The CHD among different sexes differed significantly . However, we found no statistical differences in GHD with different sexes . In terms of age correlation, a negative curve correlation existed between age and AHD among the different sexes . A negative linear correlation was found in CHD among the different sexes . There were no differences between age and minimum GHD in both sexes. The 3D CT reconstruction model can accurately measure the minimum AHD, CHD, and GHD value in vivo and is worthy of further investigation for standard clinical anatomical assessment. Aging may correlate with AHD and CHD narrowing for both sexes.Accurate measurement of the minimum distance between bony structures of the humeral head and the acromion or coracoid helps advance a better understanding of the shoulder anatomical features. Our goal was to precisely determine the minimum acromiohumeral distance (AHD), coracohumeral distance (CHD), and glenohumeral distance (GHD) in a sample of the Chinese population as an in vivo anatomical analysis. We retrospectively included 146 patients who underwent supine computed tomography (CT) examination of the shoulder joint. The minimum AHD, CHD, and GHD values were quantitatively measured using three-dimensional (3D) CT reconstruction techniques. The correlation between minimum AHD, CHD, and GHD value and age with different sexes was evaluated using Pearson Correlation Coefficient. The mean value of minimum AHD in males was greater than that in females (male 7.62 ± 0.98 mm versus female 7.27 ± 0.86 mm,  Shoulder joint stability maintenance requires a certain bone-to-bone distance to avoid bony impingement. It has been found that the variations of acromiohumeral distance (AHD), coracohumeral distance (CHD), and glenohumeral distance (GHD) are closely related to the normal function and biomechanical stability of the shoulder joint ,3,4,5,6.Biological sex-associated anatomical differences exist in the shoulder anatomical features. Giaroli et al.  and YoshThere are significant skeletal anatomical differences present in different geographical populations, therefore, the humeral head-related bone-to-bone distance could vary widely across geographic groups. Zhang et al.  measuredWe therefore retrospectively measured the minimum AHD, CHD, and GHD of the healthy shoulder joint in the supine position using our clinical data. By using 3D reconstruction techniques, the minimum AHD, CHD, and GHD in a sample of Chinese healthy shoulders among groups of different sexes would be precisely assessed. Of note, degeneration with age was thought to be the main cause of changes in the scapula-humeral head interval, we, therefore, hypothesized that a negative correlation may exist between age and AHD, CHD, or GHD with a different sex. This retrospective in vivo anatomical study would complement the knowledge of the shoulder anatomical values in a sample of the Chinese population.All procedures performed in studies involving human participants were in accordance with the ethical standards of the Third Affiliated Hospital of Sun Yat-sen University ethics committee and with the 1975 Helsinki Declaration and its later amendments or comparable ethical standards. The protocol was previously reviewed and approved by the Third Affiliated Hospital of Sun Yat-sen University Ethics and Research Committees ([2022] 02-013-01).Between January 2011 and December 2021, a total of 1713 patients underwent CT examinations of the shoulder joint. During the study period, all patients had standard shoulder radiographs and supine shoulder CT images taken during their preoperative examination. All images were retrospectively reviewed by two senior orthopedic surgeons (TZL and JHR) and findings were recorded independently. Discrepancies in interpretation were resolved by consensus. The inclusion criteria for this study were patients with normal standard shoulder radiographs and supine shoulder CT images. Exclusion criteria were patients with shoulder fractures, rotator cuff tears, shoulder instability, shoulder deformities, shoulder osteoarthritis, upper limb bone tumors, scoliosis deformities, and immunologically related disorders or those aged less than 20 years. In this research, 1399 were excluded because of the history of shoulder fractures, rotator cuff tears, shoulder instability, shoulder deformities, shoulder osteoarthritis, upper limb bone tumors, scoliosis deformities, immunology-related diseases, and poor-quality imaging. Additionally,18 patients were excluded because they were younger than 20 years of age . In addiImaging of the shoulders was acquired for each patient using a 64-row CT scanner . The CT data were accumulated in Digital Imaging and Communication in Medicine (DICOM) data format. The 3D bone surface models of the acromion, coracoid process, glenoid cavity, and proximal humerus were extracted from DICOM data using Mimics software  and measured minimum AHD, CHD, and GHD using Meshlab software  ,23,36. Op > 0.05). The means and SDs of the geometric parameters were calculated and Bar graphs were plotted to show the distributions of the parameters. Independent-samples t-tests were used to assess whether there was a difference between AHD, CHD, and GHD when the humeral head was internally and externally rotated. The same method was also used to compare differences related to sex, but differences in AHD, CHD, and GHD were evaluated separately for males and females at different ages using the Mann–Whitney U test. Age-related differences were compared using a One-Way ANOVA, if the result was significant, post hoc range tests and pairwise multiple comparisons were applied and the associated line graphs were plotted. Pearson correlation coefficients and curve estimation were used to assess the correlation between the values of AHD, CHD, and GHD with the age of the participants. The results of this study were compared with those of Eastern and Western population cohorts derived by combining several series from the published literature, and the sample sizes, means, and SDs of the individual studies are presented in the tables [p < 0.05. SPSS Statistics 22.0.0.0 software  was used for the statistical analyses. The data for AHD, CHD, and GHD for males and females showed a normal distribution using the Shapiro–Wilk test . Internal rotation of the humeral head was noted in 71 cases with a mean rotation angle of 5.96 ± 2.87° (6.15 ± 2.89° in men and 5.55 ± 2.83° in women). External rotation of the humeral head was observed in 75 cases with a mean rotation angle of 6.03 ± 2.89°  . Of the p = 0.046)  . The disp < 0.05). In the female groups, AHD values in the 20–29, the 40–49, and the 50–59 years age groups were found significantly greater than that in the >69 years age group (p < 0.05)  and in females , respectively  . In termectively .p < 0.0001) (p = 0.001) and 50–59 (p = 0.047) years old age groups  . The dise groups .p < 0.05). In the female groups, CHD values in group 30–39 years were significantly higher than the age group 50–59 years, 60–69 years, and >69 years, respectively (p < 0.05)  and in females , respectively  . Furtherectively . These rWe found that the mean GHD is 2.00 ± 0.31 mm in men and 1.96 ± 0.36 mm in women, respectively, showing no statistical difference . In addiIn the current study, we accurately measured the minimum AHD, CHD, and GHD values in vivo by using a 3D CT reconstruction model. The primary findings of this study were negative correlation existed between age and AHD, CHD with both sexes, although changes in GHD did not statistically differ by age or sex.In this study, regression analysis showed that increasing patient age was associated with the narrowing of AHD and CHD, while no significant correlation was seen with GHD. This age-related difference is most evidenced when comparing the shoulders parameters of the young with those of the elderly. Previous results indicated that the shape of the acromion does not change with age, however, the proliferation of bone spurs and aging of the ligaments at the acromion and coracoid process can lead to the narrowing of the subacromial and subcoracoid gaps with age ,38,39,40Although biological sex-associated anatomical differences exist in the shoulder anatomical features, we did not find a correlation between GHD variance and sex differences. Of note, statistical differences in sex were found only in the 30–39 age group for AHD and in the 40–49 and 50–59 age groups for CHD. In the current study, the mean values for AHD and CHD were significantly greater in men than in women. This is consistent with previous reports that sex differences should be taken into consideration in orthopedic studies ,41. In aPrevious studies have shown that there are significant skeletal anatomical differences between populations from different geographical regions ,24,25. CFrom a clinical aspect, variations in the subacromial and subcoracoid space can have an impact on the impingement of the rotator cuff tendons and bursa, which were first described by Neer  and GerbThere are some limitations to this study. Firstly, we report measurement values in 146 patients, which are not sufficient for generalization to the general population, but a multi-center study is being designed in the near future. Secondly, we did not conduct an inter-investigator reliability analysis, which may affect the reproducibility of the results. However, the study was conducted in its entirety by two orthopedic researchers and the measurement of values was entirely automated by the software. Finally, controversy still exists regarding the advantages and disadvantages of different methods of measuring the scapula-humeral head distance, and the prone position also significantly reduces inter-articular distance. Although the standard shoulder joint spacing measurement is variable, we here try a possible accurate method to measure the most realistic joint spacing to provide a theoretical basis for helping the future diagnosis of clinically shoulder joint-relevant diseases.The 3D CT reconstruction model can accurately measure the minimum AHD, CHD, and GHD values in vivo and is worthy of further investigation for standard anatomical assessment. Aging may correlate with AHD and CHD narrowing in both sexes in a sample of the Chinese population.


Bombus breviceps is an important native species of bumblebee in southern China. As a pollinator, it has the potential for domestication and commercial rearing. In this study, in order to identify the nutritional requirements for the egg-laying, hatching, and colony foundation stages, we selected three common local types of pollen, including camellia pollen, oilseed rape pollen, and apricot pollen, as well as mixtures of them, to feed B. breviceps queens to evaluate the reproductive performance, and feasibility of these pollens as a diet and the nutritional requirements. The results showed that the pollen with a higher essential amino acid content had advantages in laying time, the number of eggs laid, larval ejection, and the time of first worker emergence, while the pollen with a higher crude protein content had advantages in the time the colony took to reach ten workers. These results may guide the selection of feed in B. breviceps artificial feeding and help to explore the nutritional requirements in oviposition and the colony stage from the perspective of conventional pollen.Bombus breviceps colonies, camellia pollen, oilseed rape pollen, apricot pollen, and mixtures of two or three types of pollen in equal proportions were used to feed the queens in this study. The results showed that the camellia pollen with a higher essential amino acid content was superior to the pollen with a lower essential amino acid content in the initial egg-laying time (p < 0.05), egg number (p < 0.05), larval ejection (p < 0.01), time of first worker emergence (p < 0.05), and the average weight of workers in the first batch (p < 0.01). It took less time for colonies under the camellia pollen and camellia–oilseed rape–apricot pollen mix treatments, both with a higher crude protein content, to reach ten workers in the colony (p < 0.01). On the contrary, the queens fed apricot pollen never laid an egg, and larvae fed oilseed rape pollen were all ejected—both pollens with a lower essential amino acid content. The results emphasize that the diet should be rationally allocated to meet the nutritional needs of local bumblebees at various stages when guiding them to lay eggs, hatch, and develop a colony.Pollen is an important source of nutrition for bumblebees to survive, reproduce, and raise their offspring. To explore the nutritional requirements for the egg laying and hatching of queenright  Bombus terrestris, as the most successful commercial bumblebee in Europe, has been widely introduced into many countries for pollination [B. terrestris, as an alien species, can escape or be deliberately released during pollination, potentially spreading new diseases and parasites [B. terrestris naturalized [Bombus impatiens and Bombus occidentalis in North America [Bombus ignitus in South Korea [Bombus hypocrita and Bombus ignitus in Japan [Bombus ignitus, Bombus lucorum, Bombus pyrosoma, Bombus disciplines, Bombus lantschoolensis and Bombus patagiatus in northern China [B. terrestris [Bombus breviceps, as a native Chinese species mainly distributed in southern China [B. breviceps, having higher colony foundation rates, a large number of workers, and new virgin queens, is a promising candidate for domestication and commercial rearing [Bumblebees are important pollinators of many wild plants and crops, and they play vital roles in natural and agricultural ecosystems ,2. In relination ,4. Howevarasites ,6 and coarasites ,8, whicharasites ,10,11. Buralized ,12,13,14 America , Bombus th Korea , Bombus in Japan ,18,19, arn China ,21. Howerrestris , have norn China ,24, is arn China ,26. Rece rearing .B. terrestris microcolonies, salix and camellia pollens with a higher amino acid content led to a higher number of eggs laid, and living larvae, and a lower rate of larva ejection, respectively [B. terrestris productivity and the size and longevity of male offspring [B. impatiens workers [B. breviceps feeding did not refer to the required pollen and nutrition [B. breviceps in oviposition and the colony stage from the perspective of conventional pollen.The diet and nutritional requirements are key factors for the artificial feeding of bumblebees. In wild nature, bumblebees obtain their macronutrients including carbohydrates, proteins, and lipids from floral nectar and pollen, to meet their nutritional demands . Pollen ectively ,32. Furtectively . Moreoveffspring  and reduffspring . Pollen  workers . Nutrien workers ,37. Howeutrition ,39. TherB. terrestris [B. impatiens [The research on the nutritional requirements of bumblebees mainly focuses on microcolonies of rrestris ,40,41 ormpatiens ,42,43, wmpatiens . There ampatiens ,45; howempatiens ,46,47.B. breviceps is to find the nutrients it needs. Therefore, in this study, three types of dried pollen, including camellia pollen, oilseed rape pollen, and apricot pollen, were selected; these pollens are widely distributed in southern China and easily available. Then, these pollens, and their equivalent mixtures, were fed to B. breviceps queens to assess their performance, in order to provide a theoretical basis for the nutritional requirements of local bumblebees in reproduction and colony development.The key to artificially breeding the native bumblebee species B. breviceps in the wild were collected in the middle of the spring of 2021, in Guizhou Province, China. The queens and colonies were reared until virgin queens emerged. When the virgin queens matured, the queen mated with a drone from a different colony; method reference [B. breviceps with a similar size, used for the colony foundation, were hibernated for 60 days at 4.5 ± 0.5 °C and 70% ± 5% relative humidity [The queens of eference . The queeference , in the humidity . Sixty-thumidity .Three pre-selected commercially sourced dried pollens, all collected from honeybee colonies with pollen traps, were purchased at one time from beekeepers. The three pollens were camellia pollen, oilseed rape pollen, and apricot pollen, which were obtained as camellia pollen mix, oilseed rape pollen mix, and apricot pollen mix with high proportions of a major pollen species and lower other diverse pollen species according to palynological analysis. We obtained seven pollen treatments as the diets, based on the three single pollen mixes and mixtures of two or three single pollen mixes in equal proportion. The seven experimental treatments were as follows: C: camellia pollen mix; O: oilseed rape pollen mix; A: apricot pollen mix; C–O: 50%:50% mixture of camellia pollen mix and oilseed rape pollen mix; C–A: 50%:50% mixture of camellia pollen mix and apricot pollen mix; O–A: 50%:50% mixture of oilseed rape pollen mix and apricot pollen mix; C–O–A: 1/3:1/3:1/3 mixture of the above three single pollen mixes. Each pollen treatment was well mixed again before being broken down, and then homogenized and stored in a freezer at −20 °C, in preparation for palynological analysis and feeding treatment. One of seven pollen treatments was offered to the queens and colonies (N = 9 queens per treatment).According to Smyth , a samplThree subsamples (5–10 g) of each pollen mix treatment were tested in terms of nutritional composition, including crude protein content, lipid content, carbohydrate content, and amino acid content. The Bradford assay was used to calculate the protein concentration, and a modified assay from Van Handel and Day  was usedDuring the rearing period, the performance and state of the queens and queenright colonies were observed and recorded at a fixed time every day, including behaviors before egg hatching (nest building and egg laying), and the development of larvae, pupae, and workers. Thereinto, nine paracmeters were used to estimate colony performance for the treatment diets: (1) initial nesting time ; (2) initial egg-laying time ; (3) number of eggs laid (the number of eggs laid by queens in the first batch); (4) the duration of larval development ; (5) number of larvae ejected (the number of larvae ejected until the first worker emerged); (6) duration of pupal development ; (7) time of first worker emergence ; (8) the average weight of workers in the first batch; (9) the time it took the colony to reach ten workers . The terminology of other behaviors follows ,32.p less than 0.05 and 0.01 indicate a significant difference and an extremely significant difference, respectively. Individual missing data were replaced with the group average. Data that did not conform to the normal distribution were converted with log or sqrt transformations.One-way ANOVA in SPSS 26.0 software was used to analyze the content of major nutrients in the pollen and the nine parameters of colony performance. Tukey post hoc tests were used to confirm where significant differences occurred between treatments. The results are expressed as the mean ± SE. The generalized linear model (GLM) method was used to analyze the data among parameters in colony performance and pollen nutrients. GLM selection was based on the Akaike information criterion (AIC). GraphPad Prism 9.4 was used for illustration. Values of Camellia sp. and the remainder dominated by Brassica sp., Armeniaca sp. Lactuca sativa and Acer sp.  in all pollens ranged from 4.69 to 11.35 . The carF = 98.98, df = 6, p < 0.01; p < 0.01; p > 0.05; The essential amino acid (EAA) content ranged from 1.27% to 1.41%. The camellia pollen mix had a significantly higher EEA content than the other treatments  ranged from 0.75% to 0.92%. There was a significant difference in the NEAA content between the treatments  contents among the seven treatments . There was no significant difference between the camellia–oilseed rape pollen mix and camellia–apricot pollen mix , but with no difference from the camellia–oilseed rape–apricot pollen mix . Pollen essential amino acid content, as a predictive variable, better predicted the initial egg-laying time  and the number of eggs laid . Lipid content better predicted the development duration of larvae  and pupae . Pollen essential amino acid content, as a key variable, better predicted the time of first worker emergence  and the average weight of workers in the first batch . Moreover, pollen crude protein content significantly impacted the time the colony took to reach ten workers . The effects of total amino acid and non-essential amino acid contents and protein–lipid, carbohydrate–lipid, and carbohydrate–protein ratios on reproductive performance in each treatment were not significant (p > 0.05).The relationship between reproduction performance and pollen nutrition was analyzed based on datasets of the major nutrients and their proportions and nine parameters in the seven pollen treatments. Pollen carbohydrate content significantly impacted the initial nesting time (B. breviceps queens and to explore the nutritional needs of their growth and reproduction.Pollen contains protein, lipids, carbohydrates, sterols, vitamins, and minerals , which aB. breviceps queens and colonies. In our study, the queens feeding on the apricot pollen mix did not lay eggs, while the queens feeding on the oilseed rape pollen mix and oilseed rape–apricot pollen mix did lay eggs; nevertheless, all larvae were ejected until the queens died. Fortunately, the queens feeding on the camellia pollen mix with a higher amino acid content and higher protein content had a significant advantage in the initial egg-laying time, the number of eggs laid, duration of larval development, the number of larvae ejected, time of first worker emergence, the average weight of workers in the first batch and the time the colony took to reach ten workers. The queens feeding on the oilseed rape–apricot pollen mix had advantages in the initial nesting time, and the queens feeding on the camellia–apricot pollen mix had a distinct advantage in the duration of larval and pupal development.According to the performance of the queens and colonies feeding on different pollens, we found that not all pollen treatments can meet the growth and development needs of B. terrestris microcolony. The workers with higher protein had higher productivity, and the drones were heavier [B. terrestris colony was fed pollens containing 22%, 18%, and 14% crude protein; the results showed that the pollen group with 22% protein content had lower larval mortality and heavier weighted workers than the pollen group with 14% protein content [B. breviceps queens and colonies. On the contrary, the lower crude protein and essential amino acid contents in the apricot pollen mix cause B. breviceps queens not to lay eggs, as well as their death. However, the relationship based on the major nutrients and their proportions and the nine parameters in the pollen shows that pollen with a higher essential amino acid content has advantages in the initial egg-laying time, the number of eggs laid, time of first worker emergence, and the average weight of workers in the first batch, while a higher protein content can only accelerate the colony development. Although high-protein pollens generally contribute more to colony growth and development than low-protein pollens [The protein content is generally used to estimate the pollen quality and nutritive value for bumblebees; high-protein pollen seems to reduce the mortality of larvae and females and the larval ejection rate, and increase the offspring number and larval weight . Oilseed heavier . In anot content . In our  pollens ,59, the  pollens ,60. Ther pollens ,62.B. terrestris had the worst performance in the initial egg-laying time when feeding on rape pollen with a lower essential amino acid content; in contrast, the performance of the microcolony was better in the initial egg-laying time and the number of larvae ejected when feeding on camellia pollen with a higher essential amino acid content [Actinidia deliciosa, Cistus sp., and Salix sp., with different contents of essential amino acids were used to feed a B. terrestris microcolony. The results showed that the pollen with a higher content of essential amino acids had higher egg production, the lowest number of ejected larvae, and the heaviest pupae [Some studies have shown that the content of amino acids in pollen is the main driving factor for the normal development of bumblebees ,31, espe content . Previoust pupae , promotest pupae . Thus, qB. terrestris colony, and they found that the number of offspring with a higher protein content increased faster [Similar to some research results, our results suggest that it took a shorter amount of time for the colony to reach ten workers under the camellia pollen mix and camellia–oilseed rape–apricot pollen mix, with higher protein contents. Wynants et al. reported that protein contents of 21.8%, 21.4%, 18.4%, and 11.9% were fed to a queenright d faster . This maB. terrestris workers and 12:1 P:L for B. impatiens workers when fed in limited conditions [The lipid content of pollen is also a factor affecting the development of bumblebees . In our nditions . TherefoB. breviceps microcolony fed different sugar solutions, the pre-oviposition period of the bumblebees varied with the type of sugar syrup [The effects of carbohydrates in pollen on bumblebees have been poorly studied. In one report of a queenright ar syrup . In our ar syrup . This maB. breviceps and B. terrestris. More specifically, the mortality rate of the queens was higher compared with the female mortality of B. terrestris when raised with or without a queen [B. breviceps and B. terrestris in previous research. For example, Liang et al. and Qin reported that the average egg-laying time of B. breviceps was 13.6 days and 8.9–15.2 days at different collection sites in queenright colonies, respectively [B. terrestris queen’s average initial egg-laying time ranges from 11.3 to 12.5 days [B. terrestris, workers are often added to stimulate queen ovarian development to speed up egg production [In addition, in our study, the performance of the queens and queenright colonies was worse than that found in other studies on  a queen ,35. Some a queen ,69 or th a queen . Therefoectively ,39, but 2.5 days . The timoduction . Howeveroduction . TherefoB. breviceps colonies, such as the initial egg-laying time, the average weight of the first batch of workers, the time of first worker emergence and the time the colony takes to reach ten workers. However, it is noteworthy that our research is restricted to the main macroelement nutrients, while other nutrients are not considered, such as sterol concentration [B. breviceps in terms of growth and reproduction.To sum up, the content and proportion of major nutrients in different pollens, especially the contents of essential amino acids and protein, seem to lead to differences in the indicators of queenright ntration , fatty antration , taste [ntration , and digntration . TherefoB. breviceps different pollens from the perspective of some major nutrients. The results showed that the camellia pollen mix with a high essential amino acid content accelerated B. breviceps queens’ initial egg-laying time, increased the number of eggs and the average weight of workers in the first batch, and reduced the rate of larval ejection and the time of first worker emergence, while the camellia pollen and camellia–oilseed rape–apricot pollen mix with a high protein content helped the colony foundation of the workers. The results provide data for ascertaining the reproductive nutritional needs of local bumblebee species and, further, for protecting local bumblebee species.This study evaluated the feasibility of feeding queens


Cytochrome P450 17A1 (CYP17A1) is one of the key enzymes in steroidogenesis that produces dehydroepiandrosterone (DHEA) from cholesterol. Abnormal DHEA production may lead to the progression of severe diseases, such as prostatic and breast cancers. Thus, CYP17A1 is a druggable target for anti-cancer molecule development. In this study, cheminformatic analyses and quantitative structure–activity relationship (QSAR) modeling were applied on a set of 962 CYP17A1 inhibitors  compiled from the ChEMBL database. For steroidal inhibitors, a QSAR classification model built using the PubChem fingerprint along with the extra trees algorithm achieved the best performance, reflected by the accuracy values of 0.933, 0.818, and 0.833 for the training, cross-validation, and test sets, respectively. For nonsteroidal inhibitors, a systematic cheminformatic analysis was applied for exploring the chemical space, Murcko scaffolds, and structure–activity relationships (SARs) for visualizing distributions, patterns, and representative scaffolds for drug discoveries. Furthermore, seven total QSAR classification models were established based on the nonsteroidal scaffolds, and two activity cliff (AC) generators were identified. The best performing model out of these seven was model VIII, which is built upon the PubChem fingerprint along with the random forest algorithm. It achieved a robust accuracy across the training set, the cross-validation set, and the test set, i.e., 0.96, 0.92, and 0.913, respectively. It is anticipated that the results presented herein would be instrumental for further CYP17A1 inhibitor drug discovery efforts. Prostate cancer is highly prevalent in men worldwide. The androgen–androgen receptor axis is essential for disease progression. Thus, androgen-deprivation therapy, either medically or surgically, is a front-line prostate cancer treatment. However, after a median of 24 months of androgen-deprivation therapy, treatment resistance is inevitable, as demonstrated by a relapse in serum prostate-specific antigen levels . DehydroA series of small molecules has been reported as CYP17A1 inhibitors. Ketoconazole is the first nonsteroidal CYP17A1 inhibitor used for prostate cancer treatment, with poor selectivity and mild androgen receptor agonist effects . AbirateComputer-aided drug discoveries are indispensable operations in the entire process of drug discovery. As a multidisciplinary field, it involves data science, statistics, pharmacology, medicinal chemistry, structural biology and computational biology, etc. Ligand-based drug discoveries are common approaches used in computer-aided drug discovery. This focuses on ligands, which are chemical entities that can bind to the biological target, while information with respect to the target is not required. Ligand-based drug discovery can be applied to identify hits or to optimize hits/leads using two important approaches, namely the quantitative structure–activity/property relationship (QSAR/QSPR) and pharmacophore modeling . The forThe essence of QSAR is based on two major principles: (i) structure dictates activity and (ii) molecules with similar structures demonstrate similar bioactivities or properties. As a methodology in chemistry and drug discovery, QSAR/QSPR experienced remarkable transformations since its introduction 60 years ago. As early as the time of Corwin Hansch, QSAR/QSPR modeling operations were performed on a small number of molecules, with few molecular descriptors, employing multilinear regression. Now, thanks to the development of information technology and artificial intelligence, QSAR/QSPR has evolved with respect to the application of a large dataset, equipped with sophisticated molecular descriptors, advanced machine learning algorithms, and various validation techniques. The Organization of Economic Cooperation and Development established principles for QSAR modeling consisting of five rules: defined endpoint, unambiguous algorithms, defined applicability domain, modeling validation, and mechanistic interpretation, if possible. This involves various steps, including data compilation, data splitting, the machine learning process, an evaluation of the robustness and predictability of the model, and the mechanistic interpretation of feature importance ,8, etc.50 ≥ 8), active (8 > pIC50 ≥ 7), intermediate (7 > pIC50 ≥ 6), and inactive (pIC50 < 6), we built one QSAR classification model (model I) to reveal the interrelation between steroidal inhibitor structures and the associated activities. The structures were represented using PubChem and KlekotaRothCount fingerprints provided by the PaDEL package [In this study, we performed systematic cheminformatic analyses and machine learning modeling techniques to explore the chemical space of CYP17A1 inhibitors. By separating the activities into four categories, viz., potent  for bioactivity predictions. The findings in this study can provide guidance, leading the optimization of further CYP17A1 inhibitor drug discoveries.50 ≥ 7.0) were employed in the analysis. Results are shown in Exploratory data analysis was performed to compare nonsteroidal and steroidal molecules based on six drug-likeness descriptors, including the molecular weight (MW), the octanol–water partition coefficient (LogP), the number of hydrogen bond acceptors (nHA), the number of hydrogen bond donors (nHD), the number of rotatable bonds (nRot), and the topological polar surface area (TPSA). Only potent and active inhibitors  were performed over the distribution of the six drug-likeness properties, as mentioned in After the initial analysis, a classification QSAR model (model I) was built to predict the bioactivity class for a steroidal compound. PubChem fingerprints were employed for generating the fingerprint information of steroidal CYP17A1 inhibitors, and 12 representative classification algorithms were tested to construct the model with the best performance. The tested algorithms included the decision tree (DT), extra trees (ET), random forest (RF), gradient boost (GB), light gradient boosting machine (LGBM), extreme gradient boost (XGB), multilayer perceptron (MLP), logistic regression (LR), K-nearest neighbor (KNN), support vector machine (SVM), naïve-Bayes (NB), and Gaussian process (GP) algorithms. The descriptions for these algorithms are provided in the Materials and Methods . The perThe performances of the 12 algorithms are listed in Notably, algorithms, such as RF, DT, XGB, LGBM, GB, MLP, and GP, also performed quite well. Now, one may notice that the performance of the test set is quite good and is comparable with the performance of the cross-validation set or even the performance of the training set. This can be explained by how these data are distributed in the chemical space. Following the previous success in QSAR modeling for steroidal compounds, we planned to carry out the same modeling operation for nonsteroidal molecules. However, the chemical structures of nonsteroidal molecules are more diverse, i.e., there are many different scaffolds. Thus, the underlying scaffolds must be identified before the QSAR models can be constructed. To achieve this, we visualized the nonsteroidal scaffolds, analyzed the level of diversity, and calculated the scaffold enrichment factor.50 ≥ 7.0 (active and potent molecules) demonstrated lower scaffold diversity than those with a pIC50 < 7.0. Hence, finding more novel scaffolds for androgen receptor antagonists is desired.50) thus can be visualized for each scaffold. 50 of scaffolds with a frequency of occurrence (Murcko frequency) equal to or larger than 10. Among the 11 scaffolds in the figure, 10 of them showed datapoints with a pIC50 ≥ 7, viz., a scaffold containing active or potent compounds. In particular, some scaffolds contained only active or potent molecules, e.g., structures in the middle of the panel with all datapoints with a pIC50 ≥ 7. These scaffolds are worthy of further analyses. A complete scatter plot of the Murcko scaffold vs. pIC50 for 683 nonsteroidal inhibitors can be found in the Using Murcko scaffolds, nonsteroidal molecules can be sorted based on their structures. The distribution of the bioactivity . All molecules in this scaffold series are strong inhibitors regardless of the substitutions.Scaffolds 9 and 10. These scaffolds are steroid-like, with ring A replaced by a seven-membered ring. All molecules in these scaffolds are highly active against CYP17A1 regardless of the substitutions.Scaffold 14 only has one core fragment (14-1). Molecules with methyl groups on the R1 or R2 positions are active.Scaffold 15 only has one core fragment (15-1). All molecules in this scaffold are potent or active against CYP17A1. All functional groups at the R1 position are sulfonyl groups.Scaffolds 16, 17, 18, and 19 are similar scaffolds sharing the same cyclic skeleton. All molecules in scaffold 18 are either potent or active against CYP17A1 regardless of the substitution. For other scaffolds, compounds with fluoride-containing groups on R1 or R2 positions are active.Scaffold 20 has only one core fragment (20-1). All molecules in this scaffold are potent or active against CYP17A1, and all functional groups on the R1 position contain the carbonyl group.Based on the 20 scaffolds, we can further discuss how the functional group substitution affects the bioactivities. For this purpose, the core fragments of each scaffold are depicted in Prior to QSAR modeling for nonsteroidal inhibitors, structure–activity similarity (SAS) maps were visualized to identify activity cliffs (ACs); i.e., molecules have highly similar structures but show large activity differences. ACs are cases where the SAR is violated and thus should be avoided in QSAR modeling processes. As shown by the SAS maps of nonsteroidal compounds, viz., Now, we could construct the QSAR models for the nonsteroidal compounds. Previously, we identified 20 important scaffolds, but only 683 nonsteroidal molecular data were available for training. Hence, combining data from similar scaffolds, judged by their cyclic skeletons, for QSAR modeling is necessary. This yielded seven groups of cyclic skeletons: scaffolds 1 and 4; scaffolds 2 and 12; scaffolds 6, 7, 11, and 20; scaffold 3; scaffold 5; scaffolds 16, 17, 18, and 19; and scaffold 13. Consequently, seven QSAR models (model II to model VIII) were established for the seven groups. Again, 12 algorithms and 3 molecular fingerprints were tested and the combination with the best performance was selected as the QSAR model. The performance of the algorithms with the best choice of the fingerprint can be found in the The reader may notice that no classification QSAR models were constructed for scaffolds 8, 9, 10, 14, and 15. Scaffolds 9 and 10 were steroidal analogs with EF = 1.961, meaning that all associated molecules are active or potent. Hence, there was no point in constructing the classification model, but they are still good candidates for drug development. Scaffold 8 was a structurally unique scaffold with three tandem-fused benzene rings. Only 16 molecules belonged to this scaffold. The count of molecules was too small to build robust models. The same problem occurred for scaffold 15. As for scaffold 14, its cyclic skeleton was too simple and can be found in 433 nonsteroidal molecules with highly diversified structures. After initial modeling with the molecules, the model demonstrated significant overfitting that cannot be used for predictions.CYP17A1 inhibitors have been structurally categorized as steroidal or nonsteroidal. The steroidal inhibitors are structurally similar to the natural substrates of CYP17A1, viz., pregnenolone or progesterone, and they are often involved in the modification of the substrate’s D-ring at the C17 position. A typical steroidal CYP17A1 inhibitor is abiraterone, which is currently administered in combination with prednisone for the treatment of metastatic CRPC in men. Unfortunately, as time proceeds, resistance to abiraterone is developed in most cases. Furthermore, abiraterone-resistant tumors are also frequently resistant to subsequent treatments with enzalutamide, an androgen receptor antagonist that otherwise could confer a survival benefit that is similar to that of abiraterone . GaleterThe most challenging issue of treating CRPC with abiraterone is its resistance. Currently, there are several hypotheses to explain the emerging resistance to abiraterone, including androgen receptor splice variant up-regulation of CYP17A1; the increased expression of steroidogenic enzymes, including AKR1C3 and HSD17B3 ; 17-hydrThere are a few previous studies focusing on the structure–activity relationship of CYP17A1 inhibitors ,19. PrevAdditionally, this study identified several significant ACs and AC generators. AC generators are defined as molecules that frequently appear among AC molecule pairs that are identified by the structure–activity landscape index (SALI) values. The identification of AC generators amongst datasets are important for medicinal chemists to facilitate further lead optimizations. The two maximum common AC generators shown in The applicability domain is a theoretical region in the chemical space surrounding both the model’s descriptors and modeled response. In the construction of a QSAR model, the applicability domain of molecules plays a critical role in estimating the uncertainty in the prediction of a given set of compounds based on how similar they are to the training sets used to build the model ,20,21. TFinally, we mention that the current datasets are compiled from the ChEMBL database. These are retrospective records accumulated over years and with various sources for the bioactivities. Further prospective studies should focus on molecular enumerations based on scaffold analysis and SAR, molecular synthesis, and experimental validation. In addition, structural analysis based on ligand-protein interactions via molecular dynamic simulation could provide valuable insights into the mechanisms of action. This was a computational study of CYP17A1 inhibitors. All steps and procedures were performed using computational methods. The study’s design consists of data compilation, exploratory data analysis, Murcko-scaffold analysis, structure–activity landscape visualization, and QSAR modeling, as shown in 50 data used in this study were taken from the ChEMBL database. Originally, there were 2223 activity entries, but only 962 remained after the data cleansing procedure, which included removing redundant, unqualified, or missing data. The associated molecule structures were also processed to remove the salt and to standardize the tautomers using the PaDEL package [50 to pIC50 (−log IC50) for better visualization and data processing [50 ≥ 8, 8 > pIC50 ≥ 7, 7 > pIC50 ≥ 6, and pIC50 < 6 were labeled as potent, active, intermediate, and inactive, respectively. As shown in The ICp-values of the Mann–Whitney tests  between different groups were calculated to determine if there were any statistically significant differences. DataWarrior [In this study, exploratory data analyses of the datasets focused on the visualization of drug-likeness properties. Six drug-likeness properties were calculated, visualized, and compared between active class nonsteroidal inhibitors and active class steroidal inhibitors: MW, LogP, nHA, nHD, nRot, and TPSA. In this section, the maximal, minimal, median, mean, skewness, and kurtosis were analyzed for the descriptors, and normality tests were performed to observe whether they abide by normal distributions. Then, the aWarrior  was usedThe PCA is a dimensionality-reduction method that is used to reduce the dimensionality of large datasets by transforming large datasets into smaller datasets that still contain most of the information of the large set. In this study, the abovementioned 10 drug-likeness properties were used to perform PCA after exploratory data analysis had been performed. DataWarrior  was usedSAR is based on the idea that structure dictates activity, and molecules with similar structures demonstrate similar bioactivities. However, this relationship can be interrupted by AC, where a small change in structure can result in substantial activity loss. Since ACs capture chemical modifications that strongly influence biological activity, they are of particular interest in drug discovery and must be applied prior to any SAR modelling. In this study, SAS maps and SALI  plots weMolecular fingerprints are the representations of a complex form of molecular descriptors, which describe molecules in terms of their constitution, connectivity, and physicochemical properties. They are typically encoded by bit strings to characterize a given molecule. In this study, KlekotaRoth and PubChem fingerprints provided by the PaDEL package  were useTo improve the accuracy of the QSAR model and to avoid the overfitting, the feature selection process was performed. In particular, the correlation-based filter method was employed: features with variances that were lower than 0.1 and features demonstrating a high correlation (>0.90) were removed, thus reducing the complexity and computational resources without affecting the model’s performance. The QSAR models in this study were multiclass classification models that predict 4 bioactivities . Here, the one-vs.-rest strategy was employed for the multi-class classification task. The workflow of the QSAR modeling process is shown in iTP, iTN, iFP, and iFN denote the true positive, true negative, false positive, and false negative for class i, respectively. The accuracy is defined as the number of correct predictions divided by the number of total predictions, i.e., the trace of a confusion matrix divided by the sum of all matrix elements. For the confusion matrix shown in iTP + iFN. Hence, the accuracy reads:i runs through the classes. On the other hand, the micro-averaged recall is defined as [The performance of the QSAR multiclassification models is often evaluated via three parameters: the accuracy, the recall, and the MCC. Let fined as :Recall =i and j run through all classes. Clearly, accuracy and recall are identical in the multiclass cases, and only one needs to be reported. As one extra metric, multiclass MCC is defined as [Again, the indices fined as : MCC=∑iTWhile the values of accuracy and recall range from 0 (worst) to 1 (best), the MCC value ranges from −1 to 1. The extreme values of −1 and 1 represent a perfect misclassification and a perfect classification, respectively. In this study, the balanced steroidal dataset was further split into the training set and the test set according to a ratio of 80:20. Similarly, the same split was performed for most of the nonsteroidal dataset, except for models III and VII, where the splitting was performed with a ratio of 70:30. The size of the training set and the test set from each model is listed in 2,rnd [2, which reflects the impact of data balancing on the model’s performance. If the data are perfectly balanced, its value will be 0.5. Otherwise, this is zero. As this binary classification is out of scope of the present work, the full results are presented in the SI . This essentially entails comparing the chemical space of compounds from the training set with those from the test set using the PCA of scores plot. DataWarrior  was usedTo gain more insights from the structurally diverse 683 nonsteroidal ligands, we applied scaffold analysis to find representative molecular scaffolds. In medicinal chemistry, the molecular scaffold refers to the core structure of a molecule with preferable bioactive properties. Here, the most widely adopted scaffold definition, Murcko-Bemis scaffolding, was employed for the analysis. Murcko and Bemis dissected a molecule into four parts: ring systems, linkers, side chains, and the Murcko framework, which is a union of ring systems and linkers in a molecule . The fre50 levels so that favorable and unfavorable scaffolds could be identified and further modeled. In addition, the frequency of skeletons and scaffolds was ranked. DataWarrior was used for Murcko scaffold generation and visualization. In this study, Murcko scaffolds and cyclic skeleton systems were obtained for non-steroidal androgen receptor antagonists and compared based on pICMurcko scaffold diversity was calculated as the proportion of the number of scaffolds to the total number of molecules. 50 = 7.0  was used for EF calculations. The entire dataset of nonsteroidal molecules had 351 molecules out of 683 with a pIC50 ≥ 7.0; therefore, the proportion of active molecules of the entire dataset was 351/683 = 0.51. Furthermore, with the EF value being 1, it is indicative of the same proportion of active molecules in a given scaffold. An EF of 0 means that there are no active molecules in the given scaffold, and an EF of 1.961, with a ratio of 1 to 0.51, means that all molecules in the given scaffold are active. The scaffold EF is the ratio of the proportion of active molecules with a given scaffold to the proportion of active molecules in the entire dataset . The molhttps://github.com/GitGears/CYP (accessed on 1 January 2023). Additionally, the prediction results for the training set and the test set of each QSAR model are provided in CSV files in the Reproducibility is defined as the ‘closeness of the agreement between the results of measurements of the same measure and carried out under changed conditions of measurement’. The reproducibility of the experiment, whether in vitro or in silico, is a major concern in science and technology as it is closely related to the extensibility of knowledge and reproducibility of outputs . As thisThe inhibition of CYP17A1 has become a crucial therapeutic strategy in the treatment of prostate cancer because it reduces androgen levels, which can slow down the growth and spread of prostate cancer cells. This study employed cheminformatics and QSAR modeling for analyzing a set of 962 CYP17A1 inhibitors. In particular, one classification QSAR model was built for steroidal inhibitors, while seven models for major scaffolds with a sufficient number of molecules were built for nonsteroidal inhibitors. All eight QSAR models produced robust performances. The cheminformatic analysis results indicated that most of the 20 representative scaffolds contributed favorably to CYP17A1 inhibition, while only scaffolds 1, 2, 3, 8, and 13 were detrimental to the inhibitory bioactivity of CYP17A1. Furthermore, several functional groups are often employed to modulate the bioactivity of the scaffold, including the hydroxyl group, halogen, fluoride, sulfonamide, ketone, amide, and the methyl group. The effect depends on where the functional group is placed on the scaffold. We hope that the results presented herein will be instrumental for CYP17A1 drug discovery efforts.


Tensins are a family of focal adhesion proteins that connect the extracellular matrix (ECM) to the cytoskeletal networks, mainly through integrin receptors and their associated protein complexes. To investigate, for the first time in canine species, the expression of CTEN was evaluated in oral and cutaneous squamous cell carcinoma samples from several dog breeds and submitted to immunohistochemistry technique. The results reveal a higher level of expression associated with the highest grades of the tumor, evidencing that this protein is more expressed in the highest grades of squamous cell carcinoma, probably due to its involvement in tumor progression. In the present study, we demonstrated, for the first time, CTEN clinical significance in a canine squamous cell carcinoma, and, to our knowledge, this is also the first time that the CTEN expression pattern is linked to the canine species.p < 0.0001). These data suggest that CTEN is an oncogene in SCC of dogs and a promising biomarker and a therapeutic target for dogs affected by SCC.C-terminal tensin-like (tensin-4/TNS4/CTEN) is the fourth member of the tensin family, frequently described as displaying oncological functions, including cellular migration, invasion, adhesion, growth, metastasis, epithelial to mesenchymal transition, and apoptosis, in several different types of cancer. To investigate, for the first time, the clinical significance of CTEN in squamous cell carcinoma (SCC) of dogs, we studied a total of 45 SCC sections from various dog breeds. The mean age of the affected dogs was 8.9 ± 3.6 years. Immunohistochemistry confirmed strong cytoplasmatic CTEN expression in the basal layer of the epidermis next to the tumor. We detected high CTEN expression associated with the highest grade of the tumor (grade III) and observed 100% of immunopositivity for this tumor grading ( It was motility .Despite tensins being known to localize to focal adhesions, CTEN is also found in the cytoplasm and the nucleus, and it is in these three sub-cellular localizations that CTEN exhibits its physiological functions ,8. BeingSquamous cell carcinoma (SCC) is a malignant tumor derived from epidermal keratinocyte differentiation , commonlComparative oncology has gained increasing interest in the scientific community. Studies in animal tumors can open avenues for studies in human tumors and vice versa. Given the percentage of identity between dogs’ (ENSCAFT00845014125.1) and humans’ (ENST00000254051.11) CTEN protein (up to 84%) , it is pWith this study, we intended to evaluate CTEN expression in squamous cell carcinoma of dogs with different ages, breeds, and tumor localizations, describing for the first time not only CTEN immunoreactivity in SCC but also its pathological involvement in canine tumors.A total of 45 canine SCC histological sections were obtained from the Histopathological Laboratory of the University of Trás-os-Montes and Alto Douro archives. These tumors were excised from 45 dogs and previously fixed in 10% buffered formalin and paraffin-embedded. For each case, clinical information was recorded such as gender, age, breed, and localization  and size of the tumor. It was not possible to obtain the clinical staging or follow-up of animals included in the study.For the histopathological study, sections of tissue 4 µm thick were stained with hematoxylin and eosin. Two independent pathologists reviewed each sample. All slides were considered in this study and entire sections of the tumors were analyzed.Histopathologic diagnosis was based on the World Health Organization (WHO) histological classification of animal tumors .A Nikon Eclipse E600 microscope with a Nikon DXM1200 digital camera  was used for microscopical observations and images capture. Histological grading was assigned using an adapted version of Anneroth’s multifactorial system . The gralamina propria; III—invasion below lamina propria, including muscles. Lymphoplasmacytic infiltration was evaluated as: I—marked; II—moderate; III—slight to none.The tumor–host relationship was also evaluated, depending on the pattern of invasion, stage of invasion, and lymphoplasmacytic infiltration, scoring each parameter from 1 to 3. The pattern of invasion was considered as follows: I—pushing, well-delineated infiltrating borders; II—infiltrating solid cords, bands, and/or strands; III—small groups, cords, or single cells infiltrating. Stage of invasion was graded as: I—corresponding to carcinoma in situ and/or questionable invasion; II—distinct invasion, only involving The total points calculated were divided into three grades: grade I (score 5–10), including well-differentiated tumors; grade II (score 11–15), including moderately differentiated tumors; and grade III (+16), including poorly differentiated tumors.®, Newcastle, UK).The immunohistochemical (IHC) technique was performed in 45 canine SCC sections (3 μm thick) from formalin-fixed paraffin-embedded (FFPE) tissues to investigate CTEN’s expression. The protocol was divided into two parts and included the usage of the kit NovolinkTM Polymer Detection Systems (Leica Biosystems2O2) for 30 min and, after washing with PBS, the sections were incubated with Protein Block for 5 min. The first part of this procedure ended with the incubation of the antibody tensin-4 , clone SP83, rabbit anti-human, Invitrogen MA516355), diluted 1:100 in PBS for 24 h at 4 °C. In the second part of the protocol, we applied post-primary reagent, followed by Novolink Polymer, each for 30 min. After several washings with PBS, the samples were revealed, by incubation of 3,3-diaminobenzidine (DAB)  for 10 min and counterstained with Gill’s hematoxylin for 1 min. After washing with water, the sections were dehydrated in graded alcohols  for 3 min each, diaphanized in xylene, and then mounted with the aqueous mounting medium Entellan .In the first part, the samples were dewaxed in xylene for 15 min, followed by hydration in graded alcohols , for 5 min each. For antigen retrieval, the samples were submersed in citrate buffer solution  and heated in a 750 W microwave for 3 cycles, 5 min each. For peroxidase inhibition, we applied 3% hydrogen peroxide  software, version 24.0, in which we performed Pearson’s chi-squared test (ꭓ2) and Fisher’s exact test. The results were considered statistically significant for corporis integument (50%) [Overall, 48.9% (22 cases) were female and 42.2% (19 cases) were male, having missing data from four animals. The ages ranged from 1 to 17 years (mean = 8.9 ± 3.6 years) and their breed details are given in nt (50%) . The tumThe classification was attributed according to the criteria that were described above: 33.3% (15 cases) were well-differentiated (grade I); 37.8% (17 cases) were moderately differentiated (grade II); 28.9% (13 cases) were poorly differentiated (grade III); The expression of CTEN was observed in the basal layer of the non-tumoral epidermis next to the tumor and it was cytoplasmatic. In tumor cells, the labeling was cytoplasmatic, being noted mainly in the areas of invasion .n = 17) revealed negative CTEN expression in 11 cases (64.7%) and a positive reaction in 6 cases (35.3%) ; In total, 55.6% (25 squamous cell carcinomas) revealed negative CTEN expression and 44.4% (20 cases) were positive for this marker. Considering the well-differentiated tumors, only 5% (1 case) showed positive CTEN expression, while 93.3% (14 cases) accounted for negative CTEN expression. Moderately differentiated squamous cell carcinomas ( (35.3%) . The higp < 0.0001).Analyzing the association of CTEN immunoreactivity and the histological grading of the tumors, a significant association of the higher tumor grading (poorly differentiated tumors) with CTEN immunoreactivity was noted, which was statistically significant (p = 1); A possible association between CTEN expression and the localization of the tumors  was also evaluated. However, the differences between CTEN expression in cutaneous and oral tumors were not statistically significant .A total of 45 SCC tumor samples with different localizations and histological gradings, from dogs with distinct breeds , genderstegument . We coulThe immunohistochemical procedure revealed a cytoplasmatic staining, more predominant in the basal layer of the epidermis next to the tumor, corroborating the findings of Seo and his colleagues, in human skin . They suOur results show 44.4% positive immunoreactivity for CTEN, suggesting a strong concordance with the results found in head and neck squamous cell carcinoma in humans, which reported different levels of expression between normal tissues and SCC samples of head and neck tumors . In squap < 0.0001). A similar pattern of results was obtained in different human cancers, such as adenocarcinoma of the esophagogastric junction [Another interesting finding reported in the present study is the positive correlation between the histological grading of the tumors and CTEN expression, evidencing the aggressiveness of this marker. The highest histological grade (grade III) revealed 100% of immunopositivity , conferring motility (both invasion and migration) to tumor cells . DownregInterestingly, in canine spontaneous head and neck SCCs similar alterations were found to those described in human head and neck SCC, including dysregulation in the PI3K/AKT/mTOR and TGF-β signaling pathways, and in EMT, through increased expression of vimentin and diminished expression of E-cadherin . In caniWe acknowledge some limitations of the present work, mainly related to the retrospective nature of the study, the relatively small number of cases, and the lack of some clinical information. Nevertheless, we investigated, for the first time, CTEN’s behavior in canine SCC, and based on the correlation of positive immunoreactivity and increasing histologic grade in this study, we postulate that CTEN may be involved in tumor progression.To our knowledge, we have reported for the first time CTEN expression in canine SCC. The results from our experiment reveal a higher level of expression associated with elevated invasive behavior and higher histological grade of the tumor, evidencing that this protein is more expressed in poorly differentiated squamous cell carcinomas, probably due to its involvement in tumor progression.Overall, our results suggest that CTEN contributes to tumor progression in canine SCC, therefore revealing CTEN’s potential as a biomarker and a therapeutic target for canine SCC, as well as its possible value in histological grading of invasive fronts. However, future studies are required to focus on the protein networks displayed by CTEN and to clarify the mechanisms associated with CTEN in the carcinogenesis of squamous cell carcinoma.


The activity of microglia is fundamental for the regulation of numerous physiological processes including brain development, synaptic plasticity, and neurogenesis, and its deviation from homeostasis can lead to pathological conditions, including numerous neurodegenerative disorders. Carnosine is a naturally occurring molecule with well-characterized antioxidant and anti-inflammatory activities, able to modulate the response and polarization of immune cells and ameliorate their cellular energy metabolism. The better understanding of microglia characteristics under basal physiological conditions, as well as the possible modulation of the mechanisms related to its response to environmental challenges and/or pro-inflammatory/pro-oxidant stimuli, are of utmost importance for the development of therapeutic strategies. In the present study, we assessed the activity of carnosine on human HMC3 microglial cells, first investigating the effects of increasing concentrations of carnosine on cell viability. When used at a concentration of 20 mM, carnosine led to a decrease of cell viability, paralleled by gene expression increase and decrease, respectively, of interleukin 6 and heme oxygenase 1. When using the maximal non-toxic concentration (10 mM), carnosine decreased nitric oxide bioavailability, with no changes in the intracellular levels of superoxide ion. The characterization of energy metabolism of HMC3 microglial cells under basal conditions, never reported before, demonstrated that it is mainly based on mitochondrial oxidative metabolism, paralleled by a high rate of biosynthetic reactions. The exposure of HMC3 cells to carnosine seems to ameliorate microglia energy state, as indicated by the increase in the adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio and energy charge potential. The improvement of cell energy metabolism mediated by 10 mM carnosine could represent a useful protective weapon in the case of human microglia undergoing stressing conditions. Microglia are myeloid cells and represent the primary component of the brain immune system. Microglia are best known as the immune cells of the brain, but emerging data indicate that they also influence brain development, synaptic plasticity, neurogenesis, memory, and mood under physiological conditions . Both ex2−•), can react with RNS  to form peroxynitrite (ONOO−), which is more toxic than the parent species, and is able to cause oxidative (inflammatory) damage involving DNA, proteins, and/or lipids H+, involving both its constituent amino acids (β-Ala and His). Of note, the ability of carnosine to increase the formation of nitrite levels is also in accordance with a study carried out by Takahashi et al. in which endothelial cells were exposed to increasing concentrations of carnosine, showing enhanced nitrite production in a dose-dependent manner [2−• leading to the formation of ONOO− [−, diminishes the risks of peroxynitrite-mediated damages to fundamental macromolecules such as proteins and lipids [With this in mind, in the present study, we first investigated the effects of carnosine on HMC3 cell viability. We focused our attention on carnosine concentrations  normally used in in vitro studies, with 20 mM representing the gold standard ,64,65,66t manner . This cat manner ,74 and tof ONOO− . Therefod lipids . It shoud lipids ,77 and td lipids ,79,80. Id lipids ,80.2−•) is not always biologically damaging, with cells responding to these species in a regulated way through the redox signaling [2−• were not significantly modulated by the treatment of HMC3 cells with carnosine  ; for instance, in cancer cells, that are more glycolytic even under basal conditions, the ratio is around 8–10 [+/NADH ratio and cells rely their energy requirements mainly on glycolysis rather than on OxPHOS [+/NADH ratio (~27) along with that of ATP/ADP ratio (~13), generally ranging from 5 to 10 [The energy pattern of untreated human microglia cells showed very high concentrations of ATP  89,90,9,990,91 a ± 2.81) . In factund 8–10  and is aund 8–10 ,93,94. In OxPHOS ,96,97. T 5 to 10 ,98,99, m+/NADPH ratio has value lower than 1 (0.78 ± 0.23), suggesting that this cell line is characterized by a high rate of bio-synthetic reactions, possibly devoted to support lipid biosynthesis. Despite that, it should be taken into account that differently from microglia of the human brain, immortalized microglia  are not in their physiological environment and proliferating continuously, and thus characterized by different metabolic requirements [Conversely, compared to other cell lines in which values equal or higher than 2 were observed ,100,101,irements .2−• intracellular levels. In the present study, we also carried out, for the first time, the characterization of the energy metabolism of HMC3 microglial cells under basal (untreated) and carnosine exposure conditions, demonstrating that it is mainly based on mitochondrial OxPHOS coupled to efficient electron transfer paralleled by a high rate of biosynthetic reactions. Additionally, the exposure of HMC3 cells to carnosine led to a generalized amelioration of the microglia energy state, evaluated through the increase both in the ATP/ADP ratio and the ECP.In the present study, we provided evidence that carnosine, when used with human HMC3 microglial cells at a concentration of 20 mM, representing the gold standard in in vitro studies, led to a decrease of cell viability paralleled by an increase of IL-6 and a decrease of HO-1 in terms of gene expression. When using the maximal carnosine concentration avoiding significant changes in cell viability and relative molecular alterations (10 mM), carnosine decreased NO bioavailability, increasing its conversion into NO end-products nitrite and nitrate, without influencing OOur results suggest a therapeutic potential of carnosine and set the stage for future studies in which the neuroprotective activity of this dipeptide will be tested in the presence of pro-inflammatory/pro-oxidant stimuli in experimental models mimicking different neurodegenerative disorders, including PD and AD, characterized by microglia over-response, inflammation, oxidative stress, and energy unbalance.


VP frequency decreased with age (41% in < 3 mo to 9% in ≥ 60 mo), in contrast to AP (13% in ≥ 60 mo). Among the analyzed parameters, the best differentiating potential was shown by: C-reactive protein , procalcitonin , and CRP/procalcitonin ; a good positive predictive value  but a low negative predictive value  was observed. CAP influences hospital patient days more than the crude number of patients would suggest. On a clinical basis, BP is mainly recognized, although viral pneumonia is confirmed most often. RSV and influenza are responsible for a huge percentage of hospitalized cases, as well as M. pneumoniae in children aged ≥ 5 years. Serum inflammatory markers may help differentiate etiological factors.Community-acquired pneumonia (CAP) severely affects pediatric hospitalizations. This study assessed the contribution of CAP to hospitalizations, its etiology in relationship with age, and the inflammatory markers. Between 2013 and 2018, 1064 CAP patients were hospitalized and diagnosed with bacterial/possibly bacterial pneumonia (BP), viral/possibly viral pneumonia (VP) and atypical pneumonia (AP). The etiology was confirmed using blood/pleural fluid culture/polymerase chain reaction (PCR), rapid antigen test/PCR in nasopharyngeal swabs, or serological studies. CAP accounted for 9.9% of hospitalizations and 14.8% of patient days. BP was diagnosed in 825 (77.5%), VP in 190 (17.9%), and AP in 49 (4.6%) cases; the confirmed etiology ( Lower respiratory tract infections are the major global cause of hospital morbidity and mortality in children, and each year, they are responsible for approximately 12 million hospitalizations and 4.5 million deaths . AccordiThe global estimates report approximately 155 million cases of pneumonia each year, with considerable geographical and socioeconomic variations , and theStreptococcus pneumoniae (S. pneumoniae) and Hemophilus influenzae type B, were the most common causes of pneumonia [Mycoplasma pneumoniae (M. pneumoniae), Chlamydophila pneumoniae (C. pneumoniae), and Legionella pneumophila (L. pneumophila) are the main pathogens [Prior to the introduction of conjugate vaccines, typical bacteria, i.e., neumonia , yet pedneumonia . Viral eneumonia ,14,15,16neumonia . On the neumonia ,18,19,20athogens .Mycoplasma and Chlamydophila pneumoniae) have been suggested to induce lower PCT levels than typical bacteria [Mycoplasmatic pneumonia has also recently been raised [Children with CAP often require antimicrobial treatment, which is inherently associated with side effects and the development of antimicrobial resistance; a targeted therapy should be sought after to minimize these adverse outcomes . Difficubacteria ,36, but n raised ,38.Except for significant fluctuations and shifts in epidemiological trends, local antimicrobial stewardship and infection management guidelines need to be based upon current data; the local Polish data on the contribution of the particular etiological factors in hospitalized CAP cases, however, are scarce. Moreover, antibiotics are overused and often inappropriately prescribed, making CAP a significant public health concern in Poland . AnotherStreptococcus pneumoniae), J14 (pneumonia due to Haemophilus pneumoniae), J15 , J16 (pneumonia due to other infectious organisms), J17 (pneumonia in diseases classified elsewhere), and J18 . All the children diagnosed with CAP were eligible for the study. This was a retrospective observational study. Electronic medical charts of the patients hospitalized in the period between 2013 and 2018 (6 consecutive years) at the pediatric ward of the Bielanski Hospital, Warsaw, were reviewed, and the data were extracted into a previously prepared grid. The search included the final diagnoses of pneumonia according to the International Classification of Diseases, Tenth Revision (ICD-10): J10.0 , J11.0 , J12 , J13  in the upper respiratory tract swab, tested using rapid antigen test and/or PCR in a nasopharyngeal swab , and/or 4. positive serological tests suggesting a recent/ongoing infection . According to the final ICD-10 diagnosis and based upon the etiological factors, patients were finally divided into 3 groups: bacterial/potentially bacterial pneumonia (BP), viral/potentially viral pneumonia (VP), and atypical pneumonia (AP). All the diagnoses were made and encoded with the use of ICD-10 at the discretion of the attending pediatricians responsible for the treatment of the patient, and were not influenced by the study. When a single etiological factor was confirmed, the corresponding etiology of pneumonia was established; when more than one etiological factor was found, corresponding etiologies were diagnosed. In the other cases, the diagnosis was made upon the clinical course, use of and response to the treatment; when antibacterial treatment was considered necessary (and implemented) by a physician, potentially bacterial pneumonia was diagnosed; when a patient was successfully cured and discharged without antibiotics, potentially viral pneumonia was the diagnosis, although no viral factor was established. In fact, this subgroup of the study may also contain bacterial pneumonia cases that did not require antibiotic treatment, but this approach, in the opinion of the authors, might be used, since it translates clearly to practice—patients in this group do not require antibacterial treatment and may benefit from refraining from antibiotics. In the case of atypical pneumonia, only confirmed cases were diagnosed as AP. With regard to treatment success, all the cases were verified in terms of readmission due to the possible complication of pneumonia. Additional separate analysis was also performed in the subgroup with a confirmed etiology. The etiological factor was established if there was: 1. the growth of the pathogen in a sample from a sterile site  proven by a culture or its presence by a molecular assay , and/or 2. the presence of The primary outcome was the contribution of the particular etiological factors to the hospitalized CAP cases. The share of the specific etiological agents was presented in the following age groups: <3 months of age; 3–11 months old (mo); 12–59 months of age; and ≥5 years old (yo), i.e., ≥60 mo. Secondary end-points compared the diagnostic performance of routinely used inflammatory markers, including white blood cell count (WBC), the absolute neutrophil count (ANC), the C-reactive protein (CRP), procalcitonin, and the CRP to PCT ratio, in the differentiation between the etiological agents. Additionally, this study assessed the contribution of pneumonia to the total number of hospitalizations and the total number of patient days during the analysis period, the length of stay (LOS) in the 3 study groups, and the LOS according to the particular causative agents.Laboratory procedures:Chlamydophila pneumoniae and with the use of chemiluminescent immunoassays (CLIA)—Liaison XL —in the case of Bordetella pertussis. The RSV/influenza PCR was performed with the use of RSV Xpert Xpress Flu/RSV XC GeneXpert . All the procedures were performed in accordance with the manufacturers’ instructions. Blood was taken immediately after admission, and inflammatory markers were assessed and blood culture performed. In the case of serological studies, a blood sample was taken upon admission and/or during the hospitalization. Nasopharyngeal swabs were taken immediately after admission. WBC and ANC were counted using Sysmex XT2000i, and Sysmex XN1000/Sysmex XN550  since 10 April 2014. CRP was determined using the Cobas 6000 analyzer  with the limit of detection of 0.1 mg/L. Procalcitonin was determined using the Cobas e411 analyzer  until 4 August 2016, and using the Cobas 6000 analyzer  from 5 August 2016; the limit of detection was 0.02 ng/mL. Serological studies were performed with the use of enzyme-linked immunosorbent assay (ELISA)—Euroimmun Medizinische Labordiagnostika AG —in the case of t-test or the Mann–Whitney U test, respectively). One-way ANOVA or a non-parametric Kruskal–Wallis test with corresponding post hoc tests (Bonferroni correction or multiple rang comparison) was performed for comparisons between the different etiological groups. Inflammatory markers that were found to differ significantly between the groups  were then further tested via receiver operating characteristic (ROC) curve analysis in order to estimate its usefulness in a differential diagnosis; the area under the curve (AUC) was calculated with 95% confidence intervals (95% CI), and optimal cut-off values were calculated with the use of the Youden index; the corresponding sensitivity, specificity, positive predictive value (PPV), and the negative predictive value (NPV) were shown. In order to facilitate the comparisons between the AUC if an inverse relationship was observed , it was highlighted and the re-calculated AUC (over 0.5) was presented. Data distribution was estimated using the Kolmogorov–Smirnov test. The normally distributed data are presented as the mean ± standard deviation (SD), while skewed data are presented as the median and interquartile range (IQR), and adequate parametric or non-parametric tests were performed . The study obtained approval from the Ethics Committee at the Centre of Postgraduate Medical Education in Warsaw .A In the period between 2013 and 2018, a total number of 10,726 hospitalizations took place; finally, 1064 children  with CAP were included in the study . The medThe bacterial/possibly bacterial pneumonia was diagnosed in 825 children (77.5%), while the viral/possibly viral one in 190 (17.9%), and the atypical one in 49 (4.6%) patients. The proportion of the particular diagnoses varied among the groups; VP was recognized mainly in the youngest patients (under 3 mo) in 41% of cases, and decreased with the patients’ age: 32% in 3–11 mo, 14% in 12–59 mo, and 9% in ≥ 60 mo .On the other hand, the percentage of AP was the highest in ≥60 mo (13%), and correlated with the age . Bacterial pneumonia (confirmed or suspected) was diagnosed in the vast majority of cases, with the highest contribution in 12–59 mo (83%), and slightly lower in ≥60 mo (78%). The patients’ age differed significantly among the groups, with the youngest children in the VP group , followed by the BP , and the oldest children in the AP group . Patients with VP required a slightly longer LOS than BP patients .Mycoplasma pmeumoniae , Chlamydophila pneumoniae (8 cases), Streptococcus pneumoniae (2 cases), Bordetella pertussis (2 cases), and Streptococcus pyogenes (1 case), Streptococcus mitis/oralis (1 case), Streptococcus parasanguinis (1 case), and Staphylococcus warneri (1 case). In total, 154 cases of viral, 49 atypical, and 6 bacterial infections were confirmed. A difference between the median age was seen only between viral and atypical pneumonia (16 vs. 75.2 months), and the differences in terms of the LOS were noted.The etiological factor was established in 209 children (20%), and influenza was the leading cause , followed by RSV , 3/μL; IQR: 9–18.4), but a significant difference was observed only between the BP and the VP . The absolute neutrophil count differed between the BP  and the VP , and between the VP and the AP , but not between the BP and the AP. Among the serum inflammatory markers, procalcitonin differed among all the groups; the highest values were observed in the BP , followed by the VP , and the AP , whereas the CRP differed only between the bacterial and viral pneumonia . A statistical significance in the CRP to PCT ratio was observed among all the groups, with the highest value in the AP group , followed by the BP , and VP  (Children with BP presented the highest white blood cell count on admission (median 12.82 × 1013–83.9) . In the ROC analysis, inflammatory markers that differed between the groups were further tested to investigate the potential of a differential diagnosis. p < 0.01), followed by the ANC , WBC , CRP/PCT , and PCT   . p < 0.01), followed by CRP/PCT , and CRP . To differentiate between the typical and atypical bacteria, the PCT showed the best performance , followed by PCT , the ANC , and CRP .With regard to a viral versus atypical etiology, the CRP/PCT (reverse relationship) showed the best performance  at the cost of a low NPV .In a separate analysis, focusing on the confirmed etiological factors only, the results were comparable to those from the whole study group with regard to the possible use of corresponding inflammatory markers, although generally, higher AUC values were noted since they may have overestimated the true usefulness of the markers in clinical settings . 3/μL).The highest PCT values were observed for bacterial infection , followed by viral infection , and atypical infection , while the CRP differed only between bacterial and viral pneumonia (median 264.3 mg/L versus 8.5 mg/L). The CRP to PCT ratio differed between atypical and viral pneumonia . The white blood cell count did not differ between the groups, while the ANC was higher in children with atypical pneumonia compared to those with viral pneumonia (6.4 vs. 4.7 × 10n = 1064) corresponded to 9.92% of the total number of hospitalizations , while the contribution to the total number of patient days was almost 1.5-fold higher, and reached 14.8% . According to age, the CAP in infants accounted for 6.8%, with a 10.6% share in patient days, whereas in children aged 1 and older, the share in the number of hospitalizations and patient days reached 11.4% and 17.5%, respectively.The total burden of pneumonia was significant, affecting both the number of hospitalizations and, to a greater extent, the number of patient days due to a longer LOS (7 days versus 4.5 days in controls). The number of CAP patients . Data on using the CRP remain controversial. While some studies show a significant association between higher CRP and a bacterial etiology ,57, othep < 0.01) [Our study also confirms the observation in other studies that PCT may help to differentiate a typical CAP from an atypical one  ,38,64,65 < 0.01) , similarMycoplasma pneumoniae [p < 0.001). We observed a slightly worse performance  of PCT concentrations; lower PCT values in the course of atypical CAP were also reported by Stockmann [The CRP/PCT ratio shows a promising value in distinguishing an atypical etiology from a viral one . These results align with the Swiss study on adult patients with CAP, where a much higher CRP/PCT ratio was seen in eumoniae  than in tockmann  and by Wtockmann , while iThere are strengths and limitations to this study. First, it needs to be recognized that the study analyzed a single pediatric ward only, and any generalizations should be made with extreme precaution. Second, the etiological factor was established in only 20% of cases, which might be attributed to a lack of specific test in some of the cases ; thus, a different pathogen distribution across the age groups cannot be excluded, and this study might only provide hints regarding the etiology of the individual pneumonia cases. We did not use multiple pathogen panels, which would facilitate the detection of coinfections, especially viral ones, that need to be taken into consideration. Similarly, the usefulness of the inflammatory markers might differ in other study settings, given the obstacles met regarding the confirmation of the pneumonia etiology, and we aimed to verify a practical approach. It should be remembered that inflammatory markers in our study showed low to moderate test accuracy. In conclusion, the impact of CAP on hospital treatment is far beyond what the crude number of hospital admissions suggests. We observed that viral pneumonia decreases with age, in contrast to the percentage of atypical pneumonia, while bacterial pneumonia remained constant in all age groups. A major impact of two viruses (influenza and RSV) is clearly seen—they were confirmed in approximately 15% of the total number of CAP cases; on the other hand, the vast majority of cases were finally diagnosed as bacterial or possibly bacterial pneumonia, but only 0.6% found a laboratory confirmation with the use of the currently employed techniques. Diagnostic possibilities, although narrow, may be supported by procalcitonin levels . The CRP may also be used, yet its increase only differentiates bacterial from viral pneumonia, while the CRP/PCT ratio points to patients with an increased risk of atypical infection. Efforts to establish the etiology of CAP are crucial in order to implement adequate treatment.


To describe the body composition in preschool children and to evaluate the association with prepregnancy BMI and gestational weight gain (GWG).Children were recruited in their first year in kindergarten (3 years old) and followed up for the next 2 years. Information during pregnancy and birth was retrieved from medical records. Height, weight, fat mass, fat-free mass, and percentage of body fat (FM%) were measured through a bioelectrical impedance analysis for each child visit, and BMI, fat mass index (FMI), and fat-free mass index (FFMI) were calculated. Generalized linear mixed models (GLMMs) were used to evaluate the associations between prepregnancy weight, GWG, and adiposity indicators.2, 2.8 (±1.3) kg/m2, 12.8 (±0.7) kg/m2, and 17.2% (±5.8%), respectively. The prevalence rates of overweight and obesity in mothers before pregnancy were 16.6 and 3.2%, respectively. Mothers were divided into three groups based on GWG: appropriate , excessive , and insufficient . GLMMs analyses showed that the preschool children’s BMI, FMI, FFMI, and FM% were all significantly positively related to maternal prepregnancy BMI and GWG ; the children of mothers who were overweight/obese before pregnancy were more likely to be overweight/obese, high FMI, high FFMI, and high FM% at preschool age ; although maternal excessive GWG was not correlated with offspring’s overweight/obese (P = 0.156), the children of mothers with excessive GWG are more likely to have higher FMI, but not to be with a higher FFMI status than the children of mothers with appropriate GWG. For prepregnancy overweight/obese women, compared with the GWG-appropriate group, maternal excessive GWG was related to the risk of high FMI  and high FM% , but was not related to the risk of overweight/obese or high FFMI of the offspring at preschool age.A total of 3,329 single-birth 3-year-old children were recruited as the baseline population and were followed at 4 and 5 years old. During the 3 years of follow-up, the mean (±SD) values of BMI, FMI, FFMI, and FM% of the children were 15.6 (±1.6) kg/mFat mass index decreased with age, while FFMI increased with age among 3- to 5-year-old children. It is necessary to optimize maternal weight prior to conception and GWG management to improve the health outcomes of the offspring. ParentaPrevious studies that evaluated the consequences of prepregnancy BMI or GWG for the child mostly focused on birth outcomes or the health outcomes at a single age point, and few studies followed up on the health conditions of offsprings at multiple time points, especially lack comprehensive assessment of the children’s growth and development, including not only anthropometric indicators but also multi-time evaluation of body composition. Studies with larger sample sizes and an extended follow-up period are also warranted to generate stronger evidence and to evaluate the influence of prepregnancy weight status and GWG on the long-term body composition of children.This study prospectively followed preschool children in kindergartens for 3 years between the ages of 3 and 5 and linked their mothers’ medical records during pregnancy. This ambispective cohort study aimed to describe the body composition of preschool children and to evaluate whether and how prepregnancy weight status and GWG affect the body composition of 3- to 5-year-old children.This study was carried out by the Tianjin Women’s and Children’s Health Center and Capital Institute of Pediatrics in Tianjin from 2017 to 2020 and was approved by the IRB of Tianjin Women’s and Children’s Health Center (BGI-IRB 17116-201711). Parental written consent forms were obtained when the children were recruited in junior class.In general, children enter kindergarten at 3 years old in China, and they usually stay there for 3 years  before school. Children were recruited at kindergarten junior classes with informed consent obtained from their parents, and their mothers’ information during pregnancy was matched by retrieval from medical records. The exclusion criteria for the children were as follows: (1) any condition or chronic diseases or use of any drug known to affect growth and development; (2) acute diseases that prohibit children from participating in the physical examination; (3) twins or other multiple births; (4) mothers without GWG records in the antenatal healthcare system.By using a stratified cluster sampling method, 11 districts  were selected from the 16 municipal districts in Tianjin, the city where the prevalence of obesity in school-aged children is the highest in China. Then, 42 state-owned kindergartens were selected from 11 districts starting in 2017. Children in the junior classes of the 42 kindergartens were recruited into the cohort study, which has good population representativeness of the preschool children in Tianjin, and they were followed up and completed physical examinations in junior, middle, and senior classes from September 2017 to September 2020. Their mothers’ clinical information on antenatal and postnatal healthcare was collected from the Tianjin Hospital healthcare system, including anthropometry results collected at each follow-up phase.25–P75 was 6–8 times. GWG was calculated as weight at the last visit (within 1 week of delivery) minus the self-reported weight for the first visit at the antenatal clinic.Prepregnancy weight and height were self-reported and registered at the first visit during the 1st trimester. Maternal gestational weight was measured at antenatal clinics during each visit. The median number of repeat weight measurements per woman was 7, and PFor each child followed up in kindergarten, height was measured without shoes at each visit. Bodyweight, fat mass, fat-free mass, and percentage of body fat mass (FM%) were measured by trained nutritionists through bioelectrical impedance analysis , which measured the impedances of the body and each limb in the standing position and is suitable for children aged 3 years and older, at frequencies of 1 kHz, 5 kHz, 50 kHz, 250 kHz, 500 kHz, and 1 MHz. Children were required to be on fasting and to have an empty bladder. When in the measurement, children in light clothing stood on the platform without shoes, and held both hands at a 45-degree angle away from the body; four tactile electrodes were in contact with the palm and thumb of both hands, and the other four were in contact with the anterior and posterior aspects of the sole of both feet. BMI, fat mass index (FMI), and fat-free mass index (FFMI) were calculated for each subject as body weight, fat mass, and fat-free mass in kilograms divided by height in meters squared, respectively.2 group and ≥ 25 kg/m2 overweight/obese group).GWG was classified as insufficient, appropriate, or excessive according to the 2009 Institute of Medicine (IOM) criteria . PrepregThe Chinese gender-specific and age-specific BMI cutoffs for overweight and obesity for children from 2 to 18 years  were useData analyses were performed using SPSS 20.0  and R software (version 4.1.2). Comparisons of characteristics were conducted using one-way ANOVA or chi-square tests. The loess smooth curve of body composition indicators (95% CIs) with age during the 3-year follow-up was developed. Generalized linear mixed models (GLMMs) , 17 were2, A total of 3,822 children were recruited in their kindergarten junior class and completed physical examinations, including body composition analysis, from October 2017 to October 2018. Excluding 74 twins or other multiple-birth children and 419 mothers who had missing GWG data in medical records, 3,329 single-birth children had complete maternal GWG data in the antenatal healthcare system, and they provided the cohort baseline data. In total, 3,329 children remained participants and the other 419 children were non-participants. There were no significant differences in demographic and growth parameters between the participant and non-participant groups in the kindergarten junior class except that the FFMI showed a slight difference  kg/m2, 12.8 (±0.7) kg/m2, and 17.2% (± 5.8%), respectively. The change trends of adiposity indicators (95% CIs) with age during the 3-year follow-up are shown in During the 3 years of follow-up, the mean (±SD) values of BMI, FMI, FFMI, and FM% of the children were 15.6 (±1.6) kg/m2 and ≥25 kg/m2 groups. Parity, child gender, gestational week, and age at kindergarten during the 3-year follow-up were not significantly different among the three different GWG groups.As shown in SD = 5.8) kg/m2, and the prevalence rates of overweight and obesity of mothers before pregnancy were 16.6 and 3.2%, respectively.For maternal characteristics, the mean prepregnancy BMI was 22.5 . The chi-squared test showed that the prevalence of overweight and obesity during the three years were all significantly different between the prepregnancy BMI <25 kg/m2 and ≥25 kg/m2 groups , and the chi-squared test showed that these indicators increased with increasing GWG status .Of the children during the 3-year follow-up in kindergarten, BMI, FMI, FFMI, and FM% were all significantly different between the prepregnancy BMI <25 kg/mP < 0.001)  .P < 0.01). Although excessive maternal GWG was not correlated with offsprings’ overweight/obese (P = 0.156), the children of mothers with excessive GWG were more likely to have a higher FMI  but not correlated with the possibility of having a higher FFMI (P = 0.064) compared with the children of mothers with appropriate GWG. Compared with the children of prepregnancy BMI <25 kg/m2 mothers, the children of mothers who were overweight/obese before pregnancy were more likely to be overweight/obese , and have a high FMI/high FFMI/high FM% at preschool age   .2 and ≥ 25 kg/m2), associations between GWG groups and the adiposity indicators are shown in 2 women, compared to the GWG-appropriate group, maternal excessive GWG was not related to the risk of overweight/obesity, high FMI, or high FM% of the offspring but was related to high FFMI . GWG insufficiency was a protective factor against overweight/obesity, high FMI, and high FM%, but it was a risk factor for low FFMI . For prepregnancy overweight/obese women, compared with the GWG-appropriate group, maternal excessive GWG was not related to overweight/obese or high FFMI of the offspring at preschool age but was related to the risk of high FMI  and high FM% , which provides supporting evidence for the association between maternal weight and offsprings’ body composition at preschool age.Stratified by prepregnancy weight status , and macrosomia . GWG hasChildren with obesity before puberty can develop obesity in early adulthood, with early-life fat deposition associated with a later risk of adult obesity , 25. TheThe strength of this study is that both the continuous follow-up of child adiposity development in the 3 years of kindergarten and obstetric records during pregnancy were included in an ambispective cohort study. Some potential confounding variables were adjusted in the analysis which reveals the relatively causal link between prepregnancy BMI, GWG, and preschool child adiposity at the body composition level. Body composition and anthropometric measurements were involved in the continuous 3-year measurements, which made it possible to draw continuous lines during the 3 years in preschool children. Furthermore, maternal gestational weight was measured at antenatal clinics at each visit, and the medical records of the gestational weight improved the reliability of the GWG data.2 was 10.6%. Limited by the sample size, prepregnancy BMI of 25 kg/m2 was used to divide the mothers into two groups, which may have a certain impact on the analysis results when combined with underweight and overweight as a reference category.Our study has some limitations. First, prepregnancy weight was self-reported at the first visit to the antenatal clinic: there may be a recall bias in the self-reported weight data. Although a systematic review concluded that self-reported pregnancy-related weight has a high correlation with weight measurement , women gFat mass index decreased with age, while FFMI increased with age among 3- to 5-year-old children. Maternal overweight/obesity before pregnancy increases the risk of overweight/obesity, high FMI, high FFMI, and high FM% in preschool offspring. The preschool children of prepregnancy overweight/obese mothers with excessive GWG are more likely to have a higher FMI and FM% level but not a classified high FFMI level, which is required to remain healthy. It is necessary to optimize maternal weight prior to conception and GWG management to improve the health outcomes of the offspring.airechen@126.com.The datasets presented in this article are not readily available because the fund supporting the project does not allow the research data to be transferred to others or other institutions without consent. Requests to access the datasets should be directed to FC, The studies involving human participants were reviewed and approved by the IRB of Tianjin Women’s and Children’s Health Center (BGI-IRB 17116-201711). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.FC conceptualized and designed the study, carried out the analyses, drafted the manuscript, and reviewed and revised the manuscript. XX and GL conceptualized and designed the study, supervised data analyses, and reviewed the manuscript. TZ conceptualized and designed the study and supervised data analyses. JW, ZL, and XZ involved in data acquisition and critically reviewed and revised the manuscript. All authors critically reviewed the manuscript for interpretation and intellectual content and approved the final manuscript as submitted.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.