Objectives: Hypoparathyroidism (HP) is a rare endocrine disorder caused by parathyroid hormone (PTH) defi ciency. The PTH is a candidate gene for familial isolated hypoparathyroidism (FIH). This study aimed to investigate the pathogenicity of two novel rare variants (RVs) ofPTH through in vitro functional study. Methods: Targeted next-generation sequencing was used to identify candidate gene mutations. Clinical data were retrospectively collected. Wild-type (WT) PTH was used as a template for site-directed mutagenesis to create mutant eukaryotic expression plasmids, which were transfected into cells. Treated with or without 4-phenylbu tyric acid (4-PBA), the levels of intact PTH (iPTH) and PTH (1-84) were measured by chemiluminescence, and protein expression was assessed using Western blotting. Results: Two patients carrying PTH mutations (c.154G > A: p.Val52Ile, c.270G > T: p.Leu90Phe) were identified.Patient 1, a 45-year-old male, presented with carpal and pedal numbness, muscle cramps, and low serum calcium (1.29 mmol/L). Patient 2, a 12-year-old female, had muscle twitches, convulsions, low calcium (1.50 mmol/L), and iPTH of 4 pg/mL. The iPTH or PTH (1-84) levels in the medium transfected with mutant Val52Ile and Leu90Phe PTH decreased by 31%–38%, and 51%–96% compared to WT (allP < 0.05), which were not rescued by 4-PBA. No significant changes in intracellular PTH expression were observed. Conclusions In this study, two novel RVs of PTH(Val52Ile and Leu90Phe) were identified that may impair hormone synthesis and secretion. Our study has broadened the mutation spectrum of the PTH and shed light on potential mechanisms underlying FIH.

Objectives: Hypoparathyroidism (HP) is a rare endocrine disorder caused by parathyroid hormone (PTH) defi ciency. The PTH is a candidate gene for familial isolated hypoparathyroidism (FIH). This study aimed to investigate the pathogenicity of two novel rare variants (RVs) ofPTH through in vitro functional study. Methods: Targeted next-generation sequencing was used to identify candidate gene mutations. Clinical data were retrospectively collected. Wild-type (WT) PTH was used as a template for site-directed mutagenesis to create mutant eukaryotic expression plasmids, which were transfected into cells. Treated with or without 4-phenylbu tyric acid (4-PBA), the levels of intact PTH (iPTH) and PTH (1-84) were measured by chemiluminescence, and protein expression was assessed using Western blotting. Results: Two patients carrying PTH mutations (c.154G > A: p.Val52Ile, c.270G > T: p.Leu90Phe) were identified.Patient 1, a 45-year-old male, presented with carpal and pedal numbness, muscle cramps, and low serum calcium (1.29 mmol/L). Patient 2, a 12-year-old female, had muscle twitches, convulsions, low calcium (1.50 mmol/L), and iPTH of 4 pg/mL. The iPTH or PTH (1-84) levels in the medium transfected with mutant Val52Ile and Leu90Phe PTH decreased by 31%–38%, and 51%–96% compared to WT (allP < 0.05), which were not rescued by 4-PBA. No significant changes in intracellular PTH expression were observed. Conclusions In this study, two novel RVs of PTH(Val52Ile and Leu90Phe) were identified that may impair hormone synthesis and secretion. Our study has broadened the mutation spectrum of the PTH and shed light on potential mechanisms underlying FIH.

Objectives: Hypoparathyroidism (HP) is a rare endocrine disorder caused by parathyroid hormone (PTH) defi ciency. The PTH is a candidate gene for familial isolated hypoparathyroidism (FIH). This study aimed to investigate the pathogenicity of two novel rare variants (RVs) ofPTH through in vitro functional study. Methods: Targeted next-generation sequencing was used to identify candidate gene mutations. Clinical data were retrospectively collected. Wild-type (WT) PTH was used as a template for site-directed mutagenesis to create mutant eukaryotic expression plasmids, which were transfected into cells. Treated with or without 4-phenylbu tyric acid (4-PBA), the levels of intact PTH (iPTH) and PTH (1-84) were measured by chemiluminescence, and protein expression was assessed using Western blotting. Results: Two patients carrying PTH mutations (c.154G > A: p.Val52Ile, c.270G > T: p.Leu90Phe) were identified.Patient 1, a 45-year-old male, presented with carpal and pedal numbness, muscle cramps, and low serum calcium (1.29 mmol/L). Patient 2, a 12-year-old female, had muscle twitches, convulsions, low calcium (1.50 mmol/L), and iPTH of 4 pg/mL. The iPTH or PTH (1-84) levels in the medium transfected with mutant Val52Ile and Leu90Phe PTH decreased by 31%–38%, and 51%–96% compared to WT (allP < 0.05), which were not rescued by 4-PBA. No significant changes in intracellular PTH expression were observed. Conclusions In this study, two novel RVs of PTH(Val52Ile and Leu90Phe) were identified that may impair hormone synthesis and secretion. Our study has broadened the mutation spectrum of the PTH and shed light on potential mechanisms underlying FIH.

Objectives: Hypoparathyroidism (HP) is a rare endocrine disorder caused by parathyroid hormone (PTH) defi ciency. The PTH is a candidate gene for familial isolated hypoparathyroidism (FIH). This study aimed to investigate the pathogenicity of two novel rare variants (RVs) ofPTH through in vitro functional study. Methods: Targeted next-generation sequencing was used to identify candidate gene mutations. Clinical data were retrospectively collected. Wild-type (WT) PTH was used as a template for site-directed mutagenesis to create mutant eukaryotic expression plasmids, which were transfected into cells. Treated with or without 4-phenylbu tyric acid (4-PBA), the levels of intact PTH (iPTH) and PTH (1-84) were measured by chemiluminescence, and protein expression was assessed using Western blotting. Results: Two patients carrying PTH mutations (c.154G > A: p.Val52Ile, c.270G > T: p.Leu90Phe) were identified.Patient 1, a 45-year-old male, presented with carpal and pedal numbness, muscle cramps, and low serum calcium (1.29 mmol/L). Patient 2, a 12-year-old female, had muscle twitches, convulsions, low calcium (1.50 mmol/L), and iPTH of 4 pg/mL. The iPTH or PTH (1-84) levels in the medium transfected with mutant Val52Ile and Leu90Phe PTH decreased by 31%–38%, and 51%–96% compared to WT (allP < 0.05), which were not rescued by 4-PBA. No significant changes in intracellular PTH expression were observed. Conclusions In this study, two novel RVs of PTH(Val52Ile and Leu90Phe) were identified that may impair hormone synthesis and secretion. Our study has broadened the mutation spectrum of the PTH and shed light on potential mechanisms underlying FIH.

Objectives: Hypoparathyroidism (HP) is a rare endocrine disorder caused by parathyroid hormone (PTH) defi ciency. The PTH is a candidate gene for familial isolated hypoparathyroidism (FIH). This study aimed to investigate the pathogenicity of two novel rare variants (RVs) ofPTH through in vitro functional study. Methods: Targeted next-generation sequencing was used to identify candidate gene mutations. Clinical data were retrospectively collected. Wild-type (WT) PTH was used as a template for site-directed mutagenesis to create mutant eukaryotic expression plasmids, which were transfected into cells. Treated with or without 4-phenylbu tyric acid (4-PBA), the levels of intact PTH (iPTH) and PTH (1-84) were measured by chemiluminescence, and protein expression was assessed using Western blotting. Results: Two patients carrying PTH mutations (c.154G > A: p.Val52Ile, c.270G > T: p.Leu90Phe) were identified.Patient 1, a 45-year-old male, presented with carpal and pedal numbness, muscle cramps, and low serum calcium (1.29 mmol/L). Patient 2, a 12-year-old female, had muscle twitches, convulsions, low calcium (1.50 mmol/L), and iPTH of 4 pg/mL. The iPTH or PTH (1-84) levels in the medium transfected with mutant Val52Ile and Leu90Phe PTH decreased by 31%–38%, and 51%–96% compared to WT (allP < 0.05), which were not rescued by 4-PBA. No significant changes in intracellular PTH expression were observed. Conclusions In this study, two novel RVs of PTH(Val52Ile and Leu90Phe) were identified that may impair hormone synthesis and secretion. Our study has broadened the mutation spectrum of the PTH and shed light on potential mechanisms underlying FIH.

With the continuous exploration of microemulsions as solvents for traditional Chinese medicine extraction, polyoxyethy-lene(35) castor oil(CrEL), a commonly used surfactant, is being utilized by researchers. However, the problem of detecting residues of this surfactant in microemulsion extracts has greatly hampered the further development of microemulsion solvents. Based on the chemical structures of the components in CrEL and the content determination method of castor oil in the 2020 edition of the Chinese Pharmacopoeia(Vol. Ⅳ), this study employed gas chromatography(GC) and single-factor experiments to optimize the preparation method of methyl ricinoleate from CrEL. The conversion coefficient between the two was validated, and the optimal sample preparation method was used to process microemulsion extracts of Zexie Decoction from three batches. The content of methyl ricinoleate generated was determined, and the content of CrEL in the microemulsion extracts of Zexie Decoction was calculated using the above conversion coefficient. The results showed that the optimal preparation method for CrEL was determined. Specifically, 10 mL of 1 mol·L~(-1) KOH-methanol solution was heated at 60 ℃ for 15 min in a water bath. Subsequently, 10 mL of boron trifluoride etherate-methanol(1∶3) solution was heated at 60 ℃ for 15 min in a water bath, followed by extraction with n-hexane twice. CrEL could stably produce 20.84% methyl ricinoleate. According to this conversion coefficient, the average mass concentration of CrEL in the three batches of Zexie Decoction microemulsion extracts was 11.94 mg·mL~(-1), which was not significantly different from the CrEL mass concentration of 11.57 mg·mL~(-1) during microemulsion formulation, indicating that the established content determination method of this study was highly accurate, sensitive, and repeatable. It can be used for subsequent research on microemulsion extracts of Zexie Decoction and provide a reference for quality control of other drug formulations containing CrEL.
Polyethylene Glycols/chemistry* ; Castor Oil ; Methanol ; Surface-Active Agents/chemistry* ; Solvents ; Water/chemistry* ; Emulsions/chemistry*

ObjectiveTo optimize the extraction and purification process of Gardeniae Fructus for industrial production， and to obtain the total iridoid and total crocin extracts. MethodOrthogonal test was used to optimize the water extraction process by taking contents of geniposide， genipin gentiobioside， gardenoside， crocin-1 and crocin-2 as indicators and the decocting time， decocting times and water amount as factors. The purification process was optimized by single factor test， and four different types of macroporous adsorption resins were screened. The process conditions such as resin type， maximum loading amount， water washing amount， ethanol concentration， ethanol dosage， and flow rate of sample loading were mainly investigated. In addition， the drying methods （vacuum drying and spray drying） of the extract were investigated， and a pilot scale-up verification test was carried out. ResultThe optimal water extraction process of Gardeniae Fructus was to add 15， 10 times the amount of water for decocting twice， 1 h each time. The optimal purification process was as follows：the water extract through SP825L macroporous resin column， the amount of crude drug-the amount of resin （1∶1.5）， the sample loading flow rate of 3 BV h^-1， adding 2 BV of water to remove impurities， adding 4 BV of 30% ethanol to obtain the iridoid part， then adding 3 BV of 70% ethanol to obtain the crocin part， collecting the ethanol lotion， and drying at 70 ℃. Under these conditions， the extraction amount of total iridoids was 590.75 mg·g^-1 with the transfer rate of 70.48%， and the yield of dry extract was 8.89%. The extraction amount of total crocins was 83.37 mg·g^-1 with the transfer rate of 22.20%， and the dry extract yield was 2.60%. ConclusionThe optimized extraction and purification process is stable and feasible with high extraction rate of active components， which is suitable for the industrial extraction and purification of active parts of Gardeniae Fructus.

Objective:To analyze the clinical characteristics and molecular mechanisms of 5 cases of hypoparathyroidism caused by GATA3 gene mutation.Methods:A total of 5 childhood-onset hypoparathyroidism patients with GATA3 mutation were identified from 198 hypoparathyroidism (aged ≤18 years) from 1975 to 2021 in Peking Union Medical College Hospital. Clinical data and biochemical indices of the 5 patients were collected and analyzed retrospectively. Genetic screening was conducted by targeted next-generation sequencing (T-NGS), and bioinformatics analysis was performed to analyze the underline mechanisms.Results:The medium onset age of hypoparathyroidism of the 5 patients was 0.5 (0.1, 1.3) years old, and the time duration from onset to confirmed diagnosis of hypoparathyroidism and hypoparathyroidism- deafness-renal dysplasia syndrome was (7.0±5.2) years and (15.0±5.4) years, respectively. The clinical manifestations included carpopedal spasm accompanied by seizures (5 cases), basal ganglia calcification (5 cases), cataract (1 case), deafness (4 cases), and renal malformations or absence (2 cases). The blood calcium and blood parathormone(PTH) before treatment was (1.65±0.31) mmol/L and (4.64±2.63) ng/L, respectively. The 5 patients carried different heterozygous mutations in GATA3 gene, which caused nonsense mutations, frameshift mutations and splice site mutations, respectively. All the GATA3 gene mutations of the 5 patients are classified as pathogenic or likely pathogenic by the Clin Var database and American College of Medical Genetics and Genomics(ACMG).Conclusions:Attention should be paid to genetic diseases in patients with childhood-onset hypoparathyroidism. The possibility of hypoparathyroidism-deafness-renal dysplasia syndrome should be considered in hypoparathyroidism patients with hearing loss or renal dysplasia. GATA3 gene screening is highly recommended for the confirmation of the diagnosis.

The small size， moderate drug loading， and targeting properties of nano-preparations make them can be excellent delivery tools for drugs， genes or proteins crossing the cell or blood-brain barrier （BBB）. Currently， facilitating drug crossing BBB with innovative nano-drug delivery systems is considered as a strategic approach for the prevention， diagnosis and treatment of central nervous system （CNS） diseases. However， with the deepening of the research， the adverse reactions and toxicity of nanocarriers have gradually attracted the attention of researchers. Based on this， this paper summarized the situation of BBB-penetrating targeted nano-preparations at home and abroad in recent years from the perspective of classification of types and properties of nanocarriers， and analyzed the advantages and disadvantages of each carrier. The results showed that nano-preparations with active ingredients of traditional Chinese medicine （TCM） as carriers have become a promising way of cancer treatment， but the complexity and diversity of TCM components limited its application to a certain extent. Further studies should be strengthened to lay a foundation for the application and development of TCM nano-preparations in the field of CNS diseases.

Zexietang is derived from Jingui Yaolue （《金匮要略》）， which is composed of Alismatis Rhizoma and Atractylodis Macrocephalae Rhizoma， and has the effect of inducing diuresis and invigorating the spleen to produce water. Compared with western medicine in the treatment of related diseases， Zexietang can not only improve the curative effect， but also reduce the occurrence of adverse reactions， so as to achieve long-term stable administration. The authors sorted out and analyzed the chemical composition， pharmacological effect and clinical application of Zexietang in recent years. It was found that the main active components of Zexietang were alismol A and B， 23-acetyl-alismol B and C， atractylenolides （atractylenolide Ⅰ， Ⅱ， Ⅲ） and polysaccharides. Pharmacological experiments showed that they had diuretic， hypolipidemic， anti-inflammatory and others. And it can be used in the treatment of hypertension， hyperlipidemia， vertigo， cerebral vascular insufficiency and other diseases combined with other Chinese materia medica， and the curative effect is obvious. By summarizing the research status of Zexietang in recent years， its active components and pharmacological mechanism can be further clarified， which provides the basis for the clinical application of Zexietang and guides the direction of its further research.