Carrier-Envelope Phase-Dependent Strong-Field Excitation.

We present a joint experimental-theoretical study on the effect of the carrier-envelope phase (CEP) of a few-cycle pulse on the atomic excitation process. We focus on the excitation rates of argon at intensities in the transition between the multiphoton and tunneling regimes. Through numerical simulations, we show that the resulting bound-state population is highly sensitive to both the intensity and the CEP. The experimental data clearly agree with the theoretical prediction, and the results encourage the use of precisely tailored laser fields to coherently control the strong-field excitation process. We find a markedly different behavior for the CEP-dependent bound-state population at low and high intensities with a clear boundary, which we attribute to the transition from the multiphoton to the tunneling regime.

[Levels of human milk oligosaccharides in breast milk of mothers delivering preterm infants of different gestational ages and their effects on early growth and development].

Objective: To investigate the human milk oligosaccharides (HMOs) levels in breast milk of mothers delivering preterm infants and their effects on the early growth and development of infants. Methods: In this prospective cohort study, full-term and preterm newborns whose parents decided to breastfeed were recruited from Peking University Third Hospital between December 1, 2017 and November 30, 2018. The preterm infants were divided based on their gestational ages into extremely preterm (<28 weeks), very preterm (28-31+6 weeks) and moderate to late preterm (32-36+6 weeks) groups. Breast milk was collected from mothers at 7, 14, 28 and 120d postpartum. 368 breast milk samples were collected from 125 mothers in this study, including 54 mothers of full-term infants, 23 mothers of moderate to late preterm infants, 39 mothers of very preterm infants, and 9 mothers of extremely preterm infants. Ultra-performance liquid chromatography-mass spectrometer (UPLC-MS/MS) was used to determine the concentration of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 3'-sialyllactose (3'SL), A-tetrasaccharide (P1), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose Ⅱ (LNFP-Ⅱ) and lacto-N-fucopentaose Ⅴ (LNFP-Ⅴ). Secretor status of mothers was defined as 2'-fucosyllactose (2'FL) concentration in colostrum and transitional milk greater than 200 µg/mL. Weight gain and the occurrence of allergic diseases of infants were collected at 120 d(4 months) postpartum. The chi-square test or the Fisher's exact test was used for the comparison of categorical data between groups; Kruskal-Wallis test and Wilcoxon rank sum test were used for comparison of continuous data between groups. Nemenyi test was used for multiple comparison. Results: 79.2% (99/125) of the mothers were secretor. There were no statistical differences between groups in the secretor status of mothers (χ²=1.31,P>0.05). The total concentration of HMOs peaked at 1-2 weeks postpartum. Compared to the preterm milk, the HMOs from the term milk was trending downwards at an earlier time. In the breast milk of secretor mothers on 28 d, total concentration of HMOs significant differed among the three groups of preterm milk and the term milk, with the median value of 4 587.09,4 615.25,5 277.44,5 476.03 µg/mL, respectively (Kruskal-Wallis χ²=8.1234,P=0.044). When analyzed by the median weight gain of the infants (low vs high weight gain) at 4 months postpartum, 2'FL was significantly lower in the high weight gain group at 7 d (1 818.04 µg/mL vs 2 181.67 µg/mL, W=1 386,P=0.018), while LNT & LNnT were significantly higher (1 182.36 µg/mL vs 1 053.62 µg/mL, W=816,P=0.044). The level of 3FL at 120 d was significantly affected by presence of allergic disease in infants, breast milk from mothers of infants with allergic disease had lower 3FL than those from mothers of infants without allergic disease (256.17 µg/mL vs 286.18 µg/mL, W=564,P=0.026). Conclusions: The overall profiles of HMOs in breast milk of mothers delivering preterm infants was basically the same as that of mothers delivering term infants; individual HMOs play a role in weight gain and the development of allergic diseases in preterm infants, but the mechanism is unclear and needs further study.

[Clinical characteristics of ureaplasma urealyticum pneumonia in preterm infants with gestational age less than 32 weeks].

Objective: To investigate the clinical characteristics of ureaplasma urealyticum(UU) pneumonia in preterm infants less than 32 weeks. Methods: Preterm infants with gestational age <32 weeks who were hospitalized in neonatal intensive care unit (NICU) of Peking University Third Hospital from January 2018 to December 2019 were retrospectively analyzed. There were 105 premature infants, 63 males and 42 females. After the first diagnosis of pneumonia during hospitalization, the airway secretions were collected for UU nucleic acid detection. They were divided into UU positive group and UU negative group. Perinatal factors, comorbidities, antibiotic treatment and clinical outcomes were compared between the two groups. SPSS24.0 statistical software was used for statistical analysis. T test or chi-square test was used to compare the two groups, and logistic regression was used for multivariate analysis. Results: Among 105 cases of preterm pneumonia, 37 cases (35.2%) were diagnosed with UU pneumonia and 68 cases (64.8%) were negative for UU test. There was no significant difference in gestational age [28(27,30) weeks vs 29(28,30)weeks,Z=-0.98, P>0.05] and birth weight[(1 282.03±292.49)g vs (1 196.62±322.89)g,t=1.34, P>0.05] between the two groups. In UU pneumonia group, the rate of singleton (86.5% vs 50%,χ2=12.15), chorioamnionitis (10.8% vs 1.55%,χ2=4.61), premature rupture of membranes>12 h (32.4% vs 11.8%,χ2=5.37) and vaginal delivery rate(59.5% vs 35.3%,χ2=4.75) were higher than UU negative group (P<0.05). Further multivariate logistic regression analysis showed that vaginal delivery was an independent risk factor for UU (OR = 2.694, 95%CI: 1.113-6.525). WBC count in UU positive group was significantly higher [12.85×109/L (9.32×109/L,17.22×109/L) vs 9.06×109/L (7.06×109/L,13.37×109/L), Z=-3.01, P<0.05], and oxygen consumption time was prolonged[ (46.8±19.8)d vs (37.8±20.7)d, t=2.177,P<0.05]. The incidence of hemodynamically significant patent ductus arteriosus (29.7% vs 57.4%,χ2=6.265) and respiratory distress syndrome (54.1% vs 75.0%,χ2=4.801) in UU positive group was significantly lower than that in UU negative group (P<0.05). There was no significant difference in bacterial infection(62.2% vs 50.0%, χ2=8.826) and antibiotic(48.6% vs 47.1%,χ2=1.352) between the two groups(all P>0.05). After azithromycin treatment, the time for UU negative was (9.00±3.14) days. There was no significant difference in the incidence of bronchopulmonary dysplasia(73.0% vs 69.1%,χ2=0.036), retinopathy of prematurity(10.8% vs 26.5%,χ2=2.665), neonatal necrotizing enterocolitis(2.7% vs 1.5%,χ2=0.195), intraventricular periventricular hemorrhage (69.4% vs 72.1%,χ2=0.003) and periventricular leukomalacia (8.1% vs 8.8%,χ2=0.016) between the two groups (P>0.05). Conclusions: If premature rupture of membranes >12 h, combined with chorioamnionitis, and vaginal delivery, preterm infants less than 32 weeks are likely to have an increased risk of UU infection. UU pneumonia in preterm infants less than 32 weeks old was characterized by prolonged oxygen consumption and increased white blood cell count. Oral azithromycin treatment could effectively remove UU and improve prognosis.

[Reference ranges for Doppler echocardiographic measurements within seven days of age in preterm infants].

Objective: To establish reference ranges for Doppler echocardiography in preterm infants within 7 days after birth based on different gestational age (GA), birth weight (BW) and body surface area (BSA). Methods: This retrospective study analyzed Doppler echocardiographic measurements of 489 premature infants, who were admitted to the neonatal Intensive Care Unit of Department of Pediatrics, Peking University Third Hospital from March 2017 to February 2020. These infants were divided into four groups according to GA:<28 weeks, 28-31+6 weeks, 32-33+6 weeks and 34-36+6 weeks; and five groups according to BW:<1 000 g, 1 000-1 499 g, 1 500-1 999 g, 2 000-2 499 g and ≥ 2 500 g;and 14 groups according to BSA from 0.07-0.20 m2. The Doppler values among groups were compared by independent sample KW test, and based on which, the 95%CI were established as reference ranges. Results: Among the 489 preterm infants, males were 264 and females were 225. Their GA, BW and BSA were 32.0 (30.0,33.9) weeks, 1 700 (1 260,2 040) g and 0.13 (0.11,0.15)m2, respectively. Measurements are presented charting as 95%CI with respect to GA,BW and BSA for preterm infants aged 0-7 days. Aortic valve(AV) flow rate, mitral valve E peak (MV-E), mitral valve E/A (MV-E/A) and tricuspid valve E peak (TV-E) were all correlated with GA, BW and BSA (r = 0.263, 0.256, 0.324 and 0.114 for GA; 0.292, 0.261, 0.281 and 0.135 for BW; 0.287, 0.268, 0.312 and 0.140 for BSA, all P<0.05). Within the first 7 days after birth, the greater the GA, birth weight and BSA, the greater the AV, MV-E, MV-E/A and pulmonary valve flow rate(all P<0.05). According to the above grouping of GA, the 95%CI of AV were 48-54, 52-57, 58-63 and 60-65 cm/s, respectively; and the 95%CI of MV-E were 32-37, 33-36, 39-42 and 40-45 cm/s, respectively; and the 95%CI of MV-E/A were 0.66-0.73, 0.74-0.80, 0.81-0.90 and 0.92-1.06. And according to the above grouping of BW, the 95%CI of AV were 45-53, 49-53, 59-64, 60-66 and 56-65 cm/s, respectively; 95%CI of MV-E were 29-35, 32-36, 38-41, 40-44 and 38-46 cm/s, respectively; 95%CI of MV-E/A were 0.65-0.74, 0.74-0.81, 0.81-0.99, 0.86-0.99 and 0.84-1.07. Conclusion: The 95%CI of Doppler echocardiographic measurements established based on GA, BW and BSA could provide a reference for preterm infants aged 0-7 days.

Deexcitation Dynamics of Muonic Atoms Revealed by High-Precision Spectroscopy of Electronic K X Rays.

We observed electronic K x rays emitted from muonic iron atoms using superconducting transition-edge sensor microcalorimeters. The energy resolution of 5.2 eV in FWHM allowed us to observe the asymmetric broad profile of the electronic characteristic Kα and Kß x rays together with the hypersatellite K^{h}α x rays around 6 keV. This signature reflects the time-dependent screening of the nuclear charge by the negative muon and the L-shell electrons, accompanied by electron side feeding. Assisted by a simulation, these data clearly reveal the electronic K- and L-shell hole production and their temporal evolution on the 10-20 fs scale during the muon cascade process.

[Reference ranges for M-mode echocardiographic measurements within seven days after birth in preterm infants].

Objective: To establish reference ranges for M-mode echocardiography in preterm infants within 7 days after birth based on different gestational age (GA) and birth weight. Methods: This retrospective study analyzed M-mode echocardiographic values of 489 premature infants, who were admitted to the neonatal intensive care unit of Department of Pediatrics, Peking University Third Hospital from March 2017 to February 2020. These infants were divided into four groups according to GA:<28 weeks, 28-31+6weeks, 32-33+6weeks and 34-36+6weeks; and five groups according to birth weight:<1 000 g, 1 000-1 499 g, 1 500-1 999 g, 2 000-2 499 g and ≥2 500 g. The M-mode values among groups were compared by independent sample K-W test, and based on which, the 95% confidence interval (CI) and the Z-value reference ranges were established. Results: The gestational age of these infants was 32.0 (24.0-36.7) weeks, and the birth weight was 1 700 (650-3 180) g. The interventricular septum end-diastolic thickness (IVSd), left ventricular posterior wall end-diastolic thickness (LVPWd), left atrial diameter (LAD), left ventricular end-diastolic diameter (LVED), left ventricular end-systolic diameter (LVES), right ventricular outflow tract (RVOT) and the right ventricular end-diastolic diameter (RVED), were all correlated with GA and birth weight (r = 0.209, 0.216, 0.430, 0.608, 0.495, 0.464, 0.447; r = 0.275, 0.288, 0.445, 0.609, 0.496, 0.499, 0.464;all P<0.01). While the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) had no correlation with GA or birth weight (all P<0.05). Within the first 7 days after birth, the greater the GA and birth weight, the greater the inner diameters of the heart chambers, and the thicker the ventricular wall. The LVEF and LVFS maintained a high and stable level within the first week of life (95%CI: 67%-69%, 34%-36%). Conclusions: According to different GA and birth weight, the reference ranges for chamber diameters, interventricular septal thickness and left ventricular wall thickness within 7 days were established. The 95% CI and Z score ranges for M-mode echocardiographic measurements established based on gestational age and birth weight can provide a reliable reference for preterm infants aged 0-7 days.

[Efficacy of minimally invasive pulmonary surfactant administration in preterm infants with neonatal respiratory distress syndrome: a multicenter clinical trial].

Objective: To explore the feasibility and safety of minimally invasive surfactant administration (MISA) in preterm neonates with respiratory distress syndrome (NRDS). Methods: In this multicenter prospective randomized controlled trial, 92 preterm infants with gestation age ≤30 weeks and diagnosed with NRDS were enrolled in 8 level Ⅲ neonatal intensive care units (NICU) in Beijing-Tianjin-Hebei Region from 1(st) July 2017 to 31(st) December 2018. They were randomly assigned to minimally invasive surfactant administration (MISA) group or endotracheal intubation surfactant administration (EISA) group according to random number generated by computer. Infants in both groups received calf pulmonary surfactant preparation at a dose of 70-100 mg/kg. The data of demography, perinatal situation, medication administration, complications, clinical outcomes in the two groups were compared with Chi-square test, Student's t-test, Mann-Whitney U test or Fisher's exact test. Results: Among the 92 preterm infants, 53 were males, 39 were females; 47 were in the MISA group (25 males), and 45 were in the EISA group (28 males). The gestational age and birth weight were (29.5±1.2) weeks and (1 271±242) g in all patients, (29.5±1.4) weeks and (1 285±256) g in the MISA group, and (29.6±0.9) weeks and (1 255±227) g in the EISA group. The duration of surfactant infusion and the length of whole procedure in the MISA group were significantly longer than that in the EISA group (60 (18, 270) s vs. 50 (30, 60) s, Z=3.009, P=0.003; 90 (60, 300) s vs. 60 (44, 270) s, Z=3.365, P=0.001). For the outcomes, the incidence of hemodynamically significant patent ductus arteriosus (hsPDA) and bronchopulmonary dysplasia (BPD) were lower in the MISA group than in the EISA group (36% (17/47) vs. 67% (30/45), χ(2)=8.556, P=0.003; 26% (12/47) vs. 47% (21/45), χ(2)=4.464, P=0.035). Conclusions: Minimally invasive surfactant administration is applicable in preterm infants ≤30 weeks gestational age with NRDS. Although the length of whole procedure is longer than route endotracheal administration, the benefit of decreasing the incidences of hsPDA and BPD outweighs this demerit.

[Clinical application of vitrification freezing of oocytes].

Objective: To evaluate the effect of oocyte vitrification on embryo quality and developmental potential. Methods: From January 2014 to December 2017, 30 cases of oocytes vitrification in the reproductive center of Sir Run Run Shaw hospital were collected as the frozen group, due to failure of sperm extraction or inability to obtain sufficient sperm at the ovum pick-up-day because of oligoospermia or azoospermia. 220 cases of fresh oocytes from the same period were selected as the fresh group. The fertilization rate, embryo rate, pregnancy rate, implantation rate and live birth rate of the two groups were compared retrospectively. Results: The survival rate of oocyte resuscitation was 91.4% (180/197). In the frozen group, 24 cases were transferred with 14 pregnancies, while in the fresh group, 31 cases were transferred with 18 pregnancies. The number of 2PN fertilized eggs, 2PN embryos and 2PN high-quality embryos in the frozen group was significantly lower than that in the fresh group (3.7±2.5 vs 7.3±4.8), (3.3±2.5 vs 7.2±4.8), and (1.2±1.8 vs 2.9±2.7) (all P<0.05). The fertilization rate of the frozen group was 77.2% (115/149) and the high-quality embryo rate was 36.6% (37/101), lower than that of the fresh group 77.6% (1 637/2 109) and 40.9% (651/1 591) (P>0.05).The pregnancy rate of the frozen group was 58.3% (14/24) higher than that of the fresh group 58.1% (18/31), the implantation rate of each mature oocyte and the live birth rate of each mature oocyte were 10.8% (15/138) and 10.8% (15/138), both higher than that of the fresh group 9.6% (21/218) and 8.7% (19/218) (all P>0.05). Conclusions: Vitrification cryopreservation of oocytes may lead to a decrease in embryo quality, but embryo development potential is still considerable. Higher pregnancy rate, implantation rate and live birth rate may be obtained.

Effects of a nutrient-dense formula compared with a post-discharge formula on post-discharge growth of preterm very low birth weight infants with extrauterine growth retardation: a multicentre randomised study in China.

BACKGROUND: Post-discharge optimal growth and nutritional intake have beneficial effects for neurodevelopment in preterm very low birth weight infants (VLBWIs) with extrauterine growth retardation (EUGR). The present study aimed to compare the effects of a nutrient-dense formula (NDF) to a post-discharge formula (PDF) on post-discharge growth of preterm VLBWIs with EUGR. METHODS: Forty-eight preterm VLBWIs with EUGR at discharge were randomised to receive NDF (100 kcal per 100 mL; 2.6 g protein per 100 mL) or PDF (74 kcal per 100 mL; 1.95 g protein per 100 mL) for 1-6 months until body weight reached the 50th percentile on growth charts with corrected age. Volume, nutrient intake, anthropometry and biochemistry data were collected. RESULTS: Volume intake was lower in the NDF group than the PDF group during the first 2 months of feeding (P = 0.039 and 0.018, respectively).There were no significant differences in volume intake during months 2-6 of feeding. Energy, protein, carbohydrate and fat intake were higher in the NDF group during months 1-6 of feeding. There were no significant differences in weight, length, and head circumference Z-scores during months 1-6 between the two groups. The △length Z-score from discharge to month 6 was significantly higher in the NDF group than the PDF group (P = 0.043). No differences existed between the two groups with respect to biochemistry. CONCLUSIONS: After discharge, preterm VLBWIs with EUGR fed a NDF gain anthropometric parameter Z-scores similar to those for a PDF within 6 months of follow-up. A NDF leading to gain in length requires further follow-up.

Effects of different drainage methods on serum bile acid and hepatocyte apoptosis and regeneration after partial hepatectomy in rats with obstructive jaundice.

The aim of this study was to investigate the mechanism of hepatocyte apoptosis and regeneration after partial hepatectomy in obstructive jaundice (OJ) rats under different drainage methods of bile acid intervention. Forty male Sprague Dawley rats were randomly divided into five groups. An OJ rat model was established by the following protocols. Seven days after obstruction, an SD rats model with 70% partial hepatectomy was established by different drainage methods of OJ. Blood and liver tissue samples were collected from rats 72 h after surgery; 72 h after partial hepatectomy (PH), the liver regeneration rate, the expression of proliferating cell nuclear antigen (PCNA) and the level of mitotic index (MI) in the internal biliary drainage (IBD) group were higher than those in external biliary drainage (EBD) group (P less than 0.05). Those in the EBD group were higher compared to the OJ group (P less than 0.05). There was no significant difference among the IBD group, EBD+CA group and (SO) sham operation group (P>0.05). Bax expressions had the same trend as AI in the five groups. The expression of Bcl-2 was increased in the IBD group and EBD+CA group, which was statistically higher compared to the SO group (P less than 0.05). In conclusion, both internal and external drainage can relieve biliary obstruction. The difference in liver regeneration caused by external drainage and internal drainage may be attributed to the destruction of bile acid enterohepatic circulation, which increases hepatocyte apoptosis and affects liver regeneration.

The interaction of excited atoms and few-cycle laser pulses.

This work describes the first observations of the ionisation of neon in a metastable atomic state utilising a strong-field, few-cycle light pulse. We compare the observations to theoretical predictions based on the Ammosov-Delone-Krainov (ADK) theory and a solution to the time-dependent Schrödinger equation (TDSE). The TDSE provides better agreement with the experimental data than the ADK theory. We optically pump the target atomic species and measure the ionisation rate as the a function of different steady-state populations in the fine structure of the target state which shows significant ionisation rate dependence on populations of spin-polarised states. The physical mechanism for this effect is unknown.

Precise and Accurate Measurements of Strong-Field Photoionization and a Transferable Laser Intensity Calibration Standard.

Ionization of atoms and molecules in strong laser fields is a fundamental process in many fields of research, especially in the emerging field of attosecond science. So far, demonstrably accurate data have only been acquired for atomic hydrogen (H), a species that is accessible to few investigators. Here, we present measurements of the ionization yield for argon, krypton, and xenon with percent-level accuracy, calibrated using H, in a laser regime widely used in attosecond science. We derive a transferable calibration standard for laser peak intensity, accurate to 1.3%, that is based on a simple reference curve. In addition, our measurements provide a much needed benchmark for testing models of ionization in noble-gas atoms, such as the widely employed single-active electron approximation.

Attosecond Coherent Control of Single and Double Photoionization in Argon.

Ultrafast high harmonic beams provide new opportunities for coherently controlling excitation and ionization processes in atoms, molecules, and materials on attosecond time scales by employing multiphoton two-pathway electron-wave-packet quantum interferences. Here we use spectrally tailored and frequency tuned vacuum and extreme ultraviolet harmonic combs, together with two phase-locked infrared laser fields, to show how the total single and double photoionization yields of argon can be coherently modulated by controlling the relative phases of both optical and electronic-wave-packet quantum interferences. This Letter is the first to apply quantum control techniques to double photoionization, which is a fundamental process where a single, high-energy photon ionizes two electrons simultaneously from an atom.

Cystic fibrosis transmembrane conductance regulator is correlated closely with sperm progressive motility and normal morphology in healthy and fertile men with normal sperm parameters.

Cystic fibrosis transmembrane conductance regulator (CFTR) has been demonstrated to be expressed in mature spermatozoa and correlated with sperm quality. Sperm CFTR expression in fertile men is higher than that in infertile men suffering from teratospermia, asthenoteratospermia, asthenospermia and oligospermia, but it is unknown whether CFTR is correlated with sperm parameters when sperm parameters are normal. In this study, 282 healthy and fertile men with normal semen parameters were classified into three age groups, group (I): age group of 20-29 years (98 cases, 27.1 ± 6.2), group (II): age group of 30-39 years (142 cases, 33.7 ± 2.6) and group (III): age group of more than or equal to 40 years (42 cases, 44.1 ± 4.6). Sperm concentration, total count and progressive motility were analysed by computer-assisted sperm analysis. Sperm morphology was analysed by modified Papanicolaou staining. Sperm CFTR expression was conducted by indirect immunofluorescence staining. There was a significant positive correlation (P < 0.001) between CFTR expression and sperm progressive motility (r = 0.221) and normal morphology (r = 0.202), but there were no correlations between sperm CFTR expression and semen volume, sperm concentration, sperm total count as well as male age (P > 0.05). Our findings show that CFTR expression is associated with sperm progressive motility and normal morphology in healthy and fertile men with normal sperm parameters, but not associated with the number of spermatozoa and male age.

Controlling the XUV transparency of helium using two-pathway quantum interference.

Atoms irradiated with combined femtosecond laser and extreme ultraviolet (XUV) fields ionize through multiphoton processes, even when the energy of the XUV photon is below the ionization potential. However, in the presence of two different XUV photons and an intense laser field, it is possible to induce full electromagnetic transparency. Taking helium as an example, the laser field modifies its electronic structure, while the presence of two different XUV photons and the laser field leads to two distinct ionization pathways that can interfere destructively. This work demonstrates a new approach for coherent control in a regime of highly excited states and strong optical fields.

Laser-enabled Auger decay in rare-gas atoms.

In rare-gas atoms, Auger decay in which an inner-valence shell ns hole is filled is not energetically allowed. However, in the presence of a strong laser field, a new laser-enabled Auger decay channel can open up to increase the double-ionization yield. This process is efficient at high laser intensities, where an ns hole can be filled within a few femtoseconds of its creation. This novel laser-enabled Auger decay process is of fundamental importance for controlling electron dynamics in atoms, molecules, and materials.

Anomalous bumpy structures in the capture cross sections of antiprotons by helium.

We investigate the state-specified capture process of antiprotons by helium. Freezing one of the two electrons, we reduce this four-body rearrangement problem into a three-body problem. The capture cross sections are calculated by solving the Chew-Goldberger-type integral equation. Differing from the capture of antiprotons by hydrogen atoms, the bumpy structures are revealed in the total angular momentum dependent capture cross sections. Further analysis shows that the bumps arise from the partial channel closing due to the removal of the energy degeneracy in the antiprotonic helium.

Construction of expression vector of hTERT- hIL18 fusion gene and induction of cytotoxic T lymphocyte response against hTERT.

AIM: Recombiant human telomerase reverse transcriptase (hTERT)-human IL-18(hIL18) was constructed to investigate its expression and biological function in eukaryotic cells. DCs transfected with hTERT-IL18 acquired stronger telomerase activity and were able to elicit an hTERT-specific cytotoxic T lymphocyte CTL response in vitro. METHODS: hIL-18 gene fragment was amplified by polymerase chain reaction (PCR) and TA cloned. The hIL-18 gene was then subcloned into eukaryotic expression vector pcDNA3.1(+) containing human TERT via a linker. The sequence of gene fusion was confirmed using both restrictive enzyme digestion and DNA sequencer. The expression vector with gene fusion was transfected into 3T3 cell line with Lipofectamine 2000. ELISA, Western blot, immunofluorescence stain were performed to determine the expression properties of hTERT-hIL18 in 3T3 cells. Its biological effect on the anti-apoptosis was measured by Flow cytometry and its effect on INF-gamma expression was determined using ELISA. After preparation of dendritic cells, hTERT-hIL18, hTERT, hIL-18 expression vectors were transfected into DCs respectively by electroporation to generate hTERT-specific DCs lines. The peripheral blood mononuclear cells PBMCs were stimulated with different DCs lines to create specific CTL. The response of target cell (leukemia cell line-K562 cell) to hTERT-specific CTL was evaluated by LDH release assay. RESULTS: The human IL-18 gene fragment was amplified from the human mononuclear cells and was inserted into pcDNA3.1(+)/hTERT vector successfully. The correct sequence was proved by both restrictive enzyme digestion and sequencing. The correct open reading frame was also verified. Fusion protein of hTERT-hIL18 was effectively expressed in eukaryotic cells, which was detected by both Western-blotting and immunofluorescence stain. The expressed recombinant fusion protein induced similar levels of INF-gamma to that of native IL-18 protein. FCM assay showed that the transfected fusion protein inhibited the apoptosis, which was consistent with the effects of hTERT as a universal tumor associated antigen. CTL assay shows that hTERT- hIL18 and hTERT gene-transfected DCs stimulated T-cell responses that recognized and lysed tumor target cells of high hTERT expression, whereas DCs transfected with hIL-18 gene didn't induced the response of tumor targets lyses. CONCLUSION: The Recombinant hTERT- hIL18 fusion protein had both biological activity of hTERT and hIL-18, indicating that this rationally designed protein can be further developed as novel tumor therapeutics. DCs transfected with hTERT-IL18 gene were capable of eliciting a stronger hTERT-specific CTL response in vitro.