To statistically study the 3D shape of oesophageal cancer (EC) and its spatial relationships based on computed tomography angiography (CTA) 3D reconstruction, to determine its relationship with T‐stages, and to create an optimal T‐stage diagnosis protocol based on CTA calculation.t‐test, ROC, etc., were performed and critical values between different T‐stages were calculated. We also invited two radiologists to evaluate the measurements.Pre‐operative CTA images of 155 patients with EC were retrospectively collected and divided into four groups: T1–T4. We used Amira software to segment and 3D reconstruct the EC, oesophagus, aorta, pericardium and peripheral lymph nodes and measured their surface area, volume, major axis, minor axis, longitudinal length, roughness and relationship to the aorta of the EC. One‐way ANOVA, independent sample 3 separately (p < 0.05), and the T1–T4 volume cut‐off values were 11,712.00, 19,809.00 and 44,103.50 mm3 separately. For comparison with radiologists, the AUC value of our measurements was 0.704, which was higher than the radiologists of AUC = 0.630.There were no significant differences in EC longitudinal length, roughness score and relationship with the aorta between the different T‐stages of EC. There were significant differences in EC surface area, EC volume and mean major and minor axis among the different T‐stages. The volumes of the T1–T4 tumours were 12,934.36 ± 7739.25, 23,095.27 ± 14,975.67, 37,577.98 ± 36,085.64 and 58,579.25 ± 41,073.96 mmEC volume, major and minor axis can be used as important factors for surgeons in the T‐stage diagnosis of EC, which helps to improve prognosis and treatment decisions after CTA. Inclusion criteriap > 0.05) and were comparable  for manual segmentation of the EC images. The SEGMENTATION module of the software was used to segment EC images. The segmented structures included the tumour, normal oesophagus, pericardium, aorta, bronchi and lung tissue, creating labels for a total of seven structures. For the outlining of each tumour region, EC contours were drawn around the total tumour volume, and any pixels <−50 HU were excluded to avoid interference with the outlining of the tumour boundary from the surrounding adjacent air, fat, blood vessels , and bone. When there was uncertainty concerning the tumour area, imaging physician's judgement prevailed.2.4Once image segmentation is complete, the original and segmented images were saved separately in “.am” format. 3D models were reconstructed using the Generate Surface in Amira software, which allows a 3D model of EC to be viewed in all directions, moved, scaled and rotated. Three‐dimensional reconstruction and visualisation can directly display the tumour's location, 3D shape and spatial relationship. Based on the 3D model, we can calculate morphological parameters of the tumour, which can help doctors identify different T stages of tumour more accurately.2.5In the 3D EC reconstruction models, the surface area volume module in Amira software was used to measure the surface area and volume of the tumour and surrounding normal tissue and to analyse the volume relationships between tumour and total oesophageal at different T‐stages. The tumour‐related lengths were measured on the 3D model using Measure: line. The measurements included the EC surface area, volume, major axis , minor axis , longitudinal length, normal oesophageal surface area, volume and longitudinal length. To exclude the influence of chance on the results, four sections were randomly selected for measurement of the major and minor axis of the oesophageal tumour and normal oesophagus. Tumour roughness and contact with the aorta were assessed based on the reconstructed 3D model and coronal, sagittal and axial CTA images.2.62.6.1https://www.ibm.com/cn‐zh/spss) for relevant statistics. Basic information surrounding cases was expressed using means ± standard deviation, one‐way ANOVA was used for comparison between multiple groups, and for independent samples t‐test was used for analysis between two groups; differences were considered statistically significant at p < 0.05.The above measurement and assessment results were analysed using SPSS software  as the horizontal coordinate (X‐axis) and the true positive rate (sensitivity) as the vertical coordinate (Y‐axis).2.6.3We invited two radiologists (the first with 5 years and the second with 10 years of experience) to independently re‐evaluate the data and their results were compared with the measure based on 3D‐model predictions, using ROC curves and AUCs to evaluate.33.1A total of 155 patients with EC were enrolled in this study, and their characteristics are detailed in Tables p < 0.05), with a strong correspondence. The proportion of neurovascular invasion gradually increased with elevating T‐stage of EC, with 22.9% in T2, 26.9% in T3 and 66.7% in T4, where it can be seen that the proportion is gradually increasing. The ulcerative type accounted for the majority (61.3%), followed by the medullary type (33.5%), mainly in the T3 and T4 stages of EC; the pathological types of advanced EC were mainly ulcerative and medullary; the roughness of EC increased with the T stage of EC, and the roughness of T4 stage EC, with a score of 3 accounting for 75%. The relationship between tumour and aorta can be used to evaluate the risk level of surgery; we found that the score of the relationship between tumour and aorta in T1 was mainly 1 (71.4%), indicating a low‐risk level at surgery; the score of the relationship between tumour and aorta in T2 and T3 was 1–2, with a higher risk level at surgery than in T1; and the score of T4 was mainly 3 (62.5%), indicating greater risk. Clinical staging was judged according to TNM stage, the clinical stage of T1 was mainly (88.9%) andII(11.1%), the clinical stage of T2 and T3 was mainly concentrated in II, III and IV, the proportion of II was 68.6% and 58.1%, the proportion of III and IV in T4 was 86.7% and 13.3%. Hence, with the increase of T staging, the clinical stage also increased with it, and the severity was higher  . Tumour volumes for T1–T4 were 12,934.36 ± 7739.25, 23,095.27 ± 14,975.67, 37,577.98 ± 36,085.64, and 58,579.25 ± 41,073.96 mm3, showing an increasing relationship, in which the EC volumes of patients with EC in T1:T2, T2:T3 were statistically different ; the EC longitudinal length, roughness score and EC volume/total oesophageal volume of patients with EC; there were no statistically significant differences between the above three parameters in the T1–T4 (p > 0.05). Tumour surface area/total surface areathe of oesophagus, tumour major‐axis length/total lengththe of oesophagus, and EC relationship with the aorta in EC patients at T3–T4 were all p < 0.05  and the correlation was poor Figure .Finally, by analysing the T3 and T4 periods, we found high correlations with AUCs >0.6 for all parameters  Figure .3, with mean major axis and mean minor axis cut‐off values of 24.25, 26.28, 37.05 and 14.69, 16.91, 25.46 mm, and EC surface areas of 4008.50, 5533.50 and 10,894.50 mm2.Table 3.4We found that the model results were better overall than the radiologists' diagnoses. By comparison, radiologists predicted different T‐stages of oesophageal cancer with an AUC value of 0.630 and the accuracy of 0.760, 0.443, 0.352 and 0.167 for T1–T4 stage of EC. Our 3D model‐based measurements had an AUC value of 0.704, higher than radiologists and the accuracy of 0.673, 0.782, 0.601, 0.752 for T1–T4 stage of EC. , which agrees with Zhao et al.'s study.Our study showed that EC surface area, EC volume, mean major axis and mean minor axis were all significantly different between different EC T‐stages (p < 0.05) had greater value in predicting the T‐stage of EC; however, the length of the tumour in the longitudinal axis (p > 0.05) was not significantly different between the different stages, consistent with the findings of Zhang et al.,p < 0.05 in distinguishing different T stages, and the AUC values of the minor axis are the highest. In comparison, the minor axis has more predictive value. Simopoulos et al.In our study, we found that the mean major and minor axis of the tumour ; formal analysis (lead); methodology ; software (lead); visualization (lead); writing – original draft (lead). Xiaoqin Zhang: Methodology ; supervision ; writing – review and editing . Wei Wu: Funding acquisition ; methodology ; project administration . Jinfeng Ma: Data curation ; resources . Jincheng Chen: Data curation ; resources . Zhu Zhang: Data curation . Liqun Liu: Data curation (supporting); formal analysis (supporting). Shanshan Xu: Formal analysis (supporting). Ximei Cao: Writing – review and editing (supporting). Yi Wu: Conceptualization ; funding acquisition ; methodology ; supervision ; writing – review and editing (lead). Huilin Cui: Funding acquisition ; writing – review and editing .This study has received funding by the National Natural Science Foundation of China (31971113), the Chongqing Science and Technology Talent Project (CQYC201905037), University Funded Science and Technology Innovation Capacity Improvement Project (2019XYY14), Chongqing Scientific and Health Committee Joint Research Key Project (2022ZDXM018), Basic Research (Free Exploration) Project of Shanxi Province (2021011079‐2) and Chongqing Excellence Package Project .The authors have no conflict of interest.The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Army Military Medical University (No. (B)KY2021165). Informed consent was objectively unavailable for this study, and subjects' privacy and personally identifiable information were protected.


The emergence of antibacterial resistance (ABR) is an urgent and complex public health challenge worldwide. Antibiotic resistant genes (ARGs) are considered as a new pollutant by the WHO because of their wide distribution and emerging prevalence. The role of environmental factors in developing ARGs in bacterial populations is still poorly understood. Therefore, the relationship between environmental factors and bacteria should be explored to combat ABR and propose more tailored solutions in a specific region. Here, we collected and analyzed surface water samples from Yangtze Delta, China during 2021, and assessed the nonlinear association of environmental factors with ARGs through a sigmoid model. A high abundance of ARGs was detected. Amoxicillin, phosphorus (P), chromium (Cr), manganese (Mn), calcium (Ca), and strontium (Sr) were found to be strongly associated with ARGs and identified as potential key contributors to ARG detection. Our findings suggest that the suppression of ARGs may be achieved by decreasing the concentration of phosphorus in surface water. Additionally, Group 2A light metals  may be candidates for the development of eco-friendly reagents for controlling antibiotic resistance in the future. Due to the increasing prevalence, health effects, and economic impact of AMR, the World Health Organization has characterized AMR as “a complex global public health challenge” (p. XIX) that requires urgent action1. Countermeasures against antibacterial resistance (ABR) are of particular urgency because of severe inadequacy of alternative therapeutic options1. In 2019, it was estimated that 4.95 million deaths were associated with ABR, and 1.27 million deaths were attributable to ABR2. In a recent study that estimateed the economic burden of ABR in China, 27.45% of inpatient bacterial infections were antibiotic resistant, which is estimated to have caused a 1.5% increase in inpatient mortality rates and cost $77 billion in related societal expenses in 20173. Moreover, bacteria are capable of spreading ABR at a high rate through the exchange of antibiotic resistant genes (ARGs) by horizontal gene transfer (HGT)4. ARGs are a group of genes that encode the proteins responsible for antibiotic resistance and are frequently carried on mobile genetic elements (MGEs) like plasmids, transposons, and integrons4. Through HGT, one bacterial cell transfers its MGEs to other bacterial cells, facilitating environmental dispersal of the ARGs4. Thus, ARGs have been recognized as an emerging environmental pollutant that poses a threat to human health4.Antimicrobial resistance (AMR) refers to the process by which bacteria, parasites, viruses, and fungi evolve mechanisms that protect them from drugs7. Later studies have further identified additional non-antibiotic substances as significant contributors to the propagation of ARGs8.Earlier studies have investigated whether ABR would decrease when antibiotic use was reduced, but the results have indicated that ABR may persist due to the low cost of resistance, other biological functions of ARGs, selection at sub-minimum inhibitory concentration levesl, or compensatory evolution11. Because bacteria resist antibiotics and heavy metals through similar strategies , heavy metals may also pose a selective pressure on ARGs. Furthermore, antibiotic resistant communities in urban lakes have also been identified to be more competitive than non-resistant communities at higher temperatures, but less competitive at higher concentrations of magnesium or salinity12. Second, microplastics (MPs) have also garnered attention for their role in enriching ARGs. Zhang et al.13 reported that MPs can directly absorb ARGs onto their surfaces within matriculate systems, thereby creating an ideal platform for microbial colonization. Furthermore, the presence of MPs has been associated with an increased potential for HGT, promoting the propagation of ARGs in municipal wastewater, as noted by Cheng et al.14. In addition to MPs, various common disinfectants, such as chlorination, and the disinfection by-products they generate have been shown to exert dose-dependent effects on ARG migration by influencing the permeability of cell membranes, as demonstrated by Guo, Yuan, and Yang15. Notably, low-intensity ultrasound has also been observed to enhance the efficiency of resistant gene transfer by augmenting cell membrane permeability, as documented by Song et al.16. These findings collectively highlight the diverse ways in which non-antibiotic materials can contribute to the dissemination of ARGs, shedding light on the intricate interactions between environmental factors and antibiotic resistance. Therefore, to effectively understand and mitigate ABR in a specific region, local environmental conditions must be investigated.First, heavy metals are reported to have a co-selection effect on ARGs17. Plasmids are genetic materials that are physically separate from chromosomes and replicate independently18. Due to these features, this study employed a plasmid-centric framework19 to study the effect of environmental factors on ARG abundance. Specifically, this study treated the plasmids as the unit of population dynamics and assumed the quantity followed logistic growth. This permits the assessment of nonlinear association between ARGs and environmental factors through a sigmoid model.ARGs are mainly encapsulated in mobile genetic elements (MGE), such as plasmids20. Shanghai represents the primary geographical majority of the region with a high population density of 3823 persons/square kilometre as of 201821. The quality of drinking water in this area  is critical given that it is the primary water source for residents. Due to its economic and environmental importance, previous studies have investigated the prevalence of ARGs and patterns of antibiotic use in the area.The Yangtze River Mouth is one of the most economically developed regions in China, which includes Shanghai City, the southern part of Jiangsu Province, and the northern part of Zhejiang Province24. While  these studies  demonstrated the widespread distribution and persistence of antibiotics in this region, the environmental risk for most types of antibiotics were low25  based upon the calculated hazard quotients26. However, the environmental risks of enrofloxacin, amoxicillin, and oxytetracyclines to aquatic organisms, represented by the risk quotient method28, are distinctly higher than those in other countries23. As for ARGs, tetracycline resistance genes (tet), sulfonamide resistance genes (sul), and β-lactam were detected in waterbodies in the Yangtze Delta and surrounding regions30. For sul and tet genes, the detection frequency of sulI, sulII, tetA, tetG, tetM and tetO were markedly higher than sulIII, which was still significantly higher than tetC30. It was noted that samples with more sul genes were more likely to contain higher levels of total sulfonamides, which was similarly observed for tet genes29.In 2020, 64 out of 69 types of antibiotics monitored in the surface water in China were detected in Shanghai with concentrations ranging from a few pg/L to hundreds of ng/LIn this study, we tested surface water samples for ARGs, antibiotics, and dissolved elements to better understand what environmental factors might contribute to the prevalence of ARGs in main water bodies of the Yangtze Delta.In 2021, surface water samples were collected from sampling sites located along a watershed starting at Yangcheng Lake in Suzhou, running along the Wusong River, passing Dianshan Lake in Shanghai, running along the Huangpu River in Shanghai, and ending at an estuary at the Shanghai coastal area. The watershed connects most of the main water bodies in Suzhou and Shanghai. The sampling area contained four subregions: Dianshan Lake , Huangpu River , estuary , and Kunshan (Suzhou).Surface water samples were collected using brown HDPE bottles  that were dipped into the top 10 cm of the water body with the goal of collecting 1000 mL of water. Water temperature, salinity, and pH of the collected samples were immediately measured and recorded using a salinity and pH meter  according to the manufacturer’s instructions. Sample bottles were transported on wet ice within 3 h to Duke Kunshan University where 650 mL of each sample was filtered through a 0.45 µm glass microfiber filter membrane . The filtrate and filter membranes were stored in a − 80 °C freezer.tetA, tetC, tetE, tetK, tetL, tetA/P, tetG, tetM, tetO, tetQ, tetS, tetT, tetW, tetBP, and tetX), 4 β-lactam resistance genes , 3 sulfonamide resistance genes , 3 macrolides resistance genes , and 1 beta-lactam resistance gene (ampr). Primer sequences and cycling conditions for the quantitative PCR experiments are listed in Table Material captured by the filter membrane was reconstituted in 10 mL of ultrapure water. A TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit was used to extract 240 µL of DNA from 10 mL of stored sample, according to the manufacturer’s instructions. Quantitative PCR was used to screen extracts for ARGs using the SYBR Green Real-Time PCR assay  on a Mic qPCR Cycler , according to the manufacturer’s instructions. One sample was randomly selected from each of the four subregions. A total of 26 primers for ARGs were used to screen the four samples. The ARGs include 15 tetracycline resistance genes  nitric acid and preserved in a − 80 °C freezer. The samples were sent to the Nanjing Institute of Geography & Limnology, Chinese Academy of Sciences and measured for calcium (Ca), potassium (K), magnesium (Mg), sodium (Na), silicon (Si), silver (Ag), mecury (Hg), thallium (Tl), aluminium (Al), arsenic (As), boron (B), barium (Ba), beryllium (Be), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), nickel (Ni), phosphorus (P), lead (Pb), antimony (Sb), selenium (Se), tin (Sn), strontium (Sr), titanium (Ti), vanadium (V), and zinc (Zn) by ICP-MS 7900 , as described in Ref.Concentrations of amoxicillin and tetracycline were measured using 500 mL of stored filtrate using the Amoxicillin ELISA kit  and Tetracycline ELISA kit , according to the manufacturer’s instructions. The procedures of the two kits were technically the same. Briefly, samples and standard solutions with known antigen (amoxicillin/tetracycline) concentration were separately mixed with corresponding enzyme conjugates (enzyme-labelled antibodies) on a micro-well plate where micro-well strips were pre-coated with coupling antigens. The antigens in the sample and the coupling antigens would compete for the enzyme conjugates. After removing the mixed solution and adding TMB substrate for coloration, the enzyme conjugates bound with sample antigens were removed and only those bound with pre-coated antigens remained, which led to higher optical density (OD) values in the samples with lower antigen concentrations. The OD values of the solutions generated from samples and standard solutions were measured at a wavelength of 450 nm. All procedures above were performed in duplicate for each sample and standard solution. A standard curve of absorption versus antigen concentration was generated with the OD values from the standard solutions, and the OD values from the samples were compared to the standard curve to get the antigen concentration.26. HQ values > 1.0 indicate a potential risk to the aquatic system32. This study adopts PNEC values proposed by Bengtsson-Palme and Larsson, 2016 (0.25 μg/L for amoxicillin and 1 μg/L for tetracycline)33.To assess the potential risk of antibiotics in the environment, a hazard quotient (HQ) was used, which is the ratio between the measured environmental concentration (MEC) of the antibiotic and the predicted no-effect concentration (PNEC)34. Therefore, the influence of environmental factors can be described as:This study treated the plasmids as the unit of logistic population growth and assumed that ARG abundance is proportional to the quantity of plasmids. The fitness cost of conveying plasmids and environmental pressures were represented as coefficients on growth rate, as in Ref.As described by the Eqs. –3), the, the3), If we substitute  the Eq. :6\documeThis Eq.  allows ftetE in Sample S7 as a reference with the following formula:ARG screening data was calculated to relative abundance using the median Cq value of TetBP, ampr, and tetO qPCR data was calculated to absolute abundance using a standard curve derived by serially diluting (tenfold) synthetic genes at a range of 1012 copies/mL to 102 copies/mL. An ANOVA test was performed by the aov function in R base to explore the regional difference in ARG abundance. The samples also underwent scaled hierarchical clustering with Euclidean distance and complete linkage through the scale, dist, and hclust functions in R base.35 and Statistical Bureau of Republic of China36. Figure All figures were generated using R (version 4.0 and 4.1). Figure glm function in the base package of R. The nonlinear association was represented as the Pearson correlation coefficient between the true ARG abundance and the predicted abundance by the glm model, calculated by the cor function in R base.To assess the nonlinear association between ARG abundance and environmental factors, as described in Eq. , the vartetBP, ampr, and tetO. The ereA, ereB, and mphA ARGs were not detected in the samples. Therefore, tetBP, ampr, and tetO were selected for further study.The ARG screening results were shown in Fig. tetBP, ampr, and tetO) are shown in Fig. ampr (SD = 1.4e+06), 2.6e+07 copies/mL for tetBP (SD = 6.8e+07), and 8.8e+03 copies/mL for tetO (SD = 1.5e+04). The mean copy number at Qingpu was 1.0e+06 copies/mL (SD = 4.9e+05) for ampr, 9.5e+05 copies/mL (SD = 1.9e+06) for tetBP, and 2.8e+02 copies/mL (SD = 5.5e+02) for tetO. For Songjiang, ampr had a mean abundance of 3.8e+05 copies/mL (SD = 5.5e+05), tetBP averaged 1.1e+03 copies/mL (SD = 1.0e+03), and tetO 9.8e+05 copies/mL (SD = 1.7e+06). Finally, for Jinshan, tetBP was not detected, while ampr had 6.9e+06 copies/mL and tetO had 7.9e+05 copies/mL. Notably, an ARG hotspot (S10) was found in Yangcheng Lake, Kunshan, which contained an elevated amount of tetBP , as shown in Table As for the concentration of dissolved elements, the complete measurement results are shown in the Supplementary Data. The range of concentration for light metals is the highest among the tested elements, which is 5.90–61.3 mg/L for K, 15.3–48.2 mg/L for Na, 21.3–35.0 mg/L for Ca, 5.59–8.62 mg/L for Mg, 83.5–645 μg/L for Al, and 0–0.091 μg/L for Be. The range for non-metal elements, sorted in decreasing order, is 0–0.462 mg/L for Si, 24.3–459 μg/L for P, 99.2–139 μg/L for B, 2.22–5.01 μg/L for As, and 0–0.535 μg/L for Se. Finally, the range for heavy metals, sorted in decreasing order, is 50.7–862 μg/L for Fe, 110–257 μg/L for Sr, 15.5–207 μg/L for Mn, 0–45.9 μg/L for Ba, 0–34.5 μg/L for Ag, 0.339–11.6 μg/L for Ti, 0–11.1 μg/L for Zn, 2.46–6.18 μg/L for Mo, 0.860–5.85 μg/L for V, 0.553–4.42 μg/L for Ni, 0.333–2.92 μg/L for Pb, 0–2.19 μg/L for Sn, 0–2.08 μg/L for Cr, 0–2.07 μg/L for Cu, 0.617–1.57 μg/L for Sb, 0.070–0.643 μg/L for Co, 0–0.047 μg/L for Hg, 0–0.031 μg/L for Cd, and 0 μg/L for Tl. In general, the water quality was good for all of the tested samples , while tetracycline has a limited association with tetO or tetBP . For physical water properties, salinity has negligible association with tetBP (r = 0.06), ampr (r = 0.19), and tetO (r = 0.14), and water temperature also shows negligible association with tetBP and ampr , but moderate association with tetO (r = 0.54).Figure tetO (r ≥ 0.89). In addition, Ca and Sr have strong associations with ampr as well . Second, Cluster C, including Ti, V, Al, and Co, show weak associations with tetBP (0.47 ≤ r ≤ 0.66), and have negligible associations with tetO or ampr (r ≤ 0.24). This cluster is the most associative group of tetBP. Third, the elements in Cluster D  and Hg in Cluster B have negligible associations with all three ARGs (r ≤ 0.3). Fourth, the remaining elements in Cluster B  show relatively equal associations to all three ARGs. While Cd and Mg have moderate associations with ampr , and Mg and B have negligible associations with tetBP, the other associations are all relatively low (0.2 < r < 0.5). Last, Sb, as an outgroup in the hierarchical clustering, has a moderate association with tetO (r = 0.65), and negligible associations with tetBP and ampr .As for the dissolved elements, the variables were grouped into 4 clusters based on the Manhattan distance  of association coefficients. Cluster A, C, and D have distinct characteristics from each other. First, Cluster A includes the most associative elements, which are pH, Na, Ca, Sr, Cr, P, Si, and Mn. All of these elements have a strong association with ampr, while tetracycline is not associated with tet genes. Figure tetO and ampr from the most associative variables (r > 0.7) using the sigmoid model. The predicted sigmoid curves fit the ARG abundance well.Based on their associations, environmental factors were selected and the sigmoid relationship with ARGs and other assessed factors were visualized. Figure This study aimed to assess the nonlinear association of environmental factors and the prevalence of ARGs in main water bodies in the Yangtze River Mouth area. In summary, a high abundance of ARGs were detected and four categories of environmental factors were found to be associated with ARG abundance.29, the ARG abundance in Huangpu River detected in this study was five magnitudes higher. Specifically, historical data shows that the abundance of tetO was at a magnitude of 10 copies/mL, and other tetracycline-resistant genes were at a magnitude of 10 to 104 copies/mL29. But in this study, tetO abundance in Shanghai reached up to 106 copies/mL , one major component of the bacterial cell membrane, thus destabilizing the cell membrane and eventually causing cell death50. The disrupted cell membrane partly loses the barrier function, which can cause leakage of some cell contents and finally lead to cell death50. In addition, Ca could suppress bacterial growth through inhibiting peptidoglycan-layer (PG-layer) synthesis, which is an essential component of the bacterial cell wall51. PG-layer synthesis involves transpeptidase (TP) enzymes, including D, D-TP and L, D-TP. These TP enzymes are the targets of many antibiotic β-lactams as inhibiting their normal functions can lead to cell death by destablizing the cell walls51. Although these antibiotics are often inactivated by ARGs, calcium is hardly influenced, which can hinder the binding of L, D-TP to PG-stem by inducing the formation of L, D-TP dimers51. Therefore, since the synthesis of ARGs consumes energy but fails to promote viability, a higher concentration of Ca can decrease the relative fitness of the antibiotic resistant bacteria in the environment compared to non-resistant bacteria. Based on such mechanisms, a higher calcium concentration in the water bodies is likely to decrease the overall bacterial quantities regardless of the ARGs carried, and lead to lower concentrations of ARGs detected.One potential explanation is that these light metals can all place selective pressure on bacteria  with or without resistance genes. First, Sr has been experimentally proven to have cytotoxicity on bacteria. The cytotoxicity is achieved through induction of oxidative radicals that damage the cell membrane, or inactivation of adenosine triphosphate (ATP) synthesisThis finding, combined with previous research about Mg, reveals the possibility of developing Group 2A light metals as reagents to control ARB. For example, as one of the major elements in nature, calcium salts are of low toxicity and eco-friendly. Calcium-based reagents might be capable of suppressing antibiotic resistant bacteria in natural environments if added to natural surface water .53, except for tetracyclines53. The high flow conditions in summer might dilute the concentration of antibiotics in the surface water54, and the higher activity of microorganism and strong sunlight of summer could also result in faster degradation of antibiotics55. Considering the reported seasonal variance, the findings on antibiotic concentrations can only be generalizable to surface water of the Yangtze River Mouth in summer. Also, impacted by the outbreak of COVID-19 Delta strain in China in the summer of 2021, the sampling phase of this study was emergently terminated, ahead of schedule. The unexpected termination led to a small sample size (15 samples). The small sample size decreases statistical power and increases potential error in the results. Finally, ARG concentrations were of large standard deviations in all four subregions. Though this could be explained as indication of point sources of ARG pollution, one alternative explanation is there was degradation of DNA during the experiments.However, limitations exist in this study. It is worth noting that overall antibiotic levels in the Yangtze Delta were observed to vary in accordance to seasons, with the number of detected antibiotics and their detection frequencies being significantly lower in summer than in winterThis study detected a high abundance of ARGs in surface water samples collected from the Yangtze River Mouth. Amoxicillin, P, Cr, Mn, Ca, and Sr, assessed through the nonlinear association model, were identified as potential key contributors of ARGs in the Yangtze Delta. Reduction of ARG abundance could be realized through decreasing the concentration of phosphorus in surface water. In addition, Ca was identified to have a potential ARG suppression effect, though future studies are still needed to further evaluate this association.Supplementary Information 1.Supplementary Information 2.


Vibrio alginolyticus within 72 h by metagenomic sequencing.Vibriosis causes enormous economic losses of marine fish. The present study investigated the intestinal microbial response to acute infection of half-smooth tongue sole with different-dose V. alginolyticus for the control, low-dose, moderate-dose, and high-dose groups were 0, 8.5 × 101, 8.5 × 104, and 8.5 × 107 cells/g respectively, the infected fish were farmed in an automatic seawater circulation system under a relatively stable temperature, dissolved oxygen and photoperiod, and 3 ~ 6 intestinal samples per group with high-quality DNA assay were used for metagenomics analysis.The inoculation amount of V. alginolyticus at high, medium, and low doses caused the change of different-type leukocytes at 24 h, whereas the joint action of monocytes and neutrophils to cope with the pathogen infection only occurred in the high-dose group at 72 h. The metagenomic results suggest that a high-dose V. alginolyticus infection can significantly alter the intestinal microbiota, decrease the microbial α-diversity, and increase the bacteria from Vibrio and Shewanella, including various potential pathogens at 24 h. High-abundance species of potential pathogens such as V. harveyii, V. parahaemolyticus, V. cholerae, V. vulnificus, and V. scophthalmi exhibited significant positive correlations with V. alginolyticus. The function analysis revealed that the high-dose inflection group could increase the genes closely related to pathogen infection, involved in cell motility, cell wall/ membrane/envelope biogenesis, material transport and metabolism, and the pathways of quorum sensing, biofilm formation, flagellar assembly, bacterial chemotaxis, virulence factors and antibiotic resistances mainly from Vibrios within 72 h.The acute infections with Vibrio and that the disease could become even more complicated because of the accumulation and transfer of antibiotic-resistance genes in intestinal bacteria during the process of V. alginolyticus intensified infection.It indicates that the half-smooth tongue sole is highly likely to be a secondary infection with intestinal potential pathogens, especially species from  Orientalis, Streptococcus agalactiae infection caused an onset of mortality that was more rapid and occurred at a significantly higher rate than in fish without the secondary infection , coinfection of Flavobacterium columnare and Edwardsiella ictaluri caused significantly high cumulative mortality than a single infection with either F. columnare or E. ictaluri at the same dose of bacteria , a kind of large bottom fish with warm water, mainly distributed in the Yellow Sea and Bohai Sea of China  were obtained from commercial suppliers in Tianjin, China. The fish were temporarily reared for 2 weeks in 3,000 L aquariums with a density of 100 fish per aquarium while being fed a commercial diet with 48% protein content, 10% lipid content and 2% fiber content . Fish were fed 2.5% of their body weight at 9 a.m. and 4 p.m. each day for half an hour, and maintained under a stable photoperiod . The aquaculture system was an automatic seawater circulation system with a water exchange rate of about 65 L/h, and the dissolved oxygen concentration was 8.20 ± 0.58 mg/L in all aquariums. The water temperatures of the aquariums ranged from 23 to 25°C.2.2.Vibrio alginolyticus was obtained from the diseased half-smooth tongue sole with enteritis and ulcers on the skin surface  solution. The bacteria were re-suspended in PBS to adjust the density to 1 × 105, 1 × 108, and 1 × 1011 cells/mL. surface . To ensuV. alginolyticus, three treatment groups with different pathogen loads were studied. The inoculation amounts of the control (C), low-dose (L), moderate-dose (M), and high-dose (H) groups were 0, 8.5 × 101, 8.5 × 104, and 8.5 × 107 cells/g, respectively. The concentrated bacterial suspension was intraperitoneally administered to the fish through injection. One hundred and sixty fish (40 fish per group) were randomly selected for the pathogen challenge test. For blood and intestinal tissue sampling, 40 fish from the C, L, M, and H groups were placed in four separate aquariums measuring 1.2 m × 1 m × 1 m with an actual water volume of 720 L.To simulate different stages of proliferation of 2.3.After intraperitoneal injection, 6 fish from each group were randomly picked out and anesthetized with 100 mg/L eugenol at 24 and 72 h. Before sampling, the fish’s body was sterilized with 70% alcohol. Blood was aseptically collected from the caudal vein of fish with a disposable sterile syringe and immediately transferred into collection tubes containing heparin anticoagulant for hematology analysis. The intestinal contents, located in the sections from the foregut to the anus, were collected with a medical-grade swab, rapidly frozen in liquid nitrogen, and stored at −80°C before microbial genomic DNA extraction.2.4.The leukocytes  of each sample were determined using a UniCel DxH 800 Coulter hematology analyzer  following the manufacturer’s protocols.2.5.Total genomic DNA was extracted from the intestinal contents (approximately 200 mg for each fish) using the QIAamp DNA extraction kit for stool  following the manufacturer’s instructions. The level of DNA degradation degree and potential contamination was monitored on 1% agarose gels. DNA concentration was measured using the Qubit® dsDNA Assay Kit in Qubit® 2.0 fluorometer . The input material for the DNA sample preparations consisted of 1 μg DNA per sample. Sequencing libraries were generated using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina  following the manufacturer’s recommendations. The final 41 sample libraries of 8 groups (3 ~ 6 per group) reached grade A for metagenomic sequencing analysis. On an Illumina HiSeq platform, the library preparations were sequenced and paired-end reads were generated.2.6.2.6.1.The Raw Data obtained from the Illumina HiSeq sequencing platform using Readfq V8. All unu2.6.2.p < 0.05). The number of genes in H group at 24 h with large intra-group differences was no significantly difference with other groups (The Scaftigs (≥500 bp) were predicted for the ORF through the MetaGeneMark V2.10. Each sar groups .2.6.3.For taxonomy annotation, the DIAMOND software V0.9.9. The fun2.7.Vibrio genus in intestine, the transcriptional levels of 16S rRNA were quantitatively analyzed by qPCR with Vibrio-specific primers   (-ΔΔCt method (To quantify the levels of  CTG−3′) . When ba TGA-3′) . Total Rt method .2.8.p < 0.05 was required. All data are expressed as mean ± standard deviation.The basic information statistic, core-pan gene analysis, correlation analysis of samples, and Venn figure analysis of the number of genes were all based on the abundance of each gene in each sample in the gene catalog. The exhibition of abundance cluster heat map and non-metric multidimensional scaling (NMDS) decrease-dimension analysis were based on the abundance table of each taxonomic hierarchy by R. Principal co-ordinates analysis (PCoA) based on Bray–Curtis distances was applied to visualize the differences between microbial structures; Analysis of molecular variance (AMOVA) based on weighted-unifrac distances was used in R. Linear discriminant analysis effect Size (LEfSe) analysis was conducted with the LEfSe software (the default LDA score was 3). Permutational multivariate analyses of variance (PERMANOVA) were performed to test the significance of differences among microbial structures. The mean comparison of data among three or more groups was analyzed using a one-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons with SPSS (V18.0). For statistically significant differences, 3.3.1.p < 0.05), the atypical lymphocyte proportion was higher in the M group (p < 0.05), and the monocyte proportion was higher in the L group (p < 0.05). At 72 h, the leukocyte count, lymphocyte proportion, and atypical lymphocyte proportion were significantly lower and the neutrophil and monocyte proportions were significantly higher in the H group than those in the C group (p < 0.05). Moreover, the proportions of eosinophils and basophils increased in the H group, but no significant difference was observed compared with the C group (p > 0.05).At 24 h, leukocyte count was no significantly differences between infected groups, but the proportion of different-leukocyte types did . Compare3.2.Vibrio genus in intestine was showed that at 24 and 72 h, significantly higher levels of 16S rRNA were only found in group H than that in group C (p < 0.05)  . The effse group .3.3.Vibrio levels were relatively high in the H group at both 24 and 72 h, with distinct species at each time point , and the H group was significantly separated from other groups at 24 and 72 h  of the H group at the phylum and genus levels were significantly lower than those of the C, L, and M groups at 24 and 72 h, but no significant difference was observed in the H group at 24 and 72 h ,B. Besidme point . At genuand 72 h ,E.Vibrio in the H group was significantly higher than that in the C and L groups at 24 and 72 h, and higher than that in the M group at 24 h, but no significant difference with the M group at 72 h. The abundance of Tenacibaculum was significantly higher in the H group at 72 h than that at 24 h. The abundance of Shewanella in the H group was significantly higher than that in the C, L, and M groups at 24 and 72 h , 3 of Shewanella (r ≥ 0.66), 2 of Enterovibrio (r ≥ 0.66), 3 of Lentisphaerae (r ≥ 0.73) and 2 of Sphingomonas (r = 1) . Moreover, V. alginolyticus had extremely significant positive correlations with Shewanella fidelis, Enterovibrio nigricans, and Escherichia coli (p < 0.001) and significant positive correlations with S. waksmanii, S. amazonensis, Enterovibrio coralii, Tenacibaculum maritimum, Lentisphaerae bacterium GWF2_45_14 and GWF2_44_16 (0.001 < p < 0.05). V. alginolyticus had negative correlations with Pseudoalteromonas shioyasakiensis (r = −0.10) and Brevinema andersonii (r = −0.19) but was not significant  . The significant .3.5.Vibrio cholerae, bacterial chemotaxis, and flagellar assembly , fliC , fur , ompW , ompU , colA , toxR , toxS , hppD  and hap  were annotated, with most belonging to the H group , hyaluronan synthase (EC 2.4.1.212), chitin synthase (EC 2.4.1.16), and chitin oligosaccharide synthase (EC 2.4.1.-) .3.6.A total of 66 antibiotic-resistance genes were annotated based on the CARD database. Cluster analysis showed that these genes were divided into 49 ARO groups . The ove4.Vibrio can enter the host’s bloodstream, thereby accelerating pathogen transmission ; and V. coralliilyticus chemotaxes toward coral mucus , the Key R&D Program of Hebei Province, China (21326307D), the National Marine Genetic Resource Center, the Natural Science Foundation of Hebei Province (C2019204360) and the Science and Technology Project of Hebei Education Department (QN2020132).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.https://www.frontiersin.org/articles/10.3389/fmicb.2023.1178575/full#supplementary-materialThe Supplementary material for this article can be found online at: Click here for additional data file.Click here for additional data file.


Fetal facial profile could be measured during the early pregnancy. Its abnormalities might be associated with certain congenital malformations. We aimed to study the associations between fetal facial profile measurements with crown-rump length and congenital malformations  during early pregnancy.+ 6 weeks were enrolled. Two sonographers performed fetal facial profile measurements independently. The associations between these measurements with crown-rump length and congenital malformations were evaluated.We performed a prospective cross-sectional study between June 2019 and April 2022. Pregnant women at a gestational age between 11–13There were 406 and 25 fetuses without or with congenital malformations, respectively. Two sonographers showed satisfactory inter- and intra-observer agreements and reproducibility. The maxillary gap was only observed in 7.6% of normal fetuses, whereas all cleft lip and palate fetuses had a maxillary gap ≥ 0.8 mm. The crown-rump length was negatively correlated with frontomaxillary facial angle, inferior facial angle, and profile line distance but positively correlated with maxilla-nasion-mandible angle, facial maxillary angle, frontal space distance, and palatine maxillary diameter. These measurements showed various significant changes with different congenital malformations.Measurements of fetal facial profile in early pregnancy were feasible with satisfactory reproducibility. These measurements correlated with crown-rump length and showed significant differences with certain fetal congenital malformations.The online version contains supplementary material available at 10.1186/s12884-023-06067-6. Fetal craniofacial malformations refer to a diverse group of growth deformities in the head and facial areas in the fetus . It can + 6 weeks [Ultrasound is the main diagnostic test used to assess fetal structural malformations . Most st 6 weeks . Certain 6 weeks –11. Howe 6 weeks , 13. UndTherefore, we performed the present prospective cross-sectional study. First, we determined the agreement and reproducibility of fetal facial profile measurements by ultrasound during early pregnancy. Then, we evaluated the associations between these measurements and crown-rump length (CRL) and congenital malformations. We aimed to further provide a preliminary normal measurement reference for the Chinese population and explore the clinical implications of these abnormal measurements.+ 6 weeks; (2) were willing to receive prenatal ultrasound evaluations with clear images obtained. We targeted to study the fetuses with normal facial profile measurements or cleft lip and palate, micrognathia, or open spina bifida. Fetuses with other structural abnormalities were excluded from the study. In addition, fetuses with abnormal chromosome analysis were also excluded since these fetuses might have unique facial profiles, which was not the purpose of the present study.We performed a prospective cross-sectional study on pregnant women at the Guangdong Women and Children Hospital, China, between June 2019 and April 2022. The study protocol was approved by the hospital ethics committee. All the study participants signed the written informed consent. The inclusion criterion was Chinese women who (1) had singleton pregnancy at the gestational age between 11 weeks and 13Ultrasound equipment included GE Voluson E10, E8, and E6 and Samsung HERA W9 and Aplio i700. They were equipped with a 2-dimensional (2D) convex probe with a frequency of 2.0–5.0 mHz, 6.0–8.0 mHz, and 3.0–10.0 mHz.The ultrasound examinations followed the guidelines from ISUOG . BrieflyThe definitions for the facial profile markers are described in Fig. In addition, we also measured the CRL, which was the length from the top of the head to the bottom of the buttocks.The intra- and inter-observer agreements and reproducibility were assessed by the intraclass correlation coefficient (ICC) and Bland-Altman analysis.P < 0.05 was considered statistically significant.Based on the absence or presence of congenital malformations ,  these fe+ 2 weeks). The final congenital abnormal structure diagnoses were confirmed on the fetal autopsy.A total of 542 pregnant women received fetal ultrasound evaluations during early pregnancy. Among them, 2, 4, 3, and 1 fetuses had thickened nuchal translucency, torso abnormalities, demise, and maternal uterus malformation, respectively, and were excluded. In addition, 76 and 21 fetuses had no clear mid-sagittal plane or facial profile images, respectively, and were excluded. Four fetuses were excluded due to chromosome abnormalities (2 cases of trisomy 13 and 2 cases of trisomy 18). Finally, there were 431 fetuses included in the analysis, with 406 fetuses in the normal group and 25 in the abnormal group . Transabdominal ultrasound examinations were able to obtain satisfactory images in all pregnant women in the normal group. Four pregnant women in the abnormal group (16%) had to receive additional transvaginal ultrasound examination to obtain clear images. All the pregnant women with abnormal fetuses selected induced abortion . In the normal group, the FMF angle, MNM angle, FMA, IFA, FSD, PL distance, and PMD were 86.4°±3.1°, 7.8°±2.1°, 64.3°±3.8°, 67.1°±3.5°, -1.7 ± 1.6 mm, 3.4 ± 0.6 mm, and 6.6 ± 0.9 mm, respectively. MG was only observed in 7.6% of fetuses without congenital malformations, whereas all fetuses with cleft lip and palate had an MG ≥ 0.8 mm. Detailed measurements of fetal facial profile markers in the normal and abnormal groups are provided in the supplementary materials °, which had some variations from the reports by the other two studies in Chinese populations  , 22. We We measured the FMA with the anterior surface of the maxilla as the reference line to avoid the influence of the vomer curvature . The mea15 but similar to that reported by Li et al. (66.5°) [Our present study found an IFA of 67.1 ± 3.5° in normal fetuses, which was smaller than that reported by Ji et al. (80.2 ± 7.3°), (66.5°) . We also (66.5°) , 24, 25  (66.5°) . In the There was no previously reported FSD measurement in Chinese fetuses. Our FSD measurement was − 1.7 ± 1.6 mm, which was smaller than those from other countries  , 24. TheThe mean PL distance in this study was 3.4 ± 0.6 mm, which was slightly higher than 2.8 ± 0.5 mm reported by Ji et al . The PL The PMD measurement was never reported in Chinese fetuses previously. The PMD measured in the present study was 6.6 ± 0.9 mm. PMD was positively correlated with CRL. In 25 fetuses in the abnormal group in the present study, there were five fetuses with micrognathia and concurrent cleft lip and palate and one fetus with a case of open spina bifida and concurrent cleft lip and palate. A total of 20 fetuses with cleft lip and palate had reduced PMD, which was statistically significantly different from the normal group. None of the fetuses with normal PMD were found to have cleft lip and palate. The sensitivity and specificity of PMD for detecting cleft lip and palate were 100% and 97.8%, respectively. PMD can be used as a reliable measurement to assess the cleft lip and palate, with high sensitivity and specificity.In 2015, Chaoui et al. proposed that MG could screen for cleft lip and palate . They reThe limitations of the present study included its single-center research with a small number of abnormal cases. We used the 2D ultrasound in our fetal evaluations. It was suggested that three-dimensional ultrasound could provide a better mid-sagittal view for accurate measurements. Our measurements in the normal fetuses still had some variations from other reports conducted in the Chinese population, which might be due to body habitus differences in the different geographic areas of China. Future multicenter research is required.Measurements of fetal facial profile in early pregnancy were feasible with satisfactory reproducibility in Chinese pregnant women. These measurements correlated with crown-rump length and showed significant differences with certain fetal congenital malformations.Below is the link to the electronic supplementary material.Supplementary Material 1


The estimation of measurement uncertainty was performed along with the assessment of regression models describing the trend of measured quantities as a function of temperature to be used in simulations involving heat transfer in kidney tissue. A direct comparison of the thermal properties of the same tissue from two different species, i.e., porcine and bovine kidney tissues, with the same experimental transient hot-wire technique, was conducted to provide indications on the possible inter-species variabilities of k and α at different selected temperatures. Exponential fitting curves were selected to interpolate the measured values for both porcine and bovine kidney tissues, for both k and α. The results show that the k and α values of the tissues remained rather constant from room temperature up to the onset of water evaporation, and a more marked increase was observed afterward. Indeed, at the highest investigated temperatures, i.e., 90.0–92.8 °C, the average k values were subject to 1.2- and 1.3-fold increases, compared to their nominal values at room temperature, in porcine and bovine kidney tissue, respectively. Moreover, at 90.0–92.8 °C, 1.4- and 1.2-fold increases in the average values of α, compared to baseline values, were observed for porcine and bovine kidney tissue, respectively. No statistically significant differences were found between the thermal properties of porcine and bovine kidney tissues at the same selected tissue temperatures despite their anatomical and structural differences. The provided quantitative values and best-fit regression models can be used to enhance the accuracy of the prediction capability of numerical models of thermal therapies. Furthermore, this study may provide insights into the refinement of protocols for the realization of tissue-mimicking phantoms and the choice of tissue models for bioheat transfer studies in experimental laboratories.This experimental study aimed to characterize the thermal properties of ex vivo porcine and bovine kidney tissues in steady-state heat transfer conditions in a wider thermal interval (23.2–92.8 °C) compared to previous investigations limited to 45 °C. Thermal properties, namely thermal conductivity ( Moreover, the thermal diffusivity (α) of a tissue quantitatively represents its capability to transfer heat in relation to storing thermal energy , k is thermal conductivity [W/(m·K)], r is the distance between the two needles (6 mm), α is thermal diffusivity [mm2/s], t is time [s], ht is heating time [s] (30 s), and x) is the exponential integral, approximated using polynomials: in particular, for small arguments, it is approximated as the sum between the Euler–Mascheroni constant (γ ≈ 0.5772) and the natural logarithm of the argument, ln(x) [The container was placed into the water thermal bath to reach the desired temperature, which was kept constant during the measurement of thermal properties. The thermal bath was set to specific temperatures for each sample, and measurements of the thermal properties were performed in steady-state heat transfer conditions, i.e., only when the sample reached thermal equilibrium. The temperature range of interest set in the thermal bath was ~22 °C to >90 °C, and was divided into 10 steps for porcine kidneys and 9 steps for bovine kidneys. The TEMPOS thermal properties analyzer equipped with an SH-3 dual-needle sensor was used to measure e method ,37,38,39e method ,57. Indee method . In the t, ln(x) ,37,59.EU) were calculated. Each experimental trial concerning the measurement of the thermal properties from room to ablative temperatures was repeated three times for each tissue type, i.e., three experimental trials from room to ablative temperatures were performed for the measurement of the thermal properties of porcine kidney. Three experimental trials from baseline to ablative temperatures were also conducted for the measurement of the thermal properties of bovine tissue.For each thermal parameter, the mean value EU is defined by s, given in Equation (4), multiplied by the coverage factor fk. This coverage factor is obtained considering a confidence level of 95% for a Student’s t-distribution. Thus, having 3 measurements (2 degrees of freedom), fk = 4.30 [n is the number of experiments for each temperature (n = 3), y is the measured thermal property and The f = 4.30 .(4)s=∑ik and α is represented by an exponential curve as follows:In order to obtain the trend of the studied properties as a function of the temperature, the best-fit model was obtained for each thermal parameter. Concerning the modeling of thermal properties, the best-fit model for ® . Finally, the coefficient of determination  to ensure the measurement system was accurate and functioned correctly without any flaws. At room temperature, i.e., 22.3–26.0 °C, the results of our measurements were k = 0.379 W/(m∙K) and α = 0.189 mm2/s, while the values reported on the Certificate of Quality Assurance are k = 0.384 W/(m∙K) and α = 0.189 mm2/s. The difference of <1.5% between our measurement results on the standard material and the values reported by the manufacturer indicated the accurate reading capability of our system [Prior to proceeding with actual measurements of thermal properties in ex vivo biological tissues, measurements were conducted on a reference material provided by the manufacturer  are reported in The thermal properties of ex vivo porcine kidney tissue were measured in steady-state heat transfer conditions at different tissue temperatures, i.e., from nominal conditions at room temperature (23.9 °C) to approximate body temperature (35.4 °C), and up to hyperthermia and ablative temperatures . The attained results, in terms of the average values of k and α, reported in In order to visualize the trend of the thermal properties with increasing tissue temperatures, the data of k of porcine kidney tissue, its nominal value at room temperature was equal to 0.549 W/(m·K). A gradual increase from 23.9 °C to 46.2 °C, followed by a slight decline from 60.0 °C to 82.3 °C, can be observed. Finally, an exponential growth up to 92.8 °C was registered, with a value of 0.648 W/(m·K) at 92.8 °C. Comparing the value of k at the initial value and last value of the temperature interval  an overall increase of 18% was observed at 92.8 °C with respect to the baseline value measured at room temperature.Concerning the k values, which are represented by an exponential curve defined using Equation (6). The regression coefficients are reported in 2. The results of the residuals analysis are reported in α at different temperatures, α remained almost constant from room temperature up to 60.0 °C, showing values in the interval of 23.9–60 °C, ranging from a minimum of 0.155 mm2/s (observed at 23.9 °C) to a maximum of 0.163 mm2/s (at 46.2 °C). After 60.0 °C, the value α increased with temperature, reaching values of 0.178 mm2/s at 82.3 °C, 0.194 mm2/s at 86.6 °C, and 0.216 mm2/s at 92.8 °C, corresponding to increases, compared to the baseline values, of 14%, 25%, and 39%, respectively. α at the different temperatures along with the best-fit curve interpolating the experimental measurements. The trend of α as a function of tissue temperature was modeled using an exponential curve (Equation (6)), whose regression coefficients are reported in Concerning the measurement of k and α of ex vivo bovine tissues as a function of temperature from 23.2 °C up to 90.0 °C. The same experimental setup employed for the evaluation of the temperature dependence of the thermal properties of porcine kidney tissue was utilized for the measurement of k of bovine kidney tissue, attained using the dual-needle technique. As is observed, the value of k exponentially increased when temperatures of 90 °C were reached. At nominal conditions, i.e., room temperature, k was equal to 0.528 W/(m·K); at body temperature , the average value of k was 0.551 W/(m·K). At the maximum investigated temperature value, i.e., 90.0 °C, k reached a value of 0.703 W/(m·K), showing increases of 33% and 28% compared to the baseline values at room and body temperatures, respectively. In k is also shown. The exponential model (Equation (6)), similar to what was observed for the k of porcine kidney tissue, can be considered an accurate approximation of measured thermal property values at the different temperatures. 2R, which is equal to 0.805. The analysis of the residuals over temperature  was registered at 56.4 °C. Moreover, an increment in the tissue temperature up to 90.0 °C led to an increase in α of 21% compared to the nominal value at ambient temperature. The exponential model, for which the regression coefficients are shown in α values . Concerning the characterization of the α values a and desk attained at temperatures of 23.2–23.9 °C, 35.4–36.9 °C, 40.9–41.5 °C, 46.2–48.8 °C, 56.4–56.7 °C, 69.5–70.1 °C, 75.8–76.4 °C, and 90.0–92.8 °C for both porcine and bovine samples, along with the associated measurement uncertainty (estimated with a 95% confidence level). It is possible to notice that, for instance, at room temperature, i.e., 23.2–23.9 °C, the difference between the average values of k associated with porcine kidney tissue and those related to bovine tissue was 4%. Likewise, at the highest investigated temperature (90.0–92.8 °C), the difference in terms of the average values k between the porcine and bovine tissue was <9%. Overall, at the same tissue temperature, the results of the k of porcine tissue were not statistically significantly different from the values of k measured for the bovine kidney . This was observed for all the investigated temperatures. k data for both porcine and bovine kidney samples. For the sake of comparison between the values of α measured for porcine and bovine tissues, the bar plots showing the average values attained at different increasing temperatures are presented in k, considering the same tissue temperature, no statistically significant difference was found between the values of α measured for porcine tissue and the values recovered for bovine tissue . The exponential curves which best fit the measured values of α are shown in In The objective of the present experimental study was threefold. We devised the characterization of the thermal properties of ex vivo porcine and bovine kidney tissues from room up to supraphysiological  temperatures in order to provide quantitative values of these properties in a wider thermal interval, compared to previous investigations that were limited to 45 °C . Furtherk and α, of biological media was performed in a temperature-controlled environment by means of a dual-needle probe, capable of measuring tissue temperature and delivering a specific thermal energy to the sample. The probe was connected to a commercial thermal property analyzer designed in accordance with ISO 2008 standards and in compliance with ASTM 5334 and IEEE 442 [The measurement of the thermal properties, i.e., IEEE 442 . The samIEEE 442 ,40,62. Mk, our measurements are in agreement with values registered at room, body, and hyperthermic temperatures for ex vivo porcine renal cortex, rabbit kidney, human renal pelvis, human renal medulla, and human renal cortex  using self-heating thermistors [k for different kidney tissues investigated in the present study and other studies that used various tissue temperatures. At room temperature, our measurements resulted in average values of 0.549 W/(m·K) for porcine kidney tissue at 23.9 °C and 0.528 W/(m·K) for bovine tissue at 23.2 °C, whilst the values measured by Valvano et al. (at 23 °C) were 0.524, 0.525, 0.524, 0.525, and 0.529 W/(m·K) for ex vivo porcine renal cortex, rabbit kidney, human renal pelvis, human renal medulla, and human renal cortex, respectively [k were 0.559 W/(m·K)  and 0.551 W/(m·K) , showing a maximum percentage difference of lower than 4% compared to the values reported at 37 °C by Valvano et al. in porcine renal cortex, rabbit kidney, human renal pelvis, human renal medulla, and human renal cortex. Concerning higher temperatures, i.e., close to 45 °C, we attained average values of k = 0.573 W/(m·K)  and k = 0.541 W/(m·K) . Hence, in this case, the maximum difference between the values reported by Valvano et al. (45 °C) [k remained rather constant until the onset of water vaporization, i.e., close to 90 °C [k values of porcine and bovine tissues, the best-fit regression models interpolating experimental data were represented by exponential curves. Concerning porcine kidney tissue, at 92.8 °C, a 1.2-fold increase in the average value of k compared to its baseline value at room temperature was observed. Similarly, for bovine kidney tissue, at 90.0 °C, the average value of k was 1.3 times higher with respect to its value at nominal conditions. These results are in line with the experimental observations reported for ovine kidney tissues by Silva et al. [k measured for the porcine kidney and the values attained for bovine kidney tissue.For values of rmistors . Table 5ectively . Thus, aa et al. . Indeed,α of porcine and bovine kidney tissues, as a function of temperature , we noticed an exponential trend with increasing temperature, as also observed for the values of k. The obtained average values at room temperature were 0.155 mm2/s (at 23.9 °C) and 0.151 mm2/s (at 23.2 °C) for porcine and bovine tissues, accordingly. At room temperature, a maximum percentage difference of 14% was observed in comparison with the values measured by Valvano et al. (at 23 °C) in porcine renal cortex, rabbit kidney, human renal pelvis, human renal medulla, and human renal cortex using self-heating thermistor probes [α obtained in our investigation along with the results of other experimental trials performed on ex vivo ovine kidney, porcine renal cortex, rabbit kidney, human renal pelvis, human renal medulla, and human renal cortex [α attained in this study are slightly higher compared with the average values measured by Valvano et al. at room temperature, body temperature, and at 45 °C in porcine renal cortex, rabbit kidney, human renal pelvis, human renal medulla, and human renal cortex [α values for both porcine and bovine kidney tissues. At the highest investigated temperatures, i.e., 90.0–92.8 °C, the average α values were subject to a 1.4- and 1.2-fold increase for porcine and bovine kidney tissues, correspondingly. Also in this case, the results are in accordance with the measurements reported in ovine kidney tissue [α at 93 °C was shown compared to its nominal value at room temperature , no statistically significant differences between porcine and bovine kidney tissue were reported, despite the anatomical and structural differences between the two tissue types. This observation may be insightful for the proper selection of the tissue models for the optimization of different procedures involving heat transfer in kidney tissue. Indeed, porcine and bovine kidney tissues have been advocated as experimental models for training and medical research, also involving the optimization of thermal therapies [Concerning the comparison of the herapies . Howeverk and α of human tissue as a function of temperature, considering both diseased and healthy kidney tissues for a thorough characterization of their thermal features.In conclusion, this study presents the characterization of the thermal properties of porcine and bovine kidney tissues as a function of temperature, ranging from room up to supraphysiological thermal ranges. The provided quantitative values, as well as the best-fit regression models describing the trends of thermal properties, can be used as inputs for numerical models of thermal therapies. Indeed, mathematical equations concerning the trends of the thermal properties of kidney tissue as a function of temperature can be easily incorporated into in silico models for a more reliable description of the thermal behavior of biological tissue. Accurate information on the thermal behavior of kidneys is required to enhance the prediction capability of computational and mathematical tools toward more accurate therapy pre-planning. Moreover, the measured properties may be used for the realization of tissue-like materials for testing, validation, and the improvement of preclinical devices. Thus, the measured properties may be useful for the design of new investigations aiming to improve thermal-energy-based therapies such as catheter-based renal denervation and thermal ablation procedures for unhealthy tissue treatment. Furthermore, the inter-species variabilities analysis of the thermal properties of porcine and bovine tissue may provide insights into the refinement of protocols and choice of tissue models for bioheat transfer studies in experimental laboratories. Additionally, species-dependent specific data on the thermal properties of tissues can help realize more accurate simulation models with respect to the type of tissues chosen for experimental validation. The principal limitation of the present study is the use of ex vivo healthy animal tissue models. Thus, future in vivo studies should be conducted considering the influence of physiological phenomena such as metabolic heat and perfusion on the variance of thermal properties with temperature. In addition, in the present study, measurements were performed with the aim of preserving the structure of the kidneys and penetrating both the cortex and medulla with a dual-needle probe. Hence, the measurement of the thermal properties refers to the thermal characteristics of both these tissue structures, considered as a whole. It may be of interest, also considering possible implications for improving thermal ablation treatments, to evaluate, in future studies, possible variations in thermal properties for the different regions of kidney tissue. Furthermore, additional investigations should also be devised for the measurement of the


Patients with obesity undergoing rectal cancer surgery may have an increased risk of developing complications, though evidence is inconclusive. The aim of this study was to determine the direct impact of obesity on postoperative outcomes using data from a large clinical registry.The Binational Colorectal Cancer Audit registry was used to identify patients who underwent rectal cancer surgery in Australia and New Zealand from 2007–2021. Primary outcomes were inpatient surgical and medical complications. Logistic regression models were developed to describe the association between body-mass index (BMI) and outcomes.2, 35.4% had a BMI of 18.5–24.9 kg/m2, 37.6% had a BMI of 25.0–29.9 kg/m2, 16.7% had a BMI of 30.0–34.9 kg/m2, and 8.2% had a BMI ≥ 35.0 kg/m2. Surgical complications occurred in 27.7% of patients with a BMI of 18.5–24.9 kg/m2, 26.6% of patients with a BMI of 25.0–29.9 kg/m2 , 28.5% with a BMI of 30.0–34.9 kg/m2 , and 33.2% with a BMI ≥ 35.0 kg/m2 . Modelling BMI as a continuous variable confirmed a J-shaped relationship. The association between BMI and medical complications was more linear.Among 3,708 patients , 2.0% had a BMI < 18.5 kg/mRisk of postoperative complications is increased in patients with obesity undergoing rectal cancer surgery.The online version contains supplementary material available at 10.1007/s00384-023-04447-0. In 2017–18, an estimated 31% of Australians aged 18 and over were considered obese, defined as a body mass index (BMI) of 30 kg/mIn rectal cancer surgery, patients who have obesity may present technical challenges and are at increased risk of developing postoperative complications. In a recent Australian study by Bell et al. that included 481 patients with rectal cancer, those with obesity were found to have a significantly higher risk of surgical complications 35.6% vs 23.6%) and likelihood of requiring conversion from a minimally invasive surgical approach to open surgery (OR 4.1)  and infe.6% and lMore recent studies utilising data from large registries have more consistently demonstrated an association between obesity and adverse outcomes in patients undergoing surgery for rectal cancer. A study by Gebauer et al. of 9,920 patients using prospectively-collected data across 183 German hospitals between 2008 through 2011 showed that obesity was associated with significantly higher risk of overall postoperative (42.9% vs 36.6%) and surgical complications 33.9% vs 26.8%) . Similar.8% . SimThe aim of this study was to analyse data from the Binational Colorectal Cancer Audit (BCCA), an Australian and New Zealand clinical registry, looking for variations in outcome in patients undergoing surgery for rectal cancer between those with and without obesity. We hypothesised that patients with obesity are at higher risk of developing postoperative complications.This study used individual, de-identified data from the BCCA . The BCCPatients aged 18 years and over who underwent surgery for rectal cancer were included in the study. Rectal cancer, as defined in the registry, was a malignancy that was clinically, radiologically and/or endoscopically considered to be below the rectosigmoid junction, less than or equal to 15 cm from the anal verge. Measurement of body weight and height was not standardised and may have been self-reported in some cases. Patients with a synchronous malignant colorectal neoplasm and those who underwent synchronous resection of another organ at the time of rectal cancer surgery were excluded. After exclusions, 10,502 patients were available for analysis. As the registry’s weight and height fields were not previously mandatory, BMI data was available or able to be calculated for only 3,708 (35.3%) patients, after excluding the top and bottom 0.5% of BMI data to reduce the impact of potential outliers.This study was approved by the Human Research Ethics Committee of the South Western Sydney Local Health District.The primary outcomes of our study were inpatient surgical and medical complications. Definitions of these composite outcomes are provided in Section Our primary analysis evaluated the association of BMI with surgical and medical complications. We constructed two logistic regression models with adjustment for potential confounding factors, including age, sex, American Society of Anesthesiology (ASA) score, height of the cancer from the anal verge, neoadjuvant therapy, T and N stage, operative urgency, seniority of the person performing the surgery, method of surgical entry, and formation of a stoma. The first model treated BMI as a categorical variable according to the standard World Health Organisation (WHO) classification system of weight status. The second model treated BMI as a continuous variable by using a natural cubic spline. Further details including model specifications are provided in Section In the secondary analyses, we repeated the regression modelling for the outcomes of unplanned return to theatre, readmission within 30 days, and margin involvement. Length of hospital stay, distal margin clearance, and number of lymph nodes harvested were summarised using their medians and interquartile ranges (IQR). All analyses were performed using R software, version 3.6.1 .2 , with 925 patients (24.9%) having a BMI ≥ 30.0 kg/m2. Half the patients received neoadjuvant therapy. Two-thirds of tumours were located less than 10 cm from the anal verge. Most patients (95.5%) had an elective procedure. The proportions of patients who developed surgical and medical complications were 27.9% and 14.3%, respectively. Inpatient mortality rate was 0.5%.The characteristics of the 3,708 patients, stratified according to BMI cohort, are summarised in Table 2, 28.5% with a BMI of 30.0–34.9 kg/m2 , and 33.2% with a BMI ≥ 35.0 kg/m2 . Modelling BMI as a continuous variable showed a J-shaped relationship with risk lowest for patients with BMI between 25 and 30 kg/m2 , had ASA ≥ 3 , had an open procedure , or had proximal diversion  . After excluding wound infection from the composite outcome, the association of BMI with overall surgical complications still exhibited a J-shaped relationship .Of the 10,502 eligible patients in the BCCA registry, 6,756 64.3%) had missing weight, height and/or BMI data. The characteristics of these patients are summarised in Supplementary table .3% had mp < 0.001). Surgical complications were slightly greater in patients with missing BMI data . There were no differences in medical complications or any other clinical or oncological outcomes  for their inclusion criteria, as patients with BMI 30.0–34.9 kg/m2 had a similar rate of complications as patients with normal BMI.Our study shows that BMI is an important modifiable risk factor for complications in patients undergoing surgery for rectal cancer. Given that half of the patients in our study received neoadjuvant therapy, this would be an ideal time for interventions that help patients with obesity achieve a healthier weight. Evidence on prehabilitation in rectal cancer surgery is limited, but intervening during neoadjuvant treatment appears feasible . A speciOur study showed that patients with obesity were at higher risk of developing postoperative complications following surgery for rectal cancer. Patients who were underweight were also at greater risk. These findings may support the importance of prehabilitation programs for high-risk patients. The main limitation of the study is the large amount of missing data within the BCCA registry. Further research is required to verify our findings using more complete datasets.Supplementary file1 (PDF 183 KB)Below is the link to the electronic supplementary material.


Doryteuthis gahi) waste by a low-toxicity solvent mixture . The effect of the waste weight/solvent volume  ratio and the number of extractions (NoE) on the total lipid (TL), phospholipid (PL), and tocopherol yields and on the fatty acid (FA) profile  was investigated. As a result, an increased NoE led to an increased (p < 0.05) TL yield but a decreased (p < 0.05) proportion of PLs in the lipid extract. Additionally, a lower (p < 0.05) polyunsaturated FA/saturated FA ratio was detected by increasing the NoE. Some differences (p < 0.05) could be outlined as a result of increasing the WW/SV ratio; however, a definite trend for this extraction condition could not be concluded for any of the lipid parameters measured. Yield results were compared to those obtained by the conventional chloroform/methanol procedure. In order to attain an increased yield, the NoE required would depend on the polarity degree of the lipid molecule concerned. All ethanol/acetone extracting conditions tested led to remarkable yields for lipid compounds  and FA ratios with healthy, nutritional, and preserving properties.The study focused on the extraction of valuable lipid compounds from squid ( Marine species are known as nutrient-rich and balanced foods because of their high content of valuable constituents ,2. AmongProcessing marine species generates great amounts of by-products. FAO states that annual seafood production contributes to over 180 million tonnes ; nonetheBased on new challenges for the food industry, a wide range of eco-friendly methods are being used for lipid extraction from natural sources. Among them, ensilage , pH adjuDoryteuthis gahi) is a neritic species that is widely distributed along the Atlantic and Pacific coasts of South America [v/v) mixture as an extracting system. To enhance the extraction of valuable lipid compounds and reduce the volume of the solvent employed, the effect of the number of extractions (NoE) and the effect of the WW/SV ratio were analysed.The Patagonian squid  waste, a mixture of heads, viscera, skin, tails, etc., was obtained from SERPESBA S. L. U. . The initial squid samples were captured near the Argentinean coast in the south-west Atlantic Ocean, frozen on board (−40 °C), and transported for a 3–4-day journey to the Vigo (Spain) factory. Then, samples were thawed (overnight storage at 4 °C), the mantel was taken for commercialization purposes, and the resulting waste was pooled together and transported in refrigerated conditions (4 °C) to the laboratory, which is located in the same town.Patagonian squid  . The resulting lyophilized substrates were pooled together, minced, and used for moisture determination as well as lipid extraction by conventional and ethanol/acetone solvents according to the experimental procedure shown in Based on previous studies on the same squid species ,28, the Solvents and chemical reagents used were of reagent grade and purchased from Merck ; otherwise, the source is mentioned.−1 waste substrate.The moisture content was determined in initial and lyophilized waste (1–2 g) according to the official AOAC method 950.46B . The wei−1 dry waste.The conventional lipid extraction of lyophilized waste was carried out according to the Bligh and Dyer  method. v/v) was carried out according to the extraction conditions described in −1 for the WW/SV ratio and 1, 2, and 3 for the NoE. Each of the extraction conditions (from EC-1 to EC-9) was carried out in quadruplicate.The lipid extraction of lyophilized waste with ethanol/acetone , and the supernatant was collected. This procedure was repeated one or two more times or not repeated according to the experimental design , according to Raheja et al. . In this−1 flow rate was employed, and detection was carried out at 292/328 nm in a HPLC Waters  detector. The possible presence of α-, β-, γ-, and δ-tocopherol molecules was checked. For quantitative purposes, the content of each tocopherol present in the lipid extract was calculated with calibration curves prepared from the corresponding commercial tocopherol compound (Sigma–Aldrich) and calculated as mg·kg−1 lipids.The profile of tocopherol compounds in squid waste was determined by the method proposed by Cabrini et al. . For thi−1, from 190 °C to 210 °C at 5.0 °C·min−1, held for 13.5 min at 210 °C, and from 210 °C to 230 °C at 5.0 °C·min−1. Nitrogen at 10 psi as carrier gas and a flame ionisation detector (FID) at 250 °C were used. The qualitative analysis was carried out according to the FAME retention times and comparison to standard mixtures . Peak areas were automatically integrated, and the quantitative analysis was carried out using C19:0 FA as the internal standard. The content of each FA was calculated as g·100 g−1 total FAs.Acid-catalysed esterification and transesterification by employing acetyl chloride in methanol were applied to convert lipid extracts into fatty acid methyl esters (FAMEs). The resulting FAMEs were then subjected to gas–liquid chromatography  analysis . A fusedResults related to FA groups  and FA ratios  were calculated on the basis of the results obtained for individual FAs.n = 4). Data obtained from the different lipid composition parameters  were subjected to one-way ANOVA (p < 0.05) to analyse the effect of the WW/SV ratio and the effect of the NoE on the yields and ratios obtained in the different ethanol/acetone extracting conditions . The comparison of means was carried out using a least-squares difference (LSD) procedure.According to the experimental design described in −1. This value is similar to the one previously reported for the waste corresponding to the commercialization of the present cephalopod species [−1 value of moisture.The starting waste showed a moisture value of 83.24 ± 1.11 g·100 g species ,28. The p < 0.05) on the lipid yield obtained from the squid waste  waste by employing different low-toxicity solvents  [v/v); however, yields were lower  than in the current case. This lower lipid yield could be explained by the fact that we employed a different WW/SV ratio  than in the present study.It is concluded that the current ethanol/acetone solvent mixture can lead to satisfactory lipid recoverability provided a three-time extraction and a WW/SV ratio of 0.25 g·mLixtures) . In agreKatsuwonus pelamis) heads was studied [v/v) extraction of hoki roe when compared to the conventional Folch procedure [K. pelamis) eyeballs [Illex argentinus) viscera [Previous studies focused on the lipid extraction from different marine waste substrates by employing different low-toxicity solvents and different eco-friendly procedures. Thus, the effect of previous heating on the lipid extraction by wet reduction of skipjack tuna  [v/v) from microalga Scenedesmus obliquus was studied by applying subcritical conditions [Scenedesmus dimorphus by employing several extracting systems ; according to the high PL presence in the microalga substrate, the highest lipid yields were obtained by applying 95% ethanol .Previous research has also addressed the employment of low-toxicity solvents for the lipid extraction of microalga substrates. However, the effect of the NoE and the effect of the microalga weight/solvent ratio were not studied. An increased presence of ethanol in an acetone/ethanol extracting system led to an increased lipid yield obtained from Antarctic krill (superba) . Notablynditions ; the optnditions  carried p < 0.05) in the case of applying a WW/SV ratio of 0.25 g·mL−1.The effect of both extraction variables on the PL yield is depicted in p < 0.05) PL values were detected for a WW/SV ratio of 0.30 g·mL−1; on the contrary, a 0.20 g·mL−1 value of the WW/SV ratio led to the highest average PL content when a three-time extraction was employed.Regarding the effect of the WW/SV ratio , a definA decreased PL presence in the lipid extract by increasing the NoE value can be explained on the basis that the current ethanol/acetone mixture tested is more polar than the mixture employed in the conventional procedure. Thus, PLs would be more easily extracted than less polar lipid classes such as triacylglycerols (TAGs), waxes, cholesterol esters, etc. By repeating the extraction procedure , a more complete lipid extraction would be expected to occur according to the above-mentioned results on TL yields. This yield increase for TLs would lead to a relative decrease in the PL content in the TL extract.PL classes have been described as being important constituents of cell membranes and therefore playing a decisive role in living bodies ,40. BaseD. gahi) waste by low-toxicity solvents [−1) than in the present case was employed.Previous research also focused on the PL extraction from squid (solvents . In suchE. superba) [−1 lipids) in salmon  skins, heads, and backbones when compared to the present ones. With the aim of extracting high levels of polar-rich lipid fractions from microalga Nannochloropsis sp. biomass, a two-step extraction was carried out by Jiménez Callejón et al. [−1 lipids; i. e., PLs and glycolipids) lipid extract. The content of polar lipid compounds  showed an increase with previous heating time during the lipid extraction by a wet-rendering method of skipjack (K. pelamis) eyeballs [v/v) extraction procedure on hoki roe by comparison to the conventional Folch method [Previous studies focused on PL extraction from different marine substrates by employing eco-friendly and low-toxicity procedures. A higher PL yield (ca. 30%) was detected by applying a 1/6 ratio (acetone/ethanol) than in the case of using a higher presence of ethanol  during PL extraction from krill (superba) . By emplsuperba)  found lon et al. ; thus, aeyeballs ; thus, ah method ; in the As for PL compounds, α- and γ-tocopherol yields obtained with the current ethanol/acetone extracting mixture led to a high recovery when compared to the conventional procedure  . This hi−1, the highest α- and γ-tocopherol values (p < 0.05) in the lipid extract were obtained with a one-time extraction; on the contrary, for a WW/SV ratio of 0.30 g·mL−1, the lowest (p < 0.05) tocopherol contents were observed by applying a two-time extraction.The extractability of both tocopherol molecules showed very similar tendencies. Thus, the NoE did not lead to a definite trend. For a WW/SV ratio of 0.25 g·mL−1 value in the case of employing a one-time extraction. If two- or three-time extractions were applied, the highest average values were obtained for a WW/SV ratio of 0.20 g·mL−1.Regarding the effect of the WW/SV ratio , the hig−1) and composition variations resulting from the catching season [According to previous related studies ,44, α-toD. gahi) waste with ethanol, acetone, and ethyl acetate was studied by Rodríguez et al. [E. superba) extraction; such a study revealed an increasing tocopherol yield by increasing the acetone ratio in an acetone/ethanol extracting system. Recently, Li et al. [S. dimorphus by employing ethanol-containing systems  than in the case of using ethyl acetate/hexane , methanol/hexane , or hexane alone.Previous research focused on the extraction of tocopherol from marine substrates by low-toxicity solvents. However, the effect of the number of extractions and the substrate weight/solvent volume ratio was not analysed. Thus, tocopherol compound extraction from squid : 3.65 ± 0.04 (C14:0), 0.48 ± 0.03 (C15:0), 22.81 ± 0.26 (C16:0), 1.64 ± 0.02 (C16:1ω7), 0.84 ± 0.03 (C17:0), 3.94 ± 0.07 (C18:0), 3.61 ± 0.11 (C18:1ω9), 2.57 ± 0.09 (C18:1ω7), 0.57 ± 0.05 (C18:2ω6), 4.45 ± 0.04 (C20:1ω9), 0.47 ± 0.11 (C20:2ω6), 2.65 ± 0.03 (C20:4ω6), 0.57 ± 0.03 (C22:1ω9), 17.35 ± 0.01 (C20:5ω3), 0.26 ± 0.05 (C22:4ω6), 0.67 ± 0.04 (C24:1ω9), 0.56 ± 0.06 (C22:5ω3), and 32.90 ± 0.20 (C22:6ω3). If FA groups are considered, the following composition was obtained : 31.72 ± 0.34 (STFAs), 13.51 ± 0.21 (MUFAs), 54.77 ± 0.13 (PUFAs), 50.81 ± 0.14 , and 3.96 ± 0.10 .The FA profile of the lyophilized waste obtained by the conventional extraction procedure revealed the following composition . All EPA values were included in a very narrow range , which was very close to the value obtained with the conventional procedure . In the case of the DHA content, some differences were observed among the different ethanol/acetone conditions tested. Thus, a decreased (p < 0.05) DHA presence was detected by increasing the NoE in the case of WW/SV values of 0.25 and 0.30 g·mL−1. For the WW/SV ratio, an increased value led to an increased DHA presence (p < 0.05) in lipid extracts corresponding to two-time extractions. Similar to EPA content, DHA values for all ethanol/acetone extracting conditions were included in a narrow range ; such a range was found to be higher than the value obtained for the conventional procedure .For the EPA content, the effect of the WW/SV ratio and the NoE did not lead to significant differences (D. gahi) waste with different kinds of low-toxicity solvents  [v/v) led to values of 15.82 and 31.92 g·100 g−1 total FAs for both ω3 FAs, respectively. Such values were similar to those obtained in the conventional extraction procedure (16.05 and 31.12 g·100 g−1 FAs) and were found to be lower than those reported in −1) and that the lipid composition of the squid waste varied with the seasons.Previous related research analysed the EPA and DHA presence in lipid extracts obtained from squid (ixtures) . Thus, t−1 total FAs) were obtained by Chantachum et al. [K. pelamis) heads by a wet-reduction method; in the meantime, negligible levels of EPA were obtained in both kinds of samples. Lower EPA  and DHA  contents than in the present study were detected in three different salmon  by-products  when extracted following different eco-friendly methods  followed by pressing and centrifugation [−1 total FAs) yield was obtained from skipjack (K. pelamis) eyeballs by employing a wet-rendering method [v/v) extraction on hoki roe by comparison to the conventional Folch method [−1 total FAs) and DHA  contents than in the present study were obtained by Rodríguez et al. [I. argentinus) viscera.Previous research accounts for EPA and DHA values in lipid extracts of marine waste obtained by different kinds of eco-friendly extracting methods. Such values have been shown to depend on the species and the part of the body taken into account. Compared to current results, lower levels of DHA  of the PUFA/STFA ratio. This result is in agreement with the above-mentioned tendency obtained for the DHA presence in the ethanol/acetone extracts. No effect (p > 0.05) of the WW/SV value could be concluded on the PUFA/STFA ratio.In all present extracts obtained by the ethanol/acetone solvent mixture, PUFA/STFA average values were included in a narrow range (1.87–1.99) and were found to be higher than the one detected in the lipid extract corresponding to the conventional procedure (1.79) . An incrp < 0.05) was obtained by increasing the NoE in samples, corresponding to a WW/SV value of 0.25. Additionally, a higher (p < 0.05) ω3/ω6 value was detected in samples, corresponding to a one-time extraction when considering a WW/SV value of 0.25 g·mL−1.As for the PUFA/STFA ratio, all ω3/ω6 average values corresponding to the ethanol/acetone extraction system were included in a narrow range (12.24–13.00). Average values were found to be higher in most cases than those detected in the lipid extract obtained by the conventional procedure . A decre−1 and a three-time extraction) leading to an average value of ca. 13.The present ω3/ω6 values are in agreement with those obtained in the present squid waste by employing different low-toxicity solvent systems  . In suchThunnus thynnus) and sardine (Sardina pilchardus) by-products, while markedly lower ratios (0–2 range) were obtained for tuna (T. thynnus) liver and sea bass (Sparus aurata) and sea bream (Dicentrarchus labrax) by-products. An ω3/ω6 value of 12.5 was obtained by Ahmmed et al. [v/v) extraction on hoki roe; this value was found to be similar to the one obtained in the lipid extracts resulting from the conventional Folch method and the two-step ethanol/hexane procedure. The ω3/ω6 ratio of lipid extracts obtained by different wet-pressing procedures on squid (I. argentinus) viscera was analysed by Rodríguez et al. [Previous studies on waste substrates corresponding to the processing of other marine species have also focused on the ω3/ω6 ratio. Thus, Šimat et al.  obtainedd et al.  by emploz et al. ; as a rep < 0.05) TL yield but a decreased (p < 0.05) proportion of PLs in the lipid extract. Additionally, a lower (p < 0.05) PUFA/STFA ratio was detected by increasing the NoE. Some differences (p < 0.05) could be outlined as a result of increasing or decreasing the WW/SV ratio; however, a definite trend for this extraction condition could not be concluded for any of the valuable lipid compounds taken into account. Compared to the conventional chloroform/methanol procedure, a 94–99% TL recovery was accomplished provided a three-time extraction was carried out. Meantime, a high PL recovery (98–106%) was observed when compared to the lipid extract obtained by the conventional procedure; notably, higher average values (over 100%) were obtained if a one-time extraction was carried out. No effect (p > 0.05) of the NoE was observed on the α- and γ-tocopherol yields; remarkably, all ethanol/acetone extracts led to recovery yields similar to those obtained by the conventional procedure.An increased NoE led to an increased  mixture as a low-toxicity solvent procedure to be employed as an alternative method for the extraction of lipid compounds. Remarkably, all ethanol/acetone extracting conditions tested led to valuable yields for lipid molecules  and FA ratios (PUFAs/STFAs and ω3/ω6) with healthy, nutritional, and preserving properties. Further research is envisaged to check the suitability of the present low-toxicity solvent mixture for the extraction of valuable lipids from other kinds of marine waste substrates.The effect of two extracting conditions (NoE and WW/SV ratio) was analysed, to our knowledge for the first time, during the lipid extraction of a marine substrate by employing a low-toxicity solvent mixture. Present results confirm the suitability of the ethanol/acetone (50:50,