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γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter of the central nervous system that impacts physical and mental health. Low GABA levels have been documented in several diseases, including multiple sclerosis and depression, and studies suggest that GABA could improve disease outcomes in those conditions. Probiotic bacteria naturally produce GABA and have been engineered to enhance its synthesis. Strains engineered thus far use inducible expression systems that require the addition of exogenous molecules, which complicates their development as therapeutics. This study aimed to overcome this challenge by engineering Lactococcus lactis with a constitutive GABA synthesis gene cassette. GABA synthesizing and transport genes (gadB and gadC) were cloned onto plasmids downstream of constitutive L. lactis promoters [P2, P5, shortened P8 (P8s)] of different strengths and transformed into L. lactis. Fold increase in gadCB expression conferred by these promoters (P2, P5, and P8s) was 322, 422, and 627, respectively, compared to the unmodified strain (P = 0.0325, P8s). GABA synthesis in the highest gadCB expressing strain, L. lactis-P8s-glutamic acid decarboxylase (GAD), was dependent on media supplementation with glutamic acid and significantly higher than the unmodified strain (P < 0.0001, 125 mM, 200 mM glutamic acid). Lactococcus lactis-P8s-GAD is poised for therapeutic testing in animal models of low-GABA-associated disease., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Succinic semialdehyde dehydrogenase deficiency is a rare autosomal recessively inherited metabolic disorder of γ-aminobutyric acid catabolism manifested by intellectual disability, expressive aphasia, movement disorders, psychiatric ailments and epilepsy. Subjects with succinic semialdehyde dehydrogenase deficiency are characterized by elevated γ-aminobutyric acid and related metabolites, such as γ-guanidinobutyric acid, and an age-dependent downregulation of cerebral γ-aminobutyric acid receptors. These findings indicate impaired γ-aminobutyric acid and γ-aminobutyric acid sub-type A (GABA A ) receptor signalling as major factors underlying the pathophysiology of this neurometabolic disorder. We studied the cortical oscillation patterns and their relationship with γ-aminobutyric acid metabolism in 18 children affected by this condition and 10 healthy controls. Using high-density EEG, we recorded somatosensory cortical responses and resting-state activity. Using electrical source imaging, we estimated the relative power changes (compared with baseline) in both stimulus-evoked and stimulus-induced responses for physiologically relevant frequency bands and resting-state power. Stimulus-evoked oscillations are phase locked to the stimulus, whereas induced oscillations are not. Power changes for both evoked and induced responses as well as resting-state power were correlated with plasma γ-aminobutyric acid and γ-guanidinobutyric acid concentrations and with cortical γ-aminobutyric acid measured by proton magnetic resonance spectroscopy. Plasma γ-aminobutyric acid, γ-guanidinobutyric acid and cortical γ-aminobutyric acid were higher in patients than in controls ( P < 0.001 for both). Beta and gamma relative power were suppressed for evoked responses in patients versus controls ( P < 0.01). No group differences were observed for induced activity ( P > 0.05). The mean gamma frequency of evoked responses was lower in patients versus controls ( P = 0.002). Resting-state activity was suppressed in patients for theta ( P = 0.011) and gamma ( P < 0.001) bands. Evoked power changes were inversely correlated with plasma γ-aminobutyric acid and with γ-guanidinobutyric acid for beta ( P < 0.001) and gamma ( P < 0.001) bands. Similar relationships were observed between the evoked power changes and cortical γ-aminobutyric acid for all tested areas in the beta band ( P < 0.001) and for the posterior cingulate gyrus in the gamma band ( P < 0.001). We also observed a negative correlation between resting-state activity and plasma γ-aminobutyric acid and γ-guanidinobutyric acid for theta ( P < 0.001; P = 0.003), alpha ( P = 0.003; P = 0.02) and gamma ( P = 0.02; P = 0.01) bands. Our findings indicate that increased γ-aminobutyric acid concentration is associated with reduced sensory-evoked beta and gamma activity and impaired neuronal synchronization in patients with succinic semialdehyde dehydrogenase deficiency. This further elucidates the pathophysiology of this neurometabolic disorder and serves as a potential biomarker for therapeutic trials., Competing Interests: The authors report no competing interests., (Published by Oxford University Press on behalf of the Guarantors of Brain 2023.)

Deep sequencing of viral populations using next-generation sequencing (NGS) offers opportunities to understand and investigate evolution, transmission dynamics, and population genetics. Currently, the standard practice for processing NGS data to study viral populations is to summarize all the observed sequences from a sample as a single consensus sequence, thus discarding valuable information about the intrahost viral molecular epidemiology. Furthermore, existing analytical pipelines may only analyze genomic regions involved in drug resistance, thus are not suited for full viral genome analysis. Here, we present HAPHPIPE, a HAplotype and PHylodynamics PIPEline for genome-wide assembly of viral consensus sequences and haplotypes. The HAPHPIPE protocol includes modules for quality trimming, error correction, de novo assembly, alignment, and haplotype reconstruction. The resulting consensus sequences, haplotypes, and alignments can be further analyzed using a variety of phylogenetic and population genetic software. HAPHPIPE is designed to provide users with a single pipeline to rapidly analyze sequences from viral populations generated from NGS platforms and provide quality output properly formatted for downstream evolutionary analyses., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Neutrophils are the most abundant leukocytes in circulation and are key "first responders" in the immune response to infectious and non-infectious stimuli. Unlike other immune cells, neutrophils can mount a robust response (including a change in surface markers and the production of extracellular traps and reactive oxygen species) just minutes after sensing a disturbance. It has been speculated that, in some individuals, the activation of neutrophils inadvertently leads to the generation of anti-neutrophil cytoplasmic autoantibodies (ANCA) against particular neutrophil proteins (antigens) such as myeloperoxidase (MPO) and proteinase 3 (PR3). In these individuals, continuous ANCA-antigen interactions are thought to drive persistent activation of neutrophils, chronic immune activation, and disease, most notably, small vessel vasculitis. There are significant gaps however in our understanding of the underlying mechanisms and even the pathogenicity of ANCA given that vasculitis can develop in the absence of ANCA, and that ANCA have been found in circulation in other conditions with no apparent contribution to disease. These gaps are particularly evident in the context of human studies. Herein, we review knowledge on neutrophil-derived ANCA antigens PR3 and MPO, ANCA generation, and ANCA-antigen interaction(s) that may promote immune activation and disease., (©2020 Society for Leukocyte Biology.)

Objectives: Patients with SSADH deficiency, a disorder of chronically elevated endogenous GABA and GHB, were studied for sleep symptoms and polysomnography. We hypothesized that patients would have excessive daytime somnolence and decreased REM sleep., Design: Polysomnography and MSLT were performed on patients enrolled for comprehensive clinical studies of SSADH deficiency., Setting: Sleep studies were obtained in the sleep laboratories at CNMC and NIH., Patients: Sleep recordings were obtained in 10 patients with confirmed SSADH deficiency., Interventions: Thirteen overnight polysomnograms were obtained in 10 patients (7 male, 3 female, ages 11-27 y). Eleven MSLT studies were completed in 8 patients., Measurements and Results: Polysomnograms showed prolongation of REM stage latency (mean 272 +/- 89 min) and decreased percent stage REM (mean 8.9%, range 0.3% to 13.8%). Decreased mean sleep latency was present in 6 of 11 MSLTs., Conclusions: SSADH deficiency is associated with prolonged latency to stage REM and decreased percent stage REM. This disorder represents a model of chronic GABA and GHB accumulation associated with suppression of REM sleep.

Background: Exogenous gamma-hydroxybutyrate (GHB) increases slow-wave sleep and reduces daytime sleepiness and cataplexy in patients with primary narcolepsy., Objective: To examine nighttime sleep and daytime sleepiness in a 13-year-old girl homozygous for succinic semialdehyde dehydrogenase (SSADH) deficiency, a rare recessive metabolic disorder that disrupts the normal degradation of 4-aminobutyric acid (GABA), and leads to an accumulation of GHB and GABA within the brain., Methods: Sleep interview, nighttime polysomnography, Multiple Sleep Latency Tests, and continuous 24-hour in-lab recordings in the patient; overnight polysomnography in her recessive mother and in a 13-year-old female control., Results: During quiet wakefulness, background electroencephalographic activity was slow and composed of 7-Hz activity. Sleep stage 3/4 was slightly increased (28.1% of total sleep period, norms 15%-28%), and the daytime mean sleep latency was short in the patient (3 minutes 42 seconds, norms > 8 minutes). Stage 2 spindles were infrequent in the child (0.18/minute, norms: 1.2-9.2/minute) and her mother (0.65/minute) but normal (4.6/minute) in the control. At the beginning of the second night, a tonic-clonic seizure occurred, followed by a dramatic increase in stage 3/4 sleep, that lasted 46.3 % of the total sleep period, double the normal value. The mother showed a reduced total sleep time and rapid eye movement sleep percentage., Discussion: This suggests that a chronic excess of GABA and GHB induces subtle sleep abnormalities, whereas increased slow-wave sleep evoked by a sudden event (here an epileptic seizure) may be caused by a supplementary increase in GABA and GHB.

Background: Several methods have been published for measuring gamma-aminobutyric acid transaminase (GABA-T) activity, but these methods are either impracticable because of the use of radioisotopes or insufficiently sensitive to determine small enzyme activities in leukocyte extracts. We developed a direct and sensitive enzyme method., Methods: We developed a stable-isotope dilution method for the measurement of [15N]glutamic acid derived from [15N]GABA and alpha-ketoglutaric acid, catalyzed by GABA-T. The method for analysis of [15N]glutamic acid comprised a solid-phase extraction procedure to isolate this analyte from incubation samples. After derivatization, [15N]glutamic acid was quantified by gas chromatography-mass spectrometry relative to its 2H5-labeled internal standard. In addition to [15N]GABA, [15N]beta-alanine was a cosubstrate., Results: GABA-T-deficient lymphoblasts showed diminished enzyme activity, with both [15N]GABA and [15N]beta-alanine as substrate. Vigabatrin inhibited the enzyme activity for both substrates., Conclusions: The activity of GABA-T can be accurately determined by our procedure using 15N-labeled substrate, measuring the formation of [15N]glutamic acid. Our results with [15N]beta-alanine indicate that GABA and beta-alanine transaminases are identical.

Tiglyglycine (TG), an intermediate product of the catabolism of isoleucine, is increased in the urine of patients with beta-ketothiolase deficiency or with disorders of propionate metabolism. It is also implicated as a useful diagnostic marker in disorders of the respiratory chain. We present a method for the synthesis of TG and tiglyl[13C, 15N]glycine and the development of a stable isotope dilution mass spectrometric assay for TG. We compare data from controls with that from subjects with beta-ketothiolase deficiency and propionyl-CoA carboxylase deficiency, and with six patients with enzyme-confirmed disorders of the respiratory chain. TG was increased in the urine from all of the patient groups. The increased TG excretion did not persist in one patient with a respiratory chain defect, which suggests that, in some patients, multiple sample analysis may be necessary to identify a respiratory chain defect. This is the first urinary compound to be implicated as a potential marker of disorders of the respiratory chain.

A patient with 3-hydroxy-3-methylglutaryl-CoA lyase (HMG-CoA lyase, EC 4.1.3.4) deficiency presented consistently above-normal values of plasma amylase (EC 3.2.1.1). Activities measured were in the lower normal range in family members not proven heterozygotes and in the upper normal range in the proven heterozygotes. Heterozygosity was proven by intermediate HMG-CoA lyase activities determined in cultured fibroblasts and in lymphocytes in the parents and the paternal grandmother. Because all of the family members had diseases of the pancreas, colon, and liver, we question whether the heterozygote state contributes to the impaired function of these organs. Our findings of significantly increased amylase activities in the heterozygotes and the patient, in comparison with the other family members, support this hypothesis.

In this rapid radiochemical assay for 3-hydroxy-3-methylglutaryl-coenzyme A lyase (I) activity in cell extracts, DL-3[glutaryl-3-14C]hydroxy-3-methylglutaryl-coenzyme A is used as substrate and the radiochemical product, [3-14C]acetoacetic acid, is converted to the more stable [3-14C]-3-hydroxybutyric acid in the presence of added NADH and 3-hydroxybutyrate dehydrogenase. Substrate and product are separated and quantified by thin-layer chromatography on cellulose (solvent system: butanol/water/formic acid, 77:13:10 by vol). All reagents for the assay are commercially available. No detailed column chromatography or spectrophotometry is required. Thus the assay is suited for any clinical laboratory. Using this procedure, we studied cultured fibroblasts or lymphocytes isolated from whole blood from five patients in whom the urinary organic acid profile was suggestive of deficiency of I. Three patients had less than or equal to 18% of control I activity in fibroblast or lymphocyte extracts. The other two had activity within the normal range. In one of the latter cases, urinary excretion of three of the characteristic acids disappeared with age, and 3-hydroxyisovaleric acid excretion was within normal limits. The other case presented with urinary excretion of moderate amounts of all four metabolites and the characteristic absence of urinary ketone bodies. Evidently, confirmatory enzyme studies should be undertaken, even when the profile of urinary organic acids appears definitive for this deficiency.