DNA-Binding Proteins and Passenger Proteins in Plasma DNA-Protein Complexes: Imprint of Parental Cells or Key Mediators of Carcinogenesis Processes?

Knowledge of the composition of proteins that interact with plasma DNA will provide a better understanding of the homeostasis of circulating nucleic acids and the various modes of interaction with target cells, which may be useful in the development of gene targeted therapy approaches. The goal of the present study is to shed light on the composition and architecture of histone-containing nucleoprotein complexes (NPCs) from the blood plasma of healthy females (HFs) and breast cancer patients (BCPs) and to explore the relationship of proteins with crucial steps of tumor progression: epithelial-mesenchymal transition (EMT), cell proliferation, invasion, cell migration, stimulation of angiogenesis, and immune response. MALDI-TOF mass spectrometric analysis of NPCs isolated from blood samples using affine chromatography was performed. Bioinformatics analysis showed that the shares of DNA-binding proteins in the compositions of NPCs in normal and cancer patients are comparable and amount to 40% and 33%, respectively; in total, we identified 38 types of DNA-binding motifs. Functional enrichment analysis using FunRich 3.13 showed that, in BCP blood, the share of DNA-binding proteins involved in nucleic acid metabolism increased, while the proportion of proteins involved in intercellular communication and signal transduction decreased. The representation of NPC passenger proteins in breast cancer also changes: the proportion of proteins involved in transport increases and the share of proteins involved in energy biological pathways decreases. Moreover, in the HF blood, proteins involved in the processes of apoptosis were more represented in the composition of NPCs and in the BCP blood-in the processes of active secretion. For the first time, bioinformatics approaches were used to visualize the architecture of circulating NPCs in the blood and to show that breast cancer has an increased representation of passenger proteins involved in EMT, cell proliferation, invasion, cell migration, and immune response. Using breast cancer protein data from the Human Protein Atlas (HPA) and DEPC, we found that 86% of NPC proteins in the blood of BCPs were not previously annotated in these databases. The obtained data may indirectly indicate directed protein sorting in NPCs, which, along with extracellular vesicles, can not only be diagnostically significant molecules for liquid biopsy, but can also carry out the directed transfer of genetic material from donor cells to recipient cells.

Clinical Evaluation of VITEK MS PRIME with PICKME Pen for Bacteria and Yeasts, and RUO Database for Filamentous Fungi.

The VITEK MS PRIME (bioMérieux, Marcy-l'Étoile, France), a newly developed matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) system, alongside the VITEK PICKME pen (PICKME), offers easy sample preparation for bacteria and yeasts. The VITEK MS PRIME also offers two software platforms for filamentous fungi: the IVD database and the RUO database. Our study evaluated its identification agreement on 320 clinical isolates of bacteria and yeasts, comparing PICKME and traditional wooden toothpick sampling techniques against MicroIDSys Elite (ASTA) results. Additionally, we assessed the IVD (v3.2) and SARAMIS (v4.16) RUO databases on 289 filamentous fungi against molecular sequencing. The concordance rates for species-level identification of bacteria and yeasts were about 89.4% (286/320) between the PICKME and wooden toothpick, and about 83.4-85.3% between the VITEK MS PRIME and ASTA MicroIDSys Elite. Retesting with PICKME improved concordance to 91.9%. For filamentous fungi, species-level identification reached 71.3% with the IVD database and 85.8% with RUO, which significantly enhanced basidiomycetes' identification from 35.3% to 100%. Some strains in the IVD database, like Aspergillus versicolor, Exophiala xenobiotica, and Nannizzia gypsea, failed to be identified. The VITEK MS PRIME with PICKME offers reliable and efficient microorganism identification. For filamentous fungi, combined use of the RUO database can be beneficial, especially for basidiomycetes.

Uncommon pathogen misidentification of Herbaspirillum huttiense as Burkholderia cepacia in bacteremia: a case report.

Herbaspirillum huttiense is an opportunistic pathogen associated with rare cases of bacteremia. In this case report, H huttiense was isolated from blood samples collected from an intravenous catheter (incubated for 20.8 hours) and a peripheral vein (incubated for 14.16 hours) of a lung adenocarcinoma patient. Positive blood culture bottles were subjected to smear preparation, and Gram staining and microscopic examination revealed the presence of gram-negative rods in both aerobic bottles. We used the VITEK MS automatic microbial mass spectrometry system, VITEK 2 Compact automatic microbial analysis system, and high-throughput nucleic acid sequencing for accurate identification of the isolate. It is noteworthy that although the VITEK 2 Compact identified the isolate as Burkholderia cepacia, confirmation through VITEK MS mass spectrometry and 16S ribosomal DNA (rDNA) sequencing identified it as H huttiense. Subsequently, antimicrobial susceptibility testing was performed using the broth microdilution method, following the guidelines for nonfermenting gram-negative bacilli provided by the Clinical and Laboratory Standards Institute. This case highlights the possibility of misidentification of H huttiense as B cepacia by VITEK 2 Compact in certain situations, emphasizing the importance of considering uncommon pathogens, such as H huttiense, in the context of bacteremia in cancer patients.

Pseudomonas aeruginosa Lipid A Structural Variants Induce Altered Immune Responses.

Pseudomonas aeruginosa causes chronic lung infection in cystic fibrosis (CF), resulting in structural lung damage and progressive pulmonary decline. P. aeruginosa in the CF lung undergoes numerous changes, adapting to host-specific airway pressures while establishing chronic infection. P. aeruginosa undergoes lipid A structural modification during CF chronic infection, not seen in any other disease state. Lipid A, the membrane anchor of lipopolysaccharide (i.e., endotoxin), comprises the majority of the outer membrane of Gram-negative bacteria and is a potent toll-like receptor (TLR)4 agonist. The structure of P. aeruginosa lipid A is intimately linked with its recognition by TLR4, and subsequent immune response. Prior work has identified P. aeruginosa strains with altered lipid A structures that arise during chronic CF lung infection; however, the impact of P. aeruginosa lipid A structure on airway disease has not been investigated. Here, we show that P. aeruginosa lipid A lacks PagL-mediated deacylation during human airway infection using a direct-from-sample mass spectrometry approach on human bronchoalveolar lavage fluid. This structure triggers increased pro-inflammatory cytokine production by primary human macrophages. Furthermore, alterations in lipid A 2-hydroxylation impact cytokine response in a site-specific manner, independent of CFTR function. Interestingly, there is a CF-specific reduction in IL-8 secretion within the epithelial-cell compartment that only occurs in CF bronchial epithelial cells when infected with CF-adapted P. aeruginosa that lack PagL-mediated lipid A deacylation. Taken together, we show that P. aeruginosa alters its lipid A structure during acute lung infection and that this lipid A structure induces stronger signaling through TLR4.

Discriminatory power of MALDI-TOF MS protein profiling analysis of pork meat and meat products.

Forty different sample preparation methods were tested to obtain the most informative MALDI-TOF MS protein profiles of pork meat. Extraction by 25% formic acid with the assistance of zirconia-silica beads followed by defatting by methanol:chloroform mixture (1:1, v/v) and deposition by using the layer-by-layer method was determined as the optimum sample preparation protocol. The discriminatory power of the method was then examined on samples of pork meat and meat products. The method was able to discriminate between selected salami based on the production method and brand and was able to monitor the ripening process in salami. However, it was not able to differentiate between different brands of pork ham or closely located parts of pork meat. In the latter case, a more comprehensive analysis using LC-MS/MS was used to assess the differences in protein abundance and their relation to the outputs of MALDI - TOF MS profiling.

[Skin abscess caused by Actinomyces radingae]. / Hautabszess verursacht durch Actinomyces radingae.

We report a 77-year-old man with a skin abscess caused by Actinomyces radingae. Targeted antibiotic therapy with amoxicillin/clavulanic acid for 6 weeks resulted in clearing of the infection. A. radingae is a rare pathogenic agent of skin and soft tissue infections. As with other Actinomyces infections, the early identification of the pathogen and specific antibiotic therapy is crucial for successful resolution of the infection because of the chronic course and the long treatment time needed. Usually, A. radingae is sensitive to ß­lactam antibiotics.

Yeast Biodiversity of Karst Waters: Interest of Four Culture Media and an Improved MALDI-TOF MS Database.

Karst aquifers are a significant source of drinking water and highly vulnerable to pollution and microbial contamination. Microbiological regulations for the quality of drinking water mostly focus on bacterial levels and lack guidance concerning fungal contamination. Moreover, there is no standardised microbial analysis methodology for identifying fungi in water. Our main objective was to establish the most effective culture and identification methodology to examine yeast diversity in karst waters. We assessed the comparative efficacy of four culture media (CHROMagar Candida, dichloran glycerol 18% [DG18], dichloran rose Bengal chloramphenicol [DRBC], and SYMPHONY agar) for yeast isolation from karst water samples. Furthermore, we investigated the comprehensiveness of databases used in MALDI-TOF mass spectrometry (MALDI-TOF MS) for identifying environmental yeast species. In total, we analysed 162 water samples, allowing the identification of 2479 yeast isolates. We demonstrate that a combination of four culture media, each with distinct specifications, more efficiently covers a wide range of yeast species in karst water than a combination of only two or three. Supplementation of a MALDI-TOF MS database is also critical for analysing environmental microbial samples and improved the identification of yeast biodiversity. This study is an initial step towards standardising the analysis of fungal biodiversity in karst waters, enabling a better understanding of the significance of this environmental reservoir in relation to public health.

MALDI-TOF mass spectrometry identification and antifungal susceptibility testing of yeasts causing vulvovaginal candidiasis (VVC) in Tebessa (Northeastern Algeria).

Vulvovaginal candidiasis (VVC) alongside with antifungal resistance are becoming a major clinical problem in recent years. A prospective study aimed to evaluate the diversity of yeast strains associated with VVC in Tebessa city (northeastern Algeria) and investigate their susceptibility patterns. Over two months, yeasts were isolated on chromogenic medium from twenty-nine non-pregnant women with symptomatic VVC. The isolates were characterized with MALDI-TOF MS and antifungal susceptibility testing was performed for nine antifungal drugs using SensititreTM YeastOneTM YO10. Twenty-nine non-duplicate yeasts were recovered and the mass spectrometry profiles showed reliable scores of which four genera and five different species were identified. Candida albicans accounted for 65.5 % (n = 19) of the total number of isolates, followed by C. glabrata with 20.7% (n = 6). For the remaining non-albicans Candida (NCA) species, Kluyveromyces marxianus with 6.9% (n = 2), Pichia kudriavzevii and Saccharomyces cerevisiae with one isolate each. The antifungal susceptibilities showed wild type MICs of C. albicans to amphotericin B, azoles and echinocandins. In addition, four C. albicans isolates were resistant to flucytosine. For C. glabrata isolates, 100% non-WT phenotype was found for both posaconazole and itraconazole. For the very first time, the obtained outcomes bring out new data concerning the epidemiology of yeasts causing VVC in Algeria and their antimicrobial susceptibility profiles.

Blood Plasma Circulating DNA-Protein Complexes: Involvement in Carcinogenesis and Prospects for Liquid Biopsy of Breast Cancer.

Circulating DNA (cirDNA) is a promising tool in translational medicine. However, studies of cirDNA have neglected its association with proteins, despite ample evidence that this interaction may affect the fate of DNA in the bloodstream and its molecular functions. The goal of the current study is to shed light on the differences between the proteomic cargos of histone-containing nucleoprotein complexes (NPCs) from healthy female (HFs) and breast cancer patients (BCPs), and to reveal the proteins involved in carcinogenesis. NPCs were isolated from the blood samples of HFs and BCPs using affinity chromatography. A total of 177 and 169 proteins were identified in NPCs from HFs and BCPs using MALDI-TOF mass spectrometry. A bioinformatics analysis revealed that catalytically active proteins, as well as proteins that bind nucleic acids and regulate the activity of receptors, are the most represented among the unique proteins of blood NPCs from HFs and BCPs. In addition, the proportion of proteins participating in ion channels and proteins binding proteins increases in the NPCs from BCP blood. However, the involvement in transport and signal transduction was greater in BCP NPCs compared to those from HFs. Gene ontology term (GO) analysis revealed that the NPC protein cargo from HF blood was enriched with proteins involved in the negative regulation of cell proliferation, and in BCP blood, proteins involved in EMT, invasion, and cell migration were observed. The combination of SPG7, ADRB1, SMCO4, PHF1, and PSMG1 NPC proteins differentiates BCPs from HFs with a sensitivity of 100% and a specificity of 80%. The obtained results indirectly indicate that, in tandem with proteins, blood cirDNA is an important part of intercellular communication, playing a regulatory and integrating role in the physiology of the body.

Improved Screening of Monoclonal Gammopathy Patients by MALDI-TOF Mass Spectrometry.

Monoclonal gammopathies are a group of blood diseases characterized by presence of abnormal immunoglobulins in peripheral blood and/or urine of patients. Multiple myeloma and plasma cell leukemia are monoclonal gammopathies with unclear etiology, caused by malignant transformation of bone marrow plasma cells. Mass spectrometry with matrix-assisted laser desorption/ionization and time-of-flight detection is commonly used for investigation of the peptidome and small proteome of blood plasma with high accuracy, robustness, and cost-effectivity. In addition, mass spectrometry coupled with advanced statistics can be used for molecular profiling, classification, and diagnosis of liquid biopsies and tissue specimens in various malignancies. Despite the fact there have been fully optimized protocols for mass spectrometry of normal blood plasma available for decades, in monoclonal gammopathy patients, the massive alterations of biophysical and biochemical parameters of peripheral blood plasma often limit the mass spectrometry measurements. In this paper, we present a new two-step extraction protocol and demonstrated the enhanced resolution and intensity (>50×) of mass spectra obtained from extracts of peripheral blood plasma from monoclonal gammopathy patients. When coupled with advanced statistics and machine learning, the mass spectra profiles enabled the direct identification, classification, and discrimination of multiple myeloma and plasma cell leukemia patients with high accuracy and precision. A model based on PLS-DA achieved the best performance with 71.5% accuracy (95% confidence interval, CI = 57.1-83.3%) when the 10× repeated 5-fold CV was performed. In summary, the two-step extraction protocol improved the analysis of monoclonal gammopathy peripheral blood plasma samples by mass spectrometry and provided a tool for addressing the complex molecular etiology of monoclonal gammopathies.

Poor Sensitivity of the MALDI Biotyper® MBT Subtyping Module for Detection of Klebsiella pneumoniae Carbapenemase (KPC) in Klebsiella Species.

Rapid detection of Klebsiella pneumoniae carbapenemase (KPC) in the Klebsiella species is desirable. The MALDI Biotyper® MBT Subtyping Module (Bruker Daltonics) uses an algorithm that detects a peak at ~11,109 m/z corresponding to a protein encoded by the p019 gene to detect KPC simultaneously with organism identification by a matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-ToF MS). Here, the subtyping module was evaluated using 795 clinical Klebsiella isolates, with whole genome sequences used to assess for blaKPC and p019. For the isolates identified as KPC positive by sequencing, the overall sensitivity of the MALDI-ToF MS subtyping module was 239/574 (42%) with 100% specificity. For the isolates harboring p019, the subtyping module showed a sensitivity of 97% (239/246) and a specificity of 100%. The subtyping module had poor sensitivity for the detection of blaKPC-positive Klebsiella isolates, albeit exhibiting excellent specificity. The poor sensitivity was a result of p019 being present in only 43% of the blaKPC-positive Klebsiella isolates.

Discrimination between hypervirulent and non-hypervirulent ribotypes of Clostridioides difficile by MALDI-TOF mass spectrometry and machine learning.

Hypervirulent ribotypes (HVRTs) of Clostridioides difficile such as ribotype (RT) 027 are epidemiologically important. This study evaluated whether MALDI-TOF can distinguish between strains of HVRTs and non-HVRTs commonly found in Europe. Obtained spectra of clinical C. difficile isolates (training set, 157 isolates) covering epidemiologically relevant HVRTs and non-HVRTs found in Europe were used as an input for different machine learning (ML) models. Another 83 isolates were used as a validation set. Direct comparison of MALDI-TOF spectra obtained from HVRTs and non-HVRTs did not allow to discriminate between these two groups, while using these spectra with certain ML models could differentiate HVRTs from non-HVRTs with an accuracy >95% and allowed for a sub-clustering of three HVRT subgroups (RT027/RT176, RT023, RT045/078/126/127). MALDI-TOF combined with ML represents a reliable tool for rapid identification of major European HVRTs.

Extracellular Vesicles Contribute to the Difference in Lipid Composition between Ovarian Follicles of Different Size Revealed by Mass Spectrometry Imaging.

Follicular fluid (FF) ensures a safe environment for oocyte growth and maturation inside the ovarian follicle in mammals. In each cycle, the large dominant follicle (LF) contains the oocyte designated to be ovulated, whereas the small subordinate follicles (SFs) of the same wave will die through atresia. In cows, the oocytes from the SF, being 2 mm in size, are suitable for in vitro reproduction biotechnologies, and their competence in developing an embryo depends on the size of the follicles. FF contains proteins, metabolites, fatty acids, and a multitude of extracellular vesicles (ffEVs) of different origins, which may influence oocyte competence through bidirectional exchanges of specific molecular cargo between follicular cells and enclosed oocytes. FF composition evolves along with follicle growth, and the abundance of different lipids varies between the LF and SF. Here, significant differences in FF lipid content between the LFs and SFs within the same ovary were demonstrated by MALD-TOF mass spectrometry imaging on bovine ovarian sections. We then aimed to enlighten the lipid composition of FF, and MALDI-TOF lipid profiling was performed on cellular, vesicular, and liquid fractions of FF. Differential analyses on the abundance of detected lipid features revealed specific enrichment of phospholipids in different ffEV types, such as microvesicles (MVs) and exosomes (Exo), compared to depleted FF. MALDI-TOF lipid profiling on MVs and Exo from the LF and SF samples (n = 24) revealed that more than 40% of detected features were differentially abundant between the groups of MVs and Exo from the different follicles (p < 0.01, fold change > 2). Glycerophospholipid and sphingolipid features were more abundant in ffEVs from the SFs, whereas different lysophospholipids, including phosphatidylinositols, were more abundant in the LFs. As determined by functional analysis, the specific lipid composition of ffEVs suggested the involvement of vesicular lipids in cell signaling pathways and largely contributed to the differentiation of the dominant and subordinate follicles.

Evaluation of a Novel Benchtop Tool for Acceleration of Sample Preparation for MALDI-TOF Mass Spectrometry.

During the past decade, MALDI-TOF mass spectrometry (MS) has become a standard method for identification of bacteria and yeasts. Nonetheless, further optimization of the identification process is important to streamline workflows and save resources. This study evaluated the application of a multipurpose benchtop tool, MBT FAST Shuttle IVD, for accelerated drying of liquid assay components (matrix, formic acid, and/or sample) on a MALDI target. A total of 50 bacterial and fungal isolates were subjected to three different sample preparation procedures prior to the identification by MALDI-TOF MS: direct transfer (DT), extended direct transfer (eDT), and protein extraction (PE). Compared to conventional drying at room temperature, the preparation was performed with standardized heating of the MALDI target on the MBT FAST Shuttle. During DT, eDT, and PE, 56.7% (P < 0.001), 56.8% (P < 0.001), and 52.8% (P < 0.001) of time for matrix drying were saved by using the MBT FAST Shuttle, respectively. Applying the MBT FAST Shuttle, 57.5% (P < 0.001) of time for drying of formic acid were saved for eDT and 57.5% (P < 0.001) of time for sample drying were saved for PE. A significant improvement of the identification rates and scores was observed with MBT FAST Shuttle for eDT (P = 0.001) and PE (P = 0.008) methods, while the effect on identification quality for DT was not statistically significant (P = 0.16). In conclusion, the use of the MBT FAST Shuttle shortened the drying time of assay components by about a half for all preparation methods. Moreover, positive effect on identification success was observed.

A multiplex method for detection of SARS-CoV-2 variants based on MALDI-TOF mass spectrometry.

The recent outbreak of the coronavirus disease 2019 (COVID-19) pandemic and the continuous evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have highlighted the significance of new detection methods for global monitoring and prevention. Although quantitative reverse transcription PCR (RT-qPCR), the current gold standard for diagnosis, performs excellently in genetic testing, its multiplexing capability is limited because of the signal crosstalk of various fluorophores. Herein, we present a highly efficient platform which combines 17-plex assays with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), enabling the targeting of 14 different mutation sites of the spike gene. Diagnosis using a set of 324 nasopharyngeal swabs or sputum clinical samples with SARS-CoV-2 MS method was identical to that with the RT-qPCR. The detection consistency of mutation sites was 97.9% (47/48) compared to Sanger sequencing without cross-reaction with other respiratory-related pathogens. Therefore, the MS method is highly potent to track and assess SARS-CoV-2 changes in a timely manner, thereby aiding the continuous response to viral variation and prevention of further transmission.

Alterations in the Glycan Composition of Serum Glycoproteins in Attention-Deficit Hyperactivity Disorder.

Changes in protein glycosylation are associated with most biological processes, and the importance of glycomic analysis in the research of disorders is constantly increasing, including in the neurodevelopmental field. We glycoprofiled sera in 10 children with attention-deficit hyperactivity disorder (ADHD) and 10 matching healthy controls for 3 types of samples: whole serum, sera after depletion of abundant proteins (albumin and IgG), and isolated IgG. The analytical methods used were a lectin-based glycoprotein microarray enabling high-throughput glycan analysis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) as a standard method for the identification of glycan structures. For microarray analysis, the samples printed on microarray slides were incubated with biotinylated lectins and detected using the fluorescent conjugate of streptavidin by a microarray scanner. In the ADHD patient samples, we found increased antennary fucosylation, decreased di-/triantennary N-glycans with bisecting N-acetylglucosamine (GlcNAc), and decreased α2-3 sialylation. The results obtained by both independent methods were consistent. The study's sample size and design do not allow far-reaching conclusions to be drawn. In any case, there is a strong demand for a better and more comprehensive diagnosis of ADHD, and the obtained results emphasize that the presented approach brings new horizons to studying functional associations of glycan alterations in ADHD.

Detection of serum M-protein in acetonitrile precipitates by MALDI-TOF mass spectrometry: A novel, low-cost methodology.

BACKGROUND: Several studies have demonstrated the analytical sensitivity of MALDI-TOF mass spectrometry (MALDI-TOF MS) by immunoenrichment for M-protein analysis. We report the results of a novel, low-cost, reagent-based extraction process using acetonitrile (ACN) precipitation to enrich for κ and λ light chains which can be analysed by MALDI-TOF MS. METHODS: Institutional Ethics committee approval was obtained. Serum samples from patients with monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM), plasmacytoma, AL amyloidosis and Waldenström macroglobulinemia (WM) underwent ACN precipitation. The images obtained were overlaid on apparently healthy donor serum samples to confirm the presence of M-protein. A sample was considered positive for M-protein if there was a sharp or broad peak within the κ or λ mass/charge (m/z) range: m/z- [M + 2H]2+: 11,550-12,300 Da and λ m/z- [M + 2H]2+: 11,100-11,500 Da. Images were acquired at a m/z range of 10,000-29,000 Da. Corresponding serum protein electrophoresis (SPEP), serum immunofixation electrophoresis (IFE) and serum free light chain (sFLC) assay by nephelometry were performed for all the samples. RESULTS: Two-hundred-and-two serum samples were included in the study: MM- 184 (91%); AL amyloidosis- 2 (1%); plasmacytoma- 8 (4%); MGUS- 6 (3%) and WM- 2 (1%). All the SPEP positive samples were identified by MALDI-TOF MS. Out of 179 samples positive for M-protein by IFE, MALDI-TOF MS was positive in 176 samples (98%). Compared to IFE, the sensitivity and specificity of M-protein identification by MALDI-TOF MS were 98.3% and 52.2%, respectively. CONCLUSIONS: This study demonstrates the feasibility of qualitatively identifying M-protein without the need for antibody-based immunoenrichment, making the technique cost-effective.

Comparative Analysis of Molecular Functions and Biological Role of Proteins from Cell-Free DNA-Protein Complexes Circulating in Plasma of Healthy Females and Breast Cancer Patients.

Cell-free DNA (cfDNA) circulates in the bloodstream packed in membrane-coated structures (such as apoptotic bodies) or bound to proteins. To identify proteins involved in the formation of deoxyribonucleoprotein complexes circulating in the blood, native complexes were isolated using affinity chromatography with immobilized polyclonal anti-histone antibodies from plasma of healthy females (HFs) and breast cancer patients (BCPs). It was found that the nucleoprotein complexes (NPCs) from HF plasma samples contained shorter DNA fragments (~180 bp) than BCP NPCs. However, the share of DNA in the NPCs from cfDNA in blood plasma in HFs and BCPs did not differ significantly, as well as the share of NPC protein from blood plasma total protein. Proteins were separated by SDS-PAGE and identified by MALDI-TOF mass spectrometry. Bioinformatic analysis showed that in the presence of a malignant tumor, the proportion of proteins involved in ion channels, protein binding, transport, and signal transduction increased in the composition of blood-circulating NPCs. Moreover, 58 (35%) proteins are differentially expressed in a number of malignant neoplasms in the NPCs of BCPs. Identified NPC proteins from BCP blood can be recommended for further testing as breast cancer diagnostic/prognostic biomarkers or as being useful in developing gene-targeted therapy approaches.

Pushing the Boundaries of Structural Heterogeneity with the Lipid A of Marine Bacteria Cellulophaga.

Marine bacteria, which are often described as chemical gold, are considered an exceptional source of new therapeutics. Considerable research interest has been given to lipopolysaccharides (LPSs), the main components of the Gram-negative outer membrane. LPS and its lipid A portion from marine bacteria are known to exhibit a tricky chemistry that has been often associated with intriguing properties such as behaving as immune adjuvants or anti-sepsis molecules. In this scenario, we report the structural determination of the lipid A from three marine bacteria within the Cellulophaga genus, which showed to produce an extremely heterogenous blend of tetra- to hexa-acylated lipid A species, mostly carrying one phosphate and one D-mannose on the glucosamine disaccharide backbone. The ability of the three LPSs in activating TLR4 signaling revealed a weaker immunopotential by C. baltica NNO 15840T and C. tyrosinoxydans EM41T , while C. algicola ACAM 630T behaved as a more potent TLR4 activator.

Evaluation and clinical impact of MALDI Biotyper Mycobacteria Library v6.0 for identification of nontuberculous mycobacteria by MALDI-TOF mass spectrometry.

Major challenges in the identification of non-tuberculous mycobacteria (NTM) by MALDI-TOF MS include protein extraction protocol and updating of the NTM database. The aim of this study was to evaluate MALDI Biotyper Mycobacteria Library v6.0 (Bruker Daltonics GmbH, Bremen, Germany) for identification of clinical NTM isolates and its impact on clinical management. NTM isolates cultivated from clinical samples in 101 patients were identified simultaneously by PCR-reverse hybridization (Hain Lifescience GmbH, Nehren, Germany) as a routinely used reference molecular method and using MALDI Biotyper Microflex LT/SH after protein extraction. Each isolate was applied to eight spots, and mean scores were used in analysis. MALDI-TOF MS obtained correct identification to the species level for 95 (94.06%) NTM isolates. The majority of correctly identified isolates (92/95; 96.84%) were identified with high-confidence score of ≥1.80 and only 3.16% (3/95) with a score of <1.80. Mean value ± SD of RGM NTM isolates (2.127 ± 0.172) was statistically significant higher in comparison to SGM NTM isolates (2.027 ± 0.142) with a p value of 0.007. In comparison to PCR-reverse hybridization, discordant identification results by MALDI-TOF MS were found in six (6/101; 5.94%) NTM isolates for which clinical data were analyzed. We demonstrated a high confidence NTM identifications using Mycobacterium Library v 6.0 on routine clinical isolates. This is the first study that analyzed MALDI-TOF MS identification results of NTM isolates in the context of clinical data, and it showed that MALDI-TOF MS with its updated databases could help clarify the epidemiology, clinical characteristics, and course of infections caused by less frequent NTM species.