surgery or chemotherapy , 38. Ma There are a few limitations to this study. First, the findings are limited because of the small size of breast cancer patients and controls. Second, since the follow-up period is not long enough, less than half of the patients reached the follow-up endpoint. In addition, the clinical pathways of our patients were diverse, which may disturb the results. A prospective study with a larger size of the cohort and a longer follow-up period is necessary to validate the findings.In summary, we used an improved SE-iFISH method to codetect CTC and CTEC in peripheral blood of 89 breast cancer patients and 20 controls. The CTEC level was positively correlated with CTC, and both of them were independent of traditional cancer biomarkers such as CA15-3, CA19-9, and CA12-5. CTECs are a more effective biomarker for breast cancer screening and prognosis prediction than CTCs.


No liquid biomarkers are approved in renal cell carcinoma (RCC), making early detection of recurrence events following surgery of nonmetastatic RCC difficult. Here we develop a noninvasive blood/urine test measuring free glycosaminoglycans that can detect RCC recurrence events. No liquid biomarkers are approved in renal cell carcinoma (RCC), making early detection of recurrence in surgically treated nonmetastatic (M0) patients dependent on radiological imaging. Urine- and plasma free glycosaminoglycan profiles—or free GAGomes—are promising biomarkers reflective of RCC metabolism.To explore whether free GAGomes could detect M0 RCC recurrence noninvasively.Between June 2016 and February 2021, we enrolled a prospective consecutive series of patients elected for (1) partial or radical nephrectomy for clinical M0 RCC (cohort 1) or (2) first-line therapy following RCC metachronous metastatic recurrence (cohort 2) at Sahlgrenska University Hospital, Gothenburg, Sweden. The study population included M0 RCC patients with recurrent disease (RD) versus no evidence of disease (NED) in at least one follow-up visit. Plasma and urine free GAGomes—consisting of 40 chondroitin sulfate (CS), heparan sulfate, and hyaluronic acid (HA) features—were measured in a blinded central laboratory preoperatively and at each postoperative follow-up visit until recurrence or end of follow-up in cohort 1, or before treatment start in cohort 2.We used Bayesian logistic regression to correlate GAGome features with RD versus NED and with various histopathological variables. We developed three recurrence scores  proportional to the predicted probability of RD. We internally validated the area under the curve (AUC) using bootstrap resampling. We performed a decision curve analysis to select a cutoff and report the corresponding net benefit, sensitivity, and specificity of each score. We used univariable analyses to correlate each preoperative score with recurrence-free survival (RFS).p = 0.02). Limitations include a lack of external validation.Of 127 enrolled patients in total, 62 M0 RCC patients were in the study population . The median follow-up time was 3 months, totaling 72 postoperative visits —17 RD and 55 NED cases. RD was compatible with alterations in 14 (52%) of the detectable GAGome features, mostly free CS. Eleven (79%) of these correlated with at least one histopathological variable. We developed a plasma, a urine, and a combined free CS RCC recurrence score to diagnose RD versus NED with AUCs 0.91, 0.93, and 0.94, respectively. At a cutoff equivalent to ≥30% predicted probability of RD, the sensitivity and specificity were, respectively, 69% and 84% in plasma, 81% and 80% in urine, and 80% and 82% when combined, and the net benefit was equivalent to finding an extra ten, 13, and 12 cases of RD per hundred patients without any unnecessary imaging for plasma, urine, and combined, respectively. The combined score was prognostic of RFS in univariable analysis (hazard ratio = 1.90, Free CS scores detected postsurgical recurrence noninvasively in M0 RCC with substantial net benefit. External validity is required before wider clinical implementation.In this study, we examined a new noninvasive blood and urine test to detect whether renal cell carcinoma recurred after surgery. However, currently neither molecular nor liquid biomarkers are approved in the clinical management of RCC Glycosaminoglycans (GAGs) are structurally diverse polysaccharides. Their complex profile of sulfation and epimerization patterns—also called the GAGome—is now viewed as a nongenetic code that controls a wide range of biological functions, with many of these being active in cancer In this study, we sought to explore whether urine and plasma free GAGomes correlated with postsurgical recurrence in M0 RCC in a single-center prospective cohort study.22.1This study is reported in compliance with the STARD guidelines . The pre2.2g for 15 min at 4°C). Urine was collected in polypropylene tubes at room temperature. All samples were stored at –70°C and shipped on dry ice. The free GAGome analysis was performed at a single blinded central laboratory  using the MIRAM® Free Glycosaminoglycan Kit , wherein the absolute concentrations (in µg/ml) of 17 chondroitin sulfate (CS), heparan sulfate, and hyaluronic acid disaccharides—that is, the free GAGome features—were detected and quantified using a UHPLC-MS/MS system  following the kit instructions for use (Whole blood samples were collected in EDTA-coated tubes at room temperature and centrifuged within 15 min (2500 for use  [18]. Ou2.3Each detectable feature in the plasma and urine free GAGome was correlated with recurrence status (RD vs NED), as assessed at the same postoperative visit in which the corresponding plasma or urine sample was collected using a Bayesian mixed-effect logistic regression model . Each pr2.4The top two free GAGome features were used as inputs to train two Bayesian Additive Regression Trees (BART) models 2.5R2 (%) with empirical 95% confidence intervals (CoIs). We assessed the discrimination performance in terms of the area under the receiving operating characteristic curve (AUC) for the classification of RD versus NED according to each score versus the observed recurrence status. The goodness of fit and corresponding χ2 statistics were tested using the Hosmer-Lemeshow test. We used a decision curve analysis to determine a single test positivity cutoff for all free CS RCC recurrence scores, so that the net benefit over the base scenarios “intervention for all”  and “intervention for none”  was maximized We performed internal validation using bootstrap resampling and reported the predictive performance in terms of scaled Brier score (%) and Nagelkerke 2.6We correlated recurrence-free survival (RFS) with each preoperative score in the subset of patients with pathological diagnosis of M0 RCC from cohort 1 . A univa33.1The study design and patient flow are illustrated in N = 51, 82%), followed by five papillary RCC  and six chromophobe RCC cases. We listed other histopathological characteristics per cohort and across the study population in The median age was 63 year (interquartile range [IQR]: 56–70). The population was predominantly composed of males . The most common histological subtype was clear cell RCC , there were 122 RCC (106 ccRCC and 16 non-ccRCC) and five oncocytoma patients. We listed other population characteristics per cohort and across cohorts in the total population in In the total population  samples in a single blinded central laboratory using a standardized UHPLC-MS/MS kit. No samples were identified as outliers.3.2N = 62), we explored the correlation between free GAGomes and recurrence status using samples obtained at the same postoperative visit. The median follow-up time from curative intent surgery were 3 months . Each patient contributed on average 1.2 visits for a total of 72 visits. Throughout these, we recorded 17 (24%) RD and 55 (76%) NED cases. Plasma and urine samples were obtained, respectively, in 71 (99%) and 67 (93%) visits.In the study population . Of these, we determined that six (86%) of seven detectable features in plasma and eight (40%) of 20 detectable features in urine were compatible with RD versus NED . In gene3.3N = 67), we observed that 11 (79%) of 14 preoperative free GAGome features compatible with recurrence were also compatible with at least one histopathological variable and in the same direction of correlation  and 4-sulfated CS (in %); and for urine, unsulfated CS (in μg/ml) and 6-sulfated CS (in %). For the combined model, we used as inputs the outputs of the plasma and urine models. The predicted probability of each model was scaled between 0 and 100, and henceforth referred to as plasma, urine, or combined free CS RCC recurrence score, given that the models rely solely on free CS. For example, if the plasma free CS RCC recurrence score is >50, then the probability of RD is >50% according to the model that uses only plasma features as predictors.R2 values were 0.59 (95% CoI: 0.42–0.78), 0.64 (95% CoI: 0.49–0.81), and 0.64 (95% CoI: 0.48–0.81), respectively , 0.43 (95% CoI: 0.27–0.59), and 0.43 (95% CoI: 0.25–0.60) and the ectively . In termectively . The cal3.5p < 0.001), 81% and 80% in urine , and 80% and 82% when combined , respectively . For example, the European Association of Urology (EAU) guidelines recommend that radiological evaluations are curtailed after 3 year of postoperative follow-up with NED in those with a low risk of recurrence In the second scenario, we imagined a follow-up visit in which there is a radiological evaluation of recurrence planned . For example, the National Comprehensive Cancer Network guidelines recommend a radiological evaluation after 9 and 15 months after surgery as opposed to the EAU guidelines, indicating different willingness to intervene In 3.6N = 51), there were seven (14%) recurrences at a median follow-up time of 11 months. In the univariable Cox regression analysis (p = 0.02), but only nominal correlations in plasma  and urine . In multivariable Cox regression adjusted for Leibovich risk points, the preoperative combined free CS RCC recurrence score was nominally correlated with RFS without reaching statistical significance .In the subset of the total population with preoperative samples from cohort 1 and pathological diagnosis of M0 RCC  and a lack of external cohorts for validation. These two factors compound the risk of selection and detection biases. We believe that the prospective consecutive series cohort design of the study and the double-blind analysis of biomarker versus radiological results properly controlled for these biases. However, other factors such as inclusion of different RCC subtypes and median follow-up of <1 year may confound and limit the generalizability of the findings. The small sample size for these subsets cannot rule out that free GAGomes may not be as significantly altered in conjunction with long-term recurrences or in non–clear cell subtypes. Therefore, the generalizability of the here-reported performance of free CS RCC recurrence scores in a clinical setting should wait until evidence of external validation is produced in a more narrowly defined and larger M0 RCC population. This is, for example, investigated in the clinical study AURORAX-0087A (NCT04006405).5We conclude that plasma and urine free GAGomes were altered in association with RCC recurrence and that free CS RCC recurrence scores could substantially improve the follow-up of RCC patients after surgery. External validation of the scores is warranted prior to a wider implementation in the clinical routine.Author contributions: Jens Nielsen, Francesco Gatto and Sven Lundstam had collectively full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.Study concept and design: Gatto, Stierner, Lundstam, Nielsen.Acquisition of data: Lundstam, Stierner, Volpi, Maccari, Galeotti.Analysis and interpretation of data: Gatto, Bratulic, Limeta.Drafting of the manuscript: Gatto.Critical revision of the manuscript for important intellectual content: All authors.Statistical analysis: Gatto, Bratulic.Obtaining funding: Gatto, Nielsen.Administrative, technical, or material support: None.Supervision: Gatto, Stierner, Lundstam, Nielsen.Other: None.Financial disclosures: Jens Nielsen, Francesco Gatto and Sven Lundstam certify that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript , are the following: At the study start, Francesco Gatto and Jens Nielsen were listed as coinventors in patent applications related to the biomarkers described in this study. At the time of submission, Francesco Gatto and Jens Nielsen were shareholders in Elypta AB, which owned the abovementioned patent applications. Francesco Gatto was an employee in Elypta AB, and Jens Nielsen was a board director. Saeed Dabestani received advisory fees from Elypta AB. All other authors declare no relevant conflicts of interest.Funding/Support and role of the sponsor: This work was financially supported by the Knut and Alice Wallenberg Foundation and Cancerfonden to Chalmers University of Technology (Jens Nielsen), and by VINNOVA (#2016-00763) and Västra Götaland Region to Chalmers University of Technology (Francesco Gatto).


This inhibitory effect was comparable to the effect produced by a standard AChE inhibitor physostigmine. One of the known fenugreek constituents, diosgenin, was also tested, and it also showed an AChE inhibitory effect in a concentration-dependent manner (11-190 μg/ml). Interaction between diosgenin and AChE was further investigated by molecular docking and molecular dynamics simulations for 100 ns, which showed that diosgenin interacted with the active-site gorge of AChE through hydrophobic, pi-pi stacking, and hydrogen bonds with various amino acids of the AChE enzyme.  When tested on the baseline of isolated tissues, Tfg.Cr was devoid of any activity (stimulant or relaxant) till 10 mg/ml. This is an interesting finding, keeping in mind that the fenugreek seeds are used to alleviate constipation and diarrhoea. When Tfg.Cr was tried for any potential AChE inhibitory activity, it did show an inhibitory effect in increasing concentrations (47-380  The results show that the fenugreek extract does not possess any GI stimulant or relaxant activity even though it is used traditionally in GI motility disorders. The extract and diosgenin could inhibit the AChE enzyme pointing towards their benefit to enhance the memory. Trigonella foenum-graecum Linn. (family: Fabaceae or Leguminosae), or “fenugreek” as commonly known in English, is a widely used medicinal plant. In Urdu or Hindi, it is called “methi.” An annual herb , 2, fenuFor centuries, fenugreek has popularly been used by herbalists and traditional healers of China and South Asia in several medical conditions, specifically for gastrointestinal (GI) and neurological issues. The seeds are consumed as is or processed (boiled or roasted) for their benefit in dyspepsia, colic, flatulence, diarrhoea, dysentery, anorexia , 2, 4, gMany of the benefits of this plant are linked to the chemicals that have been isolated from it. Fenugreek is known to contain steroidal saponins like diosgenin, tigogenin, trigogenin, and fenugreekine; alkaloids like choline and trigonelline; amino acids like histidine, arginine, tryptophan, and lysine; and vitamins like nicotinic acid , 4, 16. As noted above, several experimental and clinical studies on fenugreek scientifically elucidate its traditional uses. But still, a lot more work needs to be done to discover the many hidden benefits of this herb. This was the idea behind undertaking this endeavour. A 70% aqueous methanolic crude extract was prepared. This extract was tested pharmacologically on gastrointestinal (GI) smooth muscle preparations from rabbits and rats to see if there is any smooth muscle tone modulatory activity in the GI system, a potential acetylcholine- (ACh-) like effect that would lead us to believe in its memory-enhancing effects too. A substance acting like ACh can potentially have both GI stimulant and memory-enhancing effects due to an action similar to ACh on the cholinergic receptors in the GI tract and CNS. The extract was also tested for activity against the acetylcholinesterase (AChE) enzyme in vitro. This is because fenugreek is used to enhance the memory. Although the extract did not exhibit any effect on GI smooth muscle preparations, it did show an inhibitory effect on the AChE enzyme. The AChE inhibitory activity was traced back to its known chemical ingredient, diosgenin . InteracCare was taken to avoid any suffering to the animals used in this study. Experiments were performed ethically in strict accordance with lab animal handling specifications of European Community guidelines, EEC Directive 86/609/EEC. Local rabbits  and Sprague-Dawley rats  were used in this project. These were kept in the animal quarter at the Aga Khan University. The air was pathogen-free, and the temperature was controlled at around 23°C. The rabbits and rats had free access to water, although the food was withheld a day before the experiments. Food given to the animals was made of the following: fiber, table salt, white flour, sweetener, Nutri-vet L, potassium metabisulfite, grease, seafood, and powdered milk.2, 11.90 NaHCO3, 0.42 NaH2PO4, 1.80 CaCl2, and 5.55 glucose.Standard chemicals and reagents were obtained from Sigma Company, USA. These included acetylcholine (ACh), acetylthiocholine (ATCh), 5,5-dithiobis (2-nitro), benzoic acid (DTNB), diosgenin, electric eel AChE (type VI-S), histamine, nicotine, and physostigmine. The solutions and dilutions of these were made on the day of the experiment. To make Tyrode's physiological salt solution, chemicals were purchased from Sigma, USA, and Merck, Germany. Tyrode's was constituted as follows (mM): 2.68 KCl, 136.90 NaCl, 1.05 MgClFenugreek seeds (around a kg) were acquired from a supplier in Karachi, Pakistan. This was botanically identified by Mr. S. Ahmad. A specimen was kept in the herbarium of Natural Products Research Unit, Aga Khan University, for identification and cataloguing (# TF-SE-05-04-59). For making the crude extract, methodology described previously  was used2 and 5% CO2 at physiologic temperature. All changes in contractility were noted isotonically using Harvard equipment (oscillographs and force transducers). The tissues were left to stabilize for half an hour. The advantage of using rabbit jejunum is its ability to contract spontaneously , Tfg.Cr extract (20 μl and diluted in 5% ethanol), and AChE (20 μl) were dissolved and kept for 15 min at 25°C. Then, DTNB (10 μl) was added. The interaction of chemicals initiated with the addition of 10 μl of ATCh (0.71 mM). Breakdown of ATCh was quantified by determining synthesis of 5-thio-2-nitrobenzoate anion  using SpectraMax microplate spectrophotometer . The test for the substances under study was performed at least thrice. The preliminary difference was calculated as the difference in optical density/min and used in the following determination. The test reaction contained test samples, while the control lacked test substance. For comparison, the standard AChE inhibitor was physostigmine.The enzyme inhibitory tests were done in 96-well microtiter plates. Briefly, sodium phosphate buffer  module implemented in Desmond Schrödinger package to record the protein deviation, fluctuation, compactness, and hydrogen bond contacts with their occupancies, while ligand root means square deviation (RMSD), the radius of gyration (rGyr), torsional angle, molecular surface area (MolSA), solvent accessible surface area (SASA), and polar surface area (PSA) were also calculated during the production run of MD simulated time.The investigated docked AChE-diosgenin complex was further expedited to spurt molecular dynamics (MD) production run via the Desmond simulation package. MD simulations of the AChE-diosgenin docked complex were accomplished to reveal the capability of the diosgenin inhibition in the acetylcholinesterase. Receptor topology was generated, and the SPC water model with the specified periodic boundary conditions at a distance of 1.0 nm was set to create an aqueous environment. Furthermore, the solvated receptor charges were neutralized by the addition of required Nan” is observations) and the effective concentration producing 50% inhibition (EC50) with 95% confidence intervals (CI). The graphs were constructed and analyzed utilizing the GraphPad program . Statistical comparisons were made via two-way analysis of variance (ANOVA), and unpaired Student's t-test (p < 0.05) was taken as statistically different (GraphPad program).Data are shown as mean ± standard error of the mean  and nicotine (10 μM) both elicited an immediate stimulant effect on the resting spontaneous contractions  and histamine (10 μM) both exhibited a sharp spasmogenic response on the baseline of rat ileum tissues , inhibited the ACh-degrading enzyme AChE . Tfg.Cr showed a maximum of 48.8 ± 1.3% (n = 4) inhibition of the enzyme . This action of fenugreek aligns with its folkloric consumption to enhance memory [Another reason for using the isolated tissue preparations was to see if the extract can show an ACh-like spasmogenic effect. This is because ACh is a GI stimulant and a major neurotransmitter implicated in the pathophysiology of Alzheimer's disease . Chemicayme AChE . The EC5e enzyme . This ine memory . One stue memory  reports e memory  and cogne memory .μg/ml), inhibited the AChE enzyme . The EC50 of the suppressive action of diosgenin was 27.9 μg/ml . Diosgenin showed a maximum of 53.0 ± 0.4% (n = 4) inhibition of the AChE enzyme . We have earlier reported this AChE inhibitory effect of diosgenin [To determine the responsible compound for this AChE inhibitory action, we tested diosgenin , a steroE enzyme . This acE enzyme . The max/ml 24.9-.1, n = 4ΔG binding energy threshold of −10.7 kcal/mol was observed among all the ranked poses based on the estimated interaction energy. The top ranked conformation of diosgenin according to the highest ΔG binding energy was elected for molecular investigation. Auto-Dock Vina top most ranked pose output revealed that diosgenin established hydrogen-mediated, hydrophobic, and pi-pi stacking contacts with the aromatic amino acid residues of TcAChE of the peripheral anionic site , an anionic subsite (Phe330), catalytic site (Phe331), and acyl pocket residues (Phe288 and Phe290) as illustrated in A molecular modelling study was executed to interpret the binding mode analysis of diosgenin into the possible binding cavity of the TcAChE receptor 7B2W. The docking output with putative interaction site with the highest Molecular dynamic simulation data analysis is the basic quantitative evaluation parameter to analyze the stability, instability, deviations, fluctuations, compactness, and molecular interaction contact analysis of the protein and ligand via conformational geometries and dynamic vibrations and motions.RMSD was used to record the dislocation of the atoms present within the protein and ligand during MD simulated time with the initially generated frame of the pre-MD production run. RMSD plot analyzed for the protein backbone carbon-alpha atoms and the heavy atoms of the ligand during 100 ns of MD executed production runs. RMSD plot of TcAChE receptor 7B2W-diosgenin complex as depicted in The stability of the TcAChE-diosgenin was estimated via a protein-ligand contact histogram plot to check the overall protein-ligand contacts with the active-site residues in terms of interaction fraction pattern. These protein-ligand contacts were also inspected on the simulated trajectories of 100 ns of the production run. The interaction patterns follow H-bonds, hydrophobic, ionic, and water bridging-mediated interactions. H-bonding interactions play a crucial role in drug designing, but in this contrast, hydrogen bonding occurred as 0.74% with Asp72 and <0.1% with the Tyr121 and Tyr334. Hydrophobic contacts were established with Tyr70, Phe75, Trp279, Phe331, and Tyr334 while 0.55% hydrophobic contact with Trp279 and 0.3% and 0.35% fraction contact with Phe331 and Tyr334 were noted. Trp84, Tyr121, and Tyr334 possessed weak hydrogen bonding interactions. Asp72 showed maximum H-bond occupancy throughout the simulation, but in the last 25 ns, MD run. Its contact with diosgenin was diminished and converge into the water-mediated bridging interactions. At the same time, other residues which are mediating interactions with negligible hydrogen bond occupancies were observed negligible as implemented in A timeline representation of the above-mentioned receptor-ligand histogram was further analyzed to check the total number of interaction or molecular contacts in each trajectory record frame of all amino acid residues of the TcAChE, further clarified by the timeline exhibit of active-site residues of the TcAChE-diosgenin complex. The top panel of the timeline representation, as depicted in The ligand properties were studied carefully to understand the conformational repositions utilizing the reference of pre-MD production data. For this purpose, all atoms of the diosgenin used for the detailed analysis such as RMSD, compactness or extendedness, MolSA determination via 1.4 Å probe radius, SASA, the PSA were estimated as shown in 2 to 388Å2, and the equilibrium achieved around 385Å2 is visualized in 2O solvent was also noted. The SASA denoted that the raised value from 80 to 320 Å2 until 20 ns simulation and then afterward became persistent until the end of the simulation with the range of 200Å2 (2 and N atoms. The PSA was in the range of 50Å2-60Å2 throughout the simulation, but there were visible fluctuations occurred at 46 ns to 65 ns simulation period, while the equilibrium attained around 56Å2 inspected in The RMSD of the ligand showed stability throughout the simulation, while minor fluctuations were seen at 50-55 ns after reaching the equilibrium position after 5 ns. The ligand RMSD lay around 0.25 Å to 0.8 Å, while RMSD attained around 0.6 Å during the whole simulation shown in of 200Å2 . The PSADial plot illustrated that the torsion angle variations persisted throughout the whole MD production runs. The bar plot emphasized the dial plots by indicating the probable density of the torsions via utilizing each rotatable bond present in the ligand, as depicted in Molecular docking and post-MD results revealed that diosgenin penetrated in the active-site gorge region of TcAChE and showed almost similar molecular interactions in the static and dynamic mode. The peripheral anionic site aromatic residues Trp279 and Tyr334 showed hydrophobic or pi-pi stacking contacts with diosgenin in both pre- and post-MD stages. Subsequently, new molecular interactions were also observed and mediated hydrogen bond during 100 ns MD run with the hydroxyl moiety of diosgenin via Asp72 with the distances of 1.68 Å and 2.84 Å and Ser81 with the distances of 2.93 Å. The OH group interacted with Asp72- and Ser81-mediated H-bonding interactions could be the reason for enhancing the binding affinity of diosgenin in TcAChE.These results show that although fenugreek is so popularly used traditionally in GI disorders, it exhibited neither a stimulant nor a relaxant effect on the GI smooth muscles. This is an important finding as it rules out several variables and gives valid information to other researchers to try either a different solvent system for the extraction or different animal species to investigate any potential GI effects of this herb. Isolated smooth muscle preparations not only help to elucidate GI effects but also help look for ACh-like effects, which are also relevant in Alzheimer's disease pharmacology. Although it did not affect the isolated tissues, the extract did exhibit an inhibitory effect on the AChE enzyme assay, providing a rationale for its traditional consumption in memory loss. Diosgenin, a known chemical component of fenugreek, also showed a much more potent inhibitory effect on the AChE enzyme assay, indicating that it must be the responsible compound in fenugreek for this effect. After applying in silico approaches, we concluded that diosgenin permeated the active-site gorge of TcAChE and displayed substantially identical molecular interactions in the static and dynamic states obtained from molecular docking and post-MD findings.


Fetal growth restriction (FGR) occurs in up to 10% of pregnancies and is a leading cause of perinatal mortality and neonatal morbidity. Three-dimensional ultrasonography of intracranial structure volume revealed significant differences between fetuses with FGR and appropriate for gestational age (AGA) fetuses. We aimed to compare the frontal lobe development between fetuses with FGR and appropriately grown fetuses and evaluate the impact of fetal circulatory redistribution (FCR) on frontal lobe development in fetuses with FGR.We performed a case–control study at our institution from August 2020 to April 2021. The frontal antero-posterior diameter (FAPD) and occipito-frontal diameter (OFD) were measured on the trans-ventricle view and we calculated the Z-scores for FAPD and OFD standardized for gestational age (GA) and transverse cerebellar diameter (TCD) by performing a standard regression analysis followed by weighted regression of absolute residual values in appropriately grown fetuses. We calculated the FAPD/OFD ratio as 100 × FAPD/OFD and FAPD/HC (head circumference) as 100 × FAPD/HC. To compare intracranial parameters, we randomly selected a control group of appropriately grown fetuses matched with the FGR group at the time of ultrasonography. We performed between-group comparisons of the FAPD Z-score, OFD Z-score, FAPD/OFD ratio and FAPD/HC. Similarly, we compared intracranial parameters between fetuses with FGR with and without FCR.FAPD/OFD ratio was curvilinear related to all the independent variables . Compared with appropriately grown fetuses, fetuses with FGR showed a significantly lower FAPD/OFD ratio, FAPD Z-score, and FAPD/HC. There was no significant difference in the FAPD Z-score, FAPD/OFD ratio, and FAPD/HC between FGR fetuses with and without FCR.The FAPD/OFD ratio varied during pregnancy, with a mild reduction before and a mild increase after about 33 gestational weeks. Fetuses with FGR showed reduced frontal lobe growth; moreover, fetal frontal lobe development disorders were not significantly different in fetuses with FCR.Date: 09–27-2017; Number: [2017]239. The SDs or standard errors (SEs) for the variables were reported when necessary. Statistical significance was set at nd or 3rd trimester screening in our institution. Among 141 fetuses with FGR, 88 met the inclusion criteria and had available outcome data. Finally, the case–control analyses included 88 fetuses with FGR and 352 GA-matched appropriately grown fetuses  in 437 appropriately grown fetuses, with means of 34.7, 84.5, and 40.92, respectively. Comparisons among different regression models of the FAPD and OFD based on the independent variables (GA and TCD) are shown in Table The FAPD, OFD, and FAPD/OFD ratios were normally distributed   and FAPD and OFD. However, the residuals were non-normally distributed for the cubic regression models. Therefore, we chose a logarithmic regression model for establishing the Z-score reference ranges for the FAPD and OFD. There were clear relationships of TCD with FAPD and OFD in appropriately grown fetuses (ly) Fig. . Table 3The mean FAPD/OFD ratio was 40.92 ± 1.25 in 437 appropriately grown fetuses. There was a curvilinear association between the FAPD/OFD ratio and all independent variables   than in appropriately grown fetuses (0.17 ± 1.20) (P < 0.001). The mean FAPD/OFD ratio was significantly lower in the FGR cohort (37.33 ± 1.31) than in the control cohort (40.62 ± 1.00) (P < 0.001). Table The FAPD and OFD Z-scores standardized for TCD were used for comparisons because the volume of the cerebellum is not influenced by impaired fetal growth.P = 0.383). The FAPD/HC was not significantly different between fetuses with FGR with and without FCR . Table ; P = 0.116).There was no significant difference in the distribution of FAPD/OFD ratios between fetuses with FGR with and without FCR (37.56 ± 1.16 and 37.20 ± 1.40, respectively; The intra- and interobserver ICCs for the FAPD and OFD were > 0.95, indicating good reliability. Table The FAPD/OFD ratio varied during pregnancy, with a mild reduction before and a mild increase after about 33 gestational weeks. FGR showed reduced frontal lobe growth. Moreover, based on a comparison of FGR fetuses with and without FCR, FCR may not be significantly associated with development disorders of the fetal frontal lobe.Infants born at term or preterm with birth weights lower than the AGA values are at higher risk of developmental impairments than those with birth weights AGA . NeurodeCognitive and academic deficits occur in children with FGR aged 6–8 years, even when born at term . Early ird or 6th month of fetal life. Myelination sequentially proceeds from the internal capsule to the occipital, splenium, parietal, body of corpus callosum, temporal, frontal, and genu white matter regions [Examining variation patterns in the FAPD and FAPD/OFD ratio can reveal changes in the fetal frontal lobe during pregnancy. We found that the FAPD/OFD ratio is relatively fixed; however, it showed a mild variation during fetal development. These changes suggest that growth velocities differ across different brain regions. Myelination begins from the 3 regions , 33. VarWe observed a non-significant reduction in brain hemodynamic redistribution in the frontal lobe region in FGR fetuses with FCR when compared with those without FCR. There was no difference in the FAPD Z-score and FAPD/OFD ratio between fetuses with FGR with and without FCR. We speculate that in the FGR fetuses with FCR, the frontal lobe growth is reduced but it is not more severe than without FCR. However, a systematic review published in 2014 reported that children with FGR with FCR had more severe neurodevelopmental impairment than those without FCR . For fetAlthough the delivery timing is a potential modifier of the association between FGR and neurodevelopmental impairment , 34, we Our study has several strengths. We focused on a clinically important population, namely fetuses with growth restriction with poor neurodevelopmental prognosis. The FAPD/OFD ratio, as a predictor, is flexible and convenient and can be easily obtained on the trans-ventricle view during BPD and HC measurements. The FAPD/OFD ratio has not been used to assess fetal frontal lobe development in fetuses with FGR. We also established the Z-score reference range of FAPD and varying pattern of the FAPD/OFD ratio in appropriately grown fetuses to evaluate the fetal frontal lobe in other diseases that may impair its development.th percentile. Furthermore, we have to admit that we cannot discriminate between SGA and FGR in this study.This study has several limitations. First, we did not evaluate neurodevelopment in infants with FGR. Second, a longitudinal design with repeated measurements, which may facilitate elucidation of FAPD/OFD ratio variations in fetuses with FGR, was not performed. We think we may benefit from a longitudinal analysis with different sampling moments to assess the changes of FAPD/OFD ratio. Finally, there are other societies and experts, such as the ACOG , suggestIn conclusion, this study provides a marker, FAPD/OFD ratio, for evaluating fetal frontal lobe development in appropriately grown fetuses and fetuses with FGR. Fetuses with FGR showed a markedly lower FAPD Z-score and FAPD/OFD ratio than appropriately grown fetuses. Furthermore, FCR was not associated with frontal lobe development.