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.

Trace analysis of taste and odour compounds in drinking water by stir bar sorptive extraction followed by thermal desorption - gas chromatography - mass spectrometry (SBSE-TD-GC-MS)

Because of negative tap water quality perception, people often prefer bottled water over tap water despite the higher energy consumption and production of plastic waste. Taste and odour (T&O) deviations in tap water are an important aspect of this issue and should be avoided. However, T&O compounds typically occur at low concentrations (≤ ng.L−1) and are numerous, originating from various sources, ranging from source water to kitchen taps. Consequently, unravelling T&O events is challenging and causing compounds may remain unknown. Therefore, a multi-compound method (SBSE-TD-GC–MS) was developed, optimised and validated for the simultaneous trace analysis of 45 T&O compounds in drinking water. It covers key compounds of different odour categories with a wide range of physical-chemical properties and originating from the different steps of production and distribution. The intra- and interday precision of the method was shown by relative standard deviations (RSD) lower than 15 % and 23 %, respectively, for 75 % of the measured concentrations. For most of the compounds (>75 %), the detection capability (CCβ) was below 1/3rd of their lowest reported odour threshold concentration (OTC), while for the other 6 compounds, the CCβ was within the reported OTC range. Additionally, the CCβs were comparable to or lower than those in literature. As a proof of concept, the method was used to investigate the occurrence and concentrations of T&O compounds before (surface water) and after drinking water production. The results show a clear removal of compounds related to microbial activity (e.g. geosmin, 2-methylisoborneol, 2,4,6-tribromoanisole) after treatment and an increase of compounds related to disinfection. Although rarely investigated, 3-methylbutanal showed concentrations similar to its OTC in treated drinking water, emphasizing the necessity of a broad ultra-trace analysis to ultimately prevent the occurrence of T&O compounds and guarantee tap water quality.

Fizzy extraction secondary electrospray ionization mass spectrometry for analysis of volatile solutes in high-matrix samples

Fizzy extraction (FE) is a rapid way to prepare liquid samples containing volatile organic compounds (VOCs) for analysis. The sample is first infused with a carrier gas, and subsequent rapid decompression of the sample chamber causes it to effervesce spontaneously. Secondary electrospray ionization (SESI) enables analysis of VOCs using mass spectrometry (MS). Here, we report direct hyphenation of FE with SESI-MS. In order to adjust the flow rate of the gaseous extract to match the optimum input flow rate of the ion source, flow was split with a tee junction. The following parameters have been optimized: pressure inside the sample chamber (during saturation), high voltage applied to the nano-ESI solution, temperature of desolvation line, temperature of heated block, temperature of the SESI chamber, gas pressure applied to the headspace of nano-ESI solution, and length of tubing connected to the tee junction. The optimum parameters were 150 kPa, +4.0 kV, 225 °C, 200 °C, 90 °C, 18 kPa, and 6 cm, respectively. The developed method was characterized using MS and high-speed imaging. The limits of detection for ethyl butyrate, ethyl pentanoate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate are 1.11 × 10−7 M, 4.33 × 10−7 M, 8.13 × 10−8 M, 1.09 × 10−7 M, and 2.19 × 10−7 M, respectively. The analysis time was only 2.5 min per sample (not counting the cleaning step). The method was further applied for profiling VOCs in selected dairy products. Interestingly, the FE-SESI-MS method is much more tolerant to the sample matrix (milk) than headspace vapor analysis using the same ion source.

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.

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.

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

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

Triple acquisition mass spectrometry (TRAM) combining targeted and non-targeted metabolomics in a single run

BackgroundWe introduce TRAM, a triple acquisition strategy on a high-speed quadrupole time-of-flight mass spectrometer for merging non-targeted and targeted metabolomics into one run. TRAM stands for “quasi-simultaneous” acquisition of (1) a full scan MS1, (2) top 30 data-dependent MS2 (DDA), and (3) targeted scheduled MS2 for multiple reaction monitoring (MRM) within measurement cycles of ∼ 1 second. TRAM combines the selectivity and sensitivity of state-of-the-art targeted MRM-based methods with the full scope of non-targeted analysis enabled by high-resolution mass spectrometry. ResultsIn this work, we deploy a workflow based on hydrophilic interaction liquid chromatography (HILIC). For a broad panel of metabolites, we provide chromatographic retention times, and optimized conditions as a basis for targeted MRM experiments, listing accurate masses and sum formulas for fragment ions (including fully 13C labeled analogs). Validation experiments showed that TRAM offered (1) linear working ranges and limits of quantification comparable to MRM-only methods, (2) enabled accurate quantification in SRM 1950 human plasma reference material, and (3) was equivalent to DDA-only approaches in non-targeted metabolomics. Metabolomics in human cerebrospinal fluid showcased the power of the strategy, emphasizing the need for high coverage/high throughput metabolomics in clinical studies. SignificanceAcquiring up to 30 data-dependent spectra per MS cycle while still offering gold standard absolute quantification down to low nanomolar concentrations, TRAM allows in-depth profiling and reduces required sample volume, time, cost, and environmental impact.

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 (MMP's) 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 L929 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 inhibitors 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.

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.

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.

LncRNA HOXA11-AS intercepts the POU2F2-mediated downregulation of SLC3A2 in osteoarthritis to suppress ferroptosis

BackgroundOsteoarthritis (OA) is a prevalent ailment characterized by the gradual degradation of joints, resulting in discomfort and restricted movement. The recently proposed mechanism of ferroptosis is intricately associated with the initiation and progression of OA. Our study found that the long non-coding RNA HOXA11-AS reduces ferroptosis by increasing the expression of SLC3A2 through the transcription factor POU2F2. Materials and methodsHOXA11-AS was identified through lncRNA microarray analysis, and its impact on chondrocytes and extracellular matrix was assessed using real-time quantitative PCR, western blotting, and CCK8 assays. Subsequently, overexpression of HOXA11-AS in the knee joints of mice confirmed its protective efficacy on chondrocyte phenotype in the OA model. The involvement of HOXA11-AS in regulating ferroptosis via SLC3A2 was further validated through RNA sequencing analysis of mouse cartilage and the assessment of malondialdehyde levels and glutathione peroxidase activity. Finally, a combination of RNA sequencing, pull-down assays, mass spectrometry (MS), and chromatin immunoprecipitation (ChIP) techniques was employed to identify POU2F2 as the crucial transcription factor responsible for repressing the expression of SLC3A2, which can be effectively inhibited by HOXA11-AS. ResultsOur study demonstrated that HOXA11-AS effectively enhanced the metabolic homeostasis of chondrocytes, and alleviated the progression of OA in vitro and in vivo experiments. Furthermore, HOXA11-AS was found to enhance SLC3A2 expression, a key regulator of ferroptosis, by interacting with the transcriptional repressor POU2F2. ConclusionsHOXA11-AS promotes SLC3A2 expression and inhibits chondrocyte ferroptosis, by binding to the transcriptional repressor POU2F2, offering a promising and innovative therapeutic approach for OA.

Water-soluble organic selenometabolites of alfalfa (Medicago sativa L.) green biomass-derived fractions

BackgroundTolerance of plants towards selenium, a non-essential microelement for higher plants, is a key issue when designing either the indirect (selenium-depletion from highly seleniferous soils) or directed (selenized feed production) enrichment of selenium in forages. Alfalfa (Medicago sativa L.), the well-known forage crop of the Fabaceae family, has been gaining considerable interest due to its application as a green manure, as a cover crop, or in soil remediation by nitrogen fixation. ObjectiveThe goal of our study was to assess into which selenocompounds alfalfa plants biotransform the excess selenium uptake from the soil. Selenocompounds (other than selenomethionine and inorganic forms) accumulated in the fiber and the so-called brown juice by-product fractions of processed alfalfa biomass were targeted. MethodologyInductively coupled plasma – mass spectrometry assisted multidimensional (strong anion exchange, strong cation exchange, reversed phase) orthogonal chromatographic purification was applied to supply Se-containing fractions in adequately high purity for electrospray high-resolution mass spectrometry (used for the first time for this matrix) analyses. ResultsAs a total, 30 selenocompounds (with isomers) were described, showing the abundance of the derivatives of selenohexose, selenohomolanthionine, and 2,3-dihydroxypropionic acid. Out of the 30 selenocompounds, 15 could be assigned the elemental composition, and the tentative structure of five compounds including among others deamino-2-oxo-selenohomolanthionine, deamino-hydroxy-selenohomolanthionine, and the dimer of 2,3-dihydroxypropionyl-selenohomocysteine could be presented. ConclusionsThe studied fractions arising from the standard alfalfa processing technology contained a wide variety of selenocompounds whose origin can be either the plant’s inherent Se metabolism or the processing technology itself. The importance of negative mode data acquisition has been highlighted, as out of the 30 compounds, 16 could be detected exclusively in this electrospray ionization mode.

Polycondensation, Cyclization and Disproportionation of Solid Poly(L-lactide) Trifluoroethyl Esters and the Simultaneous Formation of Extended Chain Crystals and Extended Ring Crystals

Two poly(L-lactide)s (PLAs) with a degree of polymerization (DP) of 20 or 100 were prepared by trifluoroethanol-initiated ring-opening polymerization (ROP) catalyzed by tin(II) 2-ethyl hexanoate (SnOct2). These PLAs were annealed at 140 °C or at 160 °C in the presence of SnOct2, and the changes in topology and molecular weight distribution (MWD) were monitored by matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry and gel permeation chromatography (GPC). For the PLA with a DP 20, the main reaction was polycondensation combined with higher dispersities. In the case of the DP 100, PLA polycondensation was combined with disproportionation and the formation of a new MWD maximum around m/z 3 500. In addition, extensive cyclization occurred, and the resulting cyclic PLAs crystallized separately from the linear chains in the form of extended ring crystals. These results also suggest that both extended chain and extended ring crystals posses the same crystal thickness as a result of thermodynamically controlled transesterification in the solid state.

Transcription factor PRRX1-activated ANXA6 facilitates EGFR-PKCα complex formation and enhances cisplatin sensitivity in bladder cancer

BackgroundTumor resistance to cisplatin represents a major clinical challenge, particularly in bladder cancer (BC). ANXA6 is a member of annexin family, and its role in cisplatin resistance remains unclear. This study explores ANXA6's role in promoting cisplatin sensitivity. MethodsBioinformatics analyses and clinical specimen verifications assessed the correlation between ANXA6 and cisplatin treatment. A series of assays, including CCK-8, clone formation assay, flow cytometry assays for reactive oxygen species (ROS) and apoptosis, and comet assays, were used to confirm ANXA6's role in enhancing cisplatin sensitivity and re-sensitizing resistant BC cells. Mass spectrometry, immunofluorescence, and co-immunoprecipitation experiments elucidated ANXA6's role in enhancing PKCα/EGFR complex formation and inhibiting the EGFR pathway. ChIP-PCR and dual-luciferase assays determined PRRX1's regulatory role on ANXA6 transcription. Finally, the impact of ANXA6 in vivo was evaluated using xenograft models. ResultsBioinformatics analyses showed a significant correlation between ANXA6 expression and cisplatin sensitivity. In vitro and in vivo experiments confirmed that ANXA6 was a new target for cisplatin treatment. ANXA6 overexpression not only enhanced cell viability inhibition, DNA damage and apoptosis caused by cisplatin, but also re-sensitized cisplatin-resistant cells. Mechanistically, ANXA6 promotes PKCα/EGFR complex formation, inhibiting EGFR phosphorylation and downstream AKT and ERK1/2. Moreover, PRRX1 was identified as a transcription factor promoting ANXA6 expression, thereby augmenting the cytotoxic effects of cisplatin. ConclusionOur study reveals the mechanism by which ANXA6 enhances cisplatin sensitivity and re-sensitizes resistant cells. The roles of PRRX1 and ANXA6 in cisplatin resistance offer new therapeutic targets to overcome cisplatin resistance in clinical practice.

Snake venom weakens neurovascular integrity and promotes vulnerability to neuroinflammation

Snake envenomation poses significant medical challenges, particularly in subtropical and tropical regions, with long-term impacts on neurovascular integrity and neuroinflammation remaining underexplored. This study investigates the effects of venom from four species of venomous snakes in southern China—Zhoushan Cobra (Naja atra, NA), Many-banded Krait (Bungarus multicinctus, BM), Five-paced Pit Viper (Deinagkistrodon acutus, DA), and Chinese Moccasin (Protobothrops mucrosquamatus, PM) — on the blood–brain barrier (BBB) and chronic neuroinflammation. Using mass spectrometry, we analyzed venom protein compositions, while cytotoxic effects on mouse brain endothelial cells (bEND.3) were evaluated to determine IC50 values. In vitro BBB models and in vivo experiments in C57BL/6J mice revealed that NA venom, in particular, significantly compromised BBB integrity without inducing large-scale apoptosis, leading to persistent BBB disruption characterized by increased permeability and selective degradation of extracellular matrix and tight junction proteins. Moreover, to simulate secondary infections that often occur following snakebites, we combined venom exposure with lipopolysaccharide (LPS) treatment, which exacerbated neuroinflammatory responses by intensifying microglial activation and promoting a pro-inflammatory phenotype. These findings highlight the role of snake venom in compromising neurovascular integrity and promoting vulnerability to chronic neuroinflammation, emphasizing the need for further research into venom-induced neuroinflammatory pathways and their potential as therapeutic targets.

Single-step purification and characterization of Pseudomonas aeruginosa azurin.

Azurin is a small periplasmic blue copper protein found in bacterial strains such as Pseudomonas and Alcaligenes where it facilitates denitrification. Azurin is extensively studied for its ability to mediate electron-transfer processes, but it has also sparked interest of the pharmaceutical community as a potential antimicrobial or anticancer agent. Here we offer a novel approach for expression and single-step purification of azurin in Escherichia coli with high yields and optimal metalation. A fusion tag strategy using an N-terminal GST tag was employed to obtain pure protein without requiring any additional purification steps. After the on-column cleavage by HRV 3C Protease, azurin is collected and additionally incubated with copper sulphate to ensure sufficient metalation. UV-VIS absorption, mass spectroscopy, and circular dichroism analysis all validated the effective production of azurin, appropriate protein folding and the development of an active site with an associated cofactor. MD simulations verified that incorporation of the N-terminal GPLGS segment does not affect azurin structure. In addition, the biological activity of azurin was tested in HeLa cells.

Imaging and Non-imaging Analytical Techniques Used for Drug Nanosizing and their Patents: An Overview.

Nanosizing is widely recognized as an effective technique for improving the solubility, dissolution rate, onset of action, and bioavailability of poorly water-soluble drugs. To control the execution and behavior of the output product, more advanced and valuable analytical techniques are required. The primary intent of this review manuscript was to furnish the understanding of imaging and non-imaging techniques related to nanosizing analysis by focusing on related patents. In addition, the study also aimed to collect and illustrate the information on various classical (laser diffractometry, photon correlation spectroscopy, zeta potential, laser Doppler electrophoresis, X-ray diffractometry, differential scanning calorimeter, scanning electron microscopy, transmission electron microscopy), new, and advanced analytical techniques (improved dynamic light scattering method, Brunauer-Emmett- Teller method, ultrasonic attenuation, biosensor), as well as commercial techniques, like inductively coupled plasma mass spectroscopy, aerodynamic particle sizer, scanning mobility particle sizer, and matrix- assisted laser desorption/ionization mass spectroscopy, which all relate to nano-sized particles. The present manuscript has taken a fresh look at the various aspects of the analytical techniques utilized in the process of nanosizing, and has achieved this through the analysis of a wide range of peer-reviewed literature. All summarized literature studies provide the information that can meet the basic needs of nanotechnology. A variety of analytical techniques related to the nanosizing process have already been established and have great potential to weed out several issues. However, the current scenarios require more relevant, accurate, and advanced analytical techniques that can minimize the time and deviations associated with different instrumental and process parameters. To meet this requirement, some new and more advanced analytical techniques have recently been discovered, like ultrasonic attenuation technique, BET technique, biosensors, etc. Conclusion: The present overview certifies the significance of different analytical techniques utilized in the nanosizing process. The overview also provides information on various patents related to sophisticated analytical tools that can meet the needs of such an advanced field. The data show that the nanotechnology field will flourish in the coming future.

HPLC with parallel ESI-MS and ICP-AES detection as a tool for the speciation studies of phosphovanadotungstates

The presence of different equilibrated chemical species in aqueous solutions of polyoxometalates represents a challenge for their identification, requiring the use of both separation and detection techniques. The use of reversed-phase ion-pair high-performance liquid chromatography (HPLC) in gradient elution mode resulted in optimal separation conditions. The detection was accomplished by parallel hyphenation of the inductively coupled plasma atomic emission spectrometry (ICP-AES) and electrospray ionization mass spectrometry (ESI-MS), which combined the advantages of element and molecule specific detectors. This hyphenation resulted in an unambiguous identification of the products of the synthesis of mixed-addendum Keggin-type phosphovanadotungstates of [PVxW12-xO40](3+x)- (x = 1 – 3) general formula without the isolation of the individual species.