An engineered biosensor enables dynamic aspartate measurements in living cells.

Intracellular levels of the amino acid aspartate are responsive to changes in metabolism in mammalian cells and can correspondingly alter cell function, highlighting the need for robust tools to measure aspartate abundance. However, comprehensive understanding of aspartate metabolism has been limited by the throughput, cost, and static nature of the mass spectrometry (MS)-based measurements that are typically employed to measure aspartate levels. To address these issues, we have developed a green fluorescent protein (GFP)-based sensor of aspartate (jAspSnFR3), where the fluorescence intensity corresponds to aspartate concentration. As a purified protein, the sensor has a 20-fold increase in fluorescence upon aspartate saturation, with dose-dependent fluorescence changes covering a physiologically relevant aspartate concentration range and no significant off target binding. Expressed in mammalian cell lines, sensor intensity correlated with aspartate levels measured by MS and could resolve temporal changes in intracellular aspartate from genetic, pharmacological, and nutritional manipulations. These data demonstrate the utility of jAspSnFR3 and highlight the opportunities it provides for temporally resolved and high-throughput applications of variables that affect aspartate levels.

Non-targeted metabolomics identifies biomarkers in milk with high and low milk fat percentage.

Milk is widely recognized as an important food source with health benefits. Different consumer groups have different requirements for the content and proportion of milk fat; therefore, it is necessary to investigate the differential metabolites and their regulatory mechanisms in milk with high and low milk fat percentages (MFP). In this study, untargeted metabolomics was performed on milk samples from 13 cows with high milk fat percentage (HF) and 13 cows with low milk fat percentage (LF) using ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS). Forty-eight potential differentially labeled compounds were screened using the orthogonal partial least squares-discriminant analysis (OPLS-DA) combined with the weighted gene co-expression network analysis (WGCNA) method. Amino acid metabolism was the key metabolic pathway with significant enrichment of L-histidine, 5-oxoproline, L-aspartic acid, and L-glutamic acid. The negative correlation with MFP differentiated the HF and LF groups. To further determine the potential regulatory role of these amino acids on milk fat metabolism, the expression levels of marker genes in the milk fat synthesis pathway were explored. It was noticed that L-histidine reduced milk fat concentration primarily by inhibiting the triglycerides (TAG) synthesis pathway. L-aspartic acid and L-glutamic acid inhibited milk fat synthesis through the fatty acid de novo and TAG synthesis pathways. This study provides new insights into the mechanism underlying milk fat synthesis and milk quality improvement.

Variable fragmentation and ionization of amyloid-beta epimers and isomers.

While the existence of D-amino acids in peptides and proteins has recently been accepted in higher forms of life, their roles and importance are yet to be understood. The lack of analytical methods present for such epimeric and/or isomeric analyses often limits developments in the field. Studies have shown the elevated presence of epimeric and isomeric modifications to amyloid-beta (Aß) peptides extracted from Alzheimer's disease patients. These modifications most frequently occur through aspartic acid and serine residues. Because such peptides are indistinguishable by mass alone, selective liquid chromatography tandem mass spectrometry analysis is required to differentiate such peptides. Herein, we examine MS/MS of tryptic fragments of Aß peptides containing D-Asp, L-iso-Asp, D-iso-Asp, and/or D-Ser modifications. Peptide ionizability and fragmentation are explored through selected reaction monitoring, selected ion monitoring, and product ion scan. The results show the variability of ionization and fragmentation for many "identical mass peptides" and how these differences can affect the analysis of isomeric and epimeric peptides.

[Chromatographic analysis of hard tooth tissue to determine the age of personality]. / Khromatograficheskii analiz tverdykh tkanei zuba v tselyakh opredeleniya vozrasta lichnosti.

The biochemical approach for age assessment is most appropriate in forensic medicine, as racemization of aspartic acid in bones and teeth is closely related to human biological age. The aim of the study is to assess the biochemical parameters of aspartic acid in human teeth, which can be implemented into forensic practice in Russia. Samples of dentin in amount of 20, taken from the teeth of subjects aged between 16 and 76, were examined. Chromatographic analysis of the samples was performed on a gas chromatograph using chiral column. Statistical data processing showed that the relative squared peak of D-aspartic acid has a strong correlation with human biological age. Data, obtained from the Russian population study, demonstrate the applicability of chromatography for forensic purposes. It should be noted that the approach to the racemization rate estimation in the hard tooth tissue was performed using standard laboratory equipment, which allows to easily implement this method in forensic medical practice.

An enzymatic fluorimetric assay for determination of N-acetylaspartate.

N-acetylaspartate (NAA) is an abundant metabolite in the mammalian brain and a precursor of the neuropeptide N-acetylaspartylglutamate (NAAG). The physiological role of NAA is not fully understood and requires further studies. We here describe the development of a coupled enzymatic fluorimetric assay for the determination of NAA in biological samples. Deproteinized tissue extracts are first passed through a strong cation exchange column to remove aspartate. NAA in the sample is hydrolysed by aspartoacylase and released aspartate oxidized using l-aspartate oxidase. Generated H2O2 is measured with peroxidase in a fluorimetric assay using Ampliflu Red. The limit of detection and the lower limit of quantification are 1.0 µM (10 pmol/well) and 3.3 µM (33 pmol/well), respectively, with a linear range to 100 µM. Specificity of the assay was confirmed using samples from mice deficient in NAA synthase Nat8l that were spiked with NAA. Analysis of samples from aspartoacylase-deficient mice showed a 2 to 3-fold increase in brain NAA concentration, in line with previous reports. Mice lacking NAAG synthetases had a slightly reduced (-10%) brain NAA level. Thus, the new fluorimetric enzymatic assay is useful to perform sensitive and large scale quantification of NAA in biological samples without the need for expensive equipment.

Impact of dietary supplementation with N-carbamoyl-aspartic acid on serum metabolites and intestinal microflora of sows.

BACKGROUND: N-Carbamoyl-aspartic acid (NCA) is a critical precursor for de novo biosynthesis of pyrimidine nucleotides. To investigate the cumulative effects of maternal supplementation with NCA on the productive performance, serum metabolites and intestinal microbiota of sows, 40 pregnant sows (∼day 80) were assigned into two groups: (1) the control (CON) and (2) treatment (NCA, 50 g t-1 NCA). RESULTS: Results showed that piglets from the NCA group had heavier birth weight than those in the CON group (P < 0.05). In addition, maternal supplementation with NCA decreased the backfat loss of sows during lactation (P < 0.05). Furthermore,16S-rRNA sequencing results revealed that maternal NCA supplementation decreased the abundance of Cellulosilyticum, Fournierella, Anaerovibrio, and Oribacterium genera of sows during late pregnancy (P < 0.05). Similarly, on the 14th day of lactation, maternal supplementation with NCA reduced the diversity of fecal microbes of sows as evidenced by significantly lower observed species, Chao1, and Ace indexes, and decreased the abundance of Lachnospire, Faecalibacterium, and Anaerovorax genera, while enriched the abundance of Catenisphaera (P < 0.05). Untargeted metabolomics showed that a total of 48 differentially abundant biomarkers were identified, which were mainly involved in metabolic pathways of arginine/proline metabolism, phenylalanine/tyrosine metabolism, and fatty acid biosynthesis, etc. CONCLUSION: Overall, the results indicated that NCA supplementation regulated intestinal microbial composition of sows and serum differential metabolites related to arginine, proline, phenylalanine, tyrosine, and fatty acids metabolism that may contribute to regulating the backfat loss of sows, and the birth weight and diarrhea rate of piglets. © 2022 Society of Chemical Industry.

Elevated choline in dorsolateral prefrontal cortex of lithium responders with bipolar I disorder.

INTRODUCTION: Response to lithium maintenance varies widely across patients with bipolar disorder (BD). The studies on neurochemical correlates of long-term lithium response in BD remain scant. AIM: To assess the neurochemical profile in DLPFC based on lithium response status among subjects with bipolar I disorder (BD-I) using in vivo MRS. MATERIALS AND METHOD: This was an observational study of 40 right-handed, euthymic adult participants with DSM-5 BD-I on long-term lithium maintenance with no psychiatric comorbidities (MINI 7.0). Using Alda Lithium Response Scale (LRS), a cut-off ≥ 7 for excellent lithium response, the sample was grouped into study group I for responders and group II for non-responders. All participants were assessed using NIMH Life Chart Method and IGSLI typical/atypical features scale. 1H-MRS was carried out on a 3 T MR scanner (Achieva, Phillips) using a 32-channel head coil, with a voxel placed at the left DLPFC. LC model was used to measure absolute concentrations of neurochemicals and their ratios in relation to creatine. RESULTS: Group I (n = 20) was comparable to Group II (n = 20) with respect to demographic and illness profile. The GPC/Cr+PCr ratio was significantly higher (p = 0.028) among excellent lithium responders (0.32 ± 0.20 mmol/l) compared to sub-optimal responders (0.25 ± 0.05 mmol/l). Choline-containing compounds reflect alterations in cell membrane synthesis or myelin turnover, and are a marker of overall cell density. No significant alterations were detected in NAA, glutamate, glutamine, myo-inositol and creatine. CONCLUSION: The lithium responders exhibited elevated choline (GPC) in the left DLPFC compared to non-responders.

Cognitive dysfunction and neurometabolic alternations in major depressive disorder with gastrointestinal symptoms.

BACKGROUND: Brain biochemical abnormalities have been associated with major depressive disorder (MDD) and cognitive impairments. However, the cognitive performance and neurometabolic alterations of MDD patients accompanied by gastrointestinal (GI) symptoms remain to be elucidated. We aimed to reveal the features and correlation between cognitive impairments and brain biochemical abnormalities of depressed patients with GI symptoms. METHODS: Fifty MDD patients with GI symptoms (GI group), 46 patients without GI symptoms (NGI group) and 50 demographically matched healthy controls (HCs) underwent Measurement and Treatment Research to Improve Cognition in Schizophrenia (MATRICS) Consensus Cognitive Battery (MCCB) assessments. In addition, proton magnetic resonance spectroscopy (1H-MRS) was used to obtain ratios of N-acetyl aspartate to creatine (NAA/Cr) and choline-containing compounds to creatine (Cho/Cr) in the thalamus, putamen and anterior cingulate cortex (ACC). Finally, association analysis was conducted to investigate the relationships of these measurements. RESULTS: Compared to HCs, participants in both the GI and NGI groups had significantly reduced performance in the six MCCB cognitive domains (all p < 0.05), except for reasoning and problem solving. Higher Cho/Cr ratios in the right thalamus (p < 0.05) and lower NAA/Cr ratios in the left putamen (p < 0.05) were found in the NGI group than in the GI group. The severity of GI symptoms was negatively correlated with Cho/Cr ratios in the right ACC (r = -0.288, p = 0.037). In addition, the T-scores of visual learning were negatively correlated with NAA/Cr ratios in the right ACC (r = -0.443, p = 0.001) and right thalamus (r = -0.335, p = 0.015). CONCLUSION: Our findings suggest that MDD patients with GI symptoms may exhibit greater neurometabolic alternations than those without GI symptoms, while both show similar cognitive dysfunction. In addition, neurometabolic alterations in the ACC and thalamus may underlie the neural basis of GI symptoms and cognitive impairment in MDD.

Chemical Composition Assessment of Structural Parts (Seeds, Peel, Pulp) of Physalis alkekengi L. Fruits.

In recent years there has been an extensive search for nature-based products with functional potential. All structural parts of Physalis alkekengi (bladder cherry), including fruits, pulp, and less-explored parts, such as seeds and peel, can be considered sources of functional macro- and micronutrients, bioactive compounds, such as vitamins, minerals, polyphenols, and polyunsaturated fatty acids, and dietetic fiber. The chemical composition of all fruit structural parts (seeds, peel, and pulp) of two phenotypes of P. alkekengi were studied. The seeds were found to be a rich source of oil, yielding 14-17%, with abundant amounts of unsaturated fatty acids (over 88%) and tocopherols, or vitamin E (up to 5378 mg/kg dw; dry weight). The predominant fatty acid in the seed oils was linoleic acid, followed by oleic acid. The seeds contained most of the fruit's protein (16-19% dw) and fiber (6-8% dw). The peel oil differed significantly from the seed oil in fatty acid and tocopherol composition. Seed cakes, the waste after oil extraction, contained arginine and aspartic acid as the main amino acids; valine, phenylalanine, threonine, and isoleucine were present in slightly higher amounts than the other essential amino acids. They were also rich in key minerals, such as K, Mg, Fe, and Zn. From the peel and pulp fractions were extracted fruit concretes, aromatic products with specific fragrance profiles, of which volatile compositions (GC-MS) were identified. The major volatiles in peel and pulp concretes were ß-linalool, α-pinene, and γ-terpinene. The results from the investigation substantiated the potential of all the studied fruit structures as new sources of bioactive compounds that could be used as prospective sources in human and animal nutrition, while the aroma-active compounds in the concretes supported the plant's potential in perfumery and cosmetics.

Milk metabolome reveals pyrimidine and its degradation products as the discriminant markers of different corn silage-based nutritional strategies.

The purpose of this study was to evaluate the effect of 6 different feeding systems (based on corn silage as the main ingredient) on the chemical composition of milk and to highlight the potential of untargeted metabolomics to find discriminant marker compounds of different nutritional strategies. Interestingly, the multivariate statistical analysis discriminated milk samples mainly according to the high-moisture ear corn (HMC) included in the diet formulation. Overall, the most discriminant compounds, identified as a function of the HMC, belonged to AA (10 compounds), peptides (71 compounds), pyrimidines (38 compounds), purines (15 compounds), and pyridines (14 compounds). The discriminant milk metabolites were found to significantly explain the metabolic pathways of pyrimidines and vitamin B6. Interestingly, pathway analyses revealed that the inclusion of HMC in the diet formulation strongly affected the pyrimidine metabolism in milk, determining a significant up-accumulation of pyrimidine degradation products, such as 3-ureidopropionic acid, 3-ureidoisobutyric acid, and 3-aminoisobutyric acid. Also, some pyrimidine intermediates (such as l-aspartic acid, N-carbamoyl-l-aspartic acid, and orotic acid) were found to possess a high discrimination degree. Additionally, our findings suggested that the inclusion of alfalfa silage in the diet formulation was potentially correlated with the vitamin B6 metabolism in milk, being 4-pyridoxic acid (a pyridoxal phosphate degradation product) the most significant and up-accumulated compound. Taken together, the accumulation trends of different marker compounds revealed that both pyrimidine intermediates and degradation products are potential marker compounds of HMC-based diets, likely involving a complex metabolism of microbial nitrogen based on total splanchnic fluxes from the rumen to mammary gland in dairy cows. Also, our findings highlight the potential of untargeted metabolomics in both foodomics and foodomics-based studies involving dairy products.

Effect of exogenous l-aspartate nano­calcium on root growth, calcium forms and cell wall metabolism of Brassica napus L.

Currently, l-aspartate nano­calcium (Ca(L-asp)-NPs) has been sued as a calcium supplement for humans, but its effects on plants are not well elucidated. This study aimed to investigate the effect of exogenous Ca(L-asp)-NPs on the growth of rapeseed (Brassica napus L.) for the first time. Different concentrations (0, 50, 100, 150, and 200 mg L-1) of Ca(L-asp)-NPs and 1.18 g L-1 Ca(NO3)2 were used in the nutrient solution. The results indicated that Ca2+ released from Ca(L-asp)-NPs were absorbed by the roots, and had a significant effect on plant height, root length, biomass accumulation, and root structure formation, especially on the growth and development of coarse roots at 100 mg L-1 Ca(L-asp)-NPs. Calcium (Ca) accumulation, Ca-pectinate, Ca-phosphate and Ca­carbonate, and Ca-oxalate in plant roots and leaves were positively linked with Ca(L-asp)-NPs concentration. For cell wall, Ca(L-asp)-NPs treatment increased the content of pectin, and the activity of cell wall degrading enzymes in roots, such as pectin methyl-esterase (PME), cellulose enzyme (CE), polygalacturonase (PG), and ß-galactosidase (ß-Gal). For cell membrane osmotic regulation, Ca(L-asp)-NPs promoted the accumulation of soluble sugar and soluble protein. This finding suggests that 100 mg L-1 Ca(L-asp)-NPs had the best growth-promoting effect on rapeseed. This study provides a valuable reference for exogenous Ca(L-asp)-NPs as new nano Ca supplements for plant growth.

The effects of low pH on the taste and amino acid composition of tiger shrimp.

Recent research has revealed that shrimp sensory quality may be affected by ocean acidification but we do not exactly know why. Here we conducted controlled pH exposure experiments on adult tiger shrimp, which were kept in 1000-L tanks continuously supplied with coastal seawater. We compared survival rate, carapace properties and flesh sensory properties and amino acid composition of shrimp exposed to pH 7.5 and pH 8.0 treatments for 28 days. Shrimp reared at pH 7.5 had a lower amino acid content (17.6% w/w) than those reared at pH 8.0 (19.5% w/w). Interestingly, the amino acids responsible for the umami taste, i.e. glutamate and aspartic acid, were present at significantly lower levels in the pH 7.5 than the pH 8.0 shrimp, and the pH 7.5 shrimp were also rated as less desirable in a blind quality test by 40 volunteer assessors. These results indicate that tiger shrimp may become less palatable in the future due to a lower production of some amino acids. Finally, tiger shrimp also had a lower survival rate over 28 days at pH 7.5 than at pH 8.0 (73% vs. 81%) suggesting that ocean acidification may affect both the quality and quantity of future shrimp resources.

Separation and determination of 3-hydroxyaspartate by online concentration capillary electrophoresis/laser-induced fluorescence with microwave-assisted derivatization.

A chiral analytical method was proposed based on capillary electrophoresis with laser-induced fluorescence detection coupled with microwave-assisted derivatization for the simultaneous baseline separation and sensitive detection of four stereoisomers of 3-hydroxyaspartate. The derivatization reaction of 3-hydroxyaspartate with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole was greatly accelerated by microwave irradiation. Under the optimized conditions, the derivatization yield was increased by 20% and the derivatization time was shortened by 20 min when compared with those from conventional water bath heating. In addition, the sensitivity was improved by online sample concentration methods. The detection limit of l-threo-3-hydroxyaspartate obtained by large-volume sample stacking with polarity switching was 5.3 nmol/L, which was around 1000-fold lower than that of the capillary electrophoresis/laser-induced fluorescence without stacking. The excellent analytical performance in terms of linearity and precision was also achieved. Furthermore, the developed method was successfully applied to the determination of 3-hydroxyaspartate in the spiked urine, and satisfactory recoveries were obtained ranging from 90.5 to 107.0%.

An In-vivo 1H-MRS short-echo time technique at 7T: Quantification of metabolites in chronic multiple sclerosis and neuromyelitis optica brain lesions and normal appearing brain tissue.

Magnetic Resonance Spectroscopy (MRS) allows for the non-invasive quantification of neurochemicals and has the potential to differentiate between the pathologically distinct diseases, multiple sclerosis (MS) and AQP4Ab-positive neuromyelitis optica spectrum disorder (AQP4Ab-NMOSD). In this study we characterised the metabolite profiles of brain lesions in 11 MS and 4 AQP4Ab-NMOSD patients using an optimised MRS methodology at ultra-high field strength (7T) incorporating correction for T2 water relaxation differences between lesioned and normal tissue. MS metabolite results were in keeping with the existing literature: total N-acetylaspartate (NAA) was lower in lesions compared to normal appearing brain white matter (NAWM) with reciprocal findings for myo-Inositol. An unexpected subtlety revealed by our technique was that total NAA differences were likely driven by NAA-glutamate (NAAG), a ubiquitous CNS molecule with functions quite distinct from NAA though commonly quantified together with NAA in MRS studies as total NAA. Surprisingly, AQP4Ab-NMOSD showed no significant differences for total NAA, NAA, NAAG or myo-Inositol between lesion and NAWM sites, nor were there any differences between MS and AQP4Ab-NMOSD for a priori hypotheses. Post-hoc testing revealed a significant correlation between NAWM Ins:NAA and disability (as measured by EDSS) for disease groups combined, driven by the AP4Ab-NMOSD group. Utilising an optimised MRS methodology, our study highlights some under-explored subtleties in MRS profiles, such as the absence of myo-Inositol concentration differences in AQP4Ab-NMOSD brain lesions versus NAWM and the potential influence of NAAG differences between lesions and normal appearing white matter in MS.

An investigation of glutamate quantification with PRESS and MEGA-PRESS.

Glutamate is an important neurotransmitter. Although many studies have measured glutamate concentration in vivo using magnetic resonance spectroscopy (MRS), researchers have not reached a consensus on the accuracy of glutamate quantification at the field strength of 3 T. Besides, there is not an optimal MRS protocol for glutamate measurement. In this work, both simulation and phantom scans indicate that glutamate can be estimated with reasonable accuracy (<10% error on average) using the standard Point-RESolved Spectroscopy (PRESS) technique with TE 30 ms; glutamine, however, is likely underestimated, which is also suggested by results from human scans using the same protocol. The phantom results show an underestimation of glutamate and glutamine for PRESS with long TE and MEGA-PRESS off-resonance spectra. Despite the underestimation, there is a high correlation between the measured values and the true values (r > 0.8). Our results suggest that the quantification of glutamate and glutamine is reliable but can be off by a scaling factor, depending on the imaging technique. The outputs from all three PRESS sequences (TE = 30, 68 and 80 ms) are also highly correlated with each other (r > 0.7) and moderately correlated (r > 0.5) with the results from the MEGA-PRESS difference spectra with moderate to good shimming (linewidth < 16 Hz).

Understanding the pathway and kinetics of aspartic acid isomerization in peptide mapping methods for monoclonal antibodies.

Isomerization of aspartic acid (Asp) in therapeutic proteins could lead to safety and efficacy concerns. Thus, accurate quantitation of various Asp isomerization along with kinetic understanding of the variant formations is needed to ensure optimal process development and sufficient product quality control. In this study, we first observed Asp-succinimide conversion in complementarity-determining regions (CDRs) Asp-Gly motif of a recombinant mAb through ion exchange chromatography, intact protein analysis by mass spectrometry, and LC-MS/MS. Then, we developed a specific peptide mapping method, with optimized sample digestion conditions, to accurately quantitate Asp-succinimide-isoAsp variants at peptide level without method-induced isomerization. Various kinetics of Asp-succinimide-isoAsp isomerization pathways were elucidated using 18O labeling followed by LC-MS analysis. Molecular modeling and molecular dynamic simulation provide additional insight on the kinetics of Asp-succinimide formation and stability of succinimide intermediate. Findings of this work shed light on the molecular construct and the kinetics of the formation of isoAsp and succinimide in peptides and proteins, which facilitates analytical method development, protein engineering, and late phase development for commercialization of therapeutic proteins.

Unequivocal Identification of Aspartic Acid and isoAspartic Acid by MALDI-TOF/TOF: From Peptide Standards to a Therapeutic Antibody.

Aspartic acid (Asp) to isoaspartic acid (isoAsp) isomerization in therapeutic monoclonal antibodies (mAbs) and other biotherapeutics is a critical quality attribute (CQA) that requires careful control and monitoring during the drug discovery and production processes. The unwanted formation of isoAsp within biotherapeutics and resultant structural changes in the peptide backbone may negatively impact the efficacy, potency, and safety of the molecule or become immunogenic, especially if the isomerization occurs within the mAb complementarity determining region (CDR). Herein we describe a MALDI-TOF/TOF mass spectrometry method that affords unequivocal identification of the presence and the exact position of the isoAsp residue(s) in peptide standards ranging in size from a tripeptide to a docosapeptide (22 residues). In general, the peptide bond immediately N-terminal to the isoAsp residue is more susceptible to MALDI-TOF/TOF fragmentation than its unmodified counterpart. In some of the peptides evaluated in this study, fragmentation of the peptide bond C-terminal to the isoAsp residue (the aspartate effect) is also enhanced when compared to the control. Relative quantification by MALDI-TOF/TOF of this chemical modification is dependent upon a successful reversed-phase HPLC (rpHPLC) separation of the control and modified peptides. This method has also been validated on a therapeutic mAb that contains a well-documented isoAsp residue in the heavy chain CDR3 after forced degradation. Moreover, we also demonstrate that higher energy C-trap dissociation of only the singly charged species, and not the multiply charged form, of the isoAsp containing peptide, separated by rpHPLC, results in LC-MS/MS fragmentation that is highly consistent to that of MALDI-TOF/TOF.

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity.

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant.

Neonatal brain metabolite concentrations: Associations with age, sex, and developmental outcomes.

Age and sex differences in brain metabolite concentrations in early life are not well understood. We examined the associations of age and sex with brain metabolite levels in healthy neonates, and investigated the associations between neonatal brain metabolite concentrations and developmental outcomes. Forty-one infants (36-42 gestational weeks at birth; 39% female) of predominantly Hispanic/Latina mothers (mean 18 years of age) underwent MRI scanning approximately two weeks after birth. Multiplanar chemical shift imaging was used to obtain voxel-wise maps of N-acetylaspartate (NAA), creatine, and choline concentrations across the brain. The Bayley Scales of Infant and Toddler Development, a measure of cognitive, language, and motor skills, and mobile conjugate reinforcement paradigm, a measure of learning and memory, were administered at 4 months of age. Findings indicated that postmenstrual age correlated positively with NAA concentrations in multiple subcortical and white matter regions. Creatine and choline concentrations showed similar but less pronounced age related increases. Females compared with males had higher metabolite levels in white matter and subcortical gray matter. Neonatal NAA concentrations were positively associated with learning and negatively associated with memory at 4 months. Age-related increases in NAA, creatine, and choline suggest rapid development of neuronal viability, cellular energy metabolism, and cell membrane turnover, respectively, during early life. Females may undergo earlier and more rapid regional developmental increases in the density of viable neurons compared to males.

NAA is a Marker of Disability in Secondary-Progressive MS: A Proton MR Spectroscopic Imaging Study.

BACKGROUND AND PURPOSE: The secondary progressive phase of multiple sclerosis is characterised by disability progression due to processes that lead to neurodegeneration. Surrogate markers such as those derived from MRI are beneficial in understanding the pathophysiology that drives disease progression and its relationship to clinical disability. We undertook a 1H-MRS imaging study in a large secondary progressive MS (SPMS) cohort, to examine whether metabolic markers of brain injury are associated with measures of disability, both physical and cognitive. MATERIALS AND METHODS: A cross-sectional analysis of individuals with secondary-progressive MS was performed in 119 participants. They underwent 1H-MR spectroscopy to obtain estimated concentrations and ratios to total Cr for total NAA, mIns, Glx, and total Cho in normal-appearing WM and GM. Clinical outcome measures chosen were the following: Paced Auditory Serial Addition Test, Symbol Digit Modalities Test, Nine-Hole Peg Test, Timed 25-foot Walk Test, and the Expanded Disability Status Scale. The relationship between these neurometabolites and clinical disability measures was initially examined using Spearman rank correlations. Significant associations were then further analyzed in multiple regression models adjusting for age, sex, disease duration, T2 lesion load, normalized brain volume, and occurrence of relapses in 2 years preceding study entry. RESULTS: Significant associations, which were then confirmed by multiple linear regression, were found in normal-appearing WM for total NAA (tNAA)/total Cr (tCr) and the Nine-Hole Peg Test (ρ = 0.23; 95% CI, 0.06-0.40); tNAA and tNAA/tCr and the Paced Auditory Serial Addition Test (ρ = 0.21; 95% CI, 0.03-0.38) (ρ = 0.19; 95% CI, 0.01-0.36); mIns/tCr and the Paced Auditory Serial Addition Test, (ρ = -0.23; 95% CI, -0.39 to -0.05); and in GM for tCho and the Paced Auditory Serial Addition Test (ρ = -0.24; 95% CI, -0.40 to -0.06). No other GM or normal-appearing WM relationships were found with any metabolite, with associations found during initial correlation testing losing significance after multiple linear regression analysis. CONCLUSIONS: This study suggests that metabolic markers of neuroaxonal integrity and astrogliosis in normal-appearing WM and membrane turnover in GM may act as markers of disability in secondary-progressive MS.