[Objective] To study the molecular biological mechanism for a case of ABO blood group B subtype, and perform three-dimensional modeling of the mutant enzyme. [Methods] The ABO phenotype was identified by the tube method and microcolumn gel method; the ABO gene of the proband was detected by sequence-specific primer polymerase chain reaction (PCR-SSP), and the exon 6 and 7 of the ABO gene were sequenced and analyzed. Homologous modeling of Bw.03 glycosyltransferase (GT) was carried out by Modeller and analyzed by PyMOL2.5.0 software. [Results] The weakening B antigen was detected in the proband sample by forward typing, and anti-B antibody was detected by reverse typing. PCR-SSP detection showed B, O gene, and the sequencing results showed c.721 C>T mutation in exon 7 of the B gene, resulting in p. Arg 241 Trp. Compared with the wild type, the structure of Bw.03GT was partially changed, and the intermolecular force analysis showed that the original three hydrogen bonds at 241 position disappeared. [Conclusion] Blood group molecular biology examination is helpful for the accurate identification of ambiguous blood group. Homologous modeling more intuitively shows the key site for the weakening of Bw.03 GT activity. The intermolecular force analysis can explain the root cause of enzyme activity weakening.

Objective: To explore the impacts of coal mine dust on lung function in rats, so as to provide the basis for the early prevention and treatment of coal worker's pneumoconiosis. Methods: Seventy-two SPF-grade 8-week-old male Sprague-Dawley rats were randomly divided into the coal dust group, the coal-silica dust group, the silica dust group and the control group. The rats in the first three groups of rats were administered 1 mL corresponding dust suspension into the lungs using non-exposure tracheal instillation, while the rats in the control group were administered 1 mL normal saline. Respiratory rate (f), forced vital capacity (FVC), peak expiratory flow (PEF) and dynamic pulmonary compliance (Cdyn) were measured at 1, 3 and 6 months after dust exposure. Lung tissues were collected to measure reactive oxygen species (ROS) and adenosine triphosphate (ATP) levels using corresponding ELISA kits and ATP assay kits, respectively. The relative mRNA expressions of peroxisome proliferators-activated receptor gamma coactivator 1-alpha (PGC-1α) and mitochondrial transcription factor A (TFAM) were detected using real-time fluorescent quantitative polymerase chain reaction assay. The relative protein expressions of PGC-1α and TFAM were detected using Western blotting. Results: There was no interaction between dust type and exposure duration on f (P>0.05), but there were interactions on FVC, PEF and Cdyn (all P<0.05). Compared with the control group at 6 months after dust exposure, the f of the rats in the silica dust group were increased, while the FVC and PEF of the rats in the coal-silica dust and silica dust groups were decreased, and Cdyn of the rats in the coal dust, coal-silica dust and silica dust groups were decreased (all P<0.05). There were interactions between dust type and exposure duration on ROS and ATP levels, the relative mRNA and protein expressions of PGC-1α and TFAM (all P<0.05). Compared with the control group at 3 and 6 months after dust exposure, the ROS levels in the rats in the coal dust, coal-silica dust and silica dust groups were increased, while the ATP levels, the relative mRNA and protein expressions of PGC-1α and TFAM were decreased (all P<0.05). Conclusion The lung function impairment in rats caused by different types of coal mine dust is related to PGC-1α-mediated mitochondrial biogenesis dysfunction, which leads to increased ROS levels, decreased ATP and TFAM levels.

[This corrects the article DOI: 10.1016/j.apsb.2022.05.019.].

Glycosylation modification is an important post-translational modification of proteins, which participates in regulating protein half-life, biological activity, and immunogenicity, thereby affecting their functions. Glycoproteins expressed in Pichia pastoris predominantly carry high-mannose type glycans, primarily composed of mannose residues, which starkly contrasts with the complex-type glycans synthesized by mammalian cells. This study aims to transform the high mannose glycosylation modification of P. pastoris into a hybrid glycosylation modification similar to that of mammalian cells through genetic engineering technology. We introduced the mannosidase Ⅰ gene (MDSⅠ) from Trichoderma viride and the human β-1,2-N-acetylglucosaminyltransferase I gene (GnTⅠ) into a previously constructed P. pastoris strain (∆och1) capable of producing Man₈GlcNAc₂ glycans. To precisely regulate the expression of MDSⅠ and GnTⅠ, we designed various promoter combinations, including the strong inducible AOX promoter and the constitutive GAP promoter. The receptor-binding domain (RBD, residues 377-588) of the spike protein from the Middle East respiratory syndrome coronavirus (MERS-CoV) was selected as the reporter protein for this investigation (MERS-RBD). The N-glycosylation profile of MERS-RBD was systematically analyzed using PNGase F digestion coupled with mass spectrometry. The results showed that after the knockout of och1 and the introduction of MDSⅠ and GnTⅠ genes with different promoter combinations, P. pastoris strains capable of producing GlcNAcMan₅GlcNAc₂ glycans were successfully generated. When the AOX promoter was used to control the MDSⅠ gene and the GAP promoter was used to control the GnTⅠ gene, the engineered strain exhibited the highest proportion of hybrid-type GlcNAcMan₅GlcNAc₂ glycans, which accounted for 68.38% of the total N-glycosylation. In conclusion, we successfully engineered a P. pastoris strain capable of synthesizing hybrid-type GlcNAcMan₅GlcNAc₂ glycans, establishing a foundation for subsequent research on the biosynthesis of complex-type N-glycans in P. pastoris.
Glycosylation ; Glycoproteins/genetics* ; Polysaccharides/metabolism* ; N-Acetylglucosaminyltransferases/metabolism* ; Pichia/metabolism* ; Humans ; Mannosidases/metabolism* ; Genetic Engineering ; Trichoderma/genetics* ; Recombinant Proteins/genetics* ; Saccharomycetales

Background Mitochondrial biogenesis is pivotal in coal workers' pneumoconiosis fibrosis, yet the role of the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)-nuclear respiratory factor 1 (NRF1)-mitochondrial transcription factor A (TFAM) pathway inmitochondrial biogenesis remains elusive, warranting further investigation. Objective To elucidate the role of the PGC-1α-NRF1-TFAM pathway in mitochondrial biogenesis in a rat coal workers' pneumoconiosis model through in vivo and in vitro experiments. Methods (1)n vivo: twelve SPF male SD rats (200-220 g) were randomized into a control group and a coal dust group (n=6 per group). After acclimatization, the coal dust group received 1 mL 50 mg·mL⁻¹ coal dust suspension via intratracheal instillation; the controls received saline. Lung tissues were harvested after two months for histopathology [HE, Masson, and transmission electron microscopy (TEM) ], protein and mRNA analysis, and mitochondrial DNA (mtDNA) quantification by quantitative real-time polymerase chain reaction (qPCR). (2) In vitro: rat lung type II epithelial cells (RLE-6TN) cells were exposed to coal dust (50, 100, 200, and 400 mg·L⁻¹, 24 h). CCK-8 assay determined optimal doses. Ultrastructural changes were analyzed by TEM. Cells were transfected with OE-PGC-1α (PGC-1α overexpression) or shRNA-PGC-1α plasmids (PGC-1α knockdown), and the transfection efficiency was determined by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The expression levels of alpah-smooth muscle actin (α-SMA), citrate synthase (CS), PGC-1α, NRF1, TFAM, and fibronectin (Fn) proteins and their corresponding mRNA were detected using Western blot and RT-qPCR, respectively. The relative content of mtDNA was determined by qPCR. Results In vivo: the control group lung samples exhibited soft, pink parenchyma, while the coal dust-exposed lungs showed blackened surfaces with soft texture. The histopathological evaluation revealed intact alveolar walls in the controls versus structural destruction, micro-nodules, and fibrotic areas in the coal dust group. After Masson staining, coal dust deposits were found surrounded by blue collagen fibers in the exposed lungs, but absent in the controls. The coal dust group displayed significant upregulation of fibrotic marker α-SMA and downregulation of mitochondrial biogenesis markers (CS, PGC-1α, NRF1, TFAM) and mtDNA compared to the controls (P<0.05). In vitro: coal dust exposure reduced cell density and induced morphological alterations. TEM revealed evenly distributed normal mitochondria in controls versus mitochondrial swelling, disrupted cristae, and reduced numbers in exposed cells. The mitochondrial biogenesis markers were elevated in the coal dust + OE-PGC-1α group compared to the coal dust + OE-NC group (P<0.05); in contrast, they were decreased in the coal dust + shRNA-PGC-1α group compared to the coal dust + shRNA-NC group (P<0.05). Compared to the control group, the expression levels of the fibrosis marker α-SMA mRNA and protein were increased in the coal dust group (P<0.05). Overexpression of PGC-1α reduced α-SMA expression, while downregulation of PGC-1α increased its expression (P<0.05). Conclusion Coal dust exposure induces mitochondrial dysfunction and pulmonary fibrosis in vivo and in vitro via the PGC-1α-NRF1-TFAM pathway dysregulation. Targeting this pathway may mitigate coal dust-induced fibrosis by restoring mitochondrial biogenesis.

Objective:To evaluate the efficacy and safety of hypomethylating agents (HMA) in patients with myelodysplastic syndromes (MDS) .Methods:A total of 409 MDS patients from 45 hospitals in Zhejiang province who received at least four consecutive cycles of HMA monotherapy as initial therapy were enrolled to evaluate the efficacy and safety of HMA. Mann-Whitney U or Chi-square tests were used to compare the differences in the clinical data. Logistic regression and Cox regression were used to analyze the factors affecting efficacy and survival. Kaplan-Meier was used for survival analysis. Results:Patients received HMA treatment for a median of 6 cycles (range, 4-25 cycles) . The complete remission (CR) rate was 33.98% and the overall response rate (ORR) was 77.02%. Multivariate analysis revealed that complex karyotype ( P=0.02, OR=0.39, 95% CI 0.18-0.84) was an independent favorable factor for CR rate. TP53 mutation ( P=0.02, OR=0.22, 95% CI 0.06-0.77) was a predictive factor for a higher ORR. The median OS for the HMA-treated patients was 25.67 (95% CI 21.14-30.19) months. HMA response ( P=0.036, HR=0.47, 95% CI 0.23-0.95) was an independent favorable prognostic factor, whereas complex karyotype ( P=0.024, HR=2.14, 95% CI 1.10-4.15) , leukemia transformation ( P<0.001, HR=2.839, 95% CI 1.64-4.92) , and TP53 mutation ( P=0.012, HR=2.19, 95% CI 1.19-4.07) were independent adverse prognostic factors. There was no significant difference in efficacy and survival between the reduced and standard doses of HMA. The CR rate and ORR of MDS patients treated with decitabine and azacitidine were not significantly different. The median OS of patients treated with decitabine was longer compared with that of patients treated with azacitidine (29.53 months vs 20.17 months, P=0.007) . The incidence of bone marrow suppression and pneumonia in the decitabine group was higher compared with that in the azacitidine group. Conclusion:Continuous and regular use of appropriate doses of hypomethylating agents may benefit MDS patients to the greatest extent if it is tolerated.

Objective To explore the trends and patterns of changes in airway inflammation,mitochondrial function,and arginine levels in patients with different degrees of asthma.Methods A total of 122 patients with acute asthma admitted to the Affiliated Traditional Chinese Medicine Hospital of Xinjiang Medical University from November 2016 to May 2017 were selected as the research subjects.According to the severity grading criteria,the patients were divided into the intermittent state group(n=45),mild asthma group(n=23),moderate asthma group(n=30),and severe asthma group(n=24).Another 44 healthy individuals who underwent physical examinations at the Medical Examination Center of the Affiliated Traditional Chinese Medicine Hospital of Xinjiang Medical University from November 2016 to May 2017 were selected as the healthy control group.About 18-20 mL of venous blood was extracted from each group of subjects.The levels of L-arginine(L-Arg)and asymmetric dimethylarginine(ADMA)were detected by high-performance liquid chromatography-tandem mass spectrometry,and the levels of type 1 protein arginine methyltransferase(PRMT1),dimethylarginine dimethylaminohydrolase 1(DDAH1),reactive oxygen species(ROS),thiobarbituric acid reactive substances(TBARS),8-isoprostaglandin(8-iso),lipid peroxide(LPO),and malondialdehyde(MDA)were detected by enzyme-linked immunosorbent assay.Platelets and mitochondria were extracted from each group of subjects.The platelet mitochondrial membrane potential(MMP)was measured using JC-1 flow cytometry,and the mitochondrial adenosine triphosphate(ATP)and cytochrome C oxidase(COX)activity were detected.Results The level of 8-iso in plasma of patients in the intermittent state group,mild asthma group,moderate asthma group,and severe asthma group were significantly higher than that in the healthy control group(P＜0.05);the level of 8-iso in plasma of patients in the severe asthma group was significantly higher than that in the intermittent state group,mild asthma group,and moderate asthma group(P＜0.05);the level of 8-iso in plasma of patients in the intermittent state group,mild asthma group,and moderate asthma group showed an increasing trend,but there was no statistically significant difference(P＞0.05).There was no statistically significant difference in the TBARS level in the plasma of subjects in the five groups(P＞0.05).The levels of ROS and MDA in plasma of patients in the intermittent state group,mild asthma group,moderate asthma group,and severe asthma group were significantly higher than those in the healthy control group(P＜0.05);the levels of ROS and MDA in plasma of patients in the moderate asthma group and severe asthma group were significantly higher than those in the intermittent state group and mild asthma group(P＜0.05);there was no statistically significant difference in plasma ROS and MDA levels between the intermittent state group and the mild asthma group(P＞0.05),and there was no statistically significant difference in plasma ROS and MDA levels between the moderate asthma group and the severe asthma group(P＞0.05).The plasma LPO level of patients in the intermittent state group,mild asthma group,moderate asthma group,and severe asthma group was significantly higher than that in the healthy control group(P＜0.05);the plasma LPO level of patients in the moderate asthma group and severe asthma group was significantly higher than that in the intermittent state group(P＜0.05);there was no statistically significant difference in plasma LPO level of patients in the mild asthma group,moderate asthma group,and severe asthma group(P＞0.05).The mitochondrial MMP,ATP,and COX activity in the intermittent state group,mild asthma group,moderate asthma group,and severe asthma group were significantly lower than those in the healthy control group(P＜0.05);there was no statistically significant difference in mitochondrial MMP,ATP,and COX activity of patients in the intermittent state group,mild asthma group,moderate asthma group,and severe asthma group(P＞0.05).The levels of plasma L-Arg,the ratio of L-Arg/ADMA,and DDAH1 in the intermittent state group,mild asthma group,moderate asthma group,and severe asthma group were significantly lower than those in the healthy control group(P＜0.05);there was no statistically significant difference in plasma L-Arg,the ratio of L-Arg/ADMA,and DDAH1 levels of patients in the intermittent state group,mild asthma group,moderate asthma group,and severe asthma group(P＞0.05).The plasma ADMA and PRMT1 levels showed no statistically significant difference among the five groups of patients(P＞0.05).Conclusion Asthma patients exhibit mitochondrial dysfunction and abnormal arginine metabolism at any stage of the disease.Although there is no statistically significant difference in the change trend at the cellular micro level among patients with different stages of asthma,the trend of changes in oxidative damage in the body is consistent with the aggravation of the disease.

Objective To explore the influencing factors of visual acuity recovery in patients with high myopia after posterior chamber phakic intraocular lens(ICL)implantation.Methods A prospective study was conducted on 210 pa-tients(420 eyes)with high myopia who underwent ICL implantation at the Second Affiliated Hospital of Zhengzhou Univer-sity from May 2021 to March 2023.The patients were divided into a good recovery group[best corrected visual acuity(BC-VA)recovery ≥0.3 D]and a poor recovery group(BCVA recovery＜0.3 D)based on their visual acuity recovery status three months after surgery.The baseline data of patients in the two groups were compared,and the factors affecting visual acuity recovery were analyzed using Logistic regression.The receiver operating characteristic(ROC)curve was used to an-alyze the predictive value of the Logistic regression model for poor visual acuity recovery in patients with high myopia after ICL implantation.Results Three months after surgery,149 patients(298 eyes)were in the good recovery group,and 61 patients(122 eyes)were in the poor recovery group.There were no significant differences in gender,age,years of myopi-a,body mass index,and academic performance between the two groups(all P＞0.05).The proportions of patients with corneal astigmatism＜1.30 D(55.74％),corneal diopter＜45 D(59.02％),Pittsburgh Sleep Quality Index(PSQI)＜7 points(63.93％),and average central radius of curvature[(7.82±0.27)mm]in the poor recovery group were lower than those in the good recovery group[83.89％,81.88％,85.91％,and(7.90±0.24)mm,respectively].The central flat me-ridian curvature(k1)of the anterior corneal surface[(43.27±1.43)D],steep meridian curvature(k2)of the anterior corneal surface[(44.84±1.53)D],and arch height[(628.49±67.28)μm]in the poor recovery group were higher than those in the good recovery group[(42.73±1.42)D,(44.12±1.47)D],and[(417.56±80.14)pm],with significant differences(all P＜0.05).Logistic regression analysis showed that corneal astigmatism,corneal diopter,k1,k2,arch height,and PSQI score were independent influencing factors of poor visual acuity recovery after ICL implantation in pa-tients with high myopia(all P＜0.05).ROC curve analysis showed that the area under the ROC curve for predicting poor visual acuity recovery after ICL implantation in patients with high myopia by Logistic regression model was 0.938(95％CI:0.896-0.966),the sensitivity was 83.61％,and the specificity was 91.95％(P＜0.05).Conclusion The visual acuity recovery after ICL implantation in patients with high myopia is affected by corneal astigmatism,corneal diopter,k1,k2,arch height,and PSQI score.The Logistic regression model based on these factors has high predictive value for visual acui-ty recovery after ICL implantation.

As non-invasive drug delivery systems,transdermal patches can deliver drugs through the intact skin at a fixed dose and a adjustable rate in order to product a systemic or local therapeutic effect.Focused on the key quality attributes of transdermal patches,the article illustrated the testing methods and how to control key points.It also briefly described the testing methods and standards of some other characteristics referring to pharmacopoeia,current policies and regulations in various countries,which provided a reference for pharmaceutical industries and relevant departments to improve the quality control methods and standards.

Objective:To establish a method for the determination of n-butylamine in the air of the workplace by ion chromatography.Methods:In February 2022, on-site sampling was carried out using an atmospheric sampler. N-butylamine was adsorbed by a neutral silica gel tube and then performed for qualitative and quantitative determination by ion chromatography after ultrasonic desorption with 10 mmol/L sulfuric acid solution.Results:The linear range of the method was 0.0375-100.0 μg/ml, the linear equation of the standard curve was y=0.0713 x-0.0327, the correlation coefficient was 0.9992. The detection limit of the method was 11.25 μg/L, and the lower limit of quantification was 37.50 μg/L, the lowest quantitative concentration was 0.025 mg/m 3 (in term of sampling 7.5 L). The average desorption efficiency of the method was 91.50%-95.38%, the precision was 1.10%-2.30%, the standard recovery was 83.83%-100.02%, sampling efficiency was 100.00%. Conclusion:This method is fast, sensitive and accurate, and can be used for the determination of n-butylamine in the air of workplace.