Amino Acid Residues Research Articles

Decoding of novel umami peptides from corn fermented powder and its mechanism via multisensory techniques, virtual screening, and molecular simulation approaches

This study aimed to identify umami peptides in corn fermented powder (CFP) and investigate their umami enhancing effect. Ultrafiltration and ethanol precipitation was used to separate the umami peptides in CFP. Dynamic sensory evaluations were used to identify the peptide fraction with the intense umami taste, and the peptides in the fraction were identified by nano-liquid chromatography-tandem mass spectrometry. Subsequently, ten umami-enhancing peptide candidates were screened using an integrated virtual screening strategy. Molecular docking revealed that Ser382, Ser104, Leu334, Glu338 and Glu148 of the T1R1 and T1R3 taste receptors are important amino acid residues for binding of the ten umami peptides. Three umami peptides (VDW, WGDDP, and WPAGE) exhibited the stronger binding affinity with the umami receptors. Moreover, molecular dynamics simulation revealed that the T1R1/T1R3 formed stable complexes with the three umami peptides during the simulation. Sensory evaluation indicated that the three peptides exhibited diverse taste characteristics (detection thresholds:0.0315–0.0625 mg/mL). The sigmoid curve analysis further confirmed peptides were identified as synergistically (VDW and WGDDP) or additively (WPAGE) enhancing the umami of 3 mg/mL MSG solution. This study uncovers the mechanism of umami-peptide-driven taste in fermented corn products.

Emulsions co-stabilized by Polygonatum sibiricum saponin and soy protein isolate: Physical stability, interaction mechanism, interface behavior

This study investigates the interaction mechanism between Polygonatum sibiricum saponin (PSS) and soy protein isolate (SPI) to enhance the stability of oil-in-water emulsions, laying the foundation for an efficient PSS delivery system. Initially, we evaluated the stability of oil-in-water emulsions with various PSS concentrations (0.1%, 0.2%, 0.3%, 0.5%, and 1% w/w). Results indicated that PSS enhances emulsion stability by increasing absolute zeta potential, reducing average particle size, and improving resistance to external factors. Notably, concentrations of 0.3%, 0.5%, and 1% (w/w) PSS show significant improvements in emulsion stability (p < 0.05). Subsequently, we elucidated the mechanism by which PSS enhances stability. Our findings reveal that PSS interacts with SPI, inducing structural modifications in SPI and expanding its hydrophobic regions. Additionally, PSS forms SPI-PSS complexes through hydrogen bonding with aromatic amino acid residues in SPI, thereby reducing interfacial tension and maintaining stable viscoelasticity. Comparative analysis of Lissajous plots identifies the optimal PSS to SPI ratio as 0.3% (w/w) each, resulting in a resilient interface film formed by numerous SPI-PSS complexes capable of withstanding compression or extension. Overall, our study underscores the efficacy of PSS in navigating the emulsion system and collaborating with SPI to stabilize emulsions.

Comparative analysis of the interaction mechanism of γ-globulin and hemoglobin with spherical and rod-shaped gold nanoparticles

The interaction mechanism of spherical gold nanoparticles (AuNPs) and rod-shaped gold nanoparticles (AuNRs) with γ-globulin and hemoglobin was comprehensively and comparatively analyzed. γ-Globulin and hemoglobin have high affinity with AuNPs, and with two different types of binding sites on AuNRs surface. Except hemoglobin interaction with the first binding site of AuNRs, the interaction between γ-globulin/hemoglobin and AuNPs/AuNRs is the spontaneous, endothermic and entropy-driven process, and hydrophobic interaction plays a dominant role. The molecular adsorption mechanism of γ-globulin/hemoglobin on AuNPs and AuNRs surface conforms to Langmuir model and Freundlich model, respectively. The kinetic molecular mechanism between them conforms to the pseudo-second-order model, and chemisorption is the rate-limiting step. AuNPs result in the loosening and unfolding of γ-globulin backbone. AuNRs have no significant effect on γ-globulin backbone. AuNPs/AuNRs result in no significant changes in hemoglobin structure and heme group microenvironment. AuNPs/AuNRs decrease the hydrophobicity of Trp microenvironment of γ-globulin, but there is an intramolecular energy transfer from Trp residue to Tyr residue of hemoglobin. The β-sheet of γ-globulin and the α-helix of hemoglobin reduce by increasing concentrations of AuNPs/AuNRs. Molecular docking is suggesting that the specific amino acid residues of γ-globulin and hemoglobin interaction with AuNPs/AuNRs, and validates the experimental results.

Synthesis and Antifungal Activities of Glucosamine Aromatic Derivatives Against Four Phytopathogenic Fungi of Crops.

A series of diversified glucosamine derivatives (3a-3y) was synthesized and their antifungal activity was examined against four kinds of phytopathogens, Fusarium graminearum (F. graminearum), Fusarium moniliforme (F. moniliforme), Curvularia. lunata (C. lunata), and Rhizoctonia solani (R. solani) which cause seriously economic losses worldwide by affecting crops. The compound 3o showed remarkable antifungal activity against F. graminearum with EC50 value of 3.96 μg/mL, compared to the standard drug triadimefon (10.1 μg/mL). 3D-QSAR model with the statistically recommended values (r2=0.915, q2=0.872) showed that positive charge group or bulky group in the benzyl ring was favorable for the antifungal activity. Enzyme activity assays confirmed that 3o had a moderate inhibition of trehalase with inhibition rate of 51.4 % at 5 μg/mL, which is comparable to those of commercial inhibitor validamycin A with inhibition rate of 83.3 %. Molecular docking analysis revealed that 3o also had a hydrogen bond interaction with key amino acid residue compared to validoxylamine.

Synthesis and BSA binding study of the Cu(II) complex and applications of it to enhance catalytic activity for C(Sp2)-H bond functionalization and for the synthesis of polyhydroquinolines

Among the various copper-catalyzed coupling reactions, C–S and C–N bond-forming reactions have carried off plentiful recognition due to their demand in the synthesis of molecules having biological and pharmaceutical impact. Organo-copper complex has an incontestable supremacy over the other catalytic complex due to its low cost and the use of readily accessible and stable ligands. As a consequence and in continuation of our explorative studies in this direction, an efficient and novel organo-copper complex has been synthesized and characterized by spectroscopic (NMR, FT-IR) and single crystal X-ray diffraction (XRD) methods. The synthesized stable metal complex has fascinating physical, chemical, biological and catalytic properties. In the chemical exploration refer to protein-metal interactions; we have often used Bovine Serum Albumin (BSA) protein. The synthesized copper complex has the potential to form coordinate bonds with functional groups present in BSA, together with amino acid residues and the protein backbone. The binding of metal complexes to BSA has outstanding imputation in medicinal chemistry. For enhancement of the catalytic activity of the organo-copper complex, we have evaluated it in carbon-hetero bond formation reactions where it proffered an influential tool for the establishment of C–S and C–N bonds.

Discovery of Cinnamic Acid Derivatives as Potent Anti-H. pylori Agents

Antibiotics are currently used for the treatment of Helicobacter pylori (H. pylori), which is confirmed to be the major cause of gastric disorders. However, the long-term consumption of antibiotics has already caused antibiotic resistance and side effects in vivo. Therefore, there is an emerging need for searching for safe and effective anti-H. pylori agents. Inspired by the excellent bioactivities of cinnamic acid, a series of cinnamic acid derivatives (compounds 1–30) were synthesized and determined for H. pylori inhibition. The initial screening revealed that compound 23, a 2,4-dinitro cinnamic acid derivative containing 4-methoxyphenol, showed excellent H. pylori inhibition with an MIC value of 4 μM. Further studies indicated that compound 23 showed anti-bacterial activity and had a bactericidal effect on H. pylori due to the destruction of the bacterial structure. Molecular docking analysis revealed that the 2,4-dinitro groups in cinnamic acid moiety formed hydrogen bonding with amino acid residues in an active pocket of H. pylori protein. Interestingly, the ester moiety fitted into the hydrophobic pocket, attaining additional stability to compound 23. Above all, the present study reveals that compound 23 could be considered a promising anti-H. pylori agent to treat H. pylori causing gastritis.

Enhancing the imidase activity of BpIH toward 3-isobutyl glutarimide via semi-rational design

(R)-3-Isobutylglutarate monoamide (R-IBM) is a key intermediate in the synthesis of the analgesic drug pregabalin. Recently, the imidase BpIH derived from Burkholderia phytofirmans was identified as a promising catalyst for the industrial production of R-IBM. Notably, this catalyst has the distinct advantage of achieving a 100% theoretical yield from 3-isobutyl glutarimide (IBI). In this study, homology modeling and structure alignment techniques were used to determine the substrate binding pocket of BpIH. Semi-rational design was used to analyze the amino acid residue conservation in the binding pocket region of BpIH. Interestingly, mutations of several low-conserved amino acid located 6–9 Å from the substrate significantly enhanced the catalytic activity of BpIH. Among them, the triple mutant Y37FH133NS226I (YHS-I) showed approximately a fivefold increase in enzyme activity and a significantly improved catalytic efficiency (kcat/Km). Under the same reaction time and conditions, YHS-I successfully converted IBI into R-IBM with a conversion rate of 88.87%, with an enantiomeric excess (ee) of the product exceeding 99.9%. In comparison, wild-type BpIH had a conversion rate of only 38.15%. Molecular dynamics and docking results indicated that YHS-I had higher rigidity around the mutation sites. The synergistic substitutions of Y37F, H133N, and S226I altered the interaction network within the mutation site, enhancing the protein’s affinity for the substrate and improving catalytic efficiency.Key points• 100% theoretical yield of R-IBM by BpIH compared with 50% by resolution• Semi-rational design of BpIH based on conservativity with homologous enzymes• Mutant with enzyme activity of sixfold and product ee value of 99.9%

Design, synthesis, and evaluation of chalcone derivatives as xanthine oxidase inhibitors

Xanthine oxidase (XO) is an important enzyme that catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid in the catabolism of purines in humans. This makes XO a well-recognized target in alleviating hyperuricemia. The present study adapted a structure-based drug discovery approach to develop potent and low-toxicity XO inhibitors with the chalcone skeleton. We introduced a carboxyl group and a hydroxyl group to the B ring and modified the A ring. 35 chalcone derivatives were designed and synthesized. All the 35 derivatives exhibited higher XO inhibition activities (IC50=0.064-0.559μM) compared with allopurinol (IC50=2.588μM). Their high affinity was attributed to strong hydrogen bond interactions formed between the introduced carboxyl and hydroxyl groups with key amino acid residues in XO. SAR analysis disclosed that carboxyl, hydroxyl, ethyl (12c), methylamino (12h), dimethylamino (12i), indolin (13k), and indol (13l) groups played important roles in improving the whole molecules' inhibition potency against XO. ADME predictions and cytotoxicity assays suggested their pharmacokinetic characteristics and biocompatibility were desirable. Additionally, 12c exhibited a significant hypouricemic effect on potassium oxonate-induced hyperuricemia rats after orally administrated at a dose range of 10-40mg/kg, representing a promising anti-hyperuricemia potential for further optimization and development.

Assessment of amino acid charge states based on cryo-electron microscopy and molecular dynamics simulations of respiratory complex I

The charge states of titratable amino acid residues play a key role in the function of membrane-bound bioenergetic proteins. However, determination of these charge states both through experimental and computational approaches is extremely challenging. Cryo-EM density maps can provide insights on the charge states of titratable amino acid residues. By performing classical atomistic molecular dynamics simulations on the high resolution cryo-EM structures of respiratory complex I from Yarrowia lipolytica, we analyze the conformational and charge states of a key acidic residue in its ND1 subunit, aspartic acid D203, which is also a mitochondrial disease mutation locus. We suggest that in the native state of respiratory complex I, D203 is negatively charged and maintains a stable hydrogen bond to a conserved arginine residue. Alternatively, upon conformational change in the turnover state of the enzyme, its sidechain attains a charge-neutral status. We discuss the implications of this analysis on the molecular mechanism of respiratory complex I.

Maeruines A−E, elusive indole alkaloids from stems of Maerua siamensis and their inhibitory effects on cyclooxygenases and HT-29 colorectal cancer cell proliferation

Five previously undescribed indole alkaloids, maeruines A−E (1−5), bearing imino-2H-thieno[2,3-b]indol-3(8H)-one skeleton, were obtained from the stems of Maerua siamensis. Their chemical structures were elucidated using spectroscopic techniques [NMR, MS, IR, and UV], and single-crystal X-ray diffraction. Maeruine D (4) displayed selective cyclooxygenase-2 (COX-2) inhibitory activity in vitro with an IC50 of 29.72 ± 6.36 μM. Molecular dynamics simulations revealed that maeruine D could form a stable complex with human COX-2, predominantly driven by hydrophobic interactions. In addition, five amino-acid residues including Val349, Leu352, Leu384, Val523, and Ala527 were identified as hot-spot ones, which may lead to high binding affinity and selectivity. Furthermore, it exhibited cytotoxicity against HT-29 colorectal cancer cells with an IC50 of 29.32 ± 4.76 μM, and, at 0.1−10 μM, significantly inhibited their proliferation, induced by the proinflammatory cytokine interleukin-1β (IL-1β), in a dose-dependent manner.

Hydrophilic Interaction Chromatography Coupled to Ultraviolet Photodissociation Affords Identification, Localization, and Relative Quantitation of Glycans on Intact Glycoproteins.

Protein glycosylation is implicated in a wide array of diseases, yet glycoprotein analysis remains elusive owing to the extreme heterogeneity of glycans, including microheterogeneity of some of the glycosites (amino acid residues). Various mass spectrometry (MS) strategies have proven tremendously successful for localizing and identifying glycans, typically utilizing a bottom-up workflow in which glycoproteins are digested to create glycopeptides to facilitate analysis. An emerging alternative is top-down MS that aims to characterize intact glycoproteins to allow precise identification and localization of glycans. The most comprehensive characterization of intact glycoproteins requires integration of a suitable separation method and high performance tandem mass spectrometry to provide both protein sequence information and glycosite localization. Here, we couple ultraviolet photodissociation and hydrophilic interaction chromatography with high resolution mass spectrometry to advance the characterization of intact glycoproteins ranging from 15 to 34 kDa, offering site localization of glycans, providing sequence coverages up to 93%, and affording relative quantitation of individual glycoforms.

GGN-GO: geometric graph networks for predicting protein function by multi-scale structure features.

Recent advances in high-throughput sequencing have led to an explosion of genomic and transcriptomic data, offering a wealth of protein sequence information. However, the functions of most proteins remain unannotated. Traditional experimental methods for annotation of protein functions are costly and time-consuming. Current deep learning methods typically rely on Graph Convolutional Networks to propagate features between protein residues. However, these methods fail to capture fine atomic-level geometric structural features and cannot directly compute or propagate structural features (such as distances, directions, and angles) when transmitting features, often simplifying them to scalars. Additionally, difficulties in capturing long-range dependencies limit the model's ability to identify key nodes (residues). To address these challenges, we propose a geometric graph network (GGN-GO) for predicting protein function that enriches feature extraction by capturing multi-scale geometric structural features at the atomic and residue levels. We use a geometric vector perceptron to convert these features into vector representations and aggregate them with node features for better understanding and propagation in the network. Moreover, we introduce a graph attention pooling layer captures key node information by adaptively aggregating local functional motifs, while contrastive learning enhances graph representation discriminability through random noise and different views. The experimental results show that GGN-GO outperforms six comparative methods in tasks with the most labels for both experimentally validated and predicted protein structures. Furthermore, GGN-GO identifies functional residues corresponding to those experimentally confirmed, showcasing its interpretability and the ability to pinpoint key protein regions. The code and data are available at: https://github.com/MiJia-ID/GGN-GO.

A model for polyomavirus helicase activity derived in part from the AlphaFold2 structure of SV40 T-antigen.

Enzymes termed helicases are essential for the replication of DNA tumor viruses. Unfortunately, much remains to be determined about this class of enzymes, including their structures and the mechanism(s) they employ to unwind DNA. Herein, we present the full-length structure of a model helicase encoded by a DNA tumor virus. Moreover, this AI-based structure has been analyzed in terms of its basic functional properties, such as the orientation of the helicase at replication forks and the relative locations of the amino acid residues that are critical for helicase activity. Obtaining this information is important because it permits proposals regarding how DNA is routed through these model helicases. Also presented is structural evidence that the conclusions drawn from our detailed analyses of one model helicase, encoded by one class of tumor viruses, are likely to apply to other viral and eukaryotic helicases.

Marine Bioactive Molecules as Inhibitors of the Janus Kinases: A Comparative Molecular Docking and Molecular Dynamics Simulation Approach.

A treasure trove of naturally occurring biomolecules can be obtained from sea living organisms to be used as potential antioxidant and anti-inflammatory agents. These bioactive molecules can target signaling molecules involved in the severity of chronic autoimmune diseases such as rheumatoid arthritis (RA). The intracellular tyrosine kinases family, Janus kinases (JAKs, includes JAK1, JAK2, and JAK3), is implicated in the pathogenesis of RA through regulating several cytokines and inflammatory processes. In the present study, we conducted molecular docking and structural analysis investigations to explore the role of a set of bioactive molecules from marine sources that can be used as JAKs' specific inhibitors. Around 200 antioxidants and anti-inflammatory molecules out of thousands of marine molecules found at the Comprehensive Marine Natural Products Database (CMNPD) website, were used in that analysis. The details of the interacting residues were compared to the recent FDA approved inhibitors tofacitinib and baricitinib for data validation. The shortlisted critical amino acids residues of our pharmacophore-based virtual screening were LYS905, GLU957, LEU959, and ASP1003 at JAK1, GLU930 and LEU932 at JAK2, and GLU905 and CYS909 of JAK3. Interestingly, marine biomolecules such as Sargachromanol G, Isopseudopterosin E, Seco-Pseudopterosin, and CID 10071610 showed specific binding and significantly higher binding energy to JAK1 active/potential sites when being compared with the approved inhibitors. In addition, Zoanthoxanthin and Fuscoside E bind to JAK2's critical residues, GLU930 and LEU932. Moreover, Phorbaketal and Fuscoside E appear to be potential candidates that can inhibit JAK3 activity. These results were validated using molecular dynamics simulation for the docked complexes, JAK1(6sm8)/SG, JAK2 (3jy9)/ZAX, and JAK3 (6pjc)/Fuscoside E, where stable and lower binding energy were found based on analyzing set of parameters, discussed below (videos are attached). A promising role of these marine bioactive molecules can be confirmed in prospective preclinical/clinical investigations using rheumatoid arthritis models.

Hemagglutinin and neuraminidase of a non-pathogenic H7N7 avian influenza virus coevolved during the acquisition of intranasal pathogenicity in chickens.

Polybasic amino acid residues at the hemagglutinin (HA) cleavage site are insufficient to induce the highly pathogenic phenotype of avian influenza viruses in chickens. In our previous study, an H7N7 avian influenza virus named "Vac2sub-P0", which is nonpathogenic despite carrying polybasic amino acids at the HA cleavage site, was passaged in chick air sacs, and a virus with high intravenous pathogenicity, Vac2sub-P3, was obtained. Intranasal infection with Vac2sub-P3 resulted in limited lethality in chickens; therefore, in this study, this virus was further passaged in chicken lungs, and the resultant virus, Vac2sub-P3L4, acquired high intranasal pathogenicity. Experimental infection of chickens with recombinant viruses demonstrated that mutations in HA and neuraminidase (NA) found in consecutive passages were responsible for the increased pathogenicity. The HA and NA functions of Vac2sub-P3L4 were compared with those of the parental virus in vitro; the virus growth at 40 °C was faster, the binding affinity to a sialic acid receptor was lower, and the rate of release by NA from the cell surface was lower, suggesting that these changes enabled the virus to replicate efficiently in chickens with high intranasal pathogenicity. This study demonstrates that viruses that are highly pathogenic when administered intranasally require additional adaptations for increased pathogenicity to be highly lethal to intranasally infected chickens.

Structural activity of synthesized pyrimidine-thiophene and pyrimidine-thiadiazole conjugates as anticancer agents

New pyrimidine linked thiophene conjugates 5a-d and thiadiazole conjugates 7a-d through a carboxamide bridge were synthesized and characterized by the spectral data (IR, NMR and mass). Exploration of the isolated conjugates’ characteristics using DFT methodology revealed that they had angular configurations with distinctive outline of the FMO’s belonging to the thienyl- and thiadiazolyl-pyrimidine classes. In accordance, the FMO’s energy values of these analogues disclosed reduced energy gap (ΔEH-L=3.66–3.86 eV) than the parent 4 (4.49 eV) and may be arranged as: 7c < 7d < 5c < 5d < 5a < 5b < 7b < 7a < 4. Meanwhile, the cytotoxic effectiveness of the synthesized conjugates was assessed against different cancer cell lines using IC50 values in comparison to 5-Fu, drug reference. The data showed that the acrylamide derivative 4 exhibited the most potent cytotoxicity against MCF-7 and PC3 (IC50 6.11 ± 0.10 and 7.19 ± 0.04 μM, respectively). Moreover, the carbonic anhydrases inhibitory effect on hCA IX and hCA IIX have been evaluated, using acetazolamide (as standard), showing greater impact on hCA IX compared to hCA IIX. Molecular docking has been applied to investigate the nature of the binding interactions with the target amino acid residues, and it disclosed that the pyrimidine-thiophene conjugates 5d and 5c had the highest binding scores. Finally, the SwissADME pharmacokinetic properties of synthesized conjugates indicated that the acrylamide compound 4 exhibited acceptable pharmacokinetic manners, such GI absorption, solubility, no BBB permeation, and slight CYP450 inhibition, implying a promising therapeutic candidate.

Synthesis and Anticancer Activity Assessment of Zelkovamycin Analogues.

The zelkovamycin family is a class of cyclic octapeptides with potent antibacterial and antiviral activity. Due to their unique chemical structures and excellent bioactivity, zelkovamycins have consistently attracted the interest of synthetic chemists. However, only the total synthesis of zelkovamycin and zelkovamycin G has been reported until now. The current work presents, for the first time, the synthesis of zelkovamycin analogues, along with their anticancer activity assessment. Firstly, the corresponding chain peptide based on the amino acid sequence of zelkovamycin H was synthesized using the Fmoc solid-phase peptide strategy. This was followed by cyclization under high dilution conditions to obtain compound 21, and its structure was elucidated by NMR analysis. The results confirm that compound 21 is not the natural product of zelkovamycin H. We deduced that during the synthesis of peptide 12, the D-Abu residue epimerized to the L-Abu form, leading to the formation of peptide 20, which blocked our efforts during the synthesis of zelkovamycin H. Two more analogues, 22 and 23, were synthesized by changing the structure of amino acid residues using the same strategy. The anticancer activity of analogues 21-23 against Huh-7 cells was evaluated in vitro; however, their IC50 values were >50 μM.

Flavin‐Catalyzed, Photochemical Conversion of Dehydroalanine into 4,5‐Dihydroxynorvaline

The chemical synthesis of unnatural amino acids (UAA) is a key strategy for preparing designed peptides, including pharmaceutically active compounds. Alterations of existing amino acid residues such as dehydroalanine (Dha) are particularly important since selected positions can be addressed without the necessity of a complete de novo synthesis. The intriguing UAA 4,5‐dihydroxynorvaline (Dnv) is found in a variety of naturally occurring peptides and biologically active compounds. However, no method is currently available to modify an existing peptide with this residue. We report the use of flavin catalysts and visible light irradiation for this challenge, which serves as a versatile strategy for converting Dha into Dnv. Our study shows that excited flavins are competent hydrogen atom abstraction catalysts for ethers and acetals, which allows masked 1,2‐dihydroxyethylene functionalization from 2,2‐dimethyl‐1,3‐dioxolane. The masked diol was successfully coupled to Dha residues, and a series of Dnv‐containing products is reported. A mild and orthogonal protocol for deprotection of the acetal group was also identified, allowing free Dnv‐modified peptides to be obtained. This method provides a straightforward strategy for Dnv functionalization, which is envisioned to be crucial for accessing natural products and synthetic analogues with pharmaceutical activity.

Design and validation of a method for evaluating medical device cleanliness by recovering and quantifying residual proteins on stainless plates

We recently reported a method for recovering and quantifying residual proteins bound to surfaces of various medical instruments via thermal coagulation under neutral pH and room temperature. The method effectively recovered and solubilised coagulated proteins at high temperatures in dry and humid conditions, with a protein recovery rate of > 90%. This study validated the previous method by comparing residual protein recovery from test samples using a conventional extraction solution (1% SDS, [pH 11.0]) and proposed solution (1% SDS, 10 mM TCEP, and 10 mM HEPES [pH 7.0]). To mimic soiled medical equipment, pseudo-blood-contaminated stainless steel plates were prepared. Residual protein was recovered using conventional and proposed solutions under varying temperature and humidity conditions. Quantitative protein recovery limits were determined at nine facilities. Compared with the conventional solution, the proposed solution recovered proteins more effectively from samples processed at temperatures > 60 °C. However, low recovery rates were observed for samples processed at 95 °C, possibly owing to differences in protein adhesion due to sample and plate-surface properties. Our findings present a method for quantifying residual proteins on medical instruments exposed to high temperatures during use or disinfection. Further studies should standardise test soiling conditions, materials, and solutions to evaluate cleaning methods.

Bioassay-Guided Isolation and Identification of Xanthine Oxidase Inhibitory Constituents from the Fruits of Chaenomeles speciosa (Sweet) Nakai.

Chaenomeles speciosa (Sweet) Nakai (C. speciosa) is a traditional Chinese herbal medicine that possesses not only abundant nutritional value but also significant medicinal properties. The extracts of C. speciosa fruits effectively reduce urate levels, but the specific chemical constituents responsible for this effect in C. speciosa fruits are still unknown. Therefore, this study aims to investigate and analyze the structure-activity relationships of these constituents to better understand their ability to lower uric acid. Activity-guided fractionation and purification processes were used to isolate compounds with xanthine oxidase (XO) inhibitory activity from C. speciosa fruits, resulting in three extracts: petroleum ether, ethyl acetate, and n-butanol. The ethyl acetate and n-butanol fractions showed strong activity and underwent further separation and purification using chromatographic techniques. Twenty-four compounds were isolated and identified, with nine showing potent activity, including chlorogenic acid, methyl chlorogenate, butyl chlorogenate, ethyl chlorogenate, cryptochlorogenic acid methyl ester, caffeic acid, p-coumaric acid, benzoic acid and protocatechuic acid. The docking analysis showed that these compounds interacted with amino acid residues in the active site of XO through hydrogen bonding and hydrophobic interactions. These findings suggest that these compounds help reduce uric acid in C. speciosa, supporting further investigation into their mechanism of action.