The results showed RSPN had the lowest glycemic index value if compared to BSP and SPN  and fell under food with medium GI, but the differences were not statistically significant. RSPN and SPN were classified as medium GI; however, the process of partial gelatinization followed by retrogradation during the preparation of flour used for raw material in making RSPN has successfully maintained the total phenol and anthocyanin and increased resistant starch content of the noodle Processing of purple sweet potato flour into noodle lowered the GI category, and when the flour was partially gelatinized and retrograded, the noodle had more potentiality as a functional food due to their high total phenolic and anthocyanin content.Purple sweet potato is a source of starch with high potential to be developed as a functional food. It can be boiled and consumed as a snack or processed into intermediate goods such as flour. The flour can then be modified through partial gelatinization and followed by a retrogradation process to produce resistant starch-rich purple sweet potato flour. The study was aimed at obtaining the glycemic response values of purple sweet potato products, namely, boiled purple sweet potatoes (BSP), purple sweet potato noodles (SPN), and resistant starch-rich purple sweet potato noodles (RSPN). SPN was prepared from conventional purple sweet potato flour, whereas RSPN was made from resistant starch-rich purple sweet potato flour. Moreover, water, ash, protein, fat, and carbohydrate; total phenolic, anthocyanin, and resistant starch contents; and the rate of hydrolysis of starch were evaluated. Ten subjects were involved in the estimation of glycemic response determined by the area under the curve (AUC) of the blood glucose after consuming products. Glucose syrup was used as a reference. The glycemic response data were processed using ANOVA and further tested using LSD at  Purple sweet potato is a food source rich in carbohydrates, primarily starch. As a starch source, purple sweet potatoes have a high potential to be developed as a functional food due to its bioactive components that are said to be good for health. One of the bioactive components with physiological functions is anthocyanins . Anthocyα-glucosidase enzyme [α-amylase enzyme activities [Processing purple sweet potatoes into flour has other benefits such as having more flexible utilization, saving storage space, and being available anytime without waiting for the harvest season. Another benefit is that it is easy to modify purple sweet potato flour to maintain its bioactive components and improve its physiological functions. Nurdjanah  reportede enzyme  and α-amtivities .α-amylase enzyme activity in the small intestines is inhibited, slowing down glucose digestion [Research by Sajilata et al.  states tigestion . Second,igestion .Research regarding the glycemic response of processed food made from high-carbohydrate tubers such as purple sweet potatoes is still limited. The same ingredient has different glycemic indexes when processed differently because the characteristics and physiochemical properties have changed . For exa2SO4, H2SO4, 95% alcohol, 0.02 N HCL, H3BO3, Na2CO3, gallic acid, 0.2% citric acid, KCl buffer solution, pH 4.75 sodium acetate buffer solution, pH 1.5 KCl-HCl buffer solution, phenol, KOH 4 M, pepsin, glucose, α-amylase enzyme from Aspergillus oryzae (powder), 3,5-dinitrosalicylic acid (DNS), and glucoamylase enzyme.The main material of this research was local purple sweet potatoes from a traditional market in Way Kandis, Bandar Lampung, Indonesia. Chemical substances for analysis included NaOH, HgO, KEquipment used in this study was a modified rotary drum dryer, refrigerator, peeler, and cabinet dryer. Equipment used for analysis included Kjeldahl flask, boiling flask, Soxhlet extractor, vortex, centrifuge spectrophotometer Genesys S10-UV-VIS, water bath, and blood glucose testing equipment Accu-Chek Performa.Fresh purple sweet potatoes (1 kg) were sorted, washed, drained, and boiled in boiling water (2 L) for about 30 minutes. Boiled purple sweet potatoes were then taken out, drained, peeled, and cut into 2 × 2 × 2 cm cubes to be served to respondents.Purple sweet potatoes were cleaned and grated to 1 mm thickness. Purple sweet potatoes were then dehydrated in a cabinet dryer at 60°C for about 16 hours until the water content was about 10%. Dehydrated purple sweet potatoes were processed into flour using a hammer mill and sieved through the 80-mesh sieve.Purple sweet potato flour noodles were prepared following the method of Yuliana et al.  with sliPurple sweet potatoes were cleaned, grated, and heated to 90°C for 30 minutes in a rotary drum dryer. For the cooling process, purple sweet potatoes were then cooled at 25°C for 1 hour and put into the refrigerator at 5°C for 48 hours. Cold purple sweet potatoes were dried in a cabinet dryer at 60°C for about 16 hours until the water content reached 10%, then processed into flour using a hammer mill and sieved through the 80-mesh sieve [The proximate analysis of water, ash, protein, fat, and carbohydrate by difference contents followed the AOAC (2005) method .2CO3) was added before mixing again in a vortex for 1 minute and left in a dark room for 30 minutes at room temperature. The absorbance of gallic acid reaction with the Folin-Ciocalteu reagent was then read at 760 nm wavelength. A blank form with the same procedure as the sample was prepared. The absorbance results were plotted on a standard curve for gallic acid using the linear regression equation with gallic acid concentration on the x axis and absorbance on the y axis. A standard curve for gallic acid was made by mixing 1 mg of gallic acid with distilled water , preparing a series of 0%, 20%, 40%, 60%, 80%, and 100% dilution from that gallic acid solution, and giving them the same treatment as the sample. The results were shown in the standard curve equation:Total phenolic content analysis used a modified version of the method of Nurdjanah et al. . The extThe total anthocyanin concentration was measured using the method by Giusti and Wrolstad . Two samA is absorbance, MW is molecular weight of cyanidin 3-glucoside (449), DF is dilution factor, ε is coefficient of cyanidin 3-glucoside molar extinction , and 1 is cuvette thickness.The total anthocyanin content was determined using the following equation:The resistant starch content was determined following the Goni et al.  method wα-amylase enzyme solution was added. The mixture was then incubated for 16 hours in a water bath at 37°C with constant stirring and centrifuged for 15 minutes at 3000 rpm. The resulting supernatant was thrown away, and 10 mL of distilled water was added to the residue to be centrifuged again . The supernatant was then separated. The residue was mixed together with an additional 3 mL of distilled water and added with KOH (4 M) before being incubated in a Memmert shaker water bath for 30 minutes at 25°C with constant stirring. Then, 5.5 mL HCl (2 M) and 3 mL sodium acetate (0.4 M) buffer solutions were added, and pH was adjusted to 4.75 by adding HCl (2 M).The mixture was put into a 40°C water bath for 60 minutes, then cooled at room temperature. pH was adjusted to pH 6.9 by adding NaOH (0.5 M), and 1 mL of μL of glucoamylase enzyme was added and mixed in and left in a Memmert water bath for 45 minutes at 60°C. The supernatant was separated using a centrifuge tube  and kept while 10 mL of distilled water was added to the residue and centrifuged again. The resulting residue was then thrown away. The supernatant was mixed with the previously obtained supernatant, and distilled water was added to reach 50 mL in volume. Glucose content was determined using the phenol-sulfuric acid method, and resistant starch content was determined by multiplying the glucose content by 0.9.After that, 80 α-amylase enzyme was determined following the modified method by Nurdjanah et al. [α-amylase enzyme and 3 mL of pH 7 phosphate (0.1 M) buffer solution; incubated at 37°C for 0, 60, and 120 minutes; and centrifuged at 3000 rpm for 15 minutes. The glucose content of the resulting sample was then determined using the phenol-sulfuric acid method.The hydrolysis rate of starch with h et al. . Powdere2) and in healthy conditions. The exclusion criteria included having no history of diabetes, no current digestive issues, not being in the middle of treatment, not using illegal drugs, not smoking, and not drinking alcohol. There were 10 candidates selected as respondents, and they were given a short explanation of this research before signing informed consent forms.The glycemic response was evaluated in several steps, which were (a) ethical clearance application, (b) subject candidate recruitment, (c) subject candidate selection, (d) research explanation and informed consent, and (e) glycemic response evaluation. This research was done after obtaining ethical clearance from the authorized committee of ethics. The recruitment and selection of subject candidates were done using the purposive sample method that fulfilled the inclusive and exclusive criteria. The inclusive criteria were subjects aged between 18 and 30 years of age of any gender with normal BMI  was then calculated with the following equation:After consuming each purple sweet potato products, 0.6 x axis (time) and y axis (glycemic response). The glycemic responses of ten respondents on each product were averaged to reveal the curve chart. The effect of consuming each product on the glycemic responses was analyzed for its normality, followed by the paired samples test using the software SPSS 16.0.This research was arranged in a completely randomized design with three replications. The data of proximate composition were reported for their means and standard deviation, whereas total phenol, total anthocyanin, and resistant starch content' data were then analyzed for their homogeneity and additivity using the Bartlett and Tukey tests, then processed using analysis of variance (ANOVA), and further tested using the least significant difference (LSD) test. All the cooked purple sweet potato products and pure glucose were then evaluated for their glycemic responses following the method of El  with tenIn this research, proximate analysis was completed to figure out the nutritional content of these products such as water, fat, ash, and carbohydrate by difference. One factor that affects blood glucose response is the composition of nutrition . Boiled The results of total phenolic, anthocyanin, and resistant starch contents are presented in This research revealed that the highest total phenolic content of 357.33 mg GAE/100 g was found in boiled purple sweet potatoes . This miIn this research, the total phenolic content of resistant starch-rich purple sweet potato noodles was lower than that of boiled purple sweet potatoes because in the noodle-making process, there was direct heat contact during dough steaming before making noodle sheets and dry noodle steaming. When boiling purple sweet potatoes, the skin stayed on thus avoiding direct heat contact between the water and the purple sweet potato flesh. According to Padda and Picha , the proBoiled purple sweet potatoes showed the highest amount of total anthocyanin content with 95.64 mg/100 g . MeanwhiThe higher amount of total anthocyanin content in boiled purple sweet potatoes than in other products was directly proportional to the amount of total phenolic content. Heat treatment on purple sweet potatoes is predicted to inactivate the enzymes that caused browning such as polyphenol oxidase and peroxidase , thus inBoth SPN and RSPN showed lower anthocyanin content when compared to boiled purple sweet potato anthocyanin content. This was probably because during the thermal process anthocyanins experience deglycosylation, nucleophilic attack of water, cleavage, and polymerization . These rThe result of this research revealed that boiled purple sweet potatoes had the lowest amount of resistant starch with only 2.99% . This miThe highest amount of resistant starch (14.29%) was found in resistant starch-rich sweet potato noodles. This relatively high amount was because of the partial gelatinization of purple sweet potato flour at 90°C for 30 minutes and the following retrogradation at 5°C for 48 hours. Nurdjanah and Yuliana  stated tα-amylase enzyme was inversely proportional to the resistant starch content of purple sweet potato products. High resistant starch content in food may decrease the enzyme hydrolysis rate [The highest hydrolysis rate in 120 minutes happened in boiled purple sweet potatoes with 78.56% ± 0.26%. Meanwhile, the hydrolysis rate of purple sweet potato noodles reached 66.28% ± 1.88% with the lowest number of 52.27% ± 1.40% belonging to resistant starch-rich sweet potato noodles. The rate of hydrolysis with sis rate . Resistasis rate . That ensis rate .  Figure2) and in healthy conditions. The exclusive criteria included having no history of diabetes, no current digestive issues, not being in the middle of treatment, not using illegal drugs, not smoking, and not drinking alcohol. There was a total of 10 respondent subjects selected.This research was completed after obtaining ethical clearance from the Research Ethics Committee Faculty of Medicine University of Lampung Number 2985/UN26.8/DL/2017. Respondent subject recruitment was conducted through socialization with several students from the Agricultural Technology Department University of Lampung and interviewing them regarding their health history. The weight, height, and BMI of candidates were then observed. Candidates selected to be respondent subjects must fulfill the inclusive and exclusive criteria. The inclusive criteria were subjects aged between 18 and 30 of any gender with normal BMI  consensus, the normal BMI for Indonesians was between 18.5 and 22.9 kg/m2. The preprandial blood glucose level was obtained by averaging the four tests done at minute 0, indicating no diabetes history with the numbers all under 100 mg/dL.The average age of respondent subjects was 22 years with 53.5 kg weight, 1.6 m height, and 20.83 kg/mμL. The blood glucose level was measured using the Accu-Chek Performa digital glucometer. The blood samples obtained were touched onto the censor crack at the end of the test strip, and the results were read on the screen.Blood samples were obtained using lancet pens on respondent subjects' pointer, middle, and ring fingers. The respondent subjects' fingers were first cleaned and sterilized with an alcohol swab. For this research, the volume of the blood sample needed was minimum 0.6 Respondent subjects received food intervention of pure glucose syrup as a comparison in addition to the purple sweet potato products of boiled purple sweet potatoes, purple sweet potato noodles, and resistant starch-rich purple sweet potato noodles. Each food intervention was given 3–4-day intervals for recovery. Respondent subjects fasted for around 10 hours and were tested for their preprandial blood glucose levels. The testing was done in a comfortable room and relaxed condition with no influence from physical activities.The first food consumed was 50 g of pure glucose syrup dissolved in 200 mL of water for control. Pure glucose syrup was the recommended item to act as control when determining the glycemic index values of other foods due to its standardized and uniform composition, unlike white bread. Respondent subjects needed 3 minutes to consume the glucose syrup. Blood samples were taken for blood glucose level testing at minutes 30, 60, 90, and 120 afterwards. In order, food interventions given after pure glucose syrup were boiled purple sweet potatoes, purple sweet potato noodles, and resistant starch-rich purple sweet potato noodles. The serving size for each product was equivalent to 50 g of carbohydrate by difference, as shown in The biggest serving size was for boiled purple sweet potatoes, and the smallest was for resistant starch-rich purple sweet potato noodles. This research did not consider soluble and insoluble fiber; thus, the available carbohydrate content could not be measured. The serving size in this research was determined using the carbohydrate by the difference method without revealing the total available carbohydrate to be digested by the body. The serving size of resistant starch-rich purple sweet potato noodles should have been bigger because resistant starch was not easily digested, lowering the total available carbohydrate.Boiled purple sweet potatoes were served on the second testing (day 4) with 145 g serving size per person. Respondent subjects needed about 10 minutes to consume the sample. Purple sweet potato noodles were served on the third testing (day 8) with 112.87 g serving size per person and resistant starch-rich purple sweet potato noodles on the fourth testing (day 12) with 112.87 g per person. Respondent subjects needed about 15 minutes to consume these samples, which was quite a long time, due to the less desirable taste (distinctively sweet potato-like) and the portion size that was bigger than the regular noodle soup dishes. The amount of time it took to consume these products might affect the blood glucose level results because products consumed earlier would be digested earlier, too.x axis and blood glucose level on the y axis, and a curve was formed as seen in The glycemic response was determined from how respondent subjects' blood glucose levels responded to the samples of pure glucose syrup, boiled purple sweet potatoes, purple sweet potato noodles, and resistant starch-rich purple sweet potato noodles. The data from respondent subjects' blood glucose tests were entered with time on the The peak of blood glucose increment happens around 15-45 minutes after consuming food, depending on how fast the body digests and absorbs carbohydrates. The glycemic response of food products is shown by the fluctuation curve of glucose absorption in the blood after consuming them. Food components that are absorbed quickly by the body will cause high fluctuation in blood glucose levels in a short time. The curve with the lowest peak belongs to resistant starch-rich purple sweet potato noodles, indicating the slow digestion process that causes the slow release of blood glucose until 2 hours postprandial.After the curve was formed, the incremental area under the curve was calculated. The area under the horizontal basic line of the preprandial blood glucose level was not included  The areap < 0.05) due to the different treatments in the processing of purple sweet potato products. The change in structures might affect the postprandial response to starchy food [α = 0.05) between purple sweet potato noodles and resistant starch-rich purple sweet potato noodles  revealed there were differences in the area under the curve of each product  purple sweet potato flour that was made with partial gelatinization and retrogradation process. Partial gelatinization opened the amylose and amylopectin bond structures. Then, in the cooling process, compact starch molecules were restructured and stabilized with hydrogen bonds, creating starch that was difficult to hydrolyze by digestive enzymes . ResistaProcessing starch to produce resistant starch is one way to lower glycemic response values. Resistant starch type 3, due to its special properties as a result of the retrogradation process, is not digested in the small intestine. However, after 2 hours, this type of resistant starch will be fermented in the colon resulting in short-chain fatty acids. This type of resistant starch has beneficial effects on recuperating insulin resistance and lowering blood glucose in type 2 diabetes mellitus patients. Blood glucose levels of respondent subjects after consuming these food products were also quite stable even after two hours. A smaller area under the curve indicated slower digestion of carbohydrates that resulted in slower and less glucose release, so the curve had gentler response and lower peak level of glycemic response, and vice versa.Some respondent subjects only had a minimum difference in the area under the curve for different purple sweet potato products; some did not. Each respondent subject displayed different glycemic response curves with different peaks and glycemic index value indications. These varying results were assumed to be due to several factors of physiological responses such as metabolism, stress level, physiological condition, and fasting time.The area under the curve that showcases glycemic response becomes the reference in calculating the glycemic index value of a food product. As applied in this research, the glycemic index value indications of purple sweet potato products were determined by dividing the AUC of each purple sweet potato product by the AUC of pure glucose syrup, then multiplying by 100. This equation came from the glycemic index equation; however, this research called these results shown in α-amylase enzyme directly [directly .Based on general food glycemic index (GI) values, boiled purple sweet potatoes fell under food with high GI of over 70, while both noodles fell under the medium range with 55-70. Even though both noodles were in the medium range, resistant starch-rich purple sweet potato noodles still showed the lowest GI due to the main ingredient, RS3, that was made through partial gelatinization and retrogradation process and formed starch structures that were hard to digest. Different treatments and processes in making purple sweet potato products resulted in different glycemic index value indications.α-amylase enzyme, and it was inversely proportional with their resistant starch content. Partial gelatinization of sweet potato flour has failed to reduce GI of noodles as indicated that RSPN and SPN were classified as medium GI food, but the process has successfully maintained the total phenolic and anthocyanin and increased resistant starch content of the noodle. Processing of purple sweet potato into noodles lowered the GI category, and when the flour was partially gelatinized and retrograded, the noodles had more potentiality as a functional food due to their high total phenolic and anthocyanin content.The purple sweet potato products had different GI values depending on their processing method. The RSPN had the lowest glycemic index value if compared to BSP and SPN  and fell under food with medium GI, but the differences were not statistically significant. The pattern of the GI value of the products was concomitant with the pattern of the rate of starch hydrolysis with


Surfactants are ubiquitous materials that are used in diverse formulations of various products. For instance, they improve the formulation of gel by improving its wetting and rheological properties. Here, we describe the effects of anionic surfactants on an anesthetic drug, tetracaine hydrochloride (TCH), in NaCl solution with tensiometry and UV–visible techniques. Various micellar, interfacial, and thermodynamic parameters were estimated. The outputs were examined by using different theoretical models to attain a profound knowledge of drug–surfactant mixtures. The presence of attractive interactions among drug and surfactant monomers (synergism) in mixed micelle was inferred. However, it was found that sodium dodecyl sulfate (SDS) showed greater interactions with the drug in comparison to sodium lauryl sarcosine (SLS). The binding of the drug with surfactants was monitored with a spectroscopic technique (UV–visible spectra). The results of this study could help optimize the compositions of these mixed aggregates and find the synergism between monomers of different used amphiphiles. It is often observed that the surfactant mixtures  exhibit better performance than single surfactants ,3,4,5,6.A lesser water solubility and the dissolution characteristics of a drug usually limit its bioavailability and therapeutic efficacy. The poor water-solubility of drugs may also lead to disappointing and inconstant ingesting, which aggravates the complications of bioavailability and scarcity in the delivery of drugs. In addition, excessive dosages of drugs cause side effects such as vomiting, nausea, dizziness, and fatigue ,13. The + channels on neuronal membranes and stops the transmission of the pain sensation along the nerve 2, a straight line is obtained (5 (± 0.04) and 9.09 × 104 (± 0.04) mol–1 dm3 for the SDS + TCH and SLS + TCH mixed systems, respectively. The SLS + TCH mixed system had lesser binding constant values than the SDS + TCH system. In comparison, SDS has one functional group and SLS has two functional groups. The localized positive charge on the nitrogen atom on the TCH interacts with the negative charge on the sulphonic group, thus enhancing the electrostatic attraction between the guest and host. SLS, however, has methylated amide nitrogen, so the amide bond cannot be a hydrogen bond donor, which inhibits intermolecular attraction between SLS and TCH at the palisade layer. Furthermore, the steric hindrance of the N-methyl group of SLS may make it difficult to tightly align the amphiphiles. All these behaviors of SLS are responsible for its lesser binding constant compared to SDS.The binding constant and stoichiometric ratio were estimated with the differential absorbance method represented by the Benesi–Hildebrand equation :(25)1A−Aobtained , specifyK) values, free energy change of binding could be attained with Equation (26):By using binding constant (–1 for SDS + TCH and – 28.30(± 0.2) kJmol–1 for SLS + TCH. In both mixed systems, the G values were negative, indicating that the binding process was spontaneous.The binding free energies were –30.08 (± 0.2) JmolThe negative deviation of experimentally determined cmc values with hypothetical values confirms the nonideality of current mixtures.The interaction parameter at the interface and in solution was determined to be –ve, thus validating synergism between monomers of two species at the surface and in bulk.The higher values of the ideal mole fraction of component 1  with SDS and SLS (–ve charged head group) surfactants in the presence of salt (100 mM NaCl) was analyzed with both tensiometry and UV–visible spectroscopic techniques. The following conclusions can be derived:–1. To prepare standard solutions for experiments, amphiphiles (both pure and mixed) were dissolved and accurately weighed in a 100 mM NaCl solution. The stock solutions for both techniques (surface tension and UV–vis spectrophotometer measurements) were prepared in aqueous 100 mM NaCl solutions.Tetracaine hydrochloride , an anesthetic amphiphilic drug, and sodium lauroyl sarcosine  were supplied by Molecules On (Switzerland) and used as received. Sodium chloride  and sodium dodecyl sulfate  were acquired from Sigma-Aldrich . At 298.15 K, all experiments were performed using ultra-pure, double-distilled de-ionized water with a conductivity between 1 and 2 µScm2O. Throughout all experiments, water was circulated from a thermostatically controlled water bath through the outer jacket to keep the temperature at 298.15 K.The surface tension experiments were conducted with a digital tensiometer  by using a platinum ring. The instrument was occasionally calibrated with ultra-pure distilled water. In tensiometric titration, an amphiphile stock solution was titrated into a static volume of H2O solution of TCH in a quartz cell. We measured the absorption spectra of TCH solutions with surfactants and plotted them against the wavelengths. For the measurement of the absorption spectrum of TCH solutions over the range of 200–400 nm, an Evolution 300 spectrophotometer from Thermo Scientific, Tokyo, Japan was used to record UV–visible spectra (We measured the spectra of the aqueous solutions of the drug and the drug–surfactant binary mixtures to determine the level of the binding of the drug with surfactants. As a first step, TCH in water was prepared as a stock solution in a volumetric flask. The desired concentration of surfactant solution was prepared from the aqueous TCH solution. Finally, a suitable volume of surfactant solution was added to the H spectra .


SMAD4 or BMPR1A genes. Differential diagnosis may be difficult based on histology alone, due to morphological similarities to other familial syndromes. Here we report a case of familial JP diagnosed in a 50-years woman with a familial history positive for gastrointestinal cancers and other tumor types. The patient presented with severe iron deficiency anemia and showed numerous polyps in the stomach and jejunum according to endoscopy and imaging. She underwent an intra-gastric laparoscopic removal of the major gastric polyp, followed by jejunal exploration and resection of a segment with multiple neoformations. Histological examination revealed the presence of hamartomatous polyposis. Gastric and intestinal samples were analyzed with next-generation sequencing. Molecular analysis showed that the patient harbored a germline splicing site variant of SMAD4, c.1139 + 3A > G, which was complemented by different somatic variants of the same gene in the different polyps. Immunohistochemistry for SMAD4 confirmed loss of protein expression in the polyps, with regular expression in normal cells. cDNA sequencing further confirmed the findings. We thus definitively diagnosed the woman as having JP thanks to an integrated approach based on histology, immunohistochemistry and molecular analysis. The identified variants, all previously reported as variants of unknown significance, were classified as pathogenic as they complemented each other leading to SMAD4 loss.Juvenile polyposis (JP) is a rare familial syndrome characterized by the development of numerous hamartomatous polyps of the gastrointestinal tract and by an increased risk of developing gastrointestinal cancers. It follows a pattern of autosomal dominant inheritance and is associated with germline variants of The online version contains supplementary material available at 10.1007/s10689-022-00289-x. Hamartomas have been defined as an overgrowth of cells and tissues native to the anatomic site in which they arise , 2. In tBMPR1A) and the SMAD family member 4 (SMAD4), represents a well-established molecular pathogenetic mechanism of JP. SMAD4 was the first gene reported to cause JP, and its germline variant is also associated with another condition, the hereditary hemorrhagic telangiectasia [SMAD4 and BMPR1A account for about 18–20% of JP cases each, while the remaining 60% of cases are still orphan of a genetic driver [SMAD4 variants usually feature both gastric and intestinal polyps, and a large fraction displays high-grade adenomatous lesions and malformative vessels, while cases with BMPR1A variants feature only colorectal polyps with lack of adenomatous aspects and malformative vessels [STK11 gene, also known as LKB1) [In the context of hamartomatous polyposis of the gastrointestinal tract, the two most frequent conditions are represented by JP and PJS . Althougiectasia , 15. Varc driver –18. Case vessels . On the  vessels –21. Likeas LKB1) , 23. Thias LKB1) , 8. Thisas LKB1) . DiffereSMAD4 variants. The histopathologic examination of the polyps, coupled with molecular profiling by next-generation sequencing, represents a very robust approach in this field.Of note, the correct identification of a genetic predisposition to hamartomatous polyposis, as well as a correct diagnosis among several distinctive potential entities, is becoming an urgent clinical issue with important implications for the affected patients and their relatives. Tailored surveillance should be offered to all patients with hamartomatous polyposis to manage their increased risk of cancer development. Furthermore, defining the inheritable gene alteration of each patient allows for screening and surveillance of their relatives. Here we report the paradigmatic case of a 50-years-old lady with hamartomatous polyposis with gastric and small-intestinal manifestations, diagnosed as JP based on the identification of novel pathogenic Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissues using the GeneRead DNA FFPE kit (Qiagen) according to the manufacturer’s instructions. The kit procedure includes the removal of deaminated cytosine to prevent false results in DNA sequencing. Neoplastic cellularity was evaluated by two gastrointestinal pathologists  on hematoxylin and eosin staining, and each tumor sample was manually microdissected with a fine-needle hypodermic syringe to enrich for tumor cells. Quantification of genomic DNA samples was performed with the Qubit dsDNA HS assay kit on a Qubit fluorometer (ThermoFisher) and qualification as previously described .www.agilent.com), hereinafter referred as CORE [NGS was performed using the SureSelectXT HS CD Glasgow Cancer Core assay . Forward and reverse reads from each demultiplexed sample were aligned to the human reference genome (version hg38/GRCh38) using BWA and saved in the BAM file format [CORE panel analysis started with demultiplexing performed with FASTQ Generation v1.0.0 on the BaseSpace Sequence Hub , SnpSift [https://www.ncbi.nlm.nih.gov/refseq/; last access 11/30/2020). All candidate variants were manually reviewed using Integrative Genomics Viewer (IGV), version 2.9 [Single nucleotide variants were called using Shearwater . Small . Structural rearrangements were detected using the BRASS software [Tumor mutational burden and microsatellite instability were derived from sequencing analysis and computed following the method of Papke et al. . Copy nusoftware , and vishttps://www.ncbi.nlm.nih.gov/clinvar/; last access 11/16/2020) and accepted when the record complied with the following requisites: reviewed by expert panel according to the ACMG/AMP guidelines and/or reported by multiple submitters with evaluation criteria according to the ACMG/AMP guidelines and no conflicts. When a consistent classification was unavailable or when the variant was not present in the ClinVar database, variants were evaluated in-house, according to the ACMG/AMP guidelines using also the following databases and software to gather and integrate all relevant information: My Cancer Genome , Intogen [https://www.intogen.org/search; last access 11/23/2020) and QIAGEN Clinical Insight (QCI) software .Variants were classified following the five-tier classification system recommended by the joint consensus of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) . Variant Intogen  , according to the manufacturer’s protocol. Quality and quantity of the mRNA were assessed with the Qubit RNA BR Assay Kit on the Qubit fluorophore (ThermoFisher Scientific) and with the RNA ScreenTape Analysis kit on the Agilent tape station (Agilent Technologies) according to the manufacturer’s instructions. 1 μg of mRNA from each sample was reverse transcribed in a total reaction volume of 20 μl using the Superscript VILO kit and random hexamers (ThermoFisher Scientific) according to the manufacturer’s protocol. 2 μl of reverse transcription reaction were amplified using a primer pair that targets a 425 bp region in the 3′UTR of SMAD4 mRNA, corresponding to the genomic coordinates chr18:51,084,186–51,084,611 of the hg38 release of the human genome. The primers’ sequences are: FWD = CCACCCTCCTAAGTGGTGTG; REV = CCTTCTATCAATGACAAGCA GCC. The PCR mix was denatured for 5 min at 95 °C, and then 30 cycles of amplification were performed with the following thermal profile: 94 °C × 30 s, 60 °C × 45 s, 72 °C × 45 s. The PCR products were visualized with the D1000 ScreenTape kit on the Agilent tape station (Agilent Technologies) to confirm the presence of the proper molecular weight size products. PCR products were purified with Agen-court AMPure XP beads (Beckman Coulter) and labelled with BigDye® Terminator v3.1 (Applied Biosystems). Agencourt CleanSEQ magnetic beads (Beckman Coulter) were used for post-labeling DNA fragment purification, and sequence analysis was performed on the Applied Biosystems 3130xl Genetic Analyzer.Sanger sequencing was used to confirm the loss of heterozygosis of https://www.progenygenetics.com/online-pedigree, last accessed 06/18/2021). The patient’s mother died of gastric cancer, the maternal aunt died of colon cancer, the maternal uncle died of a brain tumor, while a maternal cousin suffered from bladder cancer. Laboratory tests revealed severe microcytic anemia  and hypoalbuminemia , whilst other parameters, among which B2 microglobulin, calcitonin, Anti-parietal cell antibodies, Castle's intrinsic factor, CEA, CA19-9, CA125, CA15-3, Cyfra 21–1, neuron specific enolase, were normal. Abdominal computed tomography (CT) confirmed the presence of an expansive, non-infiltrating lesion of the body and fundus of about 11 cm. The proximal small intestine appeared tense and strained, thickened for 13 cm. Fluorine 18-labeled fluorodeoxyglucose PET/CT scan , was referred to our Tertiary Care Center after an esophagogastroduodenoscopy (EGDS) evidenced multiple hamartomatous polyps and the presence of a large villous neoplasia, 11 cm in main diameter, located in the gastric fundus. The family tree was reconstructed . The variant is recorded in dbSNP as a germline variant (rs786202607) and was never reported as a somatic variant. This variant was also reported twice in ClinVar (VCV000185983) as class 3 (variant of unknown significance) because it was absent from controls and predicted to alter exon splicing by multiple algorithms but lacked further evidence. However, the two previous ClinVar reports amount to three cases, which rise to four considering our patient. According to GnomAD data, this variant was never identified in a population of 76,111 subjects. We could not retrieve the exact number of subjects tested for JP, but a conservative estimate would be less than 20,000 considering the low prevalence of JP in the population. Under these conditions, the incidence of this variant was significantly higher in the population of patients compared to GnomAD controls (p = 0.0001), which would bring the variant to class 4 according to the ACMG/AMP criteria [Molecular analysis was performed by next-generation sequencing (NGS) on histological samples of (a) the gastric polyp, (b) the largest intestinal polyp, and (c) normal intestine adjacent to polypoid lesions. The samples were sequenced using the CORE panel, which enables the detection of small nucleotide variants and structural alterations in 174 cancer-relevant genes, plus the evaluation of gross chromosomal alterations . Resultscriteria .Table 1MSMAD4 gene which did not harbor the germline variant  in exon 12, a class 4 double base substitution resulting in a nonsense aminoacid change, p. D537_E538delinsA* that was never reported before. Given the distance from the other two variants, it was not possible to understand which copy of the SMAD4 gene was affected by this somatic event. Moreover, the somatic variant could belong to a second clone of hyperplastic cells, as supported by the different allelic frequencies of the two somatic variants.The two polypoid lesions also harbored somatic variants that were not detected in the normal control sample. The gastric lesion displayed a somatic event near to the above reported germline splice variant Table . This miant Fig. A, thus cSMAD4 variant, affecting base -6 at the splice acceptor site of exon 4  RNF43 variants, including two nonsense  and a frameshift (p.L61fs*13) variant. The two variants affecting codons 61 and 82 were near enough to verify if they were represented in the same DNA strand or not. Verification performed by visualization of the alignments with IGV confirmed that the two alterations were harbored by different DNA strands, meaning that these two nearby variants either arose in different clones or in different sister chromosomes of the same clone.The largest intestinal polyp harbored one somatic SMAD4 variants were highly suspect for being pathogenic, but no conclusion could be done based on genetic data alone, we performed immunohistochemistry for SMAD4 to assess whether the compresence of the germline and somatic variants led to abolished protein expression. IHC clearly showed SMAD4 loss of expression in the epithelial cells of both the gastric and intestinal polyps, whereas inflammatory and stromal cells showed a retained expression of the protein in the nuclei . The results  according to the ACMG/AMP criteria [Given that the germline c.1139 + 3A > G variant was predicted to alter splicing, we sequenced the cDNA from normal gastroduodenal tissue of our patient to assess whether one of the two alleles was lost at the mRNA level. Our patient was heterozygous for two SNPs (rs140241965 and rs3819122) located in the 3’UTR of the criteria .Fig. 6DNSMAD4 variants by a combination of histology, next-generation sequencing (NGS), cDNA sequencing and immunohistochemistry. This integrative approach represents a robust model to improve routine diagnostic strategies as well as the management of patients with potential familial polyposis.We herein presented a case of JP that was diagnosed and linked to pathogenic SMAD4-dependent JP [SMAD4, while no alterations were found in STK11, the PJS driver. While one somatic variant was a novel finding, the other three variants, including the germline one, were previously classified as variants of uncertain clinical significance due to lack of data. However, they were detected in patients featuring the upper gastrointestinal polyps characterizing SMAD4-dependent JP . Immunohistochemistry for SMAD4 showed that the somatic variants and the germline one complemented each other, leading to the complete loss of SMAD4 expression in the epithelial cells of the polyps, while inflammatory and stromal cells showed intact expression of the protein. Sequencing of cDNA from non-neoplastic gastroduodenal tissue of the patient showed that the presence of the germline variant caused loss of expression of one of the SMAD4 alleles. Therefore, the integration of histology, NGS, cDNA sequencing and immunohistochemistry allowed to change the classification of the three variants from class 3 (unknown significance) to class 4 (probably pathogenic), especially that of the germline splice site variant c.1139 + 3A > G. This calls for surveillance and genetic testing of relatives of the JP patients bearing these variants, also considering the high risk of gastrointestinal malignancy displayed by SMAD4-JP patients [Juvenile polyps usually present in the first two decades of life, although at least 15% are found in adults as in our case . Our patndent JP , 47, whipatients , 48. Thipatients , 48. At cDNA sequencing and immunolabeling of histological samples. This allowed not only to exclude other possible diseases, but also to address the patient’s treatment. Indeed, JP cases with SMAD4 variants feature worse symptoms, including the frequent presence of high-grade adenomatous lesions and malformative vessels compared to cases with BMPR1A variants or no variants [While germline testing has been used in the past to profile probands for JP and other polyposis syndromes, the use of a multigene NGS panel and the simultaneous analysis of germline DNA and samples from the polypoid lesions was herein demonstrated to allow a better resolution of the pathogenetic mechanism, permitting the simultaneous identification of both germline and somatic alterations, whose effects could be verified by variants . Since rRNF43 variants in the intestinal polyp we analyzed. RNF43 inactivating variants have been reported in several gastro-entero-pancreatic neoplasms, including colon adenocarcinoma, pancreatic adenocarcinoma, and intraductal papillary mucinous neoplasms of the pancreas [RNF43 is a ubiquitin E3 ligase that acts as a tumor suppressor via negative modulation of the Wnt pathway by targeting the frizzled receptor [RNF43-associated serrated polyposis cases [SMAD4 variants, and the fact that RNF43 was mutated solely in the intestinal sample, these somatic variants probably constituted a secondary event marking disease progression after the onset of the disease by SMAD4 inactivation. However, since 60% of cases still have no driver, these finding still suggest how using a NGS panel could help find new clues in orphan cases at a reasonable cost compared to heavier genetic screening approaches.The application of a comprehensive NGS panel to polyposic lesions is also attractive when considering that roughly 60% of JP cases lack a driver germline alteration –18. Indepancreas –51. RNF4receptor . Its inais cases . As for In conclusion, we showed how a combined approach featuring the integration of histology, comprehensive targeted NGS and immunohistochemistry can allow the identification of germline and somatic alterations leading to the correct diagnosis of a hamartomatous polyposis; this case represents a next-generation model to approach patients with polyposis, even opening innovative horizons for the identification of new driver variants.Supplementary Figure 1. Intra-gastric laparoscopic view of the polypoid lesion of the upper stomach (A). Intraoperative laparoscopic view of the first jejunal loop harboring multiple polypoid lesions (B) (TIF 510 kb)Supplementary Figure 2. Genotype of three control individuals for the polymorphisms rs140241965 and rs3819122 located in the 3’UTR of the SMAD4 gene, as derived from deep sequencing of DNA from gastroduodenal non-neoplastic tissue. Ctrl1 is homozygous reference compared to the hg38 version of the human genome, Ctrl2 is heterozygous and Ctrl3 is homozygous variant (TIF 1781 kb)Supplementary Figure 3. cDNA amplification of a 425bp region of SMAD4 3’UTR including polymorphisms rs140241965 (5bp deletion) and rs3819122 (A>C transversion) demonstrates loss of heterozygosis in the cDNA of a patient heterozygous for the polymorphisms and the c.1139+3A>G splice site variant. A) electropherogram of the amplicons, showing a longer amplicon for the wild-type allele and a shorter one for the variant allele. B) Densitometry of each electrophoretic band. Ctrl1: cDNA of a control individual bearing none of the variants. Ctrl2: cDNA of a control individual bearing heterozygous rs140241965 and rs3819122 but not the splice site variant. Ctrl3: cDNA of a control individual bearing homozygous rs140241965 and rs3819122 but not the splice site variant. Patient: cDNA of the patient bearing heterozygous rs140241965 and rs3819122 and the splice site variant. Despite the patient has heterozygous genotype, the cDNA only displays the longer amplicon for the wild-type allele, showing that the mature mRNA for the variant allele is not being produced (TIF 1164 kb)Supplementary Table 1: Targeted genes in the CORE sequencing assay (PDF 42 kb)Below is the link to the electronic supplementary material.


Increasingly large heat flux and high heat dissipation makes transferring the heat generated by electronic devices such as central processing units and hard disk drives difficult . Loop heThere are several excellent materials for wicks, such as nickel, titanium, polymeric, ceramic, copper and stainless steel, while those made of carbonyl nickel powder possess the highest purity, controllable porosity and pore size, appropriate mechanical properties, moderate thermal conductivity, and compatibility with various working fluids. Hence, carbonyl nickel powder is widely used to fabricate high-performance wicks ,7,8.To fabricate high permeability wicks with carbonyl nickel powder, pore forming agents and loose sintering processes are usually adopted, and a comparatively high sintering temperature, which is normally higher than 550rticular ,9,10,11.rticular  investigIn order to manufacture large capillary pressure wicks, the method of sintering after pressing carbonyl nickel powder without pore forming agents is frequently adopted. Wicks fabricated in this method are praised for their fine pores and high strength, although their porosity and permeability are somewhat low. For LHPs, the maximum pore diameter in wicks is vitally important because there is backflow of vapor from the evaporation zone to the compensation chamber through the pore with the largest diameter due to the inverse dependence of capillary pressure on pore diameter . This kiSintering processes significantly affect the pore structure and properties of porous materials. In sintering processes, sintering necks form and grow large between particles while porosity and permeability simultaneously decrease . InitialLunin and Kostornov  made cylIn this work, we fabricated large capillary pressure wicks with carbonyl nickel powder by cold isostatic pressing followed by sintering, and studied the effects of sintering temperature on their hydraulic diameter, permeability and capillary performance. In order to analyze the changes in permeability and hydraulic diameter, the porosity and volume-specific surface area were determined, and we then investigated the fracture morphology and cross-section morphology of the wicks. Additionally, we established an evolution model of hydraulic diameter and validated it using the parameter of maximum pore diameter. This work promotes understanding of the increase in pore size and permeability during sintering carbonyl nickel powder, and also provides insight into methods of fabrication for large capillary pressure wicks at an optimal sintering temperature.Commercial carbonyl nickel powder with a mean diameter of 1.78 μm was chosen as the raw material, and its bulk density was 0.35 −1 were used to determine the permeability according to ISO 4022: 2018, where nitrogen passed through samples at flow rates ranging from 50 mL·min−1 to 500 mL·min−1. Absolute alcohol, nitrogen and a digital pressure gauge with an accuracy of ±1 kPa were used to determine the bubble point pressure Permeability of green slices and wicks was measured and used together with maximum pore diameter to calculate capillary performance. Digital pressure gauges with an accuracy of −14 m2, 1.8 kPa, 0.04 mm, 0.005 g and 10 mm3, respectively. In addition, the relative uncertainty for measurement of the surface area was 5%. Using the standard error analysis method, the uncertainties of maximum pore diameter and radial sintering shrinkage ratio were 0.02 μm and 0.3%, respectively, and those of porosity and volume-specific surface area were within 0.6% and 0.18 × 106 m−1, respectively.The uncertainties for measurement of the permeability, bubble point pressure, diameter, mass and volume were estimated to be 0.05 × 10High permeability enhances the heat transfer limit of LHPs because it reduces pressure drop of fluids while filtering through wicks during the operation of LHPs . The perGrain-boundary diffusion dominates in the sintering shrinkage of wicks in the presence of low temperatures and small particle diameters . The sinTo study the change in volume-specific surface area, we observed the microstructure of green slices and wicks by SEM. Moreover, clear sintering necks formed between particles at 400ar pores . As showSurface diffusion causes the decrease in roughness of particle surfaces, and grain-boundary diffusion together with surface diffusion contributes to the formation and growth of sintering necks. In the sintering process, atoms on rough surfaces move from a convex point to a concave point due to the chemical potential gradient introduced by the curvature gradient, leading to the decrease in roughness of particle surfaces . MoreoveGrain-boundary diffusion also contributes to the growth of sintering necks. Vacant lattice points diffuse away from sintering necks and sink at the grain boundary, which results in sintering shrinkage and neck growth ,32. As sThe decrease in roughness of particle surfaces and the growth of sintering necks together contribute to the decline in volume-specific surface area . The mos2 and 0.010 μm, respectively. Using the standard error analysis method, the uncertainty of the mean pore diameter was within 0.015 μm.For a unique pore with an irregularly shaped cross section, the hydraulic diameter tion (5)  as:(5)dhThe decrease in the roughness of particle surfaces a–f smootAt sintering temperatures of 400Maximum pore diameter is defined as the maximum among all the hydraulic diameters of the most constricted part in every through pore . The studiameter . The wic−14 m2 at 400ace area  and the ace area  barely c−8 m. The capillary performance first increased to the maximum at 400−8 m. When the wicks were sintered at 400Capillary performance parameter is usually defined as the ratio of permeability to pore diameter ,35. In t of LHPs . Figure Based on the pore microstructure and porous properties of large capillary pressure wicks sintered at different temperatures, a summary is proposed:(1)The pore size first increased and then decreased with increasing sintering temperature from 200(2)The evolution model of hydraulic diameter with increasing sintering temperature was established and validated. In the contact-formation stage and the initial sintering stage (200–500(3)−14 m2 and their highest capillary performance of 1.46 × 10−8 m.The optimal sintering temperature proved to be 400


MAS: p < 0.001). ROC analysis showed a sufficient prediction of sepsis with limited specificity . The plasma levels of sDLL correlated closely with renal parameters , resulting in a good predictive performance of sDLL1 for the identification of acute kidney injury . By quantifying the plasma concentration of sDLL1, sepsis can be discriminated from the physiological postsurgical inflammatory response in abdominal and cardiac surgical patients. However, sDLL1 has only limited specificity for the detection of sepsis in cardiac surgical patients, which may be explained by impaired renal function. Based on these findings, this study identifies the predictive value of sDLL1 for the detection of AKI, making it a potential biomarker for surgical intensive care patients.The early identification of sepsis in surgical intensive care patients is challenging due to the physiological postoperative alterations of vital signs and inflammatory biomarkers. Soluble Delta-like protein 1 (sDLL1) may be a potential discriminatory biomarker for this purpose. For this reason, this study aimed to evaluate sDLL1 for the identification of sepsis in a cohort of surgical intensive care patients. This study comprises a secondary analysis of a prospective observational study including 80 consecutive patients. The study groups included 20 septic shock patients, 20 patients each undergoing major abdominal surgery (MAS) and cardiac artery bypass surgery (CABG), and 20 matched control subjects (CTRL). The surveillance period was 72 h. The plasma concentration of sDLL1 was measured with ELISA. The plasma levels of sDLL1 were significantly elevated in septic patients compared to both surgical cohorts , registration date 11.07.2018, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00013584. Despite great research efforts regarding innovative strategies for the management of sepsis, its early detection and the rapid initiation of treatment remain the most effective approaches to reduce sepsis-associated mortality2. Unfortunately, surgical patients are at risk for delayed diagnosis of sepsis because typical symptoms  are masked by postsurgical reactions. Furthermore, the established inflammatory biomarkers, such as C-reactive protein (CRP) are also altered by major abdominal or cardiovascular surgery6. To address this clinical problem, several biomarkers have been assessed but only a minority have been validated sufficiently and even fewer have been evaluated in surgical intensive care patients7. To date, no biomarker with sufficient discriminatory power for the differentiation of sepsis from postsurgical systemic inflammation has been introduced into the daily clinical routine8.Sepsis is a life-threatening complication of major abdominal and cardiovascular surgery and is associated with increased morbidity and mortality9. DLL1 is part of the Delta/Jagged family of transmembrane proteins and functions as an activator of intracellular pathways during embryonic angiogenesis and hematopoiesis11. However, it has been reported that DLL1 is also associated with cancerous and infectious diseases14. In particular, the interaction of DLL1 and human monocytes during in vitro bacterial infections raised interest in its use as a biomarker for infection, and consequently, for sepsis. It has been shown that DLL1 activates a cellular immune response via notch signaling, resulting in the release of proinflammatory cytokines14. DLL1 is further involved in sepsis-induced endothelial damage leading to a loss of its barrier function15. Since the endothelium plays an important role for the recognition of pathogens but also for vascular integrity, DLL1 might contribute to the pathological host response during sepsis16. Even though DLL1 has not yet been specifically investigated in this context, the notch signal pathway is of reasonable interest for sepsis research because it modifies T-cell dysfunction and might be associated with sepsis-induced immunosuppression19. Moreover, the pathophysiological effect of DLL1 on human monocytes is of particular interest because these cells play a pivotal role in the development of sepsis. Hildebrand et al. recognized DLL1 as a potential discriminatory biomarker for sepsis and, therefore, quantified soluble DLL1 (sDLL1) as a surrogate for DLL1 receptor activation9. sDLL1 originates from the enzymatic degradation of the DLL1 receptor and its ligand and was measurable after in vitro bacterial infection. The authors could demonstrate that the diagnostic accuracy of sDLL1 for the identification of sepsis is superior compared to the established biomarkers, namely CRP, leukocytes, and procalcitonin (PCT). The results were also remarkable because the study sample consisted of heterogenous cohorts of patients suffering from systemic inflammation from different sources, such as sepsis, major surgery, or severe trauma. Nevertheless, these results have not yet been confirmed in other studies, and furthermore, sDLL1 has not been evaluated in cardiac surgical patients thus far. For this reason, this study aimed to describe the course of sDLL1 in cardiac and major abdominal surgical patients as well as in septic shock patients and healthy controls.In 2019, Delta-like protein 1 (DLL1), a host-derived canonical Notch ligand, was identified as a novel diagnostic biomarker with promising results regarding its discriminatory value in this context20. The study is registered in the German Clinical Trials Register  and was approved by the local ethics committee . Both the original study and this secondary analysis were performed in accordance with the Helsinki Declaration, and all methods and results are presented in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines.This secondary analysis is based on a single-center, prospective, observational proof-of-concept study of 80 patients21. All surgical patients suffered postoperatively from systemic inflammatory response syndrome (SIRS)22. Furthermore, a control group  for the septic shock patients was established by matching for age, gender, and underlying health conditions .After obtaining each patient’s informed consent, 80 consecutive patients were recruited between October 2018 and March 2019. Patients were excluded if they were pregnant or nursing, younger than 18 years, had a history of severe valvular heart, autoimmune, and/or hematological disease, had recently suffered severe trauma, or were in need of immunomodulatory medication, extracorporeal membrane oxygenation, or renal replacement therapy. While septic patients (n = 20) had to meet the Sepsis-3 criteria for septic shock, the surgical study groups included either patients undergoing coronary artery bypass graft surgery  or major abdominal surgery Blood was collected at onset, 24 h, and 72 h from septic shock patients, whereas the blood of subjects in the CTRL group was only drawn once. Surgical patients were investigated pre- and postoperatively, and after 24 and 72 h. Blood was originally drawn in ethylenediaminetetraacetic acid (EDTA) tubes and stored at − 80 °C. Clinical data including the inflammatory and renal laboratory parameters were obtained from the patient data management system .The plasma concentration of sDLL1 was quantified using a commercially available enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer’s instructions. To fit measurements into the calibration curves and simultaneously reduce interfering matrix effects, all samples were diluted 1:30 (or higher if demanded by the concentration) with the supplied Assay Diluent A prior to the measurements. An ELx808 microplate reader  was used for absorbance measurements, with a subsequent automatized calculation of concentrations by the corresponding Gen5 software .p Values < 0.05 were considered statistically significant. Potential correlations between plasma sDLL1 levels and inflammatory and renal parameters were analyzed by Pearson’s correlation coefficient. Receiver operating characteristic (ROC) curve analyses were used for the calculation of area under the ROC curve (AUCROC), sensitivity, and specificity of inflammatory and renal parameters. All data were stored in an external database . Data were analyzed using R statistical software version 4.0.4 .While parametric data were described as mean and standard deviation, non-parametric data were expressed as median and interquartile range (IQR). The analysis of variations in plasma sDLL1 levels across different time points was performed by the repeated measures ANOVA test, followed by the Tukey HSD test for paired groups (septic shock group compared to matched controls). All included patients approved to their study participation.20). 70% (n = 14) of the patients in the shock group, 75% (n = 15) in the CABG group, 60% (n = 12) in the MAS group, and 70% (n = 14) in the CTRL group were male. The ages (in years) of each group (expressed as the median [IQR]) were 69 [64–74] for septic shock, 70 [62–79] for CABG, 68 [54–70] for MAS, and 69 [66–74] for CTRL. The severity of sepsis was indicated by the sepsis-related organ failure assessment score (SOFA) score , leading to an in-hospital-mortality rate of 35% (n = 7). Sepsis originated from intraabdominal infections in 60% (n = 12) of cases, whereas a pulmonary or urological source of infection was identified in 15% of cases (each n = 3) and a soft-tissue infection was detected in 10% (n = 2) of cases. MAS included Whipple’s procedure in 40% (n = 8) of patients, whereas open partial colectomy and esophagus resection were each performed in 20% (n = 4) of patients, and other types of MAS accounted for 20% (n = 4) of patients.The patient characteristics of the 80 included patients did not differ significantly regarding their age and sex , lactate plasma concentration , and inflammatory parameters .Among surgical patients in need of intensive care, the performance of plasma sDLL1 concentration was not superior to CRP but superior to leukocyte count regarding the prediction of sepsis. The number of PCT values within the surgical cohorts was too low for ROC analysis Fig. , Table 2r2 = 0.37, p < 0.0001; urea: correlation coefficient = 0.52, r2 = 0.26, p < 0.0001; glomerular filtration rate [GFR]: correlation coefficient = − 0.61, r2 = 0.37, p < 0.0001; Fig. The plasma concentration of sDLL1 showed a strong correlation with all renal parameters , the levels of sDLL1 increased more than in abdominal surgical patients, explaining its diminishing difference to the levels in septic patients after 24 h. Moreover, cardiac surgical patients also featured an elevated sDLL1 compared to the CTRL group, which was not observed in MAS patients. To summarize, regarding abdominal surgical patients, the results of this study confirm the findings of Hildebrand et al. while revealing that cardiac surgical patients differed from MAS patients in terms of higher sDLL1 plasma levels. To the best of our knowledge, the kinetics of the plasma concentration of sDLL1 have not yet been described in cardiac surgical patients and should therefore also be assessed in future studies. Second, the predictive value of sDLL1 for the detection of sepsis was sufficient, but it did not perform superiorly to CRP. This is mainly due to the limited specificity of sDLL1, which may be explained by the increase in sDLL1 levels in cardiac surgical patients. To summarize, these results indicate that the use of sDLL1 for discrimination between sepsis and physiological postoperative inflammatory reactions may be impaired in cardiac surgical intensive care patients, and leaves the question of how they differ from patients after MAS.24. Since cardiac surgical patients are at increased risk of renal failure, we also searched for a correlation between the plasma levels of sDLL1 and surrogate parameters of renal function to potentially explain the elevation of sDLL1 following CBP25. Indeed, this study was able to confirm the association of sDLL1 with renal parameters by showing a strong positive correlation between sDLL1 and the plasma creatinine and urea concentration as well as a negative correlation to the GFR. These findings indicate that CPB-induced AKI may cause increased sDLL1 plasma levels. However, despite this strong hint, the underlying causative mechanisms, such as an accumulation of sDLL1 caused by impaired renal function, have not yet been identified. Even though the interaction between sDLL1 and renal function limits its specificity for the detection of sepsis, it may be useful for the identification of AKI. With a prevalence of up to 60%, AKI represents a major burden in critically ill patients27. Even though the definition of AKI has been adapted and novel biomarkers have been introduced, its rapid identification still poses a clinical challenge. The plasma concentration of sDLL1 showed high sensitivity and specificity for the prediction of AKI stages ≥ 1 in surgical intensive care patients, independent of the presence of surgery- or CPB-induced SIRS or sepsis as the underlying condition. It performed better than urea and was comparable to creatinine and GFR. Even though novel renal biomarkers have not been investigated in this study, the high predictive value of sDLL1 is promising. For example, the Translational Research Investigating Biomarkers and End Points for Acute Kidney Injury (TRIBE-AKI) study investigated urine and plasma neutrophil gelatinase-associated lipocalin  in cardiac surgical patients and revealed only a moderate predictive performance of these well-investigated biomarkers 28. Comparable predictive capacity has also been shown in mixed cohorts of critically ill patients30. Furthermore, a recent meta-analysis reported an AUCROC of 0.75 for uNGAL for the identification of severe AKI31. Plasma concentration of sDLL1 performed even better than the product of tissue inhibitor of metalloproteinase 2 and insulin-like growth factor-binding protein-7  that has already been approved for the prediction of AKI in critically ill patients 35. However, it is worth noting that this study is the first to report the predictive value of sDLL1 in the context of renal failure. For this reason, the AUCROC cannot be directly compared to the high number of patients included in the mentioned studies and meta-analyses. Nevertheless, it justifies further research in surgical intensive care patients, especially in cardiac surgical patients. Apart from the causative mechanisms of the release of sDLL1 during renal injury, future research should focus on kinetics to evaluate sDLL1 as an early predictor of AKI.Norum et al. investigated the diagnostic value of plasma sDLL1 levels in patients suffering from dilated cardiomyopathy. Along with a good correlation of sDLL1 with disease severity and cardiac function, they identified an association with renal parameters such as creatinine and GFR14. Finally, as mentioned previously, this study does not provide insights into the underlying causative mechanisms of sDLL1 release in surgical intensive care patients.This study has some limitations. First, due to its design as a secondary analysis of an observational study, the plasma concentration of PCT was not available for all patients, making an ROC analysis and comparison to sDLL1 unfeasible. For the same reason, it was not possible to measure the concentration of sDLL1 in urine, which would be of high interest in this context. Third, only patients suffering from septic shock were included in this study. It is also worth investigating whether the identified differences between septic and surgical patients are also present in patients with a lower degree of disease severity. Furthermore, the majority of patients suffered from an abdominal source of sepsis; however, since pneumonia is a common source of sepsis in non-surgical patients, future studies should also include patients with other origins of sepsis in order to detect potential differences. Fourth, sDLL1 identifies only bacterial infections, leaving a blind gap in the detection of viral and fungal infectionsIn summary, this study could underline the potential diagnostic value of sDLL1 for the discrimination of sepsis from surgery-induced systemic inflammation within the first 24 h after intensive care unit admission. However, for the detection of sepsis, sDLL1 exhibited only limited specificity in cardiac surgical patients, which may be explained by the close association between the impaired renal parameters and increase in sDLL1. These findings have led to the identification of the high sensitivity and specificity of sDLL1 for the detection of AKI in this study, making it an interesting option for surgical intensive care patients.


These ether glycerolipids are present either as polar glycerophospholipids or neutral glycerolipids. Before studying the biological role of molecular species of ether glycerolipids, there is a need to separate and quantify total alkyl and alkenyl glycerolipids from biological samples in order to determine any variation depending on tissue or physiopathological conditions. Here, we detail the development of the first high-performance thin-layer chromatography method for the quantification of total alkyl and alkenyl glycerolipids thanks to the separation of their corresponding alkyl and alkenyl glycerols. This method starts with a reduction of all lipids after extraction, resulting in the reduction of neutral and polar ether glycerolipids into alkyl and alkenyl glycerols, followed by an appropriate purification and, finally, the linearly ascending development of alkyl and alkenyl glycerols on high-performance thin-layer chromatography plates, staining, carbonization and densitometric analysis. Calibration curves were obtained with commercial alkyl and alkenyl glycerol standards, enabling the quantification of alkyl and alkenyl glycerols in samples and thus directly obtaining the quantity of alkyl and alkenyl lipids present in the samples. Interestingly, we found a differential quantity of these lipids in shark liver oil compared to chimera. We quantified alkyl and alkenyl glycerolipids in periprostatic adipose tissues from human prostate cancer and showed the feasibility of this method in other biological matrices .Ether lipids are composed of alkyl lipids with an ether bond at the  HereThe aim of this study was to develop a simple method to assess the total quantity of alkyl and alkenyl glycerolipids separately because we believe quantifying these two forms of ether lipids is important. This was achieved by developing a procedure consisting of a first step of reduction of all lipids, resulting in the reduction of alkyl and alkenyl glycerolipids to alkyl and alkenyl glycerols, whereas their ester analogues are reduced to glycerol. This procedure is followed by a step of separation by HPTLC of alkyl and alkenyl glycerols, followed by a step of densitometric quantification of alkyl and alkenyl glycerols, directly obtaining quantities of alkyl and alkenyl glycerolipids, respectively. The HPTLC method was validated following the guidelines provided by the International Conference on Harmonization (ICH)  to assessn-2 and sn-3 position, this method starts with a reduction of all alkyl and alkenyl glycerolipids to alkyl and alkenyl glycerols, respectively , and intermediate precision was assessed over a two-month period by several operators (interday assays). RSD values under 10% were obtained for alkyl glycerol ranging from 2000 to 7000 ng/band and under 20% below 2000 ng/band. For alkenyl glycerol, the RSD was under 20% for amounts ranging from 1000 to 7000 ng/band .These results show a satisfactory precision of the proposed method. Regarding accuracy, results from the recovery studies performed according to the method described by Pinault et al.  are presFor both ether glycerol species, recoveries were between 96.39 and 100.84%, with an RSD under 5%, indicating a satisfactory accuracy of the proposed method .Band identification was confirmed by performing acid hydrolysis see  on both 1H NMR, where the multiplets at 4.3 and 5.9 ppm corresponding to the vinylic protons, as described in the literature [The alkenyl glycerol band disappeared completely after acid hydrolysis on both the alkenyl glycerol standard and the sample, whereas the alkyl glycerol band remained present on HPTLC . This reterature ,27 and aA summary of the validation parameters can be found in p < 0.05. Moreover, the coefficient of determination r2 being close to 1 in both models suggests a good fit between the experimental data and the established models.Variance homogeneity between datasets was verified by analysis of variance (ANOVA) using Cochran’s C test, showing no significant difference of variance at a 95% confidence interval . The fitDifferent types of biological samples were tested to confirm that this method is suitable for samples with a complex matrix. Oils, adipose, muscle and tumor tissues were used. For analysis of adipose, muscle and tumor tissues, a purification by solid-phase extraction (SPE) was required due to lipid composition heterogeneity and the lower amount of ether lipids compared to marine oils. Purification by SPE was carried out after total lipid extraction and reduction to eliminate the fatty alcohols formed during the reduction and to obtain samples concentrated in ether glycerols . DiffereThe quantities of alkyl lipids and alkenyl lipids per mg of total lipids in each sample can be obtained with the quantities of alkyl and alkenyl glycerols measured by densitometric analysis on the HPTLC plates. After HPTLC analysis of the lipids extracted from oils A,C, we fIn human periprostatic adipose tissue (PPAT), tissues known to contain an important amount of lipids, less than 0.5% of lipids are ether lipids, with approximately the same amounts of alkyl lipids and alkenyl lipids: around 1.3 µg of each ether lipid species per mg of total lipids B,D. ThisWe also verified that our method was suitable for the analysis of heart and tumor tissues, two interesting physiopathological tissues for ether lipids .In non-human primate heart (NHP), ether lipids were found to represent less than 5% of total lipids, with almost only alkenyl lipids in our samples tested , consistThe proposed method is well designed to be used for a lipidomic study and in combination with an HPLC-MS/MS system. After total lipid extraction, a small part of the sample is readily processable by HPLC-MS/MS to identify the lipids in the sample. In addition, quantifications of other lipid subspecies can be performed by HPTLC  on a frasn-glycerol  (ref 852650P) were obtained from Sigma-Aldrich and Avanti Polar. Vitride® (sodium bis(2-methoxyethoxy)aluminiumhydride) (ref 196193), hydrochloric acid (ref 524525), oleyl alcohol  (ref 369314), deuterated chloroform (ref 151823), tetramethylsilane (ref 87920) and butylated hydroxytoluene  were purchased from Sigma-Aldrich . All solvents used were of analytical grade.1-O-Octadecyl-rac-glycerol  (ref B402) and 1-O-1′-(Z)-Octadecenyl-254 glass plates coated with a 200 µm layer of 60 Å silica gel . Plates were developed in a twin-trough glass chamber  and carbonized on a hot plate . Results were obtained with a TLC visualizer I  working under winCATS Planar Chromatography Manager software , and densitometry was performed with VideoScan TLC/HPTLC software .An automatic TLC sampler 4  equipped with a 25 µL syringe  was used for sample application under a stream of nitrogen on 20 × 10 cm Fv/v), air-dried and activated in an oven for 30 min at 110 °C. Standard and sample solutions were then applied by spray-on technique with an ATS4 sample applicator as 10 mm wide bands, 8 mm from the bottom and 15 mm from the edge of the plate. Application was performed at a rate of 150 nL/s under a stream of nitrogen at 6 bars to enable continuous drying, thus avoiding diffusion, permitting better separation and homogenous quantification. Linearly ascending development was carried out in a twin-trough glass chamber with a petroleum ether: diethyl ether: acetic acid  mobile phase prepared weekly. The glass chamber was first pre-equilibrated with the mobile phase at room temperature for 15 min without saturation paper. Following development, plates were air-dried for 120 min under a ventilated hood before being dipped for 50 s in a 7% sulfuric acid in absolute ethanol staining solution prepared daily. Plates were air-dried for another 120 min before carbonization for the staining reaction at 140 °C for 14 min. Pictures of the plates under white light illumination were obtained using a TLC visualizer, and densitometric analysis was performed with VideoScan.Before sample application, the 20 × 10 cm glass plates pre-coated with silica gel were developed once in chloroform: methanol  with 250 µM of BHT and stored at −20 °C. Alkenyl lipids are known to have very low stability against oxidation due to their sn-1 vinyl ether bond [Standard solutions of alkyl glycerol and alkenyl glycerol used for the quantification of alkyl and alkenyl glycerolipids, respectively, were prepared at 1 mg/mL in chloroform: methanol . Graphical representations were performed by using GraphPad Prism (version 6.01) . ® . The macaque was housed under conventional conditions in the animal facility  in accordance with the latest European legislation (Directive 2010/63/UE). The animal was placed under an experimental protocol of cigarette smoke exposition conducted according to NIH guidelines for the care and use of laboratory animals. This protocol was approved by the Ethics Committee of Val de Loire Univeristé and the Ministry of Higher Education and Research (Notification: APAFIS#14748-2018041713242393 v7).A whole NHP heart exposed to cigarette smoke was sampled, washed with PBS and stored at −20 °C before lipid extraction. Heart tissue came from a euthanized healthy pre-pubescent (3 years old) female cynomolgus macaque  weighing 3 kg and obtained from BioprimPPAT was sampled during a surgical procedure in 10 prostate cancer patients treated by radical prostatectomy at Tours University Hospital. During the surgical procedures, pieces of PPAT were sampled, sent to the biological resource center and immediately frozen at −80 °C. The patients included in the study signed an informed consent . The human cancer cell line MDA-MB-435s, obtained from the American Tissue Culture Collection, was grafted in mice to obtain an orthotopic mammary-tumor model following the protocol described by Chantôme et al. [No lipid extraction was necessary for oils.v/v) and 1 volume of aqueous sodium chloride 0.73% was first put in an ultrasonic bath (Branson 5510) at room temperature for 20 min. After centrifugation , the organic layer containing the lipids was filtered on cotton, dried on anhydrous sodium sulfate and evaporated.Lipids from PPAT, mostly neutral lipids, were extracted according to a method adapted from Folch et al. . Brieflyv/v) on ice using an Ultra-Turrax homogenizer  at 24000 rpm for 30 s to 3 min per sample to obtain a homogenous sample. This solvent enables membrane disorganization, with the denaturation and precipitation of most proteins and the solubilization of lipids. After centrifugation, , the obtained supernatant was transferred to a second tube, whereas the pellet was extracted again in chloroform: methanol  to recover more lipids. Chloroform: water  was then added to both tubes for phase partitioning. The lower chloroform phases containing the lipids were then filtered on cotton, dried on anhydrous sodium sulfate and evaporated.NHP heart and tumors containing a high amount of membrane lipids were extracted according to a method adapted from Bligh and Dyer . A preliv/v) with 250 µM of BHT, closed under nitrogen and stored at −80 °C.After evaporation, total lipids were weighted to proceed immediately to lipid reduction and avoid the oxidation of alkenyl lipids. The amount of tissue used and the total lipids recovered after extraction can be found in ® was added to 10 mg of extracted lipids in 5 mL of diethyl ether: n-hexane , closed under nitrogen and reacted for 30 min at 37 °C, with vortexing for 5 s every 10 min. The reduction was stopped on ice by slowly adding 8 mL of ethanol: water , obtaining ether glycerols  and fatty alcohols , the organic layer was filtered on cotton, dried on anhydrous sodium sulfate, evaporated and diluted in 750 µL n-hexane. The amount of total lipids used for reduction and the corresponding amount of reduced lipids recovered can be found in Reduction of the ester and phosphate bonds was then carried out according to the method described by Pinault et al. . Brieflyalcohols . After cv/v; first fraction containing fatty alcohols), followed by 5 mL n-hexane: dichloromethane  and then 6 mL dichloromethane: diethyl ether  (third fraction containing reduced ether lipid species) and, in the same tube, 6 mL diethyl ether: ethyl acetate . Finally, the third fraction was concentrated under vacuum and solubilized in chloroform: methanol  before analysis by HPTLC. The solubilization and deposited volumes can be found in A purification step by SPE was performed to remove impurities after reduction. The purification conditions were optimized for amounts of reduced lipids ranging from 10 to 30 mg , dependiBiological samples were quantified by HPTLC immediately after extraction and purification to avoid oxidation of the alkenyl lipid species.v/v) and 150 µL of water were added to extract the hydrolyzed reduced lipids. The organic phase was recovered and evaporated. The samples before and after acid hydrolysis can be analyzed both on HPTLC and in 1H NMR to verify the disappearance of the alkenyl glycerol band after hydrolysis on HPTLC and the disappearance of the multiplets corresponding to the chemical shift for both vinylic protons in 1H NMR.Band identity confirmation of the alkyl and alkenyl glycerols was verified by first performing acid hydrolysis, a method specific to ether lipids adapted from the literature ,24,25. A1H NMR spectra were obtained on a 300MHz spectrometer  in deuterated chloroform (CDCl3) with 0.03% tetramethylsilane commercial standard of 1-O-1′-(Z)-Octadecenyl-sn-glycerol : 1H NMR (CDCl3) δ 5.97 , 4.29 , 3.73 , 3.52 , 2.02 , 1.26 , 0.87 .We developed a simple HPTLC method for the quantification of total alkyl and alkenyl glycerolipids thanks to the separation of alkyl and alkenyl glycerol. This method was successfully used to quantify alkyl glycerolipids and alkenyl glycerolipids in oils and adipose tissues. We showed that it could also be applied to matrices, such as muscle and tumor tissues. It should be noted that a sufficient amount of lipids is required, especially for samples containing a low amount of ether lipids.