Background::Total human immunodeficiency virus (HIV) DNA and integrated HIV DNA are widely used markers of HIV persistence. Droplet digital polymerase chain reaction (ddPCR) can be used for absolute quantification without needing a standard curve. Here, we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA.Methods::The limit of detection, dynamic ranges, sensitivity, and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat (LTR) and human CD3 gene (for total HIV DNA) and ACH-2 cells (for integrated HIV DNA). Forty-two cases on stable suppressive antiretroviral therapy (ART) were assayed in total HIV DNA and integrated HIV DNA. Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive (CD4 +) T-cell counts, CD8 + T-cell counts and CD4/CD8 T-cell ratio, respectively. The assay linear dynamic range and lower limit of detection (LLOD) were also assessed. Results::The assay could detect the presence of HIV-1 copies 100% at concentrations of 6.3 copies/reaction, and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction (95% confidence intervals [CI]: 3.6-6.5 copies/reaction) with linearity over a 5-log 10-unit range in total HIV DNA assay. For the integrated HIV DNA assay, the LLOD was 8.0 copies/reaction (95% CI: 5.8-16.6 copies/reaction) with linearity over a 3-log 10-unit range. Total HIV DNA in CD4 + T cells was positively associated with integrated HIV DNA ( r = 0.76, P <0.0001). Meanwhile, both total HIV DNA and integrated HIV DNA in CD4 + T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8 + T-cell counts. Conclusions::This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity. It can be readily adapted for measuring HIV DNA with non-B clades, and it could be beneficial for testing in clinical trials.

Compelling evidence has demonstrated the critical role of circular RNAs (circRNAs) during lung adenocarcinoma (LUAD) progression. Herein, we explored a novel circRNA, circ_0129047, and detailed its mechanism of action. The expression of circ 0129047, microRNA-665 (miR-665), and protein tyrosine phosphatase receptor type B (PTPRB) in LUAD tissues and cells was determined using reverse transcription quantitative polymerase chain reaction and Western blotting. Cell Counting Kit-8 and colony formation assays were conducted to detect LUAD cell proliferation, and western blotting was performed to quantify apoptosis-related proteins (Bcl-2 and Bax). Luciferase reporter and RNA immunoprecipitation assays were used to validate the predicted interaction between miR-665 and circ_0129047 or PTPRB.A xenograft assay was used for the in vivo experiments. Circ_0129047 and PTPRB were downregulated in LUAD tissues and cells, whereas miR-665 expression was upregulated. Overexpression of circ_0129047 suppresses LUAD growth in vivo and in vitro. Circ_0129047 is the target of miR-665, and the miR-665 mimic ablated the antiproliferative and pro-apoptotic phenotypes of LUAD cells by circ_0129047 augmentation. MiR-665 targets the 3ʹUTR of PTPRB and downregulates PTPRB expression. PTPRB overexpression offsets the pro-proliferative potential of miR-665 in LUAD cells. Circ_0129047 sequestered miR-665 and upregulated PTPRB expression, thereby reducing LUAD progression, suggesting a promising approach for preventing LUAD.

OBJECTIVE: To assess the value of copy number variation sequencing (CNV-seq) for revealing the genetic etiology of fetuses with isolated ventricular septal defect (VSD). METHODS: From December 2017 to December 2020, 69 fetuses with isolated VSD were identified at the First Affiliated Hospital of Zhengzhou University. Meanwhile, 839 similar prenatal cases were selected from public databases including Wanfang data, Wanfang Medicine, and China National Knowledge Infrastructure (CNKI) by using keywords such as "Ventricular septal defect", "Copy number variation", and "Prenatal". A total of 908 fetuses with isolated VSD were analyzed. CNV-seq was carried out for 69 fetuses. RESULTS: Among the 908 fetuses, 33 (3.63%) were found to harbor pathogenic CNVs, which included 11 chromosomal aneuploidies (1.21%) and 22 pathogenic CNVs (2.42%). The pathogenic CNVs have involved 12 genetic syndromes, with those known to involve the heart development including 5 cases of 22q11.21 deletion syndrome, 2 cases of 4q terminal deletion syndrome, and 1 case of 9q subtelomere deletion syndrome. The outcome of pregnancies for 15 fetuses with pathogenic CNVs was known, of which 12 were terminated, and 3 had spontaneous closure of the ventricular septum after birth, but 1 of them had other abnormalities. CONCLUSION Fetuses with isolated VSD have a relatively high risk for chromosomal abnormalities, for which CNV-seq should be recommended.
Female ; Pregnancy ; Humans ; DNA Copy Number Variations ; Heart Septal Defects, Ventricular/genetics* ; 22q11 Deletion Syndrome ; Fetus

OBJECTIVE: To detect variants of the MMACHC gene among 110 ethnic Han Chinese pedigrees affected with metabolic deficiency methylmalonic acidemia (MMA) of cobalamin C (cblC). METHODS: Peripheral blood samples were collected from the probands and their parents. Following DNA extraction, the coding regions of the MMACHC gene were subjected to PCR amplification, Sanger sequencing and quantitative PCR assaying. For 48 pedigrees, chorionic villus samples were taken for prenatal genetic diagnosis. RESULTS: Thirty five types of variants were detected among the 110 pedigrees, which included missense, nonsense, frameshifting, splicing variants and exonic deletions. Most variants have occurred in exons 4 (73.18%). The detection rate for c.609G>A (p.Trp203Ter) variant was the highest (33.64%), followed by c.658_660delAAG (12.27%), c.567dupT (9.09%) and c.80A>G (6.82%). Two variants, namely c.57_58insT (p.Gly20Trpfs*14) and c.505_506delAT (p.Ile169Argfs*12), were unreported previously and both were of frameshifting types. For the 48 pedigrees undergoing prenatal diagnosis, 14 fetuses were found to be normal, 24 have carried heterozygous variants, the remaining 10 have carried compound heterozygous or homozygous variants. CONCLUSION The discovery of the two novel variants has expanded the spectrum of the MMACHC gene variants among ethnic Han population. Above finding has provide a basis for the prenatal diagnosis and genetic counseling for the affected pedigrees.
Amino Acid Metabolism, Inborn Errors/genetics* ; China ; DNA ; Female ; Humans ; Mutation ; Oxidoreductases/genetics* ; Pedigree ; Pregnancy ; Prenatal Diagnosis ; Vitamin B 12/genetics*

Objective:To analyze the relationship between FAB morphological classification and World Health Organization (WHO) 2016 classification in children with acute erythroid leukemia(AEL), and to summarize the clinical features and prognosis.Methods:Clinical data of de nova childhood AEL patients from January 1, 2002 to December 31, 2019, in Pediatric Blood Disease Center, Institute of Hematology & Blood Disease Hospital were retrospectively analyzed.All of them were re-evaluated according to the WHO 2016 classification.Results:(1) A total of 20 patients were diagnosed as AEL by FAB classification.According to the criteria of WHO 2016, they were re-diagnosed as myelodysplastic syndromes (MDS)- refractory anemia with excess of blasts (11 cases), acute myeloid leukemia with MDS-related changes (3 cases), acute monocytic leukemia (1 case), and pure red leukemia (PEL, 5 cases). (2) Pathological hematopoiesis was frequently detected in bone marrow smears.Auer bodies were seen occasionally in some blasts.The most common antigen expressing were CD 117, CD 13, CD 33, CD 34, CD7, and CD 38.Karyotype analysis was performed in 18 cases successfully, involving 6 cases with abnormal karyotypes, including + 8, -7, 22p+ , t (3; 5: ? ), + 3q-, 15q-, and del (9)(q13). (3) Thirteen cases were treated by chemotherapy, and the one-course complete remission rate was 38.5%.By July 1, 2020, only 2 cases were alive without disease.The overall survival was 49 months and 11 months, respectively. Conclusions:Childhood AEL is susceptible to pathological hematopoiesis, poor response to early chemotherapy and poor prognosis.After re-evaluation according to WHO 2016 classification, most of them were diagnosed as MDS-related.Therefore, adjusting the suitable induction regimen with allogeneic hematopoietic stem cell transplantation may improve the prognosis.

Objective:To understand the dynamics of circulating Tfh (cTfh) and B cells, and their relationship in individuals with acute (AHI) and chronic HIV infection (CHI).Methods:HIV infected subjects and HIV negative healthy controllers (HC) were enrolled from the men who have sex with men(MSM) high-risk cohort at Beijing Youan hospital. Multicolor flow cytometry was used for the frequency and absolute number analysis of cTfh cell and B-cell subsets with AHI, CHI, and HC.Results:Compared to HC, AHI resulted in an increase in cTfh cell levels and persisted into CHI, however, there was no significant difference between AHI and CHI (AHI vs HC: 2.09±1.48 vs 0.26±0.38, t=5.25, P<0.001; CHI vs HC: 2.26±1.35 vs 0.26±0.38, t=6.25, P<0.001; AHI vs CHI: 2.09±1.48 vs 2.26±1.35, t=0.40, P=0.449). Among B cell subsets, activated memory (AM), tissue-like memory (TLM), and plasmablast (PB) cell levels increased [AM(AHI vs HC: 3.59±1.77 vs 0.83±0.44, t=6.65, P<0.001; CHI vs HC: 3.99±2.49 vs 0.83±0.44, t=5.46, P<0.001); TLM(AHI vs HC: 11.05±4.96 vs 1.30±0.93, t=8.45, P<0.001; CHI vs HC: 13.91±6.59 vs 1.30±0.93, t=8.28, P<0.001); PB( AHI vs HC: 3.01±2.50 vs 0.43±0.26, t=4.47, P<0.001; CHI vs HC: 1.88±1.57 vs 0.43±0.26, t=3.97, P<0.001)], whereas resting memory (RM) and na?ve mature (NM) cell levels decreased in both AHI and CHI [RM(AHI vs HC: 20.06±9.74 vs 25.43±10.91, t=1.70, P=0.040; CHI vs HC: 15.70±8.47 vs 25.43±10.91, t=3.29, P=0.003); NM(AHI vs HC: 55.71±13.88 vs 66.26±11.71, t=2.90, P=0.004; CHI vs HC: 58.33±14.47 vs 66.26±11.71, t=1.94, P=0.006)]. The levels of cTfh cells were positively correlated with those of AM ( r=0.67, P<0.001), classical memory (CM) ( r=0.59, P=0.001), RM ( r=0.47, P=0.010), and PB ( r=0.65, P<0.001), and negatively correlated with those of NM B cells in AHI ( r=-0.55, P=0.003). However, cTfh cells did not show any relationship with B cell subsets except for the positive correlation with PB ( r=0.56, P=0.003) in CHI. Conclusions:HIV infection drives the expansion of cTfh cells, which may in turn leads to perturbations of B cell differentiation during AHI stage.

Objective:To explore the laboratory characteristics and diagnostic methods for therapy-related acute megakaryocytic leukemia (t-AMKL).Methods:The data of one child with acute lymphoblastic leukemia (ALL) in the Blood Disease Hospital of Chinese Academy of Medical Sciences & Peking Union Medical College in September 2014 was retrospectively analyzed. After inducing remission for more than 43 months, the child was diagnosed as t-AMKL.Results:After the diagnosis of ALL, the child was given chemotherapy with standard childhood ALL regimen. After 43 months, t-AMKL was diagnosed by comprehensive morphology, cytogenetics, and molecular biology. Bone marrow morphology showed that the proportion of primitive cells was 0.44; flow cytometry showed the phenotype was abnormal myeloid primitive cells; the pathology result showed that the abnormal cells weakly expressed CD42b and CD61; the electron microscopy showed platelet peroxidase (PPO)-positive and myeloperoxidase (MPO)-negative; the bone marrow immunohistochemistry showed the positive rate of CD41 was 34%; the child had a complex karyotype. After reviewing his medical history, he was diagnosed as t-AMKL.Conclusion:The t-AMKL is relatively rare, and it is helpful to improve the prognosis of patients by completing the relevant examinations for early diagnosis.

Objective:To investigate the effectiveness of balloon-assisted technique for the treatment of intraprocedural aneurysmal rupture (IAR) during intracranial aneurysm coil embolization and its impact on the clinical outcomes of patients.Methods:Patients with intracranial aneurysm received coil embolization and complicated with IAR in Xijing Hospital of Air Force Medical University from January 2013 to January 2019 were enrolled retrospectively. They were divided into balloon-assisted hemostasis group and rapid packing hemostasis group according to the methods of intraoperative hemostasis. The modified Rankin Scale was used to evaluate the clinical outcomes at 3-month postoperative follow-up. A score of 0-2 was defined as a good outcome. Multivariate logistic regression analysis was used to identify the independent influencing factors of clinical outcome. Results:A total of 77 patients with IAR were enrolled, of which 46 (59.74%) used balloon-assisted hemostasis, and 31 (40.26%) used rapid packing hemostasis. In 51 patients (66.23%) with 3-month follow-up data, 32 (62.75%) had good outcomes, and 19 (37.25%) had poor outcomes. Univariate analysis showed that there were significant differences in time from IAR to treatment, time from IAR to confirmed hemostasis, postoperative Fisher grade changes, and good outcomes between the balloon-assisted hemostasis group and the rapid packing hemostasis group (all P<0.05). There were significant differences in IAR treatment methods, time from IAR to treatment, time from IAR to confirmed hemostasis, and postoperative Fisher grade changes between the good outcome group and the poor outcome group (all P<0.05). Multivariate logistic regression analysis showed that balloon-assisted hemostasis (odds ratio 0.234, 95% confidence interval 0.056-0.990; P=0.048) and time from IAR to confirmed hemostasis ≤10 min (odds ratio 0.097, 95% confidence interval 0.024-0.397; P=0.001) were the independent protective factors of the good outcomes in patients with IAR. Conclusion:Using balloon-assisted technique to treat IAR during intracranial aneurysm coil embolization can achieve satisfactory hemostatic effect and improve the clinical outcomes of patients.

Objective:Since December 2019, novel coronavirus infection has occurred in Hubei province and spread throughout the country quickly. This new crown viral pneumonia was named as coronavirus disease of 2019 (COVID-19) by WHO. However, at present, there is a high incidence of acute aortic dissection in winter and spring. How to prevent the spread of the epidemic and choose the appropriate treatment is an important topic for the patients with acute aortic dissection.Methods:From January 16, 2020 to February 26, 2020, a total of 37 of acute aortic dissection operations were carried out in several cardiovascular surgery centers in Hubei Province. There were 18 cases of Stanford type A aortic dissection and 19 cases of Stanford type B aortic dissection. There were 10 cases (55.55%) with ascending aorta replacement and 7 cases (38.89%) with Bentall procedure for aortic root surgery, and total arch replacement with stented elephant trunk implantation were performed in 14 cases (77.8%). In 19 patients with Stanford type B aortic dissection, thoracic endovascular aortic repair was performed, with the left subclavian artery chimney technique in 2 cases.Results:No deaths occurred within 30 days of hospitalization. Preoperative nucleic acid testing excluded 7 cases of novel coronavirus infection, and 3 suspected cases underwent emergency surgery. the three-level protective standard was adopted in the majority of the surgeries(62.2%, 23/37), and 11 patients were negative in the reexamination of viral nucleic acid after the operation.Conclusion:During the epidemic period, patients with acute aortic dissection should be carefully identified with actife COVID-19 before surgery. The treatment principles-" prevention and control of pneumonia epidemic should be emphasized, conservative medical management should be taken in the comfirmed cases, the selective operation should be delayed as far as possible, and the operation should be reasonable performed in critical cases" should be followed, which can save patients' lives to the greatest extent and prevent the spread of the virus.

Objective:To analyze the clinical characteristics and prognosis of mixed phenotype acute leukemia (MPAL) in children.Methods:The data of 29 children diagnosed as MPAL in the Pediatric Blood Disease Center, Institute of Hematology & Blood Disease Hospital, Chinese Academy of Medical Sciences from January 1, 2005 to December 1, 2019 were collected retrospectively. The morphology, immunophenotypes, cytogenetics, molecular biological characteristics, induction chemotherapy regimen, and prognosis were analyzed. Kaplan-Meier Method was used to draw survival curve. Log-Rank was used for univariate analysis.Results:(1) Among 29 MPAL cases, there were 1 case with KMT2A rearrangement, 1 case with BCR-ABL1, 13 cases with B/myeloid(B-M) type, 12 cases with T/myeloid(T-M) type and 2 cases with acute undifferentiated leukemia. (2) The common immunophenotypes were CD33 (23 cases, 79%), CD34 (25 cases, 86%) and HLA-DR (20 cases, 69%), and CD19 was positive in 17 cases (59%). (3) In molecular genetics analysis, 8 cases were detected to have abnormal gene fusion, including 1 case with MLL-AF4 fusion gene, 1 case with BCR-ABL1 fusion gene, 3 cases with TEL-AML1 fusion gene, 2 cases with WT1 and 1 case with FLT3-ITD. (4) In cytogenetics analysis, 27 cases obtained chromosome karyotypes, including 14 cases with abnormal karyotypes and 10 cases were complex karyotypes. (5) In treatment efficacy analysis, 27 cases received induction chemotherapy and the complete remission(CR) rate was 85%（23/27）.The 5-year disease free survival(DFS) rate was (71±10)% and 5-year overall survival(OS) rate was (74±10)%. Thirteen of 14 cases received acute lymphoblastic leukemia（ALL） induction therapy achieved CR, while 10 of 12 cases received hybrid induction therapy achieved CR. No significant difference was found in 5 year-OS rates between cases with ALL induction therapy and hybrid induction therapy ((77±15)% vs. (80±13)%, χ2=0.027， P=0.870). Conclusions:MPAL is a rare childhood leukemia and is prone to incorporate complex karyotypes. Induction therapy with ALL or hybrid regimens is a good choice to obtain favorable prognosis.