One pair of primers were designed and synthesized on the base of the cDNA sequence encoding Schizosaccharomyces pombe N-glycanase reported on the GenBank. The cDNA sequence encoding Peptide N-glycanase was cloned from the Schizosaccharomyces pombe by RT-PCR. And then the RT-PCR product was cloned into the expression vector pET-15b. The expression vector pET-15b(+)/Png1p was transformed into E. coli BL21(DE3). The results showed that the relative molecular weight of the enzyme was determined to be approximately 39 kD using SDS-PAGE. The expression products after induction and purification can catalyze the cleavage of N-linked oligosaccharides from glycoprotein coped with heat, but have no action on the native glycoprotein with the help of DTT. The percentage of deglycosylated RNase B treated with equate Png1p in different reaction temperature, pH, concentration of DTT and denatured temperature showed that the optimum temperature, the optimum pH is 30 degrees C; the optimum concentration of DTT is 10 mmol/L and the optimum denatured temperature is 100 degrees C.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Glycosylation ; Hydrogen-Ion Concentration ; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase ; biosynthesis ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Schizosaccharomyces ; enzymology ; genetics ; Temperature

As a highly prevalent global condition, myopia significantly impacts the ocular health of young individuals in China. Orthokeratology lens, as a rigid corneal contact lens, has demonstrated effective control over the progression of myopia; however, its mechanism of action remains incompletely elucidated. As one of the factors influencing visual acuity, higher-order aberrations will undergo marked changes after orthokeratology, with particular emphasis on the alterations in spherical aberrations and coma. The changes in corneal morphology induced by orthokeratology lead to significant positive increase in both spherical aberration and coma. Furthermore, the elevation of spherical aberration and coma demonstrates a negative correlation with the rate of axial length growth following orthokeratology. The interplay among spherical aberration, coma, defocus, accommodation, astigmatism, and pseudo-accommodation may constitute the underlying mechanism governing the control of myopia through orthokeratology.

Objective To investigate the peripheral retinal defocus and its influencing factors in children and adoles-cents with low to moderate myopia.Methods Totally 281 children and adolescents aged 6-15 years were included in the study,and only the right eye was selected.After cycloplegic refraction as well as axial length(AL)and average corneal curvature(AveK)measurements,the patients were divided into low myopia(LM)group(-3.00 D≤SE≤-0.50 D)and moderate myopia(MM)group(-6.00 D≤SE＜-3.00D)according to spherical equivalent(SE),and stratified compari-sons were made according to AL[AL1 group(23.00 mm≤AL≤24.00 mm),AL2 group(24.00 mm＜AL≤25.00 mm),and AL3 group(25.00 mm＜AL≤26.00 mm)]and AveK[AveK1 group(40.00 D≤ Ave K≤43.00 D)and AveK2 group(43.00 D＜AveK≤46.00 D)].Multispectral refraction tomography was used to measure the refraction difference value(RDV),in-cluding TRDV(0° to 53°),RDV-15(0° to 15°),RDV-30(0° to 30°),RDV-45(0° to 45°),RDV-15-30(15° to 30°),RDV-30-45(30° to 45°),RDV-45-53(45° to 53°),RDV-S(superior),RDV-I(inferior),RDV-T(temporal)and RDV-N(na-sal).The RDV was compared in the groups divided according to SE,AL and AveK individually,and the correlation be-tween RDV and age,SE,AL and AveK was analyzed.Moreover,the factors affecting RDV in all ranges were analyzed by multiple linear regression.Results Compared with the LM group,the MM group had significant increases in TRDV,RDV-30,RDV-45,RDV-15-30,RDV-30-45,RDV-45-53,RDV-S,RDV-I and RDV-N(all P＜0.05)and no significant differ-ence in RDV-15 and RDV-T(both P＞0.05).According to the comparisons of AL groups and AveK groups,the TRDV,RDV-30,RDV-45,RDV-15-30,RDV-30-45,RDV-45-53,RDV-S,RDV-I and RDV-N in the AL2 group were significantly higher than those in the AL1 group(all P＜0.05);the TRDV,RDV-30,RDV-45,RDV-15-30,RDV-30-45,RDV-45-53,RDV-S and RDV-N in the AL3 group were significantly higher than those in AL2 and AL1 groups,and RDV-I and RDV-T in the AL3 group were significantly higher than those in the AL1 group(both P＜0.05);the TRDV,RDV-30,RDV-45,RDV-15-30,RDV-30-45,RDV-45-53,RDV-S,and RDV-I in the Ave K1 group were significantly higher than those in the AveK2 group(all P＜0.05).The correlation analysis showed that TRDV,RDV-45,RDV-30-45,RDV-45-53,RDV-S and RDV-N were positively correlated with age and AL and negatively correlated with SE and Ave K;RDV-30,RDV-15-30 and RDV-I were positively cor-related with AL and negatively correlated with SE and AveK;RDV-T was positively correlated only with AL;RDV-15 was not correlated with age,SE,AL and AveK.Multiple linear regression analysis showed that age was the influencing factor of RDV-45-53 and RDV-S;AL was the influencing factor of TRDV,RDV-30,RDV-45,RDV-15-30,RDV-30-45,RDV-45-53,RDV-S and RDV-T;AveK was the influential factor of RDV-I;SE had no significant effect on RDV in all ranges.Conclu-sion Peripheral retinal defocus in children and adolescents with low to moderate myopia has reached hyperopic defocus,and hyperopic defocus is the least in patients with relatively short AL.Age,AL and AveK can affect peripheral retinal defo-cus in children and adolescents with low to moderate myopia,among which AL is the most important influencing factor.

Objective:To evaluate the diagnostic efficacy and practicality of the Jaundice color card (JCard) as a screening tool for neonatal jaundice.Methods:Following the standards for reporting of diagnostic accuracy studies (STARD) statement, a multicenter prospective study was conducted in 9 hospitals in China from October 2019 to September 2021. A total of 845 newborns who were admitted to the hospital or outpatient department for liver function testing due to their own diseases. The inclusion criteria were a gestational age of ≥35 weeks, a birth weight of ≥2 000 g, and an age of ≤28 days. The neonate′s parents used the JCard to measure jaundice at the neonate′s cheek. Within 2 hours of the JCard measurement, transcutaneous bilirubin (TcB) was measured with a JH20-1B device and total serum bilirubin (TSB) was detected. The Pearson′s correlation analysis, Bland-Altman plots and the receiver operating characteristic (ROC) curve were used for statistic analysis.Results:Out of the 854 newborns, 445 were male and 409 were female; 46 were born at 35-36 weeks of gestational age and 808 were born at ≥37 weeks of gestational age. Additionally, 432 cases were aged 0-3 days, 236 cases were aged 4-7 days, and 186 cases were aged 8-28 days. The TSB level was (227.4±89.6) μmol/L, with a range of 23.7-717.0 μmol/L. The JCard level was (221.4±77.0) μmol/L and the TcB level was (252.5±76.0) μmol/L. Both the JCard and TcB values showed good correlation ( r=0.77 and 0.80, respectively) and agreements (96.0% (820/854) and 95.2% (813/854) of samples fell within the 95% limits of agreement, respectively) with TSB. The JCard value of 12 had a sensitivity of 0.93 and specificity of 0.75 for identifying a TSB ≥205.2?μmol/L, and a sensitivity of 1.00 and specificity of 0.35 for identifying a TSB ≥342.0?μmol/L. The TcB value of 205.2?μmol/L had a sensitivity of 0.97 and specificity of 0.60 for identifying TSB levels of 205.2 μmol/L, and a sensitivity of 1.00 and specificity of 0.26 for identifying TSB levels of 342.0 μmol/L. The areas under the ROC curve (AUC) of JCard for identifying TSB levels of 153.9, 205.2, 256.5, and 342.0 μmol/L were 0.96, 0.92, 0.83, and 0.83, respectively. The AUC of TcB were 0.94, 0.91, 0.86, and 0.87, respectively. There were both no significant differences between the AUC of JCard and TcB in identifying TSB levels of 153.9 and 205.2 μmol/L (both P>0.05). However, the AUC of JCard were both lower than those of TcB in identifying TSB levels of 256.5 and 342.0 μmol/L (both P<0.05). Conclusions:JCard can be used to classify different levels of bilirubin, but its diagnostic efficacy decreases with increasing bilirubin levels. When TSB level are ≤205.2 μmol/L, its diagnostic efficacy is equivalent to that of the JH20-1B. To prevent the misdiagnosis of severe jaundice, it is recommended that parents use a low JCard score, such as 12, to identify severe hyperbilirubinemia (TSB ≥342.0 μmol/L).