Matrix-assisted Laser Desorption/ionization Time-of-flight Research Articles

Presence of the blaTEM Gene in Commensal Neisseria spp.: A Possible Cause for the Acquired Drug Resistance Among Pathogenic Respiratory Bacteria.

Background The oral microbiome consists of various bacterial genera, withNeisseriaspp. being a prominent part of this niche. WhileNeisseria gonorrhoeaeandNeisseriameningitidisare human-restricted pathogens, non-pathogenicNeisseriaspecies likeNeisseria sicca,Neisseria perflava, etc.,are primarily commensals that can also behave as opportunistic pathogens. With increasing penicillin resistance in commensalNeisseria, there is a concern that these bacteria might harbor resistance genes that can be transferred to other pathogens. This study aimed to characterize the blaTEM gene (encodes for the plasmid-mediated β-lactamase enzyme that hydrolyzes the β-lactam ring) of commensalNeisseriaspp. isolated from respiratory samples. Methodology The research was conducted in the Department of Clinical Microbiology at Sri Ramachandra University, Chennai. The specimens used were sputum and throat swabs, which were subjected to a series of phenotypic methods and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) for speciation. The antibiogram was determined using the Kirby-Bauer disk diffusion method, and a PCR assay was utilized to identify theblaTEMgene responsible for β-lactamase production. Results Out of 274 processed samples, 65 unique commensalNeisseriaspp. were identified. The study highlighted the presence of theblaTEMgene in 93.9% (61) of the isolates, which is responsible for β-lactamase production. All isolates exhibitedresistance to penicillin. MostblaTEM-positive commensalNeisseria spp. were susceptible to cefuroxime (83.6%), ceftriaxone (85.2%), and cefotaxime (85.2%). The high prevalence of theblaTEMgene in commensalNeisseriais alarming. The gene, found on plasmids, could potentially transfer to other related species likeNeisseria gonorrhoeaeandNeisseriameningitidis, as well as other Gram-negative bacilli. Conclusion The presence of resistance genes in commensal bacteria is of concern, as they might be reservoirs for resistance transfer to pathogenic strains. The study emphasizes the importance of continuous monitoring and deeper investigations into commensal bacteria, emphasizing the need for a broader community screening approach to understand resistance mechanisms in the normal microbiome.

Laccase-Catalyzed Synthesis of Added-Value Polymers from Cork and Grape Extracts.

The development of products from natural plant sources, including agriculture and food wastes, contributes significantly to the circular economy and global sustainability. Cork and grape wastes were employed as the primary sources in this study to obtain compounds of interest under mild extraction conditions. Laccase was applied to oxidize the cork and grape extracts, with the aim of producing value-added molecules with improved properties. Ultraviolet-visible (UV-vis) spectroscopy was assessed to monitor the oxidation process, and characterization of the end products was performed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) spectroscopy. The antioxidant and antiaging properties were evaluated by means of ABTS, DPPH, FRAP, and SPF testing. Overall, as compared to their monomeric counterparts, the polymeric compounds displayed remarkable antioxidant and antiaging characteristics after laccase oxidation, showing tremendous potential for applications in the food, pharmaceutical, cosmetic, and textile industries.

Antibacterial Susceptibility Pattern of S. maltophilia Isolates at A Tertiary Care Hospital, India

A sudden emergence of Stenotrophomonas maltophilia as a primary pathogen both in immunocompromised and immunocompetent individuals has raised a serious concern, as it is associated with significant case fatality ratio. We intended to study the clinico-microbiological profile of S. maltophilia isolates from various samples and outcome of the infections in a tertiary healthcare center, Pune, India. This is an observational cross-sectional study was conducted from January 2021 to June 2022 at Department of Microbiology of a tertiary care Centre in Pune, India. Of the 12049 samples received for culture, S. maltophilia was isolated in 57 samples. Only 42 samples with pure growth of S. maltophilia were included in the study with 15 excluded due to mixed growth. All isolates were confirmed by VITEK-MS (bioMerieux, SA, France) which uses Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) technology. Of the 42 isolates, majority were isolated from pus(28.6%) and most of patients (61.9%) were from acute health care settings. The isolates had high susceptibility to Cotrimoxazole (85.7%) and Minocycline (85.7%) and low susceptibility to Ceftazidime (45.2%). A case fatality rate of 7.1% (3/42 cases) was noted and 39 cases were discharged after complete treatment. All the three fatal cases were susceptible to levofloxacin, ciprofloxacin, cotrimoxazole and minocycline and all three fatal cases were resistant to ceftazidime. S. maltophilia has recently shown an increase in nosocomial infections especially in acute healthcare settings like ICU and other critical care wards. The isolates of the present study had high susceptibility to trimethoprim-sulfamethoxazole (TMP-SXT) and Minocycline and low susceptible to Ceftazidime.

High prevalence of Panton-Valentine Leucocidin among Staphylococcus coagulans isolated from dogs in Rio de Janeiro.

The purpose of this study was to characterize the capacity for biofilm formation, antimicrobial resistance rates, and search for genetic determinants of resistance and virulence in the species. Strains were collected from asymptomatic and infected dogs. Identification was conducted using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), antimicrobial susceptibility using disk diffusion and PCR targeting mecA. Biofilm formation was evaluated on a microtiter plate assay. A total of 27 strains were selected for whole-genome sequencing. We identified 111 Staphylococcus coagulans. The highest number was obtained from infected dogs. The highest resistance rates were observed for penicillin, gentamicin, and ciprofloxacin/erythromycin. Twelve strains were characterized as resistant to methicillin. All isolates had the ability to form biofilm and were strong producers. Among Methicillin Resistant Staphylococcus coagulans (MRSC), SCCmec types IIIA, and Vc were identified. Acquired resistance genes, such as aac(6')-aph(2''), tet(K), blaZ, qacG, qacJ, and erm(C) were found. Different virulence genes were identified. Of note, Panton-Valentine Leucocidin was highly prevalent among the isolates. Staphylococcus coagulans had a high isolation rate among infected dogs and demonstrated significant resistance to commonly used antibiotics such as penicillin and gentamicin.

Molecular and translational biology of the blood-based VeriStrat® proteomic test used in cancer immunotherapy treatment guidance

IntroductionThe VeriStrat® test (VS) is a blood-based assay that predicts a patient's response to therapy by analyzing eight features in a spectrum obtained from matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis of human serum and plasma. In a recent analysis of the INSIGHT clinical trial (NCT03289780), it was found that the VS labels, VS Good and VS Poor, can effectively predict the responsiveness of non-small cell lung cancer (NSCLC) patients to immune checkpoint inhibitor (ICI) therapy. However, while VS measures the intensities of spectral features using MALDI-TOF analysis, the specific proteoforms underlying these features have not been comprehensively identified. ObjectivesThe objective of this study was to identify the proteoforms that are measured by VS. MethodsTo resolve the features obtained from the low-resolution MALDI-TOF procedure used to acquire mass spectra for VS DeepMALDI® analysis of serum was employed. This technique allowed for the identification of finer peaks within these features. Additionally, a combination of reversed-phase fractionation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was then used to identify the proteoforms associated with these peaks. ResultsThe analysis revealed that the primary constituents of the spectrum measured by VS are serum amyloid A1, serum amyloid A2, serum amyloid A4, C-reactive protein, and beta-2 microglobulin. ConclusionProteoforms involved in host immunity were identified as significant components of these features. This newly acquired information improves our understanding of how VS can accurately predict patient response to therapy. It opens up additional studies that can expand our understanding even further.

Early diagnosis of monomicrobial Clostridioides difficile bacteremia in a patient without colitis.

Bacteremia is a rare finding among Clostridioides difficile infections. We describe a case of a 67-year-old man with resected colorectal cancer with colostomy who presented with small bowel obstruction and was admitted for lysis of adhesions. On day 8 of admission, he developed leukocytosis and raised inflammatory markers with isolation of Gram-positive bacilli in several blood cultures, which was presumptively identified through blood culture pelleting and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) as C. difficile. The diagnosis was confirmed with conventional culture and reference lab identification and the patient demonstrated clinical response with parenteral then oral vancomycin that briefly worsened when therapy was switched to parenteral metronidazole and then improved once oral vancomycin was resumed. Our case was notable in that the combination of pelleting and MALDI-TOF offered early diagnosis in this patient whose positive blood cultures were suspicious for contamination and in whom there was an absence of diarrheal illness or features of colitis on abdominal imaging. Early diagnosis is critical for the timely initiation of therapy, implementation of infection prevention and control measures and in selection of appropriate therapy for antimicrobial stewardship.

Identification of Vibrio metschnikovii and Vibrio injensis Isolated from Leachate Ponds: Characterization of Their Antibiotic Resistance and Virulence-Associated Genes.

This study aimed to evaluate the antibiotic resistance of 22 environmental Vibrio metschnikovii isolates and 1 Vibrio injensis isolate from landfill leachates in southwestern Colombia. Isolates were identified by Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight (MALDI-TOF), and 16S ribosomal RNA gene sequencing. Analysis of the susceptibility to six antibacterial agents by the Kirby-Bauer method showed susceptibility of all the isolates to ciprofloxacin and imipenem. We recorded resistance to beta-lactams and aminoglycosides, but no multidrug resistance was observed. The genome of one of the isolates was sequenced to determine the pathogenic potential of V. injensis. Genes associated with virulence were identified, including for flagellar synthesis, biofilm formation, and hemolysins, among others. These results demonstrate that landfill leachates are potential reservoirs of antibiotic-resistant and pathogenic bacteria and highlight the importance of monitoring Vibrio species in different aquatic environments.

Production and characterization of a highly stable laccase from extreme thermophile Geobacillus stearothermophilus MB600 isolated from hot spring of Gilgit Balitistan (Pakistan)

Extracellular thermophilic multicopper oxidase i.e. laccase was purified, identified and characterized from Geobacillus stearothermophilus MB600. Fast protein liquid chromatography (FPLC) of lacasse showed a prominent peak at 610 nm. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for laccase presented a molecular weight of 59.13 kDa. Analysis via Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) confirmed the presence of laccase similar to in Geobacillus thermoleovorans B23 and Geobacillus sp. LEMMY01. Laccase showed Km 0.532 mM for guaiacol and 0.307 mM for ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)). Its optimum activity was witnessed at 90°C, at pH 5.0. Laccase catalytic activity was completely inhibited by sodium dodecyl-sulfate (SDS), sodium azide (NaN3), and ethylenediaminetetraacetic acid (EDTA). Among the metal salts against Cu, Ca, Cd, and Li enhanced while Mg inhibited the enzyme activity. It was stable against Cl, I and solvents (Ethanol > Methanol > Dimethyl sulfoxide > Acetonitrile). Thermophilic laccase effectively degraded 93% of Remazol brilliant blue R, 92% Bisphenol A diglycidyle ether without mediator.

Bacillus dicomae sp. nov., a new member of the Bacillus cereus group isolated from medicinal plant Dicoma anomala.

A Gram-stain-positive, endospore-forming endophytic bacterial strain designated MHSD28T was isolated from surface-sterilized leaves of Dicoma anomala collected from Eisleben, Botlokwa, Limpopo Province, South Africa. The phenotypic and phylogenetic characteristics of strain MHSD28T were consistent with those of members within the Bacillus cereus group. Comparative analysis between this strain and its relatives confirmed that it belongs to this group and forms a monophyletic branch. The digital DNA-DNA hybridization values between strain MHSD28T and its relatives were lower than the 70 % threshold for species delineation. To further determine its phylogenetic position, multi-locus sequence analysis (MLSA) based on five concatenated housekeeping gene (gyrB, atpD, DnaK, rpoB and rpoD) sequences, phenotypic analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) biotyper identification, fatty acid and polar lipid profile analyses were carried out. Phenotypic characterization, MLSA, whole genome sequence based analyses and MALDI-TOF results placed strain MHSD28T within the B. cereus group. The major fatty acids were iso-C15 : 0 and summed feature 3 and the main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The respiratory quinone was menaquinone-7. The cell-wall peptidoglycan structure included meso-diaminopimelic acid. Considering the above results, strain MHSD28T represents a novel species of the B. cereus group, for which the name Bacillus dicomae sp. nov. is proposed. The type strain is MHSD28T (=BD 2262T=LMG 32287T=CECT 30671T).

Evaluation of RapidChek® SELECT™ for the detection of Salmonella spp. in environmental samples from a veterinary hospital

Nosocomial salmonellosis in hospitalized animals is a recognized hazard, especially in large animal clinics. A standardized culture protocol (SCP) for detecting Salmonella spp. in environmental samples using a 48-h enrichment step results in a 5-day turnaround time for negative results. The RapidChek® SELECT™ Salmonella (RCSS) test system offers detection of organisms in 22–44 h through double enrichment followed by a lateral flow immunoassay. Negative results are reported within 48 h. To determine the most sensitive and rapid method for detecting Salmonella spp. from environmental samples collected at the large animal Purdue Veterinary Hospital (LA-PVH), a preliminary study compared the performance of RCSS and a SCP when testing artificially spiked and naturally contaminated samples. An expanded study analyzed results obtained using the RCSS method to test 872 environmental samples over a 12-month period. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry was chosen as the confirmation method for RCSS-presumptive positive samples. A randomly selected subset of samples received additional confirmation by real-time PCR. Here, we reported the performance data of RCSS in terms of sensitivity, specificity, and positive predictive value using MALDI-TOF results as reference for comparison. We also provide guidelines for reporting results obtained using this system.

OR19-06 State-of-the-art MALDI Mass Spectrometry Imaging Reveals Unique Spatial Lipid Signatures in Human and Mouse Ovarian Compartments

Abstract Disclosure: N. Pascuali: None. F. Tobias: None. K. Valyi-Nagy: None. S. Salih: None. A. Veiga-Lopez: None. The incidence of infertility has been consistently increasing over the past few decades, with a high proportion of these cases caused by ovarian-related dysfunction. Metabolic disorders such as hyper- or dyslipidemia have been associated with subfertility (e.g., follicular fluid dyslipidemia is associated with lower IVF success). Lipids play a critical role in the structure and function of ovarian tissue. However, while several studies have investigated transcript- and protein-level alterations in ovarian pathologies, less is known about alterations in the lipid landscape. The first step in determining if ovarian disorders are associated with ovarian dyslipidemia - and thus use lipids as potential biomarkers of ovarian pathology - is to understand the physiological distribution of ovarian lipids. Matrix-Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry imaging (MSI) enables label-free in situ analysis of molecules such as lipids and metabolites directly within tissue sections. We aimed to begin mapping the distribution of lipids in human and mouse ovaries using high spatial resolution MALDI MSI. To this end, and after informed consent, two fresh ovarian biopsies from a 26-year-old patient were frozen, cryosectioned into 10-μm sections, and mounted onto ITO-coated slides. A set of 21-day-old mouse ovaries (n=10) were gelatin-embedded and cryosectioned. Slides were sprayed with 1,5-diaminonaphthalene as the matrix. MALDI MSI was performed using a Bruker RapifleX MALDI Tissuetyper (20-μm resolution) in the negative ion mode. Data were processed using the SCiLS Lab software. We identified for the first time in human ovaries over 100 lipid species, with most of them displaying distinct spatial distributions. Specifically, we detected 3 lipids specific to theca cells (m/z: 701.6; 1050.8; 1263.9), 6 lipids specific to granulosa cells (m/z: 279.3; 697.5; 861.7; 883.7; 911.7; 1448.0), 3 lipids specific to the stromal region (m/z 392.1; 753.5; 835.7), and one specific to luteal cells (m/z: 615.3). As for mouse ovaries, we detected 5 lipids specific to follicular cells (theca or granulosa) (m/z: 327.1: 343.2; 552.6; 632.6; 703.5), 5 lipids specific to stromal cells (m/z: 715.6; 729.6; 743.6; 771.7; 799.6), 5 lipids specific to oocytes (m/z: 778.5; 863.7; 1063.8; 1225.7; 1336.0) and 7 lipids specific to the ovarian cortex (m/z: 721.5; 748.5; 776.6; 792.6; 810.6; 869.6; 885.7). To our knowledge, this is the first evaluation of the human ovarian lipidome and characterization of region- or cell-layer-specific lipids. This approach has allowed us to demonstrate that compartments as small as oocytes (∼70 μm in diameter) have a unique lipid signature. Harnessing the unique specificity of the lipid signature per ovarian region and/or cell layer in this study could help pave the way to develop novel biomarkers of ovarian dysfunction. Presentation Date: Saturday, June 17, 2023

Milk as diagnostic fluid for udder health management.

Mastitis is the major disease affecting milk production of dairy cattle, and milk is an obvious substrate for the detection of both the inflammation and its causative infectious agents at quarter, cow, or herd levels. In this review, we examine the use of milk to detect inflammation based on somatic cell count (SCC) and other biomarkers, and for the detection of mastitis pathogens through culture-based and culture-free methods. The use of SCC at a cow or bulk milk level to guide udder health management in lactation is well-established, and SCC is increasingly used to guide selective dry cow treatment. Other markers of inflammation include electrical conductivity, which is used commercially, and markers of disease severity such as acute phase proteins but are not pathogen-specific. Some pathogen-specific markers based on humoral immune responses are available, but their value in udder health management is largely untested. Commercial pathogen detection is based on culture or polymerase chain reaction, with other tests, for example, loop-mediated isothermal amplification or 16S microbiome analysis still at the research or development stage. Matrix-assisted laser desorption ionisation time of flight (MALDI-ToF) is increasingly used for the identification of cultured organisms whilst application directly to milk needs further development. Details of test sensitivity, specificity, and use of the various technologies may differ between quarter, cow, and bulk milk applications. There is a growing array of diagnostic assays that can be used to detect markers of inflammation or infection in milk. The value of some of these methods in on-farm udder health improvement programs is yet to be demonstrated whilst methods with proven value may be underutilised.

High-throughput microfluidic chip with silica gel-C18 channels for cyclotide separation.

Over the past two decades, microfluidic-based separations have been used for the purification, isolation, and separation of biomolecules to overcome difficulties encountered by conventional chromatography-based methods including high cost, long processing times, sample volumes, and low separation efficiency. Cyclotides, or cyclic peptides used by some plant families as defense agents, have attracted the interest of scientists because of their biological activities varying from antimicrobial to anticancer properties. The separation process has a critical impact in terms of obtaining pure cyclotides for drug development strategies. Here, for the first time, a mimic of the high-performance liquid chromatography (HPLC) on microfluidic chip strategy was used to separate the cyclotides. In this regard, silica gel-C18 was synthesized and characterized by Fourier-transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H-NMR) and then filled inside the microchannel to prepare an HPLC C18 column-like structure inside the microchannel. Cyclotide extract was obtained from Viola ignobilis by alow voltage electric field extraction method and characterized by HPLC and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). The extract that contained vigno 1, 2, 3, 4, 5, and varv A cyclotides was added to the microchannel where distilled water was used as a mobile phase with 1 µL/min flow rate and then samples were collected in 2-min intervals until 10 min. Results show that cyclotides can be successfully separated from each other and collected from the microchannel at different periods of time. These findings demonstrate that the use of microfluidic channels has a high impact on the separation of cyclotides as a rapid, cost-effective, and simple method and the device can find widespread applications in drug discovery research.

Peak Detection Algorithm for Mass Spectrometry Integrating Weighted Continuous Wavelet Transform with Particle Swarm Optimization-Based Otsu

Peak detection is an important step in mass spectrometry as accurately identifying characteristic peaks is key to data analysis. In order to address the issue of false peak detection, while simultaneously ensuring accurate detection of weak and overlapped peaks, this paper introduces an improved algorithm for mass spectrometry integrating weighted continuous wavelet transform with particle swarm optimization-based Otsu (WWTPO). The algorithm applies the weighted continuous wavelet transform (WCWT) to compress the frequency spectrum signal into a smaller scale range, which allows for the acquisition of more distinct and informative peak information. Moreover, the algorithm employs the particle swarm optimization (PSO) algorithm to iteratively evaluate the optimal image segmentation threshold, which addresses the challenge of inaccurate Otsu image segmentation. The method was applied to detect simulated peaks as well as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) datasets. The performance evaluation was conducted using receiver operating characteristic (ROC) curves, F1 measure and F-scores. Through comparison with continuous wavelet transform (CWT) and genetic algorithm-based threshold segmentation (WSTGA), multi-scale peak detection (MSPD) and CWT and image segmentation (CWT-IS), the results demonstrate that WWTPO exhibits excellent performance in peak detection. The determination of 4-isopropyltoluene also demonstrates that WWTPO has excellent practical application. This method not only maintains a low false peak identification rate but also detects more weak peaks and overlapping peaks, further improving the accuracy and efficiency of peak detection in mass spectrometry.

B-179 Development of a Reflex Mass Spectrometric Workflow for Confirmation of IgE and IgD Specificity Paraproteins Utilising the EXENT® Platform

Abstract Background The EXENT® (GAM) assay is a MALDI-TOF (Matrix Assisted Laser Desorption Ionisation Time of Flight) mass spectrometry-based solution which utilises IgG, IgA, IgM, total kappa and total lambda specific paramagnetic beads, which could be used in the investigation of monoclonal proteins in multiple myeloma and other plasma cell disorders (in development by The Binding Site (TBS), part of Thermo Fisher Scientific). Rare cases of IgD and IgE myelomas will be partially typed with the current iteration of the EXENT (GAM) assay. We present here a workflow to confirm the heavy chain specificity of such paraproteins as IgD or IgE using reflex LC-MS analysis of EXENT eluates. Methods Samples known to contain IgD (2) or IgE (3) paraproteins were provided from either routine workload at the immunology laboratory at Birmingham Heartlands Hospital, UK or an internal library of historical myeloma samples. Capillary zone electrophoresis (CZE) and immunofixation (IFE) were performed on Capillarys2™ and Hydrasys2 Scan™ platforms, respectively (Sebia, France). Serum free light chain (sFLC) measurement was performed using Freelite® reagents (The Binding Site, UK) on a Cobas c501 turbidimeter (Roche, Switzerland). Samples were prepared via EXENT (GAM) assay, by separately incubating diluted serum with antisera specific for IgG, IgA, IgM, total kappa and total lambda conjugated to paramagnetic beads. After washes, purified immunoglobulins were treated to reduce and elute the light chain component. Samples were mixed with matrix, spotted onto a MALDI target plate and acquired by MALDI-TOF MS. Once light-chain specificity of the suspected IgD or IgE paraprotein was determined, repeat immuno-precipitation with the same light-chain bead was carried out. Eluted samples were neutralised, transferred to fresh tubes, reduced, alkylated and digested with trypsin for 3 h. After acidification digested samples were analysed by C18 reverse phase UPLC on a BioAccord LC-MS system (Waters, UK). Data was then screened against a database of known immunoglobulin heavy chain peptides to assess the presence of heavy chain peptides specific for each immunoglobulin isotype. Results In each sample tested, the EXENT solution identified at least one monoclonal light chain that was only seen in the total kappa or total lambda immunoprecipitations with no corresponding monoclonal peak(s) at the same mass(es) in the IgG, IgA or IgM specificities. This finding would be consistent with the presence of clonal plasma cells producing either monoclonal free light chains only, or IgD and IgE paraproteins. Subsequent LC-MS and LC-MSe (tandem MS) analysis of reduced, alkylated and tryptically digested eluates corresponding to these suspected IgD and IgE samples produced high quality peptide sequencing data. The data identified multiple IgD or IgE specific peptides in each sample and showed between 40% and 66% sequence coverage for IgD or IgE heavy-chain constant regions. Thus, confirming the presence of IgD or IgE in these samples which had been partially typed by the EXENT solution. Conclusion This data demonstrates that the EXENT (GAM) assay could potentially aid in detection of IgD and IgE paraproteins when combined with LC-MS proteomic techniques, as it allows efficient and facile confirmation of these rare heavy chain isotypes.

B-180 The EXENT® Solution Provides Evidence for High Prevalence of Multiple M-proteins in Monoclonal Gammopathies

Abstract Background Monoclonal gammopathies (MG) are characterized by excessive production of monoclonal immunoglobulin (M protein), typically consisting of a heavy chain (HC) and a light chain (LC), or a LC only. In some patients, however, more than one restricted band may be present on serum protein electrophoresis. These additional M proteins may be present at initial diagnosis or emerge later following observation or treatment, especially autologous stem cell transplantation. Immunofixation electrophoresis may not be able to differentiate between two bands of the same isotype or other sources of an additional M protein, such as polymerization, glycosylation or therapeutic monoclonal antibody. Mass spectrometry (MS) has recently emerged as an analytically sensitive technology for the detection of M proteins. The EXENT® solution (in development by The Binding Site, part of Thermo Fisher Scientific) uses immuno-purification of immunoglobulins, followed by matrix-assisted laser desorption ionization time of flight (MALDI-ToF) MS analysis. The automated, algorithm-driven analysis of the resulting mass spectra determines the isotype, the molecular mass and the quantity of M proteins. The molecular mass of the M protein serves as a “fingerprint” and allows for easy differentiation between multiple M proteins. The goal of this cross-sectional study was to assess the prevalence of biclonal and multiclonal M proteins in monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), multiple myeloma (MM), Waldenstrom macroglobulinemia (WM) and AL amyloidosis with mass spectrometry. Methods Diagnostic (baseline) samples from 271 patients (35 MGUS, 66 SMM, 125 MM, 31 WM and 14 AL amyloidosis) were analyzed with EXENT® using the EXENT® Immunoglobulin Isotypes reagents. A second M protein was considered distinct from the primary M component if it had a different HC or LC component, or, if it had the same isotype, but had a different molecular mass. Samples producing an additional high-mass peak attributed to LC glycosylation have not been considered biclonal. Results The EXENT® solution has identified a second M protein in 201 (74%) samples, although the second M protein was below the lower limit of the measuring interval in 107 (39%) cases, and only 36 (13.5%) was ≥0.2 g/L: 5 out of 35 in MGUS, 10 out of 66 in SMM, 13 out of 125 in MM, 8 out of 31 in WM and none in AL amyloidosis. Among these, the second M protein had the same isotype in 17 cases and contained a different HC or LC or both in 19 cases. IgG was the most prevalent; the distribution of kappa and lambda LC was 11:13. More than two M proteins were identified in 120 (44%) patients, in 12 (4.5%) cases with a concentration of ≥0.2 g/L. Conclusion The prevalence of two or more M proteins was higher than the previously reported 3%–6%. The higher prevalence is likely the result of the ability of the system to detect small monoclonal peaks that have previously gone unrecognized by traditional methods, and to distinguish between M proteins of the same isotype based on molecular mass.

Development of Thiol-Ene Reaction-Based HA Hydrogel with Sustained Release of EGF for Enhanced Skin Wound Healing.

This study develops a novel drug delivery system using a hyaluronic acid (HA) hydrogel for controlled release of epidermal growth factor (EGF) to enhance skin wound healing. Conventional hydrogel-based methods suffer from a burst release and limited drug delivery times. To address this, we employ bioconjugation to introduce an acrylate group to EGF, enabling chemical bonding to the HA hydrogel matrix through thiol-ene cross-linking. This approach results in sustained-release delivery of EGF based on the degradation rate of the HA matrix, overcoming diffusion-based limitations. We confirm the introduction of the acrylate group using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. We evaluated the hydrogel morphology and rheological properties following binding of acrylate-conjugated EGF to the HA matrix. Assessment of the EGF release profile demonstrates delayed release compared to unconjugated EGF. We evaluate the impact on cells through cell proliferation and scratch assays, indicating the system's efficacy. In a rat wound healing model, the sustained release of EGF from the hydrogel system promotes appropriate tissue healing and restores it to a normal state. These findings suggest that this practical drug delivery system, involving the modification of growth factors or drugs to chemically bind healing factors to hydrogels, can achieve long-lasting effects.

Comparison of Two Bacterial Characterization Techniques for the Genomic Analysis of River Microbiomes

This study compares the feasibility of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry with whole genome sequencing (WGS) for identifying bacteria in river surface water samples. We collected samples from four rivers in the Dominican Republic and used both techniques to characterize bacterial profiles. MALDI-TOF demonstrated high precision, with 86.2% similarity to WGS results, except for a few discordant cases due to database limitations. MALDI-TOF provided cost-effective and rapid identification, making it a promising alternative to WGS in resource-constrained regions. In particular, good effectiveness of MALDI-TOF in identifying bacteria with a high probability of being resistant to antibiotics was observed, which allows this technology to be used in the monitoring processes of this type of microorganism for their rapid, accurate, and low-cost identification. We found this technology to be advantageous for environmental bacterial profiling, with potential applications in understanding waterborne pathogenic bacteria. Our findings underline the relevance of MALDI-TOF in microbiology and its potential to expand its capabilities in bacterial identification and protein profiling.

A New Hemoglobin Variant: Hb Tangshan [HBA1: c.239C > T, CD79(GCG > GTG)(Ala > Val)] Detected by MALDI-TOF MS

In this report we decribed a new α-chain variant found during the measurement of hemoglobin A1c (Hb A1c) using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). MALDI-TOF MS analysis detected an α-chain variant with a mass of 15,155 Da. However, this Hb variant was not detected during Hb A1c measurement by cation-exchange high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) methods. Sanger sequencing validated the presence of a heterozygous missense mutation [HBA1: c.239C > T, CD79(GCG > GTG)(Ala > Val)]. The observed 28 Da mass difference exactly matches the theoretical mass difference (28 Da) resulting from the substitution of alanine (89.079) with valine (117.133). As this represents the initial documentation of the mutation, we named it Hb Tangshan after the proband’s residence.

Non-thermal plasma radiation-induced changes in antibiotic susceptibility and protein profile of Staphylococcus aureus.

Plasma radiation is a widely used technique for sterilization or decontamination in various industries, as well as in some healthcare settings such as dentistry. The primary aim of this study was to assess the potential of plasma radiation to create a new population of Staphylococcus aureus cells with distinct characteristics that could lead to novel healthcare challenges. A homemade non-thermal plasma apparatus was applied and the effects of plasma treatment on S. aureus ATCC25923 was assessed. Plasma radiation was applied under controlled conditions to ensure that some bacterial cells remained viable. The treatment was repeated 10 times, with each round followed by a recovery phase to collect any surviving bacterial cells. To assess the potential changes in the bacterial population, we examined the antibiotic susceptibility pattern, micro-structural characteristics using scanning electron microscopy (SEM), and total protein profile using the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) technique. The experimental results revealed slight variations in the antibiotic susceptibility patterns of certain cell wall agents (imipenem, cephalothin, and cefepime), as well as in the MALDI-TOF spectra. However, no changes were observed in the SEM images. The insufficient application of non-thermal plasma in bacterial decontamination may lead to physiological changes that could enrich or select certain subpopulations of S. aureus.