Endotoxins are common exogenous pyrogens. Excessive endotoxins in medical devices and injections can lead to serious consequences such as sepsis, septic shock, and even death. Therefore, endotoxin detection plays a crucial role in medical, pharmaceutical, and food sectors. The wide application of Limulus amebocyte lysate (LAL) has led to a sharp decline in the number of horseshoe crabs. Moreover, the LAL assay has limitations such as interbatch variations and difficulty in quantification. The recombinant factor C (rFC) assay is stable between batches, highly sensitive, and capable of quantitation, and thus it can be used as an alternative for the LAL assay. However, the high cost and complex procedures involved in producing recombinant factor C have limited the widespread application of this method. In order to simplify the preparation and reduce the production cost of recombinant factor C, this study focuses on the production of recombinant factor C based on the baculovirus expression system. Multiple measures such as a high-yield and anti-apoptotic vector qBac-IIIG, the optimal signal peptide, and the optimized codon were used to reach the goal of endotoxin detection with cell supernatant. This method simplifies the steps of protein purification. The sensitivity of the supernatant reached 0.05 EU/mL in a 1-L fermentation system, and 500 000 detecting reactions can be supported per liter of fermentation broth. This study increases the yield and activity of recombinant factor C, simplifies the procedures of protein purification, and reduces the cost, laying a foundation for the promotion and application of recombinant factor C in endotoxin detection.
Animals ; Recombinant Proteins/genetics* ; Horseshoe Crabs/chemistry* ; Baculoviridae/metabolism* ; Endotoxins/analysis* ; Protein C/biosynthesis* ; Genetic Vectors/genetics* ; Arthropod Proteins/genetics* ; Enzyme Precursors ; Serine Endopeptidases

Betacoronavirus ; isolation & purification ; pathogenicity ; Bile Acids and Salts ; metabolism ; Bile Ducts, Intrahepatic ; pathology ; virology ; Cell Culture Techniques ; Coronavirus Infections ; complications ; pathology ; Cytokine Release Syndrome ; etiology ; physiopathology ; Cytopathogenic Effect, Viral ; Epithelial Cells ; enzymology ; pathology ; virology ; Humans ; Hyperbilirubinemia ; etiology ; Liver ; pathology ; Organoids ; pathology ; virology ; Pandemics ; Peptidyl-Dipeptidase A ; analysis ; Pneumonia, Viral ; complications ; pathology ; Receptors, Virus ; analysis ; Serine Endopeptidases ; analysis ; Viral Load

Acute Kidney Injury ; etiology ; Betacoronavirus ; Coronavirus Infections ; complications ; Humans ; Kidney ; enzymology ; Pandemics ; Peptidyl-Dipeptidase A ; physiology ; Pneumonia, Viral ; complications ; Sequence Analysis, RNA ; methods ; Serine Endopeptidases ; physiology ; Single-Cell Analysis ; methods

OBJECTIVE: To observe the household environment dust mites allergens content distribution characteristics and influence factors of children with allergic rhinitis to dust mites in Wuhu. METHOD: Collect the surface dust in bedroom and living room floor, mattresses, pillows, sofa of 102 children with allergic rhinitis families. Dust mite allergen components 1 (Der f1) and house dust mites allergens 1 components (Der p1) of the dust samples were determined by enzyme-linked immunosorbent assay (ELISA). RESULT: One hundred and twenty samples were collected . In a domestic dust mites samples, with a median of M (Min and Max) said dust mite allergen levels, Der f1 and Der p1 content was 2.66 (0.03, 26.63), 3.48 (0, 03, 33.68), respectively. Der f1 was significantly less than Der p1, and the difference was statistically significant (P < 0.05). According to the classification of urban and township, there were 68 cases and 34 cases. Der f1 content in the samples was 2.91 (0.31, 26.63), 2.40 (0.08, 16.02), respectively. Der p1 content was 4.28 (0.03, 20.77), 3.88 (0.14, 33.68), respectively. The dust mites content of urban was significantly more than that of villages and towns, the difference was statistically significant (P < 0.05). Samples were also classified by gender. The dust mites allergens content in either boy's or girl's family were similar, there was no statistically significant difference (P > 0.05). CONCLUSION The household dust mites of children allergic rhinitisin Wuhu area is given priority to with Der p1, and urban dust mites are significantly more than village's and town's. Enhancing health education, controlling dust mites allergens contamination inside the bedroom, especially urban areas, are positive differences for the prevention and treatment of allergic diseases such as allergic rhinitis in children.
Allergens ; analysis ; Animals ; Antigens, Dermatophagoides ; analysis ; Arthropod Proteins ; analysis ; Bedding and Linens ; Child ; Cysteine Endopeptidases ; analysis ; Dermatophagoides farinae ; Dust ; analysis ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Male ; Rhinitis, Allergic ; epidemiology

We performed gene expression profiling in bladder cancer patients to identify cancer-specific survival-related genes in muscle invasive bladder cancer (MIBC) patients. Sixty-two patients with MIBC were selected as the original cohort and another 118 MIBC patients were chosen as a validation cohort. The expression of USP18, DGCR2, and ZNF699 genes were measured and we analyzed the association between gene signatures and survival. USP18 and DGCR2, were significantly correlated to cancer-specific death (P=0.020, P=0.007, respectively). Cancer-specific survival in the low USP18 or DGCR2 expression group was significantly longer than the high expression group (P=0.018, P=0.006, respectively). In multivariate Cox regression analysis, a combination of USP18 and DGCR2 mRNA expression levels were significant risk factors for cancer-specific death (HR, 2.106; CI, 1.043-4.254, P=0.038). Overall survival and cancer-specific survival rates in the low-combination group were significantly longer than those in the high-expression group (P=0.001, both). In conclusion, decreased expressions of USP18 and DGCR2 were significantly associated with longer cancer-specific survival, and also the combination of two genes was correlated to a longer survival for MIBC patients. Thus, the combination of USP18 and DGCR2 expression was shown to be a reliable prognostic marker for cancer-specific survival in MIBC.
Adult ; Aged ; Aged, 80 and over ; Biological Markers/metabolism ; Carrier Proteins/genetics/metabolism ; Endopeptidases/genetics/*metabolism ; Female ; Gene Expression Profiling ; Humans ; Kaplan-Meier Estimate ; Male ; Middle Aged ; Muscle Neoplasms/*secondary ; Neoplasm Invasiveness ; Neoplasm Staging ; Platelet Glycoprotein GPIb-IX Complex/genetics/*metabolism ; Predictive Value of Tests ; ROC Curve ; Regression Analysis ; Risk Factors ; Urinary Bladder Neoplasms/*diagnosis/metabolism/*mortality/pathology

OBJECTIVETo study the correlation of nucleotide polymorphism in the ubiquitin-specific protease 26 (Usp26) gene with idiopathic male infertility and its action mechanism in spermatogenesis.

METHODSBased on the WHO criteria (4th ed.), we selected 41 patients with idiopathic infertility from 150 infertile males, and enlisted 50 normal fertile men as controls. We examined the selected patients for mutations using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, and determined how and where the mutations occurred by gene sequencing.

RESULTSLow sperm concentration and poor sperm motility were found in the 41 men with idiopathic infertility. Nine (22.0%) of them exhibited changes in the Usp26 gene (P = 0.01), including compound mutations of 364insACA and 460G > A in 8 (19.5%, P = 0.01) and 1 044T > A substitution in 1 (2.4%, P > 0.05). The above three variations led to changes in the coding amino acids. No other changes were found in the remaining patients and normal fertile controls.

CONCLUSIONThe nucleotide polymorphisms of the Usp26 gene might be closely related with idiopathic male infertility, and exert negative effect on the testis function.

Case-Control Studies ; Cysteine Endopeptidases ; genetics ; Humans ; Infertility, Male ; genetics ; pathology ; Male ; Polymorphism, Single Nucleotide ; Semen Analysis

BACKGROUNDHaikou locates in tropical island with unique mite propagation. The aim of this stuy is to determine mite allergens levels in Haikou, and to investigate the prevalence of mite specific IgE-sensitization and IgE cross-reactivity between house dust mites.

METHODSAllergen and antigen concentrations against six mite species were tested by enzyme-linked immunosorbent assay (ELISA). Specific IgE concentrations and cross-inhibitions were measured with ADVIA Centaur(®).

RESULTSAllergen or antigen Dermatophagoides pteronyssinus (Der p 1), Blomia tropicalis (Blo t) and Tyrophagus putrescentia (Tyr p) were detected in dust samples. Dermatophagoides farinae (Der f 1), Lepidoglyphus destructor (Lep d 2), and Acarus siro (Aca s) were found in very few samples. Specific IgE tests showed high prevalence of sensitizations against all tested mites with high IgE levels to Der p, Der f, and Blo t. Storage mites, Blo t, Tyr p, Lep d, and Aca s, could inhibit Der p from 0 to 50%. Storage mites could inhibit Der f between 30% and 100%. Der p IgE could be inhibited by Der f with up to 90%, and vice versa. Der p could inhibit Blo t from 40% to 80%. Blo t was able to fully inhibit IgE binding to Lep d, Tyr p, and Aca s compared to partial inhibition by Der p.

CONCLUSIONSDer p is the dominating mite and has the highest specific IgE prevalence among asthmatic children. Blo t represents an important source of storage mite sensitization and some patients may be independently sensitized to both Der p and Blo t. High prevalence of sensitization to Der f may be due to IgE-mediated cross-reactivity with Der p and Blo t.

Adolescent ; Air Pollution, Indoor ; Allergens ; analysis ; Animals ; Antigens, Dermatophagoides ; analysis ; Arthropod Proteins ; analysis ; Asthma ; immunology ; Child ; Child, Preschool ; China ; Cross Reactions ; Cysteine Endopeptidases ; analysis ; Dust ; Humans ; Immunoglobulin E ; blood ; immunology ; Mites ; immunology

OBJECTIVETo explore the protective effects of mitochondrial ATP-sensitive potassium channel opener diazoxide on hyperoxia-induced apoptosis of type II alveolar epithelial cells (A549 cells) and possible mechanisms.

METHODSA549 cells were cultured in vitro and divided randomly into control, hyperoxia and diazoxide group. The hyperoxia group was exposed to a mixture of O2 (900 mL/L) and CO2 (50 mL/L) for 10 minutes, then cultured in a closed environment. The diazoxide group was pretreated with diazoxide of 100 μmol/L for 24 hrs before hyperxia induction. The cells were collected 12, 24 and 48 hrs after culture. The morphologic changes of A549 cells were observed under an inverted microscope. A549 cell apoptosis was detected by flow cytometry. The expression of Omi/HtrA2 in the endochylema of A549 cells was determined by immunohistochemistry.

RESULTSA549 cells were damaged and the changes in morphology of the cells were serious in the hyperoxia group. The apoptosis rate of A549 cells and the expression of Omi/HtrA2 in the endochylema increased in the hyperoxia group compared with the control group (P<0.05). The growth and the morphology of A549 cells were greatly improved and the cell injuries were obviously alleviated in the diazoxide group. The expression of Omi/HtrA2 in the endochylema and the apoptosis rate of A549 cells were significantly reduced in the diazoxide group compared with the hyperoxia group (P<0.05).

CONCLUSIONSDiazoxide as an opener of mitoKATP channel can reduce the expression of Omi/HtrA2 and the apoptosis rate of A549 cells, thus relieves the injury of A549 cells induced by hyperoxia.

Apoptosis ; Cells, Cultured ; Cytoprotection ; Diazoxide ; pharmacology ; High-Temperature Requirement A Serine Peptidase 2 ; Humans ; Hyperoxia ; complications ; Lung ; pathology ; Mitochondrial Proteins ; analysis ; Potassium Channels ; physiology ; Serine Endopeptidases ; analysis

OBJECTIVETo investigate the expression of serine protease Omi/HtrA2 in kidneys of postasphyxial neonatal rats, and to study the effects of Ucf-101 on apoptosis and the expression of Omi/HtrA2 in these rats.

METHODSSeventy-two neonatal Wistar rats of 7-10 days old were randomly divided into 3 groups: control, postasphyxial model, Ucf-101-treated postasphyxialThe postasphyxial model was established by normobaric asphyxiaExpression of Omi/HtrA2 was determined with streptavidin-peroxidase immunohistochemistry 2, 24 and 48 hrs after asphyxia. Terminal deoxynuleotidyl-mediated nick end labeling (TUNEL) was used to ascertain the apoptosis of renal cells.

RESULTSCompared with the control group, OmiHtrA2 expression in renal cells began to increase 2 hrs after asphyxia and peaked at 24 hrs. The expression of Omi/HtrA2 in the Ucf-101-treated postasphyxial group was significantly lower than that in the postasphyxial model group (P<0.01). TUNEL-positive cells began to increase 2 hrs after asphyxia and peaked at 24 hrs in the postasphyxial model group when compared with the control group. The number of TUNEL-positive cells in the Ucf-101-treated postasphyxial group was significantly lower than that in the postasphyxial model group at all time points (P<0.01).

CONCLUSIONSThe expression of Omi/HtrA2 in kidneys is increased in postasphyxial neonatal rats. The increased Omi/HtrA2 expression may play an important role in the development of postasphyxial renal injury. Treatment with Ucf-101 can reduce the expression of Omi/HtrA2 in kidneys of postasphyxial neonatal rats and thus reduce renal tububar epithelial cell apoptosis.

Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Asphyxia Neonatorum ; drug therapy ; metabolism ; pathology ; Female ; High-Temperature Requirement A Serine Peptidase 2 ; Humans ; Immunohistochemistry ; In Situ Nick-End Labeling ; Infant, Newborn ; Kidney ; chemistry ; Male ; Mitochondrial Proteins ; analysis ; antagonists & inhibitors ; Pyrimidinones ; pharmacology ; therapeutic use ; Rats ; Rats, Wistar ; Serine Endopeptidases ; analysis ; Thiones ; pharmacology ; therapeutic use

OBJECTIVETo develop an assay methodology for determination of lumbrukinase potency in Rongshuan capsules.

METHODThe agarose-fibrin plate assay methodology for determination of Lumbrukinase potency in Rongshuan capsules was studied including durability, specificity, linearity range, product's handling method, accuracy , repetitiveness, solution stability, recovery and statistical method. The method of parallel line assay based on quantitative responses in statistical methods for biological assays was used in the statistics of potency assay.

RESULTThe durability and specificity of assay accord with the requirement; The linearity range was 12.5 to approximately 400 U, the RSD of accuracy tests was 3.2%, the RSD of repetitiveness tests was 8.3%, the solution is stable under 4 degrees C for 72 hours, the recovery rate was 97.0% and the RSD of recovery assays was 16.5%.

CONCLUSIONThe agar-fibrin plate assay is rapidly, feasible, simple, convenient and accurate way for determining the Lumbrukinase potency. The method of parallel line assay based on quantitative responses in statistical methods for biological assays can control the error of determination.

Animals ; Biological Assay ; methods ; Capsules ; analysis ; Endopeptidases ; analysis ; Pharmaceutical Preparations ; analysis