MCTs are increasingly being used to promote ketogenesis by patients on ketogenic diet therapy, but also by people with other conditions and by the general public for the perceived potential benefits. However, consumption of carbohydrates with MCTs and untoward gastrointestinal side effects, especially at higher doses, could decrease the sustainability of the ketogenic response. This single-center study investigated the impact of consuming carbohydrate as glucose with MCT oil compared to MCT alone on the BHB response. The effects of MCT oil versus MCT oil plus glucose on blood glucose, insulin response, levels of C8, C10, BHB, and cognitive function were determined, and side effects were monitored. A significant plasma BHB increase with a peak at 60 min was observed in 19 healthy participants (24.4 ± 3.9 years) after consuming MCT oil alone, and a more delayed but slightly higher peak was observed after consuming MCT oil plus glucose. A significant increase in blood glucose and insulin levels occurred only after MCT oil plus glucose intake. The overall mean plasma levels of C8 and C10 were higher with the intake of MCT oil alone. MCT oil plus glucose consumption showed improved scores for the arithmetic and vocabulary subtests. Medium-chain triglycerides (MCTs) are fats primarily made up of fatty acids with carbon chain lengths ranging from C6-C12 ,4,5,6. HLCFAs: long-chain fatty acids; MCFAs: medium-chain fatty acids; CD36:  is a member of the class B2 scavenger receptor family; FATPs: fatty acid transport proteins; CPT1: carnitine palmitoyltransferase 1; CPT2: carnitine palmitoyltransferase 2; BHB: β-hydroxybutyrate; AcAc: acetoacetate; CAC: citric acid cycle; HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA.Nineteen healthy volunteers  were recruited by advertising at the university hospital in Muenster (UKM), Germany, and gave written, informed consent to participate in this crossover study. Inclusion criteria were age 18–30 years, being non-overweight and non-obese (≤25 BMI), not being diagnosed as diabetic, not currently or previously following a ketogenic diet, and not consuming any MCT-based supplements (MCT oil or coconut oil). Baseline characteristics of the participants are described in For test day 2 (MCT intervention), MCT was provided to participants using a commercially available MCT oil  with a 60:40 ratio of C8:C10 (0.5 g MCT/kg of body weight in 200 mL of still drinking water). We used this dosage as it was most likely to elicit an effect without unduly exposing participants to gastrointestinal discomfort, which was more likely to arise with higher MCT intake . For tesThis was a single-center study carried out at UKM, Germany. The study followed a repeated-measures design involving three separate test days at the exact same time during four consecutive weeks. All three study days were identical for each participant, as shown in 2O 1:1 (v/v), 200 µL H20, 100 µL Methyl-t-butyl ether, and 50 µL n-hexan in a 0.5 mL Eppendorf reaction vessel and centrifuged . The supernatant (50 µL) was transferred to an autosampler vial with a conical insert, and 1 µL was loaded in splitless mode onto a GC-MS  equipped with a PTV injector and a capillary column  and an autosampler AS AOC-20 s. Measuring time: 58.7 min; injector temperature: 70 °C; column oven temperature program isothermal: 2 min at 40 °C; gradient 12°C/min to 240 °C; isothermal 40 min at 240 °C. The carrier gas was helium with a constant flow of 1 mL/min. The transfer line to the ion source was held at 260 °C. The MS detector was set to selected ion monitoring (SIM) mode as m/z 60 (C8/C10) and m/z 63 . An example base peak intensity chromatogram of free fatty acids is presented in Blood samples were centrifuged for 10 min at 4 °C and plasma was stored at −80 °C until analyzed. Glucose, insulin, ketones, and C8 and C10 were measured in accordance with routine procedures at the university hospital lab. Plasma glucose was analyzed by using the enzymatic hexokinase method with an automated clinical chemistry analyzer, Cobas c702 . Plasma insulin was measured by an electrochemiluminescence immunoassay on a Cobas e801 analyzer . Plasma β-HB, AcAc, total ketones were quantified enzymatically using the Total Ketone and BHB autokit  on a Cobas integra 400 plus analyzer . The determination of plasma C8 and C10 fatty acid levels by gas chromatography-mass spectrometry was performed according to a modified method of Bartolucci et al. . For thiA questionnaire on satiety and tolerance was used immediately after t1 and t7 on each study day. The questionnaire was designed for this study, using a 0–10-point scale, from 0 = not satiated at all to 10 = very satiated. At the end of the questionnaire, participants were asked to record any side effects , and any symptoms were recorded hourly during testing.Cognitive performance was measured using the Wechsler Adult Intelligence Scale, 4th edition (WAIS-IV). The WAIS-IV is a classic tool to assess general intelligence as well as four subcategories, including verbal comprehension, fluid reasoning, working memory, and processing speed. In the current study, full WAIS-IV assessment was not employed due to the long completion time. We used two “vocabulary” subtests as part of the Verbal Comprehension Index (VCI), which is designed to measure verbal reasoning and concept formation. The vocabulary subtest required the participant to try to define up to 30 words. This test was performed at t2. The second subtest was “arithmetic,” part of the Working Memory Index (WMI), which was designed to assess the ability to sustain attention, concentrate, and exert mental control. This subtest consisted of 22 timed arithmetic questions to be solved without the use of pencil and paper, such as: “If Jo has 12 buns, he then eats 3 and gives 4 away; how many does he have left?” The arithmetic subtest was performed at t7. The vocabulary and arithmetic subtests were chosen because they are close to everyday cognitive performance and easy to administer, both loading highly on their underlying factors “verbal comprehension” (vocabulary) and “working memory” (arithmetic), with arithmetic being largely independent from verbal comprehension according to Ward et al. . Both subtests were performed under the supervision of a physician (time needed to administer the subtest: 10 min per subtest), as shown in p < 0.05. Metabolic data (plasma metabolites) did not follow a normal distribution and were therefore analyzed using non-parametric methods; the non-parametric Mann–Whitney-U/Wilcoxon test was used. The level of statistical significance was set at p < 0.05. Spearman’s correlation test was used for correlations. p < 0.05 was considered to indicate a statistically significant difference. Linear regression analysis was carried out to determine the relationship between plasma C8 and C10 levels and plasma BHB levels by using the statistical programming language R version 4.2.1.Statistical analyses were performed using GraphPad Prism version 9.3.1  and the R statistical programming language , 7 males (37%)] completed the study in full. Total median MCT intake was 32 (30–35.5) g during the MCT oil intervention, and total median glucose intake was 12.7 (11.8–13.8) g during the MCT oil plus glucose intervention. Nutritional analysis of reported pre-study meals and drinks  on each test day, calculated by a dietician, confirmed no differences in results for the individual participants . In all p = 0.003), t3 (p = 0.005), t4 (p = 0.004), t5 (p = 0.006), t6 (p = 0.005), and t7 (p = 0.03). The median (IQR) BHB at baseline was 50.8 (30.7–105.7) µmol/L, which increased to 141.2 (49.9–241.4) after 30 min, to 348.8 (204.4–408.4) µmol/L at 120 min, to 417.8 (266.4–473.7) µmol/L at 180 min, and to 465.4 (354.8–509.3) µmol/L at 240 min . Signifip = 0.11); however, the increase in BHB levels from MCT oil alone was significantly greater at 60 min compared to MCT oil plus glucose (p = 0.0004), and levels of BHB were similar at 120 min for MCT alone and MCT plus glucose (p = 0.81). Thus, the ketogenic response of MCT plus glucose was delayed compared to MCT alone but reached similar final concentrations. The difference in BHB levels was not statistically significant between MCT oil alone and MCT oil plus glucose at 30 min , t5 (p = 0.0001), t6 (p = 0.0001), and t7 (p = 0.001). The median (IQR) BHB at baseline was 73.8 (27.9–192.3) µmol/L, which increased to 374.9 (235.1–511.8) µmol/L at 120 min, 381.7 (252.6–644.2) µmol/L at 180 min, 453.7 (357.2–722) µmol/L at 240 min and 524.6 (377–617) at 300 min . SignifiThe median (IQR) glucose level at baseline was 72 (65–76) mg/dL, which slightly increased with prolonged fasting throughout the experimental period A.p = 0.0005). The median glucose level then decreased to 64 (55–72) mg/dL at 60 min and increased to 68 (63–71) mg/dL at 90 min  glucose at baseline was 68 (65–76) mg/dL, which significantly increased to 93 (85–111) mg/dL after 30 min (t 90 min A. p = 0.8). Median insulin fell to 4.7 (2.9–5.9) µU/mL by 60 min. The lowest median insulin was measured at 2.5 (0.5–3.4) µU/mL after 300 min (The median (IQR) insulin level was 4.3 (2.3–6 µU/mL) at baseline, which increased to 5.1 (1.9–6.7) µU/mL after 30 min; however, this difference was not statistically significant compared to the control intervention ( 300 min B.p = 0.0003). Median insulin decreased to 4.1 (2.7–7) µU/mL at 120 min (p = 0.002) and 300 min (p = 0.04).The median (IQR) insulin level was 4.7 (3.4–7) µU/mL at baseline, which significantly increased to 17.9 (12.6–28.1) µmol/L after 30 min compared to MCT oil intake ( 120 min B. InsuliPlasma octanoate (C8) and decanoate (C10) levels were not measured during the control intervention, which did not contain C8 or C10. Plasma octanoate (C8) and decanoate (C10) concentrations during the MCT oil and MCT plus glucose interventions are presented in The median (IQR) C8 level was 1.2 (1–1.5) µg/mL at baseline, which increased to 4.5 (1.8–12) µg/mL after 30 min and to 16.52 (7.4–27.8) µg/mL at 60 min. The median (IQR) C10 level was 0.85 (0.68–0.94) µg/mL at baseline, which increased to 1.5 (1.8–12) µg/mL after 30 min, to 2.64 (1.46–4.08) µg/mL at 60 min and to 3.21 (1.79–51.5) µg/mL at 90 min. The median (IQR) C8 level was 1.2 (0.9–1.5) µU/mL at baseline, which increased to 2.1 (1.5–6.5) µg/mL after 30 min and to 9.6 (2.3–13.8) µg/mL at 60 min. The highest median C8 level was measured at 17.1 (9.6–21.5) µg/mL after 240 min. The median (IQR) C10 level was 0.76 (0.67–0.88) µg/mL at baseline, which slightly increased to 0.97 (0.68–1.61) µg/mL after 30 min, to 1.29 (0.8–2.2) µg/mL at 60 min, and to 2.31 (1.3–3.5) µg/mL at 90 min. The highest median C10 level was measured at 3.7 (3.1–6) µg/mL after 300 min.The area-under-the-curve (AUC) for plasma BHB is shown in The area-under-the-curve (AUC) for C8 and C10 is shown in p < 0.001) and a significantly greater increase with MCT plus glucose .A linear regression analysis between plasma BHB and plasma C8 and C10 is presented in p < 0.001) and a significantly greater increase with MCT plus glucose .Linear regression analysis with plasma C10 showed a significant increase in plasma BHB levels with MCT  after MCT oil alone and MCT oil plus glucose ingestion at t1. The median satiety score decreased to 3 (a little bit satiated) in both interventions at t7. In the control intervention, the median satiety score decreased from 3 (a little bit satiated) at t1 to 1 (not satiated) at t7 (data not presented). Five participants (26.3%) experienced side effects. None of these symptoms were reported by the participants to be severe enough for study discontinuation. The most common side effects are summarized in p = 0.005); see For the vocabulary subtest, there was no significant difference between the control and the MCT oil alone intervention, nor between MCT oil alone and MCT oil plus glucose. However, a significant difference was found between the control and the MCT oil plus glucose intervention (p = 0.014); see For the arithmetic subtest, there was no significant difference between the control and the MCT oil alone intervention. However, a significant difference was found between the control and the MCT oil plus glucose intervention  alone can be difficult to maintain indefinitely, due to the need for constant attention to meal planning, weighing, and measuring, the very low carbohydrate content, and common side effects like constipation, unintended weight loss, and muscle cramps . To addrThe results of this study confirmed these previous findings. MCT oil ingestion led to elevated BHB levels (>0.5 mmol/L) in many of the participants. The highest individual BHB level was 0.9 mmol/L following a single dose of MCT oil alone. MCT oil supplementation at 0.5 g per kg of body weight in healthy participants significantly increased plasma BHB levels compared to the control intervention. The increase in ketosis was faster after intake of MCT oil alone compared to MCT oil plus glucose during the first 90 min but reached similar levels for the remainder of the study. The highest individual BHB level after MCT oil plus glucose peaked at 1 mmol/L at 300 min.Careful consideration of administering large doses of MCT together with carbohydrate is warranted, since the insulin response related to carbohydrate intake activates acetyl CoA-carboxylase, which increases the cellular concentration of malonyl-CoA, which could in turn result in more acetyl-CoA being redirected away from ketogenesis and toward lipogenesis, less oxidation of LCFAs, and more LCFAs available for esterification to form TG and cholesterol esters; see Only a few studies have investigated the effect of an accompanying meal or carbohydrate content on ketone production from MCT intake . These sIn another study, St-Pierre et al. showed that, when 20 mL (18.9 g) of MCT were given to nine healthy volunteers (mean age 34 ± 12 years), diluted in lactose-free skim milk, the ketogenic response was 2-fold stronger if the drink was given without an accompanying meal containing 55 g of carbohydrate . Freemantle et al. examined the ketogenic effect in 32 healthy adults in three different age groups , providing a very low carbohydrate (3 g) meal with MCT (71 g ± 4 g). The group measured plasma BHB levels hourly and found an increase in BHB level to 0.7 mmol/L at 1 and 2 h after administration, with a peak of 1.3 mmol/L at 6 h . The results of this study, that consuming a low-carbohydrate meal with MCT does not inhibit the ketogenic effect but slightly reduces the production of BHB, are in line with the findings of previous studies. It should be noted that this study was limited to healthy young adults aged 24.4 ± 3.9 years, whereas the other studies included adults ranging from young to elderly. The study also provides additional information, specifically that consumption of MCT (0.5 g/kg BW) with a small amount of carbohydrate (0.2 g/kg BW), typically prescribed in ketogenic diet therapy, may prolong the time required to attain the maximal plasma BHB concentration. Quantification of ketosis in a longer study day (>6 h) with higher amounts of monosaccharides, mixtures of different carbohydrates, meals, or snacks could be the focus of future studies to determine how much flexibility is possible in the implementation of a ketogenic diet with the addition of MCT oil. These new results differ from a published study by Vandenberghe et al., who assessed the influence of MCT on metabolic switching from glucose to ketones as a fuel in 10 young adults (mean age 28 ± 7 years) and in 10 older adults (mean age 65 ± 6 years) . The autTaking MCT with meals is known to improve gastrointestinal tolerability ,43. SimiA slight decrease in blood glucose was observed between t0 and t2 and in blood insulin between t1 and t4 after MCT ingestion alone. Thereafter, blood glucose remained stable, slightly above the baseline level, which implied a systemic homeostasis of gluconeogenesis . Similar2 and H2O in the citrate cycle to generate energy in the form of ATP via the mitochondrial OXPHOS system or biosynthesis of ketones [Similar to St-Pierre et al., the current study demonstrated that MCT ingestion with and without glucose resulted in higher plasma C8 and C10 levels over the 5 h test day, suggesting that spot checks of plasma C8 and C10 could be useful for monitoring compliance when one or more of these fatty acids are consumed in a longer-term study . It is k ketones ,47,48. T ketones ,46. One  ketones . Another ketones .We did not measure total triglycerides in the study; therefore, further trials should explore the short- to long-term effects of MCT intake on postprandial lipid parameters such as fasting and postprandial TG and chylomicron triglycerides to support this evidence. Since this study measured levels of octanoic acid, decanoic acid, and ketones for up to five hours, we could not determine when the levels began to decrease and returned to baseline. Results of this study and previous studies suggest that an 8-h follow-up test period or longer and studying two or more daily doses could provide more complete information on the kinetics of BHB, C8, and C10 following MCT ingestion . FurtherMCTs have repeatedly been shown to improve memory and benefit cognitive performance in healthy and diseased populations ,53,54. TThis study compared the ketogenic effect of two MCT oil interventions, with and without glucose, with the aim of assessing differential effects. In summary, we show in young, healthy adults that MCT oil supplementation alone and in combination with a small amount of glucose increased average BHB levels up to physiologically significant concentrations (>0.5 mM), along with a moderate increase in blood glucose and a large increase in insulin levels when glucose was added. MCT intervention alone resulted in a more rapid BHB response than MCT plus glucose but with similar final BHB concentrations. Plasma C8 and C10 levels were detected after both MCT interventions, suggesting that if MCT oils with less ketogenic MCFAs, e.g., only C10 and or C12, are studied, compliance could be monitored by measuring the relevant fatty acid levels during planned study visits in addition to BHB. Kinetic studies of longer duration are needed to determine how long BHB and individual MCFAs are detectable in the blood, which could help with dosing regimens when used for clinical indications and also for future study design. For MCT plus glucose consumption, we found fewer side effects and positive effects on cognitive ability, although statistical trends were also observed for MCT alone. Longer-term studies is needed to understand how MCT supplementation of meals with and without carbohydrate affects metabolic health, cognition, and other clinical outcomes in the long term for children and adults of all ages.


R)-2-propanamido)benzoic acid methyl ester (5) to understand its properties for further chemical transformations. The methyl anthranilate (2) was produced by the esterification of anthranilic acid in an acidic medium. The phthaloyl-protected alanine (4) was rendered by the fusion of alanine with phthalic anhydride at 150 °C, followed by coupling with (2) furnished isoindole (5). The characterization of products was performed using IR, UV-Vis, NMR and MS. Single-crystal XRD also verified the structure of (5) in which N-H⋯O bonding stabilizes the molecular configuration of (5), resulting in the formation of S(6) hydrogen-bonded loop. The molecules of isoindole (5) are connected in the form of dimers, and the π⋯π stacking interaction between aromatic rings further stabilizes the crystal packing. DFT studies suggest that HOMO is over the substituted aromatic ring, the LUMO is present mainly over the indole side, and nucleophilic and electrophilic corners point out the reactivity of the product (5). In vitro and in silico analysis of (5) shows its potential as an antibacterial agent targeting DNA gyrase and Dihydroorotase from E. coli and tyrosyl-tRNA synthetase and DNA gyrase from Staphylococcus aureus.The work here reflects synthesis, DFT studies, Hirshfeld charge analysis and crystal data exploration of pharmacologically important ( Isoindoles belong to an important class of heterocyclic compounds of pharmaceutical importance. The substituted isoindoles have significant therapeutic potential and remain a long-standing focus of dynamic scientific interest. It is a large group of salutary agents showing anti-HIV , antican5) was formed, which failed to be further converted into benzodiazepine 7 . Further, the coupling of ester 2 with phthalamide 4 formed an expected coupled product 5. The deprotection  followed by conversion of resulting carboxylic acid 6 to COCl2 and the intramolecular cyclization was expected to construct diazepine ring 7, which was abandoned due to the conversion of 5 back into 4 instead of the desired benzodiazepine 7  generated via the MeOH removal from [M+•] is attributed as the base peak (119 amu)   (Supplem119 amu) .2 terminal of (S)-alanine 2 to avoid interference during the coupling reaction. The N-protection of (S)-alanine 3 was carried out by the fusion of amino acid with phthalic anhydride thiazol-2-ylcarbamothioyl)-2/4-substituted benzamide had −7.4 kcal/mol binding energy against E. coli dihydroorotase. In 5) are shown against E. coli dihydroorotase and DNA gyrase. The dihydroorotase dehydrogenase enzyme is involved in de novo pyrimidine biosynthesis, while DNA gyrase is a topoisomerase involved in ATP-dependent negative super-coiling. Inhibition of either enzyme can affect the viability of E. coli. That is why these enzymes were screened as potential targets for the compound (5) to predict its mechanism of action as an antibacterial agent.Disc diffusion assay revealed that  showed very promising binding, with −8.47 KJ/mol binding energy and with 0.615 µM dissociation constant (S. aureus), the binding energy and dissociation constant were −6.77 kcal/mol and 10.79 µM, respectively  seem to agree. The Dihydroorotase enzyme from E. coli and Tyrosyl-tRNA synthetase from S. aureus are vital enzymes, and these are necessary for the viability of these bacteria. Inhibiting these enzymes affects the growth and survival of bacteria in a growth media. The tyrosyl-tRNA synthetase is involved in aminoacylation; failing that leads to impaired protein synthesis and cellular functions.Against tyrosyl-tRNA synthetase of ectively c. The tyectively , and agad ARG176 . Previou3, MeOH and EtOH were obtained from commercial sources. For crystallization and chromatography, a fraction of distilled hexane at 60–80 °C was employed. All solid chemicals were used as such.Freshly distilled analytical-grade organic solvents were used. The EtOAc, CHClε = A/cl, was used to find ε values.Gallenkamp was used to record melting points  and reported uncorrected. For UV spectra, a Thermo Spectronic (UV-1700) spectrometer was used, and IR spectra were taken on IR Prestige 21 FTIR spectrophotometer, Shimadzu (Japan) at High Tech Laboratory, UoS, Sargodha. Molar absorptivity ε and absorption maxima are both reported. Equation LR EIMS (low-resolution electron impact mass spectra) were recorded on Fisons Instrument VG Autospec mass spectrometer at ICCBS , HEJRIC, University of Karachi, Karachi, while electrospray ionization mass spectra (ESI MS) were recorded using Q-TOF Ultima API (Micromass) at BMSF , UNSW , Sydney, Australia.1H NMR spectral data are shown as follows: chemical shifts in ppm downfield with reference to TMS (δ); proton number/count; multiplicity due to neighboring protons; observed J (coupling constant) in Hertz (Hz). Multiplicities are recorded in the form of s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet), p (quintet), m (multiplet), dd (doublet of doublet), dt (doublet of triplet) and their combinations. Chemical shifts δ in the 13C NMR are reported downfield from TMS in ppm and show all identifiable carbons.The NMR spectra of our products were recorded at ICCBS, HEJRIC, University of Karachi, Karachi, Pakistan, and at the School of Chemistry, University of New South Wales (UNSW), Sydney, Australia. The For recording XRD data to elucidate the structure of crystals, Bruker Kappa APEX 11 CCD diffractometer at the Department of Physics, UoS, was used. For the radiation source, fine-focus sealed tube monochromator graphite  was used.ζ basis set def2-TZVP +•, 120 (33%) [M–OCH3]+ A, 119 (100%) [M–CH3OH]+•, 92(78%) [A–CO]+•.The anthranilic acid S)-alanine 3  was fused with phthalic anhydride  at about 155 °C for 60 min. The reaction completion was confirmed by the disappearance of reactants on TLC. The ninhydrin dip confirmed the absence of unreacted amino acids. The reaction flask was cooled to ambient temperature, and the unreacted crystallized phthalic anhydride was scratched off from the flask wall. The rest of the solid product was recrystallized from EtOH, and H2O yielded colourless prisms such as crystals of phthalamide 4 (750).The (f = 0.26 (EtOOCH3); [α]29D = –32.0  M.P. = 160 °C; log ε (λmax in nm) = 3.2003 (295.5); ύmax in cm−1 KBr: 1708 (amide C=O), 1757 (carboxylic C=O), 3230 (O–H); δH in ppm (400 MHz): 7.80–7.90 , 5.00 , 1.70  LR EIMS : 219 (21%) [M]+•, 175 (86%) [M+•–CO2], 174 (100%) [M+•–OH–CO].R2  was added to phthalamide 4  and heated at 100 °C until a clear solution was obtained. The addition of ester 2  was carried out to the reaction solution and was further heated for 5 h at 50 °C. The cooled solution was partitioned between H2O and CHCl3 (3×15 mL). The organic layer was filtered, dried over anhydrous Na2SO4 and concentrated under reduced pressure, which resulted in a pale, yellow-colored solid. It was crystallized from C2H5OH to form white-colored crystals (40%). Rf: 0.5 ; M.P. 157 °C; log ε (λmax in nm): 3.83024 (305); ύmax in cm−1: 3269 for N–H, 1770 for ester C=O, 1707 for lactam C=O, 1595 for amide C=O; δH in ppm (300 MHz): 11.61 , 8.70 , 7.98 , 7.08 , 7.88–7.91 , 7.73–7.76 , 7.53 , 5.14 , 3.72 , 1.87 ; δC in ppm (75 MHz) 168.8 (ester C=O), 2 × s, 168.0 (C1 & C3); s, 167.9 (C2′); s, 141.2 (C1″); 2 × s, 134.9 (C4a & C7a); 2 × d, 134.2 (C4 & C7); d, 132.3 (C3″); 2 × d, 131.0 (C5 & C6); d, 130.9 (C5″); d,123.0 (C6″); d, 120.5 (C4″); s, 115.3 (C2″); q, 52.3 (OMe); d, 50.1 (C1′); q 15.1(Me); LR EIMS (m/z in amu): 352 (20%) [M]+•, 320 (35%) [M–MeOH]•+, HR ESIMS [M + Na]+• (m/z in amu) for C19H16N2O5 = 375.0997 , 375.0943 (observed), 5.4 mmu variation.The SOClE. coli and S. aureus were used to assess antibacterial activities, while Ciprofloxacin (1 mg/mL) was used as a standard drug. In silico studies were performed as described previously by our group. The protein structures were downloaded from the Protein Data Bank , and YASARA Structure ver. 22.10 was used for molecular docking with parameters used before [Disk diffusion assay was performed as previously described . Clinicad before . The ligd before  and LigPd before .5 is verified by the SC-XRD technique. The molecular configuration of isoindole 5 is stabilized due to N-H⋯O bonds that form a S (6) hydrogen-bonded loop. The molecules are connected in the form of dimers by C-H⋯O bonds to make a 11) loop. The π⋯π stacking interaction between aromatic rings with inter-centroid distance range from 3.602 to 4.252 Å helps with additional stability of crystal packing. DFT calculations were performed for compound (5). These provided us with an understanding of the geometrical features. FMO analysis was performed to observe the reactivity pattern of the compound and also to calculate the energy difference between the HOMO and the LUMO. Hyperpolarizability studies showed that the compound showed a moderate non-linear optical response. DFT studies showed HOMO’s localization is localized over the substituted aromatic ring (right side), and the LUMO exists mainly over benzazole. This further suggests that the nucleophilic side of the molecule is the right side (according to 5). Preliminary studies regarding in vitro and in silico antimicrobial activities of (5) against E. coli and S. aureus shows this compound can be a good nucleus for developing a further antibacterial compound.The structure of isoindole


FUT1. These phenotypes are clinically important because subjects with these phenotypes can only accept transfusions of autologous blood or blood from subjects with the same phenotypes due to the anti-H antibody. To survey FUT1 alleles involved in Bombay and para-Bombay phenotypes, the effect of 22 uncharacterized nonsynonymous SNPs in the Erythrogene database on the αfucosyltransferase activity were examined by transient expression studies and in silico analysis using four different online software tools. Two nonfunctional alleles (FUT1 with c.503C>G and c.749G>C) and one weakly functional allele (with c.799T>C) were identified in transient expression studies, while the software predicted that the proteins encoded by more alleles including these would be impaired. Because both nonfunctional FUT1 alleles appear to link to the nonsecretor alleles, homozygotes of these alleles would be of the Bombay phenotype. The present results suggest that functional assays are useful for characterization of nonsynonymous SNPs of FUT1 when their phenotypes are not available.Rare individuals with Bombay and para-Bombay phenotypes lack or have weak expression of the ABO(H) antigens on surface of red blood cells due to no or very weak H-type αfucosyltransferase activity encoded by  Humans have two types of αfucosyltransferase, encoded by FUT1 and FUT2. FUT1 encodes the H-type αfucosyltransferase (H enzyme) which determines expression of the H antigen in the erythroid lineage, whereas FUT2 encodes a secretor-type αfucosyltransferase (Se enzyme) that controls expression of the H antigen in a variety of secretory epithelia and saliva3. FUT1 and FUT2 have approximately 70% DNA sequence similarity and are located 35-kb from each other on chromosome 19q13.36.The H blood group antigen is synthesized by αfucosyltransferase and is an essential precursor for the synthesis of A and B antigens in the presence of the corresponding A or B transferases7. The Bombay phenotype has only nonfunctional alleles of both FUT1 (h) and FUT2 (se), whereas the para-Bombay phenotype has only nonfunctional alleles of FUT1 (h) but at least one functional allele, FUT2 (Se), so that the H antigen produced by the Se enzyme is adsorbed from the serum into the erythrocytes, or has very low H enzyme activity encoded by the weak-functional FUT1 allele (Hw) in the presence of a nonfunctional FUT2 allele, resulting in negligible H antigen production9. The Bombay phenotype was first recognized by the presence of anti-H in the serum in addition to anti-A and anti-B10. Because anti-H produced by subjects with Bombay phenotype carries the risk of severe hemolytic transfusion reactions, subjects with Bombay phenotype require autologous blood donation or blood from other subjects with same phenotype11. On the other hand, anti-H produced by subjects with para-Bombay phenotype is usually not clinically significant1. Therefore, it is clinically important to correctly determine Bombay or para-Bombay phenotypes.Two H-deficient red cell phenotypes due to H enzyme deficiency have been recognized: the Bombay phenotype (H– phenotype), in which H, A, and B antigens are completely absent on erythrocytes, saliva, and body fluids, and the para-Bombay phenotype , in which the amount of H antigen  on erythrocytes is very low. H-deficient red cell phenotypes are extremely rare , whereas nonsecretors due to Se enzyme deficiency are present in about 25% of many populationsFUT1 resides only in exon 4, which encodes a 365-amino acid protein12. This structural feature of the gene makes it easy to determine the sequences, haplotypes of SNPs of the protein coding region or get expression constructs. Since the cloning of FUT14, molecular analysis of H-deficient red cell phenotypes has identified a number of nonfunctional or weak-functional FUT1 alleles14. At present, the ISBT allele table for the H blood group system  v6.1 31-MAR-2023 (https://www.isbtweb.org/resource/018h.html) lists 77 FUT1 alleles, including two functional alleles , 37 alleles involved in phenotype H + weak, i.e., para-Bombay phenotypes , and 38 alleles involved in phenotype H–, i.e., the Bombay phenotype (FUT1*01N.01-37 and FUT1*0N.01).The coding sequence of 15, but the prediction results are not always accurate. Alternatively, the enzyme activity has been experimentally determined by transient expression in cultured cells and then measurement of the αfucosyltransferase activity by using 14C-labeled fucose and its acceptor13. Another strategy to examine enzyme activity is flow cytometry of cell surface H antigens by transient expression in cultured cells, which is indirect but does not require a radioisotope16. Erythrogene v0.8 (27-Nov-2017) (http://www.erythrogene.com/17) extracted the data of blood group alleles from ISBT 018 H (FUT1FUT2) blood group alleles (older version) and the 1000 Genome Project  and matched them against blood group reference lists. Seventy-nine alleles are listed for FUT1.However, not every nonsynonymous substitution affects the function of the encoded protein, and we need to estimate the impact of each SNP through in silico analysis in the absence of information on the phenotypeFUT2, we identified two nonfunctional alleles (se) and one weak-secretor allele (Sew) by transient expression studies, but there were discrepancies between the results of transient expression studies and in silico analysis in assessing the functional impacts of each SNP on Se enzyme activity19.In the previous study of FUT1 alleles from Erythrogene that were not registered in the ISBT database and analyzed their effects on enzyme activity. In addition, three DNA samples with causal substitution  of the FUT1 (FUT1*01N.09) giving rise to the classical Indian Bombay phenotype21 were also examined to better understand the genetic background of this phenotype.In this study, with the aim of determining how many nonsynonymous substitutions affect the activity of the encoding enzyme and whether they could be responsible for the Bombay or para-Bombay phenotypes, we picked 22 All methods were carried out in accordance with relevant guidelines and regulations. The oral informed consent was obtained and the DNA samples were taken from participants . The statement for oral informed consent approved by ethical committee of Kurume University in 1999 and 2002. However, present study protocol was reviewed and approved by the ethical committee of Kurume University School of Medicine in 2022 using existing and already anonymized DNA .FUT1*01N.09, which is a nonfunctional FUT1 allele of the classical Indian Bombay phenotype21. In addition, we used genomic DNA from 58 Sri Lankan Tamils, 54 Sinhalese, and 47 Bangladeshis whose FUT2 genotypes had already been determined23.Twenty-nine genomic DNAs  were purchased from the Coriell Institute for Medical Research   and FUT1-R  were used. The 50 µL PCR reaction contained about approximately 7 ng of genomic DNA, 25 µL of PrimeSTAR Max Premix , and 250 nM of each primer. The PCR temperature conditions were 35 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 5 s, and extension at 72 °C for 7 s. Direct Sanger sequencing of the PCR products was performed using each PCR primer as the sequencing primer as described previously19. To determine the haplotypes of individuals who were heterozygous at two sites, we cloned PCR products by use of restriction sites of HindIII and XbaI into a mammalian expression vector pcDNA3.1(+) and sequenced the clones. The coding sequence of the FUT2 of the individuals who were shown to have nonfunctional FUT1 was also amplified and directly sequenced as described previously19.The nucleotide sequence is numbered from the A residue of the translation initiation codon as position number 1FUT1, transient expression experiments followed by flow cytometry analysis were performed as done with the FUT2 using an anti-H 1E3 monoclonal antibody24. In addition to the FUT1 alleles containing each SNP concerned, the effects of the wild-type allele (FUT1*01), c.725T>G (FUT1*01N.09) inserted into pcDNA3.1(+) vectors were determined. Two μg of each construct together with 60 ng of the pGL3 Promoter was transfected into 2 × 105 COS-7 cells (African green monkey kidney fibroblast-like cell) by means of TransIT-X2 . After 2 days, the cells were immunostained by using a mouse monoclonal antibody to H type 1–4 (1E3)24, followed by incubation with FITC-labeled goat anti-mouse IgM , and H antigen expression was examined using a BD Accuri C6 system  as described previously19. The experiments were repeated four times independently. The transfection efficiency in each experiment was determined by luciferase luminescence intensity and the similar transfection efficiency was confirmed by the intensity of luciferase light as described previously19.To evaluate the significance of each of nonsynonymous SNP of FUT1 on the function of the enzyme were predicted using four free software programs, MutationTaster (http://www.mutationtaster.org/)25, MutationAssessor (http://mutationassessor.org/r3/)26, PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/)27, and Sorting intolerant from tolerant (SIFT) (http://sift.jcvi.org/)28.The effects of each nonsynonymous SNP of FUT1 alleles with c.503C>G c.725T>G, and c.749G>C identified in this study were found in South Asian populations. Therefore, we attempted to screen these substitutions by high resolution melting (HRM)29. Primer3  was used to design PCR primers. For detection of c.503C>G, a forward primer  and a reverse primer  were used , and a reverse primer  were used . The 20 μL PCR reaction mixture contained 2–20 ng of genomic DNA, 10 µL of Premix Ex Taq (Probe qPCR) , 1 µL of LightCycler 480 High Resolution Melting Dye , and 125 nM of each primer. The PCR temperature conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 20 s. The products were then denatured at 95 °C for 1 min and rapidly cooled to 60 °C for 1 min allowing heteroduplex formation, and data were collected over the range from 74 to 90 °C for c.503C>G or 80–96 °C for c.725T>G and c.749G>C, increasing at 0.02 °C/s with 25 acquisitions/sec. The raw melting curve data were normalized by manual adjustment of linear regions of pre- and post-melt signals of all samples. Temperature shifting was then performed using a temperature shift threshold of default setting (5%) for detection for c.503C>G. On the other hand, temperature shifting was not performed for c.725T>G and c.749G>C to clearly separate heterozygotes of c.725T>G and c.749G>C.FUT1 of 29 individuals to survey the nonfunctional alleles of FUT1 registered in Erythrogene17. We confirmed all of the indicated SNPs in respective DNA samples in the database by direct Sanger sequencing of the FUT1 coding region. As a result, in addition to c.725T>G, we encountered 22 uncharacterized nonsynonymous SNPs: c.20G>C, c.101A>G, c.181G>A, c.220C>T, c.229C>G, c.283C>G, c.468C>G, c.503C>G, c.530T>G, c.565G>C, c.607C>T, c.625G>A, c.649G>A, c.691C>T, c.749G>C, c.796G>C, c.799T>C, c.800G>C, c.1013T>A, c.1022C>T, c.1064A>G, and c.1096T>C.First, we determined the DNA sequence of the total coding region of the FUT1 coding region with two substitutions, that is, c.35C>T and c.181G>A, c.35C>T and c.220C>T, c.35C>T and c.649G>A, c.822C>A and c.1064A>G, by subcloning them into plasmids. Sequencing of the clones revealed that only one haplotype of the four alleles differed from those listed in the database. That is, c.822C>A and c.1064A>G were on the same chromosome in the database, but each was on a different chromosome. On the other hand, consistent with the database, c.181G>A, c.220C>T, and c.649G>A were on the functional FUT1 allele with c.35C>T (FUT1*01.02). Because c.822C>A is a synonymous SNP, we performed functional analyses of the 23 alleles including c.725T>G (FUT1*01N.09) listed in Table We then determined haplotypes of four fragments of the FUT1 allele encodes a functional H enzyme or not, the αfucosyltransferase activity in transfectants of each of FUT1 expression vector was determined in previous studies20. In this study, we tried flow cytometry for measurement of H antigens expressed on the surface of culture cells using anti-H monoclonal antibody (1E3)24 because the phenotype of erythrocytes could not be demonstrated and antibody tests of serum could not be performed. The predicted amino acid change for each allele and the expression levels of H antigens on the cell surface are shown in Table For determination of whether each uncharacterized , the α1,fucosyltrFUT1 allele, FUT1*01, 28.7 ± 3.2%) were defined as activity-affecting substitutions. Among them, alleles with substitutions whose activity was less than 10% of the positive control  were considered weakly functional alleles. In addition, H-enzyme inactivating substitutions were defined as those with no difference in activity (p > 0.05) compared to the negative control (the expression level of pcDNA3.1(+) without insert, 0.7 ± 0.2%). The percentage of H antigen–positive cells transfected with pcDNA3.1 ligated with the FUT1 of c.20G>C (p.R7P), c.101A>G (p.H34R), c.35C>T (p.A12V) and c.181G>A (p.A61T), c.35C>T (p.A12V) and c.220C>T (p.P74S), c.229C>G (p.L77V), c.283C>G (p.Q95E), c.468C>G (p.D156E), c.35C>T (p.A12V) and c.530T>G (p.L177R), c.565G>C (p.D189H), c.607C>T (p.R203C), c.625G>A (p.D209N), c.35C>T (p.A12V) and c.649G>A (p.V217I), c.796G>C (p.E266Q), c.1064A>G (p.D355G), and c.1096T>C (plus10 amino acids) were not significantly different (p > 0.05) with those ligated with the positive control of the wild-type allele, FUT1*01 (28.7 ± 3.2%). On the other hand, the percentage of H antigen–positive cells transfected with pcDNA3.1 ligated with FUT1 of c.691C>T (p.R231C), c.1013T>A (p.I338N), and c.1022C>T (p.P341L) were two-third of the positive control and those of c.800G>C  or c.749G>C  was almost undetectable as was that of c.725T>G  and pcDNA3.1 without FUT1 (0.7 ± 0.2%).In this study, substitutions with significantly (p < 0.05) lower activity compared to the positive control  analyses19, the software generally tended to overestimate the impacts of the nonsynonymous SNPs we tested here.The results of predictions were not always consistent with those of expression experiments. For example, it has already been reported that p.L242R (c.725T>G), the substitution responsible for the classical Indian Bombay phenotype, is an H-deficient allele but it was classified as medium by Mutation Assessor. Of 22 amino acids substitutions, the predicted effects were matched for all software and experiments for p.A61T (c.181G>A), p.D156E (c.468C>G), p.D209N (c.625G>A) as polymorphic, neutral, benign, and tolerated substitutions and p.P168R (c.503C>G), p.L242R (c.725T>G), p.R250P (c.749G>C), p.W267S (c.799T>C) and p.I338N (c.1013T>A) as disease-causing, medium or low, damaging, and affected substitutions. On the other hand, they were mismatched for the other 14 substitutions (Table http://dgv.tcag.ca/dgv/app/variant?id=esv3644597&ref=GRCh38/hg38)31, one heterozygote of FUT1 with c.503C>G  was a compound heterozygote of FUT2*01N.02  and FUT2*0N.01  and one heterozygote of FUT1 with c.749G>C  was a homozygote of FUT2*01N.02  was a functional FUT2 homozygote. Therefore, homozygotes for this allele are considered to be of the secretor phenotype. Unfortunately, we cannot be certain because the phenotype has not been confirmed, but homozygotes for this weak-functional allele are likely to be the para-Bombay phenotype, regardless of the secretor phenotype  and one weakly functional allele (with c.799T>C) in this study. According to the 1000 Genomes Project Database, the Database of Genomic Variants  were heterozygous for FUT2*0N.01 and these two FUT1*01N.09 alleles are presumed to link to FUT2*0N.01  was functional FUT2/FUT2*01N.02 for the FUT2. Thus, in this one subject, FUT1*01N.09 does not link to FUT2*0N.01, and if it linked to functional FUT2, the homozygote for this allele would be a para-Bombay phenotype  were heterozygous for FUT2*0N.01 . In addition to c.725T>G, a causal substitution of the classical Indian Bombay phenotype, they were the only two substitutions here that completely inactivated enzyme activity. It is interesting to note that all of these were found in South Asian populations, but the reason for this is not clear at present. Classical Indian Bombay subjects with FUT1*01N.09 and FUT2*0N.01 have been reported not only in Indians, but also Bangladeshis, Pakistanis, Sri Lankans, and even in West Asian Iranians33. Thus, the causal haplotype (FUT1*01N.09–FUT2*0N.01) of the classical Indian Bombay phenotype is presumed to be present with some frequency, albeit low, and to be widespread in a broad band of South Asian populations and certain West Asian populations, while the other two nonfunctional FUT1 alleles (with c.503C>G and c.749G>C) may be restricted to relatively specific populations in South Asia. To investigate distribution of these substitutions and estimation of prevalence of Bombay or para-Bombay phenotypes, a large-scale analysis of the South Asian population is needed, and the HRM analysis used in this study is expected to be a good tool for this purpose.In this study, we also performed transient expression studies of 22 uncharacterized FUT1*01W.24 by the ISBT 018 H (FUT1FUT2) blood group alleles v6.1 31-MAR-2023. In addition, the FUT1 with c.799T>C that produces p.W267R significantly reduced H enzyme activity, and the FUT1 with c.800G>C that produces p.W267S also reduced enzyme activity by more than half compared to the wild-type enzyme. Thus, 217 V and 267W appeared to be important for H enzyme activity.The allele with c.649G>A resulting in p.V217I appears to be functional in the present transient expression studies although all in silico analyses suggest that this substitution has significant impact of encoded protein. On the other hand, the allele with c.649G>T resulting in p.V217F is listed as a weakly functional allele with the name of FUT1*01N.09 links to FUT2*0N.01 in the literature21, and in at least two 1000 Genomes Project subjects and three Tamil, Sinhalese, and Bangladeshi subjects with FUT1*01N.09 also appear to be linked to FUT2*0N.01. However, we encountered here one FUT1*01N.09 not linked to FUT2*0N.01 because the genotype of FUT2 of this subject was functional FUT2/FUT2*01N.02. It is difficult to determine the exact haplotypes of FUT1 and FUT2 in this subject because FUT1 and FUT2 are 35 kb apart on chromosome 19q13.36. Based on gene frequencies, it is likely that the c.725T>G substitution of FUT1 occurred on the chromosome with FUT2*0N.01 in South Asia. Therefore, although we cannot be certain, it is likely that FUT1*01N.09, which does not link to the FUT2*0N.01, arose by homologous recombination between chromosomes during meiosis. In any case, since only one allele has been analyzed so far, analysis of a large number of samples and family analyses will be necessary to estimate the mechanism of generation of this allele.As mentioned above, in the classical Indian Bombay phenotype, FUT1 alleles (with c.503C>G and c.749G>C) and one weak allele (with c.799T>C) in samples in the 1000 Genomes Project Database. To estimate the impact of each SNP, transient expression studies are desirable for analysis of FUT1 as well as FUT2.We identified two nonfunctional


Inguinal hernias are a common disease that may present at all ages. Adolescents are a unique patient population between children and adults. The etiology and the surgical treatment strategies of adolescent indirect hernias are not clear. Specifically, whether these kinds of hernias should be treated by high ligation or mesh repair remains controversial. We aimed to evaluate the efficacy of laparoscopic high hernia sac ligation in adolescent indirect hernia.The data of adolescent patients who underwent laparoscopic high hernia sac ligation at the The First People’s Hospital of Foshan,China, from January 2012 to December 2019 were analyzed retrospectively. Data collected included their age, gender, weight, surgical method, hernia ring diameter, operation time, postoperative recurrence rate and postoperative complications.A total of 70 patients were enrolled, including 61 males (87.14%) and 9 females (12.86%),all patients were aged 13–18 years old (mean 14.87 ± 1.56years), weighed 28-92 kg (mean 53.04 ± 10.60 kg). All 70 patients underwent laparoscopic surgery except 2 patients with irreducible hernias who converted to laparotomy.The hernia ring diameter ranged from 0.5 to 3 cm (mean 1.39 ± 0.49 cm), and was ≤ 2 cm in 68 cases (97.14%).The operative length ranged from 12 to 105 min (average 24.96 ± 12.61 min), There were 37(52.9%) right-sided hernias, 32 (45.7%) left-sided hernias, 1 (1.4%) bilateral hernia.There were 67(95.7%)reducible hernias,2(2.9%)irreducible hernias ,and 1(1.4%)incarcerated hernia.Hospital stays ranged from 1 to 5 days (mean 2.37 ± 1.05 days). Follow-ups were performed from 30-119months (mean 74.27 ± 28.14months). There were no cases of recurrence, howere, incision infection occured in 1 patient, who underwent a second operation 6 months after surgery, and 4 (5.7%) patients had complaints of intermittent pain around the ligation incision site, mostly during exercise .Laparoscopic high hernia sac ligation is feasible for treatment of adolescent indirect hernias with a hernia ring diameter of ≤ 2 cm.The online version contains supplementary material available at 10.1186/s12893-023-02048-w. Inguinal hernias are a commom disease that may present at all ages, The overall incidence of inguinal hernias in childhood ranges from 0.8–4.4% .IndirectWe retrospectively reviewed the data of adolescent patients who underwent inguinal hernia repairs from January 2012 to December 2019 at The First People’s Hospital of Foshan, Guangdong, China. The inclusion criteria for this study were: age 13–18 years;primary indirect inguinal hernia; signed informed consent by the patient’s legal guardian; and no dysfunction of vital organs, including the heart, lungs, liver, and kidneys. Exclusion criteria were: age < 13 years or > 18 years; direct hernia; femoral hernia; recurrent hernia; patients who refused to sign informed consent; and heart, lung, liver, and kidney failure. The gender, age ,weight, hernia characteristics, operative approach, operative length,length of stay, postoperative recurrence rate, and complications of the patients were collected.Postoperative complications were defined as surgical site infection, symptomatic seroma or hematoma, symptomatic hydrocele, postoperative pain.The procedures of laparoscopic high hernia sac ligation were performed with the patient in the supine position under general anesthesia. The typical process of the procedure is described as follows:The size of the hernia diameter was measured Fig. . A 2 mm Patients were followed up by the outpatient department at 1 week, 1 month and 3 months post-operation. At 3 months post-operation, patients were followed up by telephone. Patients were asked yes and no questions whether they had any residual pain at the surgical incision site, bulging in the groin, or whether they had surgical site redness and any discharge of pus. If recurrence was suspected, the patient would be called back to the outpatient department and excluded by physical examination or imaging .All patients were followed-up. The follow-up time of the entire cohort was 30-119months (mean 74.27 ± 28.14months).All statistical analyses were performed using the spss21 statistical software .Continuous data were expressed as the mean ± standard deviation.A total of 70 patients were enrolled, including 61 males (87.14%) and 9 females (12.86%); all patients were aged 13–18 years (mean14.87 ± 1.56years), and their body weight was between 28 and 92 kg(mean 53.04 ± 10.60 kg). 68 patients underwent elective laparoscopic high hernia sac ligation except 1 incarcerated hernia underwent emergency laparoscopic surgery. In addition, 2 patients underwent laparoscopic surgery converted to laparotomy because of irreducible hernias.The hernia ring diameters for the patients were between 0.5 and 3 cm (mean 1.39 ± 0.49 cm); among these hernia rings of 1 patient was 3 cm,another patient was 2.5 cm, while 68(97.14%) patients had hernia rings ≤ 2 cm .The operative lengths were between 12 and 105 min (average 24.96 ± 12.61 min). There were 37(52.9%) right-sided hernias, 32 (45.7%) leftsided hernias, and 1 (1.4%) bilateral hernia. Moreover, 24 patients(34.3%) simultaneously had contralateral hernias.There were 67 (95.7%)reducible hernias, 2 (2.9%)irreducible hernias, and 1 (1.4%)incarcerated hernia  of follow-ups, the recurrence rate were 0%. The observed positive outcome of the present series may be due to the following reasons: (1) patient selection, we included patients with small internal rings (1.39 ± 0.49cm); (2) all patients received double high hernia sac ligation without peritoneal tear. Some other studies have shown that the recurrence rates after high hernia sac ligation without mesh for adolescent hernias were 0.95-3.5%, which is also relatively low .The recuThe main complication after inguinal hernia repair is seroma, but since the high ligation of the hernia sac did not require the separation of the preperitoneal space and hernia sac, the incidence of seroma was low. No seroma occurred in the paients analyzed in this study. One case of incision infection was documented. The patient had undergone a laparoscopy converted to laparotomy because of an irreducible hernia. However, 6 months post-operation, the wound became infected due to the non-absorbable suture knots which were removed by a second operation. In particular, 4 patients 4/70,5.7%) had complaints of intermittent pain around the ligation incision site, mostly during exercise. This was similar to previous reports 0,5.7% ha. The patThere were some limitations in our study; the small number of cases, no comparative study was conducted, and the follow-up times of some patients were short compared to others. Follow-ups of patients who recently underwent surgery should be conducted over a longer period of time to observe the true recurrence rates. Prospective muti-center studies with larger sample size should be designed in the future.The results of this study suggest that laparoscopic high hernia sac ligations are effective in the treatment of adolescent indirect hernias with internal ring diameters of ≤ 2 cm. Moreover, the rates of recurrence and complications are low, and there is no need to worry about the adverse effects of using mesh. However, this study is a retrospective study with a small number of participants. Further prospective comparative studies are required.Below is the link to the electronic supplementary material.Additional File 1: The data of adolescent hernias


According to further in vitro and in vivo evaluations, 68Ga-5 showed promising properties as a PET ligand, and selective accumulation in PFKFB3-positive tumors was observed in PET images . Our results indicated that radio-metal radiolabeled aminoquinoxaline derivative, as represented by 68Ga-5, held the potential to be developed as selective PFKFB3-targeted PET tracers, and further investigation and optimization would also be required for this scaffold.Glycolysis, as a multi-step oxidation process, plays important roles in the energy supply for living cells, including malignant tumor cells. Recent studies have revealed that 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (named PFKFB3), a bifunctional enzyme in glycolysis, is upregulated in a variety of malignant solid tumors and has been regarded as a potential biomarker for the diagnosis and treatment of tumor patients. Based on the structure of selective PFKFB3 inhibitors, we designed and synthesized a radio-metal radiolabeled small molecule,  As the most important multi-step oxidation process to provide energy from glucose, glycolysis is controlled by a series of bifunctional enzymes and enables the conversion of glucose to pyruvic acid with the release of two molecules of ATP . The priPFKFB3 is one of the major isozymes of PFKFB1-4 and is frequently overexpressed in a variety of human solid tumors, including breast, lung, gastric, pancreatic, ovarian and colon cancers . In addivia specific targets by one single molecule can be achieved.  of one highly sensitive molecule, the detection, diagnosis, and treatment of tumors chieved. . For exae clinic .68Ga for in vitro and in vivo evaluations. With a similar structure of glucose, 18F-fluorodeoxyglucose (18F-FDG) is able to visualize the abnormal glucose metabolism and hence has been widely used for the diagnosis of tumors (18F-FDG mainly revels the activity of glucose transporters (GLUT) in tumor cells and tissues, the biomarker for tumor glycolysis may play more important role in tumor diagnosis, such as PFKFB3  attracted our attention . With thf tumors . Howevers PFKFB3 . Althoug lesions . Therefo68Ga radio-labeled aminoquinoxaline derivative (68Ga-5) was successfully produced and evaluated. Our results indicate that aminoquinoxaline derivatives have the potential to be developed as a “theranostic” probe for tumor diagnosis and treatment, but further comprehensive studies are needed to fully investigate the SAR of this scaffold with PFKFB3.In this study, a potent Molecular docking study was performed to predict and evaluate the binding model of compound 5 with PFKBF3. All of the calculations were based on Discovery Studio 3.1 . The protein structure was prepared with the crystal structure of PFKBF3 (PDB ID: 6IBX). The binding site was configured as a sphere with the residues that stay within 10 Å diameter from its original ligand (compound 4), which is large enough to incorporate the ATP binding pocket of the PFKBF3 interface. The GOLD program was used to perform the docking calculations.1H and 13C NMR spectra was carried out on a Bruker spectrometer  in the Analysis and Testing Center of Sichuan University. The coupling constants are calculated and presented as J with Hz (hertz), and singlet, doublet, triplet, quartet and multiple are displayed as s, d, t, q, and m, respectively. The mass spectra were obtained on an Agilent 6125 LC/MSD system. As a positive control, compound 4 was synthesized with an identical procedure from published literature, which was also modified for the synthesis of compound 5  for mpound 5 . All the™ Kinase Assay kit was also obtained from Promega.All regents used in this section were purchased from Sigma, and the Human Recombinant PFKFB3 Protein (His and GST Tag) was purchased from SinoBiological. The ADP-GloThe PFKFB3 enzymatic activity assay followed an identical procedure used in previously published literature, along with the same regents and materials .50 calculations in GraphPad Prism 5.0 software.For the cellular inhibition assay, the protocol used in this section was also from the same literature as the PFKFB3 enzymatic activity assay, with minor modifications . Briefly68Ga-5 was prepared following a general procedure for 68Ga radiolabeling (68Ge/68Ga generator (Eckert and Ziegler) was eluted with 10 mL of 0.1 N HCl solution , the first fraction (2.5 mL) showed low activity and was discarded, and the next fraction (2.0 mL) with approximately 85% radioactivity was collected for radiolabeling. In a glass vial containing 50 µg of compound 5 (lyophilized powder) was added 40 µL of 1 N sodium acetate buffer and 400 µL of generator elution. The pH value of the final reaction mixture was about 3.5–4.0. The reaction was performed under 90°C for 20 min with a block heater. The reaction mixture was then passed through a Waters C-18 September-Pak light cartridge and rinsed with 20 mL of distilled water, and the radioactivity trapped in the C-18 cartridge was then washed with 400 µL (2 × 200) of ethanol. The 68Ga-5 in ethanol was then formulated in 5 mL of saline for further evaluations. The radio-chemical purity (RCY) of the final product was assessed by an Agilent HPLC 1100 system equipped with a Phenomenex C-18 Luna column , and an FC3200 gamma detector (BioScan) was used to collect the gamma signal. The mobile phase of A (MeCN) and B (0.1% TFA in water) was used in the analysis based on a gradient method , with a flowrate set as 1 mL/min.The labeling . Briefly68Ga-5 was determined in PBS saline, 80% EtOH solution and rat serum. Briefly, 600 µL of the final 68Ga-5 solution was added to 2 mL of PBS saline, 80% EtOH solution and rat serum with each 200 µL. These solutions were then incubated in a water bath under 37°C for 4 h, and samples (200 µL) from these solutions were obtained at 1 h, 2 h, 3 h, and 4 h after incubation. Samples from PBS saline and 80% EtOH solution were loaded into the radio-HPLC for RCP analysis as described in the radiolabeling and characterization section. For samples from rat serum, 200 µL of MeCN was added to denature the plasma protein, and then the mixture was centrifuged at 5,000 RPM to precipitate the denatured protein. The supernatants of the centrifuged solution were then applied to HPLC for analysis. The RCP of all samples was calculated based on the area under the curves generated by the gamma detector.The stability of 68Ga-5 was measured and presented as the partition coefficient at pH 7.4 (Log D7.4) as described previously  in 1-octanol and in PBS saline (pH 7.4). A series of solutions were prepared and tested until a constant value of log D7.4 was obtained.The lipophilicity of eviously . Briefly6 cells per well) overnight for attachment before use. For cellular uptake, approximately 370 KBq of 68Ga-5  was added into the cells and incubated under 37°C for 1 h. At each time point, i.e., 15, 30, 45, 60 min, the medium in the well was transferred, and then the well was washed with 2 mL of cold PBS. The medium and the PBS were combined and counted for radioactivity in a gamma counter . The cells were then dissolved in NaOH (1 N), and the lysate was also counted in the gamma counter. For blocking studies, all cells were pretreated with 1 µM unlabeled compound 5 1 h before the addition of 68Ga-5, and the subsequent procedure for medium and cells was the same as described above. The uptake ratio was then calculated by the following formula: Uptake = (counts in cells)/(counts in medium and PBS) * 100%.H1975, HCC827, MKN45 and NUGC3 cells (obtained from ATCC) were investigated in this section. Briefly, cells were cultured, transferred, and plated in 60-mm culture dishes , and were administered 30 µCi of 68Ga-5 by tail vein injection under anesthesia. At the designated time points, subjects were sacrificed. The tissues of interest were collected, weighed, and counted in a gamma counter  for radioactivity. The tissue distribution was then calculated and presented as the percentage of the injected radioactivity dose per gram of tissue .All protocols and procedures in this study were approved by the animal care and use committee of Sichuan University. To evaluate the 6 cells) in the left or right axilla. The tumor models were ready to use when the tumor volume reached 500 mm3 about 10–15 days postinoculation. Small animal PET imaging studies were carried out on a micro-PET/CT system . Briefly, tumor-bearing mice were injected with approximately 3.74 MBq of 68Ga-5 or 18F-FDG with tail vein administration under anesthesia. For blocking studies, subjects were pre-injected with unlabeled compound 5 (5 mg/kg) via the tail vein 1 h before the administration of 68Ga-5 and static PET images were obtained at designated time points, i.e., 15, 30, 60, and 90 min, which were reconstructed by a 3D-OSEM algorithm and a Monte Carlo-based accurate model. Regions of interest (ROIs) for major organs were drawn directly from the PET images, and the standardized uptake value (SUV) of all ROIs was also obtained directly from static PET images.BALB/c nude mice  were subcutaneously injected with tumor cells suspended in 100 µL of PBS  were purchased from Abcam. A standard immunofluorescence procedure was performed to handle the tumor sections that were removed from the subjects after small PET imaging.via the nitrogen atom on the piperazine ring of compound 5 and was placed in the outer space of the binding pocket, suggesting a minimal impact on the inhibitory potency  quinoxalin-6-yl) amino)-N-(piperidin-3-yl) nicotinamide (compound 14). The final product for radiolabeling, 2,2′,2′′-(10-(1-carboxy-4-(3-(5-((8-(1-methyl-1H-indol-6-yl) quinoxalin-6-yl) amino) nicotinamido) piperidin-1-yl)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (compound 5), was produced by an amidation reaction with 2,2′,2′′--1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DOTA-GA anhydride). In conclusion, the compound was successfully prepared through an 8-step convergent synthesis route with a total yield of 2.51%. In addition, compound 4 was also synthesized as a positive control with an identical procedure published previously.Compound 5 was synthesized following the published literature, with minor modifications as the synthesis route presented in Regents and conditions: a. Iron powder, H2O, EtOH, 78°C, 3 h; b. 1,4-dioxane-2,3-diol, EtOH, r.t., overnight; c. Pd (dppf)Cl2, DIPEA, 1,4-Dioxane, H2O, N2, 85°C, 4 h; d. EDCI, HOBT, 4-Methylmorpholine, tert-butyl 3-aminopiperidine-1-carboxylate, r. t, 4 h; e. Iron powder, H2O, EtOH, 78°C, 4 h; f. tBuOK, Pd(OAc)2, BINAP, toluene, 100°C, overnight; g. TFA, CH2Cl2, r. t, overnight; h. DOTA-GA anhydride, Et3N, DMSO, r. t, overnight.™-based PFKFB3 activity inhibition assay and a modified procedure for the cellular inhibition assay, compound 4 and compound 5 showed similar enzymatic IC50 values for PFKFB3 inhibition and IC50 values for inhibition of F2,6BP production in HCT166 cells in vitro. As shown in the table, the IC50 values for 4 and 5 for PFKFB3 activity inhibition were 6.7 nM ± 2.3 nM and 12.5 nM ± 4.5 nM, respectively. The IC50 values for F2,6BP production in HCT116 cells were 2.3 µM ± 0.8 µM and 5.3 µM ± 1.4 µM, respectively. These results indicated that the introduction of a chelating group (DOTA) led to a negligible impact on the inhibitory activity against PFKFB3. In addition, IC50 values of 2 nM and 8.37 µM were reported for the enzymatic PFKFB3 inhibition and cellular F2,6BP production assay (According to the ADP-Gloon assay .68Ga-5 was 94.5% + 4.2% (n = 5) after an SPE purification procedure. The radiolabeling reaction and the representative HPLC diagram are presented in 68Ga-5 showed a retention time of 7.8 min, which corresponds to the UV signal of compound 5.Radiolabeling of compound 5 was performed manually with high yield under 90, pH 3.5. According to the radio-HPLC profile, the radiochemical purity (RCP) of 68Ga-5 solutions of PBS saline, 80% EtOH, and rat serum, 68Ga-5 showed strong in vitro stability, and over 90% RCP was calculated from the HPLC diagram after 4 h incubation. The log D7.4 value of 68Ga-5 was −1.65 ± 0.28, indicating a much lower lipophilicity than compound 4 .According to the HPLC results for the 68Ga-5 showed preferential accumulation in all tested cells, with uptake ratios of 2.9%, 2.64%, 2.52%, and 2.31% for NUGC3, MKN45, H1975, and HCC827 cells after 1 h of incubation., respectively, In addition, the uptake in these cells can be effectively blocked by the unlabeled precursor (2 µM of compound 4), with uptake ratios decreased to 2.44%, 1.77%, 1.76%, and 1.84%, respectively. NUGC3 showed the highest uptake ratios as well as the largest blocking efficacy (uptake decreased to 84% compared with the unblocked group). As PFKFB3 is widely expressed in almost all types of tumor cells, the uptake ratios and the corresponding blocked uptake ratios indicated that the accumulation of radioactivity in cells is selective and could be mediated by PFKFB3.As shown in 68Ga-5 showed moderate pharmacokinetic properties, and the radioactivity in the blood and other major organs slowly decreased post injection. The liver showed the highest initial accumulation of radioactivity with an uptake ratio of 9.26%ID/g at 5 min post-injection (p.i.), and the accumulation decreased to 4.77%ID/g at 120 min p.i., indicating major hepatobiliary excretion. A notable distribution of radioactivity in the kidney was also observed, with accumulation of 7.00%ID/g and 1.27%ID/g at 5 min and 120 min p.i., respectively. The accumulation in the kidney indicated minor urinary excretion of 68Ga-5. Interestingly, a higher accumulation in the lung was observed with 3.70%ID/g at 5 min p.i., perhaps due to the large radioactive particles blocked in capillaries of the lung or contamination during organ harvest. Other organs and tissues showed no abnormal accumulation of radioactivity, such as the heart, stomach, muscle, bone and brain, with accumulation data of 1.27, 1.37, 0.45, 0.68, and 0.12%ID/g at 120 min p.i., respectively. Based on the biodistribution profile of 68Ga-5, we have the confidence to perform subsequent PET imaging studies.The tissue biodistribution profile is presented in 68Ga-5 in NUGC3 tumors can be observed and maintained afterwards, with SUV values of 0.60 and 0.50 at 15 min and 90 min p. i., respectively. With the clearance of radioactivity from major organs and muscle, a higher “Tumor-to-muscle” ratio can be obtained. In addition, significant uptake in the lung was also observed with SUV values ranging from 0.51 to 0.65 for all types of tumors included in this study, which agreed with the results of biodistribution studies. However, the intestine and urinary system showed higher uptake which may resulted from the lipophilicity of 68Ga-5, as also presented in 68Ga-5, with the tumor SUV value decreasing to 0.23 at 90 min in NUGC3 models. In the same subject, the NUGC3 tumor was also clearly visualized by 18F-FDG PET with a tumor SUV value of 0.54 at 90 min p.i. 18F-FDG not only showed higher SUV values than 68Ga-5 in NUGC3 tumors (0.54 VS. 0.50 @ 90 min p.i.), but also showed higher “Tumor-to-muscle” ratios (8.28 VS. 5.00 @ 90 min p.i.). In addition, 68Ga-5 showed much higher background uptake in internal organs such as liver, kidney, spleen, stomach and small intestine, making the quality of tumor imaging is much lower than that of 18F-FDG. Tumor uptake was also confirmed in mude mice bearing MKN45, H1975 and HCC827 tumors, with SUV values of 0.36, 0.26 and 0.27 at 90 min p.i (compared with the mean SUV 0.50 for NUGC3 tumors), respectively. The “Tumor-to-muscle” ratios for NUGC3, MKN45, H1975 and HCC827 models at 90 min p.i. are 5.50, 3.27, 2.45, and 2.36, respectively. For 18F-FDG and block studies in NUGC3 tumor models, the “Tumor-to-muscle” ratios are 8.28 and 1.91 (compared with 5.50 for non-blocking group), indicating the tumor angiogenesis is success and the blocking efficacy is significant in NUGC3 tumor models.Representative static PET images at different time points and the corresponding TACs (Time-Activity Curves) are presented in In further immunofluorescent staining of tumor tissues, we observed the binding of the antibody to the tumor cells, which confirmed the positive expression of PFKFB3 in all cell lines and cancer tissues used in this study .18F-FDG and 68Ga-FAPIs have been widely used in clinical for the diagnosis of a variety of solid tumors as powerful tools, but the false positive results are also reported which may affect the diagnostic efficacy. To develop highly potent, selective and sensitive PET tracers for the complement of 18F-FDG PET and 68Ga-FAPI PET is of great importance. Aminoquinoxaline derivatives showed potent activities against PFKFB3, and our interest in this scaffold lies in the convenient conversion of the most potent molecule, compound 4, to a 68Ga-labeled PET tracer (68Ga-5) using DOTA as a chelator. 68Ga-5 was prepared with high radio-chemical yield and radio-chemical purity, and the in vitro tests indicated that 68Ga-5 had strong stability and moderate lipophilicity. The selective accumulation of 68Ga-5 was confirmed by cell uptake studies with NUGC3, HCC827, MKN45 and H1975 cells. Biodistributions of 68Ga-5 revealed its moderate pharmacokinetic property, and primary hepatobiliary excretion was observed. In micro-PET imaging studies, the highest SUV values were obtained in NUGC3 tumors (0.5 at 90 min), followed by HCC827 (0.36), MKN45 (0.27) and H1975 (0.26), which agreed with the cell uptake studies. Tumor uptake can be blocked by the pre-injection of unlabeled compound 5 (5 mg/kg), suggesting that tumor uptake is selective. Furthermore, immunofluorescence microscopy images also confirmed the positive expression of PFKFB3 in all types of tumors included in this study.As a glycolysis-related enzyme, PFKFB3 is emerging as a potential target for tumor diagnosis and treatment in that it is highly expressed in almost all tumor tissues. In contrast to specific targets that are only expressed in specific types of tumors , PFKFB3 holds the potential to be regarded as a broad-spectrum target for PET tracer development. 68Ga-5 in the detection of PFKFB3-positive tumors, relatively higher background uptake was observed in both biodistribution and PET imaging studies, which may be caused by the lipophilicity of this tracer. With a log D7.4 value of −1.6 for 68Ga-5, significant liver uptake was detected, as well as higher intestine and urinary system distribution, and further investigation and optimization should pay more attention to the physicochemical properties to accelerate the excretion of the desired compound to reduce the background uptake, and enhance the “Tumor-to-Background” ratios. Furthermore, with DOTA as a chelator for radio-metal labeling, therapeutic radionuclides can also be radiolabeled for tumor treatment via this scaffold. Although our results indicated the capability of 68Ga-labeled aminoquinoxaline derivative (68Ga-5) as a potential PFKFB3 PET tracer, and our preliminary evaluations indicated that although this scaffold showed potent activity against PFKFB3 in vitro and in vivo, further investigation and optimization, focused on the physicochemical parameters of this scaffold, would be needed to develop more successful PFKFB3 targeted PET tracer for tumor diagnosis and treatment.In conclusion, we have successfully prepared the