Genetic Analysis of Two Novel GPI Variants Disrupting H Bonds and Localization Characteristics of 55 Gene Variants Associated with Glucose-6-phosphate Isomerase Deficiency.

OBJECTIVE: Glucose-6-phosphate isomerase (GPI) deficiency is a rare hereditary nonspherocytic hemolytic anemia caused by GPI gene variants. This disorder exhibits wide heterogeneity in its clinical manifestations and molecular characteristics, often posing challenges for precise diagnoses using conventional methods. To this end, this study aimed to identify the novel variants responsible for GPI deficiency in a Chinese family. METHODS: The clinical manifestations of the patient were summarized and analyzed for GPI deficiency phenotype diagnosis. Novel compound heterozygous variants of the GPI gene, c.174C>A (p.Asn58Lys) and c.1538G>T (p.Trp513Leu), were identified using whole-exome and Sanger sequencing. The AlphaFold program and Chimera software were used to analyze the effects of compound heterozygous variants on GPI structure. RESULTS: By characterizing 53 GPI missense/nonsense variants from previous literature and two novel missense variants identified in this study, we found that most variants were located in exons 3, 4, 12, and 18, with a few localized in exons 8, 9, and 14. This study identified novel compound heterozygous variants associated with GPI deficiency. These pathogenic variants disrupt hydrogen bonds formed by highly conserved GPI amino acids. CONCLUSION: Early family-based sequencing analyses, especially for patients with congenital anemia, can help increase diagnostic accuracy for GPI deficiency, improve child healthcare, and enable genetic counseling.

Case report: Genetic analysis of a novel intronic inversion variant in the SPTB gene associated with hereditary spherocytosis.

Background: Hereditary spherocytosis (HS) is a congenital haemolytic anaemia attributed to dysregulation or abnormal quantities of erythrocyte membrane proteins. Currently, the most common erythrocytic gene, spectrin ß (SPTB), variants are located in exons and give rise to mRNA defects. However, the genetic characteristics and pathogenic mechanisms of SPTB intronic variants are not completely understood. This study aimed to analyse a rare intronic inversion variant in the SPTB gene associated with HS, and explore the impact of the variant on SPTB mRNA splicing. Method: The clinical manifestations of the patient were summarised and analysed for spherocytosis phenotype diagnosis. The pathogenic variant was identified in the proband using targeted next-generation and Sanger sequencing. RNA sequencing was performed to analyse whether SPTB gene splicing and expression were affected. Results: Targeted next-generation sequencing identified a novel disease-associated intronic inversion variant of the SPTB gene in the proband. The inversion variant was located between intron 19 and 20, and contained the entire exon 20 and partial sequences of adjacent introns. Sanger sequencing confirmed that the intronic inversion variant only appeared in the genome of the proband, not in his parents. RNA sequencing revealed that the variant could result in the skipping of exon 20 and reduced expression of SPTB mRNA. Conclusion: This study identifies a rare intronic inversion variant in the SPTB gene associated with hereditary spherocytosis. The pathogenic variant can lead to exon 20 skipping and decreased SPTB gene expression. This finding has not been previously reported in any literature. This study can expand the intronic variant spectrum of the SPTB gene, deepen our understanding of HS pathogenesis, and contribute to the genetic diagnosis and clinical management of patients.

Systematically uncovering the absorbed effective substances of Radix Scutellaria-licorice drug pair in rat plasma against COVID-19 using a combined UHPLC-Q-TOF-MS analysis and target network pharmacology.

Radix Scutellaria-Licorice drug pair (RSLDP), a frequently used herbal pair with the effect of clearing heat and detoxifying, is the commonly employed drug pair in TCM prescriptions for the treatment of COVID-19. Until now, the metabolism feature and anti-COVID-19 mechanism of RSLDP have not been fully elucidated. In this study, a sensitive and rapid method was developed for the separation and identification of the absorbed constituents of RSLDP in the rat plasma by UHPLC-QTOF-MS. Additionally, we optimized the conventional methodologies of network pharmacology and proposed a new concept called target network pharmacology (T-NP). It used the absorbed constituents and the corresponding targets to generate a compound-target network, and compared to conventional network pharmacology, it could reduce false-positive results. A total of 85 absorbed constituents were identified or tentatively characterized in dosed plasma, including 32 components in the group of Radix Scutellaria, 27 components in the group of Licorice, and 65 components in the group of RSLDP. The results showed that the compatibility of Radix Scutellaria and Licorice increased the number of components in vivo. We found that 106 potential targets among the 61 active compounds in RSLDP were related to COVID-19. And 12 targets (STAT3, AKT1, EGFR, HSP9AA1, MAPK3, JUN, IL6, VEGFA, TNF, IL2, RELA, and STAT1) could be core targets for RSLDP in treating COVID-19. Results from these targets indicate that RSLDP treatment of COVID-19 mainly involves response to chemical stress, response to oxygenates, positive regulation of cytokines, PI3K-Akt signaling pathway, AGE-RAGE signaling pathway for diabetic complications, virus-related pathways such as novel coronavirus and human cytomegalovirus infection, inflammatory immune-related pathways, and so on. The metabolism feature of RSLDP in vivo was systematically uncovered. The combined use of the T-NP method could discover potential drug targets and disclose the biological processes of RSLDP, which will clarify the potential mechanisms of RSLDP in the treatment of COVID-19.

A practical technique for rapid characterisation of ent-kaurane diterpenoids in Isodon serra (Maxim.) Hara by UHPLC-Q-TOF-MS/MS.

INTRODUCTION: The diterpenoids are the most important active constituents that contribute to the pharmacological efficacy of Isodon serra (Maxim.) Hara. Clinical studies have revealed that diterpenoids possess multiple features, e.g. antitumour, antitubercular and anti-ischemic activities. Therefore, the identification and detection of diterpenoids may be equally important for understanding the pharmacological basis of diterpenoids and enhancing the product quality control of I. serra. OBJECTIVES: The purpose of this study was to develop a practical analysis approach of rapid characterisation using ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) for the structure characterisation of the ent-kaurane diterpenoids from I. serra. METHODOLOGY: The analytical strategy was as follows: first, ent-kaurane diterpenoids were detected by a novel on-line data acquisition approach, i.e. sequential window acquisition of all theoretical fragment-ion spectra (SWATH). Second, the MS of eight ent-kaurane diterpenoids was explored, and their mass spectrum cleavage pathways were summarised and determined. Finally, the methanol extract of I. serra was studied using SWATH and identified by extracted ion chromatography (XIC). RESULTS: Compared to the traditional information-dependent acquisition (IDA) method, SWATH significantly improved the hit rate of ent-kaurane diterpenoids. With support from UHPLC separation and specific detection by tandem mass spectrometry (MS/MS), 48 ent-kaurane diterpenoids were successfully characterised and classified as ent-kaurane diterpenoids from a complex matrix. CONCLUSIONS: These combined qualitative methods were used to provide a potential approach for the characterisation of traditional Chinese medicine (TCM) and its preparations. Meanwhile, the SWATH provided a novel and reliable method for the structural characterisation of ent-kaurane diterpenoids from other complicated TCMs.

Evaluation of dynamic changes in the bioactive components in Citri Reticulatae Pericarpium (Citrus reticulata 'Chachi') under different harvesting and drying conditions.

BACKGROUND: The Citrus reticulata 'Chachi' pericarp (CRCP) is one cultivar of Citri Reticulatae Pericarpium (Chenpi), which is widely applied in medicine and food. To determine the potential value of CRCP harvested at different stages and subjected to different drying processes, the dynamic changes in the bioactive components were profiled and evaluated in this study. RESULTS: The contents of all non-volatile components, i.e. synephrine, limonin, phenolic acids and flavonoids, decreased with delayed harvest time. The volatiles thujene, α-pinene, ß-pinene, d-citronellol, d-citronellal, decanal, linalool, geraniol, l-cis-carveol, terpinen-4-ol, α-terpineol, carvacrol, perillaldehyde, methyl 2-(methylamino)benzoate and d-limonene were considered the characteristic components for distinguishing CRCP harvested at different stages. Phenolic acids, synephrine and limonin were stable at different drying temperatures; however, high-temperature drying at 60 °C induced a significant transformation in the flavonoids (especially polymethoxyflavones) and volatile substances in CRCP. CONCLUSIONS: The results suggested that most of the bioactive components declined with the growth of Citrus reticulata 'Chachi'. And it is believed that the fresh peel should be naturally sun-dried or dried at low temperature (30 or 45 °C) rather than at high temperature (60 °C) to prevent excessive loss of nutrients. © 2020 Society of Chemical Industry.

Identification of lipophilic components in Citri Reticulatae Pericarpium cultivars by supercritical CO2 fluid extraction with ultra-high-performance liquid chromatography-Q Exactive Orbitrap tandem mass spectrometry.

To systematically identify the lipophilic constituents of Citri Reticulatae Pericarpium from different cultivars, supercritical CO2 fluid extraction and ultra-high-performance liquid chromatography-Q Exactive Orbitrap tandem mass spectrometry were integrated for the component analysis of 18 batches of Citri Reticulatae Pericarpium from 12 cultivars for the first time. A total of 57 components from the supercritical CO2 fluid extracts were demonstrably or tentatively identified by the obtained parent peaks, fragment peaks, and retention times. In total, two flavonoids, six organic acids, nine coumarins, three aldehydes, seven esters, three terpenes, one limonoid, and five other compounds were detected for the first time; notably, coumarin components have not yet been reported in Citri Reticulatae Pericarpium. Furthermore, the extract constituents differed between cultivars. In particular, organic acids were more abundant in Citrus reticulata "Chachi" than in other cultivars, and pterostilbene was exclusively found in Citrus reticulata "Yichangju". The results showed that a greater variety of compounds in Citri Reticulatae Pericarpium could be extracted by supercritical CO2 fluid extraction and detected by ultra-high-performance liquid chromatography-Q Exactive Orbitrap tandem mass spectrometry. This study provides a more scientific basis for further analysis of the pharmacological activity and quality of Citri Reticulatae Pericarpium components from different cultivars.

Construction and Chemical Profile on "Activity Fingerprint" of Citri Reticulatae Pericarpium from Different Cultivars Based on HPLC-UV, LC/MS-IT-TOF, and Principal Component Analysis.

Citri Reticulatae Pericarpium (CRP), known as Chenpi (CP) in Chinese, is a medicinal food for health and fitness. In order to find out the characteristic activity chemicals distinguishing various cultivars of CRP and provide a reference for effective development of citrus resources, an "activity fingerprint" of CRP from 21 different cultivars was established based on the evaluation of antitussive and expectorant activities. There were 18 common peaks in the HPLC fingerprint, of which 3 flavonoid glycosides and 14 polymethoxyflavonoids (PMFs) were identified by LC/MS-IT-TOF. Furthermore, five characteristic chemicals were determined and similarity calculation with principal component analysis (PCA) was combined together to compare the similarity and difference among various cultivars. The results showed that some other cultivars were also similar to C. reticulata "Chachi" except for C. reticulata "Tangerina" and C. reticulata "Dahongpao" recorded in Chinese Pharmacopoeia. Most importantly, the peels of C. reticulata "Shiyueju," C. reticulata "Ponkan," C. reticulata "Tribute," and C. reticulata "Bayueju," traditionally rarely used for medicinal food, were highly similar to that of C. reticulata "Chachi" and rich in bioactive flavonoids, which can be considered the effective medicinal resources of CRP.

Synthesis and photocatalytic CO2 reduction performance of aminated coal-based carbon nanoparticles.

To obtain high-efficiency, low-cost, environmentally friendly carbon-based photocatalytic material, we synthesized coal-based carbon dots with sp2 carbon structure and multilayer graphene lattice structure by the hydrogen peroxide (H2O2) oxidation method to strip nano-scale crystalline carbon in the coal structure and link with oxygen-containing groups such as the hydroxyl group. N, S co-doped aminated coal-based carbon nanoparticles (NH2-CNPs) were then obtained by thionyl chloride chlorination and ethylenediamine passivation. The physical properties and chemical structure of the synthesized NH2-CNPs were studied and the photocatalytic CO2 reduction performance was tested. The results show that NH2-CNPs are vesicle-type spherical particles with particle size of 42.16 ± 7.5 nm and have a mesoporous structure that is capable of adsorbing CO2. A defect structure was formed on the surface of the NH2-CNPs due to the doping of N and S elements, thereby significantly improving the ability to photogenerate electrons under visible light along with the ability to efficiently separate the photo-generated carriers. The photocatalytic reduction products of CO2 over NH2-CNPs were CH3OH, CO, C2H5OH, H2 and CH4. After 10 hours of reaction, the total amount of products was 807.56 µmol g-1 cat, the amount of CH3OH was 618.7 µmol g-1 cat, and the calculated selectivity for conversion of CO2 to CH3OH was up to 76.6%.

Comparison of acoustic radiation force impulse imaging and transient elastography for non-invasive assessment of liver fibrosis in patients with chronic hepatitis B.

The aims of this study were to compare the performance of acoustic radiation force impulse (ARFI) elastography and transient elastography (TE) in the assessment of liver fibrosis in patients with chronic hepatitis B and to evaluate the impact of elevated alanine transaminase levels on liver stiffness assessment using ARFI elastography. One hundred eighty consecutive patients with chronic hepatitis B were enrolled in this study and evaluated with respect to histologic and biochemical features. All patients underwent ARFI elastography and TE. ARFI elastography and TE correlated significantly with histologically assessed fibrosis (r = 0.599, p < 0.001, for ARFI elastography; r = 0.628, p < 0.001, for TE) and necro-inflammatory activity (r = 0.591, p < 0.001, for ARFI elastography; r = 0.616, p < 0.001, for TE). Areas under the receiver operating characteristic curves for ARFI elastography and TE were 0.764 and 0.813 (p = 0.302, ≥stage 2), 0.852 and 0.852 (p = 1.000, ≥stage 3) and 0.825 and 0.799 (p = 0.655, S = 4), respectively. The optimum cutoff values for ARFI elastography were 1.63 m/s for stage ≥2, 1.74 m/s for stage ≥3 and 2.00 m/s for stage 4 in patients for whom alanine transaminase levels were evaluated. The cutoff values decreased to 1.24 m/s for ≥ stage 2, 1.32 m/s for ≥ stage 3 and 1.41 m/s for stage 4 in patients with normal alanine transaminase levels. ARFI elastography may be a reliable method for diagnosing the stage of liver fibrosis with diagnostic performance similar to that of TE in patients with chronic hepatitis B. In addition, liver stiffness values obtained with ARFI elastography, like those obtained with TE, may be influenced by alanine transaminase levels.

Microwave ablation of the liver abutting the stomach: insulating effect of a chitosan-based thermosensitive hydrogel.

PURPOSE: Collateral thermal injury can occur as a serious complication of microwave ablation. This study aimed to assess the insulating effect of a thermosensitive, chitosan-based hydrogel during the percutaneous microwave ablation of liver tissue abutting the stomach. MATERIALS AND METHODS: Thermometry needles (R1-R4) were positioned at 5-mm intervals from a thermal source to measure in vitro the temperature differences due to the hydrogel (R1 closest to the thermal source). Subsequently, two groups of eight rabbits each were injected with 10 mL of hydrogel solution or 410 ± 95 mL of 5% saline between the liver and stomach wall. A control group of eight rabbits received no ablation protection measures. The livers were ablated with 40 W for 300 s in 24 ablation zones. The severity of thermal injury to the stomach wall was assessed histologically. RESULTS: In vitro, the mean maximum temperature difference between the R1 and R2 thermometry needles was 31.3° ± 0.1 °C. When R1 was over 60 °C, the mean temperatures at R2, R3, and R4 were 29.8° ± 0.1 °C, 18.6 ± 0.3 °C, and 18.1° ± 0.1 °C, respectively. After ablation for 300 s, the maximum temperature at R2 was 48.7° ± 0.2 °C. None of the rabbits injected with gel showed any injury after ablation; however, the other two groups showed varying degrees of thermal injury. CONCLUSION: The in situ gel protected the adjacent stomach wall from injury during percutaneous microwave ablation of liver tissue. Although the present technique appears promising, further studies are necessary prior to clinical application.