A PDE3A-SLFN12 Molecular Glue Exhibits Significant Antitumor Activity in TKI-Resistant Gastrointestinal Stromal Tumors.

PURPOSE: Gastrointestinal stromal tumor (GIST), the most common mesenchymal tumor with KIT or PDGFRA driver mutations, is typically treated with tyrosine kinase inhibitors (TKIs). However, resistance to TKIs due to secondary mutations is a common challenge in advanced GISTs. In addition, there are currently no effective therapies for several other molecular subtypes, such as SDH-deficient GISTs. Therefore, novel therapeutic strategies are needed. EXPERIMENTAL DESIGN: To address this need, we tested the efficacy of a novel non-TKI compound, OPB-171775, using patient-derived xenograft models of GISTs. In parallel, we sought to elucidate the mechanism of action of the compound. RESULTS: Our study revealed that OPB-171775 exhibited significant efficacy against GISTs regardless of their KIT mutation status by inducing complex formation between phosphodiesterase 3A (PDE3A) and Schlafen family member 12 (SLFN12), which are highly expressed in GISTs, leading to SLFN12 RNase-mediated cell death. Furthermore, we identified the activation of general control non-derepressible 2 (GCN2) and its downstream response as an effector pathway of SLFN12 in mediating anticancer activity and revealed potential pharmacodynamic markers. CONCLUSIONS: These findings suggest that OPB-171775, with its significant efficacy, could potentially serve as a novel and effective treatment option for advanced GISTs, particularly those resistant to TKIs.

Loss-of-function polymorphisms in NQO1 are not associated with the development of subacute myelo-optico-neuropathy.

BACKGROUND: Subacute myelo-optico-neuropathy (SMON) is a neurological disorder associated with the administration of clioquinol, particularly at very high doses. Although clioquinol has been used worldwide, there was an outbreak of SMON in the 1950s-1970s in which the majority of cases were in Japan, prompting speculation that the unique genetic background of the Japanese population may have contributed to the development of SMON. Recently, a possible association between loss-of-function polymorphisms in NQO1 and the development of SMON has been reported. In this study, we analyzed the relationship between NQO1 polymorphisms and SMON in Japan. METHODS: We analyzed 125 Japanese patients with SMON. NQO1 loss-of-function polymorphisms (rs1800566, rs10517, rs689452, and rs689456) were evaluated. The allele frequency distribution of each polymorphism was compared between the patients and the healthy Japanese individuals (Human Genomic Variation Database and Integrative Japanese Genome Variation Database), as well as our in-house healthy controls. RESULTS: The frequencies of the loss-of-function NQO1 alleles in patients with SMON and the normal control group did not differ significantly. CONCLUSION: We conclude that known NQO1 polymorphisms are not associated with the development of SMON.

Differences in time-intensity sensory profiles of sweet taste intensity of glucose between older and young adults.

Background: To understand age-related changes in sweet taste perception in daily life, it is important to understand taste intensity at the suprathreshold level. Previous studies have attempted to characterize the temporal aspects of human taste perception in terms of time-intensity evaluations. The perception of dynamic taste intensity in older adults increases slowly for salty taste; however, there have been no previous studies on time-intensity sensory evaluation of sweet taste in older adults. We hypothesized that older adults perceive sweet taste intensity more slowly than young adults. Methods: Fifty young and 40 older adults participated in the study and glucose solutions of 0.6 M and 1.5 M were used as stimuli. The study comprised two experiments: (1) a cup tasting test (static taste perception in the mouth), and (2) a time-intensity sensory evaluation, in which the solutions were presented using a custom-made delivery system. The intra-oral device was made to fit each participant's dentition. Further, the level of gag reflex was taken into consideration for each participant in the design of the intra-oral device. A suction tube was placed across the posterior tongue near the throat to remove solution and saliva. The solution delivery system was controlled by an original computer program. Results: Older adults presented significantly different maximum intensity timing and slope for both concentrations compared with young adults (slope for 1.5 M, p < 0.01; others, p < 0.05). No significant differences were found between the older and young adults for reaction timing and maximum intensity. Conclusion: We conclude that older adults perceived sweetness more slowly than young adults, and ultimately perceived almost the same intensity as young adults. This is the first reported characterization of the time-intensity profile of sweet taste intensity of glucose in older adults. Using a standardized system enabled us to assess and compare feedback on taste intensities among different age groups in real-time. Based on this, we recommend older adults "savor" to perceive sweet tastes at the same level experienced by young adults.

Dentigerous cysts suspected the other odontogenic lesions on panoramic radiography and CT.

Dentigerous cysts are known as the second most common type of cyst in the jaws. The cyst is one of the lesions occurred frequently in the posterior body of the mandible and is often related to the unerupted third molar and forms around the crown of the unerupted tooth attaching at the cementoenamel junction. Such characteristic appearances are the diagnostic points differentiating from ameloblastoma or odontogenic keratocyst. However, it would be hard for us to diagnose it as a dentigerous cyst if the lesion does not show its typical appearance. We experienced two cases of dentigerous cysts which did not form around the crown of the unerupted tooth on radiologically. Both cysts were relatively large and resorbed adjacent teeth roots. Therefore, an ameloblastoma or an odontogenic keratocyst was suspected rather than a dentigerous cyst as the imaging diagnosis. The biopsy revealed that the lesion was a "dentigerous cyst" in one of the cases and "developmental cyst with inflammation" in another case. After the excision, the histopathological diagnosis was a dentigerous cyst with inflammation in both cases. This report shows the two cases of dentigerous cysts focusing on panoramic radiography and CT images. Also, we discuss the differential diagnosis by reconsidering those diagnostic points.

Association of Tooth Loss with Alzheimer's Disease Tau Pathologies Assessed by Positron Emission Tomography.

BACKGROUND: Deterioration of the oral environment is one of the risk factors for dementia. A previous study of an Alzheimer's disease (AD) model mouse suggests that tooth loss induces denervation of the mesencephalic trigeminal nucleus and neuroinflammation, possibly leading to accelerated tau dissemination from the nearby locus coeruleus (LC). OBJECTIVE: To elucidate the relevance of oral conditions and amyloid-ß (Aß) and tau pathologies in human participants. METHODS: We examined the number of remaining teeth and the biofilm-gingival interface index in 24 AD-spectrum patients and 19 age-matched healthy controls (HCs). They also underwent positron emission tomography (PET) imaging of Aß and tau with specific radiotracers, 11C-PiB and 18F-PM-PBB3, respectively. All AD-spectrum patients were Aß-positive, and all HCs were Aß-negative. We analyzed the correlation between the oral parameters and radiotracer retention. RESULTS: No differences were found in oral conditions between the AD and HC groups. 11C-PiB retentions did not correlate with the oral indices in either group. In AD-spectrum patients, brain-wide, voxel-based image analysis highlighted several regions, including the LC and associated brainstem substructures, as areas where 18F-PM-PBB3 retentions negatively correlated with the remaining teeth and revealed the correlation of tau deposits in the LC (r = -0.479, p = 0.018) primarily with the hippocampal and neighboring areas. The tau deposition in none of the brain regions was associated with the periodontal status. CONCLUSIONS: Our findings with previous preclinical evidence imply that tooth loss may enhance AD tau pathogenesis, promoting tau spreading from LC to the hippocampal formation.

White epidermoid cyst transformation after stereotactic radiosurgery: illustrative case.

BACKGROUND: White epidermoid cysts (WECs) are a rare type of epidermoid cyst with atypical radiological features. The epidemiological aspects and mechanisms of their onset remain unknown. Herein, the authors report a unique case of WEC transformation from a typical epidermoid cyst after stereotactic radiosurgery (SRS), confirmed by radiological and pathological findings. OBSERVATIONS: The case involved a 78-year-old man with a history of 2 surgeries for a left cerebellopontine angle typical epidermoid cyst 23 years earlier and SRS using the CyberKnife for recurrent trigeminal neuralgia (TN) 14 years earlier. The tumor with high intensity on T1-weighted imaging, low intensity on T2-weighted imaging, without restriction on diffusion-weighted imaging had gradually enlarged after SRS. Therefore, a salvage surgery was performed via a left suboccipital craniotomy, and the intraoperative findings showed a cyst with a brown, viscous liquid component, consistent with those of WECs. Histopathologically, keratin calcification and hemorrhage were identified, leading to a diagnosis of WEC. The postoperative course was uneventful, and the TN resolved. No tumor recurrence was recorded at 2 years postoperatively. LESSONS: To the best of the authors' knowledge, this is the first world case of WEC transformation from a typical epidermoid cyst after SRS, confirmed by radiological and pathological findings. Radiation effects could have been involved in this transformation.

Two pathways differentially linking tau depositions, oxidative stress, and neuronal loss to apathetic phenotypes in progressive supranuclear palsy.

Patients with progressive supranuclear palsy (PSP) frequently exhibit apathy but the neuropathological processes leading to this phenotype remain elusive. We aimed to examine the involvement of tau protein depositions, oxidative stress (OS), and neuronal loss in the apathetic manifestation of PSP. Twenty patients with PSP and twenty-three healthy controls were enrolled. Tau depositions and brain volumes were evaluated via positron-emission tomography (PET) using a specific probe, 18F-PM-PBB3, and magnetic resonance imaging, respectively. Glutathione (GSH) levels in the anterior and posterior cingulate cortices were quantified by magnetic resonance spectroscopy. Tau pathologies were observed in the subcortical and cortical structures of the patient brains. The angular gyrus exhibited a positive correlation between tau accumulations and apathy scale (AS). Although PSP cases did not show GSH level alterations compared with healthy controls, GSH levels in posterior cingulate cortex were correlated with AS and tau depositions in the angular gyrus. Marked atrophy was observed in subcortical areas, and gray matter volumes in the inferior frontal gyrus and anterior cingulate cortex were positively correlated with AS but showed no correlation with tau depositions and GSH levels. Path analysis highlighted synergistic contributions of tau pathologies and GSH reductions in the posterior cortex to AS, in parallel with associations of gray matter atrophy in the anterior cortex with AS. Apathetic phenotypes may arise from PET-visible tau aggregation and OS compromising the neural circuit resilience in the posterior cortex, along with neuronal loss, with neither PET-detectable tau pathologies nor OS in the anterior cortex.

Subchronic tolerance trials of graded oral supplementation with ornithine hydrochloride or citrulline in healthy adults.

Ornithine and citrulline are amino acids used in dietary supplements and nutritional products consumed by healthy consumers, but the safe supplementation levels of these compounds are unknown. The objective of this study was to conduct two 4-week clinical trials to evaluate the safety and tolerability of graded dosages of oral ornithine (as hydrochloride) and citrulline. Healthy male adults (n = 60, age 41.4 ± 1.5 years) completed graded dosages of either ornithine hydrochloride (3.2, 6, 9.2, and 12 g/day) or citrulline (6, 12, 18, and 24 g/day) supplement for 4 weeks with 2-week wash-out periods in between. Primary outcomes included vitals, a broad spectrum of circulating biochemical analytes, body weight, sleep quality, and mental self-assessment. In the ornithine hydrochloride supplementation group, minor increase in plasma aspartic acid and glutamic acid concentrations was observed at the highest intake dosages. In the citrulline supplementation group, minor changes in laboratory data for serum lactate dehydrogenase and plasma amino acid concentration of lysine, methionine, threonine, aspartic acid, glutamic acid, glutamine and ornithine, arginine, and citrulline itself were measured. No other changes in measured parameters were observed, and study subjects tolerated 4-week-long oral supplementation of ornithine hydrochloride or citrulline without treatment-related adverse events. A clinical, no-observed-adverse-effect-level (NOAEL) of ornithine hydrochloride and citrulline supplementation in healthy adult males was determined to be 12 g/day and 24 g/day (4 weeks), respectively.

An optimized reference tissue method for quantification of tau protein depositions in diverse neurodegenerative disorders by PET with 18F-PM-PBB3 (18F-APN-1607).

Positron emission tomography (PET) with 18F-PM-PBB3 (18F-APN-1607, 18F-Florzolotau) enables high-contrast detection of tau depositions in various neurodegenerative dementias, including Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD). A simplified method for quantifying radioligand binding in target regions is to employ the cerebellum as a reference (CB-ref) on the assumption that the cerebellum has minimal tau pathologies. This procedure is typically valid in AD, while FTLD disorders exemplified by progressive supranuclear palsy (PSP) are characterized by occasional tau accumulations in the cerebellum, hampering the application of CB-ref. The present study aimed to establish an optimal method for defining reference tissues on 18F-PM-PBB3-PET images of AD and non-AD tauopathy brains. We developed a new algorithm to extract reference voxels with a low likelihood of containing tau deposits from gray matter (GM-ref) or white matter (WM-ref) by a bimodal fit to an individual, voxel-wise histogram of the radioligand retentions and applied it to 18F-PM-PBB3-PET data obtained from age-matched 40 healthy controls (HCs) and 23 CE, 40 PSP, and five other tau-positive FTLD patients. PET images acquired at 90-110 min after injection were averaged and co-registered to corresponding magnetic resonance imaging space. Subsequently, we generated standardized uptake value ratio (SUVR) images estimated by CB-ref, GM-ref and WM-ref, respectively, and then compared the diagnostic performances. GM-ref and WM-ref covered a broad area in HCs and were free of voxels located in regions known to bear high tau burdens in AD and PSP patients. However, radioligand retentions in WM-ref exhibited age-related declines. GM-ref was unaffected by aging and provided SUVR images with higher contrast than CB-ref in FTLD patients with suspected and confirmed corticobasal degeneration. The methodology for determining reference tissues as optimized here improves the accuracy of 18F-PM-PBB3-PET measurements of tau burdens in a wide range of neurodegenerative illnesses.

A Machine Learning-Based Approach to Discrimination of Tauopathies Using [18 F]PM-PBB3 PET Images.

BACKGROUND: We recently developed a positron emission tomography (PET) probe, [18 F]PM-PBB3, to detect tau lesions in diverse tauopathies, including mixed three-repeat and four-repeat (3R + 4R) tau fibrils in Alzheimer's disease (AD) and 4R tau aggregates in progressive supranuclear palsy (PSP). For wider availability of this technology for clinical settings, bias-free quantitative evaluation of tau images without a priori disease information is needed. OBJECTIVE: We aimed to establish tau PET pathology indices to characterize PSP and AD using a machine learning approach and test their validity and tracer capabilities. METHODS: Data were obtained from 50 healthy control subjects, 46 patients with PSP Richardson syndrome, and 37 patients on the AD continuum. Tau PET data from 114 regions of interest were subjected to Elastic Net cross-validation linear classification analysis with a one-versus-the-rest multiclass strategy to obtain a linear function that discriminates diseases by maximizing the area under the receiver operating characteristic curve. We defined PSP- and AD-tau scores for each participant as values of the functions optimized for differentiating PSP (4R) and AD (3R + 4R), respectively, from others. RESULTS: The discriminatory ability of PSP- and AD-tau scores assessed as the area under the receiver operating characteristic curve was 0.98 and 1.00, respectively. PSP-tau scores correlated with the PSP rating scale in patients with PSP, and AD-tau scores correlated with Mini-Mental State Examination scores in healthy control-AD continuum patients. The globus pallidus and amygdala were highlighted as regions with high weight coefficients for determining PSP- and AD-tau scores, respectively. CONCLUSIONS: These findings highlight our technology's unbiased capability to identify topologies of 3R + 4R versus 4R tau deposits. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Immediate postnatal central hypothyroidism caused by maternal Graves' disease: Importance of early screening.

This report illustrates a case of central hypothyroidism in a newborn immediately after birth caused by maternal Graves' disease. Infants from mothers with Graves' disease require careful examination without waiting for neonatal screening results, even though the mother's thyroid function is normal at birth or the newborn does not have goiter.

Differences in dynamic perception of salty taste intensity between young and older adults.

In super-aged societies, high salt intake substantially increases the risk of stroke and cardiovascular disease. Perceived low salty taste often prompts the addition of table salt to food. However, it remains unclear how older adults perceive the nature and intensity of salty taste in the mouth and brain. We compared the perceptions of salty taste intensities of older adults with those of young adults. Participants were 74 healthy adults: 31 older (age, 60-81 years [65.0 ± 5.5 SD]) and 43 young (age, 21-39 years [25.0 ± 3.6 SD]). Our research project comprises three sequential experiments. This article reports on the first two, which were (1) static and (2) dynamic sensory evaluations of taste perceptions in the mouth. Participants assessed the taste of 0.3 M and 0.5 M sodium chloride solutions in two types of sensory evaluations: (1) a cup tasting test, in which they sipped the solution from cups, spat it out, and rated static salty taste intensity, and (2) a time-intensity sensory evaluation, in which the solutions were delivered to participants' tongues through a custom-made delivery system while they recorded dynamic taste intensities on a hand-held meter. Older adults perceived significantly lower taste intensities than young adults (p = 0.004 and p < 0.001 for 0.3 M and 0.5 M, respectively). Reaction timings for both solutions did not differ, but the slopes for both concentrations were significantly lower for older adults than for young adults (p < 0.001). Using a standardized system allowed us to evaluate and directly compare real-time feedback on taste intensities according to age. This study is the first to characterize the time-intensity profiles of salty taste intensity in older adults. Our findings show that older adults do not take longer to recognize a salty taste, but their perception of taste intensity slowly increases, and yet remains lower than that of young adults. This suggests that older adults should be aware of the tendency to add more salt to their food to compensate for their low perceptions of salty taste. We would like to suggest them to savor and chew sufficiently during eating to optimize the perceived salty taste. Furthermore, our results offer a reference for ordinary citizens' taste-intensity perceptions; our standardized system could be usefully integrated into clinical follow-up examinations and treatments.

Founder genetic variants of ABCC4 and ABCC11 in the Japanese population are not associated with the development of subacute myelo-optico-neuropathy (SMON).

BACKGROUND: Subacute myelo-optico-neuropathy (SMON) is a severe neurological disorder associated with clioquinol administration, which frequently occurred in Japan during the 1950s and 1960s. The unique genetic background of the Japanese population is considered to be strongly involved in the development of this neurological disease. Recently, genetic variants of ABCC4 (OMIM: 605250) and ABCC11 (OMIM: 607040), which are particularly common in the Japanese population, were suggested as possible genetic susceptibility factors for the development of SMON. METHODS: We analyzed 125 Japanese SMON patients who provided consent for this study. Patient DNA was collected from peripheral blood, and genetic analysis was performed for ABCC4 rs3765534 (c.2268G>A, p.Glu857Lys) and ABCC11 rs17822931 (c.538G>A, p.Gly180Arg) polymorphisms using the Sanger sequencing method and/or TaqMan PCR method. The frequency distribution of each polymorphism was compared with that in healthy Japanese people recorded in two genomic databases (Human Genomic Variation Database and Integrative Japanese Genome Variation Database), and each genotype was compared with the clinical features of patients. RESULTS: The frequencies of ABCC4 rs3765334 and ABCC11 rs17822931 polymorphisms in SMON patients and healthy Japanese people were not significantly different in the multifaceted analysis. CONCLUSION: We conclude that the ABCC4 rs3765334 and ABCC11 rs17822931 polymorphisms are not associated with the development of SMON.

Dietary Supplementation with Monosodium Glutamate Suppresses Chemotherapy-Induced Downregulation of the T1R3 Taste Receptor Subunit in Head and Neck Cancer Patients.

(Background) We investigated the effect of dietary supplementation with monosodium glutamate (MSG) on chemotherapy-induced downregulation of the T1R3 taste receptor subunit expression in the tongue of patients with advanced head and neck cancer. (Methods) Patients undergoing two rounds of chemoradiotherapy were randomly allocated to a control or intervention group (dietary supplementation with MSG at 2.7 g/day during the second round of chemotherapy). The relative expression of T1R3, a subunit of both umami and sweet taste receptors, in the tongue was assessed by quantitative polymerase chain reaction analysis. Dysgeusia was assessed with a visual analog scale and daily energy intake was evaluated. (Results) T1R3 expression levels in the tongue, taste sensitivity, and daily energy intake were significantly reduced after the first round of chemotherapy compared with before treatment. Furthermore, these parameters significantly decreased after the second round of chemotherapy, but the extent of decrease was significantly attenuated in the MSG group compared with the control group. (Conclusions) MSG supplementation suppresses chemotherapy-induced dysgeusia, possibly due to the inhibition of the T1R3-containing taste receptor downregulation in the tongue, thereby increasing energy intake in patients with advanced head and neck cancer.

Tolerable amounts of amino acids for human supplementation: summary and lessons from published peer-reviewed studies.

Amino acid supplementation may be indicated to correct for insufficient amino acid intake in healthy individuals, and in specific physiological or pathophysiological situations. However, there is a concern to not supplement beyond the tolerable upper intake level (UL) by determining parameters of no-observed-adverse-effect level (NOAEL) or lowest-observed-adverse-effect level (LOAEL) for each amino acid. Since the NOAEL and LOAEL values are at least one order of magnitude different when comparing the values obtained in rats and humans, the aim of this review is to evaluate to what extent the amino acid UL measured in the rat model, when referenced to the dietary usual consumption (UC) and dietary requirement (RQ) for indispensable amino acids, may be used as an approximation of the UL in humans. This review then compares the ratios of the NOAEL or LOAEL over UC and RQ in the rat model with the same ratios calculated in humans for the nine amino acids (arginine, serine, glycine, histidine, leucine, lysine, methionine, phenylalanine, and tryptophan) for which this comparison can be done. From the calculations made, it appears that for these 9 amino acids, the calculated ratios for rats and humans, although rather different for several amino acids, remains for all of them in the same order of magnitude. For tryptophan, tyrosine, and valine, the ratios calculated in rats are markedly different according to the sex of animals, raising the view that it may be also the case in humans.

Stress Condition on a Restricted Sodium Diet Using Umami Substance (L-Glutamate) in a Pilot Randomized Cross-Over Study.

Hypertensive patients who adopt a sodium-restricted diet have difficulty maintaining this change, and this could increase stress. On the other hand, soup rich in umami substances (dashi) was reported to reduce indexes of anxiety and stress. The objective of this study was to measure mood and physiological stress indexes during administration of a sodium-restricted diet with and without an umami substance (free L-glutamate) by a cross-over randomized, single-blind, placebo-controlled trial in Japanese female university students. The baseline was measured for 5 days followed by a sodium-restricted diet intervention phase that lasted for 10 days. The Profile of Mood States questionnaire was administered, a stress marker in saliva (chromogranin-A) was measured, and the amount of sodium intake was confirmed from 24 h urine collection samples. Results showed that the sodium reduction was verified by 24 h urine excretion. The percentage of change in the stress marker from the baseline showed that the stress level in group without the umami substance was significantly higher than that in the group with the umami substance (p = 0.013) after receiving a sodium-reduced diet for 6 or more days, indicating that stress was alleviated. This study suggested that umami substances might help to ameliorate stress during a sodium-reduced diet, especially in the initial phase.

Exposure of the cytoplasm to low-dose X-rays modifies ataxia telangiectasia mutated-mediated DNA damage responses.

We recently showed that when a low X-ray dose is used, cell death is enhanced in nucleus-irradiated compared with whole-cell-irradiated cells; however, the role of the cytoplasm remains unclear. Here, we show changes in the DNA damage responses with or without X-ray microbeam irradiation of the cytoplasm. Phosphorylated histone H2AX foci, a surrogate marker for DNA double-strand breaks, in V79 and WI-38 cells are not observed in nucleus irradiations at ≤ 2 Gy, whereas they are observed in whole-cell irradiations. Addition of an ataxia telangiectasia mutated (ATM) kinase inhibitor to whole-cell irradiations suppresses foci formation at ≤ 2 Gy. ABL1 and p73 expression is upregulated following nucleus irradiation, suggesting the induction of p73-dependent cell death. Furthermore, CDKN1A (p21) is upregulated following whole-cell irradiation, indicating the induction of cell cycle arrest. These data reveal that cytoplasmic radioresponses modify ATM-mediated DNA damage responses and determine the fate of cells irradiated at low doses.

Subchronic Tolerance Trials of Graded Oral Supplementation with Phenylalanine or Serine in Healthy Adults.

Phenylalanine and serine are amino acids used in dietary supplements and nutritional products consumed by healthy consumers; however, the safe level of phenylalanine or serine supplementation is unknown. The objective of this study was to conduct two 4-week clinical trials to evaluate the safety and tolerability of graded dosages of oral phenylalanine and oral serine. Healthy male adults (n = 60, 38.2 ± 1.8y) completed graded dosages of either phenylalanine or serine supplement (3, 6, 9 and 12 g/d) for 4 weeks with 2-week wash-out periods in between. Primary outcomes included vitals, a broad spectrum of circulating biochemical analytes, body weight, sleep quality and mental self-assessment. At low dosages, minor changes in serum electrolytes and plasma non-essential amino acids glutamine and aspartic acid concentrations were observed. Serine increased its plasma concentrations at high supplemental dosages (9 and 12 g/day), and phenylalanine increased plasma tyrosine concentrations at 12 g/day, but those changes were not considered toxicologically relevant. No other changes in measured parameters were observed, and study subjects tolerated 4-week-long oral supplementation of phenylalanine or serine without treatment-related adverse events. A clinical, no-observed-adverse-effect-level (NOAEL) of phenylalanine and serine supplementation in healthy adult males was determined to be 12 g/day.

In vitro functional analysis of four variants of human asparagine synthetase.

The loss-of-function variants of the human asparagine synthetase (ASNS) gene cause asparagine synthetase deficiency (ASNSD). Diagnosis of ASNSD requires genetic tests because a specific biochemical diagnostic for ASNSD is not available. There are a few reports describing the functional evaluation of ASNS variants. Therefore, in vitro methods are needed to evaluate the detected variants in patients. In this report, five types of human ASNS proteins (wild-type and our reported four variants: p.Leu145Ser, p.Leu247Trp, p.Val489Asp, and p.Trp541Cysfs*5) were expressed in silkworm using a baculoviral expression system. An enzymatic activity assay of ASNS was performed, and the concentration of asparagine by ninhydrin and High Performance Liquid Chromatography methods using the purified recombinant proteins was measured. We established ASNS deficient HEK293 cells using the CRISPR/Cas9 method and evaluated the growth of cells without asparagine after transduction of ASNS variants with a lentiviral expression system. The four ASNS variants displayed significantly low enzymatic activity. The ASNS deficient HEK293 cells transduced with wild-type ASNS grew without asparagine, whereas cells transduced with the variants did not grow or showed significantly slower growth than cells transduced with wild-type ASNS. Herein, we established a method for evaluating the enzymatic activity of the recombinant human ASNS variants. The results of the cell-based assay corroborated the results of the enzymatic activity. These methods should enable the evaluation of the pathogenicity of ASNS variants.