Although visual and quantitative assessments of [18F]FDG PET/CT studies typically rely on liver uptake value as a reference or normalisation factor, consensus or consistency in measuring [18F]FDG uptake is lacking. Therefore, we evaluate the variation of several liver standardised uptake value (SUV) measurements in lymphoma [18F]FDG PET/CT studies using different uptake metrics.liver, SUVpeakliver and SUVmeanliver as a function of (1) volume-of-interest (VOI) size, (2) location, (3) imaging time point and (4) as a function of total metabolic tumour volume (MTV). The impact of reconstruction protocol on liver uptake is studied on 15 baseline lymphoma patient scans. The effect of noise on liver SUV was assessed using full and 25% count images of 15 lymphoma scans.PET/CT scans from 34 lymphoma patients were used to calculate SUVmaxliver and SUVpeakliver were 38% and 16% higher compared to SUVmeanliver. SUVmaxliver and SUVpeakliver increased up to 31% and 15% with VOI size while SUVmeanliver remained unchanged with the lowest variability for the largest VOI size. Liver uptake metrics were not affected by VOI location. Compared to baseline, liver uptake metrics were 15–18% and 9–18% higher at interim and EoT PET, respectively. SUVliver decreased with larger total MTVs. SUVmaxliver and SUVpeakliver were affected by reconstruction protocol up to 62%. SUVmax and SUVpeak moved 22% and 11% upward between full and 25% count images.Generally, SUVmaxliver was most robust against VOI size, location, reconstruction protocol and image noise level, and is thus the most reproducible metric for liver uptake. The commonly recommended 3 cm diameter spherical VOI-based SUVmeanliver values were only slightly more variable than those seen with larger VOI sizes and are sufficient for SUVmeanliver measurements in future studies.SUVmeanEudraCT: 2006–005,174-42, 01–08-2008.The online version contains supplementary material available at 10.1007/s00259-022-05977-5. In the majority of cases, clinical reads are based on visual assessment of [18F]FDG uptake and distribution in lesions and across the body [the body –4.Both visual evaluation and quantitative assessments of 18F]FDG uptake require standardisation and harmonisation of the [18F]FDG PET/CT examinations in order to obtain reproducible results . To thisFFDG uptaThere is, however, no consensus nor consistency in the way liver SUV is used. For QC purposes, the EANM, PERCIST, UPICT and SNMMI recommend to evaluate the mean SUV in a 3-cm-diameter VOI placed in the upper right lobe of the (unaffected) liver. The ACRIN trial protocols recommend the use of liver SUVmean calculated from a maximum region of interest (ROI) diameter in the liver . In the The inconsistency in the definition of liver uptake and the use of various VOI definitions makes comparisons across studies, even before, during and after treatment within the same patient, difficult. Moreover, the use of SUVmax is under debate as it has been reported that it is prone to noise, showing increased upward bias with elevated noise levels, and is more sensitive to variations in image quality and reconstruction settings/protocols than SUVpeak and SUVmean , 17.The aim of this paper is therefore to perform an evaluation on the variation of several liver SUV uptake measurements arising from using different uptake metrics, different VOIs, different noise levels, at different time points across treatment  and at different tumour loads. The paper will conclude by providing some recommendations for the use of liver SUV that will improve the robustness and reproducibility of liver SUV in multicentre studies and for comparisons between studies.In this study, three study datasets were used. The first dataset (dataset 1) consisted of 34 randomly selected patients with diffuse large B cell lymphoma (DLBCL) for whom baseline, interim and EoT scans were available from the HOVON-84 study  . The latter scans were collected consecutively from ongoing clinical investigations and the use of these (anonymised) data for technical scientific purposes was waived by the VU Medical Center ethics review board. The data was used previously and are described in Kaalep et al. FDG PET/CT studies of lymphoma patients scanned at baseline using the Philips Ingenuity PET/CT system . Data were taken from ongoing routine clinical investigations, and the use of the (anonymised) data for technical scientific purposes was waived by the VU Medical Center ethics review board. Two sets of reconstructions were made. First, we reconstructed the full count image data taking all the counts acquired during our standard 2 min per bed position acquisition protocol using a 3MBq/kg FDG activity prescription (scan duration = 120 s per bed). Next, for each bed position, we used only the counts collected during the first 30 s for each bed position to generate an image with 25% of the full count, resulting in images with about 2 times higher noise level (4 times less counts gives about 2 times higher percentage noise).Finally, a third dataset was generated to explore the impact of noise on liver SUV. For this dataset, we included In the first (HOVON-84) dataset, the liver uptake in all images was analysed in several ways with respect to VOI size and location. Spherical VOIs of 1, 1.5, 2, 3 and 5 cm diameters were placed in the (unaffected) right upper lobe of the livers. In addition, spherical VOIs of 3 cm diameter were placed at different locations within the liver, as illustrated in Supplementary Fig. For the second and third datasets, we used the 3-cm-diameter VOI placed in the upper right lobe of the liver, as per EANM recommendations. SUVmax, SUVpeak and SUVmean were reported for each of the applied reconstruction protocols for dataset 2 and measured on the 120 s full and 30 s (25%) count data using dataset 3 and directly compared to demonstrate the impact of reconstruction protocol and noise on liver SUV.p ≤ 0.05) among data was based on paired t tests or Wilcoxon signed rank test, when appropriate. Associations (p ≤ 0.05) were analysed using Spearman’s rank-order correlation testing.Data is described using median values, interquartile ranges (IQR) and is presented using Tukey’s boxplots or scatter plots including generalized additive models (GAM) based trend lines. Significance of differences , while SUVpeak only increased up to 15%, although significantly   Fig. . SUVmean01) Fig. . The SUVAs presented in Fig. p < 0.05 using Spearman’s correlation testing) with again the largest intersubject variability when using SUVmax (IQR 1.0) versus SUVpeak (IQR 0.67) and SUVmean (IQR 0.62).Liver SUV as function of total MTV is illustrated in Fig. p < 0.05) and SUVpeak , but not for SUVmean .Finally, we studied the impact of reconstruction protocol  and the effect of image noise by means of scan duration on liver uptake. The liver SUVmax, and to a lesser extent the SUVpeak calculated on the EARL-1 reconstructed scans differed significantly compared to the liver uptake metrics using the other reconstructions Fig. . The higIn this study, we aimed to assess whether and to which extent VOI size and location, imaging time point related to treatment phase, tumour load, reconstruction parameters and image noise levels (e.g. scan duration) affect liver uptake measurements and to determine which image analyses approach—in terms of VOI definition and SUV metrics—would provide the most robust and reproducible assessment of liver SUV.Our findings suggest that SUVmean based on a spherical VOI of 3 cm or larger seems to provide the most robust estimate of the underlying liver SUV, showing limited or no sensitivity for VOI size, VOI location and image noise (e.g. scan duration). Moreover, SUVmean for larger diameter VOIs showed least intersubject variability, most likely because averaging over a larger VOI volume reduces the impact of image noise. This can also be seen when using a 1-cm-diameter VOI , showing a similar level of variability as that of SUVpeak. SUVmax is based on a single voxel and therefore most sensitive to noise  resThe exact location of the VOI within the liver appears to have less of an effect on the measured liver SUVs. This is advantageous as usually the VOI is placed by an observer and likely the exact placement of the VOI suffers from intra- and interobserver variability. In our study, the different evaluated locations were well separated such that the translation in VOI placement is much larger than can be expected from any observer when applying clear definitions and rules for placing the VOI, i.e. in the upper right lobe of the unaffected liver. Clearly, locations with image artifacts near the diaphragm due to breathing motion, causing a spatial mismatch between PET and CT for attenuation correction, should be avoided as this will obviously result in inaccurate liver SUV measurements.Unexpectedly, baseline liver SUVs appear to be lower than those observed at interim and EoT, regardless of the VOI and metric used. In an attempt to explain these findings, we explored the relation between liver SUV and tumour load by means of total MTV, with the hypothesis that large volume tumours (bulky and/or strongly disseminated) would act as a sink, reducing the availability of [18F]FDG to be taken off by the liver. Indeed, we observed that liver SUVs decrease with increasing total MTV suggesting that baseline tumour [18F]FDG uptake, which is intense and can have large MTVs, may result in lower liver SUV. However, when considering the actual SUVs, these baseline values align with the normal acceptable range suggested in the EANM guideline. Alternatively, it could be hypothesized that interim and EoT liver SUV is elevated due to treatment effects causing extra metabolic activity in the liver. A variety of biological factors potentially affects liver uptake measurements, which are summarized in Supplementary Table Based on the findings in this paper we conclude and propose the following recommendations: (1) liver SUVmean is most robust to VOI size, reconstruction protocol and image noise. It therefore reflects the most reproducible liver uptake metric and is recommended when liver uptake is used for QC purposes, as reference for tumour assessment and tumour segmentation thresholding or when it is used as normalisation factor. (2) SUVmax shows an upward bias compared to the other SUV metrics in liver as well as in lesions, as was repeatedly shown in several publications , 22. SUVIn this paper, we evaluated the impact of several factors that can affect the liver uptake assessment. Liver SUVmax seemed to be most sensitive to VOI size, image noise and reconstruction protocol and should therefore not be used for quality control purposes, to define tumour segmentation thresholds or as reference or normalisation value, i.e. when assessing tumour to liver ratios. Liver SUVmean was most robust against these factors, showing smallest inter-subject variability as well. The commonly recommended liver SUVmean derived from a 3-cm-diameter spherical VOI was only slightly more variable than those seen with larger VOI sizes, which does not seem to justify an urgent adjustment of current guidelines. Finally, we observed that liver uptake may differ systematically between different time points across treatment, possibly caused by the high lesional uptake and large total MTV seen at baseline [18F]FDG PET/CT in DLBCL patients and/or treatment effect which directly affects liver metabolism. This phenomenon should be kept in mind when using liver SUV for any quantitative purpose of when scoring lesion uptakes.Supplementary file1 (DOCX 64 KB)Supplementary file2 (DOCX 43 KB)Supplementary file3 (DOCX 24 KB)Supplementary file4 (DOCX 28 KB)Supplementary file5 (DOCX 61 KB)Below is the link to the electronic supplementary material.


A male patient of diabetic ketoacidosis and septic shock was started on norepinephrine infusion following which he developed bulla and subcutaneous tissue ischemia in the event of inadvertent extravasations of norepinephrine. The patient improved after management with mechanical debridement of necrosed tissue and regular dressing of the wound. The use of higher concentration of norepinephrine via peripheral intravenous route may lead to vasoconstriction and subcutaneous tissue ischemia due to inadvertent extravasations. He was started on insulin infusion, fluid resuscitation, and antibiotic. The correct positioning of peripheral intravenous (IV) cannula was assessed by flushing with 10 ml of normal saline. Owing to the perceived urgency, norepinephrine infusion (10 mcg/min) was initiated for hemodynamic support through a peripheral intravenous (PIV) line. Following hemodynamic stability after 48 h of norepinephrine PIV infusion, patient developed a large bulla on his right forearm around the peripheral intravenous site , the extravasation rate was 4% (median administered dose was 0.08 mcg/kg/min).In a study conducted by Pancaro et al., out of 14,385 patients receiving general anesthesia as well as PIV norepinephrine while undergoing surgery, five (0.035%) had an extravasation event, for an estimated risk of one to eight events per 10,000 patients. The norepinephrine infusions that extravasated were in a dose range of 0.02 to 0.05 mcg/kg/min (median administered dose was 40 mcg).The pathophysiology involves relatively higher concentration of norepinephrine in both the recipient vein and adjacent blood vessels following extravasations that lead to vasoconstriction and increased vascular permeability.It is important to grade the severity of extravasation for patient's management. It is graded as the following: grade 1 (intact skin), grade 2 , grade 3 , grade 4 (life‐threatening warranting immediate intervention), and grade 5 (death).RRP conceived the study, collected data, and wrote the manuscript. RRP read and approved the final manuscript.None.None.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.


The preservative effect of the addition of different essential oils (copaiba and oregano) on meat quality parameters and sensorial acceptability was analyzed for fresh ground beef patties over 21 days of display. Five treatments were assessed: control (CON) without antioxidants; addition of the synthetic additive butylated hydroxytoluene (BHT); addition 0.05% of copaiba essential oil (CEO); 0.05% of oregano essential oil (OEO); or blend of 0.025% copaiba and 0.025% oregano essential oils (BEO). The lowest cooking losses and greatest tenderness (P <0.05) were reached with the blend (BEO). The inclusion of oregano essential oil presented a more intense chroma (P <0.05), with the best color retained during display. Oregano essential oil (OEO) and the blend (BEO) showed the highest antioxidant activity, reducing the lipid oxidation of beef patties during display (P < 0.05). Consumers preferred the odor of beef patties with essential oils (OEO and BEO) to the CON; however, the flavor from OEO had the lowest acceptability and the worst scores for overall acceptability (P < 0.05). Patties with the blend addition (BEO) were the best scored on overall acceptability assessments. In conclusion, the oregano and copaiba essential oils blend had a good preservative effect on fresh beef patties during display and increased sensory acceptability of the product, thus being a possible alternative for replacing synthetic compounds in processed foods. Consumer demand for meat and meat products has increased quickly, mainly due to the high levels of nutrients in these products ,2. HowevTo meet industrial needs, processed products prove to be an attractive alternative, since the transformation of meat into processed products can generate nutritionally valuable by-products that can be used to add value, encouraging better use of that meat and increasing the industry profitability ,5,6.The addition of chemical preservatives can minimize or prevent lipid peroxidation, delaying the formation of toxic compounds and increasing the shelf life of foods . CurrentAs an alternative to synthetic compounds, natural substances of plant origin such as essential oils have been widely applied in the meat industry as safe alternatives and approved for use in foods by the Food and Drug Administration  that exhCopaifera spp., CEO) is widely distributed in northern South America and produced in the Brazilian Amazon rainforest [Among the many oils approved for use, copaiba essential oil  is also one of the oils widely used as food preservatives, having different active phenolic compounds, such as thymol and carvacrol, providing good antioxidant activity [Oregano essential oil , purchased directly in the slaughterhouse 24 hours after animals’ slaughter. Muscles were refrigerated and transported to the laboratory where the excess fat was removed. The beef was minced  mixed, and compressed manually with 10 g of NaCl per kg of meat. Patties (40 g each) were produced in molds 10 cm in diameter and 1 cm in height, specific for the preparation of beef patties, and randomly distributed to the five different treatments tested with four replicates of four samples per each day of analyses per treatment.Tested treatments were: a control without antioxidants (CON); beef patties containing synthetic additive butylated hydroxytoluene ; beef patties with 0.05% of copaiba essential oil (CEO), beef patties with 0.05% of oregano essential oil (OEO) and beef patties with an essential oil blend: 0.025% copaiba essential oil + blend of 0.025% oregano essential oil (BEO). Afterward, the patties were placed on polystyrene trays coated with oxygen-permeable polyethylene film. The trays were exposed in a refrigerated showcase (4 ° C), under fluorescent light , simulating conditions typical of the Brazilian market.® , and the concentration used was based on previous research [The essential oils tested were obtained from Ferquimaresearch ,12. The research , and forresearch . For BHTresearch .The samples for lipid oxidation (TBARS), antioxidant capacity , pH, color properties, cooking losses, and texture were evaluated at  to define the basic meat composition.The moisture, protein, lipid, and ash were measured in raw beef patties, according to the methods established by the Association of Official Analytical Chemists . Crude pBurger bioactive compounds extract. Beef patties extracts (1:1 w/v with methanol) were obtained using an ultra-Turrax homogenizer, followed by centrifugation and filtration. The extracts were analyzed for antioxidant activity by measuring the 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic) (ABTS) radical scavenging activities, and the total phenolic content (TPC) according to Monteschio et al. 2021 .ABTS radical scavenging assay and Folin–Ciocalteu for total phenolic content (TPC). Beef samples were mixed with methanol (1:1 w/v with methanol) to obtain beef patties extracts for antioxidant analysis. Five grams of hamburger samples were ground with methanol using an Ultra-Turrax homogenizer, followed by centrifugation and filtration . The extracts were analyzed for antioxidant activity by measuring the 2,2’-azino-bis(3-ethylbenzothiazoline- 6-sulfonic) (ABTS) radical scavenging activities, as well as the total phenolic content (TPC).For the ABTS solution, 7mM ABTS (5 mL) was mixed with 140 mM potassium persulfate (88 μL), and the mixture was incubated in the dark at 25 °C for 16 h. The ABTS-activated radical was diluted with ethanol to an absorbance of 0.70 ± 0.02 at 734 nm. The radical scavenging activity (%) was also measured at 734 nm.Beef patties extract (40 μL) was mixed with ABTS+• solution (1960 μL), and the absorbance was recorded after 6 min. The ABTS radical scavenging activity (%) was calculated as 1- (Asample t0/Asample t) × 100, where Asample t0 is the absorbance of the sample at time zero and A sample t is the absorbance of the sample at 6 min.2CO3 (28 g/L). The solutions were then incubated in the dark at 25 °C for 30 min, and the absorbance was measured at 725 nm using a spectrophotometer , and the results were expressed as milligrams of gallic acid equivalents (GAE) per gram of sample. The ABTS radical scavenging activity (%) and TPC were calculated according to Monteschio et al. 2021 [For TPC, an aliquot of each extract 125 mL) was mixed with 125 mL of Folin–Ciocalteu reagent (1:1 v/v deionized water) and 2250 mL Na mL was mThe malondialdehyde (MDA) content in beef patties was measured by the thiobarbituric acid-reactive substances (TBARS) assay with modifications . The samL*a*b* system, with a 10° view angle, obtaining lightness (L*), redness (a*), and yellowness (b*), Chroma (C*), and hue (h°) were calculated as follows:The CIELab color parameters were recorded by using a Minolta CR-400 chroma meter (Japan) under D65 illumination, aperture of 8 mm, and a closed cone set on the The pH was measured using a pH meter with a Testo 205/206 penetration probe. The pH meter was calibrated at 20 °C using standard pH 4.0 and 7.0 buffers before use.Beef patties were weighed and wrapped in aluminum foil. Each sample was cooked in a pre-heated grill  at 200 °C until the internal temperature reached 72 °C, which was monitored using an Incoterm internal thermocouple . The beef patties were removed from the heat and left at ambient temperature to cool to 25 °C. Each burger was weighed and the cooking losses were calculated each day as a percentage relative to the initial weight.2, and cross-section and texture were analyzed using a texture analyzer installed with a Warner–Bratzler blade and set with a 50-kg load cell and an operating speed of 2 mm/s, registering the maximum shear force (kg).After recording the cooked weight, beef patties were cut into rectangular pieces of 1 cmThe sensory analysis study was approved by the Research Ethics Committee involving human beings at the Federal University of Roraima (protocol numbers: 23282819.9.0000.5302), Boa Vista, State of Roraima, Brazil, Before tasting each sample, each participant completed a written consent form, In a room adequate to perform sensory analyses , 124 consumers participated in the sensory test; they were divided into 13 sessions with 10 participants per session, some consumers did not complete the test and were removed from the study. Consumers were randomly selected with quotas of gender (42% men and 58% women) and age (from 18 to 70 years) according to the Brazilian national profile.Each consumer assessed five different samples , after 1 day of display. A randomized design was used to serve the meat, avoiding carry-over and order effects. Beef patties were individually packed with aluminum foil and cooked on a pre-heated grill at 200 °C until the internal temperature reached 72 °C. Each beef patty was cut into four triangles and kept warm (50 °C) until consumer evaluation (around 10 min after cooking). Consumers scored acceptability of the following attributes of patties: odor, tenderness, flavor, and overall acceptability. Ratings were scored using a structured hedonic nine-point scale , without the middle level.The microstructure analysis was performed according to Vital et al. 2016  with modAll measurements were carried out in triplicate and data were analyzed as a factorial experiment in a completely randomized design using SPSS (version 27.0)  for Windows. The factors included were five treatments  and four display times . A general linear model ANOVA assessed treatments and displays, which were considered fixed factors, and their interactions were also considered. For the consumer, acceptability treatment was the only fixed effect evaluated and the consumer was considered a random effect, and session a blocking effect.When differences between means were statistically significant, Tukey’s test was performed, with statistical significance set at (P < 0.050).The composition of beef patties  was simiThe antioxidant capacity (ABTS and TPC) was affected by the treatments and display P <0.05, . The ABTThere was a significant decrease (P <0.05) during display until day 7, with an increase on day 14 and a further decrease until the last day of display (day 21). An interaction between the effects (treatments and storage time) was observed, as is shown in From the beginning of display (day 1) until day 14, the CON treatment showed greater antioxidant activity, decreasing on the last day of display (Day 21). BHT and CEO showed the same behavior of antioxidant capacity for display days, with higher values on day 14 between the other days . Until day 7, the OEO and BEO treatments had lower antioxidant capacities and higher values on days 14 and 21 of the display.In the TPC assay, activities similar to ABTS were observed for treatments and display days (P <0.05), showing greater antioxidant activity for OEO and BEO, lower CON, intermediate BHT, and the CEO not differing from CON and BHT. During display, the same behavior of ABTS was observed, showing a decrease in antioxidant activity until day 7, with an increase on day 14 and a further decrease until the last day of display (day 21). An interaction between the effects (treatments and display) was also observed for the CPT, as shown in Different lower-case letters indicate significant differences between treatments in the same day of display (P < 0.050). Different upper case letters indicate significant differences in the same treatment along display (P < 0.050). CON–beef patties, without antioxidants; BHT—beef patties containing hydroxytolueneobutylated; CEO-0.05% beef patties with 0.05% of Copaiba essential oil; OEO- burger with 0.05% of Oregano essential oil; BEO—blend of essential oil .Lipid oxidation measured by MDA production was affected by treatments and display time (P <0.05). The CON showed a higher MDA value than OEO and BEO and intermediate values were found for the BHT and CEO treatments. In addition, the lipid oxidation of beef patties increased significantly (P <0.05) during the cold display. When the effects of both factors were assessed, an interaction was observed between them , as showLipid oxidation was higher for the CON and BHT treatments compared to the other treatments on day 7 of display. The CON treatment showed a higher MDA value than the OEO and BEO, the BHT showed an intermediate value and the CEO treatment did not differ from the CON and BHT on day14 of display. On the last day of display (day 21), the CON presented a higher MDA value than the OEO and BEO, intermediate for the CEO and the BHT did not differ from the CEO, OEO, and BEO treatments.Different lower-case letters indicate significant differences between treatments in the same day of display (P < 0.050). Different upper case letters indicate significant differences in the same treatment along display (P < 0.050). CON–beef patties, without antioxidants; BHT—beef patties containing hydroxytolueneobutylated; CEO-0.05% beef patties with 0.05% of Copaiba essential oil; OEO- burger with 0.05% of Oregano essential oil; BEO—blend of essential oil .The color values  associated with the treatments and display are shown in . There wChroma (C*) showed a significant difference (P <0.05) between treatments, with a higher value for beef patties with the addition of essential oil of oregano, intermediate for the treatment BEO and control, and lower value for the treatment BHT and OEO.Differences were observed for display days (P <0.05) for the values of a*, b*, and C*. Higher values of a* (redness) were observed at the beginning of display (day 1) and lower on the last day (day 21), intermediates found at days 7 and 14 of display, and day 14 did not differ from days 7 and 21 of display.At the beginning of display (day 1), the values of b* (yellowness) were higher (P <0.05) and remained constant with the increase in display days. The same behavior was observed for the chroma values (C*) during the days of display of beef patties. No interactions were observed between treatments and display for color parameters.The pH showed no differences (P >0.05) between treatments; however significant changes (P <0.05) occurred in display, with higher values observed at 21 days compared to shorter display periods (1 and 7 days) and intermediate values at 14 days of display .The treatments and days of display affected cooking losses (P <0.05). They were significantly lower at the end of display than on day 1, and the BEO treatment had the lowest cooking loss value among the other treatments . No interaction between treatments and display was observed for pH, while an interaction was observed for cooking loss, as shown in Differences in the cooking loss were observed for the BEO treatment (P <0.05), which showed lower losses between the treatments evaluated at 7 days of display and for the BHT treatment at day 14 of display, which showed greater losses compared to the BEO, being that the CON and OEC treatment did not differ from BHT and the OEO and OEO treatment did not differ from CON, OEC, and BEO.On display days, the differences found (P <0.05) showed the same behavior for the CON and OEO treatments with greater cooking losses with shorter display periods (days 1 and 7) compared to the other days. The BHT and CEO treatments showed greater losses on 1 and 7 days of display, were intermediate at 14 days, and lower at 21 days of display. The BEO treatment showed higher losses on the initial display day (day 1), was intermediate at 7 days, and had lower losses on the final display periods (days 14 and 21).Regarding the tenderness of beef patties, differences (P <0.05) were observed between treatments and display periods. Softness was lower for the CON treatment than the BEO treatment, intermediate for the CEO and OEO treatments, and the BHT treatment did not differ from the CON, CEO, and OEO treatments. The shear force was greater until the seventh day of display, and from day14 onwards, there was a progressive reduction in values, indicating softer meat after a longer display (P <0.05). No interactions were observed between treatments and the display on the tenderness of beef patties .Different lower-case letters indicate significant differences between treatments in the same day of display (P < 0.050). Different upper case letters indicate significant differences in the same treatment along display (P < 0.050). CON–beef patties, without antioxidants; BHT—beef patties containing hydroxytolueneobutylated; CEO-0.05% beef patties with 0.05% of Copaiba essential oil; OEO- burger with 0.05% of Oregano essential oil; BEO—blend of essential oil .For all parameters evaluated in sensory acceptability , differences (P <0.05) were observed between treatments .The odor obtained a higher score (P <0.05) for BEO and OEO compared to CON and intermediate values for the BHT and CEO treatments. For beef patties flavor acceptability, the highest scores (P <0.05) were found in the BEO treatment and lowest in the OEO treatment, being intermediate for the CON and CEO treatments, and the BHT treatment did not differ from the CON, CEO, and BEO treatments. The tenderness of beef patties from the CON treatment had the lowest score (P <0.05) significantly different from the other treatments . The highest score for overall acceptability (P <0.05) was obtained for the BEO treatment, the lowest for the OEO treatment, and the intermediate scores were for the CON, BHT, and CEO treatments.The microstructure was examined to verify the structural properties of the processed food and the organization of the droplets of the essential oil present in the beef patties.Microstructures of meat and essential oil are shown in . Beef paRosmarinus officinalis) as an antioxidant.The percentage of fat in the patties of the current study is similar to those (which ranged from 11.84% to 13.05%) described by Borella et al. 2019  for a miCarvalho et al. 2019  observedPhenolic compounds from plants are responsible for their antioxidant properties ; those aFor the antioxidant activities evaluated by ABTS and TPC tests, beef patties containing OEO and BEO showed greater antioxidant activity compared to the CON treatment. The fact can be explained by the effectiveness of antioxidants, mainly natural antioxidants, and corroborate the work of other authors ,26,36, wIn addition, the antioxidant capacity of oregano essential oil (OEO) and the mixture of essential oils (BEO) remained high until day 14 of display by the two methods of antioxidant capacity (DPPH and TPC), indicating that the polyphenolic compounds’ assets of these oils kept their activity throughout the display. The same increase in antioxidant capacity (DPPH) during display days was observed by Cantú-Valdéz et al. 2020  when evaChanges in the oxidative state during display affect the shelf life of meat and its by-products , includiThe use of essential oils  was more effective than the CON and BHT treatments in delaying lipid oxidation after 7 days of display. This is due to the antioxidant activity of phytochemicals in the essential oil associated with the hydroxyl group attached to the aromatic ring, which is capable of donating hydrogen atoms with electrons and neutralizing free radicals ,41, with−1 according to Campo et al. 2006 [During the display period, TBARS values increased, which would be expected, as lipid oxidation is an autocatalytic reaction and, therefore, the oxidation rate increases as the reaction proceeds . Despiteal. 2006 , showingal. 2006  and copaal. 2006 .Ocimum basilicum L.) essential oil in beef burgers [Similar results were found by other authors when they incorporated sage in beef burgers during prolonged chilled storage , as well burgers  and copa burgers . The incRedness a* is an important component in the visual appeal of meat by consumers and is related to the oxygenation of myoglobin, with high values of a* are associated with the presence of oxymyoglobin, while the reduction in a* values is related to the formation of metmyoglobin ,45.In the current experiment, it was expected that redness (a*) would be progressively reduced during display, as has been previously reported in other studies ,46. ThisThe meat product with low chroma values is considered pale and clear , which m3 and amines, alkaline compounds [Zataria multiflora essential oil have been reported by Amiri et al. 2019 [During display, the pH values increased, which reflects the degree of deterioration of the meat through the breakdown of proteins for the production of free amino acids leading to the formation of NHompounds ,51, leadompounds . Previoual. 2019 ,38.The loss from cooking food is an important quality characteristic, associated with the meat yield at the moment of consumption, which can be influenced by the water retention capacity in the meat structures, providing better quality attributes to the food . Beef paThe increase in cooking losses during display is one of the main undesirable changes in quality during shelf life, which was observed in this trial and results agree with other research results with essential oil blends on the meat of crossbred heifers  and esseThe greater tenderness of the beef patties from the BEO treatment may be associated with water loss and oxidative processes during display days, and the presence of water in the product system provides a more succulent and softer product . This faThe acceptability of food by consumers establishes the future direction of the market and influences the applications of new technologies in the food processing industry ,58. In tCinnamomum zeylanicum) and marjoram (Origanum majorana L.) essential oils of Wagyu hamburgers, which despite CON showing a significantly greater shear strength than all other samples, consumers did not notice these variations.Regarding tenderness, it is possible that consumers detected a significantly higher shear force than presented in the CON treatment and reported a difference in the acceptability of beef patties from this treatment. Nascimento et al. 2020 , when evOverall acceptability usually is highly correlated with other sensorial attributes such as flavor or tenderness . In the Thymus vulgaris) in the conservation of meat products similar to hamburgers. This verified the correct and homogeneous distribution of essential oils in the beef patties’ structure.Similar characteristics were observed in previous studies with edible coatings containing essential oils of rosemary and oregano in the characteristics of beef, showing the droplets of essential oils throughout the meat . In addiThe inclusion of oregano essential oil and the blend of oregano and copaiba essential oil increased antioxidant activity and decreased lipid oxidation compared to the control treatment , with higher antioxidant activity and lower lipid oxidation of these treatments for up to 21 days of display. Oregano essential oil increased chroma values and provided reduced values during exposure days, making the meat more attractive for a longer display period. The blend of essential oils (copaiba at 0.025% and oregano at 0.025%) in the hamburgers provided a lower cooking loss than the other treatments, with lower losses in the final display periods (days 14 and 21).The mixture of essential oils also provided greater tenderness to the burgers compared to the control treatment, causing a progressive reduction in values from day 14 onwards, indicating softer meat after longer storage, in addition to improving the sensory attributes of the burgers. The essential oils studied increased the shelf life of the product, proving to be a potential option for the food industry to use natural ingredients instead of synthetic compounds to preserve the quality of processed foods. The combination of both essential oils (copaiba at 0.025% and oregano at 0.025%) would be a good alternative for this replacement of synthetic additives such as BHT because at low concentrations, they had a positive effect on shelf life in addition to increasing sensory properties evaluated by consumers.S1 File(PDF)Click here for additional data file.


Mass loss was measured after each incubation time, and digestion kinetic parameters were estimated. Thermogravimetric, differential scanning calorimetry, and intrinsic viscosity analyses were conducted on incubated samples. Generally, across treatments, mass loss was significant by 96 h with a minimum increase of 0.25% compared to 0 h but did not change thereafter. Degradation kinetics demonstrated that polymer treatments were still in the exponential degradation phase at 240 h with a maximum disappearance rate of 0.0031 %/h. Melting temperature increased, onset thermal degradation temperature decreased, and intrinsic viscosity decreased with incubation time, indicating structural changes to the polymers. Based on these preliminary findings, the first stage of degradation occurs within 24 h and PHA degrades slowly. However, further ruminal degradation studies of biodegradable polymers are warranted to elucidate maximum degradation and its characteristics.Using polyhydroxyalkanoate (PHA) materials for ruminal boluses could allow for longer sustained release of drugs and hormones that would reduce administration time and unneeded animal discomfort caused by continuous administration. The objective of this study was to determine ruminal degradability and kinetics of biodegradable polymers and blends. A proprietary PHA-based polymer, poly(butylene succinate- CHClmT. The PBSA:PHA polymers had two degradation curves after thermogravimetric analysis was conducted; the first point of degradation was determined to be the PHA component and the second point of degradation was determined to be the PBSA component. Polymers dT and mT were analyzed with fixed effects of treatment, time, and the interaction and random residual error. All data were evaluated with an alpha value of 0.05.Analyses were conducted separately to compare polymer compositions in nurdle form and to compare the PBSA:PHA melt blend in nurdle and filament formation. All statistical analyses were completed using the MIXED procedure in SAS. Polymer mass loss was analyzed with fixed effects of bag type, treatment, time, and interactions and random residual error. Disappearance kinetics parameters were analyzed with the fixed effects of bag type, treatment, the interaction, and random residual error. Some polymer treatments had two peaks within the first heating scan during differential scanning calorimetry due to multiple components; the initial peak was used for multiple comparisons of p = 0.07; p = 0.95). Mass loss increased over time for each polymer treatment as expected, though significant mass loss was generally not achieved until 96 h of incubation and did not change thereafter (Bag type did not affect the mass loss of polymer nurdles nor the PBSA:PHA blend in nurdle and filament formation (ereafter . By 96 hp < 0.0001). On average after 240 h of incubation, PHA had 0.53 ± 0.13%, PBSA had 0.49 ± 0.16%, Blend had 0.82 ± 0.20%, and Filament had 1.68 ± 0.33% total mass loss.Our polymer treatments did not differ from one another in nurdle form. Though, we observed a two-fold increase in mass loss in the Filament compared to the Blend (p = 0.02). The A fraction did not differ among polymers in nurdle composition (p = 0.50). Rates of degradation did not differ among the polymers in nurdle composition or among the PBSA:PHA blend in nurdle and filament composition . The maximum rate of disappearance achieved was 0.0031 %/h by the Blend, substantially less than what was achieved by the controls.Ruminal digestibility kinetics parameters for polymer treatments and forage controls are shown in Predicted digestibility curves of polymers using the parameters in mT) from the first heating scan of polymers after incubation for 0, 24, 96, and 240 h in rumen fluid are shown in The first melting temperature . The dT associated with the PHA component within both the Blend and Filament polymers significantly decreased from 0 to 24 h by 119.50 ± 1.03 °C and 114.00 ± 1.60 °C, respectively. For the PBSA components, the dT for PBSA significantly decreased from 0, 24, to 96 h consecutively . The dT associated with the PBSA component within both the Blend and Filament significantly decreased from 0 to 24 h by 137.00 ± 1.92 °C and 135.35 ± 0.81 °C, respectively.Both the Blend and Filament presented two onset thermal degradation temperatures after 0, 24, 96, and 240 h as well. For assessment, these two temperatures were separated into the PHA component A and PBSThe intrinsic viscosities of PHA, PBSA, and Blend after fermentation in rumen fluid for 0, 24, and 240 h in F57 bags are shown in Repetitive administration of oral drugs due to their release times induces stress to the animal by additional handling. Other potential materials should be identified and studied to replace current slow-release bolus carriers that last only three to five months . PolyhydII Incubators. Mass loss of residues was determined and used to estimate ruminal digestive kinetics. Residues additionally underwent TGA and DSC to evaluate structural alterations to materials.This is the first study to specifically evaluate the ability of a ruminal environment to breakdown a PHA-based polymer and PBSA. Proprietary PHA-based polymer nurdle, PBSA nurdle, and a melt blend of the two in nurdle and filament form underwent fermentation for up to 10 days in rumen fluid in DaisyPrevious literature has evaluated growth and health of monogastrics supplemented with PHA-based materials that indicated their degradation in the gastrointestinal tract. Specifically, aquaculture species supplemented with PHB, a particular PHA material composed of hydroxybutyrate monomers, had increased growth, decreased pH, and enrichment of the gut microbial community that indicated their degradation ,26,27. HAlthough abiotic and biotic factors differ between these studies and the present one, the literature supports trends observed in our study in which multi-polymer blends degrade more readily than individual polymers. In a mass loss study over 35 days where PHB and copolymers were buried in ground soil at 28 °C, PHB homopolymer films had the slowest rate of degradation at 0.93 mg/d and achieved 10% degradation by 7 days whereas the fastest degrading copolymer with 4-hydroxybutyrate had a 75% increase in degradation rate at 1.63 mg/d and achieved 30% mass loss at 7 days [Polymer formation may have a greater effect on degradation than composition. In a mass loss study in coastal waters over 160 days, films of PHB and a copolymer with 3-hydroxyvalerate had 58% and 54% degradation whereas their pellet counterparts had 38% and 13% degradation, respectively . AdditioNo studies have evaluated a PBSA:PHA blend for mass loss and degradation kinetics due to biodegradation, and generally studies with PBSA are limited. In one study, dog-bone shaped specimens of polybutylene succinate (PBS) and PBSA underwent biodegradation in soil and compost for 24 weeks . SamplesdT and mT within only 24 h and the minimal mass loss.Trends seen within the present study after only 240 h support studies at longer time frames to capture additional polymer degradation kinetics in the rumen. Some studies have described biodegradable polymer degradation to have a lag phase requiring microbial films to develop on the surface of polymers followed by a two-phase curve of mass loss in which rapid degradation is preceded by slower degradation for more than 10 d ,31. At omT indicates the shortening of polymer chains and increasing crystallinity of polymers in this study likely caused by preferential depolymerase degradation of amorphous material [dT indicates a reduction in the thermal stability of the polymers, which may be explained by selective depolymerization of lower molecular weight polymer molecules in the amorphous region by 24 h [Melting temperature and onset thermal degradation temperature indicate structural integrity of polymers in the face of thermal challenge. Evaluating changes in these values over time indicates structural changes in polymers due to degradation. The observed increase in material ,33. Simi by 24 h . The trePrevious studies support these findings in that PHA-based materials and blends thereof of various compositions show decreased molecular weight, increased melting temperatures, and decreased onset thermal degradation temperatures after environmental degradation by microbes ,35. StudStaphylococcus sp. from rumen fluid collected from one mature cow were capable of degrading PHB as indicated by the development of clearing zones after plating serial dilutions of rumen fluid on PHB overlay plates [We propose that the slow, linear trend of degradation observed in this study is due to the beginnings of material surface erosion by bacteria . While ay plates . Our stuy plates ,39. ThusII Incubator in vitro system is a widely accepted and reliable method for determining true ruminal digestibility of feedstuffs [Concurrently, in vivo studies must be conducted under these conditions to establish differences between in vitro and in vivo systems. It is possible that results seen in this study are undermined by the limitations of an in vitro system. Although the Daisyedstuffs , it is aBased on these preliminary findings, PHA-based and PBSA materials are degradable in the rumen and may be a viable option for slow-release ruminal boluses. Within 24 h of incubation, polymer chains were likely cleaved via abiotic and biotic factors to alter mechanical structures of polymers as supported by thermochemical analyses. However, though polymer chains were shortening, mass loss was unsubstantial by 240 h of incubation, and the proprietary PHA-based polymer had the slowest rate of degradation. Further ruminal degradation studies of biodegradable polymers for a longer duration are warranted to elucidate full degradation kinetics and the relative contributions of water-induced and enzymatic hydrolysis to degradation. These studies should be conducted with a range of ruminant hosts fed different diets and in different housing conditions to determine the general ability of all ruminants to host depolymerase-producing microbes.


Leptomeningeal metastasis (LM) refers to the dissemination of malignant cells in the subarachnoid space, pia, and arachnoid mater and is a severe condition associated with metastatic solid tumors. The most common solid tumor that develops into LM is lung cancer and the incidence increased in patients with advanced non‐small‐cell lung cancer (NSCLC) with targetable mutations. However, tissue biopsy of LM is inaccessible, leading to the paucity of genomic profiles of LM to guide targeted treatments and explore biological mechanisms. In recent years, liquid biopsy is considered a minimally invasive and dynamic method to trace the genomic alterations of cancer cells and some studies started to perform sequencing of cerebrospinal fluid (CSF) in patients with LM to reveal the targeted mutations and genomic profiles. In this review, we focused on studies performed sequencing of CSF in NSCLC patients with LM and summarized the sequencing results and their commonality. As the only way to reveal the genomic landscapes of LM, our review provided evidence that sequencing of CSF is a promising management method in LM patients to dynamically guide target therapy and monitor intracranial tumor response. Furthermore, it reveals a unique genomic profile of LM including driver genes, drug‐resistant mutations, and a number of copy number variations. Sequencing of CSF in LM patients seems to provide more comprehensive genomic information than we expected and the biological significance behind the genomic alternations needs further study. The frequency of CNVs and alternations of the reported genes. Information regarding the following characteristics was extracted: author, year, institution, topic, number of patients, treatment history of the tyrosine kinase inhibitors (TKI), sequencing platform and method, sequencing sample, paired plasma, and paired tissue. Alternated genes were analyzed per study and on a per patient basis. Alternations, including single nucleotide variations (SNVs), insertion–deletion mutations, frameshift mutations, and copy number variations (CNVs) of the reported genes, were obtained. When the mutations were not included in the above types, the data were excluded. The results of multiple sampling at the same time and different sequencing samples from one patient were considered as a whole, which were analyzed together to represent the genomic alterations of the patient. The detection rates and the concordance rates were recorded if they were reported in the original studies. If the rates were not reported explicitly, they were presented in the form of a ratio through manual extraction. We compared the alternated genes with the list of Cancer Gene Census (CGC) and pan‐lung cancer data in The Cancer Genome Atlas (TCGA) database. CGC is an ongoing effort to catalog alternated genes that have been causally implicated in cancer and explain how the dysfunction of these genes drives cancer. The data can be obtained from the websites directly. Representative cases of CSF sequencing in guiding treatment were also summarized.2.3Only descriptive statistics were used. The frequency was defined as the number of patients with detected mutations divided by the total number of patients.33.1EGFR) mutations and the otherEGFR subtypes, so the information of the two studies was merged and duplicated patients were removed. Patients with other types of primary tumors were also reported in three studiesEGFR mutations in the primary tumor and one study included patients with ALK receptor tyrosine kinase (ALK) fusions. The history of TKI treatments was illustrated in 238 patients and 192 of them were TKI exposed. Three hundred and twenty‐two patients had sequencing results from paired plasma, and 212 patients had sequencing results from paired biopsy tissues. Eight studies performed sequencing through different cancer‐related gene panels, and nine studies sequenced only predesignated genes including EGFR, KRAS proto‐oncogene (KRAS), B‐Raf proto‐oncogene (BRAF), erb‐b2 receptor tyrosine kinase 2 (ERBB2), and MET proto‐oncogene (MET). Cell‐free DNA (cfDNA) in CSF was sequenced in 14 studies, cells in CSF were sequenced in four studies, and circulating tumor cells (CTC) in CSF were sequenced in three studies. The next‐generation sequencing (NGS) and the droplet digital PCR (ddPCR) were the most commonly used methods and were used in 10 and five studies, respectively. Most studies used various Illumina platforms for NGS and BioRad QX20TM system for ddPCR  Table . Four st3.33.3.1EGFR mutations. Fourteen studies reported subtypes of EGFR mutations. The most frequent subtypes were classical sensitive exon21 L858R and exon19 del mutations, which occurred in 158/377 and 137/351 patients, and accounted for 90% (288/320) of all the activating EGFR mutations, which was similar to the prevalence in NSCLC.A total of 525 patients in 15 studies performed sequencing of CSF and 377 of them had detected The most common secondary mutation T790M was detected in 42/377 patients. Of the 42 patients, the treatment history of 34 patients were known, and 67% (23/34) patients had received TKI treatment. C797S mutations were detected in seven patients and all of them were exposed to the third generation of TKI, indicating the secondary drug‐resistant mutations in CSF were also relevant to TKI treatment Figure .3.3.2EGFR mutations, other driver genes and potential drug‐resistant mutations were also revealed in the genomic profile, including MET, ALK, ERBB2, KRAS, ROS proto‐oncogene 1 (ROS1), BRAF, ret proto‐oncogene (RET), and neurotrophic receptor tyrosine kinase 1 (NTRK1). Overall, 155 mutations of at least one of the above genes were detected in 201 patients from 11 studies (Table MET amplification (37/155). ALK was the following mutated gene and was found in 33 patients, but this result needs to be interpreted cautiously because all the enrolled patients in the study of Zheng et al were ALK‐mutated.NTRK1 (23/155), ERBB2 (18/155), and BRAF (14/155). ERBB2 amplification was found in six patients. NTRK fusions and BRAF V600E are the rare driver mutations of NSCLC, while in the sequencing results of CSF, nearly all types of NTRK1 and BRAF mutations were copy number amplifications .In addition to es Table . The mosTP53) frequently occurred in CSF, with a detection rate of around 50%.TP53 loss of heterozygosity (LOH) in CSF than in plasma .TP53 LOH or mutations, higher frequencies of genomic alternations in CNVs, EGFR, MET, and RB transcriptional corepressor 1 (RB1) were discovered. This profile was unique in CSF and might be related to resistance and the emergence of LM.Tumor protein p53 , MET, RB1, cyclin‐dependent kinase inhibitor 2A (CDKN2A), NTRK1, NK2 homeobox 1 (NKX2‐1), cyclin‐dependent kinase 4 (CDK4), fibroblast growth factor receptor 1 (FGFR1), RPTOR independent companion of MTOR complex 2 (RICTOR), BRAF, SMAD family member 4 (SMAD4), cyclin D1 (CCND1), fibroblast growth factor 3 (FGF3), cyclin‐dependent kinase 6 (CDK6), and POM121 transmembrane nucleoporin like 12 (POM121L12) , and fibroblast growth factor 19 (FGF19), and the cyclin family related genes such as CCND1, CDK4, and CDK6 showed only copy number amplifications. Since somatic CNVs affect a larger fraction of the cancer genome than any other type of alterations does, the profile of CNVs in CSF of LM patients warrants further study.Even though detecting CNVs is more challenging than SNVs due to technology issues,) Figure . Most of3.3.4EGFR, the most frequently mutated gene was TP53. Ninety‐four genes mutated in two or more patients and 25 genes mutated in more than 10 patients , olfactory receptor family 4 subfamily A member 15 (OR4A15), casein kinase 2 alpha 1 (CSNK2A1), olfactory receptor family six subfamily F member 1 (OR6F1), BMP/retinoic acid inducible neural specific 3 (BRINP3), and thrombospondin type 1 domain containing 7A (THSD7A). These genes may promote oncogenic transformation and indicate a role in LM but need extensive available evidence.In order to dig more information on genomic alternations in LM patients, we summarized the sequencing results from the studies which performed sequencing in gene panels instead of sequencing in several predefined genes. In summary, seven studies reported 120 genes with variations. Except for s Figure . Compare3.4Liquid biopsy is considered a dynamic method to trace genomic alternations and guide targeted treatment. Here, we summarized 14 representative cases Table  and mostEGFR mutations in CSF dropped, while after that, the subsequent change was the increase of T790M level in plasma, which occurred earlier than that in CSF.In the studies, a higher detection rate of T790M was found in extracranial lesions and plasma than in CSF.4In this review, we summarize the existing literature on the topic of sequencing of CSF in NSCLC patients with LM. We found sequencing of CSF performed well and revealed a unique genomic profile. We also summarized the cases that used CSF sequencing to guide treatments and suggested that it was better to combine the sequencing of plasma together.Plasma and CSF have been widely compared as samples for liquid biopsy in LM patients. Here, we found CSF performed better than plasma. The phenomenon attributes to some possible reasons. First, more interference of background gene fragments from normal blood cells existed in plasma than in CSF. Second, LM tumor cells are bathed by CSF, so the DNA fragments are released into CSF directly. However, the cells and cfDNA in CSF need to be absorbed through the arachnoid villi into the superior sagittal sinus every 4 h and then get into the systemic circulation. Third, the interference from extracranial lesions could be prevented by the blood–brain barrier and blood–CSF barrier.OR4A15 and OR6F1 belong to the olfactory receptor family, which can pair with the molecular secreted from neighboring neurons and is also highly expressed in different cancer tissues.NAV3 and BRINP3 are predominantly expressed in the nervous system and involved in neuron proliferation and regeneration. It is important to explore whether these mutations are interference of background genes in CSF or an implication of connections between LM tumor cells and neurons.Tumor metastasis is an evolutionary process, and the acquired genomic alterations of metastatic carcinoma during disease evolution induce treatment resistance and accelerate dissemination,TP53 LOH uniquely occurred in CSF rather than plasma, accompanied by more genomic mutations and CNVs. Aneuploidy, another trigger of genomic instability, is also widely detected in patients in CSF from LM patients.MET amplification, which was found associated with treatment resistance and brain metastasis in NSCLC. Most genes with CNVs also mutated, emphasizing the importance of genomic alterations in these genes and their relevant pathways. While the genes belonging to the fibroblast growth factor family and cyclin family showed a specific amplification, which indicated an enhanced function in the cell cycle and survival activities in LM.A common characteristic across studies is the number of CNVs detected in CSF, although the large fragment alternations were difficult to recognize during sequencing. We speculated that indicated the potential existence of genomic instability and might be the underlying cause of the difference in LM. EGFR changes in CSF along with treatments. Besides, mutations in CSF might provide clues to different treatment responses, such as T790M in TKI‐naive patients and bypass activations including MET amplification and ERBB2 amplification.EGFR subtypes in CSF and plasma were not simultaneous and T790M occurred earlier in plasma than in CSF after first‐ or second‐generation TKI therapy. The phenomenon may attribute to the followings: (1) the intracranial lesions were not exposed to the concentration of first‐ or second‐generation TKI that was enough to kill the cancer cells due to the obstruction of the blood–CSF barrier and (2) the blood–brain barrier and blood–CSF barrier also obstruct T790M mutations into CSF from systemic circulation. Furthermore, the difference in genomic alternations between CSF and plasma can be affected by clinical features, radiotherapy history, or TKI treatment of patients,In the representative cases, most patients improved in both brain imaging and CSF cytology after receiving TKI. Dynamic monitoring revealed mutation subtypes and AFs of TP53 LOH, a number of CNVs, and more mutated genes than plasma, indicating the potential existence of genomic instability in LM tumors. The sequencing of CSF in LM patients seems to provide more comprehensive information than we expected and the biological significance behind the genomic alternations needs further study.In conclusion, like tissue biopsy, sequencing of CSF in LM patients can guide targeted treatments and reveal resistance mutations and also provides a dynamic method to monitor intracranial tumor response. Furthermore, it reveals a unique genomic profile including mutations in driver genes and drug‐resistant genes, Tianqi Gao: Have made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; been involved in drafting the manuscript or revising it critically for important intellectual content; agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Fengxi Chen: have made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; been involved in drafting the manuscript or revising it critically for important intellectual content. Man Li: have made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; Given final approval of the version to be published. Each author should have participated sufficiently in the work to take public responsibility for appropriate portions of the content; Agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.No potential conflict of interest relevant to this article was reported.Our study is secondary research, and all data presented were from published studies. So, there is no need for ethical approval.Appendix S1Click here for additional data file.Table S1Click here for additional data file.


In situ data from the Water Quality Portal (WQP) were used for validation. The WQP serves data collected by state, federal, and tribal groups. Discrete in situ temperature data included measurements at 11,910 U.S. lakes and reservoirs from 1980 through 2015. Landsat temperature measurements could include 170,240 lakes and reservoirs once an operational product is achieved. The Landsat-derived temperature mean absolute error was 1.34°C in lake pixels >180 m from land, 4.89°C at the land-water boundary, and 1.11°C in estuaries based on comparison against discrete surface in situ measurements. This is the first study to quantify Landsat resolvable U.S. lakes and reservoirs, and large-scale validation of an operational satellite provisional temperature climate data record algorithm. Due to the high performance of open water pixels, Landsat satellite data may supplement traditional in situ sampling by providing data for most U.S. lakes, reservoirs, and estuaries over consistent seasonal intervals (even with cloud cover) for an extended period of record of more than 35 years.The United States Harmful Algal Bloom and Hypoxia Research Control Act of 2014 identified the need for forecasting and monitoring harmful algal blooms (HAB) in lakes, reservoirs, and estuaries across the nation. Temperature is a driver in HAB forecasting models that affects both HAB growth rates and toxin production. Therefore, temperature data derived from the U.S. Geological Survey Landsat 5 Thematic Mapper and Landsat 7 Enhanced Thematic Mapper Plus thermal band products were validated across 35 lakes and reservoirs, and 24 estuaries.  Several previous studies have validated the Landsat thermal bands, though most studies investigated either single or a select few waterbodies. Larger waterbodies studied include the Michigan and Ontario Great Lakes, Lake Tahoe, Lake Constance in Germany, and Chesapeake Bay.  Ontario . Barsi eeake Bay . The Embin situ data. On the other hand, the wide usage of Landsat temperature algorithms enabled by broader validation efforts would make waterbody temperature data more available to municipalities and other stakeholders lacking the resources to perform field measurements.Though substantial effort has been put forth to validate the thermal band performance of various satellites over the past several decades, there is a paucity of research that does so both at a fine spatial resolution and across a broad spatial extent relevant for management applications. Until such a broad validation is performed, reliable remote sensing of waterbody surface temperature remains restricted to individual waterbodies with available in situ stations compared to Landsat and (2) validate the water application of a U.S. Geological Survey (USGS) land surface temperature provisional product in ecologically diverse lakes, reservoirs, and estuaries.This study leverages spatially extensive lake, reservoir, and estuarine temperature datasets to provide a broad-scale validation of Landsat surface temperature data for lakes across CONUS and a variety of ecoregions. The objectives of this study are to (1) determine the number of surface lake and reservoir waterbodies sampled for temperature through traditional 2.in situ water temperature measurements, Landsat temperature data, Landsat validation with in situ data, and in situ locations for data validation.This section discusses the study methods, including national 2.1.In situ discrete measurements of temperature were accessed through the Water Quality Portal . The WQP website serves data collected by state, federal, and tribal groups across the United States from the USGS National Water Information System (NWIS), U.S. Environmental Protection Agency (U.S. EPA) Storage and Retrieval Data Warehouse (STORET), and U.S. Department of Agriculture (USDA) Sustaining The Earth’s Watersheds Agricultural Research Database System (STEWARDS). Data were selected from WQP based on the following criteria: estuary, lake, reservoir, or impoundment site type; water sample medium; physical characteristic group; and water temperature characteristic. The data were for the period 1 January 1980 through 1 January 2016 within the continental United States (CONUS). Discrete in situ measures were retained for temperature (°C) Landsat validation if they were in the upper 0.5 m of the water column. Only USGS NWIS in situ temperature measurements were used for Landsat temperature validation to maintain a consistently high-quality    to ident2.3.in situ measures were retained for validation across the CONUS.Landsat surface water temperature (SWT) was derived from the USGS land surface temperature provisional product . The lan2.4.https://shoreline.noaa.gov/data/datasheets/medres.html) was selected for estuarine temperature validation based on exceedance of National Map Accuracy Standards and an average scale of 1:70,000. For lake and reservoir validation, two sets of temperature data were evaluated: land-water boundary (<59 m from shore) data and far shore (>180 m from shore) data. Estuary data were retained and evaluated only if they were >180 m from shore. The distance of >180 m from shore was selected to avoid pixels contaminated by a land signal for both TM and ETM+. The diagonal distance of a 120 m Landsat 5 TM pixel was calculated with the equation D = R×√2, where D is the diagonal distance and R is the length of the pixel side (120 m). The distance was increased to >180 m, to account for the Landsat geolocation uncertainty of <12 m  and Landsat 5 TM (120 m) sensors. This <59 m near shore threshold ensured that any near-shore point had the probability of falling (1) within a 100% water 30 m SWT pixel adjacent to a ‘mixed’ (land and water) SWT pixel or (2) within a ‘mixed’ (land and water) 30 m SWT pixel. Statistics generated included the bias and mean absolute error (MAE). The results were presented in a star plot as a convenient way to summarize and compare performance  Medium Resolution Shoreline  NLA 2012 NHDPlusV2 waterbodies were sampled for temperature within a 100 m buffer from 1980 through 2015  occurred from April through September, with the remaining 15.0% occurring from October through March , and Landsat could provide measurements in 52.5% of these lakes, which is a large improvement compared to the 1.1% of the lakes and reservoirs with in situ sampling  were resolvable by a window width 90 m wide, a resolution equivalent to a 3 × 3-pixel array . Most (8sampling . The monsampling . Both inin situ sampling by providing data for most CONUS lakes and reservoirs (62%) over seasonal intervals, even with cloud cover, for at least 35 years. The 30 m Landsat dataset provides more measurements for smaller lakes and reservoirs than representation from traditional sampling. Programs that combine a variety of monitoring methods such as discrete in situ sites, synoptic measurements from satellites, continuous sensors, and models may improve management strategies for water quality problems  or waist depth 1 m; .4.in situ sampling with an operational high-resolution satellite platform that will provide a long-term, consistently processed climate data record. To the authors knowledge, this is also one of the largest validation efforts of temperature for inland waters across a variety of ecosystems, other than in situ temperature samples, this study demonstrates the continued importance of these in situ datasets, which permitted the robust validation of the Landsat SWT algorithm and associated performance metrics in lakes, reservoirs, and estuaries across the CONUS. Comparison of in situ water temperature measurements with Landsat-derived SWT measurements indicates that absolute errors were within 2°C, and that the stability of the Landsat algorithm is sufficient for determining seasonal trends and assessing inter-annual variability of lake, reservoir, and estuarine temperatures. The satellite datasets provide a synoptic complement to discrete in situ sampling and may be considered part of the comprehensive management toolbox for monitoring water quality changes.This is the first study to quantify and identify Landsat resolvable lakes and reservoirs across CONUS, and compare traditional The 2016 HABHRCA report identifies monitoring challenges that include sustaining monitoring programs and maintaining the consistency of methods across monitoring programs. This study provides an opportunity for water quality managers and stakeholders to have a uniform satellite dataset  and a coGlobal collections of lake, reservoir, and estuary SWT data may be possible in the near future as the NARR data were used for processing over North America, and global processing would include the Modern-Era Retrospective analysis for Research and Applications, Version 2 (MERRA-2) aerosol data for atmospheric characterization in the radiative transfer model. The results shown here demonstrated that new SWT data from satellites can be broadly applied to a vast number of lakes for monitoring and assessing changes throughout the decades.


Aedes mosquitoes in these areas was unknown. Serological surveillance of DENV in Ae aegypti is a powerful tool for early detection of dengue outbreaks and essential for developing effective control strategies. Therefore, this research aimed to examine a sample of adult Ae aegypti mosquitoes from Makkah, KSA, to detect DENV. In total, 1295 Ae aegypti mosquitoes were collected from the field from target areas of Makkah with a high incidence and prevalence of DF. The samples were divided into 259 coded pools (five mosquitoes in each) and preserved in 1.5 mL plastic tubes. The tubes were labeled, capped, and stored at−86°C until use. RT-PCR was used to detect DENV in the samples. All positive pools were confirmed by RT-PCR. The RT-PCR products were analyzed by gel electrophoresis (1.5% agarose in Tris-acetate EDTA buffer), stained with ethidium bromide, and visualized. DENV was isolated from six female Ae Aegypti collected from six pools (out of 259 pools). No other viruses were detected. Only five of the nine target localities had positive pools. Samples from the remaining four localities yielded negative results. Four DENV-positive mosquitoes were collected at the aquatic stages, and two were collected at the adult stage. These results show the circulation of DENV in adult mosquitoes and offspring, indicating vertical transmission of DENV. In conclusion, this study found that, in Makkah, DENV is circulating in dengue vectors with a high significance rate, suggesting the possibility of a dengue outbreak in the future; therefore, a sensitive surveillance system is vital to predict the outbreak and for early intervention and control.Dengue fever (DF) is endemic to Makkah and Jeddah, the Kingdom of Saudi Arabia (KSA). However, until recently, the circulation of dengue virus (DENV) in  Aedes aegypti, a mosquito species that breeds and flourishes in stagnant water containers maintained by rain or human activity (Dengue virus (DENV) is a single-stranded RNA virus of the family Flaviviridae. It is the most prominent mosquito-borne arbovirus worldwide , 2. The activity .Ae aegypti  relies on the knowledge of local DENV species distribution and vector and human behaviors that may allow mosquitoes to avoid contact with interventions. Furthermore, surveillance of DENV-infected mosquitoes provides an early warning sign for the risk of transmission in an area and indicates the predominant circulating serotype in the vector population . This maAedes mosquitoes in 42,981 potential Aedes larval breeding sites in Makkah, KSA. We found a sizeable immature population and adaptation of Ae aegypti to the arid climate of Makkah , the area within a radius of 0.5 km was carefully inspected. Each house was inspected once during the study period. Adult mosquitoes were collected using either blacklight traps set up in the selected areas for 24 h and/or manually caught using hand nets or aspirators between 4 and 6 p.m. (before dusk).For aquatic collection, larvae at different stages were collected from households with breeding sites. World Health Organization (WHO) standard entomological kits (mosquito inspection kit bags) were used to inspect and survey the mosquitoes in the aquatic stages. The sample containers were labeled and stored in a cool box at approximately 25°C. The samples were then transported to the Vector-Borne Disease Control Laboratory for species identification and further processing.Aedes mosquito species (The collected larvae and pupae were reared in the vector control rearing insectary following the Food and Agriculture Organization of the United Nations (FAO) and the International Atomic Energy Agency (IAEA) Guidelines for routine colony maintenance of  species , 17. MosAedes (Stegomyia) mosquitoes of East Africa and a pictorial key for identification of subfamilies of Culicidae and genera of Culicidae as well as resources from the WHO were used to identify the Ae aegypti . These were divided into 259 pools (five mosquitoes per pool)  , preserv2, and 5 μL DNase I enzyme) was added into the collection tube, incubated for 15 min at 20–25°C, and then 200 μL of DNase stop solution was added and centrifuged for 1 min at 10,000 rpm. The filter tube was then washed twice by adding 450 μL of wash buffer under the same centrifugation conditions, followed by centrifugation for 15 s at 13,000 rpm to remove any residual wash buffer. The high filter tube was then inserted into a nuclease-free, sterile 1.5 mL centrifuge tube, and 100 μL of elution buffer was added to elute the viral RNA by centrifugation at 10,000 rpm for 1 min.A total RNA isolation kit  was used for RNA extraction. Following the manufacturer's instructions, the mosquito samples were homogenized using a mortar and pestle  in 175 μL of RNA lysis solution buffer. Then, 350 μL of RNA dilution buffer was added to 175 μL of the lysate and mixed by inverting three to four times. The mixture was then placed in a heat block for three min at 70°C. After centrifugation at 10,000 rpm for 10 min, the clear lysate was transferred to a new 1.5 mL microcentrifuge tube containing 200 μL of 95% ethanol and mixed by pipetting. The mixture was transferred to a spin column inserted into a collection tube, and centrifuged for 1 min at 10,000 rpm. The collection tube was discarded, and the filter tube was placed in a new collection tube, 600 μL of RNA wash solution was added and then centrifuged for 1 min at 10,000 rpm. Following this, 50 μL of DNase incubation mix  (project #43409019).Ae aegypti mosquito collection is shown in Ae aegypti mosquitoes (232 caught as adults and 1063 developed from larvae into adults) were used for DENV detection.The result of DENV was detected in six of 259 pools (2.3%) .Four DENV-infected mosquitoes were collected at the aquatic stages and two were collected at the adult stage . ResultsFive of the nine target localities (55.5%) yielded positive pools, while the other four yielded negative pools .Ae aegypti shown in The number of dengue cases by localities and entomological surveillance data and indices of Our results confirm that DENV is prevalent in the Makkah region, and many studies have shown that DENV-2 is the most predominant in suspected dengue cases, followed by DENV-1 and DENV-3 , 30–32. Ae aegypti vectors, supporting this well-documented finding in endemic areas worldwide .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. 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Mules are thought to be stoic animals that generally express few signs of illness or disease. Mules used as working equids in strenuous conditions on a daily basis may not show clinical signs of disease until the disease is more advanced. The help of stable side tests and measuring variables such as acute phase proteins, which are known to elevate in a rapid manner due to infection or inflammation, may assist owners and practitioners with detecting early signs of disease in working mules. This study measured serum amyloid A (SAA), an acute phase protein, in 10 healthy working mules in Central Italy. The study found no change in SAA for mules working 8 h but mild increases in serum electrolytes, urea, and creatinine concentrations that supported a loss of body water and decreased renal blood flow in response to exercise.p < 0.05) of fieldwork was found on mules’ electrolyte profile with increased levels of sodium, chloride, and calcium, as well as on blood urea nitrogen and creatinine. After a day of fieldwork, serum sodium, chloride, calcium, urea, and creatinine concentrations were increased, supporting decreases in body water and renal blood flow. However, without comparison to a group of mules that were not exercised yet maintained under similar ambient conditions, it is uncertain whether these changes can be attributed to exercise. Further, no change in SAA concentration was found after exercise, indicating that the work performed did not result in systemic inflammation.According to FAO reports, the global mule population counts about 9 million mules. This hybrid cross of a male donkey and a female horse is mainly used for draft purposes because they are thought to be strong and hardy animals. Most consider mules to be less susceptible to disease and fatigue compared to horses. Therefore, the aim of this study was to investigate the effects of fieldwork on biochemical variables and serum amyloid A in working mules. Blood samples were collected from 10 healthy, female, working mules before and after 8 h of fieldwork. According to statistical analysis, a significant influence ( Equus mulus) is a hybrid cross of a male donkey (Equus asinus) and a female horse . The world population of mules is estimated to be close to 9 million animals, which are found primarily as a beast of burden thanks to their strength and rusticity, but more mules are being used as performance and recreational animals [The mule , high incidence of musculoskeletal disorders, skin lesions associated with the harnessing system, dental diseases, parasitosis, and behavioral disorders, such as apathetic behavior or aggressivity, are often detected in working equids such as mules .Heavy work, such as physical activity, is a commonly recognized stress factor that can perturb the homeostasis of an organism ,8. BloodThese variables can be used as tools for assessing the appropriateness of the workload they are requested to perform. The study of biochemical changes resulting from physical activity allows researchers to deeply understand the adaptations occurring within the animal’s body following physical effort and to adopt suitable measures to preserve animal welfare and health status ,11,12.It is widely accepted that physical activity elevates circulating acute phase protein (APPs) levels also in equine species ,13. TissDue to the stoic nature and limited information on blood chemistry in mules, conditions such as strenuous field work or exercise can result in damage to the body, which can go undetected until advanced stages develop and the animal is no longer able to perform its daily tasks.Mules are thought to be less susceptible to diseases and fatigue compared to horses, yet no scientific investigation has been performed so far to support this belief. In view of such considerations, the purpose of the current survey was to investigate the effects of strenuous exercise on health status and welfare by evaluating SAA and biochemical variables in working mules in Central Italy.Ten adult female mules, with a mean age (±standard deviation) of 15 ± 5 years and a mean weight (±standard deviation) of 450 ± 40 kg, were included in the study. The body condition score (BCS) of animals ranged from 2 to 3 out of 5 . Owner’sThe mules were reared in Central Italy as working animals and were kept in outdoor paddocks. The management was the same for all the mules, their diet consisted of fresh forage (grass), 8 ± 1 kg grass hay and 4 ± 0.5 kg concentrates per day, and water ad libitum.The animals were routinely used for wood transportation in a hill country 5 days per week during the spring season. Each animal carried 200 ± 10 kg of wood. The mules walked 60 km on a path with a mean slope of 15 ± 5%. Animals walked about 30 km unloaded to gather wood and then walked backloaded .Blood samples were collected, by jugular venipuncture, on the first working day of the week in the morning  at rest before taking the mules out of paddocks and in the afternoon  immediately after the work was completed. Weather conditions during the work and blood sampling were as follows: average temperature 13 °C (11–14 °C), average relative humidity 86% (76–93%), and partially cloud weather. During the work period, mules had the opportunity of eating some grass while loading the wood and had access to water four times during the daily working period. All mules were regularly vaccinated, checked for intestinal parasitosis, and treated when necessary.Animals were clinically evaluated before and after work with particular attention to musculoskeletal injuries, skin lesions, and behavioral alterations.All animal housing, care, and experimental procedures herein described were approved by the Animal Care Committee of Camerino University  and were in accordance with the standards recommended by the EU Directive 2010/63/EU for experiments on animals.g , and the obtained sera were divided into two 1.5 mL aliquots and stored at −20 °C until analysis. Sera were tested for potassium (K), sodium (Na), chloride (Cl), calcium (Ca), phosphorus (P), calcium/phosphorus ratio (Ca:P) blood urea nitrogen (Bun), γ-glutamyl transferase (GGT), glucose (Glu), creatinine (Cre), glutamic oxaloacetic transaminase (Got), serum glutamic pyruvic transaminase , alkaline phosphatase (ALP), cholesterol (Chol), triglyceride (TG), creatine kinase (CK), total bilirubin (TBil), direct bilirubin (DBil), indirect bilirubin (IBil), total protein (Tp), albumin (Alb), globulins (Glob), and albumin/globulin ratio (A/G) by using the automatic clinical chemistry analyzer BT 3500 VET plus .After collection, blood samples were placed on ice and delivered to the laboratory within 2 h. At the lab, samples were centrifuged for 10 min at 1000× Serum amyloid A (SAA) was detected in serum samples by a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) using the Tridelta PhaseTM range SAA kit . Briefly, a monoclonal antibody specific for SAA was coated onto the wells of the microtiter strips provided. The 1:2000 diluted samples and calibrators of known SAA content were incubated in micro-wells at 37 °C together with horseradish peroxidase (HRP)-labeled anti-SAA antibody. Any SAA present was captured between the coated microplate and the labeled antibody. After the washing steps, the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine was added. The resulting blue product is directly proportional to the amount of SAA present in the serum of mules. The reaction was stopped by adding a stop solution, and the intensity of color was measured at 450 nm using a microtiter plate reader. The concentrations of the test samples were derived from the calibration semi-logarithmic standard curve and were expressed as mean concentration (µg/mL) ± SE. Intra (within)-assay precision/reproducibility was 4.4–5%, whereas inter-batch assay reproducibility was 6.22–11.4%.t-tests and Mann–Whitney test were used for normally and not normally distributed data, respectively. p-values were reported to show significant differences in studied blood variables before (T0) and after (T1) the working section. Values of p < 0.05 were considered statistically significant.Data were analyzed using the statistical software Prism 8 . A Shapiro–Wilk test was used to verify the normal distribution of data. Paired Student’s All the mules were accustomed to the type of exercise and completed the working section in about 8 h. None of the animals showed any sign of disease, lameness, skin lesions, or behavioral alteration during the experimental period. Mean values and standard deviations of biochemical variables are shown in p = 0.048) ranging from 137 mEq/L (T0) to 140 mEq/L (T1), chloride (p = 0.014) ranging from 100 mEq/L (T0) to 104 mEq/L (T1), calcium (p = 0.001) ranging from 12.5 mg/dL (T0) to 13.7 mg/dL (T1), while mean phosphorus decreased (p = 0.02) from 4 mg/dL to 3.6 mg/dL, with consequent modification in Ca/P ratio (p = 0.001) that passed from 3.1 to 3.8. Significant increases in the concentrations of some biochemical variables such as Bun (p = 0.03), Cre (p = 0.0003) and Glu (p = 0.0001) have been observed as well. On the contrary, SAA did not statistically change (p = 0.85) in mules during burden work, ranging from 4.84 μg/mL at T0 to 5.41 μg/mL at T1.According to statistical analysis a significant influence of fieldwork was found on mules’ electrolyte profile with mean levels of sodium  are linked to overtraining syndrome or maladaptation to exercise in horses ,27. In tGlucose tends to decrease during and after physical activity in horses , yet thiCreatine kinase did not increase in this group of mules, demonstrating that the workload  was well tolerated and not excessive.The use of APPs in veterinary clinical chemistry as non-specific variables for monitoring inflammatory activity has been adopted during high-intensity physical activity, as the occurrence of muscle injury can trigger the APR .The physiological response to exercise differs in acute response and chronic adaptation. It has been shown that there is an analogous relationship between the APR to exercise and existing infectious and inflammatory processes so that the presence of any tissue injury promotes the release of pro-inflammatory cytokines, nitric oxide, and glucocorticoids that activate and modulate the APR and release of APPs, primarily by the liver . The detThere is a paucity of studies on SAA concentrations in mules, and preliminary data indicated a difference in SAA response in mules versus horses or donkeys , suggestThe circulating values of SAA obtained in the present investigation ranged from 4.84 μg/mL at T0 to 5.41 μg/mL at T1 in the investigated mules, suggesting that draft work did not significantly affect the APR in mules that are accustomed to and regularly trained for this type of fieldwork. These findings seem to indicate that mules can cope with strenuous exercise, which did not promote enough physiological stress to cause an increase in the concentration of SAA in mules, thus suggesting a great ability of mules to adapt to fieldwork. However, the absence of a control group might represent a limitation of the study as the differences herein reported may be related to or moderated by the temporal effect.Mules have supported man for millennia and are currently critical to the lives of rural developing countries and have taken on a new role as valued performance and recreational animals in industrial countries. There is potential for working mules to suffer from a poor harness, lack of veterinary care, improper nutrition, and low status and value, despite their usefulness. Because of this important role as working equids, the scientific community should direct attention to improving the knowledge about the assessment of mules’ welfare. Such studies can also be helpful for clinicians treating mules in developing countries and further develop their understanding of biochemical and inflammatory responses in mules.It is well known that the welfare status of working equids is generally poorer than other equids with different attitudes . Our finAlthough the authors are aware of the difficulties that can be encountered in developing countries in observing certain good practices, this work demonstrates how the correct management of animals is compatible with their use as working animals.Further studies are advocated to better evaluate other possible markers of inflammation and/or stress in these working equids to control and preserve their welfare and health status.