Objective:To investigate differences in bacterial and fungal microbiome between infected nails and healthy nails among patients with onychomycosis.Methods:Nail scraping samples were collected from infected nails and healthy nails of 31 patients with onychomycosis, who visited Dalian Dermatosis Hospital from August 2020 to July 2021. The total DNA of nail microbiota was extracted, and the V3-V4 regions of the bacterial 16S rDNA gene and the fungal internal transcribed spacer (ITS) region were amplified and sequenced using Illumina technology. The USEARCH and mothur softwares were used for data cluster analysis to obtain the operational taxonomic units (OTUs) , Wilcoxon rank sum test was used to analyze α diversity, analysis of similarities (ANOSIM) was performed to analyze β diversity, linear discriminant analysis of effect size (LEfSe) was performed to evaluate the species difference.Results:Among the 31 patients with onychomycosis, 16 were males and 15 were females. According to the age, they were divided into young group (18 - 35 years old, 10 cases) , middle-aged group (36 - 60 years old, 11 cases) , and elderly group (over 60 years old, 10 cases) . As the α-diversity analysis revealed, the infected nail group showed significantly decreased Shannon index ( W = 290, P = 0.007) , but significantly increased Simpson index ( W = 663, P = 0.010) compared with the healthy nail group, suggesting that the diversity and evenness of bacterial communities were lower in the infected nail group than in the healthy nail group; however, there was no significant difference in the diversity of fungal communities between the infected nail group and healthy nail group. The β-diversity analysis based on the unweighted-UniFrac distance matrix showed no significant difference in the fungal or bacterial community composition between the infected nail group and healthy nail group (bacterial communities: R = 0.0052, P = 0.331; fungal communities: R = 0.0036, P = 0.337) ; the β-diversity analysis based on the weighted-UniFrac distance matrix showed significant differences in the abundance of bacterial and fungal communities between the two groups (both P = 0.001) . In terms of the species composition, the bacterial flora with significantly decreased abundance in the infected nail group compared with the healthy nail group included Bacteroidetes, Proteobacteria, Betaproteobacteria, Burkholderiales, Ralstonia, Sphingomonas and Streptococcus, while the abundance of Bacilli, Bacillales and Staphylococcus was significantly higher in the infected nail group than in the healthy nail group. Compared with the healthy nail group, the fungal flora with significantly increased abundance in the infected nail group included Eurotiomycetes, Onygenales, Leotiomycetes-ord-incertae-sedis, Arthrodermataceae, Periconia, Erysiphe, Tilletiopsis, Trichophyton, Erysiphe cruciferarum, Trichophyton rubrum, Malassezia sympodialis, while the abundance of Saccharomycetes, Saccharomycetales, Saccharomycetaceae, Dothioraceae, Candida and Alternaria was significantly lower in the infected nail group than in the healthy nail group. Conclusion:The diversity and abundance of bacterial communities significantly differed between infected nails and healthy nails among patients with onychomycosis, while only the abundance of fungal communities differed between the two groups, and perhaps there was correlations between some bacterial and fungal communities.

Objective To evaluate the feasibitity of robot-assisted posterior laparoscopic modified"single-position"radical nephroureterectomy.Methods A retrospective analysis was made on 7 patients receiving robot-assisted posterior laparoscopic single-position radical nephroureterectomy between April 2022 and April 2023.The patients were in a fully healthy lateral position,and an artificial pneumoperitoneum was established.Trocars were placed at the right costal margin of the posterior axillary line,3-4 cm above the iliac crest of the midaxillary line,6-8 cm below the anterior axillary line,and 3-4 cm above the iliac crest of the midaxillary line near the outer edge of the musculus rectus abdominis,respectively.After the kidney was removed,the ureter was freed down to the iliac vessels,and then the main joint of the robot was reversed 180° for redocking.The ureter was continuously freed downwards to the bladder wall and the catheter was clamped.The bladder was opened after filling with indocyanine green and distilled water mixture.Then the fluid in the bladder was washed,the contralateral ureteral orifice was observed,the affected side of the ureter was resected,and the bladder incision was sutured by two layers with V-LOCK 2-0 sutures.The incision was extended under the right costal margin of the posterior axillary line and 3-4 cm above the iliac crest of the midaxillary line to remove the specimen.Results The operation was successfully completed in all the 7 cases.The surgical operation time was 155-263 min(mean,247.0 min)and the blood loss was 20-100 ml(mean,42.9 ml).The postoperative anal exhaust time was 14-24 h(mean,22.6 h).There were 1 case of postoperative absorption fever,2 cases of moderate anemia,and 2 cases of postoperative incision fat liquefaction.In the 2 patients with moderate anemia,one patient developed postoperative intramuscular artery rupture leading to massive bleeding and the formation of hematoma in the surgical area,with the amount of bleeding being approximately 1000 ml,and the other had moderate anemia before and after surgery.The hospital stay ranged 8-16 d(mean,11.6 d).Pathologic examinations showed high-grade uroepithelial carcer in all the patients.Postoperative follow-ups lasted 3-9 months,with a mean of 6.2 months.None had bladder tumor recurrence or distant metastasis.Conclusion Robot-assisted posterior laparoscopic modified"single-position"radical nephroureterectomy is safe and feasible.

In recent years, the genome editing technologies based on the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) have developed rapidly. The system can use homologous directed recombination (HDR) to achieve precise editing that it medicated, but the efficiency is extremely low, which limits its application in agriculture and biomedical fields. As an emerging genome editing technology, the CRISPR/Cas-mediated DNA base editing technologies can achieve targeted mutations of bases without generating double-strand breaks, and has higher editing efficiency and specificity compared with CRISPR/Cas-mediated HDR editing. At present, cytidine base editors (CBEs) that can mutate C to T, adenine base editors (ABEs) that can mutate A to G, and prime editors (PEs) that enable arbitrary base conversion and precise insertion and deletion of small fragments, have been developed. In addition, glycosylase base editors (GBEs) capable of transitioning from C to G and double base editors capable of editing both A and C simultaneously, have been developed. This review summarizes the development, advances, advantages and limitations of several DNA base editors. The successful applications of DNA base editing technology in biomedicine and agriculture, together with the prospects for further optimization and selection of DNA base editors, are discussed.
Agriculture ; CRISPR-Cas Systems/genetics* ; DNA/genetics* ; Gene Editing ; Technology

Objective:To investigate the effects of body mass index (BMI) levels at different baseline on the risk of new-onset acute pancreatitis (AP).Methods:The subjects were from the Kailuan Study Cohort and divided into 3 groups according to baseline BMI levels: BMI<24 kg/m 2, normal weight; BMI 24-28 kg/m 2, overweight; BMI≥28 kg/m 2, obesity. The incidence of new-onset AP in these three groups was analyzed. The survival curve was plotted by Kaplan-Meier method, the cumulative incidence was calculated and tested by log-rank method. Multivariate Cox proportional hazards regression model was used to calculate HR of baseline BMI levels for AP. Results:A total of 123 841 subjects were included and followed up for (11.94±2.13) years, during which, 395 cases were found with AP. The incidence of AP was 2.67 per 10 000 person years in total population, and the incidences of AP were 2.20, 2.72 and 3.58 per 10 000 person-years in the normal, overweight and obesity groups, respectively. The cumulative incidences of AP was 0.32%, 0.40% and 0.49% in normal, overweight and obesity groups, respectively, which showed a significant inter-group difference by log-rank test ( χ2=13.17, P<0.01). The results of multivariable adjusted Cox proportional hazards regression model analysis indicated that obesity group ( HR=1.45, 95% CI: 1.10-1.92) had a higher risk for AP compared with the normal BMI group. The subgroup analyses by age and sex showed that compared with the normal weight group,the HRs for AP in the obesity group was 1.58(95% CI:1.14-2.19) and 1.40(95% CI:1.03-1.90) among subjects younger than 60 years old and male subjects, respectively. After excluded onset AP within two years from baseline,with a control group from normal weight,the results of multivariate Cox proportional hazards regression model analysis indicated that the AP in the obesity group was 1.60 (95% CI: 1.18-2.15). Conclusion:Obesity may increase the risk of developing AP, particularly among young and middle-aged men.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system is a hotspot of gene editing and gene expression research, in which CRISPR/Cas13 system provides a new direction for RNA interference and editing. In this study, we designed and synthesized the corresponding gRNAs of CRISPR/Cas13a and CRISPR/Cas13b systems in non-homologous end joining (NHEJ) pathway, such as Ku70 and Lig4, and then detected the expression of ku70 and lig4 in HEK293T cells. The CRISPR/Cas13a system could efficiently knockdown the mRNA expression of ku70 and lig4 more than 50%, and CRISPR/Cas13b system also suppressed ku70 and lig4 about 92% and 76%, respectively. Also, CRISPR/Cas13a, b systems could down-regulate Ku70 and Lig4 proteins level to 68% and 53%, respectively. The study demonstrates that the CRISPR/Cas13 system could effectively knockdown the expression of RNA and protein in HEK293T cells, providing a new strategy for gene function and regulation research.
CRISPR-Cas Systems ; DNA Ligase ATP ; genetics ; Gene Expression Regulation ; genetics ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Ku Autoantigen ; genetics

Objective:To analyze the subcellular localization of family of serine hydrolases 1 (FSH1) protein in Microsporum canis. Methods:The FSH1 and enhanced green fluorescent protein (EGFP) genes were amplified by PCR using the previously constructed plasmid containing the FSH1 gene and the recombinant plasmid pCAMBIA-LRP-EGFP as the template; the vector DNA was obtained by double-enzyme digestion of the recombinant plasmid pCAMBIA-LRP-EGFP with SnaBI/KpnI. Then, the EGFP expression plasmid and Ptrcp-FSH1-EGHP-Ttrcp fusion plasmid were constructed by inserting the amplified EGFP gene and EGFP-FSH1 gene into the vector DNA respectively, and identified by PCR and sequencing. The two recombinant plasmids were transformed into Microsporum canis by an Agrobacterium tumefaciens-mediated method, and the gene EGFP and fusion gene FSH1-EGFP were expressed integratedly in Microsporum canis under the regulation by the fungal universal promoter Ptrpc and terminator Ttrpc. The cellular localization of the fusion protein was observed by laser scanning confocal microscopy. Results:The Agrobacterium tumefaciens-mediated transformation system and EGFP expression vector in Microsporum canis were successfully constructed; the fusion gene FSH1-EGFP was expressed integratedly in Microsporum canis. Laser confocal microscopy showed that fluorescence signals of the FSH1-EGFP fusion protein were concentrated in the cytoplasm and nuclei of Microsporum canis, with a granular or cluster-like appearance. Conclusion:The FSH1-EGFP fusion protein was successfully localized in the cytoplasm and nuclei of Microsporum canis, providing a basis for further clarifying the function and pathogenic mechanisms of the FSH1 gene in Microsporum canis.

Objective:To explore the correlation between the body mass index (BMI) trajectories and new-onset non-alcoholic fatty liver disease (NAFLD) so as to provide a scientific basis for the prevention and treatment of NAFLD.Methods:A total of 16388 observation subjects that met the inclusion criteria in the Kailuan study were used to form a cohort study. According to the BMI values of the observed subjects during annual physical examinations from 2006 to 2007, 2008 to 2009 and 2010 to 2011, SAS Proc Traj was used to determine four different BMI trajectories groups, namely, the low-stable medium-stable, medium-high and high-stable group. NAFLD incidence in each group was followed up during annual physical examinations from 2012 to 2013, 2014-2015 and 2016-2017. A total of 14998 observation subjects were finally included in the statistical analysis. The cumulative incidences of NAFLD differences in the four groups were compared. The Cox’s proportional hazards regression model was used to analyze the correlation between different BMI trajectories and new-onset NAFLD. One-way analysis of variance was used to compare the intergroup difference of measurement data, and pairwise comparisons were conducted. LSD test was used for the homogeneity of variance. Dunnett's T3 test was used for heterogeneity of variances. χ2 test was used to compare the count data, and the difference of NAFLD cumulative incidence rate between the different BMI trajectories groups was compared by log-rank test. Results:(1) the cumulative incidence of NAFLD was increased with the increase of BMI trajectories, which were 31%, 47%, 63%, 77%, respectively, and the difference was statistically significant ( P < 0.01). (2) after adjusting for multiple confounding factors such as age and gender with the Cox’s proportional hazards regression model, the risk of NAFLD in the BMI medium stable, medium-high, and high stable group was still 1.757 times [95% confidence interval ( CI): 1.589 ~ 1.942], 2.612 (95% CI: 2.353 ~ 2.900), 3.566 (95% CI: 3.129 ~ 4.064) of the low-stable group ( P < 0.01). Conclusion:The risk of NAFLD increases with increase of BMI trajectories, and long-term high levels of BMI are independent risk factors for the onset of NAFLD.

ObjectiveTo investigate the influence of dyslipidemia and number of items conforming to the diagnostic criteria for dyslipidemia on new-onset acute pancreatitis (AP). MethodsA prospective cohort study was performed for 99 695 on-the-job or retired workers of Kailuan Group who underwent the first physical examination from 2006 to 2007. According to the number of items conforming to the diagnostic criteria for dyslipidemia in the first physical examination, they were divided into G0 group with 69 465 workers who did not meet the diagnostic criteria for dyslipidemia, G1 group with 23 921 workers who met one item of the diagnostic criteria for dyslipidemia, G2 group with 5791 workers who met two items of the diagnostic criteria for dyslipidemia, G3 group with 500 workers who met three items of the diagnostic criteria for dyslipidemia, and G4 group with 18 workers who met four items of the diagnostic criteria for dyslipidemia. New-onset AP cases were collected once every year during follow-up, and a multivariate Cox proportional hazards regression model analysis was used to analyze the influence of dyslipidemia on new-onset AP cases. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the chi-square test was used for comparison of categorical data between groups. The Kaplan-Meier method was used to calculate the cumulative incidence rate of AP in each group, and the log-rank test was used for comparison of cumulative incidence rate between groups. ResultsThe total follow-up time of all 99 695 workers was 782 395 person-years, and since no new-onset AP cases were observed in G4 group, G4 group was combined with G3 group for analysis. The incidence rates of AP in G0, G1, G2, and G3 groups were 1.61, 2.05, 2.59, and 7.72 per thousand person-years, respectively, and the cumulative incidence rates of AP in these four groups were 1.76‰, 2.40‰, 3.12‰, and 8.39‰, respectively. The log-rank test showed a significant difference in cumulative incidence rate between groups (χ2=15.18, P=0.004 3). After adjustment for the other risk factors, the Cox model showed that the hazard ratio (95% confidence interval) for AP was 1.23 (0.88-1.73) in G1 group, 1.58 (1.09-2.10) in G2 group, and 4.90 (1.81-13.37) in G3 group. ConclusionDyslipidemia is a risk factor for new-onset AP, and the risk of AP increases with the increase in the number of items conforming to the diagnostic criteria for dyslipidemia.

Objective To determine the location of PQ-LRP protein in Microsporum canis using the enhanced green fluorescent protein(EGFP)as a marker.Methods The total RNA was extracted from Microsporum canis,and reversely transcribed into cDNA.The PQ-LRP gene was amplified by PCR using the above cDNA as the template.The fusion gene of PQ-LRP gene and EGFP gene was linked to the plasmid pCAMBIA 1300.Microsporum canis was subjected to Agrobacterium tumefaciens-mediated transformation,in order to achieve the integrated expression of the fusion gene LRP-EGFP in Microsporum canis under the regulation by the fungal universal promoter Ptrpc and terminator Ttrpc.Laser-scanning confocal microscopy was conducted to determine the cellular localization of the fusion protein.Results The expression vector pCAMBIA-LRP-EGFP was successfully constructed,and the fusion gene LRP-EGFP was expressed integratedly in Microsporum canis.Laser-scanning confocal microscopy showed that fluorescence signals of LRP-EGFP were concentrated on the cell membrane of Microsporum canis,giving a granular or cluster-like appearance.Conclusion The infusion gene LRP-EGFP can be successfully expressed in Microsporum canis,and PQ-LRP protein is located on the cell membrane of Microsporum canis.

Objective To identify the pathogenesis gene mutation of a pedigree with Cockayne syndrome.Methods The peripheral blood samples of the patient and his family members were collected and the genomic DNA was then extracted.Whole exome sequencing(WES)was performed for proband′s DNA.The disease-causing mutations were identified by bioinformatics analysis and pedigree analysis. Meanwhile,the mutations were confirmed by Sanger sequencing.Results Two novel mutations in ERCC8 gene,including c.400-2A >G and c.394_398delATGTA(p.L132fs)were identified in proband.The splicing mutation originated from his father and changed the splice acceptor site AG to GG, thus possibly caused alternative splicing.The c.394_398delATGTA(p.L132fs)frameshifting mutation was inherited from his mother.The proband′s sister also carried the same compound heterozygous mutation and had the same phenotype as proband.Conclusion The pathogenesis ERCC8 gene mutation of this pedigree with Cockayne syndrome was identified by using whole exome sequencing.