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@#Objective To construct a single-chain fragment variable(scFv)phage display library against receptor-binding domain(RBD)of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike protein(S)to screen specific scFv and identify the function.Methods m RNA was extracted from spleen cells of mice immunized with RBD protein and reversely transcribed into c DNA,with which as template,genes of the hight chain fragment of variable(VH)and light chain fragment of variable(VL)of scFv were amplified and then assembled into scFv gene fragment through splicing overlap extension PCR(SOE-PCR).The scFv gene fragment was inserted to phage vector to construct scFv phage display library.After four rounds of biopanning,the scFv gene with strong binding ability to RBD was screened and expressed recombinantly,purified and identified for biological activity.Results The constructed scFv phage library showed a titer of 6.0×10(11)pfu/m L.After four rounds of biopanning,four scFv strains with strong binding to RBD were selected,namely scFv11,scFv12,scFv25and scFv28.scFv was mainly expressed in the form of inclusion body with a relative molecular mass of about 27 000,a concentration of 2.4 mg/m L and a purity of about 90%,which bound specifically to mouse monoclonal antibody against His labeled by HRP after purification.All four scFv strains bound specifically to RBD recombinant protein,among which the other 3 scFv strains bound to the S protein of wild type and multiple mutant strains except scFv28.All four strains showed dose-dependent interaction with RBD,with affinity dynamic fitting dissociation constants(K_Ds)8.9,5.92,10.67and 2.36 nmol/L,and steady-state fitting dissociation constants(K_Ds)of 5.3,6.5,8.7 and 5.8 nmol/L,respectively.scFv11,scFv12 and scFv25 simultaneously identified three independent RBD polypeptides,including RBD2(S(11)pfu/m L.After four rounds of biopanning,four scFv strains with strong binding to RBD were selected,namely scFv11,scFv12,scFv25and scFv28.scFv was mainly expressed in the form of inclusion body with a relative molecular mass of about 27 000,a concentration of 2.4 mg/m L and a purity of about 90%,which bound specifically to mouse monoclonal antibody against His labeled by HRP after purification.All four scFv strains bound specifically to RBD recombinant protein,among which the other 3 scFv strains bound to the S protein of wild type and multiple mutant strains except scFv28.All four strains showed dose-dependent interaction with RBD,with affinity dynamic fitting dissociation constants(K_Ds)8.9,5.92,10.67and 2.36 nmol/L,and steady-state fitting dissociation constants(K_Ds)of 5.3,6.5,8.7 and 5.8 nmol/L,respectively.scFv11,scFv12 and scFv25 simultaneously identified three independent RBD polypeptides,including RBD2(S(334～353)),RBD9(S(334～353)),RBD9(S(439～458))and RBD13(S(439～458))and RBD13(S(499～518)).Homologous model of scFv constructed by online server SWISS-MODEL showed a good quality and was used for molecular docking.The interface at which scFv11 interacted with RBD only partially coincided with the interaction interface of human angiotensin converting enzyme 2(ACE2)and RBD,and the interaction interfaces of scFv12 and scFv25 with RBD were quite different from that of ACE2.Conclusion In this study,scFv specifically bound to SARS-Co V-2 RBD was screened and prepared through constructing scFv phage library against SARS-CoV-2 RBD,which provided experimental basis for further development of anti-SARS-CoV-2 drugs and detection reagents.
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Reduced protein S activity is one of the high-risk factors for venous thromboembolism.Hereditary protein S deficiency is an autosomal dominant disorder caused by mutations in the PROS1 gene.We reported a female patient with a mutation of c.292 G>T in exon 3 of the PROS1 gene,which was identified by sequencing.The genealogical analysis revealed that the mutation probably originated from the patient's mother.After searching against the PROS1 gene mutation database and the relevant literature,we confirmed that this mutation was reported for the first time internationally.
Subject(s)
Humans , Female , Protein S/genetics , Protein S Deficiency/genetics , Pedigree , MutationABSTRACT
Objective To observe the effect of specific knockdown of hepatic stellate cells (HSC) ribosomal protein S5 (RPS5) on liver fibrosis in rats. Methods The glial fibrillary acidic protein (GFAP) promoter-driven RPS5 shRNA adenovirus was established, and AdGFa2-shRPS5 and its control AdGFa2 shNC were used to transfect primary rat HSCs and hepatocytes, respectively. RPS5 was determined by Western-blot and Real Time PCR, α-SMA and type I collagen expression; the rat liver fibrosis model was established by dimethyl nitrosamine (DMN) and bile duct ligation (BDL), and intrahepatic HSC was specifically knocked down by tail vein injection of adenovirus of RPS5 levels. The pathological changes of liver tissue sections were analyzed by HE staining; the content of hydroxyproline, sections of Sirius red and Masson staining were used to evaluate collagen deposition; immunohistochemical staining was used to detect the expression of α-SMA and RPS5. Results AdGFa2-shRPS5 specifically knocked down the expression level of RPS5 in HSC and increased the expression of α-SMA and type I collagen in vitro. The in vivo results showed that in two animal models of chronic liver injury, specific knockdown of RPS5 expression in HSCs promoted HSC activation, increased the deposition of extracellular matrix, and promoted liver fibrosis. Conclusion RPS5 is essential for HSC activation and liver fibrosis, which could be a potential target for the treatment of liver fibrosis.
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Extra-hepatic portal vein obstruction (EHPVO) is an important cause of non-cirrhotic portal hypertension, in Third World countries like India. In this disorder, it results in obstruction and cavernomatous transformation of portal vein with or without the involvement of intra-hepatic portal vein, splenic vein, or superior mesenteric vein resulting in portal hypertension and esophagogastric varices. Extensive collateral circulation develops, involving paracholecystic, paracholedochal and pancreaticoduodenal veins which results in formation of ectopic varices, and portal biliopathy. Besides variceal bleeding, patients may have symptoms of portal biliopathy, hypersplenism, and growth retardation. Although the liver may appear normal, functional compromise develops in the long term. Patients with extra-hepatic portal vein obstruction are usually young and belong to India and other Asian countries. The variceal bleeding in EHPVO can be managed by endoscopic obliteration of varices, or by portosystemic shunt surgery. In this case report, we present a case of 15 year old male, with extra-hepatic portal vein obstruction due to combined deficiency of Protein C and Protein S recanalized by short-term low molecular heparin plus oral Rivaroxaban therapy
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Paciente sexo masculino de 53 años de edad sin patologías de base conocidas acude por un cuadro de 72 horas de evolución de petequias pruriginosas que inician en región interna de muslos, que progresan a ambas piernas y pies de forma simétrica, luego aumentan en número y tamaño y cambio de coloración (de rojo a purpura). Se realiza biopsia de piel (pie y brazo izquierdo). Se realizan los estudios para búsqueda de etiología, donde llama la atención dosaje de proteína S disminuida (88 %)
A 53-year-old male patient with no known underlying pathologies presented with a 72-hour history of pruritic petechiae that began in the inner thighs, progressed symmetrically to both legs and feet, then increased in number and size. and color change (from red to purple). Skin biopsy (left foot and arm) is performed. Studies are performed to search for etiology, where low protein S dosage (88%) is striking.
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A plataforma de ELISA (ensaio de imunoabsorção por ligação enzimática) tem sido amplamente utilizada para detectar anticorpos anti-SARS-CoV-2 gerados após a exposição ao vírus ou à vacinação. A amostra comumente utilizada para a realização do teste é o soro. Até o momento, nenhum estudo havia investigado a urina do paciente como amostra para detectar anticorpos específicos para o vírus SARS-CoV-2. A urina é um espécime biológico que traz vantagens significativas inerentes ao tipo de amostra, que compreende coleta não invasiva, de fácil manuseio e armazenamento. Neste trabalho, propomos um ELISA indireto in house baseado no uso de urina e proteínas recombinantes do Nucleocapsídeo (N) ou da Spike (S) do vírus SARS-CoV-2. As proteínas recombinantes (r) de SARS-CoV-2, N e as subunidades da proteína S (S-Glic, S1-NGlic e RBD-NGlic), foram avaliadas usando um painel composto por aproximadamente 200 amostras de urina e de soro. A presença de anticorpos anti-SARS-CoV-2 na urina foi detectada com sensibilidade e especificidade similares ou superiores ao soro, nas quais foram obtidos valores de sensibilidade de 94,0%, 75,0%, 81,38% e 89,66%, e especificidade de 100%, 96,0%, 96,77% e 96,77%, frente às proteínas rSARS-CoV-2 N, S-Glic, S1-NGlic e RBDNGlic, respectivamente. Dessa forma, os dados apresentados sugerem que a urina poderia ser considerada como uma potencial amostra biológica para aplicação em plataformas de imunodiagnóstico para a infecção por SARS-CoV-2, trazendo benefícios tanto no contexto individual quanto populacional.
The Enzyme-linked immunosorbent assay (ELISA) method has been widely used to detect anti-SARS-CoV-2 antibodies generated after exposure to the virus or vaccination. The sample usually used to perform the test is the serum. Thus far, no study has investigated the urine of patients as biological sample to detect specific SARS-CoV-2 antibodies. Urine is a biological specimen with significant advantages inherent to the type of sample, which comprises non-invasive collection, easy handling and storage. In this work, we propose an in house urine-based indirect ELISA using recombinant proteins from Nucleocapsid (N) and Spike (S) of the SARSCoV-2 virus. SARS-CoV-2 recombinant N and S protein subunits (Gly-S, NonGly-S1 and NonGly-RBD) were evaluated in an ELISA platform with a panel composed about 200 urine and serum samples. The presence of anti-SARS-CoV-2 antibodies in urine was detected with similar or superior sensitivity and specificity to serum, in which sensitivity values of 94.0%, 75.0%, 81.38% and 89.66% were obtained, while specificity values were of 100.0%, 96.0%, 96.77% and 96.77%, respectively, against rSARS-CoV-2 N, S-Glic, S1-NGlic and RBD-NGlic proteins. In conclusion, the data presented suggest that urine could be considered as a potential biological sample for application in immunodiagnostic platforms for SARS-CoV-2 infection, with benefits to the individual and population context.
Subject(s)
Humans , Male , Female , Urine , Immunologic Tests , Nucleocapsid Proteins , Spike Glycoprotein, Coronavirus , SARS-CoV-2 , COVID-19 , Antibodies , Viruses , Recombinant Proteins , Vaccination , Diagnostic Techniques and Procedures , Protein SubunitsABSTRACT
Objective:To investigate the clinical characteristics and etiology of pulmonary embolism in children, and to discuss the efficacy and safety of anticoagulation therapy.Methods:The data of 30 children with pulmonary embolism, who were treated with anticoagulation therapy in the Department of Pediatrics, Provincial Hospital Affiliated to Shandong First Medical University from January 2017 to December 2021, were analyzed retrospectively.The etiology, clinical characteristics, complications, outcomes and prognosis after anticoagulation treatment were analyzed.Results:There were 17 males and 13 females, with an average age of (8.95±2.58) years (age range: 4-13 years). The follow-up duration was 3-59 months.(1) The symptoms included cough in 30 cases (100.0%), fever in 29 cases (96.7%), shortness of breath in 27 cases (90.0%), chest pain in 15 cases (50.0%), hemoptysis in 9 cases (30.0%), bloody secretions under bronchoscopy but no hemoptysis in 4 cases (13.3%), and respiratory failure in 2 cases (6.7%). (2) The protopathy was Mycoplasma pneumoniae infection in 23 cases (76.7%), whose symptoms accorded with refractory Mycoplasma pneumoniae pneumonia.About 16 cases (53.3%) were positive for Mycoplasma pneumoniae drug resistance mutation 2063A>G or 2064A>G.Two cases (6.7%) had nephrotic syndrome.One case (3.3%) had purpura nephritis (nephrotic syndrome type). One case (3.3%) was lupus nephritis (nephrotic syndrome type). One case (3.3%) was hereditary protein S deficiency.One case (3.3%) had osteomyelitis and Staphylococcus aureus sepsis.One case (3.3%) had congenital heart disease.(3) Complications included limb thrombosis in 7 cases (23.3%), atrial thrombosis in 2 cases (6.7%), thoracic and abdominal deep venous thrombosis in 2 case (6.7%), cerebral infarction in 2 cases (6.7%), and splenic infarction in 1 case (3.3%). (4) Imaging examination showed that 30 children had lung consolidation/atelectasis (100.0%), and 24 cases had pleural effusion (80.0%). (5) Coagulation function examination suggested D-dimer increased to ≥ 5 mg/L in 21 cases (70.0%). (6) One case (3.3%) was given thrombolytic therapy with urokinase at the acute stage.Nine cases (30.0%) were treated with heparin/low molecular weight heparin.Twenty-one cases (70.0%) first received anticoagulation therapy with heparin/low molecular weight heparin and later took oral anticoagulant.Four cases (13.3%) were treated with Warfarin and 17 cases (56.7%) with Rivaroxaban.The anticoagulant treatment lasted 1-9 months.No recurrence of embolism or sequelae of chronic thromboembolic pulmonary hypertension was observed. Conclusions:Infection, especially Mycoplasma pneumoniae infection, is the main cause of pulmonary embolism in children.The symptoms of pulmonary embolism in children are atypical, so it is difficult to distinguish this disease from primary underlying diseases.Bronchoscopy can help find occult pulmonary hemorrhage.Unexplained shortness of breath in children of any age suggests the possibility of pulmonary embolism.Combination of clinical symptoms and necessary examination contribute to early diagnosis of pulmonary embolism.Then selection of appropriate anticoagulant drugs and timely anticoagulant therapy can improve the prognosis of children.
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Objective:To investigate the clinical characteristics and etiology of pulmonary embolism in children, and to discuss the efficacy and safety of anticoagulation therapy.Methods:The data of 30 children with pulmonary embolism, who were treated with anticoagulation therapy in the Department of Pediatrics, Provincial Hospital Affiliated to Shandong First Medical University from January 2017 to December 2021, were analyzed retrospectively.The etiology, clinical characteristics, complications, outcomes and prognosis after anticoagulation treatment were analyzed.Results:There were 17 males and 13 females, with an average age of (8.95±2.58) years (age range: 4-13 years). The follow-up duration was 3-59 months.(1) The symptoms included cough in 30 cases (100.0%), fever in 29 cases (96.7%), shortness of breath in 27 cases (90.0%), chest pain in 15 cases (50.0%), hemoptysis in 9 cases (30.0%), bloody secretions under bronchoscopy but no hemoptysis in 4 cases (13.3%), and respiratory failure in 2 cases (6.7%). (2) The protopathy was Mycoplasma pneumoniae infection in 23 cases (76.7%), whose symptoms accorded with refractory Mycoplasma pneumoniae pneumonia.About 16 cases (53.3%) were positive for Mycoplasma pneumoniae drug resistance mutation 2063A>G or 2064A>G.Two cases (6.7%) had nephrotic syndrome.One case (3.3%) had purpura nephritis (nephrotic syndrome type). One case (3.3%) was lupus nephritis (nephrotic syndrome type). One case (3.3%) was hereditary protein S deficiency.One case (3.3%) had osteomyelitis and Staphylococcus aureus sepsis.One case (3.3%) had congenital heart disease.(3) Complications included limb thrombosis in 7 cases (23.3%), atrial thrombosis in 2 cases (6.7%), thoracic and abdominal deep venous thrombosis in 2 case (6.7%), cerebral infarction in 2 cases (6.7%), and splenic infarction in 1 case (3.3%). (4) Imaging examination showed that 30 children had lung consolidation/atelectasis (100.0%), and 24 cases had pleural effusion (80.0%). (5) Coagulation function examination suggested D-dimer increased to ≥ 5 mg/L in 21 cases (70.0%). (6) One case (3.3%) was given thrombolytic therapy with urokinase at the acute stage.Nine cases (30.0%) were treated with heparin/low molecular weight heparin.Twenty-one cases (70.0%) first received anticoagulation therapy with heparin/low molecular weight heparin and later took oral anticoagulant.Four cases (13.3%) were treated with Warfarin and 17 cases (56.7%) with Rivaroxaban.The anticoagulant treatment lasted 1-9 months.No recurrence of embolism or sequelae of chronic thromboembolic pulmonary hypertension was observed. Conclusions:Infection, especially Mycoplasma pneumoniae infection, is the main cause of pulmonary embolism in children.The symptoms of pulmonary embolism in children are atypical, so it is difficult to distinguish this disease from primary underlying diseases.Bronchoscopy can help find occult pulmonary hemorrhage.Unexplained shortness of breath in children of any age suggests the possibility of pulmonary embolism.Combination of clinical symptoms and necessary examination contribute to early diagnosis of pulmonary embolism.Then selection of appropriate anticoagulant drugs and timely anticoagulant therapy can improve the prognosis of children.
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Objective: To analyze the clinical manifestations and molecular pathogenesis of 18 patients with inherited protein S (PS) deficiency. Methods: Eighteen patients with inherited PS deficiency who were admitted to the Institute of Hematology & Blood Diseases Hospital from June 2016 to February 2019 were analyzed: activity of protein C (PC) and antithrombin (AT) , PS activity were measured for phenotype diagnosis; high throughput sequencing (HTS) was used for screening of coagulation disease-related genes; Sanger sequencing was used to confirm candidate variants; Swiss-model was used for three-dimensional structure analysis. Results: The PS:C of 18 patients ranged from 12.5 to 48.2 U/dL. Among them, 16 cases developed deep vein thrombosis, including 2 cases each with mesenteric vein thrombosis and cerebral infarction, and 1 case each with pulmonary embolism and deep vein thrombosis during pregnancy. A total of 16 PROS1 gene mutations were detected, and 5 nonsense mutations (c.134_162del/p.Leu45*, c.847G>T/p.Glu283*, c.995_996delAT/p.Tyr332*, c.1359G> A/p.Trp453*, c.1474C>T/p.Gln492*) , 2 frameshift mutations (c.1460delG/p.Gla487Valfs*9 and c.1747_1750delAATC/p.Asn583Wfs*9) and 1 large fragment deletion (exon9 deletion) were reported for the first time. In addition, the PS:C of the deep vein thrombosis during pregnancy case was 55.2 U/dL carrying PROC gene c.565C>T/p.Arg189Trp mutation. Conclusion: The newly discovered gene mutations enriched the PROS1 gene mutation spectrum which associated with inherited PS deficiency.
Subject(s)
Female , Humans , Pregnancy , Antithrombin III/genetics , Genetic Testing , Mutation , Protein C/genetics , Protein S/genetics , Protein S Deficiency/geneticsABSTRACT
Portal vein thrombosis (PVT) is a rare condition in the general population that develops serious complications if left untreated for long time. We present a case of a 29-year-old woman who developed PVT due to protein S deficiency versus neonatal funiculitis. Over time, the patient developed upper gastrointestinal bleeding due to esophageal varices and hypersplenism with splenic sequestration that caused minor bleeding episodes. Laparoscopic splenectomy and proximal splenorenal shunt with distal pancreatectomy due to aneurysmal dilatations of the splenic artery were successfully performed to avoid mayor progression of portal hypertension. Patient was discharged with indefinite anticoagulation and after surgery platelets raised up to 200x103/mm3. Laparoscopic splenectomy and proximal splenorenal shunt for portal hypertension due to portal vein thrombosis is an adequate surgery procedure which should be applied in these medical cases.
La trombosis de la vena porta (TVP) es una afección poco común en la población general que desarrolla complicaciones graves si no se trata durante mucho tiempo. Presentamos el caso de una mujer de 29 años que desarrolló TVP por deficiencia de proteína S versus funiculitis neonatal. Con el tiempo, la paciente desarrolló hemorragia digestiva alta por varices esofágicas e hiperesplenismo con secuestro esplénico que provocó episodios hemorrágicos menores. La esplenectomía laparoscópica y la derivación esplenorrenal proximal con pancreatectomía distal por dilataciones aneurismáticas de la arteria esplénica se realizaron con éxito para evitar una mayor progresión de la hipertensión portal. La paciente fue dada de alta con anticoagulación indefinida y tras la cirugía se elevaron las plaquetas hasta 200x103/mm3. La esplenectomía laparoscópica y la derivación esplenorrenal proximal para la hipertensión portal por trombosis de la vena porta es un procedimiento quirúrgico adecuado que debe aplicarse en estos casos médicos.
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Objective:To investigate the expression of microRNA (miRNA, miR)-4699-3p in ovarian cancer cell lines, and observe its ability to regulate the expression of mitochondrial ribosomal protein S23 (MRPS23) and its effect on the migration and proliferation of ovarian cancer cells.Methods:Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-4699-3p in ovarian cancer cell lines (OVCAR-3, SKOV-3, HO-8910, OC3, A2780) and normal ovarian epithelial cells (IOSE80). The liposome transfection method was used to transfect miR-4699-3p mimic or negative control miR-NC to the cell line with the lowest miR-4699-3p expression, which was defined as the experimental group and the control group. qRT-PCR was used to verify transfection efficiency. Bioinformatics technology predicted the candidate target gene of miR-4699-3p, and the dual luciferase reporter gene experiment identified its ability to regulate the target gene. qRT-PCR and Western blot were used to detect the expression of target genes at the mRNA and protein levels. Cell counting method (CCK-8) and transwell migration experiment were used to detect the effect of miR-4699-3p on the proliferation and migration of ovarian cancer cells.Results:The expression of miR-4699-3p in ovarian cancer cell lines was significantly lower than that of normal ovarian epithelial cells ( P<0.05), and the lowest expression in OVCAR-3 cells ( P<0.01). After transfection of miR-4699-3p, the expression of miR-4699-3p in OVCAR-3 cells in the experimental group was significantly increased ( P<0.01), which proved that the transfection was successful. Bioinformatics technology predicted that the candidate target gene of miR-4699-3p may be MRPS23. The dual luciferase reporter gene experiment confirmed that miR-4699-3p can directly target the 3′-UTR of MRPS23 gene mRNA ( P<0.01). Compared with the OVCAR-3 cells in the control group, the mRNA and protein expression levels of the MRPS23 gene were significantly reduced, while the expressions of Twist, Slug, CDK6 and Cyclin D3 were significantly decreased ( P<0.01) in the experimental group after up-regulating miR-4699-3p expression ( P<0.01). After up-regulating miR-4699-3p expression, the proliferation ability of OVCAR-3 cells decreased ( P<0.05) and the migration ability of OVCAR-3 cells was reduced ( P<0.01). Conclusions:miR-4699-3p is under-expressed in ovarian cancer cell lines. Up-regulation of miR-4699-3p expression in OVCAR-3 cells can inhibit ovarian cancer cell proliferation and migration by interfering with MRPS23 gene expression.
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COVID-19 is a severe acute respiratory syndrome caused by a novel coronavirus, SARS-CoV- 2.COVID-19 is now a pandemic, and is not yet fully under control.As the surface spike protein (S) mediates the recognition between the virus and cell membrane and the process of cell entry, it plays an important role in the course of disease transmission.The study on the S protein not only elucidates the structure and function of virus-related proteins and explains their cellular entry mechanism, but also provides valuable information for the prevention, diagnosis and treatment of COVII)-19.Concentrated on the S protein of SARS-CoV-2, this review covers four aspects: (1 ) The structure of the S protein and its binding with angiotensin converting enzyme II (ACE2) , the specific receptor of SARS-CoV-2, is introduced in detail.Compared with SARS-CoV, the receptor binding domain (RBD) of the SARS-CoV- 2 S protein has a higher affinity with ACE2, while the affinity of the entire S protein is on the contrary.(2) Currently, the cell entry mechanism of SARS-CoV-2 meditated by the S protein is proposed to include endosomal and non-endosomal pathways.With the recognition and binding between the S protein and ACE2 or after cell entry, transmembrane protease serine 2(TMPRSS2) , lysosomal cathepsin or the furin enzyme can cleave S protein at S1/S2 cleavage site, facilitating the fusion between the virus and target membrane.(3) For the progress in SARS-CoV-2 S protein antibodies, a collection of significant antibodies are introduced and compared in the fields of the target, source and type.(4) Mechanisms of therapeutic treatments for SARS-CoV-2 varied.Though the antibody and medicine treatments related to the SARS-CoV-2 S protein are of high specificity and great efficacy, the mechanism, safety, applicability and stability of some agents are still unclear and need further assessment.Therefore, to curb the pandemic, researchers in all fields need more cooperation in the development of SARS-CoV-2 antibodies and medicines to face the great challenge.
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Objective @#To investigate the activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway molecules during the process by which kaempferol (Kae) promotes osteogenic differentiation of mouse bone marrow mesenchymal cells (BMMCs) under cyclic and uniaxial tension.@*Methods @#BMMCs isolated and cultured in vitro were subjected to uniaxial dynamic tension with a 10% shape variable. The appropriate concentration of Kae was selected by cytotoxicity testing. The endogenous mTOR signal was inhibited by pp242. Four hours after traction, alkaline phosphatase (ALP) and osteocalcin (OCN) were detected by chemical colorimetry and ELISA, and the relative concentration of intracellular calcium was detected by flow cytometry. Phosphorylation of mTOR, 4E/BP1, and ribosomal protein S6 kinases (S6K), which are the main molecules of the endogenous mTORC1 signaling pathway, and expression of osteogenic transcription factors (Runx2 and Osterix) were detected by western blotting (WB), and mRNA expression levels of the above factors were detected by qRT-PCR.@*Results @# The cytotoxicity test showed that 10 μmol/L Kae had little inhibitory effect on cell proliferation but had the strongest osteogenic ability. Four hours after stretching, Kae effectively promoted the osteogenic differentiation of BMMCs. The expression of ALP was （153.04 ± 18.72） U/mg, the expression of OCN was （1.64 ± 0.25） U. The mRNA and protein levels of Runx2 and Osterix were upregulated, and the intracellular calcium content was decreased. The mRNA and protein phosphorylation of mTOR and S6K was upregulated, and the opposite effect was observed with 4E/BP1. After pp242 was added to inhibit mTOR signaling, mTOR and S6K mRNA and protein phosphorylation were downregulated, but 4E/BP1 mRNA and protein phosphorylation was upregulated. The osteogenic differentiation of BMMCs was also significantly inhibited, mRNA and protein expression of Runx2 and Osterix were significantly downregulated, ALP and OCN expression were downregulated, and intracellular calcium content was increased. @* Conclusion@#Kae promotes osteogenic differentiation of mouse BMMCs under uniaxial dynamic tension through the mTORC1 signaling pathway.
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La trombosis se puede definir como un trastorno vascular que se presenta cuando se desarrolla un trombo que bloquea de forma total o parcial el interior de un vaso sanguíneo, ya sea una vena o una arteria. La trombofilia define a una alteración de la hemostasia que predispone al desarrollo de trombosis; sin embargo, la presencia de una trombofilia no implica necesariamente la aparición de un evento trombótico. La deficiencia de los inhibidores fisiológicos, como las proteínas C y S, constituyen factores de riesgo hereditarios y adquiridos que favorecen al desarrollo de trombosis. Se determinó la incidencia de deficiencia de estas proteínas de la coagulación en pacientes con historia obstétrica adversa, eventos de trombosis y otros eventos asociados. Se realizó un estudio longitudinal prospectivo en el período comprendido del 2011 al 2018, en un grupo de pacientes remitidos al Instituto de Hematología e Inmunología, a los que se les realizó la medición de los niveles plasmáticos de las proteínas C y S. En 133 pacientes (52,17 por ciento) se detectó deficiencia de proteína C (n=50), proteína S (n=66) o ambas (n=17). Las principales causas de realización de estos estudios fueron la historia obstétrica adversa y los eventos trombóticos y coincidieron con los eventos clínicos donde predominó alguna deficiencia. La deficiencia de proteína S tuvo mayor incidencia(AU)
Thrombosis is a vascular disorder appearing when a thrombus totally or partially blocks the inside of a blood vessel, be it a vein or an artery. Thrombophilia is a hemostatic alteration leading to thrombosis. However, the presence of thrombophilia does not necessarily imply the appearance of a thrombotic event. Deficiency in physiological inhibitors such as proteins C and S is a hereditary or acquired risk factor fostering thrombosis. Determination was made of the incidence of deficiency in these coagulation proteins in patients with an adverse obstetric history, thrombotic events and other associated disorders. A prospective longitudinal study was conducted in the period 2011-2018 of a group of patients referred to the Institute of Hematology and Immunology, who underwent measurement of their plasma levels of proteins C and S. A total 133 patients (52.17 percent) were found to be deficient in protein C (n=50), protein S (n=66) or both (n=17). The main reasons for the conduct of these studies were an adverse obstetric history and thrombotic events, which coincided with clinical disorders in which some sort of deficiency prevailed. Protein S deficiency was the most common(AU)
Subject(s)
Humans , Thrombosis , Protein S Deficiency , Protein C Deficiency , Hematology , Prospective Studies , Longitudinal StudiesABSTRACT
The pregnancy is an immunocompromised state. Thus, autoimmune diseases may affect pregnancy and get worsen during pregnancy. Here authors discuss a rare autoimmune thrombophilia disorder, protein C and S deficiency which may cause recurrent pregnancy losses by affecting haemostatic mechanisms in the body. This patient with recurrent pregnancy loss when evaluated extensively was found to have combined inherited protein C and S deficiency. It was successfully managed with thromboprophylaxis therapy, which resulted in the delivery of healthy baby. Long term anticoagulant prophylaxis should be considered weighing the risk of bleeding to thrombotic recurrence in such cases. In conclusion, combined protein C and S deficiency and that too presenting as recurrent pregnancy loss is very rare. Thrombophilia screening should be considered in cases of recurrent pregnancy losses. Adequate and appropriate thromboprophylaxis is an important part of the management of pregnant women with inherited thrombophilia.
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Abstract Introduction: Congenital protein S deficiency is a very rare disease in the population. In pregnant women it is associated with spontaneous abortion and fetal death, among other complications. Case presentation: We present the case of a 32-year-old multigravida with a 36-week pregnancy, with thromboprophylaxis with enoxaparin from the 4th week of gestation and with a diagnosis of thrombophilia-due to functional protein S deficiency-which was intervened with elective c-section under spinal anesthesia. In addition, a review of the relevant literature was conducted. Discussion: The risk of venous thromboembolism is approximately 4 to 5 times greater during gestation, and the recommendation of thromboprophylaxis in low-risk thrombophilia is based on the presence of associated risk factors. In patients receiving low molecular weight heparin (LMWH) as thromboprophylaxis, an interval of at least 12 hours after the last dose of LMWH before neuropsy and restarting the next dose after at least 4hours of spinal technique use is recommended. Conclusion: Neuroaxial techniques should be individualized and receive pre and postpartum thromboprophylaxis. In addition, non-pharmacological thromboprophylaxis measures in the perioperative period should be considered. Spinal anesthesia was effective and safe in this patient.
Resumen Introducción: La deficiencia congénita de proteína S es una enfermedad muy rara en la población. En gestantes está asociada a aborto espontáneo y muerte fetal, entre otras complicaciones. Presentación del caso: Presentamos el caso de una multigesta de 32 años con embarazo de 36 semanas, con tromboprofilaxis con enoxaparina desde la semana cuarta de gestación y con diagnóstico de trombofilia -por deficiencia de proteína S funcional-, la cual fue intervenida con cesárea electiva bajo anestesia espinal. Además, se realizó revisión de la literatura al respecto. Discusión: El riesgo de tromboembolismo venoso es aproximadamente 4 a 5 veces mayor durante la gestación, y la recomendación de tromboprofilaxis en trombofilias de bajo riesgo se basa en la presencia de factores de riesgo asociados. En pacientes que reciben Heparinas de Bajo Peso Molecular (HBPM) como tromboprofilaxis, se recomienda un intervalo de al menos 12 horas después de la última dosis de HBPM antes de la punción del neuroeje, y reiniciar la siguiente dosis después de al menos 4 horas de uso de la técnica espinal. Conclusión: Las técnicas neuroaxiales deben ser individualizadas y recibir tromboprofilaxis pre y posparto. Además, se deben tener en cuenta las medidas de tromboprofilaxis no farmacológicas en el periodo perioperatorio. La anestesia espinal fue efectiva y segura en esta paciente.
Subject(s)
Humans , Female , Pregnancy , Protein Deficiency , Protein S , Anesthesia, Spinal , Thrombosis , Cesarean Section , EnoxaparinABSTRACT
ObjectiveTo investigate the expression of phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR), 70-kDa ribosomal protein S6 kinase (p70S6K), and interferon-α (IFNα) in umbilical cord blood plasmacytoid dendritic cells (pDCs) in parturients in the immune tolerance stage of hepatitis B virus (HBV) infection versus normal parturients. MethodsA total of 20 parturients in the immune tolerance stage of HBV infection and 10 normal parturients who were hospitalized in Inpatient Department of Obstetrics in The First Affiliated Hospital of Hunan University of Chinese Medicine from October 2017 to January 2020 were enrolled as hepatitis B group and normal group, respectively. Umbilical cord blood pDCs were isolated and cultured, and CpG-A was added on day 7. The cells and the supernatant were collected after 24 hours; real-time PCR was used to measure the mRNA expression of PI3K, mTOR, and p70S6K, Western Blot was used to measure the protein expression of PI3K, mTOR, and p70S6K, and ELISA was used to measure the level of IFNα in the supernatant of pDCs. The two-independent-samples t-test was used for comparison of continuous data between the two groups. ResultsCompared with the normal group, the hepatitis B group had significantly lower mRNA and protein expression of PI3K, mTOR, and p70S6K in umbilical cord blood pDCs (mRNA expression: t=-81.04, -63.07, and -34.55, all P＜0.001; protein expression: t=-8.13, -7.75, and -6.71, all P＜0.001). The hepatitis B group had significantly lower expression of IFNα in the supernatant of umbilical cord blood pDCs than the normal group (t=-15.88, P＜0.05). ConclusionParturients in the immune tolerance stage of HBV infection have reductions in the mRNA and protein expression of PI3K, mTOR, and p70S6K and the level of IFNα in umbilical cord blood pDCs, suggesting that pDC function is inhibited.
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Objective@# To investigate the expression of the mTORC1 signaling pathway during the osteogenic differentiation of mouse bone marrow mesenchymal cells (BMMSCs) under cyclic uniaxial tension and explore its possible role.@*Methods @# The BMMSCs of mice were affected by uniaxial dynamic tensile force. Western blot was used to detect the expression changes of major molecules (mTOR, Raptor, S6K) in the endogenous mTORC1 signaling pathway at 0, 1, 2, 4, and 8 hours after stretching. Chemical colorimetry, ELISA and PCR were used to detect alkaline phosphatase (ALP), osteocalcin (OCN) and Runx2 mRNA, respectively. Then, inhibition, activation and control groups were established by administration of the drugs PP242, MHY1485 and PBS, respectively. Two hours after the stress, the expression of S6K was detected by western blot, and the expression of the osteogenic signal was continuously detected by the above methods.@*Results @#Western blot analysis showed that the main molecules of the mTORC1 signaling pathway were all expressed within 8 hours after traction, and the highest expression was 2 hours after the stress. Compared with those in the control group, the ALP activity and OCN expression decreased and the Runx2 mRNA levels increased after the mTORC1 signal pathway was inhibited (P ＜ 0.001); ALP activity and OCN expression increased after the mTORC1 signal pathway was activated, while the Runx2 mRNA levels decreased (P ＜ 0.001). @*Conclusion @#The mTORC1 signaling pathway participates in the osteogenic differentiation of mouse BMMSCs under tension. The osteogenesis of BMMSCs under cyclic uniaxial tension would be enhanced if the mTORC1 signaling pathway was activated.
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Objective: To investigate the clinical characteristics and gene mutation, and analyze the association between genotype and phenotype of hereditary protein S deficiency in a Chinese pedigree. Methods: Hereditary protein S deficiency was diagnosed in January 2016 in our hospital. A total of 26 family members were surveyed in this study. Blood samples and clinical data were collected from them, and mutations were identified by Sanger sequencing. Pathogenicity of gene mutations was predicted by protein function prediction software including SIFT, PolyPhen_2, nsSNPAnalyzer and MutPred2. Swiss Model (https://swissmodel.expasy.org/) was used to perform homology modeling of the tertiary structure of the protein S wild-type and mutant-type, and observe the impact of gene mutation on the tertiary structure of the protein. Results: Four out of 26 family members of 4 generations were clinically diagnosed with hereditary protein S deficiency. The proband presented with recurrent pulmonary embolism and venous thromboembolism of the lower extremities, and her uncle and mother had a history of venous thromboembolism. Sequencing revealed a mutation in the c.200A>C gene in the second exon of the PROS1 gene of proband and part of her families (Ⅱ2, Ⅱ6, Ⅲ4, Ⅳ2). The prediction results of this gene mutation performed by SIFT, PolyPhen_2, nsSNPAnalyzer, MutPred2 were all harmful. The results of Swiss-Model homology modeling showed that the 67th amino acid was mutated from glutamic acid to alanine because of this gene mutation. Conclusion: A gene mutation cDNA (c. 200A>T) is identified in a Chinese pedigree with hereditary protein S deficiency. This gene mutation may reduce protein S activity, which may cause recurrent pulmonary embolism and venous thromboembolism of the patients.
Subject(s)
Female , Humans , Asian People/genetics , Exons , Pedigree , Protein S Deficiency , Surveys and QuestionnairesABSTRACT
PURPOSE: Phosphorylated ribosomal S6 kinase 1 (pS6K1) is a major downstream regulator of the mammalian target of rapamycin (mTOR) pathway. Recent studies have addressed the role of S6K1 in adipogenesis. pS6K1 may affect the outcome of estrogen depletion therapy in patients with hormone-sensitive breast cancer due to its association with adipogenesis and increased local estrogen levels. This study aimed to investigate the potential of pS6K1 as a predictive marker of adjuvant aromatase inhibitor (AI) therapy outcome in postmenopausal or ovarian function-suppressed patients with hormone-sensitive breast cancer.METHODS: Medical records were retrospectively reviewed in postmenopausal or ovarian function-suppressed patients with estrogen receptor-positive and node-positive primary breast cancer. pS6K1 expression status was scored on a scale from 0 (negative) to 3+ (positive) based on immunohistochemical analysis.RESULTS: A total of 428 patients were eligible. The median follow-up duration was 44 months (range, 1–90). In patients with positive pS6K1 expression, AIs significantly improved disease-free survival (DFS) compared to selective estrogen receptor modulators (SERMs) (5 year-DFS: 83.5% vs. 50.7%, p = 0.016). However, there was no benefit of AIs on DFS in the pS6K1 negative group (5 year-DFS 87.6% vs. 91.4%, p = 0.630). On multivariate analysis, AI therapy remained a significant predictor for DFS in the pS6K1 positive group (hazard ratio, 0.39; 95% confidence interval, 0.16–0.96; p = 0.041). pS6K1 was more effective in predicting the benefit of AI therapy in patients with ages < 50 (p = 0.021) compared to those with ages ≥ 50 (p = 0.188).CONCLUSION: pS6K1 expression may predict AI therapy outcomes and serve as a potential predictive marker for adjuvant endocrine therapy in postmenopausal and ovarian function-suppressed patients with hormone-sensitive breast cancer. AIs may be more effective in patients with pS6K1 positive tumors, while SERM could be considered an alternative option for patients with pS6K1 negative tumors.