Background: The incidence and risk of urinary tract infection (UTI) in patients with type 2 diabetes mellitus (T2DM) who use sodium glucose co-transporter-2 (SGLT2) inhibitors are still controversial. Therefore, this study aimed to investigate the incidence and risk factors of using SGLT2 inhibitors, particularly in Thai patients. Methods: Electronic medication records of all patients, who started the treatment of T2DM between 1 January 2019 and 30 June 2021 at a tertiary hospital in Thailand, were reviewed. The patients were divided into SGLT2 inhibitor and non-SGLT2 inhibitor groups to compare the incidence of UTI. Results: The overall incidence rate of UTI was 33.49% in the SGLT2 inhibitor group and 11.72% in the non-SGLT2 inhibitor group. The incidence rates of UTI were not different between dapagliflozin and empagliflozin treatment . Patients treated with SGLT2 inhibitors had a 3.70 higher risk of UTI compared with those treated with non-SGLT2 inhibitors (95%CI 2.60–5.29). Moreover, the significant risk factors for UTI found in this study were gender, age, and occupation. Conclusions: This study highlighted the high incidence of UTI in patients using dapagliflozin and empagliflozin compared with non-SGLT2 inhibitors. Additionally, patients of female gender and older age had a significantly higher risk of UTI when treated with SGLT2 inhibitors, whereas those with permanent jobs had a lower risk. Sodium glucose co-transporter 2 (SGLT2) inhibitors are a novel class of oral anti-hyperglycemic agents that are used in the treatment of type 2 diabetes mellitus. The mechanism of SGLT2 inhibitors involves the inhibition of glucose reabsorption by the kidney, increasing glucose excretion and lowering blood glucose levels in patients with diabetes . HoweverIn 2015, the Food and Drug Administration of the United States (US FDA) launched a warning about the risks of serious UTI due to SGLT2 inhibitors . HoweverSeveral meta-analysis studies reported that patients with type 2 diabetes mellitus who used SGLT2 inhibitors had a significantly higher risk of UTI than those who used a placebo or other oral anti-diabetic agents ,5. ConveFurthermore, there was no report of this drug-induced UTI in Thailand. In Thailand, UTI is known to be one of the diseases that many patients tend not to go to hospitals with, because the antibiotics for the treatment of UTI can be prescribed by pharmacists in drug stores. Therefore, the underreported cases of UTI are feasible if the events are observed using only urinary analysis results. This study aimed to investigate the overall incidence of UTI related to SGLT2 inhibitors in Thai patients with type 2 diabetes mellitus, as well as its potential risk factors.This study was a retrospective cohort study. The population was divided into two groups: the study group (patients using SGLT2 inhibitors) and the control group (patients using non-SGLT2 inhibitors). The inclusion criteria were patients with the diagnosis of type 2 diabetes mellitus who were treated at Maharaj Nakhon Si Thammarat Hospital. The patients had to be first prescribed at least one oral anti-diabetic agent, either SGLT2 inhibitors or non-SGLT2 inhibitors, during the period of 1 January 2019 to 30 June 2021. The exclusion criteria were patients who used insulin, had incomplete data, passed away during the study, or refused to participate in the study by telephone. The incomplete data were defined as any participants with only the first visit for treatment (pre-treatment) or without the first visit for treatment. In addition, patients with data indicating urinary tract infection at pre-treatment were excluded.A1c), fasting blood sugar (FBS), and serum creatinine (SCr), and parameters in urine including bacterial count, white blood cell (WBC) count, and urinary glucose. Additionally, the diagnosis of UTI in the hospital with the following ICD-10 codes was recorded: N39 indicates urinary tract infection with the site not specified; N30 indicates acute cystitis; N34 indicates urethritis and urethral syndrome; N10 indicates acute tubulointerstitial nephritis.The data collection in this study was conducted in two parts. The first part was an electronic medication record (EMR) review. The EMRs of all recruited patients were reviewed in order to collect all information, including gender, age, religion, occupation, weight, height, body temperature, laboratory results, and prescribed anti-diabetic agents, as well as concurrent medication. The relevant laboratory data included parameters in the blood  after treatment with oral anti-diabetic agents. The second part asked whether the patient used to have any symptoms indicating UTI. These included acute dysuria, fever, urinary incontinence, frequency, suprapubic pain, hematuria, or pain in the lumbar region . If the patients answered positively for either of the two sections, they were asked to describe the time of the event if possible.3. Thirdly, a patient had acute dysuria with or without fever, with at least one of these symptoms: urinary incontinence, frequent urination, suprapubic pain, hematuria, and pain in the lumbar region. Lastly, a patient had the diagnosis of UTI at a medical clinic or a pharmacy store.The primary outcome of this study was the first time that patients had UTI after treatment with anti-diabetic agents. An event of UTI in this study was identified if a patient had one of the following criteria. Firstly, a patient was diagnosed by a doctor at Maharaj Nakhon Si Thammarat hospital with the ICD-10 codes N39, N30, N34, and N10. Secondly, a patient had at least one of these laboratory results post-treatment: (1) reports of bacteria in urine were few, moderate, or severe; or (2) urinary leukocytes > 10 cell/mmA1c, FBS, and SCr).The secondary outcome of this study was the time between the first drug use and the first UTI occurrence, and the risk factors of UTI investigated from patient characteristics and relevant laboratory data, including age, gender, body mass index (BMI), religion, occupation, and the laboratory results regarding levels of substances in the blood  of the UTI events and the expected relevant risk factors were investigated using Logistic regression analysis. All the statistical analyses were performed using IBM SPSS Statistics software version 28.0.0.0. . The significance threshold of all analyses was set at 2, respectively. In the other group, 144 cases (33.10%) were female, with an average age of 55.68 years and an average BMI of 25.78 kg/m2. Buddhism was the most common religion, and Metformin and Sulfonylurea were the two most common oral anti-diabetic drugs in both groups.Between 1 January 2019 and 30 June 2021, there were 853 patients with diabetes mellitus who met the inclusion criteria and were recruited for analysis. A1c in the SGLT2 inhibitors group was not different from the non-SGLT2 inhibitors group . The mean baseline FBS was also similar between both groups . Moreover, the mean baseline serum creatinine in patients treated with SGLT2 inhibitors was 0.97 mg/dL, similar to 0.86 mg/dL in patients with non-SGLT2 inhibitors. Statistical analysis of the baseline characteristics is shown in Laboratory results of both groups are also shown in p-value < 0.001). In addition, the UTI incidence rate of patients using dapagliflozin was not significantly different from that for patients using empagliflozin. Moreover, compared with patients using non-SGLT2 inhibitors, patients who were treated with SGLT2 inhibitors had a 3.71 times higher UTI risk (95%CI 2.60–5.29)  .p-value 0.222).Regarding the time of UTI events, the mean time to UTI event of patients in the SGLT2 inhibitor group was 15.92 months, and that of patients in the non-SGLT2 inhibitor group was 14.69 months. The Kaplan-Meier curve in p-value 0.031). On the other hand, permanent employees or salarymen had a 0.55 times (95%CI 0.30–0.99) decrease in UTI risk compared with people with other jobs. Other factors were not associated with UTI events, including age, BMI, religion, blood sugar levels, and serum creatinine.The results indicated that gender and occupation were the risk factors associated with UTI . In partThe odd ratio of using SGLT2 inhibitors that was adjusted with the relevant risk factors was 4.85 (95% CI 2.77–8.52). The treatments with dapagliflozin and empagliflozin delivered risks that were 4.23 and 5.29 times higher than treatment with non-SGLT2 inhibitors, respectively .The results of this study indicated an increase in UTI occurrence in patients who used SGLT2 inhibitors, i.e., dapagliflozin and empagliflozin. The UTI incidence in patients who used SGLT2 inhibitors was more than 30% compared with 12% in those who used non-SGLT2 inhibitors. In other words, using SGLT2 inhibitors increased the risk of UTI 3.7 times more than using non-SGLT2 inhibitors. Moreover, the factors that were associated with UTI in this study were gender and occupation.The incidences of UTI that were reported in this study were much higher than in several previous studies; the UTI incidences related to SGLT2 inhibitors were usually approximately reported at 3–9% ,9. FocusThe same phenomenon was observed when considering odds ratios of UTI. Patients who used SGLT2 inhibitors in this study tended to have a more than 3.70 times increase in the risk of UTI, compared with patients with non-SGLT2 inhibitors. In contrast, many meta-analysis studies reported the increased risk of UTI ranged between 1.10 to 1.30 times, compared with placebo ,5. MoreoFocusing on an individual SGLT2 inhibitor, dapagliflozin has shown increases in UTI risk in many studies. For instance, the study by Figueiredo et al. reported a significant increase in UTI of 1.18-fold, similar to the study by Puckrin et al. that reported a 1.33-fold increase ,6. AlthoThe main cause of differences in the incidence and odds ratio between this study and the previous studies might be the difference in data collection; the above-mentioned studies included only UTIs documented in hospitals, whereas in the current study, all types of UTIs were recorded. In addition, the difference in diagnostic criteria, such as signs, symptoms, self-report, and urine culture, could result in a dissimilar incidence of UTIs ,13. The The factors affecting UTI events have been widely studied, and the results in this study showed the same results. Female gender is one of the known risk factors of UTI; many studies indicated that females had higher events suggestive of UTI and had a greater tendency for developing symptomatic UTI and recurrent complications with a higher incidence of more serious complications than the male population ,15.Another factor that was found to be associated with UTI in the present study was being a permanent employee. Even though there are not many studies that indicated an association between occupation and UTI, some jobs were considered to be the risk factors of UTI, such as cleaning and driving taxis or buses ,17. On tA1c, and serum creatinine. However, unlike the present study, there were reports of increasing UTI events of some factors. For example, an elevated BMI, especially more than 30 kg/m2, was associated with an increased incidence of UTI because of the inability to exert sufficient pressure to empty the urinary bladder [According to the results of this study, other factors were not associated with the incidence of UTI, including age, BMI, religion, FBS, Hb bladder . Elevate bladder .As this study was conducted retrospectively, there were several limitations that should be highlighted. First, the collection of data from both laboratory records in the hospital to identify the UTI events might be underreported because some patients did not go to hospitals. Additionally, patients with long-term diabetes mellitus have a high risk of neurogenic bladder, so they might not recognize their symptoms of UTI and might not go to the hospital. In fact, all patients with uncontrolled diabetes mellitus should be monitored for neurogenic bladder, and if they have the disease, there are several treatments that can be used . MoreoveThe second limitation was the criteria for the diagnosis of UTI in this study, which were different from those used in the previous studies ,13, and The incidence of UTI in patients with SGLT2 inhibitor treatment was 33.49%, compared with 11.72% in patients without SGLT2 inhibitor treatment. In addition, the results indicated that dapagliflozin and empagliflozin were associated with significant increases in UTI risk, by 3.79 and 3.64 times, respectively, compared with non-SGLT2 inhibitors. Female patients should be closely monitored for UTI events, and patients who work as permanent employees might be safer when treated with SGLT2 inhibitors.


However, these devices still suffer from low power conversion efficiency (3 PSCs using a solar cell capacitance simulator (SCAPS) for the first time. The aim was to replace the conventional electron transport layer (ETL) TiO2 that reduces PSC stability under solar irradiation. Therefore, 2 and stable ZnOS as the ETLs. Perovskite light absorber parameters including Cs content, defect density, doping concentration and thickness, and the defect density at the interface were tuned to optimize the photovoltaic performance of the PSCs. The simulation results showed that the device efficiency was strongly governed by the ETL material, Cs content in the perovskite and its defect density. All the simulated devices with ZnOS ETL exhibited 3 could greatly increase its potential for practical production.Formamidinium tin iodide (FASnI Due to their carbon footprints, traditional sources of energy are major contributors to climate change and global warming, representing direct threats to the current century. Over the last two decades, there has been an international movement towards substituting the use of fossil fuels with other sources that are renewable, environmentally safe and sustainable. One of the most prominent sources of renewable energy is solar energy. Solar energy can be harnessed by photovoltaic panels, which are an alternative method to generate electricity ,2,3. Theer 25.5% ,5.In addition to their low manufacturing cost, and their high efficiency, lead-based perovskite solar cells are found to have low exciton-binding energies, high optical absorption coefficients, long diffusion lengths and tunable bandgaps ,7,8,9. IDespite the previously mentioned advantages, lead is known to be a toxic material and degradable. A solution for this latter drawback is to substitute lead with other materials (Tin (Sn)), leading to the appearance of a new era of photovoltaic cells, in particular, Sn-based perovskite solar cells. Indeed, since Sn and Pb are both in group 14 of the periodic table and have similar ion radii ,16, tin 3), Formamidinium tin iodine perovskites (FASnI3), and Cesium tin iodine perovskites (CsSnI3) [3 and FASnI3, CsSnI3 posesses the lowest 2, SnCl2 and SnBr2) to the CsSnI3 structure. It has been found that SnBr2 is the most convenient additive, increasing the In solar cell applications, three major typical Sn-based perovskites are used: Methylammonium tin iodine perovskites (MASnI(CsSnI3) . Despitear cells ,22,23. Tm et al.  investig3 is known to have a stable structure [3 solar cells is relatively low compared to Pb-based perovskite solar cells [3 is found to be more stable than MASnI3 [3, instead of MASnI3, despite the fact that both materials are sensitive to air [3 has been done [2, and SnF2 [3.Methylammonium tin iodine MASnItructure , an imprtructure , a long tructure  and a sutructure . Howeverar cells . Althougn MASnI3 . Therefoe to air . An exteeen done , all whieen done , guanidieen done , GeI2, aand SnF2 , to the 3 perovskites [3 doping. In fact, Cs added to FASnI3 can act as a reduction agent to limit the oxidation of 3 increases electron mobility by a factor of three [Previously cited works herein emphasized the common issues and limitations that arise in employment of pure FASnIovskites , namely,ovskites ,40,41,42se range ,36. Alsoar cells , hence, This work aimed to suggest possible optimization routes for efficiency improvements of the stable CAPS-1D) . Recent The studies show good agreement between the simulation results and the experimental data, indicating the reliability of SCPAS software ,46. SCAP3 and 2 and ZnOS, which recently showed promising results [In this paper, a comparative study between pure FASnI results ,52,53,54The impact of the defect density, thickness, acceptor doping concentration, electron and hole mobility of the absorber layer and the interface defect density between the perovskite from one side and the HTL (Hole Transport Layer)/ETL (Electron Transport Layer) from the other side, on the overall performance of the proposed device was studied. It was proven that an optimum In the present study, a numerical simulation was conducted on Belgium. . SCAPS an − i − p configuration of FTO/ETL/2O/Au is considered  [Numerous studies emphasize the importance of the energy level alignment between the absorber (PSC) and the ETL layer (TiOor ZnOS) ,66,67,682 ETL layers.Therefore, interface engineering and control at the ETL-perovskite interface is crucial for addressing the CBO and achieving high-efficiency planar PSCs ,33. More2 as ETL. In fact, the difference of mobility of electrons in the two ETL materials could be a direct reason for this discrepancy. As shown in 2. All simulated solar devices with ZnOS as ETL showed high 2 as ETL only showed a 2 compared to that of the perovskite could lead to a significant charge recombination in the ETL, thus resulting in unbalanced charge transfer and, consequently, low power convergence efficiency [For best assessment of the Cs content and the ETL material choice, four different parameters were investigated: the ficiency .Another crucial factor behind the outperformance of the solar cells with ZnOS as ETL was the enhanced band alignment of the ZnOS against the perovskite. In addition, 2, for different Cs content are grouped in The values of the CBO (found from Equation (4) and From 2 was used as ETL, it can be noticed from 3, and zero in the case of the pure structure. A negative CBO indicated that the CB level of ETL was lower than that of the perovskite, resulting in the formation of an energy cliff at the ETL-perovskite interface.When TiOHence, it can be noticed from 2 was used as ETL, Cs doping did not help in enhancing the performance of the PSC. On the contrary, Cs doping was found to deteriorate the photovoltaic properties, and decreased the 3, to Thus, when TiOFrom 2. Mainly, this was due to much higher electron mobility and better band alignment with the perovskite. 3. In addition, it is worth mentioning here that the In brief, regardless of the Cs content, ZnOS was found to provide better photovoltaic properties compared to TiO and Jsc . For tha, one can notice that the best structure corresponded to a Cs doping concentration of Comparing the results plotted in Table 42. Therefore, in the following stages of this study, ZnOS as ETL with a FASNI3 absorber doped with Cs at 0.75Cs0.25SnI3) was adopted.The latter result is in accordance with previous studies  suggestiIn addition to the importance of choosing the adequate ETL material and the structure of the absorber layer, defects density of this layer e output ,71. On timproves .In addition, it can be noticed from formance .However, as doping concentration continued to increase and exceeded In conclusion, it was found in this part that According to ,72, the x-axis) and PSC/HTL interface defect density (y-axis) both between Regarding FF, at PSC/HTL defect densities below Indeed, the lower the interface defects densities, the better the PSC performance was. However, taking into consideration the high cost of fabrication of devices with such low interface defect densities, it is, hence, necessary to adopt the lowest pair of defect densities leading to the best PSC performance and fabrication cost. Therefore, based on the above analysis, the optimal values of PSC/HTL and ETL/PSC defect densities were found to be 75Cs25SnI3, with different thicknesses varying up to The thickness of the light-absorbing layer was found to be of high importance to the solar cell performance. The choice of thickness is delicate. A large value maximizes current density, but minimizes the reverse saturation current, all while increasing the fabrication cost. This section is dedicated to the study of the impact of absorber layer thickness on device photovoltaic outputs. In this study, the studied absorber layer was FAQuantum efficiency (QE) indicates the capability of a solar cell to collect carriers from incident photons of a given energy/wavelength. From 3-based PSC. A normal n-i-p planar structure of FTO/ETL/2O/Au was numerically simulated and investigated using SCAPS-1D simulation software. The effect of TiO2 and ZnOS ETL on the solar cell performance was thoroughly investigated. The study proved that solar cells with ZnOS have outstanding performance, due to the high electron mobility in the ZnOS layer and excellent band alignment of ZnOS against all tested perovskites with different Cs contents.In this study, a robust and stable 3 as absorber, had, by far, the best performance among all the PSCs with TiO2 ETL. However, solar cells with The CBO between the ETL and the perovskite was mainly affected by the Cs content in the perovskite. The work herein clearly explained the significant effect of Cs doping and CBO on the electrical performance of the cells. The solar cells with pure FASnI2O/Au was obtained with the following factors: absorber defect density Furthermore, the performance of the latter device was optimized by tuning four major factors: doping concentration


Kirkwood Navel and Ruby Valencia are two spontaneous bud-mutations of the ordinary Washington Navel and Valencia late oranges characterized by the red coloration of their flesh. The purpose of this study was to analyze the physiological features, internal fruit quality, contents of relevant bioactive compounds and antioxidant capacity in the pulps of the red-fleshed fruits compared with the ordinary oranges during late development and maturation. In general, the content of sugars, organic acids, vitamin C, tocopherols, total phenolics and flavonoids, the hydrophilic antioxidant capacity and their changes during maturation were similar in the red-fleshed oranges and in the corresponding blond oranges. However, the mature Ruby fruits contained lower concentrations of sugars, malic and succinic acid and higher levels of citric acid than the ordinary Valencia. The major difference between the pulps of the Kirkwood and Ruby oranges and those of the ordinary oranges was the higher lipophilic antioxidant capacity and SOAC (singlet oxygen absorption capacity) of the former. Together, the high and unique content and composition of carotenoids in Kirkwood and Ruby may contribute to an enhanced antioxidant capacity without any detrimental effects on other fruit-quality attributes, making these varieties good sources of phytochemicals for the fresh-fruit and juice-processing citrus industries. Citrus is one of the most important commercial fruit crops worldwide in terms of production, market trade and consumption. The demand for and acceptance of citrus fruits for fresh consumption or juice production is highly variable across different national and international markets, depending on the consumer choices and preferences but, in general, it is mainly based on the external appearance, organoleptic properties, nutritional quality and health-related benefits [benefits .The consumption of citrus fruits is associated with anti-inflammatory effects, obesity control, a decrease in the incidence of cardiovascular diseases and a reduction in the risk of certain cancers ,3,4. TheCitrus sinensis) are relevant sources of various nutrients and phytochemicals, including carbohydrates, protein, lipids, vitamins, mineral elements, phenolics, limonoids and carotenoids among others [The content and composition of nutrients and bioactive and antioxidant metabolites in citrus fruits vary widely among the different species and varieties, tissues and maturation stages, influencing their quality, nutritional value and health-promoting effects . Orange g others ,7,8. Sugg others ,3. Carotg others . The divg others . Citrus g others . The flag others ,12. Flavg others .Citrus [Citrus fruits are also important sources of vitamins ,8. For eCitrus ,14. AlthCitrus . On the Citrus ,17. The Citrus . SeveralCitrus ,19,20. MCitrus ,22. AddiCitrus .Citrus, restricted to a few varieties and mutants [The accumulation of the carotene lycopene in the fruit pulp confers a particular bright-reddish coloration, but this is an unusual feature in the genus  mutants . The red mutants ,25,26,27 mutants ,29, anti mutants ,31,32 an mutants ,35,36,37The availability of new red-fleshed orange varieties is of special relevance to the provision of novel products with distinctive value for local and international markets, but also to understand the biochemical basis of lycopene accumulation in the pulps of citrus fruits and whether this feature may alter other parameters of fruit quality. We recently reported the characterization of carotenoid metabolism during development and maturation in two new red-fleshed orange mutants: Kirkwood Navel (referred in this work as Kirkwood or K) and Ruby Valencia (Ruby or R). These spontaneous bud-mutations are derived from the ordinary Navel and Valencia late oranges, respectively, and are already being commercially propagated in order to check their adaptation to Mediterranean growing and environmental conditions . The pulThe objective of this work was to conduct a comparative analysis of the chemical composition, nutritional quality and antioxidant capacity in the fruits of the red-fleshed varieties, Kirkwood and Ruby, and the corresponding standard varieties, Navel and Valencia, respectively, in order to provide reliable data on their fruit-quality characteristics under edaphoclimatic Mediterranean conditions. We report, for the first time, the evolution of internal quality-related parameters, such as sugars and organic acids, from the late stages of fruit development to full maturation, as well as the accumulation of vitamins and other bioactive compounds, such as ascorbic acid, tocopherols, total phenolics and flavonoids. Furthermore, the relevance of these changes is complemented by the analysis of the carotenoid composition in the fruit pulp at two maturity stages and the assessment of the antioxidant capacity by different in vitro assays.Citrus sinensis) genotypes, Washington Navel  and Valencia late  and those of the red-fleshed varieties, K and R, were cultivated at the Fundación ANECOOP . Washington Navel oranges are characterized by precocious maturity and bear large seedless fruits. They are the main orange group produced for fresh consumption. On the other hand, Valencia late oranges belong to the Blancas botanical group; they are characterized by the absence of navel in their fruits, late maturity and the absence of a bitter flavour in their juices [Citrus sinensis × Poncirus trifoliata), grown in the same orchard and under the same agronomical and environmental conditions [Fruits of the two ordinary sweet orange (r juices . Fruits nditions . N and K3) of the pulp containing juice vesicles without segment membranes were excised and frozen in liquid nitrogen, ground to fine powder and stored at −80 °C until analysis. Juice was extracted with a domestic electric hand squeezer , filtered through a metal filter (0.8 mm pore size), frozen in liquid nitrogen and stored at −20 °C until analysis. Fruit samples were harvested during two consecutive seasons  and data reported here are representative of one season unless otherwise indicated.At each selected stage, at least 50 fruits per variety were harvested and delivered to the laboratory. Fruits were picked up from the external canopy; they were of similar size and free of external defects. Color of the flavedo and pulp was measured and small pieces  and total titratable acidity (TA) of the juice were determined using a digital refractometer PAL-BX/ACID1 . TSS is expressed as °Brix and TA as mg citric acid/100 mL of juice; maturity index (MI) was calculated as the TSS/TA ratio. Determination of internal maturity was analyzed in fruits of the four varieties at the different harvest times, except for those harvested in August.2SO4 1.5 mM; flux, 0.6 mL/min; and oven temperature, 50 °C. For sucrose determination, a Pb column was used ; the conditions used in this analysis were: eluent, Milli-Q water; flux, 0.6 mL/min; and the column temperature was set at 50 °C. Samples were extracted twice and results are the mean of two replicates (mean ± SD).Sugars and organic acids were determined essentially as described by Pérez-Través et al. . Pulp tiL-Ascorbic acid from the pulp of the fruit was extracted and determined essentially as described in Alòs et al. , using a® Arc™, Waters, Barcelona, Spain) coupled with a fluorescence detector  and a YMC C30 column  . Identification and quantification of the different tocopherols was achieved by comparison with the retention times and peak areas of authentic standards of δ-, γ- and α-tocopherol , and the concentrations expressed as μg/g of fresh weight. Samples were extracted twice and results are the mean of two replicates (mean ± SD).Tocopherols in the pulp were extracted and quantified essentially as described in Rey et al. , using aCarotenoids were extracted and analyzed in the pulps of mature fruits of the four varieties  as described by Rodrigo et al. , using a2CO3 2% and NaOH 0.1 M) and incubated for 15 min at room temperature in darkness. Next, 50 μL of Folin–Ciocalteu reagent were added (diluted 50% with purified water) and incubated for 30 min. The absorbance was measured at 724 nm in a UV/Vis microplate spectrophotometer  and compared with a standard curve of gallic acid . Results were expressed as mg of gallic-acid equivalents (GAE) per 100 g of fresh weight. Determinations were performed per triplicate in each extract.Phenolic content was determined by the method of Folin–Ciocalteu, as described by Singleton and Rossi , with sl2. After 5 min of incubation at room temperature, 30 μL of 10% AlCl3 were added and the reaction was stopped by adding 200 μL of 1 N NaOH. Flavonoid contents were determined by UV/Vis microplate spectrophotometer comparing the absorbance at 350 nm with a standard curve of hesperidin . Results were expressed as mg of hesperidin equivalents (HesE) per 100 g of fresh weight. Determinations were performed per triplicate in each extract.Total flavonoids were determined in the same supernatant used to analyze total phenolics following the method described by Lafuente et al. . Two supFlavonoids were extracted and purified from pulp tissue as follows. Briefly, pulp tissue (0.5 g) was extracted with 5 mL of Milli-Q water centrifuged at 3000 g for 30 min at 4 °C. Next, the extract was subsequently filtered through a 0.45-micrometer PVDF filter . Flavonoid content was determined by HPLC-DAD. Separation of flavonoids was carried out with a XBridgeTM BEH C18 , maintained at 40 °C, and a secondary gradient elution with water 0.1% TFA (solvent A) and acetonitrile 0.1% TFA (solvent B) at a flow rate of 1 mL/min as follows. From 0 to 5 min 100% A, 5 to 30 min 95% A and 5% B, 30 to 33 min 50% A and 50% B and from 33 to 37 min 100% B. Wavelength was registered from 220 to 550 nm and flavonoids were quantified at 280 nm. Identification and quantification of the different flavonoids was achieved by comparison with the retention times and peak areas of authentic standards of hesperidin, narirutin, naringin, eriocitrin, dydimin and rutin . Concentrations are expressed as mg/100 g of fresh weight. Samples were extracted twice and the results are the mean of two replicates (mean ± SD).The hydrophilic antioxidant capacity (HAC) was determined by the DPPH free-radical assay , as described by Rey et al. . The ass3.6H2O and 300 mM acetate buffer, pH 3.6, at 1:1:10, v:v:v and incubated in a bath at 37 °C for 10 min. In a 96-well plate 10 μL of each sample were mixed with 290 μL of FRAP reagent and incubated at 37 °C in darkness for 30 min. Thereafter, the absorbance change of the mixture was determined in a UV/Vis microplate spectrophotometer  at 595 nm. The assay was replicated twice and a curve of ascorbic acid was used as a standard. Results were expressed as mg of ascorbic acid equivalents (AsAE) per 100 g of fresh weight.The HAC was also determined in the same supernatant used in DPPH assay by using FRAP (ferric reducing antioxidant power) assay, as described by Benzie and Strain , with so® Omega, BMG Labtech, Offenburg, Germany) at 730 nm. A calibration curve was drawn with Trolox  and results are expressed as Trolox equivalent antioxidant capacity (TEAC) per 100 g of fresh weight.The ABTS  assay was used to determine antioxidant capacity in comparison to a standard antioxidant, Trolox  . Total antioxidant capacity was quantified as described by Legua et al. , with sl1O2 radical was determined using endoperoxide  as a singlet oxygen generator and 2,5-diphenyl-3,4-benzofuran  as an UV-Vis absorption probe in a 96-well quartz glass microplate . Changes in absorbance at 413 nm were monitored for 90 min at 35 °C using a UV-Vis spectrophotometer microplate reader; α-Tocopherol  was used as a blank standard. The SOAC value was calculated by Formula (2):Analysis of SOAC in the pulp of the fruits was performed as previously described by Rey et al. . The capEach sample was analyzed in two independent assays and samples were run in triplicate in each plate.t-test (p < 0.05) using XLSTAT software 2019.3.2 . The correlations between bioactive compounds and antioxidant capacity measured by different assays were examined by Pearson’s correlation (p < 0.05). Two kinds of principal component analysis (PCA) were built. The first PCA was set up using data for vitamin C, tocopherols, total phenolics, total flavonoids, DPPH, FRAP, ABTS-H, ABTS-L by XLSTAT software. The second PCA was built with the same variables and levels of carotenoids. The data for total carotenoids for this second PCA analysis were obtained from our previous work [Results are presented as the mean of two biological replicates ± standard deviation (SD). Differences between the red-fleshed and the corresponding ordinary variety for each harvest date were analyzed by ous work , using thttps://www.biogold-em.com/assets/va---spec-sheet---kirkwood-red-navel-(print).pdf) (accessed on 19 September 2022) and Ruby Valencia (R) .pdf) (accessed on 19 September 2022), originated from spontaneous bud mutations of the blond-colored Palmer Navel and Olinda Valencia oranges, respectively, grown in commercial orchards. These varieties were developed under agronomical and environmental Mediterranean conditions, maintaining the distinctive color singularities and carotenoid complements in the pulp [The red-fleshed oranges, Kirkwood Navel (K)  of citrus-fruit development, in which the increase in pulp size is responsible for most of the growth . From OcThe main feature of K and R mutants is the inner coloration of the fruit. The internal appearance of the Navel, Kirkwood, Valencia and Ruby orange fruits is shown in The analysis of the internal quality parameters  revealed only minor differences between the K and N fruits at the initial maturity stages (October and November) . In geneThe analysis of the main sugars in the pulps of the four genotypes revealed a progressive accumulation during development and maturation . The conIn this study, we detected four major organic acids in the pulps of the four varieties evaluated . Citric In the orange fruits, the free acids increased rapidly in stage II of the fruits’ development and then declined in concentration due to the dilution caused by the increase in size and water content of the pulp , accordiA comparative gene-expression analysis indicated the downregulation of the transcription of the genes relevant to citric-acid accumulation, those related to electron transport to the TCA cycle or to the proton pump involved in the transport of citrate to the vacuole ,52. The The concentrations of ascorbic acid and tocopherols, as well as those of phenolics and flavonoids, in the pulps of the red-fleshed oranges, K and R, and the respective blond genotype, were also analyzed during development and maturation. The orange fruit is recognized as a significant source of vitamin C. The concentrations of ascorbic acid in the pulps of Navel and Valencia oranges were relatively similar and, in general, declined moderately during maturation A,B. ThesInformation about the tocopherol content in citrus fruits is very limited, and the regulation of tocopherols accumulation in fruits of different citrus species have only been investigated in recent studies ,53,54. TThe analysis of the total phenolics and flavonoids in the pulps of the four orange varieties revealed that the immature fruits (August) contained the highest concentrations, which that thereafter . PreviouAlthough the pattern of flavonoid accumulation during maturation was similar to that of the total phenolics, it is interesting that the immature fruits of the two red-fleshed oranges had lower flavonoid levels than those of the ordinary oranges G,H. MoreTo ascertain the quantitative and qualitative differences in flavonoids between the ordinary and the red-fleshed oranges, we analyzed the composition of the main flavonoids in the pulps of the mature fruits harvested in January for the Navel varieties and in April for those of the Valencia . Five flCitrus junos) [One of the limitations in the interpretation of the antioxidant capacity of citrus fruits is the determination of the relative contribution of lipophilic compounds, such as carotenoids and tocopherols, to the antioxidant capacity, since most studies focus on the antioxidant contribution of hydrophilic compounds, such as vitamin C and flavonoids ,58. In ts junos) , or a sls junos)  and orans junos) .2 = 0.79) and FRAP (r2 = 0.67). Moreover, the total phenolic and flavonoid contents displayed a good positive correlation with the DPPH  and FRAP activity   . These rctively) . This rectively) F. These ctively) H. SeveraP r2 = 0.7. MoreovOn the other hand, the analysis of the LAC, measured by ABTS (ABTS-L), revealed the highest values in the four varieties in immature fruits (August), which dropped sharply in October, after which they remained relatively constant or experienced a slight increase during maturation . Interes2 = 0.85) (2 = 0.87) and flavonoids (r2 = 0.85). Several reports have obtained an efficient extraction of phenolics and flavonoids from orange tissues using ethyl acetate as a solvent [The ABTS-L showed a significant positive Pearson’s correlation with the concentration of tocopherols (r = 0.85) . Tocophe solvent ,63. Sinc solvent ,65, as w solvent . Since c solvent ,61,66, i solvent .Overall, these results indicate that the pulp extracts of citrus fruits can effectively scavenge different types of free radicals under in vitro conditions. The variability of the assays performed suggests that multiple mechanisms and different bioactive compounds may contribute to their antioxidant capacity. For instance, Liu et al.  reported1O2) is a reactive oxygen species (ROS) generated in biological systems, which reacts with many biological targets, inducing the degradation of cellular components. Carotenoids are efficient quenchers of 1O2 in biological systems [Singlet oxygen  was carried out to explore the relationship between the contents of the different compounds  and the antioxidant activity  A,B. In tIn the second PCA analysis, including total carotenoids , the samThe analysis of the quality attributes, bioactive compounds and antioxidant activities from the last stages of fruit development to full maturity revealed that the new red-fleshed varieties of sweet orange, Kirkwood Navel and Ruby Valencia, displayed, under Mediterranean growing conditions, a pattern and timing of maturation and a harvesting date similar to those of the ordinary Navel and Valencia varieties. No relevant differences were detected in the accumulation of sugars, organic acids, phenolics, flavonoids, vitamin C or tocopherols between the red Kirkwood and the blond Navel. However, the Ruby Valencia fruits had lower levels of sugars, malic acid and succinic acid and higher levels of citric acid than the ordinary Valencia fruits. The main feature of both red-fleshed oranges was the presence of higher lipophilic antioxidant capacity (LAC) and singlet oxygen absorption capacity (SOAC) than in the ordinary oranges, which was probably due to their lycopene content and, in particular, to their high levels of phytoene and phytofluene. Taken together, the results suggest that these novel red-fleshed orange varieties offer new products to the fresh-citrus market and juice-processing industries, with improved properties primarily derived from their carotenoid content and composition, as well as their lipophilic and singlet oxygen antioxidant capacity, without any detrimental effects on their other fruit-quality attributes. Further experiments with cell cultures, in vivo systems or clinical trials would be appropriate to evaluate the additional health-related benefits of these red-fleshed orange fruits.


To study the clinical efficacy of nutritional intervention combined with muscle exercise on sarcopenia patients with femoral fracture.  From January 2019 to January 2021, a total of 100 sarcopenia patients with femoral fracture were included in this study and were divided into a control group (routine postoperative care) and a research group , 50 cases in each group. Primary clinical outcomes included sarcopenia-related indicators and functional independence assessed by activities of daily living scale (ADL). Secondary clinical outcomes included time of fracture healing and hospital stay, pain score as assessed by visual analogue scale (VAS), and nursing satisfaction. P > 0.05). After 3 months of intervention, the BMI, grip strength, calf circumference, pace, and body muscle rate of the patients in the research group were significantly higher than those in the control group (P < 0.05), while body fat rates were significantly lower than those in the control group (P < 0.05), and the capability of eating, walking, bathing, and doing housework in research group were all significantly higher than those in control group (P < 0.05). In addition, the time of fracture healing and hospital stay in research group were all significantly lower than those in control group (P < 0.05), and the VAS scores of the control group at each time point after intervention were significantly higher than those of the research group (P < 0.05). The nursing satisfaction of the patients in the research group was significantly higher than that in the control group .  Before the intervention, there was no significant difference in the indicators of sarcopenia and the indicators of functional independence assessed by ADL between the two groups ( Nutritional intervention combined with muscle exercise can help improve sarcopenia symptoms and promote fracture recovery in patients with sarcopenic femoral fractures. Sarcopenia is a syndrome of persistent loss of skeletal muscle mass, strength, and function and characterized by a marked age-dependent onset. Previous studies have found that skeletal muscle begins to age from about 40 years, with an average annual decrease in quantity and quality of about 8%, and muscle loss doubles after the age of 70 , 2. EpidImportantly, fracture patients need to stay in bed for a long time, and long-term bed rest is more prone to muscle strength loss caused by muscle reduction, and the probability of falling when getting out of bed is very likely to increase, thereby increasing the risk of femoral fracture again , 9. TherFemoral fracture is one of the common diseases in clinical surgery, characterized by severe pain, swelling, and distortion, which have a serious impact on the patient's ability to live. Sarcopenia and osteoporosis are two major risk factors for fractures in patients, especially elderly patients , 7. In tA total of 100 patients with sarcopenic femoral fractures were included in this study from January 2019 to January 2021 . The following are the inclusion criteria: (1) femoral fracture and received artificial joint replacement; (2) combined with sarcopenia; (3) aged 40-75 years; (4) mentally stable, able to communicate normally and to move independently, and voluntarily cooperate with this study. The following are the exclusion criteria: (1) complicated with severe cardiopulmonary insufficiency; (2) severe neuropsychiatric disorders; (3) inability to stand up independently; (4) impaired cognitive function; (5) severe renal insufficiency requires protein intake to be restricted; (6) patients with malignant disease or infectious disease; and (7) lost in the middle of the study, suffering from other diseases or death. All patients were informed about the study and signed the informed consent to join the study voluntarily. In addition, this study was a prospective study; the present study was approved and supervised by The Third Affiliated Hospital of Chongqing Medical University Hospital Ethics Committee.Patients in the control group were received routine care such as primary care and symptomatic care. Patients in the research group received nutritional interventions and muscle exercises in addition to routine care. Nutritional intervention and muscle exercise duration is from 10 days postoperatively to 3 months plus 10 days postoperatively. The following are the nutritional interventions: daily supplementation of 400 kcal of whole nutritional formula powder and 0.6-1.0 g/kg of whey protein powder. The following are the muscle exercises: do 30 minutes of aerobic and resistance exercise each time, 5 times a week, including brisk walking and slow walking. Muscle exercises are guided by medical staff, mainly targeting the leg muscles, with video instruction which is given once a week.Baseline data were collected for all patients recruited in this study, including gender, age, education, comorbidities , drinking, and smoking. All patients received a 4-month follow-up after surgery, including telephone follow-up every 2 weeks and unscheduled hospital admissions.2(m2); (2) in grip strength, the subject stands and relaxes, with both hands hanging down naturally; hold the grip dynamometer with one hand, and firmly clench the grip dynamometer once, and record the grip strength; (3) in pace, record the time spent walking 6 m, and calculate the pace; and (4) flex the knee joint 90, and measure the thickest part of the calf. Moreover, we used activities of daily living scale to evaluate functional independence of patients before the intervention and 3 months after the intervention. ADL scale evaluates the capability of eating, walking, bathing, and doing housework in patients, and the higher the score, the stronger the functional independence.Primary clinical outcomes included sarcopenia-related indicators and functional independence assessed by activities of daily living scale (ADL) . In detaSecondary clinical outcomes included the time of fracture healing and hospital stay, pain as assessed by visual analogue scale (VAS) at different times, and patient satisfaction with care. In detail, we used VAS to assess pain before intervention, 1 week, 2 weeks, 1 month, and 3 months after intervention. The VAS scoring standard is divided into 10 equal parts using a ruler, 0 is no pain, 1-3 is mild pain, 4-6 is moderate pain, and 7-10 is severe pain.P values are calculated using a chi-square or Fisher's exact test as appropriate. The Kolmogorov-Smirnov test was used to check whether quantitative data conformed to a normal distribution, data that conformed to a normal distribution were presented as (mean ± standard deviation), and unpaired Student's t-test was used to compare differences and calculate P values. P < 0.05 indicated significant difference.Data in the present study were analyzed by SPSS 20.0 software . Qualitative data are presented as counts (%), and P > 0.05), including gender, age, education, comorbidities , drinking, and smoking , including BMI, grip strength, calf circumference, pace, body muscle percentage, and fat percentage (P < 0.05), while body fat rate were significantly lower than those in the control group (P < 0.05) (There was no significant difference in the indicators of sarcopenia between the two groups before the intervention (rcentage . After t < 0.05) .P > 0.05), including the capability of eating, walking, bathing, and doing housework (P < 0.05)  .P < 0.05) (P < 0.05)  . However < 0.05) .P < 0.05)  ( < 0.05) .Sarcopenia, an age-dependent loss of muscle mass and function, is a common disorder in older adults and is associated with adverse health outcomes. Sarcopenia patients suffer from loss of muscle strength and mobility impairments, which reduce their quality of life, with higher risk for falls, fractures, metabolic disease, and mortality . In ChinIn this study, we found that compared with patients in the control group, patients in the research group receiving nutritional intervention combined with muscle exercise had higher BMI, grip strength, calf circumference, faster walking speed, and body muscle rate after the intervention, which suggests that nutritional intervention combined with muscle exercise can effectively improve symptoms in patients with sarcopenia. Undernutrition is an important cause of sarcopenia in middle-aged and elderly people, and it is also the main direction for the prevention and treatment of sarcopenia , 22. MalFurthermore, muscle exercise is also very important for improving symptoms in patients with sarcopenia. On the one hand, regular aerobic training can improve the activity of muscle mitochondrial enzymes, increase capillary density, promote nerve repair and motor unit mobility, thereby increasing skeletal muscle strength and exercise capacity , 29. On In this study, we also found that the postoperative recovery of the patients in the research group was better than that of the patients in the control group, including higher functional independence scores, shorter fracture healing times and hospital stay time, and lower postoperative VAS pain scores, which indicates that our intervention not only improves sarcopenia symptoms but also accelerates recovery in patients with femoral fractures. Adequate nutrition and protein supplementation can not only slow down muscle atrophy but also help increase muscle strength and help patients with femoral fractures get out of bed early to exercise , 33. EarAll in all, our results in this study indicated that nutritional intervention combined with muscle exercise could help improve sarcopenia symptoms and promote fracture recovery in patients with sarcopenic femoral fractures. However, as a single center study, there are problems such as a single research population and a small sample size. In the future, we need to expand the sample size to verify the accuracy of the results.


In this study, we explore the dynamics of expansion of two reintroduced populations of wild ungulates, Italian roe deer  and red deer (Cervus elaphus), assessing their contribution in the recovery of landscape diversity.In the past decades, the abandonment of traditional land use practices has determined landscape changes inducing reforestation dynamics. This phenomenon can be contrasted with rewilding practices, By using direct and indirect information on the two species, collected by nocturnal and diurnal surveys and camera trapping, we modelled a habitat suitability map, and estimated the density and distribution of the populations. We also performed a land use changes analysis, combining the presence of wild ungulates and livestock.C.c. italicus.We demonstrated that deer dispersed gradually from their release location, increasing in population size, and this occurred in the entire study area. Moreover, we show that areas with lower grazing density are significantly affected by forest encroachment. A possible interpretation of this result could be that wild grazers (roe deer and red deer) prefer semi-open areas surrounded by the forest. This, in association with other factors, such as domestic grazing, could be one of the main responsible in maintaining landscape mosaic typical of the Apennine mountain, confirming the value of grazers as a landscape management tool. Moreover, we show the possibility to conserve through reintroduction the vulnerable  In the past decades in Southern Europe, socio-economic demands have led to the abandonment of traditional land use practices and the migration of human populations towards urban and industrial areas , especiaIn particular, the abandonment of hinland rural areas has led to a decrease in biodiversity associated with Mediterranean landscapes  due to ae.g., large herbivores), a process also named rewilding, may contribute to restoring the natural processes  in the Romanian Carpathians (Equus ferus) in Sweden (Capreolus capreolus) (Cervus elaphus) in Corsica   and red  Corsica . In partThe goal of our work is to evaluate the effect of the reintroduction and spreading of wild deer, combined to livestock in grazing in open areas (grassland).C. c. italicus, an Italian endemic subspecies assessed as Vulnerable (VU-D2) by the International Union for Conservation of Nature , defined as the Cilento, Vallo di Diano e Alburni National Park (PNCVDA) and its immediate surroundings . The PNCFagus sylvatica) forests, interspersed with secondary grasslands  and maple trees (Acer sp.). Nowadays, these forests are populated mostly by Italian hare , the main predator in the region, expanding at a fast pace with a population currently estimated at many dozens of individuals  the habitat is dominated by beech . In addivialis; . Finallyivialis; .The abandonment can be considered as the main driver of landscape change given that in the PNCVDA the human activity in the forest  are limited and under rules.In this area, roe deer and red deer were extinct, dating back to the early 1950s, as a consequence of several factors including human persecution and habitat changes. However, roe deer and red deer were reintroduced between 2003–2006  .In order to simulate the demography of expansion of the wild ungulates, we performed a population viability analysis (PVA). The PVA was largely adopted for evaluation of management action in endangered species, using even the supplementation scenario to examine the relative benefits of alternative management actions . In reinThe PVA was performed in Vortex 10.1.6.0 software , puttingi.e., footprints, excrement, barking of trees, shrub browsing) presence information ascribable to the roe deer and red deer, by using diurnal and nocturnal surveys, with walking transects and spotlight method .To define the potential distribution for roe deer and red deer, presence data were employed in spatial elaboration by using Maximum Entropy Distribution Model MaxEnt,  in MaxEnA total of 13 spatially environmental variables were selected: elevation, aspect, slope, roads, waterways, broadleaf forests, mixed woods, conifer forests, scrublands, grasslands, tree plantations, agricultural fields, urban area.http://www.pcn.minambiente.it/mattm/), while both aspect and slope were calculated from the DTM, using “Aspect” and “Slope” GDAL functions in QGIS 3.10 (https://www.qgis.org). The remaining variables were obtained as categorical vector files from Corine Land Cover 2018 classes  from the National Geoportal . Detection zone and radius were obtained from manufacturer manuals for each camera trap model used (according to All cameras had a detection radius (r) = 20 m, except for Uovision UV575,  (2), y/t is the trapping rate , r (radius of the detection zone) and θ (the angle of the detection zone) are the camera trap detection parameters, v represents the animal movement speed   in R (R el (REM) , using “el (REM) , taking nt speed .When different models of cameras have been used for the same quadrant, radius and angle of detection zone were average. Averaged daily movements for ungulate species and livestock were obtained from the literature: 1.99 km/day for roe deer , and 2.82) selecting an “Inverse gaussian” family for roe deer and “Gamma” family for red deer, using the R package “lme4”. We considered density as a response variable, and distance from release points as a predictor variable. The latter was calculated using the algorithm Proximity (raster distance) implemented in QGIS 3.10, by setting distance units to “geographical units”. This allowed also to understand whether our deer populations are represented exclusively by the reintroduced individuals or have been enriched by other. Finally, to obtain the area affected by the cumulative presence of grazers, the livestock density was merged to roe deer and red deer density rasters.For each species, we estimated the average density for the study area as well as local densities for each surveyed cell, along gradually distant areas from the release locations. The density of each cell was interpolated using the IWD Interpolation feature in QGIS and clipped to the extent of suitable habitat for each species. To obtain the distribution of density, we fitted GLM models , in a scale of 1:100,000, with a minimum cartographic unit (MCU) of 25 ha, a geometric accuracy better than 100 m.To infer the potential effect of reintroduced wild herbivores and livestock on the landscape, we compared the land use and land cover from 2000 and 2018, considering Level III Corine Land Cover , a thirdoverlay function in QGIS Layers, cleaning polygons smaller than 0.25 ha, as these are generally misalignment artifacts or represent areas too small to have a significant impact on the landscape , indicator of loss of semi-open habitats (open area surrounded by the forest), was defined as the transformation of natural areas (class 3) into more advanced succession stages according to natural dynamics .Inverse Succession (IS), indicator of landscape opening areas, was defined as transition from natural advanced forms to less advanced states of succession, for example from woods to bushes .The polygons were subsequently rasterized to obtain files comparable with the density distributions (100 × 100 m resolution).Inverse and Spontaneous succession occurred, as well as where they not occurred. We tested the differences using Welch’s t-test, to account for different sample sizes as the number of pixels where the changes occurred different.To infer on the combined effect of wild and domestic grazers’ pressure, we compared the mean grazing density in areas where The PVA showed that in 16–19 years, the population size of roe deer expanded from 37 released individuals to an estimated population of 175.61 (SE = 9.24) while the red deer expanded from 35 to 84.28 (SE = 9.95).For roe deer, the most suitable settings for the habitat suitability model are linear + quadratic features with a regularization multiplier of four; while for red deer, linear features with a regularization multiplier of three. The test AUC for the final models is 0.880 and 0.862 for roe deer and red deer, respectively. The 10th percentile training presence threshold results in values of 0.221 for roe deer and 0.209 for red deer, while the maximum training sensitivity plus specificity threshold results in values of 0.559 for roe deer and 0.621 for red deer.The highly suitable territories for the two species are closely associated with mountainous areas , in whicAnalyses of the contributions of the variables show that the second relevant environmental variable affecting habitat suitability is the distance from agricultural meadows (10.7% and 14.9% for roe deer and red deer respectively), followed by the tree plantations distance for roe deer (9%), and by the slope (5.8%) and grasslands distance (5.6%) for red deer. All other variables show a contribution equal to or less than 5% .2 and 880 km2, respectively (2). Conversely, in red deer the largest extension is observed for highly level of suitability (642 km2) (2), reaching a value of 665 km2 considering the total suitable area.The suitable area for roe deer and red deer extends for about 687 kmectively , 2D. Con642 km2) . A spati2 of roe deer and 0.13 ind/km2 of red deer.Furthermore, considering the population size estimated by PVA and highly suitable habitat (assuming a more parsimonious approach), we estimated about 0.75 ind/kmIn total, 12 cells (with four cameras each) were surveyed, for 2876 trapping days . Roe dee2, SE = 0.37) as those estimated for red deer .The current mean densities for roe deer are three times as much   , 4B, andctively) . Distancred deer , with a 2 (SE = 0.60) (The deer share the pasture hosting a livestock mean density of 1.78 ind/km = 0.60) . The den2 (SE = 0.02), and overlaps with high suitability of wild grazers, with the exception for the Northern portion of the study area (Alburni Mountains)  .2 of Inverse Succession and 32.26 km2 of Spontaneous Succession are found in the study area (2 (IS) and 4.33 km2 (SS) are included in the suitable area for roe deer and red deer, whereas 14.59 km2 and 27.93 km2 are outside of the suitable habitat for these ungulates  = 2,175, p < 0.0001, indicating a significantly higher frequency of Spontaneous Succession outside the suitable range for the ungulates.In total, 26.30 kmudy area . Of thesngulates . A chi-sSpontaneous Succession was significantly lower compared to density in Inverse Succession (Spontaneous Succession is observed are well used by 2.41 ind/km2 (SE = 0.14) of all ungulates. Instead, the Inverse Succession sites are home of 11.92 ind/km2 (SE = 0.10) grazers. Differences between densities in SS and IS are significant using Welch’s t-test (p < 0.01).Cumulative density of ungulates and livestock in sites classified as ccession . The sitSpontaneous Succession of patches not inhabited by deer to their altitudinal levels. There was no significant difference in elevation between the areas with Spontaneous Succession  and Inverse Succession   .The population analysis of wild ungulates, formerly extinct in historical times and then reintroduced, has clearly shown a good capacity of expansion and growth.C. c. italicus that opens motivating conservation implications for this Italian endemism.The demography of the deer has reached remarkable population size if we consider the data obtained both from the population growth model (PVA) and through the survey data. This increase depends exclusively on the reintroduced individuals without any immigrants from surrounded areas, based on the observed spatial pattern of the populations. Moreover, this must also be considered in light of the growing of wolf population in the last decade , probablInterestingly, the suitable habitat for these wild ungulates is all placed in the central portion of the study area whereas the density is decreasing in the boundaries. The most possible interpretation of this observation could be that Cilento behaves like an isolated land that disconnected from other suitable areas due to the marine coastline in the south and west, and the two large anthropized valleys in the north and east , WGS 84/UTM zone 33N reference system.Coordinates of presence point for Click here for additional data file.10.7717/peerj.14492/supp-2Supplemental Information 2Cell coordinates in which species density was estimated, WGS 84/UTM zone 33N reference system.Click here for additional data file.


Breast cancer is a serious threat to female health, and its incidence varies with education level (EL). In the present study, the association between EL and the risk of developing female breast cancer was investigated.From May 2006 to December 2007, 20,400 observation subjects in Kailuan Cohort received questionnaires and were subjected to clinical examinations for data collection on baseline population characteristics, height, weight, lifestyle and past disease history. Then, these participants were followed up with from the date of recruitment to December 31, 2019. Cox proportional risk regression models were used to analyse the association between EL and the risk of developing female breast cancer.The cumulative follow-up period of 20,129 observation subjects that meet the inclusion criteria of this study was 254,386.72 person-years, and the median follow-up time was 12.96 years. During the follow-up period, 279 cases of breast cancer were diagnosed. In comparison with the low EL group, the risk of developing breast cancer was significantly higher in the medium  = 2.23 (1.12–4.64)] and high [HRs (95% CI) = 2.52 (1.12–5.70)] EL group.An increased risk of breast cancer was associated with a higher EL, and some certain factors, such as alcohol use and hormone therapy, may play a mediating role. Breast cancer poses a serious threat to female health worldwide. Based on GLOBOCAN data, in 2020 alone, the number of new cases of breast cancer reached 2.26 million, and the incidence of breast cancer ranks first among female malignant tumours . AlthougNotably, the incidence of malignant tumours varies with the education level (EL). A negative correlation has been observed between EL and the risk of developing gastric, liver, lung, and oesophageal cancer in humans , 5, the The Kailuan Study, which began in 2006, is an ongoing observational cohort study of a functional community population. The EL information of the Kailuan Study subjects was obtained at baseline (from May 2006 to December 2007). Then, continuous follow-up data, including breast cancer data, were obtained from the establishment of the cohort to the present, allowing us to study the association between EL and breast cancer. More details about the Kailuan Study can be found in the literature , 9. Subj2 (m2).Face-to-face questionnaire interviews and health examinations were performed by well-trained physicians or nurses by using a standardized protocol to gather information at baseline. These baseline data included sociodemographic characteristics , lifestyle characteristics , history of previous diseases  and physical examination data (height and weight). Participants were asked to take off their hats and shoes and wear thin clothes during the measurement. Height and weight were measured, and body mass index (BMI) was calculated as weight (kg)/heightEL was classified into three categories, namely, low EL (illiteracy and primary), medium EL (middle school) and high EL (college and above). Smoking was defined as smoking one or more cigarettes per week in a recent year, and participants were classified into never smokers, former smokers and current smokers. Alcohol drinking was defined as drinking one or more alcoholic drinks per month for no less than six consecutive months, and participants were classified into never drinkers, former drinkers and current drinkers. Physical activity was classified according to the frequency of physical activity (20 min = 1 instance of activity) performed during leisure time. The categories of physical activity include exercise occasionally (< four instances of activity per week) and frequent exercise (≥ four instances of activity per week).The follow-up period was considered from the establishment of Kailuan Cohort (May 2006 – December 2007) to December 31, 2019. The follow-up endpoint was newly diagnosed breast cancer or death in the observed subjects (whichever came first). First, information on the participant medical records was obtained through the Tangshan City health insurance system. Then, the medical histories of subjects were collected by professionally trained investigators. Pathology, imaging  and blood biochemical examination results were verified by clinicians to confirm the diagnosis of breast cancer. Tumour cases were coded according to the International Classification of Diseases-10 (ICD-10), and breast cancer was coded as C50. Information on fatal events was obtained from the Kailuan Group social insurance system.P < 0.05 (two-sided).SAS 9.4 was used to perform statistical analysis of the data. Continuous variables were expressed as mean ± standard deviation, while categorical variables were expressed as the number of cases (percentage). Differences in baseline demographic characteristics among the low, medium and high EL groups were analysed using variance analysis and Chi square test. A multivariable Cox proportional hazards regression model was applied to analyse the associations between EL and the risk of developing breast cancer. Subgroup analysis was performed according to age, BMI, physical activity and hypertension history, and the associations between EL and the risk breast cancer were explored in different subgroups. Sensitivity analysis was conducted to evaluate the consistency of our findings after excluding participants new breast cancer within 2 years of the start of the follow-up period. All statistical tests were considered statistically significant at P values < 0.0001; Table The number of female participants in the 2006 baseline survey was 20,400. A total of 116 individuals with a history of malignancy and 155 participants lacking baseline EL data were excluded, and 20,129 female participants were eventually included in the statistical analysis. The mean age of the observed subjects was 48.75 ± 11.50 years. The low, medium and high EL groups included 1,078, 16,833 and 2,218 subjects, respectively. In comparison to the medium- and high-EL groups, the mean age, BMI and the proportions of subjects with current smoking, heavy salt intake, frequent physical activity, hypertension and diabetes were higher in the low EL group, whereas the proportions of current drinkers were lower  were detected during the follow-up period. Cox proportional risk regression analysis was carried out with the occurrence of breast cancer as the dependent variable and EL difference as the independent variable. In comparison with the low EL group, model 1 was adjusted for age, while model 2 was adjusted for smoking, alcohol consumption, BMI, physical activity, salt intake, hypertension and diabetes mellitus based on model 1. The hazard ratios (HRs) and 95% confidence intervals (95% CI) for breast cancer in the medium and high EL group were 2.23(1.12–4.64) and 2.52(1.12–5.70), respectively. In the sensitivity analysis, after excluding participants who developed breast cancer within 2 years after the start of the follow-up period, the risk of breast cancer was significantly higher in the medium and high EL group . The larger study cohort and longer follow-up time aims to minimise the effect of the above missing factors, and the abovementioned variables will be increased in subsequent studies. Secondly, only baseline data of EL were used for the analysis of the association between EL and breast cancer risk, and time-dependent exposures during follow-up were not considered. Some subjects may have received on-the-job education, so changes in EL may affect the risk of developing breast cancer, which should be explored in depth in subsequent studies.An increased risk of breast cancer was associated with a higher EL. This condition was more prominent in the sensitivity analysis, and certain factors such as alcohol use and hormone therapy may play a mediating role. Breast cancer can be reduced by reducing alcohol intake, maintaining a normal BMI, controlling blood pressure, increasing physical activity and improving screening compliance.


Ganoderma lucidum is known as a medicine food homology that can ameliorate gastrointestinal diseases. To evaluate the gastroprotective effects on different Ganoderma lucidum polysaccharides (GLPs), GLP was separated into three parts with different molecular weights using 100 kDa, 10 kDa, and 1 kDa membranes. The mitigation effects of different GLPs on ethanol-induced acute gastric injury were observed in rats. After pretreatment with different GLPs, especially GLP above 10 kDa, the symptoms of gastric mucosal congestion and bleeding were improved; serum myeloperoxidase, inflammatory factor, and histamine were decreased; and antioxidant activity and defense factors (NO and EGF) were increased. Results showed that GLP with different molecular weights had a dose-dependent effect in alleviating alcohol-induced gastric injury. The underlying mechanism might be related to regulating anti-oxidation, promoting the release of related defense factors, reducing inflammatory factors, and reducing the level of histamine in serum. The current work indicated that GLPs above 10 kDa could be applied as natural resources for producing new functional foods to prevent gastric injury induced by ethanol. The gastrointestinal tract is the largest endocrine organ in the human body, and its mucosal barrier protects it from invasion factors. Gastrointestinal diseases have become a global problem, threatening 5–10% of the world’s population . Severe Dendrobium officinale [Kimura & Migo] could protect the gastric mucosa by inhibiting cell apoptosis induced by oxidative stress [Momordica charantia could reduce ethanol-induced gastric injury in rats by inhibiting gastritis and oxidative stress [Ganoderma lucidum, an important medicinal and food fungus, is mainly distributed in China, Korea, and Japan. Physiologically active substances in Ganoderma lucidum such as polysaccharides, triterpenoids, alkaloids, steroids, and ergosterol have been isolated and identified [Ganoderma lucidum polysaccharide (GLP) is one of the main resources for its pharmacological activities. Various biological activities of GLP have been extensively studied, such as anti-inflammatory, anti-tumor, anti-oxidant, anti-diabetic, and immunomodulatory activities [Dendrobium nobile could alleviate ethanol-induced histological damage, antioxidant activities, and the level of epidermal growth factor (EGF) [In recent years, many studies have focused on the development of safe and effective functional foods to regulate inflammatory response, especially some natural ingredients. These ingredients have the advantages of high safety and good anti-inflammatory effects; accordingly, they received widespread attention ,7. In ade stress . Polysace stress . Accordientified . Among ttivities . The reltivities . GLPs prtivities . GLPs witivities . Moreoveor (EGF) . To our Lentinus edodes and explored their immunomodulation. Ultrafiltration and a series of chromatographic steps were used to separate and purify the polysaccharides. In this study, ultrafiltration membranes of 100 kDa, 10 kDa, and 1 kDa were used to fractionate the crude polysaccharides of Ganoderma lucidum. Effects of GLPs with different molecular weights on repairing gastric mucosal damage would be carried out. In summary, the present results provide evidence for understanding different molecular weights and biological activities of GLPs, which can be applied as potential feedstock for functional foods and dietary supplements.Gradient alcohol precipitation and column chromatography classification are widely used to classify polysaccharides with different molecular weights , which mGanoderma lucidum was obtained from Longevity Valley Botanical Co. Ltd. . Omeprazole was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. . Superoxide dismutase (SOD), glutathione peroxidase (GPX), myeloperoxidase (MPO), nitric oxide (NO), epidermal growth factor (EGF), TNF-α, 1L-1β, IL-18, and histamine (HIS) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Nanjing Jiancheng Bioengineering Institute . All other chemicals used in this study were of analytical grade.The fruiting body of Sprague–Dawley rats (150~180 g) were purchased from Hangzhou QDKR Bioscience  and used for ethanol-induced acute gastric ulcer animal experiments. The rats were housed at a room temperature of 25 ± 1 °C with a cycle of 12 h light and 12 h dark (lights on from 6:00 am to 6:00 pm), received food and water ad libitum, and adapted to the experimental environment for 1 week. The sample was administered to experimental animals by oral gavage for 1 week, and the administration volume was the mass of rats × 0.2 mL/20 g. Omeprazole was used as the positive control group, and the administration concentration was 20 mg/kg. Referring to previous studies ,20, the Ganoderma lucidum was added with distilled water to a beaker at a ratio of 15:1 (mL: g), mixed thoroughly with a glass rod, and extracted at 90 °C for 2 h. The extracts were filtered through filter paper (20 μm), and the residue was re-extracted in accordance with the above conditions. These two filtrates were mixed to be the crude extracts of GLPs. The crude extracts were classified by a membrane with 100 kDa at 1 MPa and 700 rpm, and the retentate was concentrated by rotary evaporation. Ethanol was added to be the volume ratio of concentrate to ethanol at 1:4, sealed, and left overnight to obtain the precipitate. GLPs were divided into three grades with different molecular weights using cascade ultrafiltration membrane technology. The precipitate was redissolved with water, rotary evaporated to remove the organic solvent, and vacuum freeze-dried as GLP100. According to the same procedure, the permeate of 100 kDa was filtered with the 10 kDa membrane to obtain the sample as GLP10, and the permeate of 10 kDa was filtered with the 1 kDa membrane to obtain the sample as GLP1. This specific process is shown in The preparation of GLPs is based on our previous studies by cascade membrane technology . 200 g o−1 to 400 cm−1.The procedures of purification and evaluation of the characteristics of GLPs were carried out according to our previous study . DiffereRats were divided into groups of blank control, model control, positive control , GLP100-L , GLP100-M , GLP100-H , GLP10-L , GLP10-M , GLP10-H , GLP1-L , GLP1-M , GLP1-H . There were 10 rats in each group. After the last administration, animals were strictly prohibited from eating (no drinking water) for 24 h. Each group of experimental animals was given samples or the same volume of normal saline. One hour later, the model group and sample-treated groups were given 75% ethanol at a dose of 10 mg/kg. The blank control group was given an equal volume of normal saline. One hour later, the animals were euthanized, the entire stomach was exposed, and the pylorus was ligated. The appropriate amount of 10% formaldehyde solution was perfused and fixed for 20 min. An incision was made along the greater curvature of the stomach, and the contents were removed. Lavage of the stomach was performed to unfold the gastric mucosa, and the length and width of the bleeding point or bleeding band were measured with a vernier caliper under a stereoscopic dissecting microscope or the naked eye. A pathological section of the most severely damaged part was taken and scored. The stomach was taken from the euthanized rats immediately and washed with cold physiological saline. The stomach was laid flat on filter paper to observe the size, shape, and occurrence location of the gross gastric ulcer and erosion hemorrhagic points. The ulcer index and ulcer inhibition rate were calculated according to the Guth standard . ObservaThe hematoxylin and eosin (H&E) staining method was used for histopathological observation of the general microstructure of gastric mucosa . The gasg rpm at 4 °C. The upper clear serum was transferred to a new 1.5 mL Ep tube. The sample was stored in a refrigerator at −80 °C for subsequent biochemical indicators and ELISA detection, which was measured according to the manufacturer’s instructions. The antioxidant capacity of rats was evaluated by measuring the content of antioxidant-related enzymes  in serum. The self-healing ability of the rats was evaluated by detecting the content of EGF and NO in serum. The degree of gastric injury was evaluated by detecting the contents of HIS. The degree of inflammation in the rats was evaluated by detecting the content of inflammatory factors .The whole blood of the rats was dissected, placed in an enzyme-inactivated centrifuge tube, and centrifuged for 15 min at 6000× p < 0.05 was considered as statistically significant. The significant difference was analyzed by the letter marking method. Origin 2018 software  was used for the drawing.All data were expressed as the mean ± standard deviation. SPSS16.0 software  was used for one-way ANOVA. The significant difference between the two groups was determined by the student–Newman–Keuls test. Membrane separation as a safe and gentle classification technology was used in the classification of polysaccharides. After ultrafiltration membrane classification, three GLPs with different molecular weights were obtained . In our Hericium erinaceus and Chinese Iron Yam, have a protective effect on gastric injury [Bletilla striata polysaccharide for pretreatment, the symptoms of gastric injury were alleviated. However, as with other studies on the anti-gastritis effect of polysaccharides, this study only focused on the improvement effect of different polysaccharides on abnormal lesions of gastric mucosa.Gastric mucosal injury is characterized by gastric mucosal erosion, extending to the muscle layer to puncture the gastric wall . The ethc injury ,24. By op < 0.05), and all three polysaccharides showed good inhibition efficiency.In our study, the gastric mucosa of normal rats had no symptoms of erosion and congestion, as shown in p < 0.05). Compared with the model group, the content of SOD was increased in groups of positive drug and classified polysaccharides. It indicated that after being given 75% alcohol gavage, the stomachs were damaged and corresponding oxidative stress reaction occurred. GLPs have protective effects on mucosa in rats, and among them, GLP100-H and GLP100-M had the best protective effects. High-concentration alcohol will cause the body to produce a large amount of ROS, which will accelerate the formation and severity of stomach damage . Thus, tp < 0.05). Compared with the model group, groups of omeprazole and classified polysaccharides kept the content of GPX steady. It was demonstrated that GLPs could protect the stomach by maintaining the GPX and reducing the harm of ethanol. Except for the groups of GLP10-M, GLP1-H, and GLP1-L, the effects of other groups on GPX were significantly increased (p < 0.05). Among them, GLP100 has the best protective effect on gastric mucosa, and with a dose–response relationship.GPX is another important antioxidant enzyme in the body. GPX can prevent red blood cells from being attacked by ROS and thus being hindered from performing their normal function of oxygen transport . Furtherp < 0.05), and the NO contents in the GLP-treated groups were significantly higher than in the model group (p < 0.05). The study demonstrated that the content of NO decreased after 75% alcohol gavage, which indicated that alcohol was prone to cause the inflammation. Both the positive drug and classified GLPs could maintain NO content steadily in the groups. Among them, GLP10-M, GLP100-L, and GLP100-M have the best effect.NO is one of the important molecules that lead to pro-inflammatory effects, and many diseases are caused by overproduction of NO . The intp < 0.05). The EGF content in each treatment group was higher than in the model group. It was indicated that after 75% alcohol gavage, the stomach was damaged and EGF was decreased in serum. GLPs have protective effects on alcoholic stomach injury, in which GLP100-H had the strongest protection ability, while GLP1-H had the weakest ability. Based on the above results, it is speculated that GLPs effectively promoted the secretion of EGF, which is speculated to be the protective mechanism of GLPs on gastric mucosa.EGF is secreted in the gastrointestinal tract  with the capabilities of reducing secretion of gastric acid, maintaining structural integrity, protecting submucosal blood flow, and speeding up the healing of ulcers . EGF is p < 0.05). MPO content in the groups of positive drug and classified polysaccharides decreased compared with the model group. Among them, MPO in groups of GLP100-H and GLP100-M decreased significantly (p < 0.05). Accordingly, it was demonstrated that pretreatment with positive drugs and GLPs could inhibit gastric injury.MPO is a heme protein contained in neutrophils, which is synthesized in the bone marrow by granulocytes before entering the circulation. External stimuli can cause neutrophils to accumulate and release MPO. Ingestion of high concentrations of alcohol in the stomach will greatly increase the activity of neutrophils, and the number of such cells directly reflects the level of inflammation in the stomach . Ethanolp < 0.05) in each treatment groups. However, compared with the model group, these levels in the groups of positive drugs and the polysaccharides were significantly reduced (p < 0.05). Accordingly, GLPs could significantly inhibit the levels of inflammatory cytokines, in which GLP 100 had the best inhibitory effect. Our results indicated that GLPs could inhibit inflammatory cytokine levels , which implicated the anti-inflammatory effects of GLPs in ethanol-induced gastric injury. This is consistent with a previous study showing that polysaccharides reduce the increase in inflammatory factors induced by ethanol [Ethanol can trigger TLRs, particularly TLR4, which induces the activation of NF-κB, sequentially promoting the production of inflammatory mediators and cytokines, e.g., TNF-α and IL-1β . Once th ethanol ,24. In tp < 0.05). HIS contents in the groups of positive drugs and GLPs were significantly lower than that in the model group (p < 0.05). Accordingly, omeprazole and GLPs can significantly reduce the HIS content in serum (p < 0.05). Among them, the GLP100-H treatment had the most significant effect on reducing the HIS content in serum.The gastric mucosa contains a variety of histamines, which are stimulant of gastric acid secretion. Excessive gastric acid secretion would change the permeability of gastric mucosa and accelerate ulcers. Gastric mucosal bleeding and its degree of damage could be judged by identifying the HIS content . In FiguGLPs were classified by ultrafiltration membranes of 100 kDa, 10 kDa, and 1 kDa with corresponding molecular weights of about 322.0 kDa, 18.8 kDa, and 6.4 kDa, respectively. These classified GLPs had a protective effect on gastric mucosa in rats. In alcohol-induced gastric injury treated by GLPs in rats, antioxidant enzymes, such as SOD, GPX, and MPO, could be regulated, thereby protecting the stomach tissue. GLPs could also maintain the content of signal molecule NO and keep the level of EGF steadily to protect the gastric mucosa from gastric damage. GLPs could inhibit TNF-α, IL-1β, and IL-18 at the level of inflammatory factors, thereby inhibiting the aggravation of gastric injury. All three GLPs can significantly reduce ethanol-induced acute gastric injury in rats and have a certain dose-dependent effect. Moreover, GLP100 has a greater improvement effect on acute gastric injury than GLP10, and GLP10 has a better effect than GLP1. Therefore, the greater the molecular weight of GLPs (6.4~322.0 kDa), the better the effect on acute gastric injury. In addition, GLPs can reduce the serum HIS content in rats with acute gastric injury, which may also contribute to the protective effect of GLPs. Accordingly, different levels and parts of molecular weights of GLPs, especially that above 10 kDa, could reduce alcoholic gastric injury at various degrees. There are still many contents that are worth further study. For instance, further separation and purification of GLPs are needed to determine efficacy. The synergistic effect of the components of each GLP fraction on gastrointestinal protection, and which proportion of GLP components are more effective when mixed in proportion, are worthy of further study, so as to make the research results viable to application.


To statistically study the incidence of endometrioma and deep infiltrating endometriosis (DIE) in various anatomical sites and to illustrate the significance and potential implications for each site. Furthermore, to improve the knowledge of the community for a non-invasive diagnosis alternative to laparoscopy.This study includes 2040 patients who had been referred with clinical evidence of pelvic endometriosis. These patients had been examined and undergone transvaginal sonography by the referring gynecologists. The imaging modality used to discover and locate various anatomical locations of involvement was MRI with contrast. Two radiologists with expertise in endometriosis separately assessed the patients' MRIs and highlighted the involved areas.In total, 79.1% were positive for either endometrioma or DIE. We detected both DIE and ovarian endometrioma in 78.2% of positive cases. Isolated endometrioma or DIE was present exclusively in 13.7% and 8.1% of patients, respectively. Uterosacral ligaments were detected as the most common (73.8%) site of DIE involvements and in 2.9% of cases were the sole affected location. Interestingly, very rare independent involvement of the genitourinary tract was seen in two patients.In this study, MRI was used to assess the likely involvement sites of endometrioma and DIE, as well as the frequency of incidences in various places and their relationships over a large dataset. Understanding the possibly involved sites, their statistics, and their co-existence can provide radiologists with a roadmap for non-invasive endometriosis diagnosis and treatment planning. These principles should hopefully assist reduce under- and overdiagnosis. A single rare DIE area involvement rate could help answer unknown sources of patient complaints.Information of potentially impacted sites, their frequencies, and their co-existence prevent under/overdiagnosis.The non-endometrioma DIE rate is 10% (an important finding), which could be missed by sonography.Endometriosis is a condition in which endometrial cells develop outside the uterine cavity, causing pelvic pain. The most common sites of involvement are ovaries, tubes, and pelvic peritoneum . It is mEndometriosis infiltrates the pelvic peritoneum in three ways: superficial or non-invasive (less than 5 mm depth), ovarian or cystic (endometriomas), and deep infiltrating endometriosis (DIE)  which isLaparoscopy has long been accepted as the main modality to diagnose endometriosis, although it suffers from associated risks, limitations, and costs . One of MRI can be used in diagnosis (presence of disease) and evaluating the extension of disease  as well as its detailed mapping . ConsideSeveral research –14 have Our work is a large-scale cross-sectional study aiming to cover rare cases of pelvic endometriosis as well as the relationship between different locations' involvement. To our knowledge, this is the first comprehensive study over a large dataset up to this point to investigate the percentage of various compartment involvement detected by MRI.This is a cross-sectional retrospective study. From May 2016 to March 2019, 2704 women were referred to our institution for endometriosis evaluation and, as a result, pre-operation mapping. Patients with endometriosis complaints, suspicious findings in other imaging modalities , or known cases of endometriosis were included . The data had been routinely collected for the patients referred to the imaging center for endometriosis without any interfering protocols or processes.Before the imaging process, patients were instructed to fast for six hours and have a semi-filled bladder and an empty bowel. Just before the picture acquisition, antimuscarinic drugs were administered (Hyoscine 20 mg intravenous). 30–40 cc intravaginal gel was utilized in the case of non-virgin patients. In addition, for individuals who had no contraindications, a paramagnetic contrast agent (Magnevist 15 cc intravascular) was employed. T2-weighted and fat-suppressed T1-weighted sequences are the best MR imaging sequences for diagnosing endometriosis. The best sequences for detecting pelvic DIE, especially for evaluating fibrotic lesions, are T2-weighted sequences without fat suppression.Total imaging time was 17–20 min depending on the patient size. The acquired sequences include axial and sagittal T1-weighted, axial fat-suppressed T1-weighted, axial T2-weighted, sagittal, and coronal fat-suppressed T2-weighted, diffusion-weighted and ADC mapping, and axial and sagittal fat-suppressed T1-weighted sequences with contrast . A gynecologist who specialized in infertility and laparoscopy, as well as an expert in the field of endometriosis, referred these cases.Reporting protocol for each patient included the complete evaluation and investigation of exactly 10 compartments. The list includes the right ovary, left ovary, ovarian fossa, uterosacral/round ligaments, retrocervical region, rectal wall, cul-de-sac, kidneys/ureters, vagina, and bladder.For each patient, the involvement or the lack of involvement was identified concerning all ten categories and reported. Over the full length of this study, a total of 2704 cases of pelvic MRI were referred to our center for the assessment of pelvic pain or endometriosis. Among them, this study covers 2040 cases (age 12–65 years old with a mean age of 32.85) which were referred from a single endometriosis-specialized gynecology center and had been thoroughly re-evaluated.A 1.5 Tesla MRI machine (Siemens Avanto) had been used as the scanner, and the images were reviewed by the PACS system (INFINITT VER 5.0.0.2), using cine movie and comparative techniques.All of these patients' MRIs were examined by two radiologists by default, as requested by the referring gynecologist. Furthermore, in the event of any difference between the MRI and ultrasound, physical exams, and/or the surgery, the images were rechecked, and the reports were verified. In particular, if there was a discrepancy between the MRI imaging results and the initial diagnosis via sonography or laparoscopy, the pictures were re-evaluated, especially for the location of concern (triple checked by both radiologist and the gynecologist). Following the reevaluation and confirmation, it was discovered that these cases were only a minor portion of the dataset, and the final results were provided in their report.Among the 2040 patients that we tested for endometriosis, 1613  cases were positive at least in one site for endometrioma or deep infiltrative endometriosis. For further detailed analysis, the positive cases were identified and separated from the non-involved population (21%). Age distribution was also evaluated in the involved patients with the majority of them being in their 20 s and 30 s . Also, 19% of patients were between 40 and 49 years old. Only 4% and 2% were under 20 and over 50 years old, respectively , 1104/1613 (68.4%), and 599/1613 (37.1%) patients, respectively. Isolated cases of pelvic DIE in at least one site without any sign of endometrioma were confirmed in 131 (8.1%) patients. Retrocervial region, uterosacral ligaments, ovarian fossa, cul-de-sac and rectal wall involvements were diagnosed in 888 (55%), 1191 (73.8%), 419 (26%), 298 (18.5%) and 318 (19.7%) patients, respectively. Rare involvement of the vagina in 14 (0.9%) cases, as well as kidney, ureters, and bladder  individuals with DIE (in at least one location) and 221 (13.7%) patients without DIE.Isolated DIE involvements exclusively in the US ligaments, retrocervical region, and genitourinary system were found in 47 2.9%), 15 (0.9%), and 2 (0.1%) of the cases, respectively. Ligaments were the most common site for DIE  vs DIE groups without endometrioma Table .Endometriosis, a common gynecological illness marked by extra-uterine endometrial cells, is the most common cause of chronic female pelvic pain . SeveralMRI has been found to be accurate in the diagnosis of endometriosis in numerous investigations, and various modalities have been examined. Bazot et al. evaluated the sensitivity of MRI, TVS, and rectal endoscopic sonography in 92 patients in 2009 and found that while they are equivalent in bowel loops, MRI is superior for evaluating US ligaments and vagina [Luciana et al. reported on the accuracy of MRI in the bladder, retrocervical, ureters, rectosigmoid, and vagina in 2009 (92 cases) .3D MRI was found to be useful in the evaluation of the bladder wall and involvement of the rectosigmoid in a study of 57 cases because it facilitates the imaging of these areas .In a 2013 study (152 patients), the sensitivity and specificity of the MRI as a function of the location were evaluated and the best MRI performance was for the bladder and the worst was for the peritoneum .Rectosigmoid, ovarian, and rectovaginal endometriosis can be easily diagnosed by MRI, with an excellent inter-and intra-observer agreement .Despite the fact that TVS can be very specific in DIE diagnosis, it is not as sensitive as MRI . For womOur research looked at how endometriosis affects different compartments and how they relate to one another. In our large database, the most common and rarest forms (2 in 2048: 0.1 percent) were identified and discussed. This could help physicians better understand the causes of pelvic or flank pain, hematuria, vaginal bleeding, and infertility in their patients.Furthermore, the relationship between certain sites requires deeper investigation. For example, approximately one-fifth (17%) of rectal DIE is linked to retrocervical DIE; therefore, both locations should be thoroughly examined if one of them is affected. Furthermore, no single case of rectal wall DIE has been reported, so after a rectal wall DIE diagnosis, every portion of the pelvic should be re-evaluated.Another noteworthy observation is the existence of pelvic DIE in 8.1 percent of individuals who do not have endometrioma. Because there are not any cystic lesions in these circumstances, ultrasonography may overlook them (roughly one in ten patients with endometriosis). As a result, even if there is no endometrioma, all suspicious patients should be still investigated for DIE by MRI.The contrast could have added value for endometriosis evaluation and better implant visualization Fig. a, b, espIn a recent study , reviewiOne of the limitations of our study was the lack of pathological confirmation in a group of patients who did not undergo surgery and instead received nonsurgical medical treatments. Nevertheless, they still underwent ultrasound . Another limitation of our study is the evaluation of bowel loops in pelvic regions only, resulting in a smaller percentage relative to laparoscopic and pathologic studies covering the entire abdomen .For a large dataset based on MR imaging, the prevalence of endometrioma and DIE involvement in various pelvic compartments, as well as their statistical correlation, were assessed. Knowing the likelihood of various sections being involved as well as their co-existence is a key step in accurately diagnosing and treating endometriosis. Pretreatment DIE mapping is essential for determining the type of treatment and ensuring thorough removal, especially if a multi-specialty operation is required. Furthermore, it aids in diagnosis and prevents under- or over-diagnosis.