Lipidomic profiling of non-mineralized dental plaque and biofilm by untargeted UHPLC-QTOF-MS/MS and SWATH acquisition.

Dental plaque is a structurally organized biofilm which consists of diverse microbial colonies and extracellular matrix. Its composition may change when pathogenic microorganisms become dominating. Therefore, dental biofilm or plaque has been frequently investigated in the context of oral health and disease. Furthermore, its potential as an alternative matrix for analytical purposes has also been recognized in other disciplines like archeology, food sciences, and forensics. Thus, a careful in-depth characterization of dental plaque is worthwhile. Most of the conducted studies focused on the screening of microbial populations in dental plaque. Their lipid membranes, on the other hand, may significantly impact substance (metabolite) exchange within microbial colonies as well as xenobiotics uptake and incorporation into teeth. Under this umbrella, a comprehensive lipidomic profiling for determination of lipid compositions of in vivo dental plaque samples and of in vitro cultivated biofilm as surrogate matrix to be used for analytical purposes has been performed in this work. An untargeted lipidomics workflow utilizing a ultra-high-performance liquid chromatography (UHPLC)-quadrupole-time-of-flight (QTOF) platform together with comprehensive SWATH (sequential window acquisition of all theoretical fragment ion mass spectra) acquisition and compatible software (MS-DIAL) that comprises a vast lipid library has been adopted to establish an extensive lipidomic fingerprint of dental plaque. The main lipid components in dental plaque were identified as triacylglycerols, followed by cholesterol, cholesteryl esters as well as diacylglycerols, and various phospholipid classes. In vivo plaque is a rare matrix which is usually available in very low amounts. When higher quantities for specific research assays are required, efficient ways to produce an appropriate surrogate matrix are mandatory. A potential surrogate matrix substituting dental plaque was prepared by cultivation of in vitro biofilm from saliva and similarities and differences in the lipidomics profile to in vivo plaque were mapped by statistical evaluation post-analysis. It was discovered that most lipid classes were highly elevated in the in vitro biofilm samples, in particular diacylglycerols, phosphatidylglycerols, and phosphatidylethanolamines (PEs). Furthermore, an overall shift from even-chain lipid species to odd-chain lipids was observed in the cultivated biofilms. On the other hand, even-chain phosphatidylcholines (PCs), lysoPCs, cholesteryl esters, and cholesterol-sulfate were shown to be specifically increased in plaque samples. Graphical abstract.

Multivariate optimization of a method for the determination of fatty acids in dental biofilm by GC-MS.

AIM: Phospholipid fatty acid methyl ester (FAME) analysis offers a simple option additionally to 16S rRNA sequencing to characterize microbial communities and to monitor changes. A method was established for the characterization of dental plaque via FAME profiles. METHODOLOGY: Fatty acids were determined as FAMEs (direct, acidic transesterification) and analyzed by GC-MS using an optimized temperature gradient. The transesterification reaction was optimized using a fractional factorial central composite face-centered design. RESULTS: Optimal conditions for the transesterification in methanol/toluene: hydrochloric acid concentration 2% (w/v), reaction time 40 min, temperature 110 °C. Method validation showed satisfactory accuracy, precision and linearity. CONCLUSION: The method provides a useful tool to characterize plaque via FAME profiles and was successfully applied to samples from ten subjects demonstrating its applicability.

Automated chromatographic laccase-mediator-system activity assay.

To study the interaction of laccases, mediators, and substrates in laccase-mediator systems (LMS), an on-line measurement was developed using high performance anion exchange chromatography equipped with a CarboPac™ PA 100 column coupled to pulsed amperometric detection (HPAEC-PAD). The developed method was optimized for overall chromatographic run time (45 to 120 min) and automated sample drawing. As an example, the Trametes versicolor laccase induced oxidation of 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-dihydroxypropane (adlerol) using 1-hydroxybenzotriazole (HBT) as mediator was measured and analyzed on-line. Since the Au electrode of the PAD detects only hydroxyl group containing substances with a limit of detection being in the milligram/liter range, not all products are measureable. Therefore, this method was applied for the quantification of adlerol, and-based on adlerol conversion-for the quantification of the LMS activity at a specific T. versicolor laccase/HBT ratio. The automated chromatographic activity assay allowed for a defined reaction start of all laccase-mediator-system reactions mixtures, and the LMS reaction progress was automatically monitored for 48 h. The automatization enabled an integrated monitoring overnight and over-weekend and minimized all manual errors such as pipetting of solutions accordingly. The activity of the LMS based on adlerol consumption was determined to 0.47 U/mg protein for a laccase/mediator ratio of 1.75 U laccase/g HBT. In the future, the automated method will allow for a fast screening of combinations of laccases, mediators, and substrates which are efficient for lignin modification. In particular, it allows for a fast and easy quantification of the oxidizing activity of an LMS on a lignin-related substrate which is not covered by typical colorimetric laccase assays. ᅟ.

Laccases for biorefinery applications: a critical review on challenges and perspectives.

Modern biorefinery concepts focus on lignocellulosic biomass as a feedstock for the production of next generation biofuels and platform chemicals. Lignocellulose is a recalcitrant composite consisting of several tightly packed components which are stuck together by the phenolic polymer lignin hampering the access to the carbohydrate compounds of biomass. Certain saprophytic organisms are able to degrade lignin by the use of an enzymatic cocktail. Laccases have been found to play a major role during lignin degradation and have therefore been intensively researched with regard to potential applications for biomass processing. Within this review, we go along the process chain of a third generation biorefinery and highlight the process steps which could benefit from laccase applications. Laccases can assist the pretreatment of biomass and promote the subsequent enzymatic hydrolysis of cellulose by the oxidative modification of residual lignin on the biomass surface. In combination with mediator molecules laccases are often reported being able to catalyze the depolymerization of lignin. Studies with lignin model compounds confirm the chemical possibility of a laccase-catalyzed cleavage of lignin bonds, but the strong polymerization activity of laccase counters the decomposition of lignin by repolymerizing the degradation products. Therefore, it is a key challenge to shift the catalytic performance of laccase towards lignin cleavage by optimizing the process conditions. Another field of application for laccases is the detoxification of biomass hydrolyzates by the oxidative elimination of lignin-derived phenolics which inhibit hydrolytic enzymes and are toxic for fermentation organisms. This review critically discusses the potential applications for laccases in biorefinery processes and emphasizes the challenges and perspectives which go along with the use of this enzyme for the technical utilization of lignocellulose.

Pitfalls in optical on-line monitoring for high-throughput screening of microbial systems.

BACKGROUND: New high-throughput screening systems for microbial systems, e.g. the BioLector technology, are simple to handle and offer various options of optical online measurements. The parallelization and small scale in microtiter plates allow economical high throughput and, hence, to screen many parameters in reasonable time. Fluorescent proteins as fluorescent tags made the tracking of cellular proteins in-vivo a routine task. All these tools significantly contribute to the understanding of bioprocesses. But, there are some pitfalls which might mislead the user of such techniques. RESULTS: In this work the bacterium E. coli and the yeast K. lactis expressing the recombinant fluorescent proteins GFP, YFP, FbFP and mCherry were investigated. Cultivations were performed applying special microtiter plates with optodes for dissolved oxygen tension (DOT) and pH measurement in the BioLector system. In this way, microbial growth, protein formation, DOT and pH were monitored on-line via optical signals. During these studies it became obvious that fluorescent proteins can interfere with the optical signals leading to incorrect results. In this work these effects are characterized in detail and possibilities are presented how such adverse effects can be corrected or minimized by mathematical procedures or modification of the measuring method. Additionally, it is shown that morphological changes of cells can affect the biomass on-line monitoring via scattered light. CONCLUSIONS: The here reported phenomena refer to typical experiments in biotechnological labs. For this reason these aspects are highlighted in this work to make operators of such valuable techniques as the BioLector aware for potential pitfalls and resulting misinterpretations. With the right approach it is possible to minimize existing problems and deal with them.

Utilizing high-throughput experimentation to enhance specific productivity of an E.coli T7 expression system by phosphate limitation.

BACKGROUND: The specific productivity of cultivation processes can be optimized, amongst others, by using genetic engineering of strains, choice of suitable host/vector systems or process optimization (e.g. choosing the right induction time). A further possibility is to reduce biomass buildup in favor of an enhanced product formation, e.g. by limiting secondary substrates in the medium, such as phosphate. However, with conventional techniques (e.g. small scale cultivations in shake flasks), it is very tedious to establish optimal conditions for cell growth and protein expression, as the start of protein expression (induction time) and the degree of phosphate limitation have to be determined in numerous concerted, manually conducted experiments. RESULTS: We investigated the effect of different induction times and a concurrent phosphate limitation on the specific productivity of the T7 expression system E.coli BL21(DE3) pRhotHi-2-EcFbFP, which produces the model fluorescence protein EcFbFP upon induction. Therefore, specific online-monitoring tools for small scale cultivations (RAMOS, BioLector) as well as a novel cultivation platform (Robo-Lector) were used for rapid process optimization. The RAMOS system monitored the oxygen transfer rate in shake flasks, whereas the BioLector device allowed to monitor microbial growth and the production of EcFbFP in microtiter plates. The Robo-Lector is a combination of a BioLector and a pipetting robot and can conduct high-throughput experiments fully automated. By using these tools, it was possible to determine the optimal induction time and to increase the specific productivity for EcFbFP from 22% (for unlimited conditions) to 31% of total protein content of the E.coli cells via a phosphate limitation. CONCLUSIONS: The results revealed that a phosphate limitation at the right induction time was suitable to redirect the available cellular resources during cultivation to protein expression rather than in biomass production. To our knowledge, such an effect was shown for the first time for an IPTG-inducible expression system. Finally, this finding and the utilization of the introduced high-throughput experimentation approach could help to find new targets to further enhance the production capacity of recombinant E.coli-strains.

Brain and cognitive-behavioural development after asphyxia at term birth.

Perinatal asphyxia occurs in approximately 1-6 per 1000 live full-term births. Different patterns of brain damage can result, though the relation of these patterns to long-term cognitive-behavioural outcome remains under investigation. The hippocampus is one brain region that can be damaged (typically not in isolation), and this site of damage has been implicated in two different long-term outcomes, cognitive memory impairment and the psychiatric disorder schizophrenia. Factors in addition to the acute episode of asphyxia likely contribute to these specific outcomes, making prediction difficult. Future studies that better document long-term cognitive-behavioural outcome, quantitatively identify patterns of brain injury over development and consider additional variables that may modulate the impact of asphyxia on cognitive and behavioural function will forward the goals of predicting long-term outcome and understanding the mechanisms by which it unfolds.

Neurodevelopmental outcome of preterm infants with ventricular dilatation with and without associated haemorrhage.

This study investigated whether in preterm children who had ventricular dilatation (VD) on neonatal cranial ultrasound outcome at age 8 years was influenced by the additional presence of germinal matrix haemorrhage--intraventricular haemorrhage (GMH-IVH). Six-hundred and ninety-nine preterm infants (<33 wks' gestation, mean 29.6 wks [SD 2.1]) with either normal cranial ultrasound (n=616; 286 females, 330 males), or with VD with (n=66; 32 females, 34 males) or without (n=17; 4 females, 13 males) GMH-IVH were enrolled in the study. At age 8 years outcome was assessed in 567 (81%) of the 699 children by neurological examination, the Test of Motor Impairment (TOMI), the test of Visuo-Motor Integration (VMI), and the Wechsler Intelligence Scales for Children. Results showed that the proportion of children with disabling impairments was higher in the group with VD and GMH-IVH. Performance on TOMI and VMI (even in those without disabling impairments) was poorer in those with VD and GMH-IVH than in children with normal scans or those with VD only. Children with VD and GMH-IVH had significantly lower performance IQ than children with normal ultrasound, whereas those with VD only were not different from those with normal scans. Results suggest the presence of subtle white matter injury that has not been identified by neonatal cranial ultrasound. Although this study did not investigate biochemical markers of haemorrhage, we hypothesize that non-protein-bound iron is likely to be a contributing factor to white matter damage in preterm infants.

Long-term neurodevelopmental outcome of preterm children with unilateral cerebral lesions diagnosed by neonatal ultrasound.

OBJECTIVE: Little information is available on long-term neurodevelopmental outcome of preterm infants with unilateral cerebral lesions detected by neonatal cranial ultrasound. This study aims to investigate the long-term outcome in a cohort of very preterm infants with unilateral cerebral lesions acquired in the perinatal period. METHODS: A prospective cohort study of 668 preterm infants (<33 weeks gestation; birth years 1985-1991) at a single tertiary perinatal centre in the UK. All infants had serial cranial ultrasound examination in the neonatal period. Outcome was assessed at age 8 years with the Wechsler Intelligence Scales for Children (WISC-R), Test of Visuo-motor Integration (VMI) and the Test of Motor Impairment (TOMI). RESULTS: Of the 668 infants, 369 infants had normal ultrasound scans. Two hundred and ninety nine children had bilateral parenchymal or non-parenchymal lesions (57 left-sided, 41 right-sided, 201 bilateral). Five hundred and thirty four (79%) children attended follow-up at age 8 years. Mean Full Scale IQ (FSIQ) was 101 (SD+/-16), 93 (SD+/-17), 102 (SD+/-17) and 91 (SD+/-21) for normal, left-sided, right-sided and bilateral lesion groups respectively. In all groups verbal IQ (VIQ) was higher than performance IQ (PIQ). Scores of FSIQ, VIQ and PIQ, VMI and TOMI were significantly different between the groups. After exclusion of children with parenchymal lesions, however, the difference was only significant for the TOMI scores. In all tests, children with left-sided lesions performed poorer than children with right-sided lesions. CONCLUSIONS: In this cohort of preterm infants with unilateral cerebral lesions, verbal function was preserved over non-verbal function independently of the side of lesion. Furthermore, the results suggest that the neurodevelopmental outcome of children with left-sided lesions is less favourable than that of children with right-sided lesions.

Predictors of long-term outcome in very preterm infants: gestational age versus neonatal cranial ultrasound.

OBJECTIVES: To investigate the effect of gestational age at birth on the frequency of ultrasound-detected brain lesions in infants born at <33 weeks of gestation and to investigate whether the relationship between neonatal cranial ultrasound diagnosis and neurodevelopmental outcome at 8 years of age was independent of gestational age. METHODS: Eight hundred forty-seven infants born at <33 weeks of gestation, admitted to a single tertiary referral center between 1983 and 1988, underwent serial neonatal cranial ultrasound. At 8 years of age neurodevelopmental outcome was assessed by structured neurologic examination, psychometric tests (Wechsler Intelligence Scale for Children), tests of visuomotor integration (Beery) and motor impairment (Henderson-Stott). Infants were subdivided into a group born at <28 weeks and a group born at between 28 and 32 weeks. Neurodevelopmental outcome was analyzed for each ultrasound diagnosis. RESULTS: Hemorrhagic lesions such as germinal matrix/intraventricular hemorrhage and hemorrhagic parenchymal infarction were more frequent in infants born at <28 weeks. There was no difference in the frequency of cystic periventricular leucomalacia between the 2 groups. When neurodevelopmental outcome for each ultrasound diagnosis was compared, no significant difference was found between the 2 gestational age groups. CONCLUSION: In the gestational age range studied, adverse neurodevelopmental outcome depends primarily on the type of the intracranial lesion rather than on gestational age.