Immunomagnetic Separation Research Articles

FEN1 upregulation mediated by SUMO2 via antagonizing proteasomal degradation promotes hepatocellular carcinoma stemness

PurposeMetastasis of hepatocellular carcinoma (HCC) critically impacts the survival prognosis of patients, with the pivotal role of hepatocellular carcinoma stem cells in initiating invasive metastatic behaviors. The Flap Endonuclease 1 (FEN1) is delineated as a metallonuclease, quintessential for myriad cellular processes including DNA replication, DNA synthesis, DNA damage rectification, Okazaki fragment maturation, baseexcision repair, and the preservation of genomic stability. Furthermore, it has been recognized as an oncogene in a diverse range of malignancies. Our antecedent research has highlighted a pronounced overexpression of protein FEN1 in hepatocellular carcinoma, where it amplifies the invasiveness and metastatic potential of liver cancer cells. However, its precise role in liver cancer stem cells (LCSCs) remains an enigma and requires further investigation. MethodsTo rigorously evaluate the stemness attributes of LCSCs, we employed sphere formation assays and flow cytometric evaluations. Both CD133+ and CD133- cell populations were discerningly isolated utilizing immunomagnetic bead separation techniques. The expression levels of pertinent genes were assayed via real-time quantitative PCR (RT-qPCR) and western blot analyses, while the expression profiles in hepatocellular carcinoma tissues were gauged using immunohistochemistry. Subsequent immunoprecipitation, in conjunction with mass spectrometry, ascertained the concurrent binding of proteins FEN1 and Small ubiquitin-related modifier 2 (SUMO2) in HCC cells. Lastly, the impact of SUMO2 on proteasomal degradation pathway of FEN1 was validated by supplementing MG132. ResultsOur empirical findings substantiate that protein FEN1 is profusely expressed in spheroids and CD133+ cells. In vitro investigations demonstrate that the upregulation of protein FEN1 unequivocally augments the stemness of LCSCs. In a congruent in vivo context, elevation of FEN1 noticeably enhances the tumorigenic potential of LCSCs. Conversely, inhibiting protein FEN1 resulted in a marked reduction in LCSC stemness. From a mechanistic perspective, there exists a salient positive correlation between the protein expression of FEN1 and SUMO2 in liver cancer tissues. Furthermore, the level of SUMO2-mediated modification of FEN1 is pronouncedly elevated in LCSCs. Interestingly, SUMO2 has the ability to bind to FEN1, leading to a inhibition in the proteasomal degradation pathway of FEN1 and an enhancement in its protein expression. However, it is noteworthy that this interaction does not affect the mRNA level of FEN1. ConclusionIn summation, our research elucidates that protein FEN1 is an effector in augmenting the stemness of LCSCs. Consequently, strategic attenuation of protein FEN1 might proffer a pioneering approach for the efficacious elimination of LCSCs.

Isolation of Shiga Toxin-Producing Escherichia coli from the Surfaces of Beef Carcasses in Slaughterhouses in Japan

Shiga toxin-producing E. coli (STEC) is an important foodborne pathogen worldwide. It is necessary to control and prevent STEC contamination on beef carcasses in slaughterhouses because STEC infection is associated with beef consumption. However, the frequencies of STEC contamination of beef carcasses in various slaughterhouses in Japan are not well known. Herein, we investigated the contamination of beef carcasses with STEC in slaughterhouses to assess the potential risks of STEC. In total, 524 gauze samples were collected from the surfaces of beef carcasses at 12 domestic slaughterhouses from November 2020 to February 2023. The samples were measured for aerobic plate counts and tested for pathogenic genes (stx and eae) and major O-serogroups (O26, O45, O103, O111, O121, O145, and O157) by real-time PCR screening. Subsequently, immunomagnetic separation (IMS) was performed on samples positive for stx, eae, and at least one of the seven O-serogroups of STEC. Isolation process without IMS was performed on samples positive for stx, including those subjected to IMS. STEC O157:H7 and stx-positive E. coli other than serotype O157:H7 were isolated from 0.6% and 4.6% of beef carcass surfaces, respectively. Although the STEC O157:H7 isolation rate was low and stx-positive E. coli other than serotype O157:H7 belonged to minor O-serogroups, the results mean a risk of foodborne illness. Furthermore, a moderate correlation was observed between aerobic plate counts and detection rates of stx-positive samples by real-time PCR screening. The STEC O157:H7 isolated facilities showed higher values on aerobic plate counts and detection rates of stx-positive samples than the mean values of total samples. Therefore, these results suggest that it is important to evaluate hygiene treatments against beef carcasses for the reduction of STEC contamination risk, particularly in facilities with high aerobic plate counts.

Virulence and Antimicrobial Resistance Profiles of Shiga Toxin-Producing Escherichia coli from River Water and Farm Animal Feces near an Agricultural Region in Northwestern Mexico

Shiga toxin-producing Escherichia coli (STEC) are zoonotic enteric pathogens linked to human gastroenteritis worldwide. To aid the development of pathogen control efforts, the present study characterized the genotypic diversity and pathogenic potential of STEC recovered from sources near agricultural fields in Northwest Mexico. Samples were collected from irrigation river water and domestic animal feces in farms proximal to agricultural fields and were subjected to enrichment followed by immunomagnetic separation and plating on selective media for the recovery of the STEC isolates. Comparative genomic analyses indicated that the recovered STEC with the clinically relevant serotypes O157:H7, O8:H19, and O113:H21 had virulence genes repertoires associated with host cell adherence, iron uptake and effector protein secretion. Subsequent phenotypic characterization revealed multidrug resistance against aminoglycoside, carbapenem, cephalosporin, fluoroquinolone, penicillin, phenicol, and tetracycline, highlighting the need for improved surveillance on the use of antimicrobials. The present study indicated for the first time that river water in the agricultural Culiacan Valley in Mexico is a relevant key route of transmission for STEC O157 and non-O157 with a virulence potential. In addition, feces from domestic farm animals near surface waterways can act as potential point sources of contamination and transport of diverse STEC with clinically relevant genotypes.

Exploring the Effects of Cyclosporin A to Isocyclosporin A Rearrangement on Ion Mobility Separation.

Cyclosporin A (CycA) is a peptide secondary metabolite derived from fungi that plays a crucial role in transplantation surgery. Cyclic traveling wave ion mobility mass spectrometry (IM-MS) revealed an N → O peptidyl shift in singly protonated CycA to isocyclosporin A (isoA), whereas no such isomerization was observed for doubly protonated and sodiated molecules. CycA and isoA were able to be separated by considering doubly protonated precursors using a specific ion fragment. In parallel, sodium ion stabilization facilitated the simultaneous separation and quantitation of singly charged cyclosporin isomers with the limit of detection and coefficient of determination of 1.3% and 0.9908 for CycA in isoA and 1.0% and 0.9830 for isoA in CycA, respectively. Finally, 1H-13C gHSQC NMR experiments permitted parallel recording of up to 11 cyclosporin conformers. The ratios were determined by integrating the volume of cross-peaks of the upfield resonating hydrogen in the diastereotopic methylene group of sarcosine-3.

Cytokine secretion in stem cells of cattle infected with bovine leukaemia virus.

Bovine leukaemia virus (BLV) is a Deltaretrovirus responsible for enzootic bovine leukosis, the most common neoplastic disease of cattle. It deregulates the immune system, favouring secondary infections and changes in the blood and lymphatic tissues. Blood homeostasis depends on functional haematopoietic stem cells (HSCs). Bone marrow is populated by these cells, which express CD34+ and CD35+ surface antigens and produce and release cytokines involved in the maintenance of haematopoiesis. The aim of the study was determination of the profile of cytokine production by CD34+ stem cells of cattle naturally infected with BLV. The HSCs were generated from the blood and lymphoid organs of cows infected with BLV and healthy control cows with immunomagnetic separation and anti-CD34+ monoclonal antibodies. Isolated CD34+ cells were cultivated for two weeks with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor. The levels of IL-6, IL-10, IL-12p40, IL-12p70, interferon gamma (IFN-γ) and tumour necrosis factor alpha (TNF-α) were determined in culture fluid by flow cytometry. The expression of IL-6, IL-12p70 and TNF-α in blood HSCs was higher in BLV+ cows than in the control animals. In bone marrow HSCs of infected cows, IL-12, TNF-α and IFN-γ were more concentrated, but in these cows' spleen HSCs only expression of IL-10 was elevated. In HSCs isolated from the lymph nodes of leukaemic cows, only TNF-α secretion was lower than in control cows, the other cytokines being more potently secreted. Infection with BLV caused statistically significant differences in cytokine expression by HSC CD34+ cells.

Insight into JNK Inhibition-based Strategy for the Treatment of Alzheimer's Disease

Background: Existing drugs for the treatment of Alzheimer's disease do not meet the basic requirements. Therefore, it is necessary to search for fundamentally new targets for the discovery of drugs for Alzheimer's disease (AD). When creating approaches to stimulate neurogenesis, such a search is promising to conduct as part of the development of a strategy for targeted regulation of intracellular signal transduction in regeneration-competent cells (RCCs). Moreover, the participation of c-Jun Nterminal kinases (JNK) in the regulation of the functions of the nervous tissue and neuroglial cells is known. Objective: The aim of the work was to study the effect of the JNK inhibitor (IQ-1S) on exploratory behavior and cognitive functions in aged mice (16-month-old male C57BL/6 mice as a model of AD) in combination with the dynamics of shifts in the RCCs functioning (nervous tissue progenitors: neural stem cells (NSCs) and neuronal-committed progenitors (NCPs); neuroglial cells: astrocytes, oligodendrocytes, microglial cells). Methods: The studies were carried out on male C57BL/6 mice aged 16 months as model of Alzheimer's disease. We studied the effect of the JNK inhibitor on exploratory behavior, conditioned reflex activity, and on the functioning of RCCs in the subventricular zone of the cerebral hemispheres (SVZ). NCPs and neuroglial cells of different types were obtained by immunomagnetic separation. Results: We observed significant changes in exploratory behavior and impaired conditioned reflex activity in aged C57BL/6 mice. The administration of the JNK inhibitor led to a significant correction of age-related behavioral and memory disorders in aged mice. At the same time, against the background of JNK inhibition-based therapy, an increase in mitotic activity and the content of both NSCs and NCPs in the SVZ was noted. However, these shifts were more pronounced in committed precursors. The phenomenon of the inhibition of NSC specialization under the influence of a pharmacological agent was also revealed. In addition, the JNK inhibitor caused an increase in the secretion of neurotrophic growth factors by oligodendrocytes and microglial cells. Conclusion: The findings open up prospects for the development of JNK targeting-based approaches for the treatment of AD.

Prevalence, Virulence Profiles, and Genetic Relatedness of Escherichia coli O45 from Raw meats, Southern Thailand

Non-O157 Enterohaemorrhagic Escherichia coli (EHEC) has become a major public health concern all around the globe. This study investigated the E. coli serogroup O45 from raw meats in southern Thailand using the immunomagnetic separation approach (IMS). Forty-nine E. coli O45 strains were obtained from 13 positive meat samples (13/105), showing the prevalence of O45 contamination as 12.4%. They were not members of EHEC. Exploration of other virulence genes exhibited that fimH, responsible for bacterial adherence, was found in all strains, while astA encoding for EAST-1 toxin was 63.2% of the strains. Moreover, lpf encoding long polar fimbriae was found as 30.6%. Phylogenetic group analysis revealed that the majority of E. coli O45 belonged to group D (88%), followed by group A (8%) and group B1 (4%). However, none belonged to group B2. The intactness of stx2-phage integration sites showed that sbcB was occupied by prophages at the highest rate, followed by the Z2577 site. Antimicrobial susceptibility assay demonstrated relatively high bacterial resistance to cephalothin (78%), streptomycin (51%), cotrimoxazole (39%), tetracycline (31%), and chloramphenicol (23%). Furthermore, multi-drug resistant ability was uncovered in O45 strains at 49%. DNA profiling of E. coli O45 by BOX-PCR analyzed at 80% genetic similarity revealed 5 distinguishable clusters. More importantly, the strains from different samples and time intervals showed identical DNA fingerprints, suggesting that they may have originated from the same bacterial clone.

Magnetic capture device for large volume sample analysis.

Immunomagnetic separation (IMS) techniques employing superparamagnetic particles can successfully isolate various components from mixtures. However, their utility can be limited for large-volume samples, viscous samples, or those containing a high density of particulate matter because of the need to generate high field gradients for particle recovery. Therefore, a new class of immunomagnetic particles was devised utilizing a single, macroscopic Pyrex spinbar conjugated with biorecognition elements to address these limitations. Advantages include an inherent capacity for effective mixing, an almost instantaneous recovery of the spinbar that can be performed without expensive equipment and with no loss of magnetic particles during processing, and reduced transfer of sample matrix. As a result, spinbars can provide an effective means for IMS with large-volume assays composed of complex matrices.

Cyclable Variable Path Length Multilevel Structures for Lossless Ion Manipulations (SLIM) Platform for Enhanced Ion Mobility Separations.

Ion mobility-mass spectrometry (IMS-MS) is used to analyze complex samples and provide structural information on unknown compounds. As the complexity of samples increases, there is a need to improve the resolution of IMS-MS instruments to increase the rate of molecular identification. This work evaluated a cyclable and variable path length (and hence resolving power) multilevel Structures for Lossless Ion Manipulations (SLIM) platform to achieve a higher resolving power than what was previously possible. This new multilevel SLIM platform has eight separation levels connected by ion escalators, yielding a total path length of ∼88 m (∼11 m per level). Our new multilevel SLIM can also be operated in an "ion cycling" mode by utilizing a set of return ion escalators that transport ions from the eighth level back to the first, allowing even extendable path lengths (and higher IMS resolution). The platform has been improved to enhance ion transmission and IMS separation quality by reducing the spacing between SLIM boards. The board thickness was reduced to minimize the ions' escalator residence time. Compared to the previous generation, the new multilevel SLIM demonstrated better transmission for a set of phosphazene ions, especially for the low-mobility ions. For example, the transmission of m/z 2834 ions was improved by a factor of ∼3 in the new multilevel SLIM. The new multilevel SLIM achieved 49% better resolving powers for GRGDS1+ ions in 4 levels than our previous 4-level SLIM. The collision cross-section-based resolving power of the SLIM platform was tested using a pair of reverse sequence peptides (SDGRG1+, GRGDS1+). We achieved 1100 resolving power using 88 m of path length (i.e., 8 levels) and 1400 following an additional pass through the eight levels. Further evaluation of the multilevel SLIM demonstrated enhanced separation for positively and negatively charged brain total lipid extract samples. The new multilevel SLIM enables a tunable high resolving power for a wide range of ion mobilities and improved transmission for low-mobility ions.

Three-Minute Enantioselective Amino Acid Analysis by Ultra-High-Performance Liquid Chromatography Drift Tube Ion Mobility-Mass Spectrometry Using a Chiral Core-Shell Tandem Column Approach.

Fast liquid chromatography (LC) amino acid enantiomer separation of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives using a chiral core-shell particle tandem column with weak anion exchange and zwitterionic-type quinine carbamate selectors in less than 3 min was achieved. Enantiomers of all AQC-derivatized proteinogenic amino acids and some isomeric ones (24 in total plus achiral glycine) were baseline separated (Rs > 1.5 except for glutamic acid with Rs = 1.3), while peaks of distinct amino acids and structural isomers (constitutional isomers and diastereomers of leucine and threonine) of the same configuration overlapped to various degrees. For this reason, drift tube ion mobility-mass spectrometry was added (i.e., LC-IM-MS) as an additional selectivity filter without extending run time. The IM separation dimension in combination with high-resolution demultiplexing enabled confirmation of threonine isomers (threonine, allo-threonine, homoserine), while leucine, isoleucine, and allo-isoleucine have almost identical collisional cross-section (DTCCSN2) values and added no selectivity to the partial LC separation. Density functional theory (DFT) calculations show that IM separation of threonine isomers was possible due to conformational stabilization by hydrogen bond formation between the hydroxyl side chain and the urea group. Generally, the CCSN2 of protonated ions increased uniformly with addition of the AQC label, while outliers could be explained by consideration of intramolecular interactions and additional structural analysis. Preliminary validation of the enantioselective LC-IM-MS method for quantitative analysis showed compliance of accuracy and precision with common limits in bioanalytical methods, and applicability to a natural lipopeptide and a therapeutic synthetic peptide could be demonstrated.

Multiplatform High-Definition Ion Mobility Separations of the Largest Epimeric Peptides.

Ion mobility spectrometry (IMS) coupled to mass spectrometry (MS) has become a versatile tool to fractionate complex mixtures, distinguish structural isomers, and elucidate molecular geometries. Along with the whole MS field, IMS/MS advances to ever larger species. A topical proteomic problem is the discovery and characterization of d-amino acid-containing peptides (DAACPs) that are critical to neurotransmission and toxicology. Both linear IMS and FAIMS previously disentangled d/l epimers with up to ∼30 residues. In the first study using all three most powerful IMS methodologies─trapped IMS, cyclic IMS, and FAIMS─we demonstrate baseline resolution of the largest known d/l peptides (CHH from Homarus americanus with 72 residues) with a dynamic range up to 100. This expands FAIMS analyses of isomeric modified peptides, especially using hydrogen-rich buffers, to the ∼50-100 residue range of small proteins. The spectra for d and l are unprecedentedly strikingly similar except for a uniform shift of the separation parameter, indicating the conserved epimer-specific structural elements across multiple charge states and conformers. As the interepimer resolution tracks the average for smaller DAACPs, the IMS approaches could help search for yet larger DAACPs. The a priori method to calibrate cyclic (including multipass) IMS developed here may be broadly useful.

Real-time and visual detection of viable Salmonella in milk from remote pasture via IMS-LAMP-NALFS

Salmonella is a prevalent foodborne pathogen commonly found in dairy products. Controlling the presence of Salmonella during dairy product production is of utmost concern due to the potential for contamination at any stage of the production chain, particularly at the source. Moreover, the unique location of pastures and limited testing resources further highlight the necessity of establishing a rapid and precise detection method to ensure milk quality control and enhance overall farm safety. To address these issues, in this study, we constructed a method as well as a portable toolbox for rapid on-site detection of Salmonella. The method involves immunomagnetic separation (IMS) using streptavidin-coupled magnetic nanoparticles and biotinylated antibodies. The reaction conditions for magnetic capture and enrichment of Salmonella were optimized to efficiently remove matrix interference from milk and enhance detection sensitivity. Additionally, an improved propidium monoazide (PMAxx) treatment was employed to eliminate false positive results caused by dead bacteria. This treatment was combined with loop-mediated isothermal amplification (LAMP) and nucleic acid lateral flow strip (NALFS) (LAMP-NALFS) for on-site detection of viable bacteria in milk. The resulting IMS-LAMP-NALFA assay achieved rapid and sensitive detection of viable Salmonella, with a detection sensitivity of 1 × 102 CFU/mL in milk samples, completed within 90 min under optimal conditions. Based on this achievement, a portable toolbox was designed for easy transportation and on-site testing in pastures. It is equally applicable for rapid detection of viable Salmonella contamination in food products in resource-limited areas such as pastures, where the observed detection rate of Salmonella in unpasteurized raw milk was 7.93 % (28 out of 353 samples tested). Consequently, the IMS-LAMP-NALFA method and the associated portable toolbox represent highly effective means of enhancing the detection of viable Salmonella in dairy products.

Extending the Coverage of Lys-C/Trypsin-Based Bottom-up Proteomics by Cysteine S-Aminoethylation.

To improve the coverage in bottom-up proteomics, S-aminoethylation of cysteine residues (AE-Cys) was carried out with 2-bromoethylamine, followed by cleavage with lysyl endopeptidase (Lys-C) or Lys-C/trypsin. A model study with bovine serum albumin showed that the C-terminal side of AE-Cys was successfully cleaved by Lys-C. The frequency of side reactions at amino acids other than Cys was less than that in the case of carbamidomethylation of Cys with iodoacetamide. Proteomic analysis of A549 cell extracts in the data-dependent acquisition mode after AE-Cys modification afforded a greater number of identified protein groups, especially membrane proteins. In addition, label-free quantification of proteins in mouse nonsmall cell lung cancer (NSCLC) tissue in the data-independent acquisition mode after AE-Cys modification showed improved NSCLC pathway coverage and greater reproducibility. Furthermore, the AE-Cys method could identify an epidermal growth factor receptor peptide containing the T790 M mutation site, a well-established lung-cancer-related mutation site that has evaded conventional bottom-up methods. Finally, AE-Cys was found to fully mimic Lys in terms of collision-induced dissociation fragmentation, ion mobility separation, and cleavage by Lys-C/trypsin, except for sulfoxide formation during sample preparation.

Culture-Independent Quantification of Legionella pneumophila in Evaporative Cooling Systems Using Immunomagnetic Separation Coupled with Flow Cytometry

Legionella pneumophila are pathogenic bacteria that repeatedly occur in high concentrations in the process water of evaporative cooling systems (ECS). When released into the environment, the resulting bioaerosols can cause outbreaks with fatal consequences. The official, internationally accepted detection method for Legionella spp. in water samples is based on cultivation. However, cultivation is time-consuming and may underestimate the total count of viable L. pneumophila in ECS. Therefore, culture-independent methods are receiving attention for rapid monitoring. Cartridge-based immunomagnetic separation (IMS) coupled with flow cytometry (FCM) is an innovative, antibody-based method for the culture-independent quantification of L. pneumophila, using a panel of antibodies against serogroup (Sg) 1–15. We characterized the IMS-FCM method as a quantitative rapid test by general analytical procedures. Viable cryopreserved L. pneumophila standards were used in calibration experiments for the method. We achieved detection limits for Sg 1, Sg 4, and Sg 6 of 100, 105 and 88 viable cells per 100 mL, respectively. Furthermore, we demonstrated the practical applicability of IMS-FCM with real ECS samples and compared the performance against cultivation. Cultivation showed here no positive results, but IMS-FCM evidenced L. pneumophila in a range of 0–80,000 viable cells per 100 mL. This work demonstrates that IMS-FCM is a suitable, culture-independent, quantitative method for rapidly monitoring L. pneumophila.

Ion mobility-tandem mass spectrometry of bulky tert-butyl thiol ligated gold nanoparticles.

Gold nanoparticles (AuNPs) synthesized in the 1-3 nm range have a specific number of gold core atoms and outer protecting ligands. They have become one of the "hot topics" in recent decades because of their interesting physical and chemical properties. The characterization of their structures is usually achieved by crystal X-ray diffraction although the structures of some AuNPs remain unknown because they have not been successfully crystallized. An alternative method for studying the structure of AuNPs is electrospray ionization-ion mobility-tandem mass spectrometry (ESI-IM-MSMS). This research evaluated how effectively ESI-IM-MSMS using the commercially available Waters Synapt XS instrument yielded useful structural information from two AuNPs; Au23 (S-tBu)16 and Au30 (S-tBu)18 . The study used the maximum range of available collision energies along with ion mobility separation to measure the energy-dependence of the product ions and their drift times which is a measure of their spatial size. For Au23 (S-tBu)16 , the dissociation gave the masses of the outer protecting monomeric [RS-Au-SR] and trimeric [SR-Au-SR-Au-SR-Au-SR] staples where R = tBu, and complete dissociation of the outer layer Au and tBu groups to reveal the Au15 S8 core. For Au30 (S-tBu)18 , the dissociation products was primarily through the loss of the partial ligands S-tBu and tBu from the outer protecting layer and the loss of single Au4 (S-tBu)4 unit. These results showed the that ESI-IM-MSMS analysis of the smaller Au23 (S-tBu)16 gave information on all it major structural components whereas for Au30 (S-tBu)18 , the overall structural information was limited to the ligands of the outer layer.

Rapid and simultaneous multiepitope antigen-based detection of Enterococcus by microscale thermophoresis and immunomagnetic separation.

Generally, enterococci bacteria cause nosocomial infections and are major indicators of bacterial contamination in marine bathing beach. However, a method for the rapid and simultaneous detection of multiple pathogenic enterococci has not been developed on account of the wide variety of pathogenic enterococci and their existence in complex matrices. Immunoinformatics tools were used to design a multi-epitope antigen for the detection of various pathogenic enterococci by using the sequence of dltD gene on enterococci lipoteichoic acid (LTA) surface, which is associated with toxicological effects. The multi-epitopes included enterococci such as Enterococcus faecalis, E. gallinarum, E. raffinosus, E. durans, E. faecium, E. hirae, E. thailandicus, E. casseliflavus, E. avium, E. mundtii, E. lactis, E. solitarius, E. pseudoavium, and E. malodoratum. Microscale thermophoresis (MST) and western blot were carried out to detect the affinity between multi-epitope antigens and antibodies and between multi-epitope antibodies and bacteria. Furthermore, the detection of pathogenic enterococci was carried out by using immunomagnetic beads (IMBs) and immune chromatographic test strip (ICTS). The multi-epitope antibody had a satisfactory affinity to the antigen and enterococci. IMBs and ICTS were detected with a minimum of 101 CFU/mL and showed incompatibility for Vibrio parahemolyticus, V. vulnifcus, V. harveyi, V. anguillarum, and Edwardsiella tarda. The present study demonstrated that the multi-epitope antigens exhibited excellent specificity and sensitivity, making them highly suitable for efficient on-site screening of enterococci bacteria in marine bathing beaches.

Advances in Mass Spectrometry of Gangliosides Expressed in Brain Cancers.

Gangliosides are highly abundant in the human brain where they are involved in major biological events. In brain cancers, alterations of ganglioside pattern occur, some of which being correlated with neoplastic transformation, while others with tumor proliferation. Of all techniques, mass spectrometry (MS) has proven to be one of the most effective in gangliosidomics, due to its ability to characterize heterogeneous mixtures and discover species with biomarker value. This review highlights the most significant achievements of MS in the analysis of gangliosides in human brain cancers. The first part presents the latest state of MS development in the discovery of ganglioside markers in primary brain tumors, with a particular emphasis on the ion mobility separation (IMS) MS and its contribution to the elucidation of the gangliosidome associated with aggressive tumors. The second part is focused on MS of gangliosides in brain metastases, highlighting the ability of matrix-assisted laser desorption/ionization (MALDI)-MS, microfluidics-MS and tandem MS to decipher and structurally characterize species involved in the metastatic process. In the end, several conclusions and perspectives are presented, among which the need for development of reliable software and a user-friendly structural database as a search platform in brain tumor diagnostics.

Preliminary investigations of parasite contamination of water sources in Armenia

The intestinal protozoan parasites, Cryptosporidium and Giardia, are known to have a global distribution, infecting and causing disease in a range of hosts, including people, livestock, pets, and wildlife. However, data from some regions is very sparse. In Armenia, in the Caucasus region of West Asia, only scanty data are available, with just a few surveys on Cryptosporidium infections in livestock, and no available data on human infections or environmental contamination. As part of implementation of water analysis methods for these parasites in Armenia, 24 raw water samples and two sediment samples were analysed for these parasites using a range of approaches, including modified Ziehl-Neelsen, Lugol stain, immunofluorescent antibody test (IFAT), qPCR and, on sediment samples, immunomagnetic separation and IFAT. Results suggest substantial contamination of raw water sources and indicate the need for further targeted studies using appropriate methods and collecting data on host infections in catchment areas.

Improved biosensing of Legionella by integrating filtration and immunomagnetic separation of the bacteria retained in filters

A novel approach is presented that combines filtration and the direct immunomagnetic separation of the retained bacteria Legionella in filters, for further electrochemical immunosensing. This strategy allows for the separation and preconcentration of the water-borne pathogen from high-volume samples, up to 1000 mL. The limit of detection of the electrochemical immunosensor resulted in 100 CFU mL−1 and improved up to 0.1 CFU mL−1 when the preconcentration strategy was applied in 1 L of sample (103-fold improvement). Remarkably, the immunosensor achieves the limit of detection in less than 2.5 h and simplified the analytical procedure. This represents the lowest concentration reported to date for electrochemical immunosensing of Legionella cells without the need for pre-enrichment or DNA amplification. Furthermore, the study successfully demonstrates the extraction of bacteria retained on different filtering materials using immunomagnetic separation, highlighting the high efficiency of the magnetic particles to pull out the bacteria directly from solid materials. This promising feature expands the applicability of the method beyond water systems for detecting bacteria retained in air filters of air conditioning units by directly performing the immunomagnetic separation in the filters.Graphical abstract

Solution cathode glow discharge coupled to atmospheric pressure chemical ionization for elemental detection of S and P in organic compounds

We report a post-plasma chemical ionization approach to evaluate solution cathode glow discharge (SCGD) for S and P elemental analysis. Here, the SCGD serves as a reactor to produce chemical vapors for S and P from organic compounds containing these elements, while a corona discharge operated in negative mode is used to ionize the products. The approach creates long-lived ions in atmospheric pressure, enabling direct investigation of chemical vapor products via mass spectrometric and ion mobility separations. The investigations indicate that SCGD converts S and P to H2SO4 and H3PO4, respectively. These species are then ionized as HSO4HNO3− and H3PO4NO3HNO3− via reactions with NO3HNO3− produced by corona discharge. The response factors for P among several small molecules varies within 10% of the average response from the compounds, suggesting a reasonable species-independent characteristic. The response factors for S show larger variations among compounds, indicating a higher dependence of chemical vapor generation efficiency on analytes' chemical structures. Detection limits of 15 and 29 ng/mL are achieved for P and S detection, respectively. These figures are limited by background equivalent concentrations and low ion flux in the utilized ion mobility-time of flight mass spectrometer, indicating potential for significant improvements. In particular, the specificity of clustering for S and P-containing ions produced in this approach suggest facile analysis of S and P using quadrupole-based mass spectrometers for improved analytical performance.