The efficacy and safety of combination therapy of repeated low-level red light and defocus-incorporated multiple segments spectacle lenses for myopia control in children: the study protocol for a 12-month, randomized, parallel-controlled, and single-center clinical trial.

BACKGROUND: Myopia is increasing in prevalence worldwide. Combination therapy showed a better effect on myopia control than monotherapy. Repeated low-level red light therapy (RLRL) therapy and defocus-incorporated multiple segment (DIMS) spectacle lenses have been reported to retard myopia progression significantly. However, whether these two therapies are better than one is still unknown. The present study aims to report the study protocol of a trial designed to evaluate the efficacy and safety of combination therapy of RLRL and DIMS versus DIMS alone for reducing the progression of myopia among Chinese school-aged children. METHODS: This study is a 12-month, randomized, parallel-controlled, single-center clinical trial. We will recruit children aged 8-12 years with spherical equivalence (SE) between - 0.50 D and - 6.00 D under cycloplegia in both eyes. We will recruit 66 participants with an allocation ratio of 1:1 from our hospital. Participants in the intervention group will be treated with an RLRL therapy device twice a day from Monday to Friday at home, 3 min per session, with a minimum interval of 4 h, under the supervision of their parents/guardians. They will wear DIMS spectacles for myopia correction during the day. Participants in the control group will not receive the RLRL therapy and will only wear DIMS spectacles to correct myopia. Participants from both groups will attend the hospital every 6 months. The primary outcome is the change in axial length at 12 months. Secondary outcomes include changes in refraction under cycloplegia, optical coherence tomography (OCT), multifocal electroretinogram (mfERG), color vision, and participants' self-reporting of adverse events at 12 months. DISCUSSION: This study will report the efficacy and safety outcome of the combination therapy of RLRL and DIMS versus DIMS for school-aged children with myopia in detail. TRIAL REGISTRATION: ChiCTR2300075398. Registered 4 September 2023. https://www.chictr.org.cn/bin/project/edit?pid=200751 .

Assessing the rebound phenomenon in different myopia control treatments: A systematic review.

PURPOSE: To review the rebound effect after cessation of different myopia control treatments. METHODS: A systematic review that included full-length randomised controlled studies (RCTs), as well as post-hoc analyses of RCTs reporting new findings on myopia control treatments rebound effect in two databases, PubMed and Web of Science, was performed according to the PRISMA statement. The search period was between 15 June 2023 and 30 June 2023. The Cochrane risk of bias tool was used to analyse the quality of the selected studies. RESULTS: A total of 11 studies were included in this systematic review. Unifying the rebound effects of all myopia control treatments, the mean rebound effect for axial length (AL) and spherical equivalent refraction (SER) were 0.10 ± 0.07 mm [-0.02 to 0.22] and -0.27 ± 0.2 D [-0.71 to -0.03] after 10.2 ± 7.4 months of washout, respectively. In addition, spectacles with highly aspherical lenslets or defocus incorporated multiple segments technology, soft multifocal contact lenses and orthokeratology showed lower rebound effects compared with atropine and low-level light therapy, with a mean rebound effect for AL and SER of 0.04 ± 0.04 mm [0 to 0.08] and -0.13 ± 0.07 D [-0.05 to -0.2], respectively. CONCLUSIONS: It appears that the different treatments for myopia control produce a rebound effect after their cessation. Specifically, optical treatments seem to produce less rebound effect than pharmacological or light therapies. However, more studies are required to confirm these results.

Data independent acquisition for gas chromatographic MS/MS analysis of volatile compounds.

This study presents a novel tandem mass spectrometry (MS/MS) approach utilizing a data independent acquisition (DIA) concept specifically designed with gas chromatography-electron ionization-triple quadrupole mass spectrometry (GC-EI-QqQMS). This allows compound identification based on comparison between all the experimental MS/MS product ion spectra and the simulated library data of >1,000 MS/MS transitions of 71 compounds. The simulation data were generated by using the Competitive Fragmentation Modeling (CFM-ID) 3.0 program. The approach for calculation of the DIA MS/MS library match scores was then established and applied for identification of a range of terpenoids and oxygenated compounds in perfume. The identity of each peak was confirmed using 4-241 MS/MS transitions. The established data collection and analysis methods are expected to be useful for increased confidence in volatile compound analysis.

Dynamic Instrumental and Sensory Methods Used to Link Aroma Release and Aroma Perception: A Review.

Perception of flavor is a dynamic process during which the concentration of aroma molecules at the olfactory epithelium varies with time as they are released progressively from the food in the mouth during consumption. The release kinetics depends on the food matrix itself but also on food oral processing, such as mastication behavior and food bolus formation with saliva, for which huge inter-individual variations exist due to physiological differences. Sensory methods such as time intensity (TI) or the more-recent methods temporal dominance of sensations (TDS) and temporal check-all-that-apply (TCATA) are used to account for the dynamic and time-related aspects of flavor perception. Direct injection mass spectrometry (DIMS) techniques that measure in real time aroma compounds directly in the nose (nosespace), aimed at obtaining data that reflect the pattern of aroma release in real time during food consumption and supposed to be representative of perception, have been developed over the last 25 years. Examples obtained with MS operated in chemical ionization mode at atmospheric or sub-atmospheric pressure (atmospheric pressure chemical ionization APCI or proton-transfer reaction PTR) are given, with emphases on studies conducted with simultaneous dynamic sensory evaluation. Inter-individual variations in terms of aroma release and their relevance for understanding flavor perception are discussed as well as the evidenced cross-modal interactions.

Defocus incorporated multiple segments (DIMS) spectacle lenses increase the choroidal thickness: a two-year randomized clinical trial.

BACKGROUND: Myopia control interventions, such as defocus incorporated multiple segments (DIMS) spectacle lenses, have been adopted in school-aged children to reduce the prevalence of myopia and its complications. This study aimed to investigate the effect of DIMS spectacle lenses on subfoveal choroidal thickness (SfChT) over a period of two years, as the choroidal response to myopic control is a crucial factor in exploring its potential effect on predicting myopia progression. METHODS: This study involved a secondary analysis of our previous randomized clinical trial. Myopic school-aged children aged 8-13 years were recruited in a two-year study investigating the effect of DIMS spectacle lenses on myopia progression. The treated group received DIMS spectacle lenses (n = 78), while the control group was treated with a pair of single vision (SV) spectacle lenses (n = 80). SfChT was monitored at 1 week, 1, 3, 6, 12, 18 and 24 months post lens wear using spectral-domain optical coherence tomography and a custom made auto-segmentation algorithm utilizing convolutional neural networks. RESULTS: SfChT increased significantly after one week of DIMS spectacle lens wear compared to those wearing SV spectacle lenses (adjusted mean change relative to baseline ± SEM at one week; DIMS vs. SV, 6.75 ± 1.52 µm vs. - 3.17 ± 1.48 µm; P < 0.0001, general linear model). The thickness of choroid increased to 13.64 ± 2.62 µm after 12 months of DIMS lens wear while the choroid thinned in SV group (- 9.46 ± 2.55 µm). Choroidal changes demonstrated a significant negative association with axial elongation over two years in both the DIMS and SV groups. Choroidal change at three months significantly predicted the changes in AL at 12 months after controlling the effect of age and gender. CONCLUSIONS: Our study demonstrated a significant choroidal thickening in response to myopic defocus incorporated in a spectacle lens after one week of lens wear, sustained over the two-year study period. The results suggested that choroidal changes at three months may help predict changes in axial length after one year. Trial registration ClinicalTrials.gov. Myopia control with the multi-segment lens. NCT02206217. Registered 29 July 2014, https://clinicaltrials.gov/ct2/show/study/NCT02206217.

Optical characterisation of two novel myopia control spectacle lenses.

PURPOSE: To quantify the amount of myopic defocus, contrast modulation and other optical characteristics of two novel spectacle lenses (MiYOSMART by Hoya and Stellest by Essilor) with the inclusion of lenslets in their designs were investigated computationally and experimentally. This paper examined the hypothesis that despite the non-coaxial nature of the optics, image degradation will exist due to the fragmented nature of the base optic when imaging through the lens regions populated by lenslets. METHODS: Optical power was evaluated by computing wavefront vergence and curvature from wavefront slope measured with the Optocraft aberrometer within 1.0 and 6.0 mm apertures across MiYOSMART hexagons and Stellest rings. Point-spread functions (PSFs) were computed using physical (wave) optics and geometrical ray optics principles, and compared with experimental measurements using a 4f optical system. Simulated retinal images and modulation transfer functions (MTFs) were computed from PSF-derived optical transfer functions (OTFs). RESULTS: Mean lenslet power in MiYOSMART was +3.95 ± 0.10 D through the hexagons and +6.00 ± 0.15 D in Stellest in rings 1-5 and decreased by 0.42 D/ring reaching 3.50 D in the final one. Stellest lenslets included up to -0.015 microns of primary spherical aberration. PSFs and retinal images revealed simultaneous contributions of the base optic and lenslets. MTFs showed a decrease in contrast at low (1-10 c/deg) spatial frequencies (SFs) comparable to 0.25 D of defocus, and retention of diminished levels of contrast at higher SFs. CONCLUSIONS: Varying sagittal power and consistent curvature power across the lenslets is an identifying signature of the novel non-coaxial lens design included in both spectacle lenses. Lenslet array structure itself plays a significant role in determining image characteristics. For both lenses, the blur created by the fragmented base optic contributes to the image quality. The reduced MTFs over a wide range of spatial frequencies result in lowered image contrast.

Effect of atropine 0.01% on myopia control in children aged 6-13 years during the 2022 lockdown in Shanghai.

Purpose: To compare the myopic progression in children treated with 0. 01% atropine and those who discontinued atropine during the 2022-home quarantine in Shanghai. Methods: In this retrospective study, children aged 6-13 years with follow-up visits before (between January 2022 and February 2022) and after the lockdown (between July 2022 and August 2022) were included. Cycloplegic refraction and axial length (AL) were measured at both visits. The atropine group had continuous medication during the lockdown while the control group discontinued. The 0.01% atropine eyedrops were administered daily before bedtime. The types of spectacle lens were recorded: single vision (SV) spectacles or defocus incorporated multiple segments lenses (DIMS). Results: In total, 41 children (81 eyes) in the atropine group and 32 children (64 eyes) in the control group were enrolled. No significant difference was found in the demographic characteristics, spherical diopter, spherical equivalent (SE), AL, and follow-up time between the two groups before the lockdown in 2022 (all p > 0.1). After the home confinement, a greater myopia progression was observed in the control group (-0.46 ± 0.42 D) compared to atropine group (-0.26 ± 0.37 D; p = 0.0023). Axial elongation was also longer in the control group than that in children sustained with atropine (0.21 ± 0.17 vs. 0.13 ± 0.15 mm, p = 0.0035). Moreover, there was no significant change of spherical diopter and SE during lockdown in the atropine + DIMS combined subgroup (0.03 ± 0.033 D for spherical diopter, p = 0.7261 and 0.08 ± 0.27 D for SE, p = 0.2042, respectively). However, significant myopic shift was observed in the atropine + SV subgroup during the quarantine time (-0.31 ± 0.39 D for SE and 0.15 ± 0.16 mm for AL, both p < 0.001). Conclusion: Children treated with 0.01% atropine had slower myopia progression during the lockdown period in Shanghai compared with children discontinued. Moreover, the effect of atropine on myopic prevention can be strengthened with DIMS lenses.

The Efficacy of Defocus Incorporated Multiple Segments Lenses in Slowing Myopia Progression: Results from Diverse Clinical Circumstances.

PURPOSE: Defocus incorporated multiple segments (DIMS) spectacle lenses were reported to slow myopia progression significantly in a randomized controlled trial (RCT). The study evaluated their effectiveness in clinical settings. DESIGN: Retrospective study. PARTICIPANTS: Patient records involving use of DIMS and single-vision (SV) spectacle lenses were collected from subsidiary hospitals of Aier Eye Hospital Group. METHODS: The spherical equivalent (SE), determined by subjective refraction, was adopted to assess the myopia progression. The strategy of propensity score matching (PSM) was applied to match the confounding baseline characteristics between the 2 groups. The effectiveness was calculated based on the difference of myopia progression of these 2 approaches. MAIN OUTCOME MEASURES: Change in SE. RESULTS: Three thousand six hundred thirty-nine patients with DIMS and 6838 patients with SV spectacles were included. The age of the patients was 6 to 16 years (mean ± standard deviation: 11.02 ± 2.53 years). The baseline SE was between 0.00 and -10.00 diopters (D) (mean ± standard deviation: -2.78 ± 1.74 D). After the PSM, data on 2240 pairs with 1-year follow-up and on 735 pairs with 2-year follow-up were obtained. Significantly slower progression was seen in the DIMS group at both the 1-year (DIMS, -0.50 ± 0.43 D; SV, -0.77 ± 0.58 D; P < 0.001) and 2-year (DIMS, -0.88 ± 0.62 D; SV, -1.23 ± 0.76 D; P < 0.001) subdataset. In the 1-year subdataset, 40% and 19% showed myopia progression of no more than 0.25 D for the DIMS and SV groups, respectively (chi-square, 223.43; P < 0.001), whereas 9% and 22% showed myopia progression of more than 1.00 D for the DIMS and SV groups, respectively (chi-square, 163.38; P < 0.001). In the 2-year subdataset, 33% and 20% showed myopia progression of no more than 0.50 D for the DIMS and SV groups, respectively (chi-square, 31.15; P < 0.001), whereas 12% and 29% showed myopia progression of more than 1.50 D for the DIMS and SV groups (chi-square, 65.60; P < 0.001). CONCLUSIONS: Although the magnitude was lower than that reported in the previous RCT, this large-scale study with diversity of the data sources confirmed the effectiveness of DIMS spectacles to slow myopia progression in clinical practice. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Balancing Trade-Offs Imposed by Growth Media and Mass Spectrometry for Bacterial Exometabolomics.

The bacterial exometabolome consists of a vast array of specialized metabolites, many of which are only produced in response to specific environmental stimuli. For this reason, it is desirable to control the extracellular environment with a defined growth medium composed of pure ingredients. However, complex (undefined) media are expected to support the robust growth of a greater variety of microorganisms than defined media. Here, we investigate the trade-offs inherent to a range of complex and defined solid media for the growth of soil microorganisms, production of specialized metabolites, and detection of these compounds using direct infusion mass spectrometry. We find that complex media support growth of more soil microorganisms, as well as allowing for the detection of more previously discovered natural products as a fraction of total m/z features detected in each sample. However, the use of complex media often caused mass spectrometer injection failures and poor-quality mass spectra, which in some cases resulted in over a quarter of samples being removed from analysis. Defined media, while more limiting in growth, generated higher quality spectra and yielded more m/z features after background subtraction. These results inform future exometabolomic experiments requiring a medium that supports the robust growth of many soil microorganisms. IMPORTANCE Bacteria are capable of producing and secreting a rich diversity of specialized metabolites. Yet, much of their exometabolome remains hidden due to challenges associated with eliciting specialized metabolite production, labor-intensive sample preparation, and time-consuming analysis techniques. Using our versatile three-dimensional (3D)-printed culturing platform, SubTap, we demonstrate that rapid exometabolomic data collection from a diverse set of environmental bacteria is feasible. We optimized our platform by surveying Streptomyces isolated from soil on a variety of media types to assess viability, degree of specialized metabolite production, and compatibility with downstream LESA-DIMS analysis. Ultimately, this will enable data-rich experimentation, allowing for a better understanding of bacterial exometabolomes.

Overview on Defocus Incorporated Multiple Segments Lenses: A Novel Perspective in Myopia Progression Management.

Myopia is becoming more common across the world, affecting approximately two billion people and rising. Different kinds of therapies (optical, pharmaceutical, environmental, or behavioral) have been proposed to decrease myopia progression, but with variable results and a lack of standardization. The evidence that targeted myopic defocus inhibits eye length growth has paved the way for several contact and spectacle lense designs to induce a peripheral defocus, thus slowing myopia progression, but the perfect configuration has yet to be defined. One of the newest and more promising approaches in this field is the use of Defocus Incorporated Multiple Segments (DIMS) lenses. These lenses are built from the assumption that targeted myopic defocus, produced by 396 mid-peripheral lenslets with positive power, inhibits eye length growth. Recent studies have highlighted the effectiveness of these lenses compared to children who had worn single vision spectacle lenses, in terms of myopia control and tolerability. Despite the evidence that these lenses can help slow down the progression of myopia, the occasional mid-peripheral aberrations they can induce, as well as the overall eye strain that comes with wearing them, should not be overlooked. The aim of this review is to give attention to the advantages and the shortfalls of this new approach and to evaluate its effectiveness in clinical practice.

Mobile monitoring of VOCs and source identification using two direct-inlet MSs in a large fine and petroleum chemical industrial park.

Mobile monitoring with direct-inlet MS (DI-MS), one of the most direct and effective ways to track emission sources, can effectively serve air quality management in chemical industrial parks (CIPs). Mobile monitoring using a high mass-resolution proton-transfer-reaction time-of-flight MS (HMR-PTR-TOFMS) and single-photon ionization time-of-flight MS (SPI-TOFMS) was conducted in a large fine and petroleum CIP in eastern China for three days. The high mixing ratios of aliphatic hydrocarbons (AHs), aromatics, oxygenated VOCs (OVOCs), and nitrogenous VOCs (NVOCs) were found in the northeast, middle, north, and northeast of the fine chemical industrial zone (FCIZ), respectively. OVOCs were the most abundant VOC group in this area. Abnormal emissions of aromatics were universal throughout the CIP. We discovered 38 characteristic VOCs by the HMR-PTR-TOFMS, mainly including C6-C10 aromatics, C2-C6 carbonyls, C2-C3 organic acids, and some NVOCs. The time series and spatial distribution of the TVOCs obtained by the two DI-MSs are generally consistent. A comparison of the speciated VOCs at the TVOC peak points illustrates that the characteristic VOCs obtained by different instruments differed significantly: PTR-TOFMS showed an advantage in measuring aromatics and OVOCs; SPI-TOFMS showed an advantage in measuring aromatics and some Ahs; offline GC-MS showed an advantage in measuring AHs, aromatics, some OVOCs, and halohydrocarbons. Similarities were compared between five positive matrix factorization (PMF) model-based fingerprints of VOCs in a previous study and observed profiles of VOCs from mobile monitoring. The emission sources of the five fingerprints were identified and validated: two were widely distributed, one was a chemical reagent production factory, one was an acrylic fiber production plant, and one was a pesticide factory. This study demonstrated methods for analyzing mobile monitoring data, characterizing the VOCs in the fine and petroleum CIP, correlating the results of stationary observation and mobile monitoring, and integrating the source tracing system with DI-MSs.

Integrative Metabolomic and Lipidomic Profiling of Lung Squamous Cell Carcinoma for Characterization of Metabolites and Intact Lipid Species Related to the Metastatic Potential.

SQCC is a major type of NSCLC, which is a major cause of cancer-related deaths, and there were no reports regarding the prediction of metastatic potential of lung SQCC by metabolomic and lipidomic profiling. In this study, metabolomic and lipidomic profiling of lung SQCC were performed to predict its metastatic potential and to suggest potential therapeutic targets for the inhibition of lung SQCC metastasis. Human bronchial epithelial cells and four lung SQCC cell lines with different metastatic potentials were analyzed using gas chromatography-mass spectrometry and direct infusion-mass spectrometry. Based on the obtained metabolic and lipidomic profiles, we constructed models to predict the metastatic potential of lung SQCC; glycerol, putrescine, ß-alanine, hypoxanthine, inosine, myo-inositol, phosphatidylinositol (PI) 18:1/18:1, and PI 18:1/20:4 were suggested as characteristic metabolites and intact lipid species associated with lung SQCC metastatic potential. In this study, we established predictive models for the metastatic potential of lung SQCC; furthermore, we identified metabolites and intact lipid species relevant to lung SQCC metastatic potential that may serve as potential therapeutic targets for the inhibition of lung SQCC metastasis.

[Myopia treatment and prophylaxis with defocus incorporated multiple segments spectacle lenses]. / Myopietherapie und Prophylaxe mit "Defocus Incorporated Multiple Segments"-Brillengläsern.

Excessive axial eye growth in children and adolescents leads to progressive myopia and can result in severe ocular diseases in adulthood. Various strategies have already been developed to inhibit progression of myopia. The novel single vision lens presented in this article features the defocus incorporated multiple segments (DIMS) technology and adds an easy to use, noninvasive option to the portfolio of myopia treatment. Initial studies showed promising results with only a very low side effect profile.

Stationary monitoring and source apportionment of VOCs in a chemical industrial park by combining rapid direct-inlet MSs with a GC-FID/MS.

Fast and comprehensive monitoring of VOCs, required for air quality management in large-scale chemical industrial parks in China, cannot be accomplished by stationary measurements using conventional GC-FID or GC-MS alone due to their low temporal resolutions and limited detectable ranges. Novel direct-inlet mass spectrometry (DI-MS) has been widely applied for real-time monitoring of VOCs. To verify its applicability in industrial settings, high mass-resolution proton-transfer-reaction time-of-flight MS (HMR-PTR-TOFMS), single-photon ionization time-of-flight MS (SPI-TOFMS), together with online GC-FID/MS were simultaneously deployed at the boundary of one of the largest chemical industrial parks in eastern China. Aromatics, acetonitrile, acetic acid, ethyl acetate, aliphatic hydrocarbons, 1,2-dichloroethane, and acetone were detected as the main pollutants. These three instruments detected 12 common species, among which ethyl acetate, toluene, C8-aromatics, and methyl ethyl ketone showed similar time series and levels. Acetone, benzene, chlorobenzene, styrene, and C9-aromatics showed only similar time series. The HMR-PTR-TOFMS uniquely detected 14 species, mainly oxidized VOCs, nitriles, and amines, which greatly helps acknowledge the pollutants in the chemical industrial area. Positive matrix factorization, using the HMR-PTR-TOFMS and GC-FID/MS datasets, was used to identify eight sources. Four of the identified sources were mainly detected by the HMR-PTR-TOFMS, with pollutants mainly comprised of nitriles, amines, carbonyls, and organic acids, most of which were hazardous and/or odorous. These four sources accounted for 41.5% and 33.2% of the total VOCs and ozone formation potential, respectively. The complementary nature of GC-FID/MS and HMR-PTR-TOFMS in VOC source apportionment in industrial settings is of great practical use for advanced VOCs abatement. Thus, the high mass resolution DI-MSs are suggested to be a supplementary measurement for fence-line monitoring. Although with a relatively short period attempt, this study has wide implications for the fence-line stationary observational modes and source apportion methods combining with traditional observations.

Optical and imaging properties of a novel multi-segment spectacle lens designed to slow myopia progression.

PURPOSE: High sampling density optical metrology combined with pupil- and image-plane numerical analyses were applied to evaluate a novel spectacle lens containing multiple small zones designed to slow myopia progression. METHODS: High-resolution aberrometry (ClearWave, www.lumetrics.com) was used to sample wavefront slopes of a novel spectacle lens, Defocus Incorporated Multiple Segments (DIMS) (www.hoya.com), incorporating many small, positive-powered lenslets in its periphery. Using wavefront slope and error maps, custom MATLAB software ('Indiana Wavefront Analyzer') was used to compute image-plane point-spread functions (PSF), modulation transfer functions (MTF), simulated images and power distributions created by the dual-focus optic for different pupil sizes and target vergences. RESULTS: Outside of a central 10 mm zone containing single distance optical power, a hexagonal array of small 1 mm lenslets with nearest-neighbour separations of 0.5 mm were distributed over the lens periphery. Sagittal and curvature-based measures of optical power imperfectly captured the consistent +3.50 D add produced by the lenslets. Image plane simulations revealed multiple PSFs and poor image quality at the lenslet focal plane. Blur at the distance optic focal plane was consistent with a combination of diffraction blur from the distance optic and the approximately +3.50 D of defocus from the 1 mm diameter near optic zones. CONCLUSION: Converging the defocused beams generated by the multiple small (1 mm diameter) lenslets to a blurred image at the distance focal plane produced a blur magnitude determined by the small lenslet diameter and not the overall pupil diameter. The distance optic located in between the near-add lenslets determines the limits of the optical quality achievable by the lens. When compared to the optics of a traditional concentric-zone dual-focus contact lens, the optics of the DIMS lens generates higher-contrast images at low spatial frequencies (<7 cycles per degree), but lower-contrast at high spatial frequencies.

Assessing the Pre-Analytical Stability of Small-Molecule Metabolites in Cerebrospinal Fluid Using Direct-Infusion Metabolomics.

Metabolomics studies aiming to find biomarkers frequently make use of historical or multicenter cohorts. These samples often have different pre-analytical conditions that potentially affect metabolite concentrations. We studied the effect of different storage conditions on the stability of small-molecule metabolites in cerebrospinal fluid to aid a reliable interpretation of metabolomics data. Three cerebrospinal fluid pools were prepared from surplus samples from the Amsterdam Dementia Cohort biobank. Aliquoted pools were exposed to different storage conditions to assess the temperature and freeze/thaw stability before final storage at -80 °C: storage up to four months at -20 °C and up to one week at either 5-8 °C or 18-22 °C and exposure to up to seven freeze/thaw cycles. Direct-infusion high-resolution mass spectrometry was performed, resulting in the identification of 1852 m/z peaks. To test the storage stability, principal component analyses, repeated measures analysis of variance, Kruskal‒Wallis tests, and fold change analyses were performed, all demonstrating that small-molecule metabolites in the cerebrospinal fluid (CSF) are relatively unaffected by 1‒3 freeze/thaw cycles, by storage at -20 °C up to two months, by storage at 5-8 °C for up to 72 h, or by storage at 18-22 °C for up to 8 h. This suggests that these differences do not affect the interpretation of potential small-molecule biomarkers in multicenter or historical cohorts and implies that these cohorts are suitable for biomarker studies.

Characterization and classification of rat neural stem cells and differentiated cells by comparative metabolic and lipidomic profiling.

It is necessary to characterize and classify neural stem cells (NSCs) and differentiated cells (DCs) for potential use of NSC to treat neurodegenerative diseases. We therefore performed an analysis of NSCs and DCs using gas chromatography mass spectrometry (GC-MS) and direct infusion mass spectrometry (DI-MS) with elaborate multivariate statistical analysis for the characterization and classification of rat NSCs and DCs. GC-MS and DI-MS detected a total of 92 metabolites and lipids in NSCs and DCs, and the levels of 72 of them differed significantly between NSCs and DCs. The optimal model for partial least squares (PLS) discriminant analysis was constructed by applying 3 and 2 PLS components with a unit-variance scaling method for classifying NSCs and DCs based on the data obtained in the GC-MS and DI-MS analyses, respectively. The obtained results from PCA and PLS-DA suggest that creatinine, lactic acid, lysine, glutamine, glycine, pyroglutamic acid, PG 18:1/20:2, PS 18:0/20:2, PI 18:0/20:3, PC 16:0/20:4, PI 16:0/20:4, and PI 18:1/20:4 were the main contributors that provided distinct characteristics of NSCs and DCs. The results of this study suggest objective and complementary criteria for the characterization and classification of NSCs and DCs for potential clinical applications. Graphical abstract.

Direct-infusion based metabolomics unveils biochemical profiles of inborn errors of metabolism in cerebrospinal fluid.

BACKGROUND: For inborn errors of metabolism (IEM), metabolomics is performed for three main purposes: 1) development of next generation metabolic screening platforms, 2) identification of new biomarkers in predefined patient cohorts and 3) for identification of new IEM. To date, plasma, urine and dried blood spots are used. We anticipate that cerebrospinal fluid (CSF) holds additional - valuable - information, especially for IEM with neurological involvement. To expand metabolomics to CSF, we here tested whether direct-infusion high-resolution mass spectrometry (DI-HRMS) based non-quantitative metabolomics could correctly capture the biochemical profile of patients with an IEM in CSF. METHODS: Eleven patient samples, harboring eight different IEM, and thirty control samples were analyzed using DI-HRMS. First we assessed whether the biochemical profile of the control samples represented the expected profile in CSF. Next, each patient sample was assigned a 'most probable diagnosis' by an investigator blinded for the known diagnoses of the patients. RESULTS: the biochemical profile identified using DI-HRMS in CSF samples resembled the known profile, with - among others - the highest median intensities for mass peaks annotated with glucose, lactic acid, citric acid and glutamine. Subsequent analysis of patient CSF profiles resulted in correct 'most probable diagnoses' for all eleven patients, including non-ketotic hyperglycinaemia, propionic aciduria, purine nucleoside phosphorylase deficiency, argininosuccinic aciduria, tyrosinaemia type I, hyperphenylalaninemia and hypermethioninaemia. CONCLUSION: We here demonstrate that DI-HRMS based non-quantitative metabolomics accurately captures the biochemical profile of this set of patients in CSF, opening new ways for using metabolomics in CSF in the metabolic diagnostic laboratory.

Direct Infusion Based Metabolomics Identifies Metabolic Disease in Patients' Dried Blood Spots and Plasma.

In metabolic diagnostics, there is an emerging need for a comprehensive test to acquire a complete view of metabolite status. Here, we describe a non-quantitative direct-infusion high-resolution mass spectrometry (DI-HRMS) based metabolomics method and evaluate the method for both dried blood spots (DBS) and plasma. 110 DBS of 42 patients harboring 23 different inborn errors of metabolism (IEM) and 86 plasma samples of 38 patients harboring 21 different IEM were analyzed using DI-HRMS. A peak calling pipeline developed in R programming language provided Z-scores for ~1875 mass peaks corresponding to ~3835 metabolite annotations (including isomers) per sample. Based on metabolite Z-scores, patients were assigned a 'most probable diagnosis' by an investigator blinded for the known diagnoses of the patients. Based on DBS sample analysis, 37/42 of the patients, corresponding to 22/23 IEM, could be correctly assigned a 'most probable diagnosis'. Plasma sample analysis, resulted in a correct 'most probable diagnosis' in 32/38 of the patients, corresponding to 19/21 IEM. The added clinical value of the method was illustrated by a case wherein DI-HRMS metabolomics aided interpretation of a variant of unknown significance (VUS) identified by whole-exome sequencing. In summary, non-quantitative DI-HRMS metabolomics in DBS and plasma is a very consistent, high-throughput and nonselective method for investigating the metabolome in genetic disease.

Metabolomics Discovers Early-Response Metabolic Biomarkers that Can Predict Chronic Reproductive Fitness in Individual Daphnia magna.

Chemical risk assessment remains entrenched in chronic toxicity tests that set safety thresholds based on animal pathology or fitness. Chronic tests are resource expensive and lack mechanistic insight. Discovering a chemical's mode-of-action can in principle provide predictive molecular biomarkers for a toxicity endpoint. Furthermore, since molecular perturbations precede pathology, early-response molecular biomarkers may enable shorter, more resource efficient testing that can predict chronic animal fitness. This study applied untargeted metabolomics to attempt to discover early-response metabolic biomarkers that can predict reproductive fitness of Daphnia magna, an internationally-recognized test species. First, we measured the reproductive toxicities of cadmium, 2,4-dinitrophenol and propranolol to individual Daphnia in 21-day OECD toxicity tests, then measured the metabolic profiles of these animals using mass spectrometry. Multivariate regression successfully discovered putative metabolic biomarkers that strongly predict reproductive impairment by each chemical, and for all chemicals combined. The non-chemical-specific metabolic biomarkers were then applied to metabolite data from Daphnia 24-h acute toxicity tests and correctly predicted that significant decreases in reproductive fitness would occur if these animals were exposed to cadmium, 2,4-dinitrophenol or propranolol for 21 days. While the applicability of these findings is limited to three chemicals, they provide proof-of-principle that early-response metabolic biomarkers of chronic animal fitness can be discovered for regulatory toxicity testing.