Bmal1 regulates female reproduction in mice via the hypothalamic-pituitary-ovarian axis.

The hypothalamic-pituitary-gonadal axis (HPG) is the key neuroendocrine axis involved in reproductive regulation. Brain and muscle ARNT-like protein 1 (Bmal1) participates in regulating the metabolism of various endocrine hormones. However, the regulation of Bmal1 on HPG and female fertility is unclear. This study aims to explore the regulation of female reproduction by Bmal1 via the HPG axis in mice. Bmal1-knockout (Ko) mice were generated using the CRISPR/Cas9 technology. The structure, function, and estrous cycle of ovarian in Bmal1 Ko female mice were measured. The key genes and proteins of the HPG axis involved in regulating female reproduction were examined through transcriptome analysis and then verified by RT-PCR, immunohistochemistry, and western blot. Furthermore, the fertility of female mice was detected after intervening prolactin (PRL) and progesterone (Pg) in Bmal1 ko mice. The number of offspring and ovarian weight were significantly lower in Bmal1-Ko mice than in wild-type (Wt) mice. In Bmal1-Ko mice, ovarian cells were arranged loosely and irregularly, and the total number of follicles was significantly reduced. No corpus luteum was found in the ovaries. Vaginal smears revealed that Bmal1-Ko mice had an irregular estrus cycle. In Bmal1-Ko mice, Star expression was decreased, PRL and luteinizing hormone (LH) levels were increased, and dopamine (DA) and Pg levels were decreased. Inhibition of PRL partially recovered the estrous cycle, corpus luteum formation, and Star expression in the ovaries. Pg supplementation promoted embryo implantation in Bmal1-Ko female mice. Bmal1 Ko increases serum PRL levels in female mice likely by reducing DA levels, thus affecting luteal formation, resulting in decreased Star expression and Pg production, hindering female reproduction. Inhibition of PRL or restoration of Pg can partially restore reproductive capacity in female Bmal1-Ko mice. Thus, Bmal1 may regulate female reproduction via the HPG axis in mice, suggesting that Bmal1 is a potential target to treat female infertility.

The RNA m6A modification might participate in microglial activation during hypoxic-ischemic brain damage in neonatal mice.

BACKGROUND: The RNA m6A modification has been implicated in multiple neurological diseases as well as macrophage activation. However, whether it regulates microglial activation during hypoxic-ischemic brain damage (HIBD) in neonates remains unknown. Here, we aim to examine whether the m6A modification is involved in modulating microglial activation during HIBD. We employed an oxygen and glucose deprivation microglial model for in vitro studies and a neonatal mouse model of HIBD. The brain tissue was subjected to RNA-seq to screen for significant changes in the mRNA m6A regulator. Thereafter, we performed validation and bioinformatics analysis of the major m6A regulators. RESULTS: RNA-seq analysis revealed that, among 141 m6A regulators, 31 exhibited significant differential expression (FC (abs) ≥ 2) in HIBD mice. We then subjected the major m6A regulators Mettl3, Mettl14, Fto, Alkbh5, Ythdf1, and Ythdf2 to further validation, and the results showed that all were significantly downregulated in vitro and in vivo. GO analysis reveals that regulators are mainly involved in the regulation of cellular and metabolic processes. The KEGG results indicate the involvement of the signal transduction pathway. CONCLUSIONS: Our findings demonstrate that m6A modification of mRNA plays a crucial role in the regulation of microglial activation in HIBD, with m6A-associated regulators acting as key modulators of microglial activation.

Long-term outcomes of very early treated infantile-onset Pompe disease with short-term steroid premedication: experiences from a nationwide newborn screening programme.

BACKGROUND: Starting enzyme replacement therapy (ERT) before severe irreversible muscular damage occurs is important in infantile-onset Pompe disease (IOPD). This long-term follow-up study demonstrates our diagnostic and treatment strategies for IOPD and compares our clinical outcomes with those of other medical centres. METHODS: In this long-term follow-up study, we analysed the outcomes of very early ERT with premedication hydrocortisone in patients with IOPD. Out of 1 228 539 infants screened between 1 January 2010 and 28 February 2021, 33 newborns had confirmed IOPD in Taipei Veterans General Hospital. Twenty-six were regularly treated and monitored at Taipei Veterans General Hospital. Echocardiographic parameters, biomarkers, IgG antibodies against alglucosidase alpha, pulmonary function variables and developmental status were all assessed regularly over an average follow-up duration of 6.18±3.14 years. We compared the long-term treatment outcomes of our patients with those of other research groups. RESULTS: The average age at ERT initiation was 9.75±3.17 days for patients with classic IOPD. The average of the latest antialglucosidase alpha IgG titre was 669.23±1159.23. All enrolled patients had normal heart sizes, motor milestones, cognitive function and pulmonary function that were near-normal to normal. Compared with patients in other studies, our patients had better outcomes in all aspects. CONCLUSION: Very early ERT using our rapid diagnostic and treatment strategy enabled our patients with IOPD to have better outcomes than patients in other medical centres.

Determination of Multiple Neurotransmitters through LC-MS/MS to Confirm the Therapeutic Effects of Althaea rosea Flower on TTX-Intoxicated Rats.

Tetrodotoxin (TTX) inhibits neurotransmission in animals, and there is no specific antidote. In clinical practice in China, Althaea rosea (A. rosea flower) extract has been used to treat TTX poisoning. In this work, the efficacy of the ethyl acetate fraction extract of A. rosea flower in treating TTX poisoning in rats was investigated. A high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to determine nine neurotransmitters in rat brain tissue, including γ-aminobutyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT), noradrenaline (NE), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxyindole-3-acetic acid (5-HIAA), epinephrine (E), and tyramine (Tyn). The detoxifying effect of A. rosea flower was verified by comparing the changes in neurotransmitters' content in brain tissue before and after poisoning in rats. The assay was performed in multiple reaction monitoring mode. The quantification method was performed by plotting an internal-standard working curve with good linearity (R2 > 0.9941) and sensitivity. Analyte recoveries were 94.04-107.53% (RSD < 4.21%). Results indicated that the levels of 5-HT, DA, E, and NE in the brains of TTX-intoxicated rats decreased, whereas the levels of GABA, Tyn, and 5-HIAA showed an opposite trend, and HVA and DOPAC were not detected. The levels of all seven neurotransmitters returned to normal after the gavage administration of ethyl acetate extract of A. rosea flower to prove that the ethyl acetate extract of A. rosea flower had a therapeutic effect on TTX poisoning. The work provided new ideas for studies on TTX detoxification.

Three-dimensional relationships between condylar volume and dentoskeletal characteristics in Class II hyperdivergent female adults.

BACKGROUND: This study aimed to determine the differences among various volumes of condylar osseous patterns and the corresponding dentoskeletal characteristics based on the risk of temporomandibular disorder. METHODS: Craniofacial spiral computed tomography data of 60 Class II hyperdivergent female adults were divided into normal, resorptive, flattened, and osteophyte groups based on condylar osseous forms. The condylar volumes of each group were compared, and their correlations with the dentoskeletal characteristics were assessed in three dimensions. Pairwise least significant difference tests were used to examine individual pairwise differences between groups, and one-way analysis of variance was used to measure differences among multiple groups. Pearson correlation and Spearman rank correlation analyses were used to determine the correlation between condylar volume and dentofacial characteristics. Statistical significance was established at p < 0.05. RESULTS: The condylar volume in the normal group was significantly greater than that in the changed groups, with no significant differences between the subgroups. The decrease in condylar volume was associated with a retruded and clockwise-rotated mandible with shorter rami. Condylar volume was negatively correlated with overjet, the alveolar height of the lower anterior and posterior teeth, sagittal inclinations of the lower teeth, intermolar width of the mandibular first molars, and width between the corresponding alveolar crests. CONCLUSION: Multiple three-dimensional dentoskeletal characteristics of Class II hyperdivergent female adults are correlated with condylar bony changes, regardless of the form. These results could be helpful in indicating potential pathological changes in the temporomandibular joint and in making proper treatment plans for these patients.

The role of crystal packing on the optical response of trialkyltetrelethynyl acenes.

The electronic and optical responses of an organic semiconductor (OSC) are dictated by the chemistries of the molecular or polymer building blocks and how these chromophores pack in the solid state. Understanding the physicochemical nature of these responses is not only critical for determining the OSC performance for a particular application, but the UV/visible optical response may also be of potential use to determine aspects of the molecular-scale solid-state packing for crystal polymorphs or thin-film morphologies that are difficult to determine otherwise. To probe these relationships, we report the quantum-chemical investigation of a series of trialkyltetrelethynyl acenes (tetrel = silicon or germanium) that adopt the brickwork, slip-stack, or herringbone (HB) packing configurations; the π-conjugated backbones considered here are pentacene and anthradithiophene. For comparison, HB-packed (unsubstituted) pentacene is also included. Density functional theory and G0W0 (single-shot Green's function G and/or screened Coulomb function W) electronic band structures, G0W0-Bethe-Salpeter equation-derived optical spectra, polarized ϵ2 spectra, and distributions of both singlet and triplet exciton wave functions are reported. Configurational disorder is also considered. Furthermore, we evaluate the probability of singlet fission in these materials through energy conservation relationships.

Study on Quality Control of Compound Anoectochilus roxburghii (Wall.) Lindl. by Liquid Chromatography-Tandem Mass Spectrometry.

Compound Anoectochilus roxburghii (Wall.) Lindl. (A. roxburghii) oral liquid (CAROL) is a hospital preparation of A. roxburghii and Ganoderma lucidum (G. lucidum), which have hepatoprotective effects. Eight active components (five nucleosides/nucleobases and three triterpenoid acids) in CAROL, A. roxburghii, and G. lucidum were simultaneously detected by high-performance liquid chromatography−tandem mass spectrometry (LC−MS/MS). The multiple reaction monitoring (MRM) mode was applied for the detection of analytes. These eight compounds were separated well within 12 min and quantified using the internal standard working curve method. The method showed good linearity (R2 > 0.9935) and high sensitivity (limit of detection = 0.29 ng/mL). The analyte recovery ranged from 85.07% to 97.50% (relative standard deviation < 3.31%). The content of the target analytes in four batches of CAROL, and the raw materials of G. lucidum and A. roxburghii from the five regions was determined using this method. The contents of guanosine and ganoderic acid A in four batches of oral liquid were high and stabilized and could be recommended as quality markers (Q-marker) for CAROL. Simultaneous qualitative and quantitative analysis of nucleosides and triterpenoid acids in CAROL, A. roxburghii, and G. lucidum by LC−MS/MS based on the MRM model was reported for the first time. The proposed method provides a sensitive, rapid, and reliable approach for the quality control of Chinese medicinal products.

Optimization of S-Nitrosocaptopril Monohydrate Storage Conditions Based on Response Surface Method.

From unstable crystals to relatively stable monohydrate crystals, many researchers have been working on S-nitrosocaptopril for more than two decades. S-nitrosocaptopril monohydrate (Cap-NO·H2O) is a novel crystal form of S-nitrosocaptopril (Cap-NO), and is not only a nitric oxide (NO) donor, but also an angiotensin-converting enzyme inhibitor (ACEI). Yet, a method for long-term storage has never been reported. In order to determine the optimal storage conditions, Plackett-Burman (PB) design was performed to confirm the critical factors. Response surface methodology (RSM) was employed to determine the optimal Cap-NO·H2O storage condition, based on the rough interval determined by the path of steepest ascent experiment. The optimized storage condition was denoted as nitrogen purity of 97%, temperature of -10 °C and 1.20 g deoxidizer. In this case, a final preservation rate of 97.91 ± 0.59% could be obtained. In specific storage conditions, Cap-NO·H2O was found to be stable for at least 6 months in individual PE package, procreating a potentially applicable avenue.

Chitosan and Whey Protein Bio-Inks for 3D and 4D Printing Applications with Particular Focus on Food Industry.

The application of chitosan (CS) and whey protein (WP) alone or in combination in 3D/4D printing has been well considered in previous studies. Although several excellent reviews on additive manufacturing discussed the properties and biomedical applications of CS and WP, there is a lack of a systemic review about CS and WP bio-inks for 3D/4D printing applications. Easily modified bio-ink with optimal printability is a key for additive manufacturing. CS, WP, and WP-CS complex hydrogel possess great potential in making bio-ink that can be broadly used for future 3D/4D printing, because CS is a functional polysaccharide with good biodegradability, biocompatibility, non-immunogenicity, and non-carcinogenicity, while CS-WP complex hydrogel has better printability and drug-delivery effectivity than WP hydrogel. The review summarizes the current advances of bio-ink preparation employing CS and/or WP to satisfy the requirements of 3D/4D printing and post-treatment of materials. The applications of CS/WP bio-ink mainly focus on 3D food printing with a few applications in cosmetics. The review also highlights the trends of CS/WP bio-inks as potential candidates in 4D printing. Some promising strategies for developing novel bio-inks based on CS and/or WP are introduced, aiming to provide new insights into the value-added development and commercial CS and WP utilization.

Expression and functional analysis of lncRNAs in the hippocampus of immature rats with status epilepticus.

Long non-coding RNAs (lncRNAs) have been implicated in the regulation of gene expression at various levels. However, to date, the expression profile of lncRNAs in status epilepticus (SE) was unclear. In our study, the expression profile of lncRNAs was investigated by high-throughput sequencing based on a lithium/pilocarpine-induced SE model in immature rats. Furthermore, weighted correlation network analysis (WGCNA), gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to construct co-expression networks and establish functions of the identified hub lncRNAs in SE. The functional role of a hub lncRNA (NONRATT010788.2) in SE was investigated in an in vitro model. Our results indicated that 7082 lncRNAs (3522 up-regulated and 3560 down-regulated), which are involved in cell proliferation, inflammatory responses, angiogenesis and autophagy, were dysregulated in the hippocampus of immature rats with SE. Additionally, WGCNA identified 667 up-regulated hub lncRNAs in turquoise module that were involved in apoptosis, inflammatory responses and angiogenesis via regulation of HIF-1, p53 and chemokine signalling pathways and via inflammatory mediator regulation of TRP channels. Knockdown of an identified hub lncRNA (NONRATT010788.2) inhibited neuronal apoptosis in vitro. Taken together, our study is the first to demonstrate the expression profile and potential function of lncRNAs in the hippocampus of immature rats with SE. The defined hub lncRNAs may participate in the pathogenesis of SE via lncRNA-miRNA-mRNA network.

Classification of Flavonoid Metabolomes via Data Mining and Quantification of Hydroxyl NMR Signals.

Utilizing the distinct HMBC cross-peak patterns of lower-field range (LFR; 11.80-14.20 ppm) hydroxyl singlets, presented NMR methodology characterizes flavonoid metabolomes both qualitatively and quantitatively. It enables simultaneous classification of the structural types of 5-OH flavonoids and biogenetically related 2'-OH chalcones, as well as quantification of individual metabolites from 1H NMR spectra, even in complex mixtures. Initially, metabolite-specific LFR 1D 1H and 2D HMBC patterns were established via literature mining and experimental data interpretation, demonstrating that LFR HMBC patterns encode the different structural types of 5-OH flavonoids/2'-OH chalcones. Taking advantage of the simplistic multiplicity of the H,H-uncoupled LFR 5-/2'-OH singlets, individual metabolites could subsequently be quantified by peak fitting quantitative 1H NMR (PF-qHNMR). Metabolomic analysis of enriched fractions from three medicinal licorice (Glycyrrhiza) species established proof-of-concept for distinguishing three major structural types and eight subtypes in biomedical applications. The method identified 15 G. uralensis (GU) phenols from the six possible subtypes of 5,7-diOH (iso)flav(an)ones with 6-, 8-, and nonprenyl substitution, including the new 6-prenyl-licoisoflavanone (1) and two previously unknown compounds (4 and 7). Relative (100%) qNMR established quantitative metabolome patterns suitable for species discrimination and plant metabolite studies. Absolute qNMR with combined external and internal (solvent) calibration (ECIC) identified and quantified 158 GU metabolites. HMBC-supported qHNMR analysis of flavonoid metabolomes ("flavonomics") empowers the exploration of structure-abundance-activity relationships of designated bioactivity. Its ability to identify and quantify numerous metabolites simultaneously and without identical reference materials opens new avenues for natural product discovery and botanical quality control and can be adopted to other flavonoid- and chalcone-containing taxa.

Airway abnormalities in very early treated infantile-onset Pompe disease: A large-scale survey by flexible bronchoscopy.

Early enzyme replacement therapy (ERT) improve long-term outcomes in patients with infantile-onset Pompe disease (IOPD). Our cohort of patients with IOPD at Taipei Veterans General Hospital (TVGH) joined Taiwan Pompe newborn screening program from 2008, testing more than one million newborns until 2018. By 2010, we had established rapid diagnostic strategies. Now, the average age of ERT initiation starts at an average age of <10 days-old, the earliest group in the world. However, they still presented some airway problems. We present a retrospective study focused on airway abnormalities in these patients along 8 years of observation. Fifteen patients with IOPD, who received very early treatment at a mean age of 8.94 ± 3.75 days, underwent flexible bronchoscopy (FB) for dynamic assessment of the whole airway. Long-term clinical outcomes and relevant symptoms of the upper airway were assessed. All patients in the study had varying degrees of severity of upper airway abnormalities and speech disorders. The three oldest children (Age 94, 93, and 88 months, respectively) had poor movement of the vocal cords with reduced abduction and adduction and had silent aspiration of saliva through the glottis during respiration. This is the largest cohort study presented to date about airway abnormalities in very early treated patients with IOPD patients by FB. Despite very early treatment, we observed upper airway abnormalities in these IOPD patients. In IOPD, upper airway abnormalities seem inevitable over time. We suggest early and continuous monitoring for all IOPD patients, even with early and regular treatment.

[Expression and role of Pim1 in cultured cortical neurons with oxygen-glucose deprivation/reoxygen injury].

OBJECTIVE: To study the expression and effect of Pim1 in primary cortical neurons after hypoxic-ischemic injury. METHODS: Cortical neurons were isolated from 1-day-old C57BL/6 mice and cultured in neurobasal medium. On the 8th day of neuron culture, cells were subjected to oxygen-glucose deprivation/reoxygen (OGD/R) treatment to mimic in vivo hypoxic injury of neurons. Briefly, medium were changed to DMEM medium, and cells were cultured in 1% O2 for 3 hours and then changed back to normal medium and conditions. Cells were collected at 0 hour, 6 hours, 12 hours and 24 hours after OGD/R. Primary neurons were transfected with Pim1 overexpression plasmid or mock plasmid, and then were exposed to normal conditions or OGD/R treatment. They were named as Pim1 group, control group, OGD/R group and OGD/R+Pim1 group respectively. Real-time PCR was used to detect Pim1 mRNA expression. Western blot was used to detect the protein expression of Pim1 and apoptotic related protein cleaved caspase 3 (CC3). TUNEL staining was used to detect cell apoptosis. RESULTS: Real-time PCR and Western blot results showed that Pim1 mRNA and protein were significantly decreased in neurons after OGD/R. They began to decrease at 0 hour after OGD/R, reached to the lowest at 12 hours after OGD/R, and remained at a lower level at 24 hours after OGD/R (P<0.01). Overexpression of Pim1 significantly upregulated the protein level of Pim1. Under OGD/R conditions, the CC3 expression and the apoptosis rate in cells of the Pim1 group were significantly lower than in un-transfected cells (P<0.01). CONCLUSIONS: Hypoxic-ischemic injury may decrease Pim1 expression in neurons. Overexpressed Pim1 may inhibit apoptosis induced by OGD/R.

[Effect of irisin on hypoxic-ischemic brain damage in neonatal rats].

OBJECTIVE: To study the effect and mechanism of action of irisin on hypoxic-ischemic brain damage in neonatal rats. METHODS: A total of 248 7-day-old Sprague-Dawley rats were randomly divided into a sham-operation group, a model group, and low- and high-dose irisin intervention groups (n=62 each). The rats in the model and irisin intervention groups were given hypoxic treatment after right common carotid artery ligation to establish a model of hypoxic-ischemic brain damage. Those in the sham-operation group were given the separation of the right common carotid artery without ligation or hypoxic treatment. The rats in the high- and low-dose irisin intervention groups were given intracerebroventricular injection of recombinant irisin polypeptide at a dose of 0.30 µg and 0.15 µg respectively. Those in the model and sham-operation groups were given the injection of an equal volume of PBS. The water maze test was used to compare neurological behaviors between groups. TTC staining, hematoxylin-eosin staining and TUNEL staining were used to observe histopathological changes of the brain. Western blot was used to measure the expression of the apoptosis-related molecules cleaved-caspase-3 (CC3), BCL-2 and BAX. RESULTS: Compared with the sham-operation group, the model group had a significant increase in latency time and a significant reduction in the number of platform crossings (P<0.05). Compared with the model group, the high-dose irisin intervention group had a significant reduction in latency time and a significant increase in the number of platform crossings (P<0.05). Compared with the sham-operation group, the model group had massive infarction in the right hemisphere, with significant increases in karyopyknosis and karyorrhexis. Compared with the model group, the high-dose irisin intervention group had a smaller infarct area of the right hemisphere, with reductions in karyopyknosis and karyorrhexis. The model group had a significantly higher apoptosis rate of cells in the right cerebral cortex and the hippocampus than the sham-operation group. The high-dose irisin intervention group had a significantly lower apoptosis rate than the model group (P<0.05). At 24 and 48 hours after modeling, the sham-operation group had a significantly lower level of CC3 than the model group (P<0.05). Compared with the model group, the high-dose irisin intervention group had a significantly lower level of CC3 and a significantly higher BCL-2/BAX ratio (P<0.05). The low-dose irisin intervention group had similar laboratory markers and histopathological changes of the brain to the model group. CONCLUSIONS: Irisin can alleviate hypoxic-ischemic brain damage in neonatal rats in a dose-dependent manner, possibly by reducing cell apoptosis in the cerebral cortex and the hippocampus.

Stable clinical course in three siblings with late-onset isolated sulfite oxidase deficiency: a case series and literature review.

BACKGROUND: Isolated sulfite oxidase deficiency (ISOD) is an autosomal recessive disorder caused by a deficiency of sulfite oxidase, which is encoded by the sulfite oxidase gene (SUOX). Clinically, the disorder is classified as one of two forms: the late-onset mild form or the classic early-onset form. The latter is life-threatening and always leads to death during early childhood. Mild ISOD cases are rare and may benefit from dietary therapy. To date, no cases of ISOD have been reported to recover spontaneously. Here, we present three mild ISOD cases in one family, each with a stable clinical course and spontaneous recovery. CASE PRESENTATION: All three siblings had two novel compound heterozygous mutations in the SUOX gene (NM_000456; c.1096C > T [p.R366C] and c.1376G > A [p.R459Q]). The siblings included two males and one female with late ages of onset (12-16 months) and presented with specific neuroimaging abnormalities limited to the bilateral globus pallidus and substantia nigra. The three patients had decreased plasma homocysteine levels. They exhibited a monophasic clinical course continuing up to 8.5 years even without dietary therapy. CONCLUSION: This is the first report of mild ISOD cases with a stable clinical course and spontaneous recovery without dietary therapy. Our study provides an expansion for the clinical spectrum of ISOD. Furthermore, we highlight the importance of including ISOD in the differential diagnosis for patients presenting with late-onset symptoms, bilaterally symmetric regions of abnormal intensities in the basal ganglia, and decreased plasma homocysteine levels.

Hepatic metabolism of licochalcone A, a potential chemopreventive chalcone from licorice (Glycyrrhiza inflata), determined using liquid chromatography-tandem mass spectrometry.

The metabolism of the chemoprevention agent licochalcone A, which is a chemopreventive chalcone found in abundance in the licorice species Glycyrrhiza inflata, was investigated using human liver microsomes and human hepatocytes combined with analysis using high performance liquid chromatography-mass spectrometry (LC-MS). Five oxygenated phase I metabolites of licochalcone A were formed by human liver microsomes, including a catechol on the A-ring, two intramolecular cyclization products following epoxidation of the exocyclic alkene at position 5 of the B-ring, and two dioxygenated products. Nine phase II monoglucuronides of licochalcone A and its oxygenated phase I metabolites were formed during incubation with human hepatocytes. These included (E)-licochalcone A-4-glucuronide, (E)-licochalcone A-4'-glucuronide, (Z)-licochalcone A-4-glucuronide, glucuronic acid conjugates of all of the monooxygenated phase I metabolites, and glucuronides of the licochalcone catechol after methylation by catechol-O-methyl transferase. In addition, human hepatocytes formed one sulfate conjugate and one glutathione conjugate of licochalcone A. The structures of all major metabolites were determined using a combination of accurate mass measurement, LC-tandem mass spectrometry, LC-UV, nuclear magnetic resonance, and comparison with standards. The cytochrome P450 enzymes and UDP-glucuronosyltransferases responsible for the formation of the major metabolites were identified. Based on in vitro hepatic clearance calculations, licochalcone A is predicted to be metabolized primarily by phase II conjugation reactions. Graphical abstract Phase I and II metabolism of licochalcone A from the licorice species Glycyrrhiza inflata by human liver microsomes and hepatocytes determined using LC-MS/MS, LC-UV and NMR.

Very Early Treatment for Infantile-Onset Pompe Disease Contributes to Better Outcomes.

OBJECTIVE: To evaluate whether very early treatment in our patients would result in better clinical outcomes and to compare these data with other infantile-onset Pompe disease (IOPD) cohort studies. METHODS: In this nationwide program, 669,797 newborns were screened for Pompe disease. We diagnosed IOPD in 14 of these newborns, and all were treated and followed in our hospital. RESULTS: After 2010, the mean age at first enzyme-replacement therapy (ERT) was 11.92 days. Our patients had better biological, physical, and developmental outcomes and lower anti-rh acid α-glucosidase antibodies after 2 years of treatment, even compared with one group that began ERT just 10 days later than our cohort. No patient had a hearing disorder or abnormal vision. The mean age for independent walking was 11.6 ± 1.3 months, the same age as normal children. CONCLUSIONS: ERT for patients with IOPD should be initiated as early as possible before irreversible damage occurs. Our results indicate that early identification of patients with IOPD allows for the very early initiation of ERT. Starting ERT even a few days earlier may lead to better patient outcomes.

Chondroitin sulfate promotes activation of cathepsin K.

Cathepsin K (CatK), a major lysosomal collagenase produced by osteoclasts, plays an important role in bone resorption. Evidence exists that the collagenase activity of CatK is promoted by chondroitin sulfate (CS), a sulfated glycosaminoglycan. This study examines the role of CS in facilitating CatK activation. We have demonstrated that chondroitin 4-sulfate (C4-S) promotes autoprocessing of the pro-domain of CatK at pH ≤ 5, leading to a fully matured enzyme with collagenase and peptidase activities. We present evidence to demonstrate this autoactivation process is a trans-activation event that is efficiently inhibited by both the covalent cysteine protease inhibitor E-64 and the reversible selective CatK inhibitor L-006,235. During bone resorption, CatK and C4-S are co-localized at the ruffled border between osteoclast bone interface, supporting the proposal that CatK activation is accomplished through the combined action of the acidic environment together with the presence of a high concentration of C4-S. Formation of a multimeric complex between C4-S and pro-CatK has been speculated to accelerate CatK autoactivation and promote efficient collagen degradation. Together, these results demonstrate that CS plays an important role in contributing to the enhanced efficiency of CatK collagenase activity in vivo.

Detection of reactive metabolites using isotope-labeled glutathione trapping and simultaneous neutral loss and precursor ion scanning with ultra-high-pressure liquid chromatography triple quadruple mass spectrometry.

Metabolic activation of drugs to electrophilic species is responsible for over 60% of black box warnings and drug withdrawals from the market place in the United States. Reactive metabolite trapping using glutathione (GSH) and analysis using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) or HPLC with high resolution mass spectrometry (mass defect filtering) have enabled screening for metabolic activation to become routine during drug development. However, current MS-based approaches cannot detect all GSH conjugates present in complex mixtures, especially those present in extracts of botanical dietary supplements. To overcome these limitations, a fast triple quadrupole mass spectrometer-based approach was developed that can detect positively and negatively charged GSH conjugates in a single analysis without the need for advanced knowledge of the elemental compositions of potential conjugates and while avoiding false positives. This approach utilized UHPLC instead of HPLC to shorten separation time and enhance sensitivity, incorporated stable-isotope labeled GSH to avoid false positives, and used fast polarity switching electrospray MS/MS to detect GSH conjugates that form positive and/or negative ions. The general new method was then used to test the licorice dietary supplement Glycyrrhiza glabra, which was found to form multiple GSH conjugates upon metabolic activation. Among the GSH conjugates found in the licorice assay were conjugates with isoliquiritigenin and glabridin, which is an irreversible inhibitor of cytochrome P450 enzymes.

Altered expression of transfer RNAs and their possible roles in brain white matter injury.

Transfer RNAs (tRNAs) can regulate cell behavior and are associated with neurological disorders. Here, we aimed to investigate the expression levels of tRNAs in oligodendrocyte precursor cells (OPCs) and their possible roles in the regulation of brain white matter injury (WMI). Newborn Sprague-Dawley rats (postnatal day 5) were used to establish a model that mimicked neonatal brain WMI. RNA-array analysis was performed to examine the expression of tRNAs in OPCs. psRNAtarget software was used to predict target mRNAs of significantly altered tRNAs. Gene ontology (GO) and KEGG were used to analyze the pathways for target mRNAs. Eighty-nine tRNAs were changed after WMI (fold change absolute ≥1.5, P  < 0.01), with 31 downregulated and 58 upregulated. Among them, three significantly changed tRNAs were identified, with two being significantly increased (chr10.trna1314-ProTGG and chr2.trna2771-ProAGG) and one significantly decreased (chr10.trna11264-GlyTCC). Further, target mRNA prediction and GO/KEGG pathway analysis indicated that the target mRNAs of these tRNAs are mainly involved in G-protein coupled receptor signaling pathways and beta-alanine metabolism, which are both related to myelin formation. In summary, the expression of tRNAs in OPCs was significantly altered after brain WMI, suggesting that tRNAs may play important roles in regulating WMI. This improves the knowledge about WMI pathophysiology and may provide novel treatment targets for WMI.