Objective: To explore the intra-host genetic evolution of HIV-1 pol gene via follow-up for treatment-naïve HIV infections, and estimate the infection time with Bayesian coalescent theory, so as to support the evaluation of HIV epidemic. Methods: Five cases were recruited and followed up. The pol gene fragments were amplified for the characteristics of transmitted drug resistance ( TDR ) by RT-PCR. Bayesian coalescent theory was utilized to construct maximum clade credibility ( MCC ) tree for genetic evolution and calculate the time to the most recent common ancestor ( tMRCA ). Results: The five cases were all male, and aged from 27 to 50 years old.Five to nine sampling times were obtained from each case, and the pol gene sequences from each case formed a unique subcluster (posterior probability: 100% ), with different evolution characteristics, in the MCC tree. The three cases in primary HIV-1 infection were estimated to be infected one to five months before the first positive reaction of HIV screening, whereas the two HIV-1 diagnosed cases at first screening were extrapolated to get infected fourteen months and seven months before diagnosis, respectively. One case with acute HIV-1 infection carried TDR mutation ( M46I ) , expressing fast disease progress and quasispecies variation. Conclusions The general infection time can be estimated by analyzing the characteristics of intra-host genetic evolution of HIV-1 pol gene with Bayesian coalescent theory, and this method can help to estimate the HIV epidemic.

TMTP1, a 5-amino acid peptide NVVRQ, obtained by using the flagella peptide library screening in our previous studies, can be used for the labeling of malignant in situ and metastatic lesions, and even micro-metastases. In this study, TMTP1 was assessed for its ability to specifically target the malignant hematopoietic cells and metastatic lesions of hematological malignancies. FITC-TMTP1 was chemically synthesized. Immunofluorescence assay and competitive test were carried out to determine the specific binding capacity of TMTPl to hematological malignant cell lines, including HL60, k562, SHI-1, Jurkat, Raji, El-4 and umbilical cord blood mononuclear cells. Mononuclear cells were isolated from the bone marrow of healthy subjects and patients with chronic myeloid leukemia. Then the cells were co-clutured with TMTP1 or scrambled peptides and the binding and affinity of TMTP1 peptide to the primary cells of hematological malignancies were flow cytometrically analyzed. The binding specificity of TMTP1 to target hematological malignancies was measured in vivo by intravenous injection of FITC-conjugated TMTP1 into El-4 lymphoma-bearing mice. The results showed that TMTP1 specifically bound to the cells of a series of hematological malignancies, including HL60, k562, Jurkat, Raji, El-4 and chronic myeloid leukemia primary cells but not to bone marrow mononuclear cells from healthy subjects. By contrast, TMTP1 could bind to the metastatic foci of lymphoma originating from the EL-4 cell line while the scrambled peptide failed to do so. Moreover, the occult metastases could be identified, with high specificity, by detecting FITC-TMTP1. We are led to conclude that TMTP1, as a novel tumor-homing peptide, can serve as a marker for primary malignant and metastatic lesions for the early diagnosis of hematological malignances and a carrier of anticancer drugs for cancer treatment.

TMTP1,a 5-amino acid peptide NVVRQ,obtained by using the flagella peptide library screening in our previous studies,can be used for the labeling of malignant in situ and metastatic lesions,and even micro-metastases.In this study,TMTP1 was assessed for its ability to specifically target the malignant hematopoietic cells and metastatic lesions of hematological malignancies.FITC-TMTP 1 was chemically synthesized.Immunofluorescence assay and competitive test were carried out to determine the specific binding capacity of TMTPI to hematological malignant cell lines,including HL60,k562,SHI-1,Jurkat,Raji,El-4 and umbilical cord blood mononuclear cells.Mononuclear cells were isolated from the bone marrow of healthy subjects and patients with chronic myeloid leukemia.Then the cells were co-clutured with TMTP1 or scrambled peptides and the binding and affinity of TMTP1 peptide to the primary cells of hematological malignancies were flow cytometrically analyzed.The binding specificity of TMTP 1 to target hematological malignancies was measured in vivo by intravenous injection of FITC-conjugated TMTP1 into El-4 lymphoma-bearing mice.The results showed that TMTP1 specifically bound to the cells of a series of hematological malignancies,including HL60,k562,Jurkat,Raji,El-4 and chronic myeloid leukemia primary cells but not to bone marrow mononuclear cells from healthy subjects.By contrast,TMTP1 could bind to the metastatic foci of lymphoma originating from the EL-4 cell line while the scrambled peptide failed to do so.Moreover,the occult metastases could be identified,with high specificity,by detecting FITC-TMTP1.We are led to conclude that TMTP1,as a novel tumor-homing peptide,can serve as a marker for primary malignant and metastatic lesions for the early diagnosis of hematological malignances and a carrier of anticancer drugs for cancer treatment.

Objective:To investigate the molecular expression and pathological features of endothelial cell (EC) in a murine model of choroidal neovascularization (CNV) based on single-cell RNA sequencing (scRNA-seq).Methods:Six C57BL/6 mice aged 6-8 weeks were randomly divided into two groups, with 3 mice in each group.Bilateral eyeballs were enucleated.The choroidal tissues from the two groups were isolated by shearing the complex and scraping the choroid, respectively.Single-cell suspension was prepared by continuous digestion with trypsin/type Ⅰ collagenase at 37 ℃, and the cell viability and EC ratio were detected by flow cytometry to determine the preparation method of single-cell suspension.Another 6 mice were randomly assigned into the control group and the CNV group, with 3 mice in each group.The CNV model was induced by laser photocoagulation and single-cell suspensions were prepared 7 days after modeling.Gene expression library construction was performed using the Chromi-um (10x Genomics) instrument.High throughput sequencing was performed using the Illumina Novaseq6000 to obtain the expression matrix.The EC subpopulations were classified according to previous researches and the Cellmarker database.Pseudo-time analysis was performed in EC, revealing the gene expression matrix of different states.CNV-EC were further selected with preliminary analysis of the expression characteristics.Another 6 mice were selected to establish the CNV model and eyeball frozen sections were prepared 7 days after modeling.Expression and distribution as well as the area percentage of EC marker Pecam1, mitochondrial outer membrane proteins Tomm20 and mt-Co1, and capillary markers Kdr and Plvap were observed by immunofluorescence staining, and the vascular diameter was calculated.The use and care of animals followed the ARVO statement.This study protocol was approved by the Experimental Animal Welfare and Ethics Committee of Air Force Military Medical University (No.20200181).Results:The cell viability of the single-cell suspension prepared from choroidal-scleral fragments and choroidal scrapings was 99.4% and 99.1%, respectively, both of which met the sequencing requirements.The percentage of EC detected by flow cytometry was approximately 1.58%.The scRNA-seq result revealed that both the normal control and CNV groups contained 13 choroidal cell clusters.Compared with the normal control group, the proportions of rod/cone photoreceptor cells, EC and hematopoietic cells all increased, while the retinal pigment epithelium (RPE) and Schwan cells reduced in the CNV group.Among all clusters, EC constituted 18.4%.The pseudo-time analysis demonstrated that EC could be further divided into 4 states.The percentage of state 2 EC was 29.1% in the CNV group, which was significantly higher than 9.5% in the normal control group.Differentially expressed gene analysis showed that the expression of mitochondrion-related genes, including mt-Nd4 and mt-Atp6, were upregulated in state 2 EC, while capillary-related genes, including Kdr and Esm1, were downregulated.Immunofluorescent staining revealed that the area of Tomm20 and mt-Co1 in Pecam1-positive EC in the CNV area was (19.50±4.68)% and (4.64±2.82)%, respectively, which were both higher than (3.00±2.09)% and (0.18±0.34)% in normal area ( t=7.88, 3.84; both at P<0.01). The area of Kdr and Plvap in Pecam1-positive EC in the CNV area was (1.50±0.29)% and (0.79±0.97)%, respectively, which were both lower than (31.30±5.44)% and (10.43±2.28)% in the normal area ( t=13.40, 9.48; both at P<0.01). The vascular diameter in the CNV area was (5.52±1.85)μm, which was larger than (4.21±1.84)μm in the normal area ( t=9.57, P<0.001). Conclusions:When CNV occurs, the proportion of EC in choroid increases, and CNV-EC shows pathologic features of mitochondrial metabolic activation and loss of capillary properties, suggesting the mitochondrial activation of EC may play a role in the formation of CNV.