Infertility remains a significant public health concern. An issue with controlled ovarian stimulation (COS) is the selection of an exogenous gonadotropin (Gn) regimen, which is mainly based on urinary follicle-stimulating hormone (uFSH), recombinant follicle-stimulating hormone alfa , and human menopausal gonadotropin (HMG). In addition, most previous studies focused on the clinical pregnancy rates or live birth rates (LBR) per transfer cycle, but not on the cumulative live birth rate (CLBR) per started cycle. The CLBR, appears to be a more comprehensive and accurate universal measure of IVF treatment success. Therefore, this study aimed to compare the cumulative live birth rate (CLBR) between rFSH-alfa and uFSH regimens for ovarian stimulation.This retrospective cohort study included patients who underwent assisted reproductive technology (ART) with gonadotropin-releasing hormone (GnRH) agonist long protocol between March 2009 and December 2018. Patients were grouped according to the Gn regimen received . The main outcome was CLBR, which defined as the first live birth following the use of all fresh and frozen embryos derived from a single COS cycle.P < 0.05). There were no differences in pregnancy rate (32.7% vs. 33.8%) and LBR (25.5% vs. 26.9%) per transfer cycle (P > 0.05). No significant difference was found in clinical outcomes among the frozen embryo transfer cycles (P > 0.05). The CLBR per started cycle in the rFSH-alfa group was higher than in the uFSH group . After adjustment, rFSH-alfa was independently associated with a higher CLBR .A total of 1078 cycles were analyzed . The rFSH-alfa group was characterized by a higher number of retrieved oocytes (13.3 vs. 11.0) and transferable embryos (5.0 vs. 4.0), a higher fresh embryo transfer rate (35.0% vs. 26.3%), and a higher multiple birth rate among the fresh embryo transfer cycles (8.2% vs. 2.5%) (rFSH-alfa and uFSH have similar pregnancy rates and LBR per transfer cycle, rFSH-alfa might achieve more transferrable blastocysts and higher CLBR per started cycle compared to uFSH.The online version contains supplementary material available at 10.1186/s13048-022-01009-w. Infertility remains a significant public health concern, affecting about 15% of all couples of reproductive age . Since tOutcomes traditionally used to evaluate results of in vitro fertilization, and embryo transfer (IVF-ET) treatment include the number of embryos obtained per IVF cycle, clinical pregnancy rate and LBR per transfer cycle  . NeverthAt present, the rFSH-alfa and uFSH are widely used for COS in China , 18. StiTherefore, this study aimed to compare the clinical outcomes, particularly CLBR, between rFSH-alfa and uFSH regimens for COS in a real-world setting. The results could provide additional evidence for selecting the most optimal treatment for COS.This retrospective study included patients who underwent ART with COS at the Center for Reproductive Medicine of Subei People’s Hospital of Jiangsu Province from March 2009 to December 2018. STROBE guideline was followed in our preparation. This study was approved by the ethics committee of the Reproductive Medicine of Subei People’s Hospital of Jiangsu Province. Due to the retrospective nature, the need for informed consent was waived by the committee.The inclusion criteria were 1) underwent gonadotropin-releasing hormone (GnRH) agonist long protocol, including short-acting GnRH agonist long protocol and long-acting GnRH agonist long protocol, 2) received rFSH-alfa or uFSH for COS, 3) ≤2 previous stimulation cycles, and 4) antral follicle count (AFC) ≥5.The depot GnRH agonist long protocol was used for COS. For the long-acting long protocol, a long-acting agonist analog was given on day 2–3 of menstruation for downregulation, with blood tests for FSH, luteinizing hormone (LH), and estradiol (E2) and a vaginal ultrasound 28–35 days after downregulation to check the status of downregulation. For the short-acting GnRH agonist long protocol, a short-acting agonist analog was given on day 21 of the previous menstrual cycle or 7 days after ovulation of the previous menstrual cycle. Blood was drawn for FSH, LH, and E2, and a vaginal ultrasound was performed on days 2–3 after the return of menstruation to check the status of downregulation. If pituitary down-regulation was confirmed, exogenous Gn was given to initiate COS. The FSH received by patients during the study period included rFSH-alfa  or uFSH . The Gn initiation dose was 100–225 IU/d for patients with normal ovarian reserve function and 225–300 IU/d for patients with low reserve function. The drug dose was adjusted according to the growth rate of the follicles and hormone levels. During stimulation, the ovarian response was monitored by measuring serum E2, progesterone (P4), and LH and using serial transvaginal ultrasonographic examinations. Recombinant human choriogonadotropin (hCG) was administered until at least one follicle was observed with a diameter of ≥19 mm or two with diameters of ≥18 mm.< 20%, or normal fertilization rate < 30%; otherwise, IVF was performed. A pronucleus was observed 18–20 h after insemination. The Vitrolife serial sequential culture medium was used for embryo culture.Oocyte retrieval occurred 36–40 h after hCG injection (trigger). IVF or ICSI were performed 4–6 h after in vitro culture of the oocyte. ICSI was performed in cases of males with severe oligospermia or azoospermia requiring testicular or epididymal puncture, previous IVF fertilization failure, fertilization rate For patients with high risk of ovarian hyperstimulation syndrome (OHSS), substandard endometrial thickness or morphology, or moderate to severe hydrosalpinx, frozen embryo transfer is recommended; otherwise, fresh embryo transfer will be utilized. Fresh embryo transfer was performed 48 or 72 h after oocyte retrieval using a Cook soft catheter, while FET was performed on the day of thawing or day 1 post-thawing. The greatest endometrial thickness in the uterus’s longitudinal plane in transvaginal ultrasound was measured from one endometrial-myometrium interphase to the corresponding interphase. The measurement recorded on the day of hCG administration was used as the referral value in this study. Pregnancy was first assessed by a positive hCG test 2 weeks after oocyte retrieval and confirmed by ultrasound visualization of an intrauterine gestational sac with an embryo with heartbeats at 6 weeks gestational age.Patients’ information through the electronic medical record (EMR) were retrospectively collected, including baseline data , COS treatment and laboratory embryo culture . Clinical outcomes, including CLBR per started cycle, LBR per transfer cycle, pregnancy rate per transfer cycle, abortion rate, and multiple birth rate, were calculated.High-quality embryos were defined as grade I and II embryos, based on the size, regularity, refractive index of the cytoplasm, and the number of fragments. Grade I embryos: uniform cell size, regular shape, complete zona pellucida, uniform and clear cytoplasm without granular phenomenon, and fragmentation between 0 and 5%. Grade II embryos: slightly uneven cell size, slightly irregular shape, the cytoplasm may be granular, and fragmentation between 6 and 20%. Grade III embryos: obviously uneven cell size, obviously irregular shape, the cytoplasm may be granular, and fragmentation between 21 and 50%. Grade IV embryos: severely uneven cell size, severely granular cytoplasm, and fragmentation > 50%.Live birth was defined as the final birth of at least one living child born after 28 weeks of gestation. CLBR was defined as the first live birth following the use of all fresh and frozen embryos derived from a single COS cycle. In cases when all embryos were used up in one embryo retrieval cycle, or if a live birth was not achieved for more than 2 years after ART started, we considered that this embryo retrieval cycle finally did not achieve a live birth. LBR per transfer cycle was calculated as the number of live birth cycles divided by number of transfer cycles. Miscarriage rate was calculated as the number of miscarriage cycles divided by number of clinical pregnancy cycles. Multiple birth rate was calculated as the number of multiple live birth cycles divided by transfer cycles. Clinical pregnancy was defined as a transvaginal ultrasound 4 weeks after transplantation or 2 weeks after a positive hCG blood test indicating a gestational sac in or outside the uterine cavity. Clinical pregnancy rate per transfer cycle was calculated as the number of clinical pregnancies divided by number of transfer cycles.www.empowerstats.com, X&Y solutions, Inc. Boston, MA, USA) and R 3.6.1 (http://www.r-project.org). The continuous variables were tested for normality using the Kolmogorov-Smirnov test. The data complying with a normal distribution were presented as means ± standard deviations; otherwise, they were expressed as medians (25th–75th percentile). Categorical variables were expressed as the number of cases (percentage). The data between the two groups were compared and analyzed using non-parametric tests or the chi-square test, as appropriate. A logistic regression model was used to assess the relationship between FSH regimens and CLBR after adjusting for age, BMI, baseline FSH, and AFC. We selected confounders based on the associations reported with the outcomes of interest [P < 0.05 was considered statistically significant.All statistical analyses were performed using Empower Stats . The characteristics of the patients are shown in Table This study included 1078 ART cycles with the GnRH agonist long protocol. Of them, 314 cycles were in the rFSH-alfa group, and 764 cycles were in the uFSH group, respectively. The patients in the uFSH group were 29.4 ± 3.8 years of age and had a mean BMI of 22.1 ± 3.6 kg/mP < 0.001). As shown in Table P < 0.05).The overall Gn dose needed to achieve ovulation was significantly lower with rFSH-alfa than with uFSH . The fresh embryo transfer rate (35.0% vs. 26.3%) and the multiple birth rate in the fresh embryo transfer cycles (8.2% vs. 2.5%) in the rFSH-alfa group were higher than in the uFSH group . In the fresh transfer subgroup, there were no differences in the pregnancy rate (32.7% vs. 33.8%), the abortion rate (11.1% vs. 11.8%), and LBR (77.8% vs. 79.4%) among the groups in the fresh embryo transfer cycles . For the FET cycles, there were no differences in the clinical pregnancy rate (47.7% vs. 48.7%), abortion rate (13.6% vs. 16.3%), LBR (40.1% vs. 39.1%), and multiple birth rate (7.9% vs. 7.5%) between the two groups .The clinical outcomes were analyzed and compared between the two groups and between the fresh and frozen embryo transfer subgroups in each group, as shown in Table P = 0.0018), age , and AFC  were associated with higher CLBR in the total study population. The multivariable analysis showed that rFSH-alfa  and age  were independently associated with a higher CLBR (Table P = 0.0004) . To the best of our knowledge, this is the first real-world study that used CLBR as a main outcome on a relatively large sample size and reported significant differences between rFSH-alfa and uFSH.This study aimed to compare the clinical outcomes, particularly CLBR, between rFSH-alfa and uFSH regimens for ovarian stimulation in a real-world setting. Our main finding is that after adjusting potential confounders , rFSH-alfa was independently associated with a higher CLBR compared with uFSH , 2PN oocytes (7.0 vs. 5.0), and transferable embryos (5.0 vs. 4.0) in the rFSH-alfa group were higher than in the uFSH group. There were no differences in the incidence of OHSS (7% vs. 4.7%), pregnancy rate (32.7% vs. 33.8%), abortion rate (11.1% vs. 11.8%), and LBR (77.8% vs. 79.4%) among the groups both in the fresh and frozen embryo transfer cycles.COS is an important part of assisted reproduction, and FSH is commonly used clinically to enhance recruitment and development of ovarian follicles. Ongoing issues with uFSH derived from urine donations include batch-to-batch variability and a finite donor supply . Indeed,P < 0.05).rFSH-alfa is produced by inserting the FSH DNA into a Chinese hamster ovary cell line and has no detectable LH activity and very high specific bioactivity for FSH  , 25. HigAnother recent large  real-world study demonstrated significantly higher rates of CLBR, cumulative ongoing pregnancy, and cumulative clinical pregnancy with r-hFSH-alfa compared to human menopausal gonadotropin . The relThis study has certain limitations. First, there is a risk of selection bias due to the retrospective nature of the study. In order to reduce this effect, we used multivariable analyses and PSM to ensure higher reliability of the obtained outcome. Second, we did not collect the medical costs of two groups in the study. Future studies are warranted to provide more comprehensive evidence for clinical decision-making. Third, we only compared the CLBR between rFSH-alfa and uFSH in patients received agonist long protocol. The different FSH in antagonist protocol will need to be studied in the future. Finally, our study is single-center research. Despite the large sample size, it might not represent other regions or nationalities.In conclusion, this study reports that despite rFSH-alfa and uFSH having similar pregnancy rates and LBR in both fresh embryo transfer cycle and FET cycle, rFSH-alfa achieved more transferrable blastocysts and higher CLBR compared with uFSH. Those results contribute to the discussion on the use of exogenous Gn regimens in COS and might help in optimizing COS strategies for clinical practice.Additional file 1.


Candida albicans is an opportunistic pathogen that causes gastrointestinal (GI) candidiasis closely associated with intestinal inflammation and dysbiosis. Drug resistance, side effects of available antifungal agents, and the high recurrence of candidiasis highlight the need for new treatments. We investigated the effects of hydroethanolic extracts of licorice root (LRE) and walnut leaf (WLE) on GI colonization by C. albicans, colon inflammation, and gut microbiota composition in C57BL/6 female mice. Oral administration of LRE and WLE alone or in combination once daily for 12 days before C. albicans infection and then for 5 days after infection significantly reduced the level of C. albicans in the feces of gastrointestinal infected mice as well as colonization of the GI tract, both extracts showing robust antifungal activity. Although total bacterial content was unaffected by the extracts , the abundance of protective bacteria, such as Bifidobacterium spp. and Faecalibacterium prausnitzii, increased with the combination, in contrast to that of certain pathobiont bacteria, which decreased. Interestingly, the combination induced a more robust decrease in the expression of proinflammatory genes than either extract alone. The anti-inflammatory activity of the combination was further supported by the reciprocal increase in the expression of anti-inflammatory cytokines and the significant decrease in enzymes involved in the synthesis of proinflammatory eicosanoids and oxidative stress. These findings suggest that LRE and WLE have synergistic effects and that the LRE/WLE combination could be a good candidate for limiting GI candidiasis and associated inflammation, likely by modulating the composition of the gut microbiota.IMPORTANCE The adverse effects and emergence of resistance of currently available antifungals and the high recurrence of candidiasis prompt the need for alternative and complementary strategies. We demonstrated that oral administration of hydroethanolic extracts of licorice root (LRE) and walnut leaf (WLE) separately or in combination significantly reduced the colonization of the gastrointestinal (GI) tract by C. albicans, highlighting a robust antifungal activity of these plant extracts. Interestingly, our data indicate a correlation between LRE and WLE consumption, in particular the combination, and a shift within the gut microbiome toward a protective profile, a decrease in colonic inflammation and prooxidant enzymes, suggesting a synergistic effect. This study highlights the significant prebiotic potential of the LRE/WLE combination and suggests that the health benefits are due, at least in part, to their ability to modulate the gut microbiota, reduce inflammation and oxidative stress, and protect against opportunistic infection. Candida albicans inhabit the gastrointestinal (GI) tract of most healthy individuals  and walnut (Juglans regia L.) among other plants have been evaluated in several in vitro and in vivo studies. A propylene glycol extract of a dry powder of licorice roots inhibited the growth of C. albicansin vitro  and Gram-negative bacteria species . Only the growth of Gram-positive bacteria  was inhibited by these extracts  could correct overall gut microbial dysbiosis and fecal metabolic disorders associated with CPT-11-induced colitis in mice  and licorice root (licorice root extract [LRE]), both separately and particularly in combination, in mice with GI candidiasis with the intention also to investigate whether the observed effects could involve anti-inflammatory activity and modulation of gut microbiota.Although many studies showed the ompounds , 14–16, didiasis , 12, 17.o assays –21. The High-performance thin-layer chromatography (HPTLC) analyses identified both glycyrrhizic acid (glycyrrhizin) and formononetin in LRE . Ultra-hCandida GI infection after oral administration of LRE and WLE separately or in combination . Interestingly, the combination of the two extracts induced a more robust decline in the expression of proinflammatory genes than either extract administered separately  (Hpgds [prostaglandin D synthase] and Alox15 [12/15-lipoxygenase]) was not affected by administration of the plant extracts  . The mRNextracts .phoxp47, a cytosolic subunit of the NADPH oxidase complex, and the expression of inducible nitric oxide synthase (Nos2), the activation of which is essential for the release of reactive oxygen species (ROS), were downregulated in response to the plant extracts with a stronger effect when the two extracts were combined (Nos2 and arginase-1 toward the expression of arginase-1 (Sod2 (superoxide dismutase) and Hemox-1 (hemoxygenase 1) concentrations, the combination significantly increased expression of the antioxidant enzymes Nqo1 (NADPH quinone dehydrogenase 1), Cat  and Arg1 (arginase-1)  .C. albicans.Altogether, these data highlight the anti-inflammatory and antioxidant potential of the LRE/WLE combination in the colon during gastrointestinal infection with Candida spp. form part of the commensal microbiota in most individuals with a healthy immune system, variations in the local microenvironment, antibiotic treatment, or alterations in the immune system can favor dysbiosis and rapid proliferation of Candida, which can then become a pathogen  exerted a dominant Th1-immunological adjuvant effect  and walnut leaf (WLE), administered separately or in combination, on GI colonization by fects 11–, 24–26. nfection . In micet effect . In vitrdidiasis . Severaldidiasis , 15–17. nt route . LiquiriC. albicans infection  evaluated in the present study could be due to the presence of flavonoids, such as glabridin and liquiritigenin, and to the presence of glycyrrhizic acid (and its derivative glycyrrhetinic acid), identified by UHPLC-MS analysis, and known to increase the resistance of mice to nfection , 13.Trachyspermum ammi seeds enriched in rosmarinic acid-3-O-glucopyranoside, as well as kaempferol-(coumaroyl glucosyl)-rhamnoside and quercetin-3-O-galactoside, inhibited Candida in vitro (in vitro studies (Usnea longissima), could sensitize resistant C. albicans to fluconazole and thereby enhance the efficacy of this drug. Quercetin enhanced the destruction of C. albicans NBC099 cells by fluconazole and induced cell death. It was also found to strongly suppress the onset of virulence-enhancing processes such as biofilm formation and hyphal development, as well as phospholipase, proteinase, esterase, and hemolytic activities. The sensitization was dependent on the farnesol response generated by quercetin, farnesol being a quorum-sensing compound produced by C. albicans, that is known to regulate the expression of Candida virulence factors. In addition, taxifolin was identified as an inhibitor of the transcriptional factors (Tec1 and Rfg1) inducing the hyphal growth responsible for the invasiveness and virulence of C. albicans  roots and walnut (Juglans regia L.) leaves provided by PiLeJe Laboratoire. The preparation and phytochemical analysis of the hydroethanolic extract of walnut leaves was previously published  roots were collected in Spain in November 2017. Fresh licorice roots were extracted by 20% to 70% (vol/vol) ethanolic leaching. The extracts were then mixed and concentrated under reduced pressure  at controlled temperature (35 to 45°C). Glycerol was then added to dilute the resulting extract to a final concentration of 5.2% (wt/wt) .Licorice (Standards were diluted in ethanol 70% at a concentration of 0.4 mg/mL for glycyrrhizic acid and in methanol 0.1 mg/mL for formononetin. The LRE without glycerol (1 mL) was diluted in 3 mL of a mixture of ethanol and water (70/30 vol/vol). The resultant solution was shaken and centrifuged for 5 min at 4400 rpm. The supernatant solution was transferred into individual vials and then submitted for HPTLC analysis. In addition,1.8 g of ground licorice roots was extracted with 20 mL of ethanol and water (70/30 vol/vol). The resultant solution was sonicated for 10 min and centrifuged for 5 min at 4400 rpm. The supernatant solution was transferred into individual vials and then subjected to HPTLC analysis.HPTLC analysis was performed on 200.0 × 100.0 mm silica gel 60 F 254 HPTLC glass plates . Standard solutions and samples were applied to the plate as 6.0 mm wide bands using CAMAG Automatic TLC sampler (ATS 4). The equipment comprised a CAMAG horizontal developing chamber, a TLC plate heater, a CAMAG Derivatizer Device, a CAMAG chromatogram immersion device, a CAMAG visualizer, and VisionCATS software. The general chromatography conditions are presented in 2 data were obtained at a resolution of 17,500. Data were processed using Xcalibur software .Chromatographic analyses (UHPLC) were performed on an Ultimate 3000 RSLC UHPLC system  coupled to a binary pump (U3000 HPG-3400RS) and a diode array detector. Compounds were separated on an Uptisphere Strategy C18 column , which was controlled at 40°C. The mobile phase was a mixture of 0.1% (vol/vol) formic acid in water (phase A) and 0.1% (vol/vol) formic acid in acetonitrile (phase B). The gradient of phase A was 100% (0 min), 80% (10 min), 73% (35 min), 30% (50 min), 0% (55 min). The flow rate was 0.8 mL/min, and the injection volume was 5 μL. The UHPLC system was connected to a Q-Exactive Orbitrap  mass spectrometer, operated in negative and positive electrospray ionization mode. Source operating conditions: 3 kV spray voltage for negative mode and 3.5 kV spray voltage for positive mode; 320°C heated capillary temperature; 475°C auxiliary gas temperature; sheath, sweep, and auxiliary gas (nitrogen) flow rate 60, 18, and 4 arbitrary units, respectively; and collision cell voltage between 20 and 50 eV. Full scan data were obtained at a resolution of 35,000 whereas MSCompounds present in the LRE were characterized according to their retention times, mass spectral data, and comparison with authentic standards when available or with published data.C. albicans infection and then 5 days after infection. Control groups received only the vehicle . Esophageal and GI candidiasis was established by intraesophageal administration of C. albicans at the rate of 50 × 106 blastospores in sterile saline solution per mouse, as described previously  and the French Ministry of Higher Education, Research, and Innovation (ESRI) under permit number 5412–2016051917498658;2016 to 2020 and renewed under permit number 23558–2020011016561848;2020 to 2025 in accordance with European legal and institutional guidelines (2010/63/UE) for the care and use of laboratory animals. Female C57BL/6 mice aged 8 weeks were purchased from Janvier Labs (France). LRE and WLE were administered orally, separately (2.5 g/kg) or in combination (1.25 + 1.25 g/kg), once daily for 12 days before eviously , 48. TenC. albicans used throughout these experiments (sc-5314) was provided by the American Type Culture Collection (ATCC) and was maintained on Sabouraud dextrose agar  plates containing gentamicin and chloramphenicol. Growth from an 18 to 24 h SDA culture of C. albicans was suspended in sterile saline solution (NaCl 0.9%) for mice infection /g of stool.Stools were collected daily from day 3 to day 5 after infection, weighed, and mechanically homogenized in phosphate buffer saline (PBS). Serial dilutions of homogenates were plated on SDA plates containing gentamicin and chloramphenicol for the quantitative determination of the number of The esophagus, cecum, and colon dissected from infected mice were crushed using lysing matrix tubes . Tissue sample homogenates were resuspended in BLB lysis buffer  for 20 min at room temperature and DNA was purified using a High Pure PCR Template Preparation kit (Roche). RT-PCR was performed on a Light Cycler 480 system using Light Cycler SYBR Green I Master (Roche). For amplicon detection, the Light Cycler DNA SYBR green I kit was used as described by the manufacturer . The primers used are listed in Candida, C. albicans cell suspensions were standardized at 106 cells/mL and serially diluted samples of genomic fungal DNA (range: 100 to 106 cells/mL) were used as external standards in each run. Cycle numbers of the logarithmic linear phase were plotted against the logarithm of the concentration of template DNA to evaluate the number of yeasts present in each tissue sample homogenate and normalized to the amount of genomic β-actin.To quantify the number of Semiquantitative RT-PCR was performed with primers that amplify the genes encoding 16S rRNA from specific bacterial groups on DNA isolated from colonic mucosa to evaluate mucosa-associated bacteria colonization. Relative quantity was calculated and normalized to the amount of genomic β-actin.mRNA from colonic tissues were prepared and cDNA was synthesized according to the manufacturer’s recommendations . RT-PCR was performed on a Light Cycler 480 system with Light Cycler SYBR Green I Master Mix (Roche). Serially diluted samples of pooled cDNA were used as external standards in each run for the quantification. GAPDH was used as the housekeeping gene. The primers (Eurogentec), designed with the software Primer 3, were listed in P ≤ 0.05 were considered significant . Data represent mean values ± standard error of the mean (SEM).GraphPad Prism  was used for graph preparation and statistical evaluation. Differences between groups were assessed using ANOVA, followed by a nonparametric Mann-Whitney test. Differences with


Valorization of by-products obtained from food processing has achieved an important environmental impact. In this research, sunflower wax recovered from oil refining process was incorporated to low and high-methoxyl pectin films produced by electrospraying. Film-forming solutions and wax-added electrosprayed films were physical and structurally evaluated. The addition of sunflower wax to the film-forming solutions reduces conductivity while raising surface tension and density, whereas the type of pectin had a larger impact on viscosity, with the low-methoxyl solution having the highest value. These changes in physical solution properties influenced the film characteristics, observing thicker films with lower water vapor transmission rate (WVTR) when adding wax. Micrographs obtained by scanning electron microscopy (SEM) revealed the presence of wax particles as small spherical shapes, having a good distribution through the sectional area of films. According to X-ray diffraction (XRD), atomic force microscopy (AFM) and mechanical properties analyses, the presence of wax had an impact on the degree of crystallinity, producing a more amorphous and rougher film’s structure, without affecting the elongation percentage and the tensile stress (p>0.05). These results showed that wax addition improves the physical properties of films, while the suitability of using both pectins and the electrospraying technique was demonstrated. Plastic films made from non-renewable resources often cause harmful effects to the environment after disposal, while the current rise in consumer needs for safe, healthy, and stable foods, besides the awareness concerning the harmful environmental effects of wastes, have increased interest in using natural materials for food packaging ,2,3. ThiEdible films and coatings act as a reinforcement of the outer protective layer of foods (peel) and as a barrier to reduce the quality deterioration process, which is promoted by the migration of water vapor, oxygen, ethylene, etc. ,8. There3 and 226 × 103 g/mol [Pectin is an anionic polysaccharide that is usually present in many primary and middle layers of the cell walls of plants ,16, and 03 g/mol ,20. The 03 g/mol ,26,27,2803 g/mol ,30,31,3203 g/mol ,34. The 03 g/mol . In this03 g/mol  evaluate03 g/mol . These d03 g/mol . This te03 g/mol , fresh m03 g/mol , cracker03 g/mol  and fres03 g/mol . In prev03 g/mol ,29,31,3703 g/mol ,29,31. TThe aim of this work was to study the feasibility of producing electrosprayed films with low and high-methoxyl pectin added with recovered sunflower wax, evaluating the structure, composition, water resistance, and mechanical properties of the films as a result of the different materials used.High-methoxyl pectin (HM) from citrus peel  with a galacturonic acid content ≥74% (dried basis) and methoxy groups ≥6.7% (dried basis), partly amidated low-methoxyl citrus pectin (LM)  with esterification degree approximately 27%, recovered sunflower wax (SFW), previously obtained of waste material from sunflower oil refining process (winterization stage) and characterized [w/w to complete dissolution at room temperature. Glycerol was added at 50 g/100 g pectin and Tween 20 in a ratio 1:10 (w/w) (Tween 20:pectin) and the mixture was stirred for 10 min. Pectin and sunflower wax emulsions were performed according to the procedure described by Chalapud et al. [Control solutions (without wax) were prepared by hydrating and stirring pectin (HM or LM) with distilled water at 2% d et al.  with somPhysicochemical properties of pectin control solutions and sunflower wax emulsions such as conductivity, viscosity, density and superficial tension were evaluated as quality parameters.Density and refractive index of solutions and emulsions was evaluated according to 962.37 and 990.35 methods of the A.O.A.C. International . Surfaceσ (kΩ−1 m−1) is conductivity, R (kΩ) the resistivity, L (m) is the distance between two electrodes and A (m2) is cross-sectional area of cell.Conductivity determination was performed following two-point resistivity technique  using a −1.This parameter was performed using a HR-3 Discovery Hybrid Rheometer  and a concentric cylinder geometry with a cup (diameter = 30.36 mm) and rotor . Volume sample was 23 mL and the tests were performed by duplicate at 25 °C without pre-shear. Share rate range was between 0.01–100 s2 were obtained and were stored at room temperature inside a desiccator with a saturated solution of NaBr at 57% relative humidity (RH) before testing.Electrospraying technique was followed using the equipment and procedure described by Gaona Sánchez et al.  with somThickness was measured at five sites of the films (one in the center and four on the periphery) by using a digital micrometer model ID-C112EXB . The average value was used for calculations of mechanical and permeability properties.Structure of top surface and cross section of films was examined by SEM. Samples were covered with a thin layer of gold in a Denton Vacuum Desk II. The micrographs were obtained with a scanning electron microscope JEOL JSM 6460 LV  at an acceleration voltage of 15 kV and 1500x magnification. −1., reporting images roughness . For this evaluation, for each sample, sections of 0.5 cm × 0.5 cm were cut and two areas of 10 × 10 μm2 were scanned at a speed of 1 Hz and at a resolution of 256 × 256 pixels. The surface roughness was measured from images calculating the root square deviation of the heights (Rq) (Equation (2)) and the arithmetic mean of the absolute values of the height deviations (Ra) (Equation (3)) using the Nano Scope Analysis 1.5 program  and applying a flattening process (one degree). Determinations were performed in triplicate.iZ is the height deviation from the mean of the heights, and N is the number of points in the image. Topographic AFM images in three dimensions and at real-time resolution were obtained by an atomic force microscope  with a resonance frequency of 286–362 kHz and a force constant of 20–80 NmA is the integrated intensity of the crystalline phase and (A + B) is the total area of the diffractogram.X-ray diffractograms were obtained by a Rigaku Miniflex 600 powder diffractometer  operating at 45 kV and 40 mA. The scanning regions were in a 2θ range of 3°–35°. The degree of crystallinity (%) of films was calculated using Equation (4). The separation and integration of the areas under the crystalline and amorphous X-Ray diffraction peaks was performed as recommended by Ribotta et al. . The sof−1m−2) by dividing this ratio (g/s) by the area of tested film. WVP was calculated according to the combined Fick and Henry laws for gas diffusion through films, using Equation (5), where x is the film thickness (m) and ∆P corresponds to the vapor pressure difference inside the system (4246.9 Pa). The measurements were performed in triplicate.WVP of films was determined according to the methodology proposed by Cazón et al.  based on2), while %E (Equation (7)) by the film stripe length changes during elongation, both methodologies according to Cazón et al. [L is the film width (mm), and x is the thickness (mm).fL is the length of film at break and iL is the final length.Mechanical properties (tensile strength (TS) and elongation at break (%E)) were evaluated according to the ASTM Standard D882-09 , using an et al. . Young’s−1 silicon line. The measurements were performed at wave numbers from 100 cm−1 to 3200 cm−1 at room temperature. The spectra were recorded by applying an exposure time of 4–60 s and scanning the sample 10 to 20 times. The acquired spectra were managed by LabSpec software (Horiba Jobin Yvon) and edited using Spectragryph program version 1.1.2. The measurements were performed in triplicate.Raman analysis was performed using a LabRam HR800  coupled to an Olympus BX 41 microscope with a 100× objective and a Peltier-cooled charged coupled device detector and three excitation lines: 532 nm, 633 nm and 785 nm with output power of 43.4 mW, 86.3 mW and 56.7 mW, respectively. Raman spectra were recorded using 400 lines/mm and an emission laser of 785 nm. Calibration was determined using the 520.5 cmp < 0.05). For statistical analysis, InfoStat statistical analysis software, Version 2011 was used. Significant differences were determined using one-way ANOVA comparing all samples simultaneously. The type of pectin (high- or low-methoxyl), and the presence of wax were the variables studied. Each response (columns) was statistically analyzed and labeled with different lowercase letters provided by Fisher Test (α = 0.05) where values followed by different letters indicate significant differences , and following a shear thinning behavior as indicated by the flow index, whose values are further away from 1 than those found for solutions and emulsions with high-methoxyl pectin (HMsol and SFW-HMem), which present a behavior close to Newtonian. In this regard, Morris et al. [p > 0.05).g et al.  and Yapog et al. , who stug et al. . Densityg et al.  for highs et al.  found anp > 0.05). These results are related with the increase in density of sunflower wax added emulsions as compared to control solutions without wax inclusion.These results showed that SFW-HM and SFW-LM presented higher thickness values than control samples, without a significant effect due to the type of pectin (p < 0.05) when compared to non-wax added films. These results show that sunflower wax offered an important barrier to water vapor transference mainly due to its hydrophobic character caused by their high content of long-chain fatty alcohols and alkanes [Regarding WVTR of films, these values decreased with wax addition, finding significant differences  and sunflower waxes as small spherical shapes (arrows) that produce an irregular area with some cracks and areas that correspond to pectin matrix. These results were similar to those obtained in a previous work by Chalapud et al. , who useImages of cross-sectional area of films  showed aIn 2 groups within hydrocarbon chains that integrate into the crystalline configuration [p < 0.05). On the other hand, the high degree of crystallinity presented by LM films  than control LM films, which could be attributed to changes in the gelling mechanisms of each type of pectin, resulting in a less homogenous surface in those films elaborated with LM pectin due to the creation of junction zones. For control LM pectin films, these results are associated with a high degree of crystallinity and network structure revealed by X-ray diffraction (p < 0.05) than SFW-HM an SFW-LM. Eghbal et al. [Rq and Ra values used to evaluate the surface roughness of films are presented in fraction . Rq and fraction  with lowl et al.  reportedl et al. .p > 0.05), according to Campos et al. [p > 0.05). However, in SFW-LM films, a slightly increase in %E was observed, possibly due to interactions generated by the carboxyl groups of LM pectin [s et al.  and Harns et al. , as the M pectin .p > 0.05) and a not significant variation of Y (p > 0.05), the dispersion of wax bodies serves as focus areas, decreasing chain mobility, promoting tensile strength and acting in stiffness of films [Regarding tensile stress at break, films elaborated with high-methoxyl pectin and sunflower wax (SFW-HM) presented the highest TS value, followed by HM films, which can be related to the lower degree of crystallinity presented by SFW-HM . LM filmof films ,78. In gof films , who fouof films  and cellof films , it is pof films .−1 corresponds to asymmetric CH2 stretch and 2849 cm−1 to symmetric CH2 and CH3-CH stretching [−1 could be assigned to C-O and C-C bonds, respectively [−1 vibrations allocated to CH2 [The resulting Raman spectra of sunflower wax and films obtained by electrospraying are presented in retching ,84. In ad to CH2 . As expe−1 region. Vibrations at ~2950 cm−1 are assigned to the C-H bonds of the pyranoid ring carbons, which is associated with the presence of galacturonic acid in pectins [−1 and LM and SFW-LM at 1732 cm−1, which was assigned to C = O stretching of COOH [−1 were found in films elaborated with LM pectin  [−1 and a slight wavenumber shift between HM and LM pectins were reported by Szymańska-Chargot et al. [−1 for LM films and 851 cm−1 for HM films  . A Ramant et al. , with thHM films , suggestilms (−1 , which cPectin films with sunflower wax addition produced by electrospraying were elaborated. Solutions and emulsions were prepared and characterized. Spherical shapes along the cross section of films showing a fine entrapment of sunflower waxes non homogenously distributed into the support matrix with small voids presence and without visual destabilization phenomena were observed by SEM. AFM analysis showed a smother surface in control pectin films and a high roughness in SFW films, due to the immiscibility of lipidic particles into pectin matrix. Despite the presence of voids, and the more crystalline structure in LM films, the addition of sunflower waxes to pectin films resulted in a decrease in molecular mobility, and water molecule accessibility, and therefore reduced the rate of water vapor transmission, demonstrating that waxes are an effective water vapor flux barrier, and the formed network had good water resistance. Films elaborated with HM pectin resulted in being more resistant, less flexible and stiffer than those films from LM pectin; however, the non-homogeneity of lipidic material distribution could influence the mechanical properties of films. Raman studies indicated that the electrospraying technique did not cause structural changes in the films or chemical interactions and mean bonds and groups from raw materials were conserved. This study showed that useful results for pectin film elaboration with sunflower waxes addition by electrospraying technique presents good functional properties.


The purpose of this study was to verify whether the prognostic value of primary tumor location (PTL) for patients undergoing resection for colorectal liver metastasis (CRLM) is affected by tumor burden.2 = (maximum tumor diameter in cm)2 + (number of lesions)2. Then, the prognostic role of PTL was assessed in different TBS zones.Patients who underwent a first curative-intent surgery for CRLM from 2006 to 2017 were enrolled. The imaging tumor burden score (TBS) was calculated as TBSn = 161 (25.1%)], zone 2: TBS ≥3 to <7 [n = 343 (53.4%)], and zone 3: TBS ≥7 [n = 138 (21.5%)]. In the whole cohort, the 5-year overall survival (OS) in the RS group was worse than that in the LS group (35.6% vs. 45.4%). However, after adjustment for known prognostic confounders, the RS group was not independently associated with a poorer OS . Among patients with TBS <7, OS in the RS group was significantly shorter than that in the LS group in both univariate and multivariate analyses. The prognostic role of PTL remained significant after propensity score matching or excluding patients who received anti-EGFR agents. Conversely, the association between PTL and OS was no longer evident in patients with TBS ≥7.The patient population consisted of 524 left-sided (LS) and 118 right-sided (RS) primary tumors. The distribution of TBS in the patient cohort was: Zone1: TBS <3 [The current study demonstrates that the prognostic value of PTL varies by TBS, and RS tumors are only associated with shorter survival in patients with low or medium TBS. Colorectal cancer (CRC) is common in China and ranks fifth in cancer-related death . The livPrimary tumor location (PTL) has been identified as a prognostic factor in patients with advanced metastatic CRC . GrowingThe “tumor burden score” (TBS) was first proposed by Sasaki et al. , 16 for Patients undergoing a first liver resection for CRLM at the Hepato-Pancreato-Biliary Surgery Department I, Peking University Cancer Hospital, in 2006 and 2017 were consecutively enrolled in this study. Only patients who underwent complete resection of both primary and CRLM were included in this study. Patients were excluded if operative mortality occurred and patients lost to follow-up. The clinical information for each patient, including sex, age, primary tumor characteristics , preoperative factors , CRLM characteristics , perioperative outcomes , postoperative therapy and follow-up data, was obtained from a prospective hepatic resection database and supplemented by medical record reviews. The present study was approved by the Institutional Review Board of the hospital.The distal third of the transverse colon was defined as the boundary between the right and left colon by embryological origin . HoweverAll patients underwent routine laboratory evaluation and preoperative imaging to assess their extrahepatic disease and resectability of CRLM. Preoperative staging included contrast-enhanced CT, MRI scan or positron emission tomography/CT. Resectability was defined as macroscopic complete resection could be achieved with liver remnant volume equal to at least 30% or 40% (for patients with chemotherapy-induced liver injury) of the total estimated liver volume. Preoperative fluorouracil-based chemotherapy was recommended unless single, metachronous resectable metastases were present or the patient refused. Restaging was performed every 1–2 months during chemotherapy. Hepatectomy was performed at 4–6 weeks after chemotherapy. Portal vein embolization or ligation with two-stage hepatectomy was performed to achieve an sufficient remnant liver volume. CRLM or primary tumor samples were analyzed for RAS and BRAF mutations using techniques described previously .During the surgery, an intraoperative ultrasound scan was routinely used to assess lesions and vascular anatomy and to detect new metastases. An intermittent Pringle maneuver was also applied when deemed necessary by the surgeon. Combined resection/radiofrequency ablation (RFA) was selected when not all tumors could be removed by single hepatectomy or if the tumors were deeply embedded in the remnant liver. Routine 6-month systemic therapy was given after the surgery along with necessary adjuvant chemotherapy. All subjects were followed up every 3 months for the first 2 years and then every 6 months in subsequent years. At each follow-up, CEA measurement, liver function tests, and imaging studies, such as thoracic, pelvic and abdominal enhanced computed tomography or abdominal MRI scans, were performed to detect recurrence.2 = (maximum tumor diameter in cm)2 + (number of tumors)2  were retained in the multivariable model, and the hazard ratio (HR) and 95% confidence interval were calculated for the strength of association between OS and each variable. A 2-sided p value <0.05 was considered statistically significant. Finally, nearest neighbor propensity score matching (PSM) was conducted to reduce selection biases. Confounders were selected as variables  inconsistently distributed between RS and LS cancers in patients with TBS <7 (p < 0.10). PSM analysis was performed using the MatchIt package in R 3.5.1 with the caliper set to 0.05. SPSS Statistics 23.0  and R 3.5.1. (https://cran.r-project.org/) were used for the above analyses.TBS was calculated using the formula below: TBStumors)2 . The sizp = 0.000), a higher rate of preoperative chemotherapy (p = 0.017) and a lower chance of RAS/BRAF mutations (p = 0.000). Other clinicopathologic features were similar between the two groups.During the study period, 699 patients underwent 766 CRLM resections. Of these patients, 57 were excluded from the study population for the following reasons: 4 patients experienced postoperative mortality, 22 patients had incomplete resection, 20 patients underwent repeated resection, and 11 patients were lost to follow-up. Of the remaining 642 patients, 524 had LS (81.6%), 118 had RS (18.4%) primary tumors, and the status of RAS/BRAF was available in 634 (98.8%). Clinicopathological characteristics stratified by PTL are summarized in p = 0.026, p = 0.247). On the other hand, the lymph node status of the primary tumor , increasing TBS , extrahepatic metastasis (EHD)  and the presence of RAS/BRAF mutations  were all independently associated with a worse OS in multivariate analysis , and 1-, 3-, and 5-year survival rates were 92%, 57.2%, and 46.6%, respectively. In univariate analysis, patients with RS tumors had a markedly lower OS than those with LS tumors, with 3- and 5-year OS rates of RS and LS tumor patients of 44.7% vs. 59.3% and 35.6% vs. 45.4%, respectively ; zone 2: TBS ≥3 to <7, n = 343 (53.4%); and zone 3: TBS ≥7, n = 138 (21.5%). Among patients with high TBS (zone 3), OS did not differ between the RS and LS groups in both univariate and multivariate analyses   . We also= 0.027) .p = 0.000), EHD  and RAS/BRAF mutations  were independent risk factors for OS  than LS; thus, RS patients need to pay a higher price (more extensive therapy). For instance, among patients with limited disease (TBS <7), chemotherapy may be routinely recommended to RS patients, though a multicenter, randomized, and controlled phase 3 trial did not show an OS benefit with the combination of surgery and perioperative chemotherapy in the neoadjuvant setting. Moreover, intensified chemotherapy, such as triplet plus biologic therapy, should be considered for the treatment of RS tumors. Although no previous study has verified this combination regimen in resectable CRLM, retrospective analysis of the TRIBE trial by GONO suggested that FOLFOXIRI plus bevacizumab can effectively weaken the intrinsic aggressiveness of RS mCRC . As suchThis study had several limitations. First, this study was carried out at a single institution and employed a retrospective design. Thus, selection bias was inevitable. However, selection bias was minimized by controlling for possible competing factors in multivariate analysis and PSM. Second, as most of the patients received systemic therapy at other hospitals, the preoperative chemotherapy regimens were not standardized, and detailed information on response to preoperative chemotherapy was not available. Therefore, it is possible that a higher number of RS patients were nonresponders, thus interfering with our survival analysis because many previous studies have shown that a worse preoperative response is associated with shorter survival –30. LastIn summary, this study indicates that the impact of RS primary tumors on OS is only relevant for low and medium tumor burden profiles. Therefore, intensified systemic therapy may be needed for these patients. Conversely, PTL was not associated with long-term survival among patients with high TBS. Tumor laterality may not be a determining factor to exclude hepatectomy in this patient population.


En-bloc complete resection remains the treatment of choice for localized chest wall (CW) tumors. Titanium bars reconstruction demonstrated encouraging results with satisfactory early outcomes. However, long-term outcomes remain under-reported. The purpose of this study is to evaluate long-term outcomes after CW resection and repair with titanium devices.From June 2012 to December 2018, we retrospectively reviewed all patients with CW tumors who underwent surgical resection and repair using titanium. Long-term outcomes were assessed.n = 12) of cases. Eleven of twelve patients had an early infection (<1 year), which required material removal in six patients. Asymptomatic connector unsealing occurred in 6% (n = 4), with only one re-intervention. Titanium allergy has never been reported. Chronic chest pain  was reported in 24% of patients.We identified 87 patients who underwent CW tumor resections and titanium reconstruction. Sixty-eight patients were included in the study . There were 29 sarcomas, 20 isolated CW metastases, eight lung cancers, four breast cancers, three thymic malignancies, two sarcomatoid mesothelioma, and one desmoid tumor. Complete resection was achieved in 64 patients (94%), while R1 resection in four patients (6%). Resection involved one rib in two patients, two ribs in thirteen, three ribs in eighteen, four ribs in nine, five ribs in two, seven ribs in one, partial sternum in fifteen, and full sternum in sixteen patients. No patient experienced flail chest. The 1-year, 3-year, and 5-year overall survival rates and disease-free survivals were 82.3%, 61.4%,57.3%, and 67.6%,57.3%,52.6%, respectively. Surgical site infection occurred in 18% (CW resections with titanium reconstruction are associated with long-term survivors. Titanium devices were safe, reliable, and achieved satisfactory oncological results with low morbidity and implant-related complication rates. The prevalence of primary malignant bone and soft tissue tumors is reported from 1% to 2% of all thoracic neoplasms whereas CW metastasis or involvement from lung or breast cancers are more frequent  statement .2.1From June 2012 to December 2018, we reviewed all patients with CW tumors who underwent surgical rib and/or sternal resection and repair with the Thorib® or Trionyx® system  in the Marie Lannelongue Hospital. During this period, congenital CW deformities (as pectus excavatum or carinatum) and thoracic trauma were not included, and benign tumors, palliative surgery, and Pancoast tumors were excluded. Contraindications to curative surgical resection of CW tumors were other metastasis than the CW and N2/N3 carcinoma as determined by the preoperative workup .All patients had suspected diagnosis on computed tomography (CT), carried out for pain, thoracic mass, or the extension of the initial tumor. A magnetic resonance imaging (MRI) was systematically performed to evaluate the medullary invasion for sternal tumors. For other tumors, MRI was performed to specify the extent of the CW, pleural, and mediastinal involvement. In all cases, a positron emission tomography (PET)/CT scan confirmed a localized disease to the CW with no distant metastases. The preoperative pathologic diagnosis was obtained either by CT-guided percutaneous biopsy or surgical biopsy. Cardio-respiratory function evaluation including electrocardiogram, echocardiogram, ventilation/perfusion scan, spirometry with plethysmography, and measurement of diffusing capacity of the lung for carbon monoxide (DLCO) was performed preoperatively. Diabetes, smoking history, pack years, and body mass index (BMI) were noted. All cases were discussed in a specific tumor board before surgery.2.22.2.1To isolate primary or secondary CW tumors, a direct approach focused on the lesion was performed, with skin excision of the prior biopsy. Postero-lateral thoracotomy was preferred for non-small cell lung cancer (NSCLC) infiltrating the CW. Median sternotomy incision was performed for mediastinal tumors and sternal tumors. Resection of the involved ribs with the related intercostal muscles included one segment of the intact rib above and below the gross margin of the tumor, and if possible, at least 3 cm laterally. If the primitive was NSCLC, lobectomy was associated with the parietectomy without any extra-pleural dissection. For other cases, if there were pulmonary adherences, wedge resection was performed for an en-bloc resection. When the sternum was involved, adjacent sternocostal cartilages were removed and partial or complete sternectomy was performed. A mastectomy was associated with primary breast cancer invading the CW. All other invaded structures were resected to achieve R0 procedure. Intrathoracic exploration was always performed in order to eliminate pleural extension before removing CW.2.2.22.2.2.1All patients required CW reconstruction due to the removal of ≥1 anterolateral rib. Posterior and apical CW defects resection did not require prosthetic reconstruction for functional reasons because of the natural parietal suspension provided by the sternum, scapula, clavicula, and attached wide muscles of the thorax. However, defects that extend down to the fifth and sixth ribs may cause the impaction of the tip of the scapula, which is a source of discomfort. Reconstruction should be discussed for posterior resection larger than 10 cm. For anterolateral CW tumor, the Thorib® titanium bars system was fixed on both sides of the former rib with specific staples. Fixation on the sternocostal cartilage was avoided because of the high risk of early disjunction. If the tumor location did not allow the fixation on a bone structure, bars could be secured on the other side of the CW. For postero-lateral CW tumors when posterior fixation was impossible, the Thorib® bar was reshaped in a Z shape and fixed on the upper and lower rib , 3.2.2.2.2Trionyx® sternal plate was used for large sternal defects (>10 cm) after sub-total or total sternectomy. The Trionyx® system is made of a central plate with arms that were fixed to two or three ribs on each side of the CW. In case of partial sternectomy, such as manubriectomy, one or two Thorib® bars were inserted from each side of the CW on the second or third rib.2.2.2.3Muscular or musculocutaneous flaps were performed when there was a history of thoracic radiation therapy or when there was a large associated soft tissue resection. Pectoralis major muscle with skin advancement was mostly used for sternal resection and latissimus dorsi pedicled flap was the favorite choice for lateral rib resection. If a pedicled flap was not possible, a free flap was then performed. These procedures were performed in collaboration with the oncologic plastic surgery team. A resorbable Vicryl®  mesh was used to protect the lungs from any damage from titanium devices.2.2.2.4The reconstructing sequence was performed as follows: chest drain insertion, Vicryl® mesh suture, titanium bar insertion, and flap covering.2.3Neoadjuvant treatment was decided by a specific tumor board following the national recommendations. Similarly, adjuvant therapy was decided by a specific oncologic tumor board, depending on the pathology, the extent of the disease, the patient's comorbidities, and the postoperative course.2.4Postoperative complications were classified according to the Clavien–Dindo classification . More spOperative mortality was defined as death within the first 30 days after surgery or during the same hospital stay. Survival was calculated from the date of surgery until death or the date of last follow-up. Disease-free survival was calculated from the date of surgery until recurrence or the date of last follow-up. No patients were lost in the follow-up. Follow-up imaging studies consisted of routine chest X-ray, chest CTs, and PET/CT scans.2.5chi-square test, or Fisher's exact test for small samples. Continuous data were compared using Student's test (parametric test) or Mann–Whitney's test (non-parametric test). Overall survival rates were calculated using the Kaplan–Meier method, and a log-rank test was used to calculate survival differences between the two groups. Statistical significance was accepted as a p-value < 0.05.Statistical analysis was performed using GraphPad Prism Version 8.4.3 (471) . Categorical variables were analyzed using the 33.1Between June 2012 and December 2018, we identified 87 patients who underwent CW tumors resection and repair using Thorib® bars or Trionyx® sternal plate in our department. Nineteen patients were excluded . Therefore, CW resection and reconstruction for malignancies were performed in 68 patients (26 men and 45 women), with a mean age of 56.4 years (range 18–80). Induction chemotherapy was performed in 20 patients, induction radiation therapy was performed in one patient and two patients had both. There were 29 sarcomas (including ten radiation-induced), 20 isolate CW metastasis, eight NSCLC, four breast cancer, three thymic malignancies invading the CW, and two localized sarcomatoid mesothelioma and one desmoid tumor. Patient characteristics data are summarized in 3.2We used the Thorib® bars system in 51 patients with a mean of 1.35 bars per patient (range 1–3). The mean number of resected ribs was 2.65 (range 1–7). For the Thorib® system, tumor location was sternal in 12/51 (23%), anterior and anterolateral in 29/51 (57%), strictly lateral in 6/51 (12%), and postero-lateral in 4/51 (8%). The flow chart is available in n = 11), lobectomy (n = 10), mastectomy (n = 10), thymectomy (n = 5), pericardium (n = 3), vessels (n = 2), phrenic nerve (n = 1), diaphragm (n = 1).Musculocutaneous flap was performed in 63% of the patient (43/68). Soft tissue coverage of thoracic defect was pectoralis major muscle with skin advancement in eleven patients, musculocutaneous latissimus dorsi pedicled flap in 27 patients, or free musculocutaneous flap in six patients [deep internal epigastric perforating (DIEP) free flap or controlateral latissimus dorsi flap]. No omentoplasty was performed. Associated organs resections consisted of wedge resection (The definitive pathology examination showed complete R0 resection in 64 patients (94%) patients and R1 resection in four patients (6%). Two patients initially had an incomplete resection and had an early surgical revision with clean margins.There were no per-operative and in-hospitality deaths. There were 66/68 patients (97%) extubated within the 24 first hours. The median length of postoperative care unit and hospital stays were 1 day (range 0–4) and 11.5 days (range 4–112), respectively. For patients who required intensive care unit, the median length of stay was 7 days (range 2–100). Forty-nine percent of the patient experienced a post-operative complication more than grade I from the Clavien–Dindo classification. Among them, sixteen had a grade II (24%), two had a grade IIIA (3%), eight had a grade IIIB (12%), five had a grade IVA (7%), and two had a grade IVB (3%).3.3No patient experienced a paradoxical chest movement although respiratory failure occurred in six patients (9%) .Infection of the operative site occurred in twelve patients (18%). Most of them had an early infection, but they required removal of the bars only in 50% of the cases (six patients). Others had also surgery with washing of the operative site without removing bars or plates. No Trionyx® system had to be removed for the three infections. No seroma was detected around the bars and plates during the follow-up. One patient had a late infection , due to the skin atrophy of the muscular flap and Thorib® bar exposure. Only the exposed bar had been successfully removed.Implant failure occurred in four patients (6%) (three patients with Thorib® and one with Trionyx®). The patient with the Trionyx® system had an asymptomatic costal unsealing found in a CT during the follow-up. Only one patient with Thorib® failure needed a reintervention almost 4 years after the initial surgery. Another patient had an asymptomatic connector unsealing 8 years after the initial surgery, but due to the age of the patient and the asymptomatic fracture, we decided not to re-operate. The last patient had an implant failure associated with infection and loco-regional recurrence and has not been re-operated.p = 0.22). A sensation of corset was also reported by these patients.Chronic pain was the most common complication and occurred in 24% (16/68). The incidence did not differ between Trionyx® and Thorib® systems   and the first second of forced expiration (FEV1) were 10% lower after CW resection and reconstruction  .4. Titanium-based reconstruction allows thoracic surgeons to perform larger resections without increasing the risk of paradoxical motion with acute respiratory insufficiency. Titanium is an ideal prosthetic material, as it demonstrates high resistance to corrosion, low weight, remarkable resistance to traction forces, and high biocompatibility. CW reconstructions with titanium bars and plates have dramatically changed post-operative early outcomes. Paradoxical chest movement has become rarer, and patients are now facing long-term issues.We demonstrated that CW reconstruction using titanium bar and plate could achieve satisfactory long-term results in terms of survival and complication rate. The primary aim of CW resection for cancer is to achieve a complete en-bloc resectionIn the present study, we reported early and long-term results of a novel CW repair system called Thorib® for rib replacement and Trionyx® for sternal replacement. Our titanium system showed good results with non-paradoxical chest movement, and 97% of extubation on the day of surgery regardless of the size of chest resection. Titanium bars and plates allowed us to perform more extended surgeries, leading to a high rate of R0 resection. However, postoperative complications of such complex procedure remained relatively high with a total of 49% of grade II or more complications according to the Clavien–Dindo classification . SeriousOur 18% infection rate is higher than the previously published rate , 18. ConWe reported a 6% rate of implant failure which could be considered as low compared to the literature. A bi-centric study found a 44% rate of failure with STRATOS™ and MatrixRIB® fixation systems  with a mean follow-up of less than 2 years . In thisWe did not notice any titanium allergy, which confirms the biocompatibility of the Thorib®/Trionyx® bars and plates. Metal allergy is very rare and has been described mostly in orthopedic surgery . In the Chronic pain was the major complaint in patients with long-term survival (24%). Trionyx® plates seem to have a higher rate of chronic pain (35%) probably due to the number of resected ribs. Of importance, patients described the sensation of corset around the chest which could also explain any discomfort sensation. This sensation could be the consequence of the use of a rigid material or thoracic denervation. In 1983, LeRoux, one of the pioneers in the use of prostheses for CW reconstruction, described the characteristics of an ideal prosthetic material which should be sufficiently rigid to abolish paradox, biologically inert, tolerated and incorporated by the host, and able to be manipulated and shaped to fit a defect. Nowadays, paradox is no more an issue with titanium bars and plates, but a more flexible material may be able to reduce this sensation of CW rigidity.We have no information about possible consequences in the case of cardio-pulmonary resuscitation using chest compression. The presence of bars behind the sternum like in the Nuss procedure notably lowered compression depth by a minimum of 69% compared to a chest without bar(s) . Except 4.1This study was conducted at a single center and was retrospective. The low number of patients precluded any mortality, R1 resection, or functional outcomes statistical risk-factors analyses. However, CW tumors are rare diseases, and this report is the largest single-center series to date reporting long-term outcomes. Furthermore, CW malignancies were heterogeneous. At least, Thorib® bars have evolved in time, becoming more resistant for the second generation.5In summary, our study shows that Thorib® and Trionyx® bars and plates are safe and reliable in time, without paradoxical CW motion. Despite a high morbidity rate  secondary to extended resection and post-radiation surgery, this new titanium system allowed the surgeons to perform more extended surgeries with a high rate of complete resections. Chronic pain is a long-term issue in ¼ of alive patients. CW resection and reconstruction with titanium bars achieved a 50% survival rate at 5 years for thoracic malignancies.


The gut microbiome is important for host nutrient metabolism and ecological adaptation. However, how the gut microbiome is affected by host phylogeny, ecology and diet during sympatric speciation remain unclear. Here, we compare and contrast the gut microbiome of two sympatric blind mole rat species and correlate them with their corresponding host phylogeny, ecology soil metagenomes, and diet to determine how these factors may influence their gut microbiome. Our results indicate that within the host microbiome there is no significant difference in community composition, but the functions between the two sympatric species populations vary significantly. No significant correlations were found between the gut microbiome differentiation and their corresponding ecological soil metagenomes and host phylogeny. Functional enrichment analysis suggests that the host diets may account for the functional divergence of the gut microbiome. Our results will help us understand how the gut microbiome changes with corresponding ecological dietary factors in sympatric speciation of blind subterranean mole rats. The gut microbiome plays a vital role in digestion, energy acquisition, detoxification, immune system development, behavior , and driSpalax, belonging to the Spalacidae family, is an herbivorous subterranean mammalian rodent that lives most of its life underground  live in abutting but contrasting geologies and soils from “Evolution Plateau” eastern Upper Galilee, Israel. The slightly alkaline rendzina soil weathered from Senonian chalk at approximately 99.6 Mya, and the acid basalt soil, weathered from Pleistocene basalt, was generated from a volcanic eruption of about 1 Mya. The chalk is much drier and barren, compared to the basalt which has a clay consistency, and is wetter and muddier. There are 113 species of plants in total from the two soils, but only 28% are common in the different soil types  and pair-end (PE) reads from NCBI at https://www.ncbi.nlm.nih.gov/. PE reads were filtered by removing low-quality reads and adapters with fastp v0.20.1  were removed by SOAPaligner  and its abutting derivative basalt (6 samples) from “Evolution Plateau” (EP): in the eastern Upper Galilee, Israel, in January 2016 and were stored in liquid nitrogen immediately for DNA extraction. DNA was isolated using QIAmp DNA stool mini kit . Qualified DNA was sonicated to about 350 bp fragments, after purification, end-repair, A-tailing, and adaptor ligation were carried out and followed by PCR amplification. After quality control, libraries of each sample were sequenced with 150 bp paired-end reads. We generated ~50 million 150 bp PE raw reads from the shotgun metagenomic sequencing for each sample. Paired-end raw sequencing reads were first trimmed by SOAPnuke  to removPaligner . We alsoPaligner , and theFirst, clean reads were uploaded into the MG-RAST  server, https://www.bioinformatics.com.cn.Contigs were assembled by MEGAHIT v1.1.3  with defThe coding sequences  were predicted by Prodigal v2.6.3 , and we i = xi/Li;Step 1: Clean reads were aligned to the non-redundant gene catalog using BWA , and geni = bi/∑bi;Step 2: Calculation of the relative abundance of gene i: ai: the relative abundance of gene i.ai: the copy number of gene i from sample N.bi: the length of gene i.Li: the number of mapped reads.xThe calculations mentioned above were performed with custom Python scripts.Akkermansia muciniphila we used was generated in the present study. SNPs were detected the same as described above with the following parameters: “--maf 0.05. --hwe 0.01 and --max-missing 0.9.” Principal component analysis (PCA) was accomplished by PLINK v1.90b6.21 (FST and nucleotide diversity (π) were calculated by VCFtools in 1 kb sliding windows.First, we used kraken2 v2.1.2 to identify the most abundant species and significantly different species were identified by LEfSe; second, we downloaded their genomes from NCBI as references, but the reference genome of .90b6.21 . Neighbo.90b6.21 . Demogra.90b6.21 . FST andFlavonifractor plautii is also high in the host dwelling soil, so we compared both the gut and soil microbiome to investigate the differentiation of this species. Furthermore, admixture v1.3.0 , 10 mM DTT, 8 M urea, and 1X protease-inhibitor. The mixture was incubated on ice for 30 min and then centrifuged at 10,000 g and 4°C for 30 min. The supernatant was collected, and protein concentration was determined with a BCA protein assay kit. Protein quality was identified with SDS-PAGE.We collected eight liver samples of the host 4HCO3 (200 mM), and incubated with 10 mM DDT for 1 h at 56°C, after cooling, add 55 mM iodoacetamide (IAA) for 40 min in the dark to alkylate samples, then digested with 5 μg trypsin for 14-16 h at 37°C. Digested peptides were concentrated to about 1 mg/ml, then separated with chromatography using Eksigent 425 (AB SCIEX). Separated peptides were performed mass spectrometric analysis with Q-Exactive (Thermo Scientific).Protein samples were diluted with NHp < 0.05). Statistical analysis was conducted by Student t -test with Microsoft Excel. For proteins with missing values, data for that protein were kept if its lowest value in one group was higher than its highest value in another group. Finally, Gene ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analysis were performed to characterize the properties of the proteins identified using STRING . PCoA based on the relative abundance of all bins showed a clear separation between the chalk and basalt populations (p = 0.001998). The heat map also showed similar results . Additionally, chalk and basalt did not differ significantly in alpha diversity  populations. In both populations, the most abundant phyla were Firmicutes that accounted for 59.6% of abundance, followed by Proteobacteria (17.4%), Bacteroidetes (10.7%), ActinobacteriaI (7.61%) and others  and Candidatus Micrarchaeota (p = 0.013). At the genus level, Christensenella, Acidovorax, Bordetella, Bibersteinia and Faecalitalea were enriched in basalt; While in chalk, Providencia, Acidithiobacillus, Gimesia, Gemella and Tardiphage were enriched. The ratio of Firmicutes to Bacteroidetes (F/B ratio) was higher in the basalt compared to chalk  and mantel test  showed there was no correlation between the gut and soil microbiomes.The most dominant kingdom in both populations gut microbial communities were bacteria (over 99.5%), followed by archaea and eukaryota. For the bacterial community, 57 phyla, 99 classes, 214 orders, 469 families, 1721 genera, and 6,401 species were identified from 12 gut samples. We found 2 phyla, 8 classes, 16 orders, 24 families, 109 genera, and 313 species were significantly different between basalt and chalk . The Shannon index of basalt was higher than that of chalk (p = 0.0039). For GO terms, the non-redundant genes of basalt were enriched in growth, oxidoreductase complex, transporter activity, cation transmembrane, etc.  were also calculated for these species based on SNPs  between the host genetic matrix and the Bray-Curtis distance matrix based on the gut microbiome. The microbiome dendrogram did not reflect the host phylogeny (p < 0.05 Student t-test) that were significantly enriched in metabolic pathways such as endocytosis, biosynthesis of amino acids, endocrine and other factor − regulated calcium reabsorption, arginine and proline metabolism, vasopressin−regulated water reabsorption, and pyruvate metabolism  between the host genetic matrix and Bray-Curtis distance matrix based on the gut microbiome; this may be due to the short differentiation time of the gut microbiome (p = 0.5317) showed there was no significant correlation between the soil and gut microbiome, allowing us to reject the hypothesis that the local environment was the main factor shaping the gut microbiome.The community composition between the chalk and basalt gut microbiome was not significantly different  when comcrobiome , 13C–31Ccrobiome  illustracrobiome –11. Basicrobiome –31. The crobiome . The divcrobiome ; this iscrobiome , 2009. Tcrobiome . Both thcrobiome  and mantS. galili chalk is from Eryngium sp., poaceae roots, and Ranunculus sp. leaves; while the S. galili basalt mainly feed on geophytes, including Hordeum bulbosum, Bellevalia sp., Iris histrio, and a very small amount of Eryngium sp.  can be found at: This study was approved by Ethics Committee of College of Ecology, Lanzhou University and in accordance with the current ethical review: Complies with the ethical requirements and agree to study in accordance with this scheme (No. EAF2022013).EN and KL designed the research. ZK, FL, QD, and CT performed the research. ZK, EN, and KL wrote the paper. All authors contributed to the article and approved the submitted version.Spalax research Program. We received support for computational work from the Big Data Computing Platform for Western Ecological Environment and Regional Development and Supercomputing Center of Lanzhou University.This project was supported by the National Natural Science Foundation of China (32271691 and 32071487), National Key Research and Development Programs of China (2021YFD1200901), Science Fund for Creative Research Groups of Gansu Province (21JR7RA533), Lanzhou University’s “Double First-Class” Guided Project-Team Building Funding-Research Startup Fee for KL, Chang Jiang Scholars Program, The Fundamental Research Funds for Central Universities, LZU (lzujbky-2021-ey17), a grant from State Key Laboratory of Grassland Agro-Ecosystems , Ancell-Teicher Research Foundation for Genetic and Molecular Evolution for its constant financial support for supporting The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The aims of this study were to assess the potential associations between the serum cardiac troponin I (cTnI) concentration in healthy cats and feline characteristics, systolic blood pressure, heart rate (HR), echocardiographic measurements and storage time; and to compare cTnI concentrations in healthy cats with concentrations in cats with hypertrophic cardiomyopathy (HCM), with or without left atrial enlargement (LAE) and in cats with HCM, to assess potential associations between cTnI concentration and echocardiographic variables.Cardiac TnI was analysed using an Abbott ARCHITECT ci16200 analyser in serum from prospectively included healthy Norwegian Forest Cat , Birman (n = 33) and domestic shorthair  cats, and from 39 cats with HCM, with or without LAE.P = 0.014) and in neutered male cats than in intact females (P = 0.032). Cardiac TnI was positively associated with HR (P <0.0001). In cats with HCM, cTnI concentration was positively associated with left ventricular wall thickness and with left atrial-to-aortic root ratio . Cats with HCM had higher cTnI concentrations than healthy cats, and cTnI concentrations were higher in cats with HCM and LAE than in those with HCM without LAE .In healthy cats, higher cTnI concentrations were found in Birman cats than in NF cats (Breed and sex may affect serum cTnI concentrations in healthy cats. The cTnI concentration increased with increasing severity of HCM. An hs-cTnI assay enables the detection of cTnI concentrations in >50% of healthy humans, with a coefficient of variation (CV) of ⩽10% for the 99th percentile. In humans, different cut-off values are used for the available hs-cTnI assays because of varying capture and detection antibodies and lack of standardisation.8 Only one hs-cTnI assay has been validated in cats. In humans, male sex is positively associated with cTnI concentration, and sex-specific reference intervals (RIs) are used.911 Breed differences have been found in healthy dogs,13 and a previous report in cats with hypertrophic cardiomyopathy (HCM) indicated a breed effect. In healthy cats, cTnI concentrations have neither been associated with sex nor with breed or body condition score (BCS).14 Results in cats regarding associations between cTnI concentrations and age and body weight (BW) are conflicting.15Cardiac troponin I (cTnI) is a sensitive biomarker for myocardial injury, and high-sensitivity cTnI (hs-cTnI) assays allow detection of low concentrations of cTnI in healthy cats,17 The disease is characterised by left ventricular hypertrophy in the absence of other explanations for wall thickening, and may lead to congestive heart failure (CHF), arterial thromboembolism (ATE), arrhythmia or sudden death.1921 Using conventional cTnI assays, significantly higher concentrations have been detected in cats with HCM than in healthy cats,23 in which cTnI concentrations have been reported to be commonly below the detection limit.24 In cats with HCM, positive associations between cTnI concentrations and hypertrophy of the left ventricle (LV), and with left atrial enlargement (LAE), have been shown.223 Although cTnI is specific for the myocardium, increased cTnI concentrations are not specific for primary cardiac disease but have also been shown in cats with hyperthyroidism, hypertension, renal disease and critical illness.HCM is a common cardiac disease in cats, with a reported prevalence rate of approximately 15%.The aims of this study were to assess potential associations between serum concentration of cTnI in healthy cats and feline characteristics , systolic blood pressure (SBP), heart rate (HR) at auscultation, echocardiographic measurements and storage time. A further aim was to compare cTnI concentrations in healthy cats with concentrations in cats with HCM, with and without LAE, and in cats with HCM, to assess potential associations between serum cTnI concentration in cats with HCM and echocardiographic variables. We hypothesised that the serum concentration of cTnI in healthy cats may be influenced by feline characteristics, and that cTnI concentrations may differ between healthy cats and cats with HCM, with or without LAE.30This prospective observational study was approved by the Uppsala Animal Experiment Ethics Board, Sweden (C137/13). Client-owned cats were recruited through information distributed online, at seminars or at recruiting clinics. Cats were examined at the Evidensia Animal Clinic in Västerås, Sweden, between September 2014 and June 2017. Clinically healthy cats and cats with murmurs or previously diagnosed HCM were examined for possible inclusion. Informed written consent was obtained from the owner of each cat. The population has, in part, been described previously.Apparently healthy Norwegian Forest Cat (NF), Birman and domestic shorthair (DSH) cats aged 1–14 years, with normal echocardiograms, were included, as were cats of any breed with preclinical HCM or clinical HCM stabilised following CHF therapy. A diagnosis of HCM was based on characteristic findings on an echocardiogram, as outlined below.Cats with mean SBP >160 mmHg, or increased serum concentrations of total thyroxine (TT4), creatinine or alanine aminotransferase were excluded. Cats with decompensated CHF, ATE, congenital cardiac disease, other acquired cardiovascular disorders, equivocal findings concerning the presence of LV hypertrophy, or significant organ-related or systemic diseases other than HCM were excluded. All cats receiving medical treatment other than standard CHF treatment, standard antithrombotic treatment or medroxyprogesterone acetate were excluded. followed by a general physical examination, including assessment of BCS on a nine-point scale, echocardiography and blood sampling for haematology, biochemistry profiles, and TT4 and fructosamine levels, as previously described.All examinations were performed according to a standardised protocol in a quiet examination room by an experienced veterinarian (SH). SBP was measured indirectly using a high-definition oscillometric device , The left atrial-to-aortic root diameter ratio (LA:Ao) was measured from the right two-dimensional short-axis view. Left atrial enlargement was defined as LA:Ao ⩾1.5. End-diastolic and systolic LV dimensions  were measured from M-mode and two-dimensional images, and fractional shortening (FS) was calculated.36Echocardiographic examination was performed using an ultrasound unit  equipped with a 4–12 MHz phased-array probe, and with continuous electrocardiogram monitoring. Expected BW-dependent values for the IVSd, LVIDd and LVFWd, as well as percentage deviations from these expected values, were calculated for both healthy cats and cats with HCM according to previously generated formulas for cats. The calculated deviations and LA:Ao were used to classify cats into three groups – healthy controls, HCM without LAE and HCM with LAE – as described previously.A diagnosis of HCM was made when subjective impression of hypertrophy  with a non-dilated LV chamber was supported by increased M-mode and two-dimensional diastolic LV wall dimensions of the IVSd, LVFWd or both, as previously described. In the imprecision study, two concentrations of pooled feline serum  and two human control samples (Liquicheck Cardiac Marker LT1 and Liquicheck Cardiac Marker Cardio 2 [Bio-Rad Laboratories]) were used. Each sample was analysed three times daily for 5 days to determine within-run (CVR), between-run (CVB) and within-laboratory (CVWL) repeatability. Limit of blank (LOB) was evaluated using 20 replicates of physiological saline solution. Linearity under dilution was performed in duplicate through the dilution of two concentrations of pooled feline serum  with physiological saline solution as serial dilution and the addition of an extra sample consisting of 75% feline serum and 25% physiological saline solution. Recovery ([observed/expected concentration] × 100) was calculated. Stability was determined by keeping fresh feline serum samples with initial cTnI concentrations of 1070, 1040, 761 and 91 ng/l, respectively, at 20°C in the dark for 0, 3, 5 and 7 days, respectively, and then stored at −80°C until thawed for batched analysis. The effect of freezing and thawing was determined by assaying aliquots of fresh feline serum samples subjected to three cycles of freezing at −80°C for 24 h and thawing to room temperature for 30 mins.The validation study was set up in accordance with the Clinical and Laboratory Standards Institute document EP15-A3, evaluating precision using a one-way ANOVA.Whole blood was centrifuged and the serum stored at −80°C within 60 mins of collection. Samples were transported from the clinic to the laboratory at −80°C using a portable freezer. Serum for cTnI was batch-analysed in duplicate at an accredited laboratory at the Department of Clinical Chemistry and Pharmacology, Uppsala University Hospital, using a two-step, double-monoclonal chemiluminescent microparticle immunoassay for the detection of cTnI concentration . The reported assay interval for cTnI concentration was 2–50,000 ng/l. Concentrations <2 ng/l were assigned a value of 2 ng/l in the calculations. and JMP . A P value <0.05 was considered to be statistically significant. Descriptive statistics for continuous variables  were presented as mean ± SD. Within each of these continuous variables, multiple comparisons were performed between breeds for healthy cats, and between healthy cats and cats with HCM, using one-way ANOVA followed by Tukey’s honestly significant difference (HSD) test.Statistical analyses were performed using Rstudioinc%, LVIDdinc%, LVFWdinc%, FS% and LA/Ao, and the duration of storage at −80°C of serum samples, were analysed using univariable linear regression for continuous variables and one-way ANOVA for categorical variables. BCS was divided into normal (BCS 4–5) and overweight (BCS 6–7), and sex into four classes . Variables with a P value <0.2 were included in a linear multiple regression analysis and, as cTnI is a marker of myocardial injury, a variable for the LV wall – LVPWdinc% – was also included in the model for healthy cats. A model selection was performed, excluding covariate echocardiographic variables for the LV, using the echocardiographic variable with the lowest P value in the model. The variable with the highest P value was then removed until all remaining variables had a P value <0.05. The model with the lowest Akaike information criterion was chosen. All variables were assessed as main effects. The interactions breed × BW and breed × sex were evaluated but were not significant and thus not included in the model. All multiple regression analyses were performed after logarithmic transformation of cTnI concentrations, to have normally distributed residuals. For multiple comparisons between sex and breed categories, respectively, Tukey’s HSD test was used as implemented by the routine general linear hypotheses in the R package ‘multcomp’.Data on cTnI concentrations were presented as median and interquartile range (IQR). Potential associations between cTnI concentration in healthy cats, as well as in cats with HCM and the variables breed, sex, BCS, age, BW, SBP, HR, echocardiographic measurements IVSdComparisons of cTnI concentrations between the healthy, HCM without LAE and HCM with LAE groups were done using the Kruskal–Wallis test, followed by post-hoc Wilcoxon tests for each pair.R, CVB and CVWL were 4.0–8.6% for the feline samples and 5.1–8.4% for the human control samples. Assay results were adequately linear after dilution and the recovery was 64–116%. The LOB was 0.25 ± 0.12 ng/l. After 3 days of storage at room temperature, the concentrations of cTnI were still within 10% in comparison with the initial values .The CVSupplementary Table 1; excluded cats are detailed in The 96 healthy cats comprised Birman, NF and DSH cats. The characteristics of the healthy cats are presented in Supplementary Table 2; excluded cats are detailed in The 39 cats with HCM comprised 18 DSH and cats from 10 different breeds . Of 32 cats with HCM without LAE, one female was treated with medroxyprogesterone acetate, and three were treated with enalapril. Of the seven cats with HCM and LAE, three cats with stabilised CHF were treated with furosemide and enalapril, one of which also received clopidogrel. The characteristics of the included cats with HCM are presented in inc%. Higher cTnI concentrations were found in Birman cats than in NF cats (P = 0.014), and in neutered males than in intact females . cTnI was significantly associated with breed and sex, and positively associated with HR  and 4 bu32; inc% .P <0.0001; P = 0.0003; inc% and LA/Ao, both positively associated with cTnI (The median cTnI serum concentration in cats with HCM was 37 ng/l (IQR 17.5–81.5), and was higher in cats with HCM with LAE than in cats with HCM without LAE or in healthy cats , is a limitation. The different breed distributions may have affected the results because breed influences the cTnI concentration. Only healthy cats and cats with HCM, but no other known diseases, were included in the study. Cats with equivocal echocardiographic findings and measurements, or other comorbidities were excluded, which introduces the spectrum effect for cTnI concentration.Analytical performance of the hs-cTnI analysis allows its clinical use in cats. There was an effect of both breed and sex on the serum concentration of cTnI. In cats with HCM, cTnI concentrations increased with increasing LV wall thickness, and cats with LAE had higher cTnI concentrations than cats with HCM without LAE and than healthy cats.Click here for additional data file.The concentration of cTnI decreased in serum samples stored at 20°C. After three days, the mean decrease was 4% in comparison with the initial value. Mean decrease was 14% after 5 days and 20% after 7 days . The cTnI concentration changed from -1 to 5% after three freeze-thaw cycles.Click here for additional data file.Auscultation, basic echocardiographic and laboratory variables in 96 healthy Birman, Domestic Shorthair (DSH), and Norwegian Forest (NF) catsClick here for additional data file.Auscultation, basic echocardiographic and laboratory variables in 96 healthy cats and 39 cats with hypertrophic cardiomyopathy (HCM)Click here for additional data file.The stability of cardiac troponin I (cTnI) concentration in serum samples from three cats with hypertrophic cardiomyopathy (HCM) after storage in the dark at 20°CFrom cat one, two samples were studied (Cat 1a and Cat 1b).