Aspergillus niger is an important industrial strain which has been widely used for production of enzymes and organic acids. Genome modification of A. niger is required to further improve its potential for industrial production. CRISPR/Cas9 is a widely used genome editing technique for A. niger, but its application in industrial strains modification is hampered by the need for integration of a selection marker into the genome or low gene editing efficiency. Here we report a highly efficient marker-free genome editing method for A. niger based on CRISPR/Cas9 technique. Firstly, we constructed a co-expression plasmid of sgRNA and Cas9 with a replication initiation region fragment AMA1 (autonomously maintained in Aspergillus) by using 5S rRNA promoter which improved sgRNA expression. Meanwhile, a strain deficient in non-homologous end-joining (NHEJ) was developed by knocking out the kusA gene. Finally, we took advantage of the instability of plasmid containing AMA1 fragment to cure the co-expression plasmid containing sgRNA and Cas9 through passaging on non-selective plate. With this method, the efficiency of gene editing reached 100% when using maker-free donor DNA with a short homologous arm of 20 bp. This method may facilitate investigation of gene functions and construction of cell factories for A. niger.
Gene Editing ; Aspergillus niger/genetics* ; CRISPR-Cas Systems/genetics* ; Plasmids/genetics*

OBJECTIVE: To understand the true perception and attitude of the healthcare workers(HCWs) on the workplace violence(WPV) in a secondary Grade A hospital.METHODS: A total of 27 HCWs in a secondary Grade A hospital were selected as study subjects using a typical sampling method. Semi-structured interviews were conducted to collect the information of their perception and attitude on the types and effects of WPV, as well as on the coping measures and the work stress. The characteristics of WPV in the hospital were analyzed. RESULTS: The interviewees believed that verbal abuse was the most common type of WPV received by HCWs. Nurses and female workers were the main victims of WPV. Horizontal violence among colleagues and sexual harassment should not be ignored. Common causes of violent incidents for patients and their family members against HCWs included the fees for treatment, doctor-patient communication, patients alcohol abuse and treatment outcomes not meeting patients′ expectation. About half of the interviewees said that WPV had serious impact on their mental health. The follow-up coping measures to violent incidents were mostly to reassurance to patients with unconditional apology from the HCWs. More than half of the interviewees considered that they had acceptable workload, while some interviewees considered their work to be stressful. The interviewees suggested that improving doctor-patient communication, hospital systems, and the professional knowledge of HCWs were the recommended means to prevents and control the WPV. CONCLUSION: The perception and attitude of HCWs on WPV in this hospital are relatively scattered. Considering the complex causes, the serious consequences, and difficult coping measures, the hospitals, HCWs and all sectors of society need to take comprehensive measures to prevent WPV towards HCWs.

Objective To analyze the epidemiological characteristics of reported imported malaria cases in Zhengzhou City from 2016 to 2020, so as to provide insights into the management of imported malaria in the city. Methods All data pertaining to cases with definitive diagnosis of malaria in Zhengzhou City from 2016 to 2020 were captured from the National Notifiable Disease Report System and the Information Management System for Parasitic Disease Control in China, including individual demographic data, and malaria onset, initial diagnosis and definitive diagnosis data. All data were descriptively analyzed. The duration from malaria onset to initial diagnosis, from initial diagnosis to definitive diagnosis and from onset to definitive diagnosis was compared among cases. In addition, the diagnoses of imported malaria cases in which definitive diagnosis was made were compared with the reexaminations by Zhengzhou Municipal Malaria Diagnosis Reference Laboratory. Results A total of 302 cases with definitive diagnosis of malaria were reported in Zhengzhou City from 2016 to 2020, and all were imported cases, with Plasmodium falciparum malaria as the predominant type (230 cases, 76.2%). There were 293 malaria cases imported from Africa (293 cases, 97.0%), which mainly included Nigeria (48 cases, 15.9%), Angola (40 cases, 13.2%), and the Democratic Republic of the Congo (29 cases, 9.6%). There was no obvious seasonality found in the date of malaria onset and time of reporting malaria. The ratio of male to female malaria cases was 49.3:1, and there were 103 cases (34.1%) with the current residency address in Zhengzhou City, 193 cases (63.9%) with the current residency address in other cities of Henan Province and 6 cases (2.0%) in other provinces of China. There were 271 cases (89.7%) seeking initial diagnosis in medical institutions, and the diagnostic accuracy of malaria was 56.6% (171/302) at initial diagnosis institutions. A total of 122 cases (40.4%) sought medical care on the day of malaria onset, and 252 cases (86.4%) within 3 days; however, only 22 cases (7.3%) were definitively diagnosed on the day of onset, and 162 cases (53.6%) diagnosed within 3 days. There were no significant differences between malaria cases seeking initial diagnosis at medical institutions and disease control and prevention institutions in terms of the duration from malaria onset to initial diagnosis (Z = −1.663, P > 0.05), from initial diagnosis to definitive diagnosis (Z = −0.413, P > 0.05) or from malaria onset to definitive diagnosis (Z = −0.838, P > 0.05). The median duration (interquartile range) from initial diagnosis to definitive diagnosis of malaria was 3.00 (2.00), 3.00 (6.00), 2.00 (4.00) d and 1.00 (1.00) d among cases seeking medical care at township-level and lower, county-, city- and province-level medical institutions, and the median duration from initial diagnosis to definitive diagnosis of malaria was significantly longer among cases seeking medical care at township-level and lower medical institutions than at city (Z = −3.286, P < 0.008 33) and province-level medical institutions (Z = −9.119, P < 0.008 33), while the median duration from initial diagnosis to definitive diagnosis [1.00 (3.00) d vs. 2.00 (4.00) d; Z = −4.099, P < 0.016] and from malaria onset to definitive diagnosis [3.00 (4.00) d vs. 4.00 (5.00) d; Z = −2.868, P < 0.016] among malaria cases with the current residency address in Zhengzhou City was both shorter than in other cities of Henan Province. The diagnostic accuracy was 89.1% (269/302) among malaria cases in which definitive diagnosis was made, and the accuracy of malaria reexaminations was 94.0% (284/302) in Zhengzhou Municipal Malaria Diagnosis Reference Laboratory. Conclusions P. falciparum malaria was predominant among reported imported malaria cases in Zhengzhou City from 2016 to 2020, and these imported malaria cases were predominantly diagnosed at medical institutions; however, the diagnostic capability of malaria is poor in township-level and lower medical institutions. Strengthening the collaboration between medical institutions and disease control and prevention institutions and improving the diagnostic capability building at medical institutions are recommended to consolidate malaria elimination achivements.

Objective To establish a method for determination of escitalopram in biological samples by ultrasound-assisted ionic liquid-dispersive liquid-liquid microextraction combined with gas chromatography-tandem mass spectrometry （GC-MS/MS） and provide evidences for forensic determination of cases related to escitalopram. Methods The 1-hexyl-3-methylimidazolium hexafluorophosphate （[C₆MIM][PF₆]） was selected as an extract solvent to process biological samples. Ultrasound-assisted extraction was used on the samples. Then the samples were detected by GC-MS/MS. Results The linear range of escitalopram in blood and liver were 5.56-1 111.10 ng/mL and 0.025-5.00 mg/g, respectively. The correlation coefficient （r） were greater than 0.999, limit of detection （LOD） were 4.00 ng/mL and 2.00 μg/g, limit of quantitation （LOQ） were 14.00 ng/mL and 6.00 μg/g, respectively. The extraction recovery rates were all greater than 50%, the interday and intraday precision were less than 20%. Escitalopram was detected in blood and liver samples from the actual poisoning case by this method with a content of 1.26 μg/mL and 0.44 mg/g, respectively. Conclusion The ultrasound-assisted ionic liquid-dispersive liquid-liquid microextraction combined with GC-MS/MS is environment friendly, rapid, has good enriching effect and consumes less organic solvent and can be used for forensic determination of escitalopram related cases.
Citalopram ; Gas Chromatography-Mass Spectrometry ; Limit of Detection ; Liquid Phase Microextraction ; Tandem Mass Spectrometry

Maxillary defects result in esthetic and functional defects in patients. Several techniques are available for ma-xillary reconstruction. Herein, we present a case of maxillary reconstruction with medial femoral condyle periosteal flap by intraoral anastomosis. The characteristics of medial femoral condyle periosteal flap and the advantages of intraoral anasto-mosis are introduced.
Anastomosis, Surgical ; Esthetics, Dental ; Femur ; Humans ; Maxilla ; Reconstructive Surgical Procedures ; Surgical Flaps

Objective: To explore the expressions of transient receptor potential vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1) in the dorsal root ganglion (DRG) and their action mechanisms in the rat model of orchialgia. METHODS: The models of orchialgia were established in male SD rats by injection of 2% acetic acid into the testis. Then the number of spontaneous pain responses and withdrawal latency in the model rats were recorded by behavioral tests and the expressions of TRPV1 and TRPA1 in T13－L1 DRGs determined by RT-qPCR, Western blot and immunofluorescence staining. RESULTS: Compared with the normal control rats, the orchialgia models showed a significant increase in the number of spontaneous pain responses (0.13 ± 0.35 vs 22.63 ± 3.42, P<0.01) and a decrease in the withdrawal latency at 4 hours after injection (［12.75 ± 1.50］ vs ［4.85 ± 1.00］ s, P<0.05). The mRNA expressions of both TRPV1 and TRPA1 were observed in the membrane of the neurons in the DRG, the former increased by 1.77 times and the latter by 1.75 times that of the control (P<0.05). CONCLUSIONS The expressions of TRPV1 and TRPA1 were up-regulated in the DRG of the rat models of orchialgia, which may be involved in the allodynia and hyperalgesia of the rats.
Acetic Acid ; Animals ; Ganglia, Spinal ; metabolism ; Hyperalgesia ; chemically induced ; metabolism ; Male ; Membrane Glycoproteins ; Oxidoreductases ; Rats ; Rats, Sprague-Dawley ; TRPA1 Cation Channel ; metabolism ; TRPV Cation Channels ; metabolism ; Testicular Diseases ; chemically induced ; metabolism ; Up-Regulation

Objective·To explore the association between neuropeptide Y (NPY) gene polymorphism and schizophrenia in Chinese Han population. Methods·Four single nucleotide polymorphism (SNP) loci (rs16148, rs1859290, rs16147, and rs16478 in NPY gene) were selected and genotyped by TaqMan genotyping assay in a case-control study with 678 schizophrenia cases (case group) and 685 healthy controls (control group). The allele, genotype and haplotype frequencies distribution of the SNPs between the groups were compared with SHEsis online software. Results·The distribution of genotype frequency of locus rs16478 showed a nominal statistically difference between the adult-onset schizophrenia (AOS group) and control group (χ2=6.66, P=0.036, P correction=0.144). Under recessive inherited model, the distribution of CC genotype frequency of locus rs1859290 showed statistically difference between the male schizophrenia cases and control group (P=0.012, OR=0.97, 95％CI 0.94-0.99, P correction=0.048). The distribution of haplotype CCTA (consisted of rs16148, rs1859290, rs16147, and rs16478) frequency showed statistically difference between the male AOS group and control group (8.1％ vs 13.2％, OR=0.57, P=0.010, P correction=0.040). Conclusion·The polymorphisms in NPY gene may be associated with schizophrenia in Chinese Han population.

ObjectiveTo evaluate the effect of ejaculatory duct dilation combined with seminal vesicle clysis in the treatment of refractory hematospermia.

METHODSUsing ureteroscopy, we treated 32 patients with refractory hematospermia by transurethral dilation of the ejaculatory duct combined with clysis of the seminal vesicle with diluent gentamicin.

RESULTSThe operation was successfully accomplished in 31 cases, with the mean operation time of 32 (26－47) minutes. The patients were followed up for 6－39 (mean 23.6) months. No complications, such as urinary incontinence and retrograde ejaculation, were found after operation. Hematospermia completely disappeared in 27 cases, was relieved in 1, and recurred in 3 after 3 months postoperatively. Those with erectile dysfunction or mental anxiety symptoms showed significantly decreased scores of IIEF-Erectile Function (IIEF-EF) and Self-Rating Anxiety Scale (SAS).

CONCLUSIONSEjaculatory duct dilation combined with seminal vesicle clysis under the ureteroscope, with its the advantages of high effectiveness and safety, minimal invasiveness, few complications, and easy operation, deserves general clinical application in the treatment of refractory hematospermia.

Dilatation ; Ejaculatory Ducts ; surgery ; Genital Diseases, Male ; Hemospermia ; surgery ; Humans ; Male ; Postoperative Period ; Recurrence ; Seminal Vesicles ; surgery ; Ureteroscopy

OBJECTIVETo evaluate the safety, feasibility and the long-term outcomes of laparoscopy-assisted gastrectomy (LAG) for advanced gastric cancer (AGC).

METHODSThe clinical and follow-up data of 46 cases after LAG from June 2008 to December 2009 were analyzed, and compared with 85 cases after conventional open gastrectomy (OG) for advanced gastric cancer at the same period at our hospital. The surgical safety, postoperative recovery, complications, radical degree, survival rate were compared.

RESULTAs compared with OG group, operation time was longer in LATG group ((274 ± 78) min vs. ( 217 ± 41) min, t = 4.635, P = 0.000). Estimated blood loss in the LAG group ((254 ± 112) ml) was significantly less than in the OG group (t = 3.942, P = 0.000). Time to ambulation ((63 ± 16) hours), first flatus ((77 ± 20) hours), resumed liquid diet ((88 ± 15) hours), duration of analgesic medication ((53 ± 20) hours) and postoperative hospital stay ((11.1 ± 4.6) days) were significantly shorter in the LAG group (t = 5.549, 6.508, 9.436, 9.464 and 2.980 respectively, all P < 0.01). The distance of the proximal and distal resection margin were (5.7 ± 1.4) cm and (3.9 ± 1.5) cm in LAG group, (5.8 ± 1.1) cm and (4.7 ± 1.5) cm in OG group respectively, but the difference was not significant. The number of lymph node dissections was also similar, (30.5 ± 10.4) in LAG group and (32.6 ± 12.3) in OG group (t = 0.960, P = 0.339). The incidence of postoperative complications and mortality rate in LAG group (8.7% and 0 respectively) were also lower than in the OG group, with no statistically significant difference (P > 0.05). The mean follow-up was 31.0 months (range 6-48 months), and the cumulative survival of the 2 groups was similar (χ(2) = 1.594, P = 0.207).

CONCLUSIONSLaparoscopy-assisted gastrectomy for advanced gastric cancer is not significantly different with open surgery in surgical safety, radical degree, and survival rate. It is less traumatic and of fewer complications.

Aged ; Female ; Gastrectomy ; methods ; Humans ; Laparoscopy ; Laparotomy ; Lymph Node Excision ; Male ; Middle Aged ; Postoperative Complications ; epidemiology ; Stomach Neoplasms ; surgery ; Survival Rate ; Treatment Outcome

OBJECTIVETo evaluate the correlation between programmed death-ligand 1 (PD-L1) expression in primary lung cancer cells, tumor associated macrophages (TAM) and patients' clinicopathological characteristics.

METHODSFrom 2008 to 2010, 208 non-small cell lung cancer patients who underwent surgery or CT-guided biopsy were recruited from Huadong Hospital, Fudan University. Immunohistochemistry staining was performed to evaluate the PD-L1 expression in both primary lung cancer cells and CD68 positive TAM. The relationship between PD-L1 expression and the clinical pathology was evaluated using χ(2) test. Spearman's rank correlations were used to determine the correlation between PD-L1 expression in tumor cells and macrophages.

RESULTSPositive PD-L1 expression in primary cancer cells was found in 136 (65.3%) patients, which were negatively correlated with lymph node metastasis (P=0.009) and smoking history (P=0.036). Besides, TAM with PD-L1 expression (found in 116 patients) was positively associated with smoking history (P=0.034), well-differentiation (P=0.029) and negative lymph node metastasis (P=0.0096). A correlation between PD-L1 expression in primary tumor cells and non-small cell lung cancer associated macrophages was found (r=0.228, P=0.021).

CONCLUSIONPD-L1, secreted from TAM, might induce cancer cells apoptosis, and decrease lymph node metastasis.

Adult ; Aged ; Aged, 80 and over ; Apoptosis ; B7-H1 Antigen ; secretion ; Carcinoma, Non-Small-Cell Lung ; pathology ; secretion ; Cell Line, Tumor ; Female ; Humans ; Lung Neoplasms ; pathology ; secretion ; Lymphatic Metastasis ; Macrophages ; pathology ; secretion ; Male ; Middle Aged ; Retrospective Studies