Objective To express UL148 RNA of human cytomegalovirus (HCMV) clinical strains in vitro and to study its functions. Methods Urine of a newborn with HCMV infection was inocula-ted into human embryo lung cells. HCMV clinical strain was isolated and identified by multiplex PCR. UL148 gene was amplified and cloned into pGEM-T-Easy plasmid after double enzyme digestion. A recombi-nant plasmid was constructed and located at the downstream of the T7 promoter. The recombinant plasmid was identified by electrophoresis of the recombinant plasmid,PCR product and double enzyme product. Se-quencing analysis was used for final confirmation. UL148 was transcribed into RNA by 32P labeling. Post-translational modification sites were analyzed by bioinformatics method based on UL148 sequence characteris-tics. Results The clinical strain of HCMV was obtained in vitro. Electrophoresis and sequencing analysis confirmed the successful construction of the recombinant plasmid. UL148 RNA was transcribed in vitro by T7RNA polymerase. Post-translational modification sites showed that UL148 gene contained one cell adhe-sion sequence, one legume lectins beta-chain signature, two N-myristoylation sites, one casein kinase Ⅱphosphorylation site,seven protein kinase C phosphorylation sitse, one cAMP/cGMP-dependent protein ki-nase phosphorylation site, two N-glycosylation sites and one transmembrane region. Conclusion UL148 gene might encode a viral adhesion molecule involving in the signal transduction in host cells.

Objective: To investigate the UL148 gene function of human cytomegalovirus (HCMV) low passage clinic isolate and new strategies for anti-HCMV treatment, the DNA-based external guide sequences (EGSs) were designed to inhibit UL148 RNA expression. Methods: UL148 RNA secondary structure was analyzed by RNA structure technique, an appropriate region was chosen for DNA-based EGS57 synthesis, targeted the UL148 RNA. The M1RNA and UL148 RNA were generated by PCR for transcription in vitro. The UL148 RNA and M1RNA were transcribed in vitro under the function of T7 RNA polymerase. The UL148 was labelled by ³²P. The cleavage reactions were carried out by mixing up EGS, M1RNA with UL148 RNA for 1 h. The products were separated by urea denaturing polyacrylamide gel electrophoresis and detected with Typhoon Phosphor Imager. Results: UL148 RNA ranged from 58 to 72 sites was the binding position, and 57 was a cleavage site. EGS57 was designed and synthesized. EGS57 was combined with UL148 RNA to form the natural substrate of M1RNA. UL148 RNA and M1RNA were synthesized through T7 RNA polymerase catalyzing, and the products were conformed. After cleaving reactions, DNA-based EGS57 was shown to be able to cleave UL148 RNA efficiently in vitro by a complex with M1RNA. Conclusions UL148 RNA was cleaved efficiently by EGS57, and the cleaving site was conformed as expectation. It will provide the gene silent tool effectively for further study the function of UL148 gene.

Objective:To study the clinical phenotype and molecular genetic characteristics of a Chinese Han family with X-linked retinoschisis (XLRS), and to determine the associated gene variations.Methods:A pedigree investigation was performed.The clinical characteristics and pedigree analysis of a Han Chinese family line with XLRS was conducted in August 2021 at the Xiamen Eye Center Affiliated to Xiamen University.All patients and the carriers underwent comprehensive medical history collection and routine ophthalmological examinations, including visual acuity, non-contact tonometer, slit lamp microscope, direct ophthalmoscope, and optical coherence tomography.The proband and some patients underwent medical optometry, fundus photography or wide-angle fundus photography, and electroretinogram examination.Peripheral venous blood samples were collected from the family members, and whole exome sequencing (WES) analysis was performed on the proband samples.For variants screened by WES, the expanded verification in other patients and normal persons in the family was carried out by Sanger sequencing.Multiple bioinformatic tools were used to analyze the pathogenicity of variants.This study protocol was approved by the Ethics Committee of Xiamen Eye Center of Xiamen University (No.XMYKZX-KY-2021-012). Written informed consent forms were obtained from each subject or guardian of minors.CADD, FATHMM and other bioinformatics tools were used to analyze the pathogenicity of the variation sites.Results:The Han XLRS pedigree consisted of 8 individuals in 3 generations.Out of the 3 cases diagnosed with XLRS based on clinical evaluation, all were male.The mother of the proband was a carrier of related genes.There were 5 persons with normal phenotypes.There was no history of consanguineous marriages within the family, and the disease was shown to be intergenerational, which is consistent with the recessive inheritance of the X chromosome.None of the patients had a history of systemic disease or any other abnormal manifestations.The prevailing feature of ophthalmopathy was poor binocular vision since childhood.The proband and his younger brother had spoke split in the macula, and their grandfather showed atrophy of retinal nerve fibers.Genetic analysis revealed a hemizygous variation c. 214G>C: p.Glu72Gln in the RS1 gene in all the patients in this family.The proband's mother was heterozygous at this site, and all other phenotypically normal family members exhibited wild type at this site.This variant was predicted to be a deleterious variation and likely to cause disease based on bioinformatics analysis. Conclusions:The proband and patients in this Han Chinese family have the known c. 214G>C: p.Glu72Gln hemizygous variation of the RS1 gene and exhibit mild XLRS, which was consistent with the recessive inheritance of X chromosome.

To investigate the presence of integrated Epstein-Barr virus (EBV) DNA in the NK/T cell lymphoma (NKTCL) ge-nome and analyze the integration information in the genome of NKTCL cell lines. Methods: PCR and in situ hybridization were used to detect EBV infection in five EBV (+) NK/T samples and four EBV (-) NK/T samples provided by the biobanks of the First Affiliated Hospi-tal of Zhengzhou University. Whole-genome DNA of the samples was sequenced and subjected to bioinformatics analysis. Whole-ge-nome sequence alignment was used to identify the EBV integration sequence. BLAST analysis was used to compare EBV fasta files of the samples and EBV fasta library. CREST software was used to extract softclip reads, filter all paired reads, and enumerate their distri-bution on chromosomes. The integrated genomics viewer (IGV) was used to compare the distribution of reads in partial regions of chromosome. PCR was used to amplify the high-frequency integration region of the EBV DNA. The amplified fragments were sanger se-quenced. Results: EBV DNA and EBER expression were detected in five EBV (+) NK/T samples but not in the four EBV (-) NK/T samples. Sequencing depth, coverage depth, proportion of coverage, and proportion of alignment all met the requirements for subsequent re-search. Sequence alignment revealed that the captured sequences were viral sequences. Filtered reads were most numerous in EBV (+) NKTCL cell line SNK, YTS, and EBV (+) nasal NKTCL tissue. The reads were non-randomly enriched in chromosome 2. EBV DNA inte-gration in the 400 bp region of chr2:30234084-30234483 caused insertion or deletion in the chr2p23.1 site. Conclusions: EBV DNA is highly integrated in the chr2p23.1 site of EBV (+) NKTCL cells and may affect the expression of related genes.

An integrated segmentation method for 3D ultrasound carotid artery was proposed. 3D ultrasound image was sliced into transverse, coronal and sagittal 2D images on the carotid bifurcation point. Then, the three images were processed respectively, and the carotid artery contours and thickness were obtained finally. This paper tries to overcome the disadvantages of current computer aided diagnosis method, such as high computational complexity, easily introduced subjective errors et al. The proposed method could get the carotid artery overal information rapidly, accurately and completely. It could be transplanted into clinical usage for atherosclerosis diagnosis and prevention.

Objective To discuss the clinical efficacy and safety of hepatic arterial infusion chemotherapy(HAIC)combined with carrelizumab and sorafenib in treating advanced hepatocellular carcinoma(HCC).Methods The clinical data of 36 HCC patients,who were admitted to the Affiliated Nantong Third Hospital of Nantong University of China to receive HAIC combined with carrelizumab and sorafenib from August 2019 to August 2020,were collected.According to modified Response Evaluation Criteria in Solid Tumors(mRECIST),the objective response rate(ORR)and disease control rate(DCR)of the combination therapy were evaluated.The Common Terminology Criteria Adverse Events Version 5.0 developed by American National Cancer Institute was used to evaluate the clinical safety.Results After receiving 4 cycles of FOLFOX-HAIC,the ORR and DCR of the patients were 38.9％and 77.8％respectively.The patients were followed up for 30 months.The median progression-free survival(mPFS)was 306 days(95％CI:242.7-369.3),and the median overall survival(mOS)was 515 days(95％CI:2 482.5-547.5).After HAIC treatment,one patient was successfully changed to surgical operation.The overall incidence of adverse events were 100％.There were 9 adverse events(25％)above grade m,including severe abdominal pain(n=2,5.6％),nausea(n=1,2.8％),vomiting(n=1,2.8％),elevated alanine aminotransferase(n=3,8.3％),elevated aspartate aminotransferase(n=1,2.8％),and death due to pulmonary failure caused by severe immune-induced pneumonia(n=1,2.8％).Conclusion For the treatment of advanced HCC,HAIC combined with carrelizumab and sorafenib has better ORR and DCR with controllable safety,which provides a new option for the treatment of advanced HCC.However,studies with large sample size need to be conducted before its long-term survival benefit of patients can be further validated.

Objective:To analyze the epidemiological characteristics of untreated 16-25 years old people living with HIV-1 (PLWH) in Dehong on the China-Myanmar border during 2009 to 2017, by using molecular network method and to provide references for precise prevention and reduction of the spread of HIV-1 in Dehong.Methods:Screening people living with HIV-1, collecting blood sample and separating plasma, extracting RNA were performed to amplify HIV-1 pol sequence, construct molecular network by HIV-TRACE program and conduct statistical analysis. Results:Among the 573 infected persons in the group, 319 were Chinese (55.67%), 254 were Burmese (44.33%); 351 were males (61%), and 222 were females (39%); 404 had heterosexual transmission (HET, 70.51%), 110 people injected drugs (PWID, 19.20%), 51 men had sex with men (MSM, 8.9%); genotypes included 252 unique recombinant forms (43.98%), and 222 had circular recombinant forms (39.02%), 76 had HIV-1 C (13.26%) and 23 HIV-1 B (4.01%) infection. The 83 molecular networks constructed through HIV-TRACE involved 250 PLWH, 49% were the China-Myanmar mixed network (41/83). Myanmar citizens were at high risk of accessing the China-Myanmar mixed network ( AOR=2.676, p=0.002). Chinese male PWID network assortativity is 0.34, Myanmar male PWID was 0.14, MSM was 0.12. Conclusions:There is a continuous risk of cross-border transmission of HIV-1 in Dehong on the China-Myanmar border; attention should be paid to the mixed transmission of MSM and Myanmar male PWID populations with other transmission routes.

Objective: To investigate the expression and clinical significance of programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), and their receptor programmed cell death protein 1 (PD-1) in EBV-positive T/NK lymphoproliferative disease [Epstein-Barr virus-positive T/natural killer (NK)-cell lymphoproliferative disease, EBV(+)-T/NK-LPD]. Methods: The pathological paraffin-embedded tissues of 17 patients with EBV(+)-T/NK-LPD from the First Affiliated Hospital of Zhengzhou University from January 2013 to December 2017 were collected. These patients include 12 males and 5 females, aged 10-82 years old, the average age being 29 years, 4 people in gradeⅠ, 7 in gradeⅡ, 3 in gradeⅢ, and 3 people with hydroa vacciniforme-like lymphoproliferative disorders. Immunohistochemical SP method was used to detect the expression of PD-1, PD-L1, and PD-L2 in human EBV(+)-T/NK-LPD tissues. The relationship between PD-1, PD-L1, PD-L2 expression, and clinicopathological parameters, pathological grades and prognosis were analyzed by Fisher's exact probabilities and Spearman rank correlation. Result: After statistical analysis, the results showed that in 17 cases of tissue samples, there were 12 cases with positive PD-1 expression, 6 cases with positive PD-L1 expression and 5 cases with positive PD-L2 expression. There was no significant correlation between PD-1 and PD-L2 expression and prognosis (P>0.05). PD-L1 expression showed a positive correlation with prognosis (P<0.05). There was no significant correlation between the expression of PD-L1 and PD-L2 with age, sex, as well as LDH and Ki-67 levels (P>0.05). Moreover, there was no significant correlation of PD-1 and PD-L2 expression with pathological grade (r=0.141, r=-0.149, both P>0.05). However, there was a negative correlation between the PD-L1 expression and pathological grade (r=-0.563), and the correlation between the PD-L1 ex-pression and pathological grade was statistically significant (P<0.05). Conclusions: PD-1, PD-L1, and PD-L2 are abnormally expressed in the pathological tissues of EBV(+)-T/NK-LPD. Although there was no significant correlation between the expression of PD-1 and prognosis or pathological grade, it was significantly higher in EBV+T/NK-LPD. PD-1/PD-Ls associated signaling pathway is expected to be a potential new target for EBV(+)-T/NK-LPD immunotherapy.

Gene editing technology has been a hotspot in the field of biotechnology. CRISPR/Cas systems are efficient gene editing tools because of its specificity, simplicity and flexibility, these features enabled the rapid application of CRISPR/Cas systems in a variety of organisms. Moreover, the combination of transcriptional activator with dead Cas protein can achieve specific regulation of gene expression at the transcription level, which has made important contributions to the development of biotechnology in medical and agriculture. Overexpression of foreign genes is a common method to verify gene function and regulation. However, due to the limitation of vector capacity, it is difficult to achieve overexpression of multiple genes. CRISPR/Cas9 activation system can regulate the expression of multiple genes under the guidance of different guide RNAs to verify gene functions at the regulatory level. This review summarizes the composition of the CRISPR/Cas9 activation system and different activation strategies, and summarizes solutions for excessive activation. It may facilitate the application of CRISPR/Cas9 activation system in genetic improvement of cotton and herbicide resistance research.
Biotechnology ; CRISPR-Cas Systems/genetics* ; Gene Editing ; Phenotype ; RNA, Guide, Kinetoplastida/metabolism*