Mass Spectrometry Research Articles

Use of infrared and mass spectrometry for the discrimination of metabolites from different stages of Schinus terebinthifolia Raddi leaves

The Brazilian Peppertree (Schinus terebinthifolia Raddi) is a medicinal plant with several biological activities, the most important of which are anti-inflammatory, antimicrobial and antiproliferative. Therefore, scientific research and investigation of its bioactive compounds are required. Leaves at three development stages were collected from three different genotypes of S. terebinthifolia. Methanol extracts were prepared and analyzed by infrared and mass spectrometry. The data obtained were treated by principal component analysis (PCA). The PCA model applied to the mass spectrometry data proved to be efficient to discriminate the three stages of development, in addition to indicating a differentiated tendency in the production of secondary metabolites by the genotypes. Two principal components were needed to describe the variance of the infrared spectrometry data (81.96%), while for mass spectrometry data three principal components were needed (55.73%). Ten substances were the most influential in the separation of the groups (young, intermediate, and old leaves or differentiation between genotypes A, B, and C), with emphasis on methyl digallate, quinic acid hexoside, digallic acid, and methyl gallate. Infrared spectroscopy data allowed us to differentiate young leaves from intermediate ones and the spectral bands that most influenced this differentiation (related to the groups O–H, C–O, C=C, C–H and C=O) correlated with the substances indicated by mass spectrometry. These results showed a different distribution of compounds among the leaf development stages, providing valuable information for those aiming to isolate specific substances of biological interest.

Integration of ion mobility-quadrupole time-of-flight mass spectrometry and triple quadrupole-ion trap mass spectrometry coupled with ultra-high performance liquid chromatography for characterizing and quantitatively assay the saponins of Panax quinquefolius flower

Panax quinquefolius (American ginseng) is globally popular serving as herbal medicine or tonifying agent. Compared with the root, studies on the flower of Panax quinquefolius (PQF) in respect of the multicomponent characterization and quality evaluation is scarce. This work was designed, by feat of two liquid chromatography–mass spectrometry platforms, to characterize and quantitatively assay the saponins of PQF. Ultra-high performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS) combined with data-independent high-definition MSE (HDMSE) was developed to acquire the structure information of PQF saponins. Consequently, efficient and more accurate identification (higher identification accuracy and much lower false positives rate) of the involved saponins were accomplished by the intelligent peak annotation workflows of the UNIFI bioinformatics platform incorporated with a “Multi-dimensional Information Library of Ginsenosides” (GinMIL). Totally 128 ginsenosides were identified or tentatively characterized, and 45 thereof were identified by comparing with the reference standards. Additionally, an efficient UHPLC/QTrap-MS (triple quadrupole-ion trap mass spectrometry) method enabling quantitative assay of 21 ginsenosides in PQF was established and validated by scheduled multiple reaction monitoring (sMRM). It gave low limit of detection (0.011–0.65 pg) and limit of quantification (0.053–4.1 pg), good linearity (R2 > 0.99), intra-/inter-day precision (RSD < 13.6 %), stability (RSD < 3.5 %), repeatability (RSD < 7.5 %), and recovery (RSD < 13.5 %). It application to quantify 21 ginsenosides in 14 batches of PQF samples revealed differences in their saponin content. Conclusively, systematic multi-component characterization and multi-indicator quantitation of PQF were achieved, thus providing the methodological reference for the quality investigation of functional herbs.

Replacing traditional coffee appraisers with inductively coupled plasma - mass spectrometry (ICP-MS): From manual sensory evaluation to scientific analysis

Scientific development provides opportunities to replace many traditional manual methods to achieve more accurate results and higher efficiency. To scientifically ascertain the geographical origin of coffee, this study develops a method for the rapid determination of 16 inorganic elements in coffee using microwave digestion combined with the ICP-MS internal standard method. Principal component analysis (PCA), Fisher discriminant analysis (FDA), and Partial least squares discriminant analysis (PLS-DA) are employed to analyze 40 coffee samples from three production areas of Pu'er, Baoshan, and Wanning. The results show that the linear correlation coefficients of the 16 elements in this method are above 0.999, the detection limits are in the range of 0.0004–0.63 mg/kg, the RSD of the precision experiments are 4.5 %–13.5 %, the recovery rate of the peak experiment is 86.0 %–96.3 % with the RSD of 1.1 %–8.8 %, and the results of the standard substances are within the range of standard values. Using the discriminant analysis of inorganic elements in coffee (FDA and PLS-DA), coffee origin discrimination was realized, and six key elements (Al, Mn, Fe, Cu, Na, and Ba) are identified as effective discriminatory indexes. Accordingly, a coffee origin discrimination model is established, and the overall accuracy discrimination rate of the discrimination model are all more than 90.0 %, and FDA > PLS-DA. The findings indicated that the method has good accuracy and reliability, is suitable for analyzing and determining multiple elements in sample components as targets, and may have a positive impact on the development of related industries.

Arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria promoted changes in plant metabolism and the volatile profile of Piper callosum Ruiz & Pav

Piper callosum Ruiz & Pav. is known in the Amazon as a paregoric elixir and exists as a shrub native to the Neotropics. In traditional medicine, the tea from its leaves is used to treat pain in the digestive tract, rheumatism, and muscular aches. This study aimed to evaluate the influence of arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR) inoculation on the secondary metabolism of P. callosum. The plants were inoculated after 30 days of cultivation and kept in a greenhouse. Plant development parameters were monitored at 30-, 60-, and 90-days after inoculation (DAI) and indicated that AMF and PGPR favored plant growth throughout all stages of development. The leaf volatiles extracted by simultaneous distillation and extraction were analyzed by gas chromatography coupled with mass spectrometry and showed that phenylpropanoids and monoterpene hydrocarbons were predominant in all essential oils, revealing distinct abundance patterns across the different sampling points. The major volatile compounds identified were safrole, methyl eugenol, and β-pinene, and a reduction trend in their content was observed for safrole and methyl eugenol, especially at 90 DAI in both treatments. Principal component analysis revealed that germacrene D-4-ol and limonene were among the most influential in both treatments. Finally, the total phenolic content and the enzymatic activity of phenylalanine ammonia-lyase revealed different effect patterns for inoculation and time of evaluation on each treatment. All the data presented indicate that symbiotic inoculation can optimize the production of essential oil, which can improve the quality and productivity of P. callosum plants.

Use of a hydrosilylation reaction for the preparation of structure-controlled boroxine-based polyborosiloxanes

The structure of organosilicon vinyl-containing boroxine was confirmed by 1H nuclear magnetic resonance (NMR) spectroscopy, infrared (IR) spectroscopy and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Its thermal properties were studied using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) methods. The ability of boroxine to undergo hydrosilylation using the Karstedt's catalyst was investigated on various organosilicon substrates. Boroxine was shown to be involved in these reactions as a cross-linking agent. The formation of dynamic boroxine cross-links allowed the analysis of the reaction products by NMR spectroscopy, including the use of polyfunctional hydride-containing reagents. In all cases the addition proceeds selectively to the β-position and with complete conversion. The structure of the resulting polyborosiloxane containing 1.4 mol% of modified units was also confirmed by 1H NMR spectroscopy, and its thermal and rheological properties were studied.

Exploring the Role of Unconventional Post-Translational Modifications in Cancer Diagnostics and Therapy.

Unconventional Post-Translational Modifications (PTMs) have gained increasing attention as crucial players in cancer development and progression. Understanding the role of unconventional PTMs in cancer has the potential to revolutionize cancer diagnosis, prognosis, and therapeutic interventions. These modifications, which include O-GlcNAcylation, glutathionylation, crotonylation, including hundreds of others, have been implicated in the dysregulation of critical cellular processes and signaling pathways in cancer cells. This review paper aims to provide a comprehensive analysis of unconventional PTMs in cancer as diagnostic markers and therapeutic targets. The paper includes reviewing the current knowledge on the functional significance of various conventional and unconventional PTMs in cancer biology. Furthermore, the paper highlights the advancements in analytical techniques, such as biochemical analyses, mass spectrometry and bioinformatic tools etc., that have enabled the detection and characterization of unconventional PTMs in cancer. These techniques have contributed to the identification of specific PTMs associated with cancer subtypes. The potential use of Unconventional PTMs as biomarkers will further help in better diagnosis and aid in discovering potent therapeutics. The knowledge about the role of Unconventional PTMs in a vast and rapidly expanding field will help in detection and targeted therapy of cancer.

Bacteriocin CM175, a new high molecular weight and phage associated protein produced by Pediococcus pentosaceus CM175

Bacteriocins are proteins with antimicrobial capacity produced by different bacteria. Developing bacteriocin-based technologies could be an effective strategy to address current problems in the pharmaceutical and food industries, including limited therapeutic options against superbug infections, foodborne diseases, and food spoilage microorganisms. The lactic acid bacteria Pediococcus pentosaceus are known producers of bacteriocins. Particularly, Ped. pentosaceus strain CM175 has been reported to produce an uncharacterized bacteriocin-like inhibitory substance (BLIS) with an interesting antibacterial spectrum against Gram-positive and -negative pathogenic bacteria. The objective of the present study was to explore whether the BLIS produced by CM175 contains at least one bacteriocin, and identify it. Our results showed that the CM175 strain produced a non-previously characterized antimicrobial protein of 49 kDa identified by mass spectrometry as a phage-related protein, named bacteriocin CM175. Through fluorescence and transmission electron microscopies, it was demonstrated that bacteriocin CM175 damages the cell membrane integrity of Listeria monocytogenes through a non-lytic mechanism. Bacteriocin CM175 is the first high molecular weight and phage protein-like bacteriocin reported in Ped. pentosaceus. The results of this study open the possibility of exploring various applications directly related to the antimicrobial potential of bacteriocin CM175, including the development of antibiotics, and disinfectants.

Method development and application for multi-phenolic components in the processing of Dendrobium officinale using UPLC coupled with mass spectrometry and chemometric analysis

Dendrobium officinale (DO) is a perennial herb that has been utilized medicinal and edible purposes for thousands of years, often transforming into 'Fengdou' after drying. Despite its long-standing use, the specific phenolic components present during DO processing have remained elusive. This study established a method for the simultaneous and accurate quantification of 27 phenolic compounds during DO processing employing ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), with 8 reference-free components optimized through an online strategy. Furthermore, correlation analysis and molecular docking were conducted to further screened and validated 6 phenolics with α-glucosidase inhibitory activity, including vanillin, p-coumaric acid, 4-hydroxybenzaldehyde, resveratrol, coniferyl alcohol, and dihydropinosylvin. Notably, resveratrol exhibited the lowest binding energy at −7.7 kcal/mol among these compounds. These findings significantly contribute to the development of the deep-processing techniques for DO and the hypoglycaemic functional food industry by providing data that support the elucidation of medicinal substances in DO.

Synthesis of Chalcones, Screening In silico and In vitro and Evaluation of Helicobacter pylori Adhesion by Molecular Docking.

We synthetized 10 hydroxylated and methoxylated chalcones and evaluated them targeting MMP-9 inhibition, looking for the rate of adhesion of H. pylori in gastric cells, and then, reduction of the inflammatory response as alternative therapeutic agents for controlling the infection. Helicobacter pylori is a Gram-negative bacterium that chronically infects the human stomach, a risk factor for the development of inflammatory gastrointestinal diseases, including cancer, and is classified as a group I carcinogen. It is estimated that it infects around 45% of the global population and that the persistence of the infection is related to the adhesion of the bacteria in the gastric epithelium. The progression of gastric lesions to cancer is connected to the activation of the NF-κB and MAPK pathways, especially in cagA+ strains, which are related to increased expression of MMP-9. The activation of these metalloproteinases (MMPs) contributes to the adhesion of the bacterium in gastric cells and the evolving stages of cancer, such as enabling metastasis. Due to the increasing resistance to the current therapy protocols, the search for alternative targets and candidate molecules is necessary. In this way, controlling adhesion seems to be a suitable option since it is a crucial step in the installation of the bacterium in the gastric environment. Synthetize ten hydroxylated and methoxylated chalcones. Assess their anti-H. pylori potential, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). Evaluate their cytotoxicity in AGS cells and selectivity with L-929 cells. Analyze the results and correlate them with in silico predictions to evaluate potential anti-adhesive properties for the chalcones against H. pylori. The chalcones were synthetized by Claisen-Schmidt condensation using Ba(OH)2 or LiOH as catalysts. Predictive in silico assays in PASS Online, tanimoto similarity, ADME properties and molecular docking in MMP-9 (PDB code: 6ESM) were performed. The in vitro assays carried out were the cell viability in gastric adenocarcinoma cells (AGS) and fibroblasts (L-929) by the MMT method and anti-H. pylori, by the broth microdilution method, through the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Ten chalcones were synthesized through Claisen-Schimdt condensation with yields of 10 to 52% and characterized by 1H and 13C nuclear magnetic resonance (NMR) and mass spectrometry (MS). in silico data revealed the possibility of anti-H. pylori, anti-inflammatory, and MMP-9 inhibition for the chalcones. Chalcone 9 showed the best growth inhibition values for MIC and MBC, at 1 μg/mL and 2 μg/mL, respectively. Chalcones 14 and 15 likewise demonstrated excellent inhibitory results, being 2 μg/mL for both MIC and MBC. Additionally, 15 had the best MMP-9 inhibition score. Despite not corroborating the in silico findings, chalcones 10, 13, and 18 showed good cytotoxicity and the best selectivity indices. All compounds exhibited strong activity against H. pylori, specially 15. The predicted MMP-9 inhibition by molecular docking added to the reasonable SI and CI50 values for 15 and the satisfactory reduction in the rate of survival of the bacteria, reveals that it may be acting synergically to reduce the inflammatory response and the possibilities for developing a tumor by inhibiting both bacteria and malignant cells.

Easy-to-make peptide-decorated C18 microspin columns combined with MALDI-MS enabling high-throughput enrichment and detection of arsenic (III) ions

In this study, a user-friendly, cost-effective, and high-throughput method was developed for the selective enrichment and accurate quantification of arsenic (III) ions using peptide-decorated C18 microspin columns, integrated with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. The manufacturing process of the peptide-decorated C18 microspin columns originated from C18 StageTips packing with 3 μm C18 beads, followed by decoration with the peptide YYYCYCYYCR, which contained the arsenic (III) ion-binding motif CXCXXC, where X represents any amino acid. The microspin columns were applied to selectively capture arsenic (III) ions in environmental water by forming arsenic (III)-peptide complexes. After elution and drying, the high m/z ions from these complexes were directly desorbed and ionized with MALDI-MS by the addition of the α-CHCA matrix solution, effectively reducing interference from matrix-related ions and small molecules in the low m/z range. The developed method demonstrated a linear range from 10 μg/L to 1500 μg/L, with a limit of detection of 0.7 μg/L. Remarkably, this method enabled rapid quantification of arsenic (III) ions in environmental water, averaging under one minute per sample, using only a small volume of 50 μL. The reported method for detecting arsenic (III) ions in environmental water offers a practical approach and holds promise for analyzing other emerging toxic elements in the future.

Lipid analysis of human primary dermal fibroblasts and epidermal keratinocytes after near-infrared exposure using mass spectrometry imaging

Photobiomodulation (PBM) therapy is the application of near-infrared (NIR) exposure to injuries or lesions to (among others) improve wound healing, reduce inflammation, and decreases acute and chronic pain. However, the understanding of the molecular mechanism of PBM, more specifically the effects of NIR on skin cells is still lacking behind. Lipids are essential components of cellular membranes that are integral to skin structure and function. This study aims to elucidate the impact of NIR exposure on the skin’s lipidome by investigating the molecular effect of NIR exposure on single skin cells. Primary human dermal fibroblasts (NHDFa) and human epidermal keratinocytes (HEKa) were exposed to NIR (850nm) with a dose of 6.5J/cm2 for 5 consecutive days between 09.00 and 12.00 am. A workflow utilizing matrix-assisted laser desorption/ionization mass spectrometry imaging combined with liquid chromatography tandem mass spectrometry for lipidomics analysis was performed. This study provides evidence that adequate exposure of NIR influences lipid metabolism in NHDFa, whereas no alterations were found in HEKa. This work lays the groundwork in explaining the beneficial properties on both skin-related effects and systemic health benefits as seen in clinical studies.

Eucalyptus globulus essential oil as food preservative: Chemical composition, antimicrobial (in vitro and in situ), antibiofilm and insecticidal potential

The use of species within the Eucalyptus genus has been widespread in traditional medicine practices globally. However, concerns regarding safety and toxicity data have been prominent due to the extensive use of its essential oil, which is widely regarded for its medicinal properties and various applications. This research aimed to analyze the chemical makeup of Eucalyptus globulus essential oil (EGEO) and explore its antibacterial and insecticidal properties both in laboratory settings and real-life scenarios. Gas chromatography/mass spectrometry (GC/MS) was employed to identify the chemical constituents of EGEO, revealing 1,8-cineole (86.1 %), ο-Cymene (4.9 %), α-Pinene (3.8 %), γ-Terpinene (2.2 %), and α-Terpinene (1.6 %) as the primary components. The disc diffusion method demonstrated EGEOʼs strongest antimicrobial activity against Micrococcus luteus (p < 0.05), while minimal inhibition concentration tests indicated notable effectiveness against Streptococcus pneumoniae (MIC50 2.25 mg/mL and MIC90 2.79 mg/mL). In situ antibacterial assessments using white radish and pear food models showed heightened activity compared to contact application, particularly with the highest concentration (500 µg/L), yielding optimal results against Pseudomonas putida (p < 0.05). Additionally, the study investigated EGEO's antibiofilm properties against Salmonella enterica using mass spectrometry and the crystal violet method. In terms of insecticidal action, EGEO at 100 % concentration resulted in the mortality of 90 % of Harmonia axyridis individuals. This comprehensive investigation contributes to a deeper understanding of both the chemical composition and biological activities of Eucalyptus globulus essential oil, highlighting its potential as a source of natural compounds with promising therapeutic properties.

Fasting intact insulin by mass spectrometry is associated with metabolic dysfunction-associated steatotic liver disease in youth.

Fasting intact insulin concentrations can predict metabolic dysfunction-associated steatotic liver disease (MASLD) in adults without diabetes; however, research in youth is limited. We sought to determine whether fasting intact insulin, measured by liquid chromatography-tandem mass spectrometry, is associated with MASLD in children. This retrospective cross-sectional analysis used data and samples from children who participated in studies across 3 universities between 2014 and 2022. Key measurements included fasting intact insulin, ALT, and hepatic steatosis assessed by MRI techniques. MASLD was defined as hepatic steatosis ≥5% by MRI with at least 1 cardiometabolic risk factor. The optimal cutoff points to identify MASLD were determined by maximizing the Youden index, and the AUROC curves were compared using the DeLong test. The analysis included 184 children (28% male; 14.9 ± 2.6y; 57% Hispanic race/ethnicity; body mass index 32.5 ± 8.1kg/m2; 64% with MASLD, 43% with polycystic ovary syndrome, and 5% with other liver diseases). Fasting intact insulin and ALT levels were significantly higher in children with MASLD (p < 0.05). Fasting intact insulin was strongly associated with MASLD with an AUROC of 0.83 (0.77-0.90), sensitivity of 71%, and specificity of 85%. When combined with ALT (intact insulin × ALT [μU/mL × U/L]), the AUROC was 0.88 (0.83-0.94), with a sensitivity of 89% and specificity of 81%. The improvement in AUROC over intact insulin alone was not statistically significant (p = 0.089) but was statistically significant from ALT (p = 0.022). Optimal cutoff points for intact insulin and intact insulin × ALT were 20μU/mL and 522μU/mL × U/L, respectively. In pediatric patients, measurements of fasting intact insulin alone and combined with ALT provide a noninvasive strategy for identifying the presence of MASLD.

GACT-PPIS: Prediction of protein-protein interaction sites based on graph structure and transformer network

The prediction of protein-protein interaction sites (PPIS) is currently crucial for regulating many biological activities in cells and developing drugs for various diseases. Deep learning-based methods have been proposed for predicting PPIS, significantly reducing the manpower and time costs associated with traditional experimental methods such as yeast two-hybrid, mass spectrometry, and affinity purification. However, the predictive accuracy of these deep learning methods has not yet reached the expected level. Therefore, we introduce a model called GACT-PPIS.The design of the GACT-PPIS algorithm aims to utilize combined information from protein sequences and structures as input to predict protein-protein interaction sites. The core of GACT-PPIS utilizes an Enhanced Graph Attention Network (EGAT) with initial residual and identity mappings, along with a deep Transformer network as the basic units, supplemented by Graph Convolutional Networks (GCN), effectively aggregating information from neighboring nodes for each node. After multiple network layers, the information of the entire protein is also fused into the nodes, and the Transformer network further enhances the model's performance.Experimental results show that GACT-PPIS outperforms the most representative models in terms of Recall, F1-measure, MCC, AUROC, and AUPRC on the benchmark test set (Test-60). Additionally, on other independent test sets (UBTest-31-6), GACT-PPIS leads in terms of Accuracy, Precision, Recall, F1-measure, MCC, AUROC, and AUPRC compared to the most representative models. It is worth noting that GACT-PPIS demonstrates excellent generalization and versatility across different test sets, showcasing good performance on multiple test sets for the same trained GACT-PPIS model.

Hansen parameters and GastroPlus assisted optimized topical elastic liposomes to treat breast cancer using a novel isatin derivative

Breast cancer treatment is a global health challenge using conventional toxic chemotherapeutic agents. A novel isatin could be a promising alternative. An isatin derivative (TM-19) was synthesized and characterized using NMR (nuclear magnetic resonance), mass spectrometry, Fourier transform infrared (FTIR), and differential scanning calorimetry (DSC). HSPiP and GastroPlus predictive software were used to predict suitable solvents for solubility and in vivo oral pharmacokinetics in humans (fasted condition), respectively. Based on predictive findings, topical elastic liposomes (phosphatidylcholine as PC and span 80 by film hydration method) was prepared, characterized, and optimized using QbD (quality by design). QbD identified the impact of composition on response variables (Y1 for size, Y2 for polydispersity index, Y3 for zeta potential, and Y4 for %EE). In vitro cytotoxicity study was carried out against MCF-7 cell lines. Low molecular weight and lipophilic TM-19 was crystalline in nature (255 g/mol). GastroPlus program predicted limited in vivo dissolution and poor oral absorption (10.4 %). Results confirmed the highest TM-19 solubility in DMSO (dimethyl sulfoxide) ˃ chloroform ˃ PEG 400 (polyethylene glycol 400). Considering experimental and predicted data, topical delivery of TM-19 using optimized (desirability parameter value = 0.97) elastic liposomes (TF1) was quite promising due to optimal size (344 nm), high %EE (61.7 %), low PDI (0.45), and optimal zeta potential (− 12.34 mV). TM-19 loaded TF1 elicited concentration dependent cytotoxic effect (˃ 90 % at 10 µM) against MCF-7 as compared to TM-19 suspension.

A comprehensive assessment of the antiproliferative effects of Cymbopogon winterianus essential oil, citronellal, and citronellal complexed with β-cyclodextrin on cervical cancer cell line (HeLa)

Cymbopogon winterianus. Jowitt (syn. Cymbopogon nardus. (L.) W. Watson var. mahapengiri. Winter) (Poaceae family) is native to tropical and semitropical areas of Asia, India, and Indonesia, and is cultivated in South and Central America. In this work, the antiproliferative potential of the essential oil of C. winterianus, its major compound against the HeLa cervical cancer lineage, as well as the major compound Citronellal complexed with β-cyclodextrin (β-CO) was evaluated. By gas chromatography coupled with mass spectrometry, it was possible to identify that Citronellal (38.89 %) is the major compound in C. winterianus. In vitro assays with cervical cancer cells (HeLa), it was possible to observe the antiproliferative activity of C. winterianus essential oil (CW), with IC50 of 1.86 μg/mL, as well as of the compound Citronellal (CO), IC50 4.44 μg/mL and citronellal complexed β-cyclodextrin (β-CO), IC50 2.59 μg/mL, as well as the evaluation of cell migration and cell cycle. NIH-3T3 non-tumor cells were also used to evaluate the selectivity of these compounds in tumor cells. All tested samples showed SI > 3, confirming the highest toxicity in tumor cells. All samples tested were able to decrease cell migration and when the proportion of cells in the Sub-G0 Phase was observed, all samples increased the proportion of cells with fragmented DNA, an indication of cell death, except for CW. Thus, it can be concluded that there is a potential to be explored in the C. winterianus essential oil, Citronellal, and Citronellal complexed with β-cyclodextrin.

Kunitz-type trypsin inhibitor from durian (Durio zibethinus) employs a distinct loop for trypsin inhibition.

Kunitz-type trypsin inhibitors are ubiquitous in plants. They have been proposed to be a part of a defense mechanism against herbivores. Trypsin inhibitors also have potential applications in the biotechnology industry, such as in mammalian cell culture. We discovered that durian (Durio zibethinus) seed contains Kunitz-type trypsin inhibitors as identified by N-terminal sequencing and mass spectrometry. Eleven new trypsin inhibitors were cloned. The D. zibethinus trypsin inhibitors (DzTIs) that are likely expressed in the seed were produced as recombinant proteins and tested for trypsin inhibitory activity. Their inhibitory activity and crystal structures are similar to the soybean trypsin inhibitor. Surprisingly, a crystal structure of the complex between DzTI-4, the DzTI with the lowest inhibitory constant, and bovine trypsin revealed that DzTI-4 utilized a novel tryptophan-containing β1-β2 loop to bind trypsin. Site-direct mutagenesis confirmed the inhibitory role of this loop. DzTI-4 was not toxic to the HEK293 cells and could be used in place of the soybean trypsin inhibitor for culturing the cells under serum-free conditions. DzTI-4 was not toxic to mealworms. However, a mixture of DzTIs extracted from durian seed prevented weight gain in mealworms, suggesting that multiple trypsin inhibitors are required to achieve the antinutritional effect. This study highlights the biochemical diversity of the inhibitory mechanism of Kunitz-type trypsin inhibitors and provides clues for further application of these inhibitors.

Kaixinsan regulates neuronal mitochondrial homeostasis to improve the cognitive function of Alzheimer's disease by activating CaMKKβ-AMPK-PGC-1α signaling axis

BackgroundAlzheimer's disease (AD) is a neurodegenerative disease primarily characterized by cognitive impairments. With the intensification of population aging, AD has become a major health issue faced by the elderly. Kaixinsan, a classical traditional Chinese prescription consists of Panax ginseng C. A. Mey., Polygala tenuifolia Willd., Poria cocos (Schw.) Wolf and Acori Tatarinowii rhizoma, is commonly used in clinical for treating memory decline. However, its mechanism remains unclear, which hinders its popularization and application. MethodMorris water maze (MWM) was performed to evaluate the effect of Kaixinsan on improving learning and memory ability in SAMP8 (senescence-accelerated mouse prone 8, an AD model mice) mice. Nissl staining, TdT-mediated dUTP Nick End Labeling (TUNEL) and western blotting (Bax and Bcl-2) were used to confirm the effect of Kaixinsan on the neuronal structure and apoptosis of SAMP8 mice. ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was performed to identify the distribution components of the brain tissue after receiving Kaixinsan extraction. Based on the identified brain distribution components, the mechanism of Kaixinsan improving the cognitive function was predicted by network pharmacology. Then, using HSP60 as mitochondrial marker and RBFOX3 as neuron marker, immunofluorescence co-localization was used to confirm the effect of Kaixinsan on neuronal mitochondria quantity in SAMP8 mice. Using western blotting to detect the expression of predicted proteins (AMPK, CaMKKβ, PGC-1α and HSP90) affecting mitochondrial homeostasis. To further confirm the mechanism of Kaixinsa. The authors replicated SH-SY5Y cell injury model induced by Amyloid β - protein fragment 25-35 (Aβ25-35) and compared the effects of serum containing Kaixinsan on apoptosis of injured SH-SY5Y cells (detected by flow cytometer) and the expression level of apoptosis-associated protein (Bax and Bcl-2) and mitochondrial homeostasis related proteins (AMPK, CaMKKβ, PGC-1α and HSP90), with or without the addition of CaMKKβ inhibitor (STO-609). ResultsThe results indicate that Kaixinsan can improve the cognitive function of SAMP8 mice, alleviate the hippocampal tissue lesions and inhibit neuron apoptosis. Seventeen brain distribution components of Kaixinsan were identified. Based on the brain distribution components of Kaixinsan, the results of network pharmacology suggest that Kaixinsan may regulate mitochondrial homeostasis through the CaMKKβ-AMPK-PGC-1α signaling axis. In vivo experiments, The authors observed that Kaixinsan could reverse neuronal mitochondrial loss in SAMP8 mice by upregulating CaMKKβ, AMPK, HSP90 and PGC-1α to promote mitochondrial biogenesis and increase the number of neuronal mitochondria. Additionally, the in vitro experiments demonstrated that Kaixinsan can inhibit apoptosis of Aβ25-35 injured SH-SY5Y cells and upregulate mitochondrial homeostasis-related proteins CaMKKβ, AMPK and PGC-1α. However, in addition to CaMKKβ inhibitors, the neuroprotective effect disappeared. ConclusionThe results indicate that Kaixinsan can improve the cognitive function of SAMP8 mice by regulating CaMKKβ-AMPK-PGC-1α signaling axis to maintain mitochondrial homeostasis and inhibit neuronal apoptosis.

Deciphering the alteration of MAP2 interactome caused by a schizophrenia-associated phosphorylation

Microtubule-associated protein 2 (MAP2) is a crucial regulator of dendritic structure and neuronal function, orchestrating diverse protein interactions within the microtubule network. We have shown MAP2 is hyperphosphorylated at serine 1782 (S1782) in schizophrenia and phosphomimetic mutation of S1782 in mice (MAP2S1782E) is sufficient to impair dendritic architecture. We sought to determine how this hyperphosphorylation affects the MAP2 interactome to provide insights into the disorder's mechanisms. We investigated the MAP2 interactome using co-immunoprecipitation and mass spectrometry in MAP2S1782E and MAP2WT mice. We found that S1782E MAP2 led to a substantial disruption of protein-protein interactions relative to WT MAP2. Reduced interactions with PDZ domain-containing proteins, calmodulin-binding proteins, ribosome proteins, and kinesin proteins may all contribute to dendritic impairments induced by S1782E, and may be linked to schizophrenia pathogenesis. Interestingly, novel gain-of-function interactions with PPM1L and KLHL8 nominated these as regulators of phosphoS1782 MAP2 abundance and potential therapeutic targets in schizophrenia.

The crucial quality marker of Panax ginseng: Glycosylated modified ribonuclease-like storage protein

Panax ginseng C.A.Mey is a famous natural herbal medicine worldwide. Mountain-cultivated ginseng (MCG) and garden-cultivated ginseng (GCG) are two types of Panax ginseng. There is a significant difference in economic benefits between MCG and GCG, which can always lead to problems such as adulteration and substitution of MCG with lower-priced alternatives. We explored the quality marker of ginseng at the intact protein level and established a foundation for the quality control of ginseng. Cellulose nanocrystal assisted sample preparation combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) equipped with a high mass detector was performed to analyze intact proteins in ginseng. The results revealed that the ribonuclease-like storage protein is the most abundant protein in MCG and GCG. Meanwhile, the molecular weight of the ribonuclease-like storage protein showed great difference between different ginseng species, which is 26.2 kDa in MCG and 24.2 kDa in GCG. The ribonuclease-like storage protein glycosylation modification difference provides data support for the differentiation between MCG and GCG. This study showed that glycosylated modified ribonuclease-like storage protein can be a crucial quality marker of ginseng, facilitating the rapid distinction between MCG and GCG.