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Bacterial leaf spot of pepper (BSP), primarily caused by Xanthomonas euvesicatoria (Xe), poses a significant challenge to pepper production worldwide. Despite its impact, the genetic diversity of this pathogen remains underexplored, which limits our understanding of its population structure. To bridge this knowledge gap, we conducted a comprehensive analysis using 103 Xe strains isolated from pepper in southwest Florida to characterize genomic and type III effector (T3E) variation in this population. Phylogenetic analysis of core genomes revealed a major distinct genetic lineage associated with amylolytic activity. This amylolytic lineage was represented in Xe strains globally. Molecular clock analysis dated the emergence of amylolytic strains in Xe to around 1972. Notably, non-amylolytic strains possessed a single base pair frameshift deletion in the ⍺-amylase gene yet retained a conserved C-terminus. GUS assay revealed the expression of two open reading frames in non-amylolytic strains, one at the N-terminus and another that starts 136 base pairs upstream of the ⍺-amylase gene. Analysis of T3Es in the Florida Xe population identified variation in 12 effectors, including two classes of mutations in avrBs2 that prevent AvrBs2 from triggering a hypersensitive response in Bs2-resistant pepper plants. Knowledge of T3E variation could be used for effector-targeted disease management. This study revealed previously undescribed population structure in this economically important pathogen.IMPORTANCEBacterial leaf spot (BSP), a significant threat to pepper production globally, is primarily caused by Xanthomonas euvesicatoria (Xe). Limited genomic data has hindered detailed studies on its population diversity. This study analyzed the whole-genome sequences of 103 Xe strains from peppers in southwest Florida, along with additional global strains, to explore the pathogen's diversity. The study revealed two major distinct genetic groups based on their amylolytic activity, the ability to break down starch, with non-amylolytic strains having a mutation in the ⍺-amylase gene. Additionally, two classes of mutations in the avrBs2 gene were found, leading to susceptibility in pepper plants with the Bs2 resistance gene, a commercially available resistance gene for BSP. These findings highlight the need to forecast the emergence of such strains, identify genetic factors for innovative disease management, and understand how this pathogen evolves and spreads.

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ß-Amylase, which catalyses the release of ß-anomeric maltose from the non-reducing end of starch, is widely used in the food industry. Increasing its enzyme activity through protein engineering might improve the efficiency of food processing. To obtain detailed structural information to assist rationale design, here the crystal structure of Bacillus cereus ß-amylase (BCB) complexed with maltose was determined by molecular replacement and refined using anisotropic temperature factors to 1.26 Å resolution with Rwork/Rfree factors of 12.4/15.7 %. The structure contains six maltose and one glucose molecules, of which two maltose and one glucose are bound at sites not previously observed in BCB structures. These three new sugar-binding sites are located on the surface and likely to be important in enhancing the degradation of raw-starch granules. In the active site of BCB, two maltose molecules are bound in tandem at subsites -2 âˆ¼ -1 and +1 âˆ¼ +2. Notably, the conformation of the glucose moiety bound at subsite -1 is a mixture of α-anomeric distorted 1,4B boat and 4C1 chair forms, while those at subsites -2, +1 âˆ¼ +2 are all in the 4C1 chair forms. The O1 of the distorted α-glucose residue at subsite -1 occupies the position of the putative catalytic water, forming a hydrogen bond with OE1 of Glu367 (base catalyst), suggesting that this distorted sugar is not involved in catalysis. Together, these findings pave the way for further improving the functionality of microbial ß-amylase enzymes.

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Pueraria lobata (Willd.) Ohwi is a traditional medicinal herb that has been extensively used in Chinese medicine for various therapeutic purposes. In this study, twelve chemical constituents were isolated from the roots of P. lobata, comprising three puerosides (compounds 1-3), six alkaloids (compounds 4-9), and three additional compounds (compounds 10-12). Notably, compound 1 (4R-pueroside B) was identified as a novel compound. The structures of all compounds were elucidated using a range of spectroscopic techniques, including CD spectroscopy for the first-time determination of the absolute configurations of pueroside B isomers (compounds 1 and 2). Enzyme inhibition assays revealed that, with the exception of compound 2, all isolated compounds exhibited varying degrees of α-glucosidase and α-amylase inhibitory activity. Remarkably, compound 12 demonstrated IC50 values of 23.25 µM for α-glucosidase inhibition and 27.05 µM for α-amylase inhibition, which are superior to those of the positive control, acarbose (27.05 µM and 36.68 µM, respectively). Additionally, compound 11 exhibited inhibitory activity against α-glucosidase and α-amylase comparable to the positive control, acarbose. Molecular docking studies indicated that compound 12 interacts with the active sites of the enzymes via hydrogen bonds, van der Waals forces, and hydrophobic interactions, which likely contribute to their inhibitory effects. These findings suggest that the chemical constituents of P. lobata could be potential natural sources of α-amylase and α-glucosidase inhibitors, with compound 12 being particularly promising for further investigation.

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Calendula officinalis L. has numerous health-promoting properties due to the presence of a large number of lipophilic compounds. Their effective delivery to the body requires the use of an appropriate technique such as emulsification. So, the main purpose of this study was to understand how the profile of lipophilic compounds from pot marigold and the pro-health potential are shaped by different types of protein, oil, and drying techniques in o/w nanoemulsion. To obtain this, the profiles of carotenoid compounds and tocols were measured. Additionally, antioxidant potential and the ability to inhibit α-amylase and α-glucosidase were measured. Pea protein emulsion exhibited a higher final content of carotenoid compounds (23.72-39.74 mg/100 g), whereas those with whey protein had stronger α-amylase inhibition (487.70 mg/mL). The predominant compounds in the studied nanoemulsions were ß-carotene (between 19% and 40%), followed by α-tocopherol/γ-tocopherol. The type of proteins shaped the health-promoting properties and determined the content of health-promoting compounds.

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Type-two diabetes, characterised by insulin resistance or inadequate insulin production, is prevalent among adults. The α-amylase enzyme contributes to carbohydrate digestion, elevating postprandial glucose levels. Natural compounds like caffeic acid offer a solution. This study investigates α-amylase inhibition via in-vitro and in-silico methods, emphasising the connection between phenolic compounds and antidiabetic efficacy for in-silico analysis. Enzyme kinetics, IC50, and molecular docking examine caffeic acid's inhibitory action on α-amylase, comparing it with gallic acid and acarbose. Caffeic acid outperforms acarbose with an IC50 of 4.505 mg/mL versus 16.81 mg/mL, showcasing strong antidiabetic activity. Caffeic acid's superior 1,1-diphenyl-2-picrylhydrazyl (DPPH) inhibition (90.67%) compared to gallic acid (55.76%) indicates potent antioxidative and antidiabetic properties. Molecular docking reveals hydrogen bonding between caffeic acid and α-amylase. These insights lay the groundwork for phenolic-based diabetic therapies, offering less expensive treatment for diabetes patients.

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This study assessed the effects of increased pre-start diet density on the metabolism, crop filling, and overall performance of broilers under cold stress during their initial 14 days of life. Using 576 one-day-old Cobb500 male chicks from 27-week-old breeders, the experiment employed a 2 × 2 arrangement, varying thermal conditions (thermoneutrality or cold stress at 18 °C for 8 h) and pre-start diet composition (21.5% crude protein, 2970 kcal/kg or 22.5%, 3050 kcal/kg). The cold stress group exhibited lower cloacal temperature and decreased crop filling rate during the first two days (P < 0.05). Chick behavior was significantly affected at 1 and 5 days (P < 0.05), and corticosterone levels in serum were higher for the cold stress group at 7 days (P < 0.05). Feed intake at 7 days was lower in the high-density feed group (P < 0.05). No significant interactions were observed for feed intake, body weight gain, or feed conversion ratio at 7 and 35 days (P > 0.05). Cold stress resulted in performance losses, impacting feed conversion and the Productive Efficiency Index. The dense diet influenced performance only within the first week, with subsequent diets showing no effect, suggesting dietary manipulation alone was insufficient to mitigate cold stress-induced losses.

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Exploring the potential of natural products against diabetes and obesity is in demand nowadays. Pancreatic α-amylase and pancreatic lipase are the drug targets to minimize the absorption of glucose from starch and fatty acids from lipids, respectively. In this study, five Piper species, namely P. sarmentosum (Ps), P. wallichii (Pw), P. retrofractum (Pr), P. nigrum (Pn), and P. betle (Pb), which are commonly used as food ingredients and traditional medicines, were evaluated for their inhibitory activities against pancreatin using the microtiter plate method. Additionally, pancreatin inhibitors were identified through a cost-effective high-performance thin-layer chromatography (HPTLC)-bioautography developed using red starch and p-nitrophenyl palmitate, corresponding to anti-amylase and -lipase activities, respectively. Of the 15 samples tested, leaf samples from Pb, which had the highest total phenolic and total flavonoid contents, exhibited remarkable inhibitory activity against pancreatin, with a relative amylase inhibitory capacity (RAIC) ranging between 4.260 × 10-5 and 4.861 × 10-5 and a reciprocal half-maximal inhibitory concentration (1/IC50, PTL) of 0.390-0.510 (mg/mL)-1. Additionally, Ps samples demonstrated the second-ranked anti-pancreatin activity. Principal component analysis indicated that total phenolic content contributed to the anti-pancreatin activities of Pb samples. The anti-pancreatin bands were isolated and identified as caffeic acid, myricetin, genistein, piperine, and eugenol. Myricetin, in the roots of Ps samples, showed notable anti-pancreatin activity, which was consistent with results from the in silico prediction toward pancreatic α-amylase and pancreatic lipase. Caffeic acid and eugenol were present in Pb samples. In conclusion, the developed cost-effective pancreatin HPTLC-bioautography efficiently identified amylase and lipase inhibitors from Piper herbs, which supported the use of these plants for antidiabetes and anti-obesity.

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Alpha-amylase and beta-amylase coexist as mixtures in industrial production, and the two amylases have active synergistic effects when they approach each other. These effects are due to enhanced enzyme binding affinity for the substrate and the rate of particle hydrolysis. Here, we report the allosteric mechanism of this synergistic effect in α- and ß-amylase mixtures. The assay showed higher activity after mixing α- and ß-amylase. Molecular docking showed that α- and ß-amylase create a stable dual-enzyme complex with high binding energy, and that complex formation does not affect the exposure of respective active sites. ß-Amylase is specifically bound to the B domain of α-amylase, and the dynamic plasticity of the B domain makes it move spatially, and this adjustment leads to a more open conformation in the active site of α-amylase. Because the enzymes binding make the complex more stable, the degree to which the relative activity of the dual-enzyme complex is inhibited is significantly reduced. After enzyme hydrolysis, the products maltose and glucose accumulate and produce competitive inhibition, which explains the relative activity decrease of the later-stage dual-enzyme cooperation. Structural characterization by FT-IR and CD spectroscopy did not reveal significant changes in respective secondary structures after enzyme binding.

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The Pacific fat sleeper, Dormitator latifrons, is an omnivorous freshwater fish that primarily feeds on detritus. Our understanding of the digestive physiology of this species still needs to be completed, particularly concerning the characterization of its digestive enzymes. This information is crucial in guiding the design of diets that promote optimal digestion of this species, which has the potential for aquaculture. Thus, this study aimed to optimize enzymatic methods and characterize the digestive enzymes of the digestive tract regions: anterior region (AR), middle region (MR), posterior region (PR), and hepatopancreas (HP). Total acid protease, total alkaline protease, amylase, and lipase activities were measured. The enzymatic methods were optimized at an eco-physiological temperature of 25 °C based on extract volume, extract dilution, incubation time, pH, and CaCl2 concentration to determine specific activity (U/mg of protein). The optimal pH for acid protease (AR) was pH 2.0; while for alkaline protease, the optimal pH was between 7.5 and 11.0. For AR, chymotrypsin was pH 7.0; for the remaining digestive regions, it was pH 9.0-11.0. The optimal pH for amylase was 6.0 to 7.5 (all regions), and for lipase, it was between 7.0 and 11.0, with two apparent in vitro activity peaks (PR). HP experimental samples showed low or no chymotrypsin, amylase, and lipase activity. CaCl2 did not affect enzyme activity except for amylase and lipase (only in PR and HP, respectively). The acid proteolytic activity (pH 2.0) found in AR and the proteolytic inhibition by pepstatin suggest the presence of a stomach.

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Research and theory suggest an important role of neuroendocrine function in emotional development, particularly under conditions of elevated stress. We provide empirical data to clarify associations between alpha-amylase (AA) and cortisol as well as test the differential linkages among AA, cortisol, and symptoms of anxiety, depression, and posttraumatic stress in children. Children recruited from a low-income elevated violence community (n = 100; mean age = 10, SD = 0.64; 79% Latino; 67% received free or reduced lunch) were assessed on diurnal levels of AA and cortisol along with assessments of anxiety, depression, and posttraumatic stress symptoms (PTSS). Elevated anxiety symptoms were associated with steeper linear slopes of AA with higher levels of AA in the morning but lower levels of AA in the evening. Depression was associated with differential cubic trajectories of AA when PTSSs were included in the model. Anxiety also predicted differential cubic diurnal trends in cortisol, such that greater anxiety symptoms were associated with relatively higher levels of cortisol in the evening. Again, depression symptoms when PTSS were included predicted diurnal cubic trends with elevated depression associated with lower awakening and midday cortisol that reversed to higher evening cortisol compared to youth with fewer self-reported depression symptoms.

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Pomace is a by-product resulting from the pressing of fruits and vegetables into juices, and it is typically treated as waste. Interestingly, pomace contains minimal amounts of protein and fat but is characterized by its high polyphenol and dietary fiber contents, which may have health benefits for human physiology. Therefore, they are a potentially attractive raw material for the food industry, but to our knowledge, no smoothies with their addition have been prepared and described so far. Consequently, products derived from apple juice, incorporating different doses of fresh (6% and 12%) and dried (3% and 6%) black or red currant pomace, were formulated, and their physical properties, chemical composition, bioactive compound content, and health-promoting potential (in vitro antioxidant and antidiabetic activity) were evaluated. Additionally, the products underwent sensory assessment by consumers. The fortified beverages exhibited different physical characteristics and chemical compositions than apple juice. All smoothies were characterized by higher concentrations of anthocyanins, flavonols, and procyanidin polymers compared to the base product. Moreover, 75% of them exhibited a significantly elevated phenolic acid content as well as a higher concentration of flavan-3-ols. The majority of fresh smoothies exhibited significantly higher in vitro antioxidant capacities and increased in vitro α-amylase and α-glucosidase inhibitory effects compared to the base product. The highest ABTS activity was recorded in the variant with 6% dried black currant pomace. In turn, the smoothie with 3% dried red currant pomace had the most effective FRAP effect and, together with the product containing 12% fresh black currant pomace, ORAC antioxidant activity and α-glucosidase inhibition also. The introduction of 6% dried red currant pomace led to the creation of a beverage that most effectively inhibited α-glucosidase. The study showed that the application of various types of pomace, mainly that of black currant, into apple juice enables the development of new functional products with sensory attributes that are favorably evaluated by consumers.

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The digestive properties of starch are crucial in determining postprandial glycaemic excursions. Genistein, an active phytoestrogen, has the potential to influence starch digestion rates. We investigated the way genistein affected the digestive properties of starch in vitro. We performed enzyme kinetics, fluorescence spectroscopy, molecular docking, and molecular dynamics (MD) simulations for analysing the inhibitory properties of genistein on starch digestive enzymes as well as clarifying relevant mechanism of action. Our findings demonstrated that, following the addition of 10% genistein, the contents of slowly digestible and resistant starches increased by 30.34% and 7.18%, respectively. Genistein inhibited α-amylase and α-glucosidase, with half maximal inhibitory concentrations of 0.69 ± 0.06 and 0.11 ± 0.04 mg/mL, respectively. Genistein exhibits a reversible and non-competitive inhibiting effect on α-amylase, while its inhibition on α-glucosidase is a reversible mixed manner type. Fluorescence spectroscopy indicated that the presence of genistein caused declining fluorescence intensity of the two digestive enzymes. Molecular docking and MD simulations showed that genistein binds spontaneously to α-amylase via hydrogen bonds, hydrophobic interactions, and π-stacking, whereas it binds with α-glucosidase via hydrogen bonds and hydrophobic interactions. These findings suggest the potential for developing genistein as a pharmacologic agent for regulating glycaemic excursions.

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The foramen of Huschke or foramen tympanicum is a developmental defect in the bony part of the external auditory meatus. It occurs due to incomplete obliteration of the tympanic canal and the persistence of openings along the canal. This leads to the presence of a salivary fistula and the most common symptoms of the condition are otalgia, otorrhea, and hearing loss. We report a case of a 69-year-old female patient who presented to the ENT with symptoms of watery discharge from the left ear for 12 years. In the last two weeks, pain and itching appeared. Otoscopy showed the presence of a clear fluid in the external auditory meatus with a normal tympanic membrane. Biochemical analysis of the fluid proved the presence of amylase. Computed tomography showed a fistula between the parotid gland and the external ear canal. The diagnosis of the persistent foramen of Huschke remains difficult due to the fact that it could mimic many other conditions. The otalgia and otorrhea are symptoms that suggest a very wide differential diagnosis. Misdiagnosis and inappropriate treatment are common.

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Postprandial hyperglycemia (PPG) exacerbates endothelial dysfunction and impairs vascular function in diabetes as well in healthy people. Though synthetic drugs are available to regulate PPG, the severe gastrointestinal side effects of those medications have prompted the search for alternative treatments. Recently, some phytochemicals captured the attention because of their inhibitory effects on α-amylase to control diabetes. The aim of this study was to investigate and identify potential alpha-amylase inhibitors in C. indica and W. coagulans. This study also aims to understand one of the possible mechanisms of action of plants for their anti-diabetic activity. A total of 36 phytochemical ligands were subjected for protein-ligand docking analysis. Among the phytochemicals, Taraxerol and Epoxywithanolide-I demonstrated significant binding free energy of - 10.2 kcal/mol and - 11.9 kcal/mol respectively, which was higher than the reference acarbose with - 8.6 kcal/mol. These molecules were subjected for molecular dynamics simulation (MDS) analysis with alpha-amylase protein for a duration of 150 ns. Among the three complexes, Taraxerol and Epoxywithanolide-I complexes demonstrates strong potential as inhibitors of the target protein. MDS results were analyzed via root mean square deviation (RMSD), fluctuation of residues, potential energy, radii of gyration and solvent access surface area analysis. Taraxerol demonstrated a significantly low potential energy of - 1,924,605.25 kJ/mol, and Epoxywithanolide-I demonstrated - 1,964,113.3 kJ/mol of potential energy. RMSD plot shows that Epoxywithanolide-I has much higher stability than the other MDS complexes. Drugability and toxicity studies show that the test ligands are demonstrating strong potential as drug like molecules. The results of the study conclude that, Taraxerol of C. indica and Epoxywithanolide-I of W. coagulans are strong inhibitors of alpha-amylase enzyme and that, this is one of the possible mechanisms of action of the plants for their reported anti-diabetic activities. Further in-vitro analysis is in demand to prove the observed results.

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OBJECTIVES: We investigated salivary biomarkers of stress, more specifically, cortisol and alpha-amylase, to evaluate effects of individualized music listening (IML) in people with dementia. METHOD: Participants were N = 64 nursing home residents with dementia (meanage = 83.53 ± 7.71 years, 68.8% female). Participants were randomly assigned to either listening to their favorite music every other day for a period of six weeks (intervention), or standard care (control). Using the Saliva Children`s Swab (SCS), saliva was collected before, after, and 20 min after IML sessions at the beginning and end of the intervention period for the analysis of salivary alpha-amylase and cortisol. RESULTS: Using the SCS was feasible in people with dementia. Nevertheless, there was no effect of IML on salivary stress markers. DISCUSSION: Although using SCS was feasible, active patient engagement is required. Future studies need to corroborate findings in larger samples. TRIAL REGISTRATION: German Clinical Trials Register: DRKS00015641, ISRCTN registry: ISRCTN59052178.

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As a typical C4 plant and important crop worldwide, maize is susceptible to drought. In maize, transitory starch (TS) turnover occurs in the vascular bundle sheath of leaves, differing from that in Arabidopsis (a C3 plant). This process, particularly its role in drought tolerance and the key starch-hydrolyzing enzymes involved, is not fully understood. We discovered that the expression of the ß-amylase (BAM) gene ZmBAM8 is highly upregulated in the drought-tolerant inbred line Chang7-2t. Inspired by this finding, we systematically investigated TS degradation in maize lines, including Chang7-2t, Chang7-2, B104, and ZmBAM8 overexpression (OE) and knockout (KO) lines. We found that ZmBAM8 was significantly induced in the vascular bundle sheath by drought, osmotic stress, and abscisic acid. The stress-induced gene expression and chloroplast localization of ZmBAM8 align with the tissue and subcellular sites where TS turnover occurs. The recombinant ZmBAM8 was capable of effectively hydrolyzing leaf starch. Under drought conditions, the leaf starch in ZmBAM8-OE plants substantially decreased under light, while that in ZmBAM8-KO plants did not decrease. Compared with ZmBAM8-KO plants, ZmBAM8-OE plants exhibited increased drought tolerance. Our study provides insights into the significance of leaf starch degradation in C4 crops and contributes to the development of drought-resistant maize.

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Objective: Diabetic individuals have a higher probability of suffering from illness and death due to small blood vessel-related problems such as retinopathy, neuropathy, nephropathy, and stroke than other complications. There are many synthetic anti-diabetic agents available, but these can be expensive and have undesirable pathological effects. The enzyme α-amylase (hydrolase), catalyzes the hydrolysis of starch to maltose and glucose via the cleavage of α-1,4-glucosidic linkages. Diabetes mellitus patients may benefit from a therapeutic strategy that involves slowing the hydrolysis of starch by inhibiting the activity of α-amylase. Thus, looking for cost-effective, natural, and safe antidiabetic agents is essential. This study aims to screen phytoconstituents and evaluate the in-vitro and in-silico α-amylase inhibitory activity of the ethanolic extract of Adhatoda vasica leaves. Methods: The extraction of Adhatoda vasica leaves was performed using ethanol via the Soxhlet extraction process. Different concentrations (100 µg/mL to 1000 µg/mL) of ethanolic extract, Acarbose, and Sitagliptin, were prepared and evaluated for α-amylase inhibitory activity using the spectrophotometric method. Molecular docking (AutodockVina 1.2.0) and toxicity profiling (SToPToX web server) studies were performed. Results: The ethanolic extract of Adhatoda vasica leaves showed the highest percentage inhibition against α-amylase (56.763 ± 0.0035) at a concentration of 1000 µg/mL. The in-silico study supported this inhibitory activity. Vasicoline (C5) and Quercetin (C9), the active constitute of Adhatoda vasica, showed the best binding energies of -8.3 and -8.0 Kcal/mol, respectively against α-amylase enzyme (PDBID: 4W93). A toxicity study revealed the safety profile of the plant extract. Conclusion: It was concluded that Adhatoda vasica leaves possess some bioactive compounds that are responsible for controlling blood glucose levels, and their identification, purification, and isolation may lead to the development of new therapeutic agents with fewer side effects than the available drugs.

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Urine amylase levels are usually used to diagnose acute pancreatitis. However, there are reported cases where urine amylase levels are slightly increased in individuals without pancreatitis. Herein, we report the case of a young lady who presented with acute abdominal pain for 3 days. Her urine amylase level was 1717 U/L upon admission, and her condition was initially treated as acute pancreatitis. Unfortunately, the patient demonstrated abdominal guarding after 24 h; thus, urgent computed tomography (CT) was performed. CT revealed the presence of a dilated small bowel. She underwent emergency laparotomy, wherein a gangrenous small bowel with no evidence of saponification at the lesser sac was noted. Due to the non-specific nature of hyperamylasaemia, an alternative diagnosis other than acute pancreatitis should be considered if the clinical symptoms are not suggestive of pancreatitis or the condition worsens despite conservative management.

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Inhibitors of α-amylase have been developed to regulate postprandial blood glucose fluctuation. The enzyme inhibition arises from direct or indirect inhibitor-enzyme interactions, depending on inhibitor structures. However, an ignored factor, substrate, may also influence or even decide the enzyme inhibition. In this work, it is innovatively found that the difference in substrate enzymolysis modes, i.e., structural composition and concentration of α-1,4-glucosidic bonds, triggers the diversity in inhibitor-enzyme aggregating behaviors and α-amylase inhibition. For competitive inhibition, there exists an equilibrium between α-amylase-substrate catalytic affinity and inhibitor-α-amylase binding affinity; therefore, a higher enzymolysis affinity and concentration of α-1,4-glucosidic structures interferes the balance, unfavoring inhibitor-enzyme aggregate formation and thus weakening α-amylase inhibition. For uncompetitive inhibition, the presence of macromolecular starch is necessary instead of micromolecular GalG2CNP, which not only binds with active site but with an assistant flexible loop (involving Gly304-Gly309) near the site. Hence, the refined enzyme structure due to the molecular flexibility more likely favors the inhibitor binding with the non-active loop, forming an inhibitor-enzyme-starch ternary aggregate. Conclusively, this study provides a novel insight into the evaluation of α-amylase inhibition regarding the participating role of substrate in inhibitor-enzyme aggregating interactions, emphasizing the selection of appropriate substrates in the development and screening of α-amylase inhibitors.

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Uncontrolled hyperglycemia leads to increased oxidative stress, chronic inflammation, and insulin resistance, rendering diabetes management harder to accomplish. To tackle these myriads of challenges, researchers strive to explore innovative multifaceted treatment strategies, including inhibiting carbohydrate hydrolases. Herein, we report alkyl-ether EGCG derivatives as potent α-amylase and α-glucosidase inhibitors that could simultaneously ameliorate oxidative stress and inflammation. 4″-C18 EGCG, the most promising compound, showed multifold improvement in glycaemic management compared to acarbose, with 230-fold greater inhibition (competitive) of α-glucosidase (IC50 0.81 µM) and 3-fold better inhibition of α-amylase (IC50 3.74 µM). All derivatives showed stronger antioxidant activity (IC50 6.16-15.76 µM) than vitamin C, while acarbose showed none. 4″-C18 EGCG also downregulated pro-inflammatory cytokines and showed no significant cytotoxicity up to 50 µM in primary human peripheral blood mononuclear cells (PBMC), non-cancerous cell line, 3T3-L1 and HEK 293. The in silico binding affinity analysis of 4″-C18 EGCG with α-amylase and α-glucosidase was found to exhibit a good extent of interaction as compared to acarbose. In comparison to EGCG, 4″-Cn EGCG derivatives were found to remain stable in the physiological conditions even after 24 h. Together, the reported molecules demonstrated multifaceted antidiabetic potential inhibiting carbohydrate hydrolases, reducing oxidative stress, and inflammation, which are known to aggravate diabetes.