MALDI-TOF Analysis Research Articles

Biochemical, genetic and transcriptional characterization of multibacteriocin production by the anti-pneumococcal dairy strain Streptococcus infantariusLP90.

Streptococcus pneumoniae infections are one of the major causes of morbility and mortality worldwide. Although vaccination and antibiotherapy constitute fundamental and complementary strategies against pneumococcal infections, they present some limitations including the increase in non-vaccine serotypes and the emergence of multidrug-resistances, respectively. Ribosomally-synthesized antimicrobial peptides (i.e. bacteriocins) produced by Lactic Acid Bacteria (LAB) may represent an alternative or complementary strategy to antibiotics for the control of pneumococal infections. We tested the antimicrobial activity of 37 bacteriocinogenic LAB, isolated from food and other sources, against clinical S. pneumoniae strains. Streptococcus infantarius subsp. infantarius LP90, isolated from Venezuelan water-buffalo milk, was selected because of its broad and strong anti-pneumococcal spectrum. The in vitro safety assessment of S. infantarius LP90 revealed that it may be considered avirulent. The analysis of a 19,539-bp cluster showed the presence of 29 putative open reading frames (ORFs), including the genes encoding 8 new class II-bacteriocins, as well as the proteins involved in their secretion, immunity and regulation. Transcriptional analyses evidenced that the induction factor (IF) structural gene, the bacteriocin/IF transporter genes, the bacteriocin structural genes and most of the bacteriocin immunity genes were transcribed. MALDI-TOF analyses of peptides purified using different multichromatographic procedures revealed that the dairy strain S. infantarius LP90 produces at least 6 bacteriocins, including infantaricin A1, a novel anti-pneumococcal two-peptide bacteriocin.

Antimicrobial susceptibility pattern of Stenotrophomonas species isolated from Mexico.

Stenotrophomonas species are multi-resistant bacteria with ability to cause opportunistic infections. We isolated 45 Stenotrophomonas species from soil, sewage and the clinic with the aim of investigating their susceptibility to commonly used antimicrobial agents. The identities of isolates were confirmed with 16S rRNA gene sequence and MALDI-TOF analysis. Anti-microbial resistance, biofilm production and clonal diversity were also evaluated. The minimum inhibitory concentration technique as described by Clinical & Laboratory Standards Institute: CLSI Guidelines (CLSI) was employed for the evaluation of isolate susceptibility to antibiotics. Forty-five Stenotrophomonas species which include 36 environmental strains and 9 clinical strains of S. maltophilia were considered in this study. 32 (88.9 %) environmental strains were identified to be S. maltophilia, 2 (5.6 %) were Stenotrophomonas nitritireducens, and 2 (5.6 %) cluster as Stenotrophomonas spp. Stenotrophomonas isolates were resistant to at least six of the antibiotics tested, including Trimethoprim/Sulfamethoxazole (SXT). Environmental isolates from this study were resistant to SXT which is commonly used for the treatment of S. maltophilia infections. This informs the need for good public hygiene as the environment could be a reservoir of multi-resistant bacteria. It also buttresses the importance of surveillance study in the management of bacterial resistance.

Linear 2-Ethylhexyl Imidophosphoric Esters as Effective Rare-Earth Element Extractants

Imidophosphoric organic esters containing phosphoryl groups are potential polydentate ligands and promising extractants of rare-earth elements. For their preparation, a monophosphazene salt [PCl3=N−PCl3]+[PCl6]− and short phosphazene oligomers of the general formula [Cl–(PCl2=N)n–PCl3]+[PCl6]−, where n = 4–7, were synthesized via living cationic polymerization of Cl3P=NSiMe3 and used as starting compounds. All phosphazenes were reacted with 2-ethylhexanol to obtain the corresponding esters of imidophosphoric acids (EIPAs). The formation of imidophosphoric acids occurs due to the phosphazene-phosphazane rearrangement of –P(OR)2=N– or –P(OH)(OR)=N– units, where R = 2-ethylhexyl. The prepared EIPAs were characterized by 1H, 31P NMR, and MALDI-TOF analyses and their extractive capacity towards lanthanide ions in aqueous solutions of nitric acid was examined. The EIPAs are mixtures of mono-, di-, and trifunctional compounds of the type HxA, where x = 1–3, which can form chelate complexes of lanthanide ions [Ln(A)z], where z = 3–6, depending on the chain length. The longer chain EIPAs are more suitable for collective rare-earth elements extraction. A comparison of the extraction properties of the EIPAs with the industrially used polyalkylphosphonitrilic acid (PAPNA) was drawn.

The condensed tannins of Okoume (Aucoumea klaineana Pierre): A molecular structure and thermal stability study

In order to promote convenient strategies for the valorization of Aucoumea klaineana Pierre (Okoume) plywood and sawmill wastes industry in the fields of adhesives and composites, the total phenolic content of Okoume bark, sapwood and heartwood was measured. The molecular structure of tannins extracted from the bark was determined by Matrix Assisted Laser Desorption/Ionization Time-Of-Flight (Maldi-ToF) mass spectrometry and Fourier transform infrared spectroscopy (FTIR). The total phenolic content displayed significant difference (p = 0.001) between the bark, sapwood and heartwood which decreased as follows: 6 ± 0.4, 2 ± 0.8 and 0.7 ± 0.1% respectively. The pro-anthocyanidins content was also significantly different (p = 0.01) among the three wood wastes, and the bark was the richest in condensed tannins (4.2 ± 0.4%) compared to the sapwood (0.5 ± 0.1%) and heartwood (0.2 ± 0.2%). Liquid chromatography coupled mass spectroscopy (LC-MS) and Maldi-ToF analysis of the bark showed for the first time that Okoume condensed tannins are fisetinidin, gallocatechin and trihydroxyflavan based monomers and complex polymers obtained with glycosylated units. No free catechin or robitinidin units were detected, whereas distinctive dihydroxy or trihydroxyflavan-3-benzoate dimers were observed in the investigated condensed tannin extracts. FTIR analysis showed the occurrence of glucan- and mannan-like sugars in the condensed tannins, and Maldi-ToF highlighted that these sugars should account for ten glycosylated units chemically bonded with two fisetinidins and one gallocatechin trimer. The condensation of these polyphenols with formaldehyde led to Stiasny numbers of 83.3, 73.3 and 53.3% for the bark, sapwood and heartwood, respectively.

One-Year Old Dormant, "Non-culturable" Mycobacterium tuberculosis Preserves Significantly Diverse Protein Profile.

For adaptation to stressful conditions, Mycobacterium tuberculosis (Mtb) is prone to transit to a dormant, non-replicative state, which is believed to be the basis of the latent form of tuberculosis infection. Dormant bacteria persist in the host for a long period without multiplication, cannot be detected from biological samples by microbiological methods, however, their “non-culturable” state is reversible. Mechanisms supporting very long capacity of mycobacteria for resuscitation and further multiplication after prolonged survival in a dormant phase remain unclear. Using methods of 2D electrophoresis and MALDI-TOF analysis, in this study we characterized changes in the proteomic profile of Mtb stored for more than a year as dormant, non-replicating cells with a negligible metabolic activity, full resistance to antibiotics, and altered morphology (ovoid forms). Despite some protein degradation, the proteome of 1-year-old dormant mycobacteria retained numerous intact proteins. Their protein profile differed profoundly from that of metabolically active cells, but was similar to the proteome of the 4-month-old dormant bacteria. Such protein stability is likely to be due to the presence of a significant number of enzymes involved in the protection from oxidative stress (katG/Rv1908, sodA/Rv3846, sodC/Rv0432, bpoC/Rv0554), as well as chaperones (dnaJ1/Rv0352, htpG/Rv2299, groEL2/Rv0440, dnaK/Rv0350, groES/Rv3418, groEL1/Rv3417, HtpG/Rv2299c, hspX/Rv2031), and DNA-stabilizing proteins. In addition, dormant cells proteome contains enzymes involved in specific metabolic pathways (glycolytic reactions, shortened TCA cycle, degradative processes) potentially providing a low-level metabolism, or these proteins could be “frozen” for usage in the reactivation process before biosynthetic processes start. The observed stability of proteins in a dormant state could be a basis for the long-term preservation of Mtb cell vitality and hence for latent tuberculosis.

Ready-to-eat street food: a potential source for dissemination of multidrug-resistant Escherichia coli epidemic clones in Quito, Ecuador.

Ready-to-eat food contamination with ESBL-producing Escherichia coli is a growing health concern. Some of these strains also are epidemic clones and can cause community-associated infections that are difficult to treat. In this study, the occurrence of ESBL-producing E. coli contaminated ready-to-eat street food in Quito, Ecuador was evaluated. In total, 150 samples were collected randomly in the most crowded sites of the city. In all, 34 samples (34/150; 22·6%) were positive for total thermotolerant (44·5°C) coliforms resistant to cefotaxime. MALDI-TOF analysis identified that the E. coli was found in 20 food samples (20/34; 59%). ESBL gene blaCTX-M-55 was identified in nine isolates, blaCTX-M-15 in six isolates, blaCTX-M-14 in two isolates, and one isolate each harboured blaCTX-M-24 , blaCTX-M-65 , blaCTX-M-55 and blaCTX-M-8 . Phylogenetic groups like A and B1 were the most common, followed by groups D and B2. MLST analysis identified 12 different sequence types (STs), the most common was ST162. Recognized epidemic clonal groups ST410, ST131 and ST744 were encountered. Ready-to-eat street food is a potential way of spreading ESBL-producing E. coli epidemic clones in Quito, Ecuador. SIGNIFICANCE AND IMPACT OF THE STUDY: This study identified ESBL-producing Escherichia coli epidemic clones: ST131, ST410 and ST744 in ready-to-eat street food samples. Street food is a possible way to spread harm multidrug-resistant (MDR) E. coli strains in the community. Studies to identify the contamination sources of this kind of food are needed to tackle MDR E. coli dissemination.

The Influence of Glycans-Specific Bioconjugation on the FcγRI Binding and In vivo Performance of 89Zr-DFO-Pertuzumab.

Rationale: The overwhelming majority of radioimmunoconjugates are produced via random conjugation methods predicated on attaching bifunctional chelators to the lysines of antibodies. However, this approach inevitably produces poorly defined and heterogeneous immunoconjugates because antibodies have several lysines distributed throughout their structure. To circumvent this issue, we have previously developed a chemoenzymatic bioconjugation strategy that site-specifically appends cargoes to the biantennary heavy chain glycans attached to CH2 domains of the immunoglobulin's Fc region. In the study at hand, we explore the effects of this approach to site-specific bioconjugation on the Fc receptor binding and in vivo behavior of radioimmunoconjugates.Methods: We synthesized three desferrioxamine (DFO)-labeled immunoconjugates based on the HER2-targeting antibody pertuzumab: one using random bioconjugation methods (DFO-nsspertuzumab) and two using variants of our chemoenzymatic protocol (DFO-sspertuzumab-EndoS and DFO-sspertuzumab-βGal). Subsequently, we characterized these constructs and evaluated their ability to bind HER2, human FcγRI (huFcγRI), and mouse FcγRI (muFcγRI). After radiolabeling the immunoconjugates with zirconium-89, we conducted PET imaging and biodistribution studies in two different mouse models of HER2-expressing breast cancer.Results: MALDI-ToF and SDS-PAGE analysis confirmed the site-specific nature of the bioconjugation, and flow cytometry and surface plasmon resonance (SPR) revealed that all three immunoconjugates bind HER2 as effectively as native pertuzumab. Critically, however, SPR experiments also illuminated that DFO-sspertuzumab-EndoS possesses an attenuated binding affinity for huFcγRI (17.4 ± 0.3 nM) compared to native pertuzumab (4.7 ± 0.2 nM), DFO-nsspertuzumab (4.1 ± 0.1 nM), and DFO-sspertuzumab-βGal (4.7 ± 0.2 nM). ImmunoPET and biodistribution experiments in athymic nude mice bearing HER2-expressing BT474 human breast cancer xenografts yielded no significant differences in the in vivo behavior of the radioimmunoconjugates. Yet experiments in tumor-bearing humanized NSG mice revealed that 89Zr-DFO-sspertuzumab-EndoS produces higher activity concentrations in the tumor (111.8 ± 39.9 %ID/g) and lower activity concentrations in the liver and spleen (4.7 ± 0.8 %ID/g and 13.1 ± 4.0 %ID/g, respectively) than its non-site-specifically labeled cousin, a phenomenon we believe stems from the altered binding of the former to huFcγRI.Conclusion: These data underscore that this approach to site-specific bioconjugation not only produces more homogeneous and well-defined radioimmunoconjugates than traditional methods but may also improve their in vivo performance in mouse models by reducing binding to FcγRI.

Identification and characterization of methicillin-resistant Staphylococcus spp. isolated from surfaces near patients in an intensive care unit of a hospital in southeastern Brazil.

INTRODUCTION: Contaminated hospital environments contribute to the transmission of microorganisms associated with healthcare. Contaminated surfaces handled by patients or healthcare professionals are a source of microorganism transmission by hand. Methicillin-resistant Staphylococcus bacteria are among the main agents responsible for increasing healthcare-associated infections in Brazil and worldwide. METHODS: The objective of this study was to screen and characterize methicillin-resistant Staphylococcus spp. on surfaces near patients in an intensive care unit. Microbiological samples, collected from ten beds in an intensive care unit with five sampling sites, were inoculated into a methicillin-resistant Staphylococcus aureus chromogenic medium. MALDI-TOF and PCR analyses were used to identify the bacteria. Antimicrobial susceptibility was determined using the disk diffusion test. The presence of the mecA gene was investigated using PCR. RESULTS: We observed that 44 out of the 50 sampling sites presented grown isolates in the methicillin-resistant Staphylococcus aureus medium. The incidence of isolated microorganisms on the right side rail, left side rail, tables, infusion pump keypad, and cardiac monitor were 18.8 %, 36.7 %, 10.9 %, 2.4 %, and 31 %, respectively. The 42 isolates included in this study were identified as coagulase-negative Staphylococcus. All of these microorganisms were multidrug-resistant and mecA gene-positive. CONCLUSIONS: This study identified the presence of methicillin-resistant coagulase-negative Staphylococcus on the beds of an intensive care unit, providing evidence for the necessity of assertive actions to decrease the risk of healthcare-associated infections at the site.

First Comprehensive Report of Clinical Fusarium Strains Isolated in the State of Sao Paulo (Brazil) and Identified by MALDI-TOF MS and Molecular Biology.

The aim of this study was to compare the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), phenotypic and molecular methods for the identification of Fusarium species complexes isolated from clinical cases in the State of Sao Paulo (Brazil) between the years 2001 and 2017. Sequencing of ITS region of ribosomal DNA and elongation factor 1 alpha gene (ET1α) were used as reference method in the analysis of a total of 108 Fusarium spp. clinical strains isolated from human hosts with superficial and systemic infections. Agreement between MALDI-TOF-MS and molecular data was observed for 97 out of 108 clinical isolates (89.8%), whereas five (4.6%) and six (5.5%) clinical isolates were misidentified and were not identified by MALDI-TOF MS, respectively. ITS region sequences and MALDI-TOF MS mass spectra identified and grouped correctly most of Fusarium clinical isolates at species complex level. This investigation highlights the potential of MALDI-TOF MS technique as a fast and cost-efficient alternative for clinical Fusarium identification. However, MALDI-TOF MS requires a more accurate and larger database. This work is the first comprehensive report for Fusarium population, based on phenotypic analyses, proteomic profile by MALDI-TOF and phylogenetic analyses of Fusarium species complexes isolated from clinical cases in the State of Sao Paulo, Brazil.

-Identification of a novel intestinal phospholipase A2 from annular seabream: Insights into its catalytic mechanism and its role in biological processes

Phospholipase A2 (PLA2) is responsible for the lipid hydrolysis process. Fish PLA2 have warranted renewed interest due to their excellent properties in phospholipid digestion. We report for the first time the catalytic properties of a PLA2 secreted from the intestine of the annular seabream Diplodus annularis (IDaPLA2). The refolded IDaPLA2 was purified to homogeneity and showed a molecular mass of around 15 kDa attested by SDS-PAGE and MALDI-TOF analyses. Interestingly, IDaPLA2 revealed higher thermostability compared to mammal pancreatic sPLA2 as it was active and stable at 55 °C with specific activity of 290 U mg−1 on phosphatidylcholine (PC) as a substrate. Using the lipid monolayer technique, the activity of IDaPLA2 was found to be 21.68, 6.88 and 5.66 mol cm−2 min−1 mM−1 using phosphatidylglycerol (PG), PC and phosphatidylethanolamine (PE) monolayers, respectively, at surface pressures from 20−30 mN m−1. Interestingly, the interfacial activity of IDaPLA2 measured at higher surface pressures may highlight its ability to penetrate into phospholipid monolayers suggesting its involvement in cell lipid membrane degradation which can explain the cytotoxicity potential towards macrophage. The docking simulation data provided insights into the involvement of some key amino-acids in substrate binding and selectivity. The dynamic simulation proved the high stability of IDaPLA2. Overall, these results provide original evidence on the involvement of IDaPLA2 into the lipid hydrolysis suggesting it as a potential target in biotechnological applications.

Application of polydopamine-coated nylon capillary-channeled polymer fibers as a stationary phase for mass spectrometric phosphopeptide analysis.

Capillary-channeled polymer (C-CP) fibers are demonstrated as a selective stationary phase for phosphopeptide analysis via LC-MS. Taking advantage of the oxidative self-polymerization of dopamine under alkaline conditions, a simple system involving a dilute aqueous solution of 0.2% w/v dopamine hydrochloride in 0.15% w/v TRIS buffer, pH 8.5 was utilized to coat polydopamine onto nylon 6 C-CP fibers. Confirmation of the polydopamine coating on the fibers (nylon-PDA) was made through attenuated total reflection-FTIR (ATR-FTIR) analysis. Imaging using SEM was also performed to examine the morphology and topography of the nylon-PDA. Subsequent loading of Fe3+ to the nylon-PDA matrix was confirmed by SEM/energy dispersive X-ray spectroscopy (SEM/EDX). The Fe3+ -bound nylon-PDA fibers packed in a microbore column format were tested in the off-line preconcentration of phosphopeptides from a 1:100 mixture of β-casein/BSA digests for MALDI-TOF analysis. The packed column was also installed onto an HPLC system as a platform for the online sample clean-up and enrichment of phosphopeptides from a 1:1000 mixture of β-casein/BSA protein digests that were determined by subsequent ESI-MS analysis.

Thrombolytic Potential of Novel Thiol-Dependent Fibrinolytic Protease from Bacillus cereus RSA1.

The present study demonstrates the production and thrombolytic potential of a novel thermostable thiol-dependent fibrinolytic protease by Bacillus cereus RSA1. Statistical optimization of different parameters was accomplished with Plackett–Burman design and validated further by central composite design with 30.75 U/mL protease production. Precipitation and chromatographic approaches resulted in 33.11% recovery with 2.32-fold purification. The molecular weight of fibrinolytic protease was 40 KDa and it exhibited a broad temperature and pH stability range of 20–80 °C and pH 5–10 with utmost activity at 50 °C and pH 8, respectively. The protease retained its fibrinolytic activity in organic solvents and enhanced the activity in solutions with divalent cations (Mn2+, Zn2+, and Cu2+). The enzyme kinetics revealed Km and Vmax values of 1.093 mg/mL and 52.39 µg/mL/min, respectively, indicating higher affinity of fibrinolytic activity towards fibrin. Also, complete inhibition of fibrinolytic activity with DFP and a 2-fold increase with DTT and β-mercaptoethanol indicates its thiol-dependent serine protease nature. MALDI–TOF analysis showed 56% amino acid sequence homology with Subtilisin NAT OS = Bacillus subtilis subsp. natto. The fibrinolysis activity was compared with a commercial thrombolytic agent for its therapeutic applicability, and fibrinolytic protease was found highly significant with absolute blood clot dissolution within 4 h in in vitro conditions. The isolated fibrinolytic protease of Bacillus cereus RSA1 is novel and different from other known fibrinolytic proteases with high stability and efficacy, which might have wide medicinal and industrial application as a thrombolytic agent and in blood stain removal, respectively.

Extending the information content of the MALDI analysis of biological fluids via multi-million shot analysis.

IntroductionReliable measurements of the protein content of biological fluids like serum or plasma can provide valuable input for the development of personalized medicine tests. Standard MALDI analysis typically only shows high abundance proteins, which limits its utility for test development. It also exhibits reproducibility issues with respect to quantitative measurements. In this paper we show how the sensitivity of MALDI profiling of intact proteins in unfractionated human serum can be substantially increased by exposing a sample to many more laser shots than are commonly used. Analytical reproducibility is also improved.MethodsTo assess what is theoretically achievable we utilized spectra from the same samples obtained over many years and combined them to generate MALDI spectral averages of up to 100,000,000 shots for a single sample, and up to 8,000,000 shots for a set of 40 different serum samples. Spectral attributes, such as number of peaks and spectral noise of such averaged spectra were investigated together with analytical reproducibility as a function of the number of shots. We confirmed that results were similar on MALDI instruments from different manufacturers.ResultsWe observed an expected decrease of noise, roughly proportional to the square root of the number of shots, over the whole investigated range of the number of shots (5 orders of magnitude), resulting in an increase in the number of reliably detected peaks. The reproducibility of the amplitude of these peaks, measured by CV and concordance analysis also improves with very similar dependence on shot number, reaching median CVs below 2% for shot numbers > 4 million. Measures of analytical information content and association with biological processes increase with increasing number of shots.ConclusionsWe demonstrate that substantially increasing the number of laser shots in a MALDI-TOF analysis leads to more informative and reliable data on the protein content of unfractionated serum. This approach has already been used in the development of clinical tests in oncology.

Corynebacterium kroppenstedtii subsp. demodicis is the endobacterium of Demodex folliculorum.

Demodex spp. mites are the most complex member of the human skin microbiome. Mostly they are commensals, although their pathophysiological role in inflammatory dermatoses is recognized. Demodex mites cannot be cultivated invitro, so only little is known about their life cycle, biology and physiology. Different bacterial species have been suggested to be the endobacterium of Demodex mites, including Bacillus oleronius, B.simplex, B.cereus and B.pumilus. Our aim was to find the true endobacterium of human Demodex mites. The distinct genetic and phenotypic differences and similarities between the type strain and native isolates are described by DNA sequencing, PCR, MALDI-TOF, DNA-DNA hybridization, fatty and mycolic acid analyses, and antibiotic resistance testing. We report the true endobacterium of Demodex folliculorum, independent of the sampling source of mites or life stage: Corynebacterium kroppenstedtii subsp. demodicis. We anticipate our finding to be a starting point for more in-depth understanding of the tripartite microbe-host interaction between Demodex mites, its bacterial endosymbiont and the human host.

An improved simple method for the identification of Mycobacteria by MALDI-TOF MS (Matrix-Assisted Laser Desorption- Ionization mass spectrometry)

The aim of this study was to establish a simple method for the rapid identification of Mycobacteria species by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass spectrometry) using the Bruker MALDI-TOF Biotyper system (Bruker Daltonik, Bremen, Germany). A multicentre, prospective, and single blind study was performed in three European Hospitals, two Spanish and one UK hospital from May to August 2018. The BD BACTEC MGIT (Becton Dickinson, Berks, UK) liquid culture system was used in all three centres for the growth of Mycobacteria. When signal positive, tubes were removed from the analyser and in addition to standard laboratory procedures were subcultured on blood agar plates for MALDI-TOF analysis. Plates were incubated aerobically for 1 to 7 days at 37 °C and inspected every day. Once any growth was visible, it was transferred to the steel target plate, overlaid with 1 μl of neat formic acid and 1 μl HCCA matrix (alpha hydroxyl 4 cinnamic acid), and analysed in a Bruker Biotyper MALDI-TOF. Results given by MALDI-TOF were compared with the reference methods used for identification in the different centres. At two Spanish hospitals, identification by MALDI-TOF was only attempted on presumptive non-tuberculosis mycobacteria (NTM) and the results were initially compared with the results obtained by a commercial reverse hybridisation assay, GenoType CM/AS (Hain Lifescience, Tübingen, Germany). At the UK Hospital, identification of any presumptive mycobacteria was attempted and compared with the results obtained by whole genome sequencing (WGS). Overall in 142/167 (85%) of cases the identifications obtained were concordant; all Mycobacterium tuberculosis (MTB) isolates 43/43 (100%), 57/76 (75%) of the rapid growing nontuberculous mycobacteria (NTM), and 42/48 (85%) slow growing NTM tested were identified correctly. We report a new, easy, cheap and quick method for isolation and identification of Mycobacterium spp. without the need for additional steps or equipment and this method is in routine used in all three centres.

Synthesis and Characterization of Unsymmetrical Double-Decker Siloxane (Basket Cage).

The one-pot synthesis of an unsymmetrical double-decker siloxane with a novel structure via the reaction of double-decker tetrasodiumsilanolate with 1 equiv. of dichlorotetraphenyldisiloxane in the presence of an acid is reported herein for the first time. The target compound bearing all phenyl substituents on the unsymmetrical siloxane structure was successfully obtained, as confirmed by 1H-NMR, 13C-NMR, 29Si-NMR, IR, MALDI-TOF, and X-ray crystallography analyses. Additionally, the thermal properties of the product were evaluated by TG/DTA and compared with those of other siloxane cage compounds.

Functionalization of Bacterial Cellulose Nonwoven by Poly(fluorophenol) to Improve Its Hydrophobicity and Durability.

The present study aims to improve the hydrophobicity and durability of bacterial cellulose (BC) nonwoven by functionalization with poly(fluorophenol). To this end, laccase was first entrapped onto BC and then used to polymerize the fluorophenol {4-[4-(trifluoromethyl) phenoxy] phenol} in-situ. The polymerization of fluorophenol by laccase was confirmed through 1H NMR and MALDI-TOF analyses. The effect of poly(fluorophenol) on BC nonwoven was determined by evaluation of the surface hydrophobicity and olephobicity properties such as water contact angle (WCA), oil contact angle (OCA), surface energy and water/oil absorption time. After BC functionalization with poly(fluorophenol) (20 mM), the WCA increased from 54.5 ± 1.2° to 120 ± 1.5° while the surface energy decreased (11.58 ± 1.4 mN/m). The OCA was also increased from 46.5 ± 2.5° to 87 ± 2° along to the decrease of surface energy (8.7 ± 1.5°). X-ray photoelectron spectroscopy (XPS) analysis confirmed an increase in the fluorine content in BC from 5.27 to 17.57%. The findings confirmed the polymerization of fluorophenol by laccase and its entrapment onto a BC nanofiber structure. The durability of the functionalization with poly(fluorophenol) was confirmed by evaluating the washing fastness, tensile strength after washing and dimensional stability. The results indicate that the functionalized BC nonwoven had higher tensile strength (×10 times) and better dimensional stability (30%) than the non-functionalized BC nonwoven material.

Tannin plywood bioadhesives with non-volatile aldehydes generation by specific oxidation of mono- and disaccharides

Sodium periodate has been shown to cleave glucose by specific oxidation to yield a number of non-volatile aldehydes which can react with the phenolic compounds in tannin extract and lead to tannin cross-linking and hardening. This approach to prepare a tannin resin useable for wood adhesives can be used by either treating with periodate a mixture of tannin and glucose, or to treat glucose beforehand with periodate to cleave it and generate the aldehydes, and only afterwards to mix it with the tannin. The results obtained with these two methods are identical, but the latter method avoids oxidation of tannin. The aldehydes were generated either by direct cleavage of glucose C–C bonds carrying vicinal oriented hydroxyls, or by recombination of the aldehydes so formed by aldol condensation or by water elimination between two aldehyde groups. MALDI ToF analysis indicated that all the three types of aldehydes appeared to react with the polyphenols in the tannin. Thermomechanical analysis (TMA) and plywood bonded with the tannin + glucose + periodate mixes gave good bonding results, with dry, 24 h cold water soaking and 3 h at 63 °C shear strength values improving concomitantly to the proportion of periodate used for the oxidation step.

Description of Klebsiella spallanzanii sp. nov. and of Klebsiella pasteurii sp. nov.

Klebsiella oxytoca causes opportunistic human infections and post-antibiotic haemorrhagic diarrhea. This Enterobacteriaceae species is genetically heterogeneous and is currently subdivided into seven phylogroups (Ko1 to Ko4 and Ko6 to Ko8). Here we investigated the taxonomic status of phylogroups Ko3 and Ko4. Genomic sequence-based phylogenetic analyses demonstrate that Ko3 and Ko4 formed well-defined sequence clusters related to, but distinct from, Klebsiella michiganensis (Ko1), K. oxytoca (Ko2), K. huaxiensis (Ko8), and K. grimontii (Ko6). The average nucleotide identity (ANI) of Ko3 and Ko4 were 90.7% with K. huaxiensis and 95.5% with K. grimontii, respectively. In addition, three strains of K. huaxiensis, a species so far described based on a single strain from a urinary tract infection patient in China, were isolated from cattle and human feces. Biochemical and MALDI-ToF mass spectrometry analysis allowed differentiating Ko3, Ko4, and Ko8 from the other K. oxytoca species. Based on these results, we propose the names Klebsiella spallanzanii for the Ko3 phylogroup, with SPARK_775_C1T (CIP 111695T and DSM 109531T) as type strain, and Klebsiella pasteurii for Ko4, with SPARK_836_C1T (CIP 111696T and DSM 109530T) as type strain. Strains of K. spallanzanii were isolated from human urine, cow feces, and farm surfaces, while strains of K. pasteurii were found in fecal carriage from humans, cows, and turtles.

A MALDI-TOF analysis of gliadins and glutenins in “Astana” wheat cultivar

Targeted, quantitative metabolomics can, in principle, provide precise information on intracellular metabolite levels, which can be applied to accurate modeling of intracellular processes required in systems biology and metabolic engineering. However, quantitative metabolite profiling is often hampered by biased mass spectrometry-based analyses caused by matrix effects, the degradation of metabolites and metabolite leakage during sample preparation, and unexpected variation in instrument responses. Isotope Dilution Mass Spectrometry (IDMS) has been proven as the most accurate method for high-throughput detection of intracellular metabolite concentrations, and the key has been the acquisition of the corresponding fully uniformly (U) -13C-labeled metabolites to be measured. Here, we have prepared U-13C-labeled cell extracts by cultivating P. chrysogenum in a fed-batch fermentation with fully U-13C-labeled substrates. Towards this goal, a dynamic fed-batch model describing P. chrysogenum growth and penicillin production was used to simulate the fermentation process and design the fed-batch fermentation media. Further, a case study with extensive intracellular metabolomics data from glucose-limited cultivation of Penicillium chrysogenum under both single and repetitive glucose pulses was illustrated by using the IDMS methods with the prepared U-13C-labeled cell extracts as internal standards. In conclusion, the IDMS method can be incorporated into well-established fast sampling and quenching protocols to obtain dynamic quantitative in vivo metabolome data at the timescales of (tens of) seconds and elucidate the underlying regulatory architecture. The case study revealed gross differences between single and repeated pulses, which suggests that single pulse studies have limited value for understanding of metabolic responses in large-scale bioreactors. Instead, intermittent feeding should be favored.