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Summary: The study explores the enhanced functional and bioactive properties of Konjac Glucomannan Hydrolysate (KGMH) by partially degrading Konjac Glucomannan (KGM) using β‐mannanase over 60 min. KGM concentrations (40% and 50% w/w) were treated with 200–300 U g−1 of the enzyme over 60 min. The hydrolysis of KGM was monitored by colorimetry, with DP values ranging from 4.20 to 6.16 for 40% KGM and 4.10 to 4.60 for 50% KGM. MALDI‐TOF‐MS analysis confirmed typical oligosaccharides with DP values from 2 to 9 and some acetyl substitutions. The optimal conditions of 40% KGM with 250 U g−1 enzyme and varying hydrolysis times produced KGMHs with a wide range of DPs, demonstrating in vitro antioxidant and anti‐glycation activities. The results showed significant bioactivities (P < 0.05) positively correlated with lower DP values. This study emphasises the potential of KGMH as a novel functional food ingredient, highlighting its bioactive properties and the significant impact of DPs on the biological functionality of saccharides. [ABSTRACT FROM AUTHOR]

Iron plays vital roles in important biological processes in fish, but can be toxic in high concentrations. The information on metalloproteins that participate in maintenance of Fe homeostasis in an esocid fish, the northern pike, as an important freshwater bioindicator species, are rather scarce. The aim of this study was to identify main cytosolic constituents that sequester Fe in the northern pike liver. The method applied consisted of two-dimensional HPLC separation of Fe-binding biomolecules, based on anion-exchange followed by size-exclusion fractionation. Apparent molecular masses of two main Fe-metalloproteins isolated by this procedure were ~360 kDa and ~50 kDa, with the former having more acidic pI, and indicated presence of ferritin and hemoglobin, respectively. MALDI-TOF-MS provided confirmation of ferritin subunit with a m/z peak at 20.65 kDa, and hemoglobin with spectra containing main m/z peak at 16.1 kDa, and smaller peaks at 32.1, 48.2, and 7.95 kDa (single-charged Hb-monomer, dimer, and trimer, and double-charged monomer, respectively). LC-MS/MS with subsequent MASCOT database search confirmed the presence of Hb-β subunits and pointed to close relation between esocid and salmonid fishes. Further efforts should be directed towards optimization of the conditions for metalloprotein analysis by mass spectrometry, to extend the knowledge on intracellular metal-handling mechanisms. [ABSTRACT FROM AUTHOR]

A facile and mild method based on self-assembled lysozyme (LYZ) to fabricate bifunctional MNPs@UIO-66-Arg core-shell-satellite nanocomposites (CSSNCs) is reported for the high-efficiency enrichment of phosphopeptides. Under physiological conditions, LYZ rapidly self-assembled into a robust coating on Fe3O4@SiO2 magnetic nanoparticles (MNPs) with abundant surface functional groups, which effectively mediate heterogeneous nucleation and growth of UIO-66 nanocrystals. Well-defined MNPs@UIO-66 CSSNCs with stacked pores, showing high specific surface area (333.65 m2 g− 1) and low mass transfer resistance, were successfully fabricated by fine-tuning of the reaction conditions including reaction time and acetic acid content. Furthermore, the UIO-66 shells were further modified with arginine to obtain bifunctional MNPs@UIO-66-Arg CSSNCs. Thanks to the unique morphology and synergistic effect of Zr-O clusters and guanidine groups, the bifunctional MNPs@UIO-66-Arg CSSNCs exhibited outstanding enrichment performance for phosphopeptides, delivering a low limit of detection (0.1 fmol), high selectivity (β-casein/BSA, mass ratio 1:2000), and good capture capacity (120 mg g− 1). The mechanism for phosphopeptides capture may attribute to the hydrogen bonds, electrostatic interactions, and Zr–O–P bonds between phosphate groups in peptides and guanidyl/Zr-O clusters on bifunctional MNPs@UIO-66-Arg CSSNCs. In addition, the small stacking pores on the core-shell-satellite architecture may selectively capture phosphopeptides with low molecular weight, eliminating interference of other large molecular proteins in complex biological samples. [ABSTRACT FROM AUTHOR]

Critical septic conditions caused by infectious pathogens are one of the leading causes of death in children glob- ally. The lack of introduced algorithms for rapid assessment of the condition of these patients, the use of inflammatory markers with low specificity and sensitivity and the use of standard microbiological methods pose a risk of delaying the diagnosis and treatment in children with infectious complica- tions. The aim of this scientific review is to create and test a convenient algorithm for early assessment, diagnosis and treatment of septic conditions in children. It will be assessed the physiological condition of patients using pSOFA, PRISM III and IV scores and the risk of mortality will be calculated also. The routine biochemical parameters of inflammation, procalcitonin will be compared with the new ones. Innovative markers of inflammation will be studied - Presepsin, soluble mannose receptor (sMR) by ELISA method for research purposes. An innovative and rapid method with high sensitivity and specificity (MALDI-TOF-MS) for the species identification of the pathogens will be performed, based on microbial protein analysis, incl. direct identification of the causative agent in the blood sample within 1- 2 hours. As a result, it is expected: to create a comprehensive assessment of critical and septic conditions in children by using innovative scores for the first time in Bulgaria; to find a correlation between the effective- ness of new inflammatory laboratory markers with the old ones and to choose the most appropriate combination of markers for an early detection of inflammation in the course of sepsis development in children; to improve the diagnostic process of microbial identification by using an innovative method for early detection of the infectious pathogen associated with the septic condition. More importantly, it will help the preven- tion, prognosis and will improve the therapeutic approach in children with septic and critical conditions. [ABSTRACT FROM AUTHOR]

Background Isepamicin is a 1-N-S-a-hydroxy-b-aminopropionyl derivative of gentamicin B and the spectrum of pathogenic microorganisms covered by it and its effectiveness is similar to that of amikacin except the action of aminoglycoside inhibitor enzymes is ineffectual on it. Material and Methods We performed a prospective study in the Bacteriology section of the Department of Microbiology at a 1,600-bedded hospital in Northern India from Jan 2022 to March 2022. Isepamicin was tested for susceptibility against gram-negative bacteria, identified by routine biochemicals and matrix-assisted-desorption/ionization –time of flight-mass spectrometry (MALDI-TOF-MS) assay. The antibiotic susceptibility testing for each of the isolates was performed by Kirby Bauer's disc diffusion method, according to the CLSI 2019 guidelines. Results The majority of isolates were obtained from blood samples (50, 39.1%). Among the non-inducible Enterobacteriaceae , Escherichia coli was least susceptible to amikacin (8/27, 29.63%) and most susceptible to isepamicin (18/27, 66.67%). Klebsiella pneumoniae followed the same pattern of susceptibility as E. coli and was least susceptible to Amikacin (20/46, 43.48%) and most susceptible to isepamicin (24/46, 52.17%). Enterobacter cloacae (6/7, 85.71%) was most susceptible to both amikacin and isepamicin, followed by 71.43% (5/7, 71.43%) susceptibility to gentamicin and tobramycin each. Enterobacter aerogenes was equally 53.33% (8/15) susceptible to all antibiotics. Pseudomonas aeruginosa was the most susceptible isolate to all antibiotics (18/21, 85.71%). Conclusion Isepamicin is a potential antimicrobial agent for treating an array of gram-negative bacteria-associated infections and shows better in vitro activity than older aminoglycoside agents. [ABSTRACT FROM AUTHOR]

Polymyxin antibiotics are the last resort for treating patients in intensive care units infected with multiple-resistant Gram-negative bacteria. Due to their polycationic structure, their mode of action is based on an ionic interaction with the negatively charged lipid A portion of the lipopolysaccharide (LPS). The most prevalent polymyxin resistance mechanisms involve covalent modifications of lipid A: addition of the cationic sugar 4-amino-L-arabinose (L-Ara4N) and/or phosphoethanolamine (pEtN). The modified structure of lipid A has a lower net negative charge, leading to the repulsion of polymyxins and bacterial resistance to membrane disruption. Genes encoding the enzymatic systems involved in these modifications can be transferred either through chromosomes or mobile genetic elements. Therefore, new approaches to resistance diagnostics have been developed. On another note, interfering with these enzymatic systems might offer new therapeutic targets for drug discovery. This literature review focuses on diagnostic approaches based on structural changes in lipid A and on the therapeutic potential of molecules interfering with these changes. [ABSTRACT FROM AUTHOR]

Background: Meningitis can be attributed to bacterial, fungal, or viral agents. In this study, we demonstrate the common bacterial agents causing meningitis along with their antibiotics susceptibility pattern in patients of all age groups. Material and Methods: This retrospective, observational study was carried out in the Department of Microbiology with cerebrospinal fluid (CSF) samples collected from November 2019 to May 2022. We collected 1986 nonrepeat CSF samples from clinically suspected patients of bacterial meningitis, and clinical information about the patients was extracted from the hospital information system. Results: Out of the 1986 CSF samples included in our study, 150 (7.55%) were found to be positive on bacterial culture. Most of our patients were in the age group of 0-20 years. Common clinical manifestations observed in our patients were: high-grade fever, 87 patients (58%); severe headache, 126 patients (84%); neck rigidity, 47 patients (31.3%); altered mental status, 76 patients (50.7%) and photophobia, 83 patients (55.3%). The most commonly isolated bacteria was Acinetobacter species (30%). The mean length of hospitalization (37.76 ± 25.30), the mean total cell count, high levels of protein (mg/dl) and low levels of glucose (mg/dl) of CSF were statistically significant in meningitis caused by multidrug-resistant bacteria. Conclusion: We recognized the spectrum of pathogens causing meningitis at our center along with the antibiotic resistance pattern to guide and facilitate early treatment by primary health care professionals and family medicine practitioners. [ABSTRACT FROM AUTHOR]

Backfat trait is an important economic trait and highly heritable, but difficult to evaluate. Thus, it is of great significance to explore optimal backfat thickness of pigs by using marker-assisted selection (MAS) to speed up its breeding process and improve economic efficiency. This study aimed to investigate the relationship between genetic variations (e.g., SSRs) and backfat of Qinghai Bamei pigs using MALDI-TOF Mass Spectrometry (MALDI-TOF-MS). Herein, five alternative SSR loci (namely V1, V2, V3, V4 and V5) were selected for subsequent detection. The results suggested that 3 (141-, 143- and 145-), 3 (128-, 130- and 132-), 2 (160- and 162-), 2 (136- and 139-) and 3 (170-, 184- and 192-) alleles of V1, V2, V3, V4 and V5 were found, respectively. Subsequent analysis showed that there was linkage equilibrium among five SSRs and Hap19 (13.1%) (141-/132-/160-/139-/192-) had the highest haplotype frequency. Among these five SSR loci, V1, V2 and V3 loci were significantly associated to the backfat of Qinghai Bamei sows. These findings enriched the study of SSRs in Qinghai Bamei pigs, and (AC)n (Chr15:85485851-85485995), (AC)n (Chr10:52724583–52724713) and (TG)n (Chr4:90732644–90732802) could be utilized as the candidate locus for MAS in pig industry. Five novel SSR loci was identified in pigs through MALDI-TOF MS. V1, V2 and V3 loci was were significantly associated to the backfat of pigs. [ABSTRACT FROM AUTHOR]

To capture the structure of assembled hemagglutinin (HA) nanoparticles at single-particle resolution, HA-specific antigen binding fragments (Fabs) were labeled by fluorescent (FLR) dyes as probes to highlight the HA trimers displayed on the assembled tetravalent HA nanoparticles for a qualitative localization microscopic study. The FLR dyes were conjugated to the Fabs through N-hydroxysuccinimide (NHS) ester mediated amine coupling chemistry. The labeling profile, including labeling ratio, distribution, and site-specific labeling occupancy, can affect the imaging results and introduce inconsistency. To evaluate the labeling profile so as to evaluate the labeling efficiency, a combination of intact mass measurement by MALDI-MS and peptide mapping through LC-MS/MS was implemented. At the intact molecular level, the labeling ratio and distribution were determined. Through peptide mapping, the labeled residues were identified and the corresponding site-specific labeling occupancy was measured. A systematic comparative investigation of four different FLR-labeled 1H01-Fabs (generated from H1 strain HA specific mAb 1H01) allowed accurate profiling of the labeling pattern. The data indicate that the labeling was site-specific and semiquantitative. This warrants the consistency of single-particle fluorescent imaging experiments and allows a further imaging characterization of the single nanoparticles. [ABSTRACT FROM AUTHOR]

The acylation of a cyclodextrin‐based [3]rotaxane was implemented with anhydride reagents following a facile procedure. Methacrylic anhydride facilitated the introduction of a vinyl‐group into the rotaxane structure toward achieving a crosslinker. Interestingly, only a few methacrylic units were attached to the cyclodextrin at reaction temperatures of up to 60 °C even with an excess amount of the reagent, probably due to steric hindrance. Therefore, the number of vinyl groups on the [3]rotaxane framework was easily controlled to be approximately two units via a simple random acylation, which worked effectively as a facile synthesis route for the rotaxane crosslinker. The acylation behavior was investigated in detail by NMR and MALDI‐TOF‐MS analyses. Such a proposed approach will help overcome the trade‐off between convenience and utility associated with the mono‐modification of cyclodextrins. [ABSTRACT FROM AUTHOR]

Herein, we isolated the pathogenic strain ZZ051 from hemorrhagic channel catfish (Ictalurus punctatus). Physiological and biochemical identification, 16S rRNA gene sequence analysis, and MALDI-TOF-MS showed that the ZZ051 strain was Aeromonas veronii. After artificial infection, the diseased fish showed symptoms similar to the natural disease, and the characteristics of the bacteria reisolated from the tissues were the same as those of the original infection, indicating that the isolated strain ZZ051 was the pathogen responsible for the channel catfish disease. The ZZ051 isolate was highly sensitive to enrofloxacin but resistant to florfenicol. This study provided a theoretical basis for preventing and controlling hemorrhagic disease in channel catfish. [ABSTRACT FROM AUTHOR]

The occurrence of pathogenic bacteria has emerged as a plausible key component of summer mortalities in mussels. In the current research, four bacterial isolates retrieved from moribund Greenshell࣪ mussels, Perna canaliculus , from a previous summer mortality event, were tentatively identified as Vibrio and Photobacterium species using morpho-biochemical characterization and MALDI-TOF MS and confirmed as V. celticus, P. swingsii, P. rosenbergii , and P. proteolyticum using whole genome sequencing. These isolates were utilized in a laboratory challenge where mussels were injected with cell concentrations ranging from 105 to 109 CFU/mussel. Of the investigated isolates, P. swingsii induced the highest mortality. Additionally, results from quantitative polymerase chain reaction analysis, focusing on known virulence genes were detected in all isolates grown under laboratory conditions. Photobacterium rosenbergii and P. swingsii showed the highest expression levels of these virulence determinants. These results indicate that Photobacterium spp. could be a significant pathogen of P. canaliculus , with possible importance during summer mortality events. By implementing screening methods to detect and monitor Photobacterium concentrations in farmed mussel populations, a better understanding of the host–pathogen relationship can be obtained, aiding the development of a resilient industry in a changing environment. [ABSTRACT FROM AUTHOR]

Glycans are involved in many fundamental cellular processes such as growth, differentiation, and morphogenesis. However, their broad structural diversity makes analysis difficult. Glycomics via mass spectrometry has focused on the composition of glycans, but informatics analysis has not kept pace with the development of instrumentation and measurement techniques. We developed Toolbox Accelerating Glycomics (TAG), in which glycans can be added manually to the glycan list that can be freely designed with labels and sialic acid modifications, and fast processing is possible. In the present work, we improved TAG for large-scale analysis such as cohort analysis of serum samples. The sialic acid linkage-specific alkylamidation (SALSA) method converts differences in linkages such as α2,3- and α2,6-linkages of sialic acids into differences in mass. Glycans modified by SALSA and several structures discovered in recent years were added to the glycan list. A routine to generate calibration curves has been implemented to explore quantitation. These improvements are based on redefinitions of residues and glycans in the TAG List to incorporate information on glycans that could not be attributed because it was not assumed in the previous version of TAG. These functions were verified through analysis of purchased sera and 74 spectra with linearity at the level of R2 > 0.8 with 81 estimated glycan structures obtained including some candidate of rare glycans such as those with the N,N'-diacetyllactosediamine structure, suggesting they can be applied to large-scale analyses. [ABSTRACT FROM AUTHOR]

Petroleum hydrocarbons are one of the most well-known environmental pollutants not only for plants, and animals but also for humans because of their carcinogenic mutagenic, toxic, and teratogenic effects. Hence, various remediation technologies have been used to overcome these environmental pollutants. Among them, bioremediation is novel promising sustainable approach for treating petroleum and petroleum derivative raw material deleterious components with their effective, low-cost eco-friendly features. In this perspective, the evaluation of soil samples was collected from areas containing petroleum and petroleum derivatives from Kazanlı and Karaduvar refinery regions in Mersin-Turkey. Hence, potential hydrocarbon and petroleum degrading bacteria were identified and characterized by the MALDI-TOF MS method as well as biochemical, physiological, and morphological tests. According to the MALDI-TOF MS analysis; members of Bacillus, Stenotrophomonas, Enterobacter, Pseudomonas, Cupriavidus, Acinetobacter, and Escherichia were successfully identified as hydrocarbon and petroleum degrading bacteria. [ABSTRACT FROM AUTHOR]

During the SARS-CoV-2 pandemic, many virus-binding monoclonal antibodies have been developed for clinical and diagnostic purposes. This underlines the importance of antibodies as universal bioanalytical reagents. However, little attention is given to the reproducibility crisis that scientific studies are still facing to date. In a recent study, not even half of all research antibodies mentioned in publications could be identified at all. This should spark more efforts in the search for practical solutions for the traceability of antibodies. For this purpose, we used 35 monoclonal antibodies against SARS-CoV-2 to demonstrate how sequence-independent antibody identification can be achieved by simple means applied to the protein. First, we examined the intact and light chain masses of the antibodies relative to the reference material NIST-mAb 8671. Already half of the antibodies could be identified based solely on these two parameters. In addition, we developed two complementary peptide mass fingerprinting methods with MALDI-TOF-MS that can be performed in 60 min and had a combined sequence coverage of over 80%. One method is based on the partial acidic hydrolysis of the protein by 5 mM of sulfuric acid at 99 °C. Furthermore, we established a fast way for a tryptic digest without an alkylation step. We were able to show that the distinction of clones is possible simply by a brief visual comparison of the mass spectra. In this work, two clones originating from the same immunization gave the same fingerprints. Later, a hybridoma sequencing confirmed the sequence identity of these sister clones. In order to automate the spectral comparison for larger libraries of antibodies, we developed the online software ABID 2.0. This open-source software determines the number of matching peptides in the fingerprint spectra. We propose that publications and other documents critically relying on monoclonal antibodies with unknown amino acid sequences should include at least one antibody fingerprint. By fingerprinting an antibody in question, its identity can be confirmed by comparison with a library spectrum at any time and context. [ABSTRACT FROM AUTHOR]

Phosphate-solubilising bacteria are an important component of sustainable agriculture but their number and performance are reduced due to the presence of pesticides in soils. The potato requires a high amount of phosphorus making it a fertilizer-demanding crop. Therefore, the present study was conducted with the aim to develop pesticide-tolerant phosphate-solubilising bacterial inoculants for improving growth and phosphorus content in potato. Phosphate-solubilising bacteria were isolated from potato rhizosphere and screened for pesticide tolerance, phosphate solubilisation in the presence of pesticides and growth promotion of potato in pesticide-amended soils. Nineteen phosphate-solubilising bacteria isolated from potato rhizosphere showed phosphate solubilisation ranging from 115 to 747 µg ml−1 with significant difference among the isolates. Out of 19 isolates tested for tolerance against two commonly used pesticides in potato, two isolates showed tolerance up to 7.5 μl ml−1 (3 times the recommended dose) of chlorpyrifos and 0.75 g ml−1 (2 times the recommended dose) of carbofuran. These two pesticide-tolerant bacterial isolates also showed phosphate-solubilisation in the presence of chlorpyrifos and carbofuran, although the phosphate-solubilising ability decreased significantly with increased pesticide concentrations. The isolates were identified as Arthrobacter oxydans PR1PSB2 and Bacillus flexus PR2PSB1 based on phenotypic features, biochemical tests, and matrix-assisted laser desorption/ionisation-time of flight mass spectroscopy (MALDI-TOF–MS). The bacterial treatments Arthrobacter oxydans PR1PSB2 and Bacillus flexus PR2PSB1 alone or in combined application exhibited a significant increase in the growth parameters, phosphorus (P) content, and soil available P over the uninoculated control under greenhouse conditions in potato in the presence of pesticides. However, the plant growth promotion and P content decreased with an increase in pesticide concentrations. Pesticide-tolerant phosphate-solubilising Arthrobacter oxydans and Bacillus flexus from potato are reported for the first time. [ABSTRACT FROM AUTHOR]

Human milk oligosaccharides (HMOs) are bioactive components which play an important role in infant health. HMO composition is vulnerable to changes of maternal conditions including lactation stages and maternal phenotypes. Pregnant diseases such as gestational diabetes mellitus (GDM) are commonly found in women during pragnancy, and may cause disorder in maternal physiological metabolism which is harmful to infants. Unfortunately, anlysis of oligosaccharides from women with GDM is limited. To address this issue, we analyzed HMO compositions and profiles in breast milk from women with GDM using an established 96-well plate permethylation platform and MALDI-TOF-MS. We enrolled 127 women with GDM, and investigated HMO abundances in colostrum, transition milk, and mature milk respectively. We found that GDM affected HMO compositions in breast milk, and the level of fucosylation became higher over the course of lactation for all the women with GDM. Interestingly, the relative abundances of fucosylated HMOs in different lactation stages were affected differentially by GDM, with the most pronounced effect in colostrum. In particular, the relative abundances of H3N1F1 and H3N1F2 sharply decreased over time, showing very low levels in late lactation. These differences in our findings need further investigation to develop optimal feeding for mothers with GDM. [Display omitted] • The high-throughput 96-well plate permethylation platform was used to investigated HMO profiles for the first times. • The abundance of fucosylation of highly polymerized HMO increased over lactation in breast milk from women with GDM. • H3N1F1 and H3N1F2 levels in milk from mothers with GDM were significantly reduced in late lactation than in healthy mothers. [ABSTRACT FROM AUTHOR]

Mastitis, a prevalent and economically important disease in the dairy industry, poses substantial challenges to dairy cow health, milk quality, and farm profitability worldwide. Mastitis is predominantly caused by bacterial infections. The objective of this study was to estimate the sensitivity (Se) and specificity (Sp) of bacterial culture and the VetMAX™ MastiType Multi Kit PCR in identified clinical mastitis pathogens. A total of 396 quarter-level milk samples were collected from 396 cows with clinical mastitis on 29 farms in the subtropical dairy region of Australia between March and December 2021. These samples were cultured and tested by PCR, and analysed using Bayesian latent class analysis under the assumption of one population two tests and also of three populations two tests, by dividing the population into subpopulations based on regions. Informative priors used in the analysis were calculated from published evidence. Models were compared using the Deviance Information Criterion (DIC). Sensitivity analysis was performed to evaluate the impact of changes in priors. The most common isolates cultured and detected by PCR were Streptococcus uberis (17.4 % and 27.3 %, respectively) and Escherichia coli (12.6 % and 25.0 %, respectively). Under the assumption of one population two tests , the Se of PCR (at cycle threshold (Ct) ≤ 37) was higher than that of bacterial culture for both pathogens: for E. coli , the Se was 50.2 % (95 % posterior probability interval (PPI): 37.4; 74.1) for bacterial culture, and 93.7 % (95 % PPI: 85.5; 98.4) for PCR. For S. uberis , the Se was 50.4 % (95 % PPI: 40.9; 61.3) for bacterial culture, and 81.5 % (73.0; 88.9) for PCR. Conversely, the Sp of bacterial culture was higher than that of PCR for both pathogens: for E. coli , the Sp was 99.2 % (97.8; 100) for bacterial culture, and 95.1 % (87.8; 99.4) for PCR. For S. uberis , the Sp was 99.2 % (95 % PPI: 97.6; 100) for bacterial culture, and 96.7 % (95 % PPI: 92.1; 99.2) for PCR. Bayesian latent class analysis with three populations two tests was only performed for S. uberis. For E. coli, this could not be performed because there were no PCR-positive results in one subpopulation. Under the assumption of three populations two tests , for S. uberis, the Se was 49.6 % (40.6; 59.4) for bacterial culture, and 81.1 % (72.6; 88.6) for PCR; and the Sp for bacterial culture was 99.1 % (97.7; 100), and for PCR was 96.9 % (93.0; 99.3). The DIC for the one population two tests model was lower than the DIC for the three populations two tests model. The sensitivity analysis for the one population two tests model demonstrated that a 10 % reduction in priors led to substantial changes in Se of both bacterial culture and PCR tests for E. coli and S. uberis , with overlap percentages ranging from 80.6 % to 92.2 %. In contrast, the Sp of bacterial culture and PCR tests remained relatively stable despite changes in priors, except for the Sp of PCR test for E. coli. In summary, the VetMAX™ MastiType Multi Kit demonstrated higher Se compared to bacterial culture, suggesting its potential as a routine test for identifying mastitis pathogens in milk samples from cows with clinical mastitis. While the bacterial culture method offered higher Sp in pathogen detection; results obtained following bacterial culture and subsequent susceptibility testing remain valuable, particularly in guiding antimicrobial treatment for mastitis. [ABSTRACT FROM AUTHOR]

The manufacturing of indigo naturalis requires prolonged leaf soaking and lime stirring; the resulting indigo purity is less than 3.00% and the yield of indigo (measured in stems and leaves weight) is less than 0.50%, making it unsuitable for use in industrial procedures like printing and dyeing. An enzymatic method of creating indigo without the requirement for lime was investigated in order to generate high purity indigo. Single factor tests were performed to optimize the enzymatic preparation conditions. The findings showed that 60 °C, pH 5.5, 200 mL of leaves extract containing 0.45 mg/mL indican, and a 4:1 ratio of the acidic cellulose (activity: 9000 U/mL, liquid) to indican were the ideal parameters for enzymatic preparation. The yield of indigo was 40.32%, and the contents of indigo and indirubin were 37.37% and 2.30%, respectively. MALDI-TOF-MS in positive ion mode and UPLC-Q-TOF-MS in both positive and negative ion modes were used to analyze indigo extracts from Baphicacanthus cusia (Nees) Bremek by enzymatic preparation. It has been discovered that 13 alkaloids, 5 organic acids, 3 terpenoids, 3 steroids, 2 flavones, and 7 other compounds are present in indigo extracts. The presence of the indigo, indirubin, isorhamnetin, tryptanthrin, indigodole B, and indigodole C determined by UPLC-Q-TOF-MS was verified by MALDI-TOF-MS analysis. The enzymatic preparation of indigo extracts kept the same chemical makeup as conventional indigo naturalis. Thermal analysis and SEM morphology were used to confirm that there was no lime in the indigo extract. During the enzymatic process, Baphicacanthus cusia (Nees) Bremek was employed more effectively, increasing the yield and purity of indigo. • An enzymatic technique without the need for lime was investigated in order to produce high purity indigo. • The enzymatic preparation of indigo extracts kept the same chemical makeup as conventional indigo naturalis. • The indigo extract obtained through enzymatic production was a loose, inhomogeneous powder that contained 37.37% indigo. [ABSTRACT FROM AUTHOR]

Mussel foot proteins (Mfps) possess unique binding properties to various surfaces due to the presence of L-3,4-dihydroxyphenylalanine (DOPA). Mytilus edulis foot protein-3 (Mefp-3) is one of several proteins in the byssal adhesive plaque. Its localization at the plaque-substrate interface approved that Mefp-3 plays a key role in adhesion. Therefore, the protein is suitable for the development of innovative bio-based binders. However, recombinant Mfp-3s are mainly purified from inclusion bodies under denaturing conditions. Here, we describe a robust and reproducible protocol for obtaining soluble and tag-free Mefp-3 using the SUMO-fusion technology. Additionally, a microbial tyrosinase from Verrucomicrobium spinosum was used for the in vitro hydroxylation of peptide-bound tyrosines in Mefp-3 for the first time. The highly hydroxylated Mefp-3, confirmed by MALDI-TOF-MS, exhibited excellent adhesive properties comparable to a commercial glue. These results demonstrate a concerted and simplified high yield production process for recombinant soluble and tag-free Mfp3-based proteins with on demand DOPA modification. [Display omitted] • Production of recombinant tag-free Mefp-3 using SUMO-fusion technology. • One-pot-strategy for fusion-tag removal and peptide-bound tyrosine hydroxylation. • In vitro application of microbial tyrosinase for efficient DOPA-modification. • DOPA-containing m Mefp-3 displays excellent adsorption and tensile-shear strength. [ABSTRACT FROM AUTHOR]

In eukaryotic organisms, cell-signalling completely relies on Post Translational Modifications (PTMs) that can function as regulatory switches. Phosphorylation is a fundamental and frequently occurring PTM in almost all eukaryotes. Herein, we have studied the importance of protein phosphorylation using classical proteomic techniques in C. elegans upon bacterial infection. The differentially regulated proteins during bacterial infection were excised from SDS-PAGE (One-Dimensional) gel (TiO 2 column elutes) and subjected to MALDI-TOF-MS which ended up in identifying 220 proteins kinetically. KEGG pathway analysis of those proteins suggested that MAPK pathway was part of the innate immunity. Thus, we have characterized p38-MAPK (one of the crucial downstream MAPKs) using immunoblotting, subcellular fractionation, coimmunoprecipitation, LC-MS/MS, bioinformatics studies and qPCR. Meanwhile, KU25 strain (pmk-1 mutant) exhibited an earlier mortality during infection suggesting the crucial role of p38-MAPK during host-pathogen interaction. Interestingly, Reactome pathway analysis of p38 interactors (CoIP coupled to LC-MS/MS) revealed the involvement of various proteolytic pathway players (ubiquitination, SUMOylation and Neddylation) during bacterial infection. Further, the regulation of SUMOylation and Neddylation was identified and validated using immunoblotting and qPCR analyses, respectively. Concisely, our study indicated that bacterial infection triggers the MAPK cascade to elicit innate immunity which in turn recruits proteolytic pathways to counteract the invading pathogen. [Display omitted] • MAPK pathway is required to combat the bacterial exposure • p38 localizes into nucleus during bacterial exposure • LC-MS/MS analysis reveals the interacting partners of p38 during bacterial exposure • C. elegans innate immunity requires proteolysis pathway(s) during bacterial invasion • This study discloses the role of proteolysis pathway along with p38-MAPK during bacterial exposure [ABSTRACT FROM AUTHOR]

• Endophytic microorganisms have been considered a valuable biotechnological tool. • Endophytic from cacti are acknowledged to be a source of bioactive compounds. • Endophytic bacteria from Cereus peruvianus plants were retrieved in the current study. • Endophytic bacteria were isolated from in vivo and in vitro cultivated plants. Somaclones were indicated as the most promising source of endophytic bacteria. Bioprospecting of endophytic microorganisms has been considered a promising resource for obtaining new biomolecules with biotechnological applications. Since few studies are available on the diversity of endophytic bacteria associated with species of the Cactaceae family, current analysis isolates and characterizes the communities of endophytic bacteria associated with the species of C. peruvianus grown in vivo obtained from seed and with somaclone of the same species regenerated in vitro to prospect for their plant-growth promoting activity. Isolation of endophytic bacteria from plants obtained from seeds and from regenerated somaclone in vitro identified nine species of endophytic bacteria (Moraxela osloensis, Corynebacterium mycetoides, Micrococcus luteus, Kocuria palustris, Staphylococcus epidermidis, Staphylococcus cohnii, Pseudomonas stuzeri, Bacillus licheniformis and Microbacterium sp.) not previously described in cacti, and signalized the somaclones as the most promising source of endophytic bacteria for obtaining new biomolecules with biotechnological applications. Somaclones are indicated as the most promising source of endophytic bacteria due to their high concentration of endophytes per gram identified in fresh somaclone tissue, the identification of the highest proportion endophytic bacteria, a source of IAA, and with the highest capacity of biological nitrogen fixation, and EPS production exclusively by the endophytic bacteria isolated from the in vitro regenerated somaclone. [ABSTRACT FROM AUTHOR]

Non-digestible oligosaccharides are associated with positive health outcomes; however, hemicellulosic oligosaccharides of N. arbor-tristis have not been studied. Herein, we report production of hemicellulosic oligosaccharides from its leaves and their identification. A series of complex neutral oligosaccharides (1-16), produced by endo-(1,4)-β-D-glucanase digestion of the alkali extracted xyloglucan, have been identified by chemical, HPAE chromatographic and MALDI-mass spectrometric techniques. Additionally, xylan enriched hemicellulose-A fraction upon endo-(1,4)-β-D-xylanase digestion yield several acidic and neutral xylo-oligosaccharides (17-24), each containing a backbone of β-(1,4)-D-Xylp units substituted at position 2 with a single stub of D-Xylp or 4-O-Me-D-GlcpA residues. The structural diversity and high water solubility of these enzyme-generated oligosaccharides suggested promising health implications. [ABSTRACT FROM AUTHOR]

The problem of heavy metal pollution in nature has increased rapidly in recent years. Hexavalent chromium (Cr+6) is one of the most toxic heavy metals that cause environmental pollution. Although many studies in the literature that illuminate the stress response mechanisms of biological organisms such as bacteria, algae, and plants against heavy metals, there is limited information about revealing the protein level changes of lichen species in response to heavy metal stress. Here, we used a MALDI-TOF-based proteomic assay to determine protein level changes in Pseudevernia furfuracea after exposure to Cr+6 heavy metal stress at 6, 18 and 24 h. It was determined that expression levels of 26, 149 and 66 proteins changed in P. furfuracea. 6, 18 and 24 h after Cr+6 application compared to the control sample, respectively. We identified 9 common proteins expressed at three different time levels (6, 18, 24 h) and evaluated their protein–protein interaction profiles with the STRING tool. According to the results of the study, it was determined that the expression level of six proteins was up-regulated (OP4, KIP3, BNI5, VSP64, HSP 60, BCK1) and three proteins were down-regulated (MNS1, ABZ2, ATG4) from the expression level of nine proteins in total with Cr+6 exposure. It was determined that nine proteins were also found to be effective in biological processes such as stress signaling, transcription regulation and cellular detoxification metabolisms. To confirm the protein expression level, we analyzed the HSP60 protein by western blot assay. It has been shown that exposure to Cr+6 exposure in P. furfuracea caused an increase in HSP60 protein level compared to the control sample (non-exposed Cr+6). In this study, new knowledge are presented for the use of P. furfuracea as a biosorption agent in the removal of industrial wastes in biotechnological applications. [ABSTRACT FROM AUTHOR]

The problem of heavy metal pollution in nature has increased rapidly in recent years. Hexavalent chromium (Cr+6) is one of the most toxic heavy metals that cause environmental pollution. Although many studies in the literature that illuminate the stress response mechanisms of biological organisms such as bacteria, algae, and plants against heavy metals, there is limited information about revealing the protein level changes of lichen species in response to heavy metal stress. Here, we used a MALDI-TOF-based proteomic assay to determine protein level changes in Pseudevernia furfuracea after exposure to Cr+6 heavy metal stress at 6, 18 and 24 h. It was determined that expression levels of 26, 149 and 66 proteins changed in P. furfuracea. 6, 18 and 24 h after Cr+6 application compared to the control sample, respectively. We identified 9 common proteins expressed at three different time levels (6, 18, 24 h) and evaluated their protein–protein interaction profiles with the STRING tool. According to the results of the study, it was determined that the expression level of six proteins was up-regulated (OP4, KIP3, BNI5, VSP64, HSP 60, BCK1) and three proteins were down-regulated (MNS1, ABZ2, ATG4) from the expression level of nine proteins in total with Cr+6 exposure. It was determined that nine proteins were also found to be effective in biological processes such as stress signaling, transcription regulation and cellular detoxification metabolisms. To confirm the protein expression level, we analyzed the HSP60 protein by western blot assay. It has been shown that exposure to Cr+6 exposure in P. furfuracea caused an increase in HSP60 protein level compared to the control sample (non-exposed Cr+6). In this study, new knowledge are presented for the use of P. furfuracea as a biosorption agent in the removal of industrial wastes in biotechnological applications. [ABSTRACT FROM AUTHOR]

Several well-preserved polychrome lacquered coffins were found in Zhang Dong family's tombs in Shaanxi, China, rare in the history of archaeological excavation. In the lacquered coffins, a large amount of dark solid of suspected natural resin was unearthed with mysterious compositions and uses, exerting a tremendous fascination on archaeologists. In this work, a new method was explored for the identification of the suspected natural resin, mainly based on thermogravimetry (TG), elemental analysis (EA), scanning electron microscopy-energy dispersive spectrometry (SEM-EDS), matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and Fourier transform infrared spectrometry (FT-IR). The results suggested that the sample was mainly composed of organics with a content of 81.66% and little inorganics with a content of 2.21% by water excluded. Rosin with the main component of abietic acid (molecular formula C20H30O2) was identified as the principal component of the sample. Dehydrogenated abietic acid (DHA) and other oxides were also tested out. FT-IR confirmed the identification results. SEM revealed the rough surfaces evenly covered with holes of similar sizes. The use of this method directly obtained integral quasi-molecular ion fragments and molecular components of the sample. As a result, intricate multi-stage mass spectrometry is avoidable, which dramatically simplifies the analysis procedure. This approach is simple and effective for the identification of precious relic samples, requires no references, and has potential for the analysis of these kinds of unknown samples. Especially, for the first time, EA is used to identify natural resins from archaeological sites. [ABSTRACT FROM AUTHOR]

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) was used to analyze the polymerization distribution of xylo‐oligosaccharides from birchwood and beechwood xylans following hydrolysis by the xylanase (XynA) from Bacillus subtilis, which was obtained by recombinant expression and purified by Ni2+ affinity column chromatography. The hydrolysis products of birchwood and beechwood xylans were analyzed by thin layer chromatography (TLC) and MALDI‐TOF‐MS. In the birchwood hydrolysate, the main neutral xylo‐oligosaccharides were xylobiose (X2) and xylotriose (X3), and the acidic xylo‐oligosaccharides (polymerization degree 4‐12) were attached via a single methyl‐glucuronic acid sidechain (MeG). Both X2 and X3 were identified in the beechwood xylan hydrolysate and acidic xylo‐oligosaccharides (polymerization degree 4‐16), which were structurally similar to those in the birchwood xylan hydrolysate. Therefore, the recombinant xylanase, XynA, has the potential to produce xylobiose (X2) and xylotriose (X3) as well as acidic xylo‐oligosaccharides (MeGXn) that would be applied in food industry. Practical applications: Xylo‐oligosaccharides are a novel functional food additive, which has great potential in improving the quality of food. Xylo‐oligosaccharides with methyl‐glucuronic acid sidechains (MeG) are acidic xylo‐oligosaccharides (MeGXn), which can be applied in the preparation of drugs for the treatment of cystitis and mucopolysaccharidosis. In this study, xylanase XynA was first obtained by gene cloning and expression and then used to hydrolyze the birchwood and beechwood xylans. The polymerization distribution of xylo‐oligosaccharides generated during the enzymatic digestion was then determined by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Through enzyme hydrolysis, we are able to produce xylobiose and xylotriose for food additives. [ABSTRACT FROM AUTHOR]

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by fungi of the Ustilaginaceae family in the presence of hydrophobic carbon sources like plant oils. In the present study,we investigated the structural composition of MELs produced from castor oil using seven different microorganisms and compared them to MEL structures resulting fromother plant oils. Castor oil is an industrially relevant plant oil that presents as an alternative to currently employed edible plant oils like rapeseed or soybean oil. The main fatty acid in castor oil is the mono-hydroxylated ricinoleic acid, providing the possibility to produce novel MEL structures with interesting features. Analysis of the produced MELs from castor oil by different chromatographic and mass spectrometry techniques revealed that all seven microorganisms were generally able to integrate hydroxylated fatty acids into the MEL molecule, although at varying degrees. These novel MELs containing a hydroxy fatty acid (4-O-[2'-O-alka(e)noyl-3'-O-hydroxyalka(e)noyl-4'/6'-O-acetyl-β- D-mannopyranosyl]-erythritol) were more hydrophilic than conventional MEL and therefore showed a different elution behavior in chromatography. Large shares of novel hydroxy MELs (around 50% of total MELs) were found for the two MEL-B/C producing species Ustilago siamensis and Ustilago shanxiensis, but also for the MEL-A/B/C producer Moesziomyces aphidis (around 25%). In addition, triacylated hydroxylated MELs with a third long-chain fatty acid esterified to the free hydroxyl group of the hydroxy fatty acid were identified for some species. Overall, production of MEL from castor oil with the investigated organisms provided a complex mixture of various novel MEL structures that can be exploited for further research. [ABSTRACT FROM AUTHOR]

Seasonal allergic rhinitis (SAR) is an inflammatory process. In this pilot study, matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS)-based analyses were conducted to investigate the effects of SAR on serum N-glycome in a small dataset (n = 10 for both SAR patients and controls). It was detected that two N-glycan compositions (H6N5E2L1 and H6N5E1L2) were down-regulated in SAR patients. Additionally, five tri-antennary N-glycan traits, including both galactosylated and sialylated (A3GS and A3GL) and non-fucosylated (A3F0GE, A3F0GL, A3F0GS) ones, were decreased. Furthermore, three high-antennary and fucosylated N-glycan traits (A3F, A3EF and A4F) were increased in SAR patients compared to controls. [ABSTRACT FROM AUTHOR]

Background: Infection of Helcococcus kunzii(H. kunzii) from diabetic foot wound is rarely reported. This case report describes the infection of H.kunzii and highlights the therapeutic effect on H.kunzii from a diabetic foot wound.Case Presentation: In this study, one H. kunzii strain was isolated from a patient with diabetic foot, which was confirmed by 16S rRNA gene analysis and MALDI-TOF-MS. It is the first Chinese case of H. kunzii in a patient with diabetic foot. As a result of the lack of antibiotic sensitivity data and multiple comorbidities, antibiotics were used cautiously, and those administered during the first 3 months were ineffective. Then, vacuum sealing drainage (VSD) was applied during hospitalization; no antibiotics were used and the wound healed well.Conclusions: VSD alone may be more effective in treating diabetic feet infected with H. kunzii, which may provide reference for clinical treatment of H. kunzii infection from diabetic foot. [ABSTRACT FROM AUTHOR]

By employing the mixed matrix, we established a new MALDI-TOF MS platform for rapid and accurate identification of clinical microorganisms. [Display omitted] • A cooperative matrix was demonstrated more peaks in the analysis of bacteria. • Much more components presented in the bacterial colony can be easily identified. • The characteristic peaks identified can directly distinguish similar bacteria. • The present method is simple, rapid, reproducible and accurate. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is emerging as the most powerful tool for the identification of bacteria. Herein, we demonstrated that the capability of MALDI-TOF-MS towards the discrimination of clinical bacteria can be further dramatically improved. A cooperative matrix consisting of (E)-propyl α-cyano-4-hydroxylcinnamylate (CHCA-C3) and α-cyano-4-hydroxycinnamic acid (CHCA) was used to replace the commonly used matrix CHCA. As a result, 1.4–3.8 times more fingerprint peaks have been identified by the cooperative matrix in comparison with CHCA when six different bacteria were studied. The peaks distribute in the wider m / z range, and the spectral quality has also been significantly improved. As an example of practical applications, the direct and more reliable discrimination between Escherichia coli (E. coli) and Shigella sonnei (S. sonnei) was achieved based on their protein fingerprints, which was previously claimed to be impossible by CHCA. Thus, a promoted MALDI-TOF-MS platform towards bacterial identification has been established by utilizing the new cooperative matrix, which will exhibit great potential for many applications. [ABSTRACT FROM AUTHOR]

The formaldehyde-based adhesive is commonly used in wood industry; however, the emission of formaldehyde is a fatal defect, which endangers people's health as well as pollutes the environment. Herein, the non-toxic and low-volatility glyoxal (G) is chosen to react with dimethylolurea (DMU) to prepare a novel and excellent water-resistant wood adhesive of glyoxal-dimethylolurea (G-DMU) resin. Quantum chemical calculations, structure characterizations, and performance measurements are carried out to study the reaction mechanism, and structure-performance relations of G-DMU resin with different DMU/G molar ratios. The results show that during the synthesis of G-DMU resin, the hydroxyl compounds N-p-G-DMU and N-p-G1-DMU, as well as the carbocation intermediates C-p-G-DMU and C-p-G1-DMU are formed by the addition reactions between DMU and protonated glyoxal (p-G and p-G1). The plywood bonded with G-DMU resin with the optimum DMU/G molar ratio of 1.2:1.0 has the best water resistance. Its wet shear strength is increased by 145% (cold water) and 193% (63 °C) over the DMU/G molar ratio of 0.8: 1.0. The analyses of structure characterizations reveal that the generation of C=N, C–O–C, N–C–N, and heterocyclic rings, simultaneously, the main cross-linked components concentratedly are characterized in 400–500 Da, 500–550 Da, 698 Da, and 712 Da regions. In addition, the high molecular weight oligomers at 658–712 Da, 874 Da, 1037 Da and 1213 Da promote the formation of a highly cross-linked network during the curing process. Meanwhile, these results are in good agreement with the results of quantum chemical calculations. Thus, this work establishes the theoretical foundation for the novel glyoxal-based resin of G-DMU resin. [Display omitted] • A novel eco-friendly glyoxal-dimethylolurea (G-DMU) resin has been successfully prepared. • The molar ratio DMU/G is a key parameter to influence the properties of G-DMU resin. • N-p-G-DMU, N-p-G1-DMU, C-p-G-DMU and C-p-G1-DMU are key intermediates. • The addition reactions of G or G1 with DMU are thermodynamically unfavorable. • The high molecular weight oligomers promotes a highly cross-linked network. [ABSTRACT FROM AUTHOR]

Extremely halophilic microbes which inhabit the hypersaline environments such as marine salterns, salted food products, soda lakes, etc. are diverse in morphology, physiology and biotechnological applications. Gujarat has a very long coastline (approximately 1600 km) and there are several locations where the sea water is used for manufacturing marine chemicals. We report here the production and purification of an extracellular lipase from the archaeon Haloferax laresnii strain BVM005 (Gen Bank accession no. KP636736) that was isolated from the solar salt works at Okhamadhi, near Dwarka. This isolate had a remarkable characteristic of producing multiple extracellular hydrolyases namely, amylase, caseinase, gelatinase, lipases (hydrolyzed Tween 20, 40, 60 and 80), etc. The Haloferax larsenii lipase (HLL) produced in broth culture was precipitated by using isopropyl alcohol and further purified by reverse-phase chromatography (RPHPLC). Ultra-performance liquid chromatography (UPLC) analysis was used to determine the amino acid profiles. SDSPAGE and Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) analyses revealed that HLL was a monomer, with a molecular mass of 31kDa. Compared to Lipolase (Novozymes), HLL displayed an elevated and considerable compatibility with various commercialized laundry detergents, and wash performance analysis revealed that it could remove oil-stains effectively. HLL has a number of attractive properties that make it a potential candidate for the synthesis of non-aqueous peptides and application in detergent industry. [ABSTRACT FROM AUTHOR]

One hundred and ninety-three staphylococcal strains were isolated from samples of alaska pollock meat (Theragra chalcogramma), atlantic mackerel meat (Scomber scombrus), atlantic herring meat (Clupea harengus), and femoral muscle samples of the wild pheasants (Phasianus colchicus), wild rabbits (Oryctolagus cuniculus) and bryndza cheese. Phenotypic manifestations typical of individual staphylococcal species were detected in these isolates. Species identification was also performed by matrix-assisted-laserdesorption- ionization-mass-spectrometry on the basis of which 5 species of staphylococci were determined: S. aureus, S. epidermidis, S. capitis, S. haemolyticus, S. warneri. Subsequently, the presence of the 16S rDNA gene was confirmed by PCR assay; a specific sequence for S. aureus species; putative transcriptional regulator gene serp0107, a specific sequence of the sodA gene was used to identify S. epidermidis and to identify S. warneri, S. haemolyticus, S. capitis species. When comparing the results of identification by PCR assay and matrix-assisted-laser-desorption-ionization-mass-spectrometry, the identification at the genus level was identical in all cases (100%). However, the discrepancies in results were confirmed at the species level, where congruence for identification of isolates by PCR assay and matrix-assisted-laser-desorption-ionization-mass-spectrometry was only 80%. For these isolates, the score value ranged from 1.953 to 2.564. [ABSTRACT FROM AUTHOR]

Gluten protein composition determines the rheological characteristics of wheat dough and is influenced by variable alleles with distinct effects on processing properties. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF–MS), we determined the high-molecular weight glutenin subunit (HMW-GS) composition of 665 wheat genotypes employed in breeding programs in South Korea. We identified 22 HMW-GS alleles, including 3 corresponding to the Glu-A1 locus, 14 to Glu-B1, and 5 to Glu-D1. The Glu-1 quality score, which is an important criterion for high-quality wheat development, was found to be 10 for 105/665 (15.79%) of the studied genotypes, and included the following combinations of HMW-GS: 2*, 7 + 8, 5 + 10; 2*, 17 + 18, 5 + 10; 1, 7 + 8, 5 + 10; and 1, 17 + 18, 5 + 10. To select wheat lines with the 1Bx7 overexpression (1Bx7OE) subunit, which is known to have a positive effect on wheat quality, we used a combination of MALDI-TOF–MS and published genotyping markers and identified 6 lines carrying 1Bx7OE out of the 217 showing a molecular weight of 83,400 Da, consistent with 1Bx7G2 and 1Bx7OE. This study demonstrates that the MALDI-TOF–MS method is fast, accurate, reliable, and effective in analyzing large numbers of wheat germplasms or breeding lines in a high-throughput manner. [ABSTRACT FROM AUTHOR]

The unexplored marine bivalves: Donax cuneatus and Pitar erycina are collected and the crude tissue proteins are extracted from them by diverse extraction methods. The crude protein fraction obtained from the acidic extraction method showed the highest yield and have further considered in our study. Various process parameters for the crude peptide extraction was statistically optimized based on response surface methodology revealed that the predicted peptide concentration from the polynomial model was 17.32 mg/mL, based on of the predicted process variables, i.e., 13.36 g/mL solid/liquid ratio, 26.63 °C temperature and 18.36 min of homogenization time. Subsequently, the crude peptide fraction isolated was subjected to purification by column chromatography followed by RP-HPLC. The fraction 3a from Donax cuneatus and 5b from Pitar erycina showed the highest antibacterial activity against Proteus vulgaris (20 µg/mL), Klebsiella pneumonia (10 µg/mL) and Streptococcus pyogenes (10 µg/mL) when compared to that of standard drug chloramphenicol at 40, 20 and 10 μg/mL respectively. Besides, the lowest minimum bactericidal concentration observed against Klebsiella pneumonia and Salmonella typhi for both the purified peptides was10 µg/mL. These antimicrobial peptides 3a and 5b exhibited significantly high antibacterial activity at 2× concentration of minimum inhibitory concentration, as evidenced through the in-vitro time-kill kinetic assay. They found to suppress the growth of all pathogenic bacteria under evaluation after 4 h. In addition to the above results, MALDI ToF-MS revealed the molecular mass of the peptide fractions 3a and 5b was 10.5 kDa and 10.0 kDa, respectively. Thus, the purified antimicrobial peptides isolated from the crude protein extracts from Donax cuneatus and Pitar erycina are found to be potential against the tested dreadful pathogens and could be translated for preclinical research in the future. [ABSTRACT FROM AUTHOR]

Human breast milk is an incredibly rich and complex biofluid composed of proteins, lipids and complex carbohydrates, including a diverse repertoire of free human milk oligosaccharides (HMOs). Strikingly, HMOs are not digested by the infant but function as prebiotics for bacterial strains associated with numerous benefits. Considering the broad variety of beneficial effects of HMOs, and the vast number of factors that affect breast milk composition, the analysis of HMO diversity and complexity is of utmost relevance. Using human milk samples from a cohort of Bangladeshi mothers participating in a study on malnutrition and stunting in children, we have characterized breast milk oligosaccharide composition by means of permethylation followed by liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-MS/MS) analysis. This approach identified over 100 different glycoforms and showed a wide diversity of milk composition, with a predominance of fucosylated and sialylated HMOs over nonmodified HMOs. We observed that these samples contain on average 80 HMOs, with the highest permethylated masses detected being >5000 mass units. Here we report an easily implemented method developed for the separation, characterization and relative quantitation of large arrays of HMOs, including higher molecular weight sialylated HMOs. Our ultimate goal is to create a simple, high-throughput method, which can be used for full characterization of sialylated and/or fucosylated HMOs. These results demonstrate how current analytical techniques can be applied to characterize human milk composition, providing new tools to help the scientific community shed new light on the impact of HMOs during infant development. [ABSTRACT FROM AUTHOR]

Safety of probiotics has always been an area of concern. The present study evaluates the safety aspects of novel probiotic Bacillus species as per the guidelines recommended jointly by Indian Council of Medical Research (ICMR) and Department of Biotechnology (DBT). It also includes assessment of viability during storage since the number of viable probiotic bacteria per gram of product or per serving should be mentioned on its label. Fifteen probiotic Bacillus isolates were subjected to the study of haemolytic activity, testing for enterotoxin genes by PCR technique, antibiotic sensitivity tests by Kirby Bauer method, and identification of the isolates by 16S rRNA and MALDI-TOF-MS studies. Five of these cultures were found to be non-haemolytic which is a desirable probiotic characteristic. Three of these acid and bile tolerant isolates were tested for antibiotic sensitivity. They were found to be sensitive to most antibiotics used, thus safe for consumption, in terms of antibiotic resistance transfer to normal flora and pathogens. The three Bacillus isolates tested negative for frequently encountered enterotoxin genes in Bacillus species-- nheA, nheB, bal, hblA, entFM and bceT. As per the WHO/FAO joint working committee recommendations, minimum viable numbers of the probiotic strains after six months storage at different temperatures was evaluated using the spread plate method. One of these cultures, identified as Brevibacillus borstelensis was found to be the most stable. Its GenBank accession number is MK156349. The culture has been identified to be a non-pathogenic strain. Thus, the novel prospective probiotic bacterium proved to be safe for consumption. [ABSTRACT FROM AUTHOR]

Malignant melanoma is one of the most aggressive human tumor types, mainly due to its high invasion capability, metastatic properties, and the absence of effective treatments. Glycosylation serves a pivotal role in the migration and invasion of melanoma. However, differences in glycosylation between high and low metastatic melanoma cells and how these regulate migration and invasion by altering the expression of fucosyltransferases (FUTs) remain unclear. In the present study, matrix‐assisted laser desorption/ionization‐time‐of‐flight‐mass spectrometry (MALDI‐TOF‐MS) analysis revealed that the composition profiling of fucosylated N‐glycans differed between high metastatic C8161 and low metastatic A375P cells. Further analysis revealed that FUT4 expression was significantly increased in C8161 cells. Melanoma tissue arrays further demonstrated that FUT4 was overexpressed in metastatic samples. Altered FUT4 expression was accompanied by a change in the migration and invasion capacity of the cells. In addition, the migration and invasion potential of melanoma cells were decreased in C8161 and increased in A375P cells upon altering FUT4 expression levels by small interfering RNA or complementary DNA transfection. Furthermore, regulating FUT4 expression markedly modulated the activity of the phosphoinositide‐3‐kinase/Akt (PI3K/Akt) signaling pathway, which affected melanoma cell migration and invasion. In conclusion, FUT4 is a novel biomarker and regulator of the migration and invasion of melanoma cells and may serve as a therapeutic target for melanoma. [ABSTRACT FROM AUTHOR]

Involvement of several candidate immune regulatory players at transcriptomic levels during microbial interactions were reported by involving C. elegans as a model system for the past few years. In the present study, we have identified a wide range of phenotypical, physiological and biochemical alterations in C. elegans triggered due to S. Typhi infection using standard approaches. We performed several behavioural studies and molecular studies such as liquid-phase IEF, MALDI-MS and bioinformatics analyses. S. Typhi exerted a slow killing against C. elegans and prompted several phenotypical changes such as egg laying defects, pharyngeal arresting, and triggered functional group variations which were disclosed using FT-IR. Proteome analysis using liquid phase IEF and MALDI-ToF-Mass Spectrometry ended up with the identification of 123 proteins which contains human orthologs. Bioinformatics analysis of the MS identified proteins revealed the involvement of ubiquitination pathway which was then validated using immunoblotting. Extensive studies similar to our study with the utility of high-throughput OMICS technologies during host pathogen interactions may pave a way for the identification of biomarkers against bacterial diseases. Unlabelled Image • Comprehensive analysis of bacterial exposure in C. elegans reveals the multifaceted physiological and biochemical abnormalities. • This is one of the few studies to utilize MicroRotofor™ coupled with MALDI-MS for understanding host pathogen interactions. • The orthologous genes of MS identified C. elegans proteins are conserved in H. sapiens , D. melanogaster , M. musculus, etc. • Immune regulatory proteins identified in the study have many reported glycosylated PTM sites. [ABSTRACT FROM AUTHOR]

Insects from the Dermestidae family (Dermestes ater and Dermestes maculatus) are synanthropic insects, which are household, agricultural and warehouse pests. Their lipidomics and the insects' ability to use compounds present in their bodies to protect them against pathogens are not fully understood. Therefore, the purpose of this work was to determine the composition of compounds present in the bodies of two insect species, Dermestes ater and Dermestes maculatus, by the MALDI technique. Several free fatty acids and acylglycerols were found to be present as a result of the research. Significant differences in the composition and number of identified compounds have been shown, depending on the tested species and on the development stage. In lipids of D. ater, a greater variety of free fatty acids were found than in those of the second species. Biological studies have determined the high resistance of both species of Dermestidae to fungal infection with Conidiobolus coronatus. These results provide baseline data for further studies on the possible role of lipids. [ABSTRACT FROM AUTHOR]

BACKGROUND: Georgia's small fruit sector is important to the state's agricultural economy, and is seeking ways to promote their fruits in the health and wellness market. Based on the hot, humid, climatic conditions of the U.S. Southeast, three blackberry varieties, namely 'Navaho', 'Ouachita', and 'Kiowa', can grow, but a complete characterization of their antioxidant phenolic constituents has not been reported. OBJECTIVE: This study aims to investigate the phenolic compounds and antioxidant activities in U.S. Southeastern erect-type blackberry cultivars of commercial significance. METHODS: Phenolics from blackberries were isolated, separated, and elucidated both qualitatively and quantitatively by various chromatographic means, including HPLC–ESI–MS and MALDI–TOF–MS. RESULTS: Delphinidin-3-O-glucoside, malvidin-3-O-glucoside, quercetin-3-O-glucoside malonate, and propelargonidin B-type dimer were present in the Southeastern blackberries, which have not been found in any major U.S. cultivars. MALDI–TOF–MS analysis revealed a distinctive ellagitannin profile among the cultivars, and identified 5 additional ellagitannin compounds that were not elucidated by HPLC–ESI–MS.The detection of the propelargonidin B-type dimer revealed that the flavan-3-ols of blackberries are not limited to (epi)catechin-based compounds. CONCLUSIONS: U.S. Southeastern erect-type blackberry cultivars grown in Georgia showed distinct phenolic profiles and antioxidant activities from those of Pacific Northwestern cultivars. [ABSTRACT FROM AUTHOR]

Introduction: For fast and effective antibiotic therapy of serious infections like sepsis, it is crucial with rapid information about antibiotic susceptibility, especially in a time when the number of infections caused by multi resistant bacteria has escalated in the world. Methods: Here, we have used a semi-quantitative MALDI-TOF-MS based method for antibiotic resistance detection, MBTASTRATM, which is based on the comparison of growth rate of the bacteria cultivated with and without antibiotics. We demonstrate a new protocol where several parameters have been optimized and automated leading to reduced hands-on time and improved capacity to simultaneously analyse multiple clinical samples and antibiotics. Results: Ninety minutes of incubation at 37 °C with agitation was sufficient to differentiate the susceptible and resistant strains of E. coli and K. pneumoniae, for the antibiotics cefotaxime, meropenem and ciprofloxacin. In total, 841 positive blood culture analyses of 14 reference strains were performed. The overall sensitivity was 99%, specificity 99% and the accuracy 97%. The assay gave no errors for cefotaxime (n¼263) or meropenem (n¼289) for sensitive and resistant strains, whilst ciprofloxacin (n¼289) gave six (0.7%) major errors (false resistance) and four (0.5%) very major errors (false susceptibility). The intermediate strains showed a larger variety compared to the E-test MIC values. Conclusions: The hands-on time and the analysis time to detect antibiotic resistance of clinical blood samples can be substantially reduced and the sample capacity can be increased by using automation and this improved protocol. [ABSTRACT FROM AUTHOR]

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was employed to analyze differential serum and urine peptides in patients with small cell lung cancer (SCLC) and healthy individuals, and SCLC diagnostic classification models were constructed. Serum and urine samples from 72 patients with SCLC, age- and gender-matched with 72 healthy individuals, were divided into training and testing sets in a 3:1 ratio. Serum and urine peptides were extracted using copper ion-chelating nanomagnetic beads, and mass spectra were obtained using MALDI-TOF-MS. Peptide spectra for the training set were analyzed, and the classification model was constructed using ClinProTools (CPT). The testing set was used for blinded model validation. For training-set sera, 122 differential peptide signal peaks with a mass of 0.8–10 kDa were observed, and 19 peptides showed significantly different expression [P<0.0005; area under curve (AUC) ≥0.80]. CPT screened 5 peptide peaks (0.8114, 0.83425, 1.86655, 4.11133 and 5.81192 kDa) to construct the classification model. The testing set was used for the blinded validation, which had 95.0% sensitivity and 90.0% specificity. For the training-set urine, 132 differential peptide signal peaks with m/z ratios of 0.8–10 kDa were observed, and 8 peptides had significantly different expression (P<0.0005; AUC ≥0.80). Then, 5 peaks (1.0724, 2.37692, 2.7554, 4.75475 and 4.7949 kDa) were used for classification model construction. The testing set was used for 36 blinded validation, which had 85.0% sensitivity and 80.0% specificity. Among the differential peptides, 3 had the same significant peaks at 2.3764, 0.8778 and 0.8616 kDa, identified as fibrinogen α, glucose-6-phosphate isomerase and cyclin-dependent kinase-1, respectively. The present study highlighted the differences that exist in serum and urine peptides between patients with SCLC and healthy individuals. Serum and urine peptide diagnostic classification models could be constructed using MALDI-TOF-MS, and showed high sensitivity and specificity. [ABSTRACT FROM AUTHOR]

Stored-product insects can transfer a wide range of serious pathogens involved in human health. The close contact of these insects with the food production chain makes these species extremely dangerous as carriers of severe infections. In addition, pathogenic bacteria, such as members of Enterococcus, are often resistant to antibiotics commonly used for human therapy. Herein we identified, using Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), Enterococcus and coliform species isolated from 17 strains of different insect species associated with durable stored products. The antibiotic susceptibility of the isolated strains was also evaluated. MALDI-TOF MS revealed mainly the presence of Enterococcus (E. faecium, E. phoeniculicola and E. casseliflavus) and Enterobacter (Eb. cloacae and Eb. asburiae). E. casseliflavus was resistant to all antibiotics tested, while E. faecium and E. phoeniculicola were resistant to sulphonamides. Among E. faecium isolates, approx. 20% were found to be resistant to tetracycline, while Eb. cloacae and Eb. asburiae showed resistance to erythromycin. The current series of data clearly indicates that certain bacteria of the genera Enterococcus and Enterobacter are very common in stored-product insects, and, under certain circumstances, may seriously endanger public health, through potential introduction of antibiotic resistance. [ABSTRACT FROM AUTHOR]

Mycolicibacter kumamotonensis (M. kumamotonensis), formerly Mycobacterium kumamotonense , is a nontuberculous mycobacteria species, which was first separated from Mycobacterium terrae complex in 2006. Reports about infections caused by M. kumamotonensis are extremely rare, with most of them being lung infection. Here, we report the case of a 68-year-old man with a hobby of gardening who developed swelling in his right middle finger. He underwent surgical debridement at a previous hospital and was diagnosed with nontuberculous mycobacteria infection based on positive findings of acid-fast staining of pus obtained from the surgical specimen. He was treated with rifampicin, ethambutol, and clarithromycin, but the swelling worsened. Therefore, he was referred to our hospital for further examination and treatment. We performed a second debridement and added isoniazid to the treatment regimen, but the swelling continued to worsen. We then administered levofloxacin, but his condition did not change. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and DNA sequencing analysis confirmed M. kumamotonensis as the causative bacterium. Since the finger swelling did not improve, the patient underwent a third debridement and amikacin was added to the treatment regimen. Finally, the infection was controlled. He completed amikacin therapy and will continue treatment with the other five antibiotics for a total of 24 months. To the best of our knowledge, this is the first report of a patient with M. kumamotonensis soft tissue infection. We consider this case might provide important insights into the diagnosis and treatment of soft tissue infections caused by M. kumamotonensis. [ABSTRACT FROM AUTHOR]

Background: Group B streptococcus (GBS) is an important pathogen that is responsible for invasive infections, including sepsis and meningitis. GBS serotyping is an essential means for the investigation of possible infection outbreaks and can identify possible sources of infection. Although it is possible to determine GBS serotypes by either immuno-serotyping or geno-serotyping, both traditional methods are time-consuming and labor-intensive. In recent years, the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been reported as an effective tool for the determination of GBS serotypes in a more rapid and accurate manner. Thus, this work aims to investigate GBS serotypes by incorporating machine learning techniques with MALDI-TOF MS to carry out the identification. Results: In this study, a total of 787 GBS isolates, obtained from three research and teaching hospitals, were analyzed by MALDI-TOF MS, and the serotype of the GBS was determined by a geno-serotyping experiment. The peaks of mass-to-charge ratios were regarded as the attributes to characterize the various serotypes of GBS. Machine learning algorithms, such as support vector machine (SVM) and random forest (RF), were then used to construct predictive models for the five different serotypes (Types Ia, Ib, III, V, and VI). After optimization of feature selection and model generation based on training datasets, the accuracies of the selected models attained 54.9–87.1% for various serotypes based on independent testing data. Specifically, for the major serotypes, namely type III and type VI, the accuracies were 73.9 and 70.4%, respectively. Conclusion: The proposed models have been adopted to implement a web-based tool (GBSTyper), which is now freely accessible at http://csb.cse.yzu.edu.tw/GBSTyper/, for providing efficient and effective detection of GBS serotypes based on a MALDI-TOF MS spectrum. Overall, this work has demonstrated that the combination of MALDI-TOF MS and machine intelligence could provide a practical means of clinical pathogen testing. [ABSTRACT FROM AUTHOR]

Lipin1, a member of the lipin family, serves as a phospholipid phosphatase or a co-transcriptional regulator in lipid metabolism. Recent studies also show that lipin1 is involved in many other cellular metabolism processes. However, the clear regulatory mechanism for lipin1 is unknown. The 293T human renal epithelial cell line represents a commonly used and well established expression system for recombinant proteins. Herein, we used two-dimensional polyacrylamide gel electrophoresis (2D-GE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to explore the changes in protein expression induced by lipin1 overexpression in 293T cells. Western blotting was used to confirm one of the expression changes of related proteins. Subsequently, the function and relationship of these proteins were analyzed by bioinformatics approach. By using 2D-PAGE, approximately 152 proteins were separated and eleven proteins were found to be significantly affected by lipin1 overexpression compared to the control. Among them, three proteins (eEF-1B γ, CCT1 and CCT3) were up-regulated and other eight proteins (NDKA, Stathmin, HNRNP A1, TK, KRT1, PKM, RanBP1 and LDHB) were down-regulated. These proteins were successfully identified with peptide mass fingerprinting using MALDI-TOF-MS after in-gel trypsin digestion. The bioinformatic analysis showed that these proteins are classified into seven protein species, including transferase, cleavage enzyme, cytoskeleton protein, chaperone protein, regulatory protein, structural protein and oxidoreductase. The results highlight the potential roles of lipin1 involved in many cellular metabolism processes, including myelin synthesis, extracellular domain formation, membrane bound vesicle synthesis and companion protein T complex synthesis. [ABSTRACT FROM AUTHOR]

Obligate anaerobes exist as resident flora in various sites in humans, but they are also emphasized as endogenous causative microorganism of infections. We performed surveillance to understand the trend of drug susceptibility in obligate anaerobic bacteria in the Kinki area of Japan. In the experiment, we used 156 obligate anaerobe isolates collected from 13 institutions that participated in the Study of Bacterial Resistance Kinki Region of Japan. MALDI Biotyper was used to identify the collected strains, and among the 156 test strains, those that could be identified with an accuracy of Score Value 2.0 or more included 6 genera, 30 species, and 144 strains (Bacteroides spp. 77 strains, Parabacteroides sp. 2 strains, Prevotella spp. 29 strains, Fusobacterium spp. 14 strains, Porphyromonas spp. 2 strains, and Clostridioides difficile 20 strains), and they were assigned as subject strains for drug susceptibility testing. The drug susceptibility test was carried out by broth microdilution method using Kyokuto Opt Panel MP ANA (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan) and judged according to CLSI criteria. As a result, Bacteroides and Parabacteroides species showed good sensitivities to tazobactam-piperacillin, imipenem, metronidazole and chloramphenicol, and low sensitivities to ampicillin, cefoperazone and vancomycin. Prevotella species showed good sensitivities to sulbactam-ampicillin, tazobactam-piperacillin, cefmetazole, imipenem, doripenem and metronidazole. Susceptibility rates to other drugs were slightly different depending on the bacterial species. Both Fusobacterium spp. and Porphyromonas spp. showed high sensitivities to many drugs. C. difficile was highly sensitive to vancomycin and metronidazole, having MIC 90 s of 0.5 μg/mL and ≤2 μg/mL, respectively. [ABSTRACT FROM AUTHOR]

In this study, identification of Enterococcus spp. from sikma cheeses and determination of microbiological and chemical properties of the samples were aimed. For this purpose, 84 isolates from 20 cheese samples were obtained. Among them, 32 E. faecalis, 31 E. faecium, 13 E. durans, 4 E. gallinarum, 3 E. casseliflavus and 1 E. thailandicus were identified. According to microbiological analysis; total aerobic mesophilic bacteria, yeast and mould, coliform, Enterococcus and Lactobacillus spp. were assesed to be 7.32±0.95, 4.94±0.89, 4.02±1.88, 5.86±1.38 and 6.90±1.22 log cfu/g, respectively. Chemical analysis revealed that dry matter, moisture, protein, fat, fat in dry matter, salt, salt in dry matter, ash, acidity and pH were 55.44±3.68%, 44.56±3.68%, 23.51±2.26%, 23.42±5.22%, 41.89±5.16%, 5.51±2.13%, 10.06±5.98%, 6.88±2.62%, 0.3779±0.103% and 5.66±0.14%, respectively. In conclusion, results indicated that sikma cheeses have wide range of microbiological and chemical properties and a diverse Enterococcus microflora due to using raw milk in the production of cheeses. [ABSTRACT FROM AUTHOR]