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BackgroundA substantial body of research has demonstrated a notable impact of hot temperatures on mortality from cardiovascular diseases (CVDs). However, a paucity of studies has addressed the influence of sweltering conditions on CVD mortality.ObjectiveTo investigate the effect of sweltering conditions on mortality from CVD among permanent residents of Huizhou City, using the temperature-humidity index (THI) as an indicator.MethodsThis study employs descriptive statistics, distributed lag non-linear model (DLNM) and general algebraic modeling system (GAMs) with the THI as an indicator in order to examine the impact of sweltering conditions on the mortality of CVD among permanent residents of Huizhou City.ResultsSweltering conditions increase the risk of death from CVDs and have a cumulative lag effect. The greater the THI, the more pronounced the increase in mortality, and after a certain range, the mortality rate from CVDs increases significantly, and the effect is gender-specific. The lag effect generally peaks in 2–3 days, and the lag effect of stroke mortality is longer and deeper than that of coronary heart disease (CAD) mortality.ConclusionSweltering increased the mortality of cardiovascular diseases in Huizhou city, so we should pay attention to public health intervention strategies under sweltering.

Abstract Digital PCR (dPCR) holds immense potential for precisely detecting nucleic acid markers essential for personalized medicine. However, its broader application is hindered by high consumable costs, complex procedures, and restricted multiplexing capabilities. To address these challenges, an all‐in‐one dPCR system is introduced that eliminates the need for microfabricated chips, offering fully automated operations and enhanced multiplexing capabilities. Using this innovative oscillation‐induced droplet generation technique, OsciDrop, this system supports a comprehensive dPCR workflow, including precise liquid handling, pipette‐based droplet printing, in situ thermocycling, multicolor fluorescence imaging, and machine learning‐driven analysis. The system's reliability is demonstrated by quantifying reference materials and evaluating HER2 copy number variation in breast cancer. Its multiplexing capability is showcased with a quadruplex dPCR assay that detects key EGFR mutations, including 19Del, L858R, and T790M in lung cancer. Moreover, the digital stepwise melting analysis (dSMA) technique is introduced, enabling high‐multiplex profiling of seven major EGFR variants spanning 35 subtypes. This innovative dPCR system presents a cost‐effective and versatile alternative, overcoming existing limitations and paving the way for transformative advances in precision diagnostics.

The grinding process is one of the key factors affecting the quality of glutinous rice flour (GRF). As an emerging grinding method, semidry grinding aims to solve the problems of the high yield of wastewater in traditional wet grinding and the high content of damaged starch in dry grinding, in which the water content has a great influence on the quality of GRF. However, semidry grinding has not yet been formally put into production due to limitations such as the long time required to adjust the water content of rice grains. Therefore, this work was carried out to shorten the soaking time of glutinous rice (GR) by hot air pretreatment, and to conduct a systematic and in-depth study of the effect of water content on the quality of GRF, including water distribution, water hydration properties, thermal properties, rheological properties, and microstructure. The results showed that the GRF with higher water content had lower water solubility and higher enthalpy of pasting, which were due to the low content of damaged starch and the high degree of crystallization. The particle size of the GRF became smaller as the interaction between water and starch was enhanced and the GR was softened. In addition, the viscosity and elasticity of the GRF were also improved with an increase in water content. This work provides theoretical guidance for the improvement of semidry grinding to a certain extent.

Many α-agarases have been characterized and are utilized for producing agarooligosaccharides through the degradation of agar and agarose, which are considered valuable for applications in the food and medicine industries. However, the catalytic mechanism and product transformation process of α-agarase remain unclear, limiting further enzyme engineering for industrial applications. In this study, an α-agarase from Catenovulum maritimus STB14 (Cm-AGA) was employed to degrade agarose oligosaccharides (AGOs) with varying degrees of polymerization (DPs) to investigate the catalytic mechanism of α-agarases. The results demonstrated that Cm-AGA could degrade agarose into agarotetraose and agarohexaose. The reducing ends of agarotetraose and agarohexaose spontaneously release unstable 3,6-anhydro-α-l-galactose molecules, which were further degraded into agarotriose and agaropentose. Cm-AGA cannot act on α-1,3-glucoside bonds in agarotriose, agarotetraose, neoagarobiose, and neoagarotetraose but can act on AGOs with a DP greater than four. The product analysis was further verified by β-galactosidase hydrolysis, which specifically cleaves the non-reducing glycosidic bond of agarooligosaccharides. Multiple sequence alignment results showed that two conserved residues, Asp994 and Glu1129, were proposed as catalytic residues and were further identified by site-directed mutagenesis. Molecular docking of Cm-AGA with agaroheptose revealed the potential substrate binding mode of the α-agarase. These findings enhance the understanding of Cm-AGA’s catalytic mode and could guide enzyme engineering for modulating the production of agarooligosaccharides.

The 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) has garnered considerable attention for its ability to increase the degree of branching of starch and retard starch digestion, which has great industrial applications. Previous studies have reported that the N-terminal domain plays an important role in the expression and stability of GBEs. To further increase the catalytic ability of Gt-GBE, we constructed five mutants in the N-terminal domain: L19R, L19K, L25R, L25K, and L25A. Specific activities of L25R and L25A were increased by 28.46% and 23.46%, respectively, versus the wild-type Gt-GBE. In addition, the α-1,6-glycosidic linkage ratios of maltodextrin samples treated with L25R and L25A increased to 5.71%, which were significantly increased by 19.96% compared with that of the wild-type Gt-GBE. The results of this study suggest that the N-terminal domain selective modification can improve enzyme catalytic activity, thus further increasing the commercial application of enzymes in food and pharmaceutical industries.

Abstract Agarases are enzymes that degrade agar and agarose to produce agar oligosaccharides with multiple functional activities. Compared with β‐agarases, the natural source of α‐agarases is limited, which severely restricts the industrial application of α‐agarases. Here, we cloned and heterologously expressed an α‐agarase belonging to glycoside hydrolase family 96 named Cm‐AGA from the marine bacterium Catenovulum maritimum STB14. The production conditions of recombinant Cm‐AGA were optimized as: taking Terrific Broth (TB) (pH 6.5) with 5 g/L of fructose and 24 g/L of yeast extract H07014 as the fermentation medium, after culturing at 37°C for 2 h, isopropyl‐β‐ d‐thiogalactoside was added with a final concentration of 0.01 mM to induce for 44 h. The obtained enzyme activity was 13.81 U/ml and was about 6.6 times the initial activity. The specific activity of recombinant Cm‐AGA was 206.1 U/mg, the optimum temperature and pH were 35°C and 8.0, respectively, and the enzyme activity could be activated by Mn2+ and Ca2+. The hydrolysis product results showed that Cm‐AGA is the first reported α‐agarase with agarobiose (A2) and agarotetraose (A4) as the dominant products, suggesting the great potential of Cm‐AGA in the efficient production of agaro‐oligosaccharides with a low degree of polymerization.

Abstract Background CDK4/6 inhibitors combined with endocrine therapy has become the preferred treatment approach for patients with estrogen receptor-positive metastatic breast cancer. However, the predictive biomarkers and mechanisms of innate resistance to CDK4/6 inhibitors remain largely unknown. We sought to elucidate the molecular hallmarks and therapeutically actionable features of patients with resistance to CDK4/6 inhibitors. Methods A total of 36 patients received palbociclib and endocrine therapy were included in this study as the discovery cohort. Next-generation sequencing of circulating tumour DNA in these patients was performed to evaluate somatic alterations associated with innate resistance to palbociclib. Then the candidate biomarker was validated in another independent cohort of 104 patients and publicly available datasets. The resistance was verified in parental MCF-7 and T47D cells, as well as their derivatives with small interfering RNA transfection and lentivirus infection. The relevant mechanism was examined by RNA sequencing, chromatin immunoprecipitation and luciferase assay. Patient-derived organoid and patient-derived xenografts studies were utilized to evaluated the antitumor activity of rational combinations. Results In the discovery cohort, S6K1 amplification (3/35, 9%) was identified as an important reason for innate resistance to CDK4/6 inhibitors. In the independent cohort, S6K1 was overexpressed in 15/104 (14%) patients. In those who had received palbociclib treatment, patients with high-expressed S6K1 had significantly worse progression free survival than those with low S6K1 expression (hazard ratio = 3.0, P = 0.0072). Meta-analysis of public data revealed that patients with S6K1 amplification accounted for 12% of breast cancers. Breast cancer patients with high S6K1 expression had significantly worse relapse-free survival (hazard ratio = 1.31, P

This review presents an update on the physical, chemical, and biological properties of food allergens in plant sources, focusing on the few protein families that contribute to multiple food allergens from different species and protein families recently found to contain food allergens. The structures and structural components of the food allergens in the allergen families may provide further directions for discovering new food allergens. Answers as to what makes some food proteins allergens are still elusive. Factors to be considered in mitigating food allergens include the abundance of the protein in a food, the property of short stretches of the sequence of the protein that may constitute linear IgE binding epitopes, the structural properties of the protein, its stability to heat and digestion, the food matrix the protein is in, and the antimicrobial activity to the microbial flora of the human gastrointestinal tract. Additionally, recent data suggest that widely used techniques for mapping linear IgE binding epitopes need to be improved by incorporating positive controls, and methodologies for mapping conformational IgE binding epitopes need to be developed.

In order to improve the ability of starch to absorb menthone, corn starch was modified by enzymatic treatment (amyloglucosidase and α-amylase) combined with octenyl succinic anhydride (OSA) esterification. The oil absorption rate of starch modified by enzymatic treatment followed by OSA (P-OSA) reached 101.33%, whereas that of samples with reverse action sequences (OSA-P) was only 59.67%. The degree of substitution of OSA-P was also generally lower than that of P-OSA. At high OSA addition, OSA-P had a smaller specific surface area with fewer pores because octenyl succinic (OS) groups impeded the enzymatic treatment. Compared with OSA-P, the lamellar structure of P-OSA is sparser and less ordered. Owing to its pores, P-OSA was beneficial for the reaction to occur inside the granules, which was observed by Raman spectroscopy and laser confocal microscopy. At high OSA addition, the loading of P-OSA to menthone could reach 64.34 mg/g.

Highland barley (HB) is a nutritious crop with excellent health benefits, and shows promise as an economically important crop with diverse applications. Starch is the main component of HB and has great application potential owing to its unique structural and functional properties. This review details the latest status of research on the isolation, chemical composition, structure, properties, and applications of highland barley starch (HBS). Suggestions regarding how to better comprehend and utilize starches are proposed. The amylopectin content of HBS ranged from 74% to 78%, and can reach 100% in some varieties. Milling and air classification of barley, followed by wet extraction, can yield high-purity HBS. The surface of HBS granules is smooth, and most are oval and disc-shaped. Normal, waxy, and high-amylose HBS have an A-type crystalline. Due to its superb freeze-thaw stability, outstanding stability, and high solubility, HBS is widely used in the food and non-food industries. The digestibility of starch in different HB whole grain products varies widely. Therefore, the suitable HB variety can be selected to achieve the desired glycemic index. Further physicochemical modifications can be applied to expand the variability in starch structures and properties. The findings provide a thorough reference for future research on the utilization of HBS.

Abstract Background Krabbe disease (also known as globoid cell leukodystrophy) cause by a deficiency of the enzyme β-galactocerebrosidase (galactosylceramidase, GALC). The deficiency of GALC leads to accumulation of galactosylceramide and psychosine, the latter GALC substrate having a potential role in triggering demyelination. Typically, the disease has an infantile onset, with rapid deterioration in the first few months, leading to death before the age of 2 years. The late onset forms (late-infantile, juvenile, and adult forms) are rare with variable clinical outcomes, presenting spastic paraplegia as the main symptom. Case presentation We recruited a family with two affected individuals. The proband (Patient 1), a 25-year-old male, was presented with slow progressive symptoms, including spastic gait disturbance and vision loss since the 5th year of life. His elder sister (Patient 2), became wheelchair-bound and demented at the age of 22 years. Brain magnetic resonance imaging (MRI) showed increased signal intensity in the white matter along with the involvement of the bilateral corticospinal tracts. GALC deficiency was confirmed by biochemical analysis. DNA sequencing revealed two mutations (c.865G > C: p. G289R and c.136G > T: p. D46Y) in GALC. The clinical characteristics, brain MRI, biochemical and molecular findings led to the diagnosis of Krabbe disease. Conclusion Clinical and neuroimaged signs, positive enzymatic analysis and molecular data converged to definite diagnosis in this neurodegenerative disease.

Marine extremophiles produce cold-adapted and/or salt-tolerant enzymes to survive in harsh conditions. These enzymes are naturally evolved with unique structural features that confer a high level of flexibility, solubility and substrate-binding ability compared to mesophilic and thermostable homologs. Here, we identified and characterized an amylase, SdG5A, from the marine bacterium Saccharophagus degradans 2-40T. We expressed the protein in Bacillus subtilis and found that the purified SdG5A enabled highly specific production of maltopentaose, an important health-promoting food and nutrition component. Notably, SdG5A exhibited outstanding cold adaptation and salt tolerance, retaining approximately 30 and 70% of its maximum activity at 4°C and in 3 M NaCl, respectively. It converted 68 and 83% of starch into maltooligosaccharides at 4 and 25°C, respectively, within 24 h, with 79% of the yield being the maltopentaose. By analyzing the structure of SdG5A, we found that the C-terminal carbohydrate-binding module (CBM) coupled with an extended linker, displayed a relatively high negative charge density and superior conformational flexibility compared to the whole protein and the catalytic domain. Consistent with our bioinformatics analysis, truncation of the linker-CBM region resulted in a significant loss in activities at low temperature and high salt concentration. This highlights the linker-CBM acting as the critical component for the protein to carry out its activity in biologically unfavorable condition. Together, our study indicated that these unique properties of SdG5A have great potential for both basic research and industrial applications in food, biology, and medical and pharmaceutical fields.

The contribution of newly designed salt bridges to protein stabilization remains controversial even today. In order to solve this problem, we investigated salt bridges from two aspects: spatial distribution and evolutionary characteristics of salt bridges. Firstly, we analyzed spatial distribution of salt bridges in proteins, elucidating the basic requirements of forming salt bridges. Then, from an evolutionary point of view, the evolutionary characteristics of salt bridges as well as their neighboring residues were investigated in our study. The results demonstrate that charged residues appear more frequently than other neutral residues at certain positions of sequence even under evolutionary pressure, which are able to form electrostatic interactions that could increase the evolutionary stability of corresponding amino acid regions, enhancing their importance to stability of proteins. As a corollary, we conjectured that the newly designed salt bridges with more contribution to proteins, not only, are qualified spatial distribution of salt bridges, but also, are needed to further increase the evolutionary stability of corresponding amino acid regions. Based on analysis, the 8 mutations were accordingly constructed in the 1,4-α-glucan branching enzyme (EC 2.4.1.18, GBE) from Geobacillus thermoglucosidans STB02, of which 7 mutations improved thermostability of GBE. The enhanced thermostability of 7 mutations might be a result of additional salt bridges on residue positions that at least one of amino acids positions is conservative, improving their contribution of stabilization to proteins. Keywords: Evolutionary trace, Evolutionary stability, Salt bridge, Thermostability, Mutagenesis

Amylo-α-1,6-glucosidase (EC 3.2.1.33, AMY) exhibits hydrolytic activity towards α-1,6-glycosidic bonds of branched substrates. The debranching products of maltodextrin, waxy corn starch and cassava starch treated with AMY, pullulanase (EC 3.2.1.41, PUL) and isoamylase (EC 3.2.1.68, ISO), were investigated and their differences in substrate selectivity and debranching efficiency were compared. AMY had a preference for the branched structure with medium-length chains, and the optimal debranching length was DP 13–24. Its optimum debranching length was shorter than ISO (DP 25–36). In addition, the debranching rate of maltodextrin treated by AMY for 6 h was 80%, which was 20% higher than that of ISO. AMY could decompose most of the polymerized amylopectin in maltodextrin into short amylose and oligosaccharides, while it could only decompose the polymerized amylopectin in starch into branched glucan chains and long amylose. Furthermore, the successive use of AMY and β-amylase increased the hydrolysis rate of maltodextrin from 68% to 86%. Therefore, AMY with high substrate selectivity and a high catalytic capacity could be used synergistically with other enzyme preparations to improve substrate utilization and reduce reaction time. Importantly, the development of a novel AMY provides an effective choice to meet different production requirements.

Maltooligosaccharides are a novel type of functional oligosaccharides with potential applications in food processing and can be produced by glycosyl hydrolases hydrolyzing starch. However, the main obstacle in industrial applications is the balance between the high temperature of the process and the stability of enzymes. In this study, based on the structural information and in silico tools (DSDBASE-MODIP, Disulfide by Design2 and FoldX), two disulfide bond mutants (A211C-S214C and S409C-Q412C) of maltotetraose-forming amylase from Pseudomonas saccharophila STB07 (MFAps) were generated to improve its thermostability. The mutation A211C-S214C was closer to the catalytic center and showed significantly improved thermostability with a 2.6-fold improved half-life at 60 °C and the thermal transition mid-point increased by 1.6 °C, compared to the wild-type. However, the thermostability of mutant S409C-Q412C, whose mutation sites are closely to CBM20, did not change observably. Molecular dynamics simulations revealed that both disulfide bonds A211C-S214C and S409C-Q412C rigidified the overall structure of MFAps, however, the impact on thermostability depends on the position and distance from the catalytic center.

Globally, obesity and its metabolic complications, which are intimately linked to diet, are major public health problems. Cyclodextrins (CDs) are cyclic oligosaccharides consisting of (α-1,4)-linked D-glucopyranose units that can reduce fat bioavailability and affect metabolism by improving intestinal flora as prebiotics. We compared the effects of three CDs on preventing fat accumulation and remodeling gut microbiota in a high-fat diet-fed C57BL/6J mouse model. α-CD maximized energy expenditure by 12.53%, caused the RER value to drop from 0.814 to 0.788, and increased the proportion of fatty acid oxidation for energy supply. β-CD supplementation resulted in a marked 24.53% reduction in weight gain and a decrease in epididymal-fat-relative weight from 3.76% to 2.09%. It also minimized ectopic fat deposition and improved blood lipid parameters. γ-CD maximized the concentration of SCFAs in the cecum from 6.29 to 15.31 μmol/g. All three CDs reduced the abundance ratio of Firmicutes and Bacteroidetes to a low-fat diet level, increased the abundance of Lactobacillus and Akkermansia, and reduced the abundance of Allobaculum and Ruminococcus. These findings imply that a combination of multiple CDs may exert superior effects as a potential strategy for obesity prevention.

Potentilla parvifolia Fisch. (Rosaceae) is one of the genuine medicinal materials in Qinghai-Tibet Plateau, China. Here we report the first chloroplast (cp) genome of P. parvifolia using Illumina NovaSeq 6000 platform. The length of its complete cp genome is 152,898 bp, containing four sub-regions; a large single copy region (LSC) of 84,160 bp and a small single copy region (SSC) of 18,128 bp are separated by a pair of inverted repeat regions (IRs) of 25,305bp. The complete cp genome of P. parvifolia contains 130 genes, including 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The overall GC content of the cp genome is 37.2%. The phylogenetic analysis, based on 17 cp genomes, suggested that P. parvifolia is closely related to P. fruticosa L. and Fragaria species.