Time-of-flight Mass Spectrometry Research Articles

Hydrolytic Response of Beta-Amylase to Selected Starches when Adsorptively Immobilized on Agarose Gel

Fermentative microorganisms bring about transformative processes in food, thereby enhancing flavor and appearance indices, inhibiting undesirable components, improving shelf and storage life, as well as improving health and nutritional quality parameters. Traditional methods for identification of fermentative microorganisms involve cultivating and analyzing phenotypic characteristics, which are labor-intensive and time-consuming. Advanced molecular techniques such as polymerase chain reaction, whole gene sequencing, next generation sequencing, fluorescent in situ hybridization (FISH), Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), fourier transform infrared spectroscopy and hyper-spectral imaging offer rapid, cost-effective, and reliable identification of bacteria and fungi cultured on agar plates or in liquid media. Techniques, such as genotyping, examine the DNA sequence to determine the genetic makeup of microorganisms, alongside the grouping of isolates via application of phylogenetic tree similarity indices. Overall, modern molecular techniques have revolutionized the identification of fermentative microorganisms, providing efficient and more elaborate alternatives to traditional methods. In this review work, an attempt is made to examine the historical and analytical trends from the conventional methods to the most recent modern tools for effective identification of industrially-important fermentative microorganisms.

Recent Advances in the Identification and Characterization of Fermentative Microorganisms: An Exploratory Review

Fermentative microorganisms bring about transformative processes in food, thereby enhancing flavor and appearance indices, inhibiting undesirable components, improving shelf and storage life, as well as improving health and nutritional quality parameters. Traditional methods for identification of fermentative microorganisms involve cultivating and analyzing phenotypic characteristics, which are labor-intensive and time-consuming. Advanced molecular techniques such as polymerase chain reaction, whole gene sequencing, next generation sequencing, fluorescent in situ hybridization (FISH), Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), fourier transform infrared spectroscopy and hyper-spectral imaging offer rapid, cost-effective, and reliable identification of bacteria and fungi cultured on agar plates or in liquid media. Techniques, such as genotyping, examine the DNA sequence to determine the genetic makeup of microorganisms, alongside the grouping of isolates via application of phylogenetic tree similarity indices. Overall, modern molecular techniques have revolutionized the identification of fermentative microorganisms, providing efficient and more elaborate alternatives to traditional methods. In this review work, an attempt is made to examine the historical and analytical trends from the conventional methods to the most recent modern tools for effective identification of industrially-important fermentative microorganisms.

Characteristics of Umami Taste of Soy Sauce Using Electronic Tongue, Amino Acid Analyzer, and MALDI−TOF MS

The objective of this study was to investigate the umami characteristics of soy sauce using electronic tongue evaluation and amino acid composition and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI−TOF MS) analysis. The soy sauce peptides were isolated from soy sauce using XAD−16 macroporous resin combined with ethanol solution. The results showed that the soy sauce peptide fraction eluted by 60% ethanol (SS−60%) exhibited a prominent umami taste, and the umami scores were highly positively correlated with the amino acid nitrogen contents of soy sauces. The umami scores of SS−60% were significantly positively correlated with the contents of free amino acids. Especially, Phe showed the highest positive correlation with the umami scores. In addition, five characteristic ion peaks with m/z at 499, 561, 643, 649, and 855 were identified in the peptide mass fingerprinting. Therefore, this study provides new insights into the umami characteristics for the taste evaluation and reality identification of soy sauce.

Mobile VOC measurements in Commerce City, CO reveal the emissions from different sources

ABSTRACT Source attribution of volatile organic compounds (VOCs) can be challenging in urban areas, which have many point sources. Mobile laboratories using time-of-flight mass spectrometers (TOF-MS) can take measurements throughout areas of concern, resulting in data with high spatial resolution that can be used to more easily identify these sources. However, emissions in heavily polluted areas still undergo significant mixing over short distances, making source attribution of some compounds challenging. Positive matrix factorization (PMF) has been widely used for attributing pollutants to different sources when taking stationary measurements due to its ability to process large amounts of data into generally interpretable results. However, some limitations of PMF can impact its usefulness to mobile data; PMF is a computationally intensive process, requires some user choices in attributing factors to emissions sources, and results can be significantly impacted by chemical transformations after emission. Here, both PMF and a simpler comparative analysis method are evaluated in analyzing measurements taken in the Elyria Swansea neighborhood of Commerce City, CO. This neighborhood is located near an oil refinery, a wastewater treatment plant, local industrial shops, and major highways. PMF failed to differentiate between oil refinery emissions and traffic emissions, and had difficulties recognizing other key sources. A simpler comparative analysis showed that the refinery contributed significantly to VOC concentrations throughout the neighborhood, including air toxics such as benzene. A wastewater treatment plant contributed to methanethiol and dimethyl sulfide. Finally, a small woodshop was identified as a hyperlocal VOC source, and contributed high amounts of some VOCs, such as toluene and other solvents, in its immediate surroundings. Implications Statement: This work discusses mobile measurements of VOCs around Commerce City, CO, a heavily polluted urban area north of Denver, using a PTR-TOF-MS. Two different source attribution methods, positive matrix factorization (PMF) and comparative analysis, were evaluated in the context of mobile measurements. The results show that an oil refinery and a woodshop contributed greatly to many VOC concentrations in the Elyria Swansea residential area of Commerce City. Additional sources, such as a wastewater treatment plant, also contributed to some odorous VOCs. PMF was unable to fully describe sources based on the mobile data. Comparative analysis was useful in attributing more VOCs to different sources, but quantitative results were influenced by how the analysis is set up. These findings are relevant to the residents of Denver and regulatory bodies to better understand Denver air pollution, as well as to other mobile studies doing source attribution of VOCs.

Transmission of Brucella canis in a canine kennel following introduction of an infected dog

Brucella canis is a zoonotic pathogen and the main causative agent of canine brucellosis. In the Netherlands, B. canis had previously only been detected in individual cases of imported dogs. However, an outbreak of B. canis occurred for the first time in a cohort of autochthonous dogs in a breeding kennel in 2019. The outbreak began with a positive serological test result of an imported intact male dog showing clinical symptoms of brucellosis. Consequently, urine and blood samples were collected and tested positive for B. canis by culture, matrix-assisted laser desorption/ionization – time of flight mass spectrometry (MALDI-TOF MS) and whole-genome-sequencing (WGS). Screening of the contact dogs in the kennel where the index case was kept, revealed that antibodies against B. canis could be detected in 23 out of 69 dogs (34 %) by serum agglutination test (SAT). Of the 23 seropositive dogs, B. canis could be cultured from the urine and/or heparin samples of 19 dogs (83 %). This outbreak represents the first documented case of transmission of B. canis to autochthonous contact dogs in the Netherlands. WGS revealed all B. canis isolates belonged to the same cluster, which means the transmission of B. canis in the breeding kennel was most likely caused by the introduction of one infected dog. Comparing this cluster with data from other B. canis isolates, it also appears that characteristic clusters of B. canis are present in several endemic countries. These clusters seem to remain stable over time and may help in locating the origin of new isolates found. This outbreak showed that the international movement of dogs from endemic countries poses a threat to the canine population, while serological screening and WGS proved to be valuable tools for respectively screening and the epidemiological investigation.

New insights into the chemistry and anticancer activity of Ophiocordyceps xuefengensis

Ophiocordyceps xuefengensis (O. xuefengensis), the sister taxon of Ophiocordyceps sinensis (O. sinensis), is consumed as a “tonic food” due to its health benefits. However, little is known regarding the chemistry and bioactivity of O. xuefengensis. In this study, we characterized 80 indole-based alkaloids in the ethyl acetate fraction of O. xuefengensis by high performance liquid chromatography-quadrupole time of flight mass spectrometry (HPLC–Q–TOF–MS/MS), of which 54 indole-based alkaloids were identified as possibly new compounds. Furthermore, 29 of these compounds were established as potential anti-cancer compounds by ligand fishing combined with HPLC–Q–TOF–MS/MS. Moreover, molecular docking identified the NH- and OH- groups of these compounds as the key active groups. The present study has expanded the knowledge on the characteristic indole-based alkaloids and anti-cancer activity of O. xuefengensis.

P-315 Microbiota in endometriosis: culturomics analysis in receptive-phase endometrium

Abstract Study question Does the endometrium possess alive, culturable bacteria, and are there any microbes associated with endometriosis? Summary answer Thirty-five cultured bacterial species were identified in the receptive-phase endometrium, and an endometriosis-specific bacterial profile was detected. What is known already Endometriosis is a chronic inflammatory disease and one of the most prevalent female reproductive disorders, but its etiology remains unknown. Recently, significant attention has been directed towards studying the endometrial microbiome and its potential correlation with endometriosis. These analyses typically employ 16S rRNA gene sequencing, which detects bacterial genera but does not distinguish whether the bacteria are alive and hasn’t provided conclusive results regarding endometrial microbiome composition. Our aim is to develop a culture-based method for identifying live microbes in the endometrium and identifying bacterial species associated with endometriosis. Study design, size, duration This pilot cross-sectional study recruited twenty women from couples prior to undergoing infertility treatment at the Reproductive Unit of Universitary Hospital Virgen de las Nieves (Granada) between 2020 and 2022. Ten women were diagnosed with active endometriosis (no treatment), while ten women had male factor infertility, serving as the control group in our study. Participants/materials, setting, methods Endometrial biopsies were obtained during the mid-secretory phase, 7-9 days after ovulation (assessed with a urinary LH test), using an endometrial curette device. The biopsies were snap-frozen and stored at -80 °C until analysis. Microbiota cultures were conducted for up to 30 days using nine different media under aerobic, microaerobic, and anaerobic conditions. Matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS) was utilized for bacterial isolate identification. Main results and the role of chance In total, 19 out of 20 patients (95%) tested positive for one or more microbes, with only 1 patient (5%) showing no microbial growth. We detected thirty-five bacterial species in these mid-secretory endometrial samples. Among them, fourteen bacterial species were common in both endometriosis and control samples, while five species—Bifidobacterium bifidum, Bifidobacterium breve, Cutibacterium avidum, Lactobacillus vaginalis, and Staphylococcus warneri—were exclusively cultured from endometriosis samples. Conversely, healthy samples harbored fourteen exclusive species, including Actinomyces neuii, Corynebacterium amycolatum, Enterococcus faecalis, and Finegoldia magna, among others. Control endometrial samples, altogether, possessed significantly more bacterial species than endometriosis samples (30 vs. 19 microbial species). These findings are consistent with the initial culturomics analyses of endometrial microbiota, highlighting the method’s efficacy in identifying isolates at the species level. Notably, species such as Actinotignum schaalii, Bifidobacterium dentium, Atopobium vaginae, and Rothia dentocariosa were identified in our samples, marking the first description of these species in endometrial culturomics studies. Among all participants, the bacteria with the highest colony forming units were Cutibacterium acnes, Lactobacillus jensenii, Lactobacillus crispatus, and Lactobacillus iners. Our study confirms the presence of the endometrial microbiota and suggests microbial dysregulation in endometriosis. Limitations, reasons for caution This is a pilot study with a small sample size, requiring further investigation with a larger cohort. The developed protocol analyzed frozen samples, which may have failed to detect some of the fragile microbial species. Wider implications of the findings The identification of live culturable bacteria through culturomics in endometrial biopsies offers a reliable method for detecting endometritis-associated bacteria. Utilizing culturomics for endometrial microbiota testing shows promise in uncovering specific bacteria associated with gynecological disorders like endometriosis, and it enhances our understanding of potential pathological processes. Trial registration number not applicable

Cai's herbal tea enhances mitochondrial autophagy of type 1 diabetic mellitus β cells through the AMPK/mTOR pathway and alleviates inflammatory response.

This study investigates the therapeutic mechanisms of Cai's Herbal Tea in Type 1 Diabetes Mellitus (T1DM) mice, focusing on its effects on mitochondrial change and autophagy via the AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway. The composition of Cai's Herbal Tea was analyzed by Ultra-High Performance Liquid Chromatography-Quadrupole Time of Flight Mass Spectrometry (UHPLC-Q/TOF-MS). C57BL/6 mice and Min6 pancreatic beta cells were divided into control, diabetic mellitus (DM)/high glucose (HG), and treatment groups (low, medium, and high doses of Cai's Tea, and Metformin). Key physiological parameters, pancreatic islet health, Min6 cell morphology, viability, and insulin (INS) secretion were assessed. Small Interfering RNA-AMPK (si-AMPK) was utilized to confirm the pathway involvement. Cai's Herbal Tea improved body weight, pancreatic islet pathological injury, and INS secretion whereas reduced total triglycerides, fasting blood sugar, and Interferon gamma (INF-γ) in T1DM mice, particularly at higher doses. In Min6 cells, Cai's Tea mitigated HG-induced damage and proinflammatory response, enhancing cell viability and INS secretion. Notably, it reduced swelling and improved cristae structure in treated groups of mitochondria and promoted autophagy via the AMPK-mTOR pathway, evidenced by increased LC3II/LC3I and P-AMPK/AMPK ratios, and decreased P-mTOR/mTOR and P62 expressions in pancreatic islet β-cells. Furthermore, these effects were converted by si-AMPK interference. Cai's Herbal Tea exhibits significant therapeutic efficacy in T1DM mice by improving mitochondrial health and inducing autophagy through the AMPK-mTOR pathway in pancreatic islet β-cells. These findings highlight its potential as a therapeutic approach for T1DM management.

Red cabbage extract immobilized in bacterial cellulose film as an eco-friendly sensor to monitor microbial contamination and gamma irradiation of stored cucumbers

The aim of the present study is to develop a pH-sensing biopolymer film based on the immobilization of red cabbage extract (RCE) within bacterial cellulose (BC) to detect contamination and gamma radiation exposure in cucumbers. The results obtained show a sensitivity to pH changes for RCE in its aqueous form and that incorporated within BC films (RCE-BC), both showed color change correlated to bacterial growth (R2 = 0.91), this was supported with increase in pH values from 2 to 12 (R2 = 0.98). RCE and RCE-BC exposure to gamma radiation (0, 2.5, 5, 10, 15, 20, 25 kGy) resulted in gradual decrease in color that was more evident in RCE aqueous samples. To sense bacterial contamination of cucumbers, the total count was followed at 0, 5, 10 and 15 days in cold storage conditions and was found to reach 9.13 and 5.47 log cfu/mL for non-irradiated and 2 kGy irradiated samples, respectively. The main isolates detected throughout this storage period were identified as Pseudomonas fluorescens, Erwinia sp. Pantoea agglomerans using matrix assisted laser desorption ionization–time of flight-ms (MALDI–TOF–MS). Bacterial growth in stored irradiated cucumbers was detected by color change within 5 and 10 days of storage, after which there was no evident change. This is very useful since contamination within the early days of storage cannot be sensed with the naked eye. This study is the first to highlight utilizing RCE and RCE-BC as eco-friendly pH-sensing indicator films for intelligent food packaging to detect both food contamination and gamma preservation for refrigerator stored cucumbers.

Detection of the synthetic cathinone N,N-dimethylpentylone in seized samples from prisons

Drug use is prevalent in prisons with drugs associated with depressant effects found to be more prevalent than stimulants. Synthetic cathinones (SCats; often sold as “bath salts”, “ecstasy”, “molly”, and “monkey dust”) are the second largest category of new psychoactive substances (NPS) currently monitored by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) and are commonly used as substitutes for regulated stimulants, such as amphetamine, cocaine, and MDMA. N,N-dimethylpentylone (also known as dimethylpentylone, dipentylone, and bk-DMBDP) was detected for the first time in the Scottish prisons in seven powder samples seized between January and July 2023. Samples were analyzed using gas chromatography-mass spectrometry (GC-MS), ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-QToF-MS), and nuclear magnetic resonance imaging (NMR). Dimethylpentylone was detected alongside other drugs in four samples, including the novel benzodiazepine desalkylgidazepam (bromonordiazepam) and the synthetic cannabinoid receptor agonists (SCRAs) MDMB-INACA and MDMB-4en-PINACA.

Detection of the synthetic cathinone N,N-dimethylpentylone in seized samples from prisons

Drug use is prevalent in prisons with drugs associated with depressant effects found to be more prevalent than stimulants. Synthetic cathinones (SCats; often sold as “bath salts”, “ecstasy”, “molly”, and “monkey dust”) are the second largest category of new psychoactive substances (NPS) currently monitored by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) and are commonly used as substitutes for regulated stimulants, such as amphetamine, cocaine, and MDMA. N,N-dimethylpentylone (also known as dimethylpentylone, dipentylone, and bk-DMBDP) was detected for the first time in the Scottish prisons in seven powder samples seized between January and July 2023. Samples were analyzed using gas chromatography-mass spectrometry (GC-MS), ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-QToF-MS), and nuclear magnetic resonance imaging (NMR). Dimethylpentylone was detected alongside other drugs in four samples, including the novel benzodiazepine desalkylgidazepam (bromonordiazepam) and the synthetic cannabinoid receptor agonists (SCRAs) MDMB-INACA and MDMB-4en-PINACA.

Comparison of Head Space Solid Phase Micro Extraction with Conventional and Comprehensive Gas Chromatography Mass Spectrometry for Volatile Profiling of Irish whiskey

Whiskey is a traditional distilled product produced on the island of Ireland that is currently experiencing a major expansion in export volume. Different styles of Irish whiskey exist, however only some very limited published information exists on the volatile congener profile of the different styles of Irish whiskey. Such information is potentially indispensable from a quality, flavour and authenticity perspective. As gas chromatography is the established analytical method of choice to identify volatile congeners, this study compared headspace-solid phase microextraction (HS-SPME) using conventional gas chromatography single quadrupole mass spectrometry, to HS-SPME and HS-SPME-Arrow using comprehensive two-dimensional gas chromatography (GC×GC) modulated using reverse flow in tandem with time-of-flight mass spectrometry (TOFMS) detection. Six representative Irish whiskey samples were evaluated consisting of new make spirit and mature whiskies representing Single Malts, Pot Stills and Blends. The number of volatile congeners identified in these samples by HS-SPME/HS-SPME Arrow GC×GC-TOFMS was approximately twice that detected by conventional HS-SPME GCMS. In total 145 unique individual volatile congeners, excluding ethanol were identified, with the majority consisting of esters, but also benzenes, alcohols, aldehydes, terpenoids, furans, ketones, alkanes, alkenes, norisoprenoids, acetals, acids, lactones and phenols. The use of HS-SPME Arrow GC×GC-TOFMS significantly enhances the number of volatile congeners that can be identified and therefore provides much more information that can convey insights into product quality, consistency, flavour and also for authentication purposes.

Comparison of Vaginal Microbiota in HPV-Negative and HPV-Positive Pregnant Women Using a Culture-Based Approach

The purpose of this study was to conduct a comparative analysis of the composition of the dominant groups of vaginal microorganisms in healthy pregnant women and pregnant women infected with HPV using a microbiological culture-based method. The MALDI TOF MS method and 16S rRNA gene fragment sequencing were used to identify microorganisms isolated from healthy pregnant women (n=32) and pregnant women infected with HPV (n=24). It was found that vaginal secretion samples from both groups contained bacteria of 4 phyla: Bacillota, Actinomycetota, Pseudomonadota, Bacteroidota, and Ascomycota fungi. The most common microbial community in healthy pregnant women being CST I (p=0.0007), and CST V in pregnant women infected with HPV (p=0.0001). At the genus level, a total of 25 taxa were found in all samples, with Lactobacillus being the dominant genus overall. Escherichia (p<0.0001) and Prevotella (p=0.0001) concentrations were higher in HPV infected patients. When calculating the Pearson correlation coefficient for the phyla, it was found that Bacillota correlated negatively with HPV genotypes 16 and 51 (p≤0.05), but positively with HPV genotype 59 (p≤0.05), just like Actinomycetota (p≤0.05). Bacteroidota correlated positively with HPV genotype 56 (0.001<p<0.01), and Ascomycota correlated positively with HPV genotypes 39 and 51 (p≤0.05; 0.001<p<0.01). Pearson correlation coefficients between bacteria genera and HPV genotypes were statistically significant for the following genera: Lactobacillus, Streptococcus, Enterococcus, Gardnerella, Escherichia, Prevotella. The data obtained in our study indicates that the culture-based method is informative when assessing the qualitative and quantitative composition of the microbiota.

Further screening of SNP loci of eggshell translucency related genes and evaluation of genetic effects

Eggshell translucency is a widespread issue in the field of egg quality. Previous research has established that the heritability of eggshell translucency is relatively low or moderate. Scientists have also successfully identified SNP loci related to eggshell translucency on different chromosomes by using gene chips and single-variant GWAS. However, the specific impact of single or multiple genes on the trait of eggshell translucency remains unknown. In an effort to investigate this, we examined 170 SNPs associated with eggshell translucency obtained by our research group. We selected 966 half-sibling laying hens from 2 generations in 3 pure lines: Dwarf Layer-White, Rhode Island Red-White Strain, and Rhode Island Red. Eggs were collected from each hen over a period of 5 consecutive days, and eggshell translucency was measured using a grading method in which the hens were divided into 2 groups: an opaque group and a translucent group. We collected blood samples from the laying hens and extracted DNA. Time of flight mass spectrometry (TOF-MS) was used for genotyping to identify SNP loci that influence the trait of eggshell translucency. The results of our analysis revealed that using TOF-MS in 3 chicken strains, we were able to eliminate loci with low gene polymorphism, genetic effect contribution less than 1%, and deviation from Hardy-Weinberg equilibrium. Ultimately, 5 SNPs (Affx-50362599, rs15050262, rs312943734, rs316121113, and rs317389181) were identified on chromosomes 1, 5, and 19. Additionally, nine candidate genes (DCN, BTG1, ZFP92, POU2F1, NUCB2, FTL, GGNBP2, ACACA, and TADA2A) were found to be associated with these SNPs. No linkage disequilibrium relationship was observed between the 2 pairs of SNP loci on chromosomes 1 and 19. Based on previous studies on the formation mechanism of eggshell translucency, we hypothesize that NUCB2, FTL, and ACACA genes may be affecting the eggshell structure through different mechanisms, such as increase the water permeability or make thin of eggshell membrane, which promote moisture or part of other egg contents and ultimately lead to the formation of eggshell translucency. These findings validate and identify five SNP loci that regulate the translucency trait, and provide molecular markers for breeding non-translucent populations. Furthermore, this study serves as a reference for further investigation of the genetic regulatory mechanisms underlying eggshell translucency.

Efficient treatment of secondary treated refinery wastewater using sand biofiltration: Removal of hazardous organic pollutants

This study explored the potential of sand biofiltration for tertiary treatment of real refinery wastewater. The biofilter (2 cm (I.D.) x 15 cm (L)) operated on secondary treated refinery wastewater at flow rate of 1 mL/min had empty bed contact time (EBCT) of 47.12 min for one circulation. Maximum reduction in COD after 4, 8 and 12 times recirculation was 25 %, 52 % and 56 %; while the TOC reduction was 33 %, 43 % and 51 %, respectively, after biofilm development over 30 days. Quantification using two dimensional gas chromatography – time of flight mass spectrometry (GCxGC-TOF MS) revealed that several of the identified target compounds could not be detected in the wastewater after 12 recirculations. After 8 times recirculation, most of the compounds showed very high removal efficiency. For biofiltration over the flow rate range 2–10 mL/min, the reduction in COD and NH4+-N ranged from 62–73 % and 78–86 %, respectively, after 8 times recirculation. The nitrite concentration first increased and subsequently decreased, while the nitrate concentration continuously increased with increase in the number of recirculations. Solid phase micro-extraction (SPME) analysis of the aqueous phase using GCxGC-TOF MS and a semi-quantitative approach indicated that the removal of predominant classes of compounds was greater than 95 % after 8 times recirculation, with maximum reduction occurring in the first pass through the biofilter. Assimilable organic carbon (AOC) reduction was 98 % after 8 times recirculation. Metagenomic analysis revealed that Proteobacteria was the most dominant phylum in the biofilter. Many known polynuclear aromatic hydrocarbon (PAH) degraders, such as Sphingomonadales, Burkholderiales, Rhodobacterales and Rhodospirillales, were found in the biofilter leading to high removal efficiency of hazardous organic pollutants.

Metal binding feature of copper‒induced metallothionein from freshwater crab Sinopotamon henanense reveals its Cu‒thionein character

Sinopotamon Henanense expresses two metal‒induced metallothioneins (MTs), Cd‒induced MT and Cu‒induced MT (ShCuMT). The Cd‒induced MT has been characterized as a Cd‒thiolate MT. However, it is unknown whether ShCuMT is a Cu‒thiolate MT. In the present study, ShCuMT was expressed heterologously in Escherichia coli and purified by Ni‒NTA column and superdex‒75 column. And its metal‒binding feature was evaluated by DTNB reaction, circular dichroism spectroscopy (CD), isothermal microtitration (ITC), electrospray flight mass spectrometry (ESI‒TOF‒MS), and matrix‒assisted laser desorption ionization flight mass spectrometry (MALDI‒TOF‒MS). Bioinformatics analysis demonstrated that ShCuMT possessed the cysteine‒triplet motif of a Cu‒specific MT. Expression and purification of ShCuMT illustrated that SUMO tag used as the production system for ShCuMT resulted in a high production yield. The stability order of ShCuMT binding metal ions were Cu (Ⅰ) > Cd (Ⅱ) > Zn (Ⅱ). The CD spectrum indicated that ShCuMT binding with Cu (I) exhibited a compact thiol metal clusters structure. Besides, there emerged no a visible nickel‒thiol absorption after Ni‒NTA column affinity chromatography. The ITC results implied that Cu‒ShCuMT possessed the optimal thermodynamic conformation and the highest stoichiometric number of Cu (Ⅰ). Overall, the results suggested that SUMO fusion system is a robust and inexpensive approach for ShCuMT expression and Ni‒NTA column had no influence on metal binding of ShCuMT and Cu(Ⅰ) was considered its cognate metal ion, and ShCuMT possessed canonical Cu‒thiolate characteristics. The metal binding feature of ShCuMT reported here contributes to elucidating the structure‒function relationship of ShCuMT in S. Henanense.

Exploring molecular composition of upgraded pyrolysis bio-oil using GC×GC-(EI/PI)-TOF MS with different column set-ups

Bio-oils produced from the pyrolysis of lignocellulosic biomass are rich in oxygen, which causes instability and corrosion problems. Therefore, dedicated post-pyrolysis treatments such as hydrotreatment are required to increase the H/C ratio of the products to be used as fuel or chemicals. Here, a characterization method based on two-dimensional gas chromatography (GC×GC) was developed to highlight the evolution of the volatile fraction during hydrotreatment. Six samples obtained at different times of the hydrotreatment were analyzed. In this way, information on catalyst deactivation was obtained. The combination of information from normal and reversed phase GC×GC analysis provides a detailed characterization of the hydrotreated bio-oils. Furthermore, the use of soft photoionization (PI), in addition to conventional electron impact ionization (EI), yields a better understanding of the compound structures present in the sample. Identification and semi-quantification of the sample components indicate that the concentration of paraffins, cycloalkanes, and monoaromatics have largely decreased during hydrotreatment, while the concentration of oxygenated species and polycyclic aromatic hydrocarbons have increased. Using complementary GC methods highlights changes in the molecular composition of volatile species that correlate with catalyst performance. This approach can be used to follow the decrease in hydrodeoxygenation activity as a function of time on stream to estimate the catalyst aging during the hydrotreating process.

Expression and characterization of a novel microbial GH9 glucanase, IDSGLUC9-4, isolated from sheep rumen.

This study aimed to identify and characterize a novel endo-β-glucanase, IDSGLUC9-4, from the rumen metatranscriptome of Hu sheep. A novel endo-β-glucanase, IDSGLUC9-4, was heterologously expressed in Escherichia coli and biochemically characterized. The optimal temperature and pH of recombinant IDSGLUC9-4 were determined. Subsequently, substrate specificity of the enzyme was assessed using mixed-linked glucans including barley β-glucan and Icelandic moss lichenan. Thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) analyses were conducted to determine the products released from polysaccharides and cello-oligosaccharides substrates. The recombinant IDSGLUC9-4 exhibited temperature and pH optima of 40 °C and pH 6.0, respectively. It exclusively hydrolyzed mixed-linked glucans, with significant activity observed for barley β-glucan (109.59 ± 3.61 μmol·mg-1·min-1) and Icelandic moss lichenan (35.35 ± 1.55 μmol·mg-1·min-1). TLC and HPLC analyses revealed that IDSGLUC9-4 primarily released cellobiose, cellotriose, and cellotetraose from polysaccharide substrates. Furthermore, after 48 h of reaction, IDSGLUC9-4 removed most of the glucose, indicating transglycosylation activity alongside its endo-glucanase activity. The recombinant IDSGLUC9-4 was a relatively acid-resistant, mesophilic endo-glucanase (EC 3.2.1.4) that hydrolyzed glucan-like substrates, generating predominantly G3 and G4 oligosaccharides, and which appeared to have glycosylation activity. These findings provided insights into the substrate specificity and product profiles of rumen-derived GH9 glucanases and contributed to the expanding knowledge of cellulolytic enzymes and novel herbivore rumen enzymes in general.

Urine as Biobased Fertilizer: The Netherlands as Case Study

Humankind has grown ever more dependent on mineral fertilizer to feed a growing world population. However, nutrients are rarely recovered and recycled after consumption, leading to environmental waste issues now and potential supply issues in the future. Urine is rich in these nutrients and therefore a prime candidate as precursor for biobased fertilizers. This work presents a case study to remove contaminants – predominantly pharmaceuticals – from human urine using activated carbon adsorbents in an effort to adhere to Dutch fertilizer legislation. In the urine, three pharmaceuticals were identified by target analysis and 11 other contaminants by suspect screening using liquid chromatography quadrupole time of flight mass spectrometry (LC-QTOF/MS). These were successfully removed by treatments with both granulated and powdered activated carbon, albeit with a loss of 16-17% and 2-4% of dissolved nutrients, respectively. Further screening of inorganic contaminants and persistent organic pollutants by inductively coupled plasma mass spectrometry (ICP-MS) and gas chromatography quadrupole-orbitrap mass spectrometry (GC-Q-Orbitrap/MS) showed that all prerequisites for fertilizer status are met, paving the way for its future legal use as biobased fertilizer in the Netherlands.

Exploring the mechanism of diabetic cardiomyopathy treated with Qigui Qiangxin mixture based on UPLC-Q/TOF-MS, network pharmacology and experimental validation.

Despite its effectiveness in treating diabetic cardiomyopathy (DCM), Qigui Qiangxin Mixture (QGQXM) remains unclear in terms of its active ingredients and specific mechanism of action. The purpose of this study was to explore the active ingredients and mechanism of action of QGQXM in the treatment of DCM through the comprehensive strategy of serum pharmacology, network pharmacology and combined with experimental validation. The active ingredients of QGQXM were analyzed using Ultra-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC-Q/TOF-MS). Network pharmacology was utilized to elucidate the mechanism of action of QGQXM for the treatment of DCM. Finally, in vivo validation was performed by intraperitoneal injection of STZ combined with high-fat feeding-induced DCM rat model. A total of 25 active compounds were identified in the drug-containing serum of rats, corresponding to 121 DCM-associated targets. GAPDH, TNF, AKT1, PPARG, EGFR, CASP3, and HIF1 were considered as the core therapeutic targets. Enrichment analysis showed that QGQXM mainly treats DCM by regulating PI3K-AKT, MAPK, mTOR, Insulin, Insulin resistance, and Apoptosis signaling pathways. Animal experiments showed that QGQXM improved cardiac function, attenuated the degree of cardiomyocyte injury and fibrosis, and inhibited apoptosis in DCM rats. Meanwhile, QGQXM also activated the PI3K/AKT signaling pathway, up-regulated Bcl-2, and down-regulated Caspase9, which may be an intrinsic mechanism for its anti-apoptotic effect. This study preliminarily elucidated the mechanism of QGQXM in the treatment of DCM and provided candidate compounds for the development of new drugs for DCM.