HCEC cells were stimulated for 72 h with DAC (10 µM) or vehicle with/without the addition of TNFα (20 ng/mL) for the last 24 h. Proteins of whole-cell lysates were separated by SDS-PAGE. After incubation with a colloidal Coomassie staining solution, bands found at 50 kDa were excised, and an In-Gel Tryptic Digestion Kit (Thermo ScientificTM PIERCETM) was applied. The resulting digest was mixed 1:1 with the matrix solution (α-cyano-4-hydroxy cinnamic acid, HCCA) and 1 µl of this mixture was applied onto a steel plate for MALDI-TOF-MS analysis (Bruker Daltonic GmbH, Bremen, Germany). TOF-MS measurements were performed in the reflector mode operation over the mass range of m/z 500–5000. Further details for methodical parameters are given in the here cited literature [79 (link)].
Maldi tof ms analysis
Manufactured by Bruker
Sourced in Germany
MALDI-TOF MS analysis is a technique used for the identification and characterization of biomolecules, such as proteins, peptides, and small molecules. It employs a matrix-assisted laser desorption/ionization (MALDI) source coupled with a time-of-flight (TOF) mass spectrometer to analyze the masses of analytes. This analytical method provides high sensitivity and accuracy in determining the molecular weights of the samples.
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Lab products found in correlation
Maldi tof ms, by Bruker (2 mentions)
In gel tryptic digestion kit, by Thermo Fisher Scientific (1 mentions)
Columbia cna agar, by BD (1 mentions)
Ph fix strips, by Macherey-Nagel (1 mentions)
Api 50 chl, by bioMérieux (1 mentions)
Macconkey, by BD (1 mentions)
Xlt 4, by BD (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
Fecalswab system, by Copan (1 mentions)
Xpert carba r assay, by Cepheid (1 mentions)
Msp 96 polished steel target, by Bruker (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
C18 column, by Shimadzu (1 mentions)
Multiplex pcr kit, by Qiagen (1 mentions)
Chromatography grade ethanol, by Merck Group (1 mentions)
Seakem, by Lonza (1 mentions)
14 protocols using maldi tof ms analysis
1
Proteomic Analysis of DAC-induced Changes
2
Vaginal Bacterial and Cellular Analysis
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Three vaginal swabs were collected from each woman enrolled in this study. One swab was used for pH measurement using pH-Fix strips (Macherey-Nagel GmbH & Co. KG, Düren, Germany).
One swab was soaked in 1 ml of saline. A certain volume of this sample was cultured on BD Columbia CNA Agar containing 5% Sheep Blood (Becton Dickinson, New Jersey, USA). For G. vaginalis identification, mass-spectrometry (MALDI-TOF MS) analysis (Bruker Daltonik GmbH, Bremen, Germany) was performed. The remaining volume of the sample was used for Gram stain (Nugent’s criteria) for the diagnosis of BV. The last swab was soaked in 1 ml of saline and vortexed for at least one minute. Around 100 μL of the sample was examined under a light microscope (Olympus, Milan, Italy) to evaluate the presence of neutrophils (PMNs) and EC exfoliation. The numbers of PMNs and ECs were counted in four fields at ×400 magnification and expressed as the average number of PMNs or ECs/field, as previously described10 (link),19 (link),27 (link).
The remaining sample (900 μl) was centrifuged at 1,600 rpm for 10 min, and the cellular fraction was used for flow cytometric analysis or for assessment of EC damage or EC apoptosis.
Due to the limited amount of ECs in our samples, we were unable to perform all analyses for all samples. In each figure, we have reported the number of BV and healthy specimens used.
One swab was soaked in 1 ml of saline. A certain volume of this sample was cultured on BD Columbia CNA Agar containing 5% Sheep Blood (Becton Dickinson, New Jersey, USA). For G. vaginalis identification, mass-spectrometry (MALDI-TOF MS) analysis (Bruker Daltonik GmbH, Bremen, Germany) was performed. The remaining volume of the sample was used for Gram stain (Nugent’s criteria) for the diagnosis of BV. The last swab was soaked in 1 ml of saline and vortexed for at least one minute. Around 100 μL of the sample was examined under a light microscope (Olympus, Milan, Italy) to evaluate the presence of neutrophils (PMNs) and EC exfoliation. The numbers of PMNs and ECs were counted in four fields at ×400 magnification and expressed as the average number of PMNs or ECs/field, as previously described10 (link),19 (link),27 (link).
The remaining sample (900 μl) was centrifuged at 1,600 rpm for 10 min, and the cellular fraction was used for flow cytometric analysis or for assessment of EC damage or EC apoptosis.
Due to the limited amount of ECs in our samples, we were unable to perform all analyses for all samples. In each figure, we have reported the number of BV and healthy specimens used.
3
Isolation and Characterization of Lactobacillus Strains
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Lactobacillus strains were isolated from fermented cucumbers, which are the most popular fermented foods consumed in Poland. Juice of fermented cucumbers was seeded on MRS agar (De Man, Rogosa and Sharpe agar, BTL, Warsaw, Poland) and incubated in aerobic condition for 24 h at 37 °C. Bacteria were isolated and identified from selected colonies. Isolates which were Gram-positive and catalase-negative rods were further identified using API 50CHL (bio-Merieux, Marcy l’Etoile, France) and MALDI-TOF MS analysis (Bruker, Billerica, Massachusetts, USA). Of 26 isolates, 4 strains of Lactobacillus plantarum and 4 strains of Lactobacillus brevis were selected for further study. One of the selection criteria was the growth capacity of these strains in the presence of bile salts and gastric juice which was checked by methods described by Li et al. [22 ].
P. mirabilis (strain C7) was derived from encrusted biofilm formed on urinary catheters of long-term catheterized patients and was deposited in the bacterial strain collection at the Department of Biology of Bacteria, the University of Lodz. The method of isolation of this strain and its characteristics had been described in our previous study [31 (link)].
P. mirabilis (strain C7) was derived from encrusted biofilm formed on urinary catheters of long-term catheterized patients and was deposited in the bacterial strain collection at the Department of Biology of Bacteria, the University of Lodz. The method of isolation of this strain and its characteristics had been described in our previous study [31 (link)].
4
Isolation and Identification of Pathogenic Bacteria
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After preparation, the samples were centrifuged at 200× g for 5 min at 4 °C. One loopful of the supernatant (0.01 mL) was taken and streaked onto to selective agar, namely MacConkey (pink/red colour; Becton Dickinson, Bergen, NJ, USA), XLT4 (black; Becton Dickinson, Bergen, NJ, USA), Baird–Parker (grey/black; Oxoid, Basingstoke, UK) and Kenner KF (purple; Becton Dickinson, Bergen, NJ, USA) agar to identify E. coli, Salmonella, S. aureus and Enterococcus, respectively. For quality control of each batch, all of the selective media were incubated at 38 °C for 24 h. As positive controls, the four bacteria were directly applied to the selective agar. The isolation and identification of pathogenic colonies were performed according to each manufacturer’s instruction manual.
Twenty isolated colonies from each selective agar were streaked onto the blood agar at 38 °C and incubated for 24 h. For Gram-positive bacteria, the incubation time was 24–36 h (FAO regional antimicrobial resistance monitoring and surveillance guidelines, 2019). The isolated colonies from the blood agar were taken for MALDI-TOF-MS analysis (Bruker, Germany) at the Bacteriology Laboratory of the Kimron Institute for confirmation of their identities [44 (link)].
Twenty isolated colonies from each selective agar were streaked onto the blood agar at 38 °C and incubated for 24 h. For Gram-positive bacteria, the incubation time was 24–36 h (FAO regional antimicrobial resistance monitoring and surveillance guidelines, 2019). The isolated colonies from the blood agar were taken for MALDI-TOF-MS analysis (Bruker, Germany) at the Bacteriology Laboratory of the Kimron Institute for confirmation of their identities [44 (link)].
5
Bacterial Identification using MALDI-TOF MS
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The bacterial isolates were identi ed by observing nature of growth, cultural characteristics, Gram's staining followed by MALDI-TOF MS analysis (Bruker Daltonics, Bremen, Germany).
The protein was extracted from bacterial cultures using formic acid and analysed by MALDI-TOF MS as per the procedure recommended by Bruker Daltonics. The bacterial growth was pelleted by centrifugation.
The bacterial pellet was mixed with 1 ml of 70% ethanol and centrifuged at 13,000 g for 2 min. The supernatant was discarded. The pellet was dissolved in 25 µl of 70% formic acid and 25 µl of acetonitrile.
The mixture was centrifuged at 13,000 g for 2 min. The supernatant (1 µl) containing the bacterial extract was transferred onto the MALDI target plate and allowed to dry at room temperature. After drying the sample spot was overlaid with 1 µl of MALDI matrix (a saturated solution of a-cyano-4-hydroxycinnamic acid in 50% acetonitrile-2.5% tri uoroacetic acid) and dried in the air. Mass spectra were then acquired by Micro ex MALDI-TOF MS. MALDI BioTyper 2.0 software was used for spectral analysis and comparison with the MALDI BioTyper database. The score ≥ 2 was considered as valid for species identi cation while the score between 1.7 and 2 was considered as valid for genus identi cation. Score ≤ 1.7 was considered invalid.
The protein was extracted from bacterial cultures using formic acid and analysed by MALDI-TOF MS as per the procedure recommended by Bruker Daltonics. The bacterial growth was pelleted by centrifugation.
The bacterial pellet was mixed with 1 ml of 70% ethanol and centrifuged at 13,000 g for 2 min. The supernatant was discarded. The pellet was dissolved in 25 µl of 70% formic acid and 25 µl of acetonitrile.
The mixture was centrifuged at 13,000 g for 2 min. The supernatant (1 µl) containing the bacterial extract was transferred onto the MALDI target plate and allowed to dry at room temperature. After drying the sample spot was overlaid with 1 µl of MALDI matrix (a saturated solution of a-cyano-4-hydroxycinnamic acid in 50% acetonitrile-2.5% tri uoroacetic acid) and dried in the air. Mass spectra were then acquired by Micro ex MALDI-TOF MS. MALDI BioTyper 2.0 software was used for spectral analysis and comparison with the MALDI BioTyper database. The score ≥ 2 was considered as valid for species identi cation while the score between 1.7 and 2 was considered as valid for genus identi cation. Score ≤ 1.7 was considered invalid.
6
Prevalence of mcr-5 in Backyard Pigs
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A total of 336 faecal samples were collected from backyard pigs from 194 households across 12 villages in rural areas of Shandong Province, China, in August 2017, using the ESwab Collection and Transport System (Copan, Brescia, Italy). The village and household selection methods have been described previously.12 All samples were enriched in 1 mL of LB broth containing 10 mg/L vancomycin at 37°C overnight. Total DNA was extracted from 500 μL of each of the enriched cultures using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) and the DNA used for screening of mcr-5 by PCR, as previously reported.6 (link) The enriched samples positive for mcr-5 were inoculated onto Salmonella Shigella agar (Binhe Microorganism Reagent Co., Hangzhou, China) containing 2 mg/L colistin. One random colony of each morphotype was selected for mcr-5 gene detection. Species identification of mcr-5-positive isolates was conducted by 16S rRNA gene sequencing and MALDI-TOF MS analysis (Bruker Daltonik GmbH, Bremen, Germany).11 mcr-5-positive isolates were also subjected to antimicrobial susceptibility testing for colistin and eight other commonly used antibiotics, using the broth dilution method and interpreted according to CLSI M100-S25,13 VET01-S214 and EUCAST (version 6.0, http://www.eucast.org/clinical_breakpoints/ ). E. coli strain ATCC 25922 served as the quality control strain.
7
Bacterial Identification Using API and MALDI-TOF
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For an initial identification, a biochemically-based commercial system, manual Api 20 Strep (bioMériuex, Marcy L’Etoile, France) was performed. For the interpretation of results, the manufacturer’s guidelines were followed. Precisely, probability values of >90%, >80% indicated excellent and good levels of identification, respectively. Probability values < 60% indicates a not reliable identification.
The recovered isolates were then identified also by MALDI-TOF MS analysis (Bruker, Daltonics, Germany), to confirm Api 20 Strep results. For MALDI-TOF-MS identification, fresh colonies were used. The protocol used was the following: the bacterial colony was first inoculated in a MALDI-TOF-MS target plate. Subsequently, 1 µL of cinnamic acid matrix solution was added to the sample and dried at room temperature for ten minutes. Afterward, the target plate was placed in the equipment for MALDI-TOF-MS analysis. The identification was based on the score values released by the equipment’s instructions. Specifically, according to Bruker biotyper’s guidelines, a score of ≥2.0 indicated a highly probable species-level identification, a score of 1.70 to 1.99 indicated a secure identification to the genus level, and a score of <1.7 was interpreted as no identification.
S. zooepidemicus ATCC® 53698TM was included as positive control strain.
The recovered isolates were then identified also by MALDI-TOF MS analysis (Bruker, Daltonics, Germany), to confirm Api 20 Strep results. For MALDI-TOF-MS identification, fresh colonies were used. The protocol used was the following: the bacterial colony was first inoculated in a MALDI-TOF-MS target plate. Subsequently, 1 µL of cinnamic acid matrix solution was added to the sample and dried at room temperature for ten minutes. Afterward, the target plate was placed in the equipment for MALDI-TOF-MS analysis. The identification was based on the score values released by the equipment’s instructions. Specifically, according to Bruker biotyper’s guidelines, a score of ≥2.0 indicated a highly probable species-level identification, a score of 1.70 to 1.99 indicated a secure identification to the genus level, and a score of <1.7 was interpreted as no identification.
S. zooepidemicus ATCC® 53698TM was included as positive control strain.
8
Surveillance of Carbapenem-Resistant Gram-Negatives
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The University Hospital Città della Salute e della Scienza di Torino is a tertiary-care teaching hospital in Turin, North-western Italy. Four national hospitals (San Giovanni Battista Molinette General Hospital; CTO Trauma Orthopaedic Hospital; Regina Margherita Paediatric Hospital and Sant’Anna Maternity Hospital) belong to this facility, which counts over 2.300 beds and approximately 80,000 admissions per year. Here, a proactive surveillance program to detect intestinal colonization by CPE and carbapenem-resistant Gram-negative (CR-GN) organisms among Enterobacterales, P. aeruginosa and Acinetobacter baumannii isolates has been adopted in acute and chronic care facilities for new admissions and for inpatients on a weekly basis. Rectal swabs are collected using the FecalSwab™ system (Copan, Brescia, Italy) and inoculated on a chromogenic screening plate (Chromatic CRE medium, Liofilchem, Roseto degli Abruzzi, Italy) by automated direct plating using the WASP® instrument (Copan, Brescia, Italy). Overnight colonies are identified by MALDI-TOF MS analysis (Bruker Daltonics GmbH, Bremen, Germany), and carbapenemase production in Enterobacterales isolates is investigated by genotypic testing (Xpert Carba-R assay; Cepheid Sunnyvale, CA) and/or lateral flow immunoassay (NG-test CARBA 5; NG Biotech, Guipry, France).
9
Bacterial Identification using MALDI-TOF MS
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The bacterial isolates were identi ed by observing nature of growth, cultural characteristics, Gram's staining followed by MALDI-TOF MS analysis (Bruker Daltonics, Bremen, Germany).
The protein was extracted from bacterial cultures using formic acid and analysed by MALDI-TOF MS as per the procedure recommended by Bruker Daltonics. The bacterial growth was pelleted by centrifugation.
The bacterial pellet was mixed with 1 ml of 70% ethanol and centrifuged at 13,000 g for 2 min. The supernatant was discarded. The pellet was dissolved in 25 µl of 70% formic acid and 25 µl of acetonitrile.
The mixture was centrifuged at 13,000 g for 2 min. The supernatant (1 µl) containing the bacterial extract was transferred onto the MALDI target plate and allowed to dry at room temperature. After drying the sample spot was overlaid with 1 µl of MALDI matrix (a saturated solution of a-cyano-4-hydroxycinnamic acid in 50% acetonitrile-2.5% tri uoroacetic acid) and dried in the air. Mass spectra were then acquired by Micro ex MALDI-TOF MS. MALDI BioTyper 2.0 software was used for spectral analysis and comparison with the MALDI BioTyper database. The score ≥ 2 was considered as valid for species identi cation while the score between 1.7 and 2 was considered as valid for genus identi cation. Score ≤ 1.7 was considered invalid.
The protein was extracted from bacterial cultures using formic acid and analysed by MALDI-TOF MS as per the procedure recommended by Bruker Daltonics. The bacterial growth was pelleted by centrifugation.
The bacterial pellet was mixed with 1 ml of 70% ethanol and centrifuged at 13,000 g for 2 min. The supernatant was discarded. The pellet was dissolved in 25 µl of 70% formic acid and 25 µl of acetonitrile.
The mixture was centrifuged at 13,000 g for 2 min. The supernatant (1 µl) containing the bacterial extract was transferred onto the MALDI target plate and allowed to dry at room temperature. After drying the sample spot was overlaid with 1 µl of MALDI matrix (a saturated solution of a-cyano-4-hydroxycinnamic acid in 50% acetonitrile-2.5% tri uoroacetic acid) and dried in the air. Mass spectra were then acquired by Micro ex MALDI-TOF MS. MALDI BioTyper 2.0 software was used for spectral analysis and comparison with the MALDI BioTyper database. The score ≥ 2 was considered as valid for species identi cation while the score between 1.7 and 2 was considered as valid for genus identi cation. Score ≤ 1.7 was considered invalid.
10
MALDI-TOF-MS Analysis of Okra N-Glycans
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MALDI-TOF-MS analysis (Bruker Daltonics, Bremen, Germany) was performed to determine the detailed N-glycan structures (Zhao et al., 2018a) . The lyophilised N-glycans of the okra glycoprotein were added to 5 μL of methanol:water (MW; 1:1, v/v). Then, 1 µL of the sample mixture was deposited on the MTP target plate, and air-dried. The same volume of 20 mg/mL DHB in MW was spotted to recrystallise the glycans. The instrument was externally calibrated using peptide calibration standards. The spectrum was obtained in the positive ion mode with the maximum accelerating voltage of 25 kV. The m/z data and the structure of the N-glycans were checked using GlycoWorkbench software mainly designed to assist the annotation of MS/MS data by allowing the user to quickly define a candidate structure and test its fragmentation against a peak list (Tissot et al., 2008) .
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