Transaminases are a class of enzymes that catalyze the transfer of amino between amino acids and keto acids, playing an important role in the biosynthesis of organic amines and the corresponding derivatives. However, natural enzymes often have low catalytic efficiency against non-natural substrates, which limits their widespread applications. Enzyme engineering serves as an effective approach to improve the catalytic properties and thereby expand the application scope of transaminases. In this study, a high-throughput screening method for transaminases was established based on the fluorescent color reaction between methoxy-2-aminobenzoxime (PMA) and ketones. According to the changes in fluorescence intensity, the concentration changes of ketones could be easily monitored. The efficiency, sensitivity, and accuracy of the screening method were improved by optimization of the system. With 4-hydroxy-2-butanone as the substrate, the mutant library of the transaminase from Actinobacteria sp. was established and a mutant with increased activity was successfully obtained, which improved the production efficiency of (R)-3-aminobutanol by enzyme-catalyzed synthesis. This study laid an important foundation for efficient screening, modification, and application of transaminase.
Transaminases/metabolism* ; Fluorescent Dyes/chemistry* ; High-Throughput Screening Assays/methods* ; Ketones/metabolism* ; Actinobacteria/enzymology*

Objective:To investigate the clinicopathological characteristics, molecular features, differential diagnosis and prognosis of renal cell carcinoma (RCC) with TFEB gene amplification.Methods:A total of 113 cases of unclassified RCCs and RCCs with TFEB positive expression were collected from the Affiliated Hospital of Qingdao University and Navy 971 Hospital from January 2010 to December 2024. Eight cases of RCCs with TFEB amplification were identified using tissue microarrays, immunohistochemistry, and fluorescence in situ hybridization (FISH) techniques. The clinicopathological data and prognosis of the 8 cases were summarized, and relevant literature was reviewed.Results:Among the 8 cases, there were 5 males and 3 females. The average age was 63.4 (54, 77) year and the median age was 63.5 (59.0, 65.5) year. Seven cases were detected through physical examination, and 1 case presented with initial symptoms of metastasis to bones and lungs. The cohort included 1 biopsy specimen and 7 surgical resection specimens. The tumor diameters ranged from 2.5 to 15.0 cm. The cut surfaces of 5 cases were grayish-yellow or grayish-red, and 2 cases exhibited a colorful appearance, among which 3 cases involved renal sinus and 1 case showed invasion of the perirenal fat tissue. Microscopically, 4 cases were composed of clear cells arranged in solid sheets or acinar structures, along with varying numbers of eosinophilic cells. Two cases exhibited the morphology of high-grade eosinophilic RCC, and 1 case presented biphasic morphology with diffuse polygonal eosinophilic tumor cells and dense small cell components. The remaining 1 case exhibited the morphology of clear cell RCC. According to the WHO/ISUP nuclear grading system, 6 cases were Grade 3 and 2 cases were Grade 2. Multifocal necrosis was observed in 4 cases. In 4 surgical specimens, the tumor tissue invaded the renal parenchyma, with 2 cases showing nodular infiltration to surrounding tissues and 1 case with intravascular tumor thrombus. Immunohistochemical results showed varying degrees of TFEB nuclear positivity in 6 cases (6/8). Melanocytic markers such as Melan A (5/8) and HMB45 (3/8) were expressed at varying degrees. Cathepsin K (6/8), GPNMB (6/8), P504s (7/8) and CD10 (7/8) were positively expressed in most cases. FISH results revealed high-copy amplification of TFEB gene in 4 cases (partially showing clustered amplification) and low-copy amplification in 4 cases. During the follow-up period of 3 to 64 months of the 8 cases, 3 cases metastasized and 2 cases died of disease (both with high-copy TFEB gene amplification).Conclusions:RCC with TFEB gene amplification is rare and exhibits diverse morphological features. A common morphological characteristic of this type of tumor is a mixture of sheet-like clear cells and high nuclear grade eosinophilic cells. Combined immunohistochemical staining for TFEB, melanocytic markers, and GPNMB is helpful for the diagnosis of the tumor, and FISH detection of TFEB gene amplification is the most definitive method in diagnosing this tumor. RCC with TFEB gene amplification usually presents with strong aggressiveness and poor prognosis. Combining surgical resection with immunotherapy or VEGFR-targeted drugs might have therapeutic effects on the tumor.

Crigler-Najjar syndrome (CNS) and Gilbert syndrome (GS; OMIM: 143500) are rare autosomal recessive diseases that cause unconjugated hyperbilirubinemia due to decreased UGT1A1 enzyme activity. Crigler-Najjar syndrome type 2 (CNS2; OMIM: 606785) increases the risk of gallbladder stone formation and cholecystitis, while GS seldom causes health issues. We found a 28-year-old male patient with recurring right upper abdomen pain who experienced persistent jaundice from birth. CNS2 with gallbladder stones and cholecystitis was diagnosed after genetic testing revealed rare double homozygous mutations A(TA)₇TAA (rs3064744) and P229Q (rs35350960) in the UGT1A1 gene. After pedigree investigation, we found that the patient's parents with modestly increased bilirubin had compound heterozygous mutations A(TA)₇TAA and P229Q, which were GS. Bioinformatics analysis showed that A(TA)₇TAA is in the TATA-box region of the gene UGT1A1 promoter, affecting gene transcriptional initiation, whereas P229Q modifies protein three-dimensional structure and may be harmful. In this pedigree, double homozygous mutations have a more severe phenotype than compound heterozygous mutations. Inherited causes of hyperbilirubinemia should be suspected after ruling out biliary obstruction, and early bilirubin reduction (< 103 µmol/L (6 mg/dL)) may reduce the risk of complications like cholecystitis in CNS2 patients, though further studies with longer follow-up are needed to confirm this observation.
Humans ; Male ; Glucuronosyltransferase/genetics* ; Adult ; Crigler-Najjar Syndrome/complications* ; Cholecystitis/etiology* ; Homozygote ; Mutation ; Pedigree

Objective:To analyze the association between daily physical activity patterns and dyslipidemia among people receiving physical examination aged 40-65 years.Methods:This cross-sectional study consecutively enrolled 864 participants aged 40-65 years and met the inclusion and exclusion criteria who underwent health check-ups at the Physical Examination Center of Fuquan First People′s Hospital from March to November in 2022. The data of general characteristics, physical activity, physical examination findings, and lipid profiles were collected. The daily physical activity patterns were identified using K-means clustering analysis. The unconditional binary logistic regression was employed to explore the associations between these activity patterns and dyslipidemia, followed by subgroup analyses.Results:The physical activity of the 864 study participants (517 males and 347 females) included in the analysis was divided into 4 patterns (G1: low physical activity; G2: active commuting; G3: housework; G4: leisure exercise). Using G1 as a reference, after adjusting for confounders, G4 was negatively associated with low high density lipoprotein cholesterol (HDL-C) ( OR=0.37, 95% CI: 0.14-1.00) ( P=0.05). In the male, G3 was negatively associated with dyslipidemia ( OR=0.44, 95% CI: 0.21-0.93) and low HDL-C ( OR=0.25, 95% CI: 0.10-0.68) (both P<0.05). In the subjects aged 50 years and above, G2 was negatively associated with dyslipidemia ( OR=0.52, 95% CI: 0.30-0.90), hypertriglyceridemia ( OR=0.50, 95% CI: 0.28-0.90) and low HDL-C ( OR=0.47, 95% CI: 0.24-0.91) (all P<0.05). In those who never or occasionally stayed up late, G2 was negatively associated with hypertriglyceridemia ( OR=0.31, 95% CI: 0.13-0.75) ( P<0.05); in those who stayed up late often, G4 was negatively associated with dyslipidemia ( OR=0.33, 95% CI: 0.13-0.85) and low HDL-C ( OR=0.19, 95% CI: 0.04-0.84) (both P<0.05). In the centrally obese population, G2 was negatively associated with dyslipidemia ( OR=0.55, 95% CI: 0.35-0.88) and hypertriglyceridemia ( OR=0.54, 95% CI: 0.33-0.86) (both P<0.05). Conclusions:Association between different physical activity patterns and dyslipidemia varied among adults aged 40-65 years undergoing health check-ups. Leisure-time exercise is associated with a reduced risk of dyslipidemia, while household activities also emerges as a beneficial factor linked to lower dyslipidemia risk particularly in the male population.

Objective:To investigate the clinicopathological characteristics, molecular features, differential diagnosis and prognosis of renal cell carcinoma (RCC) with TFEB gene amplification.Methods:A total of 113 cases of unclassified RCCs and RCCs with TFEB positive expression were collected from the Affiliated Hospital of Qingdao University and Navy 971 Hospital from January 2010 to December 2024. Eight cases of RCCs with TFEB amplification were identified using tissue microarrays, immunohistochemistry, and fluorescence in situ hybridization (FISH) techniques. The clinicopathological data and prognosis of the 8 cases were summarized, and relevant literature was reviewed.Results:Among the 8 cases, there were 5 males and 3 females. The average age was 63.4 (54, 77) year and the median age was 63.5 (59.0, 65.5) year. Seven cases were detected through physical examination, and 1 case presented with initial symptoms of metastasis to bones and lungs. The cohort included 1 biopsy specimen and 7 surgical resection specimens. The tumor diameters ranged from 2.5 to 15.0 cm. The cut surfaces of 5 cases were grayish-yellow or grayish-red, and 2 cases exhibited a colorful appearance, among which 3 cases involved renal sinus and 1 case showed invasion of the perirenal fat tissue. Microscopically, 4 cases were composed of clear cells arranged in solid sheets or acinar structures, along with varying numbers of eosinophilic cells. Two cases exhibited the morphology of high-grade eosinophilic RCC, and 1 case presented biphasic morphology with diffuse polygonal eosinophilic tumor cells and dense small cell components. The remaining 1 case exhibited the morphology of clear cell RCC. According to the WHO/ISUP nuclear grading system, 6 cases were Grade 3 and 2 cases were Grade 2. Multifocal necrosis was observed in 4 cases. In 4 surgical specimens, the tumor tissue invaded the renal parenchyma, with 2 cases showing nodular infiltration to surrounding tissues and 1 case with intravascular tumor thrombus. Immunohistochemical results showed varying degrees of TFEB nuclear positivity in 6 cases (6/8). Melanocytic markers such as Melan A (5/8) and HMB45 (3/8) were expressed at varying degrees. Cathepsin K (6/8), GPNMB (6/8), P504s (7/8) and CD10 (7/8) were positively expressed in most cases. FISH results revealed high-copy amplification of TFEB gene in 4 cases (partially showing clustered amplification) and low-copy amplification in 4 cases. During the follow-up period of 3 to 64 months of the 8 cases, 3 cases metastasized and 2 cases died of disease (both with high-copy TFEB gene amplification).Conclusions:RCC with TFEB gene amplification is rare and exhibits diverse morphological features. A common morphological characteristic of this type of tumor is a mixture of sheet-like clear cells and high nuclear grade eosinophilic cells. Combined immunohistochemical staining for TFEB, melanocytic markers, and GPNMB is helpful for the diagnosis of the tumor, and FISH detection of TFEB gene amplification is the most definitive method in diagnosing this tumor. RCC with TFEB gene amplification usually presents with strong aggressiveness and poor prognosis. Combining surgical resection with immunotherapy or VEGFR-targeted drugs might have therapeutic effects on the tumor.

Objective:To analyze the association between daily physical activity patterns and dyslipidemia among people receiving physical examination aged 40-65 years.Methods:This cross-sectional study consecutively enrolled 864 participants aged 40-65 years and met the inclusion and exclusion criteria who underwent health check-ups at the Physical Examination Center of Fuquan First People′s Hospital from March to November in 2022. The data of general characteristics, physical activity, physical examination findings, and lipid profiles were collected. The daily physical activity patterns were identified using K-means clustering analysis. The unconditional binary logistic regression was employed to explore the associations between these activity patterns and dyslipidemia, followed by subgroup analyses.Results:The physical activity of the 864 study participants (517 males and 347 females) included in the analysis was divided into 4 patterns (G1: low physical activity; G2: active commuting; G3: housework; G4: leisure exercise). Using G1 as a reference, after adjusting for confounders, G4 was negatively associated with low high density lipoprotein cholesterol (HDL-C) ( OR=0.37, 95% CI: 0.14-1.00) ( P=0.05). In the male, G3 was negatively associated with dyslipidemia ( OR=0.44, 95% CI: 0.21-0.93) and low HDL-C ( OR=0.25, 95% CI: 0.10-0.68) (both P<0.05). In the subjects aged 50 years and above, G2 was negatively associated with dyslipidemia ( OR=0.52, 95% CI: 0.30-0.90), hypertriglyceridemia ( OR=0.50, 95% CI: 0.28-0.90) and low HDL-C ( OR=0.47, 95% CI: 0.24-0.91) (all P<0.05). In those who never or occasionally stayed up late, G2 was negatively associated with hypertriglyceridemia ( OR=0.31, 95% CI: 0.13-0.75) ( P<0.05); in those who stayed up late often, G4 was negatively associated with dyslipidemia ( OR=0.33, 95% CI: 0.13-0.85) and low HDL-C ( OR=0.19, 95% CI: 0.04-0.84) (both P<0.05). In the centrally obese population, G2 was negatively associated with dyslipidemia ( OR=0.55, 95% CI: 0.35-0.88) and hypertriglyceridemia ( OR=0.54, 95% CI: 0.33-0.86) (both P<0.05). Conclusions:Association between different physical activity patterns and dyslipidemia varied among adults aged 40-65 years undergoing health check-ups. Leisure-time exercise is associated with a reduced risk of dyslipidemia, while household activities also emerges as a beneficial factor linked to lower dyslipidemia risk particularly in the male population.

OBJECTIVE To analyze the types and control measures of major microorganisms in pharmaceutical water systems, so as to provide guidance for effective control of pharmaceutical water systems. METHODS The main microbial species, abundance and harmfulness of drinking water, purified water and water for injection were reviewed, and the control measures on microorganisms in pharmaceutical water were discussed. RESULTS There were differences in the main microbial types in pharmaceutical water. Burkholderia cepacia complex and Ralstonia pickettii were conditioned pathogens in pharmaceutical water, thus causing certain biological safety hazards. CONCLUSION Pharmaceutical companies can strengthen the control of microorganisms in the water system by establishing microbial databases and common microbial strain banks at all levels. Trend analysis should to be conducted based on alert limits and action limits, so as to strengthen the control of microorganisms in the water system.

OBJECTIVE To establish a rapid and accurate PCR method for detecting 24 strains of Burkholderia cepacia complex(Bcc) by comparing three detection methods of loop-mediated isothermal amplification(LAMP), SYTO 9 dye method based on polymerase chain reaction(PCR) and TaqMan probe real-time fluorescent quantitative PCR method( TaqMan probe method). METHODS According to the molecular biological information of 24 strains of Bcc in the NCBI database, multiple candidate sequence fragments unique to Bcc were screened out, and specific primer and probe that could simultaneously detect 24 strains of Bcc were designed. At the same time, the detection methods of LAMP, SYTO 9 dye method based on PCR and Taqman probe were explored, and the optimal annealing temperature was optimized and screened. The 39 experimental strains were used to verify the Bcc detection method. RESULTS LAMP method could not effectively detect Bcc, SYTO 9 dye method and TaqMan probe method could effectively detect more than 20 strains of Bcc, while TaqMan probe method had higher amplification effect, better detection sensitivity, repeatability and stability, which could meet the requirements of this study. CONCLUSION In this study, a TaqMan probe method for rapid detection of Bcc was established. Compared with LAMP method and SYTO 9 dye method, this method has the advantages of fast, simple and high sensitivity, and provides technical support for the rapid detectionof Bcc.

Objective:To prepare seven standard strains of 2019 novel coronavirus (2019-nCoV), including wild type (WT) strain, Beta variant, Delta variant, Omicron variants (BA.2, BA.5, BQ.1, XBB.1 branches), which could be used to apply for national standard strains.Methods:According to cytopathic effect (CPE), virus titer, whole-virus-genome-sequencing and detection of mycoplasma, the basic biological characteristics of clonal isolation were determined through plaque purification technology.Results:The CPE was mainly characterized by cell shrinkage and exfoliation in Vero cells after infection with seven 2019-nCoV clonal isolations (WT, Beta, Delta, Omicron, BA.2, BA.5, BQ.1 and XBB.1 branches). The result of mycoplasma detection were negative and titers of the clonal isolation from 2nd to 5th generations were stable at 10 5-10 8 TCID 50/ml; the electron microscope showed that the virions were all round or elliptical, with a diameter between 60 nm and 140 nm. The subtypes of 7 strains were identified by whole-virus-genome-sequencing and phylogenetic analysis, with genomic stability after the fifth successive generations of clonal isolation. Conclusions:The series clonal isolation of 2019-nCoV with typical CPE of coronavirus, clear morphological structure, good viral activity and stable genetic characteristics were prepared, and they could be used to apply for national standard strains.

Objective To study the correlation between expression levels of serum microRNA(miRNA)-21 and miRNA-27b and the prognosis in colorectal cancer patients.Methods A total of 60 colorectal cancer pa-tients who were treated in the hospital from January to May 2021 were selected as the observation group.An-other 60 healthy volunteers who underwent the physical examination in the hospital during the same period were selected as the control group.The expression levels of serum miRNA-21 and miRNA-27b in the two groups,the expression levels of serum miRNA-21 and miRNA-27b levels in patients with different pathologi-cal features were detected and compared,and the survival prognosis of patients with different expression levels of serum miRNA-21 and miRNA-27b were compared.Results The expression levels of serum miRNA-21 and miRNA-27b in the observation group were significantly higher than those in the control group(P＜0.05).The expression levels of serum miRNA-21 and miRNA-27b were lower in patients with TNM stage Ⅲ-Ⅳand with lymph node metastasis than those in stage Ⅰ-Ⅱ and without lymph node metastasis respectively(P＜0.05).With follow-up of 2 years,the mean survival time in the miRNA-21 high expression group was(15.36±2.32)months,and the mean survival time in the low miRNA-21 expression group was(21.54±2.46)months.The mean survival time in the miRNA-27b high expression group was(16.11±3.20)months,and the mean survival time in the miRNA-27b low expression group was mean(21.37±2.55)months.The 1 and 2-year survival rates in the miRNA-21 high expression group and miRNA-27b high expression group were significantly lower than those in the miRNA-21 low expression group and miRNA-27b low expression group,respectively(P＜0.05).Serum miRNA-21 and miRNA-27b levels were negatively correlated with the survival prognosis of patients(P＜0.05).Conclusion Serum miRNA-21 and miRNA-27b are highly expressed in pa-tients with colorectal cancer,and the expression levels of both are negatively correlated with the prognosis of patients,and they could be used for clinical monitoring of patients'condition and prognosis status,which de-serves attention.