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.

Abstract: In the present study, the compound XL-12 from our previous work was utilized as a lead compound. Through the optimization of the terminal phenyl ring, 12 target compounds were designed and synthesized. The structures of all target compounds were confirmed by 1H NMR, 13C NMR, and H RMS. In vitro enzyme activity assay showed that most compounds demonstrated significant inhibitory activity toward Bruton’s tyrosine kinase (BTK) and Janus kinase 3 (JAK3). Among them, compound I-3 exhibited moderate cell proliferation inhibitory activity toward Daudi cells and BaF3-JAK3 cells. In the evaluation of anti-inflammatory activity in vitro, compound I-3 could effectively inhibit the production of inflammatory factors IL-6; besides, it exhibited superior anti-inflammatory activity compared to ibrutinib in xylene-induced ear swelling model in mice.

Objective To provide an animal model for studying the effect of TDO2 on the function of macrophages on the occurrence and development of diseases by constructing macrophage-specific tryptophan,2,3-dioxygenase(TDO2)gene knockout mice.Methods TDO2flox/flox Lyz2-iCre+mice were constructed based on Cre/LoxP sys-tem.The genotypes of mice were identified by PCR amplification and agarose gel electrophoresis.Western blot and immunofluorescence were used to verify the effect of TDO2 knockdown in mouse macrophages.The spontaneous le-sions in major tissues and organ were observed by HE stainings.Results The results of genotype identification showed that the mice with only one band at 407 bp or 408 bp for the flox amplification product and one band at 543 bp for the Cre amplification product were TDO2flox/flox Lyz2-iCre+mice.Western blot results showed that TDO2 ex-pression in bone marrow-derived macrophages(BMDMs)of TDO2flox/flox Lyz2-iCre+mice decreased compared with TD02flox/flox mice(P＜0.01).Immunofluorescence results showed that TDO2 expression in peritoneal macrophages and BMDMs of TDO2flox/flox Lyz2-iCre+mice decreased compared with TDO2flox/flox mice.HE staining showed no sig-nificant differences in cell morphology in the liver,brain,kidney and spleen tissues of TDO2flox/flox Lyz2-iCre+mice compared to TDO2flox/f1ox mice.Conclusion TDO2flox/flox Lyz2-iCre+mice is successfully constructed,providing a more precise experimental animal model for subsequent in-depth study of the role and mechanism of TDO2-regulated macrophage activation in disease.

Objective To investigate the effects of tryptophan-2,3-dioxygenase(TDO2)on collagen-induced ar-thritis,the expression of CIA in mice and the specific mechanism by which prostaglandin E2(PGE2)inhibits the expression and activity of TDO2 and thus regulates the function of macrophages.Methods Type Ⅱ collagen in-duced the CIA model in DBA/1J mice.The ankle joint injury of CIA mice was detected by X-ray.The expression of TDO2 in ankle joint and spleen was detected by immunohistochemistry.Changes of TDO2 expression in perito-neal macrophages(PMs)were detected by qPCR and immunofluorescence.TDO2 expression was detected by small interference in RAW264.7 cells,TDO2 inhibitor 680C91,PGE2 stimulation with different concentrations(0.1,1,10 μmol/L)and EP4 receptor agonist CAY10598.qPCR and Western blot were used to detect TDO2 expression.The phagocytosis and polarization of macrophages were detected by flow cytometry.The activity of TDO2 was detec-ted by colorimetry.Results Compared with normal mice,CIA mice had larger soft tissue swelling in ankle,and increased TDO2 expression in synovium,spleen and PMs.In RAW264.7 cells,TDO2 expression was significantly inhibited after small interference,TDO2 inhibitor 680C91,PGE2 stimulation,and EP4 receptor agonist CAY10598,macrophage phagocytosis decreased,and M1/M2 ratio decreased(P＜0.05).Colorimetric results showed that the activity of TDO2 was inhibited after stimulation of PGE2 and EP4 agonist CAY10598 in RAW264.7 cells(P＜0.05).Conclusion The increased expression of TDO2 in macrophages may promote synovial injury in CIA mice,and PGE2 regulates the function of macrophages by inhibiting the expression and activity of TDO2 by ac-tivating EP4 receptor.

Objective To investigate the difference between imaging flow cytometry and immunofluorescence tech-niques for detecting Ahr entry in fibroblast-like synoviocytes.Methods Human-derived fibroblast-like synoviocytes line MH7A was cultured with 15％ DMEM.The nucleation levels of Ahr in MH7A were detected by laser scanning confocal microscopy and quantitative imaging flow cytometry(ImageStreamX Mark Ⅱ)in the blank control group,the Kyn group,and the Kyn+CH223191 group,respectively.The results of the two assay techniques were com-pared using nonparametric tests-correlation of the results.Results Compared with the blank control group,the Kyn and Kyn+CH223191 groups of MH7A cells increased Ahr nucleation ability as measured by immunofluores-cence technique and imaging flow cytometry(P＜0.05),and the three groups of results calculated by the two ex-perimental methods were consistent,with the R2 values of 0.863 8,0.928 7,and 0.901 8,respectively,which were statistically significant differences(P＜0.05).Conclusion Compared with the operation and results of im-munofluorescence experiments,imaging flow cytometry data processing is more complicated.However,the results are notably precise,avoiding the subjectivity of the experimenter and minimizing experimental inaccuracies.

Objective:To analyze the clinical characteristics of invasive klebsiella pneumoniae liver abscess syndrome (IKPLAS), and compare it with common pyogenic liver abscess (CPLA). Methods:The social demography and clinical data of inpatients with pyogenic liver abscess from January 2011 to December 2021 in the Peking University People's Hospital were collected. Based on the presence or absence of invasive infections and the results of bacterial etiology, IKPLAS was diagnosed and compared with CPLA. The general information, symptoms, past medical history, auxiliary examinations and prognosis indicators of the two groups of patients were compared.Results:Total of 172 patients with pyogenic liver abscess were collected, including 25 cases of IKPLAS. Compared with CPLA group, the proportion of fever in IKPLAS group was lower, the proportion of diabetes history was higher, the proportion of monocytes was lower, and procalcitonin and urea nitrogen were higher(all P<0.05), the proportion of multiple abscesses is higher, and the positive rate of blood culture and the cultivation of klebsiella pneumoniae are both higher (all P<0.05).A total of 9 cases (5.2%) of patients developed septic shock, of which 2 cases (1.2%) died. The IKPLAS group had a higher proportion of ICU admissions ( P<0.05),but but the difference of mortality between the two groups was not statistically significant ( P>0.05). The most common sites of invasion infection in the IKPLAS group are the lungs(22/25), brain(9/25), and eyes(9/25). Conclusions:There are differences in clinical characteristics between IKPLAS and CPLA, the most common sites of invasion infection are the lungs, brain, and eyes, but there is no difference in mortality in this study. For PLA with pathogenic Klebsiella pneumoniae, it is necessary to carefully evaluate the presence of invasive lesions and provide targeted local treatment to better improve prognosis.