OBJECTIVETo investigate the expressions of different forms of ROS fusions in Chinese patients with cholangiocarcinoma (CCA).

METHODSRT-PCR was employed to examine formalin-fixed and paraffin-embedded CCA samples from stage I-IV patients for detection of ROS fusions involving Fused in Glioblastoma (FIG), solute carrier protein (SLC34A2) and major histocompatibility complex class II invariant chain (CD74). Serpin peptidase inhibitor clade A member 1 (SERPINA1) was detected as the reference gene.

RESULTSIn all the 56 CCA samples, 80.4% (45/56) were positive for SERPINA1 expression as evaluable samples. Of these evaluable samples, none expressed the ROS fusions.

CONCLUSIONROS fusions are not common in Chinese CCA patients.

Antigens, Differentiation, B-Lymphocyte ; genetics ; metabolism ; Bile Duct Neoplasms ; metabolism ; pathology ; Carrier Proteins ; genetics ; metabolism ; Cholangiocarcinoma ; metabolism ; pathology ; Female ; Gene Expression ; Histocompatibility Antigens Class II ; genetics ; metabolism ; Humans ; Male ; Membrane Proteins ; genetics ; metabolism ; Middle Aged ; Oncogene Proteins, Fusion ; genetics ; metabolism ; Paraffin Embedding ; Protein-Tyrosine Kinases ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; Sodium-Phosphate Cotransporter Proteins, Type IIb ; genetics ; metabolism

Antigens, Differentiation, B-Lymphocyte ; genetics ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; genetics ; metabolism ; Gene Fusion ; Gene Rearrangement ; Histocompatibility Antigens Class II ; genetics ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Lung Neoplasms ; drug therapy ; genetics ; metabolism ; Protein Kinase Inhibitors ; therapeutic use ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; genetics ; metabolism ; Proto-Oncogene Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Pyrazoles ; therapeutic use ; Pyridines ; therapeutic use ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Sodium-Phosphate Cotransporter Proteins, Type IIb ; genetics

PiT2 is an inorganic phosphate (Pi) transporter whose mutations are linked to primary familial brain calcification (PFBC). PiT2 mainly consists of two ProDom (PD) domains and a large intracellular loop region (loop7). The PD domains are crucial for the Pi transport, but the role of PiT2-loop7 remains unclear. In PFBC patients, mutations in PiT2-loop7 are mainly nonsense or frameshift mutations that probably cause PFBC due to C-PD1131 deletion. To date, six missense mutations have been identified in PiT2-loop7; however, the mechanisms by which these mutations cause PFBC are poorly understood. Here, we found that the p.T390A and p.S434W mutations in PiT2-loop7 decreased the Pi transport activity and cell surface levels of PiT2. Furthermore, we showed that these two mutations attenuated its membrane localization by affecting adenosine monophosphate-activated protein kinase (AMPK)- or protein kinase B (AKT)-mediated PiT2 phosphorylation. In contrast, the p.S121C and p.S601W mutations in the PD domains did not affect PiT2 phosphorylation but rather impaired its substrate-binding abilities. These results suggested that missense mutations in PiT2-loop7 can cause Pi dyshomeostasis by affecting the phosphorylation-regulated cell-surface localization of PiT2. This study helps understand the pathogenesis of PFBC caused by PiT2-loop7 missense mutations and indicates that increasing the phosphorylation levels of PiT2-loop7 could be a promising strategy for developing PFBC therapies.
Humans ; Cell Membrane ; Mutation, Missense ; Phosphates/metabolism* ; Sodium-Phosphate Cotransporter Proteins, Type III/genetics*

OBJECTIVES: To observe the electrophysiological changes of astrocytes in the process of hyperoxia induced apoptosis and analyze the relationship between electrophysiological characteristics and morphological changes. METHODS: Astrocytes were exposed to 90% hyperoxia for 12-72 h. The electrophysiological characteristics of astrocytes in each group were detected by patch clamp technique, and the morphological characteristics of astrocytes were observed at the same time. Then the same batch of astrocytes were collected, and the expression levels of caspase-1, caspase-3, gasdermin D (GSDMD) and gasdermin E (GSDME) were detected by Western blotting. RESULTS: From 12 h to 72 h after hyperoxia exposure, the inward current was significantly lower than that of the control group (<0.05), while the outward current was significantly decreased at 12 h and increased at 48 h (<0.05). There was no significant difference between 24 h or 72 h after hyperoxia exposure and the control group (>0.05). At each time point, the morphology of cells changed correspondingly. Western blotting showed that the expression of caspase-1 was increased significantly at 24 h and decreased significantly at 72 h after hyperoxia exposure (<0.05); the expression of GSDMD was increased at 12 h and decreased gradually from 24 h to 72 h after hyperoxia exposure (<0.05); the expression of caspase-3 did not change significantly at 12 h and 24 h after hyperoxia exposure (>0.05), but began to decrease at 48 h (<0.05); GSDME increased gradually at 24 h after hyperoxia exposure (<0.05). CONCLUSIONS Under hyperoxia exposure, the ion channels of astrocytes are damaged, which can maintain the dysfunction of ion homeostasis, activate GSDME, induce the damaged cells to break away from the apoptotic pathway, and mediate the pyroptosis.
Apoptosis ; Astrocytes ; Caspase 1 ; Humans ; Hyperoxia ; Intracellular Signaling Peptides and Proteins ; Neoplasm Proteins ; Phosphate-Binding Proteins ; Pyroptosis

OBJECTIVETo describe clinical and genetic feature in a Chinese family with familial idiopathic basal ganglia calcification 3 (IBGC-3) caused by a novel mutation in the SLC20A2 gene.

METHODSClinical data was collected from a family with familial IBGC-3. All of the family members underwent cerebral CT. Potential mutation of the SLC20A2 gene were screened in the proband, 5 symptomatic patients, 5 asymptomatic family members, and 100 healthy Chinese controls. Exon 8 of the SLC20A2 gene was cloned into plasmid and sequenced.

RESULTSThere were 6 symptomatic patients (3 males and 3 females) in an autosomal dominant pedigree. The patients manifested as juvenile-onset paroxysmal kinesigenic dyskinesia, in addition to pyramidal signs in proband. 5 patients alive had calcification in bilateral basal ganglia and subcortical areas. One asymptomatic member also had calcification in the brain; and 2 cases of asymptomatic young members had bilateral globus pallidus calcification. A novel c.1086delC mutation in SLC20A2 gene has been identified in proband and 7 family members with intracranial calcification. The deletion mutation was not found in 2 family members without intracranial calcification and healthy controls members. There is no clear relationship between clinical symptoms and the severity of calcification in cerebral CT.

CONCLUSIONFamilial idiopathic basal ganglia calcification caused by the SLC20A2 gene mutation can manifest as juvenile onset paroxysmal kinesigenic dyskinesia. Further study should be done to validate the unrelated relationships between the severity of calcification in IBGC 3 cranial CT and clinical symptoms.

Adolescent ; Adult ; Basal Ganglia Diseases ; genetics ; Calcinosis ; genetics ; Child ; Female ; Humans ; Male ; Mutation ; Neurodegenerative Diseases ; genetics ; Sodium-Phosphate Cotransporter Proteins, Type III ; genetics ; Tomography, X-Ray Computed

Basal Ganglia ; pathology ; Calcinosis ; complications ; Female ; Humans ; Middle Aged ; Mutation ; Sodium-Phosphate Cotransporter Proteins, Type III ; genetics ; Supranuclear Palsy, Progressive ; etiology

OBJECTIVE: To explore whether extracellular histones aggravate acute respiratory distress syndrome (ARDS) by inducing peripheral blood mononuclear cell (PBMC) pyroptosis. METHODS: Twenty patients with ARDS admitted to Shanghai Pulmonary Hospital, Tongji University School of Medicine from April to September in 2019 were enrolled, and 20 healthy volunteers were enrolled as controls. In vivo experiment: peripheral blood samples of patients with ARDS within 24 hours after diagnosis and healthy volunteers were collected, and the levels of plasma extracellular histone, interleukins (IL-1β and IL-18) and lactic dehydrogenase (LDH) were determined by enzyme-linked immunosorbent assay (ELISA). PBMC were harvested, the expression levels of the pyroptosis associated N terminal-gasdermin-D (GSDMD-N) protein were determined by Western Blot. In vitro experiment: PBMC isolated from healthy volunteers were divided into four groups. Blank control group without any treatment; lipopolysaccharide (LPS) group was treated with 1 mg/L LPS for 4 hours; LPS+histones group was treated with 100 mg/L exogenous histones for 24 hours after LPS treatment; LPS+histone+heparin group was treated with 200 U heparin for 24 hours after LPS and exogenous histones treatment. The GSDMD-N protein expression was determined by Western Blot, and the levels of IL-1β, IL-18 and LDH in cell supernatant were determined by ELISA. Spearman test was used to test the correlation among the parameters. RESULTS: In vivo experiment results: compared with healthy control group, the GSDMD-N protein expression in PBMC of patients with ARDS was significantly increased [GSDMD-N/GAPDH: 0.136 (0.062, 0.246) vs. 0.026 (0.018, 0.036), P < 0.01], as well as the plasma levels of IL-1β, IL-18, LDH and extracellular histones [IL-1β (ng/L): 120.0 (94.2, 213.0) vs. 88.5 (82.3, 105.3), IL-18 (ng/L): 164.5 (70.8, 236.3) vs. 60.5 (52.0, 89.0), LDH (U/L): 30.9 (24.7, 39.5) vs. 19.8 (17.2, 21.5), extracellular histones (mg/L): 73.0 (42.8, 112.9) vs. 12.2 (9.6, 16.9), all P < 0.01], indicating that the PBMC of ARDS patients had significant pyroptosis and release of a large number of inflammatory factors. The oxygenation index (PaO₂/FiO₂) of ARDS patients was 135.5 (94.5, 196.0) mmHg (1 mmHg = 0.133 kPa). Correlation analysis showed that the expression of GSDMD-N protein in patients with ARDS was negatively correlated with PaO₂/FiO₂ (r = -0.935, P < 0.01) and positively correlated with IL-1β, IL-18, LDH and extracellular histones (r value was 0.844, 0.843, 0.887, 0.899, respectively, all P < 0.01). In vitro experiment results: compared with blank control group, the expression of GSDMD-N protein in PBMC and the levels of inflammatory mediators in the supernatant of the LPS group were significantly increased [GSDMD-N/GAPDH: 0.035±0.006 vs. 0.028±0.006, IL-1β (ng/L): 39.8±5.5 vs. 22.6±4.7, IL-18 (ng/L): 31.2±4.4 vs. 20.0±2.2, LDH (U/L): 51.2±7.3 vs. 36.6±7.6, all P < 0.05], indicating that LPS stimulation could increase PBMC pyroptosis and the release of inflammatory mediators. Compared with LPS group, the expression of GSDMD-N protein and the levels of inflammatory mediators of the LPS+histones group were further increased [GSDMD-N/GAPDH: 0.114±0.009 vs. 0.035±0.006, IL-1β (ng/L): 119.0±18.7 vs. 39.8±5.5, IL-18 (ng/L): 49.2±8.5 vs. 31.2±4.4, LDH (U/L): 127.8±19.8 vs. 51.2±7.3, all P < 0.01], indicating that the stimulation of LPS on PBMC could be significantly amplified by exogenous histone treatment, GSDMD-N protein expression could be up-regulated and inflammatory factor release could be promoted to further induce PBMC pyroptosis. These adverse effects of exogenous histones on PBMC could be abrogated by heparin, the expression of GSDMD-N protein and the levels of inflammatory mediators were significantly lower than those of LPS+histones group [GSDMD-N/GAPDH: 0.063±0.004 vs. 0.114±0.009, IL-1β (ng/L): 46.8±8.6 vs. 119.0±18.7, IL-18 (ng/L): 33.0±5.1 vs. 49.2±8.5, LDH (U/L): 65.4±11.0 vs. 127.8±19.8, all P < 0.05]. CONCLUSIONS Extracellular histones in plasma may aggravate ARDS by mediating PBMC pyroptosis.
China ; Histones/metabolism* ; Humans ; Interleukin-1beta ; Intracellular Signaling Peptides and Proteins ; Leukocytes, Mononuclear ; Phosphate-Binding Proteins ; Pyroptosis ; Respiratory Distress Syndrome

BackgroundIdiopathic basal ganglia calcification (IBGC) is a genetic disorder characterized by bilateral basal ganglia calcification and neural degeneration. In this study, we reported a new SLC2OA2 mutation of IBGC and reviewed relevant literature to explore the association between phenotypes and genotypes in Chinese IBGC patients.

MethodsClinical information of the proband and her relatives were collected comprehensively. Blood samples of both the patient and her father were obtained, and genetic screening related to IBGC was performed using second generation sequencing with their consent. Findings were confirmed by Sanger sequencing. Polyphen-2 was used to predict the potential association between mutations and disease. Then, we retrieved literatures of Chinese IBGC patients and explored the association between phenotype and genotype.

ResultsA novel mutation was identified through genetic testing, and it is suggested to be a damage mutation predicted by Polyphen-2. Through literature review, we found that SLC20A2 mutation is the most common cause for IBGC in China. Its hot spot regions are mainly on the 1 and 8 exons; the second common one is PDGFB where the hot spot covered a length of 220-230 bp localized on the 2 exon; moreover, Chinese IBGC patients featured early-onset, more severe movement disorder and relatively mild cognitive impairment compared with those in other countries.

ConclusionsThere is significant heterogeneity both in phenotype and genotype in Chinese IBGC patients. Further research of pathogenic mechanism of IBGC is required to eventually develop precise treatment for individuals who suffered this disease.

Asian Continental Ancestry Group ; Basal Ganglia Diseases ; genetics ; Calcinosis ; genetics ; Exons ; genetics ; Female ; Genetic Association Studies ; Humans ; Male ; Mutation ; genetics ; Neurodegenerative Diseases ; genetics ; Pedigree ; Phenotype ; Sodium-Phosphate Cotransporter Proteins, Type III ; genetics

BACKGROUNDHyperphosphatemia in renal failure has been identified as a major role in the pathogenesis of hyperparathyroidism that is independent of changes in serum calcium and 1,25(OH)(2)D(3). The aim of this study was to evaluate the expression of parathyroid Pit-1 in hyperphosphatemia-induced secondary hyperparathyroidism (SHPT) of chronic renal failure (CRF) rats.

METHODSWistar rats with CRF induced by 5/6 nephrectomy were ramdomly fed with diet containing 1.2% inorganic phosphate (Pi, high phosphate (HP) group, n = 9) or 0.2% Pi (low phosphate (LP) group, n = 9) for 10 weeks starting from the fourth week after the surgery. Another 7 nephrectomy rats with HP diet were intraperitoneally injected with phosphonoformic acid (PFA, the specific inhibitor of Pit-1, HP + PFA group) 0.15 g/kg every other day for 10 weeks starting from HP diet. Another 6 HP rats injected with the same amount of normal saline as the control of the HP + PFA group (HP + saline group). At the same time, 9 rats with sham surgery received HP diet as the controls. At the 4th week and 14th week, blood was taken for measurement of serum creatinine (SCr), serum calcium (SCa), serum phosphorus (SPi), 1,25(OH)(2)D(3) and intact parathyroid hormone (iPTH). At the 14th week, two parathroid glands (PTGs) of each rat were removed by microsurgery, one gland for immunohistochemistry analysis of proliferating cell nuclear antigen (PCNA), the other one for detection of Pit-1 by Western blotting, and for the measurement of Pit-1 mRNA and PTH mRNA by real-time quantitative polymerase chain reaction.

RESULTSIn nephrectomy rats, high dierary phosphate induced a marked increase in serum phosphate, iPTH, PTH mRNA and PCNA parathyroid cells, accompanying Pit-1 and its mRNA in parathyroid gland increased significantly. However, serum Ca and 1,25(OH)(2)D(3) remained unchanged. PFA decreased Pit-1 and its mRNA levels to reduce intact PTH, PTH mRNA and PCNA-positive parathyroid cells.

CONCLUSIONSExpression of parathyroid Pit-1 in hyperphosphatemia-induced SHPT of CRF rats was upregulated. Pit-1 may mediate the stimulation to parathyroid gland by hyperphosphatemia.

Animals ; Blotting, Western ; Hyperparathyroidism, Secondary ; etiology ; metabolism ; Hyperphosphatemia ; complications ; Immunohistochemistry ; Kidney Failure, Chronic ; complications ; Male ; Polymerase Chain Reaction ; Rats ; Rats, Wistar ; Sodium-Phosphate Cotransporter Proteins, Type III ; genetics ; metabolism

OBJECTIVEThis study aimed to investigate the susceptibility of mice with streptozotocin(STZ)-induced diabetes mellitus (TIDM) to the uptake of pentavalent inorganic arsenic (iAsV) and the possible molecular mechanism.

METHODSTIDM was induced in mice by STZ. TIDM and normal mice were treated with 15.0 mg/kg Na2HAsO4·12H2O by intragastric administration. Then, the concentrations of arsenic in various tissues were measured by atomic fluorescence spectrometry. The gene expression levels of Pit1 and Pit2 were quantified by real-time RT-PCR, and their protein levels were detected by Western blotting in mouse heart, kidney, and liver tissues.

RESULTSThe concentrations of arsenic in STZ-induced TIDM mouse tissues were higher at 2 h after intragastric administration of Na2HAsO4·12H2O. Compared with the levels in normal mice, PIT1 and PIT2, which play a role in the uptake of iAsV, were upregulated in the livers and hearts of TIDM mice. PIT1 but not PIT2 was higher in TIDM mouse kidneys. The upregulation of Pit1 and Pit2 expression could be reversed by insulin treatment.

CONCLUSIONThe increased uptake of iAsV in TIDM mouse tissues may be associated with increased PIT1 and/or PIT2 expression.

Animals ; Arsenic ; pharmacokinetics ; Diabetes Mellitus, Experimental ; metabolism ; Environmental Pollutants ; pharmacokinetics ; Gene Expression Regulation ; physiology ; Male ; Mice ; Mice, Inbred ICR ; Sodium-Phosphate Cotransporter Proteins, Type III ; genetics ; metabolism ; Transcription Factor Pit-1 ; genetics ; metabolism