BackgroundNAD+, nicotinamide adenine dinucleotide, is mostly described to associate with the aging process. We aimed to investigate the association between human whole blood NAD+ contents and aging in a relative large-scale community-based population and further to address the gender impact on this association.MethodsWe recruited 1,518 participants aged over 18 years old and free of cardiovascular and any type of cancer from the Jidong community from 2019 to 2020. Whole blood NAD+ level was measured by cycling assay and LC-mass spectroscopy assay. The chronological age and clinical data were collected using standard questionnaires. The participants were divided into five groups according to their chronological age. General liner regression model was performed to analyze the association between NAD+ contents and aging. In addition, we also conducted subgroup analysis by gender.ResultsThe mean age of included 1,518 participants was 43.0 years, and 52.6% of them were men. The average levels of whole blood NAD+ of total participants was 33.0 ± 5.5 μmol/L. The whole blood NAD+ contents in men were significantly higher than that in women (34.5 vs. 31.3 μmol/L). There was significant difference in the meat diet among NAD+ quartile groups (p = 0.01). We observed a decline trend of NAD+ contents with aging before 50 years in total participants with significant level in 40–49 years old group (β coefficients with 95% confidence interval (95% CI): −1.12 (−2.18, −0.06)), while this trend disappeared after the 50 years. In addition, this association was significantly altered by gender (p for interaction = 0.003). In men, as compared with ≤29 years group, adjusted β coefficient decreased with aging but was only significant in the ≥60 year group (β,−2.16; 95% CI, −4.16 to −0.15). In females, the level of whole blood NAD+ did not significantly differ among five age groups and without the trend as males.ConclusionsAssociation of whole blood NAD+ contents with aging significantly differed in males and females. The loss of blood NAD+ with aging only was observed in males, especially in the male middle-aged population. It is crucial to consider the gender difference in further NAD+ related studies in the future.

Transmembrane protein 30A (Tmem30a) is the β-subunit of P4-ATPases which function as flippase that transports aminophospholipids such as phosphatidylserine from the outer to the inner leaflets of the plasma membrane to maintain asymmetric distribution of phospholipids. It has been documented that deficiency of Tmem30a led to exposure of phosphatidylserine. However, the role of Tmem30a in vivo remains largely unknown. Here we found that Vav-Cre-driven conditional deletion of Tmem30a in hematopoietic cells led to embryonic lethality due to severe anemia by embryonic day 16.5. The numbers of erythroid colonies and erythroid cells were decreased in the Tmem30a deficient fetal liver. This was accompanied by increased apoptosis of erythroid cells. Confocal microscopy analysis revealed an increase of localization of erythropoietin receptor to areas of membrane raft microdomains in response to erythropoietin stimulation in Ter119−erythroid progenitors, which was impaired in Tmem30a deficient cells. Moreover, erythropoietin receptor (EPOR)-mediated activation of the STAT5 pathway was significantly reduced in Tmem30a deficient fetal liver cells. Consistently, knockdown of TMEM30A in human CD34+ cells also impaired erythropoiesis. Our findings demonstrate that Tmem30a plays a critical role in erythropoiesis by regulating the EPOR signaling pathway through the formation of membrane rafts in erythroid cells.

目的：探讨2型糖尿病（T2DM）患者血浆C反应蛋白（CRP）水平及其影响因素。方法将T2DM 患者按照年龄、性别、不同肥胖程度分组，并比较各组患者的血浆CRP水平，采用多元线性回归分析CRP的影响因素。结果 T2DM患者血浆CRP水平随着年龄、肥胖程度增加而升高，且与空腹血糖（FPG）、高密度脂蛋白胆固醇（HDL－C）、低密度脂蛋白胆固醇（LDL－C）等水平相关（P＜0．05）。结论 T2DM患者血浆CRP水平受多种因素的影响。

In the mouse embryo, the aorta-gonad-mesonephros (AGM) region is considered to be the sole location for intraembryonic emergence of hematopoietic stem cells (HSCs). Here we report that, in parallel to the AGM region, the E10.5-E11.5 mouse head harbors bona fide HSCs, as defined by long-term, high-level, multilineage reconstitution and self-renewal capacity in adult recipients, before HSCs enter the circulation. The presence of hemogenesis in the midgestation head is indicated by the appearance of intravascular cluster cells and the blood-forming capacity of a sorted endothelial cell population. In addition, lineage tracing via an inducible VE-cadherin-Cre transgene demonstrates the hemogenic capacity of head endothelium. Most importantly, a spatially restricted lineage labeling system reveals the physiological contribution of cerebrovascular endothelium to postnatal HSCs and multilineage hematopoiesis. We conclude that the mouse embryonic head is a previously unappreciated site for HSC emergence within the developing embryo.

Objective to study the role of Cramp gene in the self-renew and differentiation of hematopoietic stem cells(HSCs).Methods cells from the bone marrow,spleen and thymus were analyzed by flow cytometry.HSCs were sorted and cultured in vitro.Results Compared with wild type mice,there are no great differences in the cell percentage and function of bone marrow,spleen and thymus in Cramp over expression mice.Conclusions Compared with the wild type mice,there was no great difference in the HSCs` differentiation and single cell clone in Cramp over expression transgenic mice.

OBJECTIVE Recent studies have identified a set of serological markers for telomere dysfunction and DNA damage. The relevance of these serological markers in type 2 diabetes remains elusive. We investigated the association of serological markers (elongation factor 1α [EF-1α], stathmin, and N-acetyl-glucosaminidase) with leukocyte telomere length, a functional variant of uncoupling protein-2 (UCP2), and susceptibility of type 2 diabetes. RESEARCH DESIGN AND METHODS A total of 930 patients and 867 control subjects were recruited to examine the association between leukocyte telomere length, UCP2 variant (−886G>A), recently identified serological markers, and type 2 diabetes. Telomere length was determined by a quantitative real-time PCR–based assay. EF-1α, stathmin, and C-reactive proteins were measured by enzyme-linked immunosorbent assays. N-acetyl-glucosaminidase was measured by an enzyme activity assay. The UCP2 variant was determined by PCR and restriction enzyme digestion. RESULTS The average telomere length of type 2 diabetic patients was significantly shorter than that of control subjects. Serological N-acetyl-glucosaminidase correlates with both age and telomere length and was significantly higher in patients than in control subjects. Neither EF-1α nor stathmin showed significant difference between patients and control subjects. The UCP2–886G>A variant correlated with type 2 diabetes status but did not correlate with telomere length or the serological markers. Multivariate analysis showed that higher serological N-acetyl-glucosaminidase, shorter telomeres, and the UCP2–886G>A variant are independent risk factors for type 2 diabetes. CONCLUSIONS Serological N-acetyl-glucosaminidase, telomere length, and the UCP2–886G>A variant are independent risk factors for type 2 diabetes. Serological N-acetyl-glucosaminidase correlates with telomere length but not with the UCP2–886G>A variant.

Objective To study the role of WIP1 gene in mouse B cell and T cell development.Methods The proportion of the B cell in bone marrow and T cell in the thymus were determined by the flow cytometry.Results Although the proportion of different subsets in B cell development was normal,the total number of B cell in bone number was decreased.WIP1 gene knockout mice had defect in thymus development,the proportion of CD8/CD4 double negative cells was increased,but the CD8/CD4 double positive cells was decreased.Conclusion WIP1 gene plays an important role in the development of B cell and T cell.

Objective To investigate the influence of Exo-1 gene deletion on the engraftment of hematopoietic stem cell(HSC) transplantation by different methods into the telomerase knockout mice.Methods Bone marrow cells or KSL((c-kit +,Sca-1 +,lineage-) cells were isolated from CD45.1 mice,and then injected into recipients,which were telomer-ase gene knockout or Exo-1 and telomerase gene double knockout mice with or without irradiation.At 1 month later,pe-ripheral blood was collected and analyzed by flow cytometry.Results Low chimerisms of donor-derived cells were ob-served in recipients without irradiation or with 1 Gy,2 Gy irradiation.With 6 Gy irradiation,the chimerisms were below 50%,and died in 1 month.With 3 Gy irradiation,most mice lived with high chimerisms.Conclusions 3 Gy X-ray irra-diation gives rise to high chimerisms in telomerase knockout mice,whereas Exo-1 gene does not further increase the chimer-isms of HSC transplantation.

Objective To test the Flow fluorescent in situ hybridization(Flow-Fish) method for measuring the length of telomeres. Methods The peripheral blood mononuclear cells were separated from 3rd generation telomerase knockout mice and wild-type mice. Cells were hybridized with PNA fluorescent probe and their telomere length was analyzed by flow cytometry. To confirm the Flow-Fish method we compared the measurement of telomere length obtained with the Flow-Fish method with that determined with conventional SYBR Green fluorescence quantitative PCR assay and Q-Fish method. Results The relative telomere length measured by Flow-Fish was 0. 5345,which was accordant to the results measured by fluorescence quantitative PCR assay (0. 5717). Conclusion These results show that Flow-Fish can be used to measure specific nucleotide repeat sequences in single cells efficiently and easily.

Objective To construct lentiviral short hairpin RNA(shRNA) expression vectors and have them expressed efficiently in the hematopoietic system of mice.Methods p-CMV and p-SFFV lentiviral vectors,PAX2 and PMD2G auxiliary vectors were mixed before 293T packaging cells were cotransfected by calcium phosphate to produce lentiviral vectors to infect HSCs of mice isolated by fluorescence-activated cell sorting(FACS).The p-SFFV lentiviral vector which had higher transfection efficiency was used to infect HSCs of mice before infected cells were delivered into lethally irradiated mice.Four weeks and nine weeks after transplantation,the murine peripheral blood was harvested.Sixteen weeks after transplantation,the murine bone marrow was harvested.The proportion of donor cells was analyzed by FACS.Results The p-SFFV lentiviral vector was successfully restructured which could be effectively expressed in mouse bone marrow hematopoietic stem cells(infection efficiency was 72.4%).The lentiviral vector carrying exogenous shRNA could be expressed in the murine hematopoietic system chronically and stably(infection efficiency was 92%).Conclusion The recombinant shRNA lentiviral vector can be expressed in the murine hematopoietic system.

Objective To establish systemic expression Cramp transgenic mice,which can be used for the study of its function and effect on aging.Methods The construct was generated by inserting the murine Cramp cDNA into a vector with CMV promoter.The transgenic mice were created by the method of microinjection.The genotype of transgenic lines was identified by PCR and the expression level of the gene was determined by RT-PCR and Western blotting.Results Cramp cDNA transgenic vector was successfully constructed and Cramp transgenic mice were created.Three high expression lines were confirmed by RT-PCR and western blotting.Conclusion The systemic expression Cramp transgenic mouse was successfully established,and the Cramp gene was high expression in the tissue,such as bone marrow,spleen,and liver.The transgenic mouse could be a useful model for study its function in aging.